JOURNAL OF THE ITALIAN SOCIETY OF ANATOMIC PATHOLOGY AND DIAGNOSTIC CYTOPATHOLOGY, ITALIAN DIVISION OF THE INTERNATIONAL ACADEMY OF PATHOLOGY Periodico trimestrale - Aut. Trib. di Genova n. 75 del 22/06/1949 ISSN: 1591-951X (Online)

The GIPAD handbook of the gastrointestinal pathologist (in the Covid-19 era) - Part I

03VOL. 112

Edited by Paola Parente and Matteo Fassan SEPTEMBER 2020 Editor-in-Chief C. Doglioni G. Pelosi M. Barbareschi San Raffaele Scientific Institute, Milan University of Milan Service of Anatomy and M. Fassan F. Pierconti University of Padua Pathological Histology, Trento Catholic University of Sacred G. Fornaciari Heart, Rome Associate Editor University of Pisa M. Chilosi M.P. Foschini S. Pileri Department of Pathology, Verona Bellaria Hospital, Bologna Milano European Institute of University, Verona G. Fraternali Orcioni Oncology, Milan S. Croce e Carle Hospital, Cuneo 03Vol. 112 P. Querzoli Managing Editor E. Fulcheri St Anna University Hospital, Ferrara University of Genoa September 2020 P. N oz za L. Resta M. Guido Pathology Unit, Ospedali Galliera, University of Bari Genova, Italy University of Padua S. Lazzi G. Rindi Catholic University of Sacred Italian Scientific Board University of Siena L. Leoncini M. Brunelli Heart, Rome University of Siena E.D. Rossi University of Verona C. Luchini G. Bulfamante Catholic University of Sacred University of Verona University of Milano G. Magro Heart, Rome G. Cenacchi University of Catania A.G. Rizzo University of Bologna E. Maiorano “Villa Sofia-Cervello” Hospital, C. Clemente University of Bari Aldo Moro Palermo San Donato Hospital, Milano A. Marchetti G. Rossi M. Colecchia University of Chieti-Pescara Hospital S. Maria delle Croci, IRCCS National Cancer Institute, G. Marucci Azienda Romagna, Ravenna Milan Bellaria Hospital, Bologna P. Cossu-Rocca G. Martignoni G. Santeusanio University of Sassari IRCCS Sacro Cuore Don Calabria University of Rome “Tor Vergata G. d’Amati Hospital, Negrar, Verona L. Saragoni Sapienza University of Rome G. Negri Morgagni-Pierantoni Hospital, Forlì E. d’Amore Pathology Unit Central Hospital G. Tallini San Bortolo Hospital, Vicenza Bolzano University of Bologna A. D’Errico A. Orlandi L. Ventura S. Orsola-Malpighi Hospital, University of Rome “Tor Vergata” University of Bologna M. Paulli San Salvatore Hospital, L’Aquila P.A. Dei Tos University of Pavia; IRCCS San G. Zamboni University of Padua Matteo Hospital Foundation, Pavia University of Verona International Scientific Board Governing Board SIAPEC-IAP R. Alaggio President: Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, Pennsylvania, USA A. Sapino G. Bhagat University of Torino Columbia University, New York, USA Past President: A. Capitanio M. Truini Linköping University Hospital, Likping, Sweden Niguarda Cancer Center, Ospedale Niguarda Ca’ Granda, Milan T.V. Colby Mayo Clinic Scottsdale, AZ, USA President Elect: G. Cserni F. Fraggetta University of Szeged, Szeged, Hungary Cannizzaro Hospital, Catania, Italy S. Di Palma General Secretary: Royal Surrey County Hospital, Guilford, UK E. Bonoldi J. Fukuoka Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan Nagasaki University, Nagasaki, Japan Councillors: C. Giannini E. Bonoldi Department of Laboratory Medicine and Pathology Mayo Clinic Rochester, Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan MN, USA E. Fulcheri J. Kulka University of Genoa Semmelweis University, Budapest, Hungary A. Parafioriti T. Mentzel Gaetano Pini Hospital, Milan Dermatopathology Bodensee, Friedrichshafen, Baden-Württemberg Germany G.F. Zannoni M. Mihm Catholic University of Sacred Heart, Rome Brigham and Women’s Hospital, Harvard Medical School, Boston, Massa- chusetts, USA Representative L. Pantanowitz University/IRCCS: University of Pittsburgh Medical Center, Pittsburgh, PA, USA E. Maiorano M. Reyes-Múgica University of Bari UPMC Children’s Hospital of Pittsburgh; University of Pittsburgh School of Hospital/Individuals Medicine, Pittsburgh, PA, USA L. Molinaro F. Roncaroli City of Health and Science, Turin Imperial College of London, UK A. Skálová Junior counsellor: Charles Univesity, Plzen, Czech Republic V. L’Imperio M. Smith San Gerardo Hospital, University of Milano-Bicocca, Monza Mayo Clinic Scottsdale, AZ, USA Citology Board: S. Suster G. Fadda Medical College Wisconsin, Milwaukee, WI, USA Fondazione Policlinico Universitario A. Gemelli-IRCCS, Rome Y. Zen G. Negri King’s College Hospital & King’s College London, UK Central Hospital Bolzano Coord. National Council: Editorial Secretariat M. Guido F. Pedica University of Padua School of Medicine San Raffaele Scientific Institute, Milan L. Saragoni Morgagni-Pierantoni Hospital, Forlì F. Tallarigo Public Health Unit, COR Calabria, Crotone APOF Representative: G. Dell’Antonio San Raffaele Scientific Institute, Milan AITIC Representative: Moris Cadei ASST Spedali Civili-Unibs, Brescia

Copyright Società Italiana di Anatomia Patologica e Citopatologia Diagnostica, Divisione Italiana della International Academy of Pathology

Publisher Pacini Editore S.r.l. Via Gherardesca, 1 56121 Pisa, Italy Tel. +39 050 313011 Fax +39 050 3130300 [email protected] www.pacinimedicina.it CONTENTS PATHOLOGICA 03 September 2020

EDITORIAL The GIPAD handbook of the gastrointestinal pathologist (in the Covid-19 era) P. Parente, S. Battista, E. Unti, A. D’Amuri, A. Vanoli, L. Mastracci, M. Fassan...... 115

REVIEWS Gastro-esophageal reflux disease and Barrett’s esophagus: an overview with an histologic diagnostic approach L. Mastracci, F. Grillo, P. Parente, E. Unti, S. Battista, P. Spaggiari, M. Campora, G. Scaglione, M. Fassan, R. Fiocca...... 117

Non gastro-esophageal reflux disease related esophagitis: an overview with a histologic diagnostic approach L. Mastracci, F. Grillo, P. Parente, E. Unti, S. Battista, P. Spaggiari, M. Campora, L. Valle, M. Fassan, R. Fiocca...... 128

Neoplastic and pre-neoplastic lesions of the oesophagus and gastro-oesophageal junction F. Grillo, L. Mastracci, L. Saragoni, A. Vanoli, F. Limarzi, I. Gullo, J. Ferro, M. Paudice, P. Parente, M. Fassan...... 138

Gastritis: update on etiological features and histological practical approach G. Pennelli, F. Grillo, F. Galuppini, G. Ingravallo, E. Pilozzi, M. Rugge, R. Fiocca, M. Fassan, L. Mastracci...... 153

Precancerous lesions of the stomach, gastric cancer and hereditary gastric cancer syndromes I. Gullo, F. Grillo, L. Mastracci, A. Vanoli, F. Carneiro, L. Saragoni, F. Limarzi, J. Ferro, P. Parente, M. Fassan...... 166

Celiac disease: histology-differential diagnosis-complications. A practical approach V. Villanacci, A. Vanoli, G. Leoncini, G. Arpa, T. Salviato, L. Reggiani Bonetti, C. Baronchelli, L. Saragoni, P. Parente...... 186

Inflammatory and tumor-like lesions of the pancreas C. Luchini, M. Fassan, C. Doglioni, P. Capelli, G. Ingravallo, G. Renzulli, S. Pecori, G. Paolino, A.M. Florena, A. Scarpa, G. Zamboni...... 197

Malignant epithelial/exocrine tumors of the pancreas C. Luchini, F. Grillo, M. Fassan, A. Vanoli, P. Capelli, G. Paolino, G. Ingravallo, G. Renzulli, C. Doglioni, A. D’Amuri, P. Mattiolo, S. Pecori, P. Parente, A.M. Florena, G. Zamboni, A. Scarpa...... 210

Gastrointestinal lymphoproliferative lesions: a practical diagnostic approach M. Pizzi, E. Sabattini, P. Parente, A. Bellan, C. Doglioni, S. Lazzi...... 227

Current prognostic and predictive biomarkers for gastrointestinal tumors in clinical practice M. Fassan, A. Scarpa, A. Remo, G. De Maglio, G. Troncone, A. Marchetti, C. Doglioni, G. Ingravallo, G. Perrone, P. Parente, C. Luchini, L. Mastracci...... 248

Front cover: Different sampling techniques for duodeno-pancreatic head resections. Margins are inked indifferent colors: green - anterior surface; blue - superior mesenteric vein groove; red - uncinate process/ medial/retroperitoneal/superior mesenteric artery margin; black (minimum portion shown) - posterior surface. (page 212). PATHOLOGICA 2020;112:115-116; DOI: 10.32074/1591-951X-160

Editorial

The GIPAD handbook of the gastrointestinal pathologist (in the Covid-19 era)

Paola Parente1, Serena Battista2, Elettra Unti3, Alessandro D’Amuri4, Alessandro Vanoli5,6, Luca Mastracci7,8, Matteo Fassan9 1 Pathology Unit, Fondazione IRCCS Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy; 2 SOC di Anatomia Patologica, Azienda Sanitaria Universitaria Friuli Centrale, Udine, Italy; 3 UOC Anatomia Patologica, ARNAS Ospedali Civico-Di Cristina-Benfratelli, Palermo, Italy; 4 Anatomic Pathology Unit, “A. Perrino” Hospital, Brindisi, Italy; 5 Anatomic Pathology Unit, Department of Molecular Medicine, University of Pavia, Italy; 6 IRCCS San Matteo Hospital, Pavia, Italy; 7 Anatomic Pathology, Ospedale Policlinico San Martino IRCCS, Genova, Italy; 8 Anatomic Pathology, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genova, Italy; 9 Surgical Pathology Unit, Department of Medicine (DIMED), University of Padua, Italy

In 1996, with the publication in Pathologica of the article “Who needs a gastroenterology pathologist?” 1, Massimo Rugge, Roberto Fiocca and Cesare Bordi, the three scientific fathers of Italian gastrointestinal (GI) pathology, officially established the Italian Group of Gastrointestinal Pa- thologists (GIPAD) of the Italian Society of Anatomic Pathology and Di- agnostic Cytology (SIAPeC). This scientific group was created because Received and accepted: June 29, 2020 of the impelling need for educational proposals regarding GI diseases, Published online: 29 October, 2020 which are an important share of daily diagnostic load of our surgical pathology laboratories. Correspondence Matteo Fassan The Tradition of GIPAD has always been based on an ongoing effort to Department of Medicine (DIMED), Surgical meet the updating needs for GI and non-subspecialized pathologists: Pathology & Cytopathology Unit, University the first recommendations were published in 1999 2,3 with the purpose of of Padua, via Gabelli 61, 35121 Padua, Italy Tel. +39 049 8211312 providing a basic set of information to include in a standard GI histology Fax: +39 049 8272277 report. In 2011, GIPAD drafted GI-Pathologist Guide Lines concerning E-mail: [email protected] the most frequent and/or controversial topics in the field of GI histology diagnostic practice with interdisciplinary look: in addition to pathologists, Conflict of interest The Authors declare no conflict of interest. endoscopists, gastroenterologists, pediatricians and surgeons were in- volved 4. How to cite this article: Parente P, Battista The numerous discoveries on the physiological basis and molecular fea- B, Unti E, et al. The GIPAD hand-book of tures of neoplastic and non-neoplastic diseases in GI pathology of the the gastrointestinal pathologist (in the last 10 years, with the new WHO Classification of Tumours of Digestive Covid-19 era). Pathologica 2020;112:115-116. 5 https://doi.org/10.32074/1591-951X-160 System , required an additional updating with a clear and complete syn- thesis, comprehensive of the most important clinical correlations, useful © Copyright by Società Italiana di Anatomia Pato- for a daily consultation. For this reason, we have enthusiastically accept- logica e Citopatologia Diagnostica, Divisione Itali- ana della International Academy of Pathology ed the proposal of Dr. Mattia Barbareschi to dedicate special issues of Pathologica, the official journal of Italian pathologists, to new GI pathol- OPEN ACCESS ogy fields. The impracticality in organizing live GI meetings in the COV- ID-19 era, made this work more demanding but also more necessary for This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- all pathologists and their updating. NonCommercial-NoDerivatives 4.0 International) license: the We asked for the valuable contribution to internationally-recognized ex- work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original perts among Italian pathologists to act as coordinators of work groups, version. For further information: https://creativecommons. each focusing on a particular GI pathology topic. We are especially org/licenses/by-nc-nd/4.0/deed.en grateful to Italian Group of Endocrine Pathologists (GIPE), Italian Group 116 P. Parente et al.

of Emathology Pathologists (GIE), Italian Group of References Molecular Pathology and Predictive Medicine Pathol- 1 Rugge M, Fiocca R, Bordi C. Who Needs a Gastroenterology ogists (PMMP) and Pathologist of Italian Group for the Pathologist? [Article in Italian] Pathologica 1996;88:359-60. study of Inflammatory Bowel Disease (P-IG-IBD) for 2 Goldsmith JD, Siegal GP, Suster S, et al. Reporting guidelines for clinical laboratory reports in surgical pathology. Arch Pathol their significant contributes. Lab Med 2008;132:1608-16. https://doi.org/10.1043/1543- According to the cornerstone spirit of GIPAD we inher- 2165(2008)132[1608:RGFCLR]2.0.CO;2 ited from our scientific teachers, we hope that these 3 Ruby SG Clinician interpretation of pathology reports: confusion or comprehension? Arch Pathol Lab Med 2000;124:943-944. https:// monographies can help our colleagues in their routine doi.org/10.1043/0003-9985(2000)124<0943:CIOPR>2.0.CO;2 practice with special regards on novel topics in GI di- 4 Rugge M. “Die Grenzen meiner sprache sind die Grenzen mein- agnosis and intrigue them in GI world. This would be er Welt” - Pathologists and their readers. Dig Liver Dis 2011;43 a nice opportunity to rediscover importance of team Suppl 4:S279-81. https://doi.org/10.1016/S1590-8658(11)00114-9 5 WHO Classification of Tumours Editorial Board. WHO Classifi- work between pathologists and the other GI actors cation of Digestive System Tumours. 5th ed. Lyon: IARC press (also in COVID-19 era). 2019. PATHOLOGICA 2020;112:117-127; DOI: 10.32074/1591-951X-162

Review

Gastro-esophageal reflux disease and Barrett’s esophagus: an overview with an histologic diagnostic approach

Luca Mastracci1,2*, Federica Grillo1,2*, Paola Parente3, Elettra Unti4, Serena Battista5, Paola Spaggiari6, Michela Campora1, Giulia Scaglione1, Matteo Fassan7, Roberto Fiocca1,2 1 Anatomic Pathology, San Martino IRCCS Hospital, Genova, Italy; 2 Anatomic Pathology, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genova, Italy; 3 Unit of Pathology, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, (FG), Italy; 4 UOC Anatomia Patologica, ARNAS Ospedali Civico-Di Cristina-Benfratelli, Palermo, Italy; 5 SOC di Anatomia Patologica, Azienda Sanitaria Universitaria Friuli Centrale, Udine, Italy; 6 Department of Pathology, Humanitas Clinical and Research Center-IRCCS, Rozzano, Milan, Italy; 7 Surgical Pathology Unit, Department of Medicine (DIMED), University of Padua, Italy * These authors contributed equally

Summary The first part of this overview on non-neoplastic esophagus is focused on gastro-esoph- ageal reflux disease (GERD) and Barrett’s esophagus. In the last 20 years much has changed in histological approach to biopsies of patients with gastro-esophageal reflux Received and accepted: June 29, 2020 disease. In particular, elementary histologic lesions have been well defined and modality Published online: 29 October, 2020 of evaluation and grade are detailed, their sensitivity and specificity has been evaluated and their use has been validated by several authors. Also if there is not a clinical indication Correspondence to perform biopsies in patient with GERD, the diagnosis of microscopic esophagitis, when Luca Mastracci biopsies are provided, can be performed by following simple rules for evaluation which Anatomic Pathology Section, University of allow pathologists to make the diagnosis with confidence. On the other hand, biopsies Genova and Ospedale Policlinico San Martino, are required for the diagnosis of Barrett’s esophagus. This diagnosis is the synthesis of largo Rosanna Benzi 10, 16132 Genova, Italy endoscopic picture (which has to be provided with the proper description on extent and Tel. +39 010 5555954 with adequate biopsies number) and histologic pattern. The current guidelines and expert Fax: +39 010 5556932 opinions for the correct management of these diagnosis are detailed. E-mail: [email protected] Key words: gastro-esophageal reflux disease (GERD), microscopic esophagitis, Barrett’s https://orcid.org/0000-0003-0193-5281 esophagus, intestinal metaplasia of the cardia, histology Conflict of interest The Authors declare no conflict of interest. Introduction How to cite this article: Mastracci L, Grillo F, Parente P, et al. Gastro-esophageal reflux In the last 20 years the approach to esophageal non-neoplastic disease disease and Barrett’s esophagus: an overview has dramatically changed and improved. Various reasons can explain with an histologic diagnostic approach. Pathologica 2020;112:117-127. https://doi. this new interest for esophageal pathology, from the worldwide increas- org/10.32074/1591-951X-162 ing incidence of esophageal adenocarcinoma, which recognizes in gastro-esophageal reflux disease (GERD) and in Barrett’s esophagus © Copyright by Società Italiana di Anatomia Pato- (BE) its principal carcinogenic mechanisms, to the comprehension of logica e Citopatologia Diagnostica, Divisione Itali- ana della International Academy of Pathology the pathology of conditions such eosinophilic esophagitis (EE), to the description of new rare entities such lymphocytic esophagitis (LE) which OPEN ACCESS represent a challenge when approaching esophageal biopsies. Furthermore, changes in definitions of some conditions, refinement of This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- histologic elementary lesions and application of this knowledge in rou- NonCommercial-NoDerivatives 4.0 International) license: the tine diagnostic practice, need to be addressed and implemented by all work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original pathologists involved in the diagnosis of gastrointestinal diseases. version. For further information: https://creativecommons. This overview on non-neoplastic esophageal disease has the main aim org/licenses/by-nc-nd/4.0/deed.en to furnish a practical diagnostic approach to biopsy samples to the prin- 118 L. Mastracci et al.

cipal illnesses affecting the esophagus tract, on the more frequently reported extra-esophageal symp- basis of recent published recommendations, guide- toms 4,5. lines and expert opinions. On the basis of symptoms and endoscopic appear- ance the following conditions can be distinguished: - Erosive reflux disease (ERD): presence of symp- Normal Esophageal Mucosa toms and mucosal breaks at endoscopy; - Non-erosive reflux disease (NERD): presence of The esophagus is lined by a multilayered non-kerati- symptoms in the absence of mucosal breaks at nizing squamous epithelium. In normal individuals, endoscopy. NERD patients are reported to be 50- the basal layer (the proliferative part of the epithelium) 60% of patients with GERD; is limited to 2-3 layers of cells and occupies less than - Esophageal Disorders of Gut-Brain Interaction 15% of the total epithelial thickness. Normal papillae (EDGBI, previously named functional disorders): are shorter than 2/3 of the epithelium and inflam- these are a group of disorders with symptoms re- matory cells are sparse. Intraepithelial inflammatory lated to motility disturbances, visceral hypersen- cells are generally represented by T lymphocytes with sitivity, altered mucosal and immune functions, a mean number of 20 in Z-lines biopsies of healthy gut microbiota, and/or central nervous system controls and less numerous in more proximal sites 1. processing 6. This category includes functional Immunophenotypically normal intraepithelial lympho- esophageal chest pain, functional heartburn and cytes are predominantly CD8+ suppressor, while a reflux hypersensitivity (esophagus sensitive to the minority are CD4+ helper T cells 2. normal reflux of acidic or non-acidic material), glo- Sparse intraepithelial Langerhans cells and very few bus (sensation of foreign body in the throat) and mast cells are also present, while B lymphocytes, NK functional dysphagia (sensation of abnormal bolus cells and macrophages are limited to the submucosal transit through the esophageal body) 7. compartment. In healthy subjects, no eosinophils or neutrophils are present within the esophageal mucosa. Endoscopy picture ERD is diagnosed by endoscopy when visible breaks are seen in the esophageal mucosa near to or at the Gastro-Esophageal Reflux Disease GE junction. The Los Angeles classification 8 is the (GERD) validated and most reliable and diffuse system for grading esophageal mucosal breaks (scoring based Definition on the number and extension). The term esophagitis, Gastro-Esophageal Reflux Disease (GERD) is de- grades A to D, is used to define endoscopically detect- fined as damage of the squamous epithelium sec- able erosive lesions. NERD and EDGBI, by definition, ondary to the pathological reflux of gastric content do not show any endoscopic abnormalities. (comprising both gastric acidic substances and bile salts from duodenum-gastric reflux) in the esophagus, Biopsy site causing troublesome symptoms and/or complications. Patients with GERD are usually diagnosed on the ba- sis of symptoms and endoscopic assessment with/ Clinical picture without pHmetry and impedance, in the absence of GERD is a very common condition with a prevalence any indications for routine esophageal biopsies. This approaching 20% in western countries, while in eastern is mainly due to the fact that histology does not pro- countries, the prevalence is generally lower than 10% 3. vided additional information for patient management. The diagnosis is related to presence of typical or atyp- Despite this, esophageal biopsies can be performed ical symptoms which adversely affect the individual’s by gastroenterologists in patients with suspect- well-being. Troublesome symptoms are defined on the ed GERD complications or in patients with atypical basis of their frequency and severity: mild symptoms symptoms or functional disease in the absence of en- occurring 2 or more days a week or moderate/severe doscopic alterations. For these reasons, an accurate symptoms occurring more than 1 day a week 4. evaluation of histological lesions in order to provide Typical esophageal symptoms include: heartburn (ret- a correct diagnosis, may be important. As histologic rosternal burning sensation), regurgitation (percep- lesions in GERD are usually limited to the distal eso- tion of flow of refluxed gastric content in the mouth or phagus, sampling should include the last 2 cm above hypopharynx) and epigastric/chest pain, sometimes the Z line (2 biopsies at 2 cm and 2 biopsies on the indistinguishable from ischemic cardiac pain. Chronic esophageal side of the Z line). More proximal biopsies cough, laryngitis, asthma and dental erosion are the are less informative 9-10. GASTRO-ESOPHAGEAL REFLUX DISEASE AND BARRETT’S ESOPHAGUS 119

Histologic elementary lesions The term microscopic esophagitis refers to a group of histologic lesions, observed in patients with ERD and NERD reflux disease and also in some ‘function- al disorders’. Histologic lesions are unspecific and can also be observed in other types of esophagites and in physiological reflux. In the last 15 years, numerous studies have focused on the definition of histologic elementary lesions, sensitivity and specificity of his- tology, reproducibility and validation of diagnosis and demonstration of improvement of esophageal mu- cosal repair after surgical or medical therapy 9-20. The most informative histological lesions are the fol- lowing: - Basal cell hyperplasia (BCH) is defined as the thickness of the squamous epithelium basal layer ≥ 15% of the total thickness. The upper limit of the basal layer is defined as the level where the nu- clei of epithelial cells are separated by a distance greater than their diameter. Basal cell hyperpla- sia is graded as mild (< 30%) or marked (≥ 30%) (Fig. 1) and assessment requires well oriented samples 11,14. - Papillae elongation (PE) is defined as an elonga- tion of papillae ≥ 66% of total epithelial thickness. The upper limit of the papilla is defined as the up- per limit of the vessel along its axis. It is graded as mild (< 75%) or marked (≥ 75%) (Fig. 2) and assessment requires well oriented samples 11,14. - Dilated intercellular spaces (DIS) are irregular dila- tations of intercellular spaces, detectable as optical- ly empty bubbles or ladders. DIS are more prevalent in the lower half of the epithelium and around the papillae and must be differentiated from “stretching” artefacts and from intracytoplasmic vacuoles 11,14. DIS can be graded as small or large (in relation to the diameter of a small lymphocyte) (Fig. 3). - Inflammatory cells: intraepithelial eosinophils are present in about 50% of patients with GERD; in- traepithelial neutrophils are a rare finding (< 5%) in patients with NERD and their presence is usually Figure 1. Elementary lesion - increasing grades of severity associated with erosive disease. Both are highly in basal cell hyperplasia: A) normal, epithelium basal layer specific but suffer from low sensitivity. Intraepitheli- thickness is less than 15% of the entire thickness; B) mild al lymphocytes do not play a significant role in the basal cell hyperplasia ranges between 15% and 30%; C) diagnosis of microscopic esophagitis, nor other severe basal cell hyperplasia: basal cells occupy more than types of inflammatory cells (mast cells, Langer- 30% of whole epithelial thickness. Magnification 20x. Re- hans cells, macrophages), rarely seen in esopha- printed from ref. 10 with permission from Virchows Archiv, geal biopsies10. Springer Nature. - Erosions are characterized by the presence of necrosis with granulation tissue and/or fibrin with neutrophils; healed erosions show fibrosis/granu- lation tissue covered by thin regenerative epitheli- specificity but a low sensitivity, and, together with um in the absence of necrosis 11-14. These lesions intraepithelial neutrophils represent the most se- are mainly seen in erosive esophagitis, with a high vere lesions in the spectrum of microscopic eso- 120 L. Mastracci et al.

Figure 2. Elementary lesion - increasing grades of severity in papillae elongation: A) normal papillae occupy less than 2/3 of the total epithelial thickness; B) mild papillae elonga- tion does not exceed 75% of total epithelial thickness; C) marked papillae elongation with the upper limit of papillae approaching the epithelial surface. Magnification 20x. Re- printed from ref. 10 with permission from Virchows Archiv, Figure 3. Elementary lesion - increasing grades of severity Springer Nature. in dilated intercellular spaces (DIS): (A) in normal squamous epithelium, cells are sealed one with the other; (B) small, irregular DIS are shown close to a papilla; (C) large DIS with bubbles and ladders larger than the diameter of a small lymphocyte. Magnification 40x. Reprinted from ref. 10 with permission from Virchows Archiv, Springer Nature. GASTRO-ESOPHAGEAL REFLUX DISEASE AND BARRETT’S ESOPHAGUS 121

phagitis. Whenever they are found, microscopic obtain this goal, it is necessary to comply with recog- esophagitis can be diagnosed regardless of the nized and standardized terminology and to precisely presence of other lesions. describe endoscopic landmarks. Histologic lesions in microscopic esophagitis are ir- regularly distributed and may be focal. Consequent- Terminology ly, assessment should be made in the most affected GEJ. The term anatomic gastro-esophageal junction areas. A scoring system including multiple histologic (GEJ) identifies the proximal border of the gastric folds lesions could help to increase both sensitivity and when endoscopy is performed with minimal air-insuf- specificity of histologic findings 10. flation 31. GEJ is identified exclusively by endoscopists and this term should not be used by pathologists in Diagnosis their diagnostic report. For a practical approach to esophageal biopsies, the SCJ or Z-line. The term histologic squamo-colum- diagnosis of microscopic esophagitis should be made nar junction (SCJ) or Z-line refers to the transition when any of the following are seen: a) at least two between esophageal stratified squamous epithelium mild histologic lesions; b) at least one severe lesion; and columnar epithelium. It can be identified both by c) any one of erosions/healed erosions/intraepithelial endoscopists (due to the white color of the squamous 10,21 neutrophils . epithelium compared to the pinkish color of columnar epithelium) and by pathologists who easily identify the histologic transition between squamous and columnar Barrett’s esophagus epithelium 32. As a rule, GEJ and SCJ coincide in nor- mal subjects but a dis-alignment of the SCJ up to 1 cm Definition proximally to GEJ is also seen. Barrett’s esophagus (BE) is defined as the replace- ESEM. The term Endoscopically Suspected Esopha- ment of any portion of the esophageal normal distal geal Metaplasia, according to the Montreal definition 4 squamous epithelium by metaplastic columnar ep- defines the presence of salmon pink mucosa in the ithelium, which is clearly visible endoscopically (≥ 1 distal esophagus at endoscopy; it describes endo- cm) above the gastro-esophageal junction (GEJ). This scopically suspected columnar metaplasia related definition requires histological confirmation on esoph- to the proximal dislocation of SCJ with respect to the ageal biopsies. GEJ. This definition is common to international guide- 22-26 lines as well as Italian guidelines and expert state- Landmarks (Fig. 4) ments 21,27. 1 By visualizing two landmarks, namely the distal end 33 Clinical picture of the palisade vessels and the proximal end of the gastric folds 34 at endoscopy, it is possible to BE represents a complication of gastro-esophageal accurately delineate the GEJ and identify wheth- reflux disease, with which it shares symptoms. er there is a columnar-lined segment in the lower The prevalence of BE in the population at large re- mains uncertain. Two studies have attempted to as- esophagus. The two landmarks should coincide sess the prevalence via endoscopy screening of an at the GEJ, however different factors (peristaltic or unselected adult population, reporting respectively a respiratory movements, esophagitis, and degree of prevalence of 1.6% in the Swedish 28 and of 1.3% in air insufflation) can lead to inconsistencies between the Italian population 29. However, the limited partici- these two landmarks. In a comparative study of the pation rate remained a concern in both these studies, two methods, investigators found that the proximal since it introduced a risk of selection bias resulting in extent of the gastric folds was more accurate com- a possible overestimate of the prevalence. On the oth- pared to the palisade vessels (due to frequent lower 35 er hand, in symptomatic patients with chronic GERD, position of these compared to the GEJ) . BE prevalence is as high as 15% 30. 2 The diaphragmatic hiatus is identified as an in- dentation of the gastric folds that is apparent dur- Endoscopy picture ing upper endoscopy with inspiration. The importance of measuring the length and shape 3 The SCJ, as mentioned above, can be recognized of the columnar-lined segment using a standardized by endoscopists by the color changing from white methodology is recognized worldwide. This aids com- to salmon pink. It is recommended that all three munication between endoscopists and pathologists landmarks (or at least GEJ and SCJ), are men- so improving the level of diagnostic confidence. To tioned in every endoscopic report. 122 L. Mastracci et al.

Figure 4. Landmarks (GEJ and Z-line) and the Prague C&M system for reporting ESEM length: on the left side GEJ and Z-line coincide in normal case; on the right side Z-line is proximally dislocated with respect to GEJ and a C2M4 ESEM is represented. Artwork by Federica Grillo.

41 Report endoscopical methodology being correlated to ESEM length . The Seattle biopsy The Prague C & M system for ESEM length is widely protocol, which entails four-quadrant random biopsies used by major academic societies such as the Amer- every 2 cm in addition to targeted biopsies on mac- ican Gastroenterological Association 22, American roscopically visible lesions, is recommended at the 42-44 College of Gastroenterology 23, the British Society of time of diagnosis and at subsequent surveillance . Gastroenterology 24 and it is also recommended by the Unfortunately, this sampling protocol is not frequently Asia-Pacific consensus 25 and Australian Guidelines 26. performed in routine practice because it is lengthy and The Prague C&M classification is based on validated, poorly tolerated by patients. Targeted biopsy samples explicit, consensus-driven criteria 31 and includes as- from visible lesions should be taken before random bi- sessment of the circumferential (C) and maximal (M) opsies and distal areas should be biopsied first start- extent of ESEM. The overall reliability coefficients for ing 1-2 cm above the GEJ and advancing proximally. In endoscopic recognition of ESEM ≥ 1 cm was 0.72. patients with ESEM without confirmation of IM despite These findings have been reproduced in different adequate number of biopsies, a repeat examination patient populations 36,37 and have also been validat- could be considered in 1-2 years based on a longitu- ed in a multicenter study 38. A subtext in the Prague dinal cohort study demonstrating that around 30% of classification, records non-continuous ESEM islands these patients can be expected to demonstrate IM on (which may be found after endoscopic therapy); these a repeat examination 45. Advances in chromoendosco- last findings should also be recorded according to the py (methylene blue, indigo carmine, and acetic acid), Paris classification 39. endoscope digital enhancements (narrow-band imag- ing, i-SCAN, Fujinon intelligent chromo endoscopy), Biopsy site and enhanced magnification have not been shown to Intestinal metaplasia (IM – which in some countries be superior to the currently accepted practice of ran- is necessary for a diagnosis of BE – see paragraph dom four-quadrant biopsies at 2-cm intervals 46-48. below) can be patchy 40 and multiple biopsies are re- Biopsies of a normal or irregular z-line are not rec- quired in order to characterize ESEM, their number ommended. If biopsies are sampled from an irregular GASTRO-ESOPHAGEAL REFLUX DISEASE AND BARRETT’S ESOPHAGUS 123

Z-line, with no clear endoscopic evidence of Barrett’s, biopsies obtained. In a large retrospective study, they should then be sent to the pathologist as z-line the yield for IM was 35% if 4 biopsies were ob- biopsies and not as endoscopically suspected Barrett tained, and up to 68% after 8 biopsies were per- esophagus (ESEM) biopsy samples. formed 56. Biopsy site is also relevant for IM detec- tion, with a 94% detection rate of IM when biopsies Histologic elementary lesions are performed close to the squamo-columnar junc- Two types of columnar epithelium may replace eso- tion, even if fewer samples are taken 55. Finally, rec- phageal stratified squamous epithelium: cardiac/oxyn- ognition of IM was shown to increase cumulatively tic atrophic type and intestinal type epithelium 4. IM with follow-up: over 50% of patients who originally in BE is most commonly of an incomplete (type II or did not have IM were found to have IM at 5 years’ III) subtype comprising mucous cells and goblet cells, and over 90% at 10 years’ follow-up 57. although a complete type (type I with absorptive cells) Few studies focus on inter-observer variability in the may also be seen 49. diagnosis of BE. In particular, one study 58, reported Finding mature oxyntic epithelium on distal esophage- an inter-observer agreement of 88.3% (kappa value of al biopsies is generally a sign of hiatus hernia where- 0.41) in distinguishing columnar epithelium types. An as it represents gastric ectopia (inlet patches) when Italian study, evaluated both the inter-observer agree- found in the mild-proximal esophagus. ment in type of epithelium recognition (oxyntic/cardia What defines the histologic diagnosis of BE still re- versus intestinal) and more importantly on diagnostic mains a contentious issue. Indeed the type of colum- category assignation. While agreement for diagnosis nar mucosa necessary for BE diagnosis varies be- of BE had a moderate-substantial agreement rate tween different countries. While the American College among participants (overall K = 0.60 95% CI 0.58- of Gastroenterology - ACG 50 and Australian guide- 0.62) major problems arose when interpreting colum- lines 26 require IM as a necessary diagnostic criterium nar epithelium in an irregular z-line 32. This perfectly to diagnose BE, the British Society of Gastroenterol- describes the frequent and diffuse problems faced by ogy guidelines 24 and the Asia-Pacific consensus on practicing pathologists in esophageal biopsy assess- the management of GERD 25 both suggest that in the ment, and therefore in BE diagnosis. In our opinion context of visible columnar epithelium, IM is not a req- this point needs to be addressed further, as clear, re- uisite, and hence gastric cardiac/oxyntic atrophic type producible and accurate BE diagnoses are the first, in- metaplasia is also regarded as BE. Both points of view dispensable step for access to surveillance programs. have some merit: Ancillary techniques. There is an undeniable need for a the emphasis on IM as a defining feature of BE is novel diagnostic approaches to the evaluation and based on an increasing body of evidence which risk stratification of patients with BE. Unfortunately, has demonstrated an increased risk of neoplas- currently available ancillary techniques, including tic progression for ESEM with IM compared to histochemical and immunohistochemical markers, ESEM without IM. Among various studies, one of have little to offer over routine H&E assessment, be- the largest population-based cohort investigations cause they lack sufficient specificity for detection of demonstrated a substantially higher EAC risk in BE and dysplasia classification 59. Some such bio- subjects with columnar metaplasia with IM com- markers have been investigated and are here briefly pared with those without IM (0.38%/year vs 0.07%/ described. Alcian Blue-PAS helps distinguish cardia year, p < 0.01) 51. Furthermore, a detailed genomic from intestinal epithelium. Unfortunately, the speci- analysis comparing IM and non-IM epithelium in ficity of Alcian blue for goblet cells is generally low, 45 patients with BE reported a higher frequency particularly with respect to distinguishing goblet cells of mutations in cancer-associated genes such as from their morphologic mimics. Johnson and col- CDKN2A, WWOX, c-MYC and GATA6 in IM 52. leagues found that Alcian blue detects goblet cells However, other studies have not corroborated with similar sensitivity (100%), but lower specificity such findings 53-54. (90%), compared with H&E, owing to false-positive b IM is not necessary for BE definitionas sampling staining of esophageal mucus glands and columnar may impact on IM detection. Within the length of non-goblet cells 60. There is insufficient evidence to ESEM, IM has been shown to be patchy and gen- justify the reflexive use of Alcian blue and/or PAS on erally found with greater frequency on the squa- all esophageal biopsies because goblet cells are al- mo-columnar junction leading edge 55. This may most always identifiable on routine H&E-stained sec- lead to sampling error with misclassification of ES- tions 61. The value of immunohistochemical markers EM with IM as ESEM without IM. The yield for IM in establishing a diagnosis of BE has been diffusely correlates directly with the number of endoscopic investigated applying numerous markers of intestinal 124 L. Mastracci et al.

phenotype (CDX2, MUC2, MUC1, Villin, SOX9 and (when IM is not demonstrated in an ESEM ≥ 1 cm DAS1). Although these stains may be markers of an at index biopsies). earlier phase of intestinal differentiation, there is in- These two different diagnoses will lead to different sufficient evidence to suggest that they predict the management and follow-up. development of IM. Furthermore, their use to distin- No diagnosis of BE can be made on biopsies taken guish between IM in BE from IM of the cardiac has from < 1 cm z-line irregularities; the type of columnar been largely unsuccessful 62-65. epithelium in GEJ biopsies should be described differ- entiating between oxyntic/cardial epithelium sugges- Diagnosis tive of site-appropriate gastric mucosa and intestinal According to the previous terminology suggest- epithelium suggestive of intestinal metaplasia of the ed for BE diagnosis in the histology report by SIA- cardia. PEC-IAP 21, we suggest the use of the following ter- minology (Fig. 5): - Barrett’s esophagus with Intestinal Metaplasia Conclusions (when IM is histologically proven in an ESEM ≥ 1 cm at index biopsies); The role of histology in diagnosing microscopic eso- - Barrett’s esophagus without Intestinal Metaplasia phagitis related to GERD is still debated, however, in

Figure 5. Pictorial representation of the diagnostic algorithm taking into account endoscopic and histologic findings to correctly interpret the columnar lined esophagus. (A-C) Columnar cardia-type epithelium; (B) Columnar intestinal-type epi- thelium. BE: Barrett’s Esophagus. Artwork by Federica Grillo. GASTRO-ESOPHAGEAL REFLUX DISEASE AND BARRETT’S ESOPHAGUS 125

the last 20 years or so, histologic elementary lesions oesophagitis: clinical and functional correlates and further vali- have been more precisely described and defined. dation of the Los Angeles classification. Gut. 1999;45:172-80. https://doi.org/10.1136/gut.45.2.172 Though biopsies are currently not part of the routine 9 Zentilin P, Savarino V, Mastracci L, et al. Reassessment of the work up in diagnosing GERD, it is important to rec- diagnostic value of histology in patients with GERD, using mul- ognize and properly evaluate histologic elementary tiple biopsy sites and an appropriate control group. Am J Gas- lesions, in order to make this diagnosis only when troenterol 2005;100:2299-306. https://doi.org/10.1111/j.1572- clear damage of the squamous mucosa is seen. The 0241.2005.50209.x identification of true lesions will hopefully contribute 10 Mastracci L, Spaggiari P, Grillo F, et al. Microscopic esophagitis in gastro-esophageal reflux disease: individual lesions, biopsy in maintaining the high sensitivity and specificity that sampling, and clinical correlations. Virchows Arch 2009;454:31- histology has demonstrated to have in this setting. On 9. https://doi.org/10.1007/s00428-008-0704-8 the other hand, biopsies are mandatory in diagnos- 11 Fiocca R, Mastracci L, Riddell R, et al. Development of con- ing GERD-related complications, and in particular BE. sensus guidelines for the histologic recognition of microscopic The diagnosis of BE suffers from a low inter-observer esophagitis in patients with gastroesophageal reflux disease: the Esohisto project. Hum Pathol 2010;41:223-31. https://doi. reproducibility related to different reasons (ie differ- org/10.1016/j.humpath.2009.07.016 ent BE definition in different countries, availability of 12 Mastracci L, Grillo F, Zentilin P, et al. Cell proliferation of squa- precise endoscopic description and sampling). This mous epithelium in gastro-oesophageal reflux disease: correla- diagnosis has however a major impact both in decid- tions with clinical, endoscopic and morphological data. Aliment ing which patients need to be included in surveillance Pharmacol Ther 2007;25:637-45. https://doi.org/10.1111/j.1365- programs and in making these programs cost-effec- 2036.2006.03243.x 13 tive. For these reasons it seems to be very important Savarino E, Zentilin P, Mastracci L, et al. Microscopic esopha- gitis distinguishes patients with non-erosive reflux disease from to precisely describe the type of columnar epithelium those with functional heartburn. J Gastroenterol 2013;48:473-82. present in esophageal biopsies, to apply the algorithm https://doi.org/10.1007/s00535-012-0672-2 (histology plus endoscopy) for diagnosis, and to refer 14 Yerian L, Fiocca R, Mastracci L, et al. Refinement and repro- to the adopted current guidelines (whichever are cho- ducibility of histologic criteria for the assessment of micro- sen and referenced) in order to make this diagnosis as scopic lesions in patients with gastroesophageal reflux disease: the Esohisto Project. Dig Dis Sci 2011;56:2656-65. https://doi. clear as possible for the clinician. org/10.1007/s10620-011-1624-z 15 Fiocca R, Mastracci L, Engström C, et al. Long-term outcome References of microscopic esophagitis in chronic GERD patients treated with esomeprazole or laparoscopic antireflux surgery in the 1 Mastracci L, Bruzzone M, Pacella E et al. The contribution of LOTUS trial. Am J Gastroenterol 2010;105:1015-23. https://doi. intraepithelial inflammatory cells to the histological diagnosis of org/10.1038/ajg.2009.631 microscopic esophagitis. Esophagus 2016;13:80-7. https://doi. 16 Furnari M, Zentilin P, Mastracci L, et al. Esophageal biopsies in org/10.1007/s10388-015-0501-9 the management of GERD: complementary tool for many but 2 Lucendo AJ, Navarro M, Comas C, et al. Immunophenotypic not for all. Hum Pathol 2014;45:2512-3. https://doi.org/10.1016/j. characterization and quantification of the epithelial inflammatory humpath.2014.06.029 infiltrate in eosinophilic esophagitis through stereology: an anal- 17 Savarino E, Zentilin P, Mastracci L, et al. Light microscopy is ysis of the cellular mechanisms of the disease and the immuno- useful to better define NERD and functional heartburn. Gut logic capacity of the esophagus. Am J Surg Pathol 2007;31:598- 2014;63:368. https://doi.org/10.1136/gutjnl-2013-305955 606. https://doi.org/10.1097/01.pas.0000213392.49698.8c 18 Mastracci L, Fiocca R, Engström C, et al. The dynamics of the 3 Eusebi LH, Ratnakumaran R, Yuan Y, et al. Global prevalence oesophageal squamous epithelium ‘normalisation’ process in of, and risk factors for, gastro-oesophageal reflux symptoms: patients with gastro-oesophageal reflux disease treated with a meta-analysis. Gut. 2018;67:430-40. https://doi.org/10.1136/ long-term acid suppression or anti-reflux surgery. Aliment Phar- gutjnl-2016-313589 macol Ther 2017;45:1339-49. https://doi.org/10.1111/apt.14038 4 Vakil N, van Zanten SV, Kahrilas P, et al. The Montreal definition 19 Vieth M, Mastracci L, Vakil N, et al. Epithelial Thickness is a and classification of gastroesophageal reflux disease: a global Marker of Gastroesophageal Reflux Disease. Clin Gastro- evidence-based consensus. Am J Gastroenterol. 2006;101:1900- enterol Hepatol 2016;14:1544-51 https://doi.org/10.1016/j. 20. https://doi.org/10.1111/j.1572-0241.2006.00630.x cgh.2016.06.018 5 Pace F, Bazzoli F, Fiocca R, et al. The Italian validation of the 20 Schneider NI, Plieschnegger W, Geppert M, et al. Validation Montreal Global definition and classification of gastroesophage- study of the Esohisto consensus guidelines for the recogni- al reflux disease. Eur J Gastroenterol Hepatol 2009;21:394-408. tion of microscopic esophagitis (histoGERD Trial). Hum Pathol https://doi.org/10.1097/MEG.0b013e32830a70e2 2014;45:994-1002. https://doi.org/10.1016/j.humpath.2013. 6 Drossman DA. Functional gastrointestinal disorders: history, 21 Fiocca R, Mastracci L, Milione M, et al. Microscopic esophagi- pathophysiology, clinical features and Rome IV. Gastroenterology tis and Barrett’s esophagus: the histology report. Dig Liver Dis 2016;150:1262-79. https://doi.org/10.1053/j.gastro.2016.02.032 2011;43:S319-30. https://doi.org/10.1016/S1590-8658(11)60588-4 7 Schmulson M. How to use Rome IV criteria in the evaluation of 22 Spechler SJ, Sharma P, Souza RF, et al. American Gastroen- esophageal disorders. Curr Opin Gastroenterol 2018;34:258-65. terological Association technical review on the management of https://doi.org/10.1097/MOG.0000000000000443 Barrett’s esophagus. Gastroenterology 2011;140:e18-52. https:// 8 Lundell LR, Dent J, Bennett JR, et al. Endoscopic assessment of doi.org/10.1053/j.gastro.2011.01.031 126 L. Mastracci et al.

23 Wang KK, Sampliner RE, Practice Parameters Committee of esophagus by gastroenterology trainees: a multicenter study. the American College of Gastroenterology. Updated guidelines Gastrointest Endosc 2012;75:236-41. https://doi.org/10.1016/j. 2008 for the diagnosis, surveillance and therapy of Barrett’s gie.2011.09.017 esophagus. Am J Gastroenterol 2008;103:788-97 https://doi. 39 Endoscopic Classification Review Group. Update on the org/10.1111/j.1572-0241.2008.01835.x Paris classification of superficial neoplastic lesions in the 24 Fitzgerald RC, di Pietro M, Ragunath K, et al. British Society digestive tract. Endoscopy 2005;37:570-8. https://doi. of Gastroenterology guidelines on the diagnosis and manage- org/10.1055/s-2005-861352 ment of Barrett’s oesophagus. Gut 2014;63:7-42. https://doi. 40 Endlicher E, Rummele P, Beer S, et al. Barrett’s esophagus: a dis- org/10.1136/gutjnl-2013-305372 crepancy between macroscopic and histological diagnosis. En- 25 Fock KM, Talley N, Goh K, et al. Asia-Pacific consensus on the doscopy 2005;37:1131-5. https://doi.org/10.1055/s-2005-870409 management of gastro-oesophageal reflux disease: an update 41 Yantiss RK, Odze RD. Optimal approach to obtaining mucosal focusing on refractory reflux disease and Barrett’s oesophagus. biopsies for assessment of inflammatory disorders of the gastro- Gut 2016;65:1402-15. https://doi.org/10.1136/gutjnl-2016-311715 intestinal tract. Am J Gastroenterol 2009;104:774-83. https://doi. 26 Whiteman DC, Appleyard M, Bahin FF, et al. Australian clini- org/10.1038/ajg.2008.108 cal practice guidelines for the diagnosis and management of 42 Levine DS, Blount PL, Rudolph RE, et al. Safety of a systematic Barrett’s esophagus and early esophageal adenocarcinoma. J endoscopic biopsy protocol in patients with Barrett’s esopha- Gastroenterol Hepatol 2015;30:804-20. https://doi.org/10.1111/ gus. Am J Gastroenterol 2000;95:1152-7. https://doi.org/10.1111/ jgh.12913 j.1572-0241.2000.02002.x 27 Costamagna G, Battaglia G, Repici A, et al. Diagnosis and endo- 43 Abela JE, Going JJ, Mackenzie JF, et al. Systematic four-quad- scopic management of Barrett’s esophagus: an Italian experts’ rant biopsy detects Barrett’s dysplasia in more patients than opinion based document. Dig Liver Dis 2017;49:1306-13. https:// nonsystematic biopsy. Am J Gastroenterol 2008;103:850-5. doi.org/10.1016/j.dld.2017.08.034 44 Reid BJ, Blount PL, Feng Z, et al. Optimizing endoscopic biopsy 28 Ronkainen J, Aro P, Storskrubb T, et al. Prevalence of Barrett’s detection of early cancers in Barrett’s high-grade dysplasia. Am esophagus in the general population: an endoscopic study. Gas- J Gastroenterol 2000;95:3089-96. troenterology 2005;129:1825-31. https://doi.org/10.1053/j.gas- 45 tro.2005.08.053 Khandwalla HE, Graham DY, Kramer JR, et al. Barrett’s Esopha- gus suspected at endoscopy but no specialized intestinal meta- 29 Zagari RM, Fuccio L, Wallander MA, et al. Gastro-oesopha- plasia on biopsy, what’s next. Am J Gastroenterol 2014;109:178- geal reflux symptoms, oesophagitis and Barrett’s oesophagus 82. https://doi.org/10.1038/ajg.2013.408 in the general population: the Loiano-Monghidoro study. Gut 46 2008;57:1354-9. https://doi.org/10.1136/gut.2007.145177 Ferguson DD, DeVault KR, Krishna M, et al. Enhanced mag- nification-directed biopsies do not increase the detection 30 Johansson J, Hakansson HO, Mellblom L et al. Prevalence of of intestinal metaplasia in patients with GERD. Am J Gas- precancerous and other metaplasia in the distal oesophagus troenterol 2006;101:1611-6. https://doi.org/10.1111/j.1572- and gastro-oesophageal junction. Scand J Gastroenterol 2005; 0241.2006.00622.x 40:893-902. https://doi.org/10.1080/00365520510015692 47 Ngamruengphong S, Sharma VK, Das A. Diagnostic yield of 31 Sharma P, Dent J, Armstrong D, et al. The development and vali- methylene blue chromoendoscopy for detecting specialized dation of anendoscopic grading system for Barrett’s esophagus: intestinal metaplasia and dysplasia in Barrett’s esophagus: a the Prague C & M criteria. Gastroenterology 2006;131:1392-9. meta-analysis. Gastrointest Endosc 2009;69:1021-8. https://doi. https://doi.org/10.1053/j.gastro.2006.08.032 org/10.1016/j.gie.2008.06.056 32 Mastracci L, Piol N, Molinaro L, et al. Interobserver reproduc- 48 Horwhat JD, Maydonovitch CL, Ramos F, et al. A randomized ibility in pathologist interpretation of columnar-lined esophagus. comparison of methylene blue-directed biopsy versus conven- Virchows Arch 2016;468:159-67. https://doi.org/10.1007/s00428- tional four-quadrant biopsy for the detection of intestinal meta- 015-1878-5 plasia and dysplasia in patients with long-segment Barrett’s 33 Ogiya K, Kawano T, Ito E, et al. Lower esophageal palisade ves- esophagus. Am J Gastroenterol 2008;103:546-54. sels and the definition of Barrett’s esophagus. Dis Esophagus 49 Gottfried MR, McClave SA, Boyce HW. Incomplete intestinal 2008;21:645-9. https://doi.org/10.1111/j.1442-2050.2008.00825.x metaplasia in the diagnosis of columnar lined esophagus (Bar- 34 Sharma P, Morales TG, Sampliner RE. Short segment Barrett’s rett’s esophagus). Am J Clin Pathol 1989;92:741-6. https://doi. esophagus - the need for standardization of the definition and of org/10.1093/ajcp/92.6.741 endoscopic criteria. Am J Gastroenterol 1998;93:1033-6. https:// 50 Shaheen NJ, Falk GW, Iyer PG, et al. ACG clinical doi.org/10.1111/j.1572-0241.1998.00324.x guideline:diagnosis and management of Barrett’s esophagus. 35 Amano Y, Ishimura N, Furuta K, et al. Which landmark results in Am J Gastroenterol 2016;111:30-50. https://doi.org/10.1038/ a more consistent diagnosis of Barrett’s esophagus, the gastric ajg.2015.322 folds or the palisade vessels? Gastrointest Endosc 2006;64:206- 51 Bhat S, Coleman HG, Yousef F, et al. Risk of malignant progres- 11. https://doi.org/10.1016/j.gie.2006.04.029 sion in Barrett’s esophagus patients: results from a large popu- 36 Lee YC, Cook MB, Bhatia S, et al. Interobserver reliability in the lation-based study. J Natl Cancer Inst 2011;103:1049-57. https:// endoscopic diagnosis and grading of Barrett’s esophagus: an doi.org/10.1093/jnci/djr203. Asian multinational study. Endoscopy 2010;42:699-704. https:// 52 Bandla S, Peters JH, Ruff D, et al. Comparison of cancer-associ- doi.org/10.1055/s-0030-1255629 ated genetic abnormalities in columnar-lined esophagus tissues 37 Chang CY, Lee YC, Lee CT, et al. The application of Prague with and without goblet cells. Ann Surg 2014;260:72-80. https:// C and M criteria in the diagnosis of Barrett’s esophagus in an doi.org/10.1097/SLA.0000000000000424 ethnic Chinese population. Am J Gastroenterol 2009;104:13-20. 53 Kelty CJ, Gough MD, Van Wyk Q, et al. Barrett’s oe- https://doi.org/10.1038/ajg.2008.43 sophagus: intestinal metaplasia is not essential for can- 38 Vahabzadeh B, Seetharam AB, Cook MB, et al. Validation of the cer risk. Scand J Gastroenterol 2007;42:1271-4. https://doi. Prague C & M criteria for the endoscopic grading of Barrett’s org/10.1080/00365520701420735 GASTRO-ESOPHAGEAL REFLUX DISEASE AND BARRETT’S ESOPHAGUS 127

54 Siddiki HA, Lam-Himlin DM, Kahn A, et al. Intestinal metaplasia 61 Srivastava A, Appelman H, Goldsmith JD, et al. The use of of the gastric cardia: findings in patients with versus without Bar- ancillary stains in the diagnosis of Barrett Esophagus and rett’s esophagus. Gastrointest Endosc 2019;89:759-68. https:// Barrett Esophagus-associated dysplasia: recommendations doi.org/10.1016/j.gie.2018.10.048 55 Chandrasoma PT, Der R, Dalton P, et al. Distribution and signifi- from the Rodger C. Haggitt Gastrointestinal Pathology Soci- cance of epithelial types in columnar lined esophagus. Am J Surg ety. Am J Surg Pathol 2017;41:e8-e21. https://doi.org/10.1097/ Pathol 2001;25:1188-1193. https://doi.org/10.1097/00000478- PAS.0000000000000819 200109000-00010 62 Groisman GM, Amar M, Meir A. Expression of the intestinal 56 Harrison R, Perry I, Haddadin W, et al. Detection of intestinal metaplasia in Barrett’s esophagus: an observational comparator marker Cdx2 in the columnar-lined esophagus with and with- study suggests the need for a minimum of eight biopsies. Am out intestinal (Barrett’s) metaplasia. Mod Pathol 2004;17:1282-8. J Gastroenterol 2007;102:1154-61. https://doi.org/10.1111/j.1572- https://doi.org/10.1038/modpathol.3800182 0241.2007.01230.x 63 57 Gatenby PA, Ramus JR, Caygill CP, et al. Relevance of the de- McIntire MG, Soucy G, Vaughan TL, et al. MUC2 is a highly spe- tection of intestinal metaplasia in non-dysplastic columnar-lined cific marker of goblet cell metaplasia in the distal esophagus and oesophagus. Scand J Gastroenterol 2008;43:524-30. https://doi. gastroesophageal junction. Am J Surg Pathol 2011;35:1007-13. org/10.1080/00365520701879831 https://doi.org/10.1097/PAS.0b013e318218940d 58 Corley DA, Kubo A, DeBoer J, et al. Diagnosing Barrett’s esophagus: reliability of clinical and pathologic diagnoses. 64 Shearer C, Going J, Neilson L, et al. Cytokeratin 7 and 20 Gastrointest Endosc 2009;69:1004-10. https://doi.org/10.1016/j. expression in intestinal metaplasia of the distal oesopha- gie.2008.07.035 gus: relationship to gastro-oesophageal reflux disease. His- 59 Panarelli NC, Yantiss RK. Do Ancillary Studies Aid De- topathology 2005;47:268-75. https://doi.org/10.1111/j.1365- tection and Classification of Barrett Esophagus? Am J Surg Pathol 2016;40:e83-e93. https://doi.org/10.1097/ 2559.2005.02219.x PAS.0000000000000654 65 Zhang X, Westerhoff M, Hart J. Expression of SOX9 and CDX2 60 Johnson DR, Abdelbaqui M, Tahmasbi M, et al. CDX2 protein in nongoblet columnar-lined esophagus predicts the detection of expression compared to alcian blue staining in the evaluation of esophageal intestinal metaplasia. World J Gastroenterol Barrett’s esophagus during follow-up. Mod Pathol 2015;28:654- 2015;21:2770-6. https://doi.org/10.3748/wjg.v21.i9.2770 61. https://doi.org/10.1038/modpathol.2014.157 PATHOLOGICA 2020;112:128-137; DOI: 10.32074/1591-951X-156

Review

Non gastro-esophageal reflux disease related esophagitis: an overview with a histologic diagnostic approach

Luca Mastracci1-2*, Federica Grillo1-2*, Paola Parente3, Elettra Unti4, Serena Battista5, Paola Spaggiari6, Michela Campora1, Luca Valle1, Matteo Fassan7, Roberto Fiocca1-2 1 Anatomic Pathology, San Martino IRCCS Hospital, Genova, Italy; 2 Anatomic Pathology, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genova, Italy; 3 Pathology Unit, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy; 4 UOC Anatomia Patologica, ARNAS Ospedali Civico-Di Cristina-Benfratelli, Palermo, Italy; 5 SOC di Anatomia Patologica, Azienda Sanitaria Universitaria Friuli Centrale, Udine, Italy; 6 Department of Pathology, Humanitas Clinical and Research Center-IRCCS, Rozzano, Milan, Italy; 7 Surgical Pathology Unit, Department of Medicine (DIMED), University of Padua, Italy * These authors contributed equally

Summary Several pathological conditions, other than gastro-esophageal reflux disease and its com- plications, can affect the esophagus. While some of these can present with unspecific lesions (i.e. ulcers and epithelial damage) and require clinico-pathological correlation for Received and accepted: June 24, 2020 diagnosis (i.e. drug-induced esophagitis and corrosive esophagitis) other conditions show Published online: 29 October, 2020 distinctive histological lesions which enable the pathologist to reach the diagnosis (i.e. some specific infectious esophagites and Crohn’s disease). In this context eosinophilic Correspondence esophagitis is the condition which has been increasingly studied in the last two decades, Luca Mastracci while lymphocytic esophagitis, a relatively new entity, still represents an enigma. This over- Anatomic Pathology Section, University of view will focus on and describe histologic lesions which allow pathologists to differentiate Genova and Ospedale Policlinico San Martino, between these conditions. largo Rosanna Benzi 10, 16132 Genova, Italy Tel. +39 010 5555954 Key words: drug-induced esophagitis, esophagitis dissecans superficialis, esophageal Fax: +39 010 5556932 Crohn’s disease, lymphocytic esophagitis, eosinophilic esophagitis E-mail: [email protected]

https://orcid.org/0000-0003-0193-5281 Conflict of interest Introduction The Authors declare no conflict of interest. Gastro-esophageal reflux disease (GERD) and its complications, such How to cite this article: Mastracci L, Grillo F, as Barrett’s esophagus (BE), are a major concern when facing esopha- Parente P, et al. Non gastro-esophageal reflux geal biopsies. However, other than GERD and BE, many other patholog- disease related esophagitis: an overview ical conditions can affect the esophagus, either as the unique and typical with a histologic diagnostic approach. localization of an illness or as an expression of diseases which can affect Pathologica 2020;112:128-137. https://doi. org/10.32074/1591-951X-156 any part of the gastro-intestinal tract (i.e. Crohn disease) or also as com- plications in patients with immunodeficiency (i.e. infectious esophagitis). © Copyright by Società Italiana di Anatomia Pato- Some of these conditions, such as eosinophilic esophagitis (EE), have logica e Citopatologia Diagnostica, Divisione Itali- ana della International Academy of Pathology received much attention in the last decades in order to better define both clinico-pathological aspects and possible therapeutic approaches. Other OPEN ACCESS conditions have been only recently described (ie lymphocytic esophagi- tis) and still represent an enigma or, as stated by Ronkainen “a condition This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- in search for a disease”. Finally, there are numerous drugs and exog- NonCommercial-NoDerivatives 4.0 International) license: the enous substances which can affect the esophagus: in this setting the work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original importance of a close relationship with the gastroenterologist in order version. For further information: https://creativecommons. to obtain relevant clinical information and the knowledge of rare entities org/licenses/by-nc-nd/4.0/deed.en which can enter in the differential diagnosis are fundamental. NON GASTRO-ESOPHAGEAL REFLUX DISEASE RELATED ESOPHAGITIS 129

This overview on non-neoplastic esophageal disease Biopsy site has the main aim of furnishing a practical diagnostic Histology is considered the gold standard for iden- approach to biopsy samples in the principal illness- tifying specific infectious agents. Biopsies must be es affecting the esophagus tract, referring to the most taken on the more pronounced lesions. For HSV in- recent published recommendations, guidelines and fections, biopsies obtained at the ulcer edges are expert opinions. more informative as they permit the recognition of pathologic lesions seen in epithelial cells, while in CMV infection, biopsies from the ulcer center, where Infectious esophagitis endothelial and stromal cells are easily identified, are more reliable.

Definition Histologic elementary lesions Infectious esophagites are a group of diseases in The inflammatory background is non-specific and com- which inflammation and damage of the esophagus is posed of a mixed inflammatory infiltrate with erosion, attributable to various infectious agents. ulcer and necrotic debris. Typical lesions include rec- ognition of hyphae and spores for Candida, multinu- Clinical picture cleated giant cells and glassy eosinophilic intranuclear The clinical presentation is extremely variable ranging inclusions for HSV 5 and nucleomegaly with ‘owl’s eye’ from no symptoms to severe dysphagia, retrosternal intranuclear inclusions for CMV (Fig. 1). Histochemical chest pain and fever. stains for hyphae/spores (PAS, Grocott), mycobacteria Infectious esophagites occur more often in immu- (Ziehl Neelsen) and immunohistochemistry (CMV or nocompromised patients, and in particular in Hu- HSV) help in confirming diagnosis. man Immunodeficiency Virus (HIV) infection, in post-transplant immunosuppression and in long- Diagnosis term oral corticosteroid use. In immunocompetent When typical lesions are histologically recognized, subjects, infectious esophagites are rare and favored and eventually confirmed by histochemistry and/or im- by conditions that cause a relative weakening of the munohistochemistry, a diagnosis of specific infectious immune system (i.e. diabetes mellitus, cirrhosis, etc.) esophagitis can be made. In other cases, a clinical or functional (dysmotility) and structural (strictures) integration with microbiologic/sierologic studies is rec- disorders 1. ommended.

Endoscopy picture The endoscopic appearances vary depending on the Drug-induced esophagitis infectious agent. In Candida esophagitis, which more often affects HIV patients, white mucosal plaque-like Definition lesions and exudates tenaciously adherent to the mu- Drug-induced esophagitis can present either as pill-in- cosa and not removable with water from irrigation, are duced esophagitis or radiation and chemotherapy es- observed along the esophageal surface 2; subsequent ophagitis. disease manifestations may include friability and ul- The term pill-induced esophagitis refers to esopha- ceration. Diversely, Herpes Simplex Virus (HSV) and geal injury caused by almost 100 different types of Cytomegalovirus (CMV) infections are more frequent substances ingested in form of pills. Antibiotics are in post-transplant patients; endoscopic manifestations one of the major contributors, especially tetracycline vary from vesicles to unspecific erosions to large, and doxycycline; other major contributors are biphos- well circumscribed, volcano-like ulcerations with ex- phonates, non-steroidal anti-inflammatory drugs and udates 3,4 for HSV while CMV presents with erosions iron pills 6. Injuries occur when caustic medicinal pills and ulcers in the middle and distal portion of the es- dissolve in the esophagus rather than passing rap- ophagus. idly into the stomach 7; usually this occurs for larger Mycobacterium tuberculosis rarely affects the esoph- pills and/or in correspondence of lumen reduction or agus, even in endemic areas. Human Papillomavirus strictures which make swallowing more difficult 8. has been occasionally documented as cause of acute Radiation esophagitis affects less than 1% of patients esophagitis, while it can play a role in esophageal receiving radiation treatment for advanced stages of squamous papilloma and esophageal squamous car- common cancers such as lung, breast and esophage- cinoma. al carcinomas 9. 130 L. Mastracci et al.

Figure 1. (A) Cytomegalovirus esophagitis (magnification 40x). Nucleomegaly with intranuclear inclusions (black arrow). (B) Cytomegalovirus esophagitis (magnification 40x). Immunohistochemistry with anti-CMV antibody, showing sparse positive nuclei. (C) Herpes Simplex Virus esophagitis (magnification 10x). Multinucleated giant cells and ground glass intranuclear inclusions (black arrow) with necrotic debris and inflammation. (D) Candida Albicans esophagitis (magnification 20x). Alcian Blu PAS staining showing hyphae and spores on the surface of squamous epithelium.

Several chemotherapeutic agents (bleomycin, 5-fluo- incrementing radio-sensitizing action (as in the case rouracil, methotrexate, vincristine) may cause esoph- of doxorubicin and bleomicin) 10. ageal and oropharyngeal mucositis 10. The referral information concerning pill ingestion and type of treatment is fundamental for a correct diagno- Clinical picture sis of drug induced esophagitis. Common symptoms for pill-induced esophagitis are odynophagia, chest pain, vomiting, dysphagia and, Endoscopy picture less frequently, hematemesis 11. Perforation and fatal The endoscopic description of pill-induced esophagi- injuries have also been reported. tis can vary from erythema, which is the most frequent Radiation esophagitis generally presents with non- specific symptoms, such as dysphagia and odyno- endoscopic alteration, to erosions and ulcers which phagia, occurring within the first 2 months of treat- are present, on the whole, in about 50-60% of patients. ment. Radiation and chemotherapy esophagitis show non- Chemotherapy can directly damage the esophageal specific and non-diagnostic endoscopic changes mucosa and manifests with unspecific symptoms, or such as erythema and ulceration. NON GASTRO-ESOPHAGEAL REFLUX DISEASE RELATED ESOPHAGITIS 131

Biopsy site Diagnosis Biopsies are taken in the endoscopically affected areas. When a certain correlation with pill ingestion or chemo-radiation treatment is clinically referred, the di- Histologic elementary lesions agnosis of drug-induced esophagitis can be suggest- Biopsies show variable inflammatory changes with ed even though histological lesions are unspecific. erosion, ulcer, acute inflammation and granulation tissue. In radiation esophagitis, early damage is rep- Corrosive esophagitis resented by ballooning degeneration and edema with inflammation and vessels hyalinization (Fig. 2A). Ra- Definition diation-related atypia of endothelial and stromal cells Corrosive esophagitis is the consequence of volun- can guide diagnosis. This aspect can mimic cytomeg- tary (suicide, attempted suicide or parasuicide) or ac- alovirus cytopathic changes which need to be ruled cidental ingestion of strong alkaline or, less frequent- out through negative CMV specific immunostaining. ly, acidic substances (liquid or solid mainly in form of

Figure 2. (A) Radiation esophagitis (magnification 10x). Ballooning degeneration of squamous epithelium with edema; a vessel with hyalinized wall is present in the submucosal layer. (B) Corrosive esophagitis (magnification 10x) in a patient with accidental ingestion of sodium hydroxide (lye or caustic soda) showing necrosis and inflammation with diffuse ulceration of the mucosa and submucosa. (C) Esophagitis dissecans superficialis (magnification 40x). An intraepithelial cleft (black arrow) is visible with two strips of squamous epithelium showing a different color tone. (D) Black esophagus (magnification 10x). Autopsy finding, revealing necrosis of the esophagus with abundant neutrophils and abundant brown pigmented granules. 132 L. Mastracci et al.

crystals). Daily consumption of vinegar drinks with pH Diagnosis value lower than 5.5 has also been reported to cause Clinical correlation with type of ingested substances 12 corrosive damage in the esophagus . and timing is necessary to express diagnosis with cer- tainty. Clinical picture Clinical presentation depends on type of substance, its amount and physical form. Liquid substances affect Esophagitis dissecans superficialis the esophagus and stomach more easily, while crys- (sloughing esophagitis) tals (more difficult to swallow) provoke greater dam- age in the oral cavity, oropharynx and upper airways. Definition Patients with esophageal damage complain of stridor, drooling, dysphagia and odynophagia. In about 70% Esophagitis dissecans superficialis (EDS) is a benign, of patients there is contemporary gastric involvement self-limited, desquamative disorder of the esophagus, manifesting with epigastric pain and bleeding. Perfora- characterized by sloughing of the superficial mucosa. tion can occur within 2 weeks from ingestion 13. Clinical picture

Endoscopy picture EDS affects older women more frequently, often with Endoscopy can be safely performed up to the 4th day chronic illness and in poly-medication therapy, includ- after corrosive liquid ingestion with minimal air insuffla- ing benzodiazepines, selective serotonin re-uptake 17 tion; risk of perforation after this time set, significantly inhibitors, opiates and antiepiletics . increases. Severity of mucosal lesions on endoscopy More than half of patients are asymptomatic and EDS can be graded from 1 to 4 14,15 varying from mucosal is discovered during upper GI endoscopy for other edema and erythema (grade 1) to superficial (2A) or reasons. When symptomatic, dysphagia, abdominal deep (2B) mucosal and submucosal ulcerations, trans- pain or heartburn can be part of the symptomatic mural ulcerations with focal (3A) or extensive (3B) ne- spectrum 18. crosis and perforation (4). High grade (2B-4) damage Endoscopy picture may lead to the development of chronic complications, such as esophageal strictures and extensive fibrosis, Endoscopically, esophageal mucosa appears more frequently than low grade injuries (1-2A) 16. sloughed in streaks and patches. These features are more pronounced in the distal two thirds of the eso- Biopsy site phagus 19. Biopsies are not indicated in the acute setting. Pathol- Biopsy site ogists can face acute damage in surgical specimens (Fig. 2B) or autopsy. In chronic complications biopsies Biopsies are taken in the endoscopically affected ar- are performed in the affected area without a specific eas. protocol. Histologic elementary lesions Histologic elementary lesions The main histologic feature is the presence of an in- Acute damage. Acidic liquids cause acute coagula- traepithelial cleft which separate the squamous epi- tive necrosis, while alkaline substances provoke liq- thelium in two strips with a different color tone: the uefactive necrosis. Other than necrosis, in the acute superficial sloughed layers are intensely eosinophil- phase, small vessel thrombosis, mucosal sloughing ic, while the layers under the cleft are almost normal and bacterial overgrowth are present. (Fig. 2C). Inflammation can be present, with neutro- Chronic damage. Esophageal mucosa repair starts philic and eosinophilic infiltrates, but is not a constant after 10 days with subsequent re-epithelialization of feature; necrotic debris mixed with bacteria and fungi ulcers. Esophageal biopsies show variable degree of can be seen within the superficial sloughed epitheli- esophagitis with basal cell hyperplasia, papillae elon- um 20. gation, dilatation of intercellular space and intraep- ithelial inflammatory infiltrates with erosion/ulcers; Diagnosis hyper-parakeratosis are frequent. Repair of lesions EDS histologic diagnosis can be suggested when include scar tissue formation with marked fibrosis and there is a clear color-tone change between layers consequent strictures; in the lower esophagus this over and under the cleft; epithelium detachments, as a leads to sphincter impairment with gastro-esophageal consequence of withdrawal traumatism during biopsy reflux worsening histologic damage 13. procedures, have to be excluded. NON GASTRO-ESOPHAGEAL REFLUX DISEASE RELATED ESOPHAGITIS 133

Black esophagus (acute esophageal esophageal involvement, CD is already diagnosed in necrosis or Gurvits syndrome) its more common ileal and colonic localizations with only exceptional cases being diagnosed in the esoph- 26 Definition agus as the unique primary involved site . Black esophagus is an uncommon entity character- Clinical picture ized by a diffuse, circumferential black color of the es- ophageal mucosa. The incidence of esophageal CD is less than 2% in adults, while it can reach up to 25-40% in children. Es- Clinical picture ophageal symptoms are often mild, non-specific and Typical patients are older men with multiple comor- generally overcome by typical intestinal symptoms. bidities (including vascular disease, diabetes mellitus, Moreover, in just under 50% of patients, gastro-eso- hypertension, chronic liver, kidney and pulmonary dis- phageal CD can be completely asymptomatic 27. Up- eases, malnutrition, malignancy, or alcohol abuse) pre- per gastrointestinal CD involvement plays an impor- senting with hematemesis or melena. A combination tant role because it predicts a more severe disease of esophageal ischemia, corrosive damage caused by with a greater frequency of complications such as ob- gastro-esophageal reflux of acid and pepsin and im- struction and perforation 28. paired mucosal reparative mechanisms contribute to Endoscopy picture the mucosal damage 21,22. Recurrence or death are fre- quent and affect over a quarter of cases 23. Esophageal CD most commonly involves the mid and distal portions of the esophagus while the proximal Endoscopy picture esophagus is rarely affected. Endoscopic alterations On endoscopy, esophageal mucosa appears diffuse- range from erythema and erosions to superficial and ly black colored, sometimes with small whitish areas, deep ulcers in the majority of cases. Pseudopolyps, and circumferential extension involving the lower third fistulas and strictures may be rarely associated 26. of the esophagus with an abrupt transition at the gas- troesophageal junction and variable proximal exten- Biopsy site sion 22,24. Upper gastro-esophageal endoscopy is not routinely performed in patients with CD and a biopsy protocol Biopsy site is not suggested; biopsies are, as a rule, taken in af- The endoscopic picture is diagnostic and biopsies are fected areas. usually not required to confirm the diagnosis 22. Pa- thologists more often make the diagnosis of black es- Histologic elementary lesions ophagus on autopsy cases or on surgical specimens Histologic lesions are more commonly unspecific with when no response to medical treatment is seen. chronic inflammation, erosions, ulcers and necrotic debris. Non-caseating granulomas, epithelioid and/or Histological elementary lesions giant cells, in lamina propria are identified only in a Histology reveals complete mucosal necrosis with minority of patients (Fig. 3A), making this diagnosis abundant neutrophils above the muscularis mucos- challenging in the absence of intestinal manifestations ae which remains viable. Vessels with intravascu- of CD. lar thrombi and golden brown pigmented granules Lymphocytic esophagitis (LE; see later) has been (Fig. 2D) negative for Prussian blue and referable to associated in a quarter of cases with CD in pediatric lipofuscin complete the picture 25. patients 29. This association was not confirmed with the same incidence by other authors nor in adult- Diagnosis hood, however, even though it may be rarer, there The endoscopic aspect drives the diagnosis which is are enough reports to confirm that an association be- eventually confirmed by the presence of the histologic tween CD and LE does exist. mucosal damage. Diagnosis In patients with previously diagnosed CD, unspecific Esophageal Crohn’s disease inflammatory changes and ulcers can suggest esoph- ageal involvement, while non-caseating granulomas Definition are diagnostic. Crohn’s disease (CD) can localize in the entire gas- For first suspicions of diagnosis of CD, it is essential to tro-intestinal tract; in the vast majority of patients with exclude infectious etiology. 134 L. Mastracci et al.

Figure 3. (A) Esophageal Crohn’s disease (magnification 20x). Presence of an epithelioid granuloma within squamous epi- thelium, diagnostic for esophageal localization of Crohn’s disease in a patient with previous diagnosis in the distal ileum; (B) Eosinophilic esophagitis (magnification 40x). A high number of intraepithelial eosinophils is present, also forming microab- scesses (black arrow) in the superficial layer. (C) Lymphocytic esophagitis (magnification 40x). Marked spongiosis is present with an increase in intraepithelial lymphocytes in the peri-papillary areas. (D) Lymphocytic esophagitis (magnification 40x). Immunostains for CD3 showing a marked increase in intraepithelial T lymphocytes.

Lymphocytic esophagitis alence, varying from younger male to older female patients. Definition It is emerging that LE is a histologic pattern common Lymphocytic esophagitis (LE) was initially described to different clinical diseases: 1) in children and young- by Rubio as a novel histologic phenotype of chron- er patients, LE is frequently associated with CD 33; 2) ic esophagitis characterized by a high number of in- in older women, presenting with dysphagia, LE can be traepithelial lymphocytes surrounding papillae, asso- an expression of motility disorders 34 - adult patients ciated with marked spongiosis 30; the same pattern with LE, stratified immunophenotypically, showed a was also described in other primates 31,32. high prevalence of non-achalasia primary esophageal motility disorders with a predominant CD4+ T cell infil- Clinical picture trate 35; 3) some reports finally suggest that in adults, LE seems to be a rare condition, diagnosed in about LE with a predominant CD8+ T cell infiltrate, can be 1 every 1000 esophageal endoscopies. Different stud- expression of gastroesophageal reflux disease in ab- ies have reported a different age and sex related prev- sence of motility disorders 36. NON GASTRO-ESOPHAGEAL REFLUX DISEASE RELATED ESOPHAGITIS 135

Patients can be completely asymptomatic or present clinical and pathologic information. EoE is defined by with unspecific upper gastro-intestinal symptoms, the following criteria: symptoms related to esophageal ranging from dysphagia to reflux/heartburn, abdomi- dysfunction; ≥ 15 eosinophils per HPF; eosinophilia nal and chest pain, or nausea and vomiting 37. limited to the esophagus excluding other causes of esophageal eosinophilia 41. Endoscopic picture Endoscopy is negative in about a third of patients; in Clinical picture about another third of patients, LE can endoscopically EoE shows a white male predominance (both in chil- resemble eosinophilic esophagitis with multiple con- dren and adults) with a 6.5% prevalence in all patients centric rings giving an aspect of ‘feline esophagus’. undergoing upper endoscopy 42. Other possible observations are strictures, erythema, In pediatric patients, symptoms are heterogeneous furrows and webs 38. and include abdominal pain, nausea, reflux-like symp- toms and feeding difficulties with consequent growth Biopsy site failure. The mid esophagus is involved more frequently and In adults, EoE predominately presents with dysphagia with more pronounced endoscopic lesions than the for solid food associated with food impaction. proximal or distal part; if LE is clinically or endoscopi- Currently, two different forms of EoE are recognized cally suspected, biopsies from all the esophagus seg- on the basis of response to treatment: proton pump ments are suggested. inhibitor (PPI)-responsive EoE and steroid-responsive EoE. PPI-responsive EoE substantially differs from histologic elementary lesions GERD as it shows mast cell signature genes and the The main histologic feature is the notable increase in expression of genes involved in type 2 (Th2)-associ- intraepithelial lymphocytes in the peri-papillary areas, ated allergic inflammation, and is similar to steroid-re- associated with marked spongiosis (Fig. 3 C-D). Dif- sponsive EoE. The introduction of PPI-responsive ferent studies have tried to better define this lympho- EoE in the spectrum of EoE has changed the diag- cytic increase using cut-off values ranging from 10 to nostic algorithm of this condition 43,44. 50 per HPF with 20 lymphocytes/HPF being the com- monly used cut off. This results in a marked increase Endoscopy picture of LE diagnoses and therefore, the use of a cut off val- The clinical diagnosis of EoE does not require the ue (in particular, with a such a low count as 20/HPF, presence of endoscopic features; however, prospec- which is present in some healthy volunteers and in tive studies have identified endoscopic lesions in as reflux disease) is not recommended. The main criteria much as 93% of patients 45. Endoscopically detectable continues to be of a high number of peri-papillary lym- features vary from multiple concentric rings with ‘fe- phocytes with marked spongiosis in the absence of line esophagus’ which is the most evocative lesion, to both neutrophils and eosinophils 39. Candida Albicans strictures and narrowing of lumen. Longitudinal furrows can superimpose on LE, so attention must be paid to and white exudates complete the spectrum of possible lymphocytes even in other conditions. lesions that affect both the distal and proximal esoph- agus. Rings and strictures are the outcome of multiple Diagnosis inflammatory insults with esophageal remodeling, thus Given the variability of clinical conditions associated they can be completely absent in children 43. with this histological aspect 40, a diagnosis of com- patibility with LE is suggested in the presence of his- Biopsy site tologic hallmarks: clinicians should investigate which The recommended biopsy protocol includes at least conditions (among CD, motility disorders, GERD and six biopsies taken from the distal, middle and proximal maybe others) are responsible for the histologic le- esophagus, focusing on areas with endoscopic mu- sions. cosal abnormalities 46.

Histologic elementary lesions Eosinophilic esophagitis The main histological feature is the presence of a high number of intraepithelial eosinophils, characteristi- Definition cally forming microabscesses in the superficial layer Eosinophilic esophagitis (EoE) is a chronic allergen (Fig. 3B). At a formal count, a number of ≥ 15 eosino- driven immune mediated clinico-pathological disorder phils per HPF (standard size of 0.3 mm2) in hot spots diagnosed by clinicians taking into consideration both is required 46. 136 L. Mastracci et al.

In addition to eosinophils, basal cell hyperplasia, pa- jury: endoscopic features and clinical outcomes. Endoscopy. pillae elongation and dilatation of intercellular spaces 2005;37:740-744. https://doi.org/10.1055/s-2005-870129 12 complete the histologic picture. Chang J, Han SE, Paik SS, et al Corrosive esophageal injury due to a commercial vinegar beverage in an adolescent. Clin Endosc 2019. https://doi.org/10.5946/ce.2019.066. Epub ahead Diagnosis of print The diagnosis, as already mentioned, is performed by 13 Contini S, Scarpignato C, Caustic injury of the upper gastroin- clinicians taking into account clinical and pathological testinal tract: a comprehensive review. World J Gastroenterol. information. For this reason, finding ≥ 15 eosinophils 2013;19:3918-3930. https://doi.org/10.3748/wjg.v19.i25.3918 per HPF should lead to a report of ‘compatible with 14 Zargar SA, Kochhar R, Nagi B, et al. Ingestion of corrosive acids. EoE’ rather than ‘diagnostic for EoE’. Spectrum of injury to upper gastrointestinal tract and natural his- tory. Gastroenterology 1989;97:702-7. 15 Zargar SA, Kochhar R, Nagi B, et al. Ingestion of strong corro- sive alkalis: spectrum of injury to upper gastrointestinal tract and Conclusions natural history. Am J Gastroenterol 1992;87:337-41. 16 Kamat R, Gupta P, Reddy YR, et al. Corrosive injuries of the In conclusion, as seen in this brief overview, many upper gastrointestinal tract: a pictorial review of the imaging non-neoplastic diseases can affect the esophagus. features. Indian J Radiol Imaging 2019;29:6-13. https://doi. Endoscopic biopsies of the esophagus are often re- org/10.4103/ijri.IJRI_349_18 17 quired to establish the correct diagnosis within an Purdy JK, Appelman HD, McKenna BJ. Sloughing esophagitis is associated with chronic debilitation and medications that injure appropriate clinical context. Indeed, information on the esophageal mucosa. Mod Pathol 2012;25:767-775. https:// endoscopic and clinical findings are a fundamental doi.org/10.1038/modpathol.2011.204 basis for the interpretation of histology, which may be 18 Hart PA, Romano RC, Moreira RK, et al. Esophagitis dissecans similar in diverse settings, thus leading to a correct superficialis: clinical, endoscopic, and histologic features. Dig histopathologic diagnosis. Dis Sci 2015;60:2049-2057. https://doi.org/10.1007/s10620-015- 3590-3 19 Longman RS, Remotti H, Green PH. Esophagitis dissecans References superficialis. Gastrointest Endosc 2011;74:403-404. https://doi. org/10.1016/j.gie.2011.03.1117 1 Ahuja NK, Clarke JO. Evaluation and management of infectious 20 esophagitis in immunocompromised and immunocompetent in- Moawad FJ, Appleman HD. Sloughing esophagitis: a spectacu- dividuals. Curr Treat Options Gastro 2016;14:28-38. https://doi. lar histologic and endoscopic disease without a uniform clinical org/10.1007/s11938-016-0082-2 correlation. Ann N Y Acad Sci 2016;1380:178-182. https://doi. org/10.1111/nyas.13112 2 Mohamed AA, Lu XL, Mounmin FA. Diagnosis and treatment of 21 esophageal candidiasis: current updates. Can J Gastroenterol Khan H, Ahmed M, Daoud M, et al. Acute Esophageal Necro- Hepatol. 2019:3585136. https://doi.org/10.1155/2019/3585136 sis: a view in the dark. Case Rep Gastroenterol 2019;13:25-31. https://doi.org/10.1159/000496385 3 Itoh T, Takahashi T, Kusaka K, et al. Herpes simplex esophagitis 22 from 1307 autopsy cases. J Gastroenterol Hepatol. 2003;18:1407- Dias E, Santos-Antunes J, Macedo G. Diagnosis and man- 1411. https://doi.org/10.1046/j.1440-1746.2003.03166.x agement of acute esophageal necrosis. Ann Gastroenterol 2019;32:529-540. https://doi.org/10.20524/aog.2019.0418 4 Wang HW, Kuo CJ, Lin WR, et al. The clinical characteristics and 23 manifestations of cytomegalovirus esophagitis. Dis Esophagus Kim SM, Song KH, Kang SH, et al. Evaluation of prognostic fac- 2016;29:392-399. https://doi.org/10.1111/dote.12340 tor and nature of acute esophageal necrosis: restropective mul- ticenter study. Medicine (Baltimore) 2019;98:e17511. https://doi. 5 Maguire A, Sheahan K. Pathology of oesophagitis. Histo- org/10.1097/MD.0000000000017511 pathology. 2012;60:864-879. https://doi.org/10.1111/j.1365- 24 2559.2011.03855.x Shah J, Savlania A, Bush N, et al. Three cases of 6 an unusual cause of haematemesis: black oesopha- Bestari MB, Agustanti N, Abdurachman SA. Clindamycin-In- gus. Trop Doct 2020;14:49475519900756. https://doi. duced Esophageal Injury: Is It an Underdiagnosed entity? Clin org/10.1177/0049475519900756 Med Insights Case Rep. 2019;12:1179547619884055. https:// 25 doi.org/10.1177/1179547619884055 Jessurun J, Cui I, Aristi-Urista G. Acute (gangrenous) esopha- geal necrosis (black esophagus). A rare form of injury with spe- 7 Kikendall JW. Pill esophagitis. J Clin Gastroenterol 1999;28:298- cific histologic features and diverse clinical associations with a 305. https://doi.org/10.1097/00004836-199906000-00004 common pathogenesis. Hum Pathol. 2019;87:44-50. https://doi. 8 Panarelli NC. Other forms of esophagitis: it is not gastroesoph- org/10.1016/j.humpath.2019.02.003 ageal reflux disease, so now what do I do? Surg Pathol Clin 26 De Felice KM, Katzka DA, Raffals LE. Crohn’s Disease of the 2017;10:765-779. https://doi.org/10.1016/j.path.2017.07.001 esophagus: clinical features and treatment outcomes in the 9 Murro D, Jakate S. Radiation esophagitis. Arch Pathol Lab Med biologic era. Inflamm Bowel Dis 2015;21:2106-2113. https://doi. 2015;139:827-830. https://doi.org/10.5858/arpa.2014-0111-RS org/10.1097/MIB.0000000000000469 10 Grossi L, Ciccaglione AF, Marzio L. Esophagitis and its causes: 27 Saadah OI, Fallatah KB, Baumann C, et al. Histologically con- Who is “guilty” when acid is found “not guilty”?. World J Gas- firmed upper gastrointestinal Crohn’s disease: is it rare or are troenterol 2017;23:3011-3016. https://doi.org/10.3748/wjg.v23. we just not searching hard enough? Intest Res 2020. https://doi. i17.3011 org/10.5217/ir.2019.00091 11 Abid S, Mumtaz K, Jafri W, et al. Pill-induced esophageal in- 28 Laube R, Liu K, Schifter M, et al. Oral and upper gastrointesti- NON GASTRO-ESOPHAGEAL REFLUX DISEASE RELATED ESOPHAGITIS 137

nal Crohn’s disease. J Gastroenterol Hepatol 2018;33:355-364. an enigma a decade later. World J Gastroenterol 2017:23:949- https://doi.org/10.1111/jgh.13866 956. https://doi.org/10.3748/wjg.v23.i6.949 29 Ebach DR, Vanderheyden AD, Ellison JM, et al. Lymphocytic 39 Genta RM. Lymphocytic Esophagitis. Gastroenterol Hepatol esophagitis. A possible manifestation of pediatric upper gas- 2015;11:559-561. trointestinal Crohn’s disease. Inflamm Bowel Dis 2011;17:4549. 40 Ronkainen J, Walker MM, Aro P et al. Lymphocytic oesophagitis, https://doi.org/10.1002/ibd.21347 a condition in search of a disease? Gut 2012;61:1776. https:// 30 Rubio CA, Sjödahl K, Lagergren J. Lymphocytic esophagitis. doi.org/10.1136/gutjnl-2012-302329 A histologic subset of chronic esophagitis. Am J Clin Pathol 41 Dellon ES, Gonsalves N, Hirano I, et al. ACG clinical guideline: 2006;125:432-437. Evidenced based approach to the diagnosis and management 31 Rubio CA, Dick EJ Jr, Orrego A, et al. Incidence of lymphocytic of esophageal eosinophilia and eosinophilic esophagitis (EoE). esophagitis in baboons. In vivo 2008;22:613-616. Am J Gastroenterol 2013;108:679-692. https://doi.org/10.1038/ 32 Rubio CA, Dick EJ, Orrego A, et al The frequency of lymphocytic ajg.2013.71 and reflux esophagitis in non-human primates. Int J Clin Exp 42 Veerappan GR, Perry JL, Duncan TJ. Prevalence of eosinophilic Pathol 2008;1:531-535. esophagitis in an adult population undergoing upper endoscopy: 33 Sutton LM, Heintz DD, Patel AS, et al. Lymphocytic esophagitis a prospective study. Clin Gastroenterol Hepatol. 2009;7:420- in children Inflamm Bowel Dis 2014;20:1324-1328. https://doi. 426. https://doi.org/10.1016/j.cgh.2008.10.009 org/10.1097/MIB.0000000000000100 43 Molina-Infante J, Bredenoord AJ, Cheng E, et al. Proton pump 34 Haque S, Genta RM. Lymphocytic oesophagitis: clinicopatho- inhibitor-responsive oesophageal eosinophilia: an entity chal- logical aspects of an emerging condition. Gut 2012;61:1108- lenging current diagnostic criteria for eosinophilic oesophagitis. 1114. https://doi.org/10.1136/gutjnl-2011-301014 Gut 2016;65:524-31. https://doi.org/10.1136/gutjnl-2015-310991 35 Xue Y, Suriawinata A, Liu X, et al. Lymphocytic Esophagitis With 44 Molina-Infante J, Hirano I, Spechler SJ, et al. Clarifying mis- CD4 T-cell-predominant intraepithelial lymphocytes and primary understandings and misinterpretations about oesophageal esophageal motility abnormalities: a potential novel clinico- eosinophilia. Gut. 2017;66:1173-1174. https://doi.org/10.1136/ pathologic entity. Am J Surg Pathol 2015;39:1558-67. https://doi. gutjnl-2016-312851 org/10.1097/PAS.0000000000000493 45 Kim HP, Vance RB, Shaheen NJ, et al. The prevalence and diag- 36 Lisovsky M, Westerhoff M, Zhang X. Lymphocytic esophagitis: nostic utility of endoscopic features of eosinophilic esophagitis:a a histologic pattern with emerging clinical ramifications. Ann N Y meta-analysis. Clin Gastroenterol Hepatol 2012;10:988-996. Acad Sci 2016;1381:133-138. https://doi.org/10.1111/nyas.13260 https://doi.org/10.1016/j.cgh.2012.04.019 37 Cohen S, Saxena A, Waljee AK, et al. Lymphocytic esoph- 46 Lucendo AJ, Molina-Infante J, Arias Á, et al. Guidelines on eo- agitis: a diagnosis of increasing frequency. J Clin Ga- sinophilic esophagitis: evidence-based statements and recom- stroenterol 2012;46:828-832. https://doi.org/10.1097/ mendations for diagnosis and management in children and MCG.0b013e3182500de8 adults. United European Gastroenterol J 2017;5:335-358. https:// 38 Rouphael C, Gordon IO, Thota PN. Lymphocytic esophagitis: still doi.org/10.1177/2050640616689525 PATHOLOGICA 2020;112:138-152; DOI: 10.32074/1591-951X-164

Review

Neoplastic and pre-neoplastic lesions of the oesophagus and gastro-oesophageal junction

Federica Grillo1-2*, Luca Mastracci1-2*, Luca Saragoni3, Alessandro Vanoli4, Francesco Limarzi5, Irene Gullo6, Jacopo Ferro1, Michele Paudice1, Paola Parente7, Matteo Fassan8 1 Anatomic Pathology Unit, Department of Surgical Sciences and Integrated Diagnostics (DICS), University of Genova, Italy; 2 Ospedale Policlinico San Martino, IRCCS for Oncology and Neuroscience, Genova, Italy; 3 UO Anatomia Patologica, Ospedale G.B. Morgagni-L. Pierantoni, Forlì, Italy; 4 Anatomic Pathology Unit, Department of Molecular Medicine, University of Pavia and Fondazione IRCCS San Matteo Hospital, Pavia, Italy; 5 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST/IRCCS), Meldola (FC), Italy; 6 Department of Pathology, Centro Hospitalar Universitário de São João (CHUSJ) & Department of Pathology, Faculty of Medicine of the University of Porto (FMUP), Porto, Portugal and Instituto de Investigação e Inovação em Saúde (i3S) & Institute of Molecular Pathology and Immunology of the University of Porto (Ipatimup), Porto, Portugal; 7 Unit of Pathology, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy; 8 Surgical Pathology Unit, Department of Medicine (DIMED), University of Padua, Italy *These two authors contributed equally

Summary Received and accepted: June 29, 2020 Oesophageal and gastro-oesophageal junction (GOJ) neoplasms, and their predisposing Published online: 29 October, 2020 conditions, may be encountered by the practicing pathologist both as biopsy samples and Correspondence as surgical specimens in daily practice. Changes in incidence of oesophageal squamous cell Federica Grillo carcinomas (such as a decrease in western countries) and in oesophageal and GOJ adeno- Anatomic Pathology Unit, Department of carcinomas (such as a sharp increase in western countries) are being reported globally. New Surgical Sciences and Integrated Diagnostics modes of treatment have changed our histologic reports as specific aspects must be detailed (DICS), University of Genova and Ospedale such as in post endoscopic resections or with regards to post neo-adjuvant therapy tumour Policlinico San Martino, IRCCS for Oncology regression grades. The main aim of this overview is therefore to provide an up-to-date, easily and Neuroscience, largo Rosanna Benzi 10, available and clear diagnostic approach to neoplastic and pre-neoplastic conditions of the 16132 Genova, Italy oesophagus and GOJ, based on the most recent available guidelines and literature. Tel. +39 010 5555957 Fax: +39 010 5556392 Key words: oesophageal dysplasia, Barrett’s dysplasia, oesophageal squamous cell E-mail: [email protected] carcinoma, oesophageal adenocarcinoma, tumour regression grade

Conflict of interest The Authors declare no conflict of interest. Introduction How to cite this article: Grillo F, Mastracci L, Saragoni L, et al. Neoplastic and pre-neoplastic Oesophageal and gastro-oesophageal junction (GOJ) neoplasms, and lesions of the oesophagus and gastro-oesophageal their predisposing conditions may be encountered by the practicing pa- junction.Pathologica 2020;112:138-152. https:// doi.org/10.32074/1591-951X-164 thologist both as biopsy samples and as surgical specimens. While oe- sophageal squamous cell carcinomas are decreasing in incidence in the © Copyright by Società Italiana di Anatomia Pato- western world, a sharp increase in oesophageal Barrett’s dysplasia, oe- logica e Citopatologia Diagnostica, Divisione Itali- sophageal adenocarcinoma and GOJ cancer has been observed due to ana della International Academy of Pathology better recognition and widespread predisposing factors. New modalities OPEN ACCESS of treatment are impacting our daily reporting practice and the patholo- gist is increasingly being asked to provide reliable diagnoses necessary This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- for optimal management and surveillance. NonCommercial-NoDerivatives 4.0 International) license: the The main aim of this overview is to provide an up to date, easily available work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original and clear diagnostic approach to neoplastic and pre-neoplastic conditions version. For further information: https://creativecommons. of the oesophagus and GOJ, based on the most recent available guide- org/licenses/by-nc-nd/4.0/deed.en lines and literature. HISTOPATHOLOGY OF TUMORS OF THE OESOPHAGUS AND GASTRO-OESOPHAGEAL JUNCTION 139

Oesophageal squamous carcinoma oesophageal squamous epithelium, without invasion. Squamous dysplasia can occur anywhere in the oe- Globally, oesophageal squamous carcinoma (OSCC) sophagus and it is likely to follow the distribution of accounts for the majority of the cases of oesophageal squamous cell carcinoma 1. cancer, although its proportion, relative to oesopha- The diagnosis of precancerous lesions or OSCC in its geal adenocarcinoma, varies from country to country. early phase should be the main goal of screening, con- Oesophageal squamous dysplasia is the main pre-ne- sidering that advanced OSCC shows poor prognosis oplastic lesion. (overall survival of 38% at 1 year and 12% at 5 years after diagnosis) 2. Precancerous dysplastic lesions are Pre-neoplastic lesion - Oesophageal Squamous detectable using endoscopy as they can appear as Dysplasia (Fig. 1) small, superficial or flat lesions. Though endoscopy is Oesophageal squamous dysplasia (OSD) (also still considered gold standard for the diagnosis of dys- known as oesophageal squamous intra-epithelial ne- plasia and early OSCC, alternative approaches, such oplasia) is an unequivocal neoplastic alteration of the as exfoliative cytology, have been suggested 3.

Figure 1. (A) Oesophageal biopsy section showing low grade squamous dysplasia with only mild cytological atypia within the lower half of the epithelial thickness (magnification x40). (B) Oesophageal biopsy section showing high grade squamous dysplasia with severe cytological atypia in more than half of the epithelium (magnification x40). (C) Dysplastic squamous epitelium and invasive squamous cell carcinoma (magnification x10). (D) Invasive squamous cell carcinoma with keratin pearl formation (arrow) (magnification x20). 140 F. Grillo et al.

Histologic diagnosis of oesophageal squamous dys- noma 10. Not uncommonly, the basal layers of regener- plasia. Histologic criteria for OSD were initially de- ative squamous epithelium may undergo increase of scribed in 1970 4-6 and modified in the 1980s, based nuclear/cytoplasm ratio and simulate dysplasia. After on experiences from China 7. The diagnosis of OSD radiotherapy or chemotherapy, squamous epithelium requires the presence of both cytological and archi- may become atypical and mimic dysplasia or even tectural atypia. Cytological atypia is characterized by carcinoma. Atypical cells after therapy do not show an nuclear atypia (enlargement, pleomorphism and hy- altered nuclear/cytoplasm ratio, but reveal prominent percromasia), loss of normal cell polarity while archi- cytoplasmic vacuolization instead. In addition, similar tectural atypia is characterized by abnormal epithelial changes may also be observed in stromal cells. maturation without invasion of epithelial cells through Malignancies that mimic dysplasia include lateral the basement membrane. The two-tiered (low grade spread of invasive squamous cell carcinoma and ver- versus high grade) histological system is currently rucous carcinomas 11. Lateral spread of squamous recommended by the WHO classification of Tumours cell carcinoma may mimic dysplasia particularly if the Editorial Board 1. Low grade dysplasia is defined as a invasive component is not present in the biopsy sam- lesion involving only the lower half of the epithelium, ple 12. In these cases, nuclear hyperchromasia and with only mild cytological atypia. High-grade dysplasia pleomorphism are usually more prominent compared is diagnosed when more than half of the epithelium with high-grade dysplasia, and a clear demarcation is involved or when severe cytological atypia is pres- from carcinomatous to non-carcinomatous epithelium ent (regardless of the extent of epithelial involvement). is typically present. There are two general types of lat- High-grade dysplasia includes the group of lesions al- eral spread. One shows full-thickness replacement by so termed “carcinoma in situ” in Japan and other parts carcinoma (full-thickness type) and the other reveals of Asia 8. Overexpression of p53 and hypermethyla- only basal epithelium involvement (basal layer-type). tion of CDKN2A (P16INK-4a) have been reported in In the basal layer-type, involvement may be seen in OSD 9. only one or two layers of the epithelium. Verrucous Differential Diagnoses. Benign and malignant lesions carcinoma is an extremely rare form of malignancy can morphologically mimic squamous dysplasia. and shows an exophytic papillary growth pattern 13 These include squamous papilloma, pseudo-epithe- with only minor cytologic atypia and a blunt pushing liomatous (regenerative hyperplasia) induced by ero- invasive margin which may be difficult to interpret as sions, ulcers, or oesophagitis, multinucleated change invasive. in oesophagitis and radiation or chemotherapy ef- fect. Squamous papillomas lack cytologic atypia and Oesophageal squamous cell carcinoma show an orderly cellular maturation from the basal Definition. Oesophageal squamous cell carcinoma layer toward the surface. Pseudo-epitheliomatous hy- NOS is a malignant oesophageal epithelial neoplasm perplasia may also simulate invasive squamous cell displaying squamous cell differentiation characterized carcinoma. However, neither significant nuclear ple- by keratinocyte-type cells with intercellular bridges omorphism, nor loss of polarity are present. Distinc- and/or keratinization. It is located most commonly in tion of OSD from regenerative or reactive squamous the middle third of the oesophagus followed by the epithelium may sometimes be difficult, especially in lower third 14. biopsy specimens. Regenerative squamous epitheli- Clinical aspects. Over the past three decades, a con- um lacks significant nuclear pleomorphism, overlap- sistent decline in the rate of OSCC has been observed ping, or crowding. In addition, surface maturation is in Western Europe while a stable rate of slower de- usually present. Inflammation frequently accompanies cline has been seen in central Europe and converse- reactive squamous epithelium, so, in the presence of ly, an increase in OSCC incidence has been reported inflammation, a diagnosis of OSD should always be in Eastern Europe. In moderate and lower-risk West- made with caution. In practice, when low-grade dys- ern countries, the most important risk factors are the plasia is suspected in biopsy specimens with inflam- combination of tobacco smoking and excessive alcohol mation, the use of the term “indefinite” for squamous consumption 15; indeed, the decline in OSCC incidence dysplasia, similar to the nomenclature in Barrett’s oe- in Western Europe has been mainly attributed to the sophagus (see later) or inflammatory bowel disease, reduction in alcohol intake and smoking habits 16. could be used and follow-up with biopsies is recom- With regards to the possibility of screening for OSCC mended after treatment of the underlying oesophagi- in moderate and lower-risk countries, it would have tis. The term “atypical regenerative hyperplasia” of the to include an excessively large population at risk and oesophagus in endoscopic biopsies has been used to therefore seems impractical. Screening is therefore describe lesions that approach squamous cell carci- usually proposed to a small subgroup of patients who HISTOPATHOLOGY OF TUMORS OF THE OESOPHAGUS AND GASTRO-OESOPHAGEAL JUNCTION 141

are at a very high risk of cancer development, such - Grade 2 (moderately differentiated): has evident as those with a previous or concomitant diagnosis of cytological atypia and the cells are less ordered. head/neck squamous cell carcinoma 17, achalasia (up Mitotic figures are easily identified while keratin to 10 times risk) 18, previous radiotherapy for breast pearl formation is infrequent. cancer, previous caustic injury to the oesophagus - Grade 3 (poorly differentiated): consists predominant- and tylosis 19. In practice, no consensus has been ly of basal-like cells forming nests, which may show reached among world experts with regards to timing central necrosis. The tumour nests consist of sheets of surveillance programmes: practices are still varied, or pavement-like arrangements of tumour cells with with screening starting within 1 year after diagnosis in occasional parakeratotic or keratinizing cells. some cases, compared to 5 and 10 years in others. Furthermore, surveillance intervals also vary from 2 Subtypes of oesophageal Squamous Cell Carcinoma to 5 years 20. Verrucous squamous cell carcinoma. Most cases are Typical symptoms include new onset dysphagia, gas- encountered in the lower third of the oesophagus, as trointestinal bleeding, recurrent aspiration or emesis, a protuberant mass. The tumour comprises differenti- weight loss and/or loss of appetite. ated squamous cells with minimal cytological atypia, OSCC is most often found in older men (aged > 60 low mitotic activity and surface papillary projections. years) with male to female ratio of 3:1, although this recognizes occurs abruptly, with no intervening gran- ratio varies considerably across geographical re- ular cell layer. The invasive front is pushing and the gions 21,22. Apart from alcohol overconsumption and to- tumour is slow-growing and metastases are uncom- bacco smoking, particularly when in combination, oth- mon 30-32. er risk factors include the consumption of red meat 23 Spindle cell squamous cell carcinoma. Macroscop- and of very hot beverages 24. Genetic factors are al- ically it has a polypoid growth pattern. Histologically, so involved: a pooled analysis of three genome-wide there is a biphasic pattern of neoplastic squamous association studies found new susceptibility loci for epithelium and spindle cells. Squamous cells are OSCC 25. well to moderately differentiated or may be occasion- Treatment Options. The main factors affecting choice ally carcinoma in situ alone. The spindle cells have of treatment are tumour stage and location, histologi- high-grade malignancy, which may show osseous, cal type, patient’s performance status and comorbidi- cartilaginous, or skeletal muscle differentiation 33,34. ties 26. While surgery (transthoracic oesophagectomy Although these tumours tend to be large, the progno- Ivor-Lewis procedure) alone is the treatment of choice sis is sometimes better than that of OSCC NOS of the in limited disease (cT1-T2 cN0 M0), neo-adjuvant same size, due to their intraluminal rather than deeply therapy with chemotherapy or chemoradiotherapy has invasive growth. supplemented surgery as standard treatment for local- Basaloid squamous cell carcinoma. Unlike similar ly advanced oesophageal cancer (cT3-T4 or cN1-N3 tumours in the oropharynx, this neoplasm is not as- M0). This is mostly due to difficulty in achieving com- sociated with HPV infection 35. It shows solid or nest- plete R0 tumour resections with surgery alone, while ed growth pattern of basaloid cells, sometimes with neo-adjuvant preoperative treatment has been shown comedonecrosis and occasionally with pseudoglan- to increase R0 resections and survival rates 27,28. dular or cribriform formation. Exclusion of high-grade Macroscopic and Microscopic description. Mac- neuroendocrine carcinoma (NEC) by immunohisto- roscopically, OSCC often presents as an ulcerative chemistry is often required. Areas of squamous cell mass, especially when advanced. The most useful carcinoma in situ or OSCC NOS are relative common. macroscopic classification is provided by Japan Eso- The tumour is highly aggressive, with a worse progno- phageal Society 29. sis than OSCC NOS, even though differences are not Histology shows a typical, invasive squamous carci- statistically significant 32. noma with both vertical and horizontal growth beyond Carcinoma cuniculatum is characterized by prominent the basement membrane (Fig. 1). Histological grading surface keratin-filled sinuses lined by bland squamous is based on the degree of cytological atypia, mitotic cells. activity and presence of keratinization. A three-tiered Molecular pathways. OSCC develops by stepwise system is commonly applied: progression from histologically normal squamous mu- - Grade 1 (well differentiated): shows enlarged cells cosa to low-grade dysplasia (intra-epithelial neopla- with abundant eosinophilic cytoplasm and keratin sia) and high-grade dysplasia. pearl production. Cytological atypia is minimal and TP53 mutation is a key early driver mutation 36. Genet- the mitotic rate low. The invasive margin is pushing ic changes identified at the intra-epithelial neoplasia and the cells are well oriented. stage include aneuploidy, copy-number alterations, 142 F. Grillo et al.

changes related to the amplification of genes, such and requires exclusion of regenerative lesions. Dys- as EGFR, and the silencing of genes, such as CD- plasia is endoscopically associated with flat, raised or KN2A, due to promoter hypermethylation 36. Frequent- depressed lesions and is defined by combined archi- ly mutated genes are, however, shared at the dysplas- tectural abnormalities and cytological atypia identifia- tic stage and invasive cancer 37. While copy number ble on standard haematoxylin and eosin-stained sec- variations are not significantly increased between tions 40,41. Dysplastic glands show architectural chang- dysplastic and malignant cells, it has been suggested es (gland fusion and budding, variability in gland size, that several cancer genes (ATR, MECOM, PIK3CA, cribriform pattern) and cytological alterations (hyper- BCL6, MYC, and CCND2) are more frequently affect- chromatic and elongated nuclei, nuclear stratification) ed by copy number variations in malignant than dys- causing an increasing morphological deviation from plastic cells. Many of these commonly mutated genes the metaplastic phenotype. Cytological and architec- are shared with other squamous cell carcinomas of tural abnormalities involve the entire length of glands the head and neck and of the lung 38,39. It is therefore and typically cellular maturation toward the surface of probable that cancers that arise from similar lineages the mucosa is missing. Dysplasia confined to the crypt are more molecularly alike than cells that arise from but with surface maturation is also reported 42,43. different lineages within the same organ. This could Pathologists should report dysplasia as fitting into one mean that biomarkers and therapeutic targets for can- of four categories (namely negative for dysplasia, in- cer will be shared between tumour types based on definite for dysplasia, low grade dysplasia and high 44 cell of origin rather than anatomic location 36. grade dysplasia) . The rationale for this tiered ap- Molecular subtypes. According to The Cancer Ge- proach is to stratify patients into categories of increas- nome Atlas Research network 39, three molecular sub ing risk for development of, or concurrent presence of, 45 types are identified as follows: oesophageal adenocarcinoma (OAC) . - OSCC type 1 recognizes alterations in the NRF2 Negative for dysplasia (NEG). This diagnosis repre- pathway, which regulates adaptation to oxidative sents two different situations: a) columnar epithelium stress. This group also contains a higher frequency with no cell atypia; b) reactive (hyperplastic/regener- of SOX2 and TP63 amplifications. Geographically, ative) changes. it includes 66% of the Asian population. Indefinite for dysplasia (IND) - this term only applies to - OSCC type 2 is referred to many of the Eastern cases where the pathologist cannot decide with cer- European and South American patients. Their tu- tainty whether the lesion is hyperplastic/regenerative mours are characterized by higher rates of ZNF750 or neoplastic in nature. This may be due to inadequate and NOTCH1 mutation, inactivation of the chroma- biopsy sampling (e.g. poorly oriented biopsies that do not enable full thickness assessment) or to the pres- tin modulators KDM6A and KDM2D, CDK6 amplifi- ence of cytological atypia and/or structural alterations cation and inactivation of the PIK3CA suppressors of doubtful interpretation. It is a “provisional” diagnosis PIK3R1 and PTEN. that must be followed by short-term resampling and/ - OSCC type 3 are few and come from North Amer- or second opinion. The presence of erosion and neu- ica. Mutations in the cell cycle pathway are absent trophilic infiltrate requires additional caution in diag- in this subtype and only 1 patient had a TP53 nosing dysplasia. mutation. They all contained mutations predicted Low grade dysplasia (LGD) - this is characterized by to activate the RTK/RAS/PI3K pathway and 75% scanty architectural distortion and mild to moderate contained somatic mutations in chromatin re-mod- cytological atypia. Hyperchromatic nuclei with irregu- elling. lar contours, nuclear overlapping and crowding and At present, no molecular tests are required 1. dystrophic goblet cells may be present. High grade dysplasia (HGD) - this is characterized by both architectural abnormality and severe cytological Oesophageal adenocarcinoma atypia. Aberrant architectural features include glandu- and gastro-oesophageal junction lar crowding, branching or budding glands, villiform, adenocarcinoma cribriform, micropapillary, or cystically dilated crypt patterns. Cytological features include complete loss Pre-neoplastic lesion - Barrett’s dysplasia (Fig. 2) of cell polarity, rounded enlarged nuclei with irregu- Dysplasia in Barrett’s Oesophagus (BO) is an une- lar-thickened nuclear membranes and conspicuous quivocal neoplastic transformation of the epithelial nucleoli. Typical and atypical mitotic figures are readily cells confined within the basement membrane of the identified at all levels within the glands, as well as on metaplastic columnar epithelium within which it arises the luminal surface. HISTOPATHOLOGY OF TUMORS OF THE OESOPHAGUS AND GASTRO-OESOPHAGEAL JUNCTION 143

Figure 2. (A) Intestinal type low grade dysplasia in Barrett’s oesophagus showing scanty architectural distortion and mild to moderate cytological atypia; hyperchromatic nuclei with irregular contours, nuclear overlapping and crowding are seen (mag- nification x20). (B) Intestinal type high grade dysplasia in Barrett’s oesophagus showing both architectural abnormality and severe cytological atypia adjacent to squamous epithelium (magnification x10). (C) Foveolar type low grade dysplasia in Barrett’s oesophagus with closely packed glands with a single layer of columnar cells with no interspersed goblet cells, round/oval basal nuclei with little stratification or pleomorphism and vesicular nuclei (magnification x20). (D) Foveolar type high grade dysplasia with enlarged cells with greater pleomorphism, loss of polarity and increased mitoses (magnification x20). (E) Crypt dysplasia: dysplasia is confined to the crypt with surface epithelium maturation (magnification x20). (F) p53 immunostained section of crypt dysplasia showing p53 nuclear accumulation in the crypt areas of dysplastic epithelium but not in the surface epitheium. 144 F. Grillo et al.

The risk of progression to OAC in patients with BO olar or non-intestinal, gastric dysplasia, on the other increases with male gender, current tobacco smoking, hand, shows closely packed glands with a single layer visceral obesity 46 and Caucasian origin 47. A longer of columnar cells with few or no interspersed goblet length of BO has been associated with a higher can- cells, round/oval basal nuclei with little stratification or cer risk. A prospective cohort study 48 has shown that pleomorphism and vesicular nuclei with prominent nu- the risk of OAC increases linearly with the length of cleoli. High-grade foveolar dysplasia shows cells to be BO, with a higher risk of developing cancer in long enlarged with greater pleomorphism, loss of polarity segment BO (> 3 cm) than in short segment BO. A and increased mitoses 59. Foveolar dysplasia typically recent model for the calculation of risk of progression expresses gastric type MUC5AC while it is negative of BO, is based on several weighted factors (male for intestinal type markers, such as MUC2, CDX2 and gender, cigarette smoking, BO length and confirmed villin, which are typically expressed in intestinal type low grade dysplasia) and identifies a risk pyramid dysplasia 60. Initial data suggest that foveolar dysplasia varying from 0.13 to 2.1% patient-years increase in may be associated with a high risk of progression 61. progression (high grade dysplasia and OAC) 49. A me- ta-analysis of 57 studies, shows that the pooled annu- al incidence of OAC is 0.33% (95% CI 0.28-0.38%) 50 Adenocarcinoma of the Oesophagus and while when considering patients with short segment Gastro-Oesophageal Junction BO incidence falls to 0.19%. With regards to dysplasia to cancer risk, patients with LGD have a pooled annu- Definition. Adenocarcinoma of the oesophagus (OAC) al incidence rate of 1.7% for HGD/EAC 51, in patients and GOJ adenocarcinoma (GOJ-AC) are malignant with HGD the annual incidence rate of OAC is 7% 52. epithelial tumours which show a tubular, papillary, sol- It has long been recognised that there is inter- and id with mucin production, mucinous or, less frequently, intra-observer variability in the diagnosis of Barrett’s poorly cohesive patterns of growth. dysplasia and this particularly affects LGD and the in- Clinical aspects. OAC and GOJ-AC are the most definite category 53-55. A recent study 56 has evaluated rapidly increasing cancer types in the western world the agreement across the entire diagnostic BO spec- including North America, western Europe and Austral- trum (NEG, IND, LGD, HGD). Excellent concordance asia 62. Risk factors include age (mean age 66 years), (> 70%) has been demonstrated for non-dysplastic male gender, Caucasian ethnicity, gastro-oesoph- BO and HGD, while concordance was intermediate ageal reflux disease and BO, obesity, smoking and for LGD (46%) and poor for indefinite for dysplasia dietary factors. Unfortunately, late stage cancers still (23.5%). Ancillary tests (e.g. p53, AMACR and Ki-67 harbour a relatively poor prognosis with survival rates stains) have been advocated to aid the diagnosis of falling sharply from 1 year (approximately 50%) to 5 dysplasia. At present, conventional haematoxylin and years (15%) 63. eosin examination remains the gold standard even Nearly all cancers (95%) are found in the distal oe- though the use of p53 stain is suggested 57,58. Studies sophagus/GOJ even if infrequently, adenocarcinoma have shown that the prediction of progression of oe- can be found in the middle/upper third of the oesoph- sophageal dysplasia is improved if at least two expert agus either arising from an inlet patch area (ectopic pathologists agree on the diagnosis and increases gastric oxyntic type mucosa) 64 or from submucosal further when a greater number of pathologists con- oesophageal glands. cur 53. In day-to-day diagnostic practice, a diagnosis of Most frequent symptoms include dysphagia, anorex- dysplasia in the setting of BO requires confirmation by ia with weight loss, dyspepsia and retrosternal pain. a second pathologist, preferably with a special interest Diagnosis is based on endoscopic biopsy of the le- in gastrointestinal pathology. Considering that major sion while enlarged peri-oesophageal nodes may be inconsistencies are found when diagnosing the indefi- amenable to endoscopic ultra-sound cytologic or mi- nite category, the extension of consensus reporting to crohistologic sampling. this category appears advised. Surgery is no longer the only option as endoscopic Types of dysplasia. The two main types of dysplasia, dissection techniques permit wide dissections of mu- which may also be found admixed, are type 1 intesti- cosa or submucosa when early and endoscopically nal (adenomatous) dysplasia and type 2 foveolar or identified lesions are present. non-intestinal, gastric-type dysplasia. Macroscopic description. OAC and GOJ-AC are of- Intestinal dysplasia is the more widely recognised ten found in advanced stages as polypoid/fungating, type and is characterized by the presence of intestinal ulcerated or diffusely invasive lesions leading to stric- type columnar cells as well as goblet cells (which may tures. Early stage cancers may be found during endo- however be mucous depleted) and enterocytes. Fove- scopic surveillance and appear as slightly elevated, HISTOPATHOLOGY OF TUMORS OF THE OESOPHAGUS AND GASTRO-OESOPHAGEAL JUNCTION 145

Figure 3. Schematic representation of the Siewert macroscopic classification of gastro-oesophageal junction tumours65. Siewert Type I: Adenocarcinoma of the distal oesophagus. The tumour centre is located 1-5 cm above the gastric cardia. Siewert Type II: Adenocarcinoma of the GOJ/cardia. The tumour centre is located 1 cm above or 2 cm below the gastric cardia. Siewert Type III: Adenocarcinoma of the subcardial stomach. The tumour centre is located 2-5 cm below the gastric cardia. In the figure, 0 cm represents the gastric cardia.

flat or depressed lesions with possible nodular areas. The signet-ring cell pattern, characterized by the pres- Critical problems in distinction between distal oesoph- ence of non-cohesive cells containing intracellular mu- ageal, GOJ and proximal gastric cancer arise due to cin which pushes the nucleus to the periphery of the the absence of reliable anatomic landmarks, which cell, has been reported in approximately 7% of OAC/ may be even less readily identified due to complicat- GOJ-AC, though percentages in the literature range ing BO. In consideration of this, a macroscopic clas- between 0.46% and 26%. The main reason for these sification – the Siewert classification 65 – aids the pa- marked differences are the diverse criteria for report- thologist in describing the site of origin of the tumour. ing the signet ring cell pattern (some studies consider The Siewert classification identifies three macroscop- only tumours with > 50% signet ring cells, others con- ic types (Fig. 3): sider any percentage of signet ring cells, while other - Type I: Adenocarcinoma of the distal oesophagus. still consider signet ring cells in mucin pools as well The tumour centre is located 1-5 cm above the – see next paragraph). Signet ring cell tumours have gastric cardia. been shown to behave more aggressively and show - Type II: Adenocarcinoma of the GOJ/cardia. The worse response to neo-adjuvant treatment 67. Though tumour centre is located 1 cm above or 2 cm below still little is unknown concerning their biology and op- the gastric cardia. timal treatment strategy, the percentage of signet ring - Type III: Adenocarcinoma of the subcardial stom- cells within the tumour should be mentioned in the ach. The tumour centre is located 2-5 cm below the pathology report when they are identified. Signet ring gastric cardia. cell carcinoma, in its pure form is extremely rare in the This distinction is a fundamental moment during gross oesophagus. description as in the VIIIth edition of the TNM staging The mucinous pattern is rare and characterized by manual, Siewert types I and II are staged using the abundant pools of extracellular mucin in which car- TNM for the oesophagus/GOJ while Siewert III can- cinoma cells are present; of note is that floating ne- cers are staged with gastric cancers 66. oplastic cells may show intracellular mucin vacuoles Microscopic description. OAC/GOJ-AC show vari- (signet ring cell features), but these should not be ous patterns which are often found mixed; most fre- classified as signet ring cell cancers. quently observed growth patterns include glandular/ Poorly cohesive non signet ring pattern in OAC/GOJ- tubular, papillary, mucinous or signet ring cell features AC is morphologically similar to poorly cohesive car- (Figs. 4, 5). cinomas of the stomach, though much less frequent. The glandular/tubular growth pattern is characterized Early stage adenocarcinoma. Intramucosal adeno- by irregular, fused neoplastic glands lined by a single carcinoma (pT1a) is defined as an adenocarcinoma layer of atypical cells with evidence of mucin produc- which invades the lamina propria or the muscolaris tion (alcian blue PAS positive) while the papillary pat- mucosae but does not extend into the submucosa. tern shows structured papillae or, rarely, micropapillae. These early lesions will mostly be managed by con- 146 F. Grillo et al.

Figure 4. (A) Invasive oesophageal adenocarcinoma, tubular/glandular type which invades and undermines squamous epi- thelium (magnification x10). (B) Invasive oesophageal/GOJ adenocarcinoma, papillary type (magnification x10). (C) Invasive oesophageal/GOJ adenocarcinoma with micropapillary features (magnification x20). (D) Invasive oesophageal/GOJ adeno- carcinoma, mucinous type (magnification x10).

servative endoscopic dissections 68 considering the features on endoscopic resections include: < 500-μm low risk of nodal or distant metastases (< 2%) and the invasion in the submucosa (sm1), well/moderately dif- high risk of surgery 69. Sampling protocols are beyond ferentiated tumours, absence of lympho-vascular in- the scope of this review, although the entire endoscop- vasion and clear resection margins 71. ically resected specimen must be sampled after inking Difficulties in distinguishing between pT1a and pT1b of margins. Features which distinguish intramucosal cancers may be due to duplication of the muscularis carcinoma from high grade dysplasia include tubules mucosae, which may be seen in endoscopic resec- or small aggregates of compact back to back glands, tions with BO 72. single cells, cribriform patterns or solid sheets. Signet ring cells in intramucosal OAC are extremely rare. Tumour Regression Grading systems for Oesophageal The management of adenocarcinoma with submu- Cancer cosal invasion (pT1b) is less clear cut. Indeed pT1b Neo-adjuvant treatment is performed in advanced metastatic risk may be as high as 45% 70 however OSCC and OAC and modifications seen at histology in a subset of pT1b cancers with low risk features, comprise residual fibrous areas with dispersed neo- endoscopic resection may be sufficient (especially plastic glands or cells showing cytoplasmic modifica- in patients who are at high risk for surgery). Low-risk tions as well as mucinous changes 73. HISTOPATHOLOGY OF TUMORS OF THE OESOPHAGUS AND GASTRO-OESOPHAGEAL JUNCTION 147

Figure 5. (A) Invasive oesophageal/GOJ adenocarcinoma, poorly cohesive non signet ring cell type (magnification x40). (B) Invasive oesophageal adenocarcinoma, poorly cohesive signet ring cell type (magnification x40). (C) Invasive oesophageal/ GOJ adenocarcinoma, undifferentiated carcinoma with lymphoepithelioma-like carcinoma features including a syncytial pat- tern and prominent lymphocyte infiltration (magnification x40). (D) Invasive oesophageal/GOJ adenocarcinoma, adenosqua- mous type with squamous areas (asterisks) and glandular areas with mucin production (arrows) (magnification x20).

Different tumor regression grade (TRG) systems have surgeons and oncologists preferably in a multi-disci- been proposed for oesophageal cancer 74. The first plinary team setting. TRG grading system according to Mandard was in- Subtypes of oesophageal adenocarcinoma. While deed originally proposed for oesophageal squamous conventional adenocarcinoma of the oesophagus and cell carcinoma 75 and later applied to other cancer GOJ have been described in the above paragraph, types and sites. Since then other systems have been non-conventional histotypes of oesophageal carcino- proposed including the JSED system 76, the Chirie- mas according to the WHO 2019 classification of di- ac 77, the Swisher 78 and the Schneider 79 systems, gestive system tumours1 are rarely seen (Fig. 6). These amongst others. In these various systems categories include: i) adenoid cystic carcinoma; ii) adenosqua- vary from 3 to 5 and some systems also take into ac- mous carcinoma; iii) mucoepidermoid carcinoma, and count nodal response. The choice of which system to iv) undifferentiated carcinoma (1). Their definitions and use should be discussed between the pathologists, clinico-pathologic correlates are briefly described here. 148 F. Grillo et al.

Adenoid cystic carcinoma is defined as a carcinoma tial pattern and prominent lymphocyte infiltration, has composed of two distinct population of cells: epithelial been regarded as a subtype of undifferentiated carci- and myoepithelial cells. It accounts for only 0.1% of noma. Differential diagnoses include non-epithelial tu- all oesophageal cancers and is predominantly locat- mours, such as melanomas or lymphomas, as well as ed in the middle third of the oesophagus, with typ- poorly differentiated epithelial neoplasms (squamous ical symptoms such as dysphagia and pain being cell carcinomas, adenocarcinomas or neuroendocrine the most common. They are histologically similar to carcinomas). They usually express cytokeratins, while the salivary gland adenoid cystic carcinoma and are they are negative for p40, synaptophysin and chromo- characterized by cytokeratin, CEA and CD117/c-KIT granin-A. The prognosis is remarkably poor. positive epithelial cells in a cribriform, tubular or sol- Immunohistochemical and molecular characteristics id architecture. The myoepithelial cells on the other OAC and GOJ-AC, similarly to gastric cancer, show a hand show positivity for the myoepithelial markers CK7 positive/CK 20 negative phenotype with variable p63, smooth muscle actin and calponin, as well as CDX2 expression. for S100. Histological differential diagnoses include Though numerous studies have provided some in- the basaloid squamous cell carcinoma, which is neg- sight on the use of immunohistochemistry (MUC2, ative for smooth muscle actin and S100. Similarly to its MUC5AC, MUC 6, CDX2 etc) in the cancerogenic se- salivary gland counterpart, it is often characterized by quence from BO to OAC/GOJ-AC, the use of these perineural invasion. The staging of adenoid cystic car- markers in routine diagnosis has no prognostic impact cinoma follows that of oesophageal adenocarcinoma. and is not advocated. Other markers such as p53, Data on prognosis is scarce and these tumours seem PDL-1 85, p16 and Ki67 86 have been studied as pos- to behave aggressively with the majority of patients sible prognostic factors both in treatment-naïve, sur- having distant metastases at presentation 80. gically resected, adenocarcinoma and in the neo-ad- Adenosquamous carcinoma is defined as a carcino- juvant setting, but these markers are still a long way ma with biphasic differentiation, composed of both away from being validated for routine use. squamous cell carcinoma and adenocarcinoma, as At the present time the only useful predictive evalua- morphologically distinct components. It is commonly tions in OAC and GOJ-AC are represented by HER2 located in the middle third of the oesophagus 81, the status and mismatch repair protein (MMR)/microsat- male-to-female ratio is about 5 and the median patient ellite instability (MSI) status in relation to possible tai- age at diagnosis is 60 years. These tumours tend to lored therapeutic strategies. behave aggressively 82. HER-2 dysregulation is an early event in oesophageal Mucoepidermoid carcinoma is defined as a carcino- cancerogenesis 87 and its overexpression/amplifica- ma composed of typically admixed epidermoid, in- tion is seen in approximately 15-20% of OAC/GOJ- termediate and mucin-secreting cells. It accounts for AC cancers. HER-2 status in gastric and oesophageal less than 1% of all oesophageal malignancies, and is cancers utilises a specific immunoscoring system 88 found more frequently in males with a median age of and they often show basolateral/lateral membrane 58 years. It can be located in the middle or lower oe- staining 89. From a practical point of view it is impor- sophagus 83. The epidermoid cells are often arranged tant underline that HER-2 expression can be patchy in squamous nests with admixed intermediate and and heterogeneous. This is in particularly important mucinous cells. Its differential diagnosis should in- when HER2 evaluation is performed on endoscopic clude adenosquamous carcinoma, which lacks inter- biopsies 90: a minimum set of 5 biopsies has shown to mediate cells and show more separate components. be necessary for reliable HER2 assessment 91. However, in some cases, the differential diagnosis in MMR/MSI status, assessed either via immunostain almost impossible. The prognosis of mucoepidermoid loss of nuclear MLH1/PMS2 or MSH2/MSH6 protein carcinoma is currently unclear but some evidence of a expression or via MSI, can demonstrate an unstable more aggressive behaviour is available 83. phenotype in about 5% of cancers 92. This evaluation Undifferentiated carcinoma is a carcinoma without his- may prove useful when immunotherapy is considered tologic and immunohistochemical evidence of squa- in the advanced/metastatic setting 93. mous, adenocarcinomatous or neuroendocrine differ- The molecular landscape of OAC and GOJ-CA, is entiation. The median age of patients is 65 years, with increasingly being described as new studies identify predominance of males. The most common site in the diverse molecular subtypes. While gastric molecular lower oesophagus, often in association with Barrett’s subtypes are essentially four (chromosomal instabil- oesophagus 84. Neoplastic cells are undifferentiated, ity – CIN – cancers, microsatellite unstable cancers, with vesicular or pleomorphic nuclei. Lymphoepitheli- Epstein Barr Virus positive cancers and genomically oma-like carcinoma, which is characterized by syncy- stable cancers), oesophageal/GOJ adenocarcinomas HISTOPATHOLOGY OF TUMORS OF THE OESOPHAGUS AND GASTRO-OESOPHAGEAL JUNCTION 149

are predominantly of CIN type 94 with rare genomical- 10 Arista-Nasr J, Rivera I, Martinez-Benitez B, et al. Atypical re- ly stable cancers at the GOJ. Structural DNA varia- generative hyperplasia of the esophagus in endoscopy biopsy: a mimicker of squamous esophageal carcinoma. Arch Pathol tions and copy number changes are the main events Lab Med 2005;129:899-904. https://doi.org/10.1043/1543- in OAC and rearrangements, amplifications and de- 2165(2005)129[899:ARHOTE]2.0.CO;2 letions were shown to be more frequent than point 11 Shimizu M, Ban S, Odze RD. Squamous dysplasia and other mutations or insertions/deletions 95. The most fre- precursor lesions related to esophageal squamous cell carci- quently involved gene is TP53, while less frequently noma. Gastroenterol Clin N Am 2007;36:797-811. https://doi. org/10.1016/j.gtc.2007.08.005 mutated genes, include ARID1A, SMAD4, CDKN2A 12 Soga J, Tanaka O, Sasaki K, et al. Superficial spreading car- and ERBB2, amongst others. A better understanding cinoma of the esophagus. Cancer 1982;50:1641-5. https:// of the molecular alterations in OAC/GOJ-AC will open doi.org/10.1002/1097-0142(19821015)50:8<1641::aid- new paths for targeted treatments 96. cncr2820500830> 3.0.co;2-9 13 Biemond P, ten Kate FJ, Van Blankenstein M. Esophageal verru- cous carcinoma: histologically a low-grade malignancy but clini- cally a fatal disease. J Clin Gastroenterol 1991;13:102-7. Conclusions 14 Rustgi AK, El-Serag HB. Esophageal carcinoma. N Engl J Med 2014;371:2499-2509. https://doi.org/10.1056/NEJMra1314530 A standardized approach is essential for the histo- 15 Rossini AR, Hashimoto CL, Iriya K, et al. Dietary habits, etha- pathologic reporting of oesophageal and gastro-oe- nol and tobacco consumption as predictive factors in the de- sophageal junction (GOJ) neoplasms, and their velopment of esophageal carcinoma in patients with head and predisposing conditions. Novelties in the diagnostic/ neck neoplasms. Dis Esophagus 2008;21:316-21. https://doi. interpretation procedures, as well as new modes of org/10.1111/j.1442-2050.2007.00769.x 16 treatment, require up to date recommendations for Saftoiu A, Hassan C, Areia M., et al. Role of gastrointestinal en- doscopy in the screening of digestive tract cancers in Europe histologic reporting. : European Society of Gastrointestinal Endoscopy (ESGE) Position Statement. Endoscopy 2020;52:293-304. https://doi. References org/10.1055/a-1104-5245 17 Muto M, Hironaka S, Nakane M., et al. Association of multiple 1 WHO Classification of Tumours of the Digestive System. th5 ed. Lugol-voiding lesions with synchronous and metachronous Geneva: World Health Organization Classification of Tumours. esophageal squamous cell carcinoma in patients with head and WHO Classification of Tumours Editorial Board. Lyon: IARC neck cancer. Gastrointest Endosc 2002;56:517-21. https://doi. press 2019. org/10.1067/mge.2002.128104 2 RARECARENet Information Network on Rare Cancers. Ac- 18 Zendehedel K, Nyrèn O, Edberg A, et al. Risk of esophageal cessed: 15 Sept 2019. Available from http://rarecarenet.eu/index. adenocarcinoma in achalasia patients, a retrospective cohort php study in Sweden. Am J Gastroenterol 2011;106:57-61. https://doi. 3 Pan Q-J, Roth MJ, Guo H-Q, et al. Cytologic detection of esoph- org/10.1038/ajg.2010.449 ageal squamous cell carcinoma and its precursor lesions us- 19 Blaydon DC, Etheridge SL, Risk JM, et al. RHBDF2 mutations ing balloon samplers and liquid-based cytologyin asymptomatic are associated with tylosis, a familial esophageal cancer syn- adults in Llinxian, China. Acta Cytol 2008;52:14-23. https://doi. drome. Am J Hum Genet 2012;90:340-6. https://doi.org/10.1016/j. org/10.1159/000325430 ajhg.2011.12.008 4 Ismail-Beigi F, Horton PF, Pope CE. Histological consequences 20 Ravi K, Geno DM, Katzka DA. Esophageal cancer screening in of gastroesophageal reflux in man. Gastroenterol 1970;58:163-74. achalasia: is there a consensus? Dis Esophagus 2015;28:299- 5 Weinstein WM, Bogoch ER, Bowes KL. The normal human 304. https://doi.org/10.1111/dote.12196 esophageal mucosa : a histological rappraisal. Gastroenterol 21 Global Burden of Disease Cancer Collaboration, Fitzmaurice C, 1975;68:40-4. Dicker D, et al. The Global Burden of Cancer 2013. Jama Oncol 6 Taylor PR, Abnet CC, Dawsey SM. Squamous dysplasia-the 2015;1:505-27. https://doi.org/10.1001/jamaoncol.2015.0735 precursor lesion for esophageal squamous cell carcinoma. 22 Xie SH, Lagergen J. A global assessment of the male pre- Cancer Epidemiol Biomarkers Prev 2013;22:540-52. https://doi. dominance in esophageal adenocarcinoma. Oncotarget org/10.1158/1055-9965.EPI-12-1347 2016;7:38876-83. https://doi.org/10.18632/oncotarget.9113 7 Dawsey SM, Lewin KJ, Liu FS, et al. Esophageal morphology 23 Qu X, Ben Q, Jiang Y. Consumption of red and processed meat from Linxian, China. Squamous histologic findings in 754 pa- and risk for esophageal squamous cell carcinoma based on tients. Cancer 1994;73:2027-37. https://doi.org/10.1002/1097- a meta-analysis. Ann Epidemiol 2013;23:762-70. https://doi. 0142(19940415)73:8<2027::aid-cncr2820730803> 3.0.co;2-3 org/10.1016/j.annepidem.2013.09.003 8 Wang LD, Yanh HH, Fan ZM, et al. Cytological screening and 24 Chen Y, Tong Y, Yang C, et al. Consumption of hot beverages 15 years’ follow-up (1986-2001) for early esophageal squa- and foods and the risk of esophageal cancer: a meta-analysis mous cell carcinoma and precancerous lesions in a high-risk of observational studies. BMC Cancer 2015;15:449. https://doi. population in Anyang Country, Henan Province, Northern China. org/10.1186/s12885-015-1185-1 Cancer Detect Prev 2005;29:317-22. https://doi.org/10.1016/j. 25 Wu C, Wang Z, Song X, et al. Joint analysis of three genome- cdp.2005.06.004 wide association studies of esophageal squamous cell carcino- 9 Savant D, Zhang Q, Yang Z. Squamous Neoplasia in the Esoph- ma in Chinese populations. Nat Genet 2014;46:1001-6. https:// agus. Arch Pathol Lab Med 2020. Epub ahead of print. https:// doi.org/10.1038/ng.3064 doi.org/10.5858/arpa.2020-0058-RA 26 Lordick F, Mariette C, Haustermans K, et al. Oesophageal can- 150 F. Grillo et al.

cer: ESMO Clinical Practice Guidelines for diagnosis, treatment gus. Am J Surg Pathol 2011;35:45-54. https://doi.org/10.1097/ and follow-up. Ann Oncol 2016;27:v50-7. https://doi.org/10.1093/ PAS.0b013e3181ffdd14 annonc/mdw329 44 Rugge M, Correa P, Dixon MF, et al. Gastric Dysplasia. The Pa- 27 Van Hagen P, Hulshof MC, Van Lanschot JJ, et al. Preopera- dova International Classification. Am J Surg Pathol 2000;24:167- tive chemioradiotherapy for esophageal or junctional cancer. 76. https://doi.org/10.1097/00000478-200002000-00001 N Engl J Med 2012;366:2074-84. https://doi.org/10.1056/NEJ- 45 Anaparthy R, Sharma P. Progression of Barrett oesophagus: Moa1112088 role of endoscopic and histological predictors. Nat Rev Gastro- 28 Shapiro J, Van Lanschot JJ, Hulshof MC, et al. Neoadjuvant enterol Hepatol 2014;11:525-34. https://doi.org/10.1038/nrgas- chemoradiotherapy plus surgery versus surgery alone for oe- tro.2014.69 sophageal or junctional cancer (CROSS): long-term results of 46 Hardikar S, Onstad L, Blount PL, et al. The role of tobacco, a randomised controlled trial. Lancet Oncol 2015;16:1090-8. alcohol, and obesity in neoplastic progression to esophageal https://doi.org/10.1016/S1470-2045(15)00040-6 adenocarcinoma: a prospective study of Barrett’s esophagus. 29 Japan Esophageal Society. Japanese Classification of Esopha- PLoS ONE 2013;8:e52192. https://doi.org/10.1371/journal. geal Cancer, 11th Edition: part I. Esophagus 2017;14:37-65. pone.0052192 https://doi.org/10.1007/s10388-016-0556-2 47 Corley DA, Kubo A, Levin TR, et al. Race, ethnicity, sex and 30 Osborn NK, Keate RF, Trastek VF, et al. Verrucous carcinoma temporal differences in Barrett’s oesophagus diagnosis: a large of the esophagus: clinicopathophysiologic features and treat- community-based study, 1994-2006. Gut 2009;58:182-8. https:// ment of a rare entity. Dig Dis Sci 2003;48: 465-74. https://doi. doi.org/10.1136/gut.2008.163360 org/10.1023/a:1022572229285 48 Anaparthy R, Gaddam S, Kanakadandi V, et al. Association 31 Al-Shoha M, Nadeem U, George N, et al. Verrucous carcinoma of between length of Barrett’s esophagus and risk of high-grade the esophagus-remains a diagnostic enigma. Am J Gastroenterol dysplasia or adenocarcinoma in patients without dysplasia. Clin 2018;113:919-21. https://doi.org/10.1038/s41395-018-0065-0 Gastroenterol Hepatol 2013;11:1430-6. https://doi.org/10.1016/j. cgh.2013.05.007 32 Tripathi M, Swanson PE. Rare tumors of esophageal squa- mous mucosa. Ann N Y Acad Sci 2016;1381:122-32. https://doi. 49 Parasa S, Venalaganti S, Gaddam S, et al, Development and org/10.1111/nyas.13108 validation of a model to determine risk of progression of Barret’s Esophagus to Neoplasia. Gastroenterology 2018;154:1282-9. 33 Lyomasa S, Kato H, Tachimori Y, et al. Carcinosarcoma of the https://doi.org/10.1053/j.gastro.2017.12.009 esophagus: a twenty-case study. Jpn Clin Oncol 1990;20:99-106. 50 Desai TK, Krishnan K, Samala N, et al. The incidence of oesoph- 34 Raza MA, Mazzara PF. Sarcomatoid carcinoma of esoph- ageal adenocarcinoma in non-dysplastic Barrett’s oesophagus: agus. Arch Pathol Lab Med 2011;135:945-8. https://doi. a meta-analysis. Gut 2012;61:970-6. https://doi.org/10.1136/ org/10.1043/2010-0074-RSR.1 gutjnl-2011-300730 35 Bellizzi AM, Woodford RL, Moskauk CA, et al. Basaloid squa- 51 Singh S, Manickam P, Amin AV, et al. Incidence of esophageal mous cell carcinoma of the esophagus: assessment of high- adenocarcinoma in Barrett’s esophagus with low-grade dyspla- risk human papillomavirusand realted molecular markers. sia: a systematic review and meta-analysis. Gastrointest Endosc Am J Surg Pathol 2009;33:1608-14. https://doi.org/10.1097/ 2014;79:897-909. https://doi.org/10.1016/j.gie.2014.01.009 PAS.0b013e3181b46fd4 52 Rastogi A, Puli S, El-Serag HB, et al. Incidence of esopha- 36 Lin DC, Wang MR, Koeffler HP. Genomic and epigenomic ab- geal adenocarcinoma in patients with Barrett’s esophagus and errations in esophageal squamous cell carcinoma and implica- high-grade dysplasia: a meta-analysis. Gastrointest Endosc tions for patients. Gastroenterology 2018;154:374-89. https://doi. 2008;67:394-8. https://doi.org/10.1016/j.gie.2007.07.019 org/10.1053/j.gastro.2017.06.066 53 Curvers WL, Ten Kate FJ, Krishnadath KK, et al. Low-grade 37 Walker RC, Underwood TJ. Molecular pathways in the de- dysplasia in Barrett’s esophagus: overdiagnosed and under- velopment and treatment of oesophageal cancer. Best estimated. Am J Gastroenterol. 2010;105:1523-30. https://doi. Pract Res Clin Gastroenterol 2018;36-37:9-15. doi 10.1016/j. org/10.1038/ajg.2010.171 bpg.2018.11.013 54 Duits LC, Phoa KN, Curvers WL, et al. Barrett’s oesophagus 38 Cancer Genome Atlas Network. Comprehensive genomic char- patients with low-grade dysplasia can be accurately risk-strati- acterization of head and neck squamous cell carcinomas. Na- fied after histological review by an expert pathology panel. Gut ture 2015;517:576-82. https://doi.org/10.1038/nature14129 2015;64:700-6. https://doi.org/10.1136/gutjnl-2014-307278 39 Cancer Genome Atlas Research Network. Comprehensive ge- 55 Horvath B, Singh P, Xie H, et al. Risk for esophageal neoplasia nomic characterization of squamous cell lung cancers. Nature in Barrett’s esophagus patients with mucosal changes indefinite 2012;489:519-25. https://doi.org/10.1038/nature11404 for dysplasia. J Gastroenterol Hepatol 2015;30:262-7. https://doi. 40 Odze RD. Diagnosis and grading of dysplasia in Barrett’s oesoph- org/10.1111/jgh.12696 agus. J Clin Pathol 2006;59:1029-38. https://doi.org/10.1136/ 56 van der Wel MJ, Coleman HG, Bergman JJGHM, et al. Histo- jcp.2005.035337 pathologist features predictive of diagnostic concordance at 41 Voltaggio L, Montgomery EA, Lam-Himlin D. A clinical and his- expert level among a large international sample of patholo- topathologic focus on Barrett esophagus and Barrett-related gists diagnosing Barrett’s dysplasia using digital pathology. Gut dysplasia. Arch Pathol Lab Med 2011;135:1249-60. https://doi. 2020;69:811-22. https://doi.org/10.1136/gutjnl-2019-318985 org/10.5858/arpa.2011-0019-RA 57 van der Wel MJ, Duits LC, Pouw RE, et al. Improved diagnostic 42 Lomo LC, Blount PL, Sanchez CA, et al. Crypt dysplasia with stratification of digitized Barrett’s oesophagus biopsies by p53 surface maturation: a clinical, pathologic, and molecular study of immunohistochemical staining. Histopathology 2018;72:1015-23. a Barrett’s esophagus cohort. Am J Surg Pathol 2006;30:423-35. https://doi.org/10.1111/his.13462 https://doi.org/10.1097/00000478-200604000-00001 58 Tokuyama M, Geisler D, Deitrick C, et al. Use of p53 immunohis- 43 Coco DP, Goldblum JR, Hornick JL, et al. Interobserver vari- tochemistry in conjunction with routine histology improves risk ability in the diagnosis of crypt dysplasia in Barret Esopha- stratification of patients with Barrett’s oesophagus during routine HISTOPATHOLOGY OF TUMORS OF THE OESOPHAGUS AND GASTRO-OESOPHAGEAL JUNCTION 151

clinical care. Histopathology 2020 Online ahead of print. https:// of prominent mucin pools in the esophagus post neoadjuvant doi.org/10.1111/his.14143 chemoradiotherapy for Barrett’s-associated adenocarcinoma. 59 Brown IS, Whiteman DC, Lauwers GY. Foveolar type dysplasia Am J Surg Pathol 2006;30:28-35. https://doi.org/10.1097/01. in Barrett esophagus.. Mod Pathol 2010;23:834-43. https://doi. pas.0000174011.29816.fa org/10.1038/modpathol.2010.59 74 Klevebro F, Tsekrekos A, Low D, et al. Relevant issues in tumor 60 Khor TS, Alfaro EE, Ooi EM, et al. Divergent expression of regression grading of histopathological response to neoadjuvant MUC5AC, MUC6, MUC2, CD10, and CDX-2 in dysplasia treatment in adenocarcinomas of the esophagus and gastro- and intramucosal adenocarcinomas with intestinal and fo- esophageal junction. Dis Esophagus 2020;33:doaa005. https:// veolar morphology: is this evidence of distinct gastric and in- doi.org/10.1093/dote/doaa005 testinal pathways to carcinogenesis in Barrett Esophagus? 75 Mandard, AM, Dalibard, F, Mandard, al. Pathologic assess- Am J Surg Pathol 2012;36:331-42. https://doi.org/10.1097/ ment of tumor regression after preoperative chemoradio- PAS.0b013e31823d08d6 therapy of esophageal carcinoma. Clinicopathologic correla- 61 Mahajan D, Bennett AE, Liu X, et al. Grading of Gastric Foveolar- tions. Cancer 1994;73:2680-6. https://doi.org/10.1002/1097- Type Dysplasia in Barrett’s Esophagus. Mod Pathol 2010;23:1-11. 0142(19940601)73:11<2680::aid-cncr2820731105> 3.0.co;2-c. https://doi.org/10.1038/modpathol.2009.147 76 Japanese Society for Esophageal Diseases. Guidelines for clinical 62 Edgren G, Adami HO, Weiderpass E, et al. A global assess- and pathologic studies on carcinoma of the esophagus, ninth edi- ment of the oesophageal adenocarcinoma epidemic. Gut tion: preface, general principles, part I. Esophagus 2004;1:61-88. 2013;62:1406-14. https://doi.org/10.1136/gutjnl-2012-302412 77 Chirieac LR, Swisher SG, Ajani JA, et al. Post-therapy patholog- 63 Hur C, Miller M, Kong CY, et al. Trends in esophageal adeno- ic stage predicts survival in patients with esophageal carcinoma carcinoma incidence and mortality. Cancer 2013;119:1149-58. receiving preoperative chemoradiation. Cancer 2005;103:1347- https://doi.org/10.1002/cncr.27834. 55. https://doi.org/10.1002/cncr.20916 78 64 Orosey M, Amin M, Cappell MS. A 14-Year Study of 398 Esopha- Swisher SG, Hofstetter W, Wu TT, et al. Proposed revision geal Adenocarcinomas diagnosed among 156,256 EGDS per- of the esophageal cancer staging system to accommo- formed at two large hospitals: an inlet patch is proposed as a date pathologic response following preoperative chemora- significant risk factor for proximal esophageal adenocarcinoma. diation. Ann Surg 2005;241:810-20. https://doi.org/10.1097/01. Dig Dis Sci 2018;63:452-65. https://doi.org/10.1007/s10620-017- sla.0000161983.82345.85 4878-2 79 Schneider PM, Baldus SE, Metzger R, et al. Histomorpho- 65 Siewert JR, Stein HJ. Carcinoma of the cardia: carcinoma of the logic tumor regression and lymph node metastases deter- gastroesophageal junction - classification, pathology and extent mine prognosis following neoadjuvant radiochemotherapy of resection. Dis Esophagus 1996;9:173-82 for esophageal cancer: implications for response classifica- 66 tion. Ann Surg 2005;242:684-92. https://doi.org/10.1097/01. Liu K, Feng F, Chen XZ, et al. Comparison between gastric and sla.0000186170.38348.7b esophageal classification system among adenocarcinomas of 80 esophagogastric junction according to AJCC 8th edition: a ret- Sawada G, Moon J, Saito A, et al., A case of adenoid cystic car- rospective observational study from two high-volume institutions cinoma of the esophagus. Surg Case Rep, 2015;1:119. https:// in China. Gastric Cancer 2019;22:506-17. https://doi.org/10.1007/ doi.org/10.1186/s40792-015-0122-5 s10120-018-0890-2 81 Chen SB, Weng HR, Wang G, et al. Primary adenosqua- 67 Bleaney CW, Barrow M, Hayes S, et al. The relevance and im- mous carcinoma of the esophagus. World J Gastroenterol plications of signet-ring cell adenocarcinoma of the oesopha- 2013;19:8382-90. https://doi.org/10.3748/wjg.v19.i45.8382 gus. J Clin Pathol 2018;71:201-6. https://doi.org/10.1136/jclin- 82 Evans M, Liu Y, Chen C, et al. Adenosquamous carcinoma of the path-2017-204863 esophagus: An NCDB-Based Investigation on comparative fea- 68 Wu J, Pan YM, Wang TT, et al. Endotherapy versus surgery for tures and overall survival in a rare tumor. Oncology 2017;93:336- early neoplasia in Barrett’s esophagus: a meta-analysis. Gas- 42. https://doi.org/10.1159/000466699 trointest Endosc 2014;79:233-41.e2. https://doi.org/10.1016/j. 83 Chen S, Chen Y, Yang J, et al. Primary Mucoepidermoid Carci- gie.2013.08.005 noma of the Esophagus J Thorac Oncol 2011;6:1426-31. https:// 69 Sharma P, Shaheen NJ, Katzka D, et al. AGA clinical practice doi.org/10.1097/JTO.0b013e31821cfb96 update on endoscopic treatment of Barrett’s esophagus with 84 Singhi AD, Seethala RR, Nason K, et al. Undifferentiated Car- dysplasia and/or early cancer: expert review. Gastroenterology cinoma of the Esophagus: A Clinicopathological Study of 16 2020;158:760-9. https://doi.org/10.1053/j.gastro.2019.09.051 Cases. Hum Path 2015;46:366-75. https://doi.org/10.1016/j.hum- 70 Bollschweiler E, Baldus SE, Schroder W, et al. High rate of lymph- path.2014.11.021 node metastasis in submucosal esophageal squamous-cell car- 85 Babar L, Kosovec JE, Jahangiri V, et al. Prognostic immune cinomas and adenocarcinomas. Endoscopy 2006;38:149-56. markers for recurrence and survival in locally advanced esopha- https://doi.org/10.1055/s-2006-924993 geal adenocarcinoma. Oncotarget 2019;10:4546-55. https://doi. 71 Manner H, May A, Pech O, et al. Early Barrett’s carcinoma org/10.18632/oncotarget.27052 with “low-risk” submucosal invasion: long-term results of 86 Jacobsen F, Hohsar J, Gebauer F, et al. Loss of p16 and high endoscopic resection with a curative intent. Am J Gastro- Ki67 labeling index is associated with poor outcome in esoph- enterol 2008,103:2589-97. https://doi.org/10.1111/j.1572- ageal carcinoma. Oncotarget 2020;11:1007-16. https://doi. 0241.2008.02083.x org/10.18632/oncotarget.27507 72 Abraham SC, Krasinskas AM, Correa AM, et al. Duplication of 87 Fassan M, Mastracci L, Grillo F, et al. Early HER2 dysregu- the muscularis mucosae in Barrett esophagus: an under-recog- lation in gastric and oesophageal carcinogenesis. Histo- nized feature and its implication for staging of adenocarcinoma. pathology 2012;61:769-76. https://doi.org/10.1111/j.1365- Am J Surg Pathol 2007;31:1719-25. https://doi.org/10.1097/ 2559.2012.04272.x PAS.0b013e318093e3bf. 88 Rüschoff J, Dietel M, Baretton G, et al. HER2 diagnostics in gas- 73 Hornick JL, Farraye FA, Odze RD. Prevalence and significance tric cancer-guideline validation and development of standardized 152 F. Grillo et al.

immunohistochemical testing. Virchows Arch. 2010;457:299-307. associated adenocarcinoma. Am J Surg Pathol 2011;35:647-55. https://doi.org/10.1007/s00428-010-0952-2 https://doi.org/10.1097/PAS.0b013e31820f18a2 89 Grillo F, Fassan M, Sarocchi F, et al. HER2 heterogeneity in 93 van Velzen MJM, Derks S, van Grieken NCT, et al. MSI as a gastric/gastroesophageal cancers: from benchside to practice. predictive factor for treatment outcome of gastroesophageal ad- World J Gastroenterol 2016;22:5879-87. https://doi.org/10.3748/ enocarcinoma. Cancer Treat Rev 2020;86:102024. https://doi. wjg.v22.i26.5879 org/10.1016/j.ctrv.2020.102024 90 Grillo F, Fassan M, Ceccaroli C, et al. The reliability of endo- 94 Cancer Genome Atlas Research Network. Integrated ge- scopic biopsies in assessing HER2 status in gastric and gas- nomic characterization of oesophageal carcinoma. Nature troesophageal junction cancer: a study comparing biopsies 2017;541:169-75. https://doi.org/10.1038/nature20805 with surgical samples. Transl Oncol 2013;6:10-6. https://doi. 95 Secrier M, Li X, de Silva N, et al. Mutational signatures in esoph- org/10.1593/tlo.12334 ageal adenocarcinoma define etiologically distinct subgroups 91 Gullo I, Grillo F, Molinaro L, et al. Minimum biopsy set for HER2 eval- with therapeutic relevance. Nat Genet 2016;48:1131-41. https:// uation in gastric and gastro-esophageal junction cancer. Endosc Int doi.org/10.1038/ng.3659 Open 2015;3:E165-70. https://doi.org/10.1055/s-0034-1391359 96 Hassanabad AF, Chehade R, Breadner D, et al. Esophageal 92 Farris AB 3rd, Demicco EG, Le LP, et al. Clinicopathologic and carcinoma: towards targeted therapies. Cell Oncol (Dordr) molecular profiles of microsatellite unstable Barrett Esophagus- 2020;43:195-209. https://doi.org/10.1007/s13402-019-00488-2 PATHOLOGICA 2020;112:153-165; DOI: 10.32074/1591-951X-163

Review

Gastritis: update on etiological features and histological practical approach

Gianmaria Pennelli1, Federica Grillo2,3, Francesca Galuppini1, Giuseppe Ingravallo4, Emanuela Pilozzi5, Massimo Rugge1,6, Roberto Fiocca2,3, Matteo Fassan7, Luca Mastracci2,3 1 Surgical Pathology Unit, Department of Medicine (DIMED), University of Padua, Italy; 2 Anatomic Pathology, San Martino IRCCS Hospital, Genova, Italy; 3 Anatomic Pathology, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genova, Genova, Italy; 4 Department of Emergency and Organ Transplantation, Section of Pathological Anatomy, University of Bari Aldo Moro, Italy; 5 Department of Clinical and Molecular Medicine, UOC Anatomic Pathology, Sant’Andrea Hospital, University “La Sapienza”, Rome, Italy; 6 Veneto Tumor Registry (RTV), Veneto Regional Authority, Padua, Italy; 7 Surgical Pathology Unit, Department of Medicine (DIMED), University of Padua, Italy

Summary Gastric biopsies represent one of the most frequent specimens that the pathologist faces in routine activity. In the last decade or so, the landscape of gastric pathology has been changing with a significant and constant decline of H. pylori-related pathologies in Western countries coupled with the expansion of iatrogenic lesions due to the use of next-gener- ation drugs in the oncological setting. This overview will focus on the description of the elementary lesions observed in gastric biopsies and on the most recent published recom- mendations, guidelines and expert opinions.

Received and accepted: June 29, 2020 Key words: gastritis, H. pylori, secondary prevention, staging, endoscopy Published online: 29 October, 2020

Correspondence Francesca Galuppini Introduction Surgical Pathology Unit, Department of Medi- cine (DIMED), University of Padua, via Gabelli 61, 35121 Padova, Italy It would seem impractical to deal with the topic of gastritis without a Tel. +39 049 8218996 proper definition of this disease. The term “gastritis” derived from the E-mail: [email protected] Greek words gastḗr gastrós and defines every flogistic process affecting the stomach, confirmed by histological evaluation. In this brief overview https://orcid.org/0000-0002-3245-2322 we will see how not only the inflammatory cells present in the biopsy Conflict of interest sampling are fundamental, but also (and above all) the histological The Authors declare no conflict of interest. modifications of long-standing inflammation, possible ideal ground for the development and progression of neoplastic lesions of the stomach. How to cite this article: Pennelli G, Grillo F, Galuppini F, et al. Gastritis: update on etiological For this reason, the most important goal of histology is to distinguish features and histological practical approach. between non-atrophic and atrophic gastritis, in order to be informative Pathologica 2020;112:153-165. https://doi. about which patients require clinic/endoscopic surveillance. org/10.32074/1591-951X-163

© Copyright by Società Italiana di Anatomia Pato- logica e Citopatologia Diagnostica, Divisione Itali- Normal histology ana della International Academy of Pathology Before treating the various aspects of disease, it is important to recall OPEN ACCESS the normal anatomy of the gastric mucosa, which is fundamental in This is an open access journal distributed in accordance the understanding of how different etiologies involve different regions with the CC-BY-NC-ND (Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 International) license: the of the stomach. Gastric mucosa is divided in two main zones that differ work can be used by mentioning the author and the license, both in histological and in functional aspects: the oxyntic region (fun- but only for non-commercial purposes and only in the original dus and corpus) and the muco-secreting region (antrum and incisu- version. For further information: https://creativecommons. org/licenses/by-nc-nd/4.0/deed.en ra angularis). The oxyntic mucosa is composed of specialized glands 154 G. Pennelli et al.

containing: i) parietal (oxyntic) cells, large round or Table I the etiologic factors are summarized and be- pyramidal, highly acidophilic cells, important for in- low we will deal with the most important etiologies 2. trinsic factor production and hydrochloric acid (HCl) secretion, ii) the chief (zymogenic) cells, basophilic, Helicobacter pylori gastritis important for pepsinogen I and gastric lipase secre- The discovery of Helicobacter pylori (H. pylori) in 1982 tion; and iii) enterochromaffin-like (ECL) cells. On by Warren and Marshall 3 as the cause of gastritis in the other hand, the antral mucosa is characterized the vast majority of cases radically changed the epi- by glands rich in mucous cells, also producing pep- demiology and the clinico-pathologic approach to the sinogen II, as well as neuroendocrine G cells (gas- disease. It became evident that chronic gastritis could trin producing). be cured with eradication of H. pylori, resulting in res- In addition to these two specialized regions, the his- titutio ad integrum of the mucosa in those patients in tological transitional zones take on importance, as in whom the morphologic pattern has not developed to other organs, mainly in neoplastic pathology. The tran- atrophy. sitional zones are defined as the junctions between The association between H. pylori and gastric can- the different types of mucosa: antrum-body, body- cer has attracted great interest worldwide when the cardia, and antrum-duodenum. None of these transi- International Agency for Research on Cancer (IARC) tions is abrupt and they all involve a gradual merging identified H. pylori as a “group 1 (definite carcinogen)” of mucosal types. Although the location of the tran- in 1994 on the basis of the results of epidemiologic sitional zones is easy to determine histologically by studies 4. the finding of intermediate mucosa, their extent is less H. pylori infection is mainly acquired in childhood, up clear-cut. The most useful criteria to determine the to the age of 12 years, in developed countries mostly crossing from body into antrum is not disappearance by intra-familial transmission 5. H. pylori eradication of the parietal cells but the absence of chief cells and can reduce the risk for cancer, but this effect is largely the change from simple tubular glands in the body to confined to patients without atrophy and metaplasia; branched glands in the antrum 1. The cardiac mucosa indeed, cancer could occur more than 10 years after extends distally from the gastroesophageal junction eradication 6. For these reasons, the Kyoto global con- over a variable distance, ranging from 0.5 to 4 cm. sensus report defined the search and screening for Approximately 50% of the mucosal thickness is oc- H. pylori gastritis as appropriate at an age when new cupied by glands lined by mucin-secreting cells. The infections become less likely (> 12 years) and before glands have a tubular structure, in which cystic dilata- development of atrophic gastritis and intestinal meta- tion is commonly seen. Occasional chief and parietal plasia. This all depends on the geographical location cells can be seen, and endocrine cells are frequently and epidemiological context, taking into account the present. Moving distally from the cardia, the transi- prevalence of infection and age-related cancer inci- tional zone between body and cardia is identified as dence 7. the area where chief and parietal cells start to become At histologic exam, the bacterium may be detected abundant. In the literature, the antrum-duodenal tran- on hematoxylin and eosin but is usually more easily sitional zone has received little attention. detectable by histochemical (modified Giemsa stain- ing) and immunohistochemical techniques (Fig. 1A). The histological signs of H. pylori gastritis include a Gastritis classification diffuse or nodular lymphocytic inflammation in almost all cases, together with neutrophilic infiltrate. H. pylori The inflammatory pathology of the stomach may be may be difficult to detect in cases of extensive intesti- classified according to different criteria. One of the nal metaplasia, or during anti-secretory (PPI) therapy; most used are temporal classifiers which divided in such cases, H. pylori infection is suggested by the gastritis in acute (self-limited) and chronic (non self- features of inflammation. In diagnostic practice, any limited) forms. It is crucial to remember that this clas- semi-quantitative scoring systems (+--; ++-; +++) of sification is strictly clinical and words such as “acute” the bacterium’s density may be used but these have and “chronic” should be avoided in the histological re- no clinically significant implications and the inter- port. Indeed, almost all gastrites requiring histological observer reproducibility is low. Indeed, a distinction examination are chronic forms and the word “acute” between H. pylori negative versus positive status is used to describe neutrophilic inflammation is confus- considered adequate. ing. At the histologic level, it is preferable to use a clas- H. pylori is mainly present as a spiral-shaped bacte- sification based on etiology, considering the potential rium in gastric biopsy specimens. When influenced by role of various causes in the evolution to atrophy. In adverse factors (temperature or pH changes or use of GASTRITIS HISTOPATHOLOGICAL CLASSIFICATION 155

Figure 1 (A-B). Helicobacter pylori active gastritis (Giemsa staining). Lymphocytic inflammation and neutrophilic epithe- lium infiltration with conventional spiral-shaped H. pylori (A; 630x magnification). Dormant or stressed coccoid microorgan- isms form (arrows, B; 630 x magnification).

antibacterial drugs), non-spore-forming microorgan- of immune checkpoint inhibitors for the treatment of isms can be transformed into a latent coccoid form multiple metastatic malignancies has revealed a wide (Fig. 1B). The ability of H. pylori to transform from the spectrum of immune-related adverse events affecting spiral-shaped form to the coccoid form is one of its the gastro-intestinal tract. While colitis has been exten- most important special adaptive mechanisms allow- sively described, gastritis has only been sporadically ing it to survive extreme situations in the human or- reported. Johncilla et al. 10 characterized histological ganism, when cultivated, and to survive in the external features of this kind of gastritis identifying a series of environment 8. patterns which mimic other etiological aspects which Clinically, non-invasive diagnostic tests such as the include intraepithelial lymphocytosis and increased [13C]-urea breath test, fecal antigen test and sero- apoptotic activity, neutrophilic infiltration within glan- logical parameters (pepsinogen I, II, I/II, H. pylori anti- dular epithelium and foveolar hyperplasia. Checkpoint body) serve as surrogate markers of H. pylori gastritis inhibitors-associated gastritis is rare but must be con- and indicators of gastritis severity 9. sidered and promptly treated, as this might affect the course of the underlying oncological therapy 11. Reactive gastritis (gastropathies) As reported in Table I, this category includes: drug- Autoimmune gastritis induced gastritis; alcoholic gastritis; radiation gastritis, Autoimmune gastritis (AG) is due to an immune- gastritis due to duodenal reflux (etc.). Reactive gastri- mediated aggression targeting the parietal cells and tis shares some etiological agents (e.g. drugs) of the affects exclusively the oxyntic mucosa of the body- acute forms. Bile reflux into the stomach, due to par- fundus; it is associated with serum anti-parietal cell tial gastrectomy or dysmotility and nonsteroidal anti- and anti-intrinsic factor antibodies 12,13. Clinical signs inflammatory drugs (NSAIDs) are the most frequent of AG include hypo/achlorhydria, hypergastrinemia, injuries and may result in a broad spectrum of histo- low pepsinogen I/pepsinogen II ratio and vitamin B12- logical mucosal lesions, associated with low-grade in- deficient macrocytic anaemia 14. flammation of the gastric mucosa (gastropathies). The The disease may coexist with other immuno-mediat- chemical noxae increase the turnover of the gastric ed diseases, such as Hashimoto’s thyroiditis, insulin- epithelium, resulting in foveolar hyperplasia. Pro-in- dependent diabetes, and vitiligo and it was also re- flammatory cytokines produce vascular ectasia, ede- ported as an association with H. pylori infection with ma and muscularis mucosae hyperplasia. These enti- aggravation of the gastric picture and increased risk ties are frequently asymptomatic, but they may lead to of atrophic pangastritis. Studies using T cells from multiple erosions or ulcers, even with bleeding. Atro- patients infected with H. pylori and patients with au- phic changes are rare. Recently, the increasing use toimmune atrophic gastritis have identified molecular 156 G. Pennelli et al.

Table I. Gastritis Classification. Classification of Gastritis Acute Drugs, Stress induced, Uremia, Ischemia, Shock, Corrosive agents, Radiation, Certain food, Sepsis, Trauma, Certain infection, Acute alcoholism, Severe burns, Alkaline-Bile reflux, Major surgery Multiorgan failure, Portal hypertension, Congestive heart failure, Respiratory failure, Increase intracranial pressure. Reactive (chemical) gastropathy Endotossic (Alkaline Reflux-Bile Reflux, Uremic) Exotoxic (Drugs-NSAIDs, alchol, etc.) Stress induced Chronic Helicobacter pylori (and H. Heilmannii) Autoimmune Hp-Negative Cronic Gastritis Special Lymphocytic Collagenous Eosinophilic (food induced) Radiation Graft versus host disease (GVHD) Bacterial (Syphilis, Tubercolosis, Rickettsial Infections) Viral gastritis (CMV and HSV) Fungal gastritis (Candida, Aspergillus, Mucor, Coccidioides, Histoplasma, Cryptococcus neoformans, Pneumocystis carinii and Torulopsis glabrata) Parasitic Gastritis (Anisakis, Cryptosporidium, Ascaris lumbricoides, Giardia, Toxoplasma, Schistosoma etc.) Granulomatous Idiopathic Crohn Disease Sarcoidosis Food and Barium Granulomas Hypertrophic Ménétrier Disease, Zollinger-Ellison Syndrome, gastropathies Hypertrophic, Hypersecretory Gastropathy (with protein loss; Hp-associated) Gastric vasculopathies Ischemic, Antral Vascular Ectasia (Watermelon Stomach), Portal Hypertensive Gastropathy (Congestive Gastropathy), Varices, Angiodysplasia, Caliber-Persistent Artery (Dieulafoy Lesion); Hemodialysis-Associated Telangiectasias Gastric involvement in systemic Inflammatory Bowel Disease, Amyloid, Diabetes, Mastocytosis, Sjögren Syndrome, Hypercalcemia, diseases Siderosis mimicry between H. pylori and structural protein of plasia becomes increasingly prominent. These find- the parietal cells as the gastric H+/K+ ATPase, sug- ings are sufficiently pathognomonic for a diagnosis gesting that the infection might stimulate T cells tar- of autoimmune etiology, particularly if the antrum geting specialized corpus cells 15. The histopatholog- does not show inflammatory and atrophic “lesions”. ical changes of autoimmune atrophic gastritis can be Confirmation of diagnosis, however, still rests on the divided into three evolving phases. The early phase demonstration of antibodies directed against parietal is characterized by a multifocal, dense lymphocytic cell and intrinsic factor antigens. The end stage is and plasma cell infiltration of the oxyntic mucosa characterized by a marked replacement of oxyntic involving the entire thickness of the lamina propria glands, which might be completely absent; foveolar with an accentuation in the deeper part, often mixed hyperplasia with underlying microcystic change and with eosinophils and mast cells. Patchy destruction the constitution of hyperplastic and inflammatory pol- of individual oxyntic glands by lymphocytes might yps. Pseudopyloric, pancreatic, and intestinal meta- occur, and the parietal cells exhibit pseudo-hyper- plasia becomes wide spread and the muscularis trophic changes (PPI-like, Fig. 2A). These histologic mucosae is thickened (Fig. 2B). Although scattered features are non-specific, and the histologic report lymphoid aggregates may be seen, inflammation is should only raise the possibility of early autoim- usually minimal. Enterochromaffin-like (ECL) cells mune gastritis and recommend appropriate serologic hyperplasia (both linear and micronodular) is detect- tests 16. The second phase is characterized by diffuse able in increasing degrees from the initial to the sec- lymphoplasmacytic infiltration of the lamina propria, ond phase, while adenomatoid hyperplasia or ECL marked atrophy of oxyntic glands and increase in the cell dysplasia may affect the late phase and progress thickness of the foveolar component. Pseudopyloric into well-differentiated endocrine tumor (type I carci- metaplasia is often extensive 17, and intestinal meta- noid). GASTRITIS HISTOPATHOLOGICAL CLASSIFICATION 157

Figure 2. (A-B). Autoimmune gastritis. Early phase of autoimmune gastritis with hypertrophic glandular changes and mild lymphocytic and granulocytic infiltrate in the lamina propria (A; 200x magnification). The end stage is characterized by a marked replacement of oxyntic glands with pseudopyloric and intestinal metaplasia with mild inflammation of the lamina propria (B; 100x magnification).

“Special-type gastritis” (Fig. 3) dermatitis, and allergy to medicine food or pollen 20. Recent studies strongly support that Th-2 cytokines Lymphocytic gastritis (e.g. IL-4, IL-5 and IL-13) and chemokines such as eo- About thirty years ago, Haot et al. 18 described en- taxin play a critical role in the pathogenesis of EG 21. doscopic and microscopic features of the condition, The exact incidence of EG is difficult to estimate; the which they called lymphocytic gastritis (LG), charac- current literature suggests that EG occurs most com- terized by accumulation of lymphocytes in the gastric monly between 30 and 40 years of age, with a slightly epithelium. In fact, the lesion probably corresponds to male predominance and the prevalence data varies previously reported one (in 1945) called varioliform from 2.5 to 28/100,00022. Although symptoms of EG gastritis which is characterized by nodular and eroded are non-specific, abdominal pain and nausea/vomit- lesions running along the gastric rugae in the corpus. ing result the most frequent presenting symptoms in The diagnosis of LG is based on microscopic exami- children and adults. Young patients may also show nation of the gastric mucosa and according to current growth retardation and delayed puberty or amenor- definition the main feature is a presence of at least 25 rhea. Unlike esophageal mucosa, the lamina propria intraepithelial lymphocytes (IEL) per 100 gastric epi- of healthy stomach contains a quote of eosinophils thelial cells (Fig. 3A). Most of these lymphocytes pres- and the threshold number of gastric eosinophils re- ent CD3+ and CD8+ phenotype (Fig. 3B). H. pylori quired in EG diagnosis varies among authors. Re- infection is known to be associated with LG, reaching cently, Lwin et al. 23 recommended two main criteria from 0 up to 27% of cases. An association between for EG diagnosis: i) gastric biopsies with an average LG and celiac disease is well-established, varying density > 127 eosinophils/mm2 (or > 30 eosinophils/ from 10 to 38% 19. high power field (HPF) on microscopes equipped with wide-lens oculars (FN 22) in at least five separate Eosinophilic gastritis HPFs; ii) no known associated causes of eosinophilia Eosinophilic gastritis (EG) is part of the eosinophilic (e.g. H. pylori infection, Crohn’s disease, parasitic in- gastrointestinal disorders family which includes eosin- fections, and hematological or lymphoid disorders). ophilic esophagitis, eosinophilic gastroenteritis, and Considering clinical manifestations and depth of eo- eosinophilic enteritis/colitis. These disorders are con- sinophilic infiltration into the gastric wall, Klein et al. 24 sidered immune-mediated chronic inflammatory pro- proposed a classification of EG into three different cesses with strong associations to food allergen trig- patterns, including predominantly mucosal pattern, gers. Indeed, many patients have a history of atopic predominantly muscular pattern, and predominantly conditions including asthma, allergic rhinitis or atopic serosal pattern. Currently, treatment strategies consist 158 G. Pennelli et al.

Figure 3. (A-F). Special-type gastrites. Lymphocytic gastritis is defined by the presence of at least 25 intraepithelial lym- phocytes CD3+ per 100 gastric epithelial cells (A; hematoxylin-eosin, 200x magnification) (B; CD 3 immunostaining, 200x magnification). Cytomegalovirus (CMV) gastritis is characterized abundant granulation tissue with important inflammatory reaction (C; hematoxylin-eosin, 200x magnification) and CMV inclusions are visible in endothelial cells and also in macro- phages (D; CMV immunostaining, 630x magnification). Graft versus host disease (GVHD) gastric mucosa shows apoptotic bodies (arrows) and gland abscess (E; hematoxylin-eosin, 200x magnification). Collagenous gastritis is typically defined by the subepithelial deposition of collagen bands thicker than 10 μm and the intense inflammation response in the lamina propria (F; hematoxylin-eosin, 200x magnification). GASTRITIS HISTOPATHOLOGICAL CLASSIFICATION 159

in medical agents able to limit the inflammation (corti- more frequently. Since the disease was first reported costeroids) and in dietary therapy. in 1989, only 60 cases have been documented 28. Because of the small number of cases, no standard CMV gastritis therapy has been found. Based on case reports, two Cytomegalovirus (CMV) gastritis is the only viral infec- phenotypes of the disease (pediatric and adult) have tion with a distinct pathologic pattern in the stomach. been defined 29. The clinical symptoms of the pediatric It predominantly affects children and immunocompro- type are mainly of the upper gastrointestinal tract, in- mised patients. Usually, different sites of the gastroin- cluding abdominal pain and anemia secondary to the testinal tract are involved. Endoscopically, the gastric stomach specific inflammation and collagen deposi- mucosa may appear completely normal or show ero- tion. In contrast, the adult type is characterized by the sions and ulcers 25. Rarely, it may occur as a mass simultaneous occurrence of collagenous colitis, which called “pseudotumor”. Histologically, numerous CMV may be related to autoimmune processes and celiac inclusions may be seen less in epithelial than in en- disease. dothelial cells and also in macrophages, with little or absent inflammation. In this case, the immunological status of the patients is usually very compromised. In Morphological features: gastritis “grading” other cases, abundant granulation tissue with impor- tant inflammatory reaction can be observed (Fig. 3C), The Sydney System 30 and its Update in Houston Gas- while the CMV inclusions are difficult to see without tritis Workshop 1994 31 introduce a common terminol- immunohistochemical or in situ hybridization tech- ogy for the standardization of the histological report, niques (Fig. 3D). Eosinophilic infiltrates can also be providing the histopathological and morphological an important component of the CMV gastritis inflam- features characterizing every nosologic type of gas- mation. tritis. In order to describe the intensity and severity of the histological signs of gastritis, the use of a scale GVHD gastritis for grading was also proposed. Below we analyze the Graft versus host disease (GVHD) follows allogeneic main elementary lesions that histologically define the bone marrow transplant or transfusions, especially in gastritis landscape. immunocompromised patients. Acute GVHD occurs within the first 100 days post-transplant and mainly Gastritis intensity: mononuclear leukocyte infiltrate targets the liver, gastrointestinal tract and skin. Among In the normal gastric mucosa, only scattered chronic cases of GVHD of the gastrointestinal tract, GVHD inflammatory cells as lymphocytes and plasma cells rarely affects the upper tract 26. Patients with isolated are present in the lamina propria. Thus, every increase gastric GVHD present with nausea, vomiting, and in their count defines a chronic gastritis. Although it is upper abdominal pain without diarrhea. In the early not easy to define the exact count of mononucleate stages, the stomach appears endoscopically normal cells in the normal gastric mucosa, a number of 2 to 5 (30% to 80%) and some patients may show gastric lymphocytes, plasma cells and macrophages per high mucosal exfoliation. In full-blown disease, the stom- power microscope field or 2-3 of them between fove- ach appears variably congested and atrophic. Histo- olae is acceptable. logically, the mucosa shows apoptotic cells predomi- The increase in these numbers defines gastritis as nantly in the mucous neck regions (in both antral and mild (+--); moderate (++-) and marked (+++). oxyntic mucosa) and gland abscesses may also pres- Density of mononuclear cells in the lamina propria ent (Fig. 3E). Different degrees of inflammation and should be graded in areas far from the lymphoid folli- granular eosinophilic material may occur in the lamina cles that typically occur in H. pylori infection. propria and in the glands. Even though apoptotic bod- Lymphocytes may also be found in the glandular ep- ies may be present in small numbers, their presence ithelium, usually in the surface. The finding of a num- is diagnostic of GVHD in the appropriate setting 27. ber greater than 25/100 epithelial cells defines the pic- ture of lymphocytic gastritis as previously described. Collagenous gastritis Collagenous gastritis (CG) is a rare disease char- Gastritis activity: neutrophilic infiltrate acterized by the subepithelial deposition of collagen The presence of neutrophils (not eosinophils) in the bands thicker than 10 μm and the infiltration of in- gastric mucosa defines the “activity” of the gastritis flammatory mononuclear cells in the lamina propria which typically characterizes some etiological forms (Fig. 3F). Collagenous colitis seems to be part of the as H. pylori infection. Inflammation in the gastric mu- same disease entity, although the colonic form occurs cosa (within the lamina propria and/or the glandular 160 G. Pennelli et al.

Figure 4 (A-B). Enterochromaffin-like cells disorders in autoimmune gastritis. Linear hyperplasia of endocrine cells growing within the gastric gland (double arrow) and micronodular hyperplasia in the lamina propria (single arrow) (A; Chro- mogranin A immunostaining, 200x magnification). Adenomatoid endocrine hyperplasia (arrow) is defined as a nodule of 150- 500 mμ in diameter in the deep of the lamina propria (B; Chromogranin A immunostaining, 200x magnification).

lumen) is as defined in the previous paragraph. The proliferation” type was coined to identify this histologi- localization of the neutrophils defines the grade: neu- cal frame and pay attention to the follow-up of the pa- trophils in the lamina propria as mild (+--) activity; tient 32. neutrophils in the epithelium as moderate (++-) activ- ity and neutrophils in the glandular lumen as marked Oxyntic epithelium hypertrophy (+++) activity. Hypertrophic changes in the oxyntic gland, usually as a result of treatment with proton pump inhibitors (PPI) H. pylori density and in the early phase of autoimmune gastritis as “vi- As previously reported, for clinical management it is cariant” reaction, represent a remodeling of the epi- important to report if H. pylori is present or not. Since thelial structure due to cytoskeletal rearrangements. the absolute number of the bacteria may have a role The clinical significance is not yet well known, but it in the degree of the inflammation, in reactive glandular is recommended to report these changes in the his- hyperplasia and in lesions inducing lymphoprolifera- tological report. tive disorders, the density of H. pylori, if mentioned, should be graded as mild (+--), moderate (++-), and Fibrosis of the lamina propria and muscularis mucosae marked (+++). hyperplasia Focal fibrosis of the lamina propria may occur as scar- Foveolar hyperplasia ring after ulcer and inflammatory process, but it should Expansion of the proliferative compartment of the be distinguished from mucosa atrophy. Hyperplasia of gastric glands results in foveolar hyperplasia. All in- the muscularis mucosae is a mesenchymal response flammatory conditions and direct acute noxae of the to many noxae as duodenal reflux, but could be an an- gastric mucosa induce regenerative epithelial chang- trum remark in association with autoimmune gastritis. es, especially in association with erosions and peptic ulcers. Chemicals or infectious noxae increase the cell Endocrine cell hyperplasia turnover and lead to hyperplastic foveolae. The most Endocrine cell hyperplasia is secondary to corpus ox- important differential diagnosis of these changes is yntic atrophy which, by inducing hypo/achlorhydria sta- with dysplasia; indeed, in some contexts where the tus, causes a proliferative stimulus towards enterochro- inflammatory component is severe, it is not easy to maffin-like (ECL) cells. These cells are the precursor distinguish an atypical regeneration of the glandular lesions of the neuroendocrine tumor type I of the stom- neck from dysplastic lesions. For these reasons, the ach. ECL hyperplasia is graded as: i) linear hyperpla- term “indefinite for dysplasia, atypical foveolar hyper- sia; characterized by a chain of at least 5 cells, linearly GASTRITIS HISTOPATHOLOGICAL CLASSIFICATION 161

growing within the gastric gland; ii) micronodular hyper- tion of atrophy, which includes a non-metaplasic and plasia; defined as nodular clusters of at least 5 cells a metaplasic category (Tab. III) 34-37. The first frame- not exceeding the diameter of gastric glands (< 150 μm work is characterized by the decrease or complete in diameter), usually located within the lamina propria loss of glandular units, replaced by collagen deposi- at the deep of the oxyntic glands (Fig. 4A). Recently, tion in lamina propria, like a fibrotic scar. This situa- in the cascade towards neuroendocrine tumor, other tion causes the reduction of glandular mass, but does two lesions have been described: adenomatoid ECL not imply any change of the original cell phenotype. hyperplasia; defined as compact collections of ECL Sometimes, severe inflammation may hide the normal micronodules (150-500 μm in diameter) in the deep glands, mimicking atrophy and making difficult to as- part of the lamina propria (Fig. 4B); and ECL dysplasia, sess. These cases can be temporarily labeled as “in- described as large confluent ECL micronodules, rang- definite for atrophy” and the analysis can be delayed ing from 150 to 500 μm in diameter and characterized until the inflammation has regressed (e.g. after H. py- by the exceedance of the basement membrane with lori eradication). Diversely, in the metaplasic cathego- microinfiltration of the lamina propria and formation of ry the native glands are replaced by cells featuring new stroma 13,33 (Tab. II). a new commitment (intestinal and/or pseudopyloric). Two main forms of metaplasia are recognized: pseu- Mucosal atrophy dopyloric metaplasia (or spasmolytic polypeptide-ex- The accepted definition of gastric mucosa atrophy is pressing metaplasia, SPEM), corpus-restricted; and “loss of appropriate glands” for the sampling site. In- intestinal metaplasia (IM), potentially located in all deed, normal gastric biopsy samples show different gastric regions. Other metaplasic changes as pseudo- types of glands (muco-secreting or oxyntic), appropri- pancreatic metaplasia and clear-cell metaplasia are ate for the function of the anatomic site (antrum or not considered in this definition because they are not corpus) from which the specimen is obtained; the dis- related (to date) to any carcinogenic process. crepancy between the expected glands and what is SPEM is characterized by the substitution of oxyntic actually observed at the histologic exam represents epithelia with antral-like mucosa. SPEM (restricted to the concept of atrophy 34-37. This definition is derived the oxyntic mucosa by definition) can be more easily from an international group of pathologists (Atrophy assessed by exploiting its positive immunostaining for Club 2000) who established new diagnostic criteria Trefoil Factor 2 (TFF2) 38. for the two main phenotypes of the chronic gastritis Intestinal metaplasia (IM) may arise in native muco- (non-atrophic and atrophic) and proposed a classifica- secreting (antral) epithelia or in previously antralized

Table II. Enterochromaffin-like cells hyperplasia. Linear hyperplasia Chain of at least 5 cells, linearly growing within the gastric gland Micronodular hyperplasia Nodular clusters of at least 5 cells; < 150 μm in diameter; Located within the lamina propria Adenomatoid hyperplasia Compact collection of micronodules; 150-500 μm in diameter; Located in the deep of lamina propria ECL cells dysplasia Large confluent ECL cells micronodules; 150-500 μm in diameter; Microinfiltration of the lamina propria; Formation of new stroma

Table III. Mucosal atrophy classification. Gastric atrophy - Loss of glands appropriate for the sampling site - Type Histological lesions Scoore Non-metaplasic Glandular disapperance 1 = 1%-30% - Sclerosis of lamina propria (fibrotic scar results) Metaplasic Glandular replacement 2 = 31%-60% - Intestinal Metaplasia - Pseudopyloric Metaplasia (only oxintic mucosae) 3 = >60% 162 G. Pennelli et al.

oxyntic glands (SPEM). Different subtypes of intestinal metaplasia have been classified, based on whether the metaplastic epithelium phenotype resembles large bowel epithelia or the small intestinal mucosa 39,40.

Secondary prevention: gastritis staging

Gastric cancer (GC) is one of the most lethal epithelial malignancies, and its mortality rate prompts a global prevention strategy 41,42. Gastric mucosal inflamma- tion, mainly caused by H. pylori infection, is a preneo- plastic condition, which may promote the transition of stem cells into cancer stem cells involved in both in- testinal- and diffuse-type GCs. Along the progression into atrophic phenotypes, coincidental pathogenetic processes, even carcinogenic ones, are potentially triggered, concurring in the well-known “Correa cas- cade”, and they may contribute to processes that link the atrophic gastritis with intestinal type GC 43. Know- ing this carcinogenic process, a window of opportunity Figure 5. OLGA sample protocol for the gastritis staging. to prevent progression in carcinoma is available. While inflammation coexists with mucosal atrophy in the ma- jority of patients with intestinal GC, diffuse-type GCs do not show any coexisting atrophic changes and its approach recommends at least 5 biopsy samples from: “field of cancerization” is still not well-known. Discon- i) the greater and lesser curvatures of the distal antrum tinuing the inflammatory cascade triggered by H. pylori (A1-A2 = mucus-secreting mucosa); ii) the lesser cur- is crucial to preventing GC. For patients who have al- vature at the incisura angularis (A3), where the earliest ready developed gastric atrophy, the severity and the atrophic-metaplastic changes tend to occur; and iii) the topography (antrum, corpus or both) of the atrophic anterior and posterior walls of the proximal corpus/fun- changes correlates with cancer risk. Based on this dus (C1-C2 = oxyntic mucosa) (Fig. 5). assumption, the Operative Link Gastritis Assessment International guidelines for the secondary prevention of (OLGA) staging system defines five stages of gastritis with increasing cancer risk (stages 0-IV) based on atro- GC use the OLGA staging system to distinguish be- phy score (Tab. IV). At each mucosal compartment, the tween low-risk (stage 0-I-II) and high-risk (stage III and 44 atrophy score is calculated as average of the atrophic IV) gastritis . changes as detected in antral vs oxyntic mucosa. At the An alternative staging system, the Operative Link on single biopsy level, atrophy is graded as a percentage Gastric Intestinal Metaplasia Assessment (OLGIM) of atrophic transformations (both metaplasic and non- proposes to consider only intestinal metaplasia for the metaplasic). To obtain this type of staging, it is crucial atrophy score 45. This approach could have better inter- that multiple biopsy samples have been obtained to ex- observer reproducibility, but in some studies OLGIM plore the different mucosa compartments. The OLGA seemed to have less sensitivity in identifying patients at

Table IV. Gastritis staging. Olga Staging System Overall Atrophy score Oxintic (Corpus and Fundus) overall atrophy score 0: No atrophy 1: 1-30% of biopsy samples Score 0 Score 1 Score 2 Score 3 2: 31-60% of biopsy samples 3: >60% of biopsy samples Score 0 No Stage Stage I Stage II Stage II Mucosecreting Score 1 Stage I Stage I Stage II Stage III (Antrum and Angularis Incisura) overall atrophy score Score 2 Stage II Stage II Stage III Stage IV Score 3 Stage III Stage III Stage IV Stage IV GASTRITIS HISTOPATHOLOGICAL CLASSIFICATION 163

Table V. Histology report. Histology report Samples description 1) Number of bioptic specimens sent from the gastric antrum 2) Number of bioptic specimens sent from the gastric incisurae angularis 3) Number of bioptic specimens sent from the gastric corpus and fundus Clinical and endoscopic information • Prior histology • Active gastric ulcer • Active duodenal ulcer • Ulcer scar(s): Duodenal; Stomach • Therapy: • Family history of cancer • Etc. Histological description - Non-atrophic gastritis (intensity +--/++-/+++) of the antrum/ incisura angularis/corpus, active, Hp-related with (…morphological acces- sory lesions) (activity +--/++-/+++; Hp +--/++-/+++) (…). - Metaplasic-atrophic gastritis / Non-metaplasic atrophic gastritis (intensity +--/++-/+++) of the antrum/ incisura angularis (transitional area), active, Hp-related with (…morphological accessory lesions) (activity +--/++-/+++; Hp +--/++-/+++; Intestinal Metaplasia-IM %/ Non-metaplasic-Atrophy %)) (…) - Metaplasic and non metaplasic-atrophic gastritis (intensity +--/++-/+++) of the oxyntic mucosae, active, Hp-related with (…morpholo- gical accessory lesions) and endocrine (ECL) hyperplasia (linear/micronodular/adenomatoid) (activity +--/++-/+++-; Hp +--/++-/+++; Intestinal Metaplasia-IM %; Pseudopyloric Metaplasia %, Non-metaplasic-Atrophy %) (…) OLGA staging and etiological information A (antrum and incisura angularis overall atrophy score) 0-1-2-3; C (corpus and fundus overall atrophy score) 0-1-2-3; Olga staging: 0, I, II, III, IV- Hp-positive / Hp-negative / Morphological features suggestive for Hp / Autoimmune / Chemical Gastritis (Gastropathy) high risk of gastric cancer 46. Indeed, IM change is con- endoscopic features, and the biopsy sampling map. sidered just one of the phenotypes of atrophy involved in Serological tests (e.g. pepsinogen I/II and Gastrin 17 the cancer-prone atrophic microenvironment 47. Indeed, levels, autoantibody against parietal cell and intrinsic the clinic-pathological correlation between SPEM and factor) should be reported, if available. dysplastic lesions, have been less extensively explored The histology report should include 4 main chapters though recent findings suggest an important role in (Tab. V): i) sample description, including the number of “non-classic gastric” carcinogenesis 38,48. samples for each biopsy site; ii) clinical and endoscopic information; iii) histological description, according to the Houston modified-Sydney System gradation of the ele- Histology report mentary lesions; iv) the OLGA staging system; and finally v) information about the etiology of gastritis (i.e. H. pylori The diagnosis of gastritis is both clinical and histologic: infection; autoimmune disease, etc.) where possible. clinical information should always be given to the pa- thologist for the interpretation of the endoscopic and histological findings. The Sydney System protocol, Conclusion which requires biopsy sampling from the antral, incisura angularis and oxyntic areas, is the most widely applied Biopsy examination of the stomach represents one of standardized biopsy protocol. Additional specimens the most frequent pathology samples that the patholo- from any focal lesions should be added. Antral and in- gist faces in his/her routine activity. The sheer number cisura angularis specimens can be placed in the same of samples and the time-related factor can lead to the container and the corpus/fundus biopsies in another; perception that the diagnostic activity is monotonous this is essential to differentiate special forms of gastritis and possibly poorly informative (this may be in part due such ad as AG. Biopsy material should be handled with to the few clinical reports). As pathologists, we should care. Stains which should be used in routine practice formulate reproducible histopathologic reports that will are H&E and modified Giemsa for H. pylori detection. help the clinicians in the tailoring of secondary preven- The pathology request form should include essen- tion strategies for gastric cancer or allow a better defini- tial information such as the patient’s clinical history, tion of the etiology of gastric disease. 164 G. Pennelli et al.

References the assessment of gastric atrophy. Virchows Arch 2020 Online ahead of print. https://doi.org/10.1007/s00428-020-02814-8 1 Van Zanten SJ, Dixon MF, Lee A. The gastric transitional zones: 18 Haot J, Hamichi L, Wallez L, Mainguet P. Lymphocytic gastritis: neglected links between gastroduodenal pathology and helico- a newly described entity: a retrospective endoscopic and his- bacter ecology. Gastroenterology 1999;116:1217-29. https://doi. tological study. Gut 1988;29:1258-64. https://doi.org/10.1136/ org/10.1016/s0016-5085(99)70025-9 gut.29.9.1258 2 Hunt RH, Camilleri M, Crowe SE, et al. The stomach in health 19 Feeley KM, Heneghan MA, Stevens FM, McCarthy CF. Lympho- and disease. Gut 2015;64:1650-68. https://doi.org/10.1136/ cytic gastritis and coeliac disease: evidence of a positive as- gutjnl-2014-307595 sociation. J Clin Pathol 1998;51:207-10. https://doi.org/10.1136/ 3 Marshall BJ, Warren JR. Unidentified curved bacilli in the jcp.51.3.207 stomach of patients with gastritis and peptic ulceration. Lan- 20 Gonsalves N. Eosinophilic Gastrointestinal Disorders. Clin cet. 1984;1(8390):1311-5. https://doi.org/10.1016/s0140- Rev Allergy Immunol. 2019;57:272-85. https://doi.org/10.1007/ 6736(84)91816-6 s12016-019-08732-1 4 Schistosomes, liver flukes and Helicobacter pylori. IARC Work- 21 Caldwell JM, Collins MH, Stucke EM, et al. Histologic eosino- ing Group on the Evaluation of Carcinogenic Risks to Humans. philic gastritis is a systemic disorder associated with blood and Lyon, 7-14 June 1994. IARC Monogr Eval Carcinog Risks Hum extragastric eosinophilia, TH2 immunity, and a unique gastric 1994;61:1-241. transcriptome. J Allergy Clin Immunol. 2014;134:1114-24. https:// 5 Okuda M, Sugiyama T, Fukunaga K, et al. A strain-specific anti- doi.org/10.1016/j.jaci.2014.07.026 gen in Japanese Helicobacter pylori recognized in sera of Japa- 22 Zhang M, Li Y. Eosinophilic gastroenteritis: A state-of-the-art nese children. Clin Diagn Lab Immunol 2005;12:1280-4. https:// review. J Gastroenterol Hepatol 2017;32:64-72. https://doi. doi.org/10.1128/CDLI.12.11.1280-1284.2005 org/10.1111/jgh.13463 6 Ford AC, Forman D, Hunt RH, et al. Helicobacter pylori eradi- 23 Lwin T, Melton SD, Genta RM. Eosinophilic gastritis: histopatho- cation therapy to prevent gastric cancer in healthy asymptom- logical characterization and quantification of the normal gas- atic infected individuals: systematic review and meta-analysis of tric eosinophil content. Mod Pathol 2011;24:556-63. https://doi. randomised controlled trials. BMJ 2014;348:g3174. https://doi. org/10.1038/modpathol.2010.221 org/10.1136/bmj.g3174 24 Klein NC, Hargrove RL, Sleisenger MH, Jeffries GH. Eosinophil- 7 Sugano K, Tack J, Kuipers EJ, et al. Kyoto global consensus ic gastroenteritis. Medicine (Baltimore) 1970;49:299-319. https:// report on Helicobacter pylori gastritis. Gut 2015;64:1353-67. doi.org/10.1097/00005792-197007000-00003 https://doi.org/10.1136/gutjnl-2015-309252 25 El-Zimaity H, Choi WT, Lauwers GY, Riddell R. The differential 8 Bode G, Mauch F, Malfertheiner P. The coccoid forms of He- diagnosis of Helicobacter pylori negative gastritis. Virchows Arch licobacter pylori. Criteria for their viability. Epidemiol Infect 2018;473:533-50. https://doi.org/10.1007/s00428-018-2454-6 1993;111:483-90. https://doi.org/10.1017/s0950268800057216 26 Nomura K, Iizuka T, Kaji D, Yamamoto H, Kuribayashi Y, Kimura 9 Malfertheiner P, Megraud F, O’Morain CA, et al. Management of R, et al. Clinicopathological features of patients with acute graft- Helicobacter pylori infection--the Maastricht IV/ Florence Con- versus-host disease of the upper digestive tract. J Gastroenterol sensus Report. Gut 2012;61:646-64. https://doi.org/10.1136/ Hepatol 2014;29:1867-72. https://doi.org/10.1111/jgh.12651 gutjnl-2012-302084 27 10 Tobin JM, Sinha B, Ramani P, et al. Upper gastrointestinal mucosal Johncilla M, Grover S, Zhang X, et al. Morphological spec- disease in pediatric Crohn disease and ulcerative colitis: a blind- trum of immune check-point inhibitor therapy-associated gas- ed, controlled study. J Pediatr Gastroenterol Nutr 2001;32:443-8. tritis. Histopathology 2020;76:531-9. https://doi.org/10.1111/ https://doi.org/10.1097/00005176-200104000-00010 his.14029 28 11 Kamimura K, Kobayashi M, Sato Y, et al. Collagenous gastritis: Rovedatti L, Lenti MV, Vanoli A, et al. Nivolumab-associated ac- Review. World J Gastrointest Endosc 2015;7:265-73. https://doi. tive neutrophilic gastritis. J Clin Pathol 2020 Online ahead of org/10.4253/wjge.v7.i3.265 print. https://doi.org/10.1136/jclinpath-2020-206540 29 12 Lagorce-Pages C, Fabiani B, Bouvier R, et al. Collagenous gas- Rugge M, Fassan M, Pizzi M, et al. Autoimmune gastri- tritis: a report of six cases. Am J Surg Pathol 2001;25:1174-9. tis: histology phenotype and OLGA staging. Aliment Phar- https://doi.org/10.1097/00000478-200109000-00008 macol Ther 2012;35:1460-6. https://doi.org/10.1111/j.1365- 30 2036.2012.05101.x Price AB. The Sydney System: histological division. J Gastroenterol Hepatol 1991;6:209-22. https://doi.org/10.1111/j.1440-1746.1991. 13 Coati I, Fassan M, Farinati F, et al. Autoimmune gastritis: Pa- tb01468.x thologist’s viewpoint. World J Gastroenterol 2015;21:12179-89. 31 https://doi.org/10.3748/wjg.v21.i42.12179 Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis. The updated Sydney System. International 14 Neumann WL, Coss E, Rugge M, Genta RM. Autoimmune Workshop on the Histopathology of Gastritis, Houston 1994. Am J atrophic gastritis--pathogenesis, pathology and management. Surg Pathol 1996;20:1161-81. https://doi.org/10.1097/00000478- Nat Rev Gastroenterol Hepatol 2013;10:529-41. https://doi. 199610000-00001 org/10.1038/nrgastro.2013.101 32 Fassan M, Pizzi M, Farinati F, et al. Lesions indefinite for in- 15 Bergman MP, D’Elios MM. Cytotoxic T cells in H. pylori-related traepithelial neoplasia and OLGA staging for gastric atrophy. Am gastric autoimmunity and gastric lymphoma. J Biomed Biotech- J Clin Pathol 2012;137:727-32. https://doi.org/10.1309/AJCPEU- nol. 2010;2010:104918. https://doi.org/10.1155/2010/104918 41HTGXSJDQ 16 Junca J, de Soria PL, Granada ML, et al. Detection of early ab- 33 Vanoli A, La Rosa S, Luinetti O, et al. Histologic changes in type normalities in gastric function in first-degree relatives of patients A chronic atrophic gastritis indicating increased risk of neuro- with pernicious anemia. Eur J Haematol 2006;77:518-22. https:// endocrine tumor development: the predictive role of dysplastic doi.org/10.1111/j.0902-4441.2006.t01-1-EJH2913.x and severely hyperplastic enterochromaffin-like cell lesions. 17 Rugge M. Biologic profiles meet clinical priorities: incorporat- Hum Pathol 2013;44:1827-37. https://doi.org/10.1016/j.hump- ing pseudopyloric, and spasmolytic-expressing metaplasia in ath.2013.02.005 GASTRITIS HISTOPATHOLOGICAL CLASSIFICATION 165

34 Rugge M, Correa P, Dixon MF, et al. Gastric mucosal atrophy: in- 42 Rugge M, Genta RM, Di Mario F, et al. Gastric cancer as pre- terobserver consistency using new criteria for classification and ventable disease. Clin Gastroenterol Hepatol 2017;15:1833-43. grading. Aliment Pharmacol Ther. 2002;16:1249-59. https://doi. https://doi.org/10.1016/j.cgh.2017.05.023 org/10.1046/j.1365-2036.2002.01301.x 43 Correa P, Piazuelo MB. The gastric precancerous cas- 35 Rugge M, Pennelli G, Pilozzi E, et al. Gastritis: the histol- ogy report. Dig Liver Dis 2011;43 Suppl 4:S373-84. https://doi. cade. J Dig Dis 2012;13:2-9. https://doi.org/10.1111/j.1751- org/10.1016/S1590-8658(11)60593-8 2980.2011.00550.x 36 Rugge M, Correa P, Di Mario F, et al. OLGA staging for gastritis: 44 WHO Classification of Tumours - Digestive system tumours. 5th a tutorial. Dig Liver Dis 2008;40:650-8. https://doi.org/10.1016/j. Ed. Lyon: International Agency for Research on Cancer 2019. dld.2008.02.030 45 Capelle LG, de Vries AC, Haringsma J, et al. The staging of gas- 37 Rugge M, Meggio A, Pennelli G, et al. Gastritis staging in clinical tritis with the OLGA system by using intestinal metaplasia as an practice: the OLGA staging system. Gut 2007;56:631-6. https:// accurate alternative for atrophic gastritis. Gastrointest Endosc doi.org/10.1136/gut.2006.106666 2010;71:1150-8. https://doi.org/10.1016/j.gie.2009.12.029 38 Rugge M, Sacchi D, Graham DY, Genta RM. Secondary preven- 46 tion of gastric cancer: merging the endoscopic atrophic border Rugge M, Fassan M, Pizzi M, et al. Operative link for gastritis with OLGA staging. Gut 2020;69:1151-2. https://doi.org/10.1136/ assessment vs operative link on intestinal metaplasia assess- gutjnl-2019-319107 ment. World J Gastroenterol. 2011;17(41):4596-601. https://doi. 39 Niwa T, Tsukamoto T, Toyoda T, et al. Inflammatory processes org/10.3748/wjg.v17.i41.4596 triggered by Helicobacter pylori infection cause aberrant DNA 47 Rugge M, Sugano K, Scarpignato C, et al. Gastric cancer pre- methylation in gastric epithelial cells. Cancer Res 2010;70:1430- vention targeted on risk assessment: Gastritis OLGA staging. 40. https://doi.org/10.1158/0008-5472.CAN-09-2755 Helicobacter 2019;24:e12571. https://doi.org/10.1111/hel.12571 40 Correa P. Chronic gastritis: a clinico-pathological classification. Am J Gastroenterol 1988;83:504-9. 48 Rugge M, Genta RM, Fassan M, et al. OLGA Gastritis Staging 41 Rugge M, Fassan M, Graham DY. Clinical guidelines: Second- for the Prediction of Gastric Cancer Risk: A Long-term Follow- ary prevention of gastric cancer. Nat Rev Gastroenterol Hepatol up Study of 7436 Patients. Am J Gastroenterol 2018;113:1621-8. 2012;9:128-9. https://doi.org/10.1038/nrgastro.2012.19 https://doi.org/10.1038/s41395-018-0353-8 PATHOLOGICA 2020;112:166-185; DOI: 10.32074/1591-951X-166

Review

Precancerous lesions of the stomach, gastric cancer and hereditary gastric cancer syndromes

Irene Gullo1*, Federica Grillo2-3*, Luca Mastracci2-3, Alessandro Vanoli4, Fatima Carneiro1, Luca Saragoni5, Francesco Limarzi6, Jacopo Ferro2, Paola Parente7, Matteo Fassan8 1 Department of Pathology, Centro Hospitalar Universitário de São João (CHUSJ) & Department of Pathology, Faculty of Medicine of the University of Porto (FMUP), Porto, Portugal and Instituto de Investigação e Inovação em Saúde (i3S) & Institute of Molecular Pathology and Immunology of the University of Porto (Ipatimup), Portugal; 2 Anatomic Pathology Unit, Department of Surgical Sciences and Integrated Diagnostics (DICS), University of Genova, Italy; 3 Ospedale Policlinico San Martino, IRCCS for Oncology and Neuroscience, Genova, Italy; 4 Anatomic Pathology Unit, Department of Molecular Medicine, University of Pavia and Fondazione IRCCS San Matteo Hospital, Pavia, Italy; 5 UO Anatomia Patologica, Ospedale G.B. Morgagni-L. Pierantoni, Forlì, Italy; 6 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST/IRCCS), Meldola (FC), Italy; 7 Pathology Unit, Fondazione IRCCS Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG) Italy; 8 Surgical Pathology Unit, Department of Medicine (DIMED), University of Padua, Italy * These authors contributed equally

Received and accepted: June 30, 2020 Summary Published online: 29 October, 2020 Gastric cancer accounts for about 6% of cancers worldwide, being the fifth most frequently diagnosed malignancy and the third leading cause of cancer related death. Gastric carci- Correspondence nogenesis is a multistep and multifactorial process and is the result of the complex inter- Federica Grillo play between genetic susceptibility and environmental factors. The identification of predis- Anatomic Pathology Unit, Department of posing conditions and of precancerous lesions is the basis for screening programs and Surgical Sciences and Integrated Diagnostics early stage treatment. Furthermore, although most gastric cancers are sporadic, familial (DICS), University of Genova and Ospedale clustering is observed in up to 10% of patients. Among them, hereditary cases, related to Policlinico San Martino, IRCCS for Onco- logy and Neuroscience, Genova, Italy, largo known cancer susceptibility syndromes and/or genetic causes are thought to account for Rosanna Benzi 10, 16132 Genova, Italy 1-3% of all gastric cancers. The pathology report of gastric resections specimens therefore Tel. +39 010 5555957 requires a standardized approach as well as in depth knowledge of prognostic and treat- Fax: +39 010 5556392 ment associated factors. E-mail: [email protected] Key words: gastric cancer, gastric dysplasia, gastric adenocarcinoma, hereditary gastric Conflict of interest cancer syndromes, hereditary diffuse gastric cancer (HDGC) The Authors declare no conflict of interest.

How to cite this article: Gullo I, Grillo F, Matracci Introduction L, et al. Precancerous lesions of the stomach, gastric cancer and hereditary gastric cancer syndromes. Pathologica 2020;112:166-185. Gastric cancer accounts for about 6% of cancers worldwide, being the https://doi.org/10.32074/1591-951X-166 fifth most frequently diagnosed malignancy and the third leading cause of cancer related death, behind lung and colorectal cancer. According © Copyright by Società Italiana di Anatomia Pato- to the most recent GLOBOCAN cancer estimates, gastric cancer was logica e Citopatologia Diagnostica, Divisione Itali- ana della International Academy of Pathology responsible for over 1,000,000 new cancer cases and 783,000 deaths in 2018 1. Although there has been a steady decline in the incidence and OPEN ACCESS mortality of gastric cancer over the last 15 years, as the result of the decrease of Helicobacter pylori prevalence and better dietary habits, This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- the absolute incidence rate continues to rise, due to the advancing age NonCommercial-NoDerivatives 4.0 International) license: the of the world population. work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original Gastric cancer incidence and mortality vary substantially across countries version. For further information: https://creativecommons. and within each country. Incidence rates are elevated (up to 32 cases per org/licenses/by-nc-nd/4.0/deed.en 100,000) in Eastern and Western Asia. Zones of low incidence (< 7 cases HISTOPATHOLOGY OF TUMORS OF THE STOMACH 167

per 100,000) are Northern America, Northern Europe, imately half of the world’s population is infected with and most regions of Africa 1. In Italy, gastric cancer Helicobacter pylori, however, only a small fraction will ranks eighth among all cancers, with 12,803 new cases end up developing gastric carcinoma, suggesting that and 9,457 deaths in 2018 1. The poor clinical outcome additional factors participate in the carcinogenic pro- of gastric cancer is mainly due to late diagnosis, poor cess, including Helicobacter pylori virulence factors, response to therapeutic regimens and the highly heter- genetic susceptibility, diet, smoking, and possibly oth- ogenous nature of the disease 2. er bacteria species 10. Helicobacter pylori virulence Gastric carcinogenesis is a multistep and multifactorial factors that appear to influence the pathogenicity of process and is the result of the complex interplay be- the bacterium, as well as the risk of gastric cancer tween genetic susceptibility and environmental factors. development, include CagA (cag pathogenicity is- Risk factors predisposing to gastric cancer include land-encoded cytotoxin associated gene A) and Va- Helicobacter pylori infection, tobacco smoking, dietary cA (vacuolating cytotoxin A) 11, while polymorphisms 3 habits (high intake of salt-preserved, smoked foods, of genes involved in initiation and modulation of the red and processed meat, low intake of fresh fruit and inflammatory response, such as genes codifying IL- 4 vegetables), and Epstein-Barr virus (EBV) infection , 1β, IL-1 receptor antagonist, IL-10 and TNFα, are host as well as microbial community modifications by long- genetic susceptibility factors associated with individ- 5 term use of proton-pomp inhibitors . A number of pre- ual or familial susceptibility to carcinogenesis medi- cancerous conditions have been recognized, such as ated by Helicobacter pylori infection 12. Although the chronic atrophic gastritis and intestinal metaplasia due magnitude of risk is not uniformly defined, atrophic to Helicobacter pylori infection or autoimmunity (per- gastritis caused by autoimmunity (pernicious anemia) nicious anemia), peptic ulcer disease, gastric stump is associated with an increased risk of dysplasia and after partial gastrectomy and gastric polyps. adenocarcinoma 13, as well as neuroendocrine neo- Although most gastric cancers are sporadic, familial plasms and gastric epithelial polyps, such as intesti- clustering is observed in up to 10% of patients. Among nal-type adenomas and pyloric gland adenomas. them, hereditary cases, related to known cancer Several classification systems for chronic gastritis susceptibility syndromes and/or genetic causes are have been developed, including the Sydney classifica- thought to account for 1-3% of all gastric cancers 6,7. tion system 14, the Gastric Risk Index 15 and the Oper- The three major hereditable syndromes that primarily 16 affect the stomach are hereditary diffuse gastric can- ative Link on Gastritis Assessment (OLGA) system . cer (HDGC), gastric adenocarcinoma, proximal poly- These staging systems, particularly the five-tiered (0- posis of the stomach (GAPPS), and familial intestinal IV) OLGA system, provide a basis for predicting gas- gastric cancer (FIGC). tric cancer risk associated with atrophic gastritis and intestinal metaplasia and guide clinical surveillance 17. Well established evidence links intestinal metaplasia 18 Precancerous lesions to intestinal-type gastric cancer . Complete intesti- nal metaplasia shows goblet cells, absorptive entero- Atrophic gastritis and intestinal metaplasia cytes with luminal brush border and intestinal mucin Gastric carcinogenesis is a multistep process which (MUC2) expression. In contrast, incomplete intestinal involves, in most cases, a progression from normal metaplasia displays globet cells, absorptive cells with- mucosa through chronic gastritis (chronic inflamma- out brush border and co-expression of intestinal and 19 tion of the gastric mucosa), mucosal atrophy (loss of gastric (MUC5AC, MUC6) mucins . Reliable indica- gastric glands) and intestinal metaplasia (substitution tors of gastric cancer risk include the topographical of gastric epithelium by intestinal epithelium) to dys- extent of intestinal metaplasia and the degree of in- 20 plasia (intraepithelial neoplasia) and carcinoma. This complete-type intestinal metaplasia . sequence of events may last several years and has Another pattern of metaplasia, which is believed to been designated as the Correa’s cascade of multistep represent an alternative pathway to gastric neoplasia, gastric carcinogenesis 8. According to this model, long is pseudopyloric or spasmolytic polypeptide-express- standing inflammation is the primary pathogenic fac- ing metaplasia (SPEM), which expresses trefoil factor tor leading to gastric cancer development. family 2 (TFF2) spasmolytic polypeptide and repre- Among environmental factors leading to inflamma- sents the metaplastic replacement of oxyntic glands tion-mediated gastric cancer, Helicobacter pylori in- by mucin secreting antral-like glands. SPEM develops fection is associated with almost 90% of new cases in the gastric body and fundus and has been asso- of non-cardia gastric cancers 9 and was classified ciated with chronic Helicobacter pylori infection and as a type I carcinogen by the WHO in 1994. Approx- development of gastric cancer 21. 168 I. Gullo et al.

Gastric dysplasia Gastric dysplasia is defined as unequivocal neoplas- tic changes in the gastric epithelium, without evidence of lamina propria invasion. The diagnostic criteria are based on the presence of cellular atypia, abnormal differentiation, architectural disorganisation and in- creased mitotic activity. Endoscopically, gastric dys- plasia may present as flat, depressed or polypoid lesions (the latter may be referred to as gastric – in- testinal type and foveolar type – adenomas). It may arise de novo or may occur within pre-existing benign sporadic polyps, namely hyperplastic polyps and fun- dic gland polyps or hamartomatous polyps, such as juvenile polyps and Peutz-Jeghers polyps. On the basis of the histomorphological profile, gas- tric dysplasia may be classified as intestinal or fove- olar (gastric) type. Intestinal type dysplasia shows features resembling colonic adenomas, with tubular glands lined by columnar cells with overlapping, pseu- dostratified and penicillate nuclei, which can be hyper- chromatic and/or pleomorphic. Differentiation towards goblet cells, absorptive cells and Paneth cells may be observed. Intestinal type dysplasia shows immunore- activity for MUC2, CD10 and CDX2 22. The foveolar (gastric) phenotype is characterized by cuboidal to low columnar cells resembling gastric foveolar cells, with round to oval nuclei and clear or eosinophilic cy- toplasm. Gastric differentiation may be confirmed by MUC5AC and MUC6 expression. Using immunohis- tochemistry, hybrid or mixed cases may also occur, with both intestinal and gastric marker expression, as Figure 1. Two examples of intramucosal gastric carcinoma. well as null cases, negative for the aforementioned In contrast to high grade dysplasia, intramucosal carcinoma markers. Foveolar type dysplasia is more likely to be shows marked glandular crowding and cribriform pattern high-grade and is associated more frequently to gas- (upper image, HE, magnification 10x). The bottom image tric adenocarcinoma 22. shows a very well differentiated intramucosal carcinoma Dysplasia is graded as low grade or high grade on with crawling pattern (HE, magnification 10x). the basis of architectural distortion, nuclear and cy- toplasmic cell features and mitotic activity. In low grade dysplasia, glandular architecture is relatively preserved, cellular pleomorphism is mild or absent, presence of desmoplasia is not necessary for the nuclei maintain basal polarity and mitotic activity is not definition of stromal invasion. The distinction between markedly increased. High grade dysplastic features reactive/regenerative changes and true dysplasia include complex glandular architecture, marked cyto- may be difficult, especially in small biopsies and spec- logic atypia with large nuclei and prominent nucleoli, imens with technical artefacts. For these cases, the loss of cell polarity and frequent mitotic figures 23. Dis- term “indefinite for dysplasia” may be applied. Gastric tinction between high-grade dysplasia and intramu- dysplasia limited to the pit region, without superficial cosal intestinal adenocarcinoma may be challenging, epithelial involvement, is defined as crypt dysplasia 24. especially in small biopsy samples, and there is only limited consensus about diagnostic criteria, especial- Gastric adenomas ly between Asian and Western pathologists. Helpful A recent classification proposed by Hackeng WM et features for the diagnosis of intramucosal adenocar- al. 25. distinguishes gastric polyps according to the gas- cinoma include marked glandular crowding, cribriform tric mucosa compartment from which the gastric pol- and crawling pattern, budding, infiltration of isolated yp arises. Gastric adenomas arising from the foveolar cells and intraglandular necrotic debris (Fig. 1). The compartment include foveolar type adenomas (arising HISTOPATHOLOGY OF TUMORS OF THE STOMACH 169

Figure 2. Gastric adenomas: (a) foveolar type adenoma with low grade dysplasia (HE, left image, magnification 40x, right image, magnification 10x) showing diffuse immunoreactivity for MUC5AC (inset); (b) intestinal type adenoma associated to mucinous carcinoma invading the submucosa (arrow) (HE, left image, magnification 4x); the image on the right represents an area of intestinal-type low grade dysplasia with tubular/villous morphology (HE, magnification 10x); (c) pyloric gland ad- enoma (HE, left image, magnification 4x, right image, magnification 20x).

from foveolar epithelium without intestinal metaplasia) Gastric adenomas arising from the glandular compart- (Fig. 2a) and intestinal type adenomas (arising from fo- ment include pyloric gland adenoma (PGA) (Fig. 2c) veolar epithelium with intestinal metaplasia) (Fig. 2b). and oxyntic gland adenoma (OGA). Consistent with 170 I. Gullo et al.

their glandular histogenesis, OGAs and PGAs show diffuse immunoreactivity for MUC6. PGAs consist of closely packed tubules or dilated glands of pyloric type epithelium, lined by cuboidal/ low columnar cells with pale, clear or slightly eosino- philic cytoplasm. PGAs may occur in syndromic con- texts, namely familial adenomatous polyposis (FAP) and Lynch syndrome 26. OGAs is composed of dysplastic glands showing var- iable differentiation to chief and parietal cells. There is a morphological continuum between OGA and gastric adenocarcinoma of fundic gland type. Whether they are distinct lesions, the former representing the pre- cursor lesion of the latter, or represent a morphologi- cal spectrum of the same lesion is still debated 27.

Benign gastric polyps with possible gastric dysplasia and gastric cancer Hyperplastic polyps (HPs) are benign gastric epithe- lial lesions consisting of hyperplastic and cystically dilated foveolar epithelium, in a background of prom- inent inflammatory changes. As HPs represent a hy- perproliferative response to tissue injury, most of them arise in a background of longstanding gastric mucosal inflammation and are the prevalent polyp type in coun- tries with a high prevalence of Helicobacter pylori in- fection. Foveolar or intestinal type dysplasia and ade- nocarcinoma (intestinal type or diffuse type) may arise in about 2% of larger HPs 28 (Fig. 3). Copy number alterations and TP53 mutations are restricted to the adenocarcinoma component 29. Fundic gland polyps (FGPs) are benign gastric ep- ithelial lesions composed of disordered, expanded and cystically dilated oxyntic glands lined by pa- rietal and chief cells, as well as mucous neck ep- ithelium. FGPs are the predominant polyp type in Western countries, are associated with the use of proton pomp inhibitors and are inversely related to Helicobacter pylori gastritis 30. FGPs may develop foveolar-type dysplasia, which is usually low-grade (Fig. 4). In sporadic FGPs, dysplasia is rarely ob- served and the finding of dysplasia should raise suspicion of an inherited syndrome, especially in the case of young patients, multiple FGPs and (in the case of FAP) polyps elsewhere in the gastroin- testinal tract. In the syndromic context, dysplasia in Figure 3. Hyperplastic polyp with dysplasia (HE, upper FGPs may be observed in gastric adenocarcinoma image 4x) and intramucosal carcinoma (HE, middle image, and proximal polyposis of the stomach (see below) magnification 40x, bottom image, magnification 10x). and FAP. The genomic landscape of syndromic and sporadic FGPs is distinctive. FAP-associated FGPs may present second-hit inactivation of the APC gene but no CTNNB1 (beta-catenin) mutations, while spo- radic FGPs harbour CTNNB1 mutations and usually lack APC alterations 31. HISTOPATHOLOGY OF TUMORS OF THE STOMACH 171

Clinical features The clinical presentation of gastric cancer is mainly related to topography and stage of the disease. The majority of early gastric cancers are asymptomatic at diagnosis. Screening programs in high-risk popula- tions (Japan, Korea) have resulted in early diagnosis in asymptomatic patients and better overall survival 32. At advanced disease stage, common signs and symp- toms include dyspepsia, epigastric pain, abdominal mass and alarm symptoms (“red flags”), such as dys- phagia, significant weight loss, signs and symptoms of gastrointestinal hemorrhage and vomiting. Endoscopic examination with biopsies is the gold standard method for gastric cancer diagnosis. Image enhanced endoscopy and magnifying endoscopy may improve the detection rate of early gastric lesions 33. Accurate (TNM) staging is the cornerstone for ac- curately defining gastric cancer prognosis and ther- apeutic approaches. Compared to advanced gastric cancers, early gastric cancers have a much better prognosis, with a 5-year survival rate of > 90% after surgical resection. If untreated, 63% of early gastric carcinomas progress to advanced tumors within 5 years 34. In contrast, advanced and unresectable gas- tric cancers have a poor prognosis with an expected survival of few months. Endoscopic ultrasonography is the preferred technique for defining the depth of in- vasion into the gastric wall (pT stage). Endoscopic resection is recommended for early gastric Figure 4. Fundic gland polyp with focus of low grade, fo- cancers with low probability of metastasising to lymph veolar-type dysplasia (upper image, HE, magnification 4x; nodes. Risk factors associated with the development of (upper image, HE, magnification 20x). The patient had at- nodal metastases, for which surgery with lymph-node tenuated variant of familial adenomatous polyposis. dissection should be considered, include submucosal invasion, tumor diameter greater than 20-30 mm, vas- cular venous or lymphatic invasion, depressed or ulcer- ated macroscopic subtypes and undifferentiated histol- Gastric cancer ogy 35. Treatment for advanced gastric cancer is based on surgery and chemo-radiation therapy. For patients Definitions with unresectable gastric cancer, systemic therapy is Gastric adenocarcinoma is a malignant epithelial ne- the only approach, encompassing conventional chemo- oplasm with glandular differentiation arising from the therapy and targeted therapies. The latter include mon- gastric mucosa and represents a biologically hetero- oclonal antibodies directed against HER2, VEGFR2 geneous group of tumors with respect to etiology, his- and immune checkpoint inhibitors 36,37. According to the togenesis, morphology, and molecular features. Over- most recent European recommendations 38 the only all, gastric adenocarcinoma accounts for 90-95% of established predictive biomarker for the treatment of gastric malignancies. gastric cancer is HER2 status, evaluated by HER2 im- According to the depth of invasion in the gastric wall, munohistochemistry and ERBB2 in situ hybridization to gastric cancer is classified as early or advanced. Ear- select patients with unresectable or metastatic gastric ly gastric cancer is defined as a carcinoma limited to cancer for anti-HER2 based therapies. Heterogeneity the mucosa (pT1a) or the mucosa and submucosa in HER2 assessment in gastric cancer has been widely (pT1b), regardless of tumor size or the presence of documented 39 and this is of practical importance when lymph-node metastases. Gastric adenocarcinomas HER2 evaluation is performed on endoscopic biopsies: invading the muscularis propria and beyond (> pT2) a minimum set of 5 biopsies has shown to be neces- are defined as advanced. sary for a reliable HER2 assessment 40-41. 172 I. Gullo et al.

Emerging predictive biomarkers for selecting gastric can- pathological classifications proposed over time 47. The cer patients who may benefit from immune-checkpoint histopathological classifications most commonly used inhibitor-based immunotherapies include microsatellite include those proposed by Laurén 48 and the World instability (MSI)-high status 42, EBV infection 43, PD-L1 Health Organization (WHO) 23. expression (combined positive score ≥ 1%) 44, tumor mu- The Laurén classification 48 (Tab. I) distinguishes two tation load and density of intra-tumoral CD8+ T-cells 45. major types, intestinal and diffuse. The former, is com- Adverse prognostic factors in resectable cases in- posed of glands or papillae, while the latter shows an clude higher pT and pN stages 46, limited lymph node infiltrative growth pattern and is composed of tumor dissection, lymphatic and vascular invasion, and in- cells without cellular cohesion. Tumors presenting volvement of surgical margins. both intestinal and diffuse components are termed mixed carcinomas. Solid, poorly differentiated or un- Microscopic findings and histopathological differentiated carcinomas that do not fit in one of these classifications subtypes are placed in the indeterminate category. Gastric cancer presents a variability of morphological Despite dating back to 1965, Laurén classification is phenotypes, as reflected by the large number of histo- still relevant, as it distinguishes subtypes with distinct

Table I. Checklist for gastric cancer reporting (based on WHO Classification of Digestive System Tumors, 5th Edition and AJCC Cancer Staging Manual, 8th Edition). Procedure Endoscopic resection Partial gastrectomy: specify if proximal, distal, other Total gastrectomy Other Specimen description Endoscopic resection Dimension of mucosal surface (cm) and depth (cm) Gastrectomy Length (cm) of lesser and greater curvature Length (cm) of duodenal and oesophageal segments, if applicable Macroscopic examination Tumor not identified macroscopically Tumor location (gastric region): cardia, fundus, body, transitional zone, antrum, pylorus Tumor location (gastric curvatures and walls): lesser curvature, greater curvature, anterior wall, posterior wall Tumor size: greatest dimension (cm) or three dimensions (cm) Tumor macroscopic appearance - Borrmann type I: polypoid/fungating - Borrmann type II: ulcerated mass - Borrmann type III: infiltrative neoplasm with ulceration - Borrmann type IV: infiltrative neoplasm without ulceration Margins Endoscopic resection Mucosal margin - Involved by invasive carcinoma - Involved by dysplasia (low-grade/high-grade) - Uninvolved by invasive carcinoma or dysplasia Deep margin - Involved by invasive carcinoma - Involved by dysplasia (low-grade/high-grade) - Uninvolved by invasive carcinoma or dysplasia Gastrectomy Esophageal (proximal) margin - Involved by invasive carcinoma - Involved by dysplasia (low-grade/high-grade) - Uninvolved by invasive carcinoma or dysplasia Duodenal (distal) margin - Involved by invasive carcinoma - Involved by dysplasia (low-grade/high-grade) - Uninvolved by invasive carcinoma or dysplasia Omental (radial) margin - Involved by invasive carcinoma (greater and/or lesser omental margin) - Uninvolved by invasive carcinoma u HISTOPATHOLOGY OF TUMORS OF THE STOMACH 173

Table I. continues Gastric cancer histological Laurén classification55: subtype - Diffuse type - Intestinal type - Mixed type - Indeterminate WHO classification (major types and rare variants): -T ubular adenocarcinoma -P apillary adenocarcinoma -T ubulo-papillary adenocarcinoma -P oorly cohesive carcinoma: signet ring cell type/not otherwise specified - Mucinous adenocarcinoma - Mixed adenocarcinoma - Gastric squamous carcinoma - Gastric adenosquamous carcinoma - Gastric undifferentiated carcinoma - Gastric cancer with lymphoid stroma - Hepatoid carcinoma - Alpha-fetoprotein producing gastric cancer (adenocarcinoma with enteroblastic differentiation, yolk-sac tumor like carcinoma) - Micropapillary adenocarcinoma - Gastric adenocarcinoma of the fundic gland type - Mucoepidermoid carcinoma -P aneth cell carcinoma -P arietal cell carcinoma Histologic grade Only applicable to tubular and papillary adenocarcinoma: - Low grade - High grade Pathological stage: descriptors - m (multiple primary tumors) - r (recurrent) - y (post-treatment) Pathological stage: - pTX: primary tumor cannot be assessed primary tumor (pT) - pT0: no evidence of primary tumor - pTis: in situ SRC carcinoma, pagetoid progression of SRCs, high-grade dysplasia - pT1a: tumor invades the lamina propria or muscularis mucosae - pT1b: tumor invades the submucosa - pT2: tumor invades the muscularis propria - pT3: tumor penetrates the subserosal connective tissue without invasion of the visceral peritoneum or adjacent structures - pT4a: tumor invades the serosa (visceral peritoneum) - pT4b: tumor invades adjacent structures/organs Lymph node examination Number of lymph nodes involved - Lesser omentum - Greater omentum - Other Number of lymph nodes examined - Lesser omentum - Greater omentum - Other Ratio between lymph nodes involved and examined Pathological stage: - pNX: regional lymph node(s) cannot be assessed regional lymph nodes (pN) - pN0: no regional lymph node metastasis - pN1: metastasis in one or two regional lymph nodes - pN2: metastasis in three to six regional lymph nodes - pN3a: metastasis in seven to 15 regional lymph nodes - pN3b: metastasis in 16 or more regional lymph nodes Pathological stage: - Not applicable (pM status required only if confirmed pathologically) distant metastasis - pM1: distant metastasis(es) (specify site) Lymphovascular invasion - Not identified -P resent -C annot be determined

u 174 I. Gullo et al.

Table I. continues

Perineural invasion - Not identified -P resent -C annot be determined Treatment effect - No known presurgical therapy -P resent • Complete response (no viable cancer cells) – score 0 • Near complete response (single or rare small groups of cancer cells) – score 1 • Partial response (evident tumor regression but more than single or rare small groups of cancer cells) – score 2 • Poor or no response (no evident tumor regression) – score 3 -C annot be determined Additional findings - Helicobacter pylori infection - Chronic gastritis (lymphoid follicles, neutrophilic activity, erosion/ulceration) - Glandular atrophy - Intestinal metaplasia -D ysplasia -P olyps: specify type Ancillary studies Add any relevant ancillary study performed Comments Add any relevant comment epidemiologic settings, clinicopathologic profiles and enocarcinoma) applies primarily to tubular, papillary biological behaviors. As an example, in view of their and tubulo-papillary subtypes 23. Tubular and papillary cohesive nature, intestinal type gastric cancers have carcinomas roughly correspond to intestinal type gas- the ability to survive more easily into venous vessels tric cancers, while poorly cohesive carcinomas corre- and tend to metastasise haematogenously, while the spond to the diffuse subtype by Laurén. poorly cohesive phenotype of diffuse gastric cancer The 2019 WHO Classification of digestive system tu- tends to disseminate through peritoneal surfaces. mors stresses the importance of distinguishing differ- Mixed gastric cancer shows a poorer prognosis com- ent subtypes within the poorly cohesive carcinoma 49 pared to intestinal or diffuse types and a dual met- category, based on presence and quantity of signet astatic pattern (hematogenous metastases and peri- ring cells. By definition, a signet ring cell has an abun- 50 toneal dissemination with lymph node metastases) , dant mucin vacuole filling the cytoplasm and pushing probably because of the cumulative adverse effect of the nucleus at the cell periphery. Poorly cohesive car- the two components within a single tumor. cinomas of the signet ring cell type are composed pre- 23 The WHO classification (Tab. I) distinguishes five dominantly or exclusively (e.g. > 90%) of signet ring main histopathological subtypes of gastric cancers cells, while non-signet ring cell type (i.e. not otherwise (Fig. 5): tubular adenocarcinoma, composed of tubu- specified) poorly cohesive carcinomas are composed lar, glandular or acinar structures of variable diame- (or show a component) of poorly cohesive and infil- ter and various degrees of differentiation (some solid trating cells without a classic signet ring cell morphol- carcinomas may be classified as high-grade tubular ogy. It is important to recognise this latter subtype of adenocarcinomas); papillary carcinoma, showing fin- ger-like papillary architecture, eventually admixed with poorly cohesive gastric cancer, as it presents poorer glandular structures (tubulo-papillary phenotype); prognosis when compared to pure signet ring cell car- 51 poorly cohesive carcinoma, composed of tumor cells cinomas . isolated or in small clusters lacking cellular cohesion; The gastric cancer histopathological classification mucinous adenocarcinoma, defined by the presence proposed by the Japanese Gastric Cancer Associa- 52 of mucin pools accounting for > 50% of the tumor; and tion (JGCA) is mainly used by Asian pathologists . mixed carcinomas, presenting a distinct tubulo-pap- Noteworthy, in the last version of the JGCA and the illary and poorly cohesive component. In mixed car- WHO classifications, gastric cancer expert patholo- cinomas, the two components may be intermingled, gists have built a table showing the similarities of the adjacent, or completely separated. Providing that the two classification systems and corresponding enti- two components are clearly identified within the tu- ties. mor, there is no minimum cell percentage defining this To improve standards of gastric cancer reporting, entity. macroscopic and histological examination should fol- Grading system (low-grade or high-grade gastric ad- low a specific checklist, as presented in Table I. HISTOPATHOLOGY OF TUMORS OF THE STOMACH 175

Figure 5. Main histopathological subtypes of gastric cancer: (a) papillary and tubulo-papillary gastric adenocarcinoma (HE, magnification 10x); (b) tubular adenocarcinoma with solid (high grade) areas (HE, magnification 10x); (c) poorly cohesive gastric cancer of the signet ring cell type (HE, magnification 20x); this tiny intramucosal focus was found in a prophylactic gastrectomy specimen in a CDH1 variant carrier; (d) poorly cohesive gastric cancer not otherwise specified (HE, magnifica- tion 20x); in this case the poorly cohesive cells show pleomorphic and plasmacytoid features; (e) mucinous adenocarcinoma, with and signet ring cells floating in mucin lakes (HE, magnification 20x); (f) mixed gastric cancer (HE, magnification 20x). 176 I. Gullo et al.

Early gastric cancer as a roadmap for gastric cancer prognostic evalua- Early gastric cancer (EGC) is carcinoma limited to tion and targeted therapy approaches. However, the the gastric mucosa and/or submucosa regardless three molecular classifications overlap only partially, of lymph node status with good prognosis 52. Unfor- highlighting the need for a consensual patient strati- tunately, some EGC will have nodal metastases and fication. recent studies have focused on key parameters that The landmark study of gastric cancer molecular-based could be associated with worse prognosis 53. In par- stratification was carried out by The Cancer Genome ticular, size, depth of infiltration, and histological type Atlas (TCGA) research network 58, which defines four of tumors, as well as the distribution of nodal metas- molecular subtypes: EBV-associated gastric cancers, tases, are predictors of worse survival in this subset characterized by recurrent PIK3CA mutations, high of tumors 54. levels of DNA hypermethylation, frequent JAK2 and A dated but useful classification (see Tab. II) was in- CD274 (PD-L1) amplification and enrichment in genes troduced by Kodama 55 in 1983 that identifies growth involved in immune signalling; MSI-high gastric can- patterns of EGC and correlates them with prognosis; cer, characterized by MLH1 silencing and consequent more recent studies 56 have confirmed the importance high levels of DNA hypermethylation; genomically of this classification which should be part of the pa- stable gastric cancer, associated with a diffuse mor- thology report both in surgical specimens but more phology and recurrent CDH1 and RHOA events; gas- importantly in endoscopic resections (Penetrating A tric cancer with chromosomal instability exhibiting a growth subtype has a 10 year prognosis of 74% com- high number of TP53 mutations and amplifications of pared to 94% of non-penetrating A type). tyrosine kinase receptors. The prognostic and predic- tive value of TCGA four-tiered molecular classification Immunohistochemical biomarkers and molecular has been highlighted: EBV-associated and MSI-high subtypes gastric cancers present the best prognostic features Gastric cancer is the result of accumulated genom- and may respond to targeted immunotherapies, chro- ic damage that affects essential cellular functions for mosomal unstable tumors present a moderately poor cancer development. Multiple gene mutations, somat- prognosis but show sensitivity to chemotherapy, while ic copy number alterations, epigenetic and transcrip- genomically stable tumors show the worst prognosis tional changes have been detected in gastric cancer, and are resistant to chemotherapy 42,59. highlighting its molecular heterogeneity. Through There is partial correlation between histopathological high-throughput genomic analysis, several groups and molecular classifications. EBV-associated gastric have analyzed and deciphered the molecular altera- cancer shows the features of gastric cancer with lym- tions of gastric cancer at high resolution, attempting to phoid stroma (see below) in up to 80% of EBV+ cases achieve integrated molecular classification schemes (Fig. 6a); some cases present Crohn’s disease-like which recognise molecular entities with different mo- lymphoid reaction, characterised by the presence of lecular signatures and clinical phenotypes. These numerous lymphoid follicles with active germinal cen- classifications include the Singapore-Duke group clas- tres at the advancing edge of the tumor 60; conven- sification 57, based on gene expression profiling, and tional-type histology, with scant lymphocytic infiltrate, the molecular classifications proposed by The Cancer is observed in a minority of cases. MSI-high gastric Genome Atlas (TCGA) 58, and the Asian Cancer Re- cancers may also present abundant intratumoral and search Group (ACRG) 59, both based on the integra- peritumoral lymphocytic infiltrate. EBV infection and tive analysis of multiple genomic and proteomic data. MSI-high status represent two alternative pathways of These molecular classifications have been proposed gastric carcinogenesis and mutually exclusive gastric

Table II. Kodama’s Classification of growth patterns of early gastric cancer. Growth Patterns Small mucosal (M) Intramucosal EGCs measuring < 4 cm Small mucosal (SM) Intramucosal EGCs minimally invading submucosa measuring < 4 cm Supermucosal (M) Intramucosal EGCs measuring > 4 cm Supermucosal, (SM) Intramucosal EGCs minimally invading submucosa measuring > 4 cm PEN (penetrating) (A) EGCs massively invading submucosa with nodular pattern measuring < 4 cm PEN (penetrating) (B) EGCs massively invading submucosa with saw tooth pattern measuring < 4 cm EGC: early gastric cancer HISTOPATHOLOGY OF TUMORS OF THE STOMACH 177

Figure 6. Rare histopathological variant of gastric cancer: (a) gastric cancer with lymphoid stroma showing abundant lymphoplasmacytic infiltrate (HE, magnification 10x); this case was associated to EBV infection, as evaluated by EBER-in situ hybridization (inset); (b) hepatoid gastric carcinoma with numerous hyaline globules (HE, magnification 20x); (c) mi- cropapillary gastric carcinoma, with artefactual spaces at the periphery of the nests and inverted cell polarity (HE, 20x); (d) adenosquamous gastric carcinoma (HE, magnification 20x).

cancer molecular subtypes 61, with distinct transcrip- the backbone for building algorithms for directed and tional profiles, the former enriched by genes related in cost-effective molecular characterization. Moreover, the immune response and the latter associated with practical algorithms based on immunohistochemistry 62 mitosis and cell cycle biological terms . Genomical- and in situ hybridization can be applied in the routine ly stable gastric cancers show predominantly diffuse diagnostic practice to translate specific immunophe- type histology 58. When compared to pure signet ring notypes into molecular subgroups with prognostic cell carcinomas, poorly cohesive carcinomas classi- and predictive significance 63,64. Thus, positive in si- fied as not otherwise specified show a distinct genom- tu hybridization for EBV-encoded small RNA (EBER) ic profile, enriched by TP53, RHOA, SMAD4, BRAF and PIK3CA mutations 51. Gastric cancers with chro- distinguishes EBV-associated gastric cancer; loss of mosomal instability mostly present intestinal morphol- expression of DNA mismatch repair proteins (MLH1, ogy 58. MSH2, MSH6, PMS2) identifies most of gastric can- Overall, tumor morphology may provide insight in- cers with MSI-high status; genomically stable gastric to tumor biology and should be used as a frame for cancers are identified by the poorly cohesive mor- the identification of clinically relevant subgroups, as phology and abnormal E-cadherin immunoreactivity 178 I. Gullo et al.

(decreased membranous, dotted, cytoplasmic, or ab- sent); and p53 aberrant expression (overexpression or total loss) distinguishes a subset of chromosomal unstable gastric cancers with TP53 activation 64.

Differential diagnosis In poorly differentiated or undifferentiated gastric can- cers, in which epithelial differentiation is not morpho- logically evident, pancytokeratin and EMA immuno- histochemistry may highlight the epithelial nature of the neoplasm and distinguish it from aggressive lym- phomas, metastatic melanoma, germ cell neoplasms or other malignant neoplasms with epithelioid mor- phology. Very well differentiated gastric cancers should be dis- tinguished from gastritis cystica profunda, a benign lesion characterised by the displacement of gastric foveolar epithelium, gastric glands and mucin into the gastric wall or serosa. Gastritis cystica profunda usually develops in stomachs subjected to trauma- tism (e.g. surgery, gastroenterostomy) as the result of chronic inflammation, direct injury and ischemia 65. A helpful feature in distinguishing gastritis cystica pro- funda from adenocarcinoma is the presence of a rim of lamina propria-like stroma surrounding the cystical- ly dilated glands, sometimes associated with smooth muscle fibres from the muscularis mucosae. Gastric adenocarcinoma may coexist with gastritis cystica profundal 66 and the distinction between the two le- sions may be sometimes challenging (Fig. 7).

Rare histotypes of gastric carcinoma

Uncommon histological variants account for about Figure 7. Gastric cancer arising in a stomach with gastritis 5% of gastric cancer and according to the WHO 2019 cystica profunda: in gastritis cystica profunda gastric epi- classification of digestive system tumors 23, encom- thelium is displaced into the gastric wall (upper image, HE, pass i) squamous cell carcinoma; ii) adenosquamous magnification 4x); note the presence of lamina propria-like carcinoma; iii) and undifferentiated carcinoma. stroma surrounding the cystically dilated gastric glands; this Squamous cell carcinoma of the stomach is a car- case was associated to well differentiated tubular gastric cinoma with evidence of squamous cell differentia- adenocarcinoma (middle image, HE, 10x) with a mucinous tion, in the absence of other morphologic aspects. It component (bottom image, HE, magnification 10x). is preferentially located in upper part of the stomach and is extremely rare, accounting for less than 0.1% of gastric cancers. Thorough tumor sampling is required to exclude the presence of other components. Poten- least a quarter of the whole neoplasm to render this tial pitfalls include metastases from a squamous cell diagnosis. It is extremely rare, accounting for 0.2% of carcinoma from another organs or extension from an all gastric cancers and preferentially affects males. It is esophageal squamous cancer. It is an aggressive dis- predominantly located in the distal stomach. Immuno- ease, associated with poor patient prognosis. histochemistry for p40 may help confirm the presence Adenosquamous carcinoma of the stomach (Fig. 6d) of a morphologically suspected squamous cell com- is a malignant epithelial neoplasm composed of both ponent. Adenosquamous carcinoma is an aggressive squamous and adenocarcinomatous components. neoplasm. The squamous cell component should constitute at Gastric undifferentiated carcinoma has been recent- HISTOPATHOLOGY OF TUMORS OF THE STOMACH 179

ly recognized as a specific histotype of gastric can- fibrovascular cores within empty clefts and is associ- cer. It is an anaplastic carcinoma with no evidence of ated with a poor prognosis 73. In this subtype, epithe- any type of tumor cell differentiation. Four subtypes lial membrane antigen (EMA) and E-cadherin show are described, including i) large cell carcinoma with a distinctive inside-out staining pattern with loss of rhabdoid features, ii) pleomorphic carcinoma, iii) sar- immunoreactivity at the stroma interface. Gastric ade- comatoid carcinoma, and iv) carcinoma with osteo- nocarcinoma of fundic-gland type (chief cell predom- clast-like giant cells. Rhabdoid carcinomas account inant, parietal cell predominant, or mixed phenotype) for about 6% of gastric cancers with a solid architec- account for about 1% of early gastric cancers and has ture. Undifferentiated carcinomas are usually large, been more frequently described in Asia 27. It derives fungating masses, composed of intermediate-to-large from the so-called oxyntic-type adenoma and shows cells, often with pleomorphic elements. Pancytoket- immunoreactivity for pepsinogen I and MUC6, sug- atin is usually expressed by neoplastic cells, while gesting a predominant chief cell differentiation. This vimentin shows a characteristic perinuclear dot-like subtype is rather indolent, with a limited propensity pattern of expression. A subset of such cancers ex- to lymph node dissemination. Other types include pa- hibits loss of SMARCB1 (INI1) or SMARCA4 (BRG1) rietal cell carcinoma and Paneth cell carcinoma and expression 67. Mismatch repair protein deficiency may these are regarded as subtypes of gastric adenocar- be present. Differential diagnoses include carcinomas cinoma according to the 2019 WHO classification 23. with lymphoid stroma (a subtype of adenocarcinoma), lymphomas, sarcomas and melanomas. It is a very aggressive disease, with a dismal prognosis. Hereditary gastric cancer syndromes Carcinoma with lymphoid stroma (Fig. 6a) is also known as medullary carcinoma or lymphoepitheli- Three major hereditary autosomal dominant syn- oma-like carcinoma. It is characterized by irregular dromes affecting the stomach have been described: sheets, trabeculae, poorly developed tubular struc- hereditary diffuse gastric cancer (HDGC), gastric tures and isolated cells, embedded within a promi- adenocarcinoma and proximal polyposis of the stom- nent lymphocytic infiltrate with occasional lymphoid ach (GAPPS) and familial intestinal gastric cancer follicles. The lymphoid infiltrate can be so prominent (FIGC) (Tab. III). Moreover, several other hereditary that immunohistochemical study may be necessary to cancer syndromes are characterized by an increased confirm the epithelial nature of the tumor. It is often risk of gastric cancer, namely classic and attenuated associated with Epstein-Barr virus infection, which FAP, MUTYH-associated polyposis, Peutz-Jeghers may be identified by in situ hybridization, though as a syndrome, juvenile polyposis syndrome, Lynch Syn- similar morphology can be observed in gastric cancer drome, Li-Fraumeni syndrome, hereditary breast and with microsatellite instability 68. It is associated with a ovarian cancer syndrome, and Cowden syndrome 74. better prognosis in comparison with conventional ad- enocarcinoma. Hereditary diffuse gastric cancer (HDGC) Other types: primary gastric hepatoid carcinoma Definition. HDGC is an autosomal dominant cancer (composed by hepatocyte-like cells) (Fig. 6b), ade- syndrome defined by the presence of germline vari- nocarcinoma with enteroblastic differentiation (com- ants in CDH1 or CTNNA1 genes and characterized posed of clear cells arranged in tubulo-papillary by increased risk of diffuse (poorly cohesive) gastric structures) and yolk-sac tumor-like carcinoma share cancer and lobular breast cancer 75. Families fulfilling the immunohistochemical expression of alpha-feto- genetic testing criteria for HDGC (Tab. III) but without protein and should be distinguished from a metastatic CDH1 or CTNNA1 germline variants, should be de- hepatocellular carcinoma or a germ cell neoplasm. Al- fined as “HDGC-like” families 76. pha-fetoprotein and primitive enterocyte differentiation Disease penetrance and clinical features. HDGC pen- biomarkers, such as SALL4, glypican-3 and claudin-6 etrance in proven mutation carriers is incomplete and are expressed in adenocarcinoma with enteroblastic variable between families 77. According to recent esti- differentiation and hepatoid gastric carcinoma 69,70. Bi- mates, the risk of DGC is 42% for males and 33% for omarkers which help to distinguish between primary females while the lifetime risk of lobular breast cancer hepatoid gastric adenocarcinoma from hepatocellular ranges from 42 to 55% 76. The time course from early to carcinoma metastases include SALL4 and claudin-6 advanced HDGC is unpredictable 78 and prophylactic/ expression in hepatoid gastric cancer and loss of risk reduction total gastrectomy in early adulthood is ad- SMARCB1 (INI1) immunoreactivity in hepatocellular vised, regardless of endoscopic findings 76. Indeed, ap- carcinoma 71,72. Micropapillary carcinoma (Fig. 6c), propriate endoscopic surveillance, also with advanced shows small aggregated of neoplastic cells without imaging endoscopy, fails to detect precursor or invasive 180 I. Gullo et al.

Table III. Hereditary syndromes affecting primarily the stomach.

Recommended Histopathological Syndrome Genetic testing criteria genetic testing findings

HDGC Family criteria (first and second relatives): CDH1 Diffuse (poorly cohesive) - At least 2 cases of GC in family regardless of age, with at least one genetic analysis GC and precursor diffuse GC lesions (in situ signet - At least 1 case of diffuse GC any age and ≥1 case of LBC < 70 years CTNNA1 ring cell carcinoma, in different family members mutation analysis pagetoid spread of - At least 2 cases of lobular breast cancer in family members < 50 years signet ring cells) Individual criteria: LBC - Diffuse GC < 50 years - Diffuse GC at any age in individuals of Mãori ethnicity - Diffuse GC at any age in individuals with a personal or family history (1st degree) of cleft lip/cleft palate - History of diffuse GC and lobular breast cancer, both diagnosed < 70 years - Bilateral lobular breast cancer, diagnosed < 70 years - Gastric in situ signet ring cells and/or pagetoid spread of signet ring cells in individuals < 50 years GAPPS Essential criteria: APC promoter 1b FGPs (with dysplasia) - Phenotypic features: proximal polyposis with antral sparing; mutation analysis Hyperplastic polyps no evidence of colorectal or duodenal polyposis; > 100 polyps Hyperproliferative carpeting the proximal stomach in the index patient or > 30 polyps aberrant pits in a first-degree relative of another patient; predominantly FGPs Intestinal and foveolar and/or fundic gland-like polyps adenomas -P roband or relative with either dysplastic FGPs or GC Mixed polyps with FGP- - Mutation in the promoter 1B (YY1 binding motif) of APC gene like, adenomatous and Supportive criteria: hyperplastic features -A utosomal dominant patern of inheritance Intestinal and mixed GC - Spectrum of other histological features, including hyperproliferative aberrant pits, hyperplastic polyps, gastric-type adenomas FIGC IGCLC criteria in high incidence countries: NA Intestinal GC - Intestinal GC in three or more relatives; and - One being a first-degree relative of the other two; and -T wo or more successive generations affected; and - Intestinal GC <50 years in one or more patients; and - Exclusion of gastric polyposis. IGCLC criteria in low incidence countries: - Intestinal GC in two or more first-degree relatives; - Intestinal GC in second-degree relatives, one diagnosed < 50 years - Intestinal GC in three or more relatives at any age. Proposal of new criteria: - GC in two or more relatives at any age; and - At least one intestinal GC FIGC, Familial Intestinal Gastric Cancer; GAPPS, Gastric Adenocarcinoma and Proximal Polyposis of the Stomach; GC, Gastric Cancer; HDGC, Heredi- tary Diffuse Gastric Cancer; HNPCC, Hereditary Non polyposis Colorectal cancer; IGCLC, International Gastric Cancer Linkage Consortium. carcinoma foci in up to 80% of cases 79. At the time of have been recognised exclusively in CDH1 carriers, clinical presentation, almost the totality of affected indi- namely in situ SRCC, corresponding to the presence viduals presents with advanced and incurable disease. of signet ring cell within the basal membrane substi- In women, annual breast magnetic resonance imaging tuting normal epithelial cells, and pagetoid spread of is advised, starting at 30 years of age 76. signet ring cells, corresponding to a row of signet ring Histopathological findings. Histopathological analysis below the preserved epithelium of glands and foveo- of prophylactic (risk-reducing) total gastrectomies re- lae, but still contained within the basal membrane 82. A veals, in the majority of the cases 80, multiple and tiny proportion of intramucosal carcinoma foci from CDH1 (< 0.1 mm to 16 mm) foci of intramucosal (pT1a) sig- carriers progress unpredictably to advanced disease, net ring cell carcinoma 81 (Fig. 5c). Two intraepithelial with diffuse infiltration of the gastric wall, peritoneal precursor lesions (pTis) of signet ring cell carcinoma dissemination and metastases to distant organs. Ad- HISTOPATHOLOGY OF TUMORS OF THE STOMACH 181

vanced HDGC shows the features of poorly cohesive promoter 1B of the APC gene 87. Accordingly, GAPPS (diffuse) gastric cancer and is not distinguishable from is defined as a variant of FAP with an exclusive gastric the sporadic setting, except for the presence of mul- phenotype. Diagnostic criteria for GAPPS are listed tifocal intramucosal foci and precursor lesions in the in Table III. To consider a diagnosis of GAPPS, the mucosa distant from the tumor bulk 76. In contrast to presence of polyposis elsewhere in the gastrointesti- early HDGC, composed of bona fide signet ring cells nal tract should be ruled out. with an “indolent” phenotype, advanced HDGC shows Clinical features. GAPPS penetrance is also incom- pleomorphic, bizarre and diffusely infiltrative neoplas- plete, as proven by the evidence of normal endosco- tic cells with increased proliferation and activation pies in elderly obligate carriers 88. The age of onset of of oncogenic events 83,84. The finding of “aggressive” gastric cancer is variable, ranging from 23 to 75 years. histopathological features in endoscopic biopsy spec- Fundic gland polyposis carpeting the gastric body imens from CDH1 carriers is suggestive of advanced and fundus has been detected as early as 10 years disease and should be reported in the pathology re- of age 88. Recommendations on the management of port to prompt staging and clinical intervention 78. GAPPS should be decided on a case-by-case basis. Immunohistochemical biomarkers. Consistent with Clinical strategies encompass endoscopic surveil- biallelic inactivation of the CDH1 gene and support- lance with biopsies and/or polypectomies and prophy- ing the key role of E-cadherin loss for tumor initiation, lactic/risk-reduction gastrectomy 88. E-cadherin expression is usually abnormal in pre- Histopathological findings. GAPPS is characterized cursor and invasive cancer foci. Diverse E-cadherin by multiple fundic gland polyps carpeting the gastric staining patterns have been described in HDGC, in- body and fundus, some of which show foveolar-type cluding complete loss of expression, reduced mem- dysplasia and by the presence of hyperproliferative branous immunoreactivity and “dotted” or cytoplasmic aberrant pits, corresponding to hyper-proliferative and staining 83. It should be clarified that HDGC may show disorganized oxyntic glands around gastric pits 89. retained E-cadherin immunoreactivity and that E-cad- Other lesions include hyperplastic polyps, intesti- herin staining should not be used as a pre-screening nal-type and foveolar-type adenomas with low- and method to select patients eligible for germline CDH1 high-grade dysplasia, as well as mixed polyps with variant analysis. FGP-like, adenomatous and hyperplastic features 89. Differential diagnosis. The pathology of HDGC is Gastric adenocarcinomas are intestinal-type or unique and diagnostic expertise is needed to provide mixed-type 88. high quality diagnoses, both in biopsies and in resec- Differential diagnosis. Prolonged therapy with pro- tion specimens. Specifically, criteria for the identifica- ton-pump inhibitors could cause the development of tion of signet ring cell lesions should be strictly fol- multiple FGPs and sporadic fundic gland polyposis. lowed in order to diminish the risk of over diagnosing According to the clinical criteria to consider GAPPS nonspecific changes and mimics of signet ring cells, diagnosis (Tab. III), upper endoscopy should be re- such as globoid transformation and vacuolization of peated after discontinuation of therapy and appropri- the superficial epithelium, xanthomatous cells, and ate off-treatment interval 88. artefacts secondary to cell autolysis. Second opinion by an independent pathologist with experience in the Familial intestinal gastric cancer (FIGC) field should always be sought. Definition. Familial intestinal gastric cancer (FIGC) In HDGC patients presenting both lobular breast can- is an autosomal dominant cancer syndrome associ- cer and diffuse gastric cancer, a metastatic tumor ated with an increased risk of intestinal-type gastric should be considered and can be morphologically cancer 90. Diagnostic criteria (Tab. III) differ depending indistinguishable 85. Breast-associated immunomark- upon the incidence of gastric cancer in the population ers are oestrogen receptor, BRST-2 (GCDFP-15) and analysed. The genetic cause underlying the disease mammaglobin, while the expression of CK20 and HN- remains to be fully elucidated, although recent stud- F4A may favour a diagnosis of gastric cancer 86. ies brought up the possibility of a distinctive polygenic cause for the disease 91. Gastric adenocarcinoma and proximal polyposis of the Clinical features. The clinical phenotype of gastric stomach (GAPPS) cancer patients fulfilling the clinical criteria for FIGC Definition. GAPPS is an autosomal dominant cancer has been characterized recently 91. The lifetime risk predisposition syndrome associated with an increased of gastric cancer is 66% for both sexes and the mean risk of gastric cancer, arising in the context of poly- age at diagnosis is 72 years, approximately 10 years posis of the proximal stomach. The genetic cause of earlier than patients with sporadic intestinal-type GAPPS corresponds to germline point variants in the gastric cancers. The disease spectrum is broad, en- 182 I. Gullo et al.

compassing 18 cancer types including colorectal and areas. The Becker system is based on the percent- breast cancer. age of vital tumor tissue with no integrated nodal Histopathological findings. FIGC displays macroscop- evaluation: TRG1 - complete tumor regression (TRG ic and histopathological features that are undistin- 1a: 0% residual tumor) or subtotal tumor regres- guishable from intestinal-type sporadic gastric cancer. sion (TRG 1b: < 10% residual tumor); TRG 2 - par- tial tumor regression (10% to 50% residual tumor); TRG 3 - minimal/no tumor regression per tumor bed Post neo-adjuvant treatment (> 50% residual tumor cells with or without signs of tumor regression grade in gastric tumor regression). Recently, an international group adenocarcinoma of experts, through a Delphi survey, has proposed a 4-tiered system based on the modified Becker grad- Preoperative neo-adjuvant chemotherapy or com- ing system. The novelty of this system is the addition bined radiotherapy and chemotherapy (neo-CRT) has of the evaluation of response in metastatic lymph become the standard approach for locally advanced nodes (complete, partial, or no nodal response) and gastric carcinomas. Pathological tumor regression this seems add strength to the system 95. grading (TRG) systems, which aim to evaluate and quantify the amount of residual tumor and/or regres- sive changes following neo-CRT, should be applied to Conclusions all resections specimens. TRG scoring permits prognostic stratification of tum- The pathology report of gastric resections specimens ors, indeed, complete pathological response is signif- requires a standardized approach as well as an in icantly associated with better outcome – at least in depth knowledge of prognostic and treatment asso- some series – and this classification into prognostic ciated factors. Furthermore, the recognition of hered- classes is the basis for personalized treatment and itary conditions is important and requires cross-talk follow-up strategy. between the pathologist and clinicians.

Problems in TRG assignment References The presence of several validated classification sys- 1 Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics tems for TRG has however led to some confusion as 2018: Globocan estimates of incidence and mortality worldwide to which system should be preferentially applied. The for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394- presence of similar but not “exactly” similar TRG sys- 424. https://doi.org/10.3322/caac.21492 tems may, in part, explain why studies on the prognos- 2 Gullo I, Carneiro F, Oliveira C, et al. Heterogeneity in gastric can- tic impact of response have yielded variable results 92. cer: From pure morphology to molecular classifications. Pathobi- There are many possible reasons which explain the ology 2018;85:50-63. https://doi.org/10.1159/000473881 3 lack of a universally accepted TRG system: 1) ab- Eusebi LH, Telese A, Marasco G, et al. Gastric cancer preven- tion strategies: A global perspective. J Gastroenterol Hepatol sence of standardized different sampling methods 2020. Online ahead of print. https://doi.org/10.1111/jgh.15037 which could lead to over-diagnosis of complete patho- 4 Fukayama M, Abe H, Kunita A, et al. Thirty years of epstein- logical tumor regression and this may in part explain barr virus-associated gastric carcinoma. Virchows Arch its variable prognostic impact. Indeed, the complete 2020;476;353-5. https://doi.org/10.1007/s00428-019-02724-4 microscopic assessment of the entire ulcerated/scar- 5 Sanduleanu S, Jonkers D, De Bruine A, et al. Non-helicobacter ring area should be performed and this is absolutely pylori bacterial flora during acid-suppressive therapy: differen- mandatory if no tumor is identified in the initial blocks; tial findings in gastric juice and gastric mucosa. Aliment Phar- macol Ther 2001;15;379-88. https://doi.org/10.1046/j.1365- 2) not all classifications take into account the eval- 2036.2001.00888.x uation of response in loco-regional lymph nodes; 3) 6 Spoto CPE, Gullo I, Carneiro F, et al. Hereditary gastrointestinal there is a relatively low concordance rate among pa- carcinomas and their precursors: An algorithm for genetic testing. thologists in TRG assignment; 4) systems with a high- Semin Diagn Pathol 2018;35;170-83. https://doi.org/10.1053/j. er number of tiers (more than 4) do not offer any clear semdp.2018.01.004 cut prognostic advantage 93. 7 van der Post RS, Carneiro F. Emerging concepts in gastric neoplasia: Heritable gastric cancers and polyposis disorders. Surg Pathol Clin 2017;10;931-45. https://doi.org/10.1016/j. TRG systems for Gastric Cancer path.2017.07.011 Gastric cancer specific TRG systems have been pro- 8 Correa P. Human gastric carcinogenesis: a multistep and multifac- 94 posed starting from the 2003 Becker system which torial process--first american cancer society award lecture on can- requires the histologic assessment of the entire mac- cer epidemiology and prevention. Cancer Res 1992;52;6735-40. roscopically identifiable residual tumor or the fibrous 9 Plummer M, Franceschi S, Vignat J, et al. Global burden of HISTOPATHOLOGY OF TUMORS OF THE STOMACH 183

gastric cancer attributable to helicobacter pylori. Int J Cancer 25 Hackeng WM, Montgomery EA, Giardiello FM, et al. Morphol- 2015;136;487-90. https://doi.org/10.1002/ijc.28999 ogy and genetics of pyloric gland adenomas in familial adeno- 10 Polk DB, Peek RM Jr. Helicobacter pylori: gastric cancer and be- matous polyposis. Histopathology 2017;70:549-57. https://doi. yond. Nat Rev Cancer 2010;10;403-14. https://doi.org/10.1038/ org/10.1111/his.13105 nrc2857 26 Brosens LA, Wood LD, Offerhaus GJ, et al. Pathology and genet- 11 Ferreira RM, Machado JC, Figueiredo C. Clinical relevance of ics of syndromic gastric polyps. Int J Surg Pathol 2016;24;185- helicobacter pylori vaca and caga genotypes in gastric carcino- 99. https://doi.org/10.1177/1066896915620013 ma. Best Pract Res Clin Gastroenterol 2014;28;1003-15. https:// 27 Benedict MA, Lauwers GY, Jain D. Gastric adenocarcinoma of doi.org/10.1016/j.bpg.2014.09.004 the fundic gland type: Update and literature review. Am J Clin 12 Persson C, Canedo P, Machado JC, et al. Polymorphisms in in- Pathol 2018;149;461-73. https://doi.org/10.1093/ajcp/aqy019 flammatory response genes and their association with gastric 28 Ahn JY, Son DH, Choi KD, et al. Neoplasms arising in large gas- cancer: a huge systematic review and meta-analyses. Am J Epi- tric hyperplastic polyps: Endoscopic and pathologic features. demiol 2011;173;259-70. https://doi.org/10.1093/aje/kwq370 Gastrointest Endosc 2014;80;1005-1013.e1002. https://doi. 13 Mahmud N, Stashek K, Katona BW, et al. The incidence of neo- org/10.1016/j.gie.2014.04.020 plasia in patients with autoimmune metaplastic atrophic gastritis: 29 Takayama Y, Ono Y, Mizukami Y, et al. Comparative genome- a renewed call for surveillance. Ann Gastroenterol 2019;32;67- wide analysis of gastric adenocarcinomas with hyperplastic 72. https://doi.org/10.20524/aog.2018.0325 polyp components. Virchows Arch 2019;475;383-9. https://doi. 14 Sipponen P, Price AB. The Sydney system for classification of org/10.1007/s00428-019-02592-y gastritis 20 years ago. J Gastroenterol Hepatol 2011;26 Suppl 30 Carmack SW, Genta RM, Schuler CM, et al. The current spec- 1;31-4. https://doi.org/10.1111/j.1440-1746.2010.06536.x trum of gastric polyps: A 1-year national study of over 120,000 15 Meining A, Bayerdorffer E, Muller P, et al. Gastric carcinoma risk patients. Am J Gastroenterol 2009;104;1524-32. https://doi. index in patients infected with helicobacter pylori. Virchows Arch org/10.1038/ajg.2009.139 1998;432;311-4. https://doi.org/10.1007/s004280050171 31 Abraham SC, Nobukawa B, Giardiello FM, et al. Fundic gland 16 Rugge M, Meggio A, Pennelli G, et al. Gastritis staging in clinical polyps in familial adenomatous polyposis: neoplasms with fre- practice: the olga staging system. Gut 2007;56;631-6. https://doi. quent somatic adenomatous polyposis coli gene alterations. org/gut.2006.106666 [pii]10.1136/gut.2006.106666 Am J Pathol 2000;157;747-54. https://doi.org/10.1016/s0002- 9440(10)64588-9 17 Pimentel-Nunes P, Libânio D, Marcos-Pinto R, et al. Manage- 32 ment of epithelial precancerous conditions and lesions in the Hamashima C. Cancer screening guidelines and policy making: stomach (maps ii): European society of gastrointestinal en- 15 years of experience in cancer screening guideline develop- doscopy (esge), european helicobacter and microbiota study ment in japan. Jpn J Clin Oncol 2018;48;278-86. https://doi. group (ehmsg), european society of pathology (esp), and so- org/10.1093/jjco/hyx190 ciedade portuguesa de endoscopia digestiva (sped) guide- 33 Teh JL, Shabbir A, Yuen S, et al. Recent advances in diagnostic line update 2019. Endoscopy 2019;51;365-88. https://doi. upper endoscopy. World J Gastroenterol 2020;26;433-47. https:// org/10.1055/a-0859-1883 doi.org/10.3748/wjg.v26.i4.433 18 Spechler SJ, Merchant JL, Wang TC, et al. A summary of the 34 Tsukuma H, Mishima T, Oshima A. Prospective study of “early” 2016 James W. Freston conference of the american gastroen- gastric cancer. Int J Cancer 1983;31;421-26. terological association: Intestinal metaplasia in the esophagus 35 Pimentel-Nunes P, Dinis-Ribeiro M, Ponchon T, et al. Endoscop- and stomach: origins, differences, similarities and significance. ic submucosal dissection: European society of gastrointestinal Gastroenterology 2017;153;e6-e13. https://doi.org/10.1053/j. endoscopy (esge) guideline. Endoscopy 2015;47;829-54. https:// gastro.2017.05.050 doi.org/10.1055/s-0034-1392882 19 Reis CA, David L, Correa P, et al. Intestinal metaplasia of human 36 Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in stomach displays distinct patterns of mucin (muc1, muc2, mu- combination with chemotherapy versus chemotherapy alone for c5ac, and muc6) expression. Cancer Res 1999;59;1003-7. treatment of her2-positive advanced gastric or gastro-oesopha- 20 Leung WK, Lin SR, Ching JY, et al. Factors predicting progres- geal junction cancer (toga): a phase 3, open-label, randomised sion of gastric intestinal metaplasia: Results of a randomised controlled trial. Lancet 2010;376;687-97. https://doi.org/10.1016/ trial on helicobacter pylori eradication. Gut 2004;53;1244-9. s0140-6736(10)61121-x https://doi.org/10.1136/gut.2003.034629 37 Fuchs CS, Tabernero J, Tomasek J, et al. Biomarker analyses in 21 Goldenring JR, Nam KT, Wang TC, et al. Spasmolytic polypep- regard gastric/gej carcinoma patients treated with vegfr2-target- tide-expressing metaplasia and intestinal metaplasia: time for ed antibody ramucirumab. Br J Cancer 2016;115;974-82. https:// reevaluation of metaplasias and the origins of gastric cancer. doi.org/10.1038/bjc.2016.293 Gastroenterology 2010;138;2207-2210, 2210.e2201. https://doi. 38 Smyth EC, Verheij M, Allum W, et al. Gastric cancer: esmo clini- org/10.1053/j.gastro.2010.04.023 cal practice guidelines for diagnosis, treatment and follow-up. 22 Valente P, Garrido M, Gullo I, et al. Epithelial dysplasia of the Ann Oncol 2016;27;v38-v49. https://doi.org/10.1093/annonc/ stomach with gastric immunophenotype shows features of bio- mdw350 logical aggressiveness. Gastric Cancer 2015;18;720-8. https:// 39 Grillo F, Fassan M, Sarocchi F, et al. HER2 heterogeneity in doi.org/10.1007/s10120-014-0416-5 gastric/gastroesophageal cancers: from benchside to practice. 23 WHO Classification of tumors. Digestive system tumors. Tumors World J Gastroenterol 2016;22:5879-87. https://doi.org/10.3748/ of the stomach. WHO Classification of Tumors Editorial Board. wjg.v22.i26.5879 th 5 Ed. Lyon: IARC press 2019, pp. 59-110. 40 Grillo F, Fassan M, Ceccaroli C, et al. The reliability of endo- 24 Kim A, Ahn SJ, Park DY, et al. Gastric crypt dysplasia: a distinct scopic biopsies in assessing HER2 status in gastric and gas- subtype of gastric dysplasia with characteristic endoscopic fea- troesophageal junction cancer: a study comparing biopsies tures and immunophenotypic and biological anomalies. Histopa- with surgical samples. Transl Oncol 2013;6:10-6. https://doi. thology 2016;68;843-9. https://doi.org/10.1111/his.12860 org/10.1593/tlo.12334 184 I. Gullo et al.

41 Gullo I, Grillo F, Molinaro L, et al. Minimum biopsy set for and 5-fluorouracil. Gastroenterology 2013;145;554-65. https:// HER2 evaluation in gastric and gastro-esophageal junc- doi.org/10.1053/j.gastro.2013.05.010 tion cancer. Endosc Int Open 2015;3:E165-70. https://doi. 58 TCGA. Comprehensive molecular characterization of gastric ad- org/10.1055/s-0034-1391359 enocarcinoma. Nature 2014;513;202-9. https://doi.org/10.1038/ 42 Le DT, Durham JN, Smith KN, et al. Mismatch repair deficiency nature13480 predicts response of solid tumors to pd-1 blockade. Science 59 Cristescu R, Lee J, Nebozhyn M, et al. Molecular analysis of 2017;357;409-13. https://doi.org/10.1126/science.aan6733 gastric cancer identifies subtypes associated with distinct clini- 43 Sohn BH, Hwang JE, Jang HJ, et al. Clinical significance of cal outcomes. Nat Med 2015;21;449-56. https://doi.org/10.1038/ four molecular subtypes of gastric cancer identified by the can- nm.3850 cer genome atlas project. Clin Cancer Res 2017. https://doi. 60 Song HJ, Kim KM. Pathology of epstein-barr virus-associated org/10.1158/1078-0432.ccr-16-2211 gastric carcinoma and its relationship to prognosis. Gut Liver 44 Kulangara K, Zhang N, Corigliano E, et al. Clinical utility of the 2011;5;143-8. https://doi.org/10.5009/gnl.2011.5.2.143 combined positive score for programmed death ligand-1 expres- 61 Chiaravalli AM, Cornaggia M, Furlan D, et al. The role of his- sion and the approval of pembrolizumab for treatment of gas- tological investigation in prognostic evaluation of advanced tric cancer. Arch Pathol Lab Med 2019;143:330-7. https://doi. gastric cancer. Analysis of histological structure and molecular org/10.5858/arpa.2018-0043-OA changes compared with invasive pattern and stage. Virchows 45 Topalian SL, Taube JM, Anders RA, et al. Mechanism-driven Arch 2001;439;158-69. https://doi.org/10.1007/s004280100441 biomarkers to guide immune checkpoint blockade in cancer 62 Gullo I, Carvalho J, Martins D, et al. The transcriptomic land- therapy. Nat Rev Cancer 2016;16;275-87. https://doi.org/10.1038/ scape of gastric cancer: Insights into epstein-barr virus infected nrc.2016.36 and microsatellite unstable tumors. Int J Mol Sci 2018;19. https:// 46 Sano T, Coit DG, Kim HH, et al. Proposal of a new stage group- doi.org/10.3390/ijms19072079 ing of gastric cancer for tnm classification: International gastric 63 Setia N, Agoston AT, Han HS, et al. A protein and mrna ex- cancer association staging project. Gastric Cancer 2017;20;217- pression-based classification of gastric cancer. Mod Pathol 25. https://doi.org/10.1007/s10120-016-0601-9 2016;29;772-84. https://doi.org/10.1038/modpathol.2016.55 47 Solcia E, Klersy C, Mastracci L, et al. A combined histologic and 64 Ahn S, Lee SJ, Kim Y, et al. High-throughput protein and mrna molecular approach identifies three groups of gastric cancer expression-based classification of gastric cancers can identify with different prognosis. Virchows Arch 2009;455:197-211. https:// clinically distinct subtypes, concordant with recent molecular doi.org/10.1007/s00428-009-0813-z classifications. Am J Surg Pathol 2017;41;106-15. https://doi. 48 Lauren P. The two histological main types of gastric carcinoma: org/10.1097/pas.0000000000000756 diffuse and so-called intestinal-type carcinoma. An attempt 65 Machicado J, Shroff J, Quesada A, et al. Gastritis cystica profun- at a histo-clinical classification. Acta Pathol Microbiol Scand da: endoscopic ultrasound findings and review of the literature. 1965;64;31-49. Endosc Ultrasound 2014;3;131-4. https://doi.org/10.4103/2303- 49 Zheng HC, Li XH, Hara T, et al. Mixed-type gastric carcinomas 9027.131041 exhibit more aggressive features and indicate the histogen- 66 Choi MG, Jeong JY, Kim KM, et al. Clinical significance of esis of carcinomas. Virchows Arch 2008;452;525-34. https://doi. gastritis cystica profunda and its association with epstein-barr org/10.1007/s00428-007-0572-7 virus in gastric cancer. Cancer 2012;118;5227-33. https://doi. 50 Carneiro F. Classification of gastric carcinomas. Current Diag- org/10.1002/cncr.27541 nostic Pathology 1997;4;51-9. 67 67 Agaimy A, Rau TT, Hartmann A, et al. SMARCB1 (INI1)- 51 Kwon CH, Kim YK, Lee S, et al. Gastric poorly cohesive carcino- negative rhabdoid carcinomas of the gastrointestinal tract: clini- ma: a correlative study of mutational signatures and prognostic copathologic and molecular study of a highly aggressive variant significance based on histopathological subtypes. Histopathol- with literature review. Am J Surg Pathol 2014;38:910-20. https:// ogy 2018;72;556-68. https://doi.org/10.1111/his.13383 doi.org/10.1097/PAS.0000000000000173 52 Japanese gastric cancer association. Japanese classification of 68 Gullo I, Oliveira P, Athelogou M, et al. New insights into the gastric carcinoma. The 15th edition. 2017. Isbn: 978-4-307-20375-3. inflamed tumor immune microenvironment of gastric cancer 53 Saragoni L. Upgrading the definition of early gastric can- with lymphoid stroma: from morphology and digital analysis cer: better staging means more appropriate treatment. to gene expression. Gastric Cancer 2019;22;77-90. https://doi. Cancer Biol Med 2015;12:355-61. https://doi.org/10.7497/j. org/10.1007/s10120-018-0836-8 issn.2095-3941.2015.0054 69 Yamazawa S, Ushiku T, Shinozaki-Ushiku A, et al. Gastric cancer 54 Saragoni L, Scarpi E, Ravaioli A, et al. Early gastric cancer: with primitive enterocyte phenotype: An aggressive subgroup of clinical behavior and treatment options. Results of an Italian intestinal-type adenocarcinoma. Am J Surg Pathol 2017;41;989- Multicenter Study on behalf of the Italian Gastric Cancer Re- 97. https://doi.org/10.1097/pas.0000000000000869 search Group (GIRCG). Oncologist 2018;23:852-8. https://doi. 70 Ushiku T, Uozaki H, Shinozaki A, et al. Glypican 3-express- org/10.1634/theoncologist.2017-0488 ing gastric carcinoma: distinct subgroup unifying hepatoid, 55 Kodama Y, Inokuchi K, Soejima K, et al. Growth patterns and clear-cell, and alpha-fetoprotein-producing gastric carcinomas. prognosis in early gastric cancer. Superficially spreading and Cancer Sci 2009;100;626-32. https://doi.org/10.1111/j.1349- penetrating growth types. Cancer 1983;51:320-6. 7006.2009.01108.x 56 Saragoni L, Morgagni P, Gardni A et el. Early gastric cancer: 71 Ushiku T, Shinozaki A, Shibahara J, et al. Sall4 represents fetal diagnosis, staging, and clinical impact. Evaluation of 530 pa- gut differentiation of gastric cancer, and is diagnostically use- tients. New elements for an updated definition and classification. ful in distinguishing hepatoid gastric carcinoma from hepatocel- Gastric Cancer 2013;16:549-54. https://doi.org/10.1007/s10120- lular carcinoma. Am J Surg Pathol 2010;34;533-40. https://doi. 013-0233-2 org/10.1097/PAS.0b013e3181d1dcdd 57 Lei Z, Tan IB, Das K, et al. Identification of molecular subtypes 72 Mochizuki K, Kawai M, Odate T, et al. Smarcb1/ini1 is diag- of gastric cancer with different responses to pi3-kinase inhibitors nostically useful in distinguishing α-fetoprotein-producing gas- HISTOPATHOLOGY OF TUMORS OF THE STOMACH 185

tric carcinoma from hepatocellular carcinoma. Anticancer Res 84 Lee HE, Smyrk TC, Zhang L. Histologic and immunohistochemi- 2018;38;6865-8. https://doi.org/10.21873/anticanres.13061 cal differences between hereditary and sporadic diffuse gastric 73 Roh JH, Srivastava A, Lauwers GY et al. Micropapillary carci- carcinoma. Hum Pathol 2018;74;64-72. https://doi.org/10.1016/j. noma of stomach: a clinicopathologic and immunohistochemical humpath.2017.12.023 study of 11 cases. Am J Surg Pathol 2010;34:1139-46. https://doi. 85 Mahmud N, Ford JM, Longacre TA, et al. Metastatic lobular org/10.1097/PAS.0b013e3181e7043b breast carcinoma mimicking primary signet ring adenocarci- 74 Oliveira C, Pinheiro H, Figueiredo J, et al. Familial gastric can- noma in a patient with a suspected cdh1 mutation. J Clin Oncol cer: genetic susceptibility, pathology, and implications for man- 2015;33;e19-21. https://doi.org/10.1200/jco.2013.49.1159 agement. Lancet Oncol 2015;16;e60-70. https://doi.org/10.1016/ 86 van der Post RS, Bult P, Vogelaar IP, et al. Hnf4a immunohisto- s1470-2045(14)71016-2 chemistry facilitates distinction between primary and metastatic 75 Van der Post RS, Vogelaar IP, Carneiro F, et al. Hereditary diffuse breast and gastric carcinoma. Virchows Arch 2014;464;673-9. gastric cancer: updated clinical guidelines with an emphasis on https://doi.org/10.1007/s00428-014-1574-x germline cdh1 mutation carriers. J Med Genet 2015;52:361-74. 87 Li J, Woods SL, Healey S, et al. Point mutations in exon 1b https://doi.org/10.1136/jmedgenet-2015-103094 of apc reveal gastric adenocarcinoma and proximal polypo- 76 Blair VR MM, Carneiro F, Coit DG, et al. Hereditary diffuse sis of the stomach as a familial adenomatous polyposis vari- gastric cancer: updated clinical practice guidelines. Lancet ant. Am J Hum Genet 2016;98;830-42. https://doi.org/10.1016/j. Oncol 2020;21:e386-e97. https://doi.org/10.1016/S1470- ajhg.2016.03.001 2045(20)30219-9 88 Worthley DL, Phillips KD, Wayte N, et al. Gastric adenocarci- 77 Roberts ME, Ranola JMO, Marshall ML, et al. Comparison of noma and proximal polyposis of the stomach (gapps): a new cdh1 penetrance estimates in clinically ascertained families vs autosomal dominant syndrome. Gut 2012;61;774-9. https://doi. families ascertained for multiple gastric cancers. JAMA Oncol org/10.1136/gutjnl-2011-300348 2019;5;1325-31. https://doi.org/10.1001/jamaoncol.2019.1208 89 de Boer WB, Ee H, Kumarasinghe MP. Neoplastic lesions of gas- 78 Gullo I, Devezas V, Baptista M, et al. Phenotypic heterogeneity tric adenocarcinoma and proximal polyposis syndrome (gapps) of hereditary diffuse gastric cancer: report of a family with early- are gastric phenotype. Am J Surg Pathol 2018;42;1-8. https://doi. onset disease. Gastrointest Endosc 2018;87;1566-75. https:// org/10.1097/pas.0000000000000924 doi.org/10.1016/j.gie.2018.02.008 90 Caldas C, Carneiro F, Lynch HT, et al. Familial gastric can- 79 Mi EZ, Mi EZ, di Pietro M, et al. Comparative study of endo- cer: overview and guidelines for management. J Med Genet scopic surveillance in hereditary diffuse gastric cancer accord- 1999;36;873-80. ing to cdh1 mutation status. Gastrointest Endosc 2017. https:// 91 Carvalho J, Oliveira P, Senz J, et al. Redefinition of familial in- doi.org/10.1016/j.gie.2017.06.028 testinal gastric cancer: Clinical and genetic perspectives. J Med 80 Rocha JP, Gullo I, Wen X, et al. Pathological features of total Genet 2020. https://doi.org/10.1136/jmedgenet-2019-106346 gastrectomy specimens from asymptomatic hereditary diffuse 92 Zhu Y, Sun Y, Hu S, et al. Comparison of five tumor regression gastric cancer patients and implications for clinical manage- grading systems for gastric adenocarcinoma after neoadjuvant ment. Histopathology 2018;73;878-86. https://doi.org/10.1111/ chemotherapy: a retrospective study of 192 cases from National his.13715 Cancer Center in China. BMC Gastroenterol 2017;17:41. https:// 81 Huntsman DG, Carneiro F, Lewis FR, et al. Early gastric cancer doi.org/10.1186/s12876-017-0598-5 in young, asymptomatic carriers of germ-line e-cadherin muta- 93 Fanelli GN, Loupakis F, Smyth E, et al. Pathological tumor re- tions. N Engl J Med 2001;344;1904-9. https://doi.org/10.1056/ gression grade classifications in gastrointestinal cancers: role nejm200106213442504 on patients’ prognosis. Int J Surg Pathol. 2019;27:816-35. https:// 82 Carneiro F, Huntsman DG, Smyrk TC, et al. Model of the early doi.org/10.1177/1066896919869477 development of diffuse gastric cancer in e-cadherin muta- 94 Becker K, Mueller JD, Schulmacher C, et al. Histomorphology tion carriers and its implications for patient screening. J Pathol and grading of regression in gastric carcinoma treated with neo- 2004;203;681-7. https://doi.org/10.1002/path.1564 adjuvant chemotherapy. Cancer 2003;98:1521-30. 83 van der Post RS, Gullo I, Oliveira C, et al. Histopathological, mo- 95 Tsekrekos A, Detlefsen S, Riddell R, et al. Histopathologic tumor lecular, and genetic profile of hereditary diffuse gastric cancer: cur- regression grading in patients with gastric carcinoma submitted rent knowledge and challenges for the future. Adv Exp Med Biol to neoadjuvant treatment: results of a Delphi survey. Hum Pathol 2016;908;371-91. https://doi.org/10.1007/978-3-319-41388-4_18 2019;84:26-34. https://doi.org/0.1016/j.humpath.2018.08.028 PATHOLOGICA 2020;112:186-196; DOI: 10.32074/1591-951X-157

Review

Celiac disease: histology-differential diagnosis-complications. A practical approach

Vincenzo Villanacci1, Alessandro Vanoli2,3, Giuseppe Leoncini4, Giovanni Arpa2,3, Tiziana Salviato5, Luca Reggiani Bonetti5, Carla Baronchelli1, Luca Saragoni6, Paola Parente7 1 Institute of Pathology, Spedali Civili di Brescia, Italy; 2 Anatomic Pathology Unit, Department of Molecular Medicine, University of Pavia, Italy, 3 Anatomic Pathology Unit, IRCCS San Matteo Hospital of Pavia, Italy; 4 Pathology Unit, ASST del Garda, Desenzano del Garda, BS, Italy; 5 Department of Diagnostic, Clinic and Public Health Medicine, University of Modena and Reggio Emilia, Modena, Italy; 6 Department of Pathological Anatomy, AUSL Romagna, Morgagni-Pierantoni Hospital, Forlì, Italy; 7 Pathology Unit, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, FG, Italy

Summary Celiac disease is a multi-factorial chronic inflammatory intestinal disease, characterized by malabsorption resulting from mucosal injury after ingestion of wheat gluten or related rye and barley proteins. Inappropriate T-cell-mediated immune response against ingested gluten in genetically predisposed people, leads to characteristic histological lesions, as Received and accepted: June 24, 2020 villous atrophy and intraepithelial lymphocytosis. Nevertheless, celiac disease is a compre- Published online: 29 October, 2020 hensive diagnosis with clinical, serological and genetic characteristics integrated with his- tological features. Biopsy of duodenal mucosa remains the gold standard in the diagnosis Correspondence of celiac disease with the recognition of the spectrum of histological changes and classi- Paola Parente fication of mucosa damage based on updated Corazza-Villanacci system. Appropriate dif- Pathology Unit, Fondazione IRCCS Casa ferential diagnosis evaluation and clinical context also for the diagnosis of complications is, Sollievo della Sofferenza, viale Cappuccini 1, moreover, needed for correct histological features interpretation and clinical management. 71013 San Giovanni Rotondo, FG, Italy Tel. +39 0882 410927 Key words: celiac disease, sprue, small bowel, gluten. Fax: +39 0882 410411 E-mail: [email protected]

https://orcid.org/0000-0003-0591-6723 Introduction Conflict of interest The Authors declare no conflict of interest. Celiac disease (CD) is an immune-mediated inflammatory disorder of the small intestine occurring in genetically predisposed individuals when 1 How to cite this article: Villanacci V, Vanoli exposed to gluten . CD can occur at any age, from early childhood to A, Leoncini G, et al. Celiac disease: histology- elderly, with two peaks of onset, one shortly after weaning with gluten in differential diagnosis-complications. A practical the first 2 years of life and the other during the second or third decade approach. Pathologica 2020;112:186-196. of life with a preference for females (male/female ratio 1:2). The disease https://doi.org/10.32074/1591-951X-157 has a variable incidence, with a worldwide prevalence of about 1:100; in Europe is estimated between 0.3 and 1.2% 2,3. A correct diagnosis © Copyright by Società Italiana di Anatomia Pato- of CD requires a precise reconstruction of a puzzle, whose pieces are logica e Citopatologia Diagnostica, Divisione Itali- ana della International Academy of Pathology represented by the clinical, serological, genetic and histological aspects. The evaluation of all these factors, apart from genetics, must take place OPEN ACCESS while the patient is still on a diet containing gluten, since a gluten-free diet changes the clinical, serological and histological pattern, making it This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- impossible to recognize the characteristic aspects of disease. Nonethe- NonCommercial-NoDerivatives 4.0 International) license: the less, CD still represents an under-recognized condition, due to heteroge- work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original neous symptoms and/or poor disease awareness, and the occurrence of version. For further information: https://creativecommons. diagnostic delay ranging from 4 to 13 years has been reported by some org/licenses/by-nc-nd/4.0/deed.en authors 4-9. HISTOPATHOLOGICAL DIAGNOSIS OF CELIAC DISEASE 187

Clinical and laboratory aspects The main clinical significance of genetic testing is to exclude a diagnosis of CD in the absence of HLA-DQ2 The diagnosis of CD can be very challenging, since (and its fractions) and -DQ8 in cases of diagnostic symptoms can significantly vary from patient to patient doubt and predisposition to CD in family members of and this variability has been compared, not surpris- coeliac patients in the absence of HLADQ2 (and frac- ingly, to a chameleon 10. In 2011, the Oslo Classifica- tions) and -DQ8 14,15. tion ranked the clinical presentation of CD in classical, non classical, subclinical and refractory 11. The gold standard for CD diagnosis is represented by the com- Approach to duodenal biopsy bination of both mucosal changes and positivity of se- rological tests 12,13. The biopsies that the pathologist receives nowadays are all performed by endoscopic examination, which, in addition to the duodenum, makes it possible to ex- Serologic markers plore other districts of the gastro-intestinal tract. The following are some points which require a close work- A major role in the diagnostic process of CD is played ing relationship between the endoscopist, the endos- by serology, which allows identification of the subjects copy-room nurse, the pathology laboratory technician who should undergo intestinal biopsy; the following are and the pathologist 16. the most important tests and their relative significance: • IgA class antitransglutaminase antibodies (tTGA) are the tests with the highest sensitivity Site of the biopsy for CD (98%) with specificity estimated at around 90%. High titles of IgA class tTGA (> 5 times the Biopsy by endoscopy should be performed in the cut-off) are almost always the expression of CD; bulb (in particular this site in children) and second • IgA class antiendomysial antibodies (EMA), duodenal portion; the recommendation in this field this test has a lower sensitivity compared to IgA are at least 4 biopsies, 2 for each of sites mentioned class tTGA (90% vs. 98%), but shows an almost above 17-19. absolute specificity for CD; • IgA class antigliadin antibodies (AGA) are now an obsolete test with levels of sensitivity and spec- Orientation of the biopsy sample ificity significantly lower than tTGA and EMA, and the search for their presence is useful only in early Biopsy orientation is a crucial event for a proper his- childhood (children aged < 2 years); since they are tological assessment. We suggest the use of acetate the first antibodies to appear, they show a high- cellulose filters previously cut and in this setting it is er sensitivity than other tests in this age group. fundamental a strict relationship between endosco- Regard to the IgG class of antibodies, their use pist-nurse-technicians and pathologist. The endos- should be restricted to patients with selective IgA copist can place each mucosal sample in a straight deficiency, because only in this subgroup of pa- line and with proximal-to-distal orientation onto the tients the response is indicative for CD. cellulose acetate filter with a “clarinet beak-shaped cut” (Bio-Optica). Such a filter allows a perfect ad- herence of biopsies and, being chemically inert and Genetic testing not reactive with fixative or processing chemicals, it does not alter the quality of histological sections at CD is closely associated with histocompatibility anti- all. As the embedding phase starts, the filters are 90 gens (HLA) DQ2 and DQ8. Practically all CD patients degrees-rotated by technician, in order to ensure the are positive for one or both of these HLAs or for a best trans-sectional cut. The above described proce- fraction of the heterodimer, but genetic testing is nev- dure is time- and money-sparing, allowing a signifi- er diagnostically significant since at least 30% of the cant improvement of diagnostic accuracy. general population present the same HLAs as coeliac patients. The genetic test should be performed in cases where Stains there is a discrepancy between serology and histol- ogy and in 1st degree relatives to assess the genetic The “old mistress” haematoxylin-eosin is sufficient predisposition to CD. to assess all the necessary morphological elements 188 V. Villanacci et al.

(one or two sections can be used, if needed, for im- histopathology of CD is subdivided into different diag- munohistochemical assessment, generally for CD3 nostic categories according to the Marsh classifica- immunostain which is useful for a correct count of T tion 23. lymphocytes) 20. Type 1 or infiltrative lesion 1 Villi within normal morphological limits (normal vil- Histopathological aspects of normal and lous/crypt ratio 3:1); pathological duodenal mucosa 2 increased number of IEL (greater than 25/100 ep- ithelial cells). Normal intestinal mucosa Type 2 or hyperplastic lesion Villi: digitiform appearance with the ratio between the height of the villi and of the crypts always in favor of 1 Villi architecturally within normal morphological the villus (3:1 or more). limits (like type 1); Intra-epithelial lymphocytic infiltrate: the number 2 increased number of IEL (greater than 25/100 epi- of intraepithelial lymphocytes (T lymphocytes; IEL) is thelial cells) (like type 1); subject to individual variability. The majority of normal 3 hyperplasia of the glandular elements (regenera- subjects have less than 25 lymphocytes per 100 ep- tive aspects highlighted by the reduced mucinous ithelial cells; based on the experiences of Hayat and activity and increased number of mitoses). Veress 21,22 a count of IEL over 25/100 epithelial cells Type 3 or destructive lesion is considered pathological. The intraepithelial lym- phocyte count is very important and should always be 1 Varying degrees of villous atrophy associated with done, especially in the initial lesions, using anti-CD3 hyperplasia of glandular crypts; antibodies. 2 surface enterocytes with reduced height, irregular Glandular crypts: the crypts are comprehensive brush border and sometimes cytoplasmic vacu- of epithelial cells, endocrine cells, goblet cells and oles; Paneth cells; mitosis are in general 1 for any crypt. 3 increased number of IEL (like type 1 and 2 lesions). Lamina propria: plasma cells, eosinophils, histio- A combination of the three factors described above cytes, mast cells and lymphocytes are normally found with adeguate clinical informations (i.e. anamnesis in the lamina propria. Neutrophils are generally ab- and serological/genetic data) is consistent with a CD sent, except in cases of active duodenitis with possi- diagnosis. This classification is universally recognized ble gastric metaplasia closely related to Helicobacter for the diagnosis of CD, and extensively validated; the pylori infection; eosinophils must never be more than only point worthy of observation and critical analysis 5 per field at 40× 20. is that mild, moderate or severe atrophy (total villous flattening) are all grouped together in a single catego- Pathological intestinal mucosa ry: the type 3 lesion. The histological diagnosis of CD consists of an in- A modification to this classification has been proposed tegrated assessment of the following elementary le- by Oberhuber et al. 24 who divided the Marsh type 3 sions: lesion into three subgroups: • Increased intraepithelial T-lymphocytes: a value 3a mild villous atrophy and pathological increase of IEL; of 25 T-lymphocytes/100 enterocytes is considered 3b moderate villous atrophy and pathological increase a pathological condition also called “lymphocyto- of IEL; sis”. 3c total villous atrophy and pathological increase of • Crypt hyperplasia: extension of the regenerative IEL. epithelial crypts associated with presence of more Along the same lines, and in an attempt to simplify than 1 mitosis per crypt. and standardize the work of pathologists and facilitate • Villous atrophy: decrease in villous height, alter- the relationship between pathologists and clinicians, ation of normal crypt/villous ratio (3:1) until total a new version of the histological classification has re- disappearance of villi. This assessment requires cently been proposed by Corazza and Villanacci 25,26; proper orientation of the biopsies. in particular, the lesions that characterize CD have None of these elementary lesions is specific for CD; been divided into two categories: non-atrophic (gra- the diagnosis of CD is based on the identification of de A) and atrophic (grade B). histological alterations accompanied by clinical and Grade A lesions are characterized by normal villi but serological consistent data. On the basis of the pres- with a pathological increase in IEL. ence of one or more of these elementary lesions the Grade B lesions are further subdivided into: HISTOPATHOLOGICAL DIAGNOSIS OF CELIAC DISEASE 189

Table I. Comparison of Marsh - Corazza-Villanacci - Villanacci classification schemes. Marsh mod. Oberhuber Corazza-Villanacci Villanacci Lesions Diagnostic Criteria Lesions Lesions Type I lesion No architectural changes (villous/cript Grade A lesion A infiltrative ratio preserved) not atrophic Non atrophic type Increased IELs count (> 25/100 No architectural changes (villous/ No architectural changes (villous/cript epithelial cells) cript ratio preserved) ratio preserved) Type II lesion No architectural changes (villous/cript Increased IELs count (> 25/100 Increased IELs count (> 25/100 hyperplastic ratio preserved) epithelial cells) epithelial cells) Crypt hyperplasia (mitoses > 1/crypt) Increased IELs count (> 25/100 epithelial cells) Type III A lesion Villous atrophy (mild degree) Grade B1 lesion B destructive Crypt hyperplasia (mitoses > 1/crypt) partial atrophy Atrophic type Increased IELs count (> 25/100 Villous atrophy (mild-moderate Villous atrophy (mild-moderate- epithelial cells) degree) severe degree) Type III B lesion Villous atrophy (moderate degree) Crypt hyperplasia (mitoses > 1/ Crypt hyperplasia (mitoses > 1/crypt) destructive Crypt hyperplasia (mitoses > 1/crypt) crypt) Increased IELs count (> 25/100 Increased IELs count (> 25/100 Increased IELs count (> 25/100 epithelial cells) epithelial cells) epithelial cells) Type III C lesion Villous atrophy (severe degree) Grade B2 lesion destructive Crypt hyperplasia (mitoses > 1/crypt) total atrophy Increased IELs count (> 25/100 Villous atrophy (severe degree) epithelial cells) Crypt hyperplasia (mitoses > 1/ crypt) Increased IELs count (> 25/100 epithelial cells

Grade B1 in which the villus/crypt ratio is less than improve once the gluten-containing foodstuff is 3:1 and pathological increase of IEL is present; removed from the diet, and CD and wheat allergy Grade B2 in which the villi are no longer identifiable have been excluded” 32. The histologic characteris- and pathological increase of IEL is present. tics of NCGS are still under investigation, ranging from Recently a simplified classification with only two enti- normal histology to a slight increase in the number ties was proposed 27 (Tab. I). of T lymphocytes in the superficial epithelium of villi. Some authors described a normal number of T lym- phocytes but a peculiar disposition of this cells in small Immunohistochemistry “cluster” of 3-4 elements in the superficial epithelium, as well as the linear disposition in the deeper part of One of the key points in the CD diagnosis is the num- the mucosa together with an increased number of eo- ber of IEL, which are CD3 and CD8 positive T lympho- sinophils (> 5/HPF) in lamina propria. Further studies cytes; in pathological conditions, their number should are needed to assess these findings as specific for be more than 25 lymphocytes per 100 epithelial cells. NCGS 33 (Fig. 2). The counts can be performed reasonably well on the normal and irreplaceable hematoxylin-eosin but we suggest, especially in the initial forms, that an immu- Differential diagnosis nohistochemical assessment should always be car- ried out with monoclonal CD3 antibodies which often CD shares its duodenal histopathologic features with allows for a more accurate evaluation of T lympho- a large variety of intestinal disorders. Thus, a correct cytes (Fig. 1) 28-31. diagnosis may be reached only by an integrated eval- uation of clinical manifestations, laboratory and HLA tests and endoscopic findings, as well as histopatho- Non celiac gluten sensitivity (NCGS) logic findings. Main conditions with histologic changes that can overlap with CD and some tips for differential Non-celiac gluten sensitivity (NCGS) is “a clinical diagnosis are summarized in Table II. entity induced by the ingestion of gluten leading Common entities characterized by intraepithelial lym- to intestinal and/or extraintestinal symptoms that phocytosis without villous atrophy include several 190 V. Villanacci et al.

Figure 1. (A-B): normal duodenal mucosa; villous/crypt ratio over 3:1; number of T lymphocytes < 25 x 100 epithelial cells. (A) H&E x 10, (B) CD3 immunostain x 10. (C-D): Type 1 - Grade A lesion; normal villi but with pathological increase of T lymhocytes > 25 x 100 epithelail cells. (C) H&E x 20, (D) CD3 immunostain x 20. (E-F): mild to moderate villous atrophy Type 3A-3B - Grade B1 with pathological increase of T lymphocytes. (E) H&E x 20, (F) CD3 immunostain x 20. (G-H): severe villous atrophy Type 3C - Grade B2 with pathological increase of T lymphocytes. (G) H&E x 20, (H) CD3 immunostain x 20.

Figure 2. (A-B): normal villi; T lymphocytes < 25 x 100 epithelial cells. (A) H&E x 10, (B) CD3 immunostain x 10. (C-D): cluster of T Lymphocytes in the superficial epithelium. (C-D) CD3 immunostain x 60 red rectangle. (E-F): linear disposition of T lymphocytes in the deeper part of the mucosa. CD3 immunostain x 4 red rectangle. (G-H): eosinophils in lamina propria. HISTOPATHOLOGICAL DIAGNOSIS OF CELIAC DISEASE 191

Table II. Major CD non-neoplastic mimickers and histopathologic features useful for differential diagnosis. Increased Histopathologic tips for differential Mimicker Villous atrophy IELs diagnosis Infectious diseases Parasitic infestation Rare (in Rare (in children) Identification of parasites (e.g. Giardia); children) increased eosinophils in lamina propria HP-positive gastritis and peptic Possible Possible, mild (if present) Foveolar metaplasia of the duodenum; duodenitis increased plasma cells in lamina propria; neutrophilic infiltration in lamina propria and epithelium; changes more prominent in the bulbus; HP in gastric biopsies Tropical sprue Ye s Yes, usually low-grade Extensive ileal involvement Bacterial overgrowth Ye s Possible Mild lesions Whipple disease Rare Ye s PAS-positive macrophages in lamina propria Viral gastroenteritis or post-infectious Ye s Possible, variable grade Mucosal recovery after infection resolution changes Drugs NSAIDs Possible Rare, patchy, mild Erosions, neutrophilic infiltration in lamina propria Antineoplastic and immune Rare Possible Crypt architectural distortion; neutrophilic modulatory drugs (including immune infiltration in lamina propria; foci of checkpoint inhibitors) crypt apoptosis; involvement of other gastrointestinal tracts (gastritis, colitis) ARBs use (Olmesartan and others) Possible Frequent, variable grade Neutrophilic infiltration in lamina propria; deposition of subepithelial collagen, foci of crypt apoptosis Other immune-inflammatory conditions Collagenous sprue Ye s Frequent, variable grade Deposition of subepithelial collagen Immunodeficiencies (including Ye s Possible, variable grade Depletion of plasma cells in lamina propria, CVID) follicular lymphoid hyperplasia; concomitant giardiasis Autoimmune enteropathy Possible Yes, variable grade Neutrophilic infiltration in lamina propria; (celiac crypt apoptosis; reduction in goblet and pattern) Paneth cells; diffuse involvement of other gastrointestinal tracts (gastritis, enteritis, colitis) Crohn’s disease and ulcerative Rare Rare, patchy (if present) Erosions/ulcerations, neutrophilic colitis-associated duodenitis inflammation; crypt distortion; microgranulomas; basal plasmacytosis; ileal and colonic involvement Eosinophilic gastroenteritis Possible Possible, usually not severe Increased eosinophils in lamina propria; and food protein-sensitive involvement of other gastrointestinal tracts enteropathies (including gluten- (enteritis and colitis) sensitive enteropathy) Legend: ARB: angiotension receptor blocker; CVID: common variable immunodeficiencies; HP: Helicobacter pylori; IEL: intraepithelial lymphocyte; NSAID: non-steroidal anti-inflammatory drug; PAS: periodic acid Schiff. medications, Helicobacter pylori gastritis, duodenal cases. In addition, use of NSAIDs has been reported parasitic infestations, and autoimmune conditions. to usually cause mucosal erosions/ulcerations with in- Potential confounders which typically cause villous flammatory infiltrate composed with plasma cells and atrophy comprise olmesartan and other angiotensin neutrophils 35, while crypt architectural distortion, neu- receptor blockers, various immunomodulatory drugs, trophilic infiltration, ischemic changes, villous blunting, common variable immunodeficiency, autoimmune en- epithelial cell apoptosis in crypts and neutrophilic cryp- teropathy, Whipple disease and tropical sprue 1,34. titis were occasionally described in individuals treat- Several medications, including nonsteroidal anti-in- ed with checkpoint inhibitors or kinase inhibitors 36,37. flammatory drugs (NSAIDs), immunomodulatory and Olmesartan, an angiotensin II receptor blocker, was antineoplastic drugs, can mimic CD histologically; proved to cause partial to complete villous atrophy and however, villous atrophy is seldom described in these increased IEL, thus mimicking CD histology 38. 192 V. Villanacci et al.

Helicobacter pylori (HP) infection may determi- biopsies from other gastrointestinal sites often show nate an epithelial lymphocytosis generally with mini- histologic abnormalities and may aid for diagnosis. mal villous changes. Foveolar gastric metaplasia and Lastly, it should be remembered that forms of idio- marked neutrophilic infiltration in epithelium and/or pathic villous atrophy (villous atrophy or sprue of lamina propria may help in distinguishing HP-related unknown aetiology) may cause diagnostic challenges. peptic duodenitis from microscopic alterations of Some of these patients have spontaneous histological CD 39. Although intestinal parasitic organisms may recovery and are associated with excellent survival, show every CD histopathologic hallmark, a high num- whereas others show persistent villous atrophy, with ber of eosinophils in lamina propria should prompt the or without associated lymphoproliferative disorders 51. pathologist to search for the presence of parasites. Giardiasis, caused by Giardia lamblia, is one of the most common intestinal parasitic disease. Giardia can Complications of CD be easily identified in duodenal biopsy samples as a pear-shaped organism with two paired nuclei, located Refractory celiac disease (RCD). RCD a condition in lumen, adjacent to the epithelium. It usually does characterized by prolonged villous atrophy in duode- not determine significant histologic lesions, even if nal biopsies of a CD patient, along with malabsorption villous blunting, intraepithelial lymphocytosis and/or symptoms, despite a strict adherence to gluten-free crypt hyperplasia are rarely observed in children 40. diet (GFD) over a minimum period of 12 months 52,53. Nevertheless, the presence of villous atrophy in asso- Other causes of persistent villous atrophy or slow-to- ciation with signs of parasitic infections should hint the respond CD must be carefully excluded before making possibility of an underlying CD 41. a diagnosis of RCD. Endoscopic abnormalities such Food protein-sensitive enteropathies can also re- as mucosal erosions, ulcerations (ulcerative duode- produce CD histologic abnormalities, but they tend to no-jejunitis) or strictures may be observed. be transient or to respond to dietary allergen withdraw- It is a rare CD complication with a variable incidence al. In duodenal biopsies from patients with pernicious and prevalence. A systematic review by Rowinski and anemia, partial villous blunting and mucosal chronic Christensen54 showed a cumulative incidence of 1-4% inflammatory infiltration may be detected, along with over 10 years and a prevalence of 0.31%-0.38% in the more typical epithelial megaloblastic changes 42. CD patients, while a study based on a cohort of celiac Collagenous sprue is a rare malabsorption condition individuals in Austria reported an incidence over 25 which is often misdiagnosed as CD; however, the iden- years of 2.6% 55. Globally, RCD incidence seems to be tification of a thick subepithelial collagen type I band decreasing during the last 20 years, probably because with inflammatory cells and capillaries entrapped may of increased awareness, stricter adherence to GFD lead to a correct diagnosis 43,44. A significant, albeit and greater availability of gluten-free products 55,56. variable, fraction of cases is associated with CD and Mean age at RCD diagnosis has been reported to be may be treated with combinations of a gluten-free diet abound 63 years. Generally, the median time between and immunosuppressive therapy. the diagnosis of CD and the diagnosis of RCD is 21 Common variable immunodeficiency (CVID) en- months, although rare cases of RCD primarily diag- teropathy may mimic CD. Nevertheless, two distin- nosed at the time of first presentation of malabsorp- guishing features, usually absent in CD individuals, tion symptoms have been described 57. may be found in duodenal samples of CVID patients: Two types of RCD have been recognized on the bas- depletion in plasma cells (present in about two-third es of their clinical, histologic and molecular features. of cases) and follicular lymphoid hyperplasia 45. Fur- Type I RCD is characterized by an usual immunophe- thermore, pathologists should always search for a co- notype of IEL (i.e. retained expression of surface existing Giardia lamblia infection, as it was reported CD3, CD8 and CD103) and absence of a monoclonal in 23% of cases by Malamut et al. 46. In a minority of T cell receptor (TCR) gene rearrangement, whereas CVID patients, villous atrophy is gluten-sensitive 47. type II RCD is marked by an aberrant intraepithelial Autoimmune enteropathy, a disease characterized lymphocyte immunophenotype (i.e. > 50% of intraep- by small intestinal mucosal atrophy and circulating au- ithelial T cells lacking CD8 by immunohistochemistry toantibodies towards enterocytes and/or goblet cells, on formalin-fixed paraffin-embedded sections and/or may show an active enteritis pattern, characterized by >20-25% CD45+ T cells lacking surface CD3 on flow expansion of the lamina propria by mixed inflammation cytometry), and a monoclonal TCR gene rearrange- with neutrophil infiltrates, or a CD-like pattern 48-50. Fo- ment 54. TCR gene rearrangement clonal analysis by ci of apoptotic epithelial cells and reduction in goblet multiple polymerase chain reaction may be efficiently and Paneth cells may rarely be observed. Importantly, performed also on formalin-fixed paraffin-embedded HISTOPATHOLOGICAL DIAGNOSIS OF CELIAC DISEASE 193

Figure 3. (A-B): collagenous sprue; pathological increase in the thickness of the connective tissue band under the superficial epithelium > 10 mµ; (A) H&E x 20; (B) Trichrome stain x 20. (C-D): refractory celiac disease; pathological increase of T lymphocytes CD3 positive (C) negativity for CD8 (D). C-D x 20. (E-F): enteropathy type T cell lymphoma; (E) x 4, (F) H&E x 40; (G) CD3 immunostain x 40.

tissues. As samples from duodenal mucosa from immunophenotype (by immunohistochemistry or flow normal, CD or RCD type I patients may occasion- cytometry) and clonal analysis 58,59. Flow cytometry ally show TCR-β or TCR-γ clonality, the diagnosis seems to be better than CD8 immunohistochemistry and typing of RCD should be only made by a gas- in differential diagnosis between type I and II RCD. troenterologist after an integrated evaluation of clin- However, a recent study found that immunohisto- ical information, histology, intraepithelial lymphocyte chemical expression of a NK biomarker, NKp46, on 194 V. Villanacci et al.

T-cell surface, may help in distinguishing RCD type cytes and a subset of them has a medullary-type his- II (NKp46-positive) from RCD type I, usually show- tology 66. Importantly, they usually display a relatively ing no or few NKp46-positive T-cells 60. Histology of indolent behavior. SBC in CD patients is rarely asso- RCD type I may be indistinguishable from untreated ciated with adjacent preinvasive neoplastic/dysplastic responsive CD; however, a collagenous sprue-type lesions, which, like their invasive components, usually pattern and basal plasmocytosis are rarely noted in express nuclear β-catenin, while retaining mismatch RCD type I. Making a correct diagnosis is of pivot- repair protein expression. Recently, Giuffrida and col- al importance because type I and type II RCD have leagues found that CD-associated SBCs are often infil- very different prognosis, therapy response and rate trated by PD-1-positive T-cells and show expression of of development of lymphoproliferative malignancies. PD-L1 in neoplastic/immune cells (combined positive RCD type I has 5-year survival rates up to 95%, re- score ≥ 1) in more than one third of cases 67. sponse rate to corticosteroids of 90% and rates of Liver complications. Some CD patients may have developing enteropathy-associated T-cell lymphoma altered liver function tests and/or develop a wide (EATL) 5 year after RCD diagnosis lower than 14%; spectrum of liver diseases, encompassing cryptogen- on the other hand, RCD type II has a 5-year survival ic hepatitis, steatohepatitis, cirrhosis, as well as liver of 58%, lower response rate to corticosteroids and autoimmune disorders 68. higher rates of developing EATL 54,61. Lymphoproliferative malignancies. CD individuals, especially those with longstanding disease, have a Conclusion relative risk of developing extra-nodal non-Hodgkin lymphoma around 3-4 times higher than general pop- CD is a very common disorder affecting most people ulation 60. in the silent form. Many of these patients are identi- EATL is an aggressive malignancy complicating CD, fied through screening of at-risk groups or after ma- commonly involving jejunum and ileum and character- labsorption symptoms onset, rarely for disease-as- ized by markedly atypical malignant cells, densely in- sociated complications. CD diagnosis and its differ- filtrating both the epithelium, which typically shows se- ential diagnosis is made from integrations between vere villous atrophy, and the lamina propria, extending typical histological findings and clinical, serological below muscularis mucosae. Neoplastic cells are pos- and immunological features. Corazza-Villanacci Sys- itive for CD3 and CD103, negative for CD5 and CD4, tem is a helpful method to assess mucosal damage express CD8 variably, contain cytotoxic granule-asso- and and the response to gluten-free diet in patient ciated proteins and harbor a clonal rearrangement of follow-up. TCRγ and/or TCRβ genes 61. EATL should be distinguished from monomorphic References epitheliotrophic intestinal T-cell lymphoma (MEITL), 1 Robert ME, Crowe SE, Burgart L, et al. Statement on best composed of monomorphic, not significantly atypical, practices in the use of pathology as a diagnostic tool for ce- small- to medium-sized T cells, immunoreactive for liac disease: a guide for clinicians and pathologists. Am CD3, CD8, CD56, CD103, and TIA1 and negative for J Surg Pathol 2018;42:e44-e58. https://doi.org/10.1097/ PAS.0000000000001107 CD5, CD4, CD30. Neoplastic T cells infiltrate both the 2 Volta U, Caio G, Stanghellini V, et al. Non-celiac gluten sensitiv- lamina propria and the epithelium, causing partial or ity: questions still to be answered despite increased awarness. severe villous atrophy. Although the latest WHO clas- Cell Mol Immunol 2013;10:383-92. https://doi.org/10.1038/ sification of lymphoid neoplasms denied any associ- cmi.2013.28 ation of MEITL with CD, it was recently described in 3 Caio G, Volta U, Sapone A et al. Celiac disease: a comprehen- two CD patients 62. Both EATL and MEITL have an sive current review. BMC Medicine 2019;17:142-62. https://doi. org/10.1186/s12916-019-1380-z ominous prognosis, with a reported 5-year survival 4 63 Ludvigsson JF, Rubio-Tapia A, van Dyke CT, et al. Increasing inci- rate lower than 20% (Fig. 3). dence of celiac disease in a North American population. Am J Gas- Small bowel carcinoma (SBC). Patients with CD have troenterol 2013:108:818-24. https://doi.org/10.1038/ajg.2013.60 an increased risk of developing SBC. CD-associated 5 Mustalahti K, Catassi C, Reunanen A, et al. The prevalence of SBC was shown to arise after a median CD duration of celiac disease in Europe: results of a centralized, international 17 months in patients with a median age of 53 years mass screening project. Ann Med 2010;42:587-95. https://doi.org and to predominantly involve the jejunum. CD-asso- /10.3109/07853890.2010.505931 6 ciated SBCs harbor mismatch repair deficiency more Sanders DS, Patel D, Stephenson TJ, et al. A primary care cross-sectional study of undiagnosed adult coeliac disease. frequently in comparison with Crohn’s disease-asso- Eur J Gastroenterol Hepatol 2003;15:407-13. https://doi. ciated or sporadic SBCs 64,65. In addition, they often org/10.1097/00042737-200304000-00012 showed a high number of tumor infiltrating lympho- 7 Sanders DS, Hurlstone DP, Stokes RO, et al. Changing face of HISTOPATHOLOGICAL DIAGNOSIS OF CELIAC DISEASE 195

adult coeliac disease: experience of a single university hospi- 26 Corazza GR, Villanacci V, Zambelli C, et al. Comparison of the tal in South Yorkshire. Postgrad Med J 2002;78:31-3. https://doi. Interobserver reproducibility with different histologic criteria used org/10.1136/pmj.78.915.31 in celiac disease. Clin Gastroenterol Hepatol 2007;5:838-43. 8 West J, Fleming KM, Tata LJ, et al. Incidence and prevalence https://doi.org/10.1016/j.cgh.2007.03.019 of celiac disease and dermatitis herpetiformis in the UK over 27 Villanacci V. The histological classification of biopsy in celiac dis- two decades: population-based study. Am J Gastroenterol ease: time for a change? Dig Liver Dis 2015;47(1):2-3. https:// 2014;109:757-68. https://doi.org/10.1038/ajg.2014.55 doi.org/10.1016/j.dld.2014.09.022 9 Lo W, Sano K, Lebwohl B, et al. Changing presentation of 28 Shidrawi RG, Przemioslo R, Davies DR, et al. Pitfalls in diag- adult celiac disease. Dig Dis Sci 2003, 48:395-8. https://doi. nosing coeliac disease. J Clin Pathol 1994;47:693-4. https://doi. org/10.1023/a:1021956200382 org/10.1136/jcp.47.8.693 10 Fasano A. Celiac disease: how to handle a clinical chama- 29 Eigner W, Wrba F, Chott A, et al. Early recognition of possible leon. N Eng J Med 2003; 348:2568-70. https://doi.org/10.1056/ pitfalls in histological diagnosis of celiac disease. Scand J Gas- NEJMe030050 troenterol 2015;50:1088-93. https://doi.org/10.3109/00365521.20 11 Ludviggson JF, Leffler DA, Bai JC, et al. The Oslo Classification 15.1017835 for coeliac disease and related terms. Gut 2013;6:43-52. https:// 30 Freeman HJ. Pearls and pitfalls in the diagnosis of adult ce- doi.org/10.1136/gutjnl-2011-301346 liac disease. Can J Gastroenterol 2008;22:273-80. https://doi. 12 Caio G, Volta U, Sapone A, et al. Celiac disease: a comprehen- org/10.1155/2008/905325 sive current review. BMC Medicine 2019;17:142-62. https://doi. 31 Ravelli A, Villanacci V. Tricks of the trade: how to avoid histologi- org/10.1186/s12916-019-1380-z cal pitfalls in celiac disease. Pathol Res Pract 2012;208(4):197- 13 Ciacci C, Ciclitira P, Hadjivassiliou M, et al. The gluten-free diet 202. https://doi.org/10.1016/j.prp.2012.01.008 and its current application in coeliac disease and dermatitis 32 Fasano A, Sapone A, Zevallos V, et al. Nonceliac gluten sen- herpetiformis. United European Gastroenterol J. 2015;3:121-35. sitivity. Gastroenterology 2015;148:1195-204. https://doi. https://doi.org/10.1177/2050640614559263 org/10.1053/j.gastro.2014.12.049 14 Volta U, Granityo A, Fiorini E, et al. Usefulness of antibodies 33 Zanini B, Villanacci V, Marullo M, et al. Duodenal histological to deamidated gliadin peptides in celiac disease diagnosis and features in suspected non-celiac gluten sensitivity: new insights follow up. Dig Dis Sci 2008;853:1582-8. https://doi.org/10.1007/ into a still undefined condition. Virchows Arch 2018;473:229-34. s10620-007-0058-0 https://doi.org/10.1007/s00428-018-2346-9 15 Volta U, Tovoli F, Piscaglia M, et al. Old and new serological 34 Dai Y, Zhang Q, Olofson AM, et al. Celiac disease: updates on pa- testsfor celiac disease screening. EXP Rev Gastroenterol Hepa- thology and differential diagnosis. Adv Anat Pathol 2019;26:292- tol 2010;4:31-5. https://doi.org/10.1586/egh.09.66 312. https://doi.org/10.1097/PAP.0000000000000242 16 Serra S, Jani PA. An approach to duodenal biopsies. J Clin Pathol 35 Bjarnason I, Zanelli G, Smith T, et al. Nonsteroidal antiinflam- 2006;59:1133-50. https://doi.org/10.1136/jcp.2005.031260 matory drug-induced intestinal inflammation in humans. Gas- 17 Pais WP, Duerksen DR, Pettigrew NM, et al. How many duo- troenterology 1987;93:480-9. https://doi.org/10.1016/0016- denal biopsy specimens are required to make a diagnosis of 5085(87)90909-7 celiac disease? Gastrointest Endosc 2008;67:1082-7. https://doi. 36 Gonzalez RS, Salaria SN, Bohannon CD, et al. PD-1 inhibi- org/10.1016/j.gie.2007.10.015 tor gastroenterocolitis: case series and appraisal of “immuno- 18 Latorre M, Lagana SM, Freedberg DE, et al. Endoscopic biopsy modulatory gastroenterocolitis”. Histopathology 2017;70:558-67. technique in the diagnosis of celiac disease: one bite or two? https://doi.org/10.1111/his.13118 Gastrointest Endosc 2015;81:1228-33. https://doi.org/10.1016/j. 37 Louie CY, DiMaio MA, Matsukuma KE, et al. Idelalisib-asso- gie.2014.10.024 ciated Enterocolitis: clinicopathologic features and distinction 19 De Leo L, Villanacci V, Ziberna F, et al. Immunohistologic analy- from other Enterocolitides. Am J Surg Pathol. 2015;39:1653-60. sis of the duodenal bulb: a new method for celiac disease di- https://doi.org/10.1097/PAS.0000000000000525 agnosis in children. Gastrointest Endosc. 2018;88:521-6. https:// 38 Rubio-Tapia A, Herman ML, Ludvigsson JF, et al. Severe spru- doi.org/10.1016/j.gie.2018.05.014 elike enteropathy associated with olmesartan. Mayo Clin Proc 20 Villanacci V, Ceppa P, Tavani E, et al. Coeliac disease: the histol- 2012;87:732-8. https://doi.org/10.1016/j.mayocp.2012.06.003 ogy report. Dig Liver Dis 2011;43 Suppl 4:S385-95. https://doi. 39 Ensari A. Gluten-sensitive enteropathy (celiac disease): con- org/10.1016/S1590-8658(11)60594-X troversies in diagnosis and classification. Arch Pathol Lab Med 21 Hayat M, Cairns A, Dixon MF, et al. Quantitation of intraepithelial 2010;134:826-36. https://doi.org/10.1043/1543-2165-134.6.826 lymphocytes in human duodenum: what is normal? J Clin Pathol 40 Koot BG, ten Kate FJ, Juffrie M, et al. Does Giardia lam- 2002;55:393-4. https://doi.org/10.1136/jcp.55.5.393 blia cause villous atrophy in children? A retrospective cohort 22 Veress B, Franzen L, Bodin L, et al. Duodenal intraepithelial lym- study of the histological abnormalities in giardiasis. J Pedi- phocyte-count revisited. Scand J Gastroenterol 2004;39:138-44. atr Gastroenterol Nutr 2009;49:304-8. https://doi.org/10.1097/ https://doi.org/10.1080/00365520310007675 MPG.0b013e31818de3c4 23 Marsh MN. Grains of truth: evolutionary changes in small intesti- 41 Hanevik K, Wik E, Langeland N, et al. Transient elevation of anti- nal mucosa in response to environmental antigen challenge. Gut transglutaminase and anti-endomysium antibodies in Giardia 1990;31:111-4. https://doi.org/10.1136/gut.31.1.111 infection. Scand J Gastroenterol 2018;53:809-12. https://doi.org/ 24 Oberhuber G, Granditsch G, Vogelsang H. The histopathology 10.1080/00365521.2018.1481522 of coeliac disease: time for a standardized report scheme for pa- 42 Bianchi A, Chipman DW, Dreskin A, et al. Nutritional folic acid thologists. Eur J Gastroenterol Hepatol 1999;11:1185-94. https:// deficiency with megaloblastic changes in the small-bowel epi- doi.org/10.1097/00042737-199910000-00019 thelium. N Engl J Med 1970;282:859-61. https://doi.org/10.1056/ 25 Corazza GR, Villanacci V. Coeliac disease. J Clin Pathol. NEJM197004092821510 2005;58:573-4. https://doi.org/10.1136/jcp.2004.023978 43 Maguire AA, Greenson GK, Lauwers GY, et al. Collag- 196 V. Villanacci et al.

enous sprue: a clinicopathologic study of 12 cases. Am 56 Hussein S, Gindin T, Lagana SM, et al. Clonal T cell receptor J Surg Pathol 2009;33:1440-9. https://doi.org/10.1097/ gene rearrangements in coeliac disease: implications for diag- PAS.0b013e3181ae2545 nosing refractory coeliac disease. J Clin Pathol 2018;71:825-31. 44 Vakiani E, Arguelles-Grande C, Mansukhani MM, et al. Collag- https://doi.org/10.1136/jclinpath-2018-205023 enous sprue is not always associated with dismal outcomes: a 57 Celli R, Hui P, Triscott H, et al. Clinical insignficance of monoclonal clinicopathological study of 19 patients. Mod Pathol 2010;23:12- t-cell populations and duodenal intraepithelial t-cell phenotypes 26. https://doi.org/10.1038/modpathol.2009.151 in celiac and nonceliac patients. Am J Surg Pathol 2019;43:151- 45 Daniels GA, Lederman HM, Maitra A, et al. Gastrointestinal 160. https://doi.org/10.1097/PAS.0000000000001172 tract pathology in patients with common variable immuno- 58 Cheminant M, Bruneau J, Malamut G, et al. NKp46 is a diag- deficiency (CVID): a clinicopathologic study and review. Am nostic biomarker and may be a therapeutic target in gastroin- J Surg Pathol 2007;31(12):1800-12. https://doi.org/10.1097/ testinal T-cell lymphoproliferative diseases: a CELAC study. Gut PAS.0b013e3180cab60c 2019;68:1396-1405. https://doi.org/10.1136/gutjnl-2018-317371 46 Malamut G, Verkarre V, Suarez F, et al. The enteropathy asso- 59 Al-Toma A, Verbeek WH, Hadithi M, et al. Survival in refrac- ciated with common variable immunodeficiency: the delineated tory coeliac disease and enteropathy-associated T-cell lympho- frontiers with celiac disease. Am J Gastroenterol 2010;105:2262- ma: retrospective evaluation of single-centre experience. Gut 75. https://doi.org/10.1038/ajg.2010.214 2007;56:1373-8. https://doi.org/10.1136/gut.2006.114512 47 Biagi F, Bianchi PI, Zilli A, et al. The significance of duodenal 60 West J. Celiac disease and its complications: a time travel- mucosal atrophy in patients with common variable immunode- ler’s perspective. Gastroenterology 2009;136:32-4. https://doi. ficiency: a clinical and histopathologic study. Am J Clin Pathol org/10.1053/j.gastro.2008.11.026 2012;138:185-9. https://doi.org/10.1309/AJCPEIILH2C0WFYE 61 Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of 48 Akram S, Murray JA, Pardi DS, et al. Adult autoimmune enterop- Tumours of Haematopoietic and Lymphoid Tissues, revised. 4th athy: Mayo Clinic Rochester experience. Clin Gastroenterol Hep- ed. Vol. 2. Lyon: IARC 2017. atol 2007;5:1282-90. https://doi.org/10.1016/j.cgh.2007.05.013 62 Lenti MV, Biagi F, Lucioni M, et al. Two cases of monomorphic 49 Masia R, Peyton S, Lauwers GY, et al. Gastrointestinal biopsy epitheliotropic intestinal T-cell lymphoma associated with coeliac findings of autoimmune enteropathy: a review of 25 cases. disease. Scand J Gastroenterol 2019;54:965-8. https://doi.org/10 Am J Surg Pathol 2014;38:1319-29. https://doi.org/10.1097/ .1080/00365521.2019.1647455 PAS.0000000000000317 63 Nijeboer P, Malamut G, Mulder CJ, et al. Enteropathy-associ- 50 Villanacci V, Lougaris V, Ravelli A, et al. Clinical manifesta- ated T-cell lymphoma: improving treatment strategies. Dig Dis tions and gastrointestinal pathology in 40 patients with auto- 2015;33:231-5. https://doi.org/10.1159/000369542 immune enteropathy. Clin Immunol 2019;207:10-17. https://doi. 64 Potter DD, Murray JA, Donohue JH, Burgart LJ, Nagorney DM, org/10.1016/j.clim.2019.07.001 van Heerden JA, Plevak MF, Zinsmeister AR, Thibodeau SN. 51 Schiepatti A, Sanders DS, Aziz I, et al. Clinical phenotype and The role of defective mismatch repair in small bowel adenocar- mortality in patients with idiopathic small bowel villous atrophy: cinoma in celiac disease. Cancer Res 2004;64(19):7073-7. a dual-centre international study. Eur J Gastroenterol Hepa- 65 Vanoli A, Di Sabatino A, Furlan D, et al. Small bowel carcinomas tol 2020 Apr 10. Epub ahead of print. https://doi.org/10.1097/ in coeliac or crohn’s disease: clinico-pathological, molecular, MEG.0000000000001726 and prognostic features. A Study from the small bowel cancer 52 Malamut G, Afchain P, Verkarre V, et al. Presentation and long- italian consortium. J Crohns Colitis 2017;11:942-53. https://doi. term follow-up of refractory celiac disease: comparison of type org/10.1093/ecco-jcc/jjx031 I with type II. Gastroenterology 2009;136:81-90. https://doi. 66 Vanoli A, Di Sabatino A, Martino M, et al. Small bowel carci- org/10.1053/j.gastro.2008.09.069 nomas in celiac or Crohn’s disease: distinctive histophenotypic, 53 Hujoel IA, Murray JA. Refractory celiac disease. Curr Gastroen- molecular and histogenetic patterns. Mod Pathol 2017;30:1453- terol Rep 2020;22:18. https://doi.org/10.1007/s11894-020-0756- 66. https://doi.org/10.1038/modpathol.2017.40 8 67 Giuffrida P, Arpa G, Grillo F, et al. PD-L1 in small bowel ade- 54 Rowinski SA, Christensen E. Epidemiologic and therapeutic as- nocarcinoma is associated with etiology and tumor-infiltrating pects of refractory coeliac disease - a systematic review. Dan lymphocytes, in addition to microsatellite instability. Mod Pathol Med J 2016;63(12). 2020 Feb 17. https://doi.org/10.1038/s41379-020-0497-0 55 Eigner W, Bashir K, Primas C, et al. Dynamics of occurrence 68 Majumdar K, Sakhuja P, Puri AS, et al. Coeliac disease and of refractory coeliac disease and associated complications over the liver: spectrum of liver histology, serology and treatment re- 25 years. Aliment Pharmacol Ther 2017;45:364-72. https://doi. sponse at a tertiary referral centre. J Clin Pathol 2018;71:412-9. org/10.1111/apt.13867 https://doi.org/10.1136/jclinpath-2017-204647 PATHOLOGICA 2020;112:197-209; DOI: 10.32074/1591-951X-168

Review

Inflammatory and tumor-like lesions of the pancreas

Claudio Luchini1, Matteo Fassan2, Claudio Doglioni3,4, Paola Capelli1, Giuseppe Ingravallo5, Giuseppina Renzulli5, Sara Pecori1, Gaetano Paolino1, Ada M. Florena6, Aldo Scarpa1,7, Giuseppe Zamboni1,8 1 Department of Diagnostics and Public Health, Section of Pathology, University and Hospital Trust of Verona, Verona, Italy; 2 Surgical Pathology Unit, Department of Medicine (DIMED), University of Padua, Italy; 3 Vita e Salute University, Milan, Italy; 4 Pathology Unit, Pancreas Translational and Clinical Research Center, IRCCS San Raffaele Scientific Institute, Milan, Italy; 5 Department of Emergency and Organ Transplantation, Section of Pathological Anatomy, University of Bari Aldo Moro, Bari, Italy; 6 Department of Sciences for Promotion of Health and Mother and Child Care, Anatomic Pathology, University of Palermo, Italy; 7 ARC-NET Research Centre, University of Verona, Verona, Italy; 8 IRCSS Sacro Cuore Don Calabria Hospital, Negrar, Italy

Summary Inflammatory/tumor-like lesions of the pancreas represent a heterogeneous group of dis- eases that can variably involve the pancreatic gland determining different signs and symp- toms. In the category of inflammatory/tumor-like lesions of the pancreas, the most impor- tant entities are represented by chronic pancreatitis, which includes alcoholic, obstructive and hereditary pancreatitis, paraduodenal (groove) pancreatitis, autoimmune pancreatitis, lymphoepithelial cyst, pancreatic hamartoma and intrapancreatic accessory spleen. An Received and accepted: July 7, 2020 in-depth knowledge of such diseases is essential, since they can cause severe morbidity Published online: 29 October, 2020 and may represent a potential life-threatening risk for patients. Furthermore, in some cases the differential diagnosis with malignant tumors may be challenging. Herein we provide a Correspondence general overview of all these categories, with the specific aim of highlighting their most Aldo Scarpa important clinic-pathological hallmarks to be used in routine diagnostic activities and clini- Department of Diagnostics and Public Health, Section of Pathology, ARC-NET Research cal practice. Centre, University and Hospital Trust of Vero- Key words: chronic pancreatitis, paraduodenal pancreatitis, groove, autoimmune na, piazzale Scuro,10, 37134 Verona, Italy Tel. 0039 045 8127458 pancreatitis, pancreatic pathology E-mail: [email protected]

Conflict of interest The Authors declare no conflict of interest. Introduction Inflammatory /tumor-like lesions of the pancreas represent a heteroge- How to cite this article: Luchini C, Fassan M, Doglioni C, et al. Inflammatory neous group of diseases that can variably involve the pancreatic gland and tumor-like lesions of the pancreas. causing a composite spectrum of different signs and symptoms. An in- Pathologica 2020;112:197-209. https://doi. depth knowledge of such diseases is very important, since they can org/10.32074/1591-951X-168 cause severe morbidity and may represent a potential life-threatening © Copyright by Società Italiana di Anatomia Pato- risk for patients. For surgical pathologists, recognizing their histological logica e Citopatologia Diagnostica, Divisione Itali- features and morphological hallmarks is fundamental, since all these ana della International Academy of Pathology disorders can potentially mimic pancreatic ductal adenocarcinoma or OPEN ACCESS other malignant neoplasms. In the group of inflammatory/tumor-like lesions of the pancreas, the This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- most important categories are: chronic pancreatitis (CP), paraduodenal NonCommercial-NoDerivatives 4.0 International) license: the (groove) pancreatitis (PGP), autoimmune pancreatitis (AP), lymphoe- work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original pithelial cyst, pancreatic hamartoma and intrapancreatic accessory version. For further information: https://creativecommons. spleen. The key features of each category are summarized in Table I. org/licenses/by-nc-nd/4.0/deed.en Figure 1 shows some typical macroscopic aspects. 198 C. Luchini et al.

Table I. Main features of inflammatory and tumor-like lesions of the pancreas.

Pathological conditions Etiology Clinical issues Macroscopic features Microscopic features

Chronic pancreatitis Alcoholic pancreatitis. Clinical symptoms are Irregular fibrosis Fibrosis and pseudocysts. very similar for the three (irregular whitish area). subtypes, including Intraductal calculi of severe abdominal pain calcium carbonate and and dysfunction of both pseudocysts. exocrine and endocrine Obstructive pancreatitis. parenchyma Clear localization of Fibrosis and retention fibrosis (demarcated cysts. whitish area); multiple retention cysts. Pseudocysts very rare.

Hereditary pancreatitis. If PRSS1 and CFTR- Lipomatous atrophy vs related: progressive progressive fibrosis. lipomatous atrophy (yellowish areas); if SPINK1-associated: progressive fibrosis (withish areas). Paraduodenal (groove) Chronic obstruction of the Severe waxing Duodenal wall with Dense fibrosis of the pancreatitis minor papilla. and waning upper trabeculated appearance duodenal wall around the abdominal pain, and cystic change, minor papilla, with variably postprandial vomiting especially in the proximity extension to the groove and weight loss due to of the minor ampulla, area and the pancreatic duodenal stenosis. which can be absent or parenchyma. largely obstructed by Cysts are lined by ductal calcified, proteinaceous epithelium. material. Epicenter in the groove area. Autoimmune Type 1: part of the Men > 60 years, IgG4+; Grossly, type 1 and type Compact inflammatory pancreatitis systemic autoimmune obstructive jaundice, 2 are undistinguishable. infiltrate of T cell- immunoglobulin (Ig) G4+ vague abdominal Most common lymphocytes and plasma related disease. pain. Involvement appearance: pseudo- cells (IgG4+), fibrosis of different organs tumor aspect, whitish- specifically localized in (systemic disease). yellowish area. the periductal area, and Important response to a marked venulitis. The corticosteroid-based inflammation is centered therapy. around and within medium- to-large interlobular ducts. Type 2: autoimmune Male = female, Lymphoplasmacytic disorder, more pancreas- younger patients (4th- inflammation located in specific. 5th decade). Limited the periductal regions of to pancreas (15% of pancreatic parenchyma, patients may have presence of granulocytic concurrent inflammatory epithelial lesions. bowel disease). Important response to corticosteroid-based therapy. Lymphoepithelial Cyst Unknown. Usually, this is an Unilocular or multilocular The cystic epithelium is asymptomatic lesion, cyst, can be located multi-layered-squamous, discovered incidentally or entirely within the and is surrounded by a by imaging analysis due pancreatic gland or in the dense layer of lymphoid to unrelated reasons. periphery with exophytic tissue with prominent growth. It can reach a germinal centers. The large size (>5 cm), and adjacent pancreatic shows a irregular capsule. parenchyma is usually unremarkable. u INFLAMMATORY PANCREATIC PATHOLOGY 199

Table I. continues

Pathological conditions Etiology Clinical issues Macroscopic features Microscopic features

Pancreatic hamartoma Malformation, Variable and dependent Head of the pancreas, Small ductal structures disembryogenetic by size and location intrapancreatic mass. lined by columnar disorder. epithelial cells without atypia, surrounded by fibrous stroma and an unorganized acinar parenchyma. The presence of each component can vary, determining different morphological aspects, with ductal, stromal or acinar prominence. Intrapancreatic Unknown. Variable and dependent Brownish nodule (spleen Mature splenic tissue, with accessory spleen by size and location; appearance) surrounded a normal distribution of differential diagnosis by normal pancreas. white and red pulp. with neuroendocrine tumors.

Figure 1. Paradigmatic images of PGP and CP. PGP, solid variant: marked expansion of the groove area (asterisk) and mar- ginal involvement of the pancreatic parenchyma and of choledocus (black arrow) (A); PGP, cystic variant: diffuse presence of cysts and extensive pancreatitis of the pancreatic parenchyma (B); CP with intraductal calculi (asterisk: coledochus, black arrow: Wirsung’s duct) (C); Macroscopic translucent/pearly appearance of CP can be better appreciated on fresh tissues (asterisk: coledochus, black arrow: Wirsung’s duct) (D). 200 C. Luchini et al.

Chronic Pancreatitis (CP) area in location. Based on the etiology of the obstruc- tion, obstructive chronic pancreatitis affect equally Definition and terminology male and female, and can occur over a extensive age CP was first described in the scientific literature by distribution. Therapeutic approaches for obstructive Sir Thomas Cawley in 1788 1. He reported the histo- chronic pancreatitis aim at removing the underlying ry of a young man who died of diabetes, whose au- cause, whenever possible (e.g.: interventional endos- topsy revealed a pancreas filled with calculi. Starting copy, surgical resection) 2. Hereditary CP describes an from this landmark publication, many studies aimed entity resulting from the presence of genetic factors at clarifying the pathogenesis and pathophysiology of playing a critical role in both the susceptibility and pre- CP. This condition is now considered as a progressive, disposition for CP developing. The 3 most common fibro-inflammatory disease characterized by irrevers- germline alterations associated with chronic pancre- ible damage to the pancreas 2. Based on its diverse atitis involve the genes Serine Protease-1 (PRSS1), etiologies, CP is now grouped into three different cate- Cystic Fibrosis Transmembrane-conductance Regula- gories: i) alcoholic pancreatitis, ii) obstructive pancre- tor (CFTR) and Serine Peptidase Inhibitor Kazal type 1 atitis, and iii) hereditary pancreatitis. (SPINK1) 12-16. Germline mutations resulting in PRSS1 gain of function are responsible for a type of heredi- Clinical Issues and different etiologies tary CP that mostly involves very young patients (< 20 In Western Countries, the incidence of CP is around years old) 12. On the other hand, autosomal recessive 10 cases per 100,000, with a prevalence up to 40 cas- alterations in CFTR gene represent the most common es per 100,000 subjects 2-4. Although the symptomatic etiology of hereditary CP in children, within the cystic spectrum may be underhand and unspecific, patients fibrosis syndrome spectrum 15. Moreover, mutations of often present with severe abdominal pain and with SPINK1 gene are considered to be CP genetic mod- dysfunction of both exocrine and endocrine paren- erators, which either lower the threshold of originating chyma3. The abdominal pain usually have a classical pancreatitis or worsen its severity because of other back irradiation to the intra-scapular area. Therapeutic risk factors 2,16. strategies for chronic pancreatitis are mostly support- The common CP course, independent from these dif- ive, without resolving the disease and its symptoms ferent etiologies, in the initial stages of the disease is definitively. As a consequence and despite its low clinically articulated in recurrent episodes of abdomi- prevalence, CP represents a non-negligible cause nal pain 5-10. Over time, the pain attacks decrease in in- for both hospital admissions and overall costs for the cidence and severity, but in parallel there is a progres- public health system2. Notably, patients with CP have sive destruction of the glandular parenchyma, leading also an increased risk for developing pancreatic duct- to irreversible endocrine and exocrine failure. al adenocarcinoma. The underlying cause of chronic pancreatitis is mul- Macroscopic features tifactorial and involves a complex interaction of envi- The macroscopic features of alcoholic CP differ based ronmental, genetic, and/or other risk factors 5-10. Alco- on the different stages of the disease. Indeed, in ear- holic-CP is caused by a heavy and prolonged alcohol ly stages there is a patchy distribution of parenchy- abuse, and usually affect the pancreas of young-to- mal fibrosis, in both perilobular and and interlobular middle aged male patients. Notably, fewer than 5% location 10,11,17. Grossly, this feature usually results in of alcoholic individuals develop chronic pancreati- an accentuation of individual pancreatic lobules, with tis 2. This observation indicates that the involvement dilation of distortion of the portion of the pancreatic of other additional insults or cofactors are needed for ductal tree embedded in this fibrosis. In later stages, leading a subject to CP 7, 9 . Indeed smoking, obesity, the pancreatic parenchyma is extensively atrophic, genetic background and infectious diseases have with consequent reduction of the size of the entire been suggested as important cofactors of alcohol in gland 17. A diffuse loss of the normal pancreatic lobular this condition 7- 11 . Obstructive CP originates by an ob- architecture with replacement by diffuse fibrosis is as- struction of the Wirsung’s duct or of secondary ducts, sociated with prominent changes of pancreatic ducts, which can be caused by different entities, such as tu- including marked dilation and distortion. Intraductal mors, of which benign (less commonly) or malignant calculi of calcium carbonate are frequently present, (more commonly), ductal stones, scars, paraduodenal and pseudocysts are encountered in up to a half of wall cysts, stenosis of the papillary region and con- alcoholic-CP, representing a macroscopic hallmark of genital anomalies 10,11. Differently from alcoholic-CP, alcoholic-CP 18. obstructive CP affects the gland distal to the site of The macroscopic features of obstructive-CP can differ obstruction, being thus confined to a clearly delimited based on the underlying etiology. However, the most INFLAMMATORY PANCREATIC PATHOLOGY 201

important points regard the clear localization of the tological features mirror the gross alterations of each disease, involving the pancreatic distal to the obstruc- CP subtype. In early stage alcoholic CP, indeed, there tion, and the distal ductal dilation and distortion2. The is a perilobular and interlobular fibrosis, composed normal pancreatic architecture is lost and replaced by of spindled fibroblasts, which are dispersed in a thin a whitish fibrosis and by the presence of multiple re- wavy collagen 10,17,21. Atrophic changes regard in this tention cysts, above all in longstanding cases. Pseu- stage exocrine/acinar parenchyma. In this case, the docysts are very rare in obstructive-CP. remaining Langerhans islets can be visible in small In hereditary CP, PRSS1 and CFTR-related CP aggregates, representing a differential diagnosis with show a progressive lipomatous atrophy; conversely, neuroendocrine microadenoma /neuroendocrine tum- SPINK1-associated hereditary CP usually exhibits a ors. In such cases. an immunohistochemical analysis pattern of progressive fibrosis 2,19. for normal pancreatic hormones (including at least insulin and glucagon) will demonstrate the usual hor- Microscopic description with diagnostic criteria mones’ distribution, thus confirming the normal na- Paradigmatic microscopic images of CP are present- ture of the process. Usually there is also associated a ed in Figure 2. patchy distribution of lymphocytes (above all T-cells), Although, as described below, there are different his- plasma cells and histiocytes 21. In advanced stages, tological features that are more often seen in the differ- the dilated pancreatic ducts may show a prominent ent types of CP, it is of importance acknowledging that squamous metaplasia. There is an extensive loss of the most common and cardinal features for CP histo- exocrine and also endocrine parenchyma, with re- logic diagnosis are represented by the triad of fibrosis, placement by not only perilobular and interlobular fi- loss of acinar tissue and duct changes. The presence brosis, but also intralobular fibrosis 10,21. Pseudocysts of all the components of this triad represents the most are composed of thick, fibrous walls that lack by defini- important diagnostic criteria for CP 20. tion an epithelial cell lining, and are filled with necrotic Given that, from a microscopic point of view, the his- debris, fibrin, blood, and macrophages 17,21. Foci of fat

Figure 2. Important histological patterns of chronic pancreatitis. (A) Marked fibrosis with loss of normal pancreatic paren- chyma (original magnification 4X); (B) calcification in pancreatic duct with focal squamous metaplasia of a duct (original magnification 10X); (C) calcific plugs in pancreatic duct with ductal changes (original magnification 10X); (D) pancreatic pseudocyst: the lack of epithelial lining is evident (original magnification 4X). 202 C. Luchini et al.

necrosis characterized by partially necrotic adipose with frequent involvement of anatomic region between tissue with foamy macrophages, multinucleated giant the superior limit of the pancreatic head, duodenum, cells, and chronic inflammation may be also present 2. and common bile duct, which is the so-called “groove Similarly to the macroscopic description, the morphol- area” 24-28. PGP can presents either as a pure lesion, ogy of obstructive CP is characterized by narrowed as a result of pancreatitis of the intraduodenal pan- changes involving the pancreatic parenchyma distal to creatic parenchyma associated with the minor papilla, the obstruction. The normal pancreatic parenchyma is or associated with chronic pancreatitis of the proper replaced by a variable amount of perilobular and inter- pancreas. PGP as a pure lesion is rare compared with lobular fibrosis. The epithelium of the ductal tree may PGP associated with CP. undergo hyperplasia and/or squamous metaplasia. In advanced stages, a diffuse loss of pancreatic paren- Clinical issues chyma and a prominent fibrosis are typical. Another The vast majority of patients with PGP are mid- important aspect is represented by the presence of dle-aged men (5th decade) with a history of alcohol retention cysts, which represent dilated ducts lined by abuse and smoking 26. The clinical syndrome includes reactive cuboidal-to-columnar epithelium with intralu- an important symptomatology with severe waxing minal proteinaceous plugs and serous fluid 2,10. and waning upper abdominal pain, disordered gastric The microscopic features of hereditary CP are heter- emptying, postprandial vomiting and weight loss due ogeneous, based on the stage of the disease but also to duodenal stenosis 2,26,27. Although its pathogenesis on different etiologies. The morphology of PRSS1-re- remains not completely understood, the main hypoth- lated hereditary CP, for example, are dependent on esis attributes the onset of this disease to a chronic patient age. In advanced age, pancreas of patients obstruction of the minor papilla 24. with PRSS1 alterations show a diffuse parenchymal Imaging reveals thickening of the duodenal wall with atrophy, with extensive replacement by mature ad- associated cyst formation within the duodenum or the ipose tissue 22. Scattered endocrine islets and rare groove area, with a consequent radiographic appear- residual acinar cells, if present, are usually localized ance of a pseudocyst or of a cystic pancreatic neo- near Wirsung’s duct. The ductal epithelium may un- plasm2. More rarely, cases with minor cystic changes dergo dysplastic changes consistent with low-grade or solid lesions, often related to the sclerotic changes pancreatic intraepithelial neoplasia (PanIN) 23. On the in the periampullary region, may mimic a pancreatic other hand, in pediatric cases there is usually a central or a periampullary malignancy 27-30. The therapeutic parenchymal loss with mild chronic inflammation and strategies for PGP may be supportive/conservative perilobular and/or interlobular fibrosis 2,24. Noticeably, but, in case of a severe and longstanding symptom- the periphery of the gland usually shows a patchy pa- atology, and/or also in case of a difficult differential di- renchymal replacement with mature adipose tissue. agnosis with cancer, the first choice is represented by The fatty replacement of the pancreatic parenchyma pancreaticoduodenectomy 26-30. is not randomly located; indeed, it extends from the periphery to the central areas of the pancreas 22. Fi- Macroscopic features brosis may be present, but it is usually focal or mild. The macroscopical examination of a surgically re- Hereditary CP associated with CFTR mutations are sected specimen of the pancreatic head region with usually associated with ductal dilation, intraductal a PGP shows a duodenal wall with a trabeculated ap- mucinous plugs and periductal fibrosis. Conversely, pearance, often accompanied by cystic change, espe- hereditary-CP associated with SPINK1 mutations are cially in the proximity of the minor papilla, which can characterized by loss of pancreatic parenchyma with be absent or largely obstructed by calcified, proteina- concomitant perilobular and interlobular fibrosis. The ceous material 24-30. On cut sections, the epicenter of ductal epithelium can be atrophic, with intraluminal PGP can be identified around the minor papilla, with proteinaceous plugs and calculi 2. variable involvement of the groove area, which can be relatively gelatinous to solid, or contain cysts. In some cases, cyst formation may be prominent, measuring Paraduodenal (Groove) Pancreatits (PGP) up to several centimeters in size, mimicking intestinal duplication 27. The cysts are filled with proteinaceous Definition and terminology debris, usually have a smooth, opaque wall and may PGP (also reported as groove pancreatitis or cystic contain small calculi 2. Typically, the duodenal wall dystrophy of heterotopic pancreas) is a distinctive form and underlying pancreatic parenchyma are thickened of chronic pancreatitis occurring predominantly in and and fibrotic between Vater’s ampullary region and the around the duodenal wall (near the minor papilla), minor papilla; the duodenal mucosa often acquires a INFLAMMATORY PANCREATIC PATHOLOGY 203

nodular or cobblestone appearance 2,31. Although fi- bling traumatic neuroma is also a common finding in brosis is typically narrowed to the groove area, it may such area 27. spill into the adjacent pancreatic tissue, leading to ste- nosis of the Wirsung’s duct and of the common bile duct 2. The fibrosis has a typical translucent-pearly Autoimmune Pancreatits (AIP) appearance. It is also of interest to note that PGP is usually associated with the presence of enlarged “re- Definition and terminology active” lymph nodes/lymphadenopathy, macroscopi- AIP represents a heterogeneous disease process and cally evident, which are seen more rarely in the case is composed of 2 subtypes (type 1 and type 2). AIP is of pancreatic cancer. classified as type 1, which is often regarded as part of the systemic IgG4-related disease (IgG4-RD) that Microscopic description with diagnostic criteria may involve other organs, or as type 2, which is more The typical histological appearance of PGP is shown pancreas-specific 32-36. in Figure 3. From the histologic point of view, an important feature Clinical issues is the presence of a dense fibrosis of the duodenal AP is a unique form of chronic pancreatitis. From a wall around the minor papilla, with variable extension clinical point of view, the symptoms associated to to the adjacent structures including the soft tissue of this disease are heterogeneous; the most frequent 24,26,31 the groove area and the pancreatic parenchyma . is represented by obstructive jaundice, followed by Disperse in this fibrotic background, there are fibro- vague abdominal pain. Both type 1 and type 2 AIP blasts, myofibroblasts and smooth muscle cells. In the patients demonstrate significant response to corticos- duodenal wall, a marked, bulging Brunner gland hy- teroid-based therapy. perplasia is often present, contributing to the thicken- At clinical presentation, patients with AIP-type 1 are 2,24,27,31 ing of the intestinal wall . The cysts can be either generally men, older than 60 years, and seropositive restricted to the submucosal and muscular layers of for IgG4 33-37. It is within the spectrum of IgG4-RD that the duodenal wall, or extended to the groove region. often affects multiple organs and shares similar clinical, The internal surface is mainly lined by columnar pan- serologic and pathologic features 2,33-37. Together with creatic duct-like epithelium, which may be lost and re- the pancreas, the other organs that are more frequently placed by inflammatory granulation tissue. The cysts involved are liver, breast, lacrimal and salivary glands contain small calculi that may extravasate causing a (Küttner tumor), but may other districts may be affected, 2 foreign body giant cells reaction , which may call for as for example nasopharynx, bone marrow, extra-oc- a differential diagnosis with undifferentiated carcino- ular muscles and retrobulbar space, kidneys, lungs, 30 ma of the pancreas with osteoclast-like giant cells . lymph nodes, meninges, arteries, skin, prostate, thyroid In late stage of disease, the proper pancreas usually gland and even pericardium 34. Despite a remarkable shows atrophy, fat necrosis, chronic inflammation and response to corticosteroid-based therapeutic strate- 2,27 thick fibrosis . Prominence of nerve bundles resem- gies, patients are prone to frequent relapses 38. Conversely, AIP-type 2 affects more often younger subjects (< 4-5th decade) and is equally distributed be- tween male and females 37,38. Clinical manifestations are limited to the pancreatic region, but a variable percentage (up to 15%) of patients have inflammatory bowel disease 38. AIP-type 2 patients demonstrate a good response to corticosteroid-based therapy, with very low relapse rate 38. A subset of patients of both types AIP may have also high blood levels of the antigen CA 19-9 (up to > 12,000 U/ml) 39. This condition further complicates the clinical differential diagnosis with pancreatic can- cer, which represents the most important differential Figure 3. The histological appearance of paraduodenal diagnosis of both AI subtypes 39. groove pancreatitis is here shown. The cystic region usually Macroscopic features includes multiple cysts lined by ductal epithelium (original magnification 2X). Macroscopically, the two different subtypes of AIP are indistinguishable 37. Indeed, in both subtypes, the pan- 204 C. Luchini et al.

creatic resection specimen may present a narrowed, phenomenon results in the infolding of the ductal ep- discrete area, with a “pseudo-tumor” appearance, or ithelium with the consequent shrinkage of the ductal may show a more diffuse parenchymal enlargement, lumen. Regarding venulitis, the inflammatory infiltrate with a decrease of the physiological lobular structure, involves the venule and venous wall; in late-stage a white to yellow discoloration and a greater tissue disease, such aspects may evolve in an obliterative hardness 2,37. The pancreatic ductal tree and the com- phlebitis, with fibrosis of the lumen. Another common mon bile duct may be involved by these modifications, aspect is also represented by perineural inflammation. with a potential consequent obstruction. Lastly, there The inflammation, above all in late-stage diseases, is no presence of calculi in AP. may extend widely to the surrounding parenchyma, evocating fibrosis with secondary atrophy. In this stage, Microscopic description with diagnostic criteria the fibrotic changes become more diffuse, assuming a Paradigmatic images of AI are shown in Figure 4. whorled or storiform pattern 2,37. Lymphoid aggregates Microscopically, type 1 and type 2 AIP present with can be identified in most cases in intrapancreatic and two different and specific histologic framework, but peripancreatic tissue 42. Regarding IgG4 immunohis- there are some common aspects as well. tochemical research, this may represent a useful tool Type 1 AIP usually shows a compact inflammatory in- for supporting the diagnosis, and should highlight a filtrate, composed of T cell-lymphocytes and plasma high number of IgG4-positive plasma cells, above all cells, a fibrosis specifically localized in the periductal in early stages. A significant count is considered as area, and a marked venulitis 37,40,41. The inflammation the presence of at least 10 IgG4-positive plasma cells as a typical localization within the pancreatic paren- per high-power field; verifying that the proportion of chyma: indeed, it is centered around and within me- IgG4/IgG is assessed at least at 40-45% may be of dium-to-large interlobular ducts 2,37,40,41. This specific help in some cases 42,43. It should be highlighted that

Figure 4. Key histological and immunohistochemical patterns of autoimmune pancreatitis. (A) Marked inflammatory infil- trate is typically centered around pancreatic ducts (original magnification 10X); (B) dense inflammatory infiltrate with second- ary pancreatic parenchyma is encountered in late-stage autoimmune pancraetitis (original magnification 4X); (C) immunohis- tochemical analysis for CD138 highlights a diffuse infiltration by plasma cells (original magnification 4X); (D) immunohisto- chemical analysis for IgG4 indicates that a high number of plasma cells are also positive for IgG4 (original magnification 4X). INFLAMMATORY PANCREATIC PATHOLOGY 205

a “positive” count of IgG4 plasma cells may be pos- tent can show variable presentations: indeed, it may sible also in up to 10% of pancreatic ductal adeno- be limpid/serous but also milky-necrotic, depending carcinoma and alcoholic chronic pancreatitis 42. Thus, on the degree of keratin formation 45,46. The pre-oper- this finding should be interpreted with caution and ative diagnosis is challenging, since neither imaging correctly inserted in the pertinent clinical, radiological, nor fine-needle aspiration can established a reliable laboratory and histological framework to support the diagnosis; the most important differential diagnosis diagnosis of AI. that usually remains still open is represented by a Type 2 AIP, similar to type 1 AIP, usually shows a lym- pancreatic cystic neoplasm 46,47. From the microscop- phoplasmacytic inflammation that is electively located ic point of view (representative images are shown in in the periductal regions of pancreatic parenchyma; at Fig. 5), there is a multi-layered squamous epithelium, the same time, acinar atrophy and periductal fibrosis that can contain keratinaceous debris, surrounded by may be also present, but such findings are less pro- a dense layer of lymphoid tissue with prominent ger- nounced in type 2 AI 35-37. Notably, the peculiar and minal centers 44,45. The adjacent pancreatic parenchy- diagnostic hallmark of type 2 AI is represented by the ma is usually unremarkable, but it can occasionally presence of granulocytic epithelial lesions 35-37. They show granulomas or foci of steatonecrosis. There are are characterized by periductal acute inflammation no risk of malignant transformation but, due to the dif- consisting of aggregates of neutrophils beneath the ficulties of pre-surgical diagnosis, patients are usually ductal epithelial cells and within the lumen; such acute treated with curative surgical resection. inflammation brings to marked modifications of the Pancreatic hamartoma ductal epithelium, including cell damage, detachment and obliteration 2,35-37. Regarding IgG4 immunostain- Hamartoma is defined as a focal overgrowth of cells ing, very few positive plasma cells might be present, and/or tissues that are inborn in the organ in which or less commonly they could be totally absent 40-43. they are grown. Thus, its definition is closer to the concept of malformation rather than of a “true” neo- It is also of importance to report that, in some cases 48 of AIP, a marked inflammatory infiltrate may be seen plasm . Pancreatic hamartoma is a rare condition, of which pathologists should be aware since it can between pancreatic parenchyma and peri-pancreat- mimic a malignant tumor, sometimes with a challeng- ic adipose tissue. Notably, this feature may also be ing diagnosis. Usually, it is located in the head of the recognized by imaging, with typical radiological pan- pancreas and appears as a unique intrapancreatic creatic aspects (e.g.: mass forming lesions, “sau- mass 49, although rare cases with multiple and sepa- sage-shaped” appearance). Lastly, the evaluation of rated nodules have been described 50. From a micro- resection margins is a very important step during sur- scopic point of view, pancreatic hamartoma is com- gical resections for AIP. Indeed, pancreatic neck mar- posed of small ductal structures lined by columnar gin and biliary resection margin may be involved by epithelial cells without atypia, surrounded by fibrous the inflammatory process, and this aspect should be stroma and an unorganized acinar parenchyma, with clearly stated in the pathology report, also for thera- at least partial loss of lobular architecture 51. The pres- peutic purposes. ence of each component can vary, determining dif- ferent morphological aspects, with ductal, stromal or acinar prominence. The presence of neuroendocrine Other rare inflammatory/tumor-like islets is typically uncommon. In the case of complete/ lesions of the pancreas exclusive acinar component, acinar cell carcinoma can be ruled out thanks to some typical features of Lymphoepithelial cyst pancreatic-acinar hamartomas, including small di- The finding of a lymphoepithelial cyst in the pancreas mensions and the absence of cell atypia. Notably, a is a rare event. Usually, this is an asymptomatic le- previous consensus-paper has tried to provide relia- sion, discovered incidentally by imaging analysis due ble criteria for assessing the diagnosis of pancreatic to unrelated reasons 44. There are no associations with hamartoma, as follows: i) presence of a well-defined autoimmune or syndromic diseases, as described for mass; ii) composed of mature ductal structure and the counterpart affecting salivary glands27. From the acini with distorted architecture; iii) absence of dis- macroscopic point of view, lymphoepithelial cyst may crete islets of Langerhans 51 (Fig. 5). Typically, adja- be unilocular or multilocular, can be located or entirely cent pancreatic parenchyma is conserved. From an within the pancreatic gland or in the periphery with ex- immunohistochemical point of view, hamartomatous ophytic growth 44,45. Such cysts can reach a large size cells express all ordinary markers of the normal coun- (> 5 cm), and show an irregular capsule. The fluid con- terpart. Although morphology alone is the main diag- 206 C. Luchini et al.

nostic criterion, in some case of ductal predominance by normal pancreas. This nodule has the gross fea- the use of P53 and DPC4 staining may be of help in tures of the normal spleen. In case of large size, the ruling out a diagnosis of pancreatic ductal adenocar- center of the accessory spleen may remain reddish cinoma. The biological behavior of pancreatic hamar- also after fixation, due to the large amount of blood. toma is benign, but, due to the difficulties in pre-oper- From the microscopic point of view (representative im- ative differential diagnosis, it typically requires surgical ages are shown in Fig. 5), intrapancreatic accessory resection 27. spleen is usually composed of mature splenic tissue, showing a normal distribution of white and red pulp 52; Intrapancreatic accessory spleen CD8 is a very useful marker for the red pulp (vessels). Intrapancreatic accessory spleen is a rare finding, A very rare condition that may occur in case of intra- which occur typically in the pancreatic tail 52-55. It re- pancreatic accessory spleen is given by the internal fers to the presence of an accessory spleen growing growth of an epidermoid cyst 53. This typically affects into the pancreatic parenchyma. From a macroscopic young adults and show a multilayered squamous ep- point of view, there is a brownish nodule surrounded ithelium surrounded by splenic tissue 53. The impor-

Figure 5. Typical microscopic appearance of lymphoepithelial cysts (A, B; original magnification A: 2X, B: 10X), of intrapan- creatic accessory spleen (C, D; original magnification C:2X, D: 10X), and of pancreatic hamartomas (E, F; original magnifica- tion E:1X, F: 20X). Notably, the pancreatic hamartoma shows a ductal predominance (E, F). INFLAMMATORY PANCREATIC PATHOLOGY 207

tance of intrapancreatic accessory spleen is given by staining for example for IgG-IgG4 (AIP) or for P63 its typical differential diagnosis, which is represented (squamous content of lympho-epithelial cysts). by pancreatic neuroendocrine tumors. In some cas- Lastly, some cytological aspects should be mentioned es, fine-needle cytology with imaging can resolve this in the differential diagnosis of inflammatory and tu- diagnostic challenge, but still most cases undergo mor-like lesions of the pancreas with PDAC. Enlarged surgical resection and the diagnosis is established on and hyperchromatic nuclei and irregular nuclear mem- surgical specimens 54-55. branes support a diagnosis of PDAC. Notably, the un- differentiated PDAC variant with osteoclast-like giant cells could be misdiagnosed as PGP by cytology, due Differential diagnosis to the abundance of giant cells and necrotic-hem- orrhagic changes 30,58-60. This confirms the need of The most important differential diagnosis of inflamma- strong and reliable evidence in the difficult process of tory and tumor-like lesions of the pancreas is repre- differential diagnosis. sented by pancreatic ductal adenocarcinoma (PDAC). Different from the other inflammatory/tumor-like le- Unfortunately, in a significant proportion of cases it sions of the pancreas, the most important differential may be very difficult to differentiate inflammatory and diagnosis for intrapancreatic accessory spleen is not tumor-like lesions of the pancreas from PDAC based represented by PDAC, but by neuroendocrine tumors. on imaging, clinical presentation and laboratory mark- In some cases, fine-needle cytology with imaging can ers. Pathologists that are called to provide this distinc- resolve this diagnostic challenge. In case of persisting tion on cytology and/or fine-needle biopsy should be doubts, a strict surveillance protocol can be observed. aware that some features may be present in both in- However, many cases still undergo surgical resection, flammatory and neoplastic conditions. and the diagnosis is established on surgical speci- Histologically, the most helpful aspects in distinguish- mens with histological diagnosis. ing PDAC from reactive glands are represented by the location and structure of the glands, in addition to cy- References tological features. Reactive glands/ducts are usually 1 Cawley T. A singular case of diabetes, consisting entirely in the complete and well-demarcated, differently from neo- quality of the urine; with an inquiry into the different theories of plastic glands that are more often ruptured or incom- that disease. Lond Med J 1788;9(Pt 3):286-308. plete. The presence of “naked” glands in peri-pancre- 2 Stram M, Liu S, Singhi AD. Chronic pancreatitis. Surg Pathol Clin atic adipose tissue is very suggestive for PDAC, but 2016;9:643-59. https://doi.org/10.1016/j.path.2016.05.008 attention must be paid to the fact that the observed fat 3 Etemad B, Whitcomb DC. Chronic pancreatitis: diagnosis, clas- tissue might represent the final step of an involution sification, and new genetic developments. Gastroenterology 2001;120:682-707. https://doi.org/10.1053/gast.2001.22586 process involving acinar parenchyma (this feature is 4 Lankisch PG, Assmus C, Maisonneuve P, et al. Epidemiology of clearly distinguishable for the presence of remnant of pancreatic diseases in Lüneburg County. A study in a defined pancreatic islets) 56. Mitotic figures, necrotic luminal german population. Pancreatology 2002;2:469-77. https://doi. debris, stromal desmoplasia and aberrant DPC4 and/ org/10.1159/000064713 or P53 expression may be also of help for a definitive 5 Lowenfels AB, Maisonneuve P, Cavallini G, et al. Pancreati- PDAC diagnosis 56,57. Perineural or vascular invasions tis and the risk of pancreatic cancer. International Pancreatitis Study Group. N Engl J Med 1993;328(20):1433-7. https://doi. are both highly diagnostic of an infiltrating tumor. An- org/10.1056/NEJM199305203282001 other significant microscopic feature that can support 6 Whitcomb DC. Genetic risk factors for pancreatic disorders. the diagnosis of PDAC is the finding of neoplastic Gastroenterology 2013;144:1292-1302. https://doi.org/10.1053/j. glands in abnormal locations, such as immediately gastro.2013.01.069 adjacent to muscular blood vessels. This criterion is 7 Yadav D, Whitcomb DC. The role of alcohol and smoking in pan- very useful in the differential diagnosis with inflamma- creatitis. Nat Rev Gastroenterol Hepatol 2010;7:131-45. https:// doi.org/10.1038/nrgastro.2010.6 tory conditions, also in the case of cytostatic evalua- 8 Muniraj T, Aslanian HR, Farrell J, et al. Chronic pancreatitis, a tions. comprehensive review and update. Part I: epidemiology, etiol- Notably, it is of importance to report that ultra- ogy, risk factors, genetics, pathophysiology, and clinical fea- sound-guided fine needle biopsy is becoming a tures. Dis Mon 2014;60:530-50. https://doi.org/10.1016/j.dis- gold-standard in the pre-operative patients’ evalua- amonth.2014.11 tion for pancreatic masses. Thanks to the possibility 9 Apte MV, Wilson JS, Korsten MA. Alcohol-related pancreatic of examining a relatively significant portion of tissue, damage: mechanisms and treatment. Alcohol Health Res World 1997;21:13-20. this methodology can permit to avoid un-necessary 10 Klöppel G. Chronic pancreatitis, pseudotumors and other tumor- surgery. This kind of biopsy, furthermore, can permit like lesions. Mod Pathol 2007;20 Suppl 1:S113-S131. https://doi. in depth evaluation also with immunohistochemistry, org/10.1038/modpathol.3800690 208 C. Luchini et al.

11 Klöppel G, Maillet B. Pathology of acute and chronic pancreati- 28 Chatelain D, Vibert E, Yzet T, et al. Groove pancreatitis tis. Pancreas 1993;8:659-70. https://doi.org/10.1097/00006676- and pancreatic heterotopia in the minor duodenal papilla. 199311000-00001 Pancreas 2005;30(4):e92-e95. https://doi.org/10.1097/01. 12 Whitcomb DC, Gorry MC, Preston RA, et al. Hereditary pancre- mpa.0000161885.79373.1d atitis is caused by a mutation in the cationic trypsinogen gene. 29 Adsay NV, Basturk O, Klimstra DS, et al. Pancreatic pseudotu- Nat Genet 1996;14:141-5. https://doi.org/10.1038/ng1096-141 mors: non-neoplastic solid lesions of the pancreas that clinically 13 Whitcomb DC, Preston RA, Aston CE, et al. A gene for hereditary pan- mimic pancreas cancer. Semin Diagn Pathol 2004;21:260-7. creatitis maps to chromosome 7q35. Gastroenterology 1996;110:1975- https://doi.org/10.1053/j.semdp.2005.07.003 80. https://doi.org/10.1053/gast.1996.v110.pm8964426 30 Brosens LA, Leguit RJ, Vleggaar FP et al. EUS-guided FNA cy- 14 Lowenfels AB, Maisonneuve P, DiMagno EP, et al. Heredi- tology diagnosis of paraduodenal pancreatitis (groove pancre- tary pancreatitis and the risk of pancreatic cancer. Interna- atitis) with numerous giant cells: conservative management al- tional Hereditary Pancreatitis Study Group. J Natl Cancer Inst lowed by cytological and radiological correlation. Cytopathology 1997;89:442-6. https://doi.org/10.1093/jnci/89.6.442 2015;26:122-5. https://doi.org/10.1111/cyt.12140 31 15 Park RW, Grand RJ. Gastrointestinal manifestations of cystic fi- DeSouza K, Nodit L. Groove pancreatitis: a brief review of a brosis: a review. Gastroenterology 1981;81:1143-61. diagnostic challenge. Arch Pathol Lab Med 2015;139:417-21. https://doi.org/10.5858/arpa.2013-0597-RS 16 Witt H, Luck W, Hennies HC, et al. Mutations in the gene encod- 32 ing the serine protease inhibitor, Kazal type 1 are associated Yoshida K, Toki F, Takeuchi T et al. Chronic pancreatitis caused with chronic pancreatitis. Nat Genet 2000;25:213-216. https:// by an autoimmune abnormality. Proposal of the concept of au- doi.org/10.1038/76088 toimmune pancreatitis. Dig Dis Sci 1995;40:1561-8. https://doi. org/10.1007/BF02285209 17 Ammann RW, Heitz PU, Klöppel G. Course of alcoholic chronic 33 pancreatitis: a prospective clinicomorphological long-term study. Hamano H, Kawa S, Horiuchi A, et al. High serum IgG4 Gastroenterology 1996;111:224-31. https://doi.org/10.1053/ concentrations in patients with sclerosing pancreati- gast.1996.v111.pm8698203 tis. N Engl J Med 2001;344:732-8. https://doi.org/10.1056/ NEJM200103083441005 18 Klöppel G. Pseudocysts and other non-neoplastic cysts of the 34 pancreas. Semin Diagn Pathol 2000;17:7-15. Hirabayashi K, Zamboni G. IgG4-related disease. Pathologica 2012;104:43-55. 19 Altinel D, Basturk O, Sarmiento JM, et al. Lipomatous pseu- 35 dohypertrophy of the pancreas: a clinicopathologically dis- Zhang L, Chari S, Smyrk TC, et al. Autoimmune pancreatitis tinct entity. Pancreas 2010;39:392-7. https://doi.org/10.1097/ (AIP) type 1 and type 2: an international consensus study on MPA.0b013e3181bd2923 histopathologic diagnostic criteria. Pancreas 2011;40:1172-9. https://doi.org/10.1097/MPA.0b013e318233bec5 20 Esposito I, Hruban RH, Verbeke C, et al. Guidelines on the his- 36 topathology of chronic pancreatitis. Recommendations from the Chari ST, Kloeppel G, Zhang L, et al. Histopathologic and clinical working group for the international consensus guidelines for subtypes of autoimmune pancreatitis: the Honolulu consensus chronic pancreatitis in collaboration with the International As- document. Pancreas 2010;39:549-54. https://doi.org/10.1097/ sociation of Pancreatology, the American Pancreatic Associa- MPA.0b013e3181e4d9e5 tion, the Japan Pancreas Society, and the European Pancreatic 37 Zamboni G, Lüttges J, Capelli P, et al. Histopathological features Club. Pancreatology 2020;20:586-93. https://doi.org/10.1016/j. of diagnostic and clinical relevance in autoimmune pancreatitis: pan.2020.04.009. Epub 2020 Apr 29. a study on 53 resection specimens and 9 biopsy specimens. Vir- 21 Detlefsen S, Sipos B, Feyerabend B, Klöppel G. Fibrogen- chows Arch 2004;445:552-563. https://doi.org/10.1007/s00428- esis in alcoholic chronic pancreatitis: the role of tissue necro- 004-1140-z sis, macrophages, myofibroblasts and cytokines. Mod Pathol 38 Detlefsen S, Zamboni G, Frulloni L, et al. Clinical features and 2006;19:1019-26. https://doi.org/10.1038/modpathol.3800613 relapse rates after surgery in type 1 autoimmune pancreatitis 22 Singhi AD, Pai RK, Kant JA, et al. The histopathology of PRSS1 differ from type 2: a study of 114 surgically treated European pa- hereditary pancreatitis. Am J Surg Pathol 2014;38:346-53. tients. Pancreatology 2012;12:276-83. https://doi.org/10.1016/j. https://doi.org/10.1097/PAS.0000000000000164 pan.2012.03.055 39 23 Rebours V, Boutron-Ruault MC, Schnee M, et al. Risk of pancre- De Marchi G, Paiella S, Luchini C, et al. Very high serum levels atic adenocarcinoma in patients with hereditary pancreatitis: a of CA 19-9 in autoimmune pancreatitis: Report of four cases and national exhaustive series. Am J Gastroenterol 2008;103:111-9. brief review of literature. J Dig Dis 2016;17:697-702. https://doi. https://doi.org/10.1111/j.1572-0241.2007.01597.x org/10.1111/1751-2980.12403 40 24 Adsay NV, Zamboni G. Paraduodenal pancreatitis: a clinico- Zhang L, Chari S, Smyrk TC, et al. Autoimmune pancreatitis pathologically distinct entity unifying “cystic dystrophy of het- (AIP) type 1 and type 2: an international consensus study on erotopic pancreas”, “para-duodenal wall cyst”, and “groove histopathologic diagnostic criteria. Pancreas 2011;40:1172-9. pancreatitis”. Semin Diagn Pathol 2004;21:247-54. https://doi. https://doi.org/10.1097/MPA.0b013e318233bec5 org/10.1053/j.semdp.2005.07.005 41 Notohara K, Burgart LJ, Yadav D, et al. Idiopathic chronic pan- 25 Fléjou JF, Potet F, Molas G, et al. Cystic dystrophy of the gastric and creatitis with periductal lymphoplasmacytic infiltration: clinico- duodenal wall developing in heterotopic pancreas: an unrecognised pathologic features of 35 cases. Am J Surg Pathol 2003;27:1119- entity. Gut 1993;34:343-7. https://doi.org/10.1136/gut.34.3.343 27. https://doi.org/10.1097/00000478-200308000-00009 42 26 Casetti L, Bassi C, Salvia R, et al. “Paraduodenal” pancreatitis: Zhang L, Smyrk TC. Autoimmune pancreatitis and IgG4-related results of surgery on 58 consecutives patients from a single in- systemic diseases. Int J Clin Exp Pathol 2010;3:491-504. stitution. World J Surg 2009;33:2664-69. https://doi.org/10.1007/ 43 Zhang L, Notohara K, Levy MJ, Chari ST, Smyrk TC. IgG4- s00268-009-0238-5 positive plasma cell infiltration in the diagnosis of autoimmune 27 Basturk O, Askan G. Benign tumors and tumorlike lesions pancreatitis. Mod Pathol 2007;20:23-8. https://doi.org/10.1038/ of the pancreas. Surg Pathol Clin 2016;9:619-41. https://doi. modpathol.3800689 org/10.1016/j.path.2016.05.007 44 Adsay NV, Hasteh F, Cheng JD, et al. Lymphoepithelial cysts INFLAMMATORY PANCREATIC PATHOLOGY 209

of the pancreas: a report of 12 cases and a review of the lit- 53 Li BQ, Lu J, Seery S, Guo JC. Epidermoid cyst in intrapan- erature. Mod Pathol 2002;15:492-501. https://doi.org/10.1038/ creatic accessory spleen: a systematic review. Pancreatology modpathol.3880553 2019;19:10-6. https://doi.org/10.1016/j.pan.2018.10.008 45 Adsay NV, Hasteh F, Cheng JD, et al. Squamous-lined cysts of 54 Tatsas AD, Owens CL, Siddiqui MT, et al. Fine-needle aspiration the pancreas: lymphoepithelial cysts, dermoid cysts (teratomas), of intrapancreatic accessory spleen: cytomorphologic features and accessory-splenic epidermoid cysts. Semin Diagn Pathol and differential diagnosis. Cancer Cytopathol 2012;120:261-8. 2000;17:56-65. https://doi.org/10.1002/cncy.21185 46 Kim WH, Lee JY, Park HS, et al. Lymphoepithelial cyst of the 55 Pandey A, Pandey P, Ghasabeh MA, et al. Accuracy of appar- pancreas: comparison of CT findings with other pancreatic ent diffusion coefficient in differentiating pancreatic neuroendo- cystic lesions. Abdom Imaging 2013;38:324-30. https://doi. crine tumour from intrapancreatic accessory spleen. Eur Radiol org/10.1007/s00261-012-9910-6 2018;28:1560-7. https://doi.org/10.1007/s00330-017-5122-3 47 VandenBussche CJ, Maleki Z. Fine-needle aspiration of 56 Luchini C, Capelli P, Scarpa A. Pancreatic ductal adenocarci- squamous-lined cysts of the pancreas. Diagn Cytopathol noma and its variants. Surg Pathol Clin 2016;9:547-60. https:// 2014;42:592-9. https://doi.org/10.1002/dc.23080 doi.org/10.1016/j.path.2016.05.003 48 Goldblum J, Lamps L, McKenney J, et al. Rosai and Ackerman’s 57 Surgical Pathology. 11th ed. Philadelphia: Elsevier 2017. Hruban RH, Klimstra DS. Adenocarcinoma of the pancreas. Semin Diagn Pathol 2014;31:443-51. https://doi.org/10.1053/j. 49 Matsushita D, Kurahara H, Mataki Y, et al. Pancreatic hamar- semdp.2014.08.004 toma: a case report and literature review. BMC Gastroenterol 58 2016;16:3. https://doi.org/10.1186/s12876-016-0419-2 Luchini C, Pea A, Lionheart G, et al. Pancreatic undifferentiated carcinoma with osteoclast-like giant cells is genetically similar to, 50 Kawakami F, Shimizu M, Yamaguchi H, et al. Multiple solid but clinically distinct from, conventional ductal adenocarcinoma. pancreatic hamartomas: a case report and review of the lit- erature. World J Gastrointest Oncol 2012;4:202-6. https://doi. J Pathol 2017;243:148-54. https://doi.org/10.1002/path.4941 org/10.4251/wjgo.v4.i9.202 59 Reid MD, Muraki T, HooKim K, et al. Cytologic features and clini- 51 Yamaguchi H, Aishima S, Oda Y, et al. Distinctive histopathologic cal implications of undifferentiated carcinoma with osteoclastic findings of pancreatic hamartomas suggesting their “hamartoma- giant cells of the pancreas: an analysis of 15 cases. Cancer Cy- tous” nature: a study of 9 cases. Am J Surg Pathol 2013;37:1006- topathol 2017;125:563-75. https://doi.org/10.1002/cncy.21859 13. https://doi.org/10.1097/PAS.0b013e318283ce4c 60 Luchini C, Cros J, Pea A, et al. PD-1, PD-L1, and CD163 in 52 Kersting S, Janot MS, Munding J, et al. Rare solid tumors of pancreatic undifferentiated carcinoma with osteoclast-like giant the pancreas as differential diagnosis of pancreatic adenocarci- cells: expression patterns and clinical implications. Hum Pathol noma. JOP 2012;13:268-77. 2018;81:157-65. https://doi.org/10.1016/j.humpath.2018.07.006 PATHOLOGICA 2020;112:210-226; DOI: 10.32074/1591-951X-167

Review

Malignant epithelial/exocrine tumors of the pancreas

Claudio Luchini1, Federica Grillo2,3, Matteo Fassan4, Alessandro Vanoli5, Paola Capelli1, Gaetano Paolino1, Giuseppe Ingravallo6, Giuseppina Renzulli6, Claudio Doglioni7,8, Alessandro D’Amuri9, Paola Mattiolo1, Sara Pecori1, Paola Parente10, Ada M. Florena11, Giuseppe Zamboni1,12, Aldo Scarpa1,13 1 Department of Diagnostics and Public Health, Section of Pathology, University and Hospital Trust of Verona, Verona, Italy; 2 Anatomic Pathology, San Martino IRCCS Hospital, Genova, Italy; 3 Anatomic Pathology, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genova, Italy; 4 Surgical Pathology Unit, Department of Medicine (DIMED), University of Padua, Italy; 5 Anatomic Pathology Unit, Department of Molecular Medicine, University of Pavia, and IRCCS San Matteo Hospital, Italy; 6 Department of Emergency and Organ Transplantation, Section of Pathological Anatomy, University of Bari Aldo Moro, Bari, Italy; 7 Vita e Salute University, Milan, Italy; 8 Pathology Unit, Pancreas Translational and Clinical Research Center, IRCCS San Raffaele Scientific Institute, Milan, Italy; 9 Anatomic Pathology Unit, “A. Perrino” Hospital, Brindisi, Italy; 10 Pathology Unit, Fondazione IRCCS Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy; 11 Department of Sciences for Promotion of Health and Mother and Child Care, Anatomic Pathology, University of Palermo, Italy; 12 IRCSS Sacro Cuore Don Calabria Hospital, Negrar, Italy; 13 ARC-NET Research Centre, University of Verona, Verona, Italy

Summary Pancreatic malignant exocrine tumors represent the most important cause of cancer- Received and accepted: July 7, 2020 related death for pancreatic neoplasms. The most common tumor type in this category Published online: 29 October, 2020 is represented by pancreatic ductal adenocarcinoma (PDAC), an ill defined, stroma-rich, Correspondence scirrhous neoplasm with glandular differentiation. Here we present the relevant charac- Giuseppe Zamboni teristics of the most important PDAC variants, namely adenosquamous carcinoma, col- Department of Diagnostics and Public Health, loid carcinoma, undifferentiated carcinoma, undifferentiated carcinoma with osteoclast-like Section of Pathology, University and Hospital giant cells, signet ring carcinoma, medullary carcinoma and hepatoid carcinoma. The Trust of Verona, 37134 Verona, Italy, and other categories of malignant exocrine tumors, characterized by fleshy, stroma-poor, cir- Department of Pathology, IRCSS Sacro Cuore cumscribed neoplasms, include acinar cell carcinoma (pure and mixed), pancreatoblas- Don Calabria Hospital, 37024 Negrar, Italy toma, and solid pseudopapillary neoplasms. The most important macroscopic, histologic, Tel. 0039 045 8027618 immunohistochemical and molecular hallmarks of all these tumors, highlighting their key E-mail: [email protected] diagnostic/pathological features are presented. Lastly, standardized indications regarding gross sampling and how to compile a formal pathology report for pancreatic malignant Conflict of interest exocrine tumors will be provided. The Authors declare no conflict of interest. Key words: pancreatic cancer, pancreatic ductal adenocarcinoma, PDAC, acinar, solid How to cite this article: Luchini C, Grillo pseudopapillary F, Fassan M, et al. Malignant epithelial/ exocrine tumors of the pancreas. Pathologica 2020;112:210-226. https://doi. org/10.32074/1591-951X-167 Introduction

© Copyright by Società Italiana di Anatomia Pato- Malignant exocrine tumors of the pancreas represent the most com- logica e Citopatologia Diagnostica, Divisione Itali- ana della International Academy of Pathology mon pancreatic tumors, with pancreatic ductal adenocarcinoma (PDAC) in first place for both incidence and mortality rate. Due to the OPEN ACCESS important clinical implications that follow a diagnosis of malignant exocrine tumor of the pancreas, pathologists should be aware of the This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- most important diagnostic features and hallmarks of this group of NonCommercial-NoDerivatives 4.0 International) license: the tumors. work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original In this review, we will present this topic starting from the sampling pro- version. For further information: https://creativecommons. tocols of pancreatic resection specimens, and arriving to describe the org/licenses/by-nc-nd/4.0/deed.en most important clinical, macroscopic, histologic, immunohistochemical NEOPLASTIC EXOCRINE PANCREATIC PATHOLOGY 211

and molecular characteristics of PDAC, acinar cell free margin, to aid adequate fixation. A standardized carcinoma, pancreatoblastoma and solid pseudopap- sampling protocol may also include circumferential illary neoplasm. margin multicolor inking, which has proven to be a The most important aim of this manuscript is providing potentially important step for resection margin/surface to pathologists a serviceable and practical guideline identification 7. In the last decade, two main standard- for the correct approach to malignant exocrine tumors ized sampling protocols have been proposed, both of the pancreas, which may be encountered during with advantages and disadvantages with regards to routine diagnostic activity. Finally, a schematic list of surface/margin recognition, complexity of cut up, dis- the most important points, which should be described tinction between tumor origin (pancreatic origin ver- in the final pathology report, is provided. sus ampullary versus distal cholangiocarcinoma or cystic tumors) and lymph node yield. The protocol proposed by Verbeke, axially slices the Gross sampling specimen perpendicularly to the long axis of the du- odenum at the ampulla 8. Further slicing is continued The pancreatoduodenectomy specimen is one of the cranially and caudally, in parallel to the first slice, until most challenging surgical samples for pathologists. thin complete sections of all the pancreatic head are It requires experience and knowledge of anatomical obtained. The main advantages of this protocol are landmarks so that adequate information regarding re- the optimal evaluation of the circumferential margins/ section margins, origin/size of tumor and number of surfaces and an acceptable lymph node yield 9. lymph nodes is collected (at least 12 lymph nodes per The bi-valving protocol according to Adsay involves specimen according to the recent 2017 UICC/AJCC sectioning the specimen in two halves following the staging handbook) 1. plane between probes inserted in the pancreatic duct In patients with resectable PDAC of the pancreatic and the common bile duct 10. This method of sampling head, a R0 resection (i.e.: tumor-free resection mar- is also known as bi-valving. Prior to further section- gins) is a very important prognostic factor 2-5. Howev- ing, the inked pancreatic retroperitoneal margin (or er, R1 resection rates vary greatly between surgical superior mesenteric vessel margin) is orange peeled series and the prognostic differences between R0 and and fully submitted for histology with a bread-slicing microscopic R1 of the pancreatic neck margin/radial sampling. The bi-valved specimen is then further sec- margins have not been confirmed by all studies 2-5. tioned (either axially, multi-valved or bread loafed). The main reason for these differences is that for many Lymph node harvesting is performed by orange-peel- years non-standardized sampling protocols with little ing of the circumferential margins/surfaces and this attention to circumferential margin status have been correlates with a high lymph node yield. Further ad- performed. Along this line, sampling protocols for vantages of this technique are the optimal evaluation pancreatic specimens have been under scrutiny for of primary site of tumor (pancreatic, ampullary or some years as the importance of standardization in common bile duct) and the complete evaluation of the specimen handling and sample selection have come retroperitoneal margin. to light 6. The pancreatoduodenectomy specimen requires mul- A representative image of gross sampling is provided tiple samples, including a tumor-representative sam- in Figure 1. pling, the relationship of tumor with adjacent struc- tures (e.g. common bile duct), transection margins Pancreatoduodenectomy specimen handling and (pancreatic, common bile duct, gastric/duodenal) and, sampling protocols above all in cases of extensive tumor infiltration, the Pancreatoduodenectomy specimens may vary with circumferential resection margin(s)/surfaces. Attention regards to which organs/parts of organs are present must be paid in the case of resection for precursors of in the specimen such as: the distal portion of stomach pancreatic cancer (e.g.: intraductal papillary mucinous in Whipple’s procedure (while pylorus preserving pan- neoplasm), in which an infiltration may be very focal, createctomies will only have the duodenal bulb as the thus an extensive sampling is required 11. Along this proximal margin), gall bladder and extrahepatic bile line, an extensive sampling is also of importance in ducts, partial or complete venous resections (of the the setting of post-neoadjuvant therapy, where a pre- superior mesenteric vein or portal vein), which should cise gross definition of tumor boundaries is not often be annotated by surgeons. recognizable 12. Once the specimen is received (ideally fresh speci- Among various issues frequently encountered during mens for better evaluation and biobanking), the stom- routine sampling activity, the most frequent regard the ach/duodenum/jejunum should be opened along the potential misunderstanding about specific nomen- 212 C. Luchini et al.

Figure 1. These images show the different sampling techniques for duodeno-pancreatic head resections. Margins are inked in different colors: green - anterior surface; blue - superior mesenteric vein groove; red - uncinate process/medial/retroperitoneal/ superior mesenteric artery margin; black (minimum portion shown) - posterior surface. - Axial Slicing Technique (upper panel): (A) initially, a horizontal section perpendicular to the duodenum and passing through the ampulla is performed; (B) parallel sections are then obtained; (C) complete section of duodenum with pancreatic head, main pancreatic duct, bile duct and tumor (whitish area). The circumferential margins/surfaces are shown with respective colors. - Bi-valving Technique (lower panel): (A) the red uncinate process/medial/retroperitoneal/superior mesenteric artery margin is shaved and then bread-sliced and submit- ted entirely; (B) a horizontal section passing on a plane which passes through the probed bile duct and pancreatic duct is shown. Further sections can be parallel (or even perpendicular); (C) complete section of the bi-valved specimen showing the tumour (whitish area), which arises from the pancreas and invades the bile duct and the main pancreatic duct.

clature of circumferential margins/surfaces. Indeed, left to right orientation, while the splenic artery and the superior mesenteric vessel margin can be also vein, which run along the posterior-superior groove of named as uncinate margin; the posterior aspect of the the pancreatic body and tail, permit cranial to caudal head of the pancreas can be intended both as a mar- orientation. Inking with different colors is not manda- gin, behind the pancreatic transection margin, and as tory, but can be useful for distinguishing anterior/pos- a surface, towards the duodenum 13. Another possi- terior surfaces and the superior/inferior aspects. The ble issue includes the definition/concept of “positive” specimen should be sliced in sections, parallel to the margin, since it can represent a direct invasion of ne- transection margin. This method permits the identifi- oplastic cells on the margin, but can be also defined cation of the distance between the tumor and the tran- as a distance < 1 mm between tumor cells and the section margin, as well as aspects of adipose tissue margin 14. infiltration and splenic vessels invasion. Lymph nodes can be retrieved from the peri-pancreatic soft tissue Distal Pancreatectomy specimen handling and and from the splenic hilum; the most important site for sampling protocol lymph node sampling is along the course of splenic Distal pancreatectomy specimens do not present sig- vessels. nificant issues in orientation and landmark identifica- tion typical in pancreatic head specimens. Indeed, the Surgical resections after neo-adjuvant therapy spleen and the pancreatic transection margin permit Surgical resections after neoadjuvant therapy are be- NEOPLASTIC EXOCRINE PANCREATIC PATHOLOGY 213

coming one of the most frequent scenario in pancre- lial Neoplasm (PanIN), Intraductal Papillary Muci- atic pathology, above all in specialized centers. These nous Neoplasm (IPMN), Intraductal Oncocytic pap- resections are often warp and diffusely whitish, with illary Neoplasms (IOPN), Intraductal Tubulopapillary tumor area with blurred borders or even not visible. Neoplasm (ITPN), and Mucinous Cystic Neoplasm In such cases, an extensive sampling should be per- (MCN) 16,20-23. All these lesions can progress into the formed, and in cases of not identifiable tumor areas or invasive counterpart, which is represented by PDAC. of small tumor beds, they should be entirely included. PanIN is a microscopic, non-infiltrative epithelial ne- Sometimes surgical resections include complex ves- oplasm confined to the pancreatic ducts; it can show sel resections (e.g.: celiac trunk, splenic-portal-mes- a flat or a micropapillary epithelium 16,20. PanINs are enteric confluence), which should be correctly isolat- classified with a two-tiered grading system: low grade ed during gross sampling and described in the final and high grade dysplasia PanIN 15,24. Due to their mi- pathology report 15. croscopic nature, PanINs are asymptomatic. Notably, high-grade PanINs represent the main precurosor of PDAC 16,20. From the molecular point of view, KRAS Pancreatic ductal adenocarcinoma mutations have been described as an early event in PanIN formation and PDAC carcinogenesis, as well Definition and terminology as SMAD4 and TP53 mutations represent late events Pancreatic ductal adenocarcinoma (PDAC) is the in this model (high-grade PanIN) 16,20-23. most common type of pancreatic cancer 16. It is a ma- IPMNs are intraductal neoplasms of the pancreas, lignant neoplasm composed of infiltrating glands that with papillary mucinous epithelium. They are by defi- resembles pancreatic ducts. Synonyms include duct nition > 5 mm and can involve Wirsung’s duct (main cell adenocarcinoma, infiltrating duct carcinoma or tu- duct IPMN), branch ducts (branch duct IPMN) or both bular adenocarcinoma 16. (mixed IPMN) 16,20-23. Main duct IPMN present a higher risk of malignant transformation and of metachronous Clinical and gross features recurrences compared to branch duct 25,26. Based on About two-thirds of PDAC arise in the pancreatic the type of epithelium, IPMNs can be subdivided into head, with the rest affecting equally the body and/or intestinal, gastric and pancreatic-biliary; notably, in the the tail of the gland equally. Clinical features include current WHO classification, IOPN are considered sep- back pain, dyspepsia, weight loss and new-onset di- arately from the other IPMN subtypes 16,20. Similarly abetes; jaundice is a symptom typical of pancreatic to PanIN, the dysplasia of IPMN is also classified as head tumors. Rarely, acute pancreatitis and migrato- low- and high-grade. High-grade IPMN can progress ry thrombophlebitis may be seen in PDAC patients. to invasive PDAC, which can be indistinguishable Important risk factors for PDAC are tobacco smoking, from conventional PDAC, with the exception of colloid obesity and alcohol consumption. The highest rates carcinoma, a distinct PDAC subtype producing abun- of PDAC incidence have been reported among black dant extracellular mucin, which originates from intes- people in the USA. An increased risk for PDAC is al- tinal-type IPMN 26-28. From a genetic point of view, the so described for patients with chronic pancreatitis and most important alterations in IPMN include KRAS and diabetes mellitus, and for patients with a previously GNAS mutations, and microsatellite instability, which resected PDAC 16,17. is more common in the intestinal subtype 16,22,29. From a macroscopic point of view, PDAC are usually IOPN is a grossly exophytic, intraductal neoplasm a firm and poorly defined whitish mass with irregular composed by oncocytic glandular epithelium, which borders. Usually they infiltrate surrounding structures, is more frequent in females. The most important dis- including peri-pancreatic adipose tissue, the duode- tinctive features are the almost exclusive presence of nal wall, the distal choledocus and the ampulla of high grade dysplasia and the lack of the most frequent Vater. Cancers < 2 cm are quite rare (about 10% of somatic mutations of IPMN 16. PDAC) 16,18. The most recent TNM staging system is Macroscopically, the lesions are characterized by mainly based on tumor size, with pT1 for tumors with large, solid or papillary mass, which lack mucin. Mi- a diameter ≤ 2 cm, pT2 for tumors ranging from 2,1 croscopically, the lesion is constituted by complex, to 4 cm, pT3 for tumors > 4 cm, and pT4 for tumors arborizing papillae, lined by cuboidal cells with eosin- involving celiac axis, superior mesenteric artery, and/ ophilic granular cytoplasm, filled with mitochondria, or common hepatic artery, regardless of size 19. intracytoplasmic lumen and large nuclei. Almost all IOPNs show high grade dysplasia, whereas inva- Precursors of pancreatic ductal adenocarcinoma sive carcinoma occurs in only 30% of cases. Immu- Precursors of PDAC include: Pancreatic Intraepithe- nohistochemically, IOPNs show MUC6 and MUC1 214 C. Luchini et al.

positivity, with focal positivity for MUC5AC, whereas micro-invasive areas should be established by experi- immunostaining for MUC2 and CDX2 is restricted to enced pancreatic pathologists or at least discussed at sparse goblet cells. IOPNs lack the common somatic a multi-headed microscope. mutations of IPMN, like KRAS and GNAS, whereas they can show mutations in ERBB4 and the distinctive Microscopic description with diagnostic hallmarks PRKACA/B gene fusions. The 5-year disease-specific Most PDACs are composed of well to moderately dif- survival approaches 100%. ferentiated duct-like and glandular structures, invading ITPN are intraductal neoplasms of the pancreas, with the pancreatic parenchyma 16. They are characterized a typical tubulopapillary appearance and usually with by an irregular growth pattern, which is typically as- high-grade dysplasia 16. From the histologic point of sociated with a usually marked desmoplastic stromal view, ITPN form nodules of back-to-back tubular reaction. Indeed, PDACs exhibit an abundant stroma glands, with a large, cribriform architecture; an associ- and consequently can be considered as hypocellular ated invasive carcinoma is found in up to 70% of cas- tumors. A very important feature, which can be easily es 16. From a molecular point of view, ITPN are very used to discriminate neoplastic/infiltrative glands vs. peculiar, with no KRAS mutations; they show more normal ducts is represented by the fact that neoplas- frequently KMT2 and PIK3-pathway gene mutations tic glands are very commonly ruptured with areas of (PI3KCA, PTEN), and a subset of ITPN harbor FGFR2 epithelial denudation of the gland, while normal ducts fusions 16,20-23. show a continuous epithelial lining. Histologically, MCNs are cyst-forming and mucin-producing epithe- PDAC are graded into a 3-tiered system: well, mod- lial neoplasm; they are associated with the presence erate and poorly differentiated PDAC, based on com- of an ovarian-like sub-epithelial stroma 16,20-23. MCNs bined assessment of the degree of glandular differen- usually affect middle-aged women and arise in the tiation, mucin production, mitotic activity and nuclear body-tail region of the pancreas. They typically do not features 16. Representative images of PDAC grading communicate with the Wirsung’s duct. Similarly to Pa- are provided in Figure 2, as well as a case of PDAC nIN and IPMN, the dysplasia of MCN is also classified in the neoadjuvant setting, where no grading should into low- and high-grade. From a molecular point of be applied because it is unreliable due to iatrogenic view, KRAS, TP53 and RNF43 mutations are the most modifications of tissues. common mutations in MCN 16,20. Well-differentiated PDACs are composed of haphaz- Although there is no useful immunohistochemical ardly arranged invading and medium-size duct-like panel for the diagnosis of PanIN and MCN, for IPMN and glandular structures, with angular or irregular and ITPN there are some mucin-based stains that can shapes 16,30. Neoplastic cells are generally columnar be of much help in supporting diagnosis and identify- or cuboidal, forming a single cell layer with eosino- ing the correct histologic subtype (Tab. I) 16,20. philic cytoplasm; the nuclei are round or oval and One of the most difficult challenges in pancreatic pa- may be even four times larger than non-neoplastic thology regards the identification of foci of micro-in- nuclei 16,30. This comparison is often feasible since in filtration originating from precursor lesions. The hall- many PDACs some normal ducts are trapped within mark of an invasive component is represented by the the tumor mass. The nuclear membranes are well-de- presence of desmoplastic stroma around invasive, lineated and the nucleoli, which generally are two or atypical glands. Notably, an important diagnostic pitfall more, are often prominent; mitoses are not common. is represented by the differential diagnosis between Lymphatic and blood vessel invasion/embolization, mucin extravasation in the periphery of lesions vs. perineural infiltration as well as lymph node metas- true stromal invasion. The assessment of eventual tases represent very common findings 16,30. A pecu-

Table I. Immunohistochemical markers for IPMN/IOPN/ITPN classification. Type of lesion Subtype MUC1 MUC2 MUC5AC MUC6 CDX2 IPMN G negative negative positive negative negative PB positive negative positive positive negative INT negative positive positive negative positive IOPN positive negative positive positive negative ITPN positive negative negative positive negative Abbreviations: IPMN: intraductal papillary mucinous neoplasm; IOPN: intraductal oncocytic papillary neoplasm; ITPN: intraductal tubulo-papillary neoplasm; G: gastric; PB: pancreatic-biliary; INT: intestinal. Note: if the positivity of a marker is very intense at the immunohistochemical level and very important for diagnostic purposes, it is indicated in bold. NEOPLASTIC EXOCRINE PANCREATIC PATHOLOGY 215

Figure 2. (A,B,C) Representative images of the grading system for pancreatic ductal adenocarcinoma: G1 (A, original mag- nification 20X), G2 (B, original magnification 20X) and G3 (C, original magnification 20X). D) In the setting of neo-adjuvant therapy, pancreatic ductal adenocarcinoma should not be graded, due to the unreliability of grading for the iatrogenic modifi- cations of tissues, including therapy-induced cell atypia (original magnification 20X).

liar morphological aspect of lymph node metastasis, epithelial-mesenchymal transition 34,35. which is the extra-nodal extension of neoplastic cells Some important morphologic features represent true in the peripheral adipose tissue, has recently been as- diagnostic PDAC hallmarks. They include the irregular sociated with worse prognosis in many cancer types, structural tumor pattern, the abundant desmoplastic including PDAC 31-33. stromal reaction and the presence of “ruptured” or Moderately differentiated PDACs are similar to the “incomplete” glands 16,30. Furthermore, vascular and well-differentiated counterpart: glandular and duct-like perineural invasions are both highly diagnostic of an structures, as well as the cellular and nuclear size, how- infiltrating tumor. Another diagnostic PDAC hallmark ever, have a greater variation. Usually, tumor glands are is represented by neoplastic gland topographic loca- smaller and neoplastic cells are larger, with more mitot- tion: indeed, the infiltrating malignant glands are of- ic figures; mucin production appears to be less evident ten found in abnormal locations, such as immediately compared to well-differentiated PDAC 16,30. adjacent to muscular blood vessels 30,36. This criterion Poorly differentiated PDACs are composed of small is helpful in the differential diagnosis with chronic pan- glands with irregular shape and of individual atypical creatitis and for supporting the diagnosis of PDAC in cells; from the architectural point of view, poorly differ- case of low-atypia (e.g.: in surgical specimens after ential PDAC are densely packed tumors, sometimes neo-adjuvant therapies, where the degree of cellular with an arrangement in solid sheets and nests 16,30. atypia may be very mild) 16,30. Neoplastic cells exhibit pronounced nuclear pleomor- phism, scanty or no mucin production and high mitotic Variants of pancreatic ductal adenocarcinoma activity. Areas with neoplastic cells organized in small The 2019 WHO classification distinguishes different aggregates or infiltrating as single elements are com- PDAC variants, which are: adenosquamous carcino- mon, and represent so-called PDAC tumor budding, ma, colloid carcinoma, medullary carcinoma, poor- strictly correlated with the activation of the process of ly cohesive carcinoma/signet-ring cell carcinoma, 216 C. Luchini et al.

hepatoid carcinoma, large cell rhabdoid carcinoma, tumor cells composing at least 80% of tumor mass 16. undifferentiated carcinoma and undifferentiated carci- This variant usually originates from intestinal-type IP- noma with osteoclast-like giant cells 16. Although some MN 27,28. Immunohistochemical markers of colloid car- PDAC variants may show some peculiar macroscopic cinoma reflect its intestinal profile, with positivity for aspects, there are no definitive criteria to provide such MUC2, CK20 and CDX2 16. From a molecular point of a distinction by gross examination. At the same time, view, colloid carcinoma are enriched of GNAS mutations it has to be noted that colloid carcinoma is charac- (up to 60% of cases) and microsatellite instability 40,41. terized by large pools of mucin, and undifferentiated In pancreatic cancer, this alteration can be investigat- carcinoma with osteoclast-like giant cells frequently ed with immunohistochemistry for the mismatch repair shows foci of hemorrhage and/or necrosis. The other protein MLH1, PMS2, MSH2 and MSH6. In cases of variants do not display peculiar gross aspects. doubtful results, MSI-based polymerase chain reaction From a microscopic point of view, there are some or next generation sequencing should be performed 42. specific features that characterize the different PDAC Medullary carcinoma is a poorly differentiated carci- variants. Representative images of the most important noma with limited gland formation, a pushing border, variants are presented in Figure 3. a syncytial growth and several tumor-infiltrating lym- Adenosquamous carcinoma is composed of signifi- phocytes 41,43. This variant is usually associated with cant proportions with both ductal and squamous (at microsatellite instability, but a recent study highlighted least 30%) differentiation 16,30. The squamous compo- that it can also arise in a hypermutated, non-microsat- nent can also be highlighted with immunohistochem- ellite related genetic context, in association with POLE istry for p63/p40 34. From a molecular point of view, mutation 41-44. this variant typically harbors KRAS, TP53 and UPF1 Poorly cohesive/signet ring cell carcinoma is com- mutations 37,38. The prognosis is very poor 39. posed of at least 80% of individual poorly cohesive Colloid carcinoma is characterized by the presence of cells, often with intracellular mucin vacuoles peripher- large extracellular stromal mucin pools with suspended ally displacing the nuclei 45.

Figure 3. Representative images of the most relevant pancreatic ductal adenocarcinoma variants. (A) Adenosquamous carcinoma (original magnification 20X); (B) colloid carcinoma (original magnification 20X); (C) undifferentiated carcinoma (original magnification 20X); (D) undifferentiated carcinoma with osteoclast-like giant cells (original magnification 20X). NEOPLASTIC EXOCRINE PANCREATIC PATHOLOGY 217

Hepatoid carcinoma is an extremely rare neoplasm, but not MUC2, which has a staining pattern very sim- defined as a carcinoma in which at least 50% of cells ilar to Keratin 20 52,53. display histological and immunohistochemical evi- PDACs are usually negative for pancreatic exocrine dence of hepatocellular differentiation 16,30. They are enzymes such as trypsin, chymotrypsin and lipase, composed of large, polygonal cells resembling those and for the acinar-related marker Bcl-10, as well as of hepatocellular carcinoma. Alpha-fetoprotein, Hep- for neuroendocrine markers, including chromogranin par1, CD10 and arginase are useful immunohisto- A, synaptophysin, CD56 and CD200 16,30,49,54. Regard- chemical markers to support such diagnosis, although ing “immune-molecular” staining, the DPC4 protein is the main diagnostic criterion remains morphology 16. lost in up to 55% of PDACs, and the p53 protein is ex- Rhabdoid carcinoma is a malignant neoplasm com- pressed in most cases 16,30,49,55. These two IHC mark- posed of large cells with rhabdoid features; it frequent- ers may be very useful for the differential diagnosis ly harbors KRAS mutations/amplification, and in the between infiltrating vs. reactive glands. case of KRAS wild-type, a typical SMARCB1 (INI1) Regarding molecular pathology, the most important immunohistochemical loss has been reported at the alterations that are considered as the main genetic nuclear level 16,45. drivers of PDAC are somatic mutations affecting four Undifferentiated carcinoma is a malignant epithelial key genes: the oncogene KRAS and the tumor sup- neoplasm in which the majority of tumor mass does pressors CDKN2A, TP53 and SMAD4 56,57. KRAS is not show any definitive direction of differentiation16. the most common mutated gene in PDAC (up to 90- Different from conventional PDAC, undifferentiated 92% of cases); typical mutations occur in codons 12, carcinomas are hypercellular tumors. They can be 13 and 61 56-59. Microsatellite instability is a genetic stained with cytokeratin (CKAE1/3, CK8/18) and ex- alteration that can occur in up to 2% of PDAC; howev- press an immunohistochemical profile associated with er, it is more common in colloid and medullary variant an activated epithelial-mesenchymal transition (ex- than in conventional PDAC, where it probably repre- pression of vimentin, Twist1 and Snai2, and E-Cad- sents a percentage < 1% of all cases 41. herin loss) 16,46,47. The undifferentiated carcinoma with osteoclast-like Differential diagnosis giant cells variant (UCOGC) is composed by three The most important differential diagnoses for conven- cell types: spindle and/or pleomorphic cells, the real tional PDAC are summarized in Table II. The first to be neoplastic cells, mononuclear histiocytes and osteo- considered is represented by chronic pancreatitis 30,60. clast-like giant cells 16,46-48. Osteoclast-like giant cells From radiologic and macroscopic points of view, chron- can show over 20 nuclei each and do not have a ma- ic pancreatitis may share several features with PDAC. It lignant behavior. This variant shows a genetic profile is important to underline that chronic pancreatitis with very similar to conventional PDAC, and seems en- alcoholic or obstructive etiology usually involves the riched in SERPINA3 mutations 49. Its unique immuno- pancreas more widely than a neoplastic process, with a logic microenvironment is dominated by the presence more rubbery and with a more gritty consistency of the of tumor-stimulating histiocytes CD163-positive 50. In parenchyma than in PDAC. From a microscopic point of cases with the absence of an associated PDAC com- view, the most important aspects to support the distinc- ponent, UCOGC prognosis may be very favorable tion between chronic pancreatitis and PDAC are certain- with prolonged/long-term survivals 48-50. ly represented by the presence of vascular and neural invasion, and lymph node metastasis. Notably, the fibro- Immunohistochemistry and Molecular Pathology sis of chronic pancreatitis may mimic the desmoplastic An unequivocal immunohistochemical panel that can reaction of PDAC, and the degree of cell atypia should be used to discriminate PDAC from other extrapan- not be used as the most important criterion to rule out creatic mucin-producing adenocarcinomas does not chronic pancreatitis, where the protracted inflammation exist. However, some markers may be of help in sup- may generate reactive cell alterations. porting a PDAC diagnosis. First of all, PDACs express Autoimmune pancreatitis is another potential mim- the same keratins as the normal pancreatic duct epi- icker of PDAC, since it can present as a discrete, tu- thelium, such as keratins 7, 8, 18 and 19 16,30; indeed, mor-like lesion of the pancreas 61-67. The histological the highest sensibility and specificity is reached by hallmarks of autoimmune pancreatitis are represented the 8-18 cocktail. Keratin 20, a typical intestinal-epi- by a dense inflammation centered around pancreatic thelial marker, is not expressed by PDAC cells, with ducts, a cellular fibro-inflammatory stroma with a stori- the exception of infiltrating tumors arising from intesti- form appearance and a diffuse obliterative venulitis 61- nal-type IPMN 51. Among mucins, PDAC cells are usu- 67. A further potentially helpful analysis may include ally stained with IHC for MUC1, MUC4 and MUC5AC, an immunohistochemical evaluation of plasma cells 218 C. Luchini et al.

Table II. Most relevant differential diagnoses for pancreatic ductal adenocarcinoma (PDAC), highlighting the histological aspect against PDAC diagnosis. PDAC versus: Useful features versus PDAC Chronic pancreatitis Ductal lithiasis, dense inflammatory infiltration, no clear signs of infiltrating tumor (e.g.: nodal metastasis). Autoimmune Marked inflammation with abundant plasma cells concentrated around pancreatic ducts, obliterative venulitis, pancreatitis cellular fibro-inflammatory stroma with a storiform appearance An increased numbers of IgG4 plasma cells. Reactive glands Location of glands (the presence of “naked” glands in fat is very suggestive for PDAC), absence of mitotic figures, absence of stromal desmoplastic reaction. Precursor lesions The presence of a possible infiltrating component, which lacks in non-infiltrating precursor lesions, should be excluded through an extensive sampling. and of their IgG4+ sub-population, which is typically re. They include acinar cell carcinoma, along with increased above all in type 1 autoimmune pancrea- its mixed subtypes (mixed acinar-ductal carcinoma, titis 64-67. mixed acinar-neuroendocrine carcinoma, mixed aci- Another important differential diagnosis that can be nar-neuroendocrine-ductal carcinoma), and pancre- encountered during routine diagnostic activity is repre- atoblastoma 16. In addition to these malignant neo- sented by reactive glands. From a microscopic point of plasms, the pancreas may undergo the so-called “ac- view, the most helpful findings in distinguishing PDAC inar cystic transformation”, previously known as acinar from reactive glands are represented by the location cell cystadenoma, a non-neoplastic cystic lesion lined and the architecture of glands, in addition to the cyto- by acinar and ductal non-atypical cells, with no evi- logical aspects 16,30. The presence of “naked” glands dence of recurrence or malignant transformation. in fat is a histological hallmark of PDAC, but attention must be paid to the fact that the adipose tissue may Acinar Cell Carcinoma represent the result of adipose involution of normal pancreatic parenchyma rather than the true peri-pan- Clinical and gross features creatic adipose tissue. In this case, residual neuroen- Acinar cell carcinomas (ACC) are rare neoplasms, ac- docrine islets should be documented in fat tissue. Oth- counting for only 1-2% of all pancreatic neoplasms in er features, that can support PDAC diagnosis are the adults. The median age at ACC diagnosis is 60 years; presence of mitotic figures, necrotic luminal debris and however, it may also rarely affect children. Although stromal desmoplastic reaction. ACC is predominantly located in the pancreatic head, The suggested immunohistochemical analysis to sup- jaudice is a rare symptom. Some patients may devel- port a PDAC diagnosis in cases of doubtful histology op a lipase hypersecretion syndrome. The ACC patient include P53 and DPC4. P53 aberrant staining patterns, prognosis is poor, with a 5-year survival rate of 25%. which are strictly correlated with TP53 mutations, are Grossly, ACC are large, well-circumscribed and solid represented by a complete loss of immunostaining or mass, with a homogeneous, fleshy cut surface 70-73. by a diffuse nuclear staining in at least 60-70% of tu- Necrotic or hemorrhagic areas may be present. In rare mor cells 49,68. DPC4 aberrant staining pattern, which instances, the so-called “acinar cell cystadenocarci- is strictly associated with SMAD4 mutations, is repre- nomas”, gross appearance is entirely cystic. sented by loss of DPC4 nuclear expression 69. Rarely, PDAC can exhibit a cystic appearance, thus Microscopic description with diagnostic hallmarks the differential diagnoses include in such cases differ- Histologically, ACC is a highly cellular tumor, with ent types of cystic lesions. The cystic appearance may scarce stroma. Tumor cells show granular eosino- represent the macroscopic phenotype of a microscop- philic, PAS-diastase-positive cytoplasm and relatively ic epiphenomenon (e.g.: PDAC intraductal coloniza- uniform nuclei with a characteristic single prominent tion of a retention cyst or of a congenital cyst). eosinophilic nucleolus 16,70-72. Several architectural pat- terns (acinar, glandular, trabecular and solid), as well as the intraductal growth pattern have been described. Acinar neoplasms The intraductal growth pattern is generally associated with a better prognosis. A representative image of an Definition and terminology acinar cell carcinoma is provided in Figure 4A. Although the acinar component constitutes the vast majority of the pancreatic parenchyma, neoplasms Immunohistochemistry and molecular pathology showing acinar cell differentiation are relatively ra- Immunohistochemistry is of pivotal importance to con- NEOPLASTIC EXOCRINE PANCREATIC PATHOLOGY 219

firm the acinar cell differentiation. Trypsin, chymotrypsin TNM stage is the most important prognostic factor for and BCL-10 (clone 331.3, recognizing the COOH-termi- ACC; they are staged with the same TNM staging sys- nal portion of carboxil ester lypase) are the most sen- tem adopted for pancreatic ductal adenocarcinoma. sitive and specific immunohistochemical markers rec- ommended for diagnosis. Pancreatic ductal markers, Mixed acinar carcinomas such as cytokeratins 7 and 19, as well as hepatocellular Mixed carcinomas (mixed acinar-ductal carcinoma, markers, such as HepPar-1, glypican 3, and alphafeto- mixed acinar-neuroendocrine carcinoma, mixed aci- protein, may be expressed by ACC 16,70-72. nar-neuroendocrine-ductal carcinoma) are defined as Alterations in the Wnt/β-catenin pathway have been carcinomas composed of two or three different com- frequently found, often due to APC promoter hyper- ponents, each constituting at least 30% of tumor vol- methylation 74. Microsatellite instability has been de- ume 16. tected in about 10% of cases, while an ACC subset Mixed acinar-neuroendocrine carcinomas account harbors potentially targetable gene fusions involving for about 20% of ACC (representative images are BRAF or RAF1 42,75. provided in Fig. 4B, C, D) Most acinar-neuroendo- crine carcinomas do not display two morphologi- Differential diagnosis cally distinct components, as the neuroendocrine The main differential diagnoses include: pancreato- differentiation is only recognized by immunohisto- blastoma, pancreatic neuroendocrine neoplasm, solid chemistry. Such mixed acinar-neuroendocrine carci- pseudopapillary neoplasm, as well as medullary and nomas do not fit the diagnosis of mixed neuroendo- hepatoid variants of pancreatic ductal adenocarcino- crine-non-neuroendocrine neoplasm (MiNEN), and ma (Tab. III). should be regarded as a subtype of ACC. Indeed,

Table III. Histologic and immunohistochemical features helpful in the differential diagnosis of acinar and solid-pseudopap- illary neoplasms Acinar cell Neuroendocrine Important Tumor type/ Prominent Architectural Squamoid Necrosis Stroma marker marker markers for subtype nucleoli patterns nests expression expression diagnosis Acinar cell Yes Acinar, No Frequent Fibrous, Ye s No/focal, Bcl10, trypsin carcinoma glandular, scarce >30% in mixed trabecular, solid Pancreato- Ye s Acinar, Ye s Possible Fibrous, often Ye s Yes, often focal Bcl10, trypsin; blastoma trabecular, solid hypercellular EMA-CK5-P63 (SN) Pancreatic Rare Nesting, No Very rare Highly No Yes, diffuse and Chromogranin, NET trabecular, vascular, strong Synaptophysin, glandular, solid hyalinized Ki67 NEC/large Frequent Nesting/ No Frequent Desmoplastic- No Ye s Synaptophysin, cell trabecular, type TP53 occasional rosettes NEC/small No Diffuse sheets No Frequent Desmoplastic- No Ye s Synaptophysin, cell type TP53, Rb Medullary Ye s Solid, syncytial No Possible Lymphocyte- No No MMR proteins carcinoma rich Hepatoid Ye s Trabecular, No Possible Scarce, No Rare Hep-par1, carcinoma glandular, solid occasionally Arginase-1 lymphocyte- rich SPN No Solid, No Possible Fibrovascular, No No/focal β-catenin pseudopapillary hyalinized (nuclear), CD10, Progesteron receptor, LEF1, Vimentin, CD200 Abbreviations: NEC: neuroendocrine carcinoma; NET: neuroendocrine tumor; SN: squamoid nests; SPN: solid pseudopapillary neoplasm; MMR: mismatch repair. 220 C. Luchini et al.

Figure 4. (A) Representative microscopic image of an acinar cell carcinoma, with aspects of intraductal growth (original magnification 20X); (B,C,D) Representative images of a mixed neuroendocrine-acinar neoplasm: B: hematoxylin-eosin stain- ing (original magnification 10X), C: immunohistochemistry for synaptophysin (original magnification 20X), D: immunohis- tochemistry for trypsin (original magnification 20X); (E) representative histological image of a pancreatoblastoma, including a squamous nest, indicated with a black arrow (original magnification 20X); (F) representative microscopic image of a solid- pseudopapillary neoplasm (original magnification 10X).

their prognosis is similar to conventional ACC 72. sociated with Beckwith-Wiedenn syndrome and familial However, rare true pancreatic MiNENs composed adenomatous polyposis syndrome (FAP), type 1. of separate acinar and neuroendocrine components Grossly, they are usually large, solid tumors, with soft have been reported 76. In such cases, the neuroendo- yellowish lobules surrounded by fibrous bands on cut crine component should be graded and reported in surface. Some cases may have a cystic appearance. the final pathology report. Histologically, the neoplasm shows a lobulated ar- Mixed acinar-ductal carcinomas have either mucin de- chitecture, with highly cellular lobules separated posits or a separate component strongly resembling a by fibrous hypercellular septa. Most tumor cells are ductal adenocarcinoma, in addition to an acinar compo- morphologically similar to those of ACC and most of nent. Mixed acinar-neuroendocrine-ductal carcinomas them express acinar cell markers. The presence of a have a mixed composite morphology, with an immuno- variable number of squamoid nests, composed of ep- histochemical profile supporting the three lines of dif- itheliod-to-splindle cells showing squamous differenti- ferentiation. Mixed acinar-ductal or acinar-neuroendo- ation, is the histologic hallmark of the neoplasm and is crine-ductal carcinomas usually have a poor prognosis. crucial to establish correct diagnosis. A representative histologic image is provided in Figure 4E. Neoplastic Pancreatoblastoma cells within squamous nests may show nuclear clear- Pancreatoblastoma is a rare malignant neoplasm with ing and abnormal nuclear or cytoplasmic positivity for potential multiple lines of differentiation 16. Acinar cell β-catenin; CD200 may be also positive in the case of differentiation is usually prominent. The median patient nuclear β-catenin staining 53,77,78. age at diagnosis is 3-4 years; however, cases in adults Pancreatoblastomas may also express general mark- have been described. Pancreatoblastoma may be as- ers of neuroendocrine differentiation (including CD200) NEOPLASTIC EXOCRINE PANCREATIC PATHOLOGY 221

and AFP. Molecular features of pancreatoblastoma are macroscopically visible pseudopapillae; on the other loss of 11p and alteration in Wnt/β-catenin pathway 79. hand, cystic areas are often filled with hemorrhagic Pancreatoblastomas may have ad indolent behavior, and necrotic debris and may show calcification. Small- but the presence of metastasis and of a non-operable er lesions tend to be entirely solid, while other lesions disease represent adverse prognostic factors. may be completely cystic.

Microscopic description with diagnostic hallmarks Solid pseudopapillary neoplasms The microscopic features of SPN recapitulate its macroscopic appearance, with a mixture of solid, Definition and terminology pseudopapillary and cystic-hemorrhagic areas. A Solid pseudopapillary neoplasm (SPN) of the pan- representative histological image is provided in Fig- creas is a low grade malignant epithelial tumor with ure 4F. uncertain cellular differentiation, first described by VK Solid areas are composed of loosely cohesive mono- Frantz in 1959 80. It is a rare neoplasm, representing morphic polygonal cells admixed with capillary-sized 0.9-2.7% of all pancreatic exocrine tumors 81; none- blood vessels. In pseudopapillary areas, there is par- theless, it accounts for 30% of all pancreatic neo- tial detachment of the neoplastic cells; the remain- plasms in patients younger than 40 years of age 82. ing elements are left to surround the blood vessels, SPNs were previously called “tumors” and considered therefore forming structures resembling papillae. The to be benign or borderline lesions. They are now de- neoplastic cells are characterized by eosinophilic fined as malignant due to their demonstrated possibil- cytoplasm and uniform round-oval nuclei with gran- ity to metastasize 83,84. ular chromatin, lacking a prominent nucleolus. Small PAS-Diastase positive hyaline globules are often Clinical and gross features found within the cytoplasm of the neoplastic cells and The vast majority of cases (90%) occur in adolescent contain alpha-1 antitrypsin granules. Mitotic figures girls and in young women (mean age 28 years). Cas- are absent or rare. Multinucleated atypical giant cells es involving male patients are very rare and tend to and nuclear pleomorphism are sometimes present, occur 5-10 years later in life with a more aggressive especially in older patients, and are considered to be behavior 85,86. SPN is often an incidental finding in pa- degenerative changes rather than signs of malignan- tients undergoing physical examination or imaging for cy 90. Foamy macrophages and cholesterol crystals other indications. Up to two-thirds of cases present are other common degenerative features typical of with mass symptoms such as abdominal pain, dis- SPNs. comfort, dyspepsia, nausea, vomiting or manifesta- Foci of high-grade malignant transformation with tions related to intratumoral hemorrhage. A small but high mitotic index may be present, and are associat- significant percentage of patients present with acute ed with an extremely aggressive behavior 16. Despite hemoperitoneum 83. Tumor markers are generally nor- mal, and there is no known association with genetic its well-demarcated macroscopic appearance, SPNs or paraneoplastic endocrine syndromes16. Metastases may show infiltrative borders, with tumor cells sur- occur very rarely, mainly in the peritoneum and liver. rounded by non-neoplastic pancreas without a des- The diagnosis is confirmed by imaging studies (ultra- moplastic reaction. The presence of infiltrative borders sound, MRI, CT); SPNs appear as well-demarcated and capsular invasion are associated with local reoc- 91 neoplasms with both solid and cystic components. currence . Calcifications are often seen, as well as fluid-debris Although true tumor necrosis is rare, infarction is not levels 87,88. The long-term prognosis of SPNs after sur- uncommon, and can involve the lesion almost entire- gical resection is generally excellent. ly. Pools of red blood cells, known as “blood lakes”, SPNs may occur anywhere in the pancreas, although are typically present at the periphery of the lesion a slight preference for the body and tail region has and should be distinguished from vascular invasion been reported 89. Direct extension to surrounding or- if tumor cells are present within them 83. The stroma gans, such as duodenum, spleen or the portal tract is around the neoplastic cells is usually scant and barely rare but documented 16. SPNs tend to be solitary and visible; when abundant, it may appear myxoid or hya- well demarcated lesions, sometimes well-encapsulat- line. Calcifications are sometimes seen in the capsule. ed, with a mean diameter around 10 cm (range: 0.5-25 Metastatic lesions tend to have the same morpholog- cm). On cut sections, they typically show a mixture of ic features of the primary tumor; however, a higher solid and cystic areas. Solid areas have a characteris- degree of pleomorphism and mitotic activity may be tic yellow-light brown color and soft consistency, with present. 222 C. Luchini et al.

Immunohistochemistry and molecular pathology ceptor, CD10, vimentin and are constantly negative Over 90% of SPNs show nuclear/cytoplasmic expres- for chromogranin A (excluding a well-differentiated sion of β-catenin, a finding which is often accompa- neuroendocrine neoplasm) and BCL-10 (excluding an nied by loss of membranous expression of E-cadherin. acinar cell carcinoma). The combination of these two markers is considered useful in the diagnosis of SPNs 92. Tumor cells are frequently positive for vimentin, CD56, neuron-specif- The final pathology report for pancreatic ic enolase (NSE), alpha-1 antitrypsin, CD99. CD117 malignant/exocrine tumors (c-kit) is expressed in up to 50% of cases, in absence of KIT mutations 93. Synaptophysin may be focally pos- The final pathology report for malignant/exocrine tu- itive, while chromogranin A, trypsin, CEA and BCL-10 mors of the pancreas should include some important are consistently negative. points to both support the diagnosis and to permit an The key molecular feature of SPNs is the presence adequate oncologic staging. of somatic point mutations in exon 3 of the CTNNB1 Accordingly to the current TNM staging system, the gene, which encodes β-catenin. Such alterations are most important information for resectable PDAC is present in almost all cases and lead to defective phos- represented by tumor size and number of metastatic 21 phorylation and degradation of the β-catenin protein, lymph nodes . Furthermore, the status of all resec- which accumulates in the cytoplasm and nucleus of tion margins should be reported. the neoplastic cells 84. Mutated β-catenin loses its For tumors of pancreatic head, resection margins in- function as an adhesion molecule, an event that could clude: the pancreatic neck margin, the biliary margin, probably explain the poor cohesion of neoplastic cells. the retroperitoneal margin (posterior lamina) and the Recent evidences suggested that SPNs with malig- intestinal margins (one gastric and one intestinal in nant behavior harbor more often inactivating muta- the case of Whipple procedure). Lymph nodes should tions of epigenetic regulators (KDM6A, TET1, BAP1) be divided differentiating them into intestinal (duode- associated with metastatic disease 94. nal), peri-gastric (Whipple procedure), pancreatic-du- Mutations in genes typically found in infiltrating duct- odenal (dividing them in anterior and posterior may al adenocarcinoma, such as KRAS, CDKN2A and be helpful, although it is not required by TNM staging SMAD4 have not been reported in SPNs; less than system), peri-choloedocic and superior mesenteric 5% of cases harbor TP53 mutations 83,95,96. artery lymph nodes (in the retroperitoneal margin). Other useful parameters to be reported are represent- Differential diagnosis ed by vascular invasion, perineural invasion and adi- The main differential diagnoses are reported in Table pose tissue infiltration. For PDAC, the grading of the III. tumor and the contemporary presence of dysplastic In case of diffuse cystic degeneration, SPNs may be modifications of non-neoplastic ducts (e.g. PanIN, that radiologically and macroscopically confused with pan- supports the pancreatic primitiveness of the tumor) creatic pseudocysts. Clinical findings are important in should be reported. distinguishing these two entities, since pseudocysts For tumors of the body-tail, the status of pancreatic arise more commonly in men with a history of chronic transection margin must be reported. If tumors invade pancreatitis, whereas SPNs typically occur in young the anterior/posterior surfaces, this aspect should be women with no history of chronic disease. Microscop- mentioned in the final pathology report. Lymph nodes ically, pseudocysts are characterized by the absence should be divided differentiating them into peri-pan- of an epithelial lining and of the classic neoplastic creatic nodes (ideally identifying postero-superi- SPN cells. or nodes, which are classically located very closely Another important differential diagnosis for SPNs, to splenic vessels, and inferior nodes, although this above all in the case of solid appearance, is represent- distinction is not taken into account by TNM staging ed by well-differentiated neuroendocrine neoplasms. system) and the lymph nodes of splenic hilus. As for In this context, after a careful histologic examination, tumors affecting the pancreatic head, other useful pa- immunohistochemistry may be of great importance rameters to be reported are represented by vascular for completing this challenging distinction. Although invasion, perineural invasion and adipose tissue in- SPNs and neuroendocrine neoplasm may share the filtration. For PDAC, in addition, grading of the tumor positive expression for some markers, such as syn- and the contemporary presence of dysplastic modi- aptophysin and CD200 54, at the same time SPNs fications of non-neoplastic ducts (e.g. PanIN, that have a distinct IHC profile, including the positivity for supports the pancreatic primitiveness of the tumor) β-catenin (aberrant nuclear pattern), progesterone-re- should be reported. NEOPLASTIC EXOCRINE PANCREATIC PATHOLOGY 223

References topathology 2009;55:277-83. https://doi.org/10.1111/j.1365- 2559.2009.03376.x 1 Brierley JD, Gospodarowicz MK, Wittekind C. TNM Classification 15 Verbeke C, H äberle L, Lenggenhager D, et al. Pathology as- of Malignant Tumours. Oxfor: John Wiley & Sons, 2016 p. 274. sessment of pancreatic cancer following neoadjuvant treatment: 2 Neoptolemos JP, Stocken DD, Dunn JA, et al. Influence of re- time to move on. Pancreatology 2018;S1424-3903(18)30077-2. section margins on survival for patients with pancreatic cancer https://doi.org/ 10.1016/j.pan.2018.04.010 treated by adjuvant chemoradiation and/or chemotherapy in the 16 WHO Classification of Tumours Editorial Board. WHO Classifica- ESPAC-1 randomized controlled trial. Ann Surg 2001;234:758- tion of Tumours. 5th ed. Lyon: IARC press 2019. 68. https://doi.org/10.1097/00000658-200112000-00007 17 Luchini C, Pea A, Yu J, et al. Pancreatic cancer arising in the 3 Zhang B, Lee G, Qadan M et al. Revision of pancreatic neck mar- remnant pancreas is not always a relapse of the preceding gins based on intraoperative frozen section analysis is associated primary. Mod Pathol 2019;32:659-65. https://doi.org/10.1038/ with improved survival in patients undergoing pancreatectomy s41379-018-0183-7 for ductal adenocarcinoma. Ann Surg 2019 Dec 5. https://doi.org/ 10.1097/SLA.0000000000003503. Online ahead of print. 18 He J, Ahuja N, Makary MA, et al. 2564 resected periampul- lary adenocarcinomas at a single institution: trends over three 4 Lad NL, Squires MH, Maithel SK, et al. Is it time to stop check- decades. HPB (Oxford) 2014;16:83-90. https://doi.org/10.1111/ ing frozen section neck margins during pancreaticoduodenecto- hpb.12078 my?. Ann Surg Oncol 2013;20:3626-33. https://doi.org/10.1245/ s10434-013-3080-9 19 Amin MB, Edge S, Greene F et al: AJCC Cancer Staging Manu- al. 8th ed. Springer International Publishing 2017. 5 Verbeke CS, Leitch D, Menon KV, et al. Redefining the R1 resec- tion in pancreatic cancer. Br J Surg 2006;93:1232-7. https://doi. 20 Riva G, Pea A, Pilati C, et al. Histo-molecular oncogenesis of org/10.1002/bjs.5397 pancreatic cancer: From precancerous lesions to invasive duc- tal adenocarcinoma. World J Gastrointest Oncol 2018;10:317-27. 6 Lüttges J, Zamboni G, Klöppel G. Recommendation for the https://doi.org/10.4251/wjgo.v10.i10.317 examination of pancreaticoduodenectomy specimens re- 21 moved from patients with carcinoma of the exocrine pancreas. Zamboni G, Hirabayashi K, Castelli P, Lennon AM. Precancer- A proposal for a standardized pathological staging of pancre- ous lesions of the pancreas. Best Pract Res Clin Gastroenterol aticoduodenectomy specimens including a checklist. Dig Surg 2013;27:299-322. https://doi.org/10.1016/j.bpg.2013.04.001 1999;16:291-6. https://doi.org/10.1159/000018738 22 Meyers N, Gérard C, Lemaigre FP, Jacquemin P. Differential im- 7 Takahashi D, Kojima M, Sugimoto M, et al. Pathologic evalua- pact of the ERBB receptors EGFR and ERBB2 on the initiation of tion of surgical margins in pancreatic cancer specimens using precursor lesions of pancreatic ductal adenocarcinoma. Sci Rep color coding with tissue marking dyes. Pancreas 2018;47:830-6. 2020;10:5241. Published 2020 Mar 23. https://doi.org/10.1038/ https://doi.org/10.1097/MPA.0000000000001106 s41598-020-62106-8. 23 8 Verbeke CS, Gladhaug IP. Dissection of pancreatic resec- Bazzichetto C, Conciatori F, Luchini C, et al. From Genetic al- tion specimens. Surg Pathol Clin 2016;9:523-38. https://doi. terations to tumor microenvironment: The Ariadne’s String in org/10.1016/j.path.2016.05.001 Pancreatic Cancer. Cells 2020;9:309. Published 2020 Jan 28. https://doi.org/10.3390/cells9020309 9 Grillo F, Ferro J, Vanoli A, et al. Comparison of pathology sam- 24 pling protocols for pancreatoduodenectomy specimens. Vir- Basturk O, Hong SM, Wood LD, et al. A revised classification chows Arch 2020;476:735-44. https://doi.org/10.1007/s00428- system and recommendations from the Baltimore Consen- 019-02687-6 sus meeting for neoplastic precursor lesions in the pancreas. Am J Surg Pathol 2015;39:1730-41. https://doi.org/10.1097/ 10 Adsay NV, Basturk O, Saka B, et al. Whipple made simple for PAS.0000000000000533 surgical pathologists: orientation, dissection, and sampling of 25 pancreaticoduodenectomy specimens for a more practical and Sereni E, Luchini C, Salvia R, et al. Molecular and clinical pat- accurate evaluation of pancreatic, distal common bile duct, and terns of local progression in the pancreatic remnant following ampullary tumors. Am J Surg Pathol 2014;38:480-93. https://doi. resection of pancreatic intraductal papillary mucinous neoplasm org/10.1097/PAS.0000000000000165 (IPMN). Chin Clin Oncol 2019;8:21. https://doi.org/ 10.21037/ cco.2019.04.03 11 Adsay V, Mino-Kenudson M, Furukawa T, et al. Pathologic evalu- 26 ation and reporting of intraductal papillary mucinous neoplasms Pea A, Yu J, Rezaee N, et al. Targeted DNA Sequencing Reveals of the pancreas and other tumoral intraepithelial neoplasms of Patterns of Local Progression in the Pancreatic Remnant Fol- pancreatobiliary tract: recommendations of Verona Consensus lowing Resection of Intraductal Papillary Mucinous Neoplasm Meeting. Ann Surg 2016;263:162-77. https://doi.org/10.1097/ (IPMN) of the Pancreas. Ann Surg 2017;266:133-41. https://doi. SLA.0000000000001173 org/10.1097/SLA.0000000000001817 27 12 Verbeke C, Löhr M, Karlsson JS, Del Chiaro M. Pathology re- Felsenstein M, Noë M, Masica DL, et al. IPMNs with co-occur- porting of pancreatic cancer following neoadjuvant therapy: ring invasive cancers: neighbours but not always relatives. Gut challenges and uncertainties. Cancer Treat Rev 2015;41:17-26. 2018;67:1652-62. https://doi.org/10.1136/gutjnl-2017-315062 https://doi.org/10.1016/j.ctrv.2014.11.002 28 Scarpa A, Real FX, Luchini C. Genetic unrelatedness of co- 13 Soer E, Brosens L, van de Vijver M, et al. Dilemmas for the pa- occurring pancreatic adenocarcinomas and IPMNs challenges thologist in the oncologic assessment of pancreatoduodenec- current views of clinical management. Gut 2018;67:1561-3. tomy specimens: an overview of different grossing approaches https://doi.org/10.1136/gutjnl-2018-316151 and the relevance of the histopathological characteristics in 29 Lupinacci RM, Goloudina A, Buhard O, et al. Prevalence of mic- the oncologic assessment of pancreatoduodenectomy speci- rosatellite instability in intraductal papillary mucinous neoplasms mens. Virchows Arch 2018;472:533-43. https://doi.org/10.1007/ of the pancreas. Gastroenterology 2018;154:1061-5. https://doi. s00428-018-2321-5 org/10.1053/j.gastro.2017.11.009 14 Campbell F, Smith RA, Whelan P, et al. Classification of R1 30 Luchini C, Capelli P, Scarpa A. Pancreatic ductal adenocarci- resections for pancreatic cancer: the prognostic relevance of noma and its variants. Surg Pathol Clin 2016;9:547-60. https:// tumour involvement within 1 mm of a resection margin. His- doi.org/10.1016/j.path.2016.05.003 224 C. Luchini et al.

31 Veronese N, Nottegar A, Pea A, et al. Prognostic impact and im- 45 Agaimy A, Haller F, Frohnauer J, et al. Pancreatic undifferenti- plications of extracapsular lymph node involvement in colorec- ated rhabdoid carcinoma: KRAS alterations and SMARCB1 ex- tal cancer: a systematic review with meta-analysis. Ann Oncol pression status define two subtypes. Mod Pathol 2015;28:248- 2016;27:42-8. https://doi.org/10.1093/annonc/mdv494 60. https://doi.org/10.1038/modpathol.2014.100 32 Veronese N, Fassan M, Wood LD, et al. Extranodal extension of 46 Ishida K, Yamashita R, Osakabe M, et al. Expression of epitheli- nodal metastases is a poor prognostic indicator in gastric can- al-mesenchymal transition proteins in pancreatic anaplastic (un- cer: a systematic review and meta-analysis. J Gastrointest Surg differentiated) carcinoma. Pancreas 2019;48:36-42. https://doi. 2016;20:1692-8. https://doi.org/10.1007/s11605-016-3199-7 org/10.1097/MPA.0000000000001199 33 Luchini C, Veronese N, Pea A, et al. Extranodal extension in N1- 47 Mattiolo P, Fiadone G, Paolino G et al. Epithelial-mesenchymal adenocarcinoma of the pancreas and papilla of Vater: a system- transition in undifferentiated carcinoma of the pancreas with and atic review and meta-analysis of its prognostic significance. Eur without osteoclast-like giant cells. Virchows Arch 2020 Jul 13. J Gastroenterol Hepatol 2016;28:205-9. https://doi.org/ 10.1007/s00428-020-02889-3. Online ahead of print. 34 Karamitopoulou E, Wartenberg M, Zlobec I, et al. Tumour bud- ding in pancreatic cancer revisited: validation of the ITBCC scor- 48 Muraki T, Reid MD, Basturk O, et al. Undifferentiated carcinoma ing system. Histopathology 2018;73:137-46. with osteoclastic giant cells of the pancreas: clinicopathologic analysis of 38 cases highlights a more protracted clinical course 35 Lawlor RT, Veronese N, Nottegar A, et al. Prognostic role of high- than currently appreciated. Am J Surg Pathol 2016;40:1203-16. grade tumor budding in pancreatic ductal adenocarcinoma: a https://doi.org/10.1097/PAS.0000000000000689 systematic review and meta-analysis with a focus on epithelial 49 to mesenchymal transition. Cancers (Basel) 2019;11:113. Pub- Luchini C, Pea A, Lionheart G, et al. Pancreatic undifferentiated lished 2019 Jan 19. https://doi.org/10.3390/cancers11010113 carcinoma with osteoclast-like giant cells is genetically similar to, but clinically distinct from, conventional ductal adenocarcinoma. 36 Hong SM, Jung D, Kiemen A, et al. Three-dimensional visualiza- J Pathol 2017;243:148-54. https://doi.org/10.1002/path.4941 tion of cleared human pancreas cancer reveals that sustained 50 epithelial-to-mesenchymal transition is not required for venous Luchini C, Cros J, Pea A, et al. PD-1, PD-L1, and CD163 in invasion. Mod Pathol 2020;33:639-47. https://doi.org/10.1038/ pancreatic undifferentiated carcinoma with osteoclast-like giant s41379-019-0409-3 cells: expression patterns and clinical implications. Hum Pathol 2018;81:157-65. https://doi.org/10.1016/j.humpath.2018.07.006 37 Basturk O, Khanani F, Sarkar F, et al. DeltaNp63 expression in 51 pancreas and pancreatic neoplasia. Mod Pathol 2005;18:1193-8. Moll R, Löwe A, Laufer J, Franke WW. Cytokeratin 20 in human https://doi.org/10.1038/modpathol.3800401 carcinomas. A new histodiagnostic marker detected by monoclo- nal antibodies. Am J Pathol 1992;140:427-47. 38 Liu C, Karam R, Zhou Y, et al. The UPF1 RNA surveillance gene 52 is commonly mutated in pancreatic adenosquamous carcinoma. Nagata K, Horinouchi M, Saitou M, et al. Mucin expression pro- file in pancreatic cancer and the precursor lesions. J Hepato- Nat Med 2014;20:596-8. https://doi.org/10.1038/nm.3548 biliary Pancreat Surg 2007;14:243-54. https://doi.org/10.1007/ 39 Boecker J, Feyerabend B, Tiemann K, et al. Adenosquamous s00534-006-1169-2 carcinoma of the pancreas comprise a heterogeneous group of 53 Terada T, Ohta T, Sasaki M, et al. Expression of muc apomu- tumors with the worst outcome: a clinicopathological analysis cins in normal pancreas and pancreatic tumours. J Pathol of 25 cases identified in 562 pancreatic carcinomas resected 1996;180:160-5. with curative intent. Pancreas 2020;49:683-91. https://doi. 54 org/10.1097/MPA.0000000000001548 Lawlor RT, Daprà V, Girolami I, et al. CD200 expression is a feature of solid pseudopapillary neoplasms of the pancreas. 40 Amato E, Molin MD, Mafficini A, et al. Targeted next-generation Virchows Arch 2019;474:105-9. https://doi.org/10.1007/s00428- sequencing of cancer genes dissects the molecular profiles 018-2437-7 of intraductal papillary neoplasms of the pancreas. J Pathol 55 2014;233:217-27. https://doi.org/10.1002/path.4344 Xu JZ, Wang WQ, Zhang WH, et al. The Loss of SMAD4/DPC4 41 expression associated with a strongly activated hedgehog sig- Luchini C, Brosens LAA, Wood LD, et al. Comprehensive naling pathway predicts poor prognosis in resected pancre- characterisation of pancreatic ductal adenocarcinoma with atic cancer. J Cancer 2019;10:4123-31. Published 2019 Jul 10. microsatellite instability: histology, molecular pathology and https://doi.org/10.7150/jca.30883 clinical implications [published online ahead of print, 2020 56 Apr 29]. Gut 2020;gutjnl-2020-320726. https://doi.org/10.1136/ Waddell N, Pajic M, Patch AM, et al. Whole genomes rede- gutjnl-2020-320726 fine the mutational landscape of pancreatic cancer. Nature 2015;518(7540):495-501. https://doi.org/10.1038/nature14169 42 Luchini C, Bibeau F, Ligtenberg MJL, et al. ESMO recommen- 57 dations on microsatellite instability testing for immunotherapy Bailey P, Chang DK, Nones K, et al. Genomic analyses in cancer, and its relationship with PD-1/PD-L1 expression and identify molecular subtypes of pancreatic cancer. Nature 2016;531(7592):47-52. https://doi.org/10.1038/nature16965 tumour mutational burden: a systematic review-based approach. Ann Oncol 2019;30:1232-43. https://doi.org/10.1093/annonc/ 58 Almoguera C, Shibata D, Forrester K, et al. Most human carcino- mdz116 mas of the exocrine pancreas contain mutant c-K-ras genes. Cell 1988;53:549-54. https://doi.org/10.1016/0092-8674(88)90571-5 43 Wilentz RE, Goggins M, Redston M, et al. Genetic, immuno- histochemical, and clinical features of medullary carcinoma 59 Singhi AD, George B, Greenbowe JR, et al. Real-time targeted of the pancreas: A newly described and characterized entity. genome profile analysis of pancreatic ductal adenocarcinomas Am J Pathol 2000;156:1641-51. https://doi.org/10.1016/S0002- identifies genetic alterations that might be targeted with existing 9440(10)65035-3 drugs or used as biomarkers. Gastroenterology 2019;156:2242- 53.e4. https://doi.org/10.1053/j.gastro.2019.02.037 44 Kryklyva V, ter Linden E, Kroeze LI et al. Medullary pan- 60 creatic carcinoma due to somatic POLE mutation: a dis- Adsay NV, Bandyopadhyay S, Basturk O, et al. Chronic pancre- tinctive pancreatic carcinoma with marked long-term sur- atitis or pancreatic ductal adenocarcinoma?. Semin Diagn Pathol vival. Pancreas 2020;49:999-1003. https://doi.org/ 10.1097/ 2004;21:268-76. https://doi.org/10.1053/j.semdp.2005.08.002 MPA.0000000000001588 61 Zhang L, Chari S, Smyrk TC, et al. Autoimmune pancreatitis NEOPLASTIC EXOCRINE PANCREATIC PATHOLOGY 225

(AIP) type 1 and type 2: an international consensus study on tifies recurrent RAF fusions and frequent inactivation of DNA histopathologic diagnostic criteria. Pancreas 2011;40:1172-9. repair genes. [published correction appears in Cancer Discov https://doi.org/10.1097/MPA.0b013e318233bec5 2015 Mar;5(3):332]. Cancer Discov 2014;4:1398-1405. https:// 62 Zamboni G, Lüttges J, Capelli P, et al. Histopathological features doi.org/10.1158/2159-8290.CD-14-0617 of diagnostic and clinical relevance in autoimmune pancreatitis: 76 Klimstra DS, Rosai J, Heffess CS. Mixed acinar-endocrine car- a study on 53 resection specimens and 9 biopsy specimens. Vir- cinomas of the pancreas. Am J Surg Pathol 1994;18:765-78. chows Arch 2004;445:552-63. https://doi.org/10.1007/s00428- https://doi.org/10.1097/00000478-199408000-00002 004-1140-z 77 Ohike N, Morohoshi T. Exocrine pancreatic neoplasms of non- 63 De Marchi G, Paiella S, Luchini C, et al. Very high serum levels ductal origin: acinar cell carcinoma, pancreatoblastoma, and of CA 19-9 in autoimmune pancreatitis: Report of four cases and solid-pseudopapillary neoplasm. Surg Pathol Clin 2011;4:579- brief review of literature. J Dig Dis 2016;17:697-702. https://doi. 88. https://doi.org/10.1016/j.path.2011.03.001 org/10.1111/1751-2980.12403 78 Tanaka Y, Kato K, Notohara K, et al. Significance of aberrant 64 Detlefsen S, Zamboni G, Frulloni L, et al. Clinical features and (cytoplasmic/nuclear) expression of beta-catenin in pancreato- relapse rates after surgery in type 1 autoimmune pancreatitis blastoma. J Pathol 2003;199:185-90. https://doi.org/10.1002/ differ from type 2: a study of 114 surgically treated European pa- path.1262 tients. Pancreatology 2012;12:276-83. https://doi.org/10.1016/j. 79 Wood LD, Klimstra DS. Pathology and genetics of pancre- pan.2012.03.055 atic neoplasms with acinar differentiation. Semin Diagn Pathol 65 Zhang L, Smyrk TC. Autoimmune pancreatitis and IgG4-related 2014;31:491-7. https://doi.org/10.1053/j.semdp.2014.08.003 systemic diseases. Int J Clin Exp Pathol 2010;3:491-504. Pub- 80 Kneeland Frantz V. Atlas of tumor pathology, Section VII, Fas- lished 2010 May 25. cicles 27 and 28. Tumors of the Pancreas. Washington, D.C.: 66 Zhang L, Notohara K, Levy MJ, et al. IgG4-positive plasma cell in- Armed Forces Institute of Pathology 1959. filtration in the diagnosis of autoimmune pancreatitis. Mod Pathol 81 Kosmahl M, Pauser U, Peters K, et al. Cystic neoplasms of 2007;20:23-8. https://doi.org/10.1038/modpathol.3800689 the pancreas and tumor-like lesions with cystic features: a re- 67 Esposito I, Hruban RH, Verbeke C, et al. Guidelines on the his- view of 418 cases and a classification proposal. Virchows Arch topathology of chronic pancreatitis. Recommendations from the 2004;445:168-78. https://doi.org/10.1007/s00428-004-1043-z working group for the international consensus guidelines for 82 Konukiewitz B, Enosawa T, Klöppel G. Glucagon expression in chronic pancreatitis in collaboration with the International As- cystic pancreatic neuroendocrine neoplasms: an immunohis- sociation of Pancreatology, the American Pancreatic Associa- tochemical analysis. Virchows Arch 2011;458:47-53. https://doi. tion, the Japan Pancreas Society, and the European Pancreatic org/10.1007/s00428-010-0985-6 Club. Pancreatology 2020;20:586-93. https://doi.org/10.1016/j. 83 Hruban RH, Pitman MB, Klimstra DS. Tumors of the pancreas. pan.2020.04.009 American Registry of Pathology in collaboration with the Armed 68 Yemelyanova A, Vang R, Kshirsagar M, et al. Immunohistochem- Forces Institute of Pathology 2007. ical staining patterns of p53 can serve as a surrogate marker for 84 Abraham SC, Klimstra DS, Wilentz RE, et al. Solid-pseudo- TP53 mutations in ovarian carcinoma: an immunohistochemical papillary tumors of the pancreas are genetically distinct from and nucleotide sequencing analysis. Mod Pathol 2011;24:1248- pancreatic ductal adenocarcinomas and almost always harbor 53. https://doi.org/10.1038/modpathol.2011.85 beta-catenin mutations. Am J Pathol 2002;160:1361-9. https:// 69 Ritterhouse LL, Wu EY, Kim WG, et al. Loss of SMAD4 protein doi.org/10.1016/s0002-9440(10)62563-1 expression in gastrointestinal and extra-gastrointestinal carci- 85 Machado MC, Machado MA, Bacchella T, et al. Solid pseudo- nomas. Histopathology 2019;75:546-51. https://doi.org/10.1111/ papillary neoplasm of the pancreas: distinct patterns of onset, his.13894 diagnosis, and prognosis for male versus female patients. Sur- 70 Klimstra DS, Adsay V. Acinar neoplasms of the pancreas-A sum- gery 2008;143:29-34. https://doi.org/10.1016/j.surg.2007.07.030 mary of 25 years of research. Semin Diagn Pathol 2016;33:307- 86 Klöppel G, Morohoshi T, John HD, et al. Solid and cystic aci- 18. https://doi.org/10.1053/j.semdp.2016.05.009 nar cell tumour of the pancreas. A tumour in young women 71 Klimstra DS, Heffess CS, Oertel JE, et al. Acinar cell carcinoma with favourable prognosis. Virchows Arch A Pathol Anat Histol of the pancreas. A clinicopathologic study of 28 cases. Am J 1981;392:171-83. Surg Pathol 1992;16:815-37. https://doi.org/10.1097/00000478- 87 Dong PR, Lu DS, Degregario F, et al. Solid and papillary neo- 199209000-00001 plasm of the pancreas: radiological-pathological study of five 72 La Rosa S, Adsay V, Albarello L, et al. Clinicopathologic study of cases and review of the literature. Clin Radiol 1996;51(10):702- 62 acinar cell carcinomas of the pancreas: insights into the mor- 5. https://doi.org/10.1016/s0009-9260(96)80242-x phology and immunophenotype and search for prognostic mark- 88 Buetow PC, Buck JL, Pantongrag-Brown L, et al. Solid and pap- ers. Am J Surg Pathol 2012;36:1782-95. https://doi.org/10.1097/ illary epithelial neoplasm of the pancreas: imaging-pathologic PAS.0b013e318263209d correlation on 56 cases. Radiology 1996;199:707-11. https://doi. 73 La Rosa S, Sessa F, Capella C. Acinar cell carcinoma of the pan- org/10.1148/radiology.199.3.8637992 creas: overview of clinicopathologic features and insights into 89 Kang CM, Choi SH, Kim SC, et al. Predicting recurrence of pan- the molecular pathology. Front Med (Lausanne) 2015;2:41. Pub- creatic solid pseudopapillary tumors after surgical resection: lished 2015 Jun 15. https://doi.org/10.3389/fmed.2015.00041 a multicenter analysis in Korea. Ann Surg. 2014;260:348-55. 74 Furlan D, Sahnane N, Bernasconi B, et al. APC alterations are https://doi.org/10.1097/SLA.0000000000000583 frequently involved in the pathogenesis of acinar cell carcino- 90 Kim SA, Kim MS, Kim MS, et al. Pleomorphic solid pseudopapil- ma of the pancreas, mainly through gene loss and promoter lary neoplasm of the pancreas: degenerative change rather than hypermethylation. Virchows Arch 2014;464:553-64. https://doi. high-grade malignant potential. Hum Pathol 2014;45:166-74. org/10.1007/s00428-014-1562-1 https://doi.org/10.1016/j.humpath.2013.08.016 75 Chmielecki J, Hutchinson KE, Frampton GM, et al. Comprehen- 91 Marchegiani G, Andrianello S, Massignani M, et al. Solid pseu- sive genomic profiling of pancreatic acinar cell carcinomas iden- dopapillary tumors of the pancreas: Specific pathological fea- 226 C. Luchini et al.

tures predict the likelihood of postoperative recurrence. J Surg 94 Amato E, Mafficini A, Hirabayashi K, et al. Molecular altera- Oncol 2016;114:597-601. https://doi.org/10.1002/jso.24380 tions associated with metastases of solid pseudopapillary neo- 92 Kim MJ, Jang SJ, Yu E. Loss of E-cadherin and cytoplasmic-nu- plasms of the pancreas. J Pathol 2019;247:123-34. https://doi. clear expression of beta-catenin are the most useful immunopro- org/10.1002/path.5180 files in the diagnosis of solid-pseudopapillary neoplasm of the 95 Bartsch D, Bastian D, Barth P, et al. K-ras oncogene mutations pancreas. Hum Pathol 2008;39:251-8. https://doi.org/10.1016/j. indicate malignancy in cystic tumors of the pancreas. Ann Surg humpath.2007.06.014 1998;228:79-86. https://doi.org/10.1097/00000658-199807000- 93 Cao D, Antonescu C, Wong G, et al. Positive immunohisto- 00012 chemical staining of KIT in solid-pseudopapillary neoplasms 96 Fléjou JF, Boulange B, Bernades P, et al. p53 protein expres- of the pancreas is not associated with KIT/PDGFRA muta- sion and DNA ploidy in cystic tumors of the pancreas. Pancreas tions. Mod Pathol 2006;19:1157-63. https://doi.org/10.1038/mod- 1996;13:247-52. https://doi.org/10.1097/00006676-199610000- pathol.3800647 00006 PATHOLOGICA 2020;112:227-247; DOI: 10.32074/1591-951X-161

Review

Gastrointestinal lymphoproliferative lesions: a practical diagnostic approach

Marco Pizzi1, Elena Sabattini2, Paola Parente3, Alberto Bellan4, Claudio Doglioni5, Stefano Lazzi6 1 General Pathology and Cytopathology Unit, Department of Medicine – DIMED, University of Padova, Italy; 2 Hematopathology Unit, Sant’Orsola University Hospital, Bologna (BO), Italy; 3 Pathology Unit, Fondazione IRCCS Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy; 4 Department of Pathology, ULSS6, Camposampiero Hospital, Camposampiero (PD), Italy; 5 Department of Pathology, University Vita- Salute San Raffaele, IRCCS San Raffaele Hospital, Milano, Italy; 6 Department of Medical Biotechnology, Section of Pathology, University of Siena, Italy

Summary The gastrointestinal tract (GI) is the primary site of lymphoproliferative lesions, spanning from reactive lymphoid hyperplasia to overt lymphoma. The diagnosis of these diseases is challenging and an integrated approach based on clinical, morphological, immunohis- tochemical and molecular data is needed. To reach to confident conclusions, a stepwise approach is highly recommended. Histological evaluation should first assess the benign versus neoplastic nature of a given lymphoid infiltrate. Morphological and phenotypic anal- yses should then be applied to get to a definite diagnosis. This review addresses the key histological features and diagnostic workup of the most common GI non-Hodgkin lymphomas (NHLs). Differential diagnoses and possible pitfalls are discussed by considering distinct groups of lesions (i.e. small to medium B-cell NHLs; medium to large B-cell NHLs; T-cell NHLs; and mimickers of Hodgkin lymphoma). The key clinical and epidemiological features of each entity are also described. Received and accepted: June 29, 2020 Published online: 29 October, 2020 Key words: lymphoma, gastrointestinal lymphoma, Hodgkin lymphoma, B-cell lymphoma, T-cell lymphoma Correspondence Stefano Lazzi Department of Medical Biotechnology, Section of Pathology, University of Siena, via Introduction and clinical relevance delle Scotte 6, 53100 Siena (SI), Italy E-mail: [email protected] The gastrointestinal (GI) tract plays several immunologic functions, in- Conflict of interest cluding fine-tuning the intestinal microbiome, mediating tolerance toward The Authors declare no conflict of interest. food antigens and mounting immune responses against enteric patho- gens 1. Such functions rely on dedicated anatomical structures, widely How to cite this article: Pizzi M, Sabattini E, spread along the stomach, the small and large bowel (i.e. Peyer’s patch- Parente P, et al. Gastrointestinal lymphoprolif- es and rectal tonsil) 1,2. The physiologic and/or neoplastic expansion of erative lesions: a practical diagnostic approach. Pathologica 2020;112:227-247. https://doi. such structures gives rise to a broad spectrum of lesions, spanning from 3 org/10.32074/1591-951X-161 benign lymphoid hyperplasia to malignant B and T cell lymphomas . Gastrointestinal lymphoid proliferations are commonly found in the sur- © Copyright by Società Italiana di Anatomia Pato- gical pathology practice and their diagnosis can be challenging even logica e Citopatologia Diagnostica, Divisione Itali- ana della International Academy of Pathology for trained pathologists. This is due to their disguising and overlapping histological features, the small amount of diagnostic material and sam- OPEN ACCESS pling artifacts. Despite such limitations, histology is the gold standard for the diagnosis of GI lymphoproliferative disorders (LPDs) and guides This is an open access journal distributed in accordance 4 with the CC-BY-NC-ND (Creative Commons Attribution- the management of patients . For these reasons, when dealing with NonCommercial-NoDerivatives 4.0 International) license: the lymphoid infiltrates in the GI tract, every effort should be taken to get to work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original a diagnosis as accurate and informative as possible. version. For further information: https://creativecommons. This review will discuss the histological features of GI LPDs and org/licenses/by-nc-nd/4.0/deed.en non-Hodgkin lymphomas (NHLs), specifically addressing the key diag- 228 M. Pizzi et al.

nostic features and pitfalls of each entity. A practical ical picture, the endoscopic findings and the severity approach will be followed, by grouping GI LPDs into of histological atypia. major diagnostic categories, discussing their general features and considering their differential diagnosis. This work is intended to support the surgical pathol- Primary GI lymphomas versus secondary ogy practice. For a detailed discussion of the clinical involvement by systemic disease and pathophysiological features of each entity, the reader is referred to the many excellent reviews pub- Once the neoplastic nature of a lymphoid infiltrate has lished on this topic 5-10. been established, another important issue is defin- ing its origin. The GI tract can indeed host both pri- mary NHLs and secondary localizations of systemic The diagnosis of GI LPDs: benign versus disease. The clinical criteria distinguishing these two malignant lesions groups of neoplasms were first proposed by Dawson in 1961. A NHL is considered of primary GI origin if, The first most compelling issue concerning GI LPDs at presentation: (i) peripheral and/or mediastinal lym- is to define whether they are benign or malignant in phadenopathies are lacking; (ii) blood cell counts are nature. Clues favoring a diagnosis of lymphoma over normal; (iii) the lesion is predominantly localized with- reactive lymphoid hyperplasia include: (i) tissue ef- in the GI tract and the only affected lymph nodes are facement by confluent sheets of lymphoid cells (even adjacent to such lesion; and (iv) the liver and spleen with polyp formation), (ii) infiltration and disruption of are spared 14. glandular units (i.e. “lymphoepithelial lesions”; LELs), By applying Dawson’s criteria, primary GI NHLs ac- (iii) atypical follicles, follicular colonization or expand- count for about 30-40% of all extra-nodal lymphomas ed mantle zones, (iv) the monomorphic composition and for 1-4% of GI malignancies 15. Secondary GI of the lymphoid infiltrate, and (v) the documentation of involvement is by far more frequent, being reported cytologically or phenotypically atypical elements, sin- in 5-20% of NHLs 16. The anatomic distribution of GI gly or in aggregate. None of these findings is pathog- NHLs varies across geographical areas: the stomach nomonic of lymphoma per se, nor all lymphomas dis- is primarily involved in Western countries 17, while close each of these features. It is rather the integration the small bowel is mostly affected in Middle East re- of multiple parameters that leads to a correct diagno- gions 18. In all, gastric and small intestinal NHLs ac- sis (Fig. 1). count for 80-85% of all GI cases. Molecular biology may also support the differential Primary GI NHLs are mostly of B-cell lineage (90% of diagnosis between reactive hyperplasia and lympho- cases), spanning from indolent to very aggressive ne- ma, the former being associated with polyclonal rear- oplasms. Peripheral T-cell lymphomas are less com- rangements of the immunoglobulin and T cell receptor mon and typically associated with poor prognosis. In genes, the latter showing monoclonal gene patterns. contrast to patients with known classic Hodgkin lym- Exceptions nonetheless exist to this rule. In fact, an- phoma (cHL), great caution is required before making tigen-driven monoclonal populations are commonly a diagnosis of primary cHL in extranodal sites (stage found in inflammatory, infectious or autoimmune re- IE is extremely rare: 0.25-1% of cases). Mimickers of sponses 11 and polyclonal patterns may characterize cHL are well documented and will be addressed in highly mutant NHLs, as a result of limited primer an- a separate section of this review. Finally, the GI tract nealing and poor amplification of the monoclonal se- is frequently site of post-transplant LPDs (PTLDs). quences 12. Thus, the finding of a monoclonal peak These lesions are frequently driven by EBV infection does not necessarily imply a diagnosis of lymphoma and include benign, non-destructive lymphoid or plas- and the results of molecular biology should always be ma cell proliferations (i.e. follicular hyperplasia, plas- integrated with the clinical-pathological findings. ma cell hyperplasia, infectious mononucleosis-like Despite extensive phenotypic and molecular charac- LPDs), polymorphic and monomorphic B and T/NK- terization, in a minority of cases a definite diagnosis cell PTLDs (Tab. I). cannot be made. In such instances, the degree of un- certainty should be stated in the pathology report and Non-Hodgkin B cell lymphomas of the GI tract provisional statements of “atypical lymphoid hyperpla- For diagnostic purposes, B-cell NHLs can be grouped sia/infiltrates”, “lymphoid infiltrate of uncertain poten- in two broad disease categories: (i) tumors consisting tial/significance”, or “lymphoid infiltrate suspicious but of small to medium size B-cells; and (ii) tumors con- not certain for lymphoma” may be proposed 13. The sisting of medium to large size B-cells. The first group management of such lesions will depend on the clin- encompasses extra-nodal marginal zone lymphoma DIAGNOSIS OF GI LYMPHOPROLIFERATIVE DISORDERS 229

Figure 1. Differential diagnosis between benign and malignant GI lymphoid proliferations. The table summarizes the key morphological features favoring a diagnosis of neoplastic over reactive lymphoid infiltrate. (A) Reactive infiltrates are non- destructive and encompass well-demarcated lymphoid nodules (left figure); non-Hodgkin lymphomas are instead char- acterized by dense infiltrates occupying the entire lamina propria (right figure). (B) Reactive infiltrates are polymorphic and include variable numbers of small lymphocytes, granulocytes, histiocytes and plasma cells (left figure). Non-Hodgkin lymphomas mostly consist of monomorphic, atypical lymphocytes (right figure). (C) In reactive conditions, intra-glandular cells are mainly granulocytes and/or scattered, small lymphocytes (left figure); in non-Hodgkin lymphomas, native glands are infiltrated/effaced by neoplastic lymphocytes (“lymphoepithelial lesions”) (right figure; arrow). (H&E stain; original magnification, 10x and 20x). 230 M. Pizzi et al.

Table I. Classification of GI lymphomas Histologic subtype Predilection site B Cell Origin (90% of cases) Extranodal marginal zone lymphoma, MALT type Stomach, small intestine Alpha heavy chain disease (IPSID) Jejunum, Ileum Diffuse large B cell lymphoma, NOS Stomach, Ileocecal Burkitt lymphoma Ileocecal, stomach, small intestine Plasmablastic lymphoma Anorectum Mantle cell lymphoma Any sites Follicular lymphoma Duodenum, large intestine Posttransplant lymphoprolipherative disorder Any sites T/NK cell origin (5-9% of cases) Enteropathy associates T cell lymphoma (EALT) Small intestine Monomorphic epitheliotropic intestinal T cell lymphoma (MEILT) Small intestine NK/T-cell lymphoma, nasal type Small intestine, large intestine Periphetal T cell lymphoma, NOS Large intestine, small intestine Indolent T/NK lymphoprolipherative disorder Small intestine, large intestine Posttransplant lymphoprolipherative disorders Any sites Classical Hodgkin Lymphoma (< 5% of cases) Rectum

(ENMZL), mantle cell lymphoma (MCL), follicular lym- phoma (FL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). The second group mainly includes Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lympho- ma (HGBL) not otherwise specified (HGBL NOS), HGBL with MYC and BCL2 and/or BCL6 rearrange- ments (i.e. double/triple-hit lymphoma, DHL/THL), large B-cell lymphoma (LBCL) with IRF4 rearrange- ments, and plasmablastic lymphoma (PBL).

Small to medium size B-cell NHLs This group includes entities primarily arising in the GI tract or secondarily spreading to this site. A stepwise immunohistochemical approach supporting their diag- Figure 2. Immunohistochemical algorithm for the diagno- nosis is proposed in Figure 2. sis of small to medium-sized B-cell NHLs. Notes: *CD10- Extra-nodal marginal zone lymphomas (ENZML) are negative follicular lymphomas can be encountered; **A sim- indolent B-cell NHLs, mostly arising in the muco- ilar phenotype is shared by lymphoplasmacytic lymphoma, sa-associated lymphoid tissue (MALT) of the stom- which is not included in this review due to its exceptional ach and small bowel. A consistent number of cases occurrence in the GI tract. develop after chronic antigen stimulation by H. pylori (gastric ENZML) or C. jejuni (small intestinal ENMZL) infection 5. Alpha heavy chain disease (also known as “immunoproliferative small intestinal disease; IPSID) germinal centers (GCs) are scattered throughout the is a clinical-pathological variant of small bowel EN- tumor, often with features of GC colonization (i.e. ef- MZL, characterized by monoclonal alpha heavy chain facement of GCs and disruption of follicular dendritic secretion with no immunoglobulin light chains. IPSID cell [FDC] meshworks). Neoplastic cells also infiltrate affects adolescents and young adults of Mediterrane- the surrounding glands, producing LELs (Fig. 3A). an origin and presents with malabsorption, diarrhea, These are defined as intra-epithelial aggregates of ≥ 3 fever, and weight loss 19. marginal zone cells that disrupt the gland architecture. Histologically, ENMZLs disclose sheets of B-cells, ex- Epithelial cells of LELs frequently undergo eosinophil- panding the lamina propria with possible extension to ic degeneration (i.e. oxyphilic change). The neoplastic the muscolaris mucosae and submucosa. Reactive population consists of small to medium-sized mature DIAGNOSIS OF GI LYMPHOPROLIFERATIVE DISORDERS 231

Figure 3. Histological features of ENMZL and primary duodenal FL. (A) ENMZL presents as a dense infiltrate of small to medium B-cells, with frequent LELs (H&E, high power picture). Phenotypically, CD10 and Bcl6 are negative, while Bcl2 is diffusely positive. MNDA expression supports the diagnosis, while the Ki67 proliferation index is low. (B) Primary duodenal FL presents as superficially located lymphoid nodules with villous widening and lamina propria expansion. The neoplastic population includes numerous centrocytes with sparse centroblasts (large box, insert). CD20, CD10, Bcl6 and Bcl2 are dif- fusely positive. CD21 immunostain highlights peripheral ring-shaped FDC networks, while the Ki67 index is low-to-moderate and lacks polarization. (H&E, Giemsa and immunoperoxidase stain; original magnification 7x, 20x and 80x).

B cells with variable amounts of plasma cells and scat- dium-sized, have pale cytoplasm, slightly irregular nu- tered large B blasts. The mature cell component has clear contours, moderately dispersed chromatin and variable cytological features, spanning from marginal inconspicuous nucleoli. Monocytoid cells have larg- zone to monocytoid, centrocyte-like, small-cell and er amounts of paler cytoplasm, while centrocyte-like plasmacytic morphology. Marginal zone cells are me- cells are small to medium-sized with irregular/cleaved 232 M. Pizzi et al.

nuclei and dense chromatin. Small cell morphology is blurred. In such instances, uncertainty should be consists of mature B-cells with round nuclear contours reported through validated scoring systems, such as and clumped chromatin, resembling SLL/CLL. Plas- the Wotherspoon score (for treatment-naïve cases) 20 macytic differentiation occurs in one third of gastric and GELA score (for post-treatment evaluations) 21 ENMZL and may include plasma cells with intra-nu- (Tab. II). clear immunoglobulin inclusion (i.e. Dutcher bodies). The differential diagnosis of ENMZL includes a num- Such histological features are even more prominent in ber of entities. First, ENZML must be distinguished IPSID, where they occur together with villous widen- from other small-to-medium B-cell NHLs. As marginal ing and pseudo-atrophy. LELs are limited to residual zone markers (e.g. MNDA and IRTA1) have only lim- B-cell areas. ited sensitivity or specificity, the diagnosis of ENMZL On immunohistochemistry (IHC), ENMZLs express is often one of exclusion and requires thorough im- the pan-B cell antigen CD20 and are negative for munophenotyping (Fig. 2). In the pediatric population, CD10, Bcl6, CD23, Cyclin D1 and CD5. IgM are usu- ENMZL must also be distinguished from “atypical mar- ally positive in ENMZL, while IgA heavy chains are ginal zone hyperplasia” (AMZH) 22. This lesion occurs characteristically expressed in IPSID. Positivity for IR- in the appendix and tonsil of children and adolescents, TA1 has been reported in subsets of cases (mostly as a consequence of unknown inflammatory triggers. with monocytoid morphology) and supports the diag- Histologically, AMZH recapitulates the morphological nosis. MNDA and CD43 are also variably expressed features of ENMZL with frequent lambda-chain re- (Fig. 3A). In case of plasma cell differentiation, light striction on immunophenotyping, being nonetheless chain restriction can be documented by kappa and characterized by polyclonal rearrangements of the lambda immunostains 3. immunoglobulin genes and never progressing to lo- The diagnosis of ENMZL may prove challenging on cally-spread or systemic disease 22. When the plas- occasion. This holds particularly true for chronic in- macytic differentiation is so prominent to occupy most flammatory conditions (e.g. H. pylori-related gastritis) of the tissue sample, the risk of a misdiagnosis of or post-treatment assessments, in which the border extramedullary plasmacytoma does exist. The clinical between reactive lymphoid hyperplasia and ENMZL features and search for an associated B-cell compo-

Table II. Scoring systems for treatment-naïve and post-treatment ENMZL Wotherspoon scoring system for treatment-naïve gastric ENMZL* Score Diagnosis Histological features 0 Normal mucosa Scattered lymphocytes and plasma cells in the lamina propria; no lymphoepithelial lesions 1 Active gastritis Inflammatory lymphoid infiltrate in the lamina propria; no lymphoepithelial lesions 2 Active gastritis with lymphoid Inflammatory lymphoid infiltrate with follicles; no lymphoepithelial lesions follicles 3 Suspicious lymphoid Dense lymphoid infiltrate with follicles surrounded by small lymphocytes that infiltrate, probably reactive infiltrate diffusely the lamina propria with occasional lymphoepithelial lesions 4 Suspicious lymphoid Dense lymphoid infiltrate with follicles surrounded by marginal zone infiltrate, probably lymphoma lymphocytes that infiltrate diffusely the lamina propria and the glandular epithelium with focal lymphoepithelial lesions 5 ENMZL Dense lymphoid infiltrate effacing gastric mucosa with diffuse lymphoepithelial lesions GELA scoring system for post-treatment gastric ENMZL** Score Diagnosis Histological features CR Complete histological Scattered lymphocytes and plasma cells in the lamina propria; no remission lymphoepithelial lesions; empty or fibrotic lamina propria pMRD Probable minimal residual Lymphoid aggregates in the lamina propria, muscularis mucosae or disease submucosa; no lymphoepithelial lesions; empty or fibrotic lamina propria rRD Responding residual disease Dense periglandular lymphoid infiltrate with/without lymphoepithelial lesions; focal empty or fibrotic lamina propria NC No change (persistent Dense lymphoid infiltrate effacing gastric mucosa, mostly with lymphoepithelial ENMZL) lesions Notes: *modified form ref. 20; **modified from ref. 21. Note that molecular biology tests are not included in either scoring system. Even in post-treatment settings, monoclonal peaks per se cannot be assumed as evidence of residual disease. Nonetheless, the documentation of identical peaks in pre- and post-treatment samples is highly suggestive of pMRD/rRd. DIAGNOSIS OF GI LYMPHOPROLIFERATIVE DISORDERS 233

nent in subsequent sections usually allows for a relia- susception. Unlike primary duodenal FL, the intestinal ble diagnosis. Finally, ENMZL with increased large cell wall is deeply infiltrated by neoplastic follicles, variably fractions must be distinguished from DLBCL. By defi- associated with interstitial fibrosis. Most cases are low nition, any sheet-like proliferation of large cells (i.e. grade (G1/G2), express CD10, Bcl6 and Bcl2 and dis- aggregates > 20 large cells and/or areas with > 10% of close expanded FDC meshworks 24. The differential cohesive large cells) excludes ENMZL and prompts a diagnosis between such cases and secondary GI in- diagnosis of DLBCL. If both high and low-grade areas volvement by systemic FL relies on Dawson’s criteria are present, a diagnosis of DLBCL with accompany- (see above). ing ENMZL is advised and the percentage amount of Mantle cell lymphoma (MCL) is a biologically aggres- each component should be reported 3. sive neoplasm, accounting for about 3-10% of NHLs 3. In the GI tract, follicular lymphoma (FL) occurs as It affects middle-aged to elderly patients and presents either primary disease or (more frequently) as sec- as nodal/extra-nodal disease, with frequent GI involve- ondary involvement by systemic cases 23. Primary FL ment (15-30% of cases). In the large bowel, it may oc- of the GI tract usually arises in the duodenum, with casionally appear as multiple polypoid lesions, known possible synchronous seeding to the jejunum and ile- as lymphomatous polyposis. Histologically, classic um 23,24. Primary-duodenal FL is a disease variant with MCL consists of diffuse, vaguely nodular or (rarely) excellent prognosis and unique clinical-pathological perifollicular lymphoid infiltrates localized in the lam- features 3,23. It presents as small polypoid lesions, in- ina propria and submucosa. Unlike ENMZL, native cidentally found during upper GI endoscopy. Histolog- glands are spared, LELs are rarely found and the infil- ically, the lymphoid infiltrate is confined to the lamina trate is more deeply seated with limited effacement of propria of duodenal villi with minimal (if any) extension the superficial lamina propria. The neoplastic infiltrate to the submucosa. The duodenal wall and regional consists of small to medium-sized cells with irregu- lymph nodes are characteristically spared. Neoplas- lar nuclear contours (Fig. 4A). Blastoid, pleomorphic tic follicles are non-polarized, lack tingible-body mac- and (more rarely) small or marginal zone cell variants rophages and are devoid of mantle zones. The tumor are reported. The immunophenotype of MCL is high- population consists of small centrocytes (coarse chro- ly characteristic, with strong positivity for CD5, Cyclin matin and cleaved nuclei) and scattered centroblasts D1, and SOX11. Negativity for one of such markers (open chromatin and multiple nucleoli) (Fig. 3B). Cen- is nonetheless possible and correlates with specific trocytes and centroblasts disclose the classic FL phe- clinical-prognostic features. In particular, CD5-neg- notype (positivity for CD10, Bcl6 and Bcl2; negativity ative MCLs typically present as extra-nodal disease for CD5, Cyclin D1, CD23, MNDA and IRTA1), while (also in the GI tract) and bear more favorable prog- FDC meshworks are characteristically pushed at the nosis (Fig. 4A) 25. CD10, Bcl6 and CD23 are usually periphery of the neoplastic follicles. This feature is (yet not invariably) negative 3,26. The proliferation in- readily highlighted by CD21 and/or CD23 immunos- dex varies and should always be assessed, as values tain. The Ki-67 proliferation index is low and non-po- > 30% correlate with worse prognosis 27. Over 95% of larized (Fig. 3B). Like systemic FLs, primary-duodenal MCLs bear the t(11;14)(q13;32), which juxtaposes the cases bear the t(14;18)(q32;q21), which juxtaposes IGH with the Cyclin D1-coding CCND1 gene. In select- the BCL2 and IGH genes 3,23. Given its excellent out- ed cases, Fluorescence In Situ Hybridization (FISH) come, primary-duodenal FL must be distinguished analysis for this translocation supports the diagnosis 3 from duodenal involvement by systemic FL. The latter (Fig. 4A). Small subsets of Cyclin D1-positive cells should be favored in case of deep infiltration of the properly located in the mantle zone of otherwise re- duodenal wall, the mesentery and/or regional lymph active follicles can be discovered in inflammatory in- nodes, with preserved expanded FDC meshworks filtrates of patients with Cyclin D1-positive monoclo- and in cases lacking Bcl2 protein expression and/or nal B-cell lymphocytosis. In the absence of manifest BCL2 gene translocations. Moreover, primary-duode- lymphoma, this picture should not be diagnosed as nal FL is usually a low-grade neoplasm (i.e. G1/G2: clinically overt MCL 28. < 15 centroblasts/high-power field [HPF]). The docu- The major differential diagnosis of MCL is chronic mentation of high-grade morphology (i.e. G3A/G3B: lymphocytic leukemia/small lymphocytic lymphoma > 15 centroblasts/HPF) should prompt investigation (CLL/SLL). This is an indolent B-cell neoplasm, usu- for secondary disease 23. ally affecting elderly people with peripheral blood and/ In rare instances, primary GI FL occurs in non-duo- or nodal involvement 3. Despite CLL/SLL never oc- denal sites. These tumors account for < 4% of all GI curs as primary GI lymphoma, subtle interstitial and NHLs, usually involve the small and large bowel and intravascular infiltrates may be encountered in the present with abdominal pain, GI obstruction or intus- stomach, bowel and appendix as a result of system- 234 M. Pizzi et al.

Figure 4. Histological features of MCL and CLL/SLL. (A) MCL presents as a diffuse or vaguely nodular proliferation of mono- morphic small-sized B-cells. Rarely, LELs can be documented (large box, insert). The tumor cells express CD5, Cyclin D1 and SOX11. Rare cases with aberrant phenotypes, such as CD5-negativity (CD5 box, insert), are nonetheless encountered. Cyclin D1 over-expression is caused by CCND1 translocations, which can be documented by FISH (Cyclin D1, insert; break apart probes). CD23 is negative in tumor cells and positive in residual FDC meshworks. The Ki-67 index is usually low to moderate, despite occasional cases (mostly blastoid or pleomorphic variant, as in this figure) are highly proliferating. (B) CLL/SLL is usually an incidental finding in surgical specimens removed for other causes (e.g. colon adenocarcinoma; large box, upper left). Neoplastic lymphocytes are small, with scattered paraimmunoblasts (H&E, high power picture). They are positive for CD5 and CD23, but negative for Cyclin D1 and SOX11. The Ki67 index is low and limited to the paraimmunoblast component. (H&E, immunoperoxidase and DAPI stain; original magnification 5x, 20x and 40x, 80x).

ic dissemination. This is also true for specimens re- inflammatory milieu and appear as multiple, discrete moved for complicated diverticulitis or carcinoma: in and monotonous lymphoid nodules (Pizzi M, Bellan A; this setting, CLL/SLL cells are likely recruited by the unpublished data) (Fig. 4B). Histologically, CLL/SLL is DIAGNOSIS OF GI LYMPHOPROLIFERATIVE DISORDERS 235

characterized by an interstitial, diffuse or vaguely nod- is documented in most endemic and immunodeficien- ular infiltrate, made of small lymphocytes with round cy-associated BLs, while it is less common in sporadic nuclei and dark chromatin. Paler pseudo-follicles en- cases. Most BLs bear translocations juxtaposing MYC riched in medium-sized nucleolated paraimmunob- to one of the immunoglobulin genes (IGH, IGK and/or lasts are commonly seen. On IHC, CLL/SLL is positive IGL): the resulting t(8;14)(q24;q32) or alternative t(2;8) for CD20, CD23 and CD5, but (unlike MCL) it stains (p12;q24) and t(8;22)(q24;q11) play a key pathogen- negative for Cyclin D1 and SOX11 3 (Fig. 4B). CD200 ic role and help to confirm the diagnosis (Figure 5A). and LEF1 are also usually positive 29. By definition, BCL6 and/or BCL2 rearrangements are not present 3. Of note, about 10% of otherwise clas- Medium to large size B-cell NHLs sical BLs lacks MYC rearrangements 33,34, indicating This group includes a variety of clinically aggressive that alternative pathogenic mechanisms may occur. In entities (BL, DLBCL, HGBL NOS, DHL/THL, LBCL such cases, strict clinical, morphological and pheno- with IRF4 rearrangements and PBL), presenting with typic criteria should be applied to rule out possible BL bulky lesions, intestinal occlusion, GI bleeding and/or mimickers. perforation. They occur in immunocompetent as well The diagnosis of BL is usually straightforward. Small as immunocompromised patients 3. or poorly representative biopsy samples may none- Burkitt lymphoma (BL) is an aggressive NHL, typically theless pose the differential diagnosis with other occurring in extra-nodal sites or as leukemic disease. high-grade B-cell NHLs with medium size or blastoid Three epidemiological variants are described: (i) en- morphology (i.e. blastoid MCL, B-cell lymphoblastic demic BL, (ii) sporadic BL, and (iii) immunodeficien- leukemia/lymphoma [B-ALL], HGBL or, rarely, DLB- cy-associated BL. Endemic BL occurs in equatorial CL, and Burkitt-like lymphoma with 11q aberration). Africa, affects children and young adolescents and Negativity for CD5 and Cyclin D1 excludes blastoid presents as bulky lesions of the jaws and facial bones. MCL, while negativity for TdT (and other immaturi- Sporadic and immunodeficiency (mostly HIV)-associ- ty markers, such as CD34) argues against B-ALL 35. ated cases are reported throughout the world, have a HGBLs (NOS or DHL/THL) and DLBCL have features broad age distribution and account for about 1-2% of that more commonly diverge from the classical mor- adult lymphomas in Western countries 30. Immunode- phological, phenotypic and cytogenetic presentation ficiency-associated BL usually presents with nodal or of BL (e.g. pleomorphic or large-sized cells, negativ- leukemic disease, while sporadic BL mostly occurs in ity for CD10 and/or Bcl6; diffuse, strong positivity for the GI tract and abdominal cavity 3. Bcl2; lack of MYC translocations; occurrence of BCL2 The histological and cytogenetic features of BL are and/or BCL6 translocations) 3. Helpful immunohisto- highly characteristic. In prototypical cases, the tumor chemical algorithms have been reported in challeng- shows a diffuse, cohesive and monotonous prolifer- ing cases 36. Finally, Burkitt-like lymphoma with 11q ation of medium-sized lymphocytes with squared-off aberration is a rare, aggressive B-cell NHL, mostly borders, round nuclei, finely clumped chromatin, in- occurring at nodal sites in children, young adults and conspicuous nucleoli and scant basophilic cytoplasm in the post-transplant setting. The differential diagno- with lipid vacuoles. The latter are more readily appre- sis with BL relies on a greater degree of cytological ciable in cytological preparations or by adipophilin im- pleomorphism, a vaguely nodular (or even follicu- munostain 31. Rare (usually immunodeficiency-related) lar) growth pattern and on the less intense positivity cases disclose a more pleomorphic cytology and/or for Myc protein. MYC rearrangements are typically plasmacytoid differentiation. Irrespective of the mor- lacking, while chromosome 11q alterations (proximal phological variants, tingible body macrophages are gains and telomeric losses) are present by definition scattered throughout the lesion, imparting the classic (Figs. 5B, 6) 3,37. “starry sky” pattern (Fig. 5A). Phenotypically, tumor Diffuse large B-cell lymphomas (DLBCLs) are clinical- cells are almost invariably positive for CD10 and Bcl6 ly and biologically heterogeneous neoplasms, defined and negative (or only weakly positive) for Bcl2. The Ki- by purely morphological criteria (i.e. diffuse growth 67 proliferation index is equal (or close) to 100%. Myc pattern of large B cells). DLBCLs account for about protein is diffusely and strongly expressed in > 80% 45-50% of all primary GI NHLs and develop as either of neoplastic cells. Negative staining may nonetheless primary disease or secondary evolution of a prior low- occur in a minority of cases and does not prevent the grade lymphoma (ENMZL; FL or CLL/SLL) or MCL 38. diagnosis, if all other BL features are present 32. CD38, The identification of secondary cases is based on the MUM1, CD43 and SOX11 expression is variable, while clinical history, disease presentation, and on the his- TdT, Cyclin D1, CD23, and CD5 are consistently neg- tological documentation of a clonally related (prior or ative (Fig. 5A). Positivity for EBER in situ hybridization coexisting) indolent component. 236 M. Pizzi et al.

Figure 5. Histological features of BL and Burkitt-like lymphoma with 11q aberrations. (A) BL presents with bulky GI lesions, featuring a diffuse proliferation of monomorphic, medium-sized lymphocytes with squared-off borders and inconspicuous nucleoli. A starry sky pattern is usually present. Phenotypically, BL is positive for GC markers (CD10, Bcl6) and negative/ weakly positive for Bcl2. The Ki-67 index approximates 100% and c-Myc is strongly and diffusely expressed. MYC translo- cations are present in >90% of cases (c-Myc box, insert; break apart probes). Tumor cells are also positive for adipophilin, a marker of deranged lipid metabolism. (B) Burkitt-like lymphoma with 11q aberrations discloses more cytological hetero- geneity (H&E, insert) and weaker c-Myc expression than conventional BL. MYC translocations are not documented (c-Myc box, insert; break apart probes), while 11q aberrations (gain/loss signals at FISH) are present by definition. (H&E, Giemsa, immunoperoxidase and DAPI stain; original magnification 10x, 20x and 80x). DIAGNOSIS OF GI LYMPHOPROLIFERATIVE DISORDERS 237

Figure 6. Diagnostic flow chart of BL and its mimickers.

Despite several DLBCL variants are described, the ation index is variable, but typically exceeds 25-30%. NOS subtype is by far the most common. The tumor EBER in situ hybridization is positive in a minority of typically presents as a bulky, ulcerated lesion with ex- cases and is usually documented in elderly patients. tensive infiltration of the affected organ. Tumor cells These lymphomas fall within the WHO category of resemble centroblasts (medium to large-sized blasts “EBV-positive, DLBCL NOS” and should be diagnosed with multiple membrane-bound nucleoli), immunob- as such (Fig. 7B). EBV positivity may also be docu- lasts (large-sized blasts with a single, centrally located mented in immunodeficiency-related cases and in the nucleolus) or have anaplastic morphology. Spindle or post-transplant setting 3. signet-ring cytology is rarely found 3,38. In the GI tract, For prognostic purposes, newly diagnosed DLBCLs immunoblastic morphology is by far the most frequent should be stratified according to the putative cell of or- (Fig. 7A). igin into germinal center-derived (GCB) or non-GCB Given their biological heterogeneity, DLBCLs have types 39. In the routine practice, this is possible by apply- highly variable phenotypes. Blast cells are strongly ing IHC algorithms, including the Visco-Young, Choi and/ positive for panB-cell markers (CD20, CD19, PAX5, or Hans algorithm40-42. The latter is most widely used and CD79a), though some of them may exceptionally is based on the sequential assessment of CD10, Bcl6 be lacking, with variable expression of CD10, Bcl6, and MUM1 expression. All DLBCL cases should also MUM1, Bcl2 and Myc (Fig. 7A). CD30, CD23 and CD5 be stained for Myc and Bcl2, as double expressor cases can be occasionally positive, while Cyclin D1, SOX11 bear significantly worse prognosis and should be report- and TdT are consistently negative. The Ki-67 prolifer- ed as such (cutoffs for Myc and Bcl2 positivity: 40% and 238 M. Pizzi et al.

Figure 7. Histological features of DLBCL. (A) In the GI tract, DLBCLs present as sheets of large, atypical cells (H&E, high power picture). Most cases disclose a non-GCB phenotype (e.g. negativity for CD10, weak positivity for Bcl6, strong and dif- fuse expression of MUM1). Bcl2 and c-Myc are variably positive and their joint expression (i.e. double expressor phenotype) is associated with worse prognosis. (B) Rare cases of DLBCLs are EBV-positive. These tumors are more polymorphic than EBV-negative DLBCLs and may contain sternbergoid cells (H&E, high power picture; arrow). They are usually negative for CD10 and positive for MUM1 and CD30. Bcl6 is variable (positive in this case). (H&E and immunoperoxidase stain; original magnification 5x and 40x).

50% of neoplastic cells, respectively) 43 (Fig 7A). Of note, defined clinical and pathological settings, Myc and Bcl2 Myc/Bcl2 double expression is not a surrogate marker joint positivity should prompt FISH analyses for DHL/ of MYC and BCL2 gene rearrangements, although Myc THL, as recently stated by a consensus paper from the protein positivity in > 70% of the neoplastic cells is more Italian Hematopathology Group 45. likely sustained by MYC translocations 44. As such, in A subset of high-grade B-cell NHLs discloses ambig- DIAGNOSIS OF GI LYMPHOPROLIFERATIVE DISORDERS 239

uous features, not perfectly fitting within the BL and/ Plasmablastic lymphoma (PBL) is an aggressive or DLBCL categories. These cases (formerly known NHL with plasmacytic differentiation, most common- as “large B-cell lymphomas with features intermedi- ly arising in immunocompromised or elderly patients. ate between DLBCL and BL”) are currently referred to The tumor typically develops in extra-nodal locations as high-grade B-cell lymphomas (HGBL) and further (mostly the oral cavity and head and neck region). sub-classified into: (i) HGBL NOS and (ii) HGBL with Primary PBL of the GI tract accounts for about 20% rearrangements of MYC and BCL2 and/or BCL6 (i.e. of cases and occurs in the stomach, terminal ileum, DHL/THL) 3,46. Most often, these tumors have large- large bowel, rectum and anal canal 49. In these loca- cell or blastoid morphology (i.e. medium-sized blasts tions, the tumor presents with large, ulcerated lesions with finely dispersed chromatin and inconspicuous causing abdominal pain, bleeding, diarrhea and/or nucleoli). Their phenotype is variable and the prolifer- constipation. Monoclonal components in the periph- ation index is usually high, reflecting the heterogene- eral blood are only rarely documented. Histologically, ous (yet very aggressive) nature of these neoplasms. PBL is characterized by sheets of large cells, span- CD10, Bcl6, Bcl2 and Myc are variably expressed in ning from immunoblasts to plasmablasts (i.e. blasts HGBL NOS, while they are typically positive in DHL/ with eccentrically located nuclei, evident nucleoli and THL 3,47. TdT and Cyclin D1 are invariably negative, abundant cytoplasm). Immunoblast morphology fre- thus excluding a diagnosis of B-ALL and blastoid/ quently occurs in the oral and nasal cavity of HIV-pos- pleomorphic MCL 35. By definition, DHL/THL harbor itive patients, while plasmablastic differentiation is MYC translocations together with BCL2 and/or BCL6 more characteristic of other anatomic sites. A starry rearrangements. Instead, HGBLs NOS are not trans- sky pattern and abundant mitotic figures are common. located or feature rearrangements in only one of such A plasma cell component at various stages of matura- genes. tion is not a feature of PBL and should prompt consid- Large B-cell lymphoma (LBCL) with IRF4 rearrange- eration of alternative diagnoses (e.g. anaplastic plas- ment is a recently described lymphoid neoplasm, in- macytoma/multiple myeloma). On IHC, PBL discloses cluded as a provisional entity in the revised 4th edi- sharp plasmacytic differentiation with positivity for tion of the WHO Classification 3. The tumor primarily CD38, CD138, MUM1, IgG and either kappa or lamb- occurs in children and young adults, accounting for da chains. The Ki-67 proliferation index is very high < 1% of all high-grade B-cell NHLs. Despite Waldey- (> 80%). CD30 and EMA are frequently expressed, er ring and cervical lymph nodes are most commonly while CD79a, CD56 and c-Myc are positive in subsets affected, primary GI involvement is reported in up to of cases. CD45, CD20 and PAX5 are negative or only one third of cases 48. Histologically, LBCL with IRF4 focally positive, thus ruling out DLBCL NOS and other rearrangement consists of a monotonous prolifera- high-grade B-cell NHLs. Most PBLs (75% of cases) tion of medium to large-sized lymphocytes with open are positive for EBER in situ hybridization (Fig. 8A). chromatin. The tumor discloses a diffuse, nodular This feature and the overall clinical presentation and diffuse or purely nodular growth pattern. In the are extremely helpful in differentiating PBL from ex- latter case, neoplastic nodules are large, round and tramedullary anaplastic plasmacytoma and multiple back-to-back with absent or attenuated mantle zones, myeloma, which are rarely associated with EBV in- resembling G3 FL. A starry-sky pattern is typically fection. In some instances, however, the distinction is absent. On IHC, MUM1 is strongly and diffusely ex- not feasible and a descriptive diagnosis of “plasma- pressed. CD20 and Bcl6 are positive, while CD10 and blastic neoplasm, consisting with PBL or anaplastic Bcl2 are variable. The Ki67 proliferation index is high plasmacytoma” is advisable 3,50. In PBL, negativity for (> 30% of neoplastic cells). Cytogenetic analyses dis- ALK1 and HHV8 rules out other CD20-negative NHLs close cryptic rearrangements of IRF4, more often with with plasmablastic morphology, such as ALK1-positive the immunoglobulin genes. MYC and/or BCL2 trans- large B-cell lymphoma and extra-cavitary primary ef- locations are never documented 42. fusion lymphoma (Fig. 8B). The differential diagnosis of LBCL with IRF4 rearrange- ment includes FL (for cases with nodular growth pat- Non-Hodgkin T cell lymphomas of the GI tract tern) and DLBCL NOS (for cases with diffuse or mixed T-cell NHLs represent an absolute minority of GI lym- growth pattern). In the appropriate context (i.e. GI phoid neoplasms. Despite any T-cell neoplasm may B-cell NHL with high-grade morphology in children or virtually involve the GI tract, some entities localize young adults), the striking positivity for MUM1 should electively to the small intestine. These include: (i) prompt FISH analysis for IRF4 translocations. Their enteropathy-associated T-cell lymphoma (EATL); (ii) documentation is virtually diagnostic of LBCL with monomorphic epitheliotropic intestinal T-cell lympho- IRF4 rearrangement and excludes other entities 3,48. mas (MEITL); (iii) some cases of extra-nodal NK/T-cell 240 M. Pizzi et al.

Figure 8. Histological features and differential diagnosis of PBL. (A) PBL consists of sheets of large cells with plasmablastic/ plasmacytoid features. Tumor cells are characteristically negative for CD20 and express plasma cell markers, such as CD138 and MUM1. Light chain restriction is documented by kappa and lambda immunostains. The vast majority of cases is driven by EBV infection (positive EBER in situ hybridization). (H&E and immunoperoxidase stain; original magnification 10x, 20x and 40x). (B) Diagnostic flow chart for GI neoplasms with plasmablastic morphology.

lymphoma (ENKTL), nasal type; (iv) rare intestinal known as type 1 EATL) is a rare and very aggressive T-cell lymphomas, NOS; and (v) the so-called “indo- T-cell neoplasm, affecting patients with prior or con- lent T-cell LPDs of the GI tract”. comitant history of coeliac disease (CD). It accounts Enteropathy-associated T-cell lymphoma (formerly for < 5% of all GI NHLs and mostly occurs in Western DIAGNOSIS OF GI LYMPHOPROLIFERATIVE DISORDERS 241

countries. In Italy, it constitutes about two thirds of all pression of CD30 and positivity for T-cell cytotoxic primary GI T-cell neoplasms, with an estimated inci- markers 56. In ALCL, however, there is no history of dence among CD patients of 0.2-2/100.000/year 51,52. CD, the uninvolved intestinal mucosa lacks features of The disease typically occurs in the jejunum or ileum enteropathy and the positivity for CD30 is much more sometimes with multifocal lesions, and presents with intense and diffuse than in EATL. However, if CD fea- ulcerated, perforated or stenotic masses that cause tures are not seen on the tissue samples and there is abdominal pain, bleeding, intestinal occlusion and no history of CD, the differentiation can be challenging systemic symptoms. Patients also complain of CD-re- and CD clinical investigation should be suggested. lated symptoms 53. Monomorphic epitheliotropic T-cell lymphoma (MEITL; Histologically, EATL is characterized by a diffuse pro- formerly known as type 2 EATL) is another aggressive liferation of polymorphic atypical cells of medium to T-cell NHL, typically arising in the small bowel. Unlike large size that colonize the surface epithelium and EATL, it is not associated with CD and accounts for deeply infiltrate the intestinal wall. Anaplastic fea- the vast majority of primary GI T-cell NHLs in Asia 7. tures are documented in about 40% of cases, while Histologically, intestinal villi are distorted and widened angioinvasion, angiocentricity and necrotic areas are by sheets of monotonous medium-sized cells with commonly observed. The neoplastic population is ac- pale cytoplasm, sparse chromatin and small nucleoli. companied by a rich inflammatory infiltrate, consist- Epitheliotropism is striking, while angiotropism, ne- ing of histiocytes, eosinophils and small lymphocytes crotic areas and/or accompanying inflammatory cells (Fig. 9A). Spreading to regional lymph nodes, liver, are characteristically absent (Fig. 9B). The pheno- skin and other extra-abdominal organs is frequent. type of MEITL differs from EATL in that the neoplastic The uninvolved small intestinal mucosa discloses cells are positive for CD8 and CD56 with negativity the classical histological features of CD (variable de- for CD30. MATK, TCRγδ chains and TIA1 are usual- grees of villous atrophy with increased intra-epithelial ly positive, while perforin and granzyme B are more CD3-positive T lymphocytes) (Fig. 9A). On IHC, EATL variable 3,57 (Fig. 9B). About 25% of cases aberrantly expresses the panT cell markers CD3 and CD7, with express CD20, but negativity for PAX5, CD19, CD79a variable loss of CD2 and CD5. Neoplastic cells dis- and other B-cell markers supports the diagnosis of a close an activated cytotoxic phenotype (positivity for T-cell neoplasm 58. EBER in situ hybridization is con- TIA1, granzyme B and perforin) and are almost invari- sistently negative, thus excluding ENKTL, nasal type 3. ably positive for CD30. The latter is expressed at high- Extra nodal NK/T-cell lymphoma (ENKTL) is an er intensity in cases with anaplastic cytology. EATL is EBV-driven aggressive lymphoma, most commonly usually negative for CD4, CD8 and CD56 and does occurring in the upper respiratory tract. The GI is the not express T-cell receptor (TCR) antigens (Fig. 9A). primary site of disease in 2-7% of cases, but secondary EBER in situ hybridization is typically negative and involvement by extra-intestinal neoplasms is reported. its documentation should prompt consideration of an ENKTL, nasal type usually arises in the jejunum and immunodeficiency-related LPD or other NK/T-cell en- ileum of middle-aged males, presenting as large ul- tities (see below) 3. cerated lesions with bleeding and/or perforation. Key The main differential diagnoses of EATL include re- histological features are the prominent angiotropism, fractory CD and secondary GI involvement by system- the presence of large necrotic areas and/or ulceration ic T-cell NHLs (mostly anaplastic large cell lymphoma; without epitheliotropism and the diffuse positivity of ALCL). Refractory CD (RCD) is defined as CD not neoplastic cells for EBER (Fig. 9C). Tumor cells are responding to > 6-12 months of strictly gluten-free di- cytologically variable, spanning from relatively small to et. Two forms of RCD are described: (i) type 1 RCD, large atypical blasts with “sternbergoid” features in a which is histologically indistinguishable from classic mixed inflammatory background. Most cases disclose CD; and (ii) type 2 RCD, which is characterized by a NK phenotype, with positivity for CD56, cytoplasmic > 50% atypical intraepithelial T-cells, closely resem- CD3 and cytotoxic markers. Surface CD3, CD5, CD4 bling EATL (i.e. positivity for CD3; double negativity for and CD8 are negative, while CD2, CD7 and CD30 CD8 and CD4) 54. Several lines of evidence suggest are variably expressed 59 (Fig. 9C). Rare cases with that type 2 RCD is the precursor lesion of EATL 55. T-cell (either TCRγδ or TCRαβ) phenotype do also The differential diagnosis between the two entities re- occur 60. Irrespective of the cell lineage, EBER posi- lies on RCD being an intra-epithelial disease of most- tivity is always documented and its negativity should ly small atypical cells, whereas EATL is a large cell prompt consideration of other entities (e.g. MEITL, lymphoma and massively infiltrates the intestinal wall. PTCL NOS and indolent T-cell LPDs of the GI tract; Like EATL, ALCL is characterized by large atypical see below). Molecular biology supports the differential blasts with a defective T-cell phenotype, diffuse ex- diagnosis, as monoclonal TCR gene rearrangements 242 M. Pizzi et al.

Figure 9. Differential diagnosis of GI aggressive T/NK-cells NHLs. EATL, MEITL and ENKTL, nasal type are aggressive T/NK lymphoid neoplasms of the GI tract. (A) EATL affects celiac patients and presents as polymorphous sheets of atypical T-cells with a rich inflammatory background (eosinophils, histiocytes and small lymphocytes). The uninvolved intestinal mucosa shows signs of enteropathy (H&E, upper right picture). Tumor cells are positive for various panT-cell markers and CD30, but lack CD8 and CD56. The ki67 index is high. (B) MEITL is an aggressive epitheliotropic NHL made of monomorphic atypical cells. CD3 is positive and highlights the striking epitheliotropism of tumor cells. CD8, CD56 and TIA1 are diffusely expressed, CD30 is negative (not shown) and the Ki-67 index is high. (C) ENKTL, nasal type is characterized by prominent angiotropism and diffuse positivity for EBER (large box, insert). It typically expresses cytoplasmic CD3, CD56 and cytotoxic markers (e.g. Granzyme B). The Ki67 index is high. (H&E, Giemsa and immunoperoxidase stain; original magnification 5x, 20x and 40x).

are not documented in most ENKTL, nasal type (i.e. in the large bowel or the stomach and have a very cases of NK derivation), while are usually present in aggressive clinical course. The morphological and im- other T-cell entities. munohistochemical features are highly variable, yet Rare intestinal T-cell NHLs do not fulfill the diagnos- most cases disclose a cytotoxic phenotype and lack tic criteria of EATL, MEITL and ENKTL, nasal type. TCR expression. The differential diagnosis with sec- These tumors, currently referred to as intestinal T-cell ondary GI involvement by peripheral T-cell lymphoma lymphomas NOS, are not a specific disease entity, NOS is based on clinical data and imaging studies 61. but a diagnostic category to be used when other more In recent years, indolent T-cell LPDs of the GI tract common neoplasms are ruled out or not assessable have also been described 62. These cases mostly oc- (i.e. small biopsy samples lacking surface epithelium cur in the stomach, small intestine and large bowel or inadequate material for immunophenotyping) 3. and present with abdominal pain, weight loss, diar- These cases are not associated with CD, usually arise rhea or dyspepsia. Endoscopically, the mucosa of af- DIAGNOSIS OF GI LYMPHOPROLIFERATIVE DISORDERS 243

fected sites is thickened, polypoid or hyperemic with EBER in the large atypical blasts as well as in small superficial erosions. Histology discloses a dense, to medium-sized B cells. The background lymphoid in- lamina propria-limited, non-angioinvasive lymphoid filtrate and deep margin of the lesion mainly consists infiltrate, displacing (yet not infiltrating) the mucosal of CD8-positive T-cells (Fig. 10A). Molecular biology glands or surface epithelium 62. Epithelioid granu- shows monoclonal rearrangements of the immuno- lomas and occasional eosinophils may be present, globulin genes in about 50% of cases 65. yielding the differential diagnosis with Crohn disease. Depending on the amount and morphological features Cytologically, neoplastic cells are monomorphic and of the large atypical cells, EBVMCU enters the differ- small to medium-sized. Cases with NK phenotype dis- ential diagnosis with cHL, monomorphic/polymorphic close brightly eosinophilic cytoplasmic granules. On PTLD and EBV-positive DLBCL NOS. Distinction from IHC, most indolent T-cell LPDs have a non-activated cHL is based on the clinical presentation and on the cytotoxic phenotype (i.e. positivity for CD3, CD8 and sharp circumscription of the lesion (deep band-like TIA1; negativity for perforin and granzyme B), but margin of T cells). The positivity for EBER even in small positivity for CD4 or even NK cell markers has been lymphocytes and the B-cell phenotype of the atypical reported 63,64. Irrespective of the cell lineage, these le- cells are also not features of cHL. The differential di- sions have very low proliferation index (usually < 10% agnosis with monomorphic/polymorphic PTLD and of neoplastic cells) and a protracted clinical course, EBV-positive DLBCL NOS is more challenging and with limited response to conventional chemotherapy. largely depends on the clinical findings and the local- Molecular biology shows monoclonal TCR rearrange- ized, non-infiltrative nature of EBVMCU 66 (Fig. 10B). ments in all cases 3. Finally, small and superficial biopsies of EBVMCU may lead to the wrong impression of florid granulation Hodgkin lymphoma mimickers in the GI tract tissue associated with benign mucous ulcers. In such Primary classic Hodgkin lymphoma (cHL) is extremely instances, only a high degree of suspicion and careful rare in the GI tract. Occasional GI LPDs may nonethe- histological examination leads to the correct diagno- less feature neoplastic elements that closely resemble sis. Immunophenotyping for CD30, MUM1 and EBER Hodgkin and Reed-Sternberg (HRS) cells. EBV-pos- help highlighting the atypical cell component, thus fa- itive muco-cutaneous ulcer (EBVMCU) is the most voring EBVMCU over reactive inflammation. challenging of such entities. This recently described disease affects immunosup- pressed and elderly males and presents as large ul- Conclusions cerated lesions of the skin, oral cavity, and GI tract 3,65. In the latter site, the rectum and sigmoid colon are Non-Hodgkin lymphomas of the GI tract constitute a most commonly involved. The clinical course is usu- broad spectrum of neoplasms with variable clinical ally benign, with nearly all cases responding to re- and biological features. The management of such duction of immunosuppressive therapy 65. Radiation entities is based on an integrated diagnostic frame- and chemotherapy are also effective. Systemic spread work, considering morphological, immunophenotypic is very rare, but relapse and local progression have and molecular/cytogenetic data. As discussed in this been reported 3. review, a step-wise approach is highly recommended Histologically, EBVMCU appears as a well-demar- to reduce the spectrum of differential diagnoses and cated ulcer with a dense, polymorphic sub-epithelial to get to confident conclusions. Key features for any lymphoid infiltrate. The latter includes small lympho- diagnostic evaluation are the assessment of tumor cytes, plasma cells, histiocytes and eosinophils. Vari- cell cytology (typical vs atypical; small vs large size) able numbers of large atypical cells are also present, and lineage of differentiation (B versus T cell prolifer- resembling either immunoblasts or HRS cells (i.e. ations). This first evaluation will guide any subsequent very large cells with multi-lobated nuclei, eosinophilic ancillary test, reducing the number of unnecessary nucleoli and abundant cytoplasm). The deepest mar- (or even confounding) analyses. Several factors may gin of the lesion is sharp and consists of a band-like limit the diagnostic reliability of the biopsy findings infiltrate of reactive lymphocytes (Fig. 10A). Necrotic (sampling artifacts; little amount of diagnostic materi- areas and angioinvasion are frequently document- al; overlapping features among different entities). Pa- ed. On IHC, the atypical cells are positive for CD30, thologists should be aware of such limitations, stating MUM1, PAX5, CD79a and OCT2. Expression of CD20 all of them in the final report. This will likely limit the is variable and CD15 is found in about 50% of cas- over-interpretation of the histological findings, favor- es. CD10 and Bcl6 are usually negative. A key diag- ing the teamwork between pathologists and clinicians nostic feature is the strong and diffuse positivity for and the proper management of patients. 244 M. Pizzi et al.

Figure 10. Histological features of EBVMCU and monomorphic PTLD. (A) EBVMCU is an ulcerated, sharply demarcated lymphoid lesion with a band-like CD3-positive T-cell infiltrate at the base (large box, insert). Hodgkin/Reed Sternberg-like cells are characteristically present (high power picture, arrow) and express CD30, multiple B-cell markers (e.g. CD20, CD79a), MUM1 and EBER. CD15 is present in subsets of cases. These features need to be distinguished from cHL, whose primary involvement of the GI tract is extremely rare. (B). The differential diagnosis of EBVMCU encompasses various EBV-positive LPDs, including monomorphic PTLD. This consists of sheets of atypical blasts with variable histological features (the case reported here has lymphomatoid granulomatosis-like features with marked angiotropism). CD20, CD30 and MUM1 are typi- cally expressed, EBER positivity is strong and diffuse (large box, insert) and the Ki67 index is high. (H&E, immunoperoxidase stain; original magnification 2x, 10x, 20x and 40x). DIAGNOSIS OF GI LYMPHOPROLIFERATIVE DISORDERS 245

9 Abbrevations: Jain P, Wang M. Mantle cell lymphoma: 2019 update on the di- agnosis, pathogenesis, prognostication, and management. Am J AMZH: atypical marginal zone hyperplasia; B-ALL: Hematol 2019:94:710-25. https://doi.org/10.1002/ajh.25487. B-cell lymphoblastic leukemia/lymphoma; BL: Burkitt 10 Montes-Moreno S, King RL, Oschlies I, et al. Update on lympho- lymphoma; CD: coeliac disease; cHL: classic Hodgkin proliferative disorders of the gastrointestinal tract: disease spec- lymphoma; CLL/SLL: chronic lymphocytic leukemia/ trum from indolent lymphoproliferations to aggressive lympho- small lymphocytic lymphoma; DLBCL: diffuse large mas. Virchows Arch 2020:476:667-81. https://doi.org/10.1007/ s00428-019-02704-8 B-cell lymphoma; EALT: enteropathy-associated T-cell 11 Lo WY, Li JY, Chan YK, et al. Instability of clonality in gastric lymphoma; EBVMCU: EBV-positive muco-cutane- lymphoid infiltrates: a study with emphasis on serial biopsies. ous ulcer; ENKTL: extra-nodal NK/T-cell lymphoma; Am J Surg Pathol 2005:29:1582-92. https://doi.org/10.1097/01. ENMZL: extra-nodal marginal zone lymphoma; FDC: pas.0000188031.40836.00 follicular dendritic cell; FISH: Fluorescence In Situ; FL: 12 Theriault C, Galoin S, Valmary S, et al. PCR analysis of immu- follicular lymphoma; GC: germinal centers; GCD: ger- noglobulin heavy chain (IgH) and TcR-gamma chain gene re- arrangements in the diagnosis of lymphoproliferative disorders: minal center-derived; GI: gastrointestinal tract; HGBL: results of a study of 525 cases. Mod Pathol 2000:13:1269-79. high-grade B-cell lymphoma; HGBL NOS: high- https://doi.org/10.1038/modpathol.3880232 grade B-cell lymphoma not otherwise specified;HPF : 13 Hussein MR. Atypical lymphoid proliferations: the patholo- high-power field;HRS : Hodgkin and Reed-Sternberg gist’s viewpoint. Expert Rev Hematol 2013:6:139-53. https://doi. cells; IHC: immunohistochemistry; org/10.1586/ehm.13.4 IPSID: immunoproliferative small intestinal disease; 14 Dawson IM, Cornes JS, Morson BC. Primary malignant lym- LBCL: large B-cell lymphoma; phoid tumours of the intestinal tract. Report of 37 cases with a study of factors influencing prognosis. Br J Surg 1961:49:80-9. LELs: lymphoepithelial lesions; LPDs: lymphoprolifer- https://doi.org/10.1002/bjs.18004921319 ative disorders; MALT: mucosa-associated lymphoid 15 Loehr WJ, Mujahed Z, Zahn FD, et al. Primary lymphoma of tissue MALT; MCL: mantle cell lymphoma; MEITL: the gastrointestinal tract: a review of 100 cases. Ann Surg Monomorphic epitheliotropic T-cell lymphoma; NHLs: 1969:170:232-8 non-Hodgkin lymphomas; 16 Freeman C, Berg JW, Cutler SJ. Occurrence and prognosis of PBL: plasmablastic lymphoma; PTLDs: post-trans- extranodal lymphomas. Cancer 1972:29:252-60. https://doi. org/10.1097/00000658-196908000-00011 plant LPDs; RCD: Refractory CD; 17 Koch P, del Valle F, Berdel WE, et al. Primary gastrointestinal TCR: T-cell receptor. non-Hodgkin’s lymphoma: I. Anatomic and histologic distribution, clinical features, and survival data of 371 patients registered in References the German Multicenter Study GIT NHL 01/92. J Clin Oncol 2001:19:3861-73. https://doi.org/10.1200/JCO.2001.19.18.3861 1 Mowat AM, Agace WW. Regional specialization within the intes- 18 Salem P, el-Hashimi L, Anaissie E, et al. Primary small in- tinal immune system. Nat Rev Immunol 2014;14:667-85. https:// testinal lymphoma in adults. A comparative study of IPSID doi.org/10.1038/nri3738 versus non-IPSID in the Middle East. Cancer 1987:59:1670- 2 Brandtzaeg P, Kiyono H, Pabst R, et al. Terminology: nomencla- 6. doi:10.1002/1097-0142(19870501)59:9<1670::aid- ture of mucosa-associated lymphoid tissue. Mucosal Immunol cncr2820590925>3.0.co;2-d 2008;1:31-7. https://doi.org/10.1038/mi.2007.9 19 Al-Saleem T, Al-Mondhiry H. Immunoproliferative small intestinal 3 Swerdlow SH, World Health Organization, International Agency disease (IPSID): a model for mature B-cell neoplasms. Blood for Research on Cancer. WHO classification of tumours of hae- 2005:105:2274-80. https://doi.org/10.1182/blood-2004-07-2755 matopoietic and lymphoid tissues. Lyon: International Agency for 20 Wotherspoon AC, Doglioni C, Diss TC, et al. Regression of pri- Research on Cancer 2017. mary low-grade B-cell gastric lymphoma of mucosa-associated 4 Ghimire P, Wu GY, Zhu L. Primary gastrointestinal lymphoma. lymphoid tissue type after eradication of Helicobacter pylori. World J Gastroenterol 2011;17:697-707. https://doi.org/10.3748/ Lancet 1993:342(8871):575-7. https://doi.org/10.1016/0140- wjg.v17.i6.697 6736(93)91409-f 5 Nakamura S, Ponzoni M. Marginal zone B-cell lymphoma: les- 21 Copie-Bergman C, Gaulard P, Lavergne-Slove A, et al. Proposal sons from Western and Eastern diagnostic approaches. Pathol- for a new histological grading system for post-treatment evalu- ogy 2020:52:15-29. https://doi.org/10.1016/j.pathol.2019.08.012 ation of gastric MALT lymphoma. Gut 2003:52:1656. https://doi. 6 Duffles Amarante G, Collins G, Rocha V. What do we know about org/10.1136/gut.52.11.1656 duodenal-type follicular lymphoma? From pathological definition 22 Attygalle AD, Liu H, Shirali S, et al. Atypical marginal zone hy- to treatment options. Br J Haematol 2020:188:831-7. https://doi. perplasia of mucosa-associated lymphoid tissue: a reactive con- org/10.1111/bjh.16348 dition of childhood showing immunoglobulin lambda light-chain 7 van Vliet C, Spagnolo DV. T- and NK-cell lymphoproliferative dis- restriction. Blood 2004:104:3343-8. https://doi.org/10.1182/ orders of the gastrointestinal tract: review and update. Pathology blood-2004-01-0385 2020:52:128-41. https://doi.org/10.1016/j.pathol.2019.10.001 23 Marks E, Shi Y. Duodenal-type follicular lymphoma: a clinico- 8 Ott G. Aggressive B-cell lymphomas in the update of the 4th edi- pathologic review. Arch Pathol Lab Med 2018:142:542-7. https:// tion of the World Health Organization classification of haemato- doi.org/10.5858/arpa.2016-0519-RS poietic and lymphatic tissues: refinements of the classification, 24 Misdraji J, Harris NL, Hasserjian RP, et al. Primary follicular lym- new entities and genetic findings. Br J Haematol 2017:178:871- phoma of the gastrointestinal tract. Am J Surg Pathol 2011:35:1255- 87. https://doi.org/10.1111/bjh.14744 63. https://doi.org/10.1097/PAS.0b013e318224e661 246 M. Pizzi et al.

25 Miao Y, Lin P, Saksena A, et al. CD5-negative mantle cell lympho- classification in diffuse large B-cell lymphoma: a report from ma: clinicopathologic correlations and outcome in 58 patients. the International DLBCL Rituximab-CHOP Consortium Pro- Am J Surg Pathol 2019:43:1052-60. https://doi.org/10.1097/ gram Study. Leukemia 2012:26:2103-13. https://doi.org/10.1038/ PAS.0000000000001278 leu.2012.83 26 Pizzi M, Agostinelli C, Righi S, et al. Aberrant expression of 42 Choi WW, Weisenburger DD, Greiner TC, et al. A new immu- CD10 and BCL6 in mantle cell lymphoma. Histopathology nostain algorithm classifies diffuse large B-cell lymphoma 2017:71:769-77. https://doi.org/10.1111/his.13286 into molecular subtypes with high accuracy. Clin Cancer Res 27 Ye H, Desai A, Zeng D, et al. Smoldering mantle cell lymphoma. 2009:15:5494-5502. https://doi.org/10.1158/1078-0432.CCR- J Exp Clin Cancer Res 2017:36:185. https://doi.org/10.1186/ 09-0113 s13046-017-0652-8 43 Li L, Li Y, Que X, et al. Prognostic significances of overexpres- 28 Espinet B, Ferrer A, Bellosillo B, et al. Distinction between as- sion MYC and/or BCL2 in R-CHOP-treated diffuse large B-cell ymptomatic monoclonal B-cell lymphocytosis with cyclin D1 lymphoma: a systematic review and meta-analysis. Sci Rep overexpression and mantle cell lymphoma: from molecular pro- 2018:8:6267. https://doi.org/10.1038/s41598-018-24631-5 filing to flow cytometry. Clin Cancer Res 2014:20:1007-19. https:// 44 Ziepert M, Lazzi S, Santi R, et al. A 70% cut-off for MYC protein doi.org/10.1158/1078-0432.CCR-13-1077 expression in diffuse large B cell lymphoma identifies a high-risk 29 Zhang XM, Aguilera N. New immunohistochemistry for B- group of patients. Haematologica 2020. https://doi.org/10.3324/ cell lymphoma and Hodgkin lymphoma. Arch Pathol Lab Med haematol.2019.235556. Online ahead of print 2014:138:1666-72. https://doi.org/10.5858/arpa.2014-0058-RA 45 Di Napoli A, Remotti D, Agostinelli C, et al. A practical algorithmic 30 Piccaluga PP, De Falco G, Kustagi M, et al. Gene expression approach to mature aggressive B cell lymphoma diagnosis in the analysis uncovers similarity and differences among Burkitt double/triple hit era: selecting cases, matching clinical benefit: lymphoma subtypes. Blood 2011:117:3596-3608. https://doi. a position paper from the Italian Group of Haematopathology org/10.1182/blood-2010-08-301556 (G.I.E.). Virchows Arch 2019:475:513-18. https://doi.org/10.1007/ s00428-019-02637-2 31 Ambrosio MR, Piccaluga PP, Ponzoni M, et al. The alteration of lipid metabolism in Burkitt lymphoma identifies a novel marker: 46 Rimsza L, Pittaluga S, Dirnhofer S, et al. The clinicopathologic adipophilin. PLoS One 2012:7:e44315. https://doi.org/10.1371/ spectrum of mature aggressive B cell lymphomas. Virchows Arch journal.pone.0044315 2017:471:453-66. https://doi.org/10.1007/s00428-017-2199-7 32 Mundo L, Ambrosio MR, Raimondi F, et al. Molecular switch from 47 Li S, Desai P, Lin P, et al. MYC/BCL6 double-hit lymphoma MYC to MYCN expression in MYC protein negative Burkitt lym- (DHL): a tumour associated with an aggressive clinical course phoma cases. Blood Cancer J 2019:9:91. https://doi.org/10.1038/ and poor prognosis. Histopathology 2016:68:1090-8. https://doi. s41408-019-0252-2 org/10.1111/his.12884 33 Hummel M, Bentink S, Berger H, et al. A biologic definition of 48 Salaverria I, Philipp C, Oschlies I, et al. Translocations activating Burkitt’s lymphoma from transcriptional and genomic profiling. IRF4 identify a subtype of germinal center-derived B-cell lym- N Engl J Med 2006:354:2419-30. https://doi.org/10.1056/NEJ- phoma affecting predominantly children and young adults. Blood Moa055351 2011:118:139-47. https://doi.org/10.1182/blood-2011-01-330795 34 Leucci E, Cocco M, Onnis A, et al. MYC translocation-nega- 49 Luria L, Nguyen J, Zhou J, et al. Manifestations of gastrointes- tive classical Burkitt lymphoma cases: an alternative patho- tinal plasmablastic lymphoma: a case series with literature re- genetic mechanism involving miRNA deregulation. J Pathol view. World J Gastroenterol 2014:20:11894-11903. https://doi. 2008:216:440-50. https://doi.org/10.1002/path.2410 org/10.3748/wjg.v20.i33.11894 35 Swerdlow SH, Campo E, Pileri SA, et al. The 2016 revision of 50 Lorsbach RB, Hsi ED, Dogan A, et al. Plasma cell myeloma and the World Health Organization classification of lymphoid neo- related neoplasms. Am J Clin Pathol 2011:136:168-82. https:// plasms. Blood 2016:127:2375-90. https://doi.org/10.1182/ doi.org/10.1309/AJCPENJ68FFBRIYB blood-2016-01-643569 51 Gao Y, Kristinsson SY, Goldin LR, et al. Increased risk for non- 36 Naresh KN, Ibrahim HA, Lazzi S, et al. Diagnosis of Burkitt Hodgkin lymphoma in individuals with celiac disease and a lymphoma using an algorithmic approach--applicable in both potential familial association. Gastroenterology 2009:136:91-8. resource-poor and resource-rich countries. Br J Haematol https://doi.org/10.1053/j.gastro.2008.09.031. 2011:154:770-6. https://doi.org/10.1111/j.1365-2141.2011.08771.x 52 Corrao G, Corazza GR, Bagnardi V, et al. Mortality in pa- 37 Wagener R, Seufert J, Raimondi F, et al. The mutational land- tients with coeliac disease and their relatives: a cohort study. scape of Burkitt-like lymphoma with 11q aberration is distinct Lancet 2001:358(9279):356-61. https://doi.org/10.1016/s0140- from that of Burkitt lymphoma. Blood 2019:133:962-6. https://doi. 6736(01)05554-4 org/10.1182/blood-2018-07-864025 53 Delabie J, Holte H, Vose JM, et al. Enteropathy-associated T-cell 38 Xie Y, Pittaluga S, Jaffe ES. The histological classification of dif- lymphoma: clinical and histological findings from the internation- fuse large B-cell lymphomas. Semin Hematol 2015:52:57-66. al peripheral T-cell lymphoma project. Blood 2011:118:148-55. https://doi.org/10.1053/j.seminhematol.2015.01.006 https://doi.org/10.1182/blood-2011-02-335216 39 Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse 54 van Gils T, Nijeboer P, van Wanrooij RL, et al. Mechanisms and large B-cell lymphoma identified by gene expression profiling. Na- management of refractory coeliac disease. Nat Rev Gastroen- ture 2000:403(6769):503-11. https://doi.org/10.1038/35000501 terol Hepatol 2015:12:572-579. https://doi.org/10.1038/nrgas- 40 Hans CP, Weisenburger DD, Greiner TC, et al. Confirmation of tro.2015.155 the molecular classification of diffuse large B-cell lymphoma 55 Chander U, Leeman-Neill RJ, Bhagat G. Pathogenesis of Enter- by immunohistochemistry using a tissue microarray. Blood opathy-Associated T Cell Lymphoma. Curr Hematol Malig Rep 2004:103:275-82. https://doi.org/10.1182/blood-2003-05-1545 2018:13:308-17. https://doi.org/10.1007/s11899-018-0459-5 41 Visco C, Li Y, Xu-Monette ZY, et al. Comprehensive gene ex- 56 Inghirami G, Pileri SA, European TCLSG. Anaplastic large-cell pression profiling and immunohistochemical studies support ap- lymphoma. Semin Diagn Pathol 2011:28:190-201. https://doi. plication of immunophenotypic algorithm for molecular subtype org/10.1053/j.semdp.2011.03.002 DIAGNOSIS OF GI LYMPHOPROLIFERATIVE DISORDERS 247

57 Tan SY, Ooi AS, Ang MK, et al. Nuclear expression of MATK is a for Hematopathology. Histopathology 2014:64:171-99. https:// novel marker of type II enteropathy-associated T-cell lymphoma. doi.org/10.1111/his.12251 Leukemia 2011:25:555-7. https://doi.org/10.1038/leu.2010.295 62 Perry AM, Warnke RA, Hu Q, et al. Indolent T-cell lymphoprolifer- 58 Tan SY, Chuang SS, Tang T, et al. Type II EATL (epitheliotro- ative disease of the gastrointestinal tract. Blood 2013:122:3599- pic intestinal T-cell lymphoma): a neoplasm of intra-epithelial 3606. https://doi.org/10.1182/blood-2013-07-512830 T-cells with predominant CD8alphaalpha phenotype. Leukemia 63 Soderquist CR, Patel N, Murty VV, et al. Genetic and phenotypic 2013:27:1688-96. https://doi.org/10.1038/leu.2013.41 characterization of indolent T-cell lymphoproliferative disorders 59 Yu BH, Shui RH, Sheng WQ, et al. Primary intestinal extra- of the gastrointestinal tract. Haematologica 2019. https://doi. nodal natural killer/t-cell lymphoma, nasal type: a compre- org/10.3324/haematol.2019.230961. Online ahead of print. hensive clinicopathological analysis of 55 cases. PLoS One 64 Takeuchi K, Yokoyama M, Ishizawa S, et al. Lymphomatoid gas- 2016:11:e0161831. https://doi.org/10.1371/journal.pone.0161831 tropathy: a distinct clinicopathologic entity of self-limited pseu- 60 Pongpruttipan T, Sukpanichnant S, Assanasen T, et al. Extranod- domalignant NK-cell proliferation. Blood 2010:116:5631-7. https:// al NK/T-cell lymphoma, nasal type, includes cases of natural kill- doi.org/10.1182/blood-2010-06-290650 er cell and alphabeta, gammadelta, and alphabeta/gammadelta 65 Dojcinov SD, Venkataraman G, Raffeld M, et al. EBV positive mu- T-cell origin: a comprehensive clinicopathologic and phenotypic cocutaneous ulcer--a study of 26 cases associated with various study. Am J Surg Pathol 2012:36:481-99. https://doi.org/10.1097/ sources of immunosuppression. Am J Surg Pathol 2010:34:405- PAS.0b013e31824433d8 17. https://doi.org/10.1097/PAS.0b013e3181cf8622 61 Attygalle AD, Cabecadas J, Gaulard P, et al. Peripheral T-cell and 66 Natkunam Y, Goodlad JR, Chadburn A, et al. EBV-Positive B- NK-cell lymphomas and their mimics; taking a step forward - re- Cell proliferations of varied malignant potential: 2015 SH/EAHP port on the lymphoma workshop of the XVIth meeting of the workshop report-part 1. Am J Clin Pathol 2017:147:129-52. European Association for Haematopathology and the Society https://doi.org/10.1093/ajcp/aqw214 PATHOLOGICA 2020;112:248-259; DOI: 10.32074/1591-951X-158

Review

Current prognostic and predictive biomarkers for gastrointestinal tumors in clinical practice

Matteo Fassan1, Aldo Scarpa2,3, Andrea Remo4, Giovanna De Maglio5, Giancarlo Troncone6, Antonio Marchetti7, Claudio Doglioni8,9, Giuseppe Ingravallo10, Giuseppe Perrone11, Paola Parente12, Claudio Luchini3, Luca Mastracci13,14 1 Surgical Pathology Unit, Department of Medicine (DIMED), University of Padua, Italy; 2 ARC-NET Research Centre, University of Verona, Italy; 3 Department of Diagnostics and Public Health, Section of Pathology, University and Hospital Trust of Verona, Verona, Italy; 4 Pathology Unit, Service Department, ULSS9 “Scaligera”, Verona, Italy; 5 Department of Pathology, University Hospital of Udine, Italy; 6 Department of Public Health, Federico II University Medical School Naples, Italy; 7 Center of Predictive Molecular Medicine, Center for Excellence on Aging and Translational Medicine, University of Chieti-Pescara, Italy; 8 Vita e Salute University, Milan, Italy; 9 Pathology Unit, Pancreas Translational and Clinical Research Center, IRCCS San Raffaele Scientific Institute, Milan, Italy; 10 Department of Emergency and Organ Transplantation, Section of Pathological Anatomy, University of Bari Aldo Moro, Bari, Italy; 11 Department of Pathology, Campus Bio-Medico University, Rome, Italy; 12 Pathology Unit, Fondazione IRCCS Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy; 13 Anatomic Pathology, San Martino IRCCS Hospital,, Genova, Italy; 14 Anatomic Pathology, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genova, Genova, Italy

Summary Received and accepted: June 24, 2020 The pathologist emerged in the personalized medicine era as a central actor in the defi- Published online: 29 October, 2020 nition of the most adequate diagnostic and therapeutic algorithms. In the last decade, gastrointestinal oncology has seen a significantly increased clinical request for the inte- Correspondence Claudio Luchini gration of novel prognostic and predictive biomarkers in histopathological reports. This Department of Diagnostics and Public Health, request couples with the significant contraction of invasive sampling of the disease, thus Section of Pathology, University and Hospital conferring to the pathologist the role of governor for both proper pathologic characteriza- Trust of Verona, piazzale L.A. Scuro 10, tion and customized processing of the biospecimens. This overview will focus on the most 37134 Verona (VR) Italy commonly adopted immunohistochemical and molecular biomarkers in the routine clinical Tel. 0039 045 8124835 characterization of gastrointestinal neoplasms referring to the most recent published rec- E-mail: [email protected] ommendations, guidelines and expert opinions.

Conflict of interest Key words: prognostic markers, predictive markers, targeted therapy, molecular pathology, The Authors declare no conflict of interest. immunohistochemistry

How to cite this article: Fassan M, Scarpa A, Remo A, et al. Current prognostic and predictive Introduction biomarkers for gastrointestinal tumors in clinical practice. Pathologica 2020;112:248- 259. https://doi.org/10.32074/1591-951X-158 Personalized medicine in oncology has pinpointed a central role of pathol- ogists in the multidisciplinary team for the definition of the most adequate © Copyright by Società Italiana di Anatomia Pato- diagnostic and therapeutic algorithms 1. As a result, in the last decade, logica e Citopatologia Diagnostica, Divisione Itali- ana della International Academy of Pathology numerous novel prognostic and predictive biomarkers have been intro- duced and integrated in histopathological reports to obtain an inclusive OPEN ACCESS morphological and molecular characterization of the biospecimens. Several surgical pathology laboratories have implemented next gener- This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- ation sequencing (NGS) or multigene high-throughput technologies in NonCommercial-NoDerivatives 4.0 International) license: the their diagnostic portfolio; however, immunohistochemistry (IHC), in situ work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original hybridization (ISH) and single gene analyses still retain a central role in version. For further information: https://creativecommons. the diagnostic scenario. org/licenses/by-nc-nd/4.0/deed.en This overview will focus on the most commonly adopted immunohis- MOLECULAR LANDSCAPE OF GASTROINTESTINAL NEOPLASMS 249

tochemical and molecular biomarkers in daily clinical troesophageal adenocarcinoma should be tested for characterization of gastrointestinal neoplasms refer- HER2 status. ring to the most recent published recommendations, guidelines and expert opinions. Clinical and pathological associated features HER2 overexpression is observed in 15-20% gastro- esophageal adenocarcinomas and has no significant Gastroesophageal adenocarcinoma prognostic impact. The alteration is more common in intestinal-type ad- enocarcinomas than diffuse-type cancers, low-grade HER2 overexpression/amplification than high grade adenocarcinomas and gastroesoph- ageal junction cancers than distal gastric adenocarci- Definition and therapeutic implications nomas 4. The HER2 (ERBB2) proto-oncogene is a member of the human epidermal growth factor receptor (HER/ Diagnosis GFR/ERBB) family and encodes a transmembrane HER2 status may be clonally heterogeneous within growth factor receptor with tyrosine kinase activity. the same tumor 5,6 and thus, HER2 testing should be HER2 gene amplification leads to HER2 protein over- performed on surgical samples or at least 6 biopsy expression, which is important for cancer initiation samples 7, 8 . Moreover, in surgical samples, due to the and progression. presence of heterogeneous morphologic patterns is The anti-HER2 monoclonal antibody trastuzumab in reasonable to select more than one tissue block for combination with standard chemotherapy has signifi- analysis. There is a high degree of concordance be- cantly improved response rate and survival outcome tween primary and metastatic samples, hence, HER2 in patients harboring HER2-positive tumors (i.e. IHC testing should be performed on the most representa- 3+ or IHC 2+ and ISH+) 2,3. Moreover, other alternative tive material9. In biopsy samples, it should be kept in HER2-targeted therapeutic approaches are in clinical mind that low-grade and high-grade dysplastic lesions trials with promising results 3. Thus, advanced gas- may present HER2 overexpression/gene amplifica-

Figure 1. HER2 testing in gastroesophageal adenocarcinomas. (A) Diagnostic algorithm modified from Bartley AN, et al.(11). Tumor cell cluster is defined as a cluster of five or more tumor cells. (B) Representative immunohistochemical examples of a negative (0) case showing no reactivity in any of the tumor cells, a negative (1+) case with faint/barely perceptible mem- branous staining, an equivocal 2+ immunoreaction and a strongly and diffuse 3+ positive case. CISH examples of a HER2 non-amplified and an amplified case are also shown. 250 M. Fassan et al.

tion which can coexist with a HER2-negative invasive ded samples 19. This method localizes the viral infec- counterpart 10. Thus, an accurate combined morpho- tion to the malignant cells with a moderate to strong logical and IHC evaluation should be performed. nuclear staining. The presence of EBER-positive lym- HER2 status should be assessed first by IHC, followed phocytes within tumor samples has been described by ISH when IHC result is 2+ (equivocal). Positive and should not be considered in the definition of (i.e. 3+) or negative (0 or 1+) staining do not require EBV-positivity 18. further ISH testing 11. The IHC evaluation should be performed according the Ruschoff/Hofmann scoring system (Fig. 1) 12. Note that, in comparison to breast Colorectal adenocarcinoma (CRC) cancer, the completeness of membrane staining is in- frequent and expression is often seen in a basolateral pattern. For ISH, a ratio of HER2 signal to CEP17 sig- RAS genes mutational analysis nal of ≥ 2.0 is considered positive. The ISH analysis Definition and therapeutic implications evaluation should preferably be performed in areas marked as strongest HER2 IHC intensity. Brightfield The RAS gene family is composed of four small cyto- ISH techniques have been suggested to be superior plasmic proteins with GTPase activity: H-Ras, K-Ra- than FISH in HER2 testing for gastroesophageal ade- s4a, K-Ras4b, and N-Ras. These proteins promote nocarcinoma as they allow for easier identification of cell growth, differentiation, proliferation and survival. tumor nuclei in normal tissue 13. Mutations in the RAS genes (KRAS and NRAS) are well-recognized biomarkers of resistance to anti-EG- Epstein-Barr virus infection FR monoclonal antibodies 20-23.

Definition and therapeutic implications Clinical and pathological associated features The Epstein-Barr virus (EBV) is a DNA virus member KRAS mutations are an early event in colorectal car- of the herpes family, which has been associated with cinogenesis. In fact, there is a highly concordant rate several types of cancer, including gastric carcinoma (almost 95%) in paired primary cancers and metastat- (GC). An EBV-positive gastric cancer category based ic samples 24,25. Cancers may present a mucinous his- on its genomic and molecular features was proposed tology and are usually located in the right colon. by The Cancer Genome Atlas Research Network (TC- GA) 14. This peculiar class of GC is usually charac- Diagnosis terized by overexpression of PD-L1 and shows high KRAS is mutated in approximately 40% of cases, response rates to immunotherapy 15. mostly in exon 2 codons 12 (70-80%) and 13 (15- 20%). The remaining mutations are mainly located in Clinical and pathological associated features exon 3 codons 59-61 and in exon 4, which includes EBV infection is absent in gastric dysplasia or early codons 117 and 146. Mutations in NRAS are present GC, suggesting an EBV-specific carcinogenetic path- in approximately 3% to 5% of colorectal cancer sam- way 16. EBV is more often detected in moderate to ples particularly in exon 3 codon 61 (60%) and in exon poorly differentiated GCs, medullary histotype carci- 2 codons 12, 13 22. nomas and those involving the proximal stomach 17. NRAS mutations are typically mutually exclusive with EBV association is also noted in cancers of the gastric KRAS and BRAF mutations. stump following surgery. Tumors often present abun- Patients with CRC being considered for anti-EGFR dant infiltrating lymphocytes, CDKN2A gene silencing, therapy must be profiled for RAS mutational sta- frequent PIK3CA mutations and a significant overex- tus 26. Different methods can be used, such as muta- pression of PD-L1/PD-L2. tion-specific real-time polymerase chain reaction (RT- There is a male predominance and the prevalence PCR), Sanger sequencing, pyrosequencing, BEAM- is significantly higher among the Asian population ing technique, and next-generation sequencing, in comparison to Caucasians. EBV-associated GCs among others. On the basis of the evidence that no have a low frequency of lymph node involvement and improvement in the selection of patients for anti-EG- are characterized by an improved survival in compari- FR therapy was obtained by adjusting the mutant al- son to EBV-negative cases 18. lele fraction threshold in tissue samples from 5% (by pyrosequencing) to 1% (by NGS) 27, Colon Cancer Diagnosis Guidelines by Italian Association of Medical Oncolo- The gold standard assay for EBV is the targeting of gy (AIOM) suggests that mutational analysis should EBV-encoded RNA (EBER) by ISH in paraffin-embed- carried out by a method with a sensitivity detection MOLECULAR LANDSCAPE OF GASTROINTESTINAL NEOPLASMS 251

of 5% mutant allele fraction, at least in cases with CRCs bearing non-V600 BRAF mutations constitute high neoplastic cellularity (more than 50%) (https:// a distinct clinico-pathological subset 38. BRAF mu- www.aiom.it/wp-content/uploads/2019/10/2019_LG_ tations are grouped in activating RAS-independent AIOM_Colon-1.pdf) signaling as monomers (class 1-V600E) or as dimers (class 2-codons 597/601), and RAS-dependent with BRAF gene mutational analysis impaired kinase activity (class 3-codons 594/596) 38,39. Class 3 CRCs usually are non-mucinous, microsatel- Definition and therapeutic implications lite stable (MSS), arise on the left side of the colon The BRAF gene encodes a serine/threonine protein of younger male patients, have no peritoneal spread, kinase, which plays a role in regulating the MAPK/ are lower grade at presentation and are related to a ERK signaling pathways, affecting cell growth and more favorable overall survival (OS) rate compared to proliferation. Missense somatic mutations in the BRAF both V600EBRAF mutants and wild-type CRCs, where- gene have been found in about 8-15% of metastatic as class 2 lesions are clinically similar to V600EBRAF CRCs 28. CRCs. The most common BRAF mutation (> 90%), result- ing in a constitutive-active kinase, is a CTG → CAG Diagnosis transversion at residue 1799 (T1799A), leading to an BRAF mutational testing should be performed in amino acidic substitution from valine to glutamic acid metastatic CRCs for prognostic stratification, where- at codon 600 (p.V600E) in exon 15. as there is insufficient evidence to support its testing BRAF mutations are observed in hyperplastic polyps as a predictive molecular biomarker for response to and as an early event in the “serrated” carcinogenet- anti-EGFR inhibitors 26. The recent publication of the ic cascade 29. In the metastatic setting, BRAF-mutat- BEACON study pinpointed novel BRAF-targeting ed CRCs have a poor prognosis and do not seem therapies in this oncological setting 31. to benefit from EGFR inhibition 30. The phase III trial BRAF gene exon 15 mutational analyses can be per- BEACON has recently proved a significant survival formed as single gene analysis or in combination with advantage associated with the combination of en- the other RAS genes with high-throughput technolo- corafenib plus cetuximab or the same doublet plus gies. The VE1 clone has been demonstrated to be an binimetinib compared to current standard treatments alternative sensitive and specific immunohistochem- in BRAF-mutated tumors 31,32, paving the way for inno- ical marker for the detection of BRAF p.V600E-mu- vative BRAF-specific therapeutic options. tated CRCs 40. However, considering the clinical and therapeutic implication of non-V600 mutations, the Clinical and pathological associated features analysis of the most common exon 15 hotspots should BRAF-mutated metastatic CRCs arise in older pa- be preferred. tient (> 60 years old) and with a higher prevalence in Beyond the metastatic setting, V600EBRAF mutation is the female gender in comparison to BRAF-wild type strongly associated with (~60%) the somatic inactiva- cases, regardless of the MSI status 33-35. The proximal tion of the DNA mismatch repair machinery (MMR) colon is the preferential location. Moreover, this class genes, which is virtually absent in Lynch syndrome 41. of tumors present a unique metastatic pattern, show- Hence, somatic BRAF mutation testing has been in- ing high rates of peritoneal metastases, distant lymph cluded into the Lynch syndrome screening algorithm node metastases and low rates of lung metastases 28. (see below). However, no significant differences have been ob- served in liver or brain metastases rates 36. From a histopathological point of view, BRAF-mutat- Pancancer biomarkers ed CRCs frequently present mucinous features, poor differentiation and high stage at diagnosis 28; from the biological point of view, they mostly derived from Defective DNA mismatch repair complex (dMMR)/ serrated precursor lesions. Other less characteristic microsatellite instability (MSI) features include a higher frequency of tumor budding and signet ring cells histotype, infiltrative pattern of Definition and therapeutic implications invasion with an increased risk of lympho-vascular MMR is a highly conserved protein complex that rec- albeit not perineural invasion, different grade of Tu- ognizes and repairs erroneous short insertions, short mor Infiltrating lymphocytes (TILs) and of peritumoral deletions and single base mismatches that can arise lymphoid reaction with follicular appearance (Crohn- during DNA replication and recombination. The most like) 37. important MMR players include MLH1 (mutL homo- 252 M. Fassan et al.

logue 1), MSH2 (mutS homologue 2), MSH6 (mutS Clinical and pathological associated features homologue 6) and PMS2 (postmeiotic segregation in- Patients with dMMR/MMR tumors are more often creased 2) 42. These four proteins function in heterod- characterized by a prolonged overall survival in com- imers, namely MLH1-PMS2 and MSH2-MSH6 43,44, parison to proficient MMR (pMMR)/MSS cases 14,57,58. where MLH1 and MSH2 are obligatory partners of However, there is a negative prognostic effect in pa- these heterodimers. In fact, PMS2 and MSH6 can only tients treated with (neo)adjuvant chemotherapy 57,58. form a heterodimer with MLH1 and MSH2, respective- dMMR/MSI has been well described in several types ly. On the other hand, MLH1 and MHS2 can form het- of human cancers, most frequently in colorectal (17% erodimers with other MMR proteins, namely MSH3, among all stages), endometrial (20%), and gastric MLH3 and PMS1. An alteration in MLH1 and MSH2 (13%) adenocarcinomas 44,59, which are also the most results in subsequent proteolytic degradation of the frequently observed among Lynch syndrome patients. mutated protein and its secondary partner, PMS2 and Most dMMR/MSI tumors are characterized by a sig- MSH6, respectively 44. Conversely, mutations in PMS2 nificant intra- and peri-neoplastic lymphocytic infil- or MSH6 may not result in proteolytic degradation of tration and phenotypic heterogeneity 60. In colorectal their primary partners. adenocarcinoma, dMMR/MSI status is associated The inactivation of these genes (i.e. dMMR) can occur with mucinous histology and rare histotypes such as due to germline and/or somatic mutations or epige- medullary carcinoma and signet-ring cell adenocarci- netic silencing, resulting in the accumulation of frame- noma 61,62. Thus, in experienced hands, histopatholo- shift mutations (either through insertions or deletions) gy can significantly improve the efficacy of dMMR/MSI with a subsequent increased mutational burden. detection. This consideration introduces the concept Germline mutation(s) of the MMR genes is the hall- of the so-called “reflex test”, which can represent a mo- mark of Lynch syndrome and constitutional mismatch lecular test directly performed by pathologist based on repair deficiency (CMMRD) 45. Epigenetic silencing is a peculiar morphological feature typically associated usually represented by MLH1 gene promoter hyper- with a genetic profile (e.g.: medullary histology and methylation; secondary epigenetic silencing of MSH6 MSI). This kind of approach can greatly reduce the is observed after neoadjuvant radiochemotherapeutic overall diagnostic turnaround time in selected cas- treatments 46,47. es. On the other hand, remaining in the dMMR/MSI Microsatellites are repetitive DNA sequences that are landscape, it has to be noticed that a small subset distributed along the genome of both coding and non- (~6%) of colorectal cancers with this genetic altera- tion have no detectable dMMR/MSI-specific histologic coding regions and are particularly sensitive to DNA characteristics 62. In gastric adenocarcinoma, dMMR/ mismatching errors. The identification of microsatellite MSI status is associated with intestinal-type histo- instability (MSI; i.e. clustering of mutations in micro- type, an elderly age of onset and a distal location 63. satellites typically consisting of repeat length altera- In adenocarcinomas of the small intestine dMMR/MSI tions) is, therefore, an indirect evidence of a dMMR 48. status has been observed in 8.3% of cases 44, is as- Of note, 6-7% of MSI tumors retain MMR IHC expres- sociated with a history of celiac disease 64 and with a sion 49. Some of these cases presented an abnormal mucinous histotype 65. Among gastrointestinal tumors focal or dot-like nuclear MLH1 expression; some oth- with low prevalence of dMMR/MSI (< 5%), dMMR/MSI ers were associated with an ultramutated status due pancreatic ductal adenocarcinomas show medullary to POLE mutations and subsequent alterations in the or mucinous/colloid histology and are associated with 49 MMR machinery . a KRAS/TP53 wild-type molecular background 66,67, Importantly, for assessment tumor mutation burden, dMMR/MSI cholangiocarcinomas show papillary and novel NGS approaches have been introduced to test mucinous histotype 68. MSI in the clinic, which have also been suggested in the analysis of non-Lynch associated cancers 49-51. Diagnosis MMR screening/MSI testing has several important The use of immunohistochemistry to assess the pres- clinical implications: (i) dMMR/MSI universal screening ence or absence of MLH1, PMS2, MSH2 and MSH6 in colorectal and endometrial cancers has been rec- is recommended in all the patients with any sporad- ommended to identify Lynch syndrome families 43,52; ic cancer type belonging to the spectrum of cancers (ii) stage II/III colorectal cancers should be tested for found in Lynch syndrome (i.e. colorectal, endometrial, dMMR/MSI because they do not benefit from 5-fluoro- small intestine, urothelial,central nervous system and uracil adjuvant therapy 53; (iii) dMMR/MSI tumors are sebaceous gland) 26. Due to the high concordance eligible for immune checkpoint inhibitor therapies and rate among IHC and PCR 69, IHC analysis is usually are characterized by overexpression of PD-L1 15,54-56. preferred over microsatellite instability testing. In fact, MOLECULAR LANDSCAPE OF GASTROINTESTINAL NEOPLASMS 253

IHC has a lower turnaround time, allows to directly present in ≥ 10% tumor cells; (ii) loss, in case of com- understand the altered gene(s) and requires a limited plete loss of nuclear expression in cancer cells; (iii) amount of tissue (i.e. 4 tissue slides). ESMO recom- indeterminate, if IHC staining intensity in tumor cells is mendations discourage the use of a two-antibody (i.e. lower than the internal control or the tumor is positive PMS2 and MSH6) approach 44. in < 10% (Fig. 2) 70. Indeterminate IHC results should MMR protein expression is interpreted as (i) retained, undergo MSI testing. when a moderate to strong expression (similar to what False negative MMR immunostainings are mainly is observed in the stromal cells as internal control) is caused by pre-analytical issues, such as tissue fixa-

Figure 2. Immunohistochemical interpretation of MMR proteins in colorectal adenocarcinoma. (A) Diagnostic algorithm for MMR staining interpretation modified from Remo, et al. (43). (B and C) Heterogeneous MMR protein expression. (B) The lesion was heterogeneous for MSH2/MSH6 status and proficient for MLH1/PMS2. The microdissected areas also showed a heterogeneous status of the MSI testing. (C) A heterogeneous MSH6 staining pattern observed in a MLH1 mutated Lynch syndrome patient. (D) A case of indeterminate positivity for MMR proteins, in which the staining intensity observed in cancer cells’ nuclei is significantly lower in comparison to surrounding stromal cells. This case was MSI at molecular testing. 254 M. Fassan et al.

tion, but this can be easily recognized by the absence PD-L1 expression status of signal in the internal positive controls (stromal cells or normal mucosa) 71. Another reason to retest Definition and therapeutic implications the sample by MSI testing is the finding of aberrant Programmed death-ligand 1 (PD-L1; also known as staining patterns such as cytoplasmic, dot-like or per- CD247 or B7-H1) is one of the ligands of the pro- inuclear staining 71. False positive results (i.e. pMMR grammed cell death 1 (PD-1) receptor, a dominant but MSI) may be determined by catalytically inactive negative regulator of antitumor T cell effector func- mutated MMR proteins, which retain their antigenic tion 56. PD-L1 is induced by inflammation and is ex- integrity 71. MMR/microsatellite status heterogeneity pressed in the tumor microenvironment and on tumor has been described 15,72; in these cases, the analysis cells. The blockade of the PD-1–PD-L1 interaction with should be repeated on a representative sample of the therapeutic antibodies has emerged as an important metastatic disease. therapeutic option in tumors overexpressing PD-L1 or In colorectal adenocarcinoma (and solely in this set- tumors with an activation of T-cell immunoresponse ting!), MLH1/PMS2 negative tumors should be tested such as in case of high tumor mutation burden or EBV for BRAF p.V600E since this mutation is frequent- associated gastric cancers. In fact, anti-PD-1/PD-L1 therapies result in T cell proliferation and infiltration in- ly observed in sporadic cases 26. Another option to to the tumor, inducing a cytotoxic T cell response that identify a MLH1/PMS2 negative tumor as sporadic is leads to an objective tumor response 15,78. Apart from the evaluation of MLH1 promoter methylation 43. The colorectal cancer, in which dMMR/MSI status is the latter diagnostic approach is also extended to other preferred predictive biomarker in the selection of pa- cancer types in addition to colorectal lesions; howev- tients for immunotherapy, PD-L1 expression emerged er, MLH1 constitutional methylation should be ruled of importance for gastroesophageal cancers. FDA ap- 73 out . proved pembrolizumab (an anti PD-1 antibody) as a MSI testing is based on PCR amplification of micro- second-line standard of care therapy for patients with satellite markers. Two possible panels are currently advanced or metastatic esophageal squamous cell in use: (i) five microsatellites comprising two mon- carcinoma and PD-L1 combined positive score (CPS) onucleotide (BAT-25 and BAT-26) and three dinu- ≥ 10 79,80 and as third-line option in metastatic gastro- cleotide (D5S346, D2S123 and D17S250) repeats; esophageal junction adenocarcinomas with a PD-L1 (ii) five poly-A mononucleotide repeats (BAT-25, CPS ≥ 1 81. BAT-26, NR-21, NR-24, NR-27). Historically, loss of stability in 1 of the five microsatellite markers was Clinical and pathological associated features defined as MSI-low and loss of stability in ≥ 2 as In gastric cancer PD-L1 positivity is seen predomi- MSI-high. The term MSI-low should be abandoned nantly in the EBV-associated and dMMR/MSI tum- and MSI-low tumours should be included within mi- ors 15, although contrasting data are available on its crosatellite stable tumours 74. The pentaplex panel prognostic impact. In colorectal adenocarcinomas, of five poly-A mononucleotide repeats is the recom- high level of PD-L1 expression has been associated 82 mended panel given its higher sensitivity and spec- to a poorer prognosis . In pancreatic ductal adeno- ificity 75. Moreover, it may obviate the need for nor- carcinoma, the prognostic value of PD-L1 expression mal tissue for comparison, which is of central im- is still unclear; however, in the undifferentiated vari- portance in the analysis of small biopsies obtained ant with osteoclast-like giant cells, its expression has been correlated with a poorer prognosis 83. from cancer tissue. Of note, a recent report demonstrated that almost Diagnosis 10% of patients had been enrolled for immunothera- Immunohistochemistry represents the gold standard py in metastatic colorectal cancer with a false positive for PD-L1 expression evaluation. Pathologists should dMMR or MSI-PCR result assessed by local laborato- be aware that this analysis is significantly affected by 76 ries . Thus, both MMR-IHC and MSI-PCR have to be several factors: (i) different standardization protocols performed in assessing the eligibility to treatment with of PD-L1 assays, (ii) variability in PD-L1 antibody use immune checkpoint inhibitors. among the different Institutions 84; (iii) different PD-L1 NGS represents an appropriate alternative molecular quantification scoring systems 85; and (iv) intratumor test to assess MSI, especially in non-Lynch-associ- heterogeneity of PD-L1 expression 44. Moreover, ated tumors 77. However, NGS should be carried out PD-L1 is also expressed in pre-invasive lesions, which only in selected centers experienced in these tech- should be not considered in the evaluation 86,87. niques. PD-L1 positive controls are lung macrophages, pla- MOLECULAR LANDSCAPE OF GASTROINTESTINAL NEOPLASMS 255

centa, spleen and tonsil, whereas negative staining around 5% of KRAS/NRAS/BRAF wild type colorec- are alveolar cells, hepatocytes and normal squamous tal adenocarcinomas and HER2-targeting showed epithelium. promising results in HER2-positive tumors refracto- In gastroesophageal carcinomas, PD-L1 evaluation ry to standard of care therapies with EGFR inhib- is performed as CPS, which is the number of PD-L1 itors 99-101. HER2 assessment in colorectal cancer stained cells (i.e. tumor cells, lymphocytes, mac- is performed according the HERACLES diagnostic rophages) dived by the total number of viable tumor criteria (i.e. 2+/3+ HER2-IHC in ≥ 50% tumor cells cells, multiplied by 100 88. This is different from the confirmed by FISH) 101. Tumor Proportion Score (TPS), applied in non-small • The analysis of neurotrophic tyrosine receptor ki- cell lung carcinoma, which is the percentage of viable nase (NTRK) gene fusions has emerged as a pre- tumor cells showing partial or complete membrane dictive biomarker for the efficacy of inhibitors of the staining at any intensity. tropomyosin receptor kinase (TRK) proteins across At present, only pembrolizumab has indications re- a range of solid tumor types 102. In the gastrointesti- stricted to tumors expressing PD-L1 (beyond dMMR/ nal setting, NTRK gene fusions are extremely rare MSI status) and requires the use of a companion di- with a 0.23-0.31% prevalence in colorectal ade- agnostic, which is currently represented by the PD-L1 nocarcinomas, 0.34% in pancreatic carcinomas, IHC 22C3 pharmDx (Dako). Other three antibod- 0.25% in cholangiocarcinomas, 0.48% in appen- ies have been approved by FDA for PD-L1 IHC as- diceal adenocarcinomas and 0.31% in neuroen- say: PD-L1 IHC 28-8 pharmDx assay for nivolumab docrine tumors 103,104. Of note, NTRK gene rear- treatment, VENTANA PD-L1 IHC (SP142) assay for rangements are enriched in MLH1/PMS2 deficient atezolizumab treatment and VENTANA PD-L1 IHC and BRAF wild-type colorectal cancers, in which (SP263) assay for durvalumab. a 5.3% prevalence was described 105. Despite this relative rarity, the request for NTRK testing is in- creasing. NTRK alterations can be detected by Other current and potential biomarkers immunohistochemistry, RT-PCR and RNA-based with clinical impact NGS. • Germline and somatic mutations within the ho- • Gastrointestinal stromal tumors (GISTs) are the mologous recombination repair pathway (i.e. ATM, most common mesenchymal tumors of the gastro- BRCA1, BRCA2 or PALB2) have been observed intestinal tract and should be profiled for KIT and in pancreatic ductal adenocarcinoma and are PDGFRA due to their predictive value for tyrosine ki- associated with an increased sensitivity to plati- nase inhibitors therapies 89-91. In fact, almost all KIT/ num-based chemotherapy 106,107. Moreover, tum- PDGFRA alterations, but the PDGFRA p.D842V ors with BRCA1/2 mutations display increased mutation, are activating the tyrosine kinases. KIT/ sensitivity to PARP inhibitors which, when used PDGFRA mutations are present in around 85% of as maintenance therapy, result in a prolonged pro- GISTs, the other 10-15% cases are usually char- gression-free survival 108. acterized by mutations in SDH, NF1 or BRAF 92,93. • SMAD4 is a genetic driver of pancreatic ductal KIT/PDGFRA alterations are usually tested by di- adenocarcinoma; it is also known as DPC4 and rect sequencing and NGS technologies. is genetically inactivated in about half of pancre- • Recently, the therapeutic portfolio of biliary tract atic ductal adenocarcinomas (PDAC) 109. A reliable cancers has significantly improved with the intro- surrogate methodology to investigate its mutation- duction of targeted therapies associated with the al status is represented by immunohistochemis- molecular profile of the tumor 94. In particular, ther- try, with the loss of the nuclear expression of the apies targeting actionable genomic aberrations protein indicating the genetic inactivation. SMAD4 such as BRAF 95 or IDH1 96 mutations and FGFR2 mutations (SMAD4 immunohistochemical loss) gene fusions 97,98 have been successfully entered have been correlated with widespread metastat- clinical development with significant responses ic patterns in PDAC patients 110 and with higher and durable clinical benefit in selected patients. rates of local and distant failure in those receiv- As a result, the demand for molecular profiling in ing adjuvant chemoradiation 111. Its determination this tumor setting will rapidly increase in our clin- may be useful for planning therapeutic decisions: ical practice. FGFR2 fusions can be detected by although such situations are generally managed in RNA-based NGS panels, but also RT-PCR-based ultra-specialized pancreatic centers, the presence kits have been recently introduced into the market. of SMAD4 mutations may support radiofrequency • Amplificationof the HER2 gene characterizes ablation-based therapy 112. 256 M. Fassan et al.

Conclusions 13 Fox SB, Kumarasinghe MP, Armes JE, et al. Gastric HER2 Testing Study (GaTHER): an evaluation of gastric/gastro- esophageal junction cancer testing accuracy in Australia. We are facing molecularly-driven treatment choices Am J Surg Pathol 2012;36:577-82. https://doi.org/10.1097/ for advanced gastrointestinal cancers and histopatho- PAS.0b013e318244adbb logic diagnosis is becoming an integrated morpholog- 14 Cancer Genome Atlas Research N. Comprehensive mo- ical and molecular characterization of the biospec- lecular characterization of gastric adenocarcinoma. Nature imen. The pathologist should be aware of the novel 2014;513(7517):202-9. https://doi.org/10.1038/nature13480 therapies and how to improve the management of bio- 15 Kim ST, Cristescu R, Bass AJ, et al. Comprehensive molecu- specimens in the personalized medicine era. lar characterization of clinical responses to PD-1 inhibition in metastatic gastric cancer. Nat Med 2018;24:1449-58. https://doi. org/10.1038/s41591-018-0101-z References 16 Ribeiro J, Malta M, Galaghar A, et al. Epstein-Barr virus is ab- sent in gastric superficial neoplastic lesions. Virchows Arch 1 Fassan M. Molecular diagnostics in pathology: time for a next- 2019;475:757-62. https://doi.org/10.1007/s00428-019-02670-1 generation pathologist? Arch Pathol Lab Med 2018;142:313-20. https://doi.org/10.5858/arpa.2017-0269-RA 17 Ryan JL, Morgan DR, Dominguez RL, et al. High levels of Epstein-Barr virus DNA in latently infected gastric adenocarci- 2 Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in noma. Lab Invest 2009;89:80-90. https://doi.org/10.1038/labin- combination with chemotherapy versus chemotherapy alone vest.2008.103 for treatment of HER2-positive advanced gastric or gastro-oe- sophageal junction cancer (ToGA): a phase 3, open-label, ran- 18 Camargo MC, Kim WH, Chiaravalli AM, et al. Improved survival domised controlled trial. Lancet 2010;376(9742):687-97. https:// of gastric cancer with tumour Epstein-Barr virus positivity: an doi.org/10.1016/S0140-6736(10)61121-X international pooled analysis. Gut 2014;63:236-43. https://doi. org/10.1136/gutjnl-2013-304531 3 Pellino A, Riello E, Nappo F, et al. Targeted therapies in meta- static gastric cancer: current knowledge and future perspectives. 19 Gulley ML, Tang W. Laboratory assays for Epstein-Barr virus- World J Gastroenterol 2019;25:5773-88. https://doi.org/10.3748/ related disease. J Mol Diagn 2008;10:279-92. https://doi. wjg.v25.i38.5773 org/10.2353/jmoldx.2008.080023 4 Van Cutsem E, Bang YJ, Feng-Yi F, et al. HER2 screening 20 Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras muta- data from ToGA: targeting HER2 in gastric and gastroesopha- tions and benefit from cetuximab in advanced colorectal cancer. geal junction cancer. Gastric Cancer 2015;18:476-84. https://doi. N Engl J Med 2008;359:1757-65. https://doi.org/10.1056/NEJ- org/10.1007/s10120-014-0402-y Moa0804385 5 Grillo F, Fassan M, Sarocchi F, et al. HER2 heterogeneity in 21 Van Cutsem E, Kohne CH, Hitre E, et al. Cetuximab and che- gastric/gastroesophageal cancers: From benchside to practice. motherapy as initial treatment for metastatic colorectal cancer. World J Gastroenterol 2016;22:5879-87. https://doi.org/10.3748/ N Engl J Med 2009;360:1408-17. https://doi.org/10.1056/NEJ- wjg.v22.i26.5879 Moa0805019 6 Grillo F, Fassan M, Fiocca R, et al. Heterogeneous Her2/Neu 22 De Roock W, Claes B, Bernasconi D, et al. Effects of KRAS, expression in gastric and gastroesophageal cancer. Hum Pathol BRAF, NRAS, and PIK3CA mutations on the efficacy of ce- 2016;48:173-4. https://doi.org/10.1016/j.humpath.2015.08.023 tuximab plus chemotherapy in chemotherapy-refractory meta- 7 Gullo I, Grillo F, Molinaro L, et al. Minimum biopsy set for static colorectal cancer: a retrospective consortium analysis. HER2 evaluation in gastric and gastro-esophageal junc- Lancet Oncol 2010;11:753-62. https://doi.org/10.1016/S1470- tion cancer. Endosc Int Open 2015;3:E165-70. https://doi. 2045(10)70130-3 org/10.1055/s-0034-1391359 23 Douillard JY, Oliner KS, Siena S, et al. Panitumumab-FOLFOX4 8 Grillo F, Fassan M, Ceccaroli C, et al. The reliability of endoscop- treatment and RAS mutations in colorectal cancer. N Engl J Med ic biopsies in assessing her2 status in gastric and gastroesoph- 2013;369:1023-34. https://doi.org/10.1056/NEJMoa1305275 ageal junction cancer: a study comparing biopsies with surgi- 24 Han CB, Li F, Ma JT, Zou HW. Concordant KRAS mutations in cal samples. Transl Oncol 2013;6:10-6. https://doi.org/10.1593/ primary and metastatic colorectal cancer tissue specimens: a tlo.12334 meta-analysis and systematic review. Cancer Invest 2012;30:741- 9 Amato M, Perrone G, Righi D, et al. HER2 Status in Gastric Can- 7. https://doi.org/10.1056/NEJMoa1305275 cer: Comparison between Primary and Distant Metastatic Dis- 25 Santini D, Loupakis F, Vincenzi B, et al. High concordance ease. Pathol Oncol Res 2017;23:55-61. https://doi.org/10.1007/ of KRAS status between primary colorectal tumors and re- s12253-016-0082-5 lated metastatic sites: implications for clinical practice. On- 10 Fassan M, Mastracci L, Grillo F, et al. Early HER2 dysregula- cologist 2008;13:1270-5. https://doi.org/10.1634/theoncolo- tion in gastric and oesophageal carcinogenesis. Histopathology gist.2008-0181 2012;61:769-76. https://doi.org/10.1111/j.1365-2559.2012.04272 26 Sepulveda AR, Hamilton SR, Allegra CJ, et al. Molecular bio- 11 Bartley AN, Washington MK, Ventura CB, et al. HER2 testing markers for the evaluation of colorectal cancer: guideline From and clinical decision making in gastroesophageal adenocarci- the American Society for Clinical Pathology, College of Ameri- noma: guideline From the College of American Pathologists, can Pathologists, Association for Molecular Pathology, and American Society for Clinical Pathology, and American Society American Society of Clinical Oncology. Arch Pathol Lab Med of Clinical Oncology. Arch Pathol Lab Med 2016;140:1345-63. 2017;141:625-57. https://doi.org/10.5858/arpa.2016-0554-CP https://doi.org/10.5858/arpa.2016-0331-CP 27 Vidal J, Bellosillo B, Santos Vivas C, et al. Ultra-selection of 12 Hofmann M, Stoss O, Shi D, et al. Assessment of a HER2 metastatic colorectal cancer patients using next-generation se- scoring system for gastric cancer: results from a validation quencing to improve clinical efficacy of anti-EGFR therapy. Ann study. Histopathology 2008;52:797-805. https://doi.org/10.1111 Oncol 2019;30:439-46. https://doi.org/10.1093/annonc/mdz005 /j.1365-2559.2008.03028 28 Fanelli GN, Dal Pozzo CA, Depetris I, et al. The heterogeneous MOLECULAR LANDSCAPE OF GASTROINTESTINAL NEOPLASMS 257

clinical and pathological landscapes of metastatic Braf-mutat- 43 Remo A, Fassan M, Lanza G. Immunohistochemical evaluation ed colorectal cancer. Cancer Cell Int 2020;20:30. https://doi. of mismatch repair proteins in colorectal carcinoma: the AIFEG/ org/10.1186/s12935-020-1117-2 GIPAD proposal. Pathologica 2016;108:104-9. 29 Barras D. BRAF mutation in colorectal cancer: an update. Bio- 44 Luchini C, Bibeau F, Ligtenberg MJL, et al. ESMO recommen- mark Cancer 2015;7(Suppl 1):9-12. https://doi.org/10.4137/BIC. dations on microsatellite instability testing for immunotherapy S25248 in cancer, and its relationship with PD-1/PD-L1 expression and 30 Rowland A, Dias MM, Wiese MD, et al. Meta-analysis of BRAF tumour mutational burden: a systematic review-based approach. mutation as a predictive biomarker of benefit from anti-EGFR Ann Oncol 2019;30:1232-43. https://doi.org/10.1093/annonc/ monoclonal antibody therapy for RAS wild-type metastatic mdz116 colorectal cancer. Br J Cancer 2015;112:1888-94. https://doi. 45 Galuppini F, Opocher E, Tabori U, et al. Concomitant IDH wild- org/10.1038/bjc.2015.173 type glioblastoma and IDH1-mutant anaplastic astrocytoma in 31 Kopetz S, Grothey A, Yaeger R, et al. Encorafenib, binimetinib, a patient with constitutional mismatch repair deficiency syn- and cetuximab in braf v600e-mutated colorectal cancer. N drome. Neuropathol Appl Neurobiol 2018;44:233-9. https://doi. Engl J Med 2019;381:1632-43. https://doi.org/10.1056/NEJ- org/10.1111/nan.12450 Moa1908075 46 Indraccolo S, Lombardi G, Fassan M, et al. Genetic, epigen- 32 Van Cutsem E, Huijberts S, Grothey A, et al. Binimetinib, en- etic, and immunologic profiling of MMR-Deficient Relapsed corafenib, and cetuximab triplet therapy for patients With Braf Glioblastoma. Clin Cancer Res 2019;25:1828-37. https://doi. v600e-mutant metastatic colorectal cancer: safety lead-in re- org/10.1158/1078-0432.CCR-18-1892 sults from the Phase III beacon colorectal cancer study. J Clin 47 Bao F, Panarelli NC, Rennert H, et al. Neoadjuvant therapy Oncol. 2019;37:1460-9. https://doi.org/10.1200/JCO.18.02459 induces loss of MSH6 expression in colorectal carcinoma. 33 Clancy C, Burke JP, Kalady MF, et al. BRAF mutation is associ- Am J Surg Pathol 2010;34:1798-804. https://doi.org/10.1097/ ated with distinct clinicopathological characteristics in colorectal PAS.0b013e3181f906cc cancer: a systematic review and meta-analysis. Colorectal Dis 48 Ma J, Setton J, Lee NY, et al. The therapeutic significance of 2013;15:e711-8. https://doi.org/10.1111/codi.12427 mutational signatures from DNA repair deficiency in cancer. 34 Loupakis F, Intini R, Cremolini C, et al. A validated prognostic Nat Commun 2018;9:3292. https://doi.org/10.1038/s41467-018- classifier for (V600E)BRAF-mutated metastatic colorectal can- 05228-y cer: the ‘BRAF BeCool’ study. Eur J Cancer 2019;118:121-30. 49 Hechtman JF, Rana S, Middha S, et al. Retained mismatch re- https://doi.org/10.1016/j.ejca.2019.06.008 pair protein expression occurs in approximately 6% of microsat- 35 Loupakis F, Biason P, Prete AA, et al. CK7 and consensus mo- ellite instability-high cancers and is associated with missense lecular subtypes as major prognosticators in (V600E)BRAF mu- mutations in mismatch repair genes. Mod Pathol 2020;33:871-9. tated metastatic colorectal cancer. Br J Cancer. 2019;121:593-9. https://doi.org/10.1038/s41379-019-0414-6 https://doi.org/10.1038/s41416-019-0560-0 50 Niu B, Ye K, Zhang Q, et al. MSIsensor: microsatellite instabil- 36 Tran B, Kopetz S, Tie J, et al. Impact of BRAF mutation and micro- ity detection using paired tumor-normal sequence data. Bioin- satellite instability on the pattern of metastatic spread and prog- formatics 2014;30:1015-6. https://doi.org/10.1093/bioinformatics/ nosis in metastatic colorectal cancer. Cancer 2011;117:4623-32. btt755 https://doi.org/10.1002/cncr.26086 51 Galuppini F, Dal Pozzo CA, Deckert J, et al. Tumor mutation 37 Jang MH, Kim S, Hwang DY, et al. BRAF-Mutated Colorectal burden: from comprehensive mutational screening to the clinic. Cancer Exhibits Distinct Clinicopathological Features from Wild- Cancer Cell Int 2019;19:209. https://doi.org/10.1186/s12935-019- Type BRAF-Expressing Cancer Independent of the Microsatel- 0929-4 lite Instability Status. J Korean Med Sci 2017;32:38-46. https:// 52 Hechtman JF, Middha S, Stadler ZK, et al. Universal screening doi.org/10.3346/jkms.2017.32.1.38 for microsatellite instability in colorectal cancer in the clinical 38 Schirripa M, Biason P, Lonardi S, et al. Class 1, 2, and 3 BRAF- genomics era: new recommendations, methods, and consid- Mutated Metastatic Colorectal Cancer: A Detailed Clinical, erations. Fam Cancer 2017;16:525-9. https://doi.org/10.1007/ Pathologic, and Molecular Characterization. Clin Cancer Res s10689-017-9993-x 2019;25:3954-61. https://doi.org/10.1158/1078-0432.CCR-19- 53 Popat S, Hubner R, Houlston RS. Systematic review of micro- 0311 satellite instability and colorectal cancer prognosis. J Clin Oncol 39 Cremolini C, Di Bartolomeo M, Amatu A, et al. BRAF codons 2005;23:609-18. https://doi.org/10.1200/JCO.2005.01.086 594 and 596 mutations identify a new molecular subtype of 54 Overman MJ, Lonardi S, Wong KYM, et al. Durable clinical benefit metastatic colorectal cancer at favorable prognosis. Ann Oncol with nivolumab plus ipilimumab in dna mismatch repair-deficient/ 2015;26:2092-7. https://doi.org/10.1093/annonc/mdv290 microsatellite instability-high metastatic colorectal cancer. J Clin 40 Galuppini F, Pennelli G, Loupakis F, et al. BRAF p.V600E-spe- Oncol 2018;36:773-9. https://doi.org/10.1200/JCO.2017.76.9901 cific immunohistochemical assessment in colorectal cancer en- 55 Overman MJ, McDermott R, Leach JL, et al. Nivolumab in doscopy biopsies is consistent with the mutational profiling. His- patients with metastatic DNA mismatch repair-deficient or topathology 2017;71:1008-11. https://doi.org/10.1111/his.13315 microsatellite instability-high colorectal cancer (CheckMate 41 Parsons MT, Buchanan DD, Thompson B, et al. Correlation of 142): an open-label, multicentre, phase 2 study. Lancet Oncol tumour BRAF mutations and MLH1 methylation with germline 2017;18:1182-91. https://doi.org/10.1016/S1470-2045(17)30422- mismatch repair (MMR) gene mutation status: a literature review 9 assessing utility of tumour features for MMR variant classifica- 56 Ribas A, Wolchok JD. Cancer immunotherapy using check- tion. J Med Genet 2012;49:151-7. https://doi.org/10.1136/jmed- point blockade. Science 2018;359(6382):1350-5. https://doi. genet-2011-100714 org/10.1126/science.aar4060 42 Boland CR, Goel A. Microsatellite instability in colorectal can- 57 Guinney J, Dienstmann R, Wang X, et al. The consensus mo- cer. Gastroenterology 2010;138(6):2073-87 e3. https://doi. lecular subtypes of colorectal cancer. Nat Med 2015;21:1350-6. org/10.1053/j.gastro.2009.12.064 https://doi.org/10.1038/nm.3967 258 M. Fassan et al.

58 Smyth EC, Wotherspoon A, Peckitt C, et al. Mismatch repair dMMR status. J Immunother Cancer 2019;7:297. https://doi. deficiency, microsatellite instability, and survival: an exploratory org/10.1186/s40425-019-0788-5 analysis of the Medical Research Council Adjuvant Gastric Infu- 73 Pinto D, Pinto C, Guerra J, et al. Contribution of MLH1 consti- sional Chemotherapy (MAGIC) Trial. JAMA Oncol 2017;3:1197- tutional methylation for Lynch syndrome diagnosis in patients 203. https://doi.org/10.1001/jamaoncol.2016.6762 with tumor MLH1 downregulation. Cancer Med 2018;7:433-44. 59 Bonneville R, Krook MA, Kautto EA, et al. Landscape of Micro- https://doi.org/10.1002/cam4.1285 satellite Instability Across 39 Cancer Types. JCO Precis Oncol 74 Umar A, Boland CR, Terdiman JP, et al. Revised bethesda 2017;2017. https://doi.org/10.1200/PO.17.00073. guidelines for hereditary nonpolyposis colorectal cancer (lynch 60 Loupakis F, Depetris I, Biason P, et al. Prediction of benefit syndrome) and microsatellite instability. J Natl Cancer Inst from checkpoint inhibitors in mismatch repair deficient meta- 2004;96:261-8. https://doi.org/10.1093/jnci/djh034 static colorectal cancer: role of tumor infiltrating lymphocytes. 75 Goel A, Nagasaka T, Hamelin R, et al. An optimized pentaplex Oncologist 2020;25:481-7. https://doi.org/10.1634/theoncolo- PCR for detecting DNA mismatch repair-deficient colorectal can- gist.2019-0611 cers. PLoS One 2010;5:e9393. https://doi.org/10.1371/journal. 61 Shia J, Schultz N, Kuk D, et al. Morphological characterization of pone.0009393 colorectal cancers in The Cancer Genome Atlas reveals distinct 76 Cohen R, Hain E, Buhard O, et al. Association of primary resis- morphology-molecular associations: clinical and biological im- tance to immune checkpoint inhibitors in metastatic colorectal plications. Mod Pathol 2017;30:599-609. https://doi.org/10.1038/ cancer with misdiagnosis of microsatellite instability or mismatch modpathol.2016.198 repair deficiency status. JAMA Oncol 2019;5:551-5. https://doi. 62 Shia J, Holck S, Depetris G, et al. Lynch syndrome-associated org/10.1001/jamaoncol.2018.4942 neoplasms: a discussion on histopathology and immunohisto- 77 Nowak JA, Yurgelun MB, Bruce JL, et al. Detection of mismatch chemistry. Fam Cancer 2013;12:241-60. https://doi.org/10.1007/ repair deficiency and microsatellite instability in colorectal ade- s10689-013-9612-4 nocarcinoma by targeted next-generation sequencing. J Mol Di- 63 Mathiak M, Warneke VS, Behrens HM, et al. Clinicopathologic agn 2017;19:84-91. https://doi.org/10.1016/j.jmoldx.2016.07.010 characteristics of microsatellite instable gastric carcinomas 78 Baumeister SH, Freeman GJ, Dranoff G, et al. coinhibi- revisited: urgent need for standardization. Appl Immunohis- tory pathways in immunotherapy for cancer. Annu Rev Immu- tochem Mol Morphol 2017;25:12-24. https://doi.org/10.1097/ nol 2016;34:539-73. https://doi.org/10.1146/annurev-immu- PAI.0000000000000264 nol-032414-112049 64 Giuffrida P, Arpa G, Grillo F, et al. PD-L1 in small bowel ade- 79 Shah MA, Kojima T, Hochhauser D, et al. Efficacy and safety of nocarcinoma is associated with etiology and tumor-infiltrating pembrolizumab for heavily pretreated patients with advanced, lymphocytes, in addition to microsatellite instability. Mod Pathol metastatic adenocarcinoma or squamous cell carcinoma of the 2020; 33:1398-1409. https://doi.org/10.1038/s41379-020-0497-0 esophagus: the phase 2 KEYNOTE-180 study. JAMA Oncol 65 Jun SY, Lee EJ, Kim MJ, et al. Lynch syndrome-related small 2019;5:546-50. https://doi.org/10.1001/jamaoncol.2018.5441 intestinal adenocarcinomas. Oncotarget 2017;8:21483-500. 80 Kojima T, Muro K, Francois E, et al. Pembrolizumab versus che- https://doi.org/10.18632/oncotarget.15277 motherapy as second-line therapy for advanced esophageal 66 Luchini C, Brosens LAA, Wood LD, et al. Comprehensive char- cancer: Phase III KEYNOTE-181 study. J Clin Oncol 2019;37(4_ acterisation of pancreatic ductal adenocarcinoma with microsat- suppl):2. ellite instability: histology, molecular pathology and clinical im- 81 Fashoyin-Aje L, Donoghue M, Chen H, et al. FDA approval plications. Gut 2020;gutjnl-2020-320726. https://doi.org/10.1136/ summary: pembrolizumab for recurrent locally advanced or gutjnl-2020-320726 metastatic gastric or gastroesophageal junction adenocarci- 67 Luchini C, Capelli P, Scarpa A. Pancreatic ductal adenocarci- noma expressing PD-L1. Oncologist 2019;24:103-9. https://doi. noma and its variants. Surg Pathol Clin 2016;9:547-60. https:// org/10.1634/theoncologist.2018-0221. doi.org/10.1016/j.path.2016.05.003 82 Li Y, He M, Zhou Y, et al. The prognostic and clinicopathologi- 68 Goeppert B, Roessler S, Renner M, et al. Mismatch repair de- cal roles of PD-L1 expression in colorectal cancer: a systematic ficiency is a rare but putative therapeutically relevant finding review and meta-analysis. Front Pharmacol 2019;10:139. https:// in non-liver fluke associated cholangiocarcinoma. Br J Cancer doi.org/10.3389/fphar.2019.00139 2019;120:109-14. https://doi.org/10.1038/s41416-018-0199-2 83 Luchini C, Cros J, Pea A, et al. PD-1, PD-L1, and CD163 in 69 Latham A, Srinivasan P, Kemel Y, et al. Microsatellite Instabil- pancreatic undifferentiated carcinoma with osteoclast-like giant ity Is Associated With the Presence of Lynch Syndrome Pan- cells: expression patterns and clinical implications. Hum Pathol Cancer. J Clin Oncol 2019;37:286-95. https://doi.org/10.1200/ 2018;81:157-65. https://doi.org/10.1016/j.humpath.2018.07.006 JCO.18.00283 84 Ahn S, Lee Y, Kim JW, et al. Programmed cell death ligand-1 70 Sarode VR, Robinson L. Screening for lynch syndrome by im- (PD-L1) expression in extrahepatic biliary tract cancers: a com- munohistochemistry of mismatch repair proteins: significance parative study using 22C3, SP263 and E1L3N anti-PD-L1 an- of indeterminate result and correlation with mutational studies. tibodies. Histopathology 2019;75:526-36. https://doi.org/10.1111/ Arch Pathol Lab Med 2019;143:1225-33. https://doi.org/10.5858/ his.13901 arpa.2018-0201-OA 85 Shitara K, Ozguroglu M, Bang YJ, et al. Pembrolizumab versus 71 Shia J. Immunohistochemistry versus microsatellite instability paclitaxel for previously treated, advanced gastric or gastro- testing for screening colorectal cancer patients at risk for heredi- oesophageal junction cancer (KEYNOTE-061): a randomised, tary nonpolyposis colorectal cancer syndrome. Part I. The utility open-label, controlled, phase 3 trial. Lancet 2018;392(10142):123- of immunohistochemistry. J Mol Diagn 2008;10:293-300. https:// 33. https://doi.org/10.1016/S0140-6736(18)31257-1 doi.org/10.2353/jmoldx.2008.080031 86 Fassan M, Brignola S, Pennelli G, et al. PD-L1 expression in 72 Loupakis F, Maddalena G, Depetris I, et al. Treatment with gastroesophageal dysplastic lesions. Virchows Arch 2019. checkpoint inhibitors in a metastatic colorectal cancer patient https://doi.org/10.1007/s00428-019-02693-8 with molecular and immunohistochemical heterogeneity in MSI/ 87 Saraggi D, Galuppini F, Remo A, et al. PD-L1 overexpression in MOLECULAR LANDSCAPE OF GASTROINTESTINAL NEOPLASMS 259

ampulla of Vater carcinoma and its pre-invasive lesions. Histopa- label, phase 2 trial. Lancet Oncol 2016;17:738-46. https://doi. thology 2017;71:470-4. https://doi.org/10.1111/his.13254 org/10.1016/S1470-2045(16)00150-9 88 Kulangara K, Zhang N, Corigliano E, et al. Clinical utility of the 101 Valtorta E, Martino C, Sartore-Bianchi A, et al. Assessment of a combined positive score for programmed death ligand-1 expres- HER2 scoring system for colorectal cancer: results from a valida- sion and the approval of pembrolizumab for treatment of gas- tion study. Mod Pathol 2015;28:1481-91. https://doi.org/10.1038/ tric cancer. Arch Pathol Lab Med 2019;143:330-7. https://doi. modpathol.2015.98 org/10.5858/arpa.2018-0043-OA 102 Yoshino T, Pentheroudakis G, Mishima S, et al. JSCO-ESMO- 89 Rossi S, Gasparotto D, Miceli R, et al. KIT, PDGFRA, and ASCO-JSMO-TOS: international expert consensus recommen- BRAF mutational spectrum impacts on the natural history dations for tumour-agnostic treatments in patients with solid tu- of imatinib-naive localized GIST: a population-based study. mours with microsatellite instability or NTRK fusions. Ann Oncol Am J Surg Pathol 2015;39:922-30. https://doi.org/10.1097/ 2020;31:861-72. https://doi.org/10.1016/j.annonc.2020.03.299 PAS.0000000000000418 103 Lasota J, Chlopek M, Lamoureux J, et al. colonic adenocarci- 90 Ricci R, Saragoni L. Everything you always wanted to know nomas harboring NTRK fusion genes: a clinicopathologic and about GIST (but were afraid to ask) An update on GIST pathol- molecular genetic study of 16 cases and review of the litera- ogy. Pathologica 2016;108:90-103. ture. Am J Surg Pathol 2020;44:162-73. https://doi.org/10.1097/ 91 Corless CL. Gastrointestinal stromal tumors: what do we know PAS.0000000000001377 now? Mod Pathol 2014;27 Suppl 1:S1-16. https://doi.org/10.1038/ 104 Solomon JP, Linkov I, Rosado A, et al. NTRK fusion detec- modpathol.2013.173 tion across multiple assays and 33,997 cases: diagnostic im- 92 Pantaleo MA, Biasco G. Gastrointestinal cancer: management of plications and pitfalls. Mod Pathol 2020;33:38-46. https://doi. GIST--go beyond imatinib: treat resistant subtypes. Nat Rev Clin org/10.1038/s41379-019-0324-7 Oncol 2015;12:440-2. https://doi.org/10.1038/nrclinonc.2015.107 105 Chou A, Fraser T, Ahadi M, et al. NTRK gene rearrangements 93 Origone P, Gargiulo S, Mastracci L, et al. Molecular character- are highly enriched in MLH1/PMS2 deficient, BRAF wild-type ization of an Italian series of sporadic GISTs. Gastric Cancer colorectal carcinomas-a study of 4569 cases. Mod Pathol 2013;16:596-601. https://doi.org/10.1007/s10120-012-0213-y 2020;33:924-32. https://doi.org/10.1038/s41379-019-0417-3 94 Athauda A, Fong C, Lau DK, et al. Broadening the therapeu- 106 Waddell N, Pajic M, Patch AM, et al. Whole genomes rede- tic horizon of advanced biliary tract cancer through molecular fine the mutational landscape of pancreatic cancer. Nature characterisation. Cancer Treat Rev 2020;86:101998. https://doi. 2015;518(7540):495-501. https://doi.org/10.1038/nature14169 org/10.1016/j.ctrv.2020.101998 107 Hutchings D, Jiang Z, Skaro M, et al. Histomorphology of pan- 95 Wainberg ZA, Lassen UN, Elez E, et al. Efficacy and safety of creatic cancer in patients with inherited ATM serine/threonine ki- dabrafenib (D) and trametinib (T) in patients (pts) with BRAF nase pathogenic variants. Mod Pathol 2019;32:1806-13. https:// V600E–mutated biliary tract cancer (BTC): a cohort of the doi.org/10.1038/s41379-019-0317-6 ROAR basket trial. J Clin Oncol 2019;37(4_suppl):187. 108 Golan T, Hammel P, Reni M, et al. Maintenance olaparib for 96 Lowery MA, Burris HA 3rd, Janku F, et al. Safety and activity germline BRCA-mutated metastatic pancreatic cancer. N Engl J of ivosidenib in patients with IDH1-mutant advanced cholan- Med 2019;381:317-27. https://doi.org/10.1056/NEJMoa1903387 giocarcinoma: a phase 1 study. Lancet Gastroenterol Hepatol 109 Huang W, Navarro-Serer B, Jeong YJ, et al. Pattern of in- 2019;4:711-20. https://doi.org/10.1016/S2468-1253(19)30189-X vasion in human pancreatic cancer organoids is associ- 97 Mazzaferro V, El-Rayes BF, Droz Dit Busset M, et al. Dera- ated with loss of SMAD4 and clinical outcome. Cancer Res zantinib (ARQ 087) in advanced or inoperable FGFR2 gene 2020;canres.1523.2019. https://doi.org/10.1158/0008-5472. fusion-positive intrahepatic cholangiocarcinoma. Br J Cancer CAN-19-1523 2019;120:165-71. https://doi.org/10.1038/s41416-018-0334-0 110 Iacobuzio-Donahue CA, Fu B, Yachida S, et al. DPC4 gene sta- 98 Javle M, Lowery M, Shroff RT, et al. Phase II Study of BGJ398 tus of the primary carcinoma correlates with patterns of failure in in patients with FGFR-altered advanced cholangiocarci- patients with pancreatic cancer. J Clin Oncol 2009;27:1806-13. noma. J Clin Oncol 2018;36:276-82. https://doi.org/10.1200/ https://doi.org/10.1200/JCO.2008.17.7188. JCO.2017.75.5009 111 Herman JM, Jabbour SK, Lin SH, et al. Smad4 Loss corre- 99 Sartore-Bianchi A, Amatu A, Porcu L, et al. HER2 Positivity Pre- lates with higher rates of local and distant failure in pancre- dicts Unresponsiveness to EGFR-Targeted Treatment in Meta- atic adenocarcinoma patients receiving adjuvant chemora- static Colorectal Cancer. Oncologist 2019;24:1395-402. https:// diation. Pancreas 2018;47:208-12. https://doi.org/10.1097/ doi.org/10.1634/theoncologist.2018-0785 MPA.0000000000000985 100 Sartore-Bianchi A, Trusolino L, Martino C, et al. Dual-targeted 112 Paiella S, Malleo G, Cataldo I, et al. Radiofrequency ablation therapy with trastuzumab and lapatinib in treatment-refractory, for locally advanced pancreatic cancer: SMAD4 analysis segre- KRAS codon 12/13 wild-type, HER2-positive metastatic colorec- gates a responsive subgroup of patients. Langenbecks Arch Surg tal cancer (HERACLES): a proof-of-concept, multicentre, open- 2018;403:213-20. https://doi.org/10.1007/s00423-017-1627-0