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 II

01VOL. 113

Edited by Paola Parente and Matteo Fassan FEBRUARY 2021 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 01Vol. 113 P. Querzoli Managing Editor E. Fulcheri St Anna University Hospital, Ferrara University of Genoa February 2021 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 01 February 2021

REVIEWS Introduction to neuroendocrine of the digestive system: definition and classification F. Inzani, G. Rindi...... 1

Neuroendocrine neoplasms of the esophagus and stomach L. Mastracci, G. Rindi, F. Grillo, E. Solcia, M. Campora, M. Fassan, P. Parente, A. Vanoli, S. La Rosa...... 5

Neuroendocrine neoplasms of the duodenum, ampullary region, jejunum and ileum M. Milione, P. Parente, F. Grillo, G. Zamboni, L. Mastracci, C. Capella, M. Fassan, A. Vanoli...... 12

Neuroendocrine neoplasms of the appendix, colon and rectum M. Volante, F. Grillo, F. Massa, F. Maletta, L. Mastracci, M. Campora, J. Ferro, A. Vanoli, M. Papotti...... 19

Neuroendocrine neoplasms of the biliary tree, liver and : a pathological approach C. Luchini, G. Pelosi, A. Scarpa, P. Mattiolo, D. Marchiori, R. Maragliano, F. Sessa, S. Uccella...... 28

Histopathology of IBD Colitis. A practical approach from the pathologists of the Italian Group for the study of the (GIPAD) V. Villanacci, L. Reggiani-Bonetti, T. Salviato, G. Leoncini, M. Cadei, L. Albarello, A. Caputo, M.C. Aquilano, S. Battista, P. Parente...... 39

Histopathology of Non-IBD Colitis. A practical approach from the Italian Group for the study of the gastrointestinal tract (GIPAD) V. Villanacci, L. Reggiani-Bonetti, G. Leoncini, P. Parente, M. Cadei, L. Albarello, G. Mandelli, A. Caputo...... 54

Front cover: A duodenal gastrinoma, showing a trabecular pattern, with vascular pseudorosettes (A, hematoxylin and eosin), and tumor cell reactivity for gastrin (B, gastrin immunostaining); An ampullary-type somatostatin- producing neuroendocrine tumor, showing tubulo-acinar structures with psammoma bodies in a solid/trabecular architectural background (A, hematoxylin and eosin), and extensive somatostatin expression by tumor cells (B, somatostatin immunostaining) (page 14). PATHOLOGICA 2021;113:1-4; DOI: 10.32074/1591-951X-227

Review

Introduction to neuroendocrine neoplasms of the digestive system: definition and classification

Frediano Inzani1,3, Guido Rindi1-3 1 Anatomic Pathology Section, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Roma, Italy; 2 Anatomic Pathology Unit, Department of Woman and Child Health and Public Health, Fondazione Policlinico Universitario A. Gemelli IRCCS, Roma, Italy; 3 ENETS Center of Excellence, Roma, Italy

Summary Neoplasms characterized by the expression of markers of neuroendocrine differentiation in neoplastic cells are defined neuroendocrine. This broad definition comprises tumors found at different sites of the body with similar morphology but different behavior and genetic background. From a clinical standpoint neuroendocrine neoplasms may be functioning, when they give rise to unregulated secretion of hormones. Functioning tumors account for about one-third of neuroendocrine neoplasms. From a pathological standpoint neuroendo- crine are classified by cancer category, cancer families/classes, cancer types, cancer grade and cancer stage. The category identifies the cancer major trait and thus defined as neuroendocrine neoplasia (NEN) to comprise all families/classes of neuroen- docrine cancer. The cancer family/types are neuroendocrine tumors (NET) as well differen- tiated, and neuroendocrine carcinoma (NEC) as poorly differentiated forms. Cancer grade, Received: November 21, 2020 Accepted: November 23, 2020 based on proliferation measure by mitotic count and Ki-67%, and cancer stage, based on tumor size and invasion (T), node deposits (N) and distant metastases (M), complete the Correspondence pathological classification. Site-specific differences are the rule. Still missing is a genetic Guido Rindi classification tool to complement current pathological descriptors. Unit of Anatomic Pathology, Fondazione Policlinico Universitario A. Gemelli IRCCS- Key words: neuroendocrine neoplasia, NEN, NET, NEC, Ki-67% Università Cattolica del Sacro Cuore, largo A. Gemelli 8, 00167 Roma, Italy Tel. +39 06 30155883 Introduction Fax: +39 06 30157008 E-mail: [email protected] Neuroendocrine neoplasms represent a category of neoplasms defined Conflict of interest by neuroendocrine differentiation, at histology based on positive immu- The Authors declare no conflict of interest. nolabelling for chromogranin A and synaptophysin. Neuroendocrine ne- oplasms may arise anywhere in the body either from endocrine organs, How to cite this article: Inzani F, Rindi nerve structures or from the so-called diffuse neuroendocrine system. G. Introduction to neuroendocrine More than half are found in the gastro-entero-pancreatic (GEP) tract neoplasms and classification systems. Pathologica 2021:113:1-4. https://doi. and, though relatively rare, their incidence has been steadily increas- org/10.32074/1591-951X-227 ing in the last three decades 1,2. GEP neuroendocrine neoplasms are a distinct though heterogenous cancer category when considering their © Copyright by Società Italiana di Anatomia Pato- logica e Citopatologia Diagnostica, Divisione Itali- clinical behavior, morphology, specific types of cells involved, biological ana della International Academy of Pathology and genetic features. The classification of neuroendocrine neoplasms evolved along the last two decades to reflect our increased understand- OPEN ACCESS ing of this disease including clinical and pathological classifications. This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 International) license: the Classification principles work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original version. For further information: https://creativecommons. Clinical classification org/licenses/by-nc-nd/4.0/deed.en Clinically NENs are separated into functioning and non-functioning. 2 F. Inzani, G. Rindi

Functioning neoplasms are less frequent, account- tiated neoplasms as neuroendocrine carcinoma ing for about one-third of GEP neuroendocrine neo- (NEC) 6. At histology NETs substantially reproduce plasms 2. Functioning neuroendocrine neoplasms are the structural (when present) and cytological fea- characterized by specific clinical symptoms defined tures of their non-neoplastic counterparts. NETs are by unregulated hormonal production (e.g. hyper-in- characterized by an “organoid” structure (so defined sulinemic hypoglycemia determined by insulinoma). as resembling Langerhans islets), variably organized The largest fraction of functioning neoplasm account in nests, trabeculae and acini made by epithelial for well differentiated neoplasm, though paraneoplas- cells with usually monomorphic nuclei, rare mitoses, tic endocrine syndromes may associate with poorly in the absence of, or with only focal (spotty), necro- differentiated forms. Non-functioning neuroendocrine sis (Fig. 1. A-E). The morphology of NETs overlaps neoplasms are more frequent composed of by tumor at different anatomical sites of the digestive system. cells sometimes expressing hormones at immunohis- The site of origin may be defined only by the use of tochemistry but unable of unregulated release. Similar immunohistochemistry for site-specific transcription to any other epithelial neoplasms, clinical symptoms factors and/or hormones. Nonetheless, some growth are determined by the effects of tumor mass and patterns are more often observed for certain cell growth, including mucosal erosion, wall/nearby organs types at specific anatomical sites (e.g. solid islets for invasion and metastatic deposition 3, in turn reflecting serotonin-producing enterochromaffin cell tumors of tumor cell biology. the small intestine). The distinction between functioning and non-function- The poorly differentiated neuroendocrine neoplasms, ing neoplasms is virtually impossible based on his- NECs, are characterized by the disorderly solid pro- tology and may only be defined by clinical symptoms liferation of severely atypical cells, with abundant/ and blood tests. When considering well differentiated diffuse necrosis and evident, often atypical, mitoses neoplasms, tumor cell types composing both function- (Fig. 1 F-J). Two variants are described: the small cell ing and non-functioning neuroendocrine neoplasms type characterized by large nuclei with salt and pep- usually follow the physiological distribution of their per chromatin and a thin rim of cytoplasm (i.e. high non-neoplastic counterpart (e.g. enterochromaffin-like nucleus/cytoplasm ratio) and the large cell type with cells in gastric neuroendocrine neoplasms, somato- large nuclei with prominent nucleoli, salt and pepper statin-producing D cells in duodenum and pancreatic chromatin and abundant cytoplasm. neuroendocrine neoplasms). Cancer types Pathological classification In the digestive system, the NEN types overlap the The pathological classification includes the definitions Family/Class definitions, i.e. NET and NEC. At other of cancer category, cancer families/classes, cancer anatomical site this may not be the case, e.g. in the types, cancer grade and cancer stage (Tab. I) 4. Malig- lung well differentiated NENs are defined as carci- nancy is implicit. noid 7. Variants are foreseen, e.g. the clear cell or - ic subtypes in pancreas NET. Cancer category The category is defined as neuroendocrine neoplasia Cancer grade (NEN) to comprise all families/classes of neuroen- The current WHO classifications 6,8 adopt a three- docrine cancer. Neuroendocrine is defined when at tiers grading system (G1-G3) for NET. NEC are by de- least two markers of neuroendocrine differentiation fault G3, the grade being not to be specified to avoid (e.g. chromogranin A and synaptophysin) are both confusion vs NET G3 (Fig. 1). The currently adopted expressed in the vast majority of neoplastic cells. In grading system for NETs is based on proliferation as- case only a fraction of at least 30% of cancer cells are sessment with incremental ranges of mitosis and Ki67 neuroendocrine in nature, the cancer category is de- index (Tab. I). This tool directly stems from the Euro- fined as Mixed NEuroendocrine non-neuroendocrine pean Neuroendocrine Tumor Society (ENETS) propos- Neoplasia (MiNEN). al 9,10 that proved of prognostic significance and for this reason was subsequently adopted by WHO in 2010 11. Cancer families/classes The present grading system allows the identification of The class distinction is based on morphology and a NEN subgroup with a well differentiated morphology separates neuroendocrine neoplasms into well and and G3 proliferative values, isolating the most aggres- poorly differentiated forms (WHO 2000) 5. The well sive NET (Fig. 1). NET G3 display significant differenc- differentiated neoplasms are currently defined as es when compared to NEC in somatostatin receptor neuroendocrine tumors (NET) and poorly differen- expression (diffuse in NET, almost absent in NEC) CLASSIFICATION OF NEN 3

Figure 1. NET and NEC in the stomach: examples of high-grade NET G3 (A-E) and NEC (F-J). The NET G3 shows solid structure, in absence of necrosis in this case, and is made by relatively monomorphic cells with abundant cytoplasm, mildly irregular nuclei and moderate/severe atypia, atypical mitosis (A, center of the micrograph), in- tense immunoreactivity for chromogranin A (B), synaptophysin (C), somatostatin receptor type 2 (D) and high Ki-67 nuclear labelling (E, about 40%).The NEC, small cell type, shows a solid structure with abundant necrosis (F, lower right corner), and is made by severely atypical cells of medium-small size with scarce cytoplasm (G, AE1/AE2 dotted cytokeratin expression), diffuse chromogranin A (H, note some dots), synaptophysin (I) and sharply elevated Ki-67 nuclear labelling (J, about 90%). A, F H&E; B-E and G-J immunoperoxidase; magnification 400x.

Table I. Neuroendocrine Cancer Classification Category Type Grade Ki-67%a Mitosisb Stage NEN NET G1 ≤3 < 2 NET-specific & G2 3-20 2-20 site-dependent G3 > 20 > 20 NEC G3 > 20 > 20 non-NE cancer & site-dependent MiNENc nac nac nac nac non-NE cancer & site-dependent NEN: neuroendocrine neoplasia; NET: neuroendocrine tumor; NEC: neuroendocrine carcinoma; MiNEN: mixed neuroendocrine non-neuroendocrine neo- plasm; non-NE: non-neuroendocrine; na: not available. a MIB1 clone, nuclear expression in % of cells in areas of highest labelling; b per 2mm2; c since non neuroendocrine pure, typing and grading of MiNEN are done for the neuroendocrine component as here described and for the non-neuroendocrine compo- nent according to the specific cancer category that is observed ( or ) 6. prognosis (better in NET and invariably dismal in NEC) Cancer stage and in responsiveness to platinum-based chemother- The Tumor Node Metastasis (TNM) staging tools are apy (absent in NET, usually present in NEC). for NETs only and site-specific 12. Developed following 4 F. Inzani, G. Rindi

the ENETS proposals 9,10, these are based on NET 2 Dasari A, Shen C, Haperin D, et al. Trends in the incidence, prev- size and/or invasion for T definition, on the presence alence, and survival outcomes in patients with neuroendocrine tumors in the United States. JAMA Oncol 2017;3:1335-1342. of one or more (depending on site) node deposits for https://doi.org/10.1001/jamaoncol.2017.0589 N and on pathological evidence of distant metastases 3 Rindi G, Wiedenmann B. Neuroendocrine neoplasia of the gas- for M. NET staging tools differ from those adopted for trointestinal tract revisited: towards precision medicine. Nat Rev the other digestive cancer which otherwise are to be Endocrinol 2020;16:590-607. https://doi.org/10.1038/s41574- used for NEC 6. MiNEN are assimilated to non-neu- 020-0391-3 roendocrine cancer and are staged according to the 4 Rindi G, Klimstra DS, Abedi-Ardekani B, et al. A common clas- corresponding non-neuroendocrine cancer staging sification framework for neuroendocrine neoplasms: an Interna- tional Agency for Research on Cancer (IARC) and World Health tools depending on site. Organization (WHO) expert consensus proposal. Mod Pathol 2018;31:1770-86. https://doi.org/10.1038/s41379-018-0110-y 5 Solcia E, Klöppel G, Sobin LH. Histological typing of endocrine Conclusions tumours. 2 ed. Berlin Heidelberg: Springer-Verlag 2000. 6 WHO Classification of tumours editorial board. digestive system The current NEN classification and its pathological tumours. 5th ed. Vol. 1 Lyon: IARC Press 2019. grading and staging tools are built to emphasize the 7 Travis WD, Brambilla E, Burke AP, et al. Pathology and genetics profound difference existing between NET and NEC of tumours of the lung, pleura, thymus and heart. 4th ed. Vol. 7. families. This distinction well mirrors differences in Lyon: IARC Press 2015. 8 genetic background. NETs show a low number of Lloyd RV, Osamura R, Kloppel G, Rosai J. WHO Classification of tumours of endocrine organs. 4th ed. Vol. 10. Lyon: IARC Press gene alterations, low mutational burden and almost 2 01 7. no involvement of classic cancer drivers, while NECs 9 Rindi G, Klöppel G, Alhman H, et al. TNM staging of foregut show a high degree of gene abnormality, most often (neuro)endocrine tumors: a consensus proposal including a involving classical drivers including TP53 and Rb. Still grading system. Virchows Arch 2006:449:395-401. https://doi. missing is a genetic tool that may effectively comple- org/10.1007/s00428-006-0250-1 ment or even surrogate the proven efficacy of current 10 Rindi G, Klöppel G, Couvelard A, et al. TNM staging of midgut pathological descriptors. and hindgut (neuro) endocrine tumors: a consensus proposal including a grading system. Virchows Arch 2007:451:757-62. https://doi.org/10.1007/s00428-007-0452-1 References 11 Bosman F, Carneiro F, Hruban RH, Theise ND. Pathology and th 1 Leoncini E, Boffetta P, Shafir M, et al. Increased incidence trend genetics of tumours of the digestive system. 4 ed. Vol. 3. Lyon: of low-grade and high-grade neuroendocrine neoplasms. En- IARC Press 2010. docrine 2017;58:368-79. https://doi.org/10.1007/s12020-017- 12 Amin MB. AJCC Cancer Staging Manual. VIII ed. Berlin Heidel- 1273-x berg: Springer-Verlag 2017. PATHOLOGICA 2021;113:5-11; DOI: 10.32074/1591-951X-229

Review

Neuroendocrine neoplasms of the esophagus and stomach

Luca Mastracci1,2 , Guido Rindi3,4 , Federica Grillo1,2 , Enrico Solcia5 , Michela Campora1, Matteo Fassan6 , Paola Parente7, Alessandro Vanoli5, Stefano La Rosa8 1 Anatomic Pathology, Ospedale Policlinico San Martino IRCCS, Genova, Italy; 2 Anatomic Pathology, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genova, Italy; 3 Anatomic Pathology Section, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Roma, Italy ; 4 Anatomic Pathology Unit, Department of Woman and Child Health and Public Health, Fondazione Policlinico Universitario A. Gemelli IRCCS, Roma, Italy; 5 Anatomic Pathology Unit, Department of Molecular Medicine, University of Pavia, and IRCCS San Matteo Hospital, Pavia, Italy; 6 Surgical Pathology Unit, Department of Medicine (DIMED), University of Padua, Italy; 7 Unit of Pathology, Fondazione IRCCS Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo, FG, Italy; 8 Institute of Pathology, University Hospital and University of Lausanne, Switzerland

Summary Esophageal neuroendocrine neoplasms (E-NENs) are much rarer than other gastro- entero-pancreatic neuroendocrine neoplasms, the majority showing aggressive behav- ior with early dissemination and poor prognosis. Among E-NENs, exceptionally rare well differentiated neuroendocrine tumors (E-NET) and more frequent esophageal poorly dif- ferentiated neuroendocrine carcinomas (E-NEC) and mixed neuroendocrine-non neuro- Received: November 21, 2020 endocrine neoplasms (MiNEN) can be recognized. E-NECs usually exhibit a small cell Accepted: November 23, 2020 morphology or mixed small and large cells. Esophageal MiNEN are composed of NEC component admixed with adenocarcinoma or squamous cell carcinoma. Gastric (G) NENs Correspondence encompass a wide spectrum of entities ranging from indolent G-NETs to highly aggres- Stefano La Rosa sive G-NECs and MiNENs. Among G-NETs, ECL-cell NETs are the most common and, Institut Universitaire de Pathologie, CHUV, although composed of histamine-producing cells, are a heterogeneous group of neoplastic 25 rue du Bugnon, CH-1011 Lausanne, proliferations showing different clinical and prognostic features depending on the patient’s Switzerland clinico-pathological background including the morphology of the peri-tumoral mucosa, Tel. +41 (0)21 3147162 Fax: +41 (0)21 3147205 gastrin serum levels, presence or absence of antral G-cell , and presence or E-mail: [email protected] absence of MEN1 syndrome. In general, NET associated with hypergastrinemia show a better outcome than NET not associated with hypergastrinemia. G-NECs and MiNENs are Conflict of interest aggressive neoplasms more frequently observed in males and associated with a dismal The Authors declare no conflict of interest. prognosis.

Key words: neuroendocrine neoplasms, neuroendocrine tumor, neuroendocrine How to cite this article: Mastracci L, carcinoma, MiNEN, stomach, esophagus Rindi G, Grillo F, et al. Neuroendocrine neoplasms of the esophagus and stomach. Pathologica 2021:113:5-11. https://doi. org/10.32074/1591-951X-229 Neuroendocrine neoplasms of the esophagus © Copyright by Società Italiana di Anatomia Pato- logica e Citopatologia Diagnostica, Divisione Itali- Introduction ana della International Academy of Pathology Esophageal neuroendocrine neoplasms (E-NENs) are much rarer than OPEN ACCESS other gastro-entero-pancreatic neuroendocrine tumors accounting for only 0.04-1%. More than 4000 cases have been described in the inter- This is an open access journal distributed in accordance 1 with the CC-BY-NC-ND (Creative Commons Attribution- national literature , where E-NENs represent 0.03-0.05% of all esoph- NonCommercial-NoDerivatives 4.0 International) license: the ageal 2-4. Their incidence has been increased in the last work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original decades, probably as a secondary effect of improvement in diagnosis version. For further information: https://creativecommons. and clinician awareness. The majority of E-NENs show an aggressive org/licenses/by-nc-nd/4.0/deed.en behavior with early dissemination and poor prognosis depending on size 6 L. Mastracci, et al.

and grade. Unfortunately, the rarity of this neoplasm ferentiated neuroendocrine tumors (E-NET), esopha- has not yet permitted the prospective recruitment of geal poorly differentiated neuroendocrine carcinomas patients in clinical trials, in order to establish biological (E-NEC), and mixed neuroendocrine-non neuroendo- features and the optimal therapeutic regimen. crine neoplasms (MiNEN). E-NETs (grade 1 to 3) are extremely rare; mitotic and Ki67 cut off values for grading are analogous to those Clinical presentation applied in other sites. More than 90% of E-NENs are represented by small cell NECs, while a minority by Mean age of E-NEN patients at diagnosis is 66 years large cell NECs. In a recent case series of 69 consec- with a male prevalence (6:1 ratio). More than 50% utive MiNENs, the esophagus and gastro-esophageal of cases originate in the lower third of the esopha- junction was identified as the second commonest site gus 5. Only a small fraction of patients with E-NEN of origin (15.9% of cases) after colon-rectum 9. Among are asymptomatic and incidentally discovered during MiNEN, the most frequently reported type is the Mixed endoscopy, while most patients present with dyspha- AdenoNeuroendocrine carcinoma (MANEC). gia and weight loss. Less frequent symptoms are pain, hoarseness, and bleeding1. Given the frequent high Microscopic description grade of these neoplasms, syndrome is E-NETs are characterized by a proliferation of uniform, extremely rare, while paraneoplastic syndromes are medium sized cells with abundant cytoplasm, ovoid slightly more frequent. nuclei with dispersed ‘salt and pepper’ chromatin and Less than 1% of E-NENs are well differentiated, while inconspicuous nucleoli, growing in insular, trabecular the great majority are high grade poorly differentiated or cribriform pattern with interposed well-vascularized small cell neuroendocrine carcinomas, which present stroma. with common lymph node and distant metastases E-NECs are more frequently of the small cell subtype (31-90% of cases) 1,6. Distant metastases are usually composed of small round, ovoid or spindle-like cells located in the liver, lung and bone, while brain metas- with hyperchromatic nuclei with dense chromatin or- tases are relatively rare7. ganized in ample solid insulae or festoons, with multi- Prognostic factors are unclear because of the rarity focal necrosis and frequent mitoses (Fig. 1). A relative- of this neoplasm and the scarcity of collected data in ly small proportion of E-NECs are composed of large international literature. Age, disease extent, TNM clas- cells with basophilic cytoplasm and marked nuclear sification and type of treatment (local +/- systemic) are atypia with prominent nucleoli. the main prognostic factors affecting survival. MiNEN are usually composed of NEC admixed with E-NENs treatment is dependent on histological sub- an adenocarcinoma or squamous cell carcinoma (with type, grade and stage, but the most effective thera- the two components histologically and immunohisto- peutic algorithm has not been established. Guidelines chemically distinguishable). Less than 10 cases of Mi- suggest, in analogy with lung small cell neuroendo- NEN composed of NEC and squamous cell carcino- crine carcinoma, similar therapeutic protocols includ- ma of the esophagus are reported in the international ing chemotherapy, radiotherapy and surgery 7. Re- literature as case reports 10-11. cently, a retrospective study analyzing 250 stage I-III E-NEN patients, suggests that patients treated with surgical resection plus chemo and/or radiotherapy Immunohistochemical and molecular show a better 2 year survival than patients without markers surgical resection of the primary tumour 8. Radical surgery alone provides limited benefits. Neuroendo- Synaptophysin, chromogranin A, and CD56 immuno- crine carcinoma of the esophagus has a worse prog- histochemical stains are used to demonstrate neu- nosis in comparison to esophageal adenocarcinoma roendocrine differentiation and positivity for these and squamous cell carcinoma 5. markers varies between 60% and 100% of cases with chromogranin A expression usually focal and/or faint in E-NECs. Histological features Hormones can be expressed by neoplastic cells in- cluding serotonin, glucagon and gastrin, but in gen- Definition and terminology eral in NET. E-NENs are defined as epithelial neoplasms with pre- Alcian blu-PAS and cytocheratin 7 can be used to dominant neuroendocrine differentiation 7. In this cat- demonstrate and quantify the adenocarcinomatous egory the following are included: esophageal well dif- component in MiNEN, while p63, p40 and cytokeratin ESOPHAGEAL AND GASTRIC NEN 7

Figure 1. (A) Esophageal small cell neuroendocrine carcinoma undermining normal squamous esophageal mucosa; H&E, magnification 4x. (B) Small cell neuroendocrine carcinoma infiltrating the muscular wall of the esophagus; H&E, magnifi- cation 10x. (C) Diffuse positivity of neoplastic cells for synaptophysin, magnification 20x. (D) Faint, dot-like, positivity for Chromogranin A; magnification 20x. (E) High proliferative index, 80% (Ki-67 stain), magnification 20x.

5/6 are used to confirm the squamous component. Neuroendocrine neoplasms of the stomach No significant data on molecular biology of these tu- mors is available. Introduction

Differential diagnosis NENs of the stomach include small indolent NETs and highly aggressive NECs. They account for about The main differential diagnosis for E-NENs, in particu- 4% of all NENs with an estimated annual incidence lar E-NECs, is with metastases from lung small cell of 0.4 cases/100,000 persons in both USA and Eu- NEC. Clinical history and imaging are of particular ef- rope 12-13. Rare MiNENs are also observed. fort in this setting, since transcription factor immuno- histochemistry is not useful. Gastric neuroendocrine tumors (NETs) Importantly, MiNENs have to been distinguished from carcinoma with focal neuroendocrine differentiation ECL-cell NETs applying two main diagnostic criteria: the recognition Most gastric NETs are composed of ECL-cells and of structural features of neuroendocrine morphology are located in the corpus-fundus (oxyntic) mucosa (i.e. organoid or solid growth pattern) and the exten- (Tab. I). They are commonly separated into three dif- sion of each component (at least 30%). ferent clinical-pathological subtypes with prognostic- 8 L. Mastracci, et al.

Table I. Clinical-pathological features of gastric NENs. M:F Hyper- Acid Peritumoral ECL-cell 5-year % Grading Metastasis ratio gastrinemia secretion mucosa proliferations survival NET About 60%

ECL-cell 1:2.5 80-90% of Ye s Low or Atrophic Ye s -G1 1-3% about Type 1 NET absent gastritis -G2, rare 100% -G3, exceptional ECL-cell 1:1 5-7% of Ye s High Hypertrophic Ye s -G1 10-30% 60-90% Type 2 NET gastropathy -G2, rare Type 3 2.8:1 10-15% of No Normal No specific No -G1, rare 50% <50% NET change -G2 -G3, rare Provisional Unknown Unknown yes Low or Parietal cell Ye s Unknown Unknown Unknown ECL-cell absent hypertrophy Type 4 Provisional 1.7:1 Unknown yes Unknown PPI effects Ye s -G1 15-17% about ECL-cell 100% Type 5 G-cell Unknown 5% of NET Possible Norma or Normal No -G1 Unknown about high or chronic 100% gastritis D-cell Unknown Unknown No Normal Normal No -G1 Unknown about or chronic -G2 100% gastritis -G3, rare EC-cell Unknown Unknown No Normal Normal No -G1 Unknown Unknown or chronic -G2 gastritis -G3, rare

NEC 1 2.1 6-20% No Normal Chronic No -G3 70% 10% gastritis

MiNEN 2

ADC/SCC- 2:1 20% No Normal Chronic No see 2 55% 10% NEC gastritis ADC/SCC- Unknown Unknown No Normal Chronic No see 2 Unknown Unknown NET gastritis NET: neuroendocrine tumor; NEC: neuroendocrine carcinoma; MiNEN: mixed neuroendocrine-non-neuroendocrine neoplasm; ADC: adenocarcinoma; SCC: squamous cell carcinoma; M: male; F: female; PPI: proton pump inhibitor. 1: small and large cell types; 2: both the neuroendocrine and the non-neuroendo- crine (adenocarcinoma and/or squamous cell carcinoma) components are graded according to WHO 2019. significance, gastrin-dependent type 1 and type 2 of Type 1 proven ECL-cell nature and gastrin independent type Type 1 ECL-cell NETs are the most common type, 14-17 3 of less obvious cell type characterization . arise in a background of autoimmune chronic atrophic All gastric NETs are graded into three proliferative gastritis associated with reduction/lack of acid secre- grades, according to WHO 2019 correlating with prog- tion and consequent antral G-cells hyperplasia and nosis 16-17. The best prognostic stratification of patients hypergastrinemia. Patients are often female and may is obtained by coupling the above clinico-pathological classification and the WHO tumor proliferative grade 18. have autoantibodies against intrinsic factor and/or pa- All gastric NETs are characterized by the expression rietal cells. Due to the impaired absorption of vitamin of chromogranin A, synaptophysin and somatostatin B12, a sub-group of patients may also present macro- receptor type 2A. ECL-cell NETs are also positive for cytic anemia. VMAT2 and histidine decarboxylase (HDC). A few Tumors are generally multiple, small (< 1 cm), limited scattered cells may be immunoreactive for serotonin, to the mucosa or submucosa and located in the cor- ghrelin, somatostatin, and alpha-hCG. pus-fundus. Only larger tumors (> 1 cm) may infiltrate ESOPHAGEAL AND GASTRIC NEN 9

Figure 2. Type 1 ECL-cell NET is composed of well-differentiated cells arranged in small microlobular and/or trabecular structures (A, bottom). The peritumoral oxyntic mucosa (A, top) is atrophic with diffuse intestinal metaplasia and shows ECL- cell linear and micronodular hyperplasia, easily detected with chromogranin A immunostaining (B). Type 3 NETs is composed of well differentiated neuroendocrine cells as well, deeply invading the gastric wall (C, right). Peritumoral oxyntic mucosa is normal (C, left) without ECL-cell proliferations (D).

the muscularis propria or beyond. They are composed Type 2 of well-differentiated cells with monomorphic nuclei, in- Type 2 ECL-cell NETs account for about 5-7% of ECL- conspicuous nucleoli, and fairly abundant eosinophilic cell NETs and are observed in patients with MEN1 cytoplasm, arranged in small microlobular and/or tra- syndrome, without gender predilection. Tumors are becular structures (Fig. 2A). Mitotic activity is absent generally multiple, measure < 2 cm and arise in hy- or low and necrosis is lacking. Most cases are G1, but pertrophic-hypersecretory oxyntic mucosa due to gas- G2 and exceptional G3 cases have been described 17. trin stimulation. Hyperplastic and dysplastic ECL-cell The surrounding oxyntic mucosa is atrophic, showing proliferations are observed in the hypertrophic peritu- moral mucosa. Hypergastrinemia is caused by a du- ECL-cell hyperplasia and (Fig. 2A-B). In the odenal or, more rarely, a pancreatic gastrinoma. Pa- antral mucosa G-cells hyperplasia is the rule. tients with sporadic gastrinomas do not develop ECL- Patients with type 1 ECL-cell NETs have an excellent cell NETs 19, suggesting the MEN1 defect is required prognosis with a 10-year survival rate of more than for ECL-cell transformation upon gastrin stimulus 20. 90%. No significant difference for patient outcome was found between G1 and G2 cases, suggesting tu- Type 3 mor grade may not be the most important predictor in Type 3 NETs were traditionally considered ECL-cell this subgroup 17. The risk for lymph node metastasis NETs. However, since histamine production or typical correlates with tumor size and deep wall invasion 17. “ECL-type” secretory granules were not demonstrat- 10 L. Mastracci, et al.

ed in all cases, the designation ECL-cell has been re- strated a high incidence of mutation of the ATP4A moved in the last WHO classification 21. Type 3 NETs proton pump gene 26. Although this heritable condition account for about 10-15% of gastric NETs and are defined a specific clinical profile (younger age of on- more frequent in males than in females. Patients do not set, higher and iron deficiency anemia), associate with hypergastrinemia and/or chronic auto- it represents a proof of concept that gastrin depend- immune atrophic gastritis and may present symptoms ent ECL-cell NETs may result from the combination related to tumor growth or metastatic dissemination. of both epigenetic and predisposing genetic factors, Type 3 NETs are solitary and large lesions composed either known (MEN1 syndrome genetic changes) or of well differentiated neuroendocrine cells frequently presently unknown. This possibility requires careful invading the muscularis propria and the sub-serosa investigation, especially concerning provisional types. (Fig. 2C). Lymph node and distant metastases are not rare. Most cases are G1 or G2, but G3 NETs were Other gastric NETs described 16. The diagnosis on small biopsy may be G-cell, D-cell, and EC-cell NETs are rare (about 5% a challenge and is achieved by evaluating the peritu- of all gastric NENs) and usually non-functioning. Gas- moral mucosa which is usually normal or with minimal trin-producing G-cell NETs are generally small, locat- gastritis (Fig. 2C-D). ed in proximity of the pylorus and are composed of The prognosis of type 3 NETs depends on grade and well-differentiated neuroendocrine cells forming thin stage with a 10-year disease-specific survival of about trabeculae or gyriform structures. Neoplastic cells are 50% 16-17. positive for neuroendocrine markers, somatostatin

receptor (subtype 2A), and gastrin. Somatostatin-pro- Provisional types ducing D-cell NETs are extremely rare and located in Two additional types have recently been proposed, the antrum. They are composed of well-differentiated which are worth being considered while awaiting full monomorphic cells positive for chromogranin A, syn- clinico-pathologic and/or biochemical characteriza- aptophysin and somatostatin. In general, both G-cell tion. and D-cell NETs are indolent with excellent prognosis, Provisional Type 4 ECL-cell NETs were described in even in the presence of deep wall invasion or lymph association with achlorhydria, parietal cell hyperpla- node metastasis 13. EC-cell NETs may arise in any sia and hypergastrinemia. The gastric mucosa shows part of the stomach and are composed of well-differ- dilated oxyntic glands with inspissated secretory lumi- entiated cells with intense eosinophilic cytoplasm in nal material and parietal cells with often vacuolated nests with a peripheral palisading. Tumor cells are abundant eosinophilic cytoplasm 22-23. The pathogen- positive for general neuroendocrine markers, seroto- esis was linked to inappropriate acid secretion from nin, CDX2, and somatostatin receptor subtype 2A. functional defective parietal cells with mutated gas- tric proton pump α-subunit, antral-G cell hyperplasia Gastric neuroendocrine carcinoma (NEC) and hypergastrinemia. Only two cases were carefully Gastric NECs account for about 6-21% of gastric documented, one of which with lymph node metasta- NENs and usually arise in the antral or cardial regions. ses 23. Males are more frequently affected (male/female ra- Provisional Type 5 ECL-cell NETs arising in moderate tio of 2:1) with 65 years (range 41-76 years). Patients hypergastrinemic patients treated continuously for a generally present non-specific symptoms due to lo- long time (at least one year) with PPI, without auto- cal growth or metastatic dissemination. Usually large immune chronic atrophic gastritis, gastrinoma, and lesions, gastric NECs are small and large cell types MEN1 syndrome have recently been described 24. at histology. Neoplastic cells are positive for synap- Since these tumors are associated with a normal or tophysin while chromogranin A is either faint or ex- slightly hyperplastic peri-tumoral oxyntic mucosa, pressed with a peripheral or para-nuclear dot-like pat- they may mimic in part type 3 NETs, from which they tern. NECs may also express nuclear TTF1 or CDX2. need to be differentiated due to the different progno- Patients’ prognosis is dismal prognosis with survival sis 25. Indeed, this ECL-cell NET subtype shows a usually measured in months. better behavior that type 3 NET not requiring an ag- gressive surgical approach. Tumors are slightly more Gastric mixed neuroendocrine-non neuroendocrine frequent in males. Lymph node metastases have been neoplasm (MiNEN) described in about 15% of patients, but prognosis is MiNEN are usually large, polypoid, ulcerating or sten- excellent. otic lesions. The neuroendocrine component is often a Commentary on provisional types: a report on a famil- NEC (small or large cell subtypes) and only rarely a ial cluster of classical Type 1 ECL-cell tumors demon- NET. The non-neuroendocrine component is generally ESOPHAGEAL AND GASTRIC NEN 11

an adenocarcinoma or, rarely, a squamous cell carci- argyrophil carcinoid and the gastric neuroendocrine carcinoma: noma especially when in the cardial region. MiNENs a clinicopathologic study. 1993;104:994-1006. https://doi.org/10.1016/0016-5085(93)90266-f composed by adenocarcinoma and NEC (MANEC) ac- 15 count for about 20% of all digestive MiNENs 27. Patients Rindi G, Azzoni C, La Rosa S, et al. ECL cell tumor and poor- ly differentiated endocrine carcinoma of the stomach: prog- have a poor prognosis and the Ki67 proliferative index nostic evaluation by pathological analysis. Gastroenterology of the NEC component correlates with prognosis 27. 1999;116:532-42. https://doi.org/10.1016/s0016-5085(99)70174- Rare cases of associated with NET have al- 5 so been reported 28. 16 La Rosa S, Inzani F, Vanoli A, et al. Histologic characterization and improved prognostic evaluation of 209 gastric neuroen- docrine neoplasms. Hum Pathol 2011;42:1373-84. https://doi. 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Three subtypes of gastric https://doi.org/10.1097/PAS.0000000000001123 PATHOLOGICA 2021;113:12-18; DOI: 10.32074/1591-951X-228

Review

Neuroendocrine neoplasms of the duodenum, ampullary region, jejunum and ileum

Massimo Milione1, Paola Parente2, Federica Grillo3,4, Giuseppe Zamboni5, Luca Mastracci3,4, Carlo Capella6, Matteo Fassan7, Alessandro Vanoli8 1 1st Pathology Division, Department of Pathology and Laboratory Medicine Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; 2 Unit of Pathology, Fondazione IRCCS Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo, FG, Italy; 3 Anatomic Pathology, Department of Surgical Sciences and Integrated Diagnostics, University of Genova, Italy; 4 Ospedale Policlinico San Martino IRCCS, Genova, Italy; 5 Department of Diagnostic and Public Health, Section of Pathology, University of Verona, Verona, Italy; 6 Department of Medicine and Surgery, University of Insubria, Varese, Italy; 7 Surgical Pathology and Cytopathology Unit, Department of Medicine (DIMED), University of Padova, Padova, Italy; 8 Anatomic Pathology Unit, Department of Molecular Medicine, University of Pavia and Fondazione IRCCS San Matteo Hospital, Pavia, Italy

Summary Neuroendocrine neoplasms of the small intestine are some of the most frequently occur- ring along the gastrointestinal tract, even though their incidence is extremely variable according to specific sites. Jejunal-ileal neuroendocrine neoplasms account for about 27% of gastrointestinal NETs making them the second most frequent NET type. The aim of this Received: November 21, 2020 review is to classify all tumors following the WHO 2019 classification and to describe their Accepted: November 23, 2020 pathologic differences and peculiarities.

Correspondence Key words: ampulla, MiNEN, NEC, NET, neuroendocrine neoplasms, small intestine, Alessandro Vanoli Jejunal-ileal neuroendocrine neoplasms Anatomic Pathology Unit, Department of Molecular Medicine, University of Pavia, and IRCCS San Matteo Hospital, via Carlo Forlani- ni 16, 27100 Pavia, Italy Neuroendocrine neoplasms of the duodenum Tel. + 0382 523056 and ampullary region Fax: +0382 525866 E-mail: [email protected]

Conflict of interest Introduction The Authors declare no conflict of interest. Duodenal neuroendocrine neoplasms (Duo-NENs) are uncommon, How to cite this article: Milione M, Parente accounting for about 4% of all gastroenteropancreatic neuroendocrine P, Grillo F, et al. Neuroendocrine neoplasms of neoplasms (GEP-NENs) 1,2. They include ampullary NENs, which arise the duodenum, ampullary region, jejunum and within or around the major or minor papilla/ampulla and extra-ampullary ileum. Pathologica 2021;113:12-18. https:// doi.org/10.32074/1591-951X-228 NENs. Their incidence is increasing, likely due to improved diagnostic techniques. © Copyright by Società Italiana di Anatomia Pato- logica e Citopatologia Diagnostica, Divisione Itali- ana della International Academy of Pathology Clinical presentation OPEN ACCESS Patients with Duo-NENs may present with abdominal pain, jaundice, This is an open access journal distributed in accordance 3 with the CC-BY-NC-ND (Creative Commons Attribution- bleeding or anemia ; however, many nonfunctioning neuroendocrine NonCommercial-NoDerivatives 4.0 International) license: the tumors (NETs) are discovered incidentally. Zollinger-Ellison syndrome work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original due to a gastrinoma may occur, sometimes in the setting of multiple en- version. For further information: https://creativecommons. docrine neoplasia type 1 (MEN1) syndrome, whereas somatostatinoma org/licenses/by-nc-nd/4.0/deed.en or carcinoid syndrome are extremely rare. DUODENAL, AMPULLARY AND JEJUNO-ILEAL NENS 13

Subtypes with diffuse hyperplastic gastrin and somatostatin cell changes and multicentric gastrin-producing They include well-differentiated duodenal NETs (Duo- micro-NETs 5. Despite their usually small size (0.7- NETs), gangliocytic paragangliomas, poorly differenti- 0.8 cm), gastrinomas are more frequently associat- ated neuroendocrine carcinomas (NECs) and mixed ed with lymph node metastases in comparison with neuroendocrine-non-neuroendocrine neoplasms non-functioning gastrin-expressing Duo-NETs 6. (MiNENs). b Ampullary-type somatostatin-producing NETs (AS-NETs), also known as “somatostatinoma”, de- Duo-NETs spite the frequent lack of an associated hyperfunc- Duo-NETs are graded according to the WHO prolif- tioning clinical syndrome. They are characterized erative criteria as G1, G2 and G3; most Duo-NETs by a more or less prominent tubulo-acinar/pseu- (66-80%) are low-grade (G1) tumors, while grade 3 doglandular pattern of growth, often with psam- NETs are very rare. Three main clinico-pathologic subtypes of Duo-NETs moma bodies, and extensive (more than 50% of have been described 4 (Tab. I): tumor cells) somatostatin reactivity (Fig. 2). AS- a Gastrinoma (i.e. functioning gastrin-producing NETs represent the most common histologic sub- NETs). This tumor subtype is, by definition, asso- type among NETs of the major and minor papilla/ 4,7 ciated with Zollinger-Ellison syndrome and char- ampulla regions ; they can, however, be occa- acterized by gastrin expression of neoplastic cells. sionally found in the extra-ampullary duodenum. Duodenal gastrinomas usually show a well-defined A fraction of such neoplasms occur in patients trabecular pattern, with frequent vascular pseu- with neurofibromatosis type 1 and they show a dorosettes (Fig. 1). About 30% of duodenal gas- biallelic inactivation of NF1 gene 8. In addition to trinomas arise in patients with MEN1 syndrome; reactivity for general neuroendocrine markers and MEN1-associated gastrinomas are often coupled somatostatin, AS-NETs are, as a rule, positive for

Table I. Features of the main histologic subtypes of duodenal neuroendocrine neoplasms. Ampullary-type Ordinary somatostatin- non-functioning Gangliocytic Neuroendocrine Gastrinoma producing neuroendocrine paraganglioma carcinoma neuroendocrine tumor tumor Age at diagnosis 5th decade 5th decade 6th decade 6th decade 7th decade Hyperfunctional syndrome Always Rare No No Rare (Zollinger-Ellison (somatostatinoma (by definition) (paraneoplastic syndrome) syndrome) syndromes) Predisposing hereditary Multiple Neurofibromatosis Multiple endocrine Neurofibromatosis No syndromes endocrine type 1 (>10%), neoplasia type 1 (rare) type 1 (rare) neoplasia type 1 Pacak-Zhuang (less than 50%) syndrome (rare) Preferential location First duodenal Major/minor First duodenal portion Major/minor ampullary Major ampulla portion ampullary region region region Predominant histologic Trabecular Tubulo-acinar/ Nested/trabecular Triphasic: Solid/diffuse (poorly pattern glandular paraganglioid+spindle differentiated) cells+ganglion-like cells Extensive (> 50%) No Yes (by definition) Rare Frequent No/very rare somatostatin expression Gastrin expression Yes (by Rare (few cells) Frequent No No/rare cells definition) Pancreatic polypeptide No/rare cells No/rare cells No/rare cells Frequent No/rare cells expression Type 2A somatostatin Frequent Rare Frequent Frequent Rare receptor expression Size > 1 cm Rare Frequent Rare Frequent Frequent Lymph node metastasis Frequent Frequent Uncommon Uncommon Very frequent Distant metastasis Rare Rare Rare Extremely rare Frequent Prognosis (after resection) Good Good Good Good Bad 14 M. Milione et al.

A B

Figure 1. A duodenal gastrinoma, showing a trabecular pattern, with vascular pseudorosettes (A, hematoxylin and eosin), and tumor cell reactivity for gastrin (B, gastrin immunostaining). Note also the presence of gastrin-positive cells in normal duodenal mucosa overlying the neoplasm.

A B

Figure 2. An ampullary-type somatostatin-producing neuroendocrine tumor, showing tubulo-acinar structures with psam- moma bodies in a solid/trabecular architectural background (A, hematoxylin and eosin), and extensive somatostatin expres- sion by tumor cells (B, somatostatin immunostaining).

MUC1/EMA, frequently reactive for cytokeratin 7 ma bodies, and negativity (or only focal positivity) and negative for type 2A somatostatin receptors 4. for general neuroendocrine markers and soma- Although AS-NETs are significantly larger (me- tostatin. It should also be recalled that duodenal dian size: 1.8-2.5 cm) and have a higher lymph NECs and gangliocytic paragangliomas may also node metastatic rate (about 50% of cases) than express somatostatin; however, their cellular and ordinary non-functioning, mostly extra-ampullary, architectural features allow a straightforward dis- Duo-NETs, they display an indolent behavior, even tinction from AS-NETs. when metastatic to the liver. Differential diagnosis c Ordinary non-functioning NETs. The remaining with duodenal or ampullary is Duo-NETs showing the “canonical” NET orga- therefore of utmost clinical importance. In contrast noid architecture (nests, trabeculae, ribbons) and, to AS-NETs, adenocarcinomas show higher nucle- in addition to general neuroendocrine markers, ar atypia and mitotic activity, absence of psammo- variable expression of gastrin or, less frequent- DUODENAL, AMPULLARY AND JEJUNO-ILEAL NENS 15

ly, somatostatin 2 account for the vast majority and invasive masses (median size: 2.5 cm). They may of extra-ampullary Duo-NETs. Gastrin-produc- be separated histologically in two variants: small cell ing Duo-NETs are more frequently detected in NECs and large cell NECs. Duodenal NECs, regard- the first portion of the duodenum. Worthy of note less of histologic variant, are generally associated is that enterochromaffin-cell serotonin-express- with an advanced stage and a worse prognosis 4,12. ing NETs are exceptionally rare in the duode- More than half of ampullary NECs show loss of Ret- num, in comparison with the jejunum or ileum. inoblastoma (RB1) expression, which may be helpful In Duo-NETs, risk factors for lymph node metasta- to support the diagnosis of NEC (versus a NET G3) in sis encompass tumor size, invasion of muscularis challenging cases, while p53 overexpression occurs propria or beyond, lymphovascular invasion, and in about 30% of cases 12. grade (2 or 3), while independent prognostic fac- tors include tumor stage, tumor size (patients with Duodenal MiNENs tumors of 2 cm in diameter or larger have worse Few ampullary MiNENs, composed of a NEC compo- outcome) and lymphovascular invasion 4. nent combined with an adenocarcinoma component, each of which accounting for at least 30% of neoplas- Gangliocytic paragangliomas tic growth, have been described and most display ag- Gangliocytic paraganglioma represents a rare and gressive behavior 12. distinct tumor type, which is almost always located in the ampullary region. It is characterized by a triphasic morphology, i.e. i) an epithelioid, paraganglioma-like Neuroendocrine neoplasms neuroendocrine component (reactive for general neu- of the jejunum and ileum roendocrine markers and, frequently, for cytokeratins, somatostatin, pancreatic polypeptide and progester- one receptors), ii) a Shwannian-like spindle cell com- Introduction ponent (reactive for S100 protein and SOX10 and often for synaptophysin), and iii) a ganglion-like cell Jejunal-ileal neuroendocrine neoplasms (Je-Ile component (reactive for synaptophysin and, some- NENs) are almost exclusively represented by well dif- times, for somatostatin, S100 or cytokeratins) 4,9,10. ferentiated serotonin producing enterochromaffin cell The three components may be variably intermingled. neuroendocrine tumors (EC cell-NETs) of the terminal Despite their often pseudo-infiltrative pattern, gangli- ileum. They account for about 27% of gastrointestinal ocytic paraganglioma is considered a very-low-grade NETs making them the second most frequent NET tumor, with uncommon metastases, essentially to lo- type 13. The remaining Je-Ile NENs are mostly repre- co-regional lymph nodes. It should be mainly distin- sented by NETs producing gastrin (especially in the guished from Duo-NETs, especially from AS-NETs, jejunum) 14. Poorly differentiated NECs and MiNENs which display a greater metastatic potential, and from represent rare entities. true paragangliomas, gastrointestinal stromal tumors and ganglioneuroma. In addition to the typical tripha- sic histology, immunohistochemistry for progesterone Clinical presentation receptor and pancreatic polypeptide may help distin- guish gangliocytic paraganglioma from Duo-NET 9. About half of Je-Ile EC cell NET patients are asymp- Recently, Mamilla et al conclude that gangliocytic par- tomatic and their tumors are incidentally detected. agangliomas have a NET-like 9 immunoprofile but dif- Patients can be asymptomatic even if they may show fer from ordinary paragangliomas, almost all of which high serum neuroendocrine markers, urinary 5-hy- are cytokeratin-negative10. Gastrointestinal stromal droxyindoleacetic acid (5-HIAA) and liver metastases. tumors have a different immunophenotype, while gan- Primary tumor identification, in presence of liver me- glioneuroma lacks the epithelioid component. tastases may be difficult due to small size of primary tumor, limitations of endoscopy and standard imag- Duodenal NECs ing techniques. Cases with symptoms present with They are by definition high-grade NENs. Histological- crampy abdominal pain, due to intestinal obstruction ly, NECs are arranged in poorly formed trabeculae, and/or ischemia. The “carcinoid syndrome”, character- large and confluent nests or sheet-like growths, sim- ized by cutaneous flushing, diarrhea, bronchospasms ilar to those described in the lung or remaining gas- and fibrous thickening of endocardium and valves of troenteropancreatic tract. Most duodenal NECs arise right heart, occurs only when liver metastases are around the major ampulla 11,12, where they form large present and is detected in at most 10% patients. 16 M. Milione et al.

Subtypes ly located in the distal ileum, only 11% in the jejunum and rarely they are found in Meckel’s diverticulum. EC These are represented by WD Je-Ile NET, NECs and cell NETs are multiple (2-100 tumors) in about one MiNENs. third of cases and in familial cases. Tumor size is usu- ally small, ranging from 1 to 2 cm. These NETs ap- 1 Je-Ile NETs pear as firm white-yellow mucosal-submucosal nod- Je-Ile NETs are graded according to the WHO prolif- ules with intact or minimally eroded overlying mucosa. erative criteria as G1, G2 and G3. Most Je-Ile NETs Muscular wall and peritoneal infiltration is frequent and are low grade; grade 3 NETs are rare. Two Je-Ile NET consists either of extensive peritoneal fibrosis caused clinico-pathologic subtypes have been reported: by fibroblastic growth factors produced by the tumor or by metastatic lymph nodes fused together. EC cell a EC cell NETs NETs are composed of solid rounded nests (Fig. 3A) Pathology and Immunohistochemistry: they are most- of closely packed tumor cells, often showing peripher-

A B

C D

Figure 3. EC-cell ileal well differentiated neuroendocrine tumor. (A) The tumor cells are arranged in rounded solid nests. (B) Diffuse immunoreactivity of tumor cells for Chromogranin A with peculiar basal reinforcement (arrows). (C) Immunostain for serotonin confirms the diagnosis of EC cell NET. (D) Rare nuclei are positive for Ki-67. DUODENAL, AMPULLARY AND JEJUNO-ILEAL NENS 17

al reinforcement with eosinophilic secretory granules. related to the very low proliferative rate of these tumors. Cribriform, glandular-like and rosette-type structures The 5-year overall survival rate of patients with local- are also frequently observed. Tumor cells are uniform ized disease is 70-100% while that of patients with dis- with little or no pleomorphism and mitotic activity is tant metastases is 35-60%. Long term recurrence rate null or low in most of the cases (0 to 2/2 mm2), which is roughly 50% 20. The risk of recurrence is increased in classifies these tumors as G1. Lymphovascular and patients with nodal metastases, mesenteric invasion, perineural invasion are frequently observed. In addi- lympho-vascular invasion and perineural invasion. tion to reactivity for general neuroendocrine markers (Fig. 3B) and serotonin (Fig. 3C), EC-cell NETs ex- b Heterogeneous group of Je-Ile NETs press CDX2 and type 2A somatostatin receptors. This group comprises the subgroup of trabecular Ki-67 proliferative index is very low (0-2%) (Fig. 3D) in G1 NETs expressing gastrin, located in the jejunum, most cases classified as G1 but may be more than 2% sharing the same general behavior as their duodenal in some that are classified as G2 and more than 20% counterpart, and a second subgroup represented by in few cases classified as G3. jejunal non-hormone expressing NETs, most of large Molecular findings: genetically Je-Ile EC cell NETs size and locally invasive and frequently of G2 or G3 are characterized by lack of changes in K-Ras, p53 grade, located in the upper jejunum 14. and DNA mismatch repair, frequent whole chromo- some/whole arm losses, low mutation rate in the ge- 2 Je-Ile NECs nome and high percentage of epigenetic changes. They are by definition high-grade malignant Je-Ile Various studies reported loss of one copy of chromo- NENs and show poorly formed trabeculae, large and some 18, with a percentage of 44-100%15; however confluent nests or sheet-like growths similar to those there is no definitive evidence for driver genes in the previously described. Very few cases of this neoplasm regions involved. Gain of chromosome 14 is mainly have been reported 21. detected in progressed and metastatic lesions and indicates an unfavorable prognosis 15. The most fre- 3 Je-Ile MiNEN quent genetic mutation affecting CDKN1B (in ~8% of As far as we know no well described cases of Je-Ile tumors) has no immediate detectable impact on clini- MiNEN have been reported so far 22. cal phenotype or outcome. Compared to genetic mu- tations, epigenetic alterations are more frequent and References recurrent in EC cell NETs. More than half EC-cell NET 1 display CPG island methylation phenotype and sig- WHO Classification of Tumors Editorial Board. Digestive system tumors. WHO Classification of Tumors. 5th ed. Lyon: IARC press 16 nificant epigenetic dysregulation . Based on an inte- 2019. grated genomic analysis, including DNA methylation, 2 Jensen RT. Rindi G, Arnold R, et al. Well-differentiated duodenal gene expression and copy number variance (CNV), tumor/carcinoma (excluding gastrinomas). Neuroendocrinology. three molecular subgroups associated with significant 2006;84:165-72. https://doi.org/10.1159/000098008 difference in progression free survival after surgical 3 Rossi RE, Rausa E, Cavalcoli F, et al. Duodenal neuroendocrine resection have been identified 16. No established risk neoplasms: a still poorly recognized clinical entity. Scand J Gas- factors have been identified for sporadic Je-Ile EC-cell troenterol 2018;53:835-42. https://doi.org/10.1080/00365521.201 8.1468479 NET. Familial cases of Je-Ile EC-cell NETs, represent- 4 Vanoli, A, La Rosa, S, Klersy, C, et al. Four neuroendocrine ing about 3% of patients harboring Je-Ile NETs are tumor types and neuroendocrine carcinoma of the duodenum: distinguishable from sporadic cases for their multiple analysis of 203 cases. Neuroendocrinology 2017;104:112-25. synchronous tumors 17. The IPMK gene was found to https://doi.org/10.1159/000444803 be mutated in only one family compared to 32 oth- 5 Anlauf M, Perren A, Meyer CL, et al. Precursor lesions in patients er families with multiple EC cell Je-Ile NETs showing with multiple endocrine neoplasia type 1-associated duodenal wild type IPMK gene. gastrinomas. Gastroenterology 2005;128:1187-98. https://doi. org/10.1053/j.gastro.2005.01.058 Prognostic factors: well differentiated Je-Ile NETs, 6 Rosentraeger MJ, Garbrecht N, Anlauf M, et al. Syndromic ver- which typically include G1 tumors but rarely may be G2 sus non-syndromic sporadic gastrin-producing neuroendocrine or G3 NETs, must be staged according to the UICC tumors of the duodenum: Comparison of pathological features staging system (TNM classification of malignant tum- and biological behaviour. Virchows Arch 2016;468:277-87. https:// ors eight edition) 18. The majority (> 60%) of patients doi.org/10.1007/s00428-015-1890-9 with EC cell Je-Ile NETs present with metastatic dis- 7 Vanoli A, Albarello L, Uncini S, et al. Neuroendocrine tumors ease. Metastases are principally located in regional (NETs) of the minor papilla/ampulla: analysis of 16 cases under- lines homology with major ampulla NETs and differences from 19 lymph nodes and the liver . Notwithstanding this, pa- extra-ampullary duodenal NETs. Am J Surg Pathol 2019;43:725- tients with advanced disease show prolonged survival 36. https://doi.org/10.1097/PAS.0000000000001234 18 M. Milione et al.

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Review

Neuroendocrine neoplasms of the appendix, colon and rectum

Marco Volante*1,2, Federica Grillo*3,4, Federica Massa2, Francesca Maletta5, Luca Mastracci3,4, Michela Campora3, Jacopo Ferro3, Alessandro Vanoli6, Mauro Papotti1,5 1 Department of Oncology, University of Turin, Orbassano, Turin, Italy; 2 Pathology Unit, AOU San Luigi Gonzaga, Orbassano, Turin, Italy; 3 Anatomic Pathology, Department of Surgical Sciences and Integrated Diagnostics, University of Genova, Italy; 4 Ospedale Policlinico San Martino IRCCS, Genova, Italy; 5 Pathology Unit, “Città della Salute e della Scienza” Hospital, Turin, Italy; 6 Anatomic Pathology Unit, Department of Molecular Medicine, University of Pavia and Fondazione IRCCS San Matteo Hospital, Pavia, Italy *These two authors contributed equally

Summary Neuroendocrine neoplasms of the appendix, colon and rectum are classified according to the most recent WHO classification as neuroendocrine tumors (NET), neuroendocrine carcinomas (NEC) and mixed neuroendocrine-non neuroendocrine neoplasms (MiNENs). NECs and MiNENs are aggressive neoplasms requiring multimodal treatment strategies. By contrast, NETs are, in most cases, indolent lesions occurring as incidental findings in the appendix or as polyps in the rectum. While most appendiceal and rectal NETs are Received: November 21, 2020 considered relatively non-aggressive neoplasms, a few cases, may show a more aggres- Accepted: November 23, 2020 sive clinical course. Unfortunately, clinical/pathological characteristics to select patients at high risk of recurrence/metastases are poorly consolidated. Diagnosis is generally easy Correspondence and supported by the combination of morphology and immunohistochemistry. Differential Federica Grillo Anatomic Pathology, Department of Surgical diagnostic problems are for NECs/MiNENs with poorly differentiated adenocarcinomas, Sciences and Integrated Diagnostics, Uni- when immunohistochemical neuroendocrine markers are not obviously positive, whereas versity of Genova, Ospedale and Policlinico for NETs they are represented by the rare appendiceal tubular and clear cell variants San Martino IRCCS, largo Rosanna Benzi 10, (which may be confused with non-neuroendocrine cancers) and rectal L-cell tumors which 16132 Genova, Italy may be chromogranin negative and prostatic marker positive. Tel. +39 010 5555957 Fax: +39 010 5556392 Key words: appendix, colon, rectum, neuroendocrine, tumor E-mail: [email protected]

Conflict of interest The Authors declare no conflict of interest. Introduction

How to cite this article: Volante M, Grillo F, Massa F, et al. Neuroendocrine neoplasms Neuroendocrine neoplasms (NEN) of the appendix, colon and rectum of the appendix, colon and rectum. are primary epithelial neoplasms showing morphologic and immunophe- Pathologica 2021;113:19-27. https://doi. notypic signs of neuroendocrine differentiation. They are classified using org/10.32074/1591-951X-230 the WHO 2019 scheme, as neuroendocrine tumors (NET), neuroendo- © Copyright by Società Italiana di Anatomia Pato- crine carcinomas (NEC) and mixed neuroendocrine-non neuroendocrine logica e Citopatologia Diagnostica, Divisione Itali- 1 ana della International Academy of Pathology neoplasms (MiNEN) . Whereas NECs and MiNENs are aggressive ne- oplasms that are generally diagnosed at advanced stage and require OPEN ACCESS multimodal treatment strategies, NETs may be relatively indolent (such This is an open access journal distributed in accordance as in the appendix and rectum) or behave more aggressively (such as with the CC-BY-NC-ND (Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 International) license: the the colon). With regards to neuroendocrine neoplasms of the colon and work can be used by mentioning the author and the license, those of the rectum, though often grouped together, these neoplasms but only for non-commercial purposes and only in the original version. For further information: https://creativecommons. show vastly different incidences (though both are on the rise), geograph- org/licenses/by-nc-nd/4.0/deed.en ical distribution, treatment, histopathology and behavior. 20 M. Volante et al.

Neuroendocrine neoplasms and clinical behavior similar to those of the colon. of the appendix Specific data on appendiceal NECs and MiNENs, are, however, scarce. In a recent study, appendiceal Neuroendocrine neoplasms of the appendix are some MiNENs were associated with a worse prognosis of the most frequent neuroendocrine tumors which compared to pure NENs (including NECs) but better may be encountered by the practicing pathologist. than adenocarcinoma, but this difference was lost in Whereas NECs and MiNENs are aggressive neo- advanced stage disease 3. plasms that require extensive surgery if possible, ap- pendiceal NETs are mostly cured by appendectomy Subtypes alone. The parameters which identify the minority of Definition and classification appendiceal NET cases at risk for aggressive disease As also stated above, according to the most recent outcome are, however, not well-assessed, though WHO classification of gastrointestinal tumors, NENs they have an important impact in the definition of the in the appendix are subdivided into NETs, NECs and clinical management of patients, including indications MiNENs (Tab. I). Whereas NECs are high grade by for right hemicolectomy and/or medical therapy. Data definition, NETs are further subdivided into G1, G2 on prognostic parameters in appendiceal NETs are and G3 according to the mitotic index and Ki-67 pro- largely retrospective and frequently discordant, there- liferative index. WHO 2019 rules for grading gastroin- fore recommendations and guidelines are heteroge- testinal NENs are described elsewhere in this issue neous and in part controversial, as discussed below. and those in the appendix have no additional specific indications. Clinical presentation The incidence of appendiceal NENs is difficult to as- Pathology sess, due to heterogeneous inclusion criteria in epi- Appendiceal NETs (A-NETs) appear as whitish nod- demiological studies. NETs are by far more common ules at macroscopy. They may be either well demar- than NECs and MiNENs, and account for up to 70% cated or infiltrative, but may be easily missed at gross of all appendiceal neoplasms and represent the fifth specimen handling if of small size. It is therefore rec- most frequent gastrointestinal NET. They are a fre- ommended to embed the appendiceal tip in total, in all quent occurrence in appendectomy specimens, up to cases. Histologic findings are typical of well differenti- nearly 2% in a recent study. The great majority of ap- ated neuroendocrine lesions, with nests or trabeculae pendiceal NETs are discovered incidentally at the tip of uniform polygonal cells. Stromal fibrosis is frequent. of the appendix. When located in the mid or proximal Tumor borders can be well demarcated or show infil- portion, they may cause obstruction and appendicitis. trative growth often composed of isolated cells. Infil- The occurrence of functional tumors, characterized tration may be limited to the muscular wall of the ap- by the onset of carcinoid syndrome, is very rare and pendix or extend to the subserosa and/or the adipose associated with metastatic spread. The prognosis of tissue of the mesoappendix (Fig. 1). Perforation of the NETs is excellent with more than 90% survival proba- serosal surface or direct infiltration of the or bility at 10 years and an overall risk of metastases of other adjacent structures are rare. Perineural invasion < 10%. The clinical outcome is apparently even better is rare but vascular invasion is not infrequent; howev- in the pediatric population with extremely high survival er, when occurring in small thin-walled lymphatics or rates, irrespective of the presence of putative adverse capillaries, they should be distinguished from artifacts features and of type of surgical treatment 2. NECs and caused by shrinkage of tumor cell islets detaching MiNENs are aggressive neoplasms with a biological from the surrounding stroma. More than 80% of cases

Table I. Types of neuroendocrine neoplasms of the appendix. WHO 2019 histological types Grading groups Sub-types Hormone production NET G1 EC-cell type Serotonin G2 L-cell Type GLP1, other proglucagon peptides G3 NEC High grade by definition Small cell-type // Large cell-type MiNEN As for definition, each Mixed adenocarcinoma-NET // component to be graded Mixed adenocarcinoma-NEC independently NET: neuroendocrine tumor; NEC: neuroendocrine carcinoma; MiNEN: mixed neuroendocrine-non neuroendocrine neoplasms. APPENDICEAL AND COLORECTAL NENS 21

Figure 1. Neuroendocrine tumor of the appendix associated with acute appendicitis (a), with invasion of the mesoappendix (b), infiltrative growth, highlighted by chromogranin A immunohistochemical staining (c), but with low mitotic and prolifera- tive index (Ki-67 staining) coding for G1 tumor grade (d).

are G1 and G3 NETs are exceptional. Mitotic count is almost all cases, to an adenocarcinoma, of mucinous therefore usually very low and Ki-67 proliferation index or non-mucinous type. The neuroendocrine com- is less than 2% in the great majority of cases, inde- ponent can be either of the NET or NEC type, thus pendently of the depth of invasion of the tumor. Two defining the adenocarcinoma-NET and adenocarcino- major biological types are encountered, although they ma-NEC subtypes of appendiceal MiNENs, respec- do not possess distinct clinical behavior. The EC-cell tively. type, the most frequent and typical, produces seroto- It is important to remember that the so-called goblet nin, whereas the L-cell type, characterized by a more cell carcinoid, once classified as a mixed form of ap- prominent trabecular arrangement, produces GLP-1 pendiceal NENs, is now included into the group of ap- or other proglucagon-derived peptides. pendiceal adenocarcinomas and will not be discussed NECs are morphologically identical to their colonic in this brief overview. counterpart and are subdivided in small and large Staging. According to AJCC/UICC/WHO staging sys- cell types, although specific literature descriptions of tems 1, NECs and MiNENs are staged as for adeno- appendiceal NECs are missing due to their extreme carcinomas. A-NETs are staged based on the size rarity. and depth of invasion into: MiNENs are mixed neoplasms showing, by morphol- T1: ≤ 2 cm; ogy and immunohistochemistry, both neuroendocrine T2: > 2 and ≤ 4 cm; and non-neuroendocrine components, and each com- T3: > 4 cm or subserosal or mesoappendix invasion, ponent should be (arbitrarily) present in at least 30% irrespective of the size of the tumor; of the whole lesion and should be graded individually. T4: serosal perforation or direct invasion of adjacent The non-neuroendocrine component corresponds, in organs or structures. 22 M. Volante et al.

Figure 2. Neuroendocrine tumor of the appendix, clear cell variant, with clear granular cells (a) negative for PAS staining (b) and with diffuse granular positivity for chromogranin A (c) and nuclear positivity for CDX-2 (d).

Despite the majority of cases being of less than 2 cm to the identification of independent factors when test- in size, a relatively high proportion of cases are diag- ed at multivariable analysis. In a recent study, size nosed, even when incidental, as stage pT3 due to the > 15 mm, presence of lympho-vascular invasion and frequent occurrence of invasion outside the muscular G2 grade have been identified as independent indica- wall 4. tors for the presence of lymph node metastases 6, but Pathological variants. Variants of A-NETs include only the latter two were identified in a similar study 7 the tubular and the lipid-rich (clear cell) variant. The and neither tumor grade nor tumor size were associ- tubular variant is composed of cuboidal/columnar ated with disease-related survival in another study 8. cells with basally oriented nuclei arranged in small All these discrepancies reflect the high heterogene- glandular structures. They usually are of the L-cell by ity of study planning and case selection, which are type. The clear cell variant is characterized by nests the major biases of retrospective studies and are the of clear cells with a foamy cytoplasmic appearance, cause of different indications to surgical treatment in related to lipid accumulation 5 which does not stain for different national and international guidelines. Where- PAS (Fig. 2). Most cases are of the EC-cell type and as size > 2 cm is uniformly quoted as a strong indi- produce serotonin. cator, other parameters in cases ≤ 2 cm in size, such Prognostic parameters. Specific data on prognostic as location at the base of the appendix, R1 resec- parameters in NECs and MiNENs are very scarce and tion status, lymph-vascular invasion, invasion of the stage IV disease seems the only relevant parameter mesoappendix (including extension > 3 mm) and G2 to identify aggressive disease. The prognostic impact tumor grade, are heterogeneously considered 4 and of clinical and pathological parameters in NETs has recommendation for subsequent right hemicolectomy been more extensively investigated, but a great var- after appendectomy is to be discussed in an appro- iability of results is observed with special reference priate multidisciplinary setting. All these parameters APPENDICEAL AND COLORECTAL NENS 23

Table II. Essential and desirable parameters to be included in the pathological report*.

Essential parameters Additional parameters - WHO tumor type (NET, NEC, MiNEN) - NET Type (EC-cell, L-cell) If NEC, large cell or small cell type NET variant, if present - Mitotic index as absolute value (x2mm2)

- WHO tumor grade (G1, G2, G3) for NET - Ki-67 index as absolute value (%)

- Location (base, body, tip) - If present, extent of subserosa/mesoappendix invasion (mm)

- Size (mm) - In case of R0 resection, distance to the closest margin

- Depth of invasion (submucosa, muscolaris propria, subserosa, - In the presence of vascular invasion, describe if lymphatic or mesoappendix, adjacent structures) blood vessels and if intramural or extramural

- Visceral perforation (present/absent) - In the presence of perineurial invasion, describe if intramural or extramural and if close to the resection margins - Lymphovascular invasion (present/absent) - In the presence of positive lymph nodes, describe the presence - Perineural invasion (present/absent) or absence of extra nodal extension

- Lymph node status (# examined nodes and, if positive, # positive - Hormone or hormone receptor immunohistochemistry nodes)

- R status, and if R1 positive margin description (proximal, radial, mesenteric in case of appendectomy, proximal, mesenteric and distal in case of right hemicolectomy)

- pTNM stage (AJCC/WHO/UICC)

- Immunohistochemical markers performed and results (pan- neuroendocrine markers and Ki-67 mandatory) *: the table reflects authors’ opinion, only, and is partly based on the protocol for the examination of specimens from patients with neuroendocrine tumors of the appendix, College of American Pathologists, posting date February 2020; NET: neuroendocrine tumor; NEC: neuroendocrine carcinoma; MiNEN: mixed neuroendocrine-non-neuroendocrine neoplasms. therefore have to be mentioned in the pathology re- Molecular findings port (Tab. II) to provide the best description of each Specific molecular data in appendiceal NEC and MiN- individual case and serve as collective data for further ENs are not available. With regards to A-NETs, two re- clinical decision-making. cent papers analyzed small series of cases by means of next generation sequencing with different results. In Immunohistochemistry three cases, analyzed using a > 400 gene panel, no A-NETs are positive for conventional pan-neuroen- molecular alteration including mutations or copy num- docrine markers, such as chromogranin A and syn- ber variations, were identified 9, whereas in another aptophysin, although the former may be negative in study using targeted-next generation sequencing of L-cell type as for similar tumors in the rectal location. 50 genes, 4 of 5 cases presented more than 1 mu- In NECs, and the neuroendocrine component of Mi- tation, including TP53, PTEN, SMAD4 and EGFR 10. NEN, neuroendocrine markers may be negative or focally positive, therefore a panel – and not individual Differential diagnosis markers, only – should be used for an appropriate di- A-NETs are easily recognizable based on typical mor- agnostic procedure. Neuroendocrine phenotype-as- phological features of a well differentiated neuroendo- sociated transcription factors, such as INSM1, are crine neoplasm and appropriate immunohistochemistry also usually positive in both NETs and NECs. S100 easily confirm the diagnosis. Diagnostic pitfalls may be positive sustentacular cells are often present. Detec- represented by uncommon variants, such as the tubu- tion of hormone peptides or receptors (such as so- lar and clear cell types. The former may resemble an matostatin receptors) is not mandatory for diagnostic exocrine neoplasm (with special reference to goblet cell purposes. carcinoid which expresses neuroendocrine markers to- 24 M. Volante et al.

gether with mucin production), whereas the latter should Immunohistochemical and molecular markers be distinguished from other clear cell neoplasms such Colonic NENs express general neuroendocrine mark- as goblet cell carcinoid, granular cell tumor, or metastat- ers such as synaptophysin, chromogranin A (which ic ovarian/uterine neoplasms and renal cell carcinoma. may be absent in NECs), neuron-specific enolase NECs should be distinguished from high grade adeno- and CD56. C-NECs and MiNENs show microsatellite carcinomas with predominant solid growth, and some- instability in approximately 15% of tumors 13. times the focal positivity for neuroendocrine markers may pose problems in the differential diagnosis. Differential diagnosis C-NETs are usually easily identified at histology. Co- lonic NECs and MiNENs, on the other hand, similarly Neuroendocrine neoplasms of the colon to other sites, require thorough diagnostic workup and immunopanels for their distinction from solid, poorly Clinical presentation differentiated adenocarcinoma. Colonic-NENs represent approximately 5-7% of all well differentiated gastroenteropancreatic neuroen- docrine tumors but 25% of all gastroenteropancreatic Neuroendocrine neoplasms of the rectum neuroendocrine carcinomas. Mean age is 65 years and clinical presentation is similar to that of colonic Clinical presentation adenocarcinomas. Indeed, diagnosis is usually either The SEER database has shown a 10-fold increase in at biopsy of mass or after surgical resection and most the past 35 years of rectal NETs probably due to a patients show advanced stage at diagnosis. true rise in incidence and as a consequence of better and more numerous with widespread Subtypes use of screening endoscopy for detecting colorectal Definition and classification cancers. What is interesting is that there are important Colonic NENs share the same classification as other differences in incidence between countries. In Europe gastroenteropancreatic neuroendocrine neoplasms 1 and the USA, for example, rectal NETs are between and should be classified as: well differentiated neu- 5 and 27% of all gastrointestinal NETs while in Ko- roendocrine tumours of the colon (C-NETs) – G1/G2/ rea and Japan, incidence of rectal NETs is as high as G3, poorly differentiated neuroendocrine carcinomas 60% of all gastrointestinal NETs. Differences in classi- (NECs) and mixed neuroendocrine-non neuroendo- fication, database compilation, colonoscopic screen- crine neoplasms (MiNENs). ing programmes as well as possible ethnic diversities probably underlie this geographic variability 14. Pathology Mean age at presentation is 55-60 years and diag- C-NETs (35% of colonic NENs) typically show en- nosis is often incidental during screening colonos- terochromaffin (EC)-cell features (insular architecture, copy, though symptoms can include anal discomfort/ serotonin production and CDX2 positivity). At diagno- pain, blood in stools and change in bowel habits. sis, about 30-40% are metastatic to the liver, nodes, Some of these neoplasms are found in patients with mesentery and peritoneum. Indeed, C-NETs have a long-standing inflammatory bowel disease. Most rec- worse prognosis compared to rectal NETs and one of tal NENs are small polypoid lesions that appear as the lowest median survival rates of all NETs (5-year even, beige colored bulges of the mucosa, 5 to 10 cm survival rates according to stage are: 80% for stage I above the dentate line. Lesions are often submitted to or II; 50% for stage III, 10% for stage IV) 11. pathology following polypectomy, however major is- C-NECs (45% of colonic NENs) can be found in the sues which are still open include the optimal type of right and left colon and represent about a half of all endoscopic resection, management of such lesions colonic NENs but only 0.6% of colorectal cancers as after endoscopic resection and when other surgical a whole. Morphologically they are more often of large strategies (including surgical resection) are warrant- cell type, characterized by large sheets with ample ar- ed. eas of necrosis (Fig. 3a). Subtypes The colon is the most frequent site for MiNENs 12 and they often show a combined or collision type interface Definition and classification between components with the mixed adenoma/ad- Rectal NENs include the overwhelmingly more fre- enocarcinoma–NEC type being most often identified quent well differentiated rectal neuroendocrine tumors at histology (Fig. 3b-d) 13. (R-NETs) and the rare, poorly differentiated NECs and APPENDICEAL AND COLORECTAL NENS 25

Figure 3. Poorly differentiated neuroendocrine carcinoma of the colon (a). Mixed neuroendocrine-non neuroendocrine neo- plasm (MiNEN) (b) with poorly differentiated neuroendocrine component (yellow asterisk) and adenomatous component (green asterisk). Mixed neuroendocrine-non neuroendocrine neoplasm (MiNEN) with poorly differentiated neuroendocrine component (yellow asterisk) and adenocarcinoma with evident mucin production (black asterisk) (c). Synatophysin of MiNEN (d) showing diffuse expression in the neuroendocrine component (yellow asterisk) and no expression in the adenocarcino- matous areas (black asterisk).

MiNENs1. Further sub-classification is possible for Staging and prognosis R-NETs: they can either be L-cell (PP/PYY producing) Perhaps the most important aspects which must be tumors or EC-cell (serotonin producing) tumors. highlighted in R-NETs are prognostic and manage- ment issues. Most R-NETs show indolent behavior, Pathology with better long-term outcomes (overall 5-year sur- L-cell R-NETs are the most frequent type (sometimes vival rate of 74-88%) than NETs at other sites and they may also be encountered in the distal sigmoid); require conservative management. However, a small lesions are generally small (75-88% are < 1 cm) and percentage (10-20%) may metastasize to regional superficial and show a ribbon-like, trabecular or pseu- lymph nodes and beyond. The most important factor do-glandular architecture with bland cytology (Fig. 4). in therapeutic strategy (whether to limit treatment to EC-cell R-NETs share morphologic characteristics endoscopic resection or perform radical surgery) is with the more frequent small intestinal/colonic coun- the risk of lymph node metastasis and this is depen- terparts. dent on various factors: tumor size (< 10 mm - risk of 26 M. Volante et al.

Figure 4. Rectal well differentiated neuroendocrine tumor L-cell type (a). Ribbon-like and festooned architecture of cytologi- cally bland cells (b). L-cell rectal NET with pseudo-glandular architecture and overlying mucosa (c). Lympho-vascular inva- sion is an important prognostic factor (d).

nodal metastases is < 2%; 1-2 cm gray zone - risk of which is not detected by most common anti-chro- nodal metastases is 10-15%; > 2 cm - risk of nodal mogranin antibodies. They do not produce serotonin metastases is 60-80% and require surgery) 15; depth and are negative for the intestinal marker CDX2 how- of invasion (T1 versus T2 - muscularis propria) 16; ever they do produce, and can be stained for, gluca- resection margin (complete vs incomplete resec- gon-like peptides (GLP1) and/or peptide YY (PYY)/ tion); grade (G1 vs G2 vs G3); non L-cell origin which pancreatic polypeptide (PP), similarly to appendiceal shows worse prognosis 17; and lympho-vascular inva- counterparts. As not all pathology laboratories have sion 18. these stains, L-cell origin can be convincingly in- Characteristics of NECs and MiNENs of the rec- ferred by CDX2 and serotonin negativity. Apart from tum are similar to those of the colon and elsewhere, chromogranin A negativity, other potential staining though rectal NECs are often small cell, morphologi- pitfalls include a 70% positivity for prostatic acid cally. phosphatase (PAP). EC-cell R-NETs show overlapping immunohisto- Immunohistochemical and molecular markers chemical features to those of small intestinal origin L-cell R-NETs are positive for general neuroendo- (chromogranin A, nuclear CDX2 and serotonin pos- crine markers however they may be chromogranin itivity) while R-NECs and MiNENs overlap those of A negative, as they often produce chromogranin B other sites. APPENDICEAL AND COLORECTAL NENS 27

Differential diagnosis 7 Galanopoulos M, McFadyen R, Drami I, et al. Challenging the current risk factors of appendiceal neuroendocrine neoplasms: While differential diagnosis is not a major problem can they accurately predict local lymph nodal invasion? Results in localized disease, unusual immunostaining pat- from a large case series. Neuroendocrinology 2019;109:179-86. terns may cause confusion in the metastatic setting. https://doi.org/10.1159/000499381 Indeed, L-cell R-NETs express pancreatic markers 8 Volante M, Daniele L, Asioli S, et al. Tumor staging but not grad- such as Islet-1 (ISL1) and PAX8 which may lead the ing is associated with adverse clinical outcome in neuroendo- unsuspecting pathologist awry. Recent studies have crine tumors of the appendix: a retrospective clinical pathologic analysis of 138 cases. Am J Surg Pathol 2013;37:606-12. https:// shown that special AT-rich sequence binding protein-2 doi.org/10.1097/PAS.0b013e318275d1d7 19 (SATB2) , a transcription factor binding protein, is 9 Wen KW, Grenert JP, Joseph NM, et al. Genomic profile of ap- specific for NETs of rectal (and appendiceal) origin pendiceal goblet cell carcinoid is distinct compared to appendi- while it is not expressed in pancreatic/duodenal NETs. ceal neuroendocrine tumor and conventional adenocarcinoma. Hum Pathol 2018;77:166-74. https://doi.org/10.1016/j.hump- ath.2018.03.026 10 Park HY, Kwon MJ, Kang HS, et al. Targeted next-generation Conclusions sequencing of well-differentiated rectal, gastric, and appendiceal neuroendocrine tumors to identify potential targets. Hum Pathol Appendiceal, colonic and rectal neuroendocrine ne- 2019;87:83-94. https://doi.org/10.1016/j.humpath.2019.02.007 oplasms require accurate pathologic classification. 11 Chagpar R, Chiang Y, Xing Y, et al. Neuroendocrine tumors In particular, appendiceal and rectal NETs require of the colon and rectum: prognostic relevance and compara- thorough knowledge of criteria which are necessary tive performance of current staging systems. Ann Surg Oncol for correct patient management. Indeed, though gray 2013;20:1170-8. https://doi.org/10.1245/s10434-012-2746-z areas still abound, it is only though an accurate and 12 Milione M, Maisonneuve P, Pellegrinelli A et al. Ki67 prolifera- reproducible classification system that more scientifi- tive index of the neuroendocrine component drives MANEC prognosis. Endocr Relat Cancer. 2018;25:583-93. https://doi. cally robust studies will aid in patient selection for di- org/10.1530/ERC-17-0557 verse treatment options. 13 La Rosa S, Marando A, Furlan D, et al. Colorectal poorly dif- ferentiated neuroendocrine carcinomas and mixed adenoneu- References roendocrine carcinomas: insights into the diagnostic immuno- phenotype, assessment of methylation profile, and search for 1 WHO Classification of Tumors Editorial Board. Digestive system prognostic markers. Am J Surg Pathol 2012;36:601-11. https:// tumors. WHO Classification of Tumors. 5th ed. Lyon: IARC press doi.org/10.1097/PAS.0b013e318242e21c 2019. 14 Kojima M, Ikeda K, Saito N et al. neuroendocrine tumors of the 2 Njere I, Smith LL, Thurairasa D, et al. Systematic review and : clinicopathological features and predictive factors meta-analysis of appendiceal carcinoid tumors in children. Pe- of lymph node metastasis. Front Oncol 2016;6:173. https://doi. diatr Blood Cancer 2018;65:e27069. https://doi.org/10.1002/ org/10.3389/fonc.2016.00173 pbc.27069 15 Mani S, Modlin IM, Ballantyne G et al. Carcinoids of the rectum. 3 Onyemkpa C, Davis A, McLeod M, et al. Typical carcinoids, gob- J Am Coll Surg 1994;179:231-48. let cell carcinoids, mixed adenoneuroendocrine carcinomas, 16 neuroendocrine carcinomas and adenocarcinomas of the ap- Lee SH, Kim BC, Chang HJ et al. Rectal neuroendocrine and pendix: a comparative analysis of survival profile and predictors. L-cell tumors: diagnostic dilemma and therapeutic strategy. J Gastrointest Oncol 2019;10:300-6. https://doi.org/10.21037/ Am J Surg Pathol 2013;37:1044-52. https://doi.org/10.1097/ jgo.2018.11.08 PAS.0b013e3182819f0f 17 4 Rault-Petit B, Do Cao C, Guyétant S, et al. Current management Kim JY, Kim KS, Kim KJ et al. Non-L-cell immunophenotype and predictive factors of lymph node metastasis of appendix and large tumor size in rectal neuroendocrine tumors are as- neuroendocrine tumors: a national study from the French Group sociated with aggressive clinical behavior and worse progno- of Endocrine Tumors (GTE). Ann Surg 2019;270:165-71. https:// sis. Am J Surg Pathol 2015;39:632-43. https://doi.org/10.1097/ doi.org/10.1097/SLA.0000000000002736 PAS.0000000000000400 5 La Rosa S, Finzi G, Puppa G, et al. Lipid-rich variant of appen- 18 Sugimoto S, Hotta K, Shimoda T et al. The Ki-67 labeling index diceal well-differentiated endocrine tumor (carcinoid). Am J Clin and lymphatic/venous permeation predict the metastatic poten- Pathol 2010;133:809-14. https://doi.org/10.1309/AJCP0NWA1D- tial of rectal neuroendocrine tumors. Surg Endosc 2016;30:4239- BDZIOY 48. https://doi.org/10.1007/s00464-015-4735-3 6 Brighi N, La Rosa S, Rossi G, et al. Morphological factors related 19 Zhao LH, Chen C, Mao CY et al. Value of SATB2, ISL1, and to nodal metastases in neuroendocrine tumors of the appendix: TTF1 to differentiate rectal from other gastrointestinal and lung a multicentric retrospective study. Ann Surg 2020;271:527-33. well-differentiated neuroendocrine tumors. Pathol Res Pract https://doi.org/10.1097/SLA.0000000000002939 2019;215:152448. https://doi.org/10.1016/j.prp.2019.152448 PATHOLOGICA 2021;113:28-38; DOI: 10.32074/1591-951X-231

Review

Neuroendocrine neoplasms of the biliary tree, liver and pancreas: a pathological approach

Claudio Luchini1, Giuseppe Pelosi2,3, Aldo Scarpa1,4, Paola Mattiolo1, Deborah Marchiori5, Roberta Maragliano5, Fausto Sessa5, Silvia Uccella5 1 Department of Diagnostics and Public Health, Section of Pathology, University and Hospital Trust of Verona, Italy; 2 Department of Oncology and Hemato-Oncology, University of Milan, Italy; 3 Inter-Hospital Pathology Division, IRCCS MultiMedica, Milan, Italy; 4 ARC-NET Research Centre, University of Verona, Italy; 5 Department of Medicine and Surgery, Unit of Pathology, University of Insubria, Varese, Italy

Summary Neuroendocrine neoplasms of the pancreatobiliary tract and liver are a heterogeneous group that encompass a spectrum of entities with distinct morphological, biological and clinical features. Although in the various anatomical sub-sites of this region they show specific characteristics, these tumors, as a whole, share several etiological and clinical aspects. This review systematically addresses NENs arising in the extrahepatic bile ducts, , liver and pancreas, with the principal aim of pinpointing essential diagnostic and classification issues. In addition, the section on hepatic NENs has been expanded to Received: November 23, 2020 include metastatic disease of unknown primary site. Accepted: November 30, 2020 Key words: neuroendocrine neoplasms, pancreas, liver, biliary tract Correspondence Silvia Uccella Unit of Pathology, Department of Medicine and Surgery, University of Insubria, via O. Introduction Rossi 9, 21100 Varese, Italy Tel. +39 0332 270601 Neoplastic diseases of the pancreatobiliary tract and liver are rare but Fax: +39 0332 270600 E-mail: [email protected] potentially life-threatening. Although in the various anatomical sub-sites of this region they show specific biological and morphological features, Conflict of interest The Authors declare no conflict of interest. these tumors, as a whole, share several etiological and clinical aspects. Among pancreatobiliary and hepatic malignancies, neuroendocrine ne- How to cite this article: Luchini C, oplasms (NENs) are among the rarest subtypes. This review will sys- Pelosi G, Scarpa A, et al. Neuroendocrine tematically address NENs arising in the extrahepatic bile ducts and gall- neoplasms of the biliary tree, liver and pancreas: a pathological approach. bladder, in the liver and pancreas, with the principal aim of highlighting Pathologica 2021;113:28-38. https://doi. essential diagnostic and classification issues. In addition, the paragraph org/10.32074/1591-951X-231 on hepatic NENs has been expanded to include metastatic disease of © Copyright by Società Italiana di Anatomia Pato- unknown primary site. logica e Citopatologia Diagnostica, Divisione Itali- ana della International Academy of Pathology

OPEN ACCESS Neuroendocrine neoplasms of the biliary tree and liver

This is an open access journal distributed in accordance The liver and the intra- and extra-hepatic bile tract (including gallbladder) with the CC-BY-NC-ND (Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 International) license: the are the rarest sites of occurrence for NENs, if metastatic lesions are ex- work can be used by mentioning the author and the license, cluded. Here we will revise the available knowledge about primary NENs but only for non-commercial purposes and only in the original version. For further information: https://creativecommons. of the biliary tract and liver and discuss the diagnostic management of org/licenses/by-nc-nd/4.0/deed.en metastatic NENs in the liver. NEUROENDOCRINE NEOPLASMS OF THE BILIARY TREE, LIVER AND PANCREAS 29

Primary NENs of the extrahepatic bile EHBD NECs are frequently mixed with invasive and ducts (EHBD) and of the gallbladder (GB) pre-invasive non-neuroendocrine components, and can be called mixed neuroendocrine/non-neuroendo- Due to the rarity of these entities, clinicopathological crine neoplasms (MiNENs) 11,12,14-17, suggest that also studies on large series are lacking. Indeed, even ret- in these sites NEC may share common pathogenet- rospective analyses of large databases and national ic pathways with autoctonous adenocarcinomas, in tumor registries, as well as literature meta-analyses, analogy to MiNENs of other digestive and extra-diges- are poorly affordable, as the nomenclature in this tive locations 18,19. field is often confusing and the distinction between Macroscopically, tumor masses have a mean diame- neuroendocrine tumors (NETs) and neuroendocrine ter of 2.2 to 3 cm 2,17 in the EHBD, whereas GB NENs carcinomas (NECs) is not well defined 1,2. However, if are larger (mean diameter 3.5 to 5.6 cm) 11,20,21. In the we consider well documented case reports and case gallbladder, the most commonly involved subsite is the series, as well as reviews and meta-analysis on spe- fundus11. The histopathological appearance of GB and cific types of NENs, we can summarize the features of EHBD NETs is similar to NETs of other anatomical NETs and NECs in these sites. sites, with well differentiated neuroendocrine morphol- Overall, these neoplasms represent 0.2 to 2% of all ogy 8, and NECs may be of small or large cell type, and NENs, and around 2% of all primary malignancies can be found in the context of a MiNEN, mixed with arising in EHBD and GB 3,4. Gallbladder NENs most an adenocarcinoma or a papillary neoplasm 11,17,22. At commonly arise in women (M:F ratio = 1:4), where- immunohistochemistry, pan-cytokeratins and general as only a slight female prevalence (M:F ratio = 2:3) is neuroendocrine markers (synaptophysin and chro- seen in patients with NENs of EHBD 3. All ages can be mogranin A) are expressed 11,17,22, with variable pat- affected, and rare pediatric cases are also described 5. terns between NETs and NECs, as described in other In these sites, NECs are much more common than sites (Fig. 1) 23. Cytokeratin 7 has been reported to be NETs and mostly occur in the gallbladder, followed by consistently expressed in GB NECs, in contrast with the common distal and the common hepatic NECs of other digestive sites 17. CD117 immunostain, duct. which has also been reported to be positive in NECs Although some authors have suggested that NENs of of other sites, is found in a significant fraction of GB the biliary tree may arise from neuroendocrine cells NEC and may represent an additional marker in the of post-inflammatory metaplastic mucosa 6, the most differential diagnosis with poorly differentiated adeno- likely hypothesis is that they derive from an epithelial carcinoma and with NET and a putative therapeutic precursor that may also give rise to glandular neo- target 11. A number of hormonal products have been plastic proliferation, in analogy to other gastroenter- found in neoplastic cells of a subset of NETs, main- opancreatic NENs 7. Independently from the specific ly gastrin and serotonin 2, whereas no data regarding mechanism of cancerogenesis, the main risk factor transcription factors and other site-specific markers for gallbladder NENs is the presence of cholelithiasis is available, apart from the nonspecific expression of and cholecystitis 7, whereas no specific etiology has TTF1 in a subset of NECs 11. Ki-67-related labelling been identified for NENs of the EHBD. As concerns index is far above 20% in NECs, whereas in NETs it the pathogenetic mechanisms, little is known about may vary from up to 3% (NET G1) to more than 20% the molecular pathways underlying the development (NET G3), through cases in which it ranges between of GB and HEBD NETs, but alterations in tumor-relat- more than 3% but less than 20% 24. ed genes involved in local adenocarcinomas, such as The clinical presentation of EHBD and GB NENs TP53, KRAS and RB1, seem not to be present in these varies according to their site. EHBD NENs become neoplasms 8. An association with von Hippel-Lindau evident due to jaundice and other signs of cholesta- syndrome (VHL) has been proposed for EHBD NETs, sis, whereas GB NENs are most often asymptomat- as two cases have been reported in VHL patients 9,10. ic and are occasionally diagnosed during abdominal For NECs of these sites, a very recent paper report- imaging for nonspecific symptoms or after cholecys- ing the largest series of GB NECs published until tectomy for cholecystitis 11,17. Very rare cases become now, showed frequent loss of Rb1, hyperexpression evident with symptoms of hormone hypersecretion or of p16, and no mutation of BRAF in these cases 11. other paraneoplastic syndromes 25. The prognosis of In addition, TP53 point mutation has been found in a EHBD and GB NENs heavily depends on their mor- case 12, while the presence of microsatellite instability phological characterization. Patients with NECs of and alterations of genes involved in the ERBB path- small and large cell type have a poor outcome, with way (HMCN1 and CDH10) were reported in a case 13. an overall survival (OS) at 5 years of 19%, and AJCC These results, together with the evidence that GB and stage strongly influences prognosis 11,22. In contrast, 30 C. Luchini et al.

Figure 1. Small cell neuroendocrine carcinoma of the gallbladder. Solid growth of poorly differentiated small cells with zonal necrosis (A). Ki-67-related index is higher than 80% (B) and tumor cells are immunoreactive for synaptophysin (C) and chro- mogranin A (D). (Hematoxylin-eosin and immunoperoxidase, original magnification x100).

NETs bear a better prognosis than adenocarcinomas Among H-NENs, NETs are reported to be slightly less of the same anatomical sites, with a 10-year OS of frequent than NECs 27,30, and these latter are nearly 36% for gallbladder NETs and 80% for EHBD NETs 26. always associated with a non-neuroendocrine compo- The distinction of NENs from adenocarcinomas of the nent (, HCC) in a MiNEN 18,27. same sites is of paramount importance for establish- No definite sex prevalence has been reported and ing a correct treatment, in particular for NECs, which mean age at diagnosis in in the adulthood (around 50 are often diagnosed in advanced stages and benefit years), with very rare cases under the age of 40 30,31. from adjuvant platinum-bases chemotherapy 11. A meta-analysis of 69 cases showed that the most common intrahepatic location was the right lobe, in which half of the cases was detected 31. Primary NENs of the liver The histogenesis of hepatic NENs is controversial and it has been proposed that they may derive from ectopic Primary hepatic NENs (H-NENs) are exceedingly rare, intrahepatic pancreatic tissue 32, but it is more conceiv- representing, as a whole, less than 1% of all resected able that NETs arise from progenitor cells in intrahe- primary neoplasms of the liver 27. The distinction from patic bile ducts, whereas NECs may follow a common metastases from other primary site is important to es- pathogenetic pathway with hepatocellular carcinoma 33. tablish the treatment and the prognosis of patients 28,29. Definitive data on genetic features of H-NENs are lack- NEUROENDOCRINE NEOPLASMS OF THE BILIARY TREE, LIVER AND PANCREAS 31

ing, due to their rarity. Only single cases were studied, of symptomatic patients with symptomatic NENs revealing loss of one copy each of chromosomes 3 and have synchronous metastases at diagnosis, and up 18, and gain of 1q in a NET G2 metastatic to the orbit 34, to 20% of the cases present as metastasis from an whereas TP53 mutations, associated or not with EGFR occult primary 41. The identification of the primary site and other well-known cancer related genes were found is an important step towards the correct management in two cases of NEC 35,36. of the patient, particularly for NETs, as the therapeu- Macroscopically, H-NENs are expanding masses in tic approach may vary depending on the primary site the liver parenchyma, with a tannish cut surface that, and cell type. In addition, even when liver metastases in NECs, may show areas of necrosis and hemor- are unresectable, the surgical treatment of the prima- rhage. NETs are reported to be larger (mean diame- ry NET has been shown to have a positive impact of ter around 5 cm) than NECs (mean diameter around patient’s outcome 42. Consequently, thorough morpho- 3 cm) 27. The microscopic appearance of H-NETs is logical and immunohistochemical analyses are ex- mainly of a trabecular or pseudo-glandular growth of pected to give important clues to the recognition of the neoplastic cells with well differentiated morphological site of origin of a metastatic NET. In contrast, NECs, features 27. General neuroendocrine markers are well independent of the primary site, are currently treated expressed, as well as cytokeratins 7, 18, and 19, but with platinum-based regimens, and the role of the pa- HepPar-1, which is a site specific antibody for hepa- thologist may be limited to the distinction between a tocellular neoplasms is not expressed 27. Based on visceral NEC and a Merkel cell carcinoma of the skin, Ki-67-related proliferative index NETs may be graded, because the latter requires wide local excision, sen- but until now only G1 and G2 H-NETs have been re- tinel node biopsy and possibly radiotherapy. Of note, ported 33. H-NECs are mainly of the small cell sub- most of the diagnostic approaches discussed below type, but also large cell variant has been reported 33. have poor reliability in the context of NECs. Irrespec- Both subtypes have overlapping histopathological tive of the primary site, the liver represents the most features with NECs in other anatomical sites and are frequent location of metastatic NENs and liver biopsy typically found in MiNENs, combined with HCC 33. is the most common specimen with which the practic- However, occasional MiNENs with a cholangiocarci- ing pathologist is faced in the challenge of differential noma component have been described 37. Compared diagnosis. with NH-NETs, H-NECs show a lower expression of Digestive NETs, particularly ileal (Fig. 2) and pancre- general neuroendocrine markers and of cytokeratins. atic NETs, are the major sources of liver metastases HepPar-1 is consistently negative also in NECs 27. As among all NETs. However, also thoracic NETs may a whole, there are no specific morphological or im- not infrequently give hepatic secondary localizations. munohistochemical characteristics that may support As pure morphological features are frequently too the diagnosis of a primary H-NEN versus a metas- subtle to recognize and are also commonly obscured tasis and the practicing pathologist should always be by crash artifacts in small liver samples, immunohis- aware that a primary H-NEN is always a diagnosis of tochemistry turns out to be the corner stone of the exclusion, after careful consideration of all clinical and differential diagnosis in this setting. The wise use of a radiological information. step-wise immunohistochemical approach using tran- The clinical presentation of H-NENs may include non- scription factors, hormones and other markers, such specific abdominal symptoms, such as abdominal dis- as carcinoembryonic antigen (CEA), prostate-specific comfort or diarrhea, but a significant proportion of cas- acidic phosphatase (PSAP), and others, may be very es is asymptomatic 30. Serum liver tests are mostly in helpful to identify the unknown primary site of a meta- the normal range and circulating tumor markers have static NET 23,41. In this respect, despite the existence of no diagnostic value 30. Symptoms of hormone hyperse- a wide range of available antibodies for each putative cretion (Zollinger-Ellison syndrome, Cushing syndrome primary site (Tab. I), an algorithmic approach is desir- and hypercalcemia due to gastrin, adrenocorticotroph able to avoid waste of time and of financial resources hormone, and parathyroid hormone, respectively) have (Fig. 3). In fact, after the initial confirmation of the ep- been reported 38-40. The most important prognostic pa- ithelial and neuroendocrine nature of the proliferation rameters is the distinction between NETs and NECs using pan-cytokeratins and general neuroendocrine and the possibility of radical surgery 30. markers (synaptophysin, chromogranin A and, lately, the new marker INSM1), as well as of its well differenti- ated morphology, the use of TTF1 and CDX2 may rep- NENs metastatic to the liver resent an initial step for the triage of the possible pri- mary site. Immunoreactivity for CDX2 points towards a Virtually all NENs have metastatic potential, and 90% gastroenteropancreatic (GEP) origin, whereas TTF1 is 32 C. Luchini et al.

Figure 2. Ileal NET G2 metastatic to the liver (A) strongly immunoreactive for synaptophysin (B), chromagranin A (C) and CDX2 (D). Ki67 proliferation index is about 15% (E) and somatostatin receptor 2A shows strong membranous stain (F) (hematoxylin-eosin and immunoperoxidase, original magnification x100).

Table I. Useful immunohistochemical markers for the identification of the occult primary site of a NEN. Putative primary site Transcription factors Hormones Other markers Pituitary Pit1, SF1, Tpit, ER-a, GATA-2, PRL, GH, TSH, ACTH, FSH, LH, a-SU GATA-3 Thyroid PAX8, TTF1 Calcitonin CEA, CGRP Parathyroid GATA-3 PTH Lung TTF1, OTP Bombesin, serotonin, calcitonin Stomach CDX2 (Histamine), Serotonin, Ghrelin v-MAT2 Duodenum ISL-1, PDX-1, CDX2 Somatostain, Gastrin Pancreas ISL-1, PAX6, PDX-1, CDX2 Insulin, Glucagon, PP, Somatostatin, Gastrin, VIP, ACTH, Serotonin, Calcitonin, others Jejunum/Ileum CDX2 Serotonin v-MAT1 Appendix CDX2 Serotonin, Glucagon-like peptides Colon-rectum CDX2 PYY, Glucagon-like peptides, Serotonin Prostatic acid phosphatase Paraganglioma GATA-3 (Catecholamines) Tyrosine hydroxylase, v-MAT1, v-MAT2 reminiscent of a thoracic primary. The additional use of to an ileal, pancreatic, or colo-rectal origin, respective- the transcription factor PDX1, which has been reported ly. In addition, other markers may be of help in con- to have a certain specificity for pancreatic NETs, may firming, for example, an ileal (substance P), colo-rectal be considered, although, even in expert hands, the im- (PSAP), gastric (vesicular monoamine transporter 2, munoreaction with commercially available antibodies v-MAT2) or duodenal (gastrin, somatostatin) primary. may be difficult to evaluate. In CDX2-positive metas- In this setting, one should not forget the importance of tases, the employment of antibodies directed against using immunohistochemical panels, and not just sin- hormonal products like serotonin, pancreatic hor- gle antibodies, as no individual marker has absolute mones and glucagon-related peptides may give clues sensitivity and specificity in identifying an unknown NEUROENDOCRINE NEOPLASMS OF THE BILIARY TREE, LIVER AND PANCREAS 33

Figure 3. Practical diagnostic algorithm for the identification of the unknown primary site of a metastatic neuroendocrine tumor (NET). CKs, Cytokeratins; NE, Neuroendocrine; Syn, synaptophysin; Chrom A, chromogranin A; Sub P, substance P; Ins, insulin; Gluc, glucagon; Som, somatostatin; GLP, glucagon-like peptides; PSAP, prostate-specific acidic phosphatase; *GATA3 for breast NENs (but it is also positive in gonadotroph pituitary NETs); SSTR2A and SSTR5 to predict avidity to so- matostatin analogues-based imaging.

primary. Just as an example, the positive immunostain careful interpretation of clinical, morphologic and im- for TTF1 is not exclusive of lung NETs (carcinoids), as, munohistochemical findings. The use of a panel of in the context of NENs, medullary thyroid carcinomas approach combining cytokeratins along with anatomic (MTCs) are also TTF1-positive and, in turn, calcitonin site-related transcription factors, hormones and other expression is not an absolutely affordable marker of biomarkers can assist identifying the origin of the met- MTCs, as also lung NETs may stain positive for this astatic NEN. The power of this approach is limited in hormone. In such case, a positive stain for CEA favors the setting of poorly differentiated NENs (NECs). MTC versus lung NET 41. Liver metastases from NETs arising in rare sites (ex- tra-GEP and extra-thoracic) are possible findings and Neuroendocrine neoplasms of the even in this case immunohistochemistry may be of pancreas help (Tab. I, Fig. 1). For example, the possibility of me- tastases from pituitary NETs should not be underesti- NENs of the pancreas (PanNENs) are a heterogene- mated, particularly in the clinical context of a Cushing ous group of tumors with different histologic, molecu- syndrome 43. In this case, the use of pituitary-specific lar and clinical features. The current World Health Or- transcription factors may be useful to reach a correct ganization (WHO) diagnostic guidelines have refined diagnosis. Finally, another useful immunohistochem- their classification, which reflects more appropriately ical tool that can support the clinical search for the the different biological landscapes and prognostic im- unknown primary site of a NET is the immunostain plications 24. for somatostatin receptors (SSTRs). Indeed, a strong Definition/Terminology. The heterogeneous group membranous positivity for SSTR2A and/or for SSTR5 of neuroendocrine lesions of the pancreas has been has a good correlation with the avidity of the neo- named as pancreatic neuroendocrine neoplasm (Pan- plasm for somatostatin analogues-based imaging that NEN). The current WHO classification subdivides Pan- can identify the primary NET 44. NEN in three main categories: i) pancreatic neuroen- In conclusion, the workup of a metastatic NEN repre- docrine microadenoma (lesion < 5 mm); ii) well-differ- sents a critical responsibility of pathologists. It requires entiated pancreatic neuroendocrine tumor (PanNET), 34 C. Luchini et al.

which includes functional PanNETs (F-PanNENs; tumors with clinical evidence of hormone production, such as insulinoma, glucagonoma, gastrinoma, VIPo- ma, etc.) and non-functional PanNETs (NF-PanNET); iii) poorly differentiated pancreatic neuroendocrine carcinoma (PanNEC), featuring either small cell or large cell PanNECs 24. The last WHO classification has subdivided PanNETs into three subgroups of tumors: i) Grade 1 PanNET (PanNET G1): < 2 mitoses/2 mm2 and a Ki-67 pro- liferation index < 3%; Grade 2 PanNET (PanNET G2): 2-20 mitoses/2 mm2 or a Ki-67 proliferation in- dex of 3-20%; Grade 3 PanNET (PanNET G3): > 20 Figure 4. Example of a classical neuroendocrine microade- 2 mitoses/2 mm or a Ki-67 proliferation index > 20% noma. This category includes small (<0.5 cm) well-differenti- 24. This last category represents the main novelty in ated neuroendocrine neoplasms. Original magnification: x20. PanNENs classification, and should be distinguished from PanNEC, which in turn includes small-cell and large-cell carcinoma 24. A last category to be considered is represented by Mi- are the following: i) insulinoma: trabecular and solid NEN. Both components must represent at least 30% growth patterns, with normal pancreatic ducts often of the total tumor mass, are usually high-grade (G3) entrapped within tumor mass; a stromal deposit of is- and the non-neuroendocrine part is generally repre- let amyloid polypeptide is quite specific but very rare sented by acinar carcinoma or ductal adenocarcino- (5% of cases) 47; ii) gastrinoma: trabecular and glan- ma 18,19,24. dular growth patterns 48; iii) glucagonoma: presence Macroscopic description. Because of their small of densely arranged trabecular structures with scant size, microadenoma are rarely documented during stroma 49; iv) somatostatinoma: psammomatous cal- routine sampling. PanNETs are usually brownish le- cification are quite common, although they are more sions, with lobulated or pushing borders and soft to typical of duodenal location 50; v) serotonin-producing fleshy consistency. The vast majority of PanNETs are PanNETs: a trabecular architecture is the most com- encapsulated, at least in part, and sharply demarcat- mon pattern, and vascular / perineural invasion is fre- ed from the adjacent pancreatic parenchyma. Cystic quent even in G1 tumors 51. changes are rare but, if present, a unilocular cyst is PanNECs present distinctive histologic features and reported. Conversely, PanNECs generally show in- have been subdivided in small-cell and large-cell Pan- filtrative margins, hard consistency and brownish to NECs24. Small-cell PanNECs are characterized by dif- whitish color; typically, necrotic areas are reported. fuse sheets of cells with scant cytoplasm, round or Histopathology. A classic example of neuroendo- elongated nuclei and finely granular chromatin. As in crine microadenoma is shown in Figure 4, and para- the pulmonary counterpart, nuclear moulding may be digmatic examples of PanNETs G1, G2 and G3 and also present 24,52. Large-cell PanNECs is a more com- of PanNEC are depicted in Figure 5. By definition, mon subtype, and is composed by round to polygonal neuroendocrine microadenoma is a small and well large cells with coarse chromatin and prominent nu- differentiated neuroendocrine neoplasm. Histological- clei 24,52. Both small-cell and large-cell PanNECs show ly, NF-PanNETs display a well-differentiated growth necrotic areas, often with a comedo-like appearance. pattern, with a spectrum of architectural patterns, in- MiNEN are mixed neuroendocrine-non-neuroendo- cluding solid-nesting, paraganglioma-like, trabecular, crine neoplasms, where which each counterpart ac- gyriform and glandular aspects24,45,46. The stroma is counts at least for 30% of the entire lesion 24,53. The highly vascular, but areas with dense and hyalinized non-neuroendocrine counterpart, which is usually collagen are often present. Most of neoplastic cells represented by acinar cell carcinoma or ductal adeno- are monomorphic, cuboidal and with the classic nuclei carcinoma, reflects its conventional morphology. showing “salt and pepper” chromatin texture 24,45,46. Immunohistochemical and molecular markers. They are centrally located and polarized. In addition As neuroendocrine neoplasms, PanNETs are usually to these classical aspects, some functional PanNETs stained by pan-cytokeratins and general neuroendo- may exhibit particular features, although distinctive crine markers such as Synaptophysin and Chromo- morphological hallmarks are lacking. The more pe- granin-A, with known variability between NETs and culiar histological features for functional PanNETs NECs 23,24. Other markers that may be added to the NEUROENDOCRINE NEOPLASMS OF THE BILIARY TREE, LIVER AND PANCREAS 35

Figure 5. The entire spectrum of PanNET/NEC is shown. A-C: well differentitated PanNETs, as follows: A) PanNET G1, B) PanNET G2, C) PanNET G3; D) PanNEC (black asterisks indicate tumor necrosis). Original magnification: X10.

diagnostic PanNET algorythm, but have low accu- tated genes, with DAXX and ATRX mutations being rancy are: i) CD56, which has a high sensitivity but mutually exclusive 58. Collectively, about 60% of Pan- a low specificity, ii) CD200, which stains in the pan- NETs carry MEN1/DAXX/ATRX mutations. DAXX and creas both PanNETs and solid pseudopapillary neo- ATRX mutations have been recently associated with plasms (SPNs), iii) Islet-1, whose expression is not poor prognosis 55. Furthermore, the biological process restricted to PanNETs but is also commonly found in of alternative lengthening of telomeres (ALT), which well and poorly differentiated NENs in extrapancreat- is a telomerase-independent mechanism used by ic sites 23,24,56. In the prognostic grading of PanNETs, different tumors to maintain the telomere length thus the evaluation of Ki-67 proliferation index (using clone increasing cell replication’s potential, is activated in a MIB1) is crucial, as stated before in this review. Ki- subset of PanNETs and associated with an increased 67 may be useful also in the differential diagnosis rate of distant metastases 59. PanNECs commonly between PanNET G3 vs. PanNEC 52. In the former, bear TP53 and RB1 mutations, which are reflected by Ki-67 distribution is heterogeneous and usually shows abnormal expression patterns of the related proteins, areas with a low (< 20%) proliferation, together with so that the differential diagnosis between PanNET G3 highly proliferating areas, whereas PanNECs displays vs. PanNEC can be supported by immunohistochem- a more homogeneous staining, usually present in a istry 24,52. Indeed, an abnormal expression pattern for very high proportion of neoplastic cell nuclei (> 60%). p53 and the loss of Rb immunostain strongly corrobo- Immunohistochemical stains for hormonal products rate a PanNEC diagnosis against a PanNET G3. The (both pancreatic and ectopic) may provide additional genetic differences between PanNET and PanNEC is information for the characterization of both F-PanN- in keeping with the assumption that they are distinc- ENs and NF-PanNENs 23,57, but it has to be noticed tive and separate tumor entities 45,52. that the specific diagnosis of F-PanNET must be Differential diagnosis. Besides the importance of based on the hormone-related clinical syndrome rath- the distinction between PanNETs G3 and PanNECs, er than immunohistochemical analysis 24. the main differential diagnoses of PanNENs include PanNETs display a mutational profile that includes pancreatic non-neuroendocrine epithelial malignan- MEN1, DAXX and ATRX as the most commonly mu- cies with solid and/or organoid pattern of growth, such 36 C. Luchini et al.

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Review

Histopathology of IBD Colitis. A practical approach from the pathologists of the Italian Group for the study of the gastrointestinal tract (GIPAD)

Vincenzo Villanacci1, Luca Reggiani-Bonetti2, Tiziana Salviato2, Giuseppe Leoncini3, Moris Cadei1, Luca Albarello4, Alessandro Caputo5, Maria Costanza Aquilano6, Serena Battista7, Paola Parente8 1 Institute of Pathology, Spedali Civili, Brescia, Italy; 2 Department of Diagnostic, Clinic and Public Health Medicine, University of Modena and Reggio Emilia, Modena, Italy; 3 Pathology Unit, ASST del Garda, Desenzano del Garda (BS), Italy; 4 Pathology Unit, IRCCS San Raffaele Scientific Institute, Milano, Italy; 5 University Hospital “San Giovanni di Dio e Ruggi D’Aragona”, Salerno, Italy; 6 Institute of Pathology ASST Niguarda, Milan, Italy; 7 Institute of Pathology S. Maria della Misericordia Hospital, Udine, Italy; 8 Unit of Pathology, Fondazione IRCCS Ospedale Casa Sollievo Della Sofferenza, San Giovanni Rotondo (FG), Italy

Summary Inflammatory bowel diseases (IBDs) are lifelong disorders in which an interaction between genetic and environmental factors is involved. IBDs include two entities: Crohn’s disease (CD) and ulcerative colitis (UC); these can be adequately diagnosed and distinguished with a correct methodological approach based on communicating exhaustive clinical, endoscopic and laboratory information to the pathologist and performing adequate biop- Received: December 2, 2020 tic sampling and precise morphological signs including crypt architecture, distribution of Accepted: December 3, 2020 inflammation and granulomas, when present. IBD needs to be distinguished from non- IBD colitis, mostly at its onset. Moreover, IBDs are associated with an increased risk of Correspondence developing colorectal adenocarcinoma. In daily pathological practice, correct diagnosis Vincenzo Villanacci of IBD and its subclassification as well as a correct detection of dysplasia is imperative to Institute of Pathology Spedali Civili, establish the best therapeutic approach. Brescia, Italy E-mail: [email protected] Key words: inflammatory bowel disease, ulcerative colitis, Crohn’s disease, dysplasia, intestinal cancer Conflict of interest The Authors declare no conflict of interest.

How to cite this article: Villanacci V, Reggiani- Introduction Bonetti L, Salviato T, et al. Histopathology of IBD Colitis. A practical approach from the Inflammatory bowel diseases (IBDs) are relapsing diseases that can in- pathologists of the Italian Group for the volve any segment of the gastrointestinal tract and include two chronic study of the gastrointestinal tract (GIPAD). Pathologica 2021;113:39-53. https://doi. gastrointestinal disorders: Crohn’s disease (CD) and ulcerative colitis 1 org/10.32074/1591-951X-235 (UC) . IBDs have a multifactorial pathogenesis, which includes genetic predisposition, epithelial barrier defects, dysregulated immune respons- © Copyright by Società Italiana di Anatomia Pato- es and environmental factors which contribute to the onset of the dis- logica e Citopatologia Diagnostica, Divisione Itali- 2,3 ana della International Academy of Pathology eases and their progression . CD and UC differ from each other for epidemiological aspects, clinical presentation, endoscopic and histo- OPEN ACCESS pathological findings, and disease course; the two entities have different complications and management 1. At the onset, however, the histopatho- This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- logical finding on biopsy samples can overlap between the two enti- NonCommercial-NoDerivatives 4.0 International) license: the ties. Thus, for a correct interpretation of biopsy specimens from patients work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original with suspected IBD, detailed clinical information is essential to make a version. For further information: https://creativecommons. correct differential diagnosis and to schedule appropriate therapy and org/licenses/by-nc-nd/4.0/deed.en follow-up 4,5. On the other hand, a correct pathological approach, both 40 V. Villanacci et al.

on adequate biopsy samples and surgical specimens and B lymphocyte selection and activation, immune is useful to better recognize histopathological findings dysregulation disorders with defects in negative con- of IBD, subclassify UC or CD and exclude a non-IBD trol of innate and adaptative immune responses 15. colitis 4. These disorders, which represent key functional The aim of this paper is to provide an easy and ready- clues within inflammatory networks, can lead to IBD to-use guide for daily diagnostic practice for histo- in children younger than 6 years of age, configuring pathological diagnosis of IBD, its subclassification in the spectrum of Very-Early-Onset IBD (VEO-IBD) 16. CD and UC and for dysplasia recognition. The main VEO-IBDs are rare disorders representing a distinct epidemiological, clinical, laboratory data and serolog- genetic phenotype, but they also have some clinical ical markers that the pathologist needs to know for stigmata, such as an increased resistance to conven- a correct biopsy evaluation have also been reported. tional medical treatment and an high morbidity and The differential diagnosis with non-IBD colitis has even mortality 16. been discussed in the manuscript entitled “Histopa- thology of Non-IBD Colitis. A practical approach from the Italian Group for the study of the gastro- Clinical and laboratory features intestinal tract (GIPAD)”, which is also included in this Special Issue of Pathologica 6. Increased stool frequency (6-8 discharges per day) and decreased stool consistency, i.e. diarrhea, pro- tracted over time, at least for 4 weeks, are the most Epidemiological and genetic features common presenting symptoms of IBDs, followed by abdominal pain. Tenesmus is a clue to the presence The global prevalence of IBDs has been increasing of proctitis, and will be more suggestive of UC as is since 2000; at present, IBD affects up to 1 of 200 in- the presence of blood with mucus in feces; converse- dividuals in Western countries 7. Genome-wide asso- ly, fever is more often seen in CD than in UC, as is ciation studies have identified 200 risk loci for IBDs: weight loss 17. The presence of perianal disease, such together, these loci explain 13.1% and 8.2% of vari- as skin tags, anal fissures, and perianal fistulae are ance in disease liability in CD and UC, respectively, among the most helpful clinical findings suggestive but their clinical significance is not clear 3,8. The in- of CD 17. Besides intestinal symptoms, the so-called cidence and prevalence of UC have been increasing extraintestinal manifestations (EIM) are frequent, with worldwide over time, but no sex predominance has a prevalence ranging from 6 to 47% which consid- been described9. Between 8-14% of patients with UC erably affect morbidity and mortality in patients with have a family history of IBD, and first-degree relatives IBDs 18. The pathophysiology of EIM remains elusive, have four times the risk of developing the disease 10. although data from clinical trials demonstrating the Cigarette smoking, drugs such as oral contraceptives, efficacy of anti-tumour necrosis factor (TNF) agents hormone replacement therapy, and non-steroidal an- suggest a common pathogenic link between intes- ti-inflammatory drugs, have also been associated with tinal and extraintestinal disease activity 19. EIMs are an increased risk of UC 11. The incidence of CD has primarily associated with CD and are present in 43% surpassed that of UC in many Western regions; and of patients with CD; they can affect multiple organ the main risk factors are cigarette smoking, gut dys- systems, including the musculosketal (arthropathy, biosis, and dietary changes3. Antibiotic exposure in arthritis and ankylosing spondylitis), ocular (uveitis, childhood increases the risk of CD 11,12. Familiar inher- scleritis and episcleritis), and hepatobiliary (primary itance of CD is well recognized, with a concordance sclerosing cholangitis) systems 20. Additional EIMs rate among monozygotic twins higher for CD (50%) have also been described, such as metabolic bone than for UC (15%) 13. disease and pulmonary or deep-venous thromboem- Recently, an increasing number of monogenic diseas- bolic disease 3. Cutaneous manifestations of IBDs in- es has been described in association with IBDs14. For clude erythema nodosum (EN), pyoderma gangreno- these patients, a genetic diagnosis is of much higher sum (PG), Sweet’s syndrome, and oral lesions 21. The predictive value and clinical utility for correlate treat- association with erythema nodosum is more common ment options which are not similar such as in classical in CD, whereas pyoderma gangrenosum prevails in IBD 15. These monogenic defects can be grouped into UC 17. Treatment-induced manifestations have also the following pathogenic subtypes: epithelial barrier been observed, such as skin reactions induced by and epithelial response defects, neutropenia and de- anti-TNF therapy which can be misinterpreted as pso- fects in phagocyte bacterial killing, hyperinflammatory riasic lesions 22. However, no single IBD-related symp- and autoinflammatory disorders, disorders affecting T tom is pathognomonic of IBD or specific for CD or UC; PATHOLOGICAL DIAGNOSIS OF IBD 41

detailed questioning about the timing of the onset of binations of several markers has been attempted. symptoms is imperative to differentiate IBD from non- In patients with a diagnosis of IBD, the predictive IBD colitis and to better subclassify IBDs 5,17. value of a pANCA-negative/ASCA-positive result Numerous hematological parameters, including was 95% for CD, while that of a pANCA-positive/ white blood cell count, hemoglobin and hematocrit, ASCA-negative result was 90% for UC 23. platelet count, iron saturation, erythrocyte sedimen- tation rate (ESR) and C-reactive protein (CRP) may cover the full spectrum from normal to markedly ab- Gross features normal in IBDs, but these parameters are not spe- cific for the diagnosis of IBD 17. Fecal calprotectin is UC classically shows diffuse and continuous chronic highly specific in differentiating IBD from functional inflammation involving the rectum and spreading prox- bowel disease and very useful in monitoring disease imally, with gradually decreasing severity 4. The transi- activity, relapse, and response to therapy 3. However, tion between adjacent involved and healthy mucosa is fecal calprotectin is not specific for IBD because this sharp. The mucosa has a friable, granular appearance marker can be elevated in a variety of non-IBD con- which can progress to mucosal denudation or deep ditions, such as enteric infections, drug therapy, and penetration; the initially superficial ulcers, in severe or intestinal neoplasms 17. For this reason, clinical in- longstanding disease, may reach the muscularis mu- vestigation is essential, and an integrated approach cosae 24. Extensive ulceration with sparing of mucosal is of paramount importance. Every evaluation for islands may give rise to inflammatory pseudopolyps. suspected IBD should include a stool culture and a Unusual macroscopic distribution patterns, such as serological exam 17. rectal sparing, caecal patch and backwash ileitis, need to be recognized to avoid a misdiagnosis of CD 25. The rectum may be spared in children (30%), in adults with Serological markers fulminant colitis (13%) or in patients receiving topical and/or systemic treatment (44%) 4. Another thera- Antibody markers in IBDs can be classified into two py-related finding is patchiness, i.e. discontinuous main groups: autoantibodies, which are antibodies rather than continuous inflammation. For these rea- against intestinal and non-intestinal self-antigens, sons, clinical correlation is mandatory. Caecal patch and microbial antibodies, which are antibodies relat- consists in inflammation surrounding the appendiceal ed to microorganisms, including bacteria, yeasts and orifice associated with left-sided colitis, diagnosed in fungi. In addition, antibodies against some peptides, up to 75% of patients with distal disease 26. the origin of which remains unclear, have also been The localization of CD usually remains stable over reported 23. In clinical practice, only two markers have time, with approximately one third of patients present- demonstrated reasonable sensitivity and specificity in ing with colonic disease, one third with ileo-colic and the diagnosis of IBD and its subclassification: one third with small-bowel limited disease 3. Upper • Anti-neutrophil cytoplasm antibody (ANCA): gastro-intestinal involvement in CD refers to esopha- ANCA is an autoantibody, classified according to geal, gastric, duodenal and jejunal involvement, either two staining patterns: cytoplasmic ANCA (cANCA) isolated or together with other localizations, with a and perinuclear ANCA (pANCA). In IBD, pANCA prevalence of 35-75%, as reported in different stud- is thought to be induced by a cross-reaction with ies 27. Characteristically, macroscopic examination of intestinal bacterial antigens. pANCA is detected in a resection specimen shows a discontinuous pattern 60-70% of UC cases, 10-15% of CD cases, and of inflammation, with diseased segments frequently less than 5% of non-IBD colitis cases. Moreover, and abruptly separated by areas of uninvolved bowel patients with pANCA-positive CD exhibit a clinical (skip lesions). The serosal surface of an involved bow- phenotype resembling UC 17,23. el segment is often hyperemic and may be covered • Anti-Saccharomyces cerevisiae antibody (AS- with inflammatory exudate, and in longstanding dis- CA): ASCA is an anti-glycan antibody, a microbi- ease, serosal adhesions may occur 4. Mainly in small al antibody against the mannan found in the cell bowel CD, but less frequently also in large bowel CD, wall of baker’s yeast (S. cerevisiae) 23. The ASCA adipose tissue expands towards the antimesenteric IgG-positive rate is 60-70% in patients with CD, surface, a finding termed “fat wrapping” or “creeping 10-15% in patients with UC, and less than 5% in fat’’. The earliest grossly visible mucosal lesions are patients with non-IBD colitis 17,23. small aphthous ulcers that typically develop along the • At present, no single validated serological antibody mesenteric margin of the bowel wall (over lymphoid exists for the diagnosis of IBD, and the use of com- follicles) and are bordered by normal mucosa 28. As 42 V. Villanacci et al. aphthous ulcers coalesce, they form large deep ser- Biopsy handling piginous or linear ulcers with overhanging edematous mucosal edges giving rise to the classic “cobblestone” To better evaluate all histological parameters required appearance 4. As in UC, inflammatory pseudopolyps for a diagnosis of IBD and to distinguish UC and CD, can occur. Fistulae are more frequent in the small the use of appropriate cellulose acetate filters is highly bowel but, though relatively rare, can also occur in the recommended 29,30. In detail, after sampling the muco- colon. Free perforation, however, is exceptional. The sa, the endoscopist can place each fragment upright bowel wall becomes thickened and increasingly rigid in a straight line with proximal-to-distal orientation on- as a consequence of transmural inflammation with fi- to the cellulose acetate filter, which relies on a clarinet brosis and fibromuscular proliferation 4. beak-shaped cut for orientation (Bio-Optica, Milan, Italy). All tissue samples should be fixed immediately by immersion in 10% buffered formalin and, after ade- Endoscopic sampling quate fixation, processed and paraffin-embedded with 90°-rotation by the technician, in order to ensure the The histological examination of endoscopic biopsies best trans-sectional cut 30. Since lesions may be fo- is a crucial step in the diagnostic work-up of suspect- cal, multiple sections from each tissue sample should ed IBD patient and it is crucial in making a final diag- be examined 2,29. However, the optimal number of sec- nosis, particularly in differentiating between UC and tions to be examined in daily practice has not been es- CD and other forms of non-IBD colitis 4,29. In patients tablished yet; presently, the numbers reported in differ- 4 with suspected IBD, histological examination should ent studies vary between two and six sections . F o r be performed before initiation of treatment because daily practice, step-sections with two or three tissue drugs can induce changes in morphology 4. All the levels, each consisting of five or more sections, 2,4 different segments of the ileo-colic tract should be ex- are recommended . Staining with hematoxylin 29 tensively sampled, regardless of the endoscopic ex- and eosin (H&E) is sufficient for diagnosis . If a cel- tension of the disease 2,30. Endoscopic sampling is re- lulose acetate filter is not available, it is imperative to 2 quired also during follow-up to assess the therapeutic use separate vials for each intestinal biopsy site . efficacy in mucosal healing and for cancer preven- tion 29. Optimal sampling for newly diagnosed IBDs requires at least two biopsies in the terminal ileum Sampling of surgical resection and in each segment of the large bowel (caecum, as- A surgical specimen requires systematic sched- cending, transverse, descending, sigmoid colon and uled gross examination. Specimens, generally rectum), even if the mucosa appears endoscopically small-bowel segmentectomy or right hemicolectomy 2,31 normal . If the upper digestive tract is suspected to for CD, total colectomy for UC, should be opened be involved, esophageal, gastric and duodenal biop- longitudinally along the antimesenteric side. Macro- 31 sies are indicated . The minimum sampling during scopic aspects that constitute potential diagnostic follow-up consists in at least two biopsies in all large features, such as mucosal alterations, pseudopol- bowel tracts showing significant lesions and two bi- yps, erosions/ulcerations and fistulae, should be re- 2,30 opsies in the rectum . In case of discordance be- corded. Special attention should be paid to findings tween the initial histological diagnosis and the clinical suspicious for neoplasm, such as elevated lesions course during follow-up, it is mandatory to repeat a or masses. Wall thickness and serosal findings complete sampling to settle the question. It is also should also be described. Tissue samples for his- strongly recommended that previous biopsies be crit- tological examination should include resection mar- ically re-evaluated and that such review be comment- gins, lymph nodes, terminal ileum, ileo-cecal valve ed on in the final histological report 29. Finally, for the and appendix, if present. Seriated sampling, both detection of dysplasia during follow-up in longstand- from affected mucosa and macroscopically normal ing disease, it is recommended using high-definition mucosa, improves the diagnostic yield. The site of chromoendoscopy with targeted biopsies, with two bi- each tissue sample should be recorded 4. Details opsies every 10 cm in separate vials, submitting any about the mesentery, such as size and eventual suspicious lesion or mass separately 32. If white-light lesions, should also be recorded. A recent paper endoscopy is used, the recommendation is to take showed a reduction of surgical recurrence (over four biopsies for every 10 cm of the entire colon as 40%) when the mesentery was included in an ileo- well as targeted biopsies from any visible lesion 2. colic resection 33. PATHOLOGICAL DIAGNOSIS OF IBD 43

Key points: ular architecture can be observed at the level of the • IBDs are mutifactorial diseases. Their diagnosis is ileo-cecal valve, near the appendiceal orifice and in clinical and requires integration of clinical informa- proximity lymphoid follicles. tion, endoscopic findings and morphological fea- Superficial epithelial features tures as well as data from laboratory and imaging procedures; The superficial epithelium is composed of absorptive • no single serological test or antibody is specific for and goblet cells located in a continuous layer covering diagnosis of IBD; a combination of several markers the basement membrane, without any interruption or is needed for correct management; erosion. Pseudo-detachment of the epithelium from • pANCA is detected in 60-70% of UC cases, 10- the basal membrane or occasional neutrophils in the 15% of CD cases, and less than 5% of non-IBD co- surface can be observed as an effect of bowel prepa- litis cases; the IgG ASCA-positive rate is 60-70% in ration. patients with CD, 10-15% in patients with UC, and Epithelial cellular components less than 5% in patients with non-IBD colitis. Thus, the pANCA-negative/ASCA-positive phenotype is Other than absorptive and goblet cells, Paneth cells associated with CD, while the pANCA-positive/AS- can be observed in the right colon and transverse co- CA-negative combination is most often seen in UC; lon. In the upper and middle third of the crypts, oc- • standardized endoscopic sampling of intestinal casional apoptosis can be seen; in the lower third, mucosa and a correct handling of biopsies are mitoses are common, and immature cells can some- mandatory to improve histological diagnosis of times be seen. IBD. Similarly, a correct sampling of surgical spec- Lamina propria cellularity imens in patients without previous IBD diagnosis can yield insights. Lymphocytes and plasma cells are normally present in the lamina propria and are more abundant in the cecum, decreasing in density from the surface ep- Histopathological features of normal ithelium to the muscularis mucosae (i.e. top-heavy). intestinal mucosa Throughout the right colon, eosinophils are normally present. Primary and even secondary lymphoid fol- The diagnosis of IBD relies on the identification of in- licles can be observed, sometimes extending to the dividual histopathological lesions. Particular attention submucosa. Acute hemorrhagic foci and pseudolipo- should be placed on distinguishing true lesions from matosis are alterations caused by endoscopic trauma; alterations caused by bowel preparation or trauma, edema can be a consequence of bowel preparation. and from minimal changes that fall within the spectrum of variability of the normal colonic mucosa. Therefore, we begin by briefly describing the features of normal Hallmarks of UC colonic mucosa, based on the four major milestones of pathological changes 29. The diagnosis of UC is based on elementary histo- logical lesions that fall into the four main categories Mucosal architecture described below 4,29,30: Normal terminal ileal mucosa is characterized by the 1 Mucosal architecture: Crypt architectural abnor- ratio between the height of the villi and of the crypts, malities are a consequence of chronic inflamma- which is always in favor of the villus (2:1 or more). The tion. Crypt distortion consists in crypts that are no irregularity of the profile of the villi may be observed in longer parallel, vary in diameter and/or are dilated. correspondence of the lymphoid follicles. The normal Crypt branching represents regeneration through number of intraepithelial T lymphocytes in the villi is fission: branching of more than 10% of crypts or fewer than 4 per 100 of the enterocytes 34. presence of more than two branched crypts in a The normal colonic mucosa is exemplified by the “test well-oriented biopsy specimen with at least 2 mm tubes in a rack” analogy: a flat luminal surface with of muscularis mucosae is abnormal. Crypt atrophy crypts that are parallel, straight, homogenously sized, is defined as shortened crypts, accompanied by regularly spaced, and with the bottom reaching the an increased layer of lamina propria stroma be- muscularis mucosae. No more than 10% of the crypts tween the crypt base and the muscularis mucos- should be branching. The average crypt density is 7-8 ae. In UC, distorted crypt architecture with crypt per millimeter of mucosal length although this value branching and atrophy is present in 57-100% of may decrease in the cecum and distal rectum. Irreg- cases, increasing with the duration of disease; this 44 V. Villanacci et al.

is an important concept because in very early on- value for diagnosis and can be identified in 38% of set and, in particular, in children, the architecture patients within 2 weeks after presentation 2,4. The as- of the crypts can be nearly normal. sociation between basal plasmocytosis and eosino- 2 Superficial epithelial features and phils raises sensivity for the diagnosis of UC 35. Strong 3 Epithelial cellular components: Abnormalities immunohistochemical expression of TNFα in plasma include surface epithelial damage, such as flat- cells has been recently described in UC patients at tening, focal cell loss, erosions, and ulcers which early stage disease, with possible implications for reflect the activity of the disease. Mucin depletion, treatment approaches 38. defined as reduction in the number of goblet cells In long-standing disease, there is usually wide- and/or reduced quantity of intracellular mucin, is a spread architectural crypt distortion with Paneth cells weak diagnostic feature in UC as it also occurs in metaplasia in the left colon and rectum, and decreased other diseases, such as infectious colitis and CD. cellularity of the lamina propria. In this situation, con- Paneth cell metaplasia in the left colon, inflamma- fusing features such as patches of normal mucosa, tory polyps, hypertrophy of the muscularis mucos- discontinuous inflammation and rectal sparing, may ae, and the much less frequently observed submu- occur leading to a misdiagnosis of CD. Under medical cosal fibrosis are additional features of chronicity. treatment, the extent of colon involvement tends to de- 4 Lamina propria cellularity: The inflammatory in- crease, resulting in complete restoration of the rectal filtrate in untreated disease is limited to the mu- mucosa in 34-44% of patients. Similarity between UC cosa, and is diffuse and continuous without any and CD may result in diagnostic dilemmas, in particu- variations in intensity or skip lesions. Its severity in- lar when clinical information is not available 39. Back- creases characteristically towards the rectum and wash ileitis occurs in approximately 20% of patients particularly its lower third, which has the lowest with active ulcerative pancolitis and consists in a mild lamina propria cell density in normal conditions. degree of neutrophilic inflammation in the lamina pro- Plasma cells are predominantly observed between pria, which is often patchy in distribution and occa- the base of the crypts and the muscularis mucos- sionally associated with cryptitis, crypt abscesses and ae (basal plasmacytosis). This feature is helpful in a mild degree of villous atrophy and regenerative epi- differentiating between a first episode of UC (63%) thelial changes 25. and infectious colitis (6%). Neutrophils, which re- Active and inactive (quiescent) disease: To differ- flect disease activity, are present in the lamina pro- entiate between histologically active and inactive (qui- pria and/or invade the surface or crypt epithelium, escent) disease, many histological scores have been resulting in cryptitis or crypt abscesses (presence proposed to evaluate therapeutic efficacy, because of neutrophils within crypt lumina). Crypt abscess- endoscopic remission is not always paralleled by his- es are more common in UC (41%) than in CD tologically quiescent disease 2. The histological scores (19%). The number of eosinophils is variable, but reported in the literature have been elaborated for sci- their coexistence with basal plasmocytosis (more entific purposes and are affected by low inter-observer than 3 at the base and lateral part of crypts) make reproducibility and by complexity in their daily practice a diagnosis of UC more likely, especially at first application 2. The Nancy Index (NI) is the recommend- endoscopic sampling 35. Moreover, at first diagno- ed histological score 2. In the NI, grade 4 means that sis, a large number of eosinophils (over 60 for 10 ulceration is present; grade 3 refers to a moderate or HPF), in particular in the left colon, can be predic- severe neutrophil infiltrate with no ulceration; grade tive of non-reponse to medical therapy 36,37. Gran- 2 indicates that there are only a few neutrophils and ulomas are generally not observed and need to be no ulceration; grade 1 refers to a moderate or severe distinguished from those generated in response chronic inflammation (plasma cells, lymphocytes and/ to foreign bodies or to mucin from ruptured crypts. or eosinophils) with no neutrophil activity or ulcera- These histopathological modifications are time-re- tion; and grade 0 represents little or no chronic in- lated and do not necessarily match the endoscopic flammation with no neutrophils and no ulceration 40. aspect (Fig. 1). Moreover, a simplified score has been proposed to as- In early stage disease, the diagnosis of UC can be ses mucosal healing in IBDs 41. This score takes into challenging. Preserved crypt architecture and ab- consideration only three well-defined histological var- sence of a transmucosal inflammatory infiltrate do not iables: a) crypt abscesses; b) presence of granulation rule out early stage UC, in which the distinction from tissue or aggregates of inflammatory elements in the infectious colitis (acute self-limiting colitis) is a major superficial parts of the mucosa, indicative of erosions concern. Focal or diffuse basal plasmacytosis is the or ulcers; c) neutrophils in the lamina propria and their earliest diagnostic feature with the highest predictive anatomic localization, thus greatly limiting the subjec- PATHOLOGICAL DIAGNOSIS OF IBD 45

Figure 1. (A, B, C) Ulcerative colitis with moderate-severe activity. (D) Basal plasmocytosis with plasma cells and eosinophils (arrows). H&E A-B: 4x, C: 20x, D: 60x. (E, F, G, H) Ulcerative colitis in remission (quiescence of disease) H&E E: 10x, F-G-H: 20x. (I, L, M, N) Ulcerative colitis: surgical specimen with inflammation located in the mucosa. H&E I-L: 4x, M-N: 20x.

tive interpretation by the pathologist. In particular, the loss of tissue up to the underlying lymphoid follicle, presence of neutrophils was considered the hallmark are frequently observed. to differentiate the active from the remission/quiescent 2 Mucosal architecture alteration: Crypt distortion phase, and the expression of therapeutic efficacy, i.e. and atrophy are less pronounced than in UC and, histological mucosal healing. Though this simplified like inflammation, show a variable distribution. score needs validation, it has been was shown to be 3 Mucosal components alteration: These are gen- easier and less time-consuming in daily practice, and erally less prominent than in UC and focal or dis- can be proposed as a valid alternative 41. continuous. Pyloric gland metaplasia is related to mucosal ulceration and repair and is observed in 2-27% of ileal biopsies from patients with CD, while Hallmarks of CD is more common in ileal resection specimens. 4 Lamina propria cellularity: Focal (discontinuous) A variety of microscopic features support the diagno- chronic inflammation of variable density through- sis of CD. The features with the highest diagnostic val- out the biopsy specimen and not confined to the ue are discontinuous chronic inflammation, focal crypt superficial mucosa is characteristic of CD. One or architectural distortion and granulomas not related to more circumscribed foci of increased mononuclear crypt injury, which are observed in the small and large cell density with or without infiltration of neutrophils bowel as well as in esophageal, gastric and duodenal is generally found, on a background of normal mu- localizations 4,29,30. The main alterations are described cosa. Normal lymphoid aggregates are not consid- below. ered as focal inflammation. As in UC, the presence 1 Superficial epithelial alterations: Irregular villous of neutrophils in the lamina propria or in the epithe- architecture in ileal biopsy sample, for the discon- lium, including cryptitis and crypt abscesses, is an tinuous inflammatory infiltrate, is the most frequent hallmark of active disease. Another usual finding in and characteristic alteration. Aphthous ulcers, i.e. CD is epithelioid cell granuloma, a discrete collec- 46 V. Villanacci et al.

tion of at least five epithelioid cells, with or without They are represented by the focal penetration of multinucleated giant cells, not related to crypt inju- neutrophils and eosinophils into the surface epi- ry, and often poorly delimited. Notably, non-caseat- thelium 47,48 (Fig. 2). ing granulomas, small collections of epithelioid histiocytes with giant cells or isolated giant cells, occur also in infectious colitis (acute self-limiting IBDs in children and adolescents colitis) and parasitic infections, in particular Yersin- ia enterocolitica or pseudotuberculosis and intes- IBDs are an important cause of gastrointestinal pathol- ogy in children and adolescents and about 10-15% of tinal tuberculosis. When the ileitis is in continuity IBDs are diagnosed before the age of 18 years. Pedi- with proximal colitis, caution is needed, because atric histological features are distinctly different from these lesions may also occur in backwash ileitis. those found in adults, a finding which may complicate Neural hypertrophy (nerve fiber hyperplasia) can the diagnosis and thus delay the onset of adequate be present. Infiltration of eosinophils or T lympho- therapy. Children with untreated UC commonly present cytes into the submucosal or intramuscular plexus with subtotal or extensive colitis, but with less severe in- (ganglionitis) should be reported on surgical spec- flammation, less epithelial injury and less architectural imens, such as transmural nodular inflammation. abnormality than UC in adults. Backwash ileitis occurs When present at the resection margins, this finding with similar frequency, but children more often present is prognostic of disease recurrence 42-46. The mu- with unusual histological inflammation patterns, such cosal lesions of early CD are found primarily inthe as patchiness (21%) and rectal sparing (30%) 4. ileum. The lesions usually have a focal distribution At onset, CD in children less frequently affects ileum over a regular morphological background of villi. and is often characterized by colitis with rectal in-

Figure 2. Crohn’s disease. (A) Patchy inflammation, (B-C) granuloma, (D) granuloma. H&E A-B: 20x, C: 40x, D: 60x. (E, F, G, H) Crohn’s disease surgical specimen, inflammation penetrating the different layers of the terminal ileum H&E E: 4x, F: 10x, G: 20x, H: 40x. (I, L, M, N) Early Crohn’s disease, focal superficial inflammation in the terminal ileum. H&E I: 10x, L: 40x, M: 20x; N 40x. PATHOLOGICAL DIAGNOSIS OF IBD 47

volvement which complicates the distinction between 0-29.6%) and pouchitis, and also depends on the type CD and UC. In such a case, the presence of areas of immunosuppressive drugs used 49. The histological with inflammation alternating with areas with less (or diagnosis is currently considered the gold standard 50. without) inflammation in each of the multiple colonic Although inclusion bodies indicative of a CMV infec- biopsies should be noted, since focal distribution of tion can be detected in H&E-stained slides, immu- inflammation is highly suggestive of CD. The frequen- nohistochemistry or molecular techniques are more cy of granulomas is higher in children than in adults 4. sensitive and have a higher diagnostic accuracy 4,50. In addition, in pediatric patients with CD, upper gastro- On H&E-stained slides, CMV typically presents as intestinal involvement is more frequent than in adults, isolated cytomegalic endothelial cells and fibroblasts and focal inflammation may be found in the esopha- in granulation tissue. These are typically two to four gus, stomach, and/or duodenum 4. times larger than normal, with large amphophilic in- Peculiar and unusual histological patterns can be tranuclear inclusions, surrounded by a clear halo, observed before 6 years age configuring VEO-IBDs, and smaller cytoplasmic inclusions. However, some such as the apoptotic pattern, plasma-cells depletion, infected cells are morphologically less characteristic and moderate inflammation. The clinical manifesta- and the morphology of ganglion cells and necrotic or tions and histopathological features of each VEO-IBD apoptotic cells may resemble CMV inclusions 4. More- are very peculiar and specific and, for more details, over, due to sampling error, false-negative biopsies we refer the reader to a selected bibliography 16. are common 50. Immunohistochemistry increases the diagnostic yield in comparison with H&E staining, and its sensitivity reaches 93% 4,50. Indeterminate colitis It remains unclear whether CMV reactivation exacer- bates the course of IBD, but subclassifying patients In approximately 5% of clinically suspected IBDs, a based on the number of CMV-positive cells can im- definite diagnosis of UC or CD cannot be established, prove therapeutic management 51. Five or more viral most often due to either insufficient clinical, radiologic, inclusions constitute the high-grade CMV density endoscopic or pathological data, or overlapping fea- group 50, in which colectomy rates differed significantly tures of both disorders. Labels such as “indeterminate by the antiviral therapeutic status (44% of patients on colitis” (IC), “inflammatory bowel disease unclassified therapy vs 83% of patients without therapy) 51. Few- (IBDU)”, “chronic inflammatory bowel disease unclas- er than five inclusions constitute the low-grade CMV sified” and “chronic idiopathic inflammatory bowel density group 50, the colectomy rate of which did not disease not otherwise specified” are in use for such differ between patients who underwent antiviral thera- cases 4. The term IC should be restricted to cases py and those who did not 51. in which comprehensive histological examination of The second most frequent infection is caused by surgical specimen is possible. The term IC should be Clostridium difficile, especially in patients being treat- avoided on endoscopic biopsies, on which the term ed with steroids and/or immunosoppressors 52. Histol- IBDU is preferred when a patient with chronic colitis ogy does not allow reliable detection of bacterial su- clearly has IBD based upon the clinical history, but perinfection of the small or large intestine, because endoscopy and/or endoscopic biopsies show no de- the histologically characteristic pseudomembranous finitive features of either UC or CD 4,30. colitis is usually not present in IBD patients 4. Such evaluation, therefore, should include assessment of three sets of stool samples for all patients. Finally, Intestinal superinfection in IBD specific testing for sexually transmitted diseases, such as Neisseria gonorrheae, Herpes simplex virus, Chla- In active IBD, a superimposed bacterial or viral infec- mydia trachomatis and Treponema pallidum, should tion should always be excluded, especially in patients be considered in specific instances, and particularly using immunosuppressive drugs, with colonic involve- in patients with severe rectal symptoms 52. ment, or with a sudden exacerbation. Cytomegalovi- rus (CMV) is an opportunistic pathogen having tro- pism for inflamed tissue 49. Testing for CMV detection Pouchitis should be performed in all patients with severe colitis refractory to immunosuppressive therapy and on bi- Proctocolectomy with ileal pouch anal anastomosis opsies with prominent granulation tissue associated (IPAA) is the procedure of choice for most patients with large ulcers 4,30. In UC, the risk of CMV reactiva- with UC refractory to treatment. Usual histological tion is significantly higher than in CD (10-56.7% vs findings, present in up to 87% of biopsies from healthy 48 V. Villanacci et al. pouches, consist of chronic inflammatory changes, Differential diagnosis of IBD colitis such as architectural distortion, villous atrophy, crypt hyperplasia and infiltration of the lamina propria by At first diagnosis, IBD needs to be differentiated from mononuclear cells, eosinophils and histiocytes. Neu- non-IBD colitis which can have similar clinical and his- trophils are rarely present. Villous atrophy and crypt topathological findings. For the differential diagnosis hyperplasia are considered adaptive changes (termed between IBD and non-IBD colitis, we refer the reader colonic metaplasia). Mild ischemic changes occa- to the manuscript entitled “Histopathology of Non- sionally occur, while some patients develop features IBD Colitis. A practical approach from the Italian of mucosal prolapse, often in the anterior wall 4. The Group for the study of the gastrointestinal tract term pouchitis refers to active inflammation of the IPAA (GIPAD)”, which is also included in this special issue 6 mucosa and is considered as a primary, non-specific, of Pathologica . idiopathic inflammation of the neorectal ileal muco- Key points: sa 4. The etiology of pouchitis is unknown; however, it • A systematic approach in the histopathological probably results from an immune-mediated injury that evaluation of biopsies is the first step to establish occurs in genetically predisposed individuals. Specific the diagnosis of IBD. The four histological parame- pathological predictors of pouchitis include the pres- ters are: mucosal architecture, superficial epithelial ence of colonic superficial fissuring ulcers, severe ap- findings, epithelial cellular component and lamina 25 pendiceal inflammation, and severe pancolitis . The propria cellularity; diagnosis of pouchitis is based on a combination of • a combination of specific alterations of each of the clinical symptoms, endoscopic and histological find- four parameters can suggest a diagnosis of UC or ings 25. Histological changes may be patchy and more CD; prominent in the lower and posterior regions of the • in the early stage, it is important to differentiate IBD pouch. Consequently, multiple biopsies from these from non-IBD colitis; sites are essential for the diagnosis 4. In pouchitis, • the pathological report in the diagnosis of IBD re- patchy intraepithelial neutrophil infiltrates become quires a detailed description of mucosal alterations more numerous and cryptitis ensues, with crypt ab- as well as a definition of “active” or “inactive” dis- scesses and ulcerations. In this case, other possible ease and the CMV immunoistochemical assess- causes such as infections (particularly CMV) should ment; be considered. While in patients with acute pouchitis • IBDU is the preferred definition when endoscopic antibiotic therapy is usually effective, approximately biopsies show no definitive features of either UC 10-15% of these patients develop chronic pouchitis, or CD. with CD-like complications including perianal fistu- las and inflammation, stenosis or fistulas in the pre- pouch ileum and/or in the pouch itself. Approximately IBD-associated dysplasia 12% of patients develop medically refractory pouchitis Patients with IBD carry an increased risk of colorec- that may require immunosuppressive therapy or, ulti- tal cancer (CRC) 53. Dysplasia is the best and most mately, surgical resection of the pouch. Histology of reliable marker of increased risk of malignancy, and pouches excised for these complications may show it is defined as “histologically unequivocal neoplas- deep submucosal lymphoid aggregates and granula- tic epithelium without evidence of tissue invasion” 54. tion tissue-lined fistulous tracts. Similar changes, and The identification and grading of dysplasia is the even granulomas, have been observed in blind-end- cornerstone of management of IBD 55. Dysplasia re- ing rectal stumps left in situ after total colectomy for lated to IBD develops only in areas with chronic in- UC. The occurrence of CD-like complications and the flammation in any part of the colorectum, and is often presence of deeply located lymphoid aggregates do multifocall 53. In IBD, “flat dysplasia” refers to lesions 25 not refute a diagnosis of UC . CD should only be that are endoscopically undetectable, whereas “ele- diagnosed after IPAA surgery when re-examination of vated dysplasia” refers to endoscopically detectable the original proctocolectomy specimen shows typical lesions. Flat dysplasia is generally detected in random features of CD. The presence of inflammation and/or biopsies from endoscopically unremarkable mucosa, ulceration in the afferent limb proximal to the pouch and carries a high risk for cancer and, in this case, a often represents recurrent CD, but, interestingly, prox- synchronous adenocarcinoma is found in 42-67% of imal limb ulcers have also been described in UC pa- cases 4,53. Depending on the endoscopic appearance, tients who had been taking NSAIDs 25. the elevated lesions/dysplasia are subclassified as PATHOLOGICAL DIAGNOSIS OF IBD 49

“adenoma-like” and “non-adenoma-like”. The latter is illary architecture. In crypt dysplasia, the dysplasia is termed “colitis-associated dysplasia (CAD)”; the term limited to the crypts and does not extend to the sur- “dysplasia-associated lesion or mass (DALM)” is no face epithelium 53. The indefinite for dysplasia catego- longer recommended 56. ry is appropriate for cases in which a definite distinc- To simplify the matter, we define as ‘‘adenoma-like tion between non-neoplastic changes and dysplasia mass’ (ALM) the condition in which the dysplastic le- cannot be made, for deep and active inflammation. sion is outside the area affected by the disease, and Indeed, regenerating epithelium in active IBD often CAD as a dysplastic lesion localized in the context of shows changes overlapping with those observed in the disease 57. true dysplasia, e.g. enlarged and variably sized nuclei, Microscopic features are used to categorize the dys- nuclear hyperchromasia, stratification and pleomor- plasia, including both architectural and cytological phism, prominent nucleoli, and an increased number abnormalities, leading to four distinct categories: of mitoses 53-55. One of the hallmarks of a regenerative negative for dysplasia, indefinite for dysplasia, and process is the maturation of epithelial cell nuclei to- positive for low-grade (LGD) or high-grade dysplasia wards the luminal surface in contrast with dysplasia, (HGD) 4,53. Conventional (or intestinal type) dysplasia which extends uniformly along the crypt axis and sur- is the more recognized form of dysplasia 53,55 (Fig. 3). face epithelium with little or no surface maturation 53. Cytological alterations include nuclear enlargement Confirmation of dysplasia by an expert gastrointesti- with increased nuclear/cytoplasmic ratio, mucin de- nal pathologist has been recommended, and even the pletion, nuclear localization within the cells, LGD be- problem of poor reproducibility persists even among ing characterized by nuclei confined to the basal half experts 4,53,55. p53 immunostain overexpression may of the cells and HGD by nuclei distributed haphazardly help in distinguishing dysplasia from regenerative al- between the basal and apical halves. With progres- terations. The “all or nothing” concept means that dys- sion from LGD to HGD, the nuclei become marked- plastic epithelium should stain either strongly positive ly enlarged, pleomorphic, hyperchromatic with loss (due to impaired protein degradation) or completely of nuclear polarity, round, and oval-shaped. Mitoses negative (due to protein truncation); however, this is are frequent and, in HGD, sometimes atypical 53-55. Ar- currently a very controversial issue 53,58. chitectural features include abnormal growth pattern In addition to the adenomatous lesions, even serrat- including glandular crowding, tubular or villiform archi- ed lesions can occur in IBD patients. These are char- tecture, and the absence of normal base-to-surface acterized by a saw-toothed glandular contour and maturation 53-55. The LGD-HGD transition is charac- usually lack classic cytologic dysplasia, and include terized by greater architectural complexity, as well as sessile serrated lesion without cytological dysplasia glandular crowding and cribriform or significant pap- (SSL) and traditional serrated adenoma (TSA). Their

Figure 3. Dysplasia in IBD. (A, B, C, D) Low grade dysplasia A-B-C H&E A: 10x, B: 20x, C: 40x; D: immunostain for P53 20x. (E, F, G, H) High grade dysplasia E-F-G H&E E: 10x, F-G: 40x; H: immunostain for P53 40x. 50 V. Villanacci et al.

prevalence, anatomic distribution and rate of cytologic point of view, IBD-associated CRC develops from dysplasia development are similar to general popula- more polymorphous dysplastic lesions than sporadic tion 59. However, other forms of serrated lesions that CRC, and is often multifocal. IBD-associated CRC is do not meet the histomorphological criteria of SSP or characterized by Crohn’s like inflammatory reaction, TSA have been frequently observed in patients with tumour heterogeneity, presence of mucin and signet IBD, and their association with IBD-CRC has been ring cell differentiation, and well-differentiated tumours noticed 60. Finally, several less common non-adeno- and lack of tumor necrosis 53. The molecular pathway matous and non-serrated epithelial changes/lesions in IBD-related CRC involves alterations in key regu- have been increasingly found prior or adjacent to latory genes in the intestinal epithelium that are also IBD-CRC, and have been considered to be putative found in sporadic CRC; however, the timing of these neoplastic changes and likely variants of CAD, includ- changes is different, because of the chronic inflam- ing villous hypermucinous, goblet cell-depleted or mation that characterizes long-standing IBD 63. Unlike eosinophilic changes/lesions, in which no frank ad- sporadic CRC, loss of p53 tumor suppressor function enomatous cytologic dysplasia is appreciated 61. The and microsatellite instability (MSI) are early events in latest WHO classification of Tumours of the Digestive IBD-associated CRC, while adenomatous polyposis System briefly mentions an IBD-associated dysplastic coli (APC) loss of function is less frequent and ap- lesions classification with seven subtypes: (1) con- pears later 63. Metaplastic changes showing gastric ventional adenoma-like (intestinal, see above), (2) hy- and/or pancreatobiliary immunophenotype have fre- permucinous, (3) SSP-like, (4) TSA-like, (5) dysplasia quently been observed in dysplastic or non-dysplastic with terminal epithelial differentiation, (6) goblet cell mucosa adjacent to CD-associated carcinomas, sug- deficient/depleted and (7) serrated, NOS 54. Each of gesting a precancerous role of such lesions 64. Finally, these categories has a distinct morphological and bi- Epstein-Barr virus (EBV)-positive carcinomas have ological profile and has been shown to be associated been described in the ileum of CD patients and are with a distinct CRC subtype 62. However, there is no characterized by increased tumor-infiltrating lympho- clinical utility to further subtype IBD-associated dys- cytes (TILs), absence of MSI, and a better prognosis plasia or IBD-associated CRC 55. compared to sporadic cases 65.

Key points: IBD-associated adenocarcinoma • IBD is associated with an increased risk of neopla- sia, most frequently CRC, of which dysplasia is the Compared with the general population, IBD patients best and most reliable marker; are at increased risk of developing some cancers, • conventional/intestinal type dysplasia is the more particularly intestinal and biliary tract adenocarcino- recognized form of dysplasia and it is subclassified ma 53,63. Chronic inflammation is believed to promote into indefinite for dysplasia, LGD and HGD; carcinogenesis, also in the extra-intestinal sites and • the key features of LGD are: (i) abrupt transition with non-epithelial neoplasms as lymphomas, acute with a distinct area of glands that demonstrate a myeloid leukemia, myelodisplastic syndromes, skin lack of surface maturation, (ii) mucin depletion, (iii) cancers, and urinary tract cancers, thought to be at nuclear enlargement and hyperchromasia, and (iv) least partly imputable to immunosuppressive thera- increase in mitosis; pies 53. CRC is the most frequent neoplasm in both • the key features of HGD are: (i) greater architectur- UC and CD, arising through dysplasia. The most im- al complexity, (ii) full-thickness nuclear stratifica- portant risk factor is duration of disease, with the in- tion with loss of nuclear polarity, (iii) deep nuclear cidence of CRC being relatively rare within the first 8 enlargement and pleomorphism and (iv) atypical years of diagnosis. Minor risk factors are age at dis- mitoses; ease onset, extent, severity of disease, inflammatory • CRC in IBD patients has peculiar histological and complications, family history of CRC and associated molecular features compared to sporadic CRC. primary sclerosing cholangitis 63. Compared with spo- radic CRC, IBD-related CRC has several clinical and pathological features: it arises on average 20 years Pathological report earlier, and in CD tends to have a more proximal distribution, while in UC it shows a predominance of The pathology report should give an indication of sigmoid and right colonic localization. Moreover, can- disease localization (i.e. specify the intestinal side of cers complicating CD are often detected in bypassed disease localization) and activity, as reflected by the or excluded bowel segments. From a morphological extent of neutrophil granulocyte infiltration and epithe- PATHOLOGICAL DIAGNOSIS OF IBD 51

lial damage 2,4. In cases of UC, a distinction should be thology. J Crohns Colitis 2020;6:jjaa110. https://doi.org/10.1093/ made between inactive disease and active disease; ecco-jcc/jjaa110 3 and the latter should be graded 2,29,40,41. In CD, there is Roda G, Chien Ng S, Kotze PG, et al. Crohn’s disease. Nat Rev Dis Primers 2020;6:22. https://doi.org/10.1038/s41572-020- less evidence supporting the validity of the histologi- 0156-2 cal grading of the disease activity, but data from drug 4 Langner C, Magro F, Driessen A, et al. The histopathological trials indicate that patients showing mucosal healing approach to inflammatory bowel disease: a practice guide. Vir- have a better outcome 53. In CD, inactivity in a biopsy chows Arch 2014;464:511-27. https://doi.org/10.1007/s00428- may not reflect inactivity of IBD because of the dis- 014-1543-4 5 continuous and transmural character of the disease, Macaluso FS, Orlando A, Bassotti G, et al. How clinicians and pathologists interact concerning inflammatory bowel disease in which induces sampling error, and also because, as a Italy: an IG-IBD survey. Dig Liver Dis 2018;50:734-6. https://doi. rule, biopsy samples of the ileum are limited, whereas org/10.1016/j.dld.2018.03.020 3 the ileum may be the only bowel segment involved . 6 Villanacci V, Reggiani-Bonetti L, Leoncini G, et al. Histopatholo- Presence/absence of both dysplasia and CMV inclu- gy of Non-IBD colitis. A practical approach from the Italian Group sions needs to be reported 2. for the study of the gastrointestinal tract (GIPAD). Pathologica 2021;113:54-65. https://doi.org/10.32074/1591-951X-234 In summary, it is important to not forget that gastroin- 7 Ng SC, Shi HY, Hamidi N, et al. Worldwide incidence and testinal biopsies provide critical information for patient prevalence of inflammatory bowel disease in the 21st cen- management. 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Review

Histopathology of Non-IBD Colitis. A practical approach from the Italian Group for the study of the gastrointestinal tract (GIPAD)

Vincenzo Villanacci1, Luca Reggiani-Bonetti2, Giuseppe Leoncini3, Paola Parente4, Moris Cadei1, Luca Albarello5, Giulio Mandelli1, Alessandro Caputo6 1 Institute of Pathology, Spedali Civili, Brescia, Italy; 2 Department of Diagnostic, Clinic and Public Health Medicine, University of Modena and Reggio Emilia, Modena, Italy; 3 Pathology Unit, ASST del Garda, Desenzano del Garda, (BS), Italy; 4 Unit of Pathology, Fondazione IRCCS Ospedale Casa Sollievo Della Sofferenza, San Giovanni Rotondo (FG), Italy; 5 Pathology Unit, IRCCS San Raffaele Scientific Institute, Milano, Italy; 6 University Hospital “San Giovanni di Dio e Ruggi D’Aragona”, Salerno, Italy

Summary Non-IBD colitides (NIBDC) are intestinal diseases clinically and endoscopically overlap- ping with Inflammatory Bowel Diseases (IBD), sometimes with a similar histological pic- ture. NIBDC include entities such as infectious colitis, ischemic colitis, pseudomembra- nous colitis, eosinophilic colitis, autoimmune enterocolitis, segmental colitis associated with diverticulosis, drug-induced colitis, radiation-induced colitis, diversion colitis, and Received: December 2, 2020 microscopic colitis, this last including two entities: collagenous and lymphocytic colitis. Accepted: December 3, 2020 The knowledge of the most useful histological features and the main clinical data for each entity is mandatory in daily clinical practice, for correct pathological diagnosis and clinical Correspondence management. Alessandro Caputo University Hospital “San Giovanni di Dio Key words: colitis, infectious colitis, ischemic colitis, drug-induced colitis, microscopic e Ruggi D’Aragona”, via San Leonardo 1, colitis 84131 Salerno, Italy E-mail: [email protected]

Conflict of interest The Authors declare no conflict of interest. Introduction

How to cite this article: Villanacci V, Non-Inflammatory Bowel Disease (IBD) colitides (NIBDC) are patho- Reggiani-Bonetti L, Leoncini G, et al. logical conditions having a similar clinical presentation and endoscopic Histopathology of Non-IBD Colitis. A practical approach from the Italian Group for the appearance with Inflammatory Bowel Disease (IBD), but characterized study of the gastrointestinal tract (GIPAD). by peculiar histological features, different etiology, clinical course and Pathologica 2021;113:54-65. https://doi. org/10.32074/1591-951X-234 therapy. NIBDC include infectious colitis (IFC), ischemic colitis (ISC), pseudomembranous colitis (PMC), cosinophilic colitis (ESC), autoim- © Copyright by Società Italiana di Anatomia Pato- logica e Citopatologia Diagnostica, Divisione Itali- mune enterocolitis (AIE), segmental colitis associated with diverticular ana della International Academy of Pathology disease (SCAD), drug-induced colitis (DIC), radiation-induced colitis (RIC), diversion colitis (DVC) and microscopic colitis (MC), this latest OPEN ACCESS one consisting of two entities: lymphocytic colitis (LC) and collagenous This is an open access journal distributed in accordance colitis (CC) 1,2. with the CC-BY-NC-ND (Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 International) license: the Below the histopathological features of the most frequent NIBDCs are work can be used by mentioning the author and the license, summarized. The main differential diagnosis is with IBD, especially at the but only for non-commercial purposes and only in the original version. For further information: https://creativecommons. onset stage of IBD, for the characterization of which we refer to ‘IBD-co- org/licenses/by-nc-nd/4.0/deed.en litides’ in this special issue of Pathologica 3. NON-IBD COLITIS 55

Infectious colitis (IFC) basal plasmacytosis 6. In these cases, clinical integra- tion with serological data and coproculture are needed Being associated with global human movement due to for a correct diagnosis and identification of the patho- tourist and business purposes, infectious colitis (IFC), gens 5. On the other hand, infections due to spirochetes which is caused by enteric pathogens, is a worldwide or protozoa can be easily detected on the mucosal sur- condition 4. Moreover, IFCs are the most frequent cause face, if present. Spirochetes appear as luminal fringes of childhood death in the developing countries and the adherent to the epithelium (Fig. 1A), highlighted with second leading cause of death, after cardiovascular dis- Giemsa, Warthin-Starry histochemical stain or with ease, in the entire world 4. Many aspects of modern liv- immunohistochemistry based on specific antibodies7. ing also contribute to the transmission of enteric patho- Protozoans such as Entamoeba histolytica (Fig. 1B) gens, such as water sports, camping, and consumption are typically found clustered within necrotic debris, of raw or poorly cooked meat. Finally, the most recent and appear morphologically similar to histiocytes con- immunosuppressive therapies in oncologic field in- taining phagocytosed erythrocytes. PAS and Grocott’s crease the number of IFC cases 5. Although many IFCs stains can be used for the identification of histoplasma have a self-limited clinical course, post-infectious bowel spores 8 (Fig. 1C). Strongyloides stercoralis infection is syndrome with prolonged diarrhea or abdominal pain characterized by adult worms and larvae typically lo- can occur in some patients, which requires a more in- cated in the crypt lumina or the lamina propria, and it depth clinical investigation. Endoscopic findings range is surrounded by an inflammatory infiltrate rich in eo- from a quite normal to diffuse erythematous aspect sinophils. Cytomegalovirus (CMV)-infected cells, typi- of the intestinal mucosa; in some cases, an IBD-like cally detected in ulcer debris or in endothelial, stromal appearance with aphthous ulcers can be discovered, or epithelial cells, can be recognized morphologically raising the suspicion of Crohn’s or ulcerative colitis 5. or highlighted immunohistochemically with the specific Histologically, many of these cases show a nonspecific anti-CMV antibody (Fig. 1E) 9. It is important to remem- inflammatory pattern associated with edema and con- ber that CMV colitis can complicate IBD and make its gestion of the stroma but with no crypt distortion nor recognition difficult, and that quantification is crucial for

Figure 1. Infectious colitis. (A) Intestinal spirochetosis. The fuzzy luminal border is highlighted by immunohistochemistry for Treponema pallidum (peroxidase-diaminobenzidine, 40x). (B) Entamoeba histolytica trophozoites containing engulfed erythrocytes can be seen among neutrophils and inflammatory debris (H&E, 20x). (C) Grocott’s stain makes Histoplasma capsulatum stand out as intensely-stained small oval structures within the cytoplasm of macrophages (Grocott’s methena- mine silver stain, 20x). (D) Strongyloides stercoralis can be found lurking in the crypts (H&E, 20x). (E) CMV-infected cells stand out as markedly enlarged cells with typical “owl’s eye” intranuclear inclusions (H&E, 20x). (F) Cryptosporidium parvum forms small round bodies on the luminal border of enterocytes (H&E, 20x). 56 V. Villanacci et al. establishing the correct therapy 3. More specific, or even Pseudomembranous colitis (PMC) diagnostic, morphological patterns are characterized by granulomatous inflammation: PAS-positive schisto- Pseudomembranous colitis (PMC) is a distinctive somal eggs, at times calcified, are contained in micro- infective colitis associated with Clostridium difficile granulomas associated to fibrotic submucosal thicken- infection. Clostridium difficile is a Gram-positive an- 10 ing and lymphoid hyperplasia . Mycobacterium can be aerobic bacillus causing various mucosal alterations, identified by Ziehl-Neelsen stain or with bio-molecular histologically ranging from a mild inflammation to a PCR-based methods in caseating necrotic granuloma deeper pattern of involvement, morphologically re- surrounded by multinucleated giant cells 11. Yersinia en- sembling ischemic injury but with characteristic find- terocolitica infection causes intramural non-caseating ings 12 (Fig. 2). Cryptitis and crypt abscesses are de- granulomas often accompanied by stellate abscesses, usually located in the terminal ileum, mimicking the tectable in biopsy or surgical samples. Typical “mush- Crohn’s disease (IBD-like pattern) 5. Finally, other path- room-shaped” pseudomembranes can be found on ogens, such as Enterohemorragic Escherichia coli and the luminal surface and contain fibrin, epithelial de- Clostridium perfringens, can lead to an ischemic-like bris, and inflammatory cells intermingled with mucus. pattern with haemorrhage associated to acute inflam- Although the histological detection of these morpho- mation, marked oedema, crypt withering, and lamina logical alterations of the colon can be suggestive of propria hyalinization with mucosal necrosis 5. Clostridium difficile infection, definitive diagnosis is based on microbiological isolation of the organism Key points: and detection of the bacterial toxin 12. PMC can also 1 Inflammation in IFC is usually a non-specific pat- present without the characteristic pseudomembranes tern; IBD-like and ischemic-like patterns may also on the superficial epithelium, in particular in patients be recognized; using immunosuppressive agents 13. 2 features of chronicity, such as crypt distortion and basal plasmacytosis, are usually absent; Key points: 3 an accurate evaluation can sometimes help to identify the responsible pathogen, either directly • Characteristic mushroom-shaped pseudomem- (e.g. Entamoeba, Strongyloides) or by its effects branes composed of mucus, fibrin, neutrophils and (e.g. cytomegalovirus); necrotic cells overlie the mucosa in PMC; 4 clinical and serological integration with coprocul- • mucosal damage can range from mild to severe, ture is often imperative to discriminate between with cryptitis, crypt abscesses and gland dilation onset-stage IBD and IFC. without basal plasmocytosis.

Figure 2. Pseudomembranous colitis. (A) At low magnification, a mushroom-shaped necroinflammatory exudate overlies an acutely injured mucosa (H&E, 10x). (B) The pseudomembrane is composed of inflammatory cells, necrotic debris, mucus and fibrin. Cryptitis and crypt abscesses are evident in the mucosa (H&E, 20x). (C) The severity of the inflammation can vary widely, from a mild picture to severe and deep acute inflammation which can obscure other morphological features (H&E, 20x). NON-IBD COLITIS 57

Drug-induced colitis (DIC) as mefenamic acid, diclofenac, naproxen and pirpro- fen 15,16. The ulcerative colitis-like and the microscopic Drug-induced injury of the gastrointestinal tract is a rel- colitis patterns were reported with gold salts treatment atively frequent but often underestimated event due to for rheumatoid arthritis, diclofenac and aminogluteth- several factors, including the lack of knowledge of the imide (an antineoplastic agent), but the morphological side effects of drugs (amplified by the frequent abuse description is not always accurate 15,16. A Crohn’s dis- of some drugs, as is the case with non-steroidal an- ease-like pattern with granulomas has been reported ti-inflammatory drugs -NSAIDs-) 14. Knowledge of the with diclofenac, naproxen and clofazimine (with this lat- temporal relationships between the medication start ter, crystals can be demonstrated in the granulomas). date and the onset of symptoms is critical15. Different A graft-versus-host-disease-(GVHD) like pattern has patterns of DIC have been described: pseudomembra- also been described with Mycophenolate mofetil 15,16. nous colitis pattern, microscopic colitis pattern, IBD- The apoptotic pattern was recently described with type pattern, ischemic-type colitis pattern and, eosino- brentuximab, a monoclonal anti-CD30 antibody used philic-type colitis pattern 16. The diagnosis is essentially to treat relapses of Hodgkin lymphoma 19. Pseudomela- based on clinical suspicion and the awareness that a nosis, melanosis and pseudolipofuscinosis coli are patient is taking drugs, as in most cases of DIC co- the result of long-term use and abuse of anthraqui- lonoscopy is unremarkable. Often, endoscopy shows none-containing laxatives. In this condition, the lipo- flat or slightly elevated circular hyper- or hypochromic fuscin-type pigment is detectable in the macrophages lesions compared to the surrounding mucosa, some- of the lamina propria of the large bowel (including the times with a erythematous border, distributed along appendix). Kayexalate-sorbitol (sodium polystyrene a small vessel “cherry tree” appearance) 17. The mi- sulfonate), used for the treatment of hyperkalemia, croscopic spectrum ranges in hematoxylin and eosin has been reported to induce intestinal necrosis in ure- stained sections (H&E), from mild oedema, to fulmi- mic patients; the presence of the typical crystals is the nant colitis with severe lesions including extensive ne- hallmark of this condition 15,16. Necrosis is observed in crosis (Fig. 3) 15,16. The most common but not specific endoscopic and surgical specimens of the stomach, finding is an intense eosinophilic infiltrate 17,18. Other small intestine and colon 15. The use of cocaine and abnormalities are hemorrhages, hematomas, and related products has been associated with mesenter- epithelial apoptosis. Cyclosporin can promote villous ic thrombosis, perforation and visceral ischemia in the transformation and epithelial regeneration in ulcerative lower GI tract. Case reports have described the oc- colitis, at times associated with histological changes currence of colon ischemia in association with several that can mimic dysplasia 15,16. The acute colitis pattern amphetamines, including dextroamphetamine, methyl- has been described following treatment with laxatives, phenidate and methamphetamine, commonly known such as bisphosphonate enemas and bisacodyl, with as “speed”, “crank”, “ice” or ecstasy”. Their mechanism carbamazepine, isotretinoin, and with NSAIDs such of action is that of a sympathomimetic drug, causing

Figure 3. Drug-induced colitis. Various histological patterns. (A) Drug crystals and remnants can sometimes be seen in a colonic biopsy. In some cases, such as this one, they are surrounded by a foreign-body reaction (H&E, 20x). (B) Eosinophilic colitis can be caused by drugs. In this picture, numerous eosinophils infiltrate the lamina propria and crypt epithelium, form- ing microabscesses and sometimes degranulating (H&E, 40x). (C) Prominent apoptotic bodies can be seen in some cases of drug-induced colitis, especially with some laxatives and antineoplastic agents (H&E, 40x). 58 V. Villanacci et al.

vasoconstriction 15,16. Intestinal ischemia has been re- ia may represent a primary eosinophilic disorder or ported in patients treated with interleukin-2 (IL-2) in a secondary response to other diseases; therefore, combination with interferon, which has been linked to the diagnosis of primitive eosinophilic colitis (ESC) the ischemia also as a single agent 15,16. Ischemia was is usually made after the exclusion of other patholog- noted predominantly in the . Histopa- ical conditions and taking into consideration the site thology revealed thrombi in capillaries and venules. of the endoscopic sampling 23. In fact, eosinophils are Ergot compounds are generally safe, but, in some in- more abundant in the right colon, while they are rarely stances, colitis with bowel wall necrosis and perfora- seen in the left colon and in the rectum, with some tion or strictures have been recorded. Diuretics have individual variability due to age, geographical region, been implicated in the development of both non-occlu- climate, exposure to allergenic foods, and infective sive mesenteric ischemia and colonic ischemia. Oral agents 24. ESC is an uncommon chronic disease contraceptives have been reported to cause focal and which can affect individuals of any age; a combina- segmental colitis with aphthoid ulcers on a background tion of genetic predisposition, dysbiosis, and the en- of normal mucosa and rectal sparing 15,16. Biopsies vironment (ingested or inhaled allergens) is likely to showed focal, nonspecific ulceration on a background be its etiology; patients may report a history of allergy of a normal mucosa. Cessation of oral contraceptive (e.g. asthma, rhinitis, drug allergy and/or eczema). use resulted in prompt relief of symptoms and heal- By definition, peripheral blood eosinophilia in ESC is ing 15,16. Finally, the most recent biological agents act- absent; if present, an intestinal localization of system- ing via inhibition of key regulatory molecules are capa- ic hypereosinophilic syndrome is the correct diagno- ble of adverse gastrointestinal effects, most notably in sis 23. There is a growing evidence suggesting that the colon, as ipilimumab (a monoclonal antibody used primitive ESC is an allergic disorder mediated by Th2- in advanced melanoma) causing GVHD pattern coli- type cytokines 24. Patients with ESC have non-specif- tis, brentuximab (as described above) 19, rituximab (an ic symptoms and signs similar to IBDs, such as nau- anti-CD20 monoclonal antibody used in the treatment sea, vomiting, diarrhea and/or clinical manifestations of hematologic malignancies and rheumatologic dis- secondary to malabsorption. Most ESCs in adults are orders), and etanercept that can cause the onset of related to drug injury, which can be ascribed mainly IBD, (both Crohn’s disease and ulcerative colitis) and to NSAIDs (Fig. 3B) 23,24. Other secondary causes of microscopic colitis 20,21. tissue eosinophilia in the intestinal mucosa are infec- Before closing this paragraph, we deem it important tions, parasites, IBD, vasculitis, connective tissue dis- to recall Professor Chandrasoma: “Many of these orders, fungal infection, neoplasia, systemic disorders conditions, when encountered in biopsy or surgical of eosinophils and mast cells, and radiation effect. For resection specimens, usually receive nonspecific these reasons, the distinction between normal and pathologic diagnoses. Careful clinical correlation and pathologically increased eosinophil count is difficult, the knowledge of dosages and temporal relationship which has complicated attempts to define standard- of drug usage is required by the pathologist to even ized histological criteria for diagnosis of ESC23,24. At attempt to reach the conclusion that the pathology is the moment, there are still no widely accepted, ev- caused by drug toxicity: unfortunately, this information idence-based criteria for defining a pathological in- is rarely available to the pathologist” 22. crease in the number of eosinophils and how many high power field (HPF) have to be considered in the Key points: count. The site-specific ranges of eosinophil counts 1 The possible patterns of injury in DIC are numer- and distribution pattern of eosinophils are more use- ous and variegated; ful as a guide for when to consider an eosinophilic 2 most patterns of DIC are not specific, but they rath- disorder and prompt a search for secondary causes, er mimic another disease; than as absolute thresholds 23,24. 3 when the histological features are not convinc- Histological findings very suggestive of ESC are: de- ing and/or they do not match the clinical picture, tection of sheets or aggregates of eosinophils in the knowledge of the drugs taken by the patient is fun- lamina propria and muscolaris mucosae, as well as damental to suspect DIC. accumulation of eosinophils in the crypt epithelium 25. The cut-off value in general is over 60 x 10 HPF based on the experience of Odze in children 26 but field there Eosinophilic colitis (ESC) is no complete agreement, for example, for eosinophilic esophagitis 24. In advanced or severe disease, ascites Eosinophils are normal constituents in the lamina sometimes associated with symptoms of intestinal ob- propria of the colon. Intestinal tissue hypereosinophil- struction has been described, and the surgical speci- NON-IBD COLITIS 59

men shows an intense, transmural eosinophilic infiltra- ary (iatrogenic) of adults; and 5) paraneoplastic. The tion with serosal and perintestinal fat involvement 27. endoscopic aspects are non-specific and vary from mucosal hyperemia to scalloping of the mucosa, ul- Key points: cerations, and a mosaic-like appearance 29. Histologi- 1 ESC is a diagnosis of exclusion; cal patterns encompass: a) active duodenitis, b) celiac 2 the minimum number of eosinophils required for a disease-like pattern (characterized by villous atrophy, diagnosis of ESC is > 60 x 10 HPF in the left colon, glandular crypt hyperplasia, and marked increase in sigma and rectum; the number of T lymphocytes in the surface epithelium 3 other useful morphological features include eosin- (> 25 lymphocytes/100 epithelial cells), c) acute graft ophilic microabscesses, degranulation and exocy- versus host disease (GVHD)-like, with hyperplasia of tosis in the gland epithelium. the crypts and the presence of apoptotic bodies, and d) mixed pattern mainly prevalent in adults (Fig. 4) 30.

Autoimmune enterocolitis (AIE) Key points: 1 AIE is a group of diseases with different clinical, Autoimmune enterocolitis (AIE) is an uncommon dis- endoscopic and histological features. Careful clin- ease, clinically characterized by protracted diarrhea ical-pathological correlation is required for diagno- that can occur in both pediatric and adult patients, sis; frequently those affected by primary immunodefi- 2 in addition to active inflammation, increased crypt ciencies. Different clinical forms of the disease are apoptosis can be striking to the extent that it can recognized 28: 1) primary (pediatric); 2) syndromic resemble GVHD; (pediatric); 3) primary (sporadic) of adults; 4) second- 3 lymphocyte-predominant cases can resemble lym-

Figure 4. Autoimmune enteropathy. (A) At low power the mucosa appears reactive, with regenerative crypts and an hyper- cellular lamina propria (H&E, 2x). (B) Crypts are regenerative, with mucin depletion and surface epithelial damage (H&E, 10x). (C) On closer inspection, there are prominent apoptotic bodies in the surface and crypt epithelium (H&E, 40x). (D) Some- times, satellite lymphocytes can be seen around the apoptotic bodies (H&E, 40x). (E) CD3 immunohistochemistry reveals a greatly increased T lymphocyte population (H&E, 2x). (F) The T lymphocytes can be seen in the lamina propria as well as in crypt and surface epithelium (H&E, 20x). 60 V. Villanacci et al.

phocytic colitis, and goblet cells can be absent in within the crypt epithelium, or association of MC with cases with anti-goblet cell antibodies. giant cells and pseudomembranes are also been de- scribed 32,35.

Microscopic colitis (MC) Key points: 1 MC is an umbrella term for two diseases which Microscopic colitis (MC) is an umbrella term for two dis- share the clinical picture and endoscopic aspects: tinct diseases characterized by clinical history of chron- collagenous colitis and lymphocytic colitis; ic watery (non-bloody) diarrhea, normal endoscopic 2 CC is histologically characterized by an irregularly appearance of the ileo-colic mucosa and one of two thickened (> 10 μm) subepithelial collagen band, peculiar histopathological patterns: collagenous colitis associated with inflammatory features in the lami- (CC) and lymphocytic colitis (LC) 31. The pathogenesis na propria; of MC is still unknown, but it is likely to be multifactorial; 3 LC shows an increase in intrapithelial T lympho- moreover, abnormal immune response, impaired intes- cytes (> 20/100 epithelial cells) associated with tinal barrier function, and myofibroblast dysfunction (the lamina propria inflammation. latest in CC) are believed to play a major role32. Smoking and several drugs, in particular NSAIDs, have also been associated with the development of this disease. Finally, Ischemic colitis (ISC) MC is, for some authors, an autoimmune disease: in fact, MC is associated with other autoimmune conditions, Ischemic colitis (ISC) is an entity resulting from revers- such as rheumatoid arthritis, collagen vascular diseas- ible vascular occlusion, occurring in both older and es, thyroid disorders, and celiac disease 32. Because the young patients. In the elderly, ISC is more frequently due histological damage may be patchy and discontinuous, to vascular diseases such as hypertension, coronary there may be a significant variation between specimens artery disease, atrial fibrillation, chronic renal disease, from different portions of the bowel or even within a multi-drugs therapy and diabetes mellitus; while in young single biopsy specimen. Usually, CC is more common patients, it is more frequently associated with cocaine in the right colon and less frequent in the sigmoid and or vascular autoimmune systemic disease, including Be- rectum, whereas LC has a more ‘patchy’ distribution in hçet disease or systemic lupus erythematosus 36. Mani- the colonic mucosa 32,33. For these reasons, multiple bi- festations of ISC injury into the gastrointestinal tract are opsy sample should be obtained throughout the whole variable: only 10% of patients present with gangrenous colon and submitted in separate vials 32. The diagno- colitis, whereas most other patients have transient co- sis of CC on routine H&E stained sections is based on litis, reversible colopathy, chronic segmental colitis or the presence of a thick amorphous hyaline eosinophilic strictures, or, in a minority of cases (2.5%), fulminant band immediately beneath the superficial epithelium of pancolitis; this latter leading to surgical resection 37. the mucosa, of thickness greater than 10 µm, associat- The endoscopic appearance of ISC is fairly character- ed with inflammatory features in the lamina propria 33,34 istic in fulminant or severe disease, as well as in pa- (Fig. 5A). The mucosal architecture is well preserved. tients with chronic injury. Mild mucosal changes are Masson trichrome stain is useful for the detection of the often patchy and include areas of pale and edematous thickened collagen band (Fig. 5B). The histological diag- mucosa with scattered petechial hemorrhages. Longi- nosis of LC is based upon a diffuse increase of intraep- tudinal superficial ulcers may be evident as injury pro- ithelial T lymphocytes (IELs) (> 20 IELs per 100 epithe- gresses. Severe injury appears as gray-green or black lial cells) in the surface epithelium accompanied by an discoloration of the mucosa, often with pseudomem- increase of lamina propria inflammatory cells 31 (Fig. branes. Chronic ischemia can produce strictures, fis- 5C,D). In general, the finding of more than 20 intraepi- tulae, and mucosal atrophy or granularity that can sug- thelial lymphocytes is considered diagnostic for LC. In- gests an IBD diagnosis 37. flammation may be less prominent in the left colon, and Histological findings on H&E from bioptic samples retains the normal CD3/CD8- phenotype 33. A particular include atrophic crypts lined by small, irregular cells variant of MC is named microscopic colitis, incomplete with eosinophilic cytoplasm, dark nuclei and evident (MCi) 32. This entity is characterized by the presence of nucleoli dislocated in congested or hemorrhagic hy- clinical features of MC without the morphological criteria alinized stroma in acute cases; coagulative necrosis necessary for a diagnosis of LC or CC. Histology shows and capillary microthrombi may be found, mainly in an abnormal collagenous band < 10 μm in thickness, the acute early stage 38. Damage to capillaries and/or or an increased number of IELs < 20 per 100 epithelial medium-size vessels can be documented in autoim- cells 32,35. Unusual localization of increase IELs, such as mune disorders. Sometimes, different etiologies can NON-IBD COLITIS 61

Figure 5. Microscopic colitis. (A) In collagenous colitis, a thick and irregular collagen band underlies the surface epithelium and often entraps inflammatory cells. Surface epithelial detachment, shown here, is also typical. The underlying lamina pro- pria is inflamed and shows an increased amount of eosinophils (H&E, 20x). (B) A Masson trichrome clearly highlights the irregularly thickened subepithelial collagen band (Masson trichrome, 20x). (C) In lymphocytic colitis, numerous T lympho- cytes infiltrate the gland epithelium (H&E, 20x). (D) CD3 stain clearly highlights the pathologically increased intraepithelial lymphocytes (peroxidase-diaminobenzidine, 40x).

be added together showing ISC on bioptic samples. necrosis in acute cases, crypt and epithelial cell at- On surgical resection, areas of necrosis are usually so rophy in a hyalinized and sometimes hemorrhagic extensive that the underlying etiology is not apparent stroma in chronic cases; on histological sections. Mesenteric vessels can show 3 a wide spectrum of causes are described, with pe- abnormal size and thrombi. The wide range of etiolo- culiar clinical findings, leading to a similar histolog- gies of ISC with their clinical manifestations are very ical picture. peculiar and specific and, for more details, we invite the reader to refer to a selected bibliography 37. Radiation-induced colitis (RIC) Key points: 1 Ischemic colitis shows a predilection for the elder- Local radiation therapy, often combined with ly, but young patients with predisposing factors can chemotherapy, is one of the treatments of different also be affected; malignancies including rectal, prostatic and gyne- 2 the histological picture is dominated by coagulative cological carcinomas, leading to intestinal mucosal 62 V. Villanacci et al.

damage, often limited to the rectum, known as ra- 1 RIC is characterized by peculiar histological fea- diation-induced colitis (RIC) 39. Histology of acute tures at all stages; phase of RIC shows dilated crypts covered by re- 2 the most evident feature is represented by mark- active mucine-depleted epithelial cells, exhibiting edly dilated capillaries, with prominent endothelial bizarre nuclei and increased apoptosis (Fig. 6). A cell nuclei; transmucosal inflammation with diffuse fibrosis and 3 in acute phase, the glands show dilation, bizarre ep- dilated vessels is observed in the chronic stage 40. ithelial cell nuclei and some architectural distortion; In long-standing cases, glandular atrophy usually 4 in chronic phase, the lamina propria is hyalinized predominates; inflammation is mild to absent; and and fibrotic; and sometimes stromal cells can have fibrosis appears 39. prominent nuclei, mimicking dysplasia. Key points:

Figure 6. Radiation-induced colitis. (A) Crypt distortion, dilation and atrophy are evident (H&E, 10x). (B) Fibrosis of lamina propria, hyperplasia of crypts and dilated vascular channels (H&E, 20x). (C) Crypt atrophy, apoptotic bodies, goblet cell depletion and ectasia of small vessels are typical of radiation-induced colitis (H&E, 20x). (D) The lamina propria shows an inflammatory infiltrate that tends to decrease in long-standing cases (H&E, 20x). NON-IBD COLITIS 63

Diversion colitis (DVC) Key points: 1 Diagnosis of DVC cannot be made without surgical Diversion colitis (DVC) occurs in segments of the co- history; lon that are diverted from the fecal stream following 2 characteristic histological features include a dense surgery for congenital malformation, IBD or malignan- superficial lymphoplasmacytic infiltrate associated 41 cies . Histological pictures include diversion colitis, with prominent follicular hyperplasia; proctitis and pouchitis, characterized by atrophic mu- 3 the glands are regenerative and mucin-depleted; cosa with mucin depletion and regenerative epithelial architectural distortion is not a feature but can be hyperplasia, associated with lymphoid follicular hyper- caused by the prominent inflammatory infiltrate. plasia, and at times granuloma formation 41.

Figure 7. Segmental colitis associated with diverticular disease. (A) In this case, crypt distortion, cryptitis and basal plasma- cytosis dominate the picture (H&E, 10x). (B) Detail of the previous image. Active inflammation and cryptitis can be appreci- ated, as well as the increased number of plasma cells and eosinophils in the inflammatory infiltrate (H&E, 40x). (C, D) In this other case, inflammation is much less florid, while crypt distortion and stromal edema are predominant (H&E, 20x and 40x respectively). 64 V. Villanacci et al.

Segmental colitis associated with Acknowledgements diverticular disease (SCAD) We are grateful to Ms. Chiara Di Giorgio (Scientific Di- A small subset of patients with diverticulosis may de- rection of Foundation IRCCS Ospedale Casa Sollievo velop segmental colitis associated with diverticular della Sofferenza, San Giovanni Rotondo) for editing disease (SCAD). By definition, SCAD is a pathologi- and proofreading the manuscript. Special thanks to cal entity characterized by a chronic inflammatory re- AITIC (Italian Association of Laboratory Technicians) sponse involving the interdiverticular mucosa of the for scientific support regarding methodology and han- colonic segment involved. The rectum, by definition, dling of biopsy samples. is free of inflammation 42. The endoscopic pattern is characterized by a combination of congestion, hemor- References rhage, granularity and sometimes superficial ulceration 1 Schofield JB, Haboubi N. Histopathological mimics of inflamma- involving the sigmoid colon and sparing both rectal and tory bowel disease. Inflamm Bowel Dis 2020 Jun 18;26(7):994- proximal colonic mucosa. In the involved tract, lesions 1009. https://doi.org/10.1093/ibd/izz232 are usually confined on the crests of colonic fold and 2 Villanacci V, Reggiani-Bonetti L, Caprioli F, et al. Histopathol- sparing the orifices of diverticula. These findings repre- ogy of inflammatory bowel disease - Position statement of the Pathologists of the Italian Group for the Study of Inflammatory sent a peculiarity of the disorders and need to be care- Bowel Disease (IG-IBD) and Italian Group of Gastrointesti- fully detected, also for differential diagnosis with IBD. nal Pathologists (GIPAD-SIAPEC). Dig Liv Dis 2020;52:262-7. SCAD is histologically characterized by transmucosal https://doi.org/10.1016/j.dld.2019.11.005 chronic inflammation associated with crypt distortion 3 Villanacci V, Reggiani-Bonetti L, Salviato T, et al. 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