JOURNAL OF THE ITALIAN SOCIETY OF ANATOMIC PATHOLOGY AND DIAGNOSTIC CYTOPATHOLOGY, ITALIAN DIVISION OF THE INTERNATIONAL ACADEMY OF PATHOLOGY Periodico bimestrale - 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 III

03VOL. 113

Edited by Paola Parente and Matteo Fassan JUNE 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 03Vol. 113 P. Querzoli Managing Editor E. Fulcheri St Anna University Hospital, Ferrara University of Genoa June 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 03 June 2021

REVIEWS Benign biliary and biliary tumor precursors S. Sarcognato, D. Sacchi, M. Fassan, L. Fabris, M. Cadamuro, G. Zanus, I. Cataldo, C. Covelli, P. Capelli, A. Furlanetto, M. Guido...... 147

Cholangiocarcinoma S. Sarcognato, D. Sacchi, M. Fassan, L. Fabris, M. Cadamuro, G. Zanus, I. Cataldo, P. Capelli, F. Baciorri, M. Cacciatore, M. Guido...... 158

Autoimmune biliary diseases: primary biliary cholangitis and primary sclerosing cholangitis S. Sarcognato, D. Sacchi, F. Grillo, N. Cazzagon, L. Fabris, M. Cadamuro, I. Cataldo, C. Covelli, A. Mangia, M. Guido...... 170

Pathology of autoimmune hepatitis C. Covelli, D. Sacchi, S. Sarcognato, N. Cazzagon, F. Grillo, F. Baciorri, D. Fanni, M. Cacciatore, V. Maffeis, M. Guido...... 185

Pathology of non-alcoholic fatty liver disease I. Cataldo, S. Sarcognato, D. Sacchi, M. Cacciatore, F. Baciorri, A. Mangia, N. Cazzagon, M. Guido...... 194

Hepatocellular carcinoma: a clinical and pathological overview S.L. Renne, S. Sarcognato, D. Sacchi, M. Guido,Massimo Roncalli, L. Terracciano, L. Di Tommaso...... 203

The histomorphological and molecular landscape of colorectal and serrated lesions F. Galuppini, M. Fassan, L. Mastracci, R. Gafà, M. Lo Mele, S. Lazzi, A. Remo, P. Parente, A. D’Amuri, C. Mescoli, F. Tatangelo, G. Lanza...... 218

Mesenchymal tumours of the M. Sbaraglia, G. Businello, E. Bellan, M. Fassan, A.P. Dei Tos...... 230

Front cover: Biliary-type cirrhosis is characterized by parenchymal nodules with edema at the periphery, giving rise to the so-called halo effect (page 175); A portal tract showing a scar in a liver biopsy from a primary sclerosing cholangitis patient. Van Gieson stain confirms that the bile duct has been replaced by fibrous tissue (page 179). PATHOLOGICA 2021;113:147-157; DOI: 10.32074/1591-951X-251

Review

Benign biliary neoplasms and biliary tumor precursors

Samantha Sarcognato1, Diana Sacchi1, Matteo Fassan2, Luca Fabris3, Massimiliano Cadamuro3, Giacomo Zanus4,5, Ivana Cataldo1, Claudia Covelli6, Paola Capelli7, Alberto Furlanetto1, Maria Guido1,2 1 Department of Pathology, Azienda ULSS2 Marca Trevigiana, Treviso, Italy; 2 Department of Medicine - DIMED, University of Padova, Padova, Italy; 3 Department of Molecular Medicine - DMM, University of Padova, Padova, Italy; 4 4th Surgery Unit, Azienda ULSS2 Marca Trevigiana, Treviso, Italy; 5 Department of Surgery, Oncology and - DISCOG, University of Padova, Padova, Italy; 6 Pathology Unit, Fondazione IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo, Italy; 7 Department of Diagnostics and Public Health, Section of Pathology, University and Hospital Trust of Verona, Verona, Italy

Summary Benign biliary tumor are common lesions that are often an incidental finding in subjects who undergo medical imaging tests for other conditions. Most are true neoplasms while few result from reactive or malformative proliferation. Benign tumors have no clinical con- sequences, although the premalignant nature or potential for malignant transformation is of concern in some cases. The main practical problem for pathologists is the need to dif- ferentiate them from malignant biliary tumours, which is not always straightforward. Premalignant lesions of the bile duct have been described, although their incidence has been poorly characterized. These lesions include biliary mucinous cystic neoplasms, intra- ductal papillary neoplasms of the bile duct, and biliary intraepithelial neoplasia. In this article, histopathology of benign biliary tumors and biliary tumor precursors is discussed, with a focus on the main diagnostic criteria. Received and accepted: January 4, 2021 Key words: biliary, bile ducts, benign, neoplasms, precursors Correspondence Samantha Sarcognato piazzale Ospedale 23, 31100 Treviso Tel.: +390422328102 Introduction Fax: +390422328112 E-mail: [email protected] Bile duct neoplasms are a heterogeneous group of benign and malig- Conflict of interest nant tumors, which may arise at any point of the biliary tree. They derive The Authors declare no conflict of interest. from cholangiocytes and peribiliary glands. The anatomy of the biliary tree is divided into intrahepatic and extrahe- How to cite this article: Sarcognato S, patic portions. Intrahepatic biliary tree begins with the canals of Hering, Sacchi D, Fassan M, et al. Benign biliary which connect bile canaliculi to bile ductules, and progressively merges neoplasms and biliary tumour precursors. Pathologica 2021;113:147-157. https://doi. into a system of interlobular, septal, and major ducts, which then coa- org/10.32074/1591-951X-251 lesce to form the extrahepatic bile ducts. Interlobular bile ducts drain into septal ducts (measuring more than 100 μm in diameter), which in © Copyright by Società Italiana di Anatomia Pato- turn drain into segmental ducts (400 to 800 μm in diameter). Segmental logica e Citopatologia Diagnostica, Divisione Itali- ana della International Academy of Pathology ducts continue into the right and left hepatic ducts towards the hepatic hilum where they join, giving rise to the common hepatic duct which OPEN ACCESS finally becomes the common bile duct (7 to 11 cm long and 5 to 10 mm in diameter) after giving off the cystic duct to the . The This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- common hepatic duct, the common bile duct (i.e. choledochus), the gall- NonCommercial-NoDerivatives 4.0 International) license: the bladder, and the cystic duct are considered as extrahepatic biliary tree 1. work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original Peribiliary glands are minute structures that are distributed along the version. For further information: https://creativecommons. intrahepatic large bile ducts and the extrahepatic bile ducts 2. Intra- and org/licenses/by-nc-nd/4.0/deed.en extrahepatic bile ducts are embryologically different: the former originate 148 S. Sarcognato et al.

Table I. Classification of benign biliary tumors and precur- sors, according to the 5th edition of the WHO Classification of digestive system tumours 6. Benign biliary tumors Bile duct Biliary adenofibroma Mucinous cystic of the liver and biliary system Biliary tumors precursors Biliary intraepithelial neoplasia Intraductal papillary neoplasm of the bile ducts from the remodeling of the ductal plate, while large ducts derive from the elongation of hepatic ducts at hepatic hilum 1. Small and large biliary ducts also differ from a histo- logical point of view. In fact, small intrahepatic ducts Figure 1. A Von Meyenburg complex, made of irregularly are lined by small cuboidal cholangiocytes, while large shaped and dilated bile ducts, containing bile and protein- ducts are lined by tall cylindrical cholangiocytes and aceous debris, within a dense fibrous stroma (haematoxylin- mucin-producing cells. All these differences reflect the eosin; original magnification 20x). different pathological features and biological behavior of biliary neoplasms. Herein we provide a general overview of benign biliary development of single or multiple macroscopic . neoplasms and biliary tumour precursors, focusing on The lining cells are flattened or cuboidal, cytologically the most important clinical-pathological features, use- uniform, and lack mitoses. They show the typical chol- ful in the diagnostic routine. The classification scheme angiocyte immunophenotype, staining positive for cy- reported in the 5th edition of the WHO Classification tokeratin (CK) 7, CK8, CK18, and CK19 4. of digestive system tumors is provided in Table I. The VMCs show no significant risk for , although nomenclature used in this paper is consistent with this a few reports suggested the development of cholangio- classification. is the topic of an- carcinoma (CCA) in liver with multiple VMCs, particu- other paper in this special issue of Pathologica 3. larly in patients with genetic hemochromatosis 8,9. In the everyday practice of pathologists, it is important to differentiate VMC from CCA or metastatic adeno- Benign biliary neoplasms carcinoma, particularly in frozen sections on inciden- tal findings at surgery. The presence of dilated glands bile duct microhamartoma (von meyenburg complex) containing bile is the major clue to exclude metastasis, Bile duct microhamartoma, also called the von Mey- while the complete absence of any cytological atypia enburg complex (VMC), is a ductular liver lesion that is helpful to rule out CCA. is thought to be part of the ductal plate malformation spectrum. It shows a strong association with hepatic Bile duct adenoma fibropolycystic disease, even if most cases occur spo- Bile duct adenoma (BDA) is defined as a benign epi- radically 4,5. VMC is a common lesion, found in about thelial lesion made of a proliferation of small, normal- 5% of the general population in a large autoptic se- looking bile ducts 7. Peribiliary gland hamartoma is still ries6. Although its malformative rather than neoplastic an acceptable synonym. nature, it is reported in this section as it can be a mim- BDA is commonly observed as an incidental finding icker of a biliary malignant tumor. during abdominal surgery or autopsy, in patients with VMCs are discrete and well-defined nodules, often mul- a wide age range (1.5-99 years, mean 55 years), with tiple and usually less than 0.5 cm in their greatest di- no sex predilection 4,10,11. mension, and are classically related to portal tracts 4,7. The biological meaning of BDA is still controversial. Microscopically, a VMC is made of rounded or irregu- It has classically been considered as a reactive pro- larly shaped and dilated bile ducts, containing bile or cess due to inflammation or traumatic injury. However, eosinophilic proteinaceous debris, often with branch- BRAF V600E mutations have been described in high ing and U shapes, embedded in a dense fibrous stro- percentage of cases (> 50%), suggesting its neoplas- ma (Fig. 1). Sometimes, ductal dilation may lead to the tic nature 11-13. BENIGN BILIARY NEOPLASMS AND TUMOR PRECURSORS 149

BDAs are usually solitary and subcapsular (nearly portal tracts within the lesion, and the presence of in- 90% of the cases), and more than 90% are less than vasive features 4,7. However, it is not always possible 10 mm in size. Grossly, BDA appears as a whitish, to reach a definite diagnosis in frozen section, and well-circumscribed, unencapsulated firm nodule 11. even in paraffin-embedded samples diagnosis can be Histological diagnostic criteria include: i) a pattern- challenging. Thus, immunostainings can be of some less and disordered proliferation of small, relatively help. The keratin profile is not useful, since BDAs and uniformly shaped and spaced tubules and/or ductular pancreato-biliary cancers share the same immuno- structures with an intact basement membrane, in a phenotype, which consists in the expression of CK7, connective tissue stroma, ii) a single layer of cuboi- CK8, CK18, and CK19, with a variable expression of dal to columnar cells with regular nuclei without atypia CK20. Ki67, EZH2, p53, and p16 may help. In fact, a and mitosis, and iii) absence of infiltrative borders high proliferation index and a high expression of EZH2 (Fig. 2). Ducts in BDA show no or little lumen, with- and p53 favor carcinoma. However, it is important out dilation. The fibrous stroma can be variably dense, to keep in mind that a low proliferation index and a collagenized and hyalinized, loose or scant, and may low EZH2 and p53 expression do not exclude malig- show various degrees of chronic inflammation, includ- nancy. Interestingly, p16 is constantly express in BDA ing nodular lymphoid aggregates, particularly at the (Fig. 3), while a subgroup of CCA lacks its expression. periphery. Normal portal tracts are often enclosed within a BDA, usually near the periphery 4,7. Clear-cell and oncocytic changes have been described, but their meaning is still unknown 14,15. BDA epithelial cell phe- notype is similar to that of normal bile ducts. They can also express mucin and show a secretory gland cell phenotype, expressing the foregut epithelial antigens D10, 1F6, MUC6, MUC5AC, and TFF2 4,16,17. Malignant transformation of a BDA has never been clearly reported. The main BDA clinical issue is the possibility to misdiagnose BDA as a well-differentiat- ed CCA or a pancreatic , particularly during intraoperative consultation. In frozen sections, the differential is based on tumor size, cytological and architectural patterns, the presence of pre-existing A

B

Figure 2. Bile duct adenoma is a disordered proliferation Figure 3. Differently from cholangiocarcinoma, bile duct of small and uniform ductular structures within a connective adenoma shows a diffuse p16 positivity (A), and a wild-type tissue stroma, with no infiltrative borders. Neoplastic cells pattern of expression of p53, with only a few positive nuclei are cuboidal/columnar and lack atypia and mitosis (hema- (B) (A: p16 immunostain; original magnification 20x; B: p53 toxylin-eosin; original magnification 20x). immunostain; original magnification 20x). 150 S. Sarcognato et al.

Therefore, a negative p16 stain supports CCA diagno- cells have a biliary phenotype, expressing CK7, CK19, sis 18,19. BDA differential diagnoses also include other epithelial membrane antigen (EMA), and CA19-9 4,7. benign biliary proliferations, such as reactive ductular Premalignant changes have been reported in nearly proliferation (particularly in cirrhosis, when the prolif- a half of cases, showing epithelial and ar- eration may be nodular in shape), VMC, and biliary chitectural disarray, which consists in complex intra- adenofibroma. BDAs never show cystic changes and cystic papillary proliferation and cribriform pattern. As never contain bile in gland lumens 7. the behavior of these tumors is poorly understood, patients require close clinical follow-up. Malignant de- Biliary adenofibroma generation leads to the development of a conventional Biliary adenofibroma (BAF) is a solid epithelial benign adenocarcinoma 20,22-25. liver neoplasia composed of microcystic and tubulo- acinar glands lined by non-mucin-secreting biliary epi- Mucinous cystic neoplasm of the liver and biliary system thelial cells, embedded in a fibrous stroma 7. It is an Mucinous cystic neoplasm (MCN) of liver and bili- exceptionally rare with a potential risk ary system, formerly known as ‘hepatobiliary cystad- for malignant transformation and recurrence, if surgi- enoma’, is a -forming epithelial neoplasia with no cal excision is incomplete 20. In the literature, only 21 communication with the bile ducts, made of mucin- cases have been described, so far. producing epithelium associated with an underlying Mean age at presentation is 60 years, with a slight fe- ovarian-type stroma. MCN shows either low grade or male predominance. Most patients show abdominal high grade epithelial dysplasia, and may be associ- pain, while a few lesions are incidental findings 20. ated with an invasive carcinoma 4,7. Most MCNs are BAF is a primary epithelial tumor with a secondarily solitary intrahepatic lesions, rarely seen in the extra- induced stroma 21. Multiple clonal cytogenetic altera- hepatic bile ducts and in gallbladder 26,27. tions have been described in BAF, supporting its neo- MCNs are rare, with an estimated incidence of 1 plastic nature. Moreover, amplifications of CCND1 case/20,000-100,000 per year, consisting in less than and ERBB2 and mutations in CDKN2A were found 5% of all liver cysts. They occur almost exclusively in in cases showing aggressive behavior and malignant women, and the mean age at diagnosis is nearly 50 transformation 22. years, even if MCNs with an associated invasive carci- BAFs are typically solitary and may affect both liver noma arise in older people26-29. Patients typically show lobes. They are usually large lesions, with a wide di- symptoms, which include chronic non-specific abdom- ameter range, and are well-defined, round to oval, inal pain or discomfort and swelling, a palpable mass, whitish, and unencapsulated. On cut section, both and obstructive jaundice. In old patients, acute symp- solid and microcystic areas (with sponge-like appear- toms may suggest an invasive component 7,27. Preop- ance) can be recognized, and some lesions may show erative diagnosis may be challenging and requires a macrocystic changes 7. high degree of suspicion. Serum CA19-9 levels may Histologically, BAF is composed of both glandular and be increased, particularly in patients with an associ- stromal components. Glandular structures are typical- ated invasive component, while intracystic CA19-9 ly shaped in acini, tubules, and cysts, and show bili- and carcinoembryonic antigen (CEA) levels may be ary differentiation, similarly to VMC and BDA. Glands of help in differential diagnosis with non-neoplastic le- and cysts show variable size and shape, with dilation sions, since they are higher in MCN 27,30,31. Abdominal and branching, sometimes with complex configura- imaging shows large multilocular cystic lesions, with tions. Epithelial polypoid projections may be present. cyst-in-cyst appearance, without any communication The glandular component is lined by a cuboidal to low with the biliary tree, even though unilocular cystic le- columnar, amphophilic, non-mucin-producing epitheli- sions may be seen in about 10% of patients. Irregular um. Cells show bland round nuclei with inconspicuous thickness of the cystic wall, enhancing internal septa nucleoli, and apocrine changes can be seen. Micro- with mural nodules, and papillary projections suggest cysts are typically lined by flattened epithelium, and an invasive component 27,32. Etiology of MCN remains can be filled with eosinophilic, proteinaceous mate- unclear, even if its prevalence in middle-aged women rial and cellular debris, but they do not contain mucin. point toward a hormonal influence 30. Only occasional mitotic figures are present, and the MCN grossly appears as a multiloculated and well- proliferation index is low in both epithelial and stro- demarcated cystic lesion, with a fibrous capsule, inde- mal components. The background stroma is usually pendent from the biliary tree, even if a polypoid exten- abundant, collagenous, and contains bland myofibro- sion into bile duct lumen may be observed 33. Its inner blasts. It is frequently present a patchy chronic inflam- surface is usually smooth or trabeculated; when an matory infiltrate. As previously mentioned, epithelial invasive component is present, papillary projections BENIGN BILIARY NEOPLASMS AND TUMOR PRECURSORS 151

and white solid areas are seen. Cysts may contain clear serous, mucinous or gelatinous fluid, which can be purulent or hemorrhagic 28,34. Histological hallmarks are i) mucinous lining epithe- lium, and ii) specialized ovarian-like stroma. Epithelial cells are usually arranged in a single layer and may be columnar, cuboidal or flattened, with pale eosino- philic to mucinous cytoplasm, bland and basally ori- ented nuclei, and no mitotic activity. In some cases the epithelium may be non-mucinous, resembling the lin- ing of non-neoplastic bile ducts, and may show intes- tinal and gastric differentiation as well as squamous metaplasia. Scattered chromogranin- or synaptophy- sin-positive neuroendocrine cells can be seen. Small polypoid or papillary tufts may be seen along the in- A ner surface. Mucin can be typically demonstrated by the histochemical staining with mucicarmine and Al- cian blue. The lining cells have the staining pattern of biliary-type epithelium, with positivity for CK7, CK19, CK8, and CK18. They also express EMA, CEA, and MUC5AC, while CDX2, MUC2 and CK20 are positive in areas with intestinal differentiation. The epithelium may be ulcerated, and extravasation of the cyst fluid into the stroma or wall may occur, with the develop- ment of inflammation, xantho-granulomatous reac- tion, scarring, and calcifications 28,34,35. Importantly for diagnostic purposes, the entity-defining stroma is present in all MCNs, at least focally. Since it may be focal, an extensive sampling of the lesion is recom- mended. The stroma is hypercellular, clearly resem- B bling ovarian stroma, which, in turn, is surrounded by more collagenized fibrous tissue (Fig. 4). Stromal cells are characteristically positive for estrogen and proges- terone receptors, and α-inhibin, and may be focally lu- teinized (Fig. 4). Stromal inflammation, hemorrhage, calcification, and necrosis are common 28. Most MCNs have low/intermediate-grade dysplasia. However, nuclear pleomorphism, loss of polarity, presence of mitotic figures and multilayering of the epithelium define a high-grade dysplasia. The associ- ation with invasive adenocarcinoma, which is defined by the infiltration of the underlying stroma by tumor cells, is rare and occurs in nearly 6% of cases; it usu- ally shows tubular or tubulo-papillary patterns and a desmoplastic stromal reaction 28,36. It is strongly rec- ommended a thorough histological examination of the C surgical specimen for grading dysplasia and excluding Figure 4. Mucinous cystic neoplasm of liver and biliary system malignant transformation. is composed of a mucin-secreting lining epithelium, embedded Fine-needle aspiration (FNA) cytology is not helpful in a hypercellular specialized ovarian-like stroma (A). Stromal in MCN diagnosis, since it can only differentiate be- cells stain positive for estrogen (B) and progesterone receptors nign cyst contents from adenocarcinoma, so the final (C) (A: hematoxylin-eosin; original magnification 5x; B: estrogen diagnosis requires histological confirmation and cor- receptor immunostain; original magnification 10x; C: progester- relation with clinical history and imaging. Moreover, one receptor immunostain; original magnification 10x). FNA should be avoided if MCN is suspected, since 152 S. Sarcognato et al.

intraoperative cyst spillage may lead to peritoneal dis- The prevalence of BilIN of the bile ducts outside the semination. FNA samples usually contain aggregates setting of invasive carcinoma is not known and difficult of bland cuboidal to columnar epithelial cells, with oc- to assess. Indeed, BilIN is usually seen as an inciden- casional papillary arrangement. Different degrees of tal finding in specimens resected for other reasons, nuclear atypia might be observed, if a high-grade dys- and it is not detectable by imaging 7,40. BilIN devel- plastic or carcinomatous component is present 27,37. opment has been associated with different risk fac- The ovarian-type stromal component is typically not tors, such as lithiasis, familial adenomatous polypo- seen. The background is usually watery or with abun- sis, primary sclerosing cholangitis, choledocal cysts, dant thick mucin containing chronic inflammatory cells and anomalous confluence of pancreato-biliary ducts. and histiocytes 27,37. KRAS gene mutations are found BilIN is often found in biliary epithelium adjacent to in nearly 20% of MCNs, mostly in high-grade dysplas- invasive adenocarcinoma, as well as in cirrhotic liv- tic lesions, being uncommon (5%) in cases with low- ers with non-biliary diseases, such as alcoholic liver grade dysplasia 38. disease or non-alcoholic steatohepatitis 41,42. Persis- Many cystic lesions of the liver can mimic MCN, but tence of chronic inflammation seems to lead the onset the finding of ovarian-like stroma confirms MCN diag- and progression of neoplastic changes in biliary epi- nosis. The main differential diagnoses include simple thelium, with involvement of different molecular path- bile duct or peribiliary gland cysts, developmental ways. In fact, it is known that KRAS mutations are an cysts, hydatid cysts, microcystic serous cystadeno- early event in biliary carcinogenesis, being present in mas, and cystic liver metastases, which occur mainly almost 40% of BilINs, while TP53 mutations occur as from colorectal, ovarian, pancreatic, or lung carcino- a late molecular event 43,44. mas. Endometriosis can simulate MCN, as well. Fi- Macroscopically, BilIN are usually not evident, and nally, intraductal papillary neoplasms of the bile ducts only subtle mucosal changes can be observed, such can show a cystic appearance and be mimicker of as mucosal thickening, granularity, and change in MCN, but the absence of an ovarian-like stroma and color 7,40. the absence of connection with the biliary tree help Histologically, BilIN is classified according to the high- the pathologist in the diagnosis 4,7,27. est degree of nuclear and architectural atypia in low- The frequency of MCN malignant transformation has grade or high-grade. This classification recently re- been reported to be as high as 20% to 30%; therefore, placed the previous three-tiered classification, which surgical resection is indicated. Prognosis is excellent included BilIN-1, BilIN-2 (now low-grade BilIN), and if a complete excision is possible, with an outstand- BilIN-3 (now high-grade BilIN). In low-grade BilIN, ing recurrence-free survival. Marsupialization and biliary epithelium shows mild cytoarchitectural atypia, fenestration are considered inadequate surgical pro- with hyperchromatic nuclei with prominent nucleoli, cedures, with high risk of recurrence. Staging of MCN a mildly increased nucleus/cytoplasm ratio with mi- with an associated adenocarcinoma follows the TNM nor nuclear pseudostratification and preserved nu- classification for intrahepatic cholangiocarcinoma or clear polarity (Fig. 5). Peribiliary glands are rarely in- carcinoma of the extrahepatic bile ducts, depending volved 29,40,45-47. on the neoplastic location. Prognosis of MCN-associ- Low-grade BilIN should not be confused with mucosal ated carcinoma seems to be better than conventional reactive changes, which are much more common 40,43. intrahepatic cholangiocarcinoma 27,38,39. or regenerative changes are usually flat, although low-papillary or micropapillary architecture can be observed in association with hepatolithiasis Biliary tumor precursors or choledochal cyst. Compared with normal epithe- lium, the cellularity is only slightly increased. Two very There are two main types of premalignant lesions of the useful clues supporting the reactive nature are: i) the bile ducts, which are considered as precursors of CCA: appearance of nuclear membrane, which remains • Biliary intraepithelial neoplasia (BilIN) smooth, and ii) the presence of intraepithelial infiltra- • Intraductal papillary neoplasm of the bile ducts (IP- tion of neutrophils 48. Immunoistochemical stain may NB). be of help, since p53 overexpression supports the diagnosis of a dysplastic lesion, even if its absence Biliary intraepithelial neoplasia does not exclude it. BilIN is a microscopic, non-invasive, flat or micropapil- High-grade BilIN more frequently shows micropapil- lary lesion confined to the lumen of bile ducts. It is lary growth pattern, with taller papillae. Cells display now classified in BilIN with low-grade or high-grade severe nuclear atypia, with irregular, pleomorphic, dysplasia7. and bizarre nuclei, and a complete loss of nuclear po- BENIGN BILIARY NEOPLASMS AND TUMOR PRECURSORS 153

Figure 5. Low-grade biliary intraepithelial neoplasia shows Figure 6. High-grade biliary intraepithelial neoplasia dis- mild cytological atypia, with hyperchromatic nuclei, promi- plays severe nuclear atypia, with irregular and pleomorphic nent nucleoli, a mildly increased nucleus/cytoplasm ratio, and nuclei, several mitotic figures (arrows), and a complete loss minor nuclear pseudostratification, with preserved nuclear of nuclear polarization, with complex stratification (hema- polarity (hematoxylin-eosin; original magnification 20x). toxylin-eosin; original magnification 40x).

larity, with complex stratification (Fig. 6). An important nous neoplasm of , but in liver it is a rarer feature, which points to the diagnosis of high-grade disease 29,40,47,51,52. BilIN, is the evidence of nuclei on the luminal surface IPNB incidence varies consistently among different (i.e. loss of polarization). Overall, the lesion resem- geographical regions, representing 7-10% of all bile bles a malignant one, but with a preserved basement duct tumors in Europe and North America, and 10- membrane. Mitoses are frequent, with a markedly in- 40% in Asian cohorts 51,53. Patients with IPNB are pref- creased Ki-67 proliferation index, and involvement of erentially men with a median age of 50-70 years, who the peribiliary glands may occur. Immunohistochemi- usually show a recurrent and intermittent pain and cal stains may be of help in distinguishing low-grade cholangitis. IPNB is rarely seen in children, and no from high-grade BilINs (Table II). Epithelial cells may tendency for familial aggregation of cases has been show biliary, intestinal, or gastric phenotype 29,40,45-47. described 53-55. IPNB may be radiologically undetect- In FNA specimens, BilIN diagnosis is not considered, able, but in most of cases, cholangiography shows and when severely atypical features, similar to those filling defects in the biliary tree, due to an intraductal seen in carcinoma, are seen in biliary epithelial cells, mass, with dilatation of either proximal or distal bile it is not possible to differentiate pre-invasive from inva- ducts. The prevalent location of IPNB (intrahepatic sive lesions 49,50. versus extrahepatic bile ducts) is highly variable among studies 55. Intraductal papillary neoplasm of the bile ducts Etiology of IPNB remains unclear in most of the cas- IPNB is a premalignant lesion with intraductal papil- es, even if known risk factors are primary sclerosing lary or villous growth of biliary-type epithelium that cholangitis, hepatolithiasis, and liver fluke infection (in may show low- or high-grade dysplasia or have an Asian countries) 51,55,56. IPNB development and pro- associated invasive carcinoma 7. It is considered the gression from low- to high-grade dysplasia to invasive biliary counterpart of the intraductal papillary muci- carcinoma follow a sequential acquisition of molecular

Table II. Immunostains that may be of help in differentiating low-grade from high-grade biliary intraepithelial neoplasia. Immunostain Low-grade BilIN High-grade BilIN S100 Mildly to moderately increased Diffusely and strongly positive p53 Usually wild-type expression Frequently positive p16 Relatively preserved Decreased expression BilIN: biliary intraepithelial neoplasia. 154 S. Sarcognato et al.

cytic, and gastric-type differentiation, based on mor- phology and immunophenotype. The biliary subtype is the most common in Western countries, while the oncocytic and gastric types are very rare. Mixtures of different subtypes are observed in about 50% of all IPNBs, so their classification is based on the most prevalent component. The gastric subtype expresses MUC5AC and MUC6, the intestinal one is MUC2- positive, whilst the biliary type frequently expresses EMA (MUC1) 40,51,55,59,60. Epithelial cells lining IPNB may show variable cytoarchitectural atypia, classified into low-grade and high-grade dysplastic lesions, ac- cording to the highest degree. IPNBs with high-grade dysplasia are more frequent in extrahepatic bile ducts. The extension of neoplastic process into peribiliary Figure 7. Intraductal papillary neoplasm in a dilated bile glands, particularly in the hilar zone, is quite com- duct, with a papillary/villous growth pattern, made of fine mon, and is not considered a feature of invasion. A fibrovascular cores covered by biliary epithelial cells, with clear, frank invasion of the stroma is necessary for a diffuse low-grade and focally high-grade (asterisks) dyspla- definitive diagnosis of an associated invasive adeno- sia (Immunostain-eosin; original magnification 5x). carcinoma. About 40-80% of IPNBs show a minimal invasive component at diagnosis, most commonly a tubular adenocarcinoma, sometimes a colloid (muci- nous) adenocarcinoma 55,61. alterations involving common oncogenic pathways, in- Recently, a group of Japanese and Korean expert pa- 57 cluding KRAS, CDKN2A, and TP53 genes . thologists proposed a different classification of IPNB, Grossly, IPNB appears as a visible papillary, villous, or based on similarities with the pancreatic counterparts. polypoid, red-colored, soft mass, predominantly grow- It divides IPNB in two groups. Type 1 IPNB histological- ing in a dilated bile duct lumen, which may be cystic ly resembles intraductal papillary mucinous neoplasm or fusiform in shape. Lesions might be single and iso- of the pancreas; it typically develops in the intrahe- lated or multiple, and mucus hypersecretion can be patic bile ducts, and contains macroscopic mucinous seen. An invasive component can appear as a mass- components. Type 2 IPNB has a more complex his- forming or nodular lesion 51,55,58. tological architecture, with irregular papillary branch- Histologically, IPNBs are characterized by dilated bile ing or foci of solid-tubular components; it involves the ducts filled with papillary or villous structures with fine extrahepatic bile ducts, and is more frequently associ- fibrovascular cores covered by biliary epithelial cells, ated with invasive cancers 51,62 (Tab. III). with various amount of tubular or glandular compo- FNA samples from IPNBs are often hypercellular, with nents, and lack of an ovarian-type mesenchymal sheets of epithelial cells that are commonly arranged stroma (Fig. 7). Epithelial cells in IPNB are cuboidal in papillary structures with fibrovascular cores. Micro- or columnar, and may show intestinal, biliary, onco- glandular structures and mucin may be present, while

Table III. Features of type 1 and type 2 intraductal papillary neoplasm of the bile ducts, according to the Japan-Korea Coop- erative Study Group 61. Feature Type 1 IPNB Type 2 IPNB Preferential location Intrahepatic bile ducts Extrahepatic bile ducts Mucin secretion Frequent Rare Architecture Regular homogeneous papillae Irregular complex papillae Histological subtypes Gastric, intestinal Intestinal, pancreatobiliary Grade Mostly high grade Always high grade Stromal invasion < 50%, minimal > 80%, minimal or mild Similarity to pancreatic IPMN Similar Variable Aggressiveness Less aggressive More aggressive Outcome More favorable Worse IPNB: intraductal papillary neoplasm of the bile ducts; IPMN: intraductal papillary mucinous neoplasm. BENIGN BILIARY NEOPLASMS AND TUMOR PRECURSORS 155

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Review

Cholangiocarcinoma

Samantha Sarcognato1, Diana Sacchi1, Matteo Fassan2, Luca Fabris3, Massimiliano Cadamuro3, Giacomo Zanus4,5, Ivana Cataldo1, Paola Capelli6, Francesca Baciorri1, Matilde Cacciatore1, Maria Guido1,2 1 Department of Pathology, Azienda ULSS2 Marca Trevigiana, Treviso, Italy; 2 Department of Medicine - DIMED, University of Padova, Padova, Italy; 3 Department of Molecular Medicine - DMM, University of Padova, Padova, Italy; 4 4th Surgery Unit, Azienda ULSS2 Marca Trevigiana, Treviso, Italy; 5 Department of Surgery, Oncology and Gastroenterology - DISCOG, University of Padova, Padova, Italy; 6 Department of Diagnostics and Public Health, Section of Pathology, University and Hospital Trust of Verona, Verona, Italy

Summary represents the third leading cause of cancer-related death worldwide. Cholan- giocarcinoma (CCA) is the second most common type of liver cancer after , accounting for 10-15% of all primary liver . Both the incidence and mortality of CCA have been steadily increasing during the last decade. Moreover, most CCAs are diagnosed at an advanced stage, when therapeutic options are very limited. CCA may arise from any tract of the biliary system and it is classified into intrahepatic, perihilar, and distal CCA, according to the anatomical site of origin. This topographical classification also reflects distinct genetic and histological features, risk factors, and clinical outcomes. This review focuses on histopathology of CCA, its differential diagnoses, and its diagnostic pitfalls.

Key words: biliary, neoplasia, malignant, cholangiocarcinoma, subtypes

Introduction Received and accepted: January 4, 2021

Correspondence Cholangiocarcinoma (CCA) is a heterogeneous group of aggressive ma- Samantha Sarcognato lignancies arising from different locations within the biliary tree. Depend- piazzale Ospedale 23, 31100 Treviso ing on their anatomical site of origin, CCAs are classified into intrahe- Tel.: +390422328102 Fax: +390422328112 patic (iCCA), perihilar (pCCA), and distal CCA (dCCA), that differ for eti- E-mail: [email protected] ology, risk factors, prognosis, and clinical and therapeutic management. and tumors arising in the ampulla of Vater are not Conflict of interest The Authors declare no conflict of interest. included in this group. iCCA and pCCA taken together represent more than 90% of all CCAs worldwide 1-5. How to cite this article: Sarcognato S, Sacchi CCA is the second most common primary hepatic malignancy after he- D, Fassan M, et al. Cholangiocarcinoma. patocellular carcinoma (HCC), comprising about 15% of all primary liver Pathologica 2021;113:158-169. https://doi. tumors and 3% of all gastrointestinal cancers. CCA is a rare cancer, but its org/10.32074/1591-951X-252 incidence and mortality rates are constantly increasing worldwide during © Copyright by Società Italiana di Anatomia Pato- the past decades. Globally, CCA has an incidence rate of 0.3-6/100,000 logica e Citopatologia Diagnostica, Divisione Itali- inhabitants per year, with a mortality rate of 1-6/100,000 inhabitants per ana della International Academy of Pathology year. Specific regions, such as South Korea, China, and Thailand, show OPEN ACCESS a particularly high incidence rate, with more than 6 cases/100,000 ha- bitants per year. The peak of incidence of CCA is between the fifth and This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- the seventh decades of life, with a slight male predominance for iCCA. NonCommercial-NoDerivatives 4.0 International) license: the Some risk factors are shared by all CCA subtypes, while others are more work can be used by mentioning the author and the license, 1-5 but only for non-commercial purposes and only in the original specific for one subtype or for specific geographical regions (Tab. I). version. For further information: https://creativecommons. Regrettably, CCAs are often diagnosed at advanced stages, when thera- org/licenses/by-nc-nd/4.0/deed.en peutic options are very limited. Margin-negative resection is the most CHOLANGIOCARCINOMA 159

Table I. Main risk factors for intrahepatic and extrahepatic cholangiocarcinoma. Intrahepatic CCA Extrahepatic CCA (pCCA plus dCCA) Cirrhosis PSC Chronic pancreatitis Choledocal cysts HBV and HCV Caroli disease Alcohol consumption Choledocholitiasis and cholelithiasis NAFLD Liver fluke infection (South-eastern Asia) Hepatolithiasis IBD Haemochromatosis Diabetes and obesity Diabetes and obesity Chronic pancreatitis Smoking Gout Congenital hepatic fibrosis Smoking Chemical exposure (i.e. thorotrast) Chemical exposure (i.e.1,2-dichloropropane) CCA: cholangiocarcinoma; pCCA: perihilar cholangiocarcinoma; dCCA: distal cholangiocarcinoma; HBV: hepatitis B virus; HCV: hepatitis C virus; NAFLD: non-alcoholic fatty liver disease; PSC: primary sclerosing cholangitis; IBD: inflammatory bowel disease. important critical factor influencing prognosis, and is can only be based on histology. In particular, histologi- related to a better survival both in iCCA and pCCA/ cal confirmation of iCCA on liver biopsy is mandatory dCCAs. However, a significant proportion of patients in cases of unresectable disease, to determine subse- presents with locally advanced and unresectable dis- quent patient management 1,2,17. ease. iCCAs arising in non-cirrhotic livers show the iCCAs are usually asymptomatic in early stages. Jaun- worst prognosis 3,6-8. dice is not frequent and generally associated with an Prognosis has not significantly improved in recent advanced disease. Other non-specific symptoms, typ- years, despite a deeper understanding of CCA patho- ically seen in advanced disease, include fatigue, ab- genesis thanks to advanced technologies, such as dominal pain, malaise, nausea, anorexia, and weight DNA and/or RNA sequencing. About 40% of CCA loss. CA19-9 is characteristically elevated 1,2,4,5,17. patients show targetable genetic alterations; however, rapid translation into clinical trials is limited, mainly Pathology due to the low number of patients 9-15. Macroscopically, iCCA may show 3 different growth This review focuses on histopathology of CCA, includ- patterns, named mass forming (MF type), periductal ing its subtypes, differential diagnoses, and diagnostic infiltrating (PI type), and intraductal growing (IG type), pitfalls. with the MF type being the most common one 2,3,18. The IG type is not a recognized growth pattern of CCA by AJCC/UICC 1,2,4,5. MF type iCCAs look like nodular Intrahepatic cholangiocarcinoma mass lesions in the hepatic parenchyma; iCCAs with PI type grow longitudinally along the bile duct, as peri- The WHO classification defines iCCA as a malignant ductal nodular and sclerosing lesions, determining intrahepatic epithelial neoplasm with biliary differen- biliary strictures or obliterations, and eventually liver tiation. It represents nearly 10-20% of all CCAs and parenchymal invasion; IG type iCCAs display papillary arises from bile ductules to the second-order bile growth towards the duct lumen, representing, in a ma- ducts (i.e. segmental bile ducts), proximal to the left jority of cases, the malignant progression of an intra- and right hepatic ducts 3. ductal papillary neoplasm of the bile duct (IPNB). MF Many risk factors for iCCA are closely related to a type iCCA originates from peripheral small bile ducts chronic inflammation of the biliary epithelium and bile while PI and IG type iCCAs arise from large intrahe- stasis (Tab. I). The prevalence of some of these risk patic bile ducts 18. A few studies suggested that the factors, especially alcohol consumption and non-alco- PI type may be associated with a poor prognosis, but holic fatty liver disease, is increasing worldwide, thus the prognostic significance of growth patterns remains contributing to iCCA incidence rise. However, most of controversial 19. iCCAs occur in the absence of known risk factors and Histologically, iCCAs are usually well to moderately represent an incidental finding in around 20-25% of differentiated , with a ductal, tubu- cases during imaging studies performed for other rea- lar or cord-like pattern, and with variable, and often sons 1,2,16. abundant, fibrous stroma. Two histological subtypes of In 60-70% of patients, iCCA is mostly identified as a iCCA are recognized: the large duct type, arising in single mass. Radiologic criteria can only suggest a the large intrahepatic bile ducts near the hepatic hilus, possible iCCA diagnosis, and a definitive diagnosis and the small duct type, which mainly occurs in the 160 S. Sarcognato et al.

hepatic periphery 3,20. It is worth noting that the histo- logical subtype reflects the high molecular heteroge- neity of iCCAs and can be ascribed to different cells of origin and pathogenesis. Hepatic stem or progeni- tor cells and cuboidal cholangiocytes are the putative cells of origin of small duct type iCCAs, while large duct iCCAs seem to derive from columnar mucous cholangiocytes or peribiliary glands 1. Large duct iCCAs histologically resemble pCCA or dCCA. They are composed of large, irregular, dilated glands, embedded in an often abundant fibrous stro- ma, characterized by dense connective tissue with loose spindle cells, hyalinized or sclerotic collagen fibers, and disorganized blood vessels. Cancer cells are cuboidal or columnar, with atypical hyperchro- matic nuclei, and frequent mucus secretion (Fig. 1). Figure 2. An example of perineural invasion in a large duct Typical features of large duct iCCA include extensive intrahepatic cholangiocarcinoma (hematoxylin-eosin; origi- portal infiltration, perineural and lymphatic invasion nal magnification 10x). (Fig. 2), papillary structures, and features of intraduct- al dysplasia. Lymph node metastases are also com- mon. Large duct iCCAs often evolve from pre-invasive lesions, including biliary intraepithelial neoplasia (Bi- lIN) and IPNB. Differently from small duct iCCA, tumor cells show S100P and trefoil factor 1 expression 3,18,20 (Fig. 3). As already mentioned, small duct iCCAs show a MF type growth pattern, appearing as whitish or grey nodular lesions in the peripheral hepatic parenchyma. They are composed of small, cuboidal cells with uni- form round nuclei, arranged in small sized tubular or acinar structures, with no mucin production (Fig. 4). Less differentiated areas display solid, cord-like, or

Figure 3. A diffuse S100P expression in a large duct intra- hepatic cholangiocarcinoma (S100P immunostain; original magnification 20x).

cribriform patterns. Advanced lesions may show highly sclerotic and hypovascular central areas, with a more solid growth at the periphery. No defined precursor le- sions have been reported for small duct iCCAs 3,18,20-22. All iCCAs are graded as well-, moderately, or poorly differentiated adenocarcinomas, according to their cell morphology 3. Figure 1. Large duct intrahepatic cholangiocarcinoma is Histological subtypes of small-duct iCCA include chol- composed of large irregularly dilated glands, embedded in angiolocarcinoma (CLC) (formerly considered a sub- abundant fibrous stroma. Note neoplastic cell mucus secre- type of combined hepatocellular-cholangiocarcinoma) tion (hematoxylin-eosin; original magnification 10x). and iCCA with ductal plate malformation pattern. CLC closely resembles the ductular reaction seen CHOLANGIOCARCINOMA 161

Figure 4. Small duct intrahepatic cholangiocarcinoma is Figure 6. An intrahepatic cholangiocarcinoma with ductal composed of cuboidal cells arranged in small sized tubular plate malformation pattern, with tumor structures that look or acinar structures, with areas of solid growth pattern, and like ductal plate malformation, within a dense fibrotic stroma with no mucin production (hematoxylin-eosin; original mag- (hematoxylin-eosin; original magnification 2.5x). nification 10x).

in chronic cholangiopathies, and is diagnosed when EMA 3,23,24. iCCA with ductal plate malformation pat- more than 80% of ductular configuration is present. tern is composed of tumor structures that look like In this subtype, malignant ductular-like structures ductal plate malformation, with common presence of seem to radiate from a portal tract or surround it, in inspissated bile, in a dense fibrotic stroma (Fig. 6). a tubular, cord-like, anastomosing “antler-like” pattern, Neoplastic cells are benign-looking, resembling biliary within a dense and hyalinized fibrotic stroma (Fig. 5). epithelial cells. Like CLC, they may stain positive for Tumor cells are smaller and cuboidal, with round to CD56 (NCAM) and EMA 3,25. oval nuclei and scant cytoplasm. They often show im- iCCA variants munohistochemical expression of CD56 (NCAM) and Rare variants of iCCA include squamous or adeno- squamous carcinoma, lymphoepithelioma-like carci- noma related to Epstein-Barr virus infection, and sar- comatous carcinoma. The latter demonstrate areas of mesenchymal morphology, such as spindle or rhab- doid cell, in association with glandular areas, and are more aggressive than conventional CCAs 3,26 (Fig. 7). A putative novel variant of iCCA, mimicking a neuro- endocrine tumor, has been recently described and named cholangioblastic cholangiocarcinoma, due to the presence of blastemal-like areas within the tumor. Histologically, it shows a trabecular and solid/hepatoid growth pattern, with immunohistochemical expression of cytokeratin (CK) 7 and CK19, chromogranin A and/ or synaptophysin, and a strong and diffuse expression of inhibin A (Fig. 8). Only a few cases have been de- scribed so far, all with an aggressive clinical course, Figure 5. Cholangiolocarcinoma is made of malignant with recurrence and metastasis to the , ductular-like structures, arranged in tubular, cord-like, anas- liver, and lungs. The molecular profile of these tumors tomosing “antler-like” pattern, in a dense hyalinized fibrotic showed alterations in the TGFβ and WNT signaling stroma (hematoxylin-eosin; original magnification 10x). pathways, known to regulate ductal plate develop- ment 27. 162 S. Sarcognato et al.

A A

B B

Figure 7. A sarcomatoid intrahepatic cholangiocarcinoma, with diffuse spindle cell morphology. Numerous mitotic fig- ures are present (A). As conventional intrahepatic cholan- giocarcinoma, neoplastic cells diffusely express cytokeratin 7 (B) (A: hematoxylin-eosin; original magnification 20x; B: cytokeratin 7 immunostain; original magnification 20x).

iCCA differential diagnoses iCCA diagnosis can be challenging due to several dif- ferent mimickers, including benign and malignant le- sions 3,22. Therefore, clinical and histological features should be accurately considered dealing with an in- trahepatic mass. Several immunohistochemical stains C are available, and may help in leading to a definite Figure 8. Cholangioblastic variant of intrahepatic cholan- diagnosis in most cases. However, none is accurate giocarcinoma (A). Tumor cells diffusely express inhibin A in differentiating benign from malignant lesions, and (B) and show a granular cytoplasmic positivity for chromo- the expression of most of these markers, even the lin- granin A (C) (A: hematoxylin-eosin; original magnification eage-specific ones, frequently overlap among differ- 10x; B: inhibin A immunostain; original magnification 5x; C: ent lesions. Thus, it is highly recommended to always chromogranin A immunostain; original magnification 20x). use a panel of multiple immunohistochemical markers in clinical practice. CHOLANGIOCARCINOMA 163

iCCA versus benign bile duct lesions Morphology and clinical history is usually helpful in differentiating iCCA from benign lesions 17. However, the distinction between a well-differentiated iCCA and its benign mimics can be challenging, particularly in small biopsies with scant cellularity. All biliary lesions share the CK immunohistochemical profile, since they all stain positive for CK7 and CK19, with variable ex- pression of CK20. A high proliferative index favors iCCA; in fact Tsokos et al. found an average Ki-67 expression of 23% in iCCA versus 1.4% in all biliary benign lesions 28. However, a low Ki-67 expression does not always exclude iCCA. p53 and p16 can be used in combination to distinguish iCCA from benign A lesions, particularly bile duct adenomas (BDAs). p53 usually shows a strong and diffuse expression in ma- lignant lesions (Fig. 9A), even if no general consensus exists regarding the interpretation of staining results. p16 is constantly expressed in BDAs, but not in iCCAs. Therefore, a negative p16 staining supports iCCA di- agnosis 29 (Fig. 9B). Promising results have also been recently obtained by using DNA flow cytometry on formalin-fixed and paraffin-embedded tissue from bile duct biopsies. In this study, a high rate of aneuploidy (70%) was observed in malignant cases, while a nor- mal DNA content was found in all benign lesions 30. iCCA versus metastatic adenocarcinoma The main purpose of liver biopsy in the setting of malignancies arising in a non-cirrhotic liver is the B differential diagnosis between iCCA and metastatic tumor (Tab. II). Indeed, secondary liver cancers are Figure 9. Differently from benign lesions, intrahepatic chol- much more frequent than iCCA. The most common angiocarcinoma shows a strong and diffuse nuclear expres- neoplasms that metastasize to the liver are colorectal sion of p53 (A), and a complete absence of p16 (B) (A: p53 carcinoma, breast carcinoma, neuroendocrine tum- immunostain; original magnification 20x; B: p16 immunos- ors, lung carcinoma, and gastric carcinoma. Clinical tain; original magnification 20x). information is fundamental to guide the diagnostic ap- proach. In case of an adenocarcinoma with unclear histological features in a patient with unknown extra- hepatic primary tumors, the performance of different itivity and CK20-negativity represent the most com- immunohistochemical panels is recommended. mon immunoprofile of gastric carcinoma, although Colorectal adenocarcinoma (CRC) typically shows some cases may stain positive for CK20. Both gastric a CK20- and CDX2-positive and CK7-negative im- carcinoma and iCCA show CK19 expression. CDX2 munophenotype. CDX2 is a highly sensitive and spe- may be of help, since it is strongly expressed in about 33 cific marker of intestinal differentiation. It may be ex- 60% of gastric adenocarcinoma . pressed by iCCA, but never as diffuse and strong as in Breast cancer is usually CK7-positive and CK20-neg- CRC. Be aware that CDX2 might be negative in some ative. Estrogen/progesterone receptors, gross cystic poorly differentiated CRCs. Special AT-rich sequence- disease fluid protein-15 (GCDFP-15) and mammaglo- binding protein 2 (SATB2) is another specific marker bin are useful markers to exclude breast cancer liver for intestinal and appendiceal adenocarcinoma, with a metastasis. However, GCDFP-15 and mammaglobin higher expression in well-to-moderately than in poorly have a high specificity but low sensitivity 34. Nuclear differentiated CRC 31,32. expression of GATA3 has been recently reported as a Gastric adenocarcinoma may be difficult to differenti- sensitive marker for breast cancer 35. ate from iCCA, mostly of the large duct type. CK7-pos- As reported in the 5th edition of the WHO blue book, 164 S. Sarcognato et al.

Table II. Immunohistochemical markers useful in the differential diagnosis between intrahepatic cholangiocarcinoma and metastatic tumors. Markers iCCA CRC GA BC NEN LA PDA CK7 + - + + +/- + + CK20 -/+ + -/+ - +/- - -/+ CDX2 -/+ + +/- - (+&) - -/+ SATB2 - + - - - - - CK19 + - + - - - + Breast markers* - - - + - - - Neuroendocrine markers# - -§ - - + - - TTF1 - - - - (+&) + - Napsin A - - - - - + - *: Estrogen/progesterone receptors, gross cystic disease fluid protein-15, mammaglobin, GATA3; #: chromogranin A, synaptophysin, CD56; §: in non-neuroendocrine carcinoma; &: in metastatic neuroendocrine neoplasms, depending on their organ of origin (large bowel/lungs). iCCA: intrahepatic cholangiocarcinoma; CRC: colorectal carcinoma; GA: gastric adenocarcinoma; BC: breast carcinoma; NEN: neuroendocrine neoplasms; LA: lung adenocarcinoma; PDA: pancreatic ductal adenocarcinoma; CK: cytokeratin; SATB2: special AT-rich sequence-binding protein 2; TTF1: thyroid transcription factor 1. neuroendocrine neoplasms (NENs) may occur as pri- be also useful, but it is important to remind that not all mary liver tumors, even if extremely rare and much iCCA produce mucin. Arginase-1 is the most sensitive less common than the metastatic ones 3. Therefore, (> 90%) and highly specific marker for HCC, including to define a liver neuroendocrine tumor as primary, poorly differentiated and scirrhous HCC 38,39. However, metastasis from other organs must be thoroughly ex- it may rarely be observed in other tumors, including cluded. The differential diagnosis between metastatic some poorly differentiated cholangiocarcinoma. Again, NEN and iCCA is based on tissue expression of neu- HepPar1 has a low sensitivity in poorly differentiated roendocrine markers, such as chromogranin A, syn- HCC, as well as p-CEA and CD10, whose canalicular aptophysin, and CD56, which are usually absent or pattern of staining is classically considered a specific only focally expressed in iCCA. Primary and metastat- marker of hepatocellular differentiation 40. Glypican-3 ic NENs are histologically and immunohistochemically is an oncofetal protein expressed in most HCC, with undistinguishable, although lineage-specific markers, higher sensitivity for poorly differentiated tumors. Nev- such as CDX2 or thyroid transcription factor 1 (TTF1), ertheless, it is not a lineage marker and several tumor may be of help in defining the organ of origin in well- types may express it. Luckily, Glypican-3 expression differentiated tumors. is uncommon in iCCA, therefore it is a useful tool in Lung adenocarcinoma typically shows immunoreac- the differential diagnosis with HCC. iCCA typically tivity for CK7, TTF-1, and napsin A. TTF1 is less fre- stains with CK7 and CK19. However, while a CK7- and quently expressed in invasive mucinous adenocarci- CK19-negative tumor is unlikely to be an iCCA, their nomas and in adenocarcinomas with solid pattern, but expression does not necessarily point towards a bili- it is always absent in iCCA. ary differentiation 39. Pancreatic ductal adenocarcinoma metastatic to the liver is impossible to distinguish from large duct iCCA, iCCA versus epithelioid emangioendothelioma both by morphology and immunohistochemical pro- Epithelioid hemangioendothelioma (EHE) is a rare file 3,36,37. Clinical history and imaging must be con- malignant vascular neoplasia, which may occur in sidered. the liver. It is composed of epithelioid cells within a myxoid-hyaline or fibrous stroma 3. Neoplastic cells iCCA versus hepatocellular carcinoma may show intracytoplasmic vacuoles mimicking mucin Differential diagnosis between iCCA and HCC is usu- vacuoles of an adenocarcinoma, and the presence of ally straightforward. However, in poorly differentiated a dense fibrous stroma may lead to an incorrect di- lesions, when conventional histology does not allow agnosis of poorly differentiated iCCA. However, differ- a definite differential, a panel of immunohistochemical ently from iCCA, EHE neoplastic cells are consistently stains can be of help, and it should include hepatocyte positive for one or more endothelial markers, including markers (Arginase-1, HepPar-1), biliary cytokeratins ERG, CD31, CD34 and FLI1. The main pitfall in this (CK7 and CK19), polyclonal carcinoembryonic antigen differential is represented by the aberrant expression (p-CEA), CD10, and Glypican-3 3,21. A mucin stain may of cytokeratins in many EHEs 41,42. In a recent study, CHOLANGIOCARCINOMA 165

CK7- and panCK AE1/AE3-positivity was reported in 5/9 (56%) and 6/9 (67%) of hepatic EHEs, respec- tively 43. Thus, keeping this in mind is fundamental for preventing misdiagnosis of EHEs as iCCAs.

Perihilar and distal cholangiocarcinoma pCCA is the most common CCA (50-60% of cases) and develops from the extrahepatic biliary tree, proxi- mally to the origin of cystic duct (right and/or left he- patic duct and/or at their junction). dCCA (20-30% of all CCAs) involves the extrahepatic bile ducts, distally to the insertion of cystic duct (common bile duct, i.e. choledochus) 1-3,44. Recent guidelines recommend to A avoid the use of the terms extrahepatic-CCA for dCCA or for pCCA 1-3,44. All available studies on CCA epidemiology are based on the old CCA classification, which divided CCA into intra- and extra-hepatic; thus, data on pCCA- specific incidence and risk factors are still too scant. The most frequent symptom in pCCA and dCCA is jaundice due to biliary tract obstruction. CA19-9 is typically elevated 1,2. In these tumors, the associa- tion of contrast-enhanced magnetic resonance and magnetic resonance-cholangiopancreatography is the first diagnostic tool, due to its accuracy in dis- criminating between benign and malignant obstruc- tion, as well as in assessing the degree of biliary extension. However, like iCCA, a definitive diagno- B sis can only be based on pathologic confirmation. In particular, recent guidelines suggest endoscopic Figure 10. Perihilar (A) and distal (B) cholangiocarcinomas ultrasound-fine needle aspiration or biopsy (EUS- share similar morphological features, being characterized by FNA/B) as the first approach, followed by endoscopic well-formed irregular neoplastic glands in a desmoplastic retrograde cholangiopancreatography with brushing stroma (A: hematoxylin-eosin; original magnification 5x; B: and/or biopsy and/or cholangioscopy-guided biopsy hematoxylin-eosin; original magnification 5x). of a target lesion, when EUS-FNA/B is inconclusive, since they have been considered the most accurate techniques to obtain a final diagnosis of pCCA and dCCA 1-3,45. vascular invasion. Most of them are of pancreatobi- liary-type, but other histological patterns include the Pathology intestinal-type, the foveolar-type, the mucinous, the Tumors near the hepatic hilum and dCCA are usually signet ring cell, the clear cell, the pyloric gland, the small, since they cause early obstructive jaundice. hepatoid, and the invasive micropapillary ones 2,3,46- Frequently, the macroscopic boundaries of pCCA and 48. pCCA and dCCA are graded as well-, moderately, dCCA are blurred and difficult to determine. Grossly, or poorly differentiated adenocarcinomas, according both pCCA and dCCA may present as flat or poorly- to their cell morphology and gland formation 3. Rare defined nodular sclerosing masses or, less frequently, subtypes include squamous, adenosquamous, and as intraductal papillary tumors 2,3. sarcomatoid carcinoma. As for large duct iCCAs, pC- The vast majority of pCCA and dCCA are mucin- se- CAs and dCCAs are often preceded by pre-invasive creting adenocarcinomas characterized by widely lesions, including BilIN and IPNB 3,21,22. spaced, well-formed irregular glands and small cell In bile and brush cytology, the presence of epithelial clusters, within a desmoplastic sclerotic stroma cells with prominent nucleoli, thickening and irregular- (Fig. 10). They often show perineural and lympho- ity of nuclear membrane, and increased chromatin is 166 S. Sarcognato et al.

diagnostic for malignancy. Tumor cells may show dif- identifying about 25% of tumors with actionable ge- ferent degree of pleomorphism, mitotic activity, and nomic alterations, with potential prognostic and thera- loss of nuclear polarity. The distinction between inva- peutic implications 54. sive and in situ carcinoma is not possible on cytologi- cal smears 3,49. The distinction between pCCAs/dCCAs and reactive Cholangiocarcinoma staging periductal glands is the main differential issue, since it is not always straightforward on morphology alone. Staging for iCCA, pCCA, and dCCA is based on the Clinical history and imaging must always be consid- 8th edition of AJCC staging system 44 (Tabs. IV-VI). ered. Involvement of extrahepatic bile ducts by pan- creatic duct adenocarcinoma is indistinguishable from CCA, both by morphology and immunohistochemical Pathological report of resected profile 3,36,37. cholangiocarcinoma

A standardized approach to cancer reporting is high- Molecular background of ly recommended in resected CCAs, as in any tumor cholangiocarcinoma setting. A comprehensive and accurate pathology re- port is a prerequisite to adequate cancer staging and Many molecular alterations have been recently de- outcome prediction. Nowadays, cancer reports must scribed in CCAs, but full molecular profiling or gene include many elements necessary for clinical man- mutation analyses are not yet routinely recommend- agement, and with the advent of targeted therapies ed, since they do not currently result in any improve- and personalized medicine, its complexity is even ment in patient management 11,12,14,15,22. significantly increasing 55. It has been demonstrated Small duct type iCCA show frequent IDH1 and IDH2 that the adoption of histopathological reporting mod- mutations (10-20%), associated with poor prognosis, els lead to improvements in the reporting of key prog- and FGFR2 fusions (8-14%), associated with a bet- nostic factors by pathologists 56,57. ter prognosis, both representing possible therapeutic The International Collaboration on Cancer Reporting targets. On the contrary, KRAS and TP53 have been (ICCR) is an alliance formed by the Royal College demonstrated in large duct iCCAs, pCCAs, and dC- of Pathologists of Australasia, the Royal College of CAs. TP53 mutations are present in about 50% of Pathologists of the United Kingdom, the College of pCCAs and dCCAs and are a late pathogenic event, American Pathologists, the Canadian Partnership while KRAS mutations occur early in 20-30% of dC- Against Cancer, the European Society of Pathology, CAs. dCCAs may show MDM2 amplification in 12% and the American Society of Clinical Pathology, with of cases 50,51. Other genes frequently mutated in the aim to develop an evidence-based reporting data CCAs are those involved in chromatin remodelling, set for each cancer site (http://www.iccr-cancer.org/). such as ARID1 in pCCA and dCCA, and BAP1 in Lately, the ICCR data set for reporting liver tumors, iCCA 3,11,12,14,52. A molecular classification of CCA has including iCCA and pCCA, has been updated and it been recently proposed, with different subclasses is now freely available for worldwide use at the ICCR showing different features and prognosis 10,53 (Tab. III). website (http://www.iccr-cancer.org/datasets/pub- Lately, a multi-platform molecular characterization of lished-datasets/digestive-tract/liver). This dataset in- extrahepatic CCAs (pCCAs plus dCCAs) has been cludes items agreed to be essential to the pathologi- performed in a cohort of 189 patients, revealing four cal reporting, but additional data may be included ac- novel transcriptome-based molecular classes and cording to local needs and to guarantee clarification.

Table III. A recently proposed molecular classification of cholangiocarcinoma 53. Specific genetic alterations are related to anatomical and histomorphological classifications of cholangiocarcinoma. SBD iCCA-specific iCCA-specific Shared by LBD iCCA and eCCA# eCCA-specific# IDH1/2 EPHA2 KRAS ARID1B FGFR2 BAP1 TP53 PRKACA SMAD4 BRAF GNAS MDM2 NRAS/MRAS #: eCCA includes perihilar and distal cholangiocarcinoma. SBD: small bile duct; iCCA: intrahepatic cholangiocarcinoma; LBD: large bile duct; eCCA: extrahepatic cholangiocarcinoma. CHOLANGIOCARCINOMA 167

Table IV. TNM staging classification of intrahepatic cholangiocarcinoma44. T - Primary tumor Tx Primary tumor cannot be assessed T0 No evidence of primary tumor Tis Carcinoma in situ (intraductal tumor) T1a Solitary tumor ≤ 5 cm in greatest dimension WITHOUT VI T1b Solitary tumor > 5 cm in greatest dimension WITHOUT VI T2 Solitary tumor WITH intrahepatic VI OR multiple tumors with or without VI T3 Tumor perforating the visceral peritoneum T4 Tumor involving local extrahepatic structures by direct hepatic invasion N - Regional lymph nodes* Nx Regional lymph nodes cannot be assessed N0 NO regional lymph nodes metastasis N1 Regional lymph nodes metastasis M - Distant metastasis M0 NO distant metastasis M1 Distant metastasis *: in a regional lymphadenectomy specimen, ≥6 lymph nodes should be histologically evaluated. VI: vascular invasion.

Table V. TNM staging classification of perihilar cholangiocarcinoma44. T - Primary tumor Tx Primary tumor cannot be assessed T0 No evidence of primary tumor Tis Carcinoma in situ (intraductal tumor) T1 Tumor confined to the bile duct, extension up to the muscle layer or fibrous tissue T2a Tumor invades beyond the wall of the bile duct to surrounding adipose tissue T2b Tumor invades adjacent hepatic parenchyma T3 Tumor invades unilateral branches of the portal vein or hepatic artery T4 Tumor invades the main portal vein or its branches bilaterally OR the common hepatic artery OR unilateral second-order biliary radicals with contralateral portal vein or hepatic artery involvement N - Regional lymph nodes* Nx Regional lymph nodes cannot be assessed N0 NO regional lymph nodes metastasis N1 Metastases to 1-3 regional lymph nodes N2 Metastases to ≥4 regional lymph nodes M - Distant metastasis M0 NO distant metastasis M1 Distant metastasis *: in a regional lymphadenectomy specimen, ≥15 lymph nodes should be histologically evaluated.

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Table VI. TNM staging classification of distal cholangiocarcinoma 44. T - Primary tumor Tx Primary tumor cannot be assessed T0 No evidence of primary tumor Tis Carcinoma in situ (intraductal tumor) T1 Tumor invades bile duct wall to a depth <5 mm T2 Tumor invades bile duct wall to a depth of 5 mm up to 12 mm T3 Tumor invades bile duct wall to a depth >12 mm T4 Tumor involves the coeliac axis, the superior mesenteric artery and/or the common hepatic artery N - Regional lymph nodes* Nx Regional lymph nodes cannot be assessed N0 NO regional lymph nodes metastasis N1 Metastases to 1-3 regional lymph nodes N2 Metastases to ≥4 regional lymph nodes M - Distant metastasis M0 NO distant metastasis M1 Distant metastasis *: in a regional lymphadenectomy specimen, ≥12 lymph nodes should be histologically evaluated.

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Review

Autoimmune biliary diseases: primary biliary cholangitis and primary sclerosing cholangitis

Samantha Sarcognato1, Diana Sacchi1, Federica Grillo2, Nora Cazzagon3, Luca Fabris4, Massimiliano Cadamuro4, Ivana Cataldo1, Claudia Covelli5, Alessandra Mangia6, Maria Guido1,7 1 Department of Pathology, Azienda ULSS2 Marca Trevigiana, Treviso, Italy; 2 Anatomic Pathology Unit, University of Genova and Policlinico San Martino Hospital, Genova, Italy; 3 Department of Surgery, Oncology and Gastroenterology - DISCOG, University of Padova, Padova, Italy; 4 Department of Molecular Medicine - DMM, University of Padova, Padova, Italy; 5 Pathology Unit, Fondazione IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo, Italy; 6 Liver Unit, Fondazione IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo, Italy; 7 Department of Medicine - DIMED, University of Padova, Padova, Italy

Summary Autoimmune cholestatic liver diseases are rare hepato-biliary disorders characterized by a progressive, inflammatory destruction of bile ducts. Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are the main autoimmune cholestatic liver diseases. Both may evolve into secondary biliary cirrhosis and its complications. Therapeutic options are limited and liver transplantation remains the only definitive treatment for PBC and PSC. Most PBC and PSC patients have a typical presentation, which does not require liver Received and accepted: January 4, 2021 biopsy. However, in routine clinical practice, important variants or specific subgroups that benefit from liver biopsy for proper management may be observed. Herein, we provide a Correspondence general overview of clinical and pathological characteristic of PBC and PSC, highlighting Samantha Sarcognato the most important features for routine diagnostic practice. piazzale Ospedale 23, 31100 Treviso Tel.: +390422328102 Key words: immune-mediated cholangitis, autoimmune cholangitis, PBC, PSC, overlap Fax: +390422328112 E-mail: [email protected] variants

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

How to cite this article: Sarcognato S, Sacchi D, Grillo F, et al. Autoimmune Immune-mediated cholangiopathies are chronic cholestatic disorders biliary diseases: primary biliary cholangitis whose development is driven by auto- and allo-immunity 1,2. They include and primary sclerosing cholangitis. primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), Pathologica 2021;113:170-184. https://doi. org/10.32074/1591-951X-245 IgG4-related sclerosing cholangitis, graft-versus-host disease and he- patic allograft rejection, even if some studies also consider biliary atresia © Copyright by Società Italiana di Anatomia Pato- 1-5 logica e Citopatologia Diagnostica, Divisione Itali- as an altered immunity-associated disease . These diseases affect the ana della International Academy of Pathology biliary tree at different levels and for different extent, and they may also involve the peribiliary glands 6. OPEN ACCESS Immune-mediated cholangiopathies are characterized by an accumula- This is an open access journal distributed in accordance tion of activated auto- or allo-reactive T lymphocytes at the site of bile with the CC-BY-NC-ND (Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 International) license: the duct destruction, as the regulatory T cell is the main effector in the initia- work can be used by mentioning the author and the license, tion of the process 6-8. but only for non-commercial purposes and only in the original version. For further information: https://creativecommons. In this review we will focus on PBC and PSC, which are considered the org/licenses/by-nc-nd/4.0/deed.en main autoimmune cholestatic liver diseases. PBC AND PSC 171

Primary biliary cholangitis tatic biochemical profile: alkaline phosphatase (ALP) levels are increased to 2 or more times the upper limit PBC, formerly known as primary biliary cirrhosis, is of normal, and this increase is usually sustained for 6 a chronic inflammatory autoimmune cholestatic liver months or longer; similarly, γ-glutamyl transferase lev- disease characterized by the destruction of small in- els are increased to 5 or more times the upper limit of trahepatic bile ducts, leading to fibrosis and eventually normal. Total bilirubin is usually normal in early stages, cirrhosis and its complications 9,10. In 1950, Ahrens et while abnormal values should raise concern for ad- al. defined PBC in a series of 25 cases of diagnosed vanced disease. Serum aminotransferases might be chronic intrahepatic biliary obstruction with xanthoma- slightly elevated 9. The serological hallmark of PBC is tosis. However, Addison and Gull had described the the presence of anti-mitochondrial antibodies (AMA), very first case of PBC almost 100 years earlier 10. highly disease-specific antibodies identified in about 95% of PBC patients 9,10. AMA in a patient with raised Epidemiology ALP is diagnostic of PBC, after the exclusion of other PBC is mainly diagnosed in women (92% of patients), intrahepatic and extrahepatic causes of cholestasis. with a female:male ratio of about 10:1, and a mean AMA-negative PBC occurs in 5% of patients with an age at presentation of 55 years. Population-based epi- otherwise typical clinical, biochemical and histologi- demiological studies across Europe, North America, cal profile 3,10. The presence of isolated AMA positiv- Asia, and Australia have revealed an incidence of 0.9 ity in the absence of clinically apparent liver disease to 5.8 per 100,000 people per year. Some groups re- or abnormal liver function tests is seen in up to 0.5% ported an increasing incidence over time, but whether of otherwise healthy individuals. This finding may be this denotes a true rise or is biased by advances in di- a hallmark of preclinical disease, and up to 17% will agnosis, is still unclear 9,10. The prevalence of the dis- develop PBC over 5 years 3,10. Other autoantibodies ease ranges from 1.9 to 40.2 per 100,000 people, and are often identified in PBC patients, particularly anti- has also grown over time. The increased prevalence nuclear antibodies (ANA). Anti-Sp100 and anti-gp210 is probably attributable to a combination of better dis- ANA have a high specificity for PBC, and could be of ease recognition and data collection, and increased help in the diagnosis when AMA are negative. More- survival after the introduction of ursodeoxycholic acid over, the presence of anti-Sp100 or anti-gp210 is as- (UDCA). PBC is characterized by a significant geo- sociated with a more clinically aggressive disease 9,10. graphical discrepancy, suggesting a possible role IgM levels are often high, and this is useful in the dif- of environmental triggers in the development of dis- ferential diagnosis with autoimmune hepatitis (AIH) in ease 10. which high IgG levels are typical, while IgM levels are low. Hyperlipidemia is also a common finding 9,10. Overview on clinical and laboratory features PBC may coexist with other liver autoimmune disor- Fatigue and pruritus are the most common symptoms ders, particularly AIH (see the “Overlap variants” para- in PBC patients, and often have a quite negative effect graph). on quality of life 9,10. Patients with pruritus might have The goals of treatment and management are the pre- excoriations or bleeding as a result of chronic scratch- vention of end-stage liver disease, and the improve- ing. Melanin deposition may occur, causing hyperpig- ment of associated symptoms. The natural history mentation of the skin in up to 50% of cases. Long-term of the disease has changed substantially with the complications of the disease include osteopenia and introduction of UDCA, as it has significantly amelio- osteoporosis, hyperlipidemia and vitamin deficien- rated transplant-free survival in PBC patients, espe- cies 10. In late stages, typical signs of cirrhosis and cially when started early in the disease course 9,10,13,14. portal hypertension (spider nevi, palmar erythema, However, about 40% of patients do not have a bio- ascites, splenomegaly and muscle wasting) might be chemical response to UDCA and would probably present, as well as an increased risk of hepatocellular benefit from new therapies, such as obeticholic acid carcinoma (HCC) 9,10. In the cirrhotic stage of the dis- and off-label therapies (fibric acid derivatives and ease, radiological HCC surveillance is therefore man- budesonide) 9,10,14-16. Orthotopic liver transplantation datory 10. Patients with PBC, particularly women, have (OLT) is life-saving with excellent outcomes for those a higher likelihood of other concomitant autoimmune with decompensated cirrhosis10. Recurrent PBC after disorders, and up to 55% have an additional ongo- OLT occurs in up to 25% of patients 10. ing autoimmune process, such as autoimmune Hashi- In the last few years, efforts have been made to im- moto’s thyroiditis, Sjogren’s syndrome and Raynaud’s prove prognostic tools in PBC patients. The Global disease 3,10-12. PBC Study Group and the UK-PBC consortium de- PBC patients typically present with a chronic choles- veloped two new continuous scoring systems, the 172 S. Sarcognato et al.

GLOBE Score and the UK-PBC Risk Score, consid- ical, it is seen in only about 10% of biopsy specimens ering both measures of treatment response and pa- from PBC patients, usually at early disease stages 6,9. rameters of disease severity 10,17,18. Both showed better The florid duct lesion is characterized by a chronic, performance in the prediction of death or OLT than intense, inflammatory infiltrate which surrounds and previous criteria 9,10. eventually destroys the bile ducts 6,9,26 (Fig. 1). Inflam- In the presence of a cholestatic liver profile and AMA matory cells classically infiltrate between adjacent positivity, liver biopsy is not necessary to establish cholangiocytes, destroying the bile duct basement the diagnosis of PBC. However, biopsy is manda- membrane (Fig. 2). The inflammatory activity of PBC tory when AMA are absent, if the biochemical profile affects the small interlobular and septal bile ducts, shows a mixed cholestatic and hepatocellular pattern, with sparing of the large and extrahepatic ducts. In or when other co-existent liver diseases, such as AIH the affected bile ducts, cholangiocytes show swelling, or non-alcoholic steatohepatitis (NASH), are suspect- vacuolated or eosinophilic cytoplasm and pyknotic ed 9,10,19. Even if liver biopsy remains the gold stan- nuclei. The biliary epithelium also shows prolifera- dard, non-invasive methods to assess hepatic fibro- tive changes with stratification 27. The rupture of the sis are currently being considered, such as transient bile duct basement membrane may lead to bile duct elastography, that has demonstrated more than 90% ectasia 6. The inflammatory infiltrate consists mainly of sensitivity and specificity for detecting advanced fi- of CD4+ and CD8+ T lymphocytes, with a variable brosis in PBC patients 10. number of other inflammatory cells 6. Inflammatory cells may sometimes coalesce to form an epithelioid Pathogenesis non-caseating granuloma, which is a typical finding PBC seems to be related to complex interactions in PBC samples, particularly when intimately associ- between genetic predisposition and environmental ated with a damaged bile duct (Fig. 3). Infections and triggers. Geographical clustering of cases has been sarcoidosis are the main differential diagnoses in the reported, and different factors have been associated presence of granulomas in a liver specimen. Special with the disease, such as infectious agents, hair dyes, stains for microorganisms as well as a close correla- nail polish and cigarette smoking, even if their role has tion with clinical data are required to reach a correct not been yet defined. The prevalence of disease is diagnosis 6,10. Bile duct damage is usually associated higher in families with an affected member, and sev- with ductular reaction along the periphery of portal eral North American and European studies described tracts, mainly at early disease stages 20,28 (Fig. 4). a strong link between HLA alleles and PBC. Recent Portal tract (PT) inflammation accompanies bile duct molecular studies reported that PBC shares some risk damage. Lymphocytes are the predominant cells, but alleles with other autoimmune diseases. These risk al- plasma cells are often numerous and this should not leles seem to occur in genes associated with immune function, potentially affecting different immune path- ways, although the mechanisms by which they affect phenotype are not yet known 10,20-22. The target cell in PBC is the cholangiocyte, which seems to express T-cell ligands essential for the in- duction of biliary epithelial autolysis. Moreover, chol- angiocytes may act as antigen-presenting cells, thus amplifying the immune response 10,21,23-25. Farnesoid X receptors are nuclear hormone receptors which are essential in bile acid metabolism. They play a role in cholestatic liver diseases and may represent new potential therapeutic targets 10.

Histology None of the histological changes seen in PBC sam- ples are pathognomonic, thus histological features need to be interpreted in the light of clinical and labo- Figure 1. A florid duct lesion in a primary biliary cholangitis ratory findings, as in most liver disease settings. patient. A chronic inflammatory infiltrate made of lympho- The florid duct lesion, also referred to as non-suppu- cytes and plasma cells surrounds and destroys the bile duct rative destructive cholangitis, is the distinctive histo- (hematoxylin-eosin; original magnification 20x). logical lesion in PBC. However, even if considered typ- PBC AND PSC 173

Figure 2. Lymphocytes infiltrate between adjacent cholan- Figure 4. A portal tract showing a damaged bile duct and giocytes, destroying the bile duct basement membrane (ar- diffuse ductular reaction (cytokeratin 7 immunostain; origi- rows) (Masson’s trichrome stain; original magnification 40x). nal magnification 10x).

Figure 3. A typical epithelioid non-caseating granuloma in Figure 5. A portal tract with dense chronic inflammatory a portal tract, intimately associated with a damaged bile duct infiltrate, with numerous eosinophils (arrows), focally ar- (hematoxylin-eosin; original magnification 20x). ranged in clusters (asterisk) (hematoxylin-eosin; original magnification 40x).

be misinterpreted as a sign of AIH. A coronal arrange- Parenchymal necro-inflammatory changes, including ment of plasma cells around the bile duct has been hepatocyte necrosis, acidophilic bodies, Kupffer cell considered distinctive for PBC 6,29. Eosinophils may al- hyperplasia and sinusoidal infiltration by lymphocytes so be numerous, particularly at earlier stages (Fig. 5). and pigment-laden macrophages, may be seen, but Lymphoid follicles are often seen in portal tracts of they are generally mild in severity 9. Severe necro- PBC patients, both at early and late stages 10,20. inflammatory lobular damage should always raise the PT inflammation is often associated with interface hep- suspicion of AIH or PBC-AIH variant. atitis, which is usually mild. If interface hepatitis domi- In advanced disease stages, the toxic effect of hy- nates the histological picture, an alternative diagnosis drophobic bile acids causes the so-called feathery of AIH or PBC-AIH variant should be considered 6. degeneration of hepatocytes (Fig. 6). Bile stasis also 174 S. Sarcognato et al.

Figure 6. A liver biopsy from a primary biliary cholangitis Figure 7. Ductopenia in a patient with advanced primary patient showing diffuse hepatocellular cholestasis, associ- biliary cholangitis. Cytokeratin 7 stain shows diffuse ductu- ated with feathery degeneration of hepatocyte cytoplasm lar reaction, with no native bile ducts left (cytokeratin 7 im- (arrow) (hematoxylin-eosin; original magnification 63x). munostain; original magnification 5x).

leads to deposition of copper or copper-associated protein granules 6. As the disease progresses, bile duct changes evolve, till only remnants of biliary epithelium can be visible or identifiable by immunohistochemical stain for cyto- keratin (CK) 7. In the end, bile duct remnants disap- pear and only lymphocytic aggregates or granulomas persist. The smallest branches are always the first to disappear. Since the bile ducts typically run parallel to hepatic artery branches, the finding of unpaired arter- ies is a presumptive evidence of vanishing bile ducts and it is useful in the routine practice to evaluate the extent of bile duct loss (BDL), a parameter included in the current staging system 6 (see below). Persistent ductal and portal inflammation and bile duct damage lead to ductopenia (Fig. 7), periportal or diffuse meta- Figure 8. Cytokeratin 7 stain highlights biliary metaplasia plastic hepatocytes (Fig. 8), and portal and periportal of hepatocytes (cytokeratin 7 immunostain; original magni- collagen deposition, eventually with bridging fibrosis fication 10x). and cirrhosis development 10,20 (Fig. 9). The biliary- type fibrosis is dense, scar-like, with edema at the pe- riphery, giving rise to the typical halo effect 6. A premature ductopenic variant has been described which connect bile canaliculi to the interlobular bile in about 5-10% of patients, and it is characterized by ducts and are identifiable by CK19 immunostaining in a rapid onset of BDL in the absence of significant fi- normal liver, are decreased in number in all stages of brosis, icteric cholestasis, and a rapid progression to- PBC, suggesting that they are destroyed together with wards cirrhosis 3,30. small bile ducts 31. A study by Khan et al. found that Foci of concentric periductal fibrosis may be seen in this loss may be a very early manifestation of PBC about 20% of PBC patients, and may cause problems that precedes BDL and can be observed in liver biop- in the differential diagnosis with PSC. A cholangio- sies otherwise normal or nearly normal, defining the gram is necessary in such cases, since extrahepatic so-called ‘minimal change’ PBC 32 (Fig. 10). However, bile ducts are healthy in PBC 6. the role of this lesion in the natural history of PBC is It has been demonstrated that the canals of Hering, still debated 31,33. PBC AND PSC 175

A B

Figure 9. Biliary-type cirrhosis is characterized by parenchymal nodules with edema at the periphery, giving rise to the so-called halo effect (A). Trichrome stain shows complete dense fibrous septa, and better highlights the halo effect at the parenchyma- septum border (B) (A: hematoxylin-eosin; original magnification 5x; B: Masson’s trichrome stain; original magnification 5x).

Scheuer’s staging systems, which were widely used in the past 36,37 (Tab. I). Recently, a new approach to stage PBC has been proposed by Nakanuma et al., who also introduced the concept of PBC grading. This scoring system demonstrated the strongest prognos- tic value compared to previous methods, with a more accurate prediction of patient outcome at 10 years, particularly in the development of cirrhosis and its complications 38,39. Therefore, the use of Nakanuma’s staging system should replace the old systems in ev- eryday routine. This system establishes the grade of the disease by evaluating chronic cholangitis activ- ity and hepatitis activity, the latter determined by the presence and extent of interface and lobular hepati- tis 38 (Tab. II, Fig. 11). On the other hand, the stage of Figure 10. Minimal change primary biliary cholangitis. Cy- the disease is determined by the evaluation of two or tokeratin 19 immunostaining shows a decreased number of three criteria: the presence and extent of fibrosis, the canals of Hering in an otherwise normal portal tract (cyto- presence and extent of BDL, and the presence and keratin 19 immunostain; original magnification 20x). extent of orcein-positive granule deposition (Tab. III, Fig. 12). The third criterion is not essential, since an accurate and reproducible staging can be obtained even with the two-criteria method 38. In patients with portal hypertension, a reticulin stain can exclude the presence of nodular regenerative Differential diagnoses hyperplasia, a known complication of non-cirrhotic Bile duct damage may be seen in inflamed portal PBC 10,34. Moreover, in non-cirrhotic PBC stages, tracts of patients with viral hepatitis, particularly in small-cell change of periportal hepatocytes may oc- chronic hepatitis C, while lymphocytic cholangitis has cur, but it is considered a regenerative phenomenon also been described in viral hepatitis E. However, this rather than a pre-malignant lesion 6,35. does not represent a real problem in routine practice, since a diagnosis of viral hepatitis is easily obtained Staging systems by serological tests, and a liver biopsy is nowadays Many histological staging systems for PBC have rarely required in this setting. been developed over time, including the Ludwig and Bile duct inflammatory changes are also seen in AIH, 176 S. Sarcognato et al.

Table I. Ludwig and Scheuer’s staging systems for PBC. Stage Ludwig System Scheuer System I Portal inflammation with bile duct damage, with or without Portal inflammation with bile duct damage, with or without florid duct lesions florid duct lesions II Periportal inflammation/ Ductular reaction Interface hepatitis III Bridging fibrosis Bridging fibrosis IV Biliary cirrhosis Biliary cirrhosis

Table II. Grading of chronic cholangitis activity and hepatitis activity, according to Nakanuma’s system. Chronic cholangitis activity (CA) CA 0 (no activity) No cholangitis, mild biliary epithelial damage CA 1 (mild activity) 1 BD with evident chronic cholangitis CA 2 (moderate activity) ≥ 2 BDs with evident chronic cholangitis CA 3 (marked activity) ≥ 1 florid duct lesion Hepatitis activity (HA) HA 0 (no activity) No IH and no or minimal LH HA 1 (mild activity) IH in ≥ 10 continuous hepatocytes in 1 PT and mild to moderate LH HA 2 (moderate activity) IH in ≥ 10 continuous hepatocytes in ≥ 2 PTs and mild to moderate LH HA 3 (marked activity) IH in ≥ 20 continuous hepatocytes in ≥ 1/2 PTs and moderate LH or bridging/zonal necrosis BD: bile duct; IH: interface hepatitis; LH: lobular hepatitis; PT: portal tract.

moderate to severe necro-inflammatory lobular le- sions typical of AIH are never present 43. Plasma cells may be numerous in both PBC and AIH, although the presence of plasma cell clusters (defined as collec- tions of ≥ 5 contiguous plasma cells) in the lobule is considered highly sensitive for AIH 29,44.

Role of liver biopsy in PBC As mentioned above, liver biopsy is no longer required for the diagnosis of PBC in most cases, i.e. when a cholestatic liver profile is associated with AMA positiv- ity. As a general rule, liver biopsy should be performed only when the initial diagnostic workout is not able to identify the cause of cholestasis. Liver biopsy is clearly mandatory whenever AMA are not detected, and to rule Figure 11. Representation of grading of cholangitis and out the diagnosis of AMA-negative PBC. As reported hepatitis activity, according to Nakanuma’s system 38. above, up to 10% of patients may fall into this catego- ry 9,10. Suspected PBC-AIH variant is a further indica- tion for liver biopsy. In this setting, a histological diag- nosis is mandatory since patients with this variant may while interface hepatitis (the hallmark of AIH) and benefit from immunosuppressive therapy 3. Again, liver some degree of lobular necro-inflammation are com- biopsy is necessary whenever other co-existent liver mon in PBC6. The most important clues for a correct diseases, particularly NASH, are suspected in patients differential diagnosis are: i) BDL and ductopenia are with an established PBC diagnosis 9,10. Of note, NASH not typical AIH features, since the bile duct is not the has become prevalent in the same population at risk for target of the disease in AIH 40,41; ii) metaplastic chang- PBC, probably due to the pandemic diffusion of NASH es of hepatocytes (easily highlighted by CK7 stain) risk factors. However, a link between NASH and PBC and orcein periportal deposits are signs of chronic has not been yet demonstrated 45. Finally, liver biopsy cholestasis, and are not observed in AIH specimens, remains the gold standard in assessing hepatic fibro- unless extensive fibrosis or cirrhosis is present 42; iii) sis, even if non-invasive methods are currently being interface hepatitis is minimal to mild in PBC, and the used, with a good performance 10,19. PBC AND PSC 177

Table III. Nakanuma’s staging system. Fibrosis Score 0 No portal fibrosis or fibrosis limited to PTs Score 1 Portal and periportal fibrosis ± fibrous septa Score 2 Bridging fibrosis with variable lobular disarray Score 3 Liver cirrhosis Bile duct loss (BDL) Score 0 No BDL Score 1 BDL in <1/3 of PTs Score 2 BDL in 1/3 to 2/3 of PTs Score 3 BDL in >2/3 of PTs Deposition of orcein-positive granules (DOG) Score 0 No DOG Score 1 DOG in a few periportal hepatocytes in <1/3 of PTs Score 2 DOG in several periportal hepatocytes in 1/3 to 2/3 of PTs Score 3 DOG in many hepatocytes in >2/3 of PTs Stage 3 criteria 2 criteria Stage 1 (no progression) 0 0 Stage 2 (mild progression) 1-3 1-2 Stage 3 (moderate progression) 4-6 3-4 Stage 4 (advanced progression) 7- 9 5-6 PT: portal tract.

retrograde cholangiography in the 1970s led to an in- creasing recognition of the condition 46. PSC is associated with a considerable risk of gastro- intestinal malignancies, mainly cholangiocarcinoma and 46. Disease progression and end-stage liver disease are inevitable in most patients, and OLT is the only curative treatment available, even if post-transplant recurrence may occur 46.

Epidemiology PSC is a rare disease, as its incidence, although var- ies geographically, is as high as 1.3 per 100,000 people per year in Northern Europe. Prevalence is also vari- able, and it reaches 16.2 per 100,000 people in some studies. Both incidence and prevalence of the disease are increasing, but it is not clear whether the increase Figure 12. Representation of disease staging, according to is real or due to a better detection of disease 47,48. Al- Nakanuma’s system 38. though PSC affects both sexes and all age groups, more than 60% of patients are males, and the median age at onset is 30-40 years. Patients are usually non- Primary sclerosing cholangitis smokers, and about 2/3 have a coexistent IBD (75% ulcerative colitis) 47. PSC is a chronic cholestatic liver disease in which in- More than 50% of patients require OLT within 10-15 flammation and fibrosis lead to multifocal biliary stric- years of symptom development, as a result of reduced tures and progression to end-stage liver disease. In- quality of life and increased disease-related complica- tra- and extrahepatic bile ducts are primarily affected. tions and comorbidity 47. The close association with inflammatory bowel dis- eases (IBDs) is a hallmark of the condition, affecting Overview on clinical and laboratory features about two-thirds of the patients 46,47. PSC is insidious, and about half of patients do not Until the 1960s, most published articles about PSC have symptoms. Abnormal liver function tests are of- were case reports, but implementation of endoscopic ten the only signs at diagnosis, while hepatomegaly is 178 S. Sarcognato et al.

present in 44% of patients and splenomegaly in 39% outcomes 48. Several attempts to develop a PSC-spe- of them. When symptoms are present, they include cific prognostic model have been made over time 46. abdominal pain (20% of patients), pruritus (10%), The PSC specific revised Mayo risk score and the jaundice (6%) and fatigue (6%) 48. Even if the actual Amsterdam-Oxford Model are the most widely used risk of PSC in IBD patients is not yet known, prob- models 48,53,54. ably no more than 10% of patients with colitis will de- Caring for patients with PSC is challenging and re- velop PSC. About 10-20% of patients have dominant quires treatment of the primary liver disease and the bile duct strictures and many of them have recurrent coexisting conditions, as well as subsequent therapy bacterial cholangitis. This subgroup of patients seems for potential complications of end-stage liver dis- to have a worse survival and a higher risk of develop- ease 48,55. No effective medical therapies exist for PSC. ment of malignancies 47. Treatment guidelines for PSC are conflicting: some of Typically, ALP is elevated (two to three times the up- them support the use of UDCA, whereas others en- per limit of normal), along with aspartate and alanine dorse the use of only moderate doses 46,48. Several aminotransferases. Different non-specific autoanti- new treatments, such as obeticholic acid and simtu- bodies have been correlated with PSC, such as ANA, zumab, are under investigation and clinical trials are anti-cardiolipin autoantibodies, anti-thyroperoxidase ongoing 48,55. Patients with both PSC and IBD should autoantibodies, rheumatoid factor and anti-smooth be treated according to relevant guidelines. Annual muscle autoantibodies. Even if none is liver specific, with surveillance biopsies should be per- their detection may suggest an underlying immune formed even in patients who underwent OLT, given dysregulation 49. the constant increased risk of colon cancer. PSC pa- PSC is suspected when increased ALP levels persist tients without IBD should undergo colonoscopy every for more than 6 months, and diagnosis relies on chol- 5 years. An annual ultrasonography for the evaluation angiographic findings of bile-duct strictures detected of the gallbladder is also recommended. Because of by either magnetic resonance cholangio-pancreatog- the risk of cancer, patients with gallbladder masses of raphy or endoscopic retrograde cholangio-pancrea- any size should undergo cholecystectomy 48. tography. Exclusion of causes of secondary scleros- Given the progressive nature of PSC, 40% of patients ing cholangitis is mandatory to diagnose PSC, and will eventually require OLT. The 1-year and 5-year sur- include biliary stones, cholangiocarcinoma, biliary vival rates are 85% and 72%. However, the disorder tract surgery, Caroli’s disease, chronic biliary infec- may recur in 25% of cases 48. tion, biliary toxin exposure, chronic portal-vein throm- Small duct PSC (sdPSC) is a rare PSC variant (3- bosis, ischemic stricturing and liver diseases that can 5% of patients) that affects only small intrahepatic bile cause biliary injury, such as cholestatic drug-induced ducts, sparing the large ones. It is characterized by liver injury 47,50,51. clinical and histological features of classical large duct Magnetic resonance elastography and transient liver PSC, lacking typical cholangiographic changes in ex- elastography are non-invasive diagnostic tools to as- tra- and/or intrahepatic bile ducts (normal cholangio- sess liver fibrosis, even if their role in staging PSC is graphic findings). In a large clinical PSC study, sdPSC still under debate 48,50-52. was associated with a better prognosis compared to A variety of coexisting conditions are associated with large duct PSC, with a lower risk of OLT, liver-related PSC. Due to the frequent association with IBDs, colo- death and hepato-biliary malignancy. Nevertheless, noscopy is warranted in all patients who receive a new sdPSC may progress to large duct PSC, developing PSC diagnosis, since the risk of colon cancer among typical cholangiographic lesions 3,47,48. patients with PSC and concomitant IBD is 4 times higher than the risk among patients with IBD alone Pathogenesis and 10 times higher than the risk in the general popu- The pathogenesis of PSC is still unknown, but sev- lation 47,48. Gallbladder diseases (stones, polyps, and eral mechanistic theories have been proposed, and cancer) are also common in patients with PSC 48. both genetic and environmental factors seem to be In developed countries, PSC is the most common involved in disease initiation 46. risk factor for cholangiocarcinoma, which is 400 times Different genes have been studied, and a strong as- more frequent than in the general population. Among sociation with HLA alleles has been described 48. PSC patients, the annual risk of cholangiocarcinoma Several different possible environmental triggers have is 2%, and the 30-year cumulative incidence is 20%. been investigated over time, such as exposure to Therefore, annual ultrasonography of the liver and se- farm animals during childhood, use of contraceptive rum testing for CA19-9 are recommended 48. hormones, urinary tract infections, dietary intake and PSC is a slowly progressive disease, with variable methods of food preparation, but none was clearly as- PBC AND PSC 179

sociated with disease development. Of interest, smok- ing appears to be protective 48. The strong association between PSC and IBDs led to the “microbiota hypothesis”, supported by observa- tions both in vitro and in animal models. According to this hypothesis, microbial molecules from intestinal dysbiosis reach the liver through the portal circulation and initiate an aberrant cholangiocytic response, in- cluding the induction of senescence 23,48,56,57. An ad- ditional theory regarding PSC pathogenesis is the “gut lymphocyte homing” hypothesis. It is supposed that intestinal T cells are stimulated within intestine- associated lymphoid tissue and then recruited to the liver thanks to an abnormal expression of adhesion molecules on periportal endothelial cells. Once in the A liver, activated T cells from the intestine can initiate immune-mediated damage 23,48. It is also believed that cholangiocytes themselves may be actively involved in the pathogenesis of PSC, by secreting pro-inflammatory cytokines and mediating recruitment and stimulation of T cells or acquiring a senescent phenotype 23,24,48.

Histology The presence of an inflammatory infiltrate in a large intra- and extra-hepatic bile duct wall, with an oblit- erative concentric periductal loose fibrosis (so called onion skin fibrosis, Fig. 13), is traditionally considered the pathological hallmark of PSC. Cholangiocytes show features of degeneration and atrophy. The lesion leads to biliary strictures and eventually occlusions B (so called bile duct scars, Fig. 14) 6,47,58. Figure 14. A portal tract showing a bile duct scar (arrow) in a liver biopsy from a primary sclerosing cholangitis patient (A). Van Gieson stain confirms that the bile duct has been replaced by fibrous tissue (arrow) (B) (A: hematoxylin-eo- sin; original magnification 10x; B: Van Gieson stain; original magnification 20x).

Since needle liver biopsies usually include only the small most peripheral portal tracts, the histologic ap- pearance of PSC varies from normal liver tissue to the presence of only indirect signs of large duct ob- struction. Normal tissue may be seen at very early stages, and it does not preclude the diagnosis of PSC if clinical and radiologic findings are otherwise typical. In early disease, the changes are confined to portal tracts, with a mild mixed inflammatory cell infiltrate of Figure 13. Large intrahepatic bile ducts showing oblitera- lymphocytes, plasma cells and neutrophils, usually tive concentric periductal fibrosis, also called onion skin more intense around bile ducts. Lymphoid follicles or 6 fibrosis, in a liver biopsy from a primary sclerosing cholan- aggregates may be present . gitis patient (hematoxylin-eosin; original magnification 20x). The presence of bile duct fibrotic lesions, accompa- nied by reduced numbers of interlobular bile ducts, 180 S. Sarcognato et al.

is virtually diagnostic of PSC, but it is present in less The histologic distinction between PSC and PBC can than 20% of biopsy specimens 6. The absence of typi- be difficult, particularly in liver biopsy. However, cho- cal onion skin fibrosis does not exclude PSC diagno- lestasis-associated changes are more frequent and sis. Of note, although a typical sign of PSC, onion skin tend to appear earlier in PSC than in PBC, periductal fibrosis may be also seen in secondary sclerosing fibrosis and bile duct scars favor PSC, while granu- cholangitis. lomas are most commonly found in PBC 6. As PBC, A significant bile duct basement membrane thicken- many histologic staging systems have been devel- ing, displayed with a periodic acid-Schiff stain with oped in the past. However, the new grading and stag- diastase, has been recently reported in PSC speci- ing system proposed by Nakanuma et al. for PBC has mens (Fig. 15). It demonstrated a specificity of 95% been tested also in PSC patients, and demonstrated and a good agreement among pathologists, making it the strongest prognostic value compared to previous an important diagnostic tool, particularly in sdPSC 59. methods, even in this scenario 38,62 (see above). Recent studies in end-stage PSC livers reported vas- cular lesions, particularly enlarged arterioles with Role of liver biopsy in PSC thickened walls and arterial fibrointimal hyperpla- Magnetic resonance cholangio-pancreatography or sia (Fig. 16), but the functional significance of these endoscopic retrograde cholangio-pancreatography changes in the pathogenesis of PSC is unknown 60,61. are the gold standard for PSC diagnosis, and liver bi- Persistent portal inflammation, bile duct destruc- opsy is not indicated when cholangiographic findings tion and periportal fibrosis lead to BDL, disorganized are typical. However, liver biopsy retains an important ductular reaction, periportal or diffuse hepatocyte diagnostic role whenever a high clinical suspicion for metaplasia, bridging fibrosis and cirrhosis, as in PBC. PSC is associated to a normal cholangiogram. In such Histologically, inflammation and fibrogenesis may not cases, a diagnosis of sdPSC relies on histologic find- be closely associated, and the risk of biliary dyspla- ings. Moreover, a liver biopsy is mandatory whenever sia and malignancy seems to be unrelated to disease a PSC-AIH variant or the presence of other comor- duration or biliary fibrosis severity 47,58. Histologic bidities are clinically suspected, as in PBC 48,50-52. changes are unevenly distributed in the liver, and this may explain the high degree of sampling variability in needle and wedge liver biopsies 6. Generally, the pat- “Overlap” variants tern of cirrhosis in PSC is more irregular than in PBC, reflecting the larger caliber of the involved ducts, and PBC-AIH variant segmental cirrhosis or hemi-cirrhosis are occasionally About 10-20% of PBC patients may present AIH, ei- observed 6. ther simultaneously or consecutively, and a small

Figure 15. Periodic acid-Schiff stain with diastase shows a Figure 16. Enlarged arterioles with thickened wall and arte- moderate bile duct basement membrane thickening (arrow) rial fibrointimal hyperplasia are common findings in primary in primary sclerosing cholangitis (PAS-diastase stain; origi- sclerosing cholangitis (hematoxylin-eosin; original magnifi- nal magnification 40x). cation 40x). PBC AND PSC 181

Table IV. Paris criteria for PBC-AIH variant diagnosis. At least 2 out of 3 criteria for each disease Primary biliary cholangitis Autoimmune hepatitis ALP > 2x ULN or γGT > 5x ULN ALT > 5x ULN AMA > 1:40 IgG serum levels > 2x ULN or ASMA positivity Florid duct lesions on histology Moderate or severe IH on histology ALP: alkaline phosphatase; ULN: upper limit of normal; γGT: γ-glutamyltranspeptidase; AMA: anti-mitochondrial antibodies; ALT: alanine aminotransferase; ASMA: anti-smooth muscle antibodies; IH: interface hepatitis. proportion of patients with well-established AIH may sis, is not typical and should warrant the hypothesis of develop PBC 3,9. This condition was formerly called a PBC-AIH variant 42. Still, a variable degree of inflam- “PBC-AIH overlap syndrome”, but the use of the term mation is commonly present in PBC alone, but inter- “overlap” has been discouraged, since not all the pa- face hepatitis and hepatocellular damage is typically tients concurrently present both diseases at the same minimal, while acidophil bodies are few or absent 43. time, and replaced with “variant”, which seems more Thus, in PBC patients with moderate/severe interface proper to describe all the possible clinical scenari- hepatitis, the hypothesis of a PBC-AIH variant should os 3,63. However, the term “overlap” is still very popular be taken into account, and treatment with immuno- in the routine practice. suppressive agents should be considered 9. The im- PBC-AIH variant diagnosis is challenging in every- munohistochemical assessment of IgG and IgM has day routine, and no updated consensus criteria for its been proposed to help in the differential diagnosis be- definition exist. Yet, in clinical practice, the Paris cri- tween AIH, PBC and PBC-AIH variant, but with con- teria are still in use (Table IV), since previous studies flicting results 69,70. demonstrated a good sensitivity (92%) and specific- A reduced 5 year event-free survival in PBC-AIH ity (97%) for diagnosing this variant 3,9,64,65. However, variant patients, compared to PBC patients (56% vs these criteria were published 20 years ago, and seem 81%) 9,71 has been reported. to be very strict and largely different from the separate The therapeutic approach to PBC-AIH variant con- diagnostic criteria of each disease, with a consequent sists in treating both diseases with a combination of underestimation of affected patients 3,9. For this rea- UDCA and immunosuppression, since combination son, changes in the criteria have been proposed, but therapy proved more effective than UDCA alone in still not universally approved 3,66,67. slowing fibrosis progression 3,72,73. Liver biopsy is a fundamental tool in the diagnosis of the PBC-AIH variant, and all national and internation- PSC-AIH variant al guidelines recommend it 3,9,14,68. Of note, specimens As for PBC-AIH variant, the previously used term should be evaluated by an expert liver pathologist, “overlap” has been abandoned, since PSC-AIH vari- since there are important therapeutic implications 3. ant reflects a PSC subphenotype instead of a sepa- In fact, PBC-AIH variant should not be overdiagnosed rate clinical entity 3,63,74. The PSC-AIH variant is pres- to avoid side effects from immunosuppression in PBC ent in 6-14% of PSC patients, and seem to have less patients, though neither underestimated, to correctly concurrent ulcerative colitis 3,75. identify PBC patients who would benefit from immu- As previously stated, no defined diagnostic criteria ex- nosuppressive treatment. The typical histologic pic- ist 3. In clinical practice, PSC-AIH variant diagnosis is ture consists of PBC bile duct lesions in a classical based on typical cholangiographic or histologic char- AIH background. However, histologic biliary damage acteristics of PSC in combination with AIH features 3. in AIH has been reported in up to 24-60% of well-de- In the absence of an abnormal cholangiogram, a typi- fined AIH patients, with destructive cholangitis being cal histological AIH picture with periductal inflamma- present in 3-7% 40. Moreover, a recent study reported tion and fibro-obliterative, onion skin-type, fibrosis is that bile duct injury and ductular reaction is a frequent suggestive of a concomitant sdPSC, while the addi- phenomenon in AIH and it does not always point to- tional loss of interlobar ducts makes the whole sce- ward a PBC-AIH variant 41. Thus, some degree of bili- nario diagnostic of a sdPSC-AIH variant, which repre- ary involvement in AIH does not necessarily lead to a sents about 16% of the cases 3,76. change in the diagnosis. Nevertheless, finding histo- A different specific variant, called autoimmune scle- logic features of chronic cholestasis, such as copper rosing cholangitis, has been described in at least 50% deposition, CK7-positive periportal hepatocytes, and of children and adolescents with well-established AIH ductopenia in AIH specimens without bridging fibro- and sclerosing cholangitis manifestations 3,77,78. 182 S. Sarcognato et al.

Patients with PSC-AIH variant have a similar risk of 6 Zen Y, Hübscher SG, Nakanuma Y. Bile duct diseases. In: Burt liver disease progression compared to those with AD, Ferrell LD, Hübscher SG, eds. MacSween’s pathology of the liver. 7th ed. Philadelphia, PA: Elsevier 2018, p. 515. classic PSC, but they seem to have a lower risk of 7 3,75 Zhang W, Sharma R, Ju ST, et al. Deficiency in regulatory T cells hepato-biliary malignancy development . results in development of antimitochondrial antibodies and au- In PSC-AIH variant, treatment is based on a combina- toimmune cholangitis. Hepatology 2009;49:545-552. https://doi. tion of immunosuppression and UDCA, just like in the org/10.1002/hep.22651 PBC-AIH variant 3. 8 Sasaki M, Ikeda H, Sawada S, et al. Naturally-occurring regula- tory T cells are increased in inflamed portal tracts with cholan- giopathy in primary biliary cirrhosis. J Clin Pathol 2007;60:1102- PBC-PSC variant 1107. https://doi.org/10.1136/jcp.2006.044776 This variant is exceptionally uncommon, and less 9 European Association for the Study of the Liver. EASL Clinical than 10 cases have been described in the literature, practice guidelines: the diagnosis and management of patients so far 3,79,80. It consists in a syndrome with clinical and with primary biliary cholangitis. J Hepatol 2017;67:145-172. laboratory features of both PBC and PSC, including https://doi.org/10.1016/j.jhep.2017.03.022 10 the sdPSC variant. Because of its rarity, very few data Carey EJ, Ali AH, Lindor KD. Primary biliary cirrho- sis. Lancet 2015;386:1565-1575. https://doi.org/10.1016/ 3,80,81 are available, and its existence is still debated . S0140-6736(15)00154-3 11 Chalifoux SL, Konyn PG, Saab S, et al. Extrahepatic manifesta- tions of primary biliary cholangitis. Gut Liver 2017:11;771-780. Conclusions https://doi.org/10.5009/gnl16365 12 Floreani A, Cazzagon N. PBC and related extrahepatic diseases. The role of histology in diagnosing PBC and PSC has Best Pract Res Clin Gastroenterol 2018;34-35:49-54. https://doi. changed profoundly in the last 20 years, paralleling a org/10.1016/j.bpg.2018.05.013 13 better standardization of clinical, laboratory and im- Harms MH, van Buuren HR, van der Meer AJ, et al. Ursodeoxy- cholic acid therapy and liver transplant-free survival in patients aging diagnostic criteria. Liver biopsy is not current- with primary biliary cholangitis. J Hepatol 2019;71:357-365. ly part of the routine workup in diagnosing PBC and https://doi.org/10.1016/j.jhep.2019.04.001 PSC, but it retains a crucial role when patients have 14 Hirschfield GM, Dyson JK, Jones DEJ, et al. The British Society an “atypical” presentation or when non-invasive tests of Gastroenterology/UK-PBC primary biliary cholangitis treat- ment and management guidelines. Gut 2018;67:1568-1594. fail to reach a definite diagnosis. 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Review

Pathology of autoimmune hepatitis

Claudia Covelli1, Diana Sacchi2, Samantha Sarcognato2, Nora Cazzagon3, Federica Grillo4, Francesca Baciorri2, Daniela Fanni5, Matilde Cacciatore2, Valeria Maffeis2, Maria Guido2,6 1 Pathology Unit, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy; 2 Pathology Department Azienda ULSS 2 Marca Trevigiana, Treviso, Italy; 3 Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy; 4 Unit of Anatomic Pathology, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genova and Ospedale Policlinico San Martino IRCCS, Genoa, Italy; 5 Pathology Unit, Department of Medical Sciences, University of Cagliari, Cagliari, Italy; 6 Department of Medicine-DIMED, University of Padova, Padova, Italy

Summary Autoimmune hepatitis (AIH) is a relatively rare non-resolving chronic liver disease, which mainly affects women. It is characterized by hypergammaglobulinemia, circulating autoan- tibodies, interface hepatitis on liver histology and a favourable response to immunosup- pression. The putative mechanism for the development of autoimmune hepatitis is thought to be the interaction between genetic predisposition, environmental triggers and failure of the native immune system. AIH still remains a major diagnostic and therapeutic challenge, mainly because it is a very heterogeneous disease. Prompt and timely diagnosis is crucial since, if left untreated, AIH has a high mortality rate. Histological demonstration of hepatitis is required for the diagnosis of AIH and, therefore, liver biopsy is mandatory in the initial diagnostic work-up, before treatment. In this review, we summarize the histological features of AIH with the main aim of highlighting the most important clinical-pathological hallmarks useful in the routine diagnostic practice.

Received and accepted: December 14, 2020 Key words: autoimmune hepatitis, histology, liver biopsy, interface hepatitis

Correspondence Claudia Covelli Introduction Fondazione IRCCS Casa Sollievo della Soffe- renza, 71013, San Giovanni Rotondo, Italy Tel.:+39 0882 835505 Autoimmune hepatitis (AIH) is a chronic progressive liver disease of un- Fax: +39 0882 410411 known etiology. The pathogenesis of AIH is complex and involves inter- E-mail: [email protected] actions between tolerant liver, environmental triggers, and dysregulated Conflict of interest immunological mechanisms. Genetic factors influence an individual’s The Authors declare no conflict of interest. susceptibility to developing AIH 1,2. Originally considered a disease of young women, AIH may also occur How to cite this article: Covelli C, Sacchi D, in children and the elderly (about 30% of cases occur over 60 years of Sarcognato S, et al. Pathology of autoimmune age). Both sexes (about 30% are males) and all ethnic groups are in- hepatitis. Pathologica 2021;113:185-193. https://doi.org/10.32074/1591-951X-241 volved, and recent epidemiological studies indicate an increasing trend in AIH prevalence worldwide, especially in men 3. If untreated, AIH leads © Copyright by Società Italiana di Anatomia Pato- to cirrhosis and associated complications, with a small (1-2% per year) logica e Citopatologia Diagnostica, Divisione Itali- 4,5 ana della International Academy of Pathology but significant risk of hepatocellular carcinoma development . Charac- teristic laboratory features include hypergammaglobulinemia, with ele- OPEN ACCESS vation of immunoglobulin G (IgG) in the majority of cases, and presence of non-organ specific autoantibodies 6. This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- Diagnosis is often challenging, since no single feature is reliable. There- NonCommercial-NoDerivatives 4.0 International) license: the fore, careful clinical-pathological integration is required for the exclusion work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original of other causes of liver disease and a confident diagnosis of AIH. An version. For further information: https://creativecommons. early diagnosis is, however, critical for timely initiation of life-saving im- org/licenses/by-nc-nd/4.0/deed.en munosuppressive therapy. 186 C. Covelli et al.

Current guidelines recommend liver biopsy as a pre- also in patients with Non-alcoholic fatty liver disease requisite for the diagnosis of AIH 1. Liver biopsy is also (NAFLD): in a multicenter study in United States, sig- useful for treatment management, to determine dis- nificant titers of autoantibodies were detectable in ease severity and distinguish acute vs. chronic pres- 21% of adult NAFLD patients, with no association with entation. advanced histological features or presence of steato- This review focuses on the current role of liver biop- hepatitis 21. sy and describes characteristic histological lesions The spectrum of clinical presentation of AIH ranges which contribute to AIH diagnosis. from asymptomatic elevation of serum liver enzymes, to fulminant hepatitis. The most common clinical pres- entation is characterized by a slow disease onset and Overview on laboratory and clinical progression with non-specific complaints such as fa- features tigue and malaise, which explains why AIH is in ad- vanced stages of fibrosis at the time of diagnosis in Liver function tests typically show a hepatocellular around a third of patients. Acute onset of AIH occurs in pattern of injury, with an increase in aminotransferas- about one third of patients. It may represent an acute es, that can be mildly elevated or up to 50 times the exacerbation of unrecognized AIH with pathological upper normal value. Alanine aminotransferase (ALT) evidence of chronic hepatitis or a genuine newly de- is typically higher than Aspartate aminotransferase veloped acute AIH, without previous clinical-patholog- (AST). Cholestatic enzymes are usually normal or ical findings of chronic liver disease. Acute onset is mildly elevated unless there is an overlap with primary clinically indistinguishable from acute hepatitis of oth- biliary cholangitis (PBC) or primary sclerosing chol- er causes 22-24. Moreover, in some of these patients, angitis (PSC) 7- 13 . Increase in serum globulins (serum IgG levels may be within the normal range and ANA γ-globulin or IgG level) is evident in 90% of patients. and/or SMA may be negative at the first screening 10,25- This prevalence seems to be lower in patients with 28, thus leading to a challenging diagnosis. an acute onset, among which a proportion of cases, ranging from 25 to 39%, has normal IgG levels 14,15. IgA and IgM are usually normal, and their increased The role of liver biopsy in AIH levels should prompt the exclusion of different etiolo- gies, such as alcoholic steatohepatitis and PBC, re- Liver biopsy is recommended by the American Asso- spectively1. ciation for the Study of Liver diseases (AASLD) and Circulating autoantibodies are considered the hall- the European Association for the Study of the Liver mark of AIH and have been used to subclassify AIH (EASL) guidelines to help establish the diagnosis, ex- into type 1, or classic autoimmune hepatitis, and type clude other causes of liver disease, and guide treat- 2. Type 1 AIH is defined by the presence of antinuclear ment choice 1,26. antibody (ANA) and/or anti-smooth muscle antibody The diagnostic criteria for AIH have been codified in (ASMA). Antibody titers more than 1:40 are consid- 1993, revised in 1999 by the International Autoimmune ered significant in adults. Type 2 AIH is less common Hepatitis Group (IAHG) 7, 8 and more recently simplified and mainly affects pediatric patients. It is character- for clinical use (Table I) 9. In the simplified system, as ized by the presence of antibodies against liver/kid- in the previous ones, liver histology is included among ney microsomes (LKM1) and/or liver cytosol antigen the parameters required to confirm clinical diagnosis (LC1). The validity of such subclassification in clinical of AIH. Indeed, the system comprises four parame- practice has been questioned 1. Autoantibodies may ters: autoantibodies, serum IgG, results of viral hep- not be detected, particularly in cases of severe acute atitis work-up and AIH histology, which is coded as presentation (autoantibody negative AIH)16. Notewor- absent, typical or compatible (Table II). thy, is that the presence of autoantibodies always Once a diagnosis of AIH is made, liver biopsy is the needs careful interpretation, as they can be found in gold standard for grading and staging AIH and pro- healthy patients as well as in other, non-autoimmune, vides crucial information for patient management and hepatopathies. In asymptomatic blood donors, ANA treatment decisions29. Persistence of any degree of in- prevalence of any titer has been found to vary be- flammation, particularly interface hepatitis, and pres- tween 4% and 26%, with nearly 15% of cases positive ence of plasma cells in biopsy samples taken under at a 1:40 dilution, and up to 10% of pregnant women treatment, are strong predictors of AIH- relapse if im- are ANA positive 17-19. ANA are found in approximately munosuppression is stopped. 5% of individuals with HCV, showing titers more than Liver biopsy is particularly important for the differen- 1:100 20. The presence of autoantibodies is common tial diagnosis of AIH, as it may lead to an alternative AUTOIMMUNE HEPATITIS 187

Table I. Simplified diagnostic criteria for autoimmune hepatitis (from Hennes et al., 2009, adapt.) 9. Feature Cutoff Points ANA or SMA ≥ 1:40 1 ANA or SMA ≥ 1:80 2* or LKM ≥ 1:40 or SLA Positive IgG > Upper normal limit 1 > 1.10 times upper normal limit 2 Liver histology (evidence of hepatitis is a necessary condition) Compatible with AIH 1 Typical AIH 2 Absence of viral hepatitis Ye s 2 Interpretation of aggregated scores: ≥ 6: probable AIH; ≥ 7: definite AIH *Addition of points achieved for all autoantibodies (maximum, 2 points). ANA, Antinuclear antibody; SMA, smooth muscle antibody; LKM, liver-kidney microsomal antibody; SLA/LP, soluble liver antigen/liver pancreas antibody, AIH, autoimmune hepatitis.

Table II. Histological categories for grading histology in the simplified system for autoimmune hepatitis (from Hennes et al., 2009, adapt.) 9. Histological Categories Morphological features Typical for AIH Interface hepatitis, with lymphocytic/lymphoplasmocytic infiltrate in portal tracts extending into the lobule Emperipolesis Hepatic rosette formation (All three features have to be present) Compatible with AIH Chronic hepatitis pattern of injury with lymphocytic infiltration Lack of all the features considered typical Atypical for AIH Signs suggestive of other diagnosis AIH, Autoimmune hepatitis. etiology 30. Moreover, it may identify possible concur- rent disorders, especially fatty liver disease, consid- ering the epidemic proportion of risk factors for the metabolic syndrome. Finally, liver biopsy is consid- ered mandatory in cases of AIH with overlapping fea- tures of autoimmune biliary disorders. Liver biopsy at presentation should be performed prior to beginning treatment since immunosuppression may rapidly clear inflammation on liver biopsy, with the risk of a “false negative” diagnosis.

Histopathology of AIH

Despite the pivotal role of liver biopsy for diagnosis, no morphological feature is pathognomonic of AIH. How- Figure 1. Severe portal inflammation, mainly composed of ever, there is a characteristic picture in many patients lymphocytes, and interface hepatitis. Several necro-inflam- before treatment. The typical aspect of AIH is that of a matory foci are visible in the adjacent lobular parenchyma severe chronic hepatitis with intense portal and lobu- (H&E; original magnification 40x). lar inflammation, severe interface hepatitis, and much hepatocyte damage. Portal inflammation is composed of mononuclear cells, mainly lymphocytes, with a variable amount of make a diagnosis since they are rare/absent in about plasma cells. Some eosinophils and neutrophils may one third of cases. However, detection of plasma cells be seen (Fig. 1). Plasma cells are considered typical in clusters (Fig. 2) is highly suggestive of AIH. In a for AIH, but they are neither sufficient nor necessary to recent critical appraisal concerning the histological 188 C. Covelli et al.

Figure 2. A cluster of plasma cells is visible close to the Figure 4. Ductular reaction (arrows) around an inflamed por- portal tract (H&E; original magnification 40x). tal tract (immunostain for CK7; original magnification 20x).

31 features of AIH , the presence of plasma cell clus- ally more prominent compared to interface hepatitis ters (defined as a collection of ≥ 5 plasma cells) in of other causes 33. According to the 2008 simplified the lobule was the most sensitive diagnostic finding. criteria, the presence of interface hepatitis, even in the Immunohistochemical stains for multiple myeloma-1 absence of all the other typical features, is in agree- (MUM-1) or CD38 may help to highlight number and ment with the diagnosis of AIH 9. distribution pattern of plasma cells (Fig. 3) 32. Severe portal and interface hepatitis are usually asso- Although not specific, interface hepatitis is considered ciated with ductular reaction (DR). It consists of bile the hallmark of AIH. It is characterized by the exten- ductules with poorly defined lumina at the portal-pa- sion of portal inflammation beyond the limiting plate renchymal interface, arranged in anastomosing cords, into the adjacent lobule with damage and progres- and lined by small CK7-positive cells (Fig. 4). Ductular sive loss of hepatocytes at the portal-lobular interface reaction is a regenerative phenomenon, which repre- (Fig. 1). It is observed in up to 98% of AIH and is usu- sents proliferation and bidirectional differentiation of facultative hepatic stem cells in a variety of acute and chronic liver diseases. In AIH, DR correlates with the severity of portal-periportal inflammation, as in other liver diseases 34. Lobular changes are dominated by necro-inflamma- tory damage, ranging from spotty to confluent ne- crosis. Apoptotic bodies are commonly seen. Multi- ple necro-inflammatory foci may be associated with hepatocyte ballooning and sinusoidal inflammation, giving the appearance of lobular disarray (Fig. 5) which resembles what is seen in patients with acute viral hepatitis. Bridging necrosis (portal to portal and portal to central) is not uncommon and may repre- sent the deep extension of interface hepatitis into the lobules. Confluent necrosis and inflammation may be seen in perivenular areas. In most cases, it is associ- ated with the typical portal peri-portal inflammation. Figure 3. Immunostain with CD38 helps in identifying a However, there are few patients affected by AIH in cluster of plasma cells. This is a pediatric case with mild whom the major feature at presentation is the isolated portal inflammation within an otherwise typical clinical pre- centrilobular necro-inflammation, with spared portal sentation (original magnification 20x). tract (Fig. 6). This is thought to be an early feature of AIH that precedes overt portal-dominant (classic) AUTOIMMUNE HEPATITIS 189

Figure 5. Several foci of “spotty” necrosis, giving the ap- Figure 7. Typical hepatocyte rosette, representing a regen- pearance of lobular disarray (H&E; original magnification erative phenomenon in a heavily inflamed liver (H&E; origi- 20x). nal magnification 60x).

Figure 6. Centrilobular necro-inflammation in a case of true Figure 8. Emperipolesis (arrow) appears as a lymphocyte acute AIH presentation. Portal tracts were completely spared within the hepatocyte cytoplasm (H&E; original magnifica- in this case. Toxic damage is the main differential diagnosis tion 60x). (H&E; original magnification 40x).

represent a regenerative response to the necro-in- AIH 35. Indeed, this pattern of necrosis is commonly flammatory damage. Emperipolesis is characterized seen in patients with acute disease onset 36. In rare by the presence of a mononucleated inflammatory cell cases, a massive-panlobular necrosis may occur, (lymphocyte or plasma cell) within the cytoplasm of leading to liver failure. hepatocytes and it is reported in 65-78% of AIH cas- Much emphasis has been given to the presence of es. Emperipolesis and rosette formation have been rosettes (Fig. 7) and emperipolesis (Fig. 8) as classic considered better histological predictors of AIH, when changes of AIH. Indeed, they have been considered compared to plasma cells and interface hepatitis 37. as typical histological AIH-features in the 2008 simpli- However, such superiority is still controversial and fied scoring system for adults and they are a required it has been shown that emperipolesis is associated item to get score 2 (Tab. II)9. Rosettes are defined as with the severity of activity rather than the etiology 38. hepatocytes arranged around a central lumen and Moreover, emperipolesis is difficult to be reliably as- 190 C. Covelli et al.

sessed in routine practice with light microscopy, and it is best evaluated by electron microscopy. Bile duct injury can be identified in up to 83% of AIH patients 34,39,40, often in a PBC-like pattern, even af- ter the exclusion of an overlap syndrome. Therefore, some degree of biliary involvement in AIH does not necessarily lead to a change in diagnosis! A major problem is that how much bile duct damage is diag- nostic of biliary disease is still not established. In rou- tine practice, it is reasonable to suggest a possible concomitant biliary disease when bile duct damage is seen in the majority of portal tracts. The bile ducts are not the target of damage in AIH, and probably their in- flammatory damage represents collateral injury of the conspicuous inflammatory process, as demonstrated 34 by their subsiding after immunosuppressive therapy . Figure 9. Giant cell transformation in autoimmune hepatitis Therefore, an evident bile duct destruction (ductope- (H&E; original magnification 40x). nia) is not a feature of AIH and warrants consideration of primary biliary cholangitis. Moreover, copper-asso- ciated protein accumulation and/or CK7-positive peri- portal hepatocytes are recognized features of chronic sociated protein/CK7 stains, and demonstrated high- cholestasis and their presence should prompt to con- er sensitivity for the diagnosis of AIH. These results sider an alternative diagnosis or an overlap condi- should be validated in prospective series. tion. It is noteworthy to highlight that these features Cholestasis is usually not observed in AIH, but a mild of chronic cholestasis no longer have their diagnostic degree may be seen in cases with marked lobular in- value when severe fibrosis or cirrhosis is present. In flammation. such cases, nonspecific copper-associated protein Giant cell transformation may be rarely seen in adults accumulation and CK7 positivity can be seen, not- with AIH (Fig. 9). This represents an unusual regener- withstanding the etiology. The practical value of taking ative or degenerative hepatocyte reaction to various in consideration results of copper-associated protein injuries, and it is a common response in the newborn and CK7 stains in the histological features has been liver diseases. Post infantile giant cell hepatitis is a evaluated in a recent study 41. In this study, histological rare, non-specific subtype of hepatitis, which can be criteria codified in the 2008 simplified score proposed seen in a wide variety of inflammatory and cholestatic by the IAHG (Tab. II) were reviewed and new param- liver diseases, among which AIH represents the most eters were proposed. The new criteria (Tab. III) were common cause 42. based on interface/lobular inflammatory activity, num- Recently, PAS positive-diastase resistant hyaline ber of plasma cells, biliary features, and copper-as- droplets in Kupffer cells, resembling Russell bodies of

Table III. Proposed criteria for the histologic scoring of autoimmune hepatitis (from Balitzer et al., 2017, adapt.) 41. Histologic score Morphological features Score 0 Features not observed in AIH (e.g. florid duct lesion, bile duct loss, or copper/CK7 positivity, if applicable*) Score 1** 1. Hepatitis with mild or moderate necro-inflammatory activity, with any of the following: (a) Ishak A2 (mild/moderate interface activity) (b) Ishak B1 (focal confluent necrosis) (c) Ishak C2 (2-4 foci of lobular activity x 10) 2. CK7 and copper stains negative (if applicable*) Score 2 Hepatitic pattern with any of the following: 1. Plasma cells numerous or in clusters 2. High necroinflammatory activity with at least one of the following: (a) Ishak score A3 or higher (at least moderate interface activity) (b) Ishak B2 or higher (confluent necrosis in zone 3 or beyond) (c) Ishak C3 or higher (5 or more foci of lobular activity x 10) *Applicable only in cases without any bridging fibrosis. **Both 1 and 2 are necessary for histologic score 1, except in cases with acute presentation. AIH, Autoimmune hepatitis. AUTOIMMUNE HEPATITIS 191

plasma cells, have been described as specific feature tibodies associated to AIH are frequently found dur- in AIH 43. However, little information is known on this ing acute HEV infection. Clinicians and pathologists and, therefore, it is still controversial to consider this should be aware of this and always exclude HEV in- feature as a reliable histological sign of AIH. fection before diagnosing and treating AIH, as immu- nosuppression can lead to chronic HEV disease 47. A broad and highly variable spectrum, related to the clin- Histological grading and staging ical context, of histopathological findings can be ob- served in livers of patients infected with HEV, making Since no specific grading and staging systems for AIH the histopathological diagnosis very challenging 48. have been developed, grading of the inflammatory Immunohistochemistry for HEV pORF2 protein is a activity and staging of the fibrosis can be performed helpful histopathological tool 49. borrowing the systems used for chronic viral hepatitis such as the Scheuer 44, Ishak 45, and Metavir 46 sys- Drug induced liver injury tems. This is reasonable since both chronic viral hepa- Drug induced liver injury (DILI) represents the most titis and AIH have a similar pattern of necro-inflamma- challenging differential diagnosis, not only because it tory damage and share the morphogenesis of fibrosis. can mimic the clinical, biochemical, serological and The choice of grading/staging system is arbitrary and morphological phenotype of AIH (AIH-like-DILI), but depends on the pathologist’s preference and tradition, also because drugs may trigger latent or induce a de and on agreement with the local hepatologists. As novo AIH 50-53. The distinction between DILI and AIH in viral hepatitis, it is important to clearly specify the by histology can be extremely difficult (sometimes name of the used system in the final histological re- impossible), due to the absence of histological fea- port, without which the scores lose their significance. tures pathognomonic of either DILI or AIH. Severe Patients often consult different clinicians; therefore, portal plasma cell-rich inflammation, prominent in- this information is crucial for a proper interpretation of tralobular plasmacells and eosinophils, rosette forma- the histological report. tion, absence of cholestasis and presence of fibrosis Grading and staging have prognostic and therapeu- have been suggested as features that are in favor of tic implications and are required, since ALT values do the diagnosis of AIH 54. The absence of cirrhosis, or not properly correlate with the disease severity and advanced fibrosis, at presentation mainly suggests non-invasive tests for the assessment of fibrosis have AIH-like DILI. A detailed clinical information is crucial, not been fully validated in AIH 1. and the patient’s history should focus on recent expo- According to EASL guidelines, treatment withdrawl is sure to drugs that can induce AIH-DILI. Fortunately, considered when biochemical remission is reached, AIH-DILI usually responds to high doses of steroids as but it requires histological remission, defined as nor- severe AIH usually does, but differently from true AIH mal histology or minimal hepatitis (Hepatitis Activity that always relapses, steroid treatment can be discon- Index score/HAI < 4 or equivalent) 1. tinued without a DILI relapse 1.

Primary biliary cholangitis Differential diagnosis Differential diagnosis between AIH and classical PBC is not difficult. PBC is a chronic cholestatic syndrome Since the pattern of injury and typical features of AIH showing a distinctive serological profile of positive an- are non-specific, the spectrum of histological differen- timitochondrial antibodies (AMAs), often associated tial diagnosis is very broad and an accurate integra- with intense pruritus. Increase in serum IgM and alka- tion with all clinical and laboratory features is required line phosphatase (ALP) levels with a normal or slightly to reach a correct diagnosis. elevated bilirubin level and only a mild elevation of ALT is typically observed at earlier stages. PBC may show Viral hepatitis moderate to severe portal inflammatory infiltrate with All acute and chronic viral hepatitis can appear similar many plasma cells and even interface hepatitis, but to AIH and neither plasma cells nor the other histolog- prominent florid duct lesions and bile duct loss (not ic findings, which are more typically seen in AIH, are seen in AIH) are typically found in liver biopsies. More- sensitive or specific enough. However, viral infection over, PBC does not show a hepatitic pattern of lobular can be easily excluded by serological testing which injury, which is common in AIH. AMA-negative PBC are mandatory in all patients in whom a diagnosis of may occur in about 10% of cases, but the histological AIH is considered. A special attention should be paid scenario does not differ from the typical AMA-positive to hepatitis E virus (HEV), since non-specific autoan- PBC. As already highlighted, stains for copper-asso- 192 C. Covelli et al.

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Review

Pathology of non-alcoholic fatty liver disease

Ivana Cataldo1, Samantha Sarcognato1, Diana Sacchi1, Matilde Cacciatore1, Francesca Baciorri1, Alessandra Mangia2, Nora Cazzagon3, Maria Guido1,4 1 Department of Pathology, Azienda ULSS 2 Marca Trevigiana, Treviso, Italy; 2 Liver Unit, Department of Medical Sciences, Fondazione IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo, Italy; 3 Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy; 4 Department of Medicine-DIMED, University of Padova, Padova, Italy

Summary Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of different conditions which are characterized by hepatic steatosis in the absence of secondary causes. It is currently the most common chronic liver disease worldwide, and its estimated prevalence is about 1.5-6.5%. The only histological finding of steatosis (“simple” steatosis) represents the uncomplicated form of NAFLD, while non-alcoholic steatohepatitis (NASH) is its inflam- matory subtype associated with disease progression to cirrhosis and hepatocellular car- cinoma (HCC), and represents the major indication for liver transplantation. NASH is still a diagnostic and therapeutic challenge for clinicians and liver biopsy is currently the only accepted method to reliably distinguish NASH from “simple” steatosis. From the histological perspectives, NAFLD and NASH continue to be an area of active interest for pathologists, with a specific focus on better methods of evaluation, morphologic clues to pathogenesis, and predictors of fibrosis progression. This review focuses on histopathology of NAFLD in Received and accepted: December 14, 2020 adults, with the aim to provide a practical diagnostic approach useful in the clinical routine.

Correspondence Key words: fatty liver, steatohepatitis, non-alcoholic, metabolic syndrome Ivana Cataldo Pathology Department Azienda ULSS 2 Marca Trevigiana, Treviso, piazzale Ospedale 1, Treviso. Italy Introduction Tel.: +39 0422 32 8104 Fax. +390422322705 “Fatty liver” is a very common condition defined by the presence of in- E-mail: [email protected] tra-hepatocytes lipid droplets, commonly referred to as steatosis. It en- Conflict of interest compasses a wide spectrum of hepatic alterations with a wide range of 1 The Authors declare no conflict of interest. etiological and clinical-pathological features . Non-alcoholic fatty liver disease (NAFLD) is an umbrella term used to How to cite this article: Cataldo I, Sarcognato comprise a wide spectrum of conditions, which are characterized by S, Sacchi D, et al. Pathology of non-alcoholic hepatic steatosis in absence of secondary causes, including significant fatty liver disease. Pathologica 2021;113:194- 202. https://doi.org/10.32074/1591-951X-242 alcohol consumption, chronic use of medications and hereditary disor- ders. An international expert membership on liver pathology has recently © Copyright by Società Italiana di Anatomia Pato- suggested an update of nomenclature, and the definition of “Metabolic logica e Citopatologia Diagnostica, Divisione Itali- Dysfunction Associated Fatty Liver Disease” (MAFLD) has been pro- ana della International Academy of Pathology posed as a more appropriate term to describe liver diseases associated 2 OPEN ACCESS with known metabolic dysfunctions . Waiting for a worldwide accept- ance of this new term and a refinement of diagnostic criteria to define This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- MAFLD, the term NAFLD is used in this review. NonCommercial-NoDerivatives 4.0 International) license: the NAFLD is the most common chronic liver disease worldwide. Its esti- work can be used by mentioning the author and the license, 3 but only for non-commercial purposes and only in the original mated prevalence is around 1.5-6.5% on global population , although version. For further information: https://creativecommons. its absolute prevalence worldwide is unknown and is tightly connected org/licenses/by-nc-nd/4.0/deed.en to genetic and environmental factors 4. Its prevalence appears to be in- PATHOLOGY OF NAFLD 195

creasing, with an estimated 3.6 million of new cases The diagnosis of NAFLD requires evidence of hepatic annually. The estimated annual medical costs direct- steatosis in absence of other causes of liver fat accu- ly attributable to NAFLD exceed 35 billion euros in 4 mulation. From the clinical point of view, no specific large European countries (United Kingdom, France, physical signs can confirm the diagnosis of NAFLD. In Germany, and Italy). the practice, it is suspected when an increase in se- Simple steatosis represents the uncomplicated form rum aminotransferase levels are found in a patient with of NAFLD, while non-alcoholic steatohepatitis (NASH) features of MeS. Indeed, elevation of transaminase is is its inflammatory subtype which is associated with the most common laboratory modification in NAFLD disease progression, development of cirrhosis and, patients, usually mild (less than twice the upper lim- eventually, hepatocellular carcinoma (HCC), and a it of normal) irrespectively of the severity of disease. possible need for liver transplantation should be con- Of note, almost 80% of patients with NAFLD do not sidered. Indeed, due to the decline in hepatitis C virus show any biochemical abnormality and the diagno- patients, NASH-correlated cirrhosis is already the ma- sis can be established incidentally, even in advanced jor indication for liver transplantation. stage of disease. A number of other non-specific lab- NASH can be a diagnostic and therapeutic challenge oratory findings can be detected (such as increased for clinicians 5. Although several non-invasive tests γ-glutamyl transferase and alkaline phosphatase). have been developed, liver biopsy is currently the Elevated serum ferritin levels with normal transferrin only accepted method to reliably distinguish NASH saturation can be detected in an acute phase and it from “simple” steatosis. Moreover, from a histopatho- appears to be related with the progression towards a logical perspective, NAFLD and NASH continues to fibrotic stage 3,14. A high prevalence of low-title antinu- be an area of increasing interest for liver pathologists, clear antibody (ANA; ≤ 1:160) and anti-smooth muscle with specific focus on better methods of assessment, antibody (ASMA; ≤ 1:40) has been reported in NAFLD morphologic features for pathogenesis, and predictive patients, usually with a normal range of IgG 15. markers of fibrosis progression 6. This review focuses on histopathology of NAFLD in adults, with the aim to provide a practical diagnostic Pathology of NAFLD approach useful in the clinical routine. From a histological point of view, NAFLD patients may show “simple” steatosis (NAFL) or NASH. Although Overview on clinical aspects of NAFLD the natural history and prognostic features of NAFLD remain controversial, NAFL is largely considered a NAFLD is considered the hepatic manifestation of benign condition with minimal risk of progression or metabolic syndrome (MeS), characterized by obesi- clinical impact, while NASH represents the progres- ty, insulin resistance or type 2 diabetes, dyslipidem- sive and prognostically relevant form of this disease. ia and hypertension 7. MeS is the third death cause Therefore, the differential diagnosis between NAFL due to cardiovascular disease and extra-hepatic neo- and NASH play an important consideration in routine plasms 8,9. The criteria for MeS diagnosis are reported practice, and the efficacy of new drugs to induce reso- in Table I 10,11. NAFLD may develop in lean individuals 12. lution of NASH is considered a key endpoint in clinical Lean-NAFLD is defined as hepatic steatosis in patients trials. Distinguishing NASH from NAFL represents the with a BMI < 25 kg/m2 (or < 23 kg/m2 in Asian individu- major indication to perform a liver biopsy. Once a diag- als) in absence of “significant” alcohol intake 13. Initially nosis of NASH has been established, the information described in Asian populations, it is reported that, even about severity of disease, particularly about fibrosis among European individuals, approximately 20% of status, provides prognostic clues 16. patients are considered as lean NASH. Due to the epidemic spread of obesity, diabetes and

Table I. Criteria for Metabolic Syndrome (MeS) diagnosis defined by The National Cholesterol Educational Program Adult Treatment Panel III (NCEP-ATP) (from Grundy et al., 2005, mod.) 10. At least 3 of the 5 following criteria should be present: 1. Triglyceride level ≥ 150mg/dL or pharmacological treatment 2. High density lipoprotein (HDL) < 50 mg/dL for women and < 40 mg/dL for men, or pharmacological treatment 3. Blood pressure ≥ 130/85 mmHg or pharmacological treatment 4. Fasting blood glucose level > 100mg/dL or pharmacological treatment 5. Waist circumference ≥ 35 inches for women and ≥ 40 inches for men 196 I. Cataldo et al.

associated fatty liver disease, performing liver biopsy in all these patients is unaffordable. Current guidelines recommend biopsy for patients with NAFLD who are at increased risk of steatohepatitis and/or advanced fibrosis stage and for patients in whom any coexisting liver diseases cannot be ruled out. Much effort has been done to develop non-invasive tests to identify NASH 17,18 and algorithms have been proposed to help clinicians to decide when a liver biopsy should be per- formed17. A detailed description on non-invasive tests and clinical diagnostic algorithms is beyond the aims of this review.

“Simple” steatosis Figure 2. Microvescicular steatosis is characterized by tiny In NAFLD, steatosis is typically macrovescicular, and lipid droplets filling the hepatocyte cytoplasm (H&E; original is normally located in perivenular areas (acinar zone magnification 60x). 3) and easily recognized by a light microscope on He- matoxylin-Eosin (H&E) stain (Fig. 1). It appears as an empty and optically clear vacuole, because lipids are removed during tissue processing. Hepatocytes with a been demonstrated that ALT and AST show significant foamy appearance with numerous tiny vacuoles can changes when steatosis increases, thus, it supports be seen in microvescicular steatosis, but it is never the clinical guidelines of 5% cut-off for abnormal ste- a prominent feature (Fig. 2). Microvescicular steatosis atosis 20. Quantification of steatosis severity as mild tends to be present in more severe cases of steato- (5-33%), moderate (34-66%), and severe (> 66%) hepatitis, it is due to a mitochondrial injury and can should be specified in final pathological report. Grad- be life-threatening. As steatosis increases, its zonal ing of steatosis should be performed at low to medium distinction disappears and steatotic hepatocytes are magnification. equally distributed in all the acinar zones. Steatosis alone is unspecific and can be seen in Steatosis in more than 5% of hepatocytes is generally various condition. Alcohol abuse, drug, toxins and accepted as a working definition of a fatty liver. This is an arbitrary threshold based on the assumption that ischemic damage also share the preferential zone 3 21 minimal changes have no clinical relevance 19. It has location . Correlation with clinical information is es- sential to understand whether steatosis is related to NAFLD or to other causes.

NASH

Diagnosis of NASH requires i) steatosis (more than 5%), and ii) both lobular inflammation and balloon- ing degeneration of hepatocytes with a mainly zone 3 distribution. Other pathological features (such as portal inflammation, Mallory-Denk bodies, glycogen- ated nuclei, apoptosis, megamitochondria, iron dep- osition) may be seen, but they are neither necessary nor enough to establish a diagnosis of NASH. Hepatocyte Ballooning is defined as enlarged hepat- ocytes (more than 1.5-2 times the normal size, cor- responding approximately to a cellular diameter more Figure 1. Macrovescicular steatosis. (Hematoxylin and Eo- than 30 μm) with round (instead of polygonal) shape sin, H&E; original magnification 20x). and rarefied cytoplasm (Fig. 3). Bedossa et al., have recently considered two grade of ballooning: i) grade 1 PATHOLOGY OF NAFLD 197

Figure 3. Mallory-Denk bodies appear as cytoplasmic Figure 4. Hepatocyte glycogenosis. The hepatocyte cy- hyaline inclusions of ballooned hepatocytes (arrow). (H&E; toplasm appears homogeneously clear. It may mimic bal- original magnification 60x). looning degeneration but note that the cell contour remains polygonal. Several glycogenated nuclei are also evident (ar- row). (H&E; original magnification 20x).

(or small) ballooning characterized by a round hepato- cyte with typical pale reticulated cytoplasm with almost no variation in size compared to normal hepatocytes, hepatocyte dropout. Few plasma cells may be seen and ii) grade 2 ballooning characterized by more con- and small aggregates of neutrophils are rare, unless ventional ballooning 22. It has been questioned that this Mallory-Denk bodies are present. In case of a severe distinction can lead to an over-diagnosis of ballooning lobular inflammation, other or concomitant caus- and, subsequently, of NASH, due to a strong similar- es should be considered, mainly alcohol or drugs. ity of grade 1 ballooning with cytoplasmic glycogeno- Lipogranulomas (steatotic hepatocyte or fat droplet sis, which is a condition often observed in NAFLD. surrounded by lymphocytes, macrophages and rare A useful clue to distinguish grade 1 ballooning from eosinophils) are frequently detected in NASH (Fig. 5). cytoplasmic glycogenosis is the cell shape, which is They are not indicative of active inflammation and, rounded in case of ballooning while remains polygo- therefore, they are not considered for assessment of nal in case of glycogenosis (Fig. 4). True ballooning lobular inflammation 23. degeneration can be difficult to properly identify, and Subjects with NASH may develop fibrosis and about the inter-observer agreement is far from being perfect. 20% show a progression to cirrhosis. Typical NASH The consistency in ballooning detection is higher if the fibrosis is characterized by a sinusoidal collagen dep- enlargement of cells is considered a prerequisite for osition which usually begins in zone 3, giving rise to a diagnosis. Keratin stains can help in ballooning detec- delicate framework around single hepatocytes (Fig. 6). tion; indeed, it has been demonstrated that ballooned Periportal fibrosis develops subsequently in most hepatocytes lose CK8-18. Moreover, they are usually cases, followed by bridging (mainly central-to-portal) surrounded by collagen fibers that can be easily high- lighted by collagen stains 23. The feathery degenera- fibrosis. NAFLD cirrhosis may be indistinguishable tion associated with injury due to cholestasis (“cho- from cirrhosis due to other etiologies. Of note, stea- late stasis”) may mimic hepatocyte ballooning. The tosis may completely disappear at cirrhotic stage. In location (periportal in cholate stasis and pericentral the past, this led to a misdiagnosis of cryptogenic cir- in NASH) and the associated changes (bile ductular rhosis. Specific stains for collagen such as Masson reaction/ bile duct injury versus steatosis) make the trichrome, van Gieson, reticulin and Sirius red are rec- distinction usually easy. ommended, particularly at earlier stages 24. Lobular inflammation is usually mild and character- Most studies have shown that the stage of fibrosis is ized by small foci of inflammatory cells, mainly lym- an independent predictor of overall- and liver-related phocytes (usually a mixture of CD4+/CD8+ T lympho- mortality, regardless of the presence or severity of cytes) and macrophages, sometimes associated with other histological features 25,26. 198 I. Cataldo et al.

noma (HCC). MDBs are noted to be fewer and less developed in NASH than in alcoholic hepatitis. Their meaning as an epiphenomenon of hepatocyte injury, cytoprotective elements or a contributor to hepatocyte damage, is still unknown. MBDs formation is reversi- ble and does not reduce hepatocyte viability 27. Portal inflammation. Portal inflammation in adult NAFLD is usually minimal to mild and it is mainly composed of mononuclear cells, including lymphocytes, macrophag- es, and plasma cells. The presence of more than a mild portal inflammation should lead to considering the hy- pothesis of different diagnoses. However, more than mild portal inflammation can be seen in NASH and it correlates with a greater pathological severity of dis- ease, including advanced stages of fibrosis 28. Figure 5. Fat droplet surrounded by lymphocytes and mac- Megamitochondria are described as round or nee- rophages represents a lipogranulomas (arrow). Lipogranu- dle-shape eosinophilic PAS-diastase resistant intracy- lomas are frequently found in fatty liver; they are not con- toplasmic hepatocyte inclusions (Fig. 7). In NASH they sidered in the assessment of lobular inflammation. (H&E; are rarely seen, while they are more abundant in ALD original magnification 40x). where they have been associated with a better prog- nosis29. They can also be detected in other patholog- ical conditions (Wilson’s disease, drug-induced liver damage) and in pregnancy 9. Glycogenated nuclei optically appear as empty vacu- oles on H&E stain (Fig. 4). They are a normal finding in childhood, mainly in periportal areas, and may be seen even in young adults. When they are numerous in adult liver, glycogenated nuclei are to be consid- ered as abnormal finding. Glycogenated nuclei are common in NAFLD and NASH, and they have been associated with diabetes. They do not have any clini- cal significance 27 30.

Figure 6. NASH early stage: delicate perisinusoidal col- lagen deposition in zone 3. High quality connective tissue stains are required for a correct assessment. (Van Gieson stain; original magnification 20x).

Adjunctive lesions

Mallory-Denk bodies (MDB). As previously mentioned, MDBs (Fig. 3) may be seen within the cytoplasm of ballooned hepatocytes as clumped eosinophilic plot Figure 7. Megamitochondria: round eosinophilic intracyto- of cytoskeleton, mainly consisting of intermediate plasmic hepatocyte inclusion (arrow). (H&E; original magni- filaments CK8/18. They may be seen in other condi- fication 60x). tions including alcoholic, cholestatic liver diseases, primary biliary cholangitis and hepatocellular carci- PATHOLOGY OF NAFLD 199

Centrilobular arterial branches. The presence of min- The non-alcoholic nature of disease is often a chal- ute branches of the hepatic artery in perivenular zones lenge to determine histologically. In alcoholic hepatitis, has been described in NASH, especially when pe- hepatocytes that contain Mallory-Denk bodies tend to rivenular inflammation is present 21. This finding should be more pronounced and are often surrounded by not be misinterpreted as a portal tract. Centrizonal ar- polymorphonuclear leukocytes, a lesion referred to teries are associated with the presence of CD34-posi- as satellitosis. Prominent bilirubinostasis and scle- tive microvessel formation within a high stage fibrosis, rosing hyaline necrosis are features of ALD. In case suggesting a possible association between neo-angio- of well-compensated ALD distinction from NAFL or genesis and NASH progression to cirrhosis 31. NASH may be impossible and should relies on careful Iron deposition: Approximately one-third of patients af- clinical documentation of alcohol use. fected by NAFLD show signs of iron-overload, which may be exclusively mesenchymal, or parenchymal, or even a mixed mesenchymal/parenchymal pattern with different Grading and staging of NASH degrees of severity. The clinical impact of iron deposi- tion in NAFLD is not yet entirely clear, since all available As in chronic hepatitis, there are several staging studies show conflicting results. A recent observational and grading systems available in clinical research study provided evidence that a mesenchymal pattern as well as for the assessment in liver biopsy during of iron deposition may be of particular relevance for the the daily pathological practice. As in other settings, clinical outcome, suggesting that the pattern of iron dep- there should be a clear communication between the osition in liver biopsy may identify patients at risk from pathologist and clinical staff about the histological vascular or hepatic events, who may need closer mon- system used. itoring32. Therefore, it is good clinical practice to search A first system was proposed by Brunt et al. in 1999 33. for iron deposition and to report its eventual presence, This was a 3-tiered grading system based on the eval- pattern and severity in the pathological report. Hepatic uation of steatosis, ballooning and inflammation (lobu- siderosis can be assessed by Perls’ stain using a sim- lar and portal) (Tab. II). Regarding the staging of fibro- ple semi-quantitative score: score 0 as absent or barely sis, a scale from 0 to 4 is used as reported in Table III. discernible granules at a magnification of 40-fold (40×); Staging was based on the characteristic pattern and score 1 as barely discernible granules at a magnification evolution of fibrosis in NASH with an initial involve- of 20× but easily confirmed at 40×; score 2 as discrete ment of perisinusoidal spaces in zone 3 (stage 1) and granules resolved at 10× magnification; score 3 as dis- subsequent development of portal/periportal fibrosis crete granules resolved at a magnification of 2.5× and, (stage 2), bridging fibrosis (stage 3), and, finally, cir- finally, score 4 as massive granules visible even upon rhosis (stage 4) (Tab. IV). A prerequisite for applying 1.0× magnification 9. this system is a diagnosis of steatohepatitis.

Table II. Brunt system to grade NASH activity (from Brunt et al., 1999) 33. Grade Steatosis Ballooning Inflammation Grade 1 (mild) 1-3 (up to 66%) Minimal Lobular: 1-2 Portal: none/mild Grade 2 (moderate) 2-3 (> 33% up to 66%) Present Lobular: 2 Portal: mild-moderate Grade 3 (severe) 2-3 Marked Lobular: 3 Portal: mild-moderate Steatosis: grade 1 ≤ 33%; grade 2 > 33%, < 66%; grade 3 ≥ 66%. Inflammation: Lobular (0-3): 0 (none), 1 (< 2 foci/20x field), 2 (2-4 foci/20x field); 3 (> 4 foci/20x field); Portal (0-3): 0 (none), 1 (mild), 2 (moderate), 3 (marked).

Table III. Brunt system for staging NASH fibrosis (from Brunt et al., 1999) 33. Stage Zone 3, Sinusoidal Portal Based Bridging Cirrhosis 1 Focal or extensive 0 0 0 2 Focal or extensive Focal or extensive 0 0 3 Bridging septa Bridging septa + 0 4 ± ± Extensive + Evaluation of fibrosis is performed by Masson trichrome histochemical stain. 200 I. Cataldo et al.

Table IV. NASH Clinical Research Network (CRN) scoring system for nonalcoholic fatty liver disease (from Kleiner et al., 2005) 19. Steatosis grade Lobular Inflammation Hepatocellular ballooning 0: < 5% 0: None 0: None 1: 5-33% 1: < 2 foci/20x field 1: Mild, few 2: 34-66% 2-4 foci/20x field 2: Moderate-marked, many 3: > 66% 3: > 4 foci/20x field NAFLD activity score (NAS): 0-8 Fibrosis (evaluated with Masson trichrome stain) 0 None 1a Mild zone 3 sinusoidal fibrosis (trichrome stain to be identified) 1b Moderate zone 3 sinusoidal fibrosis (could be detected on H&E examination) 1c Portal fibrosis only 2 Zone 3 sinusoidal fibrosis and periportal fibrosis 3 Bridging fibrosis 4 Cirrhosis NASH: nonalcoholic steatohepatitis; NAFLD, nonalcoholic fatty liver disease.

Table V. Steatosis-Activity-Fibrosis (SAF) scoring system of NAFLD (from Bedossa et al., 2012) 34. Steatosis grade (S): 0-3 Hepatocyte ballooning: 0-2 Based on percentage of hepatocytes with large and/or medium size 0: none intracytoplasmic lipid droplets 1: Cluster of rounded hepatocytes with pale/reticulated cytoplasm S0: < 5% 2: Same as 1 with enlarged hepatocytes (more than twice of S1: 5-33% normal size) S2: 34-66% S3: > 66% Lobular inflammation: 0-2 Activity grade (A): 0-4 0: None Sum of score for ballooning and lobular inflammation 1: ≤ 2 foci/20x field A1 (A = 1): Mild activity 2: > 2 foci/20x field A2 (A = 2): Moderate activity A3 and A4 (A > 2): Severe activity Fibrosis stage (F) SAF score F0: no significant fibrosis S0-3; A0-4; F0-4 F1: 1a Mild zone 3 sinusoidal fibrosis 1b Moderate zone 3 sinusoidal fibrosis 1c Portal fibrosis only F2: zone 3 sinusoidal fibrosis with periportal fibrosis F3: Bridging fibrosis F4: Cirrhosis

Kleiner et al., in 2005 19 proposed a novel NAFLD Ac- for NASH diagnosis. Here, the histological diagnosis tivity Score (NAS) system. This is a modified Brunt sys- of NASH is a prerequisite to apply the NAS score. As tem, which could be applicable to any patient (adult or for fibrosis, Kleiner et al. slightly modified the original children), and to the various histological spectrum of Brunt scheme, as reported in Table V. NAFLD. NAS was defined as the unweighted sum of Steatosis-Activity-Fibrosis (SAF) scoring system was steatosis, lobular inflammation, and ballooning. In the proposed by the European Fatty Liver Inhibition of validation study, NAS scores 1 or 2 corresponded to Progression (FLIP) consortium in 2012 34. Based on negative for NASH, while a NAS scores 5-8 correlat- their observation that liver function tests (including ed with a definite diagnosis of NASH. Activity scores transaminases) did not differ when pure steatosis was 3 and 4 were mainly observed in cases that did not compared to normal liver, in SAF-score the activity fulfill the pathologists’ criteria for NASH. This led to the parameters include only ballooning and lobular in- assessment of NAS score as a diagnostic tool. How- flammation, whereas steatosis is separately assessed ever, the scores obtained from NAS system cannot be (Tab. VI). Fibrosis is assessed in a 4-tiered system as used as a surrogate of the histopathological criteria proposed by Kleiner et al. 19 (Tab. V). PATHOLOGY OF NAFLD 201

Table VI. Simplified diagnostic algorithm for NAFLD diagnosis on liver biopsy (from Bedossa et al., 2012, mod.) 34. Lobular inflammation Balloning Steatosis > 5% Diagnosis (any degree) (any degree) No Yes/No Yes/No No NAFLD Ye s Ye s No NAFL Ye s No Ye s NAFL Ye s Ye s Ye s NASH NAFLD: nonalcoholic fatty liver disease; NAFL: nonalcoholic fatty liver; NASH: nonalcoholic steato-hepatitis.

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20 Hall A, Covelli C, Manuguerra R, et al. Transaminase abnormali- 28 Brunt EM, Kleiner DE, Wilson LA, et al. Portal chronic inflam- ties and adaptations of the liver lobule manifest at specific cut- mation in nonalcoholic fatty liver disease (NAFLD): a histologic offs of steatosis. Sci Rep 2017;7:40977. https://doi.org/10.1038/ marker of advanced NAFLD-Clinicopathologic correlations from srep40977 the nonalcoholic steatohepatitis clinical research network. Hep- 21 Kleiner DE, Brunt EM. Nonalcoholic fatty liver disease: patho- atology 2009;49:809-820. https://doi.org/10.1002/hep.22724 logic patterns and biopsy evaluation in clinical research. Semin 29 Altamirano J, Miquel R, Katoonizadeh A, et al. A histologic scor- Liver Dis 2012;32:3-13. https://doi.org/10.1055/s-0032-1306421 ing system for prognosis of patients with alcoholic hepatitis. Gas- 22 Bedossa P, Consortium FP. Utility and appropriateness of the troenterology 2014;146:1231-1239 e1-6. https://doi.org/10.1053/j. fatty liver inhibition of progression (FLIP) algorithm and steato- gastro.2014.01.018 sis, activity, and fibrosis (SAF) score in the evaluation of biopsies 30 Brunt EM. Nonalcoholic steatohepatitis: definition and of nonalcoholic fatty liver disease. Hepatology 2014;60:565-575. pathology. Semin Liver Dis 2001;21:3-16. https://doi. https://doi.org/10.1002/hep.27173 org/10.1055/s-2001-12925 23 Burt AD, Lackner C, Tiniakos DG. Diagnosis and Assess- 31 Gill RM, Belt P, Wilson L, et al. Centrizonal arteries ment of NAFLD: Definitions and Histopathological Clas- and microvessels in nonalcoholic steatohepatitis. Am J sification. Semin Liver Dis 2015;35:207-220. https://doi. Surg Pathol 2011;35:1400-1404. https://doi.org/10.1097/ org/10.1055/s-0035-1562942 PAS.0b013e3182254283 24 Takahashi Y, Fukusato T. Histopathology of nonalcoholic fatty 32 Eder SK, Feldman A, Strebinger G, et al. Mesenchymal iron de- liver disease/nonalcoholic steatohepatitis. World J Gastroen- position is associated with adverse long-term outcome in non- terol 2014;20(42):15539-15548. https://doi.org/10.3748/wjg.v20. i42.15539 alcoholic fatty liver disease. Liver Int 2020;40:1872-1882. https:// doi.org/10.1111/liv.14503 25 Angulo P, Kleiner DE, Dam-Larsen S, et al. Liver fibrosis, but 33 no other histologic features, is associated with long-term out- Brunt EM, Janney CG, Di Bisceglie AM, et al. Nonalcoholic ste- comes of patients with nonalcoholic fatty liver disease. Gas- atohepatitis: a proposal for grading and staging the histologi- troenterology 2015;149:389-397 e10. https://doi.org/10.1053/j. cal lesions. Am J Gastroenterol 1999;94:2467-2474. https://doi. gastro.2015.04.043 org/10.1111/j.1572-0241.1999.01377.x 34 26 Ekstedt M, Hagstrom H, Nasr P, et al. Fibrosis stage is the stron- Bedossa P, Poitou C, Veyrie N, et al. Histopathological algo- gest predictor for disease-specific mortality in NAFLD after up to rithm and scoring system for evaluation of liver lesions in mor- 33 years of follow-up. Hepatology 2015;61:1547-1554. https://doi. bidly obese patients. Hepatology 2012;56:1751-1759. https://doi. org/10.1002/hep.27368 org/10.1002/hep.25889 27 Zatloukal K, French SW, Stumptner C, et al. From Mallory to 35 Bedossa P. Diagnosis of non-alcoholic fatty liver disease/non- Mallory-Denk bodies: what, how and why? Exp Cell Res alcoholic steatohepatitis: Why liver biopsy is essential. Liver Int 2007;313:2033-2049. https://doi.org/10.1016/j.yexcr.2007.04.024 2018;38 Suppl 1:64-66. https://doi.org/10.1111/liv.13653 PATHOLOGICA 2021;113:203-217; DOI: 10.32074/1591-951X-295

Review

Hepatocellular carcinoma: a clinical and pathological overview

Salvatore Lorenzo Renne1,2, Samantha Sarcognato3,4, Diana Sacchi3, Maria Guido3,4, Massimo Roncalli1,2, Luigi Terracciano1,2, Luca Di Tommaso1,2 1 Department of Biomedical Sciences, Humanitas University, Milan, Italy; 2 IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy; 3 Department of Pathology, Azienda ULSS2 Marca Trevigiana, Treviso, Italy; 4 Department of Medicine - DIMED, University of Padova, Padova, Italy

Summary HCC incidence rates have been rising in the past 3 decades and by 2025 > 1 million individuals will be affected annually. High-throughput sequencing technologies led to the identification of several molecular HCC subclasses that can be broadly grouped into 2 major subgroups, each characterized by specific morphological and phenotypical features. It is likely that this increasing knowledge and a more appropriate characterization of HCC at the pathological level will impact HCC patient management.

Key words: hepatocellular carcinoma, tumor microenvironment, angiogenesis, prognosis, diagnosis

Clinical background

Received and accepted: April 26, 2021 Epidemiology. HCC incidence rates have been rising in the past 3 de- cades and similar trends are expected through 2030 1. The WHO stated Correspondence in 2015 that HCC was the fifth most common cancer worldwide and the Luca Di Tommaso 2 Department of Pathology, IRCCS Humanitas second most common cause of cancer-related death . The global ob- Research Hospital, via Manzoni 56, 20089 servatory on cancer reported that in 2018 liver cancer was the sixth most Rozzano, Milan, Italy common cancer world-wide, with 841,080 new cases, and the fourth E-mail: [email protected] leading cause of cancer-related death globally 3. By 2025, > 1 million 3 Conflict of interest individuals will be affected by liver cancer annually . Over 90% of HCC The Authors declare no conflict of interest. cases occur in the setting of chronic liver disease. Cirrhosis from any etiology is the strongest risk factor for HCC 4,5. Several important risk How to cite this article: Renne SL, factors are related to HCC, among these HBV and HCV chronic infection Sarcognato S, Sacchi D, et al. Hepatocellular and NAFLD/NASH play the major role. About 3.5% of the global popula- carcinoma: a clinical and pathological 2 overview. Pathologica 2021;113:203-217. tion, 257 million persons, are chronically infected with HBV. The lifetime 6,7 https://doi.org/10.32074/1591-951X-295 risk of these persons to develop cirrhosis and/or HCC is 15% to 40% . In endemic areas, HBV is etiologically implicated in as many as 50% to © Copyright by Società Italiana di Anatomia Pato- 80% of all HCC cases, a figure decreasing to 20% in western countries 8. logica e Citopatologia Diagnostica, Divisione Itali- 2 ana della International Academy of Pathology HCV infection affects 71 million persons all over the world. With the use of direct-acting antiviral (DAA) therapy, patients with HCV infection have OPEN ACCESS been successfully treated to achieve a sustained virological response and this has resulted in a 50-80% reduction in the risk of HCC 5,9. None- This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- theless, HCV chronic infection is a major contributing factor to liver can- NonCommercial-NoDerivatives 4.0 International) license: the cers in the USA, and is associated with 50% of cases.10 NAFLD/NASH work can be used by mentioning the author and the license, but only for non-commercial purposes and only in the original has a global prevalence of 24%, with the highest rates reported in South version. For further information: https://creativecommons. America and the Middle East (≈30-35%) 11. It has been estimated that org/licenses/by-nc-nd/4.0/deed.en up to 25% of NAFLD can progress to NASH and that up to 20% of pa- 204 S.L. Renne et al.

tients with NASH have cirrhosis 12. NASH-associated In general, surgical resection or liver transplantation cirrhosis carried a 2.4% to 12.8% increased risk of is the first option to treat early-stage HCC yielding the HCC 13. Several studies have demonstrated that 25- best outcomes, with a 5-year survival of ~70-80% 4,5. 30% of NASH-associated HCC occur in the absence Resection should be offered to patients who have a of cirrhosis 14,15. Since 2010, the proportion of HCC single tumor (regardless of size), well-preserved liver attributed to NASH has rapidly increased, currently function (Child-Pugh A with total bilirubin < 1 mg/dl), representing 15-20% of cases in the West 16. no signs of portal hypertension (varices or ascites) Diagnosis. Imaging plays a critical role in HCC diag- or a hepatic venous pressure gradient (< 10 mmHg), nosis. HCC lesions are brighter than the surrounding and a preserved performance status. LT should fol- liver in the arterial phase in a CT scan or MRI and low Milan criteria: a single tumor of 5 cm or up to less bright than the surrounding parenchyma in the three nodules of 3 cm with no evidence of macrovas- venous and delayed phases, and this is due to the cular invasion or extra-hepatic manifestations 19. The differential blood supply of the tumor compared with recurrence of HCC after hepatic resection rates as the background liver. This phenomenon of ‘arterial high as 70% at 5 years, even in patients with a single enhancement and delayed washout’ has a sensitiv- tumor ≤ 2 cm 20. Recurrences can be divided into ity of 89% and a specificity of 96% for HCC and is either early (2 years), and late (> 2 years): the former regarded as the radiological hallmark of HCC 17. In likely representing the result of metastatic spread; patients with liver cirrhosis the presence of these typi- the latter de novo tumors arising in a microenviron- cal vascular hallmarks identified by quadruple-phase ment predisposed to carcinogenesis 21. The 10-year CT or dynamic contrast-enhanced MRI is sufficient recurrence rate after transplantation is 10-15% for for diagnosis without requiring histological confirma- HCC tumors within Milan criteria and 20% in those tion 4,5. A more recent radiological approach, the LI- down-staged to the Milan criteria 22. In very early- RADS (Liver Imaging Reporting and Data System) as- stage disease (tumors < 2 cm diameter), thermal ab- signs lesions > 10 mm to different categories reflecting lation has demonstrated similar outcomes to surgical the relative probability of the lesion of being benign, resection and thus may be recommended as first-line HCC, or other hepatic malignant neoplasm according treatment, specifically in light of its lesser invasive- to an enlarged the number of criteria (arterial phase ness and morbidity compared with surgery 23. Radio- enhancement, tumor size, washout, enhancing cap- frequency ablation and percutaneous ethanol injec- sule and threshold growth features) 18. AFP and other tion are effective for small tumors, but radiofrequency serum biomarkers generally have a minor role in the ablation is superior for tumors larger than 2 cm vs diagnosis of HCC. percutaneous ethanol injection 24,25. Adjuvant thera- Treatment. Therapeutic options might be limited be- pies to be used after the curative approaches, are an cause of the patient’s overall health status (cirrhosis). unmet medical need, as randomized controlled trials Nonetheless, there have been significant advances have so far yielded negative results. For intermediate- in HCC treatment over the past 10 years. Some ap- stage HCC, transarterial chemoembolization (TACE) proaches offer the chance of long-term response: they has been the most widely used treatment and the include surgical resection, orthotopic liver transplan- standard of care over the past two decades 26. TACE tation (LT), and ablative techniques such as thermal is recommended for patients who do not have vas- ablation. Other therapies attempt slowing tumor pro- cular invasion or extrahepatic spread and are not gression and include transarterial chemoemboliza- candidates for liver transplantation, surgical resec- tion (TACE), transarterial radioembolization (TARE), tion, or local ablation because of large tumor size or stereotactic body radiation therapy (SBRT), and sys- multifocal tumor 23,27, with an estimated average of temic chemotherapy. The most appropriate treatment median overall survival of ~30 months 28. Transarte- should be indicated by a multidisciplinary approach rial radioembolization (TARE) has shown efficacy in taking into consideration different patients features phase II investigations but has not been established and stratifying them in a disease stage. Currently, as a primary standard of care by guidelines 29. Ap- HCC is treated according to the Barcelona Clinic Liv- proximately 50-60% of patients with HCC, mostly at er Cancer scheme, which stratifies patients into five advanced stage, will be treated with systemic thera- categories: very-early (stage 0), early (stage A), inter- pies. This field has seen significant progress in the mediate (stage B), advanced (stage C) and terminal past 5 years. Until 2017 sorafenib was the only avail- stage (stage D) 4,5. The BCLC staging system links able standard of care for advanced HCC 30. In 2018 tumor stage, liver function, cancer-related symptoms a phase II study demonstrated the efficacy of lenva- and performance status to an evidence-based treat- tinib, which was then approved for advanced-stage ment algorithm. HCC in the first-line setting 31. In case of progression HCC CLINICOPATHOLOGICAL UPDATES 205

to single-agent regimens, regorafenib 32, cabozan- presence of unpaired arteries and pseudo-gland for- tinib 33, and ramucirumab 34 showed improved surviv- mation 41,43. At the molecular level, HG-DN, eHCC and al benefits and were therefore approved as second- pHCC are characterized by a progressive increase of line treatment. The median survival for these treat- TERT promoter mutation as a unique fingerprint 42. As ments was 8-10 months: a figure more than doubled shown in Table I, however, none of these alterations is by the recent approved combination of atezolizumab sufficient, per se, to distinguish lesions staying close (anti-PDL1 antibody) and bevacizumab (anti-VEGF at the border between dysplasia and malignancy. To antibody) 35. this aim, the international guideline recommends the use of a panel of immunohistochemical markers 4,5. In more advanced lesions, architectural alterations Pathology and cytological changes are overt and diagnosis of malignancy is not under discussion. In this setting the Gross features. Single HCC can be classified as differential diagnosis might involve cancer with mixed “Vaguely nodular” (a nodule with indistinct margins), hepatocellular and cholangiocellular differentiation or “Expanding nodular” (a round expansive nodule with metastasis (unusual in the setting of a cirrhotic liver). a distinct margin), “Multinodular confluent” (cluster of HCC histological subtyping rests on the evaluation of small and confluent nodules), “Nodular with perinodu- architectural growth patterns (microtrabecular, macro- lar extension” (extranodular growth in < 50% of the trabecular, pseudo-glandular, compact) and cytologi- circumference) and “Infiltrative” (extranodular growth cal aspects (clear cell, fatty change, cholestasis, pleo- in > 50% of the tumor circumference) 36. Single HCC morphic cells, spindle cells). It should be observed, with nodular morphology have favorable outcomes however, that several different features frequently co- compared with those with multinodular, perinodular or exist in the same lesions. HCC differentiation is grad- infiltrative growth patterns 37-40. In up to 30% of cases, ed into four grades according to the Edmondson and HCC might present with multiple, clearly separated, Steiner classification and into three according to the tumors. In this case, the number of lesions should be WHO 44. recorded and each lesion should be described in de- Studies based on high-throughput sequencing led to tail. A satellite nodule is a small nodule close (< 2 cm) the identification of several molecular HCC subclass- to the main tumor. es 42, 45-51. Regardless of the nomenclature used, HCC Microscopic and molecular features. HCC develops can be divided into 2 major subgroups (Fig. 1). The from a cirrhotic liver through a multistep sequence. non-proliferation class 51-54 is characterized by chro- This latter includes pre-neoplastic lesions, represent- mosomal stability and frequent TERT promoter muta- ed by low-grade (LG-) and high-grade (HG-) dysplastic tions. These HCCs are less aggressive and show well nodules (DN) and early neoplastic lesions, represent- to moderate histological differentiation, less frequent ed by early HCC (eHCC) and small and progressed vascular invasion and low levels of AFP 55. They are HCC (pHCC). Pre-neoplastic and early neoplastic related to non-alcoholic and alcoholic steatohepatitis lesions are characterized by progressive accumula- and HCV infection. Two distinct subgroups character- tion morphological and molecular abnormalities 41,42. ized this class: the WNT-β-catenin/CTNNB1-mutated The former are represented by a wide spectrum of subclass which drives an immune-excluded pheno- findings including increasing cell density and nucle- type with low immune infiltration 52,54,56, and the inter- ar-to-cytoplasmic ratio; loss of reticulin framework; feron subclass which presents a highly activated IL6-

Table I. Summary of the main pathological features of distinction between HGDN vs eHCC. Features HGDN eHCC Discriminatory value Portal tract + ± Low Cell density + (up to 1.5-2) + (x 2 or more) Pseudoglands ± ± Nuclear Atypia ± + Steatosis - ± Unpaired arteries ± + Medium Reticulin loss/decrease ± ± TERT promoter mutation ± + Stromal invasion - ± High 2 markers staining out of 3 - ± 206 S.L. Renne et al.

Figure 1. Classification of HCC. This scheme illustrates the correlations existing among molecular classes of HCC 50,51,53 and genetic, morphological and clinical features.

JAK-STAT signaling pathway, with a more inflamma- CTNNB1 mutated (cholestatic-) HCC (Fig. 2 A, B) tory tumor microenvironment. The other major class HCCs with mutations in CTNNB1 are well-differen- of HCC, i.e. proliferation-class, is characterized by tiated tumors, characterized by microtrabecular and high chromosomal instability, global DNA hypometh- pseudoglandular patterns, intratumor cholestasis ylation, frequent TP53 mutations, and overexpression and lack of immune infiltration 55-59. CTNNB1 en- of genes involved in the cell cycle 45,50,51. These HCCs codes β-catenin, a key intracellular transducer of the are more aggressive and show poor histological differ- Wnt signaling pathway that regulates liver physiol- entiation, high vascular invasion and increased levels ogy and zonation 60. Mutations result in β-catenin of AFP 55. The proliferation-class can be further divid- stabilization and subsequent nuclear accumulation, ed into two subclasses. The former, i.e. S1 or iCluster where it enhances cell proliferation and survival. 3 53,54, shows Wnt-TGFβ activation and immune-ex- hausted phenotype 56 and is barely recognizable at HCCs with mutations in CTNNB1 are characterized morphology; the latter, S2 or iCluster 1 53,54, displays at phenotypical level by glutamine synthetase (GS) a progenitor-like phenotype, highlighted by the ex- and nuclear β-catenin expression. Interestingly, di- pression of stem cell markers (CK19, EPCAM) and is verse mutations have been correlated with different 61 characterized by activated IGF2 and EPCAM signal- staining patterns . These tumors are characterized ing pathways 55. by the expression of genes involved in hepatocellu- According to the last WHO classification of liver tu- lar differentiation and function, such as APOB, ALB, mors, about 1/3 of all HCC can be classified into spe- HNF1A or HNF4A, and by the dysregulation of bile cific subtypes: steatohepatitic, clear cell, macrotrabe- salt transporters which contribute to their cholestatic colar massive, scirrhous, chromophobe, fibrolamellar, phenotype 57. One of these transporters, SLCO1B3, neutrophil- and lymphocyte- rich 44. In the following is responsible for the uptake of the MRI contrast section we will illustrate some of these entities. agent gadoxetic acid 62. HCC CLINICOPATHOLOGICAL UPDATES 207

A B

C D

Figure 2. Pathology of HCC. This figure illustrates some of the most typical pathological features of HCC subtypes. (A, B) CTNNB1-mutated. In this fresh specimen, HCC presents as a mass made by several cluster of small and confluent green nod- ules (cd. “Multinodular confluent HCC”) with some satellites (A); at histology the lesion is characterized by the presence of several pseudo-glandular structure filled by green material consistent with cholestasis (B). (C, D) Macrotrabecular Massive. In this fixed specimen the multiple confluent HCC nodules bulge over the surrounding flat fibrous strands (C); at microscopic level the lesion is characterized by a macrotrabecular pattern of growth (D).

Macro-trabecular-massive HCC (MTM-) HCC endothelial and peri-endothelial cells, which results in an (Fig. 2C, D) increased sensitivity to VEGFA. MTM-HCC are tumors characterized by a macrotra- Scirrhous HCC (Fig. 2E) becular (> 6 cells thick) growth pattern in > 50% of the lesion, regardless of the associated cytological features. The scirrhous subtype is characterized by an admix- They exhibit a very aggressive phenotype, with frequent ture of abundant dense stroma and neoplastic cells. satellite nodules and vascular (micro and/or macro) in- These latter frequently express, at phenotypical level, vasion. It frequently occurs in patients infected by HBV markers of progenitor or cancer stem cells, includ- and with high alpha-fetoprotein serum levels 57,63. At the ing CK7, CK19, or CD133. Accordingly, it has been genetic level, MTM-HCC often harbors TP53 mutations put forward the hypothesis that these tumors have and/or FGF19 amplification and they are characterized an intermediate molecular trait, between HCC and by angiogenesis activation, with angiopoietin 2 and vas- cholangiocarcinoma 65. Consistent with its histologi- cular endothelial growth factor A (VEGFA) overexpres- cal appearance, scirrhous HCC are characterized by sion 55,57. Angiopoietin 2 is responsible for the destabili- the activation of TGF-β pathway, with overexpression zation of established blood vessels and subsequent vas- of VIM, SNAIL, SMAD4 and TWIST and features of cular sprouting 64. It also disrupts interactions between epithelial-to-mesenchymal transition 55 65. 208 S.L. Renne et al.

E F

G H

Figure 2. Pathology of HCC. (E) Scirrhous. This HCC is characterized by abundant stroma separating neoplastic trabeculae. (F) In this HCC, neoplastic cells are characterized by diffuse steatosis and occasional cell ballooning; the tumor shows scat- tered inflammatory infiltrates. (G) Lymphocyte-rich. A rich intratumoral infiltrate of lymphocytes denotes this HCC. (H) A clear cell variant of HCC.

Steatohepatitic HCC (Fig. 2F) associated with improved overall survival, supporting This subtype is characterized by inflammatory infil- the hypothesis that the lymphocytic infiltrate plays an trates, cell ballooning, peri-cellular fibrosis and Mal- antitumor effect 67. The lymphocytes show a predomi- lory-Denk bodies 66. At phenotypical level neoplastic nance of cytotoxic CD8+ elements, with increased cells show overexpression of C-reactive protein (CRP) programmed cell death 1 ligand 1 (PD-L1) and pro- - a target gene of JAK/STAT signaling 55. These tumors grammed cell death 1 (PD1) expression 67,68. As oppo- are often well-differentiated and they associated with site to other settings, such as colon and lung cancer, gene expression profile similar to that of non-tumor where lymphocytic infiltration has been linked with mi- liver. crosatellite instability and/or high mutational 69, none of these HCCs were microsatellite instable or associ- Lymphocyte-rich HCC (Fig. 2G) ated with a higher number of somatic mutations 56,67. The latest WHO defined these HCC as lesions with lymphocytes outnumbering neoplastic cells in most Combined hepatocellular-cholangiocarcinoma fields at on H/E 44. Nonetheless a consensus defini- A subset of primary liver cancer may exhibit both tion on the cut-off value for intratumor lymphocyte hepatocytic and biliary differentiation. These bipheno- density is currently lacking. This rare variant has been typic tumors are much rarer, accounting for less than HCC CLINICOPATHOLOGICAL UPDATES 209

5% of all liver cancers 44,70.They were first described in unmet need for HCC management. Some of the 1903 by Gideon Wells and, since then, the definition of above-mentioned histopathological subtypes, MTM- this entity has changed several times. Recently an in- HCC in particular, had been proposed as predictors of ternational group proposed a consensus terminology 70 prognosis 57,63, but not validated in external cohort 59. later accepted by the WHO 44. It was recommended to By contrast, a robust prognostic significance has been call these lesions as combined hepatocellularcholan- proposed 72 and later validated 59 for a peculiar vas- giocarcinoma (cHCC-CCA) and the diagnostic criteria cular phenotype, characterized by CD34+ vessels proposed was the unequivocal presence of both hepat- encapsulating tumor cluster (VETC). VETC might be ocytic and cholangiocytic differentiation within the same present in up to 40% of HCC 59 and associates with tumor on routine H&E. Immunohistochemical markers higher attitude of tumoral cells to infiltrate vessels. In- of hepatocytic (HAS, Arginase, CD10 and polyclonal terestingly, a recent study demonstrated that this phe- CEA) and cholangiocytic (CK7, CK19) differentiation notype predicts the response to sorafenib 73. may help, but is neither necessary nor sufficient 44. A recent study showed that HCC and CCA compo- nents had very similar global gene expression pro- Real-life diagnostic issues files, thus suggesting a monoclonal origin 71. The most frequently mutated driver genes were TP53 (49% of In the daily practice, the pathologist’s diagnostic in- the cases), TERT promoter (23% of the cases), AXIN1 volvement is restricted to specific clinical settings. (10% of the cases), and KMT2D (9% of the cases), mutations that may be associated with either HCC or Liver biopsy in patients with healthy liver: diagnostic ICCA. In the same study, it was also suggested that of well differentiated hepatocellular lesions nestin might serve as a biomarker for the diagnosis In this setting the differential diagnosis should take into and prognosis of cHCC-ICC 71. consideration a benign liver lesion, mostly represented by focal nodular hyperplasia (FNH) and hepatocellular VETC (Fig. 3) adenoma (HA) and atypical HA or/and HCC. The first The availability of tissue biomarkers remains an question is whether the lesions has been adequately

A B C

D E F

Figure 3. Vascularization of HCC. (A, B, C) The images illustrate an HCC (lower right corner, A) with a rich vascular support as highlighted by a CD34 staining (B); at higher magnification CD34+ vessels show a capillary distribution; (C). (D, E, F) also this HCC (right part, D) shows a rich vascular network when stained with CD34 (E); in this cases however, CD34+ vessels encapsulate clusters of neoplastic cells (F) featuring a peculiar phenotype described as VETC 59,72. 210 S.L. Renne et al.

A B

C D

Figure 4. Diagnostic of well differentiated hepatocellular lesions in healthy liver: Focal Nodular Hyperplasia. (A) This liver biopsy illustrates an area characterized by the presence of fibrous septa: a feature which might raise the suspect of a not adequate specimen; (B) in such cases the expression of CD34 by the endothelium of sinusoids can be of great help: indeed, a diffuse increase is in keeping with lesional sampling; (C) CK7 highlights that the lesion is characterized by the presence of portal tracts; (D) GS immunostaing proves a pathognomonic strong, map-like, staining for GS. The mopho-phenoptypical findings are conclusive for an FNH.

sampled: both FNH and HA may have a subtle and Alfa (SAA). The profile GS+map-like/SAA-/CRP- supports deceptive morphology as to that their borders may be FNH while GS-/SAA+/CRP+ or GS+non-map-like/SAA+/ difficult to be clearly localized. A good tool to highlight PCR+ support I-HA. Indeed, GS immunoreactivity char- them, particularly in HA, is an endothelial cell marker acterize those I-HA with an activation of β-catenin path- (we use CD34) which will permit in the majority of the way. If pseudo-portal tracts are not detectable inside cases to discern the profile of the punched lesion. Once the lesion and GS staining is completely negative, there the lesion has been identified we usually evaluate are two alternatives: a) steatotic-HA and b) usual-HA whether pseudo-portal tracts (fibrous tissue with arteri- (u-HA). The evidence of steatosis favor a diagnosis of olar vascular structures and ductular reaction), can be steatotic-HA (S-HA) and lack of expression of Liver Fat- documented, at H/E or using a CK7, within the lesion. ty Acid Binding protein (LFABP) in tumoral hepatocytes The presence of pseudo-portal tracts suggests two will prove it. A non-steatotic and non-atypical adenoma diagnostic alternatives: a) FNH, b) Inflammatory-HA with a LFABP-/SAA-/CRP-/GS- phenotype should be (I-HA). FNH and I-HA may be distinguished in most of classified as u-HA. Finally, when the lesion lacks pseu- the cases using a panel GS, CRP and Serum Amyloid do-portal tracts, inflammatory/teleangectatic morphol- HCC CLINICOPATHOLOGICAL UPDATES 211

A B

C D

Figure 5. Diagnosis of well differentiated hepatocellular lesions in healthy liver: Steatotic . (A) This liver biopsy documents a well differentiated lesion which, at H/E, is barely detectable (right part); (B) at higher magnifica- tion lesional hepatocytes are characterized by clear nuclei; (C) CD34 staining highlights the lesion; (D) lesional hepatocytes, as compared to the surrounding, do not stain for LFABP. The morpho-phenotypical findings are conclusive for a steatotic HA.

ogy (SAA-/PCR-) and shows atypical hepatocytes and and GS warrants 100% specificity, with a sensitiv- GS expression the nodule is likely to be a β-catenin-HA ity of 49% 74,75. Sensitivity that can be increased to variant. GS antibody, when the staining is strong and 64% by introducing a further marker Clathrin Heavy diffuse, shows the highest diagnostic accuracy (abso- Chain (CHC) 76. On the other hand, the pathologist lute specificity and sensitivity). should be aware that the use of single marker alone can be misleading and should not be considered as a Liver biopsy, in patients with hepatitis/cirrhotic liver: proof of malignancy. GPC3 immunoreactivity can be diagnostic of well differentiated hepatocellular observed in a few cirrhotic cells and in up to 10% of lesions cells of HGDN. HSP70 is normally expressed by ap- In this setting the differential diagnosis rests between optotic hepatocytes, isolated periseptal hepatocytes, HGDN and eHCC. Morphological features suggested and stellate cells. Finally, GS shows peculiar pattern as useful in this differential diagnosis include the de- of staining according to different clinico-pathological crease of reticulin framework, the presence of stromal conditions 77. invasion, and the progressive transition toward CD34 expression by endothelial cells (Tab. I). However, none Liver biopsy, in patients with hepatitis/cirrhotic liver: of these can, per se, objectively separate HGDN from diagnostic of poorly differentiated lesions eHCC 41. Rather, the use of a panel of markers, name- In this setting the differential diagnosis rests between ly glypican 3 (GPC3), heat shock protein 70 (HSP70), a primary liver cancer (HCC, CC and combined HCC- 212 S.L. Renne et al.

A B

C D

Figure 6. Diagnosis of well differentiated hepatocellular lesions in healthy liver: teleangiectatic/inflammatory Hepato- cellular Adenoma. (A) This liver biopsy illustrates a well differentiated hepatocellular lesion characterized, at higher magni- fication, by teleangiectatic vessels; (B) CK7 highlights that the lesion is characterized by the presence of scattered pseudo- portal tracts; (C and D) the lesion is immunoreactive for SAA and PCR. The morpho-phenotypical findings are conclusive for telenagiectatic HA.

CC) and a metastasis. As already observed the great GPC3 and BSEP. Table II illustrates the performanc- majority of bona fide HCC are diagnosed according to es of the each of these histotype-markers when used radiological criteria and treated accordingly. Some of alone 78. these patients underwent a liver biopsy after standard treatments (surgical, ablative and medical) and before Liver resection: essential criteria enrolment in clinical studies. These cases, due to their Clinical and radiological features predicting HCC out- natural history and treatment as well, are poorly dif- come are part of the current staging system, including ferentiated, partially necrotic, or even shifted toward a the BCLC scheme, mentioned above. This information stem differentiation. Nonetheless, to be considered el- should be integrated and completed, by pathological igible for the study they need a conclusive histopatho- features when a resection is performed. The essential logical diagnosis. In this setting the pathologist might pathological criteria that should be reported are rep- benefit from a few immunohistochemical markers resented by the gross evaluation of tumor size and to prove the hepatocytic differentiation of the lesion. number and by the microscopic evaluation of tumor These include HepPar-1, Arginase-1, CD10, pCEA, type and grade, vascular invasion and the expression HCC CLINICOPATHOLOGICAL UPDATES 213

A B C

D E F

Figure 7. Diagnosis of well differentiated hepatocellular lesions in healthy liver: Atypical Hepatocellular Adenoma. (A) These biopsies document a well differentiated hepatocellular lesion (*) and the surrounding parenchyma; (B) the lesion, which is barely seen on H/E at scanning magnification, is highlighted and clearly outlined by CD34; and shows (C) strong and diffuse immunoreactivity for GS; (D) at higher magnification, the lesion is characterized by a mild degree of architectural disarrangement and cytological atypia (pseudo-glands and increased N/C ratio), (E) reticulin framework is conserved and (F) few neoplastic cells showing nuclear immunoreactivity for HSP70. The morpho-phenotypical findings are conclusive for an atypical HA.

Table II. Sensitivity of markers used to demonstrate HCC in a liver lesion. All HCC, sensitivity: All HCC, sensitivity: G3 HCC, Marker Best performance Worst performance sensitivity HepPar1 84% 70% 22-78% Arginase 96% 84% 44-89% BSEP 90% - 78% pCEA 81% 45% 78% CD10 74% 50% 67% GPC3 54% - 67% of CK19. The correct definition of the histotype enrich- do not represent the target for drugs affecting lympho- es the pathological report with prognostic information. cytes function 56,80. HCC grading systems (Edmond- The MTM histotype, has a poorer outcome 57,63 while son-Steiner and WHO, with several “home-made” var- the lymphocyte-rich HCC has a better prognosis 44. iations) strongly predict patient outcome in liver resec- The histotype might also be used as a predictive ev- tion or transplantation 81, with the worst grade driving idence. It has been shown that lymphocyte rich HCC HCC prognosis 82. Accordingly, a clinical meaningful are sustained by the presence of an active immune pathological report should indicate the predominant infiltrate 79 which makes at least questionable the use and also the worst grade (in line with what is done for of drugs designed to restore the immune function prostatic biopsy). Microscopic vascular invasion (MVI) such as immune check point inhibitors. On the other is a major prognostic feature of HCC and is associat- hand, HCC correlated to β-catenin pathway activation ed with advanced tumor stage, distant metastasis and are associated with a poor immune infiltrate and likely adverse outcome 83-85. MVI occurs at the rates of 25%, 214 S.L. Renne et al.

A A

C D

Figure 8. Diagnosis of well differentiated hepatocellular lesions in hepatitis/cirrhotic liver. (A) This biopsy document, in the setting of a chronic steatohepatitic background, the presence of a well differentiated lesion (*); (B) at higher magnification the lesion is characterized by unpaired arteries and pseudo-glandular arrangement of hepatocytes; (C) the nodule showed an diffuse and intense immunoreactivity for GS (non neoplastic parenchyma showed a laminar staining); (D) neoplastic cells are also immunoreactive for CHC. The morpho-phenotypical findings are conclusive for an eHCC.

40%, 55% and 63% in HCC < 3, 3-5, 5-6.5, and > 6.5 References 86 cm . Accordingly immunohistochemical markers sur- 1 Petrick JL, Kelly SP, Altekruse SF, et al. Future of hepatocellu- rogate of MVI are intensively investigated 87,88. Finally lar carcinoma incidence in the united states forecast through The use of stemness-related biomarkers represents 2030. J Clin Oncol 2016;34:1787-1794. https://doi.org/10.1200/ the field where the translation of molecular informa- JCO.2015.64.7412 2 tion on the clinical practice is more advanced. Sev- World Health Organization. Global Hepatitis Report 2017. Ge- neva, Switzerland: World Health Organization 2017. eral stemness-related markers have been identified 3 International Agency for Research on Cancer. GLOBO- and intensively investigated (CK19, EpCAM, CD133, CAN 2018. IARC https://gco.iarc.fr/today/online-analysis- SALL4, NCAM, OV6, CD90, nestin, CD44) and almost map?v=2020&mode=population&mode_population=contin all were associated with a more aggressive clinical be- ents&population=900&populations=900&key=asr&sex=0& cancer=11&type=0&statistic=5&prevalence=0&population_ havior. In particular HCCs with CK19 immunostaining groupearth&color_palette=default&map_scale= quantile&map_ in > 5% of tumor cells show higher recurrence rates nb_colors=5&continent=0&rotate=%255B10%252C0%255D and higher rates of lymph node metastasis 89. (2020) HCC CLINICOPATHOLOGICAL UPDATES 215

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Review

The histomorphological and molecular landscape of colorectal adenomas and serrated lesions

Francesca Galuppini1, Matteo Fassan1,2, Luca Mastracci3,4, Roberta Gafà5, Marcello Lo Mele1, Stefano Lazzi6, Andrea Remo7, Paola Parente8, Alessandro D’Amuri9, Claudia Mescoli1, Fabiana Tatangelo10, Giovanni Lanza5 1 Surgical Pathology Unit, Department of Medicine (DIMED), University of Padua, Italy; 2 Veneto Institute of Oncology IOV - IRCCS, Padua, Italy; 3 Anatomic Pathology, Ospedale Policlinico San Martino IRCCS, Genova, Italy; 4 Anatomic Pathology, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genova, Genova, Italy; 5 Department of Translational Medicine, University of Ferrara, Ferrara, Italy; 6 Department of Medical Biotechnology, University of Siena, Siena, Italy; 7 Pathology Unit, Service Department, ULSS9 “Scaligera”, Verona, Italy; 8 Pathology Unit, Fondazione IRCCS Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy; 9 Anatomic Pathology Unit, “A. Perrino” Hospital, Brindisi, Italy; 10 Department of Pathology, Istituto Nazionale Tumori, IRCCS-Fondazione “G. Pascale”, Naples, Italy

Summary The 2019 WHO classification of digestive system tumors significantly reformed the clas- sificatory definition of serrated lesions of the colorectal mucosa and added new essential diagnostic criteria for both conventional adenomas and hereditary gastrointestinal polypo- Received and accepted: February 24, 2021 sis syndromes. Histopathological examination of colorectal adenocarcinoma precursors lesions represents an important segment of daily clinical practice in a pathology depart- Correspondence ment and is essential for the implementation of current colorectal adenocarcinoma sec- Luca Mastracci ondary prevention strategies. This overview will focus on a schematic histopathological Anatomic Pathology Section, University of and molecular classification of precursor lesions arising within colorectal mucosa. Genova and Ospedale Policlinico San Marti- no, Genova, largo Rosanna Benzi 10, 16132 Key words: colorectal adenomas, KRAS, BRAF, dysplasia, serrated lesions Genova, Italy Tel.: +39 010 555 5954 Fax: +39 010 5556932 E-mail: [email protected] Introduction

Conflict of interest The Authors declare no conflict of interest. Histopathological examination of colorectal adenocarcinoma precursors lesions represents an important segment of daily clinical practice in a How to cite this article: Galuppini pathology department and is essential for the implementation of current F, Fassan M, Mastracci L, et al. The colorectal adenocarcinoma secondary prevention strategies. histomorphological and molecular landscape The 2019 WHO classification of digestive system tumors significantly of colorectal adenomas and serrated lesions reformed the classificatory definition of serrated lesions of colorectal Pathologica 2021;113:218-229. https://doi. org/10.32074/1591-951X-270 mucosa and added new essential diagnostic criteria for both conven- tional adenomas and hereditary gastrointestinal polyposis syndromes. © Copyright by Società Italiana di Anatomia Pato- This overview will focus on schematic histopathological and molecular logica e Citopatologia Diagnostica, Divisione Itali- ana della International Academy of Pathology classification of precursor’s lesions arising within the colorectal mucosa.

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This is an open access journal distributed in accordance Conventional adenomas with the CC-BY-NC-ND (Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 International) license: the work can be used by mentioning the author and the license, General definition but only for non-commercial purposes and only in the original version. For further information: https://creativecommons. Conventional adenomas are benign, premalignant neoplastic lesions org/licenses/by-nc-nd/4.0/deed.en characterized by dysplastic epithelium. They can arise throughout the COLORECTAL ADENOMAS 219

colorectal mucosa, from the ileocaecal valve to the an- A B orectal junction, and can have a pedunculated, ses- sile protuberant, slightly elevated/flat, or depressed appearance. Most lesions are clinically silent, but su- perficial erosion/ulceration is common and, thus, oc- cult bleeding may occur 1. The seminal work of Fearon and Vogelstein pinpoint- ed an adenoma-carcinoma genetic model of colorec- tal carcinogenesis 2,3 in which the normal colon muco- sa epithelium evolves into full-blow adenoma due to the alterations of a small number of driver genes, such as APC, KRAS, SMAD4 and TP53 4,5. C D Clinical picture As mentioned above, most patients are asymptomatic and can for occult bleeding is fundamental for the di- agnosis, especially in screening programs 1. Lesions with large dimension can manifest with evident bleed- ing, abdominal pain and occlusion symptoms. Secre- tory diarrhoea with electrolyte imbalance (McKittrick - Weelock Syndrome) can occur occasionally in distant large polyps.

Histologic elementary lesions Figure 1. Histological aspects of polypoid lesions. Three subtypes of conventional adenomas can be dif- Tubulo-villous adenoma with low-grade dysplasia, magnifi- ferentiated on the basis of villi formation. Despite the cation 100x (A). Tubular adenoma showing a characteristic poor intra-observer concordance in subtyping con- pseudoserrated pattern, magnification 100x (B). Sessile ser- ventional adenomas, this approach is historically ac- rated lesion of the , magnification 200x (C). cepted and used clinically 6,7. However, its prognostic Traditional serrated adenoma of the sigmoid colon, magni- role is not yet well defined. fication 100x (D).

Tubular adenomas Tubular adenomas are the most common phenotype of conventional adenomas detected during population Rare morphological subtypes screening 8. Tubular adenomas are polyps with large- Rare morphological variants of conventional adeno- ly conserved normal crypt architecture, with variable mas have been described. The most common is the elongation of the crypts and an increase in the num- Paneth cell-rich subtype, in which Paneth cells can ber of glands. The epithelium shows enlarged, hyper- be identified in > 20% of adenomas, that is more chromatic nuclei, with different degrees of nuclear common in proximal adenomas and in younger pa- atypia and stratification, with loss of nuclear polarity. tients 9,10. Squamous component (either as morules There is a pseudo-stratification and a de-differentia- or as true squamous metaplasia) is present in < 0.1% tion with decreased numbers of goblet cells. A small of adenomas 11. Clear cells are present in < 0.1% of villous component (< 25%) is acceptable in tubular adenomas 12. adenomas. Histological grading Tubulo-villous adenomas Grading of conventional adenomas is defined using a In tubulo-villous adenomas, > 25% of the architecture two-tiered scoring system that subdivided the lesions is composed of structures resembling small intestinal into low-grade dysplasia (LGD) and high-grade dys- villi, with cellular atypia similar to the tubular ones plasia (HGD). HGD is characterized by marked com- (Fig. 1A-B). plex glandular crowding and irregularity of glands, cri- briform architecture, and intraluminal necrosis. These Villous adenomas architectural features are accompanied by cytological lf > 75% of the adenoma has a villous architecture, it atypia, including substantial loss of cellular polarity, is diagnosed as villous adenoma. markedly enlarged nuclei with prominent nucleoli and 220 F. Galuppini et al.

dispersed chromatin, often with atypical and apical sile serrated lesions (SSLs), and traditional serrat- mitosis 13. Typically, the interobserver concordance is ed adenomas (TSAs). Nowadays, a significant level low, although its prognostic value is fundamental for of confusion surrounding serrated polyps in terms of follow-up and treatment of conventional adenoma. In classification and risk assessment is still present. In a identified during screening, a small area with part, this is due to confusing nomenclature, varied high-grade dysplasia within the lesion is enough to and changing pathology criteria, and uncertainties define the entire high-grade lesion. In other contexts, about prognosis. Although 25% of sporadic colorectal it is useful to report the presence of both dysplasia cancers (CRCs) arise through the serrated molecular pictures. pathway, many clinicians and pathologists still consid- er the serrated lesions as harmless hyperplastic pol- Molecular background yps. New edition of the WHO classification system has Sequencing studies have traced the evolution of most been changed the definitions of these entities and has conventional adenomas and sessile serrated polyps been increased our insight on distributions and clini- into carcinomas through one of two major pathways: cal impact of them. the chromosomal instability pathway or the microsat- ellite instability pathway. In both pathways, approxi- Clinical picture mately 25 genes that are commonly affected by so- Most serrated lesions are asymptomatic and therefore matic mutations become the major drivers of most an incidental finding at endoscopy is frequent. Endo- cancers. These genes include APC and TP53, the scopically they present as sessile polyps, with low risk most commonly mutated tumour-suppressor genes, of bleeding, so the faecal blood-based test is not an and KRAS, PI3KCA, BRAF, and NRAS, the most effective screening method 17. commonly mutated oncogenes. Approximately 85% of HPs of the distal tract are usually small (< 5 mm) and colorectal cancers are thought to evolve from conven- sessile. Proximal HPs and SSLs are poorly defined, tional adenomas through a median of approximate- sessile to flat lesions covered with a mucus cap and a ly 60 mutations per tumor that go beyond the genes rime of debris. Instead, TSA are usually broad-based that are major drivers; this process is referred to as polyps with a surface texture with a coral pattern. the adenoma-to-carcinoma sequence 14. The earli- est changes involve aberrations of the WNT signal- Histologic elementary lesions ling pathway, most frequently altering APC function, The last WHO edition extensively revised the serrated usually by mutations that truncate the APC protein lesions classification. that reduces degradation of beta-catenin allowing it to accumulate and dysregulate WNT signalling 15. The Hyperplastic polyps resultant altered morphology becomes histologically HPs consist of serrated epithelium which can cover the detectable as dysplasia. The enlargement of the dys- upper two-thirds of the funnel-shaped, evenly spaced plastic lesions occurs through accumulation of further crypts with proliferative zones confined to the basis. molecular abnormalities affecting a small number of As it can be considered a diagnosis of exclusion, and key signalling pathways such as KRAS, SMAD4 and the characteristics of SSLs are mainly observed in the PI3KCA. A small subset of adenomas acquires de- deeper parts of the crypts, the orientation of biopsies fects in DNA mismatch repair genes, sporadically due is essential for adequate diagnosis. Two variants of to hypermethylation of the MLH1 promoter, with a very HPs are recognized: the microvesicular type (MVHP) small number of cases of inherited mutations in MLH1 and the goblet cell-rich hyperplastic polyps (GCHP). or MSH2 (or rarely MSH6) in Lynch syndrome fam- GCHPs have fine morphologic alterations, such as ilies, and these may evolve into detective mismatch surface tufting and increased numbers of goblet cells. repair adenocarcinomas. MVHP are easily recognized and characterized by mi- crovesicular epithelial cells with abundant cytoplasm, with stellate lumina inside of the crypts. In the past Serrated lesions and polyps a third subtype was described (the mucin-poor type), but it is no longer considered a separate histotype be- General definition cause these lesions are considered to be caused by Colorectal serrated lesions and polyps are character- regenerative changes in other HPs 16. ized by a serrated (sawtooth or stellate) architecture of the epithelium and gland 16. Serrated polyp is like Sessile serrated lesion an “umbrella term” that includes different histological Sessile serrated lesions (SSLs) have bland cytology with and clinic entity as hyperplastic polyps (HPs), ses- variable amount of goblet cells and cells with microve- COLORECTAL ADENOMAS 221

sicular mucin droplets as HP, and crypts with prominent Traditional serrated adenoma serration. The characterizing feature of SSL is an overall TSA may have different clinical presentations: it may distortion of the crypt profile, probably resulting from al- present in the distal colon as frankly polypoid lesions terations of the proliferative zone. Crypt distortion can be or as sessile, flat lesions in the proximal tract. TSAs present in different forms, such as horizontal growth of are villous polyps with tall cells that contain promi- the crypts along the muscularis mucosa, dilated crypts nent eosinophilic cytoplasm and pencillate nuclei 16. (basal third of the crypt), and/or crypts that have serra- Ectopic crypts, defined as epithelial islets developed tions extending in the basis (Fig. 1C). According to the orthogonally to the main crypt axis and not related to updated WHO criteria, the presence of a single unequiv- the muscularis mucosa, are another typical feature ocally distorted crypt is considered diagnostic for SSL. of this lesion, although it most distinguishes the larg- Mucosal prolapse or herniation through the muscularis er and distally located TSAs (Fig. 1D). In more than mucosae (also known as inverted crypts) and lipomato- 50% of cases, an adjacent precursor lesion (HPs or sis of the lamina propria are phenomena resulted strong- SSLs) could be present. Areas of dysplasia (intestinal ly associated with SLL. The new WHO edition recom- or serrated type) could be found, but no specific sur- mends use of the term sessile serrated lesion vs other veillance guidelines currently exist for these lesions, terms, such as sessile serrated adenoma, sessile serrat- although they may represent a worst progression of ed polyp, or sessile serrated adenoma/polyp. The appli- TSA. The recent WHO edition advised to report these cation of the “at least one crypt” criterion resulted in a 7% cases separately, especially when the high-grade dys- increase in the proportion of serrated polyps classified plasia is documented. as SSLs 18. An additional benefit of this new definition is improved inter-observer agreement compared with Unclassified serrated adenoma previous WHO edition 19. Other crucial factors capable The differential diagnosis between different serrated of improving inter-observer variability are the training of lesions is not always easy, especially as diagnos- expert gastrointestinal pathologists and the orientation tic criteria are still evolving. However, there may be of biopsies. histological pictures that show mixed characteristics between either serrated and conventional polyps. In- Sessile serrated lesion with dysplasia cluded in this group are the recently described ser- Only 4 to 8% of SLLs evolve versus the dysplastic rated tubulovillous adenomas 22,23. At a genetic level, phenotype. Multiple morpholgical pattern of dyspla- polyps may switch phenotype as they accumulate ge- sia may develop, also in the same polyp. At least 3 netic changes, evolving from a serrated pathway to a different morphologic types of dysplasia have been more conventional one, which could be the basis for described: intestinal and serrated pathway and the a spectrum theory starting out with a TSA with serra- minimal deviation dysplasia. Stratification of dyspla- tion evolving into a TSA with conventional dysplasia sia into low-grade vs high-grade is not recommend- and, eventually, to a well-developed conventional ad- ed 16. The intestinal one is similar to the dysplasia ob- enoma. Nevertheless, other studies will be necessary served in conventional adenomas but is almost rare. to provide further connections in our present under- It is characterized by maintaining the expression of standing. Another recently described type of colorec- MLH1, and there seems to be no progression to CRC tal polyps showing mixed morphological features of in these lesions, especially when there is low-grade both conventional adenomas and serrated lesions is dysplasia 20. Serrated dysplasia is more common and the so called superficially serrated adenoma. This pol- is characterized by eosinophilic cytoplasm and small yp shows intermixed histological features with straight crowded glands with pronounced nuclear atypia and adenomatous gland. Unlike low-grade tubular adeno- mitotic activity. Loss of MLH1 staining is infrequent mas, however, proliferative cells localize to the middle and it can be considered an intermediate step for and lower layers of the mucosa while the superficial the evolution in TSA 21. Minimal deviation dysplasia, epithelium exhibits serration. The lesions exhibit nu- which, as the name implies, differs little from the LSS clear accumulation of β-catenin and MYC overexpres- architecture, is typically characterised by the loss of sion, suggestive of WNT pathway activation 24. MLH1. Immunohistochemical analysis for MLH1 is im- portant for determining the presence of clinically im- Molecular background portant dysplasia in SSLs because the loss of MLH1 The serrated pathway is characterized by a continuum staining confirms the presence of dysplasia. However, of genetic and epigenetic alterations that attend polyp it is a sufficient but not necessary condition and the progression, followed by histologic features. The first normal staining pattern can be retained in some cases step of the pathway is the acquisition of a mutation in of manifest dysplasia. a gene such as KRAS or in most cases BRAF. Acti- 222 F. Galuppini et al. vating mutations in BRAF result in widespread meth- Inflammatory bowel disease-associated ylation of CpG islands, representing the a CpG island dysplasia of the colorectum methylator phenotype (CIMP). CIMP results in silenc- ing of many genes, including some tumor suppressor General definition genes such as CDKN2A (which encodes P16) that oc- Dysplasia arising in inflammatory bowel disease (IBD) curs more frequently in TSAs than SSLs, in particular is an unequivocal neoplastic alteration of the colorec- in the advanced lesions with BRAF mutations 21. Hy- tal epithelium that remains confined within the base- permethylation of MLH1 promoter occurs specifically ment membrane in which it originated 16. Cancer risk in SSLs and approximately 75% of SSL with dyspla- in ulcerative colitis (UC) and Crohn disease is almost sia have microsatellite instability (MSI), resulting from equivalent for patients with similar lengths of colon in- this specific hypermethylation. Thus, immunostaining volved 30. In population-based cohorts, UC increases for MLH1 protein can identify dysplasia 25. Progres- the risk of CRC 2.4-fold. Male sex, young age at diag- sion of serrated polyps is associated with activation nosis, coexisting primary sclerosing cholangitis (PSC) of the WNT signaling pathway. TSA shows differences and extensive colitis are adverse factors for develop- from SSL, including more frequent mutations in the ing CRC 31. RNF43-ZNRF3 complex 26,27 and fusions of genes in the R-spondin family (RSPO fusions) resulting in Clinical picture down-regulation of RNF43 28. Colorectal carcinomas No clinical sign or symptoms characterized the dys- (CRCs) originating from serrated lesions typically are plasia in IBD; polypoid lesions may cause bleeding, grouped in three different patterns according to the but it is not an early symptom. Endoscopically, dys- molecular hallmarks: BRAF-mutated CRCs with high plasia is classified according to the SCENIC classifi- CIMP and MSI, mainly located in right colon and char- cation based on their appearance (visible or non-visi- 32 acterized by specific histological features as medul- ble) . Visible lesions are subdivided in polypoid (pe- lary, mucinous and signet ring. They typically show a dunculated or sessile) or non-polypoid (superficial, flat favorable prognosis. The second group of CRCs have or depressed). Other essential parameters to report BRAF mutation, high CIMP but they are MSS. The are the presence of ulceration and the features of the third group is characterized by KRAS mutations and borders. Typically, dysplastic lesions may occur in dif- MSS, although KRAS mutations are infrequent in ser- ferent tracts of the colon simultaneously. rated lesions. Histologic elementary lesions Historically, dysplasia was histologically classified us- ing the Vienna 33 or the Riddell 34 system. The latter is Post-polypectomy endoscopic the most world-wise used and subdivided the lesions surveillance in indefinite, low-grade or high-grade dysplasia com- bining cytological and architectural atypia. The most The new European CRC screening Guidelines (ES- common morphological phenotypes of IBD- related GE) updated the necessity of endoscopic follow-up in dysplasia are the intestinal (or conventional) and ser- patients with one or more polyps that were completely rated types. In low-grade dysplasia the crypts are tu- removed, on the basis of endoscopic and histologi- bular and/or villous or serrated, only with mild crowd- 29 cal risk factors . They recommend that patients with ing. Dysplastic cells usually involve both the crypt complete removal of 1 - 4 < 10 mm in size adeno- and the surface epithelium, but early cases and the mas with low grade dysplasia, irrespective of villous indefinite for dysplasia type show only involvement of components, or any serrated polyp < 10 mm without the crypts, taking the name of “crypt” or “pit” dyspla- dysplasia, do not require endoscopic surveillance sia. In high-grade dysplasia, the epithelium manifests and should be returned to screening. If a scheduled marked cytonuclear atypia with loss of cell polarity screening program is not available, repetition of colo- and mitotic figures, while the architecture becomes noscopy 10 years after the index procedure is recom- more cribriform and packaged. There are less com- mended. Colonoscopy after 3 years is suggested for mon types of dysplasia such as the: i) mucinous sub- patients with complete removal of at least 1 adenoma type; ii) the goblet cell deficient and the iii) terminal ≥ 10 mm or with high grade dysplasia, or ≥ 5 adeno- epithelial differentiation (also known as crypt cell dys- mas, or any serrated polyp ≥ 10mm or with dysplasia. plasia). Among these, the mucinous dysplasia was the A 3 - 6-month early repeat colonoscopy is recom- most investigated shows tubulovillous/villous architec- mended following piecemeal endoscopic resection of ture with tall mucinous cells representing > 50% of the polyps ≥ 20 mm. lesion. It typically shows low-grade dysplastic features COLORECTAL ADENOMAS 223

affecting the crypts with mild nuclear enlargement and kemia, lymphoma, neurofibromatosis type 1, and a hyperchromasia. wide range of other alteration 40. Because patients with LS do not develop large num- Molecular background bers of colorectal adenomas, initially the syndrome The inflammatory microenvironment is the major trig- was called as “hereditary non-polyposis CRC”. Now- ger in the IBD- associated neoplastic process. Tum- adays, this term is avoided and in the face of some- origenic transcriptional factors as NF-kB, the produc- times vague clinical criteria, diagnostics must be tions of cytokines as IL-1β, IL-6 and TNF-α and the based on the germinal identification of the mismatch actions of proteinases damage the cells, initiating repair genes alterations. neoplastic transformation. The frequent multifocality CRC with MSI has typical histological features as the of the lesions reflects this diffuse pre-neoplastic field presence of tumor-infiltrating lymphocytes, Crohn-like in which many factors cooperate in the development peri­tumoral lymphocytic reaction, high histological of dysplasia. The progression of oncogenic mutations grading, mucinous and signet-ring histotype and a that establish the inflammation-dysplasia-carcinoma medullary growth pattern 41. lmmunohistochemistry for cascade in IBD differs from the classic paradigm of the the mismatch repair proteins (MLH1, PMS2, MSH2, sporadic adenoma-carcinoma sequence. Mutations of and MSH6) is a common first step in the screening TP53 occur in 60-90% of IBD-associated CRCs and protocol CRCs for mismatch repair deficiency 42. usually it is the first gene involved in process. Other genes involved are the MYC amplifications and MLH1 Familial adenomatous polyposis 1 and RNF43 mutations. Almost 25% of IBD-related Familial adenomatous polyposis (FAP) 1 is an autoso- CRCs show high tumor mutation burden, correlated mal dominant syndrome caused by pathogenic APC with MSI and occasionally with defects in POLE 35,36. mutations. The disease is typically characterized by > 100 adenomatous polyps in the colorectum, other Genetic adenomatous syndromes extracolonic alterations (including polyps) elsewhere in the gastrointestinal tract, and desmoid tumors 16. of the colorectal tract The onset of colorectal adenomatous polyps usually occurs in the second decade of lite and patients have General definition almost 100% risk of developing CRC by the age of 45 The study of familial cancer syndromes has identified years. For this reason, total colectomy is recommend- key genes which are crucial not only for their role in ed by that age. The prevalence is 1 in 8000-10,000 genetic susceptibility to cancer, but also for the aware- and accounts for < 1% of all CRCs 43. ness they provide into the molecular pathogenesis The large bowel polyps are almost always convention- and classification also in many sporadic cancers 16. al adenomas of different subtype (tubular, tubulovil- Lynch syndrome lous, or villous), grade (low or high), and size; with not Lynch syndrome (LS) is an autosomal dominant dis- important differences with sporadic adenomas. How- ease resulting from constitutional pathogenic muta- ever, characteristic of FAP is the frequent presence of tions affecting the DNA mismatch repair genes most microadenomas identified as monocryptal or oligoc- in MLH1, MSH2, MSH6, and PMS2. ryptal adenomas. LS is characterized by predisposition to a wide variety The fundamental molecular criterion is the presence of cancers as tumors of the colorectum, endometri- of a pathogenic germline APC mutation -and this is um, stomach, small bowel, ovary, gallbladder, hepato- the gold standard for FAP diagnosis, although a small biliary tract, pancreas, urinary tract kidney, brain, and number of cases have undetectable APC mutations prostate 37,38. Sex, age, the involved gene, and history and may be considered as presumed FAP if typical of cancer are the main factors that affect risk of LS clinical features are present and molecular evidence patients. of the other conditions is absent 16. In the Muir-Torre syndrome variant, the previously described internal cancers occur together with seba- Other adenomatous polyposes ceous skin tumor 39 Constitutional mismatch repair Other adenomatous polyposes are a heterogeneous deficiency syndrome (CMMRD) is a recessive dis- group of generally, but not exclusively, inherited con- ease, characterized by biallelic mismatch repair gene ditions characterized by multiple - mutations. The affected individuals develop multiple tous polyps in which LS and FAP were excluded 16 as adenomas in the colorectal tract at a very young age described recently by AIFEG consensus statement and they are prone to develop CRC, brain tumors, leu- (exept for MUTYH associated) 44. 224 F. Galuppini et al.

MUTYH-associated polyposis but the age range is wide. Updated WHO criteria for MUTYH-associated polyposis (MAP) 45 is a constitu- SPS include: at least 5 serrated lesions or polyps tional DNA repair disorder caused by recessively in- proximal to the rectum, all ≥ 5 mm, with 2 or more herited mutations in MUTYH, involved in base exci- that are ≥ 10 mm, or more than 20 serrated lesions sion repair system. The prevalence is approximately 1 or polyps of any size distributed throughout the large in 2000. Individuals with MAP develop multiple adeno- bowel, with at least 5 proximal to the rectum.3 It is matous polyps of colorectum during adulthood, usu- important to note that any serrated polyp subtype (HP, ally in number of 10-100, but hundreds of lesions can SSL, TSA, or serrated adenoma not classified) is in- develop 46. Duodenal polyposis is observed in about cluded in the final polyp count, and that polyp count is 20% of cases, with a concomitant increased risk of cumulative over multiple . duodenal adenocarcinoma. A small proportion of patients with SPS have muta- tions in RNF43, which regulates the WNT pathway. NTHL 1-associated polyposis However, most cases of SPS are not associated with NTHL 1-associated polyposis (NAP) 47 is a consti- any specific genetic variants 49. tutional DNA repair disorder of base excision repair caused by recessively inherited mutations in NTHL1. NAP is thought to be rarer than MAP, although the Special dysplastic lesions of the exact prevalence is unknown. appendix

Polymerase proofreading-associated polyposis Neoplastic lesions of the appendix, especially those Polymerase proofreading-associaled polyposis with a mucinous phenotype, show peculiar clinical (PPAP) is caused by dominantly inherited mutations and histological characteristics that deserve a sepa- in the exonuclease domains of POLD1 and POLE 48. rate treatment. These proofreading mutations cause a deficit in the Epidemiology of these lesions is not well established, correction of mispaired bases during DNA replication. mainly because of the lack of standardized classifica- This mistake leads to a hypermutant phenotype with tions for appendiceal neoplasms. exceedingly numerous point mutations. Colorectal ad- In 2012 the Peritoneal Surface Oncology Group Inter- enomatous polyps occur during adulthood, generally national (PSOGI) adopted a consensus on diagnos- by the age of 50 years. Adenomas and CRC are sim- tic terminology comprehending serrate polyp (with ilar to sporadic tumors but they have a characteristic or without dysplasia), LAMN (low grade appendiceal hypermutant somatic mutation genotype, rich in neo- mucinous neoplasm), HAMN (high grade appendiceal antigens that now appear to be good targets for PD1/ mucinous neoplasm), mucinous adenocarcinoma, PDL1 immune checkpoint inhibitor immunotherapy. mucinous adenocarcinoma with signet ring cells, mu- cinous signet ring cell carcinoma 50,51. Hereditary mixed polyposis syndrome These lesions may present similarly, with acute ap- Hereditary mixed polyposis syndrome is caused by a pendicitis, evident cystic dilatation of appendix, evi- duplication that leads to increased and ectopic expres- dence of abdominal or pelvic mass and, eventually, sion of the BMP antagonist GREM1. Patients develop 16. a variety of colorectal polyps, including conventional HPs and TSAs show the same histological features adenomas, HPs, inflammatory polyps, prolapse-type commons common to the lesions that develop in other polyps, and lymphoid aggregates, with a high risk of parts of the large bowel, as previously described. developing CRC. Low grade Appendiceal Mucinous Neoplasm (LAMN) Other less frequent syndromes as MSH3-associated is defined as a mucinous neoplasm with low grade cy- polyposis, AXIN2-associated polyposis and immune tologic dysplasia and: i) loss of the lamina propria and deficiency-associated polyposis may cause hereditary muscularis mucosae, ii) fibrosis of the submucosa, iii) adenomatous colorectal lesions. “pushing” pattern of growth into the wall (expansile or diverticulum-like), iv) dissection of acellular mucin into Serrated polyposis syndrome the wall or v) mucin and/or neoplastic mucinous epi- Serrated polyposis syndrome (SPS) is a recently de- thelial cells outside the wall of the appendix. scribed condition of largely unknown etiology, charac- High grade Appendiceal Mucinous Neoplasm (HAMN) terized by multiple serrated polyps in colorectum and is a histological entity expected by PSOGI and is de- it is frequently associated with an increased risk of scribed as a mucinous neoplasm with the presence of CRC. high-grade cytological atypia and without infiltrative in- Most patients are diagnosed at 50-60 years of age, vasion. Nevertheless, primary appendiceal mucinous COLORECTAL ADENOMAS 225

neoplasms rarely show at the same time the presence lesion was removed 54. This selection is important to of cytological atypia and the absence of metastatic minimize both the risk of residual cancer and the risk disease presentation. In such cases comprehensive of surgery. histologic evaluation of the appendix is recommended in order to exclude an association with invasive ade- Clinical picture nocarcinoma. Endoscopic assessment of colorectal polyps and le- From a molecular point of view, mucinous appendiceal sions to predict the histologic class (i.e., adenoma vs neoplasia show high prevalence of KRAS mutations. serrated histotype) and determine the presence of Mutations in GNAS and RNF43 genes have been re- features associated with submucosal invasion are im- ported in some cases, even in association with those portant skills for the colonoscopist. of RAS. The main endoscopic classifications based on the surface pattern of the lesions are: i) Narrow Band Im- aging (NBI) International Colorectal Endoscopic Clas- Advanced adenomas sification (NICE), that classifies polyps as type 1 (ser- rated class), type 2 (conventional adenoma) and type This new term was included in the last WHO edition 16 3, which includes lesions with disruption of the sur- and refers to all adenomas ≥ or = 10 mm in size, with face pattern and vessel structure, specific (although tubulovillous or villous architecture, and/or high-grade not sensitive) for submucosal invasive cancer 55; ii) dysplasia or intramucosal adenocarcinoma. Japanese Narrow Band Imaging Expert Team Clas- The endoscopic resection of these lesions represents sification (JNET), a new NBI colorectal magnification the main activity of the screening program to prevent classification in 2014, that maintains NICE types 1 the CRC onset. and 3 but divides type 2 into JNET 2a and 2b, with Crucial is the differentiation between pseudoinvasion 2b features associated with high-grade dysplasia and and invasive cancer (or early pT1 CRC). Pseudoin- superficial submucosal invasion 56; iii) Kudo Pit Pat- vasion is a prolapse of the neoplastic epithelium into tern Classification, that evaluates colorectal polyps the polyp head or deeper, accompanied with traumat- through characterization of the pits, which are open- ic phenomena as hemorrhage, hemosiderin deposit ings for crypts, using a six-tier system. Type I and II and extracellular mucin. This differential diagnosis re- are characteristic of normal, serrated or inflammatory quires an expert panel of gastrointestinal pathologists polyps, whereas pit pattern classes III-V are consid- to ensure a correct interpretation of the morphological ered to indicate dysplastic and malignant changes 57. picture. The most important endoscopic classification systems The recommended management of adenomas with based on morphological features is the Paris classifi- high-grade dysplasia should be endoscopic resection cation which describes 3 major superficial morpholo- alone, because these lesions have no risk of residual gies with subtypes. Lesions are classified as polyps neoplasia in the bowel wall or lymph nodes after com- (type 0 I), which include both pedunculated (0-Ip) and plete endoscopic resection. sessile (0-Is) morphologies; or flat lesions (type 0 II), which consist of slightly elevated (0-IIa), flat (0-IIb), and slightly depressed (0-IIc) morphologies. Lesions Malignant polyp with the third major morphology, excavated (0-III), are rarely seen in the colon 58. General definition The term “malignant polyp” refers to a cancerized Histologic elementary lesions colorectal lesion invading the submucosa. These le- The traditional histological criteria applied in ranking sions are classified as pT1 in the TNM classification the risk of synchronous nodal metastasis are variably system 52. According to the Vienna classification sys- applied and the establishment of reliable criteria for tem, a consensus between Western and Japanese the identification of patients needing surgery is cru- pathologists for classifying gastrointestinal epithelial cial. In addition to resection margin, vascular invasion, neoplasia, the malignant polyp falls under catego- and tumor differentiation, several other histologic fea- ry 5.2 (submucosal carcinoma and beyond) 33. The tures have been proposed. The most promising are prevalence of cancer in colorectal polyps ranges from tumor budding (part of the tumour microenvironment 0.2% to 5% 53. The most important clinical goal is to and involved in epithelial-mesenchymal transition) understand if an endoscopically resected colorectal and those measuring tumor microscopic extension lesion with submucosal invasion requires surgical (i.e. depth, width, and area of the submucosal inva- resection of the colorectal segment from which the sion) (Fig. 2A). Number and type of tumor infiltrating 226 F. Galuppini et al.

A B 1 includes cancer invasion into the submucosa, but is limited to the head of the pedunculated polyp. Level 2 denotes cancer cells reaching the neck of the pe- dunculated polyp and, in level 3, cancer cells invade the stalk. Level 4 indicates cancer cells invading the submucosa below the stalk, but not the muscularis propria, and it is associated with high risk of lymph nodes metastasis 54. All malignant nonpedunculated lesions that by definition have submucosal invasion are classified as Haggitt level 4. Because endosco- pists transect pedunculated polyps through the stalk, it limits the clinical relevance of the Haggitt classifi- cation in assessment of malignant polyps resected endoscopically. Figure 2. pT1 Adenocarcinoma. Invasive pT1 adeno- carcinoma with micropapillary features originating from a Width of infiltration tubulo-villous adenoma, magnification 200x (A). Immuno- The objective approach introduced by Ueno et al. 63 in histochemical staining with anti-pancytokeratins antibody which depth and width beyond the muscularis mucos- highlights a high degree of tumor budding, magnification ae are measured represents the most useful system 200x (B). to report histologically the dimension of malignant pol- yp. if, as previously mentioned, the depth of invasion is fundamental to predict the presence of lymph node metastases, studies on the extent of the carcinoma lymphocytes (TILs) in CRC have been reported to component are not univocal 64,65. However, the main influence tumor behavior and patients’ prognosis but diagnostic protocols recognized worldwide its prog- also nodal metastasis risk in pT1 59. nostic value. Depth of cancer invasion Polypectomy resection margin Accurate measurement of the depth of invasion in ma- The width of any margin between the cancer and the lignant polyps requires specific handling of the spec- resection margin at the polypectomy site is an impor- imen which enables the cut sections to be properly tant histologic risk factor for the presence of lymph oriented for evaluation by the pathologist. For sessile node metastasis and recurrence for both pedunculat- malignant polyps, the Kikuchi classification describes ed and nonpedunculated malignant polyps 66. Europe- the depth of invasion by dividing the submucosa into an guidelines define positive polypectomy margins of three levels (SM1-3). SM1, 2, and 3 denote invasion of malignant polyps when malignant cells are detected cancer into the first one-third, second one-third, and < 1 mm of the margin 67. the deepest one-third of the submucosa, respective- ly 60. In non-polypoid lesions, the submucosa is almost Grade of tumor differentiation never represented in its entirety in the resected spec- It is well established that the risk of lymph-node me- imens, the Kikuchi system has been largely replaced tastasis is higher with high grade tumors vs low-grade by measuring the depth of submucosal invasion with ones. an optical micrometer. An invasion depth of < 1 mm is associated with a very low risk of lymph node me- Lymphovascular invasion tastasis (0-4%), provided that other adverse histologic Lymphovascular invasion, defined as presence of tu- features are absent. An invasion depth of ≥ 1 mm is mor cells within endothelial-lined channels, in the en- associated with a substantial risk of residual disease doscopic resection specimen is an independent risk in the bowel wall or lymph nodes metastasis (10-18%) factor for lymph node metastasis, although the defini- and is generally an indication for adjuvant surgical re- tion used by pathologists varies and the inter-observer section 61. variability is high 68. Depth of invasion in malignant pedunculated polyps is usually classified using the Haggitt system in 4 Tumor Budding levels 62, based on the invasive portion in the head, Tumour budding (TB), defined as a single cell or clus- neck, and stalk of the pedunculated poly. In level 0, ter up to four cells at the invasive front of colorectal dysplastic elements are limited to the mucosa. Level cancer (CRC) 63, is proposed as an additional prog- COLORECTAL ADENOMAS 227

Table I. Histological report of pT1 colorectal carcinoma. Adenocarcinoma of the (low/high grade according to WHO 2019) infiltrating the submucosa, arising in tubular/tubulovillous/villous adenoma with low/high grade dysplasia of the glands/serrated lesion. Histological features of the neoplasia Grading @ Low-grade/High grade sec. WHO 2019 Lymphovascular invasion @ Present/Absent Budding @ Present/Absent (Bd1; Bd2; Bd3 sec. ITBCC 2016) @ Count of buds Adjacent adenomatous component @ Present (conventional/serrated)/Absent Haggitt Classification @ 0; 1;2; 3; 4 Kikuchi Classification @ sm1; sm2; sm3 Depth of infiltration @ Millimeter Width of infiltration @ Millimeter Distance from the deep border of excision @ Millimeter Distance from the lateral border of excision @ Millimeter nostic factor in the 8th edition of the TNM classifica- IARC Perspective on Colorectal Cancer Screening. N Engl J Med tion published by the UICC 52. 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Virchows Arch org/10.1016/s0016-5107(03)02159-x 2019;475:665-668. https://doi.org/10.1007/s00428-019-02603-y PATHOLOGICA 2021;113:230-251; DOI: 10.32074/1591-951X-309

Review

Mesenchymal tumours of the gastrointestinal tract

Marta Sbaraglia1,2, Gianluca Businello2, Elena Bellan2, Matteo Fassan1,2, Angelo Paolo Dei Tos1,2 1 Department of Pathology, Azienda Ospedale-Università Padova, Padua, Italy; 2 Department of Medicine, University of Padua School of Medicine, Padua, Italy

Summary Mesenchymal tumours represent a heterogenous group of neoplasms encopassing benign, intermediate malignancy, and malignant entities. Sarcomas account for approxi- mately 1% of human malignancies. In consideration of their rarity as well as of intrinsic complexity, diagnostic accuracy represents a major challenge. Traditionally, mesenchymal tumours are regarded as lesions the occurrence of which is mostly limited to somatic soft tissues. However, the occurrence of soft tissue tumours at visceral sites represent a well recognized event, and the GI-tract ranks among the most frequently involved visceral loca- tion. There exist entities such as gastrointestinal stromal tumours (GIST) and malignant gastointestinal neuroectodermal tumors that exhibit exquisite tropism for the GI-tract. This review will focus also on other relevant clinico-pathologic entities in which occurrence at visceral location is not at all negligible.

Key words: mesenchymal tumours, sarcoma, gastrointestinal tract, GIST, rare tumours

Introduction

Received and accepted: June 29, 2021 Mesenchymal tumours represent a heterogenous group of neoplasm that include malignant, intermediate malignancy, and benign entities. Correspondence Marta Sbaraglia Sarcomas are extremely rare, accounting for only 1% of malignancies Department of Pathology, Azienda Ospedale- in adults and up to 10-15% of malignancies in the paediatric population. Università Padova, Padua, Italy They are characterised by a global incidence of 30-50 cases per mil- E-mail: [email protected] lion person-years and show a wide anatomic distribution. Rarely, mes- Conflict of interest enchymal lesions may occur in the gastrointestinal (GI) tract. Some of The Authors declare no conflict of interest. these entities occur almost exclusively in the GI tract whereas other sub- types, when located in the GI tract, may assume distinct morphological How to cite this article: Sbaraglia M, features. The majority of sarcomas of the GI tract are represented by Businello G, Bellan E. Mesenchymal gastrointestinal stromal tumours (GISTs), which are the most common tumours of the gastrointestinal tract. Pathologica 2021;113:230-251. https://doi. mesenchymal tumours in the stomach as well as in the small bowel. org/10.32074/1591-951X-309 Benign soft tissue neoplasms show an incidence 100-fold higher than malignant ones. This group includes very common entities such as vis- © Copyright by Società Italiana di Anatomia Pato- ceral lipomas, leiomyomas and haemangiomas, but also extremely rare logica e Citopatologia Diagnostica, Divisione Itali- ana della International Academy of Pathology entities such as glomus tumour and plexiform fibromyxoma. Importantly, both rarity and heterogeneity contribute to limit diagnostic accuracy. The OPEN ACCESS aim of this review is to describe the clinical and pathologic features of the most common mesenchymal tumours occurring in the GI tract, fo- This is an open access journal distributed in accordance with the CC-BY-NC-ND (Creative Commons Attribution- cusing on the application of immunohistochemistry and, when indicated, NonCommercial-NoDerivatives 4.0 International) license: the of molecular genetics to improve diagnostic accuracy and consequently work can be used by mentioning the author and the license, 1,2 but only for non-commercial purposes and only in the original promote the most appropriate therapeutic approach . version. For further information: https://creativecommons. org/licenses/by-nc-nd/4.0/deed.en MESENCHYMAL TUMOURS OF THE GASTROINTESTINAL TRACT 231

Gastrointestinal Stromal Tumours (GIST) dolent 9. On the other hand, NF-1 associated GISTs are typically multicentric, most often arise from the GIST represents a distinctive mesenchymal neoplasm small bowel, and also have a rather indolent course 10. sharing immunomorphologic features with the intersti- Grossly, localised GIST presents as a well circum- tial cell of Cajal. It exhibits an exclusive tropism for the scribed mass of variable size with the cut surface GI tract. In general GIST shows no sex predilection frequently showing foci of haemorrhage. On the other with a peak incidence between the 5th and 6th de- hand, advanced disease often presents as a larger le- cades, although it may occur at any age. The estimat- sion associated with multiple smaller peritoneal nod- ed annual incidence ranges from 11 to 15 cases per ules. million people, and approximately 80% of cases har- On histology, based on morphology, GISTs can be 3 bour activating mutation of KIT and PDGFRA . Paedi- categorised into three general groups: spindle cell atric GISTs represent a clinically as well as a molecu- (70%), epithelioid (20%), and mixed spindle and epi- larly distinct group, most often featuring succinate de- thelioid cell type (10%) 3. hydrogenase (SDH) genetic aberrations. Interestingly, Spindle cell GIST is composed of uniformly eosino- as demonstrated by both surgical and autoptic series, philic spindle cells with indistinct cell borders orga- submillimetric gastric GISTs (so-called microGISTs) nized in short intersecting fascicles (Fig. 1). The neo- are found in approximately 20% of the general popu- plastic cells have light eosinophilic cytoplasm, often 3 lation . Notably, the vast majority of these lesions do with indistinct cell borders. Nuclei tend to be oval and not progress into clinically meaningful lesions, but un- uniform in appearance, often with vesicular chroma- dergo instead regressive changes and calcification 4. tin. A peculiar feature, most often observed in gastric Notably, owing to its molecular characteristics GIST neoplasms, is represented by the presence of striking represents a still unsurpassed model of molecular tar- juxtanuclear cytoplasmic vacuoles. The presence of geted therapy of solid tumours. nuclear palisading is seen in a minority of cases and, The most common locations from which GIST may as it is frequently observed in both smooth muscle arise are the stomach (50-60%), followed by the small and neural tumours, may represent a potentially mis- intestine (20-30%), the large bowel (5%) and the oe- leading feature. Microcystic stromal degeneration, fi- sophagus (5%). More rarely, GISTs primitive of the brosis as well as stromal haemorrhage may represent peritoneum are reported, although most often they a prominent finding in some cases. represent pedunculated masses detached from the outer visceral wall. Important prognostic factors are Epithelioid GIST is composed of cells exhibiting abun- tumour size, mitotic activity and anatomic site, which dant eosinophilic or clear cytoplasm (Fig. 2). Nuclei are the basis for the prediction of risk of aggressive tend to be round-to-ovoid and uniform. In comparison biologic behaviour. Moreover, the risk of abdominal with spindle cell GIST, tumour cells tend to exhibit a dissemination is dramatically increased by intraopera- nested pattern of growth. Some cases may exhibit a tive tumour rupture (up to 90%). Importantly, all the aforementioned features have been incorporated into various risk assessment schemes 5-7. Localised disease is principally treated surgically, with the addition of imatinib as adjuvant therapy in high- risk patients. Advanced and metastatic disease cur- rently requires a therapy with three consecutive lines of receptor tyrosine kinase (RTK) inhibitors (imatinib, sunitinib, and regorafenib) all directed against the action of mutant KIT and PDGFRA. Importantly, the mutational status of these genes predicts response to RTK inhibitors and assumes prognostic value 8. SDH-deficient GIST represents a clinically and patho- logically distinct subgroup. It often shows propensity to arise from the stomach, to occur in children and young adults and, outside the context of Carney-Strat- akis syndrome, exhbits marked female predominance. Compared to the classic form of GIST, it shows a high- Figure 1. Spindle cell GIST. The neoplasm is composed of er risk of loco-regional lymph nodes spreading, even short fascicles of uniform spindle cells, with oval nuclei, eo- though the clinical behaviour remains distinctively in- sinophilic cytoplasm and poorly circumscribed borders. 232 M. Sbaraglia et al.

relatively often may feature a nesting, paraganglioma- like growth pattern. Prominent myxoid change can be seen rarely. Nuclear pleomorphism is not a typical fea- ture of GIST, although it can be observed in approxi- mately 2% of cases. Abrupt morphologic progression to high-grade pleomorphic sarcoma can also be rarely observed (so called dedifferentiated GIST) 12. SDH-deficient GIST not only represents a clinically distinctive entity, but also shows relatively peculiar morphologic features. A distinctive multinodular pat- tern of growth is often seen associated with a predom- inantly epithelioid morphology (Fig. 3) 13. Even if there are cases in which mitotic activity is re- markably high, in most GIST it tends to be low. In fact, mitotic count (which represents a major prognostic 2 2 Figure 2. Epithelioid GIST. The neoplasm shows round, epi- determinant) is assessed on 5 mm . The use of mm thelioid cells with oval shaped nuclei and eosinophilic to clear instead of High-Power Fields (HPF) has the advan- cytoplasm. The growth pattern is usually described as nested. tage of overcoming inconsistencies due to the use of microscope with variable aperture of the oculars. Not infrequently pathologists are confronted with biop- sies originating from post-treatment GIST. Depending on the level of response to RTKs inhibitors variable amounts of viable cells can be seen and relatively of- ten most of the tissue can be merely represented by diffusely hyalinised fibrotic tissue. In consideration of the current clinical as well as ther- apeutic implications immunophenotypic analysis has gained a major diagnostic role. Most cases are KIT (CD117) immunoreactive (Fig. 4A), even if has to be recognized that there are lesions with typical cytoar- chitectural features of GIST lacking KIT expression. This phenomenon occurs in 5-7% of cases overall and in up to 18% of gastric GIST. A significant pro- portion of KIT-negative cases contains mutations of the PDGFRA gene and tends to exhibit an epithelioid morphology 11. In this cases expression of PDGFRA Figure 3. SDH-deficient GIST. This category of GIST shows is commonly seen. The pattern of KIT expression is a typical multinodular pattern of growth. usually cytoplasmic and diffuse, however, up to half of cases will also show a dot-like accentuation of the staining. More rarely a dot-like pattern is seen in the absence of diffuse cytoplasmic staining. Approxi- striking “plasmacytoid” appearance. Epithelioid GIST mately 50% of KIT negative GIST actually express 14,15 arises most often in the stomach and are frequently DOG1 (anoctamine-1) (Fig. 4B) . In addition to KIT associated with PDGFRA gene mutations 3,11. Mixed and DOG1, GIST frequently expresses CD34 in 60- 70% of cases, smooth muscle actin in 30% of cases. cell type GIST may feature abrupt transition between Desmin and cytokeratin can be seen in less than 2% spindle cell and epithelioid areas or as an alternative of cases. SDHA and SDHB immunostaining is cur- the two cell types may be intermingled. rently regarded as extremely helpful in recognizing In approximately 10-20% of cases (almost exclusively SDH-deficient GIST. In fact, whatever the mutations in the small bowel), hyaline or fibrillary brightly eo- of SDH subunits loss of SDHB expression is seen sinophilic structures known as “skeinoid fibres” can (Fig. 5) 16. On the other hand, loss of SDHA predict be seen. These structures appear to be composed the presence of mutations in the SDHA gene. Fol- of nodular tangles of collagen and, typically, exhibit lowing therapy with TRK inhibitors non-canonical im- PAS positivity. GIST arising in the small bowel also munophenotypes can be observed, such as diffuse MESENCHYMAL TUMOURS OF THE GASTROINTESTINAL TRACT 233

A B

Figure 4. GIST. Diffuse cytoplasmatic immunopositivity for CD117 (A) and DOG1 (B) is usually observed.

factor (Steel factor) and platelet-derived growth factor A. Downstream oncogenic signalling for both KIT and PDGFRA involves the RAS/MAPK and the PI3K/AKT/ mTOR pathways. Mutations can be deletions, inser- tions and missense mutations involving exon 11 of the KIT gene (encoding for the juxtamembrane domain of the KIT receptor) in approximately 70% of GIST; exon 9 of KIT (encoding for the extracellular domain of the receptor) in less than 10%; exon 13 and 17 of KIT (encoding for the intracellular ATP-binding pocket and activation loop domains, respectively) in a small subset of cases. Approximately 10% of GIST harbour PDGFRA gene mutations involving exons 12, 14 and 18, with 70% being represented by the exon 18 D842V mutation. The D842V mutation is known for making Figure 5. SDH-deficient GIST. In this variant of GIST, the GIST primarily resistant to available RTK inhibitors. loss of expression of proteins forming the SDH complex However, the recently approved avapritinib seems to (SDHB) is observed. Note the positive built-in control repre- be effective also on this type of mutation. sented by lymphocytes and endothelial cells. Approximately 10-15% of GIST is wild type for both KIT and PDGFRA 18. They represent a family of tumours with distinctive molecular pathogenesis and, to some extent, different natural histories. Their classification expression of myogenic or epithelial differentiation is rapidly evolving as of today, and one may identify: markers 12. 1) SDH deficient GIST; 2) NF1-related GIST; 3) oth- From a genetic standpoint, GIST represents a rela- ers, including those with the BRAF V600E mutations. tively heterogeneous and complex group of lesions. Approximately one half of wild type GIST (WT GIST) Gain-of-function mutations of the oncogenes located are marked by alterations involving the SDH complex, on chromosome 4 (4q12) encoding for the type III which plays a key role in the mitochondrial respiratory receptor tyrosine kinases KIT and PDGFRA can be cell function 9. A group of them includes “paediatric” found in approximately 80% of cases. With exceed- GIST and can be associated with the Carney triad that, ingly rare exceptions they are mutually exclusive and when full blown, is characterised by the concomitant result in the constitutive activation of either KIT or occurrence of GIST, pulmonary chondromas and para- PDGFRA 17. Normally, KIT and PDGFRA, are activat- gangliomas. On the other hand, a group of SDH-defi- ed by binding of their respective ligands, i.e., stem-cell cient GIST carries mutations of the SDHA, SDHB or 234 M. Sbaraglia et al.

exhibits NF1 gene mutations that may be somatic or more frequently germline 10. As this happens in ab- sence of clinical evidence of Type 1 neurofibromatosis, they possibly represent examples of subclinical forms of the syndrome. Using a Massive Parallel Sequenc- ing approach an ETV6-NTRK3 gene fusion has been recently detected in a quadruple-negative GIST 20. As WT GIST tend to be not responsive to tyrosine kinase inhibitors (but keeping in mind that this alteration is very rare) molecular therapy targeting NTRK has been successfully applied 21.

Neoplasm with fibroblastic/ myofibroblastic differentiation Figure 6. Desmoid fibromatosis. This tumour is composed of long fascicles of uniform, cytologically bland spindle cells, Desmoid fibromatosis set in a collagenous background. Desmoid fibromatosis represents a locally aggressive, non-metastasising myofibroblastic neoplasm that may occur at extra-abdominal (60%), abdominal (25%) and intra-abdominal anatomic sites (15%) 3. Intra-ab- dominal lesions are usually located in the pelvis or in the mesentery. Mesenteric desmoids can be sporadic or be associated with a variant of familial adenoma- tous polyposis syndrome (Gardner’s syndrome). This syndrome is defined by the presence of synchronous of metachronous multiple adenomatous polyps, oste- omas, epidermic cysts, and desmoid fibromatosis 22. Intra-abdominal desmoid fibromatosis occur predomi- nantly in young adults. Macroscopically, tumours appear as a solitary mass with diameter ranging from 5 cm to 10 cm. The cut surface is whitish and hard 3. Desmoid fibromatosis typically shows poor circum- scription often with infiltration of the surrounding soft Figure 7. Desmoid fibromatosis. Nuclear expression of tissues. Microscopically, the tumour is composed of beta-catenin is a key diagnostic clue. a monotonous spindle cell proliferation arranged in long sweeping fascicles set in a collagenous back- ground (Fig. 6). A useful diagnostic clue is repre- sented by the fact that neoplastic cells often exhibit SDHC units of the SDH complex, and may be related a relatively regular distribution without nuclear over- to the Carney-Stratakis syndrome, a dominant autoso- lapping. Both cellularity and amount of collagenous mal disorder represented by association of GIST and stroma can be variable. Nuclear atypia is generally absent and mitotic figures may vary in number but are paragangliomas 13. WT GIST can occur in the context usually not numerous. Myxoid change of the stroma of NF-1, wherein the mutation of the NF1 gene leads may occasionally occur. Desmoid fibromatosis exhib- to loss of neurofibromin, and consequent activation its nuclear immunopositivity for beta-catenin (Fig. 7) 19 of the RAS pathway . Finally, the remaining SDHB- and multifocal positivity for smooth muscle actin 3. positive WT GIST are probably a basket of different Nuclear expression of beta-catenin protein repre- conditions: some were reported to have the V600E sents the consequence of mutations of the CTNNB1 mutation of BRAF or, more rarely, of HRAS, NRAS, gene that occurs in approximately 85% of sporadic PI3K. Recent experience would indicate that a signifi- cases. In Gardner syndrome-associated cases the cant proportion of so-called “quadruple negative” GIST same phenomenon is determined by the mutation of MESENCHYMAL TUMOURS OF THE GASTROINTESTINAL TRACT 235

antagonists, tyrosine-kynase inhibitors, low dose cy- totoxic chemotherapy) has been suggested for pro- gressive lesion. More recently, in consideration that in a significant subset of patients repeated surgery may lead to increased recurrence rates, whereas spontaneous regression is by contrast observed, a “wait and see” approach has been suggested unless clear clinical progression is observed 24.

Inflammatory Myofibroblastic Tumour Inflammatory Myofibroblastic Tumour (IMT) is a rare, locally aggressive and rarely metastasising mesen- chymal tumour of young adults, composed of myofi- broblasts and fibroblasts set in an inflammatory back- ground that includes plasma cells, lymphocytes and/ or eosinophils in variable amounts. IMT is most often observed in the lungs although it Figure 8. Inflammatory Myofibroblastic Tumour may rarely may occur anywhere in the body, including the ab- occur in gastrointestinal tract. In this case neoplastic cells dominal soft tissue, mesentery, omentum, and the GI involve the muscolaris mucosae of the small bowel. tract (Fig. 8) 25. Three main morphologic patterns are recognised 3. The “myxoid pattern” is characterized by loosely ar- ranged plump or spindled myofibroblasts embedded in an oedematous myxoid background rich in plasma cells, lymphocytes and eosinophils to the extent that it can mimic granulation tissue (Fig. 9). The “hyper- cellular pattern” consists of a compact proliferation of spindle cells associated with variable myxoid/collage- nous stroma and a rich inflammatory infiltrate. Finally, the “hypocellular fibrous pattern” is characterised by low cellularity, collagenous stroma and sparse inflam- matory cells. One or more of these patterns may be identified in a single IMT. Dystrophic calcifications and osseous metaplasia represent rare findings in IMT. Epithelioid inflammatory myofibroblastic sarcoma (EIMS) is a clinically aggressive form of IMT, charac- terized by epithelioid tumour cells featuring vesicular nuclei and prominent nucleoli, set in a myxoid stro- ma; neutrophils are often present 26. It occurs pre- Figure 9. Inflammatory Myofibroblastic Tumour. This tu- dominantly intra-abdominally. Immunohistochemically, mour is composed of spindle cells, set in a fibrous stroma IMT may show variable positivity for SMA (Fig. 10A), associated with a prominent inflammatory infiltrate, rich in calponin and desmin. Cytokeratins are focally posi- plasma cells, lymphocytes and eosinophils. tive in about 30% of cases. More than 50% of IMTs present ALK positivity (Fig. 10B), which is related to ALK gene rearrangement. Interestingly, ALK immuno- histochemical pattern varies according to ALK fusion the APC gene. A sharp debate surrounds the poten- partner: RNBP2-ALK generates a nuclear membrane tial prognostic meaning of CTNNB1 gene mutations pattern 27, RRBP1-ALK a perinuclear accentuated pat- (it has been suggested that the 45F mutation associ- tern 28, and CLTC-ALK a granular cytoplasmic one 29. ates with higher rates of local recurrences) that is still The diffuse cytoplasmic pattern is the most frequent unsettled 23. In the past, the therapeutic approach to and is associated to many other ALK fusion variants. desmoid fibromatosis has been mostly represented A subset of IMT present ROS1 gene rearrangements by surgical excision. The use of radiotherapy as well and are usually associated with immunohistochemi- as of systemic treatments (which include hormone cal cytoplasmatic expression of ROS1 30. Five to 10% 236 M. Sbaraglia et al.

A

Figure 11. Solitary fibrous tumour. This neoplasm shows B spindle cells organised in a “patternless” pattern, set in a collagenous background, and showing thin-walled, haeman- giopericytoma--like vessels.

Figure 10. Inflammatory Myofibroblastic Tumour. Neoplas- tic cells show variable positivity for smooth muscle actin (A) and in half of cases expression of ALK (B).

of IMT may feature the rearrangement of the NTRK3 Figure 12. Solitary fibrous tumour. Expression of STAT6 gene 31. As is the case of ALK and ROS1, NTRK3 rep- represents the most specific and sensitive diagnostic marker. resents a druggable biomarker. Approximately 25% of extrapulmonary IMT may recur, but metastases are rare (less than 5%). ALK-nega- described in different parts of the GI tract, liver and tive IMTs seem to be related to a higher frequency pancreas included 35-38. of metastasis. However, reliable prognostic indicators for IMT have not been validated yet. As mentioned Microscopically, SFT are composed by a patternless above, epithelioid IMT have a more aggressive clini- proliferation of spindle cells embedded in a collage- cal behaviour 32. nous background featuring distinctive branching, thin- walled blood vessels organized in a “staghorn” con- Solitary Fibrous Tumour figuration (so called haemangiopericytoma-like vas- Solitary fibrous tumour (SFT) is a rare neoplasm that cularization) (Fig. 11) 38. Cellular varation is typically even if originally reported as pleural-based tumour, observed in classic examples. Three additional SFT can actually occur at any anatomic site. Approximately subtypes are recognised: fat-forming SFT is charac- 80% of cases are deep-seated 33,34. SFTs have been terised by the presence of mature adipose tissue 39; MESENCHYMAL TUMOURS OF THE GASTROINTESTINAL TRACT 237

sa or restricted to the mucosa that can be ulcerated. Middle-aged adults are typically affected, with a slight female predominance 49. Small lesions are asymptomatic and most often dis- covered incidentally. Large masses are associated with abdominal pain and bleeding due to mucosa ul- ceration. Intussusception is a common presentation for small intestine tumours 49. Tumours can appear sessile or polypoid, and range in size from few millimetres to large masses. Histologi- cally, inflammatory fibroid polyp is a hypocellular lesion composed short spindled and stellate cells showing fine chromatin, small or indistinct nucleoli, and scant eosinophilic cytoplasm (Fig. 13). The stroma may be myxoid or collagenous, with a prominent mixed inflam- Figure 13. Inflammatory fibroid polyp. The neoplasm is matory infiltrate, often rich in eosinophils. Concentric composed of a proliferation of bland, spindle cells, associ- fibrosis (onion-skin) distributed around blood vessels ated with an eosinophilic-rich inflammatory infiltrate. represents a distinctive finding 3. Immunohistochemically, neoplastic cells express CD34 and rarely smooth muscle actin 50,51. KIT, DOG1, desmin, S100, SOX10, and keratins are all negative. giant cell-rich SFT features multinucleated giant cells The differential diagnosis includes IMT, leiomyoma, 40 often lining angiectoid spaces ; dedifferentiated SFT and plexiform fibromyxoma. show transition from classic SFT to a high-grade sar- Most cases, particularly when located in the small 41 coma . bowel, harbour PDGFRA gene mutation 49,51,52. Ex- Immunohistochemically, SFT is strongly positive for on 18 mutations, usually c.2525A > T (p.D842V) are CD34, bcl-2 and CD99 (70% of cases). STAT6 is the associated with a gastric location, whereas exon 12 most specific and sensitive marker (Fig. 12), being mutations are almost identified in small intestine tu- 42 expressed in almost all SFTs . STAT6 expression is mour 53. Only rare cases are associated with germline related to a specific recurrent gene fusion involving PDGFRA gene mutations 54. Inflammatory fibroid pol- STAT6 and NAB2 genes 43. yp is a benign tumour. Local recurrences and distant Approximately 12-22% of cases of SFT are malignant. metastasis have not been reported. Malignancy-associated features are high cellularity, nuclear pleomorphism, necrosis and more than 4 Plexiform Fibromyxoma mitoses per 10 HPF 44,45. The metastatic risk can be Plexiform fibromyxoma, also referred as plexiform an- assessed on the basis of mitotic count (2 or more mi- giomyxoid myofibroblastic tumour, is a benign mes- toses per mm2), patient age (55 years or more) and enchymal tumour arising almost exclusively in the tumour size 46. However, the absence of these criteria stomach. The tumour occurs in the antrum and pyloric does not exclude a possible aggressive behaviour and region of the stomach, but duodenum may be very no SFT should be regarded as benign 38. rarely involved 3,55. Age range is rather broad, and in- SFT may enter in differential diagnosis with GISTs, clude childhood with equal distribution in male and fe- schwannomas, benign smooth muscle tumours and males. Clinically, gastrointestinal bleeding due to mu- monophasic SS. Immunohistochemical analysis for cosal ulceration, weight loss and pyloric obstruction STAT6 is usually sufficient to assess the correct di- may be observed. agnosis. Macroscopically, the tumour appears like as gelati- nous or haemorrhagic multinodular mass, ranging Inflammatory Fibroid Polyp in size from few centimetres to large masses. The Inflammatory fibroid polyp is a benign neoplasm fea- tumour is typically located in the muscularis propria turing a polypoid configuration, composed of a hy- and protruding into the serosa 55,56. Histologically, the pocellular fibroblastic proliferation associated with a neoplasm features a distinctive plexiform pattern of variably prominent inflammatory infiltrate, rich in eo- growth. The nodules are composed of bland oval to sinophils. It most often arises in the stomach followed spindled cells with indistinct cytoplasm, set in a myx- by the ileum 47,48 but may involve the whole digestive oid, fibromyxoid or collagenous matrix associated tract. The tumour is typically located in the submuco- with a rich thin-walled capillary network (Fig. 14). In- 238 M. Sbaraglia et al.

ferential diagnosis includes SDH-deficient GIST, plexi- form schwannoma and inflammatory myofibroblastic tumour 55,56. KIT and PDGFRA mutation have not been reported. Molecularly, MALAT1-GLI1 fusions and GLI1 polysomy have been identified in a subset of tumours resulting in GLI1 overexpression 57. Plexiform fibromyxomas are benign lesions with no reports of recurrence or metastasis 55,56 however in some cases massive gastric bleeding has proved fa- tal.

Neoplasm with adipocytic differentiation

Lipoma Lipomas are the most common benign soft tissue tu- mours, most often arising in the soft tissues of the ex- tremities and trunk 32. Lipomas more rarely may also occur in the submucosa or subserosa along the whole Figure 14. Plexiform fibromyxoma. Proliferation of bland, gastrointestinal tract; the and the ascending spindle-shaped cells, set in a fibromyxoid stroma that shows colon being the most common sites followed by the il- 3 a network of thin-walled capillary-size blood vessels. eum, stomach and oesophagus . Rarely, gastrointes- tinal lipomas may be intramucosal, such cases may rarely be associated with Cowden syndrome 58. The reported incidence of large bowel lipomas is between 0.2% to 4.4% of endoscopies, with possible slight fe- male predilection and a peak incidence in the 6th de- cade 59. Signs and symptoms of GI lipomas are linked to size (highly variable from 2 to > 10 cm) and location. Most patients are asymptomatic, although abdominal pain, and intestinal obstruction have been reported 59. The histological appearance of lipomas consists in a proliferation of mature adipose tissue, with no cyto- logic atypia (Fig. 15) 3. Larger lesions may be accom- panied by overlying mucosal ulceration. Lipoma subtypes, such as angiolipomas have also been described to occur in the GI tract 60. Molecularly, benign lipomas harbour simple genetic alteration in about 75% of cases, among which the most common is HMGA2 gene rearrangements 61. Surgical and en- Figure 15. Lipoma. The tumour is composed of mature fat doscopic excision are the treatment of choice for large tissue without atypia and variation in size of adipocytic cells. or symptomatic gastrointestinal lipomas. Fibrous septa and histiocytes may be sometime observed. Well Differentiated/Dedifferentiated liposarcoma Liposarcomas are the most common mesenchymal malignancy and are currently classified according to creased cellularity is observed in those rare tumours the WHO 2020 as atypical lipomatous tumour (ALT)/ occurring in the duodenum. Nuclear atypia is usually well-differentiated liposarcoma (WDLPS), dediffer- entiated liposarcoma (DDLPS), myxoid liposarcoma absent and mitotic activity is generally low 56. Neo- (MLPS), pleomorphic liposarcoma (PLPS) and pleo- plastic cells express SMA and occasionally are immu- morphic myxoid liposarcoma 32. While the primary site nopositive for desmin and h-caldesmon whereas KIT, of occurrence is usually the soft tissue of the limbs, DOG1, ALK and S100 are consistently negative. Dif- trunk or retroperitoneum, primary gastrointestinal lipo- MESENCHYMAL TUMOURS OF THE GASTROINTESTINAL TRACT 239

Figure 16. Well Differentiated Liposarcoma. The tumour Figure 18. Well Differentiated Liposarcoma. Strong MDM2 is composed of atypical lipomatous proliferation with focal nuclear positivity is consistently observed in neoplastic cells nuclear atypia intersected by thick fibrous septa with hyper- including lipoblasts. chromatic atypical stromal spindle cells.

with symptoms related to anatomic location, which include dysphagia, cough, vomit, foreign body sensa- tion and weight loss. Albeit being the most common subtype of liposar- coma, in GI tract WDLPS seems to show a lower incidence than its dedifferentiated counterpart. Both are genetically driven by amplification of MDM2 and CDK4 genes that lead to the overexperession of the proteins thereof 64. Grossly the appearance of WDLPS strongly depends on the proportion of lipomatous and fibrous compo- nents and varies from a uniformly yellow mass, similar to a lipoma, to a white-greyish lobulated mass on cut surface. On histology, WDLS is composed of a mature lipoma- tous proliferation intersected by thick fibrous septa, Figure 17. Well Differentiated Liposarcoma. Numerous li- with variation in cell size and at least focal nuclear poblasts are rarely observed in WDLPS sometimes they can atypia in adipocytic and/or stromal cells (Fig. 16). Mi- be completely absent. totic figures are rare. Lipoblasts may be present in variable amounts (from many to none) (Fig. 17); how- ever, this feature is not required for the diagnosis of WDLS. Rarely, foci of heterologous metaplasia may sarcomas are much rarer with incidence at autopsy be observed 32. As mentioned MDM2 overexpression reported to be between 0.1% and 5.8% 62. Primary is consistently observed (Fig. 18) gastrointestinal liposarcomas show slight male pre- Dedifferentiated liposarcomas (DDLPS) is character- dominance and tend to occur in middle-aged individu- ised by a broad morphological spectrum. It is defined als. Liposarcomas also display a higher tropism for by the the presence of abrupt transition from well dif- the submucosal layer and muscularis propria of the ferentiatiated liposarcoma to high grade non-lipogen- oesophagus, closely followed by the stomach, small ic sarcoma (Fig. 19). Most often the dedifferentiated and large intestine 63. When arising in visceral sites, component is pleomorphic however it can be also they usually present as already voluminous polypoid composed by a monomorphic spindle cell prolifera- masses with slow growth. Patients usually present tion. Areas of heterologous differentiation (most often 240 M. Sbaraglia et al.

Figure 19. Dedifferentiated liposarcoma. The dedifferenti- Figure 21. Leiomyoma. Strong and diffuse immunopositiv- ated component is composed of spindle and pleomorphic ity for desmin is usually observed. cells set in a fibromyxoid stroma.

Epithelioid pleomorphic liposarcoma Epithelioid pleomorphic liposarcoma (EPL) repre- sents a subset of pleomorphic liposarcomas in which an epithelioid morphology predominates 67. Diagnosis is based on the detection of pleomorphic lipoblasts that unfortunately may be minimally represented. A significant proportion of cases occur in the GI tract of adults. Clinically EPL is a high-grade sarcoma with a dismal prognosis.

Smooth Muscle Neoplasms

Leiomyoma Leiomyoma is a rare benign mesenchymal tumour Figure 20. Leiomyoma. The neoplasm is composed of non- showing smooth muscle differentiation and represents atypical, eosinophilic, spindle cells, with blunt ended nuclei. approximately one third all mesenchymal neoplasms of the GI tract. Leiomyomas predominantly occur in the oesophagus, colon, and rectum and rarely arise 68 myogenic) may also be found 32. Multivisceral surgical in the stomach and small intestine . Incidental small resection is the mainstay of treatment. nodules, less than 7 mm, are recognised in oesopha- 69 Although DDPLS and WDLPS are the most common gogastric resections done for other reason . Most subtypes of liposarcomas described in the GI tract, a cases are sporadic, but exceedingly rare cases occur few case reports also described the exceptional oc- in association with Alport’ syndrome. currence of myxoid liposarcomas (MLPS) 65,66. Myxoid Histologically, the tumour is composed of fascicles of liposarcoma is composed of cytologically bland spin- spindled cells with blunt-ended nuclei set in eosino- dle to ovoid cells associated with monovacuolated li- philic fibrillary cytoplasm (Fig. 20) 32. There may be fo- poblasts, set in a myxoid stroma with prominent plexi- cal nuclear atypia, but necrosis and mitotic activity are form capillary network. Myxoid liposarcoma is MDM2 not detected. Detection of any mitotic activity should negative and characterized genetically by a DDIT3 in fact prompt consideration of malignancy. Neoplastic gene rearrangement that represent a useful diagnos- cells express desmin, SMA, h-caldesmon, and cal- tic confirmatory finding. ponin (Fig. 21). The main differential diagnosis is with MESENCHYMAL TUMOURS OF THE GASTROINTESTINAL TRACT 241

GI tract it most commonly occurs in the small intestine (40%) and colorectum (40%) and more rarely in the stomach (10%) and oesophagus (10%) 70,71. In the an- orectum and in the oesophagus LMS is relatively more common than GIST. Leiomyosarcoma occur typically in adult patients (peak incidence is in the sixth de- cade) with the exception of gastric LMS which tends to arise in younger patients (median age: 37 years) 71. Interestingly visceral LMS, particularly those arising in children, may be related to immunosuppression and aetiologically linked to Epstein-Barr Virus infection 72. In oesophageal and gastric cases, a slight male prev- alence has been described. Leiomyosarcomas may present as polypoid or lobulated intraluminal tumours or as a large solid and necrotic mass 71,73-75. Figure 22. Leiomyoma. KIT immunostaining highlights the Microscopically, LMS is composed of long intersect- hyperplasia of the interstitial cells of Cajal. ing fascicles of spindle cells featuring abundant eo- sinophilic cytoplasm and blunt ended nuclei (Fig. 23). Moderate to severe nuclear atypia and in some cases overt pleomorphism are observed. Mitoses tend to be numerous, although rare tumours with low mitot- ic counts have been reported 71,73. Necrosis may be present. Immunohistochemically the vast majority ex- press smooth muscle actin, whereas 70-80% of LMS stain with desmin and/or H-caldesmon 70. Importantly about 30% of LMS may exhibit multifocal positivity for cytokeratin and CD34 74,76. The main differential diag- nosis is with GIST, the clinicopathologic features of which have already been described. EBV-associated smooth muscle tumours can show a range of histological appearances, from lesions mim- icking leiomyomas to lesions composed entirely of small round to ovoid blue cells. These are positive for SMA, with variable desmin positivity. All show diffuse, strong staining for EBV-encoded small RNA (EBER), Figure 23. Leiomyosarcoma. The tumour is composed of which is not seen in conventional leiomyosarcoma. long fascicles of eosinophilic, spindle cells with blunt ended EBV-related LMS should be considered in immuno- nuclei. suppressed patients or in patients with multiple syn- chronous or metachronous tumours 77. Schwannoma may also enter the differential diagnosis. In the GI tract, schwannomas tend to be cellular, with peripher- GIST, which may show similar morphological features. al lymphoplasmacytic aggregates, and by immunohis- However, KIT (CD117) is consistently negative in leio- tochemistry they are diffusely positive for S100, and myomas. Focal expression of DOG1 can observed in negative for SMA and desmin. smooth muscle tumours, therefore representing a po- Leiomyosarcomas are aggressive neoplasms, with up tential diagnostic pitfall, as is the presence in leiomyo- to 80% local recurrence rate and 55-70% metastatic mas of hyperplasia of the interstitial cells of Cajal (KIT rate. Tumour size > 5 cm appears to be a negative positive) (Fig. 22). Leiomyomas of GI tract are benign prognostic factor. Interestingly, in the GI-tract, mitotic tumours, with no risk of recurrence or distant spread. counts and nuclear atypia seem not to correlate with outcome 70,71,73. Different classes of leiomyosarcomas, Leiomyosarcoma (LMS) based on gene expression, have been proposed as- Leiomyosarcoma is an extremely rare malignant neo- sociated with different outcomes; however, these data plasm showing smooth muscle differentiation. In the are not currently used for clinical decision making 78,79. 242 M. Sbaraglia et al.

Tumours with neural differentiation left colon, and are discovered incidentally at screen- ing colonoscopy. The lesions endoscopically appear as subcentrimetrical polyps. Histologically mucosal Mucosal Schwann Cell Hamartoma hamartoma is composed of intramucosal prolifera- Mucosal hamartoma is a benign neural lesion of the tion of uniform spindle cell with tapering or wavy nu- GI tract characterised by an intramucosal Schwann clei (Fig. 24). Mitotic figures and nuclear atypia are cell proliferation 80,81. This peculiar lesion of GI typi- absent. The cells immunohistochemically express dif- cally occur in adults with a median age at diagnosis fusely S100 protein (Fig. 25) 80. Mucosal hamartoma is of 60 years. A slight female predominance has been entirely benign and is not associated with syndromes observed 80. Most mucosal hamartomas arise in the such as NF1 or NF2.

Schwannoma Schwannoma is a benign spindle cells neoplasm showing schwannian differentiation, accounting for approximately 3% of all GI mesenchymal tumours, the incidence of GIST being 50 times higher. It can occur anywhere in the GI tract, but it predominates in the stomach with only rare occurrences in the lower oe- sophagus, colon, and rectum 82. Schwannoma arises in elderly patients with a female predilection. The tu- mour is located in submucosa or muscolaris propria bulging into the lumen, sometimes ulcerating the mu- cosa. It appears as solid mass or polypoid lesions typically lacking intratumoural haemorrhage and ne- crosis 83. Small size tumours are discovered inciden- tally during endoscopic procedures, whereas large masses may cause gastrointestinal bleeding due to mucosal ulceration, or mass-like symptoms. Figure 24. Mucosal hamartoma. The morphological fea- Microscopically, gastrointestinal schwannomas are ture is represented by a proliferation of bland spindle cells well-circumscribed, with a distinctive peripheral lym- set in the lamina propria, featuring wavy nuclei. phoid cuff often containing germinal centres (Fig. 26). Thick-walled or hyalinised blood vessels are less fre-

Figure 26. Schwannoma. The tumour is composed of Figure 25. Mucosal hamartoma. S100 immunopositivity is spindle cells organized in short fascicles surrounded by a consistently observed. lymphoid cuff that represents an important diagnostic clue. MESENCHYMAL TUMOURS OF THE GASTROINTESTINAL TRACT 243

observed in the GI tract is the microcystic/reticular subtype. It appears as an unencapsulated submuco- sal lesion in the stomach, small bowel, and colon 86. Microscopically it is characterised by a distinctive re- ticular pattern of growth associated with formation of microcystic structures. Neoplastic cells are strongly and diffusely positive for S100 (Fig. 28) and in most cases for GFAP and nestin 87. Most of conventional schwannoma either sporadic or associated with NF2 syndrome are as- sociated with loss of heterozygosity at NF2 but most cases of GI schwannoma lack NF2 gene alterations, suggesting that they may represent a morphological- ly and genetically distinct group of peripheral nerve sheath tumours 88. Gastrointestinal schwannomas are benign lesions, long-term follow-up shows no recur- Figure 27. Schwannoma. Thick-walled blood vessels are rences or metastases. only occasionally observed in GI-tract schwannomas. Perivascular neoplasms

Glomus Tumour Glomus tumour is a mesenchymal neoplasm com- posed of perivascular modified smooth muscle cells. Most often the tumour occurs in peripheral soft tissue, although viscera involvement has been reported in- cluding the GI tract. In the Gi tract the region of antrum is most often involved followed by oesophageal and intestinal locations 89. Glomus tumour occurs predominantly in adults, al- though a broad age distribution is reported, with strong female predilection 89. Small lesions are usually asymptomatic and represent an incidental finding. In case of larger lesions, upper GI bleeding, abdominal pain, or reflux-type symptoms may occur 90,91. Glomus tumour of the GI tract is typically a single lesion, al- Figure 28. Schwannoma. S100 is diffusely and strongly though multifocality has been described in approxi- positive. mately 10% of patients. Occurrence of multiple fa- milial glomus tumour is due to inactivating mutations in the glomulin gene (GLMN). Most sporadic cases are associated with NOTCH family gene rearrange- quent in GI schwannoma (Fig. 27) that also lacks the ments 92. BRAF gene mutations are reported in 6% typical combination of Antoni A and Antoni B areas of patients 93. generally observed in schwannomas of soft parts 84,85. Macroscopically, the tumour appears like as a well The tumour is uniformly cellular and is composed of circumscribed intramural mass, often multinodular. spindle cell, with tapering nuclei and palely eosino- Extension into the mucosa and the serosa may oc- philic cytoplasm, organised in a fascicular or whorled cur. Histologically the tumour is composed of uniform pattern of growth. Scattered cells with nuclear enlarge- round cells characterised by sharply defined cell bor- ment and degenerative pleomorphism are common. ders, moderate amount of palely eosinophilic cyto- A low mitotic count is observed, and necrosis is typi- plasm and central dark round nuclei with inconspicu- cally absent. The tumour stroma is variable from col- ous nucleoli (Fig. 29). Foci of oncocytic and epithelioid lagenous to myxoid sometimes contain foci of foamy transformation may be observed 94,95. The neoplastic histiocytes. A subtype of schwannoma relatively often cells are organised in nodules, sheets and trabeculae 244 M. Sbaraglia et al.

reports, large size, presence of spindling and nuclear atypia may represent adverse prognostic factors.

Tumours with uncertain differentiation

Malignant gastrointestinal neuroectodermal tumour/ Clear cell sarcoma of the GI Tract Malignant gastrointestinal neuroectodermal tumour (M-GNET) represents a highly aggressive mesen- chymal tumour closely related to clear cell sarcoma (CCS) of soft parts but with distinctive clinical, molec- ular and immunomorphological features. Unlike CCS, M-GNET in fact is usually negative for melanocytic markers such as melanA and HMB45 and whereas Figura 29. Glomus tumour. This neoplasm is composed classic CCS most often harbour EWSR1-ATF1 gene of epithelioid cells harbouring round nuclei and showing fusions, M-GNET is more frequently associated with sharply delineated cytoplasm, arranged around blood ves- EWSR1-CREB1 gene translocations. In consideration sels. of these distinctive features, in the GI tract the alter- native name of malignant gastrointestinal neuroecto- dermal tumour has been recently proposed 3. To date set in a hyalinised and collagenous stroma present- CCS and M-GNET are regarded as two distinct enti- ing a rich vascular network showing a typical heman- ties and classified separately by WHO classifications of both soft tissue and gastrointestinal tract, and are giopericytoma-like appearance. Neoplastic cells may included in the group of tumours of uncertain differ- occasionally show a spindled morphology. Vascular entiation 3,32. M-GNET occurs most frequently in the invasion is observed in one third of cases, a finding small bowel followed by stomach and large bowel but that seems to represent a peculiar pattern of growth virtually can arise anywhere in GI tract including the rather than a predictor of aggressive behaviour. Usu- oral cavity 97. The tumour affects predominantly adults, ally, necrosis and severe atypia are not observed, and with a peak incidence at 40 years of age. There is no the mitotic index is generally low. Malignant glomus different distribution between male and female pa- tumours exist, although the criteria for malignancy in tients. Clinical symptoms include anemia, weight loss, the GI tract are still poorly defined due to insufficient pain and, when the tumour occur in small bowel, in- data. In soft tissue deep locations, increased mitotic testinal obstruction. The M-GNET is a highly aggres- 2 activity ( > 5 mitoses/10 mm ) including atypical mitot- sive tumour with an overall mortality ranging between ic figures, and severe nuclear atypia represent criteria 35% and 75%. Recurrences and metastases can oc- 96 used to define malignancy . cur even after many years from diagnosis. Unlike the The majority of cases show a strong and diffuse ex- vast majority of sarcomas in which lymph node spread pression of smooth muscle actin, caldesmon and is exceedingly rare, in M-GNET shows nodal metasta- CD34 in variable conbination. Focal expression of ses in approximately 50% of cases 97. synaptophysin may also be observed, representing a Macroscopically, tumours appear as a well circum- potential pitfall in the differential diagnosis with neu- scribed firm nodule located into the submucosa of the roendocrine tumours, particularly when dealing with involved viscera, with a diameter that range from few small biopsies. Importantly, glomus tumour is negative centimetres to large masses. for keratin. KIT and DOG1 are also typically not report- Histologically, M-GNET are composed of macronucle- ed in glomus tumour allowing excluasion of epithelioid olated epithelioid and spindle cells, organised in large GIST that represents the most common mesenchy- nodules demarcated by thick fibrous septa 98. The mal neoplasm in the stomach. neoplastic cells show small amount of eosinophilic The vast majority of cases are benign and complete cytoplasm and only scattered neoplastic cells show resection is curative. However, few cases reported in clear cytoplasm (Fig. 30). Multinucleated giant cells literature have shown an aggressive clinical behav- are seen in half of the cases (Fig. 31). In M-GNET the iour 89,96. To date criteria to predict aggressive behav- most distinctive morphologic feature is represented by iour are not been validated. Based on isolated case the presence of pseudopapillary and/or pseudoalveo- MESENCHYMAL TUMOURS OF THE GASTROINTESTINAL TRACT 245

Figure 30. M-GNET. Malignant gastrointestinal neuroecto- Figure 32. M-GNET. Diffuse S100 immunopositivity is seen. dermal tumour is composed of a combination of neoplastic cells featuring eosinophilic and clear cytoplasm.

Figure 31. M-GNET. Multinucleated giant cells are relatively Figure 33. Synovial sarcoma. A primary gastric monopha- often seen. sic synovial sarcoma is seen.

79 lar pattern of growth . Mitotic activity is variable from SOX10. Of course lack of expression of melanocytic scattered mitotic figures to higher mitotic index with markers in M-GNET can be helpful in the differential also more than 20 mitoses/2 mm2 97,98. diagnosis, even if it is important to highlight that most The tumour cells are usually positive for S100 and SOX10 (Fig. 32), whereas lack the expression of sarcomatoid melanomas can also loose expression of melanocytic markers such as melanA and HMB45. HMB45 and melan-A. In this context, the identification Furthermore, the tumours are consistently negative of molecular alteration, characteristic for M-GNET, for KIT, DOG1, pancytokeratins and for markers of may represent the ony way to achieve the correct di- smooth muscle differentiation 98. agnosis 97. The main differential diagnosis includes metastatic malignant melanoma that shares with M-GNET some Synovial sarcoma morphological features such as the nested growth pat- Synovial sarcoma is a malignant mesenchymal neo- tern and the diffuse immunoexpression of S100 and plasm representing 10% of all soft tissue tumours 246 M. Sbaraglia et al.

Figure 34. Synovial sarcoma, monophasic. This highly cellular Figure 35. Synovial sarcoma, biphasic. Epithelial compo- neoplasm is composed of monomorphic, atypical spindle cells. nent exhibits a glandular configuration.

A B

C D

Figure 36. Synovial sarcoma. Pancytokeratins expression in monophasyc SS (A); biphasyc SS (B). Nuclear TLE1 expression is consistently observed but tends to exhibit low specificity (C). Currently the best marker is represented by the expression of SS18-SSX fusion-specific antibody (D). MESENCHYMAL TUMOURS OF THE GASTROINTESTINAL TRACT 247

which typically occurs in lower limbs of children and confirmation of SS. The main differential diagnosis of young adults 99. Synovial sarcoma of the GI tract are monophasic SS is with GIST. Histological features and extremely rare: in this context, the stomach is the most positivity for CD117 and DOG1 stainings are usually common location (Fig. 33), although SS of the oe- sufficient to support the diagnosis GIST 15. Important- sophagus, small and large bowel and liver have also ly, focal positivity for DOG1 has been reported in SS been reported 99,100. of the digestive system, representing a possible diag- Synovial sarcoma can be morphologically subdivid- nostic pitfall 105. Moreover, cytokeratin positivity has ed in monophasic, biphasic and poorly differentiated been observed in sporadic GISTs 106,107. variants 99, the monophasic one being most common S100 positive monophasic SS may enter in differen- in the GI tract 3. Both the monophasic and biphasic tial diagnosis with gastrointestinal clear cell sarco- variants are characterised by a monomorphic spindle mas (CCS) and CCS-like tumours of the GI tract 68. cell population arranged in fascicles (Fig. 34) set in a Negativity for epithelial markers and EWSR1 gene variablly collagenous matrix, and asscoiated with with rearrangements are distincive features of CCS and an haemangiopericytoma-like vascular pattern 32,99. CCS-like tumours 68,98,108. Markers of melanocytic dif- Biphasic SS in addition to the spindle cell component, ferentiation (e.g. MelanA/MART1, HMB45 and MiTF) presents an epithelial component, usually organised are commonly expressed in CCS (but not in CCS- in nests, cords or featuring overt glandular (Fig. 35), like tumors), helping in the differential diagnosis with architecture 32,99. Interestingly, a predominantly mono- SS 68. Monophasic SSs may also enter in diagnosis phasic epithelial pattern has also been described 101. with leiomyosarcomas, malignant spindle cell mela- Poorly differentiated SS (PDSS) make up the 20% nomas and spindle cell . of SS and it is further subdivided in three groups: i) However, a higher degree of pleomorphism is usually a round cells subtype with necrosis and high mitotic observed in all these lesions. Expression of smooth count, ii) a large epithelioid cell subtype, also associat- muscle markers and melanocytic markers can eas- ed with rhabdoid features, and iii) a high grade spindle ily direct the diagnosis toward leiomyosarcoma and cell subtype 102. Tumour size greater than 5 cm, pres- melanoma, respectively. Spindle cell squamous cell ence of neural and vascular invasion, p53 overexpres- carcinoma is usually characterised by a stronger ex- sion, and high proliferation index are histological fea- pression of epithelial markers and by the presence of tures associated with higher risk of tumour relapse 99. areas of conventional carcinoma 99. Immunohistochemically, SS is characterized by the Despite the fact that morphological and immunohis- expression of epithelial markers (i.e. EMA, cytokera- tochemical features most often make the diagnosis of tins) (Fig. 36A and 36B) together with CD99 and TLE1 biphasic SS relativley easy, the differential diagnosis (Fig. 36C) 99. Cytokeratin expression varies among dif- with gastroblastoma may sometimes be considered. ferent SS subtypes: while cytokeratins expression is Gastroblastoma is an extremely rare mixed epitheli- recognisable in the majority of biphasic SSs, it drops al-mesenchymal neoplasm with just a few cases re- to 60-70% in the monophasic subtype 99. Moreover, ported in the literature 109,110. The mesenchymal com- PDSS is characterised by cytokeratin expression only ponent consists of spindle cells with minimal to mild in 50% of cases 102. High molecular weight cytokera- nuclear atypia and variable mitotic activity, while the tins are more sensitive than low molecular weight cy- epithelial one is composed by epithelioid cells organ- tokeratins, but the most sensitive marker of epithelial ised in sheets, nests, or cords with no or mild nuclear differentiation is EMA 102. S100 positivity is reported atypia 109,110. Immunohistochemistry is helpful in the between 30-60% of SS, while positivity for CD34 is assessment of the correct diagnosis, since gastro- extremely rare 35,103. blastomas are negative for SSX-SYT, EMA and TLE1; SS is characterised by a translocation involving chro- moreover, gastroblastoma lacks SYT gene rearrange- mosomes X and 18. This translocation is reported only ments. in SS, and results in three alternative fusion products of the SS18 gene with either SSX1, or SSX2 or SSX4 gene 99. FISH analysis and amplification of the spe- Conclusions cific chimeric transcript by RT-PCR are effective tech- niques to detect translocation t (18; X), representing Mesenchymal tumours represent an extremely heter- ancillary tools for diagnosis of SS 99,104. ogeneous group of lesions. Visceral locations are in- Very recently a novel SS18-SSX fusion-specific anti- creasingly recognised due to refinement of diagnostic body has been developed (Fig. 36D) showing a highly criteria. Even if GIST represent by far the commonest sensitivity and specificity for SS. This specific antibody lesion (at least in the stomach), actually mesenchymal may replace molecular genetic testing for diagnostic neoplasms may occur in the GI-tract. Accurate diagno- 248 M. Sbaraglia et al.

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