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Asian Archives of 2015; Vol. 11 No.1, 1-25

Special article The Many Faces (and Origins) of in the : Pattern Approach to Diagnosis on Small Tissue Samples

Aileen Wee, MB,BS, FRCPath, FRCPA* Yong Loo Lin School of Medicine** *Professor and Senior Consultant, Department of Pathology **National University of Singapore, National University Hospital, 5 Lower Kent Ridge Road, Singapore, 119074 Email: [email protected]

Correspondence: Aileen Wee, MB,BS, FRCPath, FRCPA Received: 20 February 2015; accepted 13 March 2015

ABSTRACT Focal liver lesions can be solitary or multiple, solid or cystic, congenital or acquired, and range from , hamartomas, hyperplastic nodules and inÀ ammatory entities to tumors and tumor-like lesions. The background liver may be normal or diseased. An algorithmic approach based on pattern recognition and cell pro¿ ling is outlined for the diagnosis of focal liver lesions with glandular features in small tissue samples from ¿ ne needle aspiration and core needle . The morphological categories for lesions with glandular phenotypes are (i) glandular (ducts, and/or ) pattern, including biliary and papillary patterns, (ii) hepatocellular and epithelioid patterns; (iii) mixed epithelioid-glandular (hepatobiliary) pattern; (iv) predominant cell pro¿ les; and (v) cystic pattern. Hepatobiliary entities should be segregated before considering nonhepatobiliary conditions. The main diagnostic issues are to distinguish cystic from solid lesions, primary from metastatic , poorly differentiated adenocarcinoma from poorly differentiated hepatocellular ; and to recognize rare glandular entities, mimics and pitfalls. Assessment of sample adequacy, key cytohistological features, and diagnostic utility of ancillary tests are addressed. Close clinicopathological correlation is mandatory before rendering a ¿ nal de¿ nitive diagnosis.

Key Words: Adenocarcinoma, , core biopsy, ¿ ne needle aspiration cytology, liver, metastases 2 Aileen Wee, MB,BS, FRCPath, FRCPA, Yong Loo Lin School of Medicine

INTRODUCTION diagnostic utility of immunohistochemistry, and The anatomical and functional complexity diagnostic pitfalls and challenges are addressed. of the liver makes for a highly varied and Clinicopathological correlation is mandatory. challenging landscape of for clinical, radiological and pathological diagnosis. The liver CLINICAL PERSPECTIVE consists of hepatobiliary and other cellular In clinical practice, the initial impression of components, receives a dual blood supply, and has a a liver mass is to investigate for HCC or metastases monocyte-macrophage function. Focal liver lesions rather than ICC, unless there is a positive support- can be solitary or multiple, solid or cystic, congenital ive history such as primary sclerosing cholangitis or acquired, and may range from cysts, hamartomas, (PSC), chronic disease due to hepato- hyperplastic nodules and inÀ ammatory entities to biliary lithiasis and liver À ukes such as Clonorchis tumors and tumor-like lesions.1, 2 sinensis or Opisthorchis viverrini, or developmental Three clinicopathological scenarios come cystic anomalies.1-5 Metastatic tumors with glandular to mind when the commonplace adenocarcinoma is morphology that are commonly encountered in the addressed in the liver.3, 4 First, the liver is a common liver include those from colorectum, stomach, depository for secondaries from virtually any part , extrahepatic biliary tract, breast, lung, of the body. The majority are metastatic adenocarci- prostate and female genital tract. The background nomas which have to be distinguished from primary liver may provide important clues. Metastases are intrahepatic cholangiocarcinoma (ICC). Second, rarely found in cirrhotic . Pre-existing benign the liver can undergo with subsequent cysts may call attention to themselves with development of (HCC). adenocarcinomatous transformation. The pseudoacinar pattern in HCCs and the rare Patients may be or present combined hepatocellular-cholangiocarcinoma with advanced disease. They fall into several clini- (CHCC-CC) can be confused with adenocarcinoma. cal settings: Third, benign neoplastic and nonneoplastic biliary • Investigation for symptomatology lesions can mimic adenocarcinoma. referable to the primary hepatic tumor or liver Suf¿ ce it to say that the differential diagno- metastases such as obstructive , right ses abound when a carcinoma with putative glandular hypochondrial pain, biliary sepsis or morphology is encountered. It is imperative for • Radiological staging or surveillance for prognostic and therapeutic purposes that an recurrent or metastatic disease in known accurate cytohistological diagnosis be rendered. This patients can prove extremely challenging on small tissue • Follow-up/surveillance of at-risk patients samples from ¿ ne needle aspiration biopsy (FNAB) with pre-existing benign cystic condition or chronic and core needle biopsy (CNB). hepatobiliary disorder This article aims to provide an algorithm for • Routine health screening revealing the pattern recognition and cell pro¿ ling of focal abnormal liver function test pro¿ les, elevated serum liver lesions with putative glandular morphology. tumor markers such as CA19-9 and carcinoembry- Intrahepatic cholangiocarcinoma is taken as the onic antigen (CEA), or imaging abnormalities reference tumor. Key cytohistological features, Th e Many Faces (and Origins) of Adenocarcinoma in the Liver: Pattern Approach to 3 Diagnosis on Small Tissue Samples

RADIOLOGICAL PERSPECTIVE mucinous adenocarcinomas or post-chemotherapy | Imaging features of ICC are categorized into necrosis; and so can ICC. Imaging of biliary mass-forming (MF), periductal in¿ ltrating (PI), and intraductal papillary (IPN) may dem- intraductal growth (IG) types.3, 6 The checklist that onstrate dilated intra-/extrahepatic bile ducts with must be complied with includes exclusion of lesions single or multiple polypoid masses in the biliary tree. in other sites, presence of peripheral duct dilatation, delayed enhancement of radiocontrast dye, and hilar PATHOLOGICAL PERSPECTIVE lymphadenopathy. Cirrhosis should alert one to the Cholangiocytes lining the small-caliber bile possibility of an HCC or a CHCC-CC. The MF type duct branches are small and À attened to cuboidal with irregular borders is the commonest and cells. The becomes columnar with typically shows arterial and portal venous phase occasional mucous cells in the larger ducts. It is rim-like enhancement and slow diffusion of contrast speculated that ICCs found at these different into the tumor leading to late central enhancement. anatomical locations portray a corresponding The poorly enhanced center is due to necrosis and/ morphological topography.8-11 Those mixed or desmoplasia. Capsular retraction is characteristic. hepatobiliary (MHBC) portraying hybrid The PI and IG types usually present with obstructive features straddling and cholangiocytes jaundice. The PI type tends to simulate benign are likely attributed to hepatic stem and progenitor stricture with thickening and segmental enhance- cells in the canals of Hering.12 This phenotypic ment of the bile ducts. The IG type due to polypoid diversity may be used to diagnostic advantage in or papillary growths is uncommon and frequently distinguishing adenocarcinoma of biliary tract origin not detected on imaging as the lesions are usually from other adenocarcinomas, apart from pancreatic small. adenocarcinoma since the biliary tract is regarded Liver is the most common intra-abdominal as an anatomical extension of the pancreas.13 organ for metastases which are often multiple with Immunohistochemically, ICCs are characteristi- variable appearances depending on the primary cally CK7/CK19 positive and CK20 negative.14 site.3, 7 On (US), most metastases appear However, those closer to the hilum may acquire as hypoechoic masses. On computed tomography additional CK20 positivity. (CT), the appearance is highly varied but usually hypodense. Hyperdense metastases can be seen in DIAGNOSTIC ALGORITHM tumor and mucinous adenocarcinoma of Basic patterns and cell profi les colon when they calcify. Some metastases have non- The approach to the evaluation of small tis- speci¿ c targetoid appearance. sue samples begins with the consideration of whether Cystic lesions appear typically anechoic on the specimen is from a solid or cystic lesion to enable US, hypodense on CT, and hyperintense on T2- assessment of adequacy and representativeness. weighted magnetic resonance imaging (MRI). The morphological categories for lesions with Mucinous cystic (MCN) can resemble glandular phenotypes are (i) glandular (ducts, glands simple cysts and appear uniformly anechoic on and/or mucin) pattern, including biliary and papillary US, with a barely perceptible wall and no internal patterns, (ii) hepatocellular and epithelioid patterns; echoes. Metastases can appear cystic, especially from (iii) mixed epithelioid-glandular (hepatobiliary) 4 Aileen Wee, MB,BS, FRCPath, FRCPA, Yong Loo Lin School of Medicine

pattern; (iv) predominant cell pro¿ les; and (v) cystic comprising larger tumor cells admixed with pattern (Table 1).3 Hepatobiliary entities should be plentiful bland, small ductular cells reflecting segregated before considering nonhepatobiliary le- ductular reaction to chronic hepatic injury;21 and sions. Differential diagnoses and diagnostic pitfalls (iii) Usual adenocarcinoma type: This “background are discussed. poor-tumor cell rich” pattern comprising larger sheets of pleomorphic glandular cells exhibiting (i) Glandular (ducts, glands and/or mucin) cribriform, palisading and ductal appearances, is pattern, including biliary and papillary patterns not characteristic of ICC and requires other biliary The most important primary adenocarcino- identi¿ ers. ma in the liver is ICC although metastases are by far Variations and variants of ICC pose more common. Mixed hepatobiliary carcinomas, and challenges to diagnosis on small samples (Fig. 4). MCN and biliary IPN with their associated invasive Flat sheets, branching structures with peripheral carcinomas, are addressed in sections (iii) and (v), nuclear palisading, papillary structures with respectively. ¿ brovascular cores, 3-dimensional acinar spaces, The typical biliary epithelium comprises intracytoplasmic mucin secretion (signet ring cells), monolayered honeycomb sheets of cuboidal/colum- and adenosquamous components can be encountered nar epithelium. The equidistant, round to oval nu- solely or in combination. This striking phenotypic clei are bland with no nucleolus normally. The cell diversity is exemplified even in tissue cores. borders are indistinct. Reactive and atypical changes Histologically, the malignant cells may be arranged are indicated by the increasing presence of promi- in irregular tubular, tubulocystic, nested glandular or nent nucleoli, nuclear overlapping, and abrupt papillary configurations, and cordlike, with a nuclear enlargement, leading to a drunken honey- predilection for subepithelial zones beneath pre- comb appearance. Nuclear pleomorphism with high existing bile ducts. “Adenofibromatous” or nuclear-to-cytoplasmic ratio, nuclear membrane “pseudoangiomatoid” arrangement reminiscent of irregularities, hyperchromasia, and mitoses appear von Meyenburg complexes is unique. Transition with increasingly higher grades of biliary intraepi- from relatively bland-looking cells to thelial neoplasia (BilIN), progressing to in-situ dysplastic (BilIN)/malignant cells (carcinoma- (Fig. 1).15 in-situ) is a diagnostic clue. Papillary pattern is more Intrahepatic are likely to indicate metastases or ICC rather than the notoriously heterogeneous even over a small area rare biliary IPN. (Table 2).16-20 Classic ICCs can be graded into well, It is virtually impossible to distinguish ICC moderately and poorly differentiated adenocarcino- from metastatic pancreaticobiliary adenocarcinomas mas (Fig. 2 and 3). Three smear patterns can be rec- in the absence of intrahepatic BilIN or carcinoma- ognized on low power scanning, namely: (i) Scanty in-situ as they have similar immunopro¿ les (Fig. 5) cell type: This “background rich-tumor cell poor” (Table 3). Adenocarcinomas with “dirty” necrosis pattern typically yields sparse, easily overlooked are likely to be colorectal metastases but not exclu- tumor cells entrapped in desmoplastic stroma; (ii) sively (Fig. 6). Colorectal metastases are notoriously Ductular proliferation type: This “background known for their propensity to colonize the existing moderate-tumor cell modest” pattern is a cue for ICC intrahepatic biliary tree resulting in an intraductal Th e Many Faces (and Origins) of Adenocarcinoma in the Liver: Pattern Approach to 5 Diagnosis on Small Tissue Samples broma and serous ¿ ammatory pseudotumor with reactive biliary À duct (peribiliary hamartoma), biliary adeno pattern tumor plasia atypia tional component) • Hepatocellular carcinoma (HCC) with CK19 positivity • HCC with pseudoacinar pattern • Benign conditions such as bile duct hamartoma, • MCN and biliary IPN (malignant focus not sampled) • Pseudopapillary appearance mistaken for papillary • Papillary pattern overlooked for poorly differentiated • Ductular reaction, e.g. in scar of focal nodular hyper- • In • Gastrointestinal contaminants (EUS-FNA) hemangioendothelioma • Epithelioid • HCC (predominant hepatocellular component) • Adenocarcinoma (predominant glandular component) • carcinoma (predominant transi- Poorly differentiated static general nomas (pancreas, lung, ovary) cinoma, metastatic (prostate, pancreas, salivary glands) • Extrahepatic hepatoid carcinoma, meta- • Metastases from epithelioid variants of • Metastases from adenocarcinomas in • Metastases from pancreaticobiliary carci- • Metastases from papillary carcinomas • AFP-producing (nonhepatoid) adenocar- • Metastases from acinar adenocarcinomas tumor/carcinoma • Neuroendocrine (ICC), and variants (MCN) with associated invasive carcinoma neoplasm (IPN) with associated invasive carcinoma features • Mixed hepatobiliary carcinoma • Collision tumors (HCC and ICC) • Intrahepatic cholangiocarcinoma • Intrahepatic • Mixed hepatobiliary carcinoma • Mucinous cystic neoplasm • Biliary intraductal papillary • Mixed hepatobiliary carcinoma • ICC, and variants with hepatoid Algorithmic approach to the morphological diagnosis of adenocarcinomas occurring in liver. les Hepatobiliary lesions Nonhepatobiliary lesions fi Patterns and cell predominant pro Diagnostic considerations diagnosis Diagnostic pitfalls and differential (iii) Mixed epithelioid- glandular (hepatobiliary) pattern (i) Glandular (i) Glandular (ducts, glands ± mucin), including biliary and papillary patterns (ii) Hepatocellular (ii) Hepatocellular and epithelioid patterns Table 1 Table 6 Aileen Wee, MB,BS, FRCPath, FRCPA, Yong Loo Lin School of Medicine bropoly- ammation ¿ À ammatory processes ammatory À carcinoma or intraductal polypoid tumor cystic disease, ciliated foregut , and obstructive dilatation of bile duct sions and in • Exclude HCC • Exclude HCC • Exclude HCC • Exclude HCC • Exclude • Look for HCC and adenocarcinoma components HCC • Exclude • Exclude HCC, sarcomatoid type • Exclude HCC, sarcomatoid type • Exclude downstream obstruction due to bile duct • Reactive biliary atypia associated with in • mucin contamination in EUS-FNA GIT • Benign conditions such as bile duct cyst, • MCN and biliary IPN (malignant focus not sampled) • Reactive biliary atypia in association with cystic le- • Cystic degeneration of malignant tumors mas (pancreas, extrahepatic biliary tract, cervix, lungs) cell type) cavitating necrosis) • Metastases from adenosquamous carcino- • /carcinoma (small • cell type) Neuroendocrine carcinoma (large • Extrahepatic hepatoid carcinoma, metastatic • , metastatic • Extrahepatic hepatoid carcinoma, metastatic • carcinoma, metastatic Poorly differentiated • carcinoma, metastatic Poorly differentiated • , metastatic • • Sarcomatoid carcinoma, metastatic • Sarcoma • Cystic metastases (ovary and pancreas) • Neuroendocrine tumors/carcinoma (with cell variant carcinoma sive carcinoma opmental cysts • ICC, adenosquamous type • ICC with small cell features • ICC, high grade • ICC with hepatoid features • ICC with clear cell features; foamy • ICC with hepatoid features • Mixed hepatobiliary carcinoma • ICC, high grade • ICC, sarcomatoid type • ICC, sarcomatoid type • MCN with associated invasive • Biliary IPN with associated inva- ICC • Cystic • Carcinoma associated with devel- Algorithmic approach to the morphological diagnosis of adenocarcinomas occurring in the liver. (con.) Algorithmic approach to the morphological diagnosis of adenocarcinomas occurring in liver. les Hepatobiliary lesions Nonhepatobiliary lesions fi ammatory cell ammatory À Patterns and cell predominant pro Diagnostic considerations diagnosis Diagnostic pitfalls and differential (iv) Cell type: Squamous cell Small/ intermediate cell Large Polygonal Clear cell Oncocytic cell cell Transitional Pleomorphic cell Spindle cell Giant cell In (v) Cystic pattern Table 1 Table Th e Many Faces (and Origins) of Adenocarcinoma in the Liver: Pattern Approach to 7 Diagnosis on Small Tissue Samples broma can mimic ¿ ammation; potential À gurations. They are usually well- gurations. ¿ ed on small tissue samples; or they ¿ aged under hybrid forms, both morpho- À les. ¿ logically and immunologically. logically and immunologically. and other primary of the liver on clinical, imaging and morphological grounds. Pancreaticobiliary tract carcinomas share similar morphologies and immu- nopro to moderately differentiated adenocarcinomas despite to moderately differentiated ICCs can have additional spe- the phenotypic diversity. cial features, such as oncocytic, small cell, clear cell and trabecular features. tor cells can manifest as combined hepatocellular and cholangiocarcinoma (CHCC-CC) or cholangiolocel- lular carcinoma (of the mixed hepatobiliary group). One or two distinctively recognizable compo- nents may be identi may be camou pitfall during intraoperative frozen section assessment of clearance resection margin sists of cuboidal to columnar lined malignant glands in cribriform, nested, solid simple/complex acinar, tubular, and/or papillary con well-differentiated ICCs. well-differentiated lary neoplasm with associated invasive carcinoma have to be distinguished from cystic or mucinous ICCs, and from malignant transformation of pre-existing cystic anomalies. malignant BilIN simulate invasive well-differentiated glands. in the face of biliary/peribiliary glandular atypia (reactive) in the presence of gland hamartoma) and biliary adeno • ICC needs to be distinguished from metastases, HCC • Carcinomas arising in canals of Hering/hepatic progeni- • The former con- ICC has classic forms and variants. • Mucinous cystic neoplasm and biliary intraductal papil- • In core , resident bile ducts with high-grade • Threshold for diagnosing should be raised • Biliary duct hamartoma, bile adenoma (peribiliary antigen (CEA) brane antigen (EMA) CK7+ / CK 20- (CK7+/CK20+ for tumors nearer hilum) • CK19 • Carcinoembryonic mem- • Epithelial • MOC-31 • MUC1 • MUC2 • MUC3 attened to attened À gurations; occasional gurations; ammatory/necrotic ¿ À ne needle aspiration ¿ bromatous or pseudoangioma- ¿ ukes cuboidal and columnar cells +/- cell groups interface regions can be seen in hepatocytes or canali- culi debris, proliferating ductules, and liver À obtained appearance (phenotypic diversity) tubulocystic, nested glandular, tubular, or papillary con adeno toid pattern is unique mucous cells mucous • Heterogeneity of glandular cells within • from bland bile duct cells Transition clue to dysplastic/malignant cells is a diagnostic • Biliary intraepithelial neoplasis (BilIN) and other aberrant bile ducts in portal tracts • Insinuation of tumor cells between hepatic cell plates at interface is helpful • Bile duct or vascular dilatation at • No bile pigment in tumor cells; In • Background: • Continuous unbroken cores usually • Heterogeneity in cell pattern and • Malignant cells arranged in irregular • Malignant cells vary from ukes À ammatory/necrotic À Key morphological and immunohistochemical features of intrahepatic cholangiocarcinoma (ICC). -fetoprotein; EUS-FNA, endoscopic ultrasound-guided Į debris, proliferating ductular cell groups, and liver malignant cells groups, tubular pattern to sheets with peripheral nuclear palisading cells, often heterogeneous large-sized within groups malignant cells is a diagnostic clue mucin is less frequent can be seen within hepatocytes or in background • Cell arrangement: Small ductular • Cell character: Small, medium to • Heterogeneity of glandular cells • from bland bile duct cells to Transition • Mucous cells with cytoplasmic vacuoles are common; extracellular • No peripheral or transgressing endothelium • No bile pigment in tumor cells; In • Background: • Cellularity: Scant, modest to plentiful Adapted from Table 2.9 in Wee et al, Cytohistology of focal liver lesions, Cambridge University Press, 2014. Wee 2.9 in Table Adapted from Table 2 Table Cytology Immunohistochemistry diagnosis Pitfalls and differential AFP, 8 Aileen Wee, MB,BS, FRCPath, FRCPA, Yong Loo Lin School of Medicine origin for metastatic adenocarci- to the liver ferentiated adenocarcinoma mucinous and signet ring tubular, adenocarcinoma; and adenosqua- mous, small cell, and undifferenti- ated carcinoma biliary tract carcinomas, requiring clinical correlation can mimic benign glands biliary primary signet ring, basaloid, intestinal type, mucinous, squamous cell, and carcinoma toid or undifferentiated) • One of the most common sites • Generally well- to moderately dif- • diathesis is key clue Tumor Adenocarcinoma • Subtypes: papillary, (intestinal, diffuse); • Usually indistinguishable from adenocarcinoma • Well-differentiated • Desmoplasia favor pancreatico- Adenosquamous, • Variants: oncocytic, clear cell, hepatoid, anaplastic (pleomorphic, sarcoma- antigen (CEA) antigen (EMA) CK7 -, CK20 + • CDX2 • Villin • Carcinoembryonic • MOC-31 • MUC1 • MUC2 • MUC3 CK7 +/-, CK20 +/- No predominant pattern • CEA membrane • Epithelial • MUC1 • MUC2 • MUC5AC CK7 +/-, CK20 +/- +/- • CK5/6 • EMA • CEA • CA19-9 • CDX2 • Villin • MUC1 • with necrotic lumen enocarcinoma but marked cytological atypia • well-formed malignant glands Large necrosis”) (“dirty • Desmoplasia mucin • Intra-/extracytoplasmic • tubules to poorly differentiated Well • Signet ring or plasmacytoid cells mucin • Intra-/extracytoplasmic • Desmoplasia • ad- to poorly differentiated Well- • Architecturally well-formed glands • Periductal concentric desmoplasia mucin • Intra-/extracytoplasmic • Squamous component +/- prominent nucleolus palisaded oval nuclei hyperchromatic nuclei desmoplasia hesive glandular cells of variable pleomorphism tumor cells • Columnar epithelial cells in cohesive clusters +/- lumens • Broken end-on strips • palisaded nuclei with Cigar-shaped, mucin • Intra-/extracytoplasmic necrosis • Dirty • Columnar epithelial cells in cohesive clusters +/- lumens • Broken end-on strips with basal • Dissociated signet-ring or plasmacytoid cells with eccentric mucin • Intra-/extracytoplasmic • Necrosis • Cellularity varies with degree of • Sheets, cohesive clusters to disco- • Stromal fragments with entrapped mucin • Intra-/extracytoplasmic • Squamous elements +/- Common metastatic adenocarcinomas to the liver and their morphological immunohistochemical characteristics. Adenocarcinoma, colorectum Adenocarcinoma, stomach Adenocarcinoma, pancreas Tumor type origin Tumor and Cytology Histology Immunohistochemistry Comments Table 3 Table Th e Many Faces (and Origins) of Adenocarcinoma in the Liver: Pattern Approach to 9 Diagnosis on Small Tissue Samples adenocarcinoma can mimic benign glands clear cell, signet ring, lymphoepi- thelioma-like, and anaplastic carcinoma with neuroendocrine features • Usually indistinguishable from adenocarcinomas, pancreatic requiring clinical correlation well-differentiated • Metastatic • Desmoplasia favor pancreatico- primary biliary • adenosquamous, Papillary, Variants: sarcomatoid, (pleomorphic, carcinoma; and undifferentiated) • Liver metastases are usually clinically suspected • mucinous, and Papillary, Variants: tion factor -1 (TTF-1) (nucleus) nous type) CK7 +/-, CK20 +/- • EMA • CEA • p53 CK7 +, CK20 - transcrip- • Thyroid • EMA • CEA • CDX2 – (+ for muci- but poorly differentiated at but poorly differentiated gland, increased nuclear-to-cyto- plasmic ratio and nucleolar tion ranging from well-differetiated tubular pattern to poorly cell carcnoma large undifferentiated • to poorly differentiated Well- adenocarcinoma • Architecturally well-formed glands level cytological • Heterogeneity of cells within same prominence • Periductal concentric desmoplasia mucin • Intra-/extracytoplasmic • Squamous component +/- • Spectrum of glandular differentia- type, bordering on differentiated mucin • Intra-/extracytoplasmic desmoplasia discohesive glandular cells of tumor cells drunken honeycomb sheets, nuclear overlapping, increased nuclear-to- cytoplasmic ratio and nucleolar prominence cells arranged in monolayered sheets, 3D clusters or singly clusters from bland to obviously malignant frequently vacuolated • Cellularity varies with degree of • Monolayered sheets and clusters to pleomorphism variable • Stromal fragments with entrapped • Heterogeneity of cells within mucin • Intra-/extracytoplasmic • Squamous elements +/- • Polygonal to columnar epithlial • Lumens may be discernible in some • nuclear atypia, ranging Variable • Cytoplasm is delicate, fragile and mucin • Intra-/extracytoplasmic Common metastatic adenocarcinomas to the liver and their morphological immunohistochemical characteristics. (con.) Adenocarcinoma, and extrahepatic biliary tract Adenocarcinoma, lung Tumor type origin Tumor and Cytology Histology Immunohistochemistry Comments Table 3 Table 10 Aileen Wee, MB,BS, FRCPath, FRCPA, Yong Loo Lin School of Medicine symptomatic lymphovascular or peritoneal spread. and papillary serous carcinoma acinar carcinoma of pancreas and HCC with well-differentiated • In most cases, liver metastases are • Occult primary is rare • mucinous, Papillary, Variants: metaplastic, and medullary, • Metastatic lesions may represent • adenosquamous Papillary, Variants: • diagnoses include Differential pattern pseudoacinar c) ¿ c ¿ protein-15 +/- gesterone (PG) recep- tors +/- (not speci antigen (PSA) antigen acid • Prostatic (PAP) phosphatase • EMA • GATA3 • Gross cystic disease • Mammaglobin • EMA • CEA CK7 +, CK20 - • ES and PG receptors (c-erbB2) • HER2/neu • Vimentin • CEA • CA-125 • MUC1 CK7 +, CK20 + • EMA • CEA • CDX2 CK7 +, CK20 - • WT1 • CA-125 • EMA • Vimentin CK7 -, CK20 - • Prostate-speci CK7 +, CK20 - • (ES) and pro- pleomorphism, increased nuclear- to-cytoplasmic ratio and prominent nucleoli tion ing to Gleeson score dissociated cells to-cytoplasmic ratio and prominent nucleolus • Monomorphous appearance • Monomorphous • Sheets, small nests, cords or cells individual +/- • Tubules • cells exhibit variable nuclear Tumor • Desmoplasia adenocarcinoma • Conventional • lumens show dirty necrosis Tubular • Papillary (villoglandular) foci • +/- Squamous differentiation • Adenocarcinoma with mucin secre- • Adenocarcinoma • Papillary carcinoma, high grade • of appearances correspond- Variety • Small to medium-sized glands • Pale cytoplasm, increased nuclear- ame or cone-shaped cells À plasmic lumen) cytological atypia cells with pale/vacuolated cyto- plasm, central nuclei and distinct nucleolus. • cells (cells with intracyto- Target arrangement • Cell-in-cell • Rosettes or tight clusters of columnar cells with nuclear palisading necrosis • Dirty • Columnar epithelial cells with nuclear palisading and mucin secretion • Columnar epithelial cells with palisading nuclear • Papillary forms with high-grade • Rosette-like groups of polygonal • Monomorphous cell population • Flat, angulated groups • Single Common metastatic adenocarcinomas to the liver and their morphological immunohistochemical characteristics. (con.) , breast Gynecological tract carcinomas: Endometrial carcinoma Ovarian mucinous carcinoma Ovarian carcinoma, serous and endome- trioid Prostatic acinar adenocarcinoma Tumor type origin Tumor and Cytology Histology Immunohistochemistry Comments Table 3 Table et al. Cytohistology of focal liver lesions, Cambridge University Press, 2014. Wee 7.2 from Table Adapted from Th e Many Faces (and Origins) of Adenocarcinoma in the Liver: Pattern Approach to 11 Diagnosis on Small Tissue Samples growth pattern simulating ICC, intraductal type. terface.21 Ductular epithelial clusters occurring as CK20 versus CK7/CK19 immunoprofiling is small dark overlapping nuclei in curved double-cell helpful. However, the distinction may not be made cords or tramline con¿ gurations are often seen in in cytology material if one is not mindful of such the central scar of focal nodular , and in as an occurrence. In the presence of signet ring cell association with cirrhosis, and large regenerative and adenocarcinoma, one must ¿ rst and foremost exclude dysplastic nodules. They should not be mistaken for from a primary gastric carcinoma. Metas- cholangiolocellular carcinoma or small cell-type of tases from ductal carcinoma of breast should come to adenocarcinoma.10, 11 The threshold for diagnosing mind if the tumor cells appear rather monomorphic malignancy should be raised in the event of ¿ nding comprising dissociated plasmacytoid cells (Fig. 7). biliary or peribiliary glandular atypia (reactive or Rarely, an extrahepatic Į-fetoprotein-producing degenerative) in the presence of persistent inÀ am- adenocarcinoma, for example, from the stomach, mation, such as in inÀ ammatory pseudotumors.25, 26 may metastasize to the liver.22 Degenerative swelling of cells and nuclei coupled Acinar pattern should be distinguished from with nuclear pyknosis and vacuolated cytoplasm pseudoacinar structures which are rosette-like should not be mistaken for pleomorphism with arrangements of tumor cells about a space without increased nuclear-to-cytoplasmic ratio and hyper- true luminal brush border or mucin secretion. chromasia. Gastrointestinal contamination of endo- Common sources of acinar tumors include prostate scopic ultrasound (EUS)-FNA material is also a known and pancreas (Fig. 8). Neuroendocrine tumors and pitfall. A rare mimic of (signet ring cell) adenocarcinoma HCC often exhibit pseudoacinar pattern (Fig. 9).23-24 is epithelioid hemangioendothelioma. The individual The absence of stromal matrix, and in its place the endothelial tumor cells with intracytoplasmic spaces presence of (blood-¿ lled) sinusoidal spaces between and eccentric nuclei may or may not contain pseudoacini, confirms the trabecular-sinusoidal erythrocytes.27 In cases where the adenocarcinoma pattern of HCC. is poorly differentiated, distinction from a poorly Benign mimics of adenocarcinomas are differentiated HCC or metastases can be challeng- treacherous. Bile duct hamartoma (von Meyenburg ing. Ancillary tests may or may not be helpful. complexes), bile duct adenoma (peribiliary gland Correlation with clinical and radiological ¿ ndings hamartoma) and biliary adeno¿ broma can mimic is imperative. well-differentiated ICCs.2 The former two condi- In summary, some features that favor ICCs tions are small subcapsular tumorlike nodules de- over metastatic adenocarcinomas apart from those of tected incidentally during laparoscopy/laparotomy pancreaticobiliary origin are heterogeneity, insinua- staging for other gastrointestinal malignancies and tive invasion at host-tumor interface, accompanying sent for intraoperative frozen section consultation. ductular reaction, pseudoangiomatoid appearance, Reactive atypia in biliary epithelium and peribiliary entrapped portal tract structures, and biliary in-situ glands is often associated with cholangitis, calcu- change. Metastases generally tend to be more mono- lous disease, stenting, PSC, and À uke infestation; it morphic.4 has to be distinguished from BilIN and malignant glands. Ductular reaction represents the restoration (ii) Hepatocellular and epithelioid patterns process at the damaged hepatocellular-stromal in- Areas with hepatoid/epithelioid features that 12 Aileen Wee, MB,BS, FRCPath, FRCPA, Yong Loo Lin School of Medicine

stain positively for CK7/CK19 are not uncommon nant foci developing within cystic anomalies.34-39 in pure ICCs.28 Another consideration is MHBC Targeted placement of the biopsy needle into [see section (iii)].1, 12 A nondescript epithelioid suspicious areas under radiological guidance may pattern is often encountered in poorly differentiated enhance the diagnostic yield (Fig. 11). A cystic ICCs (Fig. 3). In the event that one can only arrive appearance can be encountered in mucin-producing at a diagnosis of a poorly differentiated carcinoma, ICCs or metastases. Cystically dilated bile ducts can not otherwise speci¿ ed, the cytohistology report may result from obstructive ICCs. Extensive cavitating include the statement that “features are not speci¿ c necrosis from post-therapy effects or otherwise can for any primary site; correlate with clinical and result in cystic imaging appearances. radiological ¿ ndings”. Diagnostic utility of immunohistochemistry (iii) Mixed epithelioid-glandular (hepatobiliary) Adenocarcinomas present the most dif¿ - pattern culty in establishing their site of origin. Metastatic An ambivalent glandular/epithelioid adenocarcinoma is one of the most common tumors pattern may represent MHBC.1, 12 In the classic encountered in the liver with the majority originat- subtype, combined hepatocellular-cholangiocarci- ing in the , pancreaticobiliary noma (CHCC-CC), one must demonstrate the cyto- tract, lung, breast and gynecological tract (Table morphological and immunohistochemical presence 3). In these times where therapeutic protocols are of malignant hepatocytes, adenocarcinoma, and standardized based on the site of tumor origin and hybrid (transitional) elements (Fig. 10).1, 29-33 Sampling their molecular pro¿ les, it is noted that a diagnosis of error is a distinct pitfall in small tissue samples with adenocarcinoma may not suf¿ ce for clinical manage- the likelihood of the tumor being labeled as HCC, ment. In such cases, clinical information, imaging adenocarcinoma or a poorly differentiated carci- studies and ¿ nally judicious use of immunohisto- noma. chemical stains can help re¿ ne the histogenesis and pinpoint the likely site of origin. (iv) Predominant cell profi les The aim of immunohistochemistry in the The variants of ICCs may display polygo- evaluation of focal liver lesions with glandular nal, oncocytic, small cell, large cell, clear cell, features is two-fold, namely (i) to distinguish pleomorphic cell, spindle cell or giant cell features.4 primary ICC from metastatic adenocarcinomas, and Squamous component may be part of an adenos- (ii) to de¿ ne the various subsets of carcinomas in the quamous carcinoma commonly encountered in the liver that can be possible differential diagnosis.40-50 pancreaticobiliary tract. The differential diagnoses The initial immunohistochemical segregation of are listed in Table 1. adenocarcinomas is based on the coordinate expres- sion of CK7 and CK20, listed as follows:14 (v) Cystic pattern • CK7 + / CK20 + pattern: Transitional Fine needle aspiration of neoplastic cysts cell carcinoma, pancreatic adenocarcinoma, biliary such as MCN and biliary IPN with or without (hilar) adenocarcinoma, and ovarian mucinous associated malignant transformation, may not yield carcinoma totally diagnostic material; likewise for malig- • CK7 + / CK20 – pattern: Lung adenocar- Th e Many Faces (and Origins) of Adenocarcinoma in the Liver: Pattern Approach to 13 Diagnosis on Small Tissue Samples cinoma, pancreaticobiliary adenocarcinoma, ductal Of the cytokeratins, mature hepatocytes stain and lobular breast carcinoma, ovarian carcinoma of with CK8 and 18 and CAM5.2 but not with CK7, 19 serous and endometrioid type, endometrial or 20 or the commonly used CK cocktail, AE1/AE3. carcinoma, and () Intermediate hepatocytes may show lighter • CK7 - / CK20 + pattern: Colorectal with CK7 but not with CK19. CK7 and 19 highlight adenocarcinoma biliary epithelium (Fig. 12). Focal CK7 positivity • CK7 - / CK20 – pattern: HCC, renal can be seen in poorly differentiated HCCs. Hence, cell carcinoma, prostatic adenocarcinoma, lung the commonly used keratin antibodies (AE1/AE3 squamous cell carcinoma, and lung small cell and CK7) can be expressed in both HCC and carcinoma adenocarcinoma, limiting their value. CK7 and 20 • No predominant pattern: Gastric adeno- are more helpful in determining the primary site carcinoma once the diagnosis of adenocarcinoma has been established (Fig. 13). Immunohistochemical stains commonly Cytokeratin 19 is expressed in bile duct employed include polyclonal carcinoembryonic epithelium but not generally expressed in hepato- antigen (pCEA), CD10, MOC-31, and the cytes. CK19 is expressed in 85% to 100% of ICCs, cytokeratins.40-43 Polyclonal CEA highlights bile whereas most HCCs are either negative or show canaliculi but not hepatocytes in normal tissue. patchy staining, with the exceptiom of ¿ brolamellar Biliary epithelial cells show diffuse cytoplasmic and HCC. Mixed hepatobiliary carcinomas may contain brush border staining. In high-grade HCC, there is a variable proportion of progenitor cells. loss of canalicular expression but acquisition of In such instances, a bi-directional differentiation of cytoplasmic staining in about 50% cases. This neoplastic progenitor cell populations is noted with staining pattern poses a challenge in distinguishing expression of CK19 as well as CK8 and 18. HCC from metastatic adenocarcinomas. It is, therefore, important that this stain is utilized keeping SUMMARY in mind the morphological differential diagnosis. The diagnostic approach is ¿ rst to evaluate CD10 shows similar staining patterns. the tissue samples blinded to the clinical and radio- MOC-31, also known as epithelial speci¿ c logical data. Only after a cytomorphological impres- antigen, is present in the cytoplasm as well as on cell sion is established should one review the available surface in almost all epithelia, except in most squa- data, before deciding on what special stains and mous epithelia, hepatocytes, renal proximal tubular panel of immunohistochemical stains to perform as cells and gastric parietal cells. It is almost always there is usually limited material. The ¿ nal diagno- (80% - 100%) expressed in ICC and metastatic sis is based on close clinicopathological correlation. adenocarcinoma from a variety of sites, including Preparation of cell blocks is very useful for ancillary but not limited to carcinomas of the pancreas, breast, tests. lung and stomach. This antibody, hence, cannot dis- tinguish ICCs from metastatic adenocarcinomas to the liver. It is, however, either not expressed or is only weakly and focally expressed in HCC.44, 45 14 Aileen Wee, MB,BS, FRCPath, FRCPA, Yong Loo Lin School of Medicine

A B

C

Figure 1 Biliary epithelial changes; FNAB and core biopsy of liver. (A) Biliary intraepithelial neoplasia (BilIN): Large À at sheet of biliary epithelium begins to lose the orderly honeycomb appearance due to variation in nuclear size and shape and some nuclear overlapping. The nuclei have coarse chromatin and small nucleolus. Papanicolaou, x200. (B) Biliary adenocarcinoma: Sheets of malignant ductal epithelium show marked disorderly growth with crowding and nuclear overlapping. The variably-shaped nuclei display nuclear membrane irregularities with folds, chromatin clumping, increased nuclear-to-cytoplasmic ratio, and occasional nucleolus. The cyto- plasm is pale and vacuolated. Papanicolaou, x200. (C) BilIN and adenocarcinoma: A resident bile duct (right) exhibits low- to intermediate grade in- traepithelial neoplasia with abrupt nuclear atypia and strati¿ cation. Malignant glands invade the vicinity which shows desmoplasia and lymphovascular invasion. Hematoxylin & eosin, x100. Th e Many Faces (and Origins) of Adenocarcinoma in the Liver: Pattern Approach to 15 Diagnosis on Small Tissue Samples

A B

C D

Figure 2 Intrahepatic cholangiocarcinoma, well-differentiated; core biopsy of liver with imprint cytology. (A) Cluster of columnar epithelium with basal nuclei from a malignant gland. May-Grünwald-Giemsa, x400. (B) Clusters and strips of malignant columnar glandular epithelium with nuclear palisading. The nuclei exhibit pleomorphism, coarse chromatin and small nucleolus. Nonneoplastic hepatocytes are seen at the right lower quadrant. Papanicolaou, x200. (C) Well-differentiated malignant glands lined by columnar epithelium amid desmoplastic stroma. Hematoxylin & eosin, x100. (D) CK7 highlights the malignant glands. Immunostain, x100. 16 Aileen Wee, MB,BS, FRCPath, FRCPA, Yong Loo Lin School of Medicine

A B

Figure 3 Intrahepatic cholangiocarcinoma, poorly differentiated; core biopsy of liver. (A) There are sinuous trabeculae of tumor cells exhibiting eosinophilic cytoplasm and round hyper- chromatic nuclei. Occasional cytoplasmic vacuoles are discernible. Tumor necrosis is present. Hematoxylin & eosin, x100. (B) Left panel. Tumor cells contain intracytoplasmic mucin vacuoles. PAS-diastase, x 400. Right panel. CK19 highlights the tumor cells. Immunostain, x200.

A B

C

Figure 4 Intrahepatic cholangiocarcinoma, variants; core biopsies of liver. (A) Papillary carcinoma shows ¿ brovascular cores lined by well-differentiated malignant columnar epithelium. Hematoxylin & eosin, x100. Th e Many Faces (and Origins) of Adenocarcinoma in the Liver: Pattern Approach to 17 Diagnosis on Small Tissue Samples

(B) Signet ring adenocarcinoma cells show eccentric hyperchromatic nuclei and intracytoplasmic mucin vacuoles accompanied by desmoplasia. Hematoxylin & eosin, x200. Inset: CK7 highlights tumor cells. Immunostain, x 400. (C) Adeno¿ bromatous variant with pseudoangiomatoid pattern of growth shows intercommunicating spaces lined by largely À attened epithelium and separated by hyalinized collagenous stroma invaded by small malignant glands. Hematoxylin & eosin, x100.

A B

C

Figure 5 Adenocarcinomas from pancreas; FNAB and core biopsy of liver metastases. (A) Sheet of well-differentiated adenocarcinoma cells shows loss of monolayered honeycomb appear- ance with crowding and nuclear overlapping. Small nucleoli are discernible. Papanicolaou, x200. (B) Mucinous pools in which À oat small groups or dissociated malignant glandular cells with enlarged nuclei, high nuclear-to-cytoplasmic ratio and intracytoplasmic mucin vacuoles. May-Grünwald-Giemsa, x200. (C) Corresponding mucinous adenocarcinoma. Hematoxylin & eosin, x100 18 Aileen Wee, MB,BS, FRCPath, FRCPA, Yong Loo Lin School of Medicine

Figure 6 Adenocarcinoma from colorectum; FNAB of liver metastases. Left panel. Tightly cohesive clusters of malignant glands amid striking “dirty necrosis” (tumor diath- esis) in the background. Papanicolaou, x40. Right panel. Broken glands represented by strips of tall malignant columnar epithelium with basal pali- sading of oval, hyperchromatic nuclei containing distinct nucleolus. Papanicolaou, x400.

A B

C

Figure 7 Ductal carcinoma from breast; FNAB and core biopsy of liver metastases. (A) Cohesive clusters of monomorphous, plasmacytoid tumor cells with eccentric round nuclei. May- Grünwald-Giemsa, x200. (B) Plasmacytoid cells show tendency to dissociation with nuclear streaking artifact. Papanicolaou, x200. Th e Many Faces (and Origins) of Adenocarcinoma in the Liver: Pattern Approach to 19 Diagnosis on Small Tissue Samples

(C) Corresponding histology shows nests of a monomorphic carcinoma with suggestion of glandular differentiation. Note mitotic activity. Hematoxylin & eosin, x200 Inset: Intense complete membrane staining with c-erbB2. Immunostain. x200.

Figure 8 Acinar adenocarcinoma from prostate; FNAB of liver metastases. Rosette-like acinar groupings of polygonal well-differentiated tumor cells with vacuolated cytoplasm and regular round nuclei. Complex capillary network holding the tumor cells together like a bunch of grapes. Papanicolaou, x200.

A B

C 20 Aileen Wee, MB,BS, FRCPath, FRCPA, Yong Loo Lin School of Medicine

Figure 9 Neuroendocrine carcinoma from pancreas; core biopsy of liver metastases with imprint cytology. (A) Loosely cohesive aggregate of regular small round tumor cells with suggestion of rosetting and nuclear molding. May-Grünwald-Giemsa, x200. (B) A cluster of tumor cells showing rosette formation with nuclear palisading. May-Grünwald-Giem- sa, x400. (C) Corresponding histology shows nested tumor cells with suggestion of pseudoacinar formation. Hematoxylin & eosin, x200 Inset: Chromogranin highlights the neurosecretory granules in the cytoplasm. Immunostain. x400.

Figure 10 Combined hepatocellular-cholangiocarcinoma; liver resection. An intimate relationship between well-differentiated adenocarcinoma composed of tubules lined by columnar epithelium and sheets of malignant hepatocytes characterized by well-de¿ ned polygonal cells dis- playing clear cytoplasm and central round nucleus with irregular nuclear contours. Representative sampling is an issue in small tissue samples which may not include both elements which are required for this diagnosis. Hematoxylin & eosin, x100 Th e Many Faces (and Origins) of Adenocarcinoma in the Liver: Pattern Approach to 21 Diagnosis on Small Tissue Samples

Figure 11 Polycystic liver disease with associated invasive adenocarcinoma; FNAB of liver. Folded sheets of crowded lining epithelial cells with loss of honeycomb appearance. The cells exhibit increasing pleomorphism with variably-shaped nuclei, nuclear membrane irregularities, coarse chromatin, dis- tinct nucleolus, and high nuclear-to-cytoplasmic ratio. Some dissociated tumor cells contain intracytoplasmic vacuoles. The background shows much necrotic debris. Papanicolaou, x100.

Figure 12 Moderately differentiated adenocarcinoma, primary versus metastasis; core biopsy of liver. CK7 highlights the adenocarcinoma. Residual ductules and bile ducts are intensely stained. No biliary identi¿ ers are discernible. Immunostain, x100. 22 Aileen Wee, MB,BS, FRCPath, FRCPA, Yong Loo Lin School of Medicine

A B

C D

Figure 13 Poorly differentiated adenocarcinoma, primary versus metastasis; FNAB and core biopsy of liver. (A) Cluster of highly pleomorphic malignant cells with high nuclear-to-cytoplasmic ratio, enlarged nuclei and occasional binucleation. May-Grünwald-Giemsa, x400. (B) Highly pleomorphic tumor cells with tendency to dissociation. The cytoplasm is fragile and vacu- olated. Note “poly bag” (left) and necrosis. Papanicolaou, x200. (C) Corresponding histology shows nests and trabeculae of tumor cells with eosinophilic cytoplasm. Hematoxylin & eosin, x100 (D) The tumor cells are immunoreactive for both CK7 (left panel) and CK20 (right panel). Hilar chol- angiocarcinoma is a possibility. Immunostains, x200. Th e Many Faces (and Origins) of Adenocarcinoma in the Liver: Pattern Approach to 23 Diagnosis on Small Tissue Samples

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