Liver and bile duct cytology by Dr. Alpha Tsui

-abundant neutrophils-granular necrotic background-organisms may be present (beware amoeba mimicking a macrophage)-small numbers of reactive hepatocytesHydatid cyst:-may show laminated cyst membrane and refractile hooklets (Ziehl-Neelsen andMasson trichrome stains positive)-generally believed that FNA is contraindicated because of potential anaphylacticreaction from intra-abdominal spillage-eosinophils may be prominent-calcification implies that the parasites are non-viableCirrhosis:-relatively scanty material from a cirrhotic nodule-usually not the monotonous uniform population of cells as in hepatocellularcarcinoma. Some are normal while others are atypical.-dispersed single hepatocytes may be seen-nuclei of hepatocytes often show variation in size with prominent nucleoli andbinucleation-lack of nuclear irregularity, hyperchromasia, anaplasia, high N/C ratio-there may be apoptotic cells-fibroblasts are variably present, intermingling with hepatocytes-fatty change may be seen-neutrophils and lymphocytes are present in variable amountsCharacteristic feature of cirrhosis is the variable polymorphicappearance of the hepatocytes within the same cell clusters. In HCC, there isusually a more uniform population of abnormal hepatocytes.Macroregenerative nodule / dysplastic nodule:-atypical hepatocytes (increased N/C ratio, hyperchromatic nuclei, prominentnucleoli) in small numbers, intermixed with many benign-appearing hepatocytes withlow N/C ratio-variability in the appearance of hepatocytes is important feature-no thickened trabeculae >2 cell thickFocal nodular hyperplasia:-fragments of fibrous connective tissue with thick-walled blood vessels and bile ductsare seen-otherwise, the appearance is similar to that in an adenomaLiver cell adenoma:-numerous closely packed 3-D groupings of hepatocytes-no bile duct epithelial cells or fibroblasts present-hepatocytes are normal appearingHepatocellular carcinoma:-usually highly cellular

-tissue fragments, tightly packed cohesive cell clusters, loose groupings, single cells-often in widened trabecular pattern (>2 cells thick), rounded islands, cell balls-endothelial rimming of the trabeculae-well-defined capillaries traversing fragments a feature-the tumour cells are “hepatocyte-like”-regular, uniform, centrally located round nuclei, increased N/C ratio, finely granularchromatin-intranuclear pseudoinclusions-nucleoli usually conspicuous-stripped atypical nuclei commonly seen in the background-intracytoplasmic bile may be found (greenish-black with Diff-Quik; green or yellowwith Pap). May see bile canaliculi, in-between the neoplastic hepatocytes.-lipofuscin and iron pigment usually absent-mitoses and multinucleation occasionally seen-cell block: widened trabeculae, rounded islands, acini. Loss of reticulin fibres (fibreloss may not be prominent in early well-differentiated tumours).-most helpful features of HCC: trabecular arrangement, high N/C ratio, intranuclearpseudoinclusions, stripped atypical nucleiWell-differentiated HCC is difficult to be distinguished from benignhepatocytes. Reticulin stain is most helpful. Poorly-differentiated HCC may mimica high grade metastatic carcinoma. Look for bile, trabeculae, sinusoidal capillaries,intranuclear pseudoinclusions, atypical stripped nuclei.Fibrolamellar HCC:-often showing cellular dyscohesion-tumour cells are large with uniform round nuclei, prominent nucleoli, low N/C ratioand abundant granular oncocytic-like cytoplasm (EM showing numerousmitochondria)-intracytoplasmic hyaline globules sometimes seen-fragments of layered collagen present-bile pigment usually not identifiedClear cell HCC:-atypical cells with abundant clear cytoplasm with vacuolation and well-defined cellborders-mucin stains negativeSmall cell HCC:-neoplastic cells with high N/C ratio with conspicuous nucleoli (nucleoli not usuallyprominent in metastatic small cell carcinoma)Sarcomatoid HCC:-large cohesive groups without forming trabeculae-pleomorphic spindle cells with large nucleoli and elongated cytoplasmAngiosarcoma:-bloody and necrotic background

-loose groupings, singly-round or ovoid nuclei-scanty cytoplasm-some cells may appear spindle shaped and have elongated nuclei-large, bizarre-appearing neoplastic cells with intracytoplasmic haemosiderin orerythrocytes may sometimes be seenMetastases:-common metastatic tumours to the liver: GIT, pancreas, breast, lung.Clues to determine primary sites:-lower gastrointestinal tract: peripheral palisading of the groups; columnar cells thatare pseudostratified; dirty necrosis in the background-stomach: signet-ring cells-breast: Indian files with nuclear moulding; targetoid inclusions-bladder: cercariform cells showing cytoplasmic process with flattened endMetastatic neuroendocrine carcinomas:-commonly from pancreas, GIT, lung-ranging from well-differentiated to poorly differentiated (small cell carcinoma)-cohesive groups, rosettes, single cells-tumour cells with enlarged round nuclei, nuclear moulding, granular chromatin,inconspicuous nucleoli and scanty to moderate amounts of granular cytoplasm-apoptotic debris and necrosis common, esp. in small cell carcinomasFALSE POSITIVES:-reactive changes from inflammation, cirrhosis-florid ductular reaction mimicking adenocarcinomaFALSE NEGATIVES:-well-differentiated hepatocellular carcinoma e.g. fibrolamellar variant with low N/Cratio tumour cellsBile ducts:Normal ductal cells:-cells from large ducts: cohesive monolayered sheets with evenly spaced nuclei, stripsof palisaded columnar cells may be seen-cells from small ducts: cuboidal and arranged in small clusters, sheets or in smallintact tubular structures-nucleus has round smooth distinct border-evenly distributed fine chromatin, inconspicuous nucleoli-nuclei throughout the sheets are similar in size with maintained polarity-abundant cytoplasm which may be vacuolatedDysplasia:-sheets containing cells with nuclear crowding and overlapping-single atypical cells not usually seen

-increased N/C ratio with mild chromatin coarsening and small nucleoliCholangiocarcinoma:-a diagnosis of exclusion. Needs to exclude metastatic adenocarcinoma clinically.-may have low cellularity as tumour shows extensive desmoplasia-cohesive groups, 3-D crowded sheets, acini and singly-high N/C ratio, nuclei with irregular nuclear membranes, coarse chromatin,prominent nucleoli-marked nuclear overlap, moulding and crowding-nuclear moulding, chromatin clumping, increased N/C ratio, loss of honeycombingmost important criteria of malignancy-cuboidal to columnar shaped cytoplasm which may be vacuolated (containing mucin)-necrotic debris less commonly seenFALSE POSITIVES:-reactive changes due to sclerosing cholangitis, stent placement, postoperative effect,parasitic infection, bile duct stones-pancreatitis-reactive papillary change, various metaplasiaFALSE NEGATIVES:-sampling error (small lesion size and difficult location)-extensive fibrosis-benign epithelium overlying lesion-subtle changes of well-differentiated adenocarcinomas-other bland-looking neoplasms e.g. mucinous and papillary intraductal lesionsPRACTICAL TIPS:1. Check clinical history, radiology (presence of stone, stent, recent ductinstrumentation, cirrhosis).2. Important to know the architecture of normal liver. The diagnosis of HCC is madeon altered architecture, much more than the cytological features of the cells.3. Presence of peripherally arranged endothelium around the trabeculae andtransversing endothelium into sheets of hepatocytes are specific features of HCCand they are absent in cholangiocarcinoma and metastatic carcinoma.4. The tumour cells in fibrolamellar HCC show low N/C ratio but the overall size ofthe cells is much larger than a normal hepatocyte.

5. Poorly differentiated HCC is difficult to be distinguished from metastatic tumours.Look for better differentiated tumour cells with hepatocyte-like features.6. If there are sufficient cells, reticulin stain done on a cell block is useful todistinguish benign from malignant hepatocellular lesions. Immunostains helpful todistinguish HCC from metastatic carcinoma are: Polyclonal CEA, CD10, Hep-Par1, AFP, CK7 and CK20.

Fig 1. Normal hepatocytes.Fig 2. Cirrhosis with reactive hepatocytes. Note random nuclear enlargement butoverall low N/C ratio.

Fig 3. Hydatid cyst. Ziehl-Neelsen positive staining hooklet.Fig 4. Hepatocellular carcinoma. Trabecular arrangement.

Fig 5. Hepatocellular carcinoma. Trabeculae, lined by sinusoids with flattenedendothelial cells (Arrow).Fig 6. Hepatocellular carcinoma. Acinar arrangement containing bile.

Fig 7. Hepatocellular carcinoma. Cells with conspicuous nuclear atypia.Fig 8. Hepatocellular carcinoma (red arrow). Compare with normal (green arrow).

Fig 9. Bile canaliculi: Diagnostic for hepatocellular carcinoma (Arrow).Fig 10. Hepatocellular carcinoma, often containing many atypical bare nuclei in thebackground.

Fig 11. Hepatocellular carcinoma with loss of reticulin fibres.Fig 12. Fibrolamellar HCC with multilayered collagen fibres (Arrow).

Fig 13. Fibrolamellar HCC. Mainly single large tumour cells with low N/C ratio andabundant cytoplasm.Fig 14. Metastatic colonic adenocarcinoma. Note palisaded edge with columnar cells(Arrows).

Fig 15. Metastatic neuroendocrine carcinoma from the pancreas. Note rosettes.Fig 16. Metastatic small cell carcinoma. Not reactive hepatocytes on the left.

ReferencesYang G, Tao L. Transabdominal fine-needle aspiration biopsy. edition. Worldscientific. 2007.Geisinger KR et al. Fine needle aspiration cytology of primary liver neoplasms.Pathol Case Reviews 1999;4(4):169-175Silverman JF et al. Fine needle aspiration cytology of small cell neoplasms of theliver. Pathol Case Reviews 1999;4(4):182-186Selvaggi SM. Biliary brushing cytology. Cytopathology 2004;15:74-79