Introduction

As the role of fine-needle aspiration biopsy (FNAB) in the evaluation of focal liver lesions has evolved, it has presented new challenges.^1,2 Advances in dynamic imaging modalities have obviated the need for tissue confirmation in clinically classic cases of hepatocellular carcinoma (HCC).^3–12 Ultrasound surveillance and serum α-fetoprotein (AFP) assessment of high-risk patients have improved the detection of small nodules (≤2 cm in diameter).^13–16 Accurate cytohistologic characterization of small, well-differentiated hepatocellular nodules on limited tissue samples is extremely challenging, but it has important therapeutic implications.

Much progress has been made in the molecular characterization of tumors by genomic, microRNA, and proteomic methods. There are promising new tools for tumor diagnosis, for predicting the prognosis and progression of disease, and for personalized, molecularly targeted anticancer therapies and chemopreventive strategies.^17–26 Because FNAB is the least invasive tool for tissue procurement, it is predicted that it will become a point of care in the management algorithm of HCC for diagnostic, therapeutic, and prognostication purposes.²⁷

Percutaneous (transabdominal) FNAB performed under ultrasound or computed tomography (CT) guidance is a safe, efficacious, and cost-effective outpatient procedure for the diagnosis of focal liver lesions.^28–30 Aspiration can also be performed during laparotomy or laparoscopy under palpation or direct vision. Endoscopic ultrasound-guided FNA (EUS-FNA) is the latest diagnostic and staging tool and is used primarily for left hepatic lobe lesions and hilar or perihilar masses, in which the needle traverses the wall of the gastrointestinal tract.³¹ Factors influencing the choice of guidance system include the size and location of the lesion, operator expertise and preference, and availability of imaging technologies.^27–29

Ultrasound provides rapid localization, flexible patient positioning, and imaging of the lesion without radiation. It is typically used for initial guidance, particularly when there are multiple lesions or large, relatively superficial lesions. CT allows optimal resolution of small lesions or lesions not visible with ultrasound, accurate localization of the needle tip immediately before sampling, improved definition of tissue components and vascularity, and more precise demonstration of the anatomic relationships of a given lesion.

EUS-FNA is a safe, accurate, and versatile technique, but it is highly operator dependent.^32–36 It allows concurrent sampling of pancreas and liver lesions, which can confirm primary and metastatic lesions in one diagnostic encounter. The technique is useful for small and deep-seated left lobe lesions that are below CT or magnetic resonance imaging (MRI) resolution or lesions that are not easily accessible by percutaneous FNAB. It improves staging of liver metastases, is good for early detection of multifocal HCC in cirrhosis, and can assess the accurate number of lesions (e.g., intrahepatic staging of HCC) for transplantation eligibility.^37,38

Percutaneous FNAB techniques include individual puncture, coaxial biopsy, and tandem needle biopsy techniques.³⁹ Multiple aspirations (as many as four passes) usually can be performed with minimal morbidity. The needle size is between 20 and 22 gauge. Aspiration needles with a guillotine mechanism enable microbiopsy cores to be procured at the same sitting. Concomitant core-needle (18-gauge) biopsies may also be performed.

Diagnostic Issues

Focal liver lesions range from cystic and inflammatory or infectious entities to benign or malignant primary and metastatic neoplasms. HCC is well known for its heterogeneity. Intrahepatic cholangiocarcinoma (CC) frequently shows nondescript features of adenocarcinoma. The liver is a common depository for metastases. Given these permutations, primary liver carcinomas can mimic many tumors and vice versa. The diagnostic issues are listed in Box 45.3.

Cytohistology of the Liver

Normal Morphology

Aspirate smears of normal liver typically show a polymorphous cell population dominated by hepatocytes, occurring singly in short rows or small clusters and intermingling with scattered bile duct epithelial cells and sparse sinusoidal endothelial cells and Kupffer cells (Fig. 45.1, A). Normal hepatocytes are large, polygonal cells with abundant granular cytoplasm and a centrally placed, round nucleus with a well-delineated nuclear membrane, granular chromatin pattern, and distinct nucleolus. Hepatocytes normally exhibit variations in cell and nuclear size (i.e., sibling polymorphism). The nucleus-to-cytoplasm ratio (N : C ratio) is approximately 1 : 3. Bile duct epithelial cells are smaller than hepatocytes and appear as flat, monolayered, honeycomb sheets of glandular epithelium (see Fig. 45.1, B). They may also reveal an on-edge picket-fence arrangement or small acinar structures. The cells have minimal pale cytoplasm and equidistant, round to oval, bland nuclei without nucleoli. Bile duct epithelium can be differentiated from bile ductular epithelium.

Elongated endothelial cells and comma-shaped Kupffer cells are almost indistinguishable from each other, especially when they appear as nuclear streaks. The thickness of liver cords can be appreciated in large sheets of cells and in fragments of liver parenchyma by focusing on the nuclear streaks in different planes of magnification (Fig. 45.2).

Pigments

Lipofuscin is a fine, golden, granular, relatively nonrefractile pigment with Papanicolaou stain and is black with Giemsa stain (Fig. 45.3). This predominantly perinuclear pigment is common in aspirates of adult livers, and its absence in the context of a mass lesion is considered suspicious for a hepatocellular neoplasm. One potential diagnostic pitfall is that lipofuscin stains similar to melanin with Fontana-Masson stain, and the sample may be mistaken for metastatic melanoma.⁷⁶

Bile pigment varies in color, texture, size, and density, but it is best recognized by its coarse, irregular, rather amorphous, nonrefractile appearance. In Giemsa-stained preparations, bile appears as a purplish-black pigment within the cytoplasm of hepatocytes or as ropey strands within bile canaliculi (Fig. 45.4). Hall stain may be used to confirm the pigment as bile.

Iron or hemosiderin may be present in hepatocytes, bile duct epithelial cells, and Kupffer cells as a coarse, golden-brown, refractile pigment with the Papanicolaou stain and brown-black with the Giemsa stain (Fig. 45.5). Malignant hepatocytes typically lose their ability to retain iron. In the setting of hemochromatosis, Perl (Prussian blue) stain is a helpful ancillary tool to highlight nonstaining tumor cells.^77,78

Steatosis

Steatosis is common in the liver, and the examiner should be aware of focal fatty changes.^79,80 Cytologically, fat may be a single, large, intracytoplasmic vacuole in a cell with an eccentric nucleus (i.e., macrovesicular steatosis) or multiple, small vacuoles in a cell with a central nucleus (i.e., microvesicular steatosis) (Fig. 45.6). Fat is best appreciated in Giemsa-stained preparations, which often reveal abundant oil globules in the background as a result of ruptured hepatocytes.

Infections

Liver abscess formations are suspected radiologically by the characteristic double target sign on CT scans.⁸¹ The most common is pyogenic abscess, in which case aspiration is performed for tissue confirmation, culture, and drainage.^82,83 Smears are dominated by acute inflammatory cells and cellular debris (Fig. 45.7). Cultures are helpful for identification of bacterial organisms. Special stains, such as Gram stain, are not usually helpful, but uniquely characteristic organisms may be readily identified on smears. For example, Actinomyces has sulfur granules (Fig. 45.8).

Smears from suspected fungal abscesses should be stained with Gomori methenamine silver and periodic acid–Schiff (PAS) stains. Amebic abscesses tend to be paucicellular with abundant amorphous granular debris, and trophozoites are rare. Necrotizing eosinophilic granulomas contain abundant eosinophils and possibly Charcot-Leyden crystals. Parasitic or fungal organisms should be sought (Fig. 45.9).

A potential diagnostic pitfall is that necrotic tumors may be associated with fever, simulating abscesses clinically and radiologically;⁸⁴ or they may become secondarily infected. If clinically warranted, aspiration cytology is recommended to avoid delay in instituting appropriate therapy.

Hydatid disease leads to cyst formation, which may become secondarily infected (see “Cystic Lesions of the Liver”). Echinococcosis is usually ruled out with clinical and imaging studies and the findings of a negative test result for specific echinococcal antibodies by serum enzyme immunosorbent assay performed before percutaneous needle aspiration.^85,86 In the event that a hydatid cyst is aspirated, the pathologist should search for protoscolices or hooklets of Echinococcus granulosus in the fluid or pus (Fig. 45.10).

Granulomatous inflammation may be caused by fungal or acid-fast organisms, but the presence of granulomas is not necessarily diagnostic of an infection. Cytologically, granulomas are composed of clusters of epithelioid histiocytes that have oval to elongated, sometimes twisted nuclei and visible but indistinct cytoplasm. Multinucleated giant cells may be present (Fig. 45.11). The characteristics of the necrotic background should also be evaluated. Gomori methenamine silver, PAS, and Ziehl-Neelsen stains may be performed on smears but are easier to perform on tissue sections.

Inflammatory pseudotumors are potential clinical, radiologic, and cytohistologic pitfalls because they mimic tumors. They may be lymphoplasmacytic, fibrohistiocytic, xanthogranulomatous, or granulomatous in nature (Fig. 45.12).^87–90 Activated lymphoid cells may be mistaken for lymphomas, atypical reactive hepatocytes for HCC, atypical reactive biliary epithelium for adenocarcinoma, atypical reactive stroma for sarcoma, and foamy histiocytes for amebic trophozoites. Immunoglobulin G4 (IgG4)–related disease may rarely manifest as a fibroblastic mass with lymphoplasmacytic infiltrates, eosinophils, IgG4-positive plasma cells, dense fibrosis of large bile ducts, and obliterative phlebitis.⁹¹

Nodular hematopoiesis is a rare diagnostic pitfall. Hematopoietic cells (especially megakaryocytes) may mimic inflammatory or malignant cells when perceived out of context.^92,93

Cystic Lesions of the Liver

The type of cyst contents should be evaluated because the presence of mucin is a helpful diagnostic clue. The next step is to establish the presence and type of epithelial lining and whether it is neoplastic or malignant.⁹⁴ Box 45.6 provides a diagnostic algorithm for cystic lesions of the liver.

Cystic Lesions with an Epithelial Lining

Bile Duct Cyst, Simple Hepatic Cyst, and Fibropolycystic Disease

In cases of bile duct cyst, simple hepatic cyst, and fibropolycystic disease, the fluid may contain isolated cells or scant strips or sheets of cuboidal to low columnar epithelium without cytologic atypia (Fig. 45.13).

Ciliated Foregut Cyst

Aspirates of foregut cysts are characterized by ciliated columnar cells and detached ciliated tufts.^95–97 The background usually contains amorphous debris and rare macrophages. The differential diagnosis includes cystic neoplasms.

Mucinous Cystic Neoplasm and Intraductal Papillary Neoplasm

Mucinous cystic neoplasms and intraductal papillary neoplasms have overlapping cytomorphologic features. The cytodiagnosis may indicate a benign cyst, papillary neoplasm, or adenocarcinoma. FNAB alone cannot differentiate the various types of hepatobiliary cysts.^94,98 Generic terms such as cystic mucinous neoplasm or neoplastic mucinous cyst may be applied if imaging studies are not available for correlation.

Mucinous cystic neoplasms typically occur as single or multiloculated, mucinous, epithelial-lined cysts surrounded by ovarian-type stroma.⁹⁹ The cyst fluid may be watery or viscous. Diagnostic aspirates likely contain degenerated cuboidal to columnar epithelial cells with bland nuclear features (Fig. 45.14).¹⁰⁰ The neoplastic epithelial cells may be arranged singly, on edge, in sheets, or as small papillary tufts. The background usually shows mucin with chronic inflammatory cells, histiocytes, and debris. The degree of cellularity varies and tends to increase with higher grades of intraepithelial neoplasia. Stromal components are not usually present. With malignant transformation, features of adenocarcinoma with or without mucin and cellular debris are often evident.¹⁰¹ The definitive diagnosis requires correlation with clinical information, imaging studies, and histopathologic confirmation of ovarian-type stroma surrounding the cyst.⁹⁴ The cyst fluid may be assayed for carcinoembryonic antigen and CA 19-9 to distinguish neoplastic from non-neoplastic liver cysts (Box 45.8).^98,102,103

Box 45.8

Mucinous Cystic Neoplasm

• Degenerated mucin-secreting cuboidal to columnar epithelium with bland nuclear features; arranged singly, on edge, in sheets or as small papillary tufts

• Background usually shows mucin with chronic inflammatory cells, histiocytes, and debris

• Mesenchymal stromal components are usually absent

• Variable cellularity, depending on grades of intraepithelial neoplasia

• Features of adenocarcinoma seen with malignant transformation

• Assay of cyst fluid for carcinoembryonic antigen and CA 19-9

Biliary intraductal papillary neoplasm is a fusiform or cystic (unilocular or multilocular) luminal dilation in communication with the biliary tract. The affected intrahepatic bile ducts are typically filled with a noninvasive papillary or villous biliary neoplasm covering delicate fibrovascular stalks, and mucin may be seen. Aspirates are typically hypercellular, comprising broad and often double-layered sheets of ductal cuboidal or columnar epithelium with a distinctive three-dimensional, complex-branching papillary configuration (Fig. 45.15).¹⁰⁴ The sheets of cells do not form a honeycomb pattern but are crowded. Phenotypes include pancreaticobiliary, intestinal, gastric, and oncocytic forms. The epithelium shows intracytoplasmic mucin vacuoles and occasional signet ring cells. The nuclei show variable abnormalities. Psammoma bodies may be identified. Intraductal papillary neoplasm with associated invasive carcinoma has the highest degree of cellularity and pleomorphism (Box 45.9).

Box 45.9

Biliary Intraductal Papillary Neoplasm

• Hypercellular smears

• Broad, double-layered sheets of ductal columnar epithelium with three-dimensional, complex-branching papillae

• Pancreaticobiliary, intestinal, gastric and oncocytic phenotypes

• Crowded sheets of cells with variable nuclear atypia

• Intracytoplasmic mucin vacuoles, including few signet ring cells

• Psammoma bodies

• Features of adenocarcinoma seen with malignant transformation

Metastases

Metastatic papillary carcinomas of the pancreas and ovary can simulate intraductal papillary neoplasms. Smears may contain numerous finger-like fibrovascular stalks lined by cuboidal or columnar epithelium with cytologic atypia.

Cystic Lesions without an Epithelial Lining

Non-neoplastic Cysts

Aspirates of non-neoplastic cysts typically reveal an abundant amount of turbid, brown fluid with inflammatory cells, macrophages, fibrin, debris, and bile or hematoidin pigment. The fluid lacks lining epithelial cells but may contain benign cells from surrounding liver parenchyma. The differential diagnosis includes inflammatory/infectious cysts, cystic hematomas, as well as true cysts and cystic neoplasms. Secondary bacterial infection may produce cyst fluid that contains abundant neutrophils and proteinaceous background debris. However, the presence of a neoplastic lining epithelium or background mucin excludes the diagnosis of a non-neoplastic cyst.

Neoplastic Cysts and Tumor-like Lesions

Cavernous Hemangioma

Cavernous hemangiomas are usually asymptomatic, and they are usually diagnosed incidentally during the workup for other conditions, such as during the process of staging a malignancy, and they can be difficult to distinguish from metastasis radiologically.^105–108 Hemorrhagic aspirates are often considered unsatisfactory. The nondiagnostic appearance of blood with loose rather than dense fibrous connective tissue and few or no background hepatocytes raises the possibility of a hemangioma in the proper clinical setting (Fig 45.16). Histologic material is crucial in establishing a specific diagnosis (Box 45.10).

Box 45.10

Cavernous Hemangioma

• Bloody with scant cellularity on smears

• Loose connective tissue; usually few to no hepatocytes

• Smears commonly nondiagnostic; specific diagnosis depends more on cell block pattern

Mesenchymal Hamartoma

Aspirates of mesenchymal hamartomas are obtained mainly in the pediatric population, with rare exceptions, and usually show clusters of epithelial and mesenchymal or spindle-shaped cells with bland-appearing nuclei admixed with loose connective tissue (Fig 45.17).^109–112 However, aspiration cytology rarely enables a definitive diagnosis, and because hepatoblastomas or malignant mesenchymal tumors are difficult to exclude confidently, cytology is of limited value (Box 45.11).

Box 45.11

Mesenchymal Hamartoma

• Hypocellular smears

• Spindle-shaped cells with bland nuclei

• Loose myxoid matrix with bile ductlike epithelial clusters

• Cytology of limited value in establishing a definitive diagnosis

Cystic Degeneration in a Benign or Malignant Tumor

Solid tumors may undergo central necrosis and cavitation, simulating cysts radiologically. A necrosis-associated tumor should raise the suspicion of an abscess.⁸⁴ Necrosis is more likely to occur in malignant lesions, such as squamous cell carcinomas, neuroendocrine tumors, and metastases from colorectal adenocarcinomas and nonkeratinizing undifferentiated carcinoma in the nasopharynx. The diagnostic cells may be obscured by the process.

Undifferentiated Embryonal Sarcoma

Although most undifferentiated embryonal sarcomas appear ultrasonographically as solid lesions, CT and MRI imaging may detect a low-attenuation mass with a cystic appearance. Most cystic structures result from liquefactive necrosis and blood clots; however, true cystic spaces may also occur.⁷⁵ Aspirates recapitulate the histologic pattern of the tumor.^113–117 Smears are usually composed of spindle cells and myxoid tissue, intermixed with pleomorphic cells ranging from small round cells to larger multinucleated giant cells. Giant cells have bizarre nuclei and massively deranged chromatin structure. They contain intracytoplasmic vacuoles and are AFP negative, PAS positive, but diastase-resistant hyaline globules (Fig. 45.18); extracellular globules may also be present. Immunostaining for α₁-antitrypsin, α₁-antichymotrypsin, and carcinoembryonic antigen is positive in tumor cells. The cytologic differential diagnoses include rhabdomyosarcoma, hepatoblastoma, undifferentiated pleomorphic sarcoma, NOS (so-called malignant fibrous histiocytoma), and poorly differentiated HCC (Box 45.12).

Box 45.12

Undifferentiated Embryonal Sarcoma

• Hypercellular smears

• Large, pleomorphic anaplastic cells with multinucleated giant cells and atypical spindle cells

• Intracytoplasmic globules: periodic acid–Schiff positive, diastase resistant

Benign hepatocellular nodules, which can be non-neoplastic or neoplastic, such as focal fatty changes, large regenerative nodules, dysplastic nodules, focal nodular hyperplasia, and hepatocellular adenomas, are discussed together because they share common cytologic features, including fatty changes (Box 45.13).^22,118,119 Unfortunately, it is not always possible to distinguish these entities on the basis of smear cytology alone. They all can mimic well-differentiated HCC.

A focal fatty change may simulate a mass lesion radiologically.^79,80 Aspirates are composed of a polymorphous population of enlarged, fat-laden, non-neoplastic hepatocytes intermingled with bile duct epithelial clusters. Oil globules may be seen in the background on air-dried smears.

Aspirates from large regenerative nodules are similar to those from cirrhosis, in which a polymorphous liver parenchymal population shows evidence of hyperplasia and hepatocellular-interface restoration (i.e., bile ductular epithelium and stromal fragments).^63,72 Hyperplastic hepatocytes occur in two-cell-thick cords and exhibit slight polymorphism and binucleated forms. Normal bile duct epithelial clusters should be differentiated from bile ductular epithelial cells, which appear as curved, narrow, double-stranded rows of cells with overlapping, dark, oval nuclei; scant or absent cytoplasm; and a high N : C ratio. Nuclear disarray may be disconcerting. Ductules can be seen emanating from the jagged edges of sizeable parenchymal fragments (Fig. 45.19). Stroma may be seen in the background. The presence of ductules should raise the diagnostic threshold for a malignant process.

Hepatocellular atypia, such as large cell and small cell changes (e.g., dysplasia), are considered neoplastic precursor lesions and may be detected in aspirates from cirrhotic livers.^{75,78,119–125} Hepatocytes with large cell changes show corresponding cellular and nuclear enlargement and mild nuclear atypia; but the normal N : C ratio (i.e., one to three or less) is maintained (Fig. 45.20). Hepatocytes with small cell changes are small and monotonous, with a subtle increase in the N : C ratio. The nuclear size is similar to that of normal hepatocytes, but there is less cytoplasm, imparting an impression of nuclear crowding (Fig. 45.21).¹²⁶

Aspirates of low-grade dysplastic nodules show hepatocytes that exhibit large cell changes, whereas those from high-grade dysplastic nodules show hepatocytes with small cell changes. Dysplastic hepatocytes tend to occur singly or in narrow cords, usually one to two cells thick. Transgressing endothelium is not a feature. It is almost impossible to distinguish a small cell change from a well-differentiated or early HCC.^126,127 The key to recognizing a large cell change as benign is the sporadic placement of these cells in a background of otherwise reactive-appearing hepatocytes and accompanied by bile ductal or ductular epithelium and stroma. Availability of histologic material for immunohistochemistry is invaluable.

Patients with cirrhosis without iron overload can accumulate iron within regenerative or dysplastic nodules, which are called siderotic nodules. Siderotic dysplastic nodules are considered to be premalignant, whereas siderotic regenerative nodules are a marker for severe viral or alcoholic cirrhosis.^73,74 Iron-free foci in patients with genetic hemochromatosis are likely to be malignant.^77,78

Focal nodular hyperplasia is characterized by a proliferation of hepatocytes surrounding a central scar containing blood vessels and bile ducts. A brisk ductular reaction is often seen at the hepatocellular-stromal interface.¹²⁸ Aspirates typically contain a polymorphous population of cells, but this varies.¹²⁹ Hyperplastic hepatocytes abound in one- to two-cell-thick cords. Bile duct epithelium is usually evident (Fig. 45.22). The presence of ductular epithelium supports the benign nature of this lesion, but this feature can be mistaken for adenocarcinoma if it is the dominant component of the aspirate. A possible diagnostic pitfall is mistaking reactive stroma for a spindle cell neoplasm.¹³⁰ A characteristic geographic immunostaining pattern is seen with the use of glutamine synthetase.¹³¹ Without radiologic demonstration of a central scar, it may not be possible to differentiate focal nodular hyperplasia from large regenerative nodules on smear cytology, because both show features of regeneration.

Aspirates of a hepatocellular adenoma are hypercellular and typically contain well-differentiated hepatocytes occurring in one- to two-cell-thick cords (Fig. 45.23). There is no significant atypia. Transgressing endothelium is common, but peripheral endothelium is lacking. The tumor cells are polygonal and have moderate granular cytoplasm. Some cells display fatty or clear cell changes and bile or lipofuscin pigment. Bile duct epithelium is absent. The reticulin framework is usually well preserved. CD34, which highlights capillarization of the sinusoids in HCC, can also positively stain adenomas, particularly in the periseptal areas. This may be a diagnostic pitfall in assessing small, fragmented liver core specimens and cell block preparations. Glypican 3 staining should be focal and weak at best.^132–134 The diagnosis requires clinical correlation. Chapter 55 provides more information on immunohistochemical markers of adenomas in tissue sections.¹³⁵

A nodule-in-nodule lesion occurs when an HCC subnodule develops within a parent dysplastic or regenerative nodule.¹³⁶ Representative FNAB samples may be a problem because of the focality of proliferative clones within these nodules. Classic aspirates reveal more than one population of lesional hepatocytes, including malignant hepatocytes and dysplastic or hyperplastic hepatocytes in the background (Fig. 45.24). The presence of biliary epithelial cells varies.