Cytologic features and histologic correlations of microacinar and microtrabecular types of well‐differentiated hepatocellular carcinoma in fine‐needle aspiration biopsy

Well‐differentiated hepatocellular carcinoma (HCC) in fine‐needle aspiration (FNA) biopsy is characterized by trabeculae three or more cells thick wrapped by peripheral endothelium. The authors encountered another pattern that did not fulfill these classic criteria for malignancy yet was proven to be HCC in clinical follow‐up. The objective of this study was to characterize the cytologic features of this pattern with histologic correlations.

lae may be only a few cells thick (Ն 3 cells thick), termed microtrabeculae, or Ͼ 10 -20 cells thick, termed macrotrabeculae. 9 The diagnostic criteria for well-differentiated HCC in FNA cytology was described as early as 1984 10 and has been confirmed by numerous studies. [11][12][13][14][15][16][17] Welldifferentiated HCC is characterized by a hypercellular smear comprised of trabeculae of three or more cells wrapped by peripheral endothelium. We encountered an unusual type of well differentiated HCC with cytologic features that did not fulfill these diagnostic criteria yet was proven to be HCC in clinical follow-up.
The objective of the current study was to accumulate a series of such cases and then characterize their cytologic and histologic features.

MATERIALS AND METHODS
All FNA biopsies of the liver were performed under ultrasound guidance by radiologists using a 10-inchlong, 22-gauge needle. In seven patients, the radiologist also performed a 14-gauge needle core liver biopsy for surgical pathology. A cytotechnologist was present on site to make Swedish-style, oval smears 18 on clear microscopic slides, which were then air dried. At least one smear from each pass was stained with Quick-Dip stain (Mecedes Medical, Sarasota, FL) for the assessment of the adequacy of sample. The remaining air-dried smears were brought back to the laboratory for Ultrafast Papanicolaou stain (Richard Allan Scientific, Kalamazoo, MI). 19 A dedicate pass comprised of several 22-gauge, fine-needle cores also was performed for the preparation of cell blocks. 20 In addition to hematoxylin and eosin staining, the cell block paraffin sections also were used for reticulin stain. 21 In difficult cases, immunohistochemical stains, including CD34, 22 polyclonal carcinoembryonic antigen (CEA), 23 Hepar-1, 24 and MIB-1, also were performed using the standard avidin-biotin-peroxidase method.
Every percutaneous FNA biopsy the first author examined at the Cytopathology Laboratory of New York University Medical Center since 1996 was documented. Of 428 liver FNAs, 123 cases were HCC and 14 cases (11.4%) were so well-differentiated that they did not meet the classic criteria of malignancy yet were proven to be HCC by long-term follow-up.

Clinical Information and Cytologic Diagnosis
The age of the 14 patients with the unusual type of well-differentiated HCC ranged from 49 to 82 years (mean, 64.4 years). Twelve patients were male and two patients were female. All but one patient had cirrhosis. Six patients had hepatitis C, one patient had hepatitis B, one patient had primary sclerosing cholangititis, and six patients had cirrhosis of uncertain etiology. Magnetic resonance images (MRI) and computed tomography (CT) scans showed solid, hypervascular masses with radiologic characteristics of HCC. Eleven patients presented with a solitary mass and three patients presented with multifocal masses. The size of the liver mass ranged from 2.5 cm to 9.5 cm (mean, 4.2 cm). Initially, the diagnoses on FNA smears were difficult, ranging from benign hepatocytes, to atypical hepatocytes, to carcinoid. The diagnoses improved as experience accumulated. Of the 14 cases, one was reported as benign hepatocytes, one was reported as carcinoma with neuroendocrine features, four were reported as hepatocellular neoplasm of borderline malignant potential, and eight were reported as welldifferentiated HCC. Follow-up showed all 14 patients had HCC. The relevant clinical history, radiologic findings, histology, FNA diagnosis and core biopsy diagnosis, and follow-up information are summarized in Table 1.

Cytologic Findings
Compared with the broad trabeculae of the classic type of well-differentiated HCC (Figs. 1A and 1B and Fig. 2A), the unusual types are comprised of microtrabeculae (Figs. 1C and 1D) and microacini (Figs. 1E and 1F). The microtrabeculae frequently are branched, with the narrowest regions only one or two cells thick, and the microacinar structure frequently comprised of as few as five or six cells. The microtrabeculae and microacini appear naked with inapparent peripheral endothelium and require CD34 antibody to demonstrate their patchy distribution (Fig. 2B). Transgressing capillaries are present but require searching (Fig. 1C). Seven cases presented with a predominant microtrabecular pattern. Four cases presented with a predominant microacinar pattern. Two cases presented with approximate equal proportions of microacinar and microtrabecular components. One case presented with numerous, single cells with eccentric cytoplasm and several nucleoli and occasional microacini, mimicking neuroendocrine carcinoma. However, the neuroendocrine markers were negative, and polyclonal CEA marked the lumen of the microacini as canaliculi (Fig. 1F, inset) in the concurrent needle core biopsy. The neoplastic hepatocytes uniformly were small with a high nucleus/cytoplasmic ratio, resulting from a reduction of cytoplasm rather than increased nuclear size. The majority of nuclei in the small hepatocytes contained small but distinct, single nucleolus. The cytologic features of each patient are listed in Table 2. The tissue fragments from all 14 nonneoplastic liver aspirates were difficult to smear apart and remained in large, cohesive tissue fragments (Fig. 1G). A few of the hepatic plates broke off from the smearing and appeared as flat, polyhedral cells with abundant, granular cytoplasm, sharp cell borders, and mostly central nuclei. The cells were heterogeneous with a variety of nuclear sizes and features (Fig. 1H). Intact reticulin fibers along a single plate of hepatocytes were present in nonneoplastic liver aspirates (Fig. 2C).

Histologic Correlations
In histology, nine cases were the compact type (Figs. 2E and 2F), two cases were the microacinar type, and three cases were the microtrabecular type of HCC. All showed a deficient reticulin framework (Fig. 2D).

DISCUSSION
In the World Health Organization classification 9,25 of HCC, there are a number of histologic variants of HCC, including the microacinar pattern, formed by the dilated canaliculi, and the compact (solid 26 ) growth pattern, formed by the trabeculae growing together, compressing the sinusoids and forming sheets of tumor cells. The majority of microacinar and microtrabecular types of HCC in cytology were the compact type of HCC in histology in this study.
The microtrabecular and microacinar types of HCC on FNA biopsy are characterized by monotony of the neoplastic hepatocytes with little variation in nuclear features, which appear normal in size, shape, and chromasia, but with marked reduction of cytoplasmic size, resulting in small hepatocytes with high a nucleus/cytoplasmic ratio as well as nuclear crowding. One to three-cell-thick, microtrabecular cell arrangements are not new in liver cancers and have been reported in 31% of children with a pure, fetal    epithelial pattern of hepatoblastoma. 9 Likewise, the microacinar pattern of HCC has been reported as an acinar pattern in many cytologic studies. 6,11,13,15,27,28 In fact, nearly all the unusual cytologic features of HCC described in this study have been reported previously in the cytologic literature, including high cellularity, 7,12,13,17,29 cell dissociation, 4 monotony, 4,7,29 narrow trabeculae, 4 uniform prominent nucleoli, 6,13 absence of macronucleoli, 4 multiple nucleoli, 13 small cell size, 4,7,29 decreased cytoplasm, 7,29 increased nucleus/cytoplasmic ratio, 4,6 -8,13 nuclear crowding, 4,8 polygonal cells resembling normal hepatocytes with central nucleoli, 14 and eccentric nuclei, 29 as well as the compact histologic type of HCC. 30 Pitman and Szyfelbein 15 reported that 2 of 35 cases of HCC (5.7%) failed to contain peripheral endothelium, and 1 case was well-differentiated HCC. The current study confirms all of these previous findings and summarizes these cytologic features in a series of 14 cases with histologic correlation (Table 3). When encountering small hepatocytes arranged in a microtrabecular and microacinar smear pattern, it is important to find out the size of the liver mass from radiology, because small cell dysplasia 25,26,31,32 has similar cytologic and histologic features. Nodules smaller than a certain size will be regarded by surgical pathologists as small cell dysplasia, and nodules larger than a certain size will be regarded as a compact type of HCC. The opinions regarding nodule size separating these two entities vary in the histopathologic literature, ranging from 1.5 cm 32 to 3.0 cm. 26 In the current study, 1.5 cm was the cut-off size to report HCC. One example that was excluded from this series was from a patient with cirrhosis and hepatitis C who presented with a solitary, 1.5 cm, hypervascular, solid nodule at the time of FNA and was diagnosed with HCC based on the diffuse macronucleoli in the small hepatocytes in microtrabecular arrangement. Serial MRIs and CT scans documented the doubling of the nodule size to 3.0 cm in 6 months and, for unknown reasons, regressed to Ͻ 1.0 cm in 6 months. The patient then underwent liver transplantation. The final diagnosis of the 0.9-cm. circumscribed nodule on the native liver was high-grade, small cell dysplasia.
FNA biopsy is particularly suitable for the diagnosis of well-differentiated HCC, because the smears immediately test the integrity of reticulin and widen the distance between trabeculae, which become conspicuous upon low-power examination (Fig. 1A). In contrast, the trabecular histologic pattern sometimes is not conspicuous, and CD34 immunostaining may be required to highlight the peripheral endothelium outlining the trabeculae ( Fig. 2A), and the status of the reticulin framework requires reticulin stain for assessment ( Fig. 2. C,D).