A Limited Immunohistochemical Panel Can Subtype Hepatocellular Adenomas for Routine Practice

Brent K. Larson, DO; Maha Guindi, MD


Am J Clin Pathol. 2017;147(6):557-570. 

In This Article

Materials and Methods

After institutional review board approval from Cedars-Sinai Medical Center, the departmental surgical pathology database was queried for HCA specimens resected from 1990 to 2013. Search terms included variants of hepatocellular adenoma, hepatic adenoma, and telangiectatic or peliotic focal nodular hyperplasia (FNH). H&E-stained sections of formalin-fixed, paraffin-embedded tissue were reviewed to confirm the diagnosis of HCA. Additional immunohistochemical and histochemical stains, including reticulin, CD34, and glypican 3, were reviewed as needed and available to confirm or refute very well-differentiated HCC and the presence of foci of HCC. Cases that were judged to represent well-differentiated HCC, HCC arising in HCA with insufficient HCA remaining for evaluation, and well-differentiated hepatocellular neoplasms that could not be definitively diagnosed were excluded from further analysis.

H&E-stained sections were assessed for the following morphologic features: telangiectatic sinusoids, pseudoportal tracts, ductular reaction, inflammatory infiltrates, macrovesicular steatosis, pseudoglandular formations, and nuclear atypia. Each feature was scored semiquantitatively as absent (0), rare (1), patchily present multifocally but in 50% or less of the total area (2), or diffusely present, comprising more than 50% of the total area (3). Features multifocally or diffusely present (score of 2 or 3) were considered positive overall for each case. An overall working morphologic diagnosis was then recorded for each case as IHCA, b-HCA, or untypable HCA. A working morphologic diagnosis of IHCA was assigned based on a score of 2 or 3 for telangiectatic sinusoids, pseudoportal tracts, ductular reaction, and/or inflammatory infiltrates. A working morphologic diagnosis of b-HCA was assigned based on a score of 2 or 3 for pseudoglandular formations and/or nuclear atypia. A score of 2 or 3 for macrovesicular steatosis produced a morphologic working diagnosis of untypable HCA. For HCAs showing an admixture of features, the working morphologic diagnosis was assigned based on the highest scores.

Serial 4-μm-thick sections of a representative block from each case were immunostained against SAA (amyloid A clone MC1 from DakoCytomation, Carpinteria, CA), GS (clone 6 from Biocare Medical, Concord, CA), and β-catenin (clone 14 from Cell Marque, Rocklin, CA). β-Catenin and GS were performed on the Ventana BenchMark ULTRA instrument (Ventana Medical Systems, Tucson, AZ). SAA staining was performed on the Leica BOND-III instrument (Leica, Buffalo Grove, IL). Staining protocols for all stains on both machines were fully automated with on-board antigen retrieval. Hematoxylin counterstaining was performed on the Sakura Tissue-Tek DRS Autostainer (Sakura, Torrance, CA). Positive controls were included in each run, including kidney with amyloidosis for SAA, HCC for GS, and colorectal adenocarcinoma for β-catenin. Cirrhotic liver was included in each run as a negative control for GS. Tonsillar tissue was included in each run as a negative control for β-catenin.

Cytoplasmic staining was assessed for SAA, and staining was scored as none or weak only, patchy (≤50%) strong staining, or diffuse (>50%) strong staining. Cytoplasmic staining was also assessed for GS, and staining patterns were labeled as none, perivenular, patchy (<50%), or diffuse (>50%) staining. The perivenular pattern was defined as staining limited to hepatic plates surrounding hepatic veins with or without similar staining at the edge of tumor nodules. Rare small islands of positive staining were allowed in the perivenular pattern. β-Catenin staining was recorded as either membranous or nuclear.

A diagnostic algorithm Table 1 was then used to assign a final, immunohistochemical diagnosis. b-HCAs were immunohistochemically defined as those HCAs showing any β-catenin nuclear positivity or diffuse GS staining regardless of the β-catenin staining pattern, with negative, weak, or patchy SAA staining. b-IHCAs were diffusely positive for SAA and diffusely positive for GS and/or showed nuclear β-catenin positivity. IHCAs were defined by diffuse SAA positivity with membranous β-catenin positivity and negative or perivenular GS staining. HCAs displaying all other staining patterns were called "untypable HCAs." Of note, this category of untypable HCAs is not equivalent to the category of unclassified HCA in the literature, which can only be assigned after extensive immunohistochemical and/or molecular investigation with markers and methods not performed in this study. For example, L-FABP is not used in this study, and L-FABP–negative HCAs would be classified as H-HCA.

Clinical and demographic information was recorded, including sex and age; body mass index (BMI); oral contraceptive use; alcohol consumption; anabolic steroid use; any components of the metabolic syndrome, including diabetes mellitus, hyperlipidemia, or hypertension; any personal or family history of inflammatory conditions (eg, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus); any history of a glycogen storage disease; the overall number of grossly or radiographically diagnosed masses; the gross size of the sampled adenoma(s); and the clinical assessment of adenoma hemorrhage or rupture.

A literature review was also performed to identify series of HCAs examined by immunohistochemical means. Publications from January 2007 to April 2016 with series comprising 20 or more adenomas were included. Studies of non-Western populations were excluded from additional analysis due to potentially significant differences in the epidemiology and etiology of HCAs. Because b-IHCAs were counted by different methods in the literature, the distribution of HCA subtypes from those reports was recalculated according to the method of this study, regardless of how they were counted in the original study. Namely, IHCA, b-IHCA, and b-HCA were counted separately. Also, H-HCAs and UHCAs from these reports were combined into an untypable HCA group for comparison to the distribution of diagnoses in this study.

Individual morphologic features were analyzed for correlation to HCA subtypes using the two-tailed Fisher exact test. Concordance of the morphologic working diagnosis and the final immunohistochemical subtype was determined using the Freeman-Halton extension of the two-tailed Fisher exact test. The association of BMI and other possible clinical indicators of inflammation with the IHCA subtype was analyzed by the two-tailed Fisher exact test. The distribution of HCA subgroups for our study set was compared with expected values based on previously published results using the χ2 goodness-of-fit test. A P value of less than .05 was considered statistically significant for each test.