Hepatocellular adenoma subtype classification using molecular markers and immunohistochemistry

Abstract

Hepatocellular adenomas (HCA) with activated ␤-catenin present a high risk of malignant transformation. To permit robust routine diagnosis to allow for HCA subtype classification, we searched new useful markers. We analyzed the expression of candidate genes by quantitative reverse transcription polymerase chain reaction QRT-PCR followed by immunohistochemistry to validate their specificity and sensitivity according to hepatocyte nuclear factor 1 alpha (HNF1␣) and ␤-catenin mutations as well as inflammatory phenotype. Quantitative RT-PCR showed that FABP1 (liver fatty acid binding protein) and UGT2B7 were downregulated in HNF1␣-inactivated HCA (P < 0.0002); GLUL (glutamine synthetase) and GPR49 overexpression were associated with ␤-catenin-activating mutations (P < 0.0005), and SAA2 (serum amyloid A2) and CRP (C-reactive protein) were upregulated in inflammatory HCA (P ‫؍‬ 0.0001). Immunohistochemistry validation confirmed that the absence of liver-fatty acid binding protein (L-FABP) expression rightly indicated HNF1␣ mutation (100% sensitivity and specificity), the combination of glutamine synthetase overexpression and nuclear ␤-catenin staining were excellent predictors of ␤-catenin-activating mutation (85% sensitivity, 100% specificity), and SAA hepatocytic staining was ideal to classify inflammatory HCA (91% sensitivity and specificity). Finally, a series of 93 HCA was unambiguously classified using our 4 validated immunohistochemical markers. Importantly, new associations were revealed for inflammatory HCA defined by SAA staining with frequent hemorrhages (P ‫؍‬ 0.003), telangiectatic phenotype (P < 0.001), high body mass index, and alcohol intake (P < 0.04). Previously described associations were confirmed and in particular the significant association between ␤-catenin-activated HCA and hepatocellular carcinomas (HCC) at diagnosis or during follow-up (P < 10 -5 ). Conclusion: We refined HCA classification and its phenotypic correlations, providing a routine test to classify hepatocellular adenomas using simple and robust immunohistochemistry. (HEPATOLOGY 2007;46:740-748.) H epatocellular adenomas (HCA) are rare benign liver tumors, most frequently occurring in women who are using oral contraception. Although HCA are mostly found as a single nodule, the presence of more than 10 in the liver indicates a specific nosological entity termed liver adenomatosis. 1 Two genetic alterations, the biallelic inactivation of hepatocyte nuclear factor 1 alpha (HNF1␣) and the activating mutation of ␤-catenin, have been described in HCA. 2,3 Recently, a comprehensive analysis of genetic, pathological, and clinical features in a series of 96 HCA enabled the identification of 4 HCA subtypes. 4 Biallelic HNF1␣ mutations defined the first group of HCA, phenotypically characterized by marked steatosis, lack of cytological abnormalities, and inflammatory infiltrates. Presence of a ␤-catenin-activating mutation defined the second group of HCA representing 15% of the cases generally characterized by a higher risk of malignant tr...

Immunohistochemical pitfalls in the diagnosis of hepatocellular adenomas and focal nodular hyperplasia: accurate understanding of diverse staining patterns is essential for diagnosis and risk assessment

“…This observation is surprising as our criteria are based on those recommended in their study. 3 For example, diffuse staining with glutamine synthetase was defined in our study by staining of 450% of tumor cells and all these cases were thus interpreted as b-catenin-activated lesions. The 50% cutoff was chosen on the basis of the results of the study by Bioulac-Sage et al 3 They had described homogeneous and heterogeneous patterns of glutamine synthetase staining within this group, but did not provide the number of cases for each category.…”

“…3 For example, diffuse staining with glutamine synthetase was defined in our study by staining of 450% of tumor cells and all these cases were thus interpreted as b-catenin-activated lesions. The 50% cutoff was chosen on the basis of the results of the study by Bioulac-Sage et al 3 They had described homogeneous and heterogeneous patterns of glutamine synthetase staining within this group, but did not provide the number of cases for each category. In our study, 2 we also observed homogenous and heterogeneous patterns within the diffuse glutamine synthetase staining group and described them as diffuse-strong and diffuse-intermediate staining, respectively; in addition, we also provided number of cases showing these two patterns within the diffuse category.…”

“…In their commentary, Bioulac-Sage et al state that 'the staining is positive (from mild to strong) or negative; diffuse positivity means close to 100% positivity and focal positivity means expression around inflammatory cells, which is not considered significant'. However, the results in their prior study 3 indicate that serum amyloid A staining was not diffuse in all cases and was described as being 'patchy and/or faint'; the number of cases with patchy and/or faint staining was not specified. We think that it is important to examine 'focal' vs 'diffuse' pattern of staining to assess how well a marker will work in the setting of a needle biopsy when only a small portion of the lesion is available for immunohistochemical evaluation.…”

“…Our description of 'perivascular and patchy staining' with glutamine synthetase corresponds at least in part to the description of 'patchy staining' with glutamine synthetase in their own studies. 3,4 Furthermore, our study is focused on a detailed description of the observed immunohistochemical staining patterns, which simulates the situation for majority of pathologists who encounter these lesions in practice. Molecular techniques were not part of our study and we have not claimed any molecular correlations based on our data.…”

“…In their original paper, details of C-reactive protein staining are not provided, whereas serum amyloid A staining has been described in detail for the diagnosis of inflammatory hepatocellular adenoma. 3 Their subsequent publications also recommend serum amyloid A or C-reactive protein for the diagnosis of inflammatory hepatocellular adenoma, and do not provide evidence for the superiority of C-reactive protein. 6,7 In our experience, serum amyloid A has better specificity than C-reactive protein for the diagnosis of inflammatory hepatocellular adenoma.…”