Hepatocellular adenoma classification: a comparative evaluation of immunohistochemistry and targeted mutational analysis

Hepatocellular adenomas (HCA) are rare benign liver tumors with the capacity to undergo malignant transformation. Epidemiologic studies report the prevalence of HCA is approximately 3–4 cases per 100,000 people in Europe and North America, [1] and lower in Asian countries [2]. The highest prevalence is described in females taking oral contraceptives, but other risk factors, such as anabolic steroid use, obesity and metabolic syndrome are described [2‐5]. Inherited syndromes such as glycogenosis type 1, maturity-onset diabetes of the young type 3 (MODY3), and familial adenomatous polyposis have also been linked to the development of HCA [3, 5, 6]. HCA can be solitary or multiple, with “liver adenomatosis” defined as presence of multiple HCA (>10) of any size involving all liver segments [7]. The majority of HCA are benign and some regress without surgical intervention. However, serious complications can occur. Notably, hemorrhage is described in up to one fourth of patients, mainly associated with large (>5 cm) lesions. Furthermore, 9 % of HCA may transform into hepatocellular carcinoma (HCC) with risk factors including male sex, androgen use, large tumors (>5 cm) and β-catenin-mutated HCA [5]. In these complicated cases, early surgical removal may improve patient outcomes [8, 9]. Therefore, there is interest in developing diagnostics to preemptively identify high-risk cases and guide clinical management. Imaging studies may identify a proportion of HCA with relatively high sensitivities and specificities, but some cannot be accurately differentiated from HCC [10, 11]. In these cases, histological evaluation becomes essential for diagnosis.

In the last decade, four subtypes of HCA have been identified based on genotype-phenotype analyses [12]. Inactivating mutations in the HNF1A (TCF1) gene causing loss of hepatocyte nuclear factor 1α (HNF-1α) expression define H-HCA, which are characterized histologically by marked steatosis and bland hepatocyte cytology [12, 13]. A second subtype, with activating mutations of β-catenin (b-HCA), is associated with an increased risk of malignant transformation into HCC [9, 12, 14‐16]. A third subtype (IHCA) is marked by inflammatory infiltrates or telangiectatic features by histology with increased serum amyloid A (SAA) and C reactive protein (CRP) expression by IHC. IHCA has been linked with activating mutations in several genes including IL6ST, FRK, STAT3, JAK1, and GNAS [17, 18]. Whole exome sequencing has also demonstrated that there is overlap between b-HCA and IHCA in some adenomas that harbor mutations in both the β-catenin and IL6ST genes [18]. Furthermore, in addition to previously reported mutations in exon 3, a smaller proportion of b-HCA carry mutations in exons 7 and 8 of CTNNB1; however no increased risk of malignant transformation was noted in these patients [18]. Finally, a small proportion of HCA does not appear to fall into any of the above categories and were considered unclassifiable by current testing approaches (UHCA).

Based on this classification, subtyping of HCA by immunohistochemistry has been used with some success [19]. Briefly, loss of liver fatty-acid binding protein (LFABP) staining results from HNF-1A mutation in H-HCA, and increased SAA immunoreactivity serves as a marker for IHCA. Upregulation of a downstream CTNNB1 target gene, glutamine synthase (GS), is seen in b-HCA, as well as nuclear β-catenin staining. It has been postulated that the immunophenotypic subtypes closely parallel specific histologic features and molecular alterations, but limitations have been observed by numerous studies and detailed studies correlating morphology, immunohistochemical profile and mutation analysis are lacking [3, 12, 20, 21]. The aim of our study was to apply the HCA classification system based on histologic features and immunohistochemical profiles and correlate the findings with molecular analysis.

Hepatocellular adenomas have a small risk of malignant transformation with estimated rates of less than 5 % [3, 25‐27]. Recent advances in the morphologic and molecular classification of HCA identified b-HCA as the subtype most commonly associated to malignant transformation. This finding warrants the use of robust and reliable approaches to identify such tumors [12, 18]. In particular, the status of CTNNB1 mutations in HCA may offer crucial information regarding management of some of these patients.

The current HCA classification scheme is based on the tumor’s morphologic and immunohistochemical characteristics and was originally validated with CTNNB1 and HNF1A gene sequencing [28]. More recently, high-throughput sequencing of a large HCA cohort led to the discovery of novel genetic alterations, [17, 18] including new hot-spots in CTNNB1 gene in the b-HCA subtype, and defects in the IL-6/JAK/STAT3 pathway in IHCA. Our study is one of the first to use a comprehensive targeted next-generation sequencing panel to evaluate the mutational status of HCA and correlate with immunohistochemistry results.

As expected, our study demonstrated HNF1A gene mutations in all analyzed H-HCA cases, confirming high accuracy of this marker in the diagnosis of H-HCA. However, our results suggest limited reliability of GS and β-catenin immunohistochemistry in predicting β-catenin mutations. The utility of both markers has been challenged by other researchers [10, 21, 22, 29, 30]. Lagana et al. showed strong GS positivity in over 50 % of HCA and Joseph et al. described patchy to diffuse GS staining in 23 % of IHCA, but no correlation with molecular studies were available in these studies [22]. In the present study, seven of 12 GS-positive cases displayed faint perivascular staining or heterogeneous positivity. Further molecular evaluation performed in 5 tumors failed to demonstrate CTNNB1 alterations in 4 cases, one of which was a biopsy specimen (case 13). These somewhat controversial staining patterns and their correlation with β-catenin activation have not been fully characterized and may be a result of localized metabolic alterations within the tumor [20‐22, 31].

In addition, we identified strong GS positivity in four β-catenin wild-type tumors, including one H-HCA. We speculate whether these tumors may have activation of an alternative pathway resulting in GS expression. Berry et al. described strong GS staining in a peliotic HCA and suggested that vascular flow alterations and hepatic parenchymal remodeling may explain increased GS expression [31, 32].

In our study, GS staining failed to identify 1 of 2 confirmed CTNNB1-mutated HCA. Low GS expression in b-HCA has been previously reported in HCA carrying the Ser45Pro mutation [33]. Interestingly, the same β-catenin mutation has been described in HCC and was not associated with nuclear β-catenin staining, suggesting that some CTNNB1 alterations may not disrupt GS and β-catenin expression patterns [34]. None of the 11 cases evaluated by whole-exome sequencing carried large CTNNB1 deletions or mutations in exons 7 or 8, the latter described in a minority of bHCA and previously shown to activate β-catenin in HCC cell lines [18]. Caution is warranted in such cases as they show weak and patchy GS staining and lack nuclear staining of β-catenin [18].

Following molecular analysis, four tumors were reclassified. One case phenotypically classified as IHCA was reclassified as bIHCA because it carried both a β-catenin mutation and an IL6ST alteration. Three cases initially categorized as b-HCA lacked CTNNB1 gene alterations. Two of them were re-subtyped as IHCA following identification of IL6ST gene alterations, all of which previously described in IHCA [17, 35]. The remaining case carried no mutations in HNF1A or any of the IHCA-related genes. In this challenging case, the diagnosis of a β-catenin-activated HCA is favored, but the lack of molecular evidence for CTNNB1 alterations suggests alternative mechanisms of β-catenin activation and GS overexpression in the tumor [36]. This adenoma was originally resected from a 7-year-old patient with no known medical conditions, who remains disease-free after over 13 years of her surgery. HCA occurrence in preadolescence is extremely rare and little is known about the natural history and pathogenesis of such lesions. Six adenomas immunophenotypically compatible with IHCA lacked identifiable mutations in IHCA-associated genes, a finding previously described by others [18, 27].

A large percentage of HCA showed atypical features (30.8 %) but no correlation with a specific HCA type was identified. Other histological features, such as mild steatosis and telangiectasia, were not reliable predictors of HCA subtype as defined by IHC and molecular analysis. In addition, we identified significant immunohistochemical overlap between the b-HCA and IHCA subtypes. Some of these “overlap” lesions, with immunohistochemical features of both IHCA and b-HCA, have been previously described as carrying CTNNB1 activating mutations [18]. Both β-catenin-mutated cases showed SAA staining and atypical histological features, but alterations in the inflammatory pathway were only present in one case.

There is a strong association between b-HCA and hepatocellular carcinoma at the time of diagnosis or during follow-up [19]. In our study, none of our cases progressed to HCC. One possible explanation is the fact that our group consisted predominantly of females on oral contraceptives, features generally associated with lower risk of malignant transformation [18]. Interestingly, one b-HCA with atypia harbored a CTNNB1 exon 3 deletion (Gly38_Thr40del) and a TP53 mutation (Ile251Phe), the latter located in a known ‘hotspot’ for cancer-related alterations and previously described in HCC [37]. For the purposes of this study, we favor a very well-differentiated HCC arising from b-HCA. Evason et al. described HCC-related genetic alterations in β-catenin-activated adenomas suggesting that some of these lesions more likely represent low-grade hepatocellular carcinomas [38]. No other HCC-related driver mutations were identified in our study set.

Immunohistochemistry remains the mainstay for HCA classification due to its relative reliability and rapid turnaround time, especially when differentiating HCA from other lesions, such as focal nodular hyperplasia and hepatocellular carcinoma [22, 39]. However, given the inherent variability in GS staining and the low sensitivity of β-catenin IHC, we believe that molecular testing should be used in all HCA cases with equivocal staining patterns, especially in high-risk patients. Our findings are limited by the small sample size and must to be confirmed by a systematic review of a larger number of well-characterized HCA including correlation of each immunohistochemical pattern with mutational profiles detected by molecular analysis.

In summary, while LFABP immunohistochemistry is reliable in identifying H-HCA, GS and β-catenin stains are weak predictors of β-catenin mutations. Our molecular analysis demonstrates that the currently proposed IHC criteria may be somewhat oversimplified. Until other reliable and specific immunohistochemical markers of malignant transformation in HCA are identified, molecular testing of LFAPB-positive HCA may be the best strategy to identify some of the lesions requiring further clinical management. In this context, next-generation sequencing offers a straightforward, highly sensitive, and accurate option for biopsies with equivocal immunohistochemical results.