Limited Immunohistochemical Panel Can Subtype Hepatocellular Adenomas for Routine Practice

Objectives: β-Catenin–activated hepatocellular adenomas have an elevated risk of harboring foci of hepatocellular carcinoma. Inflammatory adenomas also have an increased propensity for malignant transformation and are associated with a systemic inflammatory syndrome. Patients with these two adenoma subtypes benefit from excision. We assessed whether β-catenin–activated and inflammatory adenomas could be identified using a limited immunohistochemical panel.

Methods: Forty-six adenomas were assessed by morphology and β-catenin, serum amyloid A, and glutamine synthetase immunostains.

Results: Morphologic examination produced a morphologic working diagnosis of inflammatory adenoma in 25 (54%) of 46 cases, β-catenin–activated adenoma in three (7%) of 46 cases, and 18 (39%) of 46 cases of other adenomas. After immunohistochemical staining, the morphologic diagnosis was confirmed in 15 (33%) of 46 and changed in 20 (43%) of 46, for a final distribution of 16 (35%) of 46 inflammatory adenomas, four (9%) of 46 β-catenin–activated adenomas, seven (15%) of 46 β-catenin–activated inflammatory adenomas, and 19 (41%) of 46 other adenomas.

Conclusions: Inflammatory and β-catenin–activated adenomas were readily identified by immunostaining patterns. These findings reinforce the necessity of immunohistochemistry in classifying adenomas, as assessing morphology alone often provided inaccurate subclassification. β-Catenin–activated and inflammatory adenomas can be accurately diagnosed using only a limited panel of widely available immunostains.

Materials and Methods

Hepatocellular adenomas (HCAs) can be divided into four subtypes based on molecular techniques.¹ β-Catenin–activated HCA (b-HCA) has a notably increased risk of harboring foci of hepatocellular carcinoma (HCC).^2,3 Inflammatory HCA (IHCA) has abnormalities of the interleukin 6/JAK/STAT pathway.^1,4 Some IHCAs also have β-catenin activation (b-IHCAs).⁴ Hepatocyte nuclear factor 1 α-mutated HCA (H-HCA) is characterized by biallelic loss of HNF1A and a loss of expression of liver fatty acid binding protein (L-FABP).^5,6 A fourth subtype of HCA, the unclassified HCA (UHCA), is defined by a lack of known molecular abnormalities.⁵

With the discovery of these molecular subtypes has come a proliferation of immunohistochemical markers for their diagnosis, including β-catenin and glutamine synthetase (GS) for b-HCA,^6,7 L-FABP for H-HCA,⁶ and serum amyloid A (SAA) and C-reactive protein (CRP) for IHCA.^7,8 Studies have used up to seven markers to classify hepatocellular lesions.⁹ Although there is some overlap, the different subtypes also have different morphologic features, including macrovesicular steatosis in H-HCA; inflammatory infiltrates, dilated sinusoids, ductular reaction, and “pseudo-portal tracts” of arterioles embedded in dense collagen patches in IHCA; and pseudoglandular formations and nuclear atypia in b-HCA.^5,6,8

Overall, 4.2% of HCAs may undergo malignant transformation.¹⁰ However, several factors have been identified that may dramatically increase this risk. HCAs in males and lesions over 5 cm in size harbor an increased risk of malignancy, and excision is recommended.^3,11,12 Up to 46% of b-HCAs may contain malignant foci, and excision is again recommended.^2,3,5,11 IHCA alone and with concomitant β-catenin mutations (b-IHCAs) also have an independent risk of malignant transformation.^4,12,13 IHCA has also been associated with a systemic inflammatory syndrome that may be cured by excision.^6,14,15

On the other hand, the pathologic diagnosis of H-HCA and UHCA has not been shown to be of immediate therapeutic impact. H-HCAs are associated with rare familial conditions, including familial adenomatosis and maturity-onset diabetes of the young.^16,17 Therefore, they could potentially require additional clinical investigation. However, available literature on IHCA and b-HCA strongly suggests surgical excision is the treatment of choice, making these two adenoma subtypes necessary to identify at the time of tissue examination.

Results

Therefore, the current study was undertaken to specifically investigate whether IHCA and b-HCA could be diagnosed using only a limited but broadly available immunohistochemical panel that could be used in routine clinical practice. Secondarily, the correlation of clinical and morphologic features was investigated by adenoma subtype.

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.

Discussion

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.

A total of 46 specimens from 33 patients were obtained. Forty-five were surgical specimens, and one specimen was from autopsy material. Two specimens from one patient were sent from an outside institution for consultation; all others were in-house cases. Clinical characteristics of the patient cohort are summarized in Table 2. In brief, specimens from females outnumbered those from males by a ratio of 3.1:1. Age ranged from 2 to 81 years, with a median of 39 years. Nineteen female patients had a clinical history of oral contraceptive use, ranging in duration from 2 to 34 years and varying from remote to ongoing use. No patients had a definitive clinical history of anabolic steroid use. Thirteen of 25 patients for whom data were available had no history of alcohol use. Consumption by 10 was variously described as occasional, infrequent, social, or less than three drinks per week. Two patients had six or more drinks per week. BMI was available for 27 patients and ranged from 16.5 to 33.5 kg/m², with a median value of 24.9 kg/m². Thirteen patients were overweight or obese (BMI ≥25 kg/m²). Chart review revealed seven patients with diagnoses associated with metabolic syndrome. One had the complete metabolic syndrome. One patient had a personal history of an inflammatory condition (Crohn disease), and one other had a family history of an inflammatory condition (first-degree relative with ulcerative colitis). One patient had glycogen storage disease type I.

General pathologic adenoma characteristics are summarized in Table 3. HCA size on gross examination ranged from 0.2 to 24 cm, with a median of 4.5 cm. Sixteen of 27 patients for whom data were available had multiple masses on gross or radiologic examination, including one with more than 10 (adenomatosis). Four patients had hemorrhage/rupture of adenomas.

Morphologic examination produced a working morphologic diagnosis of IHCA in 25 (54%) of 46 cases, b-HCA in three (7%) cases, and untypable HCA in 18 (39%) cases. Immunohistochemical staining produced final diagnoses of IHCA in 16 (35%) cases, b-HCA in four (24%) cases, b-IHCA in seven (15%) cases, and untypable HCA in 19 (41%) cases. Image 1, Image 2, Image 3, and Image 4 illustrate the typical findings in IHCA, b-HCA, untypable HCA, and b-IHCA, respectively.

Specific morphologic features were also associated with different adenoma subtypes Table 4. Telangiectatic sinusoids, pseudoportal tracts, ductular reaction, and inflammation were present in 10 (63%), 13 (81%), four (25%), and 13 (81%) of the 16 immunophenotypic IHCAs, respectively, and in one (14%), two (29%), two (29%), and four (57%) b-IHCAs, respectively. Pseudoportal tracts, ductular reaction, and inflammatory infiltrates were significantly correlated with an immunophenotypic diagnosis of IHCA or b-IHCA (P = .0169, .0216, and .0012, respectively). The presence of ductular reaction was 100% specific for IHCA or b-IHCA, but sensitivity was poor. Neither nuclear atypia nor pseudoglandular architecture was significantly correlated with b-HCA or b-IHCA. Similarly, macrovesicular steatosis was not significantly correlated with untypable HCAs.

Five HCAs harbored foci of HCC Image 5. The size of these HCAs ranged from 3.0 to 24.0 cm, with only one being smaller than 5.0 cm. Two patients each had only one examined HCA (one b-HCA and one b-IHCA), each with foci of HCC. One patient had one benign HCA and a separate adenoma with foci of HCC (both untypable HCAs). One patient had one benign adenoma (IHCA) and two with foci of HCC (both b-HCAs). One patient had two benign adenomas (both untypable HCAs) and a separate nodule of HCC without associated HCA.

Of the nine patients with multiple adenomas confirmed by histopathologic examination, eight had HCAs that shared the same immunophenotype. Three patients had multiple IHCAs, one patient had multiple b-HCAs, and four patients had multiple untypable HCAs. The aforementioned patient with two b-HCAs harboring malignant foci was the only patient to have a separate adenoma of an entirely different subtype (IHCA).

Patients with IHCAs or b-IHCAs had a greater average BMI than patients with other types of HCAs (26.3 vs 22.9 kg/m²), although this difference was not statistically significant (P = .2519). Nine patients with IHCAs or b-IHCAs had clinical profiles that could be consistent with an inflammatory syndrome (including diabetes mellitus, hypertension, hyperlipidemia, or a diagnosed inflammatory disorder), whereas six did not. Five patients with other adenomas had a clinical profile that could be consistent with an inflammatory syndrome and seven did not, with no significant association found between an inflammatory clinical profile and IHCA/b-IHCA (P = .4495).

Nine previous studies examining immunohistochemical subtyping of HCAs were found.^6,9,18‐24 Three series were excluded from analysis, as they were small (fewer than 20 total adenomas) and/or reported on HCAs in East Asian populations.^9,20,23Table 5 presents a comparison of the rates of each subtype using this study’s limited immunohistochemical panel compared with the rates previously published using larger panels. Using a limited panel, the overall distribution of subtypes was significantly different from five of six previous studies.

References

In the current study, when analyzed independently, only pseudoportal tracts, ductular reaction, and inflammation were significantly correlated with a subtype (IHCA), and none were more than 70% sensitive. After overall morphologic evaluation and subsequent immunohistochemical staining, more than 40% of adenomas were reclassified, demonstrating the need for ancillary studies.

β-Catenin and GS staining markedly increased the number of b-HCAs identified from three to 11 (four pure b-HCAs and seven b-IHCAs), indicating that rare cytologic or architectural atypia are not sensitive enough measures to identify these subtypes. Over one-third of adenomas morphologically classified as IHCAs were reclassified as b-HCAs or untypable HCAs after immunostaining. This again reinforces the importance of β-catenin and GS staining, as b-IHCAs frequently had inconspicuous atypia. As in some previous studies, morphology of IHCA was variable,^18,20,21 with a total of 10 adenomas reclassified from IHCA to untypable HCA (n = 7) or from untypable HCA to IHCA (n = 3) after SAA staining. While some individual morphologic features were significantly associated with IHCA, they were clearly not sensitive predictors of a final immunohistochemical diagnosis. In particular, the large number of HCAs initially classified as IHCAs morphologically that were reclassified as untypable after immunostaining frequently showed one or a few of the morphologic features associated with IHCAs without showing the full complement.

A plethora of immunostains can now be used to subclassify HCAs. Negative L-FABP immunohistochemistry is diagnostic of H-HCA.⁶ Nuclear β-catenin staining and diffuse GS positivity correlate well with molecular disruptions of the β-catenin pathway.^4,6 SAA and CRP are useful for diagnosing IHCA.^7,8 Additional markers may be on the horizon, as well, such as a STAT3 immunostain.²⁹ When there is diagnostic difficulty in separating HCA from FNH and HCC, even more markers may be employed, including glypican 3, CD34, reticulin, CK7, CK19, Ki-67, and HSP70.^9,18,29,30

While many ancillary techniques exist, relatively few are available or practical in routine clinical practice. Molecular studies are the definitive method for subtyping HCAs, but they are not widely available. Bioulac-Sage and coauthors⁷ have suggested a diagnostic algorithm predominantly using immunohistochemistry and reserving molecular methods as a last resort. Several other groups have confirmed the usefulness of immunohistochemistry in subclassifying HCAs, with most studies focusing on a panel minimally including GS, L-FABP, β-catenin, SAA, and CRP.^{7,9,13,18‐22,27}

We chose our limited panel with the goal of identifying the two HCA subtypes that could benefit most from direct surgical intervention and with a focus on ancillary studies that could practically be adopted by a laboratory with a small or moderate volume of liver specimens. β-Catenin is already a widely used immunostain, showing nuclear positivity in a variety of neoplasms and fibromatoses. GS has additional use in assessing other liver nodules, aiding in the diagnosis of FNH^7,8,29 and the differentiation between benign and malignant nodules in cirrhosis.^31,32 The MC1 clone we used for immunostaining against SAA has cross-reactivity with amyloid A, a protein product of SAA metabolism that may aggregate in systemic amyloidosis. Therefore, not only can intracellular staining be used to subtype HCAs, but extracellular staining also can be used to subtype amyloid fibrils in other organs and clinical situations. CRP immunostaining is less specific for IHCA than SAA, particularly when FNH is in the differential.⁸ L-FABP immunostaining was developed as a marker of H-HCA and remains, to our knowledge, essentially restricted to this specific clinical situation. Therefore, we felt β-catenin, GS, and SAA to be an appropriate starting point.

GS is an enzyme involved in ammonia metabolism and glutamine synthesis in the liver. It is concentrated in zone 3 of the normal hepatic parenchyma, with immunostaining effectively limited to a few hepatic plates around central veins. However, in hepatic neoplasia, GS gene expression may be upregulated by the β-catenin pathway.^4,33 In this study, nuclear β-catenin positivity was patchy, and all b-HCAs or b-IHCAs with nuclear β-catenin positivity (one and three, respectively) also showed diffuse GS positivity, making GS 64% more specific for this subtype, similar to previous reports.^18,21,27 This suggests that our diagnostic algorithm could be further abridged by foregoing β-catenin staining and using only GS to detect b-HCA/b-IHCA.

Other authors have expressed difficulty interpreting GS.^8,9,22 In our series, eight cases showed less than overwhelming positivity. Three showed diffuse weak positivity, and two showed strong patchy positivity (see Image 5B). Three additional cases with atypical GS staining showed patchy strong staining in small areas of residual HCA compressed by adjacent HCC. Strong, diffuse positivity for GS and nuclear positivity for β-catenin are most strongly associated with mutations in exon 3 of CTNNB1, the gene coding for β-catenin. However, b-HCAs and b-IHCAs with newly recognized CTNNB1 mutations in exons 7 and 8 and other rare loci often show patchy and/or weak GS positivity and lack nuclear β-catenin positivity.^4,34 While these non–exon 3 mutations appear to be of lesser malignant potential, risk still exists and warrants caution until better characterized. The criterion of more than 50% glutamine synthetase staining in our algorithm is designed to capture both the classical strong diffuse pattern and the weaker, patchier staining associated with rarer mutations while excluding patterns with minimal staining not associated with β-catenin pathway upregulation.

β-Catenin–activated inflammatory HCA is not typically considered a standalone subtype, although its identification is also important due to CTNNB1 mutations and malignant potential.⁴ Because b-IHCA is primarily a molecular and immunophenotypic diagnosis without specific morphologic features, morphologic features of b-IHCA were not specifically assessed. Rather, considering b-IHCA as a subtype showing secondary changes superimposed on preexisting IHCA or b-HCA, it was included in analyses for morphologic features of IHCA and b-HCA. When included in these categories, b-IHCA accounted for 28% of all IHCAs and 64% of all HCAs with β-catenin activation, similar to distributions in the literature.^4,6,18,19,21

IHCAs have been associated with overweight/obesity and other proinflammatory conditions.^{14,15,19,20,35} Specifically, IHCAs have been associated with elevated serum CRP and gamma-glutamyl transpeptidase (GGT).^15,19,29 Recording preoperative serum CRP and GGT values was considered for this study but proved impractical due to the rarity of HCAs and the difficulty of collecting clinical information retrospectively. While patients with IHCA in our series were overweight compared with those with other HCAs, this difference did not reach statistical significance (P = .2519). When more features possibly indicating inflammation, such as components of the metabolic syndrome, were included, there was still no significant correlation with the presence of IHCA (P = .4495). This indicates that the idealized clinical profile may not be sufficiently sensitive or specific to influence the diagnosis of IHCA and reinforces the importance of ancillary pathologic studies.

IHCAs have been associated with overweight/obesity and other proinflammatory conditions.^{14,15,19,20,35} Specifically, IHCAs have been associated with elevated serum CRP and gamma-glutamyl transpeptidase (GGT).^15,19,29 Recording preoperative serum CRP and GGT values was considered for this study but proved impractical due to the rarity of HCAs and the difficulty of collecting clinical information retrospectively. While patients with IHCA in our series were overweight compared with those with other HCAs, this difference did not reach statistical significance (P = .2519). When more features possibly indicating inflammation, such as components of the metabolic syndrome, were included, there was still no significant correlation with the presence of IHCA (P = .4495). This indicates that the idealized clinical profile may not be sufficiently sensitive or specific to influence the diagnosis of IHCA and reinforces the importance of ancillary pathologic studies.