Nafis Shafizadeh

Utility and limitations of glypican-3 expression for the diagnosis of hepatocellular carcinoma at both ends of the differentiation spectrum

Glypican-3 is a heparin sulfate proteoglycan normally expressed in fetal liver and placenta, but not in normal adult liver. Preliminary studies have shown that glypican-3 can be useful for the diagnosis of hepatocellular carcinoma. We performed immunohistochemistry for glypican-3 on 80 resection cases of hepatocellular lesions to examine the utility of glypican-3 immunohistochemistry in hepatocellular carcinoma at two ends of the differentiation spectrum. Staining was compared to Hep Par 1 in poorly differentiated cases. Glypican-3 was expressed in 46 (79%) hepatocellular carcinomas (56, 83 and 89% of well, moderately and poorly differentiated respectively) and seven (64%) fibrolamellar carcinomas. Of the 16 well differentiated cases, 10 closely resembled adenoma and were diagnosed due to focal abnormalities and/or loss of reticulin. Glypican-3 expression was seen in 50% in this group. Hepatocellular carcinomas arising in cirrhotic liver were more likely to be glypican-3 positive (91 vs 57%, P=0.004). All hepatic adenomas and macroregenerative nodules were negative, and three (43%) high grade dysplastic nodules were positive. Focal staining was seen in regenerative nodules in four (11%) cirrhosis cases. Glypican-3 was significantly more sensitive than Hep Par 1 for diagnosis of poorly differentiated hepatocellular carcinomas (89 vs 63%, P=0.02). The difference was more significant when only cases with diffuse positive staining were considered (83 vs 21%, P<0.001). In conclusion, glypican-3 has high sensitivity for the diagnosis of hepatocellular carcinoma, but is less sensitive in the extremely well differentiated hepatocellular carcinoma and fibrolamellar variant of hepatocellular carcinoma. Caution should be exercised in using glypican-3 in biopsy specimens as cirrhotic nodules can show strong expression. Glypican-3 can be especially useful in the identification of poorly differentiated hepatocellular carcinoma as it has higher sensitivity compared to Hep Par 1.

Epidermal growth factor receptor and HER-2/neu status by immunohistochemistry and fluorescence in situ hybridization in adenocarcinomas of the biliary tree and gallbladder

Adenocarcinomas of the biliary tract and gallbladder are aggressive tumors with a poor prognosis. Standard chemotherapy often offers minimal benefit. Because epidermal growth factor receptor and HER-2/neu antagonists have been successfully used in adenocarcinomas from other sites, their use in cholangiocarcinoma can be potentially beneficial. This study examines the epidermal growth factor receptor and HER-2/neu expression and the epidermal growth factor receptor gene copy number in biliary tract adenocarcinomas. Fifty-one formalin-fixed, paraffin-embedded cases of adenocarcinomas (26 intrahepatic, 19 extrahepatic, 6 gallbladder) were stained with monoclonal antibodies against epidermal growth factor receptor and HER-2/neu. Fluorescence in situ hybridization analysis was performed in 37 cases using probes directed against epidermal growth factor receptor and centromeric region of chromosome 7. Epidermal growth factor receptor expression was present in 41 (80%) cases, with moderate or strong epidermal growth factor receptor staining in 30 (59%) cases. HER-2/neu was positive in 2 (4%) cases. Fluorescence in situ hybridization analysis showed gain in epidermal growth factor receptor gene copy number in 17 (46%) tumors. Of the latter, 1 showed gene amplification, whereas all others showed gain in chromosome 7, indicating balanced polysomy. Epidermal growth factor receptor overexpression by immunohistochemistry correlated significantly with epidermal growth factor receptor copy number by fluorescence in situ hybridization (P = .02). HER2/neu expression is uncommon in these tumors.

Diagnosis of well-differentiated hepatocellular lesions: role of immunohistochemistry and other ancillary techniques.

There is considerable overlap in morphologic features in well-differentiated hepatocellular lesions necessitating the use of immunohistochemistry and other techniques for diagnosis. Map-like pattern with glutamine synthetase in focal nodular hyperplasia and cytoplasmic staining with serum amyloid associated protein in inflammatory hepatocellular adenoma (HA) are useful for this distinction. The distinction of well-differentiated hepatocellular carcinoma (HCC) and HA in noncirrhotic liver is facilitated by demonstrating glypican-3 and cytogenetic changes like gains of chromosomes 1 and 8. Nuclear staining with &bgr;-catenin and/or diffuse staining with glutamine synthetase strongly favors well-differentiated HCC or HA with high risk for HCC. In a cirrhotic liver, separation of early HCC from high-grade dysplastic nodule requires identification of stromal invasion, which can be highlighted by absence of keratin 7-positive ductular reaction. Combined use of heat shock protein 70, glutamine synthetase, and glypican-3 can be useful as positivity for 2 or more of these markers has shown high specificity for HCC in early studies.

Correlation of exon 3 β-catenin mutations with glutamine synthetase staining patterns in hepatocellular adenoma and hepatocellular carcinoma.

The current clinical practice is based on the assumption of strong correlation between diffuse glutamine synthetase expression and β-catenin activation in hepatocellular adenoma and hepatocellular carcinoma. This high correlation is based on limited data and may represent an oversimplification as glutamine synthetase staining patterns show wide variability in clinical practice. Standardized criteria for interpreting diverse glutamine synthetase patterns, and the association between each pattern and β-catenin mutations is not clearly established. This study examines the correlation between glutamine synthetase staining patterns and β-catenin mutations in 15 typical hepatocellular adenomas, 5 atypical hepatocellular neoplasms and 60 hepatocellular carcinomas. Glutamine synthetase staining was classified into one of the three patterns: (a) diffuse homogeneous: moderate-to-strong cytoplasmic staining in >90% of lesional cells, without a map-like pattern, (b) diffuse heterogeneous: moderate-to-strong staining in 50–90% of lesional cells, without a map-like pattern, and (c) patchy: moderate-to-strong staining in <50% of lesional cells (often perivascular), or weak staining irrespective of the extent, and all other staining patterns (including negative cases). Sanger sequencing of CTNNB1 exon 3 was performed in all cases. Of hepatocellular tumors with diffuse glutamine synthetase staining (homogeneous or heterogeneous), an exon 3 β-catenin mutation was detected in 33% (2/6) of typical hepatocellular adenoma, 75% (3/4) of atypical hepatocellular neoplasm and 17% (8/47) of hepatocellular carcinomas. An exon 3 mutation was also observed in 15% (2/13) of hepatocellular carcinomas with patchy glutamine synthetase staining. The results show a modest correlation between diffuse glutamine synthetase immunostaining and exon 3 β-catenin mutations in hepatocellular adenoma and hepatocellular carcinoma with discrepancy rates >50% in both hepatocellular adenoma and hepatocellular carcinoma. The interpretation of β-catenin activation based on glutamine synthetase staining should be performed with caution, and the undetermined significance of various glutamine synthetase patterns should be highlighted in pathology reports.

Hepatocellular adenomas in a large community population, 2000 to 2010: reclassification per current World Health Organization classification and results of long-term follow-up

The data used for the World Health Organization classification of hepatocellular adenoma (HCA) is largely based on cases from tertiary level centers in Europe. This study examines the distribution of HCA subtypes in a large community population and determines the impact of immunohistochemistry (IHC) on reclassification, diagnosis, and management. All cases diagnosed as HCA in a large community hospital network from 2000 to 2010 were reviewed. The following immunohistochemical stains were evaluated in cases where paraffin-embedded tissue was available (n = 35): β-catenin, glutamine synthetase, serum amyloid A, C-reactive protein, liver fatty acid binding protein. Twenty-eight of 35 cases were confirmed to be HCA, 5 cases were reclassified as well-differentiated hepatocellular carcinoma, and 2 cases were reclassified as focal nodular hyperplasia. The HCA cases were further subclassified into hepatocyte nuclear factor 1α inactivated (29%), inflammatory (32%), inflammatory with β-catenin activation (3%), noninflammatory β-catenin activated (0%), and unclassified (36%). Long-term follow-up was available on 33 of 35 cases, and there were no cases of recurrence or distant metastasis. IHC can provide a definite HCA subtype in two-thirds of cases. HCA subtypes in this large community-based population differed from the prior large French studies, in that there were a greater proportion of unclassified adenomas and a virtual absence of β-catenin-activated adenomas. It is likely that most β-catenin-activated hepatocellular tumors show morphologic and reticulin staining abnormalities indicative of well-differentiated hepatocellular carcinoma. IHC for glutamine synthetase and serum amyloid A can identify cases with β-catenin activation and aid in the distinction of inflammatory adenoma and focal nodular hyperplasia.

Autoimmune hepatitis: review of histologic features included in the simplified criteria proposed by the international autoimmune hepatitis group and proposal for new histologic criteria

Simplified criteria for diagnosis of autoimmune hepatitis are based on autoantibodies, serum immunoglobulin G, histologic features, and negative viral serology. A score of 6 points is necessary for the designation of probable autoimmune hepatitis and 7 points or more for definite autoimmune hepatitis. The presence of three histologic features is required for categorizing a case as typical (2 points): interface hepatitis with portal lymphocytic/lymphoplasmacytic cells extending into lobule, emperipolesis, and rosettes. In the absence of all three features, a chronic hepatitis picture is considered compatible with autoimmune hepatitis (1 point). This study examines the validity of these histologic features for the diagnosis of autoimmune hepatitis. Clinical data and liver biopsies were reviewed for 88 autoimmune hepatitis, 20 primary biliary cholangitis, and 13 non-autoimmune acute hepatitis cases. Interface/lobular activity, number of plasma cells, copper/CK7 stains, and presence/absence of biliary features were assessed in autoimmune hepatitis and primary biliary cholangitis cases. The simplified criteria score was calculated. Modified histologic criteria were formulated on the basis of interface/lobular activity, number of plasma cells, and presence/absence of biliary features. Using the proposed histologic features, histologic score of 2 increased from 8 to 77%, while total simplified score of >6 increased from 69 to 86%. There was no increase in total simplified score for primary biliary cholangitis or non-autoimmune acute hepatitis. Rosettes and emperipolesis are difficult to interpret, and lack sensitivity and sensitivity for autoimmune hepatitis diagnosis. The current histologic criteria used in the current simplified score lead to underscoring of autoimmune hepatitis cases. The modified histologic criteria based on the inflammatory activity, extent of plasma cells, and results of copper/CK7 staining increased the histologic score in autoimmune hepatitis and led to a probable/definite diagnosis of autoimmune hepatitis in 17% of cases that would have otherwise been classified as non- autoimmune hepatitis by simplified score.

Grading and staging mucinous neoplasms of the appendix: a case series and review of the literature

The grading and staging of appendiceal mucinous neoplasms is challenging and fraught with terminology problems, but has critical prognostic and therapeutic implications. We utilized a small case series to examine the grading and staging systems of appendiceal mucinous neoplasms and outline the evidence for the new systems proposed in the upcoming 8th edition of the American Joint Committee on Cancer (AJCC) Staging Manual. We reviewed 33 cases of appendiceal mucinous neoplasms with available clinical follow-up data, 6 of which were widely disseminated in the peritoneum. An additional 4 cases with disseminated peritoneal involvement were also reviewed. A detailed review of the literature was performed with an emphasis on features associated with disease recurrence and correlation of grade with outcome. Recurrence was not seen in 64 low-grade appendiceal mucinous neoplasms (LAMNs) confined to the muscularis propria in our series (n=21) or in the literature (n=43). Of cases of LAMN with neoplastic epithelium present beyond the muscularis propria, 64% (57/89) had peritoneal disease at the time of diagnosis or follow-up. A majority of studies of disseminated appendiceal mucinous neoplasms showed significant five-year survival differences using a three-tier grading scheme. Thus, LAMNs confined to the muscularis propria are best considered as in situ tumors, as these are cured with complete excision. A three-tier system has prognostic significance and should be used for grading of disseminated appendiceal mucinous neoplasms. The conclusions of this case series and literature review provide evidence to support the changes proposed in the 8th edition of the AJCC Staging Manual.

Appendiceal goblet cell carcinoid: common errors in staging and clinical interpretation with a proposal for an improved terminology

Goblet cell carcinoid (GCC) is staged and treated as adenocarcinoma (AC) and not as neuroendocrine tumor (NET) or neuroendocrine carcinoma. The term carcinoid may lead to incorrect interpretation as NET. The aim of the study was to explore pitfalls in staging and clinical interpretation of GCC and mixed GCC-AC, and propose strategies to avoid common errors. Diagnostic terminology, staging, and clinical interpretation were evaluated in 58 cases (27 GCCs, 31 mixed GCC-ACs). Opinions were collected from 23 pathologists using a survey. Clinical notes were reviewed to assess the interpretation of pathology diagnoses by oncologists. NET staging was incorrectly used for 25% of GCCs and 5% of mixed GCC-ACs. In the survey, 43% of pathologists incorrectly indicated that NET staging is applicable to GCCs, and 43% incorrectly responded that Ki-67 proliferation index is necessary for GCC grading. Two cases each of GCC and mixed GCC-AC were incorrectly interpreted as neuroendocrine neoplasms by oncologists, and platinum-based therapy was considered for 2 GCC-AC cases because of the mistaken impression of neuroendocrine carcinoma created by use of the World Health Organization 2010 term mixed adenoneuroendocrine carcinoma. The term carcinoid in GCC and use of mixed adenoneuroendocrine carcinoma for mixed GCC-AC lead to errors in staging and treatment. We propose that goblet cell carcinoid should be changed to goblet cell carcinoma, whereas GCC with AC should be referred to as mixed GCC-AC with a comment about the proportion of each component and the histologic subtype of AC. This terminology will facilitate appropriate staging and clinical management, and avoid errors in interpretation.

Genomic profile of appendiceal goblet cell carcinoid is distinct compared to appendiceal neuroendocrine tumor and conventional adenocarcinoma

Goblet cell carcinoid (GCC) is a rare appendiceal tumor with unique morphologic features that shows glandular and neuroendocrine differentiation on immunohistochemistry. An additional component of adenocarcinoma (AC) can be present (GCC-AC). Both GCC and GCC-AC are staged and treated like AC. The histogenesis and genetic alterations underlying GCC and GCC-AC are unclear. Capture-based next-generation DNA sequencing targeting 479 cancer genes was performed on 19 appendiceal tumors: 4 GCC, 9 GCC-AC, 3 neuroendocrine tumors (NET), and 3 AC (2 conventional, 1 mucinous). Somatic coding mutations were not seen in any NET. Pathogenic (P)/likely pathogenic (LP) mutations were present in 1 GCC, 8 GCC-AC and all 3 AC cases. P/LP mutations in chromatin remodeling genes were seen in 4 (44.4%) GCC-AC cases, but not in NET, GCC or AC. In GCC-AC, P/LP mutations in ARID1A and RHOA were each present in 3 cases, and KDM6A and SOX9 mutations were each seen in 2 cases. APC and KRAS mutations were present in 1 conventional AC case, but were not observed in any GCC or GCC-AC. This limited series reveals mutations in SOX9, RHOA, and chromatin-modifier genes in goblet cell tumors, and shows that the mutational profile of GCC/GCC-AC is distinct from NET and conventional appendiceal AC.

Hepatic angiomyolipoma: mutation analysis and immunohistochemical pitfalls in diagnosis

Hepatic angiomyolipoma (AML) often shows epithelioid morphology with inconspicuous fat. Epithelioid component can mimic hepatocellular adenoma (HCA) or carcinoma (HCC). The aims of this study were to examine the expression of commonly used markers for HCA or HCC in hepatic AML and highlight pitfalls in diagnosis.