Mahtab Farzin

Loss of expression of BAP1 predicts longer survival in mesothelioma

Summary BRCA1-associated protein 1 (BAP1) is a tumour suppressor gene frequently inactivated in mesothelioma, rarely also in association with germline mutation. BAP1 mutations have been associated with improved prognosis and distinct clinicopathological features. We sought to determine the clinicopathological significance of BAP1 immunohistochemistry (IHC) in mesothelioma. We performed IHC on a tissue microarray (TMA) cohort comprising all available thoracic mesotheliomas encountered during the period 1991-2014 at our institution (n = 229). All cases were independently reviewed to confirm the diagnosis and subclassify as epithelioid, sarcomatoid or biphasic. The median age at diagnosis was 72 years; 188 (82.1%) were male; 120 (52.4%) were epithelioid (median survival 13.0 months), 67 (29.3%) sarcomatoid (median survival 5.6 months) and 42 (18.3%) biphasic (median survival 10.6 months). Negative staining for BAP1 (defined as completely absent nuclear staining in the presence of positive internal controls in non-neoplastic cells) occurred in 106 (46.3%) mesotheliomas. There was complete interobserver concordance for BAP1 IHC status. BAP1 loss was strongly associated with younger age at onset (p < 0.01) and epithelioid differentiation (p < 0.01). BAP1 loss predicted an improved median survival of 16.11 months (95% CI 12.16–20.06) versus 6.34 months (95% CI 5.34–7.34), p < 0.01. In a multivariate model including age, gender and histological type, BAP1 loss, younger age and epithelioid differentiation remained protective (all p < 0.01). If our results are confirmed by others, BAP1 IHC may have a role to predict prolonged survival or triage formal genetic testing for germline BAP1 mutation in patients presenting with mesothelioma.

Loss of expression of BAP1 is a useful adjunct, which strongly supports the diagnosis of mesothelioma in effusion cytology.

Although most mesotheliomas present with pleural effusions, it is controversial whether mesothelioma can be diagnosed with confidence in effusion cytology. Therefore, an ancillary marker of malignant mesothelial cells applicable in effusions would be clinically valuable. BRCA-1-associated protein (BAP1) is a tumor suppressor gene, which shows biallelic inactivation in approximately half of all mesotheliomas. We investigated whether loss of BAP1 expression by immunohistochemistry can be used to support a diagnosis of mesothelioma in effusion cytology. Immunohistochemistry for BAP1 was performed on cell blocks and interpreted blinded. 43 of 75 (57%) effusions associated with confirmed mesothelioma showed negative staining with positive internal controls. Of 57 effusions considered to have atypical mesothelial cells in the absence of a definitive diagnosis of mesothelioma, 8 cases demonstrated negative staining for BAP1. On follow-up six of these patients received a definitive diagnosis of mesothelioma in the subsequent 14 months (two were lost to follow-up immediately, and mesothelioma could not be excluded). Only 5 of 100 consecutive benign effusions were interpreted as BAP1 negative. One of these patients died soon after and mesothelioma could not be excluded. On unblinded review the four other patients with apparently negative BAP1 staining but no malignancy lacked convincing positive staining in non-neoplastic cells suggesting that BAP1 immunohistochemistry may have initially been misinterpreted. 47 effusions with adenocarcinoma were BAP1 positive. We conclude that loss of BAP1 expression, while not definitive, can be used to support the diagnosis of mesothelioma in effusion cytology. We caution that interpretation of BAP1 immunohistochemistry on cell block may be difficult and that convincing positive staining in non-neoplastic cells is required before atypical cells are considered negative. We also note that BAP1 loss is not a sensitive test as it occurs in only half of all mesotheliomas and cannot be used to exclude the diagnosis.

A Detailed Clinicopathologic Study of ALK-translocated Papillary Thyroid Carcinoma

Pathogenic ALK translocations have been reported in papillary thyroid carcinoma (PTC). We developed and validated a screening algorithm based on immunohistochemistry (IHC), followed by fluorescence in situ hybridization (FISH) in IHC-positive cases to identify ALK-rearranged PTC. IHC and FISH were performed in a cohort of 259 thyroid carcinomas enriched for aggressive variants. IHC was positive in 8 cases, 6 confirmed translocated by FISH (specificity 75%). All 251 IHC-negative cases were FISH negative (sensitivity 100%). Having validated this approach, we performed screening IHC, followed by FISH in IHC-positive cases in an expanded cohort. ALK translocations were identified in 11 of 498 (2.2%) of all consecutive unselected PTCs and 3 of 23 (13%) patients with diffuse sclerosing variant PTCs. No ALK translocations were identified in 36 PTCs with distant metastases, 28 poorly differentiated (insular) carcinomas, and 20 anaplastic carcinomas. All 14 patients with ALK translocations were female (P=0.0425), and translocations occurred at a younger age (mean 38 vs. 48 y, P=0.0289 in unselected patients). ALK translocation was an early clonal event present in all neoplastic cells and mutually exclusive with BRAFV600E mutation. ALK translocation was not associated with aggressive clinicopathologic features (size, stage, metastasis, vascular invasion, extrathyroidal extension, multifocality, risk for recurrence, radioiodine resistance). We conclude that 2.2% of PTCs are ALK-translocated and can be identified by screening IHC followed by FISH. ALK translocations may be more common in young females and diffuse sclerosing variant PTC but do not connote more aggressive disease.

The International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society grading system has limited prognostic significance in advanced resected pulmonary adenocarcinoma

Introduction: The International Association for the Study of Lung Cancer, the American Thoracic Society and the European Respiratory Society (IASLC/ATS/ERS) system which subclassifies lung adenocarcinoma into five distinct types has been widely adopted. We assessed the prognostic value of subclassifying adenocarcinoma in this way in consecutive patients undergoing surgery. Methods: All patients at our institution undergoing surgery for lung carcinoma between 2000 and 2010 were identified. The original pathology slides were independently reviewed and reclassified according to the 2011 IASLC/ATS/ERS grading and the American Joint Committee on Cancer (AJCC) 7th edition 2009 staging systems. Results: We identified 270 patients including 152 with adenocarcinoma histology with long-term follow-up. Using the Kaplan–Meier method, the calculated 5 year survival for each of the adenocarcinoma categories were papillary-predominant 80%, lepidic-predominant 71%, micropapillary-predominant 55%, acinar-predominant 43%, solid-predominant 39% and invasive mucinous adenocarcinoma 38%. The AJCC stage was a very strong predictor of survival (p < 0.001). The IASLC/ATS/ERS subclassification of adenocarcinoma demonstrated a trend as a prognostic marker but failed to reach statistical significance in univariate or multivariate analysis. Conclusion: Although the IASLC/ATS/ERS classification has been validated by several studies in stage I tumours, further studies of larger cohorts will be required to show prognostic value in unselected lung carcinoma undergoing surgery with curative intent.

Loss of BAP1 Expression Occurs Frequently in Intrahepatic Cholangiocarcinoma.

AbstractBRCA1-associated protein 1 (BAP1) is a deubiquitinating enzyme that functions as a tumor suppressor gene. Double hit BAP1 inactivation has been reported in a range of tumor types, including intrahepatic cholangiocarcinoma (ICC), sometimes in association with germline mutation.We performed immunohistochemistry for BAP1 on a well-characterized cohort of 211 ICC patients undergoing surgical resection with curative intent at 3 institutions based in 3 different countries. The median age at diagnosis was 65 years (range, 36.5–86) and 108 (51%) were men. Negative staining for BAP1 (defined as completely absent nuclear staining in the presence of positive internal controls in nonneoplastic cells) occurred in 55 ICCs (26%). BAP1 loss predicted a strong trend toward improved median survival of 40.80 months (95% CI, 28.14–53.46) versus 24.87 months (95% CI, 18.73–31.01), P = 0.059). In a multivariate model including age, sex, BAP1 status, tumor stage, tumor grade, lymphovascular invasion, and tumor size, female sex was associated with improved survival (hazard ratio [HR] 0.54; 95% CI, 0.34–0.85), while advanced tumor stage and lymphovascular invasion (HR 1.89; 95% CI, 1.09–3.28) correlated with decreased survival. In a multivariate analysis, high grade tumors were associated with BAP1 loss (odds ratio [OR] 3.32; 95% CI, 1.29–8.55), while lymphatic invasion was inversely associated with BAP1 loss (OR 0.36; 95% CI, 0.13–0.99).In conclusion, we observed a trend toward improved prognosis in ICC associated with absent expression of BAP1 and an association of BAP1 loss with higher histological grade and absent lymphatic invasion. Female sex was associated with improved survival while advanced tumor stage and lymphatic invasion were associated with decreased survival.

Fumarate Hydratase-deficient Uterine Leiomyomas Occur in Both the Syndromic and Sporadic Settings.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome secondary to germline fumarate hydratase (FH) mutation presents with cutaneous and uterine leiomyomas, and a distinctive aggressive renal carcinoma. Identification of HLRCC patients presenting first with uterine leiomyomas may allow early intervention for renal carcinoma. We reviewed the morphology and immunohistochemical (IHC) findings in patients with uterine leiomyomas and confirmed or presumed HLRCC. IHC was also performed on a tissue microarray of unselected uterine leiomyomas and leiomyosarcomas. FH-deficient leiomyomas underwent Sanger and massively parallel sequencing on formalin-fixed paraffin-embedded tissue. All 5 patients with HLRCC had at least 1 FH-deficient leiomyoma: defined as completely negative FH staining with positive internal controls. One percent (12/1152) of unselected uterine leiomyomas but 0 of 88 leiomyosarcomas were FH deficient. FH-deficient leiomyoma patients were younger (42.7 vs. 48.8 y, P=0.024) and commonly demonstrated a distinctive hemangiopericytomatous vasculature. Other features reported to be associated with FH-deficient leiomyomas (hypercellularity, nuclear atypia, inclusion-like nucleoli, stromal edema) were inconstantly present. Somatic FH mutations were identified in 6 of 10 informative unselected FH-deficient leiomyomas. None of these mutations were found in the germline. We conclude that, while the great majority of patients with HLRCC will have FH-deficient leiomyomas, 1% of all uterine leiomyomas are FH deficient usually due to somatic inactivation. Although IHC screening for FH may have a role in confirming patients at high risk for hereditary disease before genetic testing, prospective identification of FH-deficient leiomyomas is of limited clinical benefit in screening unselected patients because of the relatively high incidence of somatic mutations.

Reflex ALK immunohistochemistry is feasible and highly specific for ALK gene rearrangements in lung cancer

Summary Fluorescence in situ hybridisation (FISH) is considered the gold standard for the detection of ALK gene rearrangements in lung adenocarcinoma. The presence of ALK gene rearrangement predicts response to specific targeted therapy, but these rearrangements are relatively rare and FISH studies are expensive, not widely available, potentially challenging to interpret and therefore difficult to undertake in all patients with non-small cell lung cancer. We developed and then deployed into the routine clinical setting a screening program for ALK gene rearrangement in all non-small cell lung cancer patients based on immunohistochemistry (IHC) with a mouse monoclonal antibody (clone 5A4). ALK IHC was strongly positive in 12 (4%) of 307 tumours from consecutive patients. Only 10 of these cancers were initially thought to be rearranged by diagnostic FISH studies. The two tumours which were IHC positive but initially interpreted as FISH negative underwent repeat FISH testing because of the discrepancy. Repeat FISH testing confirmed the presence of ALK gene rearrangement with the discrepancy being attributable to an atypical FISH pattern. Therefore, in our experienced hands, IHC for ALK performed on initial diagnosis of lung cancer is 100% specific for the presence of ALK gene rearrangement. When ALK IHC and FISH studies are discrepant, IHC may outperform FISH. Although our study was not intended to formally assess the sensitivity of ALK IHC, the 4% rate of gene rearrangements identified by this approach is consistent with the expected incidence in our population. We conclude that reflex ALK IHC followed by confirmatory FISH testing can be readily integrated into the routine clinical setting and represents a cost effective and practical approach to screening for these clinically significant gene rearrangements.

EGFR mutant-specific immunohistochemistry has high specificity and sensitivity for detecting targeted activating EGFR mutations in lung adenocarcinoma

Aim We assessed the diagnostic accuracy of epidermal growth factor receptor (EGFR) mutant-specific antibodies for detecting two common activating EGFR mutations. Methods Immunohistochemical expression of mutation-specific antibodies against EGFR exon 19 deletion E746-A750 ((c.2235_2249del15 or c.2236_2250del15, p. Glu746_Ala750del) and exon 21 L858R point mutation (c.2573T>G, p.Leu858Arg) were assessed in a cohort of 204 resected early stage node negative lung adenocarcinomas, and protein expression was compared with DNA analysis results from mass spectrometry analysis. Results Of seven cases with L858R point mutation, six were positive by immunohistochemistry (IHC). There were three false positive cases using L858R IHC (sensitivity 85.7%, specificity 98.5%, positive predictive value 66.7%, negative predictive value 99.5%). All seven E746-A750 exon 19 deletions identified by mutation analysis were positive by IHC. Four additional cases were positive for exon 19 IHC but negative by mutation analysis. The sensitivity of exon 19 IHC for E746-A750 was 100%, specificity 98.0%, positive predictive value 63.6% and negative predictive value 100%. Conclusions Mutant-specific EGFR IHC has good specificity and sensitivity for identifying targeted activating EGFR mutations. Although inferior to molecular genetic analysis of the EGFR gene, IHC is highly specific and sensitive for the targeted EGFR mutations. The antibodies are likely to be of clinical value in cases where limited tumour material is available, or in situations where molecular genetic analysis is not readily available.

Loss of expression of BAP1 is very rare in non-small cell lung carcinoma

Germline mutations of the BAP1 gene have been implicated in a cancer predisposition syndrome which includes mesothelioma, uveal melanoma, cutaneous melanocytic lesions, renal cell carcinoma, and possibly other malignancies. Double hit inactivation of BAP1 with subsequent loss of expression of the BAP1 protein also occurs in approximately 50% of mesotheliomas. The link between BAP1 mutation and lung cancer is yet to be fully explored. We sought to assess BAP1 expression in a large cohort of lung cancers undergoing surgery with curative intent. We searched the Anatomical Pathology database of our institution for lung cancer patients undergoing surgery with curative intent between 2000 and 2010. Immunohistochemistry for BAP1 was then performed in tissue microarray format. Our cohort included 257 lung cancer patients, of which 155 (60%) were adenocarcinomas and 72 (28%) were squamous cell carcinomas, with no other subtype comprising more than 3%. BAP1 loss of expression was found in only one lung cancer. We conclude that BAP1 mutation occurs very infrequently (0.4%) in non-small cell lung cancer. Given that the pathological differential diagnosis between lung carcinoma and mesothelioma may sometimes be difficult, this finding increases the specificity of loss of expression for BAP1 for the diagnosis of mesothelioma.

Loss of BAP1 expression is very rare in pancreatic ductal adenocarcinoma

Background Pancreatic cancer is both common and highly lethal and therefore new biomarkers or potential targets for treatment are needed. Loss of BRCA associated protein-1 (BAP1) expression has been found in up to a quarter of intrahepatic cholangiocarcinomas. Given the close anatomical relationship between intrahepatic cholangiocarcinoma and pancreatic ductal adenocarcinoma, we therefore sought to investigate the frequency of loss of BAP1 expression in pancreatic ductal adenocarcinoma. Methods The records of the department of Anatomical Pathology Royal North Shore Hospital, Sydney, Australia, were searched for cases of pancreatic ductal adenocarcinoma diagnosed between 1992 and 2014 with material available in archived formalin fixed paraffin embedded tissue blocks. Immunohistochemistry for BAP1 was performed on tissue microarray sections and if staining was equivocal or negative it was confirmed on whole sections. Negative staining for BAP1 was defined as loss of expression in all neoplastic nuclei, with preserved expression in non-neoplastic cells which acted as an internal positive control. Results Loss of BAP1 expression was found in only 1 of 306 (0.33%) pancreatic ductal adenocarcinomas. This case was confirmed to demonstrate diffuse loss of expression throughout all neoplastic cells in multiple blocks, consistent with BAP1 loss being an early clonal event. All other cases demonstrated positive expression of BAP1. Conclusion We conclude that, in contrast to intrahepatic cholangiocarcinoma, loss of expression of BAP1 occurs very rarely in pancreatic ductal adenocarcinoma. Therefore BAP1 inactivation is unlikely to be a frequent driver abnormality in pancreatic adenocarcinoma.