Amy Sheen

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.

Loss of BAP1 expression is very rare in peritoneal and gynecologic serous adenocarcinomas and can be useful in the differential diagnosis with abdominal mesothelioma

Gynecologic and primary peritoneal serous carcinoma may be difficult to distinguish from abdominal mesotheliomas clinically, morphologically, and immunohistochemically. BAP1 double-hit inactivation and subsequent loss of protein expression have been reported in more than half of all abdominal mesotheliomas. We therefore sought to investigate the expression of BAP1 in serous carcinoma and explore its potential utility as a marker in the differential diagnosis with mesothelioma. We searched the computerized database of the Department of Anatomical Pathology, Royal North Shore Hospital, Australia, for all cases of gynecologic and peritoneal serous carcinomas and mesotheliomas diagnosed between 1998 and 2014. Immunohistochemistry for BAP1 was then performed on tissue microarray sections. Cases with completely absent nuclear staining in the presence of a positive internal control in nonneoplastic cells were considered negative. If staining was equivocal (eg, absent nuclear staining but no internal control), staining was repeated on whole sections. Loss of BAP1 expression was found in only 1 of 395 (0.3%) serous carcinomas but in 6 of 9 (67%) abdominal mesotheliomas (P < .001) and 131 of 277 (47%) thoracic mesotheliomas (P < .001). We conclude that BAP1 loss occurs extremely infrequently in gynecologic and peritoneal serous adenocarcinomas, whereas it is very common in mesotheliomas including abdominal mesothelioma. Therefore, although positive staining for BAP1 cannot be used to exclude a diagnosis of mesothelioma, loss of BAP1 expression can be used to very strongly support a pathological diagnosis of abdominal mesothelioma over serous carcinoma.

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.

NRASQ61R Mutation-specific Immunohistochemistry Also Identifies the HRASQ61R Mutation in Medullary Thyroid Cancer and May Have a Role in Triaging Genetic Testing for MEN2

A quarter of patients with medullary thyroid carcinoma (MTC) have germline mutations in the RET proto-oncogene indicating MEN2. Therefore genetic testing is recommended for all patients presenting with MTC. Approximately 40% of MTCs have somatic RET mutations. Somatic mutations in the RAS genes are the next most common driver mutations and appear to be mutually exclusive with germline RET mutation. The single most common somatic RAS mutation is HRASQ61R (c.182A>G), reported in 4.6% to 11% of all MTCs. Mutation-specific immunohistochemistry (IHC) initially developed to identify the NRASQ61R mutation in melanoma (clone SP174) has proven highly sensitive and specific. Because the amino acid sequences for the HRAS and NRAS proteins at codon 61 are identical, we postulated that SP174 IHC would also identify the somatic HRASQ61R mutation. IHC with SP174 was performed on a tissue microarray of 68 patients with MTC including 13 (22.8%) with molecularly confirmed MEN2. Seven (10.3%) MTCs demonstrated positive staining. Six of these patients had already undergone germline RET mutation testing as part of clinical care and were all confirmed to be wild type, excluding the diagnosis of MEN2. All SP174 immunohistochemically positive MTCs were proven to have HRASQ61R mutation (and lack KRASQ61R and NRASQ61R) by Sanger sequencing. All MEN2 patients showed negative staining. We conclude that IHC with SP174 is highly specific for the HRASQ61R mutation in MTC. Because current data suggest that this mutation is mutually exclusive with germline RET mutation, IHC may also have a role in triaging formal genetic testing for MEN2.

Loss of INI1 expression in colorectal carcinoma is associated with high tumor grade, poor survival, BRAFV600E mutation, and mismatch repair deficiency ☆,☆☆

SMARCB1 is a tumor suppressor gene that encodes for the protein INI1. SMARCB1 is commonly inactivated and INI1 correspondingly shows loss of expression in a range of malignant neoplasms including rhabdoid tumors, renal medullary carcinomas, and epithelioid sarcomas. Loss of INI1 expression has recently been reported in occasional gastrointestinal adenocarcinomas. We sought to investigate the incidence and clinicopathological significance of INI1 loss in colorectal adenocarcinoma (CRC). Immunohistochemistry for INI1 was performed in tissue microarray (TMA) format on a well-characterized and unselected cohort of CRCs undergoing surgical resection. If staining was negative or equivocal in the TMA sections, immunohistochemistry was repeated on whole sections. Focal or widespread negative staining for INI1 was identified in whole sections from 14 (0.46%) of 3051 CRCs. In 7 (50%) of 14 negative cases, the loss of staining was focal, whereas the remainder were characterized by negative staining in all neoplastic cells in whole sections. In the cases with focal staining, loss of staining was frequently found in areas of poor differentiation. Global or focal INI1 loss was strongly associated with higher histological grade, larger tumor size and poor overall survival (P<.001). We conclude that INI1 loss occurs rarely (0.46% when screened by TMA) in CRC, where it is associated with higher grade, larger tumor size, poorer survival, mismatch repair deficiency, and BRAFV600E mutation.

The epithelioid BAP1-negative and p16-positive phenotype predicts prolonged survival in pleural mesothelioma

Mesothelioma is a relatively uncommon but highly malignant neoplasm. Most patients die of disease within 1 year of diagnosis, but some have prolonged survival. Prospective identification of these longer‐term survivors may help to guide treatment. We therefore sought to investigate the role of p16 immunohistochemistry (IHC) both alone and in combination with other markers as a potential predictor of prolonged survival in mesothelioma.

Lessons learnt from implementation of a Lynch syndrome screening program for patients with gynaecological malignancy

Despite a trend towards universal testing, best practice to screen patients presenting with gynaecological malignancy for Lynch syndrome (LS) is uncertain. We report our institutional experience of a co-ordinated gynaecological LS screening program. All patients with endometrial carcinoma or carcinosarcoma, or gynaecological endometrioid or clear cell carcinomas undergo reflex four panel immunohistochemistry (IHC) for MLH1, PMS2, MSH2 and MSH6 followed by cascade somatic hypermethylation analysis of the MLH1 promoter locus for dual MLH1/PMS2 negative tumours. On the basis of these results, genetic counselling and targeted germline mutation testing is then offered to patients considered at high risk of LS. From 1 August 2013 to 31 December 2015, 124 patients were screened (mean age 64.6 years). Thirty-six (29.0%) demonstrated abnormal MMR IHC: 26 (72.2%) showed dual loss of MLH1/PMS2, five (13.9%) dual loss of MSH2/MSH6, three (8.3%) isolated loss of MSH6, and two (5.6%) isolated loss of PMS2. Twenty-five of 26 (96.1%) patients with dual MLH1/PMS2 loss demonstrated MLH1 promoter methylation. Therefore, 11 (8.9%) patients screened were classified as high risk for LS, of whom nine (81.8%) accepted germline mutation testing. Three (2.4% of total screened) were confirmed to have LS, two with germline PMS2 and one with germline MSH2 mutation. Massive parallel sequencing of tumour tissue demonstrated somatic mutations which were concordant with the IHC results in the remainder. Interestingly, the one MLH1/PMS2 IHC negative but not hypermethylated tumour harboured only somatic MLH1 mutations, indicating that universal cascade methylation testing in MLH1/PMS2 IHC negative tumours is very low yield and could be reconsidered in a resource-poor setting. In conclusion, universal screening for LS in patients presenting with gynaecological malignancy using the algorithm described above identified LS in three of 124 (2.4%) of our population. Only three of nine (33.3%) patients considered at high risk for LS by combined IHC and hypermethylation analysis were proven to have LS. Only one of the LS patients was less than 50 years of age and none of these patients would have been identified had more restrictive Amsterdam or Bethesda criteria been applied.

Inflammatory Myofibroblastic Tumors of the Female Genital Tract Are Under-recognized: A Low Threshold for ALK Immunohistochemistry Is Required

Inflammatory myofibroblastic tumor (IMT) of the female genital tract is under-recognized. We investigated the prevalence of ALK-positive IMT in lesions previously diagnosed as gynecologic smooth muscle tumors. Immunohistochemistry (IHC) for ALK was performed on tissue microarrays of unselected tumors resected from 2009 to 2013. Three of 1176 (0.26%) “leiomyomas” and 1 of 44 (2.3%) “leiomyosarcomas” were ALK IHC positive, confirmed translocated by fluorescence in situ hybridization (FISH) and therefore more appropriately classified as IMT. On review significant areas of all 4 tumors closely mimicked smooth muscle tumors morphologically, but all showed at least subtle/focal features suggesting IMT. Recognizing that the distinction between IMT and leiomyoma/leiomyosarcoma can be subtle, we then reviewed 1 hematoxylin and eosin slide from each patient undergoing surgery for “leiomyoma” from 2014 to 2017 and selected cases for ALK IHC with a low threshold. Of these, 30 of 571 (5.3%) underwent IHC. Two were confirmed to be IHC positive and FISH rearranged. Of the 6 IMTs, only 1 tumor with a previous diagnosis of leiomyosarcoma, an infiltrative margin and equivocal necrosis, metastasized. Of note it demonstrated a less aggressive clinical course compared with most metastatic leiomyosarcomas (alive with disease at 6 y). The patient was subsequently offered crizotinib to which she responded rapidly. In conclusion, IMTs may closely mimic gynecologic smooth muscle tumors. IMTs account for at least 5 of 1747 (0.3%) tumors previously diagnosed as leiomyoma and 1 of 44 (2.3%) as leiomyosarcoma. These tumors may be recognized prospectively with awareness of subtle/focal histologic clues, coupled with a low threshold for ALK IHC.

Mutation specific immunohistochemistry is highly specific for the presence of calreticulin mutations in myeloproliferative neoplasms

The identification of somatic calreticulin (CALR) mutations can be used to confirm the diagnosis of a myeloproliferative disorder in Philadelphia chromosome-negative, JAK2 and MPL wild type patients with thrombocytosis. All pathogenic CALR mutations result in an identical C-terminal protein and therefore may be identifiable by immunohistochemistry. We sought to test the sensitivity and specificity of mutation specific immunohistochemistry for pathogenic CALR mutations using a commercially available mouse monoclonal antibody (clone CAL2). Immunohistochemistry for mutant calreticulin was performed on the most recent bone marrow trephine from a cohort of patients enriched for CALR mutations and compared to mutation testing performed by polymerase chain reaction (PCR) amplification followed by fragment length analysis. Twenty-nine patients underwent both immunohistochemistry and molecular testing. Eleven patients had CALR mutation, and immunohistochemistry was positive in nine (82%). One discrepant case appeared to represent genuine false negative immunohistochemistry. The other may be attributable to a 12 year delay between the bone marrow trephine and the specimen which underwent molecular testing, particularly because a liver biopsy performed at the same time as molecular testing demonstrated positive staining in megakaryocytes in extramedullary haematopoiesis. All 18 cases which lacked CALR mutation demonstrated negative staining. In this population enriched for CALR mutations, the specificity was 100%; sensitivity 82-91%, positive predictive value 100% and negative predictive value 90-95%. We conclude that mutation specific immunohistochemistry is highly specific for the presence of CALR mutations. Whilst it may not identify all mutations, it may be very valuable in routine clinical care.

ATRX loss is an independent predictor of poor survival in pancreatic neuroendocrine tumours

Pancreatic neuroendocrine tumors (PanNETs) are rare neoplasms accounting for 1% to 2% of all pancreatic tumors. The biological behavior of PanNETs is heterogeneous and unpredictable, adding to the difficulties of clinical management. The DAXX (death domain associated protein) and ATRX (α-thalassemia/mental retardation syndrome X-linked) genes encode proteins involved in SWI/SNF-like chromatin remodeling. Somatic inactivating mutations in DAXX and ATRX are frequent in PanNETs, mutually exclusive, and associated with telomere dysfunction, resulting in genomic instability and alternate lengthening of telomeres. We sought to assess the clinical significance of the loss of the ATRX and DAXX proteins as determined by immunohistochemistry (IHC) in patients with PanNET. From an unselected cohort of 105 patients, we found ATRX loss in 10 tumors (9.5%) and DAXX loss in 16 (15.2%). DAXX and ATRX losses were confirmed mutually exclusive and associated with other adverse clinicopathological variables and poor survival in univariate analysis. In addition, ATRX loss was also associated with higher AJCC stage and infiltrative tumor borders. However, only ATRX loss, lymphovascular invasion, and perineural spread were independent predictors of poor overall survival in multivariate analysis. In conclusion, loss of expression of ATRX as determined by IHC is a useful independent predictor of poor overall survival in PanNETs. Given its relative availability, ATRX loss as determined by IHC may have a role in routine clinical practice to refine prognostication in patients with PanNET.