MTAP Expression Research Articles

MTAP and p16 IHC as Markers for CDKN2A/B Loss in Meningiomas.

Homozygous cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) loss is one of the parameters that support the designation of meningiomas as Central Nervous System (CNS) WHO grade 3 tumors. Evaluation of CDKN2A/B by sequencing or Fluorescence in situ hybridization (FISH) is costly and not always readily accessible. An immunohistochemistry (IHC)-based marker for the evaluation of CDKN2A/B loss would provide faster results at a lower cost. This retrospective study included patients diagnosed with meningioma at our institution between 2016 and 2019. Archival tumor tissue was used for analysis. MTAP immunohistochemistry (IHC) was performed at various dilutions (1:1200, 1:400, 1:200, 1:100) using two different antibodies, and p16 IHC was conducted simultaneously. These analyses were carried out at two different institutions. To determine the sensitivity and specificity of MTAP and p16 as surrogate markers for CDKN2A/B loss, CDKN2A FISH was utilized as the gold standard. Overall, 46/49 tumors showed strong MTAP staining (94%) at institution 1, and 44/49 (90%) showed either faint positive or positive results at institution 2. One grade 3 meningioma that demonstrated homozygous CDKN2A loss by FISH also showed loss of MTAP expression by IHC. One grade 2 meningioma showed regional CDKN2A loss by FISH and variable MTAP expression under different IHC conditions. MTAP expression evaluation was superior at a dilution of 1:100 with the Abnova Anti-MTAP Monoclonal antibody. P16 expression was variable and did not correlate with either MTAP expression or CDKN2A FISH results. MTAP IHC is a promising surrogate marker for the evaluation of CDKN2A status in meningiomas.

MTAP protein status is highly concordant with CDKN2A fluorescent in situ hybridization and allows stratification of the luminal subtype in muscle-invasive bladder cancer.

Loss of heterozygosity in chromosome 9p21, common in urothelial carcinoma (UC), typically involves deletion of CDKN2A and MTAP genes. MTAP loss is emerging as a promising therapeutic target and predictive biomarker in UC. This single-centrre retrospective study examined the incidence of CDKN2A deletions and MTAP loss in muscle-invasive bladder cancer (MIBC) and metastatic urothelial carcinoma (mUC), investigating their correlations with clinical, pathological, and genomic features, as well as patient outcomes. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were performed on 302 MIBC specimens and 63 biopsy-proven metachronous urothelial metastases to assess CDKN2A deletions and MTAP protein expression. CDKN2A homozygous deletion (HD), identified in 30.3% of MIBCs, and MTAP loss, found in 28.8% of MIBCs, were both significantly associated with the luminal-URO subtype, FGFR3 mutations, and normal/wildtype p53 IHC (P < 0.05). Loss of MTAP expression was significantly correlated with CDKN2A HD, with 84.0% sensitivity, 92.3% negative predictive value (NPV), 96.3% specificity, and 91.9% positive predictive value (PPV). MTAP expression was 100% concordant between primary tumours and nodal metastases. Patients with MTAP loss had a higher incidence of visceral metastases (50%) compared to bone/soft tissue (35.7%) and nodes (14.3%). Mean progression-free survival and overall survival were shorter for patients with MTAP loss, although not statistically significant. Our findings highlight CDKN2A HD and MTAP loss as prevalent genetic alterations in MIBC and mUC, particularly within the luminal-URO subtype and FGFR3-mutated, p53-normal/wildtype tumours. MTAP IHC can serve as a surrogate marker for 9p21.3 HD, highlighting its clinical relevance and potential as a therapeutic target and predictive biomarker in MIBC.

Abstract A028: MTAP deficiency is frequent and mostly homogeneous in ductal adenocarcinomas of the pancreas

Abstract Complete expression loss of S-methyl-5′-thioadenosine phosphorylase (MTAP) is caused by homozygous 9p21 deletion and results in a critical vulnerability of cancer cells towards drugs targeting multiple different pathways. MTAP deficiency is common in ductal adenocarcinoma of the pancreas, but data on its intratumoral heterogeneity - a potential obstacle for targeted therapies - are lacking. To learn more on the role and potential heterogeneity of MTAP deficiency in pancreatic adenocarcinoma, 236 primary tumors were analyzed in a tissue microarray (TMA) format by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for MTAP expression and 9p21 deletions. The cohort included 236 cancers from which 6 samples were taken from as many as possible different tumor containing primary tumor blocks and 3 samples were added from as many as possible lymph node metastases to construct a “heterogeneity TMA”. All available tumor containing blocks (average 4) from 6 selected cancers were also analyzed by IHC and FISH on whole sections. Complete absence of MTAP staining was seen in 350 (38.6%) of 907 interpretable TMA samples. 284 (98.6%) of 288 samples with a complete MTAP expression loss and FISH data had a homozygous 9p21 deletion while there were only two tumor spots with homozygous deletions within 557 samples with retained MTAP expression (99.6% concordance). On a patient level, an MTAP deficiency was found in 64 (41.0%) of 156 tumors for which at least 3 different samples were interpretable on the TMAs. 62 (96.9%) of these patients showed an MTAP loss in all interpretable samples (homogeneous MTAP deficiency). The TMA data were confirmed on whole sections in 5 of 6 patients. In the discrepant case a focal MTAP loss was identified in a tumor considered MTAP wild type based on the TMA results. It is concluded that MTAP expression loss is frequent and highly homogeneous in ductal adenocarcinomas of the pancreas. Given the high concordance rate between homozygous deletion and MTAP expression loss homozygous deletion may represent the only mechanism for MTAP deficiency in pancreatic cancer. MTAP IHC is an excellent tool for identification of MTPA deficient cases. MTAP IHC may also have considerable diagnostic value as even low-grade cancer glands can be easily identified in small biopsies from MTAP deficient cases. Considering also their aggressive clinical behavior, pancreatic adenocarcinomas may represent an ideal cancer type for studying new drugs targeting MTAP deficient cancer cells in clinical trials. Citation Format: Natalia Gorbokon, Katharina Teljuk, Frank Jacobsen, Till Krech, Till Clauditz, Stefan Steurer, Ronald Simon, Guido Sauter, Sarah Minner, Lukas Bubendorf, Thilo Hackert, Nina Schraps, Faik G Uzunoglu, Martina Kluth. MTAP deficiency is frequent and mostly homogeneous in ductal adenocarcinomas of the pancreas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A028.

The context-dependent epigenetic and organogenesis programs determine 3D vs. 2D cellular fitness of MYC-driven murine liver cancer cells.

3D cellular-specific epigenetic and transcriptomic reprogramming is critical to organogenesis and tumorigenesis. Here we dissect the distinct cell fitness in 2D (normoxia vs. chronic hypoxia) vs 3D (normoxia) culture conditions for a MYC-driven murine liver cancer model. We identify over 600 shared essential genes and additional context-specific fitness genes and pathways. Knockout of the VHL-HIF1 pathway results in incompatible fitness defects under normoxia vs. 1% oxygen or 3D culture conditions. Moreover, deletion of each of the mitochondrial respiratory electron transport chain complex has distinct fitness outcomes. Notably, multicellular organogenesis signaling pathways including TGFb-SMAD specifically constrict the uncontrolled cell proliferation in 3D while inactivation of epigenetic modifiers (Bcor, Kmt2d, Mettl3 and Mettl14) has opposite outcomes in 2D vs. 3D. We further identify a 3D-dependent synthetic lethality with partial loss of Prmt5 due to a reduction of Mtap expression resulting from 3D-specific epigenetic reprogramming. Our study highlights unique epigenetic, metabolic and organogenesis signaling dependencies under different cellular settings.

Prevalence of S-methyl-5'-thioadenosine Phosphorylase (MTAP) Deficiency in Human Cancer: A Tissue Microarray Study on 13,067 Tumors From 149 Different Tumor Types.

Loss of S-methyl-5'-thioadenosine phosphorylase (MTAP) expression is a common event in cancer leading to a critical vulnerability of cancer cells towards anti-cancer drugs. Homozygous MTAP deletions result in a complete expression loss that can be detected by immunohistochemistry (IHC). In this study, a tissue microarray containing 17,078 samples from 149 different tumor entities was analyzed by IHC, and complete MTAP loss was validated by fluorescence in situ hybridization. MTAP loss was observed in 83 of 149 tumor categories, including neuroendocrine neoplasms (up to 80%), Hodgkin lymphoma (50.0%), mesothelioma (32.0% to 36.8%), gastro-intestinal adenocarcinoma (4.0% to 40.5%), urothelial neoplasms (10.5% to 36.7%), squamous cell carcinomas (up to 38%), and various types of sarcomas (up to 20%) and non-Hodgkin lymphomas (up to 14%). Homozygous MTAP deletion was found in 90% to 100% of cases with MTAP expression loss in most tumor categories. However, neuroendocrine tumors, Hodgkin lymphomas, and other lymphomas lacked MTAP deletions. MTAP deficiency was significantly linked to unfavorable tumor phenotype in selected tumor entities and the presence of PD-L1 expression on tumor cells, absence of PD-L1 expression on immune cells, and a low density of CD8 + lymphocytes. In summary, MTAP deficiency can occur in various tumor entities and is linked to unfavorable tumor phenotype and noninflamed tumor microenvironment, but is not always related to deletions. MTAP IHC is of considerable diagnostic value for the detection of neoplastic transformation in multiple different applications.

Immune infiltrate characterization and differential gene expression by RNA-sequencing analysis in patients with oncogene-addicted NSCLC with early progression under targeted therapy.

8535 Background: Despite remarkable advances with tyrosine kinase inhibitors (TKI), patients (pts) with oncogene addicted advanced non-small cell lung cancer (aNSCLC) eventually face resistance. Moreover, some pts exhibit early TKI resistance, often linked to a reduced response subsequent TKIs. These pts might have unique biology, and identifying post-resistance treatments for them is an unmet need. Here, we explore the transcriptomic profile of pts with aNSCLC and early TKI progression, as compared to long responders. Methods: We included pts with aNSCLC harboring EGFR mutations or oncogenic fusions ( ALK, ROS1, RET, NTRK), treated with TKI at Gustave Roussy and enrolled in the prospective MATCH R trial (NCT02517892). Clinical data on treatment outcomes and survival were collected. Pts with PFS &lt; 6 months were considered as having early progression (EP), pts with PFS &gt; 18 months as long responders (LR). At least one tissue biopsy was performed at PD, on which bulk RNA sequencing was carried out. We explored the transcriptomic differences between pts with EP and LR. Differential gene expression (DESeq2), pathway enrichment studies and identification with MSigDb were performed and compared between groups. Immune cell type contents were evaluated by CIBERSORT deconvolution method. Results: From 2015 to 2022, 37 pts were included. 68% were females, median age was 55 (IQR43-83). 18 carried EGFR mutations (EP N=5, 28%; LR N=13, 72%), 19 had fusions (EP N=10, 52%; LR N=9, 48%). All pts had progressed to targeted TKI (7 first generation EGFR-TKI, 11 osimertinib, 8 crizotinib, 7 second generation ALK-TKI, 2 lorlatinib, 2 pralsetinib, 1 repotrectinib). Immune suppressive macrophages were the most represented immune cells in EGFR+ and fusion+ PD samples (33.5% and 33.2% of immune cells). The composition of the immune infiltrate was not significantly different between EP and LR in EGFR+ and fusion+ pts. DESeq2 showed that EGFR+ EP had significantly lower EPCAM(log2FC: 1.60, padj=0.01), higher SOS1 (log2FC: -1.18, padj=0.01), higher JAK2(log2FC: -1.56, padj=0.02) expression, as compared to EGFR+ LR. In the fusion+ group, EP had higher FGFR3/4 (log2FC: -3.5, padj=0.02) and lower MTAP expression (log2FC: 1.75, padj=0.03). Hallmark analysis in the EGFR+ showed higher activation of TNFα, IFNγ response, and KRAS pathways in EP (NES: -1.72, p=0.03; NES=-1.78,p=0.02; NES:-2.07, p=0.01) versus LR. No differential activation was observed in the fusion+ group. Conclusions: Immune suppressive macrophages are the most represented immune cells at PD to TKI, irrespective of the treatment duration. Pts with EP seem to have a different biology, with transcriptomic features of stemness and immune suppression in the EGFR+ pts, and lower MTAP expression in the fusion+ group. This should be further explored for the development of novel therapeutics.

Abstract B018: KDM6A and MTAP expression loss enable identification of a large fraction of low grade non-invasive urothelial neoplasia of the urinary bladder

Abstract The identification of low-grade urothelial neoplasia in cytologic samples or in biopsies with dysplastic lesions poses a particular problem for pathologists in routine diagnostics. Improved diagnosis of low-grade urothelial neoplasia could help to reduce the number of cystoscopic follow-up examinations. KDM6A, also known as UTX (ubiquitously transcribed X chromosome tetratricopeptide repeat protein) is an epigenetic regulator which is frequently mutated in urothelial carcinoma. Because KDM6A is localized on chromosome Xp11.3, and because of the male predominance in bladder cancer, many KDM6A mutations should result in a complete loss of KDM6A protein expression and become detectable by immunohistochemistry (IHC). Similarly, S-methyl-5′-thioadenosine phosphorylase (MTAP), a critical enzyme for DNA and RNA synthesis, is co-deleted with the CDKN2A tumor suppressor located on chromosome 9p21 in about 25% of bladder cancers, resulting in reduced or lost protein expression. To study the prevalence and diagnostic potential of immunohistochemical loss of KDM6A and MTAP expression, 928 tumors were analyzed by IHC in a tissue microarray format. The cohort included 628 non-invasive papillary tumors (pTa) and 300 tumors from patients who underwent radical cystectomy for muscle-invasive disease (pT2-4). A complete KDM6A expression loss occurred in 35% of 342 pTa G2 low-grade, 23% of 150 pTa G2 high-grade, 17.8% of 90 pTa G3 tumors, and 20.1% of 243 interpretable muscle invasive (pT2-4) cancers (p&amp;lt;0.0001). Within pTa tumors, there was a strong correlation between KDM6A loss and low tumor grade (p=0.0003), whereas no significant difference was found between pT3 G3 and muscle invasive cancers (p=0.8828). Complete absence of MTAP expression occurred in 11% of 297 pTa G2 low-grade, 29.6% of 125 pTa G2 high-grade, 18.4% of 76 pTa G3 tumors, and 26.3% of 217 muscle invasive (pT2-4) cancers (p&amp;lt;0.0001). A combined analysis of 702 cancers with interpretable data for KDM6A and MTAP showed that 6.6% of tumors had expression loss of both KDM6A and MTAP, 20% had KDM6A loss only, and 13% had MTAP loss only. The rate of expression loss of KDM6A and/or MTAP was markedly higher in pTa G2 low (41.2%) and in pTa G2 high (40.3%) than in pTa G3 tumors (29%). In summary, our data demonstrate that about 40% of low-grade non-invasive urothelial neoplasias – which are usually not detected by urine cytology – had a completed expression loss of either KDM6A or MTAP. In patients with these alterations, follow-up could be potentially facilitated by immunohistochemistry. Citation Format: Florian Viehweger, Neele Heckmann, Natalia Gorbokon, Henning Plage, Sebastian Hofbauer, Sarah Weinberger, Florian Roßner, Simon Schallenberg, Sefer Elezkurtaj, Maximilian Lennartz, Tim Mandelkow, Elena Bady, Margit Fisch, Thorsten Ecke, Stefan Koch, Nico Adamini, Ronald Simon, Guido Sauter, Tobias Klatte, Thorsten Schlomm, David Horst, Henrik Zecha, Martina Kluth, Sarah Minner. KDM6A and MTAP expression loss enable identification of a large fraction of low grade non-invasive urothelial neoplasia of the urinary bladder [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B018.

Loss of MTAP Expression by Immunohistochemistry Is a Surrogate Marker for Homozygous 9p21.3 Deletion in Urothelial Carcinoma

Homozygous deletion of the chromosomal region 9p21.3 is common in urothelial carcinoma (UC) and leads to loss of several genes, including CDKN2A and MTAP, resulting in loss of MTAP protein expression. Here, we aimed to explore the diagnostic potential of MTAP immunohistochemistry (IHC) as a surrogate marker for homozygous 9p21.3 deletion (9p21 homozygous deletion [HD]) in UC. MTAP status was determined by IHC on 27 UC tissue specimens with known 9p21.3 status as defined by fluorescence in situ hybridization in matched cytological specimens, by IHC and fluorescence in situ hybridization on a tissue microarray (TMA) containing 359 UC at different stages, and by IHC on 729 consecutive UC from routine practice. Moreover, we analyzed a longitudinal series of matched specimens from 38 patients with MTAP-negative recurrent UC. MTAP loss by IHC was found in all 17 patients with 9p21 HD and in 2/8 cases without 9p21 HD. In the TMA, MTAP loss was more common in metastases (53%) than in muscle-invasive (33%) and non–muscle-invasive UC (29%) (P = .03). In the consecutive series, 164/729 (22%) cases showed loss of MTAP expression. In 41 of these 164 cases (25%), loss of MTAP expression was heterogenous. We also discovered loss of MTAP expression in flat urothelium adjacent to MTAP-negative low-grade UC, suggesting true flat low-grade neoplasia that could not be diagnosed by morphology alone. Longitudinal analysis of recurrences showed persistent negative MTAP status over time in 37/38 (97%) patients. MTAP IHC can serve as a surrogate marker for 9p21 HD in UC and as a diagnostic tool to differentiate reactive urothelium from urothelial neoplasia. It also provides a unique opportunity to study clinicopathological associations and the heterogeneity of 9p21 HD across the whole spectrum of UC manifestations.

Loss of methylthioadenosine phosphorylase immunoreactivity correlates with poor prognosis and elevated uptake of 11C-methionine in IDH-mutant astrocytoma.

The proximate localization of MTAP, which encodes methylthioadenosine phosphorylase, and CDKN2A/B on Chromosome 9q21 has allowed the loss of MTAP expression as a surrogate for homozygous deletion of CDKN2A/B. This study aimed to determine whether MTAP status correlates with clinical outcomes and 11C-methionine uptake in astrocytomas with IDH mutations. We conducted immunohistochemistry for MTAP in 30 patients with astrocytoma, IDH-mutant who underwent 11C-methionine positron emission tomography scans prior to surgical resection. The tumor-to-normal (T/N) ratio of 11C-methionine uptake was calculated using the mean standardized uptake value (SUV) for tumor and normal brain tissues. Cox regression analysis was used for multivariate survival analysis. Among IDH-mutant astrocytomas, 26.7% (8/30) exhibited the loss of cytoplasmic MTAP expression, whereas 73.3% (22/30) tumors retained MTAP expression. The median progression-free survival (PFS) was significantly shorter in patients with MTAP loss than those with MTAP retention (1.88years vs. 6.80years, p = 0.003). The median overall survival (OS) was also shorter in patients with MTAP loss than in MTAP-retaining counterparts (5.23years vs. 10.69years, p = 0.019). Multivariate analysis identified MTAP status (hazard ratio (HR), 0.081) and extent of resection (HR, 0.104) as independent prognostic factors for PFS. Astrocytomas lacking cytoplasmic MTAP expression showed a significantly higher median T/N ratio for 11C-methionine uptake than tumors retaining MTAP (2.12 vs. 1.65, p = 0.012). Our study revealed that the loss of MTAP expression correlates with poor prognosis and an elevated T/N ratio of 11C-methionine uptake in astrocytoma, IDH-mutant.

Abstract 1046: Prevalence of MTAP deficiencyin human cancer: A tissue microarray study on 17,078 cancers from 149 different tumor types

Abstract The S-methyl-5′-thioadenosine phosphorylase (MTAP) gene is located at 9p21 and is often co-deleted with CDKN2A. Because homozygous MTAP deletions result in a complete expression loss, MTAP immunohistochemistry (IHC) can be used for detection of homozygous deleted cells. MTAP IHC is of diagnostic utility for the distinction of mesotheliomas (often 9p21 deleted) from benign mesothelial proliferations. Moreover, evidence is emerging that MTAP deficiency results in a critical vulnerability of cancer cells towards drugs targeting several different pathways that depend on a functional MTAP gene and also causes resistance to immune checkpoint inhibitors. To determine the prevalence of complete MTAP expression loss and to assess the diagnostic utility of MTAP IHC, a tissue microarray containing 17,078 samples from 149 different tumor entities and 608 samples of 76 different normal tissue types was analyzed by IHC. All cases with complete MTAP loss were validated by fluorescence in situ hybridization (FISH). In normal tissues, MTAP was ubiquitously expressed resulting in a cytoplasmic and nuclear MTAP staining. At least one case with a complete MTAP expression loss was observed in 87 of 149 (58%) tumor categories. MTAP staining was most commonly lost in neuroendocrine neoplasms of various sites (up to 80%), Hodgkin’s lymphoma (38%), mesothelioma (24-30%), bilio-pancreatic adenocarcinomas (9-36%), urothelial neoplasms (19-29%), malignant melanoma (14-21%), mucinous carcinoma of the ovary (27%), squamous cell carcinomas of various organs of origin (up to 24%), various types of sarcomas (up to 20%), adenocarcinoma of the lung (11%), gastro-esophageal adenocarcinomas (2.6-11%), and in various types of non-Hodgkin’s lymphomas (up to 14%). FISH validation identified homozygous 9p21 deletions in 90-98% of cases with MTAP expression loss in most tumor categories including urothelial neoplasms, mesotheliomas, and pancreatic cancers, and others. There were, however, also tumor types such as neuroendocrine tumors and Hodgkin’s lymphomas that always lacked 9p21 deletions. In summary, our data provide a comprehensive overview on MTAP expression in cancer and show a strong link between MTAP expression loss and homozygous 9p21 deletions in most tumor entities. These cancers might most benefit from cancer drugs targeting MTAP deficiency. The absence of 9p21 deletions in other entities with frequent MTAP expression loss demonstrates that other mechanisms can cause significant MTAP downregulation. Whether these tumors benefit from targeting MTAT deficiency remains to be determined. Citation Format: Natalia Gorbokon, Maximilian Lennartz, Doris Hoeflmayer, Sebastian Dwertmann Rico, Simon Kind, Viktor Reiswich, Florian Viehweger, Florian Lutz, Christoph Fraune, Andreas M. Luebke, Claudia Hube-Magg, Franziska Büscheck, Anne Menz, Ria Schlichter, Till Krech, Andrea Hinsch, Eike Burandt, Guido Sauter, Ronald Simon, Stefan Steurer, Andreas H. Marx, Patrick Lebok, David Dum, Sarah Minner, Frank Jacobsen, Till S. Clauditz, Christian Bernreuther, Martina Kluth. Prevalence of MTAP deficiencyin human cancer: A tissue microarray study on 17,078 cancers from 149 different tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1046.

Mesothelioma of Uncertain Malignant Potential (MUMP) of the Tunica Vaginalis: Proposal for Reclassification as "Complex Mesothelial Tumor of the Tunica Vaginalis".

A well-differentiated papillary mesothelial tumor (WDPMT) and malignant mesothelioma are 2 well-recognized entities arising from the testis tunica vaginalis. Another mesothelial lesion exclusively seen at this site is mesothelioma of uncertain malignant potential (MUMP)-a lesion reminiscent of WDPMT yet demonstrating variable proportions of more complex architectural patterns that could be confused with invasion. MUMP was first described in 2010 with a total of 11 cases reported to date. Herein, we describe 19 additional patients who underwent hydrocelectomy, excision, and/or orchiectomy. Novel morphologic patterns found in addition to the 2010 series include spindle cells, keloidal-type collagen, and multicystic architecture lined by bland mesothelial cells. Clinical follow-up in 9 patients for more than 1 year (1.5 to 22.5y, median 4.5y) revealed no evidence of disease recurrence or metastases. Despite greater architectural complexity, MUMP has (1) bland cytology; (2) merging in with WDPMT areas; (3) low mitotic rate and Ki-67 nuclear labeling index; (4) retention of MTAP and BAP1 expression; and (5) benign clinical follow-up. If these cases were malignant mesotheliomas, one would have expected at least some of the patients to demonstrate disease recurrence/progression without adjuvant therapy within the available follow-up time, particularly with limited resection in most patients. Thus, we propose that "mesothelioma of uncertain malignant potential" be renamed as "complex mesothelial tumor of the tunica vaginalis." Using the term "complex" draws a contrast with the simple cuboidal lining and simple papillary architecture seen in WDPMT. Also, labeling the lesion as "tumor" removes the stigmata of "uncertain malignant potential" and "mesothelioma" that are alarming to patients and clinicians, and potentially could unduly lead to more extensive surgery in an attempt at "complete" resection. At the same time, not definitively labeling the lesion as benign allows recommendations for follow-up.

Investigation of MTAP and BAP1 staining loss and P16/CDKN2A deletion in pleural cytology specimens and its role in the diagnosis of mesothelioma.

Mesothelioma is a malignant neoplasm with a poor survival rate. We aimed to investigate the importance of BAP1, MTAP (IHC), and p16/CDKN2A homozygous deletion (FISH) in cytologic material obtained from pleural effusion sampling, which is a less invasive procedure in the diagnosis of mesothelioma. Our study discussed pleural cytology samples of cases with histopathologically proven mesothelioma diagnoses between 2017 and 2022. As the control group, materials that had pleural effusion sampling for other reasons and reactive mesothelial hyperplasia were included in the study. Cell blocks prepared from these materials were subjected to fluorescent in situ hybridization for p16/CDKN2A homozygous deletion and immunohistochemistry for BAP1 and MTAP. The specificity of the P16/CDKN2A homozygous deletion in diagnosing mesothelioma is 100%. Its sensitivity is 68.75%. The specificity of BAP1 immunohistochemical nuclear expression loss is 95%, while the sensitivity is 60%. Loss of nuclear expression of MTAP alone has the lowest specificity and sensitivity, with a specificity of 86% and a sensitivity of 43%. The highest sensitivity is reached when BAP1 loss and p16/CDKN2A homozygous deletion are evaluated together, increasing to 81%. The specificity is 95%. It has been determined that any marker alone cannot be used for a definitive mesothelioma diagnosis in pleural effusion cytological specimens; however, sensitivity increases in some combinations. The combination of BAP1 immunohistochemistry and p16/CDKN2A homozygous deletion detected by FISH, which has a higher specificity and sensitivity, can be routinely used in the diagnosis of mesothelioma under the guidance of clinical and radiologic information.

Comparison of Immunohistochemistry, Next-generation Sequencing and Fluorescence In Situ Hybridization for Detection of MTAP Loss in Pleural Mesothelioma

9p21 deletions involving MTAP/CDKN2A genes are detected in diffuse pleural mesotheliomas (DPM) but are absent in benign mesothelial proliferations. Loss of MTAP expression by immunohistochemistry (IHC) is well accepted as a surrogate for 9p21 deletion to support a diagnosis of DPM. Accurate interpretation can be critical in the diagnosis of DPM, but variations in antibody performance may impact interpretation. The objectives of this study were to compare the performance of MTAP monoclonal antibodies (mAbs) EPR6893 and 1813 and to compare MTAP expression by IHC with 9p21 copy number status in DPM. Cytoplasmic expression of MTAP IHC with mAbs EPR6893 (ab126770; Abcam) and 1813 (NBP2-75730, Novus Biologicals) was evaluated in 56 DPM (47 epithelioid, 7 biphasic, and 2 sarcomatoid) profiled by targeted next-generation sequencing. 9p21 Copy number status was assessed by Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing (FACETS) analysis and also by CDKN2A fluorescence in situ hybridization in discrepant cases when material was available. MTAP mAb 1813 showed stronger immunoreactivity, more specific staining, and no equivocal interpretations compared to mAb EPR6893 which showed equivocal staining in 19 (34%) of cases due to weak or heterogenous immunoreactivity, lack of definitive internal positive control, and/or nonspecific background staining. MTAP expression with mAb 1813 showed near perfect agreement with 9p21 copy number by combined FACETS/fluorescence in situ hybridization calls (κ = 0.85; 95% CI, 0.71-0.99; P < .001). MTAP IHC with mAb 1813 was 96% sensitive, 86% specific, and 93% accurate for 9p21 homozygous deletion. The findings of this study suggest that interpretation of MTAP IHC is improved with mAb 1813 because mAb EPR6893 was often limited by equivocal interpretations. We show that MTAP IHC and molecular assays are complementary in detecting 9p21 homozygous deletion. MTAP IHC may be particularly useful for low tumor purity samples and in low-resource settings.

PATH-47. MOLECULAR MARKERS ASSOCIATED WITH SURVIVAL IN GRADE 3 MENINGIOMA

Abstract Meningiomas have historically been graded using histologic features. However, a subset of meningiomas progress in manners inconsistent with their histologic grading. While TERT promoter (TERTp) mutations and CDKN2A/B homozygous deletion were recently added to the WHO 2021 classification criterion for grade 3 meningioma, the precise prognostic implications of these and other molecular features remain to be fully characterized. In this case series, we explored whether molecular markers were associated with clinical outcomes in a single-center cohort of grade 3 intracranial meningiomas. Demographic, clinical and histopathological information were obtained from the electronic medical records of all grade 3 meningioma cases surgically treated at a tertiary center between 2007-2020. Tissue blocks were reviewed by a neuropathologist and tested for TERTp mutations, BAP1 and CDKN2A/B copy number profiles, and BAP1, p16 and MTAP expression by immunohistochemistry (IHC). Predictors of survival were identified by Cox regression. Fifteen patients (8 female, 7 male) with a median age of 64 (IQR: 48, 74) years were included. Eight (53%) tumors were classified as anaplastic, 6 (40%) as rhabdoid and 1 (6.7%) as papillary. One (7%) rhabdoid tumor exhibited BAP1 loss, while four (27%) tumors harbored TERTp mutations and 3 (20%) demonstrated homozygous CDKN2A/B loss. All three tumors exhibiting homozygous CDKN2A/B loss were MTAP-negative, while two were p16-negative and the third showed focal (&amp;lt; 50%) p16 loss by IHC. TERTp mutations and CDKN2A/B copy number status were associated with significant reductions in RFS (HR = 6.90, 95% CIs: 1.13-42.2 and HR = 12.0, 05% CIs: 1.51-95.4, respectively) and OS (HR = 8.72, 95% CIs: 1.87-40.6 and HR = 6.18, 95% CIs: 1.30-29.4, respectively). The associations between reduced OS and TERTp mutations and CDKN2A/B status remained significant after adjusting for Simpson resection grade. Our findings support using TERT p mutations and CDKN2A/B status for prognostication in grade 3 meningioma.

NIMG-65. METHYLTHIOADENOSINE PHOSPHORYLASE STATUS CORRELATES WITH CLINICAL OUTCOME AND11C-METHIONINE UPTAKE IN ASTROCYTOMA, IDH-MUTANT

Abstract PURPOSE Methylthioadenosine phosphorylase (MTAP) immuno histochemistry staining has been proposed as a surrogate of Cyclin-dependent kinase inhibitor 2A homozygous deletion (CDKN2A HD) in various types of tumors. This study was designed to determine whether MTAP status correlates with clinical outcome and uptake of 11C-methionine in astrocytoma, IDH-mutant. METHODS We conducted a MTAP immunohistochemistry staining of 30 astrocytoma, IDH-mutant patients who underwent 11C-methionine positron emission tomography scans prior to surgical resection from 2000 to 2020. The tumor-to-normal (T/N) ratio of 11C-methionine uptake was calculated by dividing the mean standardized uptake value (SUV) for the tumor by the mean SUV of the normal brain. RESULTS Eight (26.7%) of the thirty astrocytoma, IDH-mutant patients exhibited loss of cytoplasmic MTAP expression, which is accompanied with a poor prognosis. The median progression free survival (PFS) in astrocytoma, IDH-mutant patients with loss of MTAP was 1.88 years and significantly shorter than that of those with MTAP retention (6.80 years, p = 0.003). The median Overall survival in astrocytoma, IDH-mutant patients with loss of MTAP was 5.23 years, again significantly shorter than that of those with MTAP retention (10.69 years, p = 0.0191). Multivariate analysis revealed that MTAP status was an independent prognostic marker for PFS in astrocytoma, IDH-mutant patients. The median T/N ratio in tumors with loss of cytoplasmic MTAP expression was 2.12 (IQR 1.92-2.50), i.e., significantly higher than that of tumors with MTAP retention (1.65, IQR 1.23-1.94, p = 0.0116, U test). Thirteen (59.1%) of twenty-two patients with MTAP retention recurred and nine patients underwent a second surgery after radiation treatment with or without chemotherapy. Three (33.3%) of the nine recurrent patients showed loss of MTAP immunoreactivity, suggesting a role for this loss in tumor progression. CONCLUSION Our study showed MTAP status to correlate with clinical outcome and T/N ratio of 11C-methionine in astrocytoma, IDH-mutant patients.

Prognostic Value of Immunohistochemical Expression of MTAP and AKIP1 in IDH1 Mutant Astrocytoma.

Definite treatment for glioma is not exist, and with increased drug resistance, more effort should be paid to identify new prognostic biomarkers and molecular targets for therapy for glioma patients. The current study aimed to evaluate the immunohistochemical (IHC) expression of MTAP and A-Kinase Interacting Protein 1 (AKIP1) in astrocytoma and to investigate their association with the clinicopathological characters of these cases. Totally 66 cases of astrocytoma patients involved in this study. Cases underwent tumor resection and tissue sections were stained with MTAP, AKIP1 and IDH1 by IHC and evaluated in different grades of astrocytoma and their association with survival and response to therapy was investigated. High AKIP1 expression was positively correlated with treatment resistance and progressive disease. Positive IDH and retained MTAP expressions had shown better treatment response rather than negative IDH and lost MTAP. High AKIP, negative IDH and loss of MTAP expressions were significantly associated with poor survival outcome. Irrespective to grade and IDH status, the loss of MTAP immunoreactivity and high AKIP1 expression are predictive factors in astrocytoma, and they may be used as a biomarker for guiding astrocytoma management and prognosis surveillance.

Deletions of CDKN2A and MTAP Detected by Copy-Number Variation Array Are Associated with Loss of p16 and MTAP Protein in Pleural Mesothelioma.

CDKN2A deletion is a common alteration in pleural mesothelioma (PM) and frequently associated with co-deletion of MTAP. Since the standard detection method for CDKN2A deletion and FISH analysis is relatively expensive, we here investigated the suitability of inexpensive p16 and MTAP IHC by comparing concordance between IHC and OncoScan CNV arrays on samples from 52 PM patients. Concordance was determined using Cohen's kappa statistics. Loss of CDKN2A was associated with co-deletion of MTAP in 71% of cases. CDKN2A-MTAP copy-number normal cases were also IHC positive in 93% of cases for p16 and 100% for MTAP, while homozygous deletion of CDKN2A-MTAP was always associated with negative IHC for both proteins. In cases with heterozygous CDKN2A-MTAP loss, IHC expression of p16 and MTAP was negative in 100% and 71%, respectively. MTAP and p16 IHC showed high sensitivity (MTAP 86.5%, p16 100%) and specificity (MTAP 100%, p16 93.3%) for the detection of any gene loss. Loss of MTAP expression occurred exclusively in conjunction with loss of p16 labeling. Both p16 and MTAP IHC showed high concordance with Oncoscan CNV arrays (kappa = 0.952, p < 0.0001, and kappa = 0.787, p < 0.0001 respectively). We recommend combined MTAP and p16 immunohistochemistry to confirm the diagnosis of PM.

P18.06.B EVALUATION OF P16 AND MTAP IMMUNOHISTOCHEMICAL EXPRESSION AS SURROGATE MARKERS OF CDKN2A/B DELETION IN MENINGIOMAS

Abstract BACKGROUND Homozygous deletion (HD) of CDKN2A/B genes (chr. 9p21) is a molecular hallmark for meningiomas grading. However, testing is expensive and time-consuming. Immunohistochemical (IHC) evaluation of the CDKN2A (p16) and adjacent MTAP gene products has been proposed as a diagnostic surrogate of CDKN2A HD, with unsatisfactory or inconclusive results. MATERIAL AND METHODS CDKN2A status was assessed by FISH with the Zytolight SPEC CDKN2A/CEN 9 Dual Color probe (Zytovision©) in 68 meningiomas from 56 patients. A 15% cut-off of HD cells was used to define a sample as deleted. Negative (&amp;lt;10% positive cells) p16 (Ventana) and MTAP (Clone 2G4; Abnova) IHC were considered as a surrogate for CDKN2A HD. Since CDKN2A/B deletion in meningiomas is characterized by spatial and longitudinal heterogeneity, the same area selected for FISH analysis was assessed by IHC. Pearson Chi-square test for categorical variables and survival analysis with log-rank test and the Kaplan-Meier method were performed using the Stata/MP 17.0 software. RESULTS In our series, 29/56 (51.7%) patients were male, and median age at diagnosis was 69 years. Median overall survival (OS) from diagnosis was 37.9 months (27 months for HD cases and 39.3 months for non-HD). 51 (75%) samples were diagnosed as WHO grade 3 meningiomas, 11 (16.2%) as grade 2 and 6 (8.8%) as grade 1. 11 cases (16.2%) were CDKN2A-deleted by FISH. p16 IHC was negative in 25 (36.8%) cases, while MTAP IHC was negative in 24 (35.3%) cases. No correlation between these markers was observed (Cohen’s κ=-0.0202, p=0.7158). 5/11 (45.4%) deleted samples were negative for p16 staining, while 37/57 (64.9%) non-deleted samples displayed p16 positivity (sensitivity: 20%; specificity: 86.1%). MTAP IHC was negative in 8/11 (72.7%) deleted samples, whereas 41/57 (71.9%) non-deleted samples retained MTAP expression (sensitivity: 33.3%; specificity: 93.2%). Unlike p16 IHC, MTAP loss correlated with CDKN2A HD (p=0.005). To address intratumoral heterogeneity, FISH analysis was repeated on 2 samples with markedly variable p16 and MTAP stainings. For each sample, one positive and one negative area were analyzed, observing a correlation between IHC and HD FISH status. CONCLUSION MTAP IHC showed a higher correlation with CDKN2A HD compared to p16 IHC in meningiomas, even though the low sensitivity hinders its use in routine diagnostics. Nevertheless, the use of p16/MTAP IHC to select areas for FISH analysis could be considered to address intratumoral heterogeneity of CDKN2A status.

Recurrent Tumor Suppressor Alterations in Primary Pericardial Mesothelioma

Primary pericardial mesotheliomas are extremely rare, accounting for <1% of all mesotheliomas, and their molecular genetic features and predisposing factors remain to be determined. Here, we report the clinicopathologic, immunohistochemical, and molecular genetic findings of 3 pericardial mesotheliomas without pleural involvement. Three cases diagnosed between 2004 and 2022 were included in the study and analyzed by immunohistochemistry and targeted next-generation sequencing (NGS); corresponding nonneoplastic tissue was sequenced in all cases. Two patients were female and 1 was male, aged between 66 and 75 years. Two patients each had prior asbestos exposure and were smokers. Histologic subtypes were epithelioid in 2 cases and biphasic in 1 case. Immunohistochemical staining identified expression of cytokeratin AE1/AE3 and calretinin in all cases, D2-40 in 2 cases, and WT1 in 1 case. Staining for tumor suppressors revealed loss of p16, MTAP, and Merlin (NF2) expression in 2 cases and loss of BAP1 and p53 in 1 case. Abnormal cytoplasmic BAP1 expression was observed in an additional case. Protein expression abnormalities correlated with NGS results, which showed concurrent complete genomic inactivation of CDKN2A/p16, CDKN2B, MTAP, and NF2 in 2 mesotheliomas and of BAP1 and TP53 in 1 mesothelioma each, respectively. In addition, 1 patient harbored a pathogenic BRCA1 germline mutation, which resulted in biallelic inactivation in the mesothelioma. All mesotheliomas were mismatch repair proficient and showed several chromosomal gains and losses. All patients died from disease. Our study demonstrates that pericardial mesotheliomas share common morphologic, immunohistochemical, and molecular genetic features with pleural mesothelioma, including recurrent genomic inactivation of canonical tumor suppressors. Our study adds new insights into the genetic landscape of primary pericardial mesothelioma and highlights BRCA1 loss as a potential contributing factor in a subset of cases, thereby contributing to refined precision diagnostics for this rare cancer.

MTAP-ANRIL gene fusion promotes melanoma epithelial-mesenchymal transition-like process by activating the JNK and p38 signaling pathways

Gene fusions caused by cytogenetic aberrations play important roles in the initiation and progression of cancers. The recurrent MTAP-ANRIL fusion gene was reported to have a frequency of greater than 7% in melanoma in our previous study. However, its functions remain unclear. Truncated MTAP proteins resulting from point mutations in the last three exons of MTAP can physically interact with the wild-type MTAP protein, a tumor suppressor in several human cancers. Similarly, MTAP-ANRIL, which is translated into a truncated MTAP protein, would influence wild-type MTAP to act as an oncogene. Here, we found that MTAP-ANRIL gene fusion downregulated the expression of wild-type MTAP and promoted epithelial-mesenchymal transition-like process through the activation of JNK and p38 MAPKs in vitro and in vivo. Our results suggest that MTAP-ANRIL is a potential molecular prognostic biomarker and therapeutic target for melanoma.