Methylthioadenosine Phosphorylase Expression Research Articles

Different Frequency and Clinical Role for MTAP Loss in Pleural and Peritoneal Mesothelioma.

The objective of this study was to analyze the expression and prognostic role of methylthioadenosine phosphorylase (MTAP) in mesothelioma. MTAP protein expression by immunohistochemistry was analyzed in 113 mesotheliomas (60 pleural and 53 peritoneal), consisting of 36 effusions and 77 surgical specimens. MTAP expression was fully lost in 38 tumors and partially lost in 8 tumors. Loss of expression was significantly more common in effusions compared with biopsies/surgical resection specimens (20/36 vs. 26/77; P =0.017), and in pleural compared with peritoneal mesotheliomas (35/60 vs. 11/53; P <0.001). MTAP performed less robustly than BAP1 in comparative analysis of 57 tumors previously analyzed for expression of the latter protein (46 vs. 25 cases with loss of expression). In survival analysis for 69 patients with partial clinical data, male gender was significantly associated with shorter overall survival (OS; P =0.042), whereas loss of MTAP was associated with a trend for shorter OS ( P =0.058), with no prognostic role for patient age ( P =0.379) or anatomic site ( P =0.381). The association between loss of MTAP and poor OS became significant when survival analysis was limited to patients with pleural mesothelioma ( P =0.018). In conclusion, loss of MTAP expression is more frequent in pleural compared with peritoneal mesothelioma and has limited diagnostic relevance at the latter anatomic site. More frequent loss in effusion specimens suggests a role for this marker in effusion cytology. MTAP loss in pleural mesothelioma is associated with poor survival.

Methylthioadenosine phosphorylase and p16 as surrogate diagnostic markers for CDKN2A homozygous deletion in brain tumors

Objective: To examine whether immunohistochemistry of methylthioadenosine phosphorylase (MTAP) and p16 could be used to predict the CDKN2A status in various brain tumors. Methods: A total of 118 cases of IDH-mutant astrocytomas, 16 IDH-wildtype glioblastoma, 17 polymorphic xanthoastrocytoma (PXA) and 20 meningiomas diagnosed at Xuanwu Hospital, Capital Medical University, Beijing, China from November 2017 to October 2023 were collected and analyzed. The CDKN2A status was detected by using fluorescence in situ hybridization or next-generation sequencing. Expression of MTAP and p16 proteins was detected with immunohistochemistry. The association of loss of MTAP/p16 expression with CDKN2A homozygous/heterozygous deletion was examined. Results: Among the 118 cases of IDH-mutant astrocytoma, 13 cases showed homozygous deletion of CDKN2A. All of them had no expression of MTAP while 9 cases had no expression of p16. Among the 16 cases of IDH wild-type glioblastoma, 6 cases showed homozygous deletion of CDKN2A. All 6 cases had no expression of MTAP, while 3 of these cases had no expression of p16 expression. Among the 17 PXA cases, 4 cases showed homozygous deletion of CDKN2A, and the expression of MTAP and p16 was also absent in these 4 cases. Among the 20 cases of meningiomas, 4 cases showed homozygous deletion of CDKN2A. Their expression of MTAP and p16 was also absent. Among the four types of brain tumors, MTAP was significantly correlated with CDKN2A homozygous deletion (P<0.05), with a sensitivity of 100%. However, it was only significantly correlated with the loss of heterozygosity (LOH) of CDKN2A in astrocytomas (P<0.001). P16 was associated with CDKN2A homozygous deletion in IDH-mutant astrocytoma and PXA (P<0.001), but not with the LOH of CDKN2A. Its sensitivity and specificity were lower than that of MTAP. Conclusions: MTAP could serve as a predictive surrogate for CDKN2A homozygous deletion in adult IDH-mutant astrocytoma, PXA, adult IDH-wildtype glioblastoma and meningioma. However, p16 could only be used in the first two tumor types, and its specificity and sensitivity are lower than that of MTAP.

The combination of methionine adenosyltransferase 2A inhibitor and methyltransferase like 3 inhibitor promotes apoptosis of non-small cell lung cancer cells and produces synergistic anti-tumor activity

Methionine adenosyltransferase 2 A (MAT2A) mediates the synthesis of methyl donor S-Adenosylmethionine (SAM), providing raw materials for methylation reactions in cells. MAT2A inhibitors are currently used for the treatment of tumors with methylthioadenosine phosphorylase (MTAP) deficiency in clinical research. Methyltransferase like 3 (METTL3) catalyzes N6-methyladenosine (m6A) modification of mRNA in mammalian cells using SAM as the substrate which has been shown to affect the tumorigenesis of non-small cell lung cancer (NSCLC) from multiple perspectives. MAT2A-induced SAM depletion may have the potential to inhibit the methyl transfer function of METTL3. Therefore, in order to expand the applicability of inhibitors, improve anti-tumor effects and reduce toxicity, the combinational effect of MAT2A inhibitor AG-270 and METTL3 inhibitor STM2457 was evaluated in NSCLC. The results showed that this combination induced cell apoptosis rather than cell cycle arrest, which was non-tissue-specific and was independent of MTAP expression status, resulting in a significant synergistic anti-tumor effect. We further elucidated that the combination-induced enhanced apoptosis was associated with the decreased m6A level, leading to downregulation of PI3K/AKT protein, ultimately activating the apoptosis-related proteins. Unexpectedly, although combination therapy resulted in metabolic recombination, no significant change in methionine metabolic metabolites was found. More importantly, the combination also exerted synergistic effects in vivo. In summary, the combination of MAT2A inhibitor and METTL3 inhibitor showed synergistic effects both in vivo and in vitro, which laid a theoretical foundation for expanding the clinical application research of the two types of drugs.

The diagnostic utility of methylthioadenosine phosphorylase immunohistochemistry for pancreatic ductal adenocarcinoma in FNA and small biopsy specimens.

Accurate diagnosis of pancreatic lesions by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) or fine-needle biopsy can be challenging. Although surrogate immunohistochemical markers for genetic alterations associated with pancreatic ductal adenocarcinoma (PDAC) have been identified, they have modest sensitivity. Biallelic loss of CDKN2A occurs in up to 46% of PDACs, and methylthioadenosine phosphorylase (MTAP) immunohistochemistry (IHC) has been identified as a reliable surrogate marker for this alteration. The current study evaluates the utility of MTAP IHC for the diagnosis of PDAC. In total, 136 cases of EUS-FNA cell block or core biopsy targeting solid pancreatic masses were identified. MTAP IHC was performed and evaluated for complete loss of expression in neoplastic cells. These results were correlated with available clinical next-generation sequencing that was performed on a subset of cases. Complete loss of MTAP expression was identified in 23 of 80 (29%) PDACs. A subset of cases classified as suspicious (4 of 21) and atypical (4 of 22) showed MTAP loss. All morphologically indeterminate cases with MTAP loss were confirmed as PDAC on resection/additional sampling. No benign samples (n=13) showed loss of MTAP. In samples that had available clinical next-generation sequencing data (n=13), copy number loss of CDKN2A was detected in all cases that had loss of MTAP expression (n=4). Loss of MTAP was identified in approximately 30% of PDAC small biopsy specimens. As loss of MTAP expression is not expected in nonneoplastic cells, and these findings suggest that MTAP IHC can support a diagnosis of PDAC in small biopsy samples.

Epigenetic Silencing of MTAP in Hodgkin's Lymphoma Renders It Sensitive to a 2 nd Generation PRMT5 Inhibitor

Background: PRMT5 is type II arginine methyltransferase that catalyses symmetric dimethylation of arginine residues (SDMA) and plays an important role in cancer biology. By methylating a number of substrates, PRMT5 can regulate important processes such as DNA repair, RNA splicing, and cellular proliferation. In addition, PRMT5 is overexpressed in various cancer types and has been identified as a candidate for therapeutic intervention through the development of small molecules that inhibit PRMT5 methyltransferase activity. However, first generation PRMT5 inhibitors have shown limited clinical benefit mainly due to development of on-target toxicity, primarily in the bone marrow. AstraZeneca has developed a second generation PRMT5 inhibitor (AZ-PRMT5i), that selectively inhibits PRMT5 in MTAP deficient tumours while sparing MTAP proficient normal cells. MTAP (methylthioadenosine phosphorylase) is a metabolic enzyme involved in methionine salvage pathway. MTAP deficiency results in accumulation of the metabolite methylothioadenosine (MTA) in tumor cells that induces partial inhibition of PRMT5, rendering these tumors sensitive to PRMT5 inhibition. Homozygous deletion of the MTAP gene, that results in the loss of MTAP protein, has been found in approximately 15% of advanced solid tumors. Here, for the first time, we describe epigenetic silencing of the MTAP gene in Hodgkin's Lymphoma (HL) cell lines. This silencing results in the loss of MTAP protein expression thus increasing sensitivity to PRMT5 inhibition. Importantly, MTAP protein loss was also observed in primary cHL samples, opening a novel opportunity for the treatment of HL. Methods: Bioinformatic data mining of the Cancer Cell Line Encyclopedia (CCLE) dataset has been used to overlay levels of expression of MTAP mRNA with MTAP copy number and MTAP promoter DNA methylation. MTAP protein expression and activity of AZ-PRMT5i were assessed in 5 HL cell lines in vitro; efficacy of AZ-PRMT5i and levels of target engagement were tested in vivo in the L540 xenograft model. MTAP expression levels were determined by IHC analysis using 55 primary samples from cHL patients. Results: Using an unbiased analysis of the CCLE dataset, we have identified a subset of cell lines that in the absence of MTAP genetic loss were showing low levels of MTAP mRNA expression, equivalent to levels of MTAP mRNA in MTAP homozygous deleted cell lines. Interestingly, this was predominantly the case in HL cell lines where it was detected in 67% of cases (4 out of 6 cell lines). MTAP methylation profile analysis of HL cell lines revealed elevated MTAP DNA promoter methylation in cell lines with low MTAP mRNA expression. In addition, MTAP protein was not detected by Western Blot. Accordingly, lack of MTAP protein expression resulted in increased sensitivity of these cells to AZ-PRMT5i compared to a HL cell line that expressed MTAP protein. Utilizing RNA-seq for transcriptional profiling of AZ-PRMT5i on a MTAP-deficient HL cell line has identified robust changes in genes that regulate the cell cycle, DNA replication and TP53 pathways thus confirming on target activity. In addition, AZ-PRMT5i demonstrated strong dose dependent efficacy in a MTAP silenced L540 xenograft model where greater than 90% of tumour growth inhibition was detected, with no significant body weight loss. Corresponding dose-dependent inhibition of SDMA is observed in the treated tumours. Finally, IHC analysis of MTAP expression in 55 primary cHL samples has demonstrated that 46 of 55 cHL samples (84%) had absent nuclear MTAP expression. Conclusion: Here we are presenting a novel finding showing MTAP protein expression loss in the majority of tested primary cHL samples, which could provide a collateral vulnerability and novel therapeutic opportunity to 2 nd generation PRMT5 inhibitors as demonstrated in our pre-clinical models.

Phosphoinositide and redox dysregulation by the anticancer methylthioadenosine phosphorylase transition state inhibitor

Methylthio-DADMe-immucillin-A (MTDIA) is an 86 picomolar inhibitor of 5′-methylthioadenosine phosphorylase (MTAP) with potent and specific anti-cancer efficacy. MTAP salvages S-adenosylmethionine (SAM) from 5′-methylthioadenosine (MTA), a toxic metabolite produced during polyamine biosynthesis. Changes in MTAP expression are implicated in cancer growth and development, making MTAP an appealing target for anti-cancer therapeutics. Since SAM is involved in lipid metabolism, we hypothesised that MTDIA alters the lipidomes of MTDIA-treated cells. To identify these effects, we analysed the lipid profiles of MTDIA-treated Saccharomyces cerevisiae using ultra-high resolution accurate mass spectrometry (UHRAMS). MTAP inhibition by MTDIA, and knockout of the Meu1 gene that encodes for MTAP in yeast, caused global lipidomic changes and differential abundance of lipids involved in cell signaling. The phosphoinositide kinase/phosphatase signaling network was specifically impaired upon MTDIA treatment, and was independently validated and further characterised via altered localization of proteins integral to this network. Functional consequences of dysregulated lipid metabolism included a decrease in reactive oxygen species (ROS) levels induced by MTDIA that was contemporaneous with changes in immunological response factors (nitric oxide, tumour necrosis factor-alpha and interleukin-10) in mammalian cells. These results indicate that lipid homeostasis alterations and concomitant downstream effects may be associated with MTDIA mechanistic efficacy.

Methylthioadenosine phosphorylase deficiency in tumors: A compelling therapeutic target.

The methionine salvage pathway is responsible for recycling sulfur-containing metabolites to methionine. This salvage pathway has been found to be implicated in cell apoptosis, proliferation, differentiation and inflammatory response. Methylthioadenosine phosphorylase (MTAP) catalyzes the reversible phosphorolysis of 5'-methylthioadenosine, a by-product produced from polyamine biosynthesis. The MTAP gene is located adjacent to the cyclin-dependent kinase inhibitor 2A gene and co-deletes with CDKN2A in nearly 15% of tumors. Moreover, MTAP-deleted tumor cells exhibit greater sensitivity to methionine depletion and to the inhibitors of purine synthesis. In this review, we first summarized the molecular structure and expression of MTAP in tumors. Furthermore, we discussed PRMT5 and MAT2A as a potential vulnerability for MTAP-deleted tumors. The complex and dynamic role of MTAP in diverse malignancies has also been discussed. Finally, we demonstrated the implications for the treatment of MTAP-deleted tumors.

Utility of BAP1, p16, and Methylthio-Adenosine Phosphorylase Immunohistochemistry in Cytological and Histological Samples of Pleural Mesotheliomas

Introduction: In most cases, the diagnostic workup of pleural mesotheliomas (MPMs) starts with cytological examination of pleural effusion, but histology is needed to confirm the diagnosis. The introduction of BAP1 and methylthio-adenosine phosphorylase (MTAP) immunohistochemistry has become a powerful tool to confirm the malignant nature of mesothelial proliferations also in cytological specimens. The objective of this study was to determine the concordance of BAP1, MTAP, and p16 expression between cytological and histological samples of patients with MPM. Methods: Immunohistochemistry of BAP1, MTAP, and p16 was performed on cytological samples and compared with the corresponding histological specimen of 25 patients with MPM. Inflammatory and stromal cells served as positive internal control for all three markers. In addition, samples of 11 patients with reactive mesothelial proliferations served as an external control group. Results: Loss of BAP1, MTAP, and p16 expression was found in 68%, 72%, and 92% of MPM, respectively. Loss of MTAP was associated with loss of p16 expression in all cases. Concordance of BAP1 between cytological and corresponding histological samples was 100% (kappa coefficient 1; p = 0.008). For MTAP and p16, kappa coefficient was 0.9 (p = 0.01) and 0.8 (p = 0.7788), respectively. Conclusions: Concordant BAP1, MTAP, and p16 expression is found between cytological and corresponding histological samples, indicating that a reliable diagnosis of MPM can be made on cytology only. Of the three markers, BAP1 and MTAP are most reliable in distinguishing malignant from reactive mesothelial proliferations.

The cytologic diagnosis of mesothelioma: are we there yet?

Mesothelioma is a rare but highly aggressive malignancy with poor prognosis that frequently present with recurrent effusions. Establishing the diagnosis by cytology can lead to early diagnosis and treatment and consequently improve prognosis. This review examines the cytological diagnosis of mesothelioma in the context of its historical and morphologic evolution and provides an update of the current reporting systems. Clues to identify the mesothelial and malignant nature of the sample are detailed as well as the supporting ancillary tests. Cytologically, the samples are overwhelmingly cellular and malignancy is recognized by both architectural and cytological atypia. Numerous variably sized clusters and enlarged cells are easily identified, some with papillary architecture and collagen cores. Recognizing the mesothelial nature of the cells and supportive immunostains are essential to rule out the differential diagnosis of metastatic carcinomas and reactive mesothelium. Current ancillary tests such as homozygous deletion of CDKN2A, loss of BRCA1-associated protein, and methylthioadenosine phosphorylase expression can provide further support of malignancy. At this time with the aid of current ancillary tests and in the hands of cytopathologists with adequate experience with the interpretation of effusions, the diagnosis of mesothelioma can be established with accuracy in most cases.

Reduced symmetric dimethylation stabilizes vimentin and promotes metastasis in MTAP‐deficient lung cancer

The aggressive nature and poor prognosis of lung cancer led us to explore the mechanisms driving disease progression. Utilizing our invasive cell‐based model, we identified methylthioadenosine phosphorylase (MTAP) and confirmed its suppressive effects on tumorigenesis and metastasis. Patients with low MTAP expression display worse overall and progression‐free survival. Mechanistically, accumulation of methylthioadenosine substrate in MTAP‐deficient cells reduce the level of protein arginine methyltransferase 5 (PRMT5)‐mediated symmetric dimethylarginine (sDMA) modification on proteins. We identify vimentin as a dimethyl‐protein whose dimethylation levels drop in response to MTAP deficiency. The sDMA modification on vimentin reduces its protein abundance but trivially affects its filamentous structure. In MTAP‐deficient cells, lower sDMA modification prevents ubiquitination‐mediated vimentin degradation, thereby stabilizing vimentin and contributing to cell invasion. MTAP and PRMT5 negatively correlate with vimentin in lung cancer samples. Taken together, we propose a mechanism for metastasis involving vimentin post‐translational regulation.

A Combination of MTAP and p16 Immunohistochemistry Can Substitute for CDKN2A Fluorescence In Situ Hybridization in Diagnosis and Prognosis of Pleural Mesotheliomas.

Homozygous deletion (HD) of CDKN2A is one of the most frequent genetic abnormalities in pleural mesotheliomas. HD of CDKN2A by fluorescence in situ hybridization (FISH) is a reliable marker of malignancy in mesothelial proliferations; however, evaluation of CDKN2A deletion requires FISH. The 9p21 locus includes both CDKN2A and MTAP (methylthioadenosine phosphorylase); the latter is frequently codeleted with CDKN2A. To examine the question of whether immunohistochemistry for MTAP and p16, the protein product of CDKN2A, can serve as a surrogate for CDKN2A HD by FISH. A random selection of 125 pleural mesothelioma cases was divided into 3 groups for evaluation of p16 and MTAP expression compared with FISH for CDKN2A deletion: 53 with HD, 39 with heterozygous deletion, and 33 without deletion. By itself, loss of p16 nuclear expression (<1% staining) showed a high sensitivity (96%) but low specificity (43%) for CDKN2A HD by FISH. MTAP cytoplasmic expression loss (≤30% staining) showed a 97% specificity and 69% sensitivity. The combination of p16 nuclear (<1% staining) and MTAP cytoplasmic (≤30% staining) loss demonstrated both high specificity (96%) and high sensitivity (86%). Patients with retained p16 expression (≥1%) had the best prognosis, whereas a p16 (<1%)/MTAP loss combination was associated with a dismal prognosis. MTAP immunohistochemical staining is a valid surrogate marker for CDKN2A HD by FISH; however, to obtain the same accuracy as the FISH assay, a combination of nuclear p16 and cytoplasmic MTAP staining is recommended. These findings correlate with prognosis.

Clinical significance of 9P21 gene combined with BAP1 and MTAP protein expression in diagnosis and prognosis of mesothelioma serous effusion.

The diagnostic value of the 9P21 gene determined using fluorescence in situ hybridization (FISH) combined with BRCA1-associated protein 1 (BAP1) and methylthioadenosine phosphorylase (MTAP) expression detection by immunohistochemistry, was investigated in serous effusion samples of malignant mesothelioma. A total of 70 serous disease samples with serous effusion were collected from June 2017 to June 2020. Following biopsy specimen pathological diagnosis, samples were divided into malignant mesothelioma and benign mesothelioma. Differential expression of BAP1 and MTAP genes were identified in mesothelioma and mesenchymal hyperplasia. The 9P21 gene fragment was lost in mesothelioma. The positive rates of FISH, BAP1 and MTAP in biopsy specimens were 98.00, 94.00 and 90.00%. The specificity of the three were 96.00, 85.71 and 77.27%, the sensitivity were 90.00, 95.92 and 93.75%, and the positive rate of the combined detection of the three was 93.33%. The positive rate of serous fluid samples detected by the three methods (9P21 FISH probe combined with BAP1 and MTAP expression detected immunohistochemically) was 96.00, 92.00 and 88.00%, the specificity were 90.00, 77.27 and 71.43%, the sensitivity was 96.00, 93.75 and 89.80%, and the positive rate of the three combined detections was 91.33%. It was demonstrated that there was a high consistency between serous fluid samples and biopsy samples. According to clinicopathological analysis, sex, age, lesion site, Ki67 had little association with the occurrence and development of malignant mesothelioma, while asbestos exposure history was closely associated to the occurrence of mesothelioma. A high level of BAP1 gene was positively associated with the prognosis of mesothelioma, while a high level of MTAP gene was negatively associated with the prognosis of mesothelioma (P<0.05). Therefore, 9P21 FISH probe combined with BAP1 and MTAP can be used as a new method for the detection of malignant mesothelioma, and provide an important basis for the early diagnosis of mesothelioma.

MTAP loss correlates with an immunosuppressive profile in GBM and its substrate MTA stimulates alternative macrophage polarization

Glioblastoma (GBM) is a lethal brain cancer known for its potent immunosuppressive effects. Loss of Methylthioadenosine Phosphorylase (MTAP) expression, via gene deletion or epigenetic silencing, is one of the most common alterations in GBM. Here we show that MTAP loss in GBM cells is correlated with differential expression of immune regulatory genes. In silico analysis of gene expression profiles in GBM samples revealed that low MTAP expression is correlated with an increased proportion of M2 macrophages. Using in vitro macrophage models, we found that methylthioadenosine (MTA), the metabolite that accumulates as a result of MTAP loss in GBM cells, promotes the immunosuppressive alternative activation (M2) of macrophages. We show that this effect of MTA on macrophages is independent of IL4/IL3 signaling, is mediated by the adenosine A2B receptor, and can be pharmacologically reversed. This study suggests that MTAP loss in GBM cells may contribute to the immunosuppressive tumor microenvironment, and that MTAP status should be considered for characterizing GBM immune states and devising immunotherapy-based approaches for treating MTAP-null GBM.

Downregulation of MTAP promotes Tumor Growth and Metastasis by regulating ODC Activity in Breast Cancer.

5'-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway and has been reported to suppress tumorigenesis. The MTAP gene is located at 9p21, a chromosome region often deleted in breast cancer (BC). However, the clinical and biological significance of MTAP in BC is still unclear. Here, we reported that MTAP was frequently downregulated in 41% (35/85) of primary BCs and 89% (8/9) of BC cell lines. Low expression of MTAP was significantly correlated with a poor survival of BC patients (P=0.0334). Functional studies showed that MTAP was able to suppress both in vitro and in vivo tumorigenic ability of BC cells, including migration, invasion, angiogenesis, tumor growth and metastasis in nude mice with orthotopic xenograft tumor of BC. Mechanistically, we found that downregulation of MTAP could increase the polyamine levels by activating ornithine decarboxylase (ODC). By treating the MTAP-repressing BC cells with specific ODC inhibitor Difluoromethylornithine (DFMO) or treating the MTAP-overexpressing BC cells with additional putrescine, metastasis-promoting or -suppressing phenotype of these MTAP-manipulated cells was significantly reversed, respectively. Taken together, our data suggested that MTAP has a critical metastasis-suppressive role by tightly regulating ODC activity in BC cells, which may serve as a prominent novel therapeutic target for advanced breast cancer treatment.

Expression and localisation of methylthioadenosine phosphorylase (MTAP) in oral squamous cell carcinoma and their significance in epithelial-to-mesenchymal transition

Methylthioadenosine phosphorylase (MTAP) is a rate-limiting enzyme in the methionine salvage pathway, which recycles one carbon unit that is lost during polyamine synthesis back into the methionine cycle. Although MTAP deficiency has been reported in various tumours, MTAP is overexpressed and might promote oncogenesis in other cancers, including prostate and colon cancer. Currently, little is known about the MTAP status of oral squamous cell carcinoma (OSCC). In this study, we immunohistochemically examined the expression of MTAP in surgically resected oral epithelial dysplasia (OED, n=7), carcinoma in situ (CIS) (n=16), and OSCC (n=118). In the normal epithelium, MTAP was only weakly expressed in the cytoplasm of the basal layer cells. In OED, CIS, and OSCC, MTAP was uniformly expressed in the cytoplasm of the dysplastic and cancer cells. In addition to cytoplasmic MTAP expression, 45 of 118 cases (38.1%) exhibited increased nuclear expression of MTAP in the cancer cells at the invasive front. Statistical analysis showed that the concomitant nuclear and cytoplasmic expression of MTAP was associated with a high budding score (p=0.0023); poor differentiation (p=0.0044); aggressive invasion patterns (p=0.0001); and features of epithelial-to-mesenchymal transition (EMT), such as loss of E-cadherin expression (p=0.0003) and upregulated expression of vimentin (p=0.0002), slug (p=0.0002), and laminin 5 (p<0.0001). High expression of protein arginine methyltransferase 1 or 5, the functions of which are reported to be inhibited in MTAP-deficient cancer, was associated with the concomitant nuclear and cytoplasmic expression of MTAP (p<0.0001). Concomitant nuclear and cytoplasmic expression of MTAP was marginally significantly associated with worse 5-year relapse-free survival (p=0.045). These findings suggest that MTAP not only plays a role in the oncogenesis of OSCC, but that it might also make it more aggressive by inducing EMT through its activity in the methionine salvage pathway.

Loss of Methylthioadenosine Phosphorylase by Immunohistochemistry Is Common in Pulmonary Sarcomatoid Carcinoma and Sarcomatoid Mesothelioma

Differentiating malignant pleural mesothelioma from benign reactive mesothelial processes can be quite challenging. Ancillary tests such as BRCA1-associated protein 1 (BAP1) immunohistochemistry and p16 fluorescence in situ hybridization (FISH) are helpful tools to aid in this distinction. Immunohistochemistry for methylthioadenosine phosphorylase (MTAP) has recently been proposed as an effective surrogate marker for p16 FISH and is an attractive alternative test due to shorter turnaround time. There are little data regarding the specificity of MTAP loss for mesothelioma or whether it may be useful to distinguish mesothelioma from the most common entity in the differential diagnosis, sarcomatoid carcinoma. We studied well-characterized cases of sarcomatoid carcinoma (n = 34) and sarcomatoid mesothelioma (n = 62), which were stained for MTAP (clone 2G4) and BAP1 (clone C-4). Loss of MTAP expression was observed in 17 (50%) of 34 pulmonary sarcomatoid carcinomas; BAP1 expression was retained in all of the cases in which it was performed (n = 31). MTAP expression was lost in 38 (61%) of 62 sarcomatoid mesotheliomas; BAP1 was lost in 6 (10%) of 62. In the six cases with BAP1 loss, five also had loss of MTAP, while MTAP expression was retained in one. Loss of MTAP expression by immunohistochemistry is common in pulmonary sarcomatoid carcinoma, as it is present in half of cases. This rate is similar to what is observed in sarcomatoid mesothelioma (61%). Therefore, this stain is not useful to distinguish between these two malignancies. MTAP loss is more common than BAP1 loss in the setting of sarcomatoid mesothelioma (61% vs 10%, respectively).

Design, synthesis, and evaluation of potential carbamate prodrugs of 5′-methylthioadenosine (MTA)

5′-Methylthioadenosine (MTA) is a natural substrate of MTA phosphorylase (MTAP) and is converted to adenine via a salvage pathway for AMP production in normal healthy cells. The lack of MTAP expression in many solid tumors and hematologic malignancies compared to normal healthy cells has been explored in a potential therapeutic strategy to selectively target tumor cells using antimetabolites such as 5-fluorouracil (5-FU) and 6-thioguanine (6-TG) while protecting normal healthy cells with MTA. Herein, a series of carbamate prodrugs, namely the N-(alkyloxy)carbonyl-MTA derivatives 2a-f, was designed, synthesized, and evaluated as potential prodrugs of MTA. All carbamate prodrugs were stable in phosphate buffer, pH 7.4 at 37 °C. In the presence of mouse liver microsomes, the prodrugs were converted to MTA at varying rates with the hexyl and butyl carbamates 2a and 2b most readily activated (t1/2 of 1.2 and 9.4 h, respectively). The activation was shown to be mediated by carboxyesterases present in mouse liver microsomes.

Correlation of methylthioadenosine phosphorylase (MTAP) protein expression with MTAP and CDKN2A copy number in malignant pleural mesothelioma.

Methylthioadenosine phosphorylase (MTAP) immunohistochemical expression is a specific marker of CDKN2A deletion in malignant mesothelioma. However, the relationship of MTAP expression with MTAP copy number remains unexplored. Forty malignant pleural mesotheliomas were characterised by targeted next-generation sequencing (29), single-nucleotide polymorphism microarray (seven), or both (four). MTAP and CDKN2A copy numbers were correlated with MTAP expression. Twenty-seven (68%) tumours showed CDKN2A deletion (14 heterozygous; 13 homozygous), of which 20 (74%) showed MTAP codeletion (15 heterozygous; five homozygous). No tumours showed MTAP deletion without CDKN2A codeletion. Loss of MTAP expression was seen in 16 (40%) tumours, and was 75% sensitive and 95% specific for MTAP deletion, and 59% sensitive and 100% specific for CDKN2A deletion. Nine of 40 (23%) tumours showed heterogeneous MTAP staining, and the percentage of tumour cells with MTAP loss correlated with molecular detection of MTAP deletion. MTAP is frequently codeleted with CDKN2A in pleural mesothelioma. However, homozygous deletion of both genes occurs in a minority of tumours (5/40; 13%); CDKN2A deletion often co-occurs with heterozygous MTAP deletion or neutral MTAP copy number; and MTAP expression correlates inconsistently with heterozygous MTAP deletion. Correspondingly, MTAP immunohistochemistry is a highly specific but only moderately sensitive assay for CDKN2A deletion.

TRAF7 mutations and immunohistochemical study of uterine adenomatoid tumor compared with malignant mesothelioma

Adenomatoid tumors (ATs) are benign mesothelial tumors with a good prognosis and usually occur in female and male genital tracts, including in the uterus. ATs are genetically defined by tumor necrosis factor receptor-associated factor (TRAF) 7 mutations, and a high number of AT cases show immunosuppression. On the other hand, malignant mesotheliomas (MMs) are malignant mesothelial tumors with a very poor prognosis. Genetic alterations in TRAF, methylthioadenosine phosphorylase(MTAP), and BRCA-associated nuclear protein 1 (BAP1) in ATs derived from the uterus and MMs of pleural or peritoneal origin were compared by gene sequence analysis or immunohistochemical approaches. Formalin-fixed paraffin-embedded tissues derived from patients were used for immunohistochemical staining of L1 cell adhesion molecule (L1CAM), BAP1, MTAP, and sialylated protein HEG homolog 1 (HEG1) in 51 uterine AT cases and 34 pleural or peritoneal MM casesand for next-generation sequencing of the TRAF7 gene in 44 AT cases and 21MM cases. ATs had a significantly higher rate of L1CAM expression than MMs, whereas MMs had a significantly higher rate of loss of MTAP and BAP1 expression than ATs. There was no difference in the rate of HEG1 expression between the tumor types. Most of the ATs (37/44; 84%) had somatic mutations in TRAF7, but none of the MMs had somatic mutations in TRAF7 (0/21; 0%). In addition, a low number of AT cases were associated with a history of immunosuppression (9/51; 17.6%). TRAF7 mutation is one of the major factors distinguishing the development of AT from MM, and immunosuppression might not be associated with most AT cases.

Loss of 5'-Methylthioadenosine Phosphorylase (MTAP) is Frequent in High-Grade Gliomas; Nevertheless, it is Not Associated with Higher Tumor Aggressiveness.

The 5’-methylthioadenosine phosphorylase (MTAP) gene is located in the chromosomal region 9p21. MTAP deletion is a frequent event in a wide variety of human cancers; however, its biological role in tumorigenesis remains unclear. The purpose of this study was to characterize the MTAP expression profile in a series of gliomas and to associate it with patients’ clinicopathological features. Moreover, we sought to evaluate, through glioma gene-edited cell lines, the biological impact of MTAP in gliomas. MTAP expression was evaluated in 507 glioma patients by immunohistochemistry (IHC), and the expression levels were associated with patients’ clinicopathological features. Furthermore, an in silico study was undertaken using genomic databases totalizing 350 samples. In glioma cell lines, MTAP was edited, and following MTAP overexpression and knockout (KO), a transcriptome analysis was performed by NanoString Pan-Cancer Pathways panel. Moreover, MTAP’s role in glioma cell proliferation, migration, and invasion was evaluated. Homozygous deletion of 9p21 locus was associated with a reduction of MTAP mRNA expression in the TCGA (The Cancer Genome Atlas) - glioblastoma dataset (p < 0.01). In addition, the loss of MTAP expression was markedly high in high-grade gliomas (46.6% of cases) determined by IHC and Western blotting (40% of evaluated cell lines). Reduced MTAP expression was associated with a better prognostic in the adult glioblastoma dataset (p < 0.001). Nine genes associated with five pathways were differentially expressed in MTAP-knockout (KO) cells, with six upregulated and three downregulated in MTAP. Analysis of cell proliferation, migration, and invasion did not show any significant differences between MTAP gene-edited and control cells. Our results integrating data from patients as well as in silico and in vitro models provide evidence towards the lack of strong biological importance of MTAP in gliomas. Despite the frequent loss of MTAP, it seems not to have a clinical impact in survival and does not act as a canonic tumor suppressor gene in gliomas.