P16 Promoter Hypermethylation Research Articles

Abstract 6007: p16 epimutation modulates the tumor microenvironment and promotes malignancy of Apc-mutant colon cancer

Abstract Methylation of the p16 promoter resulting in mitotically stable gene silencing—known as p16 epimutation—is the most common epigenetic event in human colorectal cancer (CRC) and is also common in normal-appearing colonic mucosa of aging individuals. However, the functional role of p16 epimutation in CRC carcinogenesis is not completely understood. Here, using a mouse model of engineered p16 promoter hypermethylation, we show that p16 epimutation cooperates with mutant Apc to promote colon cancer development. Mice carrying combined Apc mutation and p16 epimutation display significantly shortened survival and increased tumor growth compared to those with Apc mutation only. Moreover, colon tumors from these animals express increased levels of programmed death-ligand 1 (Pdl1). Single-cell RNA sequencing (scRNA-seq) analysis further revealed the presence of dysfunctional cytotoxic CD8+ T cells together with Foxp3+ Tregs and γδT17 cells, which promote an immunosuppressive tumor microenvironment (TME). Lastly, we show that a combined therapy using an inhibitor of DNA methylation and a PD-L1 immune checkpoint inhibitor is more effective for improving survival in tumor-bearing mice than anti-PD-L1 treatment alone. These findings provide a mechanistic rationale for targeted epigenetic and immunotherapy in CRC patients, with the potential to improve colon cancer treatment. Citation Format: Li Yang, Xiaomin Chen, Christy Lee, Jiejun Shi, Emily B. Lawrence, Lanjing Zhang, Yumei Li, Nan Gao, Sung Yun Jung, Chad J. Creighton, Jingyi Jessica Li, Ya Cui, Sumimasa Arimura, Wei Li, Lanlan Shen. p16 epimutation modulates the tumor microenvironment and promotes malignancy of Apc-mutant colon cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6007.

A pilot study on DNA hypermethylation status in promoter region of P16 gene in patients with sporadic breast cancer

BackgroundEpigenetic modification of cancer-related genes plays a role over and above their genetic alterations and contributes to the tumor initiation and progression of breast cancer. Promoter methylation of tumor suppressor genes is one such epigenetic modification, which can be potential biomarker. In this study, promoter methylation status of p16 gene was studied in blood samples of patients with breast carcinoma. MethodsSeventy-five patients, freshly diagnosed with carcinoma of breast and 20 age and sex matched healthy control subjects were recruited for the study. DNA extracted from EDTA blood sample was bisulfite converted and subjected to methylation-specific PCR to amplify the p16 promoter region. ResultsOut of 75 patients, 25 (33%) patients showed hypermethylation in promoter region of p16 gene, which was statistically significant in comparison with the control group (p < 0.05). In subgroup analysis, lymph node involvement, cancer grade, and histopathological finding did not show any difference with methylation status of p16 promoter. ConclusionSignificant hypermethylation of p16 promoter region in the blood of histopathologically proven cases of breast cancer was observed suggesting promoter hypermethylation of p16 may be a possible mechanism accounting for sporadic carcinoma of breast.

Assessment of Aberrant Promoter Hypermethylation of RASSF1a and P16 in Endometrial Carcinoma

Endometrial carcinoma is one of the most common gynecological malignancies known to affect around 142000 women worldwide. Aberrant promoter hypermethylation of tumor suppressor genes is a common epigenetic alteration reported in cancers. In the present study we report the aberrant promoter hypermethylation of RASSF1a and p16 in 78 endometrial cancer patients. Methods: Endometrial carcinoma samples were collected after pathological examination and DNA was extracted. The DNA was subjected to sodium bisulfite modification and then used to perform methylation specific PCR. The PCR was performed using a two step procedure of nested and methylation specific PCR. The PCR product was visualized using agarose gel electrophoresis. The results obtained were correlated with various clinicopathological parameters. Chi square test was used for statistical analysis and a p-value of &lt;0.05 was considered statistically significant. Result: 60 of the 78 (76.92%) samples showed methylation for RASSF1a gene. 28 of the 78 (35.8%) samples showed methylation for p16 gene. A higher methylation frequency was observed for RASSF1a in the endometrioid and poorly differentiated histological subtypes and stage I and II of the disease. Conclusion: Aberrant promoter hypermethylation of RASSF1a and p16 is known to play an important role in endometrial cancer initiation and progression. The methylation patterns can be used as an important molecular marker for early diagnosis and prognosis of endometrial cancer. Keywords: Epigenetic alterations, DNA Hypermethylation, Endometrial cancer, RASSF1a, p16, Methylation-specific PCR.

Aberrant Promoter Hypermethylation of p16 and RASSF1a Genes in Colorectal Cancer - Significance in Young Patients.

The clinical profile of colorectal cancers (CRC) in India is different from that described in western countries. Microsatellite instability and APC mutation explain the molecular biology of up to 50% of colorectal cancers. Global genome hypermethylation may be the cause in at least 20% of cases. Few studies from India have examined the epigenetic profile of colorectal cancers. This study was designed to study aberrant promoter hypermethylation of two select tumour suppressor genes (p16, RASSF1a) in patients with colorectal cancer and their association with clinicopathologic features. A total of 41 samples including controls were collected from colorectal cancer patients. DNA was isolated from tumour tissue, and methylation-specific PCR was performed for the 2 genes. p16 and RASSF1a promoter hypermethylation was found in 26% and 48% of CRC cases, respectively. RASSF1a promoter hypermethylation was more often seen in young CRC patients aged 40years or less, and this was found to be statistically significant (p value = 0.037). RASSF1a hypermethylation is peculiar to rectal cancers and left-sided colonic tumours in young patients. Large-scale population-based studies with extensive genetic and epigenetic characterization are required for a better understanding and further validation of our findings. For individuals diagnosed with sporadic CRC, these studies on specimen might help predict prognosis and response to therapy.

MicroRNA-29a inhibits cell proliferation and arrests cell cycle by modulating p16 methylation in cervical cancer.

Cervical cancer is the second most common gynecological malignancy. Accumulating evidence has suggested that microRNAs (miRNAs) are involved in the occurrence and development of cervical cancer. The present study aimed to investigate the function and underlying molecular mechanism of microRNA (miRNA/miR)-29a in cervical cancer. Reverse transcription-quantitative PCR and methylation-specific PCR were used to examine the expression of miR-29a and methylated status of p16 promoter, respectively. Cell Counting Kit-8 analysis and flow cytometry were performed to evaluate cell viability and cycle, respectively. Dual-luciferase reporter assay was performed to verify the interaction between miR-29a and its targets. Western blot analysis was performed to detect the protein levels of DNA methyltransferases (DNMT)3A and DNMT3B. The results demonstrated that miR-29a expression was downregulated in cervical cancer tissues and cells, and negatively correlated with p16 promoter hypermethylation. Furthermore, cell experiments confirmed that miR-29a suppressed cell proliferation and induced cell cycle arrest in HeLa and C-33A cells. Mechanically, miR-29a restored normal methylation pattern of the p16 gene by sponging DNMT3A and DNMT3B. Taken together, the results of the present study demonstrated the epigenetic regulation of tumor suppressor p16 by miR-29a as a unique mechanism, thus providing a rationale for the development of miRNA-based strategies in the treatment of cervical cancer.

The prognostic impact of hypermethylation for a panel of tumor suppressor genes and cell of origin subtype on diffuse large B-cell lymphoma.

Diffuse Large B-cell lymphoma (DLBCL) is an aggressive disease with heterogeneous outcome and marked variable response to chemotherapy. We assessed promoter hypermethylation (PM) for a panel of tumor suppressor genes in 75 DLBCLs compared to 20 lymphoid hyperplasia (LH) and 30 normal control, using methylation specific PCR. Results were correlated to patients' clinic-pathological characteristics, immunophenotyping, and patients' outcome. DAPK1, RUNX3, MT1G, MGMT, CDH1 and p16 PM were detected in 38.7% (29/75), 49.3% (37/75), 46.7% (35/75), 44% (33/75), 49.3% (37/75) and 42.7% (32/75);respectively, of DLBCL patients compared to LH group (P < 0.05). Aberrant PM of RUNX3, MGMT, CDH1 and p16 was significantly higher in non-germinal central B-cell like (non-GCB) compared to GCB (58.3% vs. 33.3%, 56.2% vs. 22.2, 62.5% vs. 25.9, and 56.2% vs. 18.5%, respectively). PM of studies genes in DLBCL associated significantly with worse survival outcome and resistance to chemotherapy (P ≤ 0.01). In non-GCB group, DAPK1, MT1G, RUNX3, CDH1 and p16 PM associated significantly with reduced DFS (P ≤ 0.004) and OS (P ≤ 0.015). Multivariate analysis indicated that RUNX3 and CDH1 PM were independent prognostic factors for OS (P = 0.03 and 0.04; respectively), while DAPK1, RUNX3 and MT1G PM were independent prognostic factors for DFS (P = 0.002, 0.037& 0.007; respectively). DAPK1, RUNX3, MT1G, CDH1 and p16 PM are promising prognostic and/or predictive markers for non-GCB independent of IPI. Upregulation of those genes using new demethylating agents is a promising approach that sensitize chemoresistant DLBCL patients, especially the non-GCB subtype.

Organochlorine and organophosphorous pesticides may induce colorectal cancer; A case-control study

ObjectivesAmong the numerous agents, genetic factors and environmental elements such as pesticides have an important role in colorectal cancer (CRC) incidence. The present study aimed to investigate the probable-role of some organochlorine pesticides (OCPs) and organophosphorous pesticides (OPPs) in patients with CRC. MethodsIn this case-control study, 42 patients with CRC and 30 healthy subjects were selected. The serum levels of some OCPs (α-HCH, β-HCH, γ-HCH, 2,4 DDE, 4,4 DDE, 2,4DDT and 4,4DDT) were measured by gas chromatography (GC) method. Serum levels of malondialdehyde (MDA), and total antioxidant capacity (TAC) as well as the enzyme activity of acetylcholinesterase (AChE) and arylesterase activity of Paraoxonase-1 (PON-1) were evaluated in all participants. The methylation specific PCR (MSP) assay was used for determining the methylation status of CpG island of p16 and MGMT genes in CRC patients. ResultsThe mean serum levels of each OCPs were significantly higher in the patient group compared to the control group (P < 0.001). The AChE and arylesterase activity of PON-1 in the patient group were significantly lower than the control group (P < 0.001). The mean serum levels of MDA and TAC in the serum of the patient group were significantly higher than the control group (P < 0.001 and P < 0.002, respectively). The current findings demonstrated significantly hypermethylation of p16 promoter in CRC patients. ConclusionRegarding the higher levels of OCPs in CRC patients, along with hypermethylation of the p16 promoter gene, diminishing in AChE and PON-1 activity and increasing in oxidative stress factors, the role of OCPs and OPPs in the CRC progression in the South-East of Iran may be assumed.

Impact of catechol-O-methyltransferase gene variants on methylation status of P16 and MGMT genes and their downregulation in colorectal cancer.

Globally, colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second most commonly diagnosed cancer in females, with 1.4 million new cases and almost 694 000 deaths estimated to have occurred in 2012. The development and progression of CRC is dictated by a series of alterations in diverse genes mostly proto-oncogenes and tumor suppressor genes. In this dreadful disease disturbances different from mutations called as epigenetic regulations are also taken into consideration and are thoroughly investigated. The present study was designed to analyze the promoter hypermethylation of CpG (cytosine, followed by guanine nucleotide) islands of cyclin-dependent kinase inhibitor 2A (P16) and O-methylguanine-DNA methyltransferase (MGMT) genes and its subsequent effect on the protein expression in CRC. The impact of the common functional polymorphism of the catechol-O-methyltransferase (COMT) gene, Val158Met, on promoter hypermethylation of P16 and MGMT genes in CRC was also investigated. The study included 200 CRC cases and equal numbers of normal samples. DNA was extracted using the kit method and methylation specific-PCR was performed for analysis of the promoter hypermethylation status. Total protein was isolated form all CRC cases and western blotting was performed for P16 and MGMT proteins. The COMT Val158Met polymorphism was analyzed by a PCR-restriction fragment length polymorphism assay. Epigenetic analysis showed that unlike other high-risk regions, the Kashmiri population has a different promoter hypermethylation profile of both P16 and MGMT genes, with frequent and significant promoter hypermethylation of both in CRC. The frequency of promoter hypermethylation of both genes was significantly higher in males and was insignificantly found to be higher in stage III/IV. The degree of P16 and MGMT promoter hypermethylation increased significantly with increasing severity of the lesion. We also found a significant correlation between P16 and MGMT promoter hypermethylation and loss of protein expression in CRC. A significant association was found between COMT polymorphism (homozygous variant) and P16 methylation status. Similar results were also found for MGMT hypermethylated cases.

Promoter hypermethylation of RARβ2, DAPK, hMLH1, p14, and p15 is associated with progression of breast cancer: A PRISMA-compliant meta-analysis.

Background:Numerous studies have investigated the associations between RARβ2, DAPK, hMLH1, p14, and p15 promoter hypermethylation and clinical progression of patients with breast cancer, however the results remained uncertain due to the small sample size. Therefore, we performed a meta-analysis to explore the role of RARβ2, DAPK, hMLH1, p14, and p15 promoter hypermethylation in the susceptibility and clinical progression of breast cancer.Methods:Eligible studies were obtained by searching Medicine, Embase, Web of knowledge, and Chinese National Knowledge Infrastructure (CNKI) databases. The odds ratios (OR) and 95% confidence intervals (CI) were calculated to evaluate the associations of RARβ2, DAPK, hMLH1, p14, and p15 promoter hypermethylation with breast cancer pathogenesis. Trial sequential analysis (TSA) was applied to observe the reliability of pooled results of RARβ2 gene, and obtain a conservative required information size (RIS).Results:In primary screened 445 articles, 39 literatures with 4492 breast cancer patients were finally enrolled in the final meta-analysis. The results indicated that the frequency of RARβ2 promoter hypermethylation in case group was significantly higher than the frequency of control group (OR = 7.21, 95% CI = 1.54–33.80, P < .05). The RARβ2 promoter hypermethylation had a significant association with lymph node metastasis of breast cancer (OR = 2.13, 95% CI = 1.04–4.47, P < .05). And, the RARβ2 promoter hypermethylation was more common in the breast cancer patients of TNM III–IV stage than those patients of TNM I–II stage (OR = 1.85, 95% CI = 1.33–2.57, P < .05). In addition, the promoter hypermethylation of DAPK, hMLH1, and p14 genes were significantly associated with the susceptibility of breast cancer (for DAPK, OR = 4.93, 95% CI = 3.17–7.65; for hMLH1, OR = 1.84, 95% CI = 1.26–1.29; for p14, OR = 22.52, 95% CI = 7.00–72.41; for p15, OR = 2.13, 95% CI = 0.30–15.07).Conclusions:Our findings revealed that the RARβ2 promoter hypermethylation significantly increased the risk of breast cancer. In the meantime, the meta-analysis demonstrated that there were significant associations of RARβ2 promoter hypermethylation with lymph node metastasis and TNM-stage of breast cancer patients. In addition, DAPK, hMLH1, and p14 genes promoter hypermethylation were significantly associated with the susceptibility of breast cancer.

The hypermethylation of p16 gene exon 1 and exon 2: potential biomarkers for colorectal cancer and are associated with cancer pathological staging

BackgroundTumor suppressor gene p16 promoter hypermethylation has been widely studied in colorectal cancer (CRC), yet its clinicopathological significance remains controversial. The methylation alterations of other regions within p16 gene are still rarely researched. The present study aimed to explore the methylation changes of p16 gene body in CRC and to find whether they were associated with clinicopathological staging of CRC.MethodsPaired colorectal cancer tissues and corresponding adjacent normal tissues from 30 CRC patients were collected. The methylation levels of two CpG islands within p16 gene body, exon 1 and exon 2, were accurately assessed simultaneously by a LC-MS/MS method. The p16 protein expressions were assessed by immunohistochemistry assay. Statistical analyses were carried out using SPSS 17.0 software. Heat-map analysis was carried out by HemI 1.0 software.ResultsIn the present study, CRC tissues showed more highly methylated than adjacent normal tissues at both CpG islands of p16 gene. And exon 2 hypermethylation was higher and more frequent than exon 1. The ROC curve analysis showed that the simultaneous use of both indicators had excellent sensitivity and specificity for distinguishing CRC tissues and adjacent normal tissues. Following, the methylation level of p16 exon 1/2 was negatively related to p16 protein expression. Further correlation analysis revealed that p16 exon 1 hypermethylation was associated with N/Dukes staging (p = 0.033), and p16 exon 2 hypermethylaiton was associated with T staging (p = 0.035).ConclusionsThe p16 gene body was remarkably hyper-methylated in CRC tissues and associated with p16 protein expression and cancer clinicopathological staging. The combination of p16 exon 1 and exon 2 could better reflect the overall methylation status of p16 gene body and provide potential biomarkers of CRC.

ASSESSMENT OF A PROGNOSTIC VALUE OF CDKN2A AND TIMP3 GENE METHYLATION IN BLADDER CANCER

Promoter hypermethylation of tumor suppressor genes is one of the mechanisms of epigenetic regulation disturbance of gene expression and is often observed in different cancer types. The profile of mutational and epigenetic changes characterizes a malignant potential of a tumor, as well as its ability to invade and metastasize.The aim of the study was to determine a prognostic value of p16, p14ARF and TIMP3 gene methylation in the group of 158 bladder cancer patients. Epigenetic changes in these genes were observed with a frequency of 11.4, 0 and 10.8 %, respectively, and did not depend on clinic-morphological characteristics.A statistically significant association of p16 and TIMP3 abnormal methylation with smoking was found, indicating a possible influence of tobacco smoke carcinogens on the occurrence of these epigenetic changes. In the multivariate Cox regression analysis, p16 promoter hypermethylation was an independent predictor for bladder cancer progression (HR 6.84; 95 % CI 1.6–29.9; р = 0.011).The use of the data on the p16 methylation status may improve the accuracy of prognosis of the bladder cancer clinical course and the selection of appropriate treatment strategy.

INFLUENCE OF P16 GENE METHYLATION ON THE RISK OF PROGRESSION OF NON-MUSCLE INVASIVE BLADDER CANCER

To accurately predict the tumor behavior and individualize the treatment approach, new methods for bladder cancer (BC) prognosis are required. The most promising prognostic markers are the mutational and epigenetic changes of genes involved in maintaining cellular homeostasis. In the present study, we evaluated the influence of p16 promoter hypermethylation on the risk of recurrence, progression and disease outcome in the group of 158 BC patients. p16 epigenetic changes were found in 11.4 % of urothelial carcinomas and did not depend on clinicоmorphological characteristics. However, in the subgroup of patients with non-muscle invasive tumors, p16 abnormal methylation was significantly associated with smoking, and in the subgroup of patients with muscle-invasive BC, it was linked to a high tumor grade (G3). In the multivariate Cox regression analysis, p16 promoter hypermethylation was an independent predictor for bladder cancer progression (HR 6.84; 95 % CI 1.6–29.9; р = 0.011). The use of the data on the p16 methylation status may improve the accuracy of prognosis of the bladder cancer clinical course and the selection of appropriate treatment strategy.

Liquid-based cytology: do ancillary techniques enhance detection of epithelial abnormalities?

Cervical cancer is the fourth most common cancer in women worldwide with very high incidence in India. Liquid-based cytology (LBC) provides the use of ancillary techniques in addition to a good morphology and detection of cytologic abnormalities. The current study was designed to assess the diagnostics of P16INK4a immunoexpression, p16 promoter hypermethylation, human papilloma virus (HPV), and DNA ploidy in LBC samples with cervical precancer and cancer. A series of LBC samples categorised by Bethesda system including 22 atypical squamous cells of undetermined significance (ASC-US), 21 low-grade squamous intraepithelial lesion (LSIL), 41 high-grade squamous intraepithelial lesion (HSIL), 54 squamous cell carcinoma (SCC), and 26 controls with normal cytology were included. Ancillary techniques evaluated included P16INK4a immunoexpression, p16 promoter methylation DNA ploidy by flow cytometry, and HPV was detected using PGMY09/PGMY11 primers. The test positivity rate of p16 expression in women with ASC-US, LSIL, HSIL, and SCC was 21.1, 39.0, 67.7, and 85.4%. For the p16 methylation the corresponding test positivity rate was 36.4, 76.2, 92.7, and 92.6%. The test positive rate of HPV in women with ASC-US, LSIL, HSIL, and SCC was 45.5, 76.2, 87.8, and 92.6%. Diploid G1 and diploid S values significantly (p < 0.05 or p < 0.01) discriminate LSIL versus HSIL and LSIL versus. SCC. P16 gene promoter methylation and HPV seem more sensitive in detection of ASC-US and LSIL cytology with higher specificity. Diploid G1 and diploid S phase study provides progressive change in parameters with progression from LSIL to HSIL and SCC.

Detection of p16 Promoter Hypermethylation by Methylation-Specific PCR.

DNA methylation plays a decisive role in the regulation and control of gene expression. DNA methylation is a covalent modification, in which a methyl group is attached to the 5th carbon of the cytosine ring of a CpG dinucleotide that is located upstream from the promoter region of a gene. Promoter hypermethylation (gain of DNA methylation) of the p16 gene may cause silencing of gene expression and plays an important role in cancer. Therefore, detection of the methylation status of p16 gene is an important tool in epigenetic studies of various human cancers. The methylation-specific PCR (MSP) is the most commonly used technique for studying DNA methylation. This technique is based on bisulfite modification of DNA, which converts unmethylated cytosine (C) into uracil (U) and leaving methylated cytosine (Cm) unchanged. Here we describe the bisulfite modification of DNA samples and detection of promoter methylation of p16 gene from bisulfite-treated DNA using MSP. In MSP, modified DNA samples are subjected to PCR amplification using methylated and unmethylated specific primers for the p16 gene separately. The PCR amplified products are then analyzed in a 2.5-3% agarose gel containing ethidium bromide. The PCR amplified band generated by specific sets of primers is used to determine the methylation status of the p16 gene.

Congress Organization Committee

Among the numerous agents, genetic factors and environmental elements such as pesticides have an important role in colorectal cancer (CRC) incidence. The present study aimed to investigate the probable-role of some organochlorine pesticides (OCPs) and organophosphorous pesticides (OPPs) in patients with CRC.In this case-control study, 42 patients with CRC and 30 healthy subjects were selected. The serum levels of some OCPs (α-HCH, β-HCH, γ-HCH, 2,4 DDE, 4,4 DDE, 2,4DDT and 4,4DDT) were measured by gas chromatography (GC) method. Serum levels of malondialdehyde (MDA), and total antioxidant capacity (TAC) as well as the enzyme activity of acetylcholinesterase (AChE) and arylesterase activity of Paraoxonase-1 (PON-1) were evaluated in all participants. The methylation specific PCR (MSP) assay was used for determining the methylation status of CpG island of p16 and MGMT genes in CRC patients.The mean serum levels of each OCPs were significantly higher in the patient group compared to the control group (P < 0.001). The AChE and arylesterase activity of PON-1 in the patient group were significantly lower than the control group (P < 0.001). The mean serum levels of MDA and TAC in the serum of the patient group were significantly higher than the control group (P < 0.001 and P < 0.002, respectively). The current findings demonstrated significantly hypermethylation of p16 promoter in CRC patients.Regarding the higher levels of OCPs in CRC patients, along with hypermethylation of the p16 promoter gene, diminishing in AChE and PON-1 activity and increasing in oxidative stress factors, the role of OCPs and OPPs in the CRC progression in the South-East of Iran may be assumed.

Abstract 2395: Differentiation of carcinoma ex pleomorphic adenoma from pleomorphic adenoma by TERT promoter hypermethylation and elevated TERT mRNA expression

Abstract The purpose of this study was to determine whether i) genetic or ii) epigenetic aberrations of the TERT promoter, iii) elevated TERT mRNA level or iv) abnormal p53 expression pattern effectively distinguishes cases of carcinoma ex pleomorphic adenoma (CA ex PA) from pleomorphic adenoma (PA). PA is the most common salivary gland tumor which in rare cases progresses into an epithelial malignancy, termed CA ex PA. Fusion genes involving PLAG1, or less commonly HMGA2, are common in both PA and CA ex PA and are thought to be early genetic drivers in their development. Although the key molecular events which occur during malignant transformation of PA are still unknown, some genetic and epigenetic changes proposed to play a role include amplification of PLAG1, HMGA2, MYC, ERBB2, or chromosome 12q and aberrant promoter hypermethylation of RASSF1, especially in combination with promoter hypermethylation of p16, TERT and WT1. The importance of TERT promoter hypermethylation for malignant transformation in PA may be underappreciated since hypermethylation of that region most critical for malignancy in several cancers, termed the upstream of transcriptional start site (UTSS), has not previously been assayed in PA or CA ex PA. Point mutations or genomic rearrangement in the TERT promoter and elevated TERT mRNA levels are also characteristic of many cancers. We used a panel of 10 PA and 6 CA ex PA clinical samples to determine whether these alterations of TERT, or aberrant expression of TERT mRNA or p53 protein, could effectively distinguish CA ex PA from PA. UTSS hypermethylation in each sample was determined by treatment of genomic DNA with sodium bisulfite followed by MassARRAY and Sanger sequencing. Mutation and arrangement of TERT were screened for by Sanger sequencing and fluorescence in situ hybridization, respectively. p53 expression pattern was visualized by immunohistochemistry and TERT mRNA levels were measured by RT-qPCR. Mutation or rearrangements of the TERT promoter were not observed. However, all 6 CA ex PA samples harbored hypermethylated TERT promoter and had much higher TERT mRNA levels than the PA samples. Two of these samples also had aberrant p53 expression patterns, suggesting the presence of a TP53 missense mutation. None of the PA samples had aberrant p53 expression patterns, although 2 had hypermethylated TERT promoter and elevated TERT mRNA levels as compared with the other PA samples. The significance of this for long term outcome in these patients was unclear. Overall, these results suggest that TERT is commonly upregulated in CA ex PA and that this upregulation is often mediated through hypermethylation of the TERT promoter UTSS. Future large scale studies are warranted to determine the usefulness of TERT promoter hypermethylation for differentiating benign PA from CA ex PA, or the PAs at risk of malignant transformation. Citation Format: Seungjae Lee, Sumit Borah, Kurt Patton, Armita Bahrami. Differentiation of carcinoma ex pleomorphic adenoma from pleomorphic adenoma by TERT promoter hypermethylation and elevated TERT mRNA expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2395. doi:10.1158/1538-7445.AM2017-2395

Importance of biomarkers in glioblastomas patients receiving local BCNU wafer chemotherapy

BackgroundTo assess the influence of molecular markers with potential prognostic value to groups of patients with newly diagnosed glioblastoma patients were examined: group A with 36 patients (surgical resection plus standard combined chemoradiotherapy) and group B with 36 patients (surgical resection, standard combined chemoradiotherapy plus carmustine wafer implantation).Our aim was to determine chromosomal alterations, methylation status of MGMT, p15, and p16 (CDKN2A) in order to analyse the influence on patient survival time as well as radio- and chemotherapy responses. Promoter hypermethylation of MGMT, p16, and p15 genes were determined by MS-PCR. Comparative genomic hybridisation (CGH) analyses were performed with isolated, labelled DNA of each tumor to detect genetic alterations.ResultsAge of onset of the disease showed a significant effect on overall survival (OS) (p < 0.0001). Additional treatment with carmustine wafer (group B) compared to the control group (group A) did not result in improved OS (p = 0.562). Patients with a methylated MGMT promotor showed a significant longer OS compared to those patients with unmethylated MGMT promotor (p = 0.041). Subgroup analyses revealed that patients with methylated p15 showed a significant shorter OS when administered to group B rather than in group A (p = 0.0332). In patients additionally treated with carmustine wafer an amplification of 4q12 showed a significant impact on a reduced OS (p = 0.00835). In group B, a loss of 13q was significantly associated with a longer OS (p = 0.0364). If a loss of chromosome 10 occurred, patients in group B showed a significantly longer OS (p = 0.0123).ConclusionA clinical benefit for the widespread use of additional carmustine wafer implantation could not be found. However, carmustine wafer implantation shows a significantly improved overall survival if parts of chromosome 10 or chromosome 13 are deleted. In cases of 4q12 amplification and in cases of a methylated p15 promotor, the use of carmustine wafers is especially not recommended.The MGMT promoter methylation is a strong prognostic Biomarker for benefit from temozolomide and BCNU chemotherapy.

Aberrant promoter hypermethylation of p16, survivin, and retinoblastoma in gastric cancer.

Gastric cancer is common and can be found throughout the world. Notoriously like other cancers, gastric cancer is a consequence of cellular regulation disorder. Epigenetics, including many oncogenes or tumour suppressor genes, can provide some clues for this kind of disorder. DNA methylation, especially promoter methylation, which causes the silence/decrease of tumour related genes, has become a focus in various tumour types. The aim of this study was to investigate promoter methylation of certain genes: p16, survivin, retinoblastoma (Rb), all of which have been regarded as genes related to gastric cancer. To detect the promoter methylation of p16, survivin, and Rb genes, peripheral blood samples from 106 gastric cancer patients as well as 18 healthy individuals were collected for Methylation-Specific Polymerase Chain Reaction (MSP) analysis. According to the statistical analysis, positive methylation ratio of p16 was 72.6 % (77 cases), 6 % had methylated survivin (7 cases), and positive methylation ratio of Rb was only 17.9 % (19 cases). The differences of promoter methylation of these genes between the patients group and control group were observed: P (P16) = 5.097E-08, P (survivin) = 0.262, and P (Rb) = 0.187. In the control group, methylated p16 was found in only one case that also had a methylated Rb. However, Survivin could not be found methylated in control cases. In this study, promoter methylation was observed for the p16 gene, which was considered an early potential marker in gastric cancer. This data supports that further investigations should study Rb and survivin as candidate markers for gastric cancer (Tab. 2, Fig. 1, Ref. 35).

P15Ink4b Loss of Expression by Promoter Hypermethylation Adds to Leukemogenesis and Confers a Poor Prognosis in Acute Promyelocytic Leukemia Patients.

Purpose The p15Ink4b gene exerts its influence as an inhibitor of cyclin-dependent kinases and is frequently associated with hematological malignancies. Inactivation of this gene through DNA methylation has been found to be the most prevalent epigenetic alteration reported, with a high frequency in all French-American-British subtypes of acute myeloid leukemias, including acute promyelocytic leukemia (APL). In this study,we investigated the prognostic significance of p15 gene promoter hypermethylation and its expression in APL patients of Kashmir (North India).Materials and Methods p15 gene promoter hypermethylation was conducted by methylation-specific polymerase chain reaction, while its subsequent expression analysiswas carried out by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Results Of the 37 patients, 16 (43.2%) were found to have methylated p15 genes. Of these 16 cases, seven (43.8%) were methylated partially and nine (56.2%) were found to have complete methylation. Moreover, nine of the 37 patients (24.3%) who presented with leukocytosis at their baseline had complete p15 gene methylation as well (p < 0.05). Semiquantitative RT-PCR showed a complete loss of p15 expression in nine patients with complete methylation coupled with leukocytosis (p=0.031), while seven patients with partial methylation showed decreased p15 expression. Six patients relapsed during the maintenance phase of treatment and were found to have a completely methylated p15 gene and no p15 mRNA.Conclusion Complete methylation and loss of p15 gene expression causes susceptibility to relapse and decreased survival in APL patients. Thus, p15 promoter hypermethylation is a prospective prognostic indicator and a reliable clinical aid in assessment of patients with APL.

Differential responses of healthy and chronic obstructive pulmonary diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived PM4

While the knowledge of the underlying mechanisms by which air pollution-derived particulate matter (PM) exerts its harmful health effects is still incomplete, detailed in vitro studies are highly needed. With the aim of getting closer to the human in vivo conditions and better integrating a number of factors related to pre-existing chronic pulmonary inflammatory, we sought to develop primary cultures of normal human bronchial epithelial (NHBE) cells and chronic obstructive pulmonary disease (COPD)-diseased human bronchial epithelial (DHBE) cells, grown at the air-liquid interface. Pan-cytokeratin and MUC5AC immunostaining confirmed the specific cell-types of both these healthy and diseased cell models and showed they are closed to human bronchial epithelia. Thereafter, healthy and diseased cells were repeatedly exposed to air pollution-derived PM4 at the non-cytotoxic concentration of 5 μg/cm2. The differences between the oxidative and inflammatory states in non-exposed NHBE and COPD-DHBE cells indicated that diseased cells conserved their specific physiopathological characteristics. Increases in both oxidative damage and cytokine secretion were reported in repeatedly exposed NHBE cells and particularly in COPD-DHBE cells. Diseased cells repeatedly exposed had lower capacities to metabolize the organic chemicals-coated onto the air-pollution-derived PM4, such as benzo[a]pyrene (B[a]P), but showed higher sensibility to the formation of OH-B[a]P DNA adducts, because their diseased state possibly affected their defenses. Differential profiles of epigenetic hallmarks (i.e., global DNA hypomethylation, P16 promoter hypermethylation, telomere length shortening, telomerase activation, and histone H3 modifications) occurred in repeatedly exposed NHBE and particularly in COPD-DHBE cells. Taken together, these results closely supported the highest responsiveness of COPD-DHBE cells to a repeated exposure to air pollution-derived PM4. The use of these innovative in vitro exposure systems such as NHBE and COPD-DHBE cells could therefore be consider as a very useful and powerful promising tool in the field of the respiratory toxicology, taking into account sensitive individuals.