Quantitative Methylation Specific PCR Analysis Research Articles

Relationship of the Levels of microRNA Gene Methylation with the Level of Their Expression and Pathomorphological Characteristics of Breast Cancer.

We studied the relationship of the levels of microRNA group expression and methylation with clinical and pathomorphological parameters of breast cancer and its immunohistochemical status. Quantitative methylation specific PCR analysis showed a significant (p<0.001) increase in the methylation level of 4 microRNA genes (MIR127, MIR129-2, MIR132, and MIR148A) and a significant (p<0.001) decrease for gene MIR375 relative to paired histologically normal tissue. Real-time PCR analysis revealed a significant (p≤0.001) decrease in the expression of 4 microRNAs (miR-127-5p, miR-129-5p, miR-132-3p, and miR-148a-3p) and a significant (p≤0.001) increase in the expression of miR-375-3p. A significant (rs=-0.6--0.7, p≤0.001) relationship between changes in the expression level of miR-129-5p, miR-132-3p, miR-148a-3p, and miR-375-3p and the levels of methylation of the corresponding genes in breast cancer was showed by using Spearman's rank correlation test. Analysis of the samples with consideration of the pathophysiological characteristics of the tumor revealed two significant markers of tumor progression: MIR129-2/miR-129-5p and MIR375/miR-375-3p. Both factors, the increase in the level of MIR129-2 methylation (p<0.001) and a decrease in the expression level of miR-129-5p (p<0.001), are significantly associated (p<0.001) with stage III/IV and the absence of HER2 expression. For MIR375/miR-375-3p, on the contrary, an association of low methylation level and enhanced expression with increased Ki-67 level (>30%, p<0.05) was revealed. These findings are of interest for understanding the mechanisms of breast cancer development and can provide the basis for the diagnosis and prognosis of the course of this disease. Moreover, the revealed features can be useful for adjusting the course of treatment with consideration of the pathophysiological characteristics of the tumor.

Prognostic Significance of Immune Microenvironmental Factors in Undifferentiated Nasopharyngeal Carcinoma Patients Treated with Chemoradiotherapy

EBV-associated UNPC is characterized by an abundant immune infiltrate, which may have a prognostic relevance, considering EBV-specific immune response and microenvironmental immunesuppression. Our primary aim was to retrospectively assess the significance of immunesuppression in the UNPC microenvironment as prognostic biomarker of treatment failure in a non-endemic area population. As secondary aim we investigated the immunomodulating effects of chemoradiotherapy (CRT) monitoring the variation of EBV-specific immunity before and after treatment. We reviewed 63 consecutive patients (pts) with EBV-associated stage II-IVB UNPC undergoing radical CRT. Our policy was of administering concurrent CRT in pts with stage II disease, and sequential therapy in pts with advanced stages. The number of relapsed pts was 11 (cases), while 52 pts (controls) were free from treatment failure at a minimum follow-up of 2 years. DNA and RNA were extracted from tumor (T) and adjacent normal mucosa (M) frozen biopsies at diagnosis. We verified the expression of EBV-derived Barf1 gene in T versus M samples. We investigated the expression of the immune-related genes PD-L1, CD8, FoxP3 and IFN-g in T and M tissues by quantitative RealTime PCR, and their methylation status through quantitative methylation specific PCR. Quantitative RT-PCR analyses showed significantly higher expression levels of several immune-related genes (i.e. BARF1, CD8, IFN-g, IDO, PD-L1, and PD-1) in UNPC samples respect to healthy tissues. Interestingly, CD8 expression was significantly reduced in both tumor and healthy tissues in UNPC patients who relapsed within two years (P<0.001). Similarly, quantitative methylation-specific PCR analyses identified a hypomethylated FOXP3 intron 1 exclusively in relapsed UNPC patients. Finally, we quantified the amount of EBV- and BARF1-specific T-cells in the peripheral blood of 14 patients (7 relapses) through IFN-g ELISPOT assay, and observed a significant decrease after CRT only in relapsing patients. Altogether, our data suggest that a high level of immunosuppression (low CD8, hypomethylated FoxP3) in UNPC microenvironment may predict treatment failure and early identify those patients who could benefit from the addition of an immunotherapy improving first line CRT.

DNA methylation signature for the assessment of metastatic risk in primary renal cell cancer.

516 Background: Transition from localized renal cell cancer (RCC) to metastatic disease is associated with an immense mortality. Beside clinicopathological risk estimation, a molecular prediction of metastatic risk from primary cancers with a sufficient diagnostic accuracy is not available. We biometrically identified a candidate metastasis associated methylation signature (MAMS) in a genome-wide in silico DNA methylation analysis of TCGA data and carried out a double evaluation study using tissues from localized RCC, primary metastatic RCC, and distant metastases. Methods: Candidate MAMS was identified by genome-wide random forest analyses of TCGA methylation level 3 data aiming for classification of 230 primary RCC without distant metastases and 52 metastasized tumors. Forty-nine pyrosequencing and/or quantitative methylation specific PCR analyses were established for a total of 20 candidate methylated loci. For evaluation of MAMS, DNA was isolated and bisulfite converted from the primary RCC tissue cohort (n=187) as well as 99 distant metastases. Localized RCCs consisted of 92 pT1a/b tumors (clear cell, 73; papillary, 16; chromophobe, 2; not classified, 1) without lymph node or distant metastases. RCC samples of primary metastatic disease were obtained from 31 patients. Results: Single candidate loci showed specific hypermethylation when metastatic tissues were compared to primary RCC samples. Random forest analysis showed that MAMS including nine methylation loci achieved a sensitivity of 93% and a specificity of 89% (AUC 0.95) for differentiation of localized RCC and metastases (chi square, p = 3.4*10-29). Positive, negative likelihood, and diagnostic odds ratios were 8.6, 0.08, and 108. Using this random forest model for prediction of primary RCCs associated with distant or lymph node metastases revealed a sensitivity of 58% and specificity of 94% (AUC 0.84, p = 1.0*10-9). Positive, negative likelihood, and diagnostic odds ratios were 9.2, 0.45, and 20.5. Conclusions: A negative MAMS test result retrospectively reduced the probability of metastasis 15-fold in the localized disease cohort, suggesting a prospective evaluation for a possible clinical translation of MAMS in handling RCC patients.

Abstract 2960: DNA promoter hypermethylation of genes as potential diagnostic and prognostic biomarkers for ovarian cancer

Abstract Till recently, epithelial ovarian cancers were thought to arise from the ovarian surface epithelial cells. However, recent studies suggest that the majority of high grade serous carcinomas (HGSCs) arise from lesions in the fallopian tubes. DNA promoter hypermethylation of certain genes is often an early event in tumorigenesis, and it can be detected in blood and other bodily fluids, making it a feasible biomarker strategy for early detection of tumors. Therefore, we sought to determine the methylation profile of HGSCs as compared to fallopian tube epithelium, using the Infinium HumanMethylation 450 Array. We compared the methylation in 12 HGSC tissue samples to 15 fallopian tube epithelium samples. In addition, we compared the methylation profile among long-term survivors (more than 5 years) and short-term survivors (2-3 years) of HGSCs. Our results identified the promoter regions of HIST1H2BB, MAGI2, and EPSTI1 to be differentially methylated when comparing HGSC with normal fallopian tube samples. Furthermore, these genes were differentially methylated in HGSC patients with long-term survival when compared to patients with short-term survival. Quantitative methylation specific PCR analysis confirmed our results of differential methylation among short-term and long-term survivors, and show that these genes are also methylated in ovarian cancer cell lines. Taken together, we have identified genes that could have potential as diagnostic and prognostic biomarkers. Our findings could lead to the development of a biomarker panel for the diagnosis and/or prognosis of ovarian cancer. Citation Format: Blanca L. Valle, Elisabetta Kuhn, David Sidransky, Rafael Guerrero-Preston. DNA promoter hypermethylation of genes as potential diagnostic and prognostic biomarkers for ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2960. doi:10.1158/1538-7445.AM2015-2960

Comparative analysis of methods to determine DNA methylation levels of a tumor-related microRNA gene

Quantifying levels of DNA methylation in tumors is a useful approach for the identification of potential tumor suppressors and to find biomarkers that can be used as prognostic or therapeutic indicators. In the current study, we compared three methods commonly used for quantifying DNA methylation—bisulfite pyrosequencing, quantitative methylation-specific PCR (Q-MSP), and MethyLight—by focusing on the CpG island of the gene encoding the microRNA-34b and microRNA-34c (miR-34b/c); aberrant regulation of this miR is associated with various human malignancies, including gastric cancer. Standard curve analysis using control DNA samples demonstrated the highest quantitative accuracy in Q-MSP analysis. We also carried out methylation analysis using gastric mucosa specimens obtained from gastric cancer patients. We found a high correlation between methylation levels determined by Q-MSP and those determined by MethyLight (R2=0.952), whereas the results of bisulfite pyrosequencing and the other two methods were less well correlated (R2=0.864 and R2=0.804 for Q-MSP and MethyLight, respectively). This may reflect possible PCR bias in the pyrosequencing technique, which we show can be corrected for by applying a cubic approximate equation to the original data. Thus, although results obtained by the different DNA methylation analysis techniques are largely comparable, an appropriate correction may be necessary for stringent comparison.

Abstract 1363: Epigenetic inactivation of VGF in urothelial cell carcinoma and its potential as a non-invasive biomarker using urine

Abstract Background: Urothelial Cell Carcinoma (UCC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new epigenetic markers for UCC we scrutinized the methylation status of 19 genes that has been shown to be cancer specific methylated genes in other solid tumors in our comprehensive genome-wide discovery approach. Methods: We performed promoter methylation of 19 genes (CAMK4, CKMT1b, FKBP4, GALE, HOXB5, HPN, KRT14, LPAR2, MAL, NBR1, NDP, NF1, PDLIM3, PHKA2, PVRL1, RGS4, SGK1, VGF and ZMYM2) that are related to key molecular pathways involved in urothelial carcinogenesis including MAPK pathway or PI3K/AKT pathway. The methods we used includes bisulfite sequencing, methylation specific PCR and quantitative methylation specific PCR(QMSP). All the 19 genes and/or a particular gene were evaluated in urothelial cancer cell lines, primary tumor tissues and urine sediments. Results: By bisulfite sequencing, methylation frequencies for CAMK4, CKMT1b, FKBP4, GALE, HOXB5, HPN, KRT14, LPAR2, MAL, NBR1, NDP, NF1, PDLIM3, PHKA2, PVRL1, RGS4, SGK1, VGF and ZMYM2 were 33.3%, 42.9%, 71.4%, 100.0%, 42.9%, 71.4%, 85.7%, 28.6%, 100.0%, 16.7%, 42.9%, 100.0%, 33.3%, 16.7%, 16.7%, 33.3%, 16.7%, 57.1% and 57.1% respectively in urothelial cancer cell lines. Based on methylation frequencies and known functional evidence, CAMK4, HOXB5, MAL, RGS4, VGF and ZMYM2 were selected for re-expression analysis after 5-Aza-2′-deoxycytidine treatment of UCC cell lines. We also found that promoter methylation of these 6 genes were inversely correlated with gene expression. Since VGF gene was found to be completely methylated in the representative cell lines, we further performed QMSP analysis on 19 primary UCC tissues of different stages and grades with paired normal. Quantitative methylation values were higher in almost all the primary UCC tissues than the paired normal (student's t-test, P=0.005). In an independent cohort of 35 low grade UCC primary tissues, we performed QMSP assay. Twelve out of thirty-five (34.3%) of low grade UCC cases displayed VGF methylation that indicate VGF could be a marker for determining initiation of neoplastic transformation. To explore the potential of methylated VGF as a biomarker for non-invasive detection of UCC, we tested 20 urine samples from UCC cases and 20 urine samples from subjects without any known neoplastic diseases. VGF showed significantly high frequency of QMSP positive status (Fisher's exact test, P=0.020) in urine from UCC cases (8/20) compared to controls (1/20). Conclusion: Our selected genes from genome-wide screening approach successfully identified UCC specific methylated genes that can be determined in primary tissues and urine. Further studies are needed to understand the role of VGF in urothelial carcinogenesis and its potential for the detection of low grade UCC using urine. Citation Format: Masamichi Hayashi, Heike Bernert, Luciane Tsukamoto Kagohara, Mariana Brait, Mark Schoenberg, Trinity Bivalacqua, George Netto, Wayne Koch, David Sidransky, Mohammad O. Hoque. Epigenetic inactivation of VGF in urothelial cell carcinoma and its potential as a non-invasive biomarker using urine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1363. doi:10.1158/1538-7445.AM2014-1363

Epigenetic inactivation of VGF associated with Urothelial Cell Carcinoma and its potential as a non-invasive biomarker using urine

To identify new epigenetic markers and further characterize Urothelial Cell Carcinoma (UCC), we tested the promoter methylation (PM) status of 19 genes previously identified as cancer specific methylated genes in other solid tumors. We used bisulfite sequencing, methylation specific PCR and quantitative methylation specific PCR (QMSP) to test the PM status of 19 genes in urothelial cancer cell lines. Among the 19 genes tested, VGF was found to be completely methylated in several UCC cell lines. VGF QMSP analysis showed that methylation values of almost all the primary 19 UCC tissues were higher than the paired normal tissues (P=0.009). In another cohort, 12/35 (34.3%) of low grade UCC cases displayed VGF methylation. As a biomarker for non-invasive detection of UCC, VGF showed a significantly higher frequency of methylation in urine from UCC cases (8/20) compared to controls (1/20) (P=0.020). After treatment of cell lines with 5-Aza-2'-deoxycytidine, VGF was robustly re-expressed. Forced expression of VGF in bladder cancer cell lines inhibited cell growth. Selection of candidates from genome-wide screening approach in other solid tumors successfully identified UCC specific methylated genes.

DNA methylation biomarkers predict progression-free and overall survival of metastatic renal cell cancer (mRCC) treated with antiangiogenic therapies.

VEGF-targeted therapy increases both the progression-free (PFS) and overall survival (OS) of patients with metastasized renal cell cancer (mRCC). Identification of molecular phenotypes of RCC could improve risk-stratification and the prediction of the clinical disease course. We investigated whether gene-specific DNA hypermethylation can predict PFS and OS among patients undergoing anti-VEGF-based therapy. Primary tumor tissues from 18 patients receiving targeted therapy were examined retrospectively using quantitative methylation-specific PCR analysis of CST6, LAD1, hsa-miR-124-3, and hsa-miR-9-1 CpG islands. PFS and OS were analyzed for first-line and sequential antiangiogenic therapies using the log rank statistics. Sensitivity and specificity were determined for predicting first-line therapy failure. Hypermethylation of CST6 and LAD1 was associated with both a shortened PFS (log rank p = 0.009 and p = 0.004) and OS (p = 0.011 and p = 0.043). The median PFS observed for the high and low methylation groups of CST6 and LAD1 was 2.0 vs.11.4 months. LAD1 methylation had a specificity of 1.0 (95% CI 0.65–1.0) and a sensitivity of 0.73 (95% CI 0.43–0.90) for the prediction of first-line therapy. CST6 and LAD1 methylation are candidate epigenetic biomarkers showing unprecedented association with PFS and OS as well as specificity for the prediction of the response to therapy. DNA methylation markers should be considered for the prospective evaluation of larger patient cohorts in future studies.

Methylation of OPCML promoter in ovarian cancer tissues predicts poor patient survival

Ovarian cancer is a lethal gynecological malignancy largely due to the lack of biomarkers for early detection and treatment options. Opioid binding protein/cell adhesion molecule-like gene (OPCML) has a tumor-suppressor function in ovarian cancer, and epigenetic inactivation of OPCML induces oncogenic transformation of human ovarian surface epithelial cells. This study investigated OPCML promoter methylation levels in ovarian cancer tissues. A total of 30 normal ovarian, 85 benign ovarian tumor, and 102 ovarian cancer tissues were subjected to quantitative methylation-specific PCR analysis of OPCML methylation. Four ovarian cancer cell lines were cultured and treated with the DNA demethylation agent 5-aza-2'-deoxycytidine (5-AZA) for restoring OPCML expression. The data showed that 80 of 102 (78.4%) ovarian cancer tissues and 28 of 85 (32.9%) benign ovarian tumors had a methylated OPCML gene promoter. In contrast, there was no OPCML gene promoter methylation in any of the 30 normal ovarian samples. OPCML promoter methylation was significantly associated with an older age of the patients (p=0.022), an advanced pathological stage of ovarian cancer (p=0.023), and poor overall survival of ovarian cancer patients (p<0.001). Multivariate analysis data showed that pathological stage, age, and OPCML promoter methylation were all independent factors to predict overall survival of patients. Furthermore, 5-AZA was able to restore expression of OPCML mRNA and protein in ovarian cancer cell lines. These data indicate that detection of OPCML gene promoter methylation could be a useful biomarker for predicting the prognosis of ovarian cancer patients and disease progression.

Deregulation of let-7e in epithelial ovarian cancer promotes the development of resistance to cisplatin

Drug resistance remains a major clinical obstacle to successful treatment in ovarian cancer patients, and the evidence of microRNAs involvement in drug resistance has been emerging recently. In this report, we investigated the role of let-7e in the development of cisplatin-resistant ovarian cancer. On the cellular level, let-7e expression was significantly reduced in cisplatin-resistant human epithelial ovarian cancer (EOC) cell line A2780/CP compared with parental A2780 cell and decreased in a concentration-dependent manner in A2780, SKOV3 and ES2 cells treated with cisplatin. Overexpression of let-7e by transfection of agomir could resensitize A2780/CP and reduce the expression of cisplatin-resistant-related proteins enhancer of zeste 2 (EZH2) and cyclin D1 (CCND1), whereas let-7e inhibitors increased resistance to cisplatin in parental A2780 cells. Quantitative methylation-specific PCR analysis showed hypermethylation of the CpG island adjacent to let-7e in A2780/CP cells, and demethylation treatment with 5-aza-CdR or transfection of pYr-let-7e-shRNA plasmid containing unmethylated let-7e DNA sequence could restore let-7e expression and partly reduce the chemoresistance. In addition, cisplatin combined with let-7e agomirs inhibited the growth of A2780/CP xenograft more effectively than cisplatin alone. Diminished expression of EZH2 and CCND1 and higher cisplatin concentrations in tumor tissue of mice subjected to administration of let-7e agomirs in addition to cisplatin were revealed by immunohistochemistry and atomic absorption spectroscopy, respectively. Taken together, our findings suggest that let-7e may act as a promising therapeutic target for improvement of the sensibility to cisplatin in EOC.

Association of Promoter Methylation of VGF and PGP9.5 with Ovarian Cancer Progression

PurposeTo elucidate the role of biological and clinical impact of aberrant promoter hypermethylation (PH) in ovarian cancer (OC).Experimental DesignPH of PGP9.5, HIC1, AIM1, APC, PAK3, MGMT, KIF1A, CCNA1, ESR1, SSBP2, GSTP1, FKBP4 and VGF were assessed by quantitative methylation specific PCR (QMSP) in a training set. We selected two genes (VGF and PGP9.5) for further QMSP analysis in a larger independent validation (IV) set with available clinical data. Biologic relevance of VGF gene was also evaluated.ResultsPH frequency for PGP9.5 and VGF were 85% (316/372) and 43% (158/366) respectively in the IV set of samples while no PH was observed in controls. In 372 OC cases with available follow up, PGP9.5 and VGF PH were correlated with better patient survival [Hazard Ratios (HR) for overall survival (OS) were 0.59 (95% Confidence Intervals (CI) = 0.42–0.84, p = 0.004), and 0.73 (95%CI = 0.55–0.97, p = 0.028) respectively, and for disease specific survival (DSS) were 0.57 (95%CI 0.39–0.82, p = 0.003) and 0.72 (95%CI 0.54–0.96, p = 0.027). In multivariate analysis, VGF PH remained an independent prognostic factor for OS (HR 0.61, 95%CI 0.43–0.86, p<0.005) and DSS (HR 0.58, 95%CI 0.41–0.83, p<0.003). Furthermore, PGP9.5 PH was significantly correlated with lower grade, early stage tumors, and with absence of residual disease. Forced expression of VGF in OC cell lines inhibited cell growth.ConclusionsOur results indicate that VGF and PGP9.5 PH are potential biomarkers for ovarian carcinoma. Confirmatory cohorts with longitudinal follow-up are required in future studies to define the clinical impact of VGF and PGP9.5 PH before clinical application.

Abstract 680: In vivo immunomodulatory activity of SGI-110, a second generation hypomethylating agent, in hematologic malignancies.

Abstract Second generation DNA hypomethylating agents own pleiotropic properties supporting their clinical application in cancer therapy. Among these, SGI-110 is a dinucleotide synthesized as a deoxyguanosine combined with decitabine to protect the latter from the cytidine deaminase inactivation, thus increasing its in vivo exposure and efficacy. A randomized Phase 1-2 FIH PK/PD-guided, dose-escalation study is being conducted on MDS or AML patients, to determine safety and tolerability of SGI-110 and to establish maximum tolerated dose and biologically effective dose. Subjects were randomized to one of two SQ regimens (daily x 5 or once weekly x 3, both administered in 28-day courses). The PK profile showed efficient conversion of SGI-110 to decitabine, longer apparent half-life and lower Cmax than predicted equivalent decitabine IV doses. Here, we investigated the immunomodulatory activity of in vivo administration of SGI-110 in enrolled AML and MDS patients. The hypomethylating properties of in vivo SGI-110 on CTA-specific promoters and the resulting effects on the levels of CTA expression were analyzed in PBMC from patients. Quantitative methylation-specific PCR analyses showed that the daily regimen reduced the constitutive methylation levels in promoters of investigated CTA (i.e., NY-ESO-1 and MAGE-A1) in a dose-dependent manner. The major hypomethylation was observed in patients treated with the highest dose of SGI-110 (Cohort 7, 125 mg/m2), showing an average percentage of hypomethylation of 14.2 for NY-ESO-1-promoter (range 5.0 to 26.7) and 16.3 for MAGE-A1-promoter (range 3.7 to 35.6). In patients treated with the highest doses (Cohorts 4-6, 60 mg/m2 - 125 mg/m2) of the weekly regimen, it was 4.4 (range 0.4 to 6.2) and 2.5 (range 0.1 to 9.9) for NY-ESO-1- and MAGE-A1-promoter, respectively. The effects of the hypomethylating activity of in vivo SGI-110 on the constitutive CTA expression were evaluated by quantitative real-time RT-PCR. The induction/up-regulation of NY-ESO-1, MAGE-A1, MAGE-A3 expression were transient and observed in 11/18 (NY-ESO-1/β-actin molecules range 10-5 to 1.4*10-3), 5/18 (MAGE-A1/β-actin molecules range 10-5 to 8.2*10-5) and 6/18 (MAGE-A3/β-actin molecules range 10-5 to 2.4*10-3) patients treated with the highest doses of daily regimen (Cohorts 4-7). In weekly regimen the induction/up-regulation were detected in 10/14 (NY-ESO-1/β-actin molecules range 10-5 to 1.8*10-3), 1/14 (1.3*10-5 MAGE-A1/β-actin molecules) and 11/14 (MAGE-A3/β-actin molecules range 10-5 to 5.9*10-3) patients treated with the highest doses (cohorts 4-6). These newly identified immunomodulatory properties of SGI-110, combined with its favorable pharmacologic and pharmacokinetic features, define SGI-110 as a potentially useful agent to implement new and more effective combined chemo-immunotherapeutic approaches in patients with hematologic malignancies. Citation Format: Alessia Covre, Giulia Parisi, Hugues Jmg Nicolay, Ester Fonsatti, Elisabetta Fratta, Luca Sigalotti, Pietro Taverna, Sandra Coral, Michele Maio. In vivo immunomodulatory activity of SGI-110, a second generation hypomethylating agent, in hematologic malignancies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 680. doi:10.1158/1538-7445.AM2013-680

Immunomodulatory activity of SGI-110, a 5-aza-2'-deoxycytidine-containing demethylating dinucleotide.

Pharmacologic DNA hypomethylation holds strong promises in cancer immunotherapy due to its immunomodulatory activity on neoplastic cells. Searching for more efficient DNA hypomethylating agents to be utilized to design novel immunotherapeutic strategies in cancer, we investigated the immunomodulatory properties of the new DNA hypomethylating agent SGI-110, that is resistant to in vivo inactivation by cytidine deaminase. Cutaneous melanoma, mesothelioma, renal cell carcinoma, and sarcoma cells were treated in vitro with SGI-110. RT-PCR, quantitative RT-PCR, quantitative methylation-specific PCR, and flow cytometric analyses were performed to investigate changes induced by SGI-110 in the constitutive immune profile of cancer cells. The recognition by gp100-specific CTL of gp100-positive melanoma cells, treated or not with SGI-110, was tested by LDH release assays. SGI-110 induced/up-regulated the expression of investigated cancer/testis antigens (CTA) (i.e., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A10, GAGE 1-2, GAGE 1-6, NY-ESO-1, and SSX 1-5) in all cancer cell lines studied, both at mRNA and at protein levels. Quantitative methylation-specific PCR analyses identified a hypomethylation of MAGE-A1 and NY-ESO-1 promoters in SGI-110-treated neoplastic cells, demonstrating a direct role of pharmacologic DNA demethylation in CTA induction. SGI-110 also up-regulated the expression of HLA class I antigens and of ICAM-1, resulting in an improved recognition of cancer cells by gp100-specific CTL. Our findings show that SGI-110 is a highly attractive therapeutic agent to comprehensively increase immunogenicity and immune recognition of neoplastic cells, and provide the scientific rationale for its clinical development to design novel chemo-immunotherapeutic approaches in cancer patients.

Abstract A30: DNA methylation field defects in colorectal cancer patients.

Abstract Background: Colorectal cancer (CRC)-associated epigenetic abnormalities can develop in pre-neoplastic stages (“epigenetic field defect”) and are detectable throughout the colorectal mucosae as well as in colonic epithelial cells shed into stools. Thus, epigenetic field defects may be useful biomarkers in early detection and risk prediction of CRC in subjects without colonoscopically detectable abnormalities. We carried out a genome-wide analysis of DNA methylation field defects in CRC patients. Methods: Morphologically normal colonic tissues were subjected to the genome-wide DNA methylation microarray analysis. The analyzed tissues consisted of 6 colonic tissues from CRC cases (61-77 years of age; herein referred to as CRC_NCs) and 18 colonic tissues from control subjects (22-85 years of age) with no history of colorectal neoplasias. In assessing DNA methylation field defect, we used 9 of the 18 control tissues that were age-, gender-, and anatomical site-matched to the CRC_NCs as the matched controls (61-77 years of age). The array platform that was employed in this study measured DNA methylation using bisulfite converted sequence-specific probes and is capable of interrogating more than 480,000 individual CpG loci. Results: Principal component analysis of the microarray data revealed that age was the strongest contributing factor to the genome-wide DNA methylation variability in morphologically normal colonic tissues (corresponding to 64.6% of total variability, adjusted p-value&amp;lt;0.05). Anatomical site, followed by presence of concurrent CRC, demonstrated a significant but minor contribution. Relative to the matched controls, 8,454 (1.8%) of the tested autosomal loci were significantly differentially methylated in CRC_NCs (cutoff: Student t-test p&amp;lt;0.05 and delta beta&amp;gt;0.05). In these 8,454 loci, correlation coefficient between age and methylation was significantly but inversely associated with the T–values for differential methylation in CRC_NC (R=-0.63, p&amp;lt;1E-308). Of the 8,454 loci, 212 loci were designated as the candidate field defect biomarkers for CRC_NC detection based on the absence of aging- or anatomical site-dependent methylation. Pilot real-time quantitative methylation specific PCR analyses were performed on four hypermethylation field defect biomarker candidates using an expanded colonic tissue cohort consisting of 29 CRC_NCs and 27 matched controls. Significant or trend toward hypermethylation in CRC_NCs relative to matched controls were observed in all four tested candidates. Two candidates significantly discriminated CRC_NCs from matched controls according to the Receiver-operating characteristics curve analysis (AUC 0.77 and 0.78, p=1E-4). Conclusions: Genome-wide DNA methylation signature in morphologically normal colonic tissue is primarily defined by age, rather than presence of concurrent CRC. A large fraction of methylation field defects are suppression of aging-associated DNA methylation changes, suggesting a protective role of aging-associated DNA methylation changes against early stage carcinogenesis. A pilot quantitative PCR-based assessment in an expanded tissue cohort indicate that the methylation field defects detected by the current microarray analysis include promising candidate biomarkers for early detection of CRC patients and cases at a higher risk of developing CRC. Citation Format: Ling Li, Yulan Cheng, Saliat Ibrahim, Stephen Meltzer, Yuriko Mori. DNA methylation field defects in colorectal cancer patients. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A30. Note: This abstract was not presented at the conference.

P16 INK4A promoter hypermethylation is associated with invasiveness and prognosis of oral squamous cell carcinoma in an age-dependent manner

Oral squamous cell carcinoma (OSCC) is a common cancer worldwide that is highly lethal due to its recurrence and metastasis. Our candidate-based study aimed to link promoter CpG island hypermethylation to OSCC risk assessment. We examined the promoter hypermethylation status of p16(INK4A) (p16), glutathione S-transferase pi (GSTP1), O(6)-methylguanine-DNA methyltransferase (MGMT), death-associated protein kinase 1 (DAPK1), RAS-association domain family 1, isoform A (RASSF1A), and E-cadherin (CDH1) genes in OSCC tumors. Quantitative methylation-specific PCR analysis showed a significant increase in promoter hypermethylation of p16 and CDH1 in OSCC tumors relative to paired non-tumorous tissues. The mean age of patients with hypermethylated p16 was lesser than those without (P=0.027). Multiple logistic regression predicted patients with hypermethylated p16 have higher risks of lymph node invasion (adjusted OR=6.21, P=0.030) in young patients and distant metastasis (adjusted OR=19.23, P=0.007) in older patients. Moreover, p16 promoter hypermethylation was significantly associated with shortened disease-free survival (P=0.034) in older patients. Our results suggested that p16 hypermethylation was associated with early incidence of OSCC, increased lymph node invasion in young patients, and poor prognosis in older patients. Further, p16 hypermethylation may also be implicated in age-related tumor invasion in carcinogenesis.

DNA methylation-related vitamin D receptor insensitivity in breast cancer

Calcitriol (1α, 25(OH)-Vitamin D3) binds to the vitamin D receptor (VDR) and regulates differentiation of the normal mammary gland, may therefore be useful in breast cancer treatment or prevention.Many breast cancer cells are, however, resistant to Calcitriol. In this study, we investigated the resistance mechanism and the role of epigenetic silencing of VDR by promoter hypermethylation.Bisulfite sequencing of the VDR promoter region revealed methylated CpG islands at -700 base pairs (bp) upstream and near the transcription start site.VDR CpG islands were demethylated by 5'deoxy-azacytidine treatment, and this was accompanied by a parallel increase in VDR mRNA levels in breast cancer cell lines. Quantitative methylation-specific PCR analyses confirmed hypermethylation of these CpG islands in primary tumors, and its absence in normal breast tissue.VDR transcripts detected in breast cancers were predominantly 5'-truncated, while normal breast tissue expressed full-length transcripts. Consistent with this observation, genes containing the VDR-responsive element (VDRE), such as cytochrome p450 hydroxylases,p21, or C/EBP were underexpressed in breast cancers compared to normal breast samples. Expression of the active longer transcripts of VDR was restored with 5'deoxy-Azacytidine (AZA) treatment, with a concurrent increase in expression of VDRE-containing genes. Thus, promoter methylation-mediated silencing of expression of the functional variants of VDR may contribute to reduced expression of downstream effectors of the VDR pathway and subsequent Calcitriol insensitivity in breast cancer. These data suggest that pharmacological reversal of VDR methylation may re-establish breast cancer cell susceptibility to differentiation therapy using Calcitriol.

Abstract 4934: Promoter methylation biomarkers for breast cancer risk stratification using an unbiased whole genome approach

Abstract Background: Silencing of tumor suppressor gene expression by promoter methylation occurs frequently in breast cancer and can affect the expression of &amp;gt;100 genes per tumor. Since tumorigenesis is a multistep process and occurs over a large period of time, promoter methylation of some genes may occur very early in this process and can be used for risk stratification or occur very late and be used for early detection. The objective of our study is to identify genes that are frequently methylated in breast cancers and can be used for individualized breast cancer risk stratification. Methods and Results: We have used an unbiased whole genome approach to identify breast cancer methylation markers in addition to 18 candidate markers obtained from breast cancer literature. Six breast cancer cell lines and 6 short term benign breast primary cultures were exposed to 0.5 µM 5-aza-2′-deoxycytidine or DMSO for 5 days; total RNA was isolated and expression differences were determined by gene expression profiling using Illumina arrays. After normalization and bioinformatic analysis, 288 genes of interest were identified. The methylation status of each promoter was analyzed by methylation specific PCR (MSP) using an independent set of 10 breast cancer cell lines, 6 benign breast primary cultures, and 4 lymphocyte samples. Of the 288 genes tested, 204 genes were excluded because they were: not methylated in breast cancer cell lines (109), methylated in lymphocytes (91), or methylated in all benign breast cultures at the same intensity as the cancer samples (4). Of the remaining 84 genes, 58 genes with the highest methylation frequency in the breast cancer cell lines were analyzed by MSP with a panel of 15 primary breast cancers and 15 random periareolar fine needle aspirate (RP-FNA) samples from benign breast tissue (5 breast cancer patients, 5 unaffected Gail high-risk patients, and 5 unaffected Gail average-risk patients). Thirty seven genes were methylated in at least 4 breast cancer samples and had a cancer/RP-FNA ratio of &amp;gt;2. Unsupervised hierarchical clustering separated the genes into 3 groups based on the primary breast tumor data. One group containing 10 genes was highly methylated in all primary tumors with an average of 7.6/10 methylated genes per tumor (range 3-10). 21 genes representing all groups have been selected for quantitative MSP analysis. We are currently testing these genes in a panel of 200 RP-FNA samples including primary breast tumors and benign breast tissue from cancer and unaffected patients to identify biomarkers for breast cancer risk stratification. These data will be presented at the meeting. Conclusions: We have identified 37 candidate genes that are highly methylated in breast cancer and less frequently in benign breast tissue. Quantitative MSP analysis is underway using clinical samples to determine the best biomarkers for individualized breast cancer risk assessment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4934.

Prostate Cancer Detected by Methylated Gene Markers in Histopathologically Cancer-Negative Tissues from Men with Subsequent Positive Biopsies

The goal of this retrospective, multicenter study was to evaluate the ability of a newly developed refinement of a quantitative methylation-specific PCR assay to detect prostate cancer in histopathologically negative biopsy samples collected from men who were later positively diagnosed during a follow-up biopsy procedure. Biomarkers tested in the assay included the much-studied glutathione-S-transferase P1 gene and others reported to be frequently methylated in prostate cancer. Core biopsy tissue from subjects with serial negative biopsies served as a negative control to assess assay specificity. As a positive control, biopsy core tissue from patients histopathologically diagnosed with prostate cancer was used to gauge true marker sensitivity in known cancer-containing specimens. Testing was completed in 971 archived paraffin-embedded tissue blocks from 264 men screened for prostate cancer. More samples were initially tested, but due to the advanced age of the paraffinized tissue, DNA quality for quantitative methylation-specific PCR analysis was insufficient in 34% of the available blocks. The glutathione-S-transferase P1 gene has been confirmed as a powerful indicator of the presence of prostate cancer cells. A sensitivity of 52% was observed in the "potentially false-negative first biopsies," with a corresponding specificity of 85% and the sensitivity in biopsy tissue cores containing histopathologically confirmed prostate cancer was 95%. An even higher sensitivity can be reached with RAR-2beta (84%) and APC (72%), with respective specificities of 48% and 50%. Gene methylation was detected in initial, negative biopsy tissue in men who were later diagnosed with prostate cancer. Testing for methylation in histopathologically negative biopsies could improve the early detection of prostate cancer.

TGFβR2 aberrant methylation is a potential prognostic marker and therapeutic target in multiple myeloma

Multiple myeloma (MM) is an incurable hematological malignancy. Different studies demonstrated the occurrence of genetic and epigenetic alterations in MM. The aberrant methylation is one of the most frequent epigenetic alterations in human genome. This study evaluated the aberrant methylation status of 20 genes in 51 MM samples by quantitative methylation-specific PCR (QMSP) and compared the methylation profile with clinicopathological characteristics of the patients. The QMSP analyses showed that PTGS2 (100.0%), SFN (100.0%), CDKN2B (90.2%), CDH1 (88.2%), ESR1 (72.5%), HIC1 (70.5%), CCND2 (62.7%), DCC (45.1%) and TGFbetaR2 (39.2%) are frequently hypermethylated in MM while aberrant methylation of RARbeta (16.6%), MGMT (12.5%), AIM1 (12.5%), CDKN2A (8.3%), SOCS1 (8.3%), CCNA1 (8.3%) and THBS1 (4.1%) are rare events. There was no methylation of GSTP1, MINT31, p14ARF and RB1 in the samples tested. Hypermethylation of ESR1 was correlated positively with isotype IgA, while aberrant methylation of THBS1 correlated negatively with isotype IgG. Furthermore, hypermethylation of DCC and TGFbetaR2 were correlated with poor survival. The multivariate analysis showed ISS and TGFbetaR2 hypermethylation strongly correlated with poor outcome. This study represents the first quantitative evaluation of promoter methylation in MM and our data provide evidence that TGFbetaR2 hypermethylation, besides ISS, may be useful as prognostic indicator in this disease.

High Specificity of Quantitative Methylation‐Specific PCR Analysis for MGMT Promoter Hypermethylation Detection in Gliomas

Normal brain tissue from 28 individuals and 50 glioma samples were analyzed by real-time Quantitative Methylation-Specific PCR (QMSP). Data from this analysis were compared with results obtained on the same samples by MSP. QMSP analysis demonstrated a statistically significant difference in both methylation level (P = .000009 Mann Whitney Test) and frequencies (P = .0000007, Z-test) in tumour samples as compared with normal brain tissues. Although QMSP and MSP showed similar sensitivity, the specificity of QMSP analysis was significantly higher (93%; CI95%: 84%–100%) as compared with MSP (64%; 95%CI: 46%–82%). Our results suggest that QMSP analysis may represent a powerful tool to identify glioma patients that will benefit from alkylating agents chemotherapy.