Real-time Methylation-specific PCR Research Articles

Evaluation of Preanalytical Conditions and Implementation of Quality Control Steps for Reliable Gene Expression and DNA Methylation Analyses in Liquid Biopsies.

Liquid biopsy provides important information for the prognosis and treatment of cancer patients. In this study, we evaluated the effects of preanalytical conditions on gene expression and DNA methylation analyses in liquid biopsies. We tested the stability of circulating tumor cell (CTC) messenger RNA by spiking MCF-7 cells in healthy donor peripheral blood (PB) drawn into 6 collection-tube types with various storage conditions. CTCs were enriched based on epithelial cell adhesion molecule positivity, and RNA was isolated followed by cDNA synthesis. Gene expression was quantified using RT-quantitative PCR for CK19 and B2M. We evaluated the stability of DNA methylation in plasma under different storage conditions by spiking DNA isolated from MCF-7 cells in healthy donor plasma. Two commercially available sodium bisulfite (SB)-conversion kits were compared, in combination with whole genome amplification (WGA), to evaluate the stability of SB-converted DNA. SB-converted DNA samples were analyzed by real-time methylation-specific PCR (MSP) for ACTB, SOX17, and BRMS1. Quality control was assessed using Levey-Jennings graphs. RNA-based analysis in CTCs is severely impeded by the preservatives used in many PB collection tubes (except for EDTA), as well as by time to analysis. Plasma and SB-converted DNA samples are stable and can be used safely for MSP when kept at -80 °C. Downstream WGA of SB-converted DNA compensated for the limited amount of available sample in liquid biopsies. Standardization of preanalytical conditions and implementation of quality control steps is extremely important for reliable liquid biopsy analysis, and a prerequisite for routine applications in the clinic.

Clinical Validation of PITX2 DNA Methylation to Predict Outcome in High-Risk Breast Cancer Patients Treated with Anthracycline-Based Chemotherapy

Background: Breast cancer patients at high risk for recurrence are treated with anthracycline-based chemotherapy, but not all patients do equally benefit from such a regimen. To further improve therapy decision-making, biomarkers predicting outcome are of high unmet medical need. Methods: The percent DNA methylation ratio (PMR) of the promoter gene coding for the Paired-like homeodomain transcription factor 2 (PITX2) was determined by a validated methylation-specific real-time polymerase chain reaction (PCR) test. The multicenter study was conducted in routinely collected archived formalin-fixed paraffin-embedded (FFPE) tissue from 205 lymph node-positive breast cancer patients treated with adjuvant anthracycline-based chemotherapy. Results: The cut-off for the PITX2 methylation status (PMR = 12) was confirmed in a randomly selected cohort (n = 60) and validated (n = 145) prospectively with disease-free survival (DFS) at the 10-year follow-up. DFS was significantly different between the PMR ≤ 12 versus the PMR > 12 group with a hazard ratio (HR) of 2.74 (p < 0.001) in the validation cohort and also for the patient subgroup treated additionally with endocrine therapy (HR 2.47; p = 0.001). Conclusions: Early-stage lymph node-positive breast cancer patients with low PITX2 methylation do benefit from adjuvant anthracycline-based chemotherapy. Patients with a high PITX2 DNA methylation ratio, approximately 30%, show poor outcome and should thus be considered for alternative chemotherapy regimens.

ESR1-promoter-methylation status in primary breast cancer and its corresponding metastases.

The role of ESR1 methylation in breast cancer and its influence on disease progression is not yet fully understood. Healthy breast tissue usually does not show ESR1 promoter methylation, whereas the frequency of ESR1 methylation appears to increase in primary breast cancer and in metastatic disease. Although women with ER positive breast cancer have a good prognosis, some will relapse. We aimed to evaluate the methylation status of ESR1 in primary breast cancer and its corresponding metastases by a methylation-specific real-time PCR and to correlate the methylation status with clinical outcome. Women who were treated for primary and metastatic breast cancer were included in the study. Tumor DNA was isolated from paraffin embedded tissue sections. After bisulfite treatment ESR1 promoter methylation was analyzed by real time-MSP of each tissue sample. Kaplan-Meier-Curves were drawn for survival. In the group of patients with positive ESR1 promoter methylation in the primary breast carcinoma survival was lower compared to the group of patients without methylation (38.1months vs. 54.3months, n.s.). Seven out of 19 (37%) of those patients with positive ESR1 promoter methylation developed loss of ER expression in metastatic disease. None of the patients who had primary tumours that were ESR1 methylation negative developed ER expression negative metastatic disease. The results underline the importance of the ESR1 promoter methylation and its potential application as a predictive marker. To improve the clinical outcome of patients with metastatic disease, those with initially positive ESR1 methylation status should undergo a tissue biopsy already at the beginning of metastatic disease to identify those with loss of ER expression and thus resitance to anti-endocrine therapy.

PITX2 DNA-methylation as the first clinically validated predictive marker for anthracycline based chemotherapy in high-risk breast cancer patients

International guidelines recommend anthracycline-based chemotherapy for high-risk breast cancer patients as the standard-of-care, but not all patients do equally benefit from such a chemotherapy. The novel therascreen® PITX2 RGQ PCR assay is a quantitative in vitro methylation-specific real-time PCR test (qMSP), intended for the determination of the percent methylation ratio (PMR) in the pituitary homeobox 2 (PITX2) promoter 2 expressed by primary breast cancer tumor tissue (FFPE-material). After bisulfite exposure of extracted DNA to distinguish between methylated and non-methylated PITX2 DNA, the percent methylation ratio (PMR) of the PITX2 gene promoter 2 is quantified by qMSP. The PMR obtained does provide information to the treating physician about whether a patient is likely to respond to anthracycline-based chemotherapy. Data will be presented for 205 high-risk lymph node-positive, estrogen receptor-positive, HER2-negative breast cancer patients, treated with adjuvant anthracycline-based chemotherapy. The PITX2 pre-defined cut-off value of PMR = 12 demonstrated a statistically significant differentiation between low- and high-risk breast cancer patient for the primary endpoint DFS, also for patients treated with endocrine therapy in addition to anthracycline-based adjuvant therapy. High-risk lymph node-positive, estrogen-receptor-positive, HER2-negative breast cancer patients, with PITX2 methylation defined as low (PMR ≤ 12), are sufficiently treated with anthracycline-based chemotherapy, irrespective if treated with additional tamoxifen. High-risk lymph node-positive, estrogen receptor-positive, HER2-negative breast cancer patients with PITX2 methylation defined as high (PMR > 12) should avoid anthracycline-based chemotherapy. These patients are recommended to switch to other chemotherapy regimens, since with this PITX2-characteristics, the patient has a lower probability to respond to anthracycline-based chemotherapy.

Abstract 3655: DNA methylation analysis in liquid biopsy: A detailed study on quality control, sample storage and whole genome amplification procedures for downstream applications

Abstract Background: Studies on epigenetic alterations and especially DNA methylation in liquid biopsy samples like plasma circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) are of growing interest nowadays. To ensure the reliability of DNA methylation results prior to any clinical applications in liquid biopsy, there is an urgent need to standardize a plethora of parameters. The aim of our study was to evaluate: a) the stability of DNA methylation in plasma, b) the stability of sodium bisulfite (SB) converted DNA samples under different storage temperatures, and c) the accuracy and precision of whole genome amplification (WGA) protocols for downstream DNA methylation analysis. Methods: a) Stability of DNA methylation in plasma: Plasma from healthy donors was pooled and aliquoted in six tubes and spiked with genomic DNA (gDNA) isolated from 106 MCF-7 cells. Five aliquots were kept at -700C, while one was immediately processed for ctDNA isolation and SB treatment. Stored aliquots were processed in the same way after storage for a period of 3 months. All SB-treated DNA samples were further evaluated by real time methylation specific PCR (MSP) for ACTB and SOX17. b) Stability of SB-treated DNA: gDNA was isolated from 106 SKBR3 cells and processed for downstream SB-treatment. SB-treated DNA was split into aliquots and stored at -20°C and -70°C. One aliquot, used as control, was immediately processed for SB treatment followed by real time MSP for ACTB and BRMS1 amplification. The remaining aliquots were processed in exactly the same way after storage for different time points with a maximum period of one year. Additionally, a sample that was subjected to repeated freeze-thaw cycles was added in each run. c) Evaluation of WGA for SB-treated DNA: we evaluated two different SB conversion kits, EZ DNA Methylation-Gold (Zymo), and Epitect Fast Bisulfite, (Qiagen) in combination with downstream WGA (EpiTect Whole Bisulfitome Kit, Qiagen), using 0%, 100% methylated DNA standards and MCF-7, SKBR3 cell lines. Results: a) DNA methylation in plasma is stable at -700C for at least 3 months, since the average Cqs detected, varied from 1 to 2 cycles for ACTB and SOX17, respectively, b) Quality of SB-treated DNA is not affected by repeated freezing-thawing cycles; SB-treated DNA samples are stable when kept for up to one year both at -200C and -700C. c) Identical results by real time MSP were obtained before and after WGA amplification (Qiagen), after using either the Zymo or Qiagen kits for SB conversion. Conclusions: Plasma and SB-treated DNA samples are stable and can be safely used for multiple real time MSP assays over time when kept at -70°C. Downstream WGA of SB-treated DNA could bypass the limited amount of available sample for liquid biopsy applications. In any case, quality control checking should be included in each step of analysis. Citation Format: Sofia Mastoraki, Maria Chimonidou, Eleni Tzanikou, Evi S. Lianidou. DNA methylation analysis in liquid biopsy: A detailed study on quality control, sample storage and whole genome amplification procedures for downstream applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3655.

Abstract 1385: Galangin is an epigenetic modulator or BRCA1 and induces estrogen receptor alpha in triple negative breast cancer cells

Abstract The aryl hydrocarbon receptor (AhR) is a driver of breast cancer progression and its overexpression is often observed in triple negative breast cancer (TNBC). Activation of AhR results in epigenetic silencing of the breast cancer 1 gene (BRCA1), a necessary factor in the transcriptional activation of estrogen receptor-alpha (ERα). The purpose of this study was to examine the capacity for galangin (GAL; 3,5,7-trihydroxy-2-phenylchromen-4-one), a natural AhR antagonist, to mimic the effects of synthetic AhR antagonists alpha-naphthoflavone (αNF, 2-phenylbenzo[h]chromen-4-one) and 3'-methoxy-4'-nitroflavone (MNF; 2-(3-methoxy-4-nitrophenyl)chromen-4-one) in regards to counteracting epigenetic silencing of BRCA1 and restoring ERα protein expression. We utilized TNBC cells with hypermethylated BRCA1 and constitutively active AhR (HCC38 cells) for these experiments. After 72h in phenol-red free media, cells were treated for up to 72h against a range of GAL, αNF, and MNF concentrations in the presence or absence of estradiol (E2). Transcript levels of AhR and its downstream target genes CYP1A1 and CYP1B1 were determined by real-time polymerase chain reaction (RT-PCR). Protein levels of BRCA1, ERα, and AhR were determined by Western blot. Treatment with GAL results in stabilization of BRCA1 and ERα protein levels, similarly to αNF and MNF. To determine potential mechanisms of these observations, we are currently conducting experiments to analyze changes in BRCA1 promoter methylation using methylation-specific RT-PCR (MSP) and DNA methyltransferase 1 (DNMT1) protein levels by Western blot. These data suggest the naturally occurring flavanoid GAL may be an effective compound for modulating epigenetic repression of BRCA1 and restoring ERα levels, which may provide options for anti-hormonal therapy in ERα-negative breast cancers. Citation Format: Micah G. Donovan, Ornella I. Selmin, Donato F. Romagnolo. Galangin is an epigenetic modulator or BRCA1 and induces estrogen receptor alpha in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1385.

Abstract 3308: Urine epigenetic biomarkers for NSCLC diagnosis

Abstract Purpose: The National Lung Cancer Screening Trial showed mortality reduction on patients with NSCLC by the use of Low-dose CT screening. The study exhibits an unreasonably high false positive rate of 96.6%, which can to lead to significant morbidity and mortality from unnecessary tests. This study aim to determine if methylated promoter regions of a panel of genes that are correlated with Non-Small Cell Lung Cancer can be detected in urine from patients with lung cancer versus those without. Materials and Methods: We conducted a prospective case-control study recruiting subjects from the Lung Cancer Spore Trial. Urine and pre-operative CT scans were obtained from all patients. We processed the urine using the Methylation on Beads assay to isolate and bisulfite treat circulating DNA and then employed Quantitative Methylation Specific Real-Time PCR to detect promoter methylation status of the genes: CDO1, TAC1, HOXA7, HOXA9, SOX17 and ZFP42. Sensitivity, specificity, PPV and NPV values were calculated for each gene methylation status. Results: 34 patients were studied, including 23 patients with NSCLC and 11 patients with benign non-cancerous lesions. The sensitivity, specificity, PPV and NPV values for lung cancer detection in urine are shown in Table 1. Sensitivities ranged 43-96%, Specificities 64-91%, PPV 71-100% and NPV 40-67%. Promoter methylation of the gene panel CDO1, TAC1, HOXA7, HOXA9, SOX17 and ZPF42 has a 78% sensitivity, 91% specificity, 95% PPV and 67% NPV for lung cancer detection. Conclusion: Our study suggests that urine can provide a highly sensitive and specific non-invasive route for lung cancer detection. These results are very promising and could potentially reduce unnecessary morbidity and mortality in people undergoing screening for NSCLC by use of a non-invasive method that could be more accessible on primary care centers. However, further studies and validation with larger sample sizes and different populations are necessary before its application to clinical practice. Patients with positive gene methylation from urine samples and its accuracy for NSCLC detection.Cancer (n=23)Cancer (n=23)Control (n=11)Control (n=11)nSensitivitynSpecificityPPVNPVCDO114611919353TAC11565010010058SOX1716704648050HOXA711483737940HOXA910431919143ZPF4222969187167Panel of genes18781919567 Citation Format: Lane Lerner, Lily Zheng, Anastasia Kottorou, Chen Chen, Tomoaki Ito, Kristen Rodgers, Beverly Lee, Robert Winn, Enrico Benedetti, Tza- Huei Wang, Malcolm V. Brock, James G. Herman, Alicia Hulbert. Urine epigenetic biomarkers for NSCLC diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3308.

Abstract 4587: ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer

Abstract Background: The estrogen receptor is highly expressed in epithelial ovarian cancer and represents the main target for endocrine therapy. The ESR1 gene is frequently methylated in many types of gynecological malignancies and previous studies have shown an inverse correlation between ESR1 methylation and gene expression. Few studies attempted to investigate the role of ESR1 methylation in ovarian cancer so far, and the clinical significance of ESR1 methylation status is not as yet clear. Methods: The ESR1 methylation status was examined in primary tumors and corresponding circulating tumor DNA (ctDNA) samples of patients with high-grade serous ovarian cancer. For the detection of methylation we applied a novel highly specific and sensitive real-time methylation specific PCR (real-time MSP) assay. Two groups of primary tumor samples were recruited (training group, n=66 and validation group, n=61), along with the corresponding plasma samples (n=58) for the validation group. ESR1 methylation was also analyzed in a small group of 16 normal fallopian tube samples. Results: ESR1 was found methylated in both groups of primary tumor samples and in the corresponding plasma samples of the validation group. More specifically, ESR1 methylation was detected in 32/66 (48.5%) and 17/61 (27.9%) primary tumor samples of the training and the validation group, respectively, and in 23/58 (39.7%) corresponding plasma samples. A significant agreement between ESR1 methylation in primary tumors and paired ctDNA was observed in 40/56 (71.4%) samples of the validation group (P=0.004, k=0.360). Almost all normal fallopian tube samples (15/16) were found methylated. Interestingly, the presence of ESR1 methylation in the primary tumor samples of the validation group was nearly significantly correlated (P=0.057) with a better overall survival. Conclusions: We detected for the first time ESR1 methylation in ctDNA of patients with high-grade serous ovarian cancer. The agreement between ESR1 methylation in the primary tumors and paired ctDNA is statistically significant. Our results indicate a potential correlation between ESR1 methylation and better overall survival in high-grade serous ovarian cancer patients. Citation Format: Lydia Giannopoulou, Sofia Mastoraki, Areti Strati, Issam Chebouti, Kitty Pavlakis, Sabine Kasimir-Bauer, Evi Lianidou. ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4587.

Real-time methylation-specific PCR for the evaluation of methylation status of MGMT gene in glioblastoma.

The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene is a strong predictor for the efficacy of temozolomide chemotherapy and survival periods. However, the correlation between the extent of methylation and the difference in survival times has not been fully clarified. Simple and quantitative evaluations of the methylation status in the promotor region of the MGMT gene are expected to be worldwide standardized diagnostics. We applied real-time semi-quantitative methylation-specific polymerase chain reaction (SQ-MSP) of the MGMT gene promoter region to 84 glioblastoma patients. The SQ-MSP result showed that the ΔCt value, which represents the difference between uCt and mCt (uCt value – mCt value), is inversely correlated with overall survival. With adequate cutoff setting, this assay showed that those patients suffering from a tumor with low ΔCt (methylated) survived significantly longer than those having tumors with high ΔCt (un-methylated). The most significant difference was observed when the cutoff was set at a ΔCt of 2. Using this cutoff point, the result of MGMT immunohistochemical analysis was also significantly correlated with the methylation status examined with real-time SQ-MSP. These results collectively show that MGMT promoter methylation status actually affects patients’ survival and protein expression depending on its methylation level, and the extent of methylated CpGs would be better assessed with real-time SQ-MSP than with the standard gel-based MSP. This method is cost- and labor-saving compared with pyrosequencing, and significantly contributes to the accurate and objective prediction of patient survival.

ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer

ObjectiveEstrogen receptor, coded by the ESR1 gene, is highly expressed in epithelial ovarian cancer. ESR1 gene is frequently methylated in many types of gynecological malignancies. However, only a few studies attempted to investigate the role of ESR1 methylation and its clinical significance in ovarian cancer so far. The aim of our study was to examine ESR1 methylation status in primary tumors and corresponding circulating tumor DNA of patients with high-grade serous ovarian cancer (HGSC). MethodsESR1 methylation was detected by a highly specific and sensitive real-time methylation-specific PCR assay. Two groups of HGSC samples were analyzed: group A (n = 66 primary tumors) and group B (n = 53 primary tumors and 50 corresponding plasma samples). ResultsESR1 was found methylated in both groups of primary tumors: in 32/66 (48.5%) of group A and in 15/53 (28.3%) of group B. 19/50 (38.0%) corresponding plasma samples of group B were also methylated for ESR1. A significant agreement for ESR1 methylation was observed between primary tumors and paired plasma ctDNA samples (P = 0.004). Interestingly, the presence of ESR1 methylation in primary tumor samples of group B was significantly correlated with a better overall survival (P = 0.027) and progression-free survival (P = 0.041). ConclusionsWe report for the first time the presence of ESR1 methylation in plasma ctDNA of patients with HGSC. The agreement between ESR1 methylation in primary tumors and paired ctDNA is statistically significant. Our results indicate a correlation between the presence of ESR1 methylation and a better clinical outcome in HGSC patients.

Demethylation of GFRA4 Promotes Cell Proliferation and Invasion in Hirschsprung Disease.

Hirschsprung disease (HSCR) is congenital intestinal aganglionosis attributed to a failure to migrate and survive of neural crest-derived cells. Glial cell-derived neurotrophic factor alpha 4 (GFRA4) is expressed in the derivatives of the neural crest in the enteric nervous system, but whether it is related with HSCR still remains unclear. This study was designed to investigate its role and epigenetic mechanisms in HSCR in vitro. The expression of GFRA4 mRNA in HSCR tissues was determined using quantitative real-time PCR analysis. In this study, we found that GFRA4 expression was significantly reduced in HSCR tissues and cells through GFRA4 methylation by quantitative real-time PCR analysis, methylation-specific PCR, and bisulfite sequencing PCR. DNA methyltransferase inhibitor, 5-AzaC, concomitantly upregulated the protein levels of GFRA4, as well as DNA methyltransferase1 (DNMT1) and DNMT2 in SH-5YSY cells. Moreover, we found upregulated GFRA4 significantly promoted cell proliferation, cell cycle progression and invasion, but inhibited apoptosis in SH-5YSY cells, whereas GFRA4 knockdown caused the opposite effects in SH-5YSY cells by CCK-8, 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and Transwell assays. In conclusion, our results support that aberrant CpG hypermethylation at least partly accounts for GFRA4 silencing in HSCR, which impairs its protective role in enteric nervous system.

A novel DNA methylation panel accurately detects colorectal cancer independently of molecular pathway

BackgroundColorectal cancer (CRC) is one of the most incident cancers, associated with significant morbidity and mortality, and usually classified into three main molecular pathways: chromosomal instability, microsatellite instability (MSI) and CpG island methylator phenotype (CIMP). Currently, available screening methods are either costly or of limited specificity, impairing global implementation. More cost-effective strategies, including DNA methylation-based tests, might prove advantageous. Although some are already available, its performance is suboptimal, entailing the need for better candidate biomarkers. Herein, we tested whether combined use of APC, IGF2, MGMT, RASSF1A, and SEPT9 promoter methylation might accurately detect CRC irrespective of molecular subtype.MethodsSelected genes were validated using formalin-fixed paraffin-embedded tissues from 214 CRC and 50 non-malignant colorectal mucosae (CRN). Promoter methylation levels were assessed using real-time quantitative methylation-specific PCR. MSI and CIMP status were determined. Molecular data were correlated with standard clinicopathological features. Diagnostic and prognostic performances were evaluated by receiver operator characteristics curve and survival analyses, respectively.ResultsExcept for IGF2, promoter methylation levels were significantly higher in CRC compared to CRN. A three-gene panel (MGMT, RASSF1A, SEPT9) identified malignancy with 96.6% sensitivity, 74.0% specificity and 91.5 positive predictive value (area under the curve: 0.97), independently of tumor location, stage, and molecular pathway.ConclusionsCombined promoter methylation analysis of MGMT/RASSF1A/SEPT9 displays a better performance than currently available epigenetic-based biomarkers for CRC, providing the basis for the development of a non-invasive assay to detect CRC irrespective of the molecular pathway.

MiR-148b-3p affects the pathogenesis of adjuvant-induced arthritis rats through the direct target DNMT1

Our previous study showed that up-regulated DNA methyltransferase-1 (DNMT1) played an important role in the hypermethylation modification of SFRP4 in adjuvant-induced arthritis (AIA) rats. This work focused on the role of disordered miR-148b-3p in RA pathology and its corresponding regulatory targets. The expression of miR-148b-3p and DNMT1, and the effect of miR-148b-3p on the DNMT1 expression were determined by real-time qPCR, western blotting and double luciferase reporter genes. The role of miR-148b-3p on the SFRP4 expression, the canonical Wnt signaling and the pathology of AIA rats was investigated using real-time qPCR, western blotting and methylation-specific PCR (MSP). The results showed that the expression of miR-148b-3p was significantly decreased, the expression of DNMT1 was significantly increased and the DNMT1 was the direct target of miR-148b-3p in AIA rats compared with normal group. Transfection of miR-148b-3p mimics up-regulated the SFRP4 expression, inhibited the canonical Wnt signaling and the pathogenesis of AIA rats by targeting the DNMT1. The role of miR-148b-3p knockdown was opposite to that of miR-148b-3p overexpression. These results suggest that miR-148b-3p may influence the pathogenesis of RA with the DNMT1 as a direct target and miR-148b-3p may be a potential diagnostic and therapeutic target for RA patients.

Genome-wide hypomethylation of LINE-1 and Alu retroelements in cell-free DNA of blood is an epigenetic biomarker of human aging.

Aging associated DNA hypomethylation of LINE-1 and Alu retroelements may be a crucial determinant of loss of genomic integrity, deterioration and cancer. In peripheral blood LINE-1 hypomethylation has been reported to increase during aging, but other studies did not observe significant changes. We hypothesized that these apparently inconsistent reports might relate to differences between cellular and cell-free DNA. Using the technique of idiolocal normalization of real-time methylation-specific PCR (IDLN-MSP) for genetic imbalanced DNA specimens we obtained evidence that LINE-1 hypomethylation in cell-free DNA, but not cellular DNA from peripheral blood is an epigenetic biomarker for human aging. Furthermore, hypomethylation of cell-free DNA is more extensive in smokers, suggesting that it might be used as a surrogate marker for monitoring the improvement of smoking-induced adverse effects after cancelling smoking.

Abstract A177: Role of promoter methylation in regulation of Notch4 and JAG2 gene expression in human glioblastoma

Abstract In cancer, DNA methylation causes alteration in gene expression affecting important signal transduction pathways. Since methylation occurs at a very early stage and is reversible, hypermethylated promoters hold great promise as biomarkers for early detection and effective drug targets for gene reactivation. The Notch signaling pathway is one such important developmental pathway governing cell fate decisions. Dysregulated Notch signaling is found to have a prominent role in the development of various cancers. Glioblastoma (GBM) is the most common primary brain tumor, with a very poor prognosis despite advanced treatment facilities due to poor molecular stratification. It is therefore important to study genetic and epigenetic events leading to gliomagenesis and to guide new treatment strategies. The aim of this study was to detect Notch pathway genes potentially regulated by promoter methylation in human GBM. We used real-time PCR and quantitative methylation-specific PCR to study methylation status of underexpressed Notch pathway genes from human GBM formalin-fixed, paraffin-embedded sections. 50% of samples showed methylation at Notch4 gene promoter at levels higher than 25 % (P &amp;lt; 0.05). However, JAG2 gene promoter was estimated to be methylated at a negligible level (&amp;lt;2 %) (P &amp;lt; 0.05). Our study showed poor gene expression and high-level methylation of Notch4 gene, suggesting a possibility of epigenetic silencing. However, low JAG2 gene expression despite not being highly methylated suggests lack of correlation between gene expression and promoter methylation and the presence of alternative regulatory mechanisms. This study for the first time provides gene expression and DNA methylation profiles of Notch pathway genes from GBM patient samples. We have identified genes whose expression may be regulated by epigenetic mechanisms and thus can be used as potential biomarkers. This study will eventually give clues to improve diagnosis, prognosis, and treatment of GBM. Citation Format: Madhuri G S Aithal, Rajeswari Narayanappa. Role of promoter methylation in regulation of Notch4 and JAG2 gene expression in human glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A177.

Hypermethylated ZNF582 and PAX1 genes in oral scrapings collected from cancer-adjacent normal oral mucosal sites are associated with aggressive progression and poor prognosis of oral cancer

ObjectiveThis study assessed whether hypermethylated ZNF582 and PAX1 genes in oral scrapings are correlated with the progression and prognosis of oral squamous cell carcinoma (OSCC). Materials and methodsMethylation levels of ZNF582 and PAX1 genes in oral scrapings, collected from the cancer and adjacent normal oral mucosal sites of 80 OSCC patients before surgical cancer excision, were quantified using real-time methylation-specific PCR after bisulfite conversion. ResultsBoth the mean methylation (M)-indices of ZNF582 and PAX1 genes in oral scrapings were significantly higher at the cancer sites than at the adjacent normal oral mucosal sites (both P < .001). In the oral scrapings collected from the adjacent normal oral mucosal sites, the higher M-index of methylated ZNF582 (ZNF582m) was significantly correlated with a more advanced clinical stage (P = .04). Moreover, the higher M-index of methylated PAX1 (PAX1m) was significantly related to larger tumor size (P = .046). When the 80 OSCC patients were classified based on gene methylation tests, using the oral scrapings collected from the adjacent normal oral mucosal sites, we found a significantly shorter 3-year overall survival in ZNF582m-positive, PAX1m-positive, and ZNF582m/PAX1m-positive OSCC patients than in ZNF582m-negative (P = .02), PAX1m-negative (P = .04), and ZNF582m/PAX1m-negative OSCC patients (P = .02), respectively. Multivariate Cox regression analyses identified ZNF582m and ZNF582m/PAX1m as independent unfavorable prognostic factors. ConclusionHypermethylated ZNF582 and PAX1 genes in the oral scrapings collected from adjacent normal oral mucosal sites rather than cancer sites are associated with aggressive progression and poor prognosis of OSCC.

Hypermethylation of BEND5 contributes to cell proliferation and is a prognostic marker of colorectal cancer.

Aberrant hypermethylation of CpG islands in tumor suppressor genes (TSGs) contributes to colorectal tumorigenesis. To identify new colorectal cancer (CRC) screening marker, we investigated DNA methylation alterations in novel TSGs. Using HumanMethylation450 BeadChip arrays, CpG regions in BEND5 were the most highly methylated among all genomic regions in 26 colorectal tumors compared to paired non-neoplastic tissues from a Taiwan cohort. Therefore, BEND5 was selected for further analysis. Quantitative methylation-specific real-time PCR revealed that 86.7% (117/135) of CRC patients exhibited hypermethylated BEND5. Real-time reverse transcription PCR identified that BEND5 mRNA expression was downregulated in 68% (32/47) of the analyzed samples. BEND5 hypermethylation was associated with poor overall survival (OS) in Taiwan patients with early-stage CRC (P = 0.037). In a CRC tissue set from South Korea, OS was higher in patients with high BEND5 protein expression than in those with low BEND5 protein expression (P = 0.037) by using immunohistochemistry assays. Consistently, BEND5 hypermethylation was associated with poor OS in patients with early-stage CRC in The Cancer Genome Atlas (TCGA) data set (P = 0.003). Multivariate Cox proportional hazards regression analysis further supported that hypermethylation of BEND5 genes was significantly associated with OS in Taiwan and TCGA CRC patients (P = 0.023 and 0.033, respectively). Finally, the cell model assay with transient transfection of BEND5 or si-BEND5 knockdown indicated that BEND5 inhibited cancer cell proliferation. In conclusion, epigenetic alteration in the candidate TSG BEND5 contributes to colorectal cancer development and is a prognostic marker of CRC.

Silencing of G0/G1 switch gene 2 in cutaneous squamous cell carcinoma.

BackgroundMethylation of a CpG island (CGI; a dense cluster of CpGs) located in the 5' region of a gene suppresses that gene's transcription. The expression of G0/G1 switch gene 2 (G0S2) is potentially associated with tumorigenesis. The aim of this study is to elucidate the methylation status of the CGI located in the 5' region of G0S2 (hereinafter called 5' G0S2 CGI) in cutaneous squamous cell carcinoma (SCC).MethodsQuantitative real-time methylation-specific PCR (RT-MSP) and bisulfite sequencing were performed to evaluate the methylation statuses of cutaneous SCC and normal epithelial cell samples. Quantitative real-time reverse transcription-PCR was performed to evaluate RNA expression levels. Immunohistochemical analysis was performed to detect protein expression.ResultsG0S2 was suppressed in the five SCC cell lines with 5' G0S2 CGI methylation levels of nearly 100.0% and was expressed in the two normal cultured keratinocytes with methylation levels of almost 0.0%. G0S2 was re-expressed in SCC cell lines treated with a demethylating agent. The in vivo methylation levels of 5' G0S2 CGI as determined by RT-MSP varied widely (0.0% to 77.7%) in 17 cutaneous SCC samples and narrowly (0.1% to 7.3%) in 6 normal epidermis samples. Nine cutaneous SCC samples exhibited higher methylation levels than the highest methylation level (7.3%) of the 6 normal epidermis samples. Bisulfite sequencing showed dense methylated CpG sites within 5' G0S2 CGI in these highly methylated cutaneous SCC samples. The methylation levels of the cutaneous SCC samples did not correlate with any clinical parameters investigated or with histopathological grading.ConclusionsG0S2 is silenced by aberrant DNA methylation in a subset of cutaneous SCCs.

Performance of a methylation specific real-time PCR assay as a triage test for HPV-positive women

BackgroundHPV DNA testing as a primary screening marker is being implemented in several countries. Due to the high HPV prevalence in the screening population, effective triage strategies for HPV-positive cases are required. The aim of this study was to evaluate the performance of a methylation-specific real-time PCR assay (GynTect®) comprising six marker regions as a triage test.ResultsAn analytical sensitivity of 0.1 ng genomic DNA corresponding to 15 SiHa cells was achieved. Absolute specificity was observed in the presence of 20 ng unmethylated genomic DNA. In a clinical setting, cervical scrapes of 306 women showing abnormal colposcopy were tested for cytology, HPV positivity, and the GynTect markers ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671. Of all women, histopathological data were available. The overall sensitivity for GynTect to detect CIN3+ was 67.7% (95% CI 57.3%–77.1%) whereas sensitivity was significantly higher for women of age ≥ 30 years (p = 0.04). All cancer cases (n = 5) were detected by GynTect. The overall false positive rate (= 1-specificity) for women with no CIN was 17.4% (95% CI 12.5–23.1%), with a higher proportion among HPV-positive women (24.0%, 95% CI 16.0–33.6%). In a triage screening setting, where all women underwent HPV testing and the HPV positives in addition GynTect testing, the overall sensitivity would slightly decline but specificity would reach the maximum value of 88.7% (95% CI 83.7–92.6%).ConclusionThe GynTect® assay is a robust easy to use assay with high analytical sensitivity and specificity. Moreover, the performance of the assay based on cervical scrapes provides further evidence for the usefulness of methylation markers to detect HPV-positive women with clinically relevant disease.

Hypermethylated ZNF582 and PAX1 genes in mouth rinse samples as biomarkers for oral dysplasia and oral cancer detection.

Effective biomarkers for oral cancer screening are important for early diagnosis and treatment of oral cancer. Oral epithelial cell samples collected by mouth rinse were obtained from 65 normal control subjects, 108 patients with oral potentially malignant disorders, and 94 patients with oral squamous cell carcinoma (OSCC). Methylation levels of zinc-finger protein 582 (ZNF582) and paired-box 1 (PAX1) genes were quantified by real-time methylation-specific polymerase chain reaction after bisulfite conversion. An abrupt increase in methylated ZNF582 (ZNF582m ) and PAX1 (PAX1m ) levels and positive rates from mild dysplasia to moderate/severe dysplasia, indicating that both ZNF582m and PAX1m are effective biomarkers for differentiating moderate dysplasia or worse (MODY+) oral lesions. When ZNF582m /PAX1m tests were used for identifying MODY+ oral lesions, the sensitivity, specificity, and odds ratio (OR) were 0.65/0.64, 0.75/0.82, and 5.6/8.0, respectively. Hypermethylated ZNF582 and PAX1 genes in oral epithelial cells collected by mouth rinse are effective biomarkers for the detection of oral dysplasia and oral cancer.