qMSP Assays Research Articles

Apoptotic effect of melatonin on ER-positive breast cancer cell lines: ADGRL4 gene expression and promoter methylation.

Breast cancer (BC) is the second most common malignancy worldwide. ADGRL4, as a modulator of angiogenesis, undergoes various epigenetic modifications affecting its biological functions. In this study, we aimed to assess ADGRL4 promoter methylation status and its expression levels in primary breast tumors and to evaluate its potency as a plausible prognostic biomarker in BC. Furthermore, we evaluated the effect of melatonin on ADGRL4 expression and viability of BC cells in vitro. One hundred breast tumor tissue samples and adjacent non-tumor tissues were collected, followed by DNA isolation, bisulfite conversion, qRT-PCR, qMSP assay, and immunoblotting. In addition, four BC cell lines were treated with melatonin and subjected to ADGRL4 expression analysis and apoptosis assay. We found a significant correlation between ADGRL4 expression levels and HER2 status and stage of disease (P < 0.05). We observed a substantial attenuation in ADGRL4 promoter methylation in tumor samples compared to marginal non-tumor samples. A significantly lower expression of ADGRL4 was detected in two BC cell lines in the presence of melatonin. MCF-7 and BT474 melatonin-treated cell lines showed a significantly higher number of apoptotic cells than non-treated cells (P < 0.0001). Based on the receiver operating characteristic (ROC) curve analysis, ADGRL4 expression and ADGRL4 promoter methylation status showed moderate prognostic value. We found that melatonin has anti-cancer effects on BC cells. In addition, ADGRL4 expression can potentially be used as a prognostic biomarker in BC.

A novel blood-based epigenetic biosignature in first-episode schizophrenia patients through automated machine learning

Schizophrenia (SCZ) is a chronic, severe, and complex psychiatric disorder that affects all aspects of personal functioning. While SCZ has a very strong biological component, there are still no objective diagnostic tests. Lately, special attention has been given to epigenetic biomarkers in SCZ. In this study, we introduce a three-step, automated machine learning (AutoML)-based, data-driven, biomarker discovery pipeline approach, using genome-wide DNA methylation datasets and laboratory validation, to deliver a highly performing, blood-based epigenetic biosignature of diagnostic clinical value in SCZ. Publicly available blood methylomes from SCZ patients and healthy individuals were analyzed via AutoML, to identify SCZ-specific biomarkers. The methylation of the identified genes was then analyzed by targeted qMSP assays in blood gDNA of 30 first-episode drug-naïve SCZ patients and 30 healthy controls (CTRL). Finally, AutoML was used to produce an optimized disease-specific biosignature based on patient methylation data combined with demographics. AutoML identified a SCZ-specific set of novel gene methylation biomarkers including IGF2BP1, CENPI, and PSME4. Functional analysis investigated correlations with SCZ pathology. Methylation levels of IGF2BP1 and PSME4, but not CENPI were found to differ, IGF2BP1 being higher and PSME4 lower in the SCZ group as compared to the CTRL group. Additional AutoML classification analysis of our experimental patient data led to a five-feature biosignature including all three genes, as well as age and sex, that discriminated SCZ patients from healthy individuals [AUC 0.755 (0.636, 0.862) and average precision 0.758 (0.690, 0.825)]. In conclusion, this three-step pipeline enabled the discovery of three novel genes and an epigenetic biosignature bearing potential value as promising SCZ blood-based diagnostics.

Early detection and prognosis evaluation for hepatocellular carcinoma by circulating tumour DNA methylation: A multicentre cohort study.

Early diagnosis of hepatocellular carcinoma (HCC) can significantly improve patient survival. We aimed to develop a blood-based assay to aid in the diagnosis, detection and prognostic evaluation of HCC. A three-phase multicentre study was conducted to screen, optimise and validate HCC-specific differentially methylated regions (DMRs) using next-generation sequencing and quantitative methylation-specific PCR (qMSP). Genome-wide methylation profiling was conducted to identify DMRs distinguishing HCC tumours from peritumoural tissues and healthy plasmas. The twenty most effective DMRs were verified and incorporated into a multilocus qMSP assay (HepaAiQ). The HepaAiQ model was trained to separate 293 HCC patients (Barcelona Clinic Liver Cancer (BCLC) stage 0/A, 224) from 266 controls including chronic hepatitis B (CHB) or liver cirrhosis (LC) (CHB/LC, 96), benign hepatic lesions (BHL, 23), and healthy controls (HC, 147). The model achieved an area under the curve (AUC) of 0.944 with a sensitivity of 86.0% in HCC and a specificity of 92.1% in controls. Blind validation of the HepaAiQ model in a cohort of 523 participants resulted in an AUC of 0.940 with a sensitivity of 84.4% in 205 HCC cases (BCLC stage 0/A, 167) and a specificity of 90.3% in 318 controls (CHB/LC, 100; BHL, 102; HC, 116). When evaluated in an independent test set, the HepaAiQ model exhibited a sensitivity of 70.8% in 65 HCC patients at BCLC stage 0/A and a specificity of 89.5% in 124 patients with CHB/LC. Moreover, HepaAiQ model was assessed in paired pre- and postoperative plasma samples from 103 HCC patients and correlated with 2-year patient outcomes. Patients with high postoperative HepaAiQ score showed a higher recurrence risk (Hazard ratio, 3.33, p<.001). HepaAiQ, a noninvasive qMSP assay, was developed to accurately measure HCC-specific DMRs and shows great potential for the diagnosis, detection and prognosis of HCC, benefiting at-risk populations.

Promoter of lncRNA MORT is aberrantly methylated in colorectal cancer

Aberrant DNA methylation plays essential roles in the colorectal cancer (CRC) carcinogenesis and has been demonstrated as a promising marker for cancer early detection. In this project, methylation status of the MORT promoter was studied in CRC and their marginal tissues using qMSP assay. Furthermore, we investigated the molecular function of MORT in CRC progression using computational analysis. The results showed a high methylation level of MORT promoter in CRC tissues. By in silico analysis, we found that MORT downregulation could promote the proliferation of CRC cells via sponging of has-miR-574-5p and has-miR-31-5p, and alteration of their targets expression pattern such as MYOCD and FOXP2. In conclusion, based on our results, promoter hypermethylation of MORT might be considered as a potential biomarker for CRC detection.

DNA Hypomethylation in the TNF-Alpha Gene Predicts Rheumatoid Arthritis Classification in Patients with Early Inflammatory Symptoms.

DNA-methylation data were screened to select candidate CpGs to design qMSP assays. Eight assays were developed and tested on two early inflammatory arthritis cohorts. Logistic regression and bootstrapping were used to demonstrate the added value of the qMSP assays. Differentially methylated CpG data were screened for candidate CpG, thereby meeting the qMSP assay requirements. The top CpG candidate was in the TNF gene, for which we successfully developed a qMSP assay. Significantly lower DNA-methylation levels were observed in RA (p < 4 × 10-9), with a high predictive value (OR < 0.54/AUC < 0.198) in both cohorts (n = 127/n = 157). Regression using both datasets showed improved accuracy = 87.7% and AUC = 0.944 over the model using only clinical variables (accuracy = 85.2%, AUC = 0.917). Similar data were obtained in ACPA-negative patients (n = 167, accuracy = 82.6%, AUC = 0.930) compared to the clinical variable model (accuracy = 79.5%, AUC = 0.892). Bootstrapping using 2000 datasets confirmed that the AUCs for the clinical+TNF-qMSP model had significant added value in both analyses. The qMSP technology is robust and can successfully be developed with a high specificity of the TNF qMSP assay for RA in patients with early inflammatory arthritis. It should assist classification in ACPA-negative patients, providing a means of reducing time to diagnosis and treatment.

Genotoxicity and hypomethylation of LINE-1 induced by electronic cigarettes

Currently, the marketing of electronic cigarettes as a safe alternative to smoking has increased, which is associated with greater use of these devices, especially among young people and smokers interested in quitting tobacco cigarettes. Given the growing use of this type of product, there is a need to determine the consequences of electronic cigarettes on human health, especially since many of the compounds contained in the aerosol and liquid of these devices have a high potential to be carcinogenic and genotoxic. Additionally, many of these compounds' aerosol concentrations exceed the safe limits. We have evaluated the levels of genotoxicity and changes in DNA methylation patterns associated with vaping. We analyzed a total of 90 peripheral blood samples from a population of vapers (n = 32), smokers (n = 18), and controls (n = 32), in which the frequencies of genotoxicity were determined by the cytokinesis-blocking micronuclei (CBMN) assay and the patterns of methylation of the repetitive elements of LINE-1 through the Quantitative Methylation Specific PCR (qMSP) assay. Here we show an increase in genotoxicity levels associated with vaping habits. Additionally, the group of vapers showed changes at the epigenetic level specifically associated with the loss of methylation of the LINE-1 elements. These changes in LINE-1 methylation patterns were reflected in its representative RNA expression detected in vapers.

Circulating tumour cell combined with DNA methylation for early detection of hepatocellular carcinoma.

Background: The inadequate early detection strategies makes hepatocellular carcinoma (HCC) patients with poor prognisis. Therefore, more effective detection methods are urgently needed for early detection and early intervention of HCC. Methods: 17 cases of suspected HCC patients and 11 cases of HBV-related decompensated cirrhosis (HBV-DeCi) patients were enrolled. For each patient, 5ml blood sample was separated into circulating tumor cells (CTCs) and plasma, CTCs were stained with Diff staining for counting. Plasma was used for extracting cell free DNA (cfDNA) and then analyzed by qMSP assay. Ct values were recorded for GNB4 and Riplet as target genes and β-actin as an endogenous reference gene. Finally, clinical efficacy of CTC count combined with GNB4/Riplet methylation detection for early diagnosis of HCC was analyzed. Results: The CTC of HCC patients has pleomorphic characteristics, but it is difficult to distinguish from other blood cells with non-obviously pleomorphic of CTC. Although a small number of CTCs can also be detected in HBV-DeCi patients (control group), the number is significantly lower than that in HCC patients, the sensitivity and specificity of CTC for HCC detection were 70.6% and 90.9% (AUC = 0.81). The Ct values of GNB4 and Riplet methylation were significantly different between HCC patients and control group patients. When CTC combined with two genes, the AUC value was significantly increased to 0.98, the sensitivity was 88.2%, and the specificity was 100%. Conclusion: Our study has developed a novel test that CTC count combined with GNB4/Riplet methylation detection and showed its high performance for early diagnosis of HCC.

Aberrant expression of lncRNAs SNHG6, TRPM2-AS1, MIR4435-2HG, and hypomethylation of TRPM2-AS1 promoter in colorectal cancer.

Accumulating evidence has indicated that deregulation of lncRNAs plays essential roles in colorectal cancer (CRC) carcinogenesis. The goal of this study was to analyze the expression of lncRNAs in colorectal cancer and their association with clinicopathological variables. Bioinformatics analysis of published CRC microarray data was performed to identify the important lncRNAs. The expression levels of candidate genes were assessed in the human colon cancer/normal cell lines, CRC, adenomatous colorectal polyps, and their marginal tissues by qRT-PCR. Moreover, the methylation status of the TRPM2-AS1 promoter was studied using qMSP assay. Furthermore, we investigated the molecular mechanisms of these lncRNAs in CRC progression using in silico analysis. Microarray analysis revealed that lncRNAs SNHG6, MIR4435-2HG, and TRPM2-AS1 were upregulated in CRC. These results were validated in colon cell lines. Moreover, qRT-PCR showed that the expression levels of SNHG6 and TRPM2-AS1 were upregulated in the colorectal tumor tissues compared with their paired tissues. Nonetheless, there was no significant increase in MIR4435-2HG expression in CRC samples. Furthermore, we observed a significant hypomethylation of TRPM2-AS1 promoter and its activation in CRC tissues. By in silico analysis, we found that the lncRNAs upregulation could promote proliferation and drug resistance of colorectal cancer cells via miRNAs sponging and modulation of their targets expression. In conclusion, based on our results upregulation of SNHG6 and TRPM2-AS1, and hypomethylation of TRPM2-AS1 promoter might be considered as potential diagnostic biomarkers for CRC initiation and development.

Melatonin Suppresses ADGRL 4 Expression and Induces Promoter Methylation in Estrogen‐Responsive Breast Cancer Cells

Background Breast cancer (BC) is the second most common malignancy worldwide. Angiogenesis plays a significant role during tumor formation and progression. ADGRL4, as a modulator of angiogenesis, undergoes various epigenetic modifications affecting its biological functions. Here, we aimed to assess ADGRL4 promoter methylation and its expression level in breast tumors and to examine the effect of melatonin on ADGRL4 expression and promoter methylation in vitro. Materials and methods Fifty breast tumor and corresponding adjacent non-tumor tissue samples were collected, followed by DNA isolation, bisulfite conversion, qRT-PCR, qMSP assay and immunoblotting. Then, the effect of melatonin on the ADGRL4 expression and promoter methylation was investigated in two BC cell lines (MCF-7 and MDA-MB-231). Apoptosis assay was performed using annexin v and flow cytometry. Results ADGRL4 was overexpressed in tumors in comparison with normal tissue samples (p<0.0001). We found a significant correlation between ADGRL4 expression level and increased HER2 expression (p=0.021). We observed a significant decrease in ADGRL4 promoter methylation level in tumors as compared to marginal non-tumor samples (p<0.0001). Melatonin treatment significantly attenuated ADGRL4 expression (p<0.0001) and elevated promoter methylation (p<0.001) in MCF-7 cells, but not in MDA-MB-231 cell line. However, melatonin treatment induced apoptosis in both cell lines. Conclusion ADGRL4 was overexpressed and its promotor was hypomethylated in breast tumors. Melatonin treatment attenuated ADGRL4 expression only in estrogen-responsive BC cells (MCF-7) and induced apoptosis in BC cells.

P148 Differential methylation of the TNF gene promoter, potential for the development of a diagnostic biomarker for RA

Abstract Background/Aims Diagnosing rheumatoid arthritis (RA) early is difficult despite the use of anti-citrullinated protein antibodies in the new EULAR-2010 classification criteria, particularly for antibody-negative patients. Alterations in epigenetic patterns have been associated with RA CD4+T-cells at the TNF gene locus (1). The second aim of my PhD project was to select a CpG site to develop a quantitative methylation sensitive PCR (qMSP) assay as a diagnostic biomarker test for RA classification. Methods Patients attending an early arthritis clinic were used (with full ethical approval). An 450K Illumina methylation genome-wide dataset analysing DNA from CD4+T-cells identified CpG on TNF promoter region as a differentially methylated CpG between controls and RA patients (1). A qMSP assay was designed using a TaqMan approach (primers and probe), using a reference gene (GAPHD) for normalisation. The assay was validated in DNA extracted from PBMC. The performance of the assay for RA classification was accessed using binary logistic regression. Results We noted differences in levels of methylation in CD4+T-cells in RA, notably affecting the TNF gene promoter in about 15% of cells. We validated this using bisulphite sequencing showing about 30% of cells with differential methylation in early RA compared to controls. This allowed us to select cg11484872, cg21370522, and cg01569083 as candidates. We optimised a qMSP assay using fully methylated/un-methylated DNA. PBMC samples were retrieved from our tissue bank for 65 early RA, and 64 non-RA patients (including 11 reactive arthritis, 37 undifferentiated arthritis, and 16 psoriatic arthritis). The methylation levels (%) detected by the TNF-qMSP assay were significantly lower in RA (Δ-DM =-10.28%, p = 7x10-7). The loss of TNF promoter gene methylation was associated with RA classification (p &amp;lt; 0.0001, unadjusted OR = 0.900 (95% CI: 0.85-0.94)) with a good classification performance (AUROC = 0.76 (95% CI: 0.67, 0.84)). An adjusted regression model using the demographic and clinical parameters associated with RA classification in this group of patients (age, smoking, counts for tender and swollen joints) showed that adding TNF to the clinical model improved the model’s performance (increasing AUROC from 0.86 , to 0.91). Comparing classification accuracy, adding the TNF-qMSP resulted in an increased correct classification of 5.7 % compared to the clinical model. A similar result was obtained in the antibody negative patients, with TNF-qMSP assay improving the model’s classification from AUROC 0.91 to 0.96 and 3.3% more patients correctly classified. Conclusion The loss of DNA methylation on the TNF gene promoter identified in early RA patients provided a valuable target for a biomarker assay development. The results will need to be confirmed in a larger cohort but the TNF-qMSP assay offers good opportunities for RA versus non-RA classification particular in antibody negative disease. Disclosure R. Pitaksalee: Grants/research support; ROYAL THAI GOVERNMENT SCHOLARSHIP. P. Emery: None. R. Hodgett: None. F. Ponchel: None.

Comparative CpG methylation kinetic patterns of cis-regulatory regions of heat stress-related genes in Sahiwal and Frieswal cattle upon persistent heat stress.

The kinetic patterns of CpG methylation of the cis-regulatory region of heat stress-related genes on exposed to heat stress (at 42 °C) between the Sahiwal and Frieswal cattle was compared in the present study. Using an in vitro whole blood culture model, cells were continuously exposed to heat stress (at 42 °C) for 6 h. Methylation levels of five genes, viz., GPX1, HSP70, HSP90, c-FOS, and JUN were estimated by SyberGreen-based quantitative methylation-specific PCR (qMSP) assay. CpG methylation kinetics at different time points of heat stress (0.5, 1, 2, 4, 6 h) were analyzed using mixed ANOVA. The initial methylation level, estimated at 37 °C, of HSP70 was significantly high in the Sahiwal breed. A significant (p<0.001) time-dependent hypomethylation of an antioxidant gene (GPX1) CpG islands was detected at the acute phase of the stress. Heat shock protein gene (HSP70) showed a similar CpG methylation kinetics where the hypomethylation was prominent from 1 h and persisted up to 4 h. The heat stress responses of both Sahiwal and Frieswal cattle were identical as there was no distinctiveness in the methylation kinetics of CpG islands of studied genes. The acclimatization of Frieswal cattle-a breed developed in India over the years to the tropical climatic conditions, maybe one of the reasons for this similarity. Thus, the present study results could pave a path to understand the molecular mechanism of heat stress and adaptation of indigenous and crossbred cattle populations to the changing scenario in tropical climate conditions.

A Sensitive and Simplified Classifier of Cervical Lesions Based on a Methylation-Specific PCR Assay: A Chinese Cohort Study.

ObjectiveThe aim of this study is to assess the diagnostic and screening performance of a standardized methylation-specific real-time PCR assay targeting SOX1 and PAX1 genes for cervical cancer in a Chinese cohort.MethodsGenomic DNA was extracted from cervical exfoliated cells and converted by sodium bisulfite and then analyzed by qMSP assay. Ct values were collected for PAX1 and SOX1 as target genes and β-actin as an endogenous reference gene. The samples included 295 cervicitis, 111 LSIL (low-grade squamous intraepithelial lesion), 51 HSIL (high-grade squamous intraepithelial lesion) and 30 cervical cancer.ResultsThe Ct values decreased with the progression of cervical cancer from cervicitis, through LSIL and HSIL to cancer. The difference in Ct values between cytological grades was highly significant (p≤0.01) between grades either for PAX1 or for SOX1 except the difference between cervicitis and LSIL of SOX1. With the Ct cut-off values of PAX1 gene and SOX1 gene 38.6 and 38 and with the PAX1/SOX1 in combination, the positive rate of methylation in invasive cancer tissues was 100%, in contrast to 11.5% (95% CI: 8.67%–14.33%) in cervicitis tissues, 45.1% (95% CI: 40.68%–49.52%) in LSIL tissues, and 68.5% (95% CI: 64.37%–72.63%) in HSIL tissues. The specificity and sensitivity of differentiating tumors from cervicitis were 0.957 (95% CI: 0.939–0.975) and 1.00, respectively. The specificity and sensitivity of differentiation between cervicitis+LSIL and HSIL+cervical cancer were 0.881 (95% CI: 0.852–0.91) and 0.748 (95% CI: 0.709–0.787), respectively.ConclusionPAX1/SOX1 methylation could be translated into clinical practice for cervical neoplasia detection.

Methylomic Analysis of Ovarian Cancers Identifies Tumor-Specific Alterations Readily Detectable in Early Precursor Lesions.

High-grade serous ovarian carcinoma (HGSOC) typically remains undiagnosed until advanced stages when peritoneal dissemination has already occurred. Here, we sought to identify HGSOC-specific alterations in DNA methylation and assess their potential to provide sensitive and specific detection of HGSOC at its earliest stages. MethylationEPIC genome-wide methylation analysis was performed on a discovery cohort comprising 23 HGSOC, 37 non-HGSOC malignant, and 36 histologically unremarkable gynecologic tissue samples. The resulting data were processed using selective bioinformatic criteria to identify regions of high-confidence HGSOC-specific differential methylation. Quantitative methylation-specific real-time PCR (qMSP) assays were then developed for 8 of the top-performing regions and analytically validated in a cohort of 90 tissue samples. Lastly, qMSP assays were used to assess and compare methylation in 30 laser-capture microdissected (LCM) fallopian tube epithelia samples obtained from cancer-free and serous tubal intraepithelial carcinoma (STIC) positive women. Bioinformatic selection identified 91 regions of robust, HGSOC-specific hypermethylation, 23 of which exhibited an area under the receiver-operator curve (AUC) value ≥ 0.9 in the discovery cohort. Seven of 8 top-performing regions demonstrated AUC values between 0.838 and 0.968 when analytically validated by qMSP in a 90-patient cohort. A panel of the 3 top-performing genes (c17orf64, IRX2, and TUBB6) was able to perfectly discriminate HGSOC (AUC 1.0). Hypermethylation within these loci was found exclusively in LCM fallopian tube epithelia from women with STIC lesions, but not in cancer-free fallopian tubes. A panel of methylation biomarkers can be used to accurately identify HGSOC, even at precursor stages of the disease.

Comparative assessment of three methods to analyze MGMT methylation status in a series of 350 gliomas and gangliogliomas

MGMT promoter methylation is considered as a prognostic and predictive biomarker indicating response to chemotherapy and radiotherapy in glioblastoma. A number of different methods and platforms including pyrosequencing (PSQ), quantitative methylation-specific PCR (qMSP) and immunohistochemistry (IHC), methylation-sensitive high resolution melting (MS-HRM) and NGS (Next Generation Sequencing) have been used to detect MGMT promoter methylation in gliomas. However, controversy remains about the most appropriate method to use for analyzing MGMT status. The MGMT promoter methylation status of a total of 350 gliomas and gangliogliomas was examined using PSQ, qMSP and IHC in parallel. Using PSQ as a recommended standard method, the sensitivity, specificity, positive/negative predictive value and correlation with the other assays were calculated. Among 350 glioma and ganglioglioma cases, the MGMT promoter tested positive for methylation in 53.1%, 55.4%, and 70.3% of the cases by PSQ, qMSP and IHC, respectively. The sensitivity and specificity of qMSP were 97.8% and 92.7%, respectively. Twelve cases that tested positive for methylation using qMSP were negative according to PSQ, and four cases that were negative according to qMSP tested positive according to PSQ. The concordance rate between PSQ and qMSP was 90.8%. The sensitivity and specificity of IHC for the detection of MGMT at the protein level were 84.4% and 45.7%, respectively. The concordance rate between PSQ and IHC was 30.8%.This study demonstrated that qMSP is an effective and rapid detection method for routine use in pathology laboratories for the identification of MGMT promoter methylation. A combination of IHC and qMSP assays can provide high sensitivity and specificity for the prediction of MGMT status. A few cases that tested negative with PSQ did harbor MGMT promoter methylation, as confirmed by qMSP and sequencing, and this subgroup of patients may benefit from temozolomide.

Abstract 4666: DREAMing as a simple and low cost alternative for the assessment of methylation in ultra rare DNA

Abstract Background: Current approaches for the assessment of methylation, such as methylation-specific PCR (MSP) and next-generation bisulfite sequencing (BS-Seq) are fundamentally limited in their ability to detect and assess heterogeneous methylation patterns (epialleles) in ultra-rare (&amp;lt;0.1%) DNA. These limitations critically compromise diagnostic utility and render them ill suited for many emerging applications in cancer diagnostics, such as the analysis of methylation heterogeneity in cell-free DNA (cfDNA) and rare cell populations. We recently addressed the need for a low cost alternative to the assessment of methylation of ultra-rare DNA with the development of DREAMing (Discrimination of Rare EpiAlleles by Melt), which uses semi-limiting dilution and precise melt curve analysis to distinguish and enumerate individual copies of DNA at single copy sensitivity and single-CpG-site resolution. Here, we seek to demonstrate the advantages of the DREAMing method over conventional approaches to methylation assessment. Methods: We expand upon the underlying theory of DREAMing and provide guidelines for the development of single-copy sensitive DREAMing assays. We further elucidate methods for tailoring DREAMing assays to samples of interest and compare the performance of these assays to commonly employed techniques including quantitative MSP (qMSP) and BS-Seq. Results: Development of single-copy sensitive DREAMing assays for a number of loci associated with classic tumor-specific methylation such as CHFR and RASSF1A as well as a candidate pan-cancer locus are reported. These assays are then used to analyze methylation in cfDNA derived from the plasma of cancer-positive and healthy patients. DREAM analysis reveals that DREAMing can readily detect over an order of magnitude more epialleles when directly compared to qMSP and BS-Seq assays of the same locus. Some of the challenges associated with distinguishing potential tumor-specific aberrant methylation from background methylation are then discussed and proposed solutions are demonstrated. Lastly, methods for optimizing DREAMing assays for specific sample types are discussed. Conclusions: DREAMing is a recently introduced method for the assessment of locus-specific methylation in samples containing ultra rare target DNA. Its low cost and simplicity coupled with the ability to provide enhanced, single-copy detection of heterogeneous methylation make DREAMing an attractive option over traditional techniques for demanding specimens such as cfDNA and rare cell populations. DREAMing has potential utility in the evaluation of DNA methylation dynamics in cell populations, prenatal testing, as well as clear use in early cancer diagnostic, companion diagnostic and predictive applications. Citation Format: Thomas R. Pisanic, Pornpat Athamanolap, Brendan F. Miller, Vincent Wu, Laura Elnitski, Tza-Huei Wang. DREAMing as a simple and low cost alternative for the assessment of methylation in ultra rare DNA [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 4666. doi:10.1158/1538-7445.AM2017-4666

Abstract 3353: An epigenetic biomarker panel in HPV related oropharyngeal squamous cell carcinoma

Abstract HPV related head and neck squamous cell carcinoma (HNSCC) is known to occur in a separate patient population in comparison to tobacco related HNSCC, and also exhibits less mutational, genetic, and chromosomal alterations, and a different composition of epigenetic alterations. In particular, HPV is present in up to 70% of oropharyngeal squamous cell carcinoma (OPSCC), and is driving the increase in incidence rates of OPSCC. Given the precedent for successful screening for cervical cancer, it is attractive to consider similar population based screening for HPV related OPSCC. Here, we developed an epigenetic biomarker panel that can be combined with high risk HPV DNA detection with potential utility in screening. We performed high throughput qMBD-seq analysis on 50 primary HPV positive OPSCC samples and 25 UPPP normal samples. Subsequently, we performed an outlier analysis and ranking strategy of 100 bp segmented regions within this discovery cohort and defined 20 differentially methylated regions to be used as biomarkers for detection. Simultaneously, we validated these 20 biomarker candidates using TCGA methylation array dataset and found that the methylation levels of these 20 candidates in HPV positive OPSCC were significantly higher than those in normal samples. Moreover, we created QMSP assays for these genes, and validated EMBP1 and KCNA3 in a separate validation cohort including tissue and saliva from 46 HPV related OPSCC and 25 normal controls. The sensitivity and specificity values for EMBP1, KCNA3 or both in tissue and saliva are shown. EMBP1 has a high sensitivity in tissue and a high specificity in saliva, while KCNA3 has a high specificity in both tissue and saliva. A panel including both genes has an extremely high sensitivity. Ultimately, we defined a panel of two differentially methylation regions that has a high sensitivity in tissue. This study might serve as a diagnostic biomarker development trial for HPV OPSCC detection with potential application for development of a population based screening test. Sensitivity and specificity for HPV related OPSCC in tissue and salivaTissueTissueSalivaSalivaGeneSensitivitySpecificitySensitivitySpecificityEMBP197.7%83.3%13.3%94.7%KCNA376.7%92.0%13.3%94.7%EMBP1&amp; KCNA3100.0%84.0%13.0%84.2% Citation Format: Shuling Ren, Daria A. Gaykalova, Theresa Guo, Zubair Khan, Mizuo Ando, Sunny Haft, Joseph A. Califano. An epigenetic biomarker panel in HPV related oropharyngeal squamous cell carcinoma [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 3353. doi:10.1158/1538-7445.AM2017-3353

Abstract 499: ESR1 methylation in circulating tumor cells of patients with breast cancer

Abstract AIMS: DNA methylation is an epigenetic alteration which plays a decisive role in the regulation of signal translation processes. In our lab, we have demonstrated for the first time the epigenetic silencing of tumor and metastasis suppressor genes in CTCs through their promoter methylation. Estrogen receptor (ER) is an important prognostic biomarker and is predictive of response to endocrine therapy in breast cancer. In this study, we evaluated for the first time ESR1 methylation in CTCs of breast cancer patients. METHODS: We developed and validated a novel highly sensitive and specific qMSP assay for ESR1 methylation using commercially available DNA methylation controls and the MDA-MB-231 cell line. We further examined its performance in EpCAM-positive immune-magnetically isolated CTC fractions, followed by DNA isolation and sodium bisulfite (SB) treatment from: a) 74 operable, b) 48 metastasis- verified breast cancer patients and c) 30 healthy donors (control group). RESULTS: The developed assay is highly specific and sensitive since it can detect 0.1% methylated ESR1 sequences in the presence of 99.9% un-methylated. ESR1 was found to be methylated in 16/74 (21.6%) operable breast cancer patients, in 10/48 (20.8%) patients with verified metastasis, but only in 1/30 (3.3%) healthy donors (EpCAM-positive CTC fraction). CONCLUSIONS: The EpCAM-positive CTC fraction was found to be methylated for ESR1 in about 20% of patients with breast cancer. We will further evaluate these findings in respect to the clinical outcome of these patients, since the epigenetic silencing of ESR1 could be of important clinical significance especially for its impact on the efficacy of treatment. Citation Format: Sofia Mastoraki, Areti Strati, Maria Chimonidou, Nikos S. Malamos, Vasilis Georgoulias, Evi S. Lianidou. ESR1 methylation in circulating tumor cells of patients with breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 499.

Diagnostic Value of Methylated Human Telomerase Reverse Transcriptase in Human Cancers: A Meta-Analysis.

Human telomerase reverse transcriptase (hTERT) plays a critical role in the pathogenesis of human malignancies. Overexpression of hTERT is essential in controlling the propagation of cancer cells. The CpG island located at hTERT promoter region is subjected to methylation modification in human cancer. In this perspective article, we discussed the diagnostic value of methylated hTERT in human cancers. The definitive diagnosis of most solid tumors is based on histological and immunohistochemical features. Under certain circumstances, however, the use of methylated hTERT might be useful in overcoming the limitation of the conventional methods. Methylated hTERT showed a good diagnostic power in discriminating cancer from benign or normal tissues. Nevertheless, differences in detection method, methylation site, cancer type, and histological subtype of cancer make it difficult to evaluate the actual diagnostic accuracy of methylated hTERT. Therefore, we performed subgroup analysis to assess the effects of these factors on the diagnostic efficiency of methylated hTERT. We demonstrated that quantitative MSP (qMSP) assay offers the highest discriminative power between normal and cancer in comparison with different detection methods. In addition, the methylated sites selected by different studies had an impact on the detection performance. Moreover, the diagnostic power of methylated hTERT was affected by cancer type and histological subtype. In conclusion, the existing evidence demonstrated that methylated hTERT is effective in cancer detection. Detailed profiling of the methylation sites to local the common methylation hotspot across human cancers is warranted to maximize the diagnostic value of methylated hTERT in cancer detection.

Abstract A1-50: Genomic and epigenomic alterations in human and high-risk HPV DNA can discriminate normal from cervical dysplasia patients in urine

Abstract Human Papilloma Virus (HPV) testing is increasingly used for cervical cancer screening in conjunction with cervical cytology. Although privacy, cultural, and infrastructure issues challenge the effective implementation of HPV testing for cervical cancer screening worldwide, several countries have already implemented HPV testing in their screening protocols. There are currently no tests that can reliably identify the patients with abnormal cytology and positive oncogenic HPV results (HPV+) that need to be referred for colposcopy. A useful triage test from cytology to colposcopy must discriminate the patients with a Cervical Intraepithelial Neoplasia (CIN) lesion that are more likely to progress to cervical cancer (CIN2+), from those that are less likely to progress. We set out to identify a panel of methylated HPV and human genes that can discriminate between CIN2+ and normal/CIN1 patients, first in cytology samples and then in urine samples. The Reverse Line Blot Assay identified high-risk HPV types in study participants from a Discovery cohort (n=96): 31 women with normal cervical cytology and 65 women with dysplasia (LSIL, n=43) and (HSIL, n=22). We developed custom sequence capture pools of baits to pull down genomic (Roche SeqCap EZ) and bisulfite converted high-risk HPV DNA (Agilent SureSelect) from urine, before library prep for NGS in a Roche 454 and MiSeq instruments, respectively. We also optimzed a qPCR assay to identify high risk HPV types, and quantitative Methylation Specific PCR (qMSP) assays to examine methylated HPV and human genes, in Pap smear DNA and TrDNA samples from women with normal Pap and women with CIN1, CIN2 and CIN3 lesions (n=80). The human genes selected for the qMSP assays, ZNF516, FKBP6, and INTS1, were identified in a previously published genome-wide analysis of the cevical cancer methylome using Nimblegen 385K CpG plus Promoters oligonucleotide tiling arrays. Presence of high-risk HPV in cervical epithelium correctly classified 37% of HSIL when compared with LSIL patients, with 61% sensitivity and 22% specificity. Promoter methylation of INTS1 correctly classified 67% (31% sensitivity, 92% specificity), promoter methylation of ZNF516 correctly classified 54% (11% sensitivity, 84% specificity), promoter methylation of FKBP6 correctly classified 59% (19% sensitivity, 88% specificity), and methylation of the HPVL1 gene correctly classified 61% (22% sensitivity, 90% specificity) of HSIL patients when compared with LSIL patients. A molecular panel comprised of HPV16-L1 methylation and promoter methylation of INTS1, ZNF516 and FKBP6 correctly classified 70% of HSIL patients with an Area Under the Curve of 0.66, 44% sensitivity, 88% specificity and 72% Positive Predictive value. While HPV16-L1 methylation discriminated patients with normal cytology from women with cervical dysplasia with close to 100% sensitivity and specificity, the high-risk HPV TrDNA qPCR assay had a pre-capture and post-capture sensitivity of 79% with 50% specificity. The detection of circulating HPV DNA in urine is a cost-effective and non-invasive alternative for cervical cancer screening. We have shown that a panel of genomic and epigenomic alterations in human and high risk HPV DNA can discriminate normal from cervical dysplasia in cervical epithelium DNA and TrDNA isolated from patients seen in a high risk cervical cancer screening setting. Citation Format: Rafael Guerrero-Preston, Anne Jedlicka, Oluwasina Folawiyo, Francesca Pirini, Blanca L. Valle, Fahcina Lawson, Angelo Vergura, Gabriela Perez, Marisa Renehan, Carolina Guerrero-Díaz, Liliana Florea, Teresa Díaz-Montes, Josefina Romaguera, David Sidransky. Genomic and epigenomic alterations in human and high-risk HPV DNA can discriminate normal from cervical dysplasia patients in urine. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-50.

Abstract 3837: Innovative rapid gene methylation analysis of surgical margin tissues in head and neck cancer

Abstract Background: To secure negative surgical margins is the primary goal of surgical cancer treatment. However, tumor recurrence sometimes occurs even with histologically negative surgical margins. In order to detect submicroscopic residual cancerous cells intra-operatively, a time saving molecular biological approach is required. Methods: We established an innovative rapid Quantitative methylation PCR (QMSP) assay, which consists of remarkably time-minimized DNA extraction, bisulfite treatment and QMSP quantification. In order to assess the feasibility of completing the rapid assay during a surgical procedure, 10 serial specimens of primary head and neck cancers were evaluated by both rapid QMSP and conventional QMSP. Results: The DNA product quality of the most time saving procedure was as good as that of a conventional procedure. In the QMSP assay, target gene methylation and reference gene methylation were equally detected by both rapid and conventional methods. Finally, two frequently methylated genes in head and neck cancers, homeobox A9 (HOXA9) and endothelin receptor type B (EDNRB) were analyzed in 10 head and neck tumors, margin tissues and normal tissues. Relative QMSP values of rapid and conventional QMSP were quite similar in almost all tumors, margins and normal tissues. The average total time for the rapid QMSP procedure was less than 3 hours. Although postoperative follow-up periods are less than a year, local tumor recurrence occurred in one case whose margin tissue was QMSP positive. Conclusion: This innovative rapid QMSP yields acceptable levels of methylation detection and can be completed during the operative window. The procedure promises to become a useful clinical tool, if the methylated surgical margins are revealed to correlate with the local tumor recurrence in a larger cohort. Citation Format: Masamichi Hayashi, Rafael Guerrero-Preston, Zubair Kahn, Xiufeng Li, Julie Ahn, Marla Goldsmith, Christina Michailidi, Wayne Koch. Innovative rapid gene methylation analysis of surgical margin tissues in head and neck cancer. [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 3837. doi:10.1158/1538-7445.AM2014-3837