Cumulative Methylation Index Research Articles

Evaluating DNA Methylation in Random Fine Needle Aspirates from the Breast to Inform Cancer Risk

Introduction. Critical regulatory genes are functionally silenced by DNA hypermethylation in breast cancer and premalignant lesions. The objective of this study was to examine whether DNA methylation assessed in random fine needle aspirates (rFNA) can be used to inform breast cancer risk. Methods. In 20 women with invasive breast cancer scheduled for surgery at Johns Hopkins Hospital, cumulative methylation status was assessed in a comprehensive manner. rFNA was performed on tumors, adjacent normal tissues, and all remaining quadrants. Pathology review was conducted on blocks from all excised tissue. The cumulative methylation index (CMI) for 12 genes was assessed by a highly sensitive QM-MSP assay in 280 aspirates and tissue from 11 incidental premalignant lesions. Mann–Whitney and Kruskal Wallis tests were used to compare median CMI by patient, location, and tumor characteristics. Results. The median age of participants was 49 years (interquartile range [IQR]: 44–58). DNA methylation was detectable at high levels in all tumor aspirates (median CMI = 252, IQR: 75–111). Methylation was zero or low in aspirates from adjacent tissue (median CMI = 11, IQR: 0–13), and other quadrants (median CMI = 2, IQR: 1–5). Nineteen incidental lesions were identified in 13 women (4 malignant and 15 premalignant). Median CMI levels were not significantly different in aspirates from quadrants ( p = 0.43 ) or adjacent tissue ( p = 0.93 ) in which 11 methylated incidental lesions were identified. Conclusions. The diagnostic accuracy of methylation based on rFNA alone to detect premalignant lesions or at-risk quadrants is poor and therefore should not be used to evaluate cancer risk. A more targeted approach needs to be evaluated.

Gene methylation and cytological atypia in random fine needle aspirates for assessment of breast cancer risk: 10-year follow up in average risk women.

10520 Background: Alterations in DNA methylation occur early in tumorigenesis, and are a potential breast cancer risk biomarker. We previously reported a study where healthy volunteers underwent random fine needle aspiration (rFNA) of the breasts; cumulative methylation index of eight preselected tumor suppressor genes (CMI) was associated with the presence of cytological atypia in rFNA samples. We now report 10-year follow-up of this population, to evaluate whether increased CMI is associated with subsequent breast cancer development. Methods: 380 women, unselected by breast cancer risk, were enrolled. Demographics, breast cancer risk factors, lifetime Gail model risk estimates (Gail-LR), and %breast density were obtained at baseline. rFNA samples were assessed for cytopathology (Masood Score, MS) and CMI. Patients were contacted annually for 10 years to ascertain development of invasive or non-invasive breast cancer. In univariate analysis, log-rank test was used to compare breast cancer incidence rates between individuals with high and low baseline measures (separated by median). Area under the ROC curve was used to evaluate the cancer prediction accuracy. In multivariate analysis, the effect of CMI (after log-transformation to reduce skewness) was further studied using Cox regression model adjusting for confounding baseline variables. Results: 362 women participated in follow up. At a median follow up time of 9.5 years after rFNA sampling, 16 women developed invasive or non-invasive breast cancer. There were no significant differences between women who developed cancer and those that did not in regard to demographic factors, %breast density, MS, or Gail-LR. On univariate analysis, Gail-LR was higher in women who developed cancer (13.0 vs. 16.5, p=0.08). The largest hazard ratios were observed from high breast density (2.30, 95% CI 0.8, 6.6) and high CMI (2.26, 95% CI 0.8, 6.6, p=0.07). In breast cancer prediction, the AUC for CMI was 0.64 (95% CI 0.51, 0.77). In separate bivariable models that adjusted for age, Gail-LR, MS, and %breast density, the HR for log CMI was consistently above unity, with a p value consistently below 0.1, except for the model that included MS (see Table). Conclusions: Elevated CMI has potential as a robust predictor of future breast cancer occurrence in average risk women, even when adjusted for breast density or cytologic atypia. Our prior analysis established that CMI is not susceptible to variation with menstrual cycle phase and menopausal status. These features support its further evaluation in larger trials. Clinical trial information: NCT00896636 .[Table: see text]

Peripheral blood BRCA1 methylation profiling to predict familial ovarian cancer.

ObjectiveFamilial cancer appears at a young age and its incidence is increasing. About 12% of familial ovarian cancer cases are associated with BRCA1/2 mutations (BRCAm). In this study, we investigated BRCA1 methylation may predict ovarian cancer in those with a family history of cancer (FHC) but without BRCA1/2 mutations (BRCAwt).MethodsUsing peripheral blood DNA from 55 subjects without a history of cancer [cancer(−)] and 52 ovarian cancer patients, we examined BRCA1 promoter methylation through bisulfite sequencing of the promoter and expressed the results as the cumulative methylation index. Then, we evaluated the BRCA1 promoter methylation according to BRCA1/2 germline mutations.ResultsBRCA1 methylation was more prevalent in the BRCAm cancer(−) group than in the BRCAwt cancer(−) group and ovarian cancer patients (p=0.031 and p=0.019, respectively). In the BRCAwt cancer(−) group, BRCA1 methylation was more prevalent in those with an FHC than in those without one and in the BRCAm cancer(−) group with an FHC (p=0.001 and p<0.001, respectively).ConclusionOur data suggest a predictive role of BRCA1 methylation profile for ovarian cancer in those without a history of cancer but with an FHC. BRCA1 methylation has important implications for diagnostic and predictive testing of those with BRCAwt cancer(−) status with FHC.

Colorectal cancer-associated microbiota contributes to oncogenic epigenetic signatures

Sporadic colorectal cancer (CRC) is a result of complex interactions between the host and its environment. Environmental stressors act by causing host cell DNA alterations implicated in the onset of cancer. Here we investigate the stressor ability of CRC-associated gut dysbiosis as causal agent of host DNA alterations. The epigenetic nature of these alterations was investigated in humans and in mice. Germ-free mice receiving fecal samples from subjects with normal colonoscopy or from CRC patients were monitored for 7 or 14 wk. Aberrant crypt foci, luminal microbiota, and DNA alterations (colonic exome sequencing and methylation patterns) were monitored following human feces transfer. CRC-associated microbiota induced higher numbers of hypermethylated genes in murine colonic mucosa (vs. healthy controls' microbiota recipients). Several gene promoters including SFRP1,2,3, PENK, NPY, ALX4, SEPT9, and WIF1 promoters were found hypermethylated in CRC but not in normal tissues or effluents from fecal donors. In a pilot study (n = 266), the blood methylation levels of 3 genes (Wif1, PENK, and NPY) were shown closely associated with CRC dysbiosis. In a validation study (n = 1,000), the cumulative methylation index (CMI) of these genes was significantly higher in CRCs than in controls. Further, CMI appeared as an independent risk factor for CRC diagnosis as shown by multivariate analysis that included fecal immunochemical blood test. Consequently, fecal bacterial species in individuals with higher CMI in blood were identified by whole metagenomic analysis. Thus, CRC-related dysbiosis induces methylation of host genes, and corresponding CMIs together with associated bacteria are potential biomarkers for CRC.

Breast Hormone Concentrations in Random Fine-Needle Aspirates of Healthy Women Associate with Cytological Atypia and Gene Methylation.

Sex steroid hormones contribute to breast cancer development, but data on concentrations of these within breast tissue are limited. We performed simultaneous multiparameter measurement of breast sex steroids, breast epithelial cytology, and DNA methylation in 119 healthy women (54 pre- and 65 postmenopausal) without a history of breast cancer. Random fine-needle aspiration (rFNA) of the breast was performed simultaneously with blood collection. Breast samples were analyzed by LC/MS-MS for estrone, estradiol, progesterone, androstenedione, and testosterone. Blood samples were assayed for estradiol and progesterone by immunoassay. Cytomorphology was classified using the Masood Score, and DNA methylation of eight genes was analyzed using quantitative multiplexed methylation-specific PCR, and expressed as the cumulative methylation index (CMI). Serum and breast concentrations of estradiol and progesterone showed significant correlation (Spearman r = 0.34, Padj = 0.001 and r = 0.69, Padj < 0.0006, respectively). Progesterone concentration was significantly higher in the premenopausal breast (Padj < 0.0008), and showed a luteal surge. Breast estrone and estradiol concentrations did not differ significantly by menopause, but androstenedione concentration was higher in the breasts of postmenopausal women (P = 0.026 and Padj = 0.208). Breast androgens were significantly correlated with breast density (Spearman r = 0.27, Padj = 0.02 for testosterone) and CMI (Spearman r = 0.3, Padj = 0.038 for androstenedione). Our data indicate that future larger studies of breast steroid hormones along with other parameters are feasible. Significant associations of breast androgen concentrations with breast density and gene methylation warrant future study. Cancer Prev Res; 11(9); 557-68. ©2018 AACR.

DNA methylation profiling in MEN1-related pancreatic neuroendocrine tumors reveals a potential epigenetic target for treatment.

Epigenetic changes contribute to pancreatic neuroendocrine tumor (PanNET) development. Hypermethylation of promoter DNA as a cause of tumor suppressor gene silencing is a well-established oncogenic mechanism that is potentially reversible and therefore an interesting therapeutic target. Multiple endocrine neoplasia type 1 (MEN1) is the most frequent cause of inherited PanNETs. The aim of this study was to determine promoter methylation profiles in MEN1-related PanNETs. Methylation-specific multiplex ligation-dependent probe amplification was used to assess promoter methylation of 56 tumor suppressor genes in MEN1-related (n = 61) and sporadic (n = 34) PanNETs. Differences in cumulative methylation index (CMI), individual methylation percentages and frequency of promoter hypermethylation between subgroups were analyzed. We found promoter methylation of a large number of potential tumor suppressor genes. CMI (median CMI: 912 vs 876, P = 0.207) was the same in MEN1-related and sporadic PanNETs. We found higher methylation percentages of CASP8 in MEN1-related PanNETs (median: 59% vs 16.5%, P = 0.002). In MEN1-related non-functioning PanNETs, the CMI was higher in larger PanNETs (>2 cm) (median: 969.5 vs 838.5; P = 0.021) and in PanNETs with liver metastases (median: 1036 vs 869; P = 0.013). Hypermethylation of MGMT2 was more frequent in non-functioning PanNETs compared to insulinomas (median: 44.7% vs 8.3%; P = 0.022). Hypermethylation of the Von Hippel-Lindau gene promoter was observed in one MEN1-related PanNET and was associated with loss of protein expression. Promoter hypermethylation is a frequent event in MEN1-related and sporadic PanNETs. Targeting DNA methylation could be of therapeutic value in MEN1 patients with advanced PanNETs.

Tumor and serum DNA methylation in women receiving preoperative chemotherapy with or without vorinostat in TBCRC008

Methylated gene markers have shown promise in predicting breast cancer outcomes and treatment response. We evaluated whether baseline and changes in tissue and serum methylation levels would predict pathological complete response (pCR) in patients with HER2-negative early breast cancer undergoing preoperative chemotherapy. The TBCRC008 trial investigated pCR following 12weeks of preoperative carboplatin and albumin-bound paclitaxel+vorinostat/placebo (n=62). We measured methylation of a 10-gene panel by quantitative multiplex methylation-specific polymerase chain reaction and expressed results as cumulative methylation index (CMI). We evaluated association between CMI level [baseline, day 15 (D15), and change] and pCR using univariate and multivariable logistic regression models controlling for treatment and hormone receptor (HR) status, and performed exploratory subgroup analyses. In univariate analysis, one log unit increase in tissue CMI levels at D15 was associated with 40% lower chance of obtaining pCR (odds ratio, OR0.60, 95% CI 0.37-0.97; p=0.037). Subgroup analyses suggested a significant association between tissue D15 CMI levels and pCR in vorinostat-treated [OR 0.44 (0.20, 0.93), p=0.03], but not placebo-treated patients. In this study investigating the predictive roles of tissue and serum CMI levels in patients with early breast cancer for the first time, we demonstrate that high D15 tissue CMI levels may predict poor response. Larger studies and improved analytical procedures to detect methylated gene markers in early stage breast cancer are needed. TBCRC008 is registered on ClinicalTrials.gov (NCT00616967).

The relationship of single-strand breaks in DNA to breast cancer risk and to tissue concentrations of oestrogens

Context: Clinical study of breast cancer patients in Chicago, IL, USA.Objective: Ascertain the utility of measurements of single-strand breaks (SSB) in DNA for assessment of breast cancer risk.Methods: Fine-needle aspirates of the breast, SSB by nick translation, percent breast density (PBD), Gail model risk, cumulative methylation index (CMI), enzymes of DNA repair and tissue antioxidants.Results: DNA repair enzymes and 4-hydroxyestradiol were negatively associated with SSB; CMI and PBD were positively associated.Conclusions: Quantitative measurement of SSBs by this procedure indicates the relative number of SSBs and is related to promoter methylation, antioxidant availability and percent breast density.

Abstract P1-09-15: Tumor and serum DNA methylation in women receiving preoperative chemotherapy (PST) with or without vorinostat in primary operable HER2-negative breast cancer in TBCRC008

Abstract Background: DNA methylation is a promising prognostic and predictive biomarker of response to treatment in advanced breast cancer (Fackler MJ. Can Res 2014). We evaluated whether baseline and change in tissue and serum methylation would predict pathological complete response (pCR) in patients with HER2-negative early breast cancer treated with PST. Methods: TBCRC008 was a multicenter placebo-controlled trial that investigated pCR (no invasive cancer in breast/axilla) following 12 weeks of preoperative carboplatin and albumin-bound paclitaxel with or without the histone deacetylase (HDAC) inhibitor vorinostat in 62 patients with HER2-negative breast cancer. We performed an exploratory planned biomarker study correlating baseline and change (D15) in tumor tissue and serum methylation with pCR. Methylation was measured using cumulative methylation index (CMI) in quantitative multiplexed methylation-specific polymerase chain reaction (QM-MSP) assays; cMethDNA assay for serum (Fackler MJ. Can Res 2014) and QM-MSP for tissue (Fackler MJ. Can Res 2004). The analysis population included all patients with available methylation and pCR data. Association between CMI level (log transformed baseline, D15, change) and pCR was evaluated using univariate as well as multivariable logistic regression models controlling for treatment arm and estrogen receptor (ER) status. Additional subgroup analyses assessed association of CMI level with pCR stratified by ER status and treatment arms, respectively. Spearman's correlation coefficient was performed to evaluate the correlation between tissue and serum CMI. Results: pCR data were available for 61 of 62 patients. One patient with unknown response was treated as a non-responder. The pCR rate was similar in both arms (vorinostat 25.8%, placebo 29%). Both tissue and serum CMI data were available in 55 (baseline), 50 (D15) and 46 (change) patients. Elevated serum methylation based on a predefined threshold (6.9 units) was identified in 23/59 (39%) patients. In univariate analysis, one log unit increase in tissue CMI levels at D15 was associated with a 40% lower chance of obtaining a pCR (odds ratio, OR=0.60, 95% CI 0.37-0.97; p=0.037). The multivariable analysis did not show a statistically significant association of tissue or serum CMI with pCR, although a similar trend in magnitude towards association with pCR was observed for D15 and change in tumor tissue CMI (OR 0.67 and 0.57, p=0.129 and 0.147 respectively). Subgroup analyses suggested a significant association between tissue CMI levels at D15 and pCR in the group treated with vorinostat [tissue OR: 0.44 (0.20, 0.93), p=0.03; serum OR 0.37 (0.13, 1.07), p=0.07] but not in the placebo group. No significant correlation was observed between tissue and serum CMI. Conclusion: This is the first study to evaluate the predictive role of tissue and serum methylation (CMI) in patients with early breast cancer treated with PST ± an HDAC inhibitor. Methylation was detected more frequently in tissue than serum. Tissue CMI levels at D15 may predict poor response to this regimen. Further evaluation of the predictive role of methylation in early breast cancer is warranted. Citation Format: Connolly RM, Fackler MJ, Zhang Z, Zhou XC, Goetz MP, Boughey JC, Walsh B, Carpenter J, Storniolo AM, Watkins S, Barielson E, Sukumar S, Stearns V. Tumor and serum DNA methylation in women receiving preoperative chemotherapy (PST) with or without vorinostat in primary operable HER2-negative breast cancer in TBCRC008 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-09-15.

Monitoring of Serum DNA Methylation as an Early Independent Marker of Response and Survival in Metastatic Breast Cancer: TBCRC 005 Prospective Biomarker Study.

Purpose Epigenetic alterations measured in blood may help guide breast cancer treatment. The multisite prospective study TBCRC 005 was conducted to examine the ability of a novel panel of cell-free DNA methylation markers to predict survival outcomes in metastatic breast cancer (MBC) using a new quantitative multiplex assay (cMethDNA). Patients and Methods Ten genes were tested in duplicate serum samples from 141 women at baseline, at week 4, and at first restaging. A cumulative methylation index (CMI) was generated on the basis of six of the 10 genes tested. Methylation cut points were selected to maximize the log-rank statistic, and cross-validation was used to obtain unbiased point estimates. Logistic regression or Cox proportional hazard models were used to test associations between the CMI and progression-free survival (PFS), overall survival (OS), and disease status at first restaging. The added value of the CMI in predicting survival outcomes was evaluated and compared with circulating tumor cells (CellSearch). Results Median PFS and OS were significantly shorter in women with a high CMI (PFS, 2.1 months; OS, 12.3 months) versus a low CMI (PFS, 5.8 months; OS, 21.7 months). In multivariable models, among women with MBC, a high versus low CMI at week 4 was independently associated with worse PFS (hazard ratio, 1.79; 95% CI, 1.23 to 2.60; P = .002) and OS (hazard ratio, 1.75; 95% CI, 1.21 to 2.54; P = .003). An increase in the CMI from baseline to week 4 was associated with worse PFS ( P < .001) and progressive disease at first restaging ( P < .001). Week 4 CMI was a strong predictor of PFS, even in the presence of circulating tumor cells ( P = .004). Conclusion Methylation of this gene panel is a strong predictor of survival outcomes in MBC and may have clinical usefulness in risk stratification and disease monitoring.

Gene Methylation and Cytological Atypia in Random Fine-Needle Aspirates for Assessment of Breast Cancer Risk.

Methods to determine individualized breast cancer risk lack sufficient sensitivity to select women most likely to benefit from preventive strategies. Alterations in DNA methylation occur early in breast cancer. We hypothesized that cancer-specific methylation markers could enhance breast cancer risk assessment. We evaluated 380 women without a history of breast cancer. We determined their menopausal status or menstrual cycle phase, risk of developing breast cancer (Gail model), and breast density and obtained random fine-needle aspiration (rFNA) samples for assessment of cytopathology and cumulative methylation index (CMI). Eight methylated gene markers were identified through whole-genome methylation analysis and included novel and previously established breast cancer detection genes. We performed correlative and multivariate linear regression analyses to evaluate DNA methylation of a gene panel as a function of clinical factors associated with breast cancer risk. CMI and individual gene methylation were independent of age, menopausal status or menstrual phase, lifetime Gail risk score, and breast density. CMI and individual gene methylation for the eight genes increased significantly (P < 0.001) with increasing cytological atypia. The findings were verified with multivariate analyses correcting for age, log (Gail), log (percent density), rFNA cell number, and body mass index. Our results demonstrate a significant association between cytological atypia and high CMI, which does not vary with menstrual phase or menopause and is independent of Gail risk and mammographic density. Thus, CMI is an excellent candidate breast cancer risk biomarker, warranting larger prospective studies to establish its utility for cancer risk assessment. Cancer Prev Res; 9(8); 673-82. ©2016 AACR.

Improved amplification efficiency on stool samples by addition of spermidine and its use for non-invasive detection of colorectal cancer.

BackgroundUsing quantitative methylation-specific PCR (QM-MSP) is a promising method for colorectal cancer (CRC) diagnosis from stool samples. Difficulty in eliminating PCR inhibitors of this body fluid has been extensively reported. Here, spermidine is presented as PCR facilitator for the detection of stool DNA methylation biomarkers using QM-MSP. We examined its effectiveness with NPY, PENK and WIF1, three biomarkers which we have previously shown to be of relevance to CRC.ResultsWe determined an optimal window for the amplification of the albumin (Alb) gene (100 ng of bisulfite-treated stool DNA added of 1 mM spermidine) at which we report that spermidine acts as a PCR facilitator (AE = 1680%) for SG RT-PCR. We show that the amplification of methylated PENK, NPY and WIF1 is considerably facilitated by QM-MSP as measured by an increase of CMI (Cumulative Methylation Index, i.e. the sum of the three methylation values) by a factor of 1.5 to 23 fold in individual samples, and of 10 fold in a pool of five samples.ConclusionsWe contend that spermidine greatly reduces the problems of PCR inhibition in stool samples. This observed feature, after validation on a larger sampling, could be used in the development of stool-based CRC diagnosis tests.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0148-6) contains supplementary material, which is available to authorized users.

HPV-positive oropharyngeal squamous cell carcinoma is associated with TIMP3 and CADM1 promoter hypermethylation.

Oropharyngeal squamous cell carcinoma (OPSCC) is associated with human papillomavirus (HPV) in a proportion of tumors. HPV-positive OPSCC is considered a distinct molecular entity with a prognostic advantage compared to HPV-negative cases. Silencing of cancer-related genes by DNA promoter hypermethylation may play an important role in the development of OPSCC. Hence, we examined promoter methylation status in 24 common tumor suppressor genes in a group of 200 OPSCCs to determine differentially methylated genes in HPV-positive versus HPV-negative primary OPSCC. Methylation status was correlated with HPV status, clinical features, and patient survival using multivariate methods. Additionally, methylation status of 16 cervical squamous cell carcinomas (SCC) was compared with HPV-positive OPSCC. Using methylation-specific probe amplification, HPV-positive OPSCC showed a significantly higher cumulative methylation index (CMI) compared to HPV-negative OPSCC (P=0.008). For the genes CDH13, DAPK1, and RARB, both HPV-positive and HPV-negative OPSCC showed promoter hypermethylation in at least 20% of the tumors. HPV status was found to be an independent predictor of promoter hypermethylation of CADM1 (P < 0.001), CHFR (P = 0.027), and TIMP3 (P < 0.001). CADM1 and CHFR showed similar methylation patterns in OPSCC and cervical SCC, but TIMP3 showed no methylation in cervical SCC in contrast to OPSCC. Methylation status of neither individual gene nor CMI was associated with survival. These results suggest that HPV-positive tumors are to a greater extent driven by promotor hypermethylation in these tumor suppressor genes. Especially CADM1 and TIMP3 are significantly more frequently hypermethylated in HPV-positive OPSCC and CHFR in HPV-negative tumors.

Aberrant methylation of NPY, PENK, and WIF1 as a promising marker for blood-based diagnosis of colorectal cancer

BackgroundDNA methylation is a well-known epigenetic mechanism involved in epigenetic gene regulation. Several genes were reported hypermethylated in CRC, althought no gene marker was proven to be individually of sufficient sensitivity or specificity in routine clinical practice. Here, we identified novel epigenetic markers and assessed their combined use for diagnostic accuracy.MethodsWe used methylation arrays on samples from several effluents to characterize methylation profiles in CRC samples and controls, as established by colonoscopy and pathology findings, and selected two differentially methylated candidate epigenetic genes (NPY, PENK). To this gene panel we added WIF, on the basis of being reported in literature as silenced by promoter hypermethylation in several cancers, including CRC. We measured their methylation degrees by quantitative multiplex-methylation specific PCR (QM-MSP) on 15 paired carcinomas and adjacent non-cancerous colorectal tissues and we subsequently performed a clinical validation on two different series of 266 serums, subdivided in 32 CRC, 26 polyps, 47 other cancers and 161 with normal colonoscopy. We assessed the results by receiver operating characteristic curve (ROC), using cumulative methylation index (CMI) as variable threshold.ResultsWe obtained CRC detection on tissues with both sensitivity and specificity of 100%. On serum CRC samples, we obtained sensitivity/specificity values of, e.g., 87%/80%, 78%/90% and 59%/95%, and negative predictive value/positive predictive value figures of 97%/47%, 95%/61% and 92%/70%. On serum samples from other cancers we obtained sensitivity/specificity of, e.g, 89%/25%, 43%/80% and 28%/91%.ConclusionsWe showed the potential of NPY, PENK, and WIF1 as combined epigenetic markers for CRC diagnosis, both in tissue and serum and tested their use as serum biomarkers in other cancers. We optimized a QM-MSP for simultaneously quantifying their methylation levels. Our assay can be an effective blood test for patients where CRC risk is present but difficult to assess (e.g. mild symptoms with no CRC family history) and who would therefore not necessarily choose to go for further examination. This panel of markers, if validated, can also be a cost effective screening tool for the detection of asymptomatic cancer patients for colonoscopy.

A new noninvasive blood-test for the early detection of colorectal cancer.

348 Background: Colorectal cancer (CRC) remains a major medical problem in the world. Its early detection impacts the prognosis and the cost of treatment. Fecal occult blood test is a current noninvasive screening method for CRC detection in asymptomatic average risk individuals. It detects less than 50% of cancers and yields a high number of “normal” colonoscopies. The dysregulation of DNA methylation has been recognized as playing a crucial role during CRC development. The aim of the study was to develop a CRC-specific methylated DNA test in serum. Methods: First, the GoldenGate Methylation microarray containing 1,505 CpG loci within 807 cancer-related genes was used to study methylation patterns in CRC patients. We performed such assays on 9 tissues (4 CRC, 4 normal autologous tissues, 1 polyp), 17 blood and urine pooled samples (7 CRC, 6 polyps, 4 normal) and 20 stool samples (7 CRC, 3 polyps, 10 normal). The clinical status of each sample was assessed by colonoscopy and/or pathology findings. Among methylated genes detected in tumour tissue, stools, and blood, NPY, PENK and WIF1 genes were selected taking into account their high degree of methylation. We then carried out a clinical validation by quantitative multiplex methylation-specific PCR (QM-MSP) in two phases: firstly on 30 tissues (15 CRC, 15 homologous normal tissues) and secondly on 193 serum samples (32 CRC, 161 normal). Results: For to obtain the best accuracy of QM-MSP test, we defined for both biological sources a cumulative methylation index (CMI) by adding of the methylation percentage of each gene. At the CMI of 20.0% on tissues, our QM-MSP test allows to diagnose the CRC with the highest accuracy (100% of specificity and 100% of sensitivity). From sera, the CMI has been standardized at 2.0% giving a sensitivity and specificity for detecting CRC of 94.4% and 59.4% respectively (NPV of 92.1% and PPV of 67.8%). Conclusions: We developed a QM-MSP-based analysis of NPY, PENK, and WIF1 methylated genes in serum. This test classified correctly and with a high accuracy the healthy individuals and colon cancer patients. We propose here new sensitive serum-based epigenetic biomarkers as an effective and simple new noninvasive test for CRC screening in the average and high risk populations.

Abstract A15: Single-strand breaks in normal breast DNA are associated with measures of breast cancer risk, deficiencies in repair mechanisms, and diminished protection against oxidative processes

Abstract Background: Formation of single-strand breaks (SSBs) in DNA is a constant process, estimated to occur 10,000 times per day in each cell, either from endogenous biosynthetic errors or from endogenous or exogenous disruptive agents. Base excision repair in some cases may be impaired or may be exceeded by the rate of DNA damage, leading to cancer. Methods: 136 healthy women were recruited through the Love-Avon Army of Women and breast clinics at Northwestern and Johns Hopkins Universities. Digital or digitized mammograms were evaluated for percent density using Cumulus software. The population medians (ranges) were for age, 51 (36 to 60); BMI, 28.2 (18.7 to 50.8); life-time Gail estimate, 11.8 (5.6 to 38.3); % breast density, 16.2 (2.4 to 52.5); Masood score, 13 (0 to 18). Breast tissue was obtained by random fine needle aspiration (rFNA). A kit from Norgen Inc. was used to separate DNA, RNA, and protein from the pelleted material. A 200 ng aliquot of DNA was taken for the nick translation assay. Incorporation of free nucleotides at SSBs with dCTP labeled with 3H is catalyzed by Polymerase I from E. coli. A quality control preparation prepared from calf thymus DNA and a reagent blank without DNA was included with each set of 6 samples. Separation of free from incorporated 3H-dCTP was accomplished by gel chromatography on an 80 x 15 mm column. The concordance of interassay values was 0.96. RNA (100 ng), purified from rFNA, was reverse transcribed. qPCR reactions were carried out using the TaqMan OpenArray (Applied Biosystems) for MTUS2, XRCC1, APEX1, SOD2, and NRF1 and were normalized to the average expression of GAPDH and HPRT1. Quantitation of DNA methylation was conducted by QM-MSP from cell lysates assessing the cumulative methylation level of eleven cancer-specific genes (AKR1B1, FZD10, TM6SF1, GAS7C, ALX1, CCND2, RARB, RASSF1A, TWIST1, TMEFF2, and SCGB3A1/HIN1) and a cumulative methylation index (CMI) was calculated. Statistical methods: Demographic and gene expression data were summarized and associations between SSBs and genes of interest were assessed via the Spearman correlation. Results: Among measures of risk, SSBs (pmol/μg DNA) were associated with percent breast density (rho = 0.207, p = 0.016) and with CMI (rho = 0.198, p = 0.021). The association with genes expressed in the rFNA are shown in Table 1. Briefly, MTUS2 is a microtubule-associated tumor suppressor inhibited by PARP: when detected, the MTUS2 mRNA was associated with SSPs; XRCC1 functions as a molecular scaffold that interacts with components of the SSB repair process; APEX1 is an endonuclease involved in base excision repair; and SOD2 and NRF1 act in antioxidant functions. Increases in SSBs were associated with decreased PARP, as reflected in increased MTUS2, decreased XRCC1, and decreased antioxidant factors. Table 1.Association of SSBs with gene products related to DNA repair and antioxidant functionmRNA / N / rho / PMTUS2 / 23 / 0.496 / 0.016XRCC1 / 136 / -0.151 / 0.079APEX1 / 131 / 0.035 / 0.690SOD2 / 136 / -0.207 / 0.016NRF1 / 131 / -0.224 / 0.010 Conclusions: 1) SSBs were associated with two measures of breast cancer risk. 2) SSBs were increased under conditions in which DNA repair processes and protection against reactive oxygen species (ROS) were reduced or unchanged. 3) SSBs were not associated with increased activity of repair genes as should occur if increased DNA damaging agents were the primary cause of increased SSBs. (Supported by the Avon Foundation). Citation Format: Robert Treat Chatterton, Mathavi Sahadevan, Oukseub Lee, Hu Hong, Wang Jun, Irene Helenowski, Saraswati Sukumar, Vered Stearns, Mary J. Fackler, Seema A. Khan. Single-strand breaks in normal breast DNA are associated with measures of breast cancer risk, deficiencies in repair mechanisms, and diminished protection against oxidative processes. [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 A15.

Gene methylation in random FNA samples as biomarkers for breast cancer.

518 Background: Current methods to determine breast cancer risk are insufficiently sensitive to select women most likely to benefit from preventive strategies. We hypothesized that candidate gene promoter hypermethylation may provide an individualized risk profile. We performed a prospective study to determine whether DNA cumulative methylation index (CMI) varies by menstrual phase or menopausal status, and to correlate CMI with established risk factors. Methods: We obtained random fine needle aspiration (rFNA) samples from healthy women age 35-60 and determined their menopausal and menstrual status, lifetime Gail risk, mammographic breast density, and cytologic atypia assessed as the Masood score. We evaluated CMI of 11 candidate genes in rFNA cells using the Quantitative Multiplex Methylation-Specific PCR (QM-MSP) technique. We used Wilcoxon test and ANOVA model to compare CMI across menopausal and menstrual (follicular, mid-cycle, luteal) categories, respectively. We used linear regression model to adjust for age and BMI. Methylation scores were log-transformed in the analysis. Results: We enrolled 390 women at the Avon Breast Centers at Johns Hopkins and Northwestern, the majority through the Love/Avon Army of Women, and 380 completed study procedures. Median age 50 (36-60), mean BMI 28 (18.7-50.8), 52% were postmenopausal. Mean life-time Gail risk 14.6 (5.6-54.1), mean percent mammographic density 19.6 (2.5-72.8), and mean Masood score (N=354) 13.6 (7-18). QM-MSP analysis was completed on 229 samples. We did not observe differences in CMI among menopausal (P=0.4895) or menstrual categories (P=0.2333). There was no association between CMI and life-time Gail risk (P=0.706) or breast density (P=0.4116). We observed a significant correlation between CMI and Masood score (P=0.0167). Conclusions: CMI correlates with degree of cytologic atypia and is potentially a robust indicator of breast cancer risk since it does not vary with menstrual or menopausal status. Next, we will select genes that best reflect changes in the clinical parameters to create a gene methylation signature that will be validated in other studies and correlated with breast cancer risk.

Promoter hypermethylation patterns in fallopian tube epithelium of BRCA1 and BRCA2 germ line mutation carriers

BRCA1/2 germ line mutation carriers have a high risk of developing fallopian tube carcinoma (FTC), thought to occur through different early (p53 signatures) and later (dysplasia, intra-epithelial carcinoma) premalignant stages. Promoter hypermethylation of tumour suppressor genes is known to play a key role in (early) carcinogenesis. However, little is known about methylation in normal and (pre)malignant fallopian tube tissue. We identified 14 areas of p53 accumulation in the fallopian tubes of BRCA mutation carriers. Cells from these areas were harvested together with cells from adjacent benign appearing areas. An age-matched non-BRCA sporadic control group (n=13) and eight sporadic FTCs were included as negative and positive controls respectively. Methylation-specific multiplex ligation-dependent probe amplification was used to assess promoter methylation of 70 tumour suppressor genes in all samples. We observed a gradual increase in methylation from sporadic control tissue (median cumulative methylation index (CMI) 568.19) through normal tissue and from areas of p53 accumulation in BRCA carriers (median CMI 687.54 and 676.72) to FTC (median CMI 780.97). Furthermore, the methylation percentage of many individual tumour suppressor genes differed significantly between these groups, gradually increasing as for CMI. Between areas with and without p53 accumulation in BRCA mutation carriers no significant differences were found. In this paper, we have shown that BRCA mutation carriers display increased methylation of tumour suppressor genes in their non-malignant fallopian tube epithelium, closer to methylation levels in FTC than to normal sporadic tissue. Methylation could, therefore, play an important role in the increased risk of gynaecological malignancies in BRCA mutation carriers.

Methylation profiles of hereditary and sporadic ovarian cancer

Tumour suppressor gene silencing through promoter hypermethylation plays an important role in oncogenesis. Carcinogenesis of hereditary cancers usually differs from that of their sporadic counterparts, but methylation has hardly been studied in hereditary ovarian cancer. The aim of this study was to investigate promoter methylation of a set of common tumour suppressor genes in BRCA1-related ovarian cancer in comparison with sporadic ovarian cancer. Methylation-specific multiplex ligation-dependent probe amplification was used to assess the extent of promoter methylation of 24 different tumour suppressor genes in BRCA1-associated (n = 25) and matched sporadic ovarian tumours (n = 50). A cumulative methylation index (CMI) was calculated and differences between individual genes were analysed. There was no significant difference in cumulative methylation between BRCA1-associated and sporadic ovarian carcinomas (median CMI 108; CMI 110; P = 0.86). Also, methylation patterns of individual genes did not show distinct differences after correction for multiple comparisons. CDH13, GSTP1 and RASSF1 were frequently methylated in both sporadic and hereditary ovarian cancers. BRCA1 methylation occurred in 14% of sporadic tumours, but was not detected in BRCA1-associated tumours. CDH13, GSTP1 and RASSF1 are frequently methylated in both sporadic and BRCA1-associated ovarian cancers. Interestingly, methylation of BRCA1, while frequent in sporadic ovarian cancer, never occurred in the hereditary group. BRCA1-associated ovarian cancers mimic their sporadic counterparts in extent and pattern of promoter methylation of several common tumour suppressor genes. This finding could have implications for future chemotherapy regimens based on epigenetic changes.

Methylation profiles of endometrioid and serous endometrial cancers

Promoter methylation is a gene- and cancer type-specific epigenetic event that plays an important role in tumour development. As endometrioid (endometrioid endometrial carcinoma, EEC) and serous endometrial cancers (uterine papillary serous carcinoma, UPSC) exhibit different clinical, histological and molecular genetic characteristics, we hypothesized that these differences may be reflected in epigenetic phenomena as well. Identification of a panel of methylation biomarkers could be helpful in a correct histological classification of these two subtypes, which solely on the basis of morphology is not always easy. Methylation-specific multiplex ligation-dependent probe amplification was used to assess the extent of promoter methylation of different tumour suppressor genes in EEC and UPSC. Methylation results were correlated with histology and survival. The median cumulative methylation index of all genes was significantly higher in EEC (124) than in UPSC (93) (P<0.001). Promoter methylation of CDH13 and MLH1 was more frequently present in EEC, while CDKN2B and TP73 were more frequently methylated in UPSC. Almost 90% of EEC and 70% of UPSC could be predicted by CDH13 and TP73. In EEC, methylation of MLH1 was associated with a shorter disease-free survival (DFS; P<0.0001) and overall survival (OS; P=0.005). In a multivariate model, MLH1 methylation emerged as an additional prognostic factor to stage for DFS (P=0.002). In conclusion, promoter methylation is more common in EEC than UPSC. A panel of methylation biomarkers could be useful to distinguish between the two histological subtypes of endometrial cancer. Furthermore, methylation of MLH1 may have prognostic value in EEC.