Differential DNA methylation analysis of breast cancer reveals the impact of immune signaling in radiation therapy

Radiotherapy (RT) is a central treatment modality for breast cancer patients. The purpose of our study was to investigate the DNA methylation changes in tumors following RT, and to identify epigenetic markers predicting treatment outcome. Paired biopsies from patients with inoperable breast cancer were collected both before irradiation (n = 20) and after receiving 10–24 Gray (Gy) (n = 19). DNA methylation analysis was performed by using Illumina Infinium 27K arrays. Fourteen genes were selected for technical validation by pyrosequencing. Eighty‐two differentially methylated genes were identified in irradiated (n = 11) versus nonirradiated (n = 19) samples (false discovery rate, FDR = 1.1%). Methylation levels in pathways belonging to the immune system were most altered after RT. Based on methylation levels before irradiation, a panel of five genes (H2AFY, CTSA, LTC4S, IL5RA and RB1) were significantly associated with clinical response (p = 0.041). Furthermore, the degree of methylation changes for 2,516 probes correlated with the given radiation dose. Within the 2,516 probes, an enrichment for pathways involved in cellular immune response, proliferation and apoptosis was identified (FDR < 5%). Here, we observed clear differences in methylation levels induced by radiation, some associated with response to treatment. Our study adds knowledge on the molecular mechanisms behind radiation response.     

Association of frequent hypermethylation with high grade histological subtype in lung adenocarcinoma

Lung adenocarcinoma is classified morphologically into five histological subtypes according to the WHO classification. While each histological subtype correlates with a distinct prognosis, the molecular basis has not been fully elucidated. Here we conducted DNA methylation analysis of 30 lung adenocarcinoma cases annotated with the predominant histological subtypes and three normal lung cases using the Infinium BeadChip. Unsupervised hierarchical clustering analysis revealed three subgroups with different methylation levels: high-, intermediate-, and low-methylation epigenotypes (HME, IME, and LME). Micropapillary pattern (MPP)-predominant cases and those with MPP components were significantly enriched in HME (p = 0.02 and p = 0.03, respectively). HME cases showed a significantly poor prognosis for recurrence-free survival (p < 0.001) and overall survival (p = 0.006). We identified 365 HME marker genes specifically hypermethylated in HME cases with enrichment of “cell morphogenesis” related genes; 305 IME marker genes hypermethylated in HME and IME, but not in LME, with enrichment “embryonic organ morphogenesis”-related genes; 257 Common marker genes hypermethylated commonly in all cancer cases, with enrichment of “regionalization”-related genes. We extracted surrogate markers for each epigenotype and designed pyrosequencing primers for five HME markers (TCERG1L, CXCL12, FAM181B, HOXA11, GAD2), three IME markers (TBX18, ZNF154, NWD2) and three Common markers (SCT, GJD2, BARHL2). DNA methylation profiling using Infinium data was validated by pyrosequencing, and HME cases defined by pyrosequencing results also showed the worse recurrence-free survival. In conclusion, lung adenocarcinomas are stratified into subtypes with distinct DNA methylation levels, and the high-methylation subtype correlated with MPP-predominant cases and those with MPP components and showed a poor prognosis.     

Stratification of HPV-associated and HPV-negative oropharyngeal squamous cell carcinomas based on DNA methylation epigenotypes

While the incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been increasing in these two decades, primarily due to human papillomavirus (HPV), stratification of OPSCC into molecular subgroups showing different clinicopathological features has not been fully investigated. We performed DNA methylome analysis using Infinium 450k for 170 OPSCC cases, including 89 cases in our cohort and 81 cases reported by The Cancer Genome Atlas, together with targeted exon sequencing analysis. We stratified OPSCC by hierarchical clustering analysis using methylome data. Methylation levels of classifier markers were validated quantitatively using pyrosequencing, and area under the curve (AUC) values of receiver operating characteristics (ROC) curves were calculated. OPSCC was stratified into four epigenotypes: HPV(+) high-methylation (OP1), HPV(+) intermediate-methylation (OP2), HPV(−) intermediate-methylation (OP3) and HPV(−) low-methylation (OP4). Ten methylation marker genes were generated: five to classify HPV(+) cases into OP1 and OP2, and five to classify HPV(−) cases into OP3 and OP4. AUC values of ROC curves were 0.969 and 0.952 for the two marker panels, respectively. While significantly higher TP53 mutation and CCND1 copy number gains were observed in HPV(−) than in HPV(+) groups (p < 0.01), no significant difference of genomic aberrations was observed between OP1 and OP2, or OP3 and OP4. The four epigenotypes showed significantly different prognosis (p = 0.0006), distinguishing the most favorable OPSCC subgroup (OP1) among generally favorable HPV(+) cases, and the most unfavorable OPSCC subgroup (OP3) among generally unfavorable HPV(−) cases. HPV(+) and HPV(−) OPSCC are further divided into distinct DNA methylation epigenotypes, showing significantly different prognosis.     

Methylomics analysis identifies epigenetically silenced genes and implies an activation of β‐catenin signaling in cervical cancer

Using DNA methylation biomarkers in cancer detection is a potential direction in clinical testing. Some methylated genes have been proposed for cervical cancer detection; however, more reliable methylation markers are needed. To identify new hypermethylated genes in the discovery phase, we compared the methylome between a pool of DNA from normal cervical epithelium (n = 19) and a pool of DNA from cervical cancer tissues (n = 38) using a methylation bead array. We integrated the differentially methylated genes with public gene expression databases, which resulted in 91 candidate genes. Based on gene expression after demethylation treatment in cell lines, we confirmed 61 genes for further validation. In the validation phase, quantitative MSP and bisulfite pyrosequencing were used to examine their methylation level in an independent set of clinical samples. Fourteen genes, including ADRA1D, AJAP1, COL6A2, EDN3, EPO, HS3ST2, MAGI2, POU4F3, PTGDR, SOX8, SOX17, ST6GAL2, SYT9, and ZNF614, were significantly hypermethylated in CIN3+ lesions. The sensitivity, specificity, and accuracy of POU4F3 for detecting CIN3+ lesions were 0.88, 0.82, and 0.85, respectively. A bioinformatics function analysis revealed that AJAP1, EDN3, EPO, MAGI2, and SOX17 were potentially implicated in β‐catenin signaling, suggesting the epigenetic dysregulation of this signaling pathway during cervical cancer development. The concurrent methylation of multiple genes in cancers and in subsets of precancerous lesions suggests the presence of a driver of methylation phenotype in cervical carcinogenesis. Further validation of these new genes as biomarkers for cervical cancer screening in a larger population‐based study is warranted.     

Colorectal cancer early methylation alterations affect the crosstalk between cell and surrounding environment,tracing a biomarker signature specific for this tumor

Colorectal cancer (CRC) develops through the accumulation of both genetic and epigenetic alterations. However, while the former are already used as prognostic and predictive biomarkers, the latter are less well characterized. Here, performing global methylation analysis on both CRCs and adenomas by Illumina Infinium HumanMethylation450 Bead Chips, we identified a panel of 74 altered CpG islands, demonstrating that the earliest methylation alterations affect genes coding for proteins involved in the crosstalk between cell and surrounding environment. The panel discriminates CRCs and adenomas from peritumoral and normal mucosa with very high specificity (100%) and sensitivity (99.9%). Interestingly, over 70% of the hypermethylated islands resulted in downregulation of gene expression. To establish the possible usefulness of these non‐invasive markers for detection of colon cancer, we selected three biomarkers and identified the presence of altered methylation in stool DNA and plasma cell‐free circulating DNA from CRC patients.     

Discovery and validation of candidate host DNA methylation markers for detection of cervical precancer and cancer

Human papillomavirus (HPV) testing has been recently introduced as an alternative to cytology for cervical cancer screening. However, since most HPV infections clear without causing clinically relevant lesions, additional triage tests are required to identify women who are at high risk of developing cancer. We performed DNA methylation profiling on formalin‐fixed, paraffin‐embedded tissue specimens from women with benign HPV16 infection and histologically confirmed cervical intraepithelial neoplasia grade 3, and cancer using a bead‐based microarray covering 1,500 CpG sites in over 800 genes. Methylation levels in individual CpG sites were compared using a t‐test, and results were summarized by computing p‐values. A total of 12 candidate genes (ADCYAP1, ASCL1, ATP10, CADM1, DCC, DBC1, HS3ST2, MOS, MYOD1, SOX1, SOX17 and TMEFF2) identified by DNA methylation profiling, plus an additional three genes identified from the literature (EPB41L3, MAL and miR‐124) were chosen for validation in an independent set of 167 liquid‐based cytology specimens using pyrosequencing and targeted, next‐generation bisulfite sequencing. Of the 15 candidate gene markers, 10 had an area under the curve (AUC) of ≥ 0.75 for discrimination of high grade squamous intraepithelial lesions or worse (HSIL+) from <HSIL cytology using at least one assay. Overall, SOX1, DCC, and EPB41L3 showed the best discrimination with AUC values of ≥0.80, irrespective of methylation detection assay. In addition to verifying candidate markers from the literature (e.g., SOX1 and EPB41L3), we identified novel markers that may be considered for detection of cervical precancer and cancer and warrant further validation in prospective studies.     

Methylation of multiple genes as molecular markers for diagnosis of a small,well‐differentiated hepatocellular carcinoma

The current study was conducted to identify robust methylation markers and their combinations that may prove useful for the diagnosis of early hepatocellular carcinoma (HCC). To achieve this, we performed in silico CpG mapping, direct sequencing and pyrosequencing after bisulfite treatment, and quantitative methylation‐specific PCR (MSP) in HCC and non‐HCC liver tissues. In the filtering group (25 HCCs), our direct sequencing analysis showed that, among the 12 methylation genes listed by in silico CpG mapping, 7 genes (RASSF1A, CCND2, SPINT2, RUNX3, GSTP1, APC and CFTR) were aberrantly methylated in stages I and II HCCs. In the validation group (20 pairs of HCCs and the corresponding non‐tumor liver tissues), pyrosequencing analysis confirmed that the 7 genes were aberrantly and strongly methylated in early HCCs, but not in any of the corresponding non‐ tumor liver tissues (p < 0.00001). The results obtained using our novel quantitative MSP assay correlated well with those observed using the pyrosequencing analysis. Notably, in MSP assay, RASSF1A showed the most robust performance for the discrimination of HCC and non‐HCC liver tissues. Furthermore, a combination of RASSF1A, CCND2 and SPINT2 showed 89–95% sensitivity, 91–100% specificity and 89–97% accuracy in discriminating between HCC and non‐HCC tissues, and correctly diagnosed all early HCCs. These results indicate that the combination of these 3 genes may aid in the accurate diagnosis of early HCC. © 2009 UICC     

Clinical prognostic value of DNA methylation in hepatoblastoma: Four novel tumor suppressor candidates

Hepatoblastoma (HB) is very rare but the most common malignant neoplasm of the liver occurring in children. Despite improvements in therapy, outcomes for patients with advanced HB that is refractory to standard preoperative chemotherapy remain unsatisfactory. To improve the survival rate among this group, identification of novel prognostic markers and therapeutic targets is needed. We have previously reported that altered DNA methylation patterns are of biological and clinical importance in HB. In the present study, using genome‐wide methylation analysis and bisulfite pyrosequencing with specimens from HB tumors, we detected nine methylated genes. We then focused on four of those genes, GPR180, MST1R, OCIAD2, and PARP6, because they likely encode tumor suppressors and their increase of methylation was associated with a poor prognosis. The methylation status of the four genes was also associated with age at diagnosis, and significant association with the presence of metastatic tumors was seen in three of the four genes. Multivariate analysis revealed that the presence of metastatic tumors and increase of methylation of GPR180 were independent prognostic factors affecting event‐free survival. These findings indicate that the four novel tumor suppressor candidates are potentially useful molecular markers predictive of a poor outcome in HB patients, which may serve as the basis for improved therapeutic strategies when clinical trials are carried out.     

DNA methylation of circulating DNA: a marker for monitoring efficacy of neoadjuvant chemotherapy in breast cancer patients

Identification of biomarkers for monitoring efficacy of neoadjuvant chemotherapy in breast cancer patients is of utmost importance in individual tailoring of treatment and save from toxicity due to non-effective drugs. We hypothesized that methylation of circulating tumor-specific DNA may reflect changes in tumor burden in response to chemotherapy and help stratify responders from non-responders. The aim of this study was to evaluate the potential of methylation changes in circulating DNA to monitor treatment response of breast cancer patients. Six consecutive sera samples collected from 30 breast cancer patients undergoing neoadjuvant chemotherapy were analyzed for methylation status of a panel of five genes namely, BRCA1, MGMT, GSTP1, Stratifin, and MDR1. Among these five genes, BRCA1 methylation frequency was different among responders and non-responders groups. The correlation coefficients between total gene methylation with initial chemotherapy and tumor volume reduction were R ²?=?0.8 and R ²?=?0.05 in the responders and non-responders groups, respectively. Our findings warrant further development of this approach for monitoring response in patients undergoing neoadjuvant chemotherapy.     

Clinical validation of methylation biomarkers for optimal detection of high-grade vulvar intraepithelial neoplasia

The precursor lesions of vulvar squamous cell carcinoma (VSCC) include human papillomavirus (HPV)-associated and HPV-independent squamous neoplasia with a varying cancer risk. Our study aimed to validate the accuracy of previously identified DNA methylation markers for detection of such high-grade vulvar intraepithelial neoplasia (VIN). A large clinical series of 751 vulvar lesions, originally diagnosed as high-grade VIN, were reassessed and categorized into HPV-associated or HPV-independent vulvar disease categories. Together with 113 healthy vulvar controls, all samples were tested for 12 methylation markers with quantitative multiplex methylation-specific PCR (qMSP). Performance of individual markers and selection of an optimal marker panel for detection of high-grade VIN was determined by logistic regression analysis. SST was the best-performing individual marker (AUC 0.90), detecting 80% of high-grade VIN cases, with excellent detection of HPV-independent VIN (95%), known to have the highest cancer risk. Merely 2% of controls tested methylation positive for SST. Selection of a marker panel, including ZNF582, SST and miR124-2, resulted in a comparably high accuracy for detection of high-grade VIN (AUC 0.89). In conclusion, we clinically validated the accuracy of 12 DNA methylation markers for detection of high-grade VIN. SST, as a sole marker or in a panel, provides an optimal diagnostic tool to distinguish high-grade VIN in need of treatment, particularly HPV-independent VIN, from low-grade or reactive vulvar lesions. These findings warrant further prognostic validation of methylation biomarkers for cancer risk stratification of patients with VIN.     

DNA methylation accumulation in gastric mucosa adjacent to cancer after Helicobacter pylori eradication

Molecular irreversibleness with Helicobacter pylori (H. pylori) infection might have a role in gastric tumorigenesis after H. pylori eradication. We performed comprehensive DNA methylation profiling of gastric mucosa after H. pylori eradication with or without gastric cancer. Using four different groups of biopsies obtained from gastric body without history of H. pylori infection (Hp-), gastric body without cancer after H. pylori eradication (cancer-free body), gastric body with early gastric cancer diagnosed after H. pylori eradication (EGC body) and their paired samples from adjacent mucosa of cancer (EGC ADJ), methylation status of five candidate genes (MYOD1, SLC16A12, IGF2, RORA and PRDM5) was examined by the bisulfite pyrosequencing. An Infinium Methylation EPIC BeadChip array was also used to characterize the methylation status of greater than 850,000 CpG sites. The EGC ADJ group showed highest methylation levels of five candidate genes among the four groups of biopsies. In the gastric body (cancer-free body + EGC body), methylation levels were significantly decreased in patients with longer period after eradication, while such association was not observed in EGC ADJ group. Hyper methylated samples were associated with shorter telomere, an indicator for rapid cell turnover, and higher DNMT1 protein expression, an enzyme related to methyl transfer reaction. The genome-wide methylation analysis demonstrated strikingly higher methylation levels especially at CpG islands in the EGC ADJ group. Exclusively hypermethylated promoter CpG islands in the same group frequently coded zinc finger proteins. Our data show that DNA methylation accumulation is associated with molecular irreversibleness and gastric carcinogenesis after H. pylori eradication.     

A four-gene methylation marker panel as triage test in high-risk human papillomavirus positive patients

Cervical neoplasia‐specific biomarkers, e.g. DNA methylation markers, with high sensitivity and specificity are urgently needed to improve current population‐based screening on (pre)malignant cervical neoplasia. We aimed to identify new cervical neoplasia‐specific DNA methylation markers and to design and validate a methylation marker panel for triage of high‐risk human papillomavirus (hr‐HPV) positive patients. First, high‐throughput quantitative methylation‐specific PCRs (QMSP) on a novel OpenArray? platform, representing 424 primers of 213 cancer specific methylated genes, were performed on frozen tissue samples from 84 cervical cancer patients and 106 normal cervices. Second, the top 20 discriminating methylation markers were validated by LightCycler® MSP on frozen tissue from 27 cervical cancer patients and 20 normal cervices and ROCs and test characteristics were assessed. Three new methylation markers were identified (JAM3, EPB41L3 and TERT), which were subsequently combined with C13ORF18 in our four‐gene methylation panel. In a third step, our methylation panel detected in cervical scrapings 94% (70/74) of cervical cancers, while in a fourth step 82% (32/39) cervical intraepithelial neoplasia grade 3 or higher (CIN3+) and 65% (44/68) CIN2+ were detected, with 21% positive cases for ≤CIN1 (16/75). Finally, hypothetical scenario analysis showed that primary hr‐HPV testing combined with our four‐gene methylation panel as a triage test resulted in a higher identification of CIN3 and cervical cancers and a higher percentage of correct referrals compared to hr‐HPV testing in combination with conventional cytology. In conclusion, our four‐gene methylation panel might provide an alternative triage test after primary hr‐HPV testing.     

Commonality and differences of methylation signatures in the plasma of patients with pancreatic cancer and colorectal cancer

Profiling of DNA methylation status of specific genes is a way to screen for colorectal cancer (CRC) and pancreatic cancer (PC) in blood. The commonality of methylation status of cancer‐related tumor suppressor genes between CRC and PC is largely unknown. Methylation status of 56 cancer‐related genes was compared in plasma of patients in the following cohorts: CRC, PC and healthy controls. Cross validation determined the best model by area under ROC curve (AUC) to differentiate cancer methylation profiles from controls. Optimal preferential gene methylation signatures were derived to differentiate either cancer (CRC or PC) from controls. For CRC alone, a three gene signature (CYCD2, HIC and VHL) had an AUC 0.9310, sensitivity (Sens) = 0.826, specificity (Spec) = 0.9383. For PC alone, an optimal signature consisted of five genes (VHL, MYF3, TMS, GPC3 and SRBC), AUC 0.848; Sens = 0.807, Spec = 0.666. Combined PC and CRC signature or “combined cancer signature” was derived to differentiate either CRC and PC from controls (MDR1, SRBC, VHL, MUC2, RB1, SYK and GPC3) AUC = 0.8177, Sens = 0.6316 Spec = 0.840. In a validation cohort, N = 10 CRC patients, the optimal CRC signature (CYCD2, HIC and VHL) had AUC 0.900. In all derived signatures (CRC, PC and combined cancer signature) the optimal panel used preferential VHL methylation. In conclusion, CRC and PC differ in specific genes methylated in plasma other than VHL. Preferential methylation of VHL is shared in the optimal signature for CRC alone, PC alone and combined PC and CRC. Future investigations may identify additional methylation markers informative for the presence of both CRC and PC.     

Promoter hypermethylation in Indian primary oral squamous cell carcinoma

We evaluated promoter hypermethylation of a panel of tumor suppressor genes as a means to detect epigenetic alterations in oral squamous cell carcinomas (OSCC) of Indian‐origin and compare with North‐American head and neck squamous cell carcinomas (HNSCC). Quantitative‐methylation‐specific PCR was used to investigate the promoter methylation status of DCC, EDNRB, p16^INK4a and KIF1A in 92 OSCC, and compared to 48 paired normal tissues and 30 saliva and sera samples from healthy control subjects. Aberrant methylation of at‐least one of these genes was detected in 74/92 (80.4%) OSCC; 72.8% at EDNRB, 71.7% at KIF1A, 47.8% at p16^INK4a and 58.7% at DCC; and in 5 of 48 (10.4%) normal oral tissues. None of the saliva and sera samples from controls exhibited DNA methylation in these four target genes. Thirty‐two of 72 node positive cases harbored p16^INK4a and DCC hypermethylation (p = 0.005). Thus, promoter hypermethylation in genes analyzed herein is a common event in Indian OSCC and may represent promising markers for the molecular staging of OSCC patients. We found higher frequency of p16^INK4a methylation (47.8%) in this Indian cohort in comparison with a North‐American cohort (37.5%). In conclusion, aberrant methylation of EDNRB, KIF1A, DCC and p16^INK4a genes is a common event in Indian OSCC, suggesting that epigenetic alterations of these genes warrant validation in larger studies for their potential use as biomarkers.     

HOXA9, ISL1 and ALDH1A3 methylation patterns as prognostic markers for nonmuscle invasive bladder cancer: Array‐based DNA methylation and expression profiling

DNA methylation patterns are associated with the development and prognosis of cancer. The aim of this study was to identify novel methylation markers for the prediction of patient outcomes using microarray analysis of DNA methylation and RNA expression patterns in samples from long‐term follow‐up patients with nonmuscle invasive bladder cancer (NMIBC). A total of 187 human bladder specimens were used for microarray array or pyrosequencing (PSQ) analyses: 6 normal controls (NC) and 181 NMIBC. Tumor‐specific hypermethylated genes were selected from a data set comprising 24 matched microarray‐based DNA methylation and gene expression profiles (6 controls and 18 NMIBC), and their clinical relevance was verified by quantitative PSQ analysis. The methylation status of Homeobox A9 (HOXA9), ISL LIM homeobox 1 (ISL1) and Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) was significantly associated with decreased gene expression levels and aggressive clinicopathological characteristics. Multivariate regression analyses showed that hypermethylation of these genes was an independent predictor of disease recurrence (HOXA9, ISL1 and ALDH1A3, either alone or in combination) and progression (ISL1 and ALDH1A3, either alone or in combination) (each p < 0.05). The results of this study suggest that these novel methylation markers are independent prognostic indicators in NMIBC patients, which may facilitate the assessment of disease recurrence and progression in NMIBC patients and inform clinical decision making regarding treatment.     

Genome-wide DNA methylation profiling identifies two novel genes in cervical neoplasia

DNA methylation analysis may improve risk stratification in cervical screening. We used a pan-epigenomic approach to identify new methylation markers along the continuum of cervical intraepithelial neoplasia (CIN) to cervical cancer. Physician-collected samples (54 normal, 50 CIN1, 40 CIN2 and 42 CIN3) were randomly selected from women at a single-center colposcopy clinic. Extracted DNA was subjected to Illumina Infinium EPIC array analysis, and methylation was assessed blinded to histopathological and clinical data. CpG sites whose state of methylation correlated with lesion grade were assessed (Spearman correlation), and a weighted methylation score was calculated comparing normal to CIN3. Validation of the top selected genes was performed in an independent cohort (100 normal, 50 CIN1, 50 CIN2, 50 CIN3 and 8 cervical cancers) of new patients, referred for colposcopic examination at three hospitals, using targeted DNA methylation Illumina amplicon sequencing. The relationship between a combined weighted marker score and progression from normal through precancerous lesions and cervical cancer was compared using one-way ANOVA. Our analyses revealed 7,715 CpGs whose methylation level correlated with progression (from normal to CIN1, CIN2 and CIN3), with a significant trend of increased methylation with lesion grade. We shortlisted a bigenic (hyaluronan synthase 1, HAS1 and ATPase phospholipid transporting 10A, ATP10A corresponding to cg03419058 and cg13944175 sites) marker set; r = 0.55, p < 0.0001. Validation of the four most discriminating genes (CA10, DPP10, FMN2 and HAS1) showed a significant correlation between methylation levels and disease progression (p-value < 2.2 × 10⁻¹⁶, adjusted R² = 0.952). Translational research of the identified genes to future clinical applications is warranted.     

Integrated analysis of high‐resolution DNA methylation profiles,gene expression,germline genotypes and clinical end points in breast cancer patients

Breast cancer is a heterogeneous disease for which alterations in DNA methylation patterns have been shown to be of biological and clinical importance. Here we report on the integrated analysis of molecular alterations including the methylation status of 27 gene promoters analyzed by highly quantitative pyrosequencing, and the association to gene expression, germline genotype and clinical parameters including survival. Breast cancer specific deregulation of DNA methylation (both hyper‐ and hypomethylation) was found in twenty genes including ACVR1, OGG1, IL8 and TFF1. The methylation level in the promoter regions was significantly negatively correlated to gene expression for twelve genes (such as MST1R, ST6GAL1 and TFF1) indicating that a gain of aberrant methylation (hypermethylation) inhibits gene expression. Multiple associations between molecular and clinical parameters were identified, and multivariate statistical analysis demonstrated that methylation was more strongly associated to clinical parameters than gene expression for the investigated genes. The methylation level of BCAP31 and OGG1 showed significant association to survival, and these associations were validated in a larger patient cohort (The Cancer Genome Atlas). Our study provides evidence for the promise of DNA methylation alterations for clinical applications.     

Epigenetic markers for noninvasive early detection of nasopharyngeal carcinoma by methylation‐sensitive high resolution melting

Nasopharyngeal carcinoma (NPC) is a human malignancy that is closely associated with Epstein‐Barr Virus (EBV). Early diagnosis of NPC will greatly improve the overall survival. However, current EBV DNA marker detection still lacks the predictive value to perform well in high‐risk populations for early detection of NPC. Since aberrant promoter hypermethylation of tumor suppressor genes (TSGs) is widely considered to be an important epigenetic change in early carcinogenesis, this study identified a panel of methylation markers for early detection of NPC and also assessed the clinical usefulness of these markers with noninvasive plasma specimens instead of biopsies. MS‐HRM assays were carried out to assess the methylation status of a selected panel of four TSGs (RASSF1A, WIF1, DAPK1 and RARβ2) in biopsies, NP brushings and cell‐free plasma from NPC patients. High‐risk and cancer‐free groups were used as controls. DNA methylation panel showed higher sensitivity and specificity than EBV DNA marker in cell‐free plasma from NPC patients at early Stages (I and II) and in addition to the EBV DNA marker, MS‐HRM test for plasma and NP brushing DNA methylation significantly increased the detection rate at all NPC stages as well as local recurrence, using this selected four‐gene panel (p < 0.05). MS‐HRM assay on a selected gene panel has great potential to become a noninvasive and complementary test for NPC early and recurrent detection in combination with the EBV DNA test to increase the sensitivity for NPC detection at an early stage.     

Correlation between CpG methylation profiles and hormone receptor status in breast cancers

Introduction

Aberrant DNA methylation has been found frequently in human breast cancers, associated with the loss of expression of a number of regulatory genes for growth and correlated to clinical outcomes. The present study was undertaken to determine whether methylation of a set of growth-suppressor genes would correlate to the expression of estrogen receptors (ERs) and progesterone receptors (PRs).

Methods

We used a pyrosequencing methylation analysis to study the methylation of 12 known growth-suppressor genes in 90 pairs of malignant/normal breast tissues. We also examined the expression of ERs and PRs in those specimens by immunohistochemistry. Mutations of p53 in tumor cells were detected by direct sequencing.

Results

Twelve tumor-suppressor genes: ARHI, RASSF1A, HIN-1, RARβ2, hMLH1, 14-3-3 σ, RIZ1, p16, E-cadherin, RIL, CDH13, and NKD2 were selected for this methylation study. Five of them (RIL, HIN-1, RASSF1A, CDH13, and RARβ2) were frequently methylated in breast cancers (57%, 49%, 58%, 44%, and 17%, respectively) but not the normal breast (0–4%). Two panels of methylation profiles were defined. The methylation of the HIN-1/RASSFIA panel strongly correlated to the expression of ERs, PRs, and hormone receptors (HRs; which were defined as 'positive' if ERs and/or PRs were positive; p < 0.001). Conversely, the methylation of the RIL/CDH13 panel strongly correlated to negative ER, PR, and HR expression (p = 0.001, 0.025, and 0.001, respectively). The subset of triple-negative breast cancers (in other words, those with negative ER, PR, and HER-2/neu status) was positively associated with the methylation of the RIL/CDH13 panel and negatively associated with the HIN-1/RASSF1A panel. Mutations of p53 were found in nine breast tumors (11%), seven of which lacked methylation in both panels.

Conclusion

We have defined two panels (HIN-1/RASSFIA, and RIL/CDH13) of methylation profiles, which correlated, either positively or negatively, to HR status.