Peripheral blood DNA methylation profiles are indicative of head and neck squamous cell carcinoma: An epigenome-wide association study

Head and neck cancer accounts for an estimated 47,560 new cases and 11,480 deaths annually in the United States, the majority of which are squamous cell carcinomas (HNSCC). The overall 5 year survival is approximately 60% and declines with increasing stage at diagnosis, indicating a need for non-invasive tests that facilitate the detection of early disease. DNA methylation is a stable epigenetic modification that is amenable to measurement and readily available in peripheral blood. We used a semi-supervised recursively partitioned mixture model (SS-RPMM) approach to identify novel blood DNA methylation markers of HNSCC using genome-wide methylation array data for peripheral blood samples from 92 HNSCC cases and 92 cancer-free control subjects. To assess the performance of the resultant markers, we constructed receiver operating characteristic (ROC) curves and calculated the corresponding area under the curve (AUC). Cases and controls were best differentiated by a methylation profile of six CpG loci (associated with FGD4, SERPINF1, WDR39, IL27, HYAL2 and PLEKHA6), with an AUC of 0.73 (95% CI: 0.62–0.82). After adjustment for subject age, gender, smoking, alcohol consumption and HPV16 serostatus, the AUC increased to 0.85 (95% CI: 0.76–0.92). We have identified a novel blood-based methylation profile that is indicative of HNSCC with a high degree of accuracy. This profile demonstrates the potential of DNA methylation measured in blood for development of non-invasive applications for detection of head and neck cancer.     

hTERT promoter methylation status in peripheral blood leukocytes as a molecular marker of head and neck cancer progression

Cancer cells, including head and neck cancer cell carcinoma (HNSCC), are characterized by an increased telomerase activity. This enzymatic complex is active in approximately 80–90% of all malignancies, and is regulated by various factors, including methylation status of hTERT gene promoter. hTERT methylation pattern has been thoroughly studied so far. It was proved that hTERT is aberrantly methylated in tumor tissue versus healthy counterparts. However, such effect has not yet been investigated in PBLs (peripheral blood leukocytes) of cancer patients. The aim of this study was to analyze the hTERT gene promoter methylation status in blood leukocytes. DNA was extracted from PBL of 92 patients with histologically diagnosed HNSCC and 53 healthy controls. Methylation status of whole hTERT promoter fragment with independent analysis of each 19 CpG sites was performed using bisulfide conversion technique followed by sequencing of PCR products. Not significant (p?=?0.0532) differences in the general frequency of hTERT CpG sites methylation were detected between patients and healthy controls. However, it was discovered that some of analyzed positions (CpG islands: 1 [p?=?0.0235], 5 [p?=?0.0462], 8 [p?=?0.0343]) are significantly more often methylated in HNSCC patients than in controls. The opposite finding was observed in case of CpG position 2 (p?=?0.0210). Furthermore, closer analysis of single CpG positions revealed differences in methylation status dependent on anatomical site and TNM classification. To conclude, hTERT promoter methylation status (general or single CpG sites) would be considered as a molecular markers of HNSCC diagnostics.     

An Epigenetic Signature in Peripheral Blood Predicts Active Ovarian Cancer

Background

Recent studies have shown that DNA methylation (DNAm) markers in peripheral blood may hold promise as diagnostic or early detection/risk markers for epithelial cancers. However, to date no study has evaluated the diagnostic and predictive potential of such markers in a large case control cohort and on a genome-wide basis.

Principal Findings

By performing genome-wide DNAm profiling of a large ovarian cancer case control cohort, we here demonstrate that active ovarian cancer has a significant impact on the DNAm pattern in peripheral blood. Specifically, by measuring the methylation levels of over 27,000 CpGs in blood cells from 148 healthy individuals and 113 age-matched pre-treatment ovarian cancer cases, we derive a DNAm signature that can predict the presence of active ovarian cancer in blind test sets with an AUC of 0.8 (95% CI (0.74–0.87)). We further validate our findings in another independent set of 122 post-treatment cases (AUC = 0.76 (0.72–0.81)). In addition, we provide evidence for a significant number of candidate risk or early detection markers for ovarian cancer. Furthermore, by comparing the pattern of methylation with gene expression data from major blood cell types, we here demonstrate that age and cancer elicit common changes in the composition of peripheral blood, with a myeloid skewing that increases with age and which is further aggravated in the presence of ovarian cancer. Finally, we show that most cancer and age associated methylation variability is found at CpGs located outside of CpG islands.

Significance

Our results underscore the potential of DNAm profiling in peripheral blood as a tool for detection or risk-prediction of epithelial cancers, and warrants further in-depth and higher CpG coverage studies to further elucidate this role.     

The prognostic significance of whole blood global and specific DNA methylation levels in gastric adenocarcinoma

Background

Epigenetics, particularly DNA methylation, has recently been elucidated as important in gastric cancer (GC) initiation and progression. We investigated the clinical and prognostic importance of whole blood global and site-specific DNA methylation in GC.

Methods

Genomic DNA was extracted from the peripheral blood of 105 Omani GC patients at diagnosis. DNA methylation was quantified by pyrosequencing of global DNA and specific gene promoter regions at 5 CpG sites for CDH1, 7 CpG sites for p16, 4 CpG sites for p53, and 3 CpG sites for RUNX3. DNA methylation levels in patients were categorized into low, medium, and high tertiles. Associations between methylation level category and clinicopathological features were evaluated using χ² tests. Survival analyses were carried out using the Kaplan-Meier method and log rank test. A backward conditional Cox proportional hazards regression model was used to identify independent predictors of survival.

Results

Older GC patients had increased methylation levels at specific CpG sites within the CDH1, p53, and RUNX-3 promoters. Male gender was significantly associated with reduced global and increased site-specific DNA methylation levels in CDH1, p16, and p53 promoters. Global DNA low methylation level was associated with better survival on univariate analysis. Patients with high and medium methylation vs. low methylation levels across p16 promoter CpG sites, site 2 in particular, had better survival. Multivariate analysis showed that global DNA hypermethylation was a significant independent predictor of worse survival (hazard ratio (HR) = 2.0, 95% CI: 1.1–3.8; p = 0.02) and high methylation mean values across p16 promoter sites 1–7 were associated with better survival with HR of 0.3 (95% CI, 0.1–0.8; p = 0.02) respectively.

Conclusions

Analysis of global and site-specific DNA methylation in peripheral blood by pyrosequencing provides quantitative DNA methylation values that may serve as important prognostic indicators.     

Physical activity and global genomic DNA methylation in a cancer-free population

Changes in DNA methylation may represent an intermediate step between the environment and human diseases. Little is known on whether behavioral risk factors may modify gene expression through DNA methylation. To assess whether DNA methylation is associated with different levels of physical activity, we measured global genomic DNA methylation using bisulfite-converted DNA and real-time PCR (MethyLight) for LINE-1 in peripheral blood of 161 participants aged 45–75 years enrolled in the North Texas Healthy Heart Study and levels of physical activity using an accelerometer (Actigraph GT1M Monitor). We found that individuals with physical activity 26–30 min/day had a significantly higher level of global genomic DNA methylation compared to those with physical activity ≤10 min/day (β = 2.52, 95% CI: 0.70, 4.35). However, the association was attenuated and became statistically insignificant after multivariate adjustment (β = 1.24, 95% CI: −0.93, 3.40). There were some suggestions of a positive association between physical activity and global genomic DNA methylation in non-Hispanics (β = 1.50, 95% CI: −0.08, 3.08) that warrants further investigation.Key words: DNA methylation, physical activity, peripheral blood     

Tumor-suppressor Gene Promoter Hypermethylation in Saliva of Head and Neck Cancer Patients

Head and neck squamous cell carcinoma (HNSCC) accounts for a bulk of the oral and laryngeal cancers, the majority (70%) of which are associated with smoking and excessive drinking, major known risk factors for the development of HNSCC. In contrast to reports that suggest an inverse relationship between smoking and global DNA CpG methylation, hypermethylation of promoters of a number of genes was detected in saliva collected from patients with HNSCC. Using a sensitive methylation-specific polymerase chain reaction (MSP) assay to determine specific methylation events in the promoters of RASSF1A, DAPK1, and p16 genes, we demonstrate that we can detect tumor presence with an overall accuracy of 81% in the DNA isolated from saliva of patients with HNSCC (n = 143) when compared with the DNA isolated from the saliva of healthy nonsmoker controls (n = 31). The specificity for this MSP panel was 87% and the sensitivity was 80% (with a Fisher exact test P < .0001). In addition, the test panel performed extremely well in the detection of the early stages of HNSCCs, with a sensitivity of 94% and a specificity of 87%, and a high κ concordance value of 0.8, indicating an excellent overall agreement between the presence of HNSCC and a positive MSP panel result. In conclusion, we demonstrate that the promoter methylation of RASSF1A, DAPK1, and p16 MSP panel is useful in detecting hypermethylation events in a noninvasive manner in patients with HNSCC.     

White blood cell global methylation and IL-6 promoter methylation in association with diet and lifestyle risk factors in a cancer-free population

Altered levels of global DNA methylation and gene silencing through methylation of promoter regions can impact cancer risk, but little is known about their environmental determinants. We examined the association between lifestyle factors and levels of global genomic methylation and IL-6 promoter methylation in white blood cell DNA of 165 cancer-free subjects, 18–78 years old, enrolled in the COMIR (Commuting Mode and Inflammatory Response) study, New York, 2009–2010. Besides self-administrated questionnaires on diet and physical activity, we measured weight and height, white blood cell (WBC) counts, plasma levels of high sensitivity C-reactive protein (hs-CRP), and genomic (LINE-1) and gene-specific methylation (IL-6) by pyrosequencing in peripheral blood WBC. Mean levels of LINE-1 and IL-6 promoter methylation were 78.2% and 57.1%, respectively. In multivariate linear regression models adjusting for age, gender, race/ethnicity, body mass index, diet, physical activity, WBC counts and CRP, only dietary folate intake from fortified foods was positively associated with LINE-1 methylation. Levels of IL-6 promoter methylation were not significantly correlated with age, gender, race/ethnicity, body mass index, physical activity or diet, including overall dietary patterns and individual food groups and nutrients. There were no apparent associations between levels of methylation and inflammation markers such as WBC counts and hs-CRP. Overall, among several lifestyle factors examined in association with DNA methylation, only dietary folate intake from fortification was associated with LINE-1 methylation. The long-term consequence of folate fortification on DNA methylation needs to be further evaluated in longitudinal settings.     

Decitabine Rescues Cisplatin Resistance in Head and Neck Squamous Cell Carcinoma

Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) reduces survival. In this study we hypothesized that methylation of key genes mediates cisplatin resistance. We determined whether a demethylating drug, decitabine, could augment the anti-proliferative and apoptotic effects of cisplatin on SCC-25/CP, a cisplatin-resistant tongue SCC cell line. We showed that decitabine treatment restored cisplatin sensitivity in SCC-25/CP and significantly reduced the cisplatin dose required to induce apoptosis. We then created a xenograft model with SCC-25/CP and determined that decitabine and cisplatin combination treatment resulted in significantly reduced tumor growth and mechanical allodynia compared to control. To establish a gene classifier we quantified methylation in cancer tissue of cisplatin-sensitive and cisplatin-resistant HNSCC patients. Cisplatin-sensitive and cisplatin-resistant patient tumors had distinct methylation profiles. When we quantified methylation and expression of genes in the classifier in HNSCC cells in vitro, we showed that decitabine treatment of cisplatin-resistant HNSCC cells reversed methylation and gene expression toward a cisplatin-sensitive profile. The study provides direct evidence that decitabine restores cisplatin sensitivity in in vitro and in vivo models of HNSCC. Combination treatment of cisplatin and decitabine significantly reduces HNSCC growth and HNSCC pain. Furthermore, gene methylation could be used as a biomarker of cisplatin-resistance.     

Cardiovascular disease risk factors and DNA methylation at the LINE-1 repeat region in peripheral blood from Samoan Islanders

Lower levels of LINE-1 methylation in peripheral blood have been previously associated with risk of developing non-communicable conditions, the most well-explored of these being cancer, although recent research has begun to link altered LINE-1 methylation and cardiovascular disease. We examined the relationship between LINE-1 methylation and factors associated with metabolic and cardiovascular diseases through quantitative bisulfite pyrosequencing in DNA from peripheral blood samples from participants of the Samoan Family Study of Overweight and Diabetes (2002–03). The sample included 355 adult Samoans (88 men and 267 women) from both American Samoa and Samoa. In a model including all sample participants, men had significantly higher LINE-1 methylation levels than women (p = 0.04) and lower levels of LINE-1 methylation were associated with higher levels of fasting LDL (p = 0.02) and lower levels of fasting HDL (p = 0.009). The findings from this study confirm that DNA “global” hypomethylation (as measured by methylation at LINE-1 repeats) observed previously in cardiovascular disease is associated with altered levels of LDL and HDL in peripheral blood. Additionally, these findings strongly argue the need for further research, particularly including prospective studies, in order to understand the relationship between LINE-1 DNA methylation measured in blood and risk factors for cardiovascular disease.Key words: cardiovascular disease, HDL, LDL, LINE-1, DNA methylation, Samoa     

Analysis of methylation-driven genes for predicting the prognosis of patients with head and neck squamous cell carcinoma

To help provide evidence for prognosis prediction and personalized targeted therapy for patients with head and neck squamous cell carcinoma (HNSCC), we investigated prognosis-specific methylation-driven genes in HNSCC. Survival time data, RNA sequencing data, and methylation data for HNSCC patients were downloaded from The Cancer Genome Atlas. The MethylMix R package based on the β mixture model was utilized to screen genes with different methylation statuses in tumor tissues and adjacent normal tissues, and a total of 182 HNSCC-related methylation-driven genes were then identified. A survival prediction scoring model based on multivariate Cox analysis was developed to screen the genes related to the prognosis of HNSCC, and a linear risk model of the methylation status of six genes (INA, LINC01354, TSPYL4, MAGEB2, EPHX3, and ZNF134) was constructed. The prognostic values of the six genes were further independently explored by survival analysis combined with methylation and gene expression analyses. The 5-year survival rate in the high-risk group of patients in the test set was 30.4% (95% CI: 22.7%-40.8%) and that in the low-risk group of patients was 65.5% (95% CI: 56.1%-76.5%). The area under the receiver operating characteristic curve for the model was 0.723, which further verified the specificity and sensitivity of the model. In addition, subsequent combined survival analysis revealed that all six genes could be used as independent prognostic markers and thus might be potential drug targets. The innovative method provides new insight into the molecular mechanism and prognosis of HNSCC.     

LINE1 methylation levels in pre-diagnostic leukocyte DNA and future renal cell carcinoma risk

Higher levels of LINE1 methylation in blood DNA have been associated with increased kidney cancer risk using post-diagnostically collected samples; however, this association has never been examined using pre-diagnostic samples. We examined the association between LINE1 %5mC and renal cell carcinoma (RCC) risk using pre-diagnostic blood DNA from the United States-based, Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) (215 cases/436 controls), and the Alpha-tocopherol, Beta-carotene Cancer Prevention Study (ATBC) of Finnish male smokers (191 cases/575 controls). Logistic regression adjusted for age at blood draw, study center, pack-years of smoking, body mass index, hypertension, dietary alcohol intake, family history of cancer, and sex was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) using cohort and sex-specific methylation categories. In PLCO, higher, although non-significant, RCC risk was observed for participants at or above median methylation level (M2) compared to those below the median (M1) (OR: 1.37, 95% CI: 0.96–1.95). The association was stronger in males (M2 vs. M1, OR: 1.54, 95% CI: 1.00–2.39) and statistically significant among male smokers (M2 vs. M1, OR: 2.60, 95% CI: 1.46–4.63). A significant interaction for smoking was also detected (P-interaction: 0.01). No association was found among females or female smokers. Findings for male smokers were replicated in ATBC (M2 vs. M1, OR: 1.31, 95% CI: 1.07–1.60). In a pooled analysis of PLCO and ATBC male smokers (281cases/755controls), the OR among subjects at or above median methylation level (M2) compared to those below the median (M1) was 1.89 (95% CI: 1.34–2.67, P-value: 3 x 10^–4); a trend was also observed by methylation quartile (P-trend: 0.002). These findings suggest that higher LINE1 methylation levels measured prior to cancer diagnosis may be a biomarker of future RCC risk among male smokers.     

Association of X-ray Repair Cross Complementing Group 1 Arg399Gln Polymorphisms with the Risk of Squamous Cell Carcinoma of the Head and Neck: Evidence from an Updated Meta-Analysis

Background

Epidemiologic studies have reported the association of X-ray repair cross-complementary group 1 (XRCC1) Arg399Gln polymorphisms with susceptibility to squamous cell carcinoma of the head and neck (HNSCC). However, the results were conflictive rather than conclusive. The purpose of this study was to clarify the association of XRCC1 Arg399Gln variants with HNSCC risk.

Methods

Systematic searches were performed through the search engines of PubMed, Elsevier, Science Direct, CNKI and Chinese Biomedical Literature Database. Summary odds ratio (OR) with 95% confidence intervals (CI) was computed to estimate the strength association.

Results

Overall, we did not observe any association of XRCC1 Arg399Gln polymorphisms with HNSCC risk in total population (OR = 0.95, 95% CI: 0.76–1.19 for Gln/Gln vs. Arg/Arg, OR = 1.05, 95% CI: 0.92–1.20 for Arg/Gln vs. Arg/Arg, and OR = 1.03, 95% CI: 0.90–1.18 for Gln/Gln+Arg/Gln vs. Arg/Arg) based on 18 studies including 3917 cases and 4560 controls. In subgroup analyses, we observed an increased risk of XRCC1 399 Arg/Gln genotype for HNSCC in Caucasians (OR = 1.20, 95% CI: 1.00–1.44) and Gln/Gln genotype for larynx squamous cell carcinoma (OR = 1.63, 95% CI: 1.10–2.40). We did not observe any association between XRCC1 Arg399Gln variants and HNSCC risk in additional subgroup analyses.

Conclusion

The results from this present meta-analysis suggest that XRCC1 Arg399Gln variants may contribute to HNSCC risk among Caucasians and to the risk of larynx squamous cell carcinoma. Further, well-designed studies with larger sample sizes are required to verify our findings.     

Genome-wide methylation and expression differences in HPV(+) and HPV(?) squamous cell carcinoma cell lines are consistent with divergent mechanisms of carcinogenesis

Oncogenic human papillomaviruses (HPV) are associated with nearly all cervical cancers and are increasingly important in the etiology of oropharyngeal tumors. HPV-associated head and neck squamous cell carcinomas (HNSCC) have distinct risk profiles and appreciate a prognostic advantage compared to HPV-negative HNSCC. Promoter hypermethylation is widely recognized as a mechanism in the progression of HNSCC, but the extent to which this mechanism is consistent between HPV(+) and HPV(−) tumors is unknown. To investigate the epigenetic regulation of gene expression in HPV-induced and carcinogen-induced cancers, we examined genome-wide DNA methylation and gene expression in HPV(+) and HPV(−) SCC cell lines. We used two platforms: the Illumina Infinium Methylation BeadArray and tiling arrays, and confirmed illustrative examples with pyrosequencing and quantitative PCR. These analyses indicate that HPV(+) cell lines have higher DNA methylation in genic and LINE-1 regions than HPV(−) cell lines. Differentially methylated loci between HPV(+) and HPV(−) cell lines significantly correlated with HPV-typed HNSCC primary tumor DNA methylation levels. Novel findings include higher promoter methylation of polycomb repressive complex 2 target genes in HPV(+) cells compared to HPV(−) cells and increased expression of DNMT3A in HPV(+) cells. Additionally, CDKN2A and KRT8 were identified as interaction hubs among genes with higher methylation and lower expression in HPV(−) cells. Conversely, RUNX2, IRS-1 and CCNA1 were major hubs with higher methylation and lower expression in HPV(+) cells. Distinct HPV(+) and HPV(−) epigenetic profiles should provide clues to novel targets for development of individualized therapeutic strategies.Key words: epigenetics, human papillomavirus, HNSCC, DNA methylation, squamous cell carcinoma, gene expression, microarrays, illumina infinium humanmethylation27 beadarray     

Association between P16INK4a Promoter Methylation and HNSCC: A Meta-Analysis of 21 Published Studies

Background

The p16^INK4a is an important tumor suppressor gene (TSG) and aberrant methylation of promoter is known to be a major inactivation mechanism of the tumor suppressor and tumor-related genes. Aberrant TSG methylation was considered an important epigenetic silencing mechanism in the progression of head and neck squamous cell carcinoma (HNSCC). However, some studies have reported differences in the methylation frequencies of P^16INK4a promoter between cancer and the corresponding control group. Therefore, we conducted a meta-analysis to better identify the association.

Methods

PubMed, Ovid, ISI Web of Science, and EMBASE were searched to identify eligible studies to evaluate the association of p16^INK4a promoter methylation and HNSCC. Odds ratio (ORs) and 95% confidence intervals (95%CI) were calculated to evaluate the strength of association between p16^INK4a promoter methylation and HNSCC.

Results

A total of twenty-one studies with 1155 cases and 1017 controls were included in the meta-analysis. The frequencies of p16^INK4a promoter methylation in the cancer group were significantly higher than those in the control group (cancer group: median: 46.67%, range = 7.84%-95.12%; control group: median: 18.37%, range = 0–83.33%; respectively). The pooled odds ratio was 3.37 (95%CI = 2.32–4.90) in the cancer group versus the corresponding control group under the random-effects model.

Conclusion

This meta-analysis of 21 published studies identified that aberrant methylation of p16^INK4a promoter was found to be significantly associated with HNSCC.     

Associations between genetic variation in one-carbon metabolism and LINE-1 DNA methylation in histologically normal breast tissues

Genome-wide DNA hypomethylation is an early event in the carcinogenic process. Percent methylation of long interspersed nucleotide element-1 (LINE-1) is a biomarker of genome-wide methylation and is a potential biomarker for breast cancer. Understanding factors associated with percent LINE-1 DNA methylation in histologically normal tissues could provide insight into early stages of carcinogenesis. In a cross-sectional study of 121 healthy women with no prior history of cancer who underwent reduction mammoplasty, we examined associations between plasma and breast folate, genetic variation in one-carbon metabolism, and percent LINE-1 methylation using multivariable regression models (adjusting for race, oral contraceptive use, and alcohol use). Results are expressed as the ratio of LINE-1 methylation relative to that of the referent group, with the corresponding 95% confidence intervals (CI). We found no significant associations between plasma or breast folate and percent LINE-1 methylation. Variation in MTHFR, MTR, and MTRR were significantly associated with percent LINE-1 methylation. Variant allele carriers of MTHFR A1289C had 4% lower LINE-1 methylation (Ratio 0.96, 95% CI 0.93–0.98), while variant allele carriers of MTR A2756G (Ratio 1.03, 95% CI 1.01–1.06) and MTRR A66G (Ratio 1.03, 95% CI 1.01–1.06) had 3% higher LINE-1 methylation, compared to those carrying the more common genotypes of these SNPs. DNA methylation of LINE-1 elements in histologically normal breast tissues is influenced by polymorphisms in genes in the one-carbon metabolism pathway. Future studies are needed to investigate the sociodemographic, environmental and additional genetic determinants of DNA methylation in breast tissues and the impact on breast cancer susceptibility.     

Classification of DNA methylation patterns in tumor cell genomes using a CpG island microarray

Our group has initiated experiments to epigenetically profile CpG island hypermethylation in genomic DNA from tissue specimens of head and neck squamous cell carcinoma (HNSCC) using a microarray of 12,288 CpG island clones. Our technique, known as a methylation-specific restriction enzyme (MSRE) analysis, is a variation of the differential methylation hybridization (DMH) technique, in that it is not an array comparison of two DNA samples using methylation-specific restriction enzymes. Instead, it is a comparison of a single DNA sample's response to a methylation-sensitive restriction enzyme (HpaII) and its corresponding methylation-insensitive isoschizomer (MspI). Estimation of the reproducibility of this microarray assay by intraclass correlation (ICC) demonstrated that in four replicate experiments for three tumor specimens, the ICC observed for a given tumor specimen ranged from 0.68 to 0.85 without filtering of data. Repeated assays achieved 87% concordance or greater for all tumors after filtering of array data by fluorescence intensity. We utilized hierarchical clustering on a population of 37 HNSCC samples to cluster tumor samples with similar DNA methylation profiles. Supervised learning techniques are now being utilized to allow us to identify associations between specific epigenetic signatures and clinical parameters. Such techniques will allow us to identify select groups of CpG island loci that could be used as epigenetic markers for both diagnosis and prognosis in HNSCC.