Development of a multiplex methylation specific PCR suitable for (early) detection of non-small cell lung cancer

Lung cancer is a worldwide health problem and a leading cause of cancer-related deaths. Silencing of potential tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event in the initiation and development of cancer. Thus, methylated cancer type-specific TSGs in DNA can serve as useful biomarkers for early cancer detection. We have now developed a “Multiplex Methylation Specific PCR” (MMSP) assay for analysis of the methylation status of multiple potential TSGs by a single PCR reaction. This method will be useful for early diagnosis and treatment outcome studies of non-small cell lung cancer (NSCLC). Genome-wide CpG methylation and expression microarrays were performed on lung cancer tissues and matched distant non-cancerous tissues from three NSCLC patients from China. Thirty-eight potential TSGs were selected and analyzed by methylation PCR on bisulfite treated DNA. On the basis of sensitivity and specificity, six marker genes, HOXA9, TBX5, PITX2, CALCA, RASSF1A, and DLEC1, were selected to establish the MMSP assay. This assay was then used to analyze lung cancer tissues and matched distant non-cancerous tissues from 70 patients with NSCLC, as well as 24 patients with benign pulmonary lesion as controls. The sensitivity of the assay was 99% (69/70). HOXA9 and TBX5 were the 2 most sensitive marker genes: 87% (61/70) and 84% (59/70), respectively. RASSF1A and DLEC1 showed the highest specificity at 99% (69/70). Using the criterion of identifying at least any two methylated marker genes, 61/70 cancer samples were positive, corresponding to a sensitivity of 87% and a specificity of 94%. Early stage I or II NSCLC could even be detected with a 100% specificity and 86% sensitivity. In conclusion, MMSP has the potential to be developed into a population-based screening tool and can be useful for early diagnosis of NSCLC. It might also be suitable for monitoring treatment outcome and recurrence.     

Quantitative analysis of mRNA expression levels and DNA methylation profiles of three neighboring genes: FUS1, NPRL2/G21 and RASSF1A in non-small cell lung cancer patients

Background

Tumor suppressor gene (TSG) inactivation plays a crucial role in carcinogenesis. FUS1, NPRL2/G21 and RASSF1A are TSGs from LUCA region at 3p21.3, a critical chromosomal region in lung cancer development. The aim of the study was to analyze and compare the expression levels of these 3 TSGs in NSCLC, as well as in macroscopically unchanged lung tissue surrounding the primary lesion, and to look for the possible epigenetic mechanism of TSG inactivation via gene promoter methylation.

Methods

Expression levels of 3 TSGs and 2 DNA methyltransferases, DNMT1 and DNMT3B, were assessed using real-time PCR method (qPCR) in 59 primary non-small cell lung tumors and the matched macroscopically unchanged lung tissue samples. Promoter methylation status of TSGs was analyzed using methylation-specific PCRs (MSP method) and Methylation Index (MI) value was calculated for each gene.

Results

The expression of all three TSGs were significantly different between NSCLC subtypes: RASSF1A and FUS1 expression levels were significantly lower in squamous cell carcinoma (SCC), and NPRL2/G21 in adenocarcinoma (AC). RASSF1A showed significantly lower expression in tumors vs macroscopically unchanged lung tissues. Methylation frequency was 38–76 %, depending on the gene. The highest MI value was found for RASSF1A (52 %) and the lowest for NPRL2/G21 (5 %). The simultaneous decreased expression and methylation of at least one RASSF1A allele was observed in 71 % tumor samples. Inverse correlation between gene expression and promoter methylation was found for FUS1 (rs = −0.41) in SCC subtype. Expression levels of DNMTs were significantly increased in 75–92 % NSCLCs and were significantly higher in tumors than in normal lung tissue. However, no correlation between mRNA expression levels of DNMTs and DNA methylation status of the studied TSGs was found.

Conclusions

The results indicate the potential role of the studied TSGs in the differentiation of NSCLC histopathological subtypes. The significant differences in RASSF1A expression levels between NSCLC and macroscopically unchanged lung tissue highlight its possible diagnostic role in lung cancer in situ recognition. High percentage of lung tumor samples with simultaneous RASSF1A decreased expression and gene promoter methylation indicates its epigenetic silencing. However, DNMT overexpression doesn’t seem to be a critical determinate of its promoter hypermethylation.     

Development of a Non-Invasive Method,Multiplex Methylation Specific PCR (MMSP), for Early Diagnosis of Nasopharyngeal Carcinoma

Increasing evidence demonstrated that inactivation of tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event during carcinogenesis. Aiming at developing early diagnostic or prognostic tools for various tumors, we took an EBV-associated tumor, nasopharyngeal carcinoma (NPC), as a model and developed a powerful assay based on “multiplex methylation specific-PCR (MMSP)”. The MMSP assay was designed to detect tumor-specific methylation status of several NPC-related genes and was capable of acquiring multiplex information simultaneously through a single PCR reaction with the tiny tumor DNA derived from the direct body fluid close to the primary tumor. In this study, we collected paired nasopharyngeal (NP) swabs and NPC biopsies from 49 NPC patients and twenty noncancerous controls. A panel of markers including two EBV, and two cellular TSG markers were applied in this NPC-specific-MMSP assay. We optimized the working condition of MMSP so that it provides information equal to that from the corresponding separate PCRs. The results showed that MMSP patterns of NPC swab were largely consistent with those of corresponding biopsies and significantly distinguished themselves from those of 20 noncancerous volunteers. Among the 69 samples (49 NPCs and 20 normal controls), the sensitivity of detecting NPC from NP swabs is 98%. The specificity is as high as 100%. In conclusion, being characterized by its noninvasiveness, high reproducibility and informativeness, MMSP assay is a reliable and potential diagnostic tool for NPC. It paves the way for the development of population screening and early diagnosis approaches for various tumor types.     

Promoter hypermethylation of tumor suppressor genes in serum as potential biomarker for the diagnosis of nasopharyngeal carcinoma

Purpose: Promoter hypermethylation of tumor suppressor genes may serve as a promising biomarker for the diagnosis of cancer. Cell-free circulating DNA (cf-DNA) shares hypermethylation status with primary tumors. This study investigated promoter hypermethylation of five tumor suppressor genes as markers in the detection of nasopharyngeal carcinoma (NPC) in serum samples. Methods: cf-DNA was extracted from serum collected from 40 NPC patients and 41 age- and sex-matched healthy subjects. The promoter hypermethylation status of the five genes (RASSF1, CDKN2A, DLEC1, DAPK1 and UCHL1) was assessed by methylation-specific PCR after sodium bisulfite conversion. Differences in the methylation status of these five genes between NPC patients and healthy subjects were compared. Results: The concentration of cf-DNA in the serum of NPC patients was significantly higher than that in normal controls. The five tumor suppressor genes – RASSF1, CDKN2A, DLEC1, DAPK1 and UCHL1 – were found to be methylated in 17.5%, 22.5%, 25.0%, 51.4% and 64.9% of patients, respectively. The combination of four-gene marker – CDKN2A, DLEC1, DAPK1 and UCHL1 – had the highest sensitivity and specificity in predicting NPC. Conclusion: Screening DNA hypermethylation of tumor suppressor genes in serum was a promising approach for the diagnosis of NPC.     

Clinical significance of promoter hypermethylation of RASSF1A, RARbeta2, BRCA1 and HOXA5 in breast cancers of Indian patients

Promoter hypermethylation of genes is implicated in the pathogenesis of many cancers, including breast cancer. Herein, we analyzed the promoter methylation status of a panel of critical growth regulatory genes, RASSF1A, RARbeta2, BRCA1 and HOXA5, in 54 breast cancers and 5 distant normal breast tissues of Indian patients. The methylation data were correlated with clinicopathological characteristics and hormone receptor status to determine the impact of methylation in breast carcinogenesis. Promoter hypermethylation of RASSF1A was observed in 39/54 (72%), HOXA5 in 36/54 (67%), BRCA1 in 15/54 (28%) and RARbeta2 in 8/54 (15%) breast cancers. Our most significant findings were the association of RASSF1A methylation with nodal metastasis (p=0.05); and RARbeta2 methylation with age (all tumors in patients in the older age group were methylated, p=0.04). Further, the interactions between DNA methylation and hormone receptor biology in breast cancer cells are beginning to be clearly understood. In this context the association of HOXA5 methylation with loss of ERalpha (p=0.009) is noteworthy.     

Methylation status and protein expression of RASSF1A in breast cancer patients

Recently genetics and epigenetics alterations have been found to be characteristic of malignancy and hence can be used as targets for detection of neoplasia. RAS association domain family protein 1A (RASSF1A) gene hypermethylation has been a subject of interest in recent researches on cancer breast patients. The aim of the present study was to evaluate whether RASSF1A methylation status and RASSF1A protein expression are associated with the major clinico-pathological parameters. One hundred and twenty breast cancer Egyptian patients and 100-control subjects diagnosed with benign lesions of the breast were enrolled in this study. We evaluated RASSF1A methylation status in tissue and serum samples using Methyl specific PCR together with RASSF1A protein expression in tissues by immunohistochemistry. Results were studied in relation to known prognostic clinicopathological features in breast cancer. Frequency of RASSF1A methylation in tissues and serum were 70 and 63.3 % respectively and RASSF1A protein expression showed frequency of 46.7 %. There was an association between RASSF1A methylation in tissues, serum and loss of protein expression in tissues with invasive carcinoma, advanced stage breast cancer, L.N. metastasis, ER/PR and HER2 negativity. RASSF1A methylation in serum showed high degree of concordance with methylation in tissues (Kappa = 0.851, P < 0.001). RASSF1A hypermethylation in tissues and serum and its protein expression may be a valid, reliable and sensitive tool for detection and follow up of breast cancer patients.     

CpG island hypermethylation of multiple tumor suppressor genes associated with loss of their protein expression during rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine

The epigenetic mechanisms underlying the tumorigenesis caused by polycyclic aromatic hydrocarbons and nitrosamine compounds such as 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are currently unknown. We reported previously that dynamic changes in DNA methylation occurred during MCA/DEN-induced rat lung carcinogenesis. Here, we used the same animal model to further study the evolution of methylation alterations in tumor suppressor genes (TSGs) DAPK1, FHIT, RASSF1A, and SOCS-3. We found that none of these genes were methylated in either normal or hyperplasia tissue. However, as the severity of the cancer progressed through squamous metaplasia and dysplasia to carcinoma in situ (CIS) and infiltrating carcinoma, so methylation became more prevalent. Particularly dramatic increases in the level of methylation, the average number of methylated genes, and the incidence of concurrent methylation in three genes were observed in CIS and infiltrating carcinoma. Similar but less profound changes were seen in squamous metaplasia and dysplasia. Furthermore, methylation status was closely correlated to loss of protein expression for these genes, with protein levels markedly declining along the continuum of carcinogenesis. These results suggest that progressive CpG island hypermethylation leading to inactivation of TSGs might be a vital molecular mechanism in the pathogenesis of MCA/DEN-induced multistep rat lung carcinogenesis.     

Quantification of regional DNA methylation by liquid chromatography/tandem mass spectrometry

Promoter hypermethylation-associated tumor suppressor gene (TSG) silencing has been explored as a therapeutic target for hypomethylating agents. Promoter methylation change may serve as a pharmacodynamic endpoint for evaluation of the efficacy of these agents and predict the patient’s clinical response. Here a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been developed for quantitative regional DNA methylation analysis using the molar ratio of 5-methyl-2′-deoxycytidine (5mdC) to 2′-deoxycytidine (2dC) in the enzymatic hydrolysate of fully methylated bisulfite-converted polymerase chain reaction (PCR) amplicons as the methylation indicator. The assay can differentiate 5% of promoter methylation level with an intraday precision ranging from 3 to 16% using two TSGs: HIN-1 and RASSF1A. This method was applied to characterize decitabine-induced promoter DNA methylation changes of these two TSGs in a breast cancer MCF-7 cell line. Promoter methylation of these TSGs was found to decrease in a dose-dependent manner. Correspondingly, the expression of these TSGs was enhanced. The sensitivity and reproducibility of the method make it a valuable tool for specific gene methylation analysis that could aid characterization of hypomethylating activity on specific genes by hypomethylating agents in a clinical setting.     

Relationship between tumor DNA methylation status and patient characteristics in African-American and European-American women with breast cancer

Aberrant DNA methylation is critical for development and progression of breast cancer. We investigated the association of CpG island methylation in candidate genes and clinicopathological features in 65 African-American (AA) and European-American (EA) breast cancer patients. Quantitative methylation analysis was carried out on bisulfite modified genomic DNA and sequencing (pyrosequencing) for promoter CpG islands of p16, ESR1, RASSF1A, RARβ2, CDH13, HIN1, SFRP1 genes and the LINE1 repetitive element using matched paired non-cancerous and breast tumor specimen (32 AA and 33 EA women). Five of the genes, all known tumor suppressor genes (RASSF1A, RARβ2, CDH13, HIN1 and SFRP1), were found to be frequently hypermethylated in breast tumor tissues but not in the adjacent non-cancerous tissues. Significant differences in the CDH13 methylation status were observed by comparing DNA methylation between AA and EA patients, with more obvious CDH13 methylation differences between the two patient groups in the ER- disease and among young patients (age<50). In addition, we observed associations between CDH13, SFRP1, and RASSF1A methylation and breast cancer subtypes and between SFRP1 methylation and patient's age. Furthermore, tumors that received neoadjuvant therapy tended to have reduced RASSF1A methylation when compared with chemotherapy na?ve tumors. Finally, Kaplan Meier survival analysis showed a significant association between methylation at 3 loci (RASSF1A, RARβ2 and CDH13) and reduced overall disease survival. In conclusion, the DNA methylation status of breast tumors was found to be significantly associated with clinicopathological features and race/ethnicity of the patients.     

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.     

RASSF1A Promoter Methylation Levels Positively Correlate with Estrogen Receptor Expression in Breast Cancer Patients

The aim of this study was to investigate the relationship between the promoter methylation in five cancer-associated genes and clinicopathologic features for identification of molecular markers of tumor metastatic potential and hormone therapy response efficiency in breast cancer. The methylation levels in paraffin-embedded tumor tissues, plasma, and blood cells from 151 sporadic breast cancer patients and blood samples of 50 controls were evaluated by quantitative multiplex methylation-specific polymerase chain reaction. DNA methylation of RAS-association domain family member 1 (RASSF1A), estrogen receptor 1 (ESR1), cadherin 1, type 1, E-cadherin (CDH1), TIMP metallopeptidase inhibitor 3 (TIMP3) and spleen tyrosine kinase (SYK) genes was detected in the tumors of 124, 19, 15, 15, and 6 patients with mean levels of 48.45%, 3.81%, 2.36%, 27.55%, and 10.81%, respectively. Plasma samples exhibited methylation in the same genes in 25, 10, 15, 17, and 3 patients with levels of 22.54%, 17.20%, 22.87%, 31.93%, and 27.42%, respectively. Cumulative methylation results confirmed different spectra in tumor and plasma samples. Simultaneous methylation in tumors and plasma were shown in less than 17% of patients. RASSF1A methylation levels in tumor samples statistically differ according to tumor size (P = .029), estrogen receptor (ER) and progesterone receptor (PR) status (P = .000 and P = .004), and immunohistochemical subtype (P = .000). Moreover, the positive correlation was found between RASSF1A methylation levels and percentage of cancer cells expressing ER and PR. The direct relationship between RASSF1A promoter methylation and expression of ER could aid the prognosis of hormonal therapy response.     

Role of RASSF1A promoter methylation in the pathogenesis of hepatocellular carcinoma: a meta-analysis of 21 cohort studies

We carried out the current meta-analysis aiming to comprehensively assess the potential role of RASSF1A aberrant promoter methylation in the pathogenesis of hepatocellular carcinoma (HCC). A range of electronic databases were searched: Web of Science (1945–2013), the Cochrane Library Database (Issue 12, 2013), PubMed (1966–2013), EMBASE (1980–2013), CINAHL (1982–2013) and the Chinese Biomedical Database (CBM) (1982–2013) without language restrictions. Meta-analysis was conducted using the STATA 12.0 software. Crude risk difference (RD) with their 95 % confidence interval (95 % CI) was calculated. In the present meta-analysis, 21 clinical cohort studies with a total of 1,205 HCC patients were included. The results of our meta-analysis illustrated that the frequency of RASSF1A promoter methylation in cancer tissues were significantly higher than those of normal, adjacent and benign tissues (cancer tissues vs. normal tissues: RD = 0.63, 95 % CI 0.53–0.73, P < 0.001; cancer tissues vs. adjacent tissues: RD = 0.43, 95 % CI 0.33–0.53, P < 0.001; cancer tissues vs. benign tissues: RD = 0.48, 95 % CI 038–0.58, P < 0.001; respectively). Further subgroup by ethnicity demonstrated that RASSF1A aberrant promoter methylation was correlated with the pathogenesis of HCC among both Asians and Caucasians (all P < 0.05). The current meta-analysis suggests that RASSF1A aberrant promoter methylation may be implicated in the pathogenesis of HCC. Thus, detection of RASSF1A promoter methylation may be a helpful and valuable biomarker for diagnosis and prognosis of HCC.     

Epigenetic silencing of human T (brachyury homologue) gene in non-small-cell lung cancer

Early detection of lung cancer is challenging due to a lack of adequate biomarkers. To discover novel tumor suppressor genes (TSGs) silenced by aberrant promoter methylation, we analyzed the gene expression profiles of two lung adenocarcinoma cell lines using pharmacologic-unmasking and subsequent microarray-analysis. Among 617 genes upregulated, we selected 30 genes and investigated the methylation status of their promoters by bisulfite sequencing analysis. Aberrant methylation was detected in four genes (CRABP2, NOEY2, T, MAP2K3) in at least one lung adenocarcinoma cell lines. Furthermore, the T promoter was methylated in 60% of primary lung adenocarcinomas versus 13% of non-malignant lung tissues. Conversely, RT-PCR analysis revealed T expression was low in lung tumors, while high in normal tissues. In addition, no non-synonymous mutations related to gene silencing were found. While further analysis is warranted, our results suggest that T has the potential to be a novel candidate TSG in lung cancer.     

TBX2 acts as a potent transcriptional silencer of tumour suppressor genes through interaction with the CoREST complex to sustain the proliferation of breast cancers

Chromosome 17q23 amplification occurs in 20% of primary breast tumours and is associated with poor outcome. The TBX2 gene is located on 17q23 and is often over-expressed in this breast tumour subset. TBX2 is an anti-senescence gene, promoting cell growth and survival through repression of Tumour Suppressor Genes (TSGs), such as NDRG1 and CST6. Previously we found that TBX2 cooperates with the PRC2 complex to repress several TSGs, and that PRC2 inhibition restored NDRG1 expression to impede cellular proliferation. Here, we now identify CoREST proteins, LSD1 and ZNF217, as novel interactors of TBX2. Genetic or pharmacological targeting of CoREST emulated TBX2 loss, inducing NDRG1 expression and abolishing breast cancer growth in vitro and in vivo. Furthermore, we uncover that TBX2/CoREST targeting of NDRG1 is achieved by recruitment of TBX2 to the NDRG1 promoter by Sp1, the abolishment of which resulted in NDRG1 upregulation and diminished cancer cell proliferation. Through ChIP-seq we reveal that 30% of TBX2-bound promoters are shared with ZNF217 and identify novel targets repressed by TBX2/CoREST; of these targets a lncRNA, LINC00111, behaves as a negative regulator of cell proliferation. Overall, these data indicate that inhibition of CoREST proteins represents a promising therapeutic intervention for TBX2-addicted breast tumours.     

Novel miRNA genes methylated in lung tumors

MicroRNAs play an important role in the regulation of expression of many genes and are involved in carcinogenesis. The regulation of miRNA gene expression can involve the methylation of promoter CpG islands. In this work, the methylation of six miRNA genes (mir-107, mir-125b-1, mir-130b, mir-137, mir-375, and mir-1258) in non-small-cell lung cancer (NSCLC) was studied for the first time by methylation-specific PCR using a representative set of specimens (39 cases). Four new genes (mir-125b-1, mir-137, mir-375, and mir-1258) methylated in primary NSCLC tumors were identified with frequencies of 56, 31, 56, and 36%, respectively. The frequencies of miRNA promoter methylation in DNA of tumors and histologically normal tissues differed significantly (P ≤ 0.05 by Fisher’s test). In lung tissues of 20 donors without a history of cancer, these genes were only methylated in a few cases. It was also shown that the previously unstudied promoter CpG islands of mir-107 and mir-130b were not methylated in NSCLC. The frequencies of mir-125b-1 and mir-137 methylation were shown for the first time to correlate with NSCLC progression (clinical stage and metastasis).