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.     

Methylation-dependent fragment separation: direct detection of DNA methylation by capillary electrophoresis of PCR products from bisulfite-converted genomic DNA

Fundamental to understanding the role of cytosine (C) methylation in genomic DNA (gDNA) is the need for robust analysis methods to determine the location and degree of this modification. We report a novel method for methylation detection by denaturing capillary electrophoresis (CE) using standard fragment analysis conditions. Bisulfite treatment of gDNA will selectively deaminate C but not 5-methylcytosine (5mC). Amplicons generated from bisulfite-converted gDNA are analyzed immediately after PCR using a 6-carboxy fluorescein (6-FAM) dye-labeled primer. The amplicons from methylated and unmethylated gDNA separate based solely on base composition due to the presence of multiple C versus thymine (T) differences. By direct detection of PCR amplicons following PCR using primers that anneal independent of methylation status, the overall workflow from gDNA sample input to data analysis is relatively simple. Furthermore, the same PCR product is suitable for additional analyses such as direct sequencing, cloning and sequencing, single-base extension, and post-PCR incorporation of a modified dCTP, the latter of which allows resolution of amplicons with as little as a single C/T difference. We show the utility of this novel CE detection assay by analyzing the hypermethylated region of the fragile-X FMR1 locus.     

Hypermethylation of the Keap1 gene in human lung cancer cell lines and lung cancer tissues

Low expression of the oxidative stress sensor Keap1 is thought to be involved in carcinogenesis. However, the mechanisms responsible for inactivation of the Keap1 gene remain unknown. We investigated Keap1 expression using RT-PCR and found that it was downregulated in lung cancer cell lines and tissues when compared with a normal bronchial epithelial cell line. Treatment with 5-Aza-2′-deoxycytidine restored Keap1 expression in lung cancer cell lines, indicating the silencing mechanism to be promoter methylation. Moreover, we evaluated cytosine methylation in the Keap1 promoter and demonstrated that the P1 region, including 12 CpG sites, was highly methylated in lung cancer cells and tissues, but not in normal cells. Importantly, we found evidence that three specific CpG sites (the 3rd, 6th, and 10th CpGs of P1) might be binding sites for proteins that regulate Keap1 expression. Thus, our results suggest for the first time that Keap1 expression is regulated by an epigenetic mechanism in lung cancer.     

WT1 CpG islands methylation in human lung cancer: A pilot study

Background

CpG island hypermethylation of gene promoters and regulatory regions is a well-known mechanism of epigenetic silencing of tumor suppressors and is directly linked to carcinogenesis. Wilm’s tumor gene (WT1) is a tumor suppressor protein involved in the regulation of human cell growth and differentiation and a modulator of oncogenic K Ras signaling in lung cancer. Changes in the pattern of methylation of the WT1 gene have not yet been studied in detail in human lung cancer. In this study we compared the methylation profile of WT1 gene in samples of neoplastic and non-neoplastic lung tissue taken from the same patients.

Methods

DNA was extracted from neoplastic and normal lung tissue obtained from 16 patients with non small cell lung cancer (NSCLC). The methylation status of 29 CpG islands in the 5′ region of WT1 was determined by pyrosequencing. Statistical analysis was carried out by T test and Mann Whitney test.

Results

The mean percentage of methylation, considering all CpG islands of WT1 in the neoplastic tissues of the 16 NSCLC patients, was 16.2 ± 3.4, whereas in the normal lung tissue from the same patients it was 5.6 ± 1.7 (p < 0.001). Adenocarcinomas presented higher methylation levels than squamous cell carcinomas (p < 0,001).

Conclusions

Methylation of WT1 gene is significantly increased in NSCLC. Both histotype and exposure to cigarette smoke heavily influence the pattern of CpG islands which undergo hypermethylation.     

Comparison of bisulfite sequencing PCR with pyrosequencing for measuring differences in DNA methylation

DNA methylation strongly affects chromatin structure and the regulation of gene expression. For many years, bisulfite sequencing PCR (BSP) has served as the “gold standard” for measuring DNA methylation. However, with the evolution of pyrosequencing as a tool to evaluate DNA methylation, the need arises to compare the relative efficiencies of the two techniques in measuring DNA methylation. We provide for the first time a direct assessment of BSP and pyrosequencing to detect and quantify hypomethylation, hypermethylation, and mixed methylation of the ABCB1 promoter in various drug-sensitive and drug-resistant MCF-7 breast cancer cell lines through head-to-head experimentation. Our findings indicate that although both methods can reliably detect increased, decreased, and mixed methylation of DNA, BSP appears to be more sensitive than pyrosequencing at detecting strong hypermethylation of DNA. However, we also observed greater variability in the methylation of CpG sites by BSP, possibly due to the additional bacterial cloning step required by BSP over pyrosequencing. BSP and pyrosequencing equally detected hypomethylation and mixed methylation of DNA. The ability of pyrosequencing to reliably detect differences in DNA methylation across cell populations without requiring the cloning of bisulfite-treated DNA into bacterial expression vectors was seen as a major advantage of this technique.     

Prognostic value of RASSF1A methylation status in non-small cell lung cancer (NSCLC) patients: A meta-analysis of prospective studies

Objective: Ras association domain family 1?A (RASSF1A) has been regarded as a biomarker predicting the prognosis of non-small cell lung cancer (NSCLC), but previous findings are inconsistent. This meta-analysis of prospective studies aimed to assess the value of RASSF1A methylation in predicting the prognosis of NSCLC patients.

Methods: Studies were searched in PubMed and Web of Science. The estimates of the effects and the corresponding 95% confidence intervals (95% CIs) were used for the analyses. The overall effects of RASSF1A methylation on overall survival (OS) were estimated, after which subgroup analysis based on regions was conducted. Sensitivity analyses were conducted to restrict the studies with certain features.

Results: A total of 16 studies with 2210 participants were included in this meta-analysis. The overall analysis result indicated that RASSF1A methylation had no statistically significant effects on OS of NSCLC patients (HR?=?1.28; 95% CI 0.86–1.70), which were confirmed by the subgroup analysis. However, the sensitivity analysis indicated that RASSF1A methylation from lung cancer tissues was significantly associated with lower OS (HR?=?1.24; 95% CI 1.04–1.45).

Conclusion: RASSF1A methylation in lung cancer tissue can serve as a prognostic factor of NSCLC. More studies are needed to uncover the underlying mechanisms.     

Epigenetic therapy approaches in non-small cell lung cancer: Update and perspectives

Non-small cell lung cancer (NSCLC) still constitutes the most common cancer-related cause of death worldwide. All efforts to introduce suitable treatment options using chemotherapeutics or targeted therapies have, up to this point, failed to exhibit a substantial effect on the 5-year-survival rate. The involvement of epigenetic alterations in the evolution of different cancers has led to the development of epigenetics-based therapies, mainly targeting DNA methyltransferases (DNMTs) and histone-modifying enzymes. So far, their greatest success stories have been registered in hematologic neoplasias. As the effects of epigenetic single agent treatment of solid tumors have been limited, the investigative focus now lies on combination therapies of epigenetically active agents with conventional chemotherapy, immunotherapy, or kinase inhibitors. This review includes a short overview of the most important preclinical approaches as well as an extensive discussion of clinical trials using epigenetic combination therapies in NSCLC, including ongoing trials. Thus, we are providing an overview of what lies ahead in the field of epigenetic combinatory therapies of NSCLC in the coming years.     

Development of a rapid,reliable and simple multiplex PCR assay for early detection of transgenic plant materials

Multiplex PCR is a variant of conventional PCR which includes two or more pairs of primers in a single reaction to amplify corresponding genes simultaneously. In this study, a reliable multiplex PCR analysis protocol was established for simple and fast detection of transgenes in plant materials. Two pairs of primers, corresponding to neomycin phosphotransferase gene and 1-aminocyclopropane-1-carboxylate synthase gene, were selected for target and resident gene respectively. The method bypasses routine DNA extraction, requires only very little amount of plant tissue and produces reliable results as shown by successful discrimination of transformed and nontransformed tobacco, tomato and kumquat materials. The method facilitates early identification of transgenic buds when they are still quite small.     

Methylation profiling using degenerated oligonucleotide primer-PCR specific for genome-wide amplification of bisulfite-modified DNA

DNA methylation is one of the essential epigenetic processes that play a role in regulating gene expression. Aberrant methylation of CpG-rich promoter regions has been associated with many forms of human cancers. The current method for determining the methylation status relies mainly on bisulfite treatment of genomic DNA, followed by methylation-specific PCR (MSP). The difficulty in acquiring a methylation profiling often is limited by the amount of genomic DNA that can be recovered from a given sample, whereas complex procedures of bisulfite treatment further compromise the effective template for PCR analysis. To circumvent these obstacles, we developed degenerated oligonucleotide primer (DOP)-PCR to enable amplification of bisulfite-modified genomic DNA at a genome-wide scale. A DOP pair was specially designed as follows: first 3' DOP, CTCGAGCTGHHHHHAACTAC, where H is a mixture of base consisting of 50% A, 25% T, and 25% C; and second 5' DOP, CTCGAGCTGDDDDDGTTTAG, where D is a mixture of base consisting of 50% T, 25% G, and 25% A. Our results showed that bisulfite-modified DNAs from a cell line, cord blood cells, or cells obtained by laser capture microdissection were amplified by up to 1000-fold using this method. Subsequent MSP analysis using these amplified DNAs on nine randomly selected cancer-related genes revealed that the methylation status of these genes remained identical to that derived from the original unamplified template.     

Urinary exosomal long noncoding RNAs serve as biomarkers for early detection of non-small cell lung cancer

Objective: Increasing the efficiency of early diagnosis using noninvasive biomarkers is crucial for enhancing the survival rate of lung cancer patients. We explore the differential expression of non-small cell lung cancer (NSCLC)-related long noncoding RNAs (lncRNAs) in urinary exosomes in NSCLC patients and normal controls to diagnose lung cancer.Methods: A differential expression analysis between NSCLC patients and healthy controls was performed using microarrays. Gene ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to predict potential functions of lncRNAs in NSCLC. quantitative real-time PCR (QT-PCR) was used to verify microarray results.Results: A total of 640 lncRNAs (70 up- and 570 down-regulated) were differentially expressed in NSCLC patients in comparison to healthy controls. Six lncRNAs were detected by QT-PCR. GO term and KEGG pathway analyses showed that differential lncRNAs were enriched in cellular component organization or biogenesis, as well as other biological processes and signaling pathways, such as the PI3K-AKT, FOXO, p53, and fatty acid biosynthesis.Conclusions: The differential lncRNAs in urinary exosomes are potential diagnostic biomarkers of NSCLC. The lncRNAs enriched in specific pathways may be associated with tumor cell proliferation, tumor cell apoptosis, and the cell cycle involved in the pathogenesis of NSCLC.     

Blood cadmium levels as a marker for early lung cancer detection

BackgroundWe assessed whether blood cadmium levels were associated with incident lung cancer and could be used in the context of a screening program for early-stage lung cancer.Material and methodsWe measured blood cadmium levels among 205 lung cancer patients and 205 matched controls. Cases and controls were matched for sex, age and smoking history (total pack-years, years since cessation for former smokers).ResultsThe odds ratio for those in the highest quartile of cadmium level (versus lowest) was four-fold (OR = 4.41, 95 % CI:2.01–9.67, p < 0.01). The association was present in former smokers (OR = 16.8, 95 % CI:3.96−71.2, p < 0.01), but not in current smokers (OR = 1.23, 95 % CI: 0.34–4.38) or in never smokers (OR not defined). Among former smokers, the association was present in both early- and late-stage lung cancer.ConclusionBlood cadmium levels may be a marker to help with the early detection of lung cancer among former smokers.     

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.     

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.     

A reassessment of semiquantitative analytical procedures for DNA methylation: comparison of bisulfite- and HpaII polymerase-chain-reaction-based methods

Two techniques in particular are used to study site-specific DNA methylation: genomic sequencing after bisulfite modification and polymerase chain reaction after digestion by a methylation-sensitive endonuclease (usually HpaII). Only the former methodology assesses the methylation status of all the cytosine residues in the DNA sequence, but it is so complex and time consuming that the latter procedure, though limited to the restriction sites recognized by the endonuclease(s) used, is often preferred at least for a first analysis. In this work we investigate differences between these two techniques in the assessment of DNA methylation and offer some suggestions on how to avoid uncorrected results. Although there is substantial accordance in the results obtained using these different techniques, we observed a general overestimate for methylation levels above 30% and a general underestimate for methylation levels below this value using the HpaII/PCR technique in the study of methylation of the 5'-flanking region of the mouse myogenin gene in cultured muscle cells and mouse tissues.     

Validation of a blood protein signature for non-small cell lung cancer

Background

CT screening for lung cancer is effective in reducing mortality, but there are areas of concern, including a positive predictive value of 4% and development of interval cancers. A blood test that could manage these limitations would be useful, but development of such tests has been impaired by variations in blood collection that may lead to poor reproducibility across populations.

Results

Blood-based proteomic profiles were generated with SOMAscan technology, which measured 1033 proteins. First, preanalytic variability was evaluated with Sample Mapping Vectors (SMV), which are panels of proteins that detect confounders in protein levels related to sample collection. A subset of well collected serum samples not influenced by preanalytic variability was selected for discovery of lung cancer biomarkers. The impact of sample collection variation on these candidate markers was tested in the subset of samples with higher SMV scores so that the most robust markers could be used to create disease classifiers. The discovery sample set (n = 363) was from a multi-center study of 94 non-small cell lung cancer (NSCLC) cases and 269 long-term smokers and benign pulmonary nodule controls. The analysis resulted in a 7-marker panel with an AUC of 0.85 for all cases (68% adenocarcinoma, 32% squamous) and an AUC of 0.93 for squamous cell carcinoma in particular. This panel was validated by making blinded predictions in two independent cohorts (n = 138 in the first validation and n = 135 in the second). The model was recalibrated for a panel format prior to unblinding the second cohort. The AUCs overall were 0.81 and 0.77, and for squamous cell tumors alone were 0.89 and 0.87. The estimated negative predictive value for a 15% disease prevalence was 93% overall and 99% for squamous lung tumors. The proteins in the classifier function in destruction of the extracellular matrix, metabolic homeostasis and inflammation.

Conclusions

Selecting biomarkers resistant to sample processing variation led to robust lung cancer biomarkers that performed consistently in independent validations. They form a sensitive signature for detection of lung cancer, especially squamous cell histology. This non-invasive test could be used to improve the positive predictive value of CT screening, with the potential to avoid invasive evaluation of nonmalignant pulmonary nodules.     

Methylation of TMEFF2 gene in tissue and serum DNA from patients with non-small cell lung cancer

Lung cancer remains a global health problem with a high mortality rate. CpG island methylation is a common aberration frequently associated with gene silencing in multiple tumor types, emerging as a highly promising biomarker. The transmembrane protein with a single EGF-like and two follistatin domains (TMEFF2) is epigenetically silenced in numerous tumor types, suggesting a potential role as a potential tumor suppressor. However, the role of TMEFF2 in lung cancer remains to be fully elucidated. We explored the methylation status of TMEFF2 gene in 139 patients with non-small cell lung cancer (NSCLC) and the feasibility of detecting circulating methylated DNA as a screening tool for NSCLC using methylation-specific PCR in 316 patients and 50 age-matched health controls. TMEFF2 methylation in tumor tissues was found in 73 of the 139 NSCLCs (52.5%) and was related to gene expression. The frequency of TMEFF2 methylation was higher in females and never-smokers than in males and smokers with borderline significance (65.8% vs 47.8%, p = 0.06; 65.7% vs 48.1%, p = 0.07). Notably, in adenocarcinomas, TMEFF2 methylation was significantly more frequent in tumors without EGFR mutation than those with EGFR mutation (adjusted odds ratio = 7.13, 95% confidence interval = 2.05-24.83, P = 0.002). Furthermore, TMEFF2 methylation was exclusively detected in the serum of NSCLC patients at a frequency of 9.2% (29/316). These findings suggest that methylation-associated down-regulation of TMEFF2 gene may be involved in lung tumorigenesis and TMEFF2 methylation can serve as a specific blood-based biomarker for NSCLC.     

Diagnostic accuracy of MALDI-TOF mass spectrometry for non-small cell lung cancer: a meta-analysis

Objective: To assess the overall accuracy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying non-small cell lung cancer (NSCLC).

Methods: A comprehensive search of PubMed, EMBASE and CNKI databases as well as the reference lists from relevant articles was performed prior to July 2017. Two authors independently screened articles based on inclusion and exclusion criteria and assessed the quality of each study using the Quality Assessment of Diagnostic Accuracy Studies 2 (QADAS-2) tool. Meta-disc 1.4 and Stata12.0 software programs were used for the statistical analysis.

Results: Eleven eligible articles comprising 16 studies and representing 935 subjects were included in this meta-analysis. The pooled sensitivity and specificity were 0.84 (95% CI: 0.80–0.87) and 0.77 (95% CI: 0.74–0.80), respectively. The overall diagnostic performance as measured by the area under the curve (AUC) for the summary receiver-operating characteristic (SROC) curve was 0.9380.

Conclusions: MALDI-TOF MS has a high diagnostic accuracy for NSCLC.     

REV3L single nucleotide variants lead to increased susceptibility towards non-small cell lung cancer in the population of Jammu and Kashmir

BackgroundNon-small cell lung cancer (NSCLC) is the most common lung cancer, accounting for 80–85% of all lung cancer cases. Various genetic studies have associated REV3L (Protein reversion less 3-like) gene mutations, which encodes the catalytic subunit of error prone translesion synthesis polymerase zeta with cancer, including lung cancer; however, no such data is available from any North Indian population. In this study we attempted to screen the North Indian population of Jammu and Kashmir (J&K) for the potential role of REV3L gene polymorphisms in NSCLC.MethodsA total of four REV3L single nucleotide variants were selected for genotyping based on the available literature. The genotyping was carried out by using the TaqMan allele discrimination assay in 500 subjects (200 NSCLC patients and 300 age and sex matched healthy controls). The association of variants with NSCLC was evaluated by logistic regression.ResultsOut of the four REV3L variants genotyped; rs1002481, rs462779, and rs465646 were found significantly associated with NSCLC risk under the recessive model, with an Odds Ratio (OR) of 3.52(2.14–5.8 at 95% CI, p-value = 0.00000062), 3.7 (1.8–7.6 at 95% CI, p-value = 0.00031), and 2.2 (1.47–3.37 at 95% CI, p-value = 0.0003), respectively.DiscussionOur data supports a strong association between variants rs1002481, rs462779, rs465646 and NSCLC, indicating a potential role of these REV3L variants in increasing the risk for the development of NSCLC in the studied population. Although a first report from any Indian population, these variants have been previously reported to be associated with lung and colorectal cancers in different world populations. Our data along with the existing data supports the notation that these variants can be used as potential genetic predisposition markers.Availability of data and materialsData generated and analysed during study is not available publicly but can be made available from the corresponding author upon reasonable request.     

Glypican-5 is a tumor suppressor in non-small cell lung cancer cells

Glypican-5 (GPC5) belongs to the glypican family of proteoglycans that have been implicated in a variety of physiological processes, ranging from cell proliferation to morphogenesis. However, the role of GPC5 in human cancer remains poorly understood. We report that knockdown of GPC5 in bronchial epithelial cells promoted, and forced expression of GPC5 in non-small lung cancer (NSCLC) cells suppressed, the anchorage-independent cell growth. In vivo, expression of GPC5 inhibited xenograft tumor growth of NSCLC cells. Furthermore, we found that GPC5 was expressed predominantly as a membrane protein, and its expression led to diminished phosphorylation of several oncogenic receptor tyrosine kinases, including the ERBB family members ERBB2 and ERBB3, which play critical roles in lung tumorigenesis. Collectively, our results suggest that GPC5 may act as a tumor suppressor, and reagents that activate GPC5 may be useful for treating NSCLC.