title: Development of a multiplex methylation specific PCR suitable for (early) detection of non-small cell lung cancer.
abstract: 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.
authors.affiliation: Department of Microbiology; Tumor and Cell Biology; Karolinska Institute; Stockholm, Sweden; Department of Microbiology; Faculty of Life Sciences; University of Balochistan; Quetta, Pakistan.
authors.forename: Imran
authors.initials: I
authors.lastname: Nawaz
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment; Tianjin Lung Cancer Institute; Tianjin Medical University General Hospital; Tianjin, PR China.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Qiu
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment; Tianjin Lung Cancer Institute; Tianjin Medical University General Hospital; Tianjin, PR China.
authors.forename: Heng
authors.initials: H
authors.lastname: Wu
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment; Tianjin Lung Cancer Institute; Tianjin Medical University General Hospital; Tianjin, PR China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment; Tianjin Lung Cancer Institute; Tianjin Medical University General Hospital; Tianjin, PR China.
authors.forename: Yaguang
authors.initials: Y
authors.lastname: Fan
authors.affiliation: Department of Microbiology; Tumor and Cell Biology; Karolinska Institute; Stockholm, Sweden.
authors.forename: Li-Fu
authors.initials: LF
authors.lastname: Hu
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment; Tianjin Lung Cancer Institute; Tianjin Medical University General Hospital; Tianjin, PR China.
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Zhou
authors.affiliation: Department of Microbiology; Tumor and Cell Biology; Karolinska Institute; Stockholm, Sweden.
authors.forename: Ingemar
authors.initials: I
authors.lastname: Ernberg
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000473:pathology; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D055088:Early Detection of Cancer; D016147:Genes, Tumor Suppressor; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D059467:Transcriptome
keywords: Bisulfite treatment; DNA methylation; Expression microarray; Lung cancer; MMSP; Methylation microarray; NSCLC; PCR