Author of

• 12812

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  P3.20 - Poster Session 3 - Early Detection and Screening (ID 174)

  • Event: WCLC 2013
  • Type: Poster Session
  • Track: Imaging, Staging & Screening
  • Presentations: 1
  • Moderators:
  • Coordinates: 10/30/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 12813

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    P3.20-001 - DNA methylation profiling in NSCLC identifies putative diagnostic markers for screening and early detection of lung cancer (ID 150)

    09:30 - 09:30  |  Author(s): A. Iwamaru
    • Abstract

    Background
    Lung cancer is a leading cause of the cancer-related death in the world, and frequently presents with an advanced disease at diagnosis. Thus, early detection of lung cancer is thought as an important opportunity for decreasing mortality. It is willingly expected that novel method of early detection of lung cancer using biomarkers, such as cancer-specific up-regulation of methylation levels on genomic DNA. We hereby report that 7 CpG sites were found to be highly methylated in Non-Small-Cell-Lung Cancer (NSCLC), those which are possible promising NSCLC-specific biomarkers.

    Methods
    Samples for genome-wide DNA methylation analysis consisted of 10 surgical specimens including 8 NSCLC (3 adenocarcinomas, 2 squamous cell carcinomas, 2 bronchioalveolar carcinomas and 1 large cell carcinoma) and 2 adjacent normal lung tissues. Genomic DNAs were isolated from frozen tissue sections by using QIAamp DNA mini kit (QIAGEN) followed by bisulfite conversion with Epitect bisulfite kit (QIAGEN) according to manufacturer’s protocol. Bisulfite converted DNAs were hybridized to the Illumina Infinium HumanMethylation 450 BeadChip, which screens methylation levels of more than 450,000 CpG sites within a single array. Evaluation of the array screening data revealed a series of candidate CpG sites that are preferentially methylated in cancer samples as compared to normal controls. To further validate cancer-specific up-regulation of methylation levels at candidate CpG sites, the QIAGEN Pyromark Q24 system was employed to conduct bisulfite pyrosequencing analysis. Pyromark CpG assay probes were designed with the aid of PyroMark Assay Design Software 2.0 to encompass up to 300 bases region around the CpG site of interest, and used to carry out PCR amplification and pyrosequencing on bisulfite-converted genomic DNA templates. The analyzed samples included 136 surgical specimens consisting of 68 pairs of NSCLC and adjacent non-cancerous matched tissue, which are collected by macrodissection from paraffin-embedded sections. The 68 NSCLC samples are classified in terms of histological types as follows: 54 adenocarcinomas, 10 squamous cell carcinomas, 2 adenosquamous cell carcinomas, 2 large cell carcinomas, and in terms of cancer staging as follows: 37 of stage I, 15 of stage II, 12 of stage III and 4 of stage IV.

    Results
    Seven CpG sites were found to be highly methylated in NSCLC samples compared to adjacent controls in a statistically significant manner (two-way ANOVA, p < 0.05). The Illumina CpG cluster numbers (cg#) of the 7 CpG sites and their genomic locations in GRCh37 coordinates are as follows: L3302 (cg26917140; chr1:91184770), L3303 (cg04654530; chr2:63282702), L3304 (cg27315333; chr2:63285950), L3305 (cg17080163; chr2:123418757),　L3310 (cg15347189; chr3:187387999), L3314 (cg03148184; chr4:111562513), L3316 (cg16509851; chr4:174430614). Among them, L3303 and L3310 are located within the genomic region corresponding to orthodenticle homeobox 1 (OTX1) gene and somatostatin (SST) 1st exon, respectively.

    Conclusion
    These 7 CpG sites are possible promising NSCLC-specific biomarkers for screening and early detection of lung cancer, and our goal is the development of the technology to detect these novel biomarkers from a blood sample. This technology may eventually lead to early detection of lung cancer, which is thought as an important opportunity for decreasing lung cancer mortality.