Abstract 4228: Targeted Next-Generation Sequencing in DNA repair genes in prostate tumors.
Authors: Yadav Santosh , Makridakis Nick 
Published: August 2014

Prostate cancer is the most common malignancy and the second leading cause of cancer death in men in the United States. The exponential increase in risk of prostate cancer associated with aging may reflect the accumulation of DNA damage as a result of a series of processes including oxidative stress, inflammation or environmental carcinogens or a decrease in DNA damage-repair response capacity. DNA repair mechanisms are important for the removal of oxidative DNA compounds or DNA adducts from damaged genomic sites.

We aimed to sequence the entire coding region of all genes that participate in DNA repair pathways, in prostate tumors, and in another study to optimize % read coverage for GC-rich exons (i.e. exon-1)

For the first study, 5 primary tumors (Gleason score ≥7) and 1 matched blood DNA sample from a prostate cancer patient were used for the assessment. In this study we applied the SureSelectTM Target Enrichment System. The RNA bait library (total 129 targeted genes with bait length of 120 bp), was 0.4Mb in size and was hybridized with the genomic DNA library of tumor samples followed by Illumina Paired-End Multiplexed Sequencing. Among these genes some genes display GC-rich content in exon-1. In order to resolve coverage depth in these regions we used explored the use of TMAC (Tetramethylammonium chloride; (CH3)4N(Cl)) during hybridization in parallel with the standard method of library preparation.

On the average, 48 % of the mapped reads mapped to our target genes, regardless of tissue type (paraffin embedded or frozen). We obtained &gt; 500X of coverage, except of exon 1 of some genes, where depth was &lt;50X due to GC-rich content (&gt;70% GC). A subset of genes was somatically altered across tumors derived from different individuals, including TP53, MPG, NEIL1, NFKBIA, XRCC1, CCNE1, ERCC2, E2F1, MLH1, POLQ, OGG1, XPC, NFKB1, CDKN1A, POLB, E2F5, E2F2, ABL1, NOTCH1, AR, MRE11A, CHEK1, RB1, CCND2 etc. Analysis of GC rich exon data show that application of TMAC produces libraries with improved coverage in exon-1 in several genes including GADD45A, PARP1, POLD1, MSH2, ATR, which have GC content &gt;70%.

Although we plan to sequence 40 additional tumor samples and matched controls, these data suggest associations between DNA repair gene alterations and prostate cancer progression. Our optimizing condition significantly improves coverage of GC rich exons. This approach will be significantly helpful in tumor samples that need amplification due to low mass of DNA.

We thank the Cancer Crusaders Next generation Sequence Analysis Core for their support in downstream analysis of NGS data.

This work was supported by DOD grant PC094628, and NIH grant 8 P20 GM103518.

Citation Format: Santosh Yadav, Nick Makridakis. Targeted Next-Generation Sequencing in DNA repair genes in prostate tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4228. doi:10.1158/1538-7445.AM2013-4228