Abstract 2212: A genomic portrait of tumor progression using next-generation sequencing

Abstract

Abstract Accumulation of genetic and epigenetic alterations is thought to enable cancers to proliferate and survive more effectively than their neighboring cells, or for aggressive tumors to resist cytotoxic therapies. However, the molecular basis of tumorigenesis and chemoresistance is not fully understood. With the advent of next-generation sequencing technologies, we can determine the whole transcriptome or genome sequence of a tumor and thereby detect all the changes that may result in a particular phenotype such as resistance to cytotoxic therapies. To understand the genetic aberrations underlying the chemoresistance in neuroblastoma, we attempted to catalogue all genetic aberrations in two tumor samples before and after cytotoxic therapies using RNA-seq (including whole transcriptome and miRNA) and genomic DNA sequencing from the same neuroblastoma patient who was refractory to standard chemotherapies. First, we performed whole genome sequencing on patient's constitutional DNA to establish a genetic background. Multiple mate-paired libraries were constructed and subjected to next-generation sequencing. We acquired 53 Gb high-quality mappable data, equivalent to 16X coverage of this patient's genome. Next, whole transcriptome sequencing of the two tumors yielded total 345 million 50bp mappable reads representing 14,128 and 14,484 genes (20875 and 21330 transcripts) respectively. The average coverage for each transcript was >14, while 5297 genes for one sample and 6912 genes for the other had an average coverage of >10. With such deep sequencing, single nucleotide variant (SNV) analysis on the transcriptome sequencing data identified 9103 and 10556 exonic SNVs of total 154871 and 191033 SNVs respectively in two samples. Among the exonic SNVs, 3965 and 4470 SNVs were in the coding regions and about half of them were nonsynonymous. Using SIFT analysis, we predicted 353 and 443 SNVs to be damaging before and after chemotherapies. Of them, 165 SNVs were shared among two samples. Therefore, these shared SNVs and newly acquired SNVs after chemotherapies may contain the genetic alterations that are resistant to the standard neuroblastoma therapies in this patient. In addition, 188 SNVs were present in the original tumor but not in the refractory sample indicating genomic instability of tumors. In parallel, we performed microRNA sequencing on the two samples. While most of 987 mature microRNAs were detected at similar levels after normalized against total reads, 64 microRNAs were significantly differentially expressed before and after chemotherapies using stringent selection criteria (total read counts in two samples>500, >5 fold change, P<0.01, and FDR<0.01). These differentially microRNAs and accumulation of deleterious mutations during therapies are currently being investigated for pathway disruption that may lead to chemo-refractory disease and allow development of targeted therapies to overcome drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2212.