Single molecule RNA sequencing of formalin-fixed paraffin-embedded tissue derived from patients with lung cancer.

Abstract

10550 Background: Next generation sequencing has revolutionized the study of cancer biology by providing an unprecedented view of cancer genomes. However, high throughput sequencing has conventionally utilized fresh frozen tissue collected for research prospectively. The ability to apply this tool to formalin-fixed, paraffin-embedded (FFPE) clinical specimens, where nucleic acid integrity is not optimal and where next generation sequencing has been challenging, would enable its use to improve patient care and clinical research. Methods: Total RNA was extracted from FFPE blocks of 7 lung adenocarcinomas and matching normal lung tissue for 6 of the 7 tumors. Genotype information for selected mutation alleles in EGFR, TP53, etc. was previously determined using the SNaPshot single nucleotide polymorphism genotyping platform. Ribosomal RNA was depleted using biotinylated oligos targeting 28S and 18S ribosomal subunits. cDNA was synthesized using random hexamers. Whole transcriptome sequencing was performed using the Helicos BioSciences Heliscope single molecule sequencer. Sequences were aligned to reference human transcriptome and genome as well as a reference of mutation alleles. Results: Ribosomal RNA depletion increased mRNA yield from ~20% to ~35% of RNA species sequenced. We obtained ~10 million aligned mRNA reads per sample. We re-identified clinically relevant EGFR exon 19 deletions in two samples. In another sample, we discovered an exon 20 insertion in EGFR that was not previously appreciated. In a fourth sample, we were able to detect the presence of an EML4-ALK fusion through the integration of sequence and expression information. Conclusions: This study shows that RNA sequencing from FFPE patient samples is feasible, and in cases where gene expression is sufficient, enables the determination of clinically relevant mutations. These findings represent the initial step towards leveraging the power of next generation sequencing into a comprehensive diagnostic and clinical research tool.