Abstract LB-329: Enhancing the resolution and accelerating the pace of translational fusion characterization in oncology by RNA sequencing
Authors: Watson Lisa C. , Gross Stephen M. , Schlesinger Felix , Mai Anthony , Kellogg Mariko , Lee Steve , Attwooll Claire , Brenca Monica , Swanson David , Wong Andrew , Dei Tos Angelo P. , Haferlach Claudia , Haferlach Torsten , Kern Wolfgang , Maestro Roberta , Meggendorfer Manja , Nadarajah Niroshan , Polano Maurizio , Rossi Sabrina , Sbaraglia Marta , Charames George S. , Schroth Gary P. , DeSantis Grace 
Published: July 2016

Abstract
Chromosomal rearrangements are common markers of cancer progression across a wide range of cancer types, and therefore, identification of fusion transcripts in cancer biopsies may have potential to provide tumor-specific insight toward diagnosis, prognosis and precision treatment. Currently, routine methods for fusion detection using fluorescent in-situ hybridization (FISH) provide a low-resolution view of the aberrant fusion transcript. We describe an RNA-Seq approach designed to survey cancer fusions in a single assay by selectively enriching the cancer transcriptome using probes that target the coding regions of over 1385 cancer-associated genes.
We tested the performance of the 1385 gene, RNA-Seq Pan-Cancer panel on RNA extracted from 47 patient-derived samples from brain, sarcoma and leukemia, including blood, bone marrow, and formalin-fixed paraffin-embedded (FFPE) samples. Each sample harbored at least one orthogonally verified gene fusion transcript, previously confirmed by FISH or Reverse Transcriptase PCR (RT-PCR). RNA-Seq libraries were prepared from 10-100 ng of total RNA from blood or bone marrow and 20-200 ng total RNA from FFPE tissue and subsequently enriched by hybridization to the Pan-Cancer panel. All samples yielded sufficient library and were sequenced with 76 base-pair paired-end reads on an Illumina MiSeq at 8 samples per flow cell (∼3 million reads per sample). Sequencing data was analyzed using RNA-Seq with STAR aligner and Manta fusion caller. Using this capture-based single-assay approach, we successfully detected fusions commonly associated with leukemia (BCR-ABL1, MLL-MLLT3, MLL-AFF1, RUNX1-ETV6, EBF1-PDGFRB, TCF3-PBX1, IKZF1-PAX5), sarcoma (EWSR1-ATF1, EWSR1-FLI1, JAZF1-SUZ12, SS18-SSX, FUS-DDIT3, FUS-KLF17, YWHAE-FAM22B) and brain cancer (KIAA1459-BRAF) consistent with previously confirmed RT-PCR or FISH results. Several examples of previously unknown fusion partners or additional structural information that were not identified from the FISH or RT-PCR testing were also uncovered in this study. These cases are described in detail.
In summary, we show that selective enrichment of RNA-Seq libraries with cancer-specific probes enables detection of known and novel fusions across a broad range of cancer pathologies in a single reaction, creating new opportunities for discovery and translational cancer studies.
Citation Format: Lisa C. Watson, Stephen M. Gross, Felix Schlesinger, Anthony Mai, Mariko Kellogg, Steve Lee, Claire Attwooll, Monica Brenca, David Swanson, Andrew Wong, Angelo P. Dei Tos, Claudia Haferlach, Torsten Haferlach, Wolfgang Kern, Roberta Maestro, Manja Meggendorfer, Niroshan Nadarajah, Maurizio Polano, Sabrina Rossi, Marta Sbaraglia, George S. Charames, Gary P. Schroth, Grace DeSantis. Enhancing the resolution and accelerating the pace of translational fusion characterization in oncology by RNA sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-329.