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. 2019 Aug 1;25(15):4712-4722.
doi: 10.1158/1078-0432.CCR-19-0225. Epub 2019 Apr 26.

High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden

Ryma Benayed  1 , Michael Offin  2 , Kerry Mullaney  1 , Purvil Sukhadia  1 , Kelly Rios  1 , Patrice Desmeules  3 , Ryan Ptashkin  1 , Helen Won  4 , Jason Chang  1 , Darragh Halpenny  5 , Alison M Schram  6 , Charles M Rudin  2   6   7 , David M Hyman  6 , Maria E Arcila  1 , Michael F Berger  1 , Ahmet Zehir  1 , Mark G Kris  2   6   7 , Alexander Drilon  2   6   7 , Marc Ladanyi  8
Affiliations

High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden

Ryma Benayed et al. Clin Cancer Res. .

Abstract

Purpose: Targeted next-generation sequencing of DNA has become more widely used in the management of patients with lung adenocarcinoma; however, no clear mitogenic driver alteration is found in some cases. We evaluated the incremental benefit of targeted RNA sequencing (RNAseq) in the identification of gene fusions and MET exon 14 (METex14) alterations in DNA sequencing (DNAseq) driver-negative lung cancers.

Experimental design: Lung cancers driver negative by MSK-IMPACT underwent further analysis using a custom RNAseq panel (MSK-Fusion). Tumor mutation burden (TMB) was assessed as a potential prioritization criterion for targeted RNAseq.

Results: As part of prospective clinical genomic testing, we profiled 2,522 lung adenocarcinomas using MSK-IMPACT, which identified 195 (7.7%) fusions and 119 (4.7%) METex14 alterations. Among 275 driver-negative cases with available tissue, 254 (92%) had sufficient material for RNAseq. A previously undetected alteration was identified in 14% (36/254) of cases, 33 of which were actionable (27 in-frame fusions, 6 METex14). Of these 33 patients, 10 then received matched targeted therapy, which achieved clinical benefit in 8 (80%). In the 32% (81/254) of DNAseq driver-negative cases with low TMB [0-5 mutations/Megabase (mut/Mb)], 25 (31%) were positive for previously undetected gene fusions on RNAseq, whereas, in 151 cases with TMB >5 mut/Mb, only 7% were positive for fusions (P < 0.0001).

Conclusions: Targeted RNAseq assays should be used in all cases that appear driver negative by DNAseq assays to ensure comprehensive detection of actionable gene rearrangements. Furthermore, we observed a significant enrichment for fusions in DNAseq driver-negative samples with low TMB, supporting the prioritization of such cases for additional RNAseq.See related commentary by Davies and Aisner, p. 4586.

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Conflict of interest statement

Conflict of Interest Statement:

Ryma Benayed has received a grant from ArcherDx.

Michael Offin has received consulting fees from PharmaMar.

Charles Rudin is a consultant for Abbvie, Amgen, Ascentage, AstraZeneca, Bicycle, Celgene, Chugai, Daiichi Sankyo, Genentech/Roche, GI Therapeutics, Loxo, Novartis, Pharmamar, and Seattle Genetics, and serves on the Scientific Advisory Boards of Elucida and Harpoon.

David Hyman: Consulting or Advisory Role: AstraZeneca, Atara Biotherapeutics, Chugai Pharma, CytomX Therapeutics, Boehringer Ingelheim, Pfizer, Genetech, Bayer, Debiopharm, ArQule. Research Funding: Loxo Oncology, AstraZeneca, and Puma Biotechnology

Maria Arcila received speaker’s fees from Raindance Technologies.

Mark G. Kris has received consulting fees from AstraZeneca, Pfizer, and Regeneron. He has received honoraria for participation in educational programs from WebMD, OncLive, Physicians Education Resources, AstraZeneca, and Research to Practice. Dr. Kris is an employee of Memorial Sloan Kettering. Memorial Sloan Kettering has received research funding from Genentech Roche and PUMA Biotechnology for trials conducted by Dr. Kris. Memorial Sloan Kettering has a collaboration for the development of Watson for Oncology with IBM and receives royalties from IBM for this activity.

Alex Drilon: HONORARIA/ADVISORY BOARDS: Ignyta, Loxo, TP Therapeutics, AstraZeneca Pfizer, Blueprint Medicines, Genentech/Roche, Takeda/Ariad/Millenium, Helsinn, Beigene, BergenBio, Hengrui Therapeutics, Exelixis, Bayer, Tyra Biosciences. RESEARCH: Foundation Medicine. ASSOCIATED RESEARCH PAID TO INSTITUTION: Pfizer, Exelixis, GlaxoSmithKlein, Teva, Taiho. ROYALTIES: Wolters Kluwer. CME HONORARIA: Medscape, OncLive, PeerVoice, Physicians Education Resources, Targeted Oncology, Research to Practice. OTHER: Merck - Food/Beverage, MORE Health - Consulting

Michael Berger has received advisory board compensation from Roche and research support from Illumina.

Marc Ladanyi has received advisory board compensation from AstraZeneca, Bristol-Myers Squibb, Takeda, and Bayer, and research support from LOXO Oncology and Helsinn Healthcare.

All other authors declare no potential conflict of interest.

Figures

Figure 1.
Figure 1.. Comprehensive DNASeq and RNASeq for effective detection of gene fusions.
Gene fusion detection in the DNA using next generation sequencing (e.g. MSK-IMPACT™) requires the tiling of intronic regions known to likely harbor the genomic breakpoint. This approach is challenging when 1) the introns are too long to tile 2) the introns contain repetitive elements which don’t sequence very well or 3) genomic breakpoints take place in alternative introns not predicted by the panel design. RNA sequencing offers a more direct approach to fusion detection as the introns are removed by splicing.
Figure 2.
Figure 2.. Description of the lung adenocarcinomas cohort.
A total of 2,522 unique lung adenocarcinomas were submitted for MSK-IMPACT sequencing between January 2014 and December 2017. 589/2,522 cases were negative for driver alterations and submitted for MSK-Fusion testing. Adequate tissue for RNA extraction was only available in 275 samples. 21 cases were deemed insufficient due to low RNA quality based on the PreSeq™ assay. Out of the 254 sequenced samples, 22 failed based on their unique sequencing coverage (<50X). In total, 232 MSK-IMPACT driver negative cases had successfully undergone RNASeq using the MSK-Fusion panel for an assay success rate of 91%.
Figure 3.
Figure 3.. Driver alteration profile by comprehensive DNASeq and RNA Seq.
(A) 1,933/2,522 cases were positive for oncogenic drivers using MSK-IMPACT. RNA from the available driver negative cases (n=232) was tested using the MSK-Fusion panel. Gene fusions (n=29), METex14 mutations (n=6) and EGFRviii (n=1) were detected. (B) Schematic representation of the 15/29 gene fusions that were not expected to be detected by custom DNA-based sequencing due to panel design (C) Schematic representation of the 14/29 expected to be detected by DNASeq but only a fusion transcript was detected in the RNA.
Figure 4.
Figure 4.. Assessment of Tumor Mutation Burden in fusion positive and negative tumors.
(A) The tumor mutation burden (TMB) of 1,933 MSK-IMPACT driver-positive lung adenocarcinomas was compared between fusion positive (n=195, left) and negative (n=1,738, right) samples. Median TMB and IQ range are indicated on top of each plot. The median TMB of Fusion positive samples was significantly lower than the fusion negative ones (Mann Whitney, p<0.0001). 4 outlier TMB values were excluded from the plot but not from the statistical analysis. (B) A cohort of 232 MSK-IMPACT driver negative lung adenocarcinomas is organized by low TMB (0-5 mut/Mb, left) and higher TMB (>5 mut/Mb, right). The percentage of patients whose tumors are fusion negative and positive by RNASeq (MSK-Fusion) is indicated. The percentage of fusion-positive samples with low TMB (31%) was significantly higher when compared with the percentage of fusion-positive ones with high TMB (7%), (Mann Whitney, p<0.0001).
Figure 5.
Figure 5.. Gene fusion landscape in 2,522 lung adenocarcinomas.
Illustration of all gene fusions detected by comprehensive DNASeq (MSK-IMPACT) and RNASeq (MSK-Fusion).
Figure 6.
Figure 6.. Study summary and clinical findings.
RNASeq-based matched therapy for 10/33 patients whose tumors were found to be positive for gene fusions (9) or METex14 (1). 80% of the patients have shown clinical benefit. PR: partial response, SD: stable disease; PD: progression of disease. * Response assessment by RECIST v1.1. ** Confirmed PR
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Comment in

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