Abstract 4245: Detection of fusion genes in lung cancer biopsies of crizotinib resistant patients
Authors: Saber Ali , van der Wekken Anthonie , Kok Klaas , Terpstra Martijn M. , Mastik Mirjam F. , Timens Wim , Schuuring Ed M.D. , Hiltermann T. Jeroen , Groen Harry J.M. , van den Berg Anke 
Published: August 2015

Abstract
Purpose: A small proportion of lung cancer patients have an inversion involving the ALK gene on chromosome 2. These ALK+ patients initially respond to crizotinib, but later develop progressive disease. This resistance can be due to gatekeeper mutations in the kinase domain of the ALK gene like L1196M and G1269A, copy number gain of the EML4-ALK fusion gene, and mutations in EGFR and KRAS. We aimed at identifying fusion genes that may explain the resistance mechanism in patients progressing on crizotinib.
Materials and Methods: Frozen tissue of the resistant tumor samples was available for four EML4-ALK positive patients (Table 1). Paired end RNA sequencing was performed using the Illumina TruSeq RNA platform. DeFuse (v.0.6.1) was used to identify fusion products. Candidate fusion genes were validated by RT-PCR. In addition, we used the IGV browser to identify mutations in resistance-associated genes in the RNA-seq data.
Results: Thirteen fusions genes were identified with high confidence (Table 1). In three of the four patients we detected an EML4-ALK fusion product. Four novel fusion products with a functional ORF were confirmed by RT-PCR. Three of these fusion products were present in patient 1 (Table 1). The fourth fusion involving CLIP4-VSNL1 was detected in patient 3. Interestingly, three of the 4 fusion products observed in patient 3 involved 5 genes that are all close to the ALK locus, i.e. EML4, ALK, CLIP4, VSNL1 and MCFD2 (Table 1). In addition, we found two resistance-related mutations in the tyrosine kinase domain of the ALK gene, i.e. p.C1156Y and p.G1269A, in patients 2 and 3, respectively.
Fusion products in four crizotinib resistant lung cancer patientsPatientAgeSmokingALK immunohistochemistryTumor response (PFS in months)Fusion genesORF147CurrentNot availableNo (1.4)RAB35-ATP2A2YesSLC10A7-TTC29YesSTAB2-NUAK1YesKMT2B-AC002115.9NoPIAS1-SLC24A6NoZNF827-ARHGAP10No227None+Yes (9.2)EML4-ALKYesNRG1-RBPMSNo342None+Yes (10.4)EML4-ALKYesCLIP4-VSNL1YesMCFD2-CLIP4NoCPSF6-RBMS2No456Current+Yes (15.7)EML4-ALKYes
Conclusion: We found four new fusion products with a functional ORF using RNA sequencing of crizotinib resistant tumor samples. Further analysis of the primary tumor samples is ongoing and will reveal if these fusion genes might have been induced by the crizotinib treatment. Two of the four patients have a known resistance-inducing mutation in the ALK gene.
Citation Format: Ali Saber, Anthonie van der Wekken, Klaas Kok, Martijn M. Terpstra, Mirjam F. Mastik, Wim Timens, Ed M.D. Schuuring, T. Jeroen Hiltermann, Harry J.M. Groen, Anke van den Berg. Detection of fusion genes in lung cancer biopsies of crizotinib resistant patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4245. doi:10.1158/1538-7445.AM2015-4245