Deep RNA sequencing revealed fusion junctional heterogeneity may predict crizotinib treatment efficacy in ALK-rearranged non-small cell lung cancer

Abstract Introduction Gene fusion variants in ALK-rearranged non-small cell lung cancer (NSCLC) may predict patient outcomes but previous results have been inconclusive. Fusion isoforms co-existing in the same tumor may affect targeted therapy efficacy but have not been investigated. Methods ALK-rearranged NSCLC patients who received crizotinib treatments were recruited. Pre-crizotinib tumor tissues were analysed by the anchored multiplex PCR for targeted RNA sequencing. Kaplan-Meier and Cox regression were used to compare overall and progression-free survivals. Results Of the 51 studied subjects, EML4-ALK variant types v1, v2, v3 and others were detected in 23 (45.1%), 5 (9.8%), 19 (37.3%) and 4 (7.8%) patients, respectively. Multiple EML4-ALK RNA isoforms were detected in 24 (47.1%) tumors, and single-isoform in 27 (52.9%). The majority (16/19) of v3 tumors harboured both v3a and v3b RNA isoforms. Multiple isoforms were also detected in 8 non-v3 tumors (33.3% of all 24 multiple-isoform; five v1, two v5' and one v2). Compared with single-isoform, multiple-isoform patients had worse progression-free (hazard ratio [HR] and 95% confidence interval [CI]: 2.45 [1.06-5.69]) and overall (HR [95% CI]: 3.74 [1.26-11.13]) survivals after adjusting for potential confounders including variant type. Using the patient-derived H2228 cells known to express v3a and v3b, our single-cell PCR detected either v3a or v3b in most single cells. Treatment of H2228 cells by three ALK inhibitors showed increased ratios of v3a-to-v3b expression over time. Conclusions Intratumoral EML4-ALK isoforms may predict the efficacy of targeted therapy in ALK-rearranged NSCLC. Temporal changes of intratumoral fusion isoforms may result from differential selection pressures that a drug might have on one isoform over another. Larger studies on fusion heterogeneity using RNA sequencing are warranted.