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Background Despite the progress in the development of next‐generation sequencing (NGS), diagnostic PCR assays remain to be utilized in clinical routine due to their simplicity and low cost. Tests for 5′‐/3′‐end mRNA unbalanced expression can be used for variant‐independent detection of translocations, however, many technical aspects of this methodology require additional investigations. Methods Known ALK/ROS1 fusions and 5′‐/3′‐end unbalanced expression were analyzed in 2009 EGFR mutation‐negative non‐small cell lung cancer (NSCLC) samples with RT‐PCR tests, which were optimized for the use with FFPE‐derived RNA. Results Variant‐specific PCR tests for 4 common ALK and 15 common ROS1 translocations detected 115 (5.7%) and 44 (2.2%) rearrangements, respectively. Virtually all samples with common ALK fusions demonstrated some level of 5′/3′ mRNA ends unbalanced expression, and 8 additional NSCLCs with rare ALK fusions were further identified by PCR or NGS among 48 cases selected based on ALK expression measurements. Interestingly, NSCLCs with unbalanced 5′‐/3′‐end ALK expression but without identified ALK translocations had elevated frequency of RAS mutations (21/40, 53%) suggesting the role of RAS activation in the alternative splicing of ALK gene. In contrast to ALK, only a minority of ROS1 translocation‐positive cases demonstrated unbalanced gene expression, with both 5′‐ and 3′‐end mRNA expression being elevated in most of the samples with translocations. Surprisingly, high ROS1 expression level was also found to be characteristic for NSCLCs with activating mutations in other tyrosine kinases such as EGFR, ALK, or MET. Conclusions Comprehensive ALK analysis can be performed by the test for 5′‐/3′‐end unbalanced expression with minimal risk of missing an ALK rearrangement. In contrast, the use of the test for 5′‐/3′‐end unbalanced expression for the detection of ROS1 fusions is complicated; hence, the utilization of variant‐specific PCR assays for ROS1 testing is preferable.

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