Reliability analysis of exonic-breakpoint fusions identified by DNA sequencing for predicting the efficacy of targeted therapy in non-small cell lung cancer

Abstract

Background Diverse genomic breakpoints of fusions that localize to intronic, exonic, or intergenic regions have been identified by DNA next-generation sequencing (NGS), but the role of exonic breakpoints remains elusive. We investigated whether exonic-breakpoint fusions could predict matched targeted therapy efficacy in non-small cell lung cancer (NSCLC). Methods NSCLC samples were analyzed by DNA NGS, RNA NGS, immunohistochemistry (IHC), and fluorescence in situ hybridization. Results Using DNA NGS, kinase fusions were identified in 685 of 7148 (9.6%) NSCLCs, with 74 harboring exonic-breakpoint fusions, mostly anaplastic lymphoma kinase (ALK) fusions. RNA NGS and IHC revealed that 11 of 55 (20%) exonic-breakpoint fusions generated no aberrant transcript/protein, possibly due to open reading frame disruption or different gene transcriptional orientations. Four cases of genomic-positive but RNA/protein-negative fusions were treated with matched targeted therapy, but progressive disease developed within 2 months. Nevertheless, 44 of 55 (80%) exonic-breakpoint fusions produced chimeric transcripts/proteins, possibly owing to various alternative splicing patterns, including exon skipping, alternative splice site selection, and intron retention. Most of these genomic- and RNA/protein-positive fusion cases showed a clinical response to matched targeted therapy. Particularly, there were no differences in objective response rate (P = 0.714) or median progression-free survival (P = 0.500) between intronic-breakpoint (n = 56) and exonic-breakpoint ALK fusion subtypes (n = 11) among ALK RNA/protein-validated patients who received first-line crizotinib. Conclusions Exonic-breakpoint fusions may generate in-frame fusion transcripts/proteins or not, and thus are unreliable for predicting the efficacy of targeted therapy, which highlights the necessity of implementing RNA or protein assays for functional validation in exonic-breakpoint fusion cases.

Case Report: Efficacy of ensartinib treatment in pulmonary inflammatory myofibroblastic tumor with a rare GCC2-ALK fusion

“…RNA-based NGS is a mature mRNA that is sequenced; fusion detection at the RNA level proves that these fusions are indeed transcribed, and more fusions are detected than with DNA-level detection, however, the most important disadvantage is that RNA is not as stable as DNA, and the quality of RNA does not always meet the requirements of sequencing, especially for FFPE samples. The fusion type of the ALK gene can be preliminarily detected by using probes for DNA-based NGS; RNA and protein-level analysis may be the key to verifying the complex ALK rearrangement function in clinical practice to make the best treatment decision ( 19 – 21 ). In this case, GCC2-ALK fusion was detected by DNA-based NGS technology, and the results of the IHC profile showed ALK (+), which further verified the protein function at the protein level, providing theoretical guidance for the use of ensartinib.…”

Molecular characterization of genomic breakpoints of ALK rearrangements in non‐small cell lung cancer

“…Comparing the results from DNA‐based NGS and RNA‐based NGS, we found that exon skipping existed in ‘exon breakpoints’ cases carrying canonical or noncanonical ALK fusions. It may be reasoned that the lack of classical 3′ or 5′ accepter splice sites in the ‘exon–intron’, ‘intron–exon’ or ‘exon–exon’ structures resulted in the removal of the broken exon together with the previous intron to restore the reading frame [ 18 , 42 ]. Notably, although lacking the 5′ acceptor splice site of ALK exon 20, the actual transcript of case #P2008280124 excluded partial nucleotides and retained a portion of exon 20 through an alternative splicing signal at the RNA level rather than implementing exon skipping splicing and it resulted in two different variants (E19; del14A20 and E19; del20A20).…”

“…The most common partner gene for ALK is echinoderm microtubule‐associated protein‐like 4 ( EML4 ) [ 16 ], and other noncanonical partner genes have been identified, such as kinesin family member 5B ( KIF5B ), kinesin light chain 1 ( KLC1 ) and translocated promoter region ( TPR ) [ 17 ]. Previous studies have reported diverse genomic breakpoints of ALK rearrangements that occur in different regions (introns or exons) in NSCLC, and intronic breakpoint fusions usually result in in‐frame chimeric fusion transcripts/proteins [ 15 , 18 ]. Multiple ALK ‐fusion variants caused by variable genomic breakpoints have been reported with different sensitivities to ALK TKIs, especially in canonical EML4‐ALK fusions [ 19 ].…”

RNA sequencing steps toward the first line

“…3 With DNA sequencing, there is also a risk of reporting rearrangements that do not encode clinically actionable fusion products. 4 Guidelines from the National Comprehensive Cancer Network (NCCN) recommend that, to maximize the detection of fusion events in patients with NSCLC, RNA-based sequencing should be used if no driver oncogenes can be identified in broad panel testing (NCCN Guidelines, version 6.2022). The Guidelines further specify that RNAbased NGS may improve the detection of MET exon 14 skipping and is preferable to DNA-based NGS for the detection of RET fusions.…”

“…Li and colleagues demonstrated that RNA NGS confirmed canonical fusions in seven samples that could not be defined definitively as actionable fusions based on DNA NGS alone because the fusions had uncommon partner genes or breakpoints in intergenic regions 3 . With DNA sequencing, there is also a risk of reporting rearrangements that do not encode clinically actionable fusion products 4 …”