5′/ 3′ imbalance strategy to detect ALK fusion genes in circulating tumor RNA from patients with non-small cell lung cancer

Abstract

BackgroundDetecting an ALK fusion gene in patients with non-small cell lung cancer (NSCLC) could provide evidence to guide individualized therapy.MethodsThe 5′/3′ imbalance strategy for quantitative reverse transcription-PCR (RT-qPCR) was developed to detect ALK fusion genes in circulating tumor RNA (ctRNA) of NSCLC patients.ResultsThis method was validated in patients with the ALK fusion gene confirmed by next generation sequencing (NGS). The amount of the ALK fusion gene detected by the new method ranged from 33.2 to 987.4, (mean 315.2), in the patients confirmed to have the ALK fusion gene (+). This is much higher than the amount of fusion gene detected in the patients who are negative for the ALK fusion gene (−). The amount detected in the ALK fusion gene (−) samples ranged from 0.36 to 13.04, (mean 4.58). In 188 NSCLC patients, the specificity and sensitivity of the method was compared to that of the FISH method. About 10.64% of the patients showed higher ALK fusion gene expression, and were classified as ALK fusion gene (+). This is identical to the percentage of patients detected by the FISH method to be ALK fusion gene (+). The cutoff value for diagnosis of ALK fusion (+) is 32.9 as determined by this method.ConclusionsA new RT-PCR method using a 5′/3′ imbalance strategy was developed, with high specificity and sensitivity, for detection of the ALK fusion gene in ctRNA of NSCLC patients. This method can rapidly detect ALK fusion genes in patients, which will be helpful for guiding targeted therapy, particularly the individualized usage of TKIs in these patients.

Use of an Integrated Pan-Cancer Oncology Enrichment Next-Generation Sequencing Assay to Measure Tumour Mutational Burden and Detect Clinically Actionable Variants

“…The CHECKMATE [8][9][10] series of trials have suggested that a specific threshold of "tumour mutational burden" (TMB) must be reached in order for PD-1 blockade to become effective, although this has not been adopted formally, due to a lack of association between TMB and response, although other similar markers such as clonal TMB have shown promise. Although TMB has variable definitions, it is broadly accepted [9] as the number of missense mutations in the tumour genome, either divided by the size of the exome panel (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45) or via the size of the human genome for whole-genome sequencing (WGS) (3.3 Gb). Based on the CHECKMATE trials, the suggested TMB threshold is greater than 10 mutations (mut)/Mb, based on the objective response rates of the tumours in these studies not improving much beyond this threshold.…”

Current status and future perspectives of liquid biopsy in non-small cell lung cancer

“…Other targetable mutations including ALK, BRAF, ROS1, MEK, and HER2 have been detected in plasma from patients with NSCLC, although the sensitivity for detecting these mutations is lower than that for EGFR mutant detection [15,[49][50][51][52]. The NGS-based approaches will facilitate detection of various rare genetic mutations that could be targeted.…”

“…Highly fragmented ctDNA could result in insufficient mappable sequences to identify fusion events [51]. New technologies have been developed for detection of ALK fusion in ctDNA or ctRNA [15,[49][50][51][52]. Recently developed, hybrid capture-based NGS can retrieve large genomic fragments to whole genomes with high sequencing coverage and accurate detection of genomic alterations, including genomic re-arrangements and short variants at low AFs and copy number amplifications.…”

Gene rearrangements in consecutive series of pediatric inflammatory myofibroblastic tumors

“…We and others reported PCR tests, which utilize cDNA as a template and measure the amounts of 5′‐ and 3′‐end specific fragments of the involved tyrosine kinase 14‐16 . If the rearrangement occurs, the kinase domain of the involved gene is translocated to a highly expressed partner; therefore, the 3′‐end of the transcript is overrepresented as compared with the 5′‐end.…”

“…We and others reported PCR tests, which utilize cDNA as a template and measure the amounts of 5 ′ -and 3 ′ -end specific fragments of the involved tyrosine kinase. [14][15][16] If the rearrangement occurs, the kinase domain of the involved gene is translocated to a highly expressed partner; therefore, the 3 ′ -end of the transcript is overrepresented as compared with the 5 ′ -end. This test for unbalanced expression is potentially capable of detecting all variants of gene rearrangements, being similar in this respect to IHC and FISH.…”