Molecular testing for driver variants in oncogenes is crucial for NSCLC management to predict response to targeted therapy. In the majority of cases, NSCLC is diagnosed by trans-thoracic needle aspiration or more commonly bronchoscopy techniques resulting in small diagnostic tissue biopsies or cytological samples. As such specimens may be inadequate for molecular testing, we tested the accuracy of a novel digital molecular barcoding assay to detect actionable mutations in a single-centre cohort. A consecutive cohort of 46 specimens (19 endobronchial biopsy, EnBx; 18 transbronchial biopsy, TBBx; seven bronchoalveolar lavage, BAL; and two transbronchial needle aspirate, TBNA) were obtained ancillary to primary diagnostic specimens from 36 patients undergoing EBUS-guided bronchoscopy at The Prince Charles Hospital. Specimens containing at least 5ng DNA after standard column-based extraction methods were analysed using the NanoString SNV Solid Tumour Panel for testing of 104 somatic variants across 25 genes of clinical significance. NanoString variants calls were compared with routine clinical testing results from the primary diagnostic sample. Agreement analyses for variants common to both methods revealed the positive, negative and overall percentage agreement (PPA, NPA, OPA). One discordant case was validated using droplet digital PCR. Using NanoString, molecular analysis was feasible for 60.1% (28/46) of specimens. At least one variant was identified in 8/28 (28.6%) cases (Table 1). Two (7.1%) cases harboured dual mutations. KRAS mutations were detected in six (21.4%) cases, and EGFR in two (7.1%). Two patients would be eligible for targeted therapy. Agreement analysis for the two methods revealed PPA, NPA and OPA of 100%, 88.9% and 92.3%. In one discordant case, NanoString identified a KRAS G12C mutation and was confirmed by ddPCR with a mutant allele frequency of 5.5%. The mean time for reporting clinical mutation test results was 19.6 days. Of the 18 excluded cases with insufficient DNA, five had routine testing results for comparison however 3/5 cases cited insufficient DNA for reliable EGFR testing.Table 1Clinical and molecular characteristics of bronchoscopy samples used for molecular testingClinical molecular testingNanoStringConcordanceCase no.Sample typeHistological classificationDNA yield (μg)Mutation testing result (MAF)Mutation testing methodTAT to result (days)SNV panel resultAgreement (Yes/No)1BALNo evidence of malignancy2.81Not performedN/AN/AWTNCA2TBNAAC4.39Not performedN/AN/AWTNCA3TBBxSCC0.39Not performedN/AN/AKRAS G12RNCA4TBBxAC1.82EGFR exon 19 (L747_P753>S) del (24%)NGS TruSight21EGFR exon 19 (L747_P753>S) delYes5TBBxNo evidence of malignancy1.24Not performedN/AN/AWTNCA6TBBxNo evidence of malignancy0.62Not performedN/AN/AWTNCA7EnBxAC1.41WT for EGFR, KRAS, NRAS, BRAFNGS TruSight13WTYes8EnBxSCC0.57Not performedN/AN/AWTNCA9TBBxSCC0.53Not performedN/AN/AWTNCA10TBBxAC0.51WT for EGFR, KRAS, NRAS, BRAFNGS TruSight20KRAS G12CNo*11TBBxAC0.31EGFR L858R (12%), EGFR T790M (6%)NGS TruSight13EGFR L858R, EGFR T790MYes12TBBxNo evidence of malignancy0.69Not performedN/AN/AWTNCA13TBBxNSCLC4.26WT for EGFRcastPCR48WTYes14EnBxAC1.37KRAS G12C (10%)NGS TruSight23KRAS G12CYes15EnBxNSCLC19.6KRAS G12A (25%)NGS TruSight20KRAS G12AYes16EnBxCarcinoid1.16Not performedN/AN/AWTNCA17TBBxSCC5.4Not performedN/AN/AWTNCA18EnBxAC4.03WT for EGFR, KRAS, NRAS, BRAFNGS TruSight19WTYes19EnBxSCC2.12Not performedN/AN/AWTNCA20TBBxAC3.85WT for EGFRcastPCR14KRAS G12C, NRAS Q61KNCA21EnBxSCC6.4Not performedN/AN/AWTNCA22TBBxAC1.63WT for EGFRcastPCR20KRAS G12CNCA23EnBxAC0.234WT for EGFR, KRAS, NRAS, BRAFNGS TruSight16WTYes24BALAC0.871BRAF G466V (12%)NGS TruSight24WTYes25TBBxAC0.0918BRAF G466V (12%)NGS TruSightN/AWTYes26TBBxAC0.893WT for EGFRcastPCR13WTYes27TBBxSCC0.66Not performedN/AN/AWTNCA28EnBxAC4.4WT for EGFRcastPCR10WTYesMean2.5819.6 Open table in a new tab The performance of the NanoString platform for SNV characterisation was highly concordant with alternate clinical testing methods for those with sufficient DNA. Advantages of NanoString include its multiplex capacity, high sensitivity, low nucleic acid input, reduced turn-around time (<24hr) compared to alternate testing methods. The NanoString platform is a robust method for identification of actionable variants in NSCLC where at least 5ng of DNA is available.
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