METHODS: Aberrant transcripts of the six aforementioned oncogenes (36 transcripts in total) were examined in a molecular counting (MC) assay, which counts RNA molecules by simultaneous hybridization of several probes. Forty-one samples of surgically resected lung adenocarcinoma tissue found to express one of these aberrant oncogenic transcripts upon whole transcriptome sequencing (test cohort: n = 22) or reverse transcription polymerase chain reaction (validation cohort: n = 19) analyses were subjected to MC, after which biopsies were performed on tumor tissue samples.
RESULTS: Threshold values for the diagnosis of each of the 36 transcripts were determined in frozen and formalin-fixed paraffin-embedded samples from the test cohort. On the basis of these threshold values, the MC assay diagnosed expression of oncogenic transcripts in the validation cohort samples with 100% accuracy. The assay also accurately detected oncogenic fusions in bronchial lavage fluid and transbronchial biopsy samples.
CONCLUSIONS: The MC assay allows multiplex detection of oncogenic fusion and exon-skipped transcripts in tumor samples, including in formalin-fixed paraffin-embedded samples obtained in the clinic.