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Background  Exosomal nucleic acid (exoNA) is a feasible target to improve the sensitivity of  EGFR  mutation testing in non-small cell lung cancer patients with limited cell-free DNA (cfDNA) mutant copies. However, the type and size of target exoNA related to the sensitivity of  EGFR  mutation testing has not been explored extensively.    Methods  The type and size of target exoNA related to the sensitivity of  EGFR  mutation testing was evaluated using ddPCR. A total of 47 plasma samples was tested using short-length exoTNA (exosomal DNA and RNA) and cfDNA.    Results  The sensitivity of short-length exoTNA (76.5%) was higher than that of cfDNA (64.7%) for detecting  EGFR  mutations in NSCLC patients. In  EGFR -mutant NSCLC patients with intrathoracic disease (M0/M1a) or cases with low-copy T790M, the positive rate was 63.6% ( N  = 7/11) and 45.5% ( N  = 5/11) for short-length exoTNA and cfDNA, respectively. On average, the number absolute mutant copies of short-length exoTNA were 1.5 times higher than that of cfDNA. The mutant allele copies (Ex19del and T790M) in short-length exoTNA were relatively well preserved at 4 weeks after storage. The difference (%) in absolute mutant allele copies (Ex19del) between 0 days and 4 weeks after storage was − 61.0% for cfDNA.    Conclusion  Target nucleic acids and their size distribution may be critical considerations for selecting an extraction method and a detection assay. A short-length exoTNA (200 bp) contained more detectable tumor-derived nucleic acids than exoDNA (~ 200 bp length or a full-length) or cfDNA. Therefore, a short-length exoTNA as a sensitive biomarker might be useful to detect  EGFR  mutants for NSCLC patients with low copy number of the mutation target.

Selecting short length nucleic acids localized in exosomes improves plasma EGFR mutation detection in NSCLC patients