Ultra-short circulating tumor DNA (usctDNA): A new paradigm for ctDNA liquid biopsy.

Abstract

e20510 Background: Mononucleosomal ctDNA (mnctDNA, 157-bp, double-stranded) spearheads liquid biopsy (LB) for cancer detection, treatment and clinical care. The bottleneck of mnctDNA LB are low copy number of ctDNA and limit of detection of platform technologies. Recent discovery of ultra-short ctDNA (usctDNA, 52-54 nt, single-stranded) that harbors somatic mutations prompted us to explore the quantification and stoichiometry of usctDNA to mnctDNA in plasma and saliva of NSCLC patients. Methods: Paired plasma and saliva from 20 advanced NSCLC patients with tissue genotyped positive for L858R were processed for cfDNA extraction and quantification with Qubit and Tape Station. Digital PCR assays targeting L858R somatic mutation to generate amplicons of 57-bp (usctDNA) and 100-bp (mnctDNA) were performed by UCLA and independently by NIST. Results: Results in Table 1 revealed that three findings of usctDNA that will have potential to impact ctDNA liquid biopsy. First, the somatic mutation (L858R) is present in usctDNA. Second, The stoichiometric ratio of usctDNA to mnctDNA is similar in plasma and saliva at 1.6X. For the wild-type counterpart, the ratio is 1.1X for plasma and 0.6X for saliva. Third, the copy number of usctDNA and mnctDNA in saliva to plasma is 7.8X and 7.7X. Conclusions: Detection of tumor-associated/specific EGFR L858R somatic mutations in uscfDNA is translationally impactful, adding 1.6X of the ctDNA targets for molecular detection, effectively cumulating 2.6X of total ctDNA targets, permitting earlier cancer detection, treatment monitoring and therapy prognostication. The finding that saliva harbors 7X of the us- and mnctDNA further raises the prospect of advancing saliva ctDNA liquid biopsy for NSCLC detection as it may effectively allow 15.5X (7.8X+7.7X) ability to detect malignancy-associated ctDNA. [Table: see text]