Clinical Utility of a Novel Triplex Digital PCR Assay for Clone Monitoring in Sequential and Relapsed Pediatric...

Digital polymerase chain reaction (PCR) studies for clonal disease monitoring in B-acute lymphoblastic leukemia patients are currently limited due to the heterogeneous nature of mutations, which limit cost-effective assay designs.

In this study, 70 samples (14 relapse and 56 sequential therapy samples) were tested for 13 recurrent mutations identified on deep sequencing in our published cohort (KRAS, NRAS, NT5C2, PMS2, UHRF1, KMT2D, and TP53 genes) via a novel triplex digital PCR assay.

A total of seven major clones of NRAS [five] and NT5C2 [two] were noted in six out of 14 (43%) relapse patients, accounting for 44% of early relapses. In addition, 10 minor clones (PMS2 [two], NRAS [four], NT5C2 [three], and TP53 [one]) were noted in five out of 14 (36%) patients. In the 56 sequential therapy samples, six major clones were noted (NRAS [five], KRAS [one]) in four out of 14 (28.5%) patients, with two increasing in size in maintenance samples, leading to subsequent relapse in both cases. In addition, therapy-acquired minor clones in NT5C2 [four] and PMS2 [one] were seen to emerge in maintenance samples in four out of 14 (28.5%) patients, with concordant detection of such major and minor clones in unpaired relapse samples, indicating the need for their active surveillance during therapy. Overall, digital PCR validated NRAS and NT5C2 major clones in one-third (10 out of 27; 37%) of cases, driving nearly half of early relapses.