Ultrasensitive Duplex Sequencing of Pretreatment ABL1 Kinase Domain Mutations in Patients with Newly Diagnosed Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Background: Kinase domain (KD) mutations in ABL1 are the dominant mechanism of relapse in patients (pts) with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). Previous studies in Ph+ ALL have suggested that some pts harbor subclonal T315I mutations prior to tyrosine kinase inhibitor (TKI) treatment. However, nearly all reports have relied upon inherently error prone RT-PCR to generate template cDNA prior to mutation analysis. We hypothesized that conventional assays might over-estimate the incidence of pre-existing resistance mutations, and that improved sequencing accuracy might yield important information for risk stratification and TKI selection.

Methods: Duplex sequencing (DS) is a molecular tagging method that improves the accuracy of conventional next-generation sequencing by more than 10,000-fold, by comparing the nucleotide sequences of each strand of double-stranded molecules. ABL1 DS was performed on genomic DNA from 64 pts with newly diagnosed Ph+ ALL treated with hyper-CVAD plus a TKI. DS of exons 4-10 of ABL1 was performed to an average molecular depth of >10,000x. Among pts who relapsed, using RNA extracted from relapse samples, the KD (codons 221 through 500) of the BCR-ABL1 fusion transcript was sequenced by the Sanger method using a nested PCR approach, with a detection limit of 10-20%.

Results: The median age of the cohort was 54 years (range, 20-80 years). The TKI used was imatinib in 5 pts, dasatinib in 38 pts, and ponatinib in 21 pts. All pts achieved complete remission, and 12 pts (19%) underwent allogeneic stem cell transplantation in first remission.

A total of 115 pretreatment ABL1 KD mutations were detected among 47 pts (73%). The median number of pretreatment ABL1 KD mutations was 2 (range, 0-6 mutations). The median variant allelic frequency (VAF) of the detected somatic mutations was 0.008% (range, 0.004%-0.649%). Five mutations (4%) and 40 mutations (35%) were present at a VAF ≥0.1% and ≥0.01%, respectively. Eleven mutations known to confer resistance to at least one TKI were detected in 7 pts (11%), and included: F317L in 4 pts, E225K in 2 pts, and E225V, L384M, M244V, Q252H and T315I in 1 pt each. Five mutations were detected in ≥1 pretreatment sample (F317L in 4 pts, M244V in 3 pts, and E255K, E459K and V355V in 2 pts each). Of these 7 pts with pretreatment resistance mutations, 5 pts received a TKI known not to be sensitive to the mutation(s); 2 pts who received ponatinib had mutations at least intermediately sensitive to ponatinib (1 pt with F317L and 1 pt with both E255K and M244V mutations).

With a median duration of follow-up of 54 months (range, 1-124 months), 18 pts have relapsed. None of the 7 pts with known pretreatment resistance mutations relapsed. There was no difference in the number of pretreatment mutations between pts who relapsed and those who did not (median mutations: 1 [range, 0-4] and 2 [range, 0-6], respectively; P=0.26). Of the 18 pts who relapsed, 14 underwent Sanger sequencing for ABL1 KD mutations at the time of relapse. Clonal resistance mutations were detected at relapse in 9 pts (64% of sequenced samples): T315I in 6 pts, and F317I, V229L and V338G in 1 pt each (all in pts treated with dasatinib, except V338G in pt on imatinib). Relapse mutations were not observed in pretreatment samples in any of the pts.

Conclusions: DS identified very low level pretreatment ABL1 KD mutations in a majority of pts with newly diagnosed Ph+ ALL but these appear inconsequential. Ninety percent of mutations identified have not been described as resistance mutations, suggesting that they may be either synonymous or functionally neutral amino acid changes resulting from normal aging. There was no apparent association of these mutations and risk of relapse, even in the minority of cases in which known resistance mutations were detected at baseline. These data suggest that pretreatment testing for ABL1 KD mutations in Ph+ ALL is unlikely to affect treatment decisions.