What's Next in CML - a Prospective Study Evaluating Sanger Sequencing and Next Generation Sequencing (NGS) for BCR-ABL1 Kinase Domain (KD) Mutation Screening

Small retrospective studies have suggested that NGS may have several advantages over Sanger sequencing (seq) for BCR-ABL1 KD mutation screening in Philadelphia chromosome-positive leukemia patients (pts). However, how frequently low burden mutations can be detected in pts with Failure or Warning response to tyrosine kinase inhibitors (TKI) and which role low burden mutations play in these pts remain to be addressed prospectively in large series of unselected pts. Moreover, the implementation of routine, NGS-based BCR-ABL1 mutation screening in a molecular diagnostic lab network has never been attempted. To assess the feasibility, performance, cost, turnaround times and clinical utility of NGS, we have conducted a multicenter prospective study: 'Next in CML'.

The first phase of the study was aimed to 1) create a network of 4 reference labs sharing a standardized protocol, an optimized pipeline of data analysis and a joint database; 2) assess the accuracy and inter-lab reproducibility of results. NGS of amplicons generated by nested RT-PCR was done on Roche GS Junior instruments. A dedicated software (Amplicon Suite; SmartSeq srl) was implemented for read alignment and variant calling. Identical batches of 32 blinded cDNA samples (23 pt samples with known mutation status and 9 serial dilutions of BaF3 T315I-positive cells in BaF3 unmutated cells, simulating mutation loads of 20% to 1%) were sequenced in parallel to check individual lab performances. Of 64 expected mutations, 52 were detected (and accurately quantitated) by 4 labs, 7 by 3 labs, 4 by 2 labs and 1 by 1 lab only. Given that i) mutations that some labs failed to detect ranged between 1 and 3%, and ii) in mutation-negative samples, mutations likely to be PCR/sequencing errors were all <3%, 3% was set as lower detection limit.

The second phase of the study was aimed to apply NGS in parallel with Sanger seq in a consecutive series of prospectively collected samples from 211 chronic myeloid leukemia (CML) pts with Failure or Warning treated at 39 Hematology Centers. Pts positive for any point mutation (the 35bp insertion between exon 8 and 9 was excluded) were 42 (20%) by Sanger seq and 94 (45%) by NGS. Low burden mutations detectable by NGS were found in 52 pts negative for mutations by Sanger seq; in addition, 24 pts positive for mutations by Sanger seq were found to carry additional low burden mutations by NGS, for a total of 76 (36%) pts harboring low burden mutations. Median mutation burden was 11.1% (3%-18.8%). Eight pts had a low burden T315I and 43 pts had other IM/DAS/NIL/BOS resistant mutations: overall, 51 (24%) pts had relevant TKI-resistant low burden mutations missed by Sanger seq. The remaining 25 pts had only mutations with an unknown resistance profile. Pts positive by Sanger seq showing additional low burden mutations detectable by NGS (n=24) mainly had high/intermediate Sokal risk (n=19) and were receiving ≥2^nd line therapy (n=17). Pts negative by Sanger seq but positive for low burden mutations by NGS (n=52) mainly had high/intermediate Sokal risk (n=29), Warning response (n=29) and were receiving a reduced TKI dose (n=37). Among pts negative for mutations both by Sanger seq and NGS (n=117), 39 (33%) had loss of MMR reported as a Failure after a single RQ-PCR test only and/or a BCR-ABL1 transcript increase <1 log not confirmed by subsequent follow-up evaluations, suggesting that in several cases failure to detect mutations results from improper triggers for mutation screening.

Longitudinal follow-up, available at the time of writing for 27 pts with low burden mutations, showed that TKI-resistant mutations remain consistently detectable and tend to increase in burden at subsequent timepoints unless treatment is changed. Low burden mutations of unknown resistance profile, in contrast, do not necessarily require treatment intervention to be eliminated.

Our study demonstrates that a robust and reproducible NGS-based BCR-ABL1 KD mutation screening can successfully be implemented in national diagnostic lab networks and is feasible with turnaround times and costs comparable to those of Sanger seq. In a large, prospective series of CML pts with Failure or Warning, known IM/DAS/NIL/BOS resistant mutations were missed by Sanger seq in 24% of the pts. Low burden (≥3%) TKI-resistant mutations were found to be sufficient to drive clonal expansion, whereas more data are needed to understand the clinical significance of those with an unknown resistance profile.