BCR-ABL Mutations in Chronic Myeloid Leukemia (CML) Patients (pts) with Failure and Warning to First- and Second-Line Tyrosine Kinase Inhibitor (TKI) Therapy: What Is the Advantage of Next-Generation Sequencing (NGS) over Conventional Sequencing?

Background – Point mutations in the BCR-ABL kinase domain are associated with resistance to TKI therapy. The most recent (2013) European LeukemiaNet (ELN) recommendations have re(de)fined the criteria for failure in pts receiving 1^st-line and 2^nd-line TKI therapy and introduced the concept of warning. Assessing in how many CML patients with failure and warning mutations can be identified, especially now that more sensitive NGS-based mutation screening methods are available, would advance our knowledge of the biology of TKI resistance as well as contribute useful data to revise the ELN recommendations as to when and how BCR-ABL mutation analysis should be performed.

Aims – We aimed to determine the frequency of BCR-ABL mutations as assessed by NGS vs conventional Sanger sequencing (SS) in CML pts with failure and warning to 1^st- or 2^nd-line TKI therapy as per the latest, 2013 ELN definitions.

Methods – Between May 2013 and June 2015, 298 consecutive CML pts on TKI therapy were referred to our laboratory for BCR-ABL mutation screening by SS. One hundred and fifty-eight cases had no clinical data available, or were not in CP, or were receiving ≥3^rd-line TKI therapy, or had confirmed/ suspected nonadherence, or had experienced dose reductions for toxicity – leaving 140 pts who could be included in this study. Pts who were negative for mutations as determined by SS (n=105/140) were retrospectively reanalyzed by NGS on a Roche GS Junior, using a protocol already set up and optimized in the framework of the IRON II (Interlaboratory RObustness of NGS) international consortium. Sequencing depth allowed to achieve a lower mutation detection limit of 1% in all samples.

Results – Failures and warnings to 1^st-line therapy (imatinib, n=57; nilotinib, n=22; dasatinib, n=13) were 63 and 29, respectively. BCR-ABL mutations were found in 15/63 (24%) failures and 3/29 (10%) warnings by SS (Table 1).

NGS reanalysis of the 74 pts with no evidence of mutations by SS revealed low burden (median, 6.6%; range, 1.5-11.7%) mutations in 6 failures and 1 warning, so that, overall, 21/63 (33%) failures and 4/29 (14%) warnings turned out to have mutations (Table 1). Mutations were E462K, E279K, K262R, F359I, E255K, F317L, K378R, A399T, L364I, V280A. No compound mutation was detected.

Failures and warnings to 2^nd-line therapy (nilotinib, n=27; dasatinib, n=21) were 35 and 13, respectively. SS identified mutations in 13/35 (37%) failures and 2/13 (15%) warnings (Table 1).

NGS reanalysis of the 33 pts with no evidence of mutations by SS revealed low burden (median, 5.4%; range, 1.9-10.0%) mutations in 5 failures and 2 warnings, so that, overall, 18/35 (51%) failures and 4/13 (31%) warnings turned out to have mutations (Table 1). Mutations were T315I, E255V, F317I, E258D, P480L, Y393C, W261L, L370P, V371A, L324Q, again with no compound mutations.

	All pts	Pts positive for mutations by SS	Additional pts positive for mutations by NGS	Total pts positive for mutations
1ST-LINE FAILURES
No CyR @ 3 mo	9	1	0	1
BCR-ABL>10% @ 6 mo	9	0	0	0
mCyR @ 6 mo	1	1	0	1
BCR-ABL>1%  @ 12 mo	10	0	2	2
No CCyR @ 12 mo	2	1	0	1
Loss of CCyR	7	3	1	4
Loss of MMR	20	6	3	9
Loss of CHR	2	1	0	1
Progression to BP	3	2	0	2
Total	63	15 (24%)	6	21 (33%)
1ST-LINE WARNINGS
BCR-ABL>10% @ 3 mo	7	1	0	1
BCR-ABL>1% @ 6 mo	10	1	1	2
BCR-ABL>0.1% @ 12 mo	12	1	0	1
Total	29	3 (10%)	1	4 (14%)
2ND-LINE FAILURES
No CyR @ 3 mo	3	1	1	2
BCR-ABL>10% @ 6 mo	10	2	2	4
Loss of CCyR	7	3	0	3
Loss of MMR	6	1	2	3
Loss of CHR	4	3	0	3
Progression to BP	5	3	0	3
Total	35	13 (37%)	5	18 (51%)
2ND-LINE WARNINGS
BCR-ABL>10% @ 3 mo	6	2	0	2
BCR-ABL>0.1% @ 12 mo	7	0	2	2
Total	13	2 (15%)	2	4 (31%)

Conclusions

1) NGS allowed to identify BCR-ABL mutations in a greater proportion of cases as compared to SS. Low burden mutations included a T315I mutation in 2 pts on 2^nd-line therapy classified as warnings: this would have turned them into failures.

2) Still, a substantial proportion of cases was found to not harbor any mutation, even when using  a more sensitive NGS-based method. In particular, non-optimal achievement of the key molecular response milestones (10%, 1%, 0.1%) on 1^st-line therapy was mostly not associated with BCR-ABL mutations, indicating that other mechanisms of molecular disease persistence have to be investigated in an attempt to optimize therapeutic outcomes.

A national, multicenter study ('NEXT-IN-CML') aimed at the prospective assessment of NGS for routine BCR-ABL mutation screening of CML patients has just started.

Supported by ELN, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi)