Background - Point mutations in the BCR-ABL kinase domain are associated with resistance to TKI therapy. The most recent (2013) European Leukemia Net (ELN) recommendations have re(de)fined the criteria for failure in pts receiving 1st-line and 2nd-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 1st- or 2nd-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 ≥3rd-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 1st-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 2nd-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.

Table.
All ptsPts positive for mutations by SSAdditional pts positive for mutations by NGSTotal pts positive for mutations
1ST -LINE FAILURES  
No CyR @ 3 mo 
BCR-ABL>10% @ 6 mo 
mCyR @ 6 mo 
BCR-ABL>1% @ 12 mo 10 
No CCyR @ 12 mo 
Loss of CCyR 
Loss of MMR 20 
Loss of CHR 
Progression to BP 
Total 63 15 (24%) 21 (33%) 
1ST -LINE WARNINGS  
BCR-ABL>10% @ 3 mo 
BCR-ABL>1% @ 6 mo 10 
BCR-ABL>0.1% @ 12 mo 12 
Total 29 3 (10%) 4 (14%) 
2ND -LINE FAILURES  
No CyR @ 3 mo 
BCR-ABL>10% @ 6 mo 10 
Loss of CCyR 
Loss of MMR 
Loss of CHR 
Progression to BP 
Total 35 13 (37%) 18 (51%) 
2ND -LINE WARNINGS  
BCR-ABL>10% @ 3 mo 
BCR-ABL>0.1% @ 12 mo 
Total 13 2 (15%) 4 (31%) 
All ptsPts positive for mutations by SSAdditional pts positive for mutations by NGSTotal pts positive for mutations
1ST -LINE FAILURES  
No CyR @ 3 mo 
BCR-ABL>10% @ 6 mo 
mCyR @ 6 mo 
BCR-ABL>1% @ 12 mo 10 
No CCyR @ 12 mo 
Loss of CCyR 
Loss of MMR 20 
Loss of CHR 
Progression to BP 
Total 63 15 (24%) 21 (33%) 
1ST -LINE WARNINGS  
BCR-ABL>10% @ 3 mo 
BCR-ABL>1% @ 6 mo 10 
BCR-ABL>0.1% @ 12 mo 12 
Total 29 3 (10%) 4 (14%) 
2ND -LINE FAILURES  
No CyR @ 3 mo 
BCR-ABL>10% @ 6 mo 10 
Loss of CCyR 
Loss of MMR 
Loss of CHR 
Progression to BP 
Total 35 13 (37%) 18 (51%) 
2ND -LINE WARNINGS  
BCR-ABL>10% @ 3 mo 
BCR-ABL>0.1% @ 12 mo 
Total 13 2 (15%) 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 2nd-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 1st-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)

Disclosures

Soverini:Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Castagnetti:BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria. Bonifacio:Ariad Pharmaceuticals: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Novartis Farma: Research Funding. Saglio:Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria. Rosti:Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau. Baccarani:NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ARIAD Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Martinelli:Pfizer: Consultancy; Novartis: Consultancy, Speakers Bureau; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; AMGEN: Consultancy; MSD: Consultancy; Ariad: Consultancy.

Author notes

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Asterisk with author names denotes non-ASH members.

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