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?
Authors: Soverini Simona , De Benedittis Caterina , Castagnetti Fausto , Gugliotta Gabriele , Mancini Manuela , Specchia Giorgina , Russo Domenico , Iurlo Alessandra , Bonifacio Massimiliano , Salvucci Marzia , Intermesoli Tamara , Avanzini Paolo , Galimberti Sara , Martino Bruno , Maino Elena , Scortechini Anna Rita , Machova Polakova Katerina , Saglio Giuseppe , Pane Fabrizio , Rosti Gianantonio , Baccarani Michele , Cavo Michele , Martinelli Giovanni 
Published: October 2019

Abstract

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 mutations1ST -LINE FAILURESNo CyR @ 3 mo9101BCR-ABL>10% @ 6 mo9000mCyR @ 6 mo1101BCR-ABL>1% @ 12 mo10022No CCyR @ 12 mo2101Loss of CCyR7314Loss of MMR20639Loss of CHR2101Progression to BP3202Total6315 (24%)621 (33%)1ST -LINE WARNINGSBCR-ABL>10% @ 3 mo7101BCR-ABL>1% @ 6 mo10112BCR-ABL>0.1% @ 12 mo12101Total293 (10%)14 (14%)2ND -LINE FAILURESNo CyR @ 3 mo3112BCR-ABL>10% @ 6 mo10224Loss of CCyR7303Loss of MMR6123Loss of CHR4303Progression to BP5303Total3513 (37%)518 (51%)2ND -LINE WARNINGSBCR-ABL>10% @ 3 mo6202BCR-ABL>0.1% @ 12 mo7022Total132 (15%)24 (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.