-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

346 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?Clinically Relevant Abstract

Chronic Myeloid Leukemia: Therapy
Program: Oral and Poster Abstracts
Type: Oral
Session: 632. Chronic Myeloid Leukemia: Therapy: Treatment-Free Remission, Mutations, and Prognosis
Sunday, December 6, 2015: 5:15 PM
Valencia BC (W415BC), Level 4 (Orange County Convention Center)

Simona Soverini, PhD1, Caterina De Benedittis, PhD1*, Fausto Castagnetti, MD/PhD2, Gabriele Gugliotta, PhD1*, Manuela Mancini, PhD1*, Giorgina Specchia, Pr, MD3, Domenico Russo, MD, PhD4, Alessandra Iurlo, MD, PhD5*, Massimiliano Bonifacio, MD6*, Marzia Salvucci7*, Tamara Intermesoli, MD8*, Paolo Avanzini, MD9*, Sara Galimberti, MD10*, Bruno Martino, MD11*, Elena Maino, MD12*, Anna Rita Scortechini, MD13*, Katerina Machova Polakova14*, Giuseppe Saglio15, Fabrizio Pane, MD16, Gianantonio Rosti, MD1*, Michele Baccarani17, Michele Cavo, MD1* and Giovanni Martinelli, MD, PhD2

1"Serŕgnoli" Institute of Hematology, Bologna University School of Medicine, Bologna, Italy
2Bologna University School of Medicine, Bologna, Italy
3Department of Emergency and Organ Transplantation, Section of Hematology with Transplantation, Medical Sch, University of Bari, Bari, Italy
4Chair of Hematology, Unit of Blood Diseases and Stem Cell Transplantation, University of Brescia, Brescia, Italy
5Division of Hematology, IRCCS Maggiore Policlinico Hospital Foundation, Milano, Italy
6Department of Medicine, Section of Hematology, University of Verona, Verona, Italy
7S. Maria delle Croci Hospital, Ravenna, Italy
8Hematology and Bone Marrow Transplant Unit, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy
9Hematology, S.Maria Nuova Hospital, Reggio Emilia, Italy
10UO University Hematology, AOU Pisana, Pisa, Italy
11Hamatology Dept., Ospedali Riuniti Bianchi Melacrino Morelli, Reggio Calabria, Italy
12Ospedale dell Angelo-Ematologia, Mestre, Italy
13Azienda Ospedaliera Universitaria - Ospedali Riuniti di Ancona, Ancona, Italy
14Institute of Hematology and Blood Transfusion, University of Prague, Prague, Czech Republic
15University of Turin, Orbassano, Italy
16Department of Biochemistry and Medical Biotechnologies, Federico II University, Napoli, Italy
17Institute of Hematology, University of Bologna, Bologna, Italy

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 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.

 

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 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 .

*signifies non-member of ASH