-Author name in bold denotes the presenting author
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789 Compound BCR-ABL1 Kinase Domain Mutants: Prevalence, Spectrum and Correlation with Tyrosine Kinase Inhibitor Resistance in a Prospective Series of Philadelphia Chromosome-Positive Leukemia Patients Analyzed By Next Generation Sequencing

Program: Oral and Poster Abstracts
Type: Oral
Session: 632. Chronic Myeloid Leukemia: Therapy: TFR Failure, Resistance, and New Drug Development
Hematology Disease Topics & Pathways:
Technology and Procedures, NGS
Monday, December 3, 2018: 3:15 PM
Room 6E (San Diego Convention Center)

Simona Soverini, PhD1, Luana Bavaro1*, Margherita Martelli1*, Caterina De Benedittis, PhD2*, Alessandra Iurlo3*, Nicola Orofino, MD4*, Livio Pagano5*, Marianna Criscuolo6*, Massimiliano Bonifacio7*, Luigi Scaffidi, MD8*, Simona Sica, MD9*, Federica Sorà, MD10*, Elena Maino11*, Michela Rondoni, MD12*, Maria Antonella Laginestra, PhD13*, Francesca Lunghi14*, Anna Ermacora15*, Mariella D'adda, MD16*, Gabriele Gugliotta, MD, PhD17, Fausto Castagnetti18, Gianantonio Rosti17*, Cristina Papayannidis19, Giovanni Marconi19, Antonio Curti1*, Maria Cristina Miggiano20*, Sara Galimberti21*, Antonio Percesepe, MD, PhD22*, Fabio Stagno, MD, PhD23, Rosaria Sancetta, MD24*, Mario Annunziata25*, Franca Falzetti, MD26*, Isabella Capodanno27*, Patrizia Pregno28*, Margherita Maffioli, MD29*, Tamara Intermesoli, MD30*, Eros Di Bona, MD31*, Giovanni Caocci, MD32*, Imma Attolico, MD33*, Gianni Binotto34*, Monica Bocchia, MD, PhD35*, Emanuele Angelucci, MD36, Nicola Sgherza, MD37*, Luigiana Luciano38*, Flavio Mignone39*, Stefano A. Pileri, MD, PhD40, Giovanni Martinelli41 and Michele Cavo, MD1*

1Hematology/Oncology "L. e A. Seràgnoli", University of Bologna, Bologna, Italy
2Hematology/Oncology "L. e A. Seragnoli", University of Bologna, Bologna, Italy
3Hematology Division, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
4Hematology Unit, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico - University of Milan, Milan, Italy
5Institute of Hematology, Fondazione Policlinico A. Gemelli- IRCCS- Università Cattolica S. Cuore, Rome, Italy
6Institute of Hematology, Fondazione Policlinico A. Gemelli- IRCCS- Università Cattolica S. Cuore, Roma, Italy
7Department of Medicine, Section of Haematology, University of Verona, Verona, Italy
8Department of Medicine, Section of Hematology, University of Verona, Verona, Italy
9Hematology Department, Fondazione Policlinico Universitario A. Gemelli IRCCS - Università Cattolica del Sacro Cuore, Roma, Italy
10Fondazione Policlinico Universitario A Gemelli IRCSS, Catholic University of Rome, Rome, Italy
11Ospedale dell' Angelo, Hematology, Mestre, Italy
12Azienda Unità Sanitaria Locale della Romagna, Haematology Department, Ravenna, Italy
13Department of Experimental, Diagnostic, and Specialty Medicine, Hematopathology Section, Bologna, Italy
14Haematology and Bone Marrow Transplant Unit, San Raffaele Scientific Institute IRCCS, Milano, Italy
15Azienda Ospedaliera S Maria Angeli, Pordenone, Italy
16Department of Hematology, ASST Spedali Civili di Brescia, Brescia, Italy
17Institute of Hematology "L. and A. Seràgnoli", Department of Experimental, Diagnostic and Specialty Medicine, Bologna, Italy
18Institute of Hematology "L. e A. Seràgnoli", DIMES, University of Bologna, Bologna, Italy
19Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology "L. e A. Seràgnoli", University of Bologna, Bologna, Italy
20Hematology Unit, Vicenza, Italy
21Section of Hematology, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy
22Department of Medicine and Surgery, Medical Genetics Unit, University of Parma, Parma, Italy
23Hematology Unit, Hematology Section, Biomedical Sciences, Trecastagni, Catania, Italy
24Hematology Unit, Ospedale dell'Angelo Mestre, Venezia, Italy
25Hematology, Ospedale Cardarelli, Napoli, Italy
26Institute of Hematology, Centro di Ricerche Emato-Oncologiche (CREO), University of Perugia, Perugia, Italy
27Hematology Department, Arcispedale Santa Maria Nuova - IRCCS Reggio, Reggio Emilia, Italy
28Hematology Unit, Az Ospedaliero Universitaria Città' della Salute e della Scienza, Torino, Italy
29Hematology, Department of Medicine and Surgery, Ospedale di Circolo, ASST Sette Laghi, Varese, Italy
30Hematology and Bone Marrow Transplantation Unit, ASST Papa Giovanni XXIII, Bergamo, Italy
31Ospedale San Bortolo, UO Hematology, Vicenza, Italy
32Department of Medical Sciences and Public Health, University of Cagliari, "A.Businco" Hospital, Cagliari, Italy
33Haematology, University of Bari, Potenza, Italy
34Dept. of Medicine, Hematology and Clinical Immunology, Padua School of Medicine, PADOVA, Italy
35Chair of Hematology, University of Siena, Azienda Ospedaliera Universitaria, Siena, Italy
36Hematology and Transplant Centre, Unita Operativa Ematologia Ospedale 'A. Businco', Genova, Italy
37Division of Hematology, IRCCS Ospedale Casa Sollievo Sofferenza, San Giovanni Rotondo, Italy
38AOU Università degli Studi di Napoli "Federico II", Napoli, Italy
39Department of Science and Innovation Technology (DISIT), University of Piemonte Orientale, Alessandria, Italy
40Unit of Hematopathology, European Institute of Oncology, MILANO, Italy
41Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy, Meldola, Italy

Next-Generation Sequencing (NGS)-based BCR-ABL1 kinase domain (KD) mutation screening has been shown to enable greater accuracy and sensitivity and straightforward identification of compound mutants (CM) as compared to Sanger sequencing (seq). However, the prevalence of CMs has never been assessed in prospective studies, and although in vitro data suggest that many of them may be challenging for all tyrosine kinase inhibitors (TKIs) including ponatinib, attempts to correlate such data with in vivo responses have never been made. To address these issues, we have reviewed the results of routine NGS-based BCR-ABL1 KD mutation screening performed over the past 3 years.

Between 2015 and 2018, we have prospectively used NGS to analyze a consecutive series of 751 Ph+ leukemia patients (pts) on TKI therapy who were eligible for BCR-ABL1 KD mutation screening according to ELN/NCCN/ESMO recommendations. The study population included 664 chronic myeloid leukemia (CML) pts with failure or warning response (chronic phase [CP], n=593; accelerated or blastic phase [AP/BP], n=71) and 87 Ph+ acute lymphoblastic leukemia (ALL) pts with relapsed/refractory disease. NGS of ≈400bp amplicons generated by nested RT-PCR was performed on a Roche GS Junior (until April 2017) or on an Illumina MiSeq (from May 2017 on) using custom protocols whose accuracy, sensitivity and reproducibility was checked by national and international (EUTOS) control rounds. Read alignment and variant calling was done using the AmpSuite software (SmartSeq srl), with a lower detection limit set to 3%. Cis or trans configuration of mutation pairs, indicating CMs or polyclonality, respectively, was determined correcting for the likelihood of PCR recombination. The 35INS insertion/truncation mutant was excluded from the analysis.

NGS identified mutations in the BCR-ABL1 KD in a total of 313/664 (47%) CML pts (255/593 [43%] CP-CML and 58/71 [82%] AP/BP-CML) and 69/87 (79%) Ph+ ALL pts. Ninety-one percent of the mutations could be recognized as conferring resistance to at least one TKI on the basis of publicly available IC50 data or published reports. In 42/593 (7%) CP-CML, 6/71 (8.5%) AP/BP-CML and 12/87 (14%) Ph+ ALL pts, low burden mutations (i.e., mutations carried by a proportion of transcripts <15% – hence invisible to Sanger seq) were the only detectable mutation(s). In 21/593 (3.5%) CP-CML, 26/71 (37%) AP/BP-CML and 40/87 (46%) Ph+ ALL pts low burden mutations co-existed together with at least one dominant mutation (i.e. a mutation carried by a proportion of transcripts >15% – hence detectable by Sanger seq). Fifty-five (9.2%) CP-CML, 51 (72%) AP/BP-CML and 56 (49%) Ph+ ALL pts had ≥2 mutations (CP-CML: 1-5 mutations; AP/BP-CML: 1-6 mutations; Ph+ ALL: 1-13 mutations). Identification of CMs in pts with ≥2 mutations was fully possible (i.e., all the candidate pairs mapped within a distance of 400bp) in 71% of cases and partially possible (i.e., some, but not all the candidate pairs mapped within a distance of 400bp) in another 12% of cases. A total of 86 CMs (85 double and 1 triple) in 73 pts (21 [3.5%] CP-CML, 23 [32%] AP/BP-CML and 29 [37%] Ph+ ALL pts) could be catalogued (Figure 1A). All but two (T315I+D276G, M244V+E255K) were detected in pts who had received ≥2 TKIs and all included at least a 2nd-generation TKI-resistant mutation. The most frequent CMs were T315I+E255K, T315I+E255V, T315I+F359V, F317L+Y253H (Figure 1A). The triple CM, detected in a ponatinib-resistant pt, was F317I+Y253F+Q252H. Correlation of IC50 data with in vivo responses (the TKIs pts were clinically resistant to) confirmed only partially in vitro predictions (Figure 1B). In particular, although ponatinib was shown in vitro to be poorly effective against several CMs, only the T315I+E255V was consistently found to be associated with ponatinib failure.

In conclusion, our results in a large unselected series of TKI-resistant pts analyzed by NGS show that:

  • CMs are relatively infrequent in CP-CML, but may be a relevant issue in AP/BP-CML and Ph+ ALL;
  • among pts with multiple mutations, those who have failed 1 line of therapy have most often polyclonality, whereas those who have failed ≥2 lines of therapy may have CMs or polyclonality;
  • in vitro predictions of sensitivity and insensitivity based on IC50 data should be regarded with caution. In particular, the only compound mutant that we consistently found to be associated with ponatinib failure was the T315I+E255V.

Supported by EUTOS 2016.

Disclosures: Soverini: Novartis: Consultancy; Incyte Biosciences: Consultancy; Bristol Myers Squibb: Consultancy. Pagano: Pfizer: Speakers Bureau; Gilead: Speakers Bureau; Basilea: Speakers Bureau; Merck: Speakers Bureau; Janssen: Speakers Bureau. Gugliotta: Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Castagnetti: Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Meyers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Angelucci: Roche Italy: Other: Local (national) advisory board; Novartis: Honoraria, Other: Chair Steering Comiittee TELESTO Protocol; Celgene: Honoraria, Other: Chair DMC; Jazz Pharmaceuticals Italy: Other: Local ( national) advisory board; Vertex Pharmaceuticals Incorporated (MA) and CRISPR CAS9 Therapeutics AG (CH): Other: Chair DMC. Martinelli: Abbvie: Consultancy; Ariad/Incyte: Consultancy; Janssen: Consultancy; Novartis: Speakers Bureau; Jazz Pharmaceuticals: Consultancy; Roche: Consultancy; Pfizer: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy. Cavo: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees.

*signifies non-member of ASH