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

• Published in: Blood 2018-11, Vol.132 (Supplement 1), p.789-789
• Main Authors: Soverini, Simona, Bavaro, Luana, Martelli, Margherita, De Benedittis, Caterina, Iurlo, Alessandra, Orofino, Nicola, Pagano, Livio, Criscuolo, Marianna, Bonifacio, Massimiliano, Scaffidi, Luigi, Sica, Simona, Sorà, Federica, Maino, Elena, Rondoni, Michela, Laginestra, Maria Antonella, Lunghi, Francesca, Ermacora, Anna, D'adda, Mariella, Gugliotta, Gabriele, Castagnetti, Fausto, Rosti, Gianantonio, Papayannidis, Cristina, Marconi, Giovanni, Curti, Antonio, Miggiano, Maria Cristina, Galimberti, Sara, Percesepe, Antonio, Stagno, Fabio, Sancetta, Rosaria, Annunziata, Mario, Falzetti, Franca, Capodanno, Isabella, Pregno, Patrizia, Maffioli, Margherita, Intermesoli, Tamara, Di Bona, Eros, Caocci, Giovanni, Attolico, Imma, Binotto, Gianni, Bocchia, Monica, Angelucci, Emanuele, Sgherza, Nicola, Luciano, Luigiana, Mignone, Flavio, Pileri, Stefano A., Martinelli, Giovanni, Cavo, Michele
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2018-99-117166

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