scholarly journals Unraveling the complexity of tyrosine kinase inhibitor–resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain

Blood ◽  
2013 ◽  
Vol 122 (9) ◽  
pp. 1634-1648 ◽  
Author(s):  
Simona Soverini ◽  
Caterina De Benedittis ◽  
K. Machova Polakova ◽  
Adela Brouckova ◽  
David Horner ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Deep Sequencing ◽  
Sanger Sequencing ◽  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Key Points ◽  
Conventional Methods

Key Points UDS demonstrated that BCR-ABL KD mutations detectable with conventional methods may just be the tip of the iceberg. The information provided by conventional Sanger sequencing may not always be sufficient to predict responsiveness to a given TKI.

Ultra-Deep Sequencing of the Bcr-Abl Kinase Domain Allows Earlier Detection and More Accurate Characterization of Resistant Subclones in Philadelphia-Positive Acute Lymphoblastic Leukemia Patients Receiving Tyrosine Kinase Inhibitor-Based Therapies

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 284-284
Author(s):  
Simona Soverini ◽  
Caterina De Benedittis ◽  
Katerina Machova Polakova ◽  
Adela Brouckova ◽  
Cristina Papayannidis ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Deep Sequencing ◽  
Sanger Sequencing ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Mutation Screening ◽  
Kinase Domain ◽  
Abl Kinase

Abstract Abstract 284 Background and Aims: Selection of drug-resistant mutations in the Bcr-Abl kinase domain (KD) is a critical problem undermining the long-term efficacy of tyrosine kinase inhibitor (TKI)-based therapies in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients. Bcr-Abl KD mutation screening is routinely performed by Sanger sequencing (SS). Before the advent of ultra-deep sequencing (UDS) technologies, no method was available that could conjugate the possibility to scan the KD for the so many mutations known to be associated with TKI resistance with a sensitivity higher than that of SS. UDS technologies also allow high throughputness and accurate quantitation of mutated clones and their application in a diagnostic setting is not far to come. We used an UDS strategy for Bcr-Abl KD mutation screening in order to study the dynamics of expansion of mutated clones in Ph+ ALL patients receiving TKI-based therapies and to test the ability of UDS to highlight emerging clones harboring critical mutations. Methods: 72 samples from 25 Ph+ ALL patients who had developed resistance to one or multiple lines of TKI (imatinib, dasatinib, nilotinib, bosutinib, ponatinib) therapy were selected for this retrospective analysis. All the patients had previously been analyzed by Sanger sequencing (SS) and were known to have developed one or more TKI-resistant Bcr-Abl KD mutations on treatment. In order to reconstruct the dynamics of mutation emergence, longitudinal re-analysis of monthly collected samples was perfomed with UDS on a Roche GS Junior. UDS allowed to achieve a lower detection limit of at least 0.1% (by generating a minimum of 5,000 sequence reads/patient), as compared to 20% of SS. Results: 39 samples were known to harbor one (n=27 samples) or more (n=12 samples) TKI-resistant mutations with >20% abundance, as assessed by SS. UDS could successfully detect all the 54 mutations previously identified by SS. In addition, UDS detected one or multiple lower-level (<20%) mutations in 42/72 (58%) samples, demonstrating that in more than half of the cases SS may misclassify Bcr-Abl KD mutation status or underestimate its complexity. Lower-level mutations were indeed found both in samples that had been scored as wild-type by SS and in samples already harboring mutations with >20% abundance. The type of lower-level mutations detected by UDS could easily be accounted for by TKI exposure history, since the majority were known to be poorly sensitive either to the TKI being administered or to the previous TKI received. Overall, 44 samples turned out to carry multiple (two to five) mutations at any level, distributed in the same and/or in different subpopulations with a complex clonal architecture that UDS allowed to reconstruct. Of note, in 14/25 (56%) patients with molecularly detectable disease but not yet evidence of cytogenetic or hematologic relapse, UDS could identify emerging TKI-resistant mutations 1 to 2 months before they became detectable by SS. These outgrowing mutations were detected at 1–19% abundance in 12 patients and at 0.1–1% abundance in 2 patients. In the remaining 11 patients, dynamics of outgrow of the TKI-resistant mutations (five T315I, two Y253H, two E255K, one E255V and one F317L) was so rapid that not even strict monthly monitoring could allow to pick them up before they became dominant. Conclusions: Now that multiple options are available, Bcr-Abl KD mutation monitoring has become a precious tool for rational decision-making in order to maximize the efficacy of TKI-based regimens as induction or salvage therapy for Ph+ ALL patients. UDS proved as reliable as SS for the detection of mutations with >20% abundance and to have comparable costs. As a key advantage, UDS added precious quantitative and qualitative information on the full repertoire of mutated populations, that SS failed to appreciate in more than half of the samples analyzed. TKI-resistant mutations leading to patient relapse were not necessarily preexisting at low levels at diagnosis or at the time of switchover to another TKI, underlining the importance of regular monitoring of patients. Although TKI-resistant populations may arise and take over very rapidly, in approximately half of the patients monthly monitoring with UDS would have allowed to identify them earlier than SS and well in advance of clinical relapse, thus allowing a more timely therapeutic intervention. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Luppi:CELGENE CORPORATION: Research Funding. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

scholarly journals The 'Next-in-Cml' Study: A Prospective Multicenter Study of Deep Sequencing of the BCR-ABL1 Kinase Domain in Philadelphia Chromosome-Positive Patients with Non-Optimal Responses to Tyrosine Kinase Inhibitor Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3097-3097
Author(s):  
Simona Soverini ◽  
Caterina De Benedittis ◽  
Stefania Stella ◽  
Anna Serra ◽  
Francesca Carnuccio ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Board Of Directors ◽  
Deep Sequencing ◽  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Second Phase ◽  
Advisory Committees ◽  
Mutation Status

Abstract Introduction Some retrospective studies in tyrosine kinase inhibitor (TKI)-resistant Philadephia-positive (Ph+) leukemia patients (pts) have suggested that deep sequencing (DS) may provide a more accurate picture of BCR-ABL1 kinase domain (KD) mutation status as compared to conventional sequencing (CS). However, the frequency and clinical relevance of low burden mutations remains to be explored prospectively in large series of unselected pts. In addition, the implementation of routine BCR-ABL1 DS in multiple molecular diagnostic laboratories has never been attempted. These open issues led us to design a multi-center, multi-laboratory prospective study ('NEXT-IN-CML') aimed to assess the feasibility, performance and informativity of DS for BCR-ABL1 KD mutation screening. Aims The first phase of the study was aimed to establish a network of 5 reference labs sharing a standardized DS workflow, a joint database for clinical and mutational data storage and a common pipeline of data analysis, interpretation and clinical reporting. The second phase of the study, involving 54 Italian Hematology Units, is aimed to assess the frequency and clinical significance of low burden mutations detectable by DS by prospective collection and analysis of samples from chronic myeloid leukemia (CML) pts who exhibit failure (F) or warning (W) responses and relapsed Ph+ acute lymphoblastic leukemia (ALL) pts. Methods A PCR and an amplicon DS protocol already set up and optimized for the Roche GS Junior in the framework of the IRON II international consortium was adopted. In the first phase, 5 batches of blinded cDNA samples were prepared and shipped to evaluate individual lab performances. The batches included archival samples with known BCR-ABL1 mutation status as assessed by CS and serial dilutions of BaF3 T315I+ cells in BaF3 unmutated cells, simulating mutation loads of 20% down to 1%. In the ongoing second phase prospectively, consecutively collected CML and Ph+ ALL samples are being analyzed in parallel by CS and DS. Clinical history and follow-up data are used for correlations. Results In the first phase of the study, 312/320 amplicons were successfully generated and sequenced. A median of 124,686 (range, 48,181-170,687) high quality reads were obtained across the 5 labs. Median number of forward and reverse reads was 1,757 (range 884-7,838), with no coverage dropouts for any amplicon or index. Comparison of observed vs expected mutations showed that 76/78 evaluable samples were accurately scored. In the remaining two, the analysis software failed to detect the 35bp insertion ('35INS') commonly detectable between exons 8 and 9. Quantitation of point mutation burden was highly reproducible across the entire range of frequencies, from 100% to 1%. The second phase of the study has started in Jan 2016. As of Jul 31st, a total of 106 consecutive pts (CML, n=96; Ph+ ALL, n=10) have been enrolled. The present analysis focuses on the first 75 CML pts (60 F and 15 W), for whom sequencing results are currently available (analysis of the entire population of patients enrolled up to Nov 2016 will be presented at the meeting). Clinically actionable mutations have been detected in 10/75 (14%) pts by CS and in 20/75 pts (27%) by DS. Notably, among the 10 pts positive for clinically actionable mutations by DS but not by CS, 3 had a low burden T315I (2 F [dasatinib, imatinib] and 1 W [dasatinib]). In 5 additional pts negative for mutations by CS (3 F and 2 W), DS identified multiple low burden mutations with unknown IC50, suggesting that the cooperation of individually 'weak' mutants may be a new mechanism underlying reduced TKI efficacy. Longitudinal analysis and follow-up of pts are shaping the clinical significance of different types of low burden mutations and will be presented. Conclusions The 'NEXT-in-CML' study is demonstrating that DS of BCR-ABL1 can successfully be implemented in national lab networks and is an important step forward towards routine use of this technology. We have now adapted the protocol for both the Ion Torrent PGM and the Illumina Miseq platforms. For a minimum of 15 samples per sequencing run, DS costs are estimated to equal those of CS (cost per sample, reagents only: ≈100€ for PGM (314 chip) and Miseq (nano kit v2) vs ≈95€ for CS) with comparable turnaround times for delivery of results. Our study is also contributing useful data for the clinical interpretation of DS findings. Disclosures Soverini: Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Castagnetti:ARIAD Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Ciceri:MolMed SpA: Consultancy. Breccia:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Di Raimondo:Janssen-Cilag: Honoraria. Bassan:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees. Cavo:Millennium: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Rosti:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Baccarani:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Saglio:Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Martinelli:Ariad: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; BMS: Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; MSD: Consultancy; Genentech: Consultancy.

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