BCR-ABL1 Kinase Domain Mutation Screening Research Articles

What's Next in CML - a Prospective Study Evaluating Sanger Sequencing and Next Generation Sequencing (NGS) for BCR-ABL1 Kinase Domain (KD) Mutation Screening

What's Next in CML - a Prospective Study Evaluating Sanger Sequencing and Next Generation Sequencing (NGS) for BCR-ABL1 Kinase Domain (KD) Mutation Screening

NGS-Based Screening to Comprehensively Decipher TKIs Resistant Mutations in BCR-ABL1 Positive Leukemias

In BCR-ABL1 positive leukemia, mutations in the BCR-ABL1 kinase domain (KD) are the most common TKIs resistance mechanism. Sanger sequencing (SS) is currently the gold standard for detecting ABL1 KD mutations, but its low detection sensitivity cannot reveal mutations below 20% VAFs (variant allele frequency), and it is challenging to distinguish compound or polyclonal mutations. In recent years, the NGS-based BCR-ABL1 KD mutation screening protocol provides a powerful tool for deciphering complex patterns (compound or polyclonal mutations) and higher detection sensitivity (~2%, hotspot mutation 1%). Based on an inhouse designed NGS-based BCR-ABL1 KD mutation screening protocol, we retrospectively analyzed 348 specimens that undergone SS (ALL, n=164, CML, n=128, CML-BP, n=33, AML, n=3, other, n=20, other includes MAL, B-LBL). The BCR-ABL1 transcripts of 109 cases were 0, 184 cases were >0.1%, and the remaining 55 cases were between 0-0.1%. NGS screening results showed that the average VAF was 32.81% (range, 1.0%-100%), which was not provided by SS. Compared with SS, NGS screening provided a higher mutation-positive rate (22.13% vs. 33.05%, P =0.0013), and Gatekeeper T315I remained the most common BCR-ABL1 KD mutation (12.24% vs. 14.06%, P =0.4513). When the BCR-ABL1 transcript was 0, no mutation was detected in SS; while NGS ABL1-KD discovered mutations in 12 cases (0 vs. 11.01%, P =0.0004) with VAFs lower than 10% except for one S438F (VAF 14.9%) mutation,among them are the well-known F359V and E450G, as well as ten rare mutations, including T277A and E494K. There were also significant differences in mutations between 0 and 0.1% in BCR-ABL1 transcripts (16.36% vs 32.73%, P =0.046). However, when the BCR-ABL1 transcript >0.1%, there was no difference in mutation-positive rate between the two groups (36.96% vs. 46.20%, P=0.072). NGS screening disclosed more multiple mutations (59 vs 82, P =0.03), with significant advantages in detecting >3 mutations (3 vs 18, P = 0.0009). Compound mutations (CMs) were determined in 8 cases (8/384, 2.08%), with the incidence of 24.24% (8/33) in cases which carrying two or more mutations, of which T315I-including CMs were the most common (6/8, 75%). CMs or polyclonal mutation analysis is particularly crucial for severe patients who have received multiple consecutive TKIs treatments, and those with multiple background mutations and a higher risk of progression to clinical resistance. Our results showed that the inhouse designed NGS-based screening protocols could decipher TKIs resistant mutations more comprehensively than SS(Fig 1), and worthy of being implicated in clinical practice. Figure 1 Disclosures No relevant conflicts of interest to declare.

Prospective assessment of NGS-detectable mutations in CML patients with nonoptimal response: the NEXT-in-CML study

AbstractIn chronic myeloid leukemia (CML) patients, tyrosine kinase inhibitors (TKIs) may select for drug-resistant BCR-ABL1 kinase domain (KD) mutants. Although Sanger sequencing (SS) is considered the gold standard for BCR-ABL1 KD mutation screening, next-generation sequencing (NGS) has recently been assessed in retrospective studies. We conducted a prospective, multicenter study (NEXT-in-CML) to assess the frequency and clinical relevance of low-level mutations and the feasibility, cost, and turnaround times of NGS-based BCR-ABL1 mutation screening in a routine setting. A series of 236 consecutive CML patients with failure (n = 124) or warning (n = 112) response to TKI therapy were analyzed in parallel by SS and NGS in 1 of 4 reference laboratories. Fifty-one patients (22 failure, 29 warning) who were negative for mutations by SS had low-level mutations detectable by NGS. Moreover, 29 (27 failure, 2 warning) of 60 patients who were positive for mutations by SS showed additional low-level mutations. Thus, mutations undetectable by SS were identified in 80 out of 236 patients (34%), of whom 42 (18% of the total) had low-level mutations somehow relevant for clinical decision making. Prospective monitoring of mutation kinetics demonstrated that TKI-resistant low-level mutations are invariably selected if the patients are not switched to another TKI or if they are switched to a inappropriate TKI or TKI dose. The NEXT-in-CML study provides for the first time robust demonstration of the clinical relevance of low-level mutations, supporting the incorporation of NGS-based BCR-ABL1 KD mutation screening results in the clinical decision algorithms.

Detection of Actionable BCR-ABL1 Kinase Domain (KD) Mutations in Chronic Myeloid Leukemia (CML) Patients with Failure and Warning Response to Tyrosine Kinase Inhibitors (TKIs): Potential Impact of Next-Generation Sequencing (NGS) and Droplet Digital PCR (ddPCR) on Clinical Decision Making

Detection of Actionable BCR-ABL1 Kinase Domain (KD) Mutations in Chronic Myeloid Leukemia (CML) Patients with Failure and Warning Response to Tyrosine Kinase Inhibitors (TKIs): Potential Impact of Next-Generation Sequencing (NGS) and Droplet Digital PCR (ddPCR) on Clinical Decision Making

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

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

Next Generation Sequencing-Based BCR-ABL1 Kinase Domain Mutation Screening in De Novo and Tyrosine Kinase Inhibitor-Resistant Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia: Results of a Prospective Study

Next Generation Sequencing-Based BCR-ABL1 Kinase Domain Mutation Screening in De Novo and Tyrosine Kinase Inhibitor-Resistant Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia: Results of a Prospective Study

Present and future of molecular monitoring in chronic myeloid leukaemia.

Currently, physicians treating chronic myeloid leukaemia (CML) patients can rely on a wide spectrum of therapeutic options: the best use of such options is essential to achieve excellent clinical outcomes and, possibly, treatment-free remission (TFR). To accomplish this, proper integration of expert clinical and laboratory monitoring of CML patients is fundamental. Molecular response (MR) monitoring of patients at defined time points has emerged as an important success factor for optimal disease management and BCR-ABL1 kinase domain mutation screening is useful to guide therapeutic reassessment in patients who do not achieve optimal responses to tyrosine kinase inhibitor therapy. Deeper MRs might be associated with improved long-term survival outcomes. More importantly, they are considered a gateway to TFR. In molecular biology, novel procedures and technologies are continually being developed. More sophisticated molecular tools and automated analytical solutions are emerging as CML treatment endpoints and expectations become more and more ambitious. Here we provide a critical overview of current and novel methodologies, present their strengths and pitfalls and discuss what their present and future role might be.

Clinical Relevance of Low Burden BCR-ABL1 Mutations Detectable By Amplicon Deep Sequencing (DS) in Philadelphia-Positive (Ph+) Acute Lymphoblastic Leukemia (ALL) Patients (pts): The Type of Mutation Matters

Background and Aims- In Ph+ ALL pts treated with tyrosine kinase inhibitors (TKIs), the likelihood of acquiring TKI-insensitive mutations and the striking incidence of highly resistant T315I and compound mutants underscore the importance of BCR-ABL1 kinase domain (KD) sequence surveillance for timely and rational therapeutic reassessment.We used an amplicon DS strategy of the BCR-ABL1 KD to assess the following issues:i) whether DS allows earlier detection of emerging TKI-insensitive mutations in pts undergoing BCR-ABL1 KD mutation screening for minimal residual disease (MRD) persistence;ii) whether TKI-insensitive low burden mutations can be identified in relapsed pts with negative conventional sequencing results;iii) whether TKI-insensitive low burden mutations are necessary and sufficient to predict for treatment failure in all cases.Methods- This study was conducted in a total of 56 Ph+ ALL pts who received TKI-based therapies at our or collaborating institutions and were referred to our laboratory for MRD follow-up monitoring by RQ-PCR and for BCR-ABL1 KD mutation analysis in case of MRD positivity. These pts were divided into three different cohorts:i) 10 de novo and 24 advanced Ph+ ALL pts who relapsed and developed BCR-ABL1 KD mutations on TKI-based therapy administered 1st-line or for recurrent disease, respectively. To reconstruct the dynamics of mutation emergence, longitudinal re-analysis of monthly-collected samples from the time of hematologic relapse backwards was performed by DS. Whenever samples were available, the analysis was done back to the time of diagnosis (n=10/10) or back to the time of first or former relapse (n=15/24), respectively. Two to 6 samples were analyzed for each pt, for a total of 109 samples.ii) 14 Ph+ ALL pts who were known to be negative for mutations at the time of hematologic relapse as assessed by conventional sequencing. Relapse samples were reanalyzed by DS.iii) 8 Ph+ ALL pts with long-term relapse-free survival despite persistent or intermittent MRD positivity at multiple timepoints. Up to 5 samples were analyzed for each pt, for a total of 28 samples.DS was performed on a Roche GS Junior. Lower mutation detection limit of DS was 1%.Results- In the 34 de novo or advanced Ph+ ALL pts who were known to have acquired TKI-insensitive mutations at the time of relapse on tyrosine kinase inhibitor (TKI) therapy, longitudinal retrospective reanalysis by DS allowed mutation backtracking in 13 (41%) cases. One patient was found to harbour a low burden Y253H at diagnosis. In 3 imatinib (IM)-resistant pts who switched to dasatinib (DAS), a low burden T315I mutation was already detectable at baseline.In the 14 pts with no mutations detectable by conventional sequencing at the time of relapse on IM or DAS, low burden TKI-insensitive mutations were detected by DS in 6 (43%) cases. In 2 cases who had relapsed on DAS, a T315I and an F317L mutation, respectively, were present just below the lower detection limit of conventional sequencing (15.9% and 12.4%, respectively); in the remaining 4 pts, DS identified multiple (2 to 3) low burden mutations, all of which known to confer a moderate to high degree of insensitivity to the ongoing TKI.In the 8 pts with persistently or transiently detectable BCR-ABL1 transcripts at multiple timepoints despite stable hematologic remission, DS detected low burden mutations in 9 samples from 4 pts. However, no mutation known to be truly insensitive to the ongoing TKI could be recognized.Conclusions•MRD persistence in Ph+ ALL pts may hide emerging TKI-insensitive BCR-ABL1 KD mutations that DS may identify earlier than conventional sequencing - allowing a greater leeway before overt hematologic relapse occurs;•polyclonal resistance sustained by multiple TKI-insensitive low burden mutations may explain relapse in a proportion of cases with unmutated BCR-ABL1 KD sequences as assessed by conventional sequencing;•the type of mutation matters: detection of low burden mutations insensitive to the ongoing TKI was always found to predict/correlate with treatment failure. Detection of low burden mutations with low/unknown IC50 might explain low level MRD but does not predict for an impending relapse;•MRD-triggered, BCR-ABL1 KD mutation screening by DS may be precious for earlier and more effective use of preemptive rescue therapies.Supported by ELN, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi) DisclosuresSoverini:Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Abruzzese:BMS, Novartis, Pfizer, Ariad: Consultancy. Baccarani:ARIAD Pharmaceuticals, Inc.: 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; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cavo:Onyx: Honoraria; BMS: Honoraria; Novartis: Consultancy, Honoraria; Millenium Pharmaceuticals: Honoraria; Celgene: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Jansenn: Consultancy, Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; ROCHE: Consultancy; AMGEN: Consultancy; Ariad: Consultancy; MSD: Consultancy.

In Chronic Myeloid Leukemia Patients on 2nd-Line Tyrosine Kinase Inhibitor Therapy, Deep Sequencing at the Time of Warning May Allow Sensitive Detection of Emerging BCR-ABL1 Mutants

▪Background and Aims: Next generation amplicon-based deep sequencing (DS) on the Roche, Illumina or Ion Torrent instruments is becoming accessible to a wider and wider number of diagnostic laboratories. Although conventional sequencing is still the gold standard, DS has been hailed by many as the future of diagnostic BCR-ABL1 kinase domain (KD) mutation screening. BCR-ABL1 KD mutations are infrequent in newly diagnosed chronic myeloid leukemia (CML) patients (pts) receiving 1st-line TKI therapy, but remain a challenge in relapsed pts, who usually display a greater genetic instability. Indeed, pts already harboring BCR-ABL1 KD mutations have a higher likelihood of developing additional, dasatinib (DAS)- or nilotinib (NIL)-resistant mutations – which is defined as a 'failure’ by the 2013 European LeukemiaNet (ELN) recommendations. Taking advantage of a next-generation amplicon sequencing design and protocol set up and validated in the framework of the IRON-II international study, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse.Methods: among the imatinib (IM)-resistant CML pts who switched to 2nd-line TKI therapy and were referred to our laboratory for routine BCR-ABL1 transcript level monitoring and KD mutation screening by conventional sequencing, 51 acquired DAS- or NIL-resistant mutations after a median of 9 months (range, 3-27 months) of therapy and had leftover cDNA available at previous timepoints. To reconstruct the dynamics of mutation emergence, resequencing on a Roche GS Junior instrument was performed from the time of failure and mutation detection by conventional sequencing backwards. Runs were designed to achieve high sequencing depth, allowing reliable detection of variants down to 1% abundance. BCR-ABL1/ABL1%IS transcript levels and/or cytogenetic response, whichever available, were used to define whether the patient had an 'optimal response’, 'warning’ or 'failure’ at the time of first mutation detection by DS.Results: baseline mutation status, as assessed by conventional sequencing, was available for all the 51 CML pts included in this retrospective study; 29/51 pts were positive for BCR-ABL1 KD mutations, with switch to NIL or DAS selected accordingly. Twenty-six pts were later found to have acquired DAS-resistant mutations (T315I, n=13; F317L/V, n=10; V299L, n=3) and 25 pts were later found to have acquired NIL-resistant mutations (T315I, n=4; F359V/I/C, n=7; Y253H, n=6; E255K, n=9; one patient acquired two mutations). DS was able to backtrack the DAS- or NIL-resistant mutations to the previous sample(s) in 23/51 (45%) pts. Median mutation burden at the time of first detection by DS was 5% (range, 1-17%); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 3-9 months). In 5 cases, the mutations were traceable at baseline; in the remaining cases, correlation with response at the time mutations were first detected by DS revealed a 'warning’ according to the 2013 ELN definitions of response to 2nd-line therapy in 13 cases; an 'optimal response’ in one case; a 'failure’ in 4 cases. As a control, we used DS to explore BCR-ABL1 KD mutation status in 10 randomly selected pts with 'warning’ at various timepoints, that later turned into optimal responses; no DAS- or NIL-resistant mutations were detected.Conclusions: the 2011 ELN recommendations for mutation analysis suggest BCR-ABL1 KD to be screened by conventional sequencing in case of 'failure’ of 2nd-line TKI therapy – according to the provisional definitions available at the time. Earlier detection of emerging BCR-ABL1 KD mutations allows a greater leeway in tackling drug resistance and enhancing therapeutic efficacy. Data presented herein indicate that:1) DS may reliably pick TKI-resistant mutations earlier than conventional sequencing in a proportion of pts, and that2) the recently introduced definitions of ‘warning’ may provide a rational trigger, besides ‘failure’, for DS-based BCR-ABL1 KD mutation screening in CML pts on 2nd-line TKI therapy.A prospective cost-benefits evaluation of using DS in this and other settings is warranted, and will contribute useful information to the revision of the ELN recommendations for BCR-ABL1 KD mutation analysis.Supported by: European LeukemiaNet, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi). DisclosuresSoverini:Novartis: Consultancy; Bristol-Meyers Squibb: Consultancy; Ariad: Consultancy. Castagnetti:Novartis Farma: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Bonifacio:Amgen Inc.: Consultancy. Rosti:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Baccarani:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Pfizer: Consultancy. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Backtracking BCR-ABL1 Mutants in Philadelphia-Positive Acute Lymphoblastic Leukemia Patients Relapsing on Tyrosine Kinase Inhibitors with Deep Sequencing: Implications for Routine Mutation Testing

Background: With the release of benchtop next-generation sequencers allowing to conjugate assay sensitivity and throughput with affordability, it was envisioned that NGS could soon replace conventional methods in the routine diagnostic identification of mutations relevant for diagnostication, prognostication and therapeutic tailoring. Having recently implemented and optimized a strategy for BCR-ABL1 kinase domain (KD) mutation screening based on the Roche-454 NGS technology, we decided to take advantage of its unique capability to capture the complexity and the clonal relationships of major and minor mutated populations present in a sample to focus on Philadelphia-positive (Ph+) acute lymphoblastic leukemias (ALL), where BCR-ABL1 KD mutations frequently arise and undermine the long-term efficacy of tyrosine kinase inhibitor (TKI)-based therapies.Aims: Next generation amplicon-based deep sequencing (DS) was used i) to study the dynamics of expansion of BCR-ABL1 KD mutations in relation to BCR-ABL1 transcript levels in Ph+ ALL patients who relapse on TKIs and ii) to assess whether emerging mutations can be detected earlier than with conventional Sanger sequencing (SS).Methods: This retrospective analysis was conducted on 35 Ph+ ALL patients who relapsed and acquired BCR-ABL1 KD mutations while receiving TKI-based therapy (imatinib, dasatinib, nilotinib) either first-line (Group A; n=10) or for recurrent disease (Group B; n=25). All the patients had been referred to our laboratory for minimal residual disease (MRD) follow-up monitoring by real-time reverse transcription-polymerase chain reaction and for BCR-ABL KD mutation analysis by SS in case of MRD positivity. To reconstruct the dynamics of mutation emergence, longitudinal re-analysis of bone marrow samples (n=113) from the time of hematologic relapse back to diagnosis (Group A) or back to TKI start (Group B) was performed on a Roche GS Junior instrument. Median time lapse between samples was 6 weeks (range, 4-15 weeks). Runs were designed to achieve high sequencing depth, allowing reliable detection of sequence variants at least down to 1% abundance.Results: DS was found to provide a more accurate picture of BCR-ABL1 KD mutation status, both in terms of presence/absence of mutations and in terms of clonal complexity. DS indeed detected low level mutations, alone or in addition to SS-detectable mutations, in 14/35 samples from patients in Group A and 24/72 samples from patients in Group B. In all the cases with two or more mutations, DS uncovered polyclonal mixtures of single and compound mutants, with up to five distinct mutant subclones detectable. With very few exceptions, compound mutants were found to represent the dominant clone. Patients in Group B, who had received two or even three lines of TKI therapy, generally displayed greater complexity than patients in Group A.TKI-resistant mutants were found to undergo rapid selection in Ph+ ALL patients. In 13/35 (37%) patients, however, DS would have allowed to detect emerging mutations earlier than SS actually did and before hematologic relapse – including a Y253H mutation in a sample collected at diagnosis from a patient who relapsed eight weeks later.Detection of emerging TKI-resistant mutations by DS was accompanied by total BCR-ABL1/ABL1 transcript increase from half a logarithm to two logarithms, or preceded by a shift from undetectable to detectable BCR-ABL1 transcripts. Even low MRD levels were found to hide low level TKI-resistant mutants.Conclusions: The enhanced sensitivity as well as the possibility to identify compound mutations point to next generation amplicon-based deep sequencing as an ideal alternative to conventional sequencing for BCR-ABL1 KD mutation screening of Ph+ ALL patients receiving TKI based therapies. Cost is not a concern even for small/medium scale laboratories, considering that many samples and many genes can be pooled and analyzed simultaneously. A turnaround time of 3-4 days is generally feasible, irrespective of chemistries and instruments, allowing DS results to be practically integrated in the clinical decision algorithm. Further, prospective evaluation of DS for BCR-ABL1 KD sequence surveillance in the framework of a clinical trial of TKI therapy and MRD monitoring of Ph+ ALL patients is warranted.Supported by: European LeukemiaNet, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi). DisclosuresSoverini:Ariad: Consultancy; Bristol-Meyers Squibb: Consultancy; Novartis: Consultancy. Baccarani:Bristol-Myers Squibb: Consultancy, Honoraria, Speakers Bureau.

Sensitivity, Reproducibility and Clinical Utility Of Next-Generation Sequencing (NGS) for BCR-ABL1 Kinase Domain Mutation Screening: Results From The CML Work Package Of The Iron-II (Interlaboratory RObustness Of Next-Generation Sequencing) International Study

Background and AimsIn chronic myeloid leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) patients resistant to tyrosine kinase inhibitors (TKIs), BCR-ABL1 mutation status is an essential component of the therapeutic decision algorithm. Capillary Sanger sequencing (SS) is currently the gold standard for mutation screening of the BCR-ABL1 kinase domain (KD), despite key technical limitations including limited sensitivity and no discrimination between compound and polyclonal mutations. Benchtop next-generation sequencers have recently been introduced as potential diagnostic platforms and there is growing interest in their clinical application. In the framework of the IRON-II (Interlaboratory RObustness of Next-generation sequencing) international consortium, 10 laboratories from 7 countries (Italy, Germany, United Kingdom, Spain, Austria, Turkey, Czech Republic) have engaged in the set-up, standardization and validation of a laboratory-developed screening assay for BCR-ABL1KD mutations based on the Roche 454 amplicon deep-sequencing technology. MethodsFusion primers were designed to generate four partially overlapping amplicons by nested reverse transcription (RT)-polymerase chain reaction (PCR), the first amplification step needed to select for the translocated ABL1 allele. Fusion primers were barcoded with multiplex identifiers (MIDs) consisting of 10-base pair tags allowing multiplexing of twelve clinical samples (forty-eight amplicons) in a single NGS run. The assay was designed in a ready-to-use 96-well plate format containing lyophilized oligonucleotide primers. ResultsDifferent primer designs and primer-MID combinations were evaluated for their performances. Sequencing runs generated an average of 97,432 reads (range, 59,459-151,335). For the primer design selected for further evaluation, the coverage per amplicon ranged between 1,449 and 5,997 sequencing reads. To explore the sensitivity and accuracy of the assay, serial dilutions of BaF3 cell lines harboring four different known mutations (Y253F, E255K, T315I, M351T) into an unmutated BaF3 cell line (50%:50%; 25%:75%; 10%:90%; 5%:95%; 2%:98%; 1%:99%) were sequenced in parallel in two distinct laboratories (Bologna and Jena). In both centers, results showed a high linearity of mutation calling and accuracy of mutation detection and quantitation over the entire range of dilutions, down to 1% mutation abundance. Intra-run reproducibility and inter-run reproducibility were confirmed by a series of experiments in which a set of samples was resequenced in the same and in independent runs, respectively, with and without repetition of the RT and PCR steps. Importantly, we demonstrated that reproducibility could be maintained over a wide dynamic range of amplicon coverage (from 100 to 5,000 independent sequencing reads). A total of 554 clinical samples (2,216 amplicons) were analyzed by the 10 laboratories - including 517 clinical samples analyzed in parallel by NGS and SS and 30 clinical samples analyzed in parallel by NGS, SS and conventional pyrosequencing. Three hundred and ninety-four of 398 (99%) variants detected by SS were also detected by NGS. In addition, comparison between NGS, SS and conventional pyrosequencing results showed very good concordance with respect to the estimation of variant abundance. NGS allowed to detect additional, low level mutations (>1% but<10-15%, i.e. undetectable by SS) in 294/554 (53%) samples. In a subset of twenty randomly selected samples, low level mutations were confirmed by independent methods (restriction fragment length polymorphism or allele-specific oligonucleotide-PCR). Compound mutations as against polyclonality could be resolved in all the clinical samples harboring multiple mutations mapping 450 bp apart or closer. Longitudinal retrospective analysis of CML and Ph+ ALL clinical samples showed that NGS could have identified TKI-resistant mutations earlier than SS, thus allowing more timely therapeutic intervention. ConclusionsOur results indicate the technical feasibility, accuracy and robustness of NGS for BCR-ABL1 KD mutation screening and represent an important step forward towards its routine application in a clinical setting. An international ring trial to test inter-laboratory reproducibility of BCR-ABL1 mutation detection by NGS is now about to start. Disclosures:Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Machova Polakova:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Lion:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Hochhaus:ROCHE: Research Funding. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Molecular diagnostics in chronic myeloid leukemia

With the progress of chronic myeloid leukemia (CML) therapy, the molecular tools used to diagnose and monitor patients have become sophisticated. Despite this, a complete physical examination, complete blood count and bone marrow biopsy with metaphase karyotyping remain standard at diagnosis. Fluorescence in situ hybridization or qualitative reverse transcription polymerase chain reaction are indicated to exclude BCR-ABL1 in Philadelphia chromosome-negative patients with clinically typical CML. Bone marrow karyotyping is the gold standard for monitoring patients on imatinib until achievement of complete cytogenetic response, when quantitative polymerase chain reaction (qPCR) for BCR-ABL1 becomes the method of choice. Quantitative PCR results must be interpreted within a clinical context, the preceding results and performance characteristics of the PCR assay. Expression of qPCR results on the international scale enables comparison of results from different laboratories. BCR-ABL1 kinase domain mutation screening has added another level of complexity that informs the management of some patients with imatinib resistance. Using the entire diagnostic CML armamentarium in a rational and economic fashion can be as challenging as choosing the right treatment. The aim here is to describe what is universally accepted and what is controversial and to provide an update on emerging technologies, while trying to keep an eye on the real world outside specialized centers.