Detection Of Kinase Domain Mutations Research Articles

Detection of kinase domain mutations in BCR::ABL1 leukemia by ultra-deep sequencing of genomic DNA

The screening of the BCR::ABL1 kinase domain (KD) mutation has become a routine analysis in case of warning/failure for chronic myeloid leukemia (CML) and B-cell precursor acute lymphoblastic leukemia (ALL) Philadelphia (Ph)-positive patients. In this study, we present a novel DNA-based next-generation sequencing (NGS) methodology for KD ABL1 mutation detection and monitoring with a 1.0E−4 sensitivity. This approach was validated with a well-stablished RNA-based nested NGS method. The correlation of both techniques for the quantification of ABL1 mutations was high (Pearson r = 0.858, p < 0.001), offering DNA-DeepNGS a sensitivity of 92% and specificity of 82%. The clinical impact was studied in a cohort of 129 patients (n = 67 for CML and n = 62 for B-ALL patients). A total of 162 samples (n = 86 CML and n = 76 B-ALL) were studied. Of them, 27 out of 86 harbored mutations (6 in warning and 21 in failure) for CML, and 13 out of 76 (2 diagnostic and 11 relapse samples) did in B-ALL patients. In addition, in four cases were detected mutation despite BCR::ABL1 < 1%. In conclusion, we were able to detect KD ABL1 mutations with a 1.0E−4 sensitivity by NGS using DNA as starting material even in patients with low levels of disease.

ABL Kinase Domain Mutations in Iranian Chronic Myeloid Leukemia Patients with Resistance to Tyrosine Kinase Inhibitors.

Tyrosine kinase inhibitors (TKIs) are considered standard first-line treatment in patients with chronic myeloid leukemia. Because ABL kinase domain mutations are the most common causes of treatment resistance, their prevalence and assessment during treatment may predict subsequent response to therapy. The molecular response in Bcr-Abl1IS was tested via quantitative real-time polymerase chain reaction. We used the direct sequencing technique to discover the mutations in the ABL kinase domain. The IRIS trial established a standard baseline for measurement - (100% BCR-ABL1 on the 'international scale') and a major molecular response (good response to therapy) was defined as a 3-log reduction in the amount of BCR-ABL1 - 0.1% BCR-ABL1 on the international scale. We observed 11 different mutations in 13 patients, including E255K, which had the highest mutation rate. A lack of hematologic response was found in 22 patients, who showed a significantly higher incidence of mutations. Detection of kinase domain mutations is a reliable method for choosing the best treatment strategy based on patients' conditions, avoiding ineffective treatments, and running high-cost protocols in patients with acquired resistance to TKIs.

Novel Illumina-based next generation sequencing approach with one-round amplification provides early and reliable detection of BCR-ABL1 kinase domain mutations in chronic myeloid leukemia.

The occurrence of mutations in the BCR-ABL1 kinase domain (KD) can lead to treatment resistance in chronic myeloid leukaemia patients. Nowadays, next-generation sequencing (NGS) is an alternative method for the detection of kinase domain mutations, compared to routinely used Sanger sequencing, providing a higher sensitivity of mutation detection. However, in the protocols established so far multiple rounds of amplification limit reliable mutation detection to approximately 5% variant allele frequency. Here, we present a simplified, one-round amplification NGS protocol for the Illumina platform, which offers a robust early detection of BCR-ABL1 KD mutations with a reliable detection limit of 3% variant allele frequency.

Hybridization capture-based next generation sequencing reliably detects FLT3 mutations and classifies FLT3-internal tandem duplication allelic ratio in acute myeloid leukemia: a comparative study to standard fragment analysis

FLT3-internal tandem duplication occurs in 20–30% of acute myeloid leukemia and confers an adverse prognosis with its allelic ratio being a key risk stratifier. The US Food and Drug Administration recently approved FLT3 inhibitors midostaurin and gilteritinib in FLT3 mutation-positive acute myeloid leukemia. Historically, FLT3 was tested by fragment analysis, which has become the standard method endorsed by international guidelines. However, next generation sequencing is increasingly used at acute myeloid leukemia diagnosis given its ability to simultaneously evaluate multiple clinically informative markers. As FLT3-internal tandem duplication detection was known to be challenging by next generation sequencing and the results carry profound prognostic and therapeutic implications, it is important to thoroughly examine its performance in FLT3-internal tandem duplication detection and allelic ratio classification. In a comparative study with fragment analysis, we retrospectively reviewed our experience using a custom-designed, hybridization capture-based, targeted next generation sequencing panel. Among 7902 cases, FLT3-internal tandem duplication was detected in 335 with variable sizes (3–231 bp) and insertion sites. Fragment analysis was also performed in 402 cases, demonstrating 100% concordance in FLT3-internal tandem duplication detection. In 136 dual-tested, positive cases, 128/136 (94%) exhibited concordant high/low allelic ratio classifications. The remaining 6% showed borderline low allelic ratio by next generation sequencing. The two methods were concordant in FLT3-tyrosine kinase domain mutation detection at the hotspot D835/I836 targeted by fragment analysis. Furthermore, seven mutations which may benefit from FLT3 inhibitor therapy were detected by next generation sequencing, in regions not covered by fragment analysis. Our study demonstrates that using a hybridization capture-based chemistry and optimized bioinformatics pipeline, next generation sequencing can reliably detect FLT3-internal tandem duplication and classify its allelic ratio for acute myeloid leukemia risk stratification. Next generation sequencing also exhibits superior comprehensiveness in FLT3 mutation detection and may further improve personalized, targeted therapy in acute myeloid leukemia.

Clinical Intervention Using NGS-Based Kinase Domain Mutation Testing in CML Patients

Kinase domain (KD) mutations in the BCR-ABL1 gene are associated with resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML). Next-generation Sequencing (NGS) allows detection of low-level KD mutations (<20% VAF), although the detection of true low level KD mutation can be challenging. We have previously reported an improved single round PCR based NGS method to ensure higher accuracy for clinical use (Kizilors et al. ASH 2015). To implement this improved assay in our routine clinical setting, we obtained ISO 15189 accreditation for NGS based BCR-ABL1 KD mutation detection.We have previously shown the clinical significance of low-level KD mutation by NGS. However our study, along with previous studies, was retrospective. Since August 2014, we have been using NGS-based assay (in replacement of Sanger sequencing) for clinical intervention, allowing us to study the clinical relevance of low level mutations and monitor the kinetics of mutated clones.We herein report the outcome of patients in whom clinical intervention was made based on NGS KD mutation screening and monitoring.We analyzed the outcome of 340 consecutive Chronic Phase CML patients treated with first-line TKI at our institution and within our network (imatinib 235, nilotinib 49, dasatinib 10, unknown 46), of whom 70.42% achieved CCyR with first-line TKI. NGS-based KD mutation testing was done retrospectively until January 2014, and prospectively from that date.Overall, a mutation was found in 41(12.05%) patients at a median time of 14 months (range, diagnosis to 156 months). Twenty five different mutations were identified, including T315I (n=9), M244V (n=8), F317L (n=8) and E255K (n=6). Of 41 patients with mutated clone, 27 were found to have a mutation conferring resistance to the ongoing TKI treatment (defined as ‘clinically relevant’ mutation). In 8 patients, the mutation was sensitive to the TKI treatment received (M244V in 6 patients, M351T in one patient on imatinib and an E255V in one patient on dasatinib), two patients with intermediate resistance to the treatment received (H396R and E355G) and four patients with mutations of unknown clinical significance (Q252R, E450K and E450G). Of the 27 patients with clinically relevant mutations, 8 patients were part of our initial retrospective NGS screening study, of whom 3 had clinical intervention based on Sanger Sequencing results.From August 2014, clinical intervention was made based on KD mutations found by NGS. Eighteen patients had their TKI treatment changed because of mutations found to confer resistance to their ongoing TKI (including 2 with unknown clinical significance), of whom 14 had single mutation (F317L, n=3; T315I, n=2; Y253H, n=1; F359V, n=1; L248V, n=1; G250E, n=1; F311L, n=1; E459G, n=1; E450K, n=1; F359I, n=1; E450G, n=1). Four patients had multiple mutations, including two patients who developed mutations sequentially.Of the 18 patients who had clinical intervention, the first detection of the KD mutation was at a low-level in 13 patients.Response to change of TKI was seen in 12 patients, of whom 11 achieved a stable MR3, and one patient with a 2.2 log reduction in BCR-ABL transcript levels. One patient proceeded to allogeneic HSCT and four patients did not show significant response at last follow up. Finally, one patient had a low level Y253H mutation detected while on nilotinib which initially reduced after TKI change and then subsequently increased while gaining an additional T315I at the time of Blastic Phase transformation. Response to combining chemotherapy and ponatinib was seen with the achievement of sustained MR3 until allogeneic HSCT.Kinetics of mutated clone(s) was monitored post TKI change and all patients who responded experienced a reduction in their clone size. Among 12 patients who responded to TKI change, 3 had multiple mutations. One patient had a low level E255K mutation at 4% at 3 months despite achieving CCyR on nilotinib and subsequently developed an additional T315I mutation after 15 months upon loss of CCyR. This patient achieved a sustained MR3 upon switch to ponatinib with concomitant drop in the T315I clone size.To our knowledge, this is the first report of clinical intervention using NGS based KD mutation detection in the management of CML patients. Our findings suggest that NGS may improve clinical decision-making in these patients. A prospective clinical study using NGS (acronym CALLS) is currently ongoing in the UK. DisclosuresKizilors:Incyte: Honoraria, Research Funding. Harrington:BMS: Research Funding. Pagliuca:Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gentium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Mufti:Celgene: Research Funding. De Lavallade:Incyte: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

Regulation of resistance to tyrosine kinase inhibitors for patients with chronic myeloid leukemia

The application of tyrosine kinase inhibitors (TKI) has revolutionized the treatment of chronic myeloid leukemia (CML), but with its widespread use in clinical applications, drug resistance has become a huge challenge in clinical treatment. The development of the second and third generation of TKI, and some new drugs brought new treatment options and hopes for imatinib of the first generation of TKI resistant patients. The continuous improvement of the therapeutic effect monitoring system and the application of ABL kinase domain mutation detection provide an objective and sensitive index for the selection of TKI. Therefore, only the combination of therapeutic drugs and the corresponding monitoring system can make the patients get more reasonable and standardized treatment. In this paper, research progress of selection of treatment regimen and monitoring system of effect for CML patients who were resistance to TKI are reviewed. Key words: Leukemia, myelogenous, chronic, BCR-ABL positive; Protein-tyrosine kinases; Proteinkinase inhibitors; Molecular monitoring

Chinese expert consensus on the laboratory practice of BCR- ABL tyrosine kinase domain mutation detection（2015）

Chinese expert consensus on the laboratory practice of BCR- ABL tyrosine kinase domain mutation detection（2015）

Clinical significance of T315I ABL kinase domain mutation detection in patients resistant to imatinib mesylate therapy

Clinical significance of T315I ABL kinase domain mutation detection in patients resistant to imatinib mesylate therapy

Molecular diagnosis and monitoring in the clinical management of patients with chronic myelogenous leukemia treated with tyrosine kinase inhibitors

The well-established molecular pathogenesis of chronic myelogenous leukemia (CML) and its consequences for laboratory testing and clinical management illustrate a classic paradigm for the importance of molecular diagnostics in targeted drug therapy. The success of the tyrosine kinase inhibitor (TKI), imatinib, as the currently recommended first-line treatment of early chronic phase CML has both fueled the need for timely and reproducible molecular testing of the BCR-ABL1 fusion transcript in diagnosis and monitoring as well as necessitated the detection of kinase domain mutations that confer resistance to this agent. As, ongoing research continues to refine guidelines for monitoring residual disease in patients undergoing TKI therapy, an understanding of molecular technologies and their interpretation is critical. This review summarizes the molecular strategies that are currently employed in the initial diagnosis and subsequent management of CML patients maintained on TKI therapy.

Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis

AbstractImatinib-treated chronic myeloid leukemia (CML) patients with acquired resistance commonly have detectable BCR-ABL kinase domain mutations. It is unclear whether patients who remain sensitive to imatinib also have a significant incidence of mutations. We evaluated 144 patients treated with imatinib for BCR-ABL kinase domain mutations by direct sequencing of 40 accelerated phase (AP), 64 late chronic phase (≥ 12 months from diagnosis, late-CP), and 40 early-CP patients. Mutations were detected in 27 patients at 17 different residues, 13 (33%) of 40 in AP, 14 (22%) of 64 in late-CP, and 0 of 40 in early-CP. Acquired resistance was evident in 24 (89%) of 27 patients with mutations. Twelve (92%) of 13 patients with mutations in the adenosine triphosphate (ATP) binding loop (P-loop) died (median survival of 4.5 months after the mutation was detected). In contrast, only 3 (21%) of 14 patients with mutations outside the P-loop died (median follow-up of 11 months). As the detection of mutations was strongly associated with imatinib resistance, we analyzed features that predicted for their detection. Patients who commenced imatinib more than 4 years from diagnosis had a significantly higher incidence of mutations (18 [41%] of 44) compared with those treated within 4 years (9 [9%] of 100), P < .0001. Lack of a major cytogenetic response (MCR) was also associated with a higher likelihood of detecting a mutation; 19 (38%) of 50 patients without a MCR had mutations compared with 8 (8.5%) of 94 with an MCR, P < .0001. In conclusion, the detection of kinase domain mutations using a direct sequencing technique was almost always associated with imatinib resistance, and patients with mutations in the P-loop had a particularly poor prognosis. (Blood. 2003; 102:276-283)