Mutational Phasing: Clinical Relevance in Tyrosine Kinase Domain Mutations Using Next Generation Sequencing in Chronic Myeloid Leukemia

Abstract Background: Tyrosine kinase mutation analysis in BCR/ABL1 gene is important for management of patients with chronic myeloid leukemia. Sanger Sequencing has been the mainstay for testing with Next Generation Sequencing (NGS) now becoming the primary technology. In this study we show a comparison between NGS versus Sanger Seqencing based ABL kinase domain mutation analysis with a likely trend of clinical relevance based on a compound versus polyclonal state of mutational distribution which may also need to be considered for patient management and therapy. Methodology: A total of 213 Imatinib-resistant patients with CML for BCR-ABL1 mutation analysis were processed on both technologies.Initial blood counts were assessed and RNA was extractedfollowed by cDNA conversion. NGS libraries were prepared with 400bp multiplexed amplicons to allow optimal phasing. Results: 179 samples were negative by both technologies. A total of only 20 samples were positive and concordant by both technologies(58.2%). Mutations in 14 other samples however were only detected in NGS(41.17%). In these 14 samples (41.17%), NGS was able to detect 23 mutations with mutation frequencies of 3-28%, which were missed by Sanger. Conclusions: Moreover 11/34 patients had 2 or >2 mutations. An inhouse script delineated mutations as compound or polyclonal from NGS data. 2/11 cases demonstrated compound mutations (Mutations in the same clone) while 7/11 cases were polyclonal per NGS. Sanger sequencing cannot differentiate between polyclonal and compound mutations. 2/11 cases appeared to have polyclonal and compound mutations. 4/11 patients presented in a blast crisis or accelerated phase CML. Interestingly, most of these patients hadat leasttwo mutations and were polyclonal(3/4). Significantly previously archived samples patients with polyclonal mutations showed polyclonality at extremely low frequency percentages in initial samples. None of the single mutation patients had presented in a blast crisis or an accelerated phase. Disclosures No relevant conflicts of interest to declare.

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ABL Kinase Domain Mutation in Chronic Myeloid Leukemia - Compliance to Imatinib Matters

Blood ◽

10.1182/blood-2019-125998 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1654-1654

Author(s):

Jayachandran PK ◽

Trivadi S Ganesan ◽

Nikita Mehra ◽

Krishnarathinam Kannan ◽

Manikandan Dhanushkodi ◽

...

Keyword(s):

Next Generation Sequencing ◽

Chronic Myeloid Leukemia ◽

Mutation Analysis ◽

Myeloid Leukemia ◽

Sanger Sequencing ◽

Kinase Domain ◽

Next Generation ◽

Abl Kinase ◽

Kinase Domain Mutation ◽

Generation Sequencing

Background: Imatinib resistance mutation analysis (IRMA) or abl kinase domain mutation analysis is performed in patients with Chronic myeloid leukemia (CML) whenever the response to treatment is inadequate. We have analyzed the reports of IRMA at our centre. Methods: The clinical details of 71 patients with CML on Imatinib, who underwent IRMA testing during the period of January 2017 to March 2019 were collected from the patient records and analyzed. IRMA was performed for failure or warning or progression, anytime during the course of treatment. IRMA was done by either Sanger sequencing (n=45) or next generation sequencing (n=26, Illumina, NGS). The associations between variables were tested using Chi - Square test. Results: Median age at diagnosis of 71 patients was 44 years (Range 18 - 71 years). Males constituted 70% (n=50). At diagnosis, 92% (n=65) of patients were in chronic phase and the remainder were in accelerated phase (n=4) or blast crisis (n=2). Mutations in the abl kinase domain were detected in 26 patients (37%). Next Generation Sequencing (NGS) could identify more mutations (13/26 - 50%) compared to conventional Sanger Sequencing (13/45 - 29%), but the difference was not significant (p=0.07). NGS could identify three or more mutations in 5 patients in contrast to Sanger. All the mutations detected were those previously described except for an insertion of 35bp near the C-Terminal which was identified in 3 patients. E459K translocation was identified in 6 patients. E355G translocation was identified in 4 patients. F359V, M351T, Y253H, G250E, H396R, T315I translocations were identified in 3 patients each. Patients who were not compliant to therapy had increased frequency of mutations (14/26 - 54%) compared to those who were compliant (12/45 - 27%), which was significantly different (p=0.02). Patients who had loss of complete hematological response (CHR) had significantly higher frequency of mutations (14/21- 67%) compared to other reasons for performing the test (p=0.001). Patients who had failure to achieve targets at various time points had a significantly lower frequency of mutations (4/23 - 17%, p=0.02) compared to other reasons for performing the test. Conclusion: Patients who were not compliant for treatment were more likely to have mutations. Loss of CHR showed an increased frequency compared to other reasons. NGS could identify mutations in more number of patients. NGS identified numerically higher mutations in patients. Larger prospective data are needed to confirm these observations. Table Disclosures No relevant conflicts of interest to declare.

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BCR-ABL1 kinase domain mutation analysis by next generation sequencing detected additional mutations in chronic myeloid leukemia patients with suboptimal response to imatinib

Leukemia & Lymphoma ◽

10.1080/10428194.2021.1872074 ◽

2021 ◽

pp. 1-6

Author(s):

Esther Sathya Bama Benjamin ◽

Niveditha Ravindra ◽

Bharathi Murugan Rajamani ◽

Senthamizhselvi Anandan ◽

Bagavathi Kausalya ◽

...

Keyword(s):

Next Generation Sequencing ◽

Chronic Myeloid Leukemia ◽

Mutation Analysis ◽

Myeloid Leukemia ◽

Kinase Domain ◽

Next Generation ◽

Kinase Domain Mutation ◽

Generation Sequencing

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ID: 2036 Next-generation sequencing to evaluate frequency of BCR-ABL1 kinase domain Imatinib-resistance mutations

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.275 ◽

2017 ◽

Vol 4 (S) ◽

pp. 67

Author(s):

Chinh Q. Duong ◽

Trang T. Nguyen ◽

Lam V. Nguyen ◽

Huy Q. Pham ◽

Hien T. T. Trinh ◽

...

Keyword(s):

Drug Resistance ◽

Next Generation Sequencing ◽

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Fusion Gene ◽

Kinase Domain ◽

Imatinib Resistance ◽

Kinase Domain Mutation ◽

Generation Sequencing

Chronic myeloid leukemia is a clonal myeloproliferative neoplasm, characterized by the presence of chromosomal translocation t(9; 22)(q34; q11). This is found in over 95% of the cases and results in the BCR-ABL1 fusion gene with high tyrosine kinase activity. During the last decades, imatinib and other generation of tyrosine kinase inhibitors have been used effectively for target therapy of the disease. However, many of the drug resistance cases have been reported recently, due to the mutation within kinase domain of the BCR-ABL1 fusion gene. In this work, we performed a retrospective study of 141 imatinib-resistance chronic myeloid leukemia patients to analyze kinase domain mutation by deep sequencing. Another group of 20 untreated patients were added as control. RNA from bone marrow cells was extracted and deeply sequenced utilizing Illumina MiSeq. Bioinformatics pipeline was applied for variant calling and annotation. And the Sanger sequencing was used to validate those mutations. The results showed that nearly one-fourth of patients harboring mutations that resist to Imatinib, among those, Y253F/H, M351T, G250E, F359V/I and M244V were the most frequent mutations. There were also a number of samples harboring multiple substitutions and new variations. Thus, Next-generation sequencing could be the sensitive and effective method to detect kinase domain mutation and our results could provide further information about the drug-resistance mutation in chronic myeloid leukemia.

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Next-generation sequencing for BCR-ABL1 kinase domain mutation testing in patients with chronic myeloid leukemia: a position paper

Journal of Hematology & Oncology ◽

10.1186/s13045-019-0815-5 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 8

Author(s):

Simona Soverini ◽

Elisabetta Abruzzese ◽

Monica Bocchia ◽

Massimiliano Bonifacio ◽

Sara Galimberti ◽

...

Keyword(s):

Next Generation Sequencing ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Sanger Sequencing ◽

Kinase Inhibitors ◽

Group Discussion ◽

Kinase Domain ◽

Attractive Alternative ◽

Next Generation ◽

Generation Sequencing

AbstractBCR-ABL1 kinase domain (KD) mutation status is considered to be an important element of clinical decision algorithms for chronic myeloid leukemia (CML) patients who do not achieve an optimal response to tyrosine kinase inhibitors (TKIs). Conventional Sanger sequencing is the method currently recommended to test BCR-ABL1 KD mutations. However, Sanger sequencing has limited sensitivity and cannot always discriminate between polyclonal and compound mutations. The use of next-generation sequencing (NGS) is increasingly widespread in diagnostic laboratories and represents an attractive alternative. Currently available data on the clinical impact of NGS-based mutational testing in CML patients do not allow recommendations with a high grade of evidence to be prepared. This article reports the results of a group discussion among an ad hoc expert panel with the objective of producing recommendations on the appropriateness of clinical decisions about the indication for NGS, the performance characteristics of NGS platforms, and the therapeutic changes that could be applied based on the use of NGS in CML. Overall, these recommendations might be employed to inform clinicians about the practical use of NGS in CML.

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BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet

Blood ◽

10.1182/blood-2010-12-326405 ◽

2011 ◽

Vol 118 (5) ◽

pp. 1208-1215 ◽

Cited By ~ 332

Author(s):

Simona Soverini ◽

Andreas Hochhaus ◽

Franck E. Nicolini ◽

Franz Gruber ◽

Thoralf Lange ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Mutation Analysis ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Direct Sequencing ◽

Chronic Phase ◽

Kinase Domain ◽

Abl Kinase

AbstractMutations in the Bcr-Abl kinase domain may cause, or contribute to, resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia patients. Recommendations aimed to rationalize the use of BCR-ABL mutation testing in chronic myeloid leukemia have been compiled by a panel of experts appointed by the European LeukemiaNet (ELN) and European Treatment and Outcome Study and are here reported. Based on a critical review of the literature and, whenever necessary, on panelists' experience, key issues were identified and discussed concerning: (1) when to perform mutation analysis, (2) how to perform it, and (3) how to translate results into clinical practice. In chronic phase patients receiving imatinib first-line, mutation analysis is recommended only in case of failure or suboptimal response according to the ELN criteria. In imatinib-resistant patients receiving an alternative TKI, mutation analysis is recommended in case of hematologic or cytogenetic failure as provisionally defined by the ELN. The recommended methodology is direct sequencing, although it may be preceded by screening with other techniques, such as denaturing-high performance liquid chromatography. In all the cases outlined within this abstract, a positive result is an indication for therapeutic change. Some specific mutations weigh on TKI selection.

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Cytogenetic and Molecular Failure at 12 Months will be the Optimal Time Point for BCR-ABL1 Tyrosine Kinase Domain Mutation Analysis in Patients with Chronic Myeloid Leukemia: The Analysis Based on 2013 European LeukemiaNet Recommendation

Clinical Lymphoma Myeloma & Leukemia ◽

10.1016/j.clml.2017.09.045 ◽

2017 ◽

Vol 17 (10) ◽

pp. S17-S18

Author(s):

Hee-Je Kim ◽

Hea-Lyun Yoo ◽

Won-Sik Lee ◽

Hyeong-Joon Kim ◽

Jee Hyun Kong ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Mutation Analysis ◽

Myeloid Leukemia ◽

Kinase Domain ◽

Optimal Time ◽

Tyrosine Kinase Domain ◽

Kinase Domain Mutation ◽

Optimal Time Point ◽

Time Point

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Molecular Monitoring and Mutations in Chronic Myeloid Leukemia: How to Get the Most out of Your Tyrosine Kinase Inhibitor

American Society of Clinical Oncology Educational Book ◽

10.14694/edbook_am.2014.34.167 ◽

2014 ◽

pp. 167-175 ◽

Cited By ~ 10

Author(s):

Michele Baccarani ◽

Simona Soverini ◽

Caterina De Benedittis

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Sanger Sequencing ◽

Mutational Analysis ◽

Residual Disease ◽

Kinase Domain ◽

Response To Treatment ◽

Molecular Response ◽

Therapeutic Decisions

The course of chronic myeloid leukemia (CML) and the response to treatment with tyrosine kinase inhibitors (TKIs) are best monitored and assessed using two molecular tests: the first is real-time quantitative reverse transcription-polymerase chain reaction (RQ-PCR), which measures the size of residual disease that is expressed as BCR-ABL1% (the ratio between BCR-ABL1 and a control gene) and the other is mutational analysis by Sanger sequencing, which checks for the presence of BCR-ABL1 kinase domain point mutations. Both tests are technically demanding and require a high level of specialization and standardization. RQ-PCR, when performed on a regular basis, allows for the defining of molecular response (MR) levels as log reduction from a standardized baseline: major molecular response (MMR or MR3) that is the best predictor of survival; and the deeper molecular response (MR4, MR4.5, and MR5) that is necessary to enroll a patient in a trial aiming at treatment-free remission (TFR). Mutational analysis, to be performed in case of failure or warning by Sanger sequencing, allows for screening of the BCR-ABL1 kinase domain for mutations conferring resistance to TKIs. Since different mutations have different degrees of sensitivity to each of the currently available TKI, the knowledge of BCR-ABL1 kinase domain–mutation status is necessary for subsequent treatment choice. Optimal patient management requires that MR and mutational information be rationally interpreted at both the technical and at the biologic level, and put into context—therapeutic decisions also take into account other factors, such as age, comorbidities, side effects, compliance, and treatment-related complications.

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