Imatinib-resistant Chronic Myeloid Leukaemia Patients' BCR-ABL1 Kinase Domain Mutations in the Eastern Indian Population

doi:10.21203/rs.3.rs-4200659/v1

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Imatinib-resistant Chronic Myeloid Leukaemia Patients' BCR-ABL1 Kinase Domain Mutations in the Eastern Indian Population

https://doi.org/10.21203/rs.3.rs-4200659/v1

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Chronic myeloid leukemia is a bone marrow cancer that spreads slowly. Platelets (thrombocytes), which aid in blood clotting, white blood cells (leukocytes), which protect the body from infection, and red blood cells (erythrocytes), which supply oxygen, are all produced by normal bone marrow. In chronic myeloid leukemia, the bone marrow produces an abnormally large amount of white blood cells. At first, these cells appear to function correctly. However, as the disease progresses, the blood and bone marrow become overpopulated with immature white blood cells known as myeloblasts, also known as blasts. Overgrowth of myeloblasts hinders the generation of other blood cells, resulting in anemia (low red blood cell count) and platelet shortage. In twelve CML patients who either lost their response or did not obtain it within the allotted timeframes, we examined the pattern of kinase domain mutations. The most frequent reason to request a mutation investigation was loss of molecular responsiveness. It was discovered that four patients had identifiable mutations. We identified four mutations: E255V, L387M, T315I, and F359S in our cohort.

Molecular Genetics

Chronic Myeloid Leukaemia

Myeloblasts

Mutation

BCR-ABL1

Chronic Myeloid Leukemia (CML), a rare but impactful form of leukemia, is a hematologic malignancy that originates in the bone marrow, affecting the blood and immune system. The hallmark of CML is the aberrant development and expansion of myeloid cells, a subset of white blood cells that fight infections and preserve general health. This condition is distinguished by its unique genetic abnormality, the Philadelphia chromosome, and it typically progresses through three distinct phases: chronic, accelerated, and blast crisis. The fundamental cause of CML lies in a specific genetic mutation. The Philadelphia chromosome is created when two chromosomes, 9 and 22, swap genetic material. [1] This mutation causes this to happen. The fusion gene BCR-ABL1, which is the result of this translocation, encodes the aberrant protein tyrosine kinase. The presence of this fusion gene is a hallmark of CML and plays a central role in driving the disease. In its early, chronic phase, CML often develops silently, with many individuals experiencing no or mild symptoms. As the disease advances, patients may begin to notice symptoms such as fatigue, weight loss, night sweats, abdominal discomfort due to an enlarged spleen, bone pain, easy bruising, and an increased susceptibility to infections. These signs frequently result in a medical assessment and a CML diagnosis. A complete blood count (CBC) is one of several blood tests used to diagnose chronic myeloid leukemia (CML). The results of this test can show higher white blood cell counts. A bone marrow biopsy, however, is required to reach a conclusive diagnosis. This process validates the presence of the BCR-ABL1 fusion gene and makes it possible to identify the distinctive Philadelphia chromosome. Molecular tests are also crucial in determining the level of BCR-ABL1, which is vital for monitoring disease progression and assessing the response to treatment.

CML is staged based on the phase of the disease. In the chronic phase, the disease is in its early, more manageable stage. As it progresses, it may transition to the accelerated phase, characterized by an increased number of immature white blood cells. In the most advanced stage, blast crisis, there is a significant increase in these immature cells, often leading to a poorer prognosis. The management of CML has seen remarkable progress over the years with the development of targeted therapies known as tyrosine kinase inhibitors (TKIs). These medications, which include imatinib (Gleevec), dasatinib (Sprycel), and nilotinib (Tasigna), selectively target the aberrant BCR-ABL1 protein and, in many cases, result in long-term remission. [2] In more advanced stages or when TKIs are ineffective, a bone marrow transplant may be considered. Regular monitoring of BCR-ABL1 levels is essential in managing CML, ensuring that the disease remains under control. Early diagnosis and the availability of effective treatments have transformed CML from a life-threatening condition to a chronic but manageable disease, offering hope and improved quality of life to those affected. The competitive binding of ATP molecules to the BCR-ABL protein is inhibited by imatinib. This medication altered the course of CML treatment. With a median survival of 4.9 years in the Accelerated Phase (AP) and 8–9 years in the Chronic Phase (CP), CML patients' chances of survival have significantly increased. [3] This prompted a re-evaluation of stem cell transplantation's place in the treatment of CML. But as resistance and intolerance to imatinib become more apparent, the initial elation is fading. Research on CML patients receiving imatinib reveals that over 33% of individuals do not experience a full cytogenetic response (CCyR), and some patients develop drug resistance or are unable to handle the toxicity associated with their medications. [4] Subsequent phosphorylation is prevented, and BCR-ABL signaling is interfered with. A number of downstream signaling pathways are inhibited, such as RAS-GDP, STAT, and PI3K-AKT. Imatinib is a c kit tyrosine kinase inhibitor as well as a platelet-derived growth factor.

Aim

BCR-ABL1 Kinase Domain Mutation analysis of Imatinib-resistant Chronic myeloid leukemia Patients in the Eastern Indian Population.

Reagents:

Himedia provided the RPMI-1640, whereas Sigma Aldrich (St. Louis, USA) provided the fetal bovine serum (FBS), trisodium citrate, and Giemsa stain. Trypsin, Pen Strep (Penicillin and Streptomycin), and Colchicine were procured from (Thermo Fisher Scientific, Inc.), as well as phytohemagglutinin (PHA) from Invitrogen and KCl (Merck).

Ethical Approval:

The Ethics Committee of the Institute of Science, Banaras Hindu University (Ref: I.Sc./ECM-XII/2021-22) provided institutional ethics approval, and signed informed consent was obtained from the patient/participant for publication. After written informed consent for the study was obtained, a detailed clinical history and blood sample of each patient were collected. Any participant under the age of 18 must obtain permission from a parent or legal guardian. All information has been anonymized.

Microscopy, karyotyping, and cytogenetic analysis:

A total of 50 metaphase plates were captured under the microscope and karyotyping was done with 450G-banding resolution using an automated karyotyping workstation having Metasystem’s software (Ikaros®, Carl Zeiss® Microscopy GmbH, Göttingen, Germany). Based on guidelines from the International System of Human Cytogenetic Nomenclature, Chromosomes were analysed. The detection of the Ph-positive chromosome in metaphase plates and analyzed for karyotyping and cytogenetic abnormalities. [5]

Kinase Domain Mutation:

For this observational study, 12 out of 172 CML patients who were treated with imatinib but did not respond well to the medication were selected. Initially, patients were seen once a month thereafter, they saw a doctor every two to three weeks for a month. Every session highlighted the importance of compliance. Nested RT-PCR was used in the imatinib resistance mutation investigation to identify the ABL kinase domain area and the BCR-ABL fusion transcript. Multiple nucleotide sequencing alignments were employed as a bioinformatics tool after kinase domain alterations were assessed through the use of the fluorescence nucleotide sequencing technique. The current European LeukemiaNet criteria served as the basis for the indications for mutation analysis. An imatinib resistance mutation analysis was necessary when there was a delay in reaching predetermined milestones, a loss of hematological response (HR), cytogenetic response (CyR), or molecular response. The ELN 2013 chronic myeloid leukemia recommendations described the molecular, cytogenetic, and hematological reactions at different intervals. When a hematological, cytogenetic, or molecular response was not obtained at pre-established time intervals, ELN classified the response as unsatisfactory. Progressive sickness was characterized by loss of obtained responses as described by ELN. We screened these mutation D276G, E255K, E255V, E29K, E373G, F486S, G250E, G321E, L248V, T315I, Y253F, Y253H, E275K, E355G, E450K, F317L, F359C, F359V, H396P, H396R, L387M, M244V, M351T, Q252H, E355A, F311L, F317V, G250A, L387F, M237I, M388L, T315A, V289A, V299L, V379I, Y235H. Finally, we identified four mutations: E255V, L387M, T315I, and F359S in our cohort.

Methods for analyzing Kinase Domain mutations:

There are currently numerous technologies available for detecting and evaluating KD mutations. The sensitivity, specificity, and bias of these devices vary greatly. The clinical significance of the data provided by the methods influences the choice of the best approach. Conventional Sanger sequencing has a sensitivity of approximately 15% to 20%, but the mutation-specific quantitative polymerase chain reaction (PCR) technique can detect a mutant transcript in 1 in 10,000 BCR ABL transcripts. Targeted microarrays, liquid bead arrays, and denaturing high-performance liquid chromatography are further BCR-ABL KD mutant screening approaches that have been published. All these methods need a final confirmation via direct sequencing for uncommon cases. To measure the percentage of a mutant clone that remains after a therapy switch, two quantitative mutation detection technologies have been developed: mutation-specific quantitative PCR and PCR-based pyrosequencing. However, an international consensus panel recommended direct sequencing of the BCR-ABL transcript by the Sanger method as the best suitable screening test because the finding of few mutant clones might not have any clinical significance.

This study included twelve imatinib-resistant individuals ranging in age from 25 to 75 years. The ratio of men to women was 1.24:1. The range of the median Sokal score was (0.60–1.66) 0.88. Eight patients had intermediate or high-risk Sokal scores, while four individuals had low-risk scores. Patients were requested to undertake mutation analysis 4.4 (1-12) years after starting imatinib. Imatinib dosages were typically 400 mg per day. At the time of the mutation investigation, ten patients were in the chronic phase and two were in the accelerated phase. Eleven patients were in the chronic phase at the time of the mutation analysis, and one was in the accelerated phase. There were no patients with blast crises included. In 12 patients, mutation analysis could be performed. Mutation analysis was requested in three (%), five (%), and four (0%) patients who had lost their Haematological Response (HR), Cytogenetic Response (CyR), and Molecular Response (MR). Mutations in the kinase domain were found in 4 (%) of the individuals. Table 1 demonstrates the prevalence of different mutations in our patients. (E255V) mutations were p loop mutations, (F359S) SH2 mutations, and (T315I) Contact binding site (L387M) other mutations among the four discovered. E255V, L387M, T315I, and F359S mutations were discovered in the patients. In the T315I mutation, all first-line tyrosine kinase drugs are considered ineffective. Patients who tested negative for mutations were given imatinib 600 OD, nilotinib 400 bid, dasatinib 100 OD, or no treatment at all. As a result, the majority of patients were given larger dosages of imatinib or nilotinib. Patients with kinase domain mutations were switched to nilotinib 400 BD, dasatinib 100 OD, and imatinib 600 OD, depending on the sensitivity of the mutation found and affordability.

Two of the four patients had data on their molecular response at 12 months. Two patients had major molecular responses (MMR), one had a minor molecular response (MMR), and one had no molecular reaction.

Table 1: Identified different types of kinase domain mutations in imatinib-resistant patients

Kinase Domain Mutation	No of patients (4)
E255V	1
L387M	1
T315I	1
F359S	1

Imatinib was a seminal finding in the field of molecularly targeted therapy that altered the therapeutic landscape in CML. Imatinib resistance has recently been reported in a large number of patients, leading in inadequate or no response. The tyrosine kinase activity of the BCR-ABL transcript is the primary driving force in CML. By interacting to the ATP binding site, imatinib suppresses kinase activity. Mutations in tyrosine kinase domains (ploop, A-loop, SH-2, and SH3) prevent imatinib from properly contacting and binding to its ABL site.

Front-line TKI resistance is a common stumbling block in CML therapy. IRIS, one of the earlier trials, discovered that approximately 17% of patients develop resistance to imatinib, a first-generation TKI [6]. Later investigations were able to discover a number of mutations in the KD region of BCR-ABL1 and establish their role in causing imatinib resistance. [7]. T315I, for example, was discovered to specifically impede imatinib binding to the auto-phosphorylation site within the KD region. [8]. Several more hotspot mutations in the ATP binding sites were found, preventing imatinib from reaching BCR-ABL1. [9]. Other mutations were discovered that affect the structure of this oncoprotein. [10]. Notably, investigations utilizing next-generation targeted sequencing technologies may provide a deeper insight of the mutational landscape. [11]. Several Indian studies compared traditional Sanger sequencing with next-generation technologies to gain a better understanding of KD mutations in BCR-ABL1. [12–13]. However, no similar efforts were recorded focusing on Eastern-Indian cohorts, making the BCR-ABL1 mutational landscape elusive from this region of the country.

According to our findings, only 33% of the patients exhibited identifiable mutations in the ABL kinase domain, with M351T being the most prevalent. Four distinct mutations were found in 12 individuals, with one of our patients having T315I, which has been established to be the most common mutation in the international literature. M351T mutation is susceptible to increased imatinib, nilotinib, and dasatinib dosages. It is clinically significant because the majority of our patients cannot afford nilotinib or dasatinib. Our data shows E255V, L387M, T315I, and F359S as the most common mutation in our patients. T315I or E255V/K has been reported as the most frequent mutation in the remainder of the world. Surprisingly we did get both mutations in patients with T315I and E255V mutation. We suggest a study with a greater number of patients at different hematology centers in eastern India population to have more robust data on mutational frequency.

Conflict of interest: There were no conflicts of interest declared by any of the writers.

Ethical Approval: All procedures performed in this study were by the ethical standards of the Institute of Science, Banaras Hindu University ethical committee (Ref. No.: I.Sc./ECM-XII/2021-22).

Informed Consent Statement: All persons taking part in this study provided informed consent.

Acknowledgments: We would like to thank CML patients for their cooperation. This study was supported by a grant from the Institute of Eminence (IOE) Banaras Hindu University Varanasi. I also acknowledge the University Grand Commission, New Delhi, India for funding a Senior Research Fellow.

Author Contributions: AA wrote the first draught, conceived and designed the study, and reviewed and edited the final manuscript; NK and VT designed the experiments, performed data analysis, and reviewed the manuscript; and AK performed experiments, interpreted and analyzed data, and edited the manuscript. The contribution in its final form was approved by all authors.

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The authors declare no competing interests.

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Imatinib-resistant Chronic Myeloid Leukaemia Patients' BCR-ABL1 Kinase Domain Mutations in the Eastern Indian Population

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