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Title: The Role of Drug Transporters ABCB1, ABCG2, and SLC22A1 in Resistance to Tyrosine Kinase Inhibitors and Disease Progression in Chronic Myeloid Leukaemia
Authors: Ahmad, Nasir
Keywords: Biological & Medical Sciences
Issue Date: 2022
Publisher: Khyber Medical University, Peshawar
Abstract: Background Chronic Myeloid Leukaemia (CML) is characterized by the reciprocal translocation of Abelson murine leukemia viral oncogene homolog (ABL) and breakpoint cluster region (BCR) in haematopoietic stem cells in the bone marrow. This translocation leads to the transformation of the normal physiologically self-controlled Abl kinases into constitutively active Bcr-Abl. At the cytogenetic level, the translocation of these chromosomal parts leads to the formation of the Philadelphia (Ph) Chromosome. CML diagnosis is based on the presence of the Ph chromosome by cytogenetic and Fluorescence in situ hybridization (FISH) or BCR-ABL1 quantification using the Polymerase chain reaction (PCR). The advent of Tyrosine kinase inhibitors (TKIs) as standard care of therapy for CML and advancements in the detection as well as disease monitoring techniques have revolutionized the treatment and prognosis of CML. However, despite these advances, a considerable percentage of patients have shown resistance to the current CML therapies. This failure to treatment is associated with the subtherapeutic levels of drug accumulation inside the cell, as a result of the multidrug efflux transporter of ABC family (ABCB1 and ABCG2) and influx transporters of the SLC family (SLC22A1) transporters, in CML. Recent scientific reports have shown that mutations in some of these transporters can lead to resistance to TKIs in vitro. However, clinical reports and in vivo data are insufficient and enigmatic. Differential functions of the drug transporters due to genetic variations in their gene coding regions have been postulated. Minimal residual disease (MRD) monitoring in CML by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a challenging, yet important element to assess the molecular response and declare whether a CML patient falls into the ‘optimal’, ‘warning’, or ‘failure’ group. This study was aimed to evaluate the role of multidrug transporters (ABCB1, ABCG2, and SLC22A1) in resistance to TKIs at the genetic level and concurrent detection of V617F mutated Janus kinase 2 (JAK2) with BCR-ABL1 in Ph-positive CML patients. Methods The study comprised of two parts, cross-sectional and longitudinal. A total of 150 patients were enrolled in the cross-sectional (study-I) and 100 patients in the prospective cohort (study-II) group. Ethical approval was granted by the KMU-Ethics Board and patients’ information was recorded on a purposefully designed proforma after acquiring consent. Cytogenetic or FISH was used for diagnosis and RT-qPCR was performed for MRD monitoring. We could successfully follow 80 of the 100 enrolled patients in the study-II for 12 months. Rest of the 20 patients were lost to follow-up at different time intervals due to various reasons. Single nucleotide polymorphisms (SNPs) analysis of ABCB1 (rs1128503, rs2032582, rs1045642), ABCG2 (rs2231137, rs2231142) and SLC22A1 (rs628031, rs683369) was performed through Allele specific PCR for all these patients. Relative gene expression of ABCB1, ABCG2, and SLC22A1 was analysed by RT-qPCR to obtain fold change in transcript expression of the drug transporter genes. The 2−ΔΔC T method was used to determine the difference in expression. RT-qPCR was performed for absolute and/or relative quantification of BCR-ABL1 using both one-step and two-step PCR approach every 3 months. The identification of JAK2 V617F mutation was carried out in 103 CML patients using TaqMan probe-based and SYBR Green chemistry-based PCR in CML patients. Data was analyzed using Stata 12 and GraphPad 8. The continuous variables were compared by t-test and one-way ANOVA. The Chi-square test was used for the comparison of categorical variables. The results were considered statistically significant when the p value was less than 0.05. Results It was observed in the cohort that CML is more prevalent in the early age (16-35 years) group with a mean age of 41.25 ± 16.3 (7-75 years). Twelve percent of CML patients were in the accelerated phase while two percent of patients were in blast crisis at the time of induction in the study. Hydroxyurea (HU) was prescribed in 40.2% (n=51) of CML patients. The molecular response (MR3) was achieved in 40% of patients who were pretreated with HU plus a TKI while 60% of patients were only given TKI who achieved MR3. Imatinib (IM) and nilotinib (Nil) were prescribed in 60.6% and 39.4% of patients respectively in different daily doses (IM 400 mg> Nil 800 mg> Nil 600 mg). A better response (72.2%) was observed in a group of patients treated with Nil at a dose of 800 mg as compared to Nil at a dose of 600 mg (43.75%) and IM at a dose of 400 mg (32.55%). A total of 42.5% (30.4% in the IM group and 58.8% in the Nil group) achieved 3 log reductions (MR3) in their BCR-ABL1. In the IM group, it was observed that 10 patients switched to nilotinib, another 10 patients discontinued the treatment (for a short time) and eight patients reduced the prescribed dose. The JAK2 V617F and BCR-ABL1 p210 mutations were present simultaneously in 2 (1.94 %) Ph-positive CML patients. Splenomegaly was found in 82.68% of patients (mean size 18.6 ± 3.5). Cytogenetic analysis revealed 8 different chromosomal abnormalities each in one patient, other than the Ph chromosome. Baseline analyses showed an elevated Alkaline Phosphatase (ALP) level of 190.5 ± 120.8 U/L (normal range is 35-104 U/L) and Lactate Dehydrogenase (LDH) level of 817.9 ± 660.7 U/L (normal range is 240-480 U/L). The mean WBC was 185 x 103 /ul (range 7.9-660) and haemoglobin was 10.3 g/dl range (5-14.3). The B +ve blood group was more common (42.86%) in the CML patients. No coherence was observed between the prognostic risk scores. Overall, the SLC22A1 rs683369 wild-type (480CC) genotype, was associated with failure (n=6) to achieve the normal WBC count after 12 months. A slightly higher frequency of ABCB1 SNPs (1236TT and 2677GG) variants in the optimal response group was observed. Similarly, for ABCG2, the 421CA variant was more prevalent in the responder group as compared to CC and AA (50% vs 41% and 25%). The SLC22A1 1222GG variant was more prevalent (52%) in the optimal response group whereas, a greater percentage (40%) of patients having the SLC22A1 1222AA mutations were found in the failure group. Also, for SNP at position 480, the homo mutant GG was associated with optimal response (62.5%), and the homo wild-type genotype CC was related to the failure group (36.6%). No significant association was found between gene expression and response. However, SLC22A1 expression was 6.4 folds higher in optimal responder groups as compared to the failure groups (11.7 versus 5.3). Conclusion Based on the results, it is suggested that HU can be used for leukocytosis before the start of TKI but may not produce the optimal molecular response. Nilotinib was more potent than imatinib. Further, a higher prevalence of blood group B, increased serum ALP and LDH after further validation could be used as biomarkers in response prediction for CML prognosis. Concomitant JAK2 V617F presence in BCR-ABL1 mutated patients appeared to have no significant impact on molecular response in CML patients. Despite finding no significant association between the multidrug transporters and response to TKIs in CML patients, few patients having the wild-type (480CC) genotype (rs683369) were unable to achieve normal WBC. Furthermore, the 1236TT and 2677GG (ABCB1), 421CA (ABCG2), 1222GG, and 480GG (SLC22A1/ OCT1) mutations were more prevalent in the optimal response group while the 1222AA and 480CC mutations were more prevalent in the failure group. SLC22A1 expression was higher in the optimal response group suggesting a putative role of the influx transporter in response to TKIs
Gov't Doc #: 27011
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