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27 Jun 2019 
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Introduction
Chronic myeloid leukemia (CML) is clonal myeloproliferative neoplasia of pluripotent hematopoietic stem cells [1]. CML appears due to the reciprocal translocation between the long arms of chromosomes 9 and 22 t(9;22) (q34;q11) [2]. This translocation results from the formation of a chimeric gene, BCR-ABL1, which encodes the BCR-ABL protein with intrinsic tyrosine kinase activity that is critical of leukemogenesis [3]. The discovery of BCR-ABL protein which is required for the pathogenesis of this disease provided the rationale for the therapeutic intervention of an inhibitory agent that targets tyrosine kinase domain of the protein and blocks its phosphorylation [4].
The laboratory of Suisse Novartis who discovered that the Imatinib mesylate (IM) (trade name Gleevec®, formerly known as STI571) is the first molecule tyrosine kinase inhibitor (TKI) currently successfully used for the treatment of CML [5]. IM, also known as blocked tyrosine kinase activity of the BCR-ABL oncoprotein by the competitive inhibitor of ATP binding site [6].
Despite IM\’s striking efficacy, many researchers showed the resistance of this drug develops over time in minority patients with advanced-stage CML [7-9]. Unless some patients failed to respond (primary resistance) or lose response with this therapy (secondary resistance) which can be a consequence of multi-factors [8,9].
The resistance against IM can result from several mechanisms that can be widely divided into BCR-ABL dependent or BCR-ABL independent [10]. Overall these findings; the mutations in ABL kinase domain and/or amplification of the BCR-ABL oncogene are greatly observed the
BCR-ABL dependent mechanisms [11]. For the mechanisms of resistance of BCR-ABL independent to IM, some studies revealed that these mechanisms included decreasing intracellular concentration drug which can be due to related to bring of IM by human organic cation transporter 1 (hOCT1) or export by the P-glycoprotein (P-gp) [12,13]. The influence of efflux transporters in IM pharmacokinetics has been widely investigated, and their increased expression has been usually connected with IM response variability [12,13]. The most well-studied transporter protein is the P-gp which is a drug efflux transmembrane pump and it should the ability to eject IM from the leukemic cells [14]. This protein is an encoded by ATP-binding cassette, subfamily B, member 1 ABCB1 gene (also known MDR1 (Multi Drug Resistance 1)), and it is located in various tissues as well as in normal peripheral blood lymphocytes and bone marrow cells, that are involved in the traffic of IM outside cancer cells [15]. In a considerable number of cancers, overexpression of
Chronic myeloid leukemia (CML) is clonal myeloproliferative neoplasia of pluripotent hematopoietic stem cells [1]. CML appears due to the reciprocal translocation between the long arms of chromosomes 9 and 22 t(9;22) (q34;q11) [2]. This translocation results from the formation of a chimeric gene, BCR-ABL1, which encodes the BCR-ABL protein with intrinsic tyrosine kinase activity that is critical of leukemogenesis [3]. The discovery of BCR-ABL protein which is required for the pathogenesis of this disease provided the rationale for the therapeutic intervention of an inhibitory agent that targets tyrosine kinase domain of the protein and blocks its phosphorylation [4].
The laboratory of Suisse Novartis who discovered that the Imatinib mesylate (IM) (trade name Gleevec®, formerly known as STI571) is the first molecule tyrosine kinase inhibitor (TKI) currently successfully used for the treatment of CML [5]. IM, also known as blocked tyrosine kinase activity of the BCR-ABL oncoprotein by the competitive inhibitor of ATP binding site [6].
Despite IM\’s striking efficacy, many researchers showed the resistance of this drug develops over time in minority patients with advanced-stage CML [7-9]. Unless some patients failed to respond (primary resistance) or lose response with this therapy (secondary resistance) which can be a consequence of multi-factors [8,9].
The resistance against IM can result from several mechanisms that can be widely divided into BCR-ABL dependent or BCR-ABL independent [10]. Overall these findings; the mutations in ABL kinase domain and/or amplification of the BCR-ABL oncogene are greatly observed the
BCR-ABL dependent mechanisms [11]. For the mechanisms of resistance of BCR-ABL independent to IM, some studies revealed that these mechanisms included decreasing intracellular concentration drug which can be due to related to bring of IM by human organic cation transporter 1 (hOCT1) or export by the P-glycoprotein (P-gp) [12,13]. The influence of efflux transporters in IM pharmacokinetics has been widely investigated, and their increased expression has been usually connected with IM response variability [12,13]. The most well-studied transporter protein is the P-gp which is a drug efflux transmembrane pump and it should the ability to eject IM from the leukemic cells [14]. This protein is an encoded by ATP-binding cassette, subfamily B, member 1 ABCB1 gene (also known MDR1 (Multi Drug Resistance 1)), and it is located in various tissues as well as in normal peripheral blood lymphocytes and bone marrow cells, that are involved in the traffic of IM outside cancer cells [15]. In a considerable number of cancers, overexpression of
P-gp provides the most commonly found mechanism of multidrug resistance (MDR), designing a significant obstacle of failure of cancer chemotherapy [16,17]. MDR is a phenom that is related to reduced intracellular drug accumulation in leukemic cells resulting from enhanced drug efflux [18]. A diversity of studies have tried to determine MDR modulators which increase drug accumulation in tumor cells.
Within the past few years, the actual role of P-gp efflux pump in resistance against IM has been widely studied. A number of investigations have proposed that increased of P-gp expression is unlikely to be a primary mechanism of IM resistance in patients with CML [19,20]. However, These studies have not obtained by other investigators [21,22].
To clarify these inconsistent and/or controversial findings of these results; the roles of P-gp in IM resistance need to be further explored. In our study, we tried to find a correlation between the overexpression of P-gp and interindividual variability of IM response in CML patients.
Materials And Methods
Study design and patient selection
A retrospective case-control study on 59 Tunisian CML patients in chronic phase had designed. This study had effected in the hematology department of Hedi Chaker-Hospital University, Sfax, Tunisia on the period of June 2015 – January 2016. The patients were all adherent to IM.
Exclusion criteria of subjects were as follows: pregnant women, patients suffering from any another hematological illness, patients in accelerated phase or blastic crisis, patients who have BCR-ABL1 gene mutations.
The protocol of this study had approved according to the criteria by the declaration of Helsinki. Each patient informed consent for their participation in the study.
Definitions and Approaches to Measuring Responses to Imatinib
According to European Leukemia Network criteria 2016 (ELN) [23], the IM responses were evaluated by the BCR–ABL1 gene ratio. After one year of IM treatment, patients are considered as optimal responders to IM if the BCR–ABL1 gene ratio ≤0.1% (achieve a major molecular response (MMR)) or as IM resistant phenotype if the ratio >0.1%. IM no-responders CML patients can be defined as primary resistance (failure to achieve an optimal response) or as secondary resistance (loss of an initiated response).
Furthermore, Cortes et al. [29] reported that complete cytogenetic response (CCyR) was defined approximately equivalent to a BCR-ABL1 gene ratio ≤1% within six months from the start of IM treatment.
A complete hematologic response (CHR) is obtained when laboratory values return to normal levels at three months from the start of IM therapy.
The BCR-ABL gene mutations testing was performed exclusively on patients who showed IM resistant.
Definitions of endpoints
Demographic, clinical and biological characteristics, Sokal score, TKI treated, and therapy failure were extracted retrospectively from the patients’ files.
Sokal score was established to prognosticate the patients of CML at diagnosis [24]. Three risk groups were designated as follows: low risk (score 1.2).
Peripheral Blood Mononuclear Cell Isolation
Peripheral blood mononuclear cells (PBMCs) from CML were isolated by Ficoll-Hypaque gradient (Eurobio, France) immediately after sampling. Cells were washed once in phosphate-buffered saline (PBS). After washing, they are frozen in dimethyl sulfoxide (DMSO) and conserved at -80°C for long-term storage.
Determination of P-gp expression by flow cytometry
Expression of P-gp was examined on lymphocytes subpopulations in PBMCs by indirect immunofluorescence method [25]. Briefly, 5 .105 cells were rinsed twice in PBS plus 0.5 % bovine serum albumin (BSA) to eliminate the DMSO. Then, these cells were incubated with or without a P-gp Antibody (1:50) (Clone UIC2, GeneTex, USA) for 45 min at 4°C. After three washes, Cells were sequentially incubated with a saturating concentration (1 µg/mL) of PE anti-mouse IgG (Biotium, ) as well as FITC anti-CD45 (Beckman Coulter,) on ice for 30 min in the dark. Besides cells were washed twice in PBS/BSA 0.5% and resuspended in PBS.
Cells were analyzed on F<ACScantoTM II flow cytometer using Cell Quest software (Becton Dickinson, USA). PBMCs were gated by forward scatter (FSC-A) vs side scatter (SSC-A) as portray in Figure 1.
On the whole, the data were revealed as the ratio of the mean fluorescence intensity (MFI) of anti-P-gp, primary antibody, and PE-anti-mouse IgG, secondary antibody, divided by the MFI of cells treated just with the secondary antibody [26].
Based on previous studies, the cutoff value (MFI 1.1 = P-gp positive) was used for expression analysis of P-gp [27,28].
Statistical Analysis
Data were expressed in mean ± standard deviation (SD). Student\’s t-test was used to compare data between the two groups. Kaplan-Meier analysis and log-rank test were performed to predict the impact of molecular response and TKI treatment in P-gp expression. P-value ≤0.05 was considered statistically significant (SPSS 20).
Results
Clinical characteristics of CML patients
All patients started their treatment with IM 400 mg/day. According to ELN 2016, patients have classified into two groups. The first group comprised 27 patients that are considered as optimal responders to IM, while the second group comprised 32 patients that presented IM resistant phenotype [23]. Amid these IM no-responders CML patients, 26 patients defined as primary resistance and six patients considered as secondary resistance. The patients who failed of IM had switched to second-generation TKI as dasatinib (n=11) or nilotinib (n=21). Only 14 IM no-responders patients achieved their CCyR within six months (Table1). All patients achieved a complete hematologic response (CHR) at three months following IM therapy.
The test of BCR-ABL1 gene mutations indicated no mutation of the BCR-ABL kinase domain in IM resistant subjects.
Table 2 summarizes the baseline characteristics of both groups of patients with CML (IM responders, as well as, IM no-responders ). There were no significant differences for gender, age and Sokal score between both groups (p-value < 0.05). The age of IM responders CML patients ranged from 23 to 80 years, with a mean of 50.22±15.23 years and that of IM no-responders from 24 to 73 years, with a mean of 48.53±11.54 years. As assessed by the Sokal score at diagnostic: on the one hand, 8 patients showed a low-risk, 13 patients showed an intermediate-risk, and six patients showed a high-risk in IM responders CML patients, and on the other hand, 13 patients showed a low-risk, 19 patients showed an intermediate-risk, and neither patients showed a high-risk in IM no-responders CML patients.
We observed a significant increase in the b3a2 transcript type of BCR-ABL in IM responders as compared with IM no-responders.
Biological characteristics of CML patients
Table 3 lists the biological characteristics of CML patients. Each patient had informed with, White blood cell (WBC) count, neutrophils count, platelets count, and hemoglobin (Hb) concentration, as well as, by the spleen size at diagnosis. The comparison between the IM responders and IM no-responders CML patients showed higher levels of WBC and neutrophils in IM no-responders (p=0.005 and p=0.01, respectively). Besides, we observed a greater of the spleen size for IM no-responders than for IM responders patients (p=0.015).
P-gp expression status in CML patients
Pgp expression ranged from 0.76 to 1.43 with an MFI = 1.1 when all patients were studied. So, this value of MFI was used as the cutoff point to divide negative (MFI 1.1) P-gp expression. Two instances of a flow cytometric analysis for negative and positive P-gp expression are present in Figure 2.A and Figure 2.B respectively.
By analyzing levels of P-gp expression according to response to IM (responders vs no-responders), we found that all IM no responders CML patients exhibited P-gp overexpression (MFI >1.1), while all IM responders patients demonstrated negative expression of P-gp (MFI <1.1) (p=0.001) (Figure 3.A).
Relationship between MDR phenotype and expression of P-gp
In CML patients IM no-responders the comparison between patients achieved and not achieved their CCyR showed a high MFI of P-gp expression in patients not achieved their CCyR (p=0.001) (Figure 3.B).
Discussion
Pgp, an efflux membrane pump, is first associated with MDR phenom of tumor cell lines [30]. The Pgp expression level in clinical cancers was widely studied. Publishing researches have to focus on the relationship between the expression of Pgp and poor prognosis to chemotherapy, as well as, short-duration survival in various types of hematological malignancies [16,17]. Recently, there has been a spate of interest in the correlation of P-gp overexpression with drug resistance to IM in CML. Therefore, in this investigation, we aimed to measure P-gp expression in peripheral blood lymphocytes of patients with CML by flow cytometry.
The contribution of P-gp transporter in influencing the IM therapy response in CML patients remains unclear. However, some investigations have studied the role of P-gp in IM pharmacokinetics and showed findings controversial. In our research, it was clear that the mode of resistance to IM is proportional to overexpression of P-gp: the patients IM no-responders showed an increased expression of P-gp when compared to subjects IM-responders.
Within the past few years, the actual role of P-gp efflux pump in resistance against IM has been widely studied. A number of investigations have proposed that increased of P-gp expression is unlikely to be a primary mechanism of IM resistance in patients with CML [19,20]. However, These studies have not obtained by other investigators [21,22].
To clarify these inconsistent and/or controversial findings of these results; the roles of P-gp in IM resistance need to be further explored. In our study, we tried to find a correlation between the overexpression of P-gp and interindividual variability of IM response in CML patients.
Materials And Methods
Study design and patient selection
A retrospective case-control study on 59 Tunisian CML patients in chronic phase had designed. This study had effected in the hematology department of Hedi Chaker-Hospital University, Sfax, Tunisia on the period of June 2015 – January 2016. The patients were all adherent to IM.
Exclusion criteria of subjects were as follows: pregnant women, patients suffering from any another hematological illness, patients in accelerated phase or blastic crisis, patients who have BCR-ABL1 gene mutations.
The protocol of this study had approved according to the criteria by the declaration of Helsinki. Each patient informed consent for their participation in the study.
Definitions and Approaches to Measuring Responses to Imatinib
According to European Leukemia Network criteria 2016 (ELN) [23], the IM responses were evaluated by the BCR–ABL1 gene ratio. After one year of IM treatment, patients are considered as optimal responders to IM if the BCR–ABL1 gene ratio ≤0.1% (achieve a major molecular response (MMR)) or as IM resistant phenotype if the ratio >0.1%. IM no-responders CML patients can be defined as primary resistance (failure to achieve an optimal response) or as secondary resistance (loss of an initiated response).
Furthermore, Cortes et al. [29] reported that complete cytogenetic response (CCyR) was defined approximately equivalent to a BCR-ABL1 gene ratio ≤1% within six months from the start of IM treatment.
A complete hematologic response (CHR) is obtained when laboratory values return to normal levels at three months from the start of IM therapy.
The BCR-ABL gene mutations testing was performed exclusively on patients who showed IM resistant.
Definitions of endpoints
Demographic, clinical and biological characteristics, Sokal score, TKI treated, and therapy failure were extracted retrospectively from the patients’ files.
Sokal score was established to prognosticate the patients of CML at diagnosis [24]. Three risk groups were designated as follows: low risk (score 1.2).
Peripheral Blood Mononuclear Cell Isolation
Peripheral blood mononuclear cells (PBMCs) from CML were isolated by Ficoll-Hypaque gradient (Eurobio, France) immediately after sampling. Cells were washed once in phosphate-buffered saline (PBS). After washing, they are frozen in dimethyl sulfoxide (DMSO) and conserved at -80°C for long-term storage.
Determination of P-gp expression by flow cytometry
Expression of P-gp was examined on lymphocytes subpopulations in PBMCs by indirect immunofluorescence method [25]. Briefly, 5 .105 cells were rinsed twice in PBS plus 0.5 % bovine serum albumin (BSA) to eliminate the DMSO. Then, these cells were incubated with or without a P-gp Antibody (1:50) (Clone UIC2, GeneTex, USA) for 45 min at 4°C. After three washes, Cells were sequentially incubated with a saturating concentration (1 µg/mL) of PE anti-mouse IgG (Biotium, ) as well as FITC anti-CD45 (Beckman Coulter,) on ice for 30 min in the dark. Besides cells were washed twice in PBS/BSA 0.5% and resuspended in PBS.
Cells were analyzed on F<ACScantoTM II flow cytometer using Cell Quest software (Becton Dickinson, USA). PBMCs were gated by forward scatter (FSC-A) vs side scatter (SSC-A) as portray in Figure 1.
On the whole, the data were revealed as the ratio of the mean fluorescence intensity (MFI) of anti-P-gp, primary antibody, and PE-anti-mouse IgG, secondary antibody, divided by the MFI of cells treated just with the secondary antibody [26].
Based on previous studies, the cutoff value (MFI 1.1 = P-gp positive) was used for expression analysis of P-gp [27,28].
Statistical Analysis
Data were expressed in mean ± standard deviation (SD). Student\’s t-test was used to compare data between the two groups. Kaplan-Meier analysis and log-rank test were performed to predict the impact of molecular response and TKI treatment in P-gp expression. P-value ≤0.05 was considered statistically significant (SPSS 20).
Results
Clinical characteristics of CML patients
All patients started their treatment with IM 400 mg/day. According to ELN 2016, patients have classified into two groups. The first group comprised 27 patients that are considered as optimal responders to IM, while the second group comprised 32 patients that presented IM resistant phenotype [23]. Amid these IM no-responders CML patients, 26 patients defined as primary resistance and six patients considered as secondary resistance. The patients who failed of IM had switched to second-generation TKI as dasatinib (n=11) or nilotinib (n=21). Only 14 IM no-responders patients achieved their CCyR within six months (Table1). All patients achieved a complete hematologic response (CHR) at three months following IM therapy.
The test of BCR-ABL1 gene mutations indicated no mutation of the BCR-ABL kinase domain in IM resistant subjects.
Table 2 summarizes the baseline characteristics of both groups of patients with CML (IM responders, as well as, IM no-responders ). There were no significant differences for gender, age and Sokal score between both groups (p-value < 0.05). The age of IM responders CML patients ranged from 23 to 80 years, with a mean of 50.22±15.23 years and that of IM no-responders from 24 to 73 years, with a mean of 48.53±11.54 years. As assessed by the Sokal score at diagnostic: on the one hand, 8 patients showed a low-risk, 13 patients showed an intermediate-risk, and six patients showed a high-risk in IM responders CML patients, and on the other hand, 13 patients showed a low-risk, 19 patients showed an intermediate-risk, and neither patients showed a high-risk in IM no-responders CML patients.
We observed a significant increase in the b3a2 transcript type of BCR-ABL in IM responders as compared with IM no-responders.
Biological characteristics of CML patients
Table 3 lists the biological characteristics of CML patients. Each patient had informed with, White blood cell (WBC) count, neutrophils count, platelets count, and hemoglobin (Hb) concentration, as well as, by the spleen size at diagnosis. The comparison between the IM responders and IM no-responders CML patients showed higher levels of WBC and neutrophils in IM no-responders (p=0.005 and p=0.01, respectively). Besides, we observed a greater of the spleen size for IM no-responders than for IM responders patients (p=0.015).
P-gp expression status in CML patients
Pgp expression ranged from 0.76 to 1.43 with an MFI = 1.1 when all patients were studied. So, this value of MFI was used as the cutoff point to divide negative (MFI 1.1) P-gp expression. Two instances of a flow cytometric analysis for negative and positive P-gp expression are present in Figure 2.A and Figure 2.B respectively.
By analyzing levels of P-gp expression according to response to IM (responders vs no-responders), we found that all IM no responders CML patients exhibited P-gp overexpression (MFI >1.1), while all IM responders patients demonstrated negative expression of P-gp (MFI <1.1) (p=0.001) (Figure 3.A).
Relationship between MDR phenotype and expression of P-gp
In CML patients IM no-responders the comparison between patients achieved and not achieved their CCyR showed a high MFI of P-gp expression in patients not achieved their CCyR (p=0.001) (Figure 3.B).
Discussion
Pgp, an efflux membrane pump, is first associated with MDR phenom of tumor cell lines [30]. The Pgp expression level in clinical cancers was widely studied. Publishing researches have to focus on the relationship between the expression of Pgp and poor prognosis to chemotherapy, as well as, short-duration survival in various types of hematological malignancies [16,17]. Recently, there has been a spate of interest in the correlation of P-gp overexpression with drug resistance to IM in CML. Therefore, in this investigation, we aimed to measure P-gp expression in peripheral blood lymphocytes of patients with CML by flow cytometry.
The contribution of P-gp transporter in influencing the IM therapy response in CML patients remains unclear. However, some investigations have studied the role of P-gp in IM pharmacokinetics and showed findings controversial. In our research, it was clear that the mode of resistance to IM is proportional to overexpression of P-gp: the patients IM no-responders showed an increased expression of P-gp when compared to subjects IM-responders.
The result emphasizes the fact that CML patients with b3a2 transcript type accomplished both earlier, as well as, deeper responses to IM. Consequently, this transcript type could serve as a clinical and helpful biomarker to prophesy an optimal response to IM in patients with CML.
Conflict of interest statement
None of authors have any relevant conflicts of interest to disclose.