Effect of interaction of glutathione S-transferases (T1 and M1) on the hematologic and cytogenetic responses in chronic myeloid leukemia patients treated with imatinib

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O R I G I N A L P A P E R

Effect of interaction of glutathione S-transferases (T1 and M1) on the hematologic and cytogenetic responses in chronic myeloid leukemia patients treated with imatinib

Y. Kassogue^•M. Quachouh^•H. Dehbi^• A. Quessar^• S. Benchekroun^•S. Nadifi

Received: 13 March 2014 / Accepted: 22 May 2014 ÓSpringer Science+Business Media New York 2014

Abstract The glutathioneS-transferases (GSTs) are phase II xenobiotic metabolizing enzymes known to be involved in the detoxification of carcinogens and anticancer drugs.

Individual genetic variation linked to inherited polymorphisms ofGSTT1andGSTM1leading to a complete loss of enzyme activity could expose subjects to develop cancer or to induce drug resistance. Indeed, despite the impressive results obtained with the imatinib, some patients with chronic myeloid leukemia (CML) fail to achieve the expected results or develop resistance. The present study aimed to examine the impact ofGSTT1andGSTM1poly- morphisms on the response to imatinib in patients with CML.

Multiplex polymerase chain reaction was used to detect the genotypes ofGSTT1andGSTM1in 60 CML patients. We found that side effects were more frequent in patients car- ryingGSTT1null when compared toGSTT1present carriers (31 vs. 16.6 %; v²=6.2; p=0.013). The loss of

hematologic response was statistically greater in patients carrying the combined genotype GSTT1 present/GSTM1 present (26.3 %) when compared to GSTT1 null/GSTM1 present (12.8 %), GSTT1present/GSTM1 null (8.3 %) and GSTT1 null/GSTM1 null (0 %), (v²=18.85; p\0.001).

The complete cytogenetic response was higher in patients harboring the GSTT1 null/GSTM1 null (75 %) compared withGSTT1null/GSTM1present (55.6 %),GSTT1present/

GSTM1 null (50 %) and GSTT1 present/GSTM1 present (47.8). On the other hand, the frequency of none cytogenetic responders was more common in patients carryingGSTT1 present/GSTM1present (34.8 %) when compared to other genotype combinations (v²=20.99;p =0.05). Moreover, theGSTT1present/GSTM1present appeared to be associated with a final dose of 600 or 800 mg of imatinib, but not significantly. Based on these findings, we find that the interaction between GSTT1 and GSTM1 seems to influence treatment outcome in patients with CML. Therefore, further investigations are required to confirm these results, for better genotype–phenotype correlation.

Keywords GSTT1GSTM1 Chronic myeloid leukemiaImatinibDrug response

Introduction

Chronic Myeloid leukemia (CML), characterized by a malignant proliferation of cells from the granulocytic lineage, is a consequence of the reciprocal translocation t (9; 22) (q34; q11) or the BCR-ABL fusion gene at the molecular level. CML represents approximately 15-20 % of leukemia in adults [1, 2]. It is noteworthy that the introduction of imatinib mesylate (Gleevec), a tyrosine kinase inhibitor targeting the BCR-ABL fusion in CML treatment, Y. KassogueH. DehbiS. Nadifi (&)

Genetics and Molecular Pathology Laboratory, Medical School of Casablanca, University Hassan II, 19 Rue Tarik Ibnou Ziad, BP. 9154, Casablanca, Morocco

e-mail: nadifi@labgenmed.com Y. Kassogue

e-mail: kassoy2@yahoo.fr H. Dehbi

e-mail: hinddehbi@gmail.com

M. QuachouhA. QuessarS. Benchekroun

Department of Onco-Hematology, Ibn Rochd University Hospital, Casablanca, Morocco

e-mail: qachouhmeryem@gmail.com A. Quessar

e-mail: quessar.a@gmail.com S. Benchekroun

e-mail: medredaz@gmail.com DOI 10.1007/s12032-014-0047-z

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has improved the quality of life of patients, reducing the response time, toxicity, morbidity and mortality associated with the disease [3,4]. However, despite these commend- able results achieved with imatinib, some patients fail to reach adequate hematologic, cytogenetic and molecular responses. It has been reported that mutations or amplification of BCR-ABL kinase domain [5–7] or variation in the bioavailability of imatinib linked to inherited genetic differences of the enzymes involved in imatinib metabolism could explain interindividual differences in the treatment outcome [8,9]. Indeed, it is well established that the highly polymorphic cytochrome P450 enzymes particularly CYP3A4 plays a non-negligible role in the metabolism of imatinib with other isoenzymes as CYP1A2, CYP2D6, CYP2C9 and CYP2C19 [10]. Furthermore, other studies have shown a correlation between polymorphisms in the multidrug resistance gene (MDR1) and imatinib response [11, 12]. It was also reported that polymorphisms in the super family of glutathione S-transferases (GSTs) gene were associated with treatment outcome in patients receiving imatinib [13–15]. GSTs are phase II xenobiotic metabolizing enzymes, which participate in the transfor- mation and activation of a wide range of substrates including carcinogens, pesticides, anticancer drugs and harmful compounds from the environment by conjugating them to soluble glutathione [16]. GSTs are composed of microsomal and cytosolic or soluble GSTs; Cytosolic GST includes eight classes divided intoa(GSTA),l(GSTM),h (GSTT),p(GSTP),f(GSTZ),r(GSTS),j(GSTK) andx (GSTO) [17].GSTM1andGSTT1 belong to the class mu and theta and are located on chromosomes 1p13.3 and 22q11.2, respectively [18,19]. The detoxifying activity of GSTs enzymes protects cells from the adverse effects of xenobiotics, but can reduce the effectiveness of drugs in cancer cells, resulting in toxicity or drug resistance [20,21].

Deletions ofGSTM1andGSTT1gene leading to a complete absence of enzyme activity have been found to be associated with a better outcome in the treatment of some cancers [22,23], but other studies showed no relationship [24,25].

Considering the few data on the pharmacogenetic aspects of CML in relation to the polymorphisms in GSTs genes, we carried out the present study with the aim to investigate the impact of the genotypes of GSTT1 and GSTM1 on the response to imatinib in CML patients.

Materials and Methods

Patients

In the present study, 60 CML patients with a median age of 40.5 years range (20-74 years) at the time of diagnosis have participated. The patients were monitored at the

department of Onco-Hematology of the Ibn Rochd Uni- versity Hospital in Casablanca, Morocco, from 2009 to 2013. All patients were in chronic phase and were con- firmed with the presence of the reciprocal translocation t (9; 22) (q34; q11). All patients started with 400 mg of imatinib per day; this dose was escalated to 600 or 800 mg per day for patients who did not reach complete hematologic response (CHR) at 3 months, major cytogenetic response (MCyR) at 6 months, or complete cytogenetic response (CCyR) at 12 months. Clinical and biological data were obtained from the data file of each patient.

Hematologic and cytogenetic responses have been described by Deininger et al. [26] and Kantarjian et al. [27]. The local Ethics Committee approved the study, and all par- ticipants agreed and signed the informed consent. Four milliliters of peripheral venous blood were collected in an EDTA tube from each patient and stored at minus 20°C until DNA extraction. The salting-out method was used to extract DNA from white blood cells [28] at the laboratory of genetic and molecular diseases.

Identification ofGSTM1andGSTT1polymorphisms A multiplex polymerase chain reaction (PCR), in which BCL2gene as an internal control was used to identify the genotypes of GSTM1andGSTT1. The PCR mixture con- sisted of 100 ng of genomic DNA, 1X of 5X GoTaq Flexi Buffer (Promega), 1.5 mM of MgCl2, 0.2 mM of each dNTP, 10 pM of each primer and 0.5 U of GoTaq polymerase (Promega) completed to 25ll with molecular grade water. The forward and reverse primers forGSTT1, GSTM1 and BCL2 were previously described [29]. PCR amplification was carried out with an initial denaturation at 94°C for 5 min and then followed by 35 cycles at 94°C for 1 min, 61°C for 1 min, 72°C for 1 min and a last extension at 72°C for 7 min. DNA fragments were ana- lyzed through a 2 % agarose gel stained with 0.5lg/mL ethidium bromide. Expected product’s sizes were 480, 219 and 154 bp for GSTT1, GSTM1 and BCL2, respectively.

The presence ofBCL2 withoutGSTT1or GSTM1 or both shows their deletion.

Statistical analysis

The effect of GSTM1 and GSTT1 genotypes on hematologic, cytogenetic responses, as well as the toxicity related to imatinib was estimated by chi-square test (v²) or Fisher’s exact test. The same test was used to measure the effects of the interaction between GSTT1 andGSTM1on the response to imatinib. A value of p\0.05 was con- sidered significant. The statistical package SPSS version 16 (SPSS Inc., Chicago, IL, USA) was used for the analysis.

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Results

In the present study, we examined the impact ofGSTT1and GSTM1deletions on the response to imatinib in 60 CML patients. The CHR at 3 months showed no particular trend between GSTT1 present and GSTT1 null genotypes (85.1 vs. 92.3 %;v²=0.46; p=0.5). TheGSTM1present and GSTM1 null genotype showed similar trends in terms of hematologic response (84.8 vs. 88.9 %; v²=0.21;

p=0.5) (Table1).

The effect of interaction of different genotype combinations betweenGSTT1 andGSTM1showed that the loss of hematologic response (HRL) in patients who have already reached CHR at 3 months was significantly more common in patients carrying the GSTT1 present/GSTM1 present (26.3 %) compared with GSTT1 null/GSTM1 present (12.8 %), GSTT1 present/GSTM1 null carriers (8.3 %) and GSTT1 null/GSTM1 null (0 %) (v²=18.85;

p\0.001) (Fig.1).

However, patients carrying the GSTT1 null genotype were more likely to develop side effects related to imatinib compared with patients harboring the GSTT1 present (31 vs. 16.6 %;v²=6.2;p=0.013) (Fig.2). In the contrast, the distribution of side effects were similar between GSTM1null andGSTM1 present carriers (18.8 vs. 20 %;

v²=0.06; p=0.8) (Fig.3).

As shown in Table1, the rate of CCyR was comparable betweenGSTT1 present andGSTT1null genotype carriers (48.9 vs. 53.8 %). A similar trend was observed between

GSTM1 present and GSTM1 null genotype carriers (48.5 vs. 55.6 %). However, the frequency of none cytogenetic responders (NCyR) was higher in patients carrying the GSTT1 present genotype compared with GSTT1 null carriers (23.5 vs. 7.7 %); similar results were observed in patients carrying GSTM1 present when compared to GSTM1null carriers (27.2 vs. 11.1 %).

The effect of interaction between the two genes showed that patients carrying the double deletion GSTT1 null/

GSTM1 null were found to be more cytogenetically sen- sitive to imatinib (75 %) followed byGSTT1null/GSTM1 present carriers (55.6 %), GSTT1 present/GSTM1 null (50 %) and GSTT1present/GSTM1present (47.8). On the Table 1 Demographic characteristics and hemato-cytogenetic responses of patients to imatinib according toGSTT1andGSTM1genotypes

Parameters GSTT1 GSTM1

Present N (%) Null N (%) Present N (%) Null N (%)

Sex

Female 30 (85.7) 5 (14.3) 19 (54.3) 16 (45.7)

Male 17 (68) 8 (32) 14 (56) 11 (44)

v²=2.69; df=1;p=0.093 v²=0.02; df=1;p=0.55 Hematologic response^a

Yes 40 (85.1) 12 (92.1) 28 (84.8) 24 (88.9)

No 7 (14.9) 1 (7.7) 5 (15.2) 3 (11.1)

v²=0.46; df=1;p=0.5 v²=0.21; df=1;p=0.5

Cytogenetic response^b

Complete 23 (48.9) 7 (53.8) 16 (48.5) 15 (55.6)

Partial 8 (17) 2 (15.4) 4 (12.2) 5 (18.5)

Minor 4 (8.5) 0 (0) 1 (3) 3 (11.1)

Minimal 1 (2.1) 3 (23.1) 3 (9.1) 1 (3.7)

None 11 (23.5) 1 (7.7) 9 (27.2) 3 (11.1)

v²=9.13; df=4;p=0.058 v²=4.65; df=4;p=0.32

a Hematologic response at 3 months,^bcytogenetic response at 18 months

Fig. 1 Distribution of the loss of hematologic response (HRL) in patients, based on different genotype combinations. (Gray means absence of HRL;blackmeans the presence of HRL)

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other hand, the frequency of NCyR to imatinib was higher in patients carrying the GSTT1 present/GSTM1 present genotype (34.8 %) (Table2).

As shown in Fig.4, approximately half of patients carrying both functional genotypes of GSTT1/GSTM1 have continued with a final dose of 600 mg of imatinib. In addition, the increase of imatinib dose to 600 mg was twice as high among patients with both functional genotypes of GSTT1 and GSTM1 compared with other genotype combinations. Surprisingly, the dose of 800 mg was observed only in patients carrying at least theGSTT1present in the combination.

Discussion

The GSTs enzymes play an important role in the detoxification, which protects cells from the toxic effects of xenobiotic. Indeed, it is well known that the polymorphisms in GST enzymes are often associated with a decrease or complete absence of enzymatic activity [30].

Such variability in gene expression linked to inherited genetic differences might influence individual susceptibil- ity to diseases and the response to drugs. It was also reported that overexpression of GSTs enzymes could cause chemoresistance due to the rapid elimination of cancer drugs, resulting in an inadequate therapeutic response [31].

In this study, no significant difference was observed when examining hematologic response for GSTT1 and GSTM1. However, the interaction of GSTT1 and GSTM1 have demonstrated that HRL was more frequent in patients carrying the GSTT1 present/GSTM1 present genotype, while the double deletion GSTT1 null/GSTM1 null appeared to protect patients from HRL, but not significantly. Indeed, this finding may be explained by possible

rapid elimination of imatinib in patients harboringGSTT1 present/GSTM1present genotype, but also by the low frequency of the double deletion in our patients. Stanulla et al.

[22] have reported that theGSTT1orGSTM1deletion was Fig. 2 Distribution of side effects related to imatinib in patients

harboring GSTT1 null or GSTT1 present genotypes. (Gray means absence of HRL;blackmeans the presence of HRL)

Fig. 3 Distribution of side effects related to imatinib in patients harboring GSTM1 null or GSTM1present genotypes. (Gray means absence of side effects;blackmeans the presence of side effects)

Table 2 Effect of the interaction of different genotype combinations between GSTT1 and GSTM1 on the cytogenetic response at 18 months

Response Null^a/ null^b

Null^a/ present^b

Present^a/ null^b

Present^a/ present^b

N (%) N (%) N (%) N (%)

Complete 3 (75) 5 (55.6) 12 (50) 11 (47.8)

Partial 1(25) 0 (0) 5 (20.8) 3 (13)

Minor 0 (0) 0 (0) 3 (12.5) 1 (4.4)

Minimal 0 (0) 3 (33.3) 1 (4.2) 0 (0)

None 0 (0) 1 (11.1) 3 (12.5) 8 (34.8)

v²=20.99; df=12;p=0.050

a GSTT1,^bGSTM1

Fig. 4 Variation of the final daily dose of imatinib based on different genotype combinations of genotypes. (Gray means 400 mg; black means 600 mg;Redmeans 800 mg)

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associated with a twofold reduction, in the relapse of acute lymphoblastic leukemia after remission.

The frequency ofGSTT1null genotype was statistically higher in patients who developed side effects when compared to patients carrying the GSTT1 present genotype (Fig.2). Mossallam et al. [32] have reported that toxic death appeared to be associated withGSTT1null carriers in patients with acute myeloid leukemia. In contrast, the GSTM1 genotypes showed no particular trend (Fig.1).

Therefore, we can hypothesize that the identification of GSTT1 genotype can predict side effects in patients receiving imatinib.

We found that the rate of CCyR was greater in patients harboring GSTT1 null genotype compared with GSTT1 present carriers, as well as between GSTM1 null and GSTM1present genotypes, but not significantly. This rel- ative difference in the therapeutic response might be explained by the fact that patients carrying the functional genotype of GSTT1 or GSTM1 seem to have a shorter plasma half-life in the metabolism of imatinib compared with subjects with the deleted gene. Moreover, this finding is supported by the relatively high frequency of NCyR in subjects carrying the functional genotypes of GSTT1 or GSTM1(Table1).

The interaction between GSTT1 and GSTM1 revealed that patients bearing the double deletion of GSTT1 and GSTM1genes are more likely to achieve CCyR, unlike to patients carrying both genes at the functional state. It is noteworthy that HRL was also greater in this group of patients. Therefore, we can suggest that a short plasma half-life of imatinib might be associated with the presence of functional genotypes ofGSTT1andGSTM1(Table 2). It was also reported that overexpression of GSTs might expose patients to chemoresistance [21].

Considering the final daily dose of imatinib adminis- tered, we observed that patients bearing the combined GSTT1 present/GSTM1 present genotype were more can- didates at a final dose of 600 mg. Furthermore, theGSTT1 present/GSTM1present genotype appears to be associated with a final dose of 600 or 800 mg. Therefore, we can speculate that patients harboring the combined GSTT1 present/GSTM1present genotype might eliminate imatinib faster than other combinations, which could explain the observed resistance.

Conclusion

We looked the impact of GSTT1 and GSTM1 polymorphisms of glutathione S-transferase on CML patients receiving imatinib. It appears from the present study that patients carrying the GSTT1 present/GSTM1 present genotype are more prone to HRL. We noticed that the side

effects were more common in patients with GSTT1 null genotype. Furthermore, the frequency of CCyR was higher in patients with GSTT1 null/GSTM1null genotype. How- ever, the rate of NCyR was higher in patients harboring GSTT1 present/GSTM1 present genotype. Therefore, polymorphisms of GSTT1 and GSTM1may influence the clinical and cytogenetic outcome of patients treated with imatinib. Considering the sample size, additional studies are required to confirm our findings.

Acknowledgments The authors thank the Hassan II Academy of Science and Technology for the financial support.

Conflict of interest None.

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