|Year : 2019 | Volume
| Issue : 1 | Page : 6-13
CYP2B6 polymorphism and lipoprotein lipase expression in chronic lymphocytic leukemia: impact on the outcome of fludarabine–cyclophosphamide regimen
Ahmed M.L Bedewy MD 1, Waleed R El-Bendary2
1 Hematology Department, Medical Research Institute, Alexandria University, Alexandria, Egypt
2 Clinical Pathology Department, Military Medical Academy, Cairo, Egypt
|Date of Submission||31-Aug-2018|
|Date of Acceptance||20-Mar-2019|
|Date of Web Publication||27-Sep-2019|
Ahmed M.L Bedewy
Medical Research Institute, Alexandria University, Abraj Al-Shaker, Zaky Ragab Street, Smouha, Alexandria, 21615
Source of Support: None, Conflict of Interest: None
Background Chronic lymphocytic leukemia (CLL) is the most common type of leukemia and has a highly variable clinical course. Cyclophosphamide (CPA)-containing regimens are the standard of care for patients lacking the 17p deletion. CYP2B6 is a polymorphic cytochrome P450 isoform that converts CPA to its active form. Lipoprotein lipase (LPL) catalyzes the hydrolysis of triacylglycerol. Remarkably, a growing body of data emphasizes its role in the biology of different tumors.
Objectives This study aimed to study CYP2B6 polymorphism and LPL expression in fludarabine cyclophosphamide (FC)-treated CLL patients lacking 17p deletion.
Methods 46 treatment-naïve CLL patients negative for 17p deletion and indicated to receive chemotherapy were enrolled. CYP2B6 genotyping and lipoprotein lipase mRNA expression were assayed by Realtime PCR. FC-protocol was given then treatment-related toxicities, response, and event free survival were traced.
Results CYP2B6*6 allele was associated with lower rates of treatment-related anemia and hospital admission. The response to FC was affected only by CYP2B6 polymorphism. The event-free survival of responders was significantly higher in patients having low LPL expression.
Conclusion CYP2B6*6 infers lower CPA efficacy with lower treatment-induced side effects and increased risk of nonresponding to FC chemotherapy in CLL. LPL expression is a predictor of outcome in CLL, indicating poor survival.
Keywords: chronic lymphocytic leukemia, cyclophosphamide, CYP2B6, lipoprotein lipase
|How to cite this article:|
Bedewy AM, El-Bendary WR. CYP2B6 polymorphism and lipoprotein lipase expression in chronic lymphocytic leukemia: impact on the outcome of fludarabine–cyclophosphamide regimen. Egypt J Haematol 2019;44:6-13
|How to cite this URL:|
Bedewy AM, El-Bendary WR. CYP2B6 polymorphism and lipoprotein lipase expression in chronic lymphocytic leukemia: impact on the outcome of fludarabine–cyclophosphamide regimen. Egypt J Haematol [serial online] 2019 [cited 2019 Oct 23];44:6-13. Available from: http://www.ehj.eg.net/text.asp?2019/44/1/6/268001
| Introduction|| |
Chronic lymphocytic leukemia (CLL) is a blood and bone marrow disease that usually gets worse slowly. CLL is one of the most common types of leukemia in adults. It often occurs during or after middle age; it rarely occurs in children ,.
CLL is a type of cancer in which the bone marrow makes too many lymphocytes. Although the majority of patients respond to initial therapy, the disease is incurable and runs a clinical course characterized by repeated recurrences culminating in drug resistance ,.
Growth kinetics, response to therapy, and propensity for disease acceleration and transformation vary considerably between individual patients, and a large number of biological variables have been identified that correlate with adverse outcomes. These include advanced age, male sex, germline configuration of the immunoglobulin heavy-chain gene, mutation or deletion of TP53, and deletion of chromosome 11q ,. TP53 defects are strongly associated with chemoresistance but affect only a minority of previously untreated patients. Consequently, chemotherapy remains the backbone of modern frontline therapy for most patients ,.
Although fludarabine/cyclophosphamide (FC) regimen is active in the majority of previously untreated patients with CLL, the complete response (CR) rate is only 23–38%, implying a degree of therapy resistance in a significant proportion of patients ,. Although it is clear that the efficacy of FC is influenced by disease-related factors such as TP53 mutation/deletion, such factors do not account for all patients who fail to achieve a CR. It is therefore relevant to consider other variables that might affect the pharmacokinetics or pharmacodynamics of the FC drug combination .
CYP2B6 is a polymorphic cytochrome P450 isoform that converts cyclophosphamide (CPA) to its active form. CPA is a prodrug that requires activation to its active metabolite, 4-hydroxycyclophosphamide (4OH-CPA), to exert its cytotoxic effects. 4OH-CPA, which readily diffuses into cells, is very unstable, and spontaneously decomposes into the alkylating agent phosphoramide mustard . Among the several enzymes that are thought to convert CPA to 4OH-CPA, one of the most important is the cytochrome P450 isoform CYP2B6, which is expressed mainly in the liver ,. CYP2B6 gene is highly polymorphic, with more than 100 known single-nucleotide polymorphisms (SNPs) currently identified in humans. CYP2B6 SNPs have distinct ethnic frequencies and some have been linked to alterations in CYP2B6 expression and function ,,. There is some circumstantial evidence to suggest that CYP2B6 SNPs might be clinically important. For example, they have been implicated as possible determinants of treatment outcome in patients receiving CPA for lymphoma and breast cancer or as myeloablative conditioning before hematopoietic stem cell transplantation, although definitive evidence is lacking ,.
Given the potential for functional interaction between individual SNPs, it is more meaningful to consider the genetic variants of CYP2B6 in terms of its 37 known alleles. Most of these alleles are rare but one (*6) is particularly common, with a frequency ranging from ∼25% in Whites to 60% in Asian populations .
CYP2B6*6 is characterized by the presence of two SNPs, c.516G>T in exon 4 (Q172H; rs3745274) and c.785A>G in exon 5 (K262R; rs2279343), and displays altered expression and function compared with the CYP2B6*1 wild-type allele. Specifically, *6 carriers express significantly reduced levels of CYP2B6 mRNA and protein ,. On the other hand, most reports have shown that the *6 allele is associated with a higher rate of CPA 4-hydroxylation ,,. The prediction of the overall impact of CYP2B6*6 expression on CPA pharmacokinetics and its therapeutic efficacy/toxicity is difficult to predict. The net outcome of the reduced enzyme expression on one side and the increased specific enzyme activity on the other side determine its effect .
Microarray studies identified distinct sets of genes expressed differentially in mutated versus unmutated CLL , and lipoprotein lipase (LPL) was one of the several genes being overexpressed in unmutated cases. On the basis of these preliminary observations, together with ZAP-70 and other molecular markers, the role of LPL in CLL prognosis assessment was investigated by different groups. These studies indicated that LPL correlates closely with the clinical course and survival ,. Physiologically, LPL is expressed in the muscle, adipose tissue, macrophages, but not in normal T and B lymphocytes . Bound to the capillary endothelium and released upon heparin, LPL catalyzes the hydrolysis of triacylglycerol of the circulating chylomicrons and very low density lipoproteins. Remarkably, a growing body of recent data indicates that lipid utilization and LPL function play an important role in the survival and metabolism of different tumors, including CLL ,.
In light of these considerations, we sought to investigate the effect of CYP2B6*6 polymorphism and LPL expression on the efficacy and toxicity of FC protocol in CLL.
| Patients and methods|| |
Forty-six patients were enrolled in this study. They were diagnosed to have CLL and were indicated to receive therapy according to the criteria. Exclusion criteria were: (a) age above 65 years, (b) presence of 17p deletion by FISH study, (c) any comorbidity not permitting FC regimen and (d) patients with unevaluable response to FC regimen.
Patients received FC protocol. Fludarabine (30 mg/m2/day intravenously) and CPA (250 mg/m2/day intravenously) were given on days 1–3 every 28 days, for a maximum of six courses. The first restaging was performed after three treatment courses. Therapy was continued only if a partial response or CR was noted at that time. A second and final restaging was performed after three additional courses of treatment . Treatment-related toxicities were traced in patients. These included treatment-related anemia, thrombocytopenia, neutropenia, gastrointestinal complications, febrile episodes, and hospital admission between cycles. Clinical response was defined according to the revised guidelines of the National Cancer Institute-sponsored workshop . The procedures followed were according to the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. Confidentiality of data was assured for all the patients.
The CYP2B6 516G>T polymorphism was genotyped using a commercially available TaqMan real-time PCR SNP genotyping assay (Applied Biosystems, Carlsbad, California, USA). CYP2B6 785A>G polymorphism was assayed by TaqMan SNP genotyping assay designed to specifically detect the CYP2B6 gene without amplification of its homologous pseudogene CYP2B7. The sequences of primers and probes were as follows: TGGAGAAGCACCGTGAAACC (forward), TGGAGCAGGTAGGTGTCGAT (reverse), VIC-CCCCCAAGGACCTCMGB (wild type), FAM-CCCCAGGGACCTC-MGB (mutant) .
RT PCR analysis of LPL mRNA peripheral blood samples of CLL patients and two healthy controls were used for total RNA isolation by TRIzol (Life Technologies; Carlsbad, CA, USA) extraction method followed by reverse transcription. Quantitative PCR was performed by LightCycler 480 real-time PCR system with SYBR-Green (Roche, Germany) detection format: LPL-F: 5′-CCGCCGACCAAAGAAGAGAT-3′, LPL-R: CAATGACATTGGAGTCTGGTTCTCTC-3′, ABL-4F: 5′-GGGCTCATCACCACGCTCCA-3′, ABL-6F: 5′-CTGCCG GTTGCACTCCCTCA-3′ .
The relative LPL to ABL1 expression level was calculated by the ΔCt method (ΔCt=CtLPL−CtABL1). LPL/ABL1 expression in patients were normalized to the expression found in healthy control samples using the ΔΔCt method after efficiency validation of the target and reference gene PCRs .
All statistical analyses were carried out using SPSS (version 20, SPSS Inc., Chicago, IL, USA). Categorical data were summarized using frequency counts and tested using the χ2 or Fisher’s exact test. Continuous data were summarized using mean and SD (for age of patients). Odds ratio was calculated for risk estimation with 95% confidence interval. Kaplan–Meier survival analysis was used for the estimation of event-free survival.
| Results|| |
Our results cleared that there was no significant association between CYP2B6 polymorphism and age, sex, Binet stage, B2MG, lactate dehydrogenase (LDH), treatment-related anemia, thrombocytopenia, neutropenia, febrile episodes, gastrointestinal complications, or LPL expression. No patients with variant CYP2B6 genotype (*1/*6 and *6/*6) required hospital admission for more than 3 days in between cycles while 31% of CYP2B6*1/*1 patients underwent such admission. This was a significant association between wild-type CYP2B6 and interim hospital admission (P=0.009). CYP2B6 variant genotype was associated with a lower chance of achieving response to FC protocol (P=0.035; [Table 1]).
LPL expression equal to or above the median value was considered high (n=23), while expression levels below the median value were considered low (n=23). LPL expression was not associated with age, sex, Binet stage, treatment-related anemia, thrombocytopenia, neutropenia, febrile episodes, interim hospital admission, gastrointestinal complications, or response to FC protocol. High LPL expression was associated with elevated serum LDH and B2MG levels (P=0.008 for both; [Table 2]). The type of response to FC protocol was not associated with any of the studied parameters except for the CYP2B6 genotype where patients with the wild-type *1/1* have higher chances of achieving response to chemotherapy (P=0.035; [Table 3]). Having the *6 allele pertained a higher risk of failing to respond to FC protocol (odds ratio=4.375, 95% confidence interval: 1.046–18.265, P=0.042).
|Table 2 Lipoprotein lipase expression (high vs. low) in relation to the studied parameters|
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|Table 3 Response of the patients to fludarabine/cyclophosphamide protocol according to the studied parameters|
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Event-free survival of responders (35 patients) through a follow-up period that ranged from 14 to 28 months (median=20 months) was 62.9%. This was not affected by the CYP2B6 polymorphism (P=0.864; [Figure 1]). Event-free survival of patients with high LPL expression was only 42.1% compared with 87.5% event-free survival of patients with low LPL expression (P=0.048; [Figure 2]).
|Figure 1 Kaplan–Meier event-free survival chart according to the CYP2B6 polymorphism (P=0.864).|
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|Figure 2 Kaplan–Meier even-free survival chart according to lipoprotein lipase expression (P=0.048).|
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| Discussion|| |
In this study, we attempted to investigate the CYP2B6 polymorphism and LPL expression as possible determinants of therapeutic response to FC chemotherapy in CLL. The CYP2B6*6 variant was associated with higher FC treatment protocol-related toxicity as evidenced by hospital admission of more than three in between cycles. This finding was in concordance with the reports of Shu et al.  and Hofmann et al. . They showed that CYP2B6*6 (CYP2B6 516G>T, and 785A>G) encoded enzymes with low metabolic capability in the CPA 4-hydroxylation reaction in vivo and in vitro. Patients with CYP2B6 516T had a lower risk of grade 2–4 toxicities and that this SNP was an indicator of CYP2B6*6. The in-vitro findings of Shu et al.  confirmed that the *6 allele confers lower mRNA expression and slower CPA 4-hydroxylation, indicating that its carriers are poor CPA metabolizers.
The study of Johnson et al.  reported concordant findings with less frequent FC-related adverse events and fewer in-hospital days among *6 carriers. In this report, CYP2B6*6 allele represented a risk of unresponsiveness to the FC protocol. The above-mentioned important report by Johnson and his colleagues described consistent findings with a lower chance of achieving remission in patients having the *6 allele. Reports discussing the impact of CYP2B6*6 on CPA exist in the literature covering lymphoma, myeloma, breast cancer, and stem-cell transplantation ,. However, to our knowledge, apart from the study of Johnson et al. , no other reports studied it in CLL patients.
In this study, high LPL expression was associated with elevated serum LDH and B2MG levels. It was not associated with any other studied demographic or prognostic marker. However, its expression negatively affected the event-free survival. Literature review showed concordant reports ,,. LPL expression was believed to be a surrogate marker for the mutational status . These results agreed with those of Matrai et al.  who observed that LPL is a strong predictor of outcome in CLL, able to improve prognostic accuracy in good risk cytogenetic subgroups.
Lipid metabolism was thought to be crucially required for the sake of function and survival of CLL cells. This might elucidate why LPL expression is associated with progressive clinical course and short survival ,.
| Conclusion|| |
CYP2B6*6 infers lower CPA efficacy with lower treatment-induced side effects and increased risk of nonresponding to FC chemotherapy in CLL. LPL expression is a predictor of outcome in CLL, indicating poor survival. These findings represent CYP2B6 genotyping and LPL expression as attractive candidate markers for regular clinical use and a step toward personalized medicine to achieve the best treatment efficacy.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]