Abstract
Breast cancer is the most common cancer in women worldwide. Doxorubicin-based chemotherapy is used to treat breast cancer patients; however, neutropenia is a common hematologic side effect and can be life-threatening. The ABCB1 and SLC22A16 genes encode proteins that are essential for doxorubicin transport. In this study, we explored the effect of 2 common polymorphisms in ABCB1 (rs10276036 C/T) and SLC22A16 (rs12210538 A/G) on the development of grade 3/4 febrile neutropenia in Iranian breast cancer patients. Our results showed no significant association between these polymorphisms and grade 3/4 febrile neutropenia; however, allele C of ABCB1 (rs10276036 C/T) (p = 0.315, OR = 1.500, 95% CI = 0.679–3.312) and allele A of SLC22A16 (rs12210538 A/G) (p = 0.110, OR = 2.984, 95% CI = 0.743–11.988) tended to have a greater association with grade 3/4 febrile neutropenia, whereas allele T of ABCB1 (rs10276036) (p = 0.130, OR = 0.515, 95% CI = 0.217–1.223) and allele G of SLC22A16 (rs12210538) (p = 0.548, OR = 0.786, 95% CI = 0.358–1.726) tended to protect against this condition. In addition to breast cancer, a statistically significant association was also observed between the development of grade 3/4 febrile neutropenia and other clinical manifestations such as stage IIIC cancer (p = 0.037) and other diseases (p = 0.026). Our results indicate that evaluation of the risk of grade 3/4 neutropenia development and consideration of molecular and clinical findings may be of value when screening for high-risk breast cancer patients.
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Citation: Faraji A, Dehghan Manshadi HR, Mobaraki M, Zare M, Houshmand M (2016) Association of ABCB1 and SLC22A16 Gene Polymorphisms with Incidence of Doxorubicin-Induced Febrile Neutropenia: A Survey of Iranian Breast Cancer Patients. PLoS ONE 11(12): e0168519. https://doi.org/10.1371/journal.pone.0168519
Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute, UNITED STATES
Received: September 16, 2016; Accepted: December 1, 2016; Published: December 30, 2016
Copyright: © 2016 Faraji et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The authors received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Breast cancer is the most incident cancer type among women. Doxorubicin (DOX)-based treatments are appropriate for many adult and pediatric solid tumors (including breast cancer), leukemias and lymphomas [1]. However, optimized administration of DOX is hampered owing to some toxicities, such as hematopoietic suppression, nausea, vomiting, and cardiotoxicity [2].
DOX is a secondary metabolite produced by Streptomyces peucetius var. caesius and belongs to the family of anthracyclines [3]. DOX functions through dual mechanisms i) intercalation into DNA and disruption of the DNA repair mechanism that is mediated by topoisomerase II, and ii) releasing of free radicals resulting in the damaging of cell membranes, DNA, and proteins [4]. DOX is oxidized to a semiquinone, an unstable metabolite, which is reconverted to DOX through a pathway that releases reactive oxygen species; this can cause lipid peroxidation, membrane damage, DNA damage, oxidative stress, and cell death via induction of apoptotic pathways (Fig 1) [5].
ministration of DOX causes myelosuppression and leads to anemia, thrombocytopenia, and leukopenia. Neutropenia causes immune system suppression, exposing the patient to severe life-threating infections; it is the most serious hematologic toxicity associated with cancer chemotherapy, and its degree and duration determine the risk of infection. Prophylaxis with granulocyte-colony stimulating factor reduces the intensity and duration of chemotherapy-induced neutropenia and attenuates febrile neutropenia risk, and therefore plays a significant role in supporting myelosuppressive chemotherapy [6].
A number of factors have been shown to impact the response to DOX-based chemotherapy, including tumor stage, grade, the number of involved lymph nodes, and expression levels of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2) [7]. However, inter-patient variability has created a major obstacle for the clinical use of anticancer drugs [8]. Studies showed that these variations may be caused by differences in metabolizing enzymes and transporters associated with DOX. DOX is transported by the protein encoded by the ABCB1 gene [9–11] as well as the solute transporter encoded by SLC22A16 [12, 13]. ABCB1 (MDR1) belongs to the adenosine triphosphate binding cassette family genes [14], while SLC22A16 is a member of the organic cation transporter family [15]. Each of these transporter genes has been shown to carry genetic variations in the form of single nucleotide polymorphisms [16–18].
Since neutropenia is one of the adverse effects of DOX administration, the aim of this study was to investigate the possibility that ABCB1 (rs10276036 C/T) and SLC22A16 (rs12210538 A/G) gene polymorphisms play a role in the development of neutropenia in Iranian breast cancer patients treated with DOX-based chemotherapy.
Methodology
Patient information and neutropenia grading
In this case-controlled study, 100 women with breast cancer who were administered DOX-based neoadjuvant chemotherapy were selected as our study cohort. All patients were referred to the Department of Radiation Oncology of the 7-Tir Hospital in Tehran, and the presence of neutropenia was determined and graded based on their blood test report and according to the Common Terminology Criteria for Adverse Events Version 4.0. Two distinct groups were established.
The “case” group of 50 patients included those who were treated with DOX-based chemotherapy and had a neutrophil count ≤1.0 × 109/L and encompassed patients with grade 3/4 febrile neutropenia, and the “control” group (50 patients) included those who were treated with DOX-based chemotherapy and had a neutrophil count >1.0 × 109/L (patients with neutropenia ≤ grade 2).
Blood collection and DNA extraction
After approval of the study by the ethics committee of the National Institute of Genetic Engineering and Biotechnology (approval code IR.NIGEB.EC.1395.5.30.A) and obtaining written informed consent from all patients, 2–3 mL of whole blood was collected into tubes containing EDTA from each; samples were stored at 4°C. Genomic DNA was extracted by using the GPP Solution Kit (Gene Pajoohan Pouya Co., Iran). Extracted DNA samples were separated on 1% agarose gels to check their quality, and were then quantified by using the UV-Vis spectrophotometer (NanoDrop 2000, Thermo Scientific, USA).
Primer design, genotyping, and DNA sequencing
The amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method was used for genotyping the ABCB1 (rs10276036 C/T) and SLC22A16 (rs12210538 A/G) gene polymorphisms; the primers were designed using the WASP software (Genome Institute-BIOTEC, Thailand) and are shown in Table 1.
Primers were synthesized by Macrogen, Korea. The PCR program was as follows: 1 cycle: first denaturation at 95°C for 5 min; 35 cycles: denaturation at 95°C for 1 min, annealing at 59°C for 50 s, and extension at 72°C for 50 s; and 1 cycle: final extension at 72°C for 5 min. PCR reactions were prepared according to the standard protocols [19].
The PCR products were then separated by electrophoresis on 1.5% agarose gels at 80–100 V for 40–50 min. The gel was stained with ethidium bromide and visualized under ultraviolet light using a full high-definition camera (Canon SX710HS, Japan). To assess the accuracy of the ARMS-PCR primers, 3 PCR products representing a wild homozygote, heterozygote, and mutant homozygote of each of the studied polymorphisms were sequenced; results were analyzed by using the NCBI Blast software (Fig 2A–2H).
Fig 2A. Agarose gel image of the amplification refractory mutation system–PCR products of the ABCB1 gene polymorphism (rs10276036 C/T). 1: 50 bp DNA ladder, 2 & 3: heterozygote allele C/T, 4 & 5: homozygote allele C, 6 & 7: homozygote allele T, 8 & 9: negative control for alleles C and T respectively, Fig 2B. Electropherogram of the ABCB1 gene polymorphism (rs10276036 C/T) homozygote allele C, Fig 2C. Electropherogram of the ABCB1 gene polymorphism (rs10276036 C/T) homozygote allele T, Fig 2D. Electropherogram of the ABCB1 gene polymorphism (rs10276036 C/T) heterozygote allele C/T, Fig 2E. Agarose gel image of the amplification refractory mutation system-PCR products of the SLC22A16 gene polymorphism (rs12210538 A/G). 1: 50 bp DNA ladder, 2 & 3: heterozygote allele A/G, 4 & 5: homozygote allele A, 6 & 7: homozygote allele G, 8 & 9: negative control for alleles A and G respectively, Fig 2F. Electropherogram of the SLC22A16 gene polymorphism (rs12210538 A/G) homozygote allele A, Fig 2G. Electropherogram of the SLC22A16 gene polymorphism (rs12210538 A/G) homozygote allele G, Fig 2H. Electropherogram of the SLC22A16 gene polymorphism (rs12210538 A/G) heterozygote allele A/G.