The Effect of CYP2C19*2 (rs4244285) and CYP17 (rs743572) SNPs on Adriamycin and Paclitaxel based Chemotherapy Outcomes in Breast Cancer Patients

Background: Cytochrome P450 (CYP) are phase I metabolizing enzymes involved in detoxification of chemotherapeutic agents. Among the CYP gene family, including CYP1A1, CYP1B1, CYP2C, CYP2D, CYP2E and CYP17, their significance in cancer susceptibility is well established. However, there remains limited understanding regarding the polymorphisms of CYP2C19*2 and CYP17 and their potential correlation with chemotherapy-induced toxicity reactions in breast cancer (BC) patients. In this study we intended to identify the association of CYP2C19*2 and CYP17 gene polymorphisms on drug response as well as toxicity reactions in BC patients undergoing adriamycin/paclitaxel based chemotherapy within Indian population. Methods: Two hundred BC patients receiving adriamycin and paclitaxel chemotherapy were enrolled in this study and chemotherapy induced hematological and non-hematological toxicity reactions were noted. The polymorphisms of CYP2C19*2 (681G>A) and CYP17 (34T>C) isoforms of cytochrome p 450 gene was studied by PCR and RFLP analysis. Results: The univariate logistic regression analysis revealed significant associations between CYP2C19*2 (681 G>A) polymorphisms with hematological toxicities i.e., anemia (OR=9.77, 95% CI: 2.84-33.52; p=0.0003), neutropenia (OR=5.72, 95% CI: 1.75-18.68; p=0.003), febrile neutropenia (OR=4.29, 95% CI: 1.32-13.87; p=0.014) and thrombocytopenia (OR=5.86, 95% CI: 1.15-29.72); p=0.032) in BC patients. Additionally BC patients treated with adriamycin exhibited significant association between CYP2C19*2 polymorphism with chemotherapy induced nausea and vomiting (CINV) (OR=99.73, 95% CI: 5.70-174.64); p=0.001), fatigue (OR=83.29, 95% CI: 4.77-145.69); p=0.002), bodyache (OR=4.44, 95% CI: 1.24-15.91); p=0.021) and peripheral neuropathy (OR=12.00, 95% CI: 1.80-79.89); p=0.010. Furthermore, the regression analysis indicated an association between CYP17 with body ache (OR=2.77, 95% CI: 1.21-6.34; p=0.015) and peripheral neuropathy (OR=3.90, 95% CI: 1.59-9.53; p=0.002) in BC patients treated with paclitaxel chemotherapy. Conclusion: The findings obtained from this study illustrated significant association of CYP2C9*2 (681G>A) polymorphism with adreamicin based chemotherapy induced toxicities and CYP17 (34T>C) polymorphism with paclitaxel induced bodyache and peripheral neuropathy in BC patients.


Introduction
Breast cancer (BC) is the leading cause of cancer-related deaths among women, not only in developing countries but also in developed nations.The standard approach recommended for managing BC involves a combination of surgery followed by chemotherapy and radiotherapy.The systemic chemotherapy plays a significant role in BC treatment where combinations of anthracyclines, platinum agents and taxanes being widely adopted in the standard therapeutics of BC.Despite the

RESEARCH ARTICLE
The Effect of CYP2C19*2 (rs4244285) and CYP17 (rs743572) SNPs on Adriamycin and Paclitaxel based Chemotherapy Outcomes in Breast Cancer Patients development in standard chemotherapeutics, predicting treatment outcomes remains challenging due to deviations in patient to patient response towards chemotherapeutic drugs.While some patients can respond better to chemotherapy drugs, but others may experience toxic effects and adverse reactions.These diverse reactions can be attributed to genetic alterations in the drug metabolizing enzyme coding genes of patient.Hence, it is important to understand genetic diversity of each individual which determines the pharmacogenetic susceptibility of that individual.The inherited variations in drug metabolizing enzyme coding genes can determine the influence of chemotherapy drugs and impact of treatment outcomes [1].It is important to note that most of the chemotherapeutic drugs exhibit toxicity to normal cells potentially leading to acute or chronic reactions in cancer patients.These acute toxicity reactions are grouped into hematological (anemia, thrombocytopenia, neutropenia and febrile neutropenia) or Non-hematologic (nausea, vomiting, fatigue, peripheral neuropathy) [2,3].Although research have been carried out to explore the role of genetic variants of different pathway genes involved in drug transporters and drug metabolism, there remained a scope to discover the role of these genetic determinants in drug toxicity and response.
Phase I and phase II metabolizing enzymes are the key determinants of detoxification and elimination of chemotherapeutic agents, with cytochrome P450 (CYP) enzymes comprising the family of phase I metabolizers for most chemotherapeutic drugs.The CYP genes are highly polymorphic in nature where number of isozymes such as CYP1A1, CYP1B1, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP17 displaying functional polymorphisms and their importance in cancer susceptibility [4][5][6][7].The genetic polymorphisms of CYP genes play a crucial role in determining clinical outcomes related to treatment response and toxicity.These variant genotypes encoding metabolizing enzymes can alter the activity of drug metabolizing enzymes potentially leading to abnormal drug metabolism [8][9][10].It is also evident from earlier findings that polymorphisms of majority of CYP genes influence the clinical outcomes of chemotehepeutic drugs [11][12][13] and are associated with chemotherapy-induced toxicity reactions in BC patients [8].The genetic polymorphisms in CYP genes have significant implications for the breast carcinogenesis risk where CYP2C19 and CYP17 polymorphisms have a crucial role in hormone metabolism [14].The polymorphism of CYP2C19 is known to influence the therapeutic response towards taxane based chemotherapy [11]; however there remains a gap in our understanding regarding the therapeutic effects of doxorubicin and paclitaxel-based chemotherapy.The CYP17 polymorphism has been clinically correlated with treatment outcomes in response to hormone therapy in prostate cancer [15,16]; however there is no evidence on other chemotherapeutic drugs and their clinical outcome with other cancers.Similarly, much interest have been developed to address the functional polymorphisms of CYP17 with BC risk [17,18] but others have failed to prove such association [19][20][21] and produced conflicting results.Consequently, there is need to further investigate the clinical significance of these polymorphisms in BC susceptibility and therapeutic response.
To the best of our current understanding, there is lack of studies on the relevance of CYP gene polymorphisms in chemotherapy-related toxicities associated with Adriamycin/Paclitaxel treatment for BC or any other cancer in India.Therefore, we decided to explore potential association between variant genotypes of CYP2C19 and CYP17 genes and their influence on treatment efficacy and clinical outcomes among BC patients undergoing chemotherapy with Adriamycin and paclitaxel.To address this gap, we specifically investigated the polymorphisms of CYP2C19*2 (G>A, rs4244285) and CYP17 (T>C, rs743572), and examined their association with chemotherapy-induced toxicity reactions in breast cancer patients.

Study subjects and Clinical data
A total of 200 women (Mean age: 50.24±10.93;range: 27-78 years) with morphologicaly confirmed and histologically diagnosed BC were enrolled in this study.BC patients seeking treatment at the Department of Oncology in a tertiary care hospital (Krishna hospital and Medical Research Centre) were selected for participation.Patients were enrolled based on predefined inclusion and exclusion criteria.The inclusion criteria were; histopathology confirmation, patients diagnosed with BC planned for standard chemotherapy (Adriamycin and Paclitaxel).Patients without a pathological diagnosis, incomplete treatment, incomplete followup, comorbidities, or abnormal liver or renal function tests were excuuded from the study.Detailed clinicopathological and demographic features along with follow-up data were recorded.Among these patients, 104 were treated primarily with adriamycin followed by paclitaxel while 96 patients received paclitaxel first followed by Adriamycin.Chemotherapeutic effects were assessed after each chemotherapy cycle through blood testing and physical and visual examinations following the National Cancer Institute-Common Toxicity Criteria (NCI-CTC) [22].Informed consent was obtained from patients after explaining the purpose of their enrollment in the study.The study received ethics committee approval from Institutional Ethics Committee of Krihsna Institute of Medical Sciences before initiation.

Chemotherapy, Follow-up and Toxicity assessment
Once the patient was enrolled in the study, chemotherapy was planned based on the patient's stage and the reports of liver and renal function tests.Subsequently, patients received 4 cycles of combination chemotherapy with adriamycin and Cyclophsphamide, followed by 4 cycles of 3 weekly Paclitaxel.After completing the 1st cycle of chemotherapy in each schedule, patients were reevaluated between10th to 14th day after chemotherapy to assess chemotherapy-related toxicities.Patients were communicated about possible adverse effects and advised to report any serious side effects during scheduled follow-up.The details were meticulously recorded and graded according to NCI-CTC 4.03 criteria.During the course of chemotherapy, patients administered with chemotherapy were closely monitored for treatment response and acute toxicity.Routine blood and urine tests were conducted before each chemotherapy cycle to monitor overall health and detect chemotherapyinduced side effects.Hematological toxicities, including anemia, neutropenia, thrombocytopenia as well as nonhematological toxicities such as mucositis, chemotherapy induced nausea/vomiting (CINV), fatigue, bodyache, peripheral neuropathy carefully assessed and graded on a scale as 0, 1, 2, 3, 4.

Genotyping assays
Five milliliters (mL) of whole blood were collected from each patient using sterile EDTA containing vacutainer after obtaining informed consent.Genomic DNA extraction was carried out from the peripheral blood sample using HipurA®Blood genomic DNA miniprep purification kit.(Cat no.MB504-250PR) (HiMedia Laboratories) following the manufacturer's instructions.The polymorphisms of CYP2C19 and CYP17 was studied by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP).The PCR amplification for confirmation of polymorphisms was carried out separately in 20 micro liter (µL) reaction mixtures containing 1X PCR buffer 0.2 mM each dNTP, 10 picomole (pmol) of each primers (IDT technologies), 1U Taq DNApolymerase (GeNei, Merck Bioscience) and 100 nanogram (ng) of purified genomic DNA.The primer sequence used to amplify CYP2C19*2 (G681A) and CYP17 (T34C) along with the PCR amplification conditions are represented in Table 1.Thereafter, the amplified PCR products were subjected to restriction digestion using restriction enzymes with digestion conditions are detailed in Table 1.The resulting PCR products and restriction digestion reactions were checked by agarose gel electrophoresis in Tris-Acetate-EDTA (TAE) buffer thereafter stained with ethidium bromide (10 mg/mL) and visualized under UV-transilluminator and photographed in gel documentation system (BioRad Laboratories).

Statistical Analysis
A univariate logistic regression analysis was used to assess the association between CYP2C19*2 and CYP17 polymorphisms and the incidence of toxicity (0-1 grade vs. 2-4), expressing results as Odds Ratios (OR) and 95% confidence intervals (95% CIs).The OR was estimated to test whether any significant association existed between grade <1 toxicity caused by chemotherapy and the selected gene polymorphisms.Statistical significance was set at p < 0.05.All statistical analyses were carried out using SPSS (Version 21.0).Additionally, the association of each polymorphism with the severity of toxicities was compared to clinico-pathological and demographic information of the patients using chi-square test.

Genotype distribution of CYP2C19*2 (681 G>A) and CYP17 (34 T>C) polymorphisms and adriamycin/ paclitaxel based chemotherapy toxicity in BC patients
A total of two hundred BC patients seeking chemotherapy treatment were observed for the adverse toxicity reactions in response to standard Adriamycin followed by paclitaxel and vice versa.Out of these 200 patients, 104 were first administered Adriamycin followed by paclitaxel chemotherapy, while 96 patients received paclitaxel first followed by Adriamycin.Univariate logistic regression analysis was used to explore the association between polymorphisms of CYP2C19*2 (rs4244285) and CYP17 (rs743572) with chemotherapy-induced acute toxicity reactions in BC patients.The chemotherapy-

Association of CYP2C19*2 (G681A) and CYP17 (T34C) polymorphisms with demographic and clinic-pathological factors of BC patients
An association between genetic polymorphisms of metabolic CYP2C19*2 and CYP17 genes and the demographic and clinic-pathological characteristics of BC patients is presented in Table 6.When the demographic factors including age and body mass index of BC patients enrolled in this study are considered, the univariate logistic regression analysis showed significant negative association of variant genotype of CYP17 (T34C) with BMI >25 (OR=0.45,95% CI: 0.25-0.80;p=0.007) and there was no association of both CYP2C19*2 and CYP17 with age of BC patients.The results of analysis in present study found no association CYP2C19*2 and CYP17 polymorphisms with histopathological TNM grade > stage II whereas clinical TNM stage >2 showed significant association with CYP17 (T>C) polymorphism (OR=1.88,95% CI: 1.08-3.28;p=0.025).The univariate logistic regression analysis among genotype distribution of CYP2C19*2 (G>A) polymorphism showed significant association with ER/PR hormone receptor status of BC patients (OR=3.61,95% CI: 1.30-9.98;p=0.013) whereas the genotype distribution of CYP17 (T>C) was negatively associated (OR=0.55,95% CI: 0.31-0.98;p=0.044) with hormone receptors in BC patients.The genotype distribution of CYP2C19*2 polymorphism showed significant negative correlation (OR=024, 95% CI: 0.10-0.60;p=0.002) with Her2 receptor status in BC patients enrolled in this study.

Discussion
The chemotherapeutic approach is commonly used for BC management which can effectively kill cancer cells but it also impacts normal cells leading to toxicities or adverse effects in patients.During chemotherapy treatment, approximately 60 to 70% of the chemotherapy drugs are metabolized by cytochrome P450 enzymes, including CYP1A2, CYP1B1, CYP2B6, CYP2C9, CYP2C19, CYP3A4, and CYP3A5.Among these, CYP2C19 exhibits the highest activity.However, individual patient responses to different chemotherapy drugs carry due to diverse genetic susceptibility.Several pharmacogenomic studies have clarified that numerous metabolic pathways can influence inter-individual variability in response to chemotherapy drugs.The genetic polymorphisms of metabolic genes of individuals can determine the chemotherapy treatment outcomes, where variant alleles control the variability of drug efficacy.Genetic alterations in drug detoxification genes lead to decreased enzyme activity, which may affect the efficiency of chemotherapeutic agents and can cause apparent adverse reactions in patients.Acute toxicities are crucial, as they directly affect treatment outcomes.The CYP450 genes are highly polymorphic in nature, and common polymorphisms with a variety of isoforms, including CYP1A1, CYP1B1, CYP2C, CYP2D and CYP2E, have been associated with alterations inenzymatic activity.Genetic polymorphisms in drug metabolizing enzyme-coding CYP genes lead to inter-individual differences in drug responses, which affect chemotherapy treatment efficiency [23].CYP2C19 is a member of the CYP450 enzyme family and plays an important role in the metabolism and elimination of a diverse range of chemotherapeutic drugs [24][25][26].The CYP2C19*2 and CYP19*3 are the most commonly studied polymorphisms of CYP2C19.Several studies have reported an association of CYP2C19 polymorphisms and therapeutic outcomes in response to different chemotherapeutic agents.The association of the CYP2C19 polymorphism with hematological toxicities, including severe neutropenia, has been reported earlier in response to chemotherapeutic drugs in ovarian [27], breast [28], lung cancer [29] and non small cell lung carcinoma [30].However, CYP2C19 polymorphic variants were not assessed against Adriamycin and pclitaxel based-chemotherapy in BC patients.Very few studies have investigated the significance of CYP17 in chemotherapy associated adverse toxicities.The clinical significance of chemotherapy-induced vomiting was noted when correlated with polymorphism of CYP17 in North Indian women with BC [31].However, no significant association of CYP17 polymorphism was noted in prostate cancer patients in response to chemotherapy [32].Accordingly, for the first time, we analyzed and correlated the association of CYP17 and CYP2C19 polymorphisms with adverse reactions in BC patients treated with Adriamycin /paclitaxel chemotherapy.The results of the present study indicated that the polymorphisms of drug metabolizing enzyme coding CYP2C19 gene was significantly correlated with induction of chemotherapy induced toxicities in BC patients treated with Adreamicin treated chemotherapy.The logistic regression analysis signified that the variant heterozygous genotype of CYP2C19*2 independently showed an association with hematological toxicity response against Adryamycin-based chemotherapy in BC patients.The frequency distribution of CYP2C19*2 genotypes showed a significant deviation from Hardy-Weinberg equilibrium in BC patients treated with Adriamycin, which was in agreement with other investigated polymorphisms [12].A significant association of CYP2C19*2 was detected with Adriamycin-based chemotherapy resistance when analyzed for hematological and non-hematological toxicities.The CYP2C19*2 polymorphism was significantly associated with anemia (p=0.0003),neutropenia (p=0.003) and febrile neutropenia (p=0.014)toxicities in BC patients administered with adriamycin chemotherapy.The polymorphism of CYP17 did not show association with any of hematological adverse effects in BC patients treated with either Adreamicin or paclitaxel.The univariate regression analysis of CYP17 polymorphisms showed a significant association with body-ache (OR=2.77,95% CI: In conclusion, the findings from this study highlight significant association of CYP2C9*2 with hematological and non-hematological toxicities induced by Adriamycinbased chemotherapy in BC patients within selected population.The CYP2C19*2 polymorphic variant genotype demonstrated significant association with anemia, neutropenia and thrombocytopenia in response to Adriamycin.Additionally, the CYP17 polymorphism showed significant correlation with bodyache and peripheral neuropathy in response to paclitaxel-based chemotherapy.Notably, this study represents the first of its kind to analyze the influence of Adriamycin based chemotherapy on metabolic gene polymorphisms in BC patients.

Table 1 .
The List of Candidate ABCB Genes Selected in the Present Study with Details of PCR and RFLP Procedures Including Primers and Restriction Enzymes and Expected Products of Selected Ggenes.

Table 3 .
Univariate Analysis of Candidate SNPs of Cytochrome P450 (CYP2C1982, CYP17) Gene and Risk of Adriamycin Chemotherapy Induced Severe Toxicity of Non-Hematological Reactions in Breast Cancer Patients.

Table 4 .
Univariate Analysis of Candidate SNPs of Cytochrome P450 (CYP2C19*2, CYP17) Gene and Risk of Paclitaxel Chemotherapy Induced Severe Toxicity of Hematological Reactions in Breast Cancer Patients SNP, Single nucleotide polymorphism; OR, Odds ratio; CI, Confidence interval; Significance p< 0.05; *, Indicates significant Odds Ratio (p<0.05),p value determined based on χ2.

Table 5 .
Univariate Analysis of Candidate SNPs of Cytochrome P450 (CYP2C19*2, CYP17) Gene and Risk of Paclitaxel Chemotherapy Induced Severe Toxicity of Non-Hematological Reactions in Breast Cancer Patients

Table 6 .
Polymorphisms of CYP2C19*2, CYP17 Genes and Their association with Demographic and Clinic-Pathological Characteristics of Breast Cancer Patients OR, Odds ratio; CI, Confidence interval; Significance p< 0.05; *, Indicates significant Odds Ratio (p<0.05);pvalue determined based on χ2paclitaxel-based chemotherapy.A significant association between the CYP17 variant genotypes and potential tumor related characteristics of BC patients including TNM grade > stage II (OR=1.88,95% CI: 1.08-3.28;p=0.025) was noted in this study.Also, significant association of CYP2C19*2 with hormone receptor status was observed in the studied BC patients.