CYP2D6 and CYP2E1 Gene Polymorphisms and Their Influence on Chemotherapy Treatment Outcome and Toxicity in Breast Cancer Patients

Abstract

Background:

The majority of breast cancer cases are treated with invasive chemotherapy, which typically involves combinations of drugs as part of the standard therapeutic regimen. However, responses to chemotherapeutic agents and treatment outcomes can vary significantly among patients, and the occurrence of acute toxicity remains unpredictable. Phase I drug detoxification genes, such as those encoding cytochrome P450 enzymes, play a crucial role in the metabolism of chemotherapeutic drugs in cancer patients. Therefore, this study aimed to investigate polymorphisms in the CYP2D6 and CYP2E1 genes and their potential association with adverse reactions to doxorubicin and paclitaxel-based chemotherapy in breast cancer patients.

Materials & Methods:

Genotyping of CYP2D6*3, CYP2D6*4, CYP2D6*10, CYP2D6*17, CYP2E1*5B, CYP2E1*6, CYP2E1*7B genes was performed among 200 breast cancer patients undergoing chemotherapy, using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The association between gene polymorphisms and chemotherapy-induced toxicity was assessed using odds ratios (ORs) with 95% confidence intervals and corresponding p-values, where p ≤ 0.05 was considered statistically significant.

Results:

Following the analysis of CYP2D6 and CYP2E1 gene polymorphisms, we observed that CYP2D6*4 (rs3892097, G>A) (OR = 4.71; 95% CI: 1.93–11.46; p = 0.0006), CYP2D6*10 (rs1065852, C>T) (OR = 3.43; 95% CI: 1.43–8.21; p = 0.005), and CYP2E1*6 (rs6413432, T>A) (OR = 4.00; 95% CI: 1.66–9.61; p = 0.001) showed a positive association with severe peripheral neuropathy induced by paclitaxel-based chemotherapy in breast cancer patients. Additionally, CYP2E1*6 (rs6413432, T>A) (OR = 4.04; 95% CI: 1.72–9.50; p = 0.001) was significantly associated with paclitaxel-induced body ache in a subset of breast cancer patients.

Conclusion:

The findings from this study conclude that polymorphisms in the CYP2D6 and CYP2E1 genes are associated with peripheral neuropathy, a non-hematological toxicity reaction, in breast cancer patients receiving paclitaxel-based chemotherapy.

Introduction

Breast cancer (BC) is the most common type of invasive cancer among women, accounting for approximately 12.5% of all newly diagnosed cancer cases worldwide. It is also the second leading cause of cancer-related mortality in women. Nearly 60% of breast cancer deaths occur in economically developing regions of South America and Asia, including China, Pakistan, and India. BC is a heterogeneous and multifactorial disease characterized by diverse cellular origins and multiple molecular subtypes, with genetic factors increasingly recognized for their role in disease development [1–4]. The commonly adopted therapeutic strategy for managing breast cancer (BC) involves a combination of surgery, radiotherapy, chemotherapy, and targeted therapy. Chemotherapy is an aggressive modality used in conjunction with surgery or radiotherapy to improve treatment outcomes. Most breast cancers are managed with systemic adjuvant and neoadjuvant chemotherapy, wherein combinations of agents such as anthracyclines (doxorubicin, epirubicin), platinum compounds (cisplatin, carboplatin), and taxanes (paclitaxel, docetaxel) constitute the foundation of standard therapeutic regimens [5–7]. These chemotherapeutic drugs are primarily detoxified in the liver, where cytochrome P450 enzymes metabolize them into less toxic forms, while the kidneys aid in filtering the resulting drug metabolites. However, this detoxification process can occasionally generate reactive byproducts, contributing to chemotherapy-induced toxicity reactions, which may manifest as adverse effects on various organs and physiological systems. In breast cancer patients, these acute toxicity reactions are broadly categorized into hematological toxicities, such as anemia, neutropenia, febrile neutropenia, and thrombocytopenia and non-hematological adverse events including mucositis, chemotherapy-induced nausea and vomiting, fatigue, body ache, and peripheral neuropathy [8–10]. However, treatment responses and outcomes cannot be generalized across all patients, as the incidence and severity of acute toxicity are highly variable and often unpredictable. Acute hematological toxicities have been reported in approximately 40–80% of BC patients receiving both neoadjuvant and adjuvant chemotherapy [11]. Overall, non-hematological adverse drug reactions associated with both chemotherapy regimens have been observed in more than 80% of patients [12–13].

The cytochrome P450 enzyme system constitutes a critical component of the phase I detoxification pathway, wherein multiple CYP gene isoforms including CYP1A1, CYP1B1, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP2E1 are involved in the metabolism of various xenobiotics and therapeutic agents. The CYP450 family is highly polymorphic, and functional genetic variations in these enzymes have been associated with cancer susceptibility [14–15]. Among these, CYP2D6 and CYP2E1 encoded by the CYP2D6 and CYP2E1 genes respectively, are key phase I enzymes essential for the biotransformation of xenobiotic compounds within the human body. Number of studies have explored the functional association between polymorphisms in the CYP2D6 and CYP2E1 genes and altered susceptibility to various cancers, including esophageal and colorectal cancer [16–17], and more recently, breast [18] and bladder cancer [19]. Although direct evidence linking CYP2D6 and CYP2E1 polymorphisms to breast cancer risk remains limited, their role in the metabolism of carcinogens and endogenous hormones suggests a potential influence on patient responses to chemotherapeutic agents. Thus, the selection of these polymorphisms for their potential association with doxorubicin and paclitaxel metabolism is justified in the context of BC risk prediction and toxicity outcomes. Previous studies have also reported an association between CYP2D6 polymorphisms and breast cancer risk in the South Indian population [20]. CYP2E1 is a key enzyme involved in the metabolism of various dietary procarcinogens, including nitrosamines, heterocyclic amines, and polycyclic aromatic hydrocarbons. Polymorphisms in this gene may lead to reduced enzymatic activity, impairing carcinogen detoxification and thereby influencing individual susceptibility to increased cancer risk. The impact of genetic polymorphisms in CYP2D6 and CYP2E1 genes on BC outcomes in response to doxorubicin- and paclitaxel-based chemotherapy remains uncertain in clinical settings. Therefore, this study aimed to identify and evaluate the association of CYP2D6*3 (rs35742686), CYP2D6*4 (rs3892097), CYP2D6*10 (rs1065852), CYP2D6*17 (rs28371706), CYP2E1*5B (rs2031920), CYP2E1*6 (rs6413432), and CYP2E1*7B (rs6413420) polymorphisms with chemotherapy-induced toxicity reactions in BC patients receiving treatment with doxorubicin and paclitaxel.

Materials and Methods

Patient enrollment and Clinical Information

Two hundred (200) clinically confirmed and histologically diagnosed BC patients visiting the Medical Oncology Outpatient Department (OPD) for treatment at the Department of Oncology, Krishna Hospital & Medical Research Center, Krishna Vishwa Vidyapeeth (Deemed to be University), Karad, were enrolled based on predefined inclusion and exclusion criteria. The sample size was calculated by the formula n= [(p1xq1) + (p2 x p2)] X (Z_1-α/2) +Z_1-β)2/ (p1-p2)² with 95%confidence and 95% power; Where p1- presence of allele1, q1- absence of allele1, p2- presence of allele 2, q2- absence of allele 2, α- probability of detecting false results and β- power. The Inclusion criteria are; patients with age range of 18 to 85 years age diagnosed with BC, histopathologically confirmed, no metastasis at diagnosis, clinically localised or locally advanced tumors according to standard staging system, patients diagnosed with BC who were planned to receive adjuvant chemotherapy postoperatively with standard doxorubicin and cyclophosphamide chemotherapy followed by paclitaxel chemotherapy, and locally advanced BC patients receiving neoadjuvant chemotherapy for downstaging. The exclusion criteria are; no pathological diagnosis, male BC Patients, relapsed disease or metastasis, associated severe co-morbidities, auto immune disease, no or incomplete treatment taken, incomplete follow-up, missing or incomplete data, patients with abnormal renal or liver function tests at the time of enrollment and patients with performance score of Eastern Cooperative Oncology Group (ECOG) ≥2. The study protocol was approved by Institutional Ethics Committee of Krishna Institute of Medical Sciences for the utilization of human subjects in the research (KIMSDU/IEC/01/2018 dated 2^nd February 2018)

Chemotherapy Treatment Regimen follow up and Toxicity Assessment

Once the patient was enrolled in the study after fulfilling inclusion and exclusion criteria,written informed consent was taken and chemotherapy was planned as per the stage of the patient. Patients received 4 cycles of combination chemotherapy with doxorubicin and cyclophosphamide , followed by 4 cycles of 3 weekly paclitaxel. After receiving 1st cycle of chemotherapy in each schedule, patient was followed again between Day10 to Day14 post-chemotherapy for assessing chemotherapy related toxicities. The BC patients treated with adjuvant and neo-adjuvant chemotherapy were followed up for 1year at the regular intervals for the assessment of treatment response and toxicity evaluation. Patient were explained regarding possible adverse effects and advised to report back in case of serious side effects or report during scheduled followup and details were noted and graded as per National Cancer Institute- Common Toxicity Criteria (NCI-CTC) 4.03 criteria [21]. The patients were routinely tested for blood and urine along with complete blood count, renal function and liver function test before each chemotherapy cycle to monitor health and to check chemotherapy induced side effects. The hematological toxicities including anemia, neutropenia, thrombocytopenia and non-hematological toxicities such as mucositis, chemotherapy induced nausea/vomiting (CINV), fatigue, body ache, peripheral neuropathy were graded as 0, 1, 2, 3, 4. Both the hematological and non-hematological toxicities were documented and evaluated for their association with genetic polymorphisms of CYP2D6 and CYP2E1 genes. For comparison of BC patients with toxicity reactions (>1 grade) were considered as chemo-sensitive groups were compared to patients with ≤ 1 grade reactions.

Blood Sample Collection and Genomic DNA Extraction and Purification

Five milliliter (mL) of whole blood from 200 patients was collected in sterile EDTA containing vacutainer after receiving informed consent. Genomic DNA extraction was carried out from the peripheral blood sample using HipurA®Blood genomic DNA miniprep purification kit (HiMedia Laboratories) following the manufacturer’s instructions. This pure genomic DNA was used for genotyping assays by polymerase chain reaction (PCR) and Restriction fragment Length Polymorphism (RFLP).

Genotyping assays of CYP2D6 and CYP2E genes

The genotyping of CYP2D6*3, CYP2D6*4, CYP2D6*10, CYP2D6*17, CYP2E1*5B, CYP2E1*6, CYP2E1*7B genes was performed by PCR-RFLP. The PCR amplification were 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 DNA polymerase (GeNei, Merck Bioscience) and 100 nanogram (ng) of purified genomic DNA. The primer sequence used to amplify the CYP2D6 and CYP2E1 genes and the PCR conditions are shown in Table 1.

Table 1.

The List of Candidate CYP2D6 and CYP2E1 Genes Selected in the Present Study with Details of PCR and RFLP Procedures Including Details of Primers and Their Sequences, Restriction Enzymes and Expected base Pairs of Expected PCR Products

Gene/ Genotype	rs number	Nucleotide change	Primer Sequence (Forward/Reverse)	PCR Conditions	PCR product size	Enzyme / Digestion conditions	Dominant Wild type (bp)	Heterozygous (bp)	Recessive Mutant (bp)
CYP2D6*3 2549delA	rs35742686	(A>- (del A)	FP: 5’-ATG AGC TGC TAA CTG AGC CC -3’ RP: 5’- CCG AGA GCA TAC TCG GGA C -3’	95°C for 5 min , 30 cycles of 95°C- 30 sec, 54°C- 45 sec, 72°C- 60 sec , final extension 72°C for 5 min	270 bp	1 Unit of MspI Incubation at 37˚C for 16h	188 bp, 82 bp	188 bp, 168 bp 20 bp	168 bp, 20 bp
CYP2D6*4 1846G>A	rs3892097	(G>A)	FP: 5’-GCT TCG CCA ACC ACT CCG-3’ RP: 5’-AAA TCC TGC TCT TCC GAG GC-3’	95°C for 5 min, 30 cycles of 95°C- 20 sec, 57°C- 20 sec, 72°C- 20 sec ,final extension 72°C for 5 min	334 bp	1 Unit of BstO1  Incubation at 37˚C for 1h	230 bp, 105 bp	334 bp, 230 bp 105 bp	334 bp
CYP2D6*10 100C>T	rs1065852	(C>T)	FP: 5’- GTG CTG AGA GTG TCC TGC C -3’ RP: 5’- CAC CCA CCA TCC ATG TTT GC -3’	95°C for 5 min, 30 cycles of 95°C- 60 sec, 53°C- 60 sec, 72°C- 60 sec, final extension 72°C for 5 min	344 bp	1 Unit of HphI Incubation at 37˚C for 16h	282 bp, 62 bp	282 bp, 182 bp, 100bp, 62bp	182 bp, 100 bp 62 bp
CYP2D6*17 1023C>T	rs28371706	(C>T)	FP: 5’- CGG TGG TCG TGC CTC AAT G -3’ RP: 5’- CCC GGG TCC CAC GGA AAT CT -3’	95°C for 5 min, 30 cycles of 95°C- 30 sec, 55°C- 45 sec, 72°C- 60 sec, final extension 72°C for 5 min	167 bp	1 Unit of HphI  Incubation at 37˚C for 1h	167 bp	167 bp, 88 bp 55bp	88 bp, 55 bp
CYP2E1*5B 1293G>C	rs2031920	(G>C)	FP: 5’-ACC CCA ATG GGT GTC TGT C-3’ RP: 5’-TCA TTC TGT CTT CTA ACT GGC AAT-3’	95°C for 5 min , 30 cycles of 95°C- 30 sec, 54°C- 45 sec, 72°C- 30 sec , final extension 72°C for 5 min	576 bp	1 Unit of PstI  Incubation at 37˚C for 1h	294 bp, 284 bp	576 bp, 294 bp 282 bp	576 bp
CYP2E1*6 7632T>A	rs6413432	(T>A)	FP: 5’-AGG CTC GTC AGT TCC TGA AA -3’ RP: 5’-AAG GCA GGA GGA TGA CTT GA -3’	95°C for 5 min, 30 cycles of 95°C- 20 sec, 62°C- 25 sec, 72°C- 20 sec, final extension 72°C for 5 min	685 bp	1 Unit of DraI Incubation at 37˚C for 1h	376 bp, 309 bp	685 bp, 376 bp 309 bp	685 bp
CYP2E1*7B 71G>T	rs6413420	(G>T)	FP: 5’-CTG GAG TTC CCC GTT GTC TA-3’ RP: 5’-GGG TGA AGG ACT TGG GAA TA -3’	95°C for 5 min, 30 cycles of 95°C- 30 sec, 54°C- 45 sec, 72°C- 30 sec, final extension 72°C for 5 min	547 bp	1 Unit of DdeI  Incubation at 37˚C for 1h	301 bp, 246 bp	547 bp, 301 bp 246 bp	547 bp

Restriction Fragment Length Polymorphism

After performing PCR programme for each reaction, the PCR products were analysed by agarose gel electrophoresis in Tris-Acetate-EDTA (TAE) buffer. After confirmation of DNA amplification, each PCR product was digested with an appropriate restriction enzyme with specific conditions for genotyping. Ten micro litters of the PCR products digested at 37°C overnight with specific restriction enzymes in 20 µL reaction mixtures containing buffer supplied with each restriction enzyme (Table 1). After the overnight incubation, digestion products were separated on a 2-3% low EEO agarose (GeNei) gel at 100 V for 30 min stained with ethidium bromide and photographed with gel documentation system (BioRad). The results obtained by PCR-RFLP analysis were further validated by direct DNA sequencing of amplified PCR products of some randomly selected representative samples which also confirmed the polymorphism in selected genes (Figure 1).

Figure 1.

Representative Chromatogram of Corresponding DNA Sequence Showing Nucleotide Change Position. (A) CYP2D6*4 is represented by G>A substitution at nucleotide 1846 of exon-4. (a) A sample with Normal ‘G’ allele (b) Sample with heterozygous G/A genotype and (c) Sample with homozygous variant A/A genotype for CYP2D6*4 (rs3892097) polymorphism (B) CYP2E1*7B polymorphism with G>T substitution of SNP rs6413420 (a) A sample with normal ‘G/G’ genotype. (b) A sample with major ‘G/T’ genotype in 71 G>T and (c) sample with homozygous variant (T/T) genotype of CYP2E1*7B (rs6413420) polymorphism.

Statistical Analysis

A univariate logistic regression model was employed to assess the effect of genetic polymorphisms in the CYP2D6 and CYP2E1 genes on the incidence of chemotherapy-induced toxicity (Grade 0–1 vs. Grade 2–4). Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated using unconditional multiple logistic regression. Clinical severity of post-chemotherapy adverse effects was classified into hematological and non-hematological toxicity reactions, with Grade >1 considered clinically significant. Multivariate analysis was conducted to adjust for demographic and clinicopathological variables influencing chemotherapy toxicity. Associations between each polymorphism and the severity of toxicities were further evaluated using chi-square tests. p-values < 0.05 were regarded as statistically significant. Genotype frequency data were assessed for Hardy–Weinberg equilibrium using SPSS version 11.0 software.

Results

A total of 200 clinically confirmed BC patients were enrolled in this study, with ages ranging from 18 to 85 years and a mean age of 50.24 years. Among them, 157 patients (78.5%) were above 40 years of age. Based on body mass index (BMI), 122 women (61%) had a BMI ≤ 25, while 78 women had a BMI > 25. Regarding menopausal status, 70 patients (35%) were classified as premenopausal and 130 patients (65%) as postmenopausal for the evaluation of treatment outcomes and other clinical parameters. Of the cohort, 155 women received adjuvant chemotherapy, 31 patients underwent neoadjuvant chemotherapy, and 14 patients were administered palliative chemotherapy. Hormone receptor status, assessed via immunohistochemistry, revealed 83 patients to be ER/PR positive, 109 patients to be ER/PR negative, and 85 patients exhibited triple-negative breast cancer. Tumor size exceeded 2 cm in 95 patients (range: 2–10 cm), while 105 patients (52.5%) presented with tumors ≤ 2 cm. Clinically, 98 patients were staged at TNM stages III and IV, and histopathologically, 110 patients were classified at TNM stages III and IV (Table 2).

Table 2.

Details of Demographic and Clinico-Pathological Characteristics of Breast Cancer Patients Enrolled in the Study

Variables	Number	Percentage (%)
Total Number of patients	200
Age (Mean ± SD) years	50.24 ±10.93	(Range:18-85) Median:48
≤ 50	118	59
>50	82	41
BMI Kg/m2
<25	122	61
25-30	62	31
>30	16	8
Tobacco smoking Status
Tobacco users	109	54.5
Tobacco no users	91	45.5
Family history of Cancer
Yes	50	25
No	150	75
Tumor localization
Left breast	102	51
Right breast	98	49
Tumor size in cm
≤ 2	105	52.5
> 2	95	47.5
Histological Grade
I, II	107	53.5
III, IV	93	46.5
Clinical TNM Stage
I	4	2
II	98	49
III	80	40
IV	18	9
Histopathological TNM Stage
I	2	1
II	88	44
III	90	45
IV	20	10
Hormone Receptor Status
ER/ PR+ve	83	41.5
ER/ PR-ve	109	54.5
ER/PR/Her2+ve	6	3
ER/PR/Her2-ve	85	42.5
ER/ PR+ve Her2-ve	78	39
ER/ PR-ve Her2+ve	24	12
Chemotherapy
Adjuvant chemotherapy	155	77.5
Neo-Adjuvant chemotherapy	31	15.5
Palliative chemotherapy	14	7
Radiotherapy
Adjuvant RT	81	40.5
No Adjuvant RT	119	59.5

Genotype distribution of CYP2D6 and CYP2E1 gene polymorphisms and their association with doxorubicin based chemotherapy toxicity in BC patients

The polymorphism CYP2D6*3, CYP2D6*4, CYP2D6*10, CYP2D6*17, CYP2E1*5B, CYP2E1*6, and CYP2E1*7B genes were investigated in this study. Out of 200 BC patients, 104 patients were first administered with doxorubicin followed by paclitaxel chemotherapy whereas 96 patients were first treated with paclitaxel and then doxorubicin. The chemotherapy induced acute toxicity reactions were grouped into hematological and non-hematological toxicities and graded into grade ≤1 or >1 toxicities. Chemotherapy-induced acute toxicity reactions were categorized into hematological and non-hematological toxicities and graded as ≤1 (mild) or >1 (severe). Hematological toxicities, including anemia, neutropenia, febrile neutropenia, and thrombocytopenia, were evaluated through blood testing. Non-hematological toxicities such as mucositis, CINV, fatigue, body ache, and peripheral neuropathy were documented through physical examination. Among 104 patients who received treatment with doxorubicin followed by paclitaxel, 23 patients exhibited severe anemia (Grade >1), 25 developed severe neutropenia, 24 experienced febrile neutropenia, and 7 presented with thrombocytopenia. Severe non-hematological toxicities (Grade >1) were recorded as follows: mucositis in 16 patients, CINV in 34 patients, fatigue in 37 patients, body ache in 15 patients, and peripheral neuropathy in 5 patients following doxorubicin administration. The univariate logistic regression analysis was used to find out an association of CYP2D6*3 (rs35742686, A>T), CYP2D6*4 (rs3892097, G>A), CYP2D6*10 (rs1065852, C>T), CYP2D6*17(rs28371706, C>T), CYP2E1*5B (rs2031920, G>C), CYP2E1*6 (rs6413432,T>A), and CYP2E1*7B (rs6413420, G>T) single nucleotide polymorphisms (SNPs) with standard doxorubicin based chemotherapy induced acute toxicity reactions in BC patients. In our investigation of the correlation between genetic polymorphisms in the CYP2D6 and CYP2E1 genes and severe hematological toxicity reactions in patients undergoing doxorubicin chemotherapy, the analysis revealed significant findings. The CYP2D6*10 polymorphism exhibited a negative association (OR = 0.32; 95% CI: 0.10–0.94; p = 0.039), suggesting a protective effect against febrile neutropenia in breast cancer patients. Similarly, CYP2E1*5B showed a significant negative association (OR = 0.05; 95% CI: 0.03–0.90; p = 0.04) with neutropenia in the same patient cohort (Table 3). Similarly, when we investigated the association between genetic polymorphisms in the CYP2D6 and CYP2E1 genes and non-hematological toxicity reactions including mucositis CINV, fatigue, body ache, and peripheral neuropathy in patients treated with doxorubicin, the analysis revealed no correlation for CYP2D6*3, CYP2D6*4, CYP2D6*10, CYP2E1*5B, CYP2E1*6, and CYP2E1*7B polymorphisms. However, a significant association was observed between the CYP2D6*17 polymorphism and CINV in breast cancer patients receiving doxorubicin-based chemotherapy (OR = 2.58; 95% CI: 1.11–6.60; p = 0.026) (Table 4).

Table 3.

Univariate Analysis of Candidate SNPs of CYP2D6, CYP2E1 Gene and Risk of Doxorubicin Chemotherapy Induced Severe Toxicity of Hematological Reactions in Breast Cancer Patients

		Anemia				Neutropenia
Gene Name	Genotype	Grade≤1	Grade >1	OR (95% CI)	p	Grade ≤1	Grade >1	OR (95% CI)	p
SNP		(n=81)	(n=23)		value	(n=79)	(n=25)		value
CYP2D6*3	A/A	49	14	1 (Reference)		48	15	1 (Reference)
rs35742686	A/- + -/-	32	9	0.98 (0.38-2.54)	0.974	31	10	1.03 (0.41-2.58)	0.946
CYP2D6*4	G/G	51	13	1 (Reference)		51	13	1 (Reference)
rs3892097	G/A+A/A	30	10	1.80 (0.72-4.51)	0.203	28	12	1.68 (0.67-4.17)	0.263
CYP2D6*10	C/C	47	16	1 (Reference)		46	17	1 (Reference)
rs1065852	C/T+T/T	34	7	0.60 (0.22-1.63)	0.32	33	8	0.65 (0.25-1.69)	0.385
CYP2D6*17	C/C	50	12	1 (Reference)		46	16	1 (Reference)
rs28371706	C/T+T/T	31	11	1.47 (0.58-3.75)	0.411	33	9	0.78 (0.30-1.98)	0.608
CYP2E1*5B	G/G	79	23	1 (Reference)		77	25	1 (Reference)
rs2031920	G/C+C/C	2	0	0.67 (0.03-14.59)	0.803	28	0	0.05 (0.03-0.90)	0.042*
CYP2E1*6	T/T	51	12	1 (Reference)		48	15	1 (Reference)
rs6413432	T/A+A/A	30	11	1.55 (0.61-3.96)	0.352	31	10	1.03 (0.41-2.58)	0.946
CYP2E1*7B	G/G	74	23	1 (Reference)		72	25	1 (Reference)
rs6413420	G/T+T/T	7	0	0.21 (0.01-3.84)	0.293	7	0	0.18 (0.01-3.43)	0.26
		Febrile Neutropenia				Thrombocytopenia
		Grade ≤1	Grade >1			Grade ≤1	Grade >1
		(n=80)	(n=24)			(n=97)	(n=7)
CYP2D6*3	A/A	45	18	1 (Reference)		58	5	1 (reference)
rs35742686	A/- + -/-	35	6	0.42 (0.15-1.19)	0.104	39	2	0.59 (0.10-3.22)	0.546
CYP2D6*4	G/G	53	10	1 (Reference)		61	3	1 (reference)
rs3892097	G/A+A/A	27	14	1.85 (0.73-4.67)	0.188	36	4	2.25 (0.47-10.67)	0.303
CYP2D6*10	C/C	44	19	1 (Reference)		59	4	1 (reference)
rs1065852	C/T+T/T	36	5	0.32 (0.10-0.94)	0.039*	38	3	1.16 (0.24-5.49)	0.847
CYP2D6*17	C/C	48	14	1 (Reference)		57	5	1 (reference)
rs28371706	C/T+T/T	32	10	1.07 (0.42-2.70)	0.884	40	2	0.57 (0.10-3.08)	0.514
CYP2E1*5B	G/G	78	24	1 (Reference)		95	7	1 (reference)
rs2031920	G/C+C/C	2	0	0.64 (0.02-13.80)	0.776	2	0	2.54 (0.11-58.02)	0.557
CYP2E1*6	T/T	49	14	1 (Reference)		60	3	1 (reference)
rs6413432	T/A+A/A	31	10	1.12 (0.44-2.85)	0.797	37	4	2.16 (0.45-10.20)	0.33
CYP2E1*7B	G/G	73	24	1 (Reference)		90	7	1 (reference)
rs6413420	G/T+T/T	7	0	0.20 (0.01-3.63)	0.276	7	0	0.80 (0.04-15.49)	0.885

SNP, Single nucleotide polymorphism; OR, Odds ratio; CI, Confidence interval; * indicates significance p< 0.05; p value determined based on χ².

Table 4.

Univariate Analysis of Candidate SNPs of CYP2D6, CYP2E1 Genes and Risk of Doxorubicin Chemotherapy Induced Severe Toxicity of Non- Hematological Reactionsin Breast Cancer Patients.

		Anemia				Neutropenia
Gene Name	Genotype	Grade ≤1	Grade >1	OR (95% CI)	p value	Grade ≤1	Grade >1	OR (95% CI)	p value
SNP		(n=88)	(n=16)			(n=70)	(n=34)
CYP2D6*3	A/A	53	10	1 (Reference)		41	22	1 (Reference)
rs35742686	A/- + -/-	35	6	0.90 (0.30-2.72)	0.864	29	12	0.77 (0.32-1.80)	0.548
CYP2D6*4	G/G	56	8	1 (Reference)		43	21	1 (Reference)
rs3892097	G/A+A/A	32	8	1.75 (0.59-5.11)	0.306	27	13	0.98 (0.42-2.28)	0.973
CYP2D6*10	C/C	56	7	1 (Reference)		45	18	1 (Reference)
rs1065852	C/T+T/T	32	9	2.25 (0.76-6.61)	0.14	25	16	1.60 (0.69-3.67)	0.268
CYP2D6*17	C/C	53	9	1 (Reference)		47	15	1 (Reference)
rs28371706	C/T+T/T	35	7	1.17 (0.40-3.45)	0.765	23	19	2.58 (1.11-6.00)	0.026*
CYP2E1*5B	G/G	86	16	1 (Reference)		69	33	1 (Reference)
rs2031920	G/C+C/C	2	0	1.04 (0.04-22.85)	0.976	1	1	2.09 (0.12-34.43)	0.606
CYP2E1*6	T/T	52	11	1 (Reference)		41	22	1 (Reference)
rs6413432	T/A+A/A	36	5	0.65 (0.21-2.05)	0.469	29	12	0.77 (0.32-1.80)	0.548
CYP2E1*7B	G/G	81	16	1 (Reference)		63	34	1 (Reference)
rs6413420	G/T+T/T	7	0	0.32 (0.01-6.05)	0.454	7	0	0.12 (0.06-2.21)	0.155
		Fatigue				Bodyache
		Grade ≤1	Grade >1			Grade ≤1	Grade >1
		(n=67)	(n=37)			(n=89)	(n=15)
CYP2D6*3	A/A	43	20	1 (Reference)		53	10	1 (Reference)
rs35742686	A/- + -/-	24	17	1.52 (0.67-3.44)	0.312	36	5	0.73 (0.23-2.33)	0.602
CYP2D6*4	G/G	42	22	1 (Reference)		57	7	1 (Reference)
rs3892097	G/A+A/A	25	15	1.14 (0.50-2.60)	0.746	32	8	2.03 (0.67-6.13)	0.206
CYP2D6*10	C/C	44	19	1 (Reference)		54	9	1 (Reference)
rs1065852	C/T+T/T	23	18	1.81 (0.79-4.10)	0.154	35	6	1.02 (0.33-3.14)	0.96
CYP2D6*17	C/C	37	25	1 (Reference)		54	8	1 (Reference)
rs28371706	C/T+T/T	30	12	0.59 (0.25-1.37)	0.221	35	7	1.35 (0.44-4.05)	0.592
CYP2E1*5B	G/G	65	37	1 (Reference)		87	15	1 (Reference)
rs2031920	G/C+C/C	2	0	0.34 (0.01-7.47)	0.509	2	0	1.12 (0.05-24.66)	0.938
CYP2E1*6	T/T	40	23	1 (Reference)		55	8	1 (Reference)
rs6413432	T/A+A/A	27	14	0.90 (0.39-2.05)	0.805)	34	7	1.41 (0.47-4.25)	0.536
CYP2E1*7B	G/G	62	35	1 (Reference)		82	15	1 (Reference)
rs6413420	G/T+T/T	5	2	0.70 (0.13-3.84)	0.689	7	0	0.35 (0.01-6.53)	0.485

SNP, Single nucleotide polymorphism; OR, Odds ratio; CI, Confidence interval; * indicates significance p< 0.05; p value determined based on χ².

Correlation of genetic variants of CYP2D6 and CYP2E1 gene polymorphisms and paclitaxel based chemotherapy toxicity in BC patients

When the genotype distribution of CYP2D6*3, CYP2D6*4, CYP2D6*10, CYP2D6*17, CYP2E1*5B, CYP2E1*6, and CYP2E1*7B was examined in patients treated with paclitaxel-based chemotherapy, the analysis revealed a significant association between the CYP2E1*7B (rs6413420, G>T) single nucleotide polymorphism (SNP) and hematological toxicity, specifically anemia, in breast cancer patients (OR = 342.00; 95% CI: 33.04–354.60; p < 0.0001). In contrast, no significant associations were observed between the other genotypes and anemia or other hematological toxicities (Supplementary Table 1). Similarly, in our investigation of CYP2D6 gene polymorphisms in relation to non-hematological acute toxicities induced by paclitaxel-based chemotherapy, we observed that the variant genotypes CYP2D6*4 (rs3892097, G>A) (OR = 4.71; 95% CI: 1.93–11.46; p = 0.0006) and CYP2D6*10 (rs1065852, C>T) (OR = 3.43; 95% CI: 1.43–8.21; p = 0.005) exhibited significant associations with peripheral neuropathy, a non-hematological toxicity reaction triggered by paclitaxel chemotherapy. Similarly, analysis of CYP2E1 polymorphism revealed a positive association between CYP2E1*6 (rs6413432, T>A) and paclitaxel-induced peripheral neuropathy in BC patients (OR = 4.00; 95% CI: 1.66–9.61; p = 0.001). Likewise, our findings indicated a significant association of the same polymorphism with paclitaxel-induced body ache in BC patients (OR = 4.04; 95% CI: 1.72–9.50; p = 0.001) (Supplementary Table 2).

Association of CYP2D6 and CYP2E1 polymorphisms with demographic and clinic-pathological factors of BC patients

Upon analyzing the correlation between genetic polymorphisms in the CYP2D6 and CYP2E1 genes and the demographic and clinicopathological characteristics of BC patients, no significant association was observed between any of the CYP2D6 or CYP2E1 genotypes and the evaluated demographic or clinicopathological features. The results pertaining to the polymorphisms CYP2D6*3 (rs35742686, A>–), CYP2D6*4 (rs3892097, G>A), CYP2D6*10 (rs1065852, C>T), CYP2D6*17 (rs28371706, C>T), CYP2E1*5B (rs2031920, G>C), CYP2E1*6 (rs6413432, T>A), and CYP2E1*7B (rs6413420, G>T), and their association with the demographic and clinicopathological characteristics of BC patients are presented in Supplementary Table 3. When demographic factors, including age and body mass index (BMI), were considered for the BC patients enrolled in this study, the analysis revealed no association between CYP2D6 or CYP2E1 variant genotypes and patients’ age or BMI. The present study also found no association between CYP2D6 SNPs (rs35742686, rs3892097, rs1065852, rs28371706) or CYP2E1 SNPs (rs2031920, rs6413432, rs6413420) and histopathological TNM stage > II. However, clinical TNM stage > II showed a significant association with the CYP2E1*7B (rs6413420, G>T) polymorphism (OR = 5.05; 95% CI: 1.06–24.02; p = 0.041). The analysis of genotype distributions for CYP2D6 and CYP2E1 polymorphisms revealed no significant association with hormonal receptor status (ER/PR or HER2) in BC patients. However, our data indicated a significant correlation between the CYP2E1*7B (rs6413420, G>T) SNP and select demographic and clinicopathological characteristics of BC patients examined in this study. Furthermore, in our evaluation of the relationship between demographic and clinicopathological factors and chemotherapy-induced toxicity reactions, we observed that a higher BMI was negatively correlated with non-hematological toxicities, specifically mucositis (p = 0.01) and body ache (p = 0.003), following doxorubicin treatment. Moreover, patients with clinical and histopathological TNM staging beyond stage II exhibited a significant association with chemotherapy-induced nausea and vomiting in response to doxorubicin, with p-values of 0.046 and 0.035, respectively. However, our study did not reveal any significant correlations between demographic or clinicopathological factors and outcomes associated with paclitaxel-based chemotherapy

Discussion

Chemotherapeutic drugs used in cancer treatment are recognized for their specific targeted mechanisms of action; however, a substantial gap persists in understanding the pharmacological mechanisms of many chemotherapy drugs. Consequently, it is essential to investigate the genetic diversity of individuals undergoing chemotherapy, as it significantly influences their susceptibility to both therapeutic efficacy and adverse drug reactions. Examining single nucleotide polymorphisms in genes responsible for drug detoxification can provide valuable insights into their role in modulating chemotherapy responses. While numerous studies have explored genetic variations in drug metabolism-related genes and their associations with cancer susceptibility, limited data are available regarding their impact on chemotherapy treatment outcomes across various cancer types. Therefore, identifying key genetic polymorphisms in drug detoxification genes is essential for enhancing chemotherapeutic efficacy and minimizing toxicity in breast cancer patients. Several pharmacogenomic studies have demonstrated that patients respond differently to various chemotherapy drugs due to the diverse genetic susceptibility of each individual to treatment. The CYP genes are highly polymorphic in nature, encompassing multiple isozymes such as CYP1A1, CYP1B1, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP17, each of which exhibits functional polymorphisms. These genetic variations have notable clinical relevance, as they can alter drug efficacy and contribute to therapeutic failure and adverse toxicity effects [22–24]. Pharmacogenomic studies have demonstrated that polymorphisms in CYP450 genes can significantly influence therapeutic efficacy and treatment outcomes associated with various chemotherapeutic agents [25–26]. The drug transporter ABCB1 gene, with its C1236T and C3435T variants, has been extensively investigated for its diverse distribution among different ethnic groups. Previous research has established a link between genetic variations in the ABCB1 gene and alterations in response to a broad spectrum of chemotherapeutic agents. Numerous studies have highlighted the impact of ABCB1 gene polymorphisms on chemotherapy-induced toxicities in breast cancer patients, as demonstrated by Wu et al. [27] and Chaturvedi et al. [28]. Previous reports have demonstrated that polymorphisms in the CYP450 gene family are associated with therapeutic failure and severe chemotherapy-induced toxicity reactions [29–31]. Association studies have indicated a significant correlation between the CYP2E1 T>A (rs6413432) polymorphism and outcomes of platinum-based chemotherapy in cervical cancer patients [32]. Conversely, other studies have reported no significant impact of CYP2D6*1A, CYP2E1*6, and CYP2E1*7B polymorphisms on clinical responses to either platinum- or taxane-based chemotherapy in patients with non-small cell lung carcinoma [33–34]. Functional polymorphisms in CYP1A1, CYP1B1, CYP2B6, CYP2C8, and CYP2C9 play a crucial role in clinical outcomes by influencing drug efficacy and potentially contributing to therapeutic inefficacy and adverse toxicity across various cancers [29–33], including breast cancer [35, 36]. Similarly, the polymorphism of CYP2C19 and its association with hematological toxicities, including neutropenia, has been previously reported in response to chemotherapeutic drugs administered for ovarian [37], breast [38, 39], lung cancer [40], and non-small cell lung carcinoma [34]. In our prior study, we observed a significant association between the CYP2C19*2 (681 G>A) polymorphism and hematological toxicities including anemia, neutropenia, febrile neutropenia, and thrombocytopenia in BC patients treated with doxorubicin. Furthermore, a significant association was noted between the CYP2C19*2 polymorphism and non-hematological toxicities such as chemotherapy-induced nausea and vomiting, fatigue, and peripheral neuropathy in response to paclitaxel-based chemotherapy [41]. Unlikely, we observed a negative association between CYP1A1, CYP1B1, and CYP2C9 polymorphisms and peripheral neuropathy in BC patients treated with paclitaxel-based chemotherapy [42]. The ABCB1 and CYP2D6 genes have been shown to regulate the metabolism and pharmacokinetics of doxorubicin [43]. Polymorphisms in CYP2C9, CYP2C19, and CYP2D6 associated with poor metabolizer phenotypes demonstrated a significant correlation with decreased drug efficacy in breast cancer treatment [44]. This study examined the association of polymorphisms in drug detoxification genes (CYP2D6 and CYP2E1) with hematological and non-hematological toxicities following doxorubicin- and paclitaxel-based chemotherapy in BC patients. Analysis of seven genetic variants including CYP2D6*3 (rs35742686), CYP2D6*4 (rs3892097), CYP2D6*10 (rs1065852), CYP2D6*17 (rs28371706), CYP2E1*5B (rs2031920), CYP2E1*6 (rs6413432), and CYP2E1*7B (rs6413420) revealed a negative association of CYP2E1*5B (rs2031920) with neutropenia and CYP2D6*10 (rs1065852) with febrile neutropenia following doxorubicin-based chemotherapy. The CYP2D6*17 (rs28371706) polymorphism was significantly associated with doxorubicin-induced CINV in BC patients. Additionally, the CYP2D6*4 (rs3892097) variant genotype demonstrated a significant positive correlation with paclitaxel-induced peripheral neuropathy (OR = 4.71; 95% CI: 1.93–11.46; p < 0.0006). The findings of this study align with previous reports demonstrating a positive association between CYP2E1 polymorphism and platinum-based chemotherapy outcomes in cervical cancer patients [27]. In BC patients treated with paclitaxel, the CYP2E1*7B (rs6413420) polymorphism showed a significant association with anemia (OR = 342.00; 95% CI: 33.04–354.60; p < 0.0001). Paclitaxel-induced body ache showed a significant association with the CYP2E1*6 (rs6413432) polymorphism (OR = 4.00; 95% CI: 1.66–9.61; p < 0.001) in BC patients. In contrast, other studies reported no significant impact of CYP2D6*1A, CYP2E1*6, and CYP2E1*7B polymorphisms on platinum- or taxane-based chemotherapy response in non-small cell lung carcinoma patients [31, 33–34]. Notably, the genotype distribution of CYP2D6 and CYP2E1 exhibited significant deviation from Hardy–Weinberg equilibrium in patients experiencing paclitaxel-induced hematological and non-hematological toxicities. To our knowledge, this is the first study from India investigating the role of metabolic CYP450 gene polymorphisms in doxorubicin/paclitaxel-related chemotherapy toxicities in breast or other cancers.

Conclusion: In conclusion, the CYP2D6 polymorphism demonstrated a positive association with paclitaxel-induced peripheral neuropathy, while the CYP2E1*6 (rs6413432) polymorphism showed a significant correlation with body ache in BC patients. This is the first study of its kind to analyze the influence of doxorubicin-based chemotherapy on metabolic gene polymorphisms in BC patients. However, larger-scale studies are necessary to validate and strengthen the observed associations between genetic variants of CYP2D6 and CYP2E1 and chemotherapy outcomes in BC by increasing both the sample size and the number of SNPs included for genotyping.

Author Contribution Statement

Concept: RAG, AKG, SJB Design: KDD, RAG, Experimental Studies: RAG, KDD Clinical studies: RAG, AKG Data analysis: KDD, RAG, Statistical analysis: KDD Manuscript preparation: RAG, KDD, AKG, All authors read and approved the final manuscript..

Abbreviations

BC: Breast Cancer

BMI: Body Mass Index

CYP450: Cytochrome P450

PCR-RFLP: Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

DNA: Deoxyribose Nucleic Acid

EDTA: Ethylenediamdie Tetra acetate

µl: Microliter

CINV: Chemotherapy Induced Nausea and Vomiting

ECOG: Estern Cooperative Oncology Group

NCI-CTC: National Cancer Institute-Common Toxicity Criteria

OR: Odds Ratio

CI: Confidence Interval

SNP: Single nucleotide polymorphism

ER: Estrogen Receptor

PR: Progesterone receptor

HER2: Humen Epidermal Growth Factor Receptor