Introduction

Breast cancer is now the most frequent and aggressive female malignancy in both developed and developing countries, and it is the most frequent cause of death in women worldwide [1, 2]. Throughout the world, 2.3 million women were diagnosed with breast cancer and 685,000 died in the year 2020 [3]. In Bangladesh, there is no different scenario. According to the Global Cancer Observatory report, 13,028 new female breast cancer cases were diagnosed in 2020 [4]. According to the International Agency for Cancer Research (IARC), over 7,000 Bangladeshi females die of breast cancer every year [5].

Breast cancer treatment is complicated, requiring a combination of local and systemic treatments. Surgery and radiotherapy are examples of local therapy, while hormone therapy, targeted therapy, and chemotherapy are examples of systemic therapy [6, 7]. The number of effective treatment choices for breast cancer is growing; nevertheless, the utility of particular therapy for specific patients is yet unclear, and adverse events associated with the therapy vary greatly from patient to patient. Variations in tumor and host variables are the cause of anticancer treatment therapy’s inconsistency in terms of safety and efficacy [8, 9]. Slight changes in genotype can lead to varied protein expressions and, as a result, a diverse phenotype [10]. Pharmacogenetics is a recent branch of medicine that investigates how a person’s genetic characteristics affect their drug response. These differences have an impact on therapy response, as well as potential side effects. A rising body of evidence suggests that genes that are responsible for carcinogen/drug metabolism or DNA repair may play a key role in predicting cancer susceptibility and treatment results in individuals. The finding of single-nucleotide polymorphisms (SNPs) in the drug-metabolizing enzyme may be used to predict chemotherapy toxicity and/or efficacy, which could have major clinical ramifications [11, 12].

The anticancer medication cyclophosphamide (CPA) is one of the most extensively utilized in treating solid tumors such as breast cancer, especially in the case of adjuvant settings [13, 14]. CPA is frequently used in combination with other chemotherapy medications like adriamycin/epirubicin and 5-fluorouracil (CAF/CEF) [13]. Anemia, leukopenia, neutropenia, thrombocytopenia, and gastrointestinal disorders are common adverse drug reactions (ADRs) associated with the CPA-based combination treatment for breast cancer [15].

NQO1 is a phase-1 drug-metabolizing enzyme. NAD(P)H quinone oxidoreductase-1 (NQO1) converts quinone-based anticancer drugs to hydroquinones, preventing oxidative stress, reactive oxygen species generation, and carcinogenesis [16, 17]. NQO1 is disabled by a homozygous common missense mutation (NQO1*2 or rs1800566(T)), which has been linked to reduced enzyme activity and poor survival of women receiving adjuvant chemotherapy for breast cancer [18]. A study by Nagata et al. using AMR (Amrubicin hydrochloride) monotherapy for SCLC indicated that due to the NQO1 C609T polymorphism, treatment was related to a substantial rate of bone marrow suppression and hematologic toxicity (grade 3/4) [19]. Akhtari et al. observed in their study that NQO1 rs1800566 (TT genotype) SNP was associated with treatment response variation and erlotinib toxicity [20].

ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, and ALDH1L1 are members of the ALDH1 (aldehyde dehydrogenase 1 family). ALDH1A1 is a relatively abundant cytosolic protein of the human body that belongs to a family of NAD(P)+-dependent enzymes and is principally engaged in the biotransformation of endogenous and exogenous primary alcohols to aldehydes and weak carboxylic acids, respectively [21, 22]. This enzyme participates in the metabolism of cyclophosphamide (CPA) and helps cancer cells retain their stemness [23]. The inter-individual response to cyclophosphamide therapy has been observed to be influenced by genetic diversity in ALDHs [24, 25]. Patients heterozygous for (ALDH1A1*2 or rs615103) exhibited an elevated risk of liver damage in a limited study of breast cancer patients treated with high dose of cyclophosphamide [26]. Another study by Yao et al. discovered that SNP rs3764435 and rs63319 were linked to grade 3 and 4 hematological toxicity after AC (doxorubicin and cyclophosphamide) treatment in breast cancer patients [27]. However, studies regarding the influence of ALDH1A1 polymorphism on the treatment response and toxicity of CAF/CEF-based chemotherapy were very few and inconclusive, and none of them have looked at the rs13959 SNP.

The purpose of this research was to determine the influence of genetic polymorphisms in the ALDH1A1 (rs13959) and NQO1 (rs1800566) genes on the treatment and toxicities caused by CAF/CEF-based adjuvant (ACT) and neoadjuvant (NACT) chemotherapy in Bangladeshi breast cancer patients.

Materials and methods

Study population and research settings

This research enrolled 330 participants between March 16, 2017, to December 31, 2019. They were all diagnosed with invasive breast cancer that was confirmed histologically. All of the participants were recruited from various hospitals in Bangladesh, including Dhaka Medical College Hospital, Bangabandhu Sheikh Mujib Medical University Hospital, National Institute of Cancer Research and Hospital (NICRH), Ahsania Mission Cancer Hospital, and Delta Medical College Hospital. All of the patients got CEF/CAF-based chemotherapy, with 150 getting neoadjuvant treatment and 180 getting adjuvant treatment. Before providing a written agreement, patients were fully informed about the experimental methodology and the goal of the research. The research was conducted following Helsinki Declaration and its amendments (adopted by the 18th WMA general assembly, Helsinki, Finland, June 1964, and the last amendment in Seoul, South Korea, in October 2008). The genetic research was conducted in the Pharmacogenetic and Pharmacokinetics Laboratory of the Department of Clinical Pharmacy and Pharmacology, Faculty of Pharmacy, University of Dhaka, in collaboration with the Molecular Biology Research Laboratory, Southeast University, and QUEST Bangladesh Biomedical Research Centre.

Criteria for assessing responsiveness and toxicity

The influence of NQO1 and ALDH1A1 polymorphisms on chemotherapy treatment response was studied in 150 patients who received CEF/CAF-based NACT, whereas the toxicity effect was studied in patients (330) who received both CEF/CAF-based NACT and ACT. The Response Evaluation Criteria in Solid Tumors (RECIST): Revised RECIST guideline (version 1.1) was used to assess tumor response to treatment [28]. For data processing, two separate groups of patients were created—responder groups, including complete and partial responders and stable and progressing disease in case of non-responders. Chemotherapy toxicity was examined using the Common Terminology Criteria for Adverse Events (CTCAE v4.0) from 2009 [29]. The tumor-node-metastasis (TNM) staging method (Sixth edition) of the American Joint Committee on Cancer (AJCC) was used to evaluate both clinical stages before chemotherapy and the pathological response of the primary tumor and axillary lymph nodes following treatment. The treatment response was evaluated after 2 or 3 cycles of a planned 6-cycle regimen. Anemia, Neutropenia, and Thrombocytopenia were evaluated every 2 to 3 weeks, aligned with the chemotherapy cycles, especially 7–14 days after each cycle.

DNA extraction and genotyping

A 10 ml blood sample was taken from each patient and held at -20 °C in an EDTA-containing storage tube before DNA extraction. The simple non-enzymatic salting out approach was used to extract genomic DNA [30, 31]. Micro-volume Spectrophotometer (Genova Nano, Jenway) was used for the quantification of DNA, setting the absorbance ratio at 260/280 nm. The ALDH1A1 (rs13959) and NQO1 (rs1800566) polymorphisms were genotyped using the Polymerase Chain Reaction (PCR)-Restriction Fragment Length Polymorphism (RFLP) method.

Our research group with Primer Blast designed primers for ALDH1A1 rs13959 were 5’-TGTTGACAAGGCAGTGAAGG-3’ (forward) and 5’-CAAACGCTGAATGCTTTTGA-3’ (reverse), whereas for NQO1 rs1800566 polymorphism, 5’-AAGCCCAGACCAACTTCT-3’ (forward), 5’-ATTTGAATTCGGGCGTCTGCTG-3’ (reverse) were used designed previously [32]. For the digestion of PCR products, two units of the appropriate enzymes (BamHI for rs13959 and HInfI for rs1800566) from New England Biolabs® of the United States were used. Digested products were processed with the help of electrophoresis using 1% agarose gel.

Statistical analysis

The chi-square test was used to discover the variety of different demographic and clinicopathological parameters among different genotypes containing patients having different treatment outcomes and toxicity. Unconditional logistic regression was used to derive odds ratios (ORs) with 95% confidence intervals (CIs) to study the association between genetic polymorphisms and clinical outcomes and toxicity, and ORs were stratified (aOR) with demographic and clinicopathological parameters. p-values less than 0.05 were used to determine statistical significance. All statistical analyses were performed with SPSS 16.0 for Windows (SPSS, Chicago, IL, USA).

Results

Patient’s characteristics

Table 1 shows the relationship between ALDH1A1 (rs13959) and NQO1 (rs1800566) gene polymorphisms and clinicopathological factors such as age, histology, tumor grade, TNM stage, lymph node status, menstruation status, and hormone receptor status of patients. The characteristics of individuals with the ALDH1A1 and NQO1 polymorphisms (heterozygote and mutant homozygote) were compared to those of patients without the polymorphism (normal homozygote). Variations of these characteristics were not significantly associated with ALDH1A1 and NQO1 polymorphism (p > 0.05), according to statistical analysis using the value of p and OR (with 95% CI).

Table 1 Correlation of ALDH1A1 and NQO1 gene polymorphisms with clinicopathological characteristics
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Distribution of genotypes and alleles of rs13959 and rs1800566

Table 2 shows the distribution of different genotypes of ALDH1A1 (rs13959) and NQO1 (rs1800566) polymorphisms. The calculation of Hardy-Weinberg equilibrium shows that both polymorphisms are in the state of genetic equilibrium between responders and non-responders (For rs13959: χ2 = 1.87, p = 0.171 for responders and χ2 = 3.41, p = 0.065 for non-responders, and for rs1800566: χ2 = 1.38, p = 0.241 for responders and χ2 = 1.48, p = 0.224 for non-responders). The minor allele frequencies (MAF) observed for rs13959 in responders was 43.75% and in non-responders was 31.45% and for rs1800566 in responders was 44.32% and in non-responders was 35.48%.

Table 2 Analysis of the hardy-weinberg equilibrium of ALDH1A1 (rs13959) and NQO1 (rs1800566) polymorphisms
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Association of rs13959 and rs1800566 with response of chemotherapy

Based on the RECIST criteria, 22 (14.60%) of 330 patients had a full response, 66 (44.00%) had a partial response, 60 (40.00%) had a stable state, and 2 (1.33%) had disease progression. Univariate chi-square analyses revealed that ALDH1A1 (rs13959) polymorphism showed a significant connection (p-value < 0.05) with the response of chemotherapy in terms of TT genotype, recessive model, and allele model ((TT vs. CC: aOR = 6.40, 95% CI = 1.60 to 25.57, p = 0.007; recessive model: aOR = 6.38, 95% CI = 1.70 to 23.93, p = 0.002; allele model: p = 0.032)), as shown in Table 3. The NQO1 (rs1800566) polymorphism did not show any significant association with the treatment response.

Table 3 Effects of ALDH1A1 (rs13959) and NQO1 (rs1800566) polymorphisms on the response of chemotherapy
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Association of rs13959 and rs1800566 with toxicities of chemotherapy

The probability of ALDH1A1 (rs13959) and NQO1 (rs1800566) SNPs being associated with clinical toxicity resulting from ACT and NACT was also investigated in this study (Table 4). The CTCAE version 4.0 was used to assess the toxicity of chemotherapy in 330 patients. The results of unconditional logistic regression analysis revealed that the TT and CT + TT genotypes of NQO1 (rs1800566) were associated with a lower frequency of toxicities such as anemia [aOR = 0.34, 95% CI = 0.18 to 0.67, p = 0.006 and aOR = 0.58, 95% CI = 0.36 to 0.92, p = 0.021]; neutropenia [aOR = 0.42, 95% CI = 0.21 to 0.87, p = 0.044 and aOR = 0.57, 95% CI = 0.35 to 0.94, p = 0.027]; leukopenia [aOR = 0.33, 95% CI = 0.15 to 0.75, p = 0.010 and aOR = 0.46, 95% CI = 0.26 to 0.79, p = 0.005]; and gastrointestinal toxicity [aOR = 0.30, 95% CI = 0.10 to 0.88, p = 0.02 and aOR = 0.38, 95% CI = 0.19 to 0.77, p = 0.006]. A significant association was also observed for CT genotype in term of gastrointestinal toxicity and leukopenia, respectively [aOR = 0.42, 95% CI = 0.20 to 0.90, p = 0.02 and aOR = 0.52, 95% CI = 0.29 to 0.93, p = 0.010]. In the case of thrombocytopenia, there was no strong association.

Table 4 Effects of ALDH1A1 and NQO1 polymorphisms on the toxicities induced by chemotherapy
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In patients with the ALDH1A1 (rs13959) polymorphism, genotypes TT [aOR = 2.00, 95% CI = 1.09 to 3.67, p = 0.037] and CT + TT [aOR = 1.68, 95% CI = 1.05 to 2.69, p = 0.029] were more likely to show treatment-related anemia than those with the CC genotype. There was no significant link between this SNP and neutropenia, leukopenia, thrombocytopenia, or gastrointestinal toxicity.

Discussion

This study was performed with ALDH1A1 (rs13959) and NQO1 (rs1800566) polymorphisms in Bangladeshi breast cancer patients that looked at 330 patients who were given cyclophosphamide-based combined chemotherapy (CEF/CAF), 150 of them were given neoadjuvant chemotherapy and 180 were given adjuvant chemotherapy. This study observed a significant association of both ALDH1A1 (rs13959) and NQO1 (rs1800566) polymorphisms with treatment-associated toxicity, but a significant association in the case of treatment response was found for only ALDH1A1 (rs13959). A previous study found a poor response for this SNP [33] (Table 5).

Table 5 Previous findings of associations of chemotherapy outcomes with NQO1 rs1800566
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The SNPs NQO1 (rs1800566) was found to be strongly linked to CEF/CAF chemotherapy-related adverse events like anemia, neutropenia, leukopenia, and gastrointestinal disorders, while ALDH1A1 (rs13959) was linked to only anemia toxicity. Though the TT and CT + TT genotypes of NQO1 (rs1800566) polymorphism were associated with a lower frequency of toxicities than the wild CC genotype; however, ALDH1A1 (rs13959) polymorphism was related to the higher frequency of anemia toxicity. Besides, there was a significant correlation between treatment response and ALDH1A1 (rs13959) polymorphism but not NQO1 (rs1800566) polymorphism, which was inconsistent with other research findings [11, 19, 24]. A similar correlation was reported for overall toxicity in the case of rs1800566 [34]. This discrepancy may be because of the ethnic disparity and, more noteworthy hereditary admixture and variety in the number of inhabitants in Bangladesh. This could be attributed to the fact that the presence of multidrug resistance-associated protein 1 (MRP1), breast cancer resistance protein (BCRP) in cancer cells may cause efflux of the drug (e.g., cyclophosphamide,5-fluorouracil, adriamycin) by efflux transporter [35,36,37]. Also, highly effective nuclear DNA repair systems play a significant role in tumor cells’ ability to tolerate the cytotoxic effects of anthracyclines (Doxorubicin, epirubicin) [36].

It is well known that the majority of drug-metabolizing enzymes involved in drug metabolism activation and detoxification pathways are extremely polymorphic [38]. Several studies have found that polymorphism in these genes was linked to the likelihood of chemotherapy toxicity, particularly in the case of cyclophosphamide-based (CPA) combination therapy. CPA is the most extensively utilized in the treatment of breast cancer [14]. It is commonly used in combination with other chemotherapeutic drugs such as adriamycin and 5-fluorouracil (CAF). Although the CPA-based combination treatment is beneficial for breast cancer, it has been linked to adverse drug reactions (ADRs) such as leukopenia, neutropenia, and gastrointestinal issues. Furthermore, anticancer medicines are well-known for having a limited therapeutic window; a higher plasma concentration in the body produces toxicity, while a lower quantity diminishes the drugs’ efficacy. As a result, the function of pharmacogenomics, which aims to provide a prediction technique for severe drug toxicity, is crucial [39].

Myelosuppression is a common treatment-related complication in cancer patients [40]. It arises when cytotoxic chemotherapy disrupts the bone marrow’s rapidly dividing hematopoietic stem and progenitor cells (HSPCs), which gives rise to blood cell lineages. Myelosuppression is depicted by anemia, lymphopenia, neutropenia, and thrombocytopenia and mostly occurs in breast cancer patients [41, 42]. It is worth noticing that due to quick metabolism or inactivation by various drug-metabolizing enzymes, several clinically approved anti-cancer drugs have shown to be ineffective or resistant [43,44,45].

Our research revealed that ALDH1A1 (rs13959) SNP was strongly linked with CAF-based chemotherapy-induced anemia. The rs13959 variant may result in lower ALDH enzyme activity, resulting in decreased 4-hydroxycyclophosphamide detoxification, causing drug accumulation inside the body and enhanced myelosuppression. This enzyme participates in CPA metabolism and genetic variation of ALDH1A1 has been shown to alter inter-individual cyclophosphamide therapy response [24]. There were studies regarding ALDH expression in breast cancer, but the SNP study was limited. In different cancer cell lines, overexpression/increased activity of ALDHs has been found, and it increases the rate of cyclophosphamide metabolism and plasma clearance, resulting in poor treatment response or resistance to CPA [46] (Table 5). Our finding was supported by other previous studies. For instance, a study of gallbladder cancer discovered that ALDH1A1 rs13959T > G was related to an elevated incidence of grade 3–4 hematological toxicity [47]. Another study conducted over 250 breast cancer patients in India found that ALDH1A1 rs6151031 genotype *2/*2 was linked to higher drug toxicity in cyclophosphamide-treated breast cancer patients [24].

In this research, we found that NQO1 (rs1800566) polymorphism was strongly associated with anemia, leukopenia, neutropenia, and gastrointestinal disorders. As part of detoxification, NQO1 catalyzes the two-electron-mediated reduction of quinones to hydroquinones. The NQO1 C609T polymorphism has been shown to have a well-established and significant impact on the enzymatic activity of the synthesized protein because the modified version of the enzyme is rapidly ubiquitinated and damaged by the proteasome [48]. As a result, the homozygous variant T allele had nearly total eradication of its enzymatic activity (2–4% of wild-type activity), whereas the heterozygous variant T allele had a threefold decrease in enzymatic activity relative to the homozygous wild-type allele [48]. Hence, the presence of the polymorphism causes reduction of the enzyme’s ability to detoxify carcinogens and increases the likelihood of hematologic toxicity in susceptible individuals. Allele frequency of the NQO1 C609T polymorphism varies greatly by geography and ethnicity. Database of HapMap showed that NQO1 T allelic frequency ranges from 17.1 to 19.2% in Africa, 18.6% in Europe, and the highest 50% in Asian populations, and a large number of people possess CT and TT genotypes than the wild genome [49]. The association has also been studied in other studies and got a similar result to ours. A study conducted on acute myeloid leukemia patients treated with anthracycline showed that the genotypes of NQO1 rs1800566 were associated with thrombocytopenia and GI toxicity [50] (Table 5). Another study by Fagerholm et al. discovered that NQO1*2 homozygotes have low treatment-associated survival and a poor response to fluorouracil, epirubicin, and cyclophosphamide therapy [51].

Therefore, this study will help the researcher to estimate the frequency of polymorphism in this region and study further. These findings will help researchers to understand the association of ALDH1A1 and NQO1 better in breast cancer. Studies with a bigger sample size with more variants are required to determine the best therapies and get conclusive evidence.

Conclusion

This study revealed that the ALDH1A1 13,959 polymorphism is associated with response to chemotherapy. Besides, NQO1 rs1800566 and ALDH1A1 13,959 polymorphisms are correlated to the toxicities, including anemia, neutropenia, leukopenia, and gastrointestinal toxicity in CEA/CFA-based neoadjuvant and adjuvant chemotherapy patients. This information will aid in the prediction of clinical toxicity associated with cyclophosphamide-based chemotherapy.