Abstract
Liquid biopsy is a non-invasive method of detecting cancer-related mutations from circulating cell-free DNA (cfDNA) and has emerged as a promising alternative to traditional tissue biopsies. However, its utility has so far been limited to the detection of somatic mutations in cancer cells. Our study examined germline mutations that are associated with pharmacogenetics in 19 patients diagnosed with colorectal cancer and 12 patients with non-small cell lung carcinoma, utilizing the highly advanced and non-invasive technique of liquid biopsy, followed by subsequent next-generation sequencing for comprehensive analysis. Despite the relatively modest sample size of patients under consideration, our results indicated a noteworthy correlation between the presence of adverse effects and the identified germline mutations. We have identified the following mutations with significant implications for pharmacogenetics: MTHFR c.1286A > C, MTHFR c.665C > T, DPYD c.2194G > A, DPYD c.85C > T, XPC c.2815C > A, UMPS c.638G > C, SLC22A2 c.808T > G, EGFR c.1562G > A, ABCB1 2677 T > G, GSTP1 c.313A > G, ERCC2 c.2251A > C, and SLC19A1 c.80A > G. In addition, we observed various adverse drug reactions in our patient cohort, encompassing myelosuppression, hepatotoxicity, neurotoxicity and gastrointestinal toxicity. Liquid biopsy has the potential to revolutionize cancer diagnosis and treatment by detecting both somatic and germline mutations. The preliminary results of studies on germline mutations are promising, but further research is needed to address the remaining challenges and to establish the utility of liquid biopsy as a reliable diagnostic tool for germline mutations.
Introduction
Currently, tissue biopsy is considered the standard approach for diagnosing malignant tumours. However, it has limitations due to the potential risks of puncture injury, tumour metastasis, bleeding and infection. In comparison, genome analysis using blood, plasma or other bodily fluids is a less invasive alternative for patients, and can reveal comprehensive genetic alterations in the tumour. Additionally, repeated sampling can be easily performed to monitor the molecular changes in tumours over time. By monitoring the levels of circulating cell-free DNA (cfDNA), it is possible to detect tumours that may not be identified or confirmed through imaging exams, such as residual tumours after surgical removal [Citation1].
Tissue biopsies have traditionally been the preferred diagnostic tool in the field of oncology, with a primary focus on understanding the cancer site. However, with a better understanding of the role of DNA alterations in tumourigenesis, biopsies are now also used to assess genomic alterations and guide therapeutic decision-making. However, the heterogeneous nature of cancer limits the ability to capture its spatial and temporal heterogeneity in a single biopsy. Liquid biopsies that use circulating tumour DNA (ctDNA) derived from plasma offer an alternative approach. ctDNA can identify somatic alterations with high accuracy, similar to tissue biopsy, and can detect evolving mutations in metastatic cancer patients [Citation2].
Moreover, liquid biopsy holds promise for the detection of germline mutations. Germline mutations are inherited alterations in the DNA that can predispose individuals to develop certain types of cancer. Liquid biopsy, specifically the analysis of circulating cell-free DNA (cfDNA), allows for the identification of germline mutations associated with cancer risk. Various studies have indicated that liquid biopsy can identify germline mutations in genes linked with hereditary breast and ovarian cancer, as well as Lynch syndrome [Citation3,Citation4]. By analysing the cfDNA, it is possible to detect the presence of specific germline mutations that may contribute to the development or progression of tumours. This information can be valuable for understanding the underlying genetic factors contributing to the disease and for guiding personalized treatment strategies [Citation5]. Furthermore, liquid biopsy offers the advantage of repeated sampling over time, enabling the monitoring of germline mutations and their impact on disease progression and treatment response [Citation6]. By incorporating the analysis of germline mutations into liquid biopsy approaches, clinicians and researchers can gain a more comprehensive understanding of cancer genetics and individualize patient care accordingly.
The aim of this study was to screen liquid biopsy samples for germline mutations that potentially confer an elevated susceptibility to chemotherapy-induced toxicity among patients with colorectal cancer and patients with non-small cell lung carcinoma.
Subjects and methods
Study design
The present study involved the extraction of cell-free DNA from blood samples obtained from a cohort of 31 patients (19 diagnosed with colorectal and 12 with non-small cell lung carcinoma) using the QIAamp MinElute ccfDNA Midi Kit. The patients received medical care at the University Hospital "Sveti Georgi", Plovdiv, where blood specimens were collected in Streck tubes over a two-year period (1 May 2020–1 May 2022). Cell-free DNA was extracted from the samples following the established protocol of BioChain’s cfPure® Cell Free DNA Extraction Kit [Citation7]. The quality and quantity of the extracted DNA were evaluated by performing Agarose Gel Electrophoresis and utilizing the Qubit DNA Assay Kit in a Qubit 3.0 Fluorimeter (Life Technologies, CA, USA). Next-generation sequencing (NGS) was performed using a targeted assay specifically designed to identify genomic alterations in a comprehensive panel of 484 genes (NovoPMTM 2.0) () that are of utmost relevance for the precise diagnosis and treatment of solid tumours, as strongly recommended by current medical literature and clinical guidelines. Furthermore, an in-house bioinformatic algorithm developed by Novogene (Cambridge, United Kingdom) was utilized to predict the origin of short variant mutations (germline or somatic), based exclusively on the sequencing and analysis of tumour samples.
Table 1. A targeted pharmacogenetics panel for screening of 484 genes in cancer patients.
Results
Our primary focus was on identifying germline mutations which may pose a risk factor for increased toxicity of chemotherapy in the tested patients with colorectal cancer (CRC; ) and non-small cell lung cancer (NSCLC; ). Through our analysis, we identified several frequently occurring mutations in the following genes: MTHFR, DPYD, XPC, UMPS, SLC22A2, EGFR, ABCB1, GSTP1, ERCC2 and SLC19A1. The remaining 474 genes have not been addressed due to their lack of pharmacogenetic relevance to the specific aim of this study. They are associated with cancerogenesis which is not the focus of this paper. The comprehensive findings obtained from our cohort of patients are documented in .
Table 2. CRC patients: treatment, adverse effects and gene variants.
Table 3. NSCLC patients: treatment, adverse effects and gene variants.
Table 4. Genotype distributions for all analysed gene variants in patients with CRC and NSCLC.
Table 5. Associations between the genotypes of a particular SNP with occurrence of adverse effects after therapy in patients with CRC and NSCLC.
In the group of 19 patients diagnosed with colorectal carcinoma, 17 received chemotherapy treatment which involved the use of Oxaliplatin. Of those 17 patients, 15 were also treated with 5-Fluorouracil, 2 with Capecitabine, and only 3 received both 5-Fluorouracil and Capecitabine. Adverse reactions were observed in 14 out of the 17 patients receiving chemotherapy. The adverse reactions included anaemia, leukocytopenia, thrombocytopenia, hepatotoxicity, neurotoxicity and gastrointestinal complaints such as diarrhoea and vomiting. Among these 14 patients, 7 were identified as heterozygous for the MTHFR 1286 A > C polymorphism and none of them were found to be homozygous for the mutant allele ().
Out of the 19 patients with colorectal cancer who were tested, 17 underwent chemotherapy treatment that consisted of Oxaliplatin, with 15 of those patients also receiving 5-Fluorouracil. Of the treated patients, 7 were identified as heterozygous (TC) for the mutant allele of the MTHFR c.665C > T variant, while one was homozygous (TT). Among the carriers, six individuals exhibited adverse reactions to the chemotherapy treatment (). Moreover, the remaining patients displayed a homozygous wild-type allele genotype, despite experiencing adverse reactions. In the group of patients with non-small cell lung cancer (NSCLC), there were five carriers of the 1286 A > C variant and seven for the c.665C > T variant of the MTHFR gene. However, their treatment regimen did not include the administration of 5-fluorouracil (5-FU) ().
Out of the 19 patients diagnosed with colon cancer, 15 were administered 5-Fluorouracil, and 5 were treated with Capecitabine (2 received a combination of both drugs). Among the 17 patients who were treated with one or both chemotherapy medications, 7 carried the mutated variant of DPYD c.2194G > A (5 were heterozygous CT and 2 were homozygous TT) and all of them experienced unfavourable effects as a result of the treatment (). Among the cohort of 10 non-carrier patients, 7 individuals exhibited unfavourable reactions.
Among the 19 patients with colorectal cancer in our study, 17 received 5-Fluorouracil and/or Capecitabine. Nine of the people on fluoropyramidine-based therapy were heterozygous (TC) and two were homozygous for the TT variant of DPYD c.85C > T. Eight of the carriers (homozygous and heterozygous) experienced side effects such as hematological toxicity, neurotoxicity, hepatotoxicity, or gastrointestinal problems (diarrhoea, vomiting) (). All six individuals who did not carry the variants also manifested adverse reactions. Within our cohort of patients diagnosed with non-small cell lung cancer (NSCLC), we identified a subset of individuals who exhibited one or both variants of the DPYD gene that were found to be risk factors for toxic side effects. Notably, it is worth mentioning that these individuals were not administered chemotherapy regimens involving either 5-fluorouracil (5-FU) or capecitabine ().
Among our patients, the most commonly observed variant of the XPC gene is c.2815C > A (Lys939Gln, rs2228001), which was found in 24 out of 31 tested patients (8 with non-small cell lung cancer [NSCLC] and 16 with colorectal cancer [CRC]). Of these carriers, 10 were homozygous for the AA allele and the remainder were heterozygous (). In our NSCLC patient group (n = 12), only four were receiving cisplatin chemotherapy, of whom three were carriers (two homozygous and one heterozygous) and experienced adverse reactions. Among the six patients who received carboplatin chemotherapy, only one individual was heterozygous and three were homozygous (). There were also two carriers (one homozygous and one heterozygous) who did not suffer adverse reactions. In the CRC patient group, 17 patients received oxaliplatin chemotherapy, of whom 14 were carriers of the mutant variant (9 heterozygous and 5 homozygous), and 12 of them suffered adverse toxic reactions ().
Among our patient cohort of 31, 5-FU/Capecitabine treatment was administered to only a portion of the CRC patients, although carriers of the UMPS c.638C > G variant were found in both the CRC (10 out of 19 tested) and NSCLC (5 out of 12 tested) patients. Among the 10 UMPS variant carriers with CRC (1 homozygous for the CC allele and 9 heterozygous), 10 received 5-FU/Capecitabine treatment, and 9 experienced adverse drug effects (). Out of the seven non-carriers of the UMPS c.638G > C variant five suffered adverse reactions.
The SLC22A2 c.808T > G variant was found to be predominant in our patient cohort of colorectal and non-small cell lung cancer individuals who received platinum-based chemotherapy. Among the 17 patients with colorectal cancer who received oxaliplatin, 16 were carriers of the polymorphism, and 13 of them experienced hematological toxicity (10 homozygous for the mutant allele and 3 heterozygous) (). Despite being the only non-carrier among the patients, one individual still experienced drug toxicity (). Among the 12 patients with non-small cell lung cancer, 10 were treated with platinum-based chemotherapy: 6 received carboplatin and 4 received cisplatin. All these patients were carriers of the polymorphism, and 5 of them suffered adverse effects and were homozygous for the deleterious variant ().
In our study, out of 19 tested CRC patients, only 4 were carriers of the EGFR c.1562G > A variant, with 3 being heterozygous and 1 being homozygous. Three of these patients were treated with Bevacizumab and experienced hematological toxicity. We found two non-carriers of the EGFR variant treated with Bevacizumab and both suffered adverse reaction (). In the case of NSCLC, only 3 out of 12 tested patients were found to be heterozygous carriers of the EGFR c.1562G > A variant, and only 2 of them experienced adverse reactions to immunotherapy ().
Our study included 19 colorectal cancer patients, 12 of whom were treated with 5-FU, 2 with capecitabine, and 3 with both chemotherapeutic drugs. All but two of the patients were carriers of the mutant allele for the 2677 T > G variant of the ABCB1 gene, with 4 being homozygous and 11 being heterozygous. None of the patients suffered from hand-foot syndrome (HFS), and only one carrier experienced neutropenia as an adverse reaction (). The two non-carriers did not suffer adverse effects. Therefore, based on our initial findings, we are unable to validate the proposed correlation between this specific variant of the ABCB1 gene and the onset of hand-foot syndrome or gastrointestinal toxicity. Nevertheless, 10 of the patients treated with 5-FU/Capecitabine suffered adverse effects and were found to be carriers.
Of the 19 colorectal cancer patients included in this study, 17 were treated with Oxaliplatin, 5-FU and/or Capecitabine. Of these patients, 6 were heterozygous and 10 were homozygous for the AA allele of the GSTP1 c.313A > G variant. Among the 16 carriers of the deleterious variant, 13 experienced adverse reactions, most commonly, hematological toxicity. Only one patient was not a carrier of the c.313A > G variant of the GSTP1 gene and also suffered hematological toxicity (). Additionally, we studied 12 patients with non-small-cell lung cancer (NSCLC), 10 of whom were treated with platinum-based chemotherapy. All 10 patients were carriers of the deleterious variant, with 5 being heterozygous and 5 homozygous. Of these patients, 5 exhibited toxic chemotherapy effects ().
In our study, a small cohort of 19 patients with colorectal cancer were treated with different chemotherapy regimens: 12 were given Oxaliplatin and 5-FU, 2 received Oxaliplatin and Capecitabine, and 3 were given a combination of Oxaliplatin, 5-FU and Capecitabine. Of the 17 patients who received treatment, 14 were found to carry the c.2251A > C variant of the ERCC2 gene (13 were heterozygous and one was homozygous). Of the three non-carriers, only one suffered adverse reactions (hepatotoxicity and leucopoenia). All of these patients experienced toxic adverse effects, with hematological toxicity being the most common ().In the NSCLC group, 10 patients received platinum-based chemotherapy, out of which 6 were carriers of the deleterious variant of the ERCC2 gene (4 heterozygous and 2 homozygous). However, only 3 of these patients suffered adverse reactions from the treatment (). Out of the four non-carriers treated with platinum-based chemotherapy only two suffered drug toxicity.
In our group of tested patients with colorectal cancer (n = 19), 17 received 5-FU and/or capecitabine, and only 9 were treated with irinotecan. Of the treated patients, 9 were carriers of the SLC19A1 c.80A > G variant, with 5 being heterozygous and 4 being homozygous, and 8 were non-carriers (). All carriers and five of the non-carriers suffered adverse reactions. Among our non-small cell lung cancer patients (n = 12), 10 were treated with platinum-based chemotherapy, and 8 of them were carriers of the c.80A > G variant, with 7 being heterozygous and one being homozygous, and two were non-carriers. Four of the carriers and one of the non-carriers suffered drug toxicities ().
Discussion
MTHFR
The MTHFR gene, which encodes the methylenetetrahydrofolate reductase enzyme, plays a vital role in folate metabolism. This gene is responsible for producing an enzyme that catalyses the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate [Citation8]. The latter serves as a methyl donor in several biochemical reactions, contributing to the transfer of methyl groups to DNA, proteins and other molecules. This methylation process is crucial for gene expression, cellular differentiation and overall cellular function. Additionally, the MTHFR enzyme is involved in the synthesis of DNA and RNA [Citation9]. Mutations or variations in the MTHFR gene may give rise to a range of severe genetic disorders, including homocystinuria, anencephaly, spina bifida, and others [Citation10]. Moreover, this enzyme is also an indispensable part of the complex biochemical pathway that converts homocysteine into methionine, thereby maintaining healthy homocysteine levels in the body [Citation9]. The MTHFR 1286 A > C variant, which changes glutamic acid to alanine at position 429 of the protein chain, can reduce enzyme activity. Homozygous patients with colorectal cancer carrying the mutated variant CC have a higher risk of experiencing drug toxicity when treated with fluoropyrimidine-based chemotherapy than heterozygous subjects (CA) or non-carriers of the mutated variant (AA), as shown by studies [Citation11,Citation12]. This is particularly important in patients treated with specific anti-cancer drugs, where MTHFR plays a role in drug conversion. Kristensen et al. [Citation11] have demonstrated an association between the CC genotype and an elevated risk of chemotherapy-induced toxicity in individuals with colorectal neoplasms who are undergoing treatment with capecitabine, fluorouracil, leucovorin and oxaliplatin [Citation11].
ADRs were observed in the majority of our tested patients with colorectal cancer receiving chemotherapy, but only half of them were found to be carriers of the polymorphism in a heterozygous state. Therefore, the presence of the polymorphism in a heterozygous state might not fully account for all the observed ADRs in patients receiving chemotherapy. Additional factors and genetic variations could also play a significant role in determining individual drug responses and adverse reactions. Further research and comprehensive studies are warranted to better understand the underlying mechanisms behind these ADRs and their potential implications for personalized medicine approaches in cancer treatment.
The MTHFR c.665C > T (or 677 C > T p.Ala222Val, rs1801133) is another common gene variation identified in the studied patients. It leads to the production of an unstable enzyme that is sensitive to heat. Individuals with a homozygous TT genotype are likely to have higher levels of homocysteine and lower levels of serum folate compared to those with a heterozygous or homozygous CC genotype [Citation13]. Individuals with the TT genotype have an increased risk of experiencing harmful drug effects when treated with fluorouracil, leucovorin, and oxaliplatin for colonic neoplasms, as compared to individuals with the TC + CC genotypes [Citation14]. The findings in our tested patients suggest that while the heterozygous state of the mutant allele may be associated with some adverse reactions, there are likely other factors contributing to the observed adverse drug reactions in patients receiving chemotherapy. This observation highlights the importance of considering the MTHFR c.665C > T variant not as an isolated test but rather as an integral component of a comprehensive gene panel encompassing genes associated with the metabolism of platinum compounds and fluoropyrimidines. Further investigations are necessary to fully understand the interplay of genetic variations and other variables in determining individual drug responses.
DPYD
The DPYD gene encodes the dihydropyrimidine dehydrogenase (DPD) enzyme, which is a crucial component in the breakdown of pyrimidine nucleosides like uracil and thymine, both of which are fundamental constituents of DNA molecules [Citation15]. In addition to its involvement in pyrimidine metabolism, DPD also contributes to the processing of chemotherapy medications such as 5-fluorouracil and capecitabine. Individuals with mutations in the DPYD gene may experience a deficiency in DPD activity, which can exacerbate the toxic effects of these drugs, especially 5-fluorouracil, and can result in a severe adverse reaction known as DPD deficiency-associated toxicity [Citation16].
The DPYD c.2194G > A (DPYD*6) variant is a genetic alteration that substitutes the nucleotide guanine (G) with adenine (A) at position 2194 in the DPYD gene. Research has shown that this particular variant causes a substantial decrease in the activity of the DPD enzyme, which could place carriers at a heightened risk of experiencing adverse drug reactions to certain chemotherapy medications such as 5-fluorouracil [Citation17]. This variant is classified as a ‘loss-of-function’ variant, which means that it suppresses the activity of the DPD enzyme and may hinder the metabolism of certain medications, thus increasing the likelihood of toxicity [Citation17]. Studies show an increased risk of drug toxicity for genotypes AG + AA when treated with capecitabine or fluorouracil in people with colonic neoplasms as compared to genotype GG [Citation18–20]. The results from our study suggest that adverse reactions to chemotherapy cannot be solely attributed to the presence of the mutated variant, and other factors may contribute to the observed unfavourable effects in both carriers and non-carriers of the mutation. Further investigation is needed to understand the complexities of individual drug responses and adverse reactions in colonic cancer patients.
The DPYD c.85T > C (DPYD*9A) is another variant frequently found in our patients. However, it is unclear whether this variant is associated with a DPD deficiency clinical phenotype. Studies examining the relationship between DPYD*9A and toxicity from fluoropyrimidines, including diarrhoea and hand-foot syndrome, have produced conflicting results [Citation11,Citation21]. Some studies show an association between the genotype and clinical phenotype [Citation22], while others do not [Citation19,Citation21]. A recent study [Citation21] confirmed the correlation between the DPYD*9A genotype and fluoropyrimidine-related toxicity in patients who had the TT allele, whether heterozygous or homozygous. The results of our study suggest that adverse reactions to 5-Fluorouracil and/or Capecitabine treatment are not solely dependent on the presence of the identified variants (TC and TT). While carriers of these variants experienced side effects, it is noteworthy that adverse reactions were also observed in patients who did not carry the variants. This suggests that other factors, possibly genetic or environmental, may contribute to the development of these adverse effects. Further research is required to unravel the complex interplay between genetic variations and other factors influencing individual drug responses and side effects, enabling more personalized and effective treatment strategies in oncology.
XPC
DNA damage is a common event that can cause cell death, ageing and cancer. The Nucleotide Excision Repair (NER) pathway is a critical mechanism for defending against DNA damage, and Xeroderma pigmentosum group C (XPC) protein is a crucial player in this process. NER can be divided into two sub-pathways, Global Genome repair (GG-NER) and Transcription Coupled repair (TC-NER), with XPC playing a significant role in GG-NER [Citation23]. XPC recognizes DNA damage and facilitates the recruitment of repair machinery to eliminate the damage. Defective XPC function can lead to cancer development in both humans and mice due to accelerated accumulation of mutations. Numerous studies have shown that XPC participates in various DNA damage-induced cellular responses, including the removal of oxidative DNA damage, maintaining redox homeostasis, and regulating the cell cycle. Researchers suggest that the combination of increased sensitivity to oxidative DNA damage, disturbed redox homeostasis, and inefficient cell cycle control mechanisms may contribute to increased cancer susceptibility in oxygen-exposed tissues [Citation24].
XPC is a DNA damage recognition factor that is critically involved in early-stage DNA repair, as well as the regulation of cell cycle checkpoint and DNA damage-induced apoptosis. In vitro studies have demonstrated that XPC overexpression significantly enhances cell resistance to cisplatin [Citation25]. Conversely, in vivo studies showed that XPC gene knockdown significantly attenuated tumour growth in mice bearing tumours [Citation25]. Mutations in the XCP gene may be a risk factor for cancer development and unfavourable response in individuals undergoing chemotherapy regimens [Citation26,Citation27].
Studies on patients with bladder cancer have shown that individuals with genotypes AA and AC may be at increased risk for adverse effects, specifically neutropenia [Citation26]. In our study, the c.2815C > A (Lys939Gln, rs2228001) variant of the XPC gene was the most prevalent among our patients, with 24 out of 31 individuals tested carrying this variant. The presence of this variant was observed in both non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) patients, suggesting its potential relevance across different cancer types. These findings propose that the presence of the c.2815C > A variant of the XPC gene may influence individual responses to specific chemotherapy agents. However, the relationship between this variant and chemotherapy-induced adverse reactions appears to vary depending on the type of chemotherapy used and the specific cancer type. Further research is warranted to better understand the implications of this variant on chemotherapy response and toxicity and to identify potential personalized treatment strategies based on genetic profiles in NSCLC and CRC patients.
UMPS
Fluorouracil (5-FU) is an antimetabolite chemotherapy agent commonly used to treat several cancer types, including head and neck, pancreatic, breast, stomach and colorectal cancer. However, intrinsic or acquired resistance to 5-FU is a major limiting factor in the treatment of colorectal and other cancers. Response rates to 5-FU vary widely, from 6 to 53%, depending on dosage, schedule, combination with modifiers, and disease characteristics. Given the high usage and low-to-moderate response rates of 5-FU, the clinical relevance of 5-FU resistance is considerable [Citation27–30].
Uridine monophosphate synthetase (UMPS) expression levels have been proposed as a potentially critical factor in a tumour’s response to 5-FU and are cited as the primary means of activating the prodrug 5-FU to its active antitumour metabolites [Citation30]. Previous studies have attempted to use UMPS expression as a predictor of drug response and chemotherapy toxicity, with varying degrees of success [Citation27].
A variant associated with drug toxicity, UMPS c.638G > C, was identified in our patients. Although literature primarily associates this variant with adverse effects from tegafur and leucovorin [Citation28], some studies have commented on the link between this polymorphism and 5-FU/Capecitabine treatment [Citation29]. The results from our study highlight the importance of considering genetic variations such as the UMPS variant in treatment planning to minimize adverse drug reactions. However, they also indicate that individual drug responses are likely influenced by a combination of genetic and non-genetic factors, emphasizing the need for further research to identify additional determinants of treatment outcomes in both CRC and NSCLC patients. Such insights will aid in the development of more personalized and effective treatment strategies in the field of oncology.
SLC22A2
The SLC22A2 gene encodes for a protein known as organic cation transporter 2 (OCT2), which belongs to the solute carrier family 22 (SLC22). OCT2 is a transport protein responsible for the uptake and efflux of various organic cations, including drugs, neurotransmitters and biologically active compounds, across cell membranes [Citation31]. OCT2 is primarily expressed in the kidneys, liver and brain, where it plays a critical role in the uptake and elimination of drugs and foreign compounds. However, in cancer cells, OCT2 is often overexpressed, leading to multidrug resistance (MDR) by pumping out drugs from the cancer cells [Citation32].
Recent studies have shown that the SLC22A2 gene is overexpressed in various types of cancer, including breast, ovarian, prostate, colorectal, non-small cell lung cancer and leukaemia [Citation31]. This overexpression of OCT2 is often associated with MDR in cancer cells, leading to decreased efficacy of chemotherapy and other anticancer drugs.
One common variant of SLC22A2 that has been observed in cancer patients and associated with adverse chemotherapy effects is SLC22A2 c.808T > G. Studies have found that patients with the GG and GT genotype and non-small cell lung cancer or colorectal cancer may experience increased toxicity, specifically hepatotoxicity, when taking platinum-based compounds compared to patients with the TT genotype [Citation33,Citation34]. This polymorphism identified in our cohort shows a strong association with platinum-based chemotherapy-induced adverse effects in both colorectal and non-small cell lung cancer patients. Its prevalence among the patients receiving such treatment suggests that this genetic variation may play a crucial role in individual drug responses. The higher incidence of adverse effects in carriers, especially those who are homozygous for the mutant allele, reinforces the importance of genetic screening in identifying patients who may be more susceptible to drug toxicity.
However, the presence of drug toxicity in some non-carriers raises the possibility that additional factors, both genetic and environmental, may also contribute to adverse drug reactions. It highlights the complexity of individual drug responses and the need for a comprehensive understanding of all contributing factors to ensure safe and effective chemotherapy administration.
EGFR
The EGFR gene codes for a protein called epidermal growth factor receptor (EGFR), which is a member of the ErbB family of receptors. This transmembrane receptor plays a critical role in cell growth, proliferation, survival, differentiation and migration by binding to ligands such as epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) [Citation35].
In normal cells, EGFR is essential for regulating cell growth and proliferation, but in cancer cells, the EGFR signalling pathway can become overactive, leading to uncontrolled cell growth and proliferation [Citation35]. This can occur through various mechanisms, including mutations in the EGFR gene, amplification of the EGFR gene, and overexpression of the EGFR protein. EGFR mutations are particularly common in non-small cell lung cancer (NSCLC) and have been associated with poor prognosis and resistance to chemotherapy.
Therefore, drugs that inhibit EGFR signalling, such as tyrosine kinase inhibitors (TKIs) and monoclonal antibodies, have been developed and are being used as targeted therapies for cancer. However, recent studies have shown that a specific variant of the EGFR gene, known as EGFR c.1562G > A, may be associated with cytotoxicity of EGFR inhibitors [Citation36].
In solid tumours, the EGFR and VEGF pathways are linked, particularly with respect to angiogenesis. EGF and TGF-α both induce VEGF expression via activation of EGFR in cell culture models and have proangiogenic properties [Citation37]. The presence of the EGFR c.1562G > A variant has been found to be associated with adverse reactions to certain cancer treatments, such as Bevacizumab (VEGF), in patients with colorectal cancer (CRC) [Citation38] and immunotherapy in NSCLC patients [Citation39].
The EGFR c.1562G > A variant’s limited prevalence in both CRC and NSCLC patients in our study suggests that it may not be a common driver of drug response or adverse reactions in these cancer types. However, the observed adverse reactions in some carriers and non-carriers imply that other factors beyond this specific variant may also influence drug responses and toxicities.
The heterogeneity of drug responses among carriers of the EGFR variant in both CRC and NSCLC patients highlights the complex nature of cancer treatment responses and the potential influence of additional genetic and environmental factors. Moreover, the occurrence of adverse reactions in non-carriers emphasizes that multiple genetic and non-genetic variables likely contribute to treatment outcomes.
Given the small sample size in our study, further research with larger cohorts is necessary to confirm these findings and understand the interplay of genetic variations and other factors affecting drug responses in CRC and NSCLC patients. In personalized medicine approaches, comprehensive profiling of various genetic markers and consideration of other clinical parameters will be essential to optimize treatment strategies and enhance patient outcomes in cancer therapeutics.
ABCB1
The transporter and metabolism genes are widely studied in pharmacogenetics due to their importance in determining an individual’s response to drugs. One of the most significant genes in this regard is the ATP-binding cassette (ABCB1), also known as multidrug-resistance 1 (MDR1), which belongs to the ABC transporter superfamily and produces a protein called P-glycoprotein (P-gp). Overexpression of ABCB1 in tumours has been implicated in multidrug resistance to cancer chemotherapeutic agents [Citation40]. Several ABCB1 polymorphisms have been investigated as predictors of response to drugs, including rs1128503 (C1236T), rs2032592 (2677 T > G) and rs1045642 (C3435T) [Citation41,Citation42]. In particular, these variants have been studied in relation to their impact on the response to 5-FU or capecitabine, a 5-FU precursor, in colorectal cancer patients, with mixed results [Citation43].
According to a study conducted by E. Gonzalez-Haba et al. [Citation43], homozygous or heterozygous carriers of all three analysed single nucleotide polymorphisms (SNPs) in the ABCB1 gene have a lower probability of experiencing moderate-to-severe neutropenia and moderate-to-severe hand-foot syndrome (HFS) during treatment with capecitabine-based therapies compared to GG homozygous carriers. In contrast, the carriership of homozygous wild-type alleles for these SNPs is associated with a lower probability of experiencing moderate-to-severe diarrhoea during treatment with 5-FU-based therapies compared to homozygous TT or heterozygous carriers [Citation43].
The lack of HFS and minimal gastrointestinal toxicity observed in our study contradicts the proposed correlation between the specific variant of the ABCB1 gene and these adverse reactions. However, it is essential to consider that our study had a relatively small sample size and may not fully capture the potential effects of this genetic variant on drug responses and toxicities.
The observation that 10 patients treated with 5-FU/Capecitabine suffered adverse effects and were found to be carriers of the variant raises the possibility of other contributing factors influencing the occurrence of adverse reactions in these patients. These factors may include genetic variations in other genes, individual drug metabolism and clearance rates, and patient-specific characteristics.
Further research with larger patient cohorts and comprehensive genetic profiling is necessary to establish a clearer understanding of the relationship between the 2677 T > G variant of the ABCB1 gene and drug-induced adverse reactions. Identifying the genetic factors associated with treatment-related toxicities is crucial in tailoring personalized treatment plans and minimizing adverse effects in colorectal cancer patients undergoing 5-FU/Capecitabine therapy.
GSTP1
The GSTP1 gene produces an enzyme called glutathione-S transferase (GSTP1), which belongs to a family of detoxifying enzymes known as GSTs. GSTP1 is found in high levels in human tissues, including the liver, prostate, lung and breast. It helps to remove toxins and drugs from the body by attaching glutathione to them. GSTP1 may also be involved in the breakdown of some hormones and eicosanoids. Mutations in the GSTP1 gene have been linked to an increased risk of certain diseases, particularly cancer, although the mechanisms behind this link are not fully understood. Some studies suggest that GSTP1 may contribute to cancer development by promoting resistance to chemotherapy and radiation therapy, while others suggest that it may be involved in the carcinogenic process itself by regulating the metabolism of certain compounds [Citation42].
The c.313A > G variant of the GSTP1 gene has been extensively studied in the context of cancer. The literature shows that patients with the AA/AG genotype and cancer who are treated with fluorouracil and platinum compounds may have a higher risk of toxicity [Citation44] and increased risk for progression and decreased survival [Citation45] with platinum-based treatments with cancer as compared to patients with the GG variant. Moreover, individuals who are homozygous for the AA variant or heterozygous may have a decreased response to treatment with capecitabine, epirubicin and platinum [Citation46] as compared to those who are homozygous for the GG allele.
The high prevalence of the c.313A > G variant of the GSTP1 gene in both colorectal cancer and non-small-cell lung cancer patients in our study suggests its potential significance in influencing treatment outcomes and adverse reactions to chemotherapy.
The observation that the majority of carriers experienced adverse reactions, especially hematological toxicity, reinforces the importance of genetic screening in predicting patient susceptibility to chemotherapy-induced toxicities. Additionally, the occurrence of hematological toxicity in a non-carrier of the variant highlights the complexity of treatment responses, wherein genetic factors alone may not fully explain all adverse reactions.
More extensive studies need to explore the exact mechanisms through which the GSTP1 gene variant affects drug metabolism and response to chemotherapy. Additionally, studying larger patient cohorts with diverse genetic backgrounds and considering other clinical and environmental factors will be essential in refining our understanding of personalized medicine approaches and optimizing treatment strategies for cancer patients.
ERCC2
The ERCC2 gene, also known as XPF, encodes the ERCC2 (Excision Repair Cross-Complementation Group 2) protein, a critical component of the nucleotide excision repair (NER) pathway responsible for repairing DNA damage caused by environmental factors such as UV radiation. ERCC2 protein forms a complex with ERCC1 to create the XPF endonuclease, which recognizes and removes damaged DNA strands. Specifically, the XPF endonuclease generates a break in the DNA strand on the 3′ side of the damage, which permits other NER proteins to remove the impaired region and restore the DNA strand [Citation47]. Variations in the ERCC2 gene have been linked to increased susceptibility to colorectal, non-small cell lung cancer (NSCLC) and breast cancer, with certain inherited mutations inhibiting the cell’s ability to repair DNA damage caused by UV radiation, a major risk factor for various cancer types [Citation48].
The variant ERCC2 c.2251A > C is linked to an elevated susceptibility to colorectal, Non-Small Cell Lung Cancer (NSCLC) and breast cancer [Citation49]. The ERCC2 c.2251A > C variant results in a missense mutation, causing an alteration in the amino acid sequence of the ERCC2 protein. This alteration in the protein sequence disrupts its ability to repair DNA, thereby increasing the likelihood of DNA damage accumulation and the development of cancer. Moreover, research has indicated that this genetic variation is associated with the response to treatment and prognosis of these cancers [Citation50]. Studies suggest that a higher risk of drug toxicity, an increased risk of early relapse and decreased progression-free survival may be associated with the CC genotype in patients with Colorectal Neoplasms receiving fluorouracil and leucovorin or fluorouracil, leucovorin and oxaliplatin therapy compared to the АА genotype [Citation51,Citation52].
The presence of the deleterious variant of the ERCC2 gene in the majority of colorectal cancer patients receiving treatment suggests a potential role of this gene variant in drug response and toxicity. The observation that some non-carriers also experienced adverse reactions highlights the contribution of other genetic and non-genetic factors in determining individual drug responses.
Similarly, in the NSCLC group, while carriers of the ERCC2 variant received platinum-based chemotherapy, not all of them suffered adverse reactions, indicating that the variant may not be the sole predictor of chemotherapy-induced toxicities.
The relatively small cohort size and the complexity of treatment responses make it essential to consider other genetic variations, environmental factors, and individual patient characteristics when assessing the overall risk of adverse drug reactions.
Further research with larger patient cohorts and comprehensive genetic profiling is necessary to validate the role of the ERCC2 gene variant and identify additional factors influencing chemotherapy outcomes. Such knowledge will be critical in tailoring personalized treatment strategies and optimizing therapeutic interventions for cancer patients to minimize adverse effects and enhance treatment efficacy.
SLC19A1
The reduced folate carrier (RFC), encoded by the human solute carrier family 19, member 1 (SLC19A1) gene, plays a crucial role in transporting folates across the cell membrane. Folate is an essential B-vitamin required for DNA synthesis and repair, making it an important factor in cancer development and progression. Lower RFC expression has been linked to an increased risk of certain cancers, such as breast and colon cancer [Citation52]. In addition, individuals with lower RFC expression and genetic mutations or polymorphisms in the SLC19A1 gene are at a higher risk of developing cancer, and lower RFC expression has been associated with poor outcomes in patients with these types of cancer [Citation39].
The c.80A > G variant of the SLC19A1 gene has been shown to improve the response to various chemotherapeutic agents, such as capecitabine, fluorouracil, irinotecan and leucovorin, in people with colorectal neoplasms [Citation53]. Additionally, carriers of this variant have increased progression-free survival time when treated with platinum-based chemotherapy for lung cancer [Citation54].
The observation that both carriers and non-carriers of the c.80A > G variant experienced adverse reactions in both colorectal cancer and non-small cell lung cancer groups in our study raises questions about the variant’s relevance as a sole predictor of drug response or toxicity. These findings suggest that other genetic variations and non-genetic factors may play a more significant role in determining individual treatment outcomes. Additionally, the complexity of chemotherapy response in cancer patients is influenced by multiple genetic, environmental, and clinical factors, making it challenging to attribute drug reactions solely to a single genetic variant. Further research with larger and diverse patient cohorts is necessary to validate these findings and explore the interaction of the c.80A > G variant with other genetic and clinical factors.
Accumulating evidence shows that liquid biopsies have the potential to detect germline mutations, and this detection can have significant implications not only for the patient but also for their family members by serving as a mechanism for cancer prevention [Citation2]. Precision oncology involves using genomic information from tumours to guide therapeutic decision-making. Clinicians need to be aware of the potential to further identify germline alterations, as these can have significant therapeutic and preventive implications for both the patient and their family. As the use of liquid biopsies becomes more common in clinical practice for treatment decisions and targeted therapies, it is critical to develop an algorithm for identifying and confirming potential germline mutations identified through this testing. A standard method of analysis and interpretation of test results is essential to avoid missed opportunities for prevention in both the patient and their at-risk family members [Citation2].
Conclusions
In this study, we applied the liquid biopsy approach to screen for germline mutations potentially associated with chemotherapy-induced toxicity in colorectal and non-small cell lung cancer patients. Although the number of cancer patients included in our study was relatively small, it is noteworthy that our findings suggest that liquid biopsy is a highly sensitive tool for detecting germline variants that have pharmacogenetic importance. In this paper, we focused on discussing the most common polymorphisms found in our patient population. However, to fully understand the potential association between less frequent gene variants and their implications for cancer pharmacogenetics, a larger cohort of individuals would be needed to confirm or refute such associations.
Ethics statement
Ethics approval for this study was obtained from the Institutional Review Board (IRB) of The Scientific Ethics Committee at Medical University – Plovdiv (ref. no. C – 09-2/10.04.2020). All participants provided written informed consent prior to their inclusion in the study. The study was conducted in accordance with the ethical guidelines and regulations set forth by the IRB.
Author contributions
NM-M: Conceptualization, Data analysis and interpretation, Writing- Original draft preparation; HI: Supervision, Software, Writing- Editing; VP, GR, ZG-P: Patient selection and blood sampling; VS: Supervision, Writing- Reviewing and Editing. All authors have read and approved the final version of the paper.
ORCID detail
Nelly Miteva-Marcheva: 0000-0002-0921-9020.
Acknowledgements
The genotyping was performed by Novogene Co Ltd. (https://www.novogene.com/us-en/).
Disclosure statement
No potential conflict of interest was reported by the author(s).
Data availability statement
The anonymized data that support the findings of this study are available from the corresponding author, [NM], upon reasonable request.
Additional information
Funding
References
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