Genetic Polymorphisms of XPC, XPD, XPG Genes and their Association with Radiotherapy Induced Toxicity among Head and Neck Cancer Patients: A Hospital Based Study from Maharashtra

Background: The present study was planned to investigate possible association of single nucleotide polymorphisms (SNPs) of nucleotide excision repair (NER) genes such as XPC, XPD, XPG with acute radiation induced toxicities such as skin reactions and oral mucositis in normal tissue from head and neck cancer (HNC) patients receiving radiotherapy. Methods: Two hundred and fifty HNC patients receiving radiotherapy were enrolled in this study and the acute toxicity reactions and radiation response were recorded. Association of SNPs rs2228001 of XPC, rs238406, rs13181 of XPD and rs17655 of XPG gene with normal tissue reactions in the form of dermatitis and mucositis were studied by PCR-RFLP and direct DNA sequencing. Results: The results of univariate analysis of SNPs of XPC, XPD and XPG showed that XPC polymorphism at codon 939 of exon 15 (A>C) was not associated with dermatitis (OR=0.30, 95% CI: 0.06-1.39; p=0.125), or oral mucositis (OR=1.14, 95% CI: 0.41-3.20; p=0.793). The XPD codon 156 of exon 6 (C>A) and codon 751 of exon-23 A>C) polymorphism showed no association with radiosensitivity in HNC patients (OR=1.50, 95% CI: 0.60-3.71; p=0.080) for dermatitis, (OR=1.54, 95% CI: 0.66-3.61; p=0.312) for oral mucositis. The 1104 Asp variant genotype or allele of XPG (OR=1.35 95% CI: 0.50-3.64; p=0.541) showed no association with degree of radiotherapy associated dermatitis or mucositis (OR=0.80, 95% CI: 0.32-2.03; p=0.648) in HNC patients. The variant C allele of 2920 A/C genotype of XPC gene at codon 939 of exon 15, found protective with developing skin reactions with grade >1 (OR=0.60, 95% CI: 0.36-0.97; p=0.039) in HNC patients treated with radiotherapy. Conclusion: The results obtained in this study concluded that the SNPs rs2228001of XPC, rs238406, rs13181 SNPs of XPD and rs17655 SNP of XPG are not associated with normal tissue toxicity in HNC patients treated with radiotherapy. Radiotherapy with high radiation dose was significantly associated with oral mucositis in response to radiotherapy.


Introduction
Head and neck cancer (HNC) is a common type of cancer in India constituting 30 % of all cancers and accounting 245, 811 new cases and 130, 139 deaths in year 2022 [1].The HNC is complex which specifies

Genetic Polymorphisms of XPC, XPD, XPG Genes and their Association with Radiotherapy Induced Toxicity among Head and Neck Cancer Patients: A Hospital Based Study from Maharashtra
stress and DNA repair pathways [2].The commonly followed treatment of locoregionally advanced HNC is surgery followed by adjuvant radiation therapy or concurrent chemo-radiation [3,4].Systemic radiotherapy used in HNC treatment is known to cause significant toxicities to normal healthy cells.When radiation therapy is administered, the normal tissue adjacent to the target cancer area are exposed to radiation and resulted into common acute tissue toxicities such as mucositis, dysphagia, dermatitis [5][6][7].Large group of patients suffered with normal tissue reactions and there is no obvious justification for the severity of normal tissue reactions, however, it is usually presumed that genetic components of an individual might be accountable for Editorial Process: Submission:08/26/2023 Acceptance:01/24/2024 1 Department of Oncology, Krishna Vishwa Vidyapeeth "Deemed to be University", Taluka-Karad, Dist-Satara, Maharashtra) India. 2 Department of Molecular Biology & Genetics, Krishna Vishwa Vidyapeeth "Deemed to be University", Taluka-Karad, Dist-Satara,Maharashtra, India.*For Correspondence: hodgeneticslab@kvv.edu.inclinical radiosensitivity.Ionizing radiation directly affects DNA structure and induces DNA lesions including single strand breaks (SSBs) and double strand breaks (DSBs) in tumor cells, but also the genomic stability of normal cells is affected by radiotherapy.Nucleotide excision repair (NER) is an important pathway used by individuals for removal bulky DNA lesions formed by radiation or other chemical mutagens [8].Xeroderma pigmentosum complementation group C (XPC), Xeroderma pigmentosum complementation group G (XPG) and Human xenoderma pigmentosum complementation group D (XPD) are the major components of NER pathway functioning in the repair of ionizing radiation induced oxidative damage [9] Genetic variations in DNA double strand break repair genes such as single nucleotide polymorphisms (SNPs) could interfere with alteration of gene function, which make them suitable candidate genes for the study of genetic susceptibility of an individual towards acute normal tissue reactions after radiation therapy [10,11].Several other reports documented association of genetic variants of DNA repair genes with radiation induced adverse reactions in normal tissue of an individual in response to radiotherapy [12][13][14], however, some other reports declined their clinical significance in normal tissue toxicity [15,16].XPC and XPG genes are highly polymorphic in nature, where A>C transversion at 2920 position in exon 15 of XPC resulted into Lys939 Gln polymorphism (rs2228001) and C>G transversion at position 3507 in exon 15 of XPG gene resulted into His1104Asp polymorphism (rs17655).Similarly, XPD gene showed Arg156Arg polymorphism at codon156 of exon6, Ile199Met at codon199 of exon 8, Asp312Asn at codon312 of exon 10 and Lys751Gln at codon 751 of exon 23.Both XPC and XPD gene polymorphism reported their association with radiation induced toxicity in variety of cancers where XPC Lys939Gln (rs2228001) and XPD Lys751Gln (rs13181) were associated with radiotherapy outcomes in HNC [17,18], Non-small cell lung cancer [19] however, other studies observed different outcomes with no association of those genes with adverse effects of radiotherapy in breast [20,21], prostater [22] and lung cancer [23].However, the association between xenoderma pigmentosum group genes and their role in clinical radiosensitivity is obscure; therefore this postulation brings our interest into research to understand the genetic basis of radiation adverse effects on normal tissue during radiotherapy in HNC patients.In present study, we evaluated possible association of three genes XPC (Lys939Gln (A>C) (SNP: rs2228001), XPD (Arg156Arg, C>A, SNP: rs238406; Lys751Gln SNP: rs13181) XPG (His1104Asp, C>G SNP: rs17655) and risk of acute skin toxicity after therapeutic radiotherapy in HNC patients.Written informed consent was obtained and patients were explained about their participation in the study.The detailed demographic, pathology, clinical examination findings and three months post radiotherapy follow up information were recorded for all patients as described in Table 1.

Study Subjects and Clinical Information
Response Evaluation Criteria in Solid Tumors (RECIST) criteria was followed for the documentation of clinical and radiological responses.For determining acute normal skin toxicity effects, Patients were followed up for three months post radiotherapy treatment to review the clinical response (Partial/complete/no response), stable, progressive disease, early death from disease, toxicity or any other cause.

Tre a t m e n t o f p a t i e n t s w i t h r a d i o t h e r a p y & chemo-radiotherapy
All patients were treated using Linear accelerator (Model: Unique Performance, Make: Varian Medical System, USA) 6-Mega Volt (MV) (X-ray) with total radiotherapy dose of 60-66 Gy (2 Gy per fractions for 5 days a week) with volumetric modulated arc therapy (VMAT) technique.Patients after surgical resection having positive margins were given a dose of 66 Gy in 33 fractions.Patients with no positive margins were given 60 Gy in 30 fractions.Chemotherapy was added if clinically indicated and the drug used was Cisplatin at doses of 40 mg/m 2 every week given for 5-6 doses along with RT.

Follow up and Toxicity Assessment
Patients were followed up for three months after completion of radiotherapy for assessment of toxicity and response.Acute adverse effects (oral mucositis and skin reaction) were recorded during and after completion of radiatiotherapy according to Radiation Therapy Oncology Group (RTOG) criteria.Acute radiation toxicity effects were defined as injuries appearing from the initial day of radiotherapy treatment until 3 months after the end of radiotherapy.The post-radiotherapy toxicity effects on normal skin and oral mucosa were recorded weekly.The severity of acute radiation injury was determined by radiation oncologist and severity of acute radiation dermatitis and oral mucositis was graded according the RTOG grading system.For comparing the severity of radiation toxicity, the skin reactions were graded (>1 grade) severe skin reactions versus ≤ 1 grade skin reactions.The patients with oral mucositis grade >2 are radiosensitive group (cases) compared with ≤2 grade (controls).

DNA extraction and single nucleotide polymorphism genotyping assays
Five milliliter (mL) of whole blood from patients was collected before initiation of radiotherapy treatment.whereas 66.00 % patients receive combination of radiation therapy and chemotherapy.A total of 65.60 % patient's tumor was more than 2 cm size where as 34.40 % tumor was ≤ 2cm in size.Out of total 250 patients, 132 (52.80%) patients showed oral mucositis reactions (grade >2) (grade 3 and 4) and 47.20 % patients experienced grade ≤2 mucositis.Preliminary objective of the study was to investigate acute radiation induced toxicities such as dermatitis and mucositis in HNC patients after exposure to radiotherapy.When acute skin reactions were observed in response to radiation adverse effects, out of 250 patients Genomic DNA extraction from peripheral blood sample was carried out using HipurA®Blood genomic DNA miniprep purification kit following the manufacturer's instructions.This purified DNA was used for genotyping assays.Genotyping of XPC, XPD and XPG gene SNPs was performed by PCR-RFLP and direct DNA sequencing methods with appropriate primer sets presented in Table 2.The polymerase chain reaction amplification was performed in a total 20 micro liter (µL) reaction mixtures containing1X PCR buffer (10 mili molar (mM) Tris-HCl (pH 9.0), 50 mM KCl 1.5 mM MgCl2, 0.01% gelatin), 200 µM each dNTP, 10 picomole (pmol) of each primer listed in Table 1, 1U Taq DNA polymerase (GeNei, Merck Bioscience) and 100 nanogram (ng) of purified genomic DNA template.Each PCR reaction was performed with DNA denaturation at 95 °C for 10 minutes followed by 35 cycles of 95 °C for 30 seconds, and primer annealing for 30 seconds and extension at 72°C for 30 seconds lastly final extension at 72°C for 10 minutes for all gene amplification mixtures.After confirmation of DNA amplification, each PCR product (10 µl) was digested with an appropriate restriction enzyme at 37°C for 16 hours in 20 µL reaction mixtures containing buffer supplied with each restriction enzyme (Table 2).After the overnight incubation, digestion products were separated on 2-3% low EEO agarose (GeNei) gel at 100 V for 30 min, stained with ethidium bromide and photographed with Gel Documentation System (BioRad).

Statistical Analysis
The genotypic frequencies for NER genes (XPC, XPD and XPG) in the patient's were determined.The Odds Ratio (OR) and corresponding 95% confidence intervals (CI) were determined through unconditional multiple logistic regression.The OR estimated to test whether any association exists between the grade of acute toxicity and selected SNPs.The correlation between confounding factors and radiation toxicities were evaluated by the χ2 test.The p values <0.05 were considered as statistically significant.The event of occurrence of clinical severity of post-radiotherapy adverse effects are defined as skin reactions with >1grade and oral mucositis scored as grade >2.All statistical analyses were carried out using SPSS 11 Software.

Demographic and Clinical characteristics of study population
Two hundred and fifty patients of age range 25-85 years with median age 55 years were enrolled in the study and distribution of patients based on clinical characteristics, demographic information, histopathological grading and toxicity grades are presented in Table 1.187 patients were males and 63 were females.Majority of the patients were tobacco smokers (74.80%) as compare to non-smokers (25.20%).When we grouped the HNC patients based on primary site of cancer we observed oropharynx (42.0%), hypopharynx (

Genotype distribution of XPC, XPD and XPG gene SNPs and radiotherapy toxicity in HNC patients
The univariate analysis of genotype polymorphism of XPC, XPD and XPG genes and their association with radiation induced acute normal tissue toxicity such as skin dermatitis and oral mucositis are presented in Table 3.Based on severity of radiation toxicity we grouped patient normal tissue toxicity as grade ≤1 or >1 for skin reactions and grade ≤2 or >2 for oral mucositis as prescribed by RTOG criteria.Out of 250 patients treated with radiotherapy 63 patients showed grade >1 normal skin reactions such as bright erythema, moderate edema, patchy moist desquamation, increased pain, secretion of yellow exudates, itching and tightening of skin, and 187 patients reported grade ≤1 skin reactions including faint erythema, itching and tightening of skin.A total of 132 patients showed grade >2 oral mucositis reactions including severe pain, fibrinous mucositis, ulceration, hemorrhage and necrosis whereas 118 patients with grade ≤ 2 mucositis reactions with irritation, patchy mucositis, serosanguinitis discharge with moderate pain in response to radiation exposure.When we studied rs2228001 SNP of XPC, rs238406, rs13181 SNPs of XPD and rs17655 SNP of XPG, the results of univariate analysis showed that none of the variant genotypes of SNPs showed association with radiotherapy induced toxicity effects on normal tissue reactions.When we analysed polymorphism of XPC gene at 939 Lys/Gln of exon 15, we observed significant decrease of variant 'C' allele in patients with skin reactions with grade >1 (OR=0.60,95% CI: 0.36-0.97;p=0.039) which indicated negative association with protective effects of C allele in developing severe toxicity reactions in normal skin of the patients exposed to radiotherapy.When the polymorphism of XPD codon 156 (exon-6) and codon 751 (exon-23) were analyzed, there was no statistically significant difference between homozygous recessive or heterozygous genotypes with either of skin reactions or oral mucositis.Similarly, XPG 1104 His/Asp showed no association with any of the toxicity effects in HNC patients in response to radiotherapy treatment.

Correlation between of clinical factors and effects of radiotherapy
The relationship between acute normal skin reactions, mucositis and the confounding factors of age, gender, alcohol, tobacco consumption, chemotherapy, radiotherapy, radiation dose were evaluated and represented in Table 4. Out of 250 HNC patients investigated in this study, 63 patients showed acute radiation dermatitis (>1 grade) and 132 patients with oral mucositis (>2 grade).The tobacco smoking habit in HNC patients found to be associated with grade >2 oral mucositis (p=0.002).The radiotherapy with combination of chemotherapy also showed significant association with oral mucositis in HNC patients treated with radiotherapy (p<0.0001).We also observed that 45.45 % patients treated with 66 Gy radiation showed significant correlation with development of oral mucositis (p=0.006).None of the confounding factors showed significant correlation with development of severe skin reaction in HNC patients.

Association of XPC, XPD, XPG gene polymorphisms with histological tumor grade, tumor and node response towards radiotherapy in HNC patients
The distribution of SNPs of XPC (A2920), XPD (C22541A, A35931C) and XPG (C3507G) genotypes in 250 HNC patients was studied by logistic regression analysis.The results of univariate analysis carried out to study the association of XPC, XPD and XPG genotypes with histological tumor stage along with histopathological grade were represented in Table 5.The univariate analysis by logistic regression analysis showed that neither of rs2228001 SNP of XPC nor rs17655 SNP of XPG was associated with advanced tumor stages (T3, T4).Regarding XPD (C22541A, A35931) SNPs, no significant difference was found between early or advanced tumor stages and variant genotype distribution (p=0.207;0.396), where C/C, C/A, A/A genotype of (C22541) and A/A, A/C, C/C genotypes of (A35931C) polymorphisms were found with same frequency in early and advanced tumor stages.When rs238406 and rs13181 SNPs of XPD studied for their association with histological tumor grade, the variant C/C genotype of (A35931C) polymorphisms of XPD showed significant negative association with the advanced tumor grades (grade III and IV) (OR=0.26,95% CI: 0.12-0.56;p=0.0007).The association between genotypes of XPC, XPD, and XPG genes and the response to radiotherapy in HNC patients are shown in Table 6.None of the SNPs of XPC (rs2228001), XPD (rs238406, rs13181) and XPG (rs17655) showed significant difference for partial or no response of tumor or nodes.The genotypes of XPC, XPG or XPD showed no association with radiotherapy response in tumor or nodes after three months of evaluation.

Discussion
Head and neck cancer is a second most common cancer and leading cause of cancer-causing deaths in India.The commonly pursued treatment modality for HNC is surgery followed by chomoradiation or radiotherapy alone.Radiotherapy is an important treatment of HNC given in fractions for improving treatment outcomes.However, radiotherapy causes radiation induced toxic reactions to the normal tissue of patients.It is important to identify genetic determinants of the patients which may be associated with decreasing efficacy of radiotherapy in patients.Numerous studies have been carried out on the genetic variants of various pathway genes including DNA repair genes and their role in cancer development; however limited literature is available on their association with radiotherapy treatment outcomes.In this regard, we tried to explore the role of SNPs of BER genes XRCC1, XRCC2, XRCC3 and found no association of either of the gene with the susceptibility towards radiotherapy toxicity in HNC patients of south-western Maharashtra [24].In continuation with our earlier study on base excision repair (BER) genes, this study was intended to investigate possible association of genetic variants of NER pathway genes including XPC, XPD, XPG genes with risk of developing radiotherapy induced severe normal tissue toxicity in HNC patients.The SNPs of three NER pathway genes; XPC (2920 A>C, rs2228001), XPD (22541 C>A rs238406; 35931 A>C, rs13181), XPG (3507 C>G, rs17655) were studied, which showed no significant association with radiation induced severe skin reactions or mucositis in studied HNC patients.
To the best of our knowledge no published information available about the association of xenoderma pigmentosum group genes (XPC, XPD, XPG) polymorphisms and the increased acute tocxicity caused by radiotherapy in HNC patients.The only studies are available on association of base excision repair genes including XRCC1 and XRCC3 SNPs and their significant correlation with increased risk of developing acute normal skin reactions in response to radiotherapy in HNC patients [25][26][27][28].The significant association of polymorphism in NER genes including XPC, XPD and XPG genes with acute toxicity of radiotherapy was reported earlier for bladder [29], lung cancer [19].Few studies derived negative outcomes with no association of any of the polymorphisms in XPC or XPD genes with radiosensitivity in breast [20,21], prostate [22] and lung cancer [23].The polymorphism of XPC, XPD, XPG gene and their association with radiosensitivity is reported earlier in HNC for XPC Lys939Gln (rs2228001) and XPD Lys751Gln (rs13181) SNPs [17,18].
In contrast, our results depicted no association of SNPs of three XPC (rs2228001), XPD (rs238406; rs13181), and XPG (rs17655) with radosensitivity producing adverse where we are unable to prove association of XPC, XPD or XPG gene polymorphisms with risk of developing clinically significant skin reactions or oral mucositis in the HNC patients treated with radiotherapy from Indian population.This could be because of small sample size and few numbers of SNPs for genotyping.
In conclusion, the results obtained in this study concluded that the SNPs rs2228001of XPC, rs238406, rs13181 SNPs of XPD and rs17655 SNP of XPG are not associated with increased normal tissue toxicity HNC patients treated with radiotherapy.Further investigation of genetic variants and their association with radiotherapy response and its adverse toxicity effects need to be carried out to confirm our findings by using more number of samples and SNPs of genes for genotyping.

Table 1 .
Details of Baseline Demographic and Clinico-pathological Characteristics of Head and Neck Cancer Patients Enrolled in the Study

Table 2 .
The List of Candidate XPC, XPD, XPG Genes with Details of PCR and RFLP Procedures Including Primers and Restriction Enzymes and Expected Products of Selected Genes.

Table 3 .
Univariate Analysis of Candidate SNPs of XPC, XPD, XPG Genes and Radiation Induced Skin Reactions and Mucositis in Head and Neck Cancer Patients SNP, Single nucleotide polymorphism; OR, Odds ratio, CI, Confidential interval; Significance p< 0.05; *, Indicates significant Odds Ratio (p<0.05),p value determined based on χ2

Table 4 .
Association of Clinicopathological Factors and the Risk of Radiation Induced Acute Skin Reactions in Head and Neck Cancer Patients

Table 5 .
Association between Genotypes of XPC, XPD, XPG Genes with Tumor Stage and Tumor Grade in Head and Neck Cancer Patients SNP, Single nucleotide polymorphism; OR, Odds ratio; CI, confidence interval; Significance p< 0.05; *, Indicates significant Odds Ratio (p<0.05);p value determined based on χ2

Table 6 .
Association between Genotypes of XPC, XPD, XPG Genes with Tumor and Node Response in Head and Neck Cancer Patients Towards Radiotherapy SNP, Single nucleotide polymorphism; CR, Complete Response; PR, Partial Response; NR, No Response; RR, Risk ratio, CI, Confidence interval; Significance p< 0.05; *, Indicates significant Odds Ratio (p<0.05),p value determined based on χ2