Genetic variability in cisplatin metabolic pathways and outcome of locally advanced head and neck squamous cell carcinoma patients

Advanced head and neck squamous cell carcinoma (HNSCC) patients have been treated with cisplatin (CDDP) chemoradiation, and the variability of treatment effects has been attributed to single nucleotide variants (SNVs) in genes of metabolic pathways. This study investigated the roles of GSTM1, GSTT1, GSTP1 c.313A>G, XPC c.2815A>C, XPD c.934G>A and c.2251A>C, XPF c.2505T>C, ERCC1 c.354C>T, MLH1 c.93G>A, MSH2 c.211+9C>G, MSH3 c.3133G>A, EXO1 c.1765G>A, TP53 c.215G>C, CASP3 c.-1191A>G and c.-182-247G>T, FAS c.-1378G>A and c.-671A>G and FASL c.-844C>T SNVs in outcome of 109 patients treated with CDDP chemoradiation. Genotypes were identified in genomic DNA by PCR-based methods. Conventional criteria and tests analyzed response and survival. Patients with XPC c.2815AC or CC had 3.43 times more chances of presenting partial response or stable disease. Patients with FAS c.-671GG, GSTM1 present plus XPC c.2815AA, or plus XPD c.934GG, or plus XPD c.2251AA, or plus TP53 c.215GC or CC, and XPD c.2251AA plus XPF c.2505TT had up to 2.70 and 2.37 times more chances of presenting tumor progression and evolving to death, respectively. Our data indicate, for the first time, preliminary evidence that combined SNVs of CDDP metabolism act as independent prognostic factors and can be used to select patients for distinct treatments.


Treatment, response rate, and survival
The single daily fractionated RT (70 Gy at 2 Gy/day) with concurrent bolus CDDP (80-100 mg/m 2 ), given on days 1, 22, and 43, were administered to HNSCC patients; those with consistent side effects during treatment received CDDP at a lower dose (50-75 mg/m 2 ) 16 .Patients who failed to respond to their initial treatment regimen or relapsed received intravenous methotrexate as palliative chemotherapy 40 .RR to CDDP chemoradiation was assessed as complete response (CR), partial response (PR,) or stable disease (SD), using Response Evaluation Criteria in Solid Tumors (RECIST) guidelines version 1.1 41 .
EFS was defined from the date of diagnosis and the date of progression, relapse, or death by disease.OS was determined from the date of diagnosis and death by any causes or last follow-up.Patients were followed at 3-month intervals, and the end of the follow-up period considered for the present study was June 2022.

Statistical analysis
Single SNVs and combinations of two SNVs with biological significance were analyzed in the study.To analyze the roles of deleterious isolated and combined genotypes related to normal or greater detoxification of CDDP, reduced apoptosis of cells damaged by CDDP, and regular or greater repair of lesions induced by CDDP based on information presented in Tables 1 and S2, in the outcome of HNSCC patients was the focus of the study.

Clinicopathological aspects of patients
The median age at diagnosis of 109 HNSCC patients enrolled in the study was 56 years.Most of the patients were male, smokers, and drinkers, and had tumors located in the larynx and pharynx, moderately differentiated tumors, and tumors at advanced stages.Median BMI was within the ordinarily acceptable range, and HPV type 16 was negative in all analyzed cases (Table 2).

Response rate
The CR, PR, and SD were seen in 23.9%, 70.4%, and 5.7% of 88 available HNSCC patients, respectively.
All clinicopathological aspects and single and combined SNVs with biological significance in response to CDDP chemoradiation in 88 patients evaluated are presented in Supplementary Tables S3 and S4, respectively.
Factors with significant associations with the response rates to CDDP and RT are presented in Table 3.In univariate analysis, patients with T3 or T4 tumors, N2 or N3 nodal status, and FAS c.-1378GG genotype had 2.83, 4.77, and 2.67 times more chances of presenting PR or SD than those with T1 or T2 tumors, N1 or N2 nodal status and FAS c.-1378GA or AA genotype, respectively.In multivariate analysis, patients with T3 or T4 tumors, N2 or N3 nodal status, XPC c.2815AC or CC genotype had 3.05, 4.32, and 3.43 times more chances of presenting PR and SD than patients with the remaining aspects, respectively.Combined genotypes of analyzed SNVs did not consistently alter the response to CDDP chemoradiation.

Survival
The impact of all clinicopathological aspects and isolated and combined genotypes of detoxification, DNA repair, and apoptosis-related SNVs in the survival of 109 patients are presented in Supplementary Table S5.
Factors with significant associations with patients' survival are presented in Table 4.The median follow-up time of HNSCC patients was 22 months (range: 3-126).
At 24 months of follow-up, EFS was lower in patients with specific tumor aspects and genotypes (Kaplan-Meier estimates).In univariate Cox analysis, patients with T3 or T4 tumors, tumors at III or IV stage, ERCC1 c.

Discussion
The effects of CDDP and RT have been associated with genetic variability in distinct metabolic pathways [9][10][11][12][13][14][15][16][17][18] .Since patients may inherit defects in more than one pathway, we investigated in the current analysis of this prospective study the roles of eighteen SNVs involved in intracellular detoxification, DNA repair, and apoptosis pathways in the outcome of HNSCC patients treated with CDDP chemoradiation.We analyzed only combinations of two SNVs to obtain more consistent results.
We found that patients with large local tumors (T3 or T4) or tumors with large extensions to lymph nodes (N2 or N3) had 3.05 and 4.32 more chances of presenting PR or SD than others in multivariate Cox analysis.The worse response to therapy in these patients was expected in the study because when the tumor size advances, Table 2. Clinicopathological aspects of 109 patients with head and neck squamous cell carcinoma.N number of patients.*The number of patients differed from the total quoted in the study because it was not possible to obtain available tumor fragments and consistent information about the treatment and response rate in some cases.www.nature.com/scientificreports/cells change the epithelial-mesenchymal transition 53,54 , having chemotherapy resistance due to abnormalities of cellular drug accumulation, DNA repair, and cytosolic drug inactivation as consequences 55 .
In the present multivariate Cox analysis, we observed that patients with XPC c.2815AC or CC had 3.43 times more chances of achieving PR or SD than others.We found an association between XPD c.934G>A and haplotype of XPD c.934G>A and c.2251A>C SNVs 15 and EXO1 c.1765G>A and haplotype of EXO1 c.1765G>A and c.2270C>T SNVs 17 , but not of XPC c.2815A>C 15 , with response to CDDP chemoradiation in part of this cohort of patients (n = 88) in a previous analysis of NER or MMR pathways, respectively.It is possible the inclusion of new HNSCC patients (n = 21) in the current study may shed light on the real roles of the SNVs in response to CDDP chemoradiation.The association of XPC c.2815AC or CC with worse response to CDDP and RT was not expected in the study because the C allele of XPC c.2815A>C SNV was previously associated with lower DNA repair 22 , which seems to induce a better response to therapy.Nevertheless, Khan (2000) 56 did not demonstrate a clear difference in the rate of nucleotide excision repair in the evaluation of the A and C alleles.Thus, further functional studies are needed to define the fundamental role of XPC c.2815A>C SNV in the DNA repair of CDDP lesions.
We found in multivariate Cox analysis that patients with advanced tumor size, T3 or T4, had 2.33 more chances of presenting tumor progression, relapse of tumor or death by effects of the disease, and 1.94 more chances of evolving to death than those with localized tumors in multivariate Cox analysis.These associations were also seen in other studies [57][58][59] , and again, these results may be attributed to changes in morphology and behavior of tumor cells during tumor growth 53,54 , favoring the dissemination of tumors 60,61 and short survival 55 .
We observed in multivariate Cox analysis that patients with the FAS c.-671GG genotype had lower EFS and OS than those with the remaining genotypes and nearly two times more chances of presenting tumor progression, relapse of the tumor, or death than others.Our group previously published this result in the analysis of SNVs on genes of apoptosis pathways in the same cohort of patients 18 .The allele G was previously associated with reduced apoptosis of colorectal cancer cells because it affects the coupled binding of transcription factors SP1 and STAT1 to chromatin, altering complex recruitment for transcriptional activation 33 .It is also biologically plausible that the allele G attenuates transcriptional activation mediated by the SP1/STAT1 FAS complex in HNSCC, which in turn dampens the apoptotic pathway of FAS due to its dysregulated expression 32,62 , favoring the survival of tumor cells, tumor progression or relapse of tumor and death in patients with FAS c.-671GG genotype.Combinations of FAS c.-671A>G genotypes with other SNVs did not alter the EFS of HNSCC patients in the current analysis.The number of patients stratified by combined genotype may not have been sufficient to identify associations with patient survival.
Patients with GSTM1 present plus XPC c.2815AA, GSTM1 present plus XPD c.934GG (HR: 2.45 for EFS, HR: 2.37 for OS), GSTM1 present plus XPD c.2251AA (HR: 1.93 for EFS, HR: 1.90 for OS), GSTM1 present plus TP53 c.215GC or CC (HR: 1.98 for EFS, HR: 1.93 for OS), and XPD c.2251AA plus XPF c.2505TT (HR: 2.0 for EFS, HR: 1.87 for OS) had more chances of tumor progression, relapse of tumor or death than others, but changes in The presence of GSTM1 9 , XPD c.934GG 11,12 , and XPD c.2251AA 12 genotypes were associated with shorter EFS in previous studies conducted by other groups.XPD c.934AA genotype was associated with lower EFS (HR: 2.12) and OS (HR: 2.04), but XPF c.2505T>C did not alter the survival of 90 HNSCC patients treated with CDDP and/or RT in a previous analysis of this prospective study, which focused on SNVs on genes of NER pathway 15 .It is worth commenting that previous and current analyses were based on a small number of patients with XPD c.934AA genotype (n = 10) and were adjusted by different variables.Isolated TP53 c.215G>C SNV did not alter the survival of 109 HNSCC enrolled in previous 18 and current analyses of this prospective study.
As far as our knowledge goes, there are no studies about associations of genotypes of different pathways of CDDP metabolism with the survival of HNSCC patients treated by CDDP chemoradiation.Associations of the above-mentioned combined genotypes with short survival were expected in the study.GSTM1 present enhances CDDP detoxification of cells 4 and the alleles A of XPC c.2815A>C 22 , G of XPD c.934G>A 23 , A of XPD c.2251A>C 23 , T of XPF c.2505T>C 24 , and C of TP53 c.215G>C 29 SNVs induce greater DNA repair and less apoptosis of damaged cells, respectively, favoring higher survival of tumor cells and lower survival of HNSCC patients.It is possible that the sum or synergism of functional abnormalities, such as detoxification and apoptosis, as seen in GSTM1 present plus TP53 c.215GC or CC combined genotype, and repair double defect, as seen in XPD c.2251AA plus XPF c.2505TT is necessary to alter the survival of HNSCC cells and HNSCC patients´ survival in the current analysis, and this the most plausible explanation for the association of combined genotypes but not of isolated genotypes with the survival of patients enrolled in the recent analysis of this prospective study.
It is worth commenting that patients' survival was not substantially altered by isolated or combined genotypes of GSTP1 c.313A>G, EXO1 c.1765G>A, and MSH3 c.3133G>A SNVs in the current analysis of this study, but GSTP1 c.313GG genotype was associated with lower EFS in a previous analysis of the same cohort of patients (n = 90) 14 and EXO1 c.1765GG and MSH3 c.3133GG genotypes were associated with lower EFS and OS, respectively, in a large sample of HNSCC (n = 397) analyzed previously by our group 16 .The number of patients and statistical adjustments in previous and current analyses may explain differences in results found by our group.
In summary, our data present isolated XPC c.   www.nature.com/scientificreports/with CDD chemoradiation.We are aware that although a considerable number of patients were included in this complex and prospective pharmacogenetic study, we believe that a larger cohort of patients and additional functional analyses of XPC c.934G>A SNV in DNA repair may shed light on the roles of the SNVs in response and survival of HNSCC patients treated with CDDP chemoradiation.Thus, we believe that if our data is validated in further studies, specific SNVs on genes of CDDP metabolism can be used to select HNSCC with a high risk for unfavorable outcomes for a differentiated treatment.
2815A>C and FAS c.-671A>G SNVs, and for the first time, associations of GSTM1 with XPC c.2815A>C, XPD c.934G>A, XPD c.2251A>C and TP53 c.215G>C, and XPD c.2251A>C with XPF c.2505T>C SNVs, as independent factors for the outcome of HNSCC patients treated

Table 1 .
Functional roles of the wild and variant alleles of single nucleotide variant on genes enrolled in cisplatin metabolism.SNV single nucleotide variant, ND normal detoxification, RD reduced detoxification, NR normal repair, RR reduced repair, NA normal apoptosis, RA reduced apoptosis.

Functional analyses Wild allele Function Variant allele Function Reference
Differences between clinicopathological aspects and genotypes of SNVs in RR were analyzed by Fisher's exact test in univariate Cox analysis.Only variables with more than ten individuals in each group and presenting P-values ≤ 0.20 were included in the multivariate Cox analysis, and the logistic regression model assessed associations of variables and RR, with odds ratios (OR) values and 95% confidence intervals (95% CI).Significant results were internally validated using a bootstrap resampling study to investigate the stability of risk estimates (1,000 replications).Kaplan-Meier method, log-rank test, and univariate and multivariate Cox analyses identified variables predicting EFS and OS, with hazard ratios (HR) values and 95% CI.All variables with P-value ≤ 0.20 in univariate Cox regression analysis were included in multivariate analysis.All statistical tests were done using the SPSS 15.0 software (SPSS Incorporation, Chicago, IL, USA), and significance was achieved when P-values were ≤ 0.05.

Table 3 .
Clinicopathological aspects and single nucleotide variants in response to cisplatin chemoradiotherapy in 88 patients with head and neck squamous cell carcinoma.N number of patients, CR complete response, PR partial response, SD stable disease.*OR: odds ratio adjusted by age, tumor size, and nodal status.CI: confidence interval.The number of patients differed from the total quoted in the study because it was not possible to obtain consistent information about the response rate in some cases.Results with significant P-values (≤ 0.05) after multivariate analysis are presented in bold letters.a P boostrap = 0.02; b P bootstrap = 0.004; c P bootstrap = 0.03.

Table 4 .
Tumor aspects and detoxification, DNA-repair, and apoptosis-related single nucleotide variants in survival in 109 patients with head and neck squamous cell carcinoma.N number of patients, HR hazard ratio, CI confidence interval; *Multivariate analysis adjusted by tumor size, tumor stage and nodal status; Results with significant P-values are presented in bold letters.Only single and combined genotypes with P-value < 0.20 in univariate analysis were included in multivariate analysis.