The aim of this study was to evaluate in our local cancer patients whether various risk factors including genetic polymorphic variations in candidate genes involved in radiation response are associated with the severity of RT-induced fibrotic reactions in normal tissues. The 155 Head and Neck cancer patients included in this report had nasopharyngeal carcinoma. This cancer site is prevalent in Saudi Arabia and is ideal for this type of study because patients follow standardized treatment with curative radiation without surgery . This could be considered contribution to the radiogenomic consortium that contains mainly breast, prostate and gynecologic cancer patients .
Among the 45 genetic variations scored, univariate analysis showed significant association between grade of fibrosis and allelic frequency of 6 SNPs (ATM rs1801516, HDM2 rs2279744, HDM2 rs1196333, TGFB1 rs1800469, XRCC1 rs25487 and XRCC5 rs1051677; Table 1) and therefore, could be considered candidate for predictive markers testing. These results were further sustained as the values of the False Discovery Rate (FDR) of these SNPs (0.14 - 0.009) have satisfied the significance threshold allowed for this test (<0.20). Interestingly, apart from ATM where the variant A allele was associated with increased risk, the variant alleles of the remaining significantly associated SNPs showed decreased risk (Odds or Risk Ratios < 1) to develop severe fibrosis, and therefore, they exhibit protective effect.
In addition, Kaplan-Meier analysis on these 6 SNPs suggested that the protective alleles of 3 of these SNPs (ATM rs1801516 A, HDM2 rs2279744 G, and XRCC1 rs25487 A) significantly correlate with longer follow-up of patients. Our results suggest that on average, the presence of protective allele at the heterozygous status would increase patients’ follow-up by 51 months while homozygous status would increase this index by 77 months. Thus, these SNPs could be used as prognostic biomarkers for length of follow-up following radiotherapy, as patients harboring protective alleles have higher probability to live longer (Figure 2). Estimated median follow-up suggests that harboring one protective allele of each of these SNPs would increase survival by about 4 years, while having the 2nd protective allele would add 2 more years.
Multivariate logistic regression, that assesses the joint effect of various risk factors, has confirmed the association between HDM2 T/G rs2279744, HDM2 T/A rs1196333, TGFB1 C/T rs1800469, XRCC1 G/A rs25487, follow-up and radiosensitivity (Table 2). These are interesting results that plaid in favor of the potential use of genetic markers as predictors of normal tissue response and prognostic of follow-up. This is an important conclusion since the subject is currently a hot topic debate . For instance, a large prospective study has failed to replicate previously reported associations between individual SNP genotype and radiation toxicity . On the contrary, genome-wide associations study evaluating erectile dysfunction following radiotherapy has showed significant association not only in a gene that plays a role in male gonad function, but also in genes that relate to specific African ancestry .
This is the first study on the association between HDM2 T309G promoter (rs2279744) and radiosensitivity; previous studies were only concerned with its cancer predisposing potential . The functional polymorphic variant in the HDM2 promoter at position 309 (rs2279744) have been suggested to affect the transcriptional activator SP1 binding, thereby modulating HDM2 transcription level. The G variant has been shown to increase the affinity for Sp1, resulting in higher levels of HDM2 mRNA and protein and the subsequent attenuation of the TP53 pathway . Results presented here showed that the same variant G allele, and also the variant G allele in the neighboring HDM2 rs2279744 SNP, is associated with reduced risk to develop late normal tissues complications, a phenomenon that is dependent on the amount of cell depletion following radiotherapy. Therefore, in line with our results, it is conceivable that this HDM2 G variant allele could promote cell survival following irradiation and thus, cells would appear more radioresistant, despite the probable high risk of genomic instability due to presumably attenuated TP53. This may also have implication for the promotion of secondary cancers following radiotherapy.
In addition, this is also the 1st study to report association between XRCC5 (KU80) polymorphisms and clinical radiosensitivity. XRCC5 is a component of the non-homologous end-joining (NHEJ) to repair DNA double-strand breaks. Previously, SNPs in XRCC5 have been shown to influence cancer risk and chromosomal radiosensitivity . Our study showed that, although uncommon, the variant XRCC5 rs1051677 C allele was more frequent in the controls, thus it has a protective effect. The variant ATM rs1801516 A allele (Asn) was previously reported to be significantly associated with increased radiation sensitivity . Other studies have also showed similar association with enhanced risk of various adverse reactions after RT . On the other hand, the majority XRCC1 rs25487 allele (Arg) was associated with increased risk to develop late reactions to radiotherapy (Reviewed in ). This suggests that the variant (or minority) allele could confer higher radioresistance in favor of normal tissues involved in the radiation treatment . TGFB1 encodes for the versatile cytokine TGFB1 assumed to be involved in response to tissue injuries. Therefore, SNPs that alter protein production can results in excessive deposition of scar tissue and fibrosis. Therefore, many SNPs have been studied in the literature and the effect of haplotype needs to be clarified as co-segregation of polymorphic variations in TGFB1 gene has been suggested to play a role in radiation response [3, 5, 20].
Results presented here are encouraging and illustrates that radiation response requires the concerted action of multiple genes and, therefore, it is a complex genetically controlled trait with the outcome being determined by multitude of additive effects. In addition, the original demonstration of an association between certain SNPs and length of followup after radiotherapy suggests prognostic role for patients’ survival. The results also indicate that not all variant SNPs are risky, and some of them could be advantageous from a radiosensitivity point of view. The SNPs studied were synonymous, non-synonymous and in non-coding regions of the genome. From an evolutionary perspective, the genome is in consistent development due to environmental interactions and, in general, natural selection favors the allele of the SNP that constitutes the most advantageous genetic adaptation. It is possible that the substitutions observed frequently are likely to be neutral or favorable, whereas those observed rarely are likely to be deleterious . Although a large prospective study has failed to replicate previously reported associations between individual SNPs genotype and radiation toxicity , a genome-wide associations study evaluating erectile dysfunction following radiotherapy for prostate cancer has showed significant association not only in a gene that plays a role in male gonad development and function, but also in genes that relate to specific African ancestry that would not have been identified in a cohort of European ancestry . The genomic revolution with the advent of high-throughput techniques can help uncovering the panoply of these interacting factors at the DNA (genome), RNA (transcriptome) or protein (proteome) level. Research using genome-wide analysis tools heralds the future of individualized radiation treatment in broadly personalized medicine. In addition to predictive and prognostic testing, the products of the identified genes could become targets for innovative therapies in susceptible individuals.