Prognostic Impact of TS, MTHFR and XRCC1 Genetic Variants in 113 Patients with Myelodysplastic Syndromes
Authors: Loscocco Federica , Visani Giuseppe , Giacomini Elisa , Ruzzo Annamaria , Galimberti Sara , Voso Maria Teresa , Finelli Carlo , Ciabatti Elena , Fabiani Emiliano , Graziano Francesco , Barulli Sara , Volpe Antonio , Magro Domenico , Piccaluga Pier Paolo , Fuligni Fabio , Vignetti Marco , Fazi Paola , Piciocchi Alfonso , Clissa Cristina , Gabucci Elisa , Rocchi Marco , Magnani Mauro , Isidori Alessandro 
Published: October 2019

Abstract
Background: Several studies have suggested that genetic variability related with single nucleotide polymorphisms (SNPs) of the BER system, DNA synthesis and folate-metabolizing pathway genes could modulate DNA repair capacity. Moreover, these genes are supposed to be related to cancer risk. However, the prognostic impact of the association of individual and/or combined genetic variants in patients with myelodysplastic syndromes (MDS) remains undetermined.
Methods: We genotyped 113 MDS patients, 54 with IPSS low/int-1 receiving only best supportive care (BSC group) and 59 with IPSS int-2/high treated with azacitidine (AZA-group), for the following polymorphisms: XRCC1 194 and 399, APE1 148, XRCC3 241, TS5'-UTR (2R/3R and G/C) and 3'-UTR (6bp+/6bp-), MTHFR 677 and 1298. Genomic DNA was analyzed by High Resolution Melting assay and restriction digests of PCR products. Overall survival (OS) was calculated using the Kaplan-Meier estimate probabilities, and differences between survival curves were analyzed by the log-rank test. Multivariate analyses were performed using the Cox method.
Results: For all the target genes, the distribution of genotypes was consistent with the Hardy-Weinberg equilibrium. Among the baseline characteristics analyzed (age, sex, diagnosis according to WHO, hemoglobin) there was no statistically significant difference in the genotype distribution of studied polymorphisms. In the BSC group, the variants XRCC1 399 GG [Hazard ratio (HR)=7.07; p=0.02], -6/-6 of TS3'-UTR (HR=4.65; p=0.05), 2R/3G, 3C/3G, 3G/3G of TS5'-UTR (HR=11.44; p=0.02) and TT of MTHFR 677 (HR=67.12; p<0.001), were associated with a statistically significant adverse clinical outcome compared to variant alleles (Table 1). This is consistent with the enzymatic activity reduction attributed to these genetic variants. Multivariable regression model analysis was also performed in the AZA group for the same genetic variants. We found similar results for the association between XRCC1 399 GG(HR=5.71 p=0.002), TS3'-UTR +6/+6(HR=0.097 p=0.004), MTHFR 677 TT (HR=8.58 p<0.001) and survival, but not for SNPs in TS5'-UTR (Table 2). Finally, we performed an exploratory analysis to investigate the combined effect of the unfavorable genotypes on survival. In the BSC group, the 3-year OS was 33% for those patients with ≥2 variant alleles, as compared to 62.5%, and 100%, respectively, for those with 2 or 0/1 variant alleles. The predictive role of the adverse genotypes combination on survival was confirmed also in the AZA group, suggesting that patients with a higher number of genetic variants had a shorter survival. Interestingly, when we compared survival of patients with adverse genotypes between BSC and AZA groups, we did not find any statistically significant difference between the 2 groups (Kaplan-Meyer and Log-rank test). Therefore, we speculated that azacitidine could give a survival advantage to patients with unfavorable genetic variants, independently from IPSS at diagnosis.
Conclusion: Our study reveals, for the first time, an associations between genetic variants in TS, MTHFR and XRCC1 genes, BSC, azacitidine and survival in MDS patients. If confirmed, they could represent new prognostic markers able to provide guidance for clinical management of MDS patients. In particular, the presence of adverse genotypes could represent a biomarker to treat patients with low-risk IPSS with azacitidine, if confirmed on larger series. Further studies with larger population are needed to validate these associations, especially in SNPs with low variant allele frequency.
Table 1. Gene Genotype Hazard risk 95,0% CI forHazard Risk Lower 95,0% CI forHazard Risk Upper p value XRCC1 399 [G/G] versus [A/G-A/A] 7,072 1,295 38,619 0,024 TS5'-UTR [3G/3G, 3G/3C, 2R/3G] versus [2R/2R, 2R/3C, 3C/3C] 11,447 1,330 98,544 0,026 TS3'-UTR [Del/Del] versus [Del/Ins, Ins/Ins] 4,653 0,946 22,874 0,058 MTHFR 677 [T/T] versus [C/T-C/C] 67,125 6,409 703,081 <0,001
Table 2. Gene Genotype Hazard risk 95,0% CI forHazard Risk Lower 95,0% CI forHazard Risk Upper p value XRCC1 399 [G/G] versus [A/G-A/A] 5,713 1,904 17,142 0,002 TS3'-UTR [Ins/Ins] versus [Ins6/del6, del6/del6] 0,097 0,019 0,479 0,004 MTHFR 677 [T/T] versus [C/T-C/C] 8,587 2,749 26,828 <0,001

Disclosures
Finelli: Celgene: Other: Speaker, Research Funding; Novartis: Other: Speaker; Janssen: Other: Speaker.