Microbial translocation, toll-like and vitamin D receptor polymorphisms in blood and risk of recurrence in stage III colorectal cancer.
Authors: Messaritakis Ippokratis , Koulouridi Asimina , Sfakianaki Maria , Vogiatzoglou Konstantinos , Gouvas Nikolaos , Tsiaousis Ioannis , Xynos Evangelos , Athanasakis Elias , Mavroudis Dimitrios , Sougklakos Ioannis 
Published: June 2022

3531

Background: Microbial translocation from the intestinal lumen into the blood circulation is significantly linked to intestinal dysbiosis; thus, leading to colorectal cancer (CRC), disease progression and decreased survival. Toll-like (TLRs) and vitamin D receptors (VDRs) play essential role in immunity and gut microbiome determination. Polymorphisms in such receptors have been associated with increased CRC incidence risk and mortality. The aim was to evaluate the microbial translocation in the blood of stage III CRC patients and correlate the presence of TLR and VDR genetic variants with microbial DNA fragments at risk of CRC development and progression. Methods: A total of 132 stage III CRC patients and 100 healthy donors were enrolled in the study. Peripheral blood DNA was analyzed using PCR for the amplification of microbial DNA encoding 16S rRNA, β-galactosidase gene of Escherichia coli, glutamine synthase of Bacteroides fragilis, and 5.8S rRNA of Candida albicans. Moreover, DNA from patients and controls was analyzed using PCR and PCR-RFLP for genotyping functional polymorphisms of both TLR (TLR2, TLR4, TLR9) and VDR ( TaqI, ApaI, FokI and BsmI) genes. Results: Median age of patients was 62 years, 59.8% were males, 92.7% had a colon/sigmoid tumor, 24.4% had a right colon tumor and 99.2% had a good performance status. Microbial DNA fragments from 16S rRNA, E. coli, B. fragilis, and C. albicans were detected in 43.2%, 20.5%, 31.8% and 36.4% of patients, respectively. Significantly higher rates of all microbial fragments, but E. coli, were detected in the group of patients in comparison to healthy donors ( p < 0.001). Similarly, higher rates of both TLR and VDR genetic variants were detected in CRC patients compared to healthy donors ( p < 0.001). Moreover, individuals with homozygous mutant alleles of either TLR or VDR polymorphisms had significantly higher detection rates of microbial DNA fragments. KRAS, BRAF and MSI status were significantly correlated with TLR9 genetic variants ( p= 0.001, p= 0.013 and p= 0.011, respectively) and MSI status was significantly correlated with all four VDR polymorphisms ( TaqI, p= 0.044; ApaI, p= 0.037; FokI, p= 0.002 and BsmI, p< 0.001). Cox regression analysis revealed that BRAF mutations, histology type and ApaI genetic variants are significantly associated with shorter disease-free survival (DFS). C. albicans detection is significantly associated with shorter overall survival, and B. fragilis is an independent poor prognostic factor for decreased DFS (HR = 33.85; p = 0.018). Conclusions: The detection of higher frequencies of the TLR/ VDR genetic variants was correlated with significantly higher detection rates of microbial DNA fragments. The detection of these TLR/VDR polymorphisms and microbial DNA fragments in CRC patients highlighted their role in cancer development, progression, and patients’ survival.