BackgroundToll-like receptors (TLRs) play essential role in innate and acquired immunity, are expressed in various cell types, and are associated with altered susceptibility to many diseases, and cancers. The aim of this study was to investigate TLR2 (-196 to-174del), TLR4 (Asp299Gly and Thr399Ile) and TLR9 (T1237C and T1486C) gene polymorphisms at risk of colorectal cancer (CRC) development and progression.MethodsPeripheral blood was obtained from 397 patients with adjuvant (stage II/III, n = 202) and metastatic (n = 195) CRC. Moreover, blood samples from 50 healthy volunteers and 40 patients with adenomatous polyps were also included as control groups. DNA from patients and controls was analyzed using PCR and PCR-RFLP for genotyping functional polymorphism within TLR2, TLR4 and TLR9 genotypes.ResultsTLR2–196 to-174del/del genotype was detected in 76.6% of the patients and was significantly higher that the controls groups (p<0.001). TLR4 Asp299Gly, TLR4 Thr399Ile, TLR9 T1237C and T1486C homozygous genotypes were detected in 70.5%, 70.5%, 61.5% and 61.5% of the patients respectively, and were also significantly higher than that in the control groups (p<0.001). All polymorphisms detected were also significantly associated with the metastatic disease (p<0.001) leading to shorter overall survival (p<0.001); whereas, TLR4 Asp299Gly and Thr399Ile polymorphisms were significantly associated with KRAS mutations.ConclusionsThe detection of higher frequencies of the TLR2, TLR4 and/or TLR9 polymorphisms in CRC patients compared with the control groups highlight the role of these polymorphism in CRC development and cancer progression.

We aimed to assess if the discrepant prognostic information between Programmed Death Ligand 1 (PD-L1) protein versus mRNA expression in early breast cancer (BC) could be attributed to heterogeneity in its expression. PD-L1 protein and mRNA expression in BC tissue microarrays from two clinical patient cohorts were evaluated (105 patients; cohort 1: untreated; cohort 2: neoadjuvant chemotherapy-treated). Immunohistochemistry (IHC) with SP142, SP263 was performed. PD-L1 mRNA was evaluated using bulk gene expression and RNA-FISH RNAscope®, the latter scored in a semi-quantitative manner and combined with immunofluorescence (IF) staining for the simultaneous detection of PD-L1 protein expression. PD-L1 expression was assessed in cores as a whole and in two regions of interest (ROI) from the same core. The cell origin of PD-L1 expression was evaluated using multiplex fluorescent IHC. IHC PD-L1 expression between SP142 and SP263 was concordant in 86.7% of cores (p < 0.001). PD-L1 IF/IHC was weakly correlated with spatial mRNA expression (concordance 54.6–71.2%). PD-L1 was mostly expressed by lymphocytes intra-tumorally, while its stromal expression was mostly observed in macrophages. Our results demonstrate only moderate concordance between the various methods of assessing PD-L1 expression at the protein and mRNA levels, which may be attributed to both analytical performance and spatial heterogeneity.

Background: We have previously shown that PD-L1 confers discrepant prognostic information depending on level of expression, protein versus mRNA. This discrepancy may be due to heterogeneity in its expression, which we aimed to assess.Methods: Breast cancer samples from an adjuvant patient cohort (cohort 1; surgical specimen, pre-adjuvant) and from a prospective single-arm study of neoadjuvant chemotherapy (cohort 2; multiple timepoints during chemotherapy) were obtained and Tissue Microarrays (TMA) were constructed. Protein and mRNA PD-L1 expression were assessed in two different areas of each sample, each containing at least 50 cells. Immunofluorescence (IF) staining with anti-PD-L1 (clone SP142, Abcam) antibody was performed. A core was considered as protein PD-L1 positive when at least one cell with membranous immunostaining was detected in at least one TMA core, regardless of cell of origin (tumor or immune cell). PD-L1 mRNA was simultaneously detected using the in situ hybridization method RNAscopeÒ (Multiplex Fluorescent v2 Assay, Advanced Cell Diagnostics Inc) and quantified in a semi-quantitative manner.Results: A total of 90 patient samples were analyzed (cohort 1: 33, cohort 2: 57). At the protein level, 53% of areas in cohort 1 and 42.9% in cohort 2 had at least one PD-L1 positive cell, with a mean positivity rate of 6.11% and 3.99% cells per area, respectively. There was no evidence of heterogeneity in the protein expression between two areas of the same sample (cohort 1: Wilcoxon signed rank p¼0.171; cohort 2: p¼0.447). At the mRNA level, 69.7% of areas scored in cohort 1 and 81.3% in cohort 2 had none or very low staining (ACD score 0). PD-L1 mRNA expression scores between the two areas of each sample were statistically significantly but modestly correlated to each other both in cohort 1 (Spearman's rho¼0.40, p¼0.021) and cohort 2 (Spearman's rho¼0.40, p¼0.002). PD-L1 protein expression was strongly and statistically significantly correlated with mRNA expression (cohort 1: Spearman's rho¼0.805, p<0.001; cohort 2: Spearman's rho¼0.731, p<0.001).Conclusions: Our results showed high concordance between mRNA-based and protein-based assessment of PD-L1. No evidence was found in this study for neither spatial nor biological heterogeneity of PD-L1 expression.Clinical trial identification: NCT00957125.