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.

The current study aimed at the optimization of circulating tumor cell (CTC) enrichment for downstream protein expression analyses in non-small cell lung cancer (NSCLC) to serve as a tool for the investigation of immune checkpoints in real time. Different enrichment approaches—ficoll density, erythrolysis, their combination with magnetic separation, ISET, and Parsortix—were compared in spiking experiments using the A549, H1975, and SKMES-1 NSCLC cell lines. The most efficient methods were tested in patients (n = 15) receiving immunotherapy targeting programmed cell death-1 (PD-1). Samples were immunofluorescently stained for a) cytokeratins (CK)/epithelial cell adhesion molecule (EpCAM)/leukocyte common antigen (CD45), and b) CK/programmed cell death ligand-1 (PD-L1)/ indoleamine-2,3-dioxygenase (IDO). Ficoll, ISET, and Parsortix presented the highest yields and compatibility with phenotypic analysis; however, at the patient level, they provided discordant CTC positivity (13%, 33%, and 60% of patients, respectively) and enriched for distinct CTC populations. IDO and PD-L1 were expressed in 44% and 33% and co-expressed in 19% of CTCs. CTC detection was associated with progressive disease (PD) (p = 0.006), reduced progression-free survival PFS (p = 0.007), and increased risk of relapse (hazard ratio; HR: 10.733; p = 0.026). IDO-positive CTCs were associated with shorter PFS (p = 0.039) and overall survival OS (p = 0.021) and increased risk of death (HR: 5.462; p = 0.039). The current study indicates that CTC analysis according to distinct immune checkpoints is feasible and may provide valuable biomarkers to monitor NSCLC patients treated with anti-PD-1 agents.

The SS18-SSX fusion drives oncogenic transformation in synovial sarcoma by bridging SS18, a member of mSWI/SNF complex, to Polycomb repressive complex 1 (PRC1) target genes. Here we show that the SSX C-terminus, via its SSXRD domain, directs SS18-SSX chromatin binding independently of SS18. SSXRD specific targeting is mediated by interaction with mono ubiquitinated H2A (H2AK119ub1) and histone MacroH2A with which the fusion overlaps genome wide. Variant Polycomb Repressive Complex 1.1 (PRC1.1) acts as the main depositor of H2AK119ub1 and is therefore required for SS18-SSX occupancy. Importantly, the SSX C-terminus not only depends on H2AK119ub1 for localization but also further increases it by promoting PRC1.1 complex stability. Consequently, high H2AK119ub1 levels are a feature of murine and human synovial sarcomas. These results reveal an SSX/PRC1 autoregulatory feedback loop that reinforces fusion chromatin binding and therefore its oncogenic activity, and could play a role in a wider range of cancers and physiological settings where SSX proteins are overexpressed.