Prognostic value of toll-like receptors in colorectal cancer

Background and aims Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide with 1.9 million new cases occuring globally in 2020. The prognosis of CRC patients has improved, although 30–50% of all local colon cancer patients treated develop recurrence. Biomarkers serve as diagnostic, prognostic, and predictive tools that help to detect disease without clinical symptoms, to identify early recurrence at follow-up, and to determine which patients might need more aggressive treatment. Subtyping cancers using biomarkers can enable targeted and personalized therapy. Chronic inflammation may promote cancer development and dissemination. Notably, cancer may cause both systemic and local inflammation. CRC patients with a strong systemic inflammatory response (SIR) indicated by an elevated C-reactive protein (CRP) value exhibit a worse prognosis, whereas CRC patients with high local immune cell infiltration have a better prognosis. Furthermore, CD3-positive and CD8-positive immune cells are critical in local adaptive immune responses. Toll-like receptors (TLRs) are transmembranous proteins crucial in initiating innate and adaptive immune responses after recognizing pathogen-associated or host-originating patterns. In malignancies, TLRs play pro- and antitumorigenic roles. Thus, this dissertation project aimed to evaluate the prognostic role of different TLRs in CRC. Moreover, this project aimed to assess the possible relationship between TLRs and CRP, and between local innate (TLRs) and adaptive (CD3-positive and CD8-positive cells) immune responses in CRC. Methods Expressions of TLR2, TLR4, TLR5, and TLR7 were assessed in tumor cells through the immunohistochemistry of tissue microarray (TMA) slides from 1308 CRC patients who underwent surgery in the Department of Surgery at Helsinki University Hospital, Finland, between 1982 and 2005. The associations between the immunoexpressions of TLRs in tumor cells, clinicopathological characteristics, and survival were evaluated. Among a subgroup of 549 patients surgically treated between 1998 and 2005, the relationship between tumor cell expressions of TLR2, TLR4, TLR5, and TLR7 and plasma CRP was analyzed. Blood samples were taken preoperatively, and plasma CRP levels were measured using a high-sensitivity method. Finally, the relationship between the tumor cell expressions of TLRs and the immunoexpression of CD3-positive and CD8-positive T-cell densities in the tumor and stroma in the same samples was analyzed. A CD3–CD8 tumor–stroma index was established based on the density levels of CD3-positive and CD8-positive T cells in the tumor and stroma, resembling the well-known Immunoscore®. Results Patients with a high TLR2 expression, a high TLR5 expression, and a positive TLR7 expression exhibited a better disease-specific survival (DSS) in the cohort of 1308 CRC patients. Furthermore, stage III subgroup patients with a high TLR2 immunoexpression exhibited a better outcome. A high TLR5 value served as a positive prognostic factor among younger patients, patients with a higher pT stage, patients with lymph node–negative disease, and patients with a lower WHO grade. A positive TLR7 immunoexpression emerged as a positive prognostic factor among higher pT stage and lower WHO grade patients. In a cohort of 549 CRC patients, individuals with a high preoperative CRP level exhibited a worse DSS. Among patients with a high CRP level, those with a low TLR4 immunoexpression exhibited a better prognosis. In the low CRP subgroup, patients with a high TLR2, a high TLR5, and a positive TLR7 immunoexpression exhibited a better survival. Interestingly, TLR4 immunoexpression carried no prognostic value in the entire cohort. Furthermore, none of the TLRs emerged as an independent prognostic factor in the multivariate analyses. High expressions of tumoral and stromal CD3-positive and CD8-positive T cells associated with high expressions of TLR2, TLR4, and TLR5. Among all TLR subgroups except the negative TLR7 subgroup, patients with a low CD3–CD8 tumor–stroma index exhibited a worse prognosis. Conclusions Survival was better among patients with a high expression of TLR2, TLR5, and TLR7 in the tumor cells. By contrast, survival was worse among patients with a high CRP level. Furthermore, survival was better among patients with a low CRP level and with high tumor cell expressions of TLR2, TLR5, and TLR7, as well as among patients with a high CRP level and a low TLR4 expression. High expressions of immune cell densities in the tumor and stroma associated with high expressions of TLRs in the tumor cells. Thus, TLRs may play a prognostic role either alone or in combination with CRP or immune cell densities. However, further studies are needed to more fully understand the prognostic value of TLRs in CRC.