Antitumor immune responses are associated with the number of lymph node retrieved in colorectal cancer

Abstract

Background: Higher number of lymph nodes retrieved and prominent antitumor immune response of the primary tumor such as lymphocytic infiltration are favorable prognostic factors in colorectal cancer. The number of nodes retrieved after colorectal surgery is influenced by the extent of surgery and pathologic examination. Since lymph nodes are secondary lymphoid organs, the primary tumor may potentially influence the biologic environment around regional lymph nodes and induce reactive changes in the nodes themselves. Thus, antitumor immune response of the primary tumor is suggested as potentially relevant factors for the number of nodes. However, there is no study focusing on the relationship between antitumor immune response and the number of nodes.Purpose: The aim of this study was to evaluate the association between the number of lymph nodes retrieved and antitumor immune response of the primary tumor based on immunohistochemical (IHC) staining and quantitative cytokine assay using Bio-Plex® assay (BioRad, Veenendaal, The Netherlands).Methods: In the retrospective study, a total of 63 patients with colorectal cancer, TNM stage II and III, were enrolled. Inflammatory cell infiltration was assessed on hematoxylin and eosin staining. Molecular markers such as T cell-mediated immunity (CD3, CD8, CD45RO); inflammation (nuclear factor kappa B; NFκB, cyclooxygenase-2; COX-2); angiogenesis (vascular endothelial growth factor; VEGF) were evaluated using IHC staining. We evaluated inflammatory cell infiltration and IHC expression of CD3, CD8, and CD45RO at both the center of the tumor and the invasive margin and used a four-tiered scoring system. IHC expression of NFκB, COX-2, and VEGF was evaluated at the center of the tumor. CD3, CD8 and CD45RO scores are the sum of the scores for the center of the tumor (1 to 4) and the invasive margin (1 to 4) based on

immunohistochemical staining. High score is 5 to 8 and low score is 1 to 4. NFκB, COX2, and VEGF scores are the scores for the tumor cells (1 to 4) based on immunohistochemical staining. High score is 3 to 4 and low score is 1 to 2. In the prospective study, a total of 20 patients with stage I, II or III colorectal cancer were enrolled. Quantification of cytokines was conducted for markers of helper T cell type 1 (Interleukin (IL)-12, Interferon (IFN)-gamma, Granulocyte-macrophage colony-stimulating factor (GM-CSF), helper T cell type 2 (IL-4), proinflammatory cytokine (Tumor necorsis factor (TNF)-alpha, IL-6, IL-8), antiinflammatory cytokine (IL-10) and angiogenesis (VEGF). Paired samples (serum, normal colon and tumor tissue) were used for Bio-Plex® cytokine assay.Results: In the retrospective study, univariate analysis showed that right colon location, large tumor diameter, microsatellite instability (MSI)-high tumor type, and high inflammatory cell infiltration were significant predictors of an increased number of retrieved nodes. IHC staining demonstrated that increased expression of T cell markers such as CD3 and CD8 was associated with a higher number of nodes retrieved. On multivariate analysis, right colon location (mean nodes: 35±14, p=0.01) and high inflammatory cell infiltration (30±13, p=0.04) were independent predictors of a higher number of nodes. Survival analysis using Cox proportional hazards model showed that TNM stage III with low inflammatory cell infiltration was an independent prognostic factor for decreased cancer-specific survival (5-year rate 42.9%, hazard ratio=4.7, p=0.02) In the prospective study, univariate analysis showed that right-sided colon cancer (p=0.02) and larger tumor diameters (<0.001) were associated with a higher number of nodes retrieved. Median IL-6 level was 2.3 pg/ml in the serum. Median IL-8 level was 12 and 41 in

the serum and tumor tissue, respectively. Median GM-CSF level was 40.2 and 68.8 in the normal mucosa and tumor tissue, respectively. Median VEGF level was 69.5, 30.8, and 186 in serum, normal mucosa and tumor tissue, respectively. IL-4, IL-10, IL-12, IFN-γ, and TNF-α levels were out of detection range in most cases among the serum, normal and tumor tissues. Patients were divided into high and low groups based on median value. High serum IL-6 level was associated with a higher number of nodes (mean nodes 36±13,p=0.002). High IL-8 level in the tumor tissue was associated with a higher number of nodes (36±13, p=0.03), though GM-CSF (34±15, p=0.058) and VEGF levels (29±13, p=0.7) were not.Conclusion: High inflammatory cell infiltration in the primary tumor and high level of proinflammatory cytokines, IL-6 (serum) and IL-8 (tumor), were associated with higher number of nodes. This study suggests that antitumor immune response is associated with the number of nodes retrieved after colorectal cancer surgery. Further studies are needed to investigate detailed underlying immune reaction.