Increased intratumoral neutrophil in colorectal carcinomas correlates closely with malignant phenotype and predicts patients' adverse prognosis

Abstract

Background: Substantial evidence suggests that the presence of inflammatory cells plays a critical role in the development and/or progression of human tumors. Neutrophils are the common inflammatory cells in tumors; however, the infiltration of intratumoral neutrophils in colorectal carcinoma (CRC) and its effect on CRC patients' prognosis are poorly understood.

Methodology/principal findings: In this study, the methods of tissue microarray and immunohistochemistry (IHC) were used to investigate the prognostic significance of intratumoral CD66b+ neutrophil in CRC. According to receiver operating characteristic curve analysis, the cutoff score for high intratumoral CD66b+ neutrophil in CRC was defined when the mean counts were more than 60 per TMA spot. In our study, high intratumoral CD66b+ neutrophil was observed in 104/229 (45.4%) of CRCs and in 29/229 (12.7%) of adjacent mucosal tissues. Further correlation analysis showed that high intratumoral neutrophil was positively correlated with pT status, pM status and clinical stage (P<0.05). In univariate survival analysis, a significant association between high intratumoral neutrophil and shortened patients' survival was found (P<0.0001). In different subsets of CRC patients, intratumoral neutrophil was also a prognostic indicator in patients with stage II, stage III, grade 2, grade 3, pT1, pT2, pN0 and pN1 (P<0.05). Importantly, high intratumoral neutrophil was evaluated as an independent prognostic factor in multivariate analysis (P<0.05).

Conclusions/significance: Our results provide evidence that increased intratumoral neutrophil in CRC may be important in the acquisition of a malignant phenotype, indicating that the presence of intratumoral neutrophil is an independent factor for poor prognosis of patients with CRC.

Figure 1. ROC curve analysis was employed to determine the cutoff value for high intratumoral CD66b+ neutrophil in colorectal carcinoma.

The sensitivity and specificity for each outcome were plotted: tumor grade (P = 0.575, A), T stage (P = 0.029, B), N stage (P = 0.263, C), M stage (P = 0.002, D), clinical stage (P = 0.005, E), and survival status (P<0.001, F).

Figure 2. The presence of CD66b+ neutrophils in colorectal and lymph node metastatic CRC tissues.

(A) High intratumoral neutrophil was observed in a CRC (case 52), in which more than 60 intratumoral cells revealed positive immunostaining of CD66b (upper panel, ×100). (B) A CRC case (case 37) demonstrated low intratumoral neutrophil, in which less than 60 intratumoral cells showed immunoreactivity of CD66b (upper panel, ×100). (C) The corresponding adjacent mucosal tissue of case 52 showed low neutrophil, in which less than 60 cells revealed immunostaining of CD66b (upper panel, ×100). (D) High intratumoral neutrophil was observed in lymph node metastatic tissue, in which more than 60 intratumoral cells revealed positive immunostaining of CD66b (upper panel, ×100). The lower panels indicated the higher magnification (×400) from the area of the box in A, B and C, respectively.

Figure 3. Survival curves for 229 CRC patients according to infiltration status of intratumoral neutrophils (log-rank test).

(A) Total, probability of survival of all patients with CRC: low intratumoral neutrophil, n = 125; high intratumoral neutrophil, n = 104. (B) Stage II, probability of survival of stage II patients with CRC: low intratumoral neutrophil, n = 44; high intratumoral neutrophil, n = 32. (C) Stage III, probability of survival of stage III patients with CRC: low intratumoral neutrophil, n = 27; high intratumoral neutrophil, n = 29. (D) Stage IV, probability of survival of stage IV patients with CRC: low intratumoral neutrophil, n = 5; high intratumoral neutrophil, n = 18.

Figure 4. Survival curves according to infiltration status of intratumoral neutrophils in subsets of CRC patients with different histological grade and pT/pN status (log-rank test).

(A) Grade 2, probability of survival of grade 2 patients with CRC: low intratumoral neutrophil, n = 93; high intratumoral neutrophil, n = 81. (B) Grade 3, probability of survival of grade 3 patients with CRC: low intratumoral neutrophil, n = 20; high intratumoral neutrophil, n = 16. (C) pT2, probability of survival of pT2 patients with CRC: low intratumoral neutrophil, n = 45; high intratumoral neutrophil, n = 31. (D) pT3, probability of survival of pT2 patients with CRC: low intratumoral neutrophil, n = 66; high intratumoral neutrophil, n = 63. (E) pN0, probability of survival of pN0 patients with CRC: low intratumoral neutrophil, n = 96; high intratumoral neutrophil, n = 73. (F) pN1, probability of survival of pN1 patients with CRC: low intratumoral neutrophil, n = 29; high intratumoral neutrophil, n = 31.