Prognostic value of tumor-infiltrating lymphocytes in signet-ring cell carcinoma of the rectum and sigmoid colon.
Authors: Zhou Jiaolin , Wang Jia , Xu Yaping , Lin Guole , Wu Huanwen , Zhou Jianfeng , Cong Lin , Cui Peng , Du Haiwei , An Yang , Song Jinlei , Wang Jing , Wang Yang , Li Lifeng 
Published: June 2021

e15079 Background: Signet-ring cell carcinoma (SRCC) of rectum and sigmoid colon is an extremely rare subtype of colorectal cancer (CRC) with very poor prognosis. Tumor-infiltrating lymphocytes (TILs) signify the host immune response to tumors, which were reported to predict survival outcomes of patients with various cancer types. In this study, we aimed to characterize TILs and mutational features of SRCC of rectum and sigmoid colon as well as their correlations with the clinicopathological parameters and survival outcomes. Methods: 28 patients with stage II-IV SRCC of rectum and sigmoid colon were included, in which 12 patients had tumors with ≥50% signet-ring cells (SRCs) and 16 had tumors with <50% SRCs. Targeted next generation sequencing using a 1,021-gene panel was used to investigate the genetic alterations of tumor tissue. Multiplex immunofluorescence assays were performed to visualize TILs. TILs within cancer cell nests (iTILs) and in cancer stroma (sTILs) were counted separately. The correlations of TILs with survival outcomes were analyzed in stage II/III patients who underwent the radical resection. Results: Somatic alterations were detected in all the 28 cases. The most frequently mutated genes included TP53, APC and SMAD4, occurring in 68%, 36% and 36% of cases, respectively. BRAF mutation were detected in only one patient (3.6%). The median tumor mutational burden (TMB) was 4.80 (range, 0.96-42.24) muts/Mb. Three patients (10.7%) were with microsatellite instability-high (MSI-H) status and a high TMB of more than 10 muts/Mb. Patients with stage IV tumors have significantly lower PD-1+ CD8+ iTILs and sTILs (p=0.018 for both), CD8+ iTILs (p=0.022), and PD-1+ iTILs (p=0.013) levels than those with stage II/III tumors. Tumors with ≥ 50% SRCs showed lower levels of CD8+ sTILs than those with < 50% SRCs (p=0.046). Patients with CEA>5.0 ng/ml showed significantly lower levels of PD-1+ CD8+ iTILs than those with CEA≤5.0 ng/ml (p=0.015). Moreover, significantly lower levels of PD-1+ CD8+ sTILs (p=0.036) were observed in tumors that appeared as long circumferential thickening of the bowel wall with stenosis compared to those did not. Multivariate analysis indicated that patients with high PD-1+ CD8+ iTILs and sTILs levels had significantly better disease-free survival (DFS) than those with low PD-1+ CD8+ iTILs and sTILs levels (not reached vs. 22 months for both; p=0.008 and 0.003, respectively). High PD-1+ CD3+ sTILs levels were associated with significantly longer overall survival (OS) compared to low levels (not reached vs. 39 months, p=0.034). No correlation between MSI or TMB and DFS or OS was observed in this small cohort. Conclusions: Our results demonstrated that PD-1+ CD8+ iTILs and sTILs are powerful independent predictors of survival outcomes in patients with resectable SRCC of rectum and sigmoid colon. Further investigations in larger cohorts are needed to validate our findings.