Cytolytic Activity Score to Assess Anticancer Immunity in Colorectal Cancer

Abstract

Background: Elevated tumor-infiltrating lymphocytes (TILs) within the tumor microenvironment is a known positive prognostic factor in colorectal cancer (CRC). We hypothesized that since cytotoxic T cells release cytolytic proteins such as perforin (PRF1) and pro-apoptotic granzymes (GZMA) to attack cancer cells, a cytolytic activity score (CYT) would be a useful tool to assess anticancer immunity.

Methods: Genomic expression data were obtained from 456 patients from The Cancer Genome Atlas (TCGA). CYT was defined by GZMA and PRF1 expression, and CIBERSORT was used to evaluate intratumoral immune cell composition.

Results: High CYT was associated with high microsatellite instability (MSI-H), as well as high levels of activated memory CD4+T cells, gamma-delta T cells, and M1 macrophages. CYT-high CRC patients had improved overall survival (p = 0.019) and disease-free survival (p = 0.016) compared with CYT-low CRC patients, especially in TIL-positive tumors. Multivariate analysis demonstrated that CYT- high associates with improved survival independently after controlling for age, lymphovascular invasion, colonic location, microsatellite instability, and TIL positivity. The levels of immune checkpoint molecules (ICMs)-programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), lymphocyte-activation gene 3 (LAG3), T cell immunoglobulin and mucin domain 3 (TIM3), and indoleamine 2,3-dioxygenase 1 (IDO1)-correlated significantly with CYT (p < 0.0001); with improved survival in CYT-high and ICM-low patients, and poorer survival in ICM-high patients.

Conclusions: High CYT within CRC is associated with improved survival, likely due to increased immunity and cytolytic activity of T cells and M1 macrophages. High CYT is also associated with high expression of ICMs; thus, further studies to elucidate the role of CYT as a predictive biomarker of the efficacy of immune checkpoint blockade are warranted.

Figure 1:

Association between CAS and mutation load, MSI status, and TILs. a) CAS expression in 39 matched samples of Colon cancer vs. Normal colonic tissue. b) CAS expression in Microsatellite Instability-High (MSI-H) vs. Microsatellite stable (MSS). c) Assessment of mutational load in CAS-high vs. CAS-low tumors. d) Association between CAS and mutational events within SMGs in CRC; blue circles (APC and KRAS), the mutations significantly correlated with CAS-low (p<0.05); red circles (BRAF, ARID1A, ACVR2A, CTNNB1, MAP2K4, AXIN2, and LIFR), mutations significantly correlated with CAS-high (p<0.05); red bar, each mutational event (left column, mutation rate %). CAS-high vs. low in e) Activated memory CD4+ T-cells, f) Gamma-Delta T-cells, g) M1 Macrophages, h) Activated Natural Killer (NK) Cells and i) Resting NK Cells

Figure 2:

Survival analysis of CAS in CRC patients. Kaplan-Meier (KM) curve of a) Overall Survival and b) Disease-Free Survival in CAS-high vs. CAS-low. KM curves of c) Overall Survival and d) Disease-Free survival stratified by CAS-high and CAS-low in TIL positive patients. “TIL positivity” is determined by higher CD8+ T-cells composition than the median value within the TCGA CRC cohort. CAS and its association with immune checkpoint molecules: e) PD-1, f) PD-L1, g) CTLA4, h) LAG3, i) TIM3, and j) IDO1 and k) Treg marker FOXP3 and their effect on patient survival. High, high expression of each gene; low, low expression of each gene.

Figure 3:

Correlation between CAS and immune checkpoint molecules and Treg marker. Scatter plot by Spearman’s correlation test between CAS and a) PD-1, b) PD-L1, c) CTLA4, d) LAG3, e) TIM3, f) IDO1, and g) FOXP3. X-axis, log2(CAS+1); y-axis, log2(each gene+1).