Stratified analysis reveals chemokine-like factor (CKLF) as a potential prognostic marker in the MSI-immune consensus molecular subtype CMS1 of colorectal cancer

Philip D Dunne, Paul G O'Reilly, Helen G Coleman, Ronan T Gray, Daniel B Longley, Patrick G Johnston, Manuel Salto-Tellez, Mark Lawler, Darragh G McArt, Philip D Dunne, Paul G O'Reilly, Helen G Coleman, Ronan T Gray, Daniel B Longley, Patrick G Johnston, Manuel Salto-Tellez, Mark Lawler, Darragh G McArt

Abstract

The Colorectal Cancer (CRC) Subtyping Consortium (CRCSC) recently published four consensus molecular subtypes (CMS's) representing the underlying biology in CRC. The Microsatellite Instable (MSI) immune group, CMS1, has a favorable prognosis in early stage disease, but paradoxically has the worst prognosis following relapse, suggesting the presence of factors enabling neoplastic cells to circumvent this immune response. To identify the genes influencing subsequent poor prognosis in CMS1, we analyzed this subtype, centered on risk of relapse. In a cohort of early stage colon cancer (n=460), we examined, in silico, changes in gene expression within the CMS1 subtype and demonstrated for the first time the favorable prognostic value of chemokine-like factor (CKLF) gene expression in the adjuvant disease setting [HR=0.18, CI=0.04-0.89]. In addition, using transcription profiles originating from cell sorted CRC tumors, we delineated the source of CKLF transcription within the colorectal tumor microenvironment to the leukocyte component of these tumors. Further to this, we confirmed that CKLF gene expression is confined to distinct immune subsets in whole blood samples and primary cell lines, highlighting CKLF as a potential immune cell-derived factor promoting tumor immune-surveillance of nascent neoplastic cells, particularly in CMS1 tumors. Building on the recently reported CRCSC data, we provide compelling evidence that leukocyte-infiltrate derived CKLF expression is a candidate biomarker of favorable prognosis, specifically in MSI-immune stage II/III disease.

Keywords: chemokine-like factor; colorectal cancer; gene expression profiling; molecular stratification; relapse risk.

Conflict of interest statement

PDD: None; POR: None; HGC: None; RTG: None; DBL: None; PGJ: Previous Founder and Shareholder of Almac Diagnostics; CV6 Therapeutics: Expert Advisor and Shareholder; Chugai Pharmaceuticals: Consultant; MST: None; ML: None; DMA: None.

Figures

Figure 1. Study overview of discovery and…
Figure 1. Study overview of discovery and survival validation subsets
The data used in this study was obtained from 566 Affymetrix U133 Plus 2.0 patient transcriptional profiles accessed through the NCBI GEO accession number GSE39582. Filtering for stage II/III and complete relapse data reduced this cohort to 460 profiles. The CMS1 specific discovery subset was composed of 46 tumors which fulfilled risk filtering (see Materials and Methods) followed by differential gene expression analysis based on risk classification. The survival validation subset was composed of either the entire cohort, CMS1 specific or MSI specific subgroups of samples. For relapse-free survival analyses, only stage II and III patients were considered, giving 79 transcriptional profiles in the CMS1 or 60 transcriptional profiles in the MSI subgroups.
Figure 2. Relapse risk in recently defined…
Figure 2. Relapse risk in recently defined consensus molecular subtypes
Survival curve using Kaplan-Meier estimation comparing prognosis of CMS1-4 in untreated stage II/III CRC patients (GSE39582). RR indicates the 3 year relapse rate.
Figure 3. CKLF gene expression is associated…
Figure 3. CKLF gene expression is associated with prognosis only in CMS1
A. Survival curve using Kaplan-Meier estimation comparing CKLF levels in CMS1 stage II/III CRC patients (GSE39582) B. Survival curve using Kaplan-Meier estimation comparing CKLF levels in MSI stage II/III CRC patients (GSE39582).
Figure 4. CKLF gene expression within the…
Figure 4. CKLF gene expression within the tumor microenvironment and normal tissue
A. Box and whisker plot of CKLF expression according to specific endothelial, epithelial, fibroblast or leukocyte cell-of-origin from FACS sorted primary colorectal tumors. B. CKLF RNA-seq expression values are shown in RPKM (Reads Per Kilobase of transcript per Million mapped reads), calculated from a gene model with isoforms collapsed to a single gene. No other normalization steps have been applied. Box plots are shown as median and 25th and 75th percentiles; points are displayed as outliers if they are above or below 1.5 times the interquartile range. The data used for the analyses described in this manuscript were obtained from the Genotype-Tissue Expression (GTEx) portal (http://gtexportal.org/home) version V6 in January 2016. C. CKLF gene expression from a subset of the Atlas of Human Primary Cells cohort. Bar charts are shown as median CKLF gene expression according to specific lineage and error bars represent standard deviation. ** denotes p<0.005.

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