Immunologic Mechanisms of Colorectal Liver Metastases: Potential for Novel Therapies Open Access

Abstract

Colorectal cancer is the third most common cancer worldwide. Over half of patients with colorectal cancer (CRC) develop liver metastases. While immunotherapy is an emerging treatment for patients with solid tumors, its use among patients with CRC is limited due to poor efficacy. Furthermore, gene expression patterns of liver-specific CRC metastases remain unclear. The purpose of this study was to identify and validate a high-risk gene expression profile for patients with colorectal liver metastasis (CRCLM) to better inform prognosis and development of novel targeted therapies.  

CRCLM samples from patients who underwent complete metastatectomy from 2009-2017 at Emory University were examined. Expression profiling of extracted RNA was performed using the NanoString Immuno-Oncology (IO360) 770-gene panel. Statistical analysis using cutoffs of absolute log 2-fold change ≥1.5 and p-value ≤0.05 were performed. Patients were analyzed by extremes of outcomes: survival time in the lowest quartile, compared to those still alive at last follow-up. Four genes had higher expression in tumors from patients with worse overall survival compared to patients still alive at last follow-up: CSF1, MGMT, IL6R, and LILRB4. 

CSF1 signaling leads to M2-macrophage polarization, a well-studied anti-inflammatory and tumor-tolerant phenotype. MGMT encodes a DNA repair enzyme that repairs alkylating chemotherapy damage and is implicated in carcinogenesis and chemotherapy response. IL6R signaling affects tumor proliferation through tumor-associated macrophages, myeloid derived suppressor cells, and T cells. LILRB4 signaling from myeloid-derived suppressor cells leads to T cell anergy and tumor tolerance. On Kaplan-Meier survival analysis, increased expression relative to the median of CSF1, MGMT, and IL6R was significantly associated with poor survival (all p<0.05). LILRB4 expression trended towards significance in its association with poor survival (p=0.124). On multivariable cox regression adjusted for age, gender, and tumor sidedness, increased expression of CSF1, MGMT, and IL6R was associated with increased hazard of death (all p<0.05). LILRB4 expression did not reach statistical significance (p=0.162).

Immunohistochemical analysis was performed to validate these gene expression findings at the protein level. Consistent with our gene expression data for CSF1, we saw increased M2-macrophage polarization, a known downstream effect of its signaling, among tumors of patients with poor survival. Direct antibody staining confirmed protein expression of MGMT and LILRB4. Although analysis did not reach statistical significance for any genes of interest, immunohistochemistry did demonstrate expected phenotypic and spatial distribution of cells of interest.

These findings suggest a myeloid-predominant tumor immune microenvironment in which overexpression of genes of interest promote a pro-tumorigenic environment. 

Table of Contents

INTRODUCTION          9

METHODS       12

RESULTS          15

DISCUSSION    18

CONCLUSIONS 22

STRENGTHS AND LIMITATIONS          23

CONCLUSIONS 25

ACKNOWLEDGEMENTS          25

FIGURES          27

FIGURE 1. CONSORT DIAGRAM DELINEATING PATIENT SELECTION  27

FIGURE 2. HEAT MAP SHOWING DIFFERENTIAL GENE EXPRESSION RESULTS OF NANOSTRING ANALYSIS  27

FIGURE 3A. KAPLAN-MEIER ANALYSIS OF OVERALL SURVIVAL ASSOCIATED WITH CSF-1 EXPRESSION.      28

FIGURE 3B. KAPLAN-MEIER ANALYSIS OF OVERALL SURVIVAL ASSOCIATED WITH MGMT EXPRESSION.    28

FIGURE 3D. KAPLAN-MEIER ANALYSIS OF OVERALL SURVIVAL ASSOCIATED WITH LILRB4 EXPRESSION.    29

FIGURE 4. IMMUNOHISTOCHEMISTRY STAINING FOR CSF1. 30

FIGURE 5. IMMUNOHISTOCHEMISTRY STAINING FOR MGMT.         31

FIGURE 6. IMMUNOHISTOCHEMISTRY STAINING FOR LILRB4.          31

TABLES 32

TABLE 1. PATIENT DEMOGRAPHICS   32

TABLE 2. AGGREGATE NANOSTRING ANALYSIS DATA           33

TABLE 3. PATIENT DEMOGRAPHICS STRATIFIED BY GENES OF INTEREST     34

TABLE 4. MULTIVARIABLE COX REGRESSION: ASSOCIATION BETWEEN CSF1 EXPRESSION & OVERALL SURVIVAL  34

TABLE 5. MULTIVARIABLE COX REGRESSION: ASSOCIATION BETWEEN MGMT EXPRESSION & OVERALL SURVIVAL          34

TABLE 6. MULTIVARIABLE COX REGRESSION: ASSOCIATION BETWEEN IL6R EXPRESSION & OVERALL SURVIVAL   35

TABLE 7. MULTIVARIABLE COX REGRESSION: ASSOCIATION BETWEEN LILRB4 EXPRESSION & OVERALL SURVIVAL           35

TABLE 8. IMMUNOHISTOCHEMICAL ANALYSIS OF TARGETED ANTIBODY STAINING.           36

REFERENCES   37

About this Master's Thesis

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School	Laney Graduate School
Department	Clinical Research
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Health Sciences, Immunology Health Sciences, Oncology Health Sciences, Surgery
Keyword	immunology colorectal cancer colorectal liver metastases tumor immune microenvironment
Committee Chair / Thesis Advisor	Amita Manatunga, Emory University
Committee Members	Brian Robinson, Emory University Gregory Lesinski, Emory University

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