Genetic and Epigenetic Determinants of Response to Fluorouracil-based Adjuvant Chemotherapy in Patients With Stage III Colorectal Cancer

Study Overview
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Brief Summary
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The goal of this laboratory research is to look for genetic and epigenetic markers that can predict which patients with stage III colorectal cancer will benefit from fluorouracil-based adjuvant chemotherapy.

Detailed Description
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This is a prospective project in collecting and assessing clinical outcomes data related to molecular profiling of tumors based on samples from peripheral blood, primary tumor, and adjacent normal colorectal tissue. Objectives: Validation of predictive value of known markers CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) in the response to 5-fluorouracil-based chemotherapy in patients with stage III colorectal cancer. Exploratory evaluation of the potential predictive values of known genetic variations including, but not limited to, KRAS mutations, BRAF mutations, PIK3A mutations, and EGFR mutations, etc. Exploratory identification and evaluation of the predictive value of novel methylation aberrations identified by whole-genome bisulfite sequencing. Exploratory identification and evaluation of the predictive value of novel genetic aberrations discovered by RNA-sequencing (RNA-seq) or genome-wide association study (GWAS). Outline: Blood is collected at baseline and examined for single-nucleotide polymorphisms (SNPs) and expression level of specific gene. Tumor and corresponding normal tissue at surgical resection and assessed for gene methylations, mutations, and expressions.

Official Title
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The Identification, Validation and Implementation of Molecular Markers to Predict Response to Fluorouracil-based Adjuvant Chemotherapy in Stage III Colorectal Cancer Patients - Prospective Clinical Observational Study

Conditions
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Stage III Colorectal Cancer

Intervention / Treatment
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* Genetic: Gene mutations analysis
* Genetic: Gene methylation analysis
* Genetic: Gene expression analysis
* Genetic: SNP analysis
* Genetic: Protein expression analysis


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Requirements for tumor parameters Histologically confirmed colorectal adenocarcinoma. Stage III disease (any pT, N1-2, M0). Tumors must have been curatively resected (R0). No evidence of residual involved lymph node disease or metastatic disease at the time of registration. Requirements for patient characteristics Patient is ≥ 18 years of age on the day of consenting to the study. Eastern Cooperative Oncology Group Performance Status (ECOG PS) 0 or 1 at the time of screening. Fertile patients must use effective contraception. Patients must demonstrate ability to understand and the willingness to sign a written informed consent document. Patients must demonstrate ability to complete study questionnaires. Patients must provide written informed consent prior to performance of study-specific procedures or assessments and must be willing to comply with treatment and follow up. Required initial laboratory values Leukocytes ≥ 3,000/mm^3 Absolute neutrophil count ≥ 1,500/mm^3 Platelet count ≥ 100,000/mm^3 Creatinine ≤ 1.5 times upper limit of normal (ULN) Total Bilirubin ≤ 1.5 times ULN Aspartate aminotransferase (AST) ≤ 2.5 times ULN Alanine aminotransferase (ALT) ≤ 2.5 times ULN Exclusion Criteria: Patients with a known history of allergic reactions attributed to compounds of similar chemical or biologic composition to fluorouracil. Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection-requiring intravenous antibiotics, or psychological, familial, sociological, or geographical condition that would limit compliance with study requirements Following cardiovascular conditions within the past 6 months: Myocardial infarction, unstable angina, symptomatic congestive heart failure, serious uncontrolled cardiac arrhythmia, cerebrovascular accident or transient ischemic attack, deep vein thrombosis, other significant thromboembolic event. Known human immunodeficiency virus (HIV)-positive patients and those with known hepatitis B or C. Patients with evidence of other primary malignancies within the past 5 years, excluding adequately treated basal cell carcinoma of the skin or carcinoma in situ of the cervix. Pregnant or nursing.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 70 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Adjuvant chemotherapy<br>Samples from patients with stage III colorectal cancer who are going to receive fluorouracil-based adjuvant chemotherapy will be used for gene mutations analysis, gene methylation analysis, gene expression analysis, SNP analysis, and protein expression analysis. | Genetic: Gene mutations analysis<br>* Genomic DNA extracted from paired fresh-frozen tumor and normal tissues is used for KRAS mutations, BRAF mutations, PIK3A mutations, and EGFR mutations, etc., by sequencing and for MSI analysis by PCR and capillary electrophoresis.<br>Genetic: Gene methylation analysis<br>* Genomic DNA extracted from paired fresh-frozen tumor and normal tissues is used for specific gene methylation or CIMP status analysis by MethyLight or bisulfite sequencing. Whole-genome bisulfite sequencing will be used to identify novel candidate set of predictive markers.<br>Genetic: Gene expression analysis<br>* RNA extracted from paired fresh-frozen tumor and normal tissues or serum is used for specific gene expression analysis by real-time reverse-transcription PCR (RT-qPCR). RNA-seq will be used to identify novel candidate set of predictive markers.<br>Genetic: SNP analysis<br>* Genomic DNA extracted from serum is used for specific SNP analysis by using sequencing. GWAS with SNPs array will be used to identify novel candidate set of predictive markers.<br>Genetic: Protein expression analysis<br>* Tissue microarray made from formalin-fixed paraffin-embedded (FFPE) paired tumor and normal tissues is used for specific protein expression analysis by immunohistochemistry staining.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Time to recurrence (TTR) | Time to any event, except non-cancer-related death. All recurrences, treatment-related deaths, second same or other primary cancers, and deaths from other cancers are considered to be events. Loss to follow-up and non-cancer-related deaths are censored. Associations between TTR and genetic and epigenetic markers will be analyzed. | 3 years after surgery | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Disease-free survival (DFS) | Time to any event, irrespective of cause. All events are included, except loss to follow-up. Associations between DFS and genetic and epigenetic markers will be analyzed. | 3 years, 5 years after surgery | 
| Cancer-specific survival (CSS) | Time to death caused by the same cancer, whether due to the original tumor or to a second primary same cancer. The only event is death from the same cancer, without taking into account whether the death is caused by the primary tumor or a second same cancer. Locoregional recurrence, distant metastases, second primary same cancers, and second other primary cancers are ignored. Deaths from other cancers, non-cancer-related deaths, treatment-related deaths, and loss to follow-up are censored. Associations between CSS and genetic and epigenetic markers will be analyzed. | 3 years, 5 years after surgery | 
| Overall survival (OS) | Time to death, irrespective of cause. There is no need to specify whether the death was due to cancer. Locoregional recurrence, distant metastases, second primary colorectal cancers, and second other primary cancers are ignored. Loss to follow-up is censored. Associations between OS and genetic and epigenetic markers will be analyzed. | 3 years, 5 years after surgery |