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STATEMENT OF WORK

Trimethylamine N‐oxide (TMAO) and colorectal cancer

A. Background

There is convincing evidence for a role of red and processed meat in colorectal cancer, but the mechanisms underlying the association between red/processed meat and colorectal cancer are not clear. Colorectal cancer is the third leading cause of cancer‐related deaths in both men and women in the U.S. Elucidating the mechanism underlying the association between red/processed meat and colorectal cancer may help to identify intervention targets, which could reduce colorectal cancer morbidity and mortality. Recent studies demonstrated that trimethylamine N‐oxide (TMAO) is produced from choline and carnitine, compounds found in large quantities in red/processed meat and produced by gut bacteria. Moreover, TMAO is associated with cardiovascular disease, atherosclerosis, and inflammation. We propose that gut microbiota may be a major determinant in modifying colorectal cancer risk associated with red/processed meat intake via TMAO production. TMAO and the gut microbiota may account for inconsistent results from past studies, and may highlight the mechanism by which red/processed meat is associated with colorectal cancer. TMAO has been associated with increased inflammatory markers, which may impact cancer risk, and may also contribute to formation of the carcinogen N‐nitrosodimethylamine.

We will assess whether carnitine/choline and TMAO are associated with colorectal cancer in a case‐cohort study nested in the Alpha‐Tocopherol Beta‐Carotene Trial (ATBC). Our primary aim is to determine the association between TMAO, as a potential integrative measure of red/processed meat intake and the gut microbiota , and colorectal cancer. The overarching goal is to provide insight into the mechanism underlying the association between red/processed meat intake and colorectal cancer.

B. Purpose and Objectives of the Procurement

We propose to assess the role of carnitine/choline and TMAO in risk of colorectal cancer by assaying these compounds in sera in a case‐cohort study nested within the ATBC Trial. We will utilize serum samples from ~750 colorectal cancer cases and 750 controls in ATBC. Assays will be completed in Dr. Hazen’s laboratory at the Cleveland Clinic, which has extensive expertise in TMAO plasma measurements. We will measure serum carnitine, choline, and TMAO. TMAO will be measured by liquid chromatography‐tandem mass spectrometry (LC/MS/MS), the gold standard method for measuring TMAO. The Hazen laboratory already has panels set up to assay TMAO and many other analytes with this method. Choline and carnitine will be analyzed by stable isotope dilution LC/MS/MS analyses using established methods. For all assays, the laboratory will be blinded to case‐cohort status. Each batch will include an equal distribution of colorectal cancer cases and disease‐free individuals. 10% of the batch will include quality control samples used to calculate assay coefficients of variation.

1. Our primary aim is to determine the main effects of TMAO on colorectal cancer.

a. In order to fully explore this association, we will also investigate the associations between serum precursors of TMAO (carnitine, choline) and the disease endpoint.

Secondary aims are to:

2. Since TMAO is a biomarker only recently studied in relation to any disease, we will explore the associations that are related to the proposed role of TMAO in colorectal cancer. Specifically, we will investigate the association between meat intake and serum metabolites (TMAO, carnitine, choline). We will also explore the interrelationship between plasma TMAO, carnitine, and choline. Lastly, we will determine whether there is an association between meat intake and colorectal cancer in the overall ATBC cohort (~29,000 men; ~ 825 cases) as well as the subset in which TMAO will be measured (~700 cases).

3. Investigate, in an exploratory analysis, whether TMAO is a mediator of the association between red/processed meat and colorectal cancer.

C. Contractor Requirements

The contractor shall perform the following tasks:

1. Receive and catalog approximately 1500 serum samples. The receipt of biologic samples should be logged in a MS Excel file including the information of ID, amount of serum received, and date of receipt. To facilitate this, an excel file will be provided by the Government and will include: sample ID and volume provided. The Contractor will add the date of receipt, an internal ID, test/assay date, and values for each serum measurement of interest.

2. Analyze carnitine, choline, and TMAO in 1500 serum samples using LC/MS/MS and record the results in the Excel file above.

3. Provide a plan for determining if the replicate values are in agreement and for repeat duplicates, as needed. Any insurmountable problems in the analysis of samples shall be reported to the Project Officer within three days of when they happen.

4. Send data to the NCI.

5. The dates of service will extend 5.5 months from the date of award (est. 5/1/14‐10/15/14).

D. GOVERNMENT RESPONSIBILITES

NCI will provide the samples as specified by the laboratory. Specifications include:

1) All samples will consist of ≥250 μL of serum if possible. The exact value should be noted in the excel file provided.

2) Samples will be packed in dry ice for shipment to maintain their frozen condition, and shipped overnight.

3) Samples will be coded with an ID and will not have any personal identifiers attached.

There will be 33 batches, with a batch size of 50 (23 cases, 23 controls, 4 quality control samples).

E. REPORTING REQUIREMENTS AND DELIVERABLES

Results of the assay analyses shall be provided in an Excel spreadsheet containing sample ID, test/assay date, and test results (individual values for TMAO, carnitine, and choline for all samples and repeat measurement for replicates).

The contractor shall deliver to NCI no later than 10/15/2014, all of the above mentioned items.

F. INSPECTION AND ACCEPTANCE CRITERIA

NCI will have one month after receipt of the deliverables in which to review and accept the final data. If no comments or requests for revisions are provided within one month, the deliverables shall be considered acceptable.

G. ATTACHMENTS

None

H. CLEARANCES

We submitted a proposal for IRB approval for this study on 11/20/2013, and only coded, anonymous samples will be used.