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Questions & Answers

General Questions

What is Epi proColon®?

Epi proColon® is a molecular, colorectal cancer screening test that detects methylated Septin 9 DNA in blood. DNA methylation of the SEPT9 gene is increased in colorectal cancer. Methylated SEPT9 DNA from tumors is shed into the bloodstream and the methylation pattern is detectable in plasma by Real-Time PCR.

Why a blood test?

A blood test is a common and accepted method of testing. A colorectal cancer screening test based on a blood draw may be acceptable to screening-eligible people who have been offered but have a history of not completing CRC screening by 2008 United States Preventive Services Task Force (USPSTF) recommended methods. Even after counseling with their healthcare provider, 1 in 3 people in the US still decline screening by tests requiring stool sample collection and shipping, or more invasive tests requiring advance preparation and sedation such as colonoscopy and sigmoidoscopy. A blood test has the potential to increase screening participation in the eligible population that is currently not screened.

Who should be screened with Epi proColon®?

Epi proColon® is indicated for average-risk people, age 50 or older, who have been offered and have a history of not completing CRC screening by tests recommended in the 2008 USPSTF guidelines. These recommended methods such as colonoscopy, sigmoidoscopy and high-sensitivity fecal occult blood tests (FIT) should be offered and declined before Epi proColon® is offered as an option for screening. Epi proColon® is not intended to replace screening tests recommended by appropriate guidelines (e.g., 2008 USPSTF guidelines).

Can Epi proColon® be used to screen or monitor patients at high-risk for colorectal cancer (CRC)?

No. The Epi proColon® test is a screening test for use in patients considered to be at average-risk for acquiring CRC.

Are there any age restrictions for the test?

The test was clinically studied in a demographically diverse group of people between the ages of 50-85 who were considered to be of average-risk for colorectal cancer. CRC Guidelines generally recommend screening for all people between the ages of 50 and 75. Screening guidelines vary for people over age 75. The decision to screen people over age 75 should be made on an individualized basis in consultation with a healthcare provider.

Since this is a routine CRC screening test, how often should patients be tested?

The interval for use for Epi proColon® has not been established in clinical evaluations. In the subsequent years following a screening with Epi proColon®, people who continue to decline testing by 2008 USPSTF-recommended methods like colonoscopy and FIT, may be offered the test. Programmatic performance has not been studied.

What should a patient do if Epi proColon® is positive?

A positive test means that methylated Septin 9 DNA has been detected in the plasma sample tested. Methylated Septin 9 has been associated with the occurrence of colorectal cancer. Because Epi proColon® is not a confirmatory test for the presence of colorectal cancer, people with a positive Epi proColon® result should be counseled by their healthcare provider to undergo diagnostic colonoscopy to confirm a positive test.

NOTE: Positive test results have been observed in healthy people and in those diagnosed with chronic gastritis, lung cancer and in pregnant women. Because a colonoscopy procedure examines the interior lining of the colon and rectum, CRC is unlikely when no abnormal findings are discovered during this procedure.

What should a patient do if Epi proColon® is negative?

A negative blood test result indicates the absence of methylated Septin 9 DNA in the plasma sample tested. Because a negative test result is not confirmatory for the absence of colorectal cancer, persons should be advised to continue participating in a recommended screening program according to screening guidelines.

NOTE: Studies show that methylated Septin 9 DNA is not present in plasma from some patients with colorectal cancer and therefore, a negative test result does not guarantee absence of cancer. Detection of CRC is dependent on the amount of circulating tumor DNA in the plasma specimen and may be affected by sample collection methods, sample storage, patient factors and tumor stage.

Colorectal Cancer & Screening

Why should a person be screened for colorectal cancer?

Colorectal cancer is one of the most preventable and treatable cancers when it is found early. Based on the results of very large clinical trials, screening has been shown to reduce the mortality and incidence of colorectal cancer. Early stages of CRC are usually asymptomatic and therefore, early detection by screening can result in a greatly improved 5-year rate of survival and a more likely cure.

What are the recommended screening methods?

There are a number of screening guidelines that make recommendations for screening and which tests to use. The USPSTF recommends a number of tests for CRC screening with colonoscopy (every 10 years) and high-sensitivity fecal occult blood testing (FIT test, annually) being the most common.

The Epi proColon® Test – Background

What biomarkers are in the Epi proColon® test?

Epi proColon® is a test based on the detection of DNA methylation in a specific sequence of a promoter region of the SEPT9 gene. Epi proColon® also measures an internal control gene (Actin beta) to ensure that the total DNA amount in the sample is sufficient to interpret the test.

What is DNA methylation?

DNA methylation is a natural biological process where the cytosine base of DNA is modified by the addition of a methyl group. This modification is associated with the regulation of gene expression. Changes in DNA methylation often occur in cancer and can serve as a biomarker for finding cancer.

Can I use serum instead of plasma?

Epi proColon® was developed using plasma obtained from only BD Vacutainer® K2EDTA (lavender cap) blood collection tubes with a minimum of 3.5 mL needed to perform the test. While there are many publications describing DNA in serum, serum has not been evaluated for use with this test. Only BD Vacutainer® K2EDTA collection tubes have been validated for use with Epi proColon®.

What is cell-free circulating DNA?

Cell-free circulating DNA is the fraction of DNA that occurs in blood plasma (bloodstream).

Blood Collecting and Processing

What are the blood tube options for collecting blood specimens?

Blood should be collected according to your laboratory’s procedure for venipuncture using ONLY BD Vacutainer® K2EDTA (lavender cap) blood collection tubes. Other manufacturer’s EDTA tubes have not been validated for use with Epi proColon®.

How much blood should be collected?

Epi proColon® requires a minimum of 3.5 mL of cell-free plasma. To obtain this amount of plasma, we recommend that you collect a full 10 mL tube of blood—ensure that the evacuated tube is allowed to complete the fill volume. Some blood draw stations collect a second BD Vacutainer® K2EDTA (purple cap) blood collection tube as a backup measure.

Note: Do not freeze whole blood samples.

Are there specific plasma preparation requirements?

Plasma preparation should be performed within 4 hours after drawing blood. Whole blood collected in Vacutainer® K2EDTA (purple cap) blood collection tubes may be stored at 2°- 30°C prior to plasma preparation.

How should the plasma be stored and transported?

The plasma sample may be stored refrigerated, at 2°C to 8°C for up to 72 hours, or frozen, at -15°C to -25°C for up to 14 days. Transport and shipment of refrigerated or frozen plasma should follow the same temperature and time requirements as stated above.

Epi proColon® Test Procedure

The test kit is designed for 30 plasma samples and 2 controls (performed in triplicate). What if I have less than 30 patient samples to test?

The components of the Epi proColon® Plasma Quick Kit and PCR kit are provided in aliquots to allow multiple runs containing a smaller number of patient samples. The Epi proColon® Control kit provides sufficient Positive and Negative Control solution for 6 runs.

Is there an automated version of the test?

Epi proColon® has been approved for use as a manual test protocol for Applied BioSystems ABI 7500 Fast Dx. A laboratory may choose to validate an automated protocol for this test; however, the test performance characteristics would be not be covered by the manufacturer’s stated metrics and performance specifications.

Can the laboratory substitute test components?

No. The test has been approved for use only with Epi proColon® labeled kit components, reagents, and materials that are specified in the Instructions for Use (IFU 0008).

Can the laboratory substitute equipment?

Epi proColon® has been approved for use only with the Applied BioSystems ABI 7500 Fast Dx, as specified in the Instructions for Use (IFU 0008). Regarding other equipment utilized in the test procedure, substitutions are possible if the replacement has equivalent performance specifications to the recommended brand or equipment specification. This is particularly important for the magnetic separation device.

Can the test be performed on other PCR instruments or with other software?

The test has been approved for use on the ABI 7500 Fast DX. See IFU 0008 for information regarding appropriate SDS software versions. The use of any other instrument and software would require validation and would be considered off-label use by the manufacturer.

Can the magnetic beads be over-dried?

Yes. Do not increase drying time or temperature as over-drying of the magnetic beads might reduce bisDNA recovery. During Step 9.13, if over-drying occurs, the 10 minute drying time may need to be reduced.

Following the extraction step from plasma, if the magnetic beads appear to remain in solution or magnetic capture appears to be inefficient, what should be the next steps?

The tube should be incubated at 56°C for up to 10 min to improve capture efficiency. Following incubation at 56°C, place the tube into the DynaMag™-15 magnetic racks for another 5 to 10 min and continue processing following the Instructions for Use (IFU 0008).

Are there any special considerations when working with magnetic beads?

A homogeneous suspension of the Epi proColon® Magnetic Beads is essential for proper performance. Deviations from the specified amount of beads may lead to false results. To ensure correct magnetic bead concentration, the bottle should be mixed thoroughly just before pipetting. There should be no visible sediment at the bottom of the bottle. Ensure a homogeneous suspension between the pipetting steps.

What results are reported to the health care provider?

The test is a qualitative test and provides three reportable result outcomes: Negative; Positive; Invalid. Patient samples obtaining invalid results should not be reported to a healthcare provider. A new blood sample should be obtained and the test repeated. If invalid results are obtained a second time, please call Technical Support.