Ask the Experts About Circulating Tumor DNA in the Management of Colo-Rectal Cancer
CancerConnect is pleased to provide patients and caregivers the opportunity to ask questions about the role of Circulating Tumor DNA (ctDNA) in the Management of Cancer. We have put together a panel of leading cancer experts to answer questions and publish a forum for the exchange of information.
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John Strickler, M.D., Associate Professor of Medicine, Associate Director Clinical Research – GI Oncology, Co-Leader, Duke Cancer Institute Molecular Tumor Board, Duke University
Cancer is caused by genetic mutations, and these mutations can be detected by measuring circulating tumor DNA, or ctDNA, in the blood. Detection of ctDNA allows for personalized cancer surveillance based on an individual’s unique set of cancer mutations.
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ctDNA is currently the single most powerful predictor of colon cancer recurrence.
Despite standard-of-care treatment, more than 30% of patients with resectable colorectal cancer relapse. ctDNA analysis enables post-surgical risk stratification and can help with adjuvant chemotherapy treatment decision-making. The GALAXY clinical trial analyzed pre-surgical and post-surgical ctDNA in 1039 patients with stage II–IV resectable CRC and found that post-surgical ctDNA positivity was the most significant prognostic factor associated with recurrence risk and identified patients with stage II or III CRC who derived benefit from ACT. The results support the use of ctDNA testing to identify patients who are at increased risk of recurrence and are likely to benefit from ACT.3
Knowing if there are traces of cancer present in your body can help your oncologist decide:
- Your best initial treatment
- If you are responding to treatment
- If further cancer treatment needs to be considered
- If there are signs that the cancer has returned or progressed
Results from the the “Dynamic” clinical trial evaluating the strategy of using MRD assessment with "tumor informed" ctDNA also support the routine utilization of ctDNA MRD assessment for the management of early-stage CRC. According to study results ctDNA guided management decreased the use of chemotherapy by ~ 50% while not negatively impacting treatment outcomes.
Patients with suspected or newly diagnosed colon cancer should discuss the role of ctDNA testing prior to surgery to ensure testing is performed in a timely manner.
What is circulating tumor DNA (ctDNA)?
Circulating tumor DNA (ctDNA) is 150–200-base-pair fragments of DNA, which originate from cancer cells and are present in the bloodstream or other body fluids.
How is ctDNA different than cfDNA?
Cell-free DNA (cfDNA) is all the DNA in the bloodstream including germline DNA and tumor DNA. ctDNA is the portion of cfDNA that is derived specifically from the tumor.
How can ctDNA help manage cancer?
There are currently four clinical applications of ctDNA to guide precision medicine in patients with cancer:
- Detection of minimal residual disease (MRD) following surgery.
- Monitoring the treatment response in the metastatic setting.
- Identifying genomic drivers of therapeutic sensitivity and resistance.
- Guiding treatment strategies to overcome resistance to treatment.
How is ctDNA used for the management of early-stage cancers?
Across all stages of surgically removed cancer, detection of ctDNA following surgery is a strong predictor of cancer recurrence. The detection of ctDNA could lead clinicians to intensify therapy in certain situations. Conversely, the absence of ctDNA could provide an opportunity to minimize surveillance or adjuvant treatment.
How is ctDNA used for the management of metastatic cancer?
ctDNA can be used to monitor treatment response, identify genomic drivers of treatment sensitivity or resistance, or identify new therapies that could overcome genomic drivers of treatment resistance.
When should ctDNA be collected?
Both tissue and blood samples are used to build a patients individualized ctDNA test. Once the test is built, only blood samples are required for the periodic follow-up tests performed to monitor for MRD or recurrence. Since DNA assays require ctDNA shedding into the bloodstream, the performance of ctDNA assays is improved when blood is collected after—rather than during—active chemotherapy. For building the initial test, tissue should be sent as soon as it is available. The optimal time for the initial blood collection is 2-3 weeks after surgery (earlier is better)
Dr Strickler Discusses Role of ctDNA
Do you need to wait a certain amount of time, post surgery, to have the ctDNA performed?
It is advised that blood samples used for the Signatera assay should be collected 2 weeks following any surgery.
Can a “stored” tissue sample be used to build the Signatera assay?
Yes and this is important. The Signatera assay can be useful in the management advanced cancers and patients may not have had the assay built at the time of their original diagnosis. Tumor tissue stored as a formalin-fixed paraffin-embedded (FFPE) block or on slides can be used from the original diagnosis can be used to build the assay even when stored for several years.
How long should ctDNA testing be done? 3 years? 5 years?
The recommended surveillance period is 3 years. Physicians would only continue monitoring past 3 years if there was a clinical indication (rising CEA despite negative scan, indeterminate finding on imaging, clinical concern for recurrence, etc.)
What is the procedure for ordering a ctDNA test?
Currently ctDNA testing must be requested by a provider so the test can be sent to the company performing the test. Patients cannot order this test. Only a physician can order the test.
Is ctDNA different from NGS testing?
“Next Generation Sequencing,” or “NGS” is a platform that allows simultaneous testing of multiple molecular targets. NGS testing can be performed on tumor tissue or blood (ctDNA).
If my doctor orders NGS testing on tumor tissue, will ctDNA testing also be performed?
Not always - ctDNA is typically a separate test and will usually not be ordered together with NGS testing unless specifically requested. Providers and hospitals use a variety of different companies to perform DNA testing. Some companies, like Natera perform both ctDNA and NGS testing, while others do not.
Does insurance pay for ctDNA testing?
Signatera is covered by Medicare for: Monitoring disease progression, recurrence, or relapse for colorectal cancer and for monitoring of response to immunotherapy treatment.
Natera welcomes all commercial insurance plans and works with patients to ensure cost is not a barrier for testing. Please refer to our website for In-Network plans that we participate with or call your insurance company. An affordable cash pay rate is available for patients who do not wish to use insurance (exclusions apply). For questions or financial assistance, please contact Natera’s Patient Coordinators:
Phone: 650.489.9050 Fax: 650.412.1962
Email: oncologybilling@natera.com
Patient-Submitted Questions
What is difference between ctDNA tests for early cancer detection vs MRD?
Early detection tests are similar but not identical to MRD tests. Early detection tests are typically optimized to detect the early presence of an unknown malignancy. MRD tests are designed to detect the presence of a cancer that was previously known to be present but removed. As a general rule, an early detection test is broad (can detect multiple cancers) but is not as sensitive for 1 specific cancer. MRD tests are more sensitive for 1 specific cancer, but are not designed to detect other cancers.
What is difference between tumor-naive vs. tumor-informed CRC MRD assays. How much do we know about ct DNA as a forecasting tool?
Tumor naïve tests do not require genomic sequencing of tumor tissue. These tests rely on a standard mutation and methylation panel that is common to the cancer under study. Tumor informed tests require sequencing the patient’s tumor, and then building a custom panel to detect those tumor mutations in blood. We have no head to head studies comparing the two different approaches.
Stage 3b colon cancer. Successfully resection all margins clear, all ct scans clear. Took signetara test. Question, if comes back negative do I need adjuvant chemo?
Signatera is the most powerful prognostic factor of many but is not yet ready to be used alone to predict the role of chemotherapy. A negative test is of course very good news and should be considered in combination with other prognostic factors to help you and your doctor decide the role of chemotherapy – stage 3b is typically high risk for recurrence so most doctors will strongly consider recommending chemotherapy. The signatera test is also used post treatment to refine the role of surveillance for recurrence.
I have had nine negative Signatera tests and now a positive. What are the chances I really do have a recurrence versus a false positive?
False positives are extremely rare, but there have been reports of transient positive results that spontaneously clear. If there is any doubt, I recommend rechecking the test 4-8 weeks after the positive result to confirm. If the ctDNA result remains positive it confirms the result. Liver MRI is a great test to consider if the CT scan is clear.
How reliable is ctDNA testing during chemotherapy treatment? Should the test be performed while on chemotherapy?
ctDNA testing is less likely to be positive during chemotherapy treatment. Despite that, it can still be useful during treatment. Reduction in ctDNA is associated with favorable response to treatment and better prognosis.
Can they take a sample from the diagnostic colonoscopy to use for Signatera’s testing?
Yes a colonoscopy specimen can be used, but it is less likely to be a successful test (There is less tumor tissue present for genomic sequencing).
What clinical MRD testing is currently available for Lynch Patients?
Lynch patients are eligible for all tests on the market (early detection tests, tumor informed assays, and tumor naïve assays).
My cancer is suspicious for both colon and possibly bladder cancer. Does the Signatera assay require an accurate differentiation between these two types of cancer?
Great question - Signatera is “tumor informed” meaning it only detects the original cancer from which the biopsy was made. It is not “diagnostic” it is for “monitoring” the cancer to detect early recurrence or response to treatment. NGS testing and the tissue biopsy are required for determining the cancers site (organ) of origin.
I was diagnosed with stage 3 colon cancer. After surgery and 9 rounds of chemo I am cancer free. My CT DNA test have been negative since after the surgery and the chemo was just an added measure. If cancer were to metastasize, would it be the same DNA as my original colon cancer?
Yes if the cancer recurs it is almost always with the same mutation initially and be detected by the same ctDNA test.
Can you explain what "Higher than expected cell free DNA (cfDNA) events were found in the plasma sample. Excessive cfDNA may decrease clinical sensitivity of the test."
Higher levels of germline cfDNA are released when patients receive surgery (wound healing) or chemotherapy (cell death). Higher cell free DNA requires increased filtering and lowers the sensitivity for detecting tumor derived fragments which means the test may not be as accurate at detecting cancer when it is present. There are more false negative results.
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I had colon surgery to remove a tumor that was found to be cancerous, along with 3 lymph nodes. My first signatera test came back negative. Do I still need adjuvant chemotherapy?
Unfortunately it's not as simple as that. You either have a stage II or III cancer and Signatera can help determine the overall risk of cancer recurrence and whether adjuvant chemo might be of benefit. Signatera is the single best prognostic indicator for recurrence and a negative test is great news. Your doctor should be able to quantify your risk of recurrence and the potential benefit of adjuvant chemotherapy. For example if you have stage II and a negative Signatera chemo may only improve your risk of recurrence be a few percentage points, if stage III the benefit may be higher. Signatera is very useful for monitoring for a recurrence as part of your surveillance program after treatment and can detect a recurrence as much as 9-12 months before conventional PET and CT scans.
Would it be detected by the CT DNA test? And am I now unable to have metastatic cancer since I've had months of negative CT DNA tests?
No, there remains a risk of recurrence which is why your doctors will utilize a surveillance program consisting of ctDNA every 3-6 months and periodic CT/PET scans. The goal is to detect recurrence’s early when they are isolated and can be treated with surgery.
I was diagnosed with stage IV colon Cancer that metastasized to the lungs in Feb 2022. Treated with palliative FOLFOX + bevacizumab: May 2022 until March 2023 and had colon resection 9/22. Lungs clear of Cancer. Negative MRD/ctDNA test 10/2022, 1/2023, 3/2023, 5/2023 and now 8/2023 POS with a MTM/ml: 7.03. What does this mean? is this a High number?
It most likely means that the cancer has recurred as evidenced by the ctDNA detected in the blood-the magnitude of the number is not very important. A positive ctDNA can occur several months before a recurrence is detected by CT or PET scan. Early detection of recurrence by ctDNA is of course very disappointing but serves two purposes.
- Increased CT/PET surveillance in order to detect the recurrence when it may be surgically removed.
- Increased time to plan for “next steps” What is next best choice of therapy? Should I be considering a clinical trial and begin the search?
How does your ctDNA interact with active 5FU chemo drugs. That is, for aggressive maintenance, with daily oral Xeloda or UFT, 5FU prodrugs, beyond the chemo's inhibition of the cancer cells, how do they interact with the ctDNA probe measurement itself?
Chemotherapy does not directly interact with the probes. Chemotherapy suppresses the ctDNA in the bloodstream, making it harder to detect the cancer.
I am undergoing treatment for stage III colon cancer. Currently on adjuvant capecitabine. Signatera being checked monthly. January was 1.18 February was 0.69 May was 100.29 What does it mean when the number is going up while ON treatment? Can treatment effect results?
Rise of ctDNA levels on treatment is generally indicative of treatment failure. You should discuss restaging with your doctor with a focus on the liver (the most likely site of treatment failure).
If a Signatera Test can detect Residual Cancer 6-18 months before its visible on CAT or PET Scans, how much earlier on average does Signatera detect Cancer before a noticeable rise in CEA? I just got a 1.7 CEA but my CEA was 0.6 3 1/2 weeks ago and my Signatera negative?
I am unaware of any data that can answer that question for you. Because Signatera is more sensitive and specific than CEA doctors rely on it over CEA and in your case Signatera remains negative. Remember CEA can be detected in the absence of cancer.
Can you explain what "Positive Below Analytical Range" means on a Signatera test? This has happened twice and both time the values were under 1.
There is a complicated explanation for this from Natera, however this is technically a positive test meaning that cancer was detected in the sample but at a lower level of detection than can be accurately quantified. Sometimes the amount of material being measured by a test is less than the ability of the test to detect it. This can often be the case when looking for individual cells or parts of cancer cells. The Signatera test is best used serially (every 3 months) to look for residual cancer - in some situations the detection of ctDNA moves above and below the threshold of detection possible reflecting the bodies immune systems effort to eradicate the cancer.
Can you explain how or if chemo affects Signatera ctDNA testing? Are the results reliable when taken during chemo treatment?
Chemotherapy will suppress ctDNA shedding and decrease the odds of a “positive” result. The initial value is obtained prior to starting chemotherapy and its best used for surveillance once chemotherapy is completed.
Why do these tests sometimes miss cancer in the lungs/liver?
These tests rarely miss disease in the liver. MRD tests tend to miss disease in the lungs, because those metastases secrete less circulating tumor DNA into the bloodstream and tend to be slower growing overall (less cell turnover to release DNA)
Can CTDNA go up as part of pseudoprogression on immunotherapy?
No… pseudoprogression is associated with lower circulating tumor DNA levels, but worsening disease on imaging.
Will a patient need chemo if a positive ctDNA result happens within 8 months or resection surgery?
A positive ctDNA tells us that the cancer has recurred somewhere – but not exactly where. The first objective if ctDNA turns positive when used to monitor early stage colon cancer is to increase the frequency of PET/CT in order to find the recurrence’s exact location soon as possible. Isolated recurrences can often be treated with surgery alone, others require systemic treatment with chemotherapy or precision medicines based on NGS testing.
So first results from a Signatera test was negative. I had follow up colonoscopy in September and pet scan in September. All test were negative but my last Signatera tests came back .02 July .05 in September and .77 in December.
Unfortunately that strongly suggests the cancer is likely recurring. By using Signatera you can detect recurrence an average of 8 months before scans. This is helpful because your doctor can increase surveillance with PET or CT and potentially detect a surgically treatable recurrence.
Does a ctDNA positive test indicate a recurrence is probable or certain at some point without further treatment?
If a ctDNA test is positive, there is a strong likelihood for recurrence, but not 100% certain. The likelihood for recurrence is around 80%. Meaning, if the test is positive, the chance for recurrence is approximately 80%. However, 20% of patients who test positive will not have recurrence.
I finished 12 rounds of Oxaliplatin 7 weeks ago and I am currently on a 2 week cycle keeping the remaining drugs . I have been taking the signatera test every month now and the blood draw is on the Monday right before Tuesdays treatment. Do you know if the current therapy I am on effects the testing results? Also is there any data that shows best time spacing post chemo treatment and ctDNA testing?
Active/ effective chemotherapy will reduce ctDNA shedding and decrease the sensitivity of the test. That said, it is a very favorable prognostic sign when ctDNA is not detected (even if this happens while on chemotherapy). This is generally a sign that the treatment is working. Sensitivity would be increased in the blood draw occurs 1-2 months after the last dose of chemotherapy.
My 30 yr old daughter had stage 3 colon cancer had surgery followed by 12 chemo treatments her scan came back good but ctdna was detected. She has to wait for another scan. What kind of treatment is usually offered next?
A positive ctDNA almost always means there is a cancer recurrence – this early detection can lead to effective treatment. Your doctor will typically use a PET scan to search for the recurrence but it may take a few attempts as the ctDNA detects the cancer often several months before a CT or PET can locate it. The treatment plan will be determined based on the location of the recurrence and NGS-biomarker testing. Treatment of stage IV colon cancer.
Colon cancer occurring at such an early age raises the concern for “Lynch Syndrome” – a form of hereditary colon cancer. Your daughter should be evaluated is this has not already been performed. About Lynch Syndrome
I am 1 1/2 yrs cancer-free. I had negative ctDNA test results twice previously. My recent result is positive. I had a clean CT and will have a PET this week. How often can the ctDNA be done?
The Signatera ctDNA test can be performed as often as your doctor thinks it will be helpful. Normal surveillance testing is every 3 months.
I have early stage colon cancer and want to test for residual disease. Is Signatera or Reveal more sensitive and specific?
To this point Signatera and Reveal have not been compared head-to-head but Signatera appears to be the better test. The specificity for Reveal and Signatera is excellent and exceeds 90%. The sensitivity for Signatera is also excellent and recently published data for Reveal suggest it is inferior to Signatera. For Signatera the single post-op time point sensitivity is 65-70% and increases with each additional draw. Longitudinal sensitivity likely exceeds 90%. The longitudinal sensitivity for REVEAL is reported to be 50%.
The value of my Signatera test for early stage colon cancer never reached zero - what does that mean?
It means with almost 100% certainty that some cancer remains in your body - the doctors will increase their CT surveillance to look for recurrence because if the cancer can be found early and in a single location it is still curable. The benefit of additional chemotherapy administered at this time is unknown.
Is ctDNA testing available for endometrial or other cancers?
Tumor informed MRD detection is possible for any advanced solid tumor. The test is tumor type “agnostic”. There just needs to be enough sample to identify 16 clonal variants. The test is run by Natera and needs to be ordered by a physician.
The test is increasingly used in the management of muscle invasive bladder cancer. The development of ctDNA tests and their clinical validation is an ongoing process. Data is available for over 30 tumor types, the bulk of which is in Colon, lung, bladder and breast cancer. Medicare now covers testing for stage II - III Colorectal cancer and and draft coverage for immunotherapy monitoring in pancreas cancer is pending. More information is available at
My wife has been recently under surgery for a colon cancer (2nd stage); after 8 months from surgery she just received a negative signatera test. As she still has an inflammatory process affecting many nodules in her lungs, my question for you is the following: is the signatera negative test stating she's now totally cancer-free or it states only that she's free from the primary colon cancer ? I'd like to know if her lung nodules could be cancer or she is totally cancer free at this time following the signatera test result. Thanks for your answer...!
The signatera test is “tumor informed” meaning it detects the actual DNA from the colon cancer that was diagnosed where ever that cancer is in the body. It does not detect DNA from other cancers.
What if an initially negative ctDNA test converts to positive after surgery?
Through longitudinal assessment, if a negative ctDNA test were to become positive, this would be concerning for eventual disease recurrence (85-90%). The lead time is up to 8 months compared to CT scans and 3 months to CEA - within the 2-3 years recurrence risk would approach 100% after long-term follow up.
Can ctDNA replace CEA in Colon Cancer?
The sensitivity and specificity for ctDNA is much higher than CEA. Typically, if ctDNA is positive, it will be positive 3 months before a CEA is abnormal.
There are limitations for ctDNA as it does take more effort (sequencing and preparing a tumor-derived ctDNA assay for a patient), specific methods to draw and store the blood, where CEA can be done in most labs. Until there is more accessibility for ctDNA (MRD) for all patients, I would consider ctDNA as a complement to our available surveillance tools.
I had Signatera test then chemo for stage III colon cancer. Could this test replace CT/MRI imaging altogether? Why not just follow my ctDNA and get imaging scans if it becomes positive?
Signatera is strongly predictive of recurrence but it isn’t perfect. You could reduce the frequency of CT/MRI from a negative result, but national guidelines are still recommending scans at least annually for low risk patients with stage III disease.
Is there an understanding of what ctDNA positive means at it's minimal level. Is it like CEA in that the levels can vary or is it a yes or no result? Would a positive mean somewhere a fixed tumor is shedding cells? Any chance the nascent tumor location can be pinpointed based on resulting testing. Also, given the complexity of these tests, do Drs go on recommended treatment plans or do they make independent decisions? How many negatives would be needed to stop chemo, or, since getting chemo, are negative ctDNA less predictive? I guess these types of questions need major trials to be answered.
For MRD positivity, the assay is very sensitive and can detect as low as 0.1% of a MAF (mutant allele frequency). A positive result suggests that there is microscopic disease detected in the bloodstream. At this time, the assay is unable to specify where the cells are coming from. Since blood circulates throughout our whole body and we are testing for at one specific point in time, the tumor cells could be from anywhere within one's body.
At this time due to the test limitations (false negative rate), the results are prognostic and should be used on a case by case decision, rather than it being applicable for all. Clinical trials will help better ascertain whether MRD can be used to drive treatment decisions (intensifying chemotherapy in those with MRD positivity or de-escalating treatment in those who become MRD negative).
What tests are routinely ordered on colon cancer tissue removed during surgery?
DNA mismatch repair deficiency (MMR): Approximately 15% of stage I-III colorectal cancer diagnoses arise from the microsatellite instability (MSI) pathway, which is a consequence of deficient DNA mismatch repair (MMR). Deficient MMR (dMMR) can develop from an inherited germline mutation in a MMR gene (MLH1, MSH2, MSH6, PMS2)—Lynch Syndrome, for example—or, more commonly, can be due to epigenetic inactivation of the MLH1 gene and the CpG island methylator phenotype (CIMP). dMMR tumors are associated with a better stage-adjusted overall survival compared to proficient MMR (pMMR) tumors. MMR status may also be predictive for benefit from adjuvant chemotherapy and the type of chemotherapy patients will receive.
If I already had my cancer removed, can tissue from the surgery be obtained for ctDNA testing?
Most patients who have a cancer removed will have had tissue stored, which will be available for additional testing, including tumor somatic profiling, or to develop tumor-informed cell-free DNA profiling for MRD ctDNA assessment.
In tumor-informed assays, the primary tumor is sequenced to identify the patient-specific genomic alterations, upon which the primers for ctDNA testing in the plasma are designed to be based. Tumor-agnostic assays rely on a panel of preselected primers designed to detect known genomic alterations and epigenetic signatures relevant to CRC.
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The likelihood for recurrence is around 80%. Meaning, if the test is positive, the chance for recurrence is approximately 80%. However, 20% of patients who test positive will not have recurrence. The 20% can potentially be cured with adjuvant chemotherapy after their surgery. The 20% who don't have a reoccurrence, in spite of testing positive for ctDNA, all received adjuvant treatment and this turned them negative on the ctDNA test? Does this mean the other 80% of positive ctDNA tested patients waited for CEA, CT, or other tests to find the reoccurrence?
In general, a positive test is a strong predictor of recurrence. That said, some patients with ctDNA+ disease after surgery have their outcome changed by adjuvant chemotherapy. If the ctDNA test remains positive after adjuvant chemotherapy, additional scans should be obtained to identify the active disease. If these tests are negative, a PET-CT would be reasonable. In some cases, the lesions can be removed with curative intent.
Are clinical trials available evaluating ctDNA testing?
Numerous clinical trials are currently underway to validate the role of ctDNA in selecting patients for adjuvant chemotherapy by addressing several questions:
- Can adjuvant chemotherapy be omitted in patients who have no detectable ctDNA after surgery (treatment de-escalation)?
- Could treatment escalation help in patients with detectable, post-operative ctDNA?
- Does the clearance of ctDNA with adjuvant chemotherapy result in a cure?
- Is ctDNA a predictive biomarker for treatment efficacy?
- What is the best strategy to treat patients who continue to have detectable ctDNA after adjuvant chemotherapy?
Is ctDNA being used in Melanoma?
Circulating tumor DNA measurements before and during treatment could help guide selection of therapy for patients with BRAF V600-mutant metastatic melanoma.
Researchers assessed how well ctDNA levels prior to treatment, and at 4 weeks into treatment, predicted patient survival in 383 adults included in two melanoma clinical trials where patients were treated for BRAF V600 advanced metastatic melanoma.
Results showed that elevated baseline BRAF V600 mutation-positive ctDNA levels predicted worse survival and undetectable ctDNA at week 4 was significantly associated with improvements in survival and a delay in cancer progression. Future research should explore whether clinical decision-making, informed by ctDNA measurements, can improve patient survival compared to standard approaches, which rely primarily on radiographic scans.1
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References:
- https://www.thelancet.com/journals/lanonc/article/PIIS1470-2045(20%2930726-9/fulltext
- https://www.nejm.org/doi/full/10.1056/NEJMoa2200075
- https://www.nature.com/articles/s41591-022-02115-4
