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Prospective Study Supports Liquid Biopsy Monitoring of Colorectal Cancer Drug Response

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NEW YORK (GenomeWeb) – New data from a small prospective study has added to growing evidence that there is clinical value in monitoring patients with colorectal cancer using liquid biopsy, or circulating tumor DNA tests.

The study, published recently in the journal Oncotarget, tracked 25 patients in a trial of first-line therapy using the EGFR inhibitor cetuximab (marketed by Eli Lilly as Erbitux) and the chemotherapy regimen FOLFIRI.

Led by Manuel Hidalgo — who during the study was chief of the gastrointestinal research unit at the Spanish National Cancer Research Centre and is now chief of hematology-oncology at Beth Israel Deaconess Medical Center in Boston — the investigators found overall that ctDNA analysis accurately genotyped patients and did indeed correspond with emergence of resistance to therapy.

Moreover, the authors wrote, the results demonstrated for the first time that circulating mutation status is a valuable prognostic biomarker. Sustained wild-type status is associated with prolonged tumor response to anti-EGFR therapy, while mutation explosion events predicted an "imminent clinical deterioration in patients."

When the first EGFR inhibiting therapies were developed for colorectal cancer, it became clear that they only worked for patients with wild-type KRAS status, and then only for a limited period of time — until patients developed a KRAS mutation that made them resistant to treatment.

More recently, studies have emerged showing that ctDNA in these patients reflects the KRAS mutation status of their tumor tissue at different timepoints, and can be identified before clinical symptoms of resistance emerge.

"These were very exciting data but all retrospective and confined to a small cohort of selected patients. It was still unclear whether liquid biopsy could be useful in the 'real life' of an oncology practice," Rodrigo Toledo, the study's first author and a researcher at the Spanish National Cancer Research Center, told GenomeWeb in an email, so he and his colleagues on the study set out to do a prospective analysis.

At the time the study started in 2013, Toledo said that the most promising technology was Sysmex Inostics' emulsion-based digital PCR technology BEAMing.

Since then, many other tools have emerged that are pushing sensitivity and cost boundaries, for example the CAPP-seq technology being advanced by Roche. "However, in 2013 BEAMing was the gold standard and we decided to used it to be sure to get trustable and comparable results," Toledo said.

Of the 25 study subjects, two were excluded because they didn't have sufficient post-treatment plasma samples. Of the remaining 23, seventeen showed some clinical benefit from the cetuximab-FOLFIRI treatment, including three patients who had stable disease, eleven with a partial response, and three with a complete response.

Of the patients who did not show a clinical benefit, three carried BRAF mutations, and one carried a BRAF/PIK3CA mutation combination in their circulating DNA. A remaining two patients presented continuous disease progression but had no mutations detected in their plasma using the BEAMing technology.

"In these patients, disease progression was likely the result of a mutation in a rarer or unknown resistance-associated gene," the authors wrote.

Overall, the group saw a " perfect match" when they compared the BEAMing results to US Food and Drug Administration-approved tumor tissue mutation kits from Life Technologies and Roche.

Finally looking at patient responses, they were able to see distinct stratification based on ctDNA results, with patients who continued to show a wild-type KRAS status having the most prolonged tumor response to anti-EGFR therapy, and those with rapid development of mutations having a rapid deterioration.

"The community is very confident that liquid biopsy will become a standard clinical practice for tumor genotyping," Toledo said. And as evidence grows, the capacity of liquid biopsy to assess treatment resistance is also now becoming well established, he said.

Researchers are even exploring the use of ctDNA analysis to monitor other endpoints in colorectal cancer, for example using liquid biopsy in early-stage disease to predict which patients are likely to maintain a disease-free status after surgical removal or their tumors alone, and which may benefit from added chemotherapy treatment.

However, as the usefulness of liquid biopsy in cancer monitoring grows, an important question is whether being able to detect emerging relapse or drug resistance before it becomes evident in clinical symptoms actually improves patients' lives or outcomes.

How to act upon a liquid biopsy result showing rising levels of a cancer resistance mutation is still largely unknown, Toledo said, but some researchers are hopeful that by detecting emerging resistance at the earliest possible moment, creative strategies to switch therapies — or to give patients therapeutic "holidays" that may dampen the emergence of resistant clones — can lengthen or improve their lives.

"Our study [is] the first prospective assessment of [liquid biopsy] monitoring in mCRC to face such a 'dilemma'," he said. "And as far as I know, Patient 3 is the only proper case in this situation," he added.

Patient 3 achieved a significant reduction in the size of two lung metastases during an initial 10 months on FOLFIRI-cetuximab

At that point, because of its toxicity, the chemotherapy was withdrawn and the patient was treated using only the anti-EGFR drug.

Using BEAMing, the researchers saw an increase in KRAS G12D alleles from 0.001 percent to 0.037 percent after this switch. Then, when clinicians decided to reintroduce the FOLFIRI-cetuximab combination, there was a corresponding decrease in KRAS G12D levels to between 0.002 percent and 0.004 percent.

"The patient continued to maintain a controlled disease states at 30 months after she entered the study," the researchers reported.

Toledo noted that the researchers are now planning to run a clinical trial in which patients with rising levels of KRAS can be randomized to standard clinical practice or to the liquid-biopsy-guided arm "where we will test the efficiency of changing treatment of starting treatment holidays."

But regardless of the question of whether ctDNA monitoring can improve outcomes by prompting therapy changes, Toledo said that the results of the study present important new evidence that ctDNA could be immediately useful at least for prognostic purposes.

Most excitingly, he said, the study showed for the first time that the percent of mutations observed in cell-free DNA and the way that percentage changes can help physicians understand how likely patients are to respond well or poorly to these drugs.

During the two years of the study, the patients who maintained wild-type status all showed a relatively prolonged response to cetuximab. In contrast, cases with a rapid increase in cfDNA mutation levels showed a strikingly rapid clinical progression.

And in the middle, those with intermediate increases in cfDNA mutation levels also developed resistance, but not as precipitously.

Toledo said that he and his colleagues also hope to gather information on ctDNA mutations and clinical outcomes from a larger cohort of metastatic CRC patients to further explore the prognostic value that they saw in their initial study.

Finally, he said "as different groups worldwide have monitored cfDNA mutations of mCRC patients using BEAMing, we are going to try to organize a consortium for gathering all these data that are comparable in a way to expanding our knowledge."