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Liquid Biopsy Uncovers Drug Resistance Mechanisms in GI Cancer Missed by Tissue Biopsy


NEW YORK – A group led by researchers from the Broad Institute and Harvard Medical School found that liquid biopsy can capture a cancer's genetic heterogeneity not identified by tumor biopsies, as well as how cancer develops targeted drug resistance. 

The team therefore believes that liquid biopsy and standard tissue biopsy should be integrated to identify genetic alterations that lead to acquired resistance and eventually improve downstream therapeutic techniques.  

"The standard method is to perform tissue biopsies on the patient, but there are situations where collecting tissue biopsies is not optimal," said Massachusetts General Hospital and HMS Investigator Ryan Corcoran, co-corresponding author on a paper describing the findings and published last week in Nature Medicine

"Studying resistance with liquid biopsy enabled us to assess mutations in cell-free DNA shed from multiple tumor lesions in an individual patient, rather than just from the single biopsy's lesion," he added.

In the study, the researchers prospectively compared liquid biopsy to standard tumor biopsies in a group of 42 patients with gastrointestinal cancer that had acquired resistance to targeted therapy. 

The researchers collected about two 10-ml tubes of blood from each patient. In addition, they also collected matched tumor biopsies at the time of eventual disease progression in 23 patients. 

To identify candidate-acquired resistance mechanisms, Cocoran's team used Guardant Health's Guardant360 next-generation sequencing assay to analyze cell-free tumor DNA (cfDNA) isolated from post-progression plasma. By doing so, the group aimed to find emergent alterations not detected in pretreatment tumor or cfDNA samples. 

Performing targeted NGS sequencing in about 90 percent of cases, the researchers also used parallel whole-exome sequencing (WES) on patient blood samples that contained enough tumor content for analysis. 

The researchers saw that post-progression cfDNA analysis identified at least one previously validated resistance alteration in 32 out of 42 patients, while 17 of the 32 patients presented more than one detectable resistance alteration, indicating frequent tumor heterogeneity linked with acquired resistance. 

Corcoran explained that his team did not find specific mutations in either sample type; however, the researchers did find more than one resistance mechanism in the same biopsy.

In order to compare the effectiveness of cfDNA versus standard tumor biopsy, the team used WES, targeted NGS, or both to examine matched post-progression tumor biopsies from 23 patients and compared those to pretreatment tumor tissue. The group saw that tumor biopsy identified resistance alternations less often than cfDNA did in 11 of 23 patients, while cfDNA identified at least one resistance alteration in 20 of 23 patients, and in 76 percent of all patients. 

"When you do liquid biopsy in parallel with tumor biopsy, since cfDNA is shed from every tumor lesion in the blood, you're picking up ctDNA that are from other tumors you didn't biopsy," Corcoran noted. "We limited the mutations that we looked at to those that could be validated in the lab to be clinically relevant." 

However, Corcoran emphasized that post-progression cfDNA analyzed using NGS identified additionally clinically important resistance alterations not discovered in the matched tumor biopsies in 18 out of 23, or 78 percent of cases. 

"We [did] expect to find cases where liquid biopsy detected other mutations that simply weren't present in the tumors we biopsied," Corcoran said. "We were surprised how frequently that happened, [because] if you were to do this clinically and rely on standard tumor biopsy, you'd be missing clinical info, especially the alterations you'd want to act on clinically." 

Corocan noted that the team had instead expected to find more resistance mutations in tumor biopsies that did not show up in the blood samples. However, the researchers only identified a single resistance alteration, an EGFR mutation in the matched tissue biopsy that was not detected in matched post-progression cfDNA using the Guardant360 assay. Gad Getz, co-senior author and director of the Cancer Genome Computational Analysis Group at the Broad Institute, believes that the mutation the team saw in the tissue biopsy might reflect a subclone that was not present at detectable levels in the blood sample. 

"We know that patients would have different resistance mechanisms, but we didn't realize that the same patient would develop multiple ones," Getz explained. "It was unexpected how one-sided the result was, as we saw mechanisms only in the blood sample weren't in the tumor."

The study authors therefore believe that acquired resistance to targeted therapy in GI cancers is highly heterogeneous, often with several resistance alterations in the patients. 

"While cDNA analysis may miss rare tumor subclones, our study suggests effective representation of alterations from multiple lesions in cfDNA," the study authors noted. "However, cfDNA detection effectiveness may differ by tumor type or metastatic site, or in tumors with low rates of circulating tumor DNA shedding." 


Corcoran acknowledged that the major challenge his team encountered in the prospective study involved coordinating sample collection and analysis among the different collaborators, clinicians, and medical centers. Getz highlighted that the researchers also struggled to comprehensively examine tumor DNA in the blood samples, as not all the samples met the assay's required limit of detection threshold. 

In addition, the team also studied patients with unknown resistance mechanisms. Getz explained that the team used an algorithm developed in the study as part of a collaboration with IBM researchers to look at the clustering of samples to try and match the resistance mechanisms between different patients. 

"While there were patients we found [that had] more than one resistance mechanism, there were [also] some that had no known resistance mechanisms in their blood samples," Getz said. "More research needs to be done, but it's probably that every patient has several resistance mechanisms, and that we just need to find them." 

Getz highlighted that using liquid biopsy to track ctDNA in a patient's bloodstream allowed the team to cover tumor shedding from different parts of the body, which the researchers validated in two separate patient samples. While the researchers saw that different tumor lesions had different resistances, he noted that cfDNA assays (such as Guardant360) that go deep on a limited number of genes or whole-exomes assays that cover all genes to a low depth could potentially detect all the resistance mechanisms. Getz therefore believes that ctDNA allows researchers a better survey of tumors in the body.  

However, Corcoran acknowledged that the study involved logistical and sample limitations. While emphasizing that the study is one of the largest direct comparisons between liquid biopsy and tissue biopsies, Corcoran said that his team will need to perform additional studies to characterize the degree of heterogeneity on acquired resistance and its impact on cancers besides GI. 

While the researchers used multiple biopsies to confirm that several resistance alterations that were detected in cDNA derived from specific tumor lesions, Getz noted that multiple tumor specimens could not be collected from most patients.  

Because liquid biopsy could not identify a clear genomic driver alone, the study authors emphasized that tumor biopsy will still need to be used as the primary tool to examine acquired resistance, especially for novel or nongenetic resistance mechanisms. However, they believe that clinical implementation of post-progression liquid biopsy may still be useful in combination with tissue biopsy to examine and combat acquired resistance. 

Future work

Getz said the team expects to perform further studies exploring variants across multiple tumor types. He believes the study is crucial in understanding therapeutic resistance in all cancer. 

"In the future, having multiple mutations in the same pathway or gene will help us identify genes or pathways that are working for resistance," Getz explained. "We would leverage the fact that multiple mutations in the pathway exist to highlight the specific pathways causing resistance."

While the prospective study mainly focused on comparing approaches to diagnose new resistance mechanisms, the group saw that they could potentially act on the emerging mutations found in patients. Working outside the study, Corcoran's team placed two patients onto a new therapy that successful dealt with newly resistant mechanisms found in the study. 

"Despite [therapeutics] not being something we were evaluating in the study, we hope that liquid biopsy might provide … the ability to assess the cause of acquired resistance, which might lead us to opportunities like [treating patients] and hopefully prolonged clinical benefit," Corcoran said. 

Based on the current study's results however, Getz acknowledged that the team will need to perform additional studies that use liquid biopsy in tandem with a variety of cancer drugs. Researchers will also explore combining cancer drugs that have widely different cancer treatment mechanisms, which Getz believes will lead to a new way of applying combination therapies. 

"Not only do we want to see how many resistance mechanisms exist in the patient, but what they are exactly, and then suggest potential hypotheses to tackle the mechanisms within patients," Getz said. "We believe that eventually [combining drugs] will be one mechanism to prevent or at least delay resistance."