From ALK to T790M: The Role of Liquid Biopsy in Lung Cancer

Robert H. Carlson, MBA


March 23, 2018

The current standard of care for metastatic lung cancer is targeted therapy with a tyrosine kinase inhibitor (TKI), and the gold standard for identifying the molecular target of opportunity for the TKI is genotyping of tissue from the tumor.

But today, a "liquid biopsy" of cell-free DNA (cfDNA) shed from the tumor and collected from serum is increasingly being used to direct treatment of lung cancer, both as an adjunct to biopsy of tumor tissue after drug resistance and, in some cases, instead of tissue biopsy.

"Liquid biopsy is not replacing tissue biopsy by any means, but there are cases in which it is a valuable surrogate," said Joshua M. Bauml, MD, an assistant professor of medicine in the division of Hematology/Oncology at the University of Pennsylvania, speaking recently at the 35th Chemotherapy Foundation Symposium in New York.

Liquid biopsy should clearly be used for patients whose initial biopsy yields inadequate DNA for next-generation sequencing, Bauml said, but also for some patients who are developing resistance to a targeted therapy.

"If I have a patient whose initial biopsy is inadequate for next-generation tissue biopsy sequencing, such as from a very inaccessible lung mass or perhaps bone-only metastatic disease, and for whom a repeat biopsy would expose the patient to undue risk, a liquid biopsy provides a very nice access point to detect many of the same genetic aberrations that can be seen on a tissue biopsy," Bauml said.

Besides the risk for adverse events, other downsides of traditional tissue biopsy when compared with liquid biopsy are inconvenience and cost.

"Also, a tissue biopsy is only for one site—what if the resistance mechanism is from another site?" he said. "Oligoprogression is a major issue for patients on targeted therapy."

With the sensitivity of liquid biopsy in lung cancer at only 70% to 80% (depending upon the population and test studies), the liquid biopsy does not replace a tissue biopsy for the initial diagnosis of cancer, Bauml said. "However, once you know that you have an adenocarcinoma of the lung, for instance, and you want to know what molecular drivers are activated in that patient's tumor, then a liquid biopsy can play a role."

Patients on targeted therapies develop resistance in a predictable and reliable fashion. Despite all of the exciting data about the amazing responses to these agents, resistance is nearly inevitable.[1]

And resistance can be heterogeneous, Bauml said.

In epidermal growth factor receptor (EGFR)-mutant adenocarcinoma, patients who receive a first- or second-generation TKI such as erlotinib, gefitinib, or afatinib have about a 40% to 60% chance of developing a T790M resistance mutation. That's incredibly important to know because a patient with that mutation is likely to respond to osimertinib, the third-generation EGFR TKI approved by the US Food and Drug Administration (FDA) for metastatic EGFR T790M mutation-positive non-small cell lung cancer (NSCLC).

"In contrast, if they don't have T790M as a resistance mechanism, if they have small-cell transformation, then giving the osimertinib is the wrong move," he said.

Before the introduction of targeted therapies, biopsies at the time of progression were not usually performed, Bauml said. But now a repeat molecular assessment has become a standard of care for patients at the time of progression to look for a resistance mechanism to the first targeted therapy.

Historically, that has been done with a tissue biopsy, which involves risk for an adverse event, cost, and treatment delay. There is also concern about stewardship of the tissue and the possibility that the tissue will deteriorate if the analysis takes longer than expected.

"And nobody likes doing repeat biopsies on their patients; it's painful and hard to coordinate," he said.

Bauml said that any hospital can access liquid biopsy, and because there are multiple next-generation sequencing assays now approved by the FDA, it is quite likely that it will be easier for community physicians to perform next-generation sequencing.

"Now that cfDNA assays are readily available, extra blood tests are a lot more appealing and easier for a patient to experience, and few people would doubt the benefit of repeat molecular characterization for patients who have a targeted therapy," Bauml said. "Certainly in the EGFR space, it's an established standard of care."

There are multiple commercial and academic laboratories doing these liquid biopsies, he said. Some only look at select genes, while others do complete next-generation sequencing assays across a wide variety of genetic aberrations.

Tissue Biopsy Versus cfDNA

EGFR T790M mutations lead to resistance to most clinically available EGFR TKIs, and tissue biopsy is the gold standard to test for T790M positivity. But research has shown that if the plasma is positive for T790M, in the majority of those cases, the tissue is also positive, Bauml said.

For specific mutations such as EGFR T790M, the sensitivity in lung cancer is from 60% to 70%, but the specificity is approximately 90%, he said.

"The key point is that if you have plasma-positive disease, then that's reliable," he said, "but if you have plasma-negative disease, getting a tissue biopsy remains critical."

False positives in cfDNA analysis are rare, Bauml said. If the serum is positive for a specific mutation, but the tissue biopsy is negative, that may only mean that the tissue was taken from a site that did not have the mutation, while the serum would contain cfDNA from all tumor sites.

Response rates to therapy are very similar whether a mutation is found in the tissue or in serum. A major analysis of patients who developed T790M mutations found that the response rates for patients with T790M-positive cfDNA were almost identical to patients with T790M-positive tissue biopsies, when both study arms were treated with T790M-directed therapy.[2]

"In my clinic, if I have a patient with an easily accessible biopsy, like a supraclavicular lymph node, then at the time of resistance, I will usually do a plasma test and also a tissue biopsy because sometimes there are disparate results between them," Bauml said.

"But if the tumor is not easily accessible, a liquid biopsy is called for," he said.

False Positives Rare

The positive predictive value of cfDNA tests is very good, Bauml said. False negatives can occur, but a false positive is rare.

"In fact, we haven't seen very many reports of that at all in most of the common, targetable mutations," he said.

Tumors start out relatively homogeneous. If a patient initially has an EGFR mutation in their tumor, all of the sites of their disease have that EGFR mutation, Bauml said.

In contrast, at the time of resistance, there is heterogeneity. A liquid biopsy provides a potential advantage in that setting as it is getting some combination of cfDNA from multiple sites around the body. This can increase the chances of detecting a specific genetic aberration, Bauml said.

The value of liquid biopsy in disease resistance goes beyond EGFR.

"EGFR is where we have the most data, but we also know that in [anaplastic lymphoma kinase] (ALK) translocation, the resistance mechanisms depend not only on the presence of ALK but what drug you use. The in vitro sensitivity to the various ALK TKIs is enormously complex, and you would likely choose a different TKI on the basis of those sensitivities," Bauml said.

Tissue Is the Driver

"If there are discordant results, the tissue drives it," Bauml said. "If you have, for instance, a patient who is plasma-negative for T790M but is tissue-positive, their duration of response and progression-free survival are nearly identical to those patients who are plasma-positive and tissue-positive.

"In contrast, if you have a patient who is plasma-positive but tissue-negative, those patients tend not to do very well. If you are biopsying tumor tissue and not detecting the aberration, perhaps there is only a very small allelic fraction in the tumor."

The sensitivity of liquid biopsies is much higher than that of a tissue biopsy, he said.

"Tissue biopsies are looking for big amounts of change, whereas the plasma might be looking for small amounts within the circulating tumor DNA," he said.

Bauml said that a key question colleagues ask is what sort of resistance mechanism they should be looking for when choosing an assay to guide targeted therapy. Some assays look only at two or three genes, he said, and their advantage is that they can go faster and deeper on each of those genes, but a disadvantage is potentially missing other mechanisms of resistance.

"For instance, if you have a plasma assay that only looks at EGFR, you will miss something else, like a MET amplification," Bauml said.

One of the primary mechanisms of resistance to osimertinib appears to be MET amplification, and the treatment paradigm for MET amplification might be distinct from that of a C797S mutation, he said.

"So the question should be: What's the resistance mechanism I'm looking for, and what assay will help get me there?" Bauml said.

More Opportunities for Liquid Biopsy

There are other exciting prospects for liquid biopsy in lung cancer besides guiding treatment after resistance, including serial monitoring of response over time and assessing minimal residual disease (MRD) following treatment with curative intent.

"We are starting to understand the promise of the cfDNA platform," said Benjamin Levy, MD, an assistant professor at Johns Hopkins School of Medicine and clinical director of the Johns Hopkins Sidney Kimmel Cancer Center at Sibley Memorial Hospital.

"We can use it as a static marker to identify driver mutations and, based on that, select the appropriate targeted therapy," Levy said in a phone interview. "Monitoring of cfDNA could also be applied in a dynamic way by monitoring response over time for a patient receiving therapy."

Another potential promise of cfDNA is assessing MRD after a curative intent approach, Levy said. There has been early research showing that picking up cfDNA in patients who have been cured may indicate whether the patient will relapse at that point or later on.[3,4]

Liquid biopsy panels are able to detect more than EGFR mutations, as some test up to 75 relevant alterations, many of which are common in lung adenocarcinoma.

"Some common alterations that can be detected in serum are ALK rearrangements, BRAF mutations, ROS1 rearrangements, and even MET exon 14 skipping mutations," Levy said. "If these genetic alterations are discovered in the panel ordered, the oncologist can potentially act upon them with targeted therapy."

But there is currently no test for PD-L1 positivity using cfDNA, Levy said.

The upfront setting is also seeing more use of liquid biopsies.

"If the tissue biopsy for a patient is consistent with NSCLC, but there is not enough tissue for the genetic testing, or the tissue is exhausted for mutation testing, the options are to either rebiopsy or use the liquid biopsy to get the same information in a shorter turnaround time," Levy said.

"Five years ago, I would have said we have to rebiopsy, but now I can say let's start with the blood. I've guided many treatment decisions based on mutations found upfront," he said.

Serial liquid biopsy assessment remains experimental, however.

"There is some confusion about that; some practitioners believe they should test every month to see what the levels are, but we're not there yet. We don't recommend doing that," he said.

And using liquid biopsy to look for MRD is also not yet ready for clinical use, Levy said.

Tissue Biopsy Still the Standard

Geoffrey R. Oxnard, MD, assistant professor of medicine at the Lowe Center for Thoracic Oncology at Dana-Farber Cancer Institute, said that there is no setting where plasma genotyping "beats" tumor genotyping.

"But there are settings where it is an established screening test prior to getting a biopsy for tumor genotyping," Oxnard said in an email exchange. "The FDA has approved one plasma genotyping assay (Cobas®; Roche Diagnostics, Indianapolis, Indiana) for ungenotyped NSCLC where EGFR genotyping is needed, and for EGFR acquired resistance where EGFR T790M testing is needed. In both of these settings, plasma genotyping is a standard test used by many to avoid a biopsy if possible."

If the plasma genotyping is positive, appropriate EGFR TKI therapy can be started, he said, but if it is negative, tumor genotyping (potentially requiring biopsy) is needed.

When Tissue and Serum Biopsies Don't Agree

A recent study in prostate cancer pointed to a potential concern with liquid biopsy—conflicting genotype reports from different companies.[5]

The study was conducted to determine the reliability and potential utility of next-generation sequencing in the clinical treatment of patients with metastatic prostate cancer, investigating the congruence of reported mutations in two independent platforms.

Patients could potentially receive different treatments depending on the cfDNA platform.

The authors said that, "despite the limited sample size, our data show very low congruence for same patient-paired samples in two [Clinical Laboratory Improvement Amendments]-certified commercially available tests.... These data cannot determine which test is more accurate but suggest that reported gene alterations will not be the same across different platforms, raising the specter that patients could potentially receive different treatments depending on the cfDNA platform."

Senior study author Kenneth J. Pienta, MD, professor of urology and professor of oncology and pharmacology and molecular sciences at Johns Hopkins University School of Medicine, said that the finding shows that these assays are in need of further development.

"This is especially the case in patients with low tumor burden that may not accurately reflect what are the important mutations a tumor may be harboring," Pienta said in a telephone interview. "We want this to work, and we're working with companies to help them improve their assays. And liquid biopsies are going to get better and better at this, not only as the molecular biology techniques improve but also as bioinformatics improve."

In the meantime, liquid biopsies are not ready to replace tissue-based biopsies; and, in any event, there is no targeted therapy in prostate cancer that could be used as biopsy-guided TKI treatment as in NSCLC, he said.

These assays may miss mutations in patients who potentially don't have a lot of circulating tumor DNA.

"The concern we have is that these assays may miss mutations in patients who potentially don't have a lot of circulating tumor DNA, so you may be getting different answers based on different assays," Pienta said. "When you find a particular mutation, that's potentially great; but if you don't find it, that doesn't necessarily mean it's not there."

Bauml, commenting on the prostate cancer study, said that the findings of this study are certainly a concern, but he added that he would expect the incidence of inconsistency to be lower in lung cancer given a higher rate of circulating tumor DNA shedding in lung cancer.

"Nevertheless, with inconsistent plasma samples, I would use tissue as a tie breaker," he said.

Oxnard was also asked to comment on this study.

"There is very little rigorous data on concordance of different plasma genotyping assays," Oxnard said. "There was one attempt from AstraZeneca to study this for EGFR assays, and they generally found good concordance for EGFR mutations."[6]

But there are few concordance data for other targets, he said.

"I'm not that surprised by the prostate cancer series, although I was a little surprised that they were sending liquid biopsies at all, given the lack of any genomic targets in prostate cancer," he said, adding that he saw no clinical reason why anyone should send more than one plasma genotyping assay for a given patient.

Oxnard said that the best validated plasma genotyping assay is the Cobas EGFR assay, which is FDA approved.

"Otherwise, we need to acknowledge plasma genotyping as an imperfect shortcut on the way to tumor genotyping," he said. "Plasma genotyping should be used to hunt for a targetable genotype, so it's best established in lung cancer.

"And if negative, get a biopsy," Oxnard said. "With that basic principle, you can overcome the weaknesses of most any liquid biopsy assay."


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