Markers, methods remake the NSCLC map

Karen Titus

February 2021—Absorbing new biomarkers into lung cancer workups makes for a complicated diplomacy. How best to balance so many rivals?

Does it make the most sense for laboratories to try to do everything at once, a full-court press involving next-generation sequencing panels? Or is it more practical to add a new marker only as a new targeted therapy receives approval? Where do RNA-based assays fit in? What about IHC? When do you make the switch? Or do you? And how best to handle cell-free DNA tests (which seem to be the rogue states in all this)?

How do you weight external factors, such as reimbursement, existing equipment and capital expenditures, and physician expertise?

Driving this all are medical breakthroughs. As with all forms of statecraft, the latest incident can change everything.

For lung cancer, the most recent advance comes from the ADAURA trial (Wu Y-L, et al. N Engl J Med. 2020;383[18]:1711–1723), which showed a significant benefit of using osimertinib to treat stage IB to IIIA EGFR-mutation positive non-small-cell lung cancer. While EGFR tyrosine kinase inhibitors have long been used to treat metastatic disease, this offers a hopeful approach to treating localized resectable disease.

“The ADAURA trial is extremely impressive,” says Mary Beth Beasley, MD, professor of pathology, Icahn School of Medicine at Mount Sinai, and head of thoracic pathology, Mount Sinai Medical Center, New York. It’s exciting, too, particularly for practitioners like her who’ve spent decades waiting for gains that could offer genuine hope to patients. “When I started, there was often very little we could do for patients. It was so sad.”

Now that’s changing. “It was baby steps at first, and now it’s kind of exploded,” says Dr. Beasley.

Other meliorations include the drug capmatinib, which appears to be efficacious in treating advanced NSCLC tumors with MET exon 14 skipping mutations (Wolf J, et al. N Engl J Med. 2020;383[10]:944–957). And tumors with RET gene fusions appear to respond well to two newly approved agents: the RET inhibitor selpercatinib (Drilon A, et al. N Engl J Med. 2020;383[9]:813–824), as well as pralsetinib. EGFR insertion mutations on exon 20 wait in the wings.

But, says Dr. Beasley, “I think the ADAURA trial in particular is going to be the impetus for a potential shift, from testing just advanced stage cancers to showing a benefit in testing in earlier stage cancers.” It might also be a tipping point for how labs consider their approach to NSCLC—both the when and the how of testing.

Dr. Beasley’s own institution illustrates the challenges labs face as they try to stay current. The ADAURA trial is unlikely to change much at academic centers like hers. “We end up testing for everything,” Dr. Beasley says. “But that’s definitely not the case at all institutions or laboratories.”

Even labs at the forefront have to work to keep up. Dr. Beasley is a coauthor of the CAP/IASLC/AMP molecular testing guideline for lung cancer. “And of course, by the time we get that out, something new has already come up,” she says. No treaty, no matter how well crafted, is ever final.

For the last seven years or so, Lynette Sholl, MD, and her colleagues at Brigham and Women’s Hospital have been using a several-hundred-gene NGS panel for routine testing. Even when they didn’t know something was important, “We’ve been including those targets on our panel pretty much the entire time.” One goal was to identify variants so patients could enroll in clinical trials at Dana-Farber Cancer Institute. But doing so has also made it easier to stay abreast of clinically relevant biomarkers, says Dr. Sholl, who is associate professor of pathology, Harvard Medical School, and chief of the pulmonary pathology service and associate director of the Center for Advanced Molecular Diagnostics, Brigham and Women’s.

Awareness and action are only a start. While the lab has been able to detect RET fusions and MET exon 14 mutations, Dr. Sholl says, “We often didn’t know what we were looking for.”

“That’s one of the challenges of DNA-based testing,” she continues. “There’s a tremendous amount of diversity in the types of mutations that ultimately can lead to MET exon 14 skipping alterations.”

Dr. Sholl speaks from experience. She and her colleagues have retrospectively reviewed their data from time to time. In one study, published in 2016, they manually reviewed DNA sequences from patients who didn’t have other driver alterations “and identified MET exon 14 skipping mutations as 15- or 20-base pair deletions sitting fully in an intron, not touching an exon at all,” she says (Awad MM, et al. J Clin Oncol. 2016;34[7]:721–730). “Honestly, historically we would just ignore that kind of thing.” That was before they realized the deletion was sitting right over the polypyrimidine tract, which means it has critical implications for enabling the splicing to occur properly, she explains.

“We got a lot of insight into basic mechanisms of splicing that get altered in these tumors, and they weren’t on our radar for many years,” Dr. Sholl says. “We know to look for that now,” but many DNA-based tests may not be optimized to pick up the broad range of changes.

Sometimes pathologists aren’t “optimized,” either. “Because the diversity of alterations is so great, we rely on a preexisting knowledge base to help provide accurate annotations for variants on a panel,” Dr. Sholl says. For example, MET splice mutation tumors often have sarcomatoid morphology. “It may actually come into your system as a specimen that’s not necessarily recognized a priori as a primary lung cancer.” It may be a metastatic sarcomatoid carcinoma, with an uncertain primary site and an uncommon variant. “You can imagine the combination of events that transpires to miss the importance of one of these unusual MET splicing mutations.”

That can include overestimating the power of NGS. Not everyone fully appreciates the limitations of NGS, says Laura Tafe, MD, associate professor of pathology and laboratory medicine, Dartmouth-Hitchcock Medical Center and the Geisel School of Medicine, Dartmouth College, and assistant director of the laboratory for clinical genomics and advanced technology. “I’m sure the understanding of NGS’ capabilities is still mixed. I start out almost every single one of my talks talking about the different types of variants NGS can detect—and that not all assays are created equally.” Pathologists and their clinical colleagues need to be aware of—and even look for—the limitations and weaknesses of a particular assay, she says.

Dr. Sholl and her colleagues sometimes turn to a partner lab across town to perform RNA-based anchored multiplex PCR testing as needed. This approach is gaining a foothold in clinical labs, Dr. Sholl reports. An RNA-based assay can help pick up both MET splicing mutations as well as other fusion events—to confirm, for example, a suspected splicing variant, or in cases involving, say, a pan wild-type lung cancer with an unknown driver.

Some labs will run DNA- and RNA-based approaches in parallel. It’s a good way to immediately confirm that the DNA-variant of interest is functionally relevant, Dr. Sholl says. It also allows labs to achieve the challenging task of validating low-level fusion transcripts. (See “Study: Combined DNA-RNA testing improves detection of MET mutations,” CAP TODAY, March 2020.)

Though her lab’s MET adventures rely heavily on those two modalities, Dr. Sholl notes the growing interest in MET amplification by FISH or next-gen assays, as well as MET protein overexpression by IHC.

This part of the story is particularly complicated, she says. A number of studies (largely crizotinib-based) have shown that response to MET-targeted inhibitors correlated with the level of MET amplification. Patients with high levels—a five-to-one ratio of MET to the centromere, say—were much more likely to respond to treatment. The historic data may not have consistently looked for an underlying splice mutation, however, and Dr. Sholl suspects a subset of patients had one.

Newer studies involving capmatinib also examined amplification-positive, splice-negative patients, she says, demonstrating different response rates around a cutpoint of 10 gene copies. Capmatinib showed efficacy only in those patients whose tumors had at least 10 copies; however, the analysis did not meet prespecified levels defining significance (Wolf J, et al. N Engl J Med. 2020;383[10]:944–957). Until more data emerge, labs will wrestle with defining what level of amplification predicts response.

The other issue is that MET amplification, particularly at lower levels, can often be seen as a co-alteration, Dr. Sholl says, which upends the understanding of standalone driver alterations such as EGFR, MET, RET, etc. “That’s the Achilles’ heel of your tumor.” When patients potentially have another driver, whether it’s recognized at the time or not, “that’s going to potentially influence the outcomes to targeted therapies.”

IHC tells its own twisty tale. “The data is pretty confusing right now,” Dr. Sholl says. “We have historically seen that IHC is not a good predictor of response to some of the earlier MET inhibitors such as onartuzumab. There were some disappointing trial results using IHC as a biomarker initially” (Spigel DR, et al. J Clin Oncol. 2017;35[4]:412–420).

The question then became, could IHC be used as a surrogate for splicing mutations? Initial data suggested that IHC’s poor sensitivity for MET exon 14 skipping mutations limited its use. Recently, a Memorial Sloan Kettering study of patients receiving anti-MET therapies showed overall clinical outcomes were best in patients who had a splice mutation and also MET IHC overexpression (Guo R, et al. Clin Cancer Res. 2021;27[3]:​799–806).

Dr. Sholl says this lines up with the correlations she and her colleagues reported five years ago. The strong responders could be a unique subset of patients who have that high-level addiction to MET signaling, she says. While IHC in and of itself is not going to be an adequate screening tool, she says, it might help identify which patients are likely to benefit the most from targeted therapies.

She gets requests for MET IHC but is reluctant to perform it outside the context of a clinical trial enrollment. “We don’t know what it means in most contexts.”

As with children (so the joke goes), MET is unique but not special. With every new biomarker, “It’s the same story, writ a different way, every single time,” Dr. Sholl says.

RET, for example, is included on the next-gen panel in Dr. Sholl’s lab, which added it about five years ago as efforts increased nationally to bring profiling into routine practice at academic labs to qualify patients for RET inhibitors, among other targeted therapies. The lab was participating in the Lung Cancer Research Foundation’s Lung Cancer Mutation Consortium protocol, which involved testing patients prospectively by FISH as well as NGS. In her experience, RET FISH assays are fairly easy to interpret.

Explains Dr. Sholl: DNA-based NGS offers quite-high sensitivity. Most breakpoints within the RET introns are fairly well defined. But as with MET, RNA-based assays that look for RET transcripts are a powerful complement. And an RNA-based approach offers an advantage over FISH: It can identify a fusion transcript and determine the fusion partner bound to RET, which is important confirmatory evidence, whereas FISH stops short of identifying functionality.

Numerous studies have looked at IHC as a screening tool for RET. The majority, says Dr. Sholl, have been disappointing, showing poor sensitivity and specificity in identifying fusions with RET overexpression.

She says comprehensive testing is crucial. “I talk to people who, in their practice, never see these [newer] alterations. It’s always surprising to me, because MET alterations are three percent of lung cancer. That’s a lot. That’s like ALK, and people are used to seeing ALK all the time.”

She suspects labs have simply become more comfortable with ALK as the testing trajectory moved from FISH to IHC and eventually to widespread screening.

MET could follow, although, as noted, it lacks a good IHC option. Another complicating factor, Dr. Sholl notes, is that generating useful sequencing information for MET requires an infrastructure “that captures everything.” A number of commercially available assays do this, and more labs are bringing on RNA-based targeted panels. “That will be very helpful, with the caveat that everything needs to be interpreted in context,” she says. The RNA-based approaches run the risk of false-positives (in terms of low-level physiologic splicing) and false-negatives (in terms of poor quality RNA, assay failures, etc.).

“Persistence is probably the most important thing,” Dr. Sholl says. “If you have patients who have pan wild-type tumors, you need to keep pushing until you’re able to define the underlying drivers.”

MET affects a heterogeneous group of patients, she continues. While ALK and ROS1 mutations are unusual in tumors of patients who smoke, with MET, “it’s 50-50. You really just have to be looking for it in every context.” Age is another somewhat iffy clue. “Our experience is that there’s a bias toward age that’s higher than what we see for our average lung cancer patient—but not always,” says Dr. Sholl, who reports seeing 40-year-olds whose tumors have MET splice mutations.

The aforementioned phenotypic heterogeneity makes the puzzle harder to solve as well. In addition to sarcomatoid carcinomas, “We’re also seeing these in squamous cell carcinomas,” Dr. Sholl says. If she sees an SCC from a patient who is a light smoker, “The first thing I think of right now, based on my own biased experience, is the possibility of an underlying MET splice mutation. I’ve seen it enough times.”

EGFR is an old kid on the block, compared with MET and RET, but now worth another look.

When Dr. Sholl and colleagues saw the results of the ADAURA trial presented at ASCO in June, they were ready to implement reflexive EGFR testing for patients with stage IB through IIIA tumors.