CAP TODAY Pathology/Laboratory Medicine/Laboratory Management
Even if pathologists choose not to perform molecular testing themselves, they need to be involved in front-end testing steps, says Dr. Halling. For any type of solid tumor testing, in fact, “They have to identify the area of tumor to evaluate,” from, say, an H&E-stained slide. “They have to give an estimate of the percentage of tumor cells in the circled area that are truly tumor cells,” and not nonneoplastic cells. If the area isn’t rich enough in tumor percent, there’s risk of a false-negative result.
He offers as an example a slide from his lab with an estimated 60 percent of tumor circled (see page 52). The assay Mayo uses for microsatellite instability has an estimated sensitivity of 30 to 40 percent. “If we cannot identify an area that has at least 30 to 40 percent tumor cells for a patient, then we would not perform the testing.” It’s not uncommon, he adds, to get samples where the tumor cells are scattered and rare.
In the meantime, the steady drip of new markers—including PI3KCA and PTEN—continues. With them come a chasm and a decision: When should labs leap to next-generation sequencing?Dr. Halling and his Mayo colleagues are developing a next-gen sequencing panel that will be used not only for colon cancer but also for other solid tumors, including lung. Despite the push from various quarters to offer whole genome or whole exome sequencing, clinical labs are required to validate all their targets—a very difficult task, at least in the traditional sense of validation. “So we’re focusing on a set of genes that we know have clear clinical utility, such as KRAS and BRAF for CRC, and EGFR for lung cancer.” The final panel will likely contain 15 to 20 genes linked to targeted therapies.
It’s possible that next-gen platforms will eventually be affordable for even smaller labs, putting molecular testing within easy reach. Not only will it enable labs to test more targets, but—and this is no small consideration—it will also improve turnaround times and potentially lessen the problem of small tissue specimens.
In cases of advanced metastatic colorectal cancer, turnaround times have to be short, says Marina Nikiforova, MD, associate professor of pathology and director, molecular anatomic pathology laboratory, University of Pittsburgh Medical Center. “Five to seven days is just the amount of time clinicians can tolerate,” says Dr. Nikiforova, who says she hears anecdotal evidence of CRC send-outs taking about two weeks.
That could be seen as an argument for laboratories to set up molecular testing in-house if they haven’t already done so. Dr. Halling agrees—up to a point. If a lab has the expertise, then certainly it’s worth considering, he says. “But I don’t think this is something to dabble in.”
It’s a complex decision, says Dr. Nikiforova. Small labs might find it hard to offer advanced levels of testing and to stay on top of new analytes. On the other hand, they shouldn’t automatically reject it. Pathologists should be keeping abreast of molecular targets anyway, she says. Good commercial kits are available, and if a laboratory does any type of molecular testing, it may not be all that difficult to bring KRAS or BRAF testing in-house.
Dr. Frankel advises that labs look at their volumes before making a decision. “It’s one of those cost-benefit analyses that every lab has to look at.” At her own lab, MSI and methylation testing were initially send-outs. Now, as requests and evidence of clinical value have grown, “We feel it is absolutely worthwhile to set up. But that’s very lab-dependent. Just like any other test. Many smaller labs don’t do even immunohistochemistry that we consider second nature. You can only do what you can do. If money and time weren’t an issue, we’d all do everything, right?” She and colleagues at OSUWMC are setting up next-gen sequencing now.
Dr. Nikiforova and others are convinced that next-gen sequencing will soon eclipse more traditional molecular methods. Even several years ago, she notes, it was sufficient to test for one individual mutation, such as KRAS. “But now that’s not the case,” she says, “because more and more mutations are linked to specific targeted pathways or have specific prognostic or diagnostic information.” It’s apparent to all that the future will be filled with multiple targets.
At Pittsburgh, that future arrived last November. Instead of offering molecular testing for KRAS, BRAF, and PI3KCA for advanced colorectal cancers, the lab runs a next-gen sequencing panel of 46 cancer genes and more than 740 mutations. The cost is the same as, if not less than, what it would be to analyze all these genes by conventional techniques, Dr. Nikiforova says. “But the next-gen sequencing panel we use helps us to screen for all these mutations at once, quantitate the prevalence of mutation, and detect mutation quickly and cost-efficiently,” she says.
That should be strong incentive for smaller labs to start looking at next-gen sequencing, she says. And if they’re considering bringing molecular in-house, it might “be useful to wait a year or two, and then jump directly into these techniques.”
Even though next-gen sequencing is the future, it won’t blot out the past, Dr. Halling says. He doesn’t, for instance, see it being used for MSI testing. The pathologist who hopes next-gen sequencing will obviate the need for single-analyte assays is guilty of wishful thinking.
That’s the never-ending refrain from molecular experts, but the message may not have sunk in with everyone. Dr. Halling says he has pathologist acquaintances who think it’s sufficient to provide a result and leave interpretation to the clinician. That’s only safe to do with knowledgeable clinicians—and not all of them are knowledgeable.
Molecular results, like toddlers, should not travel solo. It’s not useful to tell clinicians a patient has a KRAS mutation, then stop. They need to be told that the mutation predicts resistance to anti-EGFR therapies; that absence of a mutation predicts the tumor may respond to therapy; that high levels of MSI indicate possibility of Lynch syndrome. “We provide a fair amount of information in our reports about what it means, and next steps to take, if next steps are necessary,” Dr. Halling says.
This can be done with boilerplate comments on a report, says Dr. Halling, though occasional free texting is required for unusual cases. A good starting point for guidance on what molecular pathology reports should look like can be found in “Clinical laboratory reports in molecular pathology” (Gulley ML, et al. Arch Pathol Lab Med. 2007;131:852–863).
With next-gen sequencing reports, says Dr. Nikiforova, labs typically categorize mutations into several levels.
The first is for a mutation with known clinical significance. For example, with a KRAS mutation, the report would include information about the mutation, links to references, and a short comment indicating the mutation is found frequently—in 40 percent of colorectal cancers—and that it does not respond to certain drugs, including cetuximab.
Another category is for mutations or variants with uncertain clinical significance. In addition to explaining that the mutations do not have well-defined responses to specific therapies, the report will again provide links to relevant literature, and comment on potential importance of the mutations to the particular genes involved.
The next category is for mutation-negative results. In each case, Dr. Nikiforova says, “We try to spell things out as much as possible to help clinicians understand what it means.” It goes without saying that she and others in the lab are also prepared to discuss results with clinicians, either via phone or face-to-face.
Labs that send out their testing might be tempted to take themselves out of the equation. Shouldn’t the brunt of interpretation fall on reference labs that provide the results?
No. Dr. Halling says explanations accompanying lab results tend to be short—clinicians don’t read long ones. So pathologists must be prepared to discuss findings. Ditto for discussing which tests to order.
He provides a few scenarios. “A pathologist should understand that if it’s a 45-year-old with colon cancer, that’s out of the ordinary. And that microsatellite instability testing should be considered. Where if it’s a 65-year-old, it’s more of a garden-variety colon cancer, and unless there’s a strong family history, they may not do that testing.
“Or they may see a certain kind of histology that suggests the possibility of Lynch syndrome,” he continues. “Or, for instance, for KRAS testing, the drug is FDA-approved for patients with advanced cancer, so it doesn’t make any sense to do KRAS testing on somebody who has early-stage colon cancer.”
Dr. Frankel says she hears from pathologists who are reluctant to do pathologist-directed testing. How can they make sure appropriate tests are ordered? “Call the clinician,” she says. “Identify the histologic features that suggest MSI in a young patient and suggest additional testing, if you’re uncomfortable ordering it yourself.”
Pathologists will also need to begin thinking about validation in broader terms, Dr. Halling says, especially with the advent of next-gen sequencing and with the FDA indicating it wants to boost oversight of LDTs. “In the past, just showing analytical validity was sufficient, and we left it up to our clinicians to decide whether they thought the test was useful. But I think it’s going to be more incumbent on the labs themselves to show that they’re clinically useful,” he says.
Molecular testing, in short, means pathologists have to engage with their clinical colleagues more, not less. That relationship should be as transparent as a Scandinavian government. At OSUWMC, Dr. Frankel says, everyone—pathologists, surgeons, oncologists, cancer genetics—is in the loop, every step of the way. Whenever a change is made to the CRC testing menu, it doesn’t take anyone by surprise. “Everyone knows what we’re doing.”
There’s a reason for that. When OSUWMC first began its colorectal screening program, she says, “We were concerned to learn that patients who were suspicious for LS were falling through the cracks. Some reports were not being followed up. That’s why you can’t do this testing unless your surgeons and oncologists are involved.” A member of the cancer genetics team is now meeting with these patients at one of their post-op visits, which has improved matters tremendously. “In order for this to work, this is not just pathology-driven,” she says.
This was also another example, she says, of how molecular testing undergoes constant change. It arrives as regularly as promises from politicians.
Pols can be ignored; colorectal cancer testing can’t. Certainly laboratories may find it cheaper, sooner rather than later, to sequence all the mismatch repair genes, and thus be tempted to view current screening and molecular methods as a waste.
“But it isn’t,” Dr. Frankel says. “In the meantime we saved lives.”
Karen Titus is CAP TODAY contributing editor and co-managing editor.