Carcinoma of unknown primary case reviewed in tumor board session

He describes the assays as robust and supported by the literature, yet he doesn’t think they’re used extensively. Cost of testing and turnaround time are likely to be the two main reasons, he said. “A thoughtful panel of immunostains in many settings would typically be less expensive to do than commercial gene expression profiling tests. Turnaround time expectations may be more challenging with send-out testing.”

When he and colleagues in molecular pathology consider molecular profiling for diagnosis, they are “thinking about detecting fusion genes and specific DNA mutations that may be disease-defining,” he said. These sets of molecular assays are most useful for sarcomas, he noted, but beyond the scope of the case presented in the session.

“On the surface alone,” he cautions, there is a pitfall: “If you send a case for molecular profiling, you have to be cognizant of the morphology because the molecular result alone could lead you down the wrong path or lead to unresolved ambiguity.” As an example, the fusion EWSR1-CREB1 is found in angiomatoid fibrous histiocytoma, and in clear cell sarcoma of tendon sheath, which is an entirely different entity. “It’s also seen in clear cell sarcoma-like tumor of the GI tract, which is a highly aggressive malignancy. So it is prudent to be cautious of the use and interpretation of molecular testing without any clinical context or morphologic context.”

Dr. Drilon said they selected NTRK for the session because there are now two FDA-approved TRK inhibitors—entrectinib and larotrectinib—for patients with TRK fusion-positive cancers of any type. At MSK, if any driver alteration is identified matching someone to an FDA-approved therapy, or a clinical trial, an email is sent to the patient’s oncologist and to the principal investigator of that trial. That’s how the patient in their case was matched for therapy. (More on that later.)

NTRK fusions aren’t structurally much different from colonical fusions involving, for example, ALK, ROS1, or RET. NTRK1, 2, and 3 encode the receptor types in kinase as TrkA, B, or C. Therefore, any of these three genes in the 3′ position, as long as there is an in-frame event that includes the kinase domain, would be viewed as an activating event.

Dr. Drilon said he tends to view NTRK fusions in two major groups. There are the rare cancers, where the frequency of TRK fusions, depending on the series one looks at, exceeds 90 percent. “And that means the identification of a TRK fusion is almost pathognomonic of these disease states.” These four main histotypes include mammary analogue secretory carcinoma, a salivary tumor that is morphologically similar to secretory breast carcinoma. “The last two histologies are congenital fibrosarcoma, which also shares some pathologic features under the microscope with congenital mesoblastic nephroma.”

The second major group of malignancies is more common and harbors TRK fusions at much lower frequencies: lung cancer, melanoma, sarcomas, and GI tumors. “For some of these, the frequencies are really low. For lung cancer, for example, we prospectively looked at our cases and found that a TRK fusion is found in about 0.2 percent of cases.”

NTRK fusions are actionable, so the assay the institution uses or sends out for must offer sufficient coverage for TRK, Dr. Drilon said. If resources are not an issue “and you are able to bill for next-generation sequencing, that would be the preferred method of identifying these cases.” The advantage of hybrid-capture NGS assays over amplicon-based testing is that “they are better poised to capture these events.”

Reverse transcription PCR is one alternative, he said, “but the pitfall is that you would need to know what you are looking for in the sequence of the 5′ and 3′ events in order to find it; you are not looking for any events that might not have been annotated in the past.”

FISH is another option, but there are three genes, so three FISH assays are needed, “which increases the amount of tissue you need,” Dr. Drilon said.

Immunohistochemistry has emerged as a practicable screening test for TRK fusions, he said. “Why? Similar to ALK fusions and ROS1 fusions, when you see a meaningful expression of either TrkA, B, or C in a tumor specimen, granted there are some lineages like smooth muscle and neuroendocrine where you might see basal expression of TRK outside of the cancer that might interfere with your results, but for all other histologies or primaries, if you have a positive IHC, there is a good likelihood that if you do follow-up testing with NGS, or another assay, you may find a TRK fusion.”

It’s sometimes thought that if a good DNA-based NGS test doesn’t find anything in a tumor sample, “there’s no driver in the cancer,” Dr. Drilon said. He and colleagues at MSK investigated to see if that was true (Benayed R, et al. Clin Cancer Res. 2019;25[15]:4712–4722). “We took lung adenocarcinomas that you know are enriched for actionable drivers. We took all cases that were deemed negative by MSK-IMPACT, and we ran those cases through targeted RNA sequencing,” using anchored multiplex PCR with the ArcherDx assay. MSK-IMPACT missed an actionable event in about 15 percent of the cases. The actionable fusions detected by targeted RNA sequencing included ALK, MET, NRG1, NTRK, RET, ROS1, among others. What will be needed moving into the future, Dr. Drilon predicts, is complementary DNA- and RNA-based NGS testing “to maximize finding these actionable drivers, including NTRK.”

There is no perfect assay, Pranil K. Chandra, DO, vice president and chief medical officer of genomic and clinical pathology services at PathGroup, Nashville, Tenn., said in a CAP TODAY interview. (He wasn’t a presenter or an attendee.) “DNA capture-based assays struggle with detecting fusion breakpoints within large intronic regions containing repetitive elements,” he says, “especially NTRK2 and NTRK3. RNA-based NGS takes advantage of natural molecular biologic processes where problematic intronic regions present in DNA have been removed by splicing.”

The RNA-based assays aren’t flawless either, Dr. Chandra says. “You need to have good RNA, and as we all know, RNA is relatively more susceptible to degradation. As a result, RNA quality can be highly variable and of poor quality, especially when extracted from formalin-fixed, paraffin-embedded tissues. This requires good internal controls to minimize the likelihood of false-negative results.”

More published data are needed, Dr. Chandra says, to underscore the advantages and drawbacks of various testing platforms, including DNA-based and RNA-based NGS and IHC. “Once we have a good understanding of sensitivity and specificity, we’ll be in a better position to optimize laboratory testing for best patient care.”

PathGroup, which provides laboratory services to about 95 hospitals and thousands of outpatient physician clients, uses Roche’s Ventana pan-TRK IHC assay as a first-line test in evaluating advanced solid tumors. They have integrated the IHC assay into all of their algorithmically driven advanced solid tumor molecular testing protocols, Dr. Chandra says. “Any positive [pan-TRK IHC] result is followed through with a more specific molecular test that looks for the presence of NTRK fusion. We use an RNA-based next-generation sequencing assay that looks for NTRK1, NTRK2, and NTRK3 fusion.”

The practice began using pan-TRK IHC testing late in the first half of 2019, and Dr. Chandra and colleagues have identified two patients with NTRK fusions. One patient had an advanced thyroid cancer, the other patient an advanced colon cancer.

In the patient case presented in the CAP19 tumor board session, because the NTRK fusion was found early, the patient was enrolled in a trial for a selective TRK inhibitor and had a complete response to the therapy. Subsequent imaging has confirmed this, Dr. Drilon said, “and she remains on therapy three years into the treatment. So a happy ending, a good outcome, for this patient.”

Dr. Drilon thinks it is useful to revisit the genomics of the tumor at resistance. If patients have on-target resistance mediated by several kinase domain mutations (TrkA: G595R, F589L, G667C, A608D; TrkB: G639R, F633L, G709C; TrkC: G623R, F617L, G696A), these patients may be eligible for second-generation TRK inhibitors currently in clinical trials, repotrectinib and LOXO-195. “In the face of off-target resistance, which we have also observed in some cases, these patients can be referred for histology-specific standard of care.” (Examples of potential mechanisms for off-target resistance are KRAS mutation, MET amplification, BRAF mutation, and IGF1R activation.) (Drilon A. Ann Oncol. 2019;30[suppl 8]:viii23–viii30.)

In closing, Dr. Graham said “There is always this underlying question that comes when you have a high-grade tumor and you are trying to figure out what it is. How much more specific can you get, and does it matter? We are practicing now in an era where it does matter,” thanks to agents that provide hope for prolonged survival.

Karen Lusky is a writer in Brentwood, Tenn.