Identifying an NTRK Gene Fusion

Larotrectinib (Vitrakvi) and entrectinib (Rozlytrek) were approved by the U.S. Food and Drug Administration in 2018¹ and 2019,² respectively, for the treatment of adult and pediatric patients with solid tumors that have a neurotrophic receptor tyrosine kinase (NTRK) gene fusion without a known acquired resistance mutation. These approvals are considered “tissue agnostic” in that their use is indicated in any tumor type harboring the target gene fusion. According to labeling,^3,4 eligibility includes the requirement that tumors are metastatic or inoperable or progressed on treatment. [Editor’s Note: Entrectinib is also indicated for adults with metastatic non–small cell lung cancer (NSCLC) whose tumors are ROS1-positive.⁴]

In recent articles about testing for NTRK gene fusions,^5,6 Jaclyn F. Hechtman, MD, observed that the efficacy of TRK inhibitors has created a need to identify patients with these rare genomic alterations who are most likely to derive benefit from these therapies. Dr. Hechtman was formerly Director of Biomarker Development at Memorial Sloan Kettering Cancer Center in New York City and is now Director of Precision Oncology Diagnostics at the Miami Cancer Institute, Miami.

What Are We Looking for? Why?

When asked to describe the target of NTRK gene fusion testing, Dr. Hechtman told JNCCN 360: “We are looking for a fusion of a gene that serves as a 5' partner to the kinase domain of NTRK1, NTRK2, or NTRK3.⁵ The gene fusion, resulting from chromosomal translocation, inversion, or deletion, results in constitutive activation of the Trk A, Trk B, or Trk C protein.”^7,8

The request for NTRK gene fusion testing may be triggered as part of diagnostic testing because a patient’s suspected or known tumor type is one known to be enriched for these specific fusions, such as secretory carcinoma or infantile fibrosarcoma.⁹ Alternatively, the request for NTRK gene fusion testing may be initiated for therapeutic purposes in the case of advanced cancer (eg, lung or colorectal), where no other mitogen-activated protein kinase pathway driver has been identified. Certain tumors, such as lung cancers with a low mutation burden¹⁰ or colorectal cancers characterized as BRAF wild-type and microsatellite instability–high,¹¹ are enriched for NTRK gene fusions.

Ideally, Dr. Hechtman pointed out, this testing would be conducted when tissue samples are newly available (such as after resection), because RNA is often degraded in older samples. However, many cases are inoperable, so the primary diagnosis is made based on biopsy results. Once the diagnosis is made, a request for molecular testing may be ordered, even after disease progression.

Ordering an NTRK Gene Fusion Test: Who Decides What to Use?

Although RNA-based next-generation sequencing (NGS) is the preferred method of identifying NTRK gene fusions, at least part of the decision about which test to use depends on the quality and amount of sample tissue available for testing. If the available tissue is scant or older, a different test, such as fluorescence in situ hybridization (FISH), may be recommended. In institutions that offer in-house molecular testing, Dr. Hechtman said, the decision regarding the assay of choice may be made by the oncologist. Often this decision is based on a conversation between the oncologist and pathologist, or it is part of a decision to assign cases with specific diagnoses or molecular findings to be evaluated with a specific assay.

Although RNA-based next-generation sequencing (NGS) is the preferred method of identifying NTRK gene fusions, at least part of the decision about which test to use depends on the quality and amount of sample tissue available for testing.

The pathologist may recommend a certain type of test, for instance, based on the type of cancer, the type of specimen submitted, or how quickly a molecular result is needed. Dr. Hechtman explained: “If the specimen is sent to a reference laboratory for molecular testing, the oncologist often specifies the requested assay yet may seek input from the surgical pathologist beforehand.”

Testing Options

There are several good testing options.^12,13 The decision about which one to use often depends on what type (and amount) of tumor tissue has been obtained as a test sample, Dr. Hechtman explained. The options are:

• Pan-Trk immunohistochemistry (IHC),¹⁴ which is often used as a screen
• NTRK1/2/3 FISH panel, which uses break-apart probes and identifies a gene rearrangement that often results in a gene fusion¹⁵
• NGS, RNA- or DNA-based.¹⁵

“RNA-based NGS is the best test,” Dr. Hechtman told JNCCN, “if you have a recently acquired specimen for NTRK testing. RNA is very labile, so the sample cannot be more than a couple of years old, and there must be sufficient tissue.” Biopsies are generally adequate, unless the amount of tissue is small, such as might be the case in lung cancer. “If the tissue sample is too small, a FISH test might be a better choice,” she continued.

Emphasizing the observation that NTRK gene fusions rarely occur simultaneously with other driver mutations, Dr. Hechtman explained: “If a patient already has been diagnosed with a KRAS or EGFR mutation in lung cancer, for example, or a KRAS or BRAF mutation or a strong driver mutation in the mitogen-activated protein kinase pathway, identification of an NTRK gene fusion is very unlikely.”^10,11

Unlike the European Society for Medical Oncology (ESMO), which has published recommendations regarding NTRK testing specifically,¹⁵ the American Society of Clinical Oncology (ASCO) recently published a paper about genomic testing for patients with advanced cancers.¹⁶ In general, NGS as a platform is recommended when possible, because there are so many targetable biomarkers, which each occur in a small percentage of patients, Dr. Hechtman noted.

NGS as a platform is recommended when possible, because there are so many targetable biomarkers, which each occur in a small percentage of patients.

“That said,” she cautioned, “results of NGS can take 2 to 3 weeks, whereas pan-Trk IHC takes only a day. FISH is also fairly quick, with results available within a few days. That means that if pan-Trk IHC shows expression, a confirmatory nucleic acid–based assay must follow.” Currently, TRK gene fusion testing is recommended within 21 different NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines, available at NCCN.org). Additional testing strategy information, including circumstances where FISH, IHC, or NGS methods might be considered, can be found in the NCCN Guidelines for Colon cancer,¹⁷ Gastric Cancer,¹⁸and Non–Small Cell Lung Cancer¹⁹(among others).²⁰

In cases of tumors with a high likelihood of harboring an NTRK gene fusion (eg, secretory carcinoma or congenital fibrosarcoma), some clinicians might consider initiating treatment with an NTRK inhibitor on the basis of IHC-positive results, commented Alexander Drilon, MD, Chief of Early Drug Development at Memorial Sloan Kettering Cancer Center in New York City and Co-Site Editor of the JNCCN 360 microsite Biomarkers: NTRK Gene Fusions. Nevertheless, the labeling for these drugs “requires the presence of the fusion—not just overexpression by IHC—so in most cases, it is best to confirm the presence of a gene fusion with DNA- or RNA-based NGS before commencing treatment,” he told JNCCN 360.

To understand the reported results, both the molecular pathologist and the treating oncologist need to be clear about what each assay is detecting. Dr. Hechtman explained: “Pan-Trk IHC detects expression of a protein, not necessarily a fusion of a gene,” and NTRK 1/2/3 FISH tests for a gene rearrangement. “This is not 100% indicative of a fusion,” she told JNCCN, “but it is used as a surrogate. In contrast, RNA-based or DNA-based NGS detects a fusion, particularly if canonical 5' partners are identified.”

No NTRK Gene Fusion? What Then?

There are other types of alterations that occur in NTRK genes that are not fusions or rearrangements. They include point and indel (insertion and deletion) mutations as well as amplifications. Dr. Hechtman described them as occurring “when there is a nucleotide switch (point mutation), deletion or insertion of a couple of nucleotides, or extra copies of the whole gene (amplification), rather than the kinase domain being fused to another gene.” Patients with point mutations, indel mutations, or amplifications are not eligible for treatment with NTRK fusion inhibitors, such as larotrectinib or entrectinib.

If the findings/data included in the report are unclear or may be interpreted in several ways, a phone call to the molecular pathologist is warranted.

Testing Dilemmas and Future Directions

“DNA-based NGS is susceptible to some of the same issues that we see with NTRK FISH testing,” Dr. Hechtman commented. “Sometimes, a rearrangement is identified on DNA-based NGS that is out of frame, does not include the kinase domain of NTRK, and/or does not include a canonical 5' partner. In the past, we’ve called these ‘structural variants of unknown significance,’” she noted. Nevertheless, such results are not necessarily actionable, and further testing is warranted.

If structural variants are observed with DNA-based NGS, we need to ask a few more questions, Dr. Hechtman pointed out. “Although we may note a rearrangement, is there an activating fusion present?” The next step, she said, would be RNA-based NGS, which will show whether a DNA-level rearrangement results in a transcribed RNA-fusion product and will often yield further answers. First, is a kinase domain of NTRK1–3 involved? Second, is it in-frame or out-of-frame? Third, which gene is the 5‘ partner of the resulting fusion?

Digging into the details, Dr. Hechtman suggested that “when we design our DNA-based NGS panels for fusion testing, we should specifically include the introns of the kinase domain, because that’s where the breakpoint in the two genes (NTRK and 5' partner) would occur. With RNA, in contrast, we do not have introns. Using DNA-based NGS works for NTRK1, but not for NTRK3, because some of the introns in NTRK3 are very long and repetitive. It is difficult to design a panel that accurately assesses these regions. We wind up designing panels that include the most common 5' partners for NTRK3, such as ETV6. However, if there is a novel partner (ie, one that is not included in the panel), then we would fail to identify the fusion.” She reiterated that introns are not a part of RNA-based NGS. “We extract the RNA and make a complementary DNA library from there, which is similar to RNA in that introns are not present. Because of that, this technique is much more likely to find these fusions, particularly for NTRK3,” she said.

For advanced cancers, as the number of detectable biomarkers increases and the repertoire of targeted therapies expands, Dr. Hechtman noted there would increasingly be a move to broader genomic testing profiles, eg, DNA-based markers such as mutations, microsatellite instability, tumor mutation burden, homologous recombination deficiency, as well as this and other actionable kinase fusions. She emphasized the message to clinicians: High-quality tissue specimens are paramount to accurate testing and precision medicine. “We are seeing a large paradigm shift to large-panel NGS, so it is critical for practitioners to obtain tissue samples that will be adequate for this level of diagnostic testing,” she concluded.

High-quality tissue specimens are paramount to accurate testing and precision medicine.

Disclosures

Jaclyn F. Hechtman, MD, is a part-time employee of and owns stock in NeoGenomics Laboratories; has received consulting fees from Seagen and Astellas; has received research funding from Bayer, Eli Lilly, and Boehringer Ingelheim; and has received speaker fees from Medscape, Illumina, and OncLive.

Alexander Drilon, MD, has received honoraria and consulting fees for advisory boards from Ignyta/F. Hoffmann–La Roche/Genentech, Loxo/Bayer/Eli Lilly, TP Therapeutics, AstraZeneca, Pfizer, Blueprint Medicines, Takeda/Ariad/Millennium, Helsinn, BeiGene, BerGenBio, Hengrui Therapeutics, Exelixis, Tyra Biosciences, Verastem, MORE Health, AbbVie, 14ner/Elevation Oncology, Remedica Ltd, ArcherDX, Monopteros, Novartis, EMD Serono, Liberum, Repare RX, and Melendi; has received institutional research funding from Pfizer, Exelixis, Taiho, Teva, GlaxoSmithKline, and PharmaMar; and has received research funding from Foundation Medicine; royalties from Wolters Kluwer; other from Merck, Puma, Merus, and Boehringer Ingelheim; CME honoraria from Medscape, OncLive, PeerVoice, Physicians Education Resources, Targeted Oncology, Research to Practice, Axis, PeerView Institute, Paradigm Medical Communications, WebMD, and MJH Life Sciences.

References 

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