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When it comes to cancer, understandably there's a lot of focus on a ‘cure,’ and the quest to find that treatment. But before we can treat cancer, we first need to be able to detect it accurately. Dr Jessica L. Davis, a pediatric pathologist at Oregon Health & Science University, has been leading research into the rapid and efficient diagnosis of NTRK fusion cancers in children using immunohistochemistry (IHC).
Tropomyosin, or tyrosine, receptor kinase (Trk) family members play a vital role within the nervous system by binding neurotrophins to affect both neuronal differentiation and survival. The neurotrophic receptor kinase 1 (NTRK1), NTRK2, and NTRK3 genes encode proteins TrkA, B, and C respectively. In some instances, an NTRK gene fuses to an unrelated gene, causing Trk signaling to occur without control, which can lead to cancer.
NTRK fusions are rare in most cancers, occurring in around 0.5–1% of common cancers, but we see them at much higher frequencies (up to or above 90%) in rare cancer types like secretory breast carcinoma and infantile fibrosarcoma1. These types of fusions are especially abundant in childhood tumors: NTRK gene fusions account almost half of all driver events in pediatric non-brainstem glioblastoma, for example2.
Thankfully, there are now treatment options emerging for NTRK fusion tumors: Trk inhibitors like the recently approved larotrectinib (VITRAKVI, Loxo Oncology Inc and Bayer), and more recently entrectinib (Genentech/Roche), which has just been granted priority review by the FDA3, are options for clinicians. However, to be eligible for these treatments, the tumor must have an NTRK gene fusion without a known acquired resistance mutation. As yet, there are no FDA-approved NTRK gene fusion tests, meaning that local laboratories carry out this testing.
Cancer in children can be an especially distressing and potentially life-threatening event. The new therapeutic agents promise to reduce the need for surgery in these very young patients. As such, being able to reach an accurate diagnosis to start the treatment as quickly as possible is vital. While molecular methods like NGS and reverse transcription polymerase chain reaction (RT-PCR) provide a more comprehensive look at the NTRK genes, these tests are expensive and slow – potentially delaying treatment4.
"In cases where you have children with rapidly growing tumors, the clinicians and oncologists need to start acting upon that right away. And results from NGS can take up to two weeks."
There is also the issue that sequencing methods can miss some novel NTRK rearrangements, which are appearing as recurrent events in a subset of pediatric mesenchymal tumors4.
In Dr Davis' lab, they have been using an anti-TrkA monoclonal antibody clone EP1058Y (Abcam) and an anti-pan-Trk monoclonal antibody clone EPR17341 (Abcam) to target human TrkA, TrkB, and TrkC proteins4. They set out to see if using IHC with these Trk antibodies could serve as a marker of NTRK fusions in mesenchymal tumors in children. Unlike NGS, which is offered by a handful of facilities, IHC can be run in most pathology labs, to generate results the same day.
RT-PCR and fluorescent in-situ hybridization (FISH) have long been the go-to assays to diagnose of infantile fibrosarcoma, yet, in this research, they did not identify the ETV6-NTRK3 fusion in four cases later confirmed by NGS. And while NGS tests are the most comprehensive assay available, not all medical insurers cover the relatively high cost, can take weeks to complete, and they need relatively large tissue volumes.
So, to see how IHC compared to these molecular methods, Dr Davis and her lab ran assays across dozens of NTRK fusion tissue samples. Looking at 79 cases, pan-Trk IHC gave a sensitivity of 96.7%, a specificity of 97.9%, a positive predictive value of 96.67%, and a negative predictive value of 97.9%. TrkA IHC in 76 cases gave a sensitivity of 100%, a specificity of 63.3%, a positive predictive value of 59.1%, and a negative predictive value of 100%. Staining was exclusively cytoplasmic in NTRK1/NTRK2-rearranged tumors with pan-Trk and TrkA IHC, while there was cytoplasmic without or without nuclear staining in NTRK3 rearrangement tumors. Tumors with NTRK3 rearrangements has generally weaker staining than NTRK 1 and 2. Dr Davis said that pan-Trk IHC was an excellent tool to help identify NTRK-rearranged tumors and that it was superior to the TrkA IHC.
"With NGS we get a little more data - we get to know what the fusion partner is, for example. But IHC is just so fast. And in a subset of cases, fast is beneficial. Time is key."
Additional research has demonstrated the sensitivity and specificity of IHC NTRK fusion in specific indications, such as in pediatric tumors, and for determining where translation has occurred for novel NTRK re-arrangements. However, it is clear that for now at least, NGS is required to detect atypical cases where the disease presentation is suggestive of NTRK rearrangement, but where IHC staining is weak or negative5–7.
In the case of pediatric mesenchymal tumors, Dr Davis proposes a best practice and common-sense approach, using the methods in a complementary fashion. NGS on every tumor is not feasible, and IHC, while imperfect, is very accessible, quick, cost-effective and tissue efficient. She proposes that we use IHC as a screening tool. In this situation, pan-Trk IHC could serve as a surrogate marker for NTRK1/NTRK2 fusions where we see moderate to strong diffuse cytoplasmic staining, and for NTRK3 fusions where there is nuclear pan-Trk staining. In instances where there is weak cytoplasmic staining with pan-Trk IHC, it will be best to validate NTRK fusion by NGS to confirm eligibility for treatment with a Trk inhibitor.
With the algorithm proposed by Dr Davis, pan-Trk IHC has the power to become an incredibly rapid and efficient screening tool for NTRK rearrangements.
“Unfortunately, NGS on every tumor, at the moment, isn't feasible for a lot of institutions. In those cases, IHC becomes an invaluable screening tool."
IHC as a diagnostic tool is an idea that has drug companies interested. In fact, Roche recently launched the first in vitro diagnostic (IVD) pan-TRK immunohistochemistry assay. IVDs like the VENTANA pan-TRK (EPR17341) Assay from Roche could eventually serve to get patients started on treatment right away, rather than having to wait for an NGS diagnosis.
Researchers like Dr Davis, and fellow pathologists, have already started talking to drug companies and regulators with the hopes of making an IHC an approved method for diagnosis a reality. But it's still early days right now. In the interim, Dr Davis remains excited by the line of research and continues to work towards better detecting NTRK fusions.
“We’ve gone from asking what these fusions are, to being able to detect them, and Trk-inhibitors represent the first breakthrough in sarcoma treatment for many, many years.”
Hopefully, this surge of interest in pediatric NTRK fusion cancers will drive the development of improved detection tools – especially for NTRK3 rearrangements, which in turn will fuel improved screening algorithms. Collaborations between life science companies, pathologists, drug companies, diagnostic developers, the FDA, and clinicians will be crucial for translating this research to the clinic. These advancements will improve the time to reach that all-important diagnosis, and, ultimately, patient outcomes.
6. Hung, Y. P., Fletcher, C. D. M. & Hornick, J. L. Evaluation of pan-TRK immunohistochemistry in infantile fibrosarcoma, lipofibromatosis-like neural tumour and histological mimics. Histopathology 73, 634–644 (2018).