In our previous publication, “Metastatic Triple Negative Breast Cancer with NTRK Gene Fusion on Tissue but not on ctDNA Molecular Profile, [1]” we discussed a case of a middle-aged female with metastatic triple negative breast cancer (TNBC) harboring an ETV6-NTRK fusion which was observed only on recent tissue molecular profiling, but not on circulating tumor DNA or years prior tissue molecular profiling. She was treated with Larotrectinib, a Tyrosine Receptor Kinase (TRK) inhibitor, with excellent response. This discovery also allowed for histologic reclassification of her tumor from invasive ductal carcinoma to secretory breast carcinoma. This case was particularly important for two reasons: 1) it highlighted differences in technological offering from available molecular profiling assays over time and between competing companies, and 2) the importance of comprehensive molecular profile in the management of metastatic breast cancer.

As a review, prior to Larotrectinib, this patient forgoes traditional chemotherapy for non-traditional herbal therapy. Interestingly, the tumor experienced a more indolent growth pattern almost 6 years following diagnosis of an aggressive metastatic TNBC. Her disease burden included widespread bilateral pulmonary and pleural lesions. With Larotrectinib, these lesions experienced deep partial responses. She was first intolerable of full dose Larotrectinib 100 mg BID due to grade 3 nausea and neuropathy. To determine her clinical maximal tolerable dose (cMTD) she was dose reduced to 25 mg BID and increased to her cMTD of 75 mg BID within 3 weeks. Since publishing this case report, the patient was maintained on 75 mg BID with good tolerance, with the exception of 20 lbs weight gain despite adequate weight loss measures. Her most recent CT-chest abdomen and pelvis scan on 12/12/2022 (13 months from the beginning of therapy) demonstrated a near complete response with 4 mm of residual pleural thickening, down from 70 mm at the beginning of the TRK inhibitor. This case provides strong support for comprehensive molecular profiling for patients with metastatic cancers.

The first NTRK gene fusion was identified in 1986 in a case of colorectal cancer. Here, Martin-Zanca et al. observed a chimeric fusion oncogene resulting from an intrachromosomal rearrangement at 1q22-23 [2].  Despite the identification of other NTRK fusions throughout the years, their role as a therapeutic target was not fully explored until 2013 when it was identified in lung tumor samples assayed by NGS and FISH [3]. NTRK genes encode the tropomyosin kinase receptor (TKR). There are three main genes, NTRK1, NTRK2, and NTRK3, that encode for three separate receptors TKRA, TKRB, and TKRC. These genes are located on three separate chromosomes, 1, 9, and 15 respectively. They contribute to the functioning of the nervous system via their activation by neurotrophins [4,5]. Tyrosine Kinase Proteins, when inappropriately transcribed, can result in constitutively activated kinase function, necessary for their inevitable oncogenic potential [6].

NTRK fusions function as a driver of tumorigenesis in many tissue types making it a tumor agnostic and age independent biomarker and therapeutic target. Overall, these genomic alterations are rare. Only 0.31% of adult tumors and 0.34% of pediatric tumors contained an NTRK fusion mutation with the most common being NTRK3 fusions. NTRK fusions are more commonly detected in secretory carcinoma of the breast, salivary gland carcinoma, and infantile fibrosarcoma. However, lower incidence of NTRK fusions have been noted in melanoma, glioma, colon carcinoma, thyroid carcinoma, head and neck squamous cell carcinoma, non-secretory breast carcinomas, and lung carcinomas. For example, compared to 90% of secretory breast cancers, only about 3.3% of lung cancers harbor an NTRK fusion. The most common NTRK fusion partners are ETV6 – NTRK3, LMNA-NTRK1, and TPM3 – NTRK1 [7]. Several tumor specific NTRK fusions also exist including fusions that disrupt pathways including phosphoinositide-3-kinase signaling, other tyrosine kinase receptors, cell cycle components and mitogen activated protein kinase (MAPK). Additional NTRK abnormalities including mutations, amplification and mRNA overexpression were seen in up to 14% of samples across adult and pediatric tumors [3,7]. It is still not fully understood whether TRK inhibitors have anti-cancer activity in other non-fusion related NTRK alterations.

Primary and secondary resistance to TRK inhibitors have been noted but are not very well understood. Broadly, these can occur utilizing both on-target or off-target mechanisms of action. For example, on target point mutations within the NTRK kinase domain can interfere with the binding of these tyrosine kinase inhibitors. Whereas off-target development of upstream mutations in genes encoding mitogen-activated protein kinase (MAPK), MET, BRAF, and KRAS are also possible. Off-target mechanisms of resistance poses greatest challenge and may require combination therapeutic strategies to overcome [7,8]. Second generation TKIs have been developed with hopes of circumventing this resistance mechanisms. Next generation TRK inhibitors such as selitrectinib, repotrectinib, and taletrectinib may retain activity following the development of on target resistant mutations to first generation TRK inhibitors. Selitrectinib and reprotectinib have shown activity against solvent front acquired mutations while talectrectinib and SIM1803–1A exhibit more broad activity to solvent front and additional mutations to NTRK fusions [9,10].

Given the positive clinical implication for tumors harboring NTRK-fusion, detection of these alterations when the exist is imperative. Testing modalities to detect an NTRK fusion mutation includes genomic sequencing such as next generation sequencing (NGS), real time PCR (RT-PCR), immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). IHC being the most widespread and can be most easily adopted but its sensitivity for detection can vary.  Whereas NTRK 1 and 2 stains at a high sensitivity, NTRK 3 staining is only about 79% [6]. Furthermore, IHC antibody binding specificity and sensitivity varies based on tumor type. FISH is more commonly used for solid tumors and requires specific break-apart probes for each NTRK gene. While these are readily available, they hold some technical difficulty, especially in NTRK 1 fusion mutations which tend to be intrachromosomal. RT-PCR provides more difficulty in determining the exact fusion mutation and thus far has been limited to the ETV6-NTRK fusion. Lastly, DNA-based genomic sequencing such as NGS can be an effective way of examining known fusion mutations, but limitations exist based on gene and intron length. Additionally, this approach is not ideal for detection of novel fusions [6,11].

The leading genomic tumor profiling companies are FoundationOne, Tempus, Caris, Paradigm, NantOmics and GuardanT360. When comparing their platforms, GPS Cancer NantOmics assesses the greatest number of genes [12]. Regarding NTRK fusion detection, DNA based tissue sequencing companies MSK-IMPACT and FoundationOne comprehensively cover NTRK 1 and 2 but only exons of NTRK3. The latter poses a limit as most fusion mutations occurs at introns. The most utilized ctDNA assay, GuardanT360, can only identify fusions in NTRK1 but can identify point mutations in both NTRK1 and 3. RNA sequencing appears to provide the widest ability to detect for NTRK fusion mutations with companies like Tempus and Caris providing detection of fusions in NTRK 1, 2 and 3 [12,13]. When utilizing both DNA and RNA detection assays, fusions are identified 65% of the time, as opposed to 11% with just DNA and 23% with just RNA [14]. While genomic profiling is a necessary component of managing metastatic malignancies, the choice of testing agency can greatly impact the detection of clinically important genomic alterations.

The use of Larotrectinib, proved extremely effective at disease response and improving the patient’s quality of life. Larotrectinib is a selective tyrosine receptor kinase inhibitor for the tropomyosin related kinase coded for by the NTRK gene fusion [15]. It, along with another TRK inhibitor, Entrectinib, enjoys tumor agnostic FDA approval for those harboring TRK-fusion mutations. In our case, an ETV6-NTRK fusion mutation was discovered on tissue from a lung metastatic lesion via an NGS 324-gene comprehensive molecular profile. The predicted fusion transcript included exons 1-5 of ETV6 and exons 15-19 of NTRK3, including the entire kinase domain and was highly reproducible. Importantly, a 592-gene NGS comprehensive panel performed 3 years prior, and ctDNA performed concurrent with the 324-gene assay failed to detect this mutation. The latter may be because at the time of testing using the 592 panel in 2018, NTRK-fusion testing was not being offered. Additionally, the lack of detection on concurrent ct-DNA may have been limited by low ctDNA shedding. In a study of colorectal cancer, lung metastasis only had lower rates of ctDNA shedding. This suggests that lung lesions can have a low ctDNA shedding rate and could explain the discrepancy in our case since our patient only had lung limited metastatic disease [16]. Larotrectinib approval was based in part on a 2018 study showing an overall response rate (ORR) of 75%, including 13% complete responses and 62% partial responses at 1 year. At 1 year, responses were noted in 86% of patients and 55% of the patients remained progression free. This study included 12 different cancer types, many of which are considered rare cancers [17]. The most common adverse events occurring in at least 15% of patients were increased liver enzymes (AST or ALT), dizziness, constipation, nausea and fatigue. Entrectinib by contrast, in a similar study reported an ORR of 57% with 7.4% patients achieving complete responses. While active against NTRK fusions, Entrectinib is also effective for ROS1 fusion positive lung cancers with an ORR of 78% [18]. The use of Larotrectinib over Entrectinib for this patient was based on the adverse event profile and possible superior anti-tumor activity though no direct comparison study has been completed [19,20].

As discussed, in the case of our patient from the paper “Metastatic Triple Negative Breast Cancer with NTRK Gene Fusion on Tissue but not on ctDNA Molecular Profile,” molecular profiling can be the key to finding an effective therapeutic option for resistant metastatic triple negative breast cancer. Molecular profiling, especially with the combination of DNA and RNA sequencing, increases the number of mutations that are observed in NTRK genes and can determine if the patient has an NTRK gene fusion mutation which can be targeted using a TRK inhibitor. Larotrectinib and entrectinib are some of the traditional TRK inhibitors used while new ones are emerging on the market to account for methods of TRK inhibitor resistance.