Real-world challenges in undertaking NTRK fusion testing in non-small cell lung cancer
Highlight box
Key findings
• NTRK fusions are rare genomic alterations in NSCLC that pose a diagnostic challenge relating to insufficient “tissue” and “tools”.
What is known and what is new?
• In this study we found NRTK fusions are a rare occurrence in NSCLC consistent with other studies. Screening with IHC followed by confirmation of positivity with sequencing is recommended.
• With the discovery of increasing number of targetable genomic alterations, this real-world study highlights some of the challenges experienced with often only small tumor samples available for analysis as well as determining the most appropriate methodology.
What is the implication, and what should change now?
• Testing methodologies and algorithms need to be developed based on local resourcing while also considering the limited amount of tissue available for analysis.
Introduction
Treatments targeting oncogenic mutations have demonstrated high response rates and improved outcomes in patients with non-small cell lung cancer (NSCLC) (1). The tropomyosin-receptor kinase (TRK) receptor family plays an essential role in the development and function of the nervous system and comprise of three transmembrane proteins: TRKA, TRKB, and TRKC, encoded by the neurotrophic tropomyosin-receptor kinase (NTRK)1, NTRK2, and NTRK3 genes, respectively (2). Chromosomal rearrangements of these genes cause activation and/or overexpression of TRK receptors resulting in activation of downstream oncogenic pathways, establishing NTRK as a major target for therapy (2). NTRK gene fusions have been reported across a wide range of solid tumour types as the primary oncogenic driver, however their frequency is low in more common cancers (3). Larotrectinib, a specific TRK inhibitor, and entrectinib, a multi-kinase TRK inhibitor, have been approved by the US Food and Drug Administration and European Medicines Agency as cancer agnostic drugs for patients with solid tumours harbouring a NTRK fusion. Both these agents have demonstrated impressive overall response rates and tolerability in large basket studies that enrolled different types of NTRK fusion-positive tumors (4,5).
Despite the excellent treatment outcomes, the challenge for NTRK rearrangements remains a diagnostic one, due to the rarity of the alteration and the multiple approaches developed to identify NTRK rearrangements. While RNA-based next-generation sequencing (NGS) is the diagnostic tool of choice, turn-around time, cost and pathologist expertise are some of the challenges which need to be considered. In this study we retrospectively evaluated the incidence of NTRK fusions on a lung carcinoma cohort by screening with immunohistochemistry and followed by molecular analysis of all positive samples. The study highlights some of the challenges faced in a real-world setting in screening for these alterations. We present this article in accordance with the STROBE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-113/rc).
Methods
This is a retrospective, single-site study in which archival formalin-fixed paraffin embedded (FFPE) tissue from histologically diagnosed NSCLC between 2018 and 2020 were sectioned and screened by immunohistochemistry (IHC) for NTRK rearrangements using the VENTANA® pan-TRK (EPR17341) assay. IHC-positive cases were analyzed by fluorescent in-situ hybridisation (FISH) to confirm the presence of the fusion.
For immunohistochemistry, four-micron sections of the FFPE block were cut and prepared for staining. A senior pathologist (KA) reviewed tumor histology and reported on NTRK staining. Immunohistochemistry was performed on the Benchmark Ultra platform (Ventana Medical Systems, Tucson, AZ) using the VENTANA® pan-TRK (EPR17341) assay as per manufacturer’s instructions. The staining patterns, intensity (none/0, weak/1+, moderate/2+, strong/3+) and percentage of stained tumor cells were evaluated. Samples with 2+ or 3+ immunoreactivity in at least 1% of tumour cells was considered as positive.
Specimens scored positive were further analyzed by FISH to confirm the presence of a NTRK fusion. Unstained tissue sections were dewaxed in Hurstsol (Hurstchem, Australia), then treated in 10 mM sodium citrate buffer at 98 ℃ then incubated with pepsin (2.5 mg/mL) at 37 ℃. The sections were hybridized overnight at 37 ℃ separately with the NTRK1 (Z-2167-200), NTRK2 (Z-2205-200) and NTRK3 (Z-2206-200) probes (Zytovision, Germany) and analyzed using Olympus BX53 microscope and the Cytovision Software. A positive disruption was noted when there was a separation of the orange and green signals of at least one signal dot diameter and in greater than 10% of nuclei. However, due to the high occurrence of intrachromosomal translocation involving NTRK1 or NTRK3, an additional criterion was applied in that only one orange-green signal set show the split while the other orange-green signal should not appear separated.
As per routine standard of care, all patients during the study period underwent reflex testing for at least an epidermal growth factor receptor (EGFR) mutation, ALK and ROS1 rearrangement and PDL1. Correlative clinicopathologic parameters were also collected. Data collected included: baseline patient demographics, smoking history, tumor histology and stage, PDL1 and oncogene status and treatment history.
Statistical analysis
Patient characteristics and IHC outcomes were summarised using mean, standard deviation (or medians and ranges where appropriate), range if continuous, or using percentages if categorical.
Ethical statement
The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Austin Hospital Research and Ethics committee (No. H2006/02394) and individual consent for this retrospective analysis was waived.
Results
Clinical-pathological features
A total of 289 samples were analyzed and included 81 cytology cell blocks, 30 resection specimens and 178 biopsies. For the entire cohort, the median age was 70.9 (range, 35–93) years, with 42% [121] female and 77% [223] with a current or prior smoking history. The majority of patients had stage IV disease (83%) and tumors of adenocarcinoma histology (69%).
Of the samples analyzed, 10 (3.5%) cases had NTRK expression on IHC. The median age of patients with NTRK-expression on IHC was 74.9 (range, 44–88) years, majority male (70%) and 70% were current or former smokers. Of the tumors with NTRK expression, seven (70%) were of adenocarcinoma histology, and one each of squamous cell carcinoma, large-cell neuroendocrine and not otherwise specified histologies (Table 1). PDL1 expression was ≤50% in five cases (50%). Concurrent EGFR mutations were detected in three samples (30%), with two cases also showing a PIK3CA E542K mutation and MET amplification respectively (Table 2).
Table 1
| Baseline characteristics | N=10 |
|---|---|
| Age, year, median age [range] | 74.9 [44–88] |
| Sex | |
| Male | 7 |
| Female | 3 |
| Ethnicity | |
| Caucasian/White | 7 |
| Asian | 3 |
| History of tobacco use | |
| Never | 2 |
| Current or previous | 7 |
| Not known | 1 |
| Stage at diagnosis | |
| II | 1 |
| III | 1 |
| IV | 8 |
| Histological subtype | |
| Adenocarcinoma | 7 |
| Squamous cell carcinoma | 1 |
| Non-small cell lung carcinoma not otherwise specified | 1 |
| Large cell neuroendocrine carcinoma | 1 |
| Lines of treatment | |
| No prior lines | 2 |
| ≥1 prior lines | 2 |
| ≥2 prior lines | 5 |
| Not known | 1 |
IHC, immunohistochemistry; NTRK, neurotrophic tropomyosin-receptor kinase.
Table 2
| Patient | Histology | Stage | Tumor tissue | Concurrent mutations | PD-L1 (%) | IHC staining pattern | IHC staining intensity | NTRK FISH | Nucleic acid testing |
|---|---|---|---|---|---|---|---|---|---|
| 1 | Adeno | III | FNA | EGFR ex19 del± PIK3CA E542K | 0 | Cytoplasmic | 100% 2+ | Negative* | Not done* |
| 2 | LCNE | IV | Core biopsy | Pan WT | 1 | Cytoplasmic | 20% 2+, 40% 1+ | Negative* | Not done* |
| 3 | Adeno | IV | Core biopsy | Pan WT | 80 | Cytoplasmic | 10% 2+, 60% 1+ | Negative* | Not done* |
| 4 | Adeno | IV | Core biopsy | Pan WT | 0 | Paranuclear | 30% 3+ | Negative* | Not done* |
| 5 | Adeno | IV | Core biopsy | EGFR L858R± | NA | Cytoplasmic | 40% 2+, 40% 1+ | Negative* | Not done* |
| 6 | Adeno | IV | Core biopsy | Pan WT | 90 | Cytoplasmic | 30% 2+, 50% 1+ | Negative* | Not done* |
| 7 | SqCC | II | Core biopsy | Pan WT | 20 | Cytoplasmic | <5% 3+, 90% 2+ | Negative* | Not done* |
| 8 | Adeno | IV | Core biopsy | EGFR L858R± MET amplification | NA | Cytoplasmic | 10% 2+, 70% 1+ | Negative* | Not done* |
| 9 | NOS | IV | Resection | Pan WT | 80 | Cytoplasmic | 5% 3+, 20% 2+, 15% 1+ | Negative* | Not done* |
| 10 | Adeno | IV | Core biopsy | Pan WT | 2 | Cytoplasmic | 100% 3+ | Not done* | EML4-NTRK fusion |
*, insufficient tissue; ±, T790M mutation not detected. IHC, immunohistochemistry; NTRK, neurotrophic tropomyosin-receptor kinase; PD-L1, programmed death-ligand 1; FISH, fluorescence in situ hybridization; Adeno, adenocarcinoma; FNA, fine needle aspirate; EGFR, epidermal growth factor receptor; LCNE, large cell neuroendocrine; WT, wild-type; SqCC, squamous cell carcinoma; NA, data not available; NOS, not otherwise specified.
Immunohistochemistry analysis
Altogether six cases demonstrated moderate staining (score 2+) and four cases (40%) showed strong cytoplasmic and membranous staining (score 3+) (Figure 1). In nearly all cases staining was cytoplasmic, with one case demonstrating paranuclear dot-like staining. In the one patient found to have a NTRK fusion on molecular analysis the percentage of positive tumour cells in the evaluated sections demonstrated diffuse and homogenous positivity in all cancer cells, i.e., 100% staining (Table 2).
Molecular analysis
The cases expressing NTRK were sent for molecular analysis to confirm the presence of a fusion. Of these, one case (patient 10) had a NTRK fusion, EML4-NTRK3 gene fusion, detected by NGS (Trailblaze Pharos, Ignyta, San Diego, CA, USA) on the STARTRK2 clinical trial (ClinicalTrials.gov number NCT02568267). No tissue was left over from this case for FISH analysis. The additional nine cases had insufficient tumor material for nucleic acid sequencing and therefore FISH was performed instead. In all these cases no NTRK rearrangements were detected (Figure 2).
Clinical history
Patient 10, is of Asian descent and a life-long non-smoker, who presented with a cough. A CT guided biopsy of a metastatic liver lesion confirmed an adenocarcinoma of lung origin. The patient was commenced on platinum-doublet chemotherapy but unfortunately developed disease progress after two cycles of treatment. Further analysis of the tumor tissue by NGS on the STARTRK2 clinical trial (ClinicalTrials.gov number NCT02568267) demonstrated a EML4-NTRK3 gene fusion. The patient was commenced on entrectinib, which he took for 12-month at which point further disease progression was seen in the liver. A repeat liver biopsy demonstrated a NTRKG623R compound mutation. The patient was enrolled onto the clinical trial Trident-1 (ClinicalTrials.gov number NCT03093116) where he received repotrectinib for almost two years. The patient is now off study and receiving chemoimmunotherapy with bevacizumab.
Discussion
In this study we found NRTK fusions are extremely rare in NSCLC, which is in concordance with other studies (2,6). Despite identifying a number of cases with high protein expression (3.5%), only one case of NTRK gene fusion using molecular techniques was detected. This highlights the requirement of confirmation of IHC positivity with genomic sequencing to determine fusion partners and precise breakpoints (7). In this study high NRTK expression did not correlate with sex, age, smoking history or tumour histology. We did observe a numerically higher rate of NTRK expression in tumours with low PDL1 expression; which has also been previously reported (8).
Several testing methodologies to identify NTRK fusions are available each with their own advantages and disadvantages and are utilised based on available resources. While nucleic acid-based sequencing, in particular NGS, is the diagnostic of choice, pan-TRK IHC has shown to have high sensitivity and specificity particularly for NTRK1 and NTRK2 fusions in NSCLC (9,10), in addition to being cheap, widely accessible and quick. Screening with IHC is in line with international recommendations (11-13), and can be particularly useful in cancers with low prevalence of NTRK gene fusions and in laboratories where molecular sequencing methods are not readily available or NTRK testing is not part of routine workflow. Currently, two monoclonal antibodies are used for detecting specific proteins include EPR17341 (Ventana® and Abcam®; used in this study) and A7H6R (Cell Signaling®) with comparable performance (14). In this study, the majority of IHC positive cases were adenocarcinomas and staining typically cytoplasmic with a heterogenous pattern. With exception of the case in which a NTRK fusion was detected by NGS, all other cases rarely demonstrated positive staining in more than 80% of tumor cells. We undertook FISH analysis for nine out of 10 cases as there was insufficient tumor quantity to perform RNA sequencing in these cases. While FISH is highly sensitive for fusions with canonical breakpoints, certain NTRK1 fusions due to small inversion or deletions may be missed by interphase FISH analysis. In addition, a positive FISH result does not provide information on the functional significance nor fusion partner and a false negative FISH result may occur as some of the NTRK1 and NTRK3 fusion partners are intrachromosomal. As all tumors included in this study were not analyzed using RNA-sequencing the rate of IHC false-negative cases is not known, especially involving NTRK3 (7). Furthermore, the NTRK antibody binds both the wild-type as well as the fusion protein, therefore, strong staining may indicate either expression of the wild-type protein or the presence of a TRK fusion protein (15).
NTRK fusions are typically present in a mutually exclusive manner with other oncogenic mutations and fusions (3). In this study, concurrent EGFR sensitising mutations were identified in almost a third of cases with high NTRK expression including one patient with a concurrent EGFR L858R mutation and MET amplification. Whether EGFR mutations or indeed other driver mutations lead to false positive NTRK IHC staining is not known. Tumors harboring EGFR mutations have shown to express ROS1 mRNA at levels comparable to those of tumors with ROS1 rearrangement leading to false positive ROS1 IHC (16). RTK fusions, including TPM3-NTRK1 fusion, have been identified as resistance mechanisms to first, second and third generation EGFR tyrosine kinase inhibitors (TKIs) (2). Combination treatments using EGFR TKIs with other kinase inhibitors such as osimertinib with alectinib or osimertinib and pralsetinib have shown to be successful in overcoming acquired resistance of ALK and RET fusions to EGFR TKIs (17).
This study has limitations in being a single-centre retrospective study and our ability to perform appropriate confirmatory molecular analysis was restricted by tumor quantity, which reflects some of the real world challenges faced with NTRK fusion testing. Despite the relative low number of cases examined, this study included a range of histological subtypes and tumor stages and highlights some of the challenges with NRTK testing in a real-world setting where small tumor samples (biopsies and cytology specimens) are most commonly available.
Conclusions
In conclusion, NTRK fusions are rare but targetable genomic alterations that pose a diagnostic rather than therapeutic challenge and require testing methodologies and algorithms developed based on local resourcing. While screening with IHC followed by confirmation of positivity with sequencing is a potential strategy, this study as well as others have shown high protein expression does not imply the presence of NTRK1–3 gene fusions.
Acknowledgments
Funding: Bayer (Australia) provided funding to support the analysis for the study (Ref: SM_NTRK_July21).
Footnote
Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-113/rc
Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-113/dss
Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-113/prf
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-113/coif). SP served on the advisory board of Astra Zeneca, received speaking honoraria from Astra Zeneca, Roche and MSD, and received research funding from Roche outside the submitted work. The other authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Austin Hospital Research and Ethics committee (No. H2006/02394) and individual consent for this retrospective analysis was waived.
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.
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