Antitumor Activity of Larotrectinib in Esophageal Carcinoma with NTRK Gene Amplification.

BACKGROUND: Increasing knowledge about the genomic changes underpinning cancer development and growth has led to a rapidly expanding number of individualized therapies that specifically target these changes in a patient's tumor. Here we present a case report of a patient with metastatic esophageal carcinoma whose tumor harbored NTRK1 gene amplification and who received targeted systemic therapy with larotrectinib. At initial diagnosis, the patient presented with tumor obstruction of the middle esophagus, simultaneous liver and lung metastases, UICC IV and WHO performance status 3.
MATERIALS AND METHODS: The solid tumor genomic profiling test FoundationOne CDx (F1CDx) was used to detect clinically relevant genomic alterations that, in turn, might identify a targeted therapeutic approach if suggested by the findings. The patient was then treated with larotrectinib and had subsequent follow-up biopsies.
RESULTS: Simultaneous biopsies of the primary tumor and liver lesions identified a metastatic squamous cell esophageal carcinoma. Comprehensive genomic profiling obtained from liver metastases identified numerous genomic alterations including amplification of NTRK1. Owing to the reduced performance status of the patient, chemotherapy could not be applied and was denied. Although larotrectinib is only approved for the treatment of cancers with NTRK gene fusions, treatment was started and led to a shrinkage of the primary tumor as well as the liver and lung metastases within 6 weeks according to RECIST criteria accompanied by tumor marker decrease. The NTRK1 gene amplification was below the limit of detection in a subsequent liver biopsy.
CONCLUSION: The use of comprehensive genomic profiling, specifically F1CDx, enabled the selection of a targeted therapy that led to a rapid reduction of the tumor and its metastases according to RECIST criteria. This case suggests that larotrectinib is not only effective in NTRK fusions but may be efficacious in cases with gene amplification. KEY POINTS: Advances in precision medicine have revolutionized the treatment of cancer and have allowed oncologists to perform more individualized therapy. This case shows that larotrectinib could also be effective in cases of NTRK amplification of cancer. Today, there is only limited knowledge about NTRK alterations in squamous epithelial carcinoma of the esophagus. Longitudinal tumor sequencing during the course of the disease may allow for the detection of a molecular genetic cause once the tumor progresses. Additional actionable gene alterations may then be identified, which may provide the rationale for a therapy switch.

Introduction

Cancer has long been categorized and treated based on its anatomic origin and localization. However, with the development of clinically available and robust comprehensive genomic sequencing assays, genomic driver alterations that are involved in the tumor development and progression could be detected and allow personalized therapies of actionable gene alterations.

NTRK gene fusions represent one of the most important molecular changes with known oncogenic and transforming potential 1. Gene fusions lead to transcription of chimeric TRK oncoproteins that are constitutively active and serve as oncogenic drivers in a wide variety of cancers. Therefore, NTRK gene fusions are currently being investigated in several tumor types as targets for cancer therapy 2.

Regarding a treatment of NTRK gene fusions, several TRK inhibitors have been developed, including larotrectinib. Larotrectinib is an orally available selective inhibitor of the TRK receptor family that has shown significant clinical benefit in pediatric and adult patients with NTRK gene fusion in recent years and is now approved in the European Union (EU) and the U.S. 3, 4. NTRK gene amplification has shown to result in TRK overexpression as well 5. However, knowledge on the efficacy of targeted therapy for NTRK gene amplification is yet rare. To our knowledge, there has been only one patient described so far who harbored an NTRK1 gene amplification and who had a partial response after treatment with larotrectinib 6. This patient was described in a multicenter, open‐label, phase I dose‐escalation study, which investigated larotrectinib in adult patients with solid tumors 6.

Esophageal cancer remains a major cause of cancer‐related mortality worldwide and is associated with a poor prognosis in both the locally advanced and metastatic setting 7, 8. The majority of patients with esophageal cancer suffer from the metastatic disease at the time of diagnosis or relapse after surgery or chemotherapy 9. Esophageal cancer includes two main subtypes: oesophageal squamous cell carcinoma and oesophageal adenocarcinoma 10. The standard therapy for patients with advanced/metastatic squamous cell carcinoma of the esophagus is palliative chemotherapy, usually consisting of cisplatin and a fluropyrimidine. The aim of this therapy is solely to improve the quality of life 11, 12. Although this therapy has a life‐prolonging effect in adenocarcinoma, the effect of treatment in squamous cell carcinoma is not assured 12. The efficacy of targeted therapies has so far only been shown for adenocarcinoma of the esophagus 12.

In this case report, we present the case of a patient with metastatic squamous cell esophageal carcinoma with NTRK1 gene amplification who received targeted therapy with larotrectinib. In a search of 879 cases with squamous cell carcinoma of the esophagus identified in the Foundation Medicine database, NTRK1 fusions were detected in none and gene amplification in two cases (0.2%). Therefore, this case report is of outstanding relevance. Furthermore, to our knowledge, this is merely the second published case of a patient with NTRK gene amplification who received larotrectinib.

Clinical Presentation

The patient was a 71‐year‐old male who presented in December 2018 at the Oncology Center with dysphagia, dyspnea, cough, swallowing disorders, and weight loss of 20 kg within 3 months. The patient was in a poor general condition with Eastern Cooperative Oncology Group (ECOG) performance status of 3 and had no other pre‐existing conditions. He did not consume alcohol or nicotine and had no previous tumors. Esophagogastroduodenoscopy showed a stenosing esophageal carcinoma in the middle third of the esophagus with an extension of 10–21 cm from the upper row of teeth. Computed tomography (CT) showed bilateral lung metastases, and contrast‐enhanced abdominal ultrasound sonography and CT showed multiple liver metastases. A tumor biopsy from the esophagus and a sonographically controlled liver biopsy were performed to confirm the diagnosis. The results confirmed the diagnosis of both a squamous cell carcinoma of the esophagus and liver metastases consistent with esophageal origin (Fig. 1). Because of swallowing disorders and stenosis, an esophageal stent was implanted on February 10, 2019 (Fig. 2).

Figure 1

Tumor histology. Hematoxylin and eosin staining showed a solid nodular tumor mass in the liver (A; — 100 μm) with a brown‐colored desmoplastic collagen reaction (B; — 100 μm) using the Gomori staining. Immunohistochemistry showed a strong positivity for CK5 and p63 (C and D; — 200 μm) to sustain a squamous differentiation of the metastasis. There was a prominent proliferation marked by Ki‐67 (E; — 200 μm) in tumor formation. Fluorescence in situ hybridization analysis for NTRK 3 showed no translocation (F; — 50 μm)

Figure 2

Esophageal stent before therapy with larotrectinib (left) and 6 weeks after treatment (right).

Genotyping Results and Interpretation of the Molecular Results

FoundationOne CDx Test

Approval for this test, including a waiver of informed consent and a Health Insurance Portability and Accountability Act waiver of authorization, was obtained from the Western Institutional Review Board (Protocol 20152817). Informed consent of the patient was obtained to perform genomic profiling. The DNA was isolated from the histological tumor enrichment and its quality and quantity were further investigated. Subsequently, relevant areas of the genome were further enriched by hybrid capture (Agilent SureSelect custom kit) and examined after ligation of adaptors by next‐generation sequencing (NGS; FoundationOne CDx). FoundationOne CDx is a solid tumor genomic profiling test that detects clinically relevant mutations in cancer‐associated genes and provides a comprehensive molecular tumor profile 13, 14, 15, 16.

Sequencing Results

The conventional histology examination from February 2019 identified a metastatic squamous cell esophageal carcinoma (Fig. 1). Genomic alterations of CCND1, EGFR, MET, PIK3CA, CCND2, CDK6, HGF, SOX2, CCND3, CDKN2A/B, EPHB4, FGF19, FGF3, FGF4 MCL1, NTRK1, PIM1, TP53, and VEGFA were detectable in the examined tumor tissue (primary tumor and liver) and are summarized below (Table 1). Among the strongly amplified genes, several showed much higher initial amplification levels than NTRK1 (EGFR, PIK3CA, CCND1, FGF19, FGF3, and FGF4). The microsatellite status was stable and the tumor mutational burden was classified as intermediate with 10 Muts/Mb. Furthermore, genomic variants of unknown significance were detected but did not have a clinical impact.

Table 1

Sequencing results

Gene variation	Initial liver biopsy 02/19		Second liver biopsy 05/19
Microsatellite status	MSS		MSS
Tumor mutational burden	Intermediate (10 mut/1MB)		Intermediate (6 mut/1MB)
Estimated tumor content	35.5%		40.8%
Short variant
Gene	Substitution	MAF	Gene	Substitution	MAF
TP53	P278T	0.42	TP53	P278T	0.37
CNV
Gene	Alteration	Copy number	Gene	Alteration	Copy number
CCND1	Amplification	40	CCND1	Amplification	21
EGFR	Amplification	126	EGFR	Amplification	65
MET	Amplification	13	MET	Amplificationa	7
PIK3CA	Amplification	45	PIK3CA	Amplification	20
CCND2	Amplification	8
CDK6	Amplification	10	CDK6	Amplificationa	6
HGF	Amplification	13	HGF	Amplificationa	7
SOX2	Amplification	14	SOX2	Amplification	8
CCND3	Amplification	12	CCND3	Amplificationa	7
CDKN2A/B	Loss	0	CDKN2A/B	Loss	0
EPHB4	Amplification	8
FGF19	Amplification	39	FGF19	Amplification	20
FGF3	Amplification	39	FGF3	Amplification	20
FGF4	Amplification	41	FGF4	Amplification	20
MCL1	Amplification	8
NTRK1	Amplification	8
PIM1	Amplification	14	PIM1	Amplification	8
VEGFA	Amplification	12	VEGFA	Amplificationa	7

Equivocal.

Abbreviations: CNV, copy number variation; MAF, Mutant Allel Frequency; MSS, microsatellite stable.

A fluorescence in situ hybridization (FISH) assay for NTRK1, 2, and 3 fusions was also performed. FISH confirmed the results of NGS. Specifically, there was an absence of NTRK fusions but a presence of an NTRK1 amplification with a copy number of 8 (Table 1).

After the failure of larotrectinib in May 2019, a subsequent liver biopsy continued to display strong amplification events in the abovementioned genes. However, the total number of amplicons decreased by half, consistently within the analyzed genes (Table 1).

Patient Update

On February 4, 2019, therapy with larotrectinib was initiated. The patient received a standard dose of 2 × 100 mg larotrectinib orally. Side effects did not occur during treatment. Larotrectinib was not yet approved in the EU when the patient was treated but was imported from the U.S. A spiral CT scan done on January 4, 2019, before the treatment initiation showed multiple lung and liver metastases. Two metastases in the left lung segment I/II and in segment VI and two metastases in liver segment VII were considered target lesions for tumor assessment according to RECIST 1.1. With respect to tumor response, the sum of the longest diameters of target lesions was 67.5 mm at baseline. Several pathologically enlarged pre‐ and paracaval lymph nodes were still present. ECOG performance status improved to 1 and the patient recorded a weight gain of 9 kg. He was able to leave the clinic and continue via self‐care. The pathological follow‐up examination performed on April 17, 2019, by esophagogastroduodenoscopy confirmed the decline of the tumor, as the esophageal re‐biopsy was tumor‐free (Fig. 2). The duodenal mucosa, the antral mucosa, and the corpus mucosa presented without morphologically detectable pathological findings.

On March 22, 2019, a response assessment was undertaken with a CT of the thorax and abdomen. Compared with the examination carried out on February 4, 2019, there was now improvement of the findings, with regression in size of the multiple pulmonary metastases accompanied by tumor marker (squamous cell carcinoma [SCC]) decrease (Figs. 3, 4). The sum of the longest diameters of the target lesions decreased to 33 mm, which constitutes a partial response according to RECIST criteria. The pleural effusions had receded.

Figure 3

Computed tomography of lung (upper panel) and liver (lower panel) before treatment with larotrectinib and 6 weeks later, showing partial remission of liver and lung metastases.

Figure 4

Tumor marker value SCC over time.

Abbreviation: SCC, squamous cell carcinoma.

On May 11, 2019, another CT scan and a liver biopsy with contrast medium–guided ultrasound (SonoVue) were performed as a result of a rise of the tumor marker SCC antigene (Fig. 4). A progressive disease of liver and lung metastases was observed. Results of the rebiopsy of the liver revealed that the NTRK1 amplification was now below the limit of detection, which could explain the disease progression. The patient refused conventional chemotherapy. He received crizotinib after approval by his health insurance because of the presence of a MET amplification. The treatment resulted in stable disease for another 3 months.

Functional and Clinical Significance of Gene Alterations in Esophageal Cancer

NTRK 1, NTRK 2, and NTRK 3 encode for the transmembrane proteins TRK A, B, and C, respectively. These tropomyosin receptor kinase proteins are expressed in human neuronal tissue and play an important role in the physiology of the development and function of the nervous system 2.

In numerous malignancies, mutations in NTRK gene family have been confirmed, although only fusions, in‐frame deletions, and splice variations have been identified as oncogenic 1. Very little has been reported about NTRK gene alterations in esophageal carcinoma. Considering our findings, NTRK1 alterations in squamous cell carcinoma occur at an extremely low frequency of 0.2%, as identified in the Foundation Medicine database. In two separate studies describing the frequency of NTRK fusions in esophageal carcinoma, there was only one case of NTRK3 fusion defined within 100 biopsies and, respectively, no NTRK fusions among 185 biopsies 17, 19. However, despite the low NTRK fusion and amplification findings, TRK A was observed to be overexpressed in 9/20 cases of esophagus cancer 18. Therefore, TRK‐specific protein kinase inhibitors might be considered an appropriate therapy.

Discussion

With larotrectinib, entrectinib, and selitrectinib (formerly known as LOXO‐195), three TRK inhibitors are either already available or still in clinical development. NTRK1, 2, and 3 genes fuse with unrelated genes, which results in ligand‐independent activation of the protein kinase domain of the TRK receptor. Whereas the activity of larotrectinib and entrectinib in NTRK gene fusions is established, their role on NTRK amplification is unclear 3, 20, 21. Nonfusion NTRK alterations, for example, mutation or amplification, have been associated with a lack of response with some NTRK inhibitors 2, but recently, in a multicenter, open‐label, phase I dose‐escalation study investigating larotrectinib in adult patients with solid tumors, one patient harbored an NTRK1 gene amplification and had a partial response with larotrectinib, which was the rationale for treatment decision. The described patient had a single, small target lesion (11 mm) that shrank by 5 mm (45.5%) and the duration of response was 3.7 months 5, which is similar to the response outcome observed in our patient. We also found a high amplification of EGFR in our sample, and this patient could have been treated with anti‐EGFR therapy as well. According to the abovementioned publication, the molecular tumor board decided on a treatment with larotrectinib in this particular case, and we saw a short but significant tumor shrinkage according to RECIST criteria classified as partial remission.

Similar to ERBB2 gene amplification, which is a well‐established class of driver gene alteration in breast and gastric cancer, protein overexpression of NTRK amplified tumors is inconsistent. Recently, NTRK amplification (copy number ≥4) was reported to result in a protein overexpression in 14.8% of patients 22. To our knowledge, this is the second report that describes a short but significant tumor response of larotrectinib in NTRK amplified tumors. Whereas larotrectinib is very specific for inhibition of TRK A, B, and C kinase domain, entrectinib is also active in ROS1 and ALK fusion 21. The high specificity of larotrectinib for TRK A, B, C protein kinase could explain the fact that the substance seemed to be active in NTRK amplified cancers, whereas for entrectinib, the aforesaid efficacy was not assessed 23.

Conclusion

Results of the liver rebiopsy after larotrectinib failure revealed that there was a loss of the NTRK1 amplification, hand in hand with a significant decrease of copy number of other observed genes as well. Because of the fact that the initial amplification copy number of certain genes (EGFR in particular, followed by PIK3CA, CCND1, FGF19, FGF3, and FGF4) was much higher than that of NTRK1 (5‐ to 15‐fold), it could be speculated that some of these genes were later driving the clonal expansion of the tumor cells, influencing its possible mechanism of acquired resistance. Additionally, the mechanisms of resistance and progression in this patient may include the presence or emergence of NTRK‐associated coalterations, which were commonly discerned in genes that are involved in PI3K signaling, tyrosine kinase families, cell‐cycle machinery, and MAPK pathways. Additional investigation is needed to elucidate whether these genes mediate resistance to NTRK inhibition and if cotargeting them augments anti‐NTRK antitumor activity. This way, we could explain only the short‐lived patient's response to larotrectinib. In spite of finding an appropriate driver alteration, further mRNA‐based clinical studies should be conducted.

Glossary of Genomic Terms and Nomenclature

NTRK1: neurotrophic receptor tyrosine kinase 1RK

CCND1: cyclin D1

CCND2: cyclin D2

CCND3: cyclin D3

EGFR: epidermal growth factor receptor

PIK3CA: phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha

CDK6: cyclin dependent kinase 6

HGF: hepatocyte growth factor

SOX2: SRY‐box transcription factor 2

CDKN2A/B: cyclin dependent kinase inhibitor 2A/B

EPHB4: ePH receptor B4

FGF19: fibroblast growth factor 19

FGF3: fibroblast growth factor 3

FGF4: fibroblast growth factor 4

MCL1: MCL1 apoptosis regulator

PIM1: Pim‐1 proto‐oncogene, serine/threonine Kinase

TP53: tumor protein P53

VEGFA: vascular endothelial growth factor A

Author Contributions

Conception/design: Dirk Hempel, Thomas Wieland, Beate Solfrank, Vera Grossmann, Johanna Steinhard, Andrea Frick, Louisa Hempel, Thomas Eberl, Andreas Gaumann

Provision of study material or patients: Dirk Hempel, Thomas Wieland, Beate Solfrank, Vera Grossmann, Johanna Steinhard, Andrea Frick, Louisa Hempel, Thomas Eberl, Andreas Gaumann

Manuscript writing: Dirk Hempel, Thomas Wieland, Beate Solfrank, Vera Grossmann, Johanna Steinhard, Andrea Frick, Louisa Hempel, Thomas Eberl, Andreas Gaumann

Final approval of manuscript: Dirk Hempel, Thomas Wieland, Beate Solfrank, Vera Grossmann, Johanna Steinhard, Andrea Frick, Louisa Hempel, Thomas Eberl, Andreas Gaumann

Disclosures

Vera Grossmann: Foundation Medicine Germany GmbH (E). The other authors indicated no financial relationships.

(C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (ET) Expert testimony; (H) Honoraria received; (OI) Ownership interests; (IP) Intellectual property rights/inventor/patent holder; (SAB) Scientific advisory board