BRAF V600E Mediates Crizotinib Resistance and Responds to Dabrafenib and Trametinib in a ROS1-Rearranged Non-Small Cell Lung Cancer: A Case Report.

Crizotinib, a multitargeted MET/ALK/ROS1 tyrosine kinase inhibitor, has been approved for the treatment of ROS1 fusion-positive non-small cell lung cancers (NSCLCs). However, "on-target" or "off-target" resistance alterations often emerge that confer the drug resistance. Patients with ROS1-rearranged NSCLC who develop crizotinib resistance, especially those acquiring "off-target" resistance mutations, still lack effective therapeutic options for after crizotinib treatment. Herein, we reported a patient with stage IVb lung adenocarcinoma harboring ROS1 fusion, who acquired a BRAF V600E and lost the ROS1 fusion after progression on crizotinib. It was deduced that the V600E may originate from a subclone with an extremely low fraction that was independent of ROS1 fusion-positive cells. The patient was subsequently treated with dabrafenib and trametinib combination and achieved a partial response lasting for more than 6 months. Our study revealed that BRAF V600E can confer the crizotinib resistance in ROS1 fusion-positive NSCLC and presented the first case showing that the treatment with dabrafenib and trametinib can serve as an effective option for later-line treatment for this molecular-defined subgroup. KEY POINTS: Patients with ROS1-rearranged non-small cell lung cancer (NSCLC) who acquire "off-target" resistance mutations to crizotinib still lack effective therapeutic options for after crizotinib treatment. This report describes the case of a patient with ROS1-rearranged NSCLC who acquired a BRAF V600E and lost the ROS1 fusion after crizotinib failure. The case was subsequently treated with dabrafenib and trametinib combination and achieved a partial response lasting for more than 6 months. This is the first article reporting that treatment with dabrafenib and trametinib may serve as an effective option for later-line treatment for patients harboring resistant BRAF V600E.

Introduction

ROS1 encodes a protooncogene receptor tyrosine kinase. Rearrangements of ROS1 gene occur in 1%–2% of non‐small cell lung cancers (NSCLCs) [1], characterizing a distinct molecular subgroup. The retained ROS1 kinase domain fused with a partner gene often confers constitutive activation of the tyrosine kinase domain, and therefore drives oncogenesis [2]. Crizotinib, a multitargeted MET/ALK/ROS1 tyrosine kinase inhibitor (TKI), has demonstrated a promising objective response rate of 72%, median progression‐free survival (PFS) of 19.3 months, and overall survival of 51.4 months in patients with advanced ROS1 fusion–positive NSCLC [3] and now has been approved for the treatment of this molecularly‐defined subgroup of NSCLCs.

Despite remarkable antitumor activity and survival advantage seen with crizotinib in patients with ROS1 fusion–positive NSCLC, drug resistance often arises. The acquisition of “on‐target” resistance mutations in ROS1 has been found as the significant cause of resistance to crizotinib [4, 5]. A novel generation of TKIs against these ROS1 mutations has been developed to overcome crizotinib resistance in ROS1 fusion–positive NSCLCs and is currently under clinical investigation, including brigatinib, cabozantinib, ceritinib, etc. On the other hand, resistance mechanisms by activating bypass signaling pathways are less commonly reported [5, 6]. Patients who develop crizotinib resistance by acquiring these “off‐target” resistance mutations still lack efficient therapeutic options for later‐line treatment. Herein, we report a patient with stage IVb lung adenocarcinoma harboring ROS1 fusion, who acquired BRAF p.V600E after progression on crizotinib. She was subsequently treated with dabrafenib and trametinib combination and achieved a durable partial response.

Case Presentation

The patient's treatment history and clinical course are summarized in Figure 1. In January 2019, a female patient, aged 44 years, was referred to our hospital and presented with coughing for 1 month as well as headache and dizziness for 2 weeks. She had no history of smoking or drinking. Her father had died from esophageal cancer. The patient had an Eastern Cooperative Oncology Group performance status (ECOG PS) score of 2. An enhanced chest computed tomography (CT) scan revealed a soft tissue mass measuring 4.6 cm × 2.9 cm on the right lower lobe (Fig. 2A), accompanied by mediastinal and right lung hilum lymphadenopathy. Brain magnetic resonance imaging showed multiple enhancing nodules and masses in bilateral cerebral hemispheres and the right cerebellar hemisphere, with the largest lesion measuring 2.9×2.6 cm (Fig. 2B). The abdomen enhanced CT and bone scanning detected no evidence of metastasis. The histopathological test of the biopsied pulmonary lesion indicated adenocarcinoma. The patient was diagnosed with a stage IVb (cT2bN2M1c) disease. Real‐time polymerase chain reaction (RT‐PCR) performed with the biopsy sample identified a ROS1 fusion; no EML4‐ALK fusion was detected. Amplification refractory mutation system polymerase chain reaction (ARMS PCR) was also performed but did not detect any mutations in EGFR exons 18–21, or BRAF V600.

Figure 1

The timeline and treatment history of the patient. Abbreviations: NGS, next‐generation sequencing; OR, objective response; PFS, progressive‐free survival; PR, partial response; RT‐PCR, real‐time polymerase chain reaction; SD, stable disease.

Figure 2

Responses to crizotinib treatment. (A): Lung lesion before treatment. (B): Brain metastatic lesions before treatment. (C): Lung lesion 5 months after treatment. (D): Brian lesions 5 months after treatment.

Subsequently, the patient was treated with crizotinib (250 mg b.i.d.). Local radiotherapy was performed for brain metastatic lesions (GTV‐T 50 Gy/10f), primary pulmonary lesions (GTV‐T 60 Gy/20f), and regional lymph nodes (GTV‐N 48 Gy/24f), respectively. The patient subsequently had symptoms alleviated. In May 2019, imaging tests revealed shrunken lung (Fig. 2C) and brain lesions (Fig. 2D) indicating a partial response (PR). The disease remained stable until January 2020, when the patient started to present with pain in her back. Imaging tests showed progression disease on brain lesions, stable disease (SD) on lung lesions, and the presence of a new metastatic lesion on the lumbar vertebra. Local radiotherapy was subsequently administrated for brain and lumbar vertebra lesions. The patient initiated a combinatorial regimen of crizotinib (250 mg b.i.d.) and bevacizumab (400 mg every 3 weeks) in February 2020 and achieved SD. Next‐generation sequencing (NGS) was performed with the plasma sample collected in March 2020 using a 168‐gene panel (Burning Rock Biotech, Guangzhou, China) but did not detect any mutation. After a PFS of 4 months, the patient's coughing and dyspnea symptoms were aggravated and the ECOG PS score increased to 3. Imaging tests revealed a mass (3.2 cm in diameter) on the right lower lobe and multiple nodules in both lungs (Fig. 3A) and bilateral pleural effusion and new lesions in bilateral frontal lobes (Fig.e 3B). The histopathological test of pleural effusion identified malignant cells. NGS with plasma and pleural effusion samples both identified BRAF p.V600E (allele frequency (AF), 0.06% in plasma; 2.85% in pleural effusion), BRIP1 p.S1162* (AF, 1.53% in plasma; 5.68% in pleural effusion), and TP53 p.D281E (AF, 1.17% in plasma; 6.93% in pleural effusion). ROS1 fusion was not detected.

Figure 3

Responses to dabrafenib and trametinib combinatorial treatment. (A): Lung lesions before treatment. (B): Brain metastatic lesions before treatment. (C): Lung lesions 4 months after treatment. (D): Brian lesions 4 months after treatment.

In June 2020, the patient started a combinatorial treatment of dabrafenib (75 mg b.i.d.) and trametinib (2 mg q.i.d.) and achieved PR 4 months later based on the shrinkage of lung nodules (Fig. 3C) and frontal lobe lesions (Fig. 3D). The coughing and dyspnea symptoms were largely relieved, and the ECOG PS improved to 1. NGS was performed with the patient's plasma sample collected in December 2020, when she remained as PR, and the results revealed the disappearance of BRAF p.V600E, retaining of TP53 p.D281E (AF, 48.72%), and the emergence of NF1 p.H1672fs (AF, 0.36%). The disease remained stable as of submission of the manuscript. The patient achieved a PFS of 6 months and counting.

Discussion

Several point mutations in the ROS1 gene have been identified in crizotinib‐resistant ROS1‐rearranged NSCLCs [4], with ROS1‐G2032R being the most common resistance mutation [7]. Besides, activation of bypass signaling pathways has also been linked to crizotinib resistance. It has been shown that epidermal growth factor receptor (EGFR) pathway activation can mediate crizotinib resistance by increasing the dependence on EGFR activity and reducing the dependence on ROS1 activity [8, 9]. Similarly, an activating mutation (p.D816G) in the KIT Proto‐Oncogene, Receptor Tyrosine Kinase (KIT) signaling pathway also serves as a ROS1‐independent mechanism of resistance [10]. Cargnelutti et al. revealed that activation of the RAS pathway due to KRAS Proto‐Oncogene (KRAS) amplification conferred the resistance to ROS1 inhibitors including crizotinib [11].

In the present study, we identified the acquisition of BRAF p.V600E after crizotinib progression in a ROS1 fusion–positive NSCLC. BRAF encodes a key molecule in the MAP/ERK kinase signaling pathway, located downstream of ROS1. BRAF p.V600E has been known as an oncogenic driver observed in 1%–2% of NSCLCs [12]. Of note, in our case, ROS1 fusion was neither detected in the plasma nor the pleural effusion after crizotinib resistance, whereas BRAF p.V600E was detected at a lower frequency of 0.06% in plasma and 2.85% in pleural effusion, compared with that of other mutations (>1% in plasma and >5% in pleural effusion). The results suggested that the BRAF p.V600E was more likely to originate from a subclone with an extremely low fraction that was independent of ROS1 fusion–positive tumor cells. During the crizotinib treatment, the growth and proliferation of the cells with ROS1‐fusion were inhibited, and cells with BRAF p.V600E were selected and evolved into the major resistance clone. Loss of EML4‐ALK fusion has been reported in ALK‐positive NSCLCs after crizotinib failure [13, 14]. Similarly, in vitro study and primary clinical evidence suggest that loss of activating EGFR mutation contributes to acquired resistance to EGFR TKI in lung cancer [15]. Therefore, the loss of activating alteration ROS1‐fusion may also confer crizotinib resistance in our case. However, considering the lack of a rebiopsy after crizotinib resistance for confirmatory tissue testing and technical limitations of DNA sequencing on detecting complex genomic rearrangements, the possibility of false‐negative results for ROS1 fusion detection cannot be excluded.

The combined therapy with the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib has demonstrated promising efficacy and manageable side effects in BRAF V600E–mutant NSCLC [12] and has recently been approved for clinical use. After the identification of BRAF p.V600E, our case was switched to the treatment with dabrafenib plus trametinib and showed a durable response along with the disappearance of BRAF V600E in plasma. To date, only two case reports have described the appearance of BRAF V600E as a mechanism mediating crizotinib resistance in ROS1‐rearranged NSCLC [16, 17]. However, one patient switched to the dabrafenib single agent but passed away 15 days later, and the other died 11 days after the initiation of dabrafenib and trametinib therapy. Different from our case, these two patients retained detectable ROS1 fusion after crizotinib resistance. Thus, it might be thought that combining a ROS inhibitor might be beneficial in such cases. Similarly, Meng et al. reported two EGFR‐mutated cases acquiring BRAF V600E after osimertinib resistance, who received dabrafenib and trametinib combined with osimertinib. One of them achieved a durable response and the other progressed quickly [18]. Notably, our study reported the first case of crizotinib‐induced BRAF V600E responding to the treatment with dabrafenib and trametinib in ROS1‐rearranged NSCLC.

In conclusion, our study revealed that BRAF V600E can confer the crizotinib resistance in ROS1 fusion–positive NSCLC and presented the first case suggesting that the treatment with dabrafenib and trametinib can serve as an effective option for later‐line treatment for this molecular‐defined subgroup.

Glossary of Genomic Terms and Nomenclature

NSCLC: non‐small cell lung cancers

TKI: tyrosine kinase inhibitor

PFS: progression‐free survival

NGS: next‐generation sequencing

OR: objective response

PR: partial response

SD: stable disease

RT‐PCR: real‐time polymerase chain reaction

ECOG PS: Eastern Cooperative Oncology Group performance status

CT: computed tomography

ARMS PCR: amplification refractory mutation system polymerase chain reaction

ROS1: ROS proto‐oncogene 1, receptor tyrosine kinase

KIT: KIT proto‐oncogene, receptor tyrosine kinase

KRAS: KRAS proto‐oncogene

Author Contributions

Conception/design: Juan Li, Wenxiu Yao

Collection and/or assembly of data: Qifeng Wang, Jun Ge

Data analysis and interpretation: Yuke Tian

Manuscript writing: Juan Li

Final approval of manuscript: Juan Li, Qifeng Wang, Jun Ge, Yuke Tian, Wenxiu Yao

Disclosures

The authors indicated no financial relationships.