Unusual Resistance Mechanisms in a Case of ROS 1-Rearranged NSCLC: A Case Report.

The unprecedented growth of the high-throughput next-generation sequencing has facilitated the identification of rare oncogene fusions such as ROS1 for NSCLC. ROS1 rearrangement has been observed in only 2% of cases of NSCLC and has been successfully targeted using various tyrosine kinase inhibitors including crizotinib. However, the on-target and off-target mechanisms of the resistance are still vague. Here, we report a case of a patient with ROS1 rearranged NSCLC presenting primary resistance to crizotinib.

Introduction

ROS1-rearranged NSCLC is rare, yet actionable, and is treated with tyrosine kinase inhibitors (TKIs). However, similar to other TKIs, resistance often ensues—the mechanisms of which are still unclear with both on-target and off-target mechanisms reported (Table 1). This case depicts unusual primary resistance mechanisms to crizotinib in a patient with ROS1-rearranged NSCLC.

Table 1

Various Resistance Mechanisms (On-Target and Off-Target Mechanisms) in ROS1-Rearranged NSCLC

On-Target Resistance Mechanisms	Off-Target Mechanisms
G2032R	EGFR
D2033N	HER2
L2026M	KIT
S1986F/Y	KRAS
	BRAF
	MEK
	MET

Case Presentation

A 42-year-old man, ex-smoker, presented with cough and neck swelling and was diagnosed with ROS1-rearranged (detected by fluorescence in situ hybridization) stage IV NSCLC adenocarcinoma with metastases to lymph nodes and bone. Magnetic resonance imaging of the brain was normal. He was initially treated with crizotinib 250 mg twice daily. The patient tolerated the treatment well without any treatment delays. Positron emission tomography–computed tomography re-evaluation revealed progressive disease in bones and lymph nodes after 3 months. Repeat biopsy from the new metastatic node was subjected to next-generation sequencing (Supplementary Data), which revealed SDC4-ROS1 fusion along with new subclonal variants in RET (p.C634Y) and HRAS (p.G12S). The same subclones were tracked in two-fortnightly–spaced blood samples using liquid biopsy and revealed increased allele frequency of the RET subclone, with persistent ROS1 fusion and HRAS alteration. The patient was treated with lorlatinib 100 mg per day, to which he exhibited partial response in lung and lymph nodes. He is currently on lorlatinib for 6 months with grade 2 hypercholesterolemia. To determine the primary versus secondary mode of resistance, next-generation sequencing was done on diagnostic tumor block, which revealed the same RET subclone, although HRAS was not detected.

The evolution of clones is depicted in Figure 1.

Figure 1

Graph illustrating evolution of the RET and HRAS clones along with their respective variant allele frequencies. Liquid 1 and liquid 2 were done fortnightly after disease progression on crizotinib. NGS, next-generation sequencing; VAF, variant allele frequency.

Discussion

ROS1-rearranged NSCLC is known to respond to TKI therapy including crizotinib, as reported in PROFILE 1001 (disease control rate: ∼90% and progression-free survival: ∼19.2 mo). However, in our case, there was rapid progression and resistance ensued. On-target kinase domain mutations G2032R and D2033N, and off-target activation of the bypass EGFR, KIT signaling are well-known mechanisms of resistance to crizotinib.

Activation of the RAS pathway (KRAS or NRAS mutations) has been described in vitro as both primary and secondary ROS1 resistance mechanisms; however, the same has not been studied for HRAS. HRAS is more frequently found in NSCLC cases with squamous morphology, and in anecdotal case reports of adenocarcinoma portending an aggressive disease course, distinct from this case.

In this patient, a subclonal variant in the intracellular domain of RET gene (p.C634Y) was detected, which has been proven oncogenic in medullary thyroid cancers as germline and also in a few sporadic cases. In a study, one patient with ROS1-rearranged NSCLC was detected to have a copy number gain in RET posttherapy; however, the same was not seen in our case. The possibility of contamination from germline DNA was ruled out orthogonally from DNA extracted from the cell pellet of the blood sample. The same has not been reported in cases of NSCLC as ROS1 fusions are mutually exclusive with RET alterations.

A thorough literature search for RET as a resistance mechanism to crizotinib revealed no reports, hence, this requires functional and in vitro studies for the lucid understanding of the biological processes.

Conclusion

The patient exhibits a primary resistance to crizotinib with unusual mechanisms not reported in the literature. Further elucidation for both RET and HRAS is needed to better understand the biology to institute appropriate therapy.

CRediT Authorship Contribution Statement

Ullas Batra: Conceptualization, Methodology, Software, Writing - reviewing and editing of the article.

Shrinidhi Nathany, Mansi Sharma: Data curation, Writing and preparation of the original draft, Software, Data validation.

Sakshi Mattoo, Joslia T. Jose: Laboratory techniques.

Anurag Mehta: Supervision.