Different effects of crizotinib treatment in two non‐small cell lung cancer patients with SDC4 :: ROS1 fusion variants

Abstract

The possibility of stratifying patients according to differences in ROS proto‐oncogene 1 (ROS1) fusion partners has been discussed. This study aimed to clarify the clinicopathological differences between two SDC4::ROS1 positive NSCLC cases who had different responses to crizotinib. Cytology and pathology samples from two NSCLC cases with SDC4::ROS1 who were diagnosed and treated with crizotinib at Nihon University Itabashi Hospital were obtained. Case 1 has been well‐controlled with crizotinib for over 5 years, but case 2 was worse and overall survival was 19 months. Sequencing analysis of ROS1 fusion genes was performed by reverse‐transcription‐PCR and Sanger's sequencing methods. In addition, thyroid transcription factor (TTF)‐1, ROS‐1, Ki67, and phosphorylated extracellular signal‐regulated kinase (pERK)1/2 expression were investigated using immunohistochemistry. Sequencing analysis showed SDC4 exon2::ROS1 exon 32 (exon33 deleted) in case 1, and coexistence of SDC4 exon2::ROS1 exon 34 and SDC4 exon2::ROS1 exon35 in case 2. The Ki67 index was not different, but ROS1 and pERK1/2 expression levels tended to be higher in the tumor cells of case 2 than in case 1. Therapeutic response to crizotinib and patients' prognosis in ROS1 rearranged NSCLC may be related to the activation of ROS1 signaling, depending on ROS1 and pERK1/2 overexpression status, even if the ROS1 fusion partner is the same.

Patient prognosis in ROS1 rearranged NSCLC may be related to the activation of ROS1 signaling, depending on ROS1 and pERK1/2 overexpression status, even if the ROS1 fusion partner is the same.

INTRODUCTION

In non‐small cell lung cancer (NSCLC), Ros proto‐oncogene 1 (ROS1) is known to be a minor driver gene (1%–2% of NSCLCs) ¹ that encodes receptor tyrosine kinase (RTK). ROS1‐rearrangement with chromosome translocation drives tumorigenesis via the constitutively activated ROS1 kinase domain and cell transformation. ² ROS1‐rearranged NSCLC is typically diagnosed in younger, females, and nonsmokers, ¹ , ³ , ⁴ and treated with crizotinib. ¹ , ² ROS1 fusion genes contain many partner genes, which are mainly CD74 antigen (CD74, 42%–47%), ezrin (EZR, 15%–17%), SDC4 (7%–14%), and solute carrier family 34, member 1 (SLC34A2, 9–12%). ⁵ ROS1 rearranged NSCLC patients with CD74::ROS1 have been shown to have a worse response to crizotinib treatment than non‐CD74::ROS1 patients. ⁶ Therefore, it is necessary to investigate the usefulness of patient stratification by ROS1 fusion partner genes. ⁶ We experienced two NSCLC cases with SDC4::ROS1, a rare fusion variant, who had different crizotinib treatment effects. This study aimed to investigate the clinicopathological differences between two SDC4::ROS1 positive NSCLC cases who had responded to crizotinib and those who had not.

METHODS

Patients

From 2009 to 2019, three ROS1 rearranged NSCLC patients out of 153 NSCLC patients who were diagnosed at Nihon University Itabashi Hospital were detected (1.96%) by OncoGuide AmoyDx ROS1 RT‐PCR Kit (Amoy Diagnostics Co., Ltd) and reverse transcription‐polymerase chain reaction (RT‐PCR) methods. ³ Two patients with SDC4::ROS1 were identified, with different crizotinib treatment responses. The summary of the two patients is shown in Table 1. Formalin‐fixed and paraffin‐embedded (FFPE) transbronchoscopic lung biopsy specimens and cytology samples after diagnosis were obtained for immunohistochemistry (IHC) and genetic analysis. This study was performed following the ethical standards of the institutional and national research committees and the Declaration of Helsinki. This study was approved by the institutional review board of Nihon University Itabashi Hospital (RK‐170425‐01) and written informed consents were waived from IRB approval.

TABLE 1.

Clinicopathological features of two NSCLC cases with SDC4::ROS1.

Case	Age at diagnosis	Sex	Smoking	Complain	Location	TNM	Clinical stage	Treatment	Effects	Metastasis	OS
1	50s	Female	Never	Cough and sputum	Left lingual region	cT3N3M0	cIIIB	Crizotinib (250 mg, 2 times/day)	PR	–	>5 years
2	60s	Male	Smoker	Cough and cervical lymphadenopathy	Cervical and mediastinal lymph node	cTXN3M1c	cIVB	Crizotinib (250 mg, 2 times/day)	PD	Skin and abdominal cavity	19 months

Abbreviations: NSCLC, non‐small cell lung cancer; OS, overall survival; PD, progressive disease; PR, partial response; TNM, factors of tumor, lymph node, and metastasis.

Immunohistochemistry

Immunohistochemistry was performed using a mouse monoclonal anti‐TTF‐1 antibody (clone 8G7G3/1, Agilent Technologies, Inc.), a rabbit monoclonal anti‐ROS1 antibody (clone SP384, Roche Diagnostics K.K.), a mouse monoclonal anti‐Ki67 antibody (clone MIB‐1, Agilent Technologies) and a rabbit monoclonal antiphosphorylated extracellular signal‐regulated kinase (pERK)1/2 antibody (Thr202/Tyr204, D13.14.4E XP; Cell Signaling Technology Inc.). As antigen retrieval, samples were boiled in citrate buffer (pH 6.0) for Ki67 and pERK1/2, and in target retrieval solution (TRS, pH 9.0; Agilent Technologies) for TTF‐1 and ROS1 at 97°C for 30 min. After cooling, cellular peroxidase was blocked with the 3% hydrogen peroxide solution for 10 min at RT. Samples were washed with phosphate‐buffered saline (PBS), and then the slides were incubated with the primary antibody for 30 min at RT. After washing with PBS, samples were reacted with Simple Stain MAX‐PO (Multi) (Nichirei Bioscience Inc.) for 30 min at RT. After washing, samples were visualized with 3,3‐diaminobenzidine tetrahydrochloride (for 10 min at RT) and counterstained with hematoxylin.

RT‐PCR and sequencing analysis

Total RNA was extracted from FFPE biopsy sections using an RNeasy FFPE kit (Qiagen GmbH), and from pleural effusion using acid guanidine thiocyanate‐phenol‐chloroform methods. Both genomic DNA elimination and cDNA synthesis were performed using a QuantiTect reverse transcription kit (Qiagen).

Reverse transcription‐PCR was performed using the AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific, Inc.) and the respective primers specific for ROS1 fusion genes (CD74::ROS1, SLC34A2::ROS1, TPM3::ROS1, SDC4::ROS1, EZR::ROS1, LRIG3::ROS1, and FIG::ROS1). ³ These amplicons were analyzed by Sanger's sequencing that was performed using the ABI PRISM BigDye Terminator version 3.1 cycle sequencing kit (Thermo Fisher Scientific Inc.) and ABI 3730xl DNA Analyzer (Thermo Fisher Scientific Inc.). The complete methods are shown in Supplementary Materials.

RESULTS

Cytological and histopathological findings

A cluster of cells with strong nuclear atypia with binding properties was observed in cytology samples from the left bronchoalveolar lavage fluid in case 1 (Figure 1a), and pericardial effusion in case 2 (Figure 1b). In hematoxylin and eosin (H&E) stained biopsy tissues (Figure 1c–e), tumor cells had a solid growth pattern, distinct nucleoli, and round‐like nuclei. Positive TTF‐1 expression suggested that these tumors were NSCLC favor adenocarcinoma (Figure 1f–h).

FIGURE 1.

Cytological and histological findings. Papanicolaou stained cytology samples from the left bronchoalveolar lavage fluid in case 1 (a), and pericardial effusion in case 2 (b) show a cluster of cells with strong nuclear atypia with binding properties. Hematoxylin and eosin (H&E) stained biopsy tissues in case 1 (c), in case 2 before treatment (d), and in case 2 after treatment (e) show tumor cells that have a solid growth pattern, distinct nucleoli, and round‐like nuclei. Immunohistochemical TTF‐1 positive expression of tumor cells in case 1 (f), in case 2 before treatment (g), and in case 2 after treatment (h) are shown. Bar 50 μm.

ROS1 fusion and protein expression

Sequencing analysis using amplicons of RT‐PCR (Figure 2a) for ROS1 fusion detection revealed SDC4 exon2::ROS1 exon 32 (but exon33 completely deleted) in case 1, and coexistence of SDC4 exon2::ROS1 exon34 and the SDC4 exon2::ROS1 exon35 in case 2 (Figure 2b, and long versions are shown in Supporting Information). Immunohistochemical ROS1 expression was diffusely positive in the nucleus of tumor cells, and intensity was stronger in case 2 than in case 1 (Figure 2c).

FIGURE 2.

Sequences of ROS1 fusion gene and ROS1 protein expression. (a) Agarose gel electrophoresis of PCR products show the bands of SDC4::ROS1 fusion genes. (b) Sequencing analysis using DNA samples extracted from each band revealed SDC4 exon2::ROS1 exon 32 (but exon33 completely deleted) in case 1, and coexistence of SDC4 exon2::ROS1 exon34 and the SDC4 exon2::ROS1 exon35 in case2. (c) Immunohistochemical ROS1 expression was positive in case 1, and stronger in case 2 before and after treatment. Bar 50 μm.

Amplification and signal transduction

Index of an amplification marker Ki67 was significantly higher in case 1 (82.3 ± 8.15%) than in case 2 (55.3 ± 2.46%, p = 0.011), as shown on the left side of Figure 3. On the other hand, nucleic expression of pERK1/2 was significantly higher in case 2 (69.3 ± 4.93%) than in case 1 (45.6 ± 14.4%, p = 0.045) as shown on the right side of Figure 3.

FIGURE 3.

Ki67 index and immunohistochemical pERK1/2 intensity. Immunohistochemical Ki67 expression in the tumor nucleus and Ki67 index are shown on the left side of the figure. Immunohistochemical pERK1/2 expression in the tumor nucleus and the positive ratio of pERK1/2 are shown on the right side of the figure. Bar 50 μm.

DISCUSSION

The possibility of stratifying patients according to differences in ROS1 fusion partners has been discussed. However, the two NSCLC patients with SDC4::ROS1 reported in the present study showed quite different responses to crizotinib and clinical outcomes. Although their ROS1 partner genes were the same SDC4 exon2, ROS1 gene status and ROS1 IHC expression level differed. In case 1, the response to crizotinib has been good and tumor progressions have not been detected in over 5 years. While, in case 2, the coexistence of two types of SDC4::ROS1, and the crizotinib response and patient's prognosis were worse, despite no acquired resistance mutations like ROS1 G2032R. ⁷ Combined ROS1 fusion variants have often been reported, but their effects are unclear. ⁸ Our results showed that the case with combined SDC4::ROS1 fusion had higher ROS1 and pERK1/2 expression and worse prognosis. Sato et al. have reported that ROS1 fusions may accelerate cell transformation through ERK/mitogen‐activated protein kinase (MAPK) pathway activation and worsen patient prognosis. ⁹ pERK1/2 overexpression may involve SDC4::ROS1 in the dynamics of MAPK pathway factors, like BRAF and MEK. ¹⁰ In our previous study, higher pERK1/2 expression was related to chemotherapeutic resistance and worse prognosis in patients with advanced NSCLC. ¹¹ However, the development of ERK1/2 inhibitors is relatively limited, although some clinical trials are underway. ¹² , ¹³ , ¹⁴ , ¹⁵ Our results may suggest the need for further basic and clinical study on ERK as a cause of unsuccessful treatment and therapeutic target, in addition to studying the effects of entrectinib.

AUTHOR CONTRIBUTIONS

All authors contributed to the study's conception and design. Study designs; Yuta Ohishi and Yoko Nakanishi Analysis; Yukari Hirotani, Yuta Ohishi, and Yoko Nakanishi Cytological analysis; Atsuko Suzuki Histological analysis; Tomoyuki Tanino, Haruna Nishimaki‐Watanabe, Hiroko Kobayashi, Fumi Nozaki, Sumie Ohni, and Xiaoyan Tang Clinical data collection and analysis; Kentaro Hayashi, Yoshiko Nakagawa, Tetsuo Shimizu, Ichiro Tsujino, and Noriaki Takahashi Draft writing; Yuta Ohishi and Yoko Nakanishi Revised manuscript; Shinobu Masuda and Yasuhiro Gon All authors read and approved the final manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflicts of interest to report regarding the present study.

PATIENT CONSENT STATEMENT

The requirement for patient consent was waived by IRB because this was a retrospective observational study.

Supporting information

Figure S1. Total sequencing results of SDC4 exon2‐ROS1 exon 32 in case 1.

Figure S2. Total sequencing results of SDC4 exon2‐ROS1 exon 34 in case 2.

Figure S3. Total sequencing results of SDC4 exon2‐ROS1 exon 35 in case 2.

Ohishi Y, Nakanishi Y, Hirotani Y, Suzuki A, Tanino T, Nishimaki‐Watanabe H, et al. Different effects of crizotinib treatment in two non‐small cell lung cancer patients with SDC4 :: ROS1 fusion variants. Thorac Cancer. 2024;15(1):89–93. 10.1111/1759-7714.15168

DATA AVAILABILITY STATEMENT

The authors confirm that the data supporting the findings of this study are available within the article.