Detection of Novel NRG1, EGFR, and MET Fusions in Lung Adenocarcinomas in the Chinese Population

BRIEF REPORT

Detection of Novel NRG1, EGFR, and MET Fusions in Lung Adenocarcinomas in the Chinese Population Yunjian Pan, MD, PhD,a,c Yang Zhang, MD,a,c Ting Ye, MD, PhD,a,c Yue Zhao, MD,a,c Zhendong Gao, MD,a,c Hui Yuan, PhD,a,c Difan Zheng, MD,a,c Shanbo Zheng, MD,a,c Hang Li, MD,a,c Yuan Li, MD, PhD,b,c Yan Jin, MD, PhD,b,c Yihua Sun, MD, PhD,a,c Haiquan Chen, MD, PhDa,c,d,e,* a

Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, Shanghai, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China c Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, China d State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, 200433, China e Institute of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, 200032, China b

Received 2 June 2019; revised 17 July 2019; accepted 19 July 2019 Available online - 2 August 2019

ABSTRACT Introduction: Multiple oncogene fusions beyond ALK receptor tyrosine kinase (ALK), RET, and ROS1 fusion has been described in lung cancer, especially in lung adenocarcinomas without common oncogenic mutations. Molecular inhibitors have been developed and proved effective for patients whose tumors harbor these novel alterations. Methods: A consecutive series of surgically resected lung adenocarcinomas were collected and profiled using an enrichment strategy to detect nine common oncogenic driver mutations and fusions concerning EGFR, KRAS, HER2, BRAF, MET, ALK, RET, ROS1, and FGFR. Driver-negative cases were further analyzed by a comprehensive RNA-based nextgeneration sequencing (NGS) fusion assay for novel fusions. Results: In total, we profiled 1681 lung adenocarcinomas, among which 255 cases were common driver–negative. One hundred seventy-seven cases had sufficient tissue for NGS fusions screening, which identified eight novel fusions. NRG1 fusions occurred in 0.36% of all lung adenocarcinoma cases (6 of 1681 cases), including 4 CD74-NRG1–positive cases, 1 RBPMS-NRG1–positive case, and 1 novel ITGB1NRG1–positive case. Furthermore, another 2 novel fusions were also detected, including 1 EGFR-SHC1 fusion and 1 CD47-MET fusion, both of which were in-frame and retained the functional domain of the corresponding kinases. No fusion event was detected for NTRK, KRAS, BRAF or HER2 genes in this cohort. Detailed clinicopathologic data showed that invasive mucous adenocarcinoma (three of eight cases) and acinar-predominant adenocarcinoma (three of eight cases) were the most prevalent pathologic subtypes among novel fusions.

Conclusions: Fusions affecting NRG1, EGFR, and MET were detected in 0.48% of unselected lung adenocarcinomas, and NRG1 fusions ranked the most prevalent fusions in common driver-negative lung adenocarcinomas from Chinese population. RNA-based NGS fusion assay was an optional method for screening actionable fusions in common driver-negative cases.
2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved. Keywords: NRG1 fusion; EGFR fusion; MET fusion

Introduction Lung cancer remains the cancer type with the highest mortality rates worldwide, with lung adenocarcinoma being the main histopathologic subtype.1,2 The treatment of lung adenocarcinoma has dramatically changed in the past decade due to the identification of an increasing number of actionable oncogenic abnormalities. By now,

*Corresponding author. Dr. Pan, Zhang, and Ye contributed equally to this work. Disclosure: The authors declare no conflict of interest. Address for correspondence: Haiquan Chen, MD, PhD, Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, 270 DongAn Road, Shanghai 200032, China. E-mail: [email protected] ª 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved. ISSN: 1556-0864 https://doi.org/10.1016/j.jtho.2019.07.022

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tyrosine kinase inhibitors (TKIs) have become one of the standard treatment options for patients with advanced NSCLC harboring EGFR mutations and ALK receptor tyrosine kinase (ALK) fusions.3-7 In addition, with the advances in diagnostic technologies, more and more additional oncogenic aberrations were identified and added to the list of targetable driver events, such as RET, ROS1 fusions and MET exon 14 skipping mutations, all of which could be treated with existing TKIs.8-10 Furthermore, recent genomic studies have revealed NTRK, NRG1, FGFR, EGFR, MET, BRAF, and ERBB2 fusions as novel actionable driver events in a small fraction of NSCLC cases.11-16 In particular, a molecular agent LOXO-101 (known as larotrectinib) was approved by the U.S. Food and Drug Administration (FDA) last year to treat patients with solid tumors harboring NTRK fusions, after showing a startling 75% overall response rate (ORR).17 Considering the high morbidity and mortality of lung cancer, therefore, the discovery of infrequent and yet pathologically relevant fusions can prove critical for the development of new therapies for lung cancer and even other cancer types harboring the same fusion events. Lung adenocarcinomas without common drivers accounted for 16.7% of lung adenocarcinoma cases in our previous study.18 Oncogenic drivers were predominantly found mutually exclusive and driver-negative lung adenocarcinomas were regard as an enriched collection for detecting novel drivers. In our center, to facilitate individualized treatment and molecular research, surgically resected lung adenocarcinoma samples were genotyped consecutively for common driver events, including EGFR, KRAS, HER2, BRAF, ALK, RET, ROS1, MET, and FGFR. Findings from our previous studies have drawn the most detailed mutation spectrum of lung adenocarcinomas from Chinese populations.18-21 Based on previous published data, this study was designed to characterize the distribution of novel fusions in a series of common driver-negative samples lung adenocarcinomas using a RNA-based next-generation sequencing (NGS) fusion assay (Archer FusionPlex Assays-Lung Panel) designed to detect 14 genes with known fusions in lung cancer.

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excluded. All cases were re-reviewed by two pathologists for confirmation of tumor subtype and tumor content (Drs. Li and Jin). Tumor and normal samples were snapfrozen in liquid nitrogen at the time of resection and stored in the tissue bank of FUSCC until use. This study was approved by the institutional review board of the Shanghai Cancer Center (IRB# 090977-1) and Human Genetics Resources Administration of China (Approval number: 2017-389). All patients underwent surgery and provided written informed consent. Clinicopathologic data were prospectively collected including age at diagnosis, sex, smoking status, TNM stage, pathologic subtype, and follow-up information.

DNA and RNA Preparation Surgically resected samples were collected and stored in liquid nitrogen until use as described in previous studies.8,19 Genomic DNA and RNA were extracted as per standard protocols (RNeasy Mini Kit, and QiAamp DNA Mini Kit, Qiagen, Hilden, Germany). Total RNA samples were reverse transcribed into single-stranded cDNA using RevertAid First Strand cDNA Synthesis Kit (Fermentas, St Leon-Rot, Germany).

Reverse Transcriptase and Sanger Sequencing We designed reverse transcriptase polymerase chain reaction (RT-PCR) primers to cover mutation hot-spot regions of common driver genes. EGFR (exons 18 to 22), HER2 (exons 18 to 21), KRAS (exons 2 to 3), BRAF (exons 11 to 15), and MET (exons 13 to 15) genes were PCR amplified using cDNA. Sanger sequencing was used to analyze the amplified products. In previous studies, we have developed quantitative real-time reverse transcriptase PCR (qRT-PCR)–based fusion detection methods for ALK, ROS1, and RET translocations. Multiple RT-PCR and fluorescence in situ hybridization techniques were also performed to validate the positive results from the qRT-PCR procedure. We also designed RTPCR primers to cover known fusion partners of FGFR1/ 2/3 and NRG1.

FusionPlex Assay and Targeted NGS Analysis

Methods Patients and Sample Collection Frozen lung adenocarcinoma samples were collected from patients who underwent surgical resection at the Department of Thoracic Surgery, Fudan University Shanghai Cancer Center (FUSCC), Shanghai, China, from October 2007 to January 2015. Eligible patients were required to have a minimum of 50% of tumor cells and sufficient tissue for comprehensive mutational analyses. Patients who received neoadjuvant chemotherapy were

For novel fusion detection affecting 14 genes (EGFR, KRAS, BRAF, MET, ALK, RET, ROS1, NRG1, FGFR1/2/3, and NTRK1/2/3), anchored multiplex PCR was performed, followed by targeted NGS using the ArcherDx FusionPlex Kit. In all, 250 ng of RNA was used as input for NGS library construction. Library preparation and RNA quality check were performed following the Archer FusionPlex Protocol for Illumina (ArcherDX Inc., Boulder, Colorado). Libraries were sequenced on a Miseq sequencer (Illumina, San Diego, California). Analysis of sequencing data was performed using an in-house developed pipeline,

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consisting of four steps: (1) sequencing library structure filtering; (2) merging of paired-end reads into single-end reads; (3) splicing-aware alignment and prediction of fusion break-points; and (4) realignment for expression quantification (pipeline available upon request). Fusion candidates were reported with a minimum of five valid fusion reads and a minimum of three unique start sites, excluding paralogous gene pairs.

Results Common Driver Mutations and Fusion Detection in Lung Adenocarcinomas A total of 1681 lung adenocarcinoma cases were previously sequenced for nine common driver mutations and fusions including EGFR, KRAS, HER2, BRAF, MET, ALK, RET, ROS1, and FGFR. To improve the efficiency of novel fusion detection, we used an enrichment strategy to focus on a collection of common driver-negative tumors in this study (Fig. 1). EGFR and KRAS mutations were detected as the first two steps of the screening process, respectively, before a comprehensive detection for other drivers. There were 1076 EGFR kinase domain mutations, 132 KRAS mutations, 36 HER2 kinase domain mutations, 20 BRAF mutations, 22 MET exon 14 skipping mutations, 91 ALK fusions, 25 RET fusions, 18 ROS1 fusions, and 6 FGFR fusions (Fig. 1). Among ALK/RET/ ROS1 fusions, we reported one novel ALK fusion pattern, SPECC1L-ALK, for the first time (Supplementary Fig. 1). In this cohort, common driver-negative lung

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adenocarcinomas accounted for 15.2% (255 of 1681) of all lung adenocarcinomas from the Chinese population.

Clinicopathologic Characteristics of Patients With Common Driver-Negative Lung Adenocarcinomas After excluding nine common mutations and fusions, a total of 177 cases with sufficient tumor tissue were enrolled for RNA-based NGS fusion test after RNA quality check. A summary of patient demographics is listed in Table 1. The median age at diagnosis was 61.0 years (range, 30 to 80 years). In this cohort, patients tend to be male patients (126, 71.2%) and smokers (111, 62.7%). The numbers of patients in stages I to IV were 86 (48.6%), 27(15.3%), 62(35.0%), and 2 (1.1%) respectively. The most common histologic subtype was acinar predominant (44.6%), followed by solid predominant subtype (34.5%).

Novel Actionable Fusions in Common DriverNegative Cases In this procedure, 8 fusions (8 of 177 fusions, 4.5%) were detected, including 6 NRG1 fusions (including four CD74-NRG1, one ITGB1-NRG1 fusion, and one RBPMSNRG1 fusion), one EGFR fusion (EGFR-SHC1), and one MET fusion (CD47-MET) (Fig. 2). Of these new detected fusions, three fusions (ITGB1-NRG1, EGFR-SHC1, and CD47-MET) have not been reported previously. Pathologic analysis showed that invasive mucous adenocarcinoma (3 of 8) and acinar-predominant adenocarcinoma (3 of 8) were Table 1. Clinicopathologic Characteristics of 177 Lung Adenocarcinoma Patients Without Common Drivers Variables Age, years <60 60 Sex Male Female Smoking Current/former Nevera TNM s tage I II III IV Subtype Lepidic predominant Acinar predominant Papillary predominant Micropapillary predominant Solid predominant Invasive mucinous adenocarcinoma

Figure 1. The flow chart of enrichment strategy to screen driver-negative lung adenocarcinomas (LUAD).

a

Number

%

72 105

40.7 59.3

126 51

71.2 28.8

111 66

62.7 37.3

86 27 62 2

48.6 15.3 35.0 1.1

11 79 12 4 61 10

6.2 44.6 6.8 2.3 34.5 5.6

Never smokers are patients who smoked less than 100 cigarettes in their lifetime.

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Figure 2. Diagram of fusions detected in lung adenocarcinomas. A, EGFR-SHC1 fusion. B, ITGB1-NRG1 fusion. C, RBPMS-NRG1 fusion. D, CD47-MET fusion.

the most prevalent pathologic subtypes among novel fusions. Five patients with new detected fusions were never-smokers. No NTRK, KRAS, BRAF, HER2 fusions were found in our cohort. Detailed characteristics of patients with detected fusions are summarized in Table 2.

Discussion Recent advances in genome sequencing technologies have led to an increase in the discovery of novel and therapeutically targetable genomic alterations in NSCLC. In lung adenocarcinoma, assessing for canonical mutations or fusion affecting EGFR, ALK, ROS1 is now the accepted standard of care worldwide for patients harboring these specific targets. However, chemotherapy remains a mainstay of first-line treatment for patients with advanced or

metastatic lung adenocarcinoma having no actionable mutations. In this study, by using a comprehensive RNAbased NGS fusions assay, which has been proved efficient in identifying novel fusions, we identified recurrent NRG1 fusions, as well as EGFR and MET fusions in lung adenocarcinomas from patients who do not harbor any previously identified oncogenic alterations.22 NRG1 fusions have been described in multiple solid tumors, including NSCLC (0.3%).23 We also discovered recurrent NRG1 fusions that arise from a somatic genomic event, including one novel partnership involving ITGB1. To our knowledge, ITGB1, also known as CD29, has not been reported previously as a fusion partner of NRG1 as well as other receptor tyrosine kinases; thus, this is the first report of novel ITGB1-NRG1

Table 2. Individual Clinicopathologic Data of Patients Harboring Novel Fusions Sample ID 2154 2789 1671 3346 2580 2685 2841 3685 a

Sex Male Female Female Male Male Female Female Male

Age, Years 61 50 37 63 77 69 62 70

Smoking Status Current/former Nevera Never Current/former Never Never Never Current/former

Fusion Type b

EGFR-SHC1 CD47-METb ITGB1-NRG1b RBPMS-NRG1 CD74-NRG1 CD74-NRG1 CD74-NRG1 CD74-NRG1

Stage

Subtype

RFS

RFS-E

OS

OS-E

IIIA IA IIIA IA IA IA IV IIB

IMA Acinar IMA Acinar Lepidic IMA Solid Acinar

24.0 67.0 16.9 37.1 67.1 65.0 1.0 25.0

1 0 1 0 0 0 1 1

74.4 67.0 78.0 37.1 67.1 65.0 10.3 50.0

0 0 1 0 0 0 1 1

Never-smokers are patients who smoked less than 100 cigarettes in their lifetime. Novel fusions reported for the first time. ID, identification number; RFS, recurrence-free survival; RFS-E, recurrence-free survival-event; OS, overall survival; OS-E, overall survival-event; IMA, invasive mucous adenocarcinoma.

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fusion. CD29, which is encoded by the ITGB1 gene in humans, is a cell surface receptor and involved in cell adhesion and recognition in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response, and metastatic diffusion of tumor cells.24,25 Although functional experiment was not conducted, we could infer from the in-frame structure of ITGB1-NRG1 fusion that this novel fusion may drive tumorigenesis as shown in CD74-NRG1 fusion. NRG1 fusions were found in 0.36% of all lung adenocarcinoma cases (6 of 1681 cases), which is similar with published data from another Asian population cohort.13 A previous study has reported that CD74-NRG1 fusion occurred specifically in invasive mucinous lung adenocarcinomas of never-smokers.26 In our cohort, patients with NRG1 fusion could also present in other subtypes of adenocarcinoma including solid, acinar, and lepidic. In light of the oncogenic mechanism by inducing ERBB2-ERBB3 heterodimers leading to PI3K/AKT pathway activation, NRG1 fusion could be inhibited by multitude available drugs targeting ERBB2, ERBB3, or their downstream pathways.27 It has been reported that two patients harboring SLC3A2-NRG1 and CD74-NRG1 fusion responded to afatinib treatment and the duration of response was 12 months and 10 months, respectively.28,29 Kim et al.30 reported that two patients with NRG1 fusions benefited from lumretuzumab, a monoclonal anti-HER3 antibody, in combination of erlotinib with at least 16 weeks of progression-free survival.30 Considering the effective inhibition by ERBB family inhibitors, NRG1 fusion may represent a therapeutic opportunity for common driver-negative patients. Our research also shows that EGFR fusion and MET fusion could present in lung adenocarcinomas from Chinese population. Distinct EGFR-RAD51 fusion has previously been observed in 4 of 10,000 patients with lung adenocarcinomas from Caucasians.15 Plenker et al.31 reported that MET fusions occurred in 0.5% of lung adenocarcinomas (2 of 337 cases). This is the first documentation of patient with EGFR-SHC1 fusion in the Chinese population. This is also the first report of a CD47-MET fusion case with lung adenocarcinoma. As EGFR-RAD51 fusions and MET fusions were markedly sensitive to EGFR TKIs and crizotinib, respectively, clinicians should pay more attention to the existence of EGFR and MET fusion.15,32-34 Generally, EGFR and MET gene fusion are relatively rare but promising molecular targets for individualized target therapies of lung adenocarcinomas. Detection of rare but novel fusions in this study highlights the necessity for adjusting our strategy from traditional tools such as Sanger sequencing to the comprehensive NGS test. Refinements in the fusion assay may also enable the discovery of clinically targetable novel fusions in the future.

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In the current study, no NTRK fusions were detected in this cohort, although NTRK fusions have been reported in NSCLC from the Chinese population.35 We suppose that several potential reasons may help to explain the result. First, NTRK fusion is a relatively rare molecular event in patients with lung adenocarcinomas from the East Asian population. Nakamura et al.36 from Japan reported that they only detected one NTRK fusion (ETV6-NTRK3) in 2088 non-squamous lung cancers. Second, we used surgically resected samples for fusion detection in this study (mostly at early stage), whereas other reports may have used biopsy tissues (mostly at advanced stage). Third, one limitation of this study is that enrichment strategy may miss fusions with concurrent common mutations, although double drivers occurred rare in lung adenocarcinomas. NTRK fusions may coexist with other oncogenic events, which may be underestimated by our enrichment strategy. In conclusion, fusions affecting NRG1, EGFR, and MET were detected in 0.48% of unselected lung adenocarcinomas, and NRG1 fusions ranked the most prevalent fusions in common driver negative lung adenocarcinomas from the Chinese population. RNA-based NGS fusion assay was an optional method for screening actionable fusions in common driver-negative cases.

Acknowledgments This study was supported by the National Natural Science Foundation of China (grant numbers: 81601994, 81772466, and 81572253); Shanghai Shenkang Hospital Development Center City Hospital Emerging Cutting-edge Technology Joint Research Project (SHDC12017102), and Shanghai Municipal Health Commission Key Discipline Project (2017ZZ02025). This project was also funded by Eli Lilly and Company through a project-based research agreement. The authors thank Yiqun Helen Li and Weiguo Xu from Eli Lilly and Company for their excellent work for this study.

Supplementary Data Note: To access the supplementary material accompanying this article, visit the online version of the Journal of Thoracic Oncology at www.jto.org and at https://doi. org/10.1016/j.jtho.2019.07.022.

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