Identification of a Novel MAN1A1-ROS1 Fusion Gene Through mRNA-based Screening for Tyrosine Kinase Gene Aberrations in a Patient with Leiomyosarcoma
Identification of a Novel MAN1A1-ROS1 Fusion Gene Through mRNA-based Screening for Tyrosine Kinase Gene Aberrations in a Patient with Leiomyosarcoma
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DOI：
10.1097/corr.0000000000001548
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发表时间：
2021-04-01
影响因子：
4.2
通讯作者：
Saito, Tsuyoshi
中科院分区：
文献类型：
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作者：
Suehara, Yoshiyuki;Kohsaka, Shinji;Saito, Tsuyoshi
背景软组织肉瘤是一组罕见的异质性恶性肿瘤。晚期软组织肉瘤预后差，有效的系统治疗尚未建立。酪氨酸激酶越来越多地被用作各种癌症和软组织肉瘤的治疗靶点。虽然复杂核型肉瘤通常比易位相关肉瘤（融合基因肉瘤）携带更多潜在的可操作的遗传改变，但基于我们的数据库回顾，我们发现平滑肌肉瘤和恶性外周神经鞘瘤比其他非易位软组织肉瘤具有更低的潜在靶点频率。我们推测平滑肌肉瘤和恶性周围神经鞘瘤都可能包括在任何独特的易位中。此外，如果酪氨酸激酶失衡，特别是融合基因，发生在平滑肌肉瘤和恶性外周神经鞘瘤患者，酪氨酸激酶抑制剂可能是这种肉瘤的药物开发目标。在这项研究中，我们使用了酪氨酸激酶筛选系统，可以检测酪氨酸激酶基因的5 '和3'侧之间的mRNA的不平衡，以确定潜在的新的治疗软组织肉瘤的酪氨酸激酶靶点。问题/目的（1）在软组织肉瘤患者的肿瘤中是否存在新的治疗性酪氨酸激酶靶点，这些靶点可以通过关注激酶结构域5'和3'端之间的不平衡表达的mRNA筛选来检测？(2)是否可以通过RNA测序和逆转录PCR（RT-PCR）验证潜在靶点？(3)潜在的融合基因是否会在体外试验中转化细胞？(4)具有已鉴定的融合基因的小鼠肿瘤是否会对针对该靶点的治疗药物产生反应？方法我们使用mRNA筛选来寻找可能具有治疗潜力的新酪氨酸激酶靶点。使用功能分析，我们验证了所确定的融合基因是否是软组织肉瘤的良好治疗候选基因。此外，使用体内测定，我们评估了抑制融合的激酶活性是否具有治疗潜力。研究资格是基于患有高级别梭形细胞和非易位肉瘤的患者，包括平滑肌肉瘤、恶性外周神经鞘瘤和高级别粘液纤维肉瘤。在2015年至2019年期间，在顺天堂大学医院接受手术切除治疗的172名软组织肉瘤患者中，有72名患者患有高级别非易位肉瘤。该分析主要针对平滑肌肉瘤和恶性周围神经鞘肿瘤，并且在研究开始时存在分析量限制（试剂限制），共计24份样本。我们从东京医科齿科大学的样本库中收集了额外的样本，以增加研究肉瘤的数量。因此，在本研究中，共收集了15个平滑肌肉瘤样本、5个恶性周围神经鞘瘤样本和4个高级别粘液纤维肉瘤样本，以达到24名患者的样本量。为了鉴定酪氨酸激酶融合基因，我们设计了一种基于NanoString的检测方法，（NanoString Technologies Inc，西雅图，WA，USA）在两个点查询关于90种酪氨酸激酶的转录物的表达平衡：将肿瘤的RNA与NanoString探针杂交，并分析激酶结构域的5'端和激酶结构域内或激酶结构域的3'端的异常值的表达比率。激酶结构域的3'至5'端。通过基于NanoString的测定确定的这些阳性肿瘤中的假定新融合事件通过RNA测序和确证性RT-PCR证实了酪氨酸激酶融合基因。还进行了由体外和体内测定组成的功能分析，以阐明所鉴定的酪氨酸激酶基因融合是否与致癌能力和药物反应相关。结果我们发现一名90岁女性平滑肌肉瘤中ROS 1转录本激酶结构域3'端至5'端的异常表达率。通过RNA测序从她的大腿肿瘤中鉴定出一种新的MAN 1A 1-ROS 1融合基因，并通过实时PCR证实。在功能测定中，MAN 1A 1-ROS 1重排在3 T3细胞中显示出强的转化潜力。此外，在体内试验中，克唑替尼（一种ROS 1抑制剂）以剂量依赖性方式显著抑制MAN 1A 1-ROS 1诱导转化细胞的生长。结论通过酪氨酸激酶的筛选，为软组织肉瘤的治疗提供了新的靶点。我们发现了一个新的MAN 1A 1-ROS 1融合基因，可能是平滑肌肉瘤患者的治疗靶点。这项研究表明，mRNA筛选系统可能有助于软组织肉瘤的治疗选择的发展。
Background Soft tissue sarcomas are a heterogeneous group of rare malignant tumors. Advanced soft tissue sarcomas have a poor prognosis, and effective systemic therapies have not been established. Tyrosine kinases are increasingly being used as therapeutic targets for a variety of cancers and soft tissue sarcomas. Although complex karyotype sarcomas typically tend to carry more potentially actionable genetic alterations than do translocation-associated sarcomas (fusion gene sarcomas), based on our database review, we found that leiomyosarcoma and malignant peripheral nerve sheath tumors have lower frequencies of potential targets than other nontranslocation soft tissue sarcomas. We theorized that both leiomyosarcoma and malignant peripheral nerve sheath tumors might be included in any unique translocations. Furthermore, if tyrosine kinase imbalances, especially fusion genes, occur in patients with leiomyosarcomas and malignant peripheral nerve sheath tumors, tyrosine kinase inhibitors might be a drug development target for this sarcoma. In this study, we used a tyrosine kinase screening system that could detect an imbalance in mRNA between 5'- and 3'-sides in tyrosine kinase genes to identify potential novel therapeutic tyrosine kinase targets for soft tissue sarcomas. Questions/purposes (1) Are there novel therapeutic tyrosine kinase targets in tumors from patients with soft tissue sarcomas that are detectable using mRNA screening focusing on imbalance expressions between the 5' and 3' end of the kinase domain? (2) Can potential targets be verified by RNA sequencing and reverse transcription PCR (RT-PCR)? (3) Will potential fusion gene(s) transform cells in in vitro assays? (4) Will tumors in mice that have an identified fusion gene respond to treatment with a therapeutic drug directed at that target? Methods We used mRNA screening to look for novel tyrosine kinase targets that might be of therapeutic potential. Using functional assays, we verified whether the identified fusion genes would be good therapeutic candidates for soft tissue sarcomas. Additionally, using in vivo assays, we assessed whether suppressing the fusion's kinase activity has therapeutic potential. Study eligibility was based on a patient having high-grade spindle cell and nontranslocation sarcomas, including leiomyosarcoma, malignant peripheral nerve sheath tumor, and high-grade myxofibrosarcoma. Between 2015 and 2019, of the 172 patients with soft tissue sarcomas treated with surgical resection at Juntendo University Hospital, 72 patients had high-grade nontranslocation sarcomas. The analysis was primarily for leiomyosarcoma and malignant peripheral nerve sheath tumors, and there was a limitation of analysis size (reagent limitations) totaling 24 samples at the start of the study. We collected additional samples from a sample bank at the Tokyo Medical and Dental University to increase the number of sarcomas to study. Therefore, in this study, a total of 15 leiomyosarcoma samples, five malignant peripheral nerve sheath tumors samples, and four high-grade myxofibrosarcoma samples were collected to achieve the sample size of 24 patients. To identify tyrosine kinase fusion genes, we designed a NanoString-based assay (NanoString Technologies Inc, Seattle, WA, USA) to query the expression balances regarding transcripts of 90 tyrosine kinases at two points: the 5' end of the kinase domain and within the kinase domain or 3' end of the kinase domain.The tumor's RNA was hybridized to the NanoString probes and analyzed for the expression ratios of outliers from the 3' to 5' end of the kinase domain. Presumed novel fusion events in these positive tumors that were defined by NanoString-based assays were confirmed tyrosine kinase fusion genes by RNA sequencing and confirmatory RT-PCR. Functional analyses consisting of in vitro and in vivo assays were also performed to elucidate whether the identified tyrosine kinase gene fusions were associated with oncogenic abilities and drug responses. Results We identified aberrant expression ratios regarding the 3' to 5' end of the kinase domain ratios in ROS1 transcripts in a leiomyosarcoma in a 90-year-old woman. A novel MAN1A1-ROS1 fusion gene was identified from her thigh tumor through RNA sequencing, which was confirmed with real-time PCR. In functional assays, MAN1A1-ROS1 rearrangement revealed strong transforming potential in 3T3 cells. Moreover, in an in vivo assay, crizotinib, a ROS1 inhibitor, markedly inhibited the growth of MAN1A1-ROS1 rearrangement-induced transformed cells in a dose-dependent manner. Conclusion We conducted tyrosine kinase screening to identify new therapeutic targets in soft tissue sarcomas. We found a novel MAN1A1-ROS1 fusion gene that may be a therapeutic target in patients with leiomyosarcoma. This study demonstrates that the mRNA screening system may aid in the development of useful therapeutic options for soft tissue sarcomas.