Sequencing the Osteosarcoma Kinome

骨肉瘤激酶组测序

基本信息

批准号：
8554718
负责人：
Su-Young Kim
金额：
$ 0.56万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

BackgroundOsteosarcoma is the most common pediatric bone cancer in the United States. Due to advances in surgery and chemotherapy, cure rates for patients with localized disease approach 70%. However, long-term survival for patients with metastatic disease is only 25% and multiple attempts at intensification of chemotherapy have not improved this dismal survival rate. These figures point to the need to develop new therapeutic regimens.A different treatment approach is to utilize inhibitors that target activated tyrosine kinases. Imatinib, which targets ABL and KIT, has been the paradigm for such treatment. Patients with CML have activation of ABL due to a BCR-ABL translocation and patients with GIST have activating mutations in KIT. Both groups of patients have had remarkable responses when treated with Imatinib. A similar approach to treat patients with osteosarcoma does not exist because there has not been a systematic examination of mutations in tyrosine kinases.Our goal is to sequence every gene in osteosarcoma tumor samples to try to identify activating mutations. In 2009, in preparation for this project, we identified tumor specimens collected from patients previously treated at the NIH under Pediatric Oncology Branch protocols. Approval was granted from the Office of Human Subjects Research to perform sequencing analysis using these samples. We isolated DNA and RNA and went on to determine the quality of these samples. At the same time a small portion of the tumor was sent for sectioning and H+E slides were examined with a pediatric pathologist, verifying the presence of tumor and the diagnosis of osteosarcoma.Results from 2011In 2011, in preparation of evaluation of patient samples for sequencing, we utilized a panel of well-established osteosarcoma cell lines and evaluated them by comparative genomic hybridization. In addition, we also analyzed cell lines that were recently derived at the NIH from patients at the time of their definitive resection. The results suggested that all of our samples were consistent with osteosarcoma and that they were of sufficient purity to undergo next generation sequencing.Plans for 2012In 2012, in collaboration with members of Dr. Paul Meltzers laboratory, we will perform next generation sequencing of osteosarcoma cell lines. The results of this analysis will better prepare us for sequencing of DNA obtained from patient tumor samples.Significance and GoalsThe successful completion of the above objectives may reveal the mechanism, or mechanisms, that underlie the initiation and progression of osteosarcoma. The finding of genetic changes will then allow us to perform molecular and biological evaluations to determine if they are relevant targets that merit further evaluation.

背景骨肉瘤是美国最常见的儿童骨癌。由于手术和化疗的进步，局部疾病患者的治愈率接近70%。然而，转移性疾病患者的长期生存率仅为 25%，多次尝试强化化疗并没有改善这种惨淡的生存率。这些数字表明需要开发新的治疗方案。一种不同的治疗方法是利用针对活化酪氨酸激酶的抑制剂。伊马替尼以 ABL 和 KIT 为靶点，一直是此类治疗的典范。 CML 患者由于 BCR-ABL 易位而激活 ABL，GIST 患者则激活 KIT 突变。两组患者在接受伊马替尼治疗后均取得了显着的缓解。治疗骨肉瘤患者的类似方法并不存在，因为尚未对酪氨酸激酶的突变进行系统检查。我们的目标是对骨肉瘤肿瘤样本中的每个基因进行测序，以尝试识别激活突变。 2009 年，为了准备该项目，我们鉴定了从先前在 NIH 根据儿科肿瘤科方案接受治疗的患者身上收集的肿瘤样本。人类受试者研究办公室批准使用这些样本进行测序分析。我们分离出 DNA 和 RNA，并继续确定这些样本的质量。同时将肿瘤的一小部分送去切片，并由儿科病理学家检查 H+E 载玻片，验证肿瘤的存在和骨肉瘤的诊断。2011 年的结果2011 年，准备对患者样本进行评估测序后，我们利用了一组成熟的骨肉瘤细胞系，并通过比较基因组杂交对其进行了评估。此外，我们还分析了 NIH 最近从患者进行最终切除时获得的细胞系。结果表明，我们所有的样本都与骨肉瘤一致，并且具有足够的纯度进行下一代测序。 2012年计划2012年，与Paul Meltzers博士实验室成员合作，我们将进行骨肉瘤细胞的下一代测序线。该分析的结果将更好地帮助我们对从患者肿瘤样本中获得的 DNA 进行测序。意义和目标成功完成上述目标可能会揭示骨肉瘤发生和进展的机制。遗传变化的发现将使我们能够进行分子和生物学评估，以确定它们是否是值得进一步评估的相关目标。