Blood-based detection of BRAF and NRAS DNA as biomarkers in patients with stage I

摘要 黑色素瘤在美国仍然是一种高度病态的疾病。由于发病年龄相对较小， 黑色素瘤的“生命年损失”在主要实体瘤中仅次于乳腺癌， 恶性肿瘤。尽管在早期发现方面取得了进展，但黑色素瘤死亡人数仍在继续增加， 上升. 5年生存率很低，只有3%-14%的患者有远处转移性疾病（分期 （四）实现这一里程碑。此外，在美国，唯一常用的疾病活动性血清学标志物是 US（血清乳酸脱氢酶）检测疾病进展的敏感性较差，导致临床医生 依靠昂贵的成像研究来监测疾病。总体而言，每年治疗黑色素瘤的估计成本 在美国超过31亿美元。从2002年开始激活丝氨酸-苏氨酸激酶的突变 BRAF在原发性和转移性黑色素瘤中的检出率很高，随后的体外和动物实验中， 模型实验证明BRAF是黑色素瘤中的致癌基因。V600 E替代品是 突变热点，占黑素瘤中鉴定的BRAF突变的90%以上。年初 一项第二代小分子抑制剂Vemurafenib的III期随机试验的结果 BRAFV 600 E蛋白的表达，显示了6个月时无病生存率和总生存率的改善。审判是 仅限于具有BRAFV 600 E的转移性黑色素瘤患者，如通过基因分型确定的。 活检标本。因此，患者转移性黑色素瘤的BRAF基因型的知识将是一个重要的参考。 这是做出正确治疗决策的重要步骤。根据我们的临床试验经验，肿瘤基因分型 如果难以获得用于分析的转移性肿瘤标本，则通常需要1-2周或更长时间。在 此外，我们和其他人发现，单个患者的多处转移可能与患者的预后不一致。 BRAF突变（即一个肿瘤是突变型，来自同一患者的第二个肿瘤是野生型）。a基因分型 因此，来自个体患者的单个肿瘤活检可能会无意中使一些患者不适合接受治疗。 BRAF抑制剂，否则可能受益。我们已经申请了一项高度敏感的 检测患者血液样本中突变BRAF DNA的方法。初步数据显示， 这种方法在黑色素瘤患者中是可行的，因为基于血液的检测结果与黑色素瘤患者的 肿瘤的BRAF基因型。在目前的提案中，我们将与我们的行业合作伙伴Molecular MD 进一步优化我们的血液为基础的方法，并严格证明其效用，作为一个分子， 转移性黑色素瘤基因型的诊断生物标志物和可 区分无进展生存期较长与较短的患者， 用抑制突变BRAF的新药治疗。

Abstract Melanoma remains a highly morbid disease in the United States. With a relatively young age of onset, the toll of melanoma in terms of "life-years lost" is second only to breast cancer among the major solid tumor malignancies. Despite advances in early detection, the number of deaths from melanoma has continued to rise. Five-year survival rates are dismal, with only 3%-14%% of patients with distant metastatic disease (stage IV) achieving that milestone. In addition, the only commonly used serologic marker of disease activity in the US, serum lactate dehydrogenase, has a poor sensitivity to detect disease progression leading clinicians to rely on expensive imaging studies to monitor disease. Overall, the annual estimated costof treating melanoma in the United States is over $3.1 billion. Beginning in 2002 activating mutations in the serine-threonine kinase BRAF were identified at high rates in primary and metastatic melanoma, and subsequent in-vitro and animal model experiments demonstrated BRAF to be an oncogene in melanoma. The V600E substitution is a mutation hotspot, accounting for greater than 90% of the BRAF mutations identified in melanoma. Earlier this month the results of a Phase III randomized trial of Vemurafenib, a second generation small molecule inhibitor of the BRAFV600E protein, demonstrated improved disease-free and overall survival at 6 months. The trial was limited to patients with metastatic melanoma tumors that had the BRAFV600E as determined by genotyping of a biopsy specimen. Thus, knowledge of the BRAF genotype of a patient's metastatic melanomas will be an essential step for proper therapeutic decision-making. Based on our clinical trial experience, tumor genotyping often takes 1-2 weeks or longer if there are difficulties obtaining a metastatic tumor specimen for analysis. In addition, we and others have found that multiple metastases from individual patients may be discordant for the BRAF mutation (i.e. one tumor is mutant, a second tumor from the same patient is wild-type). Genotyping of a single tumor biopsy from individual patients, therefore, may inadvertently render some patients ineligible for a BRAF inhibitor who might otherwise derive benefit. We have patented and licensed a highly sensitive approach to detect mutant BRAF DNA in patient blood samples. Our Preliminary Data demonstrate that this approach is feasible in melanoma patients, as the results of blood-based testing are highly associated with the BRAF genotype of the tumor. In the current proposal we will work with our industry collaborator, Molecular MD to further optimize our blood-based methodology, and rigorously demonstrate its utility as both a molecular diagnostic biomarker of metastatic melanoma genotype and response-predictive biomarker that can differentiate between patients that will have a longer versus shorter duration of progression-free survival while under treatment with the new agents that inhibit mutant BRAF.