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，死亡人数 在黑色素瘤的“生命损失年”中，仅次于乳腺癌的主要实体肿瘤 恶性肿瘤。尽管在早期发现方面取得了进展，但死于黑色素瘤的人数仍在继续 站起来。五年存活率令人沮丧，只有3%-14%的患者患有远处转移疾病(分期 四)实现这一里程碑。此外，唯一常用的疾病活动性血清学标志物 美国，血清乳酸脱氢酶，对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，治疗黑色素瘤的年度估计成本 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 在原发和转移性黑色素瘤以及随后的体外和动物实验中，BRAF被鉴定为高比率 模型实验证明BRAF是黑色素瘤的致癌基因。V600E替代产品是 突变热点，占黑色素瘤中已发现的BRAF突变的90%以上。今年早些时候 第二代小分子抑制剂维莫拉非尼的第三阶段随机试验结果 BRAFV600E蛋白，在6个月后显示出改善的无病和总存活率。审判是 仅限于通过基因分型确定具有BRAFV600E的转移性黑色素瘤患者 活组织检查样本。因此，了解患者转移性黑色素瘤的BRAF基因将是一种 正确作出治疗决策的必要步骤。根据我们的临床试验经验，肿瘤基因分型 如果难以获得转移性肿瘤标本进行分析，通常需要1-2周或更长时间。在……里面 此外，我们和其他人发现，个别患者的多处转移可能与 BRAF突变(即一个肿瘤是突变的，第二个来自同一患者的肿瘤是野生型)。A亚型的基因分型 因此，个别患者的单个肿瘤活检可能会无意中使一些患者不符合资格 BRAF抑制剂，否则可能会受益。我们已经为一种高度敏感的 检测患者血液样本中突变BRAF DNA的方法我们的初步数据表明，这 这种方法在黑色素瘤患者中是可行的，因为基于血液的测试结果与 肿瘤的BRAF基因。在目前的计划中，我们将与我们的行业合作伙伴分子MD合作 进一步优化我们基于血液的方法学，并严格证明其作为一种分子 转移性黑色素瘤的诊断生物标记物和反应预测生物标记物 区分无进展生存期较长和较短的患者 正在用抑制突变的BRAF的新试剂治疗。

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.