Blood-based detection of BRAF DNA as a biomarker in metastatic melanoma patients

描述(申请人提供)：黑色素瘤在美国仍然是一种高度病态的疾病。黑色素瘤发病年龄相对较小，在主要实体肿瘤恶性肿瘤中，黑色素瘤的死亡人数仅次于乳腺癌。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上升。目前转移性黑色素瘤的治疗方案大多无效，远处转移性疾病(IV期)的5年生存率仅为3%-14%。此外，美国唯一常用的疾病活动性血清学标志-血清乳酸脱氢酶-对检测疾病进展的敏感性较差，导致临床医生依赖昂贵的成像研究来监测疾病。总体而言，美国每年治疗黑色素瘤的估计成本超过31亿美元。从2002年开始，在原发和转移性黑色素瘤中发现了丝氨酸-苏氨酸激酶BRAF的高活性突变，随后的体外和动物模型实验证明BRAF是黑色素瘤的致癌基因。V600E替换是一个突变热点，在黑色素瘤中发现的突变中，V600E替代占90%以上。2009年年中，BRAFV600E蛋白的第二代小分子抑制剂PLX4032的临床试验的初步结果显示，大多数携带BRAF V600E突变的患者有反应。目前，PLX4032的临床试验仅限于可以获得肿瘤活检并检测到突变的患者。根据我们的临床试验经验，如果难以获得转移性肿瘤标本进行基因分型，目前肿瘤基因分型需要1-2周或更长时间。此外，我们和其他人发现，来自单个患者的多个转移可能与BRAF突变不一致(即一个肿瘤是突变的，来自同一患者的第二个肿瘤是野生型)。因此，对单个患者的单个肿瘤活检进行基因分型可能会无意中使一些患者没有资格使用BRAF抑制剂，否则可能会受益。为了克服这些限制，我们开发了一种快速、高度敏感的方法来检测患者血液样本中突变的BRAF DNA。我们的初步数据表明，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肿瘤的BRAF基因高度相关。目前的提案寻求进一步优化我们的方法，并严格证明其作为分子诊断工具的实用性。此外，我们将测试基于血液的BRAF DNA检测作为疾病活动性的生物标志物的实用性。使用血液样本对患者的实体肿瘤进行基因分型是癌症医学中一种高度创新的方法，不仅有可能改变黑色素瘤的临床实践，而且还有可能改变其他实体肿瘤患者的护理，如甲状腺、结肠癌、卵巢癌和肺癌，这些患者具有显著的BRAF突变率。 公共卫生相关性：最近，针对突变的BRAF激酶的一种基于机制的治疗剂已显示出对肿瘤具有突变的转移性黑色素瘤患者的有效性。我们开发了一种快速、高灵敏度的方法来检测患者血液样本中突变的BRAF DNA。目前的提案试图严格证明这些检测作为分子诊断工具的临床实用价值，这些工具有可能改变黑色素瘤的临床实践，并有可能应用基于机制的治疗方法在其他实体肿瘤中应用。

DESCRIPTION (provided by applicant): 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. Current treatment options for metastatic melanoma are largely ineffective with dismal 5-year survival rates of only 3%-14%% for distant metastatic disease (stage IV). 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 cost of 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 mutations identified in melanoma. In mid 2009 preliminary results from a clinical trial of PLX4032, a second generation small molecule inhibitor of the BRAFV600E protein, showed responses in the majority of patients carrying the BRAF V600E mutation. Currently, enrollment into clinical trials with PLX4032 is restricted to patients in whom a tumor biopsy can be obtained, and in which the mutation is detected. Based on our clinical trial experience, tumor genotyping currently takes 1-2 weeks or longer if there are difficulties obtaining a metastatic tumor specimen for genotyping. 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. To overcome these limitations we have developed a rapid, 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. The current proposal seeks to further optimize our methodology, and rigorously demonstrate its utility as a molecular diagnostic tool. In addition, we will test the utility of blood-based detection of BRAF DNA as a biomarker of disease activity. Using a blood sample to genotype a patient's solid tumor is a highly innovative approach in cancer medicine and has potential to change clinical practice not only in melanoma but also in the care of patients with other solid tumors such as thyroid, colon, ovarian and lung cancers which have significant rates of BRAF mutation. PUBLIC HEALTH RELEVANCE: Recently, a mechanism-based therapeutic agent targeting the mutant BRAF kinase has shown effectiveness in metastatic melanoma patients whose tumors have the mutation. We have developed a rapid, highly sensitive approach to detect mutant BRAF DNA in patient blood samples. The current proposal seeks to rigorously demonstrate the clinical utility of these assays as molecular diagnostic tools that have the potential to change the clinical practice in melanoma and potentially in other solid tumors in which mechanism-based therapies may be applied.