Validation of circulating tumor DNA assays for detection of metastatic melanoma

项目摘要 利用液滴数字PCR（ddPCR）的高灵敏度、特异性和定量能力 平台，该项目旨在分析和临床验证ddPCR检测7黑色素瘤相关的热， 用作切除的转移性肿瘤患者中疾病复发的基于血液的生物标志物的点突变 黑素瘤在美国，唯一常用的监测疾病复发的血清学标志物是 切除转移性黑色素瘤患者的血清乳酸脱氢酶（LDH）水平较低， 灵敏度和特异性。随着新的，有效的治疗全身性疾病的辅助应用， 在这种情况下，一种可以识别早期疾病复发的敏感和特异性生物标志物可以促使临床医生 在患者肿瘤负荷较低时改变患者管理。最近，高度敏感和特异的 技术，如ddPCR，已经揭示癌症患者具有异常高水平的无细胞， 肿瘤相关DNA（ctDNA）在其血浆中循环，通常与转移性 肿瘤负荷我们利用ddPCR进行了一项初步研究，以检测突变型BRAF和NRAS的变化， 接受BRAF治疗的不可切除的IV期黑色素瘤患者的ctDNA水平- 靶向治疗或免疫检查点阻断。对于这个应用程序重要的是，我们发现，在当时， 在治疗开始时，ctDNA是比LDH更敏感的低疾病负荷标志物。ctDNA 在全身治疗开始时，71%的RECIST评分< 5cm的患者的水平升高， LDH，仅8%的患者升高。虽然这些令人鼓舞的结果是通过 检测5种最常见的黑素瘤相关BRAF和NRAS突变中的1种的测定， 这5种突变总共仅存在于55% - 65%的患者中。近日，2款功能性、热点 端粒酶逆转录酶基因（TERT）启动子区的突变在许多肿瘤中被发现。 癌症包括黑色素瘤。我们的初步研究还发现这些TERT突变在 黑色素瘤，当评估BRAF和NRAS突变时，86%的患者具有7种突变中的1种或多种， BRAF、NRAS或TERT突变。在本申请中，我们将分析验证ddPCR 检测BRAF、NRAS和TERT中的7种常见突变，并检验ctDNA水平升高 通过这7种ddPCR测定中的1种测定的水平将预测切除的转移性肿瘤的放射学复发。 患者在常规成像扫描之前。开发可准确检测疾病的血浆生物标志物 具有高灵敏度的进展可以增强或取代每3周获得的常规放射学扫描。 几个月后，根据血浆ctDNA水平的升高进行扫描。它也可以奠定基础 对于一项临床试验，比较了所有切除患者的辅助治疗与选择患者 当基于ctDNA水平的升高出现微转移性疾病的证据时进行治疗。

Project Summary Leveraging the high sensitivity, specificity and quantitative capability of the droplet digital PCR (ddPCR) platform, this project seeks to analytically and clinically validate ddPCR assays for 7 melanoma-associated hot- spot mutations for use as blood-based biomarkers of disease recurrence in patients with resected metastatic melanoma. In the United States the only commonly used serologic marker to monitor disease recurrence in patients with resected metastatic melanoma is serum lactate dehydrogenase (LDH), which suffers from low sensitivity and specificity. With new, effective therapies for systemic disease being applied in the adjuvant setting, a sensitive and specific biomarker that can identify early disease recurrence could prompt clinicians to change patient management while the patient has a low tumor burden. Recently, highly sensitive and specific technologies, such as ddPCR, have revealed that cancer patients have abnormally high levels of cell-free, tumor-associated DNA (ctDNA) circulating in their plasma that generally correlate positively with metastatic tumor burden. We conducted a pilot study utilizing ddPCR to detect changes in mutant BRAF and NRAS ctDNA levels in patients with unresectable stage IV melanoma who were undergoing treatment with BRAF- targeted therapies or immune checkpoint blockade. Importantly for this application, we found that at the time of treatment initiation, ctDNA was a much more sensitive marker of low disease burden than LDH. ctDNA levels were elevated in 71% of patients with RECIST scores < 5cm at the start of systemic treatment compared to LDH, which was only elevated in 8% of patients. While these encouraging results were obtained with assays that detect 1 of the 5 of the most common melanoma-associated BRAF and NRAS mutations, collectively these 5 mutations are present in only 55% - 65% of patients. Recently, 2 functional, hotspot mutations in the promoter region of the telomerase reverse-transcriptase gene (TERT) were identified in many cancers including melanoma. Our preliminary studies have also found these TERT mutations frequently in melanoma and when evaluated with BRAF and NRAS mutations, 86% of patients have 1 or more of the 7 BRAF, NRAS or TERT mutations in their melanomas. In this application, we will analytically validate ddPCR assays for the 7 common mutations in BRAF, NRAS and TERT, and test the hypothesis that a rise in ctDNA levels, as measured by 1 of these 7 ddPCR assays will predict radiographic recurrence in resected metastatic patients prior to routine imaging scans. Development of plasma biomarkers that can accurately detect disease progression with high sensitivity can augment or replace the routine radiographic scans obtained every 3 months with scans ordered in response to a rise in the plasma ctDNA level. Also it could lay the groundwork for a clinical trial comparing the utility of adjuvant therapy for all resected patients versus selecting patients for treatment when evidence of micrometastatic disease emerges based on a rise in ctDNA levels.