Validation of circulating tumor DNA assays for detection of metastatic melanoma

 描述(由申请人提供)：利用Droplet数字聚合酶链式反应(DdPCR)平台的高灵敏度、特异性和定量能力，该项目寻求从分析和临床上验证ddPCR对7个黑色素瘤相关热点突变的分析和临床验证，用于作为转移性黑色素瘤切除患者疾病复发的血液生物标记物。在美国，唯一常用的监测转移性黑色素瘤患者疾病复发的血清学标志物是血清乳酸脱氢酶(LDH)，其敏感性和特异性较低。随着新的、有效的系统性疾病治疗方法在辅助环境中的应用，一种可以识别早期疾病复发的敏感和特异的生物标记物可能会促使临床医生在患者肿瘤负担较低的情况下改变患者的治疗方法。最近，高度敏感和特异的技术，如ddPCR，揭示了癌症患者血浆中循环的异常高水平的无细胞肿瘤相关DNA(CtDNA)，通常与转移的肿瘤负担呈正相关。我们利用ddPCR进行了一项试点研究，以检测接受BRAF靶向治疗或免疫检查点阻断治疗的无法切除的IV期黑色素瘤患者中突变的BRAF和NRAS ctDNA水平的变化。对于这一应用来说，重要的是，我们发现在治疗开始时，ctDNA是比LDH更敏感的疾病负担低的标志。在系统治疗开始时，RECIST评分<5 cm的患者中有71%的患者ctDNA水平高于LDH，后者仅在8%的患者中升高。虽然这些令人鼓舞的结果是通过检测与黑色素瘤相关的5种最常见的BRAF和NRAS突变中的1种获得的，但这5种突变总共只在55%-65%的患者中存在。最近，端粒酶逆转录酶基因(TERT)启动子区域的两个功能性热点突变在包括黑色素瘤在内的许多癌症中被发现。我们的初步研究也发现这些TERT突变在黑色素瘤中很常见，当用BRAF和NRAS突变评估时，86%的患者在他们的黑色素瘤中有7个BRAF、NRAS或TERT突变中的一个或多个。在这项应用中，我们将对BRAF、NRAS和TERT中的7种常见突变进行分析验证，并检验这7种ddPCR中的1种检测ctDNA水平的升高将在常规成像扫描之前预测切除转移患者的放射学复发的假设。能够以高灵敏度准确检测疾病进展的血浆生物标志物的开发，可以增加或取代每3个月获得一次的常规放射扫描，以响应血浆ctDNA水平的上升而订购扫描。此外，它还可以为临床试验奠定基础，比较所有切除患者辅助治疗的有效性。 而不是在ctDNA水平上升的基础上出现微转移疾病的证据时选择患者进行治疗。

 DESCRIPTION (provided by applicant): 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.