Validation of circulating tumor DNA assays for detection of metastatic melanoma

 描述（由申请方提供）：利用液滴数字PCR（ddPCR）平台的高灵敏度、特异性和定量能力，该项目旨在分析和临床验证7种黑素瘤相关热点突变的ddPCR检测，用作切除转移性黑素瘤患者疾病复发的血液生物标志物。在美国，监测切除转移性黑素瘤患者疾病复发的唯一常用血清学标志物是血清乳酸脱氢酶（LDH），其灵敏度和特异性较低。随着新的、有效的全身性疾病治疗方法在辅助治疗中的应用，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的情况下改变患者管理。最近，高度敏感和特异性的技术，如ddPCR，已经揭示了癌症患者在其血浆中循环的无细胞肿瘤相关DNA（ctDNA）水平异常高，其通常与转移性肿瘤负荷呈正相关。我们利用ddPCR进行了一项初步研究，以检测接受BRAF靶向治疗或免疫检查点阻断治疗的不可切除的IV期黑色素瘤患者中突变型BRAF和NRAS ctDNA水平的变化。对于该应用重要的是，我们发现在治疗开始时，ctDNA是比LDH更敏感的低疾病负荷标志物。在全身治疗开始时，71%的RECIST评分< 5cm的患者ctDNA水平升高，而LDH仅在8%的患者中升高。虽然这些令人鼓舞的结果是用检测5种最常见的黑素瘤相关BRAF和NRAS突变中的1种的测定法获得的，但这5种突变总共仅存在于55% - 65%的患者中。最近，在包括黑色素瘤在内的许多癌症中发现了端粒酶逆转录酶基因（TERT）启动子区的2个功能性热点突变。我们的初步研究还发现这些TERT突变在黑色素瘤中频繁发生，当用BRAF和NRAS突变进行评估时，86%的患者在其黑色素瘤中具有7种BRAF、NRAS或TERT突变中的1种或多种。在本申请中，我们将对BRAF、NRAS和TERT中7种常见突变的ddPCR检测进行分析验证，并检验以下假设：通过这7种ddPCR检测中的1种检测，ctDNA水平升高将预测切除的转移性患者在常规成像扫描前的放射学复发。开发能够以高灵敏度准确检测疾病进展的血浆生物标志物，可以增强或取代每3个月获得的常规放射学扫描，并根据血浆ctDNA水平的升高进行扫描。它也可以为临床试验奠定基础，比较所有切除患者的辅助治疗效用 与选择患者进行治疗相比，当基于ctDNA水平的升高出现微转移性疾病的证据时。

 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.