Using femtosecond pump-probe microscopy to improve detection of early-stage metastatic melanoma

黑色素瘤是一种起源于黑素细胞的皮肤癌，由于其高转移潜力，是迄今为止最危险的皮肤癌类型。2019年，美国预计约有10万例新的黑色素瘤发病，约7,000人预计将死于黑色素瘤，这些数字与大多数西方国家相当;以德国为例，每年约有21,000例新的黑色素瘤发病，约3,000例死亡。黑色素瘤的原发性肿瘤从0到IV“分期”; II期及以下的肿瘤没有检测到转移，并且在切除后几乎总是不治疗。然而，更多的人死于黑色素瘤后，第一阶段的诊断比第四阶段的诊断，因为目前的诊断金标准（组织病理学评价，并在适当的情况下，淋巴结活检）是不适合评估转移的潜力。十年前，测量转移潜力是没有用的，因为没有有效的治疗方法。从那时起，辅助治疗取得了很大的进步，但仅被批准用于转移性黑色素瘤（III-IV期）。毒性、副作用和耐药性发展目前限制了辅助治疗的广泛使用，但如果应用于具有高转移风险的早期肿瘤，如果有合理的标记物用于识别它们，它们可能会更有效。在这里，我们提出了一个光学飞秒泵浦-探测显微镜原发肿瘤活检识别黑色素瘤转移的高风险。黑色素（肿瘤固有色素）的超快电子和振动动力学分析提供了一个强大的生物标志物。由杜克大学沃伦实验室进行的初步回顾性研究表明，黑色素的特征性泵-探测信号随着转移潜能的增加而改变。我们将进一步发展泵浦-探针显微镜在黑色素瘤的临床应用。首先，我们将描述肿瘤固有色素黑色素的光谱特性，并制定指标来量化其转移潜力的特性。我们将建立一个紧凑的交钥匙显微镜，可以部署在诊断实验室和病理学家操作，作为最后一步，我们将使用大约600个存档的黑色素瘤活检样本（来自杜克医学中心），以严格基准我们的方法。该技术可以通过协助病理学家量化黑色素瘤的转移潜力来直接影响患者的治疗和预后。它可以指导淋巴结活检的适当使用，从而避免不必要的诊断性外科手术，从长远来看，这项技术有可能提供可靠的生物标志物，以决定哪些早期，高风险的黑色素瘤应该接受辅助治疗。

Melanoma, the skin cancer originating from melanocytes, is by far the most dangerous type of skin cancer because of its high metastatic potential. In 2019, about 100,000 new melanoma incidences are expected in the United States, and about 7,000 are expected to die from it. These numbers are comparable to most western countries; in Germany, for example, about 21,000 new incidences and about 3,000 deaths are caused by melanoma per year. Primary tumors of melanoma are “staged” from 0 to IV; tumors staged II and below do not have detected metastases and are almost always left untreated after excision. Yet, more people die from melanoma after a stage I diagnosis than after a stage IV diagnosis, because the current diagnostic gold standard (histopathological evaluation and, where appropriate, lymph node biopsy) is not suitable to assess the metastatic potential. A decade ago, measuring metastatic potential would have been not useful, because no effective treatments were available. Since then, adjuvant therapies have made great strides, but are only approved for melanoma with metastases (stage III-IV). Toxicity, side effects, and resistance development currently limit the broad use of adjuvant therapies, but they could be much more effective if applied to early stage tumors with high risk of metastases, given a reasonable marker for identifying them. Here we propose optical femtosecond pump-probe microscopy of primary tumor biopsies for identification of melanoma with a high risk for metastases. Analysis of the ultrafast electronic and vibrational dynamics of melanin, the tumor-intrinsic pigment, provides a powerful biomarker. Preliminary retrospective studies, done by the Warren lab at Duke University, have shown that the characteristic pump-probe signature of melanin changes with increased metastatic potential. We will further develop pump-probe microscopy of melanoma with the goal of clinical application. First, we will characterize the spectroscopic properties of the tumor-intrinsic pigment melanin and develop metrics to quantify their properties to metastatic potential. We will build a compact and turn-key microscope that can be deployed in a diagnostic lab and operated by pathologists and, as a last step, we will use around 600 archived melanoma biopsy samples (from the Duke Medical Center) to rigorously benchmark our method. This technology can directly impact patient treatment and prognosis by assisting pathologists in quantifying the metastatic potential of melanoma. It could guide appropriate usage of lymph node biopsy, thereby avoiding unnecessary diagnostic surgical procedures, and, in the long run, this technology has the potential to deliver a reliable biomarker to decide which early stage, high-risk melanoma should be treated with adjuvant therapy.