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，美国预计将新增约10万例黑色素瘤病例，预计约有7000人死于黑色素瘤。这些数字与大多数西方国家相当；例如，在德国，每年约有2.1万例新发黑色素瘤病例和约3000例死亡病例。原发黑色素瘤的分期从0到IV；II期及以下的肿瘤没有发现转移，几乎总是在切除后不进行治疗。然而，在I期诊断后死于黑色素瘤的人比在IV期诊断后死于黑色素瘤的人更多，因为目前的诊断金标准(组织病理学评估和适当的淋巴结活检)不适合评估转移潜力。十年前，测量转移潜能是没有用的，因为没有有效的治疗方法可用。从那时起，辅助治疗取得了长足的进步，但仅被批准用于有转移的黑色素瘤(III-IV期)。毒副作用和耐药性的产生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，如果给出一个合理的标记来识别它们，它们可能会更有效。在这里，我们提出了光学飞秒泵浦探测显微镜的原发肿瘤活检组织，以确定黑色素瘤转移的高风险。对黑色素的超快电子和振动动力学的分析，提供了一个强大的生物标记物。杜克大学沃伦实验室进行的初步回顾研究表明，黑色素的特征泵-探头特征随着转移潜能的增加而变化。我们将以临床应用为目标，进一步发展黑色素瘤的泵浦探测显微镜。首先，我们将表征肿瘤固有黑色素的光谱特性，并开发度量标准来量化它们的转移潜能。我们将建造一个紧凑的交钥匙显微镜，可以部署在诊断实验室中，由病理学家操作，作为最后一步，我们将使用大约600个黑色素瘤活组织检查样本(来自杜克医学中心)来严格基准我们的方法。这项技术可以帮助病理学家量化黑色素瘤的转移潜力，从而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一个可靠的生物标志物，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.