Imaging Nonlinear Absorption of Biomarkers for Improved Detection of Melanoma

描述(由申请人提供)：本申请涉及广泛的挑战领域生物标记发现和验证(03)和特定挑战主题成像生物标记(03-AR-104)。我们希望检测与皮肤癌发病相关的重要生物标志物，特别是黑色素瘤。具体地说，我们将应用一种新的成像技术(瞬时吸收显微镜)来成像正在形成的皮肤损伤中的黑色素和血红蛋白，无论是固定皮肤还是活皮肤。其目标是在黑色素瘤的早期阶段无创地发现它，并减少皮肤镜检查和组织病理学检查中的假阳性和假阴性。传统的黑色素瘤检测和诊断是一个两级过程，从目测或皮肤镜检查可疑葡萄胎开始，最后切除可疑组织，在显微镜下检查以确认诊断。然而，这种方法面临着两个根本性的挑战。首先是肉眼难以辨别黑色素瘤和良性葡萄胎之间的区别；大多数黑色素瘤都是高度色素的，即使使用皮肤镜，医生也看不到表面下很远的地方。这个问题在很大程度上可以通过切除、HE染色和病理学家的检查来解决，尽管患者通常会出现太多的葡萄胎，无法以这种侵入性的方式进行切除和检测。第二个挑战是，即使是训练有素的观察者，组织病理学的假阴性和假阳性仍然是一个严重的问题。假阴性延误了治疗并增加了死亡率；事实上，黑色素瘤的误诊是美国癌症医疗事故索赔的第二大常见原因(仅次于乳腺癌)。假阳性通过不必要、昂贵和侵入性的程序(包括前哨淋巴结活检和系统辅助治疗)推高了医疗保健成本，并可能使患者无法获得健康或人寿保险。因此，更准确的检测和诊断可能会对患者的生存和医疗成本的降低产生非常大的影响。现有的显微镜方法不能很好地应对这些挑战。反射共聚焦扫描激光显微镜(RCSLM)已经商业化，但由于它不针对特定的生物标志物，因此对比度(散射)缺乏特异性。传统的多光子显微镜可以以微观分辨率成像到组织深处，但有色病变是一个可怕的目标，因为产生的光被重新吸收(而且无论如何，来自黑色素的荧光非常弱)。这里我们使用的是非线性瞬时吸收显微镜，它没有保留多光子显微镜的分辨率优势，但需要样品在新的波长下产生光。这种方法已经存在了几十年，但PI实验室最近的工作利用了先进的飞秒脉冲整形和脉冲序列调制方法来显著提高灵敏度-从而使其能够以适度的功率成像组织。他和他的研究小组已经将这项技术从基础科学转移到临床应用。靶向生物标志物对黑色素瘤的诊断具有重要意义。有很好的证据表明，在黑色素瘤的发展过程中，黑素和真黑素含量之间的局部比例发生了变化；在这里，我们通过显微镜(在黑素小体和黑素细胞中)、在组织中的深度和非侵入性地测量这种分布，显著改善了以前的工作。也有证据表明，微血管和氧合作用(氧合和脱氧血红蛋白，我们也可以无创和深入地成像)与转移潜能相关。总体而言，该项目的研究团队包括化学家、激光技术专家、病理学家和皮肤科医生，以便真正收集产生临床影响所需的专业知识。例如，PI是分子成像和超快激光物理学的先驱(美国物理学会激光科学部当选主席)，但也是化学、放射学和生物医学工程的教授。在第一个目标中，我们专注于用我们的新方法和最好的传统方法来分析未识别的切除的鼹鼠。随着这项工作的发展，我们确定了与癌症发展最相关的分子特征，我们将继续进行新鲜切除的皮肤研究，以验证安全的功率限制(尽管我们目前使用的激光功率比现有商业系统更少)。最后，我们将进入活体动物模型(裸鼠身上移植了诱导性损伤的人类皮肤)，在那里我们还将测量血流和局部组织氧合，作为侵略性新陈代谢的可能标志。到资助期结束时，我们将准备好为体内黑色素瘤的发展提供壮观和新颖的见解，并为人类的临床工作做好准备。 公共卫生相关性：我们提出了一种新的成像技术，该技术可以在发生皮肤病变时对特定的癌症生物标志物进行成像，在不切除的情况下非侵入性地检测早期黑色素瘤，并减少组织病理学中的假阳性和假阴性。减少假阴性将减少癌症死亡；减少假阳性将降低总体医疗成本。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area Biomarker Discovery and Validation (03) and specific Challenge Topic Imaging Biomarkers (03-AR-104). We desire to detect important biomarkers associated with the onset of skin cancers, with a particular emphasis on melanoma. Specifically, we will apply a novel imaging technology (transient absorption microscopy) to image melanins and hemoglobins in developing skin lesions, in both fixed and live skin. The goals are to noninvasively detect melanomas developing in their earliest stages and to reduce false positives and false negatives in both dermoscopy and histopathology. Conventional melanoma detection and diagnosis is a two-tier process that begins with visual or dermoscopic inspection of suspicious moles and ends with the removal of suspected tissue that is examined in a microscope to confirm diagnosis. However, this approach faces two fundamental challenges. The first is the difficulty in visually detecting the differences between melanoma and benign moles; most melanomas are highly pigmented, and even using a dermoscope, doctors cannot see far beneath the surface. This problem is largely addressed by excision, H&E staining, and examination by a pathologist, although patients generally present far too many moles to excise and test in such an invasive manner. The second challenge is that false negatives and false positives from histopathology remain a serious problem even with trained observers. False negatives delay treatment and increase mortality; in fact misdiagnosis of melanoma is the second most common reason for cancer malpractice claims in the United States (after breast cancer). False positives drive up the cost of healthcare with unnecessary, expensive, and invasive procedures (including sentinel lymph node biopsy and systemic adjuvant therapy) and may make it impossible for the patient to obtain health or life insurance. Thus, more accurate detection and diagnosis could have a very large impact on patient survival and health care cost reduction. Existing microscopy methods are not well suited to deal with these challenges. Reflectance confocal scanning laser microscopy (rCSLM) has been commercialized for this application, but the contrast (scattering) suffers from a lack of specificity as it does not target a specific biomarker. Conventional multiphoton microscopy can image deep into tissue with microscopic resolution, but pigmented lesions present a horrible target because the generated light is reabsorbed (and in any event the fluorescence from melanin is extremely weak). Here we use nonlinear transient absorption microscopy, which does not retains the resolution advantage of multiphoton microscopy but does require the sample to generate light at a new wavelength. Such methods have been around for decades, but recent work in the PI's lab has exploited advanced femtosecond pulse shaping and pulse train modulation methods to dramatically increase the sensitivity- thus making it feasible to image tissue with modest powers. He and his research group have migrated this technology from basic science to clinical applications. The targeted biomarkers are significant for melanoma diagnosis. There is good evidence that the local ratio between pheomelanin and eumelanin contents are altered in developing melanoma; here we dramatically improve on previous work by measuring this distribution microscopically (in melanosomes and melanocytes), at depth in tissue, and noninvasively. There is also evidence that microvascularity and oxygenation (oxy- and deoxyhemoglobin, which we can also image noninvasively and at depth) correlates with metastatic potential. Overall, the research team in this project includes chemists, laser technologists, pathologists and dermatologists in order to truly gather the expertise needed to make a clinical impact. For example, the PI is a pioneer in molecular imaging and ultrafast laser physics (and Chair-Elect of the Division of Laser Science of the American Physical Society), but also a professor in Chemistry, Radiology and Biomedical Engineering. In the first aim, we focus on analyzing de-identified excised moles with our new methods and with the best conventional methods. As this work evolves, and we determine the molecular signatures which best correlate with cancer development, we will progress to freshly excised skin studies, in order to validate safe power limits (even though we currently use less laser power than existing commercial systems). Finally, we progress to a live animal model (human skin with induced lesions grafted on nude mice), where we will also measure blood flow and local tissue oxygenation as likely markers of aggressive metabolism. By the end of the grant period, we will be poised to provide spectacular and novel insight into the development of melanoma in vivo, and be ready for human clinical work. PUBLIC HEALTH RELEVANCE: We propose a novel imaging technology which can image specific cancer biomarkers in developing skin lesions, to noninvasively detect early melanomas without excision and to reduce false positives and false negatives in histopathology. Reducing false negatives would reduce cancer fatalities; reducing false positives reduces overall healthcare costs.