FLUORESCENCE BASED DETECTION AND DIAGNOSIS OF MALIGNANCY

拟议研究的长期目标是建立一种非侵入性的实时临床能力，用于早期检测、诊断、监测和筛查癌前病变和恶性病变。基于激光诱导荧光（LIF）技术表征5-氨基乙酰丙酸（ALA）诱导的原卟啉IX（PpIX）荧光和内源性荧光，所提出的方法是独特的，通过利用相同的激发波长（405 nm）将来自这2种类型的荧光的发射组合成一个最大信息量的诊断测量事件。使用局部ALA将最大限度地减少手术时间，副作用，并确保患者接受这种技术。虽然这项临床建议的重点是口腔内发育不良和癌症，结果将直接转化为身体的许多其他领域。所获得的信息也适用于光动力治疗方式。具体目标：为了（1）表征健康人口腔粘膜、异型增生和鳞状细胞癌中ALA诱导的和内源性荧光，（2）确定健康、异型增生和恶性口腔组织中ALA转化为PpIX的基于时间的药代动力学，（3）建立将来自仓鼠模型的光动力学数据转化为人的基础，（4）鉴定共存口腔病理对LIF的影响，（5）评估具有白斑临床表现的病症之间基于LIF的区分（6）比较LIF与常规测量的诊断能力。人类健康，癌前和恶性口腔粘膜将进行分析，使用低光水平荧光冷冻显微镜活检60例患者。ALA将作为乳霜或漱口水使用15-90分钟。在活检之前。激发将在405 nm处发生，PpIX检测在635 nm处发生，自发荧光检测在470 nm处发生。将确定ALA应用和PpIX荧光测量之间的最佳间隔，以使肿瘤：健康组织荧光比率最大化。将荧光特征与组织学状态进行比较。将识别与异型增生、癌共存的病症以及临床上类似白斑、癌的病理的荧光特征。将确定LIF的选择性、特异性和预测能力。这项工作将为非侵入性、实时、体内识别活检的需要提供基础，从而最大限度地减少手术取样，并允许早期治疗。对高危人群进行快速、非专家筛查将成为可能。

Long-term goal of the proposed research is to establish a non-invasive real-time clinical capability for early detection, diagnosis, monitoring and screening of pre-malignant and malignant lesions. Based on laser-induced fluorescence (LIF) techniques for characterization of 5-aminolevulinic acid (ALA)- induced protoporphyrin IX (PpIX) fluorescence and of endogenous fluorescence, the proposed approach is unique in combining emissions from these 2 types of fluorescence into one maximally informative diagnostic measurement event by utilizing the same excitation wavelength (405nm). Use of topical ALA will minimize procedure duration, side-effects and ensure patient acceptance of this technique. Although this clinical proposal focuses on intra-oral dysplasia and carcinoma, results will translate directly to many other areas of the body. The information gained is also applicable to photodynamic therapeutic modalities. Specific aims: To (l) characterize ALA-induced and endogenous fluorescence in healthy human oral mucosa, dysplasia and squamous cell carcinoma, (2) determine time-based pharmacokinetics of ALA conversion to PpIX in healthy, dysplastic and malignant oral tissue (3) establish a basis for translation of photodynamic data from the hamster model to humans (4) identify effects of co-existing oral pathologies on LIF, (5) evaluate LIF-based differentiation between conditions with a leukoplakic clinical presentation (6) compare diagnostic capability of LIF with conventional measures. Human healthy, premalignant and malignant oral mucosa will be analyzed using low-light level fluorescence cryomicroscopy in biopsies from 60 patients. ALA will be applied as a cream or mouthrinse for 15-90 mins. prior to biopsy. Excitation will occur at 405nm, PpIX detection at 635nm, autofluorescence detection at 470nm. The optimum interval between ALA application and PpIX fluorescence measurement to maximize tumor:healthy tissue fluorescence ratio will be identified. Fluorescence characteristics will be compared with histological status. Fluorescence characteristics of conditions co-existing with dysplasia, carcinoma and of pathologies clinically resembling leukoplakia, carcinoma will be identified. Selectivity, specificity and predictive capability of LIF will be established. This work will provide the basis for non-invasive, real-time, in vivo identification of the need for biopsies, thus minimizing surgical sampling, and permitting early treatment. Fast, non-specialist screening of at risk populations will become possible.