FLUORESCENCE BASED DETECTION AND DIAGNOSIS OF MALIGNANCY

本研究的长期目标是建立一种无创的实时临床能力，用于早期发现、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）荧光和内源荧光，该方法的独特之处在于，利用相同的激发波长（405nm），将这两种荧光的发射结合在一起，形成一个最大信息量的诊断测量事件。局部使用ALA将最大限度地减少手术时间和副作用，并确保患者接受这项技术。虽然这个临床建议侧重于口腔内发育不良和癌，但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:(1)表征健康人口腔黏膜、发育不良和鳞状细胞癌中ALA诱导的内源性荧光；(2)确定健康、发育不良和恶性口腔组织中ALA转化为PpIX的时间药代动力学；(3)为将仓鼠模型的光动力学数据转化为人类奠定基础；(4)确定共存的口腔病理对LIF的影响。(6)比较LIF与常规方法的诊断能力。本文将使用微光荧光冷冻显微镜对60例患者的活组织切片进行人类健康、癌前和恶性口腔黏膜的分析。ALA将作为乳霜或漱口水使用15-90分钟。活检前。激发发生在405nm， PpIX检测在635nm，自身荧光检测在470nm。将确定ALA应用与PpIX荧光测量之间的最佳间隔，以最大限度地提高肿瘤与健康组织的荧光比率。荧光特性将与组织学状态进行比较。荧光特征的条件下共存的不典型增生，癌和临床病理类似白斑，癌将被识别。建立LIF的选择性、特异性和预测能力。这项工作将为非侵入性的、实时的、活体的活检鉴定提供基础，从而减少手术取样，并允许早期治疗。对高危人群进行快速、非专业的筛查将成为可能。

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.