Optical imaging of a CD44-based oral-rinse with fluorescent detection to visualize oral cancer

描述(申请人提供)：口腔鳞状细胞癌(包括口腔和口咽癌)占所有影响这些组织的癌症的90%以上。在所有主要癌症中，口腔癌的五年生存率最低，为62.7%。从历史上看，与这种癌症相关的死亡率特别高，因为它被发现的时间较晚(III期或IV期)，通常是当它扩散到淋巴结或更深的结构时。晚期癌症死亡率高，治疗费用高，发病率高。目前口腔癌诊断的“金标准”是先进行体检和目测，然后进行活检。不幸的是，由于诊断的延误，大多数病例被诊断为晚期，即使经过包括放疗、手术和化疗在内的积极治疗方案，存活率也在20%-40%左右。然而，如果及早诊断(I/II期)，五年存活率可以高达80%-90%，这意味着显著节省了医疗成本。因此，迫切需要一种改进的口腔癌早期检测方法，以挽救生命、改善临床结果并降低治疗成本。我们开发了一种独特的、易于使用的、针对口腔癌的、基于肿瘤起始标志物CD44和总蛋白的快速侧向流动护理点(POC)测试，可以准确地评估患者患口腔癌的风险。OncAlert横向流动试纸从8500万烟酒使用者中识别出这些人患早期或发展为口腔癌的风险最大。然而，这些早期病变通常是肉眼看不见的，因此在非常早期疾病的情况下，仅凭试纸并不能指示异常细胞在哪里。为了满足这一迫切的医疗需求，在这一应用中，我们将开发一种成像设备，它将包括一种简单的冲洗和唾液，其中抗CD44探针结合到不可吸收的微球上，该微球将结合到表达异常高水平CD44的细胞上。然后，可以通过照射特定波长的光来可视化结合的探针，以便可以识别非常早期的损伤。如果成功，这有望在视觉症状显现之前就及早发现和干预Orl癌，从而解决临床上尚未满足的关键需求。我们将实现以下特定目标：在特定目标#1中，我们将测试一个使用膜上荧光探针检测CD44升高细胞的体外模型。我们将确定这种潜在的显像剂是否识别细胞表面的CD44。为了做到这一点，我们将培养特征良好的细胞系，这些细胞系已经被设计为过表达或低表达CD44。在特定目标#2中，我们将在人类肿瘤外植体中测试该系统。我们将确定这种结合物在人类致癌过程中的识别效果。我们将在手术切除后立即在体外使用人体上消化道标本。我们将使用抗CD44荧光标记的珠子分析10例正常切缘的口腔癌组织和10例良性病变(慢性扁桃体炎)切除的患者组织。

DESCRIPTION (provided by applicant): Oral squamous cell carcinomas (including those of the mouth and oropharynx) comprise more than 90% of all cancers affecting these tissues. Of all the major cancers, oral cancer has one of the worst five-year survival rates of 62.7%. Historically, the death rate associated with this cancer is particularly high because it is discovered late (stage III or IV), often when it has spread to the lymph nodes or deeper structures. Late stage cancers have high mortality, are expensive to treat and have high morbidity. The current "gold standard" for oral cancer diagnosis is a physical and visual examination followed by biopsy. Unfortunately, due to delays in diagnosis, most cases are diagnosed in late stage with a survival rate around 20-40% even after aggressive treatment regimens including combinations of radiation, surgery and chemotherapy. However, if diagnosed early (stage I/II), the five-year survival-rate can be as high as 80-90%, representing a significant healthcare cost savings. Thus, there is a pressing need for an improved oral cancer early detection test which will save lives, improve clinical outcome and reduce treatment costs. We have developed a unique, easy to use, oral cancer specific, rapid lateral flow point-of-care (POC) test based on the tumor initiating marker CD44 and total protein that accurately assesses patient's risk for oral cancer. The OncAlert lateral flow test strip identifies those individuals a most risk of harboring early or developing oral cancer from the 85 million tobacco and alcohol users. However, often these early lesions can be invisible to the naked eye, so the test strip alone will not indicate where the abnormal cells are in cases of very early disease. To answer this pressing medical need, in this application, we will develop an imaging device that will involve a simple rinse and spit solution with an antiCD44 probe bound to non-absorbable microspheres that will bind to cells expressing abnormally high levels of CD44. The bound probe can then be visualized by shining a light of specific wavelength so that very early lesions can be identified. If successful, this is expected to enable early detection and intervention of orl cancer even be- fore visual symptoms are revealed, thus addressing a crucial clinically unmet need. We will carry out the fol- lowing specific aims: in Specific Aim #1 we will test an in-vitro model for detecting CD44 elevated cells using fluorescent probes on membranes. We will determine whether this potential imaging agent recognizes CD44 on the surface of cells. To do this we will culture well-characterized cell lines that have been engineered to overexpress or underexpress CD44. In Specific Aim #2 we will test the system in tumor explants from humans. We will determine how well the conjugate identifies carcinogenesis in humans. We will use human up- per aero-digestive tract specimens ex vivo, immediately following surgical resection. We will analyze 10 oral cancer tissues with surrounding normal margin and 10 patient tissues that were excised for benign conditions (chronic tonsillitis) using the anti-CD44 fluorescently labeled beads.