Specific Detection of Cervical Cancers Using Cytometry-Based Molecular Diagnostic

描述（由申请人提供）：宫颈癌是世界范围内发病率和死亡率仅次于乳腺癌的最常见恶性肿瘤。在美国和其他工业化国家，全民使用宫颈细胞学检查（巴氏试验）与宫颈癌发病率和死亡率的急剧下降有关。尽管取得了这样的成功，宫颈病变的细胞学诊断仍然存在一个长期存在的问题，即在低级别细胞学异常的患者中，对具有临床意义的高级别病变的特异性较低。因此，每年有超过400万妇女接受细胞学诊断，需要进一步评估以排除高度不典型增生或癌症的可能性。在大多数情况下，进一步的评估不能确定低级别细胞学异常患者潜在的高级别病变。虽然HPV检测可以在一些患者的分类中发挥重要作用，但它对一些细胞学诊断并不有用。使情况更加复杂的是，简单的高风险hpv检测并不能预测潜在的高级别病变，因为感染并不能表明临床显著的宫颈病变。该项目的长期目标是应用新兴技术开发高通量的基于细胞的分析，具有合适的特异性，以识别宫颈黏膜的高级别癌前病变和恶性病变。本项目使用的方法将采用、测试和验证基于细胞术的分子诊断方法，以检测假阴性宫颈标本。在先前拨款阶段的幌子下，应用p16INK4a和Mcm5（宫颈癌生物标志物）的蛋白表达与高通量流式细胞术和细胞分选已被用于识别和捕获宫颈标本中的罕见癌细胞。此外，一种多重HPV基因分型试验已经实施，以分析这种方法分离的罕见细胞。重要的是，工作流程已经使用当前病理检测协议的通用样品制备实现。本应用程序中应用的技术和方法将通过集成的工作流程实施，具有高度自动化，以评估进一步翻译的可行性，以适应临床需求并提高全球护理标准。最终目标是建立一种有潜力取代宫颈细胞学玻片的初级检测方法，具有更高的灵敏度、更少的主观性和更少的劳动强度。

DESCRIPTION (provided by applicant): Cervical cancer is second to breast cancer as the most common form of malignancy in both incidence and mortality for women worldwide. The population-wide utilization of screening cervical cytology (Pap tests) has been associated with a dramatic decrease in morbidity and mortality from cervical cancer in the United States and in other industrialized nations. Despite this success, the cytologic diagnosis of cervical lesions is plagued by a persistent problem of low specificity for clinically significant high-grade lesions in patients with low-grade cytologic abnormalities. As a result, over four million women each year receive a cytologic diagnosis that requires further evaluation to rule out the possibility of high-grade dysplasia or cancer. In most cases, further evaluation does not identify underlying high-grade lesions in patients with low-grade cytologic abnormalities. Although HPV testing can play an important role for the triage of some patients, it is not useful for several cytologic diagnoses. Complicating the situation is that simple detection of high risk HPVs does not predict an underlying high grade lesion, since infections do not indicate clinically significant cervical lesions. The long-term goal of this project is to apply emerging technology to develop a high-throughput cell-based analysis with suitable specificity to identify high grade premalignant and malignant lesions of the cervical mucosa. The methods to be used in this project will employ, test and validate the approach of cytometry-based molecular diagnostics to detect false negative cervical specimens. Under the guise of the previous grant phase, an application of protein expression of p16INK4a and Mcm5 (cervical cancer biomarkers) with high- throughput flow cytometry and cell sorting has been used to identify and capture the rare cancerous cells in cervical specimens. Furthermore, a multiplexed HPV genotyping assay has been implemented to analyze the rare cells isolated in this approach. Importantly, the work flow has been implemented using common sample preparation with current pathology testing protocols. The technology and methodology being applied in this application will be implemented using an integrated workflow, with substantial automation, to assess feasibility of further translation to accommodate clinical need and to improve the standard of care worldwide. The ultimate goal is to establish a primary assay with potential to supplant slide based cervical cytology with greater sensitivity, less subjectivity, and less labor intensiveness. PUBLIC HEALTH RELEVANCE: This proposed project will apply new technology, for detection of abnormal cervical cells, to clinical samples from previous Pap tests. We will use automated analysis of protein content of cells to isolate abnormal cells, after which automated DNA analysis will determine whether cancer-causing human papillomavirus types are present. These results will be compared to the Pap smear and biopsy results of the same samples in order to determine how well our test can detect early stages of cervical cancer.