Development of a FISH-Based HPV-Associated Cancer Detection Test

描述(申请人提供)：高危型人乳头瘤病毒(HPV)类型在人类癌症中的致病作用已经确定，特别是对于包括宫颈癌和肛门癌在内的肛门生殖区域的癌症。这类癌症的病因学过程被认为涉及HPV感染、持续感染、进展为癌症的直接前驱，以及最终的侵袭(后两个步骤需要额外的宿主致癌事件)。尽管基于细胞学样本中异常细胞和HPV类型的宫颈癌筛查计划取得了成功，但仍迫切需要识别更多的生物标志物，以提高检测癌前病变/癌症的敏感度，从而为美国每年约2,000,000接受阴道镜检查以跟进异常细胞学的妇女提供额外的分诊。3q的获得以及较少程度的5p和20q的获得是HPV相关癌症中常见的基因组异常，因此代表着HPV相关癌症进展的潜在生物标记物。在目前的应用中，癌症遗传公司建议开发和验证一种基于荧光原位杂交(FISH)的HPV相关癌症检测测试(FHACT)，以检测宫颈和肛门细胞学标本中的三种基因组异常。第一阶段；具体目标1：确认宫颈癌前病变和癌细胞学标本中3Q、5P和20Q的获得。200份宫颈细胞学标本将被提交给使用探针的四色FISH，以检测3Q(红色)、5P(绿色)和20Q(金色)的增益，以确认与细胞学严重程度的相关性，并提供对癌前病变/癌症检测的灵敏度的初步估计。如果敏感性与细胞学相当，则将启动第二阶段。第二阶段；具体目标1：验证宫颈细胞学标本中FHACT自动扫描的有效性。FHACT的扫描/评分程序将自动进行，以适应增加的产量。第二阶段；具体目标2：优化1,000份宫颈细胞学标本的FHACT。FHACT的敏感性和特异性将在1,000个宫颈细胞学样本的整个数据集上确定，并在优化截断、探针组合和要评分的细胞核数量后与仅通过细胞学进行比较。第二阶段；具体目标3：优化1,000份肛门细胞学标本的FHACT。将对肛门细胞学标本进行与上述类似的研究，其中针对高危人群的标准护理筛查计划尚未制定。样本将从国家癌症研究所的Castle和Wentzensen博士那里获得，他们正在领导旨在改善癌症预防筛查计划和识别HPV相关癌症的生物标记物的研究。该项目的总体目标是开发和验证一种可靠、敏感和特定的基于FISH的测试，与标准细胞学和HPV分型一起，将显著有助于更准确地检测癌前病变/癌症，影响HPV相关癌症筛查计划中的护理标准建议。与公共健康相关：尽管宫颈癌预防筛查项目取得了成功，但美国仍有许多女性每年都会接受基于异常巴氏试验和致癌病毒试验的医疗程序，后来发现这些检查是不必要的，而且成本高昂，因为这些测试缺乏敏感性。因此，在目前的提案中，将开发和验证一种基于基因的癌症前期和癌症检测测试，该测试使用残留的巴氏或肛门测试材料。在宫颈癌和肛门癌预防筛查计划中实施这项测试应该提供额外的分诊，以确定哪些患者需要医疗随访，哪些患者不需要，从而减少不必要和昂贵的医疗程序的执行，最终影响低资源国家此类计划的人口覆盖率。

DESCRIPTION (provided by applicant): A causative role of high-risk human papillomavirus (HPV) types in human cancer has been established, particularly for those of the anogenital region including cervical and anal cancers. The etiologic course of such cancers is thought to involve HPV infection, persistence of infection, progression to an immediate precursor of cancer, and finally invasion (the latter two steps requiring additional host oncogenic events). Despite the success of cervical cancer screening programs based on the appearance of abnormal cells in cytology specimens and HPV-type, there is a great need to identify additional biomarkers to increase the sensitivity with which precancer/cancer are detected, so providing additional triage of the approximately 2,000,000 women each year in the US who undergo colposcopy for follow-up of abnormal cytology. Gain of 3q and to a lesser extent gain of 5p and 20q, are genomic abnormalities commonly detected in HPV-associated cancers, and as such represent potential biomarkers of HPV-associated cancer progression. In the current application, Cancer Genetics, Inc. proposes to develop and validate a fluorescence in situ hybridization (FISH)-based HPV- associated cancer detection test (FHACT) to detect the three genomic abnormalities in cervical and anal cytology specimens. Phase I; Specific Aim 1: Confirmation of gain of 3q, 5p, and 20q in cervical precancerous and cancer cytology specimens. 200 cervical cytology specimens will be submitted to four- color FISH using probes to detect gain of 3q (red), 5p (green), and 20q (gold), to confirm association with cytologic severity and to provide an initial estimate of the sensitivity for precancer/cancer detection. If the sensitivity is comparable to that for cytology alone, then Phase II will be initiated. Phase II; Specific Aim 1: Validation of automated scanning for the FHACT in cervical cytology specimens. The scanning/scoring procedure for the FHACT will be automated in order to adapt the assay for increased output. Phase II; Specific Aim 2: Optimization of the FHACT in 1,000 cervical cytology specimens. The sensitivity and specificity of the FHACT will be determined for the entire dataset of 1,000 cervical cytology specimens and compared with that by cytology alone, after optimization of cut-offs, probe combinations, and numbers of nuclei to score. Phase II; Specific Aim 3: Optimization of the FHACT in 1,000 anal cytology specimens. Comparable studies to those above will be performed for anal cytology specimens, where standard-of-care screening programs have as yet to be developed for at-risk populations. Specimens will be obtained from Drs. Castle and Wentzensen, at the National Cancer Institute who are leading studies aimed at improving cancer prevention screening programs and identifying biomarkers of HPV-associated cancers. The overall goal of the project then is to develop and validate a robust, sensitive, and specific FISH-based test that together with standard cytology and HPV-typing will significantly contribute to more accurate detection of precancer/cancer, impacting standard-of-care recommendations in HPV-associated cancer screening programs. PUBLIC HEALTH RELEVANCE: Despite the success of cervical cancer prevention screening programs, there are many women in the US, who annually undergo medical procedures based on an abnormal Pap test and oncogenic virus test, which are later found to be unnecessary and costly, due to the lack of the sensitivity of these tests. Thus, in the current proposal a genetic-based precancer and cancer detection test is to be developed and validated, that uses residual Pap or anal test material. Implementation of this test into cervical and anal cancer prevention screening programs should provide an additional triage to identify those patients who require medical follow-up versus who do not, thereby reducing the performance of unnecessary and costly medical procedures, ultimately impacting population coverage of such programs in low resource countries.