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

描述（由申请人提供）：已确定高危型人乳头瘤病毒（HPV）在人类癌症中的致病作用，特别是肛门生殖器区域的癌症，包括宫颈癌和肛门癌。这种癌症的病因过程被认为涉及HPV感染、感染持续、进展为癌症的直接前体和最终侵袭（后两个步骤需要额外的宿主致癌事件）。尽管基于细胞学标本中异常细胞的出现和HPV类型的宫颈癌筛查计划取得了成功，但仍然非常需要鉴定额外的生物标志物以增加检测癌前病变/癌症的灵敏度，因此每年为美国约2，000，000名接受阴道镜检查以随访异常细胞学的妇女提供额外的分类。3q的增加以及在较小程度上5 p和20 q的增加是HPV相关癌症中常见的基因组异常，因此代表HPV相关癌症进展的潜在生物标志物。在本申请中，Cancer Genetics，Inc.建议开发和验证基于荧光原位杂交（FISH）的HPV相关癌症检测测试（FHACT），以检测宫颈和肛门细胞学样本中的三种基因组异常。I期;具体目标1：确认宫颈癌前病变和癌细胞学标本中3q、5 p和20 q的增加。将200份宫颈细胞学标本进行四色FISH，使用探针检测3q（红色）、5 p（绿色）和20 q（金色）的增益，以确认与细胞学严重程度的相关性，并提供癌前病变/癌症检测灵敏度的初步估计。如果灵敏度与单独细胞学检查的灵敏度相当，则将启动II期。第II阶段;具体目标1：验证宫颈细胞学标本中FHACT的自动扫描。FHACT的扫描/评分程序将自动化，以适应增加输出的检测。II期;具体目标2：在1，000份宫颈细胞学标本中优化FHACT。将针对1,000份宫颈细胞学标本的整个数据集确定FHACT的灵敏度和特异性，并在优化临界值、探针组合和待评分细胞核数量后，与单独细胞学进行比较。II期;具体目标3：在1，000份肛门细胞学标本中优化FHACT。将对肛门细胞学标本进行与上述研究相似的研究，其中尚未为高危人群制定标准治疗筛查计划。标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确定HPV相关癌症生物标志物的研究。该项目的总体目标是开发和验证一种稳健、灵敏和特异的基于FISH的检测方法，该方法与标准细胞学和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标准治疗建议。公共卫生相关性：尽管宫颈癌预防筛查计划取得了成功，但美国仍有许多妇女每年接受基于异常巴氏试验和致癌病毒试验的医疗程序，由于这些试验缺乏敏感性，后来发现这些程序是不必要的，而且费用高昂。因此，在目前的提案中，将开发和验证基于遗传的癌前病变和癌症检测测试，其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该测试应提供额外的分类，以识别需要医疗随访的患者与不需要的患者，从而减少不必要和昂贵的医疗程序的性能，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.