Aberrant DNA Methylation as a Biomarker of HSIL in Liquid-based Pap Tests

描述(申请人提供)：在美国，目前使用巴氏(Pap)试验进行宫颈癌筛查的计划旨在早期发现和治疗癌前高级别鳞状上皮内病变(HSIL)。在每年超过350万在巴氏试验中有可疑或轻微细胞学异常的妇女中，近50万人患有潜在的HSIL。这些发现突出了巴氏试验单独用于检测HSIL的局限性。对于可疑的非典型鳞状细胞病例，致癌HPVDNA检测不是一种有效的分诊检测方法，也不能排除HSIL(ASC-H)和低级别SIL(LSIL)，因为致癌HPV的高患病率(&gt；80%)，因此，所有ASC-H和LSIL的妇女都被转诊至阴道镜检查。过度治疗的风险和与这一管理策略相关的不必要的成本强调了额外辅助检查的必要性，以改进巴氏试验中HSIL的检测。肿瘤抑制基因(TSG)的DNA启动子异常甲基化是一种表观遗传学改变，有助于肿瘤的进展。在50%的癌前病变HSIL和宫颈癌中已检测到TSGs的甲基化，如DAPK1和IGSF4。肿瘤特异性甲基化图谱的鉴定以及敏感和特异的基于聚合酶链式反应的检测方法的可用性，强调了其作为分子生物标记物的潜在用途。这项应用的总体目标是确定TSGs的DNA甲基化异常是否可以作为液体巴氏试验中HSIL的分子生物标记物，以提高HSIL的早期发现。本研究的具体目的是：1)建立一种甲基化特异性定量聚合酶链式反应(Q-MSP)方法，将HSIL与LsIL和阴性的液体巴氏试验区分开来；2)用Q-MSP方法确定可疑的和轻度异常的液体巴氏试验对HSIL的TSG甲基化是否阳性。对于目标1，我们将使用实时荧光定量聚合酶链式反应结合标准的MSP方法和基于残留液体的巴氏试验来建立包括DAPK1和IGSF4在内的TSG的Q-MSP检测方法。对于目标2，我们将优化的Q-MSP方法应用于经活检证实为HSIL的ASC-H和Lsil残余液体巴氏试验，并使用统计方法分析其检测性能。在巴氏试验中帮助检测HSIL的分子生物标记物的开发，给诊断和管理带来了挑战，为未来的临床试验奠定了基础，并可能对宫颈癌筛查产生重大影响。

DESCRIPTION (provided by applicant): In the United States, current screening programs for cervical cancer using the Papanicolaou (Pap) test are aimed at early detection and treatment of precancerous high-grade squamous intraepithelial lesions (HSIL). Of the greater than 3.5 million women who have equivocal or mild cytologic abnormalities on Pap tests each year, nearly 0.5 million have underlying HSIL. These findings highlight the limitations of the Pap test alone for the detection of HSIL. Testing for oncogenic HPV DNA is not an effective triage test in equivocal cases of atypical squamous cells, cannot exclude HSIL (ASC-H) and low-grade SIL (LSIL) due to the high prevalence of oncogenic HPV (>80%) and, as a result, all women with ASC-H and LSIL are referred to colposcopy. The risk of overtreatment and unnecessary costs related to this management strategy underscore the need for additional ancillary tests to improve the detection of HSIL in Pap tests. Aberrant DNA promoter methylation of tumor suppressor genes (TSGs) is an epigenetic alteration that contributes to tumor progression. Methylation of TSGs, such as DAPK1 and IGSF4, has been detected in >50% of precancerous HSIL and cervical cancers. Identification of tumor-specific methylation profiles and the availability of sensitive and specific PCR-based detection methods underlie its potential use as a molecular biomarker. The overall goal of this application is to determine if aberrant DNA methylation of TSGs can serve as a molecular biomarker of HSIL in liquid-based Pap tests to improve early detection of HSIL. The specific aims of this study are to 1) Develop a quantitative methylation-specific PCR (Q-MSP) assay for TSGs that distinguishes HSIL from LSIL and negative liquid-based Pap tests and 2) Determine if equivocal and mildly abnormal liquid-based Pap tests with underlying HSIL are positive forTSG methylation by the Q-MSP assay. For aim 1, we will use real-time quantitative PCR combined with standard MSP methods and residual liquid- based Pap tests to develop a Q-MSP assay for TSGs, including DAPK1 and IGSF4. For aim 2, we will apply the optimized Q-MSP assay to ASC-H and LSIL residual liquid-based Pap tests that have biopsy-confirmed HSIL and use statistical methods to analyze the assay performance. Development of a molecular biomarker that aids in the detection of HSIL in Pap tests that present diagnostic and management challenges forms the basis for future clinical trials and could have a major impact on cervical cancer screening.