HPV INTEGRATION AS A BIOMARKER FOR CIN BEHAVIOR

描述（由申请人提供）宫颈人乳头瘤病毒（HPV）感染可导致鳞状上皮内瘤变，其可通过增加可引起宫颈鳞状细胞癌的低分化阶段进展。绝大多数宫颈癌与高危HPV类型有关。癌症被认为是由HPV E6蛋白使p53失活和HPV E7蛋白使pRB失活引起的。p53是基因组的监护人，保护免受DNA损伤，pRB是细胞周期中的主要G1检查点。高风险HPV癌蛋白消除这些活性可导致突变的积累并最终导致癌症。鳞状上皮内病变（SIL）进展为癌症的早期事件尚未得到很好的理解。然而，据推测，病毒基因组的物理状态的变化（游离型到整合型）可能会加速癌症的发展。无法以前瞻性方式检验这一假设。然而，高级别癌前病变SIL对局部化疗药物或安慰剂的反应可能为病毒基因组状态与临床反应之间的关系提供见解。约150例高级别SIL患者随机接受安慰剂或局部全反式维甲酸（atRA）治疗。在基线和第12周（研究结束）对受试者进行活检，以确定临床疾病。与接受安慰剂治疗的受试者相比，接受局部atRA治疗4天的受试者更有可能恢复正常或不可治疗的疾病状态（p=0.05）。我们推测，在对atRA无反应的患者中，高比例的SIL含有来自整合的HPV的转录物。这些转录物比来自附加型病毒DNA的转录物更稳定，并且在具有整合序列的细胞中病毒一蛋白的水平更高。atRA可能不能将癌蛋白的水平降低到足够低以诱导含有整合的HPV DNA的那些病变的病变消退。我们将确定从基线和第12周的福尔马林固定活检中获得的逆转录RNA中5'（E7）和3'（E5）序列的比例。如果E7：E5的比率接近1，则序列是附加型的; E7：E5 >1，则由于病毒DNA的整合，E5序列代表性不足。证明atRA耐药SIL含有来自整合的HPV DNA的转录本，将提供一个强大的生物标志物，以识别那些可能失败的非消融治疗和宫颈癌进展风险增加的妇女。

DESCRIPTION (provided by applicant)Human papillomavirus (HPV) infection of the cervix can result in squamous intraepithelial neoplasia that can progress through increasing less differentiated stages that can give rise to squamous cell cancer of the cervix. The vast majority of cervix cancers are associated with high risk HPV types. Cancer is thought to result from inactivation of p53 by HPV E6 and pRB by HPV E7 proteins. p53 is the guardian of the genome and protects against DNA damage and pRB is a major G1 check point in the cell cycle. Abrogation of these activities by high risk HPV oncoproteins can result in the accumulation of mutations and eventually cancer. Early events in the progression of squamous intraepithelial lesions (SIL) to cancer are not well understood. However, it has been postulated that a change in the physical state of the viral genome (episomal to integrated form) may precipitate cancer development. Testing this hypothesis in a prospective manner cannot be done. However, the response of high grade premalignant SIL to topical chemotherapeutic agents or placebo may provide insights into the relationship between state of the viral genome and clinical response. Approximately 150 patients with high grade SIL were randomized to placebo or topical all trans retinoic acid (atRA). The subjects were biopsied at baseline and week 12 (end of study) to determine clinical disease. Subjects treated with topical atRA for four days were significantly more likely to revert to normal or non-treatable disease state than subjects getting placebo (p=0.05). We hypothesize that SIL in a high proportion of patients that failed to respond to atRA contain transcripts derived from integrated HPV. These transcripts are more stable than those derived from episomal virus DNA and the level of viral oneoproteins is higher in cells with integrated sequences. atRA may not be able to decrease the levels of oncoproteins sufficiently low to induce lesion resolution in those lesions containing integrated HPV DNA. We will determine the ratios of 5' (E7) and 3' (E5) sequences in reversed transcribed RNA obtained from formalin-fixed biopsies taken at baseline and week 12. If the ratio of E7:E5 approximates 1 then sequences are episomal; E7:E5 >1 then E5 sequences are underrepresented due to integration of virus DNA. Demonstration that atRA-resistant SIL contain transcripts derived from integrated HPV DNA would provide a powerful biomarker to identify those women who would likely fail nonablative therapy and who would be at increased risk of progression to cervical cancer.