HPV INTEGRATION AS A BIOMARKER FOR CIN BEHAVIOR

宫颈的人乳头瘤病毒（HPV）感染可导致鳞状上皮内瘤变，可通过越来越多的低分化阶段发展为宫颈鳞状细胞癌。绝大多数宫颈癌与高危HPV类型有关。癌症被认为是由p53被HPV E6和pRB被HPV E7蛋白失活引起的。p53是基因组的守护者，防止DNA损伤，pRB是细胞周期中G1的主要检查点。高风险的HPV癌蛋白破坏这些活性会导致突变的积累，最终导致癌症。鳞状上皮内病变（SIL）向癌症发展的早期事件尚不清楚。然而，据推测，病毒基因组物理状态的变化（从个体到整合形式）可能导致癌症的发生。以一种前瞻性的方式检验这一假设是不可能的。然而，高级别癌前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。大约150名高级别SIL患者被随机分配到安慰剂组或局部全反式维甲酸组（atRA）。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。局部atRA治疗4天的受试者比安慰剂组更容易恢复到正常或不可治疗的疾病状态（p=0.05）。我们假设，在对atRA没有反应的患者中，高比例的SIL含有来自整合HPV的转录本。这些转录本比来自附体病毒DNA的转录本更稳定，并且在具有整合序列的细胞中，病毒单蛋白的水平更高。atRA可能无法将癌蛋白水平降低到足以诱导含有整合HPV DNA的病变消退的水平。我们将测定5‘ （E7）和3’ （E5）序列在基线和第12周福尔马林固定活检中获得的逆转录RNA中的比例。如果E7:E5的比值接近于1，则序列是独立的；由于病毒DNA的整合，E7:E5 >1然后E5序列代表性不足。证明抗阿特拉的SIL含有来自整合HPV DNA的转录本，将提供一个强有力的生物标志物，以识别那些可能无法接受非消融治疗的妇女，以及那些将有增加发展为宫颈癌风险的妇女。

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.