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Human Papillomavirus infection is not a major risk factor for ESCC and GCA in high risk area in China.

Human Papillomavirus infection is not a major risk factor for ESCC and GCA in high risk area in China. Wenqiang Wei et.al Dept. of Cancer Epidemiology, Cancer Institute / Hospital CAMS Genetic Epidemiology Branch, DCEG, National Cancer Institute

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Human Papillomavirus infection is not a major risk factor for ESCC and GCA in high risk area in China.

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  1. Human Papillomavirus infection is not a major risk factor for ESCC and GCA in high risk area in China. Wenqiang Wei et.al Dept. of Cancer Epidemiology, Cancer Institute / Hospital CAMS Genetic Epidemiology Branch, DCEG, National Cancer Institute Dept. of Epidemiology, Bloomberg School of Public Health, The Johns Hopkins University Dept. of Applied Tumor Biology, Institute of Pathology, University of Heidelberg

  2. Outline • Background • Studies from Our Group

  3. Background • Esophageal squamous cell carcinoma (ESCC) is the fourth leading cause of cancer death in China. • HPV infection has been suggested to have a role in the etiology of ESCC. HPV vaccine given the potential for prevention through use of the vaccine if HPV is involved. • But…., the results of previous studies have not been consistent.

  4. Background • The argument support for HPV involvement in esophageal carcinogenesis: • biologic similarities between the esophagus and the cervix (a squamous mucosal surface) • shared exposure of esophageal and oral epithelium, as an association between HPV and tonsillar cancer has already been established • HPV detection in esophageal cancer tissues and precursor lesions • In vitro evidence of HPV-induced transformation of esophageal cells.

  5. Background • The argument against the involvement of in esophageal carcinogenesis: • high variability in HPV-positivity among esophageal cancer cases(0%-100%) • Some have argued that any true association between HPV and esophageal cancer would not vary geographically since HPV is a common infection worldwide • inconsistent serologic risk estimates, with estimates from the largest studies tending toward the null • lack of documented associations between ESCC and sexual behavior, immunosuppression, or previous HPV-associated malignancy.

  6. 2. Studies from Our Group • Cross sectional study • Prospective study • Multi-center Lab Study

  7. Cross sectional study • Objective: HPV infection distribution in High risk area of ESCC in Linxian, China. • Balloon cytology • HC-II methods (13 HR,5 LR types)

  8. Cross sectional study

  9. Conclusion from Cross sectional study • High-risk HPV types in 12% of subjects with no evidence of squamous dysplasia and a similar proportion of individuals with mild, moderate and severe dysplasia. • Suggest that HPV infection is not a major risk factor for ESCC in this high-risk population. No association between HPV infection and the neoplastic progression of esophagealsquamous cell carcinoma: Result from a cross-sectional study in a high-risk regionof China. International Journal of Cancer: 119: 1354–1359, 2006

  10. Prospective study • Baseline serum sample, ELISA assays for detecting antibodies • prospectively examined potential associations between HPV 16, HPV 18 and HPV 73 and the occurrence of upper gastrointestinal cancers • Case and control subjects were selected from the 29,584 participants of the Linxian General Population Trial. • Prediagnostic serum samples from 99 cases of ESCC, 100 cases of GCA, 70 cases of GNCA, and 381 age- and sex- matched controls were selected for this study.

  11. Prospective study • Fewer than 15% of ESCC, GCA, or GNCA cases were positive for each HPV type, and no significant associations were found. • Do not support a major role for these HPV types in the etiology of esophageal and gastric cancers in Linxian. International Journal of Cancer: 119: 579-584, 2006

  12. Summary of the potential reason from previous study • The variability may comes from: • Small study sizes • inter-laboratory variability • differences in laboratory detection methods • contamination during sample collection or laboratory processing

  13. Multi-center lab study

  14. Objective The overall objective is to test the hypothesis that HPV is involved in the pathogenesis of ESCC/GCA in Linxian, China. • To determine the prevalence of HPV DNA in ESCC/GCA • To determine the activity of HPV in HPV DNA positive cases

  15. Methods • Subjects recruitment • ESCC /GCA cases finished the informed consent, Demographic data were abstracted from medical records. • Blood sample and resection specimen collection followed by rigorous sterile procedures • Specimen Collection and Processing • Careful sterile technique, including sterile equipment, gloves, and sleeve covers, was used to prevent contamination while processing specimens from ESCC/GCA cases.

  16. Methods • 3. HPV DNA Testing • Frozen tissue - the Roche HPV Linear Array Assay. • Formalin-fixed paraffin embedded tissue specimens - SPF10 LiPA25 version 1 • 4. Evaluation of HPV Oncogene Activity • Cases positive for HPV DNA - immunohistochemical analysis of p16INK4a over-expression.

  17. Result - ESCC Table 1. Distribution of characteristics among 272 esophageal squamous cell carcinoma cases in Linxian, China

  18. Patients came from 13 of the 31 Provinces of China including both the high-risk Taihang mountain region (27.6%) and other areas (72.4%).

  19. Result - ESCC Table2 The prevalence of HPV infection on ESCC • @ The β-globin signal was absent for 5 cases (1.8%). 267cases have the adequacy of the DNA • # One male case HPV89 weakly positive. • $ One female for HPV16 and one male for HPV31.

  20. Result - ESCC • None of the three HPV DNA-positive cases exhibited p16INK4a protein over-expression. • The HPV16-positive case was sero-negative for HPV16 E6 and E7 antibodies. * No role for human papillomavirus in esophageal squamous cell carcinoma in China.Int J Cancer. 2010 Jul 1;127(1):93-100

  21. Result - GCA Table 2. Distribution of characteristics among 144 GCA in Linxian, China

  22. Result - GCA • The β-globin signal was strong for 71% of cases (n =102), weak for 4% (n = 6), and absent for 25% (n = 36).Thus, β-globin, and therefore DNA quality, was adequate in 75% (108 of 144) of cases. • Among the 108 cases with adequate β-globin, all were negative for HPV DNA by Linear Array and E6/E7-based PCR (100%; 95% CI 97-100%). *The gastric cardia is not a target for human papillomavirus-induced carcinogenesis. Cancer Epidemiol Biomarkers Prev. 2010 Apr;19(4):1137-9. Epub 2010 Mar 23.

  23. Conclusion • This study provides the most definitive evidence that HPV is not involved in ESCC carcinogenesis • HPV DNA contamination cannot be ruled out as an explanation for high HPV prevalence in ESCC tissue studies with less stringent tissue collection and processing protocols. • HPV infection does not contribute to gastric cardia carcinogenesis in this part of China.

  24. Acknowledgement • NCI NIHCI/H CAMS • Dr. Christian C. Abnet Dr. Youlin Qiao Dr. Sanford M. Dawsey Dr. Wen Chen Dr. Philip R. Taylor Dr. Jian-Song Ren Dr. Farin Kamangar Dr. Jian-Bing Wang Dr. Jill Koshiol Dr. Zhi-Wei Dong Dr. Mark J. Roth Dr. Philip E. Castle, • University of Heidelberg • Dr. Magnus von Knebel Doeberitz • Svetlana Vinokourova, PhD • The Johns Hopkins University • Dr. Patti Gravitt, PhD • Dr. Rapheal Viscidi, MD

  25. Thanks for you time

  26. 高发区林县拉网细胞中HPV感染情况

  27. Using PGMYprimer, detect 37HPV types. • High risk: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, 26, 53, 66 • Low risk: 6, 11, 40, 42, 54, 55, 61, 62, 64, 67, 69, 70, 71, 72, 81, 83, 84, IS39, CP6108 Linear Array Kit

  28. HPV PCR 3x8 μm HPV PCR BU 3x 8 μm 样品准备：病理组织切片（三明治法） 阴性对照 3x 4 μm HE 1x 4 μm HE 1x 4 μm p16 1x 4 μm 食管癌病理组织 • An HPV negative quality control block was cut between every 10 ESCC blocks

  29. SPF10 PCR 系统 • 14种高危型别：16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 • 11种低危型别： 6, 11, 34, 40, 42, 43, 44, 53, 54, 70, 74 • 通用引物，扩增50多种HPV型别 • 蜡块提取DNA，容易断裂。PCR片段(65bp)不易受DNA降解的影响 Kleter B. et al. Journal of Clinical Microbiology, 1999; 37(8):2508-2517

  30. LiPA HPV分型检测 • LiPA HPV分型检测技术是以逆杂交为基础。 • 使用SPF10引物扩增HPV基因组L1区，随后合成的生物素酰化的扩增子变性，与特异的寡聚核苷酸探针杂交。 • 杂交和冲洗后，加入抗生物素蛋白链菌素结合的碱性磷酸酶，与先前的杂交物结合。与BCIP/NBT色原体孵育后产生紫色沉淀物。可以检测 25种 HPV型别。

  31. LiPA HPV Genotyping 6 6 16 11 35 53 52 18 11 51 66 43 66 70 56 11 16 54 35 42 51 68 70 单个型别感染 多重感染 高危 HPV 型别: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 低危 HPV 型别: 6, 11, 34, 40, 42, 43, 44, 53, 54, 70, 74

  32. 免疫组化检测 p16INK4a 过表达 • Dako: CIN tec™ p16INK4a Histology Kit • 石蜡包埋活检组织：4μm • 质量控制: ★ 使用CICMAS的肿瘤组织作为阳性对照，正常组织为阴性对照。 ★ 其中一个样品组织切片加阴性对照试剂。

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