The use of viral load as a surrogate marker in predicting disease progression for patients with early invasive cervical cancer with integrated human papillomavirus type 16 Surasak Wanram, PhD; Temduang Limpaiboon, PhD; Chanvit Leelayuwat, PhD; Pissamai Yuenyao, MD; Donald G. Guiney, MD; Viraphong Lulitanond, PhD; Patcharee Jearanaikoon, PhD OBJECTIVE: The purpose of this study was to assess the effectiveness of the use of human papillomavirus type 16 (HPV16) physical status and viral load in combination to predict clinical outcome during cervical development. STUDY DESIGN: A follow-up study was monitored in association with HPV integration and viral load in 121 cervical samples with the use of multiplex quantitative polymerase chain reaction. RESULTS: A significant increase of viral load was found earlier from pre- invasive to invasive groups compared with normal groups, except with clinical staging and clinical outcome. High occurrence of integrated HPV16 was observed in preinvasive (27/44 samples) and invasive cervical carcinoma (40/68 samples). Cervical progression was observed signifi- cantly in most preinvasive (18/27 samples) and invasive cases (25/40 samples) that were infected with integrated HPV. Integrated HPV16 with significant viral load can be used as a predictive marker for tumor progres- sion in the early stage of invasive cervical carcinoma. CONCLUSION: Integrated HPV16 in combination with viral load is a predictive indicator for tumor progression in early invasive stage but not in preinvasive and advanced invasive stage. Cite this article as: Wanram S, Limpaiboon T, Leelayuwat C, et al. The use of viral load as a surrogate marker in predicting disease progression for patients with early invasive cervical cancer with integrated human papillomavirus type 16. Am J Obstet Gynecol 2009;201:79.e1-7. BACKGROUND AND OBJECTIVE Cervical cancer (CXCA) is the second most common malignancy among women worldwide. Human papilloma- virus type 16 (HPV16) is considered to be the major risk factor in the develop- ment of cervical lesions and CXCA. The integration of HPV into the host genome is a major step in cervical carcinogenesis. It promotes the disruption of HPV early genes, such as E1 and E2, which leads to the upregulation of E6 and E7 oncopro- tein expression in cervical lesions. The detection of HPV16 and its viral load reportedly are associated with a higher grade of cervical intraepithelial neoplasia (CIN) and CXCA. However, the usefulness of these markers in the prediction of clinical outcomes in prein- vasive cervical lesions and invasive CXCA has not been studied. The aim of our study was to evaluate the potential use of physical status and viral load of HPV16 DNA in the follow-up eval- uation of preinvasive and invasive CXCA that involves HPV16 infection. MATERIALS AND METHODS In 2005, we collected samples from 221 subjects who were participating in a pro- spective study. All samples were examined blindly by 2 pathologists. They identified preinvasive lesions in 87 cases: 38 cases with CIN I (mild dysplasia), 11 cases with CIN II (moderate dysplasia), 22 cases with CIN III (severe dysplasia), and 16 cases with carcinoma in situ and 31 normal cases. Of the 103 cases of invasive CXCA that were identified, 80 cases were squa- mous cell carcinoma, and 23 were adeno- carcinoma. In staging according to the In- ternational Federation of Gynecology and Obstetrics criteria, 17 cases were at stage I, 43 cases were at stage II, 38 cases were at stage III, and 5 cases were at stage IV. To increase the sensitivity for the detec- tion of the presence of HPV DNA, we am- plified the HPV L1 consensus region by MY11/MY09 followed by GP5 + /GP6 + . Samples that were positive for HPV DNA were analyzed further for HPV16 with E6/E7 type-specific nested multiplex poly- merase chain reaction (PCR). The physical status of HPV16 DNA was determined by quantitative PCR (qPCR) with the use of a Taqman probe. Two HPV16 plasmid DNA clones that con- tained an insert fragment of E2 and E6 were diluted at different copy numbers and used for generating standard curves. E2 and E6 in clinical samples were extrap- olated from these curves. The E2/E6 ratio was calculated for discriminating episomal and integrated form. A copy number of E6 is represented for the HPV16 viral load. For the episomal state, we assumed that E2 and E6 are present in an equimolar pro- portion, whereas E2 is deleted or absent in the integrated state. An E2/E6 ratio of  1 suggests the presence of the integrated form. From the Departments of Clinical Chemistry (Drs Wanram, Limpaiboon, and Jearanaikoon) and Clinical Immunology (Dr Leelayuwat), the Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand; the Departments of Obstetrics and Gynecology (Dr Yuenyao) and Microbiology (Dr Lulitanond), Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; and the Department of Medicine, University of California, San Diego, School of Medicine, La Jolla, CA (Dr Guiney). This study was supported by the Faculty of Associated Medical Sciences, Centre for Research and Development of Medical Diagnostic Laboratories, and Khon Kaen University Research Affairs, Khon Kaen University, Khon Kaen, Thailand. S.W. holds a scholarship from the Commission on Higher Education, Ministry of Education, Thailand. 0002-9378/free © 2009 Mosby, Inc. All rights reserved. doi: 10.1016/j.ajog.2009.03.013 www.AJOG.org Basic Science: Gynecology Research JULY 2009 American Journal of Obstetrics & Gynecology 79