SHORT REPORT HPV16 semiquantitative viral load and serologic biomarkers in oral and oropharyngeal squamous cell carcinomas Aime ´e R. Kreimer 1,2 * , Gary M. Clifford 1 , Peter J.F. Snijders 3 , Xavier Castellsague ´ 4 , Chris J.L.M. Meijer 3 , Michael Pawlita 5 , Raphael Viscidi 6 , Rolando Herrero 7 and Silvia Franceschi 1 for the International Agency for Research on Cancer (IARC) Multicenter Oral Cancer Study Group 1 International Agency for Research on Cancer, Lyon, France 2 Cancer Prevention Fellowship Program, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 3 Vrije Universiteit Medical Center, Amsterdam, The Netherlands 4 IDIBELL, Institut Catala` d’Oncologia (ICO), L’Hospitalet de Llobregat, Spain 5 Deutsches Krebsforschungszentrum, Heidelberg, Germany 6 Stanley Division of Developmental Neurovirology, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, MD, USA 7 Instituto Costarricense de Investigacio´n y Ensen˜anza en Nutricio´n y Salud, San Jose´, Costa Rica A considerable subset of oropharyngeal squamous cell carcinomas (SCCs) are positive for human papillomavirus (HPV); however, delineating etiologically-associated HPV infections from SCCs with concurrent HPV infection unrelated to tumorigenesis is challenging. Viral load assessment in biopsy specimens may help facilitate such differentiation. HPV16 viral load and serologic markers were assessed among oral and oropharyngeal cases from a multinational study conducted by the International Agency for Research on Cancer (IARC). HPV16 viral load, measured semi- quantitatively by PCR-enzyme immunoassay, was dichotomized as high or low based on the median optical density value. Serologic antibodies to HPV16 virus-like particles (VLPs) and to HPV16 E6 and E7 proteins were measured by ELISA. Compared to HPV DNA-negative cases (n = 852), HPV16 DNA-positive cases with high viral load (n = 26) were significantly more likely to originate in the oropharynx (odds ratio [OR], 12.0; 95% confidence interval [CI], 5.2–27.5) and, after adjustment for tumor site (AdjOR), have anti- bodies against HPV16 VLPs (AdjOR, 14.6; 95% CI, 6.0–35.6), E6 (AdjOR, 57.6; 95% CI, 21.4–155.3) and E7 (AdjOR, 25.6; 95% CI, 9.3–70.8). HPV16 DNA-positive cases with low viral load (n = 27) were more commonly oropharyngeal (OR, 2.7; 95% CI, 1.1–6.2) and seropositive for HPV16 VLPs (AdjOR, 2.7; 95% CI, 1.1–6.9), E6 (AdjOR, 3.0; 95% CI, 0.7–14.0) and E7 (AdjOR, 3.5; 95% CI, 0.7–16.3), compared to HPV DNA-negative cases; the associations, however, were neither as strong nor as significant as the associa- tions for high viral load. As there appears to be a strong asso- ciation between HPV16 serologic markers and viral load, in the absence of data on serologic markers, HPV16 viral load may be used to help delineate the subset of HPV16 DNA-positive oral and oropharyngeal cancers that may be the consequence of HPV infection. ' 2005 Wiley-Liss, Inc. Key words: human papillomavirus; HPV16 viral load; oral cancer; oropharyngeal cancer Numerous studies have provided consistent evidence that human papillomavirus (HPV), the necessary cause of cervical can- cer, is present in tumor biopsies from approximately 20-50% of oropharyngeal squamous cell carcinomas (SCCs) and a smaller subset of oral SCCs. 1–4 Among HPV DNA-positive oropharyngeal SCCs, 90% are positive for HPV16. 1–4 Nonetheless, HPV DNA detection in tumor biopsies may not be sufficient evidence of cau- sation. HPV16 DNA from tumor specimens analyzed jointly with markers of expression of the viral oncogene E6, mutational pat- terns of the cancer suppressor gene TP53 and levels of allelic loss, have helped identify a subset of these cancers that may be the con- sequence of HPV infection. 1,2,5–8 HPV viral load, a measure of the amount of viral DNA in biopsy specimens, alone or in conjunction with well-characterized HPV serologic assays, may clarify the role of HPV among oral and oropharyngeal cases. Antibodies against HPV E6 and E7 are markers of an invasive HPV-associated malignancy 9,10 and are rarely present among individuals with HPV DNA-negative oral and oropharyngeal tumors. 1 Antibodies against HPV virus-like particles (VLPs) are considered a marker of cumulative, lifetime HPV infection, 11–15 and are associated with HPV-related disease, but not as strongly as E6 and E7 antibodies. While these markers do not allow for inferences on causality, evaluation of the associa- tions between high and low viral load with HPV16 serologic markers among HPV16 DNA-positive and -negative oral and oro- pharyngeal SCCs may delineate the subset more likely the result of HPV infection. Viral load assessment may also compensate for the less than optimal sensitivity in each of the HPV serologic markers currently available. Material and methods Oral and oropharyngeal SCC cases from the International Agency for Research on Cancer (IARC) multinational case- control study of HPV and oral cancer were selected for this analy- sis. The methods and main results of the parent study have been previously reported. 1 Briefly, incident cases with oral and orophar- yngeal SCC were recruited from referral centers and hospitals in 9 countries (Australia, Canada, Cuba, Italy, India, Northern Ireland, Poland, Spain and Sudan) from 1996 to 1999. Following informed consent, biologic samples were collected prior to cancer treatment *Correspondence to: National Institutes of Health, National Cancer Institute, 6120 Executive Blvd., Suite T-41, Bethesda, MD 20892-7361, USA. Fax: 301.480.9939. E-mail: kreimera@mail.nih.gov Received 23 July 2004; Accepted after revision 8 October 2004 DOI 10.1002/ijc.20872 Published online 1 February 2005 in Wiley InterScience (www.interscience. wiley.com). The following investigators contributed to the study: Nubia Mun ˜oz, IARC, Lyon, France; Javier Pintos, McGill University, Montreal, Canada; Frank Kee, Queen’s University Belfast, Belfast, Northern Ireland, United Kingdom; Leticia Ferna ´ndez, Instituto Nacional de Oncologı ´a y Radiobio- logı ´a, Havana, Cuba; Ali Idris, Toombak and Smoking Research Center, Khartoum, the Sudan; Marı ´a Jose ´ Sa ´nchez, Escuela Andaluza de Salud Pu ´blica, Granada, Spain; Adoracio ´n Nieto, Facultad de Medicina, Seville, Spain; F. Xavier Bosch, Institut Catala ` d’Oncologia, Barcelona, Spain; Renato Talamini, Centro di Riferimento Oncologico di Aviano, Aviano, Italy; Alessandra Tavani, Instituto di Ricerche Farmacologiche ‘‘Mario Negri’’, Milan, Italy; Ulrich Reidel, Deutsches Krebsforschungszentrum, Heidelberg, Germany; Jolanta Lissowska, Cancer Center, Warsaw, Poland; Barbara Rose, Sydney Head and Neck Cancer Institute, Royal Prince Alfred Hospital, Sydney, Australia; Hema Sridhar, Kidwai Memorial Insti- tute of Oncology, Bangalore, India; Prabha Balaram, Regional Cancer Centre, Trivandrum, India; Thangarajan Rajkumar, Cancer Institute (WIA), Chennai, India. Int. J. Cancer: 115, 329–332 (2005) ' 2005 Wiley-Liss, Inc. Publication of the International Union Against Cancer