HPV Testing and Cervical Screening in the UK Alex Sargent HPA Manchester

Human Papillomaviruses Mainly infect the anogenital tract ( approx 40 genotypes) Quite often asymptomatic LOW RISK (20 Types including types 6 and 11) Anogenital warts Very rarely found in cancers HIGH RISK (approx 14 types) Precursor lesions (CIN) cervical cancer Most malignancies of the anogenital tract

99.7% of cervical cancers are directly linked to previous infection with a High Risk HPV type Walboomers et al 1999

High-risk HPV Prevalence (N=24,510) Percentage of HR HPV Age Group Kitchener et al 2006

HR HPV Type by Cytology Grade % of Type Specific HPV Prevalence Cytology Grade

MRI Involvement in HPV Malignant progression of laryngeal papilloma associated with human papilloma virus type 6 (HPV-6) DNA. A P Zarod, J D Rutherford, and G Corbitt. J Clin Pathol. 1988; 41: 280–283 Anal human papillomavirus and anal cancer. Tilston P. J Clin Pathol. 1997; 50:625-34 A study of anal intraepithelial neoplasia in HIV positive homosexual men. Lacey HB, Wilson GE, Tilston P, Wilkins EG, Bailey AS, Corbitt G, Green PM. Sex Transm Infect. 1999; 75:172-7 Natural history of cervical human papillomavirus infection in women during their first sexual relationship. Woodman CB, Collins S, Winter H, Bailey A, Ellis J, Prior P, Yates M, Rollason TP, Young LS. Lancet. 2001; 357:1831-6

More recent work has been our involvement in the ARTISTIC Trial Designed to investigate the value of incorporating HPV testing in to the English cervical cancer screening programme • Kitchener at al. Br J Cancer 2006; 95(1): 56-61 • Sargent et al. Br J Cancer 2008; 98(10):1704-9 • Kitchener et al Lancet Oncol. 2009;10(8):748. • Kitchener at al Health Technol Assess. 2009; 13(51):1-150, iii-iv • Sargent et al. J Clin Microbiol. 2010; 48(2), 554-8 • Kitchener at al Eur J Cancer. 2011; 47(6):864-71

ARTISTIC Trial Main findings • Primary cervical screening with combined LBC and HPV testing resulted in only a small reduction in the detection of high grade disease at the next screening round compared to LBC alone • HPV testing, either for triage or as the initial screening test triaged by cytology, would be cost effective with no loss of sensitivity • The screening interval could be increased from 3 to at least 6 years • No significant adverse psychosocial effects were detected

NHS HPV Triage Pilot Studies Studies showed A reduction of inadequate smears (from 9% conventional cytology to 1-2% LBC) • 46% (1680/3681) BNC & 83% (1507/1825) mild dyskaryosis were HPV positive • The rate of repeat smears fell by 74% (70% for BNC & 87% for mild) • Rate of referral to colposcopy more than doubled (increased from 15%-44% for BNC & 37% - 80% for mild) • Direct referral of HPV positive women to colposcopy may lead to increased detection of CIN2+ Legood. BMJ 2006; 332:79-83 Moss. BMJ 2006; 332:83-85

Sentinel Sites project Funded by the NHS Cervical Screening Programme HPV Triage March 2008 -2011, 6 cytology centres (approx. 10% screening population in England) acted as sentinel sites for HPV triage In the event of borderline or mild dyskaryosis cytology, residual material is tested for HR HPV using the Hybrid Capture 2 test (≥2RLU/Co) HPV Positive women are referred to colposcopy HPV Negative women are returned to routine recall (HPV testing has a high NPV) Virology testing centralised in Manchester Virology lab and Bristol Cytology lab

Sentinel Sites project Test of cure In the event of an abnormal cytology report post-treatment, women are referred for colposcopy (standard practice) In the event of a normal cytology report, residual material is tested for high risk HPV by HC2 (≥2 RLU/Co) HPV negative women are referred for 3-year recall ( rather than annual recall for 10 years) and HPV positive women referred for colposcopy HPV-directed referral strategy is of considerable benefit to women in terms of reducing anxiety, uncertainty and the need for repeat smears, as well as reducing work load in cytology Kitchener et al. BJOG 2008; 115:1001-1007

HR HPV Positivity Rate by Referral Site

Improvements to the NHS CSP Increased identification of high grade CIN and increased specificity in women undergoing HPV triage HPV triage offers immediate referral to colposcopy for those who may have significant disease & rapid return to routine recall for women unlikely to have significant disease due to high NPV of HPV testing HPV ToC offers rapid return to routine recall for treated women (approx. 80%) Reduced repeat testing will give rise to savings in primary care and laboratories

HPV TESTING NEW TECHNOLOGIES STUDY .

Qiagen HC2 assay Clinically regarded as the “Gold Standard” Approved cervical specimens include Cytyc ThinPrep PreservCyt® solution & SurePath preservative fluid Signal amplification DNA screening assay Targets 13 HR types Semi-automated system available No internal control for sample integrity Known issues regarding cross-reactivity

Qiagen Hybrid Capture 2 (HC2) Test

The Rapid Capture™ System Courtesy of Digene

ALTERNATIVE HPV TESTS

Basic Methodologies HPV Detection performed by molecular assays - Signal Amplification (HC2 ; Cervista) - Nucleic Acid Amplification (PCR ; TMA ; NASBA)

Screening assays Genotyping assays Designed to detect the group of High Risk HPV Genotypes Some assays also have limited genotyping capacity for types 16 and 18 Genotyping assays Designed to Genotype the majority of the HPV types infecting the Genital Tract-particularly the High Risk Genotypes Usually based on either a line blot assay format or micro-array system As yet of questionable value in the cervical screening programme

Some Commercial Screening Assays Qiagen HC2 GenProbe APTIMA Roche AMPLICOR Roche Real Time Abbott Real Time Hologic Cervista Norchip Bio-Tools Gen ID Genomica

Clinical Validation • In the case of HPV infections there is a big difference between analytical sensitivity and clinical sensitivity /specificity • Meijer CJ et al have recently developed guidelines for high-risk HPV test requirements for primary cervical screening and validation guidelines for candidate HPV assays Int J Cancer 2009 Feb 1 124 (3) 516-20

Guidelines for HPV Testing The sensitivity of the candidate test for CIN2+ should be at least 90%** of the sensitivity of the HC2 assay to Detect Clinical Disease The specificity of the candidate test for CIN2+ should be at least 98% of the specificity of the HC2 assay **For our study the minimum sensitivity has been raised to 95%

New Technologies Study Aim • To demonstrate non-inferiority of any new test relative to Qiagen Hybrid Capture 2, in terms of both sensitivity and specificity for detection of high grade disease (≥CIN2) • To assess the clinical utility of the test for triage of low grade cytology • New tests were assessed at Bristol HPA and Manchester HPA - 2500 SurePath LBC and 2500 ThinPrep LBC

Commercial Assays Under Evaluation New Test Surepath Thinprep Roche Cobas 4800  Abbott rt HPV Gen-probe HPV APTIMA ++ Hologic Cervista HPV ++ non-CE marked

Conclusions All assays so far have proved non-inferior to the HC2 assay Genprobe (SurePath) and Hologic are still under evaluation New tests are highly automated All assays have internal controls Abbott and Roche tests can simultaneously detect and identify types 16 and 18

Possible Future Setting up of sentinel sites to pilot primary HPV testing in cervical screening Use of self sampling to improve coverage of the cervical screening programme

Acknowledgements Prof Henry Kitchener and the ARTISTIC Team Prof Julietta Patnick and members of the NHSCSP Members of the Primary Screening Group Andrew Bailey and Staff in Virology, Manchester Royal Infirmary