1. HPV Testing and Its Impact on Cervical Cancer Screening John M. Douglas, Jr., MD Denver Public Health

2. Genital HPV Infection Over 100 HPV types identified; > 30 genital types Low-risk types (6,11) –genital warts and early Pap smear changes (atypia, LSIL) Higher-risk types (16,18,31,33,35,others) –“flat lesions” (SIL) and both early/late Pap smear changes All types cause subclinical infection

3. HPV and Anogenital Cancer Genital HPV –found in 95% of cervical cancers (and majority of anal, vulvar, vaginal, penile cancers) In vitro studies –interaction between “high-risk” HPV proteins and cellular oncogenes

4. Steps in HPV Induced Cervical Cancer HPV infection Persistent HPV infection Cellular dysregulation High grade CIN Invasive cancer Immunologic factors Co-carcinogens

5. HPV testing and cervical cancer prevention: how to “leverage” the association? Triage Screening? Management? Other??

6. HPV Testing Methods Non-amplified tests commercially available in U.S. since late 80s –Virapap/Viratype (dot blot assay for HPV 6/11, 16/18, 31/33/35) –HPV Profile (dot blot assay for HPV 6/11/42/43/44, and 16/18/31/33/35/45/51/52/56) –Hybrid Capture-I (tube-based solution hybridization assay for same HPV types as Profile, sensitivity threshhold, >10pg/ml) –Hybrid Capture-II (microtiter-based solution hybridization assay; same HPV types as HC-I plus 39/58/59/68) Amplified tests (polymerase chain reaction--PCR) using various primers (most commonly MY09/MYll) are more sensitive but not available in standardized formats

7. Bethesda System WNL - within normal limits; includes reactive and reparative changes ASC (ASCUS)--atypical squamous cells (of undetermined significance); borderline category of equivocal lesions LSIL--Low-grade squamous intraepithelial lesions; combines condylomatous atypia with mild dysplasia (CIN 1) HSIL--High-grade squamous intraepithelial lesions; combines moderate/severe dysplasia (CIN 2 & 3) Squamous Cell Carcinoma

8. HPV Testing for Triage of Low- grade Pap Smear Abnormalities Low-grade abnormalities (ASCUS, AGUS, LSIL) occur in 5- 10% of the 50 million Pap smears in the U.S. annually Most represent processes which will revert to normal without therapy, but minority of smears with ASCUS (5- 15%) and LSIL (10-20%) will have high-grade lesions (CIN2/3) on biopsy (accounting for the majority of CIN2/3 lesions in some settings) Cost of conservative management (colposcopy/biopsy) $ 3- 4 billion per year. New generation commercial HPV tests have high sensitivity (90%) and specificity similar to follow-up Pap smear (65%) Use unlikely to prevent cancer, but could save money; cost- effectiveness analyses will be critical

9. Hybrid Capture-II Assay in Triage of 973 Women with ASCUS--Kaiser Study

10. Kaiser Study: Conclusions HPV testing of ASCUS is highly sensitive and moderately specific for HSIL Similar results for AGUS HPV testing for LSIL reported to be less specific and maybe useful only in women 40+ Testing for HPV with either STM or PreservCyt had similar results Cost-effectiveness analysis pending

11. ALTS STUDY DESIGN Multi-center, NCI clinical trial to determine optimal management of ASCUS, LSIL Patients (>18, not pregnant or immunocompromised, no previous abative therapy) with Pap smear with ASCUS or LSIL in past 6 mos, enrolled at four clinical centers (n=3488) Randomization to 1 of 3 arms –Immediate colposcopy (colpo, biopsy, RX if CIN2/3) –HPV Triage (to colpo if HPV+ or HSIL on Pap) –Conservative management (to colpo if HSIL) Baseline evaluation with Thin-prep, HPV test by HC-II, cervicography (to result in colposcopy if “suspicious for cancer”); 6-monthly FU for 2 yrs with Thin-prep Pap and other study tests; to colpo if HSIL on Pap and RX if CIN2/3 on biopsy

12. Performance of HPV testing vs. Repeat Pap for ASCUS ALTS study*Kaiser study** (n=2324; 11.4% CIN 2/3) (n=973; 6.6% CIN 2/3) HPV test Repeat Pap HPV test Repeat Pap Sensitivity 96%85% 89% 76% Specificity nana 64% 64% PPV 20%17% 15% 13% NPV 99%96% 99% 97% % referred 56%58% 40% 39% *Modified from Solomon D et al. JNCI 2001: 93: 293-9 **Modified from Manos MM et al. JAMA 1999; 281: 1605-10.

13. ALTS Study: LSIL 642 women with LSIL, 532 (83%) HPV+ HPV detection rate same for HC-2 and PCR in 210 samples (86%); some evidence of non-specificity in HC-2 assay High HPV+ rate limits utility of testing for triage of LSIL and this part of ALTS prematurely ended ALTS Group. JNCI 2000; 92: 397

14. HPV Testing for Triage of ASCUS: Issues to Consider Logistics: requires liquid-based Paps or collection of sample transport media, so most appealing in settings using liquid- based testing Cost-effectiveness: will vary depending on local factors (costs, CIN 2/3 and HPV prevalence, FU issues) Counseling: issues of diagnosing an STD for patients and partners

15. HPV Testing in Cervical Cancer Screening Possible use in primary screening under evaluation, either with/instead of Pap smear, especially in older women –to enhance sensitivity combined with Pap smear (of greatest value to reduce screening interval) –as single method in resource-poor settings (eg, potentially cheaper and easier to QA) –as a self-collected sample for outreach settings or clinical situations where screening speculum exam not feasible Proposal to FDA to use in combination with liquid- based Pap smear (“DNA Pap”) pending

16. HPV Screening Studies

17. HPV testing: applications for the future? CIN1: assessment of those who need RX vs. FU without RX? CIN 2/3: post-RX assessment of those who need earlier FU or colposcopy? Anal cancer screening?? Screening exposed sexual partners?? Screening for infection????

18. Top 10 Questions Among Patients with Genital HPV Infection (information from ASHA HPV line) 1. How, when, or from whom did I get HPV? 2. Will HPV affect a pregnancy or a baby? 3. Can a person get or give HPV through oral sex or from hands? 4. How can I get tested for HPV? 5. Will I always have HPV? 6. How can I prevent giving or getting HPV? 7. Can partners re-infect each other? 8. Does HPV cause cervical cancer? 9. What should I tell my partner about HPV? 10. What are best RX options?

19. The 20 minute HPV curbside consult generated by HPV testing 34 yo grad student with LSIL and equivocal (+, -, -, +) HPV test; wants individual typing, referred to public health by private GYN worries: cancer>STD>perinatal long conversations with fiancee who is fortunately very understanding has been all over web: still confused “Has HPV testing helped your life?”: (long pause) “I think so…” “How?”: (longer pause) “...made me more likely to take good care of my health (eg, exercise if exams)…”

21. Triage of ASCUS in a Public Hospital Setting (Shlay et al. ObGyn 2000; (96:410) Patients 30 (n=76) (n=119) CIN 2-3 % 10.5% 5.9% % referred 49% 20% Sensitivity 100% 86% Specificity 57% 84% PPV 22% 25% NPV 100% 99%

22. Triage of ASCUS in a Public Hospital Setting: Cost Comparisons (Shlay et al. ObGyn 2000; (96:410) StrategyCost/CIN2-3 case detected Colpo all$4875 HPV triage (STM vial)$4809 HPV triage (liquid Pap)$4308 Costs: Colpo/biopsy $375; HPV test $48.50; STM/storage $7.50; Liquid Pap extra $5.60

23. Hybrid Capture-II Assay in Triage of ASCUS, by Age--Kaiser study

24. Progress in Diagnostics

25. Hybrid Capture ® 2 HPV DNA Test Cervical Sampler ™

26. Hybrid Capture-II Assay in the Triage of ASCUS Pap Smears: Kaiser Study 46,000 routine Pap smears from four N. CA Kaiser centers Cervical sample with broom for conventional Pap smear; residual rinsed into PreservCyt fluid for HPV testing and Thin- Prep smear Cervical sample with conical brush into STM for HPV testing HPVtesting by Hybrid Capture-II assay (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58; cut-off > 1.0 pg/ml); 89% agreement between STM and PreservCyt Study nurses attempted to contact women with ASCUS (n=1632) or greater: 1340 (82%) returned a mean of 67 days after initial Pap, 995 (61% enrolled), and 973 (60%) had follow- up Pap smear, biopsy, and HPV result. CIN 2/3 detected in 64 (6.6%) and HPV in 384 (39.5%) Unpublished data. Manos et al

27. Psychosocial Impact of HPV Infection (Clarke. Int J STD AIDS 1996; 7:197)

28. Epidemiology of Genital HPV Infection True incidence/prevalence difficult to define Incidence estimated to be 5 million/year (ASHA) and approximately 500,000 initial office visits for genital warts in U.S. annually (NDTI) HPV detectable in 20-50% young sexually active women, rates appear to be similar in men Infection chronic and of variable course, with an estimated prevalence of 24-40 million in U.S.

29. Association of Cervical Cancer and STDs Positive association with: a)Age of onset of sexual activity b)Multiple sexual partners c)Unstable marital relationships d)Male sexual partners with penile cancer or with prior consorts with cervical cancer e)Presence of STD’s: syphillis, gonorrhea, trichomonas, HSV-2, chlamydia, CMV, HPV

30. HPV-Associated Squamous Intraepithelial Lesions (SIL) A synonym for dysplasia/CIN Categorized as –low-grade SIL (LSIL)--HPV-associated changes, CIN 1 –high-grade SIL (HSIL)--CIN 2/3, CIS Can involve anus, vulva, vagina, penis as well as cervix

31. Diagnosis of Cervical SIL Cytology –non-invasive, inexpensive –sensitivity low with single smear, enhanced with repeat –liquid media approach may enhance sensitivity, but reduce specificity Colposcopy/biopsy –recommended to follow-up HSIL –management of ASCUS and LSIL controversial (e.g., colposcopy, repeat Pap, HPV testing)

32. New Developments in HHPV testing Availability: HPV testing for ASCUS covered by over 30 BCBS plans in US (55 million members)

33. Hybrid Capture ® 2 HPV DNA Test Specimen Collection Cervical sampler ™ specimen storage and transport Transport medium –Digene specimen collection kit, or –Cytyc preservcyt ® transport medium Cervical samples –Storage and shipment up to 2 weeks at 2-30º C –Remaining storage at 2-8º C for up to 1 week –Long term storage at - 20º C for up to 3 months

34. HPV Prevalence by Referral Pap Smear and Age--Kaiser Study