CERVICAL CANCER: EPIDEMIOLOGY, ROLE OF HPV TESTING AND HPV VACCINES Ritu Nayar, MD, MIAC Associate Professor of Pathol

1.  CERVICAL CANCER: EPIDEMIOLOGY, ROLE OF HPV TESTING AND HPV VACCINES Ritu Nayar, MD, MIAC Associate Professor of Pathology Director of Cytopathology Laboratory Northwestern University Chicago, USA

2. Cervical Cancer Statistics  Second most common cancer among women worldwide. Globally every year 470,000 new cases are diagnosed, and 230,000 deaths occur, 80% are in low resource countries Incidence highest in sub-Saharan Africa, Melanesia, Latin America and the Caribbean Age standardized rates of 18-33/ 100,000 Largest single cause of years of life lost (YLL) from cancer in developing countries For 2006 USA estimates: 9,700 new cases and 3,700 cancer related deaths Total Resident Pop. (July 2005 est.): 296,496,649 Population density: 79.6 people per sq mi Mean center of population: 3 mi east of Edgar Springs in Phelps County, Mo. Males: 138,053,563 (49.1% of pop.) Females: 143,368,343 (50.9% of pop.) White: 211,460,626 (75.1% of pop.) Black: 34,658,190 (12.3% of pop.) Asian: 10,242,998 (3.6% of pop.) American Indian and Alaska Native: 2,475,956 (0.9% of pop.) Hispanic/Latino2: 35,305,818 (12.5% of pop.) Native Hawaiian and Other Pacific Islander: 398,835 (0.1% of pop.) Median age: 35.3 Metropolitan population: 225,981,679 Nonmetropolitan population: 55,440,227 Families: 71,787,347 Average family size: 3.14 Homeownership (2004): 69.0% of pop. Married couples (2003): 57,320,000 Never married: 48,200,000 Divorced: 19,800,000 Widowed: 13,700,000 Total Resident Pop. (July 2005 est.): 296,496,649 Population density: 79.6 people per sq mi Mean center of population: 3 mi east of Edgar Springs in Phelps County, Mo. Males: 138,053,563 (49.1% of pop.) Females: 143,368,343 (50.9% of pop.) White: 211,460,626 (75.1% of pop.) Black: 34,658,190 (12.3% of pop.) Asian: 10,242,998 (3.6% of pop.) American Indian and Alaska Native: 2,475,956 (0.9% of pop.) Hispanic/Latino2: 35,305,818 (12.5% of pop.) Native Hawaiian and Other Pacific Islander: 398,835 (0.1% of pop.) Median age: 35.3 Metropolitan population: 225,981,679 Nonmetropolitan population: 55,440,227 Families: 71,787,347 Average family size: 3.14 Homeownership (2004): 69.0% of pop. Married couples (2003): 57,320,000 Never married: 48,200,000 Divorced: 19,800,000 Widowed: 13,700,000

3. Evaluation of Cervical Cancer  Screening in the US As of June 2006, as many as 82% of women in the USA have been screened with a Pap test in the past 3 years Estimated that 50% of women diagnosed with cervical cancer have never been screened, and an additional 10% have not been screened in the previous 5 years Screening programs are not reaching women of lower socio economic status, without regular access to health care, uninsured, recent immigrants

4. Cervical Cancer Mortality Disparities Worldwide, ratio of mortality to incidence is 55% GEOGRAPHIC DISPARITIES: Urban-rural gradient RACIAL AND ETHNIC DISPARITIES SOCIOECONOMIC STATUS (SES) RELATED DISPARITIES Incidence rises with increasing poverty and decreasing SES across all racial ethnic groups Screening programs are not reaching women of lower socio economic status, without regular access to health care, uninsured, recent immigrants The same populations of women at higher risk for cervical cancer mortality are also at risk for other screenable and treatable conditions Deep south- alabama and missisippi appalachia: kentucky, west virginiaDeep south- alabama and missisippi appalachia: kentucky, west virginia

5. Cervical Cancer Trends CELL TYPE Squamous cell carcinoma comprises about 75% of cervical cancer; adenocarcinoma about 25% In the USA, proportion of adenocarcinoma relative to SCCa doubled between 1973 and 1996 Adenocarcinoma: HPV appears to be major risk factor. Long term hormonal contraception is a co-factor, but smoking is not. ADCA incidence higher in areas with low incidence of cervical cancer Improvement is needed in sampling, interpreting and treatment of endocervical adenocarcinoma

6. HPV: Cervical Cancer HPV infection over 100 types of HPV recognized >40 types infect the genital area/ cervix �High risk� or oncogenic types 13 subtypes in HC 2, of which 16, 18, 31, 45 cause most cancer worldwide Additional 5 subtypes are probably carcinogenic Most high risk viruses cause low risk lesions Low risk HPV types: Associated with benign epithelial proliferations, not invasive cancer condyloma acuminatum-types 6 & 11

7. Established and Possible Co-factors in Cervical Carcinogenesis Established Cofactors Smoking HIV co-infection High parity Long term oral contraceptive use Probable cofactors Herpes simplex 2 co-infection Chlamydia trachomatis co-infection Diet and nutrition Immunosuppression

8. Role of HPV in Cervical Cancer Prevention and Screening Triage test following an ambiguous cervical cytology (ASCUS) Management of abnormal cytology The key of course though is HOW to translate our understanding of the role of HPV in cervcal cancer pathogenesis into prevention strategies. The evolution of the role of HPV in screening and prevention may be a model for other biomarkers in other tumor models. Data from ALTS showed that presence of HPV DNA identified women at risk for an underdiagnosed HG lesionThe key of course though is HOW to translate our understanding of the role of HPV in cervcal cancer pathogenesis into prevention strategies. The evolution of the role of HPV in screening and prevention may be a model for other biomarkers in other tumor models. Data from ALTS showed that presence of HPV DNA identified women at risk for an underdiagnosed HG lesion

9. ALTS: ASCUS Conclusions HPV triage (sensitivity 92% with 53% of women referred to colposcopy) detects as much CIN 3 as immediate colposcopy spares approx half of women the cost and anxiety of colposcopy Repeat cytology (sensitivity 95% with 67% of women referred to colposcopy) safe option at a threshold of ASCUS women must be compliant with follow-up visits however, the trade off of sensitivity with specificity is not as favorable as with HPV testing.

10. HPV testing: Role In Management of Abnormal Cytology Patient Monitoring During colposcopy in ASC-H, AGC, if lesion not seen Post colposcopy follow up of LSIL when lesion not detected Post LEEP to ascertain cure and/or rule out recurrence Laboratory quality control ASC-US 40%-60% LSIL 75%-85% HSIL >90% Distinguish endocervical vs endometrial adenocarcinoma AGC-- HPV may stratify risk, but overall low rate of AGC and high PPV limit impact of triageAGC-- HPV may stratify risk, but overall low rate of AGC and high PPV limit impact of triage

11. Role of HPV in Cervical Cancer Prevention and Screening More specific markers Type specific testing Multiple HPV types Viral load The key of course though is HOW to translate our understanding of the role of HPV in cervcal cancer pathogenesis into prevention strategies. The evolution of the role of HPV in screening and prevention may be a model for other biomarkers in other tumor models. Data from ALTS showed that presence of HPV DNA identified women at risk for an underdiagnosed HG lesionThe key of course though is HOW to translate our understanding of the role of HPV in cervcal cancer pathogenesis into prevention strategies. The evolution of the role of HPV in screening and prevention may be a model for other biomarkers in other tumor models. Data from ALTS showed that presence of HPV DNA identified women at risk for an underdiagnosed HG lesion

12. Role of HPV in Cervical Cancer More specific molecular markers Cell cycle linked marker p16 Monoclonal antibody detects over expression of p16 INK4a Applicable to cytology and biopsies Proliferation markers Switch from a virally productive to cellular proliferative phenotype correlates with progression to HSIL Ki 67 combined with p16 The key of course though is HOW to translate our understanding of the role of HPV in cervcal cancer pathogenesis into prevention strategies. The evolution of the role of HPV in screening and prevention may be a model for other biomarkers in other tumor models. Data from ALTS showed that presence of HPV DNA identified women at risk for an underdiagnosed HG lesionThe key of course though is HOW to translate our understanding of the role of HPV in cervcal cancer pathogenesis into prevention strategies. The evolution of the role of HPV in screening and prevention may be a model for other biomarkers in other tumor models. Data from ALTS showed that presence of HPV DNA identified women at risk for an underdiagnosed HG lesion

13. Role of HPV in Cervical Cancer Prevention and Screening Type specific HPV testing HPV 16 Absolute risk of CIN3 approaches 40% at 5 yrs of persistence Increasing evidence of value of HPV 16/18 typing for triage of low grade cytologic lesions and post colposcopy follow up Type specific assays likely to be FDA approved Currently only PCR is type specific The key of course though is HOW to translate our understanding of the role of HPV in cervcal cancer pathogenesis into prevention strategies. The evolution of the role of HPV in screening and prevention may be a model for other biomarkers in other tumor models. Data from ALTS showed that presence of HPV DNA identified women at risk for an underdiagnosed HG lesionThe key of course though is HOW to translate our understanding of the role of HPV in cervcal cancer pathogenesis into prevention strategies. The evolution of the role of HPV in screening and prevention may be a model for other biomarkers in other tumor models. Data from ALTS showed that presence of HPV DNA identified women at risk for an underdiagnosed HG lesion

14. Multiple HPV Types and Risk of Cervical CaTrottier H, et al. Cancer Epidem Biomarkers Prev ; July 2006 2462 Brazilian women in the Ludwig-McGill study of natural history of HPV/ cervical neoplasia PCR typing of cervical specimens, 4 years follow up Conclusions: Infections with multiple HPV types seem to act synergistically in cervical carcinogenesis Implications for management of lesions and prediction of outcome of HPV infections

15. Significance of HPV Viral Load  and Multiple HPV Infections Viral load is directly proportional to risk for subsequent cytologic abnormality but NOT a reliable risk factor for histologic CIN3 or cancer C/W current biologic understanding that productive viral infections cause mildly abnormal (ASC-US, LSIL) morphology. Castle PE et al, Cancer 2002; 95: 2145-51 More than 20-30% women have infections with >1 HPV type, regardless of stage of pathology HSIL risk markedly increased with number of HPV types Excess risk for multiple infections, remained after exclusion of women infected with HPV 16, high risk HPV types, or persistent infections Trottier H, et al. Cancer Epidem Biomarkers Prev ; July 2006 2462 Brazilian women in the Ludwig-McGill study of natural history of HPV/ cervical neoplasia: PCR typing of cervical specimens, 4 years follow up.Conclusions:Infections with multiple HPV types seem to act synergistically in cervical carcinogenesis Implications for management of lesions and prediction of outcome of HPV infections 2462 Brazilian women in the Ludwig-McGill study of natural history of HPV/ cervical neoplasia PCR typing of cervical specimens, 4 years follow up Conclusions: Infections with multiple HPV types seem to act synergistically in cervical carcinogenesis Implications for management of lesions and prediction of outcome of HPV infections 2462 Brazilian women in the Ludwig-McGill study of natural history of HPV/ cervical neoplasia: PCR typing of cervical specimens, 4 years follow up.Conclusions:Infections with multiple HPV types seem to act synergistically in cervical carcinogenesis Implications for management of lesions and prediction of outcome of HPV infections 2462 Brazilian women in the Ludwig-McGill study of natural history of HPV/ cervical neoplasia PCR typing of cervical specimens, 4 years follow up Conclusions: Infections with multiple HPV types seem to act synergistically in cervical carcinogenesis Implications for management of lesions and prediction of outcome of HPV infections

16. Role of HPV in Cervical Cancer Prevention and Screening Primary Screening Test The key of course though is HOW to translate our understanding of the role of HPV in cervcal cancer pathogenesis into prevention strategies. The evolution of the role of HPV in screening and prevention may be a model for other biomarkers in other tumor models. Data from ALTS showed that presence of HPV DNA identified women at risk for an underdiagnosed HG lesionThe key of course though is HOW to translate our understanding of the role of HPV in cervcal cancer pathogenesis into prevention strategies. The evolution of the role of HPV in screening and prevention may be a model for other biomarkers in other tumor models. Data from ALTS showed that presence of HPV DNA identified women at risk for an underdiagnosed HG lesion

17. American College of Obstetrics and Gynecology (ACOG) Screening GuidelinesACOG Practice Bulletin # 45 August 2003 Start screening approximately 3 years after the onset of vaginal intercourse; or no later than age 21 For women <30: annual screening (Am Cancer Society: annual if conventional smears, or every two years using liquid- based cytology) For adolescents - have other reasons for gynecologic care even if no Pap For women under 30 - higher likelihood of acquiring hi risk HPV -- premalignant cervical disease should be ruled out before extending interval. For adolescents - have other reasons for gynecologic care even if no Pap For women under 30 - higher likelihood of acquiring hi risk HPV -- premalignant cervical disease should be ruled out before extending interval.

18. ACOG & ACS Screening GuidelinesACOG Practice Bulletin # 45 August 2003 For women > 30 If cytology is negative x 3, screen every 2-3 years combined HPV testing and cytology for screening; if negative/negative then extend interval to every 3 years Why limit to women > 30 ? Why increase interval of screening ? Why add HPV testing? � increased sensitivity cp to cytology alone (studies show 95% sensitivity for CIN3), but also see decreased specificity. Approx 5-15% of women 30+ are HPV positive. Why add HPV testing? � increased sensitivity cp to cytology alone (studies show 95% sensitivity for CIN3), but also see decreased specificity. Approx 5-15% of women 30+ are HPV positive.

19. Outcome Of HPV Positive/Pap Negative Women In Primary ScreeningCastle PE et al, Cancer 2002; 95: 2145-51 Approximately 5% - 15% of women > 30 will be HPV +/ cytology negative Absolute risk of a subsequent abnormal Pap among oncogenic HPV-DNA positive, cytologically negative women Approx 1 in 6 (15%) women with a positive HPV test will develop a detectable cytologic abnormality (>/= ASC) within 5 yrs in a typical clinical setting with yearly screening Why add HPV testing? � increased sensitivity cp to cytology alone (studies show 95% sensitivity for CIN3), but also decreased specificity. Approx 5-15% of women 30+ are HPV positive. 2511 women in Oregon Kaiser study (of total 23,702) Women with c-/hpv+ followed for 57 mths had a incidence of 16.8% >ASC, 6.4% LSIL,, 2.2% >hsil versus hpv �ve / cyto-ve women had > Asc 4.2%, > Lsil 1.1% and > hsil 0.3%. Thus baseline HPV DNA test is a marker for a subsequent abnormal Pap Viral loads unlikely to be useful for predicting future CIN3 / ca among cyto neg women due to sig overlap in viral loads for any cytologic outcome. Why add HPV testing? � increased sensitivity cp to cytology alone (studies show 95% sensitivity for CIN3), but also decreased specificity. Approx 5-15% of women 30+ are HPV positive. 2511 women in Oregon Kaiser study (of total 23,702) Women with c-/hpv+ followed for 57 mths had a incidence of 16.8% >ASC, 6.4% LSIL,, 2.2% >hsil versus hpv �ve / cyto-ve women had > Asc 4.2%, > Lsil 1.1% and > hsil 0.3%. Thus baseline HPV DNA test is a marker for a subsequent abnormal Pap Viral loads unlikely to be useful for predicting future CIN3 / ca among cyto neg women due to sig overlap in viral loads for any cytologic outcome.

20. Interim Management GuidelinesDual Screening Neg HPV- ASC-US HPV- LSIL HPV- Neg HPV+ ASC-US HPV+ LSIL HPV+ HSIL HPV+/- Screen in 3 years Cyto in 12 mth Colpo (2006- likely to be conservative) HPV & Cyto in 6-12 mth Colpo Colpo Colpo Dual screening with HPV and cytology leads to various combinations of test results. Women who test double negative are at very low risk for CIN3 within the next 3 years Approximately 5-15% of women 30+ will be HPV DNA+ and cytology negative. For these women, there isS a higher risk of high grade dx, but still too low to refer directly to colpo. Current interim guidance is to repeat cytology and HPV in 6-12 months. ASCUS HPV- women, we know from ALTS are at 1.4% risk of CIN3 and may be followed with repeat cytology in 12 months. LSIL HPV- is a combination of test results that would not occur with current triage; but with dual testing we will encounter such a combination. In older women, HPV- LSIL may indicate greater likelihood of a FP cytology and these women may be a low risk for CIN3 (NEED MORE DATA)Dual screening with HPV and cytology leads to various combinations of test results. Women who test double negative are at very low risk for CIN3 within the next 3 years Approximately 5-15% of women 30+ will be HPV DNA+ and cytology negative. For these women, there isS a higher risk of high grade dx, but still too low to refer directly to colpo. Current interim guidance is to repeat cytology and HPV in 6-12 months. ASCUS HPV- women, we know from ALTS are at 1.4% risk of CIN3 and may be followed with repeat cytology in 12 months. LSIL HPV- is a combination of test results that would not occur with current triage; but with dual testing we will encounter such a combination. In older women, HPV- LSIL may indicate greater likelihood of a FP cytology and these women may be a low risk for CIN3 (NEED MORE DATA)

21. Cost Analysis For HPV Testing In Screening For Cervical Cancer For women aged 30 yrs or more, every 2 to 3 year screening strategy using either HPV DNA testing in combination with cytology for primary screening OR Cytology with reflex testing for equivocal results Will provide a greater reduction in cancer and is less costly than annual conventional smears Goldie S, Obstet Gynecol 2004; 103; 619-631 A state transition mathematical model was used to simulate the natural history of hpv and cx cancer Annual conv cyto- $ 2457 over a lifetime of a woman, decreases cx ca by 89% VS trinneal LBP lifetime with reflex HPV $ 1358 per woman and decreases cx ca by 90%. Triennial LBP with HPV combined test dec cx ca by 90-92% and is 30% less costly than annual conv cyto ( $ 1735 per woman )A state transition mathematical model was used to simulate the natural history of hpv and cx cancer Annual conv cyto- $ 2457 over a lifetime of a woman, decreases cx ca by 89% VS trinneal LBP lifetime with reflex HPV $ 1358 per woman and decreases cx ca by 90%. Triennial LBP with HPV combined test dec cx ca by 90-92% and is 30% less costly than annual conv cyto ( $ 1735 per woman )

22. HPV Vaccine Goals Multivalent Long lasting protection, preferably without booster doses Suitable for developing countries, with minimal financial and logistic demands on the health care system Low production cost, long shelf life, require single dose, administered orally or via nasal spray rather than by injection

23. Prophylactic HPV Vaccine Administered before children become sexually active, ideally both genders Would work primarily by stimulating antibody mediated immunity, thus inducing neutralizing antibodies at mucosal surfaces ? Maintaining sustained antibody levels at mucosal surfaces over prolonged periods of time may be difficult Impact on prevalence on cervical cancer will be prolonged

24. Prophylactic HPV Vaccines Viral Like Particles (VLP) vaccines Generated by recombinant technology VLP�s produced by cloning the major viral capsid genes (L1) from different HPV types, inserting them into yeast or bacculovirus vectors and then producing large amounts of the L1 proteins of each HPV type separately in eukaryotic tissue culture system. The recombinant proteins are purified and self-assemble into VLPs that appear structurally similar to HPV virions but lack DNA and RNA so are non- infectious and non-oncogenic

25. HPV 16 L1 VLP Proof of Principle Efficacy Trial Placebo controlled trial of 2392 ; 16-23 year old women 3 intramuscular doses of HPV16 L1 VLP vaccine with alum adjuvant or placebo Mean duration of follow-up 17.4 months 100% protection against HPV16 infection and cytologic abnormalities Protection was type-specific 22 new HPV infections (non HPV 16) in placebo arm and 22 in vaccine arm

26. HPV Vaccines Merck�s Gardisal, licensed in June 2006 Approved for females, 9-26 years of age Protects against HPV 16,18, 6,11 Cost is US $360 for 3 doses GSK�s Cervarix, licensure in USA by FDA likely in early-mid 2007 Protects against HPV 16, 18 HPV Cross protection against HPV 45 and 31 (Harper, Lancet 2006)

27. First Generation HPV Vaccines Viral Like Particles (VLP) vaccines Both vaccines are highly immunogenic- produce much higher level of neutralizing antibodies than natural infection In clinical trials, virtually 100% recipients seroconvert and protection against persistent HPV 16/18 infection is 100% at 5 years

28. Implications of HPV Vaccines Females are not protected if they have been infected with HPV prior to vaccination HPV vaccine does not protect against less common HPV types, not included in the vaccine Duration of protection, need for booster doses, not certain Routine vaccination of 11-12 year old girls will take decades to show a discernable effect on the incidence of cervical cancer; screening needs to continue for other women

29.  Primary Prevention with  HPV Vaccines Wright et al. Vaccine 24S3 2006 Mathematical models of cervical cancer predict Vaccine for HPV 16/18 will reduce but not eliminate the risk for cervical cancer In countries with cervical cancer screening programs, vaccines may significantly decrease HPV 16/18 associated CIN2/3 lesions and invasive cancer Age of vaccination is likely to be influential on the relative benefits and costs of primary prevention Adolscent prior to sexual activity-greatest long term impact but challenges in 3 doses/ wide spread coverage Older- more likely compliance but likely already exposed In coutries without screening impact high Poorest countries- vaccinateadolescent, older hpv and cyto if pos Brazil- vaccinate young girls, 2 screens between 35-45 If only 9-13 yrs- will take 20 yrs before any impact on cer ca seen. Full effects- 30-40 years Hpv testing not good measure of infection- pos result threshold set to detect cervical neoplasia us acip says 13- 26 years also vaccinateeAdolscent prior to sexual activity-greatest long term impact but challenges in 3 doses/ wide spread coverage Older- more likely compliance but likely already exposed In coutries without screening impact high Poorest countries- vaccinateadolescent, older hpv and cyto if pos Brazil- vaccinate young girls, 2 screens between 35-45 If only 9-13 yrs- will take 20 yrs before any impact on cer ca seen. Full effects- 30-40 years Hpv testing not good measure of infection- pos result threshold set to detect cervical neoplasia us acip says 13- 26 years also vaccinatee

30. Implications of HPV Vaccines Impact on adenocarcinoma Castellsague X, et for IARC multicenter cervical cancer study group. JNCI, March 2006 Potential to prevent 86% of adenocarcinoma worldwide, without large differences across regions Preventive potential larger than that for squamous cervical cancer, which is estimated to be about 70%

31. Issues Related to Global HPV Vaccine Implementation Cost Acceptability Prioritization among other major health problems (malaria, TB, AIDS, etc) Education of consumers, physicians

32. Ongoing modifications to improve on the current HPV vaccine Goals of vaccine modifications: To protect against more HPV types To improve vaccine implementation in the developing world: needle-less vaccination To add a therapeutic component: to induce regression of prevalent HPV infection in addition to preventing new infection

33. Therapeutic HPV Vaccine Help women infected with HPV Help prevent low-grade disease progression Cause existing lesions to regress Control the spread of metastatic cancer Prevent recurrence of cervical cancer after treatment Must prompt cell-mediated immunity in order to be effective, since antibodies cannot reach and eliminate the virus once it has incorporated into host cells

34. Current Predictions about Role of Cervical Cytology In the near future cytology still needed for primary screening With or without HR-HPV testing Follow up of LSIL and post treatment HSIL patients In the future it is likely that HR-HPV testing Will be primary screening test, followed by either cytology or type specific HPV typing for triage of positive tests Will be more useful for post treatment follow up