1. TYPE-SPECIFIC HUMAN PAPILLOMAVIRUS IN ENDOCERVIX, VAGINA, AND PERINEUM; IMPLICATIONS FOR VAGINAL SELF- COLLECTION Robert G. Pretorius, MD, Jerome L. Belinson, MD, You-Lin Qiao, MD, Ph.D., He Wang, MD, Jennifer S. Smith, Ph.D., M.P.H., Jing Li, MD, Frank J Taddeo, Ph.D., Shangying Hu, MD, and Raoul J. Burchette, MS Supported by Preventive Oncology International™; The Cancer Institute/Hospital, Chinese Academy of Medical Sciences, Beijing, China; Taussig Cancer Center of The Cleveland Clinic Foundation; and Merck, Inc.

4. SENSITIVITY AND SPECIFICITY FOR ≥CIN 2 OF VAGINAL SELF-COLLECTED AND ENDOCERVICAL SPECIMENS TESTED FOR HR-HPV WITH hc2 Sensitivity: Cervix=96.8% vs. Self=87.5%, p<.001 Specificity: Cervix=79.7% vs. Self=77.2%, p<.001 Belinson JL et al. Int J Cancer 2003;13(6):819-26 ≥CIN 2 ≤CIN 1 Self-Collection Positive 328 1850 2178 Self-Collection Negative 47 6272 6319 375 8122 8497 Endocervix Positive 363 1652 2015 Endocervix Negative 12 6470 6482 375 8122 8497

5. SHANXI PROVINCE CERVICAL CANCER SCREENING STUDY III Multi-center, population-based, cross sectional study May 2006-April 2007 Rural Provinces of Henan, Xinjiang, Shanxi, China Urban centers in Beijing and Shanghai, China Not pregnant, no prior radiation, hysterectomy, previous treatment for cervical cancer, or positive HIV 16-54 years of age

6. SHANXI PROVINCE CERVICAL CANCER SCREENING STUDY III Consent obtained Blood drawn and stored at -84 degrees C Vaginal self-collection: Conical-shaped brush inserted high into the vagina rotated three times and withdrawn Physician-collected perineal, lower vagina, upper vagina, and endocervical specimens Cervical cytology specimen

7. SHANXI PROVINCE CERVICAL CANCER SCREENING STUDY III Endocervical and vaginal self-collected specimens were tested for HR-HPV with hc2. Liquid-based cervical cytology prepared by AutoCyte® Linear Array® type specific HPV assay on women with positive HR-HPV testing by hc2 in endocervical or self- collected specimens, a random sample of 3.4% of women with negative hc2, and those with ≥CIN 2 Colposcopy and P.O.I. 5-biopsy protocol if HR-HPV by hc2 in the endocervical or self-collected specimens or cytology other than normal or ASC-US

8. SHANXI PROVINCE CERVICAL CANCER SCREENING STUDY III 2,625 participating women with no missing HPV tests 395 of the 405 women eligible for colposcopy and biopsy had this evaluation 47 of 2,625 (1.8%) women had ≥CIN 2

9. SENSITIVITY AND SPECIFICITY FOR ≥CIN 2 OF VAGINAL SELF-COLLECTED AND ENDOCERVICAL SPECIMENS TESTED FOR HR-HPV WITH hc2 Sensitivity: Cervix=97.9% vs. Self=80.9%, p=.008 Specificity: Cervix=90.2% vs. Self=88.6%, p=.001 [both McNemar test] ≥CIN 2≤CIN 1 Self-Collection Positive 38 294 332 Self-Collection Negative 9 2284 47 2578 2625 Endocervix Positive 46 253 299 Endocervix Negative 1 2325 2326 47 2578 2625

10. HPV BY LINEAR ARRAY IN FALSE NEGATIVE SELF-COLLECTED OR ENDOCERVICAL HR-HPV BY hc2 HR-HPV: hc2 HR-HPV Pos Linear Array HPV Neg HR-HPV Neg LR-HPV Pos False Negative Self-collection Endocervix 88.9% (7/9) 100.0% (1/1) 22.2% (2/9) 0.0% (0/1) 0.0% (0/9) 0.0% (0/1)

11. MEAN SIGNAL STRENGTH (RLU/CO) BY hc2 IN 34 WOMEN WITH ≥CIN 2 WITH POSITIVE HR-HPV BY hc2 IN ENDOCERVICAL, UPPER AND LOWER VAGINAL, AND SELF- COLLECTED SPECIMENS 1 688.2 vs. 118.0, p<.001, 2 688.2 vs. 51.1, p<.001, 3 688.2 vs. 273.5 p=.004, 4 118.0 vs. 51.2, p=.015 [all paired t-test, two tail] EndocervixUpper Vagina Lower Vagina Vaginal Self- collection Mean Strength 688.2 1,2,3 118.0 1,4 51.2 2,4 273.5 3

12. HPV BY LINEAR ARRAY AND MEAN SIGNAL STRENGTH (RLU/CO) BY hc2 IN FALSE POSITIVE SELF-COLLECTED OR ENDOCERVICAL HR-HPV BY hc2 1 252.1 vs. 7.4, p<.001, 2 252.1 vs. 66.9, p<.001, 3 7.4 vs. 66.9, p=.04 4 293.1 vs. 4.3, p<.001, 5 293.1 vs. 96.1, p<.001, 6 4.3 vs. 96.1, p=.02 [all t-test, two-tail, unequal variances] HR-HPV: hc2 HR-HPV Pos Linear Array HPV Neg HR-HPV Neg LR-HPV Pos False Positive Self-collection Mean Strength Endocervix Mean Strength 74.8% (220/294) 252.1 1,2 77.8% (196/253) 293.1 4,5 15.3% (45/294) 7.4 1,3 13.4% (34/253) 4.3 4,6 9.9% (29/294) 66.9 2,3 9.1% (23/253) 96.1 5,6

13. OF 220 WOMEN WITH SELF-COLLECTED SPECIMENS FALSE POSITIVE HR-HPV BY hc2 AND FALSE POSITIVE HR-HPV BY LINEAR ARRAY 55 (25%) HAD HR-HPV BY LINEAR ARRAY SOLELY IN THE VAGINA (i.e.. HR-HPV BY LINEAR ARRAY IN ENDOCERVIX WAS NEG) Endocervix HR-HPV Negative Endocervix HR-HPV Positive 16 or 18 HPV 30.9% 1 (17/55) 28.5% 1 (47/165) Mean Strength 21.1 2 329.1 2 1 30.9% vs. 28.5%, p=.7 [Chi-Square], 2 21.1 vs. 329.1, p<.001 [t-test, two-tail, unequal variances]

14. WEIGHTED PREVALENCE OF HR-HPV AND LR-HPV BY LINEAR ARRAY IN FIVE ANOGENITAL SITES 1 5.1% vs. 19.8%, p=.0004 [method of Raoul.J.Burchette@kp.org] EndocervixUpper Vagina Lower Vagina PerineumSelf- collection HR-HPV 9.5% (250/2625) 14.2% (371.6/2625) 14.0% (366.8/2625) 14.8% (389.5/2625) 12.0% (315.4/2625) LR-HPV 5.1% 1 (132.8/2625) 12.9% (338.3/2625) 19.8% 1 (520.2/2625) 14.3% (375.7/2625) 7.6% (197.8/2625)

15. PROPORTION OF TYPE 16 OR 18 HPV AMONG LINEAR ARRAY HR-HPV POSITIVE SPECIMENS Endocervix Upper Vagina Lower Vagina PerineumSelf- collected 16/18 HPV 39.6% 1 (99/251) 39.5% 1 (122/310) 39.5% 1 (120/305) 40.9% 1 (108/264) 38.0% 1 (108/284) 1 39.6% vs. each other proportion, all p≥.7 [Chi-Square]

16. SENSITIVITY FOR ≥CIN 2 OF HR-HPV BY hc2 AND HR-HPV BY Linear Array FOR FIVE ANOGENITAL SITES 1 97.9% vs. 85.1%, p=.03, 2 97.5% vs. 46.8%, p<.001, 3 97.9% vs. 80.9%, p=.008, 4 95.7% vs. 80.9%, p=.02. [all McNemar test] Anogenital SiteSensitivity HR-HPV hc2 Sensitivity HR-HPV Linear Array Endocervix 97.9% 1,2,3 (46/47) 100.0% (47/47) Upper Vagina 91.5% (43/47) 97.9% (46/47) Lower Vagina 85.1% 1 (40/47) 95.7% (45/47) Perineum 46.8% 2 (22/47) 91.5% (43/47) Self-collection 80.9% 3,4 (38/47) 95.7% 4 (45/47)

17. CONCLUSIONS The lower sensitivity for ≥CIN 2 and higher number of false negative vaginal self-collected specimens tested for HR-HPV by hc2 appears secondary: 1. To the lower viral loads of HR-HPV found in the true positive self-collected specimens.

18. CONCLUSIONS The lower specificity for ≥CIN 2 and higher number of false positive vaginal self-collected specimens for HR-HPV tested by hc2: 1.Appears secondary to the 25% of false positive HR-HPV by hc2 and Linear Array present solely in the vaginal self-collection. 2. To a lesser extent appears secondary to cross- reaction of hc2 with LR-HPV present in excess in the vagina 3.Is not secondary to a higher proportion of type 16 or 18 HPV in the endocervix

19. CONCLUSIONS The sensitivity for ≥CIN 2 of HR-HPV testing of vaginal self-collected specimens might be increased to that approaching HR-HPV testing of endocervical specimens by: 1.Using a HR-HPV assay with a lower cut point (which will decrease the specificity of the screening test further) or 2.Using a vaginal self-collecting device which helps women obtain a larger specimen from the upper vagina

20. LIMITATIONS OF SPOCCS III Verification bias secondary to colposcopy and biopsy for only 395 of 2,625 participating women Endpoint of ≥CIN 2 rather than CIN 3 or cancer Vaginal specimens are probably contaminated by endocervical cells Linear Array tests for specific HPV type on only a random 71 of 2,228 women with negative HR-HPV testing by hc2 in endocervical and vaginal self- collected specimens Signal strength, a semiquantative measure of viral load, by hc2 rather than by Linear Array

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