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.

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<.001Specificity: 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

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

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• Vaginal self-collection: Conical-shaped brush inserted high into the vagina rotated three times and withdrawn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

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

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

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 2284

47 2578 2625

Endocervix Positive 46 253

299

Endocervix Negative 1 2325 2326

47 2578 2625

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)

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

1688.2 vs. 118.0, p<.001, 2688.2 vs. 51.1, p<.001, 3688.2 vs. 273.5 p=.004, 4118.0 vs. 51.2, p=.015 [all paired t-test, two tail]

Endocervix Upper Vagina

Lower Vagina

Vaginal Self-collection

Mean Strength 688.21,2,3 118.01,4 51.22,4 273.53

HPV BY LINEAR ARRAY AND MEAN SIGNAL STRENGTH (RLU/CO) BY hc2 IN FALSE

POSITIVE SELF-COLLECTED OR ENDOCERVICAL HR-HPV BY hc2

1252.1 vs. 7.4, p<.001, 2252.1 vs. 66.9, p<.001, 37.4 vs. 66.9, p=.044293.1 vs. 4.3, p<.001, 5293.1 vs. 96.1, p<.001, 64.3 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) 252.11,2

77.8% (196/253) 293.14,5

15.3% (45/294) 7.41,3

13.4% (34/253) 4.34,6

9.9% (29/294) 66.92,3

9.1% (23/253) 96.15,6

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 21.12 329.12

130.9% vs. 28.5%, p=.7 [Chi-Square], 221.1 vs. 329.1, p<.001 [t-test, two-tail, unequal variances]

WEIGHTED PREVALENCE OF HR-HPV AND LR-HPV BY LINEAR ARRAY IN

FIVE ANOGENITAL SITES

15.1% vs. 19.8%, p=.0004 [method of Raoul.J.Burchette@kp.org]

  Endocervix Upper Vagina

Lower Vagina

Perineum Self-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)

PROPORTION OF TYPE 16 OR 18 HPV AMONG LINEAR ARRAY HR-HPV

POSITIVE SPECIMENS

Endocervix Upper Vagina

Lower Vagina

Perineum Self-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)

139.6% vs. each other proportion, all p≥.7 [Chi-Square]

SENSITIVITY FOR ≥CIN 2 OF HR-HPV BY hc2 AND HR-HPV BY Linear Array

FOR FIVE ANOGENITAL SITES

197.9% vs. 85.1%, p=.03, 297.5% vs. 46.8%, p<.001, 397.9% vs. 80.9%, p=.008, 495.7% vs. 80.9%, p=.02. [all McNemar test]

Anogenital Site Sensitivity 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)

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.

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

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

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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