Transcript HPV genotype attribution in anal intraepithelial neoplasia

HPV genotype attribution of anal
neoplasia in HIV-positive MSM:
estimating the preventable fraction
and disease misclassification
Vikrant V. Sahasrabuddhe, MBBS, MPH, DrPH
Hormonal and Reproductive Epidemiology Branch
Division of Cancer Epidemiology and Genetics
National Cancer Institute
National Institutes of Health
Rockville, MD, USA
XIX International AIDS Conference, Washington, D.C.
July 25, 2012
I have no conflicts of interest to declare
Co-Authors
Vikrant V. Sahasrabuddhe1, Philip E. Castle2,
Stephen Follansbee3 , Sylvia Borgonovo 3, Brandon
J. LaMere3, Diane Tokugawa3, Teresa Darragh4,
Sean Boyle5, Mark Sadorra 5, Scott Tang5,
Nicolas Wentzensen1
1
National Cancer Institute, Rockville, MD, USA, 2American Society for Clinical
Pathology, Washington, DC, USA, 3Kaiser Permanente Northern California,
San Francisco, CA, USA, 4University of California at San Francisco, San
Francisco, CA, USA, 5Roche Molecular Systems, Pleasanton, CA, USA
Background
• Reducing the burden of HPV-related cancers
in HIV+ populations is an important global
public health agenda
• HIV+ MSM are at exceedingly high-risk of
HPV-induced anal cancer and its precursorhigh-grade anal intraepithelial neoplasia
(HGAIN)
Background
Potential approaches to prevention of anal cancer in HIV+ MSM
Primary prevention: HPV prophylactic vaccination
Licensed vaccines:
Bivalent HPV vaccine: HPV16/18 (Cervarix®, GSK)
Quadrivalent HPV vaccine: HPV16/18/6/11 (Gardasil®, Merck)
Investigational vaccine:
Nonavalent HPV vaccine: HPV16/18/6/11/31/33/45/52/58 (V503, Merck)
No efficacy trials to-date in HIV+ MSM
Safety & immunogenicity data are reassuring
Quadrivalent vaccine licensed for HIV+/MSM/immunocompromised males:
ages 9 to 26 years
Ref: CDC MMWR February 3, 2012 / 61(04);1-7
Background
Potential approaches to prevention of anal cancer in HIV+ MSM
Secondary prevention: screening and treatment of HGAIN
Screening by anal cytology (Pap)
Diagnosis by high-resolution anoscopy + biopsy
Treatment of HGAIN [e.g., by ablation/infrared coagulation]
No consensus guidelines for anal cancer screening
Clinicians in specialized centers offer anal cancer screening
– strong similarities between anal and cervical cancer and their precursors
& demonstrated success of cervical cancer prevention program
– high proportion of HGAIN lesions can be treated using office-based
procedures
– anal screening and treatment in targeted populations shown to be costeffective
Ref: Palefsky 2012, Goldstone et al 2007, Stier et al 2008, Singh et al 2009, Fox et al 2010, Goldie et al 2000
Background
Challenges in HPV clinical epidemiology studies in HIV+
MSM with a bearing on clinical practice
– Anal cytology and high-resolution anoscopy are suboptimally sensitive how to minimize disease
misclassification?
– Multiple HPV infections and multiple carcinogenic HPV
infections are very common how to attribute etiologic
fraction to individual HPV genotypes?
Study Objectives
– Characterization of HPV genotype distribution
across AIN categories in HIV+ MSM
– Unsupervised hierarchical clustering to explore
histology-cytology disease groups based on
distribution of carcinogenic HPV genotypes
– Attribution modeling to estimate fractions of HGAIN
preventable by HPV vaccination
Methods
• Study design
– Cross-sectional study at Kaiser Permanente Northern California
(KPNC)-Anal Cancer screening clinic in San Francisco, California
• Study population
– n=363 HIV+ MSM participating in KPNC HMO plan
• Study investigations
– Anal sample by wetted, flocked nylon swab
• HPV genotyping: Roche Linear Array HPV (LA-HPV) PCR assay
• Anal cytology
– Digital anorectal examination
– High resolution anoscopy (HRA) with anal biopsy (histology)
Results
Participant characteristics
HPV results available
342/363
Demographic & behavioral
Age (Mean; +SD)
53 (+9.2)
Age at first anal sex (Mean; +SD)
21 (+6.4)
Proportion with 40+ lifetime anal sexual partners
39.1%
Proportion with 3+ anal partners in last 6 months
15.1%
Condom use during anal sex
80.6%
Ever smoker
55.2%
Clinical
Current CD4+ <350/µl
19.1%
Current CD4+ <200/µl
3%
HIV-1 Viral load <75 copies/ml
90%
Taking cART
93.4%
Results
HPV genotype prevalence and distribution (n=342)
• High prevalence of HPV genotypes & carcinogenic
HPV genotypes
– Any HPV: 94.4%
– Any carcinogenic HPV: 75.4%
– 36 of 37 detectable genotypes present
• High proportion with multiple genotypes & multiple
carcinogenic genotypes
– Multiple HPV genotypes: 85.4%
– Multiple carcinogenic HPV genotypes: 47.4%
‘Carcinogenic’ HPV: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68; Ref: Bouvard et al 2009 Lancet Oncol
Results
Cytology & HRA-Histology
Cytology
NILM
ASC-US
LSIL
ASC-H
HSIL-AIN2
HSIL-AIN3
= 154
= 33
= 80
= 13
= 19
= 43
HRA-Histology
Normal
HRA
Normal
Histology
= 92
=81
AIN1
AIN2
AIN3
=106
= 41
= 22
AIN: Anal intraepithelial neoplasia; NILM: Negative for intraepithelial lesions or malignancy
ASC-US: Atypical squamous cells of undetermined significance
ASC-H: Atypical-squamous cells cannot exclude high-grade squamous intraepithelial lesion
LSIL: low-grade squamous intraepithelial lesions ; HSIL: high-grade squamous intraepithelial lesions ; HSIL-AIN2: HSIL-favor AIN2 ; HSIL-AIN3: HSIL-favor AIN3
Normal HRA: No Biopsy taken; NILM includes cases with no cytology results were available but for which HRA/histology results were available
Methods
Unsupervised hierarchical clustering
• Categorization: Separated participants into 11 anal disease
categories by crossing cytology/HRA/histology results
• Clustering: Simultaneous clustering of both disease
combinations and HPV genotypes
– Complete linkage and Euclidean distance matrix
– Software:
• Cluster 3.0 for clustering of HPV frequency arrays
• Java TreeView for heatmap and dendrogram visualization
Results
Heatmap & Dendrograms:
Unsupervised Hierarchical Clustering
Results
Clusters of Anal Disease Categories by Unsupervised Hierarchical
Clustering
Cluster 1
Cluster 3
Cluster 2
Results
Disease ascertainment for genotype attribution: Creation
of composite cytology-histologic endpoints (n=342)
Cytology
HRA/Histology
NILM
ASC-US
LSIL
ASC-H
HSIL-AIN2 HSIL-AIN3
Normal HRA
61
19
6
Normal Histology
50
20
5
AIN1 Histology
33
49
12
AIN2 Histology
AIN3 Histology
31
56
Results
Disease ascertainment for genotype attribution: Creation
of composite cytology-histologic endpoints (n=342)
Cytology
HRA/Histology
NILM
ASC-US
LSIL
ASC-H
HSIL-AIN2 HSIL-AIN3
Normal HRA
‘No AIN’
Normal Histology
AIN1 Histology
111
(32.4%)
AIN1
121
(35.4%)
AIN2
54
(15.8%)
AIN2 Histology
AIN3 Histology
HGAIN (AIN2 + AIN3) prevalence = 110 out of 342
= 32.2% (95%CI: 27.4-37.3)
AIN3
56
(16.4%)
Results
Composite disease categories and HPV prevalence
Total
‘No AIN’
AIN1
AIN2
AIN3
HGAIN
342
111
121
54
56
110
Any HPV
323
(94.4%)
99
(89.2%)
115
(95%)
53
(98.1%)
56
(100%)
109
(99%)
Carcinogenic
HPV
258
(75.4%)
57
(51.4%)
95
(78.5%)
51
(94.4%)
55
(98.2%)
106
(96.4%)
HPV16
96
(28.1%)
10
(9.0%)
29
(24.0%)
22
(40.7%)
35
(62.5%)
57
(51.8%)
Multiple
Carcinogenic
HPV
162
(47.4%)
29
(26.1%)
57
(47.1%)
36
(66.7%)
40
(71.4%)
76
(69.1%)
‘Carcinogenic’ HPV: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68
All p for trend<0.01
(‘No AIN’ to AIN3)
Methods
Attributing HGAIN fraction to individual HPV genotypes
• Low-end estimate
– Prevalence in cases with single carcinogenic HPV
genotype
• Irrespective of other non/possible carcinogenic genotypes
• High-end estimate
– Prevalence in HGAIN cases with any HPV genotype
• Irrespective of multiple genotypes
• Hierarchical attribution model estimate
– HPV genotypes are attributed to cases by hierarchical
frequency of carcinogenic genotypes in HGAIN
Methods
Attribution modeling: assumptions and caveats
• HPV vaccination would happen before exposure
to HPV genotypes included in the vaccines
• Cross-genotype protection and synergy between
genotypes not considered
• Full immune response to HPV VLP vaccines
Results
Estimation models of HPV vaccine preventable fractions of HGAIN
(n=110)
Bivalent vaccine
Quadrivalent vaccine
Nonavalent vaccine
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Low-end estimate:
cases with single
carcinogenic HPV
Bivalent vaccine
(16/18)
Quadrivalent vaccine
(16/18/6/11)
Nonavalent vaccine
(16/18/6/11/31/33/45/52/58)
Hierarchical
attribution model
estimate
High-end estimate:
cases with any HPV
36.7%
56.4%
61.8%
(95%CI: 21.9-54.5)
(95%CI: 47.0-65.3)
(95%CI: 52.5-70.3)
40.0%
56.4%
70.0%
(95%CI: 24.6-57.7)
(95%CI: 47.0-65.3)
(95%CI: 60.9-77.8)
86.7%
89.1%
92.7%
(95%CI: 70.3-94.7)
(95%CI: 81.9-93.7)
(95%CI: 86.3-96.3)
Conclusions
• Genotype attribution models suggest that HPV prophylactic vaccines may
potentially prevent a large fraction of HGAIN in HIV-positive MSM
• HGAIN is clinically & etiologically heterogeneous in HIV-positive MSM
• HPV16 is the most common and etiologically dominant genotype in HIVpositive MSM
• Combining cytology and histology endpoints may improve disease
ascertainment in cross sectional studies in HIV-positive MSM
• Molecular characterization by HPV genotyping or biomarkers may
reduce misclassification of HGAIN in HIV-positive MSM
Future directions
• Methodology: Analytical framework needs to be replicated and
for use in evaluation and comparison of attribution fractions and
disease ascertainment across populations and across disease
sites.
• Informing prevention clinical trials: Model results can be used to
inform the design of future efficacy trials of HPV vaccines in HIV+
individuals and trials of best approaches to anal cancer
screening.
• Prospective cohort studies: Risk of HGAIN associated with
clusters of anal disease categories and HPV genotypes in HIVpositive MSM needs to be evaluated prospectively.
Acknowledgments
*Study participants
NCI/NIH
• Nicolas Wentzensen
• Louise Brinton
• Katherine McGlynn
• Lauren Schwartz
• Lauren Wilson
• Mark Sherman
• Saloni Nayar
• Jennifer Loukissas
ASCP
• Philip Castle
UCSF
• Teresa Darragh
• Joel Palefsky
KPNC
• Steven Follansbee
Roche
• Sean Boyle
Vanderbilt
• Sten Vermund
Funding:
•Intramural Research Program of the National Cancer Institute (NCI), National
Institutes of Health (NIH), Department of Health and Human Services (DHHS)
• Oak Ridge Institute of Science and Education (ORISE) Research Participation Program
Thank you for your attention
Extra slides
Results
Distribution of cytology-HRA-histology (n=342)
Cytology
NILM1
ASC-US
LSIL
ASC-H
Normal HRA2
61
8
11
4
2
6
Normal Histology
50
5
15
3
2
6
AIN1 Histology
33
13
36
4
8
12
AIN2 Histology
7
6
13
2
3
10
AIN3 Histology
3
1
5
0
4
9
HRA/Histology
Normal HRA: No Biopsy taken
AIN: Anal intraepithelial neoplasia
NILM: Negative for intraepithelial lesions or malignancy
ASC-US: Atypical squamous cells of undetermined significance
ASC-H: Atypical-squamous cells cannot exclude high-grade squamous intraepithelial lesion
LSIL: low-grade squamous intraepithelial lesions
HSIL: high-grade squamous intraepithelial lesions
HSIL-AIN2: HSIL-favor AIN2
HSIL-AIN3: HSIL-favor AIN3
1
HSIL-AIN2 HSIL-AIN3
Category includes cases with no cytology results available,
for which HRA/histology results were available
2 Normal HRA results indicate that no Biopsy was taken
Results
High prevalence of multiple HPV genotypes across AIN disease categories
80%
0
1
2
3
4
5+
60%
All HPV genotypes
40%
Median: 4
Range: 0-14
IQR: 2-6
20%
0%
No AIN
Multiple types % 62.2%
AIN1
AIN2
AIN3
86%
94.4%
92.9%
60%
Carcinogenic HPV genotypes
50%
0
40%
Median: 1
Range: 0-7
IQR: 1-3
1
2
3
4
5+
30%
20%
10%
0%
No AIN
Multiple types %
26.1%
AIN1
47.1%
AIN2
AIN3
62.2%
71.4%
Results
Clusters of Anal Disease by Unsupervised Hierarchical Clustering
Cytology
HRA/Histology
NILM
ASC-US
Normal HRA
LSIL
ASC-H
HSIL-AIN2 HSIL-AIN3
Cluster 3
Normal Histology
AIN1 Histology
Cluster 2
AIN2 Histology
Cluster 1
AIN3 Histology
Cluster 1 {ASC-H/HSIL-AIN2 cytology and abnormal HRA} + {AIN2+ histology}
Cluster 2 {<=LSIL cytology and <=AIN1 histology} + {NILM cytology/normal HRA}
Cluster 3 {Normal HRA + <=ASC-H/HSIL-AIN2cytology}
Results
Participant characteristics by dendrogram clusters of anal disease
Dendrogram cluster
Cluster 1 Cluster 2 Cluster 3
Mean Age (years)
53
55
53
HPV +
99.0%
93.5%
92.5%
Carcinogenic HPV+
96.9%
80.0%
64.8%
Multiple (2+) Carcinogenic HPV+
71.4%
58.1%
34.7%
HPV16
55.1%
12.9%
17.8%
Results
Differences in HPV genotypes prevalence in HGAIN by CD4 counts
CD4<350 CD4>=350
(n=63)
(n=266)
p-value
Any HPV
100%
98.7%
0.99
Any Carcinogenic HPV
100%
94.9%
0.57
HPV16
70.4%
43.6%
0.03
Bivalent HPV vaccine
genotypes
77.8%
55.1%
0.04
Quadrivalent HPV vaccine
genotypes
81.5%
61.1%
0.15
Nonavalent HPV vaccine
genotypes
92.6%
92.3%
0.99
Bivalent HPV vaccine genotypes: HPV16, 18 ; Quadrivalent HPV vaccine genotypes: HPV 16, 18, 6, 11; Nonavalent HPV
vaccine genotypes: HPV16, 18, 6, 11, 31, 33, 45, 52, 58
Results
Clusters of Carcinogenic HPV genotypes by Unsupervised
Hierarchical Clustering
Non-16
carcinogenic
HPV
Cluster 1
Cluster 2
HPV16
Results
Comparison of main findings with other published research
Evidence from this study in
HIV+ MSM
Pooled evidence in HIV+ MSM
Carcinogenic HPV
prevalence
75.4%
(95%CI: 70.6-79.7)
73·5 %
(95%CI: 63·9–83·0)
HGAIN prevalence
32.2%
(95%CI: 27.4-37.3)
(Composite diagnosis)
29·1%
(95%CI: 22·8–35·4)
(Variable methods of diagnosis)
Range of preventable fraction
of HGAIN from this study in
HIV+ MSM
Vaccine trial efficacy against
HGAIN in HIV- MSM
Prevention by
quadrivalent HPV
vaccine
(16/18/6/11)
(Machelek et al 2012 Lancet Oncol)
(Palefsky et al 2011 NEJM)
70.0%
74.9%
(95%CI: 60.9-77.8)
(95%CI: 8.8 to 95.4)
(Hierarchical attribution model) (Per-protocol efficacy population)