HPV Genotypes among 5683 Young Women in Guanacaste, Costa Rica ACCEPTED

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1 JCM Accepts, published online ahead of print on 7 March 2007 J. Clin. Microbiol. doi: /jcm Copyright 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Comparison of the SPF 10 /LiPA System to the HC2 Assay for Detection of Carcinogenic HPV Genotypes among 5683 Young Women in Guanacaste, Costa Rica Mahboobeh Safaeian PhD 1*, Rolando Herrero MD PhD 2, Allan Hildesheim PhD 1, Wim Quint PhD 4, Enrique Freer MD 3, Leen-Jan Van Doorn PhD 4, Carolina Porras MQC 2, Sandra Silva MQC 3, Paula González MD 2, M. Concepcion Bratti MD 2, Ana Cecilia Rodriguez MD MS 1, Philip Castle PhD MPH 1, For the CVT Group 1 Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 2 Proyecto Epidemiológico Guanacaste, Fundación INCIENSA, Costa Rica 3 Universidad de Costa Rica, Costa Rica 4 Delft Diagnostic Laboratory, Delft, The Netherlands *Corresponding author: Division of Cancer Epidemiology and Genetics Hormonal and Reproductive Epidemiology Branch National Cancer Institute 6120 Executive Boulevard, Suite 550 Rockville, MD Phone: (301) Fax: (301) safaeianm@mail.nih.gov Financial Disclosure Statement The Costa Rican Vaccine Trial is a longstanding collaboration between investigators in Costa Rica and NCI. The trial is funded by NCI and conducted in agreement with the Ministry of 1

2 Health of Costa Rica. Vaccine was provided for our trial by GSK Biologicals, under a Clinical Trials Agreement with NCI. GSK also provided support for aspects of the trial associated with regulatory submission needs of the company. NCI and Costa Rica Investigators make final editorial decisions on this presentation and subsequent publications; GSK has the right to review/comment. Short title: Comparison of the SPF 10 /LIPA System to the HC2 Assay 2

3 32 Affiliations of the Costa Rican Vaccine Trial (CVT) group are as follows: Proyecto Epidemiológico Guanacaste, Fundación INCIENSA, San José, Costa Rica Mario Alfaro (Cytologist) Manuel Barrantes (Field Supervisor) M. Concepcion Bratti (co-investigator) Fernando Cárdenas (General Field Supervisor) Bernal Cortés (Specimen and Repository Manager) Albert Espinoza (Head, Coding and Data Entry) Yenory Estrada (Pharmacist) Paula Gonzalez (co-investigator) Diego Guillén (Pathologist) Rolando Herrero (co-principal Investigator) Silvia E. Jimenez (Trial Coordinator) Jorge Morales (Colposcopist) Lidia Ana Morera (Head Study Nurse) Elmer Pérez (Field Supervisor) Carolina Porras (co-investigator) Ana Cecilia Rodriguez (co-investigator) Maricela Villegas (Clinic M.D.) University of Costa Rica, San José, Costa Rica 3

4 Enrique Freer (Director, HPV Diagnostics Laboratory) Jose Bonilla (Head, HPV Immunology Laboratory) Sandra Silva (Head Technician, HPV Diagnostics Laboratory) Ivannia Atmella (Immunology Technician) Margarita Ramírez (Immunology Technician) United States National Cancer Institute, Bethesda, MD, USA Pam Gahr (Trial Coordinator) Allan Hildesheim (co-principal Investigator) Douglas R. Lowy (HPV Virologist) Mark Schiffman (Medical Monitor) John T. Schiller (HPV Virologist) Mark Sherman (QC Pathologist) Diane Solomon (Medical Monitor & QC Pathologist) Sholom Wacholder (Statistician) SAIC, NCI-Frederick, Frederick, MD, USA Ligia Pinto (Head, HPV Immunology Laboratory) Alfonso Garcia-Pineres (Scientist, HPV Immunology Laboratory) 77 4

5 78 Womens and Infants Hospital, Providence, RI, USA Claire Eklund (QC Cytology) Martha Hutchinson (QC Cytology) Delft Diagnostics Laboratory, The Netherlands Wim Quint (HPV DNA Testing) Leen-Jan van Doorn (HPV DNA Testing) 5

6 The objective of this analysis was to compare performance of two human papillomavirus (HPV) DNA detections assays, hybrid-capture 2 (HC2) and SPF 10, for detection of carcinogenic HPV. Data are from the enrollment visit of women who participated in the randomized, double-blind, placebo-controlled phase III HPV16/18 Vaccine Trial in Guanacaste, Costa Rica. We compared results from HC2 and SPF 10 /LiPA testing of cervical specimens. Since the LiPA detection system does not distinguish between HPV68 (targeted by HC2) and HPV73 (not targeted by HC2), for SPF 10 /LiPA, we defined carcinogenic HPV as the 12 HC2 targeted types (16,18,31,33,35,39,45,51,52,56,58,59), HPV68/73, and the HC2 cross-reactive, carcinogenic HPV66. Kappa values and performance for detecting cervical abnormalities were ascertained. Paired observations were available from 5683 sexually active, young women (median age 21). Carcinogenic-HPV prevalence was 35% (n=1962) by HC2 and 35% (n=2003) by SPF 10 /LiPA. There were no differences in prevalence of carcinogenic HPV types by HC2 and SPF 10 /LiPA among women with normal, atypical squamous cells of undetermined significance, and high- grade squamous intraepithelial lesion cytology. Among women with low-grade squamous intraepithelial lesion cytology, HC2 was more likely to test positive than SPF 10 /LiPA for carcinogenic HPV (87% vs. 79%, respectively, p=0.001) as a result of HC2 cross reactivity with HPV types 40, 43, 44, 53, 54, 60, 70 and 74. Crude agreement between the two assays was 88%, with a kappa of 0.75 (95% confidence limits: ). We observed very good agreement between HC2 and SPF 10 /LiPA for carcinogenic HPV detection. 6

7 Introduction: In the 1990s, data from multiple, international epidemiologic studies established infection with a group of ~15 human papillomavirus (HPV) types as a necessary cause of cervical cancer (2,27). The steady increase in the fraction of cervical cancers attributable to HPV infection, from the low estimates reported in epidemiologic studies from a decade earlier, was achieved by reduction in misclassification due to the measurement errors caused by sub-optimal HPV-DNA tests (3,7,8,23). Now that the causal relationship between HPV infection and cervical cancer is certain, HPV- based prevention strategies are becoming increasingly important. In some countries HPV DNA assays are used as an adjunct to cytology to identify women at risk of cervical cancer who require preventive treatment. Recently, trials of prophylactic HPV vaccines have shown high efficacy in preventing new and persistent infections with the HPV types in the vaccine formulation (most notably HPV types 16 and 18 that together cause ~70% of cervical cancer worldwide) (10,26). Thus, to study cervical cancer prevention within the context of vaccines requires accurate detection of type-specific incident and persistent HPV infection associated with cancer and precancerous lesions. Beyond the detection of HPV16 and HPV18, other types must be identified accurately as well. Important secondary aims of ongoing clinical trials of vaccines are whether they confer protection against HPV types besides those in the formulations and whether reducing frequency of the two most carcinogenic genotypes as a result of vaccination could lead to increased frequency of other HPV types

8 At present there is no gold-standard for HPV detection, however, there is one FDA approved molecular assay the Hybrid Capture 2 (HC2; Digene Corporation, Gaithersburg, MD), which collectively targets 13 carcinogenic HPV types, with the limitation that it does not provide information about type-specific HPV genotypes. On the other hand, PCR-based assays are able to amplify most genital HPV genotypes in a single PCR reaction with the added benefit of type- discrimination following the amplification. Commonly used PCR primers are consensus primers GP5+/6+, MY9/11 and PGMY9/11 which respectively amplify 150 (GP5+/6+) and 450 (PGMY and MY9/11) base-pair (bp) region within the conserved L1 open reading frame (ORF) encoding the major capsid protein. A newer primer set (SPF 10 ) was developed that amplifies a 65 bp region in the same L1 ORF region (15) as the other primers mentioned. Because of its shorter amplification product, it is thought to be more analytically sensitive but possibly less specific for HPV detection compared to other DNA based assays, with a potential to amplify at least 54 HPV types. Genotype identification is achieved by using a reverse line probe assay (LiPA). We are using the SPF 10 /LiPA system as part of the HPV Vaccine Trial in Costa Rica (CVT). CVT is a phase III randomized efficacy trial of a HPV 16 and 18 vaccine, with the primary endpoint being the reduction in HPV16 and HPV18-related CIN2 and CIN3. The secondary aims of the trial (e.g., the effect of vaccination on viral persistence of the targeted and nontargeted carcinogenic HPV types) will also be dependent on the results of the SPF 10 /LiPA assay. However, large formal evaluations of SPF 10 /LiPA system are lacking, with only one report comparing performance of SPF 10 /LiPA with the HC2 test among 138 women attending 8

9 colposcopy clinic (18). Thus, we sought to compare detection of HPV DNA by SPF 10 /LiPA system to HC2 in paired samples collected during the enrollment phase of a community-based, randomized, double-blind, placebo-controlled phase III HPV16/18 Vaccine Trial in Guanacaste, Costa Rica. Methods: Study population: Data are from the enrollment visit of women who participated in CVT. As mentioned in the introduction, the primary aim of CVT is to independently assess the efficacy of an HPV16/18 vaccine manufactured by GlaxoSmithKline for prevention of precancerous lesions (defined for the trial as cervical intraepithelial neoplasia grade 2 [CIN2], grade 3 [CIN3], or adenocarcinoma in situ [AIS]) and invasive cervical cancer). Enrollment began in June 2004 and ended in December Study participants were women between years old, in good general health, without a history of chronic conditions that required treatment, willing to use birth control during the vaccination period, and living without plans of imminent departure from the study area. Approximately one-third of females identified in a previous census fulfilled the inclusion criteria and participated in the study At enrollment, women provided written, informed consent and a urine pregnancy test was performed. Prior to randomization, women were also administered a questionnaire that inquired about demographics, sexual and contraceptive use, reproductive history, cigarette use and family history of cancers. A detailed medical questionnaire was also administered, medical and pelvic exam was conducted on all consenting, sexually experienced women, during which cervical cells 9

10 were collected and placed in 20 ml of liquid cytology medium (PreservCyt; Cytyc Corporation, Marlborough, MA) for liquid-based cytology (ThinPrep; Cytyc Corporation) and for HPV detection using SPF 10 and HC2. To minimize the chance of carryover, cytologic slides were prepared after withdrawing two 0.5 ml aliquots, one for SPF 10 and one for confirmatory HPV testing in the future. Aliquots destined for PCR were stored in a liquid nitrogen tank, while the remaining PreservCyt samples were kept at room temperature (~20 o C) until used to make liquid cytology slides, to test for carcinogenic HPV, Chlamydia trachomatis, and Neisseria gonorrhea. All testing was done masked to the results of other tests or cytology. This analysis was based on the enrollment, pre-vaccination specimens from women entering the vaccine trial. All study protocols were reviewed and approved by the NCI and Costa Rican Institutional Review Boards. HPV detection and genotyping: Hybrid Capture 2: HC2 is an FDA-approved, commercially-available HPV test which collectively targets 13 carcinogenic HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) without distinguishing the HPV type present. HC2 is a signal amplification assay that uses a technique which combines antibody capture of HPV DNA and RNA probe hybrids and chemiluminescent signal detection. Additionally, because of genetic relatedness (19,21,24), other cancer-associated types such as HPV66 and HPV53 are also detected by HC2. The HC2 assay was performed according to the manufacturer s instructions in a laboratory at the University of Costa Rica in San Jose on residual PreservCyt samples. HC2 results were missing for 185 (3.2%) samples, mainly due to insufficient specimen volume. 10

11 SPF 10 /LIPA System: Total DNA was isolated from 200 µl of a PreservCyt aliquot drawn prior to ThinPrep preparation using the MagNA Pure LC instrument (Roche Diagnostics, Almere, the Netherlands), using the Total DNA isolation kit (Roche Diagnostics). DNA was eluted in 100 µl of water. Each DNA extraction run contained positive and negative controls to monitor the DNA isolation procedure. A 10 µl aliquot of extracted DNA was used for each SPF PCR reaction. The SPF 10 PCR primer set was used to amplify a broad spectrum of HPV genotypes as described earlier (14,15). Briefly, this primer set amplifies a small fragment of 65 bp from the L1 region of HPV. Reverse primers contain a biotin label at the 5 end, enabling capture of the reverse strand onto streptavidin coated microtiter plates. Captured amplimers are denatured by alkaline treatment, and the captured strand is detected by a defined cocktail of digoxigenin-labeled probes, detecting a broad spectrum of HPV genotypes. This method is designated HPV DNA enzyme immunoassay (DEIA), providing an optical density value. If the SPF 10 - DEIA yielded a borderline value (75-100% of the cut-off value), the SFP 10 PCR was repeated and retested by DEIA. Each DEIA run contained separate positive, borderline and negative controls. The broad spectrum SFP10 primers can recognize at least 54 HPV types The same SPF 10 amplimers (from SPF 10 /DEIA positive samples) were used to identify the HPV genotype by reverse hybridization on a line probe assay (LiPA), containing probes for 25 different HPV genotypes (SPF 10 HPV LiPA version 1, manufactured by Labo Bio-medical Products, Rijswijk, the Netherlands, detecting HPV 6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68/73, 70 and 74). Each LiPA run contained negative and 11

12 positive controls. Since the inter-primer regions of HPV 68 and 73 are identical, the LiPA system can not distinguish between HPV 68 and 73; hence they are assigned as HPV68/73. SPF 10 /LiPA results were available on all samples. Because the CVT is focused on HPV16 and HPV18, type-specific (TS) PCR primer sets were also used to selectively amplify HPV16 (TS16) and HPV18 (TS18) for 2513 specimens that tested positive by SPF 10 but did not contain HPV16 or HPV18 using LiPA (25). The type- specific primers were based on those described by Baay et al (1); they generate an amplimer of 92 and 126 bp for HPV16 and HPV18, respectively. Amplimers from the TS-PCRs were detected by DEIA, similar to the method for SPF 10 amplimer detection. Statistical analysis: The primary outcome was HPV prevalence determined by HC2 and SPF 10 /LiPA. It was necessary to adjust for the differences in HPV genotypes targeted by the two assays. The HC2 assay collectively targets 13 carcinogenic HPVs (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) although additional types like HPV66 also have been shown to be detected sensitively in practice (4,22). The SPF 10 PCR-DEIA can detect more than 50 HPV types, whereas the genotyping system (LiPA) can only identify 25 different HPVs: 11 non-carcinogenic (6, 11, 34, 40, 42, 43, 44, 53, 54, 70, and 74) and 14 carcinogenic (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68/73). Hence to compare SPF 10 to HC2, we defined HPV detection by SPF 10 at 3 levels; 1) PCR+: Detection of all amplified HPV genotypes by DEIA without distinguishing which genotype(s) are present, 2) LiPA+: Detection of at least one of the 25 low and high risk types, and 3) Carcinogenic+: Detection of one of the 14 carcinogenic HPV types. 12

13 The agreement between the two assays was determined by unweighted kappa (κ) statistics, and 95% confidence intervals (CI), which calculates percent agreement beyond that expected by chance alone. The non-parametric test for matched data (McNemar s χ 2 test) was used to determine whether the proportion of samples classified as positive by HC2 and negative by SPF 10 /LiPA was equal to the proportion of samples classified as negative by HC2 and positive by SPF 10 /LiPA samples. In an effort to investigate the reasons for discordant assay findings, we compared the 307 HC2- positive, SPF 10 /LiPA carcinogenic HPV-negatives to the 348 HC2-negative, SPF 10 /LiPA carcinogenic HPV-positives. Using two way tabulations and Pearson s Chi-square test we compared women with discordant tests on selected demographic and behavioral factors. Additionally we compared selected medical findings from pelvic examination. Furthermore, because discordance between HC2 and SPF 10 could be due to differential viral quantity required by the two assays, we used the semi-quantitative RLU/pc values from the HC2 assay as a proxy measure for HPV viral burden to investigate whether lower viral quantity explained why some HC2-positive samples were classified as negative by SPF 10 PCR system. RLU/pc values were categorized based on quartiles among HC2-positive samples: 1-4, 5-29, , and >268 RLU/pc. Among HC2-negatives, we investigated frequency of HPV types missed Results: Of the 7466 women enrolled in the trial, 1598 were excluded from this analysis because a pelvic exam was not performed on them mainly because they were virgins (1592). Median age was 21 years (interquartile range [IQR]: 19-23), median age at first sex was 17 years (IQR: 15-18). 13

14 Forty-four percent (n=2574) were single, 52% (n=3050) were married, and 3.4% (n=198) reported being separated, divorced or widowed. Carcinogenic HPV detection: Among the 5683 paired observations, 2856 women (50%) were PCR+, 2398 (42%) were LIPA+; and of these, 2003 (35%) were carcinogenic+. By comparison, 1962 women (35%) were positive for carcinogenic HPV as detected by HC2. Crude agreement between the two assays for carcinogenic HPV detection was 88%, and the kappa was 0.75 (95% CI: ), indicating very good agreement. Three hundred and seven (5.40%) were HC2-positive but carcinogenic- negative by SPF 10 /LiPA system. Similarly, 348 (6%) were carcinogenic-positive by SPF 10 /LiPA system but HC2 negative (McNemar s p-value=0.11) (Table 1). Table 2 presents comparison of selected characteristics between the SPF 10 /LiPA and HC2 discordant groups. The purpose of this analysis was to investigate whether one group of discordant was more likely to have characteristics which have been shown to be associated with HPV positivity. In multivariate model, HC2-negative, SPF 10 /LiPA carcinogenic HPV-positives were older (p=0.04), and more likely to have Chlamydia infection (p=0.03) compared to HC2- positive, SPF 10 /LiPA carcinogenic negatives. However, there were no differences based on lifetime sexual partners, marital status, and pregnancies, strong factors for HPV positivity To investigate the negative results by SPF 10 and/or LiPA among HC2-positives, we examined the HC2 signal strength, RLU/pc value (a semi-quantitative measure for HPV viral burden (13)), among the HC2-positive sub-groups (Table 3). Among the 104 SPF 10 PCR negative group, 70 14

15 (67%) had HC2-RLU/pc values between 1-5 and, similarly, 96 (47%) of carcinogenic negatives had HC2-RLU values of 1-5. By comparison, only 19% of the HC2-positive samples had RLU/pc values of 1-5 (p<0.001 compared to either). Table 4 presents carcinogenic HPV types among SPF 10 PCR-positive samples by HC2 status. HC2 identified as positive between 80% (HPV 52) to 93% (HPV 59) of specimens with types in its pooled probe. Eighty eight percent of HPV 16 and 18 positives by SPF 10 /LiPA were called positive by HC2. Among participants with a single HPV infection, HC2 prevalence was highest among 12 of the 13 types targeted by the HC2 probe ranging from 54% (HPV 68/73) to 96% (HPV 59). As previously noted (22), HC2 prevalence was also high for HPV 66 (67%), a type not targeted by the HC2 probe. We observed evidence of HC2 cross-reactivity with several other non-carcinogenic HPV types. The extent of cross-reactivity (among those with single HPV type only) for these types ranged from 8% (HPV43) to 49% (HPV70). Carcinogenic HPV detection by HC2 and SPF 10 system, stratified by cytology, is presented in Figure 1. For all cytologic interpretations, the assays performed similarly except for LSIL. For the 568 women with LSIL, 79% were positive by SPF 10 /LiPA and 87% by HC2 (p=0.001). When we accounted for the previously reported HC2 cross-reactive types (4,22), which we also observed in this analysis (HPV 6, 11, 40, 53, and 70), 86% of the LSIL were positive by SPF 10 /LiPA. Furthermore, considering that kappa values are somewhat dependent on prevalence rates, and since HPV prevalence differs by age, marital status and lifetime sexual partners, we examined 15

16 assay agreement by age group, marital status, and lifetime number of partners. Kappas were similar by age group, marital status, and by lifetime numbers of sexual partners (data not shown). Type-Specific Testing for HPV16 and HPV18: Among the 5868 women with SPF 10 /LIPA results, 403 (6.9%) were positive for HPV16 and 145 (2.5%) were positive for HPV18, with 16 (0.3%) infected by both based on SPF 10 /LiPA. Thus, 564 (9.6%) women were infected by HPV16 and/or HPV18. Of the 2937 SPF 10 -DEIA positives, 471 (16%) were negative for HPV types detected by LiPA. An additional 2450 were SPF 10 /DEIA-positive but either HPV16 or 18 negative using LiPA. This resulted in 2921 additional type-specific test with TS 16 and/or 18 primers, of which 95 (3.2%) were found to be either HPV16-positive (n=69) or HPV18-positive (n=27) and 1 positive for both HPV 16 and 18. Combining both SPF 10 /LIPA and TS testing, 488 (8.3%) were positive for HPV16 and 188 (3.2%) were positive for HPV18, with 30 (0.5%) infected by both. Thus, 646 (11%) women were infected by HPV16 and/or HPV18. SPF 10 /LiPA detected 86% of HPV16 and 86% of HPV18 infections detected by both SPF 10 /LiPA and TS testing. Eighty one of the 95 (85%) samples with positive TS PCR were HC2-positive (compared with 88% of any specimens that tested positive for HPV16 and/or HPV18 and 80% of specimens tested singly positive for HPV16 or HPV18 by SPF 10 /LiPA); among the HC2-positives, 33 (35%) of the TS PCR positives had RLU/pc values above Discussion: 16

17 In this large, population based cohort of young women, there was good agreement for detecting carcinogenic HPV types using HC2 and SPF 10 /LiPA system. Further stratification by cytology, age, and marital status, revealed similar kappa values among the different strata. From a research perspective, both SPF 10 and HC2 were comparable in detecting carcinogenic HPV, and from a clinical perspective, HPV prevalences determined by both tests were comparable among those with normal, ASCUS and HSIL cytology. Unlike our results, a recent study from the Netherlands found poor agreement between HC2 and SPF 10 /LiPA (crude agreement 0.70 and kappa 0.40) (18) with carcinogenic HPV prevalence of 36% by HC2 compared with 50% by SPF 10 /LiPA system. The sample size for that study was relatively small (n=138 at two 6 monthly interval resulting in 276 data points) and consisted of women with low grade cervical changes or women attending pre- and post-treatment for cervical intraepithelial neoplasia, whereas our analysis consisted of younger, healthy women from the general population. It is unclear why there are such marked differences between the two studies and what methodologic differences, if any, could explain such differences. Importantly, both studies showed similarly high sensitivity for HSIL by either the HC2 or SPF 10 /LiPA assay. Our findings confirmed the previously reported cross-reactivity of HC2 with other HPV types not targeted in its probe set (4,19,20), using different HPV amplification and typing systems than the previous reports. Interestingly we confirmed that HC2 detects HPV66 (a carcinogenic type not targeted) more frequently than HPV68/73, at least one of which (HPV68) is targeted by the HC2 probe. This is similar to other studies which also observed that, among the targeted types, HC2 detection is weakest for HPV68 (9,22). This might account for the equivocal data regarding 17

18 the carcinogenicity of HPV68 (5,12). However if 50% of HPV68/73 are HPV 68+ and 50% are HPV 73+, and HC2 detected 50%, this is consistent with 100% detection of HPV 68 and 0% detection of HPV 73. We were also able to show that there was modest degree of HC2 crossreactivity with HPV types 70 and 53, and to a lesser extent with types 6, 40, 11, 74, 44, 54, and 43. When we compared misclassification of disease outcomes among those negative by both HC2 and SPF 10 /DEIA, we observed that both assays missed similar numbers of cytologic abnormalities; however, there was a tendency for more LSIL to be HC2-positive compared to carcinogenic SPF 10 /LiPA. Further evaluation revealed that this was mainly attributed to the cross-reactivity of HC2 to other, non-carcinogenic HPV types (40, 43, 44, 53, 54, 60, 70 and 74) detected in the LSIL samples, which can also cause LSIL lesions (16). Discordance between the two assays based on the 14 carcinogenic HPV types could be explained by a couple of factors. In general, HC2-positive, SPF 10 /LiPA negatives had lower viral quantity than those positive by both assays, suggesting that sampling error due to low HPV viral load may be one reason for HC2-positive, SPF 10 negative findings at the PCR level. Further, comparing the discordants by HC2 and SPF 10 /LiPA system, we found that there were no differences between the discordant groups comparing factors that are critical for HPV positivity. Women whose samples were carcinogenic negative (by SPF 10 /LiPA) but HC2-positives were older and more likely to have had concurrent Chlamydia infection compared with the carcinogenic positive (by SPF 10 /LiPA) but HC2-negatives. However, this did not provide further clarification 18

19 regarding reasons for, or directionality of the discordance, suggesting that there is some degree of misclassification of HPV status using either assay. The HC2 test has been shown to be highly sensitive and specific for clinical outcomes (6), and its cross-reactivity with other HPV types rather than compromising, probably contributes to its high sensitivity but probably results in false positives. However, use of HC2 is limited in some applications because it does not provide information on type-specific HPV infection. The type- specific information is valuable in HPV natural history studies and specifically in the context of vaccine efficacy trials where the most relevant end-points recommended by a FDA vaccine advisory panel for determining vaccine efficacy are a reduction in the incidence of vaccine type- specific persistent infections and associated moderate to severe cervical intraepithelial neoplasia, approximated in this study by HSIL or worse by cytology (17). Such desired type-specific associations are not facilitated by HC2; however, PCR based assays and detections systems such as SPF 10 /LiPA allow for type-specific HPV determination. Interestingly the SPF 10 consensus primers can potentially co-amplify 54 different HPV types in one PCR reaction. However its analytic sensitivity, especially in the presence of multiple HPV types of varying viral loads can possibly be reduced where types can compete for limited PCR primers. This possibility might have been demonstrated by the additional HPV16 and HPV18- positive results when type-specific HPV16 and HPV18 primers were used to selectively amplify SPF 10 -DEIA-positive, LiPA-negatives and all HPV16 and HPV18-negative samples. An additional 69 samples were detected as HPV16 and 27 as HPV18 positive. From a clinical perspective, missing these additional HPV16 and 18 was not plainly detrimental in our 19

20 population sample because all 6 HSIL that were HPV16-positive and the 2 HSIL that were HPV18 positive only by the TS test, were also positive for at least one other carcinogenic HPV type, and were hence identified because of the pooling of the data. However, more investigation is warranted to evaluate the effect of competition between types within consensus PCR assays, especially if the competition is not random and rather may be dependent on factors such as HPV type and viral quantity. One limitation of SPF 10 /LiPA system especially in comparative studies to HC2, was that the LiPA detection system does not differentiate between HPV68 (a type targeted by the HC2 probe) and 73 (a type not targeted by the HC2 probe). By including the 68/73 in our comparison category, we may have artificially made a more robust category, especially if HPV73 is more prevalent in this cohort than HPV68. However, data from a previous natural history study in the same region (Guanacaste, Costa Rica) (11), showed small but equal prevalence of both HPV 68 and 73 in that population (0.3% and 0.5% respectively). Hence, it is unlikely that inclusion of 73 in the LiPA14 category would have strongly influenced our results and led to conclusions of equal performance of SPF 10 /LiPA compared to HC2. A strength of this study is that it is unlikely to suffer from selection bias. Both assays were performed on samples derived from one specimen, and the same participant, at the same time, and collected in the same media thus reducing the chance that differences could be attributed to procedural differences, or other host factors. Another strength of this study was its large, population-based sample of women

21 In conclusion, we observed good agreement between HC2 and SPF 10 /LiPA system for detection of carcinogenic HPV. We suggest that the SPF 10 /LiPA assay is a robust assay for studying the natural history of type-specific HPV and vaccine-related outcomes. 21

22 431 Table 1: Agreement between HC2 & SPF 10 /LiPA: HC2 Negative Positive Kappa % McNemar s Carcinogenic HPV Negative 3680 (64.75) Positive 2003 (35.23) LiPA Negative 3285 (57.80) Positive 2398 (42.20) SPF 10 /DEIA PCR Negative 2827(49.74%) Positive 2856 (50.66) 3721 (65.48%) 3373 (59.4) 348 (6.1) 3081 (54.2) 640 (11.3) 2723 (47.9) 998 (17.6) 1962 (34.52%) 307 (5.4) 1655 (29.1) 204 (3.6) 1758 (30.9) 104 (1.8) 1858 (32.7) (95% CI) 0.75 Agreement Test % % < % < Comprised of 13 types targeted by HC2 (16,18,31,33,35,39,45,51,52,56,58,59,68), and 66. Comprised of 6,11,16,18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68/73, 70, and

23 438 Table 2: Characteristics of assay discordant samples Age Education Less than elementary Elementary or more Marital Status SPF 10 /LiPA Carcinogenic negative & HC2- positive (n=307) 175 (57.0) 132 (43.0) 88 (28.9) 217 (71.2) SPF 10 /LiPA Carcinogenic positive & HC- negative (n=348) 162 (46.6) Univ. p-value MV p-value 186 (53.5) (30.9) 239 (69.1) Married Single Widowed/divorced Lifetime partners 131 (43.0) 164 (53.8) 10 (3.3) 164 (47.4) 164 (47.4) 18 (5.2) (34.5) 117 (34.0) 23

24 2 90 (29.6) 87 (25.3) 3 4+ Number: pregnancy Chlamydia No Yes 61 (20.1) 48 (15.8) 143 (46.9) 101 (33.1) 41 (13.4) 20 (6.6) 261 (85.0) 46 (15.0) 54 (15.7) 86 (25.0) (39.0) 121 (35.0) 70 (20.2) 20 (5.8) (77.5) 78 (22.5)

25 439 Table 3: HC2 RLU values for samples positive by HC2 but negative by SPF 10 and/or LiPA: SPF HC2+ Negative Positive 104 LiPA-Negative 100 Carcinogenic HPV-Negative 203 Carcinogenic HPV-Positive * (67.3) 47 (47.0) 96 (47.3) 309 (18.7) (23.1) 36 (36.0) 63 (31.0) 418 (25.3) (8.7) 14 (14.0) 36 (17.7) 446 (27.0) (1.0) 3 (3.0) 8 (3.9) 482 (29.1) *Comprised of 13 types targeted by HC2 (16,18,31,33,35,39,45,51,52,56,58,59,68), and 66 25

26 442 Table 4: HC2 Test Results Among SPF 10 Carcinogenic HPV-Positive Samples: Any Infection Single Infection * HC2+ HPV Type N=5683 HC2+ N = (65.2) * Normal N=992 * ASC N=88 * LSIL * HSIL N (%) N (%) N (%) N (%) N (%HC2+) N (%HC2+) N (%HC2+) N (%HC2+) HPV (7.2) 360 (88.2) 165 (11.6) 132 (83.0) 73 (75.3) 12 (80.0) 23 (100.0) 23 (100.0) HPV (2.8) 138 (87.9) 55 (3.9) 43 (78.2) 28 (70.0) 3 (100.0) 5 (100.0) 7 (100.0) HPV (4.7) 236 (89.4) 88 (6.2) 67 (80.7) 47 (75.8) 4 (100.0) 11 (91.7) 5 (100.0) HPV33 87 (1.5) 79 (90.8) 33 (2.3) 26 (81.3) 16 (76.0) 2 (67.7) 5 (100.0) 3 (100.0) HPV35 99 (1.7) 85 (85.9) 31 (2.2) 25 (80.7) 13 (72.0) 1 (100.0) 5 (83.3) 6 (100.0) HPV (3.9) 196 (89.1) 82 (5.7) 64 (80.0) 35 (70.0) 3 (100.0) 22 (95.7) 3 (100.0) HPV (2.2) 105 (84.0) 43 (3.0) 31 (73.8) 23 (67.7) 2 (100.0) 5 (100.0) 1 (100.0) HPV (5.4) 261 (85.3) 110 (7.7) 89 (82.4) 48 (75.0) 7 (87.5) 29 (93.6) 4 (100.0_ HPV (6.8) 309 (80.5) 129 (9.0) 90 (72.6) 63 (65.6) 6 (85.7) 11 (100.0) 9 (100.0) HPV (4.1) 207 (88.8) 82 (5.7) 65 (81.3) 38 (74.5) 1 (33.3) 26 (100.0) 0 (0) HPV (3.1) 162 (91.2) 64 (4.5) 56 (90.3) 25 (83.3) 9 (90.0) 14 (100.0) 8 (100.0) N=263 N=80 26

27 HPV (1.8) 96 (93.2) 45 (3.2) 42 (95.5) 29 (93.6) 4 (100.0) 8 (100.0) 1 (100.0) HPV68/ (3.2) 142 (78.0) 61 (4.3) 33 (54.1) 23 (48.9) 3 (75.00) 4 (57.1) 3 (100.0) HPV (3.6) 163 (79.1) 90 (6.3) 58 (66.7) 25 (48.1) 5 (100.0) 26 (92.9) 1 (100.0) HPV6 131 (2.3) 83 (63.4) 36 (2.5) 10 (27.8) 3 (13.0) 0 6 (66.7) 1 (100.0) HPV11 66 (1.2) 39 (59.1) 12 (0.8) 2 (16.7) 1 (14.3) 0 1 (25.0) 0 HPV34 15 (0.3) 8 (53.3) 2 (0.1) HPV40 57 (1.0) 36 (63.2) 11 (0.8) 2 (18.2) 1 (14.3) 0 1 (33.3) 0 HPV42 12 (0.2) 3 (25.0) 2 (0.1) HPV43 78 (1.4) 43 (55.1) 25 (1.8) 2 (8.3) 1 (7.7) 0 1 (14.3) 0 HPV44 85 (1.5) 35 (41.2) 36 (2.5) 4 (11.4) 3 (9.4) 0 1 (50.0) 0 HPV (4.1) 154 (65.8) 65 (4.6) 28 (43.8) 15 (31.9) 0 13 (86.7) 0 HPV (1.9) 40 (37.4) 59 (4.1) 5 (8.8) 3 (6.1) 0 2 (40.0) 0 HPV (2.5) 101 (71.1) 56 (3.9) 26 (49.1) 21 (43.8) 2 (100.0) 3 (100.0) 0 HPV (2.0) 58 (50.4) 47 (3.3) 6 (13.3) 4 (10.0) 0 2 (50.0) *40/1429 missing HC2 results, 30 missing among normal, 2 missing among ASCUS/ASC-H, 7 missing among LSIL, and 1 missing among HSIL. Includes both ASC-US and ASC-H Bold represent HC2 carcinogenic types. 27

28 Figure 1: Comparison of Clinical Performance for Cytologic Interpretation by HC2 and SPF 10 PCR System: Prevalence HSIL (n=177) LSIL (n=568) ASC-H (n=80) ASCUS (n=151) Normal (n=4857) Cytology PCR+ LiPA+* Carcinogenic+** *Comprised of 25 types detected by LiPA (6,11,16,18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68/73, 70, and 74) **Comprised of 13 types targeted by HC2 (16,18,31,33,35,39,45,51,52,56,58,59,68), and HC2+ 28

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