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1.  Optimal Positive Cutoff Points for careHPV Testing of Clinician- and Self-Collected Specimens in Primary Cervical Cancer Screening: an Analysis from Rural China 
Journal of Clinical Microbiology  2014;52(6):1954-1961.
careHPV, a lower-cost DNA test for human papillomavirus (HPV), is being considered for cervical cancer screening in low- and middle-income countries. However, not a single large-scaled study exists to investigate the optimal positive cutoff point of careHPV test. We pooled data for 9,785 women participating in two individual studies conducted from 2007 to 2011 in rural China. Woman underwent multiple screening tests, including careHPV on clinician-collected specimens (careHPV-C) and self-collected specimens (careHPV-S), and Hybrid Capture 2 on clinician-collected specimens (HC2-C) as a reference standard. The primary endpoint was cervical intraepithelial neoplasia grade 3 or more severe (CIN3+) (n = 127), and secondary endpoint was CIN2+ (n = 213). The area under the curves (AUCs) for HC2-C and careHPV-C were similar (0.954 versus 0.948, P = 0.166), and better than careHPV-S (0.878; P < 0.001 versus both). The optimal positive cutoff points for HC2-C, careHPV-C, and careHPV-S were 1.40, 1.74, and 0.85, respectively. At the same cutoff point, careHPV-C was not significantly less sensitive and more specific for CIN3+ than HC2-C, and careHPV-S was significantly less sensitive for CIN3+ than careHPV-C and HC2-C. Raising the cutoff point of careHPV-C from 1.0 to 2.0 could result in nonsignificantly lower sensitivity but significantly higher specificity. Similar results were observed using CIN2+ endpoint. careHPV using either clinician- or self-collected specimens performed well in detecting cervical precancer and cancer. We found that the optimal cutoff points of careHPV were 2.0 on clinician-collected specimens and 1.0 on self-collected specimens.
PMCID: PMC4042749  PMID: 24671789
2.  Analytic and Clinical Performance of cobas HPV Testing in Anal Specimens from HIV-Positive Men Who Have Sex with Men 
Journal of Clinical Microbiology  2014;52(8):2892-2897.
Anal human papillomavirus (HPV) infections are common, and the incidence of anal cancer is high in HIV-infected men who have sex with men (MSM). To evaluate the performance of HPV assays in anal samples, we compared the cobas HPV test (cobas) to the Roche Linear Array HPV genotyping assay (LA) and cytology in HIV-infected MSM. Cytology and cobas and LA HPV testing were conducted for 342 subjects. We calculated agreement between the HPV assays and the clinical performance of HPV testing and HPV genotyping alone and in combination with anal cytology. We observed high agreement between cobas and LA, with cobas more likely than LA to show positive results for HPV16, HPV18, and other carcinogenic types. Specimens testing positive in cobas but not in LA were more likely to be positive for other markers of HPV-related disease compared to those testing negative in both assays, suggesting that at least some of these were true positives for HPV. cobas and LA showed high sensitivities but low specificities for the detection of anal intraepithelial neoplasia grade 2/3 (AIN2/3) in this population (100% sensitivity and 26% specificity for cobas versus 98.4% sensitivity and 28.9% specificity for LA). A combination of anal cytology and HPV genotyping provided the highest accuracy for detecting anal precancer. A higher HPV load was associated with a higher risk of AIN2/3 with HPV16 (Ptrend < 0.001), HPV18 (Ptrend = 0.07), and other carcinogenic types (Ptrend < 0.001). We demonstrate that cobas can be used for HPV detection in anal cytology specimens. Additional tests are necessary to identify men at the highest risk of anal cancer among those infected with high-risk HPV.
PMCID: PMC4136172  PMID: 24899025
3.  Age-specific occurrence of HPV16- and HPV18-related cervical cancer 
The age-specific of occurrence of cervical cancer related to human papillomavirus genotypes HPV16 and HPV18, the two targeted by current HPV vaccines, is not well described. We therefore used data from two large, tissue-based HPV genotyping studies of cervical cancer, one conducted in New Mexico (USA) (n = 744) and an international study restricted to cancers (n = 1,729) from Europe, North America, and Australia to represent those regions with widely available cervical cancer screening facilities. HPV results were categorized as HPV16 or HPV18 positive (HPV16/18) versus other HPV genotype. We observed a decreasing proportion of HPV16/18-positive cancers with increasing age in the international study (ptrend < 0.001) and New Mexico study (ptrend < 0.001). There was no heterogeneity in the relationship between age of diagnosis and the proportion of HPV16/18-positive cancers between studies (p = 0.8). Combining results from the two studies (n = 2,473), the percentages of HPV16/18-positive cases were 77.0% (95%CI: 75.1%-78.9%) for women less than 65 years old and 62.7% (95%CI: 58.4%-66.9%) for women aged 65 and older (p < 0.001). In women who are under the age of 25 and have been vaccinated before becoming sexually active, the cervical cancer incidence is expected to be approximately 3.5 per million by 2020. HPV vaccination against HPV16/18 may have a greater impact on cervical cancers in women under 65 than in women aged 65 and older. These data will inform the age-specific impact of HPV vaccination and its integration with cervical cancer screening activities.
PMCID: PMC4306595  PMID: 23632816
4.  Three-Year Risk of Cervical Precancer and Cancer After the Detection of Low-Risk Human Papillomavirus Genotypes Targeted by a Commercial Test 
Obstetrics and gynecology  2014;123(1):49-56.
To investigate the risk of cervical precancer and cancer associated with detection of human papillomavirus (HPV) 6, 11, and 42.
We used data from the New Mexico HPV Pap Registry. A stratified sample of 59,644 residual cervical cytology specimens from a population of 379,000 underwent HPV genotyping. We measured the 3-year cumulative incidence of cervical intraepithelial neoplasia grade 2 or more severe (CIN2+) and grade 3 or more severe (CIN3+) after detection of single HPV6, 11, or 42 infections or single or multiple infections by of HPV6, 11, or 42 (“HPV6, 11, 42, or combinations”)(n=581).
The overall prevalence of a single infection of HPV6, 11, or 42 was 0.8% (95% confidence interval [95%CI]: 0.7%, 0.9%). The 3-year risks of CIN2+ and CIN3+ after HPV6, 11, 42, or combinations infections (n = 581) were 0.4% (CI: 0.1%, 0.7%) for CIN2+ and 0.0% for CIN3+ (nota bene, no confidence interval was calculable because no events occurred), respectively. By comparison, the 3-year risks of CIN2+ and CIN3+ after a negative HPV result (n = 27,522) were 0.2% (95%CI: 0.1%, 0.2%) and 0.1% (95%CI: 0.0%, 0.1%), respectively.
Detection of HPV6, 11, 42, or combinations in the absence of high risk HPV types does not identify women at increased 3-year risk for cervical precancer. Testing for HPV6, 11, 42, or combinations of those types should be discontinued as it has no proven benefit to patients.
PMCID: PMC3926945  PMID: 24463663
5.  Clinical Human Papillomavirus Detection Forecasts Cervical Cancer Risk in Women Over 18 Years of Follow-Up 
Journal of Clinical Oncology  2012;30(25):3044-3050.
To describe the long-term (≥ 10 years) benefits of clinical human papillomavirus (HPV) DNA testing for cervical precancer and cancer risk prediction.
Cervicovaginal lavages collected from 19,512 women attending a health maintenance program were retrospectively tested for HPV using a clinical test. HPV positives were tested for HPV16 and HPV18 individually using a research test. A Papanicolaou (Pap) result classified as atypical squamous cells of undetermined significance (ASC-US) or more severe was considered abnormal. Women underwent follow-up prospectively with routine annual Pap testing up to 18 years. Cumulative incidence rates (CIRs) of ≥ grade 3 cervical intraepithelial neoplasia (CIN3+) or cancer for enrollment test results were calculated.
A baseline negative HPV test provided greater reassurance against CIN3+ over the 18-year follow-up than a normal Pap (CIR, 0.90% v 1.27%). Although both baseline Pap and HPV tests predicted who would develop CIN3+ within the first 2 years of follow-up, only HPV testing predicted who would develop CIN3+ 10 to 18 years later (P = .004). HPV16- and HPV18-positive women with normal Pap were at elevated risk of CIN3+ compared with other HPV-positive women with normal Pap and were at similar risk of CIN3+ compared with women with a low-grade squamous intraepithelial Pap.
HPV testing to rule out cervical disease followed by Pap testing and possibly combined with the detection of HPV16 and HPV18 among HPV positives to identify those at immediate risk of CIN3+ would be an efficient algorithm for cervical cancer screening, especially in women age 30 years or older.
PMCID: PMC3732003  PMID: 22851570
6.  Risk Factors for Anal HPV Infection and Anal Precancer in HIV-Infected Men Who Have Sex With Men 
The Journal of Infectious Diseases  2013;208(11):1768-1775.
Background. Carcinogenic human papillomaviruses (HPVs) cause a large proportion of anal cancers. Human immunodeficiency virus (HIV)–infected men who have sex with men (MSM) are at increased risk of HPV infection and anal cancer compared with HIV-negative men. We evaluated risk factors for HPV infection and anal precancer in a population of HIV-infected MSM.
Methods. Our study included 305 MSM at an HIV/AIDS clinic in the Kaiser Permanente Northern California Health Maintenance Organization. Logistic regression was used to estimate associations of risk factors comparing men without anal HPV infection; men with anal HPV infection, but no precancer; and men with anal precancer.
Results. Low CD4 count (<350 cells/mm3) and previous chlamydia infection were associated with an increased risk of carcinogenic HPV infection (odds ratio [OR], 3.65; 95% confidence interval [CI], 1.28–10.40 and OR, 4.24; 95% CI, 1.16–15.51, respectively). History of smoking (OR, 2.71 95% CI, 1.43–5.14), duration, recency, and dose of smoking increased the risk of anal precancer among carcinogenic HPV-positive men but had no association with HPV infection.
Conclusions. We found distinct risk factors for anal HPV infection and anal precancer. Risk factors for HPV infection and anal precancer are similar to established risk factors for cervical cancer progression.
PMCID: PMC3814839  PMID: 23908478
anal cancer; human papillomavirus (HPV); human immunodeficiency virus (HIV); men who have sex with men (MSM); anal intraepithelial neoplasia (AIN)
7.  Comprehensive Control of Human Papillomavirus Infections and Related Diseases 
Vaccine  2013;31(0 8):I1-31.
Infection with human papillomavirus (HPV) is recognized as one of the major causes of infection-related cancer worldwide, as well as the causal factor in other diseases. Strong evidence for a causal etiology with HPV has been stated by the International Agency for Research on Cancer for cancers of the cervix uteri, penis, vulva, vagina, anus and oropharynx (including base of the tongue and tonsils). Of the estimated 12.7 million new cancers occurring in 2008 worldwide, 4.8% were attributable to HPV infection, with substantially higher incidence and mortality rates seen in developing versus developed countries. In recent years, we have gained tremendous knowledge about HPVs and their interactions with host cells, tissues and the immune system; have validated and implemented strategies for safe and efficacious prophylactic vaccination against HPV infections; have developed increasingly sensitive and specific molecular diagnostic tools for HPV detection for use in cervical cancer screening; and have substantially increased global awareness of HPV and its many associated diseases in women, men, and children. While these achievements exemplify the success of biomedical research in generating important public health interventions, they also generate new and daunting challenges: costs of HPV prevention and medical care, the implementation of what is technically possible, socio-political resistance to prevention opportunities, and the very wide ranges of national economic capabilities and health care systems. Gains and challenges faced in the quest for comprehensive control of HPV infection and HPV-related cancers and other disease are summarized in this review. The information presented may be viewed in terms of a reframed paradigm of prevention of cervical cancer and other HPV-related diseases that will include strategic combinations of at least four major components: 1) routine introduction of HPV vaccines to women in all countries, 2) extension and simplification of existing screening programs using HPV-based technology, 3) extension of adapted screening programs to developing populations, and 4) consideration of the broader spectrum of cancers and other diseases preventable by HPV vaccination in women, as well as in men. Despite the huge advances already achieved, there must be ongoing efforts including international advocacy to achieve widespread—optimally universal—implementation of HPV prevention strategies in both developed and developing countries.
This article summarizes information from the chapters presented in a special ICO Monograph ‘Comprehensive Control of HPV Infections and Related Diseases’ Vaccine Volume 30, Supplement 5, 2012. Additional details on each subtopic and full information regarding the supporting literature references may be found in the original chapters.
PMCID: PMC4062073  PMID: 24229716
HPV; Cervical cancer; Anal cancer; Penile cancer; Vaginal cancer; Vulvar cancer; Oropharyngeal cancer; Screening; HPV vaccination; HPV testing; Prevention
8.  The influence of type-specific human papillomavirus infections on the detection of cervical precancer and cancer: A population-based study of opportunistic cervical screening in the United States 
There are limited data on the prospective risks of detecting cervical precancer and cancer in United States (US) populations specifically where the delivery of opportunistic cervical screening takes place outside managed care and in the absence of organized national programs. Such data will inform the management of women with positive screening results before and after widespread human papillomavirus (HPV) vaccination and establishes a baseline preceding recent changes in US cervical cancer screening guidelines. Using data reported to the statewide passive surveillance systems of the New Mexico HPV Pap Registry, we measured the 3-year HPV type-specific cumulative incidence of cervical intraepithelial neoplasia grade 2 or more severe (CIN2+) and grade 3 or more severe (CIN3+) detected during real-world health care delivery across a diversity of organizations, payers, clinical settings, providers and patients. A stratified sample of 47,541 cervical cytology specimens from a screening population of 379,000 women underwent HPV genotyping. Three-year risks for different combinations of cytologic interpretation and HPV risk group ranged from <1% (for several combinations) to approximately 70% for CIN2+ and 55% for CIN3+ in women with high-grade (HSIL) cytology and HPV16 infection. A substantial proportion of CIN2+ (35.7%) and CIN3+ (30.9%) were diagnosed following negative cytology, of which 62.3 and 78.2%, respectively, were high-risk HPV positive. HPV16 had the greatest 3-year risks (10.9% for CIN2+,8.0% for CIN3+) followed by HPV33, HPV31, and HPV18. Positive results for high-risk HPV, especially HPV16, the severity of cytologic interpretation, and age contribute independently to the risks of CIN2+ and CIN3+.
PMCID: PMC4020996  PMID: 24226935
cervical intraepithelial neoplasia (CIN); cervical cancer; human papillomavirus (HPV); HPV vaccine effectiveness; cervical screening effectiveness; US opportunistic cervical screening; cytology; Pap test
9.  Lower cost strategies for triage of human papillomavirus DNA-positive women 
Using human papillomavirus (HPV) testing for cervical cancer screening in lower-resource settings (LRS) will result in a significant number of screen-positive women. This analysis compares different triage strategies for detecting cervical precancer and cancer among HPV-positive women in LRS. This was a population-based study of women aged 25–65 years living in China (n = 7,541). Each woman provided a self-collected and two clinician-collected specimens. The self-collected and one clinician-collected specimen were tested by two HPV DNA tests—careHPV™ and Hybrid Capture 2; the other clinician-collected specimen was tested for HPV16/18/45 E6 protein. CareHPV™-positive specimens were tested for HPV16/18/45 DNA. HPV DNA-positive women underwent visual inspection with acetic acid (VIA) and then colposcopic evaluation with biopsies. The performance for detection of cervical intraepithelial neoplasia grade 3 or cancer (CIN3+) among HPV DNA-positive women was assessed for different triage strategies: HPV16/18/45 E6 or DNA detection, VIA, colposcopic impression, or higher signal strength (≥10 relative light units/positive control [rlu/pc]). The percent triage positive ranges were 14.8–17.4% for VIA, 17.8–20.9% for an abnormal colposcopic impression; 7.9–10.5% for HPV16/18/45 E6; 23.4–28.4% for HPV16/18/45 DNA; and 48.0–62.6% for higher signal strength (≥10 rlu/pc), depending on the HPV test/specimen combination. The positivity for all triage tests increased with severity of diagnosis. HPV16/18/45 DNA detection was approximately 70% sensitive and had positive predictive values (PPV) of approximately 25% for CIN3+. HPV16/18/45 E6 detection was approximately 50% sensitive with a PPV of nearly 50% for CIN3+. Different triage strategies for HPV DNA-positive women provide important tradeoffs in colposcopy or treatment referral percentages and sensitivity for prevalent CIN3+.
What's new?
The careHPV™ test is a novel technology for primary cervical cancer screening of women from lower-resource settings. However, triage strategies are needed to identify which HPV-positive women are at highest risk of cervical precancer and cancer. Here, multiple viable and affordable strategies to manage HPV-positive women depending on local requirements and resources are identified, based on evaluation of the performance of different triage strategies for developing countries. The different strategies for women who test positive for HPV DNA provide important tradeoffs in colposcopy or treatment referral percentages and sensitivity for cervical intraepithelial neoplasia grade 3 or cancer (CIN3+).
PMCID: PMC4232922  PMID: 24248915
HPV; triage; cervical cancer; careHPV; developing countries; E6
10.  A Comparison of Cervical and Vaginal Human Papillomavirus 
Sexually transmitted diseases  2007;34(11):849-855.
We wanted to compare detection of a broad spectrum of human papillomavirus (HPV) types detected in cellular specimens from the vagina and cervix, which could provide information about the potential of each anatomical site for harboring infection. Previous studies have failed to present data on or detect a broad spectrum of HPV genotypes and/or have not carefully sampled the vagina, instead relying on self-collection that is likely contaminated with cervical cells.
Study Design
We conducted follow-up study of 353 women who had participated in study of HPV and cervical neoplasia in Costa Rica. We collected paired cervical and vaginal specimens; vaginal specimens were collected from the fornix to minimize cervical contamination. Specimens were tested in a masked fashion for >40 HPV types using a MY09/MY11 PCR method and type-specific dot blot hybridization.
The prevalence for any carcinogenic HPV type in vaginal and cervical specimens was similar (P = 0.3). However, the prevalence for any HPV type in vaginal specimens was greater than in cervical specimens (P = 0.0002), primarily due to a twofold increased vaginal prevalence of HPV types of the α3/α15 phylogenetic species (e.g., HPV61) (P <0.00005).
Carcinogenic HPV types appeared to have a similar affinity for vaginal and cervical epithelium, but noncarcinogenic HPV types of the α3/α15 phylogenetic species may have a tropism for vaginal epithelium.
PMCID: PMC3962831  PMID: 17621246
11.  Comparison of Human Papillomavirus Detections in Urine, Vulvar, and Cervical Samples from Women Attending a Colposcopy Clinic 
Journal of Clinical Microbiology  2014;52(1):187-192.
While urine-based sampling for human papillomavirus (HPV) is being explored as a simple and noninvasive approach for cervical cancer screening, data comparing HPV genotyping in urine and those in cellular sampling of the cervix and vulva, and their correlation with rigorously confirmed cervical disease status, are sparse. We performed HPV genotyping on voided-urine and clinician-collected vulvar and cervical samples from 72 women undergoing colposcopy. Although urine-based HPV carcinogenic HPV detection was lower (58.3%) than cervical (73.6%) and vulvar (72.1%) detection (P = 0.05 and 0.07, respectively), the agreement of urine HPV with cervical and vulvar HPV was moderate (kappa = 0.55) and substantial (kappa = 0.62), respectively. Urine-based carcinogenic HPV detection had a clinical sensitivity of 80.8% (95% confidence interval [CI] = 60.7 to 93.5) and a specificity of 53.3% (95% CI = 37.9 to 68.3) for diagnosing cervical intraepithelial neoplasia grades 2/3 (CIN2/3) on histology; 90.0% of CIN3 was positive for urine HPV. The corresponding sensitivity and specificity values for vulvar sampling were 92% (95% CI = 74 to 99) and 40.5% (95% CI = 25.6 to 56.7), and those for cervical sampling were 96.2% (95% CI = 80.4 to 99.9) and 40% (95% CI = 25.7 to 55.7), respectively. HPV16 was the most common carcinogenic genotype detectable in 25% of urine, 33.8% of vulvar, and 31.9% of cervical samples overall, with prevalence increasing with cervical disease grade, regardless of the sampling method. Stronger cervical HPV PCR signal strengths were associated with increased frequency of urine HPV detection. In summary, the relatively lower detection rates but comparable clinical performance of urine-based HPV sampling underscore the need for larger studies to evaluate urine-based sampling for cervical cancer screening, epidemiologic studies, and postvaccination HPV disease surveillance.
PMCID: PMC3911475  PMID: 24197879
12.  Chromosomal gains measured in cytology samples from women with abnormal cervical cancer screening results 
Gynecologic oncology  2013;130(3):10.1016/j.ygyno.2013.06.005.
Chromosomal gains at 3q26, 5p15 and 20q13 have been described in cervical precancer and cancer. We evaluated a novel fluorescence in situ hybridization (FISH) assay that detects gains at these three loci simultaneously as a possible biomarker for detecting cervical precancer.
Chromosomal copy numbers at 3q26 (3q), 5p15 (5p), 20q13 (20q) and the centromere of chromosome7 (cen7) in liquid-based cytology specimens from 168 women enrolled in the Biopsy Study were determined by FISH. The number of cells with ≥3 or ≥4 signals for a genomic locus was enumerated and diagnostic test performance measures were calculated using receiver operating characteristic (ROC) analyses. Sensitivity and specificity values were determined for the detection of CIN2+ and/or HSIL.
The median number of cells with ≥3 signals increased with the severity of cervical lesion for each genomic locus (p-trend<0.02 for each locus). ROC analysis for the number of cells with ≥3 signals resulted in area under the curve values of 0.70 (95% CI: 0.54-0.86), 0.67 (0.52-0.83), 0.67 (0.51-0.83) and 0.78 (0.64-0.92) for 3q, 5p, 20q and cen7, respectively, for the detection of CIN2+ and/or HSIL. Positivity for gains at multiple loci resulted in only slightly better test performance measures than those for the individual probes for four distinct combinations of probes.
Chromosomal gains at 3q, 5p, 20q and cen7 are associated with severity of cervical lesions. Further studies are required to quantify risk stratification of FISH assays for cervical cancer screening.
PMCID: PMC3833871  PMID: 23769811
cervical cancer; genomic gains; 3q; FISH; HPV
13.  Human papillomavirus genotyping, human papillomavirus mRNA expression, and p16/Ki-67 cytology to detect anal cancer precursors in HIV-infected MSM 
AIDS (London, England)  2012;26(17):2185-2192.
Anal cancer incidence is high in HIV-infected MSM. Screening for anal intraepithelial lesions and cancers is performed at specialized clinics and relies on high-resolution anoscopy (HRA) and anal cytology. Both approaches have limited reproducibility and sensitivity for detecting anal cancer precursors. We evaluated biomarkers for human papillomavirus (HPV)-related disease in a population of HIV-infected MSM.
A cross-sectional screening study with passive follow-up included 363 MSM followed at a HIV/AIDS clinic. All men had anal cytology samples taken and were evaluated using HRA and anal biopsies. Using a composite endpoint of biopsy results and cytology, we compared the performance of HPV16/18 genotyping, HPVE6/E7 mRNA expression, and p16/Ki-67 cytology to detect high-grade anal intraepithelial neoplasias (AINs).
For all biomarkers analyzed, there was a significant trend of increasing percentage of men testing positive with increasing severity of disease (P< 0.001). HPV DNA testing had the highest sensitivity for anal intraepithelial neoplasia grade 2 and anal intraepithelial neoplasia grade 3 (AIN3), followed by p16/Ki-67, HPVE6/E7 mRNA testing, and HPV16/18 genotyping. The highest Youden's index was observed for HPVE6/E7 mRNA testing, followed by HPV16/18 genotyping, p16/Ki-67 cytology, and HPV DNA testing. Increasing the threshold for positivity of p16/Ki-67 to five or more positive cells led to significantly higher specificity, but unchanged sensitivity for detecting AIN3.
Molecular features of anal disease categories are similar to those of corresponding cervical lesions. Biomarkers evaluated for cervical cancer screening may be used for primary anal cancer screening or to decide who should require immediate treatment vs. expectant management.
PMCID: PMC4144865  PMID: 23018436
anal cancer screening; HIV; human papillomavirus; human papillomavirus mRNA; MSM; p16
14.  Risk of precancer determined by HPV genotype combinations in women with minor cytologic abnormalities 
Studies suggest that testing for individual HPV genotypes can improve risk stratification in women with minor cytological abnormalities. We evaluated genotyping for HPV16, HPV16/18, and HPV16/18/45 in carcinogenic HPV-positive women with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) cytology.
For women enrolled in the ASCUS-LSIL Triage Study (ALTS), we calculated the age-stratified (<30 and 30+ years) positivity, and cumulative risk over two years of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) when testing positive or negative for three genotype combinations: HPV16, HPV16/18, and HPV16/18/45.
Among women with ASCUS cytology, HPV16 positivity was 17.1% and increased to 22.0% (P<.001) for HPV16/18 and 25.6% (P<.001) for HPV16/18/45. Among women with LSIL cytology, HPV16 positivity was 21.1% and increased to 30.0% (P<.001) for HPV16/18 and 34.0% (P=.017) for HPV16/18/45. Regardless of cytology and age group, the greatest risk difference between test-positives and test-negatives was observed for HPV16 with decreasing risk stratification for HPV16/18 and HPV16/18/45. However, testing negative for any of the three combinations while being positive for another carcinogenic type still implied a 2-year risk of CIN3+ of 7.8% or greater.
Although genotyping for HPV16, 18, and 45 provided additional risk stratification in carcinogenic HPV-positive women with minor cytological abnormalities, the risk among genotype-negative women was still high enough to warrant immediate colposcopy referral.
HPV genotyping in HPV-positive women with minor cytological abnormalities will likely not alter clinical management. Adding HPV45 to genotyping assays is not warranted.
PMCID: PMC3687215  PMID: 23603204
HPV testing; cervical intraepithelial neoplasia; cervical cytology; risk stratification
15.  Clinical Evaluation of the Cartridge-Based GeneXpert Human Papillomavirus Assay in Women Referred for Colposcopy 
Journal of Clinical Microbiology  2014;52(6):2089-2095.
High-risk human papillomavirus (hrHPV) testing is now being introduced as a potential primary screening test for improved detection of cervical precancer and cancer. Current U.S. Food and Drug Administration-approved tests are batch tests that take several hours to complete. A rapid, non-batch test might permit point-of-care (POC) testing, which can facilitate same-day screen and management strategies. For a non-batch, random-access platform (GeneXpert; Cepheid, Sunnyvale, CA), a prototype hrHPV assay (Xpert) has been developed where testing for 14 hrHPV types can be completed in 1 h. In the first clinical evaluation, Xpert was compared to two validated hrHPV tests, the cobas HPV test (cobas, Roche Molecular Systems) and Hybrid Capture 2 (hc2, Qiagen), and to histologic outcomes using specimens from colposcopy referral populations at 7 clinical sites in the United States (n = 697). The sensitivity of Xpert for cervical intraepithelial neoplasia grade 2 or more severe diagnoses (CIN2+) (n = 141) was equal to that of cobas (90.8% versus 90.8%, P = 1) and greater than that of hc2 (90.8% versus 81.6%, P = 0.004). Xpert was more specific than cobas (42.6% versus 39.6%, P = 0.02) and less specific than hc2 (42.6% versus 47.7%, P < 0.001). Similar results were observed for cervical intraepithelial neoplasia grade 3 or higher (CIN3+) (n = 91). HPV16 detection by Xpert identified 41.8% of the CIN2+ specimens with a positive predictive value (PPV) of 54.6%. By comparison, HPV16 detection by cobas identified 42.6% of the CIN2+ specimens with a PPV of 55.0%. hrHPV detection by the Xpert demonstrated excellent clinical performance for identifying women with CIN2+ and CIN3+ that was comparable to that of currently available clinically validated tests.
PMCID: PMC4042758  PMID: 24719440
16.  Human Papillomavirus Genotyping After Denaturation of Specimens for Hybrid Capture 2 Testing: Feasibility Study for the HPV Persistence and Progression Cohort† 
Journal of virological methods  2007;146(1-2):80-85.
Human papillomavirus (HPV) genotyping could be clinically useful, depending on the results of large, prospective studies like the HPV Persistence and Progression cohort. The cohort is based on genotyping and follow-up of Hybrid Capture-positive women at Kaiser Permanente, Northern California. HPV DNA testing by Hybrid Capture 2 requires denaturation with alkali, possibly damaging the DNA for optimal PCR-based genotyping. A feasibility study was conducted on paired aliquots of anonymized specimens from 100 women with low-grade intraepithelial lesion cytology. Test aliquots were left in denaturant for 10 or 18 hours at 4°C and then neutralized; comparison aliquots were not denatured but diluted to match the timing, temperature, concentration and salt conditions of the treated specimens. The masked aliquots were tested using a commercialized PCR-based assay that detects of 37 HPV genotypes. There was no overall effect of treatment on test positivity or number of types. HPV16 was marginally more likely to be detected in untreated versus treated aliquots (P = 0.09) but HPV45 was marginally more likely to be detected in treated than untreated aliquots (P = 0.07), suggesting that these differences represented chance (intra-test variability). It can be concluded that residual Hybrid Capture-positive specimens can be accurately genotyped by PCR after Hybrid Capture 2 processing.
PMCID: PMC2683347  PMID: 17673302
Cervical Cancer; Human Papillomavirus (HPV); HPV genotypes; Cervical Precancer; Cervical Intraepithelial Neoplasia Grade 3 [CIN3]; screening; PCR
17.  Comparative risk of high-grade histopathology diagnosis following a CIN1 finding in endocervical curettage vs. cervical biopsy 
No evidence-based clinical management recommendations exist for women with an endocervical curettage (ECC) cervical intraepithelial neoplasia (CIN) grade 1 (CIN1) result when the concurrent cervical biopsy is not high-grade. For women with these pathology findings, we assessed their short-term risk of high-grade histopathology diagnosis in the Calgary Health Region where ECC was routinely performed.
Materials and Methods
We analyzed pathology and colposcopy reports from 1902 referral colposcopies where both ECC and biopsies were normal or CIN1. We calculated the short-term risk of CIN2 or more severe (CIN2+) detected 12–24 months after colposcopy. Pearson chi-square tests or Fisher’s exact tests were used to compare risks of a CIN2+ diagnosis between combinations of test results and strata of risk factors.
The short-term risk of CIN2+ was the same following a CIN1 biopsy and CIN1 ECC (4.9% of 1389 vs. 5.0% of 359, respectively, P=.37). Compared to low-grade referral cytology, the risk of CIN2+ following high-grade cytology was elevated significantly for CIN1 ECC (13.3% vs. 3.3%, P<.01) and non-significantly for CIN1 biopsy (7.1% vs. 4.6%, P=.12).
Following low-grade cytology, the short-term risk of a high-grade histologic diagnosis in women with either CIN1 ECC or biopsy is equivalent, suggesting similar management. A CIN1 ECC may warrant different management in the context of high-grade referral cytology.
PMCID: PMC3608705  PMID: 23343702
cervical intraepithelial neoplasia; colposcopy; curettage; diagnosis; endocervical sampling
18.  Five-year risk of recurrence following treatment of CIN2, CIN3, or AIS: performance of HPV and Pap cotesting in post-treatment management 
Journal of lower genital tract disease  2013;17(5 0 1):S78-S84.
After excisional treatment, CIN2+ can recur. It is not clear how many negative post-treatment Pap or cotest results are needed to assure adequate safety against CIN2+, prior to return to extended retesting intervals.
We observed 5-year risks of CIN2+, and outcomes for 3 follow-up management strategies after treatment (Pap alone, HPV alone, and cotesting) for 3273 women aged 25 and older who were treated for CIN2, CIN3, or AIS between 2003–2010 at Kaiser Permanente Northern California.
Five-year risks of recurrent CIN2+ after treatment varied both by antecedent screening test result and the histology of the treated lesion. The risk ranged from 5% for CIN2 preceded by HPV-positive/ASC-US or LSIL to 16% for CIN3/AIS preceded by AGC/ASC-H/HSIL+ (p<0.0001). However, after post-treatment negative tests, risks were lowered and similar regardless of antecedent screening test and histology of treated disease. The 5-year recurrent CIN2+ risk associated with a negative post-treatment cotest (2.4%) was lower than that of an HPV-negative test alone (3.7%, p=0.3) or Pap-negative result alone (4.2%, p=0.1). Two negative post-treatment tests of each kind conferred slightly lower 5-year CIN2+ risk than one (Pap-negative: 2.7% vs. 4.2%, p=0.2; HPV-negative: 2.7% vs. 3.7%, p=0.7; HPV-negative/Pap-negative: 1.5% vs. 2.4%, p=0.8). The 5-year CIN2+ risk associated with 2 negative cotests of 1.5% (95%CI 0.3% to 7.2%) approached the 0.68% risk associated with a negative Pap test during routine screening.
Women with antecedent AGC/ASC-H/HSIL+ Pap results or those treated with CIN3/AIS had a substantial risk of developing CIN2+ after treatment. Based on the principle of “benchmarking to implicit risk thresholds”, after negative test results following treatment, no subgroup of women achieved risk sufficiently low risk to return to 5-year routine screening. However, negative cotests after treatment provided more reassurance against recurrent CIN2+ than either negative Pap tests or HPV tests alone.
PMCID: PMC3616418  PMID: 23519309
Human Papillomavirus (HPV); cancer prevention; Pap; cervical intraepithelialneoplasia (CIN); Hybrid Capture 2 (HC2); post-treatment; test of cure
19.  Benchmarking CIN3+ risk as the basis for incorporating HPV and Pap cotesting into cervical screening and management guidelines 
Journal of lower genital tract disease  2013;17(5 0 1):S28-S35.
In 2012, the United States Preventive Services Task Force (USPSTF) and a consensus of 25 organizations endorsed concurrent cytology and HPV testing (“cotesting”) for cervical cancer screening. Past screening and management guidelines were implicitly based on risks defined by Pap-alone, without consideration of HPV test results. To promote management that is consistent with accepted practice, new guidelines incorporating cotesting should aim to achieve equal management of women at equal risk of cervical intraepithelial neoplasia grade 3 and cancer (CIN3+).
We estimated cumulative 5-year risks of CIN3+ for 965,360 women aged 30–64 undergoing cotesting at Kaiser Permanente Northern California 2003–2010. We calculated the implicit risk thresholds for Pap-alone and applied them for new management guidance on HPV and Pap cotesting, citing 2 examples: HPV-positive/ASC-US and HPV-negative/Pap-negative. We call this guidance process “benchmarking”.
LSIL, for which immediate colposcopy is prescribed, carries 5-year CIN3+ risk of 5.2%, suggesting that test results with similar risks should be managed with colposcopy. Similarly, ASC-US (2.6% risk) is managed with 6–12 month follow-up and Pap-negative (0.26% risk) is managed with 3-year follow-up. The 5-year CIN3+ risk for women with HPV-positive/ASC-US was 6.8% (95%CI 6.2% to 7.6%). This is greater than the 5.2% risk implicitly leading to referral to colposcopy, consistent with current management recommendations that HPV-positive/ASC-US should be referred for immediate colposcopy. The 5-year CIN3+ risk for women with HPV-negative/Pap-negative was 0.08% (95%CI 0.07% to 0.09%), far below the 0.26% implicitly required for a 3-year return and justifying a longer (e.g., 5-year) return.
Using the principle of “equal management of equal risks,” benchmarking to implicit risk thresholds based on Pap-alone can be used to achieve safe and consistent incorporation of cotesting.
PMCID: PMC3616419  PMID: 23519302
Human Papillomavirus (HPV); cancer prevention; Pap; cervical intraepithelial neoplasia (CIN); Hybrid Capture 2 (HC2)
20.  Five-year risk of CIN3+ and cervical cancer for women who test Pap-negative, but are HPV-positive 
Journal of lower genital tract disease  2013;17(5 0 1):S56-S63.
Current US guidelines for cotesting recommend that the large numbers of women who test Pap-negative, but HPV-positive, return in 1 year, and those who remain HPV-positive or have LSIL (or worse) Pap be referred for colposcopy. However, the performance of these guidelines in routine clinical practice has not been evaluated.
We estimated cumulative 5-year risks of CIN3+ for 32,374 women aged 30–64 with HPV-positive/Pap-negative cotest results at Kaiser Permanente Northern California during 2003–2010.
The 5-year CIN3+ risk following an HPV-positive/Pap-negative cotest result, which was found in 3.6% of women, was 4.5% (95%CI 4.2%–4.8%). The 5-year cancer risk was 0.34% (95%CI 0.26% to 0.45%) and half of the cases were adenocarcinoma. Overall, 47% of the women remained HPV-positive upon return (median 415 days after baseline), a percentage that varied little over ages 30–64. At the return following a baseline HPV-positive/Pap-negative result, almost every repeat cotest result predicted greater subsequent 5-year CIN3+ risk than the same cotest result had at baseline (HPV-positive/LSIL: 9.2% vs. 6.1%, p=0.01; HPV-positive/ASC-US: 7.9% vs. 6.8%, p=0.2; HPV-positive/Pap-negative: 7.4% vs. 4.5%, p<0.0001; HPV-negative/LSIL: 1.7% vs. 2.0%, p=0.8; HPV-negative/ASC-US: 2.9% vs. 0.43%, p=0.0005; HPV-negative/Pap-negative: 0.93% vs. 0.08%, p<0.0001).
Using the principle of “equal management of equal risks”, HPV-positive/Pap-negative women had subsequent CIN3+ risk consistent with risk thresholds for a 1-year return. However, upon returning in approximately 1 year, about one-half of women will be referred for colposcopy due to continued HPV positivity or Pap abnormality. Clinicians should keep in mind that cotest results at the return following a baseline HPV-positive/Pap-negative finding are riskier than the same baseline cotest results in the general population, supporting intensified clinical management of return cotests.
PMCID: PMC3616446  PMID: 23519306
Human Papillomavirus (HPV); cancer prevention; Pap; cervical intraepithelial neoplasia (CIN); Hybrid Capture 2 (HC2); prospective cohort
21.  Five-year risk of CIN3+ and cervical cancer for women with HPV-positive and HPV-negative high-grade Pap results 
Journal of lower genital tract disease  2013;17(5 0 1):S50-S55.
High-grade Pap results (e.g., AGC, ASC-H, and HSIL) predict high cancer risks, resulting in referral to colposcopy without HPV triage. However, new guidelines recommending cotesting for women 30 and older imply that clinicians will often receive the HPV test result concurrently for high-grade Pap results. We examined whether HPV testing provides useful risk stratification in this context.
From a cohort of 965,360 women age 30–64 undergoing cotesting at Kaiser Permanente Northern California, we estimated 5-year risks of cervical cancer and CIN3+ for AGC (2,074 women), ASC-H (1,647 women), and HSIL (2,019 women) according to HPV test results.
HPV positivity of AGC Pap results was 25% and decreased with age (30–34: 44% vs. 60–64: 17%, p<0.0001), while HPV-positivity of ASC-H and HSIL were much higher (71% and 94% respectively) and decreased less with age. Even for these high-grade Pap results, 5-year CIN3+ risks differed substantially between HPV-positive and HPV-negative women (AGC: 33% vs. 0.93%, p<0.0001; ASC-H: 25% vs. 3.5%, p<0.0001; HSIL: 49% vs. 30%, p=0.006). However, except for AGC, cervical cancer risks differed less between HPV-positive and HPV-negative women (AGC: 9.0% vs. 0.37%, p<0.0001; ASC-H: 2.5% vs. 2.1%, p=0.8; HSIL: 6.6% vs. 6.8%, p=0.7).
The risks of CIN3+ for HPV-negative high-grade Pap results were lower than for HPV-positive high-grade Pap results, especially for AGC. However, by the principle of “equal management of equal risks”, all HPV-negative high-grade Pap results had cancer risks high enough to warrant colposcopy, confirming that there is no current role for HPV triage of high-grade Pap results.
PMCID: PMC3616447  PMID: 23519305
Human Papillomavirus (HPV); cancer prevention; baseline; cervical intraepithelial neoplasia (CIN); Hybrid Capture 2 (HC2); screening guidelines; HSIL; AGC; ASC-H
22.  Estimating 5-year risk of CIN3+ to guide the management of women aged 21–24 
Journal of lower genital tract disease  2013;17(5 0 1):S64-S68.
Current US national guidelines recommend beginning screening at age 21 using Pap tests only, with cotesting starting at age 30. To inform the management of Pap test abnormalities among women aged 21–24, who have extremely low cancer risks, we compared risks of CIN3+ for women aged 21–24 versus 25–29 or 30–64.
We estimated 5-year risks of CIN3+ for Pap test results, with HPV triage of ASC-US, for 133,947 women aged 21–24, compared with 135,382 women age 25–29 and 965,360 women age 30–64, between 2003–2010 at Kaiser Permanente Northern California.
There were 3 cancers diagnosed during follow-up in women aged 21–24. Following high-grade Pap results (0.6% of Pap results), 5-year CIN3+ risks for women aged 21–24 were comparable to those aged 25–29 and 30–64 (AGC: 6.9% vs. 14% vs. 8.5%, p=0.8; ASC-H: 16% vs. 24% vs. 18%, p=0.8; HSIL: 28% vs. 28% vs. 47%, p=0.4). Following LSIL, 5-year CIN3+ risk was lower for ages 21–24 (3.0%) than ages 25–29 (5.0%, p=0.01) or ages 30–64 (5.2%, p=0.0002). Although 5-year CIN3+ risk for HPV-negative/ASC-US was similar across all 3 groups (0.57% vs. 0.59% vs. 0.43%, p=1), risk for HPV-positive/ASC-US was lower for age 21–24 (4.4%) than 25–29 (7.1%, p<0.0001) or 30–64 (6.8%, p<0.0001).
Women aged 21–24 had almost zero cancer risk, and positive Pap test results predicted low CIN3+ risk except for the 0.6% of Pap results that were high-grade. The generally low risk supports conservative management of women aged 21–24.
PMCID: PMC3616448  PMID: 23519307
Human Papillomavirus (HPV); cancer prevention; Pap; cervical intraepithelial neoplasia (CIN); Hybrid Capture 2 (HC2); young women
23.  Follow-up testing post-colposcopy: Five-year risk of CIN2+ after a colposcopic diagnosis of CIN1 or less 
Journal of lower genital tract disease  2013;17(5 0 1):S69-S77.
The majority of women referred for colposcopy are not diagnosed with CIN2+ but, nonetheless, are typically asked to return much sooner than their next routine screening interval in 3-5 years. An important question is how many subsequent negative Pap results, or negative Pap and HPV cotest results, are needed prior to returning to an extended retesting interval.
We estimated 5-year risks of CIN2+ for 3 follow-up management strategies after colposcopy (Pap-alone, HPV-alone and cotesting) for 20,319 women aged 25 and older screened from 2003-2010 at Kaiser Permanente Northern California who were referred for colposcopy but for whom CIN2+ was not initially diagnosed (i.e., “Women with CIN1/negative colposcopy”).
Screening results immediately antecedent to CIN1/negative colposcopy influenced subsequent 5-year CIN2+ risk: women with an antecedent HPV-positive/ASC-US or LSIL Pap had a lower risk (10%) than those with antecedent ASC-H (16%, p<0.0001) or HSIL+ (24%, p<0.0001). For women with an antecedent HPV-positive/ASC-US or LSIL, a single negative cotest approximately 1 year post-colposcopy predicted lower subsequent 5-year risk of CIN2+ (1.1%) than 2 sequential negative HPV tests (1.8%, p=0.3) or 2 sequential negative Pap results (4.0%, p<0.0001). For those with an antecedent ASC-H or HSIL+ Pap, 1 negative cotest after 1 year predicted lower subsequent 5-year risk of CIN2+ (2.2%) than 1 negative HPV test (4.4%, p=0.4) or 1 negative Pap (7.0%, p=0.06); insufficient data existed to calculate risk after sequential negative cotests for women with high grade antecedent cytology.
After CIN1/negative colposcopy followed by negative post-colposcopy tests, women did not achieve sufficiently low CIN2+ risk to return to 5-year routine screening. For women with antecedent HPV-positive/ASC-US or LSIL, a single negative post-colposcopy cotest reduced risk to a level consistent with a 3-year return. For women with antecedent ASC-H or HSIL+, no single negative test result sufficed to reduce risk to a level consistent with a 3-year return.
For women with CIN1/negative colposcopy and antecedent HPV-positive/ASC-US or LSIL, a single negative post-colposcopy cotest reduced risk to a level consistent with a 3-year return.
PMCID: PMC3616505  PMID: 23519308
Human Papillomavirus (HPV); cancer prevention; Pap; cervical intraepithelial neoplasia (CIN); Hybrid Capture 2 (HC2); colposcopy
24.  Five-year risk of CIN3+ and cervical cancer for women with HPV testing of ASC-US Pap results 
Journal of lower genital tract disease  2013;17(5 0 1):S36-S42.
New screening guidelines recommend that HPV-negative/ASC-US results be considered as equivalent to HPV-negative/Pap-negative results, leading to rescreening in 5 years. However, despite ample research data, the routine clinical performance of HPV testing of women with ASC-US has not been adequately documented.
We estimated 5-year risks of CIN3+ and cancer for 2 groups between 2003-2010 at Kaiser Permanente Northern California: 27,050 women aged 30-64 who underwent HPV and Pap cotesting and had an ASC-US Pap result, and 12,209 women aged 25-29 who underwent HPV triage of ASC-US.
Five-year risks of CIN3+ and of cancer for women aged 30-64 testing HPV-negative/ASC-US and for 923,152 women testing Pap-negative alone were similar although statistically distinguishable (CIN3+: 0.43% vs. 0.26% (p=0.001); Cancer: 0.050% vs. 0.025% (p=0.1, respectively)). The cancer risk increase for HPV-negative/ASC-US versus Pap-negative alone was confined to women aged 60-64 (0.26% vs. 0.035%, p=0.3). Five-year risks of CIN3+ and of cancer for women with HPV-negative/ASC-US were substantially higher than those for women testing HPV-negative/Pap-negative (CIN3+: 0.43% vs. 0.08% (p<0.0001); Cancer: 0.050% vs. 0.011% (p=0.003, respectively)). For women aged 30-64 testing HPV-positive/ASC-US, 5-year risks of CIN3+ and cancer were slightly higher than for the 9,374 women with LSIL (CIN3+: 6.8 % vs. 5.2% (p=0.0007); Cancer: 0.41% vs. 0.16% (p=0.04)). Similar patterns were seen for women aged 25-29.
Women with HPV-negative/ASC-US had similar risk as women testing Pap-negative alone, but had higher risk than women testing HPV-negative/Pap-negative. Based on the principle of “equal management of equal risks”, our findings support equal management of women with HPV-negative/ASC-US and those with Pap-negative alone, except for exiting women from screening because cancer risks at ages 60-64 may be higher for HPV-negative/ASC-US. Our findings also support managing HPV-positive/ASC-US and LSIL similarly.
Women testing HPV-negative/ASC-US have similar risk of CIN3+ or cancer as women testing Pap-negative alone, but have higher risk than women testing HPV-negative/Pap-negative.
PMCID: PMC3616508  PMID: 23519303
Human Papillomavirus (HPV); cancer prevention; Pap; cervical intraepithelial neoplasia (CIN); Hybrid Capture 2 (HC2); ASC-US
25.  Five-year risks of CIN2+ and CIN3+ for women with HPV-positive and HPV-negative LSIL Pap results 
Journal of lower genital tract disease  2013;17(5 0 1):S43-S49.
LSIL Pap results do not typically lead to HPV testing. HPV triage is not cost-effective because most cases are HPV-positive. However, under new national guidelines recommending cotesting for women ages 30–64, clinicians will increasingly receive the HPV test result for LSIL Pap results. Some authors have suggested that HPV triage might be effective at older ages, when the percentage of HPV positivity among women with LSIL declines.
We estimated 5-year risks of CIN2+ and CIN3+ for 9,033 women aged 30–64 who had both an HPV test and an LSIL Pap result.
HPV-positivity of LSIL decreased only slightly with age (30–34: 88% vs. 60–64: 72%, p<0.0001). The 5-year risks of CIN2+ and CIN3+ for women aged 30–64 testing HPV-positive/LSIL were larger than those for women testing HPV-negative/LSIL (CIN2+: 19% vs. 5.1%, p<0.0001; CIN3+: 6.1% vs. 2.0%, p<0.0001). The 5-year CIN3+ risk in HPV-negative/LSIL women was similar to that for women with ASC-US Pap without knowledge of HPV test results (2.0% vs. 2.6%, p=0.4).
HPV-negative/LSIL posed lower risk than other Pap results that guidelines currently recommend for referral to immediate colposcopy. By the principle of “equal management of equal risks,” women with HPV-negative/LSIL might reasonably be managed similarly to those with ASC-US Pap results without knowledge of HPV testing, i.e., re-testing at 6–12 months, rather than immediate colposcopy. Although the HPV test result for LSIL Pap results provides actionable information to clinicians who screen with cotesting, the high HPV-positivity of LSIL at even the oldest ages suggests the lack of cost-effectiveness of HPV triage of LSIL for clinicians who do not use routine cotesting.
PMCID: PMC3637971  PMID: 23519304
Human Papillomavirus (HPV); cancer prevention; cytology; cervical intraepithelial neoplasia (CIN); Hybrid Capture 2 (HC2); LSIL

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