Brandon J. LaMere

Methylation of HPV18, HPV31, and HPV45 Genomes and Cervical Intraepithelial Neoplasia Grade 3

BACKGROUND Persistent infections with carcinogenic human papillomavirus (HPV) types are the necessary cause of cervical cancer. We recently demonstrated that the HPV16 genome is strongly methylated in cervical precancer compared with transient infections. However, the extent of methylation in other HPV types and its role in progression to cancer is poorly understood. METHODS We analyzed whole-genome methylation patterns of the three next most carcinogenic HPV genotypes: HPV31 (closely related to HPV16), and two other closely related types, HPV18 and HPV45. DNA was extracted from cervical cytology specimens from 92 women with precancer and 96 women infected with HPV31, HPV18, or HPV45, but who had no cytological or histological abnormalities. After bisulfite modification, genome-wide pyrosequencing was performed covering 80-106 sites. We calculated differences in median methylation, odds ratios, areas under the curve, and Spearman rank correlation coefficients for methylation levels between different sites. All statistical tests were two-sided. RESULTS For all three HPV types, we observed strongly elevated methylation levels at multiple CpG sites in the E2, L2, and L1 regions among women with cervical intraepithelial neoplasia grade 3 compared with women with transient infections. We observed high correlation of methylation patterns between phylogenetically related types. The highest areas under the curve were 0.81 for HPV31, 0.85 for HPV18, and 0.98 for HPV45. Differential methylation patterns in cervical intraepithelial neoplasia grade 3 patients with multiple infections suggest that methylation can clarify which of the infections is causal. CONCLUSIONS Carcinogenic HPV DNA methylation indicates transforming HPV infections. Our findings show that methylation of carcinogenic HPV types is a general phenomenon that warrants development of diagnostic assays.

Human Papillomavirus Genotype Attribution and Estimation of Preventable Fraction of Anal Intraepithelial Neoplasia Cases Among HIV-Infected Men Who Have Sex With Men

BACKGROUND The prevention of human papillomavirus (HPV)-induced anal cancer in high-risk populations such as human immunodeficiency virus (HIV)-infected men who have sex with men (MSM) remains an urgent priority, given rising incidence rates despite widespread antiretroviral therapy use. METHODS HPV genotypes and anal disease prevalence, by cytology and histopathologic findings, were evaluated among 363 HIV-infected MSM. We modeled fractions of high-grade anal intraepithelial neoplasia (HGAIN) attributable to individual carcinogenic HPV genotypes and estimated the range of the proportion of HGAIN cases potentially preventable by prophylactic HPV vaccines. RESULTS HPV16 was the most common genotype overall (26.4% of cases) and among HGAIN cases (55%). Prevalence of multiple (≥ 2) carcinogenic HPV genotypes increased from 30.9% in cases of AIN grade <1 to 76.3% in cases of AIN grade 3 (P(trend) < .001). The fractions of HGAIN cases attributable to carcinogenic HPV16/18 targeted by currently licensed bivalent and quadrivalent HPV vaccines ranged from 12% to 61.5%, and the fractions attributable to carcinogenic HPV16/18/31/33/45/52/58 targeted by an investigational nonavalent HPV vaccine ranged from 39% to 89.4%. CONCLUSIONS Our analytical framework allows estimation of HGAIN cases attributable to individual HPV genotypes in the context of multiple concurrent HPV infections, which are very common among HIV-infected MSM. Our results suggest that licensed and investigational HPV prophylactic vaccines have the potential to prevent a substantial proportion of HGAIN cases in this population.

Comparison of the cobas Human Papillomavirus (HPV) Test with the Hybrid Capture 2 and Linear Array HPV DNA Tests

ABSTRACT The cobas human papillomavirus (HPV) test (cobas) was recently approved by the U.S. Food and Drug Administration (FDA) and identifies HPV16 and HPV18 separately as well as detecting a pool of 11 HR-HPV genotypes (HPV31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -68) and also HPV66. We compared cobas, Linear Array (LA), and Hybrid Capture 2 (HC2) assays for detection of carcinogenic HPV DNA, and cobas and LA for detection of HPV16 and HPV18 DNA, among the first 1,852 women enrolled in the HPV Persistence and Progression Cohort (PaP Cohort) study. Specimens were tested by all 3 assays 1 year after an HC2-positive result. In 1,824 specimens with cobas results, cobas had an 85.9% agreement with HC2 and 91.0% agreement with LA for carcinogenic HPV detection. When results between cobas and HC2 disagreed, cobas tended to call more women HPV positive (P < 0.01). Categorizing cobas and LA results hierarchically according to cancer risk (HPV16, HPV18, other carcinogenic HPV genotypes, or carcinogen negative), there was a 90% agreement for all categories of HPV (n = 1,824). We found good agreement between the two U.S. FDA-approved HPV tests, with discrepancies between the two assays due to specific characteristics of the individual assays. Additional studies are needed to compare HC2 and cobas for detecting and predicting CIN3 to understand the clinical implications of the discrepant test results between the two tests.

Human papillomavirus genotyping, human papillomavirus mRNA expression, and p16/Ki-67 cytology to detect anal cancer precursors in HIV-infected MSM

Objective: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. Methods: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). Results: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 Youdens 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. Conclusion: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.

Human Papillomavirus (HPV) Genotypes in Women with Cervical Precancer and Cancer at Kaiser Permanente Northern California

Background: The human papillomavirus (HPV) Persistence and Progression Cohort is a natural history study of carcinogenic HPV positive women. Here, we present the HPV genotypes found in first ∼500 cases of cervical intraepithelial neoplasia grade 3 (CIN3) or more severe disease (CIN3+) diagnosed at the study baseline. Methods: Women aged 30 and older were screened for cervical cancer using Pap smears and tested for carcinogenic HPV using Hybrid Capture 2 (HC2; Qiagen). We randomly selected women who tested HPV positive and were diagnosed with CIN3+ (n = 448) or without CIN3+ (<CIN3; n = 830). Residual cervical Pap specimens were HPV genotyped using a MY09/11 L1-targeted PCR method. Results: Among HC2-positive women, HPV16 (48.9%), HPV31 (9.2%), and HPV18 (8.5%) were the most common HPV genotypes in CIN3+. There was a decrease at older ages in the fraction of CIN3 (Ptrend = 0.006), adenocarcinoma in situ (AIS) (Ptrend = 0.08), and CIN3/AIS (Ptrend = 0.002) associated with HPV16. Compared to the other carcinogenic HPV genotypes in aggregate, HPV18 was strongly associated with CIN3+ in women with a normal Pap [odds ratio (OR) = 5.7, 95% CI = 1.2–26] but not in women with abnormal Pap (OR = 1.3, 95% CI = 0.74–2.3). Conclusions: HPV16 is more strongly associated with cervical precancer diagnosed in younger women (vs. older women). HPV18 infections were linked to precancerous lesions that were missed by cytology. Impact: The progression timeline of HPV16 differs from other carcinogenic HPV genotypes, which may impact the use of HPV16 detection in the management of HPV-positive women. Cancer Epidemiol Biomarkers Prev; 20(5); 946–53. ©2011 AACR.

Interrater agreement of anal cytology

The majority of anal cancers are caused by persistent infections with carcinogenic human papillomaviruses (HPV). Similar to cervical carcinogenesis, the progression from HPV infection to anal cancer occurs through precancerous lesions that can be treated to prevent invasion. In analogy to cervical cytology, anal cytology has been proposed as a screening tool for anal cancer precursors in high‐risk populations.

A comparison of human papillomavirus genotype-specific DNA and E6/E7 mRNA detection to identify anal precancer among HIV-infected men who have sex with men.

Background: Human papillomavirus (HPV) RNA detection is reportedly more specific for the detection of anogenital precancer than HPV DNA but it is unknown whether this is due to detection of RNA or due to HPV genotype restriction. Methods: A total of 363 human immunodeficiency virus (HIV)–positive men who have sex with men had two anal cytology samples taken and were evaluated using high-resolution anoscopy and biopsies of visible lesions. Anal specimens were tested for E6/E7 RNA for five carcinogenic HPV genotypes (HPV16, 18, 31, 33, and 45) and tested for the DNA of 13 carcinogenic HPV genotypes. Results: DNA testing was more likely to be positive than RNA testing (53% vs. 48%; P = 0.02) for the same five HPV genotypes in aggregate. When restricted to five HPV genotypes targeted by the RNA test, the sensitivity to detect anal precancer was the same for DNA and RNA (81%), whereas RNA was more specific than DNA (65% vs. 58%; P = 0.007). In comparison, DNA detection of all 13 carcinogenic HPV genotypes was more sensitive (96% vs. 81%; P = 0.001) but much less specific (65% vs. 33%; P < 0.001) as compared with RNA detection of the five HPV genotypes. Conclusion: After controlling for HPV genotypes, RNA was only slightly more specific than DNA detection for anal precancer. Impact: DNA or RNA testing for a subset of the most carcinogenic HPV genotypes may be useful for distinguishing between those HPV-positive men at higher and lower risk of anal precancer and cancer. Cancer Epidemiol Biomarkers Prev; 22(1); 42–9. ©2012 AACR.

Impact of 6-month frozen storage of cervical specimens in alkaline buffer conditions on human papillomavirus genotyping.

The impact of 6-month storage of cervical specimens under alkaline conditions that occurs as the result of Hybrid Capture 2 testing on human papillomavirus (HPV) genotyping is not well documented. To examine this issue, 143 frozen hc2-positive specimens in specimen transport medium were selected at random from each of the following groups: specimens stored for 6 months, 4 months, and 2.5 months under alkaline pH (pH 12-13) and specimens stored 1 month at neutral pH (pH 6-7) as controls. Specimens were tested in a masked fashion for 20 HPV genotypes (HPV6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82) using a prototype, research-use-only GP5+/6+ L1 consensus PCR method and multiplex hybridization using Luminex xMAP for detection of specific HPV genotypes One control specimen had missing test results. There were no statistical differences in the number of HPV genotypes detected, number of carcinogenic HPV genotypes detected, or in the signal strength among HPV-positive results across groups. Six-month frozen storage of cervical specimens at alkaline pH had little impact on testing for HPV genotypes among hc2-positive women using this HPV genotyping method.

A study of borderline positive Hybrid Capture 2 tests in the Kaiser Permanente Northern California cervical screening program: evidence against retesting.

Infection with one or more high-risk HPV (hr-HPV) types is a necessary step in the development of cervical cancer, making its detection a useful tool for cervical cancer screening and prevention (Bosch et al., 2002). Hybrid Capture 2 (HC2, Qiagen, Gaithersburg, MD) was the first FDA-approved and is still the most widely used laboratory test for the presence of high-risk HPV DNA as a predictor of who is or is not at risk for cervical cancer and pre-cancer (defined here as cervical intraepithelial neoplasia grade 2 or worse). While HC2 has remarkable sensitivity for cervical intraepithelial neoplasia grade 2 or worse, the transient nature of most hr-HPV infections reduces its specificity as most infections are benign and will resolve without progressing to cervical intraepithelial neoplasia grade 2 or worse. This can be problematic for cervical cancer screening algorithms in which HPV positivity triggers more intensive follow-up; in such algorithms, good specificity is important for reducing the number of costly colposcopic follow-up visits (Cuzick et al., 2008). Although manufacturer specifications indicate a relative light unit (RLU) ratio compared with positive control of 1.0 as the threshold for determining positivity, reclassifying HC2 specimens that are equivocal and fall just above the positive cut-off is one potential way to improve the assay’s specificity (Rijkaart et al., 2010; Rebolj et al., 2011; Sasieni and Castanon, 2011). Some researchers have looked at increasing the RLU cut-off point because HC2 results at this level predict lower risk than higher values (Lorincz et al., 2002). However, equivocal results are not that common and raising the cut-point to 2.0 or 3.0 may only yield a modest improvement in specificity at some cost to sensitivity; i.e., misclassification of women with cervical intraepithelial neoplasia grade 2 or worse lesions whose RLU ratios are equivocal (Rijkaart et al., 2010). Rather than raising the cut-point, a more conservative approach is to repeat the HC2 test on specimens with equivocal results and determine the final test result based on a collective assessment of the test results (Knoepp et al., 2007), however this method requires additional laboratory time and cost to implement, weighing against any savings it yields from reducing the number of colposcopic examinations. In 2001 Kaiser Permanente Northern California added the HC2 assay to its cervical cancer screening program as a triage test for women with a cytologic diagnosis of atypical squamous cells of undetermined significance and, in 2003, as a concurrent test (“cotest”) with cytology for women 30 and older. In the cotesting scheme, women have been managed as follows: 1) women who are HPV-positive with a cytologic diagnosis of atypical squamous cells of undetermined significance, or have more severe (regardless of the HPV test results) are referred for immediate colposcopy; 2) women who test HPV-negative and have cytology of atypical squamous cells of undetermined significance, or who test HPV-positive with negative cytology are rescreened in one year; and 3) if both tests are negative, women are rescreened in three years. For cotests with borderline positive hr-HPV results, interpretation of the repeat test results is used to determine the final hr-HPV test result and hence the need for rescreening visits versus colposcopy. At the time that HC2 was introduced into the Kaiser Permanente Northern California cervical cancer screening algorithm, based on concern over the meaning of borderline-positive HC2-positive results, it was decided that any specimens with borderline or “equivocal” HC2 results, defined as a RLU ratio greater than or equal to 1.0 but less than 3.0, would be re-tested two additional times. The final hr-HPV result was determined by the majority results of the three tests i.e., only one of the two repeats needed to be positive to have the final positive hr-HPV result. To assess the utility of Kaiser Permanente Northern California’s re-testing scheme for equivocal HC2 specimens, HC2 data, cytology results, and histological outcomes (less than cervical intraepithelial neoplasia grade 2, cervical intraepithelial neoplasia grade 2 or worse) were compiled for patients undergoing routine cervical cancer screening at Kaiser Permanente Northern California.