Raymond J. Apple

Determinants of Genital Human Papillomavirus Detection in a US Population

This study investigated the association of selected demographic and behavioral characteristics with the detection of low-risk, high-risk, and uncharacterized genital human papillomavirus (HPV) in women attending clinic for routine nonreferral gynecologic health care. Cervical specimens obtained from 3863 women 18-40 years old (mean, 28 years) with no history of high-grade cervical disease were analyzed for 38 HPV types. Overall, HPV prevalence was 39.2%. The prevalence of high-risk, low-risk, and uncharacterized HPV types was 26.7%, 14.7%, and 13.0%, respectively. As expected, the characteristics most strongly associated with overall HPV detection were age and numbers of lifetime and recent sex partners. Low-risk, high-risk, and uncharacterized HPV detection increased with increasing numbers of sex partners. There was a decline in high-risk and low-risk HPV detection with increasing age but little change in uncharacterized HPV detection. These results suggest that the uncharacterized HPV types have a different natural history than either low-risk or high-risk HPV types.

High-Risk Human Papillomavirus Testing in Women With ASC-US Cytology

This study evaluated the clinical performance of the cobas 4800 HPV Test (Roche Molecular Systems, Pleasanton, CA) for high-risk human papillomavirus (HR-HPV) testing with individual HPV-16/HPV-18 genotyping in women 21 years or older with atypical squamous cells of undetermined significance (ASC-US). Women (N = 47,208) were recruited in the United States during routine screening, and liquid-based cytology and HPV testing were performed. The ASC-US prevalence was 4.1% (1,923/47,208), and 1,578 women underwent colposcopy with valid results. The cobas 4800 HPV Test demonstrated performance comparable to the Hybrid Capture 2 test (QIAGEN, Gaithersburg, MD) for the detection of cervical intraepithelial neoplasia (CIN) grade 2 or worse and grade 3 or worse. HPV-16/HPV-18+ women had a greater absolute risk of CIN 2 or worse compared with pooled HR-HPV+ and HR-HPV- women (24.4%, 14.0%, and 0.8%, respectively). The cobas 4800 HPV Test is clinically validated for ASC-US triage. HPV-16/HPV-18 genotyping can identify women at highest risk for high-grade cervical disease, and this additional risk stratification may be used in formulating patient management decisions.

The HLA Class I A Locus Affects Susceptibility to Type 1 Diabetes

Human leukocyte antigen A (HLA-A) genotypes were determined for samples from 283 multiplex, Caucasian, type 1 diabetes families from the Human Biological Data Interchange (HBDI) using an immobilized probe assay. Distribution of HLA-A alleles transmitted to patients was significantly different from that in affected family-based controls (AFBAC) (p = 0.004). Transmission disequilibrium test (TDT) analysis revealed differential transmission of several HLA-A alleles from parents to affected offspring. HLA class II DRB1 and DQB1 loci were also typed, allowing assignment of HLA-A alleles to haplotypes and calculation of linkage disequilibrium values. Some of the apparent effects of HLA-A alleles on type 1 diabetes susceptibility were attributable to linkage disequilibrium with DR and DQ alleles, although others were not. The differences in frequencies between patients and controls of alleles A*0101, A*2402, and A*3002 could not be explained by linkage disequilibrium alone. Our results suggest an important role for class I antigens in modulating susceptibility to type 1 diabetes.

Human leukocyte antigen class I and II alleles and risk of cervical neoplasia: results from a population-based study in Costa Rica.

To examine human leukocyte antigen (HLA) involvement in the development of all grades of cervical neoplasia, a nested case-control study of 10,077 women in Guanacaste, Costa Rica, was conducted. Participants had invasive cervical cancer, high-grade squamous intraepithelial lesions (HSILs; n=166), or low-grade squamous intraepithelial lesions (LSILs); were positive for human papillomavirus (HPV) with no evidence of cervical neoplasia (n=320); or were HPV negative with no evidence of cervical neoplasia but with a history of high-risk sexual behavior (n=173). Compared with women who were HPV negative, women with HLA-DRB1*1301 were associated with decreased risk for cancer/HSILs (odds ratio [OR], 0.4; 95% confidence interval [CI], 0.2-0.7) and for LSILs/HPV (OR, 0.6; 95% CI, 0.3-0.9). Women with both HLA-B*07 and HLA-DQB1*0302 had an 8.2-fold increased risk for cancer/HSILs (95% CI, 1.8-37.2) and a 5.3-fold increased risk for LSILs/HPV (95% CI, 1.2-23.7). These results support the hypothesis that multiple risk alleles are needed in order to increase risk for cervical neoplasia, but a single protective allele may be sufficient for protection.

Reproducibility of HPV 16 and HPV 18 viral load quantitation using TaqMan real-time PCR assays

A reproducibility study was designed to assess within-assay, between-day, and interlaboratory variability of three real-time PCR assays targeting HPV 16, HPV 18, and the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pseudogenes. Fifteen HPV 16 and fifteen HPV 18 cervical swab samples were amplified in triplicate by GAPDH and HPV 16 and by GAPDH and HPV 18 assays, respectively. All samples were amplified undiluted and at a 1:10 dilution on 2 separate days in the same laboratory, and the same samples were amplified in a separate laboratory. HPV 16 and HPV 18 normalized viral load is reported as the number of HPV genomes per 20000 GAPDH copies. The analytic specificity of the HPV 16 and 18 assays was 100 and 97%, respectively. The intraclass correlation coefficients (ICC) were 0.99, 0.97, and 0.98 for HPV 16, HPV 18, and GAPDH, respectively, indicating that the variability due to experimental error was very low. Ten-fold differences in viral load could be readily discriminated across a six order of magnitude dynamic range (ca. 5-5x10(6) copies). Power of discrimination was increased at higher target concentrations (>5000 copies). The correlation of normalized HPV 16 and 18 viral load was high between the two laboratories (Spearman rho (rho)=0.96 and 0.87, respectively). These HPV 16 and HPV 18 quantitative PCR assays with GAPDH normalization are reproducibly quantitative over a broad linear dynamic range allowing for application in epidemiologic studies for measurement of viral load.

Consistent associations of HLA class I and II and transporter gene products with progression of human immunodeficiency virus type 1 infection in homosexual men

Polymorphic products of genes in the HLA region contributing to variability in the course of human immunodeficiency virus type 1 (HIV-1) infection were identified by screening 375 Caucasian seroconverters who were aggregated from 3 cohorts. AIDS-free time was related to numerous (15) class I alleles, alone or in conjunction with transporter protein variants, to homozygosity at the A or B locus, and to alleles of two class II haplotypes. A prognostic scoring algorithm derived from the 3 cohorts captured multiple HLA contributions to protection or to risk (relative hazard=0.57-60 per unit increase in score, all P<<.001). The impact of HLA was strong and appeared independent of the effects of chemokine receptor/ligand polymorphisms and antiretroviral treatment. The algorithm also predicted divergent rates of CD4+ cell decline in 2 other groups, totaling 227 seropositive persons (P=.06 - <.001). Confirmation of these relationships should encourage investigation of HIV-1 antigen processing and presentation mediated by polymorphisms in the HLA region.

Variation of the killer cell immunoglobulin-like receptors and HLA-C genes in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barrvirus (EBV)-associated malignancy. Previous studies have shown that NPC is associated with specific human leukocyte antigen (HLA) alleles which function in adaptive immunity to present viral and other antigens to the immune system. The role of innate immunity in NPC development is unknown. To determine whether innate immunity is associated with NPC, a case-control study was conducted among 295 Taiwanese NPC cases (99% EBV seropositive) and 252 community controls (29% EBV seropositive). Using high-resolution genotyping, we evaluated the variation of HLA class I alleles and killer cell immunoglobulin-like receptor (KIR) alleles. Located on the surface of natural killer (NK) cells and a subset of T cells, inhibitory KIRs diminish NK cytolysis of target cells upon binding to their HLA class I ligands and activating KIRs are thought to stimulate NK destruction of target cells. Our results suggest that an increasing number of activating KIRs may be associated with increasing NPC risk, particularly in individuals seropositive for anti-EBV antibodies known to be linked to NPC susceptibility (Ptrend = 0.07). Among EBV-seropositive individuals, carriers of ≥5 activating KIRs had a 3.4-fold increased risk of disease (95% confidence interval, 0.74-15.7) compared with individuals with no functional activating KIRs. In contrast, there was no clear evidence of risk associated with increasing numbers of inhibitory KIRs. When evaluating HLA-Cw alleles, we observed that carriers of HLA-Cw*0401 alleles were at a significantly reduced NPC risk (odds ratio, 0.46; 95% confidence intervals, 0.23-0.92), an effect that could not be explained by linkage disequilibrium with other NPC-associated HLA alleles. Our results suggest that KIR-mediated activation may be associated with NPC risk. As this finding is consistent with a recent report examining cervical cancer, a malignancy caused by human papillomavirus, the data raises the possibility that KIRs, and more generally innate immunity, may be involved in the pathogenesis of viral-associated cancers.

A Study of Genotyping for Management of Human Papillomavirus-Positive, Cytology-Negative Cervical Screening Results

ABSTRACT The effective management of women with human papillomavirus (HPV)-positive, cytology-negative results is critical to the introduction of HPV testing into cervical screening. HPV typing has been recommended for colposcopy triage, but it is not clear which combinations of high-risk HPV types provide clinically useful information. This study included 18,810 women with Hybrid Capture 2 (HC2)-positive, cytology-negative results and who were age ≥30 years from Kaiser Permanente Northern California. The median follow-up was 475 days (interquartile range [IQR], 0 to 1,077 days; maximum, 2,217 days). The baseline specimens from 482 cases of cervical intraepithelial neoplasia grade 3 or cancer (CIN3+) and 3,517 random HC2-positive noncases were genotyped using 2 PCR-based methods. Using the case-control sampling fractions, the 3-year cumulative risks of CIN3+ were calculated for each individual high-risk HPV type. The 3-year cumulative risk of CIN3+ among all women with HC2-positive, cytology-negative results was 4.6%. HPV16 status conferred the greatest type-specific risk stratification; women with HC2-positive/HPV16-positive results had a 10.6% risk of CIN3+, while women with HC-2 positive/HPV16-negative results had a much lower risk of 2.4%. The next most informative HPV types and their risks in HPV-positive women were HPV33 (5.9%) and HPV18 (5.9%). With regard to the etiologic fraction, 20 of 71 cases of cervical adenocarcinoma in situ (AIS) and adenocarcinoma in the cohort were positive for HPV18. HPV16 genotyping provides risk stratification useful for guiding clinical management; the risk among HPV16-positive women clearly exceeds the U.S. consensus risk threshold for immediate colposcopy referral. HPV18 is of particular interest because of its association with difficult-to-detect glandular lesions. There is a less clear clinical value of distinguishing the other high-risk HPV types.

Interlaboratory variation in the performance of liquid‐based cytology: Insights from the ATHENA trial

Although it is recognized that cervical cytology is highly subjective, and that there is considerable interlaboratory variation in how slides are evaluated, little is known as to how this impacts the performance of cytology. In the ATHENA trial, liquid‐based cytology specimens from 46,887 eligible women ≥21 years of age were evaluated at four large regional US laboratories, providing a unique opportunity to evaluate the impact of interlaboratory variations on the performance of cervical cytology. All women with abnormal cytology (atypical squamous cells of undetermined significance or higher) were referred to colposcopy, as were all high‐risk human papillomavirus (hrHPV)–positive women ≥25 years of age and a random subset of those ≥25 years of age who were negative by both hrHPV testing and cytology. Sociodemographics, risk factors for cervical disease, and prevalence of cervical intraepithelial neoplasia (CIN) were similar across the laboratories. There were considerable differences among the laboratories both in overall cytological abnormal rates, ranging from 3.8 to 9.9%, and in sensitivity of cytology to detect CIN grade 2 or worse (CIN2+), from 42.0 to 73.0%. In contrast, the hrHPV positivity rate varied only from 10.9 to 13.4%, and the sensitivity of hrHPV testing from 88.2 to 90.1%. These observations suggest that hrHPV testing without cytology should be considered as the initial method for cervical cancer screening.

The Interplay of Age Stratification and HPV Testing on the Predictive Value of ASC-US Cytology Results From the ATHENA HPV Study

We have previously shown that human papillomavirus (HPV) genotyping, using the cobas HPV Test (Roche Molecular Systems, Pleasanton, CA), can be used to identify women with atypical squamous cells of undetermined significance (ASC-US) at the highest risk for cervical intraepithelial neoplasia (CIN) grade 2 or worse. We investigated the impact of age stratification on the risk of CIN 2 or worse in women with ASC-US and the performance of HPV genotyping in different age strata. The sensitivity of the cobas HPV Test was 93.3% in the 21- to 29-year-old age group and 67.7% in the 40 years or older group, most likely owing to pathologic misclassification of CIN 2 or worse in older women. The prevalence of CIN 2 or worse in younger women was nearly 4-fold that detected in older women and was predominantly HPV-16-related. Age-specific evaluation of ASC-US cytology in conjunction with HPV genotype status enables more effective risk assessment and could be used in clinical management.