Cytology-based screening for invasive cervical cancer (ICC) lacks sensitivity and specificity to discriminate between cervical intraepithelial neoplasia (CIN) likely to persist or progress from cases likely to resolve. Genome-wide approaches have been used to identify DNA methylation marks associated with CIN persistence or progression. However, associations between DNA methylation marks and CIN or ICC remain weak and inconsistent. Between 2008–2009, we conducted a hospital-based, case-control study among 213 Tanzania women with CIN 1/2/3 or ICC. We collected questionnaire data, biopsies, peripheral blood, cervical scrapes, Human papillomavirus (HPV) and HIV-1 infection status. We assessed PEG3 methylation status by bisulfite pyrosequencing. Multinomial logistic regression was used to estimate odds ratios (OR) and confidence intervals (CI 95%) for associations between PEG3 methylation status and CIN or ICC. After adjusting for age, gravidity, hormonal contraceptive use and HPV infection, a 5% increase in PEG3 DNA methylation was associated with increased risk for ICC (OR = 1.6; 95% CI 1.2–2.1). HPV infection was associated with a higher risk of CIN1-3 (OR = 15.7; 95% CI 5.7–48.6) and ICC (OR = 29.5, 95% CI 6.3–38.4). Infection with high risk HPV was correlated with mean PEG3 differentially methylated regions (DMRs) methylation (r = 0.34 p<0.0001), while the correlation with low risk HPV infection was weaker (r = 0.16 p = 0.047). Although small sample size limits inference, these data support that PEG3 methylation status has potential as a molecular target for inclusion in CIN screening to improve prediction of progression.
We present the first evidence that aberrant methylation of the PEG3 DMR is an important co-factor in the development of Invasive cervical carcinoma (ICC), especially among women infected with high risk HPV. Our results show that a five percent increase in DNA methylation of PEG3 is associated with a 1.6-fold increase ICC risk. Suggesting PEG3 methylation status may be useful as a molecular marker for CIN screening to improve prediction of cases likely to progress.
This study examines risk factors for persistent cervical intraepithelial neoplasia (CIN) and whether human papillomavirus (HPV) testing predicts persistent lesions.
Materials and Methods
Women with histologically diagnosed CIN 1 or CIN 2 (n = 206) were followed every 3 months without treatment. HPV genotyping, plasma levels of ascorbic acid, and red blood cell folate were obtained. Cervical biopsy at 12 months determined the presence of CIN. Relative risk (RR) was estimated by log-linked binomial regression models.
At 12 months, 70% of CIN 1 versus 54% of CIN 2 lesions spontaneously regressed (p< 0.001). Levels of folate or ascorbic acid were not associated with persistent CIN at 12 months. Compared to HPV negative women, those with multiple HPV types (RRs ranged from 1.68 to 2.17 at each follow-up visit) or high-risk types (RRs range = 1.74 to 2.09) were at increased risk for persistent CIN; women with HPV 16/18 had the highest risk (RRs range = 1.91 to 2.21). Persistent infection with a high-risk type was also associated with persistent CIN (RRs range = 1.50 – 2.35). Typing for high-risk HPVs at 6 months only had a sensitivity of 46% in predicting persistence of any lesions at 12 months.
Spontaneous regression of CIN 1 and CIN 2 occurs frequently within 12 months. HPV infection is the major risk factor for persistent CIN. However, HPV testing cannot reliably predict persistence of any lesion.
cervical intraepithelial neoplasia; human papillomavirus
Human papillomavirus type 58 (HPV-58) exists in a relatively high prevalence in certain parts of the world, including East Asia. This study examined the T-cell response to HPV-58 L1, E6, and E7 peptides among women with cleared infection, cervical intraepithelial neoplasia grade 2 (CIN2) or CIN3, or invasive cervical cancer (ICC). Peptides found to be reactive in the in vitro peptide binding assay or mouse-stimulating study were tested with a gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay to detect peptide-specific responses from the peripheral blood mononuclear cells (PBMC) collected from 91 HPV-58-infected women (32 with cleared infection, 16 CIN2, 15 CIN3, and 28 ICC). Four HLA-A11-restricted HPV-58 L1 peptides, located at amino acid positions 296 to 304, 327 to 335, 101 to 109, and 469 to 477, showed positive IFN-γ ELISPOT results and were mainly from women with cleared infection. Two HLA-A11-restricted E6 peptides (amino acid positions 64 to 72 and 94 to 102) and three HLA-A11-restricted E7 peptides (amino acid positions 78 to 86, 74 to 82, and 88 to 96) showed a positive response. A response to E6 and E7 peptides was mainly observed from subjects with CIN2 or above. One HLA-A2-restricted E6 peptide, located at amino acid position 99 to 107, elicited a positive response in two CIN2 subjects. One HLA-A24-restricted L1 peptide, located at amino acid position 468 to 476, also elicited a positive response in two CIN2 subjects. In summary, this study has identified a few immunogenic epitopes for HPV-58 E6 and E7 proteins. It is worthwhile to further investigate whether responses to these epitopes have a role in clearing an established cervical lesion.
To determine whether the diagnosis of cervical intraepithelial neoplasia grade 1 (CIN1) increases the risk of cervical intraepithelial neoplasia grade 3 (CIN3) above what is observed for human papillomavirus (HPV) infection.
Using data from the atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) triage study (ALTS), we compared the 2-year cumulative risk of CIN3 for women with an enrollment diagnosis of CIN1 (n = 594) (median age = 23 years) compared with those with negative histology or no biopsy taken at colposcopy (“no CIN1,” n = 570) (median age = 24 years). Baseline cervical specimens were tested for carcinogenic HPV by a clinical HPV test and HPV genotypes by polymerase chain reaction. Logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI) as a measure of association of enrollment status, including CIN1 compared with no CIN1 diagnosis, with 2-year worst outcomes of CIN3.
The two-year risks of CIN3 were 10.3% (95%CI: 7.9%–13.0%) for women with CIN1, 7.3% (95%CI: 4.6%–10.9%) for negative histology, and 6.4% (95%CI: 3.8%–9.9%) for women referred to colposcopy and no biopsies were taken (p = 0.1). The 2-year risk of CIN3 for women positive for HPV16, HPV18, or other carcinogenic HPV genotypes was 19.1%, 13.9%, and 5.7%, respectively, and did not differ significantly by the baseline cytology interpretation (ASCUS or LSIL). Taking HPV genotypes into account, having a CIN1 (compared with no CIN1) was not a risk factor for developing CIN3 (OR = 0.99, 95%CI: 0.54–1.8).
A CIN1 diagnosis does not represent a significant risk factor for CIN3 above the risk attributed to its molecular cause, genotype-specific HPV infection.
Even though HPV 16 is the most common HPV genotype associated with cancerous lesions of the cervix, only a fraction of HPV 16 infected women are diagnosed with pre-cancerous lesions of the cervix. Therefore, molecular changes in HPV 16 rather than infections per se may serve as better screening or diagnostic biomarkers. The purpose of the study was to evaluate whether methylation status of specific regions of the HPV E6 gene promoter and enhancer is independently associated with the likelihood of being diagnosed with higher grades of cervical intraepithelial neoplasia (CIN 2+).
The study included 75 HPV 16 positive women diagnosed with CIN 2+ or ≤ CIN 1. Pyrosequencing technology was applied to quantify methylation at 6 cytosine guanine dinucleotide (CpG) sites of the HPV 16 E6 promoter and enhancer. CIN 2+ (yes/no) was the dependent variable in logistic regression models that specified the degree of methylation of the CpG sites of the HPV 16 E6 gene as the primary independent predictors. All models were adjusted for demographic, lifestyle, known risk factors for cervical cancer and circulating concentrations of “cancer-protective” micronutrients.
The odds of being diagnosed with CIN 2+ was 79% lower when the degree of methylation of the HPV 16 enhancer and promoter sites were ≥9.5% (OR= 0.21; 95% CI, 0.06–0.79; P=0.02).
Results suggested that CpG methylation is independently involved in the biology of HPV-16 as well as in the development of higher grades of CIN.
HPV 16; methylation; cervical; neoplasia
To estimate the risks of cervical intraepithelial neoplasia-3 among women aged 13 to 24 years of age who were referred for abnormal cytology while receiving care in a large health maintenance organization.
At the time of referral, women had a colposcopic examination and biopsy if needed. Histology was sent to a centralized laboratory. Women were interviewed for risk behaviors. Data analysis included multinomial logistic regression analysis to compare 3 groups: CIN-3 to CIN-1 or less CIN-3 to CIN-2, and CIN-2 to CIN-1 or benign.
CIN-3 was found in 6.6% (95% CI = 4.6 - 8.6%) of the 622 women referred and no cancers were detected. Risk for CIN 3 compared to CIN 1 or less included HPV 16 or 18 (odds ratio [OR] 30.93 [95% confidence interval (CI); 6.95, 137.65]), high-risk, non-16/18 HPV (OR 6.3 [95% CI; 1.3, 29.4]), and time on oral contraceptives (OR 1.36 per year of use [95% CI ; 1.08, 1.71]).
Our data support conservative care for adolescents and young women with abnormal cytology since CIN-3 was rare, and cervical cancer was never found. HPV-16 or 18 were strongly associated with for CIN-3, and testing for these types may be warranted for triage of abnormal cytology in this age group.
Identification of associations between global DNA methylation and excess body weight (EBW) and related diseases and their modifying factors are an unmet research need that may lead to decreasing DNA methylation-associated disease risks in humans. The purpose of the current study was to evaluate the following; 1) Association between the degree of peripheral blood mononuclear cell (PBMC) L1 methylation and folate, and indicators of EBW, 2) Association between the degree of PBMC L1 methylation and folate, and insulin resistance (IR) as indicated by a higher homeostasis model assessment (HOMA-IR).
The study population consisted of 470 child-bearing age women diagnosed with abnormal pap. The degree of PBMC L1 methylation was assessed by pyrosequencing. Logistic regression models specified indicators of EBW (body mass index–BMI, body fat–BF and waist circumference–WC) or HOMA-IR as dependent variables and the degree of PBMC L1 methylation and circulating concentrations of folate as the independent predictor of primary interest.
Women with a lower degree of PBMC L1 methylation and lower plasma folate concentrations were significantly more likely to have higher BMI, % BF or WC (OR = 2.49, 95% CI:1.41–4.47, P = 0.002; OR = 2.49, 95% CI:1.40–4.51, P = 0.002 and OR = 1.98, 95% = 1.14–3.48 P = 0.0145, respectively) and higher HOMA-IR (OR = 1.78, 95% CI:1.02–3.13, P = 0.041).
Our results demonstrated that a lower degree of PBMC L1 methylation is associated with excess body weight and higher HOMA-IR, especially in the presence of lower concentrations of plasma folate.
Persistent infection with oncogenic human papillomaviruses (HPV) plays a central etiologic role in the development of squamous carcinomas of the cervix and their precursor lesions, cervical intraepithelial neoplasias (CIN). We carried out a prospective observational cohort study evaluating known, quantifiable prognostic variables of clinical behavior in women with high-grade cervical lesions.
Our study cohort included healthy women with high-grade cervical lesions (CIN2/3) with residual visible lesions after colposcopically directed biopsy. We prospectively followed 100 women over 15 weeks before standard resection. HPV typing was done using PCR and a reverse line blot detection method.
The rate of spontaneous histologic regression, defined as (CIN1 or less at resection) was 28%. The overall rate of HPV infection was 100%. HPV16 was identified in 68% of the lesions. Women with HPV16 only were significantly less likely to regress, compared with women with HPV types other than HPV16 (odds ratio, 0.342; 95% confidence interval, 0.117-0.997; P = 0.049). In the cohort with HPV16 only, patients who had an HLA*A201 allele had similar outcomes to those who did not carry A201. However, among patients with HPV types other than HPV16, the HLA*A201 allele interaction was significant; patients with HLA*A201 were the least likely to resolve.
CIN2/3 lesions associated with HPV16 alone are significantly less likely to resolve spontaneously than those caused by other types. Interactions among HPV type, HLA type, and regression rate support a role for HLA-restricted HPV-specific immune responses in determining disease outcome.
Invasive cervix cancer (ICC) is the second most common malignant tumor in women. Human papillomavirus 16 (HPV16) causes more than 50% of all ICC and is a major cause of cervix intraepithelial neoplasia (CIN). DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides. Such epigenetic modifications are associated with changes in DNA-protein interactions and gene activation. This study examined the association of viral and host genomic methylation patterns and cervix neoplasia.
Exfoliated cervical lavage samples positive for HPV16 from women with and without cytomorphic changes of infection (n=46), CIN2 (n=12), and CIN3+ (n=27) were used to interrogate the methylation patterns of the HPV16 L1 gene and upstream regulatory region (URR), five host nuclear genes (TERT, RARB, DAPK1, MAL, and CADM1), and mitochondrial DNA (mtDNA). DNA isolated from exfoliated cervicovaginal cells was treated with bisulfite, specific regions of the viral and host genome were PCR amplified and CpG methylation was quantified using EpiTYPER and pyrosequencing.
Methylation at 14 of the tested CpG sites within the HPV16 L1 region were significantly higher in CIN3+ compared to HPV16 genomes from women without CIN3+. In contrast, only 2 out of 16 CpG sites in HPV16 URR, 5/5 in TERT, 1/4 in DAPK1 and 1/3 mtDNA, and 2/5 in RARB were associated with increased methylation in CIN3+.
These results indicate that increased methylation of CpG sites in the HPV16 L1 ORF is associated with CIN3+ and thus, may constitute a potential biomarker for precancerous and cancerous cervix disease.
cervical cancer; human papillomavirus; methylation
To evaluate whether mandatory fortification of grain products with folic acid in the US is associated with changes in histone methylation in cells involved in cervical carcinogenesis.
Cervical specimens obtained before (1990 to 1992) and after mandatory folic acid fortification (2000 to 2002) were used to examine the degree of histone methylation (H3 Lys-9) by immunohistochemistry. 91 women (51 before and 40 after fortification) were diagnosed with cervical intraepithelial neoplasia (CIN) grade 3 or carcinoma in situ (CIS) and sections utilized in the study also contained normal, reactive or metaplastic cervical epithelium, CIN 1 or CIN 2. 64 women (34 before and 30 after fortification) were free of CIN and these sections contained only normal or reactive cervical epithelium. Immunohistochemical staining for H3 Lys-9, its assessment in different cell or lesion types and data entry were blinded for fortification status. For each cell type or lesion category we used PROC MIXED in SAS with the specimen identifier as a random effect and the robust variance estimator to estimate age- and race-adjusted intensity score for H3 Lys-9 in the pre- and post-fortification periods.
Degree of H3 Lys-9 methylation was significantly higher (P < 0.0001) in ≥CIN 2 lesions (CIN 2, CIN 3 and CIS) than in ≤CIN 1 lesions (CIN 1, normal, reactive and metaplastic), in both pre- and post-fortification CIN 3/CIS specimens. Age- and race-adjusted mean H3 Lys-9 score was significantly higher in all cell or lesion types in CIN 3/CIS specimens obtained in the post-fortification period compared to pre-fortification period (P < 0.05, all comparisons). In contrast, in specimens obtained from women free of CIN, Lys-9 methylation in normal/reactive cervical epithelium was significantly lower in post-fortification specimens than in pre-fortification specimens (P = 0.03).
Higher levels of Lys-9 methylation in ≥CIN 2 compared to ≤CIN 1 lesions suggest that higher Lys-9 methylation is associated with progression of lower grade CIN to higher grade CIN. Higher Lys-9 methylation in cervical tissues of women diagnosed with CIN 3 in the post-fortification period than in pre-fortification period suggest that fortification may adversely affect histone methylation in already initiated cells. Lower Lys-9 methylation in normal/reactive cervical cells of women free of CIN in the post-fortification period than pre-fortification on the other hand suggests that fortification is likely to protect against initiation of carcinogenic process in the cervix. These results suggest that mandatory fortification with folic acid in the US seems to have different effects on cancer depending on the stage of carcinogenesis. Because this is the first study to report folic acid fortification-associated differences in histone methylation and because of the limitations inherent to the approach we have taken to demonstrate these differences, validation of the results in other study populations or with other techniques for assessing histone methylation is necessary.
folic acid; fortification; histone methylation; cervix
Human papillomavirus (HPV) typing and quantitation by polymerase chain reaction was performed on exfoliated cells from 133 women referred for colposcopy because of an abnormal smear. High levels of HPV 16 correctly predicted cervical intraepithelial neoplasia (CIN) grade II-III in 93% of its occurrences, but only 59% of cases of CIN III were associated with high levels of this type. Eighty-four per cent of CIN III lesions contained high levels of at least one of HPV types 16, 18, 31, 33 and 35, but the other types were less specific for CIN III than HPV 16. Overall HPV testing compared favourably with cytology for predicting high-grade CIN lesions, but it would appear that some combination of the two modalities will produce better performance than either alone. In particular, HPV testing appears to be helpful in determining which women with mildly abnormal smears have high-grade underlying lesions in need of immediate referral for colposcopy.
Cervical mucosal expression of cytokines involved in mediating cellular immunity is believed to influence the persistence of human papillomavirus (HPV) infection, a necessary prerequisite for the development of cervical intraepithelial neoplasia (CIN). Additionally, regulatory T (Treg) cells are increasingly understood to be important modulators of cellular immunity. Using quantitative RT-PCR, we measured, in cross-sectional design, the cervical mRNA expression of IFN-γ, IL-10, and IL-12, as well as the Treg transcription factor Forkhead box P3 (Foxp3), in a cohort of young women representing CIN 1, 2, and 3 as well as benign histology. Higher levels of IFN-γ and IL-10 were significantly (p≤0.05) associated with decreased odds of having high-grade cervical disease (CIN 2 or 3) in multivariate logistic regression models. In contrast, higher levels of mucosal Foxp3 expression were associated with increased odds of having CIN 2 or 3 (p=0.004). In a multivariate model including cervical infection with HPV16 and/or another high-risk HPV type, Foxp3 remained higher in the CIN 2/3 group, but the difference was notably less significant (p=0.05). These findings support a model in which diminished cellular immunity in the cervical mucosa and mucosal enrichment of Treg cells both contribute to the development of high-grade lesions.
cervical intraepithelial neoplasia; papillomavirus infections; cytokines; regulatory T cells; mucosal immunology
Prevalence estimates of cervical intraepithelial neoplasia (CIN) among HIV-infected women in India have been based on cervical cytology, which may have underestimated true disease burden. We sought to better establish prevalence estimates and evaluate risk factors of CIN among HIV-infected women in Pune, India using colposcopy and histopathology as diagnostic tools.
Previously unscreened, non-pregnant HIV-infected women underwent cervical cancer screening evaluation including standardized diagnostic colposcopy by a gynecologist. Histopathologic confirmation was conducted among consenting women with clinical suspicion of CIN. The prevalence of CIN was evaluated by a composite diagnosis based on colposcopy and histopathology results. Multivariable ordinal logistic regression analysis was conducted to determine independent predictors of increasing severity of CIN.
The median age of the n = 303 enrolled HIV-infected women was 30 years (interquartile range, 27–34). A majority of the participants were widowed or separated (187/303, 61.7%), more than one-third (114/302, 37.7%) were not educated beyond primary school, and nearly two-thirds (196/301, 64.7%) had a family per capita income of <1,000 Indian Rupees (∼US$22) per month. Cervical high-risk HPV-DNA was detected in 41.7% (124/297) of participants. The composite colposcopic-histopathologic diagnoses revealed no evidence of CIN in 220 out of 303 (72.6%) women, CIN1 in 33/303 (10.9%), CIN2 in 31/303 (10.2%), CIN3 in 18/303 (5.9%) and 1 (0.3%) woman was diagnosed with ICC. Thus, over a quarter of the participants [83/303: 27.7% (95% CI: 22.7–33.1)] had ≥CIN1 lesions and a sixth [50/303: 16.5% (95% CI: 12.2–21.9)] had evidence of advanced (≥CIN2) neoplastic disease. The independent predictors of increasing severity of CIN as revealed by a proportional odds model using multivariable ordinal logistic regression included (i) currently receiving antiretroviral therapy [adjusted odds ratios (aOR): 2.24 (1.17, 4.26), p = 0.01] and (ii) presence of cervical high-risk HPV-DNA [aOR: 1.93 (1.13, 3.28), p = 0.02].
HIV-infected women in Pune, India have a substantial burden of cervical precancerous lesions, which may progress to invasive cervical cancer unless appropriately detected and treated. Increased attention should focus on recognizing and addressing this entirely preventable cancer among HIV-infected women, especially in the context of increasing longevity due to antiretroviral therapy.
The triage of women with high-risk (HR) human papillomavirus (HPV)-positive smears for atypical squamous cells of undetermined significance (ASC-US) to colposcopy is now an integrated option in clinical guidelines. The performance of cobas 4800 HPV and that of Hybrid Capture 2 (HC2) for HR HPV DNA detection in cervical samples in PreservCyt were compared in 396 women referred to colposcopy for ASC-US. Of these, 316 did not have cervical intraepithelial neoplasia (CIN), 47 had CIN1, 29 had CIN2 or CIN3 (CIN2+), and 4 had CIN of unknown grade. HR HPV was detected in 129 (32.6%) and 149 (37.6%) samples with HC2 and cobas 4800 HPV, respectively (P = 0.15). The clinical sensitivities and specificities for detecting CIN2+ were 89.7% (95% confidence interval [CI], 72.8 to 97.2%) and 66.7% (95% CI, 61.7 to 71.3%) with cobas 4800 HPV and 93.1% (95% CI, 77.0 to 99.2%) and 72.2% (95% CI 67.4 to 76.5%) with HC2. The performance of cobas 4800 HPV was similar to that of HC2 for identifying women with ASC-US who would benefit the most from colposcopy.
To estimate the fraction of cervical intraepithelial neoplasia-2 (CIN-2) that might regress if untreated using data from the atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) triage study (ALTS).
We compared the cumulative occurrence of CIN-2 (n = 397) and CIN-3 or worse (n = 542) diagnosed by the Pathology Quality Control Group in three trial arms— immediate colposcopy, human papillomavirus (HPV) triage, and conservative management— over the 2-year duration of the ALTS trial. A non-parametric test of trend was used to test for differences in the number of CIN-2 cases relative to number of CIN-3 or worse cases across study arms, with an increasing percentage of women referred to colposcopy at baseline.
There were no significant differences in the cumulative 2-year cumulative CIN-3 or worse diagnoses by study arm (10.9%, conservative management; 10.3%, HPV; 10.9%, immediate colposcopy) (ptrend = 0.8) but there was a significant increase in CIN-2 diagnoses (5.8%, conservative management; 7.8%, HPV triage; 9.9%, immediate colposcopy) (ptrend < 0.001) in the study arms with increasing number of women referred to colposcopy at baseline. The relative differences in CIN-2 by study arm among women who tested HPV16 positive at baseline were less pronounced (ptrend = 0.1) than women who tested positive for other high-risk HPV genotypes (ptrend = 0.01).
There was evidence that approximately 40% of undiagnosed CIN-2 will regress over 2 years but CIN-2 caused by HPV16 may be less likely to regress than CIN-2 caused by other high-risk HPV genotypes.
Cervical intraepithelial neoplasia (CIN) is greatly increased in women infected with sexually transmitted Human Papillomaviruses (HPVs) and who are co-infected with Human Immunodeficiency Virus (HIV) infection. Factors associated with promotion of HPV to CIN in these women include degree of immunosuppression and preventable behavioral factors such as tobacco smoking and psychological stress. Interventions such as cognitive behavioral stress management (CBSM) decrease stress and modulate disease activity in HIV-infected men though effects have not been established in HIV-infected women. This study examined the effects of CBSM on life stress and CIN in HIV+ minority women.
Participants were 39 HIV+ African American, Caribbean and Hispanic women with a recent history of an abnormal Papanicolaou smear. Participants underwent colposcopic examination, psychosocial interview, and peripheral venous blood draw at study entry and 9 months after being randomly assigned to either a 10-week CBSM group intervention (n = 21) or a one-day CBSM workshop (n = 18).
Women assigned to the 10-week CBSM intervention reported decreased perceived life stress and had significantly lower odds of CIN over a 9-month follow-up, independent of CIN at study entry, HPV type, CD4+CD3+ cell count, HIV viral load, and tobacco smoking. Women free of CIN at follow-up reported decreases in perceived stress over time while those with CIN reported increases in perceived stress over the same period.
Although preliminary these findings suggest that stress management decreases perceived life stress and may decrease the odds of CIN in women with HIV and HPV.
HIV; HPV; Cervical cancer; Stress; Stress Management
Essentially all squamous cervical cancers and their precursor lesions, high grade cervical intraepithelial neoplasia (CIN2/3), are caused by persistent human papil-lomavirus (HPV) infection. However, not all CIN2/3 lesions progress to cancer. In a brief, observational study window monitoring subjects with CIN2/3 from protocol entry (biopsy diagnosis) to definitive therapy (cervical conization) at week 15, in a cohort of 50 subjects, we found that 26% of CIN2/3 lesions associated with HPV16, the genotype most commonly associated with disease, underwent complete histologic regression. Nonetheless, HPV16-specific T cell responses measured in peripheral blood obtained at the time of study entry and at the time of conization were marginally detectable directly ex vivo, and did not correlate with lesion regression. This finding suggests that, in the setting of natural infection, immune responses which are involved in elimination of cervical dysplastic epithelium are not represented to any great extent in the systemic circulation.
Human papillomavirus (HPV); Cervical dysplasia; Regression; Systemic immune response
Previous studies have suggested an association between human papillomavirus type 16 (HPV16) genome methylation and cervical intraepithelial neoplasia grade 3 (CIN3) (ie, cervical precancer) and cancer, but the results have been inconsistent.
We designed a case–control study within a large prospective cohort of women who underwent multiple screenings for cervical cancer in Guanacaste, Costa Rica. Diagnostic specimens were collected at the time of CIN3 diagnosis (n = 30 case subjects) and persistent HPV16 infection (persistence; n = 35 case subjects), prediagnostic specimens at the first HPV16-positive screening visit (n = 20 CIN3 case subjects; n = 35 persistence case subjects), and control specimens from women with infection clearance within 2 years (n = 34 control subjects). DNA extracted from specimens (cervical cells) was analyzed for methylation levels at 67 CpG sites throughout the HPV16 genome using pyrosequencing. Benjamini–Hochberg method was used to account for multiple testing. Associations between methylation levels and risk of CIN3 or persistence were assessed using logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals (CIs).
Increased methylation in diagnostic vs control specimens at nine CpG sites, three in each L1, L2, and E2/E4 genomic regions, was associated with an increased risk of CIN3 (third tertile [high] vs first and second tertiles combined [low], OR = 3.29 [95% CI = 1.16 to 9.34] to 11.12 [95% CI = 2.29 to 76.80]) and persistence. High methylation at three of these CpG sites was associated with a much higher risk when combined compared with low methylation at these sites (OR = 52, 95% CI = 4.0 to 670). In prediagnostic vs control specimens, increased methylation at a CpG site (nucleotide position 4261) in L2 was associated with an increased risk of CIN3.
In this HPV16-infected cohort, increased methylation of CpG sites within the HPV16 genome before diagnosis and at the time of diagnosis was associated with cervical precancer.
HPV infrequently persists and progresses to cervical cancer. We examined host genetic factors hypothesized to play a role in determining which subset of individuals infected with oncogenic human papillomavirus (HPV) have persistent infection and further develop cervical pre-cancer/cancer compared to the majority of infected individuals who will clear infection.
We evaluated 7140 tag single nucleotide polymorphisms (SNPs) from 305 candidate genes hypothesized to be involved in DNA repair, viral infection and cell entry in 416 cervical intraepithelial neoplasia 3 (CIN3)/cancer cases, 356 HPV persistent women (median: 25 months), and 425 random controls (RC) from the 10,049 women Guanacaste Costa Rica Natural History study. We used logistic regression to compute odds ratios and p-trend for CIN3/cancer and HPV persistence in relation to SNP genotypes and haplotypes (adjusted for age). We obtained pathway and gene-level summary of associations by computing the adaptive combination of p-values. Genes/regions statistically significantly associated with CIN3/cancer included the viral infection and cell entry genes 2′,5′ oligoadenylate synthetase gene 3 (OAS3), sulfatase 1 (SULF1), and interferon gamma (IFNG); the DNA repair genes deoxyuridine triphosphate (DUT), dosage suppressor of mck 1 homolog (DMC1), and general transcription factor IIH, polypeptide 3 (GTF2H4); and the EVER1 and EVER2 genes (p<0.01). From each region, the single most significant SNPs associated with CIN3/cancer were OAS3 rs12302655, SULF1 rs4737999, IFNG rs11177074, DUT rs3784621, DMC1 rs5757133, GTF2H4 rs2894054, EVER1/EVER2 rs9893818 (p-trends≤0.001). SNPs for OAS3, SULF1, DUT, and GTF2H4 were associated with HPV persistence whereas IFNG and EVER1/EVER2 SNPs were associated with progression to CIN3/cancer. We note that the associations observed were less than two-fold. We identified variations DNA repair and viral binding and cell entry genes associated with CIN3/cancer. Our results require replication but suggest that different genes may be responsible for modulating risk in the two critical transition steps important for cervical carcinogenesis: HPV persistence and disease progression.
High-risk human papillomavirus (hrHPV) is the primary cause of cervical cancer. As Chlamydia trachomatis is also linked to cervical cancer, its role as a potential co-factor in the development of cervical intraepithelial neoplasia (CIN) grade 2 or higher was examined.
The placebo arms of two large, multinational, clinical trials of an HPV6/11/16/18 vaccine were combined. A total of 8441 healthy women aged 15–26 years underwent cervicovaginal cytology (Papanicolaou (Pap) testing) sampling and C trachomatis testing at day 1 and every 12 months thereafter for up to 4 years. Protocol-specified guidelines were used to triage participants with Pap abnormalities to colposcopy and definitive therapy. The main outcome measured was CIN.
At baseline, 2629 (31.1%) tested positive for hrHPV DNA and 354 (4.2%) tested positive for C trachomatis. Among those with HPV16/18 infection (n=965; 11.4%) or without HPV16/18 infection (n=7382, 87.5%), the hazard ratios (HRs) associated with development of any CIN grade 2 according to baseline C trachomatis status were 1.82 (95% CI: 1.06 to 3.14) and 1.74 (95% CI 1.05 to 2.90), respectively. The results were comparable when only the 12 most common hrHPV infections were considered, but the excess risk disappeared when the outcome was expanded to include CIN grade 3 or worse.
Further studies based on larger cohorts with longitudinal follow-up in relation to the C trachomatis acquisition and a thorough evaluation of temporal relationships of infections with hrHPV types, C trachomatis and cervical neoplasia are needed to demonstrate whether and how in some situations C trachomatis sets the stage for cervical carcinogenesis.
NCT00092521 and NCT00092534.
In a retrospective case-control study biopsy specimens of cervical intraepithelial neoplasia (CIN) lesions from 47 women in whom invasive cancer subsequently developed (cases) and from 94 control subjects in whom CIN was diagnosed within 6 months of the diagnosis for the matched case subject but invasive disease did not develop were tested for human papillomavirus (HPV) DNA with tissue in-situ hybridization. There were no significant differences in the frequency of detection of HPV DNA between the two groups. In a cross-sectional survey the prevalence of HPV DNA was found to be 11% in specimens without CIN, 27% in those with CIN I, 49% in those with CIN II and 56% in those with CIN III. The positivity rates for HPV 16/33 DNA increased with the severity of CIN, but this was not observed for HPV 6/11 and 18 DNA. A comparison of the results of the case-control and cross-sectional studies suggested that the younger cohort of women had higher prevalence rates of HPV DNA than the older cohort.
The Papanicolaou test generates pain and embarrassment, and cytology screening has limited sensitivity for detection of cervical neoplasia. These factors urge the use of another screening test that can overcome these limitations. We explore a completely noninvasive method using detection of human papillomavirus (HPV) DNA in women's menstrual blood (MB). The participants were divided into 3 cohorts: (i) 235 patients with cervical intraepithelial neoplasia 3 (CIN 3) (n = 48), CIN 2 (n = 60), CIN 1 (n = 58), or condyloma acuminatum (CAC) (n = 69) before treatment or remission; (ii) from the first cohort of patients, 108 CIN 3 or CIN 2 patients after treatment and 62 CIN 1 or CAC patients after remission; and (iii) 323 apparently normal subjects (ANS) without any cervical disease. The HPV genotypes of the infected patients were confirmed by direct sequencing. Quantitative real-time PCR (QRT-PCR) was used to measure the MB HPV16 load for 15 infected patients. Results showed that the sensitivity, specificity, and positive and negative predictive values for detection of MB HPV DNA in samples from patients with CIN or CAC were 82.8%, 93.1%, 90.0%, and 87.9%, respectively. Moreover, MB HPV DNA was found in samples from 22.2% of CIN 3 or CIN 2 patients after treatment, 0.0% of CIN 1 or CAC patients after remission, and 8.1% of ANS, 4 of whom were found to have CIN 1 or CAC. Furthermore, QRT-PCR showed that the normalized MB HPV16 DNA copy numbers in samples from patients with CIN 1 to CIN 3 were significantly increased. These preliminary results suggested that MB HPV DNA is a potential noninvasive marker for these premalignant cervical diseases.
An early detection of precursor lesions of cervical cancer will help to eliminate the worldwide burden of cervical cancer.
This exploratory study aimed to identify, by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), serum protein profiles that distinguish cervical intraepithelial neoplasia grades CIN 1 or lower (≤CIN 1) from CIN 2+ among 127 women infected with human papillomavirus (HPV) 16. Of these 127 women, 25 and 23 were diagnosed with CIN 2 or CIN 3, respectively (cases), and 79 were diagnosed with ≤CIN 1 (non-cases). Serum protein profiles were generated by MALDI-TOF-MS. A total of 95 m/z peaks were tested for association with case status by two racial groups, African American (AAs) and Caucasian American (CAs).
Overall, 2 protein peaks identified by our study demonstrated higher specificity for identifying CIN 2+ than previously published studies. An increasing intensity of [m/z 4459] was associated with a higher risk of being a case, regardless of race with a specificity of 58% for CIN 2 and a specificity of 75% for CIN 3. An increasing intensity of [m/z 4154] was not only associated with a higher risk of being a case only among CAs, but also had an opposite effect among AAs.
Identification of specific proteins associated with the peaks detected in serum and development of antibody-based tests such as ELISA should lead to the development of race-specific, non-invasive and cost effective screening tests with higher specificity for identifying HPV 16 associated CIN 2+.
serum mass spectrometry; cervical intraepithelial neoplasia; race
To evaluate alternative cervical cancer screening methods, digital colposcopy and collection of cervical exfoliated cells for liquid-based cytology (LBC) and hybrid capture 2 (HC2) testing were performed among 2562 women aged 15–59 years in three study sites in the People's Republic of China (rural Shanxi province, Shenyang city in Liaoning province and Shenzhen city in Guangdong province). Visual inspection with acetic acid (VIA) was also evaluated independently from colposcopy. A total of 74 cases of histologically confirmed cervical intraepithelial neoplasia grade 2 or worse (CIN2+) were identified, and 16 CIN2+ cases were imputed among unbiopsied women to correct for verification bias. Corrected sensitivity for CIN2+ was 37% for VIA, 54% for colposcopy, 87% for LBC with a threshold of atypical cells of undetermined significance (LBC⩾ASCUS), 90% for HC2, 84% for LBC using HC2 to triage ASCUS and 96% for positivity to LBC⩾ASCUS or HC2. For VIA, sensitivity was much lower among women ⩾40 years (12%) than those aged ⩽39 years (50%). Specificity varied from 77% for positivity to LBC⩾ASCUS or HC2, up to 94% for LBC using HC2 to triage ASCUS. In conclusion, LBC, HC2 and their combinations performed well, whereas VIA missed a majority of CIN2+, particularly in older women. Digital colposcopy performed better than VIA, but still missed nearly half of CIN2+ in this study.
cervical cancer; screening; human papillomavirus; China
Defining type-specific human papillomavirus (HPV) infections within cervical tissues is important for understanding the pathogenesis of cervical neoplasia and assessing the effectiveness of prophylactic vaccines with limited type-specific spectra. We compared HPV DNA-testing results from 146 matched exfoliated-cell and formalin-fixed-tissue specimens collected by cervicovaginal lavage (CVL) within 90 days of each other from women with histologically confirmed cervical intraepithelial lesions (CIN). The CVL specimens were HPV typed using a MY09/11 L1 consensus primer PCR method followed by dot blot hybridization. The tissue specimens were HPV typed using an SPF10 line probe assay HPV detection system. Of the 146 specimen pairs with evidence of CIN in the tissue, 91.8% were positive for one or more HPV types in both the tissue and cellular specimens. Tissue sections were more likely to be HPV negative (P < 0.01). Typing directly from tissue sections resolved multiple infections detected in exfoliated cells to a single HPV type in only 46.9% of cases. Combined use of both specimen types to attribute lesions to HPV type 16 (HPV-16) and/or -18 led to 43.1% attributed to HPV-16 and/or -18 by both specimen types and 19.9% attributed to HPV-16 and/or -18 by one, but not both, specimen types. Unambiguous attribution of cervical lesions to a single, specific HPV type remains a difficult proposition. Use of multiple specimen types or the development of highly sensitive and robust in situ hybridization HPV-testing methods to evaluate the certainty of attribution of lesions to HPV types might provide insights in future efforts, including HPV vaccine trials.