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
Sexually transmitted carcinogenic Human Papillomavirus (HPV) infections are extraordinarily prevalent worldwide. However, most incident HPV infections clear within a few years, whereas a small minority persists to invasive cancer. Recent studies indicate that detection of methylated viral DNA may distinguish women with cervical intraepithelial neoplasia grade 2+ (CIN2+) from those with a carcinogenic HPV type infection that shows no evidence of CIN2+. Several studies have reported a positive association between methylation of CpG sites in the L1 gene and CIN2+, while there are inconclusive results regarding methylation of CpG sites in the Upstream Regulatory Region (URR). In this review, we summarize the current state of knowledge on HPV DNA methylation in cervical carcinogenesis, and discuss the merits of different methods used to measure HPV DNA methylation. To follow the promising leads, we suggest future studies to validate the use of methylated carcinogenic HPV DNA as a predictive and/or diagnostic biomarker for risk of cervical cancer among HPV-positive women.
human papillomavirus; methylation; cervical cancer; biomarker; epigenetics
Infection with human papillomavirus (HPV) is associated with uterine cervical intraepithelial neoplasia (CIN) and invasive cancers (ICC). Approximately 80% of ICC cases are diagnosed in under-developed countries. Vaccine development relies on knowledge of HPV genotypes characteristic of LSIL, HSIL and cancer; however, these genotypes remain poorly characterized in many African countries. To contribute to the characterization of HPV genotypes in Northeastern Tanzania, we recruited 215 women from the Reproductive Health Clinic at Kilimanjaro Christian Medical Centre. Cervical scrapes and biopsies were obtained for cytology and HPV DNA detection.
79 out of 215 (36.7%) enrolled participants tested positive for HPV DNA, with a large proportion being multiple infections (74%). The prevalence of HPV infection increased with lesion grade (14% in controls, 67% in CIN1 cases and 88% in CIN2-3). Among ICC cases, 89% had detectable HPV. Overall, 31 HPV genotypes were detected; the three most common HPV genotypes among ICC were HPV16, 35 and 45. In addition to these genotypes, co-infection with HPV18, 31, 33, 52, 58, 68 and 82 was found in 91% of ICC. Among women with CIN2-3, HPV53, 58 and 84/83 were the most common. HPV35, 45, 53/58/59 were the most common among CIN1 cases.
In women with no evidence of cytological abnormalities, the most prevalent genotypes were HPV58 with HPV16, 35, 52, 66 and 73 occurring equally. Although numerical constraints limit inference, findings that 91% of ICC harbor only a small number of HPV genotypes suggests that prevention efforts including vaccine development or adjuvant screening should focus on these genotypes.
Human papillomavirus (HPV); cervical intraepithelial neoplasia (CIN); invasive cervical cancer (ICC)
To determine the prevalence of HPV and cervical neoplasia among HIV-infected women in southwestern China.
Cervical cytology, HPV detection by Hybrid Capture-2™ assay, and diagnostic colposcopy were followed by cervical biopsy if indicated. Logistic regression analysis was used to analyze associations between HPV co-infection and cervical intraepithelial neoplasia (CIN), and HIV-related clinical and laboratory parameters.
Colposcopic-histopathologically proven CIN2+ lesions were present in 7/83 (8.4%) HIV-infected women. Nearly half (41/83, 43%) were co-infected with carcinogenic HPV genotypes. HPV co-infection was higher in women with colposcopic-histopathologically proven CIN2+ lesions than women with
HIV/AIDS care and treatment programs should integrate effective cervical cancer prevention services to mitigate the risk of invasive cervical cancer among HIV-infected women in China.
China; HIV/AIDS; HPV; cervical cancer; screening; prevention
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.
Purpose of the review
The incidence of human papillomavirus (HPV)-related cancers is increased among people with HIV infection compared with the general population. This review will describe recent findings in HPV-associated cancer incidence since the introduction of antiretroviral therapy (ART), HPV/disease prevalence at sites other than cervix and anus, and recent data on screening and treatment of anal intraepithelial neoplasia (AIN).
Consistent with high prevalence of anogenital HPV infection, cervical intraepithelial neoplasia (CIN) and AIN in HIV-positive men and women, new data show that the incidence of cervical cancer has not declined since the introduction of ART and that the incidence of anal cancer is rising. Several studies also highlight high rates of HPV infection and HPV-associated disease at sites other than the cervix and anus, including the penis and mouth. Treatment methods for AIN have been described and show reasonable efficacy.
New data imply that the problem of HPV-related cancers will not decline among HIV-positive men and women in the ART era, highlighting the need to perform studies to determine if screening and treatment of AIN will prevent development of anal cancer. Recent data show progress in both of these areas.
Human papillomavirus; anal cancer; cervical cancer; anal intraepithelial neoplasia; cervical intraepithelial neoplasia
Epidemiologic studies have reported the under-representation of cervical precancerous lesions caused by human papillomavirus (HPV) types 18 and 45 (HPV18/45) compared to the proportion of cervical cancers attributed to these HPV types. We investigated the timing of diagnosis of histologic cervical intraepithelial neoplasia grade 3 or worse (CIN3+) using data from the ASCUS-LSIL Triage Study (ALTS). Of the 2,725 women who underwent enrollment colposcopy 412 of 472 (87.3%) diagnosed with histologic CIN3+ over the 2-year duration of ALTS could be assigned to a HPV type or group of types and were included in this analysis. Eighty-four percent of HPV16-positive CIN3+ were diagnosed at enrollment, compared with 57% of HPV18/45-positive CIN3+, and 58% of CIN3 positive for other carcinogenic HPV types at enrollment. In contrast, only 8% of HPV16-positive CIN3+ were diagnosed at exit, while 31% were HPV18/45-positive and 22% were positive for other carcinogenic types at study exit (p<0.001).. These results indicate the under-representation of HPV18/45 in pre-cancers while HPV16-associated CIN3+ is diagnosed much earlier. Whether the under-representation of 18/45 may be due to occult pathology needs further investigation.
HPV genotypes; precancerous cervical lesions; timing of diagnosis
Background. Viral load may influence the course of human papillomavirus type 16 (HPV-16) infection.
Methods. This case-control study was nested within the 2-year Atypical Squamous Cells of Undetermined Significance and Low-Grade Squamous Intraepithelial Lesion Triage Study, in which women were followed semiannually for HPV and cervical intraepithelial neoplasia (CIN). Case patients (n = 62) were women diagnosed with CIN3 following HPV-16–positive detection at a follow-up visit. HPV-16–positive controls (n = 152) without CIN2 or CIN3 were matched to cases based on the follow-up visit in which viral load was measured. Real-time polymerase chain reaction was used for HPV-16 DNA quantification.
Results. The risk of CIN3 increased with increasing HPV-16 DNA load at the follow-up visit (odds ratio, 1.63; 95% confidence interval, 1.33–1.99 per 1 log10 unit increase); the association was not affected by whether HPV-16 was present at enrollment. When HPV-16 was present at both enrollment and follow-up, viral load remained high among cases (P = .77) but decreased substantially among controls (P = .004). Among women with HPV-16 found initially during follow-up, viral load in the first HPV-16–positive sample was associated with short-term persistence; load was higher in those with infection, compared with those without infection, 1 visit after the initial positivity (P = .001).
Conclusions. Viral load of newly detected infections and changes in viral load predict persistence and progression of HPV-16 infections.
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.
Human papillomavirus (HPV) vaccines containing types 16 and 18 are likely to be effective in preventing cervical cancer associated with these HPV types. No information currently exists in Botswana concerning the HPV types causing precancerous or cancerous lesions. Our goal was to determine the prevalence of HPV types associated with precancerous cervical intraepithelial neoplasia (CIN) stages 2 and 3 in HIV-infected women in Gaborone, Botswana. HIV-infected women referred to our clinic with high-grade intraepithelial lesion on the Pap smear were enrolled in the study. HPV typing was only performed if the histopathology results showed CIN stage 2 or 3 disease using linear array genotyping (CE-IVD, Roche Diagnostics).One hundred HIV-infected women were identified with CIN stages 2 or 3 between August 11, 2009 and September 29, 2010. Eighty-two of 100 women enrolled had coinfection by multiple HPV subtypes (range, 2 to 12). Of the remaining 18 women, 14 were infected with a single high-risk subtype and 4 had no HPV detected. Overall, 92 (92%) women were infected with at least 1 high-risk HPV subtype, and 56 were coinfected with more than 1 high-risk HPV type (range, 2 to 5). Fifty-one (51%) women had HPV subtypes 16, 18, or both. HPV 16 and 18 are the most common types in HIV-infected women with CIN 2 or 3 in Gaborone, Botswana, suggesting that the implementation of HPV vaccination programs could have a significant impact on the reduction of cervical cancer incidence. However, given the relative lack of knowledge on the natural history of cervical cancer in HIV-infected women and the significant prevalence of infection and coinfection with other high-risk HPV types in our sample, the true impact and cost-effectiveness of such vaccination programs need to be evaluated.
HIV; HPV; CIN; Cervical cancer
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.
Infection load and the integration of human papillomaviruses (HPV) have been implicated as determinants for oncogenesis, but whether variation among different HPV types exists remains unclear. We investigated 91 women infected with HPV type 52 (HPV-52), a type that is rare worldwide but common in East Asia. The median viral load increased with the severity of the lesion (248 copies/cell equivalent for normal/cervical intraepithelial neoplasia [CIN] grade 1, 402 copies/cell equivalent for CIN 2, 523 copies/cell equivalent for CIN 3, and 1,435 copies/cell equivalent for invasive cancer). The proportion of specimens with integration increased significantly with the severity of the lesion (P < 0.001). The viral load was associated with the physical status of the viral genome, with higher levels for the pure episomal form (P = 0.001). Infection status should be considered when interpreting viral load data for HPV-52, as single infections with this HPV type were found to have marginally higher viral loads than coinfections (P = 0.051). All except one sample had E2 disruption restricted to only a part of the gene. Integration is a critical step in HPV-52-induced carcinogenesis. The profile of E2 disruption was different from that described for HPV-16, with the amino-terminal region being most frequently involved. Selecting the appropriate E2 region for amplification is critical in studying the integration of HPV-52. In summary, the HPV-52 viral load and the integrated proportion increased with the severity of the cervical lesions but had a different pattern than that of HPV-16.
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
Early detection of precancerous cells in the cervix and their clinical management is the main purpose of cervical cancer prevention and treatment programs. Cytological findings or testing for high risk (HR)-human papillomavirus (HPV) are inadequately sensitive for use in triage of women at high risk for cervical cancer. The current study is an exploratory study to identify candidate surface-enhanced laser desorption/ionization (SELDI) time of flight (TOF) mass spectrometry (MS) protein profiles in plasma that may distinguish cervical intraepithelial neoplasia (CIN 3) from CIN 1 among women infected with HR-HPV. We evaluated the SELDI-TOF-MS plasma protein profiles of HR-HPV positive 32 women with CIN 3 (cases) and 28 women with CIN1 (controls). Case-control status was kept blinded and triplicates of each sample and quality control plasma samples were randomized and after robotic sample preparations were run on WCX2 chips. After alignment of mass/charge (m-z values), an iterative method was used to develop a classifier on a training data set that had 28 cases and 22 controls. The classifier developed was used to classify the subjects in a test data set that has six cases and six controls. The classifier separated the cases from controls in the test set with 100% sensitivity and 100% specificity suggesting the possibility of using plasma SELDI protein profiles to identify women who are likely to have CIN 3 lesions.
cervical; neoplasia; protein profiles; SELDI
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
The control arm of PATRICIA (PApillomaTRIal against Cancer In young Adults, NCT00122681) was used to investigate the risk of progression from cervical HPV infection to cervical intraepithelial neoplasia (CIN) or clearance of infection, and associated determinants.
Methods and Findings
Women aged 15-25 years were enrolled. A 6-month persistent HPV infection (6MPI) was defined as detection of the same HPV type at two consecutive evaluations over 6 months and clearance as ≥2 type-specific HPV negative samples taken at two consecutive intervals of approximately 6 months following a positive sample. The primary endpoint was CIN grade 2 or greater (CIN2+) associated with the same HPV type as a 6MPI. Secondary endpoints were CIN1+/CIN3+ associated with the same HPV type as a 6MPI; CIN1+/CIN2+/CIN3+ associated with an infection of any duration; and clearance of infection. The analyses included 4825 women with 16,785 infections (3363 womenwith 6902 6MPIs). Risk of developing a CIN1+/CIN2+/CIN3+ associated with same HPV type as a 6MPI varied with HPV type and was significantly higher for oncogenic versus non-oncogenic types. Hazard ratios for development of CIN2+ were 10.44 (95% CI: 6.96-15.65), 9.65 (5.97-15.60), 5.68 (3.50-9.21), 5.38 (2.87-10.06) and 3.87 (2.38-6.30) for HPV-16, HPV-33, HPV-31, HPV-45 and HPV-18, respectively. HPV-16 or HPV-33 6MPIs had ~25-fold higher risk for progression to CIN3+. Previous or concomitant HPV infection or CIN1+ associated with a different HPV type increased risk. Of the different oncogenic HPV types, HPV-16 and HPV-31 infections were least likely to clear.
Cervical infections with oncogenic HPV types increased the risk of CIN2+ and CIN3+. Previous or concomitant infection or CIN1+ also increased the risk. HPV-16 and HPV-33 have by far the highest risk of progression to CIN3+, and HPV-16 and HPV-31 have the lowest chance of clearance.
Little is known about the dynamics of human papillomavirus (HPV) infection and subsequent development of high-grade cervical intraepithelial neoplasia (CIN2/3), particularly in women >30 years of age. This information is needed to assess the impact of HPV vaccines and consider new screening strategies. A cohort of 1728 women 15–85 years old with normal cytology at baseline was followed every 6 months for an average of 9 years. Women with squamous intraepithelial lesions were referred for biopsy and treatment. The Kaplan–Meier method was used to estimate the median duration of infection and Cox regression analysis was undertaken to assess determinants of clearance and risk of CIN2/3 associated with HPV persistence. No difference in the likelihood of clearance was observed by HPV type or woman's age, with the exception of lower clearance for HPV16 infection in women under 30 years of age. Viral load was inversely associated with clearance. In conclusion, viral load is the main determinant of persistence, and persistence of HPV16 infections carry a higher risk of CIN2/3.
human papillomavirus; persistence; risk; cervical intraepithelial neoplasia
There are few large case series describing the HPV genotypes found in women diagnosed with rigorously reviewed cervical intraepithelial neoplasia grade 3 (CIN3), cervical precancer.
The Atypical Squamous Cells of Undetermined Significance (ASCUS) and Low-Grade Squamous Intraepithelial Lesion (LSIL) Triage Study (ALTS) was a clinical trial to evaluate the best management strategies for women with equivocal (ASCUS) or mildly abnormal (LSIL) Pap tests. During enrollment and the two-year follow-up, 608 women had a histopathologic diagnosis of CIN3 and PCR-based HPV genotyping results. The genotyping results were ranked hierarchically according to cancer risk: HPV16 > other carcinogenic HPV > non-carcinogenic HPV > PCR negative.
Among the 608 women diagnosed with CIN3, 601 (98.8%) cases were positive for any HPV genotype and 95.4% for any carcinogenic HPV. HPV16 (59.9%), HPV31 (18.1%), HPV52 (14.8%), HPV51 (14.0%), and HPV18 (13.2%) were the 5 most common HPV genotypes detected. Younger age, consensus histologic confirmation, smoking, and multi-parity increased the likelihood of testing HPV 16 positive. Specifically, HPV16-positive CIN3 occurred at a younger age than CIN3 positive for other carcinogenic HPV genotypes (median of 23.5 years versus 25 years, respectively, p = 0.0003, Kruskal-Wallis).
HPV16-positive CIN3 was more commonly diagnosed in younger women (vs. older women), with consensus diagnosis (vs. some disagreement between reviewers), and in smokers (vs. non-smokers), and less commonly diagnosed in multi-parous women.
In populations vaccinated against HPV16 (and HPV18), the median age of CIN3 in women with ASC-US and LSIL cytology should shift to older ages, possibly permitting later age at first screening.
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.
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.
Histological and immunocytochemical features of cervical intraepithelial neoplasia (CIN) associated with HPV 6 and HPV 16, either singly or in combination, were studied in 48 cases. Features of HPV infection (koilocytosis, binucleation, multinucleation, giant irregular nuclei and individual cell dyskeratosis) were present in high prevalence in both HPV 6 and HPV 16 associated CIN. Abnormal mitoses seemed to be a good indicator of CIN and were present in about 50% of cases of CIN associated with either HPV 6 or HPV 16 infection. This finding provides no support for the view held by some investigators that associated HPV 16 infection can be predicted by the presence of abnormal mitoses. Expression of HPV antigen was shown in about 40% of cases with a slight, but not significantly, higher prevalence in cases of combined HPV 6 and HPV 16 infection. Conventional histology and immunocytochemistry could not distinguish CIN associated with HPV 6 from CIN associated with HPV 16 infection.
Women with high-grade cervical intraepithelial neoplasia (HGCIN) frequently present with multiple cervical lesions, and multiple concomitant Human papillomavirus (HPV) genotype infections. To elucidate HPV genotype attribution in different regions on the cervix, we performed molecular mapping of cervical disease in women with HGCIN. Thirteen subjects referred to colposcopy for abnormal cervical cancer screening results were included. A cervical smear and biopsies from four different areas on the cervix were collected. HPV genotyping using Linear Array (for cytology) or SPF-10-LiPA25 (for histology) were performed in 13 smears, 52 whole sections from biopsies, and 138 tissue regions isolated with laser capture microdissection (LCM). Twelve subjects had a diagnosis of CIN3 and one subject had a diagnosis of CIN2 based on the worst histology found in four biopsies. Eight of the 13 smears (62%) showed multiple genotype infections. Four of 13 women (31%) had multiple HPV infections in their biopsies. After performing LCM-PCR, only one woman (8%) had two different carcinogenic HPV types in morphologically distinct, but colliding HGCIN lesions. HPV16 was identified as the causal type in all women with HPV16 in cytology. A large proportion of other HPV types found in cervical smears were not detected at the tissue level. Using tissue-based genotyping and LCM-PCR analysis, we were able to attribute an individual HPV type to each area of CIN lesions. We demonstrate that HPV16 is even more etiologically dominant than previously thought, based on various genotype attribution models.
CIN; HPV; colposcopy; LCM; genotyping
Host genetic factors might affect the risk of progression from infection with carcinogenic human papillomavirus (HPV), the etiologic agent for cervical cancer, to persistent HPV infection, and hence to cervical precancer and cancer.
We assessed 18,310 tag single nucleotide polymorphisms (SNPs) from 1113 genes in 416 cervical intraepithelial neoplasia 3 (CIN3)/cancer cases, 356 women with persistent carcinogenic HPV infection (median persistence of 25 months) and 425 randomly selected women (non-cases and non-HPV persistent) from the 10,049 women from the Guanacaste, Costa Rica HPV natural history cohort. For gene and SNP associations, we computed age-adjusted odds ratio and p-trend. Three comparisons were made: 1) association with CIN3/cancer (compared CIN3/cancer cases to random controls), 2) association with persistence (compared HPV persistence to random controls), and 3) progression (compared CIN3/cancers with HPV-persistent group). Regions statistically significantly associated with CIN3/cancer included genes for peroxiredoxin 3 PRDX3, and ribosomal protein S19 RPS19. The single most significant SNPs from each gene associated with CIN3/cancer were PRDX3 rs7082598 (Ptrend<0.0001), and RPS19 rs2305809 (Ptrend=0.0007), respectively. Both SNPs were also associated with progression.
These data suggest involvement of two genes, RSP19 and PRDX3, or other SNPs in linkage disequilibrium, with cervical cancer risk. Further investigation showed that they may be involved in both the persistence and progression transition stages. Our results require replication but, if true, suggest a role for ribosomal dysfunction, mitochondrial processes, and/or oxidative stress, or other unknown function of these genes in cervical carcinogenesis.