Evaluation of type-specific HPV persistence and high-risk HPV viral load quantitation in HPV positive women under 30 with normal cervical cytology

The persistence of high-risk HPV (HR-HPV) infection is necessary for the development of cervical intraepithelial neoplasia. The aim of this study was to evaluate if HR-HPV typing and HPV16, 18, 31, and 33 quantitation are predictive for type-specific infection persistence and/or the development of CIN in women under 30 with normal cervical cytology. Young women (under 30) attending a family planning clinic who were HPV positive with normal cervical cytology were included. HPV genotyping was assessed by MY09/MY11 PCR, sequencing, phylogenetic analysis, and cloning when necessary. HR-HPV viral load was quantified using duplex real-time PCR. Study patients were offered for a second smear and HR-HPV detection and quantitation after 12 months. HR-HPV was identified in 43 (21.9%) of the 199 included women. Of these, 39 patients had a second cervical sample taken within a mean interval of 11.7 months (8.8-18.3 months). The mean HR-HPV 16, 18, 31, and 33 initial viral load was 1.9 × 10(6) copies/million cells. The level of viral load did not reveal any significant association with type-specific HR-HPV persistence or the subsequent development of cervical intraepithelial neoplasia. Only HPV16 infection was significantly more likely to persist (91.7% vs. 33.1%, P=0.001) and to develop CIN (33.3% vs. 3.7%, P=0.025). In women under 30 with normal cytology, HR-HPV viral load is common and is not predictive of HPV persistence or the development of cervical intraepithelial neoplasia. HPV16 positive women are significantly more likely to have persistent infection and to develop cervical intraepithelial neoplasia.     

DNA image cytometry and human papillomavirus (HPV) detection help to select smears at high risk of high-grade cervical lesions

Three samples were submitted from women undergoing routine screening (n=910): two smears (one for routine cytology and one for DNA image cytometry) and a scrape for human papillomavirus (HPV) testing. DNA histograms were classified as suspect in cases of aneuploidy, polyploidy, and/or diploidy with a high proliferation rate. Follow-up was available in 239 cases. The primary end-point was the presence of a high-grade squamous intraepithelial lesion (HGSIL) at biopsy. Seventy women (7.7%) had a high-risk (HR) HPV infection and a suspect DNA profile. In 77 women with cytological abnormalities, 28 HGSILs were detected: four with a prior diagnosis of ASCUS (all HR-HPV infected including three with a suspect DNA profile), three with smears evocative of LGSIL (all with HR-HPV infection and a suspect DNA profile), and 21 with smears evocative of HGSIL (all with HR-HPV infection and 20 with a suspect DNA profile). During the follow-up period, out of 239 women with a cytologically normal smear at first entry, five developed a HGSIL; all were HR-HPV-positive and four had a suspect DNA profile at the first smear. HR-HPV detection alone gives a sensitivity of 100% for the detection of HGSIL, with a specificity of 84.3%, whereas DNA measurement associated with HPV testing significantly enhances the specificity to 95.4%. Thus, the combination of HPV testing and DNA measurement provides a highly sensitive and specific evaluation of the risk of HGSIL on cervical smears.     

Physical status of the E2 human papilloma virus 16 viral gene in cervical preneoplastic and neoplastic lesions.

BACKGROUND: Integration of human papilloma virus (HPV) 16 DNA is considered an important genetic change in cervical lesion progression towards ICC. The viral E2 gene is often disrupted by this process, releasing suppression of viral E6/E7 oncogenes, a key factor for oncogenic progression. OBJECTIVES: To evaluate the physical status of HPV 16 E2 gene in cervical preneoplastic and neoplastic lesions and its relation with lesion severity. STUDY DESIGN: A sensitive PCR approach for the detection of an intact E2 HPV 16 gene in infected epithelial cells from the cervix with low grade squamous intraepithelial lesion (LGSIL), high grade squamous intraepithelial lesion (HGSIL) and invasive cervical carcinoma (ICC) diagnosis was applied. The correlation between gene disruption and lesion stage was examined. RESULTS: Sixty-two LGSIL, 39 HGSIL and 24 ICC samples were analyzed. Fifty-seven LGSIL [92%], 13 HGSIL [33%] and 4 ICC [17%] showed results compatible with an intact E2 gene, while 5 LGSIL [8%], 26 HGSIL [67%] and 20 ICC [83%] samples gave no signal. CONCLUSIONS: HPV 16 E2 gene disruption showed a positive correlation with cervical lesion progression, particularly from LGSIL to HGSIL. Although additional genetic events are very likely to be needed for HGSIL to ICC progression, the E2 gene disruption is a putative early marker to consider in the prognostic analysis of HPV 16 chronically infected women.     

Comparison of AMPLICOR and Hybrid Capture II assays for high risk HPV detection in normal and abnormal liquid-based cytology: use of INNO-LiPA Genotyping assay to screen the discordant results.

The study was aimed to evaluate the feasibility of detecting human papillomavirus (HPV) in women with normal or abnormal cervical smears using the Roche Amplicor MWP HPV Test. We compared by AMPLICOR Test and Hybrid Capture II (HCII) Test, the prevalence of HR-HPV in 470 cervical samples including 55 samples with WNL cytology, 208 ASC-US, 193 LGSIL and 14 HGSIL. Samples with discordant results were retested with INNO-LiPA Genotyping HPV Test v2. The HR-HPV positivity in WNL cytology samples was similar (21.8%) by AMPLICOR and HCII. In ASC-US, the HPV positivity was 42.3% by both tests. In LGSIL, HPV positivity was 66.3% and 66.8% by AMPLICOR and HCII, respectively. In HGSIL, 92.8% of samples were positive by AMPLICOR and 85.7% by HCII. The agreement of both tests was 96.2% with a Kappa value of 0.92. Eighteen cases were discordant: 9 HCII positive/AMPLICOR negative and 9 HCII negative/AMPLICOR positive. The INNO-LiPA test revealed HPV positivity in every case. Interestingly, all HCII+/AMPLICOR- samples were found to harbour HPV53. As for the HCII-/AMPLICOR+ samples, 8 demonstrated a multiple infection with HR 16- and/or 18- and/or 56-phylogenetically related HPV types. Moreover, two of these samples were co-infected with HPV6 and two other with HPV54. By using consensus HR-HPV as our reference HPV positivity, the sensitivity (96.6%) and specificity (100%) of AMPLICOR was similar to that of HCII Test. The AMPLICOR HPV Test is sensitive, specific, feasible and appropriate for routine HPV detection.     

Type-specific human papillomavirus detection in cervical smears in Romania

Although Romania has one of the highest incidence of cervical cancer in Europe (30 new cases/100 000 women), little is known about the distribution of the human papillomaviruses (HPV) genotypes in this population. We seek to determine the distribution of HPV genotypes in women with normal and abnormal cervical cytology. We analyzed 460 cervical cytology specimens from women who self-referred to the gynecologic clinic. HPV was detected and genotyped using the commercially available INNOLiPA (INNOGENETICS NV) kit based on the reverse hybridization principle. HPV DNA was detected in 279 cases (60.7%) with a median age of 32.9 years. In HGSIL (High Grade Squamous Intraepithelial Lesion) cytology, the presence of HPV DNA was confirmed in 82.7% of cases. The most frequent high-risk genotype was HPV16, found in 32.6% of HPV-positive samples. The next common high-risk genotypes were HPV18, HPV31 and HPV51. Our findings on the distribution and frequency of the HPV genotypes in Romanian population confirmed the utility of the current available HPV vaccines, HPV16 and 18 being detected in 28.7% of cases in the investigated area.     

Evaluation of HPV-16 and HPV-18 genotyping for the triage of women with high-risk HPV+ cytology-negative results

The ATHENA (Addressing THE Need for Advanced HPV Diagnostics) HPV study evaluated the clinical usefulness of the cobas HPV Test (Roche Molecular Systems, Pleasanton, CA) for high-risk human papillomavirus (HR-HPV) testing (14 HR types) and individual HPV-16/HPV-18 genotyping in women undergoing routine cervical cytology screening in the United States. For the study, 47,208 women were recruited, including 32,260 women 30 years or older with negative cytology. All women with positive results for HR-HPV (n = 4,219) plus a subset of HR-HPV- women (n = 886) were referred for colposcopy and biopsy. The overall prevalence of HR-HPV was 6.7% and of HPV-16/HPV-18 was 1.5%. Cervical intraepithelial neoplasia grade 2 (CIN 2) or worse was found in 1.2% of women examined. The estimated absolute risk of CIN 2 or worse in HPV-16+ and/or HPV-18+ women was 11.4% (95% confidence interval [CI], 8.4%-14.8%) compared with 6.1% (95% CI, 4.9%-7.2%) in HR-HPV+ and 0.8% (95% CI, 0.3%-1.5%) in HR-HPV- women. These analyses validate the 2006 American Society of Colposcopy and Cervical Pathology guidelines for HPV-16/HPV-18 genotyping, which recommend referral to colposcopy of HPV-16/HPV-18+ women with negative cytology.     

Expression of folate receptors and heterogeneous nuclear ribonucleoprotein E1 in women with human papillomavirus mediated transformation of cervical tissue to cancer

AIMS: Folate receptors (FRs) mediate cellular uptake of folates in many cancer cells and in folate deficiency heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) mediates translational upregulation of FR in cultured cervical cancer cells. hnRNP-E1 can also interfere with human papillomavirus 16 (HPV-16) viral capsid protein synthesis (and thereby HPV proliferation) in vitro. This study aimed to evaluate prospectively the relevance of FR and hnRNP-E1 expression in the normal cervix, cervical dysplasia, and cancer. METHODS: Cervical tissues from 12 women with normal histology and 69 consecutive women with varying grades of cervical dysplasia and cancer were prospectively evaluated for immunohistochemical expression of FR, hnRNP-E1, proliferating cell nuclear antigen (PCNA), and HPV. There were 22 women with low grade squamous intraepithelial lesions (LGSIL), 22 with high grade squamous intraepithelial lesions (HGSIL), and 25 with invasive cervical carcinoma. RESULTS: Among normal subjects, 100% and 92% expressed hnRNP-E1 and FR, respectively. FR expression decreased from 91% in LGSIL to 68% and 64% in women with HGSIL and cancer, respectively. Similarly, hnRNP-E1 expression decreased from 86% in LGSIL to 68% and 40% in HGSIL and cancer, respectively. There was a highly significant positive correlation between the extent of FR and hnRNP-E1 expression, and an inverse correlation between HPV infection and hnRNP-E1 expression during progression of cervical dysplasia to cancer. CONCLUSION: These results are consistent with a hypothesis that reduced hnRNP-E1 expression may be permissive for HPV proliferation and progression to cervical cancer, and support the need for prospective longitudinal studies of hnRNP-E1 expression in HPV-16 infected women.     

Intracellular Human Papillomavirus E6, E7 mRNA Quantification Predicts CIN 2+ in Cervical Biopsies Better Than Papanicolaou Screening for Women Regardless of Age

Context.-Cervical cancer screening in women younger than 30?years relies on cervical cytology because of the poor performance of human papillomavirus (HPV) DNA testing in this age group. Objectives.-To determine the performance of in-cell HPV E6, E7 mRNA quantification (HPV OncoTect) for the detection of high-grade cervical intraepithelial neoplasia in women younger than 30?years. Design.-We analyzed 3133 cytology specimens from a screening population of women aged 19-75?years investigate HPV OncoTect as a triage/secondary screening test for atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) cytology in women younger than 30?years. Test results were compared to histology in 246 cases. Results.-The sensitivity of E6, E7 mRNA was 89% for CIN 2+ and 100% for CIN 3+ lesions in women 30?years and older. In women younger than 30?years, the sensitivity of E6, E7 mRNA for CIN 2+ lesions was 88% for CIN 2+ and 92% for CIN 3+ lesions. Abnormal cytology (≥ASCUS) exhibited a sensitivity of 89% for CIN 2+ and 100% for CIN 3+ in women 30?years and older and 96% sensitivity for CIN 2+ and 93% sensitivity for CIN 3+ in women younger than 30. The specificity of E6, E7 mRNA was >80% for CIN 2+ and CIN 3+ in both groups of women compared to a specificity of abnormal cytology of <10% for CIN 2+ and CIN 3+ in both groups. Conclusions.-HPV OncoTect demonstrates a performance that would be effective for ASCUS/LSIL triage in women including those younger than 30?years.     

High-risk human papillomavirus testing in women with ASC-US cytology: results from the ATHENA HPV study

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.     

Human papillomavirus infection of the cervix uteri in women attending a Health Examination Center of the French social security

Since human papillomavirus (HPV) is the central causal factor in cervical cancer, understanding the epidemiology of this infection constitutes an important step towards development of strategies for prevention. Six hundred and fifty seven cervical samples were tested for HPV using PCR with consensus primers (MY09/MY11), by genotyping (restriction and sequencing analyses) and by cervical cytology, from women who attended a Health Examination Center of the French social security. Women with no cervical smear as well as women with cytological abnormalities within the last 3 years were recruited. HPV DNA was detected in 7.3% of the women (5.3% for high-risk, 2.4% for low-risk, and 0.5% for unknown risk types) including 6 (0.9%) mixed infections. Fifteen different genotypes were detected, of which genotypes 16 (22.2%), 58 (13.0%), 18 (11.1%), 30 (9.2%), and 33 (9.2%) were the most prevalent. In age group 17-25 years, we found the highest frequencies for both any (22.1%) and high-risk (14.7%) HPV, and prevalences gradually decreased with age. 5.2% of low-grade squamous intraepithelial lesion, 0.3% of high-grade squamous intraepithelial lesion, and 1.2% of atypical squamous cells of undetermined significance were found. The frequencies of high risk and all HPV types were significantly higher in squamous intraepithelial lesions than in those with normal and reactive/reparative changes (P < 0.0001). The prevalence of high-risk HPV in the atypical squamous cells of undetermined significance/low-grade squamous intraepithelial lesion group (28.6%) was significantly higher than in the normal and reactive/reparative changes groups (3.4%) (P < 0.0001). HPV detection was associated with younger age, single marital and non-pregnant status (P < 0.0001), premenopausal status (P = 0.0004), and contraception (P = 0.0008). Marital status (OR 4.5; 95% CI = 2.3-9.0) and tobacco consumption (OR 3.0; 95% CI = 1.6-5.7) were predictive independent factors of HPV infection. The French system of Health Examination Centers might be of interest for following women regularly, especially those with a low socioeconomic status.     

Single-cell cytokine analysis allows detection of cervical T-cell responses against human papillomavirus type 16 L1 in women infected with genital HPV

Specific types of human papillomavirus (HPV) are known to play a causal role in the development of cervical cancer, with human papillomavirus type 16 (HPV-16) identified as the predominant type. Despite this, little is known about cervical immune responses to this pathogen. The aim of this study was to assess the feasibility of cervical cytobrush sampling and single-cell cytokine staining to investigate cervical lymphocyte-specific cytokine responses to HPV-16 antigens. Of eighteen women recruited into the study, five were HPV DNA positive at the cervix (current exposure) and a further five had circulating antibodies to HPV-16 (previous exposure). Cervical lymphocytes, isolated from the five HPV DNA-positive women, two HPV DNA-negative controls, and one woman with circulating HPV-16 antibodies were assessed for HPV-specific responses using intracellular staining for interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). We demonstrate that both CD4(+) and CD8(+) cervical T lymphocytes, harvested from noninfected and infected subjects, produce these cytokines in response to nonspecific stimulation. However, antigen-specific (HPV-16 L1) IFN-gamma production by CD4(+) and CD8(+) cervical T lymphocytes is only detectable in women exposed currently or previously to HPV-16. This is the first time that antigen-specific cytokine responses of mucosal lymphocytes, obtained from a site of HPV infection, have been demonstrated. This finding clearly illustrates the use of intracellular cytokine staining for investigation of low precursor frequency single-cell antigen-specific responses in lymphocytes harvested from mucosal sites with HPV infection.     

Role of HPV DNA,HPV mRNA and cytology in the follow‐up of women treated for cervical dysplasia

The aim of this study was to assess the role of cytology, human papilloma virus (HPV) DNA and human papilloma virus messenger RNA (HPV mRNA) assays in detecting cervical intraepithelial neoplasia grade 2+ (CNi 2+) (recurrences/persistence) during the follow‐up of women after treatment of cervical intraepithelial lesion. This cross‐sectional study was performed among 43 women treated for cervical intraepithelial neoplasia (CIN) between January 2014 and January 2017 at the Department of Obstetrics and Gynecology of Spedali Civili's Hospital, Brescia, Italy. Pap smear and cervical samples for HPV tests were collected during the follow‐up visit. Furthermore, colposcopy was always performed in order to find out the persistence/recurrence of the disease. A cervical biopsy was collected when necessary. Cervical samples obtained were tested for HPV DNA using the INNO‐LiPa HPV assay and for HPV mRNA using the APTIMA assay. The mean age of enrolled women was 42.5 years. Among the treated patients, more than 50% of women revealed the absence of high risk HPV DNA and HPV mRNA. We found the persistence of the disease cervical intraepithelial neoplasia grade 2 (CIN 2) only in one woman. The sensitivity of cytology, HPV DNA and HPV mRNA in detecting disease was satisfactory (100%), while the specificity was quite different for the three tests: 64.2, 52.4 and 78.9%, respectively. The HPV mRNA test has higher specificity with respect to cytology and HPV DNA, avoiding the referral to unnecessary colposcopy with an improvement of costs/benefits for healthcare system. However, given the small size sample, this study should be considered as a pilot for future larger studies.     

Frequencies and role of regulatory T cells in patients with (pre)malignant cervical neoplasia

Oncogenic human papillomavirus (HPV)-infection is crucial for developing cervical cancer and its precursor lesions [cervical intraepithelial neoplasia (CIN)]. Regulatory T cells (T(regs)) might be involved in the failure of the immune system to control the development of HPV-induced cancer. We investigated frequencies, phenotype and activity of T(regs) in patients with cervical neoplasia. CIN and cervical cancer patients showed increased CD4(+)/CD25(high) T cell frequencies in peripheral blood and CD4(+) T cell fraction. These CD4(+)/CD25(high) T cells represent T(regs) as demonstrated by their low proliferation rate, low interferon (IFN)-gamma/interleukin (IL)-10 ratio, high expression of CD45RO, GITR, CTLA-4, forkhead box P3 (FoxP3) and low CD45RA expression. Moreover, in HPV16(+) cervical cancer patients, in-vitro depletion of CD25(+) T cells resulted in increased IFN-gamma T cell responses against HPV16 E6- and E7 peptides. Thus, increased frequencies of T(regs) in cervical cancer patients may indeed suppress HPV-specific immunity. Longitudinal analysis of CD4(+)/CD25(high) T cell frequencies in patients showed a modest decline 1 year after curative surgery or chemoradiation. This study demonstrates increased frequencies and suppressive activity of T(regs) in cervical cancer. These results imply that T(regs) may suppress the immune control of cervical neoplasia and furthermore that suppression of immunity by T(regs) will be another hurdle to overcome in therapeutic immunization strategies against cervical neoplasia.     

Viral load and genomic integration of HPV 16 in cervical samples from HIV-1-infected and uninfected women in Burkina Faso

The relationships between human papillomavirus type 16 (HPV 16) viral load, HPV 16 integration status, human immunodeficiency virus type 1 (HIV-1) status, and cervical cytology were studied among women enrolled in a cohort of female sex workers in Burkina Faso. The study focused on 24 HPV 16-infected women. The HPV 16 viral load in cervical samples was determined by real-time PCR. Integration ratio was estimated as the ratio between E2 and E6 genes DNA copy numbers. Integrated HPV16 viral load was defined as the product of HPV 16 viral load by the integration ratio. High HPV 16 viral load and high integration ratio were more frequent among women with squamous intraepithelial lesions compared with women with normal cytology (33% vs. 11%, and 33% vs. 0%, respectively), and among women with high-grade squamous intraepithelial lesions compared with women without high-grade squamous intraepithelial lesions (50% vs. 17%, and 50% vs. 11%, respectively). High HPV 16 DNA load, but not high integration ratio, was also more frequent among HIV-1-positive women (39% vs. 9%; and 23% vs. 18%, respectively). The absence of statistical significance of these differences might be explained by the small study sample size. High-integrated HPV 16 DNA load was significantly associated with the presence of high-grade squamous intraepithelial lesions (50% vs. 5%, P = 0.03) in univariate and multivariate analysis (adjusted odds-ratio: 19.05; 95% confidence interval (CI), 1.11-328.3, P = 0.03), but not with HIV-1 or other high-risk HPV types (HR-HPV). Integrated HPV 16 DNA load may be considered as a useful marker of high-grade cervical lesions in HPV 16-infected women.     

p16(INK4a) as a complementary marker of high-grade intraepithelial lesions of the uterine cervix. I: Experience with squamous lesions in 189 consecutive cervical biopsies

AIM: To test the usefulness of p16(INK4a) immunostaining for improving the diagnostic accuracy of cervical punch biopsies referred to a routine laboratory setting during the investigation of women with abnormal Papanicolaou smears. METHODS: A total of 188 consecutive and unselected colposcopically directed cervical biopsies and a single contemporaneous cervical polyp were accessioned prospectively over a 3-month period, step-serially sectioned and examined by H&E and immunostained for p16(INK4a). The clinical context, results of concurrent Papanicolaou smears/ThinPrep slides and Digene hybrid capture tests for high-risk human papillomavirus (HPV) subtypes, as well as follow-up cervical smears/ThinPrep, biopsies and loop excisions of transformation zones or cone biopsies were all correlated with the morphological and immunohistochemical findings. RESULTS: Seventy-seven biopsies (40.7%) displayed a high-grade squamous intraepithelial lesion (HGSIL; cervical intraepithelial neoplasia [CIN] 2-3), 27 (14.3%) showed a low grade squamous intraepithelial lesion (HPV +/- CIN1) and 85 (45%) showed a range of non-dysplastic (inflammatory or reactive) changes. Diffuse strong parabasal immunostaining for p16(INK4a), suggestive of integrated high-risk HPV DNA into the host genome, was observed in 81 biopsies (42.9%, including the cervical polyp) and correlated (>90%) with HGSIL in the H&E sections. Only one case revealed irreconcilable discordance between the histological features and this strong parabasal immunostaining pattern. Focal and weaker midzonal or superficial p16(INK4a) immunostaining, suggestive of episomal HPV infection, was noted in 19 biopsies (10%) and these biopsies exhibited a range of histological changes but predominantly low grade squamous intraepithelial lesion (LGSIL). No staining of the squamous epithelium was seen in 89 biopsies (47.1%). Again, only one case revealed irreconcilable discordance between the histological features and this negative immunostaining pattern. On review of all cases where discordant results were noted between the H&E appearances and expected p16(INK4a) immunostaining, we found 26 cases (13.7%) in which this discordance prompted justifiable modification of the original diagnosis. CONCLUSIONS: Thus, within a routine diagnostic laboratory, p16(INK4a) immunostaining appears to be a very useful adjunctive test in the examination of colposcopically directed cervical biopsies, in the diagnostic cascade of women investigated for abnormal Papanicolaou smears. It is possible, as further data accumulate concerning the importance of integration of high-risk HPV DNA into the host cell genome and the reliability with which this can be identified by p16(INK4a) immunostaining, that this will become the diagnostic 'lesion of interest', replacing the subjective histological grading of cervical dysplasia, in the management of such patients; i.e., the discriminatory watershed between continued surveillance and active intervention.     

Detection and typing of human papillomavirus in biopsy and cytological specimens by polymerase chain reaction and restriction enzyme analysis: A method suitable for semiautomation

Screening for high-risk human papillomavirus (HPV) types allows the detection of women at a high risk of cervical squamous carcinomas, thereby defining a subset of patients targeted for more intensive screening and follow-up. Thirty-four cervical biopsy specimens and isolated cells from cervical smears of normal women or women diagnosed with high-grade intraepithelial lesion (HGSIL) were screened for the presence of HPV by in situ hybridization (ISH) and/or by polymerase chain reaction (PCR). The exact HPV type was determined using a novel restriction typing method. The detection of HPV was facilitated greatly by the use of a PCR-enzyme-linked immunosorbent assay (ELISA)-based method. HPV was detected by PCR in 32% of the biopsy specimens, whereas only 23% had a positive staining by ISH. In one case, a double infection was detected by ISH as well as by PCR. In two cases, the presence of HPV was detected by both methods but the exact type was different. Analyzing cells isolated from cervical smears by the PCR-ELISA technique or by PCR followed by agarose gel electrophoresis, HPV was detected only in patients with HGSIL and not in the control group. The PCR system is more sensitive than conventional ISH, and the PCR-ELISA system presented in this study is efficient in screening large series of cytological samples. Furthermore, this system allows exact HPV typing on the microtiter plate. These innovations may allow the application of HPV detection and typing as a routine screening method to identify patients with a high risk of developing cervical neoplasia. © 1996 Wiley-Liss, Inc.     

Viral load and physical status of human papillomavirus (HPV) 18 in cervical samples from female sex workers infected with HPV 18 in Burkina Faso

Viral DNA load and physical status might be predictive of either high‐grade cervical lesions or disease progression among women infected by human papillomavirus (HPV) 16, but these virological markers have rarely been studied in HPV 18 infections. The relationships between HPV 18 DNA load, viral genome physical status and cervical squamous intraepithelial lesions were analyzed among female sex workers infected with HPV18 in Burkina Faso. HPV 18 E2 and E6 genes were quantitated by real‐time PCR. Among 21 women infected with HPV 18, 67% of whom were HIV‐1‐seropositive, 11 (52.4%) had a normal cytology, 8 (38.1%) had low‐grade squamous intraepithelial lesions, and 2 (9.5%) had high‐grade squamous intraepithelial lesions. Total viral load and integrated viral load were higher in women with squamous intraepithelial lesions than in women with normal cytology (P = 0.01 for both parameters). Total viral load and integrated viral load were higher in HIV‐1‐seropositive women than in those who were not infected with HIV (P = 0.01, and P, 0.01, respectively). Total viral load or integrated viral load >1,000 copies/ng of DNA were more frequent in women with squamous intraepithelial lesions than in women with normal cytology (7/10 vs. 1/11; P = 0.007) and in HIV‐1‐seropositive women (8/14 vs. 0/7 in HIV‐uninfected women; P = 0.02). Both HPV 18 DNA and integrated DNA loads might represent markers of cervical lesions. Prospective evaluations are needed to establish the value of these parameters to predict high‐grade lesion or lesion progression. J. Med. Virol. 81:1786–1791, 2009. © 2009 Wiley‐Liss, Inc.     

Chromosome fragility in lymphocytes of women with cervical uterine lesions produced by human papillomavirus.

We studied 30 women with cervical lesions that showed human papillomavirus infection (HPV). Cervical HPV infection was diagnosed by cytology, histology, immunohistochemistry, and electron microscopy, as well as by DNA viral hybridization in situ with 6, 11, 16, and 18 HPV types. Three groups of patients were studied: 15 women infected by HPV of 6 and 11 types with koilocytic lesions and benign evolution, 15 women infected by HPV of 16 and 18 types with koilocytic lesions and malignant evolution, and 15 normal women without cervical lesions who served as controls. For each group, chromosome fragility was studied in peripheral blood lymphocytes. Aphidicolin (AP) was used as a clastogenic agent at a concentration of 0.12 microM. There were significant differences (p less than 0.001) between the control population and the patients affected by HPV. There were also significant differences (p less than 0.001) between the two groups infected with HPV. Our findings support the concept that chromosome fragility could serve as a cytogenetic marker to measure evolution, prognosis, and treatment of cervical lesion associated with HPV.     

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.     

Molecular detection of human papillomavirus in women with minor-grade cervical cytology abnormalities.

BACKGROUND: Human papillomavirus (HPV) has been shown to be the major risk factor for the development of cervical carcinoma, the second most common cancer among women worldwide. Cervical cytology has been the main screening tool for detection of premalignant lesions in last 50 years. OBJECTIVE: The utility of a molecular assay for detection of HPV in cervical smears was evaluated. STUDY DESIGN: A total of 466 women with minor-grade cervical cytology abnormality supposed to be produced by HPV were included. Patients were classified into three groups: Patients with reactive changes, patients with cervical intraepithelial neoplasia grade 1 (CIN 1), and patients with cervical intraepithelial neoplasia grade 2 (CIN 2). In all patients, another cervical swab was obtained and tested for the HPV genome using the Digene Hybrid Capture II. This assay is able to distinguish between high-risk and low-risk HPV types. RESULTS: Based on cytology results, 44 patients showed reactive changes, 250 patients displayed CIN 1, and 172 patients displayed CIN 2. With the molecular assay, HPV was detected in 289/466 (62%) patients. The high-risk HPV type was present in 263 (56.4%) patients and the low-risk type in 26 (5,5%) patients. In 25% of patients with reactive changes, the HPV genome was detected. Corresponding rates for patients with CIN 1 and CIN 2 were 55 and 81%, respectively. CONCLUSION: Molecular detection of HPV should additionally be used to cytology in patients whose cervical smears display reactive changes, CIN 1, or CIN 2. The employed assay allows identification of patients who are at risk for development of high-grade cervical lesions.