Stefania Cesari

Cervical infections by multiple human papillomavirus (HPV) genotypes: Prevalence and impact on the risk of precancerous epithelial lesions

A large proportion of human papillomavirus (HPV) infections is sustained by multiple genotypes. The effect of multiple infections on the risk of cervical intraepithelial neoplasia (CIN) and the potential efficacy of vaccine on these infections are controversial. We performed viral typing by SFP10‐LIPA on a consecutive series of 1,323 women undergoing colposcopy, 69% of whom had cervical biopsy, and correlated CIN severity with the type and number of HPVs. Overall prevalence of HPV‐DNA was 68.9%, 97.3% in CIN1, and 98.1% in CIN≥2. HPV positivity correlated with younger age (35.9 vs. 37.3 years, P = 0.026) and history of CIN (P < 0.001). Multiple types were detected in 44.2% of cases, including 63.1% CIN1 and 80.8% CIN≥2. Twenty‐three different types were detected, HPV‐16, 31 and 52 being the most frequent. Infections by HPV‐6, 11, 16, or 18 occurred in 59.4% of CIN1 and 71.3% of CIN≥2. Number of viral types and class of oncogenic risk were linearly correlated with CIN severity (P < 0.0001) by univariate and multivariate analyses controlling for age and history of CIN. The effect of the number of HPV types was maintained after exclusion from the model of infections by HPV‐6, 11, 16, and 18. Frequency, distribution, and clinical correlates of multiple HPV infections highlight the importance of assessing individual types in the management and the prediction of outcome of women with abnormal baseline cytology and point to potential limitations in current vaccine strategies. J. Med. Virol. 81:703–712, 2009

Clustering patterns of human papillomavirus genotypes in multiple infections.

Many human papillomavirus (HPV) infections are sustained by multiple viral genotypes whose effect on the risk of cervical intraepithelial neoplasia (CIN) is unknown. The study investigated whether specific HPV types or species may affect the likelihood of multiple infections and have a clustered distribution in a consecutive series of 681 women with a histological diagnosis of CIN. HPV typing was performed by the SPF(10)-LIPA assay; associations were evaluated by loglinear analysis of multiple contingency tables after stratification by age and CIN grade. HPV prevalence was 99.4% with a 72.1% rate of coinfection. The risk of coinfection was higher for types 6, 11, 16, 18, 31, 33, 51, 52, 56. Significant interactions were found for species A7-A9-A10, A6-A9 and A7-A10. Coinfection by types 31-35-56, 16-51-52, 16-18 and 51-52 was more frequent than expected. Interactions between viral species and HPV 16-18 were maintained among CIN1, whereas interactions of 16-51-52 and 31-51-56 were significant only in CIN> or =2. Interactions between species and types were lost among women younger than 32 years. Significant clustering of HPV types and species occurs among women with CIN. This has implications for the assessment of the oncogenic potential and the prevention of HPV infections.

Validation of the SPF10 LiPA human papillomavirus typing assay using formalin-fixed paraffin-embedded cervical biopsy samples

ABSTRACT Lower levels of performance of human papillomavirus (HPV) typing assays in studies using formalin-fixed paraffin-embedded (FFPE) tissue compared to those using exfoliated cervical cells have been reported. The interpretation of current studies is limited by bias in inclusion criteria, sample matching, and methods of cell collection. We aimed to validate FFPE tissue for typing by the use of the SPF10 LiPA assay, comparing cervical scrapings to punch and cone biopsy specimens. We examined 165 paired cervical scraping and FFPE punch biopsy samples, and 66 paired FFPE punch and cone biopsy samples. HPV typing was performed using the SPF10 LiPA assay. Kappa statistics were used to measure interrater agreement. The overall agreement with respect to HPV status was 100%. For 74.5% of subjects (kappa = 0.6147), the same numbers of HPV types were detected in scraping and biopsy specimens. The overall positive typing agreement was 95.4% (range, 93.4 to 97.3) for 441 out of 484 individual HPV type analyses. Agreement was good for HPV-39, -42, -43, and -70 (kappa = 0.6506 to 0.7166), excellent for HPV-6, -16, -18, -31, -33, -35, -40, -51, -52, -56, -58, and -66 (kappa = 0.8499 to 0.9665), and absolute for HPV-11, -44, -45, -53, and -68. In 43.9% of cases (kappa = 0.247), the same numbers of HPV types were found in punch and cone biopsy specimens. Overall positive agreement for typing was 86.8% (range, 82.5 to 91.1) for 204 out of 266 individual HPV type analyses. More infections by HPV-18, -33, -51, and -52 were detected in cone specimens. HPV typing by SPF10 LiPA performed equally well for cervical scraping specimens and standard pathological material. Some viral types are preferentially detected in cone specimens, likely reflecting better sampling of diseased epithelium and endocervix tissue.

Time trends of human papillomavirus type distribution in Italian women with cervical intraepithelial neoplasia (CIN)

OBJECTIVE It is assumed that the circulation of HPV types in a population is stable over time although there are limited historical data to support this view. The existence of possible cohort effects in the circulation of HPV types has major implications for vaccination strategies and risk assessment in HPV-infected women. We analysed archival biopsy samples of cervical intraepithelial neoplasia (CIN) to study the distribution of HPV types in Northern Italy over the years 1985-2007. METHODS DNA from formalin-fixed paraffin-embedded cervical biopsies from the years 1985-87 (67 samples) and 1995-97 (92 samples) was HPV-typed by the SPF-(10) Lipa assay. Cases were compared with 159 control biopsies from the years 2005-07 matched by patient age and CIN grade. Quantitative PCR was used to compare titres of HPV sequences in DNA extracted from biopsies of the three periods. Type-specific PCR was used to confirm HPV51 and 52 typing by SPF-(10) Lipa. RESULTS HPV51, 52, 53, 56, 58, and 66 were markedly under-represented or undetectable in samples from past periods whereas they represented 5.7-30.8% of present infections. Frequency of multiple HPV infections and high-risk infections (p=0.0001) also increased in recent years. The main changes occurred over the last decade. Infections by HPV16, 18, were three times more frequent 20 years ago than today (p=0.012). Loss of amplifiable HPV sequences over prolonged storage was not observed. Type-specific PCR confirmed all HPV51 and 52 infections. CONCLUSIONS Secular trends in the distribution of HPV types among women with CIN may occur in specific populations.

A prenatal case of duplication with terminal deletion of 5p not identified by conventional cytogenetics

A 4-year long prospective study referring to secondtrimester termination of pregnancy due to fetal malformation revealed chromosomal anomalies in about 16% of the 57 cases with congenital malformation diagnosed by ultrasound examination and confirmed by fetal autopsy (Akgun et al., 2007). It is possible that many of the fetuses thought to have a normal karyotype, have instead cryptic chromosomal deletions or duplications that cannot be detected by conventional cytogenetic techniques. In fact, Mefford et al. (2007) found that 6% of autopsy samples from 155 fetuses (128 with normal karyotype, and 27 without any karyotype information) with well-defined developmental pathologies had cryptic imbalances detected through an array-based comparative genomic hybridization (array-CGH) containing bacterial artificial chromosomes (BACs) targeted to rearrangement hotspots. Here we report a 24-year-old woman who underwent chorionic villus sampling, after a diagnosis of cystic hygroma in the first trimester of pregnancy. Although chromosome analysis on cells from both short and long term cultures preparation at a resolution of about 320–400 bands did not reveal any numerical or structural rearrangement (Figure 1), array-CGH analysis on fetal blood after termination of pregnancy revealed a de novo large rearrangement on the short arm of one chromosome 5 with a deletion of at least 25 Mb of the short arm of the chromosome 5 and a contiguous duplication of 20.3 Mb (Figure 1). Thus, the karyotype of the fetus was 46,XY,del(5)(pter-p14.1),dup(5)(p14.1p11). Microsatellite analysis demonstrated the paternal origin of the rearranged chromosome 5 with maternal alleles present only in the deletion region. The area of paternal allelic peaks in the duplication region was double in respect to the maternal alleles (Table 1). The woman was referred for cytogenetic evaluation between the 12th and the 13th week of pregnancy

Evaluation of the HPV typing INNO-LiPA EXTRA assay on formalin-fixed paraffin-embedded cervical biopsy samples

BACKGROUND The HPV genotyping line probe assay INNO-LiPA EXTRA allows the detection of a wider spectrum of viral types compared to the earlier V2 version of the assay. Its performance in formalin-fixed paraffin-embedded tissues is unknown. OBJECTIVES To test the EXTRA assay in HPV genotyping of paired cervical scrapings and corresponding FFPE biopsy specimens. STUDY DESIGN Paired samples from 188 women with abnormal cytology were examined using the INNO-LiPA HPV genotyping assay, version EXTRA. The assay can simultaneously detect 18 high-risk, 7 low-risk, and 2 unclassified HPV types. Kappa statistics were used to measure interrater agreement between groups. RESULTS The evaluation of paired cervical scraping and biopsy samples gave a 100% overall agreement for HPV status and of 72.9% (kappa 0.6554) for the number of infecting HPVs. 392 out of 507 individual HPV types were concordant, corresponding to a positive agreement rate of 87.2% (95% CI 84.1-90.3). As to the individual genotypes, the agreement was absolute for HPV 45, 68, 73 (kappa 1), excellent for HPV 6, 11, 16, 18, 31, 35, 39, 44, 51, 52, 53, 54, 56, 69/71 and 82 (kappa 0.7796-0.9714), good for HPV26, 33, 43, 58, 66 and 74 (kappa 0.6768-0.7449), and poor for HPV 59 and 40. These agreement values were comparable to those obtained with the V2 assay. CONCLUSIONS The EXTRA assay provided excellent performance in HPV typing on FFPE samples comparable to the earlier version of the test despite higher complexity and increased coverage of types.

Multiple Papillomavirus Infection and Size of Colposcopic Lesions Among Women With Cervical Intraepithelial Neoplasia.

Objective The aim of the study was to evaluate the association between the size of cervical lesions as detected by colposcopy and multiple human papillomavirus (HPV) infection in subjects with cervical intraepithelial neoplasia (CIN). Methods A case series of 898 subjects with CIN diagnosed by histopathology and infected by high-risk HPV. Human papillomavirus genotypes were identified using the INNO-LIPA genotyping system. Results The rates of CIN 1, CIN 2, and CIN 3+ lesions were 53.1% (477/898), 14.1% (127/898), and 32.7% (294/898), respectively. Among CIN lesions diagnosed by loop electrosurgical excision procedure or by cold-knife conization, the rates of multiple as compared with single HPV infections increased from 31.7% (59/186) in lesions covering 0% to 25% of the cervix to 39.2% (40/102), 41.9% (13/31), and 48.9% (45/92) in those covering 26% to 50%, 51% to 75%, and more than 75% of the cervix, respectively (&khgr;2 for trend = 7.9; p = .005). In ordered logistic regression, after correction for confounders, odds ratios (ORs) of larger cervical lesions were higher in multiple as compared with single infections (OR = 1.82; 95% CI = 1.24–2.66; p = .002). This association was confirmed among subjects infected by HPV 16 (OR = 2.45; 95% CI = 1.14–5.26; p = .02) and in CIN 3+ lesions (OR = 2.43; 95% CI = 1.23–4.80; p = .01). Conclusions Multiple high-risk HPV infection is associated with larger cervical lesions as detected by colposcopy. This association was confirmed among subjects infected by HPV 16 and in CIN 3+ lesions.

Outcome of Persistent Low-grade Cervical Intraepithelial Neoplasia Treated With Loop Electrosurgical Excision Procedure

Objective The aim of the study was to evaluate the outcome of persistent (≥2 years) low-grade cervical intraepithelial neoplasia (CIN 1) treated with loop electrosurgical excision procedure (LEEP). Materials and Methods A study of 252 subjects with persistent biopsy-confirmed CIN 1 diagnosed after low-grade squamous intraepithelial lesions or atypical squamous lesions of undetermined significance on Papanicolaou test and treated with LEEP. Post-LEEP follow-up cytological, colposcopic, and molecular diagnostic examinations were scheduled at 6 months, 1 year, and yearly thereafter. Results The 252 subjects enrolled had a total number of 1,008 visits per colposcopies (median = 3, range = 1–7) during a median post-LEEP follow-up of 25 months (range = 12–121). The cumulative incidence of CIN 2+ at 2 years and at 3 years of follow-up was 2.3% (4/176) and 5.5% (7/128), respectively, or 1.7 cases (95% CI = 1–2.8) per 100 woman-years. Low-grade cervical lesions during post-LEEP follow-up were diagnosed in 70 subjects (27.8%) or 10 cases (95% CI = 7.9–12.6) per 100 woman-years. Overall, persistent and multiple high-risk HPV infections during follow-up were associated with increased rates of CIN persistence or progression. Conclusions Women with persistent CIN 1 after atypical squamous lesions of undetermined significance/low-grade squamous intraepithelial lesion treated with LEEP had a low rate of progression to CIN 2+ but remained at a high risk of low-grade cervical abnormalities during follow-up. This information should be taken into account when deciding on the treatment strategy and counseling women with persistent CIN 1.

Diagnostic accuracy of colposcopy in relation to human papillomavirus genotypes and multiple infection

OBJECTIVE The aim of this study is to evaluate the diagnostic accuracy of colposcopy for cervical intraepithelial neoplasia grade 3 or worse (CIN3+) in relation to the detection of human papillomavirus (HPV) type 16 and multiple HPV infection. METHODS A cohort study of 2526 subjects attending a colposcopic service because of cytological abnormalities. HPV genotypes were identified using the INNO-LIPA genotyping system. RESULTS The final colposcopic/pathological diagnoses were as follows: 1282 (50.8%) negative, 709 (28.1%) CIN1, 169 (6.7%) CIN2, 318 (12.6%) CIN3 and 48 (1.9%) invasive cervical cancer, respectively. Among women with ASCUS/LSIL, assuming any colposcopic abnormality as a cut-off, there were no significant differences in the sensitivities (83.8%, 95% CI=76-89.6 as compared to 84.1%, 95% CI=73.2-91.1, p=0.9) and ROC curves (0.61, 95% CI=0.58-0.65 as compared to 0.59, 95% CI=0.54-0.64, p=0.5) in the detection of CIN3+ lesions between subjects with single and multiple high-risk infection, and between subjects infected by HPV16 (83.1%, 95% CI=73.7-89.7, ROC=0.59, 95% CI=0.54-063) or other high-risk HPVs (84.7%, 95% CI=75.6-90.8, ROC=0.62, 95% CI=0.58-0.66, p=0.8 and p=0.6 compared to HPV16). After correction for confounders, the odds ratios of CIN3+ associated with any abnormal colposcopic findings were 2.47 (95%CI=1.44-4.23, p=0.001) among HPV16 positive, 3.34 (95% CI=2.16-5.42, p<0.001) among other high-risk HPVs and 1.3 (95% CI=0.72-2.48, p=0.36) among subjects with negative/low-risk HPVs. CONCLUSION In routine clinical practice, multiple infection or HPV16 positivity did not affect colposcopic accuracy in the diagnosis of CIN3+ lesions. The sensitivity of colposcopy was poor among subjects who were uninfected or infected by low-risk HPV genotypes.