Southern blot and dot blot hybridisation compared to PCR for the detection of human papillomavirus DNA in biopsies of the uterine cervix from women with dysplasia

The aim of this study was to compare the sensitivity of Southern blot (SB) and dot blot (DB) hybridisation with polymerase chain reaction (PCR) for the detection of HPV in cervical biopsies from samples with differing histology. One hundred and forty seven women with cervical dysplasia had biopsies performed; one sample was analyzed for HPV DNA from types 6/11, 16, and 18 by SB, DB and PCR (L1 consensus primer and type specific probes) while an adjacent sample was examined histologically. The histology of the samples was normal in 40 (27%), squamous metaplasia in 25 (17%), inflammation 2 (1%) HPV infection 24 (17%), cervical intraepithelial neoplasia (CIN) grade I in 11 (7%), CIN II in 18 (12%), CIN III in 22 (15%), while 5 (3%) had invasive cancer. The number of biopsies positive for HPV DNA from types 6/11, 16, and 18, using the different hybridisation methods was 56 (38%) by dot blot, 57 (39%) by Southern blot hybridisation and 66 (45%) by PCR. When the L1 consensus primer was used 100 (68%) specimens were positive by PCR. The sensitivity of SB and DB hybridisation, as compared with PCR (type specific probes 6/11, 16, 18) was greater in biopsies with abnormal histology (histological grades of HPV infection and greater, as a group) (sensitivity of SB 83%, DB 74%) than those with normal and metaplastic change (as a group) (sensitivity of SB 44%, DB 35%) (P < 0.005 for SB and DB) (inflammation excluded from analysis). This study demonstrated that the sensitivity of SB and DB hybridisation, relative to PCR is greater in samples with abnormal histology than in samples with normal histology.     

Prevalence of human papillomavirus among Croatian women attending regular gynecological visit

Human papillomavirus (HPV) infection has been identified as major risk factor for cervical intraepithelial neoplasia (CIN) and invasive cervical cancer. About 40 HPV viral types are commonly found in the genital tract. Most HPV infections resolve spontaneously, while persistent infection with oncogenic types, namely HPV 16 and 18 is necessary for CIN to occur and progress to cancer. Cervical screening is presently based on the Pap smear that is designed to diagnose precancerous lesions and cervical cancer The aim of this study was to investigate the prevalence of HPV DNA and to determine HPV types distribution among 361 women attending regular gynecological visit. There were 205 women (29+/-8 years old) without determined abnormal cervical lesions and 156 women (34+/-15 years old) with abnormal Pap smear; low grade squamous intraepitehelial lesions (LSIL, n=69), high grade squamous intraepithelial lesions (HSIL, n=72) and atypical squamous cells of undetermined significance (ASCUS, n=15). HPV DNA detection and genotyping was performed by Hybrid Capture 2 assay and additionally by consensus and type-specific primers directed PCR. The overall prevalence of high-risk HPV (hrHPV) in women with abnormal Pap smears was 67.9% (106/156), of which in ASCUS 33.4% (5/15), LSIL 62.3% (43/69) and HSIL 80.6% (58/72). In HPV positive specimens, HPV 16 was found as predominant type in 60.4% cases, followed by HPV 31 (8.5%), HPV 33 (6.6%) and HPV 18 (3.7%). In the group of women without obvious cervical changes the overall hrHPV prevalence was 35.6% with HPV 16 found in 43.8% cases, followed by HPV 31 (17.8%), HPV33 (9.5%) and HPV18 (6.8%). In both study groups, women with and without cervical lesions, the prevalence of HPVof indeterminate type was 14.2% and 13.7%, respectively. Our results indicate that cervical intraepithelial lesions are largely associated with HPV type 16, followed by HPV types 31, 33, 18 and HPV of indeterminate type. Although there is a significant difference in hrHPV DNA prevalence among two groups, no significant differences between particular hrHPV types distribution were observed.     

Neutralizing antibodies against oncogenic human papillomavirus as a possible determinant of the fate of low-grade cervical intraepithelial neoplasia

To determine whether neutralizing antibodies (NAs) against HPV16 is responsible for a higher regression rate of low-grade cervical intraepithelial neoplasia (CIN1), we investigated an association between the presence of the NAs and the fate of the HPV16-related CIN1. All the women examined in this study had HPV16 positive cervix. The women were allocated into four groups by their cervical pathology, i.e., non-pathological (n:7), CIN1 (n:37), CIN2/3 (n:19), and cervical cancer (n:13). Their sera were tested for the presence of NAs against HPV16 by an in vitro assay using HPV16-pseudovirions. As for the CIN1 cases, clinical regression of the lesions were compared between NA-positive and NA-negative groups. Copy number of HPV16-DNA in smear samples was measured by quantitative PCR. The incidence of the presence of the NAs in the women with a non-pathological cervix (85.7%) was significantly higher than in the CIN1 cases (21.5%), the CIN2/3 cases (15.7%), and the cervical cancer cases (0%) (p<0.0001). The regression of the CIN1 lesion was closely associated with the presence of the N As (p=0.0002). The presence of the NAs was associated with low-level copy number of the viral DNA relative to the NA-negative group (p=0.05). The presence of the NAs against HPV16 was associated with a higher regression rate of HPV-related CIN1 lesions. The NAs seem to have a role in deterring HPV-related cervical lesions from progressing to CIN2/3 by inhibiting the infection with de novo replicated HPV. This study further suggests that HPV vaccine to induce the NAs may be effective in eliminating CIN lesions, especially in the NA-negative cases.     

Genital human papillomavirus testing by in situ hybridization in liquid atypical cytologic materials and follow-up biopsies

OBJECTIVE: To describe cases of HPV testing by DNA in situ hybridization performed on atypical cervicovaginal samples collected by a liquidsed method that were negative for HPV DNA on cytology but revealed cervical intraepithelial neoplasia on follow-up biopsies. STUDY DESIGN: Three hundred ninety-five consecutive SurePath atypical squamous cells of undetermined significance (ASC-US) cytologic samples from asymptomatic, reproductive-age women were tested for human papillomaviruses (HPVs) by the in situ hybridization (ISH) method (Ventana Inform HPV Test, Tucson, Arizona, U.S.A). One hundred (25%) cases underwent follow-up colposcopic biopsy within 3 months of cytology. All the tests (cytology, ISH, histology) were independently evaluated without knowledge of the other tests. RESULTS: One hundred twenty-two (33%) cytologic samples were positive for HPVs. Of a total of 100 (HPV positive and negative) follow-up biopsies, 55 were positive for cervical intraepithelial neoplasia (CIN). Fourteen cases of biopsy-proven CIN tested negative for all HPV types in the prior cytologic samples. Retesting of the 14 CIN tissues by ISH was negative in 10, positive for HPV in 2 and inconclusive in 2. CONCLUSION: There is a small but significant (14%) false negative rate with HPV testing by the Ventana ISH method. Clinically suspicious cases should be followed even if an HPV test is negative.     

Detection of human papillomavirus types by the polymerase chain reaction and the differentiation between high-risk and low-risk cervical lesions

By means of a consensus polymerase chain reaction (PCR) method, the prevalence of HPV types was determined in cervical biopsies from 137 women referred to the gynecological outpatient clinic for colposcopy because of an abnormal cervical smear. The prevalence of HPV was 80.3%. There was a statistically highly significant rise in the prevalence of the oncogenic HPV types (16, 18, 31, 33) with increasing severity of cervical intraepithelial neoplasia (CIN I to III), indicating a role for these HPV types in the pathogenesis of cervical cancer. The prevalence of other HPV types decreased significantly with the severity of the lesion, suggesting that these HPV types play a less significant role in this process. These data indicate that HPV typing with PCR may be a valuable tool for distinguishing between highrisk and low-risk cervical lesions. Furthermore, our results suggest that the detection of HPV types by consensus PCR in the cervix of patients with an abnormal smear but without histologically detectable CIN is a useful tool for predicting which of these patiens will eventually develop CIN. Finally, a relatively low percentage (3%) of HPV double infections is reported in this study.     

Detection of human papillomavirus types by the polymerase chain reaction and the differentiation between high-risk and low-risk cervical lesions.

By means of a consensus polymerase chain reaction (PCR) method, the prevalence of HPV types was determined in cervical biopsies from 137 women referred to the gynecological outpatient clinic for colposcopy because of an abnormal cervical smear. The prevalence of HPV was 80.3%. There was a statistically highly significant rise in the prevalence of the oncogenic HPV types (16, 18, 31, 33) with increasing severity of cervical intraepithelial neoplasia (CIN I to III), indicating a role for these HPV types in the pathogenesis of cervical cancer. The prevalence of other HPV types decreased significantly with the severity of the lesion, suggesting that these HPV types play a less significant role in this process. These data indicate that HPV typing with PCR may be a valuable tool for distinguishing between high-risk and low-risk cervical lesions. Furthermore, our results suggest that the detection of HPV types by consensus PCR in the cervix of patients with an abnormal smear but without histologically detectable CIN is a useful tool for predicting which of these patients will eventually develop CIN. Finally, a relatively low percentage (3%) of HPV double infections is reported in this study.     

Human papillomavirus in invasive cervical cancer and cervical intraepithelial neoplasia 2 and 3 in Venezuela: A cross-sectional study

BackgroundThis study investigated the distribution of human papillomavirus (HPV) types in invasive cervical cancer (ICC), cervical intraepithelial neoplasia 2 (CIN2) and cervical intraepithelial neoplasia 3 (CIN3) in Venezuela.MethodsParaffin-embedded samples from 329 women from 29 medical centers of the 24 states of Venezuela were analyzed to determine the distribution of HPV types for ICC, CIN2, and CIN3, the prevalence of single and multiple infection, and the association of HPV types with severity of lesion, comparing CIN2 versus CIN3+ (CIN3 and ICC). The samples were analyzed with the polymerase chain reaction (PCR) followed by reverse hybridization for the identification of HPV types.ResultsHPV was identified in 95/96 ICC specimens (98.9%), in 142/149 CIN3 (95.3%) and in 78/84 CIN2 samples (92.8%). The most common types for ICC and CIN3 were: HPV16, 18, 31, and 33, and for CIN2 were HPV16, 31, 51, 52, and 18. HPV single infection was found in 82.1% of ICC cases, in 79.4% of CIN2 cases, and in 77.4% of CIN3 cases. HPV16 was identified as a single infection more frequently in women with CIN3+ than in those with CIN2 (68.6% versus 46.7%, P = 0.002), and HPV16 or HPV18 types were more prevalent in CIN3+ than in CIN2 (73.4% versus 50%, P = 0.0006).Conclusionthis is the first study of the distribution of HPV types in ICC, CIN2, and CIN3 conducted throughout the territory of Venezuela. HPV16 and HPV18 were the most frequent HPV types identified in single and multiple infections in both ICC and CIN3 groups, and are associated with severity of lesion. The knowledge of the distribution of HPV types would allow organization of an HPV-DNA-based screening test, and consideration of the implementation of prophylactic vaccination in Venezuela.     

Human Papillomavirus 16 Non-European Variants Are Preferentially Associated with High-Grade Cervical Lesions

HPV16 accounts for 50–70% of cervical cancer cases worldwide. Characterization of HPV16 variants previously indicated that they differ in risks for viral persistence, progression to cervical precancer and malignant cancer. The aim of this study was to examine the association of severity of disease with HPV16 variants identified in specimens (n = 281) obtained from a Cervical Pathology and Colposcopy outpatient clinic in the University Hospital of Espírito Santo State, Southeastern Brazil, from April 2010 to November 2011. All cytologic and histologic diagnoses were determined prior to definitive treatment. The DNA was isolated using QIAamp DNA Mini Kit and HPV was detected by amplification with PGMY09/11 primers and positive samples were genotyped by RFLP analyses and reverse line blot. The genomes of the HPV16 positive samples were sequenced, from which variant lineages were determined. Chi² statistics was performed to test the association of HPV16 variants between case and control groups. The prevalence of HR-HPV types in <CIN1, CIN2 and CIN3+ were 33.7%, 84.4% and 91.6%, respectively. Thirty-eight of 49 (78%) HPV16 positive samples yielded HPV16 sequence information; of which, 32 complete genomes were sequenced and an additional 6 samples were partially sequenced. Phylogenetic analysis and patterns of variations identified 65.8% (n = 25) as HPV16 European (E) and 34.2% (n = 13) as non-European (NE) variants. Classification of disease into CIN3+ vs. <CIN3 indicated that NE types were associated with high-grade disease with an OR = 4.6 (1.07–20.2, p = 0.05). The association of HPV16 NE variants with an increased risk of CIN3+ is consistent with an HPV16 genetically determined enhanced oncogenicity. The prevalence of genetic variants of HPV16 is distributed across different geographical areas and with recent population admixture, only empiric data will provide information on the highest risk HPV16 variants within a given population.     

Cervical smears in assessment of the natural history of human papillomavirus infections in prospectively followed women

The value of cervical (Papanicolaou) smears in monitoring the natural history of cervical human papillomavirus (HPV) infections was assessed in a series of 513 women prospectively followed since 1981. On each clinic visit, the patients were subjected to colposcopy accompanied by cervical smears and/or punch biopsies. The latter were analyzed by light microscopy for concomitant cervical intraepithelial neoplasia (CIN) and by transmission electron microscopy (TEM) for HPV particles as well as for HPV structural proteins. The stromal immunocompetent cell (ICC) infiltrates were phenotypically characterized using monoclonal antibodies for T-cell subsets, NK and K cells and Langerhans cells. HPV DNA typing was accomplished by Southern blot, spot and in situ hybridization using probes for HPV 6, 11, 16, 18 and 31. Lesions showing only changes consistent with HPV infection (HPV-NCIN) were associated with less severe atypia in cervical smears than were lesions with coexistent CIN (HPV-CIN). Normal smears were observed, however, in 24.7% of the cases with HPV-NCIN lesions, in 11.5% of cases with HPV-CIN I lesions but only exceptionally in cases with HPV-CIN II and III lesions (2.2% and 3.3%). The percentages of the different ICC phenotypes did not correlate with the atypia in cervical smears, but there was a shift towards the lower values of the T-helper/T-suppressor (OKT4+/OKT8+) cell ratio in parallel with increasing atypia. The possibility of latent HPV infection was suggested by the detection of viral particles, HPV antigens and HPV DNA in lesions shedding normal cells in the smears. The high-risk HPV types 16 and 18 were associated with the highest frequency of severely atypical cells; in the majority of cases, the low-risk types HPV 6 and 11 presented with less severe atypia. The first cervical smear seems to be of value as a predictor of the natural history of HPV lesions, as indicated by the fact that regression was inversely and progression directly related to initial cellular atypia. The present results confirm the intimate association between HPV infections and CIN. Although the biologic potential of the HPV infections seems to be dependent on multiple factors, routine cervical smears, because of their potential value in monitoring the natural history of this infection, should constitute an important means in the prospective follow-up of these patients.     

Comparison of cytologic and acridine-orange flow-cytometric detection of malignant cells in human body cavity fluids

Flow cytometrically (FCM) derived DNA and RNA profiles were studied in acridine orange (AO)-stained body cavity fluid (BCF) specimens obtained from 78 patients with various solid tissue and hematologic malignancies. The ploidy (DNA index), RNA content (RNA index), proliferative activity (% S + G2M) and DNA and RNA scattergram patterns were tested "double-blind" against the cytologic scoring of specimens as malignant, benign or reactive. It was determined that expression of an "abnormal" RNA index (greater than or equal to 2.8) and an elevated proliferative activity (% S + G2M greater than or equal to 7.4) was dependent on the presence of malignancy; 21 of 22 specimens having those abnormal indices had DNA aneuploidy and were cytologically scored as positive. The AO FCM sensitivity and specificity for detecting malignant cells (when measured against cytology scoring) were 61% and 90%, respectively, using the "abnormal" RNA index and % S + G2M cut-offs together with the cellular DNA aneuploidy marker. By supplementing the cytologic scoring with AO FCM DNA and RNA features, the sensitivity for detecting malignant cells was 94%, as compared to 72% for cytology alone. Two specimens gave false-positive FCM results: a tuberculous effusion with a tetraploid subpopulation and a reactive mesothelial proliferation that was diploid and negative cytologically. Scoring for malignancy based on the visual pattern of the DNA and RNA FCM scattergrams, while showing good correlation for aneuploid specimens, in some cases failed to identify diploid disease. The results demonstrate the usefulness of FCM DNA and RNA analysis for supplementing cytologic examination of BCF specimens for the purpose of detecting malignant cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

宫颈癌病变组织人乳头瘤病毒感染率的免疫细胞化学与DNA杂交的比较观察

近年来人乳头瘤病毒（ＨＰＶ）被认为与宫颈癌的发生有密切关系。该实验报道了用分子杂交技术（在严格条件下）检测了宫颈活检组织：慢性宫颈炎、宫颈上皮内瘤（ＣＩＮ）、宫颈癌和宫颈正常组织中１６型乳头瘤病毒（ＨＰＶ－１６）ＤＮＡ的阳性率,并将其与用免疫细胞化学方法（ＰＡＰ）所得结果进行了比较。结果显示１３％（２／１５）的慢性宫颈炎、１７％（２／１２）的ＣＩＮ、５６％（５１／９１）的宫颈癌含有ＨＰＶ－１６ＤＮＡ,而正常组织为阴性;ＨＰＶ属抗原仅见于３５％（６／１７）的ＣＩＮ,其他受检组织均为阴性。结果提示用ＰＡＰ法检查ＨＰＶ－１６的抗原,所得阳性率不及ＤＮＡ杂交所得阳性率高;但它作为一种简便、快速的方法用于福尔马林固定、石腊切片材料,显示ＨＰＶ属抗原,借以筛选多种不同型的ＨＰＶ对ＣＩＮ或其他组织中ＨＰＶ的增殖性感染有一定的价值。     

Comparison of the clinical performance of an HPV mRNA test and an HPV DNA test in triage of atypical squamous cells of undetermined significance (ASC‐US)

M. Waldstrom and D. Ornskov
Comparison of the clinical performance of an HPV mRNA test and an HPV DNA test in triage of atypical squamous cells of undetermined significance (ASC‐US) Objective: The effect of triaging women with atypical squamous cells of undetermined significance (ASC‐US) with human papillomavirus (HPV) DNA testing has been well documented. New tests detecting HPV E6/E7 mRNA are emerging, claiming to be more specific for detecting high‐grade disease. We evaluated the clinical performance of two HPV tests: the Linear Array HPV genotyping test (LA) detecting HPV DNA from 37 oncogenic and non‐oncogenic HPV types and the Aptima HPV assay detecting E6/E7 mRNA from 14 oncogenic HPV types. Methods: We identified 369 consecutive PreservCyt samples diagnosed with ASC‐US tested for HPV DNA using the LA test. The Aptima HPV test was performed on residual material in the same vial. Follow‐up of 325 women was available. The gold standard used was histologically confirmed cervical intraepithelial neoplasia (CIN) grade 2+ or 3+. Results: LA and Aptima HPV assays were positive in 44.3% and 31.7% of the cases, respectively. The concordance was 81.2%. The two tests had identical sensitivity for detecting CIN3+ [92.6% (95% CI, 75.7–99.1)] but the Aptima HPV assay showed a significantly better specificity of 73.8% (95% CI, 68.5–78.7) versus 60.1% (95% CI, 54.3–65.7) for LA for detecting CIN3+. When using CIN2+ as the gold standard the sensitivity for LA was higher than for the Aptima HPV assay [93.8% (95% CI, 82.8–98.7) versus 87.5% (95% CI, 74.8–95.3)], but the specificity was higher for the Aptima HPV assay: 78.0% (95% CI, 72.6–82.7) versus 64.3% (95% CI, 58.3–69.9). Conclusions: Both tests showed good and equal clinical sensitivities for detecting CIN3+, but the Aptima HPV assay had significantly higher specificity for detecting CIN2+ and CIN3+ in women aged 30 years or older with ASC‐US.     

PCR diagnosis of HPV in cervical biopsies of CIN and invasive neoplasia formerly diagnosed as HPV negative

OBJECTIVE: To evaluate whether the polymerase chain reaction (PCR) enhances the diagnosis of human papillomavirus (HPV) in biopsies of the uterine cervix with cervical intraepithelial neoplasia (CIN) or invasive neoplasia. STUDY DESIGN: Samples of 71 paraffin-embedded cervical tissue blocks from patients seen in the period 1997-1998 were analyzed. Samples were selected according to age (18-60 years old) and an active sexual life and divided in to 3 groups: test (samples with CIN or invasive neoplasia and a negative HPV diagnosis), positive controls (samples with CIN or invasive neoplasia and a positive HPV diagnosis) and negative controls (samples without CIN or invasive neoplasia and a negative HPV diagnosis). Samples were subjected to DNA extraction and PCR for HPV detection. RESULTS: PCR analysis matched the colposcopic and cytopathologic diagnoses in the positive and negative controls. However, 77% of samples in test group were HPV positive. CONCLUSION: CIN, an invasive neoplasm, is associated with the presence of HPV. Colposcopy and cytopathology are efficient but not sufficient to identify HPV. Thus, despite the high cost, PCR can be used as an additional examination, in women with cervical lesions.     

Outcomes of women with atypical squamous cells of undetermined significance and high-risk human papillomavirus DNA

OBJECTIVE: To study the risk of high-grade squamous intraepithelial lesion (HSIL) and cervical intraepithelial neoplasia (CIN) 2 or 3 in women with human papillomavirus (HPV)-positive atypical squamous cells of undetermined significance (ASCUS) cytology over a 2-year period using the 2001 Bethesda System and ThinPrep Paps. STUDY DESIGN: In 2002, 846 patients with ThinPrep cervical cytology having an ASCUS interpretation and positive for high-risk HPV DNA were identified. A cohort of 514 (60.8%) patients with follow-up by repeat cytology, cervical biopsy or both was included in the study. Patient age was 12-81 years, with a median of 25 years. RESULTS: There were 291 women (56.6%) with negative status by cytology, HPV testing or biopsy with a median interval of 8.5 months, and an additional 174 patients (33.9%) had persistent ASCUS, positive HPV DNA or low-grade SIL/CIN 1. Finally, 49 patients (9.5%) had CIN 2 or 3, with a median interval of 8.5 months. CONCLUSION: Our study suggests that HSIL or CIN 2 or 3 will be detected in 1 in 10 women with HPV-positive index ASCUS cervical cytology at initial colposcopy or within a 2-year follow-up period.     

Cervical intraepithelial neoplasia: Telomerase activity and splice pattern of hTERT mRNA

Cervical cancers are characterized by the persistence of human papilloma virus (HPV) genome that is found in tissue samples starting from the early stages of tumor progression. Just like in other tumors, the activation of telomerase was observed in cervical carcinomas, but information about its expression was controversial. The aim of this study is to find possible correlations between the presence of HPV sequences, activity of telomerase and expression of different spliced forms of hTERT RNA in cervical intraepithelial neoplasias (CIN). The results show that HPV DNA is present in 60% of normal tissue adjacent to CIN lesions and up to 84% in CIN samples. Telomerase activity was found in 28% of adjacent normal tissue and in 68% of CIN II–III. hTERT RNA that encodes an active enzyme was present almost in all CIN samples. Variations in levels of telomerase activity are possibly not regulated by the splicing forms of hTERT mRNA with deletions.     

IFN-gamma and IL-12B polymorphisms in women with cervical intraepithellial neoplasia caused by human papillomavirus

The Human Papillomavirus (HPV) is a sexually transmitted organism associated with Cervical Intraepithelial Neoplasia (CIN) and cervical cancer, the second main cause of malignancy in women worldwide. The virus itself, however, is not enough to cause lesions on the cervix. Several studies suggest that some polymorphic sites changes the cytokines levels and influence the cancer development in HPV infected patients. In this study, we evaluated the presence of functional polymorphisms at +874 (T/A) IFNG and +1188 (A/C) IL-12B genes in cervical smears samples from 76 healthy women and 162 women, HPV positive, with CIN lesion--CIN I (45), CIN II (55), CIN III (53) and cervical cancer (9)--in Brazilian population. There was no significant differences in genotype (p = 0.4192) and allele (p = 0.370; OR = 1.20) distributions between CIN patients and control groups on IFNG allelic polymorphism. Moreover, for IL-12B gene, there was a significant difference in genotype (p = 0.015) and allele distribution (p = 0.014; OR = 0.5754) between the groups. When samples were stratified according to grade of cervical lesion, the AA genotype and A allele were less frequent in the group with low-grade cervical lesions than in group with high-grade cervical lesions (p = 0.0036 and p = 0.0010; OR = 0.3819, respectively), suggesting that the C allele (mutant) may protect against the emergence of CIN lesions and its progression.     

Association between HLA DQB1 * 03 and cervical intra-epithelial neoplasia.

BACKGROUND: Cervical intraepithelial neoplasia (CIN) and cervical cancer have been shown to be strongly associated with infection by human papillomavirus (HPV). However, other factors may be contributory in the progression from normal epithelium to CIN and cervical cancer, since not all women with HPV infection develop disease. Recently, it was demonstrated that there is a high risk for cervical cancer and CIN in women with HLA DQB1 * 03 (RR = 7.1, p < 0.0009) (1). Subsequent reports have been conflicting, due to sample size, genetic heterogeneity and differences in the techniques employed for the detection of HLA DQB1 * 03. MATERIALS AND METHODS: DNA from cervical smears of 178 women with CIN and 420 controls with normal cervical cytology was analyzed by polymerase chain reaction (PCR) with type-specific primers for HPV 16, 18, 31, and 33. The DNA from test and control samples were also analyzed by a novel PCR technique, which mutates the first base of codon 40 (DQ alleles) from T to G to create an artificial restriction site for an enzyme Mlu I that distinguish DQB1 * 03 from other alleles and are confirmed by digestion of amplified DNA with Mlu I. Further analysis of individual DQB1 * 03 alleles was performed using PCR and allele-specific primers. RESULTS: One hundred forty-four (34%) out of 420 controls (all HPV 16, 18, 31, or 33 negative and normal cytology), 37/66 (56%) of CIN I and 72/112 (64%) of CIN III were positive for DQB1 * 03 (trend test, p < 0.001, chi 2 = 37.3). A significant association was observed between DQB1 * 03 and CIN (odds ratio 3.03; 95% CI 2.11-3.45). Of women with CIN, 131/178 (73.5%) had HPV (types 16, 18, 31, or 33) infection. There was a significant association between DQB1 * 03 and presence of HPV (odds ratio 3.43; 95% CI 2.25-5.10). Homozygosity for DQB1 * 03 was more strongly associated with CIN than heterozygosity (odds ratios 4.0 and 2.63, respectively); and for the presence of HPV (odds ratio 4.47; 95% CI 2.58-7.77). HLA DQB1 * 0301 was the most strongly associated allele with CIN and HPV (odds ratios 2.53 and 2.63, respectively). CONCLUSIONS: HLA DQB1 * 03 is associated significantly with CIN and may be permissive for HPV infection. Further analysis of class II HLA typing in CIN is necessary to evaluate this association.     

Prevalence of human papillomavirus genotypes in cervical cancer and precursor lesions

There are no data obtained in biopsy material on the prevalence of human papillomavirus (HPV) and HPV genotypes in Croatian women with cervical carcinoma and precursor lesions. Therefore, the prevalence of HPVand HPVgenotypes was investigated in archival material of cervical carcinoma and precursor lesions kept at Department of Pathology, School of Medicine, University of Rijeka. DNA was isolated from formalin fixed, paraffin embedded tissue, histologically classified as cervical intraepithelial neoplasia (CIN) III (n =43), squamous cell carcinoma (SCC) (n =54) and adenocarcinoma (ADC) (n =40). HPV testing was performed bypolimerase chain reaction (PCR) using generic and genotype specific primers. The prevalence of HPV DNA was 93.02%, 92.59%, and 92.5% in CIN III, SCC and ADC, respectively. In CIN III and SCC, HPV-16 was the most common high-risk genotype, identified in 65% and 52%, followed by HPV-18 in 22.5% and 28% of cases, respectively. HPV-18 showed a statistically significant prevalence in ADC (67.6%) as compared with SCC (chi(2)=9.924; p_ 0.01). Study results revealed a high prevalence of HPV-DNA in examined cervical lesions (>90%). HPV-16 predominated in SCC and HPV-18 in ADC. Single infection was more frequently present than multiple infections in all three histological groups.     

Improved rate of high-grade cervical intraepithelial neoplasia detection in human papillomavirus DNA hybrid capture testing

BACKGROUND: Numerous studies have established a link between human papillomavirus (HPV), squamous intraepithelial lesions (SIL) and carcinoma of the cervix. Testing for HPV DNA in addition to cytology in screening programs for cervical cancer has been suggested to increase detection rates. STUDY DESIGN: HPV DNA testing (performed by hybridization antibody capture assay I or II), cytology and biopsy (performed within 1 month of each other) were retrospectively reviewed for a series of 155 women. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of HPV testing vs. cytology were calculated using biopsy as the gold standard. These values were also calculated in a subgroup of 37 individuals older than 35 years. RESULTS: The sensitivity, specificity, PPV and NPV of DNA hybrid capture HPV testing for detecting high-grade cervical intraepithelial neoplasia (CIN) were 86%, 44%, 26% and 93%, respectively. The respective values for cytology detection of high-grade CIN were 17%, 97%, 56% and 82%. CONCLUSION: HPV testing was significantly more sensitive for detecting high-grade CIN than cytology (86% vs. 17%). Our data support immediate colposcopy and biopsy, rather than follow-up Papanicolaou testing, if the test for HPV DNA is positive for an intermediate- to high-risk type.