Applicability of liquid-based cytology to the assessment of DNA content in cervical lesions using static cytometry

OBJECTIVE: To evaluate the nuclear DNA content of cervical lesions in liquid-based cytologic specimens prepared for static cytometry. STUDY DESIGN: The DNA content of cervical lesions was evaluated in cervical samples prepared with the Autocyte PREP liquid-based cytology system (TriPath Imaging Inc., Burlington, North Carolina, U.S.A.). A series of 47 samples stained with the Papanicolaou method (chronic cervicitis, n = 15; cervical intraepithelial neoplasia [CIN] 1, n = 25; CIN 2, n = 5; CIN 3, n = 2) were collected from consecutive women enrolled in an ongoing screening study at Leonor Mendes de Barros Hospital, S?o Paulo, Brazil, in 2002. Each residual sample was processed according to the Feulgen-thionin method (TriPath Imaging). Ploidy evaluation was performed using the CAS 200 image analysis system and Quantitative DNA Measurement software 3.0 (version 8.1) (Becton Dickinson, San Jose, Califoria, U.S.A.). Cellular ploidy was analyzed from atypical nuclei, and the DNA index was obtained using histograms for interpretation. RESULTS: All chronic cervicitis cases were diploid. Of the CIN 1 cases, 44% were diploid, 12% tetraploid, 32% aneuploid and 12% polyploid (diploid plus tetraploid). CIN 2 lesions were diploid in 60% and aneuploid in 40% of cases, whereas all CIN 3 lesions (100%) were aneuploid. CONCLUSION: The liquid-based cytologic samples proved to be suitable and highly useful for DNA analysis by image cytometry, which was capable of discriminating CIN 3 lesions from CIN 1 and 2 but not CIN 1 from 2 lesions. Aneuploidy was closely associated with CIN 3 lesions.     

Distribution of High-Risk Human Papillomavirus Genotypes among HIV-Negative Women with and without Cervical Intraepithelial Neoplasia in South Africa

Objective

Large studies describing the profile of high-risk Human papillomavirus (hrHPV) genotypes among women in sub-Saharan Africa are lacking. Here we describe the prevalence and distribution of hrHPV genotypes among HIV-negative women in South Africa, with and without cervical intraepithelial neoplasia (CIN).

Methods

We report data on 8,050 HIV-negative women, aged 17–65 years, recruited into three sequential studies undertaken in Cape Town, South Africa. Women had no history of previous cervical cancer screening. Cervical samples were tested for hrHPV DNA using the Hybrid Capture 2 (HC2) assay and all positive samples were genotyped using a PCR-based assay (Line Blot). Women underwent colposcopy and biopsy/endocervical curettage to determine CIN status. The prevalence and distribution of specific hrHPV genotypes were examined by age and CIN status.

Results

Overall, 20.7% (95% CI, 19.9–21.6%) of women were hrHPV-positive by HC2, with women with CIN having the highest rates of positivity. Prevalence decreased with increasing age among women without CIN; but, a bimodal age curve was observed among women with CIN. HPV 16 and 35 were the most common hrHPV genotypes in all age and CIN groups. HPV 45 became more frequent among older women with CIN grade 2 or 3 (CIN2,3). Younger women (17–29 years) had more multiple hrHPV genotypes overall and in each cervical disease group than older women (40–65 years).

Conclusion

HPV 16, 35, and 45 were the leading contributors to CIN 2,3. The current HPV vaccines could significantly reduce HPV-related cervical disease; however, next generation vaccines that include HPV 35 and 45 would further reduce cervical disease in this population.     

553 例女性宫颈高危型HPV 感染与宫颈病变的相关性研究

目的:研究女性生殖道高危型人乳头瘤病毒(high risk human papilloma virus,hrHPV)感染现状及子宫颈上皮内瘤样病变(cervical intraepithelial neoplasia,CI N)的现患率,并分析该人群年龄与HPV感染及宫颈癌前病变的相关性以及hrHPV病毒载量与宫颈病变之间剂量-效应关系。方法:对553例30岁-78岁有性生活史的女性进行以人群为基础的横断面调查。对所有接受筛查的妇女均行宫颈HPV检测及电子阴道镜下病理活检,并以病理结果作为诊断子宫颈病变的金标准。资料采用EXCEL整理,利用x2检验和非条件Logistic回归分析危险因素和CI N的关系。结果:553例受检对象总体阳性检出率为18.0%。hrHPV感染情况在不同年龄段分布有显著差异,hrHPV病毒载量越高宫颈病变的程度越重。结论:不同年龄段hrHPV感染情况不同,hrHPV病毒载量与宫颈病变之间存在剂量-效应关系。     

Discrimination between precancerous and cancerous lesions of the uterine cervix by DNA measurements on tissue sections

Morphologically typical uterine cervical biopsies were separated into normal cervices, condylomas and cervical intraepithelial neoplasias (CIN) grades I, II and III. At least 100 nuclei per lesion were measured on 4 micron Feulgen-stained sections using a Zeiss microspectrophotometer, with a variant of the plug method used to compute the nuclear DNA content. DNA distribution histograms were then decomposed into subsets of diploid, tetraploid, octoploid and aneuploid cells. The decomposition, which assumed a log-normal model of polydiploidy distribution, led to the identification of six indices: (1) the percentage of diploid cells, (2) the percentage of tetraploid cells, (3) the percentage of octoploid cells, (4) the percentage of aneuploid cells with DNA contents less than tetraploidy, (5) the percentage of aneuploid cells with DNA contents between tetraploidy and octaploidy and (6) the percentage of aneuploid cells with DNA contents greater than octoploidy. These indices, along with the mean nuclear radius, the 5c exceeding rate and the 2c deviation index, generated a nine-dimensional space. Two methods of discriminant analysis on this space showed discriminating powers of 78.22% and 87.13%, respectively, as compared to the original diagnoses. The most discriminating variable in both analyses appeared to be the percentage of octoploid cells.     

553例女性宫颈高危型HPV感染与宫颈病变的相关性研究

目的：研究女性生殖道高危型人乳头瘤病毒（high risk human papilloma virus,hrHPV）感染现状及子宫颈上皮内瘤样病变（cervical intraepithelial neoplasia,CI N）的现患率,并分析该人群年龄与HPV感染及宫颈癌前病变的相关性以及hrHPV病毒载量与宫颈病变之间剂量-效应关系。方法：对553例30岁-78岁有性生活史的女性进行以人群为基础的横断面调查。对所有接受筛查的妇女均行宫颈HPV检测及电子阴道镜下病理活检,并以病理结果作为诊断子宫颈病变的金标准。资料采用EXCEL整理,利用x2检验和非条件Logistic回归分析危险因素和CI N的关系。结果：553例受检对象总体阳性检出率为18.0%。hrHPV感染情况在不同年龄段分布有显著差异,hrHPV病毒载量越高宫颈病变的程度越重。结论：不同年龄段hrHPV感染情况不同,hrHPV病毒载量与宫颈病变之间存在剂量-效应关系。     

Diagnostic and prognostic significance of image cytometric DNA ploidy measurement in cytological samples of cervical squamous intraepithelial lesions

D. Demirel, N. Akyürek and I. Ramzy
Diagnostic and prognostic significance of image cytometric DNA ploidy measurement in cytological samples of cervical squamous intraepithelial lesions Objective: To study the DNA ploidy pattern of uterine cervical squamous intraepithelial lesions (SILs) and its diagnostic and prognostic significance. Methods: The study included 31 cases of SIL: 11 low‐grade (LSIL) and 20 high‐grade (HSIL). Feulgen–pararosaniline staining was performed on previously Papanicolaou‐stained smears and a DNA image cytometric study was performed. An internal reference was used to calibrate the samples. Results: All 31 cases of SIL, either LSIL or HSIL, were non‐diploid. Of the 11 cases of LSIL, four were tetraploid and seven were aneuploid, whereas, of the 20 cases of HSIL, four were tetraploid and 16 were aneuploid. Stemline aneuploidy was not a significant discriminator between LSIL and HSIL (P = 0.32). Based on single‐cell analysis, HSIL cases had significantly higher DNA content than LSIL cases (P < 0.01). When a mean of 30% or more was used for the 6c‐exceeding event (6cEE) value, the sensitivity and specificity to indicate HSIL were 83% and 64%, respectively, with a positive predictive value (PPV) of 81% and negative predictive value (NPV) of 65%. All HSIL cases were cervical intraepithelial neoplasia grade 2 or worse (CIN2+) on biopsy. In addition, cases which showed recurrence had more DNA content by single‐cell analysis than those with an indolent clinical behaviour: P = 0.04 and P = 0.03 for LSIL and HSIL, respectively. Conclusions: Image cytometric DNA analysis is a useful technique for diagnostic and prognostic purposes in uterine cervical SIL when appropriate ‘c’ values are used in single‐cell analysis. We propose that a >6c DNA content of 30% is useful as a cut‐off level for predicting cases with CIN2+ in DNA image cytometry of cervical smears.     

宫颈异倍体细胞Ki67和P16蛋白的表达

目的通过测定宫颈上皮同一异倍体细胞内Ki67和P16蛋白的表达,了解宫颈病变的严重程度。方法将宫颈细胞经DABI,Ki67和P16蛋白的免疫荧光处理,通过不同的激发光测定着色的细胞数。结果在8例宫颈病变中（2例宫颈炎,1例CIN1,2例CIN2,2例CIN3及1例宫颈癌）,除2例宫颈炎病例外,其余6例均可见〉5c的异倍体细胞。在6例中共发现有55个〉5c异倍体细胞,其中有38个Ki67阳性细胞（69％）和12个P16阳性细胞（21．8％）。10个Ki67和P16共阳性的细胞（18．2％）。结论高级别宫颈鳞状上皮内瘤变中异倍体细胞,可出现Ki67和P16蛋白阳性表达。     

DNA ploidy and cell cycle analysis in ear malignant melanoma by flow and image cytometry.

Sixteen ear malignant melanomas (MM) were studied for ploidy and cell cycle analysis by flow and image cytometry. The results were compared with clinical (age, sex, stage), histologic (depth of invasion, level, type) and prognostic (recurrence, death) parameters. Single nuclear suspensions were obtained from fixed, paraffin-embedded tumor and adjacent normal tissue processed separately according to Hedley's technique. These, a "spiked" specimen of normal tissue and tumor, and a spleen diploid control were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, California, U.S.A.). Feulgen-stained Cytocentrifuge preparations of nuclear suspensions of normal, MM and diploid spleen were analyzed with the CAS 200 Image Analyzer (Cell Analysis Systems, Inc., Elmhurst, Illinois, U.S.A.) against commercial calibration rat hepatocytes defined as diploid. Six (37.5%) MM were diploid, and 10 (62.5%) were aneuploid; 8 (90%) were hypodiploid, for a high frequency. There were no statistically significant correlations between clinical, pathologic, prognostic or cell cycle analysis parameters and ploidy, although poor prognostic features tended to be in aneuploid lesions.     

Heat shock protein 27 and p16 immunohistochemistry in cervical intraepithelial neoplasia and squamous cell carcinoma

Heat shock protein 27 (hsp27) is expressed by squamous cell carcinoma of the uterine cervix. Results from an earlier study by our group indicted that hsp27 may be a diagnostic marker for cervical intraepithelial neoplasia (CIN) and carcinoma. p16 expression is known to be elevated in intraepithelial uterine cervical cancer and grades 2 and 3 lesions (CIN2, CIN3), but has also been reported to be negative in 5-20% of cervical cancer and CIN lesions. The aim of our study was to confirm immunohistochemically the expression of hsp27 and p16 in cervical lesions. Formalin-fixed, paraffin-embedded cervical tissue specimens obtained between 2002 and 2010 were investigated for hsp27 and p16 expression. Positive staining was detected for hsp27 in 63% of normal cervical tissues, 47% of CIN1 lesions, 75% of CIN2 lesions, 92% of CIN3 lesions, and 100% of squamous cell carcinomas (SCC); the corresponding rates for p16 positivity were 29, 47, 67, 92, and 75%, respectively. Positive staining for both hsp27 and p16 was observed in 6% of normal cervical tissues and in 19% of CIN1, 18% of CIN2, 85% of CIN3, and 75% of SCC specimens. Hsp27 or p16 positivity had a sensitivity of 95.6 or 84.7% and a specificity of 37.2 or 70.5%, respectively, for the identification of CIN3 or SCC lesions; when both hsp27 and p16 were assessed, both the sensitivity and specificity were improved. In conclusion, both hsp27 and p16 immunohistochemistry is a useful tool for the diagnosis of CIN3 lesions or cervical SCC.     

Immunohistochemical Expression of RAGE and Its Ligand (S100A9) in Cervical Lesions

Altered expressions of receptor for advanced glycation end-products (RAGE) and its ligand (S100A9) are observed in many cancers and play a key role in inflammation-associated cancer. In our previous study, by two-dimensional gel electrophoresis followed by mass spectrometry, the expression of S100A9 protein was found to increase in squamous cervical cancer compared with adjacent normal cervical tissues. Therefore, in the present study we observed the expressions of S100A9 and RAGE in 30 chronic cervicitis, 50 cervical intraepithelial neoplasia (CIN), and 40 squamous cervical cancer (SCC) using immunohistochemical analysis and analyzed the differential expression and possible role of S100A9 and RAGE in cancer development. Immunohistochemical findings were as follows: the expressions of S100A9 and RAGE were demonstrated in chronic cervicitis, CIN, and SCC. Moreover, their expressions were gradually increasing as the tumor progressed. In SCC, the staining scores of S100A9 and RAGE were significantly higher in well-differentiated tumors compared to moderately and poorly differentiated tumors. The expression of S100A9 in epithelial cells exhibited a positive correlation to RAGE expression in chronic cervicitis, CIN, and SCC. There were no significant difference of S100A9 immunoreactivity in stromal cells among chronic cervicitis, CIN, and SCC. Moreover, there was no correlation between S100A9 immunoreactivity in stromal cells of SCC and clinicopathological parameters. Finally, double immunohistochemistry illustrated that RAGE and S100A9 co-express in SCC. In conclusion, RAGE binds its ligand (S100A9), which plays an important role in the development of SCC. In addition, the expressions of S100A9 and RAGE in SCC tumor cells were closely associated with histological differentiation.     

Cytology and Human Papillomavirus Co-Test Results Preceding Incident High-Grade Cervical Intraepithelial Neoplasia

Objective

High-risk HPV (hrHPV) and cytology co-testing is utilized for primary cervical cancer screening and for enhanced follow-up of women who are hrHPV-positive, cytology negative. However, data are lacking on the utility of this method to detect pre-cancer or cancer in community-based clinical practice. This study describes cytology and hrHPV results preceding high-grade cervical intraepithelial neoplasia, adenocarcinoma in situ, or cervical cancer (i.e., CIN2+) in an integrated health system employing routine co-testing among women aged 30 years and older.

Methods

We conducted a cross-sectional analysis of adult female members of Kaiser Permanente Northern California (KPNC) with incident CIN2+ between July 2008 and June 2009. The primary outcome was the proportions of cytologic diagnoses and hrHPV co-test results preceding a diagnosis of CIN2+. Cervical cytology and hrHPV testing results were abstracted from electronic medical records.

Results

Of 1283 CIN2+ cases among adult women, 880 (68.5%) were among women aged 30 years and older and 145/880 (16.5%, 95% CI 14.1–19.1) had only normal cytology during the 12 months prior to diagnosis. Furthermore, 133/880 (15.1%, 95% 12.9–17.7) were preceded by only normal cytology and persistent hrHPV infection (at least 2 positive hrHPV tests) during the 6–36 months preceding CIN2+ diagnosis.

Conclusions

Incident CIN2+ is frequently preceded by normal cytology and persistent hrHPV infection among women aged 30 years and older; screening strategies that employ HPV testing and cytology may improve the detection of CIN2+ compared with cytology alone.     

Outcome of SurePath™ cervical samples reported as borderline nuclear change by cytological subtype and high‐risk HPV status

N. Gupta, N. Dudding, J. Crossley, S.J. Payyappilly and J.H.F. Smith
Outcome of SurePath? cervical samples reported as borderline nuclear changes by cytological subtype and high‐risk HPV status Background: The average borderline rate in cervical cytology samples for English laboratories was 3.8% with the range being 2.0–6.8% at the time of the present study, which was undertaken in order to determine the association between different subtypes of borderline nuclear change (BNC), high‐grade cervical intraepithelial neoplasia (CIN) and high‐risk human papillomavirus (hrHPV) status. Materials and methods: Of 68 551 SurePath^TM cervical samples reported in one laboratory over a period of 2 years, 2335 (3.4%) were reported as BNC. hrHPV status was known in 1112 cases (47.6%). The outcome was known only for women with hrHPV‐positive BNC, who were recommended for colposcopy under the National Health Service Cervical Screening Programme sentinel site protocol. Women with hrHPV‐negative BNC were returned to 3‐yearly recall. The cases were subdivided into BNC, high‐grade dyskaryosis cannot be excluded (B‐HG; 105 cases); BNC with koilocytosis (B‐K; 421 cases); BNC with other features of HPV (B‐HPV; 160 cases); and BNC, not otherwise specified (B‐NOS; 426 cases) and were correlated with the histological outcome where available. Results: The study population age ranged from 23 to 65 years. Cases that tested positive for hrHPV by Qiagen HCII assay comprised 78.1%, 81.0%, 73.1% and 67.8% of B‐HG, B‐K, B‐HPV and B‐NOS categories, respectively. CIN2 or worse (CIN2+) was found in 64.6%, 10.0%, 19.7% and 20.1% of hrHPV‐positive cases of B‐HG, B‐K, B‐HPV and B‐NOS, respectively, which was significantly higher in the B‐HG category (P < 0.001) and lower in the B‐K category compared with B‐NOS (p < 0.001) and B‐HPV (p = 0.006) respectively. CIN3+ comprised 55.6%, 6.3%, 26.3% and 19.1% of biopsies in the same categories, respectively. Conclusions: Subtyping BNC is useful, especially B‐K and B‐HG, which, respectively, had the lowest and highest rates of detection of both CIN2+ and CIN3+, confirming that koilocytosis is likely to be associated with transient HPV infection. Women with B‐HG should be referred to colposcopy in the absence of HPV triage.     

Correlation of immunochemical detection of HPV L1 capsid protein in pap smears with regression of high-risk HPV positive mild/moderate dysplasia

OBJECTIVE: To immunostain Pap smears of high-risk (hr) HPV DNA-positive early squamous lesions for detecting HPV L1 protein. STUDY DESIGN: Routinely stained archival slides from 84 mild and moderate hrHPV DNA-positive dysplasias were immunostained using a panreactive HPV L1 antibody. Follow-up smears were taken from women with remission for a mean period of 22.8 months (range, 6-46). Conization was done in patients with persistence or progression (3 and 48 patients, respectively) after a mean time of 12 months (range, 9-48). RESULTS: Twenty-nine of 84 smears (34.5%) had positively stained squamous epithelial cell nuclei. In 9 of 29 (31%) women progressive disease occurred (2 cervical intraepithelial neoplasia [CIN] 2 and 7 CIN 3 lesions on conization) 20 (69%) had remission. Of the 55 L1-negative cases, 13 (23.6%) had remission, 42 (76.4%) progressed (3 CIN 2, 38 CIN 3, 1 microinvasive carcinoma). The difference in follow-up between L1 positive and negative cases was statistically significant (chi2 test, p< or =0.001). CONCLUSION: Low and moderate dysplastic squamous lesions without immunochemically detectable HPV L1 protein are significantly more likely to progress than are L1-positive cases. Immunochemical L1 capsid detection in routine Pap smears thus offers prognostic information about early dysplastic lesions.     

Prospective clinical study comparing DNA flow cytometry and HPV typing as predictive tests for persistence and progression of CIN I/II.

A cohort of 70 consecutive women at a university hospital colposcopy clinic with untreated CIN I and CIN II (CIN I/II) confirmed by cytology and histology was followed for 1 year in the setting of a prospective trial. In the lesions, the presence of DNA from HPV types was examined by restriction fragment length polymorphism (RFLP) analysis. Aneuploid cell lines were demonstrated by aneuploid histograms generated by high-resolution DNA flow cytometry. HPV type 16 infection and the existence of aneuploid cell lines proved to be significant risk factors for CIN I/II lesions to persist or progress to CIN III in the 1-year follow-up period in the same cohort of patients. The relative risks and 95% confidence intervals (CI) were 1.81 (1.44-2.76) for aneuploid cell lines and 1.74 (1.10-2.76) for HPV type 16 infection in CIN I/II lesions. As a predictive diagnostic test for CIN I/II lesions to persist or progress, the specificity and positive predictive value (PPV) for aneuploid histograms were 100% (CI, 73.5-100%) and 100% (CI, 86.8-100%), respectively. The low sensitivity of 27.3% (CI, 14.9-42.8%) restricted the clinical application of the test, leaving 32 of 44 women with persisting or progressing CINI/II with diploid histograms. HPV type 16 positivity by FRLP had a PPV of 68.4% (CI, 43.5-87.4%) as a prognostic test. Six of 19 HPV 16 infected women showed complete remission of their CIN lesion. A combination of the two tests did not provide any additional information.     

Comparison of image analysis with flow cytometry for DNA content analysis in pigmented lesions of the skin.

The DNA ploidy of 85 melanocytic skin lesions was determined by flow cytometry (FCM) and interactive image analysis (IA) using nuclear extracts of paraffin-embedded tissue. Of the 85 lesions analyzed, 43 were malignant melanomas in different stages of evolution, 15 were dysplastic nevi, 11 were Spitz nevi, and 16 were other types of nevi. Some of the last had features of congenital nevi. Within the melanoma category, there was 42% aneuploidy by FCM versus 56% by IA. Of those melanomas aneuploid by FCM, all but one were aneuploid by IA. All dysplastic nevi, 10/11 Spitz nevi and 15/16 other nevi were diploid by both methods. One of the 16 nevi from the "other types" category was tetraploid by IA but diploid by FCM. A single Spitz nevus was tetraploid by FCM but diploid by image analysis. While our results suggest that interactive IA is potentially a more sensitive method than FCM for detecting aneuploidy in cutaneous pigmented lesions, it remains to be shown whether this will translate into better prognostic assessment of the biologic behavior of melanocytic neoplasms than provided by flow cytometric ploidy analysis.     

ProExC在宫颈癌及其癌前病变中的表达及意义

目的：探讨ProExC在宫颈癌及其癌前病变中的表达及意义。方法：应用免疫组织化学SP法检测ProExC在不同宫颈病变组织芯片中的表达。结果：ProExC在宫颈炎、C1NⅠ、CINⅡ、CINⅢ及宫颈癌中的阳性表达率分别是25％,51．7％,77．9％,90％,93．8％,且其表达在宫颈癌及CINⅡI级病变与宫颈炎及CINI级病变相比较差异具有统计学意义（P〈0．05）,ProExC在CINⅠ级组织中表达低于CINⅡ级,差异具有统计学意义（P〈0．05）,ProExC在浸润性宫颈癌中的表达与临床病理因素无关（P〉0．05）。结论：ProExC反映了细胞的增殖活性,促进宫颈癌的发生发展,可作为诊断及监测预后的指标。     

Residual Disease and HPV Persistence after Cryotherapy for Cervical Intraepithelial Neoplasia Grade 2/3 in HIV-Positive Women in Kenya

Objective

To assess residual cervical intraepithelial neoplasia (CIN) 2/3 disease and clearance of high-risk (hr) human papillomavirus (HPV) infections at 6 months after cryotherapy among HIV-positive women.

Design

Follow-up study.

Methods

79 HIV-positive women received cryotherapy for CIN2/3 in Nairobi, Kenya, and underwent conventional cytology 6 months later. Biopsies were performed on high grade cytological lesions and hrHPV was assessed before (cervical cells and biopsy) and after cryotherapy (cells).

Results

At 6 months after cryotherapy CIN2/3 had been eliminated in 61 women (77.2%; 95% Confidence Interval, (CI): 66.4–85.9). 18 women (22.8%) had residual CIN2/3, and all these women had hrHPV at baseline. CD4 count and duration of combination antiretroviral therapy (cART) were not associated with residual CIN2/3. CIN3 instead of CIN2 was the only significant risk factor for residual disease (odds ratio, OR vs CIN2 = 4.3; 95% CI: 1.2–15.0) among hrHPV-positive women after adjustment for age and HPV16 infection. Persistence of hrHPV types previously detected in biopsies was found in 77.5% of women and was associated with residual CIN2/3 (OR = 8.1, 95% CI: 0.9–70). The sensitivity, specificity, and negative predictive value of hrHPV test in detecting residual CIN2/3 were 0.94, 0.36, and 0.96 respectively.

Conclusions

Nearly one quarter of HIV-positive women had residual CIN2/3 disease at 6 months after cryotherapy, and the majority had persistent hrHPV. CD4 count and cART use were not associated with residual disease or hrHPV persistence. The value of hrHPV testing in the detection of residual CIN2/3 was hampered by a low specificity.     

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.     

A prospective study of human papillomavirus (HPV) testing to resolve uncertainty in colposcopy

J. Bowring, R. Albrow, A. Fisher, G. Downey, J. Cullimore, J. Patnick, P.G. Walker and H.C. Kitchener
A prospective study of human papillomavirus (HPV) testing to resolve uncertainty in colposcopy Objective: UK colposcopy services are seeing increased workloads, a large proportion of which are follow‐up appointments. The English Cervical Screening Programme HPV Special Interest Group identified five subcategories of colposcopy clinic patients who often require prolonged follow‐up regimes for low‐grade abnormalities. Human papillomavirus (HPV) testing has a high negative predictive value, meaning that HPV‐negative women are at very low risk of underlying disease. Our objectives were to quantify the number of HPV‐negative women in each study subcategory and to evaluate the number who could potentially be discharged from colposcopy on the basis of their results. Methods: Four colposcopy clinics prospectively identified women according to five categories over 12 months. All women underwent cytological testing and high‐risk HPV (hrHPV) testing using the Hybrid Capture 2 test. Management outcomes and decisions based on a knowledge of the HPV status were recorded. Results: Data available on 755 women showed that 422/755 (55.9%) and 260/755 (34.4%) had persistent cervical intraepithelial neoplasia grade 1 (CIN1) (Category 1) or a minor abnormality following treatment (Category 2), respectively. In Categories 1 and 2, 51.7% and 60.2%, respectively, were hrHPV negative. The rates with biopsies of CIN2 or worse (CIN2+) across the two categories were 3/355 (0.8%) and 21/291 (7.0%) for hrHPV‐negative and hrHPV‐positive women, respectively. Conclusion: The incorporation of hrHPV testing within organized cervical screening programmes has been widely accepted. hrHPV testing for the clinical scenarios outlined in this study detects women who are hrHPV negative and therefore at low risk of underlying disease, potentially reducing anxiety and inconvenience for women and costs to colposcopy services.     

DNA quantification is technically feasible and of value in cervical smear samples: possible applications for determination of progression in low grade dyskaryosis

This study evaluates the feasibility of DNA analysis of cervical intraepithelial neoplasia III (CIN III) lesions on cervical smear and formalin-fixed paraffin-embedded tissue (FFPET) blocks with a view to extending this type of analysis to milder grades of dyskaryosis. DNA ploidy was determined by image analysis using a CAS 200 Image Analyser. Seventeen patients with a diagnosis of CIN III were studied. Results show that all smear and tissue samples were non-diploid with nine aneuploid and eight tetraploid lesions. In 6/7 patients whose smears and corresponding biopsies were examined there was complete agreement as to the DNA profile. We conclude that DNA quantification is technically feasible in archival, routinely prepared cervical smears. This technique should now be applied to CINI and CINII cervical smears to determine if it is of value in identifying those lesions that will progress to CIN III. This study is particularly timely with the possibility in the near future of estimation of ploidy by image analysis using instruments such as the Highly Optimized Microscope Environment (HOME) system.