Cytophotometric analysis of cervical intraepithelial neoplasia grade III, with and without synchronous invasive squamous cell carcinoma

Cytophotometric analysis was performed in nuclei retrieved from paraffin-embedded cervical tissue from 57 cases of CIN III. CIN III lesions of patients without invasive squamous cell carcinoma (N = 37) were regarded to represent a mixture of progressive and nonprogressive lesions. The CIN III lesions of patients with a synchronous invasive squamous cell carcinoma (N = 20) were regarded as representing truly progressive precursor lesions (CIN.INV). Twenty-one photometric features describing geometrical, density, and texture characteristics were extracted from the digitized nuclear images. Statistical analysis of cytophotometric data indicated significant differences between the group of CIN III lesions and CIN.INV lesions. A cluster analysis, using one co-occurrency texture feature (S-HOMOG), one density feature (S-DI), and two geometrical features (S-AREA and M-CIRC), showed that two clusters (C1 and C2) were present in the total group of CIN III and CIN.INV lesions. The vast majority of CIN.INV lesions was member of one and the same cluster C1. The CIN III group appeared to consist of a mixture of two clusters, 54% C1 and 46% C2 lesions. Of patients 45 years or younger, the majority (62%) of CIN III lesions had feature values, corresponding with those of cluster C1, and as such possibly with a potentially progressive course. In patients older than 45 years the percentage of CIN III lesions with C1 feature values was 27%.     

DNA ploidy and cytophotometric analysis of cervical intraepithelial neoplasia grade III and invasive squamous cell carcinoma

Cervical intraepithelial neoplasia grade III (CIN III) and squamous cell carcinoma (INV) were examined using DNA ploidy and cytophotometric analysis. Based on hysterectomy, exconisation, and biopsy material from 69 patients in two age categories, analysis was performed in nuclei isolated from selected areas of paraffin-embedded tissue. High percentages of DNA-diploidy in INV lesions were found mainly in the group of patients age 45 years or younger. CIN III lesions in women age 46 or older demonstrated high percentages of DNA-aneuploidy. DNA-polyploidy was most frequent in CIN III lesions in the younger age category. The results of cytophotometric analysis indicated that the overall mean values of 16 nuclear photometric features discriminated significantly between the whole groups of CIN III (n = 37) and INV (n = 32). On an individual patient level, however, the mean feature values showed a large overlap. Based on the results of a stepwise linear discriminant analysis of patient mean values, a combination of geometrical and run-length texture features was used to discriminate between CIN III and INV lesions. The correct classification rate was highest in the category of patients in the older age category. The results of this study indicate age related differences in CIN III and invasive squamous cell carcinoma, and they may be of help in assessing cytophotometric features in the study of progressive and non-progressive CIN lesions.     

Clinical and pathological heterogeneity of cervical intraepithelial neoplasia grade 3

Objective

Cervical intraepithelial neoplasia grade 3 (CIN3), the immediate cervical cancer precursor, is a target of cervical cancer prevention. However, less than half of CIN3s will progress to cancer. Routine treatment of all CIN3s and the majority of CIN2s may lead to overtreatment of many lesions that would not progress. To improve our understanding of CIN3 natural history, we performed a detailed characterization of CIN3 heterogeneity in a large referral population in the US.

Methods

We examined 309 CIN3 cases in the SUCCEED, a large population-based study of women with abnormal cervical cancer screening results. Histology information for 12 individual loop electrosurgical excision procedure (LEEP) segments was evaluated for each woman. We performed case-case comparisons of CIN3s to analyze determinants of heterogeneity and screening test performance.

Results

CIN3 cases varied substantially by size (1–10 LEEP segments) and by presentation with concomitant CIN2 and CIN1. All grades of CINs were equally distributed over the cervical surface. In half of the women, CIN3 lesions were found as multiple distinct lesions on the cervix. Women with large and solitary CIN3 lesions were more likely to be older, have longer sexual activity span, and have fewer multiple high risk HPV infections. Screening frequency, but not HPV16 positivity, was an important predictor of CIN3 size. Large CIN3 lesions were also characterized by high-grade clinical test results.

Conclusions

We demonstrate substantial heterogeneity in clinical and pathological presentation of CIN3 in a US population. Time since sexual debut and participation in screening were predictors of CIN3 size. We did not observe a preferential site of CIN3 on the cervical surface that could serve as a target for cervical biopsy. Cervical cancer screening procedures were more likely to detect larger CIN3s, suggesting that CIN3s detected by multiple independent diagnostic tests may represent cases with increased risk of invasion.     

Establishment of keratinocyte colonies from small-sized cervical intraepithelial neoplasia specimens

The size of human cervical intraepithelial neoplasia (CIN) biopsies is usually very small and standard methods do not allow an adequate number of keratinocytes to be isolated for culturing purposes. In this study, a new approach to establish keratinocyte cultures from small CIN a tissue fragments was developed. Neoplastic specimens and corresponding normal tissues, which were used as controls, were digested with collagenase. Tissue‐derived fibroblasts and keratinocytes were co‐cultured in calcium and serum medium. Single keratinocyte colonies from primary cultures were expanded using a culture medium optimized in our laboratory. Primary keratinocyte colonies, as well as expanded colonies, were tested for epithelial and cervical markers such as 5, 14, 17, and 19 keratins, and p63 by immunofluorescence. Our results indicate that a variable number of primary keratinocyte colonies could be detected in neoplastic cultures, depending on the grade of cervical lesions from which the colonies originated. Single colonies, when cultured with our new medium, grew at a high rate with uniform size and morphology for some passages. Epithelial and p63 markers were expressed in keratinocyte colonies, as well as in expanded colonies. In conclusion, our study reports a rapid and easy culturing system which enables keratinocyte colonies from minute cervical tumor tissues to be obtained. Moreover, using the new culture medium, keratinocyte colonies can be expanded at a high proliferative rate. J. Cell. Physiol. 227: 3787–3795, 2012. © 2012 Wiley Periodicals, Inc.     

Long‐term cytological and histological outcomes in women managed with loop excision treatment under local anaesthetic for high‐grade cervical intraepithelial neoplasia

Y. L. Woo, C. Badley, E. Jackson and R. Crawford Long‐term cytological and histological outcomes in women managed with loop excision treatment under local anaesthetic for high‐grade cervical intraepithelial neoplasia Objective: This study examines the impact of excision margin status after large loop excision of the transformation zone (LLETZ) under local anaesthetic for high‐grade cervical intraepithelial neoplasia (HG‐CIN) on the cytological and histological outcomes up to 5 years after treatment. Methods: Prospective cytological and histological data were obtained by examination of the colposcopy database at Addenbrooke’s Hospital, Cambridge, UK. All women aged between 19 and 50 years who underwent treatment for HG‐CIN by LLETZ under local anaesthetic were included in the study. Patients without follow‐up data were excluded from the study. The excision margin status was correlated with the subsequent cytological and histological outcomes. Results: A series of 967 women with CIN2 and CIN3 underwent LLETZ excision under local anaesthetic. Overall, 42% of women had disease present at the excision margin following LLETZ. Women with CIN3 were more likely than those with CIN2 to have an involved excision margin (P < 0.0001). Cytological recurrence was highest at 12 months (16%) and did not correlate with the CIN grade or excision margin status. Histological recurrence/persistence was also highest at 12 months follow‐up (15%) and this correlated with grade of CIN and margin status (P < 0.0001). Conclusions: Histological recurrence/persistence correlates with grade of CIN and excision margin status. Management of HG‐CIN in an outpatient setting under local anaesthetic is safe, cost effective and yields a favourable long‐term outcome.     

Flow cytometric classification of biopsy specimens from cervical intraepithelial neoplasia

The distribution of single-cell DNA content was investigated in biopsy specimens from the human cervix of 121 women suspected of having intraepithelial neoplasia. Comparison of the results of the histopathological examination with the ploidy level showed that all normal specimens were diploid. Thus, no false-positive results occurred. Most of the specimens classified as mild and moderate dysplasia were diploid as well. Aneuploid cell populations occurred in 78% of the lesions classified as severe dysplasia and carcinoma in situ. The ploidy level distribution permitted a natural division of the aneuploid cell populations into two groups with DNA indices either above or below 1.5. The importance of the aneuploidy in carcinogenesis is discussed.     

DNA ploidy analysis of cervical condyloma and intraepithelial neoplasia in specimens obtained by punch biopsy

This study analyzed the feasibility of, and the strategy for, DNA ploidy analysis of cervical condyloma and intraepithelial neoplasia by a computerized digital imaging system. Paraffin-embedded tissue provided satisfactory single-cell preparations for DNA ploidy analysis after enzyme digestion and additional procedures. Negative endocervical curettings and normal squamous mucosa were used as internal diploid controls. With suitable controls, 21 (81%) of the 26 aneuploid lesions were identified as such in the single-cell preparations. The remaining five lesions (not recognized as aneuploid in the single-cell preparations) had ploidy levels between 2.08n and 2.30n and required DNA measurements on 12-microns sections. Criteria for these DNA measurements were defined: specimens intended for DNA ploidy analysis should contain abnormal epithelium of at least 3 mm to 4 mm in greatest dimension and should be accompanied by diploid controls, such as endocervical curettings or normal ectocervical squamous mucosa. With a combination of single-cell preparations and 12-microns tissue sections, it was possible to accurately determine the DNA ploidy patterns of the cervical lesion specimens obtained by punch biopsies. Available evidence suggests that ploidy analysis can provide useful diagnostic and prognostic information.     

Clonality analysis of archival cervical smears. Correlation of monoclonality with grade and clinical behavior of cervical intraepithelial neoplasia

OBJECTIVE: To establish a polymerase chain reaction (PCR)-based clonality assay for archival cervical smears and examine its value in the detection of cervical intraepithelial neoplasia (CIN) and prediction of its clinical behavior. STUDY DESIGN: Dyskaryotic cells were microdissected from archival cervical smears of 33 cases and subjected to PCR-based clonality analysis of the androgen receptor gene. High-risk HPV subtypes were screened by PCR. RESULTS: Monoclonal patterns were found in 9/9 CIN 3 and 15/21 CIN 2, while polyclonal patterns were observed in the remaining 6 CIN 2 and 3/3 CIN 1. All patients with monoclonal CIN lesions, including 15 CIN 2, showed recurrence of the disease despite treatment. The original CIN 2 and recurrent CIN lesion in each of the 6 examined cases showed the same monoclonal pattern, suggesting a clonal link. In contrast, the patients with polyclonal CIN 1 or 2 became negative and remained disease free. High-risk HPV subtypes were found in all monoclonal CIN lesions, including 9 CIN 3 and 15 CIN 2, and in 4/6 polyclonal CIN 2 but not in CIN 1 lesions. CONCLUSION: Clonality analysis of cervical smears is potentially valuable in the identification of true neoplastic cells and prediction of clinical behavior of CIN 2 lesions.     

Plasma proteome analysis of cervical intraepithelial neoplasia and cervical squamous cell carcinoma

Although cervical cancer is preventable with early detection, it remains the second most common malignancy among women. An understanding of how proteins change in their expression during a particular diseased state such as cervical cancer will contribute to an understanding of how the disease develops and progresses. Potentially, it may also lead to the ability to predict the occurrence of the disease. With this in mind, we aimed to identify differentially expressed proteins in the plasma of cervical cancer patients. Plasma from control, cervical intraepithelial neoplasia (CIN) grade 3 and squamous cell carcinoma (SCC) stage IV subjects was resolved by two-dimensional gel electrophoresis and the resulting proteome profiles compared. Differentially expressed protein spots were then identified by mass spectrometry. Eighteen proteins were found to be differentially expressed in the plasma of CIN 3 and SCC stage IV samples when compared with that of controls. Competitive ELISA further validated the expression of cytokeratin 19 and tetranectin. Functional analyses of these differentially expressed proteins will provide further insight into their potential role(s) in cervical cancer-specific monitoring and therapeutics.     

Cervical intraepithelial neoplasia grade III (CIN III) and invasive cervical carcinoma: the yawning gap revisited and the treatment of risk

In a 3-year study of the population of Southampton and south-west Hampshire there were 10 times as many cases of CIN III compared with invasive squamous carcinoma (700 compared with 70). The peak incidence of CIN III per 1000 screened women years was in those aged 25-29 years, which was 20 years earlier than the peak incidence of invasive cervical cancer per 1000 women years at risk. Ninety percent of CIN III was diagnosed in women under 50 years. There were 14 cases of cervical glandular intraepithelial neoplasia grade III (CGIN III), three coexisting with CIN III, all in women aged under 50 years: the gap between intraepithelial and invasive lesions was not seen for glandular neoplasia. Although referral was for at least moderate dyskaryosis in 86.8% of women with CIN III or CGIN III, most had been screened previously, either having had mild abnormalities requiring repeat cytology (39.8%) or negative cytology (34.5%). Only 12 women aged > or = 50 years had previous negative cytology: 21.4% compared with 35.6% of women aged < 50 years (P = 0.034). The results of this study suggest that the best opportunity for preventing invasive squamous cell carcinoma lies in screening women aged 20-39 years when the incidence of CIN III in the screened population is highest and before the peak incidence of invasive disease. The results also indicate the importance of repeated screening and follow up of minor cytological abnormalities in the detection of CIN III. The benefit of screening must be regarded as a treatment of risk, since it is almost certain that a high proportion of CIN III regresses or persists unchanged.     

Quantitative DNA analysis of low grade cervical intraepithelial neoplasia and human papillomavirus infection by static and flow cytometry.

Quantitative deoxyribonucleic acid (DNA) analysis of cervical biopsy specimens from 26 women with cytological, colposcopic, and histological evidence of mild cervical atypia consistent with cervical intraepithelial neoplasia grade I, reactive atypia, or human papillomavirus infection alone or in combination was performed in a comparative evaluation of Feulgen microspectrophotometry, the fast interval processor image analysis system, and flow cytometry. The fast interval processor image analysis system showed a distinct advantage over the other methods, being faster and allowing the operator to see the cells that were selected for measurement. The three methods of measurement together showed that the DNA content of at least 2% of the cells measured exceeded 5C (C being the haploid amount of DNA in a normal cell and 2C representing the diploid complement of a normal cell) in all cases of cervical intraepithelial neoplasia grade I and reactive atypia and in 87% of those reported as showing human papillomavirus infection alone. In contrast, the DNA content of cervical biopsy specimens from the transformation zone of 11 normal controls did not exceed 4C. This study shows the value of using a DNA threshold--that is, the "5C exceeding rate"--to distinguish between normal and neoplastic appearances of the cervix. These results support the view that cervical infection by human papillomavirus is a true precursor of neoplasia.     

The correlation between the grade of dyskaryosis on cervical smear, grade of cervical intraepithelial neoplasia (CIN) on punch biopsy and the final histological diagnosis on cone biopsies of the cervix

This study was carried out to assess how reliably a punch biopsy of the cervix predicts the maximum grade of CIN present and whether a colposcopically directed punch biopsy is more reliable than cytology in predicting the grade of intraepithelial neoplasia present in the cervix. The grade of CIN in 107 cone biopsy specimens was compared with the grade of CIN and dyskaryosis in punch biopsies and smears from the same patients. Exact correlations were identified between the highest grade lesions on cone biopsy and those in 63% of punch biopsies and 49% of cervical smears. fie conclude that punch biopsy provides a more reliable estimate of the highest grade of CIN present in a subsequent cone biopsy than cervical cytology, but nonetheless fails to give a consistent estimate of the final grade of CIN in a significant percentage of cases.     

Conservative management of intraepithelial cervical neoplasia.

In a prospective trial cryotherapy was performed in 164 patients with preinvasive cervical neoplasia, most of whom desired future childbearing. Their disease had been evaluated by repeat cytology, colposcopy and colposcopically directed punch biopsies, with endocervical curettage when necessary. This conservative treatment eradicated the disease in 147 (89.6%) of the patients. The remaining 17 underwent complete reinvestigation. The focal residual disease in 12 was successfully treated by conservative means--repeat cryotherapy, focal electrocautery or punch biopsy. The other five required either cone biopsy or hysterectomy because of more extensive lesions.     

宫颈上皮内瘤变与宫颈微生物群落结构的相关性

目的分析宫颈上皮内瘤变与宫颈微生物群落结构的相关性。方法选取2017年1月至2018年10月于我院就诊的253例女性进行回顾性分析,根据是否患有宫颈上皮内瘤变分为CIN组(86例)及对照组(167例)。采集纳入对象的宫颈菌群并提取细菌基因组DNA,对细菌16S rRNA V3、V4区片段进行PCR扩增,并采用Illuminate Miseq测序平台对PCR产物进行测序,分析两组对象宫颈微生物群落结构,并分析患者宫颈微生物群落结构与宫颈上皮内瘤变的相关性。结果两组对象宫颈微生物群落的Simpson指数及Shannon指数比较差异无统计学意义(t=1.474、1.636,P0.05),而CIN组的Chao指数及ACE指数均高于对照组(t=9.213、10.420,P0.05)。16S rRNA分析显示放线菌是宫颈部位的主要菌群。CIN组女性宫颈微生物群落中放线菌、奇异菌属、放线菌门、阴道奇异菌及奇异变形菌占主要优势(LDA4log10),对照组中杆菌、厚壁菌门等占主要优势。Spearman相关分析示卷曲乳杆菌、惰性乳杆菌、格氏乳杆菌、詹氏乳杆菌与宫颈上皮内瘤变呈负相关,而加特纳菌、阴道奇异菌、普雷沃氏菌、粪球菌与宫颈上皮内瘤变呈正相关(均P0.05)。结论 CIN女性宫颈菌群与健康女性存在明显差异,其中卷曲乳杆菌、惰性乳杆菌、格氏乳杆菌、詹氏乳杆菌与宫颈上皮内瘤变呈负相关,而加特纳菌、阴道奇异菌、普雷沃氏菌、粪球菌与宫颈上皮内瘤变呈正相关。     

Mean nuclear volume in cervical intraepithelial neoplasia and carcinoma

OBJECTIVE: To correlate three-dimensional nuclear size (mean nuclear volume) estimated by the stereologic intercept methodfor objective classification of cervical intraepithelial neoplasia (CIN) and carcinoma. STUDY DESIGN: In this retrospective study a total number of 29 CIN cases (8 cases of CIN 1, 10 cases of CIN 2 and 11 cases of CIN 3) and 10 cervical squamous cell carcinoma cases were selected. Mean nuclear volume (MNV) of all cases was measured with an image cytometer (Leica, Cambridge, England) using Quantimet 600 software (Leica). Nuclear point resection method was adopted to measure nuclear volume. Mean intercepted diameter of at least 50 nuclei was measured randomly. MNV was correlated with the histologic grade and diagnosis. RESULTS: MNV of CIN 1, 2, 3 and carcinoma cases was 291.72, 403.33, 711.45 and 893 microm3, respectively. ANOVA test results showed that MNV of CIN 1 and 2 was significantly lower than that of CIN 3 and invasive carcinoma (P < .000). MNV of CIN 3 was also significantly lower than that of carcinoma cases (P <.05). CONCLUSION: The findings suggest that estimates of MNV on conventional histopathology slides provide objective and useful criteria for relatively subjective histopathologic grading.     

DNA changes in progressive cervical intraepithelial neoplasia.

Cervical intra-epithelial neoplasm (CIN) is treated as a progressive lesion, even though most CIN will not progress to invasive cancer if left untreated. This study focussed on DNA-cytometric analysis of cytologic smears of patients who had developed invasive cancer after initial smears showing CIN. The first part of the study aimed at describing the DNA-cytometric changes in these progressive ('malignant') CIN lesions. In the second part a cluster analysis was performed on 'malignant' CIN III lesions and CIN III lesions, with 'unknown' malignant potential. The results indicated that 'malignant' CIN lesions developed high DNA-index (DI) values during malignant transformation, as demonstrated by increasing mean DI values, a high percentage of DNA-aneuploidy and 2.5c Exceeding Rates. Furthermore, a trend-like pattern of texture feature values occurred in 'malignant' CIN lesions with increasing severity. These findings provide objective quantitative confirmation of the evolution of nuclear changes during malignant transformation. Cluster analysis showed that it was possible, using a set of four cytometric features, to subdivide the 'unknown' CIN III lesions into a cluster of lesions with feature values similar to the vast majority of the 'malignant' CIN III lesions, and a second cluster of lesions with feature values dissimilar to 'malignant' CIN III. It is argued that the profile of 'malignant' CIN has become clearer and that the results of this study may serve as a basis for a more objective cytopathologic subdivision of premalignant CIN. It may be justified to follow up patients whose lesions do not yet fit this 'malignant' profile. Not treating the non-progressive lesion group will avoid putting these patients at risk.