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.     

Chromatin texture in high grade prostatic intraepithelial neoplasia and early invasive carcinoma

OBJECTIVE: To evaluate individual nuclei from high grade prostatic intraepithelial neoplasia (PIN) lesions with early invasive carcinoma foci in the area of microinvasion and in the gland in which the microinvasion originated. STUDY DESIGN: High-resolution, digitized images of nuclei from defined locations were recorded and segmented, and karyometric variables were computed. These included a set of 93 features, which form a nuclear signature characterizing the spatial and statistical distribution of the nuclear chromatin. Nuclei in the glandular epithelium were recorded sequentially, along the basal cell layer, at increasing distances from the point of microinvasion and by random selection in the region of microinvasion. RESULTS: At a distance > 60 nuclear locations from the point of microinvasion, the nuclear signatures corresponded to those seen in high grade PIN. Between 40 and 20 nuclear locations removed from the microinvasion focus the signatures began to change gradually until at a distance of 15-5 locations they strongly resembled the signatures seen in adenocarcinoma. The total optical density decreased to values seen in adenocarcinoma, and the nuclear chromatin had finer granularity. While nuclei in high grade PIN followed a widely dispersed total optical density distribution suggestive of wide-ranging aneuploidy, the nuclei in the region of microinvasion exhibited a less dispersed and bimodal total optical density distribution. CONCLUSION: The chromatin texture signatures showed a clear trend: there was an obvious attenuation as the measured nuclei approached the microinvasion area. The decrease in total optical density at the microinvasion might suggest the emergence of one or two clones that can be responsible for the invasive phenotype.     

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.     

DNA methylation changes detected by methylation-sensitive amplified polymorphism in two contrasting rice genotypes under salt stress

DNA methylation,one of the most important epigenetic phenomena,plays a vital role in tuning gene expression during plant development as well as in response to environmental stimuli.In the present study,a rnethylation-sensitive amplified polymorphism (MSAP) analysis was performed to profile DNA methylation changes in two contrasting rice genotypes under salt stress.Consistent with visibly different phenotypes in response to salt stress,epigenetic markers classified as stable inter-cultivar DNA methylation differences were determined between salttolerant FL478 and salt-sensitive IR29.In addition,most tissue-specific DNA methylation loci were conserved,while many of the growth stage-dependent DNA methylation loci were dynamic between the two genotypes.Strikingly,salt stress induced a decrease in DNA methylation specifically in roots at the seedling stage that was more profound in IR29 than in the FL478.This result may indicate that demethylation of genes is an active epigenetic response to salt stress in roots at the seedling stage,and helps to further elucidate the implications of DNA methylation in crop growth and development.     

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%.     

Pirimiphos-methyl resistance in two stored product mites, Acarus siro and Acarus farris, as detected by impregnated paper bioassay and esterase activity assays

The response to pirimiphos-methyl, in one strain of Acarus farris and two strains of Acarus siro, was assessed using an impregnated filter paper bioassay and by the selection of adults following exposure to pirimiphos-methyl. It was concluded that one of the strains of A. siro was resistant to pirimiphos-methyl and that a major resistance mechanism was involved. The second strain of A. siro gave a response similar to that of a laboratory strain unexposed to organophosphates and was considered to be susceptible. The A. farris strain responded to selection at the ED50 but not at the ED99, and it was concluded that a minor resistance mechanism is present in this strain. Assays of esterase activity were used to attempt to identify the biochemical mechanisms involved in the resistance detected by the bioassays. The A. farris and susceptible A. siro strains showed similar levels of esterase activity but the esterase activity of the resistant A. siro strain was significantly greater. An increase in esterase activity followed selection of both the A. farris strain and the resistant A. siro strain. An acetylcholinesterase assay showed no significant difference between the susceptible and pirimiphos-methyl selected strains of A. siro. The results suggest that esterases are involved in the resistance to pirimiphos-methyl found in A. siro and A. farris but that in A. siro, at least, other mechanisms may also be present.     

Influence of cytology development on frequency of pre-cancerous lesions and cervical cancer in east Croatia, 1978-2001

We analyzed the influence of cytology development to frequency of precancerosa (Cervical intraepithelial neoplasia grade 3 - CIN 3) and cervical cancer. The number of examined Pap smears increased significantly in the analyzed period. The calculated linear trend shows an average four-year increase of 17,283.35 smears. The number of detected CIN 3 increased accordingly. The calculated linear trend shows an average four-year increase by 45.03 and decrease of the rate of 0.07. A slight increase in cervical cancer was also noticed. The calculated linear trend shows an average four-year increase in cancer by 1.6 and decrease in the rate of 0.51. Analyses of cervical cancer by stages showed an increase in number of stage I and decrease of other stages. The calculated linear trend shows an average four-year increase of stage I by 4.94 or decrease in other stages of cervical cancer of 3.29 respectively. The rate of cervical cancer stage I decreased by 0.11 and of other stages by 0.39. Further analyses of the stage I showed that the linear trend of IA stage had an average four-year increase by 5.40 and decrease in stage IB of 0.48. The rate of the cervical cancer stage IA increased by 0.02, whereas the rate of IB decreased by 0.13. Cytology development in our country has resulted in detection of higher number of CIN 3 and the earliest stage (IA) of cervical cancer whereas the number of other stages (IB, II, III, IV) has decreased. However, a total number of cervical cancers haven't changed over the whole period. Thus, it is obvious that opportunistic program of detection, which has been using in Croatia, could not decrease frequency of cervical cancer. In order to achieve it, well-organized national program of detection is needed.     

宫颈病变患者阴道微生态与高危型HPV感染及宫颈癌相关增殖基因表达的相关性

目的探讨宫颈病变患者阴道微生态与高危型HPV感染及宫颈癌相关增殖基因表达的相关性。方法选择2018年1月至2019年1月间在我院确诊为原发性宫颈癌的患者50例作为宫颈癌组,在我院诊断为宫颈糜烂的患者78例作为宫颈糜烂组,同期在我院进行体检的健康女性100例作为正常对照组。对比3组研究对象的阴道微生态失调率、高危型HPV感染率以及宫颈癌组、宫颈糜烂组患者宫颈病灶组织中宫颈癌相关增殖基因(Prdx4、Nek2、Fhit、BLCAP)mRNA表达量的差异。采用Pearson检验分析宫颈癌患者阴道微生态失调率与高危型HPV感染及宫颈癌相关增殖基因表达的相关性。结果宫颈癌组、宫颈糜烂组患者的阴道微生态失调率、高危型HPV感染率高于正常对照组,其中宫颈癌组患者这两项指标水平高于宫颈糜烂组(均P0.05)。宫颈癌组患者宫颈病灶组织中Prdx4、Nek2 mRNA表达量高于宫颈糜烂组,Fhit、BLCAP mRNA表达量低于宫颈糜烂组(均P0.05)。相关性分析发现,宫颈癌患者阴道微生态失调率与高危型HPV感染率呈正相关,与癌基因(Prdx4、Nek2)mRNA表达量呈正相关,与抑癌基因(Fhit、BLCAP)mRNA表达量呈负相关(均P0.05)。结论宫颈癌患者阴道微生态失调率较高,可能与高危型HPV感染及癌细胞增殖旺盛密切相关。     

Cytological aspects of uterine cervical adenocarcinoma, adenosquamous carcinoma and combined adenocarcinoma-squamous carcinoma: appraisal of diagnostic criteria for in situ versus invasive lesions

Cytological aspects of uterine cervical adenocarcinoma, adenosquamous carcinoma and combined adenocarcinoma-squamous carcinoma: appraisal of diagnostic criteria for in situ versus invasive lesions
This paper reports the cytological findings based on air-dried smears in a retrospective series of 143 cases of endocervical adenocarcinoma, combined adenocarcinoma-squamous carcinoma and adenosquamous carcinoma drawn from the files of the BC Cancer Registry. Cervical cytology smears were available before biopsy in 131 patients, but in 18 cases the cytology showed no abnormality. Malignant changes or high-grade atypia of glandular and/or squamous cells (defined as moderate or severe dyskaryosis) were detected in 103 cases. In 46 cases, only a high-grade squamous abnormality was detected. Low-grade glandular and/or squamous lesions were detected in nine cases and one showed atypical endometrial-type glands. The cervical smears of 64 cases were reviewed in detail to determine the important cytomorphological criteria of in situ and invasive adenocarcinoma in air-dried smears, the technique used for preparing PAP smears in British Columbia. Endocervical cells were absent in four cases. Numerous (>10) groups of glandular cells were present in 51 cases. Important clues to the diagnosis of adenocarcinoma included crowding of nuclei, stratification of nuclei, loss of polarity, syncytial balls and papillary groups of glandular cells, nuclear enlargement, nuclear pleomorphism, and the presence of free-lying atypical glandular cells. Nuclear hyperchromatism, chromatin pattern, nuclear borders, nuclear membranes, and numbers and morphology of nucleoli were not helpful criteria in our material. Criteria enabling reliable distinction between in situ and invasive adenocarcinoma and/or mixed adenocarcinoma-squamous carcinoma could not be established.     

Relative accuracy of cervical and anal cytology for detection of high grade lesions by colposcope guided biopsy: a cut-point meta-analytic comparison

Background

We recently reported, using a receiver operating characteristic area metric, the first meta-analytic comparison of the relative accuracy of cervical and anal cytology in detecting moderate or severe histopathologic lesions by magnification directed punch biopsy. The aim of the present research was to meta-analytically examine cut-point specific operating characteristics (sensitivity, specificity) of cervical and anal cytology in detecting high grade squamous intraepithelial lesion (HSIL) histopathology by colposcope directed punch biopsy.

Methodology/Principal Findings

The primary eligibility requirement was availability of tabulated cytology (normal, atypical cells of unclear significance [ASCUS], low grade squamous intraepithelial lesion, HSIL or atypical squamous cells cannot rule out high grade [ASC-H]) and biopsy (Conclusions/SignificanceUsing a cytology cut-point of HSIL or ASC-H, anal cytology is less sensitive but comparably specific to cervical cytology. However, using a cut-point of ASCUS, differences in accuracy were of borderline significance.     

DNA ploidy in breast lesions. A comparative study using two commercial image analysis systems and flow cytometry

DNA ploidy determinations on a series of 24 breast specimens were performed independently utilizing flow cytometry (FCM) and two separate commercially available computerized image analysis systems for image cytometry (ICM). The tissues analyzed were obtained from 20 carcinomas, 2 benign neoplasms and 2 benign reductive procedures. The results showed a close correlation between the DNA indices (DIs) obtained by all methods in 14 of the 24 cases. In four cases, all methods showed aneuploid peaks, but with differing DIs. In six cases (two benign and four malignant) FCM showed diploidy while ICM showed peridiploid cell populations. The results obtained with the two image analysis systems were in agreement for 20 of the 24 cases. ICM is an acceptable alternative to FCM for reproducible ploidy analysis. ICM-based measurements have the advantage of the visual discrimination of abnormal cells and therefore may have a greater sensitivity in identifying small aneuploid populations. Populations with DIs in the range of 1.0 to 1.3 need to be assessed carefully in ICM-based determinations due to the potential that these "aneuploid" peaks may represent shifted diploid populations.     

Cellular damage in spermatozoa from wild-captured Solea senegalensis as detected by two different assays: comet analysis and Annexin V–Fluorescein staining

Improved sperm analysis in Senegalese sole ( Solea senegalensis ) males could provide some clues to reproduction constraints that have been restricting the introduction of this species in the aquaculture market. More accurate methodologies such as sperm membrane and DNA stability surveying might help overcome this problem. In this study spermatozoon cells were analysed in thirteen individual samples using single-cell gel electrophoresis to assess DNA fragmentation, and the Annexin V-FITC assay to assess cell apoptosis. Both techniques revealed significant individual differences. Sperm samples showed 10–50% DNA fragmentation (DNA_t). All the males presented apoptotic spermatozoa ranging from 1.4% to 20%. Annexin V proved to be an appropriate tool in differentiating between subpopulations of necrotic, apoptotic and viable cells in this species. The comet assay revealed that some individuals had a high percentage of cells with high DNA fragmentation. The Annexin V assay also revealed a high percentage of necrotic cells in some individuals. These two factors, despite not being correlated, could explain the low sperm quality of Senegalese sole males in captivity.     

Ergovaline toxicity on Caco-2 cells as assessed by MTT,alamarBlue, and DNA assays

The exact mechanisms of fescue toxicity in animals have yet to be established, but it has been associated with an inability to thrive. Ergovaline is the major ergopeptine alkaloid associated with fungal infections of tall fescue. Gastrointestinal (GI) toxicity of ergovaline (10(-11) to 10(-4) M) was evaluated in Caco-2 cells (mimicking the GI epithelium) beginning on days 1, 8, and 18 of culture. Acute and chronic toxicity was assessed after 24 and 72 h of exposure. Treatment periods were chosen to study undifferentiated, semidifferentiated, and completely differentiated cells. Cell loss and metabolic activity were assessed by thiazolyl blue reduction (3-(4,5-dimethylthiozole-2-yl)-2,5,-biphenyl tetrazolium bromide [MTT], mitochondrial succinate dehyrdogenase activity), alamarBlue assay (cytochrome oxidase activity), and deoxyribonucleic acid (DNA) quantitation. Undifferentiated cells were sensitive to 1 x 10(-4) M ergovaline after acute exposure (from 52 to 74% of control values depending on assay). After 72 h of exposure to 1 x 10(-4) M ergovaline, in all three assays, treatment means were reduced to approximately 10% of the control means. By day 11 in culture, ergovaline toxicity to cells had decreased. With 24 h exposure, an apparent paradoxical increase in MTT was seen at some concentrations. This increase in MTT was also found in fully differentiated cells (day 21), whereas alamarBlue activity decreased. No change in DNA was found until 72 h of exposure, when DNA was reduced approximately 12% over most concentrations. These findings indicate differentiation state-dependent sensitivity of Caco-2 cells to ergovaline, potential problems of the MTT assay as an indicator of cellular toxicity, and usefulness of alamarBlue assay over DNA assay for toxicity assessment.     

Homonucleotide stretches in chromosomal DNA of Campylobacter jejuni display high frequency polymorphism as detected by direct PCR analysis

Homopolymeric nucleotide tracts have been previously identified in the genome sequence of Campylobacter jejuni 11168 [Parkhill et al., Nature 403 (2000) 665-668]. These tracts are believed to regulate contingency genes but as yet no phenotypic variation has been identified associated with many of these genes. To investigate homopolymeric tracts for genes for which there is no observable phenotype, a method was designed to visualise profiles of the various tract lengths directly at the genomic level by means of PCR and denatured polyacrylamide gel electrophoresis. Six of the seven contingency genes investigated displayed variation in the length of the respective homonucleotide tracts. Surprisingly, each contingency gene gave a typical peak profile that represented a conserved size distribution of polymorphic forms. For each gene studied, peak profiles were conserved between strains of C. jejuni. Duplicated genes, containing homonucleotide stretches, displayed locus-specific peak distributions for each gene copy. Contingency genes were polymorphic within single colonies, and the observed complex peak profiles suggested a frequency of slippage several orders of magnitude higher than reported for other organisms. No G7 (or C7) stretch was ever observed, and their absence from the complete genome suggests strong selection against their presence. In view of the predictable outcome of the process leading to these polymorphisms, it is hypothesised that the formation and/or selection of these tracts is not a random process, but is driven by as yet unknown mechanism(s). High-frequency polymorphism of these genes may be a mechanism by which C. jejuni survives selection bottlenecks between opportunities for growth within a host.