Detection of cancer clones in human gastric adenoma by increased DNA-instability and other biomarkers

An immunohistochemical differential staining of cancerous cells with anti-cytidine antibody after denaturation of nuclear DNA by acid hydrolysis with 2N HCl at 30 degree C for 20 min (DNA-instability test) has been used as a marker of malignancy. The test was applied to bioptic tissues of human gastric polyp assessed histopathologically as foveolar hyperplastic polyp (13 cases), mild (58 cases), moderate (86 cases), and severe (20 cases) dysplasia, and adenocarcinomas (14 cases). The serial sections of the same tissues were also subjected to immunohistochemical staining for Ki67, p53, DNA-fragmentation factor (DFF45), and basic fibroblast growth factor (bFGF). The DNA-instability test was positive in 14 (100%) adenocarcinoma cases, 20 (100%) severe dysplasia cases, 52 (60.5%) moderate dysplasia cases, and 12 (20.7%) mild dysplasia cases, indicating malignancy. All foveolar hyperplastic polyps were negative to the DNA-instability testing. Furthermore, the percentage of glands positive in the DNA-instability test steadily increased in going from mild (10%), to moderate (40%), to severe (100%) dysplasia, and adenocarcinoma (100%). All other biological markers tested in the present study showed significantly higher values in the adenoma glands, being positive to DNA-instability testing, irrespective of the dysplasia grade, as compared to those in the adenoma glands that were negative to DNA-instability testing. Furthermore, the former values were comparable to those in adenocarcinoma. These results indicate that cancer cell clones are already present at the adenoma stages showing a positive DNA-instability test, enhanced proliferative activity, p53 mutation, induction of DFF45 and bFGF. These factors allow cancer cell proliferation, producing heterogeneous subclones due to DNA-instability, enhancing their survival by escaping apoptosis, and providing abundant nutrients during the early-stage progression of gastric cancer. Based on these findings, we herein propose the concept of "procancer" (as opposed to "pre-cancer") as being a unique stage during the course of carcinogenesis and cancer progression. We designate the term to cancer clones at the very early stages of malignant progression that do not show distinguishable morphological atypia but do show positive DNA-instability testing and positive staining for various biomarkers such as Ki67, p53, DFF45, and bFGF. We also define the abnormal positive staining of these biomarkers, including the DNA-instability test as "functional atypia", compared to the ordinary morphological atypia.     

Detection of cancer clones in human colorectal adenoma as revealed by increased DNA instability and other bio-markers

An immunohistochemical differential staining of cancerous cells with anti-cytidine antibody after denaturation of nuclear DNA by acid hydrolysis with 2N HCl at 30 degrees C for 20 min (DNA-instability test) has been used as a marker for malignancy. The test was applied to bioptic tissues of human colorectal polyps assessed histopathologically as hyperplastic polyp (11 cases), tubular adenoma of mild (68 cases), moderate (102 cases), and severe (46 cases) dysplasia, and adenocarcinoma (30 cases). The serial sections of the same tissues were also subjected to immunohistochemical staining for Ki67, p53, DNA-fragmentation factor 45 (DFF45) and vascular endothelial growth factor (VEGF). The DNA-instability test was positive in 30 (100%) adenocarcinoma cases, 46 (100%) severe dysplasia adenoma cases, 36 (35.29%) moderate dysplasia adenoma cases, and 8 (11.76%) mild dysplasia adenoma cases, indicating malignancy. All hyperplastic polyps were negative to the DNA-instability test. Furthermore, the percentage of glands positive in the DNA-instability test steadily increased in going from mild (10%), to moderate (35%), to severe (100%) dysplasia, and adenocarcinoma (100%). All other biological markers tested in the present study showed significantly higher values in those adenoma glands that were positive to the DNA-instability test, irrespective of the dysplasia grade, as compared to the markers in the adenoma glands that were negative to DNA instability testing. Furthermore, the former values were comparable to those in adenocarcinoma. The results indicate that cancer cell clones are already present at the adenoma stages showing positivity to DNA instability testing, enhanced proliferative activity, p53 mutation and induction of DFF45 and VEGF, at a time when the degree of morphological atypia are not yet large enough for them to be identified as cancer. These factors promote cancer cell proliferation, produce heterogeneous subclones due to DNA instability, enhance their survival by escaping apoptosis, and provide abundant nutrients by neovascularization during the early-stage progression of colorectal cancer.     

AgNOR counts in cervical smears under normal and other cytopathologic conditions

OBJECTIVE: To investigate the diagnostic value of AgNOR counts in cervical smears in the process of cervical carcinogenesis and in discriminating the different grades of squamous intraepithelial lesion (SIL). STUDY DESIGN: Silver nitrate staining for AgNOR counts was performed in 50 cervical smears of cytologically diagnosed normal, inflammatory, low grade SIL (LSIL) (mild dysplasia), high grade SIL (HSIL) (moderate and severe dysplasia) and squamous cell carcinoma. The smears were derived from the ongoing routine outpatient cytology screening at Queen Mary's Hospital, Lucknow, India. RESULTS: In normal and inflammatory smears, the number of AgNOR dots varied from 1 to 2, in mild dysplasia from 2 to 4, in moderate dysplasia from 4 to 6 and in severe dysplasia from 6 to 8. Frank cervical carcinoma cases revealed 8-10 dots. Thus, a progressive increase in AgNOR counts was observed when the severity of pathologic lesions increased. Statistical analysis revealed a significant difference in AgNOR counts between normal and inflammatory smears, but it was highly significant between inflammatory and LSIL cases, between LSIL and HSIL, and between severe dysplasia and frank malignancy. CONCLUSION: This study underscored the diagnostic importance of AgNOR counts, especially in discriminating between LSIL and HSIL of the cervix. Another study is under way to assess the potentiality of AgNOR counts as tumor markers in cervical carcinogenesis.     

Immunohistochemical detection of early-stage carcinogenesis of oral leukoplakia by increased DNA-instability and various malignancy markers

The degree of DNA instability as determined by immunohistochemical staining with anti-single-stranded DNA antibody after acid hydrolysis (the DNA instability test) was used as a marker of malignancy. The test was applied to tissues of oral leukoplakia assessed histopathologically as hyperplasia (38 cases), mild (12 cases), moderate (11 cases) and severe (8 cases) dysplasia, and invasive squamous cell carcinoma (SCC, 20 cases). Tissues were subjected to immunohistochemical staining for proliferating cell nuclear antigen (PCNA), p53, DNA-fragmentation factor 45 (DFF45), analysis of various AgNORs parameters, and triple immunostaining for vascular endothelial growth factor (VEGF), CD34, and PCNA. The DNA instability test was positive in 20 (100%) SCC cases, 8 (100%) severe dysplasia cases, 8 (72.7%) moderate dysplasia cases, 6 (50.0%) mild dysplasia cases, and 9 (23.7%) hyperplasia cases, indicating malignancy. The proportion of lesions positive for PCNA, p53, DFF45, and values of AgNORs parameters steadily increased from hyperplasia to mild, moderate and severe dysplasia, and SCC, especially in those showing positive DNA instability test, indicative of malignancy. Based on these results, 44.9% of leukoplakia were malignant tissues, namely carcinoma in situ. The proportion of PCNA-positive vascular endothelial cells in the vicinity of VEGF-positive epithelial lesion was significantly higher than that of negative DNA instability lesions, as revealed by immunohistochemical triple staining for VEGF, CD34, and PCNA. Our results suggest that increased DNA instability, enhanced proliferative activity, p53 mutation, and induction of DFF45 and VEGF may allow cancer cell proliferation, enhance their survival by escaping apoptosis, and provide abundant nutrients during early-stage carcinogenesis of oral leukoplakia.     

P16INK4a point mutations and promoter hypermetylation in bronchial preneoplastic lesions

The aim of this study was to determine p16INK4a point mutations and promoter hypermethylation in tumour cells and bronchial preneoplastic lesions in 32 surgically resected lungs due to primary squamous cell carcinoma. P16 point mutations were detected in 1 (3%) and promoter hypermethylation in 12 (31%) of 32 squamous cell carcinomas. The status of p16 was further characterized in 38 premalignant lesions including squamous metaplasias without dysplasia, squamous metaplasias with mild, moderate and severe dysplasias and 4 carcinomas in situ. No p16 point mutations have been found in premalignant or CIS lesions. Methylation of p16 was detected in 1 of 8 (12.5%) cases of squamous metaplasias without dysplasia, in 1 of 10 (10%) cases of squamous metaplasias with mild dysplasia, in 1 of 9 (11%) cases of squamous metaplasia with moderate dysplasia and in 2 of 7 (28.5%) cases of severe dysplasias, as well as in 1 of 4 (25%) carcinomas in situ. This investigation indicates that P16INK4a supressor gene point mutations are rather late event and inactivation of this gene by promoter hypermethylation is early and likely critical in bronchial cancerogenesis.     

Immunoexpression of tenascin as a predictor of the malignancy potential of oral leukoplakia associated with a tobacco habit

Oral leukoplakia is a morphological alteration of tissue that is an early indicator for malignancy. Tenascin (TN) is a large hexameric extracellular matrix (ECM) protein with anti-adhesive properties that fosters cell migration during development, wound healing and tissue remodeling; it is present in small amounts in adult tissues. Overexpression of TN in a pathological condition may be either a cause or a consequence of the disease. We evaluated the efficacy of TN for early prediction of tobacco-associated oral cancers. We studied retrospectively 95 cases of oral leukoplakia, including mild, moderate and severe cases, using immunohistochemistry for TN. We evaluated the intensity, area and pattern of TN expression. Greater intensity and area of TN expression was observed in mild and severe dysplasia than in moderate dysplasia. Most cases showed a reticular pattern of expression, especially in mild and moderate dysplasia; a fibrillar pattern was more evident in severe dysplasia. We also observed homogeneous expression pattern in some cases. TN is a marker for dysplastic changes in epithelium and its expression may be helpful for predicting the malignancy potential of tobacco-associated oral leukoplakia.     

Detection of non-papillary, non-invasive transitional cell G1 carcinoma as revealed by increased DNA instability and other cancer markers

The method to reveal DNA-instability as demonstrated by immunohistochemical staining with anti-cytidine antibody after acid hydrolysis (DNA-instability test) was used as a marker of malignancy. The test was applied to paraffin-embedded sections taken from 15 urinary bladders, renal pelvic cavities, and ureters bearing multiple carcinoma in situ (CIS) and totally 31 papillary urothelial cancers. The serial sections of the same tissues were also subjected to immunohistochemical staining for PCNA, p53, DFF45, and VEGF. The DNA-instability test was positive in 100% cancer lesions irrespective of the grades, and apparently normal urothelium, and hyperplastic and dysplastic urothelial lesions also showed the areas with clones positively stained with DNA-instability testing, and the percent numbers of positive areas in them were 28.3%, 37.7%, and 61.5%, respectively. These clones, which were present in apparently normal urothelium and in hyperplastic and dysplastic urothelial lesions, showed higher percent values of PCNA-positive-cells, in comparison to the values estimated in the areas with negatively stained DNA-instability testing, and the former values were statistically not different from those in carcinoma lesions. Furthermore, the percent numbers of areas positive for p53, DFF45, and VEGF, with positive DNA-instability testing were also much higher than those with negative DNA-instability testing in apparently normal urothelium, and hyperplastic and dysplastic urothelial lesions, and the former values were again comparable to those in cancer lesions with no statistical differences. These clones were regarded as already being malignant and should be the direct precursors of progressed cancer lesions. They will make progression through two different pathways, one to papillary non-invasive G1 cancers by neovascularization induced by paracrine secretion of VEGF, and another to flat CIS G2 without secretion of VEGF; thus the clones should be regarded as non-papillary, non-invasive Gl TCC, or CIS G1. It should be always taken into account that the probability for apparently normal urothelium, and hyperplastic and dysplastic urothelial lesions to contain cancer clones, will be high already, especially in tumor-bearing bladders.     

Expression of CA IX in dysplasia adjacent to surgical resection margins of oral squamous cell carcinoma

Carbonic anhydrase (CA) IX is a hypoxia marker located almost exclusively in tumor cells. We analyzed the expression of this marker in dysplastic lesions adjacent to the surgical resection margin in patients with oral squamous cell carcinoma. We investigated 70 archived tumors, 36 of which showed dysplasia adjacent to the surgical margin. We used tissue microarray technology to perform an immunohistochemical study of CA IX expression. We found 12 (33.3%) cases of mild dysplasia (10 negative, 2 positive for CA IX), five (13.9%) cases of moderate dysplasia (3 negative, 2 positive for CA IX), 1 (2.8%) case of severe dysplasia (negative for CA IX) and 18 (50%) cases of carcinoma in situ (10 negative, 8 positive for CA IX). In cases of intense expression of CA IX in the tumor, the same distribution of positive and negative cases was observed in all degrees of dysplasia (mild, moderate, severe), although cases of carcinoma in situ tended to be CA IX positive.     

Ethnic patterns of cytologic abnormalities in cervical smears in suriname, a high-risk area for cervical cancer

OBJECTIVE: To determine the prevalence of cytologic abnormalities in cervical smears from women attending the first organized screening program in Suriname and to compare the prevalences in 4 Surinamese ethnicities with different cervical carcinoma incidences. STUDY DESIGN: Cervical scrapes were taken from women with 4 different ethnicities: Maroons, Amerindians, Javanese and Hindustani. Papanicolaou staining and cytologic screening were performed on 807 cervical smears. RESULTS Cervical cytologic abnormalities were seen in 13.4%, of which 8.1% (62 of 764) had atypical changes, 2.6% (20 of 764) had mild and 2.6% (20 of 764) had moderate and severe dysplasia/carcinoma in situ (CIS). The cytologic abnormalities varied between the ethnicities: 42.1% (83 of 197) in the Maroons and 2.3% (4 of 176), 5.0% (9 of 183) and 3.0% (6 of 208) in the Javanese, Amerindians, and Hindustani, respectively. CONCLUSION: The high prevalence of moderate and severe dysplasia/CIS in all ethnicities correlates with the high cervical carcinoma incidence in Suriname. A significantly higher prevalence of mild abnormalities in the Maroons was observed; it did not reflect the relatively low cervical cancer incidence in this ethnicity. However, this can be explained by the possibility that these women have a different sexual lifestyle, leading to a higher prevalence of     

DNA/nuclear protein content in the evaluation of cell cycle modifications during colon carcinogenesis

OBJECTIVE: To investigate the colorectal adenomacarcinoma sequence by biparametric DNA/nuclear protein flow cytometry with the aim of evaluating cell cycle modifications during carcinogenesis. STUDY DESIGN: Paraffin-embedded specimens of 27 adenomas with mild/moderate dysplasia, 20 adenomas with severe dysplasia/intramucosal adenocarcinomas, 28 adenocarcinomas and 14 normal colon mucosa specimens were analyzed by biparametric DNA/nuclear protein content flow cytometric analysis in order to evaluate cell cycle modifications during colorectal carcinogenesis. RESULTS: The mean G0-G1A fraction of the cell cycle was 50.6% (SD +/- 17.2), 25.7% (SD +/- 15.1), 27.8% (SD +/- 11.7) and 29% (SD +/- 13.8) for normal mucosa, adenomas with mild/moderate dysplasia, adenomas with severe dysplasia and adenocarcinomas, respectively. The difference between normal mucosa and the other groups was statistically significant (P < .05), while no significant differences were detectable between adenomas with different degrees of dysplasia and adenocarcinomas. CONCLUSION: Our results show a decrease in G0-G1A in adenomas with mild/moderate dysplasia, suggesting that modification of the cell cycle may represent an early step in colon carcinogenesis, and they support the hypothesis that disregulation of cell cycle-controlling genes is an early event in the adenoma-carcinoma sequence.     

DNA-cytometric diagnosis of prospective malignancy in borderline lesions of the uterine cervix

Interactive DNA cytometry was used for the diagnosis of prospective malignancy in 48 smears with borderline lesions (mild and moderate dysplasias) of the uterine cervix. In addition, 183 smears with benign squamous epithelia, 38 with carcinoma in situ and 7 with invasive squamous carcinoma were also measured. Nuclear Feulgen-DNA measurements were performed using various methods, and the resulting data were analyzed by an algorithm for a DNA-cytophotometric diagnosis of malignancy. The results were compared with the data on follow-up and subsequent histologic studies in these cases. There was no false-positive diagnosis in the 183 benign smears and only 1 false-negative diagnosis in the 76 histologically proven squamous-cell carcinomas, which yields a specificity of 100% and a sensitivity of 98.6%. The sensitivity for the detection of subsequent histologically proven malignancy in cases with cytologically mild or moderate dysplasia amounted to 97%. In 13 borderline cases, there was a mean interval of 21 months between the taking of the cytologic smear on which the DNA diagnosis of malignancy was made and the date on which the histologic confirmation of malignancy was made. In 17% of the cytologically dysplastic cases, the DNA diagnosis of malignancy was not verified by subsequent histologic investigation. These results indicate that interactive DNA cytometry is able to detect prospective malignancy in smears from borderline lesions of the uterine cervix with a high sensitivity.     

Effect of the special properties of monolayer cell preparations for automated cervical cytology on visual evaluation and classification. With an estimation of the number of cells required to be screened

Monolayer preparations used in cell image analysis show some peculiarities as compared with conventional cytologic smears, such as homogeneous distribution of cells, distinct appearance of cells and a reduced number of background elements. However, for use in gynecologic cytology, monolayer preparations must be accessible to visual examination and classification. To investigate the consequences of the special features of these preparations on the strategy of visual evaluation, we estimated the minimum number of cells needed for a diagnostic decision. Cell counts were made of gynecologic monolayer preparations from 50 women with no suspicion of malignancy and 50 women with invasive squamous-cell carcinoma of the uterine cervix and its precursors. Our results showed that the more serious the lesion, the lower the number of cells needed for a diagnostic decision. The highest mean values of numbers of cells needed for an effective diagnosis were estimated in cases of mild and moderate dysplasia (734 cells) and in non-suspicious cases (731 cells). The number of cells needed did not exceed 1,700 in any case. The false-negative and false-positive rates were 6% and 2%, respectively, including the cases of mild to moderate dysplasia.     

应用单克隆抗体CL－2、CL－4对大肠癌、癌旁病变的免疫组化研究

应用抗人结肠癌单克隆抗体ＣＬ－２,ＣＬ－４,对２０５例大肠癌及癌旁病变进行了免疫组化研究。ＣＬ－２相应抗原在移行粘膜,轻、中、重度非腺瘤异型增生,大肠癌的阳性率分别为３７．６％、６３．２％、８６．７％、９０．９％及８６．８％,阳性率呈递增趋势;ＣＬ－４相应抗原的阳性率依次是３９．１％、５７．９％、７３．３％、８１．８％及７７．６％;４０例正常大肠粘膜均阴性。结果表明,ＣＬ－２、ＣＬ－４都是对大肠癌阳性率较高的标记物,但用来鉴别癌与异型增生意义不大;一部分大肠癌可能来源于非腺瘤途径;移行粘膜不同程度的肿瘤相关抗原的表达,反映了其潜在的恶性性质,但其程度低于异型增生,文中,对其临床意义进行了讨论。     

Clinical evaluation of the EA-rosette test in the early detection of cervical cancer

It was previously found that a negative EA-rosette test, showing EA-rosette-forming cells in a cervical cell suspension, excluded the presence of cells of invasive carcinoma (predictive value of 99.9%). This study on 2,462 patients confirmed the applicability of the EA-rosette test in screening for precancerous as well as cancerous lesions. In 98.6% of the cases of dysplasia, carcinoma in situ and invasive carcinoma, the cervical cell suspensions contained EA-rosette forming cells (the rosette test was positive). With a negative EA-rosette test, the probability of missing a specimen with class III cytology (mild/moderate dysplasia) was 1.4%, of missing one with class IV cytology (severe dysplasia/carcinoma in situ) was 0.8% and of missing one with class V cytology (invasive carcinoma) was 0.25%. The predictive value of a negative EA-rosette test was 98.6%. The false-negative rate for negative EA-rosette tests was 3.7% for invasive carcinoma, 17.5% for carcinoma in situ and severe dysplasia and 41.4% for mild to moderate dysplasia.     

Comparative histochemical study of Bowen's disease and actinic keratosis: preserved normal basal cells in Bowen's disease

The degree of DNA-instability as revealed by immunohistochemical staining with anti-cytidine antibody after acid hydrolysis (DNA-instability test) has been recently used as a marker of malignancy. This technique was applied to examine 17 skin tissue samples of Bowen's disease, 47 of actinic keratosis, 15 of squamous cell carcinoma, 5 of seborrheic keratosis, and 10 of normal skin. All benign neoplastic cells of seborrheic keratosis and normal epidermal cells were negative. On the other hand, all cancer cells were positive with the DNA-instability test, indicating their malignancy, but all basal cells in Bowen's disease were completely negative. Compatible with this result, the basal cells in Bowen's disease were characteristically normal as evident in other histochemical examinations. Thus, they were negative with p53 immunohistochemistry, with normal signals of chromosome 17 in situ hybridisation and argyrophilic nucleolar organiser region, and showed slightly enhanced proliferative activity as revealed by proliferating cell nuclear antigen immunohistochemistry. Immunohistochemical staining with 34 beta E12 (monoclonal antibody against cytokeratins 1, 5, 10, and 14), which stains all normal epidermal keratinocytes including basal cells, showed that only the basal cells of Bowen's disease stained strongly and homogeneously, while all cancer cells in the upper layers of Bowen's disease and all layers of actinic keratosis were only sporadically or weakly stained. Staining with 34 beta B4 (monoclonal antibody against cytokeratin 1), which recognises the whole epidermis except for the basal layer in the normal epidermis, showed that the basal cells in the Bowen's disease were completely negative, and lower layer cells in the actinic keratosis and upper layer cells in Bowen's disease were only sporadically stained positive, although the superficial layer cells in actinic keratosis stained strongly and homogeneously. Our findings clearly indicate that the basal cells in Bowen's disease are normal. In support of this conclusion, the same cells showed normal morphology on electron microscopy with preserved basement membrane, although the latter was often damaged in actinic keratosis.     

Prediction of cervical neoplasia diagnosis groups. Discriminant analysis on digitized cell images

The purpose of this study was to develop discriminant analysis models for predicting cervical dysplasia/neoplasia case diagnoses using cytometric features derived from the digital image analysis of cell monolayers. The data base consisted of 925 cells from 27 cases diagnosed either as moderate dysplasia (n = 10), severe dysplasia (n = 5), carcinoma in situ (n = 8) or invasive carcinoma (n = 4) on both tissue biopsy and monolayer preparations. Cell features examined were cell diameter, nuclear diameter, nuclear mean optical density (OD), nuclear integrated OD (IOD), nuclear OD standard deviation, normalized IOD, nuclear texture and nuclear-cytoplasmic ratio. Features derived from cells visually classified as moderate dysplasia correctly predicted the case diagnosis of moderate dysplasia versus more severe disease for 85% of the cells. Prediction models using summary measures (mean and variance) derived from all visually classified abnormal cells within each case correctly separated all cases into their respective diagnostic categories. These findings suggest that dysplastic cells in a cytologic sample have features that collectively reflect the tissue diagnosis, regardless of the visual differences among the cells. Such information has potential use for diagnosis and possibly for prognosis.     

A comparison of the frequency of sperm chromosome abnormalities in men with mild,moderate, and severe oligozoospermia

Infertile men undergoing intracytoplasmic sperm injection have an increased frequency of chromosome abnormalities in their sperm. Men with low sperm concentration (oligozoospermia) have an increased risk of sperm chromosome abnormalities. This study was initiated to determine whether men with severe oligozoospermia (<10(6) sperm/ml) have a higher frequency of chromosome abnormalities in their sperm compared with men with moderate (1-9 x 10(6) sperm/ml) or mild (10-19 x 10(6) sperm/ml) oligozoospermia. Multicolor fluorescence in situ hybridization analysis was performed using DNA probes specific for chromosomes 13, 21, X, and Y (with chromosome 1 as an autosomal control for the sex chromosomes). Aneuploidy and disomy frequencies were assessed from a total of 603,011 sperm from 30 men: 10 in each of the categories. The mean frequencies of disomy for the patients with mild, moderate, and severe oligozoospermia were 0.17%, 0.24%, and 0.30%, respectively, for chromosome 13 and 0.22%, 0.44%, and 0.58%, respectively, for chromosome 21. For the sex chromosomes, the mean frequencies of disomy for mild, moderate, and severe oligozoospermia were 0.25%, 1.04%, and 0.68%, respectively, for XY, 0.047%, 0.08%, and 0.10%, respectively, for XX, and 0.04%, 0.06%, and 0.09%, respectively, for YY. The frequencies for diploidy also increased from 0.4% for mild to 1.20% for moderate to 1.24% for severe oligozoospermia. There was a significant inverse correlation between the frequency of sperm chromosome abnormalities and the sperm concentration for XY, XX, and YY disomy and diploidy. These results demonstrate that men with severe oligozoospermia have an elevated risk for chromosome abnormalities in their sperm, particularly sex chromosome abnormalities.     

Proliferating activity in oral dysplastic leasions and squamous cell carcinomas

The aim of the study was the quantitative analysis of AgNORs in oral squamous cell carcinomas as well as in dysplastic epithelial changes accompanied and not accompanied by oral squamous cell carcinomas. AgNOR proteins were visualized in histological slides using silver impregnation technique according to D. Ploton. In each sample 100 cell nuclei were assessed. The study used 54 cases of proliferating oral epithelial changes divided into 3 groups: group I consisting of 13 cases of dysplastic lesions not accompanied by oral squamous cell carcinomas; group II (a total of 18 cases) containing dysplastic lesions situated in the vicinity of oral carcinomas and group III (23 cases) with oral squamous cell carcinomas. Statistically significant differences were found between groups with mild dysplasia and groups with severe dysplasia as well as squamous cell carcinomas. Statistical analysis did not show any differences in the number of AgNORs between squamous cell carcinomas and epithelial lesions with severe dysplasia. Our results demonstrate that the analysis of AgNORs expression can serve only as an additional parameter to evaluate the potential of malignant transformation.     

Apoptosis and apoptosis related proteins in chronic viral liver disease

Background: Apoptosis may be an important mechanism of hepatocyte death in chronic viral liver disease. Methods: We studied apoptosis in liver biopsies from 30 patients with chronic viral hepatitis and 8 patients with viral cirrhosis by the TUNEL method. 12 cases of non-alcoholic steatohepatitis and 12 cases of primary biliary cirrhosis were used as non-viral disease controls. Immunohistochemical expression of p53, p21/waf1, bcl-2 and mdm-2 proteins was also studied in the same patients. Results: A statistically significant increase of apoptotic liver cells was found in severe chronic viral hepatitis (5.3 ± 0.3%), cirrhosis (3.4 ± 0.5%) and PBC (4.4 ± 0.4%) cases compared to patients with non-alcoholic steatohepatitis (0.8 ± 0.3%). The expression of p53 protein was increased in the cases of viral cirrhosis and in chronic severe viral hepatitis whereas in the cases of chronic mild hepatitis, PBC and non-alcoholic steatohepatitis we found no expression of p53. P21/waf1 expression was increased in severe chronic hepatitis, cirrhosis and PBC cases compared to mild hepatitis and non-alcoholic steatohepatitis cases. However no induction of mdm-2 was observed in the subgroups of chronic liver disease. Bcl-2 was expressed only in epithelium of bile ducts and mononuclear cells of the portal tracts and liver lobules. A weaker Bcl-2 expression was noted in the epithelium of bile ducts of 7/12 PBC cases. Conclusion: Our results provide evidence of increased apoptosis in severe chronic viral liver disease, suggesting that apoptotic cell death might be involved in the pathogenesis of hepatocellular damage of viral hepatitis and cirrhosis. Furthermore we analysed part of the apoptotic pathways implicated in the above process and found an increased expression of p21/waf1, probably p53 mediated, without overexpression of the apoptosis inhibiting bcl-2 and mdm-2 proteins. By contrast p21/waf1 overexpression in PBC seems to be propagated by a p53 independent mechanism.