Msh2, Mlh1, Fhit, p53, Bcl-2, and Bax expression in invasive and in situ squamous cell carcinoma of the uterine cervix.

To analyze relevant factors and their effects on neoplastic progression in cervical carcinoma, a panel of genetic markers was studied. Paraffin-embedded tissue sections were obtained from 37 patients with carcinoma of the uterine cervix, 14 noninvasive squamous cell carcinomas (NISCCs), and 23 invasive squamous cell carcinomas (ISCCs). Immunoreactivity of Msh2, Mlh1, Fhit, p53, Bcl-2, and Bax proteins was examined by immunohistochemical staining with appropriate antibodies. Positive staining of Msh2 was detected in 13 of 14 (92.9%) NISCCs and in 13 of 23 (56.5%) ISCCs (P < 0.02). Mlh1 immunoreactivity was observed in 10 of 14 (71.4%) NISCCs and in 8 of 23 (34.8%) ISCCs (P < 0.04). Overexpression of p53 protein was found in 4 of 14 (28.6%) NISCCs and in 16 of 23 (69.6%) ISCCs (P < 0.02). Bcl-2 overexpression was detected in 2 of 14 (14.3%) NISCCs and in 15 of 23 (65.2%) ISCCs (P < 0.003). No significant difference in the two types of lesion was found for Bax and Fhit expression. The relationship between Mlh1, Msh2, and p53 protein expression was significant (P < 0.001 and P < 0.001, respectively), as was that between Fhit and Bax immunoreactivity (P < 0.02). In conclusion, we consider that altered expression of Msh2, Mlh1, p53, and Bcl-2 may be a critical event during cervical cancer progression, whereas Fhit may be a component of a proapoptotic pathway.

Aneuploid polyclonality in image analysis.

Solid tumors such as colorectal adenocarcinomas consist of biologically diverse cell subpopulations. Nuclear DNA content of tumor cells in colorectal carcinomas may be studied with different techniques of intranuclear DNA quantification. In the current study, the DNA ploidy of samples obtained from 68 patients with colorectal carcinoma (age ranging from 46 to 86 years, mean age 66 years), treated with radical surgery, between the years 1992 and 1995 was analyzed. DNA ploidy was assessed using a CAS 200 image analyzer and was evaluated on neoplastic tissue and undamaged healthy mucosa obtained from the edges of the surgical resection. Approximately 150-300 cells were analyzed for each sample. The aim of this study was to evaluate the prognostic significance of the polyclonal cases correlated with lymph node infiltration and disease free-survival. The pathological stage according to the TNM classification was compared to ploidy: an increase in multiple stemlines was observed in stage III cases, i.e., a progression towards aneuploidy and multiple stemlines was significantly associated with lymphatic metastasis (p<0.0003). Concerning distant metastasis, we found a correlation between stage IV and polyclonality. A significant correlation was observed between disease-free survival and aneuploid and polyclonal cases (p<0.0053). In polyclonal cases a nine fold greater relapse risk compared to the non-polyclonal cases was observed (p<0.0004). In two cases, the adeno-carcinoma of the sigma was polyclonal and its hepatic metastasis contained the predominant aneuploid clone with the same cytometric characteristics (DNA index) of the original lesion.

Asbestos bodies in the sputum of workers exposed to environmental pollution.

The diseases related to asbestos exposure (pulmonary fibrosis, broncogenic carcinoma and mesothelioma) are of widespread interest and involve different socio-economic groups of subjects. Since these pathologies have a wide diffusion in the industrial world, we carried out an investigation on two populations occupationally exposed to air pollution and asbestos fibre inhalation (164 traffic policemen of the municipal district of Rome and 218 railwaymen) and on a control group (119 residents in a rural district of Perugia) for the detection of asbestos bodies in the sputum. The results obtained from traffic policemen and railwaymen workers differred significantly from those of the control group. The presence of asbestos bodies in traffic policemen seems to be determined by a strong synergetic effect between gaseous urban pollution, cigarette smoking habits and asbestos dust arising from car brakes and building materials, whereas, in railwaymen it seems to be more directly correlated to exposure.

Overexpression of NDP kinase nm23 associated with ploidy image analysis in colorectal cancer.

The nm23 gene was originally identified by differential hybridization between two murine melanoma cell sublines with low and high metastatic potential. Nm23 is localized on chromosome 17q21.3-22. Allelic deletions of chromosome 17 have been related to the progression of colorectal carcinomas. We have evaluated and compared the expression of nm23 NPD kinase protein using an immunohistochemical method and DNA ploidy evaluation with image analysis. This study was performed on 20 patients, who underwent surgery for colorectal carcinoma. Patients were followed up during the period from 1992 to 1994. Results have shown an association between the parameters obtained for the nm23 NPD kinase protein expression, and aneuploid DNA and neoplastic progression. The expression of nucleoside diphosphate (NDP) kinase mm23 has been reported to be inversely related to the metastatic potential of experimental cells in human breast cancer. A relationship between the positivity in protein expression of gene product in the allele nm23 H1 and the state of the lymph nodes has also been found.

DNA ploidy and HPV subtypes in cervical smears of HIV-sero-positive and negative patients.

Epidemiological studies have demonstrated that cervical HPV infection and precancerous lesions of the cervix are more common in HIV-seropositive patients. However little is known about the natural history of these lesions in this population. In the present study cervical smears from 36 patients, 18 HIV-seropositive women and 18 matched controls were evaluated with the aim of quantifying morphological alterations and to evaluate DNA ploidy and HPV subtypes. Cervical lesions in HIV-seropositive patients were diploid in 50% of the cases compared to 25% in controls. The only HPVs identified by ISH were types 16/18 and no significant differences were observed in the control population. In contrast, cytological evidence of HPV infection and dysplastic changes was greatly increased in smears from HIV patients compared to HIV-seronegative women. Less than 5% of the cells showed HPV associated changes in controls while 10% to 30% of the cells were affected in HIV-patients. We suggest that the Papanicolaou test should be effective for detecting cervical disease and for a close follow-up of this population. Moreover, while additional studies with larger population groups and different population bases are needed, these findings are suggestive of the possible use of morphological criteria for the identification of HIV-seropositive subjects.

Image analysis in multisample biopsy after ileal pouch-anal anastomosis.

Pouchitis in ileal anal anastomosis represents an important clinical complication after restorative proctocolectomy. Acute and chronic inflammation of the reservoir is a frequent event sometimes associated with villous atrophy and colonic metaplasia. After ileal pouch anastomosis, twenty-one patients affected by ulcerative colitis were studied. An image analyzer CAS 200 (Becton Dickinson) was utilized to evaluate the DNA intranuclear content in every biopsy. In two cases abnormal DNA distribution was observed, and in one case a poliploid pattern was seen. Abnormal DNA distribution was also present in colonic metaplasia. Therefore, image analysis for the detection of DNA aneuploidy may be of additional value together with histologic parameters in follow up, in order to exclude transformation of the ileal mucosa in neoplastic epithelia.

Nm23-H1 protein, DNA-ploidy and S-phase fraction in relation to overall survival and disease free survival in transitional cell carcinoma of the bladder.

OBJECTIVES: To explain the overall survival (OS) and disease free survival (DFS) in relation to nm23-H1 protein, DNA-ploidy and S-phase fraction (SPF) in transitional cell carcinoma of the bladder. PATIENTS AND METHODS: Ninety-four samples were obtained from patients with transitional cell carcinoma of the bladder examined between 1994 and 1996. The patients were underwent cistectomy or surgical biopsy and the material was histologically evaluated according to World Health Organization classification. Nm23-H1 protein expression in immunohistological staining and DNA ploidy, S-phase fraction by flow cytometric were performed. RESULTS: The correlation between OS and staging, grading, DNA-ploidy and S-phase was significant; whereas the overall survival and nm23-H1 protein, was not significant. The relationship between DFS and stage, DNA-ploidy and S-phase had a significant value. The correlation between DFS and age, sex, grading and nm23-H1 protein was not significant. There was no significant difference in age, sex, stage, grading, DNA-ploidy and SPF distribution between patients with nm23-H1 positive bladder cancer and those with nm23-H1 negative tumours. CONCLUSION: In our study, multivariate analysis showed that stage, ploidy and SPF were the strongest prognostic factors in predicting disease-free survival and prolonged survival, while nm23-H1 expression was not related to disease progression and/or prolonged survival. This expression, therefore, does not appear to be an independent prognostic factor in bladder cancer, although a still larger number of patients and a longer follow-up period are now needed for a definitive assessment of the prognostic significance of nm23-H1 expression.

Curschmann's spirals in sputum of subjects exposed daily to urban environmental pollution.

Curschmann's spirals have been observed for more than 100 yr in the sputum of patients affected by lung cancer, asthma, chronic bronchitis, or in asymptomatic smokers. However, to date, their clinical significance and pathogenesis have not been completely explained. Curschmann's spirals in the sputum of two populations were studied: 164 traffic policemen of Rome and 119 subjects living in a rural district near Perugia. Of these two groups, only the first were exposed daily to urban environmental pollutions. Spirals were detected in the sputum of 29 traffic policemen and in 3 subjects from the group not exposed daily to urban environmental pollutants. Between these two groups of subjects the difference was statistically significant (P < 0.001). Therefore, daily exposure to urban atmospheric pollution may play a role in the production of Curschmann's spirals. Our two groups were also divided into smokers and nonsmokers to evaluate if cigarette smoking could influence the presence of spirals in sputum. Our study confirms the association of spirals in the sputum of smokers but also indicates their presence in individuals who never smoked but were exposed to environmental pollutants. We also noticed that exposure to pollution and cigarette smoking may have a synergistic effect on the formation of spirals in the respiratory tract.

Papnet-assisted cytological diagnosis intensifies the already marked variability among cytological laboratories.

OBJECTIVE: The main objective was to assess the sensitivity, specificity and reliability of PAPNET-assisted diagnosis in comparison with conventional screening. SETTING: Seven Italian and one English University or Research Institutes, and a random sample of an other 20 Italian Laboratories of the Italian National Health Service (INHS) provided the cervical smears. METHODS: During the training phase every center examined in rotation four sets of slides for a total of 300 representative slides. Afterwards, 900 "positive" slides were added to the 3,100 slides which were collected consecutively without any selection or exclusion. The eight main centers were divided into four couples and each couple of centers examined 775 slides with the PAPNET system, "blindly" to the original diagnosis. An expert cytopathologist (M.A.) of the National Institute of Health (NIH) reassessed 40% of the slides with an original negative diagnosis to evaluate the false negative rate. Two expert NIH cytopathologists (M.A., G.M.) re-examined all slides where a disagreement had been observed between the original and one or both of the study diagnoses. The main analyses concerned the following three main categories: WNL and unsatisfactory for evaluation; ASCUS, AGUS and LSIL; HSIL and carcinoma. A special algorithm was devised to define the reference diagnosis for sensitivity and specificity assessment. RESULTS: Laboratories, even belonging to the same couple, classified as "no review" a very different proportion of slides ranging from 35% to 74%. The index of kappa agreement between the members of couples examining the same sets of slides was low or very low, ranging from 0.30 to 0.03. The sensitivity of the review classification was particularly low in some laboratories. Surprisingly, only a small correlation was observed between the sensitivity of the review classification and the proportion of slides classified as "review". The "tentative" diagnosis on PAPNET tiles of the "review" slides was almost as reliable as the microscopic diagnosis. In the overall performance, there were many significant differences among the eight laboratories. The best laboratory had a sensitivity of 95% and a specificity of 96%. At least three laboratories displayed unacceptably low sensitivity and one a very low specificity. CONCLUSION: Altogether these results seem to confirm that there are wide differences among cytological laboratories per se, and that these differences are intensified by the use of an instrument like PAPNET. The huge variation in performance may be explained by differences in basic skills and by different training, but it is difficult to understand exactly what could have been done to reduce it.

Assessment of risk factors and human papillomavirus (HPV) related pathogenetic mechanisms of CIN in HIV-positive and HIV-negative women. Study design and baseline data of the HPV-PathogenISS study.

OBJECTIVES: In women with HIV-associated immunosuppression, HPV infections have an increased risk of progression to high-grade cervical intraepithelial neoplasia (CIN). With the HAART-induced prolonged survival and more protracted clinical course of AIDS, progression of CIN to cervical cancer (CC) has become a clinically relevant issue, and the mechanisms responsible for HIV-HPV interactions need further elucidation. The study design and analysis of the baseline data of our new project are presented. MATERIAL AND METHODS: This project is a combination of a prospective cohort study of HIV- and HIV+ women, and a retrospective analysis of CIN lesions and cervical cancer. Up to the present, 244 women have been enrolled (17 HIV+) and subjected to epidemiological interview, colposcopic examination, sampling for HPV testing and typing (PCR, InnoLiPA), and HPV serology. The retrospective series of biopsies were analysed for 13 biomarkers (monitoring key molecular events) using immunohistochemistry and tested for HPV by PCR and TaqMan. RESULTS: HIV- and HIV+ women differ in their exposure status to many of the key epidemiological risk factors of cervical cancer, the most significant ones being number of sexual partners (p = 0.0001), age at onset of sexual activity (p = 0.002), and contraception (yes-no) (p = 0.009). The differences in the baseline clinical observations are less dramatic; HIV-positive women had more frequent HSIL PAP tests (p = 0.040), CIN2 or higher in cervical biopsy (p = 0.049), and external genital warts (p = 0.019). The factors predicting intermediate endpoint markers of cervical cancer, i.e., HSIL PAP smear, ATZ2 in colposcopy, and high-grade CIN in biopsy were analysed in univariate and multivariate regression models. All factors significant in univariate analysis were entered in the multivariate model; HIV-status and Pap smear history maintained their independent predictive power of the HSIL Pap test. The most powerful predictor of ATZ2 colposcopy was HSIL in Pap test. Only the HSIL Pap test and ATZ2 colposcopy remained significant independent predictors of high-grade CIN (p = 0.0001 and p = 0.008, respectively) in the multivariate model. CONCLUSIONS: The three intermediate endpoint markers are closely interrelated, but predicted in part by different covariantes in the causal pathway to cervical cancer. To elucidate whether the increased risk of HIV-positive women to high-grade CIN is due a) to their different exposure status to the risk factors, b) to the direct effects of HIV, or c) to molecular interactions between HIV and HPV, we need to complete these analyses separately in HIV+ and HIV- women.

Carbon and hemosiderin-laden macrophages in sputum of traffic policeman exposed to air pollution.

In this study, we evaluated (a) quantitative modifications of dust cells and siderocytes and (b) qualitative modifications (i.e., cellular changes and inflammatory infiltrate) in sputum of 164 traffic policemen occupationally exposed to environmental pollution in an urban area. Such modifications were correlated with time of exposure and smoking habits. Seventy-three (45%) of the policemen were smokers, and a control group of 119 nonexposed individuals (24% smokers) who resided in a rural district near Perugia, Italy, were examined. The sputa, which was collected for 3 d, were smeared on glass slides stained according to Papanicolaou's and Perl's methods. The mean numbers of dust cells in the sputa policemen and the rural population were 103.8 and 12.48, respectively (p < .0001). The numbers of dust cells were highest in policemen who smoked (relative risk = 3.95; p < .006). The mean numbers of siderocytes in policemen and the rural population were 0.99 and 5.31, respectively (not statistically significant, Mann-Whitney test). The difference between the number of subjects with hemosiderin-laden macrophages in traffic policemen and in the control group was small, but it was significant statistically (p < .004). In traffic policemen, it was related to smoking and time of exposure to air pollution, whereas in the control population it was related to heart-pulmonary diseases. In this study, we observed a synergistic effect between smoking and environmental pollution. In addition, less than 5 y of exposure can caused significantly increased (a) numbers of macrophages and (b) inflammatory infiltrate in sputa. The results of the cytological diagnosis revealed a statistically significant difference between policemen and the rural population (p < .001). The cytological exam of sputa and correlations with smoking within each population were not statistically significant. In conclusion, macrophage count in sputum is a sensitive and reproducible method for investigators to verify pulmonary changes that may be observed before they can be measured by functional tests.

p16(INK4A) expression is related to grade of cin and high-risk human papillomavirus but does not predict virus clearance after conization or disease outcome.

The role of p16(INK4A) as a marker of HR-HPV and in the diagnosis of CIN has been well established, but its predictive value in the clearance of the virus after CIN treatment and its use as a prognostic marker of cervical cancer has not been studied. A series of 302 archival samples, including 150 squamous cell carcinomas (SCCs) and 152 CIN lesions, were subjected to immunohistochemical staining for p16(INK4A) and HPV testing using PCR with three primer sets (MY09/11, GP5/GP6, SPF). Follow-up data were available of 88 SCC patients, and 67 of the CIN lesions had been followed-up with serial PCR after conization. HR-HPV types were closely associated with CIN (OR 19.12; 95%CI 2.31-157.81) and SCC (OR 27.25; 95%CI 3.28-226.09). There was a significant linear relationship between the lesion grade and intensity of p16(INK4A) staining (p = 0.0001). The expression of p16(INK4A) was also closely related to HR-HPV (p = 0.0001). p16(INK4A) staining was a 100% specific indicator of CIN, with 100% PPV, and showed 83.5% sensitivity and 80.1% PPV in detecting HR-HPV. However, p16(INK4A) staining did not predict clearance/persistence of HR-HPV after treatment of CIN. Similarly, despite a slightly more favorable survival in women with strong/intense p16(INK4A) staining in univariate analysis, p16(INK4A) expression was not an independent prognostic predictor in multivariate survival (Cox) analysis. After adjustment for p16(INK4A) staining, HR-HPV, histological grade, International Federation of Gynecology and Obstetrics (FIGO) stage, and age, only the last two were significant prognostic predictors (p = 0.0001 and p = 0.003, respectively). The present data confirm the role of p16(INK4A) as a highly specific marker of CIN and HR-HPV type, but expression of this protein does not seem to be of any prognostic value in cervical cancer or in predicting the clearance of HR-HPV after treatment of CIN. We speculate that different subgroups of cervical cancer are characterized by aberrant p16(INK4A)/cyclin D/Rb pathways that are due to different mechanisms that can be mutually exclusive.

Variation in the assessment of adequacy in cervical smears.

OBJECTIVE: To assess the interobserver reproducibility of the diagnosis of 'adequacy' of cervical smears according to the Bethesda System criteria in cervical smears. STUDY DESIGN: 358 cervical smears were obtained from three Italian cytopathological centres in 1998-99. All centres provided consecutively collected smears. The cervical smears were independently and blindly assessed by four cytologists. The screening was performed using a 10x objective and an additional evaluation of the percentage of cellularity was performed using a 4x objective. RESULTS: The proportion of smears assessed by the four cytologists as 'adequate' ranged from 60% to 70%, the proportion of 'satisfactory for evaluation but limited by' ranged from 27% to 38%, and the proportion of 'inadequate smears' ranged from 2% to 4%. Full agreement in the assessment of smear adequacy was observed in 311 slides and disagreement was observed only in 47. The category 'inadequate smear' was less reliable than the other two; however, the kappa value observed was acceptable. CONCLUSION: The present study shows that it is possible to achieve a high reproducibility in the assessment of smear adequacy, at least among expert cytologists who follow the Bethesda System criteria strictly.

Performance of cytology and colposcopy in diagnosis of cervical intraepithelial neoplasia (CIN) in HIV-positive and HIV-negative women.

As part of an extensive multi-institutional DIANAIDS-HIV-HPV-SIL project run in Italy (co-ordinated by ISS), the present study compares the performance (sensitivity, specificity, agreement) of routine cervical smear cytology with that of colposcopy in the detection of histologically-confirmed CIN lesions in 37 HIV-positive and 21 HIV-negative women, belonging to the DIANAIDS cohort of 459 women. All women were subjected to a cervical smear, colposcopy and biopsy, making possible the pairwise comparison of these techniques. In the whole series of HIV-positive and HIV-negative women, cytology had a sensitivity of 86.9% and specificity of 83.3%, the sensitivity of grade 2 abnormality on colposcopy against histology being 82.6% and specificity, 33.3%. No statistically significant difference was observed in the performance of Pap smears between the HIV-positive and HIV-negative women. The sensitivity of cytology was 89.7% vs 82.4% and the specificity, 75% vs 100%. For colposcopy, the sensitivity was 79.3% vs 88.2% and the specificity, 75% vs 50%. These data suggest that cervical Pap smear cytology is a highly sensitive and specific diagnostic tool in the clinical monitoring of lower genital tract pathology in HIV-positive women. Colposcopy, on the other hand, proved to be a somewhat less accurate diagnostic tool in these women.