Repeat processing of residual ThinPrep Pap tests: sampling of the vial may not be invariably homogeneous.

OBJECTIVE: To re-evaluate the reproducibility of additional slides prepared from residual cervical ThinPrep (TP) samples. STUDY DESIGN: Sixty paired specimens (conventional smears and direct-to-vial TP) were studied. Up to 10 additional slides were prepared from each TP vial. All slides were reviewed for adequacy of material, presence of abnormal cells and presence of normal flora or other pathogens. The additional TP slides were further evaluated for the presence of diagnostic elements which were not found on the conventional smear and primary TP slide. RESULTS: Abnormal cells found on the primary TP slide were also identified on all additional slides in 48/50 cases (96%) with squamous cell lesions. The distribution of material on TP slides was evaluated as homogenous in 51 cases (85%) and as non-homogenous in 9 (15%). Using the primary slides (conventional smear and TP) as a reference, additional diagnostic cells upgrading the cytologic diagnosis were found on the repeat slides in 7 cases (11.7%) and fungi consistent with Candida in 3 (5%). CONCLUSION: Repeat processing of residual cervical TP samples may not be an invariably reproducible procedure and the first slide may not be necessarily representative of the specimen as a whole. Nevertheless, both primary and repeat TP slides seem to be extremely effective in detecting a lesion (regardless of grade) in abnormal cases. The exact impact of non-homogeneous sampling of the vial on the diagnostic accuracy of the TP method should be further investigated.

Prognostic value of DNA analysis of prostate adenocarcinoma: correlation to clinicopathologic predictors.

The ability to accurately predict tumor behavior and patient survival is a problem in managing patients with prostate cancer. DNA ploidy provides important information for the evaluation of the prognosis of prostate cancer. The aim of this study was to investigate the DNA ploidy in imprints from prostate adenocarcinomas in a group of 70 patients in relation to Gleason score, tumor differentiation, stage and PSA serum levels. The DNA content was studied in Feulgen-stained imprint smears through the image analysis technique using a SAMBA 2005 Image analyzer. According to our measurements, a strong correlation was observed between DNA ploidy status and tumor differentiation (p<0.001). A statistically significant difference was found between DNA aneuploidy and increased pretreatment PSA serum levels (>4 ng/ml) (p<0.001), as well as between ploidy pattern and stage of the disease (p<0.001). Our results conclude that DNA ploidy status appears to be an additional marker in the field of prognosis of prostatic adenocarcinoma and could provide useful information on the potential behavior of prostate cancer.

Changes of chromosomes 1, 3, 6, and 11 in metastatic effusions arising from breast and ovarian cancer.

This cytogenetic study deals with cell material obtained from 15 pleural fluids from 11 patients with breast cancer and 27 ascitic fluids from 16 patients with ovarian cancer; in addition, 8 pleural, 5 ascitic, and 1 pericardial fluid from patients with tuberculosis, liver cirrhosis, and heart insufficiency, were studied. Using mainly direct methods, as well as short-term cell cultures, the chromosome spreads were GTG-banded. Cancerous biopsies showed a plethora of numerical and structural chromosome anomalies and exhibited broad aneuploidy. Chromosomes participating more often in numerical and structural aberrations were 1, 3, 6, 7, 8, 9, 11, 12, and 17. This study provides further cytogenetic evidence for the involvement of these chromosomes in breast and ovarian malignancy.

Static cytometry and neural networks in the discrimination of lower urinary system lesions.

OBJECTIVES: To investigate the potential of morphometry and artificial intelligence tools for the discrimination of benign and malignant lower urinary system lesions. METHODS: The study group included 50 cases of lithiasis, 61 cases of inflammation, 99 cases of benign prostatic hyperplasia, 5 cases of in situ carcinoma, 71 cases of grade I transitional cell carcinoma of the bladder (TCCB), and 184 cases of grade II and grade III TCCB. Images of voided urine smears stained by the Giemsa technique were analyzed by a custom image analysis system. The analysis gave a data set of features from 45,452 cells. A learning vector quantizer (LVQ)-type neural network (NN) was used to discriminate benign from malignant cells on the basis of the extracted morphometric and textural features. The data from 13,636 randomly selected cells were used as a training set and the data from the remaining 31,816 cells made up the test set. Similarly, in an attempt to discriminate at the patient level, 30% of the cases randomly selected were used to train an LVQ NN and the remaining 329 cases were used for the test. RESULTS: The application of the LVQ NN enabled the correct classification of 95.42% of the benign cells and 86.75% of the malignant cells, giving an overall accuracy rate of 90.63%. At the patient level, the LVQ NN enabled the correct classification of 100% of benign cases and 95.6% of malignant cases, giving an overall accuracy rate of 97.57%. CONCLUSIONS: NNs combined with image analysis offer useful information in the discrimination of benign and malignant cells and lesions of the lower urinary system.

Expression of p53, bcl-2 and heat shock protein (hsp72) in malignant and benign ovarian tumours.

The occurrence of p53, bcl-2 and heat shock protein (HSP) expression in ovarian tumours was examined and the correlation was investigated between the expression of these proteins and other disease parameters, including FIGO stage, histological subtype, tumour differentiation and steroid hormone receptor status. We analysed p53, bcl-2 and HSP expression in 100 smears of patients with epithelial ovarian carcinomas, 16 smears of patients with borderline malignancy and 20 smears of patients with benign ovarian neoplasms by using immunocytochemical techniques. There were 29 patients with stage I disease, 24 with stage II disease, 40 with stage III disease and seven with stage IV disease according to the FIGO classification. The sensitivities and specificities of bcl-2, p53 and HSP for malignancy were 53% and 40%, 43% and 80%, and 37% and 90%, respectively. HSP was statistically significantly associated with malignant rather than benign tumours. Significant association was also observed between bcl-2 and p53, and p53 and HSP. The association of HSP with malignant tumours is confined to the premenopausal group of patients and in this group by itself there is also a significant association between p53 and malignancy. HSP and p53 were associated with undifferentiated carcinomas, bcl-2 and p53 expression is reduced as disease stage progresses in serous carcinomas and bcl-2 expression is increased as disease progresses in endometrioid carcinomas. There was no significant association between bcl-2 and ER/PR status. In conclusion, HSP has a high specificity for malignant ovarian tumours, bcl-2 and p53 have only moderate to low sensitivity and specificity. Changes in the frequency of bcl-2 and p53 overexpression between FIGO I and FIGO III stage disease of different ovarian carcinomas indicate a different role of these substances in cellular survival mechanisms in different carcinomas. bcl-2 probably is associated with cell proliferation but not with differentiation.

DNA content and proliferation activity in superficial transitional cell carcinoma of the bladder.

The identification of new prognostic parameters in Superficial Transitional Cell Carcinoma of the Bladder (STCCB) is important since conventional methods are often insufficient for prognostic purposes. We studied the proliferation activity and the DNA ploidy status of 60 pTa and pT1 Superficial Transitional Cell Carcinoma of the Bladder in relation to grade and recurrence rate. The proliferative activity was investigated by measuring the PCNA expression in paraffin embedded tissue sections. The DNA content was studied in Feulgen stained imprints by image analysis technique using a SAMBA 2005 analyser. According to our measurements a statistically significant difference was found in PCNA expression among tumors grade I, grade II, grade III (F = 5.43, p < 0.001), between tumors of the same grade with, and without recurrence (p < 0.001); and between recurrent and non-recurrent tumors (T58 = -6.03, p < 0.001). A statistically significant difference was also observed concerning the DNA-index among grade I, grade II and grade III (F = 4.81, p < 0.01), and between recurrent and non-recurrent tumors concerning the DNA DNA ploidy status (DNA-euploid vs. DNA-aneuploid tumors) (X2 = 24.96, p < 0.001). The recurrence status is also strongly influenced by the proliferation rate and the DNA ploidy status of tumors (X23 = 41.19, p < 0.001). No cases recurrence were found in the group of DNA-euploid tumors with PCNA. expression lower than 30%, in contrast a very high percentage of recurrence in patients with DNA-aneuploid tumors with PCNA expression higher than 30%. Although a small proportion of cases could not be included in me previous categories, STCCB may be classified in to main groups concerning the risk of recurrence. In keeping with this view of proliferation rate and DNA ploidy status could provide useful information, on the potential malignancy of Superficial Transitional Cell Carcinoma of the Bladder. However further studies are required to establish the clinical utility of these parameters.

Moc-31, fibronectin and CEA in the differential diagnosis of malignant effusions: an immunocytochemical study.

In discriminating benign and malignant origins of cytologically suspicious effusion smears a panel of antibodies against carcinoembryonic antigen (CEA), Fibronectin (F) and MOC-31 was used with immuno-cytochemical techniques. One hundred and thirty seven effusions were studied of which 107 had a malignant and 30 a benign aetiology as determined by clinical and histological examination. Cytologically 24 were diagnosed as benign, 97 as malignant and 14 as suspicious. Staining for F was positive in all effusions of benign and 3 of malignant origin. MOC-31 was positive in 95 (88.8%) of effusions of malignant origin but none of benign origin. Positive CEA was observed in 43% of effusions of malignant origin and in 10 of benign origin. The combination of MOC-31 positivity measured the sensitivity and specificity of the cytological examination in cases where the cytological examination result was suspicious as did F positivity improve the sensitivity for a benign origin of the effusion. Positivity or negativity for CEA is less valuable than the other parameters.

Cathepsin D immunoreactivity in ovarian cancer: correlation with prognostic factors.

In view of the somewhat inconclusive nature of the reports of the role of Cathepsin D (Cath D) in ovarian carcinoma and its relationship with various other parameters of malignancy the present study was performed to aid in the further clarification of this role. One hundred freshly resected primary ovarian carcinomas of various histological types were studied for ER, PR and Cath D status and the results examined with respect to menopausal status, histology, size and lymph node invasion. In our series Cath D positivity was more frequent in serous than in other types of ovarian cancer but this Cath D positivity was not related to the frequency of lymph node invasion regardless of the size of the tumor. Nor was any association observed between Cath D positivity and ER or PR status of the tumors or the menopausal state of the patients. The reported prognostic value of Cath D, ER and PR is discussed as well as the distinction between tumor invasion by lymphatic channels and direct interstitial infiltration. It was concluded that Cath D may not play a role in the former mode but, as might be expected from its proteolytic properties, in local spread by means of tissue destruction.

An immunohistochemical study of normal human neonate and adult parotid gland tissue. Detection of lysozyme, lactoferrin, a1-antichymotrypsin, a1-antitrypsin and carcinoembryonic antigen.

The immunohistochemical detection and distribution of lysozyme (Ly), Lactoferrin (Lf), a1-Antichymotrypsin (a1-AChy), a1-Antitrypsin (a1-AT) and Carcinoembryonic antigen (CEA) were studied in neonate and adult parotid gland tissue, using the peroxidase-antiperoxidase (PAP) method. Ly stain in neonates extended into acini, intercalated ducts and occasional cells of large ducts, whereas in adults Ly was usually confined to the intercalated ducts. The distribution pattern of Lf in neonates varied considerably between individual glands showing three staining patterns. Most of the intercalated ducts, some groups of acini and rare striated duct cells were positive for Lf in adults. a1-AChy and a1-AT in neonates were positive mainly in the large ducts, whereas staining for a1-AChy and a1-AT in adults frequently extended into some intercalated duct cells, although less intensively. Finally, CEA in neonates was localized in the lumina and luminal membranes of the acini, in intercalated ducts, and less frequently in the large ducts. In adults CEA was present predominantly in the lumina and luminal membranes of the intercalated duct cells. These differences may suggest an immunohistochemical postnatal differentiation of the parotid gland.

Diagnostic approach of effusion cytology using computerized image analysis.

The objective of this study is to investigate whether image cytometry is a sensitive and specific method for the differential diagnosis of equivocal cells in routine cytology of effusion smears. One hundred four effusion smears were studied from routine cytologic material. Cytologically 56 (53.8%) of the smears were classified as malignant, 26 (24%) as suspicious and 22 (21.1%) as benign. Two morphometric variables (nuclear major axis length and nuclear area) of the nuclei were measured by an image analysis system. Higher values for the area were found for malignant rather than benign and suspicious cells (p < 0.0005 and p < 0.005 respectively). The same result was extracted for the nuclear major axis length values (p < 0.0005 and p < 0.0005 respectively). Values of nuclear major axis length and nuclear area didn't differ significantly between benign and suspicious cells (p = 0.071 and p = 0.066 respectively). The results show that the range of the values for suspicious cells is closer to the range of the benign cells. Cytomorphometry of the effusion smear cells may provide important information for the differentiation of atypical mesothelial cells from malignant adenocarcinoma cells.

Telomerase expression as a marker in prostate cancer: correlation to clinicopathologic predictors.

The aim of this study was to investigate by an in situ hybridization procedure the Telomerase expression as a marker in prostate cancer and to correlate these results with several prognostic factors concerning this cancer. Imprint smear samples were obtained from 70 prostates removed from patients who underwent radical prostatectomy for prostate adenocarcinoma. Telomerase expression in cancerous prostate smears was studied using an in situ hybridization procedure. The results were correlated with prognostic factors such as pathologic staging, Gleason grading, PSA serum levels and tumour differentiation. Positive Telomerase expression was detected in 88.6% prostate cancer smears. Telomerase expression was significantly correlated with the Gleason score (p < 0.001), tumour differentiation (p < 0.001) and PSA serum levels (p = 0.002). The distribution of Telomerase expression according to histopathological staging was not statistically significant (p < 0.56). In conclusion Telomerase expression could be a marker indicating the malignant potential of prostate cancer.

Correlations between nuclear/cytoplasmic area ratio and classification of cervical smears.

The aim of this study was to assess the nuclear/cytoplasmic (N/C) ratio using computerized image analysis of cervical smears with intraepithelial neoplasia (CIN) grade I to III associated or not with cellular changes of human papillomavirus (HPV) in an attempt to determine if this method is more sensitive for the estimation of the grade of CIN. One hundred and ten cervical smears from women with a mean age 35.03 years were studied. The cytological diagnosis was as follows: CIN I + HPV (11), CIN II + HPV (11), CIN II + HPV (8), CIN I (7), CIN II (6), CIN III (8), Ca (22), HPV (32), CIN I-II + HPV (2) and CIN II-III + HPV (3). All cases were histologically examined: 93 cases were in agreement and 17 were under- or overestimated cytologically. The morphometric study of cervical smears was carried out by image analysis. Data were analysed by one way analysis of variance followed by Bonferroni test of multiple comparisons. Statistically significant differences were detected between the three grades of CIN or CIN HPV or only HPV (p<0.0001). The results demonstrated that the N/C ratio measured by image analysis on precancerous lesions of cervical smears could be considered as an additional tool for the classification of cervical smears, especially in determining the discrepancies between cytological and histological diagnoses.

Immunodetection of neuron-specific enolase and keratin in cytological preparations as an aid in the differential diagnosis of lung cancer.

The results of neuron-specific enolase (NSE) and keratin immunodetection in cytological specimens of sputum secured from 41 patients with lung cancer are presented. All 19 cases of small-cell carcinoma showed intense immunoreactivity for NSE. No such immunoreactivity was found in 21 of 22 cases of non-small-cell carcinoma. The single positive result was from a case of large-cell undifferentiated carcinoma. All 10 cases of squamous-cell carcinoma showed immunoreactivity for keratin. The 19 cases of small-cell carcinoma showed no such reactivity. Our findings indicate that immunostaining for NSE and keratin is a valuable aid when a definite diagnosis of small-cell carcinoma of the lung can not be made on the basis of conventional cytologic features.

p53 and bcl-2 protein expression in non-small-cell lung carcinoma.

The diagnostic significance of p53 and bcl-2 proteins in epithelial non-small-cell lung cancers was examined, and the relationship between these proteins expression and other disease parameters, including stage of the disease and tumor differentiation, were studied. We analyzed p53 and bcl-2 proteins expression in 60 imprint smears of freshly resected lung tumors (37 squamous and 23 adenocarcinomas) using the immunocytochemical technique. There were seven patients with stage I disease, 24 with stage II, 23 with stage IIIa, and six with stage IIIb disease, according to the International Staging System classification. Sixteen of the tumors were bcl-2 positive and 25 were p53 positive. Twenty tumors were negative for both bcl-2 and p53 (33.3%). Statistical analysis showed no association between the incidence of p53 or bcl-2 positivity. Adenocarcinoma or squamous carcinoma analysis showed significant associations between p53 positivity and poor differentiation and advanced disease stage as well as bcl-2 and early disease stage and well-differentiated tumors. There was also an association between the stage of the disease and the degree of differentiation of the tumors. In conclusion, bcl-2 positivity must be considered a good prognostic sign. On the other hand, p53 positivity seems to indicate, even in tumors at a relatively early stage, that a serious aggressive tumor which will not be easily eradicated is present.

Prognostic significance of p53, bcl-2 and EGFR in carcinoma of the endometrium.

OBJECTIVE: To evaluate the part played by several parameters in the prognosis of patients with endometrial carcinoma. STUDY DESIGN: Eighty imprint smears from fresh endometrial tumor specimens were studied immunocytochemically for the expression of p53, bcl-2 and epidermal growth factor receptor. Also, the presence of estrogen receptor (ER) and progesterone receptor (PR) in the tumor tissue was measured. The data obtained were related to survival, and associations were sought between the parameters studied. RESULTS: Strong associations were found between advanced stage, high grade, lymph node metastases at diagnosis, nonendometrioid histology and p53 expression with poor survival. Bcl-2 expression was associated with good five-year survival. ER expression was associated marginally with good five-year survival, but PR expression was not. A strong association was found between p53 and advanced disease, stage and lymph node metastases at diagnosis. An association between EGFR positivity and survival was not found. CONCLUSION: p53 Expression of uterine tumors is an independent and strong indicator of poor prognosis. Even patients with stage I and II disease at surgery who have p53-positive tumors must be considered at high risk.

Expression of pS2 protein and estrogen and progesterone receptor status in breast cancer.

OBJECTIVE: To investigate pS2 protein expression and estrogen (ER) and progesterone receptor (PR) status of imprints from surgical biopsies of breast cancer cases in relation to the histologic grade of malignancy. STUDY DESIGN: The study group consisted of 50 cases of primary breast carcinomas. For the demonstration of pS2 protein expression an immunocyto-chemical avidin-biotin complex technique was applied. Monoclonal antibody pS2 was used as the primary antibody, diaminobenzidine as the chromogene and hematoxylin as the counterstain. For the evaluation of ERs and PRs, a biochemical method was applied. RESULTS: Sixty-two percent of breast cancer cases showed positive expression of pS2. Of the 31 pS2+ cases, 74% had positive ERs and PRs. A statistically significant difference was observed between pS2 protein expression, ER+, PR+ and histologic grade of malignancy (P < .001). CONCLUSION: Two groups of breast cancer cases can be distinguished: one group that is ER+, PR+ and pS2+, with low malignancy potential, and another group that is ER-, PR- and pS2-, with high malignancy potential.

P53 protein expression and DNA ploidy in common epithelial tumors of the ovary.

OBJECTIVE: To investigate p53 protein expression and DNA content in imprints from surgical biopsies of common epithelial tumors of the ovary. STUDY DESIGN: The study was based on 60 cases of epithelial tumors of the ovary (15 benign, 3 border-line and 42 malignant). For the demonstration of p53 protein, immunocytochemical staining with the avidin-extravidin technique was performed using monoclonal antibody p53 DO-7. DNA content was measured by image cytometry after Feulgen staining. RESULTS: There was a strong correlation between p53 expression and aneuploidy, with the difference between diploid and aneuploid tumors statistically significant (P < .001). A correlation was found between DNA ploidy, histologic grade and clinical stage (P < .001 and P < .05), respectively. There was no correlation between DNA ploidy and histologic type (P = .89). No correlation was observed between p53 protein expression and grade or clinical stage of the tumors. Nevertheless, a correlation of p53 expression between early (I, II) and advanced stages (III, IV) (P < .05) was observed. All benign and borderline tumors were diploid and did not express p53 protein. CONCLUSION: The results of the present study and the data in the literature stress the value of p53 expression and DNA ploidy in assessing the malignant potential of common epithelial ovarian cancers. However, the clinical application of these data requires further study.

p53 protein expression in benign and malignant breast lesions.

OBJECTIVE: To investigate p53 protein expression in imprints from benign and ductal breast carcinoma cases in relation to the histologic grade of malignancy and clinical stage. STUDY DESIGN: The study group consisted of 60 cases of primary ductal breast carcinomas and 20 benign lesions. For the demonstration of p53 protein expression, an immunocytochemical avidin-extravidin complex technique was applied. Monoclonal antibody p53 was used as the primary antibody, diaminobenzidine as the chromogen and hematoxylin as the counterstain. RESULTS: Forty-five percent of breast cancer cases showed positive expression of p53. A statistically significant difference in p53 protein expression was observed between grade 1, 2 and 3 carcinomas and stage I, II and III cases. All benign lesions were negative for p53 protein expression. CONCLUSION: Immunocytochemical p53 protein expression in cytologic material is a simple method that can be applied in routine cytologic laboratories for the identification of genetic alterations in primary ductal breast cancer.

Additional slides from residual ThinPrep Pap tests: Of potential diagnostic benefit in equivocal cases?

The aim of this study was to further evaluate the diagnostic significance of additional slides prepared from residual ThinPrep (TP) Pap Tests. Up to 10 repeat slides were prepared from 105 residual TP cervical samples. All additional slides were evaluated for the presence of diagnostic elements which were not found on the primary TP slide. After the evaluation of the repeat slides, an upgraded diagnosis was noted in 15 cases (14.3%). The reclassified cases included: three negative cases reclassified as two ASC-US and as one LSIL, seven cases of ASC-US reclassified as six LSIL and as one HSIL, and five cases of LSIL reclassified as HSIL. The highest rate (7/15 cases, 46.7%) of cases with an upgraded diagnosis was noted in the ASC-US diagnostic category. Our results suggest that repeat processing of residual TP cervical samples may represent an adjunctive diagnostic tool for a more accurate classification of ASC-US cases. Nevertheless, the practical value of this approach seems to be limited by its significant cost and its uncertain effectiveness.