Cytodiagnosis of endometrial carcinoma and hyperplasia on imprint smears with additional immunocytochemistry using Ki-67 and p53 biomarkers.

OBJECTIVE: It is said to be difficult to interpret the different endometrial lesions by cytomorphology; however, evaluation of the microarchitecture of the cell clumps and application of immunocytochemistry can improve diagnostic accuracy. The aim of the present study was to evaluate cytolomorphological features and correlate them with the histological diagnosis of benign and malignant endometrial lesions, and to investigate certain immunocytochemical biomarkers to achieve a more accurate cytodiagnosis. METHODS: In the present study, we graded the cytomorphology on imprint smears of 35 low-grade endometrial endometrioid carcinomas compared with 23 cases of endometrium ranging from disordered proliferative to benign hyperplastic. Additionally, 10 cases of high-grade endometrial carcinoma and 11 cases of atrophic endometrium were evaluated. Ki-67 and p53 biomarkers were applied to the cytological smears. RESULTS: A total cytological score less than six, resulting from nuclear overlapping, nuclear/cytoplasmic ratio, the presence of a branched pattern, vesicular cytoplasm and loss of cohesiveness, distinguished all the cases of disordered proliferative and benign hyperplastic endometrium from low-grade endometrioid carcinomas of endometrium (P < 0.0001). The application of different cut-off values for Ki-67 and p53 helped differentiate certain endometrial lesions in our study. The integration of the immunocytochemical score of Ki-67 and p53 into the cytological score resulted in a final score that was also diagnostically useful. CONCLUSIONS: The results of the present study demonstrated that evaluation of certain cytological features along with specific immunocytochemical findings could improve the accuracy of endometrial cytodiagnosis but our findings need to be tested in a routine clinical situation, using pre-operative cytological samples, to ascertain whether the diagnostic criteria are reproducible.

Prognostic value of EZH2, paxillin expression and DNA ploidy of breast adenocarcinoma: correlation to pathologic predictors.

PURPOSE: The objective of this study was to examine the association of EZH2 and paxillin expression and DNA ploidy status with pathological parameters of breast cancer, aiming to correlate tumor phenotype with its malignant behavior. METHODS: EZH2 and paxillin expression and DNA ploidy were evaluated in imprint smear samples obtained from 105 breast tumors after surgical removal. RESULTS: Increased expression of paxillin was associated with p53 expression (p=0.005), Ki-67 expression (p=0.018) and EZH2 expression (p<0.0001). EZH2 expression correlated with estrogen receptor (ER) and progesterone receptor (PR) status (p=0.01 and p=0.035, respectively), and expression of p53 and Ki-67 (p=0.007 and p<0.0001, respectively). Aneuploid tumors were significantly correlated with poor differentiation (p=0.000), stage of disease (p=0.000), size of the primary tumor (p=0.015), presence of nodal metastasis (p=0.001), ER status (p=0.008), cerbB2 status (p=0.012), and expression of Ki-67 (p=0.001) and EGFR (p=0.018). Multivariate analysis of ploidy results using paxillin and EZH2 expression as dependent variables revealed that aneuploid tumors were associated with disease stage and grade of differentiation, cerbB2 expression and EZH2 expression. CONCLUSION: Our results show that aneuploid tumors, EZH2 expression and paxillin expression correlate with more aggressive phenotype of breast cancer.

Expression of Smad4, E-cadherin and beta-catenin in advanced colorectal cancer: a retrospective study.

PURPOSE: To correlate the expression of E-cadherin and beta-catenin with alterations of expression of Smad4 in advanced colorectal cancer (CRC). METHODS: Tissue specimens from 75 colorectal cancer cases (Dukes stage C and D) were tested for Smad4, E-cadherin and beta-catenin by the Avidin-Biotin immunoperoxidase method. The results were correlated with patients' clinicopathological parameters. RESULTS: Smad4 expression was lost or reduced in roughly 1 out of every 3 Dukes C and D CRCs. Association of Smad4 expression with other clinicopathological parameters was not noted. Association of expression of E-cadherin with other clinicopathological parameters was not noted, apart from tumor location. Expression of beta-catenin was not associated with clinicopathological parameters. Lack of expression of Smad4 was associated with lack of expression of both E-cadherin (<0.000) and beta-catenin (p<0.000). As regards the relation between E-cadherin and beta-catenin, the expression of each seemed to parallel the expression of the other (p<0.000). Beta-catenin was overexpressed in 68.5% of the specimens studied. CONCLUSION: Clinically advanced CRC is associated with a reduced or complete lack of expression of Smad4. Ecadherin and beta-catenin are expressed in parallel with each other and also with Smad4. This tumor suppressor role of Smad4 by affecting both E-cadherin and beta-catenin may indicate a novel pathway for metastatic tumor via cellular reshaping. The precise underlined mechanism(s) and the clinical significance of these findings remain to be determined.

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.

The value of TOP2A, EZH2 and paxillin expression as markers of aggressive breast cancer: relationship with other prognostic factors.

INTRODUCTION: The immunocytochemical expression of topoisomerase II alpha (TOP2A), enhancer of zeste homologue 2 (EZH2) and paxillin has recently gained increasing attention. Although previous studies have commented on the clinical usefulness of these markers, their role remains controversial. AIM: The purpose of the study was to investigate the expression of TOP2A, EZH2 and paxillin in relation to classic prognostic parameters and their significance as prognostic markers in imprints of resected breast carcinomas. METHODS: Imprint smears from 55 patients who underwent surgical treatment for primary carcinoma in our department between 2005 and 2006 were studied immunocytochemically with the use of TOP2A, EZH2 and paxillin antibodies. RESULTS: The expression of TOP2A correlated with higher histologic grade, tumor size and negative PR expression. High intensity staining for EZH2 expression was associated with higher histologic grade, negative ER and PR expression and positive Ki-67 expression. The expression of paxillin showed no correlation with estrogen/progesterone and HER2 expression nor with tumor grade and stage. CONCLUSION: Our data indicate that TOP2A and EZH2 expression are related to a more aggressive tumor phenotype. The expression of paxillin failed to correlate with any of the studied clinicopathologic factors. Further studies are needed to verify these results.

Differential rates of proliferation and apoptosis in nasal polyps correspond to alterations in DNA spatial distribution and nuclear polarization as observed by confocal microscopy.

The study aimed to investigate the expression of p53, Bcl-2 and Ki-67 in relation to the histologic and nuclear qualitative and spatial characteristics of chronic rhinosinusitis with polyposis (CRP). Imprint smears obtained from surgically removed nasal polyps of 20 patients were studied. The polyps were classified according to their histological characteristics as: hyperplasia (simple and pronounced) and squamous metaplasia. The expression of p53, Bcl-2 and Ki-67 was assessed by immunocytochemistry. DNA spatial distribution and nuclear orientation were studied by staining with propidium iodide and examined by confocal microscopy. Positive immunoreaction for p53, Ki-67 and Bcl-2 was observed in 50, 65, and 50% of polyp's smears, respectively. For each diagnosis, the rates were simple hyperplasia 60, 80 and 30%, pronounced hyperplasia 80, 100 and 40%, metaplasia 0, 0 and 100%, respectively. Abnormal chromatin distribution and nuclear disorientation was observed in three cases of pronounced hyperplasia combined with positive immunoreaction for Ki-67 and p53 and negative immunoreaction for Bcl-2. CRP demonstrated different proliferation and apoptotic rates, according to their histology. Nuclear characteristics observed by confocal microscopy are associated with the immunocytochemical markers of proliferation and apoptosis.

Imprint cytology of vacuum-assisted breast biopsy specimens: a rapid diagnostic tool in non-palpable solid lesions.

OBJECTIVE: Imprint cytology provides a rapid preliminary diagnosis shortly after the completion of breast biopsy. This study aims to assess the validity of imprint cytology for the pre-operative diagnosis of non-palpable mammographic solid lesions excised by vacuum-assisted breast biopsy (VABB). METHODS: Seventy-two women with non-palpable Breast Imaging Reporting and Data System 3 and 4 mammographic solid lesions without microcalcifications underwent VABB on the stereotactic Fischer's table with 11-G Mammotome vacuum probes. Imprint samples were examined (Diff-Quick stain, modified Papanicolaou stain and May-Grünwald-Giemsa). The cores were dipped into a CytoRich Red Collection fluid for a few seconds in order to obtain samples with the use of the specimen wash. After the completion of cytological procedures, the core was prepared for routine pathological study. The pathologist was blind to the preliminary cytological results. The cytological and pathological diagnoses were comparatively evaluated. RESULTS: The sensitivity of the cytological imprints for cancer was 90%. The specificity of the method for cancer diagnosis was 100%. Two precursor lesions were present in the material: one case of atypical ductal hyperplasia, which was successfully detected, and one case of lobular neoplasia, which escaped detection. The cytological imprints were inadequate in four out of 72 cases (5.6%), but none of them were included within the malignant subgroup. CONCLUSIONS: Imprint cytology seems to be an important adjunctive tool in the management of patients with non-palpable mammographic solid lesions. Its very satisfactory sensitivity and optimal specificity could establish its use in general clinical practice.

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.

Immunocytochemical detection of P-glycoprotein in the management of malignant effusions.

P-glycoprotein (P-gp), a cell membrane protein, has been found in multidrug-resistant cancer cells. A total of 104 smears from patients with breast-cancer-associated pleural effusions and ovarian-cancer-related peritoneal effusions were studied for P-gp with the antibody C-219 and the avidin-biotin-immunoperoxidase method. Samples were taken before and 3 and 7 days after intracavitary bleomycin therapy and reaccumulation of effusion was assessed at 30 days. Smears that were P-gp-negative by the 7th day were associated with a good 30-day response to bleomycin in the majority of cases, while P-gp-positive smears were associated with a significant reaccumulation of fluid at 30 days. P-gp status is a valuable prognostic indicator of response to intracavitary bleomycin treatment in effusions from breast or ovarian 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.

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.

An immunocytochemical study of ras and myc oncoproteins and EGFr expression in pleural effusion smears.

Pleural effusion smears from 112 patients with either benign or malignant lung disease were investigated for the expression of EGFr and the oncogene proteins myc p64 and ras p21. The streptavidin-biotin peroxidase technique was used. In the studied malignant group of effusions both EGFr and ras have greater sensitivity in the detection of a malignant process than does routine cytological examination though EGFr was less specific. The combination of positive cytology and 3 positive markers is highly specific for a malignant process (90%). Myc and ras had a 100% sensitivity in squamous cell carcinomas but an overall specificity of only 67.3% and 66.6% respectively. The differences in myc and ms positivity, between squamous cell and adenocarcinoma effusion smears were highly significant (p <0.005). All effusion smears associated with undifferentiated carcinomas were ras positive and 2 of them were myc and EGFr positive.

An immunohistochemical analysis of angiogenesis in invasive breast cancer with correlations to clinicopathologic predictors.

BACKGROUND: There is evidence that angiogenesis plays an important role in the biologic aggressiveness of breast carcinomas and might be used as a prognostic marker. MATERIALS AND METHODS: In a series of 140 invasive mammary carcinomas, microvessels were highlighted immunohistochemically using two endothelial markers, factor VIII-related antigen (FVIIIRA) and CD31. Cases were divided into high and low microvessel density groups according to the highest number of microvessels found in each tumour's most vessel-dense part. The data was statistically analysed with regard to classic clinicopathologic prognosticators (i.e., histologic type and grade, nuclear grade, tumour size, stage, lymph node status and steroid receptor immunoexpression) by univariate and multivariate analysis. RESULTS: Both markers' counts displayed just a weak skewness. Interestingly, CD31 angiogenesis grade was not influenced by any of the prognostic indicators assessed. FVIIIRA immunoreactivity was significantly affected only by nuclear grade (p = 0.041) in logistic regression analysis. Infiltrating lobular carcinomas frequently demonstrated higher FVIIIRA-positive microvessel densities than ductal invasive carcinomas, at least in the subgroup of patients with absence of nodal metastases and in those patients with highly oestrogen-dependent tumours. CONCLUSIONS: The lack of relation between angiogenesis and either disease stage or lymph node metastasis indicates that this process may be necessary, but not sufficient alone for breast cancer spread.

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.

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.

Immunocytochemical localization of Cathepsin D and CA 125 in ovarian cancer.

OBJECTIVE: To study the expression of Cathepsin D (Cath D) and CA 125 antigens and ER and PR receptors on freshly obtained surgical specimens of ovarian carcinomas and their relationship with menopausal status, tumor histology, primary tumor size and lymph node invasion. METHODS: The tumors obtained from 100 women were measured and cut in half. The cut surface of one half was pressed against glass slides which were air dried and stained using the Avidin-Biotin peroxidase method for Cath D and CA 125 antigens. The slides were viewed under the light microscope for the characteristic brown granules in the cytoplasm or membrane of the malignant cells. The other half of the tumor was subjected to routine histological examination and part used for the demonstration of ER and PR receptors. The results were analyzed using chi 2 analysis. RESULTS: Cath D positivity was as common as CA 125 positivity. Cath D positivity is more frequently associated with serous carcinomas than with others. No relationship was observed between ER/PR positivity and Cath D or CA 125 positivity. CONCLUSION: The high incidence of Cath D positivity makes it a possible complementary method for following up ovarian carcinoma patients especially those who are CA 125 negative.

Antigen expression of alveolar macrophages in smokers and patients with lung diseases.

Alveolar macrophage function was studied immunocytochemically using three monoclonal antibodies--macrophage CD 68 KP 1 (M), protein CD 11C (P), and anti-elastin (EL)--and three polyclonal antibodies--lysozyme (LZ), alpha-1-antitrypsin (AAT), and alpha-1-antichymotrypsin (AACT). The material for study was smears obtained from bronchial washings from 15 healthy persons and 60 patients with respiratory infections or primary or secondary malignant lung infiltration. Eight of the healthy group and 40 of the patient group were smokers (SM). The percentage of cells obtained from the washings which were macrophages was also measured. The intensity of staining reactions for each of the six antigens was noted and in general more intense staining was noted in smokers than in non-smokers. More intense staining was observed in patients with pulmonary infections (group II PI) and metastatic pulmonary infiltrations (group IV MP Ca) than in controls (group IC), while patients with primary lung cancer (group III PP Ca) had highly reduced staining reactions. The number of macrophages was similarly increased in all groups in comparison with the IC group for non-smokers and in all groups except III PP Ca for smokers. It is concluded that smoking, pulmonary infections, and metastatic infiltration of the lung are associated with an increase in the number and activity of alveolar macrophages, while patients with primary lung cancer have an increase in the number of macrophages which are functionally incompetent.