Metastasis is specific for malignant tumors and its control is one of the most important problems in the design of therapies for cancer patients. Loss or reduction of E-cadherin expression and/or beta-catenin expression plays a casual role in tumor progression and metastasis and is associated with poor prognosis.The purpose of the study is to investigate the expression of E-cadherin and beta-catenin and their significance as independent prognostic markers in imprints of resected nonsmall cell lung cancer (NSCLC).Imprint smears from 70 patients who underwent surgical lung resection for primary carcinoma were studied. As control group was used imprints of physiological tissues.Histologically 47 (67.1%) of the tumors were squamous cell carcinomas and 23 (32.9%) were adenocarcinomas. Tumors stage was I in 29 (41.4%), II in 13 (18.6%), III in 24 (34.3%) and IV in 4 (5.7%).Positive expression for E-cadherin was observed in 44.29% of malignant smears vs 85.71% for control group (P = 0.011). For beta-catenin, positive expression was observed in 42.86% malignant cases vs 85.71% for control group (P = 0.008). Positive expression of E-cadherin and beta-catenin was observed in moderate and well differentiated tumors (P < 0.0001 for both respectively). Positive E-cadherin and beta-catenin expression was observed in 70.6% and 76.5% of the cases with negative lymph-node metastasis (P < 0.0001 for both respectively). There was no statistically significant association between histological type, tumor stage, pleural invasion, tumor size (P > 0.05 for all) and E-cadherin/beta-catenin expression.Reduced E-cadherin or beta-catenin negative expression relates to dedifferentiation and progression of NSCLC. Diagn. Cytopathol. 2010;38:419-424. (C) 2010 Wiley-Liss, Inc.

Apoptosis induced in primary cultures of rat hepatocytes by transforming growth factor β1(TGF-β1) was greatly attenuated by inhibitors (5 μM) of calpain I and calpain II, respectively. Both inhibitors prevented the TGF-β-elicited increase of nucleosomal DNA fragments and the occurrence of DNA-breaks in the TUNEL reaction. The detrimental effect of TGF-β on cell viability measured by the WST-1 test was strongly reduced by calpain inhibitors. Calpain II > I inhibitors suppressed spontaneous DNA cleavage in hepatocytes during culture and prevented the appearance of immunocytochemically visible TGF-β (APAAP staining), which occurs in untreated parenchymal cell cultures. The data show that inactivation of calpains attenuates both the TGF-β-elicited and the spontaneous apoptosis of cultured hepatocytes; the latter effect is likely due to the suppression of endogenous TGF-β activation. It is suggested that calpains participate in these processes.

We investigated the prognostic significance of cyclooxygenase-2 (COX-2) and survivin in ovarian carcinoma. Imprint smears were obtained from 100 ovarian carcinoma specimens and were studied immunocytochemically for the expression of COX-2 and survivin. The results were correlated with several clinicopathological parameters, including 5-year survival. Increased COX-2 staining pattern correlated with a non-mucinous histological type (p=0.008), increased stage (p<0.0001), high histological grade (p<0.0001), and reduced survival rates (p<0.00001). Survivin expression was strongly associated with increased stage (p<0.0001), increased histological grade (p<0.0001), and reduced survival (p<0.00001). Elevated survivin expression also correlated significantly with pre-menopausal status (p=0.033). In addition, COX-2 and survivin staining patterns correlated strongly with one another (p<0.0001). However, on multivariate analysis, an independent prognostic value was found only for tumor stage and grade. The findings of our study indicate that the increased expression of COX-2 and survivin in ovarian cancer is associated with one another and with several adverse clinicopathologic parameters, including reduced survival, thus suggesting a role of these molecules in disease progression. Further investigations of the exact prognostic and therapeutic implications of COX-2 and survivin expression are strongly warranted.

Aims and background: Metallothioneins are a family of metal-binding cysteine-rich proteins that play an important role in cellular processes such as proliferation and apoptosis, protection against oxidative stress and metal ion homeostasis and detoxification. Recent findings suggest that metallothioneins might play a significant role in the development and progression of prostate cancer. It has been also demonstrated that Ki-67 expression may have prognostic value for disease-free survival in cases of prostate carcinoma.Study design: Imprint smears samples obtained from 70 patients immediately after radical prostatectomy for prostatic carcinoma were immunostained with monoclonal antibodies against metallothioneins and Ki-67. Metallothionein expression was correlated with Ki-67 immunostaining, Gleason score, stage, preoperative prostate-specific antigen levels and biochemical recurrence.Results: Metallothionein expression was shown to correlate strongly with Gleason score (P < 0.001) and significantly with pathological staging and Ki-67 immunostaining (P < 0.001, P < 0.05, respectively). In contrast, no significant association between metallothioneins and preoperative PSA was demonstrated. Both of the studied markers (metallothioneins and Ki-67) correlated with recurrence (P = 0.009, P = 0.006, respectively).Conclusions: The present findings support the independent predictive value of metallothioneins and Ki-67 in prostate cancer. However, additional data are needed in order to reveal the factors that influence the expression of metallothioneins in epithelial neoplastic cells and clarify their mechanism of action.

Mutations of the PTEN, p53, and beta-catenin genes are the most frequent molecular defects in endometrial carcinomas. The aim of this study was to investigate their prognostic significance in this form of cancer. Imprint smears were obtained from 80 fresh endometrial tumor specimens and studied immunocytochemically for the expression of PTEN, p53, and beta-catenin proteins. The staining pattern was correlated with several well-established prognostic parameters, including 5-year survival. Positive staining of p53 was significantly correlated with increased stage (P < 0.0001), lymph node metastases (P = 0.001), and a nonendometrioid histology (P = 0.001). On the contrary, positive beta-catenin expression was significantly associated with decreased stage (P = 0.002), decreased grade (P = 0.007), and a negative lymph node status (P = 0.023). PTEN positivity was correlated with decreased stage (P = 0.002) and negative lymph nodes (P = 0.008). All the three markers affected survival significantly in univariate analysis but only beta-catenin had an independent prognostic impact. An independent prognostic significance was also shown for PTEN in the stage I subgroup of patients. The results of our study indicate that loss of beta-catenin expression is a strong and independent predictor of an unfavorable outcome in patients with endometrial carcinoma. Loss of PTEN may also be associated with a worse prognosis in patients with early-stage disease.

DEAF-1 is a transcription factor that maps to human chromosomal region 11q15-5 a region that undergoes loss of heterozygozity (LOH) in prostate tumours. The aim of this study was to investigate the expression of DEAF-1 protein in prostate carcinoma cell smears in relationship with clinicopathological parameters.

Hypoxia-inducible factor-1a (HIF-1a) is a transcription factor that plays a major role in contributing to cancer progression. Few reports have identified the existence of single nucleotide polymorphisms in exon 12 of the HIF-1a gene in some carcinomas and in hormone refractory prostate carcinomas. The aim of this study was to investigate the HIF-1a protein expression in the prostates of men with benign hyperplasia (BPH) or carcinomas (Pca) in order to evaluate the malignant potential role of these diseases.

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.

The cell proliferation markers p120, Ki-67 and proliferating cell nuclear antigen (PCNA) recognize nuclear antigens. The expression of these proteins by immunostaining methods was reported to be of value in determining the prognosis of patients with malignant diseases.In this study, we evaluated the prognostic significance of the expression of nuclear antigens p120, PCNA and Ki-67 in prostate cancer and compared the results with other prognostic factors.Imprint smear samples obtained from 70 patients immediately after radical prostatectomy for prostatic carcinoma were immunostained with monoclonal antibodies against p120, Ki-67 and PCNA. The immunostaining results were correlated with Gleason score, tumour differentiation, stage and prostatic specific antigen (PSA) levels.Our findings demonstrate that p120, Ki-67 and PCNA expression in prostatic carcinoma smears, correlated significantly with the degree of Gleason score (P < 0.001). When combining p120, Ki-67 and PCNA positivity with tumour differentiation there was a significant association among these parameters (P < 0.001). Overexpression of p120, Ki-67 and PCNA, was also associated with increased PSA serum levels (>4 ng/ml) (P < 0.001). The distribution of p120, Ki-67 and PCNA expression in prostate carcinomas was not statistically significant for Ki-67 (P = 0.69) and p120 (P = 0.22) but was significant for PCNA (P < 0.001) as far as the histological stage (T2a, T2b, T2c, T3a). P120, Ki-67 and PCNA expression had significant prognostic value for disease-free survival.Our results conclude that nuclear antigens p120, Ki-67 and PCNA appear to be additional markers in the field of prognosis of prostatic carcinoma.

WD repeat domain 1 (WDR1), a protein that assists cofilin-mediated actin filament disassembly, is overexpressed in the invading front of invasive ductal carcinoma (IDC), but its implication of overexpression and how to be regulated have not been studied. In our study, we demonstrated that STAT3 bound to the 5′ upstream sequence (− 1971 to − 1964), a putative promoter region, of WDR1 gene, and its activation induced WDR1 overexpression in breast cancer cells. The exogenous overexpression of WDR1 increased the migration of MDA-MB-231, which was attenuated by WDR1 knockdown. In the analysis of breast cancer patients, WDR1 overexpression was associated with a shorter distant metastasis-free survival (DMFS), more specifically in basal-like tumors.

The testicular cystic structure and the abrupt morphological changes that the fish testis undergoes during the reproductive cycle (RC) make it an interesting model for studying the regulation of spermatogenesis, in particular the role of matrix metalloproteinases (Mmps). The gilthead seabream is a seasonal breeding teleost whose testis undergoes drastic remodeling events, especially during the post-spawning stage when a massive infiltration of a immune cell type, the acidophilic granulocytes, occurred. Bearing this in mind, we studied the gilthead seabream testis gelatinolytic activities involved in migration and tissue remodeling and its regulation by endocrine, immune and tissue stimuli. Thus, we demonstrated that the germinal epithelium of the testis showed gelatinolytic activity during spermatogenesis and post-spawning but not during resting, when only scarce interstitial cells were stained. Moreover, the precursor and mature forms of two gelatinases, Mmp2- and Mmp9-like, were active in the gonad, whose activities were up-regulated by 17β-estradiol (E2) but not by lipopolysaccharide (LPS)/bacterial DNA (VaDNA) in testicular cell suspensions. E2 and LPS/VaDNA also up-regulated a variety of cytokines and chemokines. We also cloned mmp9, mmp13, tissue inhibitors of Mmps (timp)-2a and timp2b genes and found that all of them were expressed in the gonad in a RC stage-dependent manner. Interestingly, mmps and timps were highly expressed by the testicular acidophilic granulocytes. Moreover, in these cells, the gelatinolytic activity seemed to correspond to the precursor and mature forms of putative Mmp2 and Mmp9 gelatinases, while the main gelatinolytic activity seemed to correspond to the mature form of Mmp2 in head–kidney acidophilic granulocytes. Finally, although none of the stimuli used were able to induce the gelatinolytic activity of Mmp9-like in head–kidney acidophilic granulocytes, the expression of mmp9, timp2a and timp2b were all up-regulated by LPS/VaDNA in these cells, while only mmp9 and timp2a expression increased upon stimulation with gelatin.

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.

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.

p53 protein expression and oestrogen and progesterone receptor status in invasive ductal breast carcinomas The p53 protein expression and oestrogen and progesterone receptors status was investigated in correlation to the grade of malignancy of primary breast carcinomas. Our material constituted imprints from surgical biopsies of 75 invasive ductal breast cancer cases. The p53 protein expression was investigated immunocytologically using the monoclonal antibody p53 DO-7 (DAKO). A biochemical DCC method was applied for the detection of oestrogen and progesterone receptors for all tumours. Fifty-one percent of breast cancer cases were p53 protein positive. A statistically significant association of p53 protein expression and high tumour grade was found (chi2=23.72, d.f.=2, P < 0.001). A statistically significant association was also found between oestrogen and progesterone receptor positive cases and the grade of malignancy (P < 0.001). A negative association between p53 protein expression and oestrogen (ER) and progesterone receptors (PgR) positivity was found. From our results it appears that it is possible to distinguish from grade II tumours two subgroups of cases, one with low malignancy potential and p53 (-), ER (+), PgR (+), and another subgroup with high malignancy potential and phenotype p53 (+), ER (-), PgR (-). The last subset of patients could actually benefit from adjuvant therapy.

OBJECTIVE To investigate p53 protein expression and proliferative activity in imprints of tumor biopsies from superficial transitional cell carcinoma of the bladder in relation to the histologic grade of malignancy and recurrence status. STUDY DESIGN The study group consisted of 70 cases of superficial transitional cell carcinoma of the bladder. In order to investigate p53 protein expression and Ki-67 expression, an immunocytochemical avidin-extravidin complex technique was performed using monoclonal antibodies p53 D0-7 and proliferating cells correspondingly. RESULTS Thirty-seven percent of superficial transitional cell carcinoma cases showed positive expression of p53 protein. No correlation was found between p53 protein expression and grade of malignancy (P = .45). p53 Protein expression was statistically correlated with a high Ki-67 labeling index (LI) (P < .001) and recurrence status (P < .001). Forty-seven percent of cases showed a Ki-67 LI > 25%. No correlation was found between a high Ki-67 LI and grade of malignancy (P = .703). A significant difference in high Ki-67 LI between recurrent and nonrecurrent tumors of the same grade (P < .001) and between recurrent and nonrecurrent tumors was found independently of grade (P < .001). CONCLUSION These results on cytologic material could provide useful information on the biologic behavior of superficial transitional cell carcinoma of the bladder at the time of diagnosis.