[Show abstract][Hide abstract]ABSTRACT: The mammalian E2F family of transcription factors comprises a group of 8 proteins, which either activate or repress transcription of numerous target genes, playing a role in cell-cycle progression and apoptosis. We have collectively investigated the immunohistochemical expression of E2F1, E2F2, and E2F4 transcription factors and their relation to cell kinetic parameters using serial section analysis in a series of 100 cases of human colorectal adenocarcinomas. E2F1 and E2F4 expressed nuclear immunopositivity in all cases. The range of their expression was 2% to 80% (mean 21%±15%) and 2% to 90% (mean 66%±20%), respectively. E2F2 was expressed in 41 cases at low levels (range, 1% to 5%, mean 2%±9%). A statistically significant direct association between E2F4 and cell proliferation, as expressed by high levels of Ki-67 labeling index, was shown. A mutually exclusive immunostaining pattern between E2F1 and E2F4 and a direct correlation of E2F1 and apoptosis were also highlighted. Our results point to a possible direct tumor-promoting role for E2F4 in the context of colorectal carcinogenesis. The inverse immunohistochemical relationship between E2F1 and E2F4 indicates a possible mechanistic interlink in colorectal cancer. Low expression of E2F2 may reflect functional redundancy between members of the E2F family, in this case between E2F1 and E2F2.
Article · Jun 2012 · Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry
[Show abstract][Hide abstract]ABSTRACT: Vascular endothelial growth factor (VEGF) seems to be a critical molecule in cervical carcinogenesis. We aimed to investigate the possible associations between hormonal factors and VEGF expression in cervical epithelial cells from postmenopausal women.
A total of 105 healthy postmenopausal women (aged 45-68 years old) attending a university menopause clinic were enrolled in this cross-sectional study. Pap smears were derived from current users of 17β-estradiol 1 mg + norethisterone acetate 0.5 mg (n = 28), tibolone 2.5 mg (n = 23), raloxifene HCl 60 mg (n = 21) and women not receiving treatment (n = 33). VEGF immunostaining was evaluated in squamous, glandular and metaplastic cells, using a semiquantitative method (rating scale: 0-3).
Concerning endogenous hormones, higher Δ4-androstenedione levels were associated with more intense VEGF immunostaining in glandular (p = 0.041) and metaplastic cells (p = 0.004). Hormone therapy and raloxifene did not induce any changes in VEGF immunoreactivity in the examined cells. In contrast, tibolone administration was accompanied by diminished VEGF presence in metaplastic cells (p = 0.016 vs. controls).
Our findings may in part reflect the molecular processes contributing to the safe profile of hormone therapy, tibolone and raloxifene in cervical carcinogenesis.
[Show abstract][Hide abstract]ABSTRACT: Overexpression of Epidermal Growth Factor Receptor (EGFR) and also of cell cycle control proteins, such as cyclin D1 is a frequent event in squamous cell carcinoma of the larynx (LSSC). Our aim was to correlate their protein levels with telomerase catalytic subunit (h-TERT) expression. Using tissue microarray technology, fifty-five paraffin embedded histologically confirmed primary LSSCs and also ten dysplastic lesions were cored at a diameter of 1.5 mm. Immunohistochemistry (IHC) was performed by the use of anti-EGFR, anti-cyclin D1, and anti-h TERT monoclonal antibodies. Chromogenic in situ hybridization (CISH) analysis was also applied using EGFR gene and chromosome 7 probes, respectively. EGFR, cyclin D1 and h-TERT protein overexpression was observed in 48/55 (87.2%), 19/55 (34.5%) and 21/55 (38.1%) carcinoma cases, respectively. EGFR protein expression was statistically associated with grade (P=0.01), and also with stage (P=0.001) of the examined tumors. Borderline statistical significance was assessed correlating overall cyclin D1 expression to h TERT expression (P=0.06). Simultaneous up regulation of the three proteins was established in 7/55 (12.7%) cases, correlated to the stage of the tumors (P=0.05). EGFR gene amplification was observed in 7/65 (10.7%) carcinomas and dysplasias, whereas chromosome 7 aneuploidy was detected in 4/65 (6.1%) of those cases.Simultaneous up regulation of EGFR, cyclin D1 and h TERT proteins correlates with advanced stage in LSCC. EGFR gene amplification and not only protein over expression maybe is the eligible criterion for targeted therapeutic strategies in those patients.
[Show abstract][Hide abstract]ABSTRACT: Background:
Several studies showed serum markers elevation as a result to coronary angiography. We investigated the effect of diagnostic coronary angiography (DCA) on the development of systemic inflammatory response syndrome (SIRS) and on whole blood cytokine production capacity after ex-vivo LPS stimulation.
In this observational study, clinical characteristics and serum cytokines of the patients were recorded at baseline and at 2, 6, 12, and 24h after DCA. Peripheral blood was collected at baseline and at 2, and 24h for complete blood count, coagulation profile and ex-vivo (100 microl) stimulation with LPS (500 pg) for subsequent cytokine measurement. Values are expressed as median+/-IQR and were compared using Wilcoxon's signed rank test with Bonferroni adjustment.
We included 23 male patients (mean age 52.0+/-18.0 years) undergoing DCA. None of the patients developed clinical or laboratory signs of SIRS. Serum IL-6 significantly increased at 12h. There was a significant decrease in TNF-alpha production after ex-vivo LPS stimulation of whole blood at 2 and 24h compared to baseline (median+/-IQR; 716.0+/-319.0; 576.0+/-715.0 vs. 1154+/-844.0 pg/ml; respectively) suggesting that DCA may cause transient endotoxin tolerance.
DCA is related to increased serum IL-6 levels but does not cause clinical SIRS. Development of SIRS after DCA is indicative of other in origin complication. DCA is associated with immune cells hyporesponsiveness, possibly through monocyte depression, expressed as decreased TNF-alpha production after whole blood stimulation with LPS ex vivo.
[Show abstract][Hide abstract]ABSTRACT: The liquid-based cytology (ThinPrep) is an alternative to the long-used conventional cytology that received Food and Drug Administration approval in 1996. The purpose of the current study was to determine the diagnostic cytomorphologic criteria of ThinPrep cytology and a comparison of the accuracy of conventional and ThinPrep cytology in investigation of patients with epithelial ovarian cancer. We investigated the advantages of liquid-based cytology and the potential role of the DNA ploidy in investigation of patients with the ovarian cancer. One hundred and five consecutive body cavity fluids (32 ascitic fluids, 42 free peritoneal fluids and 31 peritoneal washings) were diagnosed cytologically by both the conventional and the ThinPrep methods. The differences between ThinPrep and conventional cytology were evaluated for a variety of parameters including cellularity, cytologic morphology, specimen preparation, screening time, cytologist preference, and the impact on final diagnosis. Consequently, DNA ploidy analysis was performed on the ThinPrep specimens. The estimation of DNA ploidy was based on such parameters as the DNA index, the ploidy balance, the degree of aneuploidy, the degree of hyperploidy and the proliferation index . According to our findings, results through the TP method were considerably improved compared with those of the conventional method. Sensitivity was improved to 90.47%, negative predictive value to 72.41% and diagnostic accuracy to 92.37%. Furthermore, these findings were in excellent correlation with the final clinical diagnosis (p >0.05). According to the results of DNA ploidy analysis, 64 cases were aneuploids (60.95%), while 41 cases were euploids (39.04%). Furthermore, with the addition of the DNA ploidy analysis in our material, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were improved to 97.62%, 100%, 100%, 91.3% and 98.09%, respectively. ThinPrep method was found to provide better cytological details, significantly improved the diagnostic accuracy of the cytological diagnosis of ovarian cancer, reduced the screening time, and permitted the valuable application of current techniques of static DNA cytometry. Furthermore, the additional application of DNA ploidy further improved the diagnostic accuracy, confirming the fact that there are changes in submicroscopical level, which are difficult to be recognized with the simple microscopical examination. Based on these findings of automatic cytology, it is possible to improve our diagnostic criteria.
[Show abstract][Hide abstract]ABSTRACT: Vascular endothelial growth factor (VEGF) expression may act as a sensitive measure of the angiogenic potential of a lesion. Furthermore, VEGF has been implicated in the pathogenesis of cystic tumors and inflammatory odontogenic cysts. Thus, we studied the expression of VEGF in the epithelium of odontogenic keratocyst (OK) in association with cell proliferation and apoptosis.
Forty-two cases of OK, 26 cases of dentigerous cyst (DC), and 15 cases of residual cyst (RC) were retrospectively examined by immunohistochemistry for VEGF, Ki67/Mib-1 and anti-caspase-3. For VEGF and caspase-3, the intensity of immunostaining was qualitatively assessed, while for the evaluation of Ki67 the average number of positively stained nuclei in 10 high-power microscopic fields (x 400) was calculated.
The VEGF expression was stronger in OK when compared with DC (P < 0.007). The rate of nuclear Ki67 expression in OK was significantly higher than that in DC (P < 0.001) and RC (P < 0.001). Cytoplasmic caspase-3 expression was statistically more intense in RC than in OK (P = 0.001) or DC (P < 0.001). A statistically significant correlation was seen in OK for Ki67 (P < 0.001) and VEGF (P = 0.023), but not for caspase-3. Multiple regression analysis revealed a linear relationship between VEGF and Ki67.
The VEGF was expressed in the epithelium of OK, DC, and RC with a variable intensity, and in OK VEGF expression was related to Ki67. It is suggested that VEGF expression by the odontogenic epithelium is not induced solely by inflammation.
Full-text Article · May 2009 · Journal of Oral Pathology and Medicine
[Show abstract][Hide abstract]ABSTRACT: Epithelial cadherin (E-cadherin), a transmembrane glycoprotein involved in calcium-dependent homophilic cell-cell adhesion, is expressed aberrantly during cervical carcinogenesis. E-cadherin expression and putatively implicated predictors in healthy women remain a rather under-investigated area. The objective of this study is to evaluate the possible associations between E-cadherin expression and reproductive/lifestyle factors in cervical epithelial cells from postmenopausal women.
A total of 105 healthy postmenopausal women (aged 45-68 years old) attending a university menopause clinic were enrolled in this cross-sectional study. Pap smears were derived and E-cadherin immunostaining was evaluated in squamous, glandular and squamous metaplastic cells, using a semi-quantitative method (rating scale: 0-3). Reproductive and lifestyle factors were obtained from patients' chart review.
In squamous cells, women with a history of 0-1 deliveries presented with a higher score vs women with 2-4 deliveries (P = 0.003). Social drinkers and women drinking alcohol daily exhibited a higher E-cadherin immunostaining score in squamous cells vs non-drinkers (0.96 +/- 0.72 vs 0.56 +/- 0.65, P = 0.004). A higher dietary calcium intake was marginally correlated with a lower staining score in squamous cells (0.94 +/- 0.78 for low, 0.71 +/- 0.70 for average, 0.45 +/- 0.52 for high consumption, P = 0.073).
E-cadherin expression seems to be associated with reproductive history and lifestyle habits in squamous cervical cells from healthy postmenopausal women. E-cadherin might participate in the molecular mechanisms underlying the role of parity as a risk factor for cervical cancer.
Article · Jan 2009 · Journal of Obstetrics and Gynaecology Research
[Show abstract][Hide abstract]ABSTRACT: To establish a potential correlation between renal and systemic production of vascular endothelial growth factor (VEGF) protein after prolonged ischemia in a renal ablation model under normothermic and hypothermic conditions.
38 uninephrectomized New Zealand rabbits were divided into 5 groups. The rabbits of each group underwent partial nephrectomy under 90 and 60 min of warm and 90 and 120 min of cold ischemia, except for the sham group (S), which served as control. Serum creatinine (SCr) and blood-urea-nitrogen (BUN) levels were assessed. On the 15th postoperative day (POD), the animals were euthanized and the remaining kidneys were evaluated. VEGF immunohistochemistry and serum Western blot analysis were performed.
In comparison to the control group, groups 60W, 90C and 120C showed 1.6-, 1.14- and 1.75-fold decreases, respectively, while the production of VEGF was significantly declined by 7.4-fold in group 90W (p < 0.05). Immunohistochemistry revealed prominent VEGF staining in the above-mentioned three groups, while in group 90W staining was negative. Serum biochemistry and microscopic evaluation verified the same differentiation.
Renal and serum VEGF seem to have an analogous expression under conditions of prolonged ischemia. VEGF is overexpressed in hypothermic conditions compared to warm ischemia exceeding 60 min. Hypothermia can be more advantageous in a procedure applying prolonged ischemia.
Article · Apr 2008 · American Journal of Nephrology
[Show abstract][Hide abstract]ABSTRACT: The aim of this study was to investigate the patterns of renal function recovery during partial nephrectomy (PN) on an experimental solitary kidney rabbit model and establish the upper tolerable time limits of applied ischemia. Forty-eight New Zealand rabbits underwent an open right nephrectomy and after 30 days, the animals were clustered into five groups (A, B, C, D, E). The first four groups received an open left PN, under different types of ischemia. Groups A (n = 8) and B (n = 10) were subjected to 90 and 60 min of warm ischemia (WI), respectively, while groups C (n = 10) and D (n = 10) received 90 and 120 min of cold ischemia (CI) with ice-slush cooling. Group E (n = 10) served as sham group. Serum determinations of creatinine (SCr) and BUN were recorded preoperatively and on postoperative days (POD) 1, 3, 6 and 15. The animals were euthanized and the remaining kidneys were harvested and evaluated microscopically. The type and duration of ischemia were statistically significant parameters (P < 0.001). Groups B, C and D exhibited a similar pattern of recovery from trial initiation to the 15th POD (P = 0.788 and P = 0.068, respectively). Group A was extremely differentiated, with 100% mortality caused by uremia. The microscopic findings were consistent to the serum biochemistry. In our solitary kidney rabbit model, the upper limits of tolerable WI seem to be set on 60 min. CI can safely preserve the model's renal function--even up to 120 min.
[Show abstract][Hide abstract]ABSTRACT: Deregulation of apoptotic pathways in cutaneous malignant melanoma appears to be correlated with chemoresistance and poor prognosis. Furthermore, telomerase (especially h-TERT) expression induces proliferation and also represents a potential target for vaccination regarding some types of malignancies.
Using tissue microarrays (TMA) technology, 25 paraffin-embedded tissue samples of histologically confirmed malignant melanomas were cored at a diameter of 2 mm and re-embedded into one recipient block (final TMA density 24/25-96%). Immunohistochemistry (IHC) was performed by the use of anti-bcl-2, anti-caspase 3, anti-caspase 8 and anti- h-TERT antibodies. Protein expression levels were evaluated using a computerized image analysis system (CIA). SPSS (chi square test and inter-rater kappa) was used for statistical analysis.
Strong protein expression was observed in 1/24 (4.1%), 1/24 (4.1%), 2/24 (8.2%), and 4/24 (16.4%) cases regarding h-TERT, caspase 3, caspase 8 and bcl-2, respectively. Moderate was observed in 7/24 (29.1%), 8/24 (32.2%), 5/24 (20.2%), and 8/24(32.2%) cases, whereas reduced or absent expression demonstrated 16/24 (65%), 15/24 (60.2%), 17/24 (68.5%), and 12/24 (50 %) cases. Statistical significance was assessed correlating age to caspase 3 (p=0.05), Breslow's thickness to telomerase (p=0.013) and to bcl-2 (p=0.053), Clark's level to telomerase (p=0.008) and to bcl-2 (p=0.022), and finally ulceration to telomerase expression (p=0.007).
bcl-2 and telomerase expression are correlated to critical parameters of malignant melanoma, affecting its biological behavior. Furthermore, downregulation of proteins such as caspases 3/8, which normally induce apoptosis, is perhaps associated with resistance of the applied chemotherapeutic strategies in this type of malignancy.
Article · Oct 2007 · Journal of B.U.ON.: official journal of the Balkan Union of Oncology
[Show abstract][Hide abstract]ABSTRACT: Mutational activation of KRAS and BRAF proto-oncogenes contributes to the development of many human cancers. Current research on gynecological cancer and specifically in cervical and endometrial cancer is focused on the mechanisms of their mutational activation.
In view of the paucity of data on their mutation frequency and the status of BRAF in these two types of gynecological cancer, we performed a systematic molecular study in 114 clinically and histologically well-defined malignant tumors of uterine cervix and endometrium and correlated the mutation status of KRAS and BRAF with the age at diagnosis and with tumor grade, stage or histological type.
Direct sequence analysis of the PCR products of KRAS and BRAF genes was used to screen for known activating mutations.
In 67 cases of endometrial cancer, six KRAS mutations (8.9%) were found, four at codon 12 (5.9%) and two at codon 13 (2.9%), while no mutation was detected at codon 61. Most of the mutations occurred in surgical stage I and in the endometrioid adenocarcinoma subtype. We also detected three KRAS point mutations (6.3%) in the 47 cervical cancer samples, two at codon 12 (4.2%) and one at codon 13 (2.1%), while there was no mutation at codon 61. On the contrary, no mutation was identified in BRAF exon 15 for either endometrial or cervical cancer samples at position V600, which represents the most frequently mutated site of BRAF in human cancer. There was no association between KRAS mutations with either histological type, tumor grade or stage. Interestingly, however, KRAS mutation status in endometrial cancer was strongly associated with increased age at diagnosis (P < 0.001).
Our data document (a) the absence of BRAF mutations in cervical and endometrial cancer, despite the mutation status of KRAS, (b) suggest that KRAS mutations reflect an early event in endometrial carcinogenesis and (c) imply that BRAF activation is involving alternative pathways in these two types of cancer.
[Show abstract][Hide abstract]ABSTRACT: To investigate (1) the feasibility of preparing cell blocks by inverted filter sedimentation (IFS-CB) from endometrial samplings processed by the ThinPrep (TP) technique (Cytyc Corp., Boxborough, Massachusetts, U.S.A.), and (2) the possibility of increasing the diagnostic accuracy of TP endometrial cytology by examining the tissue architecture as an adjunctive method of detecting endometrial lesions.
Three hundred one endometrial samplings were obtained, using the Endogyn endometrial device (Biogyn S. n.c., Italy), from perimenopausal and postmenopausal women. The endometrial samplings were collected in a vial with liquid fixative for the TP processing. One TP slide was prepared from each case. If adequate material remained in the vial after the TP slide preparation, it was processed for IFS-CB preparation.
IFS-CB preparation was processed in 263 cases (87%) with adequate material. Diagnoses on IFS-CB preparations obtained by endometrial sampling matched those of the hysterectomy specimens. The addition of IFS-CB histology to the cytologic diagnosis by TP increased the diagnostic accuracy of endometrial cytology to 96.3% and 100% for benign/atrophic endometrium and adenocarcinoma, respectively (p = 0.39 and 0.46). In hyperplasia without atypia and hyperplasia with atypia, the diagnostic accuracy increased significantly, to 96% and 95.3%, respectively (p = 0.037 and < 0.001).
This study illustrates the merit of linking TP cytology with direct endometrial sampling, including small tissue fragments and material adequate for IFS-CB preparation. TP cytology provides an accurate cytologic diagnosis and the possibility of IFS-CB preparation, which could be a valuable diagnostic adjunct to TP cytology.