VEGF and Id-1 in pancreatic adenocarcinoma: prognostic significance and impact on angiogenesis.

OBJECTIVES: The significance of vascular endothelial growth factor (VEGF) and inhibitor of differentiation/DNA synthesis (Id-1) in tumor neoangiogenesis and tumor progression in pancreatic ductal adenocarcinoma (PDAC) is still unclear. Given the central role of VEGF in cancer angiogenesis and the inconclusive results on Id-1 expression in PDAC, it is of great interest to investigate whether Id-1 and VEGF expression are associated with angiogenesis and prognosis in PDAC. METHODS: Paraffin-embedded specimens from 60 consecutive patients with PDAC were immunostained for VEGF, Id-1 and CD34 and staining quantification was assessed by Image analysis system. The correlations among the expression of individual angiogenic factors and microvessel density (MVD), clinicopathologic features and clinical prognosis were analyzed. RESULTS: Id-1 and VEGF Positive Activity Indices (PAIs) closely correlated with each other. MVD positively correlated with both Id-1 and VEGF expression. More advanced T and N status correlated with more intense expression of Id-1, VEGF and higher MVD. With regard to prognostic significance higher Id-1 PAI (adjusted HR = 1.69, 95%CI: 1.10-2.59, p = 0.017), higher VEGF PAI (adjusted HR = 2.66, 95%CI: 1.09-6.50, p = 0.032), and MVD (adjusted HR = 1.55, 95%CI: 1.27-1.88, p < 0.001) were associated with poorer survival. CONCLUSIONS: VEGF and Id-1 overexpression were found to be associated with high MVD and emerged as adverse prognostic factors in terms of patient survival in PDAC. The potential of selective anti-angiogenic targeting therapy for pancreatic malignancies should prompt further validation of the present findings in studies encompassing larger samples and more elaborate techniques.

Laryngeal dysplasia: a long-term follow-up study.

PURPOSE: To assess the progression of precancerous laryngeal lesions to squamous cell carcinoma (SCC), defined by specific histopathological criteria, in patients with longterm follow-up. METHODS: Patients with laryngeal dysplasia, followed/ treated between 1985 and 2008, were retrospectively evaluated and classified according to the World Health Organization classification system (WHO). The investigated outcome parameters were progression of dysplasia to SCC, time interval to malignant transformation and continuation of smoking as potential risk factors. RESULTS: Fifty-nine patients were studied. Progression of dysplasia to SCC between the first and the final histological examination was statistically significant (p<0.0001). Malignant transformation appeared in 29 patients (49.2%). Serious dysplasia was more likely to progress to SCC (64.8%) compared to mild (41.7%) or moderate (44.4%) (p<0.0001). However, the time interval needed in these 29 cases to progress to cancer was not statistically related to the initial histological diagnosis. Continuation of smoking did not affect the progression of disease. However, the mean time from dysplasia to laryngeal cancer was much longer in patients who quitted smoking (33.5 months) vs those who continued smoking (19.5 months), with a marginal statistical difference (p=0.057). CONCLUSION: All patients with laryngeal dysplasia should be followed up at regular intervals. The progression of dysplasia to SCC did not seem to be directly related to the continuation of smoking or not. However, large long-term follow- up studies taking into account the degree of exposure (e.g. time of exposure, number of cigarettes) are needed in order to clarify risk factors and proper management. Consensus guidelines in diagnosis, follow-up, and treatment would improve substantially the current clinical practice.

PTEN expression in non small cell lung carcinoma based on digitized image analysis.

PURPOSE: HER2 depended signalling pathway is dereg-ulated in a subset of non small cell lung carcinoma (NSCLC). The tumor suppressor gene PTEN (10q21) regulates the HER2/PI3K/Akt signalling pathway. Our aim was to evaluate PTEN protein expression in NSCLC based on a quantitative analysis method correlating also the results with clinicopathological parameters. METHODS: Protein expression was determined by immunohistochemistry (IHC) in 61 paraffin-embedded cases of patients with NSCLC. Digital image analysis (staining intensity levels) was performed in the corresponding immunostained slides. RESULTS: Loss of PTEN expression was observed in 24 (39.34%) cases, low expression in 29 (47.54%) and overexpression in 8 (13.12%) cases. Multivariate analysis determined that PTEN overexpression was associated with lower risk to develop metastases (p=0.05). CONCLUSION: PTEN deregulation is a relatively frequent genetic event in NSCLC, associated with progressive metastatic process in those patients. Because of binding to the ErbB2 receptor, trastuzumab stabilizes and activates PTEN gene, and loss of its expression negatively affects the response rates in such patients.

Deregulation of EGFR/VEGF/HIF-1a signaling pathway in colon adenocarcinoma based on tissue microarrays analysis.

PURPOSE: Overexpression of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) in colon adenocarcinoma (CA) is a frequent event, whereas specific deregulation mechanisms in the corresponding signaling pathways remain under investigation. Our aim was to co-evaluate their expression correlated to the hypoxia inducible factor 1alpha (HIF-1a), which activates the transcription of VEGF gene. METHODS: 60 paraffin-embedded primary CAs were cored at 1.5 mm diameter and transferred to the microarray block. Immunohistochemistry (IHC) was performed using anti-EGFR, -VEGF, and -HIF 1a monoclonal antibodies. Concerning EGFR, quantitative evaluation was based on a semi-automated analysis system. Chromogenic in situ hybridization (CISH) was performed using EGFR gene and chromosome 7 centromeric probes. RESULTS: Protein overexpression was observed in 13/60 (21.6%), 45/60 (75%) and 7/60 (11.6%) cases regarding EGFR, VEGF, and HIF 1a, respectively. CISH analysis detected 4/60 (6.6%) EGFR gene amplified cases, whereas chromosome 7 aneuploidy was identified in 11/60 (18.3%) cases. Significant associations raised correlating stage to chromosome 7 (p=0.024), HIF 1a expression to tumor anatomical location (p=0.019) and also VEGF to HIF 1a expression (p=0.001), whereas EGFR expression was not associated to EGFR gene copies. CONCLUSION: According to our results, chromosome 7 instability is correlated to advanced disease, whereas a significant subset of CAs demonstrates an alternative, non- HIF 1a depended mechanism of VEGF overexpression. Furthermore, EGFR protein overexpression does not predict a specific gene deregulation mechanism.

Piriformis syndrome--an attempt to understand its pathology.

OBJECTIVE: Gross anatomy of the hip rotators and histology of the sciatic nerves in adult cadavers were studied, aiming to the identification of possible pathologic changes related to the piriformis syndrome (PS). MATERIAL: 50 cadavers were dissected; in 17 cases with macroscopical findings the sciatic nerves were harvested (34 sciatic nerves; 17 cadavers). History of low back or leg pain was not available. METHOD: Site anatomy and additional findings at the harvesting sites were recorded, such as anatomical variations, adhesions, hematomas etc. All nerves were additionally microscopically analyzed. In cases with findings at the dissection, the contralateral unaffected nerves served as controls. All the dissected nerves were conserved in 10% formalin solution, embedded in paraffin, stained with Hematoxylin and Eosin (H&E) and immunolabeled with antibodies against Neurofilament (NF). RESULTS: Both the H&E staining as well as the performed immunohistochemistry showed, to a variable degree, significant alterations in the structure of the affected nerves compared to the controls. CONCLUSIONS: These findings both in the local anatomy and sciatic nerve correspond to lesions that are expected in PS. Nevertheless, since this was a cadaveric study, unassociated to a certain pain patient's history, results should be considered and interpreted as an indication of a sciatic nerve injury in PS.

Comparative study of the immunohistochemical expression of metalloproteinases 2, 7and 9between clearly invasive carcinomas and "in situ" trophoblast invasion.

KEYWORDS: Matrix metalloproteinases (MMPs) are endopeptidases considered to participate in the transient invasive property of trophoblastic cells during embryo implantation and placentation. The same molecules play an important role in the invasive and metastatic potential of cancer cells. The aim of this study was to compare the immunohistochemical expression of MMP2, 7and 9between clearly invasive carcinomas and "in situ" trophoblast invasion in an effort to illuminate their distinct roles in uncontrolled and controlled invasion.
We performed an immunohistochemical analysis of 45 clearly invasive carcinomas of various organs (colorectal, gastric, breast, pulmonary, renal) and 40 first trimester gestation specimens (before the 9th week of gestation). The markers expression was evaluated semiquantitavely, seperately in cancer parenchymal and gestational trophoblastic cells as well as cancer stromal and decidual cells, according to apercentage scale (0 %, 50% of cells) and according to staining intensity (0, +, ++, +++).
MMP9 was expressed more often in the malignant parenchymal as well as in the malignant stromal component of carcinomas than in the trophoblastic (p=0, 0118) and decidual (p=0,017) component of gestations respectively. Although all carcinomas and almost all gestation specimens stained for MMP2 and MMP7, the immunostaining for both molecules was statistically more extensive and intense in trophoblasts and decidual cells by comparison to cancerous elements.
In conclusion, although there seems to be adirect link between cancer invasion and MMP9 immunohistochemical expression, the role of MMP2 and MMP7 appears to be more complicated underlining the complexity of the mechanisms involved in cancer spreading.

Myoepithelial carcinoma of the retromolar area in a young female. A rare case report.

We present a case of a 34-year-old woman with myoepithelial carcinoma of the retromolar area. Myoepithilial carcinoma is a rare tumor of small salivary glands most usually located in the parotid gland. The major differential diagnosis of myoepithelioma is from pleomorphic adenoma. Little is known about the clinical and biological behavior and the prognosis of myoepithelial carcinoma and there is no consensus for its treatment. It is considered a low-grade malignancy; it sometimes shows aggressive behavior and may locally recur. Our patient was treated successfully with wide-local resection and remained free of disease for 6 months.

Evaluation of caspase-3 and caspase-8 deregulation in tongue squamous cell carcinoma, based on immunohistochemistry and computerised image analysis.

AIMS: To investigate the potential role of caspase-3 and caspase-8 protein expression in the biological behaviour of tongue squamous cell carcinoma. MATERIALS AND METHODS: We conducted immunohistochemical analyses of 87 specimens of primary tongue squamous cell carcinoma, using monoclonal anti-caspase-3 and anti-caspase-8 antibodies. A digital image analysis assay was also performed in order to evaluate the results. RESULTS: Reduced expression of caspase-8 and -3 proteins was observed in 30/87 (34.5 per cent) and 79/87 (90.5 per cent) cases, respectively. Cox regression analysis showed no prognostic significance for the association between overall protein expression of either marker and survival probability (p = 0.174 for caspase-3; p = 0.608 for caspase-8). Interestingly, the size of the examined tumours was strongly correlated with survival status (p = 0.024). CONCLUSIONS: Simultaneous deregulation of caspase-8 and -3 is a frequent event in tongue squamous cell carcinoma. Activation of caspase-3, which is predominantly down-regulated, may be a crucial process for induction of apoptosis and response to therapeutic strategies.

Adrenaline attenuates the acute lung injury after intratracheal lipopolysaccharide instillation: an experimental study.

Endotoxin is a major cause of endotoxinemia, sepsis, and pneumonia due to gram-negative bacteria. Experimental endotoxin administration via the tracheal route has been extensively used to study the biological and pathophysiologic pathways of inflammation. In particular, experimental endotoxin instillation in the respiratory tree has allowed an extended research with regard to the local response of the lungs to the pathogenic stimulus. This study aims (a) to define early events in the inflammatory cascade and (b) to evaluate the efficacy of adrenaline to ameliorate the acute pulmonary inflammation in vivo after administration of intratracheal lipopolysaccharide (LPS) in an in vivo animal model. Two groups of animals were used for that purpose, a control group (single LPS administration) and a study group (subcutaneous adrenaline infusion following LPS administration). We found that mononuclear recruitment, along with an increased population of CD4+ T lymphocytes, is an early event during the course of LPS-challenged inflammation. In the study group, we determined that adrenaline mediated the lung inflammation in a statistically significant degree. By the use of immunohistochemistry, we identified (1) an increased population of CD4+ T lymphocytes in the inflammatory infiltrate, further endorsing the hypothesis that T-helper lymphocytes, along with macrophages, secrete cytokines which amplify the inflammatory response, and (2) an upregulation of ICAM-1 expression, suggesting an important role in the early pathogenesis of LPS-induced acute lung injury. Our study establishes that systemic adrenaline administration after LPS instillation may ameliorate the inflammatory lung response in vivo.

Altered expression of adhesion molecules in inflammatory cervical smears.

OBJECTIVE: The aim of the present study was to evaluate the expression of pan-cadherin and beta-catenin in cervical smears with various types of infectious agents. PATIENTS AND METHODS: Cervical smears obtained from 53 women, aged 21-65 years, with a diagnosis of specific inflammation were examined in our study. Eighteen subjects were infected by Candida albicans, 18 by Gardnerella vaginalis, nine by Bacteroides spp. and eight by Chlamydia trachomatis. All infectious agents found in the smears were at the same time confirmed by the microbiological laboratory methods. We performed a biotin-streptavidin-peroxidase immunocytochemical method using anti-beta-catenin (Clone 12F7) and anti-pan-cadherin (pan, polyclonal) antibodies. RESULTS: Aberrant expression of pan-cadherin was found in the cytoplasmic membrane of glandular, metaplastic, superficial and intermediate squamous cells in all types of infections. With regard to beta-catenin, this was expressed in majority (90%) of glandular and metaplastic cells in all types of infections and in a small proportion (15%) of superficial and intermediate squamous cells in infections caused by C. albicans and G. vaginalis. CONCLUSION: Our data show that infectious agents may cause alterations in the expression and distribution of these adhesive molecules, which can be recognized in cervical smears. Additional studies in larger sets of patients should help clarify this issue further.

Extracellular regulated kinase-2 immunoreactivity increases in parallel with cervical intraepithelial neoplasia grade in cervical neoplasia.

The cell cycle control system includes cyclins, cyclin-dependent kinases (CDK), and their inhibitors (CDK1). Extracellular regulated kinase (ERK1/2) (p44 and p42 mitogen-activated protein kinases [MAPKs]) is a component of the MAPK pathway, which is associated with cyclin D1 and CDK. It is a critical signaling system for the induction of cell proliferation, differentiation, and cell survival. The aim of this study was to investigate the usefulness of ERK2 expression as a marker of biological aggressiveness complementary to cervical intraepithelial neoplasia (CIN) grade as well as to compare its expression in preinvasive lesions with that in invasive carcinoma. Paraffin-embedded sections of 146 CIN lesions (32 CIN I, 49 CIN II, and 43 CIN III) and 22 invasive cervical carcinomas (13 squamous and 9 adenocarcinomas) were used for the standard immunohistochemical procedure with the application of the ERK2 monoclonal antibody. ERK2 staining displayed a cytoplasmic and nuclear pattern. The staining intensity was gradually increased according to the severity of the dysplastic lesions; ERK2 immunoreactivity was significantly increased in high-grade dysplastic lesions (CIN II and CIN III) and invasive carcinomas by comparison to low-grade dysplastic lesions (CIN I) (P < 0.001). When high-grade lesions were separately assessed, the differences between each one of them and CIN I retained their statistical significance: CIN II versus CIN I (P < 0.001) and CIN III versus CIN I (P < 0.001). In conclusion, our study found a direct relationship between the increasing grade of the dysplastic cervical lesions and the intensity of ERK2 staining, thus implying a role of ERK2 as an early event in cervical carcinogenesis.

Tissue detection of natural killer cells in laryngeal carcinoma.

Natural Killer (NK) cells have gained much attention as potential cells in antitumor immune defense mechanisms. In a group of 31 patients with surgically treated squamous cell laryngeal carcinoma, NK cell presence was semiquantitatively assessed by means of immunohistochemistry. A panel of three monoclonal antibodies including anti-CD16, was applied on frozen tissue sections. High CD 16+ cell presence was more frequently detected in poorly differentiated carcinomas (in 6 out of 14 cases) by comparison to carcinomas of high to moderate degrees of differentiation (in 1 out of 16 cases, p=0.031). No other clinicopathological variable appeared to influence NK cell presence in the examined specimens. No relation between NK cell detection and relapse-free survival emerged. Poorly differentiated laryngeal cancer cells appear to trigger off a greater NK cell tissue response than well and moderately differentiated cancer cells; however, the potential prognostic impact of this observation remains to be established.

Topoisomerase I protein expression in primary colorectal cancer and recurrences after 5-FU-based adjuvant chemotherapy.

PURPOSE: Our aim was to investigate whether chemotherapy with 5-FU induces an alteration in the levels of topoisomerase I (topo I) in colorectal neoplastic tissues METHODS: Twenty-five colorectal cancer patients were included in our study; these had undergone surgical resection of the primary tumor, received post-operatively 5-FU-based adjuvant chemotherapy and then suffered from recurrences. In a standard three-step immunohistochemical procedure, a monoclonal antibody to topo I was applied in both specimens from each patient (one from the primary location and a second one from the recurrence). Statistical analysis was subsequently performed. RESULTS: Malignant cells from the recurrences displayed a statistical significant increase, concerning the levels of topoisomerase I, by comparison with the primary tumors (P=0.01). The increase in topo I levels did not demonstrate significant correlations with Duke's stage (Fisher's Exact Test P value=0.496), differentiation grade (P value=0.661), localization (P value=0.072), patient sex (P value=0.434), nor with relapse free interval (P value=0.493). There was a statistically significant relationship between the age of patients and increase in topo I levels (P=0.011). CONCLUSIONS: Topo I expression may be part of the malignant cells' phenotype in recurrent colorectal carcinomas, suggesting a potential role for Topo I in the acquisition of a metastatic phenotype. The increase of topo I immunohistochemical status is likely to be attributed to 5-FU and given the fact that high levels of topo I correlate with sensitivity to camptothecin, advanced colorectal cancer patients seem to benefit from topo I targeted anticancer drug therapy.

Population-based study of human papillomavirus infection and cervical neoplasia in Athens, Greece.

The aim of our study is to describe the prevalence of the different HPV types in women with pre-neoplastic lesions of the cervix in Greece. Cervical scrapes from 841 women were obtained for both cytological evaluation and analysis for the presence of HPV DNA. PCR was performed on specimens from these 841 women. The Pap test results were normal or showed benign cellular changes in 45.8% of the women, atypical squamous cells of undetermined significance (ASCUS) in 23.2%, low-grade squamous intra-epithelial lesion (LSIL) in 27.9% and high-grade squamous intra-epithelial lesion (HSIL) in 3.1%. HPV DNA was demonstrated in 23.6% of cytologically normal women. We detected HPV in 60% of the total samples. Of these, HPV-16 was the most common HPV DNA detected. Interestingly, HPV-58 was inversely correlated with positive cytological findings. A clear pattern of decreasing prevalence of HPV with age was also observed. Our results indicate that HPV infections, especially those with HPV-16, represent a significant public health concern in Greece.

Metastatic low-grade endometrial stromal sarcoma of clitoris: report of a case.

Low-grade endometrial stromal sarcoma (ESS) is an uncommon neoplasm, which has a highly recurrent nature. A review of the literature revealed that only one case of low-grade ESS, arising within the vulva from a focus of endometriosis, has been previously published. We describe an additional case of low-grade ESS arising within the vulva and to the best of our knowledge the first report of low-grade ESS metastasized to clitoris. A 46-year-old woman was admitted to our hospital due to a heavy uterine bleeding. A physical examination revealed a lesion in clitoris, which exhibited a densely cellular mesenchymal neoplasm on microscopy. On the basis of the pathologic features alone, a differential diagnosis of a low-grade ESS and cellular leiomyoma was considered. Seven months later, the patient presented again with excessive uterine bleeding and a total hysterectomy was performed. A tumor of white-tan, whorled appearance was found. Its features were suggestive of low-grade ESS. Taking into account the possible extrauterine location of an ESS and reviewing the first case, a diagnosis of rare low-grade ESS metastasized to clitoris was made.

Evaluation of epidermal growth factor receptor gene and chromosome 7 alterations in squamous cell carcinoma of the larynx, using chromogenic in situ hybridization on tissue microarrays.

AIMS: To identify subgroups of patients with squamous cell carcinoma (SCC) of the larynx, characterized by the specific deregulation mechanism of the epidermal growth factor receptor (EGFR) gene, and to evaluate EGFR protein expression levels and correlate these with biological and clinicopathological parameters. MATERIALS AND METHODS: Using tissue microarray technology, 50 formalin-fixed, paraffin-embedded primary laryngeal SCCs were cored and re-embedded into one block. Immunohistochemistry and chromogenic in situ hybridization were performed. RESULTS: Epidermal growth factor receptor protein over-expression was observed in 27/50 (54 per cent) cases and was statistically associated with tumour grade (p=0.028). Epidermal growth factor receptor gene alterations were identified in 5/50 (10 per cent) cases, which demonstrated amplification (n=4) and deletion (n=1). Chromosome 7 instability was detected in 8/50 (16 per cent) cases. CONCLUSIONS: Epidermal growth factor receptor over-expression is a frequent event in SCCs, but it does not predict a specific molecular mechanism of gene deregulation for targeted therapeutic strategies via monoclonal antibodies.

Cell proliferation and apoptosis culminate in early stages of oral oncogenesis.

Markers of cell proliferation (Ki-67 antigen) and apoptosis (Bax, Bcl-2) were studied in an experimental system of induced oral carcinogenesis in Syrian golden hamsters. Thirty-seven animals were divided into one control group and three experimental groups, which were treated with a carcinogen and sacrificed at 10, 14 and 19 weeks after treatment. The histological status of the lesions in the three experimental groups corresponded well with tumour advancement (from oral mucosal dysplasia to moderately differentiated squamous cell carcinoma). Tumour sections were studied using monoclonal antibodies against Bax, Bcl-2 and Ki-67 proteins. Pro-apoptotic Bax expression maintained high levels during all stages of oral carcinogenesis. Anti-apoptotic Bcl-2 expression decreased significantly in dysplastic and early invasion lesions and consequently increased almost to normal tissue level in consequent stages. Finally, Ki-67 expression increased sharply in initial stages of oral carcinogenesis, but significantly decreased in later stages.

Epidermal growth factor receptor and proliferating cell nuclear antigen expression in urine ThinPrep specimens.

OBJECTIVE: To evaluate proliferating cell nuclear antigen (PCNA) and epidermal growth factor receptor (EGFR) expression in urine ThinPrep (TP) specimens, to compare these findings with clinical and histological features and to determine whether these immunomarkers are predictive of clinical stage. PATIENTS AND METHODS: The TP processed urine samples and the corresponding tissue sections from 42 patients with newly diagnosed bladder cancer (18 non-muscle invasive and 24 muscle invasive) were included in our study. Urine was collected for cytological evaluation before transurethral resection. Tumour grade and clinical stage were assessed from the transurethral resection specimens. The EGFR and PCNA expression was obtained by an automated immunostainer. RESULTS: There was a remarkable concordance in the expression of both antibodies in TP smears and tissue sections. No significant association was detected for any of the immunomarkers examined with regard to tumour grade. The EGFR expression as well as grade of malignancy were significantly associated with stage of disease (P = 0.0001). PCNA was not found to be a significant predictor of stage (P = 0.210). CONCLUSION: Our data suggest that the evaluation of grade of malignancy and EGFR immunopositivity can be considered as reliable predictors of disease stage in urine TP specimens.