Vasiliki Bravou

A three-dimensional breast software phantom for mammography simulation

This paper presents a methodology for three-dimensional (3D) computer modelling of the breast, using a combination of 3D geometrical primitives and voxel matrices that can be further subjected to simulated x-ray imaging, to produce synthetic mammograms. The breast phantom is a composite model of the breast and includes the breast surface, the duct system and terminal ductal lobular units. Coopers ligaments, the pectoral muscle, the 3D mammographic background and breast abnormalities. A second analytical x-ray matter interaction modelling module is used to generate synthetic images from monoenergetic fan beams. Mammographic images of various synthesized breast models differing in size, shape and composition were produced. A preliminary qualitative assessment performed by three radiologists and a quantitative evaluation study using fractal and grey-level histogram analysis were conducted. A comparative study of extracted features with published data has also been performed. The evaluation results indicated good correlation of characteristics between synthetic and actual radiographs. Applications foreseen are not only in the area of breast imaging experimentation but also in education and training.

ILK over‐expression in human colon cancer progression correlates with activation of β‐catenin, down‐regulation of E‐cadherin and activation of the Akt–FKHR pathway

Integrin‐linked kinase (ILK) has been implicated in the development and progression of several human malignancies. However, the role of ILK in human colon cancer progression is not well established, neither have its possible in vivo downstream effectors in the disease been identified. We studied, by immunohistochemistry, ILK, β‐catenin, E‐cadherin, p‐Akt and p‐FKHR protein expression in 125 primary colon carcinomas and 45 corresponding lymph node metastases. ILK was expressed in 98.4% of the primary tumours and in 100% of metastatic lesions. The levels of ILK expression correlated strongly with tumour invasion, tumour grade and stage and were significantly higher in metastatic tumours. Activation of β‐catenin, down‐regulation of E‐cadherin and activation of the Akt–FKHR pathway correlated significantly with both ILK expression and tumour progression parameters. In conclusion, our results suggest that ILK may have an important role in progression of human colon cancer, possibly through in vivo regulation of β‐catenin, E‐cadherin and Akt pathways. Our study also provides some evidence implicating p‐FKHR in human colon carcinogenesis and ILK signalling. Copyright

Overexpression of hedgehog pathway molecules and FOXM1 in non-small cell lung carcinomas

The hedgehog (HH)-signaling pathway is implicated in developmental processes and its aberrant activation in adult tissues has been associated with malignancy. The aim of this study was to determine the expression pattern of HH-signaling molecules in non-small cell lung carcinomas (NSCLC), as well as the involvement of the transcription factor FOXM1, that controls cell proliferation, in this process. Paraffin-embedded tissue sections of 80 NSCLC cases and adjacent non-neoplastic lung parenchyma were immunohistochemically analyzed with anti-SHH, anti-Patched1 (PTCH1), anti-Smoothened (SMO), anti-GLI1, anti-GLI2 and anti-FOXM1 antibodies. Correlations of HH molecules with clinicopathological parameters and FOXM1 expression were evaluated. All the HH-signaling molecules examined were overexpressed in NSCLC compared with the adjacent non-neoplastic lung parenchyma. HH pathway activity and expression of PTCH1 and SMO were significantly higher in squamous cell carcinomas compared to other NSCLC histological types. Activation of HH pathway and PTCH1 expression were correlated with tumor grade being higher in low grade tumors. There was a significant correlation of lymph node metastases with expression of SMO in all NSCLC histological types and with nuclear GLI1 immunolocalization only in adenocarcinomas. Overexpression of FOXM1 in NSCLC was also significantly correlated with PTCH1, SMO and GLI1 expression. In conclusion, HH-signaling pathway is activated in NSCLC and correlates with histological type, prognostic parameters of the tumors as well as with the increased expression of FOXM1.

Bone regulatory factors NFATc1 and Osterix in human calcific aortic valves

BACKGROUND Emerging evidence suggests that calcific aortic valve stenosis constitutes an active process sharing common features with atherosclerosis and bone formation. To further support this hypothesis, we investigated the expression of bone regulatory factors in calcified aortic valves. METHODS-RESULTS Formalin-fixed, paraffin-embedded tissue samples of human aortic tricuspid valves (n=54) were used from patients undergoing valve replacement for calcific, non-rheumatic aortic stenosis. As controls, fourteen aortic tricuspid valves (n=14) were obtained at autopsy from patients without clinical and morphological aortic valve lesions. Sections from both stenotic and normal aortic valve leaflets were studied immunohistochemically. Interstitial cells in stenotic valves showed intense expression of Sox9, Runx2 and Osterix (Osx) whereas NFATc1 was expressed in interstitial and inflammatory cells. In addition, NFATc1 expression correlated significantly with Osx (r=0.458, p<0.001) and Runx2 (r=0.387, p<0.001). Finally, there was accumulation of activated interstitial cells, T lymphocytes and macrophages as well as intense neoangiogenesis in pathological leaflets. CONCLUSIONS The presence of NFATc1 and Osx in our material lends further support to the hypothesis that during the process of aortic valve calcification there is expression of osteoblastic phenotypes by valvular cells.

The survivin -31 snp in human colorectal cancer correlates with survivin splice variant expression and improved overall survival

Background: Survivin is involved in the regulation of cell division and survival, two key processes in cancer. The majority of studies on survivin in colorectal cancer (CRC) have focused on protein expression and less is known about the expression of survivin splicing variants or survivin gene polymorphisms in CRC. In the present study, the mRNA levels of the five known isoforms of survivin as well as survivin protein were assessed in matched normal and neoplastic colorectal tissue. Moreover, the 9386C/T and −31G/C polymorphisms were investigated. Methods: Quantitative RT-PCR was used to assess mRNA levels in fresh/frozen tissue samples. Protein levels were immunohistochemically evaluated on formalin-fixed paraffin-embedded tissue sections. Individuals were genotyped using real time PCR. Results: Expression of all 5 survivin splice variants as well as survivin protein was elevated in colorectal carcinomas compared to normal tissue. Specific splice variant expression differentially correlated with clinicopathological parameters. Furthermore, both snps correlated with splice variant levels or their ratios in colorectal carcinomas while the −31G/C snp may be related to CRC development and improved overall survival. Conclusion: Our results support a role of survivin in colorectal carcinogenesis while the −31G/C snp may constitute a marker of survival.

Integrin-linked kinase (ILK) expression in human colon cancer

Sir, In a recent study, Marotta et al (2003) reported integrin-linked kinase (ILK) overexpression and dysregulation of ILK signalling in sporadic human colon cancer. It was concluded that the dysregulation of ILK signalling is an important early event in the development of the disease. We studied ILK expression in 84 human colorectal tumours (four adenomas and 80 carcinomas) by immunohistochemistry in order to assess whether ILK is involved in the development and progression of human colorectal carcinoma. Paraffin-embedded tissue samples were retrieved from the files of the Departments of Pathology, ‘Agios Andreas’ General Hospital, Patras, Greece and University Hospital of Ioannina, Ioannina, Greece. Clinicopathologic parameters were obtained from the pathology reports. Carcinomas were graded as: well, moderately and poorly differentiated on the basis of the degree of gland formation and staged according to the Astler Coller staging system. Immunohistochemical analysis was carried out using a standard streptavidin–biotin–peroxidase technique. Primary polyclonal anti-ILK antibody was obtained from Upstate Biotechnology (dilution 1 : 500), and immunodetection was performed with StrAvigen Multilink Immunodetection system B-SA (Biogenex) using DAB as the chromogen. Negative and positive controls were used in the study. The immunostaining intensity was evaluated by light microscopy and scored as negative (−), weak (+), moderate (++) and strong (+++). Statistical analysis was performed with SPSS 10 for Windows. Relationships between ILK expression and clinicopathologic parameters were evaluated by one-way ANOVA and Tukey test post-hoc analysis. P-values<0.05 were considered to be significant. There was no ILK immunoreactivity in the normal colonic epithelium (Figure 1A, B), while the majority of adenomas (75%) and in situ carcinomas (83.3%) were ILK positive (Figure 1C, D). All invasive carcinomas were positive (Figure 1E, F). The levels of expression were significantly higher in invasive compared with noninvasive lesions (P 90% of tumour cells were stained (diffuse pattern) and ILK immunostaining was confined to the cytoplasm. Figure 1 Integrin-linked kinase expression in human colorectal tumours. (A) Normal colonic epithelium with no ILK immunoreactivity (× 200). (B) Integrin-linked kinase-positive cancerous crypts compared with ILK-negative normal crypts (× 200). ( ... Figure 2 Integrin-linked kinase expression increases with tumour grade. (A) A well-differentiated colorectal carcinoma demonstrating weak (+) ILK expression (× 200). (B) Moderate levels of expression (++) in a moderately differentiated ... Table 1 Integrin-linked kinase expression in human colorectal tumours. Correlation with clinicopathologic parameters Our results seem to indicate that, in addition to being involved in the initiation of colon carcinogenesis, as suggested by Marotta et al (2003), ILK may also be implicated in the progression, invasiveness and metastatic potential of colorectal cancer. Thus, ILK may prove to be a useful prognostic marker for these tumours.

Enhancement of TGF-β Signaling Responses by the E3 Ubiquitin Ligase Arkadia Provides Tumor Suppression in Colorectal Cancer

TGF-β signaling provides tumor protection against colorectal cancer (CRC). Mechanisms that support its tumor-suppressive properties remain unclear. The ubiquitin ligase Arkadia/RNF111 enhances TGF-β signaling responses by targeting repressors of the pathway for degradation. The corepressors SnoN/Ski, critical substrates of Arkadia, complex with the activated TGF-β signaling effectors Smad2/3 (pSmad2/3) on the promoters of target genes and block their transcription. Arkadia degrades this complex including pSmad2/3 and unblocks the promoter. Here, we report that Arkadia is expressed highly in the mouse colonic epithelium. Heterozygous Akd(+/-) mice are normal but express less Arkadia. This leads to reduced expression of several TGF-β target genes, suggesting that normal levels of Arkadia are required for efficient signaling responses. Critically, Akd(+/-) mice exhibit increased susceptibility to azoxymethane/dextran sodium sulfate carcinogen-induced CRC, as they develop four-fold more tumors than wild-type mice. Akd(+/-) tumors also exhibit a more aggressive pathology, higher proliferation index, and reduced cytostasis. Therefore, Arkadia functions as a tumor suppressor whose peak expression is required to suppress CRC development and progression. The accumulation of nuclear SnoN and pSmad2, along with the downregulation of TGF-β target genes observed in Akd(+/-) colon and tumors, suggest that tumor-suppressing properties of Arkadia are mediated by its ability to derepress TGF-β signaling. Consistent with this likelihood, we identified mutations in primary colorectal tumors from human patients that reduce Arkadia function and are associated with the accumulation of nuclear SNON. Collectively, our findings reveal that Arkadia enhances TGF-β signaling responses and supports its tumor-suppressing properties in CRC.

ILK expression in human basal cell carcinoma correlates with epithelial-mesenchymal transition markers and tumour invasion

Papanikolaou S, Bravou V, Gyftopoulos K, Nakas D, Repanti M & Papadaki H
(2010) Histopathology 56, 799–809
ILK expression in human basal cell carcinoma correlates with epithelial–mesenchymal transition markers and tumour invasion

TGF-β repressors SnoN and Ski are implicated in human colorectal carcinogenesis

Background: The TGF-β signaling repressors SnoN and Ski have been critically implicated in human cancer. Methods: To explore the role of SnoN and Ski in the development and progression of colorectal cancer we examined their protein expression profile by immunohistochemistry in a series of human colorectal adenomas, carcinomas and lymph node metastases. The mRNA expression of SnoN was also quantified by Real-Time RT-PCR. Results: SnoN and Ski were overexpressed both in adenomas with severe dysplasia and colorectal carcinomas. Protein expression was cytoplasmic and nuclear with predominant cytoplasmic localization. The subcellular localization was related differently to pathologic variables of colorectal carcinomas. Although there was no significant association of protein levels with tumor invasion and metastasis, a significant correlation of nuclear SnoN and Ski with β-catenin pathway was observed. Moreover, SnoN mRNA did not differ in carcinomas as compared to normal control and there was no correlation between SnoN protein and mRNA levels. Conclusion: Our findings suggest that SnoN and Ski exert oncogenic effects in human colorectal carcinogenesis and their overexpression is implicated in early stage disease.

Loss of inhibitor of growth (ING-4) is implicated in the pathogenesis and progression of human astrocytomas.

Inhibitor of growth 4 (ING‐4) is a tumor suppressor gene that interacts with nuclear factor‐kappaB (NF‐κB) and represses its transcriptional activity. Several lines of evidence suggest that the tumor suppressor gene ING‐4, the transcription factor NF‐κB and its target genes matrix metalloproteases MMP‐2, MMP‐9 and urokinase plasminogen activator (u‐PA) are critically involved in tumor invasion. The aim of the present study was to investigate immunohistochemically the expression pattern of ING‐4, NF‐κB and the NF‐κB downstream targets MMP‐2, MMP‐9 and u‐PA in human astrocytomas from 101 patients. We found that ING‐4 expression was significantly decreased in astrocytomas, and ING‐4 loss was associated with tumor grade progression. Expression of p65, a NF‐κB subunit, was significantly higher in grade IV than in grade III and grade I/II tumors, and a statistical significant negative correlation between expression of ING‐4 and expression of nuclear p65 was noticed. MMP‐9, MMP‐2 and u‐PA were overexpressed in human astrocytomas. Of note, astrocytomas of advanced histologic grades (grade III, IV) displayed significantly higher expression levels of these proteins compared to tumors of lower grades (grade I, II). Collectively, our data suggest an essential role for ING‐4 in human astrocytoma development and progression possibly through regulation of the NF‐κB‐dependent expression of genes involved in tumor invasion.