Sox11 expression in astrocytic gliomas: correlation with nestin/c-Met/IDH1-R132H expression phenotypes, p-Stat-3 and survival.

BACKGROUND: Sox11 is a transcription factor expressed in foetal and neoplastic brain tissue, including gliomas. It has been shown to suppress the tumourigenicity of glioma stem cells in vivo, thereby being hypothesised to function as a tumour suppressor. METHODS: We investigated the expression of Sox11 in 132 diffuse astrocytomas in relation to the regulator cell marker nestin, c-Met and IDH1-R132H, which have shown to be differentially expressed among the molecular subgroups of malignant gliomas, as well as to an inducer of astrocytic differentiation, that is, signal transducer and activator of transcription (p-STAT-3), clinicopathological features and survival. RESULTS: Sox11 immunoreactivity was identified in all tumours irrespective of grade, but being correlated with p-STAT-3. Three out of seven cases showed partial Sox11 promoter methylation. In >50% of our cases neoplastic cells coexpressed Sox11 and nestin, a finding further confirmed in primary glioblastoma cell cultures. Furthermore, nestin, c-Met and IDH1-R132H expression differed among grade categories. Cluster analysis identified four groups of patients according to c-Met, nestin and IDH1-R132H expression. The c-Met/nestin high-expressor group displayed a higher Sox11 expression. Sox11 expression was an indicator of favourable prognosis in glioblastomas, which remained in multivariate analysis and validated in an independent set of 72 cases. The c-Met/nestin high-expressor group was marginally with shorter survival in univariate analysis. CONCLUSIONS: We highlight the importance of Sox11 expression as a favourable prognosticator in glioblastomas. c-Met/nestin/IDH1-R132H expression phenotypes recapitulate the molecular subgroups of malignant glioma.

pERK activation in esophageal carcinomas: clinicopathological associations.

MAPK (mitogen-activated protein kinase) pathway is considered a control regulator in various malignant tumors but its role in esophageal carcinomas remains elusive. In our study, we examined the possible prognostic significance of MAPK pathway in human esophageal cancer. We searched for mutations in exons 18-21 of EGFR gene, codons 12 and 13 of K-RAS gene and exon 15 of B-RAF gene by high resolution melting analysis (HRMA) and pyrosequencing in 44 esophageal carcinomas. Immunohistochemistry was performed in 29 cases in order to evaluate expression levels of pERK (extracellular-signal regulated kinase). In one laser microdissected squamous cell carcinoma, a somatic K-RAS mutation at codon 12 was detected, whereas none of the cases displayed mutations in EGFR and B-RAF genes. Elevated nuclear as well as cytoplasmic pERK expression (100% and 62% of cases respectively) was observed independently of EGFR and B-RAF mutational status. Increasing pERK nuclear and cytoplasmic expression as well as the intensity of nuclear staining was found to be significantly correlated with tumor grade in univariate and multivariate statistical analysis. Our findings depict the presence of activated ERK despite the low frequency of upstream alterations, implicating ERK activation in the acquisition of a more aggressive phenotype in esophageal cancer.

Analysis of PIK3CA and B-RAF gene mutations in human astrocytomas: association with activation of ERK and AKT.

OBJECTIVE: The analysis of the presence of PIK3CA and B-RAF gene mutations in relation to ERK and AKT activation in diffusely infiltrating astrocytomas, in order to determine their potential role in tumor aggressiveness. METHODS: Polymerase chain reaction-single strand confirmation polymorphism (PCR-SSCP) and sequencing analysis were used for PIK3CA and B-RAF gene mutation detection. pERK and pAKT expression were examined by immunohistochemistry. RESULTS: PIK3CA mutations were found in 2 (3%) cases of glioblastomas whereas none of these cases displayed mutations in exon 15 of B-RAF gene. Neither low grade astrocytomas nor anaplastic astrocytomas revealed any mutations in these genes. Nuclear and cytoplasmic pERK immunoreactivity was displayed in 100% and 82% of cases, respectively. pERK nuclear expression was positively correlated with pERK cytoplasmic expression (p = 0.0067). Moreover, pERK nuclear expression increased in parallel with tumor grade (II, III v/s IV, p = 0.0262). Nuclear and cytoplasmic pAKT immunoreactivity was displayed in 97% and 100% of cases, respectively. Similarly, pAKT nuclear expression was positively correlated with pAKT cytoplasmic expression (p = 0.0074). pAKT cytoplasmic expression increased with increasing tumor grade (II,III v/s IV, p = 0.0930), although the latter relationship was of marginal significance. pAKT cytoplasmic expression was also positively correlated with pERK nuclear expression (p = 0.0156). CONCLUSIONS: Our study reports the low frequency of PIK3CA and B-RAF mutations in astrocytomas, despite the presence of activated ERK and AKT proteins. Moreover, the correlation of pERK nuclear and pAKT cytoplasmic expression with tumor grade suggests the possible crucial role of the activation of these proteins in human gliomagenesis.

Minichromosome maintenance proteins 2 and 5 in non-benign epithelial ovarian tumours: relationship with cell cycle regulators and prognostic implications.

Minichromosome maintenance proteins (MCM) have recently emerged as novel proliferation markers with prognostic implications in several tumour types. This is the first study investigating MCM-2 and MCM-5 immunohistochemical expression in a series of ovarian adenocarcinomas and low malignant potential (LMP) tumours aiming to determine possible associations with clinicopathological parameters, the conventional proliferation index Ki-67, cell cycle regulators (p53, p27(Kip1), p21(WAF1) and pRb) and patients' outcome. Immunohistochemistry was applied in a series of 43 cases of ovarian LMP tumours and 85 cases of adenocarcinomas. Survival analysis was restricted to adenocarcinomas. The median MCM-2 and MCM-5 labelling indices (LIs) were significantly higher in adenocarcinomas compared to LMP tumours (P<0.0001 for both associations). In adenocarcinomas, the levels of MCM-2 and MCM-5 increased significantly with advancing tumour stage (P=0.0052 and P=0.0180, respectively), whereas both MCM-2 and MCM-5 increased significantly with increasing tumour grade (P=0.0002 and P=0.0006, respectively) and the presence of bulky residual disease (P<0.0001 in both relationships). A strong positive correlation was established between MCM-2 or MCM-5 expression level and Ki-67 LI (P<0.0001) as well as p53 protein (P=0.0038 and P=0.0500, respectively). Moreover, MCM-2 LI was inversely correlated with p27(Kip-1) LI (P=0.0068). Finally, both MCM-2 and MCM-5 were associated significantly with adverse patients' outcome in both univariate (> or =20 vs >20%, P=0.0011 and > or =25 vs <25%, P=0.0100, respectively) and multivariate (P=0.0001 and 0.0090, respectively) analysis. An adequately powered independent group of 45 patients was used in order to validate our results in univariate survival analysis. In this group, MCM-2 and MCM-5 expression retained their prognostic significance (P<0.0001 in both relationships). In conclusion, MCM-2 and MCM-5 proteins appear to be promising as prognostic markers in patients with ovarian adenocarcinomas.

c-FLIP expression in colorectal carcinomas: association with Fas/FasL expression and prognostic implications.

AIMS: Disruption of apoptotic cell death has been implicated in tumour aggressiveness in colonic carcinogenesis. The Fas-Fas ligand (FasL) system is involved in the execution of apoptosis induced by the immune system. c-FLIP protein constitutes an inhibitor of Fas and other (TRAIL) death receptor-mediated apoptosis. The aim of this study was to investigate the simultaneous expression of Fas, FasL and c-FLIP in relation to standard clinicopathological parameters and patients' outcome in colorectal cancer. METHODS AND RESULTS: Levels of Fas, FasL and c-FLIP protein expression were quantified immunohistochemically in paraffin-embedded tissues from 90 patients. Immunopositivity was detected for Fas, FasL and c-FLIP in 71%, 35.5% and 68.8% of cases, respectively. Concurrent expression of Fas/FasL was seen in 28 samples (31%), of which 24 (85.7%) also displayed c-FLIP positivity (P = 0.04). c-FLIP overexpression (> 10%) tended to prevail marginally in higher stage tumours (P = 0.09). Additionally, FasL and c-FLIP adversely affected survival on both univariate (P = 0.001 and P = 0.0024, respectively) and multivariate analysis [hazard ratio (HR) 3.491, P = 0.005 and HR 2.960, P = 0.036, respectively]. CONCLUSIONS: The frequent expression and coexpression of Fas, FasL and c-FLIP in colorectal carcinoma implicates c-FLIP as an inhibitor of the Fas-FasL-induced death pathway in these tumours. Moreover, c-FLIP conveys independent prognostic information in the presence of classical prognosticators.

Microsatellite instability in sporadic and inherited colon adenocarcinomas from Greek patients: correlation with several clinicopathological characteristics.

BACKGROUND AND AIMS: Microsatellite instability seems to play a significant role in colorectal carcinogenesis, as it is reported to occur in HNPCC patients as well as in a proportion of sporadic cases. The aim of this study was to examine the presence of microsatellite instability in relation to other commonly observed genetic abnormalities and clinicopathological characteristics of sporadic and inherited colorectal cancers. METHODOLOGY: One hundred and three sporadic colorectal adenocarcinomas and 9 adenocarcinomas from HNPCC patients were histologically evaluated. The presence of microsatellite instability was investigated at six loci. K-ras and p53 mutations, p53 LOH, hMLH1 expression and methylation status were examined as well. Statistical analysis was performed to define possible correlations of the observed genetic alterations with the clinicopathological characteristics of the analysed tumors. RESULTS: High-grade microsatellite instability was found in 14% of sporadic adenocarcinomas and in 78% of adenocarcinomas from HNPCC patients. K-ras and p53 mutations were found in 29% and 28% of sporadic adenocarcinomas respectively and in 0% and 22% of the 9 HNPCC cases. A statistically significant correlation was noticed in sporadic tumors between the presence of MSI-H and tumor location at the proximal colon, as well as with the female gender. CONCLUSIONS: Sporadic MSI+ colon adenocarcinomas seem to represent a distinct entity with a unique profile of genetic changes, different from those observed in HNPCC or MSI negative sporadic tumors.

An unexpected wide population variation of the G1733A polymorphism of the androgen receptor gene: data on the Mediterranean region.

The androgen receptor (AR) has been proposed as a candidate gene for several cancers (breast, prostate, uterine endometrium, colon, and esophagus). Ethnicity is considered an associated risk factor for some of these cancers. Several case-control genetic studies have been focused in samples of the main ethnic groups, but little is known about the distribution of risk polymorphisms in current populations with accurate ethnic and/or geographic origins. The A allele of the G1733A polymorphism of the AR gene has been associated with increased risk of prostate cancer. We provide data from this marker in 12 samples from 7 Mediterranean countries such as Spain, Italy (Sardinia), Greece, Turkey, Morocco, Algeria, and Egypt. A sample from Ivory Coast has also been analyzed. The A allele distribution shows a frequency in the Ivory Coast population (65.17%) that contrasts with the low values found in Northern Mediterraneans (mean average value of 13.98%). North African populations present two-times higher frequencies (average value of 27.19%) than Europeans. The wide population variation range found for the A allele strengthens the potential interest of further screening as a baseline to the design of future preventive and population health programs.

Mononucleotide markers of microsatellite instability in carcinomas of the urinary bladder.

AIMS: To determine the presence of microsatellite instability (MSI) and to assess the expression of the human mismatch repair (MMR) gene products hMLH1 and hMSH2 in primary transitional cell carcinomas (TCCs) of the urinary bladder in relation to clinico-pathological parameters. METHODS: Seventy-two cases of primary TCC were screened for the presence of alterations in MSI markers by molecular techniques and evaluated immunohistochemically for the expression of hMLH1 and hMSH2 proteins. Clinical data were available in 70 cases. The percentage of MSI rose to 16.6%. RESULTS: Reduced (<20%) hMLH1 expression was closely related to the presence of MSI (p=0.0004). Neither MMR proteins nor MSI was associated with grade, stage, papillary status. Clinical outcome analysed as a function of MSI did not show significant differences in terms of both disease-free and overall survival. Reduced hMLH1 expression was a significant predictor of shorter disease-free survival in univariate and multivariate analysis. CONCLUSIONS: The presence of MSI is not related to classical clinico-pathological parameters in TCCs, nor does it appear to be of prognostic significance. hMLH1 was an important indicator for recurrence.

The frequency and distribution of mast cells in pleomorphic adenomas of salivary glands.

AIM: To investigate whether the frequency and distribution of mast cells (MCs) in pleomorphic adenomas (PAs) of major and minor salivary glands justifies the suggestion that there exists an association between MCs and mucoid stromal changes in PAs. METHODS: The material consisted of 22 cases of pleomorphic adenoma (eight arising in major and 14 in minor salivary glands) and a control group represented by five cases of monomorphic adenoma (MA). Representative 3-microm thick, paraffin-embedded sections were stained with H&E and Azur A. Computer-aided image analysis was performed in order to evaluate the relative surface area occupied by epithelial and connective tissue components, as well as the absolute number of MCs. RESULTS: According to our findings, PAs from minor salivary glands contain significantly greater numbers of mast cells compared with tumours from major glands. Additionally, the distribution of MCs within the stromal connective tissue appeared not to be random. CONCLUSION: It is possible that differences in the pattern of connective tissue might influence the actual concentration of MCs and that these differences are responsible for the observed variations between major and minor gland PAs.

p53 mutations detection in urinary bladder cancer in the Greek population: application of the NIRCA assay.

The aim of the present study was to assess the use of an alternative molecular approach for p53 mutation detection and to investigate the usefulness of p53 as a prognostic indicator in bladder cancer. We applied the NIRCA assay, which consists of two-step PCR amplification, transcription of the amplified sequence, hybridisation of the transcripts and treatment with RNAses which recognizes mismatches due to the presence of mutations. Results of molecular analysis are correlated with immunohistochemical findings, standard clinopathological parameters and survival. p53 mutations were detected in 42.4% of the 66 examined TCCs cases. We could not demonstrate any statistical relationship between the presence of p53 mutation and p53 protein overexpression, and tumor stage or grade. A trend towards higher mutation rate in higher grade tumours was observed, although this failed to reach statistical significance. Despite the observation that the alterations of p53 gene are associated features of aggressive phenotype of transitional cell carcinomas they do not seem to offer additional prognostic information.

Androgen receptor status in nontumoral and malignant human colorectal tissues.

Data on androgen receptor (AR) status of nontumoral and malignant human colorectal tissues are compared using ligand binding assay in 22 patients who underwent surgery for colorectal cancer at the "M. Ascoli" Cancer Hospital Centre in Palermo, Sicily. In nontumoral tissues, ARs were predominantly (67%) positive, with 25% of cases having a 0/+ status. Conversely, malignant tissues showed only 32% of cases with a positive (+/+) AR status, with a proportional increase of 0/+ cases (from 25% to 55%); the extent of AR-negative (0/0) cases remained fairly constant (8-9%). Overall, our evidence indicates that nontumoral colorectal tissues have a predominantly positive (+/+) AR status and that this condition shifts towards a significant decrease of AR-positive cases in cancer tissues. Studies on the relation between status of sex steroid receptors and specific biomolecular markers in human colorectal tumors are currently being carried out in our laboratories.

A modified mutagenic PCR-RFLP method for K-ras codon 12 and 13 mutations detection in NSCLC patients.

Evidence from many investigators has shown that mutations in the first exon of K- ras gene occur at elevated frequencies in lung, pancreatic and colon carcinoma and seem to be of prognostic importance. The aim of this study was to develop an effective method for the detection of K- ras mutations in codons 12 and 13 in non-small-cell lung cancer (NSCLC) patients in order to investigate correlation with clinical outcome. DNA was extracted from tumour and neighbouring non-neoplastic lung tissues from 70 patients and screened for codon 12 and 13 mutations. We applied a mutagenic PCR-restriction fragment length polymorphism for both codon 12 and 13 mutation detection. Codon 12 mutation was identified in 20% of NSCLC patients, whereas no codon 13 mutation was detected. As expected, the respective non-neoplastic tissues exhibited no mutations. We observed an increased codon 12 mutation prevalence in adenocarcinoma comparing to other types of carcinomas. Follow-up for 29 patients with a mean time of 12 months indicates an increased relapse rate in NSCLC patients with the K- ras codon 12 mutation. Furthermore, a trend towards increased percentage of mutant samples was observed in the advanced stage group of patients. We provide evidence that our approach is a fast and reliable method for screening K- ras exon 1 mutations in tumour samples from NSCLC patients.

Genetic alterations involved in the development of gallbladder carcinomas from Greek patients.

BACKGROUND/AIMS: The genetic pathways of gallbladder cancer are not yet well defined since the contribution of genetic abnormalities clarified in other organs remains questionable. METHODOLOGY: We investigated a group of 22 gallbladder carcinomas from Greek patients with regard to p53 mutations, bax and TGF-beta RII alterations--as indicators of microsatellite instability. The findings were correlated to the presence of ras mutations, patients' clinicopathologic features and survival. PCR-SSCP analysis was performed for the detection of p53 mutations in conserved domains IV and V. RESULTS: In five tumors p53 mutations were detected; none of them was ras mutated. Although these tumors were characterized by flat morphology, low histologic grade and rather advanced stage, no statistical correlation could be determined. No indications of microsatellite instability were found. CONCLUSIONS: Ras and p53 genes do not appear to cooperate during gallbladder cancer, at least as far as the flat type of cancer is concerned. p53 alterations are likely to take part in the de novo pathway of gallbladder carcinogenesis.

Signet ring basal cell carcinoma. A case study emphasizing the differential diagnosis of neoplasms with signet ring cell formation.

Signet ring cells are cells in which the nucleus is crescentically compressed to the cellular border so that the cells look like signet rings. Due to the pluripotential nature of the basal cells of the epidermis, basal cell carcinoma displays many histopathological variants. We herein report the rare case of a middle-aged woman who had a basal cell carcinoma on the skin of the upper lip. The neoplasm was predominantly composed of cells with signet ring configuration. Histochemically, the latter were mucin-negative. Immunohistochemistry demonstrated intracytoplasmic reactivity for cytokeratin MNF116 with strong staining intensity, as well as for smooth muscle actin. The signet ring tumor cells were S100 protein-negative and carcinoembryonic antigen-negative. The lack of ploidy abnormality as well as of molecular alterations in K-ras and p53 genes may explain in part the non-aggressive biological behavior of the present tumor. Because of potential diagnostic difficulties, the pathologist should be aware of this unusual form of basal cell carcinoma. A brief review of the literature on the differential diagnosis of signet ring cell cutaneous tumors is presented.

Atherosclerosis of the carotid artery: absence of evidence for CMV involvement in atheroma formation.

Several studies suggest that certain viral and bacterial pathogens may contribute to the process of atherogenesis. However, this relation between infectious agents and atherosclerosis has not yet been established with certainty. The aim of this study was to investigate the presence of CMV in carotid endarterectomies from 40 patients suffering from atherosclerosis using immunohistochemistry and the polymerase chain reaction (PCR). None of the specimens examined gave a positive result, indicating absence of CMV particles or CMV DNA sequences in the walls of carotid arteries. This finding suggests it is possible that CMV infection may not play a major role in the formation of atheroma. Therefore, further investigation is required in order to clarify the etiology of atherosclerosis.

Morphological-histochemical study of intestinal carcinoids and K-ras mutation analysis in appendiceal carcinoids.

Intestinal carcinoids are potentially malignant neoplasms. Their histogenesis and pathogenesis are currently uncertain. The morphological and histochemical characteristics of twenty intestinal carcinoids are studied. The primary sites of three mucin-producing tumors were examined by electron microscope. Furthermore 11 appendiceal carcinoids were analysed by the polymerase chain reaction (PCR) for the detection of ras and p53 point mutations. Microscopically all carcinoids were of mixed type. Focal mucin production was evident in three carcinoids that metastasised to regional lymph nodes. HID-Alcian blue staining proved that mucin in both primary and secondary foci did not belong to the sulphated group. The secretory granules and mucin droplets found in a single neoplastic cell suggest that carcinoids of the small intestine and some of the appendix arise from the endoderm. Neither ras nor p53 mutations were detected. It seems that ras oncogenes are probably not involved in the pathogenesis of appendiceal carcinoids.

Isolation and characterization of a novel bladder cancer cell line: inhibition by epidermal growth factor.

A novel continuous cell line, designated BC3c, was established from a surgical biopsy of an invasive solid transitional cell carcinoma of the bladder derived from an 82-yr-old Caucasian female. BC3c cells were near-triploid bearing multiple structural and numerical chromosome anomalies. The epithelial origin of the cancer cells was indicated by the expression of cytokeratins 8 and 19 as well as by the absence of mesenchymal markers. Polymerase chain reaction-restriction-fragment length polymorphisms and single-strand conformation polymorphism mutation detection assays did not reveal any mutations in H-ras codon 12 and K-ras codons 12 and 13. In addition, no mutation in specific hot-spot codons of the p53 gene and no accumulation of the p53 protein were observed. BC3c cells grew rapidly in vitro, even in the absence of exogenous growth factors, because they were found to stimulate their growth in an autocrine manner. BC3c cells were found to express the epidermal growth factor-receptor (EGF-r) abundantly, but in contrast to other established bladder cancer cell lines, human recombinant epidermal growth factor inhibited the cells' proliferation in vitro. These features render the newly established bladder cancer cell line BC3c a useful tool for further experimentation.

Determination of CMV placentitis. Diagnostic application of the polymerase chain reaction.

Cytomegalovirus (CMV) infection constitutes an important cause of intrauterine death. In the present study CMV infection of placentas resulting from intrauterine deaths was assessed by immunohistochemistry and by the polymerase chain reaction (PCR). Among 32 cases of chronic villitis examined, 7 were found by PCR to be associated with CMV infection, although light microscopic examination revealed only 3 of them, while 4 had shown positive immunohistochemical staining. In conclusion, CMV may be considered to be a relatively common cause of placentitis, and PCR is a helpful tool in confirming the nature of the disease.

Detection of ras oncogene point mutations and simultaneous proliferative fraction estimation in gallbladder cancer.

Gallbladder cancer is notorious for its poor clinical evolution; so, a study of parameters with any prognostic potential is of particular interest. In this study, we investigated 23 adenocarcinomas regarding both the presence of point mutations in the ras gene family and the quantitative expression of Proliferating Cell Nuclear Antigen (PCNA). We respectively used molecular biology techniques and immunohistochemistry. Our results were related to several clinicopathologic determinators as well as to patients' survival. Mutations in codon 12 of the K-ras gene were detected in four gallbladder neoplasms (17%). This specific type of mutation is likely to be partially involved in this organ's tumourigenesis, particularly since no H-ras codon 12 or K-ras codon 13 (aspartic acid) mutations were detected in any of our specimens. PCNA immunoreactivity was generally limited in all tumours studied except for those morphologically characterized as being particularly aggressive. Notably, the four tumours in which K-ras mutation was detected, demonstrated relatively high PCNA positive immunoexpression in their malignant cells. This finding reinforces a possible correlation between the presence of ras mutations and increased cell growth. Staging was the only factor which was statistically associated with survival (p < 0.01). Therefore, the poor evolution of this cancer is probably due to late diagnosis and not related to a model of increased biologic aggressiveness.

Immunohistochemical expression of C-myc oncogene, heat shock protein 70 and HLA-DR molecules in malignant cutaneous melanoma.

The clinical course of malignant melanomas is frequently unpredictable, although a number of prognostically useful variables can be identified. There is a need for additional markers of prognostic value. In a series of 60 malignant cutaneous melanomas, we analysed the immunohistochemical expression of c-myc proto-oncogene, heat shock protein 70 (HSP70) and HLA-DR molecules in order to investigate their prognostic significance. C-myc, HSP70 and HLA-DR were expressed in 43.3%, 56.6% and 38.3% of all melanoma cases, respectively. Advanced Clark levels (Clark III-V) were significantly associated with c-myc expression rate (P < 0.05), HSP70 detection (P < 0.01) and HLA-DR positivity (P < 0.01). Increased Breslow thickness (> 1.5 mm) was related to HLA-DR expression (P < 0.05). High mitotic rate was closely associated with c-myc positivity (P < 0.05), while HSP70 and HLA-DR expression separately correlated to clinical stage of the disease (P < 0.05). The evaluation of these variables may be of immunological and prognostic significance. They were found to be associated with melanocyte subpopulations of the vertical growth phase which are arguably characterized by an increased invasive potential.