S. Staibano

Effect of Helicobacter pylori infection and its eradication on cell proliferation, DNA status, and oncogene expression in patients with chronic gastritis

BACKGROUND Helicobacter pylori, the main cause of chronic gastritis, is a class I gastric carcinogen. Chronic gastritis progresses to cancer through atrophy, metaplasia, and dysplasia. Precancerous phenotypic expression is generally associated with acquired genomic instability. AIM To evaluate the effect of H pylori infection and its eradication on gastric histology, cell proliferation, DNA status, and oncogene expression. METHODS/SUBJECTS Morphometric and immunohistochemical techniques were used to examine gastric mucosal biopsy specimens from eight controls, 10 patients withH pylori negative chronic gastritis, 53 withH pylori positive chronic gastritis, and 11 with gastric cancer. RESULTS All patients with chronic gastritis were in a hyperproliferative state related to mucosal inflammation, regardless of H pyloriinfection. Atrophy was present in three of 10 patients withH pylori negative chronic gastritis and in 26 of 53 with H pylori positive chronic gastritis, associated in 18 with intestinal metaplasia. DNA content was abnormal in only 11 patients with atrophy and H pylori infection; eight of these also had c-Myc expression, associated in six cases with p53 expression. Fifty three patients withH pylori positive chronic gastritis were monitored for 12 months after antibiotic treatment: three dropped out; infection was eradicated in 45, in whom cell proliferation decreased in parallel with the reduction in gastritis activity; atrophy previously detected in 21/45 disappeared in five, regressed from moderate to mild in nine, and remained unchanged in seven; complete metaplasia disappeared in 4/14, and markers of genomic instability disappeared where previously present. In the five patients in whomH pylori persisted, atrophy, metaplasia, dysplasia, and markers of genomic instability remained unchanged. CONCLUSIONS ChronicH pylori infection seems to be responsible for genomic instability in a subset of cases of H pylori positive chronic atrophic gastritis; eradication ofH pylori infection can reverse inflammation and the related atrophy, metaplasia, and genomic instability.

Survivin expression in oral squamous cell carcinoma

A series of 110 cases of oral squamous cell carcinoma (SCC) together with six lymph node and one distant metastatic lesions was analysed for expression of survivin, a recent apoptosis inhibitor, by immunohistochemistry and Western blotting. In total, 91 cases (82.7%) of carcinoma and all metastasis (seven cases, 100%) were positive for survivin expression, with weighted survivin scores ranging from 1 to 4. In contrast, normal oral epithelium did not express survivin. There was no significant correlation between survivin expression and age, sex, tumour size, the presence of lymph node and distant metastases. Survivin expression was increased in poorly differentiated tumours, even if differences were not statistically significant. In contrast, when analysed for prognostic significance, patients with low survivin expression had statistically significant better survival rates than the group with high survivin expression (P<0.05). These data suggest that survivin expression may identify cases of oral SCC with more aggressive and invasive phenotype.

The prognostic significance of tumor angiogenesis in nonaggressive and aggressive basal cell carcinoma of the human skin

Basal cell carcinoma (BCC) is currently the most common cutaneous cancer found in humans. Although it generally shows a relatively benign course (BCC1), some cases of BCCs show aggressive behavior, rapidly infiltrating deeper structures, or metastasizing (BCC2). Until now, the traditional histological diagnostic criteria have failed to discriminate unequivocally between BCC1 and BCC2. Therefore, there is still a need to find reliable prognostic indicators that correlate with outcome and may detect patients at high risk for BCC recurrence, or metastasis and death. Recent studies have suggested that there is a significant correlation between tumor angiogenesis, expressed as the microvessel density within and toward a tumor, tumor aggressiveness, and the overall survival of patients with solid tumors. In this study, the authors examined the angiogenic rate in human cutaneous BCCs, to establish if it correlates with their biological behavior. Vessels were highlighted by immunocytochemical staining for FVIII-related antigen in formalin-fixed, paraffin-embedded tissues. All BCC2 samples of this series showed a significantly higher microvessel count than did BCC1. The existence of a significant discrepancy between the neovascularization in BCC1 and BCC2 suggests that the angiogenetic process may be an important step in the acquisition of the aggressive (malignant) phenotype in human. BCCs. The findings of the present study seem to establish a correlation between tumor vascularization and clinicobiological parameters of aggressiveness in BCC. Considering the emerging studies on the possible clinical use of substances interfering with the angiogenetic process, it is possible that the local therapy for BCCs could become less destructive, with consequent improvement in the quality of life of these patients, apart from the prolongation of the overall survival. From this view-point, the assay of microvessel density might be helpful in selecting patients with cutaneous BCCs at high risk for recurrence or metastasis, who could benefit ab initio from additional therapies and closer follow-up.

Cyclooxygenase-2 expression in oral squamous cell carcinoma

Cyclooxygenase (COX), the key enzyme in prostaglandin cascade, is expressed in two isoforms: the constitutive COX-1 and the inducible COX-2. Hyper-expression of COX-2 has been implicated in the pathogenesis of colon-rectal cancer in humans but it appears to play a significant role as a tumour progression factor also in other forms of human cancer, including oral cancer. The aim of this study was to analyze the expression of COX-2, at the protein level, in 45 cases of oral squamous cell carcinoma. Standard immunohistochemical streptavidin-biotin peroxidase analysis was carried out with a highly specific antibody against human COX-2 and cell specific markers, in 45 oral squamous cell carcinomas. Our study revealed a moderate to high COX-2 expression in 35 out of the 45 oral squamous cell carcinoma specimens (77.8%). COX-2 expression appeared particularly abundant in the superficial ulcerated layers of relatively well differentiated carcinomas. However, we were unable to assess any statistically significant association between COX-2 hyper-expression and tumor site, tumor grading, tumor size, presence of lymph node metastases, tumor stage and age at onset, respectively. Interestingly, COX-2 expression was detected not only in areas of epithelial dysplasia adjacent to the primary layers (86% of the cases) but also in normal-appearing epithelium at the boundaries of squamous cell carcinomas (77%), indicating a possible involvement in tumour progression by the apparently normal tissue surrounding the lesion. Moreover, intense COX-2 staining was observed in endothelial cells of intra-tumour vessels and extra-tumour vessels adjacent to the tumour nests, in a high proportion of cases (82%). COX-2 positivity was associated with CD34 and VEGF positivity, indicating that these vessels were probably neo-formed. From this study, as well as from other works, it appears that COX-2 is over-expressed in this important human malignancy. However, further studies are necessary to understand the exact magnitude of this over-expression and, mostly, the possible role of COX-2 in the pathogenesis and progression of oral cancer.

Loss of oestrogen receptor β, high PCNA and p53 expression and aneuploidy as markers of worse prognosis in ovarian granulosa cell tumours

Aims:u2002 Ovarian granulosa cell tumour (OGCT) is a sex‐cord stromal tumour with a general trend toward late relapse and/or metastasis. However, mortality rate corrected for long‐term follow‐up shows that about 50% of patients die within 20u2003years of diagnosis. Classical clinicopathological parameters are unable to predict the biological behaviour of OGCT. The involvement of a recently characterized subtype of oestrogen receptor, ERβ, in ovarian carcinogenesis has been hypothesized.

OPN/CD44v6 overexpression in laryngeal dysplasia and correlation with clinical outcome

Laryngeal dysplasia is a common clinical concern. Despite major advancements, a significant number of patients with this condition progress to invasive squamous cell carcinoma. Osteopontin (OPN) is a secreted glycoprotein, whose expression is markedly elevated in several types of cancers. We explored OPN as a candidate biomarker for laryngeal dysplasia. To this aim, we examined OPN expression in 82 cases of dysplasia and in hyperplastic and normal tissue samples. OPN expression was elevated in all severe dysplasia samples, but not hyperplastic samples, with respect to matched normal mucosa. OPN expression levels correlated positively with degree of dysplasia (P=0.0094) and negatively with disease-free survival (P<0.0001). OPN expression was paralleled by cell surface reactivity for CD44v6, an OPN functional receptor. CD44v6 expression correlated negatively with disease-free survival, as well (P=0.0007). Taken as a whole, our finding identify OPN and CD44v6 as predictive markers of recurrence or aggressiveness in laryngeal intraepithelial neoplasia, and overall, point out an important signalling complex in the evolution of laryngeal dysplasia.

Nucleolar organizer regions in aggressive and nonaggressive basal cell carcinoma of the skin

Nucleolar organizer regions (NOR) were investigated on routine paraffin‐embedded histologic sections of 11 aggressive basal cell carcinomas that recurred and/or metastasized (BCC2) and 11 nonaggressive basal cell carcinomas (BCC1). The absolute number of NOR per nucleus was higher in BCC2 than in BCC1, and their distribution pattern was also different. In fact, the means of argyrophilic staining of NOR (AgNOR) counts in the two groups of tumors by two observers were 6.56 with a SD of 1.98 for the nonaggressive and 9.48 with a SD of 2.12 for the aggressive basal cell carcinomas. A statistical analysis of these data using the Students t test confirmed these observations (t = 64.49). Problems in the evaluation of NOR and possible comparison with other experiences are also discussed. The authors conclude that a quantitative as say of AgNOR and perhaps their distribution pattern may provide information useful to recognize BCC2 and hence may be of help in their prognostic prediction.

Up-regulation of heparin binding epidermal growth factor-like growth factor and amphiregulin expression in Helicobacter pylori-infected human gastric mucosa

BACKGROUNDnHost response plays a major role in pathogenesis of Helicobacter pylori-induced gastroduodenal disease including adenocarcinoma of distal stomach. Epidermal growth factor-related growth factors are important modulators of gastric homeostasis in normal and damaged gastrointestinal mucosa.nnnAIMnTo evaluate expression of heparin binding epidermal growth factor and amphiregulin in antral mucosa of Helicobacter pylori-infected and non-infected dyspeptic patients and to correlate levels of heparin binding-epidermal growth factor and amphiregulin mRNA with mitogenic activity of gastric epithelial cells.nnnMETHODSnA total of 10 Helicobacter pylori-infected and 15 Helicobacter pylori non-infected (10 with and 5 without gastritis) dyspeptic patients were studied. Diagnosis of Helicobacter pylori infection was based on rapid urease test and histology. Heparin binding-epidermal growth factor and amphiregulin mRNA expression in antral mucosa were assessed by reverse transcriptase-polymerase chain reaction. Protein expression and localization of both peptides were determined by immunohistochemistry. Mitogenic activity of antral gastric mucosa was assessed by determination of proliferating cell nuclear antigen labelling index by immunohistochemistry.nnnRESULTSnHeparin binding-epidermal growth factor and amphiregulin mRNA expression increased in Helicobacter pylori-infected vs Helicobacter pylori non-infected patients. Heparin binding-epidermal growth factor and amphiregulin immunostaining was more intense and deeper in gastric gland compartment in infected mucosa than in non-infected mucosa. Increase in heparin binding-epidermal growth factor and amphiregulin mRNA expression significantly correlated with increase in proliferating cell nuclear antigen labelling index.nnnCONCLUSIONSnHelicobacter pylori gastritis is associated with up-regulation of heparin binding-epidermal growth factor and amphiregulin which correlates with increased mitogenic activity of gastric mucosa. Increased heparin binding-epidermal growth factor and amphiregulin expression is postulated to contribute to reparative response of gastric mucosa to Helicobacter pylori infection.

Effects of gonadotropin-releasing hormone agonists on uterine volume and vasculature and on the immunohistochemical expression of basic fibroblast growth factor (bFGF) in uterine leiomyomas.

We investigated the effect of the GnRH agonist (GnRH-a) on the uterine volume and on the immunohistochemical expression of basic fibroblast growth factor (bFGF) and the vasculature of leiomyomas. Twenty-five women were treated with leuprorelin acetate for 3 months; 46 untreated patients were enrolled as a control group. The uterine volume was measured by ultrasonography. After myomectomy or hysterectomy, the immunoexpression of bFGF and the endothelial marker, CD34, was studied and compared in treated and untreated leiomyomas. Uterine volume decreased after therapy. The number of cells expressing bFGF and the vascularity were diminished in treated leiomyomas. Reduction in the blood supply might be responsible, in part, for uterine-volume shrinkage after GnRH-a therapy.

Genetic analysis of oral squamous cell carcinoma by cDNA microarrays focused apoptotic pathway

We investigated mRNA expression of the genes involved in the apoptotic mechanism in oral squamous cell carcinoma (OSCC) by cDNA microarray. The aim of this study was to identify genes mainly involved in tumorigenesis, comparing the difference of gene expression in neoplastic and non-neoplastic tissues. Eight frozen samples of OSCC and the corresponding normal oral mucosa were treated to obtain mRNA. The mRNA extracted from these specimens was converted into cDNA and analyzed with “SuperArray GEArray Q Series Human Apoptosis Gene Array kit”. Our results showed that in OSCC there is a different expression of CRADD, FADD, ATM and APAF-1 genes compared to normal mucosa. Real-Time PCR, and Western blot analysis were performed on a separate cohort of patients in order to confirm the results obtained by DNA microarray. Our analysis of apoptotic process through microarray technology confirmed that different molecules could be responsible or favour the imbalance of apoptosis in cancer tissues. Microarray technology has made it possible to analyze the expression of multiple genes in a single experiment. However, most commercial array kits, designed to include as many genes as possible, produce a vast amount of data that often is difficult to interpret. In addition, the cost of equipment is often prohibitive. In contrast, the focused kit used was a complete, affordable and effective method to improve knowledge of molecular specific pathways.