Fortunata Vasaturo

Survivin, bcl-2, bax, and bcl-X Gene Expression in Sentinel Lymph Nodes From Melanoma Patients

PURPOSE The expression of apoptosis-related genes, such as survivin, bcl-2, bcl-X, and bax, has been evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry in sentinel lymph nodes (SLNs) from melanoma patients and then correlated to the outcome of patients. PATIENTS AND METHODS Thirty-six SLNs were examined. After RNA extraction, an RT-PCR followed by Southern blot hybridization was performed to detect survivin, bcl-2, bcl-X, and bax mRNA. bcl-2, survivin, and bax gene expression was evaluated, whenever possible, also by immunohistochemistry at the protein level. RESULTS We found a significant correlation (P <.005) between survivin expression and outcome of patients; in fact, 61.5% of patients expressing survivin gene progressed or died because of the disease, whereas 38.5% are currently disease-free. Among patients negative for survivin expression, 100% are disease-free after a median follow-up time of 52.9 months. We did not find a significant correlation between bcl-2, bax, and bcl-X gene expression and outcome of patients. In fact, these genes were found equally expressed in patients with disease progression and in disease-free patients. CONCLUSION Our findings show a variable expression of apoptosis-related genes in SLNs of melanoma patients; more interestingly, we found that survivin expression correlates to outcome of patients in a statistically significant way, whereas the expression of other genes, such as bcl-2, bax, and bcl-X, did not seem to correlate to progression of disease. We suggest that the detection of survivin gene expression by RT-PCR in SLNs may be a useful prognostic indicator.

Prognostic significance of cyclooxygenase-2 (COX-2) and expression of cell cycle inhibitors p21 and p27 in human pleural malignant mesothelioma

Background: A study was undertaken to analyse the potential prognostic value of the immunohistochemical expression of cyclooxygenase-2 (COX-2) and p27 in 29 malignant mesotheliomas already screened for the expression of p21 and p53. Methods: Immunohistochemistry was used to determine the expression of COX-2 and p27. The correlation with survival of these factors and of p21 and p53 expression was assessed by univariate and multivariate analyses. Results: A positive statistically significant correlation was found between p27 and p21 expression (p<0.0001), but there was a negative correlation between COX-2 expression and both p27 (p = 0.001) and p21 (p<0.0001). No statistically significant correlation was recorded between p53 and all the other immunohistochemical parameters. Univariate analysis showed that overall survival was strongly influenced by p21, p27, and COX-2 expression, but multivariate Cox regression analysis showed that the only immunohistochemical parameter to influence overall survival of patients with mesothelioma was COX-2. Conclusions: These findings suggest that COX-2 expression may be a useful prognostic parameter for mesothelioma.

Surviving acute myocardial infarction: survivin expression in viable cardiomyocytes after infarction

Background: Apoptosis is a key feature in postinfarction remodelling leading to progressive myocyte loss. Both proapoptotic and antiapoptotic factors contribute to the delicate balance between death and survival. The survivin pathway has emerged as essential in the control of apoptosis, although its role in heart disease is unknown. Aim: To evaluate survivin expression after acute myocardial infarction (AMI). Methods: Survivin expression was assessed immunohistochemically in the peri-infarct and remote viable myocardium in 17 consecutive patients who died 1–30 weeks after AMI and in four control hearts. Results: Survivin was expressed by myocytes in the peri-infarct area in eight patients and in the remote region in 13 patients. The rate of survivin expression after AMI was significantly higher in the remote versus peri-infarct regions and compared with control hearts. Its expression was inversely associated with the presence of dilated cardiopathy and of apoptosis, independently from the gross pathology infarct size. Conclusions: Survivin myocardial expression after AMI may be associated with the survival of at risk myocardium and may be indicative of more favourable remodelling after AMI. These findings identify a potential new target for the treatment of postinfarction remodelling.

Cyclo-oxygenase-2 (COX-2) expression at the site of recent myocardial infarction: friend or foe?

Background: Cyclo-oxygenase-2 (COX-2) is induced in cardiomyocytes only in response to stress, such as ischaemia. Objective: To assess COX-2 expression at the site of recent myocardial infarction. Methods: COX-2 expression was evaluated by specific immunostaining in cardiomyocytes from 23 subjects who died 10–60 days after acute myocardial infarction. The relation between COX-2 myocardial expression and apoptotic rate was investigated. Cardiomyocyte apoptotic rate was defined as the number of cells co-expressing in situ end labelling of DNA fragmentation (TUNEL) and immunostaining for activated caspase-3. Results: COX-2 expression was found in cardiomyocytes at the site of infarction in nine of 23 cases (39%). It was associated with fivefold higher apoptotic rates (median 17.9% (interquartile range 11.0–25.4%) v 3.7% (0.6–12.8%); p  =  0.016), and apoptotic rate increased progressively from mild to intense COX-2 staining (p for trend 0.009). COX-2 expression co-localised with TUNEL nuclear staining in myocytes, and there was a high concordance between COX-2 and hypoxia induced factor 1-α staining (78%, p  =  0.021) and between COX-2 and bax (83%, p  =  0.014). Subjects showing myocardial COX-2 expression were more likely to have enlarged hearts (p  =  0.050), and intense COX-2 staining was strictly associated with symptomatic heart failure (p  =  0.035). Conclusions: COX-2 is expressed in cardiomyocytes in nearly 40% of cases at the site of recent acute myocardial infarction, even late after the index event. Its expression was associated with extremely high apoptotic rates. These findings suggest a potential cause–effect link between COX-2 expression and enhanced myocardial apoptosis in ischaemic cardiomyopathy.

Cyclo-oxygenase-2 (COX-2) inhibition reduces apoptosis in acute myocardial infarction

Department of Medicine, Virginia Commonwealth University, Richmond, VA USA A. Abbate (A. Abbate, G. W. Vetrovec); Institute of Cardiology, Catholic University, Rome, Italy (G. G. L. Biondi-Zoccai, A. Severino, M. Campioni, G. Liuzzo, F. Crea, L. M. Biasucci); Department of Vascular Pathology, Istituto Dermopatico Immacolata, Rome, Italy (F. Limana, M. C. Capogrossi, S. Straino); Section of Oncology, Campus Bio-Medico University, Rome, Italy (D. Santini); Department of Experimental Medicine and of Pathology, Universita degli Studi “La Sapienza”, Rome, Italy (S. Scarpa, F. Vasaturo); Centro Cardiologico Fondazione Monzino, Milano, Italy (A. Germani); Department of Biochemistry and Biophysics “F. Cedrangolo”, Section of Pathologic Anatomy, Second University of Naples, Naples, Italy (A. Baldi)

Fibronectin and laminin expression in sentinel lymph nodes of patients with malignant melanoma

SIR, Malignant melanoma (MM) has shown an alarming increase in incidence over the last few decades; currently it represents roughly 5% of skin cancers and 1% of all malignant tumours, with an annual increase in incidence and mortality of 2–3% during the last 30 years. Sentinel lymph node (SLN) biopsy has greatly improved the staging and prognostic evaluation of primary cutaneous MM; in particular, the Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR), which is able to detect melanoma cells expressing tyrosinase mRNA, offers an additional technique to the histopathological examination. The remodelling of cell adhesion molecules during the process of tumour progression is known to play a key functional role in cancer progression and metastasis. To date, although there have been several attempts to identify molecular markers among cell adhesion molecules in melanoma lesions and in peripheral blood from patients with MM, few studies have focused on the involvement of cell adhesion molecules in SLNs of such patients. Fibronectin and laminin are two adhesion molecules that have been shown to be involved in melanoma progression even though their role in SLNs has been poorly investigated. Recently, our group investigated the molecular profile of SLNs, suggesting a role as prognostic indicators for survivin, tyrosinase, MIA and Mart-1 expression. In this study we investigated the role of fibronectin and laminin mRNA expression as prognostic indicators in SLNs of patients with MM and correlated the results to progression of disease in a median follow-up of 51Æ5 months. The presence of capsular naevus cells in SLNs was tested and the positive samples were excluded from the study. We analysed 72 SLNs from 48 patients (mean age 58Æ3 years) with MM; informed consent was obtained from each patient. There were 22, 20 and six patients with stage I, II and III disease, respectively. Sections of each lymph node were stained with haematoxylin and eosin, and immunohistochemistry was performed by using antibodies to HMB-45 antigen and S100 protein that were detected with the avidinbiotin-peroxidase technique. Negative controls were obtained by using normal animal serum instead of specific primary antibodies. The results are shown in Table 1. After surgical excision the SLNs were frozen in liquid nitrogen and stored at –80 C until use in the RT-PCR assay. One microgram of total RNA extracted from the frozen tissues was reverse transcribed in a final volume of 20 lL, then 5 lL of cDNA was amplified in PCR buffer containing 25 pmol each of upstream and downstream specific primers. All the recommended precautions were taken to avoid the possibility of false-positive results. Each RT-PCR experiment included a sample without RNA as negative control and RNA extracted from the SK-MEL 5 melanoma cell line as positive control. The amplification products were separated by electrophoresis on 2% agarose gel: those showing specific amplification products were considered positive. The suitability of all samples was investigated by an RT-PCR with b-actin-specific primers. Laminin expression was found to be positive in 45 of 72 and negative in 27 of 72 samples examined. Positive expression was associated with death or disease progression in 20 of 45 cases, whereas negativity was associated with disease progression in 16 of 27 cases; 11 of 27 remain disease free. Fibronectin gene expression was found in 61 of 72 specimens: positivity was associated with disease progression in 26 of 61 positive samples, while 35 of 61 were disease free. Eleven of 72 samples did not have fibronectin gene expression: negativity was associated with death or disease progression in 10 of 11 cases, whereas one of 11 is currently disease free. The features of patients and the results obtained by the RT-PCR assay are listed in Table 1. Agarose gel electrophoresis of RT-PCR products is shown in Figure 1. In order to verify the statistical significance of our results we first determined the correlation between overall survival, Clark level and fibronectin and laminin expression by the Kaplan– Meier method. We failed to find a statistically significant correlation for the expression of laminin (P = 0Æ57); in contrast, a significant correlation was found between the Clark level and overall survival (P = 0Æ0036) and between negativity for fibronectin and overall survival (P = 0Æ0024). The log-rank test showed a strong correlation between absence of fibronectin expression and Clark level (Spearman correlation P = 0Æ35). The Cox proportional hazards model was applied to the multivariate survival analysis and demonstrated that absence of fibronectin expression and Clark level are statistically significant negative prognostic factors (P = 0Æ012 and P = 0Æ030, respectively).

EXTRACELLULAR MATRIX REMODELLING IN A MURINE MAMMARY ADENOCARCINOMA TRANSFECTED WITH THE INTERFERON‐ALPHA1 GENE

The rejection of interferon alpha1 gene‐transfected mammary adenocarcinoma cells (TSA‐IFNα) injected into syngeneic BALB/c mice was accompanied by an unusual stromal reaction and marked CD8‐positive T‐lymphocyte involvement. To investigate the biological background of this reaction, the possibility was evaluated that an interaction between TSA‐IFNα and stromal cells might remodel the extracellular matrix (EM). When fibroblasts were co‐cultured with TSA‐IFNα or treated with exogenous IFNα, there was no change in their replication rate or collagen synthesis. By contrast, their fibronectin (FN) production and release were increased, resulting in enhanced fibroblast chemotaxis. These findings were mirrored by increased FN staining in the peritumoural and tumoural areas in vivo. IFNα thus determines increased FN production and hence massive local recruitment and activation of fibroblasts, with a modification of the EM. The several activities of IFNα should thus be considered prior to its employment in clinical trials.

DIFFERENTIATING AND BIOCHEMICAL EFFECTS OF A REDUCTION OF INTRACELLULAR GTP LEVELS INDUCED BY MYCOPHENOLIC ACID (MPA) IN HUMAN NEUROBLASTOMA (NB) CELL LINES

Purpose of the present study was to investigate in human neuroblastoma (NB) cell lines the effects of GTP depletion induced by mycophenolic acid (MPA) (the most specific IMP dehydrogenase inhibitor): 1. on biopterin biosynthesis, using cytotoxic drug concentrations in short-term experiments; 2. on cellular differentiation and extracellular matrix remodelling, using drug concentrations below those inducing apoptosis.1