A multimarker study of degenerative aortic valve disease: stenoinsufficiency shows more indices of bad prognosis.

OBJECTIVES: It was the aim of this study to assess the pathophysiological, prognostic role of aortic regurgitation (AR) in the 'mixed pictures' of degenerative aortic valve stenoinsufficiency (ASI) by a multimarker clinical approach. METHODS: We enrolled 112 consecutive surgical PATIENTS: 19 with pure valve stenosis (PAS), 39 with mild regurgitation, 29 with severe regurgitation, and 25 controls with annulo-ectatic AR. All underwent complete echocardiography, carotid ultrasound and aortic/coronary multislice computed tomography calcium score evaluation. We determined tissue semiquantitative osteopontin, metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs) and circulating brain natriuretic peptide. We evaluated major adverse cardiac events and cardiovascular early, long-term mortality after bioprosthetic valve implantation. RESULTS: Tissue calcification, carotid and coronary atherosclerotic disease were prevalent in PAS versus ASI and AR patients. The multislice computed tomography calcium score (Agatston) was comparable between PAS and ASI (PAS 3,507.3 + 2,442.6; mild AR 4,270.7 + 2,213.5; severe AR 3,568.5 + 1,823.4), but much lower in AR (1,247.8 + 2,708.6). In ASI, a plasma/tissue 'profibrotic' MMP/TIMP balance prevailed, with circulating and echocardiographic indices of myocardial dysfunction. Percentages of major adverse cardiac events and early, long-term mortality were higher in ASI. CONCLUSIONS: In ASI, different, still unknown, genetic and dysplastic factors could work synergically with cardiovascular risk factors, determining a much more adverse myocardial and valve remodeling, resulting in worse clinical outcome.

Epigenetic and copy number variation analysis in retinoblastoma by MS-MLPA.

Retinoblastoma is the most common primary intraocular malignancy in children. Two step inactivation of RB1 (M1-M2) represents the key event in the pathogenesis of retinoblastoma but additional genetic and epigenetic events (M3-Mn) are required for tumor development. In the present study, we employed Methylation Specific Multiplex Ligation Probe Assay to investigate methylation status and copy number changes of 25 and 39 oncosuppressor genes, respectively. This technique was applied to analyse 12 retinoblastomas (5 bilateral and 7 unilateral) and results were compared to corresponding normal retina. We identified hypermethylation in seven new genes: MSH6 (50%), CD44 (42%), PAX5 (42%), GATA5 (25%), TP53 (8%), VHL (8%) and GSTP1 (8%) and we confirmed the previously reported hypermethylation of MGMT (58%), RB1 (17%) and CDKN2 (8%). These genes belong to key pathways including DNA repair, pRB and p53 signalling, transcriptional regulation, protein degradation, cell-cell interaction, cellular adhesion and migration. In the same group of retinoblastomas, a total of 29 copy number changes (19 duplications and 10 deletions) have been identified. Interestingly, we found deletions of the following oncosuppressor genes that might contribute to drive retinoblastoma tumorigenesis: TP53, CDH13, GATA5, CHFR, TP73 and IGSF4. The present data highlight the importance of epigenetic changes in retinoblastoma and indicate seven hypermethylated oncosuppressors never associated before to retinoblastoma pathogenesis. This study also confirms the presence of copy number variations in retinoblastoma, expecially in unilateral cases (mean 3 ± 1.3) where these changes were found more frequently respect to bilateral cases (mean 1.4 ± 1.1).

p53 Arg72Pro and MDM2 309 SNPs in hereditary retinoblastoma.

The tumor suppressor p53 and its negative regulator MDM2 have crucial roles in a variety of cellular functions such as the control of the cell cycle, senescence, genome stability and apoptosis, and are frequently deregulated in carcinogenesis. Previous studies have highlighted the contribution of the common functional polymorphisms p53 p.Arg72Pro and MDM2 309SNP to the risk of both common cancers and Li-Fraumeni syndrome. Their possible role in retinoblastoma has recently been addressed by Castéra et al, who however only studied the MDM2 309SNP. Here, for the first time, we analyzed both single nucleotide polymorphisms (SNPs) in a case-control study of 111 Italian hereditary retinoblastoma patients. We found a significant association of the p53 Pro/Pro genotype with the disease (odds ratio=3.58, P=0.002). The MDM2 309SNP showed a weak negative association of allele G that deserves further investigation. These findings further support the hypothesis that genetic variability of the p53 pathway contributes to the individual susceptibility to retinoblastoma, as shown for Li-Fraumeni syndrome and a variety of non-hereditary cancers.

Aortic valve disease and gamma-glutamyltransferase: accumulation in tissue and relationships with calcific degeneration.

OBJECTIVE: Degenerative aortic valve disease is characterized by some of the histological features of atherosclerotic lesions. Gamma-glutamyltransferase (GGT) has been recently implicated in pathogenesis of atherosclerosis, as well as in modulation of cells involved in calcium metabolism. We aimed to evaluate the possible implication of this enzyme activity in aortic valve disease. METHODS: GGT immunohistochemistry was performed on valve leaflets of 64 patients with aortic valve stenosis undergoing valve replacement. Fractional GGT activity in plasma and tissue was analysed in a subgroup of cases by molecular exclusion chromatography. RESULTS: A close association was found between tissue extracellular GGT staining and lipid deposits (p<0.0001). GGT was expressed by CD68-positive cells around neovessels, as well as by MMP-9- and TRAP-positive multinucleated cells in the vicinity of bone metaplasia areas. Total plasma GGT levels were associated with low HDL-c (p=0.028) and high triglycerides (p=0.017). Total GGT activity in tissue was negatively correlated with the extent of valves calcification (p=0.03). Both serum and tissue GGT levels were negatively associated with severity of valve stenosis, as judged by peak transvalvular pressure gradients (p<0.0003 and p<0.002, respectively). CONCLUSIONS: Accumulation of GGT activity inside the lipid component of valves leaflets suggests a common mechanism of lesion shaping underlying both atherosclerosis and degenerative aortic valve disease. Moreover, the finding of GGT expression in cells with an osteoclast-like phenotype, and its negative correlation with both valves calcification and degree of valvular stenosis lend additional support to the recently envisaged involvement of GGT in the homeostasis of calcified tissues.

Array comparative genomic hybridization in retinoma and retinoblastoma tissues.

In retinoblastoma, two RB1 mutations are necessary for tumor development. Recurrent genomic rearrangements may represent subsequent events required for retinoblastoma progression. Array-comparative genomic hybridization was carried out in 18 eye samples, 10 from bilateral and eight from unilateral retinoblastoma patients. Two unilateral cases also showed areas of retinoma. The most frequent imbalance in retinoblastomas was 6p gain (40%), followed by gains at 1q12-q25.3, 2p24.3-p24.2, 9q22.2, and 9q33.1 and losses at 11q24.3, 13q13.2-q22.3, and 16q12.1-q21. Bilateral cases showed a lower number of imbalances than unilateral cases (P = 0.002). Unilateral cases were divided into low-level (< or = 4) and high-level (> or = 7) chromosomal instability groups. The first group presented with younger age at diagnosis (mean 511 days) compared with the second group (mean 1606 days). In one retinoma case ophthalmoscopically diagnosed as a benign lesion no rearrangements were detected, whereas the adjacent retinoblastoma displayed seven aberrations. The other retinoma case identified by retrospective histopathological examination shared three rearrangements with the adjacent retinoblastoma. Two other gene-free rearrangements were retinoma specific. One rearrangement, dup5p, was retinoblastoma specific and included the SKP2 gene. Genomic profiling indicated that the first retinoma was a pretumoral lesion, whereas the other represents a subclone of cells bearing 'benign' rearrangements overwhelmed by another subclone presenting aberrations with higher 'oncogenic' potential. In summary, the present study shows that bilateral and unilateral retinoblastoma have different chromosomal instability that correlates with the age of tumor onset in unilateral cases. This is the first report of genomic profiling in retinoma tissue, shedding light on the different nature of lesions named 'retinoma'.

Activated Akt as an indicator of prognosis in gastric cancer.

The immunohistochemical expression of phosphorylated (activated) Akt (pAkt) in 50 advanced gastric carcinomas has been analyzed and the results correlated with age, sex, location in the stomach, histotype, stage, survival, mitotic and apoptotic index, some cell cycle regulators (cyclin D1, cyclin E, p34/cdc2, p27/kip1), and cell proliferation. There was a statistically significant direct correlation between pAkt expression (both cytoplasmatic and nuclear) and depth of infiltration of the tumor, number of infiltrated lymph nodes and p34/cdc2 expression, and between prevalently nuclear pAkt and cyclin D1 and cyclin E. Conversely, there was a significant inverse correlation between nuclear pAkt and apoptotic index and between cytoplasmatic and nuclear pAkt and patient survival. No correlation was found between pAkt and sex, age, tumor location, histotype, mitotic index, and cell proliferation. These findings suggest that pAkt may be considered an indicator of tumor progression and patient survival in gastric cancer.

Genomic differences between retinoma and retinoblastoma.

INTRODUCTION: Genomic copy number changes are involved in the multi-step process transforming normal retina in retinoblastoma after RB1 mutational events. Previous studies on retinoblastoma samples led to a multi-step model in which after two successive RB1 mutations, further genomic changes accompany malignancy: 1q32.1 gain is followed by 6p22 gain, that in turn is followed by 16q22 loss and 2p24.1 gain. Retinoma is a benign variant of retinoblastoma that was initially considered a tumor regression, but recent evidences suggest that it rather represents a pre-malignant lesion. Genetic studies on retinoma tissue have rarely been performed. MATERIALS AND METHODS: We investigated by Real-Time qPCR, copy number changes of candidate genes located within the 4 hot-spot regions (MDM4 at 1q32.1, MYCN at 2p24.1, E2F3 at 6p22 and CDH11 at 16q22) in retina, retinoma and retinoblastoma tissues from two different patients. RESULTS: Our results demonstrated that some copy number changes thought to belong to early (MDM4 gain) or late stage (MYCN and E2F3 gain) of retinoblastoma are already present in retinoma at the same (for MDM4) or at lower (for MYCN and E2F3) copy number variation respect to retinoblastoma. CDH11 copy number is not altered in the two retinoma samples, but gain is present in one of the two retinoblastomas. DISCUSSION: Our results suggest that MDM4 gain may be involved in the early transition from normal retina to retinoma, while MYCN and E2F3 progressive gain may represent driving factors of tumor progression. These results also confirm the pre-malignant nature of retinoma.

Biological features (inflammation and neoangiogenesis) and atherosclerotic risk factors in carotid plaques and calcified aortic valve stenosis: two different sites of the same disease?

Neoangiogenesis and inflammation have a pivotal role in atherosclerosis. Observations support the hypothesis that calcified aortic valve stenosis is an inflammatory process, similar to atherosclerosis in tissue features and risk factors. We studied 2 groups of cases: 47 were affected by hemodynamic atherosclerotic carotid plaque (group 1) and 35 by severe calcified aortic valve stenosis (group 2). We compared the groups for atherosclerosis risk factors, morphologic features, and immunohistochemical phenotypes. In both groups, men, smokers, and hypertensive subjects prevailed, and histologic analysis showed an elevated score for T-lymphocyte infiltrates, neoangiogenesis, calcium, and sclerosis. Adhesion molecule expression was present in both lesions. Expression of intercellular adhesion molecule 1 correlated with inflammatory infiltrates (group 1, P = .0007; group 2, P = .06). Neoangiogenesis also correlated with inflammatory infiltrates (group 1, P = .035; group 2, P = .045). In valves, neoangiogenesis correlated with calcium (P = .048). Carotid plaque and calcified valve stenosis showed common risk factors and biologic hallmarks of a chronic inflammatory process. Inflammation and neoangiogenesis have a crucial role in plaque evolution and in the progression of aortic valve stenosis.

Neoangiogenesis, T-lymphocyte infiltration, and heat shock protein-60 are biological hallmarks of an immunomediated inflammatory process in end-stage calcified aortic valve stenosis.

OBJECTIVES: We investigated the main biomolecular features in the evolution of aortic stenosis, focusing on advanced lesions. BACKGROUND: "Degenerative" aortic valve stenosis shares risk factors and inflammatory similarities with atherosclerosis. METHODS: We compared nonrheumatic stenotic aortic valves from 26 patients undergoing surgical valve replacement (group A) and 14 surgical control patients (group B). We performed semiquantitative histological and immunohistochemical analyses on valve leaflets to measure inflammation, sclerosis, calcium, neoangiogenesis, and intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression. We assessed heat shock protein 60 (hsp60) gene expression as an index of cellular stress and C-reactive protein, erythrocyte sedimentation rate, and fibrinogen as systemic inflammatory markers. RESULTS: In group A valves, we found a prevalence of calcium nodules surrounded by activated inflammatory infiltrates, neovessels, and abundant ICAM-1, VCAM-1, and hsp60 gene expression. Specimens from group B were negative for all of these markers, except 2 of 14 positivity for hsp60. The presence of active inflammatory infiltrates correlated with an abundance of thin neovessels (p < 0.01) and hsp60 gene expression (p = 0.01), whereas neoangiogenesis correlated with inflammation (p = 0.04), calcium (p = 0.01), and hsp60 gene expression (p = 0.04). CONCLUSIONS: "Degenerative" aortic valve stenosis appears to be a chronic inflammatory process associated with atherosclerotic risk factors. The coexistence of neoangiogenesis, T-lymphocyte infiltration, adhesion molecules, and hsp60 gene expression indicates an active immunomediated process in the final phases of the disease.

Apoptosis of cardiomyocytes in explanted and transplanted hearts. Comparison of results from in situ TUNEL, ISEL, and ISOL reactions.

We assessed the efficiency of detecting myocyte apoptosis within human hearts using in situ enzymatic reactions in paraffin-embedded tissue samples: in situ end labeling (ISEL), terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), and in situ oligoligation (ISOL). The reactions were carried out in explanted hearts (idiopathic dilatative cardiomyopathy, n = 6; ischemic heart disease, n = 3) and in endomyocardial biopsy specimens (EMBs; n = 32) obtained from transplanted human hearts. The results were verified by DNA laddering. The ISOL reaction led to a significantly (P = .027) smaller number of false-positive results (2/41 [5%]) compared with assessment by ISEL (9/41 [22%]) or TUNEL (9/41 [22%]). Only 1 ISEL+ apoptotic cardiomyocyte was found in specimens from explanted hearts. Among the EMBs, 1 specimens had TUNEL+ apoptotic cardiomyocytes and 1 specimen had ISEL+ apoptotic cardiomyocytes. This implies that verifying results by independent methods must be used for TUNEL and ISEL techniques. A smaller number of false-positive results makes interpretation of ISOL results easier, although the sensitivity of this reaction remains to be established.