Cristian Scatena

A mouse mammary tumor virus env-like exogenous sequence is strictly related to progression of human sporadic breast carcinoma.

A viral etiology of human breast cancer (HBC) has been postulated for decades since the identification of mouse mammary tumor virus (MMTV). The detection of MMTV env-like exogenous sequences (MMTVels) in 30% to 40% of invasive HBCs increased attention to this hypothesis. Looking for MMTVels during cancer progression may contribute to a better understanding of their role in HBC. Herein, we analyzed HBC preinvasive lesions for the presence of MMTVels. Samples were obtained by laser microdissection of FFPE tissues: 20 usual-type ductal hyperplasias, 22 atypical ductal hyperplasias (ADHs), 49 ductal carcinomas in situ (DCISs), 20 infiltrating ductal carcinomas (IDCs), and 26 normal epithelial cells collateral to a DCIS or an IDC. Controls included reductive mammoplastic tissue, thyroid and colon carcinoma, and blood samples from healthy donors. MMTVels were detected by fluorescence-nested PCR. DNA samples from the tissues of nine patients were analyzed by real-time quantitative PCR, revealing a different viral load correlated with stage of progression. Furthermore, as never previously described, the presence of MMTVels was investigated by chromogenic in situ hybridization. MMTVels were found in 19% of normal epithelial cells collateral to a DCIS or an IDC, 27% of ADHs, 82% of DCISs, and 35% of IDCs. No MMTVels were found in the control samples. Quantitative PCR and chromogenic in situ hybridization confirmed these results. These data could contribute to our understanding of the role of MMTVels in HBC.

Sparc-like protein 1 is a new marker of human glioma progression.

High-grade gliomas (glioblastomas) are the most common and deadly brain tumors in adults, currently with no satisfactory treatment available. Apart from de novo glioblastoma, it is currently accepted that these malignancies mainly progress from lower grade glial tumors. However, the molecular entities governing the progression of gliomas are poorly understood. Extracellular and membrane proteins are key biomolecules found at the cell-to-cell communication interface and hence are a promising proteome subpopulation that could help understand the development of glioma. Accordingly, the current study aims at identifying new protein markers of human glioma progression. For this purpose, we used glial tumors generated orthotopically with T98G and U373 human glioma cells in nude mice. This setup allowed also to discriminate the protein origin, namely, human (tumor) or mouse (host). Extracellular and membrane proteins were selectively purified using biotinylation followed by streptavidin affinity chromatography. Isolated proteins were digested and then identified and quantified employing 2D-nano-HPLC-MS/MS analysis. A total of 23 and 27 up-regulated extracellular and membrane proteins were identified in the T98G and U373 models, respectively. Approximately two-thirds of these were predominantly produced by the tumor, whereas the remaining proteins appeared to be mainly overexpressed by the host tissue. Following extensive validation, we have focused our attention on sparc-like protein 1. This protein was further investigated using immunohistochemistry in a large collection of human glioma samples of different grades. The results showed that sparc-like protein 1 expression correlates with glioma grade, suggesting the possible role for this protein in the progression of this malignancy.

Endothelial progenitor cell homing in human myocardium in patients with coronary artery disease

Endothelialprogenitorcells(EPCs)aremobilizedfrombonemarrowinto peripheral blood, contributing to the revascularization of ischemicareas, to endothelial repair and to the physiological maintenance ofvascularization. EPC mobilization and homing have been primarilylinked to ischemia and inflammation presence [1]. EPCs are directlycorrelated with endothelial function and inversely correlated withcardiovascular risk factors and atherosclerosis progression [2].EPClevelshavealsobeencorrelatedtoprognosisevaluationincardiovascu-lar disease [3].Regarding EPC correlation with coronary artery disease (CAD)presence and severity, an inverse relationship with CAD severity, inde-pendent of traditional risk factors, was demonstrated by EPC colonycounting [4],whileahighnumberofEPCsassociatedwithCADandcorre-latedwithstenosisseveritywereshownby flowcytometry [5].Recentlyanew protocol, adapted from the standardized ISHAGE protocol for hema-topoieticstemcells,hasbeendevelopedforEPClevelevaluationtoenablecomparison of clinical and laboratory data [6].While thepresence of circulatingEPCshasbeenwidely evaluated indifferent diseases, few studies tried to evaluate the presence of EPCs inhuman vital myocardium.TheaimofourstudywastoinvestigateEPClevelsbothinperipheralbloodand inmyocardium in thesame patients atthesame time, evalu-atingthecorrelationwith CADpresence. In bothsampleswequantifiedCD34

Role of circulating endothelial progenitor cells in the reparative mechanisms of stable ischemic myocardium

BACKGROUND Mobilization of endothelial progenitor cells (EPCs) into circulation from bone marrow in patients with acute myocardial infarction has strong scientific evidence; less is known about EPC mobilization in patients with stable coronary artery disease (CAD). The aim of this study was to investigate the association of stable ischemic heart disease with EPC levels in tissue and blood. METHODS Fifty-five consecutive patients admitted to a single treatment center for valve or coronary artery bypass grafting (CABG) surgeries were included in the study. Blood samples were collected in the morning before surgery and analyzed by flow-cytometry to determine peripheral EPC levels (EPC/ml). Tissue EPC (CD34+VEGFR2+) levels were assessed on a right atrial appendage segment. RESULTS Mean age was 76±5years, 48% were men, and 53% had CAD The number of CD34+ VEGFR2+ cells in the tissue of patients with CAD was significantly higher (p<0.005) and circulating EPC showed a tendency to be reduced by approximately 20% in peripheral blood of patients with CAD when compared to those without CAD. CONCLUSION Patients with stable CAD had higher EPC density values (EPC/mm2) and were more likely to have lower EPC blood levels when compare with normal controls.

CD57 and γδ T-cell receptor expression in nodal metastatic spread of melanoma

Eur J Clin Invest 2012; 42 (5): 575–576

MicroRNAs distribution in different phenotypes of Aortic Stenosis

Aortic valve stenosis (AVS) represents a cluster of different phenotypes, considering gradient and flow pattern. Circulating micro RNAs may reflect specific pathophysiological processes and could be useful biomarkers to identify disease. We assessed 80 patients (81, 76.7–84 years; 46, 57.5%females) with severe AVS. We performed bio-humoral evaluation (including circulating miRNA-1, 21, 29, 133) and 2D-echocardiography. Patients were classified according to ACC/AHA groups (D1-D3) and flow-gradient classification, considering normal/low flow, (NF/LF) and normal/high gradient, (NG/HG). Patients with reduced ejection fractionwere characterized by higher levels of miRNA1 (p = 0.003) and miRNA 133 (p = 0.03). LF condition was associated with higher levels of miRNA1 (p = 0.02) and miRNA21 (p = 0.02). Levels of miRNA21 were increased in patients with reduced Global longitudinal strain (p = 0.03). LF-HG and LF-LG showed higher levels of miRNA1 expression (p = 0.005). At one-year follow-up miRNA21 and miRNA29 levels resulted significant independent predictors of reverse remodeling and systolic function increase, respectively. Different phenotypes of AVS may express differential levels and types of miRNAs, which may retain a pathophysiological role in pro-hypertrophic and pro-fibrotic processes.

Doxycycline, an Inhibitor of Mitochondrial Biogenesis, Effectively Reduces Cancer Stem Cells (CSCs) in Early Breast Cancer Patients: A Clinical Pilot Study

Background and objectives: Cancer stem cells (CSCs) have been implicated in tumor initiation, recurrence, metastatic spread and poor survival in multiple tumor types, breast cancers included. CSCs selectively overexpress key mitochondrial-related proteins and inhibition of mitochondrial function may represent a new potential approach for the eradication of CSCs. Because mitochondria evolved from bacteria, many classes of FDA-approved antibiotics, including doxycycline, actually target mitochondria. Our clinical pilot study aimed to determine whether short-term pre-operative treatment with oral doxycycline results in reduction of CSCs in early breast cancer patients. Methods: Doxycycline was administered orally for 14 days before surgery for a daily dose of 200 mg. Immuno-histochemical analysis of formalin-fixed paraffin-embedded (FFPE) samples from 15 patients, of which 9 were treated with doxycycline and 6 were controls (no treatment), was performed with known biomarkers of “stemness” (CD44, ALDH1), mitochondria (TOMM20), cell proliferation (Ki67, p27), apoptosis (cleaved caspase-3), and neo-angiogenesis (CD31). For each patient, the analysis was performed both on pre-operative specimens (core-biopsies) and surgical specimens. Changes from baseline to post-treatment were assessed with MedCalc 12 (unpaired t-test) and ANOVA. Results: Post-doxycycline tumor samples demonstrated a statistically significant decrease in the stemness marker CD44 (p-value < 0.005), when compared to pre-doxycycline tumor samples. More specifically, CD44 levels were reduced between 17.65 and 66.67%, in 8 out of 9 patients treated with doxycycline. In contrast, only one patient showed a rise in CD44, by 15%. Overall, this represents a positive response rate of nearly 90%. Similar results were also obtained with ALDH1, another marker of stemness. In contrast, markers of mitochondria, proliferation, apoptosis, and neo-angiogenesis, were all similar between the two groups. Conclusions: Quantitative decreases in CD44 and ALDH1 expression are consistent with pre-clinical experiments and suggest that doxycycline can selectively eradicate CSCs in breast cancer patients in vivo. Future studies (with larger numbers of patients) will be conducted to validate these promising pilot studies.

Cutaneous Nodules and Erythematous Plaques on the Extremities

A healthy man in his 20s presented with a several-month history of asymptomatic, slightly erythematous cutaneous nodules, measuring 1.5 to 3.0 cm, over the calcanea (Figure, A). Physical examination revealed violaceous plaques, measuring 2 to 3 cm, on the extensor surface of the knees, bilaterally (Figure, B). Excisional biopsy specimens were obtained (Figure, C and D). Erythematous calcaneal nodules A Violaceous knee plaques B Calcaneal nodule specimen C Plaque specimen D

Abstract 1120: Glioblastoma whole transcriptome analysis: molecular mechanisms related to recurrence-free survival (RFS)

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction. Diffusely infiltrating astrocytomas represent the most frequent intracranial neoplasms, with the glioblastoma (GB) as the most malignant phenotypic endpoint. Surgical resection, radiotherapy and adjuvant chemotherapy currently represent the standard of care for this disease. From a practical perspective, controlling growth and spreading of the recurrence may have a great impact on disease-free survival. The invasive nature of glioblastoma cells represents a major cause of therapeutic failure, so clarification of the molecular mechanisms associated with cellular migration and invasion is crucial to allow better prediction of these patient. Our study had the intent to provide novel information on glioblastoma behavior, with regard to the type of genetic changes involved in length of recurrence free survival time. Materials and Methods. We used whole-transcriptome RNA sequencing (Ion Proton system) to analyze 12 primary formalin fixed, paraffin embedded GBs specifically selected for different length of time of first recurrence. Results. A total of 83 genes resulted to have a statistical significant differential expression, which allowed us to distinguish three distinct groups of tumors with different recurrence free survival times: the short term group (S) with recurrence free survival time less than 6 months (n = 6), the medium group (M) between 16 and 23 months (n = 3), and the long term group (L) with more than 25 months (n = 3). Gene expression comparison analysis identified 51, 21, 26 significant genes between L/S, L/M and S/M respectively. Interestingly the highest number of genes differentialy expressed was found between the two extreme groups (S vs L). Two genes, BOD1L1 and GLUD2 were identified as significantly upregulated in the L group. Conclusions. The analysis of the whole transcriptome of GBs, with different recurrence free survival time, revealed that many of the genes that seem to be related to time of recurrence, after first surgery, are involved in the epigenetic landscape of the transcriptional potential of the cell such as histone and miRNA expression dis-regulation. Two genes were confirmed such as BOD1L1, which is mostly unknown and seems to be involved in the bio-orientation of chromosomes, and the mitochondrial enzyme GLUD2 which has been recently reported as involved in glioma growth. Moreover, some of the upregulated genes, in the group with the worst prognosis, had been related to the Rho family of GTPases which are key mediators of GB invasion. More thorough analyses are ongoing. A comprehensive better understanding of the molecular mechanisms underlying GB recurrence free survival would make a long step forward in the improvement of the clinical management of patients with such deadly disease. Citation Format: Sara Franceschi, Francesca Lessi, Paolo Aretini, Francesco G. Carbone, Cristian Scatena, Marco La Ferla, Valerio Ortenzi, Riccardo Vannozzi, Generoso Bevilacqua, Antonio G. Naccarato, Chiara M. Mazzanti. Glioblastoma whole transcriptome analysis: molecular mechanisms related to recurrence-free survival (RFS). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1120. doi:10.1158/1538-7445.AM2015-1120

Endothelial progenitor cells recruitment correlate with coronary artery disease severity

Background: Despite many studies investigated the level and function of peripheral blood Endothelial Progenitor Cells (EPCs), less scientific evidence concerning the presence and the role of EPCs in human myocardium exists. Our study aimed to investigate EPC density in atrial appendage, a well known source of different stem cells, and EPC blood levels in patients (pts) with coronary artery disease (CAD) undergoing to bypass and in pts undergoing to Isolated Valve Surgery (IVS) with no CAD.