Evaluation of Diagnostic Accuracy of Celiac Disease screening tests in children

A. Polimeno, G. Frulio, A. Zambrotta, D. Palmieri, P. Carandente Giarrusso

Celiac disease (CD) is an autoimmune-enteropathy caused by the ingestion of -containing grains in genetically susceptible individuals. Small bowel biopsy is the gold standard for CD diagnosis (1) but serological tests are the first step in ascertaining a diagnosis of CD. Tests for CD in children include anti-tissue IgA (anti-tTGA), anti-Endomysial IgA antibodies (EMA) and anti-Deamidated Peptides IgA and IgG antibodies (a-DGP IgA/IgG) tested only in children younger than 2 years (2). In this context, it’s noteworthy that new ESPGHAN (European Society of Pediatric Gastroenterology Hepatology and Nutrition) Guidelines suggest exclusively anti-tTGA as initial approach to symptomatic subjects, also in children (3). So we compared the diagnostic accuracy of anti-tTGA and a-DGP IgA/IgG in children younger than 2 years. We selected 378 subjects (357 non CD subjects and 21 CD subjects diagnosed by duodenal biopsy) younger than 2 years from the Pediatric Clinic, University Hospital “Federico II” of Naples; for all subjects we performed anti-tTGA, a-DGP IgA and a-DGP IgG by -Linked Immunosorbent Assay (ELISA). We determined the correlation between anti-tTGA and a-DGP IgA/IgG by Spearman’s correlation coefficient. Anti-tTGA significantly correlate with a-DGP IgA (ρ=0,383; p<0,0001) and a-DGP IgG (ρ=0,260; p<0,0001). Moreover, we evaluated the diagnostic accuracy of a-DGP IgA, a-DGP IgG and anti-tTGA tests by Receiver Operator Characteristic (ROC) curves. Anti-tTGA (sensitivity=100%; specificity=100%) are more sensitive and specific than a-DGP IgA (sensitivity=85%; specificity=99,4%) and a-DGP IgG (sensitivity=90%; specificity=95,5%). Our results suggest that anti-tTGA, because of high diagnostic accuracy, may be used as initial screening test for CD, also in children younger than 2 years, according to new ESPGHAN Guidelines. A-DGP IgA and a-DGP IgG could be used as additional screening tests in children younger than 2 years, negatives for anti-tTGA, but in whom the clinical symptoms give rise to a strong suspicion of CD.

References: 1-Rubio-Tapia A., Murray J.A. Celiac Disease. Curr Opin Gastroenterol. 2010 March; 26(2):116- 122. 2-Panetta F., Torre G., Colistro F., Ferretti F., Daniele A., Diamanti A. Clinical accuracy of anti- tissue transglutaminase as screening test for celiac disease under 2 years. Acta Paediatr. 2011 May; 100(5)728-31. 3-Husby S., Koletzko S., Korponay-Szabó I.R., Mearin M.L., Phillips A., Shamir R., Troncone R., Giersiepen K., Branski D., Catassi C., Lelgeman M., Mäki M., Ribes-Koninckx C., Ventura A., Zimmer K.P.; ESPGHAN Working Group on Diagnosis; ESPGHAN Gastroenterology Committee; European Society for Pediatric Gastroenterology, Hepatology, and Nutrition. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of celiac disease. J Pediatr Gastroenterol Nutr. 2012 Jan; 54(1):136-60. Role of the non-coding regions of the CFTR gene in cystic fibrosis Felice Amato, Ausilia Elce, Sonia Giordano, Manuela Scorza, Renato Liguori, Federica Zarrilli, Rossella Tomaiuolo, Giuseppe Castaldo

Mutation epidemiology is crucial for cystic fibrosis (CF) diagnosis and counselling. About 6-7% of alleles from CF patients do not bear mutations in the coding regions of the Cystic Fibrosis Transmembrane Regulator (CFTR) disease gene; in these patients, mutations may be present in non-coding, regulatory regions of the gene as i) intronic regions (particularly in high conserved sequences); ii) the promoter region or iii) the area at the 3’ UTR of the gene, that is the target of microRNA regulation. We study these regions by gene sequencing in CF patients with one or both unidentified mutations after the analysis of CFTR coding regions, and in CF patients with a different clinical expression of the disease to evaluate if mutations in such regions may have a role in modulating CF clinical expression. Our analysis revealed so far: i) a dozen of mutations (most novel) in the large promoter area of 6000 bp at the 5’ of the gene; expression studies in four cell lines demonstrated that a half of such mutations may have a pathogenic effect; ii) a series of mutations in 52 conserved sequence tags (CSTs), i.e., intronic regions with a high homology between humans and mouse. Expression studies revealed the some of these mutations are able to increase or decrease the CFTR activity; iii) finally, we studied the 1500 bp region at the 3’ UTR of the gene, identifying three mutations. One of this has a pathogenic role, enhancing the interaction of CFTR with two inhibitory miRNAs. At our knowledge, this is the first example of such pathogenic mechanism in a monogenic disorder. To conclude: the sequencing of non coding regions may improve the detection rate of molecular analysis in cystic fibrosis, and may help to reveal novel pathogenic mechanisms of CF.

References Castaldo G, Tomaiuolo R (2013) What is the role of the non-coding regions of the CFTR gene in cystic fibrosis? Expert Rev Respir Med 7: 327-329.

Giordano S, Amato F, Elce A, Monti M, Iannone C, Pucci P,.Seia M,.Angioni A,.Zarrilli F,Castaldo G and Tomaiuolo, R. (2013).. Molecular and functional analysis of the large 5' promoter region of CFTR gene revealed pathogenic mutations in CF and CFTR-related disorders. J Mol Diagn 15: 331-340.

Amato F, Seia M, Giordano S, Elce A, Zarrilli F, Castaldo, G and Tomaiuolo, R.(2013). Gene mutation in microRNA target sites of CFTR gene: a novel pathogenetic mechanism in cystic fibrosis? PLoS One 8: e60448.

Molecular approaches for the rapid diagnosis of bacterial and fungal infections

De Gregorio E, Iula VD, Roscetto E, Pulcrano G, Catania MR.

Respiratory and bloodstream infections represent severe and potentially fatal diseases demanding rapid diagnostic methods for detection of the responsible microorganisms. Classic microbiological diagnostics are time-consuming and characterized by a high rate of negative results, not only for fastidious, slow-growing, and/or nonculturable microorganisms but also for easy-to-culture pathogens. Culture-independent molecular methods improve the diagnostic outcome in terms of speed of analysis, sensibility and specificity and allow a prompt therapeutic treatment. Our research team is interested in studying molecular methods for rapid diagnosis of fungal and bacterial infections. A first approach concerns the direct identification of pathogens from biological samples by real time PCR. Primers and probes has been designed on: i) 16S and 23S rDNA and ITS region respectively; ii) repetitive coding sequences specific for different bacterial species (1). Another aspect of our research concerns the rapid identification of fungi and bacteria by MALDI- TOF mass spectrometry(MS) directly from blood cultures to greatly shorten laboratory times (2). Based on characteristic spectra obtained from intact cells, MALDI-TOF MS is characterized by low operational costs and allows a highly discriminatory identification of bacteria, yeasts and filamentous fungi starting from colonies. More recently MALDI-TOF MS is used to distinguish epidemiologically related isolates up to detect micro-evolutionary changes (3). MALDI-TOF MS in not only much easy and less time consuming than other typing methods, but it requires a limited amount of colonies, so representing a fast and reliable method for typing strains. MALDI-TOF MS is also used for detection of carbapenems and their degradation products from both Gram-negative isolates and directly from bloodstream cultures. These two applications will benefit patient care by optimizing antimicrobial therapies and defining outbreaks.

1) Di Nocera PP, De Gregorio E, Rocco F. (2013) GTAG- and CGTC-tagged palindromic DNA repeats in prokaryotes. BMC Genomics 31: 522.

2) Pulcrano G, Iula DV, Vollaro A, Tucci A, Cerullo M, Esposito M, Rossano F, Catania MR. (2013) Rapid and reliable MALDI-TOF mass spectrometry identification of Candida non-albicans isolates from bloodstream infections. J Microbiol Methods 94: 262-6.

3) Pulcrano G, Roscetto E, Iula VD, Panellis D, Rossano F, Catania MR. (2012) MALDI-TOF mass spectrometry and microsatellite markers to evaluate Candida parapsilosis transmission in neonatal intensive care units. Eur J Clin Microbiol Infect Dis 31: 2919-28.

First and second generation Human Anti-tumor Immunoagents

Chiara D’Avino, Gennaro Riccio, Rolando Paciello and Claudia De Lorenzo

Immunotherapy is of great interest today as an effective strategy to fight cancer. An attractive target for immunotherapy is ErbB2, a tyrosine kinase receptor overexpressed on many carcinoma cells of different origin with a key role in the development of malignancy. Herceptin, a humanized anti-ErbB2 , is effective in the therapy of breast carcinoma, but it engenders cardiotoxicity and many cancer patients are resistant to Herceptin-treatment. Novel human antitumor immunoconjugates were engineered in our laboratory by isolation through phage display technology of novel human antibody fragments, such as Erbicin, a human anti-ErbB2 scFv, and their fusion with either a human RNase or the Fc region of a human IgG1 (Erb-hcAb). Erbicin-Derived Immunoagents (EDIA) were found to be selectively cytotoxic for ErbB2-positive cancer cells in vitro and vivo. Furthermore, EDIA recognize an epitope different from that of Herceptin, are active on some Herceptin-resistant cancer cells both in vitro and in vivo, and are not cardiotoxic. Indeed, EDIA, differently from Herceptin, do not show toxic effects in vitro on rat and human cardiomyocytes, and do not impair cardiac function in vivo in a mouse model. Second generation EDIA were also obtained by the fusion of either Erb-hcAb with human pancreatic RNase (Erb-hcAb-RNase) or by the conjugation of Erbicin with a ribonuclease inhibitor-resistant variant of HP-RNase (Erb-HP-DDADD-RNase) . Both the novel immunoRNases retain the enzymatic activity of HP-RNase and specifically bind to ErbB2-positive cells. Moreover, the novel EDIA are endowed with an effective and selective cytotoxic action for ErbB2-positive tumor cells more potent than that of the first generation ImmunoRNases. Thus, EDIA could fulfil the therapeutic need of cancer patients ineligible to Herceptin treatment due to cardiac dysfunction and to Herceptin-resistance. Similar immunoagents will be also produced by using other human scFvs isolated by phage display and directed against different tumor targets.

References 1) De Lorenzo C., Arciello A., Cozzolino R., Palmer D. B., Laccetti P., Piccoli R., and D'Alessio G. Cancer Res. 2004, 64, 4870-4874. 2) Riccio G., Esposito G., Leoncini E., Contu R., Condorelli G., Chiariello M., Laccetti P., Hrelia S.,D'Alessio G., De Lorenzo C. FASEB J. 2009, 23, 3171-3178. 3) Riccio G, D'Avino C, Raines RT, De Lorenzo C. A novel fully human antitumor immunoRNase resistant to the RNase inhibitor. Protein Eng Des Sel. 2013, 26, 243-248. Molecular basis and diagnosis of familial dyslipidemias. Expression and regulation of candidate genes in human atherosclerotic plaques

Maria Donata Di Taranto, Maria Nicoletta D’Agostino, Antonietta D’Angelo, Anna Ruotolo, Carola Giacobbe, Annalisa Scotto di Frega, Francesco Salvatore, Giuliana Fortunato

Dyslipidemias are a heterogeneous group of diseases characterized by the alteration of the different lipid particles. Familial Hypercholesterolemia (FH) and Familial Combined Hyperlipidemia (FCH) are the most frequent dyslipidemias. FH is a monogenic disease caused by mutations in the LDL receptor (LDLR), in apolipoprotein B100 (APOB) and in proprotein convertase subtilisin/kexin type-9 (PCSK9) although in around 20% of patients the genetic cause has not been identified. The group is involved in the following studies: - molecular analysis of the genes LDLR, ApoB, PCSK9(1) - development of assays performed for the functional in vitro analysis of LDLR(2) - molecular analysis of the other genes recently identified as associated to the disease: cholesterol 25-hydroxylase (CH25H), insulin induced gene-2 (INSIG2), dolichol Kinase (DOLK) in the patients without mutations in the candidate genes. - verifying the hypothesis that the disease has a likely polygenic cause in patients with a clinical diagnosis of FH but no detected mutation. To support the above hypothesis in collaboration with Centre for Cardiovascular Genetics, University College London we study the most common SNPs reported as significantly associated with LDL-C by Global Lipid Genetic Consortium. FCH is a multifactorial polygenic lipid disorder probably caused by an overproduction of Apo- B100 and a decreased metabolism of VLDL particles. The study of FCH includes the sequencing of genes related to lipid metabolism and the identification of SNPs that can be used to predict the disease. Atherosclerosis is a multi-factorial disease associated with an oxidative and inflammatory status. We study the expression of genes involved in development of atherosclerotic plaques(3). Our research aims at studying the intricate network of interactions between atherosclerosis, oxidation and through in vitro experiments on human vessel cells. In order to perform retrospective diagnostic-experimental studies, the group is engaged in the storage of cell samples at the CEINGE Biobank.

References

1-Romano M, Di Taranto MD, D'Agostino MN, Marotta G, Gentile M, Abate G, Mirabelli P, Di Noto R, Del Vecchio L, Rubba P, Fortunato G. Identification and functional characterization of LDLR mutations in familial hypercholesterolemia patients from Southern Italy. Atherosclerosis. 2010; 210:493-6.

2-Romano M, Di Taranto MD, Mirabelli P, D'Agostino MN, Iannuzzi A, Marotta G, Gentile M, Raia M, Di Noto R, Del Vecchio L, Rubba P, Fortunato G. An improved method on stimulated T- lymphocytes to functionally characterize novel and known LDLR mutations. J Lipid Res. 2011;52:2095-100.

3-Di Taranto MD, Morgante A, Bracale UM, D'Armiento FP, Porcellini M, Bracale G, Fortunato G, Salvatore F. Altered expression of inflammation-related genes in human carotid atherosclerotic plaques. Atherosclerosis. 2012;220:93-101.

Identifying potential functional impact of mutations associated with cardiomyopathies may reduce the risk of fatal arrhythmias and sudden cardiac death. Frisso Giulia, Detta Nicola, Coppola Pamela, Farina Alessandro, Mazzaccara Cristina, Salvatore Francesco.

Research Collaborators: R. Calabrò, G. Limongelli; B. Sarubbi: UOC Cardiologia Seconda Università di Napoli D. Bonaduce: Dipartimento di Scienze mediche traslazionali, Università degli Studi di Napoli, Federico II S. Betocchi, P. Perrone Filardi M. Santomauro: Dipartimento di Scienze biomediche avanzate, Università degli Studi di Napoli, Federico II

Ventricular arrhythmias are the major cause of sudden cardiac death (SCD). The etiology of these life-threatening arrhythmias can be classified according to whether structural heart disease is present, including dilated cardiomyopathy, hypertrophic cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy. Most of the nonstructural causes are cardiac channelopathies: acquired and congenital long QT syndromes, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia, and short QT syndrome. Since 2006 we analyzed about 450 independent cardiomyopathy patients, including 55 athletes with borderline signs/symptoms, and identified about 20 novel mutations. To evaluate the mutation segregation in the family, 520 relatives were analyzed and 52% of asymptomatic relatives carried a mutation [1]. Subsequently to the genetic analysis, mutants were functionally characterized to define their pathogenic role. Up to now, we functionally studied by patch clamp 5 SCN5A-mutants, 1 HERG- mutant and 1 KCNQ1-mutant. Here, we show 2 interesting cases of SCN5A-related mutations (p.N1472del and p.R376C) and 1 case of digenic heterozygosity KCNQ1 (p.R583H)/KCNH2 (p.C108Y). The mutant p.N1472del showed both gain- and loss-of-function alterations, and increased persistent current, causing a mixed pathological phenotype [2]. The p.R376C mutation was detected in a young athlete, going to obtain sports eligibility. This mutation showed a critical reduction of the channel activity compared to the WT channel and to the mutant p.R376H, previously associated to SCD. Thus, competitive activity was discouraged [2]. We also show an interesting case of digenic heterozygosity KCNQ1/KCNH2. The homozygous mutant KCNH2-p.C108Y led to a non-functional KCNH2 channel, whereas, in the heterozygous condition, it induced a dominant negative effect. On the contrary, mutant KCNQ1-p.R583H had no significantly differences compared to WT. Our results suggest that mutation p.C108Y is responsible for LQTS, whereas the mutant p.R583H probably play a modulator role. In conclusion, the functional characterization of mutations is useful to define the genotype- phenotype correlation, and also, it may help clinicians in the management of patients harbouring specific mutations.

Research made in collaboration with CEINGE-Biotecnologie Avanzate, Naples, Italy References 1. Sarubbi B, Frisso G, Romeo E, Evangelista E, Cordella A, D'Alto M, Santarpia G, Russo MG, Salvatore F, Calabrò R. Efficacy of pharmacological treatment and genetic characterization in early diagnosed patients affected by long QT syndrome with impaired AV conduction. Int J Cardiol. 2011 May 19;149(1):109-13. 2. Detta N, Frisso G, Zullo A, Sarubbi B, Cozzolino C, Romeo E, Wang DW, Calabrò R, Salvatore F, George AL jr. Novel deletion mutation in the cardiac sodium channel inactivation gate causes long QT syndrome. International Journal of Cardiology 2013; 165:362-5 3. Detta N, Frisso G, Limongelli G, Marzullo M, Calabrò R, Salvatore F. Genetic analysis in a family affected by sick sinus sundrome may reduce the risk of sudden death in a young agonist athlete. Submitted to International Journal of Cardiology; Inherited metabolic diseases: from molecular basis to functional characterization of novel variants

Frisso Giulia, Cozzolino Carla; Romanelli Roberta and Salvatore Francesco

Research Collaborators: G. Andria, G. Parenti. E. Del Giudice: Dipartimento di Scienze mediche traslazionali, Università degli Studi di Napoli, Federico II

Since January 2009 we provided a service in Campania Region for the molecular diagnosis of inborn errors of metabolism. Until now, 100 molecular diagnosis have been performed on 76 clinically affected patients, 11 newborns, identified by expanded metabolic newborn screening, and 13 prenatal diagnosis. Genetic analysis was performed by mutation screening of genes associated with organic acidemias, aminoacidopathies, fatty acids β-oxidation defects and other metabolic disorders, including deficit of fructose-1,6-bisphosphatase (FBP), methylenetetrahydrofolate reductase deficiency (MTHFR), Smith- Lemli-Opitz syndrome (SLOS) and glutathione synthetase deficiency (GSD). By molecular analysis, we have found 22 novel mutations in 19 patients: 15 missense, 1 nonsense, 2 splicing mutations, 2 frameshift and 2 large deletions, one of them confirmed by MLPA analysis [1]. So far, we have carried out the functional analysis of two novel missense mutations, by in vitro enzymatic assays. c.439A>T (p.N147Y) was identified in ACADSB gene, associated with short branched chain amino acid deficiency (SBCADD), in compound heterozygosity with c.443C>T (p.T148I) variant. Cells transfected with both mutant constructs showed decrease mRNA levels and reduced enzymatic activity than wild-type. However, enzyme activity was partially restored in a pool of the two altered , which is indicative of chimeric tetramer formation and/or of a protein crosstalk recovery mechanism [2]. Mutation c. G355A (p.D119N) was identified in FBP1 gene, associated with fructose-1,6- bisphosphatase (FBP). In vitro assays demonstrated that mutated FBPase protein is enzymatically less active than the wild-type. We also characterized the novel splicing variant c.254+1g>a, found in HADHB gene, in 1 child affected by mitochondrial trifunctional protein deficiency (MTPD). mRNA analysis showed the skipping of exon 5 with a 45bp in-frame deletion [3]. Functional studies allowed us to better understand the pathogenetic role of new mutations in rare metabolic disease: it is very important for correct genetic counseling, prenatal diagnosis, definition of genotype-phenotype relationship and targeted therapeutic interventions.

Research made in collaboration with CEINGE-Biotecnologie Avanzate, Naples, Italy

References 1. Catanzano F, Ombrone D, Di Stefano C, Rossi A, Nosari N, Scolamiero E, Tandurella I, Frisso G, Parenti G, Ruoppolo M, Andria G, Salvatore F. “The first case of mitochondrial acetoacetyl-CoA deficiency identified by expanded newborn metabolic screening in Italy: the importance of an integrated diagnostic approach”. J Inherit Metab Dis. 2010;33 2. Frisso G, Cozzolino C, Ombrone D, Scolamiero E, Caruso U, Di Stefano C, Parenti G, Andria G, Ruoppolo M, Salvatore F. “A new case of the rare short branched chain acyl-CoA deficiency: clues to the pathogenetic molecular of this disease”. Submitted to Clinical Chemistry and Laboratory Medicine. 3. G. Terrone, M. Ruoppolo, N. Brunetti Pierri , C. Cozzolino, E. Scolamiero, G. Parenti, A. Romano, G. Andria, F. Salvatore, G. Frisso “Recurrent rhabdomyolysis due to a fatty acid oxidation disorder”. In press on Neurology. CGH ARRAY FOR THE IDENTIFICATION OF A COMPOUND HETEROZYGOUS MUTATION IN CYP1B1 GENE IN A PATIENT WITH A SUSPECT PRIMARY CONGENITAL GLAUCOMA

Barbara Lombardo, Carlo Ceglia, Marina Tarsitano, Andrea Vitale, Carmen Munno, Francesco Verdesca, Lucio Pastore

Primary congenital glaucoma (PCG) is the most common childhood glaucoma caused by developmental defect in the trabecular meshwork and anterior chamber angle of the eye. Mutations in the CYP1B1 gene have been observed in PCG patients for about a decade; in fact, mutations in CYP1B1 are the predominant cause of autosomal recessive inherited PCG. CYP1B1 gene product is an enzyme that catalyze reactions involved in drugs metabolism and synthesis of cholesterol, steroids and other lipids. To date, several mutations have been identified in patients and families with PCG (1,2). However, diagnosis can be difficult to perform in cases with atypical clinical presentation. A high resolution array comparative genomic hybridization (a-CGH) 4X180K was performed on a patient with a form of glaucoma that eluded clinical diagnosis. The a-CGH used contains 170,334 oligonucleotide probes that cover the whole genome with an average spatial resolution of 13 kb. We identified in the patient a heterozygous deletion in 2p22.2 of approximately 118 kb involving CYP1B1. The mutation was inherited from the father who does not show any sign of PCG. The deletion observed was further confirmed by quantitative real time PCR. Subsequently, DNA sequencing was performed to detect possible mutations on the other allele and revealed the presence of a transition G>A at position g.5975 (c.182 G>A; amino acid substitution: p. Gly61Glu) inherited from the mother. This case report underline the relevance of a-CGH screening in patients with possible congenital disorders with atypical clinical presentation in order to achieve a molecular characterization and identify genetic causes of diseases and pinpoint possible therapeutic interventions.

1)Reis LM, Tyler RC, Zori R, Burgess J, Mueller J, Semina EV. Ophthalmic Genet. 2013 Sep 11. [Epub ahead of print]. “A Case of 22q11.2 Deletion Syndrome with Peters Anomaly, Congenital Glaucoma, and Heterozygous Mutation in CYP1B1”.

2) Kelberman D, Islam L, Jacques TS, Russell-Eggitt I, Bitner-Glindzicz M, Khaw PT, Nischal KK, Sowden JC. Ophthalmology. 2011 Sep;118(9):1865-73. “CYP1B1-related anterior segment developmental anomalies novel mutations for infantile glaucoma and von Hippel's ulcer revisited”.

Strategies and Products for Prevention, Early Diagnosis, and Bio-Immunological Treatments of UV Irradiation-Induced Skin Tumors:

Additive/synergistic effects of chemo/immunotherapy in combination with PhotoDynamic Therapy

Palumbo G., Crescenzi E., Gentile F.

This project, studying melanoma cell biology by various angles is aimed to define new strategies of therapy based on the combination of PDT1 with targeted, nano-carried chemotherapy and innovative immunotherapy. The better therapeutic combination will be established on the basis of the experimental findings. The study will be conducted using human cell lines and suitable animal models.

G. Palumbo - A first approach will simply analyze the effects of 5-ALA-PDT in combination with molecules endowed of anti-VEGF properties (Dehydrodidemnine, Vatalanib, Sorafenib, KRN-951, Bortezomib).

E. Crescenzi - A second approach will analyze the “role of premature senescence” in melanoma, in endothelial and melanoma-endothelial cells co-culture (previously shown to be a valuable tool in the molecular dissection of melanoma- stromal interaction2), to discover new settings able to block angiogenesis and proliferation. Aims: a) Evaluation of drug-induced senescence in melanoma cell lines and in co-culture of melanoma and endothelial cells, following single/combined treatments (antiangiogenic agents and PDT); b) Characterization of cytokines/chemokines secretion in senescent melanoma cells; c) Understanding the role of SASP in modulating the interaction between senescent melanoma and endothelial cells.

F. Gentile - A third approach considers a “glycomic approach to the modulation of tumor-initiating and tumor- sustaining abilities of melanoma cells expressing Melanoma Chondroitin Sulfate Proteoglycan (MCSP)”, a hallmark of a subset of melanoma cells with tumor-initiating/sustaining capacities, whose immunotargeting fully eradicated melanoma lesions3. CS units have a documented role in extravasation, matrix adhesion/migration and establishment. Aim: evaluate the effects of treatments interfering with chondroitin addition and sulfation to MCSP on the ability of HMW-MAA+ (high molecular weight Melanoma-Associated Antigen) cells isolated from human melanomas to produce stable melanoma lesions by subcutaneous inoculation in NIH-III mice. Treatments will include: enzymatic deglycosylation, substrate inhibition of key , such as xylosyl and sulfotransferases, as well as cytokine-mediated immunomodulation and siRNA/miRNA-mediated regulation of their expression.

References 1. Postiglione I., Chiaviello A., Aloj SM and Palumbo G. 5-aminolaevulinic acid/Photo-Dynamic Therapy and Gefitinib in Non Small Cell Cancer cell lines: a potential strategy to improve Gefitinib therapeutic efficacy” 2013 Cell Prolif. 2013 Aug;46(4):382-95 2. Crescenzi E, De Palma R, Leonardi A. Senescence and NFκB: A trojan horse in tumors? Oncoimmunology. 1:1594- 1597, 2012. 3. Schmidt P, Kopecky C, Hombach A, Zigrino P, Mauch C, Abken H (2011) Eradication of melanomas by targeted elimination of a minor subset of tumor cells. Proc Natl Acad Sci USA 108: 2474-2479.

PEGylated helper- dependent adenoviral vector for gene therapy of Hyperlipidemias

E Leggiero, D Astone, V Cerullo, B Lombardo, C Mazzaccara, G Labruna, L Sacchetti, F Salvatore, M Croyle and L Pastore

Contents Helper-dependent adenoviral (HD-Ad) vectors are very promising for gene therapy applications; however, their administration induces acute toxicity that impairs the possibility of a safe clinical admistration (1). We previously observed that PEGylation of HD-Ad vectors strongly reduces the acute response in murine and primate models (2, 3). In order to evaluate whether PEGylated HD-Ad vectors (PEG-HD-Ad) can combine safety and correction of pathological phenotypes, we administered a vector expressing the human apolipoprotein A-I (hApoA-I) to low-density lipoprotein (LDL)-receptor-deficient mice (a model for familial hypercholesterolemia) fed a high- cholesterol diet. Mice were treated with high doses of either HD-Ad-expressing apoA-I or its PEGylated version; PBS was used as control. Twelve weeks later, LDL levels were lower and high-density lipoprotein (HDL) levels higher in mice treated with either PEGylated or unmodified HD-Ad vector compared to untreated mice. After terminal sacrifice, areas of atherosclerotic lesions were much smaller in the vector-treated mice compared to control animals. Moreover, the increase in pro-inflammatory cytokines (IL-6, IL-12 and TNF-alfa) was extremely reduced with an improved the toxicity profile in mice treated with the PEGylated vector compared to mice treated with the unmodified HD-Ad vector. This data indicates that the reduction in toxicity achieved with the PEGylation of HD-Ad vectors is independent from the transgene expressed and does not impair the correction of pathological phenotypes in animal models. In conclusion, these data further supports the possibility of clinical applications of PEGylated HD-Ad vectors for correction of genetic disease.

References

1) Pastore L, Belalcazar LM, Oka K, Cela R, Lee B, Chan L et al. Helper-dependent adenoviral vector-mediated long-term expression of human apolipoprotein A-I reduces atherosclerosis in apo E-deficient mice. Gene 2004; 327: 153–160

2) Croyle MA, Le HT, Linse KD, Cerullo V, Toietta G, Beaudet A et al. PEGylated helper- dependent adenoviral vectors: highly efficient vectors with an enhanced safety profile. Gene Ther 2005; 12: 579–58

3) Wonganan P, Clemens CC, Brasky K, Pastore L, Croyle MA. Species differences in the pharmacology and toxicology of PEGylated helper-dependent adenovirus. Mol Pharm 2011; 8: 78– 92.

A four year of Point-of Care Testing project at the Azienda Ospedaliera University of Naples Federico II

M. Savoia and M. Galdiero

Point of Care Testing (POCT) is diagnostic testing performed near the patient. The implementation of POCT at the University of Naples Federico II required much effort of a multidisciplinary management group consisting of laboratory and an administration staff. The sites targeted for POCT are areas in which rapid response improved patient care: operating room; critical care units; paediatrics and nephrology. 10 blood gas analysis instrumentations were acquired (Rapid Point 405, Siemens) and in the last year 15 instruments (Rapid Point 500, Siemens). The test panel + + - consisted of pH, pCO2, pO2, Na , K , Cl , iCa, Hct, glucose, tHb and co-oximetry panels, lactate and unconjugated bilirubin. All instruments are connected with the central laboratory information system. The employed software (RAPID Comm, Siemens) allows verification and monitoring of the activity of each single analyzer, on-line control of calibration and quality control of all parameters. The samples analyzed are: about 30000 in 2009 and 2010; about 38000 in 2011 and 2012 and about 10% increase in the first six months of 2013. The laboratory management of the all materials used allowed for a 8% reduction of the foreseen annual costs. Quality management systems require: continuous improvement of testing; analysis of quality control results; correlations of POCT tests with those performed in the laboratory; training and education programmes for clinicians and nurses. The POCT project has shown many advantages: immediate real-time analysis; decrease of preanalytic error due to length of time a blood sample sits in the sample tube; good collaboration among laboratorists and nurses and pathologists; decreasing Therapeutic Turnaround Time; augmented clinical decision making and thus greater convenience for the patient. Our experience points out that management of the entire POCT process is essential to ensure quality and patient safety.

References: NCCLS document C46-A Vol.21,No 14, 2001 INTERNATIONAL STANDARD : ISO/FDIS 22870

Genetic basis and diagnostic approach to celiac disease and monogenic diabetes

Tinto N, Capuano M, Galatola M, Pirozzi D, Cola A, Sacchetti L. Research collaborators: Acquaviva F, Franzese A, Greco L, Iafusco D, Pinelli M.

Celiac disease (CD) is an immune mediated enteropathy caused by permanent sensitivity to gluten in genetically susceptible individuals. HLA DQ2/DQ8 haplotypes confer the highest estimated heritability (~35%) reported so far; however, their presence is a necessary but not sufficient condition for the development of CD. We demonstrated a miRNA-mediated gene regulation in the small intestine of CD patients; particularly, we highlighted a correlation of miR-449a over expression with a reduced NOTCH1 pathway and a decreased differentiation of goblet cells (1). These results support the idea that miRNAs may play a role in the pathogenesis of CD. According to these results and considering that the gold standard for the CD diagnosis is still the invasive intestinal biopsy, we are evaluating the miRNAs expression pattern in blood samples of CD patients and controls in order to identify a new simple diagnostic marker for CD. Monogenic diabetes is an heterogeneous group of disorders characterized by pancreatic β-cell dysfunction, caused by mutations in at least seven genes. We previously studied 218 subjects with clinical suspect of MODY and identified 71 GCK-, 10 HNF1α- , and 1 HNF4α-MODY patients. To confirm the association between the genetic defects and GCK-MODY insurgence, we performed both the in silico modeling and the functional characterization of GCK mutations (2). In addition, we contributed to validate a diagnostic flow chart (7-iF) based on specific clinical and biochemical features, to support clinicians in discriminating GCK-MODY from other diabetic forms and in selecting the patients to address to the genetic test, this 7-iF could result in improving GCK-MODY diagnosis and also in reducing the costs of inappropriate therapies (3). In progress, we are developing, in collaboration with gynecologists, a diagnostic flow chart for the early detection of MODY during pregnancy. Research made in collaboration with CEINGE-Biotecnologie Avanzate

Bibliography

1) Capuano M, Iaffaldano L, Tinto N, Montanaro D, Capobianco V, Izzo V, Tucci F, Troncone G, Greco L, Sacchetti L. MicroRNA-449a overexpression, reduced NOTCH1 signals and scarce goblet cells characterize the small intestine of celiac patients. PLoS One. 2011;6(12):e29094 2) Capuano M, Garcia-Herrero CM, Tinto N, Carluccio C, Capobianco V, Coto I, Cola A, Iafusco D, Franzese A, Zagari A, Navas MA, Sacchetti L. Glucokinase (GCK) mutations and their characterization in MODY2 children of southern Italy. PLoS One. 2012;7(6):e38906. 3) Pinelli M, Acquaviva F, Barbetti F, Caredda E, Cocozza S, Delvecchio M, Mozzillo E, Pirozzi D, Prisco F, Rabbone I, Sacchetti L, Tinto N, Toni S, Zucchini S, Iafusco D and Italian Study group on diabetes of the Italian Society of Pediatric Endocrinology and Diabetology (ISPED). Identification of candidate children for maturity-onset diabetes of the young type 2 (MODY2) gene testing: A seven-item clinical flowchart (7-iF). PLoS One 2013, Under review.

Epithelial-stromal cells interaction as a model to test targeted therapy in

Micaela Montanari, Maria Letizia Cataldo, Luigi Coppola, Maria Rita Carbone, Agostina Nardone, Sara Corvigno, Mario Giuliano, Bianca Maria Veneziani

Our aim is to identify therapeutic targets based on the features of each individual breast tumor. Primary and metastatic breast cancer tissues may contain cancer stem cells that sustain tumor growth and relapse after therapy. It has been proposed that specific targeting of these highly tumorigenic cells, often resistant to cytotoxic chemotherapy and radiation, along with the bulk of the tumor, will increase the likelihood of response and prolong survival after treatment. Based on these findings, we used a cell biological approach to provide a limitless model for identifying patients’ tailored therapeutic targets. We established an integrated tissue bank of frozen samples and clinical database of breast tumors and developed an in vitro model of co-culturing epithelial mesenchymal tumor cells (1). On the co-cultures, the combined treatment with aromatase inhibitors (AI) targeting stroma and RTKI targeting epithelial cells inhibits epithelial cell proliferation enhancing the efficacy of drugs (2). To identify the molecular features of individual tumor specimen, we generated mammospheres, isolated CD44+/CD24low breast cancer stem cells, sorted subpopulations of patients’ breast cancer stem cells and performed a multiparametric analysis on a panel of 28 selected stem/progenitor markers (3). Currently, from patients relapsed, we are isolating and propagating circulating tumor cells to monitor the changes of the progressing tumor cells and test new drugs. The comparison of the molecular profile with the respective primary tumors would help to clarify whether different subtypes of breast cancers have common lineage origins, or differing cancer stem cells may explain the different histotype, phenotype, their invasive potential, and, as a consequence, clinical outcome and treatment response.

1: Veneziani BM, Criniti V, Cavaliere C, Corvigno S, Nardone A, Picarelli S, Tortora G, Ciardiello F, Limite G, De Placido S. In vitro expansion of human breast cancer epithelial and mesenchymal stromal cells: optimization of a coculture model for personalized therapy approaches. Mol Cancer Ther. 2007 Dec;6(12 Pt 1):3091-100. PubMed PMID: 18089705.

2: Cavaliere C, Corvigno S, Galgani M, Limite G, Nardone A, Veneziani BM. Combined inhibitory effect of formestane and herceptin on a subpopulation of CD44+/CD24low breast cancer cells. Cancer Sci. 2010 Jul;101(7):1661-9. doi: 10.1111/j.1349-7006.2010.01593.x. Epub 2010 Apr 19. PubMed PMID: 20491779.

3: Leccia F, Nardone A, Corvigno S, Vecchio LD, De Placido S, Salvatore F, Veneziani BM. Cytometric and biochemical characterization of human breast cancer cells reveals heterogeneous myoepithelial phenotypes. Cytometry A. 2012 Nov;81(11):960-72. doi: 10.1002/cyto.a.22095. Epub 2012 Jul 12. PubMed PMID: 22791584.