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1.  dMyc Functions Downstream of Yorkie to Promote the Supercompetitive Behavior of Hippo Pathway Mutant Cells 
PLoS Genetics  2010;6(9):e1001140.
Genetic analyses in Drosophila epithelia have suggested that the phenomenon of “cell competition” could participate in organ homeostasis. It has been speculated that competition between different cell populations within a growing organ might play a role as either tumor promoter or tumor suppressor, depending on the cellular context. The evolutionarily conserved Hippo (Hpo) signaling pathway regulates organ size and prevents hyperplastic disease from flies to humans by restricting the activity of the transcriptional cofactor Yorkie (yki). Recent data indicate also that mutations in several Hpo pathway members provide cells with a competitive advantage by unknown mechanisms. Here we provide insight into the mechanism by which the Hpo pathway is linked to cell competition, by identifying dMyc as a target gene of the Hpo pathway, transcriptionally upregulated by the activity of Yki with different binding partners. We show that the cell-autonomous upregulation of dMyc is required for the supercompetitive behavior of Yki-expressing cells and Hpo pathway mutant cells, whereas the relative levels of dMyc between Hpo pathway mutant cells and wild-type neighboring cells are critical for determining whether cell competition promotes a tumor-suppressing or tumor-inducing behavior. All together, these data provide a paradigmatic example of cooperation between tumor suppressor genes and oncogenes in tumorigenesis and suggest a dual role for cell competition during tumor progression depending on the output of the genetic interactions occurring between confronted cells.
Author Summary
One of the major challenges of developmental biology and cancer research is to get a better understanding of how different signals regulate proper organ growth and prevent tumor formation. Even though there is a strong correlation between tumor progression and Myc family misexpression or Hippo signaling pathway malfunction, the relationship between these organ growth regulators remains unclear. Here, we demonstrate that the Hippo signaling pathway controls the transcription of Drosophila dmyc. Furthermore, we show that the misregulated expression of dMyc in Hippo mutant cells elicits their proliferative expansion at the expense of normal surrounding cells. These findings reveal a molecular mechanism of cooperation between oncogenes and tumor suppressor genes that favors both tumor progression and wild-type tissue elimination. Additionally, our findings indicate a dual role for cell competition during the tumour progression depending on the cellular context.
doi:10.1371/journal.pgen.1001140
PMCID: PMC2944792  PMID: 20885789
2.  The lethal giant larvae tumour suppressor mutation requires dMyc oncoprotein to promote clonal malignancy 
BMC Biology  2010;8:33.
Background
Neoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. Precancerous cells are often removed by cell death from normal tissues in the early steps of the tumourigenic process, but the molecules responsible for such a fundamental safeguard process remain in part elusive. With the aim to investigate the molecular crosstalk occurring between precancerous and normal cells in vivo, we took advantage of the clonal analysis methods that are available in Drosophila for studying the phenotypes due to lethal giant larvae (lgl) neoplastic mutation induced in different backgrounds and tissues.
Results
We observed that lgl mutant cells growing in wild-type imaginal wing discs show poor viability and are eliminated by Jun N-terminal Kinase (JNK)-dependent cell death. Furthermore, they express very low levels of dMyc oncoprotein compared with those found in the surrounding normal tissue. Evidence that this is a cause of lgl mutant cells elimination was obtained by increasing dMyc levels in lgl mutant clones: their overgrowth potential was indeed re-established, with mutant cells overwhelming the neighbouring tissue and forming tumourous masses displaying several cancer hallmarks. Moreover, when lgl mutant clones were induced in backgrounds of slow-dividing cells, they upregulated dMyc, lost apical-basal cell polarity and were able to overgrow. Those phenotypes were abolished by reducing dMyc levels in the mutant clones, thereby confirming its key role in lgl-induced tumourigenesis. Furthermore, we show that the eiger-dependent Intrinsic Tumour Suppressor pathway plays only a minor role in eliminating lgl mutant cells in the wing pouch; lgl-/- clonal death in this region is instead driven mainly by dMyc-induced Cell Competition.
Conclusions
Our results provide the first evidence that dMyc oncoprotein is required in lgl tumour suppressor mutant tissue to promote invasive overgrowth in larval and adult epithelial tissues. Moreover, we show that dMyc abundance inside versus outside the mutant clones plays a key role in driving neoplastic overgrowth.
doi:10.1186/1741-7007-8-33
PMCID: PMC2877678  PMID: 20374622
3.  Immunotherapy with low-dose recombinant interleukin 2 after high-dose chemotherapy and autologous stem cell transplantation in neuroblastoma. 
British Journal of Cancer  1998;78(4):528-533.
The purpose of this study was to evaluate in a phase I-II trial whether low doses of recombinant human interleukin 2 (rHuIL-2) over a prolonged period of time are safe and effective in eradicating or controlling minimal residual disease in children with neuroblastoma given high-dose chemotherapy (HDCT) and autologous stem cell transplantation (ASCT). From January 1992 to July 1996, 17 consecutive patients, with either stage IV or relapsed neuroblastoma, were enrolled. Patients received rHuIL-2 after a median time interval (min-max) of 105 days (56-153) after HDCT and ASCT. The protocol consisted of 2 'priming' courses of rHuIL-2 at escalating doses administered intravenously at 72-h intervals, followed by 'maintenance' with 11 monthly and six bimonthly boosting 5-day courses administered subcutaneously on an outpatient basis. At April 1997, 7 out of the 17 patients had completed the treatment schedule, four had discontinued treatment because of toxicity and four because of relapse; the remaining two patients are still on treatment, having completed 15 courses. Expansion of T lymphocytes, together with an increase in both natural killer cells and in activated T lymphocytes was evidenced. After a median (min-max) follow-up time of 30 (16-64) months, 12 out of 17 patients are alive and well. Two patients relapsed and died 14 and 35 months after transplant. Three patients are alive after having relapsed at 41, 21 and 13 months. The actuarial 2-year event-free survival and overall survival are 67% and 92% respectively. Intermittent administration of low doses of rHuIL-2 given for a long period of time is well tolerated and seems capable of controlling minimal residual disease after HDCT and ASCT in children with high-risk neuroblastoma.
PMCID: PMC2063104  PMID: 9716039
4.  Spleen nodules: a potential hallmark of Visceral Leishmaniasis in young children 
BMC Infectious Diseases  2014;14(1):620.
Background
Visceral leishmaniasis (VL) is a severe disease caused by Leishmania infantum in the Mediterranean basin, and is associated with considerable morbidity and mortality. Infantile VL may begin suddenly, with high fever and vomiting, or insidiously, with irregular daily fever, anorexia, and marked splenomegaly. Delays in diagnosis of VL are common, highlighting the need for increased awareness of clinicians for VL in endemic European countries.
Case presentation
We report 4 cases of young children in northern Italy presenting with persistent fever of unknown origin and diagnosed with VL by serological and molecular methods. At the time of diagnosis, these patients showed an unusual echographic pattern characterized by multiple iso-hypoechoic nodules associated with splenomegaly.
Conclusion
We suggest that detection of spleen nodules represents a signature of VL in infants, thus helping to diagnose systemic Leishmania infantum infection in children.
doi:10.1186/s12879-014-0620-2
PMCID: PMC4270008  PMID: 25496484
Visceral leishmaniasis; Hemophagocytic lymphohistiocytosis; Abdominal ultrasonography
5.  Assessment of the effect of sphingosine kinase inhibitors on apoptosis,unfolded protein response and autophagy of T-cell acute lymphoblastic leukemia cells; indications for novel therapeutics 
Oncotarget  2014;5(17):7886-7901.
Sphingosine 1-phosphate (S1P) is a bioactive lipid that is formed by the phosphorylation of sphingosine and catalysed by sphingosine kinase 1 (SK1) or sphingosine kinase 2 (SK2). Sphingosine kinases play a fundamental role in many signaling pathways associated with cancer, suggesting that proteins belonging to this signaling network represent potential therapeutic targets. Over the last years, many improvements have been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL); however, novel and less toxic therapies are still needed, especially for relapsing and chemo-resistant patients. Here, we analyzed the therapeutic potential of SKi and ROMe, a sphingosine kinase 1 and 2 inhibitor and SK2-selective inhibitor, respectively. While SKi induced apoptosis, ROMe initiated an autophagic cell death in our in vitro cell models. SKi treatment induced an increase in SK1 protein levels in Molt-4 cells, whereas it activated the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) pathway in Jurkat and CEM-R cells as protective mechanisms in a sub-population of T-ALL cells. Interestingly, we observed a synergistic effect of SKi with the classical chemotherapeutic drug vincristine. In addition, we reported that SKi affected signaling cascades implicated in survival, proliferation and stress response of cells. These findings indicate that SK1 or SK2 represent potential targets for treating T-ALL.
PMCID: PMC4202168  PMID: 25226616
T-cell acute lymphoblastic leukemia; sphingosine kinase inhibitors; apoptosis; autophagy; unfolded protein response
6.  Genomic profiling in Down syndrome acute lymphoblastic leukemia identifies histone gene deletions associated with altered methylation profiles 
Leukemia  2011;25(10):1555-1563.
Patients with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) have distinct clinical and biological features. Whereas most DS-ALL cases lack the sentinel cytogenetic lesions that guide risk assignment in childhood ALL, JAK2 mutations and CRLF2 overexpression are highly enriched. To further characterize the unique biology of DS-ALL, we performed genome-wide profiling of 58 DS-ALL and 68 non-Down syndrome (NDS) ALL cases by DNA copy number, loss of heterozygosity, gene expression, and methylation analyses. We report a novel deletion within the 6p22 histone gene cluster as significantly more frequent in DS-ALL, occurring in 11 DS (22%) and only two NDS cases (3.1%) (Fisher’s exact p = 0.002). Homozygous deletions yielded significantly lower histone expression levels, and were associated with higher methylation levels, distinct spatial localization of methylated promoters, and enrichment of highly methylated genes for specific pathways and transcription factor binding motifs. Gene expression profiling demonstrated heterogeneity of DS-ALL cases overall, with supervised analysis defining a 45-transcript signature associated with CRLF2 overexpression. Further characterization of pathways associated with histone deletions may identify opportunities for novel targeted interventions.
doi:10.1038/leu.2011.128
PMCID: PMC4107887  PMID: 21647151
Down syndrome; acute lymphoblastic leukemia (ALL); histone; JAK2; CRLF2
7.  A Unique Four-Hub Protein Cluster Associates to Glioblastoma Progression 
PLoS ONE  2014;9(7):e103030.
Gliomas are the most frequent brain tumors. Among them, glioblastomas are malignant and largely resistant to available treatments. Histopathology is the gold standard for classification and grading of brain tumors. However, brain tumor heterogeneity is remarkable and histopathology procedures for glioma classification remain unsatisfactory for predicting disease course as well as response to treatment. Proteins that tightly associate with cancer differentiation and progression, can bear important prognostic information. Here, we describe the identification of protein clusters differentially expressed in high-grade versus low-grade gliomas. Tissue samples from 25 high-grade tumors, 10 low-grade tumors and 5 normal brain cortices were analyzed by 2D-PAGE and proteomic profiling by mass spectrometry. This led to identify 48 differentially expressed protein markers between tumors and normal samples. Protein clustering by multivariate analyses (PCA and PLS-DA) provided discrimination between pathological samples to an unprecedented extent, and revealed a unique network of deranged proteins. We discovered a novel glioblastoma control module centered on four major network hubs: Huntingtin, HNF4α, c-Myc and 14-3-3ζ. Immunohistochemistry, western blotting and unbiased proteome-wide meta-analysis revealed altered expression of this glioblastoma control module in human glioma samples as compared with normal controls. Moreover, the four-hub network was found to cross-talk with both p53 and EGFR pathways. In summary, the findings of this study indicate the existence of a unifying signaling module controlling glioblastoma pathogenesis and malignant progression, and suggest novel targets for development of diagnostic and therapeutic procedures.
doi:10.1371/journal.pone.0103030
PMCID: PMC4106866  PMID: 25050814
8.  Atopic dermatitis phenotypes in childhood 
Background
Atopic dermatitis (AD) is a chronic inflammatory skin disease and can be the first step of the atopic march.
Objective
In this retrospective study, we analysed the immunological and clinical patterns of AD in a group of children affected by the disease since their first years of life, in order to evaluate if and how these patterns can change over time, and to identify biomarkers that can possibly correlate with the clinical phenotype.
Methods
We enrolled Caucasian children with diagnosis of AD performed by a specialist on the basis of Hanifin and Rajka’s criteria and with a first clinical and laboratory evaluation before 5 years of age. Patients were divided in 2 groups: IgE-associated AD (with or without allergic respiratory diseases) and non-IgE-associated AD.
Results
Among 184 patients enrolled in this study, at the beginning 30/184 were classified as having non-IgE-associated AD, but during follow-up, at the median age of 36 months, 15 patients became allergic. All 15 patients who switched from the non-IgE to the IgE-associated form had a significantly earlier onset of AD than those who did not switch. Dust mite sensitization seem to be the best biomarker (OR 2.86) to predict the appearance of allergic respiratory diseases.
Conclusion
IgE-associated AD is more frequent in childhood than non-IgE-associated AD. These two phenotypes are different in the age of onset and in the remission patterns. In the first years of life, it is important to distinguish the different phenotypes in order to evaluate possible allergic related conditions.
doi:10.1186/1824-7288-40-46
PMCID: PMC4063231  PMID: 24887188
Atopic dermatitis; Phenotypes; Allergy; Childhood; Atopic march
9.  Indoleamine 2,3-dioxygenase 1 (IDO1) activity in leukemia blasts correlates with poor outcome in childhood acute myeloid leukemia 
Oncotarget  2013;5(8):2052-2064.
Microenvironmental factors contribute to the immune dysfunction characterizing acute myeloid leukemia (AML). Indoleamine 2,3-dioxygenase 1 (IDO1) is an interferon (IFN)-γ-inducible enzyme that degrades tryptophan into kynurenine, which, in turn, inhibits effector T cells and promotes regulatory T-cell (Treg) differentiation. It is presently unknown whether childhood AML cells express IDO1 and whether IDO1 activity correlates with patient outcome.
We investigated IDO1 expression and function in 37 children with newly diagnosed AML other than acute promyelocytic leukemia. Blast cells were cultured with exogenous IFN-γ for 24 hours, followed by the measurement of kynurenine production and tryptophan consumption. No constitutive expression of IDO1 protein was detected in blast cells from the 37 AML samples herein tested. Conversely, 19 out of 37 (51%) AML samples up-regulated functional IDO1 protein in response to IFN-γ. The inability to express IDO1 by the remaining 18 AML samples was not apparently due to a defective IFN-γ signaling circuitry, as suggested by the measurement of signal transducer and activator of transcription 3 (STAT3) phosphorylation. Co-immunoprecipitation assays indicated the occurrence of physical interactions between STAT3 and IDO1 in AML blasts. In line with this finding, STAT3 inhibitors abrogated IDO1 function in AML blasts. Interestingly, levels of IFN-γ were significantly higher in the bone marrow fluid of IDO-expressing compared with IDO-nonexpressing AMLs. In mixed tumor lymphocyte cultures (MTLC), IDO-expressing AML blasts blunted the ability of allogeneic naïve T cells to produce IFN-γ and promoted Treg differentiation. From a clinical perspective, the 8-year event-free survival was significantly worse in IDO-expressing children (16.4%, SE 9.8) as compared with IDO-nonexpressing ones (48.0%, SE 12.1; p=0.035).
These data indicate that IDO1 expression by leukemia blasts negatively affects the prognosis of childhood AML. Moreover, they speak in favor of the hypothesis that IDO can be targeted, in adjunct to current chemotherapy approaches, to improve the clinical outcome of children with AML.
PMCID: PMC4039144  PMID: 24903009
Acute myeloid leukemia; IDO1; immune escape; regulatory T cells
10.  Molecular Imaging of Neuroblastoma Progression in TH-MYCN Transgenic Mice 
Purpose
TH-MYCN transgenic mice represent a valuable preclinical model of neuroblastoma. Current methods to study tumor progression in these mice are inaccurate or invasive, limiting the potential of this murine model. The aim of our study was to assess the potential of small animal positron emission tomography (SA-PET) to study neuroblastoma progression in TH-MYCN mice.
Procedure
Serial SA-PET scans using the tracer 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG) have been performed in TH-MYCN mice. Image analysis of tumor progression has been compared with ex vivo evaluation of tumor volumes and histological features.
Results
[18F]FDG-SA-PET allowed to detect early staged tumors in almost 100 % of TH-MYCN mice positive for disease. Image analysis of tumor evolution reflected the modifications of the tumor volume, histological features, and malignancy during disease progression. Image analysis of TH-MYCN mice undergoing chemotherapy treatment against neuroblastoma provided information on drug-induced alterations in tumor metabolic activity.
Conclusions
These data show for the first time that [18F]FDG-SA-PET is a useful tool to study neuroblastoma presence and progression in TH-MYCN transgenic mice.
doi:10.1007/s11307-012-0576-9
PMCID: PMC3594000  PMID: 22777578
Neuroblastoma; Small animal PET; [18F]FDG; TH-MYCN mice
11.  Next Generation Sequencing Improves the Accuracy of KRAS Mutation Analysis in Endoscopic Ultrasound Fine Needle Aspiration Pancreatic Lesions 
PLoS ONE  2014;9(2):e87651.
The use of endoscopic ultrasonography has allowed for improved detection and pathologic analysis of fine needle aspirate material for pancreatic lesion diagnosis. The molecular analysis of KRAS has further improved the clinical sensitivity of preoperative analysis. For this reason, the use of highly analytical sensitive and specific molecular tests in the analysis of material from fine needle aspirate specimens has become of great importance. In the present study, 60 specimens from endoscopic ultrasonography fine needle aspirate were analyzed for KRAS exon 2 and exon 3 mutations, using three different techniques: Sanger sequencing, allele specific locked nucleic acid PCR and Next Generation sequencing (454 GS-Junior, Roche). Moreover, KRAS was also tested in wild-type samples, starting from DNA obtained from cytological smears after pathological evaluation. Sanger sequencing showed a clinical sensitivity for the detection of the KRAS mutation of 42.1%, allele specific locked nucleic acid of 52.8% and Next Generation of 73.7%. In two wild-type cases the re-sequencing starting from selected material allowed to detect a KRAS mutation, increasing the clinical sensitivity of next generation sequencing to 78.95%. The present study demonstrated that the performance of molecular analysis could be improved by using highly analytical sensitive techniques. The Next Generation Sequencing allowed to increase the clinical sensitivity of the test without decreasing the specificity of the analysis. Moreover we observed that it could be useful to repeat the analysis starting from selectable material, such as cytological smears to avoid false negative results.
doi:10.1371/journal.pone.0087651
PMCID: PMC3913642  PMID: 24504548
12.  MYCN is a novel oncogenic target in pediatric T-cell Acute Lymphoblastic Leukemia 
Oncotarget  2013;5(1):120-130.
MYCN is an oncogene frequently overexpressed in pediatric solid tumors whereas few evidences suggest his involvement in the pathogenesis of haematologic malignancies. Here we show that MYCN is overexpressed in a relevant proportion (40 to 50%) of adult and pediatric T-cell acute lymphoblastic leukemias (T-ALL). Focusing on pediatric T-ALL, MYCN-expressing samples were found almost exclusively in the TAL1-positive subgroup. Moreover, TAL1 knockdown in T-ALL cell lines resulted in a reduction of MYCN expression, and TAL1 directly binds to MYCN promoter region, suggesting that TAL1 pathway activation could sustain the up-regulation of MYCN. The role of MYCN in T-ALL was investigated by peptide nucleic acid (PNA-MYCN)-mediated transcriptional silencing of MYCN and by siRNAs. MYCN knockdown in T-ALL cell lines resulted in a reduction of cell viability, up to 50%, while no effect was elicited with a mismatch PNA. The inhibitory effect of PNA-MYCN on cell viability was due to a significant increase in apoptosis. PNA-MYCN treatment in pediatric T-ALL samples reduced cell viability of leukemic cells from patients with high MYCN expression, while no effect was obtained in MYCN-negative blast cells. These results showed that MYCN is frequently overexpressed in pediatric T-ALL and suggested his role as a candidate for molecularly-directed therapies.
PMCID: PMC3960194  PMID: 24334727
pediatric T-ALL; MYCN; peptide nucleic acid; TAL1; TGF-β inhibitors
13.  Acute Promyelocytic Leukemia (APL): Comparison Between Children and Adults 
The outcome of adults and children with Acute Promyelocytic Leukemia (APL) has dramatically changed since the introduction of all trans retinoic acid (ATRA) therapy. Based on the results of several multicenter trials, the current recommendations for the treatment of patients with APL include ATRA and anthracycline-based chemotherapy for the remission induction and consolidation, and ATRA combined with low-dose chemotherapy for maintenance. This has improved the prognosis of APL by increasing the complete remission (CR) rate, actually > 90%, decreasing the induction deaths and by reducing the relapse rate, leading to cure rates nowadays exceeding 80% considering both adults and children.1–9 More recently the combination of ATRA and arsenic trioxide (ATO) as induction and consolidation therapy has been shown to be at least not inferior and possibly superior to ATRA plus chemotherapy in adult patients with APL conventionally defined as non-high risk (Sanz score).10
Childhood APL has customarily been treated on adult protocols. Data from several trials have shown that the overall outcome in pediatric APL appears similar to that reported for the adult population; however, some clinical and therapeutic aspects differ in the two cohorts which require some important considerations and treatment adjustments.
doi:10.4084/MJHID.2014.032
PMCID: PMC4010611  PMID: 24804005
14.  Next-Generation Sequencing of Lung Cancer EGFR Exons 18-21 Allows Effective Molecular Diagnosis of Small Routine Samples (Cytology and Biopsy) 
PLoS ONE  2013;8(12):e83607.
Selection of lung cancer patients for therapy with tyrosine kinase inhibitors directed at EGFR requires the identification of specific EGFR mutations. In most patients with advanced, inoperable lung carcinoma limited tumor samples often represent the only material available for both histologic typing and molecular analysis. We defined a next generation sequencing protocol targeted to EGFR exons 18-21 suitable for the routine diagnosis of such clinical samples. The protocol was validated in an unselected series of 80 small biopsies (n=14) and cytology (n=66) specimens representative of the material ordinarily submitted for diagnostic evaluation to three referral medical centers in Italy. Specimens were systematically evaluated for tumor cell number and proportion relative to non-neoplastic cells. They were analyzed in batches of 100-150 amplicons per run, reaching an analytical sensitivity of 1% and obtaining an adequate number of reads, to cover all exons on all samples analyzed. Next generation sequencing was compared with Sanger sequencing. The latter identified 15 EGFR mutations in 14/80 cases (17.5%) but did not detected mutations when the proportion of neoplastic cells was below 40%. Next generation sequencing identified 31 EGFR mutations in 24/80 cases (30.0%). Mutations were detected with a proportion of neoplastic cells as low as 5%. All mutations identified by the Sanger method were confirmed. In 6 cases next generation sequencing identified exon 19 deletions or the L858R mutation not seen after Sanger sequencing, allowing the patient to be treated with tyrosine kinase inhibitors. In one additional case the R831H mutation associated with treatment resistance was identified in an EGFR wild type tumor after Sanger sequencing. Next generation sequencing is robust, cost-effective and greatly improves the detection of EGFR mutations. Its use should be promoted for the clinical diagnosis of mutations in specimens with unfavorable tumor cell content.
doi:10.1371/journal.pone.0083607
PMCID: PMC3871524  PMID: 24376723
16.  DUAL INHIBITION OF CLASS IA PHOSPHATIDYLINOSITOL 3-KINASE AND mTOR AS A NEW THERAPEUTIC OPTION FOR T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA 
Cancer research  2009;69(8):10.1158/0008-5472.CAN-08-4884.
Recent investigations have documented that constitutively activated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL) where it strongly influences growth and survival. These findings lend compelling weight for the application of PI3K/Akt/mTOR inhibitors in T-ALL. However, our knowledge of PI3K/Akt/mTOR signaling in T-ALL is limited and it is not clear whether it could be an effective target for innovative therapeutic strategies. Here, we have analyzed the therapeutic potential of the dual PI3K/mTOR inhibitor, PI-103, a small synthetic molecule of the pyridofuropyrimidine class, on both T-ALL cell lines and patient samples, which displayed constitutive activation of PI3K/Akt/mTOR signaling. PI-103 inhibited the growth of T-ALL cells, including 170-kDa glycoprotein overexpressing cells. PI-103 cytotoxicity was independent of p53 gene status. PI-103 was more potent than inhibitors which are selective only for PI3K (Wortmannin, LY294002) or for mTOR (rapamycin). PI-103 induced G0/G1 phase cell cycle arrest and apoptosis which was characterized by activation of caspase-3 and -9. PI-103 caused Akt dephosphorylation, accompanied by dephosphorylation of the Akt downstream target, glycogen synthase kinase-3β. Also mTOR downstream targets were dephosphorylated in response to PI-103, including p70S6 kinase, ribosomal S6 protein, and 4E-BP1. PI-103 strongly synergized with vincristine. These findings indicate that multi-targeted therapy towards PI3K and mTOR, alone or with existing drugs, may serve as an efficient treatment towards T-ALL cells which require upregulation of PI3K/Akt/mTOR signaling for their survival and growth.
doi:10.1158/0008-5472.CAN-08-4884
PMCID: PMC3836286  PMID: 19351820
PI3K/Akt/mTOR signaling; apoptosis; caspases; drug resistance; combination therapy
18.  DHH-RHEBL1 fusion transcript: a novel recurrent feature in the new landscape of pediatric CBFA2T3-GLIS2-positive acute myeloid leukemia 
Oncotarget  2013;4(10):1712-1720.
Childhood Acute Myeloid Leukemia (AML) is a clinically and genetically heterogeneous malignant disease. Despite improvements in outcome over the past decades, the current survival rate still is approximately 60-70%. Cytogenetic, recurrent genetic abnormalities and early response to induction treatment are the main factors predicting clinical outcome. While the majority of children carry recurrent chromosomal translocations, 20% of patients do not show any recognizable cytogenetic alteration and are defined to have cytogenetically normal AML (CN-AML). This subset of patients is characterized by a significant heterogeneity in clinical outcome, which is influenced by factors only recently started to be identified. In this respect, genome-wide analyses have been used with the aim of defining the full array of genetic lesions in CN-AML. Recently, through whole-transcriptome massively parallel sequencing of seven cases of pediatric CN-AML, we identified a novel recurrent CBFA2T3-GLIS2 fusion, predicting poorer outcome. However, since the expression of CBFA2T3-GLIS2 fusion in mice is not sufficient for leukemogenesis, we speculated that further unknown abnormalities could contribute to both cancer transformation and response to treatment. Thus, we analyzed, by whole-transcriptome sequencing, 4 CBFA2T3-GLIS2-positive patients, as well as 4 CN-AML patients. We identified a new fusion transcript in the CBFA2T3-GLIS2 -positive patients, involving Desert Hedgehog (DHH), a member of Hedgehog family, and Ras Homologue Enrich in Brain Like 1 (RHEBL1), a gene coding for a small GTPase of the Ras family. Through the screening of a validation cohort of 55 additional pediatric AML patients, we globally detected DHH-RHEBL1 fusion in 8 out of 20 (40%) CBFA2T3-GLIS2- rearranged patients. Gene expression analysis performed on RNA-seq data revealed that DHH-RHEBL1 –positive patients exhibited a specific signature. These 8 patients had an 8-year overall survival worse than that of the remaining 12 CBFA2T3-GLIS2- rearranged patients not harboring DHH-RHEBL1 fusion (25% vs 55%, respectively, P =0.1). Taken together, these findings are unprecedented and indicate that the DHH-RHEBL1 fusion transcript is a novel recurrent feature in the changing landscape of CBFA2T3-GLIS2 -positive childhood AML. Moreover, it could be instrumental in the identification of a subgroup of CBFA2T3-GLIS2 -positive patients with a very poor outcome.
PMCID: PMC3858557  PMID: 24127550
pediatric acute myeloid leukemia; cytogenetically normal acute myeloid leukemia; whole-transcriptome massively parallel sequencing; CBFA2T3-GLIS2 fusion transcript; DHH-RHEBL1 fusion transcript
19.  454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples 
OncoTargets and therapy  2013;6:1057-1064.
Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues.
doi:10.2147/OTT.S42369
PMCID: PMC3741083  PMID: 23950653
colorectal cancer; targeted therapy; KRAS mutations; next-generation sequencing; real-time polymerase chain reaction; pyrosequencing
20.  Ewing Sarcoma of the Bone in Children under 6 Years of Age 
PLoS ONE  2013;8(1):e53223.
Background
Ewing Sarcoma Family Tumours (ESFT) are rare in early childhood. The aim of this study was to report the clinical characteristics and outcome of children under 6 years of age affected by ESFT of the bone in Italy.
Methods
The records of all the children diagnosed with osseous ESFT in centres members of the Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP) from 1990 to 2008 were reviewed. The Kaplan–Meier method was used for estimating overall and progression-free survival (OS, PFS) curves; multivariate analyses were performed using Cox proportional hazards regression model.
Results
This study includes 62 patients. An axial primary localization was present in 66% of patients, with the primary site in the chest wall in 34%. Fourteen (23%) patients presented metastatic disease. The 5-year OS and PFS were 73% (95% confidence interval, CI, 58–83%) and 72% (95% CI 57–83%) for patients with localized disease and 38% (95% CI 17–60%) and 21% (95% CI 5–45%) for patients with metastatic disease. Metastatic spread, skull/pelvis/spine primary localization, progression during treatment and no surgery predicted worse survival (P<0.01), while patients treated in the last decade had better survival (P  = 0.002). In fact, the 5-year OS and PFS for patients diagnosed in the period 2000–2008 were 89% (95% CI 71–96%) and 86% (95% CI 66–94%), respectively.
Conclusion
The axial localization is the most common site of ESFT in pre-scholar children. Patients treated in the most recent period have an excellent outcome.
doi:10.1371/journal.pone.0053223
PMCID: PMC3561359  PMID: 23382839
21.  Definition of miRNAs Expression Profile in Glioblastoma Samples: The Relevance of Non-Neoplastic Brain Reference 
PLoS ONE  2013;8(1):e55314.
Glioblastoma is the most aggressive brain tumor that may occur in adults. Regardless of the huge improvements in surgery and molecular therapy, the outcome of neoplasia remains poor. MicroRNAs are small molecules involved in several cellular processes, and their expression is altered in the vast majority of tumors. Several studies reported the expression of different miRNAs in glioblastoma, but one of the most critical point in understanding glioblastoma miRNAs profile is the comparison of these studies. In this paper, we focused our attention on the non-neoplastic references used for determining miRNAs expression. The aim of this study was to investigate if using three different non-neoplastic brain references (normal adjacent the tumor, commercial total RNA, and epileptic specimens) could provide discrepant results. The analysis of 19 miRNAs was performed using Real-Time PCR, starting from the set of samples described above and the expression values compared. Moreover, the three different normal RNAs were used to determine the miRNAs profile in 30 glioblastomas. The data showed that different non-neoplastic controls could lead to different results and emphasize the importance of comparing miRNAs profiles obtained using the same experimental condition.
doi:10.1371/journal.pone.0055314
PMCID: PMC3558478  PMID: 23383149
22.  Citrus Allergy from Pollen to Clinical Symptoms 
PLoS ONE  2013;8(1):e53680.
Allergy to citrus fruits is often associated with pollinosis and sensitization to other plants due to a phenomenon of cross-reactivity. The aims of the present study were to highlight the cross-reactivity among citrus and the major allergenic pollens/fruits, throughout clinical and molecular investigations, and to evaluate the sensitization frequency to citrus fruits in a population of children and adults with pollinosis. We found a relevant percentage of sensitisation (39%) to citrus fruits in the patients recruited and in all of them the IgE-mediated mechanism has been confirmed by the positive response to the prick-to-prick test. RT-PCR experiments showed the expression of Cit s 1, Cit s 3 and a profilin isoform, already described in apple, also in Citrus clementine pollen. Data of multiple sequence alignments demonstrated that Citrus allergens shared high percentage identity values with other clinically relevant species (i.e. Triticum aestivum, Malus domestica), confirming the possible cross-allergenicity citrus/grasses and citrus/apple. Finally, a novelty of the present work has been the expression of two phospholipaseA2 isoforms (PLA2 α and β) in Citrus as well as in Triticum pollens; being PLA2 able to generate pro-inflammatory factors, this enzyme could participate in the activation of the allergenic inflammatory cascade.
doi:10.1371/journal.pone.0053680
PMCID: PMC3537725  PMID: 23308273
23.  Harnessing the PI3K/Akt/mTOR pathway in T-cell acute lymphoblastic leukemia: Eliminating activity by targeting at different levels 
Oncotarget  2012;3(8):811-823.
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant hematological disorder arising in the thymus from T-cell progenitors. T-ALL mainly affects children and young adults, and remains fatal in 20% of adolescents and 50% of adults, despite progress in polychemotherapy protocols. Therefore, innovative targeted therapies are desperately needed for patients with a dismal prognosis. Aberrant activation of PI3K/Akt/mTOR signaling is a common event in T-ALL patients and portends a poor prognosis. Preclinical studies have highlighted that modulators of PI3K/Akt/mTOR signaling could have a therapeutic relevance in T-ALL. However, the best strategy for inhibiting this highly complex signal transduction pathway is still unclear, as the pharmaceutical companies have disclosed an impressive array of small molecules targeting this signaling network at different levels. Here, we demonstrate that a dual PI3K/PDK1 inhibitor, NVP-BAG956, displayed the most powerful cytotoxic effects against T-ALL cell lines and primary patients samples, when compared with a pan class I PI3K inhibitor (GDC-0941), an allosteric Akt inhibitor (MK-2206), an mTORC1 allosteric inhibitor (RAD-001), or an ATP-competitive mTORC1/mTORC2 inhibitor (KU-63794). Moreover, we also document that combinations of some of the aforementioned drugs strongly synergized against T-ALL cells at concentrations well below their respective IC50. This observation indicates that vertical inhibition at different levels of the PI3K/Akt/mTOR network could be considered as a future innovative strategy for treating T-ALL patients.
PMCID: PMC3478458  PMID: 22885370
acute leukemia; targeted therapy; signal transduction modulators; PI3K/PDK1; vertical inhibition
24.  Unbalance of intestinal microbiota in atopic children 
BMC Microbiology  2012;12:95.
Background
Playing a strategic role in the host immune function, the intestinal microbiota has been recently hypothesized to be involved in the etiology of atopy. In order to investigate the gastrointestinal microbial ecology of atopic disease, here we performed a pilot comparative molecular analysis of the faecal microbiota in atopic children and healthy controls.
Results
Nineteen atopic children and 12 healthy controls aged 4–14 years were enrolled. Stools were collected and the faecal microbiota was characterized by means of the already developed phylogenetic microarray platform, HTF-Microbi.Array, and quantitative PCR. The intestinal microbiota of atopic children showed a significant depletion in members of the Clostridium cluster IV, Faecalibacterium prausnitzii, Akkermansia muciniphila and a corresponding increase of the relative abundance of Enterobacteriaceae.
Conclusion
Depleted in key immunomodulatory symbionts, the atopy-associated microbiota can represent an inflammogenic microbial consortium which can contribute to the severity of the disease. Our data open the way to the therapeutic manipulation of the intestinal microbiota in the treatment of atopy by means of pharmaceutical probiotics.
doi:10.1186/1471-2180-12-95
PMCID: PMC3404014  PMID: 22672413
25.  Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation 
PLoS ONE  2012;7(4):e36084.
The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes.
doi:10.1371/journal.pone.0036084
PMCID: PMC3340405  PMID: 22558339

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