Search

In a previous study, the whole transcriptome of the vastus lateralis muscle from sedentary elderly and from age-matched athletes with an exceptional record of high-intensity, life-long exercise training was compared—the two groups representing the two extremes on a physical activity scale. Exercise training enabled the skeletal muscle to counteract age-related sarcopenia by inducing a wide range of adaptations, sustained by the expression of protein-coding genes involved in energy handling, proteostasis, cytoskeletal organization, inflammation control, and cellular senescence. Building on the previous study, we examined here the network of non-coding RNAs participating in the orchestration of gene expression and identified differentially expressed micro- and long-non-coding RNAs and some of their possible targets and roles. Unsupervised hierarchical clustering analyses of all non-coding RNAs were able to discriminate between sedentary and trained individuals, regardless of the exercise typology. Validated targets of differentially expressed miRNA were grouped by KEGG analysis, which pointed to functional areas involved in cell cycle, cytoskeletal control, longevity, and many signaling pathways, including AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), which had been shown to be pivotal in the modulation of the effects of high-intensity, life-long exercise training. The analysis of differentially expressed long-non-coding RNAs identified transcriptional networks, involving lncRNAs, miRNAs and mRNAs, affecting processes in line with the beneficial role of exercise training.

Background CBFA2T3-GLIS2 is a fusion gene found in 17% of non-Down syndrome acute megakaryoblastic leukemia (non-DS AMKL, FAB M7) and in 8% of pediatric cytogenetically normal acute myeloid leukemia (CN-AML, in association with several French-American-British (FAB) subtypes). Children with AML harboring this aberration have a poor outcome, regardless of the FAB subtype. This fusion gene drives a peculiar expression pattern and leads to overexpression of some of Hedgehog-related genes. GLI-similar protein 2 (GLIS2) is closely related to the GLI family, the final effectors of classic Hedgehog pathway. These observations lend compelling support to the application of GLI inhibitors in the treatment of AML with the aberration CBFA2T3-GLIS2. GANT61 is, nowadays, the most potent inhibitor of GLI family proteins. Methods We exposed to GANT61 AML cell lines and primary cells positive and negative for CBFA2T3-GLIS2 and analyzed the effect on cellular viability, induction of apoptosis, cell cycle, and expression profile. Results As compared to AML cells without GLIS2 fusion, GANT61 exposure resulted in higher sensitivity of both cell lines and primary AML cells carrying CBFA2T3-GLIS2 to undergo apoptosis and G1 cell cycle arrest. Remarkably, gene expression studies demonstrated downregulation of GLIS2-specific signature genes in both treated cell lines and primary cells, in comparison with untreated cells. Moreover, chromatin immunoprecipitation analysis revealed direct regulation by GLIS2 chimeric protein of DNMT1 and DNMT3B, two genes implicated in important epigenetic functions. Conclusions Our findings indicate that the GLI inhibitor GANT61 may be used to specifically target the CBFA2T3-GLIS2 fusion gene in pediatric AML. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0396-0) contains supplementary material, which is available to authorized users.

Introduction: Intraductal papillary mucinous neoplasm (IPMN) is the most common cystic precancerous lesion of pancreatic cancer (PDA). IPMNs can progress from low to high-grade dysplasia, to invasive PDAC, but exact data regarding cancer risks are limited. PDAC is the fourth leading cause of cancer death . The overall 5-year survival is 6%, but survival of patients with early stage PDAC is significantly better. AIM: We used an approach coupling high resolution cytogenetic analysis (Affymetrix Oncoscan FFPE Array) with a clinically-oriented bioinformatic interpretation of data to understand what are the most relevant pathways altered in precursor lesions. Materials and methods: High resolution cytogenetic analysis was performed in 20 formalin fixed paraffin embedded samples of IPMNs by Oncoscan FFPE assay. Genomic data were analyzed for copy number gains and losses, loss of heterozygosity and for a panel of recurrent somatic mutations. Results were validated by qPCR and FISH analysis. Results: Twenty samples, including 14 mixed-type IPMNs (70%), 4 branch-duct IPMNs (20%) and 2 main-duct IPMNs (10%), were collected. We identified micro-invasive carcinoma (< 1 mm) and invasive carcinoma in 25% and in 10% of IPMNs respectively. By our high resolution cytogenetic analysis we observed that 8 IPMNs (40%) presented a nearly normal karyotype (< 4 copy number alterations), while 12 IPMNs (60%) showed a complex karyotype (> 10 alterations). In the latter subgroup were detected specific copy number gains of TERC and c-MYC oncogenes. Interestingly we noticed that TERC and c-MYC overexpression, present in 92% and 45% of complex karyotype samples respectively, were only observed in high-grade IPMNs, suggesting a possible role in a progression to malignancy. Oncoscan data were confirmed by Real Time and FISH analysis. Conclusions: Those results suggest a role of TERC and c-MYC overexpression as a possible biomarkers in the early identification of patients with complex karyotype IPMNs in order to better stratification of cystic lesions that could require surgery

Context. Despite pancreatic cancer (PC) genomic char- acterization, true advances in the development of prog- nosis classification and new therapeutic strategies have yet to come. Objective. We aimed to better understand genomic al- terations of invasive phenotype in order to improve pa- tient selection for treatment options. Methods. We analyzed 37 PC samples by either whole transcriptome or exome sequencing performed at 75- 100 bpx2 on an Illumina platform, matched with normal DNA to identify somatic events. Calls of single nucleo- tide variants and InDels were annotated with 1000 ge- nomes allele frequencies, dbSNP149 rsIDs, Exac and EVS using Oncotator. We detected the pathogenicity of the emerging variants by previous knowledge and bio- informatic mutation-prediction tools. Results. We highlighted 43 recurrently altered genes involving several pathways including chromatine re- modelling and DNA damage repair. The analysis limited on early stage patients (40% of samples) showed how a poor prognosis was significantly associated with a higher number of known PC mutations (pValue=0.047). The subgroup with better overall survival (>25 months) harbors an average of 24 events instead of the one with an overall survival

// Sandra Durante 1, * , Silvia Vecchiarelli 2, * , Annalisa Astolfi 1 , Elisa Grassi 2 , Riccardo Casadei 3 , Donatella Santini 4 , Riccardo Panzacchi 4 , Claudio Ricci 3 , Salvatore Serravalle 5 , Giuseppe Tarantino 1 , Mirella Falconi 6 , Gabriella Teti 6 , Valentina Indio 1 , Andrea Pession 5 , Francesco Minni 3 , Guido Biasco 1, 2 , Mariacristina Di Marco 2 1 Giorgio Prodi Cancer Research Centre, University of Bologna, Bologna, Italy 2 Department of Experimental, Diagnostic and Specialty Medicine University of Bologna, Sant’Orsola-Malpighi Hospital, Bologna, Italy 3 Department of Medical and Surgical Sciences, University of Bologna, Sant’Orsola-Malpighi Hospital, Bologna, Italy 4 Pathology Unit, Sant'Orsola-Malpighi Hospital, Bologna, Italy 5 Department of Medical and Surgical Sciences, “Lalla Seragnoli” Hematology-Oncology Unit, University of Bologna, Bologna, Italy 6 DIBINEM—Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy * These authors contributed equally to this work Correspondence to: Annalisa Astolfi, email: annalisa.astolfi@unibo.it Keywords: PDA, IPMN, chromosome 3, NGS, PIK3CA Received: April 11, 2016 Accepted: August 10, 2016 Published: August 22, 2016 ABSTRACT Background: Intraductal papillary mucinous neoplasm (IPMN) is the most common cystic preneoplastic lesion of pancreatic cancer. We used an approach coupling high resolution cytogenetic analysis (Affymetrix Oncoscan FFPE Array) with clinically-oriented bioinformatic interpretation of data to understand the most relevant alterations of precursor lesions at different stages to identify new diagnostic markers. Results: We identified multiple copy number alterations, particularly in lesions with severe dysplasia, with 7 IPMN with low-intermediate dysplasia carrying a nearly normal karyotype and 13 IPMN with complex Karyotype (> 4 alterations), showing high grade dysplasia. A specific gain of chromosome arm 3q was found in IPMN with complex Karyotype (92%). This gain of 3q is particularly interesting for the presence of oncogenes such as PIK3CA, GATA2 and TERC that are part of pathways that deregulate cell growth and promote disease progression. Quantitative PCR and FISH analysis confirmed the data . Further demonstration of the overexpression of the PIK3CA gene supports the identification of this alteration as a possible biomarker in the early identification of patients with IPMN at higher risk for disease progression. Materials and methods: High resolution cytogenetic analysis was performed in 20 formalin fixed paraffin embedded samples of IPMN by Oncoscan FFPE assay. Results were validated by qPCR and FISH analysis. Conclusions: The identification of these markers at an early stage of disease onset could help to identify patients at risk for cancer progression and new candidates for a more specific targeted therapy.

Dermatofibrosarcoma protuberans (DFSP) is a rare and indolent cutaneous sarcoma. At times, a fibrosarcomatous transformation marked by a more aggressive clinical behavior may be present. We investigated the natural history and the molecular bases of progression from classic DFSP to the fibrosarcomatous form (FS-DFSP), looking, retrospectively, at the outcome of all patients affected by primary DFSP treated at our institution from 1993 to 2012 and analyzing the molecular profile of 5 DFSPs and 5 FS-DFSPs by an integrated genomics approach (whole transcriptome sequencing, copy number analysis, FISH, qRT-PCR, IHC). The presence of fibrosarcomatous features was identified in 20 (7.6%) patients out of 263 DFSP. All cases were treated with macroscopic complete surgery. A local relapse occurred in 4 of 23 patients who received a microscopic marginal surgery (2 classic DFSP, 2 FS-DFSP), while metastasis affected 2 patients, both FS-DFSP (10% of FS-DFSP), being the first event. DFSP evolution to FS-DFSP was paralleled by a transcriptional reprogramming. The recurrent loss of chromosome 22q appeared to contribute to this phenomenon by promoting the expression of epigenetic regulators, such as EZH2. Loss of the p16/CDKN2A/INK4A locus at 9p was also observed in two FS-DFSP metastatic cases. Implications: FS-DFSP is a rare subgroup among DFSP, with a 10% metastatic risk, that was independent from local recurrence and that was not observed in DFSP, that were all cured by wide surgery. Chromosome 22q deletion might play a role in FS-DFSP, and p16 loss may convey a poor outcome. EZH2 dysregulation was also found and represents a druggable target. Mol Cancer Res; 14(9); 820–9. ©2016 AACR.

Background Blastic plasmacytoid dendritic cell neoplasm (BPDCN) and myeloid sarcoma (MS) are two extremely rare and aggressive hematological diseases. Both malignancies most commonly arise with skin lesions with or without extramedullary organ involvement before leukemic dissemination. Given its rarity and less known biology, in some cases with defective/ambiguous phenotype distinguish between these two entities may be challenging for pathologists and clinicians.1 Can the machine learning predictive model help to solve this diagnostic question? Here we performed the first study of microRNA (miRNA) profiling of BPDCN and MS in order to 1) Discover new molecular features selectively driving BPDCN respect to MS 2) Develop a machine learning based-prediction tool useful for discriminating BPDCN and MS. Methods We performed miRNA profiling (NanoString Technologies) of cutaneous biopsies of 16 BPDCN and 23 MS cases. Using Supervised Analysis, we identified 49 miRNAs differentially expressed that were randomly validated by qRT-PCR and next interrogated by functional enrichment analysis. Finally, a machine learning algorithm based on Linear Discriminant Analysis2 was applied to identify candidate miRNAs able to discriminate BPDCN from MS cases. Results In line with the overlapping clinical features of BPDCN and MS, the molecular profiling of these two diseases was proved to be extremely similar. Unsupervised Analysis well demonstrated that the miRNA profiles of the two malignancies are closely related and indeed, BPDCN and MS cases cluster together. When a Supervised Analysis was applied, we identified a set of 49 miRNAs differentially expressed in BPDCN respect to MS, 25 down- and 24 up-regulated. Of relevance, down-regulated miRNAs were predicted to be markedly involved in the apoptosis regulation of BPDCN. Machine learning predictive model identified a set of 12 miRNAs (5 up and 7 down) able to discriminate cutaneous BPDCN from MS. Conclusion This is the first miRNA profiling study in BPDCN and MS that showed how strongly these two diseases overlap at molecular level. Despite their similarity, BPDCN cases displayed a set of miRNAs significantly down-regulated when compared to MS, with possible dysregulation of cell death pathway. Of practical interest, we designed a machine learning predictive model based on the expression of 12 miRNAs, which alone may be applied to distinguish between the two hematological diseases. This tool, if validated in a larger set of cases, may help to differentiate BPDCN and MS in cases with defective/ambiguous phenotype. References 1. Weltgesundheitsorganisation. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th edition. (Swerdlow SH, Campo E, Harris NL, et al., eds.). Lyon: International Agency for Research on Cancer; 2017. 2. Laginestra MA, Piccaluga PP, Fuligni F, et al. Pathogenetic and diagnostic significance of microRNA deregulation in peripheral T-cell lymphoma not otherwise specified. Blood Cancer J. 2014;4:259. doi:10.1038/bcj.2014. Citation Format: Maria Rosaria Sapienza, Fabio Fuligni, Federica Melle, Valentina Tabanelli, Valentina Indio, Alessandro Pileri, Lorenzo Cerroni, Francesco Bacci, Giovanna Motta, Maria Antonella Laginestra, Saveria Mazzara, Luciano Cascione, Alessandro Laganà, Claudio Agostinelli, Manuela Ferracin, Elena Sabattini, Carlo Croce, Stefano Pileri. Development of a miRNA-based prediction tool to discriminate cutaneous blastic plasmacytoid dendritic cell neoplasm from cutaneous myeloid sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1417.

Background: Gastrointestinal stromal tumors (GISTs) represent a worldwide paradigm of target therapy. The introduction of tyrosine kinase inhibitors has deeply changed the prognosis of GIST patients, however, the majority of them acquire secondary mutations and progress. Unfortunately, besides tyrosine-kinase inhibitors, no other therapeutic options are available. Therefore, it is mandatory to identify novel molecules and/or strategies to overcome the inevitable resistance. In this context, after promising preclinical data on the novel PI3K inhibitor BYL719, the NCT01735968 trial in GIST patients who had previously failed treatment with imatinib and sunitinib started. BYL719 has attracted our attention, and we comprehensively characterized genomic and transcriptomic changes taking place during resistance. Methods: For this purpose, we generated two in vitro GIST models of acquired resistance to BYL719 and performed an omic-based analysis by integrating RNA-sequencing, miRNA, and methylation profiles in sensitive and resistant cells. Results: We identified novel epigenomic mechanisms of pharmacological resistance in GISTs suggesting the existence of pathways involved in drug resistance and alternatively acquired mutations. Therefore, epigenomics should be taken into account as an alternative adaptive mechanism. Conclusion: Despite the fact that currently we do not have patients in treatment with BYL719 to verify this hypothesis, the most intriguing result is the involvement of H19 and PSTA1 in GIST resistance, which might represent druggable targets.

Extraskeletal myxoid chondrosarcoma (EMC) is an extremely rare soft tissue sarcoma, marked by a translocation involving the NR4A3 gene. EMC is usually indolent and moderately sensitive to anthracycline-based chemotherapy. Recently, we reported on the therapeutic activity of sunitinib in a series of EMC cases, however the molecular target of sunitinib in EMC is unknown. Moreover, there is still the need to identify alternative therapeutic strategies. To better characterize this disease, we performed whole transcriptome sequencing in five EMC cases. Peculiarly, in one sample, an in-frame deletion (c.1735_1737delGAT p.D579del) was identified in exon 11 of KIT. The deletion was somatic and heterozygous and was validated both at DNA and mRNA level. This sample showed a marked high expression of KIT at the mRNA level and a mild phosphorylation of the receptor. Sanger sequencing of KIT in additional 15 Formalin Fixed Paraffin Embedded (FFPE) EMC did not show any other mutated cases. In conclusion, exon 11 KIT mutation was detected only in one out of 20 EMC cases analyzed, indicating that KIT alteration is not a recurrent event in these tumors and cannot explain the EMC sensitivity to sunitinib, although it is an actionable mutation in the individual case in which it has been identified.

Despite the genomic characterization of pancreatic cancer (PC), marked advances in the development of prognosis classification and novel therapeutic strategies have yet to come. The present study aimed to better understand the genomic alterations associated with the invasive phenotype of PC, in order to improve patient selection for treatment options. A total of 30 PC samples were analysed by either whole transcriptome (9 samples) or exome sequencing (21 samples) on an Illumina platform (75X2 or 100X2 bp), and the results were matched with normal DNA to identify somatic events. Single nucleotide variants and insertions and deletions were annotated using public databases, and the pathogenicity of the identified variants was defined according to prior knowledge and mutation-prediction tools. A total of 43 recurrently altered genes were identified, which were involved in numerous pathways, including chromatin remodelling and DNA damage repair. In addition, an analysis limited to a subgroup of early stage patients (50% of samples) demonstrated that poor prognosis was significantly associated with a higher number of known PC mutations (P=0.047). Samples from patients with a better overall survival (>25 months) harboured an average of 24 events, whereas samples from patients with an overall survival of

BACKGROUND: Achalasia is a rare motility disorder characterized by myenteric neuron and interstitial cells of Cajal (ICC) abnormalities leading to deranged/absent peristalsis and lack of relaxation of the lower esophageal sphincter. The mechanisms contributing to neuronal and ICC changes in achalasia are only partially understood. Our goal was to identify novel molecular features occurring in patients with primary achalasia. METHODS: Esophageal full-thickness biopsies from 42 (22 females; age range: 16-82 years) clinically, radiologically, and manometrically characterized patients with primary achalasia were examined and compared to those obtained from 10 subjects (controls) undergoing surgery for uncomplicated esophageal cancer (or upper stomach disorders). Tissue RNA extracted from biopsies of cases and controls was used for library preparation and sequencing. Data analysis was performed with the "edgeR" option of R-Bioconductor. Data were validated by real-time RT-PCR, western blotting and immunohistochemistry. KEY RESULTS: Quantitative transcriptome evaluation and cluster analysis revealed 111 differentially expressed genes, with a P ≤ 10-3 . Nine genes with a P ≤ 10-4 were further validated. CYR61, CTGF, c-KIT, DUSP5, EGR1 were downregulated, whereas AKAP6 and INPP4B were upregulated in patients vs controls. Compared to controls, immunohistochemical analysis revealed a clear increase in INPP4B, whereas c-KIT immunolabeling resulted downregulated. As INPP4B regulates Akt pathway, we used western blot to show that phospho-Akt was significantly reduced in achalasia patients vs controls. CONCLUSIONS & INFERENCES: The identification of altered gene expression, including INPP4B, a regulator of the Akt pathway, highlights novel signaling pathways involved in the neuronal and ICC changes underlying primary achalasia.

Long non-coding RNAs (lncRNAs) are ncRNAs more than 200 nucleotides long that participate to a wide range of biological functions. However, their role in cancer is poorly known. By using an NGS-based approach we analyzed the intragenic and poliA-lncRNAs in hepatocellular carcinoma (HCC) and we assayed the relationships between their deregulated expression and clinical-pathological characteristics. The expression profile of lncRNAs was studied in a discovery series of 28 HCC and matched cirrhosis and was validated in an independent cohort of 32 HCC patients both in tissue and serum. The correlation between lncRNA expression and clinical-pathological variables, EMT markers and putative sponged microRNAs level were investigated. Functional experiments were performed in HCC-derived cell lines to clarify the role of selected lncRNAs in HCC. A panel of deregulated lncRNAs differentiated HCC from cirrhotic tissue. CASC9 and LUCAT1 were up-regulated in a subset of HCC-derived cell lines and in half of HCCs which displayed a lower recurrence after surgery. LUCAT1 and CASC9 silencing increased cell motility and invasion capability in HCC cells and influenced the EMT phenotype. LUCAT1 was demonstrated to directly sponge the onco-miR-181d-5p. Both LUCAT1 and CASC9 were secreted in exosomes, and higher circulating CASC9 levels were associated with tumor size and HCC recurrence after surgery, suggesting its potential usage as putative non-invasive prognostic biomarker of recurrence.

Cardiac tumors are a very rare condition. Mostly, they are benign tumors (75%), with myxomas being the most frequent. The remaining 25% are malignant; either primary malignant sarcoma or secondary metastases. Given the small number of cases reported and the lack of prospective and randomized clinical trials, the level of evidence for the optimal multimodal treatment of primary cardiac sarcomas is very low and the optimal imaging diagnostic workup is not well established. In particular,18F-FDG-PET/CT is not yet included in routine diagnosis of cardiac masses. Here, we report four illustrative clinical cases and a review of the literature on the current available data on the role of18F-fluorodeoxyglucose PET/CT imaging in cardiac tumors.

A wait and see approach for desmoid tumors (DT) has become part of the routine treatment strategy. However, predictive factors to select the risk of progressive disease are still lacking. A translational project was run in order to identify genomic signatures in patients enrolled within an Italian prospective observational study. Among 12 DT patients (10 CTNNB1-mutated and 2 wild type) enrolled from our institution only two patients (17%) showed a progressive disease. Tumor biopsies were collected for whole exome sequencing. Overall, DT exhibited low somatic sequence mutation rate and no additional recurrent mutation was found. In the two wild type (WT) cases, two novel alterations were detected: a complex deletion of APC and a pathogenic mutation of LAMTOR2. Focusing on WT DT subtype, deep sequencing of CTNNB1, APC and LAMTOR2 was conducted on a retrospective series of 11 WT DT using a targeted approach. No other mutation of LAMTOR2 was detected, while APC was mutated in two cases. Low-frequency (mean reads of 16%) CTNNB1 mutations were discovered in five samples (45%) and two novel intra-genic deletions in CTNNB1 were detected in two cases. Both deletions and low frequency mutations of CTNNB1 were highly expressed. In conclusion, a minority of DT is WT for either CTNNB1, APC or any other gene involved in the WNT pathway. In this subgroup novel and hard to be detected molecular alterations in APC and CTNNB1 were discovered, contributing to explain a portion of the allegedly WT DT cases.

Background Intrahepatic cholangiocarcinoma (iCCA) is a liver neoplasm with few treatment options and a dismal prognosis. To date, the only curative option remains the surgery. New insights concerning its molecular pathogenesis and genetic heterogeneity are recently coming to light and are promising to widen the treatment landscape. Methods We evaluated data of the first 39 patients with iCCA included in the “Next Generation Sequencing in Intrahepatic Cholangiocarcinoma” study (EtherBil study, NCT02184871) from May 2014 to Dec 2017. For each patient enrolled, we performed whole exome sequencing (“WES)” on fresh frozen tumor tissue and peripheral blood DNA. All variants that were unique in the tumor sample were called as somatic. We used OncoKB ( http://oncokb.org/ ) to define the clinical actionability of the somatic mutation identified (Chakravarty D. et al. JCO Precision Oncology 2017). Results A total of 33 actionable/potentially actionable mutations were identified in 17 genes. IDH1 (5/39, 12.8%), FGFR2 (4/39, 10.2%), IDH2 (3/39, 7.6%), KRAS (3/39, 7.6%), PIK3CA (3/39, 7.6%), NF1 (3/39, 7.6%) were the genes with the higher frequency of actionable/potentially actionable mutations. Remarkably, 20 out of 39 patients (51.2%) harbored at least one potentially actionable mutation. Furthermore, 2 out of 39 tumors were found to be ultra-hypermutated hypermutated (≥100 Mut/Mb). The MSI-H/MMRd phenotype in the latter was confirmed by PCR and MLH1, PMS2, MSH2, and MSH6IHC immunohistochemistry. Conclusions Given the limited armory currently available in advanced unresectable iCCA patients, these data can be helpful to consolidate new understanding aimed at opening up future treatment options. Finally, we report the data of MSI-H/MMRd prevalence in our iCCA series having regard to the new insight on the immunotherapy in this subgroup of patients. Clinical trial identification NCT02184871; First posted: July 9, 2014. Legal entity responsible for the study Sant’Orsola-Malpighi Hospital. Funding Programma Strategico Uni-Regione "Innovations in Onco-Hepatology and Onco-Haematology" (WP3-1MED3BRA-BRANDI, WP4-1MED3BRA-BRANDI). Disclosure All authors have declared no conflicts of interest.

// Maristella Saponara 1,* , Milena Urbini 2,* , Annalisa Astolfi 2 , Valentina Indio 2 , Giorgio Ercolani 3 , Massimo Del Gaudio 3 , Donatella Santini 4 , Maria Giulia Pirini 4 , Michelangelo Fiorentino 5 , Margherita Nannini 1 , Cristian Lolli 1 , Anna Mandrioli 1 , Lidia Gatto 1 , Giovanni Brandi 1 , Guido Biasco 1,2 , Antonio Daniele Pinna 3 and Maria Abbondanza Pantaleo 1,2 1 Department of Specialized, Experimental, and Diagnostic Medicine, Sant’Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy 2 Interdepartmental Centre of Cancer Research “G. Prodi”, University of Bologna, Bologna, Italy 3 Department of General and Emergency Surgery and Organ Transplantation, S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy 4 Pathology Unit, S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy 5 Laboratory of Molecular Oncologic and Transplantation Pathology, S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy * These authors have contributed equally to the work Correspondence to: Maria Abbondanza Pantaleo, email: // Keywords : GIST, CCND2, NGS, PTEN, CDKN2C, DMD Received : May 04, 2015 Accepted : October 05, 2015 Published : November 02, 2015 Abstract About 85% of GISTs are associated with KIT and PDGFRα gene mutations, which predict response to tyrosine kinase inhibitors. Although the outcomes in patients affected by GIST have dramatically improved, tumor progression control still remains a challenge. The aim of this study is the genomic characterization of individual metastatic KIT-exon 11-mutant GIST to identify additional aberrations and simultaneous molecular events representing potential therapeutic targets. Seven patients with metastatic GIST were studied with whole transcriptome sequencing and copy number analysis. Somatic single nucleotide variations were called; however, no shared mutated genes were detected except KIT. Almost all patients showed loss of genomic regions containing tumor suppressor genes, sometimes coupled with single nucleotide mutation of the other allele. Additionally, six fusion transcripts were found and three patients showed amplifications involving known oncogenes. Evaluating the concordance between CN status and mRNA expression levels, we detected overexpression of CCND2 and EGFR and silencing of CDKN2A, CDKN2C, SMARCB1, PTEN and DMD. Altered expression of these genes could be responsible for aberrant activation of signaling pathways that support tumor growth. In this work, we assessed the effect of Hedgehog pathway inhibition in GIST882 cells, which causes decrement of cell viability associated with reduction of KIT expression. Additional genomic alterations not previously reported in GIST were found even if not shared by all samples. This contributes to a more detailed molecular understanding of this disease, useful for identification of new targets and novel therapeutics and representing a possible point of departure for a truly individualized clinical approach.

// Annalisa Astolfi 1, 2 , Fraia Melchionda 2 , Daniela Perotti 3 , Maura Fois 2 , Valentina Indio 1 , Milena Urbini 1, 2 , Chiara Giusy Genovese 1 , Paola Collini 4 , Nunzio Salfi 5 , Marilina Nantron 6 , Paolo D’Angelo 7 , Filippo Spreafico 8 , Andrea Pession 2 1 “Giorgio Prodi” Cancer Research Center, University of Bologna, Bologna, Italy 2 Pediatric Hematology and Oncology Unit, S.Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy 3 Unit of Molecular Bases of Genetic Risk and Genetic Testing, Department of Preventive and Predictive Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 4 Soft Tissue and Bone Pathology, Histopathology, and Pediatric Pathology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 5 Pathology Unit, S.Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy 6 Department of Pediatric Hematology and Oncology, Istituto G. Gaslini, Genova, Italy 7 Pediatric Hematology and Oncology Unit, A.R.N.A.S. Civico, Di Cristina and Benfratelli Hospital, Palermo, Italy 8 Pediatric Oncology Unit, Department of Hematology and Pediatric Onco-Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy Correspondence to: Fraia Melchionda, e-mail: fraia.melchionda@aosp.bo.it Keywords: CCSK, whole transcriptome sequencing, BCOR Received: August 10, 2015 Accepted: September 28, 2015 Published: October 22, 2015 ABSTRACT Purpose: Clear cell sarcoma of the kidney (CCSK) is a rare pediatric renal tumor that is frequently difficult to distinguish among other childhood renal tumors due to its histological heterogeneity. This work evaluates genetic abnormalities carried by a series of CCSK samples by whole transcriptome sequencing (WTS), to identify molecular biomarkers that could improve the diagnostic process. Methods: WTS was performed on tumor RNA from 8 patients with CCSK. Bioinformatic analysis, with implementation of a pipeline for detection of intragenic rearrangements, was executed. Sanger sequencing and gene expression were evaluated to validate BCOR internal tandem duplication (ITD). Results: WTS did not identify any shared SNVs, Ins/Del or fusion event. Conversely, analysis of intragenic rearrangements enabled the detection of a breakpoint within BCOR transcript recurrent in all samples. Three different in-frame ITD in exon15 of BCOR, were detected. The presence of the ITD was confirmed on tumor DNA and cDNA, and resulted in overexpression of BCOR. Conclusion: WTS coupled with specific bioinformatic analysis is able to detect rare genetic events, as intragenic rearrangements. ITD in the last exon of BCOR is recurrent in all CCSK samples analyzed, representing a valuable molecular marker to improve diagnosis of this rare childhood renal tumor.

The aim of the current study was to implement whole transcriptome massively parallel sequencing (RNASeq) and copy number analysis to investigate the molecular biology of pancreatic ductal adenocarcinoma (PDAC). Samples from 16 patients with PDAC were collected by ultrasound‑guided biopsy or from surgical specimens for DNA and RNA extraction. All samples were analyzed by RNASeq performed at 75x2 base pairs on a HiScanSQ Illumina platform. Single‑nucleotide variants (SNVs) were detected with SNVMix and filtered on dbSNP, 1000 Genomes and Cosmic. Non‑synonymous SNVs were analyzed with SNPs&GO and PROVEAN. A total of 13 samples were analyzed by high resolution copy number analysis on an Affymetrix SNP array 6.0. RNAseq resulted in an average of 264 coding non‑synonymous novel SNVs (ranging from 146‑374) and 16 novel insertions or deletions (In/Dels) (ranging from 6‑24) for each sample, of which a mean of 11.2% were disease‑associated and somatic events, while 34.7% were frameshift somatic In/Dels. From this analysis, alterations in the known oncogenes associated with PDAC were observed, including Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations (93.7%) and inactivation of cyclin‑dependent kinase inhibitor 2A (CDKN2A) (50%), mothers against decapentaplegic homolog 4 (SMAD4) (50%), and tumor protein 53 (TP53) (56%). One case that was negative for KRAS exhibited a G13D neuroblastoma RAS viral oncogene homolog mutation. In addition, gene fusions were detected in 10 samples for a total of 23 different intra‑ or inter‑chromosomal rearrangements, however, a recurrent fusion transcript remains to be identified. SNP arrays identified macroscopic and cryptic cytogenetic alterations in 85% of patients. Gains were observed in the chromosome arms 6p, 12p, 18q and 19q which contain KRAS, GATA binding protein 6, protein kinase B and cyclin D3. Deletions were identified on chromosome arms 1p, 9p, 6p, 18q, 10q, 15q, 17p, 21q and 19q which involve TP53, CDKN2A/B, SMAD4, runt‑related transcription factor 2, AT‑rich interactive domain‑containing protein 1A, phosphatase and tensin homolog and serine/threonine kinase 11. In conclusion, genetic alterations in PDCA were observed to involve numerous pathways including cell migration, transforming growth factor‑β signaling, apoptosis, cell proliferation and DNA damage repair. However, signaling alterations were not observed in all tumors and key mutations appeared to differ between PDAC cases.

L-Asparaginase (L-Asp) is an enzyme that catalyzes the hydrolysis of L-asparagine to L-aspartic acid, and its depletion induces leukemic cell death. L-Asp is an important component of treatment regimens for Acute Lymphoblastic Leukemia (ALL). Sensitivity to L-Asp is due to the absence of L-Asparagine synthetase (ASNS), the enzyme that catalyzes the biosynthesis of L-asparagine. ASNS gene is located on 7q21.3, and its increased expression in ALLs correlates with L-Asp resistance. Chromosome 7 monosomy (-7) is a recurrent aberration in myeloid disorders, particularly in adverse-risk Acute Myeloid Leukemias (AMLs) and therapy-related myeloid neoplasms (t-MN), that leads to a significant downregulation of the deleted genes, including ASNS. Therefore, we hypothesized that -7 could affect L-Asp sensitivity in AMLs. By treating AML cell lines and primary cells from pediatric patients with L-Asp, we showed that -7 cells were more sensitive than AML cells without -7. Importantly, both ASNS gene and protein expression were significantly lower in -7 AML cell lines, suggesting that haploinsufficiency of ASNS might induce sensitivity to L-Asp in AMLs. To prove the role of ASNS haploinsufficiency in sensitizing AML cells to L-Asp treatment, we performed siRNA-knockdown of ASNS in AML cell lines lacking -7, and observed that ASNS knockdown significantly increased L-Asp cytotoxicity. In conclusion, -7 AMLs showed high sensitivity to L-Asp treatment due to low expression of ASNS. Thus, L-Asp may be considered for treatment of AML pediatric patients carrying -7, in order to improve the outcome of adverse-risk AMLs and t-MN patients.

Myoepithelial neoplasms (MN) are rare and not well-circumstanced entities displaying a heterogeneous spectrum of genetic abnormalities, including EWSR1, FUS and PLAG1 rearrangements. However, in the remaining MN no other fusion gene has been described and knowledge concerning secondary acquired molecular alterations is still poor. Therefore, we screened 5 cases of MN of the soft tissue by RNA sequencing with the aim of identifying novel fusion transcripts. A novel SRF-E2F1 fusion was detected in two cases: one was negative for other fusions while the other showed also the presence of FUS-KLF17. The fusion was validated through independent techniques and, in both cases, SRF-E2F1 was detected only in a subclone of the tumoral mass. SRF-E2F1 maintained the coding frame, thus leading to the translation of a chimeric protein containing the DNA-binding domain of SRF and the trans-activation domain of E2F1. Moreover, ectopical expression of SRF-E2F1 demonstrated that the chimeric transcript is functionally active and could affect tumor growth. Occurrence in two cases and biological relevance of the two genes involved suggest that the SRF-E2F1 fusion might become a helpful diagnostic tool. Further biologic studies are needed to better assess its role in MN biology.

Background: Several clinical studies demonstrated the efficacy and safety of combining azacitidine with lenalidomide in Myelodysplastic Syndromes (MDS), but the molecular implications of this therapy (i.e. gene mutations and microRNA profiling) are still under investigation. Moreover, phosphoinositide-specific phospholipase C (PI-PLC) beta1 is modulated in MDS cells treated with azacitidine, whereas lenalidomide, restoring a normal erythropoiesis, can affect PI-PLCgamma1 pathways. Purpose: This study firstly aimed to assess the effect of azacitidine and lenalidomide combination therapy on the expression of inositide-dependent signalling, focusing particularly on PI-PLC isoenzymes. Moreover, an extensive analysis on gene mutations and microRNA expression profiling was also performed. Patients and Methods: This study included 44 patients diagnosed with high-risk MDS who were given azacitidine and lenalidomide. Paired samples, before and during treatment, were analyzed. Firstly, we quantified the expression of PI-PLC isoenzymes by Real-Time PCR and immunocytochemical experiments. Moreover, we carried out cell cycle analyses and studied both PI-PLCbeta1 methylation status and the expression of Globin genes. In addition, the frequency of recurrent gene mutations associated with myeloid malignancies was evaluated, using an Illumina Cancer Myeloid Panel, and the expression of microRNAs was assessed using an Affimetrix array. Results: In our case series, 34/44 patients completed at least 6 cycles of treatment and were clinically evaluable, with an overall response rate of 76.5% (26/34 cases). Firstly, the therapy affectedthe MDS cell cycle. Moreover, the expression of PI-PLCs and Beta-Globin could be associated with a favourable clinical response to the combination therapy, hinting at a specific contribution of lenalidomide on erythroid activation, whilst the frequent demethylation of PI-PLCbeta1 promoter could be specifically linked to azacitidine. At baseline, the most frequent gene mutations were ASXL1 (59%), TET2 (41%), RUNX1 (35%) and SRSF2 (29%). Interestingly, all patients showing SRSF2 mutations evolved into AML. Moreover, all patients maintaining a stable disease during the therapy had increasing variant allele frequencies, showing a genetic instability. Finally, microRNA analysis revealed specific clusters associated with the treatment: 14 microRNAs were overexpressed and 28 were under-expressed as compared to baseline levels. Interestingly, down-regulation of 5 microRNAs was significantly associated with the lack of response. Conclusions: Our results show that the combination of azacitidine and lenalidomide can affect PI-PLC signalling, possibly regulating MDS cell cycle, myeloid and erythroid differentiation. Moreover, the therapy can induce a change in the specific gene mutation and microRNA profile that, if confirmed by larger studies, could be important to better evaluate the response to this therapy.

Background Extraskeletal myxoid chondrosarcoma (EMC) is a rare soft tissue sarcoma, marked by NR4A3 rearrangement. Herein we report on the activity of sunitinib in a series of 10 patients, strengthening what initially observed in two cases. Patients and methods From July 2011, 10 patients with progressive metastatic translocated EMC have been consecutively treated with sunitinib 37.5 mg/day, on a named-use basis. In an attempt to interpret the activity of sunitinib in EMC, genotype/phenotype correlations were carried out by fluorescence in situ hybridization (FISH) analyses. Moreover, transcriptome, immunohistochemical and biochemical analyses of a limited set of samples were performed focusing on some putative targets of sunitinib. Results Eight of 10 patients are still on therapy. Six patients had a Response Evaluation Criteria in Solid Tumours (RECIST) partial response (PR), two were stable, two progressed. Positron emission tomography (PET) was consistent in 6/6 evaluable cases. One patient underwent surgery after sunitinib, with evidence of a pathologic response. At a median follow-up of 8.5 months (range 2–28), no secondary resistance was detected. Median progression free survival (PFS) has not been reached. Interestingly, all responsive cases turned out to express the typical EWSR1–NR4A3 fusion, while refractory cases carried the alternative TAF15–NR4A3 fusion. Among putative sunitinib targets, only RET was expressed and activated in analysed samples. Conclusions This report confirms the therapeutic activity of sunitinib in EMC. Genotype/phenotype analyses support a correlation between response and EWSR1–NR4A3 fusion. Involvement of RET deserves further investigation.

Despite the genomic characterization of pancreatic cancer (PC), marked advances in the development of prognosis classification and novel therapeutic strategies have yet to come. The present study aimed to better understand the genomic alterations associated with the invasive phenotype of PC, in order to improve patient selection for treatment options. A total of 30 PC samples were analysed by either whole transcriptome (9 samples) or exome sequencing (21 samples) on an Illumina platform (75X2 or 100X2 bp), and the results were matched with normal DNA to identify somatic events. Single nucleotide variants and insertions and deletions were annotated using public databases, and the pathogenicity of the identified variants was defined according to prior knowledge and mutation-prediction tools. A total of 43 recurrently altered genes were identified, which were involved in numerous pathways, including chromatin remodelling and DNA damage repair. In addition, an analysis limited to a subgroup of early stage patients (50% of samples) demonstrated that poor prognosis was significantly associated with a higher number of known PC mutations (P=0.047). Samples from patients with a better overall survival (>25 months) harboured an average of 24 events, whereas samples from patients with an overall survival of

Motivation: Targeting peptides are the most important signal controlling the import of nuclear encoded proteins into mitochondria and plastids. In the lack of experimental information, their prediction is an essential step when proteomes are annotated for inferring both the localization and the sequence of mature proteins. Results: We developed TPpred a new predictor of organelle-targeting peptides based on Grammatical-Restrained Hidden Conditional Random Fields. TPpred is trained on a non-redundant dataset of proteins where the presence of a target peptide was experimentally validated, comprising 297 sequences. When tested on the 297 positive and some other 8010 negative examples, TPpred outperformed available methods in both accuracy and Matthews correlation index (96% and 0.58, respectively). Given its very low–false-positive rate (3.0%), TPpred is, therefore, well suited for large-scale analyses at the proteome level. We predicted that from ∼4 to 9% of the sequences of human, Arabidopsis thaliana and yeast proteomes contain targeting peptides and are, therefore, likely to be localized in mitochondria and plastids. TPpred predictions correlate to a good extent with the experimental annotation of the subcellular localization, when available. TPpred was also trained and tested to predict the cleavage site of the organelle-targeting peptide: on this task, the average error of TPpred on mitochondrial and plastidic proteins is 7 and 15 residues, respectively. This value is lower than the error reported by other methods currently available. Availability: The TPpred datasets are available at http://biocomp.unibo.it/∼valentina/TPpred/. TPpred is available on request from the authors. Contact: gigi@biocomp.unibo.it Supplementary information: Supplementary data are available at Bioinformatics online.

// Riccardo Masetti 1, * , Ilaria Castelli 1, * , Annalisa Astolfi 2 , Salvatore Nicola Bertuccio 1 , Valentina Indio 2 , Marco Togni 1, 3 , Tamara Belotti 1 , Salvatore Serravalle 1 , Giuseppe Tarantino 2 , Marco Zecca 4 , Martina Pigazzi 5 , Giuseppe Basso 5 , Andrea Pession 1, # , Franco Locatelli 6, 7, # 1 Department of Pediatrics “Lalla Seragnoli”, Hematology-Oncology Unit, University of Bologna, Bologna, Italy 2 Interdepartmental Centre of Cancer Research “G. Prodi”, University of Bologna, Bologna, Italy 3 Current address: Stem Cell Group, University College London Cancer Institute, University College London, London, United Kingdom 4 Department of Pediatric Hematology Oncology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy 5 Department of Woman and Child Health, Laboratory of Hematology-Oncology, University of Padova, Padova, Italy 6 Department of Pediatric Hematology-Oncology, IRCCS Ospedale Bambino Gesu, Rome, Italy 7 University of Pavia, Pavia, Italy * These authors have contributed equally to this work # Both authors have shared co-senior authorship Correspondence to: Riccardo Masetti, email: riccardo.masetti@gmail.com Keywords: pediatric acute myeloid leukemia, acute myeloid leukemia relapse, whole-exome massively parallel sequencing, SETD2 mutation, FLT3-TKD mutation Received: April 19, 2016 Accepted: July 10, 2016 Published: July 22, 2016 ABSTRACT Despite significant improvement in treatment of childhood acute myeloid leukemia (AML), 30% of patients experience disease recurrence, which is still the major cause of treatment failure and death in these patients. To investigate molecular mechanisms underlying relapse, we performed whole-exome sequencing of diagnosis-relapse pairs and matched remission samples from 4 pediatric AML patients without recurrent cytogenetic alterations. Candidate driver mutations were selected for targeted deep sequencing at high coverage, suitable to detect small subclones (0.12%). BiCEBPα mutation was found to be stable and highly penetrant, representing a separate biological and clinical entity, unlike WT1 mutations, which were extremely unstable. Among the mutational patterns underlying relapse, we detected the acquisition of proliferative advantage by signaling activation (PTPN11 and FLT3-TKD mutations) and the increased resistance to apoptosis (hyperactivation of TYK2). We also found a previously undescribed feature of AML, consisting of a hypermutator phenotype caused by SETD2 inactivation. The consequent accumulation of new mutations promotes the adaptability of the leukemia, contributing to clonal selection. We report a novel ASXL3 mutation characterizing a very small subclone (

Aim: Currently, little is known about differences in miRNA expression between KIT/PDGFRA mutant and KIT/PDGFRA wild-type (WT)-SDH deficient gastrointestinal stromal tumors (GIST). This prompted us to perform an integrated multiple expression profile of miRNA and mRNA, constructing an original miRNA–mRNA regulatory network in KIT/PDGFRA WT-SDH deficient GIST patients. Patients & methods: Analyses were carried out on KIT/PDGFRA mutant versus KIT/PDGFRA WT-SDH deficient GIST. Genome-wide miRNA and gene-expression analysis were performed using Agilent Human miRNA microarray and Affimetrix array, respectively. Results: Three potential regulatory networks (IGF1R → miR-139-5p/miR-455/let-7b, cyclin-dependent kinase 6 (CDK6) → miR-139-5p/let-7b and CD44 → miR-330-3p) were identified. Conclusion: The miR-139-5p, 455-5p and let-7b signature, in particular, may represent an important therapeutic target in KIT/PDGFRA WT-SDH deficient GIST, usually characterized by IGF1R overexpression.

Approximately 10%-15% of gastrointestinal stromal tumors (GISTs) in adults do not harbor any mutation in the KIT or PDGFRA genes (ie, KIT/PDGFRA wild-type GISTs). Recently, mutations in SDHB and SDHC (which encode succinate dehydrogenase subunits B and C, respectively) but not in SDHA and SDHD (which encode subunits A and D, respectively) were identified in KIT/PDGFRA wild-type GISTs. To search for novel pathogenic mutations, we sequenced the tumor transcriptome of two young adult patients who developed sporadic KIT/PDGFRA wild-type GISTs by using a massively parallel sequencing approach. The only variants identified as disease related by computational analysis were in SDHA. One patient carried the homozygous nonsense mutation p.Ser384X, the other patient was a compound heterozygote harboring a p.Arg31X nonsense mutation and a p.Arg589Trp missense mutation. The heterozygous nonsense mutations in both patients were present in germline DNA isolated from peripheral blood. Protein structure analysis indicates that all three mutations lead to functional inactivation of the protein. This is the first report, to our knowle dge, that identifies SDHA inactivation as a common oncogenic event in GISTs that lack a mutation in KIT and PDGFRA.

Background and Rationale. Azacitidine (AZA) is a standard first-line therapy in high-risk MDS. Also its combination with Lenalidomide (LEN) has been tested, but its molecular effect is still under investigation. Here we analyzed the effect of AZA+LEN therapy on gene mutations and microRNA expression in MDS patients. Patients and Methods. This study included 44 high-risk MDS patients treated with AZA (75 mg/m2/day, days 1-5, sc) and LEN (10 mg/day, days 1-21, orally) every 4 weeks. Patients showing complete remission (CR), partial remission (PR) or any hematologic improvement were considered as responders, while patients showing stable disease or disease progression were considered as non responders. Molecular analyses were performed at baseline and during the therapy. Gene mutations were studied by an Illumina Cancer Myeloid Panel and an Ion Torrent specific panel, whereas microRNAs expression was assessed using an Affymetrix miRNA 4.0 array. Results. 34/44 patients were considered evaluable for response, with an overall response rate of 76.25% (26/34 cases). 13 patients showed a positive response within the 4th cycle (T4) and maintained it at T8; 9 patients showed a positive response within T4 and lost response at T8; 4 patients responded after T4 and maintained the response at T8; 8 patients never responded. Molecular analyses were performed on serial samples (baseline, T4 and T8) available for 30 patients. Results from the Illumina analysis on cancer myeloid genes showed that 3/30 cases had no mutations at all, all other cases showed mutations both at baseline and during the therapy. The most frequently mutated genes were ASXL1 (14 cases = 47%), TET2 (11 cases = 37%), RUNX1 (8 cases = 27%) and SRSF2 (5 cases = 17%). All samples with a decreasing variant allele frequency (VAF) had a favourable response at T8 (CR, marrow CR or PR), while none of the non responders showed a decreasing VAF. Ion Torrent analysis of 24 inositide-specific genes showed that all patients had mutations both at baseline and during the therapy. Interestingly, all patients responding at T4 and losing response at T8, as well as cases that did not respond, acquired the same 3 point mutations at T8, affecting respectively PIK3CD (D133E), AKT3 (D280G) and PLCG2 (Q548R) genes. Patients responding at T4 and losing response at T8 showed these mutations even at T4. Kaplan-Meier analyses revealed that the presence of these mutations was significantly associated with a decreased duration of therapy (39.5 vs 8.5 months; p As for microRNA profiling, paired analysis between responders and non responders showed specific clusters of up- or down-regulated microRNAs. Interestingly, unpaired analysis on patients responding at T4 and losing response at T8 showed 18 up- and 11 down-regulated microRNAs, like miR-3613-3p and miR-6757-5p, whose predicted targets are our 3 genes among the others. Also in patients never responding to the therapy there was a specific cluster of 3 up- and 12 down-regulated microRNAs and, interestingly, 7 of these microRNAs, like miR-4786-5p or miR-6853-3p, targeting our 3-gene cluster among the others, were altered also in patients losing response. Conclusions. Our results show that the presence of a common cluster of point mutations affecting 3 inositide-specific genes (PI3KCD, AKT3, PLCG2, all regulating cell proliferation), is significantly associated with loss of response to AZA+LEN therapy. Moreover, also a cluster of 7 microRNAs, targeting our 3 genes among the others, is associated with unfavourable outcome. Further studies are warranted to confirm these data, to further analyze the role of this 3-gene cluster and to identify the specific targets for the dysregulated microRNAs identified. Disclosures Gobbi: Janssen: Consultancy; Amgen: Consultancy; Ariad: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfister: Membership on an entity's Board of Directors or advisory committees. Cavo:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Finelli:Janssen: Consultancy, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Novartis: Consultancy, Speakers Bureau.

11577Background: Desmoid-type fibromatosis (DT) are characterized by molecular alterations of CTNNB1 or APC genes. These abnormalities have been described in ∼85% of DT using Sanger sequencing. Rec...

11578Background: ES is a rare soft tissue sarcoma with two variants, i.e., the proximal and the distal. Retrospective data on the activity of anthracycline- and G-based regimens are available. EZH2...

e23547Background: EMC is an extremely rare soft tissue sarcoma, marked by a translocation involving NR4A3 gene. EMC are usually indolent and generally are moderately sensitive to anthracycline-base...

11561Background: EMC is a rare sarcoma mostly originating from soft tissues. EMC carries a specific translocation, involving NR4A3, which is fused more often with EWSR1 and less frequently with oth...

Gastrointestinal stromal tumors (GIST) carrying the D842V activating mutation in the platelet-derived growth factor receptor alpha (PDGFRA) gene are a very rare subgroup of GIST (about 10%) known to be resistant to conventional tyrosine kinase inhibitors (TKIs) and to show an indolent behavior. In this study, we performed an integrated molecular characterization of D842V mutant GIST by whole-transcriptome and whole-exome sequencing coupled with protein–ligand interaction modelling to identify the molecular signature and any additional recurrent genomic event related to their clinical course. We found a very specific gene expression profile of D842V mutant tumors showing the activation of G-protein-coupled receptor (GPCR) signaling and a relative downregulation of cell cycle processes. Beyond D842V, no recurrently mutated genes were found in our cohort. Nevertheless, many private, clinically relevant alterations were found in each tumor (TP53, IDH1, FBXW7, SDH-complex). Molecular modeling of PDGFRA D842V suggests that the mutant protein binds imatinib with lower affinity with respect to wild-type structure, showing higher stability during the interaction with other type I TKIs (like crenolanib). D842V mutant GIST do not show any actionable recurrent molecular events of therapeutic significance, therefore this study supports the rationale of novel TKIs development that are currently being evaluated in clinical studies for the treatment of D842V mutant GIST.

Purpose: To report on imatinib mesylate (IM) in patients with metastatic dermatofibrosarcoma protuberans (DFSP)/fibrosarcomatous (FS)-DFSP and on the impact of the treatment on tumor biology. Experimental design: Ten consecutive patients treated with IM from 2007 to 2015 for a metastatic relapse from DFSP/FS-DFSP were identified. FISH analysis for COL1A1-PDGFB was performed. Two IM-treated and 4 naive FS-DFSP were transcriptionally profiled by RNAseq on HiScanSQ platform. Differential gene expression was analyzed with edgeR (Bioconductor), followed by hierarchical clustering and Principal Component Analysis. Results: All cases featured fibrosarcomatous in the metastasis and retained the COL1A1-PDGFB . Best RECIST response was: 8 partial response, 1 stable disease, and 1 progressive disease. Median progression-free survival was 11 months. Five patients received surgery after IM and all relapsed. IM was restored in 4 patients with a new response. After IM, the most upregulated genes included those encoding for immunoglobulins and those affecting functions and differentiation of endothelial cells. Pathway enrichment analysis revealed upregulation in genes involved in antigen processing and presentation, natural killer–mediated cytotoxicity, and drug and xenobiotics metabolism. Conversely, a significant down-regulation of kinase signaling pathways was detected. Conclusions: All metastatic cases were fibrosarcomatous. Most patients responded to IM, but PFS was shorter than reported in published series which included both DFSP and FS-DFSP. All patients operated after IM had a relapse, suggesting that IM cannot eradicate metastatic cases and that the role of surgery is limited. Transcriptional profile of naive and posttreatment samples pointed the contribution of immune infiltrates in sustaining the response to IM. Clin Cancer Res; 22(4); 837–46. ©2015 AACR .

Background Gastrointestinal stromal tumors (GIST) recently have been recognized as a genetically and biologically heterogeneous disease. In addition to KIT or PDGFRA mutated GIST, mutational inactivation of succinate dehydrogenase (SDH) subunits has been detected in the KIT/PDGFRA wild-type subgroup, referred to as SDH deficient (dSDH). Even though most dSDH GIST harbor mutations in SDHx subunit genes, some are SDHx wild type. Epigenetic regulation by DNA methylation of CpG islands recently has been found to be an alternative mechanism underlying the lack of SDH complex in GIST. Case presentation We report a particular case of dSDH GIST, previously analyzed with microarrays and next-generation sequencing, for which no molecular pathogenetic events have been identified. Gene expression analysis showed remarkable down-modulation of SDHC mRNA with respect to all other GIST samples, both SDHA-mutant and KIT/PDGFRA-mutant GIST. By a bisulfite methylation assay targeted to 2 SDHC CpG islands, we detected hypermethylation of the SDHC promoter. Conclusion Herein we report an additional case of dSDH GIST without SDHx mutation but harboring hypermethylation in the SDHC promoter, thus confirming the complexity of the molecular background of this subtype of GIST. Electronic supplementary material The online version of this article (doi:10.1186/s12881-015-0233-7) contains supplementary material, which is available to authorized users.

Background Next generation sequencing (NGS) technology has been rapidly introduced into basic and translational research in oncology, but the reduced availability of fresh frozen (FF) tumor tissues and the poor quality of DNA extracted from formalin-fixed, paraffin-embedded (FFPE) has significantly impaired this process in the field of solid tumors. To evaluate if data generated from FFPE material can be reliably produced and potentially used in routine clinical settings, we performed whole exome sequencing (WES) from tumor samples of Gastrointestinal stromal tumors (GIST), either extracted FF or FFPE, and from matched normal DNA. Methods We performed whole exome enrichment and sequencing at 100bp in paired end on four GIST samples, either from FFPE or fresh-frozen tissue, and from matched normal DNA. Results The integrity of DNA extracted from FFPE was evaluated by a modified RAPD PCR method, thus identifying high quality (HQ) and low quality (LQ) FFPE. DNA library production and exome capture was feasible for both classes of FFPE, despite the smaller yield and insert size of LQ-FFPE. WES produced data of equal quality from FF and FFPE, while only HQ-FFPE yielded an amount of data comparable to FF samples. Bioinformatic analysis showed that the percentage of variants called both in FF and FFPE samples was very high in HQ-FFPE, reaching 94-96 % of the total number of called variants. Classification of somatic variants by nucleotide substitution type showed that HQ-FFPE and FF had similar mutational profiles, while LQ-FFPE samples carried a much higher number of mutations than the FF counterpart, with a significant enrichment of C > T/G > A substitutions. Focusing on potential disease-related variants allowed the discovery of additional somatic variants in GIST samples, apart from the known oncogenic driver mutation, both from sequencing of FF and FFPE material. False positive and false negative calls were present almost exclusively in the analysis of FFPE of low quality. On the whole this study showed that WES is feasible also on FFPE specimens and that it is possible to easily select FFPE samples of high quality that yield sequencing results comparable to the FF counterpart. Conclusions WES on FFPE material may represent an important and innovative source for GIST research and for other solid tumors, amenable of possible application in clinical practice. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1982-6) contains supplementary material, which is available to authorized users.

The genomic landscape of children with acute myeloid leukemia (AML) who do not carry any cytogenetic abnormality (CN-AML) is particularly heterogeneous and challenging, being characterized by different clinical outcomes. To provide new genetic insights into this AML subset, we analyzed through RNA-seq 13 pediatric CN-AML cases, corroborating our findings in an independent cohort of 168 AML patients enrolled in the AIEOP AML 2002/01 study. We identified a chimeric transcript involving NUP98 and PHF23, resulting from a cryptic t(11;17)(p15;p13) translocation, demonstrating, for the first time, that NUP98-PHF23 is a novel recurrent (2.6 %) abnormality in pediatric CN-AML.

Background Intragenic deletions of the dystrophin-encoding and muscular dystrophy-associated DMD gene have been recently described in gastrointestinal stromal tumor (GIST), rhabdomyosarcoma (RMS) and leiomyosarcoma (LMS). We evaluated the copy numbers and gene expression levels of DMD in our series of GIST patients who were already studied with wide genome assays, to investigate more fully a correlation between dystrophin status and disease annotations. Findings Our study highlighted a recurrent intragenic deletion on chromosome X, involving the DMD gene that codes for human dystrophin in GIST patients. Of 29 KIT/PDGFRA mutant GIST samples, 9 (31%) showed deletions of the DMD gene, which were focal and intragenic in 8 cases, and involved loss of an entire chromosome in one case (GIST_188). DMD loss was seen in only 5 patients with metastasis, whereas 18 out of 20 patients with localized disease had wild-type DMD (P = 0.0004, Fisher exact test). None of the 6 KIT/PDGFRA WT GIST showed DMD alterations. Conclusions Our study confirms the presence of DMD deletions only in KIT/PDGFRA mutant GIST and this event is almost associated with metastatic disease. These findings are, of course, quite preliminary but support development of potential therapeutic strategies that target and restore DMD function in the treatment of metastatic GIST.

Pediatric cytogenetically normal acute myeloid leukemia (CN-AML) is a heterogeneous subgroup of myeloid clonal disorders that do not harbor known mutations. To investigate the mutation spectrum of pediatric CN-AML, we performed whole-transcriptome massively parallel sequencing on blasts from 7 CN-AML pediatric patients. In 3 patients we identified a recurrent cryptic inversion of chromosome 16, encoding a CBFA2T3-GLIS2 fusion transcript. In a validation cohort of 230 pediatric CN-AML samples we identified 17 new cases. Among a total of 20 patients with CBFA2T3-GLIS2 fusion transcript out of 237 investigated (8.4%), 10 patients (50%) did not belong to the French-American-British (FAB) M7 subgroup. The 5-year event-free survival for these 20 children was worse than that for the other CN-AML patients (27.4% vs 59.6%; P = .01). These data suggest that the presence of CBFA2T3-GLIS2 fusion transcript is a novel common feature of pediatric CN-AML, not restricted to the FAB M7 subtype, predicting poorer outcome.

KIT/PDGF receptor-α (PDGFRA) wild-type (WT) gastrointestinal stromal tumors (GIST) are characterized by an overexpression of IGF-1 receptor (IGF1R) at the mRNA and protein level. More recently, germline and somatic mutations in succinate dehydrogenase (SDH) subunits A, B and C have been identified in KIT/PDGFRA WT sporadic GIST. Until now, the molecular basis of IGF1R overexpression in KIT/PDGFRA WT GIST has not been explained. In this brief report we investigate the status of the SDH complex at the genomic and protein level in relation to IGF1R expression at the mRNA and protein level in seven KIT/PDGFRA WT sporadic GIST patients. We found that IGF1R was upregulated in all patients harboring SDH mutations or displaying a SDH dysfunction, with respect to KIT/PDGFRA WT GIST without SDH mutations. Western blot analysis confirmed that all patients with an upregulation of IGF1R mRNA had detectable IGF1R protein expression. This report would suggest that IGF1R overexpression in KIT/PDGFRA WT GIST could be driven by the loss-of-function of the SDH mitochondrial complex.

Background: About 10%-15% of GISTs do not harbour KIT/PDGFRA mutations (wild-type, WT). We analyzed the tumor transcriptome of two sporadic young adults WT GISTs patients using massively parallel sequencing approach in order to identify novel genetic mutations. Methods: Patients were a 28 years-old female (GIST_07) and a 30 years-old male (GIST_10). Primary tumors were in the stomach, multi-centric in GIST_07 and uni-centric in GIST_10, and presented metastases at diagnosis. RNA-Seq paired-end libraries were sequenced on the Illumina GAIIx system using a paired-end strategy at a read length of 75bp. Bioinformatic analysis highlighted 9 candidate genes common to both patients. Mutations in these genes were then filtered with SNP&GO, a tool suited to compute the likelihood of a mutation to be disease-related or not, depending on the protein sequence and its functional annotation. The identified candidate gene was analyzed by Sanger sequencing on tumor (T) and peripheral blood (PB) DNA from GIST_07 and GIST_10 patients, and on additional 8 tumor and 3 PB DNA from 8 sporadic WT GISTs patients. Immunohistochemistry (IHC) was also performed. Results: Only mutations in the sequence of SDHA were identified as disease-related with a high reliability index. GIST_07 tumor DNA was homozygous for an exon 9 p.S384X nonsense mutation; GIST_10 patient showed compound heterozygosity for a germinal exon 2 p.R31X nonsense mutation and a somatic exon 13 p.R589W missense mutation.Heterozygous nonsense mutations were already present in germline DNA in both patients. All mutations lead to functional inactivation of the protein. IHC revealed that both patients showed the down regulation of SDHA. The expanded study showed that 2/10 carried SDHA mutations in the tumor: compound heterozygosity for an exon 9 p.G419R and exon 13 p.E564K mutations in one patient, and a somatic heterozygous exon 5 p.R171C mutation in the other. Conclusions: Recently, SDHB and SDHC mutations were also reported in these patients. This the first study that identifies mutations on SDHA, in 4/10 WT GIST patients. This gene should be evaluated in larger seriesand be added in the sequencing screening of WT GISTs.

4048 Background: A deeper knowledge of the pancreatic cancer (PDAC) biology is needed to improve the prognosis of the disease. Methods: 17 PDAC samples were collected by ultrasound-guided biopsy used for DNA and RNA extraction. 14 samples were analyzed by high resolution copy number analysis (CNA) on Affymetrix SNP array 6.0 and with segmentation algorithm against a reference of 270 Ceu HapMap individuals (Partek Genomic Suite). 17 samples were analyzed by whole transcriptome massively parallel sequencing, performed at 75x2 bp on a HiScanSQ Illumina platform. An average of 7, 3x107 reads per sample were generated, with a mean read depth of 50X. Single nucleotide variants (SNVs) were detected with SNVMix2 and compared with genetic variation databases (dbSNP, 1000genomes, Cosmic). Non-synonimous SNVs were analyzed with the predictors SNPs and GO and PROVEAN. Results: CNA results in 9/14 samples exhibited both macroscopic and cryptic cytogenetic alterations, with a mean of 10 CNA per patient. Most frequent gains were observed in 18q11.2 involving GATA6 (3/14) and 19q13 targeting AKT2 (3/14) while hotspot deletions were found on 18q21 (7/14), 17p13 (6/14), 9p21.3 (6/14), 15q (5/14) and 1q35 (4/14). RNAseq showed that samples exhibited a mean of 145 (range: 61-240) non-synonimous SNVs, of which 16 on average are potentially disease-related. Merging copy number and RNAseq data we highlighted the major oncogenic hits of PDAC, confirming the prevalence (14/17) of KRAS mutations, in one case also NRAS (G13D), and the three oncosuppressor CDKN2A (mutated in 3 cases and deleted in 6 cases, in hetero- or homozygosity), SMAD4 (altered by point mutation or gene deletion in 7/14), and TP53 (lost in 6/14 and mutated in 5/17). The signaling pathways affected were: KRAS/MAPK, TGFbeta and integrin signaling, proliferation and apoptosis, DNA damage response, and epithelial to mesenchymal transition. Moreover we found new oncogenic alterations, such as HMGCR, that displayed mutations in 17% of the analyzed patients (3/17). Conclusions: NGS combined with high resolution cytogenetic analysis can improve the understanding of pancreatic carcinogenesis.

Introduction and aim: Childhood cytogenetically normal acute myeloid leukemia (CN-AML) is a subgroup of pediatric AML lacking any known cytogenetic and abnormality. CN-AML accounts for 20% of pediatric AML and exhibits a very heterogeneous response to treatment and, consequently, variable outcome. With the aim of providing new insights into the molecular lesions of this subset of AML, we analyzed, through RNA-seq, 13 cases of pediatric CN-AML, corroborating our findings in an independent cohort of 152 AML patients enrolled in the AIEOP AML 2002/01 clinical trial. Methods: Paired-end RNA-seq (75×2) was performed on PoliA(+) RNA extracted from blasts at diagnosis. ChimeraScan, deFuse and FusionMap algorithms were used for chimeric transcript detection. Results: An alteration that was present in 2 out 13 cases was a chimeric transcript involving the genes nucleoporin 98kDa (NUP98) and PHD finger protein 23 (PHF23), resulting from a cryptic translocation t(11;17)(p15;p13). Both patients showed an in-frame fusion between exon 13 of NUP98 and exon 4 of PHF23 that was confirmed by RT-PCR analysis and Sanger sequencing. To date, the cryptic translocation t(11;17)(p15;p13) was never described before in pediatric AML and has been reported only once in an adult AML patient (Reader, et al. Leukemia 2007). In both our patients the same breakpoint in PHF23 gene at the beginning of exon 4 was present, which was different from that present in the adult patient. To assess the incidence of NUP98-PHF23 fusion in pediatric CN-AML, then we studied by RT-PCR and Sanger sequencing a validation cohort of 152 CN-AML children negative for the most common recurrent cytogenetic and molecular lesions, i.e. those involving MLL, CBFB, NPM1 and FLT3. Overall, 2 out of the 152 CN-AML cases were found positive for NUP98-PHF23, demonstrating that this genomic aberrancy is not rare in pediatric CN-AML (tentative frequency 2.4%). Fluorescence in situ hybridization analysis confirmed the cryptic chromosomal translocation t(7;11)(p15;p13) leading to the fusion between NUP98 and PHF23 in all cases. Conclusions: Lately, genome-wide approaches have been widely used to investigate the mutational landscape of CN-AML in adults. However, the genetic profile of childhood CN-AML still needs to be defined. Here, for the first time, we report the identification of a NUP98-PHF23 chimeric transcript in pediatric CN-AML. Together with recently published data demonstrating that NUP98-PHF23 promoted leukemogenesis and the observation that treatment with inhibitors of PHD-domain-mediated H3K4me3 binding, such as disulfiram (an FDA-approved drug), could selectively killed cells expressing NUP98-PHF23 (Gough, et al Cancer Discovery 2014), our findings enforce the role of epigenetic regulators in pediatric AML and suggest screening for this fusion gene at diagnosis in CN-AML pediatric patients. Citation Format: Marco Togni, Riccardo Masetti, Martina Pigazzi, Annalisa Astolfi, Daniele Zama, Valentina Indio, Salvatore Serravalle, Elena Manara, Valeria Bisio, Sergio Rutella, Franca Fagioli, Giuseppe Basso, Andrea Pession, Franco Locatelli. NUP98-PHF23 is a novel fusion gene in pediatric cytogenetically normal acute myeloid leukemia. [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 LB-211. doi:10.1158/1538-7445.AM2015-LB-211

10553 Background: We report on IM activity in patients (pts) with metastatic DFSP/FS-DFSP and on the gene expression profile (GEP) analysis of pure DFSP versus FS-DFSP, aimed at identifying prognos...

4127 Background: Advances in chemotherapy, represented by FOLFIRINOX and Gemcitabine plus Nab-paclitaxel regimens, have resulted in a modest outcome improvement and Gamcitabine-based chemotherapy remains the treatment of choice especially in metastatic disease. Therefore, there is an urgent need to develop accurate markers of pre-invasive pancreatic neoplasms in order to help disease diagnosis at an earlier stage and to apply the best treatment. We aimed to better understand PDAC biology and genomic alterations of invasive phenotype by whole genome sequencing (RNAseq and WES). Methods: We analyzed 27 PDAC samples together with their normal counterpart by HiScanSQ Illumina platform (15/27 by RNASeq and 12/27 by WES). All data were mapped with BWA on hg19, SNVs were called with either SNVMix2 (RNA-seq) or MuTect (WES) while Indels were called with GATK. All variants were filtered on dbSNP, 1000genomes and compared with Cosmic data base. Non-synonymous SNVs were analyzed with PROVEAN and SIFT to predict muta...

10540 Background: Mutations of the receptors KIT and PDGFRA in GIST are the oncogenetic events of disease as well as the targets of molecular therapies. Within the PDGFRA mutations, the D842V mutation in exon 18 confers in vitro and in vivo resistance to imatinib. Next generation sequencing techniques may completely dissect all the possible somatic mutations and genomic rearrangements in order to identify novel therapeutic targets in this patients population. Methods: Five patients with gastric GIST were analyased (3 M, 2 F; mean age 65,5 years, range 51-77). The tumor dimension ranged between 3 and 15 cm, MI < 2 and 8 /50 HPF. No metastases were present in all cases. The KIT and PDGFRA anlysis showed a D842V mutation in exon 18 of PDGFRA in all cases. Whole transcriptome sequencing was performed with Illumina HiScanSQ platform with a paired-end strategy (75x2). After performing quality controls, the short reads were mapped with Tophat-Botie pipeline against the human reference genome (HG19). The variations, such as Single nucleotide variants (SNVs) and InDels, were called by SNVMix2 (a software suited for SNVs discovery in tumor samples) implementing an accurate filtering procedures developed in our laboratory. Two predictors of mutations effect at protein level (SNPs&GO and Provean) were employed in order to prioritize the emerging variation. Results: An average of 206 coding non-synonymous variants were highlighted in the five GIST samples, of which ~ 30% were predicted as deleterious with at least one predictor. In addition to PDGFRA D842V mutation, in all five patients were found mutations on different receptor tyrosine kinases, such as FGFR4 and DDR2. Moreover three out of five patients harboured mutations on members of the MDR/TAP subfamily that are involved in multidrug resistance, in particular on ABCB1, ABCB4 and on ABCB6 genes. Other mutations were found on the hedgehog and MAPK signaling pathway and on SNF/SWI chromatin remodeling complex. Conclusions: New molecular events have been identified in PDGFRA D842V mutant GIST. These data should be validated in larger series and the role of these mutations as therapeutic targets should be further investigated.