Search

The developmental receptor NOTCH plays an important role in various human cancers as a consequence of oncogenic mutations. Here we describe a novel mechanism of NOTCH-induced tumor suppression involving modulation of the deacetylase SIRT1, providing a rationale for the use of SIRT1 inhibitors to treat cancers where this mechanism is inactivated because of SIRT1 overexpression. In Ewing sarcoma cells, NOTCH signaling is abrogated by the driver oncogene EWS-FLI1. Restoration of NOTCH signaling caused growth arrest due to activation of the NOTCH effector HEY1, directly suppressing SIRT1 and thereby activating p53. This mechanism of tumor suppression was validated in Ewing sarcoma cells, B-cell tumors, and human keratinocytes where NOTCH dysregulation has been implicated pathogenically. Notably, the SIRT1/2 inhibitor Tenovin-6 killed Ewing sarcoma cells in vitro and prohibited tumor growth and spread in an established xenograft model in zebrafish. Using immunohistochemistry to analyze primary tissue specimens, we found that high SIRT1 expression was associated with Ewing sarcoma metastasis and poor prognosis. Our findings suggest a mechanistic rationale for the use of SIRT1 inhibitors being developed to treat metastatic disease in patients with Ewing sarcoma. Cancer Res; 74(22); 6578–88. ©2014 AACR.

Giant Cell Tumor (GCT) of Bone is an intramedullary tumor histologically composed by spindle-shaped mononuclear stromal cells, that are the neoplastic and proliferative component of the lesion, and osteoclast-like multinucleated giant cells, that express receptor activator of nuclear factor κB (RANK). The mononuclear stromal cells express RANK ligand, a mediator of osteoclast activation. GCT represents 5% of all bone tumors and 20% of all benign tumors. It usually affects young adults and 2-3% is metastatic, generally to the lung. Curettage is the preferred surgery, while current therapeutic treatment is represented by Denosumab, an anti-RANK-L monoclonal antibody that blocks RANKL binding to RANK, thereby stopping the ‘vicious cycle’ involved in the biological process of GCT. Identification of circulating biomarkers could provide tools for monitoring the effectiveness of therapy and to better understand its biological mechanism. Low molecular weight (LMW) serum proteins or protein fragments, considered to be a rich source of new potential biomarkers and often masked by the presence of abundant proteins, were detected in serum of 25 GCT patients before and after 9 months of treatment with Denosumab, using the hydrogel nanoparticle technique followed by Mass Spectrometry (MS). 25 healthy sera were also analyzed. Hydrogel core-shell nanoparticles selectively entrap LMW proteins by size exclusion and affinity chromatography, protecting them from degradation and amplifying their concentration for subsequent MS detection. Statistical differences in LMW protein abundance between the three groups (GCT pre-treatment, GCT post-treatment, and healthy) were analyzed using the Wilcoxon test or Mann-Whitney U test as appropriate, with the Benjamini-Hochberg False Discovery Rate for multiple test correction. Proteomic analysis revealed 14 differentially expressed analytes in sera from patients pre- and post-treatment, including Complement factor H, Immunoglobulin kappa variable 4-1, Insulin-like growth factor-binding protein 4, Insulin-like growth factor II, Beta-2-glycoprotein 1 and Immunoglobulin lambda constant 6, while 6 proteins were unaltered by the treatment. Furthermore, 52 proteins showed differential abundance in GCT patients before treatment compared to the healthy group, including several (Vitamin D binding protein, Ceruloplasmin, CD5 antigen-like, Serum amyloid A-4 protein, Complement factor D and Apolipoprotein B-100) that we previously identified in a smaller subset of GCT patients and controls, validating our former results. In conclusion, using a noninvasive proteomic technique, we have identified candidate serum biomarkers that could be useful to monitor Denosumab therapy in patients with GCT of bone, for patient management and to better understand its molecular mechanism. Citation Format: Amalia Conti, Alessandra Luchini, Gastone Castellani, Enrico Giampieri, Laura Pazzaglia, Serena Pollino, Giovanna Magagnoli, Emanuela Palmerini, Lance L. Liotta, Piero Picci, Kerry J Rhoden, Maria Serena Benassi. Potential biomarkers for monitoring therapy with Denosumab in patients affected by giant cell tumor of bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 421.

Ewing sarcoma (EWS), a pediatric tumor predominantly occurring in children of European ancestry, is characterized by the EWSR1-FLI1 fusion oncogene. We performed a genome-wide association study (GWAS) of 749 EWS cases and 1,378 unaffected individuals of European ancestry. Our study replicated previously reported susceptibility loci at 1p36.22, 10q21.3, and 15q15.1 and identified new loci at 6p25.1, 8q24.23, 20p11.22, and 20p11.23 (P-values Citation Format: Mitchell J. Machiela, Thomas G.P. Grünewald, Didier Surdez, Stephanie Reynaud, Olivier Mirabeau, Eric Karlins, Rebeca Alba Rubio, Sakina Zaidi, Sandrine Grossetete-Lalami, Stelly Ballet, Eve Lapouble, Valérie Laurence, Jean Michon, Gaelle Pierron, Heinrich Kovar, Nathalie Gaspar, Udo Kontny, Anna González-Neira, Piero Picci, Javier Alonso, Ana Patino-Garcia, Nadège Corradini, Neal D. Freedman, Nathaniel Rothman, Casey L. Dagnall, Laurie Burdett, Kristine Jones, Michelle Manning, Kathleen Wyatt, Weiyin Zhou, Meredith Yeager, David G. Cox, Robert N. Hoover, Javed Khan, Gregory T. Armstrong, Wendy M. Leisenring, Smita Bhatia, Leslie L. Robison, Uta Dirksen, Markus Metzler, Wolfgang Hartmann, Konstantin Strauch, Thomas Kirchner, Andreas E. Kulozik, Lindsay M. Morton, Lisa Mirabello, Margaret A. Tucker, Franck Tirode, Stephen Chanock, Olivier Delattre. Genome-wide association study identifies multiple new loci associated with Ewing sarcoma susceptibility [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A13.

Osteosarcoma (OS) and Ewing's sarcoma (EWS) are the two most common pediatric solid tumors, after brain tumors. Multimodal treatments have significantly improved prognosis in localized disease but outcome is still poor in metastatic patients, for whom therapeutic options are often inadequate. Preclinical drug testing to identify promising treatment options that match the molecular make-up of these tumors is hampered by the lack of appropriate and molecularly well-characterized patient-derived models. To address this need, a panel of patient-derived- xenografts (PDXs) was established by subcutaneous implantation of fresh, surgically resected OS and EWS tumors in NSG mice. Tumors were re-transplanted to next mice generations and fragments were collected for histopathological and molecular characterization. A model was considered established after observing stable histological and molecular features for at least three passages. To evaluate the similarity of the model with primary tumor, we performed a global gene expression profiling and tissue microarrays (TMA), to assess tumor specific biomarkers on tissues from OS/EWS tumors and their PDXs (1st and 3rd passage). Moreover, we verified the feasibility of these models for preclinical drug testing. We implanted 61 OS and 29 EWS samples: 14/38 (37%) primary OS and 9/23 (39%) OS lung metastases successfully engrafted; while among EWS, 5/26 (19%) primary samples and 1/3 (33%) metastases were established. Comparison between patient samples and PDXs, highlighted that histology and genetic characteristics of PDXs were stable and maintained over passages. In particular, correlative analysis between OS and EWS samples and their PDXs was extremely high (Pearson's r range r=0.94-0.96), while patient-derived primary cultures displayed reduced correlation with human samples (r=0.90-0.93), indicating that in vitro adaptation superimpose molecular alterations that create genetic diversion from original tumors. No significant differentially expressed gene profile was observed from the comparison between EWS samples and PDXs (fold change > 2, adjusted p Citation Format: Maria Cristina Manara, Giordano Nicoletti, Manuela Ferracin, Camilla Cristalli, Alberto Righi, Mauro Magnani, Michela Pasello, Piero Picci, Patrizia Nanni, Pier-Luigi Lollini, Katia Scotlandi. Collection of patient-derived xenografts (PDX) to study the biology and therapy of bone sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2079.

Ewing sarcoma (EWS) is a rare pediatric tumor predominantly occurring in children of European ancestry and is characterized by the pathognomonic EWSR1-FLI1 fusion oncogene. To identify germline susceptibility loci associated with EWS risk, we performed a genome-wide association study (GWAS) meta-analysis of 749 EWS cases and 1,378 unaffected individuals of European ancestry from sample collections within the Institut Curie, National Cancer Institute and the Childhood Cancer Survivor Study. Our study replicated previously reported susceptibility loci at 1p36.22, 10q21.3 and 15q15.1 as well as identified novel loci at 6p25.1, 8q24.23, 20p11.22 and 20p11.23 (P-values Citation Format: Mitchell J. Machiela, Thomas G. Grünewald, Didier Surdez, Stephanie Reynaud, Olivier Mirabeau, Eric Karlins, Rebeca Alba Rubio, Sakina Zaidi, Sandrine Grossetete-Lalami, Stelly Ballet, Eve Lapouble, Valérie Laurence, Jean Michon, Gaelle Pierron, Heinrich Kovar, Nathalie Gaspar, Udo Kontny, Anna González-Neira, Piero Picci, Javier Alonso, Ana Patino-Garcia, Nadège Corradini, Neal D. Freedman, Nathaniel Rothman, Casey L. Dagnall, Laurie Burdette, Kristine Jones, Michelle Manning, Kathleen Wyatt, Weiyin Zhou, Meredith Yeager, David G. Cox, Robert N. Hoover, Javed Khan, Gregory T. Armstrong, Wendy M. Leisenring, Smita Bhatia, Leslie L. Robison, Uta Dirksen, Markus Metzler, Wolfgang Hartmann, Konstantin Strauch, Thomas Kirchner, Andreas E. Kulozik, Lindsay M. Morton, Lisa Mirabello, Margaret A. Tucker, Franck Tirode, Stephen J. Chanock, Olivier Delattre. Multiple new susceptibility loci identified in genome-wide association study of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2970.

Survival rates for osteosarcoma, the most common primary bone cancer, have changed little over the past three decades and are particularly low for patients with metastatic disease. To date, few prognostic factors have been identified to be associated with survival in patients with osteosarcoma. We conducted an international, multi-institutional genome-wide association study (GWAS) to identify germline genetic variants associated with overall survival in 632 patients with osteosarcoma. Subjects were genotyped as part of our previously published osteosarcoma susceptibility GWAS, including 523 patients of >80% European ancestry and 109 patients from Brazil. We performed a time-to-event analysis and estimated hazard ratios (HR) and 95% confidence intervals (CI) using Cox proportional hazards models, with and without adjustment for metastatic disease. Cox models were adjusted for age at diagnosis, gender, significant principal components, and study/center. SNPs that reached P Citation Format: Roelof Koster, Orestis A. Panagiotou, William A. Wheeler, Eric Karlins, Julie M. Gastier-Foster, Silvia Regina Caminada de Toledo, Antonio S. Petrilli, Adrienne M. Flanagan, Roberto Tirabosco, Irene L. Andrulis, Jay S. Wunder, Nalan Gokgoz, Ana Patiño-Garcia, Fernando Lecanda, Massimo Serra, Claudia Hattinger, Piero Picci, Katia Scotlandi, David M. Thomas, Mandy L. Ballinger, Richard Gorlick, Donald A. Barkauskas, Logan G. Spector, Margaret Tucker, Belynda Hicks, Meredith Yeager, Robert Hoover, Stephen J. Chanock, Sharon A. Savage, Lisa J. Mirabello. Genome-wide association study identifies the GLDC/IL33 locus associated with survival of osteosarcoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2077.

Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most used drugs for osteosarcoma chemotherapy. By using a series of CDDP-resistant variants, which were established from the U-2OS and Saos-2 human osteosarcoma cell lines, we found that CDDP resistance was mainly associated with the increase of both the intracellular level and enzymatic activity of glutathione S-transferase P1 (GSTP1). On the basis of these findings, we evaluated the clinical effect of GSTP1 in a series of 34 high-grade osteosarcoma patients and we found that the increased expression of GSTP1 gene was associated with a significantly higher relapse rate and a worse clinical outcome. These indications prompted us to assess the in vitro effectiveness of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), a promising new anticancer agent that is a highly efficient inhibitor of GSTP1. NBDHEX was tested on a panel of 10 human osteosarcoma cell lines and 20 variants of the U-2OS or Saos-2 cell lines that were resistant to CDDP, doxorubicin, or methotrexate. NBDHEX proved to be very active on the vast majority of these cell lines, including those with higher GSTP1 levels and enzymatic activity. Drug combination studies showed that NBDHEX can be used in association with CDDP and provided useful information about the best modality of their combined administration. In conclusion, our findings show that GSTP1 has a relevant effect for both CDDP resistance and clinical outcome of high-grade osteosarcoma and that targeting GSTP1 with NBDHEX may be considered a promising new therapeutic possibility for osteosarcoma patients who fail to respond to conventional chemotherapy. [Cancer Res 2008;68(16):6661–8]

Identification of new drugs is strongly needed for sarcomas. Insulin-like growth factor-I receptor (IGF-IR) was found to provide a major contribution to the malignant behavior of these tumors, therefore representing a very promising therapeutic target. In this study, we analyzed the therapeutic potential of a novel kinase inhibitor of IGF-IR, NVP-AEW541, in Ewing's sarcoma, osteosarcoma, and rhabdomyosarcoma, the three most frequent solid tumors in children and adolescents. NVP-AEW541 inhibits IGF-I-mediated receptor activation and downstream signaling. Ewing's sarcoma cells were generally found to be more sensitive to the effects of this drug compared with rhabdomyosarcoma and osteosarcoma, in agreement with the high dependency of this neoplasm to IGF-IR signaling. NVP-AEW541 induced a G1 cell cycle block in all cells tested, whereas apoptosis was observed only in those cells that show a high level of sensitivity. Concurrent exposure of cells to NVP-AEW541 and other chemotherapeutic agents resulted in positive interactions with vincristine, actinomycin D, and ifosfamide and subadditive effects with doxorubicin and cisplatin. Accordingly, combined treatment with NVP-AEW541 and vincristine significantly inhibited tumor growth of Ewing's sarcoma xenografts in nude mice. Therefore, results encourage inclusion of this drug especially in the treatment of patients with Ewing's sarcoma. For the broadest applicability and best efficacy in sarcomas, NVP-AEW541 may be combined with vincristine, actinomycin D, and ifosfamide, three major drugs in the treatment of sarcomas.

In children and adolescents, osteosarcoma (OS) is the most common malignant bone tumor. OS occurs in certain cancer predisposition syndromes at a higher than expected frequency. However, outside of these rare syndromes, OS is significantly more common and its genetic etiology remains poorly understood. We conducted an evaluation of rare exonic variants in 545 unselected OS cases compared with 1061 cancer-free controls using whole-exome sequencing of blood or buccal cell DNA to estimate the prevalence and burden of rare deleterious germline variants. We assessed potentially pathogenic rare variants in 126 established cancer predisposing genes (CPG) and known somatically mutated genes. We also surveyed the exome for genes with a higher burden of rare variants in 342 EUR cases with 994 EUR controls sequenced at the same time. Rare genetic variants, defined by a MAF A total of 14.5% of cases had a predicted pathogenic or high impact variant in an autosomal dominant CPG. A significantly higher pathogenic rare variant burden was present in EUR cases compared with EUR controls (Pburden=3.7x10-05). TP53 had the highest prevalence of pathogenic mutations (5% of EUR cases; Pburden=1.05x10-09). CDKN2A, MEN1, MLH1, MUTYH, PALB2 and VHL had a significantly higher rare variant burden in the EUR cases. 7.5% of cases had a predicted pathogenic or probably pathogenic variant in an autosomal recessive CPG (Pburden=5.4x10-03). Additionally, two males had a pathogenic variant in the X-linked genes, DKC1 and GPC3. In total, 21% of cases had a predicted pathogenic or high impact variant in a CPG (Pburden=2.4x10-07). Exome-wide analysis identified two novel genes with significantly higher rare variant burdens in the cases compared to controls. In conclusion, several CPGs and two novel genes, not previously associated with OS, had an enrichment of rare variants in OS and warrant further follow-up. Our results indicate that a clinically significant fraction of OS cases may harbor one or more mutations worthy of consideration for further investigation and genetic counseling. Citation Format: Roelof Koster, Bin Zhu, Meredith Yeager, Michael Dean, Matthew Gianferante, Lei Song, Joshua Sampson, NCI DCEG Cancer Genomics Research Laboratory, Julie Gastier-Foster, Richard Gorlick, Silvia Regina Caminada de Toledo, Antonio Petrilli, Ana Patiño-Garcia, Fernando Lecanda, Massimo Serra, Claudia Hattinger, Piero Picci, Katia Scotlandi, Adrienne Flanagan, Roberto Tirabosco, Maria Amary, Nilgün Kurucu, Inci Ergurhan Ilhan, Neriman Sari, Mandy Ballinger, David Thomas, Donald Barkauskas, Belynda Hicks, Margaret Tucker, Neil Caporaso, Robert Hoover, Stephen Chanock, Sharon Savage, Lisa Mirabello. Whole-exome sequencing identifies a high frequency of germline deleterious variants in cancer predisposition genes in individuals with osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4871. doi:10.1158/1538-7445.AM2017-4871

Synovial sarcoma (SS) is a rare tumor, with dismal survival when metastatic. SS contains a characteristic translocation (X;18)(p11;q11), representing the fusion of SYT on chromosome 18 with either SSX1, SSX2, or rarely SSX4 on chromosome X The resulting fusion genes appear to be mutually exclusive and concordant in primary and metastatic tumours. New prognostic and predictive factors are needed. Chemokine receptor 4 (CXCR4) is a seven-transmembrane G protein-coupled chemokine receptor and it is the chemokine receptor most commonly expressed in tumour cells, involved in cell migration and invasion, as well as angiogenesis. microRNAs (miRNAs) are involved in post-transcriptional gene expression regulation and control important physiological processes like development, cell differentiation and cell signaling. Altered expression of miRNAs is strongly correlated with the malignant phenotype and there is data reporting a strong association between microRNA expression, patient age and STS prognosis. By three different databases (miRBase, TargetScan , miRanda) and by literature data we identified two miRNA regulators of CXCR4, miR-133b and miR-494. The expression of these miRNAs was evaluated by RT-PCR in 42 SS primary samples stored at Rizzoli biobank. 20 tissues from non oncologic patients were used as control. Our results showed a significant lower expression of miR-133b and miR-494 (respectively p=0.0005 and p=0.001) when compared to non tumor tissue. In vitro study on SW982 cell line with miR-133b precursor show a CXCR4 downregulation and a decrease of cell proliferation. Our preliminary data confirmed a miR-133b and miR-494 downregulation in SS. Correlation with clinical data, with CXCR4 expression and in vitro studies also with miR-494 in several SS cell lines are on-going to better investigate their role as potential prognostic and therapeutic markers. Citation Format: Laura Pazzaglia, Serena Pollino, Mattia Vitale, Amalia Conti, Piero Picci, Maria Serena Benassi. miRNA expression as potential biomarker for synovial sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2534. doi:10.1158/1538-7445.AM2017-2534

Rare and highly aggressive adult soft tissue sarcomas (STS), leiomyosarcoma (LMS) and undifferentiated pleomorphic sarcoma (UPS), have complex genomics characterized by a multitude of rearrangements, amplifications and deletions. Our previous data (Guled et al., Genes Chromosomes Cancer, 2014) showed 21 common differentially expressed miRNAs in UPS and LMS samples when compared to mesenchymal stem cells. On the base of these results, we performed target analysis by using bioinformatic tools (Diana-microt v3.0, Targetscan Vert release 5.2 and Pictar-Vert) and we identified 12 miRNAs potentially implicated in tumor development and progression. The expression of the selected miRNAs was evaluated by TaqMan microRNA Array in 59 high grade STS primary samples (27 LMS and 32 UPS) stored at Rizzoli biobank. Ten tissues from non oncologic patients were used as control. Our results showed a significant lower expression of miR-1, miR-133b and miR-152 (respectively p = 0.0045, p = 0.0027 and p = 0.0016) in tumors when compared to nontumor tissue. Three different target prediction databases (miRBase, TargetScan, miRanda) recognized MET as common target gene of miR-1, mir-133b and miR-152 and KIT as target of miR-152. MET and KIT gene expression was validated in our series of STS by RT-PCR by using 2−ΔΔCT comparative method. The data demonstrated higher mean expression levels in STS than in control (respectively 2.08 and 0.15 for KIT; 18527.9 and 2324.9 for MET) without significant differences between two histotypes. Our preliminary results confirmed MET and KIT as miRNA targets. Correlation with clinical data and in vitro studies with miRNA precursor transfected cell lines are on-going to better investigate their role as potential prognostic and therapeutic markers. Citation Format: Laura Pazzaglia, Chiara Novello, Amalia Conti, Irene Quattrini, Serena Pollino, Piero Picci, Maria Serena Benassi. miRNA expression as potential biomarker for leiomyosarcoma and undifferentiated pleomorphic sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1969.

Background: Tissue is alive and reacting to ex vivo stress during “cold ischemia time”. Tumor cells react to the absence of vascular perfusion, hypoxia, acidosis, and accumulation of cellular waste, which can critically mask cancer related signaling network data. Standard formalin fixation of calcified tissue requires decalcification with formic/nitric acid solution, which greatly interferes with downstream molecular analysis. We developed a non-formalin fixative (TheraLin) for preserving biomarker molecules and tissue histomorphology in one step, which greatly facilitates the analysis of phosphoprotein signaling pathways in calcified tumor samples. Methods: Fifty specimens of neoplastic bone were collected at the Istituto Ortopedico Rizzoli (Bologna, Italy). Matched 0.5 cm samples were fixed in TheraLin preservative; or fixed in 4% buffered formalin and decalcified with formic/nitric acid solution. Specimens were processed into paraffin blocks and 5 μm tissue sections cut onto glass slides. Enriched tumor cell populations were procured using Laser Capture Microdissection. Reverse Phase Protein Microarrays were constructed with the microdissected tumor cells and probed with >40 antibodies to unmodified or phosphorylated proteins. Immunohistochemistry (>200 stains in total) was performed on routine clinical protein targets and a subset of phosphorylated proteins. Results: TheraLin fixation greatly facilitated molecular profiling of bony tissues by eliminating the need for decalcification (18.5 hour average reduction in processing time) while maintaining or improving histomorphology (>85% of cases) and immunohistology (>75% of cases). In addition, we were able to demonstrate full compatibility with laser capture microdissection, reverse phase protein microarrays and downstream analysis of cell signal protein activation. Protein extractability from microdissected material was significantly increased (>2.5 fold, p Conclusion: TheraLin obviates the need for decalcification of bone tissue while simultaneously preserving histomophology and immunohistochemistry. TheraLin enables reliable quantification of phosphorylated signal transduction proteins, the substrates of kinase drug targets, and greatly facilitates tumor cell enrichment compared to standard formalin fixation and decalcification. Citation Format: Claudius Mueller, Lance A. Liotta, Antonella Chiechi, Piero Picci, Marco Alberghini, Lorenzo Memeo, Vincenzo Canzonieri, Virginia Espina. A new frontier for molecular profiling of neoplastic bone tissue. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3877.

Ewing's sarcoma (EWS) is an aggressive tumor of children and young adults that requires intensive treatment. The search for new prognostic factors is very important to choose the most appropriate therapy and to better understand the biology of the disease for the development of new therapeutic tools. We found that Xg, a thus far poorly described molecule and member of the CD99 family, is expressed in EWS cell lines and EWS primary tumors. Immunohistochemical analysis confirmed the expression of Xg in 24% of patients. We found that Xg expression in EWS defines a subgroup of patients with worse prognosis compared with those with Xg-negative localized tumors, indicating a clinical relevance of Xg expression in EWS. Forced expression of Xg in an EWS cell line upregulated cell migration and invasion in vitro. Furthermore, knockdown of Xg expression with specific short hairpin RNA significantly reduced migration and invasion of EWS cells. Consistent with these data, in vivo xenotransplant studies in nude mice revealed that Xg expression increased the incidence and the number of metastases of EWS cells. Thus, Xg expression is associated with lower overall survival in EWS patients with localized tumors and is implicated in metastasis. Cancer Res; 70(9); 3730–8. ©2010 AACR.

Caveolin-1 (Cav-1) is highly expressed in normal osteoblasts. This article reports that Cav-1 down-regulation is part of osteoblast transformation and osteosarcoma progression and validates its role as oncosuppressor in human osteosarcoma. A survey of 6-year follow-up indicates a better overall survival for osteosarcoma expressing a level of Cav-1 similar to osteoblasts. However, the majority of primary osteosarcoma shows significantly lower levels of Cav-1 than normal osteoblasts. Accordingly, Met-induced osteoblast transformation is associated with Cav-1 down-regulation. In vitro, osteosarcoma cell lines forced to overexpress Cav-1 show reduced malignancy with inhibited anchorage-independent growth, migration, and invasion. In vivo, Cav-1 overexpression abrogates the metastatic ability of osteosarcoma cells. c-Src and c-Met tyrosine kinases, which are activated in osteosarcoma, colocalize with Cav-1 and are inhibited on Cav-1 overexpression. Thus, Cav-1 behaves as an oncosuppressor in osteosarcoma. Altogether, data suggest that Cav-1 down-modulation might function as a permissive mechanism, which, by unleashing c-Src and Met signaling, enables osteosarcoma cells to invade neighboring tissues. These data strengthen the rationale to target c-Src family kinases and/or Met receptor to improve the extremely poor prognosis of metastatic osteosarcoma. [Cancer Res 2007;67(16):7675–85]

Introduction: Ewing Sarcoma (EWS), the second most common bone tumor occurring in children and young adults, is a clinically very aggressive tumor with high metastatic tendency. EWS therapy is still firmly based on chemotherapy and development of chemoresistance is a major cause of disease progression. The mechanisms of chemoresistance are, however, still poorly defined. In this study, we analyzed the prognostic value of 21 ABC transporters with different mechanisms of action. Materials and methods: Gene expression of 21 ABC transporters was determined by RT-PCR in two different series of EWS patients with primary localized tumor at diagnosis (training set: 30 pts; validation set: 109 pts) and in human EWS cell lines. Association with patient outcome was determined by log-rank test and Kaplan-Meier survival curves (EFS and OVS). Sensitivity to conventional chemotherapeutic agents, such as Doxorubicin, Vincristine, Etoposide, Actinomicin-D and Ifosfamide, was tested in a representative panel of human EWS cell lines. Results: Among the 21 ABC transporters that were analyzed in the training set, expression of ABCA2, ABCA6, ABCA7, ABCB9 and ABCC4 was found to be associated with differential prognosis. Of these genes, only ABCA6 and ABCA7 were confirmed to be significant indicators of EWS progression when the analysis was extended to a new series of 109 patients with localized primary tumor (validation set): high expression of ABCA6 or ABCA7 was associated with a better prognosis. Correlative in vitro studies on EWS cell lines showed that the high expression of ABCA7 was not associated with higher response to conventional drugs, indicating a more complex role in the regulation of EWS aggressiveness. Conclusions: The study validated the impact of ABCA6 or ABCA7, the still poorly understood members of ABC transporters, on EWS progression. However, since high expression of these two transporters is related to better prognosis rather that worse, a different role from the canonical function of drug extruders should be supposed. We are currently testing the association between ABCA7 expression and ceramide levels, which are known to induce cell apoptosis, and macrophage-mediated phagocytosis as indicated in the literature (Kielar D. et al. J Invest Dermatol. 2003 Sep;121(3):465-74; Jehle AW et al. J Cell Biol. 2006 Aug 14;174(4):547-56). Funded by: AIRC to KS IG 14049. Citation Format: Michela Pasello, Marilù Fanelli, Valentina Mularoni, Sonia Ciotti, Piero Picci, Massimo Serra, Katia Scotlandi. Expression levels of ABCA6 or ABCA7 predict primary Ewing sarcoma progression at diagnosis. [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 589. doi:10.1158/1538-7445.AM2015-589

Soft Tissue Sarcoma (STS) are rare mesenchymal tumors, accounting for less than 1% of adult and 7-10% of pediatric cancers respectively. They are very complex and approximately include 50 different benign and malignant histotypes. Identification of circulating cancer biomarkers improves early detection and is important in determining patient risk stratification, in monitoring tumor progression and therapy response, and in planning new treatment. However, at present there are no laboratory tests that allow reliable early STS diagnosis and consequent prognosis. Discovery of new serum protein biomarkers is especially needed for patients with advanced/metastatic STS for whom the outcome remains strongly unfavorable. Proteomic technologies are used for global profiling and identification of disease-associated markers in biological fluids, such as serum. The low-molecular-weight (LMW) proteome ( In order to determine whether low-abundant serum proteins/peptides can be indicative of advanced disease, we analyzed 53 high-grade STS sera divided in discovery (34 sera) and validation (19 sera) subsets by using poly(N-isopropylacrylamide-co-vinylsulfonic acid) hydrogel core-shell nanoparticles with incorporated Cibacron Blue F3G-A. These nanoparticles selectively entrap LMW proteins/peptides on the bases of size exclusion and affinity chromatography. These nanoparticles protect the analytes from enzymatic degradation and amplify the concentration for mass spectrometry detection. 13 benign STS and 20 healthy sera were used as control. The most significant protein, IGFBP7, was then validated in a wider series of cases including 59 high-grade STS sera by ELISA assay and 86 paraffin-embedded high-grade primary STS by immunohistochemistry. Mass spectrometry data revealed 17 proteins/peptides common to discovery and validation subsets with significance differences in abundance when compared to benign and/or normal sera. The function of selected biomarkers was predominantly related to calcium-binding proteins (MGP), immune response (Ceruloplasmin, Lactotransferrin), coagulation process (Serpin C1) and metabolism (TGFBI). Moreover, as confirmed by ELISA assay, expression of insulin-like growth factor binding protein 7 (IGFBP7) involved in cell signaling pathway progressively increased in healthy, benign and malignant tumors. With immunohistochemistry, tissue IGFBP7 had a significantly higher expression in metastatic than in non metastatic STS. Our data suggest that proteomic technology is able to identify circulating biomarkers associated to STS malignant phenotype and IGFBP7 in paired primary specimens may discriminate STS patients with increased risk of metastatic progression. Citation Format: Amalia Conti, Claudia Fredolini, Davide Tamburro, Giovanna Magagnoli, Weidong Zhou, Lance A. Liotta, Piero Picci, Alessandra Luchini, Maria Serena Benassi. Identification and validation of novel candidate circulating biomarkers in high-grade soft tissue sarcoma. [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 590. doi:10.1158/1538-7445.AM2015-590

The identification of reliable indicators of prognosis, which may allow the stratification of patients according to different risk at diagnosis isan important aspect of translational research in Ewing sarcoma (ES). In this paper, we validated our previous evidence showing how expression of miR34a in ES tumor samples at diagnosis was signficantly associated with tumor progression (Nakatani F. J Pathol 2012). Here we analyzed a different series of speciments derived from very controlled and homogeneously treated non-metastatic ES patients, and we compared evaluation of miR34a by RT-PCR using frozen samples with that obtained by in situ hybridization on paraffin-embedded samples . The median follow-up of the 109 EWS patients was 90 months (range 9 to 241 months). 68 patients (63%) remained continuously free of disease and 41 (38%) relapsed. Expression of miR34a was evaluated by qRT-PCR using TaqMan miRNA assay kit and/or by in situ hybridization ISH optimization kit from Exiqon. High expression of miR34a was significantly related to better EFS and OVS : 5-year EFS was 74% for the 55 patients with high expression of miR34a and was 51% for the 54 patients with low expression (p = 0.004); 5-year OVS was 83% in high-expressors and 58% in low-expressors . By multivariate Cox regression analysis, the value of low miR-34a expression as well as of non-total necrosis and high LDH were all confirmed as independent risk factors associated with poor outcome. miR34a was also evaluated by ISH on TMA. miRNA expression by ISH correlates with that obtained by RT-PCR (r=0.59, p Citation Format: Katia Scotlandi, Maria Teresa Marino, Andrea Grilli, Piero Picci, Alessandra Carè, Claudio Tripodo, Stefano Ferrari. miR34a: A valuable indicator of differential outcome of Ewing sarcoma patients with complex functions. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A42.

Introduction: Ewing sarcoma (ES) is a high-grade rare malignant bone tumor in children and adolescents. This disease is primarily characterized by: i) Chromosomal translocations that fuse EWS gene and a gene of the ETS family of transcription factors (around 85% EWS-FLI1 and 10% EWS-ERG, respectively) which are the oncogenic drivers of this tumor; ii) High expression of CD99, a cell surface glycoprotein virtually present in all of ES cells. EWS-FLI1 and CD99 were reported to have divergent roles in modulating ES malignancy and neural differentiation In this study we investigated the possible role of nuclear factor-kappa B (NF-kB) signaling pathway on the neural differentiation of ES cells and its relationship with EWS-FLI1 and CD99. Materials and Methods: Transcriptional activity of several transcription factors (AP1, SRE, CREB, E2F and NF-kB) were evaluated by gene reporter assay. Using a knockdown approach in stable and transient conditions we silenced CD99, EWS-FLI1 and NF-kB itself, alone or in combination, in three ES cell lines (TC-71, IOR/CAR and ASP14). NF-kBp65 was also transiently overexpressed in TCshCD99 and CARshCD99 clones with pCMVp65 construct. Neural differentiation was detected evaluating β-III tubulin by immunofluorescence microscopy. Results: NF-kB was found heterogeneously expressed in a panel of ES cell lines. Stable CD99 silencing was found to decrease NF-kB activity (pValue Conclusion: Our findings support the concept that inhibition of NF-kB is able to induce terminal neural differentiation of ES cells. This identifying NF-kB as a potential therapeutic target against Ewing sarcoma progression. (Supported by AIRC_2013_IG 14049 and Liddy Shriver Foundation) Citation Format: Selena Ventura, Chiara Manzalini, Dave Aryee, Piero Picci, Heinrich Kovar, Katia Scotlandi. Dualistic regulation of NF-kB signaling by CD99 and EWS-FLI1 in Ewing sarcoma: impact on cell differentiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 481. doi:10.1158/1538-7445.AM2014-481

Rare and highly aggressive adult soft tissue sarcomas, leiomyosarcoma (LMS) and undifferentiated pleomorphic sarcoma (UPS), contain complex genomics characterized by a multitude of rearrangements, amplifications and deletions. Since differential diagnosis between the two sarcoma histotypes continues to remain a challenge, we attempted to identify a specific signature useful for improving the differential diagnosis, and we performed miRNA profiling on series of LMS and UPS samples stored at Rizzoli biobank. First, we investigated the different miRNA expression profile in 20 primary sarcoma samples, 10 high-grade LMS and 10 UPS, MFH-like, using miRNA array technique. The top 5 differentially expressed (p Citation Format: Laura Pazzaglia, Mohamed Guled, Ioana Borze, Maria Serena Benassi, Piero Picci, Sakari Knuutila. Different miRNAs expression in leiomyosarcoma and undifferentiated pleomorphic sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5196. doi:10.1158/1538-7445.AM2014-5196

Introduction: CD99 is a transmembrane glycoprotein which is physiologically involved in several biological processes, such as cell migration, adhesion, and differentiation. High levels of CD99 have been reported in mesenchymal stem cells and osteoblasts. In contrast, the molecule has been shown to be absent in osteosarcoma (OS), in which it acts as a tumour suppressor. The molecular mechanisms responsible for its functions are however still poorly defined. In this study we used bioinformatic studies to integrate gene expression and miRNA data to identify crucial mediators. Methods: Forced expression of CD99 was stably induced in Saos-2 OS cell line. Microarray analyses of gene or miRNA expression were performed and integrated by using different bioinformatic approaches. TargetScan, Miranda, Diana-microT and PicTar prediction databases were used to identify miRNA targets. Results: CD99-overexpressing cells displayed a massive down-regulation of gene expression. Only 19 probes were found to be significantly up-regulated in CD99 transfectants compared to parental cells. In contrast, 231 probes were found to be down-regulated, thus suggesting gene modulation by epigenetic mechanisms. We thus examined miRNA profile of these cells. A significant up-regulation of miR-1225-3p, miR-1238, miR-1305, miR-191*, miR-34a, and miR-425* was observed in CD99 overexpressing cells. Enrichment analyses pointed out an involvement of TGFβ signalling. Interestingly, SMAD-2 and -4, which emerged as targets of miR-34a, were among the main down-modulated genes. miR34 mimics confirmed the association between miR34a and SMAD-4. Functional studies are on-going to establish associations with osteosarcoma malignancy. Conclusions: Besides inhibiting osteosarcoma malignancy, CD99 expression in osteosarcoma cells determines massive gene expression repression, likely due to changes in miRNA profile. Up-regulation of miR-34a together with down-regulation of TGFβ signalling mediators seem to be critical players. Studies are granted by AIRC 10452 and Ministry Health RF-2009 to KS. Citation Format: Marika Sciandra, Andrea Grilli, Maria Teresa Marino, Maria Cristina Manara, Piero Picci, Katia Scotlandi. Integrated bioinformatic analysis to elucidate molecular mechanisms associated with reversal of osteosarcoma malignancy induced by CD99. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2894. doi:10.1158/1538-7445.AM2013-2894

Osteosarcoma (OS), the most common primary malignant bone tumor, has a peak incidence during the pubertal growth spurt. OS occurs at increased frequency in several inherited cancer syndromes (e.g., Li-Fraumeni and Rothmund Thomson syndromes). A limited number of common SNPs associated with OS have been identified in biologically plausible pathways (e.g., growth or DNA repair). We developed an international, multi-institutional study in order to conduct the first genome-wide association study (GWAS) of OS. Participating subjects provided informed consent through local IRBs. Control subjects were cancer free adults derived from large studies in prior GWAS at the NCI. Genotyping of all cases was conducted using the Illumina OmniExpress SNP microarray chip platform in 2 phases. The first phase analyzed 910 osteosarcoma cases. After quality control filtering and assessment of population substructure, 596 cases of European (EUR) ancestry were advanced to the primary GWAS analysis (Phase 1A). We combined data from 2,703 previously genotyped controls drawn from 3 EUR cohorts. There was no evidence for significant population substructure differences between OS cases and controls. The association analysis (1-degree of freedom trend test) was adjusted for study, sex and 4 eigenvectors. The top 30 SNPs (P The fixed-effects meta-analysis of all 941 OS cases and 3,291 controls identified 2 regions that achieved genome-wide significance. The first, rs1906953 on 6p21.3, is associated with susceptibility to OS (P = 8.1x10-9, odds ratio [OR] 1.57, 95% confidence interval [CI] 1.35-1.83). rs1906953 is in intron 7 of GRM4. GRM4 is expressed in osteoblasts and osteoclasts and involved in glutamate signaling, which is suggested to be involved in cell differentiation and regulation of bone formation. The second signal resides in an intergenic region on chromosome 2p25.2; rs7591996 (P = 1.0 x 10-8, OR 0.72, 95% CI 0.65-0.81). Another SNP in this region, rs10208273, is moderately correlated with the first signal (r2= 0.32) and approached genome-wide significance (P = 2.9 x 10-7, OR 1.35, 95% CI 1.21-1.52). A third locus in the ADAMTS6 gene on 5q12.3 is promising but does not yet achieve genome-wide significance: rs17206779 (P = 5.1x10-7, OR 0.75, 95% CI 0.68-0.84). Notably, variants in genes encoding members of the ADAMTS protein family have been associated with height, a known OS risk factor. We have conducted the first GWAS of OS and identified several novel loci associated with OS risk. Further investigation of these loci will advance understanding of OS etiology and biology. Citation Format: Sharon A. Savage, Lisa Mirabello, Zhaoming Wang, Julie Gastier-Foster, Richard Gorlick, Chand Khanna, Adrienne M. Flanagan, Roberto Tirabosco, Irene L. Andrulis, Jay Wunder, Nalan Gokgoz, Ana Patino-Garcia, Luis Sierrasesumaga, Fernando Lecanda, Nilgun Kurucu, Inci Ergurhan Ilhan, Neriman Sari, Massimo Serra, Claudia Hattinger, Piero Picci, Logan Spector, Donald A. Barkauskas, Neyssa Marina, Siliva Caminada de Toledo, Sergio Petrilli, Maria Fernanda Amary, Dina Halai, David Thomas, Chester Douglass, Paul Meltzer, Kevin Jacobs, Charles C. Chung, Sonja I. Berndt, Mark P. Purdue, Neil E. Caporaso, Margaret Tucker, Nathaniel Rothman, Maria Teresa Landi, Debra T. Silverman, Peter Kraft, David J. Hunter, Nuria Malats, Manolis Kogevinas, Sholom Wacholder, Rebecca Troisi, Lee Helman, Joseph F. Fraumeni, Jr., Meredith Yeager, Robert Hoover, Stephen J. Chanock. Genome-wide association study identifies novel loci associated with osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4593. doi:10.1158/1538-7445.AM2013-4593

OS is the most frequent primary bone tumor in children and young adults.The mammalian Target of Rapamycin (mTOR) and the Extracellular Regulated Kinases (ERK)1/2 pathways have been shown to cooperate in survival advantage of OS. Inhibition of this signaling can be a rational pharmacological strategy to impinge on OS progression. This study pursued the dual blockage of ERK1/2 and mTOR pathways by combining the multikinase inhibitor SOR with the rapamycin analog EV. The synergistic anti-profilerative effect was found against 5 out 7 OS cell lines (combination index Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-366. doi:1538-7445.AM2012-LB-366

Osteosarcoma (OS) is the most common primary bone malignancy in children and young adults. It displays a complex karyotype and a broad range of molecular abnormalities; however most of these alterations are not constantly detected in the majority of osteosarcoma tumors. Although its precise origin still remains unknown, increasing findings suggest OS should be regarded as a differentiation disease caused by genetic and epigenetic changes which interrupt osteoblast differentiation from mesenchymal stem cells (MSCs). Few sporadic data link CD99 molecule to osteoblast functions. It is a 32 kDa surface glycoprotein involved in several biological processes. We have previously demonstrated that CD99 acts as a tumor suppressor in osteosarcoma where it is either undetectable or present at very low levels. Being expressed in osteoblasts (OBs), the normal counterpart, we tested the hypothesis that CD99 may contribute to osteoblastic differentiation. Its role was assessed, first of all, in physiological conditions. We provided evidence that CD99 is present in MSCs, considered the cells of origin for OS, and its expression increases during osteoblastogenesis and osteoblast maturation evaluated in MSCs as well as in OBs. To study the function of CD99 in OS we took advantage of two osteosarcoma cell lines, Saos-2 and OS7, and their respective clones overexpressing the glycoprotein. Grown in osteogenic conditions for 21 days, these cells were able to differentiate and mineralize, as underlined by typical genes (collagen I, alkaline phosphatase and osteocalcin) as well as by specific stainings for matrix mineralization. In presence of the molecule, cells were able to restore a normal phenotype, following the normal osteogenic program, thus allowing transition toward the latter step of differentiation. An effective proliferation arrest was reached in association with withdrawal from cell cycle in G1 phase, reduction of cyclin A, high expression of p21, and massive apoptosis induction. The strong and prolonged activation of ERK in presence of CD99 suggested the involvement of MAP kinase in differentiation, as it was detected in MSCs under differentiating conditions. After PD98059 treatment, ERK inhibition really attenuated osteoblastic features in CD99 expressing cells. In conclusion, these results give new insight into the role of the glycoprotein in osteoblastic differentiation. Its forced expression helps to overcome the impaired osteoblastogenesis that occurs in OS cells. These data can open new avenue for the understanding of mechanisms regulating OS pathogenesis and progression and they also suggest a possible new therapeutic approach to OS based on modulating tumor differentiation (grant from Italian Association for Cancer Research). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5340. doi:10.1158/1538-7445.AM2011-5340

Bone metastasis is a main cause of cancer associated pain and mortality. The activation of osteoblasts and osteoclasts by cancer cells generates a vicious cycle of bone destruction and increased tumor growth, causing pain, fractures and spinal cord compression. In this study the microenvironment of human bone metastasis is being examined from a new point of view: the neuroreticular axis. The sympathetic nervous system regulates the homing and survival of bone marrow immune stem cells within a structural bone network called the neuroreticular complex. The neuroendocrine axis permits stem cells to survive in the bone microenvironment, regulates B and T cells and influences mesenchymal stem cells (MSC). Estrogen/androgen ratio is a fundamental regulator of bone development and turnover, inducing osteoblast or osteoclast differentiation. Platelet-derived serotonin (5-HT) is involved in bone metabolism via release from small intestine enterochromaffin cells to induce bone lysis or neuronal release, supporting bone consolidation. The aims are a) identify activated kinase pathway interconnections in each bone metastasis from different primary tumors to identify predictive/prognostic biomarkers, b) identify targets for molecular targeted therapies. 142 bone metastases frozen samples from different types of primary carcinomas (13 head and neck, 45 urogenital, 16 gastro enteric, 14 breast, 25 lung, 7 melanoma and 22 unknown carcinomas) and 24 from primary sarcomas were lysed using Covaris adaptive focused acoustic technology. Levels of 100 post-translationally modified cell signaling kinases were quantified by Reverse Phase Protein Microarray (RPMA). RPMA provides a quantitative “circuit map” of pathways that are activated/deactivated in a sample of tumor cells obtained from a biopsy. The metastasis originating from different primary tumors showed similar levels of cell signaling across tissue types for the majority of proliferation, invasion and adhesion pathway proteins analyzed. Sex hormone receptor expression was found to be similar among the metastasis samples from different carcinoma types, while bone metastasis from sarcomas showed a statistically significant higher expression compared to those originating from carcinomas (ERα Ser118 p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5180. doi:10.1158/1538-7445.AM2011-5180

Clinical outcomes of Ewing sarcoma family of tumors (ESFT) were improved after the introduction of multimodal therapies including intensive chemotherapy. However current therapeutic approaches still leave us the problems including short-term toxicities of chemotherapy and serious late adverse effects; second malignancy and fatal cardiac failure even for long survivors. Therefore, we are in the urgent need to develop the novel biomarkers for an appropriate stratification of patients at diagnosis to establish more effective and less invasive therapy for all ESFT patients. MicroRNAs (miRNAs) are newly-recognized small non gene-coding RNA family that regulate gene expression through translational suppression. More recently, they have been increasingly accepted as playing crucial roles in pathogenesis of most human malignancy as well as normal cell differentiation. However, little is known regarding to the possible role of miRNAs on sarcoma including ESFT. Here we focus on the whole profiling of the miRNA expressions in ESFT for the aim to elucidate the novel miRNA biomarkers in the treatment of ESFT. Randomly selected 34 primary biopsy samples of ESFT have been analyzed for complete miRNA expression profiles. All of the patients were treated at Rizzoli Institute from 1991 to 2008 with multimodal therapies. Microarray and statistical analyses were carried out using Agilent's miRNA microarray kit (v2) and GeneSpring GX analysis software, respectively. To identify miRNAs associated with the prognosis of ESFT patients we first compared the expression profiles of 21 patients who relapsed within 5 years with 13 patients who remained free of disease throughout the follow-up. As a result, we identified 16 miRNAs differentially expressed between the two groups (fold change > 1.5, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 167. doi:10.1158/1538-7445.AM2011-167

IGF-1R represents a promising therapeutic target, particularly in sarcomas. Identification of patient selection criteria and understanding the potential mechanisms involved in development of resistance will be crucial for an appropriate and successful design of clinical trials. Here we evaluated efficacy of the anti-IGF-IR monoclonal antibody (HAb) AVE1642, in a panel of sarcoma cell lines and we developed a model of resistance to the anti-IGF-1R human antibody AVE1642 or to the small tyrosine kinase inhibitor (TKI) NVP-AEW541 starting from two highly sensitive Ewing's sarcoma cell lines to identify indicators of intrinsic and adaptive resistance to anti-IGF1R therapies. Cells made resistant to AVE1642 showed same growth ability either in monolayer and in anchorage-independent condition respect to sensitive cells. Notably, resistant cells retained the same level of sensitivity to doxorubicin, vincristine, and ifosfamide as the parental cell lines. Annotation analysis highlighted that the insulin pathway seems to be significant in resistant cells. In fact, these cells showed higher proliferative response to insulin, in keeping with a switch towards insulin pathways sustaining proliferation and malignancy, rather than metabolism. Molecular characterization demonstrate that enhanced IR-A formation and IGF-II production are heavily involved in native and acquired resistance to IGF targeted therapies. This finding was extended to cells resistant to the small tyrosine kinase inhibitor NVP-AEW541. We also show that tumors with low IGF-1R:IR-A ratio are unlikely to greatly benefit from anti-IGF-1R therapies and that the efficacy of anti-IGF-1R therapies should be evaluated in relationship to the IR-A:IGF-1R ratio in cancer cells. Our findings demonstrate a role for IR-A in eliciting intrinsic and adaptive resistance to anti-IGF1R therapies and provide evidences supporting IR-A as an important target in sarcoma therapy. Future trials with these highly selective drugs should involve preselection of patients with tumors that express low levels of IR-A and IGF-2. Studies are supported by grants from the Italian Association for Cancer Research, the Italian Ministry of Health, the Italian Ministry of Research and Instruction and the EU project Eurobonet. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 728. doi:10.1158/1538-7445.AM2011-728

We have developed a workflow using protein kinase signal pathway mapping technology to identify key cell signaling signatures in bone metastasis samples dependent and independent of primary tumor histotype. There is little known about molecular alterations during the metastatic process, especially to the bone, as well-preserved frozen tissue sources are limiting. The activation state of kinase-driven signal networks contains important information about disease pathogenesis and the state of kinase-associated therapeutic targets. Proteomic molecular profiling can provide a means to identify activated pathways or specific protein endpoints that may be potential drug targets or molecules useful for diagnosis/prognosis in bone metastasis pathology. The data set consists of metastatic bone tissue (n=166) and matched normal tissue (n=115) with corresponding clinical data. 142 bone metastasis were from different types of primary carcinomas: 13 head & neck, 45 urogenital, 16 gastroenteric, 14 breast, 25 lung, 7 melanoma and 22 unknown carcinomas. 24 bone metastasis were from primary sarcomas. The objectives are a) identify activated kinase pathway interconnections in each bone metastasis from different primary tumors to identify predictive/prognostic biomarkers, and b) correlate this pathway information with the matched normal tissue. Our workflow overcomes 3 major limitations to analyzing bone tissue: 1) the presence of contaminating blood in the tissue, 2) the need to completely homogenize the bone, and 3) the need to verify tissue histomorphology for each sample. Many bone metastasis tissue samples are lytic and embody a large amount of blood. In order to overcome the blood contamination problem, we developed an original method using the DNA concentration in each sample to normalize protein expression data, since red blood cells are devoid of a nucleus. Data normalization by DNA staining was very stable, consistent with sample cell number, and did not cross-react with RNA or proteins. Samples were completely homogenized using CryoPrep™ and Adaptive Focus Acoustic™ (AFA) technology by Covaris, with very good protein extraction efficiency. A portion of every sample was decalcified, paraffin embedded and Hematoxylin/Eosin stained to verify the tumor cell percentage in the tissue (>90%). Reverse phase protein arrays were used to quantitatively map 150 key proteins involved in bone metabolism, tumor-host interactions, hormone response, growth-proliferation, stress-inflammation, and adhesive-cytoskeletal proteins. Our workflow overcomes the difficulties in processing and analyzing bone metastasis samples. We obtained a proteomic profile that enables elucidation of molecular mechanisms leading to bone metastasis formation and its clinical manifestations. This is a fundamental step for developing new effective therapeutic strategies to treat bone metastasis pathology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2239.

Wnt signaling pathway is involved in normal embryonic development and has a majoir role in development and progression of cancer. If the role of wnt pathway is well characterized in carcinoma, its involvement is under study in sarcoma. Starting from an experimental model constituted by stable transfectants of short-hairpin RNA of CD99, characterized by reduced malignancy, real Time PCR analysis suggested a down-modulation of Wnt signaling. In fact, TC-71 cells deprived of CD99 showed increased expression of some inhibitors of the Wnt pathway (DKK1, sFRP1) and reduction in some ligands (Wnt 2 and 11). Since TCsiCD99 are less malignant than parental cells, these preliminary data indicated a possible relationship between Wnt pathway and EWS aggressiveness. Gene expression profiling of 30 primary EWS tumors confirmed these indications: low expression of Frizzled (Fzd) 2,3, LRP5/6 and low expression of some Wnt ligands (Wnt3, 11) are significantly associated with better prognosis. Validations of these associations at protein level by evaluation of Fzd2, Fzd3 and beta-catenin in EWS tissue arrays confirmed that lower levels of Fzds are associated with better prognosis, while no beta-catenin nuclear localization was found in any of the 103 cases examined. In contrast, around 40% of patients showed high expression of beta-catenin at the membrane level and these patients showed better prognosis. No activation of canonical-wnt pathway was confirmed also in the experimental model, with non nuclear localization of the protein. It is well known that beta-catenin is an essential co-activator of the canonical Wnt pathway but it also plays a critical structural role in cadherin-based adhesions. In EWS, beta-catenin membrane associated localization suggested its major role in cell-cell adhesion. Indeed, direct and significant associations were observed with other adhesion proteins, such as ZO-1, desmoplakin and desmoglein (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3406.

CD99 is a transmembrane 32 KDa protein, whose expression is constantly associated to Ewing's Sarcomas Family of Tumors (EWSFT), a class of child bone tumors with particular poor prognosis. The monoclonal antibody 0662, raised against CD99, triggers massive and rapid cell death of Ewing Sarcoma's cells in vitro and in vivo, through a non-canonical, non caspase-dependent apoptosis. To elucidate the underlying molecular mechanism, we evaluated by phosphor- and micro-arrays specific pathways triggered by CD99 engagement in 6647 Ewing Sarcoma cell line. Despite its role in physiological and cancer scenery has not been yet fully elucidated, CD99 appears to be sufficient to modulate many important processes involved in: I) adhesion; II) migration and metastasis; III) cell-cycle regulation and cell signaling; IV) apoptosis and death receptor signaling. These data are consistent with our previous analysis of 0662 effects in vivo and in vitro: anti-CD99 monoclonal antibody triggers, indeed, homotypic aggregation and a rapid death response of ES cells, thus resulting in growth inhibition and reduced metastatic/colony-forming potential. As pointed out by array studies and subsequent western blotting validations, upon 0662 treatment, the oncosuppressor protein p53 appears to be readily activated by phosphorilation on serine 15. We therefore evaluated p53 and MDM2 activation and sub-cellular localization upon CD99 engagement both in p53 WT (LAP35 and WE68) or mutated (6647 and TC-71) cell lines: WT (LAP35 and WE68) or mis-sense point mutated p53 (6647: S241F) cell lines display a higher sensitivity to 0662 if compared to p53 truncated cell lines (TC71). Consistently we demonstrated that whereas 0662 readily induces p53 canonical targets (p21, BAX) in p53 expressing cell lines, no increase in p21 levels is detectable in TC71 p53TRUNC cells. We also demonstrated that, upon 0662 treatment, decreased or delocalized MDM2 levels may account both for p53 activation and sustained IGF-IR signaling, since both p53 and IGF1-Rß are targeted by the ubiquitine ligase for degradation. Since most Ewing Sarcoma patients display unmodified p53 status, its functional inactivation -e.g. through MDM2/MDM4 overexpression- may account for gained tumor aggressiveness, or progressed disease. Nonetheless, involvement of TP53 might give reason for higher effectiveness we reported against local tumors and metastases in the combined employment of CD99 monoclonal antibodies with Doxorubicin and may support 0662 mAb treatment in association with other drugs targeting e.g. the p53 regulators MDM2-MDM4. (grants from EU project Eurobonet and Italian Association for Cancer Research) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 671.