The identified "protective" and "risk" factors, as well as the proposed classification system, represent helpful tools for clinical management and follow-up of patients with multiple hereditary exostoses; moreover, homogeneous cohorts of patients, useful for studies on the pathogenesis of multiple hereditary exostoses, have been identified.

Multiple osteochondromas (MO) is an autosomaldominant skeletal disorder characterized by the formation of multiple cartilage-capped protuberances. MO is genetically heterogeneous and is associated with mutations in the EXT1 and EXT2 genes. In this study we describe extensive mutation screening in a set of 63 patients with clinical and radiographical diagnosis of MO. Denaturing high-performance liquid chromatography analysis revealed mutations in 43 patients. Additional deletion analysis by fluorescence in situ hybridization and a newly developed multiplex ligation-dependent probe amplification probe set identified one patient with an intragenic EXT1 translocation , three patients with a partial EXT1 deletion , and one patient with a partial EXT2 deletion. Thirty-six patients harbored an EXT1 mutation (57%) , and 12 had an EXT2 mutation (19%). We show that our optimized denaturing high-performance liquid chromatography/sequencing/multiplex ligation-dependent probe amplification protocol represents a reliable and highly sensitive diagnostic strategy for mutation screening in MO patients. Clinical analysis showed no clear genotype-phenotype correlation in our cohort of MO patients.

Standard therapy of osteosarcoma (oS) and ewing sarcoma (eW) rests on cytotoxic regimes, which are largely unsuccessful in advanced patients. preclinical models are needed to break this impasse. A panel of patient-derived xenografts (pDX) was established by implantation of fresh, surgically resected osteosarcoma (OS) and Ewing sarcoma (EW) in NSG mice. Engraftment was obtained in 22 of 61 OS (36%) and 7 of 29 EW (24%). The success rate in establishing primary cell cultures from OS was lower than the percentage of pDX engraftment in mice, whereas the reverse was observed for eW; the implementation of both in vivo and in vitro seeding increased the proportion of patients yielding at least one workable model. the establishment of in vitro cultures from PDX was highly efficient in both tumor types, reaching 100% for EW. Morphological and immunohistochemical (SATB2, P-glycoprotein 1, CD99, caveolin 1) studies and gene expression profiling showed a remarkable similarity between patient's tumor and pDX, which was maintained over several passages in mice, whereas cell cultures displayed a lower correlation with human samples. Genes differentially expressed between OS original tumor and PDX mostly belonged to leuykocyte-specific pathways, as human infiltrate is gradually replaced by murine leukocytes during growth in mice. In EW, which contained scant infiltrates, no gene was differentially expressed between the original tumor and the PDX. A novel therapeutic combination of anti-CD99 diabody C7 and irinotecan was tested against two EW PDX; both drugs inhibited PDX growth, the addition of anti-CD99 was beneficial when chemotherapy alone was less effective. The panel of oS and eW pDX faithfully mirrored morphologic and genetic features of bone sarcomas, representing reliable models to test therapeutic approaches. Osteosarcoma (OS) and Ewing sarcoma (EW), the two most common primary tumors of bone, are high-grade malignant neoplasms with very aggressive behavior and high tendency to form metastasis; they arise frequently in children and remain prominent among teenagers and young adults 1-4 .

Osteochondroma, the most common benign bone tumor, may occur as a sporadic lesion or as multiple neoplasms in the context of multiple osteochondromas syndrome. The most severe complication is malignant transformation into peripheral secondary chondrosarcoma. Although both benign conditions have been linked to defects in EXT1 or EXT2 genes, contradictory reports are present in the literature regarding the requirement of their biallelic inactivation for osteochondroma development. A major limitation of these studies is represented by the small number of samples available for the screening. Taking advantage of a large series of tissues, our aim was to contribute to the definition of a genetic model for osteochondromas onset and transformation. EXT genes point mutations and big deletions were analyzed in 64 tissue samples. A double hit was found in 5 out of 35 hereditary cases, 6 out of 16 chondrosarcomas and 2 recurrences; none of the 11 sporadic osteochondromas showed two somatic mutations. Our results clearly indicate that, in most cases, biallelic inactivation of EXT genes does not account for osteochondromas formation; this mechanism should be regarded as a common feature for hereditary osteochondromas transformation and as an event that occurs later in tumor progression of solitary cases. These findings suggest that mechanisms alternative to EXT genetic alteration likely have a role in osteochondromas pathogenesis.

This work describes the setup of a shared platform among the laboratories of the Alleanza Contro il Cancro (ACC) Italian Research Network for the identification of fusion transcripts in sarcomas by using Next Generation Sequencing (NGS). Different NGS approaches, including anchored multiplex PCR and hybrid capture-based panels, were employed to profile a large set of sarcomas of different histotypes. The analysis confirmed the reliability of NGS RNA-based approaches in detecting sarcoma-specific rearrangements. Overall, the anchored multiplex PCR assay proved to be a fast and easy-to-analyze approach for routine diagnostics laboratories.

Multiple osteochondroma (MO) is a rare skeletal disease characterized by the formation of multiple benign cartilage-capped bone tumors; in 1-5% of patients, a malignant transformation into peripheral chondrosarcoma may occur. This disorder is characterized by a large spectrum of germline mutations scattered along EXT1/EXT2 genes, the presence of a significant percentage of patients without alterations in EXT genes, and a large phenotypic variability. The molecular basis of MO genetic and clinical heterogeneity, including the causes underlying malignant transformation, is currently unknown. This leads to the lack of appropriate diagnostic/prognostic markers as well as of therapeutic options. Recently, specific microRNAs (miRNAs) were reported to be involved in chondrogenesis and inflammatory cartilage diseases. We therefore hypothesized a role for microRNAs in cartilaginous tumors and investigated microRNA expression in osteochondroma and normal cartilage tissues to evaluate whether they could affect osteochondromas onset and/or clinical manifestations. Our results indicate that miRNAs differentially expressed in MO samples may hamper the molecular signaling responsible for normal differentiation of chondrocytes, contributing to pathogenesis and clinical outcome. Although further studies are needed to validate our observations and to identify targets of miRNAs, this is the first study reporting on miRNA expression in growth plate and its comparison with pathological conditions.

Osteogenesis imperfecta (OI) is a connective tissue disorder mostly characterized by autosomal dominant inheritance. Over 1,100 causal mutations have been identified scattered along all exons of genes encoding type I collagen precursors, COL1A1 and COL1A2. Because of the absence of mutational hotspots, Sanger sequencing is considered the gold standard for molecular analysis even if the workload is very laborious and expensive. To overcome this issue, different prescreening methods have been proposed, including DHPLC and biochemical studies on cultured dermal fibroblasts; however, both approaches present different drawbacks. Moreover, in case of patients who screen negative for point mutations, an additional screening step for complex rearrangements is required; the added causative variants expected from this approach are about 1-2%. The aim of this study was to optimize and validate a new protocol that combines quantitative PCR (qPCR) and high-resolution melting (HRM) curve analysis to reduce time and costs for molecular diagnosis. Results of qPCR-HRM screening on 57 OI patients, validated by DHPLC-direct sequencing and multiplex ligation-dependent probe amplification (MLPA), indicate that all alterations identified with the mentioned methodologies are successfully detected by qPCR-HRM. Moreover, HRM was able to discriminate complex genotypes and homozygous variants. Finally, qPCR-HRM outperformed direct sequencing and DHPLC-MLPA in terms of rapidity and costs.

Glycosaminoglycans were extracted from both young rabbit growth plate (GRP) and articular (ART) cartilage tissues and enzymatically treated to selectively eliminate chondroitin sulfates and hyaluronic acid. The procedure avoided any fractionation step that could enrich the extract with over- or under-sulfated species. Isolated heparan sulfate (HS) was characterized by mono- and bidimensional nuclear magnetic resonance (NMR) spectroscopy to quantify their specific structural features and/or by mass spectrometry to establish the disaccharide composition. Both GRP and ART HSs, despite differing in their yield (GRP at least 100 times greater than ART), exhibited a surprisingly high degree of sulfation. Quantitative two-dimensional heteronuclear single-quantum coherence-NMR analysis of GRP HS revealed unusually high N-sulfated glucosamine and 2-O-sulfated iduronic acid contents, similar to heparin. The unique pentasaccharide sequence of the binding site for antithrombin was also detected in a significant amount. High-performance liquid chromatography mass spectrometry analysis of the enzymatic digests with a cocktail of heparin lyases of both cartilaginous HSs confirmed the NMR results. As well as the discovery of an unusual HS structure in the two different types of rabbit cartilage, the feasibility of the analytical method adopted here has been demonstrated within this study. Such a method can be used to isolate and analyze HS from both normal and pathologic tissues. Characterization of healthy and pathological HS structures will contribute to improve the understanding of diseases related to malfunctions of HS biosynthesis and/or metabolism.