Friday 16th November Osteosarcoma Poster discussion L 1425144 M 1426725 N 1427271 O 1433633 P 1425094 Bass Hassan University of Oxford Sir William Dunn School of Pathology Oxford Sarcoma Service at the Oxford University Hospitals Trust Oxford Cancer and Haematology Centre Nuffield Orthopaedic Centre Osteosarcoma Genomics- Highly Complex The HALLMARKS Paper Presentation Summary Adjuvant chemotherapy plus early >90% necrosis : OS=57% at 25 years miR-133a suppression improves outcome in mice Caprin 1 promotes SaOS-2 cell metastasis in mice Intercalary allografts not recommended Need for a chemotherapy trial age >40 year RNA Interference miRNA Families and targets L 1425144 Micro-RNA expression and functional profiles of osteosarcoma Kobayashi et al National Cancer Research Centre, Tokyo 24 fresh frozen OS 8 cell lines miRNA microarrays (933) anti-miR library Targets proteins identified (liquid chromatography and mass spec- 2DICAL) • Blocking let-7 suppressed proliferation • Main targets Serpin H1 and vimentin L 1425144 24 OS samples microRNA microarray analysis Global expression of miRNAs in OS samples Frequency of miRNA expression Flow diagram Anti-miR inhibitors library, MTT assay In 8 OS cells Identification of miRNAs regulating OS cell proliferation Proteomic approach (2DICAL) In U2 cells Sample 108 commonly expressed miRNAs in OS samples 515 miRNAs 108 miRNAs 100 75 50 25 0 OS03 OS13 OS17 OS18 OS20 OS25 OS26 OS27 OS28 OS32 OS35 OS36 OS38 OS44 OS45 OS46 OS48 OS49 OS50 OS51 OS52 OS64 OS65 OS67 200 400 600 800 Number of miRNAs (n=939) Identification of miRNA targeted protein Index L 1425144 Survey of miRNAs regulating OS cell proliferation Change of ATP production (%) U2 NOS1 MNNGHOS SaOS2 9N2 HsOS1 80 60 40 20 0 -20 -40 Inhibited miRNA 3N1 MG63 Identification of proteins regulated by let-7a family miRNAs 1,093.924m/z (1,093.824~1,094.024) Vimentin let-7a let-7a let-7f let-7f let-7g let-7g let-7i let-7i NC1 NC1 NC2 NC2 Serpin H1 NC2 NC1 -Actin- let-7i -Actin- let-7g Serpin H1- let-7f let-7a NC2 NC1 let-7i let-7g let-7f let-7a Vimentin- M 1426725 Evaluation of the role of metadherin in osteosarcoma metastasis Zhu et al University of Texas MD Anderson Cancer Centre Metadherin (MTDH) Expression is Up-regulated in Osteosarcoma Cells A B C M 1426725 MTDH Knockdown Decreases Cell Motility and Invasiveness Invasion Migration Blockade of Cell Surface MTDH Impedes Migration and Invasion Invasion Migration M 1426725 Knockdown of MTDH Inhibits Pulmonary Metastasis of Osteosarcoma Cells A C B D IHC-MTDH N 1427271 Targeting liposomal nanoparticles to a mouse model of osteosarcoma Vaghasia et al UCLA, Los Angeles Federman N. Targeting liposomes toward novel pediatric anticancer therapeutics. Pediatr Res. 67(5), 514-9 ALCAM (CD 166) has been previously shown to be highly expressed in osteosarcoma cell lines Previous in vitro work demonstrated targeting and improved cytotoxicity in ALCAM targeted nanoparticles when compared to a conventional liposomal Doxorubin. Federman N. Enhanced growth inhibition of osteosarcoma by cytotoxic polymerized liposomal nanoparticles targeting the alcam cell surface receptor. Sarcoma. 2012; 2012:126906. N 1427271 Osteosarcoma cell lines KHOS and MNNG were transduced with a CMV-luciferase-puromycin lentiviral plasmid N 1427271 O 1433633 Prediction of a patient survival by MMP1 and P16 immunoreactivity In osteosarcoma tumour samples before and after neoadjuvant chemotherapy Robl et al Zurich Switzerland Methods: Tissue micro array (TMA): samples of 86 patients, taken before (biopsy, Bx) and after neoadjuvant chemotherapy (resection, Rx) to analyze 36 known tumor marker proteins Only non-necrotic tissue sections were graded (controlled by a pathologist) semi-quantitative grading performed in MATLAB1 Mean values of grades (Bx and Rx) were used for further analysis: downregulation / no change / upregulation “proportion of change” (PoC) = (downregulation + upregulation) / total # of samples 1Ruifrok, AC, Johnston, DA. (2001). Anal Quant Cytol Histol, 23:291-299. O 1433633 Results 30 P16 Neuropilin2 Irx4 CXCR7_11G8 CCN1 MMP1 CD44 CXCR4 Caprin Pten Vimentin • • • • All markers showed a change in expression before and after neoadjuvant chemotherapy The PoCs ranged from 3 to 87 % based on samples fulfilling all criteria: -Bx and Rx available -no metastasis at diagnosis -neoadjuvant chemotherapy Number of Patients • Downregulation No change Upregulation 25 20 15 10 5 0 PoC (87%) (84%) (79%) (77%) (73%) (73%) (69%) (67%) (67%) (67%) Fig. 1: Markers ranked according to the largest PoC in immunoreactivity (Bx vs Rx). (59%) O 1433633 Results • If correlated with 5-YS (overall five-year survival) only expression changes of P16 (P=0.044) and MMP1 (P=0.012) showed a significant correlation P16-immunoreactivity (Ntotal = 37 patients) MMP1-immunoreactivity (Ntotal = 33 patients) 20 15 10 5 0 25 deceased alive Number of patients Number of patients 25 20 deceased alive 15 10 5 0 Fig. 2: Change in immunoreactivity (Bx vs. Rx) of P16 and MMP1 in non-metastatic OS patients related to patient status after five years. P 1425094 Micro RNA expression profiling indentifies mir-15B as a regulator of multi-drug resistance in human osteosarcoma cell lines Duann et al MGH, Boston Values of expression P<0.01 KHOS KHOSR2 U-2OS U-2OSMR SKOV-3 SKOV-3TR P 1425094 U-2OSMR Absorbance U-2OSMR+ miR-15b 1nM U-2OSMR+ miR-15b 5nM U-2OSMR+ miR-15b 10nM U-2OSMR+ miR-15b 20nM Doxorubicin (μM) U-2OSMR Absorbance U-2OSMR+ nonspecific miR precursor 5nM U-2OSMR+ nonspecific miR precursor 10nM U-2OSMR+ nonspecific miR precursor 20nM Doxorubicin (μM) P 1425094 Wee1 target gene miR-15b precursor (nM) KHOSR2 U-2OSMR 0 1 5 10 20 miR- precursor nonspecific control 40 10 20 40 Wee1 98kD Actin 42kD Wee1 98kD Actin 42kD microRNA Thank you to all the speakers, poster presenters and organisers