1. Friday 16 th 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

2. Osteosarcoma Genomics- Highly Complex

3. The HALLMARKS

4. 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

5. RNA Interference miRNA Families and targets

6. 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

7. 24 OS samples 108 commonly expressed miRNAs in OS samples Identification of miRNA targeted protein Identification of miRNAs regulating OS cell proliferation microRNA microarray analysis Anti-miR inhibitors library, MTT assay In 8 OS cells Proteomic approach (2DICAL) In U2 cells Flow diagram 515 miRNAs 108 miRNAs Number of miRNAs (n=939) Global expression of miRNAs in OS samples L 1425144

8. Change of ATP production (%) Inhibited miRNA Survey of miRNAs regulating OS cell proliferation L 1425144

9. Serpin H1- Vimentin- 1,093.924m/z (1,093.824~1,094.024) Vimentin  -Actin- Serpin H1 let-7alet-7flet-7glet-7iNC1NC2let-7alet-7flet-7glet-7iNC1NC2 let-7a let-7f let-7g let-7i NC1 NC2 let-7a let-7f let-7g let-7i NC1 NC2  -Actin- Identification of proteins regulated by let-7a family miRNAs

10. 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 C B

11. MTDH Knockdown Decreases Cell Motility and Invasiveness Blockade of Cell Surface MTDH Impedes Migration and Invasion InvasionMigration InvasionMigration M 1426725

12. Knockdown of MTDH Inhibits Pulmonary Metastasis of Osteosarcoma Cells AB CD IHC-MTDH M 1426725

13. 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.

14. Osteosarcoma cell lines KHOS and MNNG were transduced with a CMV-luciferase-puromycin lentiviral plasmid N 1427271

16. 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 MATLAB 1 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 1 Ruifrok, AC, Johnston, DA. (2001). Anal Quant Cytol Histol, 23:291-299.

17. 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 Results Fig. 1: Markers ranked according to the largest PoC in immunoreactivity (Bx vs Rx). Vimentin Pten Caprin CXCR4 CD44 MMP1 CCN1 CXCR7_11 G8 Irx4 Neuropilin2 P16 PoC (87%) (84%) (79%) (77%) (73%) (73%) (69%) (67%) (67%) (67%) (59%) O 1433633

18. 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 Fig. 2: Change in immunoreactivity (Bx vs. Rx) of P16 and MMP1 in non-metastatic OS patients related to patient status after five years. Down- regulation No change Up-regulation No change Results O 1433633

19. 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 KHOS U-2OS KHOS R2 U-2OS MR P<0.01 KHOS KHOS R2 U-2OS U-2OS MR SKOV-3 SKOV-3 TR Values of expression

20. Absorbance Doxorubicin (μM) U-2OS MR U-2OS MR + miR-15b 1nM U-2OS MR + miR-15b 5nM U-2OS MR + miR-15b 10nM U-2OS MR + miR-15b 20nM Doxorubicin (μM) Absorbance U-2OS MR U-2OS MR + nonspecific miR precursor 5nM U-2OS MR + nonspecific miR precursor 10nM U-2OS MR + nonspecific miR precursor 20nM P 1425094

21. Wee1 98kD 0 1 5 10 20 40 miR-15b precursor (nM) U-2OS MR 10 20 40 miR- precursor nonspecific control Actin 42kD Wee1 98kD Actin 42kD KHOS R2 P 1425094 Wee1 target gene

22. microRNA

23. Thank you to all the speakers, poster presenters and organisers