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Expression of Groucho / Transducin-Like Enhancer of split protein distinguishes synovial sarcoma

Expression of Groucho / Transducin-Like Enhancer of split protein distinguishes synovial sarcoma from malignant peripheral nerve sheath tumor. TO Nielsen, BP Rubin, CC Ruttan, S Liu, S Subramanian, M van de Rijn.

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Expression of Groucho / Transducin-Like Enhancer of split protein distinguishes synovial sarcoma

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  1. Expression of Groucho / Transducin-Like Enhancer of split protein distinguishes synovial sarcoma from malignant peripheral nerve sheath tumor TO Nielsen, BP Rubin, CC Ruttan, S Liu, S Subramanian, M van de Rijn University of British Columbia, University of Washington, and Stanford University

  2. SS gene expression data • Body segmentation • Retinoic acid metabolism • Mesenchymal (incl. nerve) differentiation • Epithelial differentiation • Proliferation genes: Wnt pathway • Receptor tyrosine kinases: EGFR, FGFR3

  3. Differential Diagnosis on small biopsy: cellular, non-pleomorphic spindle cell neoplasm • Fibrous • Fibrosarcoma • cellular Fibromatosis • cellular SFT (HPCT) • Nerve Sheath • MPNST • cellular Schwannoma • Smooth Muscle • Leiomyosarcoma • Other Entities • metaplastic Carcinoma • Melanoma • Monophasic SS • EWS / PNET

  4. SALL2: (+) 88% of SS 80% MPNST Immunomarkers to distinguish Synovial Sarcoma from Malignant Peripheral Nerve Sheath Tumor keratins (+) 65% of SS 20% MPNST nuclear β-catenin: 50% of SS 0% MPNST

  5. Molecular testing • options incl. cytogenetics, RT-PCR, FISH • slower, more expensive; specialized centres Immunohistochemistry • no IHC stains are both highly sensitive and specific • focal staining for keratins and EMA helpful -- but often not present on core bx, and can be found in MPNST

  6. SAM (supervised) analysis on 42k cDNA microarrays : best discriminators of SS (N=16) vs. MPNST (N=32) TLE1 (transducin-like enhancer of split-1), a transcriptional repressor of Wnt/-catenin signaling

  7. Identification of potential target genes of SYT-SSX based on strong differential overexpression in the expression profile of synovial sarcoma (Affy chips) Saito T and Ladanyi M (MSKCC) CTOS Nov 2004 Top 5 ranked by p-value

  8. “TLE1, WNT5A, FZD1, and TLE4 were ranked among the top 50 discriminating genes for the synovial sarcoma …” TLE in SS: support from other expression profiling studies “Synovial sarcomas were characterized by genes expressed in early developmental pathways involving WNT and notch signaling, including TLE1, FZD1, WNT5A, and JAG2.”

  9. anti-TLE antibodies • 10 commercial antibodies identified, all polyclonal and none rated to work for IHC • Antibody from original paper describing TLE family (Stifani S et al Nature Genet 1992; 2:119-127) obtained from Dr. Stefano Stifani, MNI, McGill University • C597.4A = Rat monoclonal anti-human TLE, recognizing C-terminal WD40 domain (90% AA identity across TLE 1-4)

  10. Score 1 (weak) <50% of cells with moderate or weak nuclear stain Score 3 (strong) >50% of cells with intense nuclear staining Staining conditions: 1:2 dilution, Ventana automated stainer, HIER in EDTA pH 8.0 Score 2 (moderate) intense in 10-50% or well above background in >50% Score 0 (negative)

  11. Sarcoma Tissue Microarrayscollaboration between UBC, Stanford, UW, CCF, OHSU Total cases arrayed: 839 (including 73 SS and 88 MPNST) New TMA: MPNSTs & related tumors

  12. PRIMARY IMAGES POSTED AT- www.gpecimage.ubc.ca pan-TLE mAb gives intense and/or diffuse nuclear staining in 59/71 (83%) of synovial sarcoma cases

  13. Even moderate nuclear staining for TLE is very rare in MPNST (4 / 88 = 4.5%)

  14. TLE on nerve sheath TMA

  15. TLE on Synovial Sarcoma TMA

  16. TLE immunostaining: summary

  17. FLY HUMAN “Transducin-Like”: has WD40 protein interaction domain Notch membrane TLE1 TLE2 TLE3 TLE4 “Enhancer of split”: domains with 70-90% AA identity to groucho cleaved, activated Notch cyto- plasm transduces cell-cell interaction signals, inhibiting cell fate decisions Transducin-like (groucho) bHLH TF (Hes) Enhancer of split mastermind nucleus

  18. TLE: a transcriptional corepressor SYT β-catenin TLE recruits, assembles repressor complex

  19. Synovial sarcoma “green genes” may be inhibited by TLE

  20. The HDAC inhibitor depsipeptide is more effective than doxorubicin against synovial sarcoma apoptosis & necrosis in spheroid assays, > doxorubicin Growth inhibition in subQ xenograft mouse models

  21. CONCLUSIONS TLE for diagnosis: sensitive and specific for SS TLE in SS oncogenesis: supports a central role for transcriptional repressors of differentiation Implications of TLE for SS treatment: supports preclinical data on value of HDAC inhibitors

  22. ACKNOWLEDGEMENTS • McGill University • Stefano Stifani • Cleveland Clinic • Foundation • Erinn Downs-Kelly • John Goldblum • University of • British Columbia • Wanda Kwan • Joanna Lubieniecka • Neal Poulin • Oregon Health • Sciences University • Chris Corless Funded by the Terry Fox Foundation

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