Identification of a binding site on retinal transducin alpha for the ...
Expression of Groucho / Transducin-Like Enhancer of split protein distinguishes synovial sarcoma
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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
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SS gene expression data•Body segmentation•Retinoic acid metabolism
•Mesenchymal (incl. nerve) differentiation
•Epithelial differentiation
•Proliferation genes: Wnt pathway
•Receptor tyrosine kinases: EGFR, FGFR3
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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
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SALL2: (+) 88% of SS80% MPNST
nuclear β-catenin:50% of SS0% MPNST
keratins (+) 65% of SS 20% MPNST
Immunomarkers to distinguish Synovial Sarcoma
from Malignant Peripheral Nerve Sheath Tumor
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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
Molecular testing
• options incl. cytogenetics, RT-PCR, FISH• slower, more expensive; specialized centres
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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
1 ZIC2 || Zic family member 2 (odd-paired homolog, Drosophila) || Hs.369063 || 117460
2 SSX1 || Synovial sarcoma, X breakpoint 1 || Hs.434142 || 331190
3 RIPK4 || Receptor-interacting serine-threonine kinase 4 || Hs.517310 || 100073
4 EFNB3 || Ephrin-B3 || Hs.26988 || 103884
5 TACSTD1 || Tumor-associated calcium signal transducer 1 || Hs.692 || 318393
6 TLE1 || Transducin-like enhancer of split 1 (E(sp1) homolog, Drosophila) || Hs.197320 || 318320
7 TACSTD1 || Tumor-associated calcium signal transducer 1 || Hs.692 || 108810
8 CRA || Myotubularin related protein 11 || Hs.425144 || 113187
9 HSRG1 || MON1 homolog B (yeast) || Hs.513743 || 315796
10 SHANK2 || SH3 and multiple ankyrin repeat domains 2 || Hs.268726 || 225192
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Identification of potential target genes of SYT-SSX based on strong differential overexpression in the
expression profile of synovial sarcoma (Affy chips)
Top 5 ranked by p-value
Saito T and Ladanyi M (MSKCC)CTOS Nov 2004
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TLE in SS: support from other expression profiling studies
“TLE1, WNT5A, FZD1, and TLE4 were ranked among the top 50 discriminating genes for the synovial sarcoma …”
“Synovial sarcomas were characterized by genes expressed in early developmental pathways involving WNT and notch signaling, including TLE1, FZD1, WNT5A, and JAG2.”
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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)
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Score 3 (strong) >50% of cells with intense nuclear staining
Score 0 (negative)
Score 1 (weak) <50% of cells with moderate or weak
nuclear stain
Score 2 (moderate) intense in 10-50% or well above background in >50%
Staining conditions: 1:2 dilution, Ventana automated stainer,
HIER in EDTA pH 8.0
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Sarcoma Tissue Microarrayscollaboration between UBC, Stanford, UW, CCF, OHSU
Total cases arrayed: 839 (including 73 SS and 88 MPNST)
MPNST (NF-1 patients) 44MPNST (sporadic) 24
neurofibroma (localized) 8neurofibroma (plexiform) 24
neurofibroma (diffuse) 11schwannoma (celluar) 7schwannoma (typical) 15
perineurioma 4melanoma (spindle cell) 10
clear cells sarcoma 5DFSP with fibrosarcoma 9
synovial sarcoma 15
New TMA: MPNSTs & related tumors
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pan-TLE mAb gives intense and/or diffuse nuclear staining in 59/71 (83%)
of synovial sarcoma cases
PRIMARY IMAGES POSTED AT- www.gpecimage.ubc.ca
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Even moderate nuclear staining for TLE is very rare in MPNST (4 / 88 = 4.5%)
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TLE on nerve sheath TMA
0 0 0 0 U 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 01 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 U 0 0 0 0 1 0 0 0 0 0 0U 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 1 1 1 0 0 0 0 0 U0 0 0 0 0 0 0 0 0 0 0 0 2 1 U 0 1 1 0 0 1 0 1 1 0 0 0 00 0 0 0 0 0 1 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 1 0 0 0 01 1 U 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 2 1 1 2 2 0 00 0 1 1 0 0 2 1 1 0 1 1 0 0 0 0 0 0 U 1 0 0 X 0 1 1 1 U1 1 0 0 0 0 0 0 0 0 0 0 U 0 0 0 1 1 0 1 X U 0 0 0 0 1 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 M M 0 0 0 0 *3* *3* 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 U 1 U 0 0 0 0 0 0 1 1 1 1 1 1 2 2 2 2 1 1
1 2 2 2 0 0 1 1 1 1 1 1 0 02 1 *3* *3* U U 2 2 *3* *3* *3* *3* 2 *3*
MPNSTs (NF1-associated and sporadic) *3* *3* *3* *3* *3* *3* *3* *3* 2 U *3* *3* 2 2neurofibromas (localized, plexiform and diffuse) *3* X X *3*schwannomas (cellular and typical)spindle cell malignancies (clear cell sarcoma, melanoma, DFSP/FS)synovial sarcoma
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TLE on Synovial Sarcoma TMA
2 2 2 1 X *3* U *3* U 0 synovial sarcoma 0 0 0 0 0*3* 2 *3* *3* *3* *3* *3* U U 0 other sarcomas: 0 0 1 *3* 02 1 0 X U *3* *3* X X U EWS,LMS, LGFMS 2 2 2 2 2
*3* *3* *3* *3* U U 2 *3* 2 *3* MPNST 0 1 2 2 2SFT & HPCT
1 1 *3* *3* U U U 0 X *3* 0 0 0 0 02 *3* *3* *3* X 2 2 X X 0 0 0 0 0 02 2 1 2 *3* *3* U X X *3* 0 U 0 0 U2 2 0 1 *3* M 2 2 *3* 0 0 0 0 0
*3* *3* *3* 1 U 2 2 2 2 U 0 0 0 0*3* *3* 2 2 U 0 0 0 0 0*3* *3* *3* *3* M 1 1 0 0 *3*
0 0 X
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Usually positive for TLE: NO STAINING SEENSynovial sarcoma 59/71 (83%) MFH (57)
neurofibromas (43)liposarcomas (34)
Occasionally positive for TLE: DFSP (17)(typical) schwannoma: 5/16 (31%) fibromatosis (16)cellular schwannoma: 2/7 (29%) rhabdomyosarcomas (14)SFT-HPCT: 5/20 (25%) angiosarcomas (12)
endometrial stromal sarcomahemangioendothelioma
Rarely positive for TLE: melanomaEwing sarcoma: 1/6 granular cell tumorCarcinosarcoma: 1/7 glomusclear cell sarcoma: 1/8 myxomaGIST: 2/35 leiomyomaMPNST: 4/88 osteosarcomaleiomyosarcoma: 1/41 LGFMS
TLE immunostaining: summary
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FLY HUMANNotchmembrane
cyto-plasm
Enhancerof split
mastermind
cleaved, activated
Notch
nucleus
TLE1TLE2TLE3TLE4
Transducin-like(groucho)
bHLH TF (Hes)
“Transducin-Like”: has WD40 protein interaction domain
“Enhancer of split”: domains with 70-90% AA identity to groucho
transduces cell-cell interaction signals, inhibiting cell fate decisions
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TLE recruits, assembles repressor complex
SYT
TLE: a transcriptional corepressor
β-catenin
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Symbol Gene name FunctionPresence of binding sites
in promoter
RUNX1 Hes1 TCF/LEF
Fer1L3 myoferlin membrane regeneration and repair YES YES noCTSS cathepsin S surface display of MHC II in myoblasts YES YES YESITGB2 β2 integrin cell adhesion-mediated signaling YES no YES
IGALS9 galectin 9 cell adhesion, differentiation; tumor suppressor YES YES YESHLA-DMA MHC II, DM α peptide loading of MHC class II YES YES YES
C7 complement 7 membrane attack complex YES no YESIL16 interleukin 16 pleiotropic cytokine YES no YES
RNASE6 RNase A k6 RNA degradation YES YES YESCD4 CD4 antigen T-helper cell surface YES YES YES
CYBB Cyt.b-245 phagocytic NADPH-oxidase YES YES YESCASP1 caspase 1 inflammation and apoptosis YES YES YESPARP14 ADPr pmase14 DNA repair; tags proteins YES YES YES
IFI 35 INF-induced35 Transcription factor YES YES YESARHGDIB Rho GDI β Rho signaling YES YES YESRARRES3 RARresponse3 tumor suppressor; antiproliferative YES YES YES
AIF1 allograft infl. 1 vascular smooth muscle cell migration YES YES YES
Synovial sarcoma “green genes” may be inhibited by TLE
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apoptosis & necrosis in spheroid assays, > doxorubicin
Growth inhibition in subQ xenograft mouse models
The HDAC inhibitor depsipeptide is more effective than doxorubicin against synovial sarcoma
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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
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Funded by the Terry Fox Foundation
ACKNOWLEDGEMENTS
University of British Columbia• Wanda Kwan• Joanna Lubieniecka• Neal Poulin
Cleveland Clinic Foundation• Erinn Downs-Kelly• John Goldblum
Oregon Health Sciences University• Chris Corless
McGill University• Stefano Stifani