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

SS gene expression data•Body segmentation•Retinoic acid metabolism

•Mesenchymal (incl. nerve) differentiation

•Epithelial differentiation

•Proliferation genes: Wnt pathway

•Receptor tyrosine kinases: EGFR, FGFR3

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

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

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

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

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

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.”

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)

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

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

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

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

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

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

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

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

TLE recruits, assembles repressor complex

SYT

TLE: a transcriptional corepressor

β-catenin

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

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

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

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