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ERB1-B4, NA17 and MAGE gene expression analysis in 83 soft-tissue and bone tumors

ERB1-B4, NA17 and MAGE gene expression analysis in 83 soft-tissue and bone tumors. S. Piperno-Neumann, P. de Cremoux, G. de Pinieux, P.Anract, D.Robert, C.Tran-Perennou, A. Babinet, V.Laurence, B. Tomeno, P. Pouillart, O. Lantz Institut Curie and Hopital Cochin, Paris, France.

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ERB1-B4, NA17 and MAGE gene expression analysis in 83 soft-tissue and bone tumors

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  1. ERB1-B4, NA17 and MAGE gene expression analysis in 83 soft-tissue and bone tumors S. Piperno-Neumann, P. de Cremoux, G. de Pinieux, P.Anract, D.Robert, C.Tran-Perennou, A. Babinet, V.Laurence, B. Tomeno, P. Pouillart, O. Lantz Institut Curie and Hopital Cochin, Paris, France

  2. Sarcoma patients: 20-30% poor clinical outcome - limited role for conventional therapies - need for new (biological) strategies Sarcoma: good model for immunotherapy - cytogenetic translocations→fusion proteins - tumor antigens recognized by CTLs, classified in differentiation Ag : restricted distribution to specific organ or tissue type : tyrosinase, Melan-A , gp-100 in melanocytes and melanoma tumor-associated Ag : expressed in tumor cells, silent in normal cells except testis: MAGE, BAGE, LAGE, NA17... INTRODUCTION

  3. MAGE-A family genes - Chr X-linked genes - Activated in melanoma, lung, head and neck, bladder cancer - 14-24% MAGE mRNA positive in sarcomas (Van den Eynde,1997) - Protein expression : Mage 3 mAb 57B (Jungbluth, 2000, 2002) 7/10 -14/22 Synovial sarcoma 0/16 LMS, LPS, MFH • NA 17 - Cryptic promoter leading to the transcription of an intronic sequence of Gn T-V gene (N-acetyl glucosaminyl transferase V gene) - Expressed in 50% cutaneous /99% uveal melanoma - Never been reported in sarcoma

  4. MATERIALS • 83 tumor samples from March 2002 to March 2005 23 bone sarcomas 38 soft-tissue sarcomas 22 mesenchymal benign tumors • Tumor sample status 48 surgical biopsies 31 surgical resections 4 post-chemotherapy resections

  5. METHODS(1) • Aliquotes were immediately immersed in RNA later® before storage at 4°C, then frozen in liquid nitrogen • RNAs were extracted using guanidium isothiocyanate method • cDNAs were prepared using Superscript II reverse transcriptase (Life Technology, Inc) • Then PCR was realised • First, semi-quantitative RT-PCR for MAGE gene analysis • Real-time quantitative PCR analysis (Taqman 7700) on the available cDNAs for erbB1 to erbB4 genes • Results were expressed as a percentage of a standard curve of cDNA obtained from reference melanoma cell lines (SK 23, MZ2, LB23, LB373-Ludwig Institute), and ΔΔCt for erbB analysis.

  6. METHODS(2) • Retrospective analysis • Central histological review - tumoral tissue evidence in tested samples - confirmation of diagnosis - type, subtype and grade classification according to FNCLCC criteria • Clinical data patient age, sex, tumor site, metastatic status at diagnosis

  7. Sex M/F 46/37 Metastatic disease 18 Primary Bone Sarcomas 23 Osteosarcoma 8 Chondrosarcoma 6 PNET/Ewing 6 Leiomyosarcoma 2 Indifferenciated 1 Benign Tumors 22 Bone 11 Soft tissue 11 Median age (yrs) 46 (15-83) Tumor site : extremities 66/axial 17 Soft tissue sarcomas 38 Liposarcoma 8 Leiomyosarcoma 6 Synovial sarcoma 5 Fibrosarcoma 2 Pleomorphic 8 Desmoid Tumor 4 Rhabdomyosarcoma 2 MPNST 1 Clear-cell sarcoma 1 Epithelioid sarcoma 1 PATIENT AND TUMOR CHARACTERISTICS (N=83)

  8. RESULTS (1) Frequency of tumor-specific antigens by multimarker RT-PCR

  9. COMMENTS • 34/61 sarcoma samples (56%) : heterogeneous expression of at least one NA 17 or MAGE gene • ≥3 genes expression ● 39% bone sarcomas 24% soft-tissue sarcomas 5% benign tumors ● 75% osteosarcomas /50% liposarcomas ● 28% non metastatic /39% metastatic sarcoma patients • Low NA 17 mRNA expression level in sarcomas

  10. RESULTS(2) High expression level of erb B1-B4 mRNA : results are classified by quartile (0, +, ++, +++) ++ and +++ are considered as high expression.

  11. COMMENTS • 8/12 STS : erbB1 alone or with erbB2 /B4 fibrosarcoma 3/4 erbB1 liposarcoma 2/3 erbB2-B4 synovial sarcoma 2/3 erbB1-B2 • Bone sarcoma : 3/7 erbB1 osteosarcoma 2/2 erbB1 Ewing 0/3 chondrosarcoma 1/2 erbB1

  12. CONCLUSION • This work contributes to define the expression profile of multiple biological markers that provide target antigens for specific immunotherapy in sarcomas. • MHC class-I antigens loss of expression in sarcomas ? • Correlation with protein expression and gene expression profiles? • From a clinical point of view, our data suggest that osteosarcoma and liposarcoma patients could be eligible for a future vaccination trial combining Mage peptides and immunological adjuvant.

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