ERB1-B4, NA17 and MAGE gene expression analysis in 83 soft-tissue by 03qZ8Xyu

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									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
                      INTRODUCTION
   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...
   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
                                transcription of an intronic sequence
- Cryptic promoter leading to the
  of Gn T-V gene (N-acetyl glucosaminyl transferase V gene)
- Expressed in 50% cutaneous /99% uveal melanoma
- Never been reported in sarcoma
                     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
                   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.
                   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
               PATIENT AND TUMOR
              CHARACTERISTICS (N=83)
   Sex       M/F        46/37      Median age (yrs)         46 (15-83)
   Metastatic disease   18         Tumor site : extremities 66/axial 17


Primary Bone Sarcomas 23         Soft tissue sarcomas        38
Osteosarcoma               8     Liposarcoma                 8
Chondrosarcoma             6     Leiomyosarcoma              6
PNET/Ewing                 6     Synovial sarcoma            5
Leiomyosarcoma             2     Fibrosarcoma                2
Indifferenciated           1     Pleomorphic                 8
                                 Desmoid Tumor               4
                                 Rhabdomyosarcoma            2
Benign Tumors              22    MPNST                       1
Bone                       11    Clear-cell sarcoma          1
Soft tissue                11    Epithelioid sarcoma         1
                                      RESULTS (1)
                         N    NA 17 Mage 1 2      3   4   6    10   C2 Lage 1 2          1 gene   >3 genes

SARCOMAS                 61                                                             34(56%) 18(30%)

Bone sarcomas            23    11      6      6   7 4     6    6     6      3       6   15(65%)   9(39%)

Osteosarcoma              8     5      4      5   5 3     4    4     4      2       4   7(87%)    6(75%)

Ewing                     6     2      -      -   -   -   -     -    -      -       -   2(33%)       0

Soft-tissue sarcomas     38    15      4      5   8 6     4    5     3      4       10 19(50%)    9(24%)

LPS                       8     3      1      1   1 2     -    2     -      4       4   4(50%)    4(50%)

LMS                       6     2      -      -   1 -     -     -    -      -       -   2(33%)       0

BENIGN TUMORS            22     4      3      -   2 -     -     -    2      -       -   8(36%)     1(5%)



                       Frequency of tumor-specific antigens by multimarker RT-PCR
                      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
                                 RESULTS(2)

                            N         erb B1          erb B2           erb B3          erb B4
Sarcomas                    19           11               6                -               3
Bone                         7            3                -               -               -
Soft-tissue                 12            8               6                -               3
Benign tumors               12            3               7                1               -
Bone                         9            3               5                -               -
Soft-tissue                  3            -               2                1               -


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