Analysis of PRAME Protein Expression in Normal Lymphoid Tissues and during B Ontogenesis.

PRAME (Preferentially Expressed Antigen in Melanoma) gene was originally isolated in melanoma. A significant increase in the number of PRAME transcripts has been demonstrated in hematologic malignancies such as acute myeloid and lymphoid leukemias, multiple myeloma and chronic lymphoproliferative diseases. Furthermore, our group generated an anti-PRAME monoclonal antibody (MoAb) and by quantitative flow cytometry has demonstrated that PRAME protein was aberrantly expressed in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma. However, the expression of this antigen in normal lymphoid tissues and during B cells ontogeneis has not been characterized. To address this question, PRAME protein expression was studied by flow cytometry in peripheral blood (PB, n=15) and bone marrow (BM, n=6) from healthy donors, lymphonodes (n=4) and spleen (n=4) from patients submitted to lymphonode excision or splenectomy for non malignant diseases. First, we determined in which hematopoietic lineage PRAME was expressed by concomitantly staining PB, BM, lymphonode and spleen mononuclear cells (MCs) with anti-PRAME and a panel of MoAbs specific to B(CD19)/ T(CD3)/ NK(CD16/56), monocytic(CD14) and granulocytic(CD33) markers. PRAME was detected exclusively in CD19⁺ cells. The median percenatge of PRAME positive cells was 5,31% (2,55–12,34%), 13,01% (8,47–38,15%), 12,79% (3,15–23,06%) and 17,5% (12,67–27,43%) in PB, BM, lymphonode and spleen MCs, respectively. Amongst CD19⁺ cells, we have observed that PRAME was expressed by 42,39% (16,16–75,72%), 16% (13–69,5%), 15,16% (5,49–41,20%) and 48,82%(12,67–58,89%) in PB, BM, lymphonode and spleen, respectively. To establish in which stage of B ontogenesis PRAME was expressed on, cell suspensions stained with anti-CD19 were submitted to positive magnetic separation and labeled with anti-PRAME, CD5, CD27, CD38, CD34, CD10 and IgD MoAbs. PRAME⁺/CD19⁺ cells were CD5⁻, CD27⁺, CD38⁺, CD34⁻, CD10⁻ and IgD⁺, thus suggesting that PRAME is expressed by the memory B cell compartment of the normal lymphoid tissues. This study defines PRAME as a B cell antigen that may accompany the neoplastic clone proliferation of mature B cell neoplasms. Although PRAME is mainly an embryonic antigen, expressed by carcinomas of immature phenotype, it is expressed by mature B cells in normal and pathological lymphoid tissues. Our findings suggest that maturational events occurring at the germinal center of lymphoid follicles affects PRAME expression.