PRAME Protein Is Aberrantly Expressed in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma.

Abstract

PRAME (Preferentially Expressed Antigen in Melanoma) gene is located on chromosome 22 (22q11.22) and is expressed at low levels by normal adrenal, ovarian and endometrial cells. In contrast, its expression has been demonstrated at high levels in several types of cancers, such as in acute myeloid and lymphoid leukemias and multiple myeloma . We recently have reported that PRAME is also expressed in lymphoproliferative diseases and its transcripts were detected in 26 out 58 patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Nevertheless, the expression of PRAME protein in normal and neoplastic tissues is unknown. In order to address this question, we produced a monoclonal antibody against PRAME protein. A 562-nt fragment of the PRAME cDNA that includes the region that codes for the immunogenic 9-peptide was clone in pCR2.1-TOPO vector in DH5 alpha cells and subcloned into a pET24a expression vector, and the 196-amino acid peptide was expressed in BL21(DE3) cells as His-taged protein. The monoclonal antibody (MoAb) against PRAME was generated from splenocytes from mice immunized with the purified peptide. We then analyzed PRAME expression by flow cytometry in samples of peripheral blood (PB, n=15), bone marrow (BM, n=3) from healthy donors; in tonsils (n=3) from patients submitted to tonsilectomy for non malignant diseases and in PB samples from 26 CLL and 7 MCL patients. PRAME positive cells represented less than 15% of cells from normal PB, BM and tonsil cells. In contrast, 25 out of 26 CLL and 6 out of 7 MCL cases presented more than 20% of PRAME + cells [mean 59% (range:20–95%) and 75% (20–94%) for CLL and MCL, respectively]. The lymphoid cells from normal BM and tonsils that expressed PRAME were CD19+CD10+ CD27+ CD38± TdT− cIgM− CD5− suggesting that PRAME is expressed early during lymphoid ontogenesis and that its expression in CLL and MCL cells is aberrant. Furthermore, PRAME expression in normal lymphocytes was dimmer than in CLL and MCL cells. To quantify PRAME expression we evaluated PRAME Specific Antibody Binding Capacity (SABC) by a quantitative flow cytometry (QFC) method. The mean values of SABC were of 10,339 sites per cell (range 3,075 to 24,665) and of 14,191 sites per cell (range 5,059 to 20,679) for CLL and MCL, respectively. In contrast, in normal PB lymphocytes SABC values ranged from 1,628 to 1,781 sites per cell. Even in the two cases of CLL and MCL that had less than 20% PRAME+ cells, PRAME SABC was 3,075 and 12,347 sites per cell respectively, therefore significantly higher than the observed in normal PB lymphocytes. To evaluate the sensitivity of the QFC method, we established a cut off value for PRAME SABC based on the highest value detected in normal PB cells and then serially diluted tumor MCL cells marked with PRAME in normal PB lymphocytes. Based exclusively on PRAME expression, we were able to detected neoplastic MCL cells up to a 1:1000 dilution. In conclusion, PRAME protein is strongly expressed in the neoplastic clone in most patients with CLL and MCL. This finding supports the suggestion that this antigen may be further explored as a target for diagnostic, to detect minimal residual disease detection and for therapeutic approaches.