Discriminative HMGA2 Isoform Expression in CD15+ Granulocytic Cells in Myeloid Metaplasia with Myelofibrosis (MMM).

MMM is a myeloproliferative disorder characterized by extramedullary hematopoiesis and reactive myelofibrosis. Recently, HMGA2 dysregulation has been demonstrated in 2 MMM patients showing 12q15 rearrangement and confirmed in 25 consecutive MMM patients without cytogenetic abnormalities (Andrieux, 2004). HMGA2 proteins belong to the high mobility group A (HMGA) family of architectural transcription factors regulating the expression of several genes. As MMM is a clonal disorder of CD34⁺ hematopoietic progenitors, we analyzed HMGA2 expression in peripheral blood sub-populations of 5 MMM patients and 7 healthy donors to determine in which sub-population HMGA2 was dysregulated.

RNA was extracted from peripheral blood mononuclear cells (PBMC) and CD15⁺ granulocytic cells (PBCD15⁺) separated through Ficoll centrifugation or from immunomagnetically selected circulating CD34⁺ cells (PBCD34⁺). Real-time quantitative PCR (RQ-PCR) using Taqman technology was performed on cDNA. As different isoforms were described in malignancies, we used two primer sets : the first one allowing the amplification of all HMGA2 isoforms (exon 1 to 3) (HMGA2 1–3), the second one allowing the amplification of the full length HMGA2 isoform (exon 1 to 5)(HMGA2 1–5).

In healthy donors and in MMM, PBMC HMGA2 expression levels were heterogeneous, depending of the cellular sub-population purity. HMGA2 1–3 or HMGA2 1–5 were both expressed in MMM and normal PBCD34⁺ cells, but with a higher expression level for HMGA2 1–3 as compared to HMGA2 1–5. Furthermore, both HMGA2 1–3 and HMGA2 1–5 expression levels were significantly increased in PBCD34⁺ MMM patients (p<10⁻⁶) compared to healthy donors. In MMM, HMGA2 expression level was significantly increased (p<10⁻⁵) in PBCD15⁺ as compared to PBCD34⁺. Moreover, PBCD15⁺ HMGA2 1–3 expression level was significantly higher in MMM patients compared to PBCD15⁺ from healthy donors (p<10⁻⁷). A persistence of HMGA2 1–5 expression was only observed in MMM PBCD15⁺ but was undetectable neither in normal PB neutrophils (purity>98%) nor in PB neutrophils from other myeloproliferative disorders (Polycythemia Vera and Essential Thrombocythemia).

To determine if HMGA2 level was modified during hematopoietic differentiation, we quantified HMGA2 1–3 and 1–5 isoform expression on purified healthy donor PB CD34⁺ and MMM CD34⁺ before and after culture with specific lineage growth factors (12 day-culture). Primary results showed that both HMGA2 isoform expression levels were higher during granulocytic differentiation. Our results demonstrate that HMGA2 1–5 isoform is discriminately overexpressed in MMM PBCD15⁺. The persistence of this HMGA2 full length expression in MMM myeloid lineage could be considered as a marker of the disease.