Novel PDGFRA Point Mutations in Hypereosinophilic Syndrome Induce Growth Factor Independence and Leukemia in Vitro and In Vivo.

Abstract 767

Considerable progress has been achieved in our understanding of the pathogenesis of hypereosinophilic syndrome (HES) and chronic eosinophilic leukemia (CEL) by identification of constitutively activated tyrosine kinase fusion genes, e.g. FIP1L1-PDGFRA or ETV6-PDGFRB. However, the overall incidence of those fusion genes in HES/CEL is below 15%, and the molecular pathogenesis of the remaining cases remains elusive. We therefore established generic quantitative RT-PCR assays (RQ-PCR) to detect overexpression of 3'-regions of PDGFRA or PDGFRB as a possible indicator of an underlying fusion gene or point mutation. Patients with known fusion genes involving PDGFRA (n=5, 51 patients) or PDGFRB (n=5; 7 patients) showed significantly increased normalized expression levels compared to 191 patients with fusion gene-negative eosinophilia or healthy individuals (PDGFRA/ABL: 0.73 vs. 0.0066 vs. 0.0064, p<0.0001; PDGFRB/ABL: 196 vs. 3.8 vs. 5.85, p<0.0001). In all patients with significantly increased expression levels who were negative for fusion genes, functionally relevant regions of PDGFRA were sequenced. Several novel mutations (R481G, I562M, H570R, M628T, L705P, G729D) as well as a double mutation (H650Q and R748G) were identified. When cloned into 32D cells, M628T, H650Q, and R748G mutants separately induced growth factor-independent proliferation and clonogenic growth, and this was associated with constitutive phosphorylation of downstream targets STAT5, ERK, and AKT. Low doses of imatinib antagonized all of these effects in vitro. M628T and R748G but not H650Q 32D cell mutants induced acute leukemia after injection into congenic C3H/HeJ mice, similar to FIP1L1-PDGFRA. Interestingly, these two mutants showed a significantly higher propensity to invade the lymph nodes than the FIP1L1-PDGFRA fusion. Oral administration of imatinib to injected mice significantly decreased leukemic growth in vivo and significantly prolonged survival of the recipients. In conclusion, we demonstrate that novel point mutations of the PDGFRA gene found in patients with HES/CEL induce growth factor independence and leukemia in vitro and in vivo and suggest that these patients may benefit from treatment with imatinib.

*CE, PE, AR, and SK contributed equally to this work.