Sensitive detection of FIP1L1-PDGFRA fusion transcripts by real-time quantitative RT-PCR (RQ-PCR) reveals achievement of molecular remission in chronic eosinophilic leukemia treated with low-dose imatinib therapy

The FIP1L1-PDGFRA fusion gene generated by a cryptic interstitial deletion at 4q12 is a recurrent molecular lesion in idiopathic hypereosinophilic syndrome (HES), that forms a basis for diagnosis of chronic eosinophilic leukemia (CEL). This disease entity is particularly important to recognize, since presence of the FIP1L1-PDGFRA fusion predicts a favorable response to molecularly targeted therapy in the form of imatinib, with clinical remissions being achieved with lower doses than are required in BCR-ABL+ chronic myeloid leukemia (CML). In order to improve our understanding of the biology of CEL and to provide a tool to improve the management of patients with this disorder we have developed real-time quantitative reverse transcriptase PCR (RQ-PCR) assays for the FIP1L1-PDGFRA fusion. Taking into account the marked heterogeneity in upstream breakpoints within the FIP1L1 gene, RQ-PCR assays were designed for cases with FIP1L1 breakpoints leading to fusion of exon 9, 10, 11, 12 or 13 to PDGFRA exon 12. FIP1L1-PDGFRA expression was compared to that of validated Europe Against Cancer endogenous control genes - ß2microglobulin (B2M) and ABL. Serial dilution of the FIP1L1-PDGFRA+ EOL1 cell-line in fusion gene negative filler cells (HL60) revealed an assay sensitivity of 1 in 105. While identification of the FIP1L1-PDGFRA fusion using conventional RT-PCR approaches can be problematic, RQ-PCR analysis undertaken in diagnostic samples from 31 patients with FIP1L1-PDGFRA+ CEL (median age 53, 31–64 years) revealed that, in the majority, the fusion transcript was expressed at relatively high level (median deltaCt FIP1L1-PDGFRA - B2M, 12.2; median deltaCt FIP1L1-PDGFRA - ABL, 2.3). The FIP1L1-PDGFRA fusion was expressed at comparable level in blood and marrow at diagnosis of CEL. Serial monitoring was undertaken in 17 patients following initiation of imatinib 100mg/d. Overall, 8/8 evaluable patients achieved at least a 3-log reduction in FIP1L1-PDGFRA fusion transcript level within the first year of therapy. In follow-up samples affording a sensitivity of at least 1 in 1000, PCR negativity by quantitative and conventional nested RT-PCR was documented in 8/17 patients following a median of 21 weeks of imatinib (4–64 weeks); in two cases profound PCR negativity (i.e. at a sensitivity level of at least 1 in 105) was documented following 13 weeks and 2 years of imatinib, respectively. Overall, these data demonstrate that CEL with the FIP1L1-PDGFRA fusion is uniquely sensitive to imatinib therapy. This contrasts with BCR-ABL+ CML, in which molecular remission is generally not achieved - a phenomenon that has been postulated to reflect the persistence of a primitive quiescent stem cell population that is resistant to this agent. Understanding the biological basis for the differences in molecular response to imatinib in CML and CEL, may yield further improvements in molecularly-targeted therapeutic approaches