Extreme Variability of FIP1L1-PDGFRalpha Transcripts In CEL: Analysis of 32 Patients Enrolled In HES0203 Italian Clinical Trial and Correlation with Clinical and Molecular Response After 5 Years Follow-up

Recent molecular studies of atypical Philadelphia negative chronic myeloproliferative disorders (CMPDs) have produced the identification of more than 40 fusion genes. The presence of FIP1L1-PDGFRA (F/P, cytogenetically invisible) have been associated more frequently with diagnosis of CEL. As this is the second most common fusion gene targeted by imatinib (IM) after BCR-ABL1, it is expected to monitoring response at molecular level as in CML, but from the analysis of major series of cases, it emerges that the diversity of F/P fusion transcripts is much more complex. We conducted a prospective phase II multicenter study of HES to explore the activity and safety of IM 400 mg in the treatment of patients affected by HES, apart from molecular status. A total of 33 patients with F/P were enrolled and now we report on the clinical and molecular follow up of 32 of them. We also report about the genomic aspects of the variability of fusion transcripts with some statistical correlations between molecular characteristics and phenotype of disease.

The primary end-point of the study was to assess the clinical anti-proliferative activity of IM in HES. Patients received IM 400 mg once daily for the first year, than the dose could be modified. In patients with F/P rearrangement, the presence of the transcripts was assessed and monitored by nested RT-PCR every 3 months until negativity, and then every 6 months. After RNA extraction, RT-PCR was performed and direct sequencing was done using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems) and an ABI PRISM 377 DNA Analyzer.

All 32 patients with F/P rearrangement had prompt responses to IM. Molecular and hematologic responses (MR, HR) were maintained with continuous therapy, molecular relapse were detected when IM was stopped for different reasons, but second and third MR was obtained with IM resumption. Deep molecular analysis revealed that all the 32 fusion transcripts were different at genomic level. Eight different FIP1L1 exons (exons 9 to 15 and 18) were found to be fused to PDGFRA exon 12 that was truncated in all cases to a variable degree. In 5 cases more than 1 band was evident for the same sample, and in these cases sequencing of c-DNA was performed for each band, identifying genomic variability also in the same patient, at different time point. Following the molecular classification proposed by Walz et al, we identified transcript type A in 4/27 cases(44%), type B in 11/27 cases (41%) and type C in 3/27 cases (15%). The distribution of genomic breakpoint reflects those reported by Walz et al, with the majority of breakpoints located in FIP1L1 intron 10, 11 and 13. Others location were rare. All genomic PDGFRA breakpoints were found in exon 12 (55% in position 83/84, 30% in 100/101, 11% in 43/44, 4% in 25/26, where position +1 represents the first base in exon 12).

After 5 years of follow-up, with this large series of patients we can confirm very high sensibility of F/P to IM, the necessity of continuous therapy and absence of acquired resistance. Our molecular data strictly confirm the data previously reported by others. Clinical correlation between this heterogeneity and phenotype of disease and response to imatinib therapy is not clear with present data and require largest studies. Primers and probes for quantitative RT-PCR have to be designed for every single patients for the complexity and variability in F/P transcripts.

Pane:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Saglio:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Baccarani:Novartis, Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.