Summary The hypereosinophilic syndromes (HES) include a group of heterogeneous diseases characterized by the persistent increase of the number of eosinophils in blood and bone marrow. Few cases of paediatric hypereosinophilia (pHES) have been described in the literature. Early identification of pHES that may evolve towards a lymphomyeloproliferative disease is relevant in light of prognostic and therapeutic implications. Molecular features of 10 pHES patients were analysed at presentation and during their clinical course, including analysis of BCR‐ABL1 and FIP1L1/PDGFRA fusion genes, quantitation of WT1 gene copy number and clonality of T‐cell receptor (TCR) and immunoglobulin heavy chain (IGH). All patients had normal karyotype and germline TCR configuration. Five children showed IGH clonality at presentation: of these, two developed a B non‐Hodgkin lymphoma and a B‐lineage acute lymphocytic leukaemia at six and 12 months respectively, two spontaneously reverted to a polyclonal IGH profile during the follow‐up, and the last one persisted with pHES without B‐clonal evolution after 19 months. One patient had a PDGFRA/FIP1L1 fusion and achieved hematologic and molecular remission after imatinib therapy. IGH rearrangement was observed to be a frequent molecular feature of pHES and may precede B‐cell clonal expansion and evolution into B‐cell malignancies in children.

SummaryFLT3 internal tandem duplication (ITD) mutations are frequently detected at diagnosis in cytogenetically normal acute myeloid leukaemia (CN-AML) and predict unfavourable outcome. FLT3 ITD is an unstable aberration and may be lost or acquired at relapse. Recent whole genome sequencing studies have suggested that FLT3 ITD + ve AML relapse may evolve from small subclones undetectable at diagnosis by routine polymerase chain reaction (PCR). We developed a patient-specific real-time quantitative-PCR (RQ-PCR) to implement FLT3 ITD detection in six AML patients whose blasts carried wild-type FLT3 at diagnosis and who relapsed with FLT3 ITD by routine PCR. Patient-specific forward primers were designed after cloning and sequencing the FLT3 ITD in each case. The assay allowed retrospective detection of FLT3 ITD in diagnostic samples of 4/6 cases and to establish the kinetics of clonal evolution preceding relapse. After conventional chemotherapy, all patients had early relapse despite having been classified as NPM1 + ve/FLT3 ITD À ve at presentation, with shorter remissions being observed in four patients re-classified as FLT3 ITD + ve by the new assay.Notably, FLT3 ITD clone became detectable by conventional PCR in three patients tested during remission after initial treatment. Our data underscore the need of identifying low FLT3 ITD levels, which are probably associated with relapse in otherwise good prognosis CN-AML.

The genotype nucleophosmin mutated and FLT3-ITD negative is characterized by high bax/bcl-2 ratio and favourable outcome in acute myeloid leukaemia Nucleophosmin gene (NPM1) mutations generate an elongated nucleophosmin (NPM) protein that localizes aberrantly in the cytoplasm (NPMc+) and characterize 50-60% of adult acute myeloid leukaemias (AML) with normal karyotype . NPMc+AML shows increased white blood cell (WBC) count, high frequency of FLT3 internal tandem duplications (ITDs) and CD34 negativity (Falini et al, 2007). Döhner et al (2005) observed better complete remission rate (CR) and favourable overall survival (OS) in the NPM1-mutated (mt)/FLT3-ITD negative subgroup. Gale et al (2008) identified FLT3-ITD+NPM1-mt patients as an intermediate prognostic subset. Recently, Schlenk et al (2008) confirmed that the genotype of mutant NPM1 without FLT3-ITD and younger age were significantly associated with CR. Previously we reported that bcl-2 and bax apoptotic proteins are independent predictors of response and survival in AML (Del Poeta et al, 2003). Therefore, we hypothesized that NPM1-mt AML show a high level of spontaneous apoptosis as detected by an elevated bax/bcl-2 ratio, thus explaining their better outcome. Therefore, we undertook a retrospective analysis of the prognostic impact of NPM1 mutations and FLT3-ITD in correlation with bax/bcl-2 ratio within a cohort of 222 adult non-M3 AML patients receiving intensive chemotherapy regimens. Patients and methods SummaryNucleophosmin gene (NPM1) mutations characterize acute myeloid leukaemia (AML) with normal karyotype and frequently co-exist with FLT3 internal tandem duplications (ITD). We evaluated bcl-2, bax, NPM1 and FLT3-ITD in 222 AML patients. Bax/bcl-2 ratio >0AE35 and NPM1 without FLT3-ITD were significantly associated (P = 0AE0001). NPM1-mutated (mt)/FLT3-ITD negative patients showed a higher complete remission (CR) rate (90%, P = 0AE0002) and a longer overall survival (OS, P = 0AE00007). NPM1-mt/FLT3-ITD negative plus bax/bcl-2 > 0AE35 subset showed a very high CR rate (96%), very long OS (P = 0AE00005) and diseasefree survival (P = 0AE004). The favourable prognosis of NPM1-mt/FLT3-ITD negative patients might be explained by a higher bax/bcl-2 ratio.

NADP-dependent enzyme isocitrate dehydrogenase (IDH) mutations, IDH1 and IDH2, have been described in acute myeloid leukemia (AML) using next generation sequencing approaches. IDH2 mutations are heterozygous; they alter a single arginine residue at position 140 or 172 and have distinct prognostic significance. The current detection methods of IDH2 mutations are laborious and time consuming as they require DNA sequencing. Herein, we report a new allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) method to detect the IDH2 mutations. Analysis of leukemic DNA samples from 120 AML patients enabled to identify IDH2 mutations in 22 cases which were confirmed by direct DNA sequencing. Of these, 17 harbored IDH2 (R140Q) and 5 IDH2 (R172K) mutations. Serial dilution experiments showed that the assay enable to detect mutations in 10⁻³ dilutions. Our ASO-PCR method appears useful for routine diagnostic screening of these prognostically relevant alterations in AML and may be conveniently included in the diagnostic workup.