Mutations in CCAAT/enhancer binding protein α (CEBPA) occur in 5–10% of cases of acute myeloid leukemia. CEBPA-double-mutated cases usually bear biallelic N- and C-terminal mutations and are associated with a favorable clinical outcome. Identification of CEBPA mutants is challenging because of the variety of mutations, intrinsic characteristics of the gene and technical issues. Several screening methods (fragment-length analysis, gene expression array) have been proposed especially for large-scale clinical use; although efficient, they are limited by specific concerns. We investigated the phenotypic profile of blast and maturing bone marrow cell compartments at diagnosis in 251 cases of acute myeloid leukemia. In this cohort, 16 (6.4%) patients had two CEBPA mutations, whereas ten (4.0%) had a single mutation. First, we highlighted that the CEBPA-double-mutated subset displays recurrent phenotypic abnormalities in all cell compartments. By mutational analysis after cell sorting, we demonstrated that this common phenotypic signature depends on CEBPA-double-mutated multi-lineage involvement. From a multidimensional study of phenotypic data, we developed a classifier including ten core and widely available parameters. The selected markers on blasts (CD34, CD117, CD7, CD15, CD65), neutrophil (SSC, CD64), monocytic (CD14, CD64) and erythroid (CD117) compartments were able to cluster CEBPA-double-mutated cases. In a validation set of 259 AML cases from three independent centers, our classifier showed excellent performance with 100% specificity and 100% sensitivity. We have, therefore, established a reliable screening method, based upon multidimensional analysis of widely available phenotypic parameters. This method provides early results and is suitable for large-scale detection of CEBPA-double-mutated status, allowing gene sequencing to be focused in selected cases.

© F e r r a t a S t o r t i F o u n d a t i o nphic alleles of the C3 gene and of the complement receptor 1 (CR1) gene. Methods PatientsSeventy-two patients with hemolytic PNH who had received eculizumab treatment were analyzed after a median follow-up of 52 months (range, 11-98 months). Patients with evidence of bone marrow failure (reticulocytes ≤60x10 9 /L, platelets ≤50x10 9 /L, neutrophils ≤1x10 9 /L) were not included in this series because this condition may affect the clinical response to eculizumab regardless of how well hemolysis is controlled. Peripheral blood samples were collected for clinical testing, flow cytometry (GPI-molecules and C3-binding) 7 and genotyping after the patients had signed an informed consent form approved by the respective Institutional Review Board. As a criterion of response to eculizumab we used the one that is most stringent and objective: namely RBC transfusion at any time after the first 6 months on eculizumab treatment. Of the 72 patients 60 were transfusiondependent before eculizumab: of these 60, those who on eculizumab received no further transfusion during follow-up have been classified as good responders; those who on eculizumab received one or more RBC transfusion at any time during follow-up have been classified as suboptimal responders. Twelve patients started eculizumab before having received any RBC transfusions. Of these, the ten patients who on eculizumab remained transfusion-independent have been classified as good responders because their hemoglobin level increased, on average, by 2.2 g/dL; the remaining two patients required RBC transfusions on eculizumab and have been classified as sub-optimal responders. Only one patient, who had hemolytic PNH at the time of starting eculizumab and who had already been classified as a sub-optimal responder, became aplastic during follow-up. GenotypingThe polymorphism rs2230199 C>G of the C3 gene was investigated by a newly designed tetra-primer amplification refractory mutation system-polymerase chain reaction method.11 Three polymorphisms of the CR1 gene were genotyped by restriction fragment length (RFLP) analysis:12 HindIII RFLP (intron 27); His1208Arg (exon 22); Pro1827Arg (exon 33). Kinetics of C3 binding in vitroRBC and sera were promptly separated from peripheral blood freshly collected from PNH patients. The RBC were then incubated with sera containing eculizumab as previously described. 13,14 Briefly, a 2% suspension of RBC from PNH patients was incubated at 37°C with a pool of ABO-compatible sera from patients on eculizumab, and the complement alternative pathway was activated by mild acidification (HCl 0.016 M). At serial time points (15,30, 60, and 120 min) after complement activation the fraction of GPI-negative RBC with newly bound C3 fragments was measured by flow cytometry (AccuriC6, Becton Dickinson, NJ, USA) after staining with anti-CD59 Alexa647 (Mem43, Serotec, UK) and anti-C3d-neoantigen (A250, Quidel, CA, USA); secondary staining was performed with phycoerythrin-labeled polyclonal rabbit-an...

BackgroundC5 blockade by eculizumab prevents complement-mediated intravascular hemolysis in paroxysmal nocturnal hemoglobinuria (PNH). However, C3-bound PNH red blood cells (RBCs), arising in almost all treated patients, may undergo extravascular hemolysis reducing clinical benefits. Despite the uniform deficiency of CD55 and of CD59, there are always two distinct populations of PNH RBCs, with (C3+) and without (C3−) C3 binding.MethodsTo investigate this paradox, the phenomenon has been modeled in vitro by incubating RBCs from eculizumab untreated PNH patients with compatible sera containing eculizumab, and by assessing the C3 binding after activation of complement alternative pathway.ResultsWhen RBCs from untreated patients were exposed in vitro to activated complement in the context of C5-blockade, there was the prompt appearance of a distinct C3+ PNH RBC population whose size increased with time and also with the rate of complement activation. Eventually, all PNH RBCs become C3+ to the same extent, without differences between old and young (reticulocytes) PNH RBCs.ConclusionsThis study indicates that the distinct (C3+ and C3−) PNH RBC populations are not intrinsically different; rather, they result from a stochastic all-or-nothing phenomenon linked to the time-dependent cumulative probability of each individual PNH red cell to be exposed to levels of complement activation able to trigger C3 binding. These findings may envision novel approaches to reduce C3 opsonization and the subsequent extravascular hemolysis in PNH patients on eculizumab.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-017-0496-x) contains supplementary material, which is available to authorized users.

Germ-line mutation rate has been regarded classically as a fundamental biological parameter, as it affects the prevalence of genetic disorders and the rate of evolution. Somatic mutation rate is also an important biological parameter, as it may influence the development and/or the course of acquired diseases, particularly of cancer. Estimates of this parameter have been previously obtained in few instances from dermal fibroblasts and lymphoblastoid cells. However, the methodology required has been laborious and did not lend itself to the analysis of large numbers of samples. We have previously shown that the X-linked gene PIG-A, since its product is required for glycosyl-phosphatidylinositol-anchored proteins to become surface bound, is a good sentinel gene for studying somatic mutations. We now show that by this approach we can accurately measure the proportion of PIG-A mutant peripheral blood granulocytes, which we call mutant frequency, ƒ. We found that the results are reproducible, with a variation coefficient (CV) of 45%. Repeat samples from 32 subjects also had a CV of 44%, indicating that ƒ is a relatively stable individual characteristic. From a study of 142 normal subjects we found that log ƒ is a normally distributed variable; ƒ variability spans a 80-fold range, from less than 1×10−6 to 37.5×10−6, with a median of 4.9×10−6. Unlike other techniques commonly employed in population studies, such as comet assay, this method can detect any kind of mutation, including point mutation, as long as it causes functional inactivation of PIG-A gene. Since the test is rapid and requires only a small sample of peripheral blood, this methodology will lend itself to investigating genetic factors that underlie the variation in the somatic mutation rate, as well as environmental factors that may affect it. It will be also possible to test whether ƒ is a determinant of the risk of cancer.

Myelodysplastic Syndromes (MDS) are clonal neoplasms where stem/progenitor cells endowed with self-renewal and capable of perpetuating the disease have been demonstrated. It is known that oxygen tension plays a key role in driving normal hematopoiesis and that hematopoietic stem cells are maintained in hypoxic areas of the bone marrow (BM). Hypoxia could also regulate leukemic/dysplastic hematopoiesis. We evaluated the stem cell potential of MDS cells derived from the BM of 39 MDS patients and selected under severe hypoxia. MDS cells rescued from hypoxia-incubated cultures were subjected to stem and progenitor cell assays in vitro, as well as to hematopoietic reconstitution assay in NOD-SCID mice. Incubation in severe hypoxia of cells explanted from MDS patients selected a cell subset endowed with stem cell potential, as determined in vitro. This occurred only from the BM of patients classified as IPSS low/INT-1 risk. Transplantation into NOD-SCID mice confirmed using an in vivo model that severe hypoxia selects a cell subset endowed with stem cell potential from bone marrow mononuclear cells (BMMC). derived from patients belonging to the IPSS low/int-1 risk group. Data here reported show that cells endowed with stem cell potential and capable of adapting to hypoxia and escaping hypoxia-induced apoptosis exist within MDS cell populations.