Digital Droplet PCR is a Specific and Sensitive Tool for Detecting IDH2 Mutations in Acute Myeloid LeuKemia Patients.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) interfere with cellular metabolism contributing to oncogenesis. Mutations of IDH2 at R140 and R172 residues are observed in 20% of acute myeloid leukemias (AML), and the availability of the IDH2 inhibitor Enasidenib made IDH2 mutational screening a clinical need. The aim of this study was to set a new quantitative polymerase chain reaction (PCR) technique, the drop-off digital droplet PCR (drop-off ddPCR), as a sensitive and accurate tool for detecting IDH2 mutations. With this technique we tested 60 AML patients. Sanger sequencing identified 8/60 (13.5%) mutated cases, while ddPCR and the amplification refractory mutation system (ARMS) PCR, used as a reference technique, identified mutations in 13/60 (21.6%) cases. When the outcome of IDH2-mutated was compared to that of wild-type patients, no significant difference in terms of quality of response, overall survival, or progression-free survival was observed. Finally, we monitored IDH2 mutations during follow-up in nine cases, finding that IDH2 can be considered a valid marker of minimal residual disease (MRD) in 2/3 of our patients. In conclusion, a rapid screening of IDH2 mutations is now a clinical need well satisfied by ddPCR, but the role of IDH2 as a marker for MRD still remains a matter of debate.

Morphologic and immunophenotypic features of a case of acute monoblastic leukemia with unusual positivity for Glycophorin-A.

Acute monoblastic leukemia (AMoL) is characterized by cells with highly undifferentiated morphology. Cytochemistry with non-specific esterases is negative in up to 20% of cases. Immunophenotyping by flow cytometry has an essential role in diagnosing such a subtype of leukemia and a multiparametric approach with a wide monoclonal antibody panel is necessary. We describe a case of AMoL with morphology resembling either plasma blasts or very immature erythroblasts. Diagnosis was made by alpha-naphtyl-acetate esterase staining and with immunophenotyping, which was made with a wide monoclonal antibody panel. Blasts were positive for monocytic markers. Most of leukemic cells, however, were positive for Glycophorin-A. The presence of Glycophorin-A, which is considered as a specific marker of the erythroid lineage, has never been reported previously in cases of AMoL. This peculiar immunophenotype might be interpreted as deriving from a common myelo-erythroid precursor undergone leukemic transformation.

Combination of CD157 and FLAER to Detect Peripheral Blood Eosinophils by Multiparameter Flow Cytometry.

The identification of eosinophils by flow cytometry is difficult because most of the surface antigens expressed by eosinophils are shared with neutrophils. Some methods have been proposed, generally based on differential light scatter properties, enhanced autofluorescence, lack of CD16 or selective positivity of CD52. Such methods, however, show several limitations. In the present study we report a novel method based on the analysis of glycosylphosphatidylinositol (GPI)-linked molecules. The combination of CD157 and FLAER was used, since FLAER recognizes all GPI-linked molecules, while CD157 is absent on the membrane of eosinophils and expressed by neutrophils. Peripheral blood samples from normal subjects and patients with variable percentages of eosinophils (n = 31), and without any evidence for circulating immature myeloid cells, were stained with the combination of FLAER-Alexa Fluor and CD157-PE. A FascCanto II cytometer was used. Granulocytes were gated after CD33 staining and eosinophils were identified as CD157(-)/FLAER(+) events. Neutrophils were identified as CD157(+)/FLAER(+) events. The percentages of eosinophils detected by this method showed a very significant correlation both with automated counting and with manual counting (r = 0.981 and 0.989, respectively). Sorting assays were carried out by a S3 Cell Sorter: cytospins obtained from CD157(-)/FLAER(+) events consisted of 100% eosinophils, while samples from CD157(+)/FLAER(+) events were represented only by neutrophils. In conclusion, this method shows high sensitivity and specificity in order to distinguish eosinophils from neutrophils by flow cytometry. However, since CD157 is gradually up-regulated throughout bone marrow myeloid maturation, our method cannot be applied to cases characterized by immature myeloid cells.

Modifications in B-Lymphocyte Number and Phenotype in the Course of Pregnancy in a Woman with Persistent Polyclonal B-Cell Lymphocytosis: A Flow Cytometric Study.

Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare clinical condition, characterized by a persistent, generally moderate lymphocytosis, generally due to stimulation of central memory B-lymphocytes, and by a moderate increase of polyclonal IgM. In some patients, slight or moderate splenomegaly is observed. A variable percentage of circulating, bone marrow and splenic lymphocytes display an abnormal nucleus (generally bilobated) or are binucleated. The clinical course is benign in most cases and transformation into splenic B-cell lymphoma occurs in few cases. In the current paper we report the first case of pregnancy in PPBL. Our patient became pregnant 18 months after diagnosis. In the course of pregnancy, a marked down-regulation of lymphocytosis (from 6 × 10(9)/L to 2.1 × 10(9)/L) and a decrease in B-lymphocyte number was observed (from 3.6 × 10(9)/L to 1 × 10(9)/L), mainly due to a marked reduction in the percentage and absolute number of central memory B-cells. Such modifications were similar to those described in normal pregnant women. One year after the delivery of a healthy female baby, the number of total lymphocytes and B-lymphocytes showed an inverse behavior, with a new expansion of central memory B-cells. Our case shows that a normal pregnancy can occur in patients with PPBL and that pregnancy can induce marked modifications in B-lymphocyte kinetics and phenotype.

Kinetics of hematogones in bone marrow samples from patients with non-Hodgkin lymphomas treated with rituximab-containing regimens: a flow cytometric study.

Treatment with rituximab, either alone or in combination with antiblastic drugs, causes significant depletion of circulating B-lymphocytes and modifications of B cell maturation in the bone marrow. In the present study, we analyzed the kinetics of hematogones in bone marrow samples from 55 patients suffering from non-Hodgkin lymphomas and treated with rituximab-containing regimens. Maturation arrest at the level of stage 2 hematogones, along with complete depletion of naïve, mature B-lymphocytes, was observed as short-term effects (2 months after completion of chemo-immunotherapy). Further bone marrow samples, obtained 12 months after the last rituximab infusion in 21 patients undergoing long-term follow-up and treated with rituximab maintenance therapy, showed complete normalization of B-lymphocyte ontogeny. Hypogammaglobulinemia developed in 26 patients, and was still observed in nine of the 21 patients undergoing long-term follow-up. Our study provides novel data on hematogone kinetics in the setting of patients with non-Hodgkin lymphomas treated with chemo-immunotherapy containing rituximab and with rituximab maintenance. Our observations show that hypogammaglobulinemia can persist in a significant percentage of patients, despite complete recovery of B-lymphocyte ontogeny.