Assessment of distribution of CD34 epitope classes in fresh and cryopreserved peripheral blood progenitor cells and acute myeloid leukemic blasts.

So far several reports have described changes in the expression of surface antigens in progenitor cells and blasts following cryopreservation. However, there are no data on the effects of cryopreservation on the expression of the three CD34 epitope classes, and on their relationship with the clonogenic capacity of PBPC collected by leukapheresis. DESIGN AND METHODS: In order to analyze the effects of freezing/thawing procedures (Eth 80C storage for 3 months) and use of dimethylsulfoxide (DMSO) on the immunophenotype profile and colony production of peripheral blood progenitor cells (PBPC) in apheresis products derived from 20 patients with stage 0-III non-Hodgkin's lymphoma (nHL), a flow cytometry study was undertaken using different CD34 monoclonal antibodies (MoAbs) capable of recognizing the 3 epitope classes of CD34 molecule (class III: HPCA-2/FITC, HPCA-2/PE, 581/FITC, 581/PE; class II: Q-Bend 10/PE; class I: ICH3/PE, BI3C5-PE, Immu-133-PE). CD34 epitope expression was also analyzed in thawed CD34+ blasts obtained from 14 patients with acute myeloid leukemia (AML), who were analyzed using a larger number (#17) of CD34 epitope class I, II, and III reactive MoAbs. RESULTS: Under our experimental conditions it was found that class III and class II CD34 epitopes (differentially resistant to enzymatic cleavage with neuraminidase, chymopapain and glycoprotease) are better preserved than class I epitope Eth sensitive to degradation Eth after cell exposure to cryoprotectant DMSO and the freezing- thawing procedures. Results further showed a concomitant decrease in class I CD34+ counts and in BFU-E colony production. A significant increase in CD34 antigen expression levels (i.e. antibody binding capacity, ABC) by cryopreserved cells stained with CD34 epitope class III, and class II reactive MoAbs was also documented, while no changes after cryopreservation were noted using class I-reactive MoAbs. The slight increase in the percentage of CD34+ cells detected after frozen storage was correlated to a concomitant decrease in the number of more mature myeloid cells (CD15+, CD13+, CD33+). Compared to pre-cryopreservation values, a slight reduction in class I CD34 epitope expression was also found in thawed CD34+ AML blasts. INTERPRETATION AND CONCLUSIONS: As far as the reduction of class I CD34 epitope is concerned, it may be hypothesized that the freezing procedure, use of DMSO, and/or lysis methodology may either damage a CD34 subset, or induce distinct alterations of the CD34 glycoprotein, possibly determining a reduction in their immunoreactivity with some CD34 MoAbs. In conclusion, this study has shown that exposure to the cryoprotectant DMSO and the freezing/thawing procedures modifies the distribution of CD34 epitopes as well as the clonogenic capacity of PBPCs from nHL patients, and CD34+ blasts from AML. These findings need to considered when selecting CD34 MoAbs for enumeration and positive selection of stem/progenitor cells for research and clinical purposes

Response to ibrutinib of a refractory IgA lymphoplasmacytic lymphoma carrying the MYD88 L265P gene mutation

In 2014 a 66-year-old woman presented with anemia and an IgAk monoclonal spike. Bone marrow (BM) biopsy showed 80% lymphocytes and lymphoplasmacytoid cells. Computed Tomography (CT) scan documented neither adenopathy nor splenomegaly. Diagnosis of IgA lymphoplasmacytic lymphoma was made. After three lines of treatment, progressive disease with adenopathies, splenomegaly, and ascites were documented on a CT scan. Our patient developed thrombocytopenia, transfusion-dependent anemia, and clinical deterioration. We performed genetic studies of peripheral blood lymphocytes with the NGS approach. Given the identification of MYD88 L265P mutation, in February 2018 our patient started ibrutinib off-label. lib and PLT improved from day +35. In July 2018 no ascites and 50% reduction of adenopathies and spleen were shown on a CT scan. In April 2019 the patient was still on ibrutinib with transfusion independence and good performance status

CD116 (GM-CSF-R)

Human CD116 is the alpha subunit of granulocytemacrophage colony stimulating factor receptor (GMCSFR, also called colony stimulating factor 2 receptor, alpha) that binds GM-CSF with low affinity. The molecule was termed CD116 at the 5th International Workshop on Leukocyte Differentiation Antigens (IWLDA, Boston, USA, 1993). The beta subunit (CD131), which is also shared with the IL3 and IL5 receptors, has no detectable binding affinity for GM-CSF on it’s own but is necessary for high affinity binding when associated with the alpha subunit and plays a fundamental role in signal transduction. Monoclonal antibodies (MoAbs) against CD116 (extracellular domain) are used for phenotyping various cell populations possibly contributing to the diagnosis and therapy of acute myeloid leukemia (AML). In AML, GM-CSFR is detectable in 60-70% of cases and particularly in M4 and M5 FAB subvarieties. The number of receptors expressed by AML cells is sometimes significantly higher than that in normal hematopoietic cells, suggesting the possibility of using this marker as a useful tool for the monitoring of minimal residual disease. Cellular CD116 expression was documented in > 50% M0 AML. Since the activity of GM-CSF on hemopoietic cells depends upon its binding to specific cell surface receptors, we have previously hypothesised that the clinical use of GM-CSF in AML patients could be optimized by a dynamic analysis of the number and the affinity status of GM-CSFR in leukemic blasts and normal hemopoietic cells

In vitro assessment of bone marrow endothelial colonies (CFU-En) in non Hodgkin’s lymphoma patients undergoing peripheral blood stem cell transplantation

The distribution and functional characteristics of in vitro bone marrow (BM) endothelial colonies (CFU-En) were studied in 70 non-Hodgkin's lymphoma (NHL) patients in different phases of the disease to explore the association between CFU-En growth and angiogenesis, and between the number of CFU-En and the presence of hematopoietic and mesenchymal progenitor cells. The mean number of CFU-En/10(6) BM mononuclear cells seen in remission patients was significantly higher than that seen in newly diagnosed patients (P=0.04), and in normal subjects (P=0.008). Patients with low-grade NHL in remission displayed a higher CFU-En value compared with high-grade NHL (P=0.04). In the autograft group (40 patients), a significant reduction of CFU-En number was detected in the first 4-6 months after transplantation. In remission patients, the CFU-En number positively correlated with the incidence of BM colony-forming unit granulocyte-macrophage (CFU-GM) (P=0.013) and CFU-multilineage (CFU-GEMM) hematopoietic colonies (P=0.044). These in vitro data show that CFU-En numbers increase following standard-dose chemotherapy, thus providing a rationale for further investigating the effects of different cytostatic drugs on BM endothelial cells growth and function

CD 123 (Interleukin 3 receptor alpha chain)

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CD123 (Interleukin 3 receptor a chain).

CD 123 is the interleukin 3 receptor alpha chain and is expressed on different type of myloid hematopoietic cell

Functional and immunophenotypic charcteristics of isolated CD105+ and fibroblasts+ stromal cells from acute myloid leukaemia: Implication for their plasticity along the endothelial lineage.

Functional and immunophenotypic charcteristics of isolated CD105+ and fibroblasts+ stromal cells from acute myloid leukaemia: Implication for their plasticity along the endothelial lineage

Human bone marrow endothelial-colony growth before and after stem cell transplantation in non-hodgkin’s lymphoma patients.

Human bone marrow endothelial-colony growth before and after stem cell transplantation in non-hodgkin’s lymphoma patients