Genomic Confirmation of the P-IIIe Subclass of Snake Venom Metalloproteinases and Characterisation of Its First Member, a Disintegrin-Like/Cysteine-Rich Protein.

Disintegrin-like/cysteine-rich (DC) proteins have long been regarded just as products of proteolysis of P-III snake venom metalloproteinases (SVMPs). However, here we demonstrate that a DC protein from the venom of Vipera ammodytes (Vaa; nose-horned viper), VaaMPIII-3, is encoded per se by a P-III SVMP-like gene that has a deletion in the region of the catalytic metalloproteinase domain and in part of the non-catalytic disintegrin-like domain. In this way, we justify the proposal of the introduction of a new subclass P-IIIe of SVMP-derived DC proteins. We purified VaaMPIII-3 from the venom of Vaa in a series of chromatographic steps. A covalent chromatography step based on thiol-disulphide exchange revealed that VaaMPIII-3 contains an unpaired Cys residue. This was demonstrated to be Cys6 in about 90% and Cys19 in about 10% of the VaaMPIII-3 molecules. We further constructed a three-dimensional homology model of VaaMPIII-3. From this model, it is evident that both Cys6 and Cys19 can pair with Cys26, which suggests that the intramolecular thiol-disulphide exchange has a regulatory function. VaaMPIII-3 is an acidic 21-kDa monomeric glycoprotein that exists in at least six N-glycoforms, with isoelectric points ranging from pH 4.5 to 5.1. Consistent with the presence of an integrin-binding motif in its sequence, SECD, VaaMPIII-3 inhibited collagen-induced platelet aggregation. It also inhibited ADP- and arachidonic-acid-induced platelet aggregation, but not ristocetin-induced platelet agglutination and the blood coagulation cascade.

Recognition of Philadelphia chromosome-like acute lymphoblastic leukemia as part of routine diagnostic work-up.

INTRODUCTION: Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) is a biologically and clinically challenging subtype of B-cell ALL which has been incorporated into the 2016 revision of the World Health classification of acute leukemia. It is independently associated with poor outcome. As it can only be reliably detected by expression profiling, it is difficult to diagnose with routine methods. Its recognition has become of greater importance due to prognostication and even more due to the new diagnostic options given by targeted therapies. There is still no standardized diagnostic test enabling its prompt recognition. Here, we introduce our approach how to detect it by combination of widely available techniques. METHODS: 179 ALL patients diagnosed in our center during the last 8 years were included. Data on immunophenotype and cytogenetics were used to select patients with potentially Ph-like ALL (65/179). CRLF2 gene rearrangement (CRLF2-r) was tested by FISH in 59/65 patients, and next-generation sequencing was done by Archer FusionPlex ALL kit in 34 patients. TSLPR expression was determined in 20 patients. RESULTS: Philadelphia chromosome-like aberrations were confirmed in 9 patients. In 10% of tested samples, CRLF2-r was confirmed. Due to a lack of material, NGS was done only in a half of potentially Ph-like cases. In 10%, other Ph-like fusions were found by NGS. CONCLUSIONS: The obtained frequencies, and genetic and patients' characteristics are in concordance with the literature data, ensuring a reliable detection of this challenging ALL subtype. The proposed algorithm allows detection of Ph-like ALL at reasonable cost and acceptable workload.

Hematopoietic progenitor cell counting can optimize peripheral blood stem cell apheresis process.

INTRODUCTION: Monitoring of stem cell concentration in transplantation settings is crucial to determine the optimal time of apheresis and is currently based on enumeration of CD34+ cells by flow cytometry. No surrogate marker has replaced CD34+ cell enumeration to date. The aim of this study was the evaluation of the hematopoietic progenitor cell (HPC) parameter of the Sysmex XN-1000 analyzer in terms of optimizing the peripheral blood stem cell apheresis process. MATERIALS AND METHODS: Results of flow cytometric CD34+ cell and Sysmex HPC count were compared in 208 preharvest samples and 139 apheresis products. RESULTS: HPC and CD34+ cell counts showed significant differences in the multiple myeloma (MM) group. The correlation between preharvest HPC and CD34+ cell counts was good in the MM group (rho = .613) and strong in the lymphoma group (rho = .802), allogeneic donors (rho = .923), and other group of samples (rho = .816). The HPC positive cutoff demonstrating 100% specificity and positive predictive value for MM patients was high for ≥20/µL and ≥10/µL CD34+ cell counts, and therefore of limited value. The HPC negative cutoff demonstrating 100% sensitivity and negative predictive value was approximately <4/µL, irrespective of diagnosis. CONCLUSIONS: Based on proposed HPC positive cutoffs (≥31/µL in the lymphoma group and ≥11/µL in the other group of samples), routine HPC enumeration could improve the workflow by replacing CD34+ cell counting in allogeneic donors as well as non-MM patients. Furthermore, based on proposed HPC negative cutoff (<4/µL), CD34+ cell counting could be fully omitted in donors and patients that are not adequately mobilized.

CD56 Expression Is an Important Prognostic Factor in Multiple Myeloma Even with Bortezomib Induction.

OBJECTIVES: In this retrospective study, we evaluated the impact of CD56, CD117, and CD28 expression on clinical characteristics and survival in newly diagnosed myeloma patients treated with bortezomib-based induction therapy. METHODS: We analyzed 110 myeloma patients. Immunophenotype was determined using panels consisting of CD19/CD38/CD45/CD56/CD138 and CD20, CD28, and CD117 were used additionally. All samples were tested for recurrent chromosomal aberrations. RESULTS: CD56, CD117, and CD28 expression rates were 71, 6, and 68%, respectively. The lack of CD56 expression was associated with light chain myeloma. The lack of CD117 expression was associated with elevated creatinine levels (p = 0.037). We discovered the correlation between CD 28 expression and female gender. The median progression-free survival (PFS) for patients with revised International Staging System stage 2 disease with CD56 expression or the lack of CD56 expression was 20.5 vs. 13.8 months (p = 0.03). In patients undergoing autologous hematopoietic stem cell transplantation (aHSCT), we found no difference in PFS and overall survival regarding the CD56 expression. We found no impact of CD117 and CD28 expression on PFS in patients regarding aHSCT. CONCLUSIONS: Induction treatment incorporating bortezomib diminishes the negative impact of the lack of CD117 expression and aberrancy of CD28 but does not overcome the negative impact of the lack of CD56 expression.

Selective apoptosis of multiple myeloma cells in primary samples induced by arsenic trioxide.

OBJECTIVES: Currently, multiple myeloma (MM) is an incurable disease. Despite the fact that arsenic trioxide (ATO) shows promising results in vitro, data from treatment of patients with MM are disappointing. Due to these discrepancies, we compared the efficacy and selectivity of ATO at two different concentrations in samples from MM patients. METHODS: The extent of apoptosis induced by 2 and 5 µM ATO was evaluated by flow cytometry using annexin V. 34 diagnostic bone marrow samples obtained from MM patients were analysed. RESULTS: 5 µM ATO efficiently induced apoptosis in primary samples. Besides efficacy, also selectivity of action on MM cells in comparison to remaining haematopoietic cells was demonstrated for 5 µM ATO but not for 2 µM ATO. DISCUSSION: Our study on primary samples confirmed that ATO has a potential role in therapeutic management of MM. Further controlled studies on MM patients are needed.

Evaluation of multiple myeloma cell apoptosis in primary bone marrow samples.

BACKGROUND: Although different approaches have been proposed to selectively determine multiple myeloma (MM) cells in a heterogeneous population of bone marrow (BM) cells, studies on plasma cells from primary samples of MM patients are still challenging. This is partially due to difficulties in obtaining a suitable amount of sample, but even more due to uneven infiltration of BM by MM cells. When the apoptotic effect of different agents on MM plasma cells is studied, evaluation is additionally complicated by morphological changes induced by apoptosis. We introduce a modified gating approach combining specific antibodies and exclusion of cellular interferences. METHODS: The extent of apoptosis induced by arsenic trioxide and camptothecin was evaluated by flow cytometry using annexin V and propidium iodide (PI) after selective labelling of plasma cells with CD38 and CD138 antibodies. We selectively analysed MM plasma cell apoptosis combining CD38/CD138-positivity and exclusion of cellular interferences. RESULTS: Thirty BM samples from newly diagnosed MM patients were analysed. We compared the proportion of cells in different phases of apoptosis obtained by gating on a CD38/CD138-positive population only and by the novel approach. The proportion of cells in early apoptosis evaluated by the modified gating technique was significantly higher for both inductors. CONCLUSIONS: The introduced gating approach can increase the reliability of selective evaluation of MM plasma cell apoptosis in primary samples. The modified method can further be implemented for the analysis of various processes in plasma cells by flow cytometry.