Celia Suriu

Bortezomib-induced peripheral neuropathy is related to altered levels of brain-derived neurotrophic factor in the peripheral blood of patients with multiple myeloma

Loss of nerve growth factor-mediated neuronal survival has recently been proposed as a candidate mechanism underlying bortezomib-induced peripheral neuropathy (BIPN) (Broyl et al, 2010). However the literature does not reveal any data from patients that can support this hypothesis. Brain-derived neurotrophic factor (BDNF) is a neuronal growth factor that is crucial for neuronal survival and repair (Hofer & Barde, 1988). We report on alterations in BDNF peripheral blood levels and the development of BIPN in patients with multiple myeloma (MM). Following approval of the Ethics Committee and obtaining patient’s informed consent, peripheral neuropathy was assessed and graded in 25 MM patients using the abbreviated version of the Total Neuropathy Score (TNS) tool (Argyriou et al, 2008). These patients were examined at diagnosis and after receiving four courses of a bortezomib-based regimen given intravenously either weekly or biweekly (Table I). Patients that developed TNS ≥ 2 during treatment were determined to have BIPN and three patients with TNS ≥ 2 at diagnosis were excluded from the study. At the time of neurological examination, a venous blood sample was obtained and soluble BDNF (sBDNF) level was determined by a commercial enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA) in platelet-poor-plasma. One of our patients passed away after two courses of bortezomib. Eight of the remaining patients (36%) developed BIPN. We found lower levels of sBDNF in the plasma of patients that developed BIPN as compared to patients that did not develop BIPN [BDNF level standard deviation (SD) in BIPN 2 164 0 721 vs. 4 62 0 61 ng/ml in non-BIPN; P = 0 007; Fig 1A]. By analysing the difference between BDNF levels at diagnosis and after four courses of treatment of each patient, we demonstrated that sBDNF level was reduced in patients developing BIPN while it remained stable or even minimally elevated in patients that did not develop BIPN (difference in BDNF SD in BIPN 1 668 0 670 vs. 0 405 0 708 ng/ml in non-BIPN P = 0 02) (Fig 1B). Platelets are thought to be responsible for the homeostasis of BDNF in the blood as they take up, store and secrete BDNF (Fujimura et al, 2002). As we did not observe differences in the platelet counts of patients that developed BIPN compared to patients that did not develop BIPN (data not shown), we reasoned that decreased sBDNF levels in the plasma of patients with BIPN may result from lack of secretion of BDNF from the platelets. To test this hypothesis, we determined the content of BDNF in the platelets of patients that developed BIPN relative to those who did not develop BIPN by two different methods. First, platelets that were collected and stored in 20°C at the time of the patients’ examination were lysed and the BDNF immunoreactive band was determined by gel electrophoresis using specific rabbit polyclonal anti-human BDNF mAb (Abcam, Cambridge, MA, USA). Second, BDNF and the platelet activation marker CD62p were determined in freshly isolated platelets of patients with BIPN and patients without BIPN using flow cytometry. Briefly, 20 ll of platelet-rich-plasma from each patient was stained with phycoerythrin-cyanin 5 (PE-CY5) Anti human CD41a monclonal antobody (mAb) (Beckmann Coulter, Brea, CA, USA) and PE anti human CD62p mAb (Biolegends, San Diego, CA, USA) or fixed permeabilized (Invitrogen, Frederick, MD, USA) and stained with ATTO-488 Anti human proBDNF pAb (Alomone Labs Ltd., Jerusalem, Israel). The intensity of surface CD62p or intracellular BDNF signal was determined in 10 000 platelets. Analysis of BDNF in platelets by gel electrophoresis suggested that BDNF content was increased in platelets of patients who developed BIPN as compared to platelets of patients that did not (mean BDNF expression level post-treatment relative to pre-treatment SD in BIPN 4 33 1 15-fold vs. 1 67 0 57-fold in non-BIPN P = 0 02) (Fig 1C). Flow cytometric analysis confirmed the increase of BDNF content in platelets of patients with BIPN, as detected by higher intensity of proBDNF compared to platelets of patients without BIPN [mean fluorescence intensity (MFI) of BDNF SD in BIPN 43 12 6 04 vs. 18 267 10 162 ng/ml in non-BIPN P = 0 009]. As expected, the increased BDNF signal in platelets of patients with BIPN coincided with lower

Efficacy and safety of salvage therapy using Carfilzomib for relapsed or refractory multiple myeloma patients: a multicentre retrospective observational study

Carfilzomib has been established in previous years as a treatment for patients with relapsed and/or refractory multiple myeloma (RR‐MM). A retrospective multicentre study to evaluate the clinical use of carfilzomib for RR‐MM outside of a clinical trial setting was conducted by our group. One hundred and thirty‐five patients were included. All patients had been previously exposed to bortezomib and 93% had also been treated with lenalidomide. The vast majority of patients received carfilzomib as part of a two‐ or three‐drug combination. The overall response rate was 47·2%. Multivariate analysis revealed bortezomib resistance, lenalidomide resistance and albumin <35 g/l to negatively impact the likelihood of achieving response. The median duration of response was 8·4 months, and was significantly higher in patients receiving three‐drug combination and patients presenting without extramedullary disease. The median progression‐free survival and overall survival for the entire cohort was 4·9 months (95% confidence interval [CI] 3·8–6·4) and 12·2 months (95% CI 9‐not reached), respectively. Toxicity was manageable, although treatment‐related death was seen in 5% of patients. In the setting of progressive multiple myeloma, carfilzomib in a combination regimens yields effective results with a manageable toxicity.

Hairy cell leukemia-variant without typical morphology and with Near-tetraploid DNA Content

A 75-year-old male was referred to our medical center due to abdominal pain. Clinical record showed splenomegaly 4 years prior to his visit and a mild microcytic anemia during the last 2 years. Physical examination showed splenomegaly without lymphadenopathy. Complete blood count showed anemia (RBC 4.21 3 10/mL, Hg 10.3 g/DL, MCV: 76.5), normal leukocyte count 4.93 3 10/mL (with normal differential), and thrombocytopenia (107 3 10/mL). Bone marrow (BM) aspirate that was sent for flow cytometry showed lymphocyte clusters that accounted for 30% of the total nucleated cell component and Kappa restricted B cells that accounted for 30% of the total lymphocytes. DNA analysis that we performed showed near tetraploid DNA content (DI 5 1.95) coexpressing the B cell marker CD20. Further immuno-phenotyping of the B cells showed bright expression of CD45, positive expression of FMC7, CD22, CD23, CD79b, and CD200 and negative expression of CD5, CD10, and CD43. Detection of HCL-relevant antigens showed positive expression of CD11c and CD123, dim CD103 expression and negative expression of CD25. Staining with LAIR1(CD305) showed bright expression. BM biopsy was mild hyper-cellular with increased small to intermediate size lymphocytes showing diffuse interstitial pattern and minimal nodular pattern. CD45 was positive in 30% of cells, part of which was positive to CD20 and CD79a and part to CD3 and CD5. The percentage of leukemic cell out of the total hematopoietic cellularity in the bone marrow biopsy was 10%. TRAP immunoreactivity was inconclusive. Flow cytometry analysis of the peripheral blood (PB) showed the malignant B cells with incidence of 42.1% among the total lymphocyte component (901 leukemic cells/mL) and identical immuno-phenotype and aneuploidy as observed in the BM aspirate (Fig. 1a–d). A search for pathological cells in the blood smear showed medium to large size cells with prominent nucleolus and wide cytoplasm that lack visible hairy branches (Fig. 1g). Due to uncertain diagnosis, the extremely enlarged spleen (2.48 kg, 28 cm long as determined by ultrasound 2 month before splenectomy) was removed from the patient. Flow cytometry analysis of the spleen biopsy showed lymphocyte cluster that accounted for 85% of the total nucleated cell component and Kappa restricted B cells that accounted for 45% of the total lymphocytes. The B cells in the spleen showed immunophenotype and aneuploidy similar to the BM aspirate and the peripheral blood (PB) (Fig. 1e,f). Histological assessment of the spleen showed little and condensed white pulp with massive lymphocyte infiltration in the red pulp. The lymphocytes were medium size with irregular cell nuclei, partly folded with clumping chromatin; some of them with prominent nucleoli. The lymphocytes were positively stained with CD20, CD79a, PAX-5, CD23, and TRAP (Fig. 1h). Immunoreactivity to Annexin-1A, DAB44, BCL-6, MUM-1, CD10, CD5, and P53 were negative. Ki67 was positive in 30% of the lymphocytes and the larger atypical cells represented the greater fraction of the proliferating cells. Immunoreactivity to cMYC and BCL-1 were positive only in a few cells. Molecular analysis of the spleen biopsy showed negative t(11:14) translocation, wild type BRAF (exon 15 V600E) and MEK1/MAP2K1 were determined to be without mutation. An unbiased search for the clonal immunoglobulin heavy chain (IGH) gene rearrangement harbored by the tumor was performed. This analysis revealed the tumor IGH gene rearrangement to contain a 100% unmutated VH4-34 variable gene. Flow cytometry analysis of BM aspirate that was taken from the patient one month after splenectomy showed lymphocyte clusters that accounted for 26% of the total nucleated cell component and Kappa restricted B cells that accounted for 36% of the total lymphocytes. The malignant B cells showed similar immuno-phenotype to the cells at diagnosis and they were clearly recognized by their near-tetraploid peak. BM sample that was sent for cytogenetics showed no abnormality in the 19 cells in metaphase that were screened. Nevertheless, BM sample that was sent for FISH analysis detected cells with nuclear atypia that showed four signals for centromeres 2, 4, 8, 10, 15, and 17 as well as four MLL and four IGH signals. Analysis of centromere 9 showed three signals. By contrast, p53 was found to be with only 2 signals, suggesting a deletion of p53 in the atypical cells with the neartetraploid DNA. Sequencing of TP53 exons 4–9 showed no mutations. The immuno-phenotype of the cells by flow cytometry in our case fits with those of HCL-variant as determined by absence of CD25 (1). As was previously shown (2,3), the wild-type BRAF gene that we found in our case gives another molecular support to the diagnosis of HCL-variant. HCL-variant is difficult to differentiate from splenic diffuse red pulp small B-cell lymphoma. However, in our case we have several evidences that are in favor of HCL-variant and against splenic diffuse red pulp small B-cell lymphoma. First, the cells in our case are medium size whereas in splenic diffuse red pulp small B-cell lymphoma cell size should be small monomorphous and with common villous cytology. Second, TRAP staining was determined to be positive in the spleen in our case and according to WHO classification usually not present in splenic diffuse red pulp small B-cell lymphoma. To the best of our knowledge, our case represents the first documentation of HCL-variant with near-tetraploid DNA content in vivo. We found this case important to be reported as this neartetraploid DNA content was associated with atypical morphological appearance of the cells as shown by lack of typical Cytometry Part B (Clinical Cytometry) 94B:169–171 (2018)

Absolute lymphocyte count as a prognostic marker in newly diagnosed multiple myeloma patients.

Sir, Multiple myeloma (MM), a malignant disorder of plasma cells, is the second most common hematologic malignancy. Different prognostic strategies are used to stratify patients at the time of the diagnosis. The survival of MM patients has improved significantly in the last decade. This improvement can be attributed especially to the use of novel agents in the therapy of MM patients [1]. These include especially two classes of drugs: the immunomodulatory drugs (thalidomide, lenalidomide, and pomalidomide) and proteosome inhibitors (bortezomib, carfilzomib). Several studies suggest a prognostic significance for the absolute lymphocyte count (ALC), as a surrogate marker of the immune status [2], at diagnosis and after autologous stem cell transplantation (ASCT) [3] in patients with myeloma and that the quantitative numbers of CD19 cells at MM diagnosis are associated with superior survival [4]. Myeloma cells survival is dependent on microenvironmental interactions in their niche. Recently, the role of tumor-associated macrophages (TAM) was studied also in the pathogenesis of multiple myeloma. It was shown that macrophages support myeloma cell growth, viability, and drug resistance [5]. Increased peripheral blood absolute monocyte count (AMC) has been reported to be a poor prognostic factor in malignant lymphoproliferative disorders and also in solid tumors [6, 7]. The relationship between the number of circulating monocytes and clinical outcomes was mainly attributed to their role as precursors of TAM. A prognostic significance was also reported for the LMR (lymphocyte/monocyte ratio) in newly diagnosed myeloma patients [8] and also at the start of the conditioning regimen of autologous stem cell transplantation (ASCT) [9]. Until now, to our best knowledge, the statistical value of these measures has not been verified in patients treated with novel agents. We retrospectively evaluated these simple measurable markers, the AMC, the ALC, and the LMR at diagnosis of multiple myeloma patients in our hospital and evaluated their prognostic value in patients treated with novel agents. This study included new multiple myeloma patients who were diagnosed between 2006 and 2014 at Galilee Medical Center, Nahariya, Israel. The diagnosis was made according to the IMWG guidelines for symptomatic MM. Approval for review of the records was obtained from the Institutional Review Board of the Galilee Medical Center. AMC and ALC were obtained at diagnosis from routine complete blood count calculated by Advia 2120 hematology analyzer (Simens). We also collected demographic and clinical characteristics of the patients from their records. Quantity data were described by median and range. Qualitative data were described by frequencies and percentages. Comparisons of quantity data between groups were examined by independent sample t-test or Wilcoxon ranks sum test, as appropriate. Correlation of qualitative variables was examined by chi-squared test or Fisher’s exact test, as appropriate. Overall survival (OS) was measured from the time of diagnosis to the date of death or last follow-up. Univariate and multivariate survival analysis was performed using Cox Regression Model and Kaplan–Meier method with Log Rank (Mantel-Cox) test. All P-values represented were one-sided, and a pvalue<0.05 was considered statistically significant. Sixty-two newly diagnosed MM patients were evaluated. The median age was 69 years (range: 46–88 years); 35 (56.5%) patients were male and 27 (43.5%) female. The paraprotein type was as follows: 33 (55%) IgG, 14 (23.3%) light chain and 13 (21.7%) IgA; 15 (25%) patients were ISS I, 16 (26.7%) were ISS II, 29 (48.3%) were ISS III. Forty (64.5%) patients were treated with chemotherapy, 16 (25.8%) were treated with chemotherapy followed by ASCT, and 6 (9.7%) patients had conservative therapy. Fifty-one patients (82%) were treated with novel agents at diagnosis. The median follow-up for the whole cohort after the diagnosis was 28.2 mo (range 0.5–98.8 months). At the time of writing, 41 (66.1%) of patients were living. The optimal cutoff for the ALC and AMC, and the LMR was set at 1600/lL,

Elevated serum BDNF levels are associated with favorable outcome in CLL patients: Possible link to CXCR4 downregulation

Increased chemokine C-X-C receptor 4 (CXCR4) expression is related to unfavorable outcome in chronic lymphocytic leukemia (CLL). Brain-derived neurotrophic factor (BDNF) is a neuronal growth factor that has been shown previously to interact with CXCR4 in neuronal cells. Here, we studied the in vitro effect of BDNF on CXCR4 expression and chemotaxis toward stromal derived factor-1 (SDF-1) in freshly isolated CLL cells. We also explored the correlations between serum BDNF levels in CLL patients and disease characteristics and clinical course. Incubation of CLL cells with recombinant BDNF (50 ng/mL) resulted in a downregulation of CXCR4 surface expression and atenuated chemotaxis toward SDF-1. Higher serum BDNF levels were associated with a mutated immunoglobulin heavy chain variable (IGHV) gene, an early clinical stage, and a stable clinical course. Our findings suggest that increased circulating blood BDNF may be associated with a favorable effect in CLL. However, the exact mechanism of this favorable effect should be investigated further.

Treatment patterns and clinical outcomes in high‐risk newly diagnosed multiple myeloma patients carrying the 17p deletion: An observational multi‐center retrospective study

Del17p is a genomic imbalance occurring in ∼7%‐10% of myeloma at diagnosis newly diagnosed myeloma patients (NDMM) and comprises a poor prognostic factor. The goal of this study is to analyze real world data and outcomes among NDMM patients carrying 17p deletion. We report an observational, retrospective, multicenter study. Sixty consecutive patients diagnosed with multiple myeloma in the 8 participating centers diagnosed between 1/2008 and 1/2016 proven to carry 17p deletion by means of fluorescence in situ hybridization (FISH) were identified. Most received a bortezomib‐based induction, over half underwent autologous hematopoietic cell transplantation (HCT); 30% of the patients gained early access to new novel agents via clinical trials, access programs or private insurance. Overall response rate (ORR) after induction was 85%; 94% for transplant eligible (TE); and 75% for transplant ineligible (NTE), and declined in subsequent treatment lines, 64% achieved ≥ VGPR. Median overall survival (OS) was 43 months; median progression free survival (PFS) was 11 months, 19 months for TE and 7 for NTE. In multivariate analysis: higher M‐Spike, presence of extramedullary disease, and >50% of cells baring del17p were associated with adverse PFS; Autologous HCT and higher hemoglobin were associated with longer PFS; OS was 59 months for patients with early access to newer agents. Older age and higher M‐Spike levels were associated with adverse OS, Autologous HCT was associated with favorable OS, 59.7 vs 28.7 months for NTE patients. Despite the improvement achieved with autologous HCT and new novel agents, the prognosis of patients with 17p deletion is still inferior, emphasizing the need for novel approaches.

A Puzzling “Switch” in Blood Type Following Blood Transfusion

Luiza Akria, M.D., Judith Chezar, Ph.D., Simona Zisman-Rozen, Ph.D., Eyal J. Scheinman, Ph.D., Zeev Zonis, M.D., Yoav Hoffmann, M.D., Tzipora Falik-Zaccai, M.D., Limor Kalfon, Ph.D., Michael Weiss, M.D., Andrei Braester, M.D., Celia Suriu, M.D., Masad Barhoum, M.D., Amir Kuperman, M.D., and Ety Shaoul, Ph.D. Blood Bank and Molecular Hematology Laboratory, Pediatric Intensive Care Unit, Cytogenetic Laboratory, Department of Surgery, Hematology, and Pediatrics, Galilee Medical Center, Nahariya; Faculty of Medicine in the Galilee, Bar Ilan University, Safed, Israel