Feasibility and safety of exercise training and nutritional support prior to haematopoietic stem cell transplantation in patients with haematologic malignancies.

BACKGROUND: Prehabilitation with regular exercise and nutritional care for patients undergoing surgeries for malignant disease was recently introduced to increase physiologic reserve prior to the procedure, accelerate recovery and improve outcomes. This study aimed to investigate the feasibility and safety of combined exercise training and nutritional support in patients with haematologic malignancies prior to haematopoietic stem cell transplantation (HSCT). METHODS: In this single-arm pilot study, 34 HSCT candidates were enrolled at least two weeks before admission for the procedure. Patients performed aerobic exercises at least 4 days per week for 20-30 min and strength exercises 3 days per week for 10-20 min. They received daily supplements of whey protein (0.3-0.4 g/kg body weight) and oral nutritional supplements if needed. The primary endpoints were feasibility (acceptability > 75%, attrition < 20%, adherence > 66%) and safety. The secondary endpoints were fat-free mass (FFM), muscle strength, physical performance and health-related quality of life (HRQoL) at HSCT. RESULTS: The rate of acceptability, attrition and adherence to aerobic exercise, strength exercise and protein supplement consumption was 82.4, 17.8, 71, 78 and 80%, respectively. No severe adverse events were reported. Twenty-eight patients participated in the study for a median of 6.0 weeks (range, 2-14). They performed aerobic exercises 4.5 days per week for 132 min per week and strength exercises 3.0 times per week. Patients consumed 20.7 g of extra protein daily. At the end of the programme, we recorded increases of 1.1 kg in FFM (p = 0.011), 50 m in walking distance in the 6-min walking test (6MWT) (p < 0.001), 3.3 repetitions in the 30-s chair-stand test (30sCST) score (p < 0.001) and 2.6 kg in handgrip strength (p = 0.006). The EORTC QLQ-C30 scores improved by 8.6 (p < 0.006) for global health status, 8.3 (p = 0.009) for emotional functioning, and 12.1 (p = 0.014) for social functioning. There was less fatigue, nausea and insomnia (p < 0.05). CONCLUSIONS: Our study shows that a multimodal intervention programme with partially supervised exercise training combined with nutritional support prior to HSCT is feasible and safe. Patients showed improvements in FFM, physical performance and HRQoL. Additional research is needed to assess the possible positive effects of such interventions.

Multiple Myeloma Treatment in Real-world Clinical Practice: Results of a Prospective, Multinational, Noninterventional Study.

BACKGROUND: The present prospective, multinational, noninterventional study aimed to document and describe real-world treatment regimens and disease progression in multiple myeloma (MM) patients. PATIENTS AND METHODS: Adult patients initiating any new MM therapy from October 2010 to October 2012 were eligible. A multistage patient/site recruitment model was applied to minimize the selection bias; enrollment was stratified by country, region, and practice type. The patient medical and disease features, treatment history, and remission status were recorded at baseline, and prospective data on treatment, efficacy, and safety were collected electronically every 3 months. RESULTS: A total of 2358 patients were enrolled. Of these patients, 775 and 1583 did and did not undergo stem cell transplantation (SCT) at any time during treatment, respectively. Of the patients in the SCT and non-SCT groups, 49%, 21%, 14%, and 15% and 57%, 20%, 12% and 10% were enrolled at treatment line 1, 2, 3, and ≥ 4, respectively. In the SCT and non-SCT groups, 45% and 54% of the patients had received bortezomib-based therapy without thalidomide/lenalidomide, 12% and 18% had received thalidomide/lenalidomide-based therapy without bortezomib, and 30% and 4% had received bortezomib plus thalidomide/lenalidomide-based therapy as frontline treatment, respectively. The corresponding proportions of SCT and non-SCT patients in lines 2, 3, and ≥ 4 were 45% and 37%, 30% and 37%, and 12% and 3%, 33% and 27%, 35% and 32%, and 8% and 2%, and 27% and 27%, 27% and 23%, and 6% and 4%, respectively. In the SCT and non-SCT patients, the overall response rate was 86% to 97% and 64% to 85% in line 1, 74% to 78% and 59% to 68% in line 2, 55% to 83% and 48% to 60% in line 3, and 49% to 65% and 36% and 45% in line 4, respectively, for regimens that included bortezomib and/or thalidomide/lenalidomide. CONCLUSION: The results of our prospective study have revealed great diversity in the treatment regimens used to manage MM in real-life practice. This diversity was linked to factors such as novel agent accessibility and evolving treatment recommendations. Our results provide insight into associated clinical benefits.

Effects of Repetitive Transendocardial CD34+ Cell Transplantation in Patients With Nonischemic Dilated Cardiomyopathy.

RATIONALE: Preclinical data in heart failure models suggest that repetitive stem cell therapy may be superior to single-dose cell administration. OBJECTIVE: We investigated whether repetitive administration of CD34+ cells is superior to single-dose administration in patients with nonischemic dilated cardiomyopathy. METHODS AND RESULTS: Of 66 patients with dilated cardiomyopathy, New York Heart Association functional class III, and left ventricular ejection fraction (LVEF) <40% enrolled in the study, 60 were randomly allocated to repetitive cell therapy (group A, n=30) or single-cell therapy (group B, n=30). Patients received G-CSF (granulocyte colony-stimulating factor) for 5 days, and 80 million CD34+ cells were collected by apheresis and injected transendocardially. In group A, cell therapy was repeated at 6 months. All patients were followed for 1 year, and the primary end point was the difference in change in LVEF between the groups. At baseline, the groups did not differ in age, sex, LVEF, NT-proBNP (N-terminal pro-B-type natriuretic peptide), or 6-minute walk test distance. When directly comparing groups A and B at 1 year, there was no significant difference in change in LVEF (from 32.2±9.3% to 41.2±6.5% in group A and from 30.0±7.0% to 37.9±5.3% in group B, P=0.40). From baseline to 6 months, both groups improved in LVEF (+6.9±3.3% in group A, P=0.001 and +7.1±3.5% in group B, P=0.001), NT-proBNP (-578±211 pg/mL, P=0.02 and -633±305 pg/mL, P=0.01), and 6-minute walk test (+87±21 m, P=0.03 and +92±25 m, P=0.02). In contrast, we observed no significant changes between 6 months and 1 year (LVEF: +2.1±2.3% in group A, P=0.19 and +0.8±3.1% in group B, P=0.56; NT-proBNP: -215±125 pg/mL, P=0.26 and -33±205 pg/mL, P=0.77; 6-minute walk test: +27±11 m, P=0.2 and +12±18 m, P=0.42). CONCLUSIONS: In patients with dilated cardiomyopathy, repetitive CD34+ cell administration does not seem to be associated with superior improvements in LVEF, NT-proBNP, or 6-minute walk test when compared with single-dose cell therapy. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02248532.

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.

CpG-ODN Stimulation in Population Screening Monoclonal B-Cell Lymphocytosis.

BACKGROUND: Synthetic CpG-oligodeoxynucleotides (CpG-ODN) induce proliferation in normal and malignant lymphocytes B (LyB). This effect is widely exploited in CLL conventional chromosome banding analysis (CBA), which has become reliable only after the cultivation of CLL LyB using CpG-ODN stimulation in combination with IL-2. Monoclonal B-cell lymphocytosis (MBL) differs from CLL mainly in clone size. Only cytogenetic data on recurrent chromosomal aberrations analyzed by fluorescence in situ hybridization (FISH) are available for population screening MBL (sMBL). In sMBL the clone of malignant LyB is typically below 10/µL. We compared CpGODN stimulation in healthy donors and in individuals with sMBL. METHODS: LyB and MBL LyB count were determined by flow cytometry in 15 samples from healthy subjects and 12 MBL cases. Mitotic indices were determined and CBA was done after cultivation of samples by CpG-ODN + IL2. In MBL samples, FISH analysis was performed on isolated LyB. RESULTS: MBL LyB clones in sMBL cases presented less than 1% of WBC and up to 33% of LyB. The MBL group was therefore compared to the group of healthy donors. Although normal and MBL group did not differ in WBC, overall LyB, and normal LyB count, a significantly higher mitotic index was observed in MBL samples (p = 0.0139). We were able to accomplish CBA in all samples which revealed a normal karyotype in all but one case. In this particular sMBL case FISH performed on isolated LyB showed 5% trisomy 12 which was later confirmed by CBA on CpG stimulated blood sample in 15% of metaphases. CONCLUSIONS: Our study, which was done on MBL cases obtained by population screening, confirmed that CpGODN preferentially induced proliferation in MBL LyB over normal LyB. Therefore, CBA can also be successfully accomplished in sMBL and can be used to additionally confirm clonality as well as to improve sensitivity of FISH analysis. Due to coexistence of comparable size of normal and malignant LyB, MBL can serve as a model for exvivo studying of LyB stimulation by CpG-ODN.

Effects of Transendocardial CD34+ Cell Transplantation on Diastolic Parameters in Patients with Nonischemic Dilated Cardiomyopathy.

We sought to evaluate the physiological background and the effects of CD34+ cell transplantation on diastolic parameters in nonischemic dilated cardiomyopathy patients (DCM). We enrolled 38 DCM patients with NYHA class III and LVEF < 40% who underwent transendocardial CD34+ cell transplantation. Peripheral blood CD34+ cells were mobilized by G-CSF, collected via apheresis, and injected transendocardially in the areas of myocardial hibernation. Patients were followed for 1 year. At baseline, estimated filling pressures were significantly elevated (E/e' ≥ 15) in 18 patients (Group A), and moderately elevated (E/e '< 15) in 20 patients (Group B). The groups did not differ in age (54 ± 9 years vs. 52 ± 10 years; p = .62), gender (male: 85% vs. 78%; p = .57), or LVEF (31 ± 7% vs. 34 ± 6%; p = .37). When compared to Group B patients in Group A had more segments with myocardial scar (4.9 ± 2.7 vs. 2.7 ± 2.9; p = .03), myocardial hibernation (2.2 ± 1.6 vs. 0.9 ± 1.1; p = .02), and longer average local relaxation time on electroanatomical mapping (378 ± 41 ms vs. 333 ± 34 ms, p = .01). During follow-up there was an improvement in diastolic parameters in Group A (E/e': from 24.3 ± 12.1 to 16.3 ± 8.0; p = .005), but not in Group B (E/e': from 10.2 ± 3.7 to 13.2 ± 9.1; p = .19). Accordingly, in Group A, we found an increase in 6-minute walk distance (from 463 ± 83 m to 546 ± 91 m; p = .03), and a decrease in NT-proBNP (from 2140 ± 1743 pg/ml to 863 ± 836 pg/ml; p = .02). In nonischemic DCM, diastolic dysfunction appears to correlate with areas of myocardial scar and hibernation. Transendocardial CD34+ cell transplantation may improve diastolic parameters in this patient cohort. Stem Cells Translational Medicine 2017;6:1515-1521.

Mobilization with cyclophosphamide reduces the number of lymphocyte subpopulations in the leukapheresis product and delays their reconstitution after autologous hematopoietic stem cell transplantation in patients with multiple myeloma.

BACKGROUND: Autologous hematopoietic stem cell transplantation is considered the standard of care for younger patients with multiple myeloma. Several mobilization regimens are currently used, most commonly growth factors alone or in combination with chemotherapy. The aim of our study was to investigate the differences in lymphocyte subpopulation counts between three different mobilization regimens on collection day, in the leukapheresis product and on day 15 after autologous hematopoietic stem cell transplantation. PATIENTS AND METHODS: In total 48 patients were prospectively enrolled in three different mobilization regimens; (i) filgrastim (20), (ii) pegfilgrastim (19) and (iii) cyclophosphamide + filgrastim (9). Lymphocytes, CD16+/56+ natural killer and CD4+/CD25high T regulatory cells were determined by flow cytometry. RESULTS: We found a statistically significant difference between the mobilization regimens. Cyclophosphamide reduced lymphocyte and natural killer (NK) cell counts on collection day (lymphocytes 1.08 × 109/L; NK cells 0.07 × 109/L) compared to filgrastim (lymphocytes 3.08 × 109/L; NK cells 0.52 × 109/L) and pegfilgrastim (lymphocytes 3 × 109/L; NK cells 0.42 × 109/L). As a consequence lymphocyte and NK cell counts were also lower in the leukapheresis products following cyclophosphamide mobilization regimen (lymphocytes 50.1 × 109/L; NK cells 4.18 × 109/L) compared to filgrastim (lymphocytes 112 × 109/L; NK cells 17.5 × 109/L) and pegfilgrastim (lymphocytes 112 × 109/L; NK cells 14.3 × 109/L). In all mobilization regimens T regulatory cells increased 2-fold on collection day, regarding the base line value before mobilization. There was no difference in T regulatory cell counts between the regimens. CONCLUSIONS: Mobilization with cyclophophamide reduces the number of mobilized and collected lymphocytes and NK cells as compared to mobilization with growth factors only and results in their delayed reconstitution following autologous hematopoietic stem cell transplantation. We found no difference between filgrastim and pegfilgrastim mobilization.

Imaging and 1-day kinetics of intracoronary stem cell transplantation in patients with idiopathic dilated cardiomyopathy.

BACKGROUND: Stem cell transplantation is an emerging method of treatment for patients with cardiovascular disease. There are few studies completed or ongoing on stem cell therapy in patients with idiopathic dilated cardiomyopathy (IDCM). Information on stem cell homing and distribution in the myocardium after transplantation might provide important insight into effectiveness of transplantation procedure. AIM: To assess early engraftment, retention and migration of intracoronarily transplanted stem cells in the myocardium of patients with advanced dilated cardiomyopathy of non-ischaemic origin using stem cell labeling with (99m)Tc-exametazime (HMPAO). MATERIALS, METHODS: Thirty-five patients with IDCM and advanced heart failure were included in the study. Autologous hematopoietic (CD34+) stem cells were harvested by peripheral blood apheresis after bone marrow stimulation, labeled with (99m)Tc-HMPAO, tested for viability and injected into coronary vessel supplying areas of myocardium selected by myocardial perfusion scintigraphy as dysfunctional yet viable. Imaging was performed 1h and 18h after transplantation. RESULTS: Myocardial stem cell retention ranged from 0 to 1.44% on early and 0-0.97% on delayed imaging. Significant efflux of stem cells occurred from site of delivery in this time period (p<0.001). Stem cell viability was not affected by labeling. CONCLUSION: Stem cell labeling with (99m)Tc-HMPAO is a feasible method for stem cell tracking after transplantation in patients with IDCM.

Efficacy of CD34+ Stem Cell Therapy in Nonischemic Dilated Cardiomyopathy Is Absent in Patients With Diabetes but Preserved in Patients With Insulin Resistance.

UNLABELLED: We evaluated the association of diabetes and insulin resistance with the response to cell therapy in patients with nonischemic dilated cardiomyopathy (DCM). A total of 45 outpatients with DCM received granulocyte colony-stimulating factor for 5 days. CD34(+) cells were then collected by apheresis and injected transendocardially. Twelve patients had diabetes mellitus (DM group), 17 had insulin resistance (IR group), and 16 displayed normal glucose metabolism (no-IR group). After stimulation, we found higher numbers of CD34(+) cells in the IR group (94 ± 73 × 10(6) cells per liter) than in the no-IR group (54 ± 35 × 10(6) cells per liter) or DM group (31 ± 20 × 10(6) cells per liter; p = .005). Similarly, apheresis yielded the highest numbers of CD34(+) cells in the IR group (IR group, 216 ± 110 × 10(6) cells; no-IR group, 127 ± 82 × 10(6) cells; DM group, 77 ± 83 × 10(6) cells; p = .002). Six months after cell therapy, we found an increase in left ventricular ejection fraction in the IR group (+5.6% ± 6.9%) and the no-IR group (+4.4% ± 7.2%) but not in the DM group (-0.9% ± 5.4%; p = .035). The N-terminal pro-brain natriuretic peptide levels decreased in the IR and no-IR groups, but not in the DM group (-606 ± 850 pg/ml; -698 ± 1,105 pg/ml; and +238 ± 963 pg/ml, respectively; p = .034). Transendocardial CD34(+) cell therapy appears to be ineffective in DCM patients with diabetes. IR was associated with improved CD34(+) stem cell mobilization and a preserved clinical response to cell therapy. SIGNIFICANCE: The present study is the first clinical study directly evaluating the effects of altered glucose metabolism on the efficacy of CD34(+) stem cell therapy in patients with nonischemic dilated cardiomyopathy. The results offer critical insights into the physiology of stem cell mobilization in heart failure and possibly an explanation for the often conflicting results obtained with stem cell therapy for heart failure. These results demonstrate that patients with dilated cardiomyopathy and diabetes do not benefit from autologous CD34(+) cell therapy. This finding could serve as a useful tool when selecting heart failure patients for future clinical studies in the field of stem cell therapy.

Exceptions in patterns of arsenic compounds in urine of acute promyelocytic leukaemia patients treated with As2O3.

Arsenic trioxide (As(III) in solution) has been shown to be the most active single agent in combating acute promyelocytic leukemia (APL). It is metabolized and excreted via urine as monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and As(V), along with excess As(III). In our study eight APL patients were treated (intravenously) with 0.15 mg As2O3/kg/day. During the therapy As(III) and its metabolites were followed in pre- and post-infusion urine using HPLC for separation followed by on-line detection using hydride generation-atomic fluorescence spectrometry. Five patients had a normal excretion pattern of residual arsenic compounds in morning pre-infusion urine, with 15-25% of As(III), 35-55% of DMA, 25-30% of MMA and 1-5% of As(V), while three patients showed unexpected exceptions from typical excretion patterns of arsenic compounds (i) a high DMA/MMA ratio (factor 5.3), (ii) severe As(III) oxidation (10.2% As(III) converted to As(V)) or (iii) the presence of an excessive amount of As(III) (average 30.4% of total arsenic). Intriguing was the occurrence of post-infusion oxidation of As(III) to As(V) observed in almost all patients and being especially high (>40%) in patient with increased residual As(V). Results indicate that arsenic metabolites patterns can be unpredictable. Observed high levels of un-metabolised As(III) are a warning signal for side effects and for routine determination of arsenic metabolites during first days of treatment. High or low percentages of MMA or DMA did not show any observable effect on treatment results, while clear presence of post-infusion As(V) supports theoretical claims of in vivo oxidation (detoxification) of As(III) to As(V) associated with various metabolic processes.

Increased red cell distribution width is associated with poor stem cell mobilization in patients with advanced chronic heart failure.

Parameters associated with poor CD34(+) stem cell mobilization in advanced chronic heart failure (CHF) patients were investigated. Forty-four CHF patients underwent bone marrow stimulation with granulocyte colony stimulating factor. Poor cell mobilization presents in 32% of patients. Poor and good mobilizers did not differ significantly regarding age, gender, left ventricular ejection fraction, kidney or liver function and exercise capacity. Significant differences were found regarding NT-proBNP levels and red cell distribution width (RDW). Increased RDW was the only independent predictor of poor CD34(+) stem cell mobilization on multivariable analysis and may serve as a biomarker of poor stem cell mobilization in CHF patients.

Intracoronary transplantation of CD34(+) cells is associated with improved myocardial perfusion in patients with nonischemic dilated cardiomyopathy.

BACKGROUND: We investigated the effects of intracoronary transplantation of CD34(+) cells on myocardial perfusion in patients with nonischemic dilated cardiomyopathy (DCM). METHODS AND RESULTS: We enrolled 21 patients with DCM (left ventricular ejection fraction [LVEF] <40%, New York Heart Association functional class III) who underwent peripheral stem cell mobilization with granulocyte-colony stimulating factor (G-CSF). CD34(+) cells were collected by means of apheresis. Patients underwent myocardial perfusion imaging, and CD34(+) cells were injected in the coronary artery supplying viable segments with reduced myocardial perfusion and regional dysfunction. Myocardial perfusion imaging was repeated 6 months later. Clinical response to stem cell therapy was predefined as a change in LVEF >5%. The majority of patients were men (81%) with an overall mean age 53 ± 9 years, LVEF 25 ± 5%, and 6-minute walking distance 354 ± 71 m. Myocardial perfusion defects at rest were observed in 86% of patients and were more common in the left anterior descending territory (50%). At 6 months' follow-up, there was a significant improvement in rest myocardial perfusion scores (6.3 ± 5.8 vs 3.1 ± 4.3; P < .001), LVEF (25 ± 7% vs 29 ± 8%; P = .005), and 6-minute walking distance (354 ± 71 m vs 404 ± 91 m; P < .001). Responders to stem cell therapy had lower summed rest perfusion score at both baseline (3.2 ± 3.0 vs 9.1 ± 6.3; P = .015) and follow-up (1.0 ± 1.5 vs 5.0 ± 5.1; P = .028). CONCLUSIONS: CD34(+) cell transplantation may lead to improved myocardial perfusion in patients with nonischemic DCM. Patients with less severe myocardial perfusion defects at baseline may have an increased likelihood to respond to intracoronary CD34(+) cell transplantation.

Effects of transendocardial CD34+ cell transplantation in patients with ischemic cardiomyopathy.

BACKGROUND: We investigated the effects of transendocardial CD34(+) cell transplantation in patients with ischemic cardiomyopathy. METHODS AND RESULTS: In a prospective crossover study, we enrolled 33 patients with ischemic cardiomyopathy with New York Heart Association class III and left ventricular ejection fraction <40%. In phase 1, patients were treated with medical therapy for 6 months. Thereafter, all patients underwent transendocardial CD34(+) cell transplantation. Peripheral blood CD34(+) cells were mobilized by granulocyte colony stimulating factor, collected via apheresis, and injected transendocardially in the areas of hibernating myocardium. Patients were followed up for 6 months after the procedure (phase 2). Two patients died during phase 1 and none during phase 2. The remaining 31 patients were 85% men, aged 57±6 years. In phase 1, we found no change in left ventricular ejection fraction (from 25.2±6.2% to 27.1±6.6%; P=0.23), N-terminal pro B-type natriuretic peptide (from 3322±3411 to 3672±5165 pg/mL; P=0.75) or 6-minute walk distance (from 373±68 to 411±116 m; P=0.17). In contrast, in phase 2 there was an improvement in left ventricular ejection fraction (from 27.1±6.6% to 34.9±10.9%; P=0.001), increase in 6-minute walk distance (from 411±116 to 496±113 m; P=0.001), and a decrease in N-terminal pro B-type natriuretic peptide (from 3672±5165 to 1488±1847 pg/mL; P=0.04). The average number of injected CD34(+) cells was 90.6±7.5×10(6). Higher doses of CD34(+) cells and a more diffuse distribution of transendocardial cell injections were associated with better clinical response. CONCLUSIONS: Transendocardial CD34(+) cell transplantation may be associated with improved left ventricular function, decreased N-terminal pro B-type natriuretic peptide levels, and better exercise capacity in patients with ischemic cardiomyopathy. These effects seem to be particularly pronounced in patients receiving diffusely distributed cell injections and high-dose cell therapy. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01350310.

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.

Absorption and elimination of imatinib through the rat intestine in vitro.

Imatinib is a potent selective inhibitor of tyrosine kinases and is used primarily in the treatment of chronic myeloid leukemia and the gastrointestinal stromal tumour. Although, it is well established that imatinib is a substrate of several transport proteins which are also active in the intestinal mucosa, the mechanisms of imatinib intestinal absorption and elimination were not systematically investigated yet. To do that, we used a Sweetana-Grass type of diffusion chambers with segments of rat intestine as a model of the intestinal mucosa, measured the permeability coefficients of imatinib and its major metabolite (N-desmethyl imatinib) in both directions with and without specific and general inhibition of active transport, and calculated the efflux ratios. The results show that the good bioavailability of imatinib is highly likely achieved by its active absorption from the intestine and that its active elimination through the intestinal mucosa is mediated by a synergistic activity of organic cation transporter 1 in the basolateral membrane and the added activity of two efflux proteins (P-glycoprotein and breast cancer resistant protein) in the apical membrane of enterocytes of the rat ileum. Interestingly, it was found that N-desmethyl imatinib is only transported by P-glycoprotein.

Concurrent rearrangements of BCL2, BCL3, and BCL11A genes in atypical chronic lymphocytic leukemia.

The most frequent chromosomal aberrations with the well established prognostic meaning in chronic lymphocytic leukemia (CLL) are +12, del(11q), del(13q), and del(17p). Less common translocations lead to deregulation of genes primarily due to juxtaposition with IGH gene. We present a case of CLL patient with atypical morphology and an aggressive course of disease. In spite of aggressive treatment including allogeneic hematopoietic stem cell transplantation disease progressed into a rare cutaneous Richter's syndrome. Trisomy 12 was found as a sole chromosomal change at initial cytogenetic analysis of lymphoma cells. At progression, besides trisomy 12 three concomitant balanced translocations t(2;14)(p13;q32), t(14;19)(q32;q13), and t(18;22)(q21;q11) were found. The same karyotype was confirmed in cells aspirated from skin infiltrates at Richter transformation. Atypical cytological features, trisomy 12, and a progressive course of disease observed in our case are typical for CLL with each of particular Ig translocations that were concomitantly found in CLL for the first time. Similar to "double hit" lymphoma concurrent rearrangements may be relevant also in CLL.

Comparison of transendocardial and intracoronary CD34+ cell transplantation in patients with nonischemic dilated cardiomyopathy.

BACKGROUND: In an open-label blinded study, we compared intracoronary and transendocardial CD34(+) cell transplantation in patients with nonischemic dilated cardiomyopathy. METHODS AND RESULTS: Of the 40 patients with dilated cardiomyopathy, 20 were randomized to receive intracoronary injection and 20 received transendocardial CD34(+) cell delivery. In both groups, CD34(+) cells were mobilized by filgrastim, collected via apheresis, and labeled with technetium-99m radioisotope for single-photon emission computed tomographic imaging. In the intracoronary group, cells were injected intracoronarily in the artery supplying segments of greater perfusion defect on myocardial perfusion scintigraphy. In the transendocardial group, electroanatomic mapping was used to identify viable but dysfunctional myocardium, and transendocardial cell injections were performed. Nuclear single-photon emission computed tomographic imaging for quantification of myocardial retention was performed 18 hours thereafter. At baseline, groups did not differ in age, sex, left ventricular ejection fraction, or N-terminal pro-brain natriuretic peptide levels. The number of CD34(+) cells was also comparable (105 ± 31 × 10(6) in the transendocardial group versus 103 ± 27 × 10(6) in the intracoronary group, P=0.62). At 18 hours after procedure, myocardial retention was higher in the transendocardial group (19.2 ± 4.8%) than in the intracoronary group (4.4 ± 1.2%, P<0.01). At 6 months, left ventricular ejection fraction improved more in the transendocardial group (+8.1 ± 4.3%) than in the intracoronary group (+4.2 ± 2.3%, P=0.03). The same pattern was observed for the 6-minute walk test distance (+125 ± 33 m in the transendocardial group versus +86 ± 13 m in the intracoronary group, P=0.03) and N-terminal pro-brain natriuretic peptide (-628 ± 211 versus -315 ± 133 pg/mL, P=0.04). CONCLUSIONS: In patients with dilated cardiomyopathy, transendocardial CD34(+) cell transplantation is associated with higher myocardial retention rates and greater improvement in ventricular function, N-terminal pro-brain natriuretic peptide, and exercise capacity compared with intracoronary route. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01350310.

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.

Monitoring of imatinib targeted delivery in human leukocytes.

The success of imatinib therapy in chronic myeloid leukemia is highly influenced by its active transport into target cells. However, the methodology for analytical evaluation of intracellular drug concentration is rare and usually reliant upon the use of radioactively labeled drugs. More specifically, there is no published method available in the literature for the determination of imatinib concentration in granulocytes. To gain further insight into the intracellular drug uptake a very reliable two-stage sample concentration procedure was devised and coupled with a sensitive ultra-high performance liquid chromatography tandem mass spectrometry. The reliability of this sample preparation and sensitivity of the analysis was confirmed by a successful validation of all necessary method parameters to an impressive lower limit of quantification of 0.5 ng imatinib per 10(6) cells still at the signal to noise ratio of 670. The usefulness of the method is further improved with only 6 mL of blood being necessary for patient analysis. The method has been applied to blood samples of 13 CML patients treated with imatinib and all the measured intracellular drug concentrations were within the validated range. These and further measurements will enable the research of factors which may, besides blood plasma concentration, influence the individual's response to imatinib therapy. Furthermore, individualisation of dosing based on the directly measured targeted drug delivery could be possible.

Rare manifestation of the thrombotic microangiopathy in a patient with sarcoidosis, common variable immunodeficiency and large B-cell non-hodgkin lymphoma: case report.

58-year old Caucasian woman was diagnosed with sarcoidosis. Low immunoglobulin levels were found and common variable immunodeficiency (CVID) was diagnosed 1year later. Laboratory tests and clinical course at this time revealed thrombotic microangiopathy (TMA). Therapeutic plasma exchange was started and her clinical status and laboratory parameters improved. According to CVID she received human immunoglobulin intravenously. Four months later we noticed swelling of the parotid glands and generalized lymphadenopathy. Histology of cervical lymph node confirmed large B-cell non-Hodgkin lymphoma (B-cell NHL). To the best of our knowledge, TMA complicating the course of sarcoidosis, CVID and B-cell NHL has never been reported.