The value of routine bone marrow biopsy in patients with diffuse large B-cell lymphoma staged with PET/CT: a Danish-Canadian study.

BACKGROUND: The added diagnostic and prognostic value of routine bone marrow biopsy (BMB) in patients with diffuse large B-cell lymphoma (DLBCL) undergoing positron emission tomography combined with computed tomography (PET/CT) staging is controversial. PATIENTS AND METHODS: Patients with newly diagnosed DLBCL who underwent both staging PET/CT and BMB were retrospectively identified in British Columbia, Aalborg, and Copenhagen. Original written PET/CT and pathology reports were retrospectively reviewed to determine Ann Arbor stage and outcomes, with and without the contribution of BMB. RESULTS: A total of 530 patients were identified: 146 (28%) had focal bone marrow (BM) lesions on PET/CT and 87 (16%) had positive BMB. Fifty-two of 146 patients (36%) with positive PET/CT had a positive BMB [39 DLBCL, 13 indolent non-Hodgkin lymphoma (iNHL)], while 35 of 384 patients (9%) with negative PET/CT had positive BMB (12 DLBCL, 23 iNHL). BMB upstaged 12/209 (6%) of stage I/II patients to stage IV, although this was the case for only 3 (1%) patients with DLBCL in the BMB. PET/CT identified BM involvement by BMB with sensitivity 60%, specificity 79%, positive predictive value 36%, and negative predictive value 91%. Concordant histological involvement of the BM by DLBCL was associated with worse overall survival and progression-free survival than discordant or no involvement in univariate and multivariate analyses. CONCLUSIONS: In patients with DLBCL, staging PET/CT can miss BM involvement with concordant DLBCL (less common) or discordant iNHL (more common). Routine BMB does not add relevant diagnostic or prognostic value over PET/CT alone in the majority of patients with DLBCL.

Management of relapsed multiple myeloma: recommendations of the International Myeloma Working Group.

The prognosis for patients multiple myeloma (MM) has improved substantially over the past decade with the development of new, more effective chemotherapeutic agents and regimens that possess a high level of anti-tumor activity. In spite of this important progress, however, nearly all MM patients ultimately relapse, even those who experience a complete response to initial therapy. Management of relapsed MM thus represents a vital aspect of the overall care for patients with MM and a critical area of ongoing scientific and clinical research. This comprehensive manuscript from the International Myeloma Working Group provides detailed recommendations on management of relapsed disease, with sections dedicated to diagnostic evaluation, determinants of therapy, and general approach to patients with specific disease characteristics. In addition, the manuscript provides a summary of evidence from clinical trials that have significantly impacted the field, including those evaluating conventional dose therapies, as well as both autologous and allogeneic stem cell transplantation. Specific recommendations are offered for management of first and second relapse, relapsed and refractory disease, and both autologous and allogeneic transplant. Finally, perspective is provided regarding new agents and promising directions in management of relapsed MM.

Performance comparison of Affymetrix SNP6.0 and cytogenetic 2.7M whole-genome microarrays in complex cancer samples.

The Affymetrix cytogenetic 2.7M whole-genome microarray (Cyto2.7M) detects genomic aberrations. The Cyto2.7M array has increased coverage in regions with cancer-related genes, ~4-fold reduced processing time, and 5-fold reduced input requirements (100 ng) compared to the commonly used Affymetrix SNP6.0 genome-wide microarray (SNP6.0). We set out to compare the performance of these microarrays on cancer samples containing complex genomic changes. We analyzed genomic DNA from 8 lymphoma samples and 1 blood sample using both SNP6.0 and Cyto2.7M microarrays. We compared the arrays with respect to 4 parameters, including detection of copy number variations (CNV), CNV boundaries, the actual copy number (CN) assigned to the aberrations, and loss of heterozygosity. The CN state of selected regions was validated by quantitative PCR. Very high consistency between arrays on all parameters tested was observed, hence only 30 of 224 aberrations disagreed on the CN state, corresponding to a total of ~12 Mb or 0.06% of the analyzed base pairs. Thus, the SNP6.0 and Cyto2.7M arrays are equally well suited to detect genomic aberrations in complex samples such as cancer samples. With reduced processing time and lower input requirements, the Cyto2.7M array enables genomic analysis of samples where only limited DNA is available.

In a high-dose melphalan setting, palifermin compared with placebo had no effect on oral mucositis or related patient's burden.

This randomized-controlled trial studied the efficacy of palifermin in a chemotherapy-only, high-dose Melphalan (HDM) transplant setting, to reduce oral mucositis (OM) and its sequelae measured by patient-reported outcomes (PRO) and medical resource use. Palifermin, relative to placebo was given either pre-/post-HDM or pre-HDM in patients with multiple myeloma (MM) undergoing auto-SCT at 39 European centers. Oral cavity assessment (WHO) and PRO questionnaires (oral mucositis daily questionnaire (OMDQ) and EQ 5D) were used in 281 patients (mean age 56, ± s.d.=8 years). 57 patients received placebo. One hundred and fifteen subjects were randomized to pre-/post-HDM receiving palifermin on 3 consecutive days before HDM and after auto-SCT and 109 patients were randomized to pre-HDM, receiving palifermin (60 µg/kg/day) i.v. for 3 consecutive days before HDM. There was no statistically significant difference in maximum OM severity. Severe OM occurred in 37% (placebo), 38% (pre-/post-HDM) and 24% (pre-HDM) of patients. No significant difference was observed with respect to PRO assessments or medical resource use, but more infections and fever during neutropenia were reported in pre-/post-HDM vs placebo (for example, 51 and 26%). To conclude, palifermin was unable to reduce OM or OM-related patient's burden in MM transplant patients.

Competition between clonal plasma cells and normal cells for potentially overlapping bone marrow niches is associated with a progressively altered cellular distribution in MGUS vs myeloma.

Disappearance of normal bone marrow (BM) plasma cells (PC) predicts malignant transformation of monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) into symptomatic multiple myeloma (MM). The homing, behavior and survival of normal PC, but also CD34(+) hematopoietic stem cells (HSC), B-cell precursors, and clonal PC largely depends on their interaction with stromal cell-derived factor-1 (SDF-1) expressing, potentially overlapping BM stromal cell niches. Here, we investigate the distribution, phenotypic characteristics and competitive migration capacity of these cell populations in patients with MGUS, SMM and MM vs healthy adults (HA) aged >60 years. Our results show that BM and peripheral blood (PB) clonal PC progressively increase from MGUS to MM, the latter showing a slightly more immature immunophenotype. Of note, such increased number of clonal PC is associated with progressive depletion of normal PC, B-cell precursors and CD34(+) HSC in the BM, also with a parallel increase in PB. In an ex vivo model, normal PC, B-cell precursors and CD34(+) HSC from MGUS and SMM, but not MM patients, were able to abrogate the migration of clonal PC into serial concentrations of SDF-1. Overall, our results show that progressive competition and replacement of normal BM cells by clonal PC is associated with more advanced disease in patients with MGUS, SMM and MM.

Priming with r-metHuSCF and filgrastim or chemotherapy and filgrastim in patients with malignant lymphomas: a randomized phase II pilot study of mobilization and engraftment.

SCF has been shown to synergize with G-CSF to mobilize CD34(+) PBPCs. In this study we report results from this combination after a phase II trial of 32 patients with malignant lymphoma randomized to receive recombinant methionyl human SCF (ancestim, r-metHuSCF) in combination with recombinant methionyl human G-CSF (filgrastim, r-metHuG-CSF) (experimental arm A) or routine chemotherapy plus filgrastim (conventional arm B). The primary objective was to evaluate the side effects and toxicity during priming and mobilization. The secondary objectives were efficacy by the level of blood-circulating PBPCs, the number of harvest days and the time to three-lineage engraftment after autografting. First, during priming 5 patients had 8 serious events, 4 in each arm. A summary of all adverse events revealed 30 (94%) patients suffering from 132 events of all grading. Second, neutropenia and thrombocytopenia was documented in arm B. Third, 9/14 (64%) patients in arm A reached the target of 5 million CD34(+) cells/kg body weight (bw) compared with 13/15 (87%) in arm B. The results represent the first randomized trial of growth factor plus chemotherapy priming and indicate that a formal phase III trial very unlikely may challenge chemotherapy plus r-metHuG-CSF priming in candidates for high-dose therapy.

Increased level of both CD4+FOXP3+ regulatory T cells and CD14+HLA-DR⁻/low myeloid-derived suppressor cells and decreased level of dendritic cells in patients with multiple myeloma.

Patients with multiple myeloma (MM) suffer from a general impaired immunity comprising deficiencies in humoral responses, T-cell responses as well as dendritic cell (DC) function. Thus, to achieve control of tumour growth through immune therapy constitutes a challenge. Careful evaluation of the immune status in patients with MM seems crucial prior to active immune therapy. We evaluated the proportion of both, DC, Treg cells and myeloid-derived suppressor cells (MDSC) in peripheral blood from patients with MM at diagnosis and in remission as well as patients with monoclonal gammopathy of undetermined significance (MGUS). We found that the proportion of both myeloid (m) DC and plasmacytoid (p) DC in patients at diagnosis was lowered compared to control donors, while only the proportion of pDC in patients in remission and with MGUS was significantly lower than in controls. The proportion of CD4+FOXP3+ Treg cells was increased in patients at diagnosis and not in patients in remission or with MGUS. Also, Treg cells from patients with MM were functionally intact as they were able to inhibit proliferation of both CD4 and CD8 T cells. Finally, we observed an increase in the proportion of CD14+HLA-DR⁻/low MDSC in patients with MM at diagnosis, illustrating that this cell fraction is also distorted in patients with MM. Taken together, our results illustrate that, both mDC, pDC, Treg cells and MDSC are affected in patients with MM underlining the fact that the immune system is dysregulated as a consequence of the disease.

Human peripheral blood B-cell compartments: a crossroad in B-cell traffic.

A relatively high number of different subsets of B-cells are generated through the differentiation of early B-cell precursors into mature B-lymphocytes in the bone marrow (BM) and antigen-triggered maturation of germinal center B-cells into memory B-lymphocytes and plasmablasts in lymphoid tissues. These B-cell subpopulations, which are produced in the BM and lymphoid tissues, recirculate through peripheral blood (PB), into different tissues including mucosa and the BM, where long-living plasma cells produce antibodies. These circulating PB B-cells can be classified according to their maturation stage into i) immature/transitional, ii) naïve, and iii) memory B-lymphocytes, and iv) plasmablasts/plasma cells. Additionally, unique subsets of memory B-lymphocytes and plasmablasts/plasma cells can be identified based on their differential expression of unique Ig-heavy chain isotypes (e.g.: IgM, IgD, IgG, IgA). In the present paper, we review recent data reported in the literature about the distribution, immunophenotypic and functional characteristics of these cell subpopulations, as well as their distribution in PB according to age and seasonal changes. Additional information is also provided in this regard based on the study of a population-based cohort of 600 healthy adults aged from 20 to 80 years, recruited in the Salamanca area in western Spain. Detailed knowledge of the distribution and traffic of B-cell subsets through PB mirrors the immune status of an individual subject and it may also contribute to a better understanding of B-cell disorders related to B-cell biology and homeostasis, such as monoclonal B-cell lymphocytosis (MBL).

Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy.

OBJECTIVE: Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly used for MSC cultivation in animal studies simulating clinical stem cell therapy. MATERIAL AND METHODS: Human mononuclear cells (MNCs) were isolated from BM aspirates by density gradient centrifugation and cultivated in a GMP-accepted medium (EMEA medium) or in one of four other media. RESULTS: FACS analysis showed that the plastic-adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation with vascular endothelial growth factor (VEGF) increased expression of the characteristic endothelial genes KDR and von Willebrand factor; the von Willebrand factor and CD31 at protein level as well as the capacity to develop capillary-like structures. CONCLUSIONS: We established culture conditions with a GMP compliant medium for MSC cultivation, expansion and differentiation. The expanded and differentiated MSCs can be used in autologous mesenchymal stromal cell therapy in patients with ischaemic heart disease.

Antigen-specific T-cell immunity in multiple myeloma patients is restored following high-dose therapy: implications for timing of vaccination.

The present study analyses the influence of high-dose chemotherapy (HD) and autologous stem cell transplantation on natural and vaccine induced specific immunity in multiple myeloma patients. Peripheral blood was collected from six multiple myeloma (MM) patients at serial time points in connection with treatment and during a follow-up period of 3 months. T-cell response to cytomegalovirus (CMV), varicella zoster virus (VZV) and tetanus toxoid (TT) was determined by flow cytometry analysis for CD69, TNFalpha, IFNgamma, IL-4 expression and cell proliferation. At diagnosis and prior to induction chemotherapy TNFalpha expressing T cells in 5/6 patients were found specific for CMV, 3/6 for VZV and 4/6 for TT. Serial analyses during treatment conclude impaired immune response, however, 3 months post-transplantation all but one patient had regained cytokine expressing CD8(+) T cells specific for CMV, VZV and TT. The highest percentages of cytokine responding T cells were observed after stimulation with CMV antigen. A striking observation was the low cytokine reactivity (close to zero) measured in G-CSF mobilized blood at the time of leukapheresis. In spite of a general reduction of the CD4/CD8 ratio following transplantation, recovery of antigen specific CD4(+) T cells reactivity generally occurred prior to CD8(+) recovery and often to a higher level. In conclusion, the study demonstrates that natural as well as vaccine induced specific immunity present prior to HD was regained after stem cell transplantation, hence identifying a possible window for future vaccination trials.

Magnetic resonance investigation of bone marrow following priming and stem cell mobilization.

PURPOSE: To evaluate application of MRI and magnetic resonance spectroscopy (MRS) to monitor bone marrow cellularity during pretransplant priming with chemotherapy and hematopoietic growth factor (HGF) administration. MATERIALS AND METHODS: A total of 10 lymphoma and myeloma patients, in remission following induction therapy and considered eligible for high-dose therapy and autologous stem cell transplantation, were included in the study. MR investigation was scheduled four times: at study entry, and one, two, and four weeks following priming. Priming with cyclophosphamide and recombinant human granulocyte colony-stimulating factor (rhG-CSF) started the day after study entry. MR parameters studied in a region of interest were as follows: bone marrow intensity on short-time inversion-recovery (STIR) turbo spin-echo (TSE; thus STIRTSE) and on T1-weighted TSE (T1TSE) images, T2 value for fat component, T2 value for water component, water/fat ratio (W/F), T1 value for fat component, and T1 value for water component. RESULTS: The results did not support the hypothesis that hematopoietic expansion quantitated and monitored by MR correlates to the level of mobilized progenitor cells. CONCLUSION: The results indicate that release of stem cells is a more complex phenomenon than hematopoietic expansion and reduction of fat tissue in bone marrow.

Acute lymphoblastic leukemia in adolescents between 10 and 19 years of age in Denmark--secondary publication.

INTRODUCTION: Data seem to indicate that young adults with acute lymphoblastic leukemia (ALL) have a better survival when treated with pediatric protocols compared with adult ALL protocols. The purpose of the study was to report the clinical characteristics and outcome of all children and young adults 10-19 years of age diagnosed with ALL in Denmark between 1992 and 2001. MATERIAL: The study includes 99 patients 10-19 years of age with ALL in Denmark during a ten year period found in the complete NOPHO (Nordic Society of Pediatric Hematology and Oncology) registry and through the Danish Cancer Registry and local pathology databases. Data were retrieved by reviewing medical charts of the patients. A total of 61 children (10-14 years) treated on pediatric protocols and 38 young adults (15-19 years) were diagnosed with ALL. Data were reported as of January 1st 2005. RESULTS: There were no difference with respect to the distribution of T-ALL, CNS-leukemia, total white blood count and high risk chromosomal abnormalities between the two groups. There was a statistical significant lower event free survival (p<0.01) and lower overall survival (p<0.01) in young adults compared with 10-14 year-old children (0.38 vs 0.60 and 0.47 vs 0.67). There were more transplant-related deaths in the young adults. Higher treatment intensity in children may be an additional explanatory factor. Children received more prednisone, vincristine and high-dose methotrexate than young adults. CONCLUSION: Young adult patients with ALL might benefit from therapy with pediatric NOPHO ALL protocols.

Use of postmenopausal hormone replacement therapy and risk of non-Hodgkin's lymphoma: a Danish population-based cohort study.

Use of postmenopausal hormone replacement therapy (HRT) has been hypothesised to be associated with a reduced risk of non-Hodgkin's lymphoma (NHL), but the epidemiologic evidence is conflicting. To examine the risk of NHL in HRT users aged 40 and older, we conducted a cohort study in the County of North Jutland, Denmark (population 0.5 million) using data from population-based health registries for the period 1989-2002. We computed age-standardised NHL incidence rates and used Cox regression analysis to compute the relative risk (RR) and corresponding 95% confidence intervals (CI) of NHL among HRT users compared with non-users, adjusting for age and calendar period. The number of prescriptions redeemed (1, 2-4, 5-9, 10-19, or 20 or more prescriptions) was used as a proxy for duration of HRT. We identified 40 NHL cases among HRT users during 179 838 person-years of follow-up and 310 NHL cases among non-users during 1 247 302 person-years of follow-up. The age-standardised incidence rates of NHL were 25.7 per 100,000 among HRT users and 24.2 per 100,000 among non-users, yielding an adjusted RR of 0.99 (95% CI: 0.71-1.39). Our data did not support an association between HRT use and risk of NHL.

Quality assessment of autografting by probability evaluation: model estimation by clinical end-points in newly diagnosed multiple myeloma patients.

BACKGROUND: Pre-transplant clinical evaluation of autografting is an important step in predicting post-transplant support, complications and safety. Today, unfavorable outcomes such as early death or graft failure are rare, making them unsuitable for quality assessment of supportive autografting. However, end-points constructed from frequently occurring clinical events may estimate clinically relevant prognostic models. METHODS: The present retrospective analysis was based on two consecutive clinical trials in the Nordic area including up to 640 newly diagnosed multiple myeloma patients. RESULTS: In the model, the efficacy (time on antibiotics and use of transfusions) was influenced by pre-transplant variables, including sex, nationality, serum creatinine, hemoglobin, disease stage at diagnosis, response following induction therapy, length of priming and average graft CD34+ cell number per day of harvest. The toxicity end-point (time to blood cell recovery) was influenced by nationality, marrow plasma cell percentage, serum creatinine, M-component isotype, response to induction therapy, length of priming and graft CD34+ cell number. The safety (early disease recurrence or death) was influenced by serum creatinine, hemoglobin, treatment response and CD34+ cell number. DISCUSSION: In conclusion, the model illustrates that intervention strategies in quality assessment of autografting may benefit from probability estimates of graded clinical end-points.

Impaired functionality and phenotypic profile of dendritic cells from patients with multiple myeloma.

Multiple myeloma (MM) is a B cell cancer characterized by clonal proliferation in the bone marrow and impaired immunity. Because MM is an incurable malignancy, efficient consolidation is needed urgently. Targeting clonotypic B cells by idiotype vaccination has proved the principle to be effective and indicated that future strategies, including dendritic cell-based vaccination, could be a suitable approach. However, as MM patients suffer from a general impaired immunity, which may include dendritic cells (DCs), a careful evaluation of phenotypic traits and functionality of DCs from MM patients is necessary before an efficient vaccine can be developed. This study determined the number, phenotypic profile and functionality of myeloid and plasmacytoid DCs purified directly from blood from MM patients at diagnosis. A reduced number and lower expression of human leucocyte antigen (HLA) molecules was observed on both myeloid and plasmacytoid DCs in MM patients compared to healthy controls. Also, the expression of CCR5, CCR7 and DEC205 was lower in MM patients compared to normal donors. In addition, the capacity to stimulate allogeneic T cell proliferation and to stimulate cytokine production was decreased, suggesting that DCs from these patients are functionally impaired. Finally, the analysis of samples following chemotherapy and transplantation demonstrated an increased expression of HLA molecules, suggesting that this time-point is optimal for harvest and use in vaccination.

Changes in circulating mesenchymal stem cells, stem cell homing factor, and vascular growth factors in patients with acute ST elevation myocardial infarction treated with primary percutaneous coronary intervention.

OBJECTIVE: To investigate the spontaneous occurrence of circulating mesenchymal stem cells (MSC) and angiogenic factors in patients with ST elevation acute myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (PCI). DESIGN: In 20 patients with STEMI, blood samples were obtained on days 1, 3, 7, 14, 21, and 28 after the acute PCI. Fifteen patients with a normal coronary angiography formed a control group. MSC (CD45-/CD34-), plasma stromal derived factor 1 (SDF-1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2) were measured by multiparametric flow cytometry and enzyme linked immunosorbent assay (ELISA). RESULTS: Circulating CD45-/CD34- cells were significantly decreased on day 7 compared with day 3. Cell counts normalised one month after the acute onset of STEMI. The changes were mainly seen in patients with a large infarction. Plasma SDF-1 increased significantly from day 3 to day 28, and VEGF-A and FGF-2 increased significantly from day 7 to day 28. CONCLUSIONS: Spontaneous sequential fluctuations in MSC and the increase in vascular growth factor concentrations after STEMI suggest that the optimal time for additional stem cell therapy is three weeks after a myocardial infarction to obtain the maximum effects by stimulating endogenous growth factors on the delivered stem cells.

Microalbuminuria is associated with impaired glomerular permselectivity in lymphoma patients.

Slightly increased urinary albumin excretion is frequently found in patients with lymphoma and other malignancies but the pathophysiological mechanisms have yet to be clarified. In this study, parameters of renal function in lymphoma patients with microalbuminuria were evaluated. Sixty-seven patients with histologically proven diffuse large B-cell lymphoma were included in the study at diagnosis. Urinary albumin excretion was measured by immunoturbidimetry and microalbuminuria was defined as an excretion rate between 20 and 200 microg/min. Glomerular function was further estimated by renal clearance of creatinine and IgG, and the IgG/IgG4 charge selectivity index. Tubular function was evaluated by renal clearance of beta(2)-microglobulin. The median value of IgG clearance was increased in the microalbuminuric patients (0.22 versus 0.18 microl/min; p = 0.03). The median selectivity index was significantly lower in patients with microalbuminuria (1.0 versus 2.2; p<0.0001). Urinary albumin excretion was correlated with both the renal clearance of IgG (p<0.0001) and the selectivity index (p<0.0001). These data suggest that a slightly elevated level of urinary albumin excretion in a population of patients with aggressive lymphoma reflects altered glomerular permselectivity probably due to a defect in charge selectivity. The glomerular sieving dysfunction may be associated with an inflammatory response to the malignancy. Further studies are needed to validate the clinical impact of the renal parameters in lymphoma patients.

Phenotypic and functional characterization of clinical grade dendritic cells generated from patients with advanced breast cancer for therapeutic vaccination.

Dendritic cells (DC) are promising candidates for cancer immunotherapy. However, it is not known whether in vitro-generated monocyte-derived DC from cancer patients are altered compared with DC from healthy donors. In a clinical phase I/II study, monocyte-derived DC were generated in vitro utilizing granulocyte macrophage colony-stimulating factor and rh-interleukin-4 (IL-4) and used for cancer immunotherapy. In this study, we tested the effect of various maturation cocktails and performed a comparative evaluation of the DC phenotype and functional characteristics. Polyriboinosinic polyribocytidylic acid (Poly I:C) + tumour necrosis factor-alpha (TNF-alpha) induced significant IL-12 p70 secretion, which was increased after addition of a decoy IL-10 receptor. The lymph node homing chemokine receptor CCR-7 expression was induced by TNF-alpha + IL-1beta + IL-6 + prostaglandin E2 but was not induced by Poly I:C + TNF-alpha. In general, DC from patients had an intermediate maturity phenotype with a significantly higher expression of CD40 and CD54 compared with healthy donors. In vitro analyses showed an unimpaired capacity of the patient-derived DC for antigen-specific (cytomegalovirus, tetanus and keyhole limpet haemocyanin) T-cell stimulation, whereas the allostimulatory capacity of patient-derived DC was significantly decreased. These data suggest that patient-derived DC are more differentiated but are less sensitive to maturation-inducing agents than DC obtained from healthy individuals.

Multiple platelet defects identified by flow cytometry at diagnosis in acute myeloid leukaemia.

Summary Previous findings of megakaryocytic hypogranulation and dysmegakaryocytopoietic features in acute myeloid leukaemia (AML) strongly indicate defects in platelet production. The bleeding tendency of these patients may result from dysregulated platelet production, resulting in thrombocytopenia as well as qualitative platelet defects. The present study examined platelet function at diagnosis in 50 AML patients by whole blood flow cytometry. Following in vitro platelet agonist stimulation, platelet activation markers were analysed and compared with 20 healthy individuals. To detect recent in vivo platelet activation, plasma soluble P-selectin (sP-selectin) was measured. Flow cytometric analysis of platelet activation markers demonstrated reduced CD62P [35.6 vs. 118.5 x 10(3) molecules of equivalent soluble fluorochrome (MESF); P < 0.0001], CD63 (11.3 vs. 50.7 x 10(3) MESF; P < 0.0001), and PAC-1 (41.5 vs. 90.5%; P = 0.0001) while reductions in CD42b were abnormal (45.6 vs. 70%; P < 0.0001). sP-selectin levels were similar in patients and healthy controls (0.04 vs. 0.27 fg/platelet; P = 0.84). The presented data indicate that AML pathogenesis may result in multiple platelet defects, involving adhesion, aggregation, and secretion and demonstrate that flow cytometry is a feasible method for platelet function analysis in patients with thrombocytopenia.