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© The Authors., Given its myeloid-restricted expression, myeloperoxidase (MPO) is typically used for lineage assignment (myeloid vs. lymphoid) during acute leukaemia (AL) diagnostics. In the present study, a robust flow cytometric definition for MPO positivity was established based on the standardised EuroFlow protocols, the standardised Acute Leukaemia Orientation Tube and 1734 multicentre AL cases (with confirmed assay stability). The best diagnostic performance was achieved by defining MPO positivity as ≥20% of the AL cells exceeding a lymphocyte-based threshold. The methodology employed should be applicable to any form of standardised flow cytometry., The co-ordination of this study was supported by the EuroFlow Consortium. The EuroFlow Consortium received support from the FP6-2004-LIFESCIHEALTH-5 programme of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP). The EuroFlow Consortium is part of the European Scientific Foundation for Hemato-Oncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA).

RAN, ZHX2 and RCBTB2 (CHC1L) expression was evaluated by quantitative real time reverse transcription polymerase chain reaction in plasma cells from 85 monoclonal gammopathies: 58 symptomatic multiple myeloma (MM) (52 untreated, six relapsed), eight smouldering MM, five monoclonal gammopathy of undetermined significance, four plasma cell leukaemias and 10 myeloid cell lines. ZHX2 was weakly expressed in high-risk/proliferative disease compared to low-risk or indolent disease. High ZHX2 expression was associated with better response and longer survival after high-dose therapy. RCBTB2 expression was weaker in hyperdiploid versus non-hyperdiploid cases while RAN was more expressed in symptomatic MM and cell lines.

The diagnosis of 'rare diseases', such as mastocytosis, remains a challenge. Despite this, the precise benefits of referral of mastocytosis patients to highly specialized reference centres are poorly defined and whether patients should be managed at non-specialized versus reference centres remains a matter of debate. To evaluate the quality and efficiency of diagnostic procedures performed at the reference centres for mastocytosis in Spain (REMA) versus other non-reference centres, we retrospectively analysed a series of 122 patients, for the overall degree of agreement obtained for the World Health Organization (WHO) diagnostic and classification criteria betwen the referring and REMA centres. Our results showed that not all WHO diagnostic criteria were frequently investigated at the referring centres. Among the five WHO diagnostic criteria, the highest degree of agreement was obtained for serum tryptase levels [median 90% (95% confidence interval 84-96%)]; in turn, the overall agreement was significantly lower for the major histopathological criterion [80% (72-89%)], and the other three minor criteria: cytomorphology [68% (56-80%)] immunophenotyping of BM mast cells [75% (62-87%)] and detection of the KIT mutation [34% (8-60%)]. Referral of patients with diagnostic suspicion of mastocytosis to a multidisciplinary reference centre improves diagnostic efficiency and quality., This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS) and RTICC of the Instituto de Salud Carlos III, Ministry of Economy and Competitivity, Madrid, Spain (grants PI11/02399 and RD12/0036/0048, FEDER); Ayudas a Proyectos de Investigacion en Salud de la Fundacion Mutua Madrileña 2014; Ayuda de la Asociacion Española de Enfermos de Mastocitosis (AEDM 2014); and Fundacion Ramon Areces, Madrid, Spain (grant CIVP16A1806). AM was supported by RTICC.

Despite the effects of BCR ABL on cell proliferation, no study has compared the proliferative rate of different haematopoietic cell compartments from chronic myeloid leukaemia (CML) with those of normal bone marrow (NBM). We comparatively analysed the cell cycle distribution and BCR/ABL expression in different compartments of BM cells from 15 CML and 11 NBM. Overall, our results showed similar proliferative indices in CML patients and NBM. However, CD34+ myeloid precursors from CML patients displayed an increased proportion of S + G2/M-phase cells (P = 0.04), while no significant differences were found between CML and NBM for other BM cell subsets analysed. In BM cells separated by fluorescence-activated cell sorting, decreasing levels of BCR/ABL mRNA were found from CD34+/CD38+ myeloid precursors to myeloblasts; BCR/ABL expression increased afterwards with a peak at the myelocyte/metamyelocyte stage, decreasing in the more mature band/neutrophil compartment. Unexpectedly, BCR/ABL gene expression showed an inverse correlation with the proportion of S + G2/M-phase cells (R = -0.33; P = 0.04). These results suggest that in CML, BCR/ABL expression is associated with an increased proliferation of CD34+ myeloid haematopoietic progenitor cells but not of other more mature myeloid precursors, as confirmed by the observation of an inverse correlation between the amount of BCR/ABL transcripts and the proportion of S + G2/M-phase cells.

Depending on their stage of maturation and other factors, mast cell (MC) subsets differ from each other in terms of the expression of complement-associated antigens. This study analysed the expression of various complement-related cell surface antigens (CD11b/CR3, CD11c/CR4, CD35/CR1, CD55/DAF, CD59/MIRL, CD88/C5aR) on bone marrow mast cells (BMMC) in patients suffering from systemic mastocytosis (SM), other haematological diseases and non-haematological disorders (control groups). Expression of complement-associated cell surface antigens was analysed by flow cytometry. There were clear immunophenotypic differences between BMMC obtained from patients with SM and those from the control subjects: the percentage of patients expressing surface CD11c, CD35 and CD88 was significantly higher in patients with SM (76%, 100%, 54%) than in the control subjects (58%, 11%, 18%) (P < 0.05). In addition, the levels of CD11c, CD35 and CD88 expressed per MC (sites per cell) were significantly higher (P < 0.05) in SM than in the control group. Expression of the complement regulatory molecules CD55 and CD59 was detected in BMMC in all patients analysed. However, the levels of CD59 per BMMC were higher in patients with SM as compared with the control subjects, which could help to explain the formation of BMMC aggregates in the former group of individuals. Together, our results showed that BMMC in systemic mastocytosis overexpressed the cell surface membrane receptors involved in binding of complement components and complement-mediated cell activation. Whether this pathological expression of complement receptors is of pathophysiological significance remains to be determined.

Currently, no information is available regarding the influence of the different CD34+ cell subsets infused on the haematopoietic recovery, following non-myeloablative allogeneic peripheral blood stem cell transplantation (allo-PBSCT). We have explored, in a group of 13 patients receiving non-myeloablative allo-PBSCT from human leucocyte antigen-identical sibling donors, the influence of the total dose of CD34+ haematopoietic progenitor cells (HPC) infused, compared with that of the different CD34+ HPC and CD34- leucocyte subsets in the leukapheresis samples, on both engraftment and clinical outcome. The overall numbers of total CD34+ HPC (P = 0.002) and myelomonocytic-committed CD34+ HPC infused (P = 0.0002) were strongly associated with neutrophil recovery (> 1 x 109 neutrophils/l), the latter being the only independent parameter influencing neutrophil recovery. Regarding long-term engraftment, only the number of immature CD34+ HPC infused/kg correlated with the duration of hospitalization in the first 2 years after discharge (r = -0.75, P = 0.005). Both the overall amount of CD34+ HPC and the number of myelomonocytic CD34+ HPC infused showed a significant influence on the risk of graft-versus-host disease (GVHD). Thus, the overall probability of GVHD was 100%vs 25% for patients receiving >/= 5 x 106 CD34+ HPC or >/= 3.5 x 106 of myelomonocytic-committed CD34+ HPC vs lower doses (P = 0.013). None of the other CD34+ and CD34- cell subsets analysed correlated with development of GVHD. In summary, our results suggest that in non-myeloablative allo-PBSCT, high numbers of CD34+ HPC, especially the myelomonocytic-committed CD34+ progenitors, lead to rapid neutrophil engraftment. However, they also strongly impair clinical outcome by increasing the incidence of GVHD.

Summary. In order to gain further insights into the role of the p16 gene in cell cycle regulation and the prognostic implications of its inactivation, we investigated the methylation status of the p16 gene in 98 untreated patients using a polymerase chain reaction assay based on the inability of some restriction enzymes to digest methylated sequences. Forty-one patients showed a p16 methylated gene (42%). The percentage of S-phase plasma cells (PC) in these patients was almost three times higher than in those with an unmethylated p16 gene (4·16% ± 3·37%vs 1·5% ± 1·41%, P

Summary. Twelve patients with smouldering or indolent multiple myeloma (MM) received 12 courses of intravenous pamidronate as a single agent to evaluate both the antitumour and bone metabolism effects. One patient achieved minor response, eight had stable disease, and three – all indolent MM – showed disease progression. Serum interleukin 6 (IL-6), IL-1β and Oncostatin-M remained stable throughout the study, while tumour necrosis factor-α increased. Bone density significantly increased after four and 12 courses compared with baseline. Markers for bone resorption and bone formation decreased with treatment. These results suggest that pamidronate treatment reduces bone turnover in smouldering or indolent MM, but has no significant antitumour effect.

Haematological, immunophenotypic and cytogenetic characteristics were analysed in 241 patients with acute myeloid leukaemia (AML) M0, including 58 children. Children < 3 years and adults between 60 and 70 years of age were most frequently affected. Immunophenotyping showed a heterogeneous phenotype. Anti-myeloperoxidase was positive in about half of the patients. Cytogenetic data were available from 129 (54%) patients. A normal karyotype was found in only 24%. Most of the abnormalities were unbalanced and the chromosomes 5, 7, 8 and 11 were the most frequently affected. Survival data were available from 152 treated patients (63%). The median overall survival for all patients was 10 months, 20 months for children (n = 36), 10 months for the young adult group (n = 50) and 7 months for the elderly patients (n = 66) (P = 0.09). Karyotype was not a prognostic factor influencing survival. AML M0 shows the immunological characteristics of early progenitor cells, but the expression of the different markers and cytogenetic abnormalities is heterogeneous. The prognosis is poor compared with other de novo AML and similar to that of AML with multilineage dysplasia or AML following myelodysplastic syndromes.

Sensitive techniques for monitoring minimal residual disease (MRD) in multiple myeloma (MM) are needed to evaluate the effectiveness of new intensive treatment strategies. The aim of the present study was to explore the applicability and sensitivity of flow cytometry immunophenotyping and DNA ploidy studies for the investigation of residual myelomatous plasma cells (PC) in MM patients. Bone marrow (BM) samples from 61 untreated MM patients were immunophenotypically analysed with a panel of 21 monoclonal antibodies, using a high-sensitive method based on a two-step acquisition procedure through a SSC/CD38+++-CD138+‘live-gate’. Overall, in 87% of MM cases, PC displayed an aberrant phenotype at diagnosis. The most important aberrant criteria were: antigen over-expression of CD56 (62%), CD28 (16%) and CD33 (6%) and asynchronous expression of CD117 (28%), sIg (21%) and CD20 (10%). DNA aneuploidy was found in 62% of cases. The simultaneous use of these two techniques allowed the detection of aberrant/aneuploid PC in 95% of the cases. Based on dilutional experiments, the detection limit of both techniques ranged from 10−4 to 10−5. In 29 stem cells harvests and 19 BM samples obtained 3 months after autologous transplantation, we have investigated the presence of residual myelomatous PC; they were detected in 44% of the stem cell collections and in 61% of the BM samples obtained after transplant. The percentage of pathological PC did not significantly change during the days of harvest. In summary, the present study shows that the combined use of immunophenotyping and DNA ploidy studies is a suitable approach for MRD investigation in MM patients based on their applicability (95% of cases) and sensitivity (up to 10−5).

We have analysed the quantitative expression of surface CD69 antigen on human mast cells (MC), from both normal and pathological bone marrow (BM) samples, using flow cytometry. Our major aim was to analyse whether CD69 is constitutively expressed by normal BMMC and to explore the possible differences between CD69 expression by BMMC from normal controls and patients suffering from different pathological conditions. The constitutive expression of surface CD69 was clearly demonstrated in BMMC; however, systemic mast cell disease (SMCD) patients showed significantly higher levels of surface CD69 expression than healthy controls (P 0.05). From the other groups of patients, monoclonal gammopathy of unknown significance (P = 0.04), myelodysplastic syndromes (P = 0.03) and paediatric mastocytosis (P = 0.003) cases showed a significantly higher expression of surface CD69 as compared to normal subjects. In summary, our findings show that the CD69 antigen is overexpressed in SMCD patients.

Immunophenotypic investigation of minimal residual disease (MRD) has traditionally been based on the investigation of phenotypic aberrants at diagnosis to be used later as a target for MRD detection. This approach has several shortcomings (it is only applicable to patients with aberrant phenotypes, requires a diagnostic sample, and is patient-specific) and therefore a search for simpler alternatives is warranted. The present study is based on the hypothesis that in precursor-B-ALL patients the persistence of residual leukaemic cells may induce abnormalities in the precursor-B-cell compartment in bone marrow (BM) and these could be used as a criteria to predict relapse. These abnormalities may include: (1) the presence of an increase in the frequencies of immature B cells (CD34+/CD19+ or CD20−/CD19+) or (2) the existence of an altered B-cell differentiation pathway due to a blockade or to the presence of B cells outside the normal pathway. A total of 180 BM samples from 45 consecutive precursor-B-ALL patients who achieved morphological complete remission (CR) were analysed by multiparametric flow cytometry. Our results show that a significant increase in immature B-cell subsets or an altered B-cell differentiation predicts a high relapse rate (P

The in vitro growth characteristics of a large series of acute myeloid leukaemia (AML) patients and their relationship with other clinical and biological disease characteristics were analysed. Patients with AML were studied, 181 with de novo AML and 45 with secondary AML (24 myelodysplastic syndrome, sAML-MDS, 21 myeloproliferative disorder, sAML-MPD). Leukaemic colony forming units (L-CFU) were assayed by plating peripheral blood (PB) blast cells in methyl-cellulose and using LCM-PHA as stimulant. In each case parallel cultures were made with and without stimulating factors. Plating efficiency (PE) was defined as the number of clusters plus colonies/10(5) cells plated. Autonomous growth (AG) was the number of colonies plus clusters growing without stimulant. The autonomous proliferative index (API) was calculated as the number of clusters + colonies without stimulating factor divided by the number of clusters + colonies with stimulating factor. No significant differences in the PE between de novo and secondary AML were found. Autonomous growth was significantly higher in sAML-MPD. The FAB subtype M3 leukaemias displayed a significantly greater PE and a significantly lower API when compared with the other FAB subgroups (P=0.0002). Upon analysing the relationship with the immunophenotype, only CD33 expression showed a significant relationship with the in vitro growth pattern; CD33+ cases displayed a higher PE (P=0.0002) and AG (P=0.0003) than CD33- cases. When patients were grouped according to the level of rh123 efflux (MDR1) it was observed that cases with >30% elimination showed a higher AG and API than those with 0.05) displayed a significantly shorter overall survival as compared with patients with API

Haemopoietic growth factors (HGFs) have been shown to accelerate recovery from severe neutropenia after autologous bone marrow transplantation (ABMT) but their effect on immune reconstitution is not well defined. The present study compares, through randomized trial, the in vivo effect of GM-CSF and G-CSF administration on the immune recovery of patients who underwent ABMT. For that purpose, we have sequentially analysed 14 different T, B and NK lymphoid cell subsets using appropriate dual staining during the first year following transplant (days +6, +17, +31, +66, +90, +120, +180, +360). 24 patients with lymphoproliferative disorders (20 lymphomas and four multiple myelomas) and who had undergone ABMT were included in the study. The median age was 43 years (range 22-62 years). All lymphoma patients were homogenously conditioned with BEAM. Our results show that both GM-CSF and G-CSF aid T-cell (CD3+/alpha beta) recovery though their contribution varies depending on the T-cell subset analysed. G-CSF contributed to a significantly faster recovery of CD8+ cells (P = 0.03). The CD8+ cell regeneration was produced mainly by activated cells (CD38+/HLA-DR+) which lacked the CD11b antigen. In contrast, GM-CSF favoured the regeneration of CD4+ cells (through both the CD45RO+ and CD45RA+ subset), leading to a higher CD4+:CD8+ ratio (P = 0.007). No statistically significant differences were detected in the three groups of patients as regards both the recovery of NK cells and NK activity. Furthermore, the use of HGF did not seem to exert a significant influence on the recovery of B lymphocytes. This recovery was based on the CD5+ subpopulation that showed a rapid rise after the first month. We suggest that G-CSF and GM-CSF not only influence myeloid recovery, but also regeneration of the immune system after ABMT.

In this study the incidence of DNA aneuploidy in a large series of untreated multiple myeloma (MM) patients was assessed in order to determine its clinical and prognostic significance. A total of 156 MM patients were included in the study. DNA measurements were performed in all cases at diagnosis using two different flow cytometry methods: (1) propidium iodide (PI) staining on isolated nuclei, and (2) CD38/PI double staining on whole cells. The DNA ploidy status was correlated with the most relevant clinical and haematological disease characteristics. From the 156 cases analysed, 91 (58%) were aneuploid (56% hyperdiploid and 2% hypodiploid). The correlation between the two techniques on the detection of DNA aneuploidy was excellent, although CD38/PI double staining would be preferable in cases with < 5% of DNA aneuploid plasma cells (PC). Upon comparing the clinical and haematological disease characteristics of hyperdiploid versus diploid cases, the former group was characterized by a lower age, reduced incidence of anaemia, lower beta 2M levels, higher proliferative activity within the residual normal haemopoietic cells, increased expression of CD56 antigen in PC, and higher proportion of PB CD4+ T cells. In contrast, diploid cases had a higher expression of the CD10, CD20 and CD15 antigens and greater numbers of PB CD56+CD3- NK cells (P < 0.05). Circulating PC were identified in six cases, all being diploid. Overall survival was significantly longer in hyperdiploid compared to diploid MM (P = 0.02). These results show that over 50% of MM patients are aneuploid, almost all of them being hyperdiploid. This characteristic is associated with better prognosis.

The presence of Ig and TCR gene rearrangement has been reported to occur in ANLL. However, most of the studies have been performed in short series of patients and, in general, not all rearranging genes have been included. We have investigated the configuration of immunoglobulin (Ig) and T-cell receptor loci (TCR) in a series of 160 untreated patients with de novo acute non-lymphoblastic leukaemia (ANLL) and correlated the results with the morphological and immunophenotypic characteristics of blast cells. IGH gene rearrangement was detected in 16/160 cases analysed (10%) and IGK was rearranged in half of them. The incidence of cases displaying TCRB, TCRG and TCRD rearrangements was 5.6%, 13.8% and 13%, respectively. Concomitant recombinatorial events including different Ig and/or TCR genes were frequently detected. Gene rearrangement was not related to the stage of cell differentiation within the myeloid lineage assessed both by morphological and immunophenotypic criteria. Regarding the correlation with the presence of lymphoid related markers, the only relevant association was between the expression of CD7 antigen and TCRG and TCRD gene rearrangement. Our results show that the incidence of gene rearrangement in ANLL may be slightly higher than previously suspected, and that it is not associated with early stages of cell differentiation nor to the expression of lymphoid markers.

We really enjoyed reading the recently published excellent review on myeloma concepts (Hajek et al, 2013). It was well timed for the presentation of myeloma data at the American Society of Hematology (ASH) 2013 meeting in New Orleans. Mainly Spanish data (Mateos et al 2013) is further translating the concept of heterogeneity and plasticity of multiple myeloma plasma cells into clinical practice. At the end of his comprehensive review Professor Hajek and his team recommended that ‘ .. . To improve outcomes of future therapy, it is necessary to take into account the plasticity of MIC (myeloma-initiating cells) and altering dominance of genetically distinct subclones that occur as results of previous treatment.’ We fully agree with this statement, but to make future therapy more efficient, even curative, we have to involve the patient’s immune system. Myeloma evolution happens within and in very close interaction with the plasma cell microenvironment, particularly in close relationship with the compartments of the patient’s immune system. In this regard, the question of whether the patient’s immune system is the innocent bystander in this process is very much ignored in all current concepts or if it plays a key role in the initiation process of myeloma pathogenesis, remains unsolved and largely unexplored. In an attempt to obtain further insight into the potential role of the immune system in the long-term control of multiple myeloma, we have recently investigated the distribution and phenotype of different compartments of the immune system in paired peripheral blood and bone marrow samples from myeloma patients with long-term disease control, having received no treatment for more than 10 years, with that of age-matched healthy adults (Pessoa de Magalh~aes et al, 2013). Interestingly, the immune system capacity and repertoire of these myeloma patients was much stronger than that of the age-matched healthy adult group and other myeloma patients who failed to retain complete remission over time. Of note, in this study, long-term disease control was not restricted to patients with a complete response, because some reverted to a monoclonal gammopathy of undetermined significance (MGUS)-like profile after therapy, still not developing symptomatic disease for more than 10 years. A more detailed analysis of the different immune cell compartments showed increased numbers of cytotoxic T and natural killer (NK) cells and recovered dendritic cell counts, together with decreased percentages of regulatory T cells in the bone marrow; this was associated with recovered B-cell counts, including all B-cell compartments, in peripheral blood, and bone marrow as well as plasma cells. These results, together with the recurrent observation of the highly adverse prognostic impact of decreased normal plasma cell numbers in the bone marrow of myeloma patients, highlight the association of a preserved vs deteriorated immune system in long-term control vs malignant progression of mono