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Purpose: The aim of this study was to optimise the yield of metaphases in mesenchymal stromal cells (MSC) in vitro cultures and to study the karyotype of MSC expanded in good manufacturing practice (GMP) conditions for clinical use. Background: MSC are being increasingly used in clinical trials for a number of diseases. Biosafety demonstration in all cases is mandatory. Unfortunately, current standard karyotyping methods fail to obtain enough number of evaluable metaphases. Methods and materials: In the present work, to optimise the yield of metaphases in MSC expanded in vitro, we have tested several conditions by modifying colcemid concentration (we have tested 0·01, 0·05 and 0·1 µg mL−1) and exposure time (during 5, 15 and 24 h). We further applied these optimised conditions to 61 MSC expansions in GMP conditions for clinical use. Results: Our results show that the highest number of metaphases was obtained when MSC were incubated with 0·05 µg mL−1 of colcemid overnight (15 h), compared to the remaining experimental conditions. In most cases (59/61 cases) enough number of metaphases was obtained. And what is more relevant, only in one case a karyotypic abnormality was found (trisomy of chromosome 10), and cells were subsequently discarded for clinical use. Conclusion: We describe here an optimal method to obtain enough number of metaphases for karyotype analysis of in vitro expanded MSCs, what is essential for their clinical use in cell therapy programmes. [ABSTRACT FROM AUTHOR]

The frequencies of human leukocyte antigen (HLA) class I and class II specificities and haplotypic associations were determined in 1940 unrelated donors from Castilla y León and compared with other Iberian, Mediterranean and European populations. Specificities were determined using polymerase chain reaction reverse sequence-specific oligonucleotide or polymerase chain reaction sequence-specific primer techniques. In the analysis, 19, 29 and 13 specificities were found for HLA-A, -B and -DRB1, respectively, with HLA-A*02 (26%), - A*01 (11%), - B*44 (16%), - B*35 (10%), - DRB1*07 (16%) and - DRB1*13 (14%) showing the highest frequencies. In addition, 10 common HLA-A-B-DRB1 haplotypic associations were observed, A*01-B*08-DRB1*03 (3%) and A*29-B*44-DRB1*07 (3%) being the most frequent ones. These findings indicate that the population of Castilla y León is genetically equidistant from the Portuguese and other Spanish populations and shares a common origin with other Iberian populations, in which European, Mediterranean and North African genetic components are present; this is in agreement with the historical and genetic background of the population. These data contribute to a better understanding of the genetic structure of the Iberian Peninsula and provide a healthy control population from our region that should be useful for the study of disease associations. [ABSTRACT FROM AUTHOR]

Few diseases have a prognosis worse than Hodgkin’s lymphoma (HL), patients relapsing after autologous or allogeneic stem cell transplantation. Here, we report two highly refractory patients with HL who successfully responded to a combination of thalidomide, cyclophosphamide and dexamethasone (ThaCyDex). Despite the use of a very large number of different drugs (>5 different schemes) including high-dose therapy and autologous and allogeneic stem cell transplantation, both patients proved to be suffering from a highly resistant disease. Fortunately, they finally responded to the ThaCyDex combination, achieving sustained complete remission that would support the running of a trial within this setting. [ABSTRACT FROM AUTHOR]

The human leukocyte antigen (HLA) system could play an essential role in multiple myeloma (MM) disease control. This report describes the results comparing HLA-DRB1 phenotypic frequencies in 181 MM patients (53 smoldering/indolent MM and 128 symptomatic MM patients) and healthy individuals. Higher DRB1*01 phenotypic frequencies were found in the smoldering patients compared with symptomatic MM patients (38% vs 14%, P = 0.001) and with the healthy individuals (38% vs 22%, P = 0.01). Additionally, higher DRB1*07 phenotypic frequencies were found in symptomatic MM compared with control population (38% vs 28%, P = 0.01). The present data suggest that HLA-DRB1*01 individuals may have a better ability to efficiently present myeloma-related antigens to immunocompetent cells, which could favor a better immune response against the tumor. This would translate into a more appropriate disease control associated with more indolent disease and prolonged survival. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fluorescence in situ hybridisation (FISH) is an effective technique for the cytogenetic analysis of Waldenström macroglobulinemia (WM), but the potential impact of molecular cytogenetics on disease evolution and as a prognostic marker is still unknown. Deletion of the long arm of chromosome 6 (6q—) is the most frequent cytogenetic abnormality in WM. This study analysed the prevalence of this aberration in 102 WM patients, and correlated it with disease characteristics. The incidence of 6q21 deletion was 7% by conventional cytogenetics and 34% when analysed by FISH (54% when cytoplasmic immunoglobulin M-FISH was used). Patients with deletion of 6q displayed features of adverse prognosis, such as higher levels of β2-microglobulin and monoclonal paraprotein and a greater tendency to display anaemia and hypoalbuminemia. Interestingly, there was a correlation between the presence of 6q deletion and the International Staging System prognostic index (incidence of 6q) among patients stratified in stages 1, 2 and 3 was 24%, 42% and 67% respectively). Those patients diagnosed with smouldering WM who displayed the abnormality showed a trend to an earlier requirement of treatment. Finally, the survival analysis did not show differences between the two groups of patients, probably due to the short follow up of our series. [ABSTRACT FROM AUTHOR]

Melphalan–prednisone (MP) has been the gold standard treatment for more than 30 years in elderly multiple myeloma (MM) patients. In order to assess whether the combination of dexamethasone with melphalan (MD) could improve on the efficacy of MP, we have carried out a randomized trial comparing both treatment approaches. A total of 201 patients≥70 years old were included in the study. The overall response rate was similar after six cycles (MP: 67·9%versusMD: 64·5%) and after 12 cycles (MP: 49·4%versusMD: 46·1%). However, the proportion of complete responses (CR) was higher in the MD arm, particularly after 12 cycles (MD: 22·4%versusMP: 9·1%;P < 0·05). There was no significant difference in event-free survival (MP: 15·9 monthsversusMD: 23·3 months). The median overall survival in both arms was almost identical (MP: 29·4 monthsversusMD: 27·2 months;P = 0·63). No significant differences in haematological toxicity were observed, but non-haematological toxicity was significantly higher in the MD arm. According to these results MP remains as the gold standard for treatment of MM and should be the reference for comparison of new therapeutic approaches involving novel agents. [ABSTRACT FROM AUTHOR]

Background: Almost all automated hematology cell analyzers use methods based on either the impedance (PLTi) or the optical (PLTo) properties of the cells for performing platelet counts. To improve the accuracy of platelet counts in peripheral blood (PB), the use of CD61 (GPIIIa) MoAbs (ImmunoPLT method) has recently been introduced in an automated hematology blood-analyzer system (Cell-Dyn 4000, Abbott Diagnostics).Study Design and Methods: A comparative evaluation was made of the accuracy and precision of the three methods currently available in the Cell-Dyn 4000 automated hematology cell analyzer for counting the number of platelets per microliter of PB in a total of 47 patients with chemotherapy-induced thrombocytopenia. A flow cytometric PB platelet count was also performed in parallel and used as an external reference.Results: PB platelet counts showed a good correlation among the PLTo, CD61-ImmunoPLT, and flow cytometric methods. In contrast, the PLTi procedure usually provided an overestimation of the number of platelets per microliter. Although a good correlation was observed between the flow cytometric reference method and both the ImmunoPLT and PLTo methods, the highest degree of agreement was found for the ImmunoPLT techniques (94% vs. 67%). A comparative analysis of the PLTo and CD61-ImmunoPLT methods with regard to their value for predicting platelet transfusion needs on the basis of specific flow cytometric platelet count thresholds showed a good correlation when the cutoff level of 10,000 platelets per microL was used. In contrast, at the threshold of 20,000 platelets per microL, slight differences were observed between the PLTo and CD61-ImmunoPLT procedures for predicting transfusion needs.Conclusion: Such results indicate that, if the CD61-ImmunoPLT method is used in the platelet transfusion decision-making process, unnecessary platelet transfusions could be avoided in up to 17.5 percent of persons with a PLTo count of <20,000 platelets per microL. [ABSTRACT FROM AUTHOR]

Several studies have focused on investigation of the optimal salvage regimen to induce maximum response before autologous stem cell transplantation (ASCT) in patients with relapsed or refractory Hodgkin's disease (HD). However, in most of these studies, the follow-up is relatively short. In the present study, we report on long-term results of 55 consecutive patients with HD who received Mini-BEAM [BCNU (carmustine), etoposide, cytarabine, melphalan] as salvage therapy before ASCT. Eleven patients were refractory to front-line therapy, 17 were partial responders, and 27 patients had relapsed from HD. Twenty-eight patients achieved complete response, and 18 achieved partial response with a median of two cycles of Mini-BEAM, giving a total response rate of 84%. Significant factors predicting poor response (P < 0·05) were: initial treatment with MOPP (mechloroethamine, oncovin, procarbazine, prednisolone), ≥ two previous chemotherapy regimens and three disease characteristics at Mini-BEAM treatment: presence of B symptoms, extranodal involvement or low serum albumin. However, only the last two factors retained independent influence on multivariate analysis. In total, 45/55 patients have been transplanted. Median follow-up after Mini-BEAM administration for living patients is 68 months. At the time of reporting, 31 out of 55 patients (56·4%) are still alive, 21 patients (38%) have relapsed, three (5·4%) have developed secondary neoplasias, and five have died of other complications not related to disease progression. The actuarial 7-year overall survival (OS) was 52%, the progression-free survival (PFS) 54% and the event-free survival (EFS) 36%. The response to Mini-BEAM was the most important prognostic factor for predicting the long-term probability of surviving the disease: none of the eight patients who did not respond to Mini-BEAM were alive at 3 years. On multivariate analysis, response to Mini-BEAM and extranodal involvement before Mini-BEAM had a significant influence on OS. Our results show the safety and efficacy of Mini-BEAM before ASCT for refractory or relapsed HD patients. [ABSTRACT FROM AUTHOR]

Considers monocyte counts as an index of haemopoietic reconstruction after peripheral blood stem cell transplantation. Information on fluorescent reticulocytes and the number of monocytes as an indicator of engraftment; Predictive value of monocyte and granulocyte recovery; Importance of early determination of engraftment in critical management decisions.

We report on a case of pyridoxine refractory hereditary sideroblastic anaemia (HSA) in a 19-year-old man who underwent peripheral blood stem cell transplantation (PBSCT) from his HLA-identical brother. By using short tandem repeat polymorphism, 100% donor cells were observed in peripheral blood on day +21; bone marrow showed mixed chimaerism from day +21 to day +221, when 100% cells of donor origin were observed. The patient developed extensive chronic graft-versus-host disease with favourable response to treatment. When the haemoglobin range was normal, a programme of phlebotomies reduced serum ferritin levels. Three years after transplantation, the patient has an ECOG rating of 0, with completely normal haemoglobin values (15 g/dl). To our knowledge, this is the first PBSCT reported in a case of hereditary sideroblastic anaemia. [ABSTRACT FROM AUTHOR]

Reports on the use of thalidomide for relapsed/refractory multiple myeloma. Effect of the increase of dosages every week; Tolerance of the treatment; Comparison of the progress of patients.

Responds to comments made by Nowak and colleagues on an article concerning the limitations of DNA quantification for minimal residual disease detection in multiple myeloma. Cases in which aneuploid plasma cells display a difference in their DNA contents; Usefulness of DNA cell content measurements and immunophenotyping; Reason for the simultaneous staining of the samples.

Melphalan & Prednisone (MP) is considered as the standard therapy for Multiple Myeloma (MM) patients not eligible for high-dose therapy. Here, we report the results of a phase I-II study to evaluate the feasibility and efficacy of the association of PLD to the conventional MP regimen during the first six cycles of the front-line therapy for untreated MM patients older than 70. Thirty patients were included in the study with a median age of 77 years (71-84) and a M/F ratio of 17/13. The phase I of the study demonstrated that the maximum tolerable dose of PLD in this setting was 30 mg/m(2), so it was the final dose evaluated in the study. Twenty-nine patients were valuable for response, which was: complete in 4 (14%) partial in 15 (52%) minor/no changes in 7 (24%) and progressive in 3 (10%). The median progression free survival (PFS) was 24 months. The median overall survival (OS) has not been reached yet, with a 3-year probability for OS and PFS of 52 and 37%, respectively. Haematological toxicity was frequent but usually weak/moderate (grades 1 & 2 of the WHO scale) and it was resolved only with dose delays. Infection was a relatively frequent event (30% of patients), but only in 4 cases it was of grade 3. No cases of palmar-plantar erythrodysesthesia were observed. In conclusion, pegylated liposomal doxorubicin can be safely added to the other chemotherapeutic drugs in the treatment of elderly MM patients, which can be very useful for patients in whom novel agents are not tolerated or inefficient.

Melphalan-prednisone (MP) has been the gold standard treatment for more than 30 years in elderly multiple myeloma (MM) patients. In order to assess whether the combination of dexamethasone with melphalan (MD) could improve on the efficacy of MP, we have carried out a randomized trial comparing both treatment approaches. A total of 201 patients >/=70 years old were included in the study. The overall response rate was similar after six cycles (MP: 67.9%versus MD: 64.5%) and after 12 cycles (MP: 49.4%versus MD: 46.1%). However, the proportion of complete responses (CR) was higher in the MD arm, particularly after 12 cycles (MD: 22.4%versus MP: 9.1%; P < 0.05). There was no significant difference in event-free survival (MP: 15.9 months versus MD: 23.3 months). The median overall survival in both arms was almost identical (MP: 29.4 months versus MD: 27.2 months; P = 0.63). No significant differences in haematological toxicity were observed, but non-haematological toxicity was significantly higher in the MD arm. According to these results MP remains as the gold standard for treatment of MM and should be the reference for comparison of new therapeutic approaches involving novel agents.

We report on a series of Spanish patients with acute lymphoblastic leukaemia in whom the t(12;21) [TEL/AML1] translocation could not be identified with two sensitive techniques: reverse transcript-polymerase chain reaction (RT-PCR) and fluorescence in-situ hybridization (FISH). 101 cases were analysed: 38 children (29 B-cell precursor; nine T-cell precursor) and 63 adults (48 B-cell precursor; 15 T-cell precursor). Specific RT-PCR to amplify the TEL/AML1 fusion transcript was negative in all 101 cases. Moreover, all 38 paediatric samples were also negative by interphase FISH analysis for the presence of the TEL/AML1 fusion. These results suggest the existence of geographic/race variations in the genotype of acute lymphoblastic leukaemia (ALL).

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 < 0.001), chronic lymphocytic leukaemia (P = 0.001), monoclonal gammopathy of unknown significance (P < 0.001), multiple myeloma (P < 0.001) patients, and myelodysplastic syndromes (P = 0.002). Furthermore, almost no overlap between the levels of CD69 expression on BMMC was observed between SMCD cases and the remaining groups of individuals except for the paediatric mastocytosis group (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<0.01) and a shorter disease-free survival (P<0.01). Moreover, abnormalities in either of these two criteria detected at specific time points during follow-up (end of induction, maintenance, or after treatment) were associated with a significantly shorter disease-free survival (P<0.01). In summary, the investigation of abnormalities in B-cell differentiation is a relatively simple and cheap approach for predicting relapse in precursor-B-ALL patients.

Bcl-2 protein plays a major role in the prevention of programmed cell death of differentiating cells. In the present study, the expression of cytoplasmic bcl-2 by human Bone Marrow Mast Cells (BMMC) from both normal and pathological bone marrow samples was examined. A total of 35 subjects corresponding to 9 healthy volunteers, 8 cases of adult indolent systemic mast cell disease (SMCD), 4 cases of pediatric mastocytosis (PM), 11 cases of hematological malignancies (HM), 2 cases of reactive bone marrow, and 1 case of mast cell leukemia (MCL) were analyzed. The expression of bcl-2 was studied using quantitative three-color flow cytometry. We also studied the molecular configuration of the bcl-2 gene and other relatives by Southern blot and polymerase chain reaction (PCR) in the MCL case. Bcl-2 expression was detected in BMMC from all samples analyzed. No significant differences on the expression of bcl-2 were detected between BMMC from healthy subjects and patients with SMCD, PM, HM, and reactive bone marrow. By contrast, bcl-2 protein was overexpressed in BMMC from MCL patient without gene rearrangement. Our results show that bcl-2 protein was constitutively expressed by BMMC. BMMC from MCL display overexpression of bcl-2, which could not be related to molecular rearrangements involving the bcl-2 gene. The expression of this protein by mature MC may play a role in the prevention of MC apoptosis and thus help to explain the long survival of these cells. The overexpression of bcl-2 by BMMC in MCL may help to explain their resistance to chemotherapy-induced apoptosis.

In the present study the incidence of abnormal karyotypes and the number and proportion of abnormal metaphases obtained in multiple myeloma (MM) using three culture conditions were compared: unstimulated culture (72 h), IL-6/GM-CSF-stimulated culture (120 h) and IL-4-stimulated culture (120 h). The three types of culture conditions were assessed simultaneously on bone marrow samples from 30 consecutive myeloma patients. In addition DNA content (DNA ploidy and cell cycle) was analysed by flow cytometry. The number of MM samples with clonal karyotypic abnormalities was higher after IL-4-stimulated cultures (53%) than it was after IL-6 + GM-CSF (37%) and unstimulated (30%) cultures. The benefit of IL-4 was also observed in cases with low numbers of plasma cells in the bone marrow, in early clinical stages and in untreated patients. In those cases in whom clonal chromosomal abnormalities were detected by the three culture methods. the cytogenetic findings were always identical. According to our results the addition of IL-4 to the cultures of bone marrow cells in MM increases the number of abnormal metaphases.

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 <30% (P=0.03). Finally we found that patients with higher API (>0.05) displayed a significantly shorter overall survival as compared with patients with API<0.05 (P=0.04). The in vitro study properties of clonogenic cells produces relevant clinical information of leukaemic cell biology in AML patients.

No previous studies have been reported on human alcoholism in which the pattern of cytokine secretion by natural killer (NK) cells is explored. The goal of the present study was to evaluate the role of NK cells in the production of cytokines in patients with chronic alcoholism, analyzing at the same time the possible relationship between cytokine production and both alcoholic liver disease and ethanol (EtOH) intake. A total of 30 chronic alcoholic patients-11 without liver disease [alcoholics without liver disease (AWLD) group] and 19 diagnosed of alcoholic liver cirrhosis-were included in this study. Twenty-five age- and sex-matched healthy volunteers were analyzed as controls. Production of interferon (IFN)-gamma, tumor necrosis factor-alpha (TNF-alpha), and interleukin (IL)-6 was performed on NK-enriched peripheral blood mononuclear cells (PBMC) after stimulation with IL-2 and IFN-alpha. In AWLD patients, the production of TNF-alpha was significantly reduced, compared with normal controls, under both IFN-alpha (p < 0.01) and IL-2 (p < 0.05) stimulation. In patients with cirrhosis, TNF-alpha production by PBMC enriched in NK cells varied depending on the EtOH intake status at the moment of evaluation. Accordingly, an increased concentration of this cytokine was detected in the supernatants of cirrhotic patients and active EtOH intake, particularly after IFN-alpha stimulation (p < 0.05); whereas, in patients with at least 1 year of alcohol withdrawal, TNF-alpha levels remained within normal range. The results on the production of IL-6 and IFN-gamma in AWLD and cirrhotic patients showed that only cirrhotic patients with a prolonged EtOH withdrawal period display abnormal production. Accordingly, in this group of patients, a significantly increased release of IL-6 was observed after both IFN-alpha and IL-2 stimulation (p < 0.01 and p < 0.05, respectively). By contrast, a lower IFN-gamma production (p < 0.005) was detected with respect to the control group. Our results point to the existence of an abnormal cytokine secretion by NK cells from chronic alcoholism patients, which depend on both the existence of liver disease and the status of EtOH intake.

The surface expression of CD117 on plasma cells (PCs) from normal individuals and patients with multiple myeloma (MM) has been analysed using triple-stained cells for flow cytometry. In addition, the clinical significance of CD117 expression in MM patients and its possible value for the evaluation of minimal residual disease was explored. A total of 11 healthy volunteers and 56 untreated MM patients were included in the study. The expression of CD117 was analysed by flow cytometry, using simultaneous staining with the MAbs BB4, CD117 and CD38. Cell acquisition was performed in two consecutive steps using a live gate drawn on SSC/CD38 cells and a total of 300,000 events were acquired. For data analysis, the Paint-a-Gate Plus software (Becton Dickinson) was used. PCs were identified according to their strong reactivity for CD38 and their positivity for BB4, as well as by their light scatter distribution. Dilution experiments of CD117+ myelomatous PCs with normal bone marrow (BM) cells were performed in order to assess the sensitivity level of the technique for detection of CD117+ residual PCs. None of the PCs from normal BM samples showed reactivity for the CD117 antigen. In contrast, CD117 antigen was present in 18/56 MM patients (32%), the proportion of positive cells in these cases being as high as 92.1 +/- 9%. Therefore, within PC lineage the c-Kit antigen would be restricted to the myelomatous population and thus could be considered as a 'tumour-associated marker' for monitoring minimal residual disease in about one third of MM patients. Dilution experiments indicate that the detection limit with this marker would be 10(-4) (one myelomatous PC/10(4) normal BM cells). Upon comparing the clinical and haematological disease characteristics of CD117-positive and CD117-negative cases, no significant differences were found.

In this study the expression of 'classically' considered lymphoid-associated antigens (CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, and CD22) was explored both in peripheral blood (PB) and bone marrow (BM) mast cells (MC) in a case of systemic mast cell disease (SMCD) by means of using multiple stainings and a direct immunofluorescence technique. CD2 and CD22 were expressed in both PB and BM MC, all the remaining lymphoid-associated markers were negative. Our results suggest that the reactivity for both CD2 and CD22 in PB and BM MC would be aberrant.

In the present study, seven normal human bone marrow samples from healthy volunteers have been analysed in order to investigate the immunophenotypic characteristics of the normal CD117+ cells and their utility for the detection of minimal residual disease in 71 acute myeloid leukaemia patients. Our results show that most of normal BM CD117+ cells coexpress the HLADR and the myeloid associated CD33 antigen. In addition, almost half of CD117+ cells are CD34+, these cells displaying a different FSC/SSC distribution when compared to the CD117+/CD34- cells. No CD117+/CD15+ and CD117+/CD10+ cells were detected and very few CD117+ cells (< 1 x 10(-3) expressing the HLADR-/CD34-, CD33+/HLADR- and CD34+/HLADR- phenotypes were found to be present in normal BM. In contrast, from the 71 AML patients analysed, 34 had CD117+/CD15+ blast cells and eight had the CD117+ phenotypes detected at low frequencies (< 1 x 10(-3)) in normal BM. In summary, the present study shows that the use of the CD117 antigen in different monoclonal antibodies combinations may be of great help for the detection of minimal residual disease in a high proportion of AML cases, especially in those patients displaying the CD117+/CD15+ phenotype, because cells coexpressing both antigens in normal BM, if present, are at very low frequencies.

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.

The prognosis and long-term results of a group of 57 acute megakaryoblastic leukaemia (M7-AML) patients was analysed from a multicentre perspective. Ages ranged from 4 to 83 years, median 49 years; 30 were males and 27 were females. The median follow-up time was 7 months, range 1-24 months. Early exits occurred in 12 cases, their median age being 71 years. Forty-five patients were treated with combined aggressive chemotherapy (CT) (n = 26) or low-dose cytarabine (LD-AraC) (n = 19). The following results were obtained with combined CT or AraC, respectively. Complete remission (CR) rates were 73% and 84%, 12-month survival (SV) were 37% and 26%, 24-month SV were 12% and 11%, median SV 10 and 4 months, and relapse rates (RR) were 68% and 94%. These differences were not statistically significant. Irrespective of the treatment modality, the results were better for children (n = 10) than for adults (n = 35): RR rates were 90% and 74%, median SV: 7 and 5 months, 12-month SV: 40% and 22%, 24-month SV; 30% and 9%, and RR: 78% and 81%, respectively; these differences also were not statistically significant. In addition, a literature review of 42 patients from 18 previous reports is presented, including seven cases treated with allogeneic bone marrow transplantation (BMT). The best results were obtained with BMT: 12 and 24 month SV was 86% and the RR was 0%. On the above-mentioned basis, we feel that children and young adults with M7-AML should be offered BMT. In patients over 60 years old or not eligible for aggressive chemotherapy or BMT, an interesting possibility would be the use of LD-AraC which allows a high CR rate, followed by a classical consolidation regimen in order to prevent early relapses.

In a uniform series of 170 untreated myeloma patients (MM) we investigated the distribution of T cell subsets in peripheral blood (PB) and their relationship with the most relevant disease characteristics, including survival. CD4 cells were significantly decreased both in percentage and absolute numbers (P less than 0.0001). On the other hand, the CD8 cells only showed a slight increase in relative numbers. Upon correlating the abnormalities in the distribution of T cells with other clinical and biological disease characteristics the most remarkable correlation was with survival. A low number of CD4 cells (less than 700 x 10(6)/l) was associated with both an advanced clinical stage and a shorter survival (20 v. 43 months, P = 0.01). Moreover, a significant correlation also exists between the decrease in CD4 cells and both high beta 2-microglobulin (beta 2M) levels and anaemia. On the other hand, no relationship was found with the type of M-component nor with the plasma cell phenotype. Finally multivariate analysis showed that the number of CD4 cells add independent prognostic information to other well-established tests for the assessment of disease outcome in patients with multiple myeloma.

The expression of both natural-killer (NK)-associated and activation antigens was studied by flow cytometry in the peripheral blood of 47 untreated multiple myeloma (MM) patients. A significant increase in both absolute and relative numbers of CD57 positive cells as well as in the proportion of CD16 and CD11b cells was observed in patients with MM, specially in those in early stages of the disease (clinical stages I and II), suggesting a possible surveillance mechanism in response to an emerging malignant clone. Additional double stainings showed that strong CD16+ NK cells coexpress the CD56, CD11b, and CD2 antigens, while they lacked CD3, CD5, and WT31 antigens. Moreover, the previously reported increase in CD8 cells present in MM would be mainly due to a subset of CD8 cells that coexpress the CD57 Ags. The expression of activation antigens, especially CD38, was increased in peripheral blood lymphocytes of MM patients, the differences reaching statistical significance both in absolute and relative numbers in those cases with high numbers of CD16 NK cells and thus suggesting that these cells are functionally activated. These results reveal the existence of an increase in NK and activated cells in the peripheral blood of myeloma patients that may reflect a host's immunological mechanism in an attempt to modulate tumor cell growth.

We have studied phenotypic and clinical features in a consecutive series of 45 patients with chronic myelogenous leukaemia (CML) in blast crisis (BC). In addition, in 22 of these patients we have analysed the genotypic characteristics including immunoglobulin, T-cell receptor (TCR) and major breakpoint cluster region (M-bcr) gene organization. The granulomonocytic and megakaryoblastic lineages are the most commonly involved in these BC of CML (33% and 33% of cases, respectively); only 18% of our cases displayed a lymphoid phenotype. Moreover, both morphological and immunophenotypic studies revealed the frequent coexistence of two or three cell populations, especially when the megakaryoblast component is involved. The lymphoid BC displayed the highest incidence of complete remissions although this was not associated with a longer survival. Only minor differences between the different myeloid subgroups were observed. Immunoglobulin heavy chain (IgH) gene rearrangement was found in five of the six lymphoid BC and in one myeloid BC. Only one case showed k light chain gene rearrangement. In all but one myeloid BC the TCR-beta gene was in germline configuration. The TCR-gamma gene was rearranged in all lymphoid and one myeloid BC, while TCR-delta gene rearrangement was detected in 67% and 16% of the lymphoid and myeloid BC, respectively. Most of the lymphoid BC (4/5) had the M-bcr breakpoint in subregion 3, while the myeloid BC had the breakpoint either in subregion 2 or 3. No differences between the different myeloid phenotypic subgroups were observed in relation to breakpoint.

The value of the immunophenotypical subtypes and individual markers was compared with classical parameters in the prognosis of 150 patients with acute lymphoblastic leukemia (ALL). Regarding the immunophenotype, common-ALL had a better prognosis than T-ALL in the children's group. However, in adults the situation was different since both null and T-ALL patients had longer survival rates than the common pre-B group. Moreover, several individual markers add interesting prognostic information, either in ALL as a whole group or within the different immunophenotypes. Thus, the expression of CD10 and TdT had a significantly favourable influence in the outcome of the whole series of patients; within the T-ALL, those cases positive for CD10 also had a longer median survival (33 versus 17 months). In addition, in the common ALL patients group the expression of a relatively mature B marker--CD20--appeared to have a favourable prognosis (27 versus 13 months). Other non lineage specific markers, such as CD9 and CD38 did not seem to influence survival. Regarding the more conventional parameters, our data suggest that the classical age prognostic classification in children (less than 15 years) and adults can be improved using two cut-off points at 11 and 35 years. Moreover, the multivariate analysis showed that this variable, together with FAB morphology and WBC counts were the best combination of parameters for predicting survival. The present study shows that although the immunophenotype helps us in understanding the biological heterogeneity of ALL, having also prognostic implications, there are other clinical and hematological features that yield stronger prognostic information.

T-cell subsets defined by monoclonal antibodies (OKT3, OKT4, and OKT8) were analyzed in 117 patients with monoclonal gammopathies--69 multiple myeloma (MM) (30 untreated and 39 treated), 14 Waldenström's macroglobulinaemia (WM), and 34 essential monoclonal gammopathy (EMG) patients. The percentage and absolute numbers of total T-lymphocytes (E+, OKT3+ cells) were within the normal range in all groups except for the treated MM patients, in which a decrease in the absolute number could be observed. The percentages of OKT4+ cells were significantly lower in MM (35 +/- 1.7) than in EMG patients (43 +/- 2) and controls (50 +/- 2). In contrast, OKT8 cells correspondingly increased in MM (38 +/- 1.6) compared with EMG patients (29 +/- 1) and controls (27 +/- 1). The OKT4/OKT8 ratio was lower in MM than that in EMG patients and controls (p less than 0.01) and was shown to be one of the four most significant variables in a linear discriminant analysis used to distinguish between MM and EMG groups. The MM patients in clinical stage III as well as Bence-Jones myeloma patients showed a more pronounced OKT4/OKT8 imbalance. The treatment did not influence the percent distribution of T-cell subpopulations. The patients with WM exhibit an alteration in the distribution of the T-cell subsets similar to the MM patients with a T4/T8 ratio of 1.1 +/- 0.1. This imbalance was more pronounced in WM patients with monoclonal B-lymphocytes in peripheral blood (leukaemic phase of WM). The functional significance of the altered T-cell subsets in MM and WM patients remains to be established, though it is probable that such an imbalance plays an important role in regulating these B-cell proliferations.

The immunological phenotype of diseases involving the last step of B cell differentiation--multiple myeloma (MM, 38 patients) and Waldenström's macroglobulinaemia (WM, 12 patients)--was analysed with a panel of monoclonal antibodies (McAb) as well as conventional markers. Most of the bone marrow plasma cells (80%) from MM patients reacted with the McAb OKT10, FMC8 and FMC48. Plasma cells were consistently negative with FMC7, Leu-1 and mouse rosettes. Ia, B1 and SIg were expressed in a minority of plasma cells (less than 30%) in half of the cases. The circulating neoplastic cells from five MM patients showed a more immature phenotype, with a higher reactivity for OKIa, B1 and increased SIg and a lower expression of CIg, than bone marrow plasma cells. The malignant cells of WM patients differed from those of MM in the reactivity with FMC7, being positive in 10 out of 11 cases, and in their high expression of B1, Ia and SIg with a predominant mu+ phenotype. Mouse rosettes and Leu-1 were positive in one case; OKT10 was positive in three out of five WM patients studied. This phenotype indicates that WM cells correspond to an earlier stage of B cell differentiation than MM plasma cells. The McAb J5 was positive in three out of six MM and two out of four WM analysed. The antigenic differences observed in MM and WM patients support the notion that the cells of the neoplastic clone are able to undergo a certain degree of differentiation.