Search

Although new agents have been approved for the treatment of MDS, the only curative approach is allogeneic hematopoietic stem cell transplantation (HSCT) and thus, in particular circumstances this procedure has been proposed as a treatment option for low risk patients. We have retrospectively analyzed the results of HSCT in 291 patients from the Spanish MDS registry with special attention to low risk MDS (LR-MDS) in order to define the variables that could impact their clinical evolution after transplantation. At 2 years OS was 51% and EFS was 50% (95% CI 0.7-4.5 years for OS and 95% CI 0.1-3.9 years for EFS). Among 43 LR-MDS, transplant-related mortality was 28%. At 3 years, OS was 67% (95% CI 264.7-8927.2 days for OS) and EFS was 64% (95% CI 0-9697.2 days for EFS). In the multivariate analysis only cytogenetics retained statistical significant effect on both OS (p=.047) and EFS (p=.046). Conditioning regimen could improve outcome among this subset of patients (OS 86% and RFS 100% for patients receiving RIC regimen). The present study confirms that specific disease characteristic as well as transplant characteristics have a significant impact on transplant outcome. Regarding low risk patients a non-myeloablative conditioning would be preferable especially in cases without high-risk cytogenetics. Copyright (C) 2014 Elsevier Ltd. All rights reserved.

Immunophenotypic alterations of bone marrow myeloid cell compartments in multiple myeloma patients predict for myelodysplasia-associated cytogenetic alterations

An atypical case of lymphoproliferative disorder in which T- and B-cell antigens were coexpressed in the neoplastic cells is described. The disease was characterised by hepatosplenomegaly, lymphadenopathy, a low WBC (5 X 10(9)/l) and bone marrow infiltration. The predominant cell population (greater than 70%) comprised lymphoid cells with a range of nuclear irregularities and included some blast cells. 90% of the peripheral blood lymphocytes showed a mature T-helper phenotype (E+, T11+, T3+, T4+, T8-, T6-, TdT-) with coexpression of the specific B-markers B1 and FMC7, in 90% and 50% of cells, respectively. HLA-DR antigens were present in 55% of cells while surface and cytoplasmic immunoglobulins (Ig) were detected in less than 10% of cells. Molecular investigations with appropriate probes showed evidence of T-cell receptor gene rearrangement but no rearrangement for the genes of the Ig-heavy and -light chains. Cytogenetic studies revealed a translocation t(10;19) (p12; q13) in all the metaphases analyzed. This case demonstrates that the study of neoplastic cells with a battery of monoclonal antibodies may disclose the existence of a hitherto unrecognised lymphoid cell population with atypical expression of B- and T-cell antigens. On the other hand, the presence of T-cell receptor gene rearrangement indicates that this is a T-cell disorder with the aberrant co-expression of specific B-cell markers.

Damage to the stem cell progenitors caused by the chemotherapy received in patients diagnosed with non-Hodgkin's lymphoma (NHL) may be an important factor limiting progenitor cell mobilization. The aim of the present analysis was to evaluate the effect of the chemotherapy on the different progenitor cell subpopulations obtained in the leukapheresis. For this purpose, a combination of immunophenotype and functional assays has been performed in 26 mobilized peripheral blood (PB) samples from NHL patients and 36 healthy donors. The different progenitor subpopulations analyzed by flow cytometry significantly correlated with the corresponding populations assessed by functional assays in both healthy donors and NHL patients (p0.05, r0.5). The number of committed CFU-GM was similar in both groups (p=0.246), but we found significant decrease in the number of BFU-E and more immature progenitors in PB from NHL patients as compared to donors (p0.05). Moreover, the number of total CFU was significantly lower in NHL patients (p=0.007). Accordingly, CD34+ cells (p=0.018) and CD34+ subpopulations was decreased in NHL patients. Nevertheless, CD90 and CD34 intensity was significantly higher within CD34+ cells from NHL patients as compared to donors. However, although numerically reduced non-committed CD34+ cells are more immature in chemotherapy mobilized NHL patients. In summary, our results show that all NHL hematopoietic progenitors, analyzed by both immunophenotypical and functional approaches, are impaired in leukapheresis products.

The aim of this study was to identify circulating endothelial progenitor cells (EPC) with colony-forming capacity and compare them with the monocytic-macrophage lineage.Forty-two healthy donors were analyzed. EPC were cultured with VEGF and b-FGF. Sequential studies were performed on days +7 (colonies) +21 and +35. Monocytic cells were cultured using the same conditions as EPC until day +21 or alternatively by adding IGF.The number of EPC colonies was higher in BM than in mobilized or steady-state PB. Using EPC medium, monocytic cells formed cord-like structures but no colonies. However, colonies grew when IGF was added to the medium. By immunocytochemistry, colonies showed CD45, CD31 and lysozyme but no vWF. Colonies were CD4+, CD13+dim, CD14+, CD15++, CD16-/+dim, CD31+dim, CD33+dim, CD45+, CD105-/+dim, lysozyme+ and VE-cadherin+, and constantly negative for CD34, CD133 and KDR, when flow cytometry was used. The immunophenotype of pre-cultured and cultured monocytes was similar to that described for EPC.Our results suggest that the so-called 'EPC' obtained at 7 days of culture belong to the monocyte-macrophage lineage, as they share immunophenotypic and molecular features.

Mobilisation with G-CSF in healthy donors promotes a high but temporal deregulation of genes

Recombinant® interferon α (r-IFN-α) has been shown to be an effective drug for chronic myeloid leukaemia (CML). However, higher response rates can be achieved using cytarabine along with r-IFN-α. YNK01 is a derivative of cytosine arabinoside for oral administration. So far, the only published experience with continuous YNK01 was in advanced CML (10 cases). We have performed a pilot study to evaluate the efficacy and toxicity of the combined therapy r-IFN-α and daily oral YNK01 in patients with newly diagnosed Ph+ CML. Ten previously untreated patients were included in the study. Among those patients evaluable for cytogenetic response, 87% (seven out of eight) reached a major cytogenetic response with four reaching complete cytogenetic response (50%). The most significant side-effects were gastrointestinal. Macrocytic anaemia was observed in three patients. In conclusion, continuous oral administration of YNK01 in combination with IFN-α is safe and can result in high-cytogenetic response rates.

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

Licencia Creative Commons Reconocimiento-No comercial.-- et al., Hematopoietic stem cell transplantation (HSCT) using umbilical cord blood (UCB) progenitors is increasingly being used. One of the problems that may arise after UCB transplantation is an impaired engraftment. Either intrabone (IB) injection of hematopoietic progenitors or mesenchymal stem cell (MSC) coadministration has been proposed among the strategies to improve engraftment. In the current study, we have assessed the effects of both approaches. Thus, NOD/SCID recipients were transplanted with human UCB CD34+ cells administered either intravenously (IV) or IB, receiving or not bone marrow (BM)-derived MSCs also IV or IB (in the right femur). Human HSC engraftment was measured 3 and 6 weeks after transplantation. Injected MSCs were tracked weekly by bioluminescence. Also, lodgment within the BM niche was assessed at the latter time point by immunofluorescence. Our study shows regarding HSC engraftment that the number of BM human CD45+ cells detected 3 weeks after transplantation was significantly higher in mice cotransplanted with human MSCs. Moreover, these mice had a higher myeloid (CD13+) engraftment and a faster B-cell (CD19+) chimerism. At the late time point evaluated (6 weeks), human engraftment was higher in the group in which both strategies were employed (IB injection of HSC and MSC coadministration). When assessing human MSC administration route, we were able to track MSCs only in the injected femurs, whereas they lost their signal in the contralateral bones. These human MSCs were mainly located around blood vessels in the subendosteal region. In summary, our study shows that MSC coadministration can enhance HSC engraftment in our xenogenic transplantation model, as well as IB administration of the CD34+ cells does. The combination of both strategies seems to be synergistic. Interestingly, MSCs were detected only where they were IB injected contributing to the vascular niche., This study was supported in part by a grant from Gerencia Regional de Salud de Castilla y León (ref. GRS/222/A/08) and by a grant from Consejería de Educación de la Junta de Castilla y León (ref. HUS003A10-2). S.C. was supported by Junta de Castilla y Leon (FPI grant EDU/1878/2006).

Post-transplant complications.-- et al., Transplantation-associated thrombotic microangiopathy (TA-TMA) is a feared complication of allogeneic hematopoietic SCT (HSCT) owing to its high mortality rate. The use of calcineurin inhibitors or sirolimus (SIR) for GVHD prophylaxis has been suggested as a potential risk factor. However, the impact of tacrolimus (TAC) and SIR combinations on the increased risk of TA-TMA is currently not well defined. We retrospectively analyzed the incidence of TA-TMA in 102 allogeneic HSCT recipients who consecutively received TAC plus SIR (TAC/SIR) (n=68) or plus MTX (TAC/MTX)±ATG (n=34) for GVHD prophylaxis. No significant differences were observed in the incidence of TA-TMA between patients receiving TAC/SIR vs TAC/MTX±ATG (7.4% vs 8.8%, P=0.8). Only grade III-IV acute GVHD, previous HSCT and serum levels of TAC >25 ng/mL were associated with a greater risk of TA-TMA. Patients developing TA-TMA have significantly poorer survival (P

This study describes a new method for the shultaneous assessment of the distribution of a cell population in the GO/Gl, s, and G2N cell-cycle phases by using multiparameter flow cytometry and single staining based on BrdUrd incorporation. Both the K562 cell line and PHA-stimulated peripheral blood lymphocytes (PBL) were analyzed. Cells were cultured in the presence of BrdUrd for 30 min prior to cell harvesting. Once collected, cells were exposed to ultraviolet light for 5 min and then fixed immediately in 70% ethanol (-20°C) for at least 30 min. Once fixed, the cells were placed for 30 min at 37°C in the presence of terminal deoxynucleotidyl transferase (TdT) and dUTP labeled with digoxigenin; they were then stained with FITC-labeled anti-digoxigenln. Our results show that GO/Gl, S, and G2N cell populations can be clearly discriminated according to FITC fluorescence and light-scatter parameters. In this way, S-phase cells can be identified by their FITC staidng. From the cells which were negative for anticould be resolved in a forward scatter, side scatter, and fluorescence pulse-width three-dimensional plot; the values obtained for GO/Gl cells were lower than those obtained for G2/M cells in all three parameters. Multiparameter analysis of PBL stained for two surEdce antigens (CD3 and CD8) and for BrdUrd by direct or indirect TdT method permitted cell-cycle analysis of Merent subpopulations, including CD3 + /CDS + , CD3 + /CD8-, CD3-/CDS + , and CD3-/CD8-. 6 1996 wiley-~iss, hc.

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.

[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. © 2012 The Authors. Transfusion Medicine © 2012 British Blood Transfusion Society., This project had been partially supported by a grant from Ministerio de Ciencia e Innovación (reference code PLE2009-0094).

The membrane phenotype of AML clonogenic cells (L-CFU) was analyzed in 19 AML patients using an in vitro culture technique after a complement-mediated lysis assay employing a panel of six monoclonal antibodies (McAb) -HLA-DR, FMC56 (CD9), FMC27 (CD9), CD14, CD15, CD41a-. Our results show that L-CFU has a heterogeneous but immature phenotype lacking on the expression of differentiation antigens (CD14, CD15, CD41a). In addition, we observed that the L-CFU phenotype is different from that of the whole blast cell population. Interestingly, L-CFU showed a higher expression of HLA-DR antigens with respect to their progeny. Upon analyzing whether the L-CFU phenotype was related to both the morphological and immunological features of AML blast cells, it was observed that, while there is no correlation with the FAB classification, there was a partial relationship between the immunological phenotype of AML blast cells and that of L-CFU. Accordingly, the more immature AML cases showed a more differentiated L-CFU phenotype (HLA-DR+, CD9+, FMC27+) when compared with cases with a more mature blast cell phenotype. These results suggest that those AML cases with a relatively immature myeloblastic phenotype may arise from a progenitor cell that has undergone partial differentiation and that is unable to acquire myeloid differentiation antigens, while those AML cases with mature blast cells might emerge from a very early L-CFU that has the capacity to undergo a greater degree of differentiation.

AIM: To evaluate the validity of the colony forming unit-granulocyte macrophage (CFU-GM) assay for predicting relapse in patients with acute myeloid leukaemia (AML). METHODS: The study population comprised 32 patients with AML in remission, followed for a median of 18 months. A mean of four studies was carried out per patient. Three patterns of in vitro growth based on the number of CFU-GM in normal bone marrow were defined: 1 = normal (normal number of CFU-GM and a cluster:colony ratio < 2); 2 = hypoplastic (low number of CFU-GM and a cluster:colony ratio < 2); 3 = anomalous (low or normal number of CFU-GM and a cluster:colony ratio > 2). RESULTS: Eleven patients relapsed, all of whom had previously displayed an abnormal CFU-GM pattern: anomalous in nine and hypoplastic in two. The remaining 25 patients were in complete remission at the time of writing, 16 of whom had a normal growth pattern. The other nine had anomalous (eight patients) or hypoplastic (one patient) growth. The latter may be false positive results. The in vitro growth pattern was not constant during follow up analysis. All 15 patients in whom the growth pattern switched from abnormal to normal remain in complete remission. By contrast, of the five cases in whom the pattern changed from normal to abnormal, three have relapsed and the other two had other indicators of relapse. The growth pattern remained unchanged in the remaining 16 patients. CONCLUSION: The present data show that the sequential investigation of the CFU-GM growth pattern may be of value in predicting relapse in patients with AML.

Studies evaluating the effects of previous chemotherapy on stem-cell yield and hematological recovery after autologous peripheral-blood progenitor-cell transplantation (PBPCT) have shown conflicting results. We have retrospectively analyzed 103 consecutive lymphoma patients treated with the BEAM regimen and autologous PBPCT. The impact of the different chemotherapeutic drugs (cumulative doses) on stem-cell yield and transplant-related toxicity was investigated. Highly significant differences in platelet recovery (20 x 10(9)/l) were observed between patients receiving less or more than 750 mg/m(2) of etoposide before transplant (15 vs 29 days, P=0.001), and between patients receiving less or more than 1.2 x 10(6)/kg CD34(+) cells (27 vs 14 days, P0.001). Differences in neutrophil engraftment between groups were not clinically significant. Pre-transplant cumulative doses of etoposide750 mg/m(2) were associated with low CD34(+) cell collections on multivariate analysis. The actuarial incidence of transplant-related mortality (TRM) was 14% at 5 years. Pre-transplant cumulative doses of etoposide350 mg/m(2) and previous administration of procarbazine were found to be independent prognostic factors for TRM.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for patients with myelodysplastic syndromes (MDSs). We have analyzed the outcome of 81 patients who underwent an allogeneic transplant from an HLA-identical sibling donor. The overall survival (OS) was 31% and disease-free survival was 30% at 5.8 years. Transplant-related complications were the cause of death in 44% and disease progression in 16% of patients. Acute graft-versus-host disease (aGVHD) grades II-IV occurred in 32 cases (39%). Extensive chronic GVHD (cGVHD) was observed in 27% of patients. When the log-rank test was performed, we observed that patients transplanted more than 6 months after diagnosis, and those transplanted with bone marrow (BM) displayed a shorter survival (P=0.009 and 0.005, respectively). Patients who developed cGVHD showed a trend towards better OS (P=0.07). Patients receiving BM had a higher incidence of aGVHD (65 vs 50%) and less cGVHD (52 vs 30%), although the differences did not reach statistical significance. Moreover, patients who received PB-HSC displayed a faster engraftment (P=0.000) and showed a significantly lower early transplant-related mortality (14 vs 42%; P=0.006) and longer OS (P=0.005). In summary, our results show that hematopoietic transplantation should be performed as soon as possible in MDS patients and that PB is preferable to BM as a source of HSC.

Hemorrhagic cystitis (HC) is a common complication following hemopoietic stem cell transplantation (HSCT), its incidence ranging from 7 to 52% of all patients. Late occurring HC frequently results from viral infections. We describe a patient who developed severe polyomavirus-associated HC, which responded dramatically to a single dose of intra-muscular vidarabine. Previous studies show an improvement in HC with vidarabine therapy, but to date only the intravenous route of administration has been described and responses described take from several days to weeks. This report confirms the safety and efficacy of vidarabine administered intramuscularly when used in patients with an adequate platelet count, thereby making its use feasible when intravenous vidarabine is not available.

Pathological rupture of the spleen is a rare but well recognized complication in hematological malignancies. Early clinical recognition of this life-threatening complication is necessary for rapid intervention. Here, we report on the case of a 26-year-old woman with acute promyelocytic leukemia who presented rupture of the spleen on day +2 of treatment with ATRA plus idarrubicin. In patients with acute leukemia, the presence of a painful abdomen and a sudden drop in hemoglobin levels, should alert of a possible splenic rupture, even without additional symptoms. This would facilitate an early treatment intervention with no modification to the chemotherapy schedule.

Summary Background Atypical chronic myeloid leukemia (aCML) is an infrequent chronic myeloproliferative disorder characterized by leukocytosis, absence of Philadelphia chromosome or BCR-ABL rearrangement, and marked myeloid dysplasia. Some cases have an absolute monocytosis but can be distinguished from chronic myelomonocytic leukemia (CMML) by the presence of a higher percentage (> 15%) of circulating immature granulocytes. Patients and methods In a series of 11 patients with a diagnosis of aCML according to the FAB proposals we have analyzed the most relevant clinical, hematological and cytogenetic characteristics. Results The median age was 65 years (16–84). All but one case showed, at time of diagnosis, leukocytosis (median WBC was 36 × 109/l), 55% had moderate anemia and 36% had thrombocytopenia. Most cases had marked dysplasia, particularly in the granulocytic lineage (82% of the cases), and all cases showed bone marrow red hypoplasia. Cytogenetic abnormalities were present in 9 out of the 11 patients. Trisomy 8 was observed in three cases and other clonal chromosomal abnormalities included deletions of 5q, 13q, 17p, 12q, and llq as well as a t(6;8)(p23;q22) translocation. Fluorescence in situ hybridization (FISH) studies failed to demonstrate ETV-6 gene involvement. The median survival time from diagnosis was only 14 months (range 3–56 months). Conclusions These data suggest that a CML is a rare disease which is characterized by leukocytosis, with dysgranulo-poiesis, BM erythroid hypoplasia, chromosomal, though not recurrent, abnormalities and poor prognosis.

Secondary myelodysplastic syndromes (MDS) are increasingly being reported after autologous transplantation. Transient dysplastic changes have also been observed after this type of treatment. However, to the best of our knowledge no systematic morphological analysis has been performed to determine the influence of stem cell transplantation on bone marrow morphology. In 53 patients undergoing autologous transplantation, we evaluated the bone marrow, before and 6 and 12 months after the transplant, in order to analyze the appearance of dyshemopoietic changes, assessed according to a pre-established score. We also studied 25 bone marrow samples obtained at the time of diagnosis, prior to treatment, but we did not find morphological atypia. Six months after transplant, cellularity and thrombopoiesis had decreased in 38% and 49% of patients respectively, although 1 year after the process they were normal in most cases. Myelodysplasia was already present in bone marrow before transplantation and continued to be in evidence for a long time afterwards. This suggests that chemotherapy and radiotherapy used prior to transplantation are responsible for dysplastic changes. The myeloid line was the most affected with 100% of patients showing dysgranulopoiesis 1 year after autografting. Cytopenias were observed in 51% and 44% of patients 6 and 12 months after transplantation. Moreover, concomitant presence of cytopenia and myelodysplasia was observed in 37.7% of patients at 6 months after transplantation and 25% at 12 months, and therefore they could be diagnosed with MDS. These data contrast with the incidence of secondary MDS reported in earlier publications. According to these findings, the value of the French-American-British Co-operative Group criteria for the diagnosis of MDS following autologous transplantation is questionable. Moreover, since dyshemopoietic features are almost always present after autologous transplant, morphological criteria are not useful for early recognition of patients with secondary MDS after transplantation.

In the present paper, we evaluate tolerability, outcome and prognostic factors in patients with poor prognosis non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD) when uniformly treated with BCNU, etoposide, cytarabine and melphalan (BEAM) and autologous stem cell transplant (ASCT). On hundred and forty-eight patients with NHL (n = 112) or HD (n = 36) received BEAM followed by infusion of bone marrow (n = 55), peripheral blood stem cells (n = 79) or both (n = 14). Twenty-eight patients had low-grade lymphoma (LGL), 68 intermediate- and 16 high-grade lymphoma (IGL). Within the NHL group, 21 patients were in 2nd or subsequent complete remission (CR) at transplant, 34 had sensitive disease and 11 resistant disease; 46 patients were transplanted in 1st CR due to the presence of > or = 2 adverse prognostic features at diagnosis or to a slow CR. Of the HD patients at transplant 17 had active disease, 16 were in > or = 2 CR and three in 1st CR. The overall percentage of toxic deaths was 5.4%, while in the group of patients transplanted with PBSC it was only 1.3%. NHL patients: 78% were in CR following ASCT, including 25 out of 45 patients (56%) who were transplanted with active disease. Only two of the 11 patients transplanted with resistant disease achieved CR. Incidence of overall survival (OS) and disease-free survival (DFS) at 3 years was 65 and 75%, respectively. As far as histology was concerned, OS was significantly better for patients with LGL in comparison with IGL (88 vs 56%) (P = 0.002). DFS was significantly higher for patients transplanted in first CR or first partial remission (PR) than it was for those transplanted in a later CR or PR (86 vs 53%) (P = 0.02). Multivariate analysis for OS showed that histology, bulky disease, poor performance status at transplant and achievement of CR were independent prognostic factors. In addition, a high number of infused MNC was associated with poor DFS. HD patients: 30 (83%) were in CR after transplantation, with 25 maintaining CR at the end of the study. Only one of the four patients transplanted with resistant disease reached CR. Incidence of OS and DFS at 3 years was 78 and 81%. DFS was similar for patients transplanted with early or late relapse (95 and 93%). With multivariate analysis, the only independent variable for OS was CR after transplant. In conclusion, the present results demonstrate the efficacy and low toxicity of the BEAM regimen in high-risk lymphoma patients with sensitive disease. Other strategies should be investigated for patients with refractory lymphoma.

The efficacy of currently available treatments for Hodgkin's disease (HD) has led to a substantial modification in the prognosis of this disease; nevertheless there is still a group of patients that cannot be cured with conventional treatments and who will be candidates for alternative therapy. In the present work we analysed the prognostic influence of the most relevant clinico-biological characteristics of HD in a consecutive series of 137 patients diagnosed and treated in a single institution. Univariate analyses identified six variables with significant prognostic influence, both on achieving complete remission (CR) and overall survival (OS); LDH > 320 U ml-1, age > 45 years, stages IIB, III and IV, extranodal involvement, alkaline phosphatase > 190 UI dl and ESR > 40 mm h. In addition, Hb < 12.5 gr dl-1 and abdominal disease were statistically relevant for CR while a poor performance score (ECOG > or = 2) affected a lower survival. In the multivariate analysis only LDH, age and the clinical stage retained a significant prognostic influence for achieving CR, while the two first factors above, together with performance status were the variables with independent prognostic value with respect to OS. Moreover, only LDH > 320 U ml-1 had prognostic influence in the probability of relapse and disease free survival (DFS), both in the univariate and multivariate analyses. According to the three independent factors obtained in the multivariate analysis for CR (LDH, age and stage) a predictive model was established that allows the stratification of patients into two prognostic groups: one with poor prognosis that includes patients with the three adverse prognostic factors, or two if one of them was elevated LDH, and the other with good prognosis that includes the remaining patients. This model was also able to separate two independent groups of patients with respect to OS and to DFS. In conclusion, the present study shows that LDH is one of the most important prognostic factors in HD.

Acute myeloblasts leukemia (AML) is known to have highly variable clinical and biological characteristics. In recent years the availability of monoclonal antibodies (MAbs) specific for the different myeloid lineages and their differentiation stages, together with the development of new highly sensitive techniques such as flow cytometry, have both provided new insights into the biology of AML and contributed to more accurate diagnosis and classification of these leukemia patients (Ball 1990; Drexler and Minowada 1986; Goasguen and Bennett 1990; Ludwig and Thiel 1990; Neame et al. 1986; Pessano et al. 1984; San Miguel et al. 1986b; Second MIC Cooperative Group Study 1988; Van der Reijden et al. 1983). Although there is only a limited amount of data available, some studies (Ball et al. 1991; Campos et al. 1989; Geller et al. 1990; Griffin et al. 1986; San Miguel et al. 1989; Schwarzinger et al. 1990) suggest that the immunophenotype might also be of help in the prognostic evaluation of AML patients.

The blast cells in acute leukemia following myelodysplastic syndrome (MDS) have classically been assumed to be of myeloid lineage (Tricot et al. 1985). However, our previous preliminary data (San Miguel et al. 1986b) as well as several case reports have shown that other cell lineages, including the lymphoid, may occasionally be involved in these leukemia’s (Barton et al. 1980; Berneman et al. 1985; Eridani et al. 1985). Moreover, to the best of our knowledge the only broad molecular genetic study in MDS patients is that reported by Wainscoat et al. (1988), showing an absence of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement. However, the appearance of new cell clones in the acute transformation of MDS may induce molecular changes that so far have not been analyzed.

We demonstrate that HEL, a human erythroleukemic cell line, has numerous megakaryocytic markers which were markedly enhanced following the addition of the inducers dimethyl sulfoxide or 12-O-tetradecanoylphorbol-13-acetate to the culture medium. Ultrastructural and cytochemical studies showed: (i) the presence of organelles morphologically resembling the platelet alpha-granules; and (ii) a peroxidase activity with the same characteristics as that specifically found in platelets. The platelet alpha-granule proteins (von Willebrand factor, platelet factor-4 and beta-thromboglobulin) were immunologically detected in the HEL cell cytoplasm and their amounts increased after induction. Of particular interest was the presence of platelet membrane proteins. A monoclonal antibody specific for glycoprotein Ib bound to HEL cells. Platelet membrane glycoproteins IIb and IIIa were identified on intact cells using specific antibodies in a binding assay or in cell lysates using either crossed immunoelectrophoresis or an immunoblotting procedure following SDS-polyacrylamide gel electrophoresis. Most HEL cells also expressed the platelet alloantigen PIA1. All of the platelet membrane proteins were present in higher amounts after induction. Glycophorin A, specific for the erythroid lineage, was also detected on HEL cells. Thus, while confirming the presence of erythroid markers, our studies provide evidence that the HEL cell line also expresses platelet antigens. As such, HEL cells represent a unique system with which to study the biosynthesis of platelet-specific proteins and glycoproteins.

Between 25 to 50 per cent of MDS patients progress to an acute leukaemia, classically assumed to be of myeloid lineage (Tricot et al., 1985). However, recently two cases of MDS evolving to ALL have been described (Barton et al., 1980; Berneman et al., 1985).

Summary Several mouse monoclonal antibodies (MoAbs) considered specific for the myeloid lineage recognize the same carbohydrate structure (3-fucosyl-N-acetyl-lac-tosamine) which is similar to the murine antigen SSEA-I. We have investigated the expression of this antigen with six different well-characterized murine IgM MoAbs on normal, leukaemic, and cultured cells by immunofluorescence and immuno-electron microscope cytochemistry. The cells were studied before and after neuraminidase treatment since epitopes recognized by these MoAbs may be masked by sialic acid. Among the recognizable normal marrow or blood cells, all these MoAbs specifically labelled the granulocytic lineage from the promyelocyte to the polymorph. After neuraminidase treatment, monocytes became labelled. All the other lineages remained unstained. Several cell lines were studied. Six of eight lymphoblastoid cell lines were stained by these MoABs; reactivity was increased by neuraminidase. One Burkitt cell line and two T cell lines were also found to be positive. These antibodies were tested on leukaemic cells. In acute non-lymphocytic leukaemia they usually labelled promyelocytes, more mature granulocytic and monocytic precursors but did not label myeloblasts; after neuraminidase treatment, these myeloblasts became stained. No labelling was observed on leukaemic proerythroblasts and promegakaryoblast before and after neuraminidase treatment except in one case of promegakaryoblastic leukaemia in which the SSEA-I antigen and platelet peroxidase were expressed in the same cell. In addition, six cases of common acute lymphoblastic leukaemia were studied; the blasts became positive after desialylation. Two examples of T cell acute leukaemia were essentially negative. We conclude, therefore, that the reactivity of haemopoietic cells with these MoAbs alone does not represent a criterion sufficient to sustain their myeloid origin since the SSEA-I antigen may be expressed at the surface of all cell lineages in the early phases of haemopoietic differentiation.

This study was designed to compare the cytochemical pattern with the immunologic phenotype in 108 cases of acute myeloblastic leukemia (AML) classified according to the French-American-British (FAB) criteria. Special attention was paid to the cases where discrepancy existed between these approaches and to a group of 11 patients considered as unclassifiable mainly because a second cell population--megakaryoblastic--was detected. Three types of discrepancies were observed: cases with typical morphologic characteristics and cytochemistry but devoid of lineage-specific antigens; these mainly include poorly differentiated leukemias (eight M1, four M2, and eight M5a), suggesting that the cytochemical enzymes are earlier myeloid markers than the currently available monoclonal antibodies; cases in which immunologic characteristics were discordant with morphologic characteristics and cytochemistry; these include two M2 cases positive for monocytic monoclonal antibodies (CD14); six M5b cases positive for granulocytic monoclonal antibodies (CD15); and seven M4 cases lacking in CD14 or CD15 antigens; cases with discrepancies between morphologic characteristics and cytochemistry and in which the immunologic markers permitted the correct assessment of cell lineage (six cases). These results show that the classification of these patients is better achieved by a combined morphologic, cytochemical, and immunologic approach.