Optimisation of mesenchymal stromal cells karyotyping analysis: implications for clinical use.

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.

In leukapheresis products from non-Hodgkin's lymphoma patients, the immature hematopoietic progenitors show higher CD90 and CD34 antigenic expression.

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 (p<0.05, r>0.5). 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 (p<0.05). 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.

Next Generation Flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma.

Flow cytometry has become a highly valuable method to monitor minimal residual disease (MRD) and evaluate the depth of complete response (CR) in bone marrow (BM) of multiple myeloma (MM) after therapy. However, current flow-MRD has lower sensitivity than molecular methods and lacks standardization. Here we report on a novel next generation flow (NGF) approach for highly sensitive and standardized MRD detection in MM. An optimized 2-tube 8-color antibody panel was constructed in five cycles of design-evaluation-redesign. In addition, a bulk-lysis procedure was established for acquisition of ⩾107 cells/sample, and novel software tools were constructed for automatic plasma cell gating. Multicenter evaluation of 110 follow-up BM from MM patients in very good partial response (VGPR) or CR showed a higher sensitivity for NGF-MRD vs conventional 8-color flow-MRD -MRD-positive rate of 47 vs 34% (P=0.003)-. Thus, 25% of patients classified as MRD-negative by conventional 8-color flow were MRD-positive by NGF, translating into a significantly longer progression-free survival for MRD-negative vs MRD-positive CR patients by NGF (75% progression-free survival not reached vs 7 months; P=0.02). This study establishes EuroFlow-based NGF as a highly sensitive, fully standardized approach for MRD detection in MM which overcomes the major limitations of conventional flow-MRD methods and is ready for implementation in routine diagnostics.

CD34 + cell dose and outcome of patients undergoing reduced-intensity-conditioning allogeneic peripheral blood stem cell transplantation.

While in bone marrow allogeneic transplantation the infusion of high doses of progenitor stem cells has a favourable impact on outcome, due to a faster hematopoietic and immune recovery, in the peripheral blood allo-setting the infusion of a high number of CD34 cells increases the risk of extensive chronic graft vs. host disease (cGVHD). This higher incidence of extensive cGVHD has an adverse impact on outcome due to a higher transplant related mortality, specially among patients receiving T-cell depleted allogeneic transplantation with myeloablative conditioning. By contrast, patients undergoing reduced intensity conditioning regimen may benefit from increasing higher CD34 + cell doses, especially those categorized as high risk according to disease status at transplant. Thus, the source of progenitors cells, type of conditioning and GVHD prophylaxis, among other factors, may influence the effect of the progenitor cell dose on outcome after allogeneic transplant.

Prognostic value of immunological markers in acute myeloblastic leukemia.

In order to investigate the prognostic value of immunologic markers together with the most relevant clinical and hematological disease characteristics in acute myeloblastic leukemia (AML), the reactivity of blast cells from 102 patients with AML was analyzed with a panel of twenty monoclonal antibodies. The univariate analysis showed that five parameters had an adverse effect on both complete remission (CR) and survival: advanced age (greater than 60 years), anemia (hemoglobin concentration (Hb) less than 10g/dl), the expression of the antigens detected by the anti-megakaryocytic antibodies (CDw41/CDw42), the monocytic antibodies (CD14), and the CD9 (FMC56, FMC8) antigen. In addition, the failure to obtain CR had a significant adverse effect on survival (p less than 0.0001). Multivariate analysis showed that only age and Hb had a significant influence on CR while for predicting survival the most important independent prognostic factors were: CR, age, number of platelets and reactivity with the CD9 antibody. These results show that immunological markers could represent a valuable tool in the assignment of risk categories in AML patients.

Concomitant chronic lymphocytic leukemia (CLL) and acute myeloid leukemia. Complete remission of CLL achieved with high-dose cytosine arabinoside.

The concomitant presentation of acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) is rare with so far only eleven cases reported. In addition, the achievement of complete remission (CR) in CLL is exceptional and generally not assessed by immunophenotypical investigations. We report a case with a simultaneous occurrence of AML and CLL in which, in addition to the complete remission of AML, an eradication of the CLL clone was obtained following high-dose cytosine arabinoside. The immunophenotypic investigation of minimal residual disease showed that less than 1 x 10(-3) CD19+/CD5+ B-cells remained in bone marrow.

Differences in genetic changes between multiple myeloma and plasma cell leukemia demonstrated by comparative genomic hybridization.

To analyze the genomic differences between multiple myeloma (MM) and plasma cell leukemia (PCL), a total of 30 cases were studied by comparative genomic hybridization (CGH). In five cases with a low proportion of plasma cells (PC) in bone marrow, an enrichment of PC was performed by using immunomagnetic beads conjugated with the monoclonal antibody B-B4. In 24 out of the 25 MM (96%) and in all five PCL (100%) patients DNA copy number changes were identified by CGH analysis; in the MM case without chromosomal imbalances, the immunomagnetic enrichment of PC had failed. The most recurrent changes in MM patients were gains at chromosomes 15q (48%), 11q (44%), 3q (40%), 9q (40%) and 1q (36%). By contrast, all PCL patients showed gains in 1q. Losses of chromosomal material were significantly more frequent in PCL than in MM patients (P = 0.03): losses on 13q in 80% of PCL vs 28% of MM; and on chromosome 16 in 80% vs 12%, respectively. In addition, PCL patients showed losses of 2q and 6p that were not present in MM. The CGH data show differences in chromosomal imbalances between MM and PCL.

Expression of NK and lymphoid-associated antigens in blast cells of acute myeloblastic leukemia.

In the present study, the expression of two NK-associated antigens (CD56 and CD16) together with six 'classically' considered lymphoid-related markers (TDT,CD19,CD10,CD7,CD2,CD4) has been analyzed by appropriate dual combinations in 265 acute myelogenous leukemia (AML) patients. Among the lymphoid markers, CD4 and CD7 were those most frequently expressed by AML blast cells (58% and 21.6%, respectively) while the incidence of positivity for the other markers was lower: CD19 (7.8%), CD10 (10.9%), CD2 (11.4%), and TDT (11.3%). Regarding NK-associated antigens, CD56 was present in 41% of AML cases analyzed whereas CD16 was detected in only 23%. All but one of the CD16+ cases coexpressed the CD56 antigen. The expression of these antigens was not associated with the degree of cell differentiation assessed either by morphological or immunophenotypical criteria, with the exception of the correlation observed between monocytic leukaemias and the expression of the CD4, CD56, and CD16 antigens. Regarding the prognostic value of the markers investigated, CD56 expression was associated with a tendency for a better outcome whereas CD7 was the only antigen that had an adverse influence on the survival of AML patients.

Prognostic value of S-phase white blood cell count in B-cell chronic lymphocytic leukemia.

Eighty previously untreated patients with B-cell chronic lymphocytic leukemia (B-CLL) were analyzed to study the proliferation rate of their peripheral blood (PB) leukocytes to determine its relationship with the extension of the disease and its value in discriminating among patients with similar tumor cell mass. The 80 B-CLL patients were distributed into two different groups according to the absolute count of PB S-phase leukocytes: a low proliferative group (less than 1 x 10(9)/I) of 48 patients and a high proliferative group (greater than or equal to 1 x 10(9)/I) of 32 patients. The high proliferative group displayed a higher incidence of splenomegaly (p less than 0.005), hepatomegaly (p less than 0.08), anemia (p less than 0.02) and thrombocytopenia (p less than 0.03) as well as a higher lymphocytic infiltration both in PB (p less than 0.0004) and in bone marrow (BM) (p less than 0.003). These patients also showed a higher incidence of a diffuse pattern of BM involvement (p less than 0.04), advanced clinical stages [stage III/IV (p less than 0.03) and group C (p less than 0.04)] and infections (p less than 0.0008) together with significantly lower IgG (p less than 0.03) and IgM (p less than 0.03) serum levels. Regarding the immunophenotype, there was a greater percentage of either CD19+ (p less than 0.06) and CD19+ CD5+ (p less than 0.05) B-cells, together with a greater reactivity for both the CD25 (p less than 0.04) and CD9 (p less than 0.08) antigens in the high proliferative group. According to the prognostic value of the PB S-phase leukocyte count it was seen that patients with low S-phase white blood cell (WBC) numbers displayed a significantly higher survival (p less than 0.03). In addition, multivariate analysis revealed that the S-phase WBC count, although partially related to other clinical and biological prognostic factors, displayed an important independent value in predicting early deaths in patients with B-CLL.

In leukemic hematopoiesis CD34 antigen does not have the same significance as it does normal hematopoiesis.

The aim of the present study was to analyze whether or not leukemic clonogenic cells are restricted to the CD34+ cell fraction and to investigate the effect of IL-3 and G-CSF on blast cell populations dissected according to their CD34 reactivity. For this purpose 34 patients were studied. Patients were classified into three groups according to CD34 antigen expression: (1) cases in which all blast cells (100%) were positive for the CD34 Ag (n = 9); (2) cases in which all blast cells lacked the expression of this antigen (n = 10); and (3) patients in whom both, CD34 positive and negative blast cell subsets coexisted (n = 15). In 15 cases immunomagnetic cell selection was performed and two subpopulations were separated: one, phenotypically more immature (CD34+), and another, theoretically more differentiated (CD34-/33+). In addition, in three cases both CD34+ and CD34- blast cell subpopulations were sorted using a FACStar flow cytometer. Blast colony assays were performed using 0.9% methylcellulose and two different recombinant human hematopoietic growth factors (HGFs), IL-3 and G-CSF, were used as growth stimulants. Either, a single or a combination of the growth factors was added to cultures. Colony formation was observed in both 100% positive or 100% negative cases for the CD34 antigen as well as in the CD34+ and CD34- cell fractions separated by immunomagnetic selection or flow cytometry. The effect of G-CSF and IL-3 on both cell fractions was as follows: cases with a uniform population according to CD34 expression (100% positive or negative) showed a better growth response with IL-3 especially for the CD34+ cases (87% vs 40% of CD34+ and CD34- cases, respectively). Within the CD34-/33+ selected fractions, IL-3 tended to induce a higher proliferative response than G-CSF while the opposite was found within the CD34+ cell selected fractions. In contrast it was observed that both IL-3 and G-CSF induced a higher PE on the CD34- blast cells (both selected and 100% negative), although the difference was not statistically significant. The existence of a possible synergistic effect (SE) between HGFs was also explored. Overall, a synergistic growth was observed in nine out of the 13 selected cases studied and this effect could be seen in both CD34- or CD34+ blast cell fractions. The analysis of the complete phenotypic characteristics of these cells revealed that cell fractions showing SE were more immature according to the expression of CD15 and HLA-DR antigens. We can conclude that in leukemic hematopoiesis, CD34 antigen expression does not have the same significance as it does in normal hematopoiesis since clonogenic cells are not restricted to the CD34+ acute myeloid leukemia (AML) blast cell fraction. Moreover, our study shows that the heterogeneous response to HGFs observed in AML patients may be associated with the existence of immunophenotypically different blast cell subsets.

Effect of cumulative etoposide doses on the outcome of autologous peripheral-blood progenitor-cell transplantation for lymphoma.

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, P<0.001). Differences in neutrophil engraftment between groups were not clinically significant. Pre-transplant cumulative doses of etoposide >750 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 etoposide >350 mg/m(2) and previous administration of procarbazine were found to be independent prognostic factors for TRM.

Peripheral blood is safer than bone marrow as a source of hematopoietic progenitors in patients with myelodysplastic syndromes who receive an allogeneic transplantation. Results from the Spanish registry.

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.

Results of autologous transplantation in lymphoma are not improved by increasing the dose of etoposide in the BEAM regimen: a single-centre sequential-cohort study.

We have undertaken a retrospective sequential-cohort analysis of 131 lymphoma patients treated with the BEAM regimen and autologous stem cell transplantation, to compare BEAM at standard doses (sBEAM; n = 67 from May 1990 to April 1995) and BEAM with escalated etoposide dose from 800 to 1600 mg/m(2) (eBEAM; n = 64 from May 1995 to June 1999). Transplant-related mortality and incidence of secondary malignancies were similar in both groups. Disease progression was significantly lower in indolent lymphoma (IL) patients receiving eBEAM (7 vs 43%), although survival was comparable due to a higher toxic mortality in the eBEAM group. The 5-year event-free survival and overall survival were better in Hodgkin's disease (HD) patients treated with eBEAM (70 and 77%, respectively) compared to sBEAM (58 and 69%, respectively), but the difference was not statistically significant. In aggressive lymphomas, no difference was detected between groups. Our results indicate that while escalation of the etoposide doses in the BEAM conditioning regimen does not appear to improve outcome, encouraging results in IL and HD may warrant further studies.

Factors that influence long-term hematopoietic function following autologous stem cell transplantation.

The aim of the present study was to assess which factors influence hematopoietic function long term after transplantation. For this purpose, we have analyzed a series of 79 patients who underwent autologous transplantation. None of them received any further chemotherapy or radiotherapy after transplant. All patients were disease-free 1 year after autologous transplantation. Late impairment of hematopoietic function was defined as the presence of non-transient peripheral blood cytopenias, detected 6 and 12 months after autografting. Before transplantation, 38.7% of patients showed peripheral blood cytopenias. Six and 12 months after transplantation, cytopenias presented in 44.2% and 42.4% of patients, respectively. Cases displaying cytopenias 6 months after transplantation had received a significantly lower dose of CFU-GM and CD34+ cells than patients without cytopenias (P = 0.012 and P = 0.04, respectively). The same correlation, with even higher statistical significance, was observed 12 months after transplant (P = 0.007 and P = 0.005). Alkylating agents and radiotherapy administered prior to transplantation and age did not seem to influence the presence of permanent cytopenias. The incidence did not vary significantly according to the stem cell source (bone marrow or peripheral blood). The number of CFU-GM and CD34+ cells infused was the most important factor for maintenance of adequate hematopoiesis.

Diagnosis of secondary myelodysplastic syndromes (MDS) following autologous transplantation should not be based only on morphological criteria used for diagnosis of de novo MDS.

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.

Intra-muscular vidarabine therapy for polyomavirus-associated hemorrhagic cystitis following allogeneic hemopoietic stem cell transplantation.

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.

Quality of life assessment in patients undergoing reduced intensity conditioning allogeneic as compared to autologous transplantation: results of a prospective study.

The aim was to analyze quality-of-life (QOL) during the first year post transplant in 47 patients undergoing reduced-intensity conditioning (RIC) allotransplantation, and to compare these with a similar subgroup of patients receiving autologous stem cell transplantation (ASCT). We used self-reported questionnaires. Each answer scored from 0 (not at all) to 4 (very much), with higher scores indicating worse functioning. Mean value of physical categories among RIC transplants ranged between 1.23 and 0.77 indicating that patients scored very low for physical symptoms. Patients undergoing ASCT had higher scores in questionnaires performed early after transplant and then gradually improved (P < 0.001). Overall, when we compared physical functioning scores, allo-RIC did significantly better (P = 0.049). Nevertheless, while allo-RIC scores were significantly better for the first three questionnaires, ASCT patients did better in the last two questionnaires. These findings are in accordance with the toxicities observed in both subgroups which are lower in the RIC group early after transplant. No significant differences were observed between either subgroup for any of the functional, social/ family, psychological distress and satisfaction with doctor/nurse relationship items. We have observed similar QOL among patients undergoing RIC-allo as compared to ASCT although GVHD remains an important 'event' in QOL.

Clinical significance of CD34+ cell dose in long-term engraftment following autologous peripheral blood stem cell transplantation.

The number of CD34+ cells has been described as the best parameter for predicting the quality of engraftment in peripheral blood progenitor cell (PBPC) transplantation in the early post-transplant period. In this study we have determined the optimal number of CD34+cells in order to maintain engraftment in the long term in a series of 100 patients receiving autologous PBPC transplantation. Based on our previous experience on the speed of early hematopoietic recovery, four subgroups of patients were established: patients infused less than 0.75 x 106/kg CD34+ (n = 9), 0.75 to 1.25 (n = 24), 1.25 to 2.0 (n = 37) and more than 2.0 (n = 30). These groups were designated as low, intermediate-low, intermediate-high and high CD34 groups, respectively. Transitory loss of neutrophil engraftment was observed in 67%, 30%, 16% and 6% of patients in the four mentioned CD34 groups respectively, with statistically significant differences between the different groups. Significant differences were also observed between the low CD34 group and the rest of the groups as regards platelet and red blood cell transfusion requirements, fever episodes, days of hospitalization and antibiotic requirements throughout the first year. Our results show that the dose of CD34+ cells influences engraftment also in the late post-transplant period, and correlates with transfusion and antibiotic requirements, fever episodes and days of hospitalization during the first year post-transplant.

BEAM chemotherapy followed by autologous stem cell support in lymphoma patients: analysis of efficacy, toxicity and prognostic factors.

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.

Discrepancies between morphologic, cytochemical, and immunologic characteristics in acute myeloblastic leukemia.

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.