Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.

Post-treatment bone marrow residual disease > 5% by flow cytometry is highly predictive of short progression-free and overall survival in patients with Waldenström's macroglobulinemia.

To assess the value of bone marrow (BM) assessment by flow cytometry FCM after therapy in the clinical outcome of WM patients, we analyzed 42 WM patients who were evaluated before and after therapy. Patients were studied with a panel that always included the CD19, CD22, CD25, and κ/λ light chain immunoglobulin monoclonal antibodies. The mean of abnormal B-cells in the pre-therapeutic BM was 17.8% ± 12.1%, which decreased was after therapy to 5.4% ± 0.7% (P = .049). A linear correlation was seen between the better quality of response and the reduction in the tumor B-lymphocyte counts at the BM, since the ratio of abnormal B cells between pre and posttherapy BM was 1172.17, 221.64, 3.37, 1.03, and 0.56 for responses complete, partial, minor, stable disease and progression, respectively (P < .001). Intensive and rituximab-containing therapies correlated with deeper tumor cell reductions. Finally, the B-cell decrease correlated with the better overall and progression-free survival.

Chimerism and minimal residual disease monitoring after reduced intensity conditioning (RIC) allogeneic transplantation.

Since graft-versus-leukemia (GVL) is the main weapon for disease eradication after reduced intensity conditioning (RIC) allogeneic SCT, the availability of sensitive and specific techniques to monitor changes in tumor load after transplant are especially helpful. These minimal residual disease techniques would allow an early intervention in the event of low tumor burden, for which immunotherapy is highly effective. Some authors have found an association between persistence of MRD, mixed chimerism and risk of relapse. Nevertheless, data from the literature remain contradictory and further correlations should be established, especially in RIC transplants. In this study we have analyzed the impact of MRD and chimerism monitoring on the outcome of 34 patients undergoing RIC allogeneic SCT who were considered poor candidates for conventional transplantation due to advanced age or other concurrent medical conditions. At day +100 25 (75%) patients reached complete remission (CR), there were five (15%) partial responses and three patients progressed. Incidence of grade 2-4 aGVHD and extensive cGVHD were 35% and 58%, respectively. Sixteen percent of patients developing aGVHD relapsed as compared to 47% in those without aGVHD (P = 0.03) and also 10% of patients developing cGVHD relapsed as compared to 50% relapses in those without cGHVD (P = 0.03). Four patients (12%) died due to early (n = 1) and late (n = 3) transplant-related mortality. After a median follow-up of 15 months, 24 out of the 34 patients remain alive. Projected overall survival and disease-free survival at 3 years are 68% and 63%, respectively. Early chimerism analysis showed 67% of patients with complete chimerism (CC) in bone marrow (BM), 86% in peripheral blood (PB), 89% in granulocytes and 68% in T lymphocytes. On day +100, these figures were 68%, 79%, 90% and 73%, respectively, and on day +180 there were 83% patients with CC in BM, 100% in PB, 100% in granulocytes and 100% in T lymphocytes. We observed a trend to a higher incidence of relapse in patients with mixed chimerism (MC) as compared to patients with CC. MRD monitoring by flow cytometry and/or RT-PCR analysis was performed in 23 patients. MRD assessment on days +21 to +56 after transplant allowed identification of patients at risk of relapse. In this sense, seven out of 12 patients (58.3%) who had positive MRD on days +21 to +56 relapsed as compared to none out of 11 patients who had negative MRD (P = 0.002). Of the seven patients with criteria to monitor MRD who relapsed after transplant, all but one remained MRD positive until relapse. By contrast, 10 patients remained MRD negative and all of them are in continuous CR. In nine additional patients, persistence of MRD or mixed chimerism was observed after transplant and withdrawal of cyclosporin with or without DLI was performed. Only two out of these nine patients relapsed. MRD clearance was preceded by CC and GVHD. In conclusion, in our study we found that RIC allogeneic transplantation can be used in patients considered poor candidates for conventional transplantation due to advanced age or other concurrent medical conditions with both low toxicity and low transplant-related mortality. Simultaneous studies of both chimerism and MRD are a useful tool in order to predict risk of relapse in patients undergoing RIC transplants and so can be helpful for individualizing treatment strategies after transplant.

Incidence of phenotypic aberrations in a series of 467 patients with B chronic lymphoproliferative disorders: basis for the design of specific four-color stainings to be used for minimal residual disease investigation.

Multiparameter immunophenotypic analysis of neoplastic cells has proven to be of great help for the investigation of minimal residual disease in acute leukemias; however, its utility has not been systematically explored in B cell chronic lymphoproliferative disorders. The aim of the present study was to investigate the incidence of phenotypic aberrations in a series of 467 consecutive leukemic B cell chronic lymphoproliferative disorders through the comparison of the phenotypic characteristics of tumor vs normal peripheral blood (n = 10) and bone marrow (n = 10) B cells, in order to explore the applicability of this strategy for minimal residual disease monitoring. An additional goal of our study was to evaluate the sensitivity of multiparameter flow cytometry for the detection of minimal residual disease in leukemic B cell chronic lymphoproliferative disorders through dilutional experiments (n = 19). From the patients analyzed 382 corresponded to B cell chronic lymphocytic leukemia/small lymphocytic lymphoma (353 typical and 29 atypical); five to prolymphocytic leukemia; 13 to hairy cell leukemias; 12 to lymphoplasmacytic lymphomas; 14 to splenic marginal zone lymphomas; 22 were follicular lymphomas; and 19 mantle cell lymphomas. The following triple stainings were systematically applied to both normal and leukemic samples: FMC7/CD5/CD19, CD22/CD23/CD19, CD103/CD25/CD19, CD10/CD11c/CD19 and sIg/sIg(lambda)/CD19. Overall, 98% of the leukemic B cell chronic lymphoproliferative disorders cases displayed aberrant phenotypes at diagnosis with no significant differences being found between cases analyzed in peripheral blood vs bone marrow samples. The most common types of aberrant criteria detected included asynchronous antigen expression (92%) and antigen over-expression (54%); abnormally light scatter characteristics were found in 17% of the cases. Most of the cases studied (90%) displayed four or more phenotypic aberrations. Once patients were divided according to the different diagnostic subgroups, the overall incidence of aberrant phenotypes ranged from 79 to 80% among atypical B cell chronic lymphocytic leukemia/small lymphocytic lymphoma and prolymphocytic leukemia to 97% of follicular lymphoma and 100% of typical B cell chronic lymphocytic leukemia/small lymphocytic lymphoma, hairy cell leukemia, lymphoplasmacytic lymphomas, splenic marginal zone lymphomas and mantle cell lymphomas. Based on the aberrant phenotypes detected unique four-color stainings could be built for the specific identification of aberrant phenotypes. These include CD22/CD23/CD19/CD5 and sIg(kappa)/sIg(lambda)/CD19/CD5 for lymphocytic leukemia/small lymphocytic lymphoma and prolymphocytic leukemia, CD103/CD25 or CD22/CD19/CD11c for hairy cell leukemia, FMC7/CD22/CD19/CD103 and sIg(kappa)/sIg(lambda)/CD22/CD19 for splenic marginal zone lymphomas, CD22/CD23/CD19/CD10 for follicular lymphomas and CD10/CD22/CD19/CD5 for mantle cell lymphomas. Serial dilutional experiments showed that the sensitivity level of immunophenotyping ranges between 10(-4) and 10(-5). In summary, the present study shows that immunophenotypic analysis allows the identification of aberrant phenotypes in 98% of leukemic B cell chronic lymphoproliferative disorders and these phenotypes can be used for minimal residual disease monitoring with a sensitivity limit of 10(-4)-10(-5).

Multiparametric analysis of apoptotic and multi-drug resistance phenotypes according to the blast cell maturation stage in elderly patients with acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: Acute myeloid leukemia (AML) is a heterogeneous group of malignant diseases, often characterized by coexistence of more than one subpopulation of blast cells. Multiparametric flow cytometry immunophenotyping has proven to be a reliable and sensitive approach for the discrimination of myeloid blast cells from residual normal cells present in bone marrow samples from AML patients and, at the same time, allows the identification of different maturation compartments among myeloid blasts. Therefore, it provides a unique tool for assessing apoptotic and multidrug resistance (MDR)-associated phenotypes in individual subsets of leukemic cells. DESIGN AND METHODS: The aim of the present study was to explore the simultaneous expression of proteins related to both apoptosis (APO2.7, bcl-2, bax) and multidrug resistance (MDR1, MRP, LRP) in the different blast cell subpopulations detected at diagnosis in a group of 72 elderly patients with AML. In addition, we included 5 bone marrow samples from healthy adult donors in the analysis. RESULTS: Immature blast cells (CD34+: subset I) showed a significantly higher level of bcl-2 expression (p <0.0001) together with a lower reactivity for APO 2.7 (p=0.02) as compared to the other more mature CD34- cell subsets. The expression of Bax parallelled that of APO 2.7, although the difference between immature CD34+ blast cells and the mature blast cell subsets did not reach statistical significance (p=0.18). These results translated into a significantly (p<0.0001) higher bcl-2/bax ratio for the CD34+ blast cells as compared to that of the two CD34- blast cell subpopulations. Regarding the expression of the multidrug resistance-associated proteins Pgp and MRP, CD34+ blast cells displayed a greater expression of both proteins as compared to the more mature CD34- AML blast cells, but differences according to maturation stage of AML blast cells did not reach statistical significance. In contrast, LRP expression was significantly lower in the more immature CD34+ blast cell subset than in the more mature ones (p=0.01). INTERPRETATIONS AND CONCLUSIONS: As far as normal bone marrow is concerned our results suggest that all blast cell subpopulations are more protected from apoptosis than their normal counterparts. We conclude that in elderly patients with AML the more immature blast cells are more resistant to apoptotic processes, which could explain why, when AML relapses, the blast cells frequently display a more immature phenotype than that observed at diagnosis. Contradictory results in multidrug resistance profile support the hypothesis that failure to respond to chemotherapeutic drugs in AML is a multifactorial phenomenon.

BIOMED-I concerted action report: flow cytometric immunophenotyping of precursor B-ALL with standardized triple-stainings. BIOMED-1 Concerted Action Investigation of Minimal Residual Disease in Acute Leukemia: International Standardization and Clinical Evaluation.

The flow cytometric detection of minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemias (precursor-B-ALL) mainly relies on the identification of minor leukemic cell populations that can be discriminated from their normal counterparts on the basis of phenotypic aberrancies observed at diagnosis. This technique is not very complex, but discordancies are frequently observed between laboratories, due to the lack of standardized methodological procedures and technical conditions. To develop standardized flow cytometric techniques for MRD detection, a European BIOMED-1 Concerted Action was initiated with the participation of laboratories from six different countries. The goal of this concerted action was to define aberrant phenotypic profiles in a series of 264 consecutive de novo precursor-B-ALL cases, systematically studied with one to five triple-labelings (TdT/CD10/CD19, CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19 and CD19/CD34/CD45) using common flow cytometric protocols in all participating laboratories. The use of four or five triple-stainings allowed the identification of aberrant phenotypes in virtually all cases tested (127 out of 130, 98%). These phenotypic aberrancies could be identified in at least two and often three triple-labelings per case. When the analysis was based on two or three triple-stainings, lower incidences of aberrancies were identified (75% and 81% of cases, respectively) that could be detected in one and sometimes two triple-stainings per case. The most informative triple staining was the TdT/CD10/CD19 combination, which enabled the identification of aberrancies in 78% of cases. The frequencies of phenotypic aberrations detected with the other four triple-stainings were 64% for CD10/CD20/CD19, 56% for CD34/CD38/CD19, 46% for CD34/CD22/CD19, and 22% for CD19/CD34/CD45. In addition, cross-lineage antigen expression was detected in 45% of cases, mainly coexpression of the myeloid antigens CD13 and/or CD33 (40%). Parallel flow cytometric studies in different laboratories finally resulted in highly concordant results (>90%) for all five antibody combinations, indicating the high reproducibility of our approach. In conclusion, the technique presented here with triple-labelings forms an excellent basis for standardized flow cytometric MRD studies in multicenter international treatment protocols for precursor-B-ALL patients.

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.

[Fulminant angiopathy caused by allogenic transplantation of bone marrow with cerebral involvement: clinico-pathologic correlation]. / Angiopatía fulminante por trasplante alogénico de médula ósea con afectación cerebral: correlación clínico-patológica.

We present a 41 woman who underwent an allogeneic bone marrow transplantation as a treatment for a multiple myeloma. After presenting graft-versus-host disease, hyperbilirubinemia, cyclosporine toxic levels, renal failure and peripheral schistocytosis, she developed complex partial seizures and bilateral hypodensity lesions in occipital lobes. Fulminant microangiopathy was diagnosed; though promptly treated with immunoglobulins and fenitoin, she died in few days. Necropsy showed diffuse thrombotic disease and a haematoma in left occipital lobe. We review the types of thrombotic microangiopathy described up to now, and the different pathogenic theories related to them. According to our reviewing of the literature, this is the first case of fulminant thrombotic microangiopathy ever described in Spanish language.

Prognostic value of immunophenotypic detection of minimal residual disease in acute lymphoblastic leukemia.

PURPOSE: The identification of immunophenotypic aberrancies through multiparametric flow cytometry makes the differentiation between normal and leukemic cells relatively simple and quick, and is therefore an attractive method for the investigation of minimal residual disease (MRD). In this report, we have analyzed the impact on relapse and relapse-free survival (RFS) of detecting immunophenotypical aberrant cells in acute lymphoblastic leukemia (ALL) patients in cytomorphologic complete remission (CR). MATERIALS AND METHODS: Two hundred eleven bone marrow (BM) samples from 53 consecutive ALL (37 precursor B-ALL and 16 T-ALL) patients were analyzed. The only selection criteria were to have at least one aberrant immunophenotypic feature at diagosis and to have achieved cytomorphologic CR after induction therapy. For MRD detection, all follow-up samples were analyzed with triple labelings using a two-step acquisition procedure, in which 106 cells were screened for the possible persistence of residual leukemic cells with the same phenotypic aberrancy as that identified diagnosis. RESULTS: Patients who displayed a gradual increase in MRD levels showed a higher relapse rate (90% v22%; P < .00001) and shorter median RFS (12 months v not reached; P < .0001) than those with stable or decreasing MRD levels. This adverse prognostic influence also was observed when children and adults, as well as B-ALL and T-ALL patients, were analyzed separately. An MRD level > or = or greater than 10(-3) discriminated two risk groups of ALL patients with significantly different relapse rates and RFS at all treatment phases (end of induction, consolidation, maintenance, and out of treatment). CONCLUSION: Multiparametric flow cytometry of MRD in ALL patients is a valuable tool for relapse prediction and for the identification of a cohort of patients with very poor prognosis.

Immunophenotypic analysis of CD19+ precursors in normal human adult bone marrow: implications for minimal residual disease detection.

BACKGROUND AND OBJECTIVE: Normal B-cell differentiation has been characterized extensively, but discrepancies persist regarding the exact sequence of antigen expression. Few systematic studies focusing on identification of the minor or undetectable B-cell subsets in normal human bone marrow (BM) which are frequently found in leukemic cells have been performed. Such studies could help to monitor minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemia (precursor-B-ALL). The aim of the present study was to analyze the sequence of antigen expression among normal human CD19+ B cells from adult BM. Our major goal was to identify infrequent and undetectable B-cell phenotypes that could be used for the detection of MRD in patients with precursor-B-ALL. DESIGN AND METHODS: Adult BM samples from a total of 33 healthy volunteers were analyzed using triple stainings, and measured by flow cytometry. A sensitive method based on the two-step acquisition procedure was used for the identification and characterization of cells present at very low frequencies. RESULTS: Five different subsets of CD19+ cells were identified in normal BM samples according to their degree of maturation: 1) CD19+/CD34+/CD10-/CD20-/CD22dlm+ (0.5 +/- 0.4% B cells); 2) CD19+/CD34-/CD10++/CD20-/CD22dlm+ (3.4 +/- 2.7%); 3) CD19+/CD34-/CD10+/CD20-/CD22dlm+ (3.5 +/- 2.2%); 4) CD19+/CD34-/CD10+/CD20+,++/CD22dlm+ (21 +/- 11%), and 5) CD19+/CD34-/CD10-/CD20++/CD22+ (73 +/- 19%). We observed that several B-cell phenotypes are frequent among precursor-B-ALL, but are infrequent or undetectable in normal human B cell differentiation. Accordingly, in all normal BM samples analyzed, less than 4 x 10(-5) cells co-expressed CD19 and CD117; CD20strong+/CD34+ and CD22strong+/CD34+ events were found at frequencies less than 5 x 10(-4), while CD20+/CD34+ phenotypes were found in less than 1 x 10(-3) BM cells. Although both CD19+/CD13+ and CD19+/CD33+ events were found at frequencies of up to 3 x 10(-3), they never formed a well-defined population of cells and therefore these latter phenotypic patterns could also be of use for MRD investigation in CD13+ and/or CD33+ precursor-B-ALL cases. INTERPRETATION AND CONCLUSIONS: Our results show that in adult BM normal B-cells display constant patterns of maturation as regards both their phenotypic characteristics and their relative distribution. Abnormalities in these patterns provide a potentially useful tool for monitoring MRD in precursor-B-ALL patients who achieve cytomorphologic complete remission.

Functional expression of MDR-1 in acute myeloid leukemia: correlation with the clinical-biological, immunophenotypical, and prognostic disease characteristics.

Up till now, clinical data on the possible prognostic influence of multidrug resistance (MDR) in hematological malignancies have been inconsistent, probably due to technical pitfalls. Moreover, in most studies qualitative information on the presence/absence of MDR-1 expression has been used instead of quantitative results. In addition, results usually refer to the total BM population and not specifically to blast cells. In the present study we analyzed the expression of MDR-1 in a series of 50 newly diagnosed de novo AML using a double-staining technique: (a) monoclonal antibodies for the specific identification of blast cells and (b) the rhodamine-123 efflux assay, which allows a quantitative and calibrated measurement of MDR-1 function. Expression of MDR-1 was correlated with clinical, biological, and immunophenotypical disease characteristics. All patients were uniformly treated according to the AML 87/91 protocols of the Spanish Pethema Cooperative Group; the median age was 51 +/- 19 years and the FAB distribution was as follows: 2 M0, 9 M1, 9 M2, 12 M3, 11 M4, 5 M5, and 2 M6 cases. Upon grouping the 50 AML patients analyzed according to the level of rh123 elimination, it was observed that those cases with > or = 30% decrease in rh123 fluorescence displayed higher WBC counts (9 +/- 12 vs 37 +/- 73 x 10(9)/l, p = 0.02) and platelet numbers (94 +/- 11 vs 35 +/- 25 x 10(9)/l, p = 0.02), together with a higher incidence of extramedullary involvement (35% vs 24%, p = 0.02). Half of the patients (47%) displaying a low rh123 elimination (< 30%) showed M3 morphology, while among the 33 patients with a higher rate of rh123 elimination (> or = 30%), only four (12%) corresponded to the M3 morphological subtype (p = 0.0006). From the immunophenotypic point of view, a low rate of rh123 elimination was associated with a lower expression of HLADR antigen (p = 0.003) and a higher expression of CD117 (p = 0.01). Regarding the possible prognostic influence of MDR1 expression, we found that a high rate of rh123 elimination (> 30%) was associated with a tendency towards poor disease outcome, illustrated by both a lower complete remission rate with the first cycle of chemotherapy (36% vs 56%) and a lower median disease-free survival (22 months vs median DFS not reached), although differences did not reach statistical significance (p = 0.1 in both comparisons). This data shows that although MDR-1 can be a relevant parameter in the evaluation of AML patients, larger series of patients using appropriate techniques for specifically analyzing the MDR of blast cells will be necessary in order to establish the final clinical value of this parameter.

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.

[Activity of various hematopoietic growth factors on acute myeloblastic leukemia blasts]. / Acción de algunos factores de crecimiento hematopoyéticos sobre los blastos de leucemia aguda mieloblástica.

PURPOSE: Acute myeloblastic leukaemia (AML) cells respond to some extent to the action of haemopoietic growth factors (HGF). This work is aimed to analyse the effect of IL-3, GM-CSF and G-CSF on the proliferative and self-replication capabilities of AML cells and to correlate such response with the morphological and immunological characteristics of the blast cells. PATIENTS AND METHODS: Mononucleated cells from the peripheral blood of 26 AML patients were incubated in liquid medium without serum in presence of IL-3 and GM-CSF alone and in combination with G-CSF. Cell proliferation was evaluated by determination of the increase in the number of cells harvested after incubation with the HGF. The proliferative response was studied in 8 cases by flow cytometry analysis of the cell cycle (number of cells in S-phase). The capability of IL-3 and GM-CSF to induce self-replication of the leukaemic clonogenic cell (CFU-L) was analysed in 9 cases by techniques of replanting on semi-solid medium. RESULTS: Cell proliferation was induced with IL-3 in more cases than with GM-CSF (50% vs 28%); furthermore, the stimulus induced by IL-3 was also more evident, as the number of cells harvested was greater than that corresponding to GM-CSF (2.1 +/- 1.1 x 10(6) cell/mL vs 1.4 +/- 1.4 x 10(6) cells/mL, p = 0.10). Although no clear correlation was seen between the proliferative response to HGF and the immunologic or morphologic subtype of the blast cells, no response to GM-CSF was detected in those cases classified as M2 nor in the CD15-positive leukaemias. A synergistic effect of G-CSF and IL-3 or GM-CSF was found in only 20% of the cases. Both IL-3 and GM-CSF augmented the number of cells in S phase in all the cases analysed; this increase was higher in M1 and M2 with respect to the leukaemias with monocytic component. CONCLUSIONS: IL-3 and GM-CSF induced self-replication of CFU-L in a half of the cases. This effect was not dependent upon the kind of stimulating agent used (IL-3 or GM-CSF), and appeared unrelated to the morphologic or immunologic characteristics of the leukaemia.

Phenotypic analysis of CD34 subpopulations in normal human bone marrow and its application for the detection of minimal residual disease.

In the past, studies on CD34+ cells have been based on the use of monoclonal antibodies conjugated with different fluorochromes that show different fluorescence intensity and yield variable results. Moreover, most of these studies have neither specifically focused on adult human BM samples nor have they used combinations to explore specifically the phenotype of myeloid committed CD34+ cells. The aim of the present study has been to characterize the normal human CD34+ precursor cells from adult BM in order to identify missing or extremely rare phenotypes that can be used for detecting minimal residual disease (MRD) in patients with AML. For this purpose we have utilized the fluorochrome conjugates that provide the most sensitive signals for identifying low antigenic expression, and the technique has been adapted to the characterization of cells present at very low frequencies. Normal human BM samples from 13 adult healthy volunteers have been analyzed using triple stainings at flow cytometry. The mean percentage of CD34+ cells detected was 0.72 +/- 0.33%; these cells displayed an heterogeneous light-scatter distribution. Most CD34+ cells coexpressed CD38 (96.7 +/- 5.7%), HLADR (81.6 +/- 14.0%), CD33 (84.7 +/- 18.3%), CD13 (84.6 +/- 16.2%) and CD71 antigens (65.5 +/- 9.1%). In addition, almost half of CD34+ cells were CD117+ (60 +/- 26.8%). Only a small proportion of CD34+ cells coexpressed CD4 (15.5 +/- 11.7%, CD36 (31.7 +/- 6.2%), CD61 (16.3 +/- 12.9%), CD41 (6.5 +/- 5.5%) or the lymphoid associated markers CD10 (18.6 +/- 11.8%) and CD19 (12.3 +/- 13.2%). Reactivity for the CD15 antigen was observed in a small population of CD34+HLADR+ cells (11.6 +/- 11.2%) although its intensity of expression was lower than that of the more mature granulocytic cells. No CD34+ cells displayed CD14, CD65, CD20, strong CD22, CD3 and CD56 antigens. Accordingly, most adult bone marrow CD34+ cells appeared to be committed to the myeloid lineage (CD13+/CD33+) and displayed an intermediate-to-large FSC/SSC while the lymphoid-committed CD34+ cells (CD19+, CD10+) were in a minority with low FSC/SSC values. By triple marker stainings several phenotypes of CD34+ precursor cells were found to be either undetectable or present at very low frequencies (< 1 x 10(-3)) in the normal human adult bone marrow. These data may be of great value for defining leukemia 'associated' phenotypes used to detect minimal residual disease in adult acute leukemia patients.

[Clonogenic cells of acute leukemia secondary to myeloproliferative and myelodysplastic syndromes]. / Estudio de las células clonogénicas de las leucemias agudas secundarias a síndromes mieloproliferativos y mielodisplásicos.

PURPOSE: To assess if the in vitro behaviour of leukaemic colony-forming units (CFU-L) from secondary leukaemias is similar to that of the de novo acute myeloblastic leukaemia. An attempt was also made to verify if such behaviour correlated with the characteristics of the disease or with the cell-surface markers of the leukaemic population. PATIENTS AND METHODS: The study was carried out on 21 patients with secondary acute leukaemia (12 had previous myelodysplastic syndrome, MDS-AL, and 9 had previous chronic myeloproliferative syndromes, MPS-AL). Peripheral blood mononucleated cells were cultured on methyl-cellulose with MCL-PHA stimulation. The dishes were examined after 7 days of incubation in humid 37 degrees C environment with 5% CO2. Direct or indirect immunofluorescence was used for the immunophenotypic analysis and the patients were divided in two groups: 1) immature phenotype, which include those cases expressing only precursor (CD34) or pan-myeloid (CD33/13) antigens, and (2) mature phenotype, comprising the caes with granulomonocytic (CD15, CD14), erythroid (glycophorin) or megakaryocytic (CD61) differentiation. The statistical analysis was done with the BMDP programme. RESULTS: Up to 95% of the secondary acute leukaemias proliferate in vitro, as opposed to 82% of the de novo ones, the difference not being significant (p = 0.14). Successful cell showing was clearly superior in the former (p = 0.02), mostly due to higher proliferation of the MPS-AL cells. Neither the clinico-biologic characteristics of the patients nor the phenotype of the blast cells correlated with the in vitro behaviour of CFU-L. Only CD19 antigen expression and nuclear TdT provided a lesser in vitro growth (p = 0.05 and p-0.06, respectively). CONCLUSION: In general terms, secondary leukaemias, especially MPS-AL, show higher in vitro growth than de novo acute myeloblastic leukaemias.

Acute lymphoblastic leukemia (ALL): detection of minimal residual disease (MRD) at flow cytometry.

In the present study the usefulness of a method combining multiple staining direct immunofluorescence technique together with flow cytometry in order to predict relapse in ALL is analyzed in a group of 47 patients (11 T-ALL and 36 B-ALL). Results show that this method can be applied to at least two-thirds of all ALL patients being specially useful for the T-ALL cases (100% vs 56%) as this corresponding to the incidence of "aberrant" phenotypes. The detection of an increase in the percentage of bone marrow cells displaying "aberrant" phenotypes in two consecutive samples from the same patient is of great help on predicting relapse (sensitivity of 92% and specificity of 75%).