Biomedical subjects

A Orfao

Publications and source records attributed to A Orfao.

At least 19 recordsLinked to original sources

PubMed1998

Indolent systemic mast cell disease in adults: immunophenotypic characterization of bone marrow mast cells and its diagnostic implications.

The aim of the present study was to explore the diagnostic value of the immunophenotypic analysis of bone marrow mast cells (BMMC) in indolent systemic mast cell disease (SMCD) patients. For that purpose, a total of 10 SMCD patients and 19 healthy controls were analyzed. Our results show that BMMC from SMCD are different from normal BMMC with regard to both their light scatter and immunophenotypic characteristics. Accordingly, forward light scatter (FSC), side (90 degrees) light scatter (SSC), and baseline autofluorescence levels were higher in BMMC from indolent SMCD patients than they were in control subjects. From the immunophenotypic point of view, the most striking findings were the constant expression of CD2 (P = .0001), CD25 (P = .0001), and CD35 (P = .06) molecules by BMMC from SMCD patients, markers that were absent from all normal controls. In contrast, CD71, absent in BMMC from indolent SMCD, was positive in BMMC from normal subjects. Although, slight differences between BMMC from SMCD patients and normal controls were found in several other markers, they did not reach statistical significance. In conclusion, our results show that simultaneous assessment of FSC/SSC and reactivity for the CD117, CD2, CD25, CD33, and CD35 forms the basis for the immunophenotypic characterization of BMMC from SMCD in adults and should be integrated with clinical and morphologic studies for the diagnosis of the disease.

Adult↗

PubMed1998

Report on the first Latin American Consensus Conference for Flow Cytometric Immunophenotyping of Leukemia.

On October 16, 1996, the first Latin American Consensus Conference for the Immunophenotyping of Leukemia took place in Puebla, Mexico, with representatives from 10 countries of the region and two external consultants. This document summarizes the major conclusions for which scientific consensus was achieved. The purpose of disseminating these guidelines to the international community is based on the potential interest for other countries with similar social conditions and economical restrictions.

Antibodies, Monoclonal↗

PubMed1998

Demonstration of donor origin of CD34+ HLA-DR- bone marrow cells after allogeneic peripheral blood transplantation with a long follow-up.

Peripheral blood progenitor cells (PBPC) are increasingly being used to perform allogeneic transplants (allo-PBPCT). An important issue regarding allo-PBPCT is the potential for long-term engraftment of human PBPC. A subset of bone marrow (BM) cells displaying the immunophenotype CD34+ HLA-DR- has functional properties associated with pluripotential stem cells. We studied the origin (donor vs recipient) of CD34+ HLA-DR- hematopoietic cells from patients having received allo-PBPCT and with a long follow-up (14+ to 21+ months). Chimeric status was determined after amplification by polymerase chain reaction (PCR) of short tandem repeat sequences (PCR-STR). Four patients (acute myeloid leukemia (n = 3), acute lymphoid leukemia (n = 1) were studied. CD34+ HLA-DR- cells from bone marrow aspirates were isolated by flow cytometry cell sorting. The mean percentage of CD34+ cells among the total nucleated BM cells from the four patients was 0.6+/-0.2% (mean +/- s.d.) (range, 0.31-1.27%). The CD34+ HLA-DR- cells accounted for 1.54+/-0.54 (range, 0.9-2.05%) of the CD34+ BM cells. The purity of the CD34+ HLA-DR- cells analyzed after sorting was higher than 94% in all sorted fractions. PCR-STR of these cells showed donor origin in all patients. The origin of CD34+ HLA-DR- bone marrow cells in patients treated with allo-PBPCT has not so far been analyzed. These results provide further evidence that G-CSF-mobilized PBPC contains cells which are capable of sustained long-term engraftment.

Adult↗

PubMed1998

Minimal number of circulating CD34+ cells to ensure successful leukapheresis and engraftment in autologous peripheral blood progenitor cell transplantation.

BACKGROUND: The number of peripheral blood (PB) CD34+ cells has been widely used to monitor the timing of leukapheresis for autologous transplantation. However, no cutoff value for CD34+ cells in PB has been defined as a guideline for the identification of patients in whom the harvest would be effective and those in whom there was a high probability of failure. STUDY DESIGN AND METHODS: The present study investigated the best threshold of CD34+ cells in PB for successful harvesting and engraftment, using 263 PB samples with their corresponding leukapheresis components. In addition, that measure has been compared to other commonly used criteria such as the white cell count, the number of mononuclear cells, and the number of colony-forming units-granulocyte macrophage in PB. RESULTS: Time to engraftment of both granulocytes and platelets was significantly influenced by the number of CD34+ cells transfused, but all patients receiving > or = 0.75 x 10(6) CD34+ cells per kg achieved engraftment within a reasonable number of days (> 0.5 x 10(9)/L granulocytes by Day 11 and > 20 x 10(9)/L platelets by Day 13). A clear correlation between the number of CD34+ cells per microL in PB and of CD34+ cells per kg collected was found at each apheresis (r = 0.9, p < 0.0001). Moreover, the number of CD34+ cells per microL measured in PB the day the first leukapheresis was initiated displayed an excellent correlation with the total amount of CD34+ cells per kg finally collected (r = 0.81, p < 0.0001). On the basis of the regression curve obtained and the clinical engraftment results, it was found that the presence of > 5 CD34+ cells per microL in PB ensured a good yield from the harvest in 95 percent of patients and would avoid an unsuccessful harvest in 81 percent of cases. CONCLUSION: A dose of only 0.75 x 10(6) CD34+ cells per kg guarantees hematopoietic recovery within a reasonable number of days. To initiate a leukapheresis from which enough progenitor cells may confidently be obtained, a minimum of 5 CD34+ cells per microL in PB is required.

Antigens, CD34↗

PubMed1997

cAMP and Ca2+ involvement in the mitochondrial response of cultured fetal rat hepatocytes to adrenaline.

The effect of adrenaline on the control of respiratory activity of mitochondria from fetal hepatocytes in primary culture was studied. In the absence of adrenaline, the respiratory control ratio (RCR) of mitochondria increased during the first 3 days of culture due to a decrease in the rate of state 4 respiration. The presence of adrenaline in the incubation medium further increased the mitochondrial RCR through a decrease in the rate of respiration in state 4 and to an increase in the respiration rate in state 3. The effect of adrenaline was mimicked by dibutyryl-cAMP, forskolin, and isobutyl methyl xanthine. All these compounds increased cAMP concentrations, suggesting that cAMP may be involved in the effect of adrenaline. The increase in intracellular free Ca2+ concentrations caused by phenylephrine, vasopressin, or thapsigargin was also accompanied by an increase in the RCR, suggesting that both phenomena are associated. Dibutyryl-cAMP also increased free Ca2+ concentrations, suggesting that the effects of cAMP may be mediated by free Ca2+ concentrations. Adrenaline, dibutyryl-cAMP, phenylephrine, vasopressin, and thapsigargin promoted adenine nucleotide accumulation in mitochondria; this may be an intermediate step in the activation of mitochondrial respiratory function. These results suggest that the stimulatory effect of adrenaline on mitochondrial maturation in cultured fetal rat hepatocytes may be exerted through a mechanism in which both cAMP and Ca2+ act as second messengers. It is concluded that the effect of adrenaline on mitochondrial maturation is exerted by both alpha- and beta-adrenergic mechanisms and is mediated by the increase in adenine nucleotide contents of mitochondria.

Adenine Nucleotides↗

PubMed1997

Murine hematopoietic reconstitution after tagging and selection of retrovirally transduced bone marrow cells.

A major problem facing the effective treatment of patients with cancer is how to get the specific antitumor agent into every tumor cell. In this report we describe the use of a strategy that, by using retroviral vectors encoding a truncated human CD5 cDNA, allows the selection of only the infected cells, and we show the ability to obtain, before bone marrow transplantation, a population of 5-fluouracil-treated murine bone marrow cells that are 100% marked. This marked population of bone marrow cells is able to reconstitute the hematopoietic system in lethally irradiated mice, indicating that the surface marker lacks deleterious effects on the functionality of bone marrow cells. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD5-expressing cells. Nevertheless, a significant proportion of the hematopoietic cells no longer expresses the surface marker CD5 in the 9-month-old recipient mice. This transcriptional inactivity of the proviral long terminal repeat (LTR) was accompanied by de novo methylation of the proviral sequences. Our results show that the use of the CD5 as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, which can entirely reconstitute the hematopoietic system in mice. These results should now greatly enhance the power of studies aimed at addressing questions such as generation of cancer-negative hematopoiesis.

Animals↗

PubMed1997

Glucocorticoids regulate L-fucose glycoconjugates in rat pancreatic zymogen granules.

Lectin-binding studies were performed on rat pancreatic zymogen granules to investigate the influence of glucocorticoid levels on saccharide membrane composition. The following animal groups were used: (1) control rats; (2) rats treated with hydrocortisone (1, 10 and 25 mg/kg/day) for 1, 3 and 8 days; (3) postadrenalectomized rats at days +1, +3 and +8; and (4) adrenalectomized rats receiving hydrocortisone therapy (10 mg/kg/day) for 8 days. By flow cytometry, fluoresceinated (FITC) lectins were used to measure the amount of Concanavalin A (Con A) (specific for D-mannose), wheat germ agglutinin (WGA) (specific for N-acetyl-D-glucosamine) and sialic acids and Tetragonolobus purpureus (TP) (specific for L-fucose) bound to individual zymogen granules from two subpopulations, Z1 and Z2, identified on the basis of their forward and side scatter properties. The molar ratio of the different FITC-lectins revealed significant differences in the glycoconjugate composition of Z1 and Z2 granules, the Z1 granules showing higher ratios of N-acetyl-D-glucosamine:L-fucose and N-acetyl-D-glucosamine:D-mannose, both in control, adrenalectomized and hydrocortisone-treated rats. It was also observed that N-acetyl-D-glucosamine and/or sialic acids were more abundant than L-fucose and D-mannose in the zymogen granule membrane. Z1 and Z2 granules had different glycosylation patterns. Neither adrenalectomy nor hydrocortisone treatments varied the Con A binding to zymogen granules. An increase in WGA binding was only induced by administration of very high doses of hydrocortisone (25 mg/kg/day) for 8 days, an effect not directly related to glucocorticoids. In contrast, a correlation between the FITC-TP labelling and glucocorticoid levels can be established, so that, in a time-dose dependent way, an increase was observed in zymogen granules of rats treated with hydrocortisone while a decreased TP binding was found in adrenalectomized rats-an effect which was reversed with hydrocortisone therapy. Therefore, glucocorticoids exert a direct influence on the saccharide composition of rat pancreatic zymogen granules, regulating the amount of L-fucose glycoconjugates, with Z2 granules more sensitive than Z1 ones.

Acetylglucosamine↗

PubMed1997

[Syndrome of abnormal chromatin clumping in leucocytes with a high fraction of bone marrow cells in S-phase and in vitro autonomous growth].

Syndrome of abnormal chromatin clumping in leucocytes syndrome (ACCLS) is an uncommon entity which shares clinical and biological features with the myelodysplastic (MDS) and chronic myeloproliferative syndrome. In fact, as some authors consider ACCLS a new type of MDS, others maintain that it is in Ph'negative/bcr-abl negative chronic myeloid leukaemia. A new case of ACCLS appeared in a 68 year old woman, who presented with anaemic symptoms, bleeding and recurrent infections, and a haematological picture including progressive macrocytic anemia, thrombocytopenia and leuco-erythroblastosis. Marrow hypercellularity with granulocytic hyperplasia, and mature granulocytes presenting nuclear hyposegmentation and large peripheral blocks of chromatin separated by clear zones were the characteristic features of this case. No cytogenetic abnormalities were found and DNA flow-cytometry content was normal (euploid), supporting the thought that a disequilibrium exists in the hetero-chromatin/eucromatin ration in AACLS. Reverse PCR for bcr-abl transcripts was negative. The cell-cycle-phase analysis showed a high fraction of S-cells in the bone marrow (27%) in contrast to a very low S-phase (0.2%) in the peripheral blood, pattern that is different from both CMML and CML. In vitro clonogenic assays showed a high colony forming capacity and a certain grade of autonomous proliferation of the bone marrow cells, which is reminiscent of the CMML growth behaviour in culture. The patient was treated with vitamin D3, low dose Ara-C, prednisone and hydroxyurea until her demise, fifteen months after diagnosis. In total, the patient received 47 units of packed cells and 114 of platelet concentrates, and was transfused only when she presented anaemic or hemorrhagic symptoms. These clinical and haematological features suggest that ACCLS is a distinct entity that should be considered a sixth type of MDS, beside CMML, with which it has much in common.

Aged↗

PubMed1997

A BCR-ABL(p190) fusion gene made by homologous recombination causes B-cell acute lymphoblastic leukemias in chimeric mice with independence of the endogenous bcr product.

BCR-ABL(p190) oncogene is the result of a reciprocal translocation between chromosomes 9 and 22 and is associated with B-cell acute lymphoblastic leukemia (B-ALL) in humans. Current models expressing the BCR-ABL(p190) chimeric gene fail to consistently reproduce the phenotype with which the fusion gene is associated in human pathology, mainly due to the difficulty of being expressed in the appropriate cell type in vivo. We have used here homologous recombination in ES cells to create an in-frame fusion of BCR-ABL(p190) that mimics the consequences of the human chromosomal translocation by fusion of BCR-ABL coding sequences into the bcr endogenous gene. The chimeric mice generated with the mutant embryonic stem cells systematically develop B-ALL. Using these chimeric mice, we further show that BCR-ABL oncogene does not require the endogenous bcr product in leukemogenesis. Our results show that BCR-ABL(p190) chimeric mice are a new model to study the biology of the BCR-ABL oncogene and indicate the efficacy of this strategy for studying the role of specific chromosome abnormalities in tumor development.

Animals↗

PubMed1997

Immunophenotyping investigation of minimal residual disease is a useful approach for predicting relapse in acute myeloid leukemia patients.

A high complete remission rate is currently achieved in patients with acute myeloid leukemia (AML). However, many patients eventually relapse due to the persistence of low numbers of residual leukemic cells that are undetectable by conventional cytomorphologic criteria (minimal residual disease [MRD]). Using immunophenotypic multiparametric flow cytometry, we have investigated in sequential studies (diagnosis and follow-up) the impact of MRD detection on the outcome of 53 AML patients that had achieved morphologic remission with standard AML protocols and displayed at diagnosis an aberrant phenotype. Patients were studied at diagnosis with a panel of 35 monoclonal antibodies in triple staining combinations for detection of aberrant or uncommon phenotypic features. According to these features, a patient's probe was custom-built at diagnosis for the identification of possible residual leukemic cells during follow-up. The level of MRD at the end of induction and intensification therapy correlated with the number of relapses and relapse-free survival (RFS). Thus, patients with more than 5 x 10(-3) residual cells (5 residual cells among 1,000 normal bone marrow [BM] cells) identified as leukemic by immunophenotyping in the first remission BM showed a significant higher rate of relapse (67% v 20% for patients with less than 5 x 10(-3) residual cells; P = .002) and a lower median RFS (17 months v not reached; P = .01). At the end of intensification, with a cut-off value of 2 x 10(-3) leukemic cells, AML patients also separated into two distinct groups with relapse rates of 69% versus 32% (P = .02), respectively, and median RFS of 16 months versus not reached (P = .04). In addition, overall survival was also significantly related to the level of residual cells in the marrow obtained at the end of induction and particularly after intensification therapy (P = .008). Furthermore, we have explored whether residual disease was related with the functional expression of multidrug resistance (MDR-1) at diagnosis as assessed by the rhodamine123 assay. Patients with > or =5 x 10(-3) residual leukemic cells at the end of induction therapy had a significantly higher rhodamine-123 efflux (mean, 56% +/- 24%) than those with less than 5 x 10(-3) residual cells (mean, 32% +/- 31%; P = .04). Finally, multivariate analysis showed that the number of residual cells at the end of induction or intensification therapy was the most important prognostic factor for prediction of RFS. Overall, our results show that immunophenotypical investigation of MRD strongly predicts outcome in patients with AML and that the number of residual leukemic cells correlates with multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Analysis of IL-2 and IL-6 binding to peripheral blood lymphocytes in Graves disease: relationship with disease activity.

Growing evidence points to the involvement of cytokines in the pathogenesis of some autoimmune diseases. To investigate the possible role of interleukin 2 (IL-2) and interleukin 6 (IL-6) on the pathogenesis of Graves disease (GD), the binding of both exogenous IL-2 and IL-6 and the expression of the IL-2 receptor subunit p55 (CD25) were evaluated by flow cytometry in peripheral blood T and B cells from 70 GD patients, in different states of the disease, and from 19 age- and sex-matched healthy volunteers. Serum levels of total T3 and T4, of free T4, and of anti-TSH receptor antibodies were also simultaneously determined. All GD patients displayed significantly increased numbers of B cells bound to IL-2. Hyperthyroid untreated GD patients had significantly higher numbers of T and B cells expressing the IL-2 receptor subunit p55 as compared to euthyroid patients in long-term remission. In addition, serum anti-TSH receptor antibody levels were directly correlated with the absolute numbers of T cells bound to IL-2 (r = 0.565, P < 0.05) and to IL-6 (r = 0.653), P = 0.02) in the hyperthyroid untreated patients, but not in long-term remission euthyroid GD patients or in patients treated with methimazole. The serum levels of total T3 and free T4 were significantly correlated with the absolute numbers of circulating T cells binding IL-2 (r = 0.720, P < 0.01 and r = 0.783, P < 0.002, respectively) as well as with the absolute numbers of circulating T cells binding IL-6 (r = 0.671, P < 0.02 and r = 0.626, P < 0.02, respectively). The serum levels of total T3 were also correlated with both the absolute numbers of B cells binding to IL-2 (r = 0.586, P < 0.05) and to IL-6 (r = 0.757, P < 0.001). These findings suggest that IL-2 and IL-6 may play a role in the pathogenesis of GD.

Adult↗

PubMed1997

Cholecystokinin regulates glycoprotein membrane composition of rat pancreatic zymogen granules.

Lectin-binding studies were performed on rat pancreatic zymogen granules to investigate the alterations in the carbohydrate membrane composition under both chronic CCK stimulation and long-term CCK blockade for 3, 7 and 15 days. By flow cytometry using FITC-WGA--which specifically binds to N-acetylglucosamine and sialic acid--we measured the amount of WGA molecules bound to each individual granule. Parallel studies on pancreatic secretion were also carried out. CCK treatment displayed a differential effect on two zymogen granule subpopulations (Z1 and Z2) identified by flow cytometry on the basis of their light scatter properties: no effects on Z2 zymogen granules were observed in CCK-treated rats, while Z1 granules showed a significant increase in WGA binding at day + 7 which coincides with an increase in protein secretion in response to the hormone. On the contrary, a significant decrease in the amount of WGA receptors was observed in zymogen granule membrane of both the Z1 and Z2 subsets of rats subjected to a long-term CCK blockade. Again, these changes parallel to the reduction observed in protein secretion. Our results suggest that glycoconjugates of zymogen granule membrane involved in CCK-regulated exocytosis contain N-acetylglucosamine and sialic acid residues whose quantities are regulated by CCK.

Animals↗

PubMed1997

Sequential immunophenotypic analysis of mast cells in a case of systemic mast cell disease evolving to a mast cell leukemia.

The immunophenotypic characteristics of both bone marrow (BM) and peripheral blood (PB) mast cells (MC), from a patient suffering from an aggressive systemic mast cell disease (SMCD), were sequentially analyzed by flow cytometry using direct immunofluorescence. Analysis was carried out at diagnosis, during clinical response induced by interferon alfa-2h/prednisone therapy, and later at relapse. Our results show that together with the CD117 and IgE characteristic markers, at diagnosis BM MC showed strong expression of CD11c, CD13, CD29, CD33, CD44, CD45, CD63, and CD71, and they were also positive for CD2, CD22, CD25, and CD54 although at a lower level. PB MC displayed similar immunophenotypic characteristics although they had a lower expression of CD11c, CD25, CD33, CD63, CD69, and CD71 with a higher reactivity for CD117. Unlike BM MC, PB MC were weakly positive for CD41a and CD61. Sequential studies showed decreased numbers of both BM and PB MC during clinical response associated with a higher expression of the CD29 and CD54 adhesion molecules. In turn, clinical relapse was related to increased numbers of PB and BM MC together with lower CD2, CD11c, CD45, and and CD54 expression and a higher reactivity for the CD117 and CD25 antigens. CD2 had become negative at the last follow-up study. In addition, an increased proportion of S-phase MC was observed at relapse. These findings suggest that the assessment of the quantitative expression of cell-adhesion molecules and growth-factor receptors together with cell cycle studies of mast cells could be of value for monitoring therapy and predicting clinical outcome in aggressive SMCD.

Aged↗

PubMed1997

Decreased natural killer cytotoxic activity in chronic alcoholism is associated with alcohol liver disease but not active ethanol consumption.

Chronic alcohol intake is associated with an increased incidence of certain neoplasms. Natural killer (NK) cells have been considered to be involved in control tumor development and growth. The goal of the present study was to contribute to a better understanding of the effects of ethanol (EtOH) per se on the NK-cell population. Both patients with chronic alcoholism without liver disease (AWLD) and subjects with alcohol-induced cirrhosis (ALC) were carefully selected for this study. Immunophenotypical and functional studies of peripheral blood (PB) NK-cells were performed during active EtOH intake and after 3 months of a withdrawal period. In the AWLD group a significant increase in number of NK-cells (CD3-/CD56+) (P < .05) associated with a parallel increase in NK-cell lytic activity (P < .01) was observed. In addition, the number of cytotoxic T cells displaying the CD3+/CD56+ phenotype as well as CD8-/CD57+ NK-cell subset was also increased (P < .01 and P < .001, respectively). By contrast, in ALC patients with active EtOH intake (ALCET group), although a significant increase in the number of NK PB lymphocytes was observed (P < .05), NK lytic activity was depressed (P < .05), suggesting the existence of a decreased lytic activity/NK-cell. After 3 months of EtOH withdrawal, PB mononuclear cells (PBMC) from the AWLD group patients still displayed an increased NK cytolytic activity; in addition, the number of PB NK-cells (CD3-/CD56+ and CD8-/CD57+) and CD3+/CD56+ PB T cells continued to be increased. Independently of the duration of withdrawal period, in ALC patients EtOH withdrawal was followed by a slight decrease in the NK lytic activity of PBMC with respect to the values in active alcoholism phase; slight differences observed in the NK lytic activity in ALC patients who quit drinking could be related to the tendency to decrease of the number of NK-cells toward normal values. Furthermore, although an increase in NK cytotoxic activity after stimulation of PBMC with interleukin-2 (IL-2) was observed in ALC, it did not reach the levels observed in healthy subjects. Overall, our results show that the behavior of PB NK-cell population in chronic alcoholism is different according to both the moment of EtOH consumption and the existence or not of ALC. Alcohol by itself induced an increase in the number and lytic activity of NK-cells. By contrast, the NK cytolytic activity is constantly depressed in the stage of alcoholic cirrhosis, supporting the notion that immunosurveillance mechanisms may be affected in these patients.

Adult↗

PubMed1997

Association between trisomy 8 and the immunophenotype of blast cells from acute leukemias secondary to a myelodysplastic syndrome or chronic myeloproliferative disorders.

In the present study we have used FISH to analyze the incidence of trisomy 8 in acute leukemias following either a primary myeloproliferative disorder (MPD) or a myelodysplastic syndrome (MDS) and correlated it with both the immunophenotype and the cell-cycle distribution of the leukemic blast cells. Six of the 21 (28%) acute leukemias studied displayed trisomy 8 by FISH. The number of trisomic cells in these cases ranged from 20 to 84%, with a mean of 46 +/- 24%. Trisomy 8 was associated with a homogeneous population of leukemic cells, phenotypically characterized by CD34+/HLADR+/CD13+/CD33+/CD11b-/ CD15-/CD14-. No significant differences were observed on the proliferative rate of cases with trisomy 8, as compared with blast cells from the remaining patients. Overall, our findings suggest that in acute leukemias secondary to MPD or MDS, trisomy 8 is associated with a blockade of myeloid maturation at an early step of the differentiation process.

Acute Disease↗

PubMed1997

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.

Adult↗

PubMed1997

Detection of the Mbcr/abl translocation in chronic myeloid leukemia by fluorescence in situ hybridization: comparison with conventional cytogenetics and implications for minimal residual disease detection.

The correlation between the detection of the Philadelphia chromosome by conventional cytogenetics and the identification of Mbcr/abl translocation by fluorescence in situ hybridization (FISH) in both metaphase and interphase cells is prospectively analyzed in a group of 21 chronic myeloid leukemia (CML) patients. To gain insight into the sensitivity and specificity of the detection of the bcr/abl translocation by FISH, a group of 10 healthy volunteers was also studied. Our results show that for the detection of bcr/abl translocation in CML patients, FISH is more sensitive than conventional cytogenetics because it detects significantly higher proportions of cells carrying the translocation both in metaphase (P < .0002) and interphase nuclei (P < .003). Moreover, in the metaphases of the controls analyzed, no bcr/abl+ chromosome was detected that makes the colocalization of bcr and abl signals in the CML patients highly specific. Conversely, in control interphase nuclei, a small proportion of cells (ranging between 0% and 3%, mean value of 1.7% +/- 0.9%) displaying colocalization of both signals is usually detected. This limits, at least for the moment, the routine use of FISH for the detection of minimal residual disease in CML patients at levels lower than 10(-1).

Adult↗

PubMed1997

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.

Adolescent↗