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PRAME is a tumor associated antigen (TAA) of particular interest since it is widely expressed by lymphoid and myeloid malignancies. Several studies have associated high PRAME RNA levels with good prognosis in acute myeloid leukemia (AML). PRAME expression is regulated at the epigenetic level. For this reason inhibitors of DNA methylation, such as 5-azacytidine, can modulate the expression of this TAAs. In the current study we analyzed the effect of 5-azaC on the expression of PRAME in blasts versus CD34+ cells from healthy donors in an attempt to increase its expression, thus inducing a potential target for therapeutic strategies., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Acute myeloid leukemia (AML) is a clonal disorder characterized by the accumulation of myeloid blasts in the bone marrow. Here, we report the effects of the novel histone deacetylase inhibitor panobinostat (LBH589) in combination with doxorubicin on AML cells. Panobinostat exhibited potent anti-AML activity in all AML cell lines tested and in primary AML cells from patients (IC(50)<20 nM). In addition, panobinostat potentiated the action of several standard-of-care anti-AML compounds, particularly, doxorubicin. The molecular effects induced by panobinostat and doxorubicin treatment were investigated by analyzing gene expression, cell cycle, apoptosis and signaling pathways. Analyses of gene expression profiles identified 588 genes whose expression was exclusively affected by the combination of panobinostat and doxorubicin. The combination induced AML cell death by an increase in the mitochondrial outer membrane permeability and release of cytochrome c from the mitochondria, resulting in caspase-dependent apoptosis and accompanied by the upregulation of Bax, Bak and, particularly, Bad. The drug combination provoked a strong activation of a DNA damage response, indicating that this combination may trigger cell death by a mechanism that induced DNA double-strand breaks. These data indicate that the combination of panobinostat and doxorubicin may be an effective therapy for the treatment of AML.

Bone marrow samples from 43 adult patients with de novo diagnosed acute myeloid leukemia (AML)--10 acute promyelocytic leukemias (APL) with t(15;17), four AML with inv(16), seven monocytic leukemias and 22 nonmonocytic leukemias--were analyzed using high-density oligonucleotide microarrays. Hierarchical clustering analysis segregated APL, AML with inv(16), monocytic leukemias and the remaining AML into separate groups. A set of only 21 genes was able to assign AML to one of these three classes: APL, inv(16) and other AML subtype without a specific translocation. Quantitative RT-PCR performed for 18 out of these predictor genes confirmed microarray results. APL expressed high levels of FGF13 and FGFR1 as well as two potent angiogenic factors, HGF and VEGF. AML with inv(16) showed an upregulation of MYH11 and a downregulation of a gene encoding a core-binding factor protein, RUNX3. Genes involved in cell adhesion represented the most altered functional category in monocytic leukemias. Two major groups emerged from the remaining 22 AML: cluster A with 10 samples and cluster B with 12. All the eight leukemias that were either refractory to treatment or that relapsed afterwards were assigned to cluster B. In the latter cluster, CD34 upregulation and serine proteases downregulation is consistent with a maturation arrest and lack of granulocytic differentiation.

Background and Objectives: Molecular analysis has contributed to the identification of several non-random chromosomal translocations, such as t(15;17), t(8:21), inv(16)/t(16;16) and 11q23 abnormalities, typically associated with acute myeloid leukemia (AML). The identification of these chromosomal abnormalities helps not only to define different AML subtypes with distinct prognoses and treatments but also to monitor the disappearance of malignant cells after treatment. Recent reports suggest that the frequency of these alterations may differ according to geographic distribution. However, most of these reports focus on just one or two genetic alterations, which may lead to some selection bias. Appropriate epidemiological studies should be based on unselected consecutive series of patients in which all relevant genes are simultaneously analyzed. The aim of the present study was to explore whether or not the incidence of genetic lesions in Spanish AML patients differs from that reported in other countries., Design and Methods: In a series of 145 consecutive un-selected adult patients with AML we simultaneously analyzed the presence of 4 genetic abnormalities, PML/RARalpha for t(15;17), AML1/ETO for t(8;21), CBFbeta/MYH11 for inv(16)/t(16;16) and rearrangements of the MLL gene for 11q23 abnormalities. AML were classified using the new World Health Organization (WHO) classification for hematologic malignancies. The techniques used were standardized according to the recommendations of the European BIOMED-1 Concerted Action., Results: The PML/RARalpha transcript was present in 34 patients (23.4%) (23 were bcr1, 2 bcr2 and 9 bcr3). The AML1/ETO fusion transcript was detected in only 2 cases (1.4%) both with M2 morphology, but 29 other cases with M2 morphology were negative. CBFbeta/MYH11 transcript was present in 9 cases (6.2%) eight of them displaying M4Eo morphology. Finally, 5 cases (3.5%) showed rearrangements of theMLL gene. Our results differ from those reported from the United States and North/Central Europe, particularly regarding the incidence of t(15;17) and t(8;21) translocations. In Spain the frequency of t(15;17) is higher while that of t(8;21) is lower., Interpretation and Conclusions: These data add epidemiological information about geographic heterogeneity of such chromosome aberrations in AML and would contribute to the design of specific screening strategies adapted to the incidence in each country.

Background and Objective: Rearrangements of the short arm of chromosome 12 have been described in different hematologic malignancies. Some of these abnormalities showed a rearrangement of the ETV6 gene. We studied the 12p region in one case with a t(8;12)(q12;p13) by fluorescence in situ hybridization (FISH)., Design and Methods: We have identified a chromosome translocation, t(8;12)(q12;p13) in two patients with myeloid disorders; one with acute myelogenous leukemia (AML) and one with refractory anemia (RA). FISH studies with specific probes (cosmids and YACs) for the 12p region were used to investigate one case., Results: FISH studies demonstrated hemizygous loss of the ETV6 and CDKN1B regions and two copies of the CCDN2 locus, as a result of the balanced translocation and an additional copy of the der(8)., Interpretation and Conclusions: Myeloid diseases with t(8;12)(q12;p13) have an interstitial deletion of 12p, including the ETV6 and CDKN1B regions. A duplication of CCDN2 locus can also be found.

In the present review we analyse the current knowledge about the growth properties of AML progenitor cells and their relationship with other clinico-biological characteristics of the disease. Leukaemic colony forming unit L-CFU is considered to be the clonogenic cell in AML and more immature than the blast cell population. Our studies have shown that in leukaemic hematopoiesis colony forming cells can exist among both cell fractions CD34+ and CD34-. Optimal "in vitro" proliferation of L-CFU is dependent upon the addition of exogenous growth factors. However, it has been observed that leukaemic progenitor cells frequently display a certain degree of autonomous proliferation. In order to quantify the "in vitro" behaviour of L-CFU, we have explored 3 parameters: 1) plating efficiency (PE); 2) autonomous growth (AG); and 3) autonomous proliferative index (API) which was calculated as AG divided by PE and we have correlated them with other clinico-biological data. According to the FAB classification we could observe that patients with M3 subtype showed an higher PE than other AML subgroups and a significantly lower API. Regarding CD34 expression we observed that AG was enhanced in CD34+ cases and also in those showing a higher rh123 elimination. In order to determine whether PE could condition clinical evolution, we analysed this parameter in a large series of patients but failed to demonstrate any relationship. By contrast, we observed that patients who displayed a higher API showed a shorter survival than patients with lower API (18% vs 48% surviving at 3 years). We have also shown that abnormalities in the CFU-GM growth pattern could be associated with risk the of relapse in AML patients; a switch from normal to abnormal "in vitro" growth should alert us. But for the assessment of the real value of these analyses sequential follow-up studies are mandatory. In summary, cell culture studies contribute not only to a better understanding of leukaemic hematopoiesis but may also contribute to better disease monitoring.

Background and Objective: Rapid identification of AML patients carrying the t(15;17) translocation for treatment decision-making is currently made on the basis of morphologic screening. However, the existence of both false positives and negatives highlights the need for more objective methods of screening AML cases and further molecular confirmation of the t(15;17) translocation., Design and Methods: In the present study we analyzed a total of 111 AML cases in order to investigate whether immunophenotyping based on the assessment of multiple-stainings analyzed at flow cytometry could improve the sensitivity and specificity of morphologic identification of acute promyelocytic leukemia (APL) carrying the t(15;17) translocation. FISH analysis was used as a complementary technique for cases in which morphology and molecular biology yielded discrepant results., Results: Concordant results between morphology and RT-PCR were found in 102/111 (91.8%) cases: 34 patients had M3/PML-RARalpha+ and 68 non-M3/PML-RARalpha- disease. Nine cases showed discrepants results. Multivariate analysis showed that the best combination of immunologic markers for discriminating between M3/PML-RARalpha+ and non-M3/PML-RARalpha- cases was that of the presence of heterogeneous expression of CD13, the existence of a single major blast cell population, and a characteristic CD34/CD15 phenotypic pattern (p<0.02). A score system based on these parameters was designed, and the 34 M3/PML-RARalpha+ cases showed a score of 3 (presence of the 3 phenotypic characteristics). In contrast, only 1 out of the 68 (1.3%) non-M3/PML-RARalpha- cases had this score, most o these latter cases (53/68, 78%) scoring either 0 or 1. Therefore, among these cases, immunophenotyping showed a sensitivity of 100% and a specificity of 99% for predicting PML/RARalpha gene rearrangements. Of the 9 cases in which morphology and molecular biology results were discrepant, four cases displayed M3 morphology without PML/RARalpha rearrangements by RT-PCR. In only one of these 4 cases did the immunophenotype score 3, this being the only FISH positive case. From the remaining five discrepant cases (non-M3 morphology while positive for PML/RARalpha) two cases had a phenotypic score of 3 and were FISH positive while the other three were negative by FISH. Upon repeating RT-PCR studies, two of these latter three cases became negative., Interpretation and Conclusions: Our results show that immunophenotyping may be of great value for quick screening of APL with PML/RARalpha rearrangements.

The in vitro growth characteristics of a large series of acute myeloid leukaemia (AML) patients and their relationship with other clinical and biological disease characteristics were analysed. Patients with AML were studied, 181 with de novo AML and 45 with secondary AML (24 myelodysplastic syndrome, sAML-MDS, 21 myeloproliferative disorder, sAML-MPD). Leukaemic colony forming units (L-CFU) were assayed by plating peripheral blood (PB) blast cells in methyl-cellulose and using LCM-PHA as stimulant. In each case parallel cultures were made with and without stimulating factors. Plating efficiency (PE) was defined as the number of clusters plus colonies/10(5) cells plated. Autonomous growth (AG) was the number of colonies plus clusters growing without stimulant. The autonomous proliferative index (API) was calculated as the number of clusters + colonies without stimulating factor divided by the number of clusters + colonies with stimulating factor. No significant differences in the PE between de novo and secondary AML were found. Autonomous growth was significantly higher in sAML-MPD. The FAB subtype M3 leukaemias displayed a significantly greater PE and a significantly lower API when compared with the other FAB subgroups (P=0.0002). Upon analysing the relationship with the immunophenotype, only CD33 expression showed a significant relationship with the in vitro growth pattern; CD33+ cases displayed a higher PE (P=0.0002) and AG (P=0.0003) than CD33- cases. When patients were grouped according to the level of rh123 efflux (MDR1) it was observed that cases with >30% elimination showed a higher AG and API than those with <30% (P=0.03). Finally we found that patients with higher API (>0.05) displayed a significantly shorter overall survival as compared with patients with API<0.05 (P=0.04). The in vitro study properties of clonogenic cells produces relevant clinical information of leukaemic cell biology in AML patients.

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.

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

The aim of the present study was to analyze the incidence of AML cases displaying more than one blast cell subpopulation by immunophenotype at diagnosis, since, any of them, although minimal, can be responsible for the relapse. For this purpose we have prospectively investigated the immunophenotype of blast cells from 40 de novo AML patients at diagnosis with a large panel of monoclonal antibodies in double and triple staining combinations analyzed at flow cytometry. The discrimination between the different cell populations was based on: (1) the existence of aberrant phenotypes; (2) differences in light-scatter characteristics; and (3) the expression of differentiation-associated antigens (CD34, CD117, HLADR, CD33, CD15, CD14, CD11b and CD4). More than one blast cell subpopulation was identified in 34 patients (85%), two subpopulations in 12 patients (30%), three in three cases (7.7%), four in 13 patients (32.5%) and five populations in six cases (15%). The most common criteria for discrimination of blast cell subpopulations was based on the expression of maturation-associated antigens and, interestingly, the blast subpopulations defined by higher reactivity for myeloid differentiation-associated markers had a more mature FSC/SSC pattern. In 53% of the patients at least one of the subpopulations identified was minimal (< 10% of the total leukemic cells). Regarding the existence of aberrant phenotypes three situations were observed: (1) none of the subpopulations had antigenic aberrations (10 cases); (2) coexistence of normal and aberrant subpopulations (five cases); and (3) all the subpopulations displayed aberrant phenotypes (19 cases). In 17 of the 23 patients (74%) who had two or more blast cell subpopulations with phenotypic aberrations, at least one aberrant criteria was common to all the subpopulations; this criteria by itself would permit the simultaneous identification of all subpopulations in minimal residual disease (MRD) studies. In the remaining cases the investigation of MRD should be based on the phenotypic characteristics of each subpopulation.

In acute non-lymphoblastic leukemia (ANLL) progenitor cells frequently display a certain degree of autonomous growth. The aim of the present work was to analyze the autonomous proliferative capacity of leukemic progenitors in both de novo and secondary to myeloproliferative disorders (MPD) and myelodysplastic syndromes (MDS), acute myeloid leukemias and to correlate with clinical and biological characteristics of the disease. Clonogenic assays with and without leukocyte conditioned medium with PHA (LCM-PHA) were performed and the autonomous proliferation index (API) calculated in a series of 50 patients (34 de novo ANLL, eight secondary to MPD and eight secondary to MDS). Patients were divided into two groups according to their API, low (< or = 0.4) or high (> 0.4). Autonomous growth was observed in 84% of cases studied (82% in de novo ANLL, 75% secondary to MDS and 100% secondary to MPD). The group with the highest API (29 patients) had increased levels of hemoglobin (P = 0.006) and platelets (P = 0.01). A high API was also associated with an immature phenotype of blast cells (P = 0.02). Upon analyzing the de novo ANLL separately we observed that a high API correlated with high Hb values (P = 0.02), a lower rate of complete remission (42% vs 61%) and a lower survival rate (medium of 3 vs 10 months). These findings suggest that the capacity for autonomous proliferation can condition the clinical and biological profile of the disease.

In the present study the DNA cell content of 205 de novo acute myeloblastic leukemia (AML) patients is analyzed at flow cytometry in order to determine both the incidence of DNA aneuploidy in AML patients and the clinical and biological characteristics of AML aneuploid cases. All technical procedures were performed in accordance with the proposed guidelines of the DNA Cytometric Consensus Conference. Our results show that the incidence of DNA aneuploidy is quite low (4.8%), with most cases (n = 8) hyperdiploid and only a small proportion (n = 2) hypodiploid. No major differences were detected between the aneuploid and diploid cases with respect to the clinical and prognostic disease characteristics. Regarding the immunophenotype of the blast cells, the aneuploid cases displayed both a higher incidence of immature myeloblastic phenotype and a greater expression of lymphoid-associated antigens. In summary, our results show that AML patients display a quite low incidence of DNA aneuploidy and that despite the fact that these cases do not display particular clinical characteristics, they show an association with the expression of lymphoid-related markers.

The presence of Ig and TCR gene rearrangement has been reported to occur in ANLL. However, most of the studies have been performed in short series of patients and, in general, not all rearranging genes have been included. We have investigated the configuration of immunoglobulin (Ig) and T-cell receptor loci (TCR) in a series of 160 untreated patients with de novo acute non-lymphoblastic leukaemia (ANLL) and correlated the results with the morphological and immunophenotypic characteristics of blast cells. IGH gene rearrangement was detected in 16/160 cases analysed (10%) and IGK was rearranged in half of them. The incidence of cases displaying TCRB, TCRG and TCRD rearrangements was 5.6%, 13.8% and 13%, respectively. Concomitant recombinatorial events including different Ig and/or TCR genes were frequently detected. Gene rearrangement was not related to the stage of cell differentiation within the myeloid lineage assessed both by morphological and immunophenotypic criteria. Regarding the correlation with the presence of lymphoid related markers, the only relevant association was between the expression of CD7 antigen and TCRG and TCRD gene rearrangement. Our results show that the incidence of gene rearrangement in ANLL may be slightly higher than previously suspected, and that it is not associated with early stages of cell differentiation nor to the expression of lymphoid markers.

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.

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

Aims: To analyze the immunophenotype of blast cells in patients with acute leukaemia after polycythemia vera, together with the most relevant clinical and haematological disease characteristics., Methods: The immunophenotype was analysed in nine patients by immunofluorescence flow cytometry using a panel of 15 monoclonal antibodies. The DNA content of blast cells was determined using Vindelov's technique., Results: The most relevant clinical and haematological disease characteristics included: the presence of enlarged spleen and liver by 56% and 67%, respectively; a moderate degree of leucocytosis with thrombocytopenia while haemoglobin was normal in 50% of patients. All patients received alkylating agents or hydroxyurea, or both. Interestingly, the chronic phase in patients receiving this latter drug was shorter. All cases showed a myeloid phenotype, four of them reactive only to early myeloid antigens (CD13/33); in the remaining cases the blast cells displayed granulomonocytic (CD14+, CD15+), erythroid (CD71 ), or megakaryocytic (CD61+, CD41+) markers. Coexpression of lymphoid related antigens (CD7, TdT, or CD19) was also detected. The morphological assessment of blast cells agreed with the immunophenotyping in five out of the nine cases. Blast cells from all six patients analysed displayed a diploid DNA content and the proportion of S-phase cells ranged from 0.4% to 4%., Conclusions: These findings suggest a pluripotential stem cell with myeloid commitment as the target cell of acute leukaemia after polycythemia vera.

We studied the effects of 1,25-dihydroxyvitamin D3 and other metabolites of vitamin D3 on the maturation in liquid culture and on colony formation in semisolid media of marrow and buffy coat cells from patients with myeloid leukemias and from normal individuals. After incubation with 1,25-dihydroxy-vitamin D3, a proportion of both normal and leukemic myeloid cells resembled cells of the monocyte-macrophage lineage; these cells expressed alpha-naphthylacetate esterase and were able to phagocytize and kill candida organisms. When granulocyte-macrophage progenitor cells (CFU-GM) were incubated with 1,25-dihydroxyvitamin D3, the number of monocyte-macrophage colonies was increased and the number of granulocyte colonies was reduced; megakaryocyte colony formation (CFU-Mk) was inhibited substantially; and there was no effect on erythroid (BFU-E) or multilineage (CFU-GEMM) progenitor cell colony formation. We propose that 1,25-dihydroxyvitamin D3 may induce cells that are normally committed to differentiate along the granulocytic pathways to differentiate instead along the monocyte-macrophage pathway. If these in vitro observations reflect the in vivo activity of 1,25-dihydroxyvitamin D3, it may be involved in the modulation of collagen deposits in the bone marrow.

The blast cells from nine patients with an overt acute leukaemia following a previous myelodysplastic syndrome (MDS) are analyzed with a panel of monoclonal antibodies as well as by morphological and cytochemical criteria. By integrating the results obtained with these three approaches the leukaemia in 6 patients was assessed as myeloid-granulocytic and/or monocytic-, in two as mixed- megakaryoblastic/myeloid- and in one as lymphoid. A good correlation between morphology, cytochemistry and immunological markers was observed in 7 out of the 9 cases. In three cases a noteworthy percentage of J5+ cells was detected. The exceptional finding of lymphoid as well as megakaryocytic and myeloid transformations suggests that the target cell for these leukaemias could be a pluripotent stem cell.

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.

CFU-L is considered as the clonogenic cell of acute non-lymphoblastic leukaemias (ANLL). Twenty-five malignant myelogenous hemopathies (5 blast crisis and 20 "de novo" ANLL) were studied in order to assess the proliferative capability of these cells and its relationship to FAB types and the achievement or not of complete remission (CR). The proliferative capability was assessed by culture on methylcellulose using conditioned leucocyte medium stimulated with phytohaemagglutinin as stimulating agent. Cell proliferation was observed in 21 cases, reaching 100% of the blast crisis and 80% (i.e., 16 cases) of the "de novo" ANLL. As regards the FAB types, it was found that the leukaemias with monocytic cells showed higher cell-growth than those of granulocytic lineage (90%, vs 60%). Of the patients not achieving CR (12 cases) colonies were formed in 66.6% (8 cases), while of the 8 patients who attained CR only in 3 (37.5%) were colonies observed. It was concluded that the FAB types of ANLL show different proliferative capability, which might influence the prognosis of the disease.

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.

The immunological phenotype of blast cells in 102 patients with acute myeloid leukaemia (AML) was analysed with a panel of 20 monoclonal antibodies and the enzyme terminal transferase, and correlated with the FAB classification. Although a partial correlation between these two approaches could be observed, almost every morphological group contained patients from more than one immunological phenotype. The M1 and M5a leukaemias showed the most undifferentiated phenotype, often lacking in specific myelomonocytic antigens. The M3 formed a uniform group defined as My7+, Ia-, FMC8+, a phenotype which was also observed in two cases of the microgranular variant. The granulocytic (CDw15) and monocytic (CDw14) antibodies crossreacted with some M5b and M2 leukaemias, respectively. Compared with M5a, the M5b leukaemias showed a large increase in the expression of CDw14 antigen, confirming the validity of the morphological differentiation. Glycophorin-A was present in four out of five M6 leukaemias. TdT activity was demonstrated in 10% of AML cases, with a higher incidence among the monocytic variants: M4 and M5-. Eleven AML were considered as unclassifiable according to the FAB criteria and in seven of them a megakaryoblastic cell population (GP IIb/IIIa+, GPIb+) was demonstrated; this confirms the need to include the subgroup of megakaryoblastic leukaemias within the AML. Finally, a possible immunological classification for AML is proposed.

Blast cells from eight out of 71 patients diagnosed with acute myeloid leukemia (AML) by morphological, cytochemical, and immunological criteria showed TdT activity. Their distribution according to the FAB classification was one M1, one M2, one M4, two M5a, one M5b, one M6, and one undifferentiated case. The TdT+ AML cases did not show major clinical and hematological differences when compared with the classical TdT- AML patients. Other phenotypical aberrations in the expression of membrane antigens, apart from the presence of nuclear TdT, were not observed in these TdT+ cases after study with a large panel of monoclonal antibodies. A higher incidence of TdT+ cases was found among the monocytic variants of AML (M4 and M5)--four cases--than in the granulocytic variants (M1, M2, and M3)--2 cases. These TdT+ cases should be distinguished from mixed leukemias by double labeling techniques, assessing in the TdT+ AML the coexpression of TdT and myeloid markers in individual cells as shown in four of our cases.