High expression of urokinase plasminogen activator receptor (UPA-R) in acute myeloid leukemia (AML) is associated with worse prognosis

Urokinase-type plasminogen activator receptor (UPA-R; CD87) is a membrane protein responsible for plasmin expression on cells facilitating cellular extravasations and tissue invasions. We studied the expression of the UPA-R on bone marrow (BM) cells of 93 patients with acute myeloid leukemia at first diagnosis and 8 healthy probands as controls by FACS analysis using phycoerythrin (PE)-conjugated antibodies. A case was defined as UPA-R-positive (UPA-R+) if >20% of the gated cells expressed UPA-R. Whereas none of the 8 healthy BM samples was positive for the UPA-R, 32 (34%) of the 93 AML samples were UPA-R+. Expression of UPA-R was heterogeneous in different FAB types, however, with the highest expression rates in monocytic subtypes (FAB M4/M5): 18%/19%/30% of UPA-R+ cases were found in M1/M2 or M3, and 58%/80% of cases with M4 or M5 were UPA-R+. Proportions of UPA-R+ cells varied between 1% and 98% of the mononuclear cell fractions, with the highest proportions in M4/M5 subtypes (on average 27%/40% UPA-R+ cells) and the lowest expression in AML M2 (11% UPA-R+ cells). The density of expressed UPA-R, estimated as mean channel fluorescence activity, was highest in cases with AML M1 (mFI: 124) followed by M4 and M5 (mFI: 78/77) and lowest in AML M2 (mFI: 43). In sAML, higher proportions of UPA-R+ cases (8 of 18; 44%) compared to pAML (24 of 75; 32%) were found as well as higher proportions of UPA-R+ cells (27% vs. 19%). Separating our patients' cohort in cytogenetic risk groups, we could not detect significant differences in the UPA-R expression profiles. For evaluations of the clinical course of AML, only patients treated by the AML-CG protocol (n = 65) were included. In the group of patients who did not respond to AML-CG therapy, significantly higher proportions of UPA-R+ cells (31% vs. 14%, P = 0.0015, t-test) were found. By evaluating a cut-off value for the percentage of positive cells that allows the most significant separation and differentiation between cases with shorter or longer relapse-free survival times, we could show that patients with >26.5% UPA-R-positive cells were characterized by a significantly higher risk for relapse compared to cases with <26.5% positive cells (P = 0.05). In summary, our data show a high expression of the UPA-R in AML, especially in (myelo)monocytoid subtypes. Cases with higher proportions of UPA-R+ cells were characterized by a significant lower remission rate after AML-CG therapy and a higher risk for relapse. Although prospective trials are still lacking, UPA-R is a prognostically relevant factor independent from the karyotype. UPA-R positivity may identify subtypes of AML associated with a more aggressive clinical course. Thus due to lower remission probabilities in UPA-R+ cases, a more intensive induction therapy regimen could be considered.     

Flow cytometric analysis of intracellular CD6 8 molecule expression in normal and malignant haemopoiesis

Summary. CD68 molecules are heavily glycosylated lysosomal membrane constituents of unknown function with strong expression in monocytes and macrophages. Using flow cytometry, we quantified expression levels of CD68 molecules in normal and malignant haemopoietic cells. CD68 molecules are intensely expressed in the cytoplasm and weakly on the surface of mature CD14⁺ monocytes. CD68 expression seems to start very early during granulo-monopoietic differentiation. Virtually all myeloperoxidase (MPO)⁺ bone marrow cells coexpress CD68 and similar proportions of CD34⁺ progenitor cells weakly express CD68 or MPO molecules. During further differentiation, CD68 expression is strongly up-regulated in early MP0⁺ precursor cells which lack lactoferrin (LF) and CD14 molecules. Compared to these, more mature MPO⁺LF⁺ bone marrow and peripheral blood granulocytes express considerable lower levels of CD68. In-line with this broad expression, all investigated acute myeloid leukaemia (AML) cases, classified as FAB M1-M5, were CD68 positive, and compared to normal CD34⁺ bone marrow cells, CD34⁺ AML blast cells expressed increased levels. CD68 expression is, however, not restricted to cells of myeloid origin, because a subset (40 – 15%, n = 6) of CD19⁺ peripheral blood B-lympho-cytes and 50% of B-ALL are also weakly positive. In contrast, normal CD3+ lymphocytes lack (<3%. n = 6) CD68 and only low proportions (6 – 3%, n= 6) of CD56⁺ NK cells are CD68⁺. Also, all investigated T-ALL cases (n = 6) lacked CD68.     

Comparative quantitative expression of bcl-2 by normal and leukaemic myeloid cells

Summary. Expression of the bcl-2 oncoprotein by AML blasts has previously been demonstrated to be heterogenous with high levels of bcl-2 expression being associated with a low complete remission rate and poor survival. We have quantified bcl-2 expression in AML blasts in relation to expression of the CD34 antigen and in comparison to CD34-positive cells from normal bone marrow. When expressed as molecules of equivalent soluble fluorochrome (MESF) per cell, AML blast cell bcl-2 expression varied from 11.1 to 99.9 × lO³ (median 39.4 × 10³, n=56) with 28.5% of patients expressing high MESF values (>50 × 10³) and 16% of patients expressing low MESF values (<20 × 10³), the remainder expressing intermediate values. There was no significant difference between intensity of bcl-2 expression and FAB classification in the de novo AML cases, and there was no significant differences between de novo and secondary AML cases. Blasts from CD34⁺ AML patients expressed significantly higher levels of bcl-2 (mean MESF 43.6 × 10³, n =36) than CD34^? AML patients (mean MESF 31.7 × 10³, n=19). In five cases of CD34⁺ AML, bcl-2 expression was determined on purified CD34⁺ and CD34^? blast cell populations. In all cases CD34⁺ blasts were found to express significantly higher bcl-2 MESF values compared to the CD34^? fraction. Purified CD34⁺ cells from normal bone marrow consistently expressed high levels of bcl-2 (MESF >75 × 10³ n = 4), which was comparable to that found on CD34⁺ AML cells. Our results suggest that the poor prognosis previously associated with AML blasts expressing the CD34 antigen may in part be related to high expression of bcl-2. Also the ability to measure bcl-2 in AML blasts quantitatively by flow cytometry and to categorize patients into discrete groups may be of value as a prognostic indicator in AML.     

Adult biphenotypic acute leukaemia: an entity with poor prognosis which is related to unfavourable cytogenetics and P-glycoprotein over-expression

Biphenotypic acute leukaemia (BAL) patients represented 8% of the 287 de novo consecutive adult acute leukaemias (23 BAL, 230 acute myeloid leukaemia (AML) and 34 acute lymphoblastic leukaemia (ALL)) referred to our department during the last 4-year period. Of these 23 BAL patients, 14 patients showed myeloid morphology and nine cases lymphoid morphology according to FAB criteria. There were no differences between lymphoid and myeloid BAL according to clinical and biological presentation and treatment outcome. We confirm the poor prognosis of BAL when compared to AML or ALL seen during the same period of time, in terms of complete remission (47%, 62% and 82% respectively, BAL v AML, NS and BAL v ALL, P  = 0.006) and 4-year overall survival (8.1%, 25.8% and 23.8% respectively, BAL v AML, P  = 0.05 and BAL v ALL, P  = 0.003). Comparing adult BAL patients with AML patients, we found an increase in poor prognostic factors: CD34⁺ phenotype (82% v 60% respectively, P  = 0.03), unfavourable karyotype (60% v 20%, P  < 0.0001) and Pgp over-expression by RT-PCR (0.705 v 0.107, P  < 0.0001) and flow cytometry (0.824 v 0.391, P  = 0.0001). MRP and LRP were not found to be poor prognostic factors. Comparing BAL patients with ALL patients, we found also an increase in poor prognostic factors: age (51 v 39, P  = 0.003) and CD34⁺ phenotype (82% v 50%, P  = 0.02). We conclude that BAL patients need a more aggressive treatment procedure, including high-dose AraC or the use of Pgp modulators for first-line therapy.     

The value of detecting surface and cytoplasmic antigens in acute myeloid leukaemia

The immunophenotype of leukaemia cells from 60 patients with acute myeloid leukaemia (AML) was analysed with the APAAP technique using a panel of anti-myeloid and lymphoid associated monoclonal antibodies (McAb). Cells from all cases, including three with negative cytochemical features, were labelled by at least one of the anti-myeloid McAb CD13, anti-myeloperoxidase (anti-Mpo), and/or CD14. The most sensitive marker was CD13, since it was positive in 90% of cases. In two out of three AML cases defined as M0-AML, CD13 was expressed in the cytoplasm but not on the membrane; in these three cases peroxidase (Mpo) was not detected by conventional cytochemistry, but could be demonstrated in all of them using the McAb anti-Mpo. The simultaneous expression of CD14 and CD68 McAb was often confined to the M4 and M5 FAB AML subtypes (92% cases) as compared to the others: M1, M2, M3 (18% cases). Lymphoid antigens were rarely positive (TdT+: 13%, CD7+: 15%, CD19+: 5%) and none of the AML cases were CD3+ or CD10+. By contrast, CD4 was expressed in blasts from 44% of cases and this was not restricted to AML with a monocytic component (M4, M5) but also found in other subtypes. There were no significant differences in the clinical or prognostic features according to the positivity or negativity with TdT and CD4. By contrast, expression of CD7 was associated with refractoriness to the treatment or short complete remission duration, although the number of patients is too small to draw firm conclusions. Our findings support the clinical and diagnostic relevance of immunophenotypic studies in AML.     

Effect of thrombopoietin on proliferation of blasts from CD7-positive acute myelogenous leukaemia

We investigated the effect of thrombopoietin (TPO) on the growth of leukaemic blasts from 30 acute myelogenous leukaemia (AML) patients according to the surface expression of CD7 and CD34: 10 patients were CD7 positive (CD7⁺), nine were CD7 negative/CD34⁺(CD7^?/CD34⁺) and the remaining 11 were CD7^?/CD34^?. Significant growth response of leukaemic blasts to TPO was observed in 10/10 CD7⁺, 5/9 CD7^?/CD34⁺ and 2/11 CD7^?/CD34^? AML cases using ³H-thymidine incorporation. Synergistic stimulatory effects of TPO with stem cell factor (SCF), interleukin-3 (IL-3), granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor were observed in both TPO-responding cases (9/17) and TPO-non-responding cases (8/13). In a leukaemic blast colony assay, significant growth response to TPO was observed in 5/6 CD7⁺ and 4/17 CD7^? AML cases examined. However, the effect of TPO on the growth of CD7⁺ leukaemic blasts was not so potent as that of IL-3 and SCF, both of which support the proliferation of primitive haemopoietic progenitors. Expression of c-mpl (TPO receptor) was significantly higher in CD7⁺ AML cases than in CD7^? cases, suggesting a relationship between expression of c-mpl and proliferative response to TPO. These data indicate that CD7⁺ leukaemic blasts express functional TPO receptors and proliferate in response to TPO. These observations also imply that CD7 expression on AML blasts may indicate involvement of leukaemic progenitors at an early stage of multipotent haemopoietic stem cells.     

Variability in responsiveness to lovastatin of the primitive CD34+ AML subfraction compared to normal CD34+ cells

In the present study, we questioned whether the cholesterol synthesis inhibitor lovastatin potentiates the cytotoxicity of chemotherapeutic agents in the primitive CD34⁺ subpopulation of acute myeloid leukemia (AML) cells. AML mononuclear cells (n = 17) were sorted in CD34⁺ and CD34⁻ fractions and compared to normal CD34^+/− cells (n = 7). The percentage of surviving cells upon exposure to lovastatin (25–100 μM) and/or chemotherapeutics (cytarabin or daunorubicin) was determined with a luminescent cell viability assay. The results demonstrate that the primitive CD34⁺ subpopulation of normal and AML cells displayed a higher sensitivity to lovastatin than the more mature CD34⁻ subpopulation. The combination of lovastatin and chemotherapeutics resulted in a more pronounced inhibitory effect on both subpopulations. In contrast to the homogeneous results in normal CD34⁺ cells, a distinct heterogeneity in lovastatin sensitivity was found in AML samples. Therefore, a group of normal (n = 11) and abnormal (n = 6) responders were identified based on a reduced or increased cell survival compared to normal CD34⁺ cells. This distinction was not only observed in the CD34⁺ AML subfraction but also in CD34⁺CD38⁻AML cells. In the abnormal responder group, 50% of patients presented with unfavorable cytogenetics and significant higher peripheral blast cell counts, which coincided with poor treatment results. In summary, the findings indicate that the primitive subfraction of CD34⁺ AML cells is in the majority of cases affected by lovastatin treatment, which is potentiated when combined with chemotherapeutics. Heterogeneity of the response observed in AML patients allowed identification of a subgroup with poor prognosis.     

Diagnostic application of monoclonal antibody KB90 (CD11c) in acute myeloid leukaemia

Summary The expression of membrane CD11c by leukaemic blast cells was examined (indirect immunorosetting) in 75 cases of acute leukaemia (myeloid, n=60; lymphoid, n=15) and evaluated as a potential marker for the diagnostic discrimination between monocytic (AMML-M4 and AMoL-M5) and non-monocytic (M1, M2 and M3) AML subtypes. Preliminary studies of normal bone marrow cells indicated that CD11c expression was not restricted to cells of monocytic lineage but was also present, with apparent lower density, on significant proportions of mature and immature granulocytes. Examination of acute myeloid leukaemia (AML) subtypes revealed that the non-monocytic leukaemias (n=33) were CD11c^–, defined as <30% positive cells, whereas all but one of the AMML-M4 (n=13) and AMoL-M5 (n=14) cases were CD11c⁺. All 15 cases of lymphoblastic leukaemia (ALL) showed <5% CD11c⁺ blasts. Membrane CD11c expression was also compared to the more widely used markers of monocytic differentiation; cytoplasmic alpha-naphthyl acetate esterase (ANAE) and membrane CD14 expression. This analysis showed that all 13 AMML-M4 leukaemias studied, including seven cases that were CD14^– and eight that were ANAE^–, were CD11c⁺. In addition, the AMoL-M5 cases (all of which were ANAE⁺) could be phenotypically subdivided into CD11c⁺ CD14⁺ (n=9), CD11c⁺ CD14^– (n=4) and CD11c^– CD14^– (n=1) subgroups. The study also confirmed that the discriminitive ability and sensitivity of the immunorosetting procedure for the detection of membrane CD11c compared favourably to immunofluorescent staining intensities as measured by flow cytometry.     

Down-regulation of bcl-2 in AML blasts by all-trans retinoic acid and its relationship to CD34 antigen expression

High levels of expression of the bcl-2 oncoprotein in acute myeloblastic leukaemia (AML) cells have been associated with low complete remission rates and poor survival. The sensitivity of AML blasts to drugs such as Ara-C can be increased by the down-regulation of bcl-2 expression by antisense oligonucleotides. All-trans retinoic acid (ATRA) has been reported to increase the sensitivity of AML cell lines to Ara-C and to induce differentiation in the HL60 promyelocytic cell line, with both effects being accompanied by a decrease in bcl-2 expression. Using flow cytometry and a monoclonal antibody to bcl-2, we have investigated the effects of ATRA (1 μM ) on bcl-2 expression in the blast cells of 25 AML patients and the K562 cell line after incubation for 72 or 24 h, respectively. Using Kolmogorov-Smirnov statistical analysis where a D value of > 0.12 was statistically significant, we found that in 8/25 AML samples and the K562 cells there was a significant decrease in bcl-2 protein expression after incubation with ATRA (D value range 0.14–0.44). The mean peak fluorescence (MPF) values for the bcl-2 levels of the ATRA responders (n = 8) was reduced to 35.5 ± 6.9 following incubation with ATRA compared to 47.6 ± 8.2 (mean ± SEM) for control samples incubated in the absence of ATRA (P = 0.014). There was no significant difference between the baseline bcl-2 molecules of equivalent soluble fluorochrome (MESF) levels in the ATRA responders (48.9 ± 5.7, n = 8) and the non-responders (41.3 ± 3.9, n = 17) (mean ± SEM) (P = 0.28). The down- regulation of bcl-2 expression by ATRA was particularly associated with CD34-negative AML and of the eight AML patients’ cells that responded to ATRA by down-regulating bcl-2, seven were CD34 negative (P<0.05). Our data suggest that the addition of ATRA to combination chemotherapy would increase the chemosensitivity of some patients with AML, particularly CD34-negative AML, due to down-regulation of bcl-2 expression.     

The phenotype of L-CFU and its correlation with the immunological characteristics of the blast cell population in AML

Summary 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.     

In vitro growth in acute myeloblastic leukaemia: relationship with other clinico-biological characteristics of the disease

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/10⁵ 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.     

Expression of the leucocyte common antigen (LCA,CD45) isoforms RA and RO in acute haematological malignancies: possible relevance in the definition of new overlap points between normal and leukaemic haemopoiesis

Summary. The membrane expression of CD45RA and CD45RO on fresh leukaemic cells taken from 529 cases of acute haemopoietic malignancies, including 117 B-origin acute lymphoblastic leukaemia (B-origin ALL), 3 7 T-origin acute lymphoblastic leukaemia (T-origin ALL), 297 de novo acute myeloid leukaemia (AML), 42 refractory anaemia with excess of blasts in transformation (RAEB-T) and 36 myeloid blastic phase of chronic myelogenous leukaemia (CML-BP-my), was analysed. B-origin ALLs were characterized by the lack of the RO isoform along with the consistent presence of RA. Conversely, a differential expression of the two isoforms was detected in different subsets of T-origin ALL, in that T-stem cell leukaemias (T-SCL: CD7⁺, CD4^?, CD8^?, CD1^?) preferentially expressed CD45RA whereas conventional T-acute lymphoblastic leukaemias (T-ALL: CD7⁺, CD4⁺ and/or CD8⁺ and/or CD1+) were consistently marked by CD45RO. Within myeloid malignancies, most of AMLs displayed CD45RA, while a substantial group of CML-BP-my preferentially exhibited CD45RO. As a general rule, a reciprocal exclusion of the two isoforms was observed in AML as well as in ALL. Nevertheless, a frequent coexpression of CD45RA and CD45RO was observed in CD14⁺ AML. In vitro treatment with all-trans retinoic acid (ATRA) was able to promote a switch from CD45RA to CD45RO expression in 2 7 de novo AML, independently from morphological subtyping. To our knowledge, this is the first report on CD45 isoform expression in a large series of patients with acute leukaemia. The knowledge of the differential expression of CD45RA and CD45RO can ameliorate our classificative approach to haematological malignancies, as well as disclose new multiple overlap points between normal and leukaemic cell differentiation.     

Targeting of acute myeloid leukaemia by cytokine‐induced killer cells redirected with a novel CD123‐specific chimeric antigen receptor

Current therapeutic regimens for acute myeloid leukaemia (AML) are still associated with high rates of relapse. Immunotherapy with T‐cells genetically modified to express chimeric antigen receptors (CARs) represents an innovative approach. Here we investigated the targeting of the interleukin three receptor alpha (IL3RA; CD123) molecule, which is overexpressed on AML bulk population, CD34⁺ leukaemia progenitors, and leukaemia stem cells (LSC) compared to normal haematopoietic stem/progenitor cells (HSPCs), and whose overexpression is associated with poor prognosis. Cytokine‐induced killer (CIK) cells were transduced with SFG‐retroviral‐vector encoding an anti‐CD123 CAR. Transduced cells were able to strongly kill CD123⁺ cell lines, as well as primary AML blasts. Interestingly, secondary colony experiments demonstrated that anti‐CD123.CAR preserved in vitro HSPCs, in contrast to a previously generated anti‐CD33.CAR, while keeping an identical cytotoxicity profile towards AML. Furthermore, limited killing of normal monocytes and CD123‐low‐expressing endothelial cells was noted, thus indicating a low toxicity profile of the anti‐CD123.CAR. Taken together, our results indicate that CD123‐specific CARs strongly enhance anti‐AML CIK functions, while sparing HSPCs and normal low‐expressing antigen cells, paving the way to develop novel immunotherapy approaches for AML treatment.     

Prognostic importance of immunophenotyping in adults with acute myelocytic leukaemia: the significance of the stem-cell glycoprotein CD34 (My 10)

A series of 23 monoclonal antibodies reactive with normal lymphoid and myeloid cells at various stages of differentiation were used to characterize 96 adult patients with acute myelocytic leukaemia (AML), concentrating on the possible role the expression of these antigens may have in predicting response to intensive chemotherapy. Only the expression of CD34 (P = 0.008) and HLA-DR (P = 0.035) was significant in predicting response to therapy; patients with leukaemic cells expressing CD34 (My10) had a complete remission (CR) rate of 59% compared to 87% for those with blasts not expressing the antigen. In a multivariate analysis predicting for CR, the expression of CD34, the disease category (de novo AML versus secondary AML [SAML] or a history of antecedent haematological disorder [AHD]), and WBC were significant covariates. Adjusting for disease category and WBC, patients with CD34-positive AML were one-third as likely to enter CR as with those with disease not expressing the antigen (P = 0.066). Comparison of clinical characteristics between the 58 patients whose leukaemia expressed CD34 and the 33 which were CD34-negative found that patients with CD34-positive AML had a higher incidence of SAML and AHD, a lower WBC at diagnosis, and a more frequent incidence of chromosomal abnormalities involving chromosomes 5 and/or 7. Twenty-eight of these patients also had immunophenotyping performed at relapse. Patients who presented with CD34-positive AML, and entered remission, and then relapsed all recurred with CD34-positive leukaemia; there was no case of CD34-positive AML at diagnosis relapsing with CD34-negative disease. In addition, there were patients presenting with CD34-negative AML and then relapsing with CD34-positive AML. These results suggest that intensive cytoreductive therapy is ineffective against CD34-positive AML. Patients who present with CD34-positive AML may require different therapeutic approaches to completely eradicate their disease.     

Early expression of MCS2 (CD13) in the cytoplasm of blast cells from acute myeloid leukaemia

The expression of two myeloid antigens identified by the monoclonal antibodies (McAb) MCS2 (CD13) and MY9 (CD33) was investigated in 136 cases of leukaemia. MCS2 was positive in blast cells of 78 of 88 (88.5%) and MY9 in 51 of 81 (64%) cases of acute myeloid leukaemia (AML) and chronic granulocytic leukaemia in myeloid blast crisis. One or other McAb, or both, were positive in all but 2 (2.3%) of these cases. MCS2 was more sensitive than MY9 to detect blasts of the myeloid lineage due to its most frequent reactivity in the cytoplasm of fixed cells by the immunoperoxidase (IP) technique compared with its membrane expression on cell suspensions by immunofluorescence (IF). MY9 was not suitable for tests on fixed cells. MCS2 was positive by IP but not by IF in 24% of AML, but the reverse was not observed. This suggests that the antigen detected by MCS2 is expressed in myeloblasts first in the cytoplasm and later on the cell membrane, pattern which is similar to that of the early antigens CD3 and CD22 in T and B lineage lymphoblasts, respectively. MCS2 was always positive in FAB types of AML-involving myeloblasts (M1-M4), including cases of undifferentiated morphology (M0), whilst MY9 was more frequently positive in monocytic leukaemia (M5). On the other hand, MCS2 was positive in 4 of 33 cases of acute lymphoblastic leukaemia and MY9 in 1. We conclude that both McAb, particularly MCS2, contribute to the better characterisation of myeloid leukaemias but that other tests are required to clarify the nature of the blasts when unexpected reactivities are observed.     

Haemopoietic defect and decreased expansion potential of bone marrow autografts from patients with acute myeloid leukaemia in first remission

Autologous bone marrow (BM) transplantation for acute myeloid leukaemia (AML) in complete remission (CR) is frequently followed by a slow haemopoietic recovery. We assessed the haemopoietic capacity of purified BM stem cell (CD34⁺DR^?) and progenitor cell (CD34⁺DR⁺) populations from patients with AML in CR, and compared these data with those of normal BM. The feasibility of ex vivo expansion in stroma-conditioned medium supplemented with cytokines was also investigated. The number of CFU-GM produced by initial patient CD34⁺DR^? cells was decreased compared to normal, whereas these values were similar to normal for CD34⁺DR⁺ cells. BFU-E, HPP-CFC and LTC-IC were reduced for both patient CD34⁺DR^? and CD34⁺DR⁺ subpopulations. In contrast to normal, the patient CD34⁺DR^? fraction was not enriched in LTC-IC. CFU-GM expansion from patient CD34⁺DR^? cells was poor and decreased after 14 d of culture. No HPP-CFC expansion could be observed for patient cells. LTC-IC were below the level of detection after 14–21 d of expansion culture of CD34⁺DR^? patient cells, whereas they were variably maintained or expanded for normal cells. After expansion culture, cytogenetic and/or FISH analyses did not reveal the anomalies present at diagnosis, regardless of the cell subpopulation analysed. In conclusion, BM cells of patients with AML in CR show a profound defect at the level of a stem cell enriched population. No meaningful ex vivo expansion could be obtained in culture conditions allowing for a significant expansion from a normal stem cell population.     

Expression of co-stimulatory and adhesion molecules and chemokine or apoptosis receptors on acute myeloid leukaemia: high CD40 and CD11a expression correlates with poor prognosis

The expression of adhesion and co-stimulatory molecules, and chemokine and death receptors such as tumour necrosis factor (TNF) and FAS on acute myeloid leukaemia (AML) may influence the biology of the disease and response to chemotherapy and immunotherapy. In this study, we analysed the expression of these molecules in 99 AML patients using monoclonal antibodies and flow cytometry, and correlated the expression with French-American-British (FAB) classification and survival. The following molecules were studied: the co-stimulatory molecules CD80, CD86 and CD40; the adhesion molecules CD11a-c, CD31, CD43, CD50, CD54, CD102, CD58 and CD62L; the chemokine receptor CXCR4; and the death receptors TNFR1 and TNFR2 and FAS. The expression of all molecules was significantly higher in the M4/M5 FAB subgroups except for CD80, CD43, CD54 and CD62L. The AML M3 subgroup had a significant lower expression of CD11a (P = 0.02) and CD11c (P = 0.03). Five-year survival was significantly shorter in cases of high CD40 expression [> 20% positive cells, relative risk (RR) 2.56, P = 0.02] or high CD11a expression (> 80% positive cells, RR 2.6, P = 0.03). This effect was most prominently present in the AML M4/M5 FAB subgroups. We conclude that the expression levels of adhesion and co-stimulatory molecules, CXCR4 and apoptosis-receptors are predominantly FAB subtype-related with high CD40 and CD11a expression as poor prognostic factors.     

Unravelling the relevance of CLEC12A as a cancer stem cell marker in myelodysplastic syndrome

Evidence of distinct disease propagating stem cells in myelodysplastic syndrome (MDS) has emerged in recent years. However, immunophenotypic characterization of these cancer stem cells remains sparse. In acute myeloid leukaemia (AML), we have previously described aberrant expression of the C‐type lectin domain family 12, member A (CLEC12A) as a stable and reliable marker of leukaemia blasts and as a tool for assessing minimal residual disease. Furthermore, CLEC12A has been proposed as a promising marker of leukaemic stem cells in AML. The role of CLEC12A in MDS, however, remains to be elucidated. In this study, we found CLEC12A aberrantly expressed on the CD34⁺CD38^? cell compartment in 71% (22/31) of MDS patients, distributed across all Revised International Prognostic Scoring System risk groups. We showed that the CD34⁺CD38^?CLEC12A⁺ cells were indeed malignant and possessed functional stem cell properties in the long‐term colony‐initiating cell assay. As opposed to reported findings in AML, we showed that cancer stem cells from MDS samples derived from both CLEC12A positive and negative CD34⁺CD38^? subpopulations. Due to the absence of CLEC12A on normal haematopoietic stem cells, CLEC12A stem cell immunophenotyping may contribute to diagnosing and monitoring MDS patients and could furthermore add knowledge about disease propagating cells in MDS.