Flow cytometric assessment of CD15+CD117+ cells for the detection of minimal residual disease in adult acute myeloid leukaemia

There is little information available regarding immunophenotypic monitoring of minimal residual disease (MRD) in acute myeloid leukaemia (AML). We investigated leukaemic cells co‐expressing CD15 and CD117 (CD15+CD117+) in 72 adult AML cases at diagnosis. In 22 cases (31%) with various AML subtypes, more than 5% of leukaemic cells showed the CD15+CD117+ phenotype (range 5.22–55.48%). These 22 cases were younger and had a higher complete remission (CR) rate than the other AML cases, but the CD15+CD117+ cell percentage at diagnosis showed no correlation with the CR duration among the 72 cases. The CD15+CD117+ cell percentage showed a range of 0.00–0.08% in bone marrow cells from 10 haematologically normal subjects. We also investigated CD15+CD117+ cells in sequential bone marrow samples from 17 AML patients who achieved CR and who had had more than 5% CD15+CD117+ leukaemic cells at diagnosis. Because the CD15+CD117+ cell percentage varied among these AML cases, we calculated the percentage of MRD {MRD% = [CD15+CD117+ cells (%) in each sequential marrow sample] ÷ [CD15+CD117+ cells (%) at diagnosis of the corresponding case] × 100}. A high MRD% after 10 months of CR was significantly associated with a short CR duration (P = 0.0004), whereas continuation of a well‐reduced MRD% was associated with a long CR duration. The leukaemic cells conserved the CD15+CD117+ phenotype in all of the eight cases who relapsed. Flow cytometric monitoring of CD15+CD117+ cells is simple and can be applied to a substantial fraction of AML cases. This monitoring may be useful for predicting relapse of adult AML.     

Identification of the CD33-related Siglec receptor, Siglec-5 (CD170), as a useful marker in both normal myelopoiesis and acute myeloid leukaemias

Sialic acid-binding immunoglobulin-like lectin (Siglec)-5 or CD170 is a CD33-related receptor, containing cytoplasmic immune receptor-based tyrosine signalling motifs, that has previously been reported to be myeloid-specific like CD33 and thus may be useful in the characterization of both normal and malignant haemopoiesis. This study showed that Siglec-5 had a distinct expression pattern to CD33 both on normal myeloid cells and in acute myeloid leukaemia (AML). In normal bone marrow and cord blood, myeloid cells predominantly expressed Siglec-5 at the later stages of granulocytic differentiation. Siglec-5 was not expressed at significant levels by CD34+ progenitors either from bone marrow or mobilized peripheral blood. During in vitro myeloid differentiation of cord blood purified CD34+ cells, Siglec-5 was upregulated later than CD33. Siglec-5 expression remained absent or very low on cultured CD34+ cells, unlike CD33, which was present on almost all CD34+ cells by day 4. However, analysis of blasts from 23 patients with AML revealed aberrant expression of Siglec-5 with CD34 in 50% (seven of 14) of patients with CD34+ AML; 61% (14 of 23) of AML cases were positive for Siglec-5 with an increased frequency in the French-American-British subtypes M3-5 (80%) compared with M0-2 (25%). All 13 acute lymphoblastic leukaemic (ALL) samples tested, including a CD33+ ALL, were Siglec-5 negative. These results support the further evaluation of Siglec-5 antibodies in the diagnosis and monitoring of AML.     

Clinical significance of residual disease during treatment in childhood acute myeloid leukaemia

In children with acute myeloid leukaemia (AML), morphological and karyotypic studies cannot precisely assess response to treatment, and less than one-third of patients have genetic markers for molecular studies of residual disease. We determined the usefulness of a four-colour flow cytometric strategy developed in our laboratory to study residual disease. We first compared the immunophenotypes of AML cells obtained from 54 children at diagnosis with those of cells from 59 normal or regenerating bone marrow samples. Forty-six of the 54 AML cases (85.2%) had immunophenotypes that allowed detection of 0.1-0.01% residual leukaemic cells. Of 230 bone marrow samples obtained from those 46 patients during and off treatment, 61 (26.5%) had >/= 0.1% AML cells by flow cytometry. We found that core binding factor-associated AML had a significantly better early treatment response. Mean (+/- standard error) 2-year survival estimate was 33.1 +/- 19.1% for patients with >/= 0.1% AML cells by flow cytometry after induction therapy, but 72.1 +/- 11.5% for those with < 0.1% AML cells (P = 0.022); overt recurrence of AML within the subsequent 6 months was significantly more likely in the former group. The assay described here holds promise for guiding the choice of post-remission treatment options in children with AML.     

Primitive acute myeloid leukemia cells with long-term proliferative ability in vitro and in vivo lack surface expression of c-kit (CD117)

A hierarchy of progenitor cells is thought to exist in human acute myeloid leukemia (AML), with only the most primitive cells capable of proliferating to maintain the malignant clone. To further characterize this AML cell hierarchy, we evaluated the coexpression of CD34 and c-kit (CD117) on cells that are capable of long-term proliferation in vitro and in vivo.AML cells were sorted for coexpression of CD34 and c-kit (CD117) using two c-kit monoclonal antibodies (mAbs), clones 95C3 and 104D2. Sorted subfractions were evaluated for the ability to produce colony-forming units (CFU) for up to 8 weeks in suspension culture (SC) and for the capacity to repopulate NOD/SCID mice.When expression of c-kit on blood cells from 19 AML patients at diagnosis was compared using both mAbs, expression defined by 104D2 (34% +/- 6% c-kit(+)) was somewhat higher than that defined using 95C3 (18% +/- 4%). AML cells were sorted for coexpression of CD34 and c-kit using both c-kit mAbs, and the subfractions were assayed in vitro and in vivo. Whereas the majority of AML blast cells lacked expression of CD34, most AML cells capable of proliferating to produce CFU after 4 to 8 weeks in SC were CD34(+)/c-kit(-). Cultures of sorted CD34(+)/c-kit(-) cells, supplemented with steel factor, were composed of a large proportion (18% to 87%) of CD34(+)/c-kit(+) cells after 1 week, suggesting that either c-kit expression was upregulated or CD34(+)/c-kit(+) cells were produced. Moreover, the CD34(+)/c-kit(-) subfraction was found to be capable of responding to steel factor alone to produce CFU after 4 weeks in SC. In most AML patients tested (11/15), the only sorted subfraction capable of engrafting NOD/SCID mice was CD34(+)/c-kit(-). The CD34(+)/c-kit(+) subfraction from only 2 of the 15 patients and CD34(-) cells from 3 patients also engrafted the NOD/SCIDs. Only the CD34(+)/c-kit(+) subfraction of normal bone marrow engrafted.These studies suggest that primitive AML cells capable of long-term proliferation in vitro and NOD/SCID repopulation differ from primitive normal progenitor cells in their lack of surface expression of c-kit.     

Oral azacitidine modulates the bone marrow microenvironment in patients with acute myeloid leukaemia in remission: A subanalysis from the QUAZAR AML-001 trial

Oral azacitidine (Oral-AZA) maintenance therapy improved relapse-free (RFS) and overall survival (OS) significantly versus placebo for AML patients in remission after intensive chemotherapy (IC) in the phase 3 QUAZAR AML-001 study. Immune profiling was performed on the bone marrow (BM) at remission and on-treatment in a subset of patients with the aim of identifying prognostic immune features and evaluating associations of on-treatment immune effects by Oral-AZA with clinical outcomes. Post-IC, increased levels of lymphocytes, monocytes, T cells and CD34 + CD117+ BM cells were prognostically favourable for RFS. CD3+ T-cell counts were significantly prognostic for RFS in both treatment arms. At baseline, high expression of the PD-L1 checkpoint marker was identified on a subset of CD34 + CD117+ BM cells; many of which were PD-L2+. High co-expression of T-cell exhaustion markers PD-1 and TIM-3 was associated with inferior outcomes. Oral-AZA augmented T-cell numbers during early treatment, increased CD4+:CD8+ ratios and reversed T-cell exhaustion. Unsupervised clustering analysis identified two patient subsets defined by T-cell content and expression of T-cell exhaustion markers that were enriched for MRD negativity. These results indicate that Oral-AZA modulates T-cell activity in the maintenance setting of AML, and these immune-mediated responses are associated with clinical outcomes.     

Lack of expression of the chondroitin sulphate proteoglycan neuron-glial antigen 2 on candidate stem cell populations in paediatric acute myeloid leukaemia/abn(11q23) and acute lymphoblastic leukaemia/t(4;11)

It has increasingly been acknowledged that only a few leukaemic cells possess the capability to renew themselves and that only these self-renewing leukaemic stem cells are able to initiate relapses. Therefore, these leukaemic stem cells should be the target cells for therapy and for minimal residual disease (MRD) detection. Because of its presence on blasts of 11q23-rearranged high-risk leukaemic patients, neuron-glial antigen 2 (NG2) is thought to be a valuable marker for detecting leukaemic stem cells. Six acute myeloid leukaemia (AML)/abn(11q23) and three acute lymphoblastic leukaemia (ALL)/t(4;11) samples were analysed by four-colour flow cytometry for NG2 expression on primitive cell populations. Candidate leukaemic cell populations were defined by the antigen profiles CD34+CD38- in AML and CD34+CD19-CD117+ in ALL. Surprisingly, in all patients these candidate stem cell populations were shown to lack expression of NG2. Instead, a correlation between the expression of the myeloid differentiation marker CD33 and increasing levels of NG2 on maturing cells could be demonstrated. Similarly, in ALL patients CD34+CD19+ cells showed a higher expression of NG2 mRNA compared with CD34+CD19-. Thus, NG2 appears to be upregulated with differentiation and not to be expressed on primitive disease-maintaining cells. This hampers the clinical use of NG2 as a therapeutic target and as a sensitive marker for MRD detection.     

CD117 expression in gammopathies is associated with an altered maturation of the myeloid and lymphoid hematopoietic cell compartments and favorable disease features

Aberrant CD117 expression is associated with a favorable outcome in multiple myeloma. We analyzed 106 patients with symptomatic multiple myeloma (n=50), smoldering multiple myeloma (n=38) and monoclonal gammopathy of undetermined significance (n=18) to elucidate biological features of CD117⁺ versus CD117⁻ monoclonal gammopathies. CD117⁺ (mono)clonal plasma cells were detected in 30% symptomatic multiple myeloma, 45% smoldering multiple myeloma and 72% monoclonal gammopathy of undetermined significance patients. CD117 expression was associated with higher percentages of normal bone marrow plasma cells, CD117⁺ myeloid precursors and CD38⁺ B lymphocytes in all monoclonal gammopathies. Conversely, the number of bone marrow CD34⁺ myeloid cells and peripheral blood neutrophils was reduced among CD117⁺ multiple myeloma but not monoclonal gammopathy of undetermined significance patients.CD117 expression by (mono)clonal plasma cells is associated with uniquely altered patterns of production of hematopoietic bone marrow cells with decreased peripheral blood neutrophil counts and persistence of normal residual bone marrow plasma cells.     

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

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

Identification of P-selectin glycoprotein ligand-1 as a useful marker in acute myeloid leukaemias

Immunophenotyping is considered to be less valuable in the diagnosis of acute myeloid leukaemias (AML) compared with acute lymphoid leukaemias. Here, we present data on the use of quantitative flow cytometry (QFC) of P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and three-colour immunophenotyping including CD162 staining in the identification of myeloid precursors in AML. Analysis of normal peripheral blood (n = 20) and normal bone marrow (n = 5) samples and on 20 samples from de novo M1, M2, M4 and M5 AML patients demonstrated that PSGL-1 is differentially expressed on various mature and immature leucocyte subsets. It was found by QFC that neutrophils expressed 26500 +/- 4500 and monocytes 47200 +/- 9900 copies of PSGL-1 on their surface, whereas AML blasts from M1 and M2 AML patients expressed significantly less PSGL-1 (12 000 +/- 5300) than mature neutrophils (P < 0.001). In M4 and M5 leukaemias, however, the amount of PSGL-1 on monocytic precursors is displayed in a fairly broad range that is not significantly different from that of mature monocytes (P = 0.084). Using three-colour immunophenotyping PSGL-1-dim staining was co-expressed with CD7 and C34 positivity and PSGL-1 staining intensity on immature myeloid cells paralleled with CD45 expression. This would imply a differential expression of PSGL-1 during myeloid haematopoietic development and suggests that quantification of surface PSGL-1 may aid in differentiating myeloblasts from monoblasts by immunophenotyping in different AML subsets.     

CXCR4 chemokine receptors (CD184) and alpha4beta1 integrins mediate spontaneous migration of human CD34+ progenitors and acute myeloid leukaemia cells beneath marrow stromal cells (pseudoemperipolesis)

Marrow stromal cells play an important role in regulating the development and proliferation of haematopoietic stem cells (HSC) within the marrow microenvironment. However, the molecular mechanisms of stem cell-stromal cell interactions are not fully understood. We observed that mobilized peripheral blood and cord-blood-derived CD34+ progenitor cells, or CD34+ acute myeloid leukaemia (AML) cells spontaneously migrated beneath marrow stromal cells, an in vitro migration phenomenon termed pseudoemperipolesis. In contrast, the CD34+ myeloid leukaemia cell line, Kasumi-1, did not display pseudoemperipolesis. Cord blood CD34+ cells had a higher capacity than granulocyte-colony-stimulating-factor-mobilized CD34+ cells for pseudoemperipolesis (28.7 +/- 12%vs 18.1 +/- 6.1% of input cells within 24 h, mean +/- SD, n = 8), whereas 9.4 +/- 12.6% (mean +/- SD, n = 10) of input AML cells displayed this phenomenon. Pseudoemperipolesis of CD34+ progenitor and AML cells was significantly inhibited by pertussis toxin and antibodies to the CXCR4 chemokine receptor (CXCR4, CD184), but not control antibodies. Moreover, CD34+ and AML cell migration was significantly inhibited by a CS1 peptide that blocks alpha4beta1 integrin binding, but not by a control peptide, in which the fibronectin binding motif was scrambled. Pseudoemperipolesis was associated with an increased proliferation of migrated CD34+ progenitor cells but not AML cells within the stromal layer, demonstrated by cell cycle analysis and cell division tracking. We conclude that alpha4beta1 integrin binding and CXCR4 chemokine receptor activation are prerequisites for the migration of CD34+ haematopoietic progenitors and AML cells beneath marrow stromal cells. These observations suggest a central role of marrow stromal cells for HSC trafficking and homing within the marrow microenvironment.     

Four-fold staining including CD45 gating improves the sensitivity of multiparameter flow cytometric assessment of minimal residual disease in patients with acute myeloid leukemia

Multiparameter flow cytometry (MFC) is capable of quantifying minimal residual disease (MRD) in acute myeloid leukemia (AML). Its broad application, however, is limited by a lack of sensitivity in about 20% of patients. CD45 gating may improve sensitivity. A broad panel of four-fold combinations of monoclonal antibodies including CD45 in each was used to define leukemia-associated aberrant immunophenotypes (LAIP), to define their sensitivities in normal bone marrow samples, and to compare results to data obtained without CD45 gating using triple staining. In 45 patients, a LAIP was defined, 11 normal bone marrow samples were analyzed as controls. The median percentage of LAIP-positive AML cells with and without CD45 gating was 21.95% (range, 3.31-82.52%) and 20.52% (range, 3.22-81.94%). The median percentage of LAIP-positive normal bone marrow cells ranged from 0.01 to 0.42% (median, 0.02%) and 0.02 to 0.58% (median, 0.15%) with and without CD45 gating. The difference of LAIP-positive cells between AML and normal bone marrow samples amounted to a median of 3.08 log (range, 1.22-4.01) and 2.28 log (range, 1.12-3.34) with and without CD45 gating. CD45 gating improves the sensitivity of MFC-based MRD monitoring in AML by 1 log.     

CD34-expressing human thymocyte precursors proliferate in response to interleukin-7 but have lost myeloid differentiation potential

CD34 is a marker for pluripotent stem cells also present on lineage- committed hematopoietic progenitors from bone marrow and a subpopulation of immature thymocytes. To characterize these early immature thymocytes, we have studied 24 pediatric thymus samples for CD34/7 expression. Three subpopulations could be defined from these T- cell receptor (TcR-) immature thymocytes: CD34+7++ (12.0 +/- 5.8), CD34- 7++ (12.6 +/- 8.6), and CD34-7+ (71.5 +/- 17.0%). CD7++ represents upregulation of this antigen and is expressed by cells of a blast-like morphology. Three-color flow cytometric analysis of these three subsets suggests the following ordered differentiation sequence: CD34+7++1-4-8- 45RA+-->CD34+7++1+ 4+8-45RA+/- -->CD34-7++1+4+8-+45RO+-->CD34- 7+1++4+8+45RO+. Early immature thymocyte cell division is essential in the thymus to generate a large number of precursors before the initiation of the selection process. We observed that both CD2 as well CD28 activation pathways were inefficient to serve as costimulant with phorbol ester 12-O-tetradecanoyl phorbol 13-acetate or interleukin-2 (IL-2) to induce the proliferation of the three CD34/7 subsets isolated by cell sorting. However, whereas IL-1, IL-2, IL-3, IL-4, granulocyte colony-stimulating factor, and granulocyte-macrophage colony- stimulating factor were ineffective, IL-7 was a potent cytokine, alone or in synergy with stem cell factor (SCF) to induce immature thymocyte proliferation. The proliferation induced by IL-7 or IL-7 + SCF is restricted to the CD34+ cells and, after 4 or 8 days of culture with IL- 7, some CD34+7++ acquire the expression of CD4 and/or CD8, but remain CD3/TcR-. We also tested the myeloid differentiation capacity of these CD34 immature thymocytes. Using two different approaches, myeloid colony formation in methylcellulose and limiting dilution analysis in the presence of myeloid growth factors, we were unable to detect myeloid differentiation capacity from CD34+ early thymocytes, whereas CD34+7+ from bone marrow contained about 10% of the clonogenic cells present in the CD34+7- fraction. Together, these data support the concept that thymic CD34+7++ represents the earliest thymic subset of fully committed T-lineage cells, capable of proliferating specifically to IL-7.     

Exclusive expression of G-CSF receptor on myeloid progenitors in bone marrow CD34+ cells

Although granulocyte colony-stimulating factor (G-CSF) has been reported to act on cells of neutrophilic lineage, the administration of G-CSF to induce the mobilization of various haematopoietic progenitors into the circulation. We analysed the expression of receptors for G-CSF (G-CSFR) on human bone marrow and G-CSF-mobilized peripheral blood CD34+ cells, and examined the proliferation and differentiation capabilities of sorted CD34+G-CSFR+ and CD34+G-CSFR- cells using methylcellulose clonal culture. Flow cytometric analysis showed that G-CSFR was expressed on 14.9 +/- 4.9% of bone marrow CD34+ cells, most of which were included in CD34+CD33+ and CD34+CD38+ cell fractions. In clonal cultures, CD34+G-CSFR+ cells produced only myeloid colonies, whereas CD34+G-CSFR- cells produced erythroid bursts, megakaryocyte and multilineage colonies. When incubated with the cytokine cocktail for 5 d, CD34+G-CSFR- cells generated CD34+G-CSFR+ myeloid progenitors. In G-CSF-mobilized peripheral blood, CD34+ cells contained 10.8 +/- 5.8% of G-CSFR+ cells, most of which were also myeloid progenitors, although CD34+G-CSFR- cells contained a substantial number of myeloid progenitors. These results indicated that the expression of G-CSFR on CD34+ cells is restricted to myeloid progenitors, suggesting that the specific activity of G-CSF on myelopoiesis depends on the exclusive expression of its receptor on myeloid progenitors, and that the mobilization of various haematopoietic progenitors is not a direct effect of G-CSF in humans.     

CD56 and PGP expression in acute myeloid leukemia: impact on clinical outcome

BACKGROUND AND OBJECTIVES: Overexpression of P-glycoprotein (PGP), a multidrug-related (MDR) protein, is one of the most important factors responsible for reduced drug sensitivity in acute myeloid leukemia (AML). Recently, we demonstrated that the presence of CD56 antigen, an isoform of the neural adhesion molecule, in AML cells is a negative independent prognostic factor for the achievement of complete remission (CR) and correlates with shorter survival. Since in our previous report we observed a more frequent PGP expression in CD56+ patients, we hypothesized that the reduced response to chemotherapy in this group of patients was due to increased PGP-mediated drug efflux. To confirm this hypothesis in this study PGP and CD56 expression on AML cells was correlated with other clinical and biological features and treatment response. DESIGN AND METHODS: Immunophenotypic analysis, including evaluation of CD56 and PGP expression, was performed using multiparameter flow cytometry on fresh and/or cryopreserved blast cells, obtained after informed consent, from bone marrow and/or peripheral blood of 143 consecutive newly diagnosed AML cases at the time of diagnosis. Samples expressing CD56 in at least 15% or more cells were considered as positive (CD56+). PGP expression was expressed as a mean fluorescence index (MFI) i.e. as the ratio of sample mean fluorescence channel and the isotypic control mean fluorescence channel. RESULTS: Overall results showed that 67/143 cases were PGP-/CD56-, 23/143 were PGP+ /CD56+, 40/143 were PGP+/CD56- and the remaining 13/143 were PGP-/CD56+. CD56+ and PGP+ on AML cells significantly reduced the CR rate (83% in the PGP-/CD56- group vs 60% in the PGP-/CD56+ group, 46% in the PGP+/CD56- group and 58% in the PGP+/CD56+ group, p = 0.002). In addition we observed a significantly higher proportion of total failures in patients expressing PGP or CD56 compared to in the group not expressing either (73% vs 27%, respectively; p = 0.0001). CD56 and PGP overexpression influenced the overall survival: in fact, the median survival of CD56+ and PGP+ patients ranged from 10 to 23 months, while the actuarial survival of CD56-/PGP- patients at 5 years is 52% (p = 0.023). INTERPRETATION AND CONCLUSIONS: Our data underline the independent negative prognostic role of PGP and CD56 expression in acute myeloid leukemia. Since the mechanism by which CD56 reduces drug sensitivity is still unknown, further investigations are required.     

The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells

In CD34(+) acute myeloid leukemia (AML), the malignant stem cells reside in the CD38(-) compartment. We have shown before that the frequency of such CD34(+)CD38(-) cells at diagnosis correlates with minimal residual disease (MRD) frequency after chemotherapy and with survival. Specific targeting of CD34(+)CD38(-) cells might thus offer therapeutic options. Previously, we found that C-type lectin-like molecule-1 (CLL-1) has high expression on the whole blast compartment in the majority of AML cases. We now show that CLL-1 expression is also present on the CD34(+)CD38(-) stem- cell compartment in AML (77/89 patients). The CD34(+)CLL-1(+) population, containing the CD34(+)CD38(-)CLL-1(+) cells, does engraft in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with outgrowth to CLL-1(+) blasts. CLL-1 expression was not different between diagnosis and relapse (n = 9). In remission, both CLL-1(-) normal and CLL-1(+) malignant CD34(+)CD38(-) cells were present. A high CLL-1(+) fraction was associated with quick relapse. CLL-1 expression is completely absent both on CD34(+)CD38(-) cells in normal (n = 11) and in regenerating bone marrow controls (n = 6). This AML stem-cell specificity of the anti-CLL-1 antibody under all conditions of disease and the leukemia-initiating properties of CD34(+)CLL-1(+) cells indicate that anti-CLL-1 antibody enables both AML-specific stem-cell detection and possibly antigen-targeting in future.     

hMICL and CD123 in combination with a CD45/CD34/CD117 backbone – a universal marker combination for the detection of minimal residual disease in acute myeloid leukaemia

Real‐time quantitative polymerase chain reaction (qPCR) has been extensively validated for the detection of minimal residual disease (MRD) in acute myeloid leukaemia (AML). Meanwhile, multicolour flow cytometry (MFC) has received less attention because the so‐called leukaemia‐associated immunophenotypes (LAIPs) are generally of lower sensitivity and specificity, and prone to change during therapy. To improve MRD assessment by MFC, we here evaluate the combination of human Myeloid Inhibitory C‐type Lectin (hMICL, also termed C‐type lectin domain family 12, member A, CLEC12A) and CD 123 (also termed interleukin‐3 receptor alpha, IL3RA) in combination with CD34 and CD117 (KIT), as an MRD assay in pre‐clinical and clinical testing in 69 AML patients. Spiking experiments revealed that the assay could detect MRD down to 10^?4 in normal bone marrow with sensitivities equalling those of validated qPCR assays. Moreover, it provided at least one MFC MRD marker in 62/69 patients (90%). High levels of hMICL/CD123 LAIPs at the post‐induction time‐point were a strong prognostic marker for relapse in patients in haematological complete remission (P < 0·001). Finally, in post induction samples, hMICL/CD123 LAIPs were strongly correlated (r = 0·676, P = 0·0008) to applied qPCR targets. We conclude the hMICL/CD123‐based MFC assay is a promising MRD tool in AML.     

Bcl-2 expression in acute myelogenous leukemia: the relation to myeloid antigen expression and response to therapy in Iranian patients

Product of Bcl-2 gene prolongs survival of hematopoietic cells by inhibition of programmed cell death. The aim of this study is to examine the expression of the bcl-2 protein in a group of patients with AML and its relation to clinical features and response to therapy. Slides from the bone marrow or peripheral blood of 70 patients with AML were assessed for the expression of bcl-2 by immunocytochemistry. The expression of myeloid and non-lineage associated markers was detected by indirect immunofluorescence method. Correlation between bcl-2 and markers expression and patients characteristics was determined. More than 20% positivity for bcl-2 was found in 22 (31.4%) patients. Bcl-2 expression showed an association with M4 and M5 subtypes (p<0.01) and was correlated with clinical parameters including WBC and platelet count, extramedullary disease and Hb level. Bcl-2 expressing cells were significantly higher in CD15(+) and CD13(+) patients and lower in CD11b(+) and CD33(+) cases (p<0.001). Complete remission (CR) rate was significantly lower in cases with 20% or more bcl-2 positivity than others (24.4% v 75.6%). A shorter CR duration was observed in bcl-2+ patients when compared with bcl-2- ones (571+/-50 versus 850+/-17 days)(p=0.0001).The expression of bcl-2 was also associated with shorter survival (p=0.0001). Survival time for bcl-2+ patients was 831+/-44 days versus 1119+/-17 days for bcl-2- ones. CD11b and CD33 positivity was associated with longer survival whereas CD13 and CD15 positivity was correlated with lower survival (p<0.007). In multivariate analysis bcl-2 positivity was associated with poor survival. Bcl-2 expression showed a prognostic value in our patients indicating that even despite of some differences in treatment regimen, immunocytochemical analysis of this marker is still a simple and inexpensive method for evaluation of prognosis in AML patients. Bcl-2 expression may be related to the expression of differentiation associated markers.     

Different expression of adhesion molecules on CD34+ cells in AML and B-lineage ALL and their normal bone marrow counterparts.

The expression of adhesion molecules on CD34+ cells in acute myeloid leukemia (AML) and B-lineage acute lymphoblastic leukemia (B-lineage ALL) was compared with that on the myeloid and B-lymphoid CD34+ cells in normal bone marrow. Bone marrow aspirates of 10 patients with AML, 8 patients with B-lineage ALL and of 6 healthy volunteers were examined. The phenotype of the CD34+ cells was determined with a double immunofluorescence method and flow cytometry. CD34+ cells in AML and B-lineage ALL showed a lower expression of VLA-2 and VLA-3 and a higher expression of ICAM-1 and LFA-3 than their normal bone marrow counterparts. AML CD34+ cells had less L-selectin but more VLA-5 on their surface membrane than normal myeloid CD34+ cells. B-lineage ALL CD34+ cells showed an overexpression of LFA-3. In individual patients deficiencies or over-expression of the beta1 integrin chain, VLA-4, PECAM-1 or HCAM also occurred. An abnormal adhesive capacity of the leukemic cells may influence their proliferation, their localisation and apoptosis. An aberrant expression of adhesion molecules may be used for the detection of minimal residual leukemia in these patients.