L. Del Vecchio

All-Trans Retinoic Acid (ATRA) and the Regulation of Adhesion Molecules in Acute Myeloid Leukemia

A review of recent information on the expression and the ATRA-driven modulation of cell surface adhesion molecules of acute myelogenous leukemia blast cells is presented. Cytofluorometric studies on fresh blast cells have demonstrated that CD11a, CD11b CD11c, CD15, CD45RO and CD54 expression is significantly lower in acute promyelocytic leukemia (APL) than is acute myeloid leukemia of other subtypes (AML). In vitro treatment with ATRA dramatically modifies the adhesion phenotype of APL blast cells, promoting a consistently striking up-regulation of CD11b, CD11c, CD15, CD65, CD54, and CD38. Which is in general, poorly demonstrable in AML. The behaviour of CD15s is variable and fully independent from CD15 and CD65 in induction experiments, suggesting a differential enzyme regulation within the selectin ligand system. ATRA is capable, in both APL and AML, of producing a switch from the high- (RA) to the low- (RO) molecular weight isoform of CD54, Moreover, treatment with this retinoid exerts a negative regulation of the membrane expression of CD49e, CD58 and CD11a in APL as well as in AML. Of particular interest is the fact that the negative effect on CD1 1a expression generates an asynchronous phenotype in APL (CD11a-, CD11b+, CD15+), undetectable on normal maturing myeloid cells. In the last part of this review the possible implications of adhesion molecule modulation in the pathogenesis of ATRA syndrome are discussed.

ALL-TRANS RETINOIC ACID PROMOTES A DIFFERENTIAL REGULATION OF ADHESION MOLECULES ON ACUTE MYELOID LEUKAEMIA BLAST CELLS

Summary. In the present study we investigated the membrance expression of selectin ligands (CD15/Lex, CDw65/VIM2, CD15s/sLex), β2 integrins (CD11a/LFA‐1, CD11b/Mac‐1) and CD45 phosphatase isoforms (CD45RA, CD45RO on leukaemic cells from 28 patients with acute myeloid malignancies cultured with and without all‐trans retinioc acid (ATRA). Within each adhesion system, ATRA was bale to differentially regulate distinct molecules. Furthermore, it was able to exert effects specific for acute promyelocytic leukaemia (APL) blast cells, as well as to induce a series of non‐cytotype‐restricted phenotypic changes. An impressive feature of ATRA induction was a simultaneous increase in the expression of CD15, CDw65 and CD11b on leukaemic promyelocytes. The sialylated antigen CD15s, however, Showed results independent from the other two carbohydrates (CD15 and CDw65), suggesting a differential enzymatic regulation within the selectin ligands system.

Critical role of multidimensional flow cytometry in detecting occult leptomeningeal disease in newly diagnosed aggressive B-cell lymphomas.

Among histological aggressive non-Hodgkin lymphomas (NHL), the overall risk of central nervous system (CNS) relapse is approximately 5%, a figure which is too low to offer prophylaxis to all patients. The aim of this work is to demonstrate the utility of flow cytometry (FCM) in detecting occult leptomeningeal disease in this subtype of NHL. We studied cerebrospinal fluid (CSF) involvement in 42 newly diagnosed aggressive NHL patients at risk for CNS involvement. We used multicolour FCM to detect CSF infiltrating neoplastic cells. Among the 42 patients studied, 11 had CSF involvement as detected by FCM. Of these, only four were also positive for conventional morphology (p=0.046). These results designate that FCM as the first choice technique in NHL CSF clinical cell analysis.

Flow cytometry analysis of acute promyelocytic leukemia : the power of 'surface hematology'

An article by Breccia et al.1 published on Leukemia highlighted one remarkable aspect of acute promyelocytic leukemia (APL): the development of thrombosis in patients treated with all-trans retinoic acid (ATRA) and chemotherapy is significantly associated with specific biological features. These characteristics included CD2 and/or CD15 expression, bcr3 promyelocytic leukemia (PML)/retinoic acid receptor (RAR) transcript type, FLT3 gene internal tandem duplications and elevated white blood cell (WBC) count. We were impressed by the strength that flow cytometry studies retain 20 years after the pioneer papers establishing the antigenic features consistently noted to characterize APL.2, 3 Starting from this article, this editorial aims to provide a series of considerations about the impact of flow cytometry on clinical hematology, with particular accent on APL.

Stem cell factor receptor (c‐kit, CD117) is expressed on blast cells from most immature types of acute myeloid malignancies but is also a characteristic of a subset of acute promyelocytic leukaemia

Investigating 208 patients with acute haematological malignancies, we found that stem cell factor receptor (SCFR) was expressed on high numbers of blast cells from the vast majority of patients (93%) with refractory anaemia with excess of blasts in transformation. SCFR was also detected in 62% of AMLs, in which it was directly associated to the expression of CD7, interleukin 6 receptor and CD34, and inversely to that of CD11b and CD14. SCFR‐positive cases were preferentially represented in AML‐M1 (70%) and in AML‐M2 (83%) subsets, whereas only 45% of the remaining samples (M3–M4–M5) exhibited SCFR positivity. Interestingly, 50% of cases with acute promyelocytic leukaemia expressed SCFR and this molecule was heterogenously regulated by in vitro treatment with all‐trans retinoic acid.

Characterization of two novel cell lines, DERL-2 (CD56 + /CD3 + /TCRγδ + ) and DERL-7 (CD56 + /CD3 − /TCRγδ − ), derived from a single patient with CD56 + non-Hodgkin's lymphoma

Two novel IL2-dependent cell lines, DERL-2 and DERL-7, were established from a patient with hepatosplenic γδ T cell lymphoma. This patient presented, at diagnosis, two discrete populations of CD56+ cells, one TCRγδ+, the second lacking T cell-restricted antigens. The cell lines derived displayed features corresponding to the two cellular components of the disease: DERL-2 was CD56+/CD3+/TcRγδ+ while DERL-7 was CD56+/CD3−/TcRγδ−. Along with CD56, the two cell lines shared the expression of CD7, CD2, CD158b and CD117. Karyotype analysis showed that both cell lines were near-diploid, with iso-7q and loss of one chromosome 10. In addition, DERL-2 showed 5q+ in all metaphases analyzed, while DERL-7 revealed loss of one chromosome 4. Genotypically, both cell lines shared the same STR pattern at nine loci and demonstrated an identical rearranged pattern of the T cell receptor genes β, γ and δ, with respect to the original tumor cells. These data indicated that both cell lines and the original neoplastic populations were T cell-derived and arose from a common ancestor. Among a large panel of cytokines tested, only SCF was able to substitute IL2 in supporting cell proliferation. Moreover, SCF and IL2 acted synergistically, dramatically enhancing cell growth. These cell lines may represent a model to further analyze the overlap area between T and NK cell malignancies, and may provide new information about the synergistic action of IL2 and SCF on normal and neoplastic T/NK cells.

Evaluation of the platelet counting by Abbott CELL‐DYN® SAPPHIRE™ haematology analyser compared with flow cytometry

The Abbot Cell‐Dyn Sapphire is a new generation haematology analyser. The system uses optical/fluorescence flow cytometry in combination with electronic impedance to produce a full blood count. Optical and impedance are the default methods for platelet counting while automated CD61‐immunoplatelet analysis can be run as selectable test. The aim of this study was to determine the platelet count performance of the three counting methods available on the instrument and to compare the results with those provided by Becton Dickinson FACSCalibur flow cytometer used as reference method. A lipid interference experiment was also performed. Linearity, carryover and precision were good, and satisfactory agreement with reference method was found for the impedance, optical and CD61‐immunoplatelet analysis, although this latter provided the closest results in comparison with flow cytometry. In the lipid interference experiment, a moderate inaccuracy of optical and immunoplatelet counts was observed starting from a very high lipid value.

JURL-MK1 (c-kit(high)/CD30-/CD40-) and JURL-MK2 (c-kit(low)/CD30+/CD40+) cell lines: 'two-sided' model for investigating leukemic megakaryocytopoiesis.

Two novel cell lines (JURL-MK1 and JURL-MK2) have been established from the peripheral blood of a patient in the blastic phase of chronic myelogenous leukemia. The cells grow in a single cell suspension with doubling times of 48 h (JURL-MK1) and 72 h (JURL-MK2). Cytogenetic analysis has shown that JURL-MK1 is hypodiploid whereas JURL-MK2 is near triploid and that both cell lines retain t(9;22). Moreover, JURL-MK1 and JURL-MK2 have a bcr/abl-fused gene with the same junction found in the patient’s fresh cells, and both cell lines express the b3/a2 type of hybrid bcr/abl mRNA. The morphology and immunophenotype of these cell lines are reminiscent of megakaryoblasts. In both lines, a limited but consistent percentage of cells expresses gpIIbIIIa (CD41a), gpIIIa (CD61) and CD36, with no expression of gpIb (CD42b), glycophorin A, hemoglobin and CD34. Both cell lines are clearly positive for CD33, CD43, CD45RO and CD63, while CD13, CD44, CD54, CD30 and CD40 are specific features of JURL-MK2. Among cytokine receptors, CD117/SCF-R is strongly displayed by a large fraction of JURL-MK1 cells but is hardly detectable on about 20% JURL-MK2 cells. Both cell lines are clearly positive for CD25/IL2Rα, while a marked expression of CD116/GM-CSF-R and CDw123/IL3Rα is restricted to JURL-MK2. Induction of cell differentiation in vitro has demonstrated that TPA is able to modulate the JURL-MK1 phenotype, causing an increased expression of platelet-associated antigens. The JURL-MK2 phenotype is easily modulated by both TPA and DMSO, which cause an increased expression of CD41a and CD117 accompanied by a decreased expression of CD30. Proliferation studies demonstrated that JURL-MK1 cell growth is enhanced by stem cell factor, while JURL-MK2 proliferation is unaffected by this cytokine. JURL-MK1 and JURL-MK2 are two novel cell lines with divergent biological features, representing a ‘two-sided’ model for investigating new aspects of megakaryocytopoiesis.

Polychromatic flow cytometry analysis of CD34+ hematopoietic stem cells in cryopreserved early preterm human cord blood samples.

During the last decades, extended characterizations were performed of human full‐term cord blood (hTCB) cells, but little information is available on human early preterm cord blood (hEPCB) hematopoietic stem cells (HSCs). In our study, we analyzed by flow cytometry 19 hEPCB and 17 hTCB samples. First, we observed that the percentage of CD34PosCD45Dim cells was higher in hEPCB compared with hTCB and that it decreased during 16th–20th week of pregnancy. Within the CD34PosCD45Dim population, we examined the expression of CD29, CD31, CD38, CD90, CD117, CD133, CD135, CD200, CD243, and CD338. We found that CD135 intensity and CD243Pos cells percentage were lower in hEPCB compared with hTCB. As to CD38, we observed that hEPCB samples were richer in undifferentiated CD34PosCD45DimCD38Neg HSCs compared with hTCB counterparts. We also compared the expression of the above‐mentioned molecules in undifferentiated and committed HSCs residing in hEPCB and hTCB. In particular, although CD34PosCD45DimCD38Pos HSCs from both hEPCB and hTCB expressed relatively higher amounts of CD29, CD71, and CD135 compared with CD34PosCD45DimCD38Neg cells, a higher expression of CD31 was restricted to CD34PosCD45DimCD38Pos cells from hEPCB samples, and a higher expression of CD117 was demonstrated in CD34PosCD45DimCD38Pos cells from hTCB samples. Moreover, our data showed that CD34PosCD45Dim cell population from hEPCB displayed higher percent of undifferentiated CD38NegCD133Pos cells compared with hTCB samples. Finally, analyzing monocytes and lymphocytes within the two samples, we observed that T‐cell percentages were higher in hTCB, whereas B‐cell percentages were higher in hEPCB. We, therefore, studied the B‐cell lineage maturation and found a higher percent of pro‐B and pre‐B cells in hEPCB compared with hTCB samples. Taken together, these results evidence the hematopoietic peculiarity of hEPCB, potentially useful for highlighting early steps of human hematolymphopoiesis as well as for developing novel strategies of stem cell‐based therapy.

Miniaturized flow cytometry-based BCR-ABL immunoassay in detecting leptomeningeal disease

Despite central nervous system (CNS) prophylactic programs limit leptomeningeal involvement in acute lymphoblastic leukemia (ALL), it can still occur in a restricted percentage of cases. The exact risk rate remains still unknown, and several factors are associated with an increased probability to develop CNS involvement. Among them, Philadelphia (Ph)-positive genotype seems to play a relevant role. Recently, a flow cytometric assay to detect BCR-ABL protein has been developed, but little is known about its possible employment in leptomeningeal disease. Here, we show the miniaturized application of the original assay for BCR-ABL oncoprotein detection in cerebrospinal fluid (CSF) samples.