Diverse effects of anti-CD44 antibodies on the stromal cell-mediated support of normal but not leukaemic (CML) haemopoiesis in vitro

We have identified three non-cross-reacting anti-human CD44 monoclonal antibodies that have significant positive or negative (or no) effects on normal human haemopoiesis in the long-term culture (LTC) system. These effects manifested as increases or decreases in the number of LTC-initiating cells (LTC-IC), and the number of colony-forming cells (CFC) recovered from cultures in which either unseparated or highly purified CD34⁺CD38⁻ normal marrow cells were placed on pre-established normal marrow feeder layers in the presence or absence of each antibody. The effects seen were rapid and sustained, and dependent on the presence of a preformed feeder layer. Interestingly, the same anti-CD44 antibodies had no effect on the maintenance of leukaemic (Ph⁺) progenitors (from patients with chronic myeloid leukaemia) when these cells were cultured on preformed feeder layers established from normal marrow. CD44 appears to be part of a mechanism by which stromal elements can regulate primitive normal haemopoietic cells but not their leukaemic (Ph⁺) counterparts.     

Blast Crisis of Chronic Myeloid Leukaemia (CML): II. CELL SURFACE MARKER ANALYSIS OF 'LYMPHOID'AND MYELOID CASES

SUMMARY. Fourteen cases of Philadelphia chromosome (Ph¹) positive chronic myeloid leukaemia in blast transformation have been investigated using cell surface markers. Morphologically eight cases were lymphoid and the remainder myeloid in appearance. All cases were negative with surface markers for thymocytes and T and B lymphocytes. Five of the lymphoid cases reacted with an antiserum specific for acute lymphoid leukaemia (ALL) of non-T non-B type and were also weakly reactive with a lymphocyte reactive antiserum. A sixth patient, whose blast cells were anti-ALL negative (ALL^-) at presentation, subsequently developed central nervous system leukaemia with anti-ALL positive (ALL⁺) blast cells in the CSF. In all cases the leukaemic blast cells showed greatly diminished expression of cholera toxin receptors when compared to granulocytic cells from the chronic phase of CML. This parallels weak or negligible expression of the cholera toxin receptor in ALL and AML.
These results suggest that the blastic phase of CML may involve different cellular derivatives of a pluripotential stem cell in which the primary malignant/genetic changes reside. The blast crisis of CML can therefore be heterogeneous with respect to cellular expression and in a significant proportion of patients involves a cell which is by membrane markers and morphological criteria indistinguishable from that seen in the common form of ALL. In these cases the Philadelphia chromosome may be the only distinguishing cellular characteristic.     

Heterogeneous Blast Cell Crises in Philadelphia Negative Chronic Granulocytic Leukaemia

S ummary . A case of Philadelphia negative chronic granulocytic leukaemia (Ph^1- CGL) is described showing features only previously demonstrated in Ph¹⁺ disease. These features include: (1) lymphoid blast crisis, determined by morphology and immunological marker analysis; (2) dual blast cell populations that can be distinguished both morphologically and by immunological markers; (3) clonal evolution, as shown by the emergence of chromosome markers and in one of the cell lines a change in membrane phenotype. These changes were apparently associated with the emergence of a relatively drug resistant subclone of leukaemic cells. This study demonstrates that the lymphoid blast crisis of CGL, and its sequelae, can occur in Ph^1- cases. It is similar in respect to morphology, enzyme, and membrane markers and responsiveness to vincristine and prednisolone therapy to the lymphoid blast crisis seen in Ph¹⁺ CGL. This suggests that the Philadelphia chromosome is a clonal marker only, and its presence is not directly related to the subsequent clinical course of the disease.     

Involvement of the Erythroid Series in Blastic Crisis of Chronic Myeloid Leukaemia. FURTHER EVIDENCE FOR THE PRESENCE OF PHILADELPHIA CHROMOSOME IN ERYTHROBLASTS

I nvolvement of the erythroid series in chronic myeloid leukaemia may be evidenced haematologically by a normochromic anaemia with anisocytosis, the presence of circulating normoblasts, the presence of bizarre normoblasts in the marrow and peripheral blood, and rarely the presence of sideroblasts. The Philadelphia (Ph¹) chromosome has been assumed to be present in the erythrocyte as well as the granulocyte series on good but circumstantial evidence and it is generally agreed that the leukaemic process probably affects the basic stem cell of the red cells, granulocytes and platelets.
When blastic crisis occurs there is an increase in the percentage of blast cells in marrow and peripheral blood, and anaemia, thrombocytopenia and resistance to therapy accompany the apparently irreversible downward clinical course. Aneuploid cell lines emerge (Hammouda, Quaglino and Hayhoe, 1964). We are here presenting evidence that aneuploid Ph¹-positive cell lines are also found in the erythroid cells in blastic crisis and therefore that when this occurs the essential change may also affect the basic stem cell. In a case of chronic myeloid leukaemia seen recently, a sideroblastic phase was found terminally and it was possible to combine standard cytogenetic methods with Perls's prussian blue stain for iron and demonstrate siderotic granules in the cytoplasm surrounding Ph¹-positive aneuploid chromosome spreads. We thus prove the erythroid nature of these spreads, and incidentally provide more direct evidence than offered hitherto, for the presence of the Ph¹ chromosome in the erythroid cells.     

Minimal residual disease after allogeneic bone marrow transplantation for chronic myeloid leukaemia: a metaphase-FISH study

Metaphase-FISH was adopted for the detection of proliferating Philadelphia-positive (Ph⁺) residual leukaemic cells in 25 patients with chronic myeloid leukaemia treated with allogeneic bone marrow transplantation (BMT). Patients were followed up during their clinical remission for 4–50 months (median 17 months) after BMT. 80 bone marrow samples were studied. For most of the cases no fewer than 1000 metaphases were analysed. Six patients (24%) showed residual Ph⁺ cells during the first 6 months and two others by the end of the first year after BMT. Three patients relapsed during the study and in two of them residual Ph⁺ cells were detected during the first 6 months after BMT. In 17 patients no Ph⁺ cells were detected at any stage of follow-up and 16 (94.1%) of them continue in complete clinical and haematological remission. Our results indicate that metaphase-FISH is a reliable tool in the quantitation of proliferating residual leukaemic cells. We suggest that consecutive findings of equal amounts of residual leukaemic cells do not necessarily predict a relapse. However, their presence calls for follow-up at shorter intervals where an increasing number of these cells predicts an ensuing relapse.     

Annotation: CELLULAR CHANGES IN CHRONIC MYELOID LEUKAEMIA

Since the original observation by Wachstein (1946), numerous investigations have demonstrated that the vast majority of patients with chronic myeloid leukaemia have very low alkaline-phosphatase activities in their polymorphonuclear leucocytes. The observation of a high leucocyte alkaline-phosphatase (LAP) activity in Down's syndrome (Alter et al, 1962), where the karyotype contains an extra G chromosome, and the fact that patients with chronic myeloid leukaemia (CML) have a partially deleted G chromosome, the so-called Philadelphia chromosome (Ph¹), at one time led to the hypothesis that a specific gene regulating LAP activity was present in a G chromosome, possibly no. 21 (Alter et al, 1963). The fact that LAP activity increases during successful treatment of CML and even returns to the normal range in a few cases (Hayhoe & Quaglino, 1958; Xefteris et al, 1961; Linke & Löffler, 1963; Elves et al 1963) might be explained if a non-leukaemic clone of cells were activated under treatment. The bone marrow remains, however, entirely Ph¹-positive in remission (Tough et al, 1963). Moreover, the observation by Hammouda et al (1964) of persisting Ph¹-positivity in patients with high LAP activity in blast crisis of CML was incompatible with the existence of a simple causal relationship between the Ph¹ deletion and depression of LAP activity.     

Clonal Origin of the Philadelphia Chromosome and Chronic Myeloid Leukaemia: Evidence from a Sex Chromosome Mosaic

S ummary . A male patient aged 69 yr with chronic myeloid leukaemia was a constitutional XY/XXY sex chromosomal mosaic as indicated by a positive sex chromatin in neutrophils and cytogenetic studies of lymphocytes cultured with phytohaemagglutinin. He was of normal phenotype and intelligence. In a bone-marrow aspirate taken when the patient was in the acute phase of leukaemia the 46, XY cell line carried the Ph¹ chromosome, whereas the 47, XXY cell line did not. Two further cell lines were considered to have been derived by clonal evolution of the 46,XY,Ph¹-positive line, although one of them possessed ambiguous features. The results support a clonal origin of the Ph¹ chromosome, and presumably also of chronic myeloid leukaemia. An interesting feature of the bone marrow was that the 46, XY, Ph¹-positive cell line had apparently replaced the normal 46, XY cells but not the 47, XXY cells.     

Analysis of Tie receptor tyrosine kinase in haemopoietic progenitor and leukaemia cells

We generated a panel of monoclonal antibodies against the extracellular domain of the Tie receptor tyrosine kinase and studied its expression in human haemopoietic and tumour cell lines and in samples from leukaemia patients. Most of the erythroblastic/megakaryoblastic (6/8), 2/7 myeloid and 3/6 B-lymphoblastic leukaemia cell lines were Tie-positive. The erythroblastic/megakaryoblastic leukaemia cell lines also expressed the related Tie-2/Tek gene and, surprisingly, its recently cloned ligand gene angiopoietin-1, which was located in chromosome 8q23.1. In addition, 16% of freshly isolated leukaemia samples were Tie positive. Peripheral blood mononuclear cells were Tie negative, but a few Tie positive cells were found in immunoperoxidase staining of mobilized peripheral blood stem cells. Long-term culture of isolated umbilical cord blood CD34⁺Tie⁺ and CD34⁺Tie⁻cells indicated that the Tie⁺ fraction contained a slightly higher frequency of cobblestone area forming cells (CAFC). Thus, Tie is expressed on haemopoietic progenitor cells and some leukaemic blasts. The coexpression of Tie-2 and angiopoietin-1 in megakaryoblastic leukaemia cell lines suggests the existence of an autocrine ligand/receptor signalling loop in these cells.     

Telomerase activity in acute myelogenous leukaemia: clinical and biological implications

We examined telomerase activity in myeloid leukaemic cell lines, normal haemopoietic cells, and leukaemic blasts from acute myelogenous leukaemia (AML) patients. Normal bone marrow mononuclear (BMNC) cells expressed low telomerase activity. Higher telomerase activity was detected in 10 myeloid leukaemic cell lines compared to normal BMNC cells. Treatment with 1,25(OH)₂D₃, and vitamin D₃ analogues, EB1089 and KH1060, reduced telomerase activity in vitamin D₃-sensitive HL-60 cells, whereas vitamin D₃ insensitive K562 cells did not change its activity. This down-regulation of telomerase activity by EB1089 was associated with induction of p21 protein. The rank order of telomerase activity was leukaemic CD34⁻ cells > leukaemic CD34⁺ cells > normal CD34⁻ cells > normal CD34⁺ cells. Telomerase activity was positive in all of the AML patients tested; however, heterogeneity of telomerase activity was found amongst this group. Therefore we compared telomerase activity with clinical response. Unexpectedly, we found that a higher rate of complete remission was noted in AML patients with higher telomerase activity. No association between telomerase activity and biological parameters including percentage of S-phase, cytotoxicity to cytosine arabinoside and percentage of CD34⁺ cells in AML blasts was found. These results suggest that telomerase activity in AML patients is detected with high frequency, but is heterogenous. Expression level of telomerase activity may have a clinical implication in AML patients regarding clinical response.     

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/Le^x, CDw65/VIM2, CD15s/sLe^x), 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.
In spite of the well-recognized expression of CD11a throughout all stages of normal myeloid differentiation, APL blast cells were found to virtually lack LFA-1 expression. Moreover, ATRA was unable to promote and up-regulation of this antigen in APL, while inducing a frequent down-modulation in non-APL cases constitutively expressing this antigen. In APL cases ATRA generated an asynchronous phenotype (CD15⁺, CDw65⁺, CD11b⁺, CD11a^-), undetectable on normally maturing myeloid cells, but consistent with the concept that incomplete differentation, in terms of surface molecule expression, can be sufficient to obtain therapeutic results.     

Expression of the leptin receptor in human leukaemic blast cells

The leptin receptor is a member of the cytokine receptor superfamily, and is expressed in CD34 haemopoietic stem cells. We examined expression of the leptin receptor in fresh human leukaemia cells. Northern blot analysis showed the leptin receptor was expressed in leukaemic cells from patients with acute myeloblastic leukaemia, acute lymphoblastic leukaemia and chronic myeloid leukaemia (CML). In CML, higher expression was observed in blast crisis than in chronic phase. The expression of leptin receptor decreased during in vitro differentiation of leukaemic blast cells. It appeared that expression of the leptin receptor was associated with immature leukaemic blast cells. Our findings may indicate the possibility that leptin has some role in leukaemia.     

A novel surface marker (B203.13) of human haemopoietic progenitors, preferentially expressed along the B and myeloid lineages

We used a monoclonal antibody (mAb) (B203.13, IgM) generated from a mouse immunized with the human B/myeloid bi-phenotypic B1b cell line, to analyse haemopoietic cells. The antigen recognized by this mAb is expressed on most adult and umbilical cord blood CD21⁺ B cells, at minimal density on mature monocytes, and is undetectable on granulocytes, T, natural killer (NK) cells, and erythrocytes. Within umbilical cord blood and adult bone marrow haemopoietic progenitor cells, the B203.13 mAb recognized a surface marker, present on progenitor cells of several haemopoietic lineages, that was transiently expressed on early erythroid and T/NK progenitors, and was preferentially maintained on cells of the B and myeloid lineages. Within the CD34⁺ cells, B203.13 was expressed on early committed myeloid (CD33⁺) and erythroid (CD71^dim) progenitor cells, as confirmed in colony formation assays. The mAb also reacted with cells of B and myeloid chronic leukaemias and cell lines. These data define B203.13 mAb as a novel reagent useful for the characterization of haemopoietic progenitors and leukaemias.     

Acute leukaemia with mixed lymphoid and myeloid phenotype

S ummary Three children with acute leukaemia had blasts that expressed both lymphoid and myeloid markers. The blasts met immunological criteria for acute lymphoblastic leukaemia (ALL)—common ALL antigen⁺, HLA-DR⁺, terminal deoxynucleotidyl transferase⁺–but their cytochemical features, including positive myeloperoxidase and Sudan black B, were those of acute nonlymphoblastic leukaemia (ANLL) as defined by the French-American-British Group. 30% of the blasts from one of two patients tested reacted with a monoclonal antibody specific for nonlymphoid cells (MCS-2). The wide overlap in the percentages of blasts expressing lymphoid or myeloid markers indicates that some leukaemic cells in each child had a mixed phenotype. There were no consistent cytogenetic findings, and the Philadelphia chromosome was not present. Complete remission was induced by treatment effective for either ALL (two patients) or ANLL. These three cases appear to represent a rare leukaemia subtype that we have designated acute leukaemia with mixed lymphoid and myeloid phenotype. Its recognition may be important in treatment, since two patients achieved remission with standard therapy for ALL. These cases demonstrate further the phenotypic heterogeneity that may be seen in leukaemic cell differentiation.     

Successful autografting in chronic myelogenous leukaemia using Philadelphia negative blood progenitor cells mobilized with rHuG-CSF alone in a patient responding to alpha-interferon

Several non-randomized studies suggest a possible survival advantage for chronic myelogenous leukaemia (CML) patients treated with an autologous stem-cell transplantation. Due to the possible contribution of residual leukaemic cells present in the inoculum in post-transplant relapse, several methods are being evaluated to eliminate neoplastic cells or to select 'normal' (Ph¹ negative) progenitor cells for autografting. Recently, several studies have shown that Ph¹ negative blood progenitor cells can be mobilized by rHuG-CSF alone in patients who have a cytogenetic response to alpha-interferon (IFN). We describe the first case, as far as we are aware, of a CML patient responding to IFN autografted by using blood progenitor cells collected by rHuG-CSF alone.     

Observations on the in vitro Growth of Peripheral Leucocytes from Patients with Myeloproliferative Disorders

In 1960 Moorhead, Nowell, Mellman, Battips and Hungerford described a method for obtaining chromosome preparations by culturing peripheral blood leucocytes. This work has provoked intensive studies of human chromosomes in many disorders. Studies on leukaemia and related conditions were not long in appearing, and in 1960 Nowell and Hungerford described an abnormality of one of the small acrocentric chromosomes in two male patients with chronic myeloid leukaemia. The abnormal chromosome was later found to occur in the majority of patients with chronic myeloid leukaemia and was named the Philadelphia (Ph¹) chromosome. In the first studies this chromosome was sometimes absent from the peripheral blood cultures stimulated with phytohaemagglutinin but present in the marrow. However, there are cases of chronic myeloid leukaemia where no Ph¹ chromosome can be found (Tough, Jacobs, Court-Brown, Baikie and Williamson, 1963; Krauss, Sokal and Sandberg, 1964).
Studies on non-leukaemic myeloproliferative disease indicated no constant chromosomal abnormality (Sandberg, Ishihara, Crosswhite and Hauschka, 1962; Goh and Swisher, 1964). While carrying out chromosome studies on patients with myeloproliferative disorders it was noticed that there was considerable variation in the number of cells in mitosis when cells from different patients were grown in vitro (Pegrum, 1965). An investigation has therefore been carried out on the growth of cells from 28 patients with myeloproliferative disorders, using 15 blood donors as controls, in an attempt to evaluate these differences. The cells synthesizing DNA prior to division were identified at intervals of 3, 5 and 7 days by autoradiography following exposure to tritiated thymidine.     

SLEx expression of normal CD 34 positive bone marrow haemopoietic progenitor cells

Summary. We investigated sialylated Lewis x (sLe^x) antigen expression on CD 34 positive (CD 34⁺) haemopoietic progenitors in the bone marrow of eight healthy volunteers using monoclonal antibodies. We found that in all the samples examined, CD 34⁺ bone marrow progenitors strongly expressed the sLe^x antigen. This contradicts previous publications which reported sLe^x expression on malignant blast cells but not on normal CD 34⁺ progenitor cells.     

Evaluation of the prognostic relevance of L-selectin and ICAM1 expression in myelodysplastic syndromes

Objectives:  An aberrant pattern of expression of l -selectin and intercellular adhesion molecule 1 (ICAM1) may characterise CD34⁺ blast cells in myelodysplastic syndromes (MDS) and secondary acute myeloid leukaemia (sAML).
Methods:  In a three-colour flow cytometric assay, we evaluated the expression of l -selectin and ICAM1 on CD34⁺ blast cells from the bone marrow (BM) of 66 MDS patients; for the purpose of comparison CD34⁺ blast cells of 18 sAML and CD34⁺ stem cells of 17 normal donors were also analysed.
Results:  The ratio of l -selectin/ICAM1 expression was identified as a parameter correlated with the percentage of BM blast infiltration and the time to leukaemic progression among MDS patients. In fact, the values of l -selectin/ICAM1 ratio were inversely correlated with the BM blast infiltration ( r  = –0.34, P  = 0.004). Furthermore, MDS patients with a baseline ratio <1 had a higher leukaemic progression rate (41% vs. 19%, P  = 0.008); the actuarial risk of disease progression for this subgroup of MDS patients was also higher (64% vs. 11% at 2 yr, P  = 0.002). Furthermore, in two patients a decrease of the ratio was observed when overt leukaemic transformation occurred; conversely, restoration of a normal ratio was observed in two patients after a chemotherapy-induced remission.
Conclusion:  (i) l -selectin is defective in the stem cell compartment of MDS and sAML, whereas ICAM1 is overexpressed; (ii) the ratio of their expression has a prognostic role; and (iii) a ratio <1 significantly predicts progression to overt leukaemia in MDS patients.     

Role of vascular endothelial growth factor (VEGF) and placenta-derived growth factor (PlGF) in regulating human haemopoietic cell growth

Vascular endothelial growth factor (VEGF) and placental derived growth factor (PlGF) stimulate cell proliferation and differentiation by binding to their specific receptors, Flk-1/KDR and Flt-1 respectively. Flk-1/KDR-deficient murine embryos manifest failure of blood-island formation and vasculogenesis. The aim of this study was to directly evaluate the importance of VEGF, PlGF/Flt-1 and Flk-1/KDR receptor ligand interactions in regulating normal and malignant human haemopoiesis. Addition of VEGF and PlGF failed to enhance survival or cloning efficiency of human haemopoietic progenitors isolated from adult bone marrows, fetal livers or cord blood samples. This finding may be explained by the apparent absence of mRNA encoding Flt-1 and Flk-1/KDR receptors on stem cell rich CD34⁺ c-kit-R⁺ Rh123^low cells. Further studies revealed that Flt-1 R mRNA, but not Flk-1/KDR mRNA was first detectable in the more mature cells isolated from haemopoietic colonies. Accordingly, VEGF receptors are either absent, or expressed at very low level, on human haemopoietic stem/progenitor cells. Of interest, normal and malignant human haemopoietic cells appeared to secrete VEGF protein. However, in contrast to normal haemopoietic progenitors, VEGF co-stimulated HEL cell proliferation as well as CFU-GM colony formation from ∼15% of the chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) patients studied. Therefore, although VEGF appeared to have minimal effects on normal haemopoietic cell growth it would appear to drive malignant haemopoietic cell proliferation to some degree. Of more importance, however, we speculate that VEGF may play an very important role in leukaemogenesis by stimulating growth of vascular endothelium, thereby providing a sufficient blood supply to feed the growing haematological tumour.     

Haemopoietic progenitor cell differentiation: flow cytometric assessment in bone marrow and thymus

Summary. We have recently shown that expression of any of the lineage-associated molecules CD2, CD7, CD10, CD19 or CD33 does not ensure lineage-commitment of CD34⁺ progenitor cells. Further, normal progenitor cells and leukaemic blast cells have been shown to coexpress molecules associated with more than one haemopoietic lineage. Five-dimensional flow cytometric analysis of normal bone marrow cells was exploited to investigate the hypothesis of a developmental stage in haemopoiesis comprising CD34⁺ cells coexpressing CD2, CD5, CD7, CD10, CD19 and CD33 or any combination of these molecules. We report on a subpopulation of CD34⁺ bone marrow cells constituting < 5% of the CD34⁺ cells and characterized by extensive coexpression of several molecules associated with the B lymphoid, T lymphoid and myeloid lineages. There is every probability that some cells display the CD34+ CD2⁺ CD5⁺ CD7⁺ CD10⁺ CD19+ CD33+ phenotype. Studies on postnatal thymocytes suggest that this may be the phenotype or one of a few phenotypes of a candidate thymus-seeding progenitor cell population. Finally, our findings that CD34⁺ as well as CD34⁺ CD5⁺ thymocytes can be driven into non-T-lymphoid differentiation by cytokines, support the notion that the thymus is seeded by uncommitted progenitors.