Acute lymphoblastic leukemia: a study of 25 cases by scanning electron microscopy.

Cells from 25 cases of acute lymphoblastic leukemia (ALL) were studied under the scanning electron microscope (SEM). In 24 of the cases, the vast majority of circulating leukaemic cells had few microvilli. Villous cells were rarely encountered and prominent ridge-like profiles and ruffled membranes were not seen. Only six cases were studied by immunological techniques and four of the cases were of the null type while in two the cells bore detectable T-markers. It seems that ALL is almost always associated with the presence of cells with few microvilli in the peripheral circulation, differing in this respect from most cases of CLL. Although circulating leukaemic lymphocytes with few microvilli are sometimes seen in CLL, the most frequent cell type encountered is a more villous lymphocyte. Differences between leukaemic cells from patients with ALL, CLL and non-lymphoblastic leukaemias are discussed. It appears that SEM may help to distinguish lymphoblastic and nonlymphoblastic leukaemic cells in many instances and can be used as a useful adjunct to other modes of microscopy in the diagnosis of acute leukaemia.     

Serological detection of B-cell ("Ia-like") antigens in serum of normal donor and in some sera of leukaemic patients

Some sera from normal donors (1/18) and from leukaemic patients (2/7 with acute myeloid leukaemia [AML], 1/4 with chronic lymphatic leukaemia [CLL], 0/3 with acute lymphoblastic leukaemia [ALL]; with high numbers of leukaemic cells expressing Ia-like p28,33 antigen on the leukaemic cell surface) inhibited the complement mediated cytotoxic activity of highly specific xenogenous anti Ia-like sera (which were prepared by immunization of rabbits with insoluble membrane fractions of B-type lymphoid lines) at a titre 1:4 or less. This effect was not observed with antisera directed against other membrane marker determinants (e.g. T lymphocyte specific antigens). These results suggest that at least a small proportion of membrane bound Ia-like antigens can be released from cell surfaces and in some patients these Ia-like moieties are detectable (by sensitive inhibition assays) in the serum.     

Prognostic significance of neoplastic cell proliferation parameters in human haematological malignancies.

High percentage of neoplastic cells in S, G2 and M phases of cell cycle is unfavourable prognostic sign in human haematological malignancies. In chronic leukaemias (CML and CLL) it is true for peripheral blood leukaemic cells, in non-Hodgkin lymphomas--for lymph node cells, in multiple myeloma--for bone marrow plasma cells. In acute leukaemia results are controversial: some authors found a correlation between proliferation parameters of bone marrow blast cells while others did not. These parameters correlate positively with the rate of complete remission and negatively with its duration. It is concluded that proliferation parameters of neoplastic cells may be used for individual prognosis in patients with haematological tumours especially in combination with other biological and clinical prognostic markers.     

Studies on the lipid composition in serum and lymphocytes in chronic leukemia]

In 33 patients with chronic lymphatic leukaemia (CLL) the lipids were examined quantitatively and qualitatively in the serum and in the lymphocytes. Besides the 5 main categories of lipids, lysolecithin, sphingomyelin, lecithin and cephalin as well as cholesterol ester subfractions were determined according to their degree of saturation of their fatty acids. Patients suffering from CLL were found to have a moderately raise of serum triglycerides and an increase of cholesterol esters with simple unsaturated fatty acids. Simultaneously the fraction of ester with poly-unsaturated fatty acids is lowered. Leukaemic lymphocytes reveal in general a lowered total lipid content. It mainly consists of phosphatides and free cholesterol. Moreover, the leukaemic lymphocytes contrary to normal ones reveal a lowered content of cholesterol esters, diglycerides and triglycerides.     

CSF‐1R inhibition disrupts the dialog between leukaemia cells and macrophages and delays leukaemia progression

Research in the last few years has revealed that leukaemic cells can remodel the bone marrow niche into a permissive environment favouring leukaemic stem cell expansion. Tumour‐associated macrophages (TAMs) are prominent components of the tumour microenvironment and play an important role in the onset and progression of solid tumours. However, little is known about their role in the development of acute lymphoblastic leukaemia (ALL). Using a unique mouse model of T‐ALL induced by injection of EL4 T‐cell lymphoma cells to syngeneic C57BL/6 mice, we report herein that ALL leads to the invasion of leukaemia‐associated monocyte‐derived cells (LAMs) into the bone marrow and spleen of T‐ALL mice. Furthermore, we found that leukaemia cells could polarize bone marrow–derived macrophages (BMDMs) into LAMs. In turn, LAMs were able to protect leukaemia cells from drug‐induced apoptosis in vitro. Therapies targeted against the TAMs by inhibiting colony stimulating factor‐1 receptor (CSF‐1R) have emerged as a promising approach for cancer treatment. In this study, we demonstrate that CSF‐1R inhibition inhibits the viability of BMDMs, blocks LAMs polarization and reduces the abundance of LAMs in T‐ALL mice. In vivo, combination treatment of CSF‐1R inhibitor and vincristine (VCR) dramatically increased the survival of T‐ALL mice and delayed leukaemia progression compared with VCR monotherapy. Finally, these data reinforce the role of microenvironments in leukaemia and suggest that macrophages are a potential target for the development of novel therapeutic strategies in T‐ALL.     

Leukaemic peripheral blood plasma and bone marrow plasma: comparison of influence on lymphocyte proliferation

Abstract. Peripheral blood plasma from some children with untreated acute lymphoblastic leukaemia (ALL) exerted an inhibitory effect in vitro on phytohaemagglutinininduced lymphocyte transformation of normal peripheral blood lymphocytes. This occurred at concentrations beyond that required for optimal response as judged by reduction of blast cell formation and tritiated thymidine and tritiated uridine incorporation into DNA and RNA, respectively. In contrast, bone marrow plasma from these patients was non-inhibitory or contained significantly less inhibitory activity. Bone marrow plasma from the majority of healthy controls was superior to their peripheral blood plasma in enhancing phytohaemagglutinin-induced mitogenesis. The difference between an individual's bone marrow- and peripheral blood-derived plasma in enhancing proliferation of patient and healthy control cells was significantly greater amongst the patients than the healthy control group; this was attributed mainly to the increased inhibitory activity of ALL peripheral blood plasma compared with normal plasma. Medium conditioned by phytohaemagglutinin-stimulated normal peripheral blood lymphocytes was effective in neutralizing the inhibitory activity of ALL peripheral blood plasma. Taken together, these in vitro results are at least suggestive that in vivo , in healthy subjects, the rapidly proliferating cells in the bone marrow and the 'resting' blood cells in the circulation may be under the influence of a fine balance of different types and/or levels of humoral growth stimulatory and inhibitory factors and that in ALL an unstable balance of these factors exists. The decreased proliferation of circulating blast cells compared with bone marrow blasts in ALL may be attributed, at least in part, to exposure to the different levels of inhibitor(s) in the circulation and bone marrow as demonstrated in vitro by our results.     

Microenvironment‐induced PIM kinases promote CXCR4‐triggered mTOR pathway required for chronic lymphocytic leukaemia cell migration

Lymph node microenvironment provides chronic lymphocytic leukaemia (CLL) cells with signals promoting their survival and granting resistance to chemotherapeutics. CLL cells overexpress PIM kinases, which regulate apoptosis, cell cycle and migration. We demonstrate that BCR crosslinking, CD40 stimulation, and coculture with stromal cells increases PIMs expression in CLL cells, indicating microenvironment‐dependent PIMs regulation. PIM1 and PIM2 expression at diagnosis was higher in patients with advanced disease (Binet C vs. Binet A/B) and in those, who progressed after first‐line treatment. In primary CLL cells, inhibition of PIM kinases with a pan‐PIM inhibitor, SEL24‐B489, decreased PIM‐specific substrate phosphorylation and induced dose‐dependent apoptosis in leukaemic, but not in normal B cells. Cytotoxicity of SEL24‐B489 was similar in TP53‐mutant and TP53 wild‐type cells. Finally, inhibition of PIM kinases decreased CXCR4‐mediated cell chemotaxis in two related mechanisms‐by decreasing CXCR4 phosphorylation and surface expression, and by limiting CXCR4‐triggered mTOR pathway activity. Importantly, PIM and mTOR inhibitors similarly impaired migration, indicating that CXCL12‐triggered mTOR is required for CLL cell chemotaxis. Given the microenvironment‐modulated PIM expression, their pro‐survival function and a role of PIMs in CXCR4‐induced migration, inhibition of these kinases might override microenvironmental protection and be an attractive therapeutic strategy in this disease.     

Specificities of heterologous antisera against human leukaemia cells. 1. Reactions against leukaemia cells.

Rabbit or goat antisera directed to ALL, CLL, AML and CML cells were investigated in cytotoxicity tests with different leukaemia and normal cells as targets. After absorptions with erythrocytes and spleen cells from allogeneic donors the antisera killed only leukaemia cells. There was no reaction with remission leukocytes or blood leukocytes from normal donors. Anti-ALL-Sera reacted in 35 out of 49 tests with ALL cells from 13 patients. Apparently the ALL antisera which were directed to the T cell subtype of ALL preferentially affected ALL cells of this subtype. Cross reactions with cells from CLL, AML and CML were not found. Anti-CLL-sera reacted in 10 out of 12 tests with CLL cells from 4 donors, and in 4 out of 20 tests with ALL cells from 7 donors and also with the cells of a CML patient. AML cells from two patients were not killed. Antisera against AML and CML showed extensive cross reactions with cells of myelocytic and lymphocytic leukaemias. Absorption tests demonstrated the presence of two antibody specificities in AML antisera, one of which being directed to a common antigen of AML and ALL cells and another against an antigen of myelocytic leukaemia cells.     

Autologous responses to human leukaemic cells in mixed leucocyte culture

Summary Cryopreserved leukaemic blasts and remission non-T cells from 22 patients with acute leukaemia (15 lymphocytic, 7 non-lymphocytic) were tested as stimulators of autologous remission T cells and normal allogeneic T cells in primary and secondary MLC. In most cases the autologous response elicited by leukaemic cells was less than or equal to that elicited by remission non-T cells. However, T cells from 2 patients in long-standing first remission from ANLL displayed greater proliferation in response to leukaemic blasts than to remission non-T cells in both primary and secondary MLC. The results are suggestive of sensitization of these 2 patients to leukaemia-specific antigens, but other possible explanations are discussed. Abbreviations used: MLC, mixed leucocyte culture; ANLL, acute non-lymphocytic leukaemia; ALL, acute lymphoblastic leukaemia; AMLR, autologous mixed lymphocyte reaction; NK cells, natural killer cells; MNC, mononuclear cells     

The behaviour of 6-keto PGF1 alpha and TXB2 concentrations in the culture supernatant of chronic lymphocytic leukaemia cells

Prostaglandins have been implicated as possible modulators of the proliferation and differentiation of neoplastic cells. The aim of this work was to determine 6-keto-PGF1 alpha and TXB2 concentrations in the blood plasma and in the supernatant of 96 hour PHA stimulated and unstimulated leukaemic cell cultures of chronic lymphocytic leukaemia patients. 62 patients with CLL classified to the 1st or 4th stage according to RAI, and 23 healthy individuals were investigated. The blastogenic transformation was measured by the standard tritiated thymidine method. The quantity of 6-keto-PGF1 alpha and TXB2 was estimated by the isotopic method using a RIA-kit. In the 4th stage of CLL a low value of blastogenic transformation was observed, whereas in the 1st stage, the values were similar to those of the control group. It was shown that in the 4th stage of the disease an increase in the 6-keto-PGF1 alpha concentrations occurs in the blood plasma and the culture supernatant together with a significant decrease in TXB2 in the culture supernatant, whereas in the 1st stage a significant decrease in the 6-keto-PGF1 alpha as compared with those of the control group is noted. These results may indicate on antagonistic action of PGI2 and TXB2 in leukaemia cell proliferation.     

Low-expression of E-cadherin in leukaemia cells causes loss of homophilic adhesion and promotes cell growth

E-cadherin (epithelial cadherin) belongs to the calcium-dependent adhesion molecule superfamily and is implicated in the interactions of haematopoietic progenitors and bone marrow stromal cells. Adhesion capacity to bone marrow stroma was impaired for leukaemia cells, suggesting that a breakdown of adhesive mechanisms governed by an adhesion molecule may exist in leukaemic microenvironment. We previously found that E-cadherin was low expressed in primary acute leukaemia cells compared with normal bone marrow mononuclear cells. In this study, we investigate the functional importance of low E-cadherin expression in leukaemia cell behaviours and investigate its effects in the abnormal interaction of leukaemic cells with stromal cells. After expression of E-cadherin was restored by a demethylating agent in leukaemia cells, E-cadherin-specific adhesion was enhanced. Additionally, siRNA (small interfering RNA)-mediated silencing of E-cadherin in Raji cells resulted in a reduction of cell homophilic adhesion and enhancement of cell proliferation and colony formation. These results suggest that low expression of E-cadherin contributes to the vigorous growth and transforming ability of leukaemic cells.     

Generation of cytotoxic T lymphocytes from peripheral blood of leukaemic patients

Peripheral blood mononuclear cells from 13 patients with acute leukaemia were used to establish long-term interleukin-2-dependent cytotoxic T lymphocytes. Cells were grown in RPMI medium containing interleukin-2 (IL-2, 100 U/ml) and 2.5% conditioned medium prepared by activating normal lymphocytes with phytohaemagglutinin. Proliferation of IL-2-dependent CD3-positive lymphocytes was seen in 1 of 2 acute lymphocytic leukaemia cases (ALL), 1 of 4 acute myelogeneous leukaemia cases (AML) (M1) and 8 of 8 more differentiated AML. In 2 cases with detectable leukaemic cell markers (1 ALL and 1 AML) passageable cells were developed, that expressed normal T cell phenotypes (namely CD3, CD4, and CD8) at the expense of leukaemic cells. In 1 of 2 cases, long-term IL-2-cultured cells showed specific cytotoxic activity against autologous leukemic cells. The percentage killing against autologous and two allogeneic target cell lines at a 50/1 effector/target (E/T) ratio was 42%, 9% and 19% respectively. Similarly the cytotoxic activity of IL-2 activated from 4 different individuals against conventional tumour targets K562 and Daudi at a ratio of 50/1 was 29%–68% (median=55%) and 34%–78% (median=61%) respectively. It was also found that this killing potential of the activated cells was maintained for as long as culture was continued (median 23 days, range 17–75 days). The mechanism(s) of T cell proliferation at the expense of leukaemic blast cells in the case of a minority of leukaemic patients and the possible clinical therapeutic potential of these cells following in vitro IL-2 activation deserve further investigation.     

Granulocytopoiesis in children with acute lymphoblastic leukaemia.

Numbers, proliferative potential, and differentiative capacity of bone marrow granulocyte-macrophage precursor cells were studied in 130 children with acute lymphoblastic leukaemia (ALL), including 77 children in an acute phase of the disease and 53 in remission. Bone marrow samples from 65 children without haematopoietic abnormalities were used as controls. The numbers of clonogenic precursors were found to be below normal in all phases of ALL, particularly during the acute period when the bone marrow was heavily infiltrated with leukaemic cells. It is shown that the decreases in the numbers and proliferative potential of the precursor cells during the acute phases was associated with the effects of leukaemic blast cells, but that in remission the observed reduction in the precursor cell pool was due to the cytostatic effect of therapy. The differentiative capacity of clonogenic granulocyte and macrophage precursors was not altered in children with ALL.     

Correlation between cholesterol esterification, MDR1 gene expression and rate of cell proliferation in CEM and MOLT4 cell lines

A positive correlation between cholesterol esterification and growth rate potential was previously found in our laboratory during the growth of CEM and MOLT4 lymphoblastic cells. In the current study, we investigated whether the rates of cholesterol esters synthesis correlate with changes of acyl-CoAcholesterol acyltransferase (ACAT) mRNA levels and of other genes implied in cholesterol biosynthesis and uptake, such as 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase and low density lipoprotein (LDL) receptor. The results showed that the more rapid growing CEM cells had lower levels of expression of HMGCoA-reductase and LDL receptors compared to MOLT4. By contrast, ACAT mRNA levels were higher in CEM cells, further supporting the concept of a possible involvement of cholesterol esters in the regulation of cell growth and division. In this study, high levels of cholesterol esterification and of expression of ACAT gene were also associated with a markedly increased expression of multidrug resistance (MDR1) gene, suggesting that MDR1 activity might contribute to regulate the rate of cell growth and division by modulating intracellular cholesterol ester levels.     

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose–response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.     

Immunological study of the T- and B-lymphocytes in normal cows and in chronic lympholeukemia

T- and B-lymphocyte populations from lymphoid organs and tissue of normal cattle and cattle with chronic lymphocytic leukaemia (CLL) were studied. Comparative studies of surface properties, quantitative parameters and heterogeneity of main T- and B-cell populations were performed. It must be noted that proliferation of B-lymphocytes, bearing surface IgM in blood, lymph nodes and spleen is closely connected with the progression of leukaemic process. An increased number of B-lymphocytes (2-3 times) with the receptors for complement was found. The proportion of T mu and T gamma cell subsets in CLL cows is distorted. The T gamma cell subset in T-cell suspensions from blood and spleen in CLL cows prevails in comparison with that in controls. The number of T mu cells in blood and lymph nodes in CLL is decreased.     

Inhibition of bromodomain and extra‐terminal proteins increases sensitivity to venetoclax in chronic lymphocytic leukaemia

The development of drugs able to target BTK, PI3k‐delta and BCL2 has dramatically improved chronic lymphocytic leukaemia (CLL) therapies. However, drug resistance to these therapies has already been reported due to non‐recurrent changes in oncogenic pathways and genes expression signatures. In this study, we investigated the cooperative role of the BCL2 inhibitor venetoclax and the BRD4 inhibitor JQ1. In particular, we found that JQ1 shows additional activity with venetoclax, in CLL cell lines and in ex vivo isolated primary CD19⁺ lymphocytes, arguing in favour of combination strategies. Lastly, JQ1 is also effective in venetoclax‐resistant CLL cell lines. Together, our findings indicated that the BET inhibitor JQ1 could be a promising therapy in CLL, both as first‐line therapy in combination with venetoclax and as second‐line therapy, after the emergence of venetoclax‐resistant clones.     

Compactin (ML-236B) reduces the content of filipin-cholesterol complexes in the plasma membrane of chronic lymphocytic leukemia cells

The polyene antibiotic filipin was used to visualize the presence and distribution of cholesterol in the plasma membrane of glutaraldehyde-fixed human chronic lymphocytic leukemia (CLL) cells. Both compactin (ML-236B), a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and 25-hydroxycholesterol reduced the content of filipin-cholesterol complexes in the plasma membrane of CLL cells grown in media supplemented with either 15% delipidized horse serum or 15% normal (whole) horse serum. The reduction due to compactin was reversed by the concomitant addition of mevalonolactone. The ability of compactin to reduce the relative cholesterol content (as judged by filipin labeling) in CLL cells grown in lipoprotein-containing (normal) serum suggest that either CLL cells are different from other cells in that they predominantly utilize endogenously synthesized cholesterol for incorporation into the plasma membrane, or that a separate pool of endogenously synthesized cholesterol provides cholesterol for the plasma membrane.     

Cholesterol efflux from cells enriched with cholesteryl esters by incubation with hypercholesterolemic monkey low density lipoprotein

The mass efflux of free and esterified cholesterol was studied in skin fibroblasts loaded with cholesterol by incubation with low density lipoproteins (LDL) isolated from normal or hypercholesterolemic cynomolgus monkeys. Cells incubated with hypercholesterolemic LDL accumulated 2-3 times more cholesteryl ester than did cells incubated with the same amount of normal LDL. Cholesteryl oleate was the principal cholesteryl ester species to accumulate in cells incubated with both normal and hypercholesterolemic LDL. Efflux of this accumulated cholesterol was absolutely dependent on the presence of a cholesterol acceptor in the culture medium. Lipoprotein-deficient serum (LPDS) was the most potent promoter of cholesterol efflux tested, with maximum efflux occurring at LPDS concentrations greater than 1.5 mg protein/ml. Upon addition of efflux medium containing LPDS, there was a reduction in both the free and esterified cholesterol concentration of the cells. Greater than 90% of the cholesteryl esters that were lost from the cells appeared in the culture medium as free cholesterol, indicating that hydrolysis of cholesteryl esters preceded efflux. Efflux was not inhibited by chloroquine, however, suggesting a mechanism independent of lysosomes. Loss of cellular free cholesterol was maximum by 6 hr and changed very little thereafter up to 72 hr. Cholesteryl ester loss from cells decreased in a log linear fashion for efflux periods of 6-72 hr, with an average half-life for cholesteryl ester efflux of 30 hr, but with a range of 20-50 hr, depending upon the specific cell line. The rate of efflux of cellular cholesteryl esters was similar for cells loaded with normal or hypercholesterolemic LDL. In cells loaded with cholesteryl esters, cholesterol synthesis was suppressed and cholesterol esterification and fatty acid synthesis were enhanced. During efflux, cholesterol synthesis remained maximally suppressed while cholesterol esterification decreased for the first 24 hr of efflux, then plateaued at a level approximately 5-fold higher than control levels, while fatty acid synthesis was slightly stimulated. There was little difference in the rate of efflux of individual cholesteryl ester species. There was, however, the suggestion that reesterification of cholesterol principally to palmitic acid occurred during efflux. Since the rate of cellular cholesteryl ester efflux was similar regardless of whether the cells had been loaded with cholesterol by incubation with normal LDL or hypercholesterolemic LDL, the greater accumulation of cholesterol in cells incubated with hypercholesterolemic LDL cannot be explained by differences in rates of efflux.-St. Clair, R. W., and M. A. Leight. Cholesterol efflux from cells enriched with cholesteryl esters by incubation with hypercholesterolemic monkey low density lipoprotein.