The Chronic Leukemias: A Review of Disease Manifestations and the Aims of Therapy

Certain aspects of the chronic leukemias that may influence future therapeutic trials are reviewed. In chronic lymphocytic leukemia (CLL), there is minimal mitotic activity in lymphoid tissues; indolent, long-lived lymphocytes, unresponsive to antigenic or phytohemagglutinin (PHA) stimulation accumulate. In many patients, erythroid precursors fail to proliferate despite the stimulus of a severe anemia, but a proliferative response can be initiated by prednisone. We need to know how the normal proliferative responses of these cells are modified, because the correction of these abnormalities would relieve most of the disease manifestations. CLL may not be a neoplastic disorder. In chronic myelogenous leukemia (CML), the leukocyte doubling time shortens as the disease duration lengthens; a significant correlation between this time and survival is demonstrated. Before therapy designed to eliminate the Ph¹-positive (Philadelphia chromosome) stem cell is tried, we need to know whether a normal hematopoietic stem cell exists in Ph¹-positive CML.     

Blood lymphocyte volumes and diameters in patients with chronic lymphocytic leukemia and normal controls

The electronic modal lymphocyte volumes of 151 patients with chronic lymphocytic leukemia (CLL) and 305 normal controls were determined by the hydrodynamically focused multi-channel Coulter TF analyser. The mean volumes of the normally distributed groups were 166 +/- 19.3 (range 126-216) fl in patients with CLL and 206 +/- 14.4 (range 126 +/- 246) fl in normal controls. The calculated cell diameters were 6.8 (6.2-7.4) micron and 7.3 (6.8-7.8) micron respectively. Our data do not support previous reports about relations between cell size and clinical stages of the Rai and Binet classifications.     

Galactose-1-phosphate uridylyltransferase activity in chronic lymphocytic leukemia.

Galactose-1-phosphate uridylytransferase (E.C.2.7.12) activity was measured in both lymphoid and erythroid cells from patients with chronic lymphocytic leukemia (CLL). Decreased enzyme activity was found in both cell types using two assay methods. The results suggest the presence of an inhibitor of the enzyme in CLL patients. A correlation between decreased uridyl transferase activity and glycogen accumulation in CLL is postulated.     

DNA-synthesizing T and non-T cells in chronic lymphocytic leukemia.

In 21 patients with chronic lymphocytic leukemia (CLL) and in 8 hematologically normal persons the number of DNA-synthesizing peripheral blood lymphocytes was investigated by autoradiographic techniques. The lymphocytes were differentiated by EN-rosette tests into T and non-T lymphoid cells. The results show a normal number of proliferating T lymphoid cells and an increased number of proliferating non-T lymphoid cells in clinical stages O-I. Stages III-IV demonstrate a significant increase of the proliferation rate of both T and non-T lymphoid cells. The possible pathogenetic factors and the prognostic value of these results are discussed.     

Fourier analysis of nuclear and cytoplasmic shape of blood lymphoid cells from healthy donors and chronic lymphocytic leukemia patients

The folding rates of the contours of nuclei and entire lymphoid cells were analyzed by Fourier analysis of the shapes. Smears of peripheral blood from healthy subjects and from patients with chronic lymphocytic leukemia (CLL: type B, stage zero) were routinely prepared and stained. The shapes of lymphoid cells from CLL patients revealed a higher folding rate (from fifth to tenth harmonics) than did those of lymphocytes from healthy subjects. Accordingly, the roughness coefficient (describing the folding rate of the surface) for CLL cells was 0.036, as compared to 0.028 for the cells of healthy subjects. The shapes of nuclei of CLL lymphoid cells also had a higher folding rate than did those of lymphocytes from healthy subjects, but a significant difference was found only for the highest harmonic calculated (the tenth harmonic); the respective roughness coefficients for nuclei were 0.037 and 0.033.     

CXCL12 is a costimulator for CD4+ T cell activation and proliferation in chronic lymphocytic leukemia patients

Activated T cells from patients with chronic lymphocytic leukemia (CLL) provide survival and proliferative signals to the leukemic clone within lymphoid tissues. Recruitment of both, CLL cells and T lymphocytes, to this supportive microenvironment greatly depends on CXCL12 production by stromal and myeloid cells. CXCL12 also supplies survival stimuli to leukemic B cells, but whether it exerts stimulatory effects on T lymphocytes from CLL patients is unknown. In order to evaluate the capacity of CXCL12 to increase CD4⁺ T cell activation and proliferation in CLL patients, peripheral blood mononuclear cells were cultured with or without recombinant human CXCL12 or autologous nurse-like cells, and then T cell activation was induced by anti-CD3 mAb. CXCL12 increases the proliferation and the expression of CD25, CD69, CD154, and IFNγ on CD3-stimulated CD4⁺ T cells from CLL patients, similarly in T cells from ZAP-70⁺ to ZAP-70^? patients. Autologous nurse-like cells establish a close contact with CD4⁺ T cells and increase their activation and proliferation partially through a CXCR4-dependent mechanism. In addition, we found that activated T cells in the presence of CXCL12 enhance the activation and proliferation of the leukemic clone. In conclusion, CXCL12 production by lymphoid tissue microenvironment in CLL patients might play a key dual role on T cell physiology, functioning not only as a chemoattractant but also as a costimulatory factor for activated T cells.     

Interactions between bone marrow stromal microenvironment and B-chronic lymphocytic leukemia cells: Any role for Notch,Wnt and Hh signaling pathways?

B-cell chronic lymphocytic leukemia (CLL), which is the most common lymphoproliferative disorder, displays characteristics consistent with a defect in programmed cell death and exhibit prolonged survival of affected cells in vivo. When recovered from peripheral blood or lymphoid tissues of patients and cultured in vitro, CLL malignant cells rapidly undergo spontaneous apoptosis. CLL B-cells co-culture with different adherent cell types, collectively referred to as stromal cells, induces leukemia cell survival, migration, and drug resistance. In addition, such survival-promoting microenvironments can rescue leukemia cells from cytotoxic therapy, giving way to disease relapse. Quite surprisingly considering that many anti-cancer drugs, including γ-secretase inhibitors, Cyclopamine and Quercetin, were reported to block Notch, Wnt, and Hedgehog anti-apoptotic signaling pathways respectively, the link between the latter anti-apoptotic pathways and bone marrow stromal cells in CLL has been pointed out only recently. Data concerning the pathogenesis of CLL have been critically reviewed in regards to the growing body of evidence indicating deregulations of Notch, Wnt and Hedgehog anti-apoptotic signaling pathways in the stromal microenvironment of affected cells.     

Decreased L system amino acid transport and decreased gamma-glutamyl transpeptidase are independent processes in human chronic lymphocytic leukemia B-lymphocytes

The L system of amino acid transport is markedly diminished in chronic lymphocytic leukemia (CLL) B-lymphocytes, with a maximal velocity less than 15% that of normal B-lymphocytes. Another membrane-associated function, the activity of the ectoenzyme, gamma-glutamyl transpeptidase (GGT), is diminished in CLL B-cells to 30% that of normal B-cells. In addition to its transpeptidase activity, a role for GGT has been postulated in the transport of amino acids. In the present report, the possible relationship of these two physiologic functions CLL B-cells was studied. The L system transport defect in CLL is restored by phorbol ester-induced cell maturation; following incubation with 0.15 microM tetradecanoyl phorbol acetate (TPA) for 17 hours, the L system initial velocity showed a 20-fold increase. In contrast, there was no significant effect on GGT activity with cell maturation. Furthermore, an antibody which diminished GGT activity by 50% in lymphoid cells did not inhibit L system transport. Thus, the impaired L system amino acid transport and GGT activity appear to be independent processes in CLL B-cells.     

Expression of ERp5 and GRP78 on the membrane of chronic lymphocytic leukemia cells: association with soluble MICA shedding

MICA is a ligand of the activating receptor NKG2D, expressed by NK and T cells. MICA expression is induced in cancer cells favoring their elimination by the immune system; however, many advanced tumors shed soluble MICA (sMICA), which impairs NKG2D-mediated cytotoxicity. ERp5 and GRP78 are endoplasmic reticulum-resident proteins that are translocated to the surface of epithelial tumor cells where they interact with MICA and are involved in sMICA shedding. In this study, we analyze the role of ERp5 and GRP78 in sMICA shedding in chronic lymphocytic leukemia (CLL). Immunofluorescence and flow cytometry analyses showed that ERp5 and GRP78 were significantly expressed on the surface of B cells and leukemia cells, but they were not expressed on T cells. The expression of ERp5 and GRP78 was significantly higher in leukemia cells than in B cells from controls. ERp5 and GRP78 co-localized with MICA on the surface of leukemia cells and the levels of expression of ERp5 and GRP78 correlated with the level of expression of membrane-bound MICA in CLL patients. Associated with higher expression of membrane-bound ERp5 and GRP78, serum sMICA levels were approximately threefold higher in patients than in controls. Elevated sMICA levels in CLL patients were associated with the down-modulation of NKG2D surface expression on CD8 T cells. Finally, pharmacological inhibition of B cell lines and stimulated leukemia cells showed that ERp5 activity is involved in sMICA shedding in CLL. In conclusion, these results uncover a molecular mechanism which regulates MICA protein shedding and immune evasion in CLL.     

Stimulation of Autochthonous Lymphocytes by Cells from Normal and Leukaemic Lines

THE mixed lymphocyte reaction (MLR) can possibly be regarded as an in vitro form of an in vivo phenomenon reflecting the recognition of “non-self” tumour specific or neo-antigens on the surface of lymphoid cells. A reaction similar to the normal MLR but of greater magnitude occurs when irradiated lymphoid cells from lymphoblastoid cell lines (LCL) are added to freshly isolated peripheral lymphocytes from allogeneic individuals^1,2. The intense stimulation which occurred in every case when irradiated cells from various LCL were added to lymphocytes from a large number of individuals³ suggested the presence of extra surface determinants on the cells, which are not present on normal freshly isolated cells. We have investigated whether freshly isolated lymphoid cells could detect and respond to extra antigenic determinants on the surface of cell lines derived more than 3 months earlier from their own lymphoid cells.     

Intraclonal cell expansion and selection driven by B cell receptor in chronic lymphocytic leukemia

The mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes utilized by chronic lymphocytic leukemia (CLL) clones defines two disease subgroups. Patients with unmutated IGHV have a more aggressive disease and a worse outcome than patients with cells having somatic IGHV gene mutations. Moreover, up to 30% of the unmutated CLL clones exhibit very similar or identical B cell receptors (BcR), often encoded by the same IG genes. These "stereotyped" BcRs have been classified into defined subsets. The presence of an IGHV gene somatic mutation and the utilization of a skewed gene repertoire compared with normal B cells together with the expression of stereotyped receptors by unmutated CLL clones may indicate stimulation/selection by antigenic epitopes. This antigenic stimulation may occur prior to or during neoplastic transformation, but it is unknown whether this stimulation/selection continues after leukemogenesis has ceased. In this study, we focused on seven CLL cases with stereotyped BcR Subset #8 found among a cohort of 700 patients; in six, the cells expressed IgG and utilized IGHV4-39 and IGKV1-39/IGKV1D-39 genes, as reported for Subset #8 BcR. One case exhibited special features, including expression of IgM or IgG by different subclones consequent to an isotype switch, allelic inclusion at the IGH locus in the IgM-expressing cells and a particular pattern of cytogenetic lesions. Collectively, the data indicate a process of antigenic stimulation/selection of the fully transformed CLL cells leading to the expansion of the Subset #8 IgG-bearing subclone.     

Human T cell differentiation antigens and correlation of their expression with various markers of T cell maturation.

Two sets of differentiation antigens are demonstrated on human T cells by using 11 heterologous anti-human antisera raised against various normal and malignant T cells. The two antigenic determinants from the first set of differentiation antigens are expressed only on thymus cells and on T lymphoblasts, whereas the two antigenic determinants from the second set are expressed on blood T cells, Sezary cells, T.CLL cells, and thymus cells. Four T cell phenotypes are thus defined; two phenotypes are expressed only by T lymphoblasts, whereas the other two phenotypes are expressed both by normal and malignant T cells. Moreover, a clear-cut relationship exists between the four T cell antigenic phenotypes and two other markers of T cell differentiation: terminal deoxynucleotidyl transferase and peanut agglutinin. Two phenotypes are linked with the presence of TdT, one phenotype is linked with the affinity for PNA, and the fourth phenotype is correlated with the absence of both markers.     

In vitro culture studies of granulocyte/macrophage and erythroid progenitor cells in lymphoproliferative disorders

Granulocyte/macrophage progenitor cells (CFU-GM) and erythroid progenitor cells (BFU-E) have been assayed in peripheral blood (PB) and/or bone marrow (BM) from 12 patients with acute lymphocytic leukemia (ALL), 16 patients with chronic lymphocytic leukemia (CLL) and 31 patients with various forms of non-Hodgkin lymphoma (NHL) without BM involvement. Progenitor cell growth in PB and BM from the NHL patients did not differ statistically from controls (p greater than 0.1). CFU-GM and BFU-E per ml PB were markedly increased in ALL and CLL patients (p less than 0.001) while CFU-GM and BFU-E per plated BM cells from these patients were severely depressed (p less than 0.001). Lymphoblasts from one ALL patient failed to inhibit CFU-GM and BFU-E-derived colony growth from control PB mononuclear cells. The high levels of circulating progenitor cells in ALL and CLL patients clearly distinguish them from other cytopenic hematological malignancies, in which decreased progenitor cell levels have been demonstrated previously (acute myeloid leukemia, hairy cell leukemia). The cause of this finding and its pathophysiological implication still remains to be established.     

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.     

Mitogen induced cellular cytotoxicity (MICC) in multiple myeloma.

Mitogen induced cellular cytotoxicity (MICC) was noted to be markedly increased in patients with multiple myeloma as compared to normal controls and to patients with chronic lymphocytic leukemia (CLL). Enhanced MICC was present at various effector-to-target cell ratios and at several mitogen concentrations. Removal of adherent, phagocytic cells by carbonyl iron, glass wool, or rayon columns abolished the MICC response from the peripheral blood of both multiple myeloma patients and normal controls. Thus, the effector cell mediating MICC may be monocytic in origin and closely resembles the suppressor cell for immunoglobulin synthesis described in patients with multiple myeloma. Our data suggest that the MICC assay with chicken red blood cells as targets may provide a convenient method for identifying pathologic conditions where this cytotoxic effector cell population plays an active role.     

Expansion of NK Cells and Reduction of NKG2D Expression in Chronic Lymphocytic Leukemia. Correlation with Progressive Disease

The immune system may mediate anti-tumor responses in chronic lymphocytic leukemia (CLL) which may affect disease progression and survival. In this study, we analyzed the immune characteristics of 99 consecutive previously diagnosed CLL patients and 50 healthy controls. The distribution of lymphocyte subsets at diagnosis was retrospectively analyzed. Compared with controls, leukemia patients showed an expansion of NK and CD8 T cells at diagnosis. The relative number of CD8 T cells at diagnosis was associated with time to treatment, suggesting that CD8 T cells may modify disease progression. The distribution of lymphocyte subsets was analyzed again when patients were enrolled in this study. The median time since these patients were diagnosed was 277 weeks. Compared with diagnosis, the absolute number of CD8 T cells significantly decreased in these patients, reaching similar values to healthy controls; however NK cells kept significantly elevated overtime. Nevertheless, NK cells showed an impaired expression of NKG2D receptor and a defective cytotoxic activity. This down-regulation of NKG2D expression was further enhanced in patients with advanced and progressive disease. Additionally, membrane NKG2D levels significantly decreased on CD8 T cells, but a significant increase of NKG2D+CD4+ T cells was observed in CLL patients. The cytotoxic activity of NK cells was diminished in CLL patients; however the treatments with IL-2, IL-15, IL-21 and lenalidomide were able to restore their activity. The effect of IL-2 and IL-15 was associated with the increase of NKG2D expression on immune cells, but the effect of IL-21 and lenalidomide was not due to NKG2D up-regulation. The expansion of NK cells and the reversibility of NK cell defects provide new opportunities for the immunotherapeutic intervention in CLL.     

Analysis of translational products of Friend strain of spleen focus-forming virus.

We have analyzed normal rat kidney cells nonproductively infected with the Friend strain of spleen focus-forming virus (SFFV) for the presence of murine leukemia virus-specific type C viral proteins. SFFV was found to code for the p15 and p12 proteins of Friend-murine leukemia virus as determined by immunological typing of their antigenic determinants. Molecular-size analysis of p15 and p12 proteins in SFFV nonproductively infected normal rat kidney cells indicated that these proteins are translated as parts of polyprotein molecules. The apparent molecular weights of the polypeptides bearing p12 antigenic determinants revealed the presence of translational products of the SFFV genome that could not be accounted for by know type C virus helper structural proteins.     

Histone deacetylase inhibitors potentiate TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in lymphoid malignancies

New therapies are required for chronic lymphocytic leukemia (CLL), an incurable disease characterized by failure of mature lymphocytes to undergo apoptosis. Activation of cell surface death receptors, such as via TRAIL receptor ligation, may provide a novel therapeutic target for various malignancies. However, CLL and other lymphoid malignancies are resistant to TRAIL. We report that low concentrations of histone deacetylase (HDAC) inhibitors, such as depsipeptide, which alone failed to induce apoptosis, markedly sensitize CLL cells and other primary lymphoid malignancies to TRAIL-induced apoptosis. These combinations caused little or no toxicity to normal lymphocytes. HDAC inhibitors sensitized resistant cells to TRAIL-induced apoptosis by facilitating formation of an active death-inducing signalling complex (DISC), leading to the rapid activation of caspase-8. The facilitated DISC formation also occurred in the absence of TRAIL-R2 upregulation. Thus, the combination of HDAC inhibitors and TRAIL may be valuable in the treatment of various hemopoietic malignancies.