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In cancer or hematologic disorders, chemokines act as growth- or survival factors, regulating hematopoiesis and angiogenesis, determining metastatic spread and controlling leukocyte infiltration into tumors to inhibit antitumor immune responses. The aim was to quantify the release of CXCL8, −9, −10, CCL2, −5, and IL-12 in AML/MDS-pts' serum by cytometric bead array and to correlate data with clinical subtypes and courses. Minimal differences in serum-levels subdivided into various groups (e.g. age groups, FAB-types, blast-proportions, cytogenetic-risk-groups) were seen, but higher release of CXCL8, −9, −10 and lower release of CCL2 and −5 tendentially correlated with more favorable subtypes (<50 years of age, <80% blasts in PB). Comparing different stages of the disease higher CCL5-release in persisting disease and a significantly higher CCL2-release at relapse were found compared to first diagnosis – pointing to a change of 'disease activity' on a chemokine level. Correlations with later on achieved response to immunotherapy and occurrence of GVHD were seen: Higher values of CXCL8, −9, −10 and CCL2 and lower CCL5-values correlated with achieved response to immunotherapy. Predictive cut-off-values were evaluated separating the groups in 'responders' and 'non-responders'. Higher levels of CCL2 and −5 but lower levels of CXCL8, −9, −10 correlated with occurrence of GVHD. We conclude, that in AML-pts' serum higher values of CXCL8, −9, −10 and lower values of CCL5 and in part of CCL2 correlate with more favorable subtypes and improved antitumor'-reactive function. This knowledge can contribute to develop immune-modifying strategies that promote antileukemic adaptive immune responses. [ABSTRACT FROM AUTHOR]

We examined the bone marrow of 45 patients with MDS at the time of diagnosis and in the course of the disease by means of Southern blot analysis and cytogenetic studies to detect and evaluate clonal markers and their implication on the prognosis of the disease and the response to treatment. All patients were enrolled in an EORTC study and received low-dose Ara-C with or without growth factors according to the study protocol. Thirty patients (67%) were characterized by different clonal markers, such as various gene rearrangements (eg Ig-JH, tcR-beta, bcr, GM-CSF, G-CSF or IL-3) and/or chromosomal markers at the time of diagnosis or early in the course of the disease. In 23 of 30 cases that could be studied in the course of the disease, a statement about the clonal situation was possible: in three cases (8%) the clonal situation did not change, in nine cases (39%) at least a transient reduction of clonal cells could be demonstrated, suggesting partial or complete response to therapy. In eight cases (35%) a change for the worse could be seen. In four cases (17%) involvement of multiple clones could be demonstrated with the clones exhibiting different susceptibilities to treatment. Clinical evaluation showed that patients without clonal markers at diagnosis had a better prognosis as compared to patients who presented with clonal markers. We suggest that clonality analysis at diagnosis and in the course of the disease will be a useful tool to study the biology and response to treatment in MDS. [ABSTRACT FROM AUTHOR]

Dendritic cells (DC) and T-cells are mediators of CTL-responses. Autologous (from patients with acute myeloid leukaemia (AML) or myelodysplasia (MDS)) or allogeneic (donor)-T-cells stimulated by DC leu , gain an efficient lysis of naive blasts, although not in every case. CXCL8, -9, -10, CCL2, -5 and Interleukin (IL-12) were quantified by Cytometric Bead Array (CBA) in supernatants from 5 DC-generating methods and correlated with AML-/MDS-patients' serum-values, DC-/T-cell-interactions/antileukemic T-cell-reactions after mixed lymphocyte culture (MLC) and patients' clinical course. The blast-lytic activity of T-cells stimulated with DC or mononuclear cells (MNC) was quantified in a cytotoxicity assay. Despite great variations of chemokine-levels, correlations with post-stimulation (after stimulating T-cells with DC in MLC) improved antileukemic T-cell activity were seen: higher released chemokine-values correlated with improved T-cells' antileukemic activity (compared to stimulation with blast-containing MNC) - whereas with respect to the corresponding serum values higher CXCL8-, -9-, and -10- but lower CCL5- and -2-release correlated with improved antileukemic activity of DC-stimulated (vs. blast-stimulated) T-cells. In DC-culture supernatants higher chemokine-values correlated with post-stimulation improved antileukemic T-cell reactivity, whereas higher serum-values of CXCL8, -9, and -10 but lower serum-values of CCL5 and -2 correlated with post-stimulation improved antileukemic T-cell-reactivity. In a context of 'DC'-stimulation (vs serum) this might point to a change of (CCL5 and -2-associated) functionality from a more 'inflammatory' or 'tumor-promoting' to a more 'antitumor'-reactive functionality. This knowledge could contribute to develop immune-modifying strategies that promote antileukemic (adaptive) immune-responses., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Expression of CD137 ligand (4-1BBL), a member of the TNF family of proteins, has been reported on several types of APCs, various carcinoma cells, and can be induced on activated T cells. In this study, we report that the soluble ligand was released constitutively at low levels from leukocytes and at higher levels following cellular activation. Release from cells was blocked by addition of a metalloproteinase inhibitor which concomitantly caused the accumulation of 4-1BBL on the cell surface. In addition, we show that a soluble form of 4-1BBL was present at high levels in the sera of some patients with various hematological diseases, but only at low levels in healthy donors. Soluble 4-1BBL was active in that it competed with recombinant 4-1BBL for binding to the 4-1BB receptor and was able to costimulate IL-2 and IFN-gamma release from peripheral T cells. These results indicate that the release of soluble 4-1BBL from the cell surface is mediated by one or more sheddases and likely regulates 4-1BB-4-1BBL interactions between cells in vivo. Cleavage of 4-1BBL to an active soluble form would alter both proximal and distal cellular responses, including cell survival and costimulatory or inflammatory responses, that are mediated through the 4-1BB pathway. This, in turn, would likely alter disease progression or outcome.

Objectives: The persistence of clonal cells after chemotherapy, or a re-emerging of clonal cells in remission (CR) or at relapse in patients with acute myeloid leukemia (AML) was studied to assess the prognostic significance of the amount of clonal DNA in predicting the clinical outcome., Methods: Clonal rearrangements in the gene sequences of retinoic acid receptor (RAR) alpha, major breakpoint cluster region (M-bcr), immunoglobulin (Ig)-JH, T-cell receptor (TcR) beta, myeloid lymphoid leukemia or cytokines (GM-CSF, G-CSF, IL-3) detected in bone marrow samples from 37 patients with primary AML (pAML) or secondary AML (sAML) were investigated. A relative increase or decrease of clonal DNA in the course of AML was evaluated by comparing the optical densities of DNA bands of the rearranged genes and the total amount of DNA., Results: High amounts of clonal DNA were detectable at diagnosis, during persisting disease and at relapse (Ø 39%, 35%, or 38% of total DNA, respectively), compared to 20% in complete remission (CR). Amounts of clonal DNA (except for Ig-JH gene rearrangements) were of prognostic significance at diagnosis, patients with less than 33% clonal DNA were characterized by significantly longer relapse-free survival times (all cases: p = 0.01; pAML: p = 0.002). Patients in CR exhibiting less than 5% (all cases) or 15% (pAML) clonal DNA showed longer relapse-free survival times (p = 0.08 or p = 0.03, respectively). Vice versa, significantly higher amounts of clonal DNA (all cases 51% vs. pAML 54%) could be detected in cases studied at diagnosis who relapsed in the following 5 months (all cases p = 0.01) or 14 months (pAML p = 0.007). Significantly higher amounts of clonal DNA (33%) could be detected in cases studied in CR who relapsed in the following 4 months (all cases p = 0.002 or pAML p = 0.006, respectively). Moreover, we could prove disease progression on a cellular level months before the clinical onset of sAML after a period of MDS., Conclusions: Clonal, gene-rearranged DNA is regularly detectable at diagnosis and during persisting AML, in CR and at relapse. However, the presence, rather than the amount of clonal DNA detectable in CR is predictive for relapse. These data might indicate the significance of gene rearrangement analyses in the course of AML to identify cases with a high risk of relapse, independently from the karyotype.

We have already shown that cytokine cocktails (IL-1beta, IL-3, IL-6, SCF, GM-CSF) and/or lymphokine-activated killer (LAK) cells can reduce the amounts of clonal, CD34-positive mononuclear bone marrow cells (BM-MNC) in acute myeloid leukemia (AML). In addition, the influence of those cocktails and/or LAK cells on the clonogenic potential of AML BM-MNC was investigated. BM colonies cultured in agar during different stages of the disease were immunophenotyped in situ: 17 patients at diagnosis, 14 patients in complete remission, 8 patients at relapse, 8 healthy donors. A significant reduction in leukemic cells and colonies positive for CD34 after in vitro culture of BM-MNC with cytokine cocktails was achieved with all samples obtained at diagnosis (n = 8, p < 0.01), in 6 of 8 cases in complete remission but only in 2 of 6 cases at relapse. Cytokine cocktails stimulated granulopoiesis as well as B and T lymphopoiesis. Colonies with leukemic phenotype could never be detected in healthy BM. A significant reduction in leukemic colonies was achieved by coculture of BM-MNC (uncultured or cytokine precultured) with autologous LAK cells in all 4 cases at diagnosis and in 1 case at relapse. An additive effect of in vitro cytokine preincubation of BM samples on the leukemia-reducing effect of LAK cells could be demonstrated in all samples studied (p < 0.001; diagnosis: n = 10, relapse: n = 3, complete remission: n = 7). Patients had a better prognosis if CD34-positive colonies in AML could be reduced by cytokine incubation (p = 0.03) or coculture with autologous LAK cells in vitro (p = 0.04). Our data show that cytokines as well as LAK cells alone and in combination can reduce, however not eliminate clonogenic AML cells. Such mechanisms might be responsible for maintaining stable remissions in AML., (Copyright 2001 S. Karger AG, Basel)

We studied the influence of autologous lymphokine-activated-killer (LAK) cells on the survival of clonal and CD34-positive bone marrow (BM) cells from patients with acute myeloid leukemia (AML) in a coculture assay in vitro. (1) LAK cells were grown in the presence of IL-2, in some cases additionally with IL-6. (2) These cytotoxic cells were cocultured with (untreated or cytokine pretreated) AML-BM cells obtained at different stages of the disease. Therefore BM cells were (a) either frozen in liquid nitrogen or (b) precultured for 14 days with cytokines: IL-1beta, IL-3, IL-6, erythropoietin (EPO), stem cell factor (SCF) with ('Cytok1') or without granulocyte macrophage colony stimulating factor (GM-CSF) ('Cytok2') or with no added cytokines ('ISC/FCS') as a control. (3) Southern blot analysis was used to detect clonal BM cells. At diagnosis, 76 of 151 cases (50%) studied showed clonal gene rearrangements in marker genes. (4) Southern blot analysis and flow cytometry were used to compare the amount of clonal and CD34 positive BM cells before and after coculture procedures. Coculture experiments with untreated BM and autologous LAK cells led to a reduction of clonal cells in 2 of 5 cases at diagnosis, in 11 of 17 BM samples in complete remission but not in the one case studied at relapse. Similar results were found if precultured AML cells (with or without cytokines) were cocultivated with LAK cells. However the cytotoxic effect of LAK cells was more pronounced if cytokines (especially GM-CSF and SCF) were comprised. Our data indicate, that (1) clonality in AML can be demonstrated by Southern blot analysis; (2) CD34 positive cells in AML are clonal, gene rearranged cells; (3) clonal cell populations persist in BM during complete remission and relapse in most of the patients; (4) incubation of AML-BM cells with LAK cells lead to a reduction of clonal, rearranged cells in 11 of 17 AML cases in complete remission, but only in 2 of 6 cases at diagnosis or relapse; (5) AML cells can be sensitized to theLAK cell treatment by preincubation of AML-BM cells with cytokines (IL-1beta, IL-3, IL-6, SCF, EPO and GM-CSF) or by adding SCF to the coculture conditions. Southern blot analysis and flow cytometry are appropriate methods to detect and quantify leukemic disease. Cytokines and LAK cells synergize to kill AML blasts in vitro. This is a feasible approach to immunotherapy of AML and merits further investigations.

The clinical course of chronic myeloid leukemia (CML) is highly variable and therefore it is difficult to predict the duration of the chronic phase. We studied the immunological expression of maturation patterns in 62 cases of CML (30 cases in clinical/cytological blast crisis (BC), 32 cases in clinical/cytological chronic phase (CP) by means of a double marker enzyme immuno assay (DM-EIA). Immunological findings were supplemented by Southern blots using Ig-JH-, TCRbeta- and bcr-probes. Patients in BC (n = 30) expressed high proportions of CD10, CD20, CD33, CD34 and low degrees of a mature myeloid marker (CD15). Myeloid BC bone marrow (BM) cells showed a high degree of coexpression of unusual, lineage restricted markers: 25% of CD15-positive cells also expressed markers like CD10, CD20 or CD34. In contrast, BM cells in lymphoid BC did not show this coexpression. In CP two groups were distinguished immunologically: concordant cases which were immunologically normal (n = 14) and discordant cases (n = 18) which showed increased proportions of unusual, lineage restricted markers and double labelled cells (e.g. CD15/CD34). The latter group developed clinical BC earlier during further follow up (p = 0.009). Cases of lymphoid BC (n = 11)--in contrast to acute lymphoblastic leukemia (ALL) patients (n = 21)--did not show coexpression of CD15/CD10, CD20, CD34. These data show that blast clones can be detected in CML-CP by characteristic immunological maturation defects several months before the clinical onset of BC. Moreover, the lymphoid "blasts" of CML-BC represent a relatively differentiated lymphoid population of cells which can be distinguished from ALL by their lack of coexpression of unusual, lineage restricted markers.

We report the case of a 49-year-old woman with acute myeloid leukemia (AML, M5b). The leukemic cells expressed blast as well as myelomonocytic antigens and were characterized by a clonal gene rearrangement of the immunoglobulin (Ig) JH gene. During the course of the disease in clinical/cytological complete remission (CR) the persistence of leukemic cells was shown by surface marker analyses on bone marrow (BM) cells or BM clones grown in agar. Moreover, clonal leukemic cells could be detected by Southern blot analyses indicating the persistence of blasts. Occasionally BM cells obtained in CR showed a cytologically, immunologically differentiated phenotype, but nevertheless DNA-rearranged cells indicated differentiated blasts ('clonal remission'). Five years after diagnosis the patient presented with an isolated pleural effusion. Southern blot analysis identified the original leukemic clone because pleural cells showed the same clonal Ig gene rearrangement as the cells at diagnosis. The patient died 6 years after diagnosis during her fourth relapse of AML. This shows the value of Southern blot analyses, immunophenotyping and culture techniques to monitor the course of leukemic tumor burden even if it is not cytologically identifiable.

Acute myeloid leukemia (AML) is usually thought to represent a monoclonal disease. Among 45 cases of newly diagnosed AML we found rearranged bands in 14 cases using the Southern blot methodology and immunoglobulin (Ig) joining region (JH) or T-cell receptor (TCR beta) probes. In three patients, the findings indicated an oligoclonal disease. One case was characterized by several bands in the JH blot, some of which reappeared at different time points during remission. A second case had monoclonally rearranged Ig-JH sequences in the bone marrow but exclusively germline configuration in DNA from peripheral blood cells despite the presence of 84% blasts. A third case was characterized by two different, Ig-JH and c mu gene rearranged cell populations at diagnosis but relapsed with a germline pattern without reappearance of the previous clones. These data indicate that AML may differentiate along different lineages with predominant appearance of one or the other subclone in the course of the disease.