Immune-monitoring of myelodysplastic neoplasms: Recommendations from the i4MDS consortium.

Advancements in comprehending myelodysplastic neoplasms (MDS) have unfolded significantly in recent years, elucidating a myriad of cellular and molecular underpinnings integral to disease progression. While molecular inclusions into prognostic models have substantively advanced risk stratification, recent revelations have emphasized the pivotal role of immune dysregulation within the bone marrow milieu during MDS evolution. Nonetheless, immunotherapy for MDS has not experienced breakthroughs seen in other malignancies, partly attributable to the absence of an immune classification that could stratify patients toward optimally targeted immunotherapeutic approaches. A pivotal obstacle to establishing "immune classes" among MDS patients is the absence of validated accepted immune panels suitable for routine application in clinical laboratories. In response, we formed International Integrative Innovative Immunology for MDS (i4MDS), a consortium of multidisciplinary experts, and created the following recommendations for standardized methodologies to monitor immune responses in MDS. A central goal of i4MDS is the development of an immune score that could be incorporated into current clinical risk stratification models. This position paper first consolidates current knowledge on MDS immunology. Subsequently, in collaboration with clinical and laboratory specialists, we introduce flow cytometry panels and cytokine assays, meticulously devised for clinical laboratories, aiming to monitor the immune status of MDS patients, evaluating both immune fitness and identifying potential immune "risk factors." By amalgamating this immunological characterization data and molecular data, we aim to enhance patient stratification, identify predictive markers for treatment responsiveness, and accelerate the development of systems immunology tools and innovative immunotherapies.

The protein kinase C agonist PEP005 increases NF-kappaB expression, induces differentiation and increases constitutive chemokine release by primary acute myeloid leukaemia cells.

Acute myeloid leukaemia (AML) cells show constitutive release of several chemokines that occurs in three major clusters: (I) chemokine (C-C motif) ligand (CCL)2-4/chemokine (C-X-C motif) ligand (CXCL)1/8, (II) CCL5/CXCL9-11 and (III) CCL13/17/22/24/CXCL5. Ingenol-3-angelate (PEP005) is an activator of protein kinase C and has antileukaemic and immunostimulatory effects in AML. We investigated primary AML cells derived from 35 unselected patients and determined that PEP005 caused a dose-dependent increase in the release of chemokines from clusters I and II, including several T cell chemotactic chemokines. The release of granulocyte-macrophage colony-stimulating factor and hepatocyte growth factor was also increased. CCL2-4/CXCL1/8 release correlated with nuclear factor (NF)-kappaB expression in untreated AML cells, and PEP005-induced chemokine production was associated with further increases in the expression of the NF-kappaB subunits p50, p52 and p65. Increased DNA binding of NF-kappaB was observed during exposure to PEP005, and the specific NF-kappaB inhibitor BMS-345541 reduced constitutive chemokine release even in the presence of PEP005. Finally, PEP005 decreased expression of stem cell markers (CD117, CXCR4) and increased lineage-associated CD11b and CD14 expression. To conclude, PEP005 has a unique functional pharmacological profile in human AML. Previous studies have described proapoptotic and T cell stimulatory effects and the present study describes additional T cell chemotactic and differentiation-inducing effects.

Nuclear factor-kappaB signaling: a contributor in leukemogenesis and a target for pharmacological intervention in human acute myelogenous leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy with only 40%-50% long-term survival even for younger patients who can receive the most aggressive therapy. For elderly patients who only receive palliative treatment, the median survival is only 2-3 months. Inhibition of the nuclear factor-kappaB (NF-kappaB) transcription factor family is one of the therapeutic strategies that are considered in AML. NF-kappaB is an important regulator of several biological processes that are involved in leukemogenesis, including proliferation, differentiation, autophagy, and apoptosis. Constitutive NF-kappaB activation has been detected in AML cells and NF-kappaB inhibition is therefore a possible therapeutic strategy in AML. Multiple pharmacological agents have shown inhibitory effects against NF-kappaB signaling pathways, including proteasome inhibitors as well as the more-specific agents that are directed against various steps of this signaling pathway. Recent studies strongly suggest that primary human AML cells (including AML stem cells) are susceptible to NF-kappaB inhibition, but this therapeutic approach should possibly be combined with other therapeutic agents to achieve a combined effect both on NF-kappaB transcriptional activity, tumor suppressor-induced signaling, and stress-induced pathways. The clinical documentation with regard to the efficiency and safety of NF-kappaB inhibition is still limited, but experimental evidence strongly suggests that NF-kappaB inhibition should be further investigated in human AML.

Circulating T cells derived from acute leukemia patients with severe therapy-induced cytopenia express a wide range of chemokine receptors.

Normal T cells can mediate antileukemic reactivity after allogeneic stem cell transplantation and T cell targeting immunotherapy is now considered for patients receiving conventional chemotherapy. This antileukemic reactivity is most effective in patients with a low leukemia cell burden, and this burden is expected to be lowest early after transplantation/chemotherapy when patients are cytopenic. Local T cell recruitment will then be essential for the efficiency of the antileukemic response. In this context, the authors compared the chemokine receptor expression for T cells derived from healthy individuals and acute myelogenous leukemia patients with therapy-induced cytopenia after conventional chemotherapy or allogeneic stem cell transplantation. Circulating CD3(+) T cells showed the same chemokine receptor expression for all three groups: CCR1(low), CCR2(low), CCR3(low), CCR4(intermediate), CCR5(intermediate), CCR7(low/intermediate), CXCR2(low), CXCR3(intermediate), and CXCR4(high). Thus, only minor differences between the groups were observed when comparing individual receptors, and we therefore conclude that the chemokine receptor profiles of circulating CD3(+) T cells show no qualitative and only minor quantitative differences for these three groups.

Anticancer immunotherapy in combination with proapoptotic therapy.

Induction of immune responses against cancer-associated antigens is possible, but the optimal use of this strategy remains to be established and especially the combination of T cell therapy and the use of new targeted therapeutic agents should be investigated. The design of future clinical studies then has to consider several issues. Firstly, induction of anticancer T cell reactivity seems most effective in patients with low disease burden. Initial disease-reducing therapy including surgery, irradiation and conventional or new targeted chemotherapy should therefore be used, preferably through induction of immunogenic cancer cell death. Secondly, after the induction phase effector T cells will induce cancer cell apoptosis mainly through the intrinsic apoptosis-regulating pathway. The effect of this anticancer immune reactivity should be strengthened by the administration of chemotherapy that mediates additional proapoptotic signalling through the external apoptosis-initiating pathway, blocking of anti-apoptotic signalling or inhibition of survival signalling. Thirdly, conventional chemotherapy and new targeted therapy have direct immunosuppressive effects on the T cell system, but even patients with severe chemotherapy-induced lymphopenia have an operative T cell system and immunotherapy may therefore be initiated immediately or early after disease-reducing therapy when the cancer cell burden is expected to be lowest. Finally, chemotherapy toxicity on human T cells is not a random process, and one should especially focus on the possibility to strengthen anticancer immune reactivity through chemotherapy-induced elimination or inhibition of immunosuppressive regulatory T cells. All these issues need to be considered in the design of future clinical studies combining chemotherapy and immunotherapy.

In vitro induction of a dendritic cell phenotype in primary human acute myelogenous leukemia (AML) blasts alters the chemokine release profile and increases the levels of T cell chemotactic CCL17 and CCL22.

Immunotherapy is now considered in acute myelogenous leukemia (AML). A dendritic cell (DC) phenotype can be induced in primary human AML cells by in vitro culture in the presence of various cytokine combinations. The aim was to investigate whether this phenotypic alteration is associated with altered chemokine release. AML cells were cultured according to four protocols that have been characterized in detail for AML-DC induction: (1) granulocyte-macrophage colony-stimulating factor (GM-CSF) + interleukin-4 (IL-4) days 1-14 and tumor necrosis factor-alpha (TNF-alpha) for days 6-14, (2) GM-CSF + IL-4 + TNF-alpha + FMS-like tyrosine kinase 3-ligand (Fl3-L) for 8 days, (3) GM-CSF + IL-4 + TNF-alpha + Flt3-L + stem cell factor (SCF) + transforming growth factor-beta1 (TGF-beta1) for 8 days, and (4) 25 Gy gamma-irradiation combined with culture in the presence of GM-CSF + SCF + IL-3 for 4 days. Significantly increased AML-DC release of CCL17 and CCL22 was observed for protocols 1, 2, and 3, whereas effects on CCL2-5, CXCL8, and CXCL10 differed in all protocols. Neutralization studies using a transwell migration assay demonstrated the increased level of CCL17 and CCL22 release was important for AML-DC chemotaxis of normal T cells. Induction of a dendritic AML cell phenotype is associated with an altered chemokine release profile. Detailed characterization of chemokine release should be included in future studies of AML-DC vaccination.

Cyclin B1 is commonly expressed in the cytoplasm of primary human acute myelogenous leukemia cells and serves as a leukemia-associated antigen associated with autoantibody response in a subset of patients.

OBJECTIVES: Aberrant expression of cyclin B1, a cell cycle regulator, is related to prognosis in various human malignancies. Additionally, cytoplasmic expression of cyclin B1 in epithelial malignancies is associated with a specific T-cell response and presumably also a humoral immune response. We therefore investigated (i) whether a similar expression pattern could be detected in native human acute myelogenous leukemia (AML) cells and (ii) whether cyclin B1 specific antibodies could be detected in AML. METHODS: AML cell expression of cyclin B1 was analyzed by flow cytometry and confocal microscopy. Humoral immune response in AML patient sera against cyclin B1 was analyzed by ELISA. RESULTS: AML cell expression of cyclin B1 was detected for all 42 patients; but the percentage of cyclin B1 positive cells showed a wide variation between patients. Confocal microscopy demonstrated that 32/42 (76%) patient samples showed abnormal cytoplasmic expression. Furthermore, the cytoplasmic expression was maintained after 14 d of in vitro culture and differentiation of the AML cells towards a dendritic cell phenotype. Cyclin B1 specific serum antibodies could be detected for seven of 65 patients with untreated AML. CONCLUSIONS: Our studies demonstrate that primary human AML cells show aberrant cytoplasmic expression of cyclin B1 for a majority of patients and a specific humoral immune response was also detected for a subset of patients with untreated leukemia.

Subclassification of patients with acute myelogenous leukemia based on chemokine responsiveness and constitutive chemokine release by their leukemic cells.

BACKGROUND AND OBJECTIVES: Chemokines are soluble mediators involved in angiogenesis, cellular growth control and immunomodulation. In the present study we investigated the effects of various chemokines on proliferation of acute myelogenous leukemia (AML) cells and constitutive chemokine release by primary AML cells. DESIGN AND METHODS: Native human AML cells derived from 68 consecutive patients were cultured in vitro. We investigated AML cell proliferation (3H-thymidine incorporation, colony formation), chemokine receptor expression, constitutive chemokine release and chemotaxis of normal peripheral blood mononuclear cells. RESULTS: Exogenous chemokines usually did not have any effect on AML blast proliferation in the absence of hematopoietic growth factors, but when investigating growth factor-dependent (interleukin 3 + granulocyte-macrophage colony-stimulating factor + stem cell factor) proliferation in suspension cultures the following patient subsets were identified: (i) patients whose cells showed chemokine-induced growth enhancement (8 patients); (ii) divergent effects on proliferation (15 patients); and (iii) no effect (most patients). These patient subsets did not differ in chemokine receptor expression, but, compared to CD34- AML cells, CD34+ cells showed higher expression of several receptors. Chemokines also increased the proliferation of clonogenic AML cells from the first subset of patients. Furthermore, a broad constitutive chemokine release profile was detected for most patients, and the following chemokine clusters could be identified: CCL2-4/CXCL1/8, CCL5/CXCL9-11 (possibly also CCL23) and CCL13/17/22/24/CXCL5 (possibly also CXCL6). Only the CCL2-4/CXCL1/8 cluster showed significant correlations between corresponding mRNA levels and NFkB levels/activation. The chemotaxis of normal immunocompetent cells for patients without constitutive chemokine release was observed to be decreased. INTERPRETATION AND CONCLUSIONS: Differences in chemokine responsiveness as well as chemokine release contribute to patient heterogeneity in AML. Patients with AML can be classified into distinct subsets according to their chemokine responsiveness and chemokine release profile.

T lymphocyte chemotactic chemokines in acute myelogenous leukemia (AML): local release by native human AML blasts and systemic levels of CXCL10 (IP-10), CCL5 (RANTES) and CCL17 (TARC).

T cell targeting immunotherapy is now considered in acute myelogenous leukemia (AML), and local recruitment of antileukemic T cells to the AML microcompartment will then be essential. This process is probably influenced by both intravascular as well as extravascular levels of T cell chemotactic chemokines. We observed that native human AML cells usually showed constitutive secretion of the chemotactic chemokines CXCL10 and CCL5, whereas CCL17 was only released for a subset of patients and at relatively low levels. Coculture of AML cells with nonleukemic stromal cells (i.e., fibroblasts, osteoblasts) increased CXCL10 and CCL17 levels whereas CCL5 levels were not altered. However, a wide variation between patients in both CXCL10 and CCL5 levels persisted even in the presence of the stromal cells. Neutralization of CXCL10 and CCL5 inhibited T cell migration in the presence of native human AML cells. Furthermore, serum CCL17 and CXCL10 levels varied between AML patients and were determined by disease status (both chemokines) as well as patient age, chemotherapy and complicating infections (only CCL17). Thus, extravascular as well as intravascular levels of T cell chemotactic chemokines show a considerable variation between patients that may be important for T cell recruitment and the effects of antileukemic T cell reactivity in local AML compartments.

Antiangiogenic therapy in acute myelogenous leukemia: targeting of vascular endothelial growth factor and interleukin 8 as possible antileukemic strategies.

Acute myelogenous leukemia (AML) is an aggressive disorder with an overall disease-free survival of 40-50% even for the younger patients under 60 years of age who can receive the most intensive treatment. The median age at the time of diagnosis is 60-65 years, and the large majority of elderly patients usually receive less intensive chemotherapy or only supportive therapy due to the high treatment-related mortality when using intensive therapy for elderly individuals. Thus, there is a need for new therapeutic approaches to improve the treatment in younger patients and to make AML-directed therapy with acceptable toxicity possible in elderly individuals. Angiogenesis seems to be important both for leukemogenesis and susceptibility to intensive chemotherapy, and antiangiogenic strategies are therefore considered for the treatment of AML. The two proangiogenic mediators vascular endothelial growth factor (VEGF) and interleukin 8, (IL-8, also referred to as CXCL8) seem to be important in human AML: VEGF is released at increased levels due to interactions between AML cells and neighboring nonleukemic cells, whereas IL-8 is released at high levels by native human AML cells. Thus, VEGF as a therapeutic target in AML is suggested both by experimental and clinical observations, whereas IL-8 as a target is mainly suggested by experimental evidence. In the present review we describe and discuss (i) the angioregulatory network of soluble mediators in AML, including both the systemic levels and local release by native human AML cells; and (ii) various therapeutic approaches to target VEGF and IL-8. Although single angioregulatory mediators can be targeted, it should be emphasized that the final effect of soluble mediators on angioregulation is determined by a complex angioregulatory network that varies between AML patients, and the final effect of targeting single mediators may therefore differ between patient subsets.