Sigi Kay

The CXCR4 antagonist AMD3100 impairs survival of human AML cells and induces their differentiation

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, participate in the retention of acute myeloblastic leukemia (AML) cells within the bone marrow microenvironment and their release into the circulation. AML cells also constitutively express SDF-1-dependent elastase, which regulates their migration and proliferation. To study the molecular events and genes regulated by the SDF-1/CXCR4 axis and elastase in AML cells, we examined gene expression profiles of the AML cell line, U937, under treatment with a neutralizing anti-CXCR4 antibody or elastase inhibitor, as compared with non-treated cells, using DNA microarray technology. Unsupervised hierarchical clustering analysis demonstrated similar gene expression profiles of anti-CXCR4 antibody or elastase inhibitor-treated cells, as compared with control. Pathway and functional analysis showed a greater tendency toward differentiation in cells under either one of both treatment modalities. Thus given, we further analyzed the effects of CXCR4 inhibition on AML cell growth and differentiation using the antagonist AMD3100. AMD3100 arrested proliferation in AML cell lines and triggered changes that mimicked differentiation, including morphological changes and the expression of myeloid differentiation antigens. Inhibition of elastase also triggered the differentiation of AML cells. Our study defines a new role for the SDF-1/CXCR4 axis in the regulation of leukemic cell survival and differentiation.

Quantitative flow cytometry of ZAP-70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome.

ZAP‐70 has emerged as a potential pivotal prognostic marker for patients with chronic lymphocytic leukemia (CLL), which could replace immunoglobulin heavy chain mutation status. Although several flow cytometry assays have been described for assessing ZAP‐70 in CLL, certain technical and scientific issues remain unsolved, which have prevented results of this crucial test from being reported, even in the best routine flow cytometry laboratories. In this report, we aimed to solve some of these issues by providing a computerized quantitative flow cytometric assay for ZAP‐70 within the entire CLL population, which would be easy to perform and enable standardization between laboratories.

T-cell ZAP-70 overexpression in chronic lymphocytic leukemia (CLL) correlates with CLL cell ZAP-70 levels, clinical stage and disease progression

T-cell ZAP-70 overexpression in chronic lymphocytic leukemia (CLL) correlates with CLL cell ZAP-70 levels, clinical stage and disease progression

Mesenchymal stromal cells revert multiple myeloma cells to less differentiated phenotype by the combined activities of adhesive interactions and interleukin-6

Multiple myeloma is characterized by the malignant growth of immunoglobulin producing plasma cells, predominantly in the bone marrow. The effects of primary human mesenchymal stromal cells on the differentiation phenotype of multiple myeloma cells were studied by co-culture experiments. The incubation of multiple myeloma cells with bone marrow-derived mesenchymal stromal cells resulted in significant reduction of the expression of the predominant plasma cell differentiation markers CD38 and CD138, and cell surface immunoglobulin light chain. While the down-regulation of CD138 by stromal cells was completely dependent on their adhesive interactions with the multiple myeloma cells, interleukin-6 induced specific down-regulation of CD38. Mesenchymal stromal cells or their conditioned media inhibited the growth of multiple myeloma cell line, thereby reducing the overall amounts of secreted light chains. Analysis of primary multiple myeloma bone marrow samples reveled that the expression of CD38 on multiple myeloma cells was not affected by adhesive interactions. The ex vivo propagation of primary multiple myeloma cells resulted in significant increase in their differentiation markers. Overall, the data indicate that the bone marrow-derived mesenchymal stromal cells revert multiple myeloma cells to less differentiated phenotype by the combined activities of adhesive interactions and interleukin-6.

Absolute monocyte count trichotomizes chronic lymphocytic leukemia into high risk patients with immune dysregulation, disease progression and poor survival

Peripheral absolute monocyte count (AMC) has been reported to correlate with clinical outcome in different types of cancers. This association may relate to alteration in circulating monocytic subpopulations and tumor infiltrating macrophages. In this study we evaluated the clinical significance of peripheral AMC in 80 treatment naive patients with CLL. Measurement of AMC was based on direct morphological enumeration, due to our findings that complete blood count data may yield incorrect monocytes enumeration values in CLL. The median AMC in patients with CLL was within normal limits, however the AMC range exceeded the values of healthy individuals. The AMC trichotomized patients into 3 distinct sub-groups with different characteristics and outcomes. High AMC patients were younger and had higher absolute lymphocytes count, while patients with low AMC had prominent immune dysregulation (lower serum IgA levels, susceptibility to infections and a tendency for positive direct anti-globulin test). The low and high AMC patients had a shorter time to treatment compared to the intermediates AMC subgroups, whereas low AMC was associated with increased mortality caused by infectious complications. In conclusion, AMC quantification during the disease course classifies CLL patients into subgroups with unique clinical features and outcomes.

Unexpected detection of monoclonal B-cell lymphocytosis in a HLA-matched sibling donor on the day of allogeneic stem cell transplantation for a patient with chronic lymphocytic leukaemia: clinical outcome

Chronic lymphocytic leukaemia (CLL) remains an incurable disease but is responsive to conventional chemo-immunotherapy. Allogeneic stem cell transplantation (allo-SCT) is currently the only potentially curative modality of therapy for CLL, achieving this via the graft-versus-leukaemia effect (Kharfan-Dabaja, et al, 2007). In the last decade it has been shown that healthy individuals with lymphocyte counts <5Æ0 · 10/l can harbour monoclonal B-lymphocytes, without any clinical evidence of disease. Such cases are now defined as monoclonal B-cell lymphocytosis (MBL) and diagnosed by flow-cytometry, when cells with a CLL phenotype are evident. MBL occurs in more than 3% of adults with normal blood counts, mostly in older individuals, and is more frequent in first degree relatives of patients with CLL (13Æ5%) (Rawstron, et al, 2002a, Rawstron, et al, 2002b). The biology of MBL is still not well understood but most cases remain stable for a long time, whereas others evolve into CLL but only a minority require treatment (Fung, et al, 2007, Hardy, et al, 2007). Recently, a large prospective study on MBL showed that the B-cell count at presentation is an independent factor predicting for progression, and that 15% of CLL-phenotype MBL subjects with initial lymphocyte counts of >4Æ0 · 10/l progressed to CLL during 6Æ7 years of follow-up (Rawstron, et al, 2008). These individuals carried an annual risk of needing chemotherapy of 1–2%. MBL was also found in 15% of potential sibling donors who were screened for CLL patients requiring transplant (Del Giudice, et al, 2009). Here, we report the clinical outcome of an alloSCT performed in a patient with CLL where his human leucocyte antigen (HLA)-matched sibling sister donor was unexpectedly found to have MBL on the actual day of stem cell collection. In 2001, a 58-year-old male was diagnosed with Binet stage C, CLL. He was treated with a fludarabine-containing regimen and achieved a partial remission. Two years later, there was rapid clinical progression from which he achieved a good partial response after retreatment with a combination of chlorambucil, mitoxantrone and prednisone. In March 2005, after completing further therapy for a second progression and subsequent splenectomy, he underwent a reduced intensity allo-SCT with cells from his healthy asymptomatic HLAidentical sister, who was 66 years old, had no history of previous diseases and had a normal blood cell count (leucocytes, 6Æ4 · 10/l; absolute lymphocyte count, 2Æ7 · 10/l, haemoglobin, 14Æ2 g/l; platelet count, 2Æ23 · 10/l). Peripheral blood stem cells were harvested after 4 d of granulocyte colony-stimulating factor administration (10 lg/kg/d). However unexpectedly, on the day of transplantation, while evaluating the collected donor cells by routine flow cytometry to assess the number of CD34 and CD3 cells present, we detected a relatively high percentage of B-cells, with a CLL-type phenotype: CD5,CD19,CD20,CD23, surface k+,CD38 and ZAP70 (whereas the recipient CLL cells were k+, CD38 and ZAP70). The patient and the donor were both immediately informed about these unexpected findings and gave consent to continue with the procedure, as the conditioning regimen with fludarabine, melphalan, and alemtuzumab had already been given. The transplant was uneventful and on day 90 there was mixed chimerism with 84% donor cells. As planned, donor lymphocyte infusions (DLI) were given to the host, to maintain stable mixed chimerism of >90% donor cells and subsequently he achieved complete remission. In November 2008, 3 years after transplantation, the leucocyte count increased to 16Æ700 · 10/l, with 75Æ6% lymphocytes and we then started to determine whether the relapse was of patient or donor origin. The peripheral blood lymphocytes had a typical CLL phenotype but were also CD38 and IGHV sequencing revealed that the CLL cells were IGHV4-30-1/IGHV4-31 with a 100% germline homology. The donor’s cells were a different B-cell clone, of IGHV1-18 with 97Æ3% germline homology, while fluorescent in situ hybridization analysis revealed mixed cell populations, one with XY (62Æ5%) and another with XX (38Æ5%) chromosomes. However, only cells of the male recipient carried the 13q14 deletion. Short tandem repeat (STR) DNA analysis of all the peripheral blood cells showed mixed chimerism (55% of donor origin). CLL cells then were purified using CD19 magnetic microbeads and STR analysis showed that they were clearly of patient origin (Fig 1). As the relapse was apparently host-derived, the patient was treated with interferon-a and cyclophosphamide and donor stem cells and DLI was added. Currently, 13 months after this therapy, the patient has normal blood counts with no evidence of CLL and retains a complete donor genotype by STR. During this entire period, the donor has been healthy and still has normal blood cell counts without any progression. Donor cell leukaemia (DCL) is rare following allo-SCT (Flynn and Kaufman 2007) and most of the reported cases in Correspondence

Reciprocal changes in regulatory T cells and Th17 helper cells induced by exercise in patients with chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is one of the most commonly diagnosed lymphoid malignancies in Western countries [1]. Although currently incurable, better understanding of the pathophysiology of CLL and the pivotal role played by immune regulation in disease progression has paved the way for the introduction of immunomodulatory drugs as part of therapy for CLL. CD4 CD25 high regulatory T cells (Tregs) are involved in suppressing host immunity. FoxP3 plays a key role in Treg function and is a specifi c marker for these cells [2]. Transforming growth factor (TGF) β , together with interleukin (IL)-2, regulates the signaling pathways that control Treg suppressive activity [2,3]. Elevated Treg counts were reported in patients with solid tumors, providing an additional mechanism explaining reduced immunity in these patients [2]. Increased numbers of Tregs were also demonstrated in patients with CLL, with the highest levels evident in untreated patients or in patients with progressive disease [4]. T helper 17 (Th 17) cells are powerful proinfl ammatory, IL-17-producing CD4 lymphocytes with a possible role in anti-tumor immune-surveillance [5]. Th e CD4 T cell lineage shows great plasticity, and suppressive Tregs can diff erentiate into Th 17 eff ector cells in the presence of TGF β and IL-6 [6].

Lymphocyte activation gene 3: a novel therapeutic target in chronic lymphocytic leukemia

A novel therapeutic approach in cancer, attempting to stimulate host anti-tumor immunity, involves blocking of immune checkpoints. Lymphocyte activation gene 3 (LAG3) is an immune checkpoint receptor expressed on activated/exhausted T cells. When engaged by the major histocompatibility complex (MHC) class II molecules, LAG3 negatively regulates T-cell function, thereby contributing to tumor escape. Intriguingly, a soluble LAG3 variant activates both immune and malignant MHC class II-presenting cells. In the study herein, we examined the role of LAG3 in the pathogenesis of chronic lymphocytic leukemia, an MHC class II-presenting malignancy, and show that chronic lymphocytic leukemia cells express and secrete LAG3. High levels of surface and soluble LAG3 were associated with the unmutated immunoglobulin variable heavy chain leukemic subtype and a shorter median time from diagnosis to first treatment. Utilizing a mechanism mediated through MHC class II engagement, recombinant soluble LAG3-Ig fusion protein, LAG3-Fc, activated chronic lymphocytic leukemia cells, induced anti-apoptotic pathways and protected the cells from spontaneous apoptosis, effects mediated by SYK, BTK and MAPK signaling. Moreover, LAG3 blocking antibody enhanced in vitro T-cell activation. Our data suggest that soluble LAG3 promotes leukemic cell activation and anti-apoptotic effects through its engagement with MHC class II. Furthermore, MHC class II-presenting chronic lymphocytic leukemia cells may affect LAG3-presenting T cells and impose immune exhaustion on their microenvironment; hence, blocking LAG3-MHC class II interactions is a potential therapeutic target in chronic lymphocytic leukemia.

Divergence in CD19-Mediated Signaling Unfolds Intraclonal Diversity in Chronic Lymphocytic Leukemia, Which Correlates with Disease Progression

Emerging data on intraclonal diversity imply that this phenomenon may play a role in the clinical outcome of patients with chronic lymphocytic leukemia (CLL), where subsets of the CLL clone responding more robustly to external stimuli may gain a growth and survival advantage. In this study, we report intraclonal diversity resolved by responses to CD19 engagement in CLL cells, which can be classified into CD19-responsive (CD19-R) and -nonresponive subpopulations. Engagement of CD19 by anti-CD19 Ab rapidly induced cellular aggregation in the CD19-R CLL cells. The CD19-R CLL cells expressed higher surface levels of CD19 and c-myc mRNA, exhibited distinct morphological features, and were preferentially abolished in rituximab-treated patients. Both subpopulations reacted to sIgM stimulation in a similar manner and exhibited similar levels of Akt and Erk phosphorylation, pointing to functional signaling divergence within the BCR. CD19 unresponsiveness was partially reversible, where nonresponding CD19 cells spontaneously recover their signaling capacity following incubation in vitro, pointing to possible in vivo CD19–signaling attenuating mechanisms. This concept was supported by the lower CD19-R occurrence in bone marrow–derived samples compared with cells derived from the peripheral blood of the same patients. CLL patients with >15.25% of the CD19-R cell fraction had a shorter median time to treatment compared with patients with <15.25% of CD19-R cell fraction. In conclusion, divergence in CD19-mediated signaling unfolds both interpatient and intraclonal diversity in CLL. This signaling diversity is associated with physiological implications, including the location of the cells, their responses to anti-CLL therapeutics, and disease progression.

Rapid in vivo testing of drug response in multiple myeloma made possible by xenograft to turkey embryos

Background:The best current xenograft model of multiple myeloma (MM) in immune-deficient non-obese diabetic/severe-combined immunodeficient mice is costly, animal maintenance is complex and several weeks are required to establish engraftment and study drug efficacy. More practical in vivo models may reduce time and drug development cost. We recently described a rapid low-cost xenograft model of human blood malignancies in pre-immune turkey. Here, we report application of this system for studying MM growth and the preclinical assessment of anticancer therapies.Methods:Cell lines and MM patient cells were injected intravenously into embryonic veins on embryonic day 11 (E11). Engraftment of human cells in haematopoietic organs was detected by quantitative real-time polymerase chain reaction, immunohistochemistry, flow cytometry and circulating free light chain.Results:Engraftment was detected after 1 week in all embryos injected with cell lines and in 50% of those injected with patient cells. Injection of bortezomib or lenalinomide 48 h after cell injection at therapeutic levels that were not toxic to the bone marrow dramatically reduced MM engraftment.Conclusion:The turkey embryo provides a practical, xenograft system to study MM and demonstrates the utility of this model for rapid and affordable testing therapeutics in vivo. With further development, this model may enable rapid, inexpensive personalised drug screening.