BCL-2 Inhibitor ABT-737 Effectively Targets Leukemia-Initiating Cells with Differential Regulation of Relevant Genes Leading to Extended Survival in a NRAS/BCL-2 Mouse Model of High Risk-Myelodysplastic Syndrome.

During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.

BCL-2 inhibition with ABT-737 prolongs survival in an NRAS/BCL-2 mouse model of AML by targeting primitive LSK and progenitor cells.

Myelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1(+)/c-Kit(+), and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS-AML transformation and survival of leukemic cells.

Novel fusion between the breakpoint cluster region and platelet-derived growth factor receptor-alpha genes in a patient with chronic myeloid leukemia-like neoplasm: undetectable residual disease after imatinib therapy.

Rare patients suffering from myeloid neoplasms share clinical and cytological features indistinguishable from chronic myeloid leukemia (CML) but lack the BCR-ABL1 fusion gene. Several studies provide evidence that alterations in genes encoding tyrosine kinase receptors such as the platelet-derived growth factor receptor (PDGFR) may be involved in the pathogenesis of these disorders. Here we describe a patient with a rare CML-like disease in whom we identified a novel in-frame BCR-PDGFRA rearrangement joining BCR exon 17 to PDGFRA exon 13, resulting in overexpression of PDGFRA. The design of a specific quantitative PCR assay to monitor the molecular response during treatment with imatinib, a multitargeted tyrosine kinase inhibitor (TKI) with activity against ABL, c-Kit, and PDGFRA revealed an outstanding disease control with durably undetectable BCR-PDGFRA transcripts. Multiple TKIs are currently available yet with distinct target profiles; thus, accurate molecular diagnosis and monitoring tools are essential to establish tailored treatments and assess response to therapy in this type of rare hematological malignancy.

Splenic marginal zone lymphoma: current knowledge and future directions.

Splenic marginal zone lymphoma (SMZL), along with extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) and nodal marginal zone lymphoma (NMZL), share a common origin from the "marginal zone." However, these three entities display different clinical characteristics, reflecting probable biological variations according to the organ and cellular origin. Within the past decade, new data have been reported regarding pathogenic mechanisms as well as therapeutic advances. Clinically, SMZL presents as an indolent and disseminated disease at diagnosis, with a specific clinical presentation that includes predominantly splenomegaly, and in half of patients, autoimmune manifestations. Establishing the diagnosis may be difficult, especially distinguishing SMZL from other low-grade lymphomas, such as small B-cell lymphomas; however, recent findings have contributed to a better characterization of the disease, and the criteria for diagnosis have been improved. Therapeutic approaches consist of splenectomy or immunochemotherapy, but there is no consensus regarding the best treatment, except when SMZL is associated with hepatitis C virus infection. In this article, we review the current knowledge on the biological findings, clinical features, and therapeutic approaches for SMZL.

Non-MALT marginal zone lymphoma.

PURPOSE OF REVIEW: Non-MALT marginal zone lymphoma regroups two subtypes of lymphoma, the splenic marginal zone lymphoma (SMZL) and the nodal marginal zone lymphoma (NMZL). Although they share a common cell of origin from the 'marginal zone', they display different clinical characteristics, reflecting probable biological variations according to the organ. RECENT FINDINGS: Within the past decade, new data regarding pathogenic mechanisms as well as therapeutic advances have been reported. SUMMARY: SMZL and NMZL often present with disseminated disease at diagnosis, with specific clinical presentation, SMZL with predominant enlarged splenomegaly and NMZL with disseminated nodal involvement. Diagnosis may be difficult among the small B-cell lymphomas and criteria for diagnosis have been recently improved. The therapeutic approaches comprise splenectomy for SMZL, and immunochemotherapy for both of SMZL and NMZL, but with no consensus about the best treatment, except when associated with hepatitis C virus. This review addresses the current knowledge on the biological findings, clinical features and therapeutic approaches for the individual SMZLs and NMZLs.

ZAP-70 protein expression and CD38 positivity in B-cell chronic lymphocytic leukemia.

BACKGROUND: The clinical course, disease progression, and survival of B-cell chronic lymphocytic leukemia (B-CLL) have been correlated with immunoglobulin heavy-chain variable region mutation status. The biologic parameters 70-kDa zeta-associated protein (ZAP-70) and CD38 expression are easier and faster surrogate markers for mutational status. OBJECTIVE: To assess retrospectively ZAP-70 expression in B-CLL cells using flow cytometry and examine its relationship with CD38 expression and the median time from diagnosis to initial therapy. METHODS: Ninety-four unselected patients who had their follow-up in the outpatient clinic from 2004 to 2005 were reviewed for immunophenotyping ZAP-70 and CD38 expression. Direct immunolabeling with clone 2E3.2, isotype IgG2a, enabled easy quantification of ZAP-70 by flow cytometry in association with CD38 expression; in addition, the mean fluorescence intensity ratio (MFIR) of CD19+CD5+ B-CLL cells compared to an isotype control monoclonal antibody was determined. RESULTS: ZAP-70 expression levels in B-CLL cells varied widely (0.3-99%). The median time to therapy was significantly shorter for the 54 patients with 20% or more ZAP-70+ cells (30 months) than for the 40 patients with less than 20% ZAP-70+ cells (median time to treatment not reached). The optimal MFIR for classifying patients as ZAP-70+ was 2. Thirty-two patients had a threshold of ZAP-70+CD38+ greater than 30%, with a median time from diagnosis to treatment of 19 months. Regardless of CD38 expression level, CD38 and ZAP-70 expressions were significantly associated. The median interval from diagnosis to initial therapy was 16.2 months for ZAP-70+CD38+ patients, 60 months for ZAP-70+CD38- or ZAP-70-CD38+ patients, and had not yet been reached for ZAP-70-CD38- patients. CONCLUSION: The association of ZAP-70+CD19+CD5+ B-CLL cells and percentage of CD38+CD19+CD5+ B-CLL cells evaluated by flow cytometry provide reliable methods that could be introduced into a routine diagnostic B-CLL panel to predict outcome.

Telehematology: a pilot experience of cytological diagnosis of acute myeloid leukemia via the Internet. A GOELAMS study.

Although modern communication technology is well developed, telehematology does not readily lend itself to practical laboratory use. Multicenter therapeutic protocols may offer preferential opportunities. The cytologists of the AML-2001 protocol established an innovative organization to demonstrate the reliability of the diagnostic assessment of acute myeloid leukemia through a rapid and decentralized exchange of information via the internet and to define the conditions optimizing expert diagnosis. Telediagnosis appears to be a powerful tool for cytological review and other issues.

GEP analysis validates high risk MDS and acute myeloid leukemia post MDS mice models and highlights novel dysregulated pathways.

BACKGROUND: In spite of the recent discovery of genetic mutations in most myelodysplasic (MDS) patients, the pathophysiology of these disorders still remains poorly understood, and only few in vivo models are available to help unravel the disease. METHODS: We performed global specific gene expression profiling and functional pathway analysis in purified Sca1+ cells of two MDS transgenic mouse models that mimic human high-risk MDS (HR-MDS) and acute myeloid leukemia (AML) post MDS, with NRASD12 and BCL2 transgenes under the control of different promoters MRP8NRASD12/tethBCL-2 or MRP8[NRASD12/hBCL-2], respectively. RESULTS: Analysis of dysregulated genes that were unique to the diseased HR-MDS and AML post MDS mice and not their founder mice pointed first to pathways that had previously been reported in MDS patients, including DNA replication/damage/repair, cell cycle, apoptosis, immune responses, and canonical Wnt pathways, further validating these models at the gene expression level. Interestingly, pathways not previously reported in MDS were discovered. These included dysregulated genes of noncanonical Wnt pathways and energy and lipid metabolisms. These dysregulated genes were not only confirmed in a different independent set of BM and spleen Sca1+ cells from the MDS mice but also in MDS CD34+ BM patient samples. CONCLUSIONS: These two MDS models may thus provide useful preclinical models to target pathways previously identified in MDS patients and to unravel novel pathways highlighted by this study.

DNA-mediated adjuvant immunotherapy extends survival in two different mouse models of myeloid malignancies.

We have previously shown that a specific promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) DNA vaccine combined with all-trans retinoic acid (ATRA) increases the number of long term survivors with enhanced immune responses in a mouse model of acute promyelocytic leukemia (APL). This study reports the efficacy of a non-specific DNA vaccine, pVAX14Flipper (pVAX14), in both APL and high risk myelodysplastic syndrome (HR-MDS) models. PVAX14 is comprised of novel immunogenic DNA sequences inserted into the pVAX1 therapeutic plasmid. APL mice treated with pVAX14 combined with ATRA had increased survival comparable to that obtained with a specific PML-RARA vaccine. Moreover, the survival advantage correlated with decreased PML-RARA transcript levels and increase in anti-RARA antibody production. In HR-MDS mice, pVAX14 significantly improved survival and reduced biomarkers of leukemic transformation such as phosphorylated mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1. In both preclinical models, pVAX14 vaccine significantly increased interferon gamma (IFNγ) production, memory T-cells (memT), reduced the number of colony forming units (CFU) and increased expression of the adapter molecule signalling to NF-κB, MyD88. These results demonstrate the adjuvant properties of pVAX14 providing thus new approaches to improve clinical outcome in two different models of myeloid malignancies, which may have potential for a broader applicability in other cancers.

Successful rituximab treatment of an EBV-related lymphoproliferative disease arising after autologous transplantation for angioimmunoblastic T-cell lymphoma.

Rituximab treatment of B-cell lymphoproliferative disease following transplantation is being evaluated. We describe an Epstein-Barr virus-related B-cell lymphoma that developed in a 55-year-old woman, one year after autologous transplantation for relapsing angioimmunoblastic T-cell lymphoma. Complete remission was achieved after four cycles of rituximab and reduced-dose CHOP. This case is discussed in the context of severe immunodepression. Monoclonal anti-CD20 antibodies might restore a balance between T-cell immunosurveillance and EBV proliferation in B-cells,

Long-term follow-up analysis of 100 patients with splenic marginal zone lymphoma treated with splenectomy as first-line treatment.

Splenectomy is considered as one of the first-line treatments for symptomatic patients with splenic marginal zone lymphoma (SMZL). Between 1997 and 2012, 100 hepatitis C virus-negative patients with SMZL were treated by splenectomy as first-line treatment. At 6 months, all patients but three recovered from all cytopenias. The median lymphocyte count at 6 months and 1 year was 11.51 × 10(9)/L and 6.9 × 10(9)/L, respectively. Median progression-free survival (PFS) was 8.25 years. The 5-year and 10-year overall survival (OS) rates were 84% and 67%, respectively. Histological transformation occurred in 11% of patients, and was the only parameter significantly associated with a shorter time to progression (p = 0.0001). Significant prognostic factors for OS were age (p = 0.0356) and histological transformation (p = 0.0312). In this large retrospective cohort, we confirmed that splenectomy as first-line treatment in patients with SMZL corrected cytopenias and lymphocytosis within the first year and was associated with a good PFS.

Localization of the NRAS:BCL-2 complex determines anti-apoptotic features associated with progressive disease in myelodysplastic syndromes.

We have previously demonstrated that two prognostic features of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML), mutant NRAS and over-expressing BCL-2, cooperate physically and functionally in vivo. Screening of MDS patient bone marrow (BM) identified NRAS:BCL-2 co-localization in 64% cases, correlating with percentage BM blasts, apoptotic features and disease status (p<0.0001). Localization of the complex at the plasma membrane or the mitochondria correlated with disease and apoptosis features in MDS patients, whilst caspase-9 mediated mechanism was elucidated in vivo and in vitro. The intensity and localization of the RAS:BCL-2 complex merits further evaluation as a novel biomarker of MDS.

Spontaneous abrogation of the G2DNA damage checkpoint has clinical benefits but promotes leukemogenesis in Fanconi anemia patients.

DNA damage checkpoints in the cell cycle may be important barriers against cancer progression in human cells. Fanconi anemia (FA) is an inherited DNA instability disorder that is associated with bone marrow failure and a strong predisposition to cancer. Although FA cells experience constitutive chromosomal breaks, cell cycle arrest at the G2 DNA damage checkpoint, and an excess of cell death, some patients do become clinically stable, and the mechanisms underlying this, other than spontaneous reversion of the disease-causing mutation, are not well understood. Here we have defined a clonal phenotype, termed attenuation, in which FA patients acquire an abrogation of the G2 checkpoint arrest. Attenuated cells expressed lower levels of CHK1 (also known as CHEK1) and p53. The attenuation could be recapitulated by modulating the ATR/CHK1 pathway, and CHK1 inhibition protected FA cells from cell death. FA patients who expressed the attenuated phenotype had mild bone marrow deficiency and reached adulthood, but several of them eventually developed myelodysplasia or leukemia. Better understanding of attenuation might help predict a patient's clinical course and guide choice of treatment. Our results also highlight the importance of evaluating the cellular DNA damage checkpoint and repair pathways in cancer therapies in general.

Mutual benefits of B-ALL and HLDA/HCDM HLDA 9th Barcelona 2010.

The B-cell panel of the ninth HLDA was applied in a multicentre fashion to cryopreserved cells from 46 patients with acute lymphoblastic leukemia. The reagents were aliquoted and shipped to volunteer participants from the French Groupe d'Etude Immunologique des Leucémies (GEIL). All samples were tested in flow cytometry, and the results collected as of the strength of labeling of the leukemic clone as negative, weak or strong. Among the 64 antibodies tested, the strongest and most frequent staining was observed for CD305 (LAIR), CD229 (Ly9), CD200 (OX-2) and, to a lesser extent, CD361 (EVI2b). Details of the observations, and information about the molecules tested are provided in the manuscript as well as a summary table.

Normal levels of peripheral CD19(+) CD5(+) CLL-like cells: toward a defined threshold for CLL follow-up -- a GEIL-GOELAMS study.

BACKGROUND: The development of flow cytometry as a useful tool for the detection of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) is potentially hampered by the fact that a normal subset of B-cells with a similar immunophenotype is present in the peripheral blood. This subset of CLL-like cells is not well defined in terms of frequency. METHODS: Here, we performed a multicenter study with a panel of four-color antibody combinations possibly useful for the detection of MRD in CLL, to establish the levels of normal CLL-like cells in 49 healthy controls. ROC curves established the upper level of such cells at 4 × 10(-4) . The two best combinations were further applied to 419 samples from 117 treated CLL patients. RESULTS: The combinations CD19/CD5/CD43/CD79b and CD19/CD5/CD81/CD22 appeared very robust and well correlated to enumerate normal CLL-like cells in a lysis no-wash approach. In follow-up samples from CLL patients, they disclosed only 9.8% of the samples within the normal range. In more than 90% of the cases, it was thus possible to report confidently on the absence or presence of MRD in these patients. CONCLUSIONS: This manuscript reports on the frequency of CD19(+) CD5(+) B-cells in normal peripheral blood and confirms the combinations recommended by the European research initiative on CLL as being performing to assess remaining CLL cells above a threshold of 4 × 10(-4) white blood cells.

BCL-2 and mutant NRAS interact physically and functionally in a mouse model of progressive myelodysplasia.

Myelodysplastic syndromes (MDS) are clonal stem cell hematologic disorders that evolve to acute myeloid leukemia (AML) and thus model multistep leukemogenesis. Activating RAS mutations and overexpression of BCL-2 are prognostic features of MDS/AML transformation. Using NRASD12 and BCL-2, we created two distinct models of MDS and AML, where human (h)BCL-2 is conditionally or constitutively expressed. Our novel transplantable in vivo models show that expression of hBCL-2 in a primitive compartment by mouse mammary tumor virus-long terminal repeat results in a disease resembling human MDS, whereas the myeloid MRP8 promoter induces a disease with characteristics of human AML. Expanded leukemic stem cell (Lin(-)/Sca-1(+)/c-Kit(+)) populations and hBCL-2 in the increased RAS-GTP complex within the expanded Sca-1(+) compartment are described in both MDS/AML-like diseases. Furthermore, the oncogenic compartmentalizations provide the proapoptotic versus antiapoptotic mechanisms, by activating extracellular signal-regulated kinase and AKT signaling, in determination of the neoplastic phenotype. When hBCL-2 is switched off with doxycycline in the MDS mice, partial reversal of the phenotype was observed with persistence of bone marrow blasts and tissue infiltration as RAS recruits endogenous mouse (m)BCL-2 to remain active, thus demonstrating the role of the complex in the disease. This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex. The colocalization of BCL-2 and RAS in the bone marrow of MDS/AML patients offers targeting either oncogene as a therapeutic strategy.