Systemic in vivo lentiviral delivery of miR-15a/16 reduces malignancy in the NZB de novo mouse model of chronic lymphocytic leukemia.

Similar to human chronic lymphocytic leukemia (CLL), the de novo New Zealand Black (NZB) mouse model has a genetically determined age-associated increase in malignant B-1 clones and decreased expression of microRNAs miR-15a and miR-16 in B-1 cells. In the present study, lentiviral vectors were employed in vivo to restore miR-15a/16, and both the short-term single injection and long-term multiple injection effects of this delivery were observed in NZB. Control lentivirus without the mir-15a/16 sequence was used for comparison. We found that in vivo lentiviral delivery of mir-15a/16 increased miR-15a/16 expression in cells that were transduced (detected by GFP expression) and in sera when compared with control lentivirus treatment. More importantly, mice treated with the miR-expressing lentivirus had decreased disease. The lentivirus had little systemic toxicity while preferentially targeting B-1 cells. Short-term effects on B-1 cells were direct effects, and only malignant B-1 cells transduced with miR-15a/16 lentivirus had decreased viability. In contrast, long-term studies suggested both direct and indirect effects resulting from miR-15a/16 lentivirus treatment. A decrease in B-1 cells was found in both the transduced and non-transduced populations. Our data support the potential use of systemic lentiviral delivery of miR-15a/16 to ameliorate disease manifestations of CLL.

Selective requirement for c-Myc at an early stage of V(alpha)14i NKT cell development.

Valpha14 invariant (Valpha14i) NKT cells are a subset of regulatory T cells that utilize a semi-invariant TCR to recognize glycolipids associated with monomorphic CD1d molecules. During development in the thymus, CD4(+)CD8(+) Valpha14i NKT precursors recognizing endogenous CD1d-associated glycolipids on other CD4(+)CD8(+) thymocytes are selected to undergo a maturation program involving sequential expression of CD44 and NK-related markers such as NK1.1. The molecular requirements for Valpha14i NKT cell maturation, particularly at early developmental stages, remain poorly understood. In this study, we show that CD4-Cre-mediated T cell-specific inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biological activities, selectively impairs Valpha14i NKT cell development without perturbing the development of conventional T cells. In the absence of c-Myc, Valpha14i NKT cell precursors are blocked at an immature CD44(low)NK1.1(-) stage in a cell autonomous fashion. Residual c-Myc-deficient immature Valpha14i NKT cells appear to proliferate normally, cannot be rescued by transgenic expression of BCL-2, and exhibit characteristic features of immature Valpha14i NKT cells such as high levels of preformed IL-4 mRNA and the transcription factor promyelocytic leukemia zinc finger. Collectively our data identify c-Myc as a critical transcription factor that selectively acts early in Valpha14i NKT cell development to promote progression beyond the CD44(low)NK1.1(-) precursor stage.

Gene expression signatures delineate biological and prognostic subgroups in peripheral T-cell lymphoma

Peripheral T-cell lymphoma (PTCL) encompasses a heterogeneous group of neoplasms with generally poor clinical outcome. Currently 50% of PTCL cases are not classifiable: PTCL-not otherwise specified (NOS). Gene-expression profiles on 372 PTCL cases were analyzed and robust molecular classifiers and oncogenic pathways that reflect the pathobiology of tumor cells and their microenvironment were identified for major PTCL-entities, including 114 angioimmunoblastic T-cell lymphoma (AITL), 31 anaplastic lymphoma kinase (ALK)-positive and 48 ALK-negative anaplastic large cell lymphoma, 14 adult T-cell leukemia/lymphoma and 44 extranodal NK/T-cell lymphoma that were further separated into NK-cell and gdT-cell lymphomas. Thirty-seven percent of morphologically diagnosed PTCL-NOS cases were reclassified into other specific subtypes by molecular signatures. Reexamination, immunohistochemistry, and IDH2 mutation analysis in reclassified cases supported the validity of the reclassification. Two major molecular subgroups can be identified in the remaining PTCL-NOS cases characterized by high expression of either GATA3 (33%; 40/121) or TBX21 (49%; 59/121). The GATA3 subgroup was significantly associated with poor overall survival (P = .01). High expression of cytotoxic gene-signature within the TBX21 subgroup also showed poor clinical outcome (P = .05). In AITL, high expression of several signatures associated with the tumor microenvironment was significantly associated with outcome. A combined prognostic score was predictive of survival in an independent cohort (P = .004).

RANKL expression, function, and therapeutic targeting in multiple myeloma and chronic lymphocytic leukemia.

Bone destruction is a prominent feature of multiple myeloma, but conflicting data exist on the expression and pathophysiologic involvement of the bone remodeling ligand RANKL in this disease and the potential therapeutic benefits of its targeted inhibition. Here, we show that RANKL is expressed by primary multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release of soluble RANKL was observed exclusively with multiple myeloma cells and was strongly influenced by posttranscriptional/posttranslational regulation. Signaling via RANKL into multiple myeloma and CLL cells induced release of cytokines involved in disease pathophysiology. Both the effects of RANKL on osteoclastogenesis and cytokine production by malignant cells could be blocked by disruption of RANK-RANKL interaction with denosumab. As we aimed to combine neutralization of RANKL with induction of antibody-dependent cellular cytotoxicity of natural killer (NK) cells against RANKL-expressing malignant cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc fusion proteins with modified Fc moieties. The latter displayed similar capacity compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis in vitro, but also potently stimulated NK cell reactivity against primary RANKL-expressing malignant B cells, which was dependent on their engineered affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as attractive tools to neutralize the detrimental function of RANKL while at the same time potently stimulating NK cell antitumor immunity.

Haematopoietic stem cell transplantation: activity in Switzerland compared with surrounding European countries.

Haematopoietic stem cell transplantation (HSCT) is a highly specialised procedure used to treat malignancies of the lymphohaematopoietic system as well as some acquired and inherited disorders of the blood. This analysis by the Swiss Blood Stem Cell Transplantation Group, based on data from 2008-2011, describes, treatment rates in Switzerland for specific indications and compares this with data from Germany, France, Italy and the Netherlands, corrected for the size of the population. Differences in transplant rates, in rates for particular indications, and in the use of specific transplant technologies such as use of unrelated donors, use of cord blood or mismatched family donors are described. These data are put in correlation with donor availability from international registries and with number of transplant teams and number of procedures per team all corrected for population size.

Cytogenetic characterization of childhood acute lymphoblastic leukemia in Nicaragua.

BACKGROUND: Within the frame of a twinning programme with Nicaragua, The La Mascota project, we evaluated in our study the contribution of cytogenetic characterization of acute lymphoblastic leukemia (ALL) as prognostic factor compared to clinical, morphological, and immunohistochemical parameters. METHODS: All patients with ALL treated at the only cancer pediatric hospital in Nicaragua during 2006 were studied prospectively. Diagnostic immunophenotyping was performed locally and bone marrow or blood samples were sent to the cytogenetic laboratory of Zurich for fluorescence in situ hybridization (FISH) analysis and G-banding. RESULTS: Sixty-six patients with ALL were evaluated. Their mean age at diagnosis was 7.3 years, 31.8% were >or=10 years. Thirty-four patients (51.5%) presented with hyperleucocytosis >or=50 x 10(9)/L, 45 (68.2%) had hepatosplenomegaly. Immunophenotypically 63/66 patients (95%) had a B-precursor, 2 (3%) a T- and 1 (1.5%) a B-mature ALL. FISH analysis demonstrated a TEL/AML1 fusion in 9/66 (14%), BCR/ABL fusion in 1 (1.5%), MLL rearrangement in 2 (3.1%), iAMP21 in 2 (3.1%), MYC rearrangement in 1 (1.5%), and high-hyperdiploidy in 16 (24%). All patients but two with TEL/AML1 fusion and high-hyperdiploidy were clinically and hematologically in the standard risk group whereas those with poor cytogenetic factors had clinical high-risk features and were treated intensively. CONCLUSIONS: Compared to Europe, the ALL population in Nicaragua is older, has a higher proportion of poor prognostic clinical and hematological features and receives more intensive treatment, while patients with TEL/AML1 translocations and high-hyperdiploidy are clinically in the standard risk group. Cytogenetics did not contribute as an additional prognostic factor in this setting.

Species selectivity of mixed-lineage leukemia/trithorax and HCF proteolytic maturation pathways.

Site-specific proteolytic processing plays important roles in the regulation of cellular activities. The histone modification activity of the human trithorax group mixed-lineage leukemia (MLL) protein and the cell cycle regulatory activity of the cell proliferation factor herpes simplex virus host cell factor 1 (HCF-1) are stimulated by cleavage of precursors that generates stable heterodimeric complexes. MLL is processed by a protease called taspase 1, whereas the precise mechanisms of HCF-1 maturation are unclear, although they are known to depend on a series of sequence repeats called HCF-1(PRO) repeats. We demonstrate here that the Drosophila homologs of MLL and HCF-1, called Trithorax and dHCF, are both cleaved by Drosophila taspase 1. Although highly related, the human and Drosophila taspase 1 proteins display cognate species specificity. Thus, human taspase 1 preferentially cleaves MLL and Drosophila taspase 1 preferentially cleaves Trithorax, consistent with coevolution of taspase 1 and MLL/Trithorax proteins. HCF proteins display even greater species-specific divergence in processing: whereas dHCF is cleaved by the Drosophila taspase 1, human and mouse HCF-1 maturation is taspase 1 independent. Instead, human and Xenopus HCF-1PRO repeats are cleaved in vitro by a human proteolytic activity with novel properties. Thus, from insects to humans, HCF proteins have conserved proteolytic maturation but evolved different mechanisms.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma.

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Autologous stem cell transplantation: leukapheresis product has anti-angiogenic effects in vivo correlating with neutrophil-derived VEGFR1.

BACKGROUND: High-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) is used for the treatment of hemato-oncologic malignancies. In this study, we measured the effect of HDC/ASCT on plasma concentrations of antiangiogenic soluble vascular endothelial growth factor receptor 1 (sVEGFR1) and of leukapheresis products (LP) and patient serum on chick chorioallantoic (CAM) angiogenesis. MATERIALS AND METHODS: VEGFR1- and CD34-expressing cells of leukapheresis products were analyzed by flow cytometry. Alternatively spliced isoforms of VEGFR1 mRNA were quantified using reverse transcription PCR. RESULTS: Plasma concentrations of sVEGFR1 decreased after HDC, but significantly increased after ASCT. In the CAM assay, sera of patients elicited a proangiogenic effect before and after HDC, but a strong antiangiogenic response after ASCT, comparable to that of bevacizumab at therapeutic concentrations. LP contains high concentrations of sVEGFR1, and high density of VEGFR1(+) neutrophilic granulocytes, in which mRNA expression is shifted toward the soluble VEGFR1 isoform. CONCLUSION: Neutrophil-derived antiangiogenic sVEGFR1 within the LP may contribute to the therapeutic efficacy of ASCT.

Glucagon-like peptide-1-(7-36) amide, oxyntomodulin, and glucagon interact with a common receptor in a somatostatin-secreting cell line.

Glucagon-like peptide-1(7-36)amide (tGLP-1), oxyntomodulin (OXM), and glucagon are posttranslational end products of the glucagon gene expressed in intestinal L-cells. In vivo, these peptides are potent inhibitors of gastric acid secretion via several pathways, including stimulation of somatostatin release. We have examined the receptors through which these peptides stimulate somatostatin secretion using the somatostatin-secreting cell line RIN T3. tGLP-1, OXM, and glucagon stimulated somatostatin release and cAMP accumulation in RIN T3 cells to similar maximum levels, with ED50 values close to 0.2, 2, and 50 nM and 0.02, 0.3, and 8 nM, respectively. Binding of [125I]tGLP-1, [125I]OXM, and [125I]glucagon to RIN T3 plasma membranes was inhibited by the three peptides, with relative potencies as follows: tGLP-1 > OXM > glucagon. Whatever the tracer used, the IC50 for tGLP-1 was close to 0.15 nM and was shifted rightward for OXM and glucagon by about 1 and 2-3 orders of magnitude, respectively. Scatchard analyses for the three peptides were compatible with a single class of receptor sites displaying a similar maximal binding close to 2 pmol/mg protein. In the hamster lung fibroblast cell line CCL39 transfected with the receptor for tGLP-1, binding of [125I]tGLP-1 was inhibited by tGLP-1, OXM, and glucagon, with relative potencies close to those obtained with RIN T3 membranes. Chemical cross-linking of [125I]tGLP-1, [125I]OXM, and [125I]glucagon revealed a single band at 63,000 mol wt, the intensity of which was dose-dependently reduced by all three peptides. These data suggest that in the somatostatin-secreting cell line RIN T3, OXM and glucagon stimulate somatostatin release through a tGLP-1-preferring receptor. This suggests that some biological effects, previously described for these peptides, might be due to their interaction with this receptor.

High-dose therapy and autologous hematopoietic stem cell transplant in T-cell lymphoma: a single center experience.

Abstract We report here the long-term outcome of autologous stem cell transplant in peripheral T-cell lymphoma (PTCL). Forty-three consecutive patients with PTCL diagnosed between 2000 and 2011 were treated with high-dose chemotherapy (HDCT) and autologous stem cell transplant (ASCT) in our center. Diagnoses included PTCL-not otherwise specified (n = 19), anaplastic large cell lymphoma (n = 11), angioimmunoblastic T-cell lymphoma (n = 5), enteropathy-associated T-cell lymphoma (n = 5) and other rare subtypes (n = 3). Thirty-six patients with a median age of 50 years (range 22-65) were transplanted in first response and seven after relapse. After a median follow-up of 63 months, estimated overall survival at 12 years was 40%, progression-free survival at 12 years was 34% and event-free survival at 12 years was 30%. On univariate analysis, age less than 50 years and no B symptoms at diagnosis were significantly associated with prolonged overall and progression-free-survival. HDCT/ASCT for peripheral T-cell lymphoma can lead to long-term survival for patients responding to induction chemotherapy.

Canonical WNT signaling in hematopoiesis and leukemia

SummaryCanonical Wnt signaling is crucial for embryonic development and the homeostasis of certain adult tissues such as the gut and the skin. The role of canonical Wnt signaling in hematopoiesis is still debated. The expression of a dominant-active β-catenin in hematopoietic stem cells (HSCs) enhances the self-renewal capacity of HSCs but is detrimental for long-term hematopoiesis. In contrast, loss of function experiments show that absence of β- and γ-catenin does not impair steady-state hematopoiesis. It has been argued that the inducible deletion of β-catenin using the IFN-responsive Mx promoter may somehow influence stem cell fate. Herein we used the constitutive deletion of β-catenin specifically in hematopoietic cells to show that the absence of β- catenin, as well as γ-catenin deletion, does not impair normal hematopoiesis and self-renewal capacity of HSCs.Dysregulation of canonical Wnt signaling is causal for several types of cancer, including colon carcinoma or breast cancer. Recently, it was found that Wnt signal transduction was upregulated in certain leukemias. Based on these data, we have investigated whether β- and γ-catenin play a role for the induction of leukemias by oncogenic BCR-ABL translocation product. We show that the induction of B-ALL (B cell acute lymphocytic leukemia) is strongly reduced in the absence of γ-catenin, while the induction of CML (chronic myeloid leukemia) occurs at a normal rate. In the combined absence of β- and γ-catenin the induction of both CML and B-ALL is essentially blocked. Consistent with these data others have found that β-catenin is essential for the induction of CML by BCR-ABL.Collectively, we find that β- and γ-catenin are dispensable for normal hematopoiesis but essential for the development of BCR-ABL induced leukemias. These findings suggest that the canonical Wnt pathway may represent a promising target for the therapy of leukemia.RésuméLa voie de signalisation canonique Wnt est essentielle pour le développement embryonnaire ainsi que l'homéostasie de certains tissus adultes, comme les intestins et la peau. Le rôle de la voie canonique Wnt pour l'hématopoïèse est encore incertain. D'un coté l'expression d'une forme active de β-catenine dans les cellules souches de la moelle augmente leur potentiel d'auto- renouvellement mais est préjudiciable pour l'hématopoïèse à long terme. Par contre, l'absence de β- et γ-catenine n'empêche pas le déroulement normal de l'hématopoïèse. La façon dont est supprimée β-catenine, en utilisant le promoteur IFN-inductible Mx, pourrait influencer le sort des cellules souches. Ici nous détruisons β-catenine spécifiquement dans les cellules hématopoïétiques de manière constitutive et montrons que, en combinaison avec l'absence de γ-catenine, l'absence de β-catenine n'affecte pas le déroulement normal de l'hématopoïèse et la capacité des cellules souches de la moelle à se renouveler.Plusieurs sortes de cancers, comme celui du colon ou du sein, sont parfois dus à une dérégulation de la voie canonique Wnt. Récemment, certaines leucémies ont présenté une activation du signal Wnt. A partir de ces données, nous avons examiné si β- et γ-catenine jouent un rôle dans l'induction des leucémies causées par le produit de translocation BCR-ABL. Nous avons montré que l'induction de la leucémie aiguë lymphoïde de cellules Β (LAL-B) est grandement diminuée en l'absence de γ-catenin, alors que l'induction de la leucémie myéloïde chronique (LMC) n'est pas affectée. En l'absence des deux catenines, l'induction des deux leucémies LAL-B et LMC est presque complètement bloquée. En confirmation de nos données, un autre groupe a montré que β-catenine est essentielle pour le développement de la LMC. Ensemble, ces données nous montrent que β- et γ-catenine ne sont pas nécessaires pour l'hématopoïèse normale, mais essentielle pour le développement des leucémies induites par BCR-ABL. Cela suggère que la voie de signalisation canonique Wnt est une cible prometteuse pour de futures thérapies.

Behavior of nuclear matrix proteins during camptothecin-induced apoptosis in HL-60 human leukemia cells.

In this study we focused our attention on the behavior of four nuclear matrix proteins during the various stages of apoptosis in the HL-60 cell line exposed to the DNA topoisomerase I inhibitor, camptothecin. We have examined the following antigens by immunocytochemical techniques: (i) the 180-kDa nucleolar isoform of DNA topoisomerase II; (ii) a 126-kDa polypeptide of nuclear bodies; (iii) a 125-kDa protein; and (iv) a 160-kDa polypeptide which are known to be components of the matrix inner network. Indirect immunofluorescence experiments were performed to follow these nuclear matrix antigens during apoptosis. Moreover, the ultrastructural localization of both 125- and 160-kDa proteins was investigated by electron microscope immunocytochemistry with gold-conjugated secondary antibodies. While the antibody to the nucleolar isoform of DNA topoisomerase II gave a fluorescent pattern that was well-maintained until the late phases of apoptosis, the other three nuclear antigens showed marked modifications in their distribution. A common feature, particularly evident for 125- and 160-kDa proteins, was their absence from cap-shaped chromatin marginations, whereas they were present in the areas of remaining decondensed chromatin. The 126-kDa polypeptide concentrated progressively in an irregular mass at the opposite side of the crescentic caps and then broke up in fine spots. The 125- and 160-kDa proteins localized in the nucleolus and precisely within certain granules which are known to appear in the nucleolar area after camptothecin administration. These results show that, in addition to the well-known chromatin changes, nuclear organization undergoes other rearrangements during the apoptotic process.

Health-related quality of life in long-term survivors of relapsed childhood acute lymphoblastic leukemia.

BACKGROUND: Relapses occur in about 20% of children with acute lymphoblastic leukemia (ALL). Approximately one-third of these children can be cured. Their risk for late effects is high because of intensified treatment, but their health-related quality of life (HRQOL) was largely unmeasured. Our aim was to compare HRQOL of ALL survivors with the general population, and of relapsed with non-relapsed ALL survivors. METHODOLOGY/PRINCIPAL FINDINGS: As part of the Swiss Childhood Cancer Survivor Study (SCCSS) we sent a questionnaire to all ALL survivors in Switzerland who had been diagnosed between 1976-2003 at age <16 years, survived ≥5 years, and were currently aged ≥16 years. HRQOL was assessed with the Short Form-36 (SF-36), which measures four aspects of physical health and four aspects of mental health. A score of 50 corresponded to the mean of a healthy reference population. We analyzed data from 457 ALL survivors (response: 79%). Sixty-one survivors had suffered a relapse. Compared to the general population, ALL survivors reported similar or higher HRQOL scores on all scales. Survivors with a relapse scored lower in general health perceptions (51.6) compared to those without (55.8;p=0.005), but after adjusting for self-reported late effects, this difference disappeared. CONCLUSION/SIGNIFICANCE: Compared to population norms, ALL survivors reported good HRQOL, even after a relapse. However, relapsed ALL survivors reported poorer general health than non-relapsed. Therefore, we encourage specialists to screen for poor general health in survivors after a relapse and, when appropriate, specifically seek and treat underlying late effects. This will help to improve patients' HRQOL.