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Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a very rare disease that currently lacks genomic and genetic biomarkers to assist in its clinical management. We performed whole-exome sequencing (WES) of three BPDCN cases. Based on these data, we designed a resequencing approach to identify mutations in 38 selected genes in 25 BPDCN samples. WES revealed 37-99 deleterious gene mutations per exome with no common affected genes between patients, but with clear overlap in terms of molecular and disease pathways (hematological and dermatological disease). We identified for the first time deleterious mutations in IKZF3, HOXB9, UBE2G2 and ZEB2 in human leukemia. Target sequencing identified 29 recurring genes, ranging in prevalence from 36% for previously known genes, such as TET2, to 12-16% for newly identified genes, such as IKZF3 or ZEB2. Half of the tumors had mutations affecting either the DNA methylation or chromatin remodeling pathways. The clinical analysis revealed that patients with mutations in DNA methylation pathway had a significantly reduced overall survival (P=0.047). We provide the first mutational profiling of BPDCN. The data support the current WHO classification of the disease as a myeloid disorder and provide a biological rationale for the incorporation of epigenetic therapies for its treatment.

Currently, multiple myeloma (MM) patients are broadly grouped into a non-hyperdiploid (nh-MM) group, highly enriched for IgH translocations, or into a hyperdiploid (h-MM) group, which is typically characterized by trisomies of some odd-numbered chromosomes. We compared the micro RNA (miRNA) expression profiles of these two groups and we identified 16 miRNAs that were downregulated in the h-MM group, relative to the nh-MM group. We found that target genes of the most differentially expressed miRNAs are directly involved in the pathogenesis of MM; specifically, the inhibition of hsa-miR-425, hsa-miR-152 and hsa-miR-24, which are all downregulated in h-MM, leads to the overexpression of CCND1, TACC3, MAFB, FGFR3 and MYC, which are the also the oncogenes upregulated by the most frequent IgH chromosomal translocations occurring in nh-MM. Importantly, we showed that the downregulation of these specific miRNAs and the upregulation of their targets also occur simultaneously in primary cases of h-MM. These data provide further evidence on the unifying role of cyclin D pathways deregulation as the key mechanism involved in the development of both groups of MM. Finally, they establish the importance of miRNA deregulation in the context of MM, thereby opening up the potential for future therapeutic approaches based on this molecular mechanism.

Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here, we analyzed the therapeutic effect of LBH589, a class I-II HDAC inhibitor, in acute lymphoblastic leukemia (ALL). In vitro, LBH589 induced dose-dependent antiproliferative and apoptotic effects, which were associated with increased H3 and H4 histone acetylation. Intravenous administration of LBH589 in immunodeficient BALB/c-RAG2(-/-)γc(-/-) mice in which human-derived T and B-ALL cell lines were injected induced a significant reduction in tumor growth. Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2(-/-)γc(-/-) mice was established, allowing continuous passages of transplanted cells to several mouse generations. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving vincristine and dexamethasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with vincristine and dexamethasone. Our results show the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL.

The AML1-ETO fusion protein, which is present in 10-15% of cases of acute myeloid leukemia, is known to repress myeloid differentiation genes through DNA binding and recruitment of chromatin-modifying proteins and transcription factors in target genes. ChIP-chip analysis of human hematopoietic stem/progenitor cells transduced with the AML1-ETO fusion gene enabled us to identify 1168 AML1-ETO target genes, 103 of which were co-occupied by histone deacetylase 1 (HDAC1) and had lost the hyperacetylation mark at histone H4, and 264 showed a K9 trimethylation at histone H3. Enrichment of genes involved in hematopoietic differentiation and in specific signaling pathways was observed in the presence of these epigenetic modifications associated with an 'inactive' chromatin status. Furthermore, AML1-ETO target genes had a significant correlation between the chromatin marks studied and transcriptional silencing. Interestingly, AML1 binding sites were absent on a large number of selected AML1-ETO promoters and an Sp1 binding site was found in over 50% of them. Reversible silencing induced by the fusion protein in the presence of AML1 and/or Sp1 transcription factor binding site was confirmed. Therefore, this study provides a global analysis of AML1-ETO functional chromatin modifications and identifies the important role of Sp1 in the DNA binding pattern of AML1-ETO, suggesting a role for Sp1-targeted therapy in this leukemia subtype.

Mantle cell lymphoma (MCL) pathogenesis is still partially unexplained. We investigate the importance of microRNA (miRNA) expression as an additional feature that influences MCL pathway deregulation and may be useful for predicting patient outcome. Twenty-three MCL samples, eight cell lines and appropriate controls were screened for their miRNAs and gene expression profiles and DNA copy-number changes. MCL patients exhibit a characteristic signature that includes 117 miRNA (false discovery rate <0.05). Combined analysis of miRNAs and the gene expression profile, paired with bioinformatics target prediction (miRBase and TargetScan), revealed a series of genes and pathways potentially targeted by a small number of miRNAs, including essential pathways for lymphoma survival such as CD40, mitogen-activated protein kinase and NF-kappaB. Functional validation in MCL cell lines demonstrated NF-kappaB subunit nuclear translocation to be regulated by the expression of miR-26a. The expression of 12 selected miRNAs was studied by quantitative PCR in an additional series of 54 MCL cases. Univariate analysis identified a single miRNA, miR-20b, whose lack of expression distinguished cases with a survival probability of 56% at 60 months. In summary, using a novel bioinformatics approach, this study identified miRNA changes that contribute to MCL pathogenesis and markers of potential utility in MCL diagnosis and clinical prognostication.

Clinical and biological studies on nodal marginal zone lymphoma (NMZL) are hampered by the lack of specific diagnostic markers and the low reproducibility of this diagnosis. A comparative expression-profiling study has shown a set of markers to be differentially expressed in NMZL compared with follicular lymphoma (FL), including myeloid cell nuclear differentiation antigen (MNDA), a nuclear protein expressed by myeloid cells and a subset of B-cells. The aim of this study was to characterize the expression of MNDA in normal and reactive human tissue, and in a large series of non-Hodgkin's B-cell lymphomas, with particular emphasis on NMZL and FL. Our results showed that MNDA is expressed in normal tissue by a subset of the marginal zone B cells. They also showed MNDA expression in subgroups of chronic lymphocytic leukemia, mantle-cell lymphoma, and diffuse large B-cell lymphoma, but MNDA was especially expressed by lymphomas derived from the marginal zone, such as mucosa-associated lymphoid-tissue lymphoma, splenic marginal-zone lymphoma and NMZL. MNDA expression was rarely observed in FL, a characteristic that is of potential value in distinguishing between NMZL and FL. MNDA expression is thus a useful tool for the recognition of NMZL.

We have carried out a high-resolution whole genome DNA profiling analysis on 100 bone marrow samples from a consecutive series of de novo acute myeloid leukemia (AML) cases. After discarding copy number changes that are known to be genetic polymorphisms, we found that genomic aberrations (GA) in the form of gains or losses of genetic material were present in 74% of the samples, with a median of 2 GA per case (range 0-35). In addition to the cytogenetically detected aberration, GA were present in cases from all cytogenetic prognostic groups: 79% in the favorable group, 60% in the intermediate group (including 59% of cases with normal karyotype) and 83% in the adverse group. Five aberrant deleted regions were recurrently associated with cases with a highly aberrant genome (e.g., a 1.5 Mb deletion at 17q11.2 and a 750 kb deletion at 5q31.1). Different degrees of genomic instability showed a statistically significant impact on survival curves, even within the normal karyotype cases. This association was independent of other clinical and genetic parameters. Our study provides, for the first time, a detailed picture of the nature and frequency of DNA copy number aberrations in de novo AML.

Disciplines such as morphology, immunophenotyping and genetics widely contributed over decades to the understanding of the cellular mechanisms of cancer. To obtain a greater insight into the complex processes of tumorigenesis, scientists have joined their efforts to combine many of the available techniques. Here, we report on the development of a FICTION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a tool for the Investigation of Neoplasms) technique that allows a simultaneous detection of immunophenotypic markers and genetic aberrations on routinely processed lymphoma samples. As the antigen retrieval method seems to play an important role in the final results, we tested the pressure-cooking method at different times (2, 4 and 8 min) using three different buffers (EDTA, Tris-EDTA and citrate), resulting in improved sensitivity for the detection of both immunophenotypic markers and genetic aberrations. We also applied this method to different types of lymphoma using double immunofluorescence assays (including CD30, CD20, CD8 monoclonal antibodies) and several fluorescence in situ hybridization probes to demonstrate that the FICTION technique could be easily applied on paraffin sections in different combinations for the diagnosis and research of cancer.