GeneChip analyses point to novel pathogenetic mechanisms in mantle cell lymphoma

The translocation t(11;14)(q13;q32) is the genetic hallmark of mantle cell lymphoma (MCL) but is not sufficient for inducing lymphomagenesis. Here we performed genome-wide 100K GeneChip Mapping in 26 t(11;14)-positive MCL and six MCL cell lines. Partial uniparental disomy (pUPD) was shown to be a recurrent chromosomal event not only in MCL cell lines but also in primary MCL. Remarkably, pUPD affected recurrent targets of deletion like 11q, 13q and 17p. Moreover, we identified 12 novel regions of recurrent gain and loss as well as 12 high-level amplifications and eight homozygously deleted regions hitherto undescribed in MCL. Interestingly, GeneChip analyses identified different genes, encoding proteins involved in microtubule dynamics, such as MAP2, MAP6 and TP53, as targets for chromosomal aberration in MCL. Further investigation, including mutation analyses, fluorescence in situ hybridisation as well as epigenetic and expression studies, revealed additional aberrations frequently affecting these genes. In total, 19 of 20 MCL cases, which were subjected to genetic and epigenetic analyses, and five of six MCL cell lines harboured at least one aberration in MAP2, MAP6 or TP53. These findings provide evidence that alterations of microtubule dynamics might be one of the critical events in MCL lymphomagenesis contributing to chromosomal instability

Mutational Profiling of Kinases in Human Tumours of Pancreatic Origin Identifies Candidate Cancer Genes in Ductal and Ampulla of Vater Carcinomas

BACKGROUND: Protein kinases are key regulators of cellular processes (such as proliferation, apoptosis and invasion) that are often deregulated in human cancers. Accordingly, kinase genes have been the first to be systematically analyzed in human tumors leading to the discovery that many oncogenes correspond to mutated kinases. In most cases the genetic alterations translate in constitutively active kinase proteins, which are amenable of therapeutic targeting. Tumours of the pancreas are aggressive neoplasms for which no effective therapeutic strategy is currently available. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a DNA-sequence analysis of a selected set of 35 kinase genes in a panel of 52 pancreatic exocrine neoplasms, including 36 pancreatic ductal adenocarcinoma, and 16 ampulla of Vater cancer. Among other changes we found somatic mutations in ATM, EGFR, EPHA3, EPHB2, and KIT, none of which was previously described in cancers. CONCLUSIONS/SIGNIFICANCE: Although the alterations identified require further experimental evaluation, the localization within defined protein domains indicates functional relevance for most of them. Some of the mutated genes, including the tyrosine kinases EPHA3 and EPHB2, are clearly amenable to pharmacological intervention and could represent novel therapeutic targets for these incurable cancers

VDJSeq-Solver: In Silico V(D)J Recombination Detection Tool

In this paper we present VDJSeq-Solver, a methodology and tool to identify clonal lymphocyte populations from paired-end RNA Sequencing reads derived from the sequencing of mRNA neoplastic cells. The tool detects the main clone that characterises the tissue of interest by recognizing the most abundant V(D)J rearrangement among the existing ones in the sample under study. The exact sequence of the clone identified is capable of accounting for the modifications introduced by the enzymatic processes. The proposed tool overcomes limitations of currently available lymphocyte rearrangements recognition methods, working on a single sequence at a time, that are not applicable to high-throughput sequencing data. In this work, VDJSeq-Solver has been applied to correctly detect the main clone and identify its sequence on five Mantle Cell Lymphoma samples; then the tool has been tested on twelve Diffuse Large B-Cell Lymphoma samples. In order to comply with the privacy, ethics and intellectual property policies of the University Hospital and the University of Verona, data is available upon request to [email protected] after signing a mandatory Materials Transfer Agreement. VDJSeq-Solver JAVA/Perl/Bash software implementation is free and available at http://eda.polito.it/VDJSeq-Solver/

Absence of TCL1A expression is a useful diagnostic feature in splenic marginal zone lymphoma.

Splenic marginal zone lymphoma (SMZL) is a low-grade lymphoma showing a rather nonspecific immunophenotype. Gene expression profiling studies suggested that TCL1A could be a marker of SMZL, but reported data are conflicting. We evaluated TCL1A expression in a series of spleen and bone marrow samples involved by SMZL and correlated the findings with other immunophenotypical, morphological, and clinical data. In addition, we evaluated the expression of TCL1A in a series of spleens and lymph nodes involved by lymphomas that might mimic SMZL (13 nodal marginal zone lymphomas (NMZL), 39 follicular lymphomas (FL), 30 B-cell chronic lymphocytic leukemias (B-CLL), 31 mantle cell lymphomas (MCL), 1 lymphoplasmacytic lymphoma) and 15 bone marrow specimens involving hairy cell leukemia (HCL). TCL1A staining was negative in 24/31 cases of SMZL (77 %); 27/31 MCL and all B-CLL were positive for TCL1A; 32/34 cases of nodal FL (96 %) and all five splenic FL were positive for TCL1A, although at a lower intensity. Eight of 13 NMZL were positive for TCL1A, often showing a heterogeneous staining pattern. All HCL samples were strongly positive for TCL1A. No correlation was found between the pattern of splenic infiltration, TCL1A expression, and the clinical course. TCL1A-positive SMZL showed a higher rate of DBA44 staining compared to the negative ones, and this difference was statistically significant (Fisher test, single-tailed, p\u2009=\u20090.0397). Our data support the use of TCL1A in the panel of diagnostic markers used in the differential diagnosis of splenic low-grade B-cell lymphoma; a possible prognostic value, however, needs a larger series to be established

Signal transduction pathways of mantle cell lymphoma: a phosphoproteome-based study.

Mantle cell lymphoma (MCL) is an incurable hematologic malignancy whose pathogenesis is only partly understood. The aim of the present study was to define a \u201ccore phosphoproteome\u201d in MCL cell lines that is representative of primary MCL in order to improve knowledge of the signal transduction pathways involved in its tumorigenesis.We have analyzed phosphorylated proteins in several MCL cell lines by immobilized metal affinity chromatography and separation by 2-D PAGE, followed by RP-HPLC coupled with MS/MS identification. These data were correlated with information on copy number gains obtained by SNP-chip analysis. Several of the proteins identified could be linked to a specific signal transduction pathway, and have been recently recognized as important players in MCL pathogenesis, such as nuclear factor-kappaB (NF-kB) and phosphoinositide-3 kinase-mammalian target of rapamycin (PI3K-mTOR). However, our data also implicate a number of novel proteins and pathways in the pathobiology of MCL, one of which is mitochondrial signaling. A second-level analysis identified MAPK1, CK2, CK1, PKCzeta, and PKCepsilon as candidate upstream molecules. Our study provides new insights in MCL pathogenesis and helps to form the basis for testing new target-specific therapeutics. Received: January 28, 200

EXPRESSION AND FUNCTIONAL ACTIVITY OF THE TNFR-SUPERFAMILY MEMBER DEATH RECEPTOR 3 IN CHRONIC LYMPHOCYTIC LEUKEMIA B CELLS ACTIVATED BY THE BCR

Background: Growing evidences suggest a dynamic balance of B-cell chron- ic lymphocytic leukemia (B-CLL) cells between the blood and lymphoid tissues, which represent permissive niches for cell proliferation and survival. Within lymphoid tissue sites, molecules of the tumor necrosis factor (TNF) superfam- ily have been shown to play a supportive role and contribute to the pathogen- esis of B-CLL. Thereby, investigating expression and functions of novel TNFR- superfamily members in B-CLL would allow us to gain deeper insights into molecular crosstalk within leukemic microenvironments. Death receptor (DR) 3 is a TNFR-superfamily member expressed in lymphocyte-enriched tissues that binds to the TNF-like ligand 1A (TL1A), a TNF superfamily member. The TL1A/DR3 axis is implicated in regulatory mechanisms of adaptive immune response under physiological and pathological settings. Further evidence for the implication of TL1A in regulatory mechanisms arises from our recent data show- ing an inhibitory function of TL1A on B-cell proliferation. In leukemia cells, DR3 expression and functions have not been explored so far. CLL patients and to explore the possible role of TL1A/DR3 axis in the disease. Methods: B cells were purified from PBMC of 37 B-CLL patients by negative selection with magnetic beads. DR3 surface expression was measured by flow cytometry at baseline and following stimulation with F(ab’)2 anti-human IgM con- jugated to latex microspheres. DR3 expression was confirmed by western blot and immunofluorescence analysis on B-CLL lymph nodes. DR3 function was studied by MTT assay and Annexin V assay. TL1A serum levels were measured by ELISA. Results: Under basal conditions CLL B cells in vitro expressed low levels of DR3. Stimulation of the B cell receptor (BCR) with anti-IgM antibodies induced a sta- tistically significant increase of DR3 expression in CLL B cells (p<0.001). Induced DR3 expression showed great variability amongst B-CLL cells (variance (σ2)=6.38). Flow cytometry data were confirmed by Western blot analysis. The relevance of these findings was further confirmed by immuofluorescence analy- sis of B-CLL lymph-node specimens showing that in vivo high levels of DR3 were expressed by a number of B-CLL cells. To assess whether the anti-IgM-induced DR3 molecule was functionally active in CLL B cells, we examined the ability of DR3 to modulate their metabolic activity. Stimulation of DR3 with TL1A in the presence of BCR engagement showed that TL1A induced an equal or greater than 25% decrease of metabolic activity in 37.5% of B-CLL cell samples. In these samples the modulation is not due to reduced survival, as assessed by Annexin V assay. No change in metabolic activity was observed following TL1A treatment, in the absence of anti-IgM. Higher levels of DR3 expression were significantly associated with early-stage (Rai 0) disease (p=0.019) and higher serum levels of TL1A were significantly associated with early-stage (Rai 0) disease (p=0.023) and absence of CD38 expressions (p=0.035). CLL cells activated by the BCR stimulation express DR3 and TL1A reduces meta- bolic activity in some B-CLL cells. Herein, our data show a novel regulatory role for TL1A that, in the presence of antigen stimulation, may modulate leukemic cell metabolism through DR3 in early-stage B-CLL and suggest that homeostatic functions of TL1A may influence the clinical course of B-CLL disease

Aberrant Wnt/beta-catenin pathway activation in idiopathic pulmonary fibrosis

To investigate the molecular events that may underpin dysfunctional repair processes that characterize idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), we analyzed the expression patterns of beta-catenin on 20 IPF/UIP lung samples, together with two downstream target genes of Wnt signaling, cyclin-D1, and matrilysin. In 18 of 20 cases of IPF/UIP investigated on serial sections, nuclear beta-catenin immunoreactivity and abnormal levels of cyclin-D1 and matrilysin were demonstrated in proliferative bronchiolar lesions (basal-cell hyperplasia, squamous metaplasia, bronchiolization, honeycombing). The nature of these lesions was precisely defined using specific markers (DeltaN-p63, surfactant-protein-A, cytokeratin-5). Interestingly, nuclear beta-catenin accumulation was also demonstrated in fibroblast foci in most (16 of 20) IPF/UIP samples, often associated with bronchiolar lesions. Similar features were not observed in normal lung and other fibrosing pulmonary diseases (diffuse alveolar damage, organizing pneumonia, nonspecific interstitial pneumonia, desquamative interstitial pneumonia). Sequence analysis performed on DNA extracted from three samples of IPF/UIP did not reveal abnormalities affecting the beta-catenin gene. On the basis of these findings new models for IPF/UIP pathogenesis can be hypothesized, centered on the aberrant activation of Wnt/beta-catenin signaling, with eventual triggering of divergent epithelial regeneration at bronchiolo-alveolar junctions and epithelial-mesenchymal-transitions, leading to severe and irreversible remodeling of the pulmonary tissue

A 6-GENE EXPRESSION SIGNATURE IN MANTLE CELL LYMPHOMA: RESULTS FROM THE FONDAZIONE ITALIANA LINFOMI (FIL)-MCL-0208 TRIAL

Background: The aggressive clinical behavior of mantle cell lymphoma (MCL) is mainly attributed to the t(11;14)(q13;q32) traslocation and cyclin D1 (CCND1) overexpression. Nevertheless, a certain degree of clinical/biological heterogeneity has been reported. Aims: To identify MCL subsets with peculiar clinical/biological features in the context of a cohort of homogeneously treated MCL patients. Methods: The study used gene expression profiling (GEP) and quantitative real-time PCR (qRT-PCR) validations in peripheral blood (PB, n=46) and formalin fixed paraffin embedded (FFPE, n=42) samples from 82 MCL cases enrolled in the Fondazione Italiana Linfomi (FIL)-MCL-0208 randomized clinical trial (high-dose therapy followed by autologous transplantation). Results: i) Unsupervised and supervised analyses. GEP from 27 PB samples were analyzed by principal component analysis (PCA) and divided in two subgroups named PCA1 (14 cases) and PCA2 (13 cases). Supervised analysis, according to PCA classification, identified a gene expression signature of 902 probes (700 up-regulated). Gene Set Enrichment Analysis (GSEA) demonstrated a significant enrichment of five B Cell Receptor (BCR)-related gene sets, these genes being constitutively over-expressed in PCA2 samples. ii) Identification of a \u201cPCA2-type\u201d gene signature. By merging the lists of differentially expressed genes and the BCR signaling related genes according to GSEA, a group of 14 genes, all overexpressed in PCA2 cases, was obtained. Among these genes, 6 genes, AKT3, BCL2, BTK, CD79B, PIK3CD, and SYK, were selected for further validations. iii) Generation of a 6-gene prediction model. These 6 genes were analyzed by qRT-PCR and utilized to generate a prediction model by using 17 cases as training cohort and 10 cases as validation cohort (all from PB samples). By this approach, 10/10 cases of the validation cohort were correctly assigned according to the PCA2/PCA1 classification. qRT-PCR was then utilized to classify 19 additional cases (10 PCA2 cases) not employed in GEP. Overall, in the 46 cases, 23 cases were classified as PCA2 by the GEP/qRT-PCR approach. iv) Clinical/biological correlations. N