Identification of novel candidate target genes in amplicons of Glioblastoma multiforme tumors detected by expression and CGH microarray profiling

BACKGROUND: Conventional cytogenetic and comparative genomic hybridization (CGH) studies in brain malignancies have shown that glioblastoma multiforme (GBM) is characterized by complex structural and numerical alterations. However, the limited resolution of these techniques has precluded the precise identification of detailed specific gene copy number alterations. RESULTS: We performed a genome-wide survey of gene copy number changes in 20 primary GBMs by CGH on cDNA microarrays. A novel amplicon at 4p15, and previously uncharacterized amplicons at 13q32-34 and 1q32 were detected and are analyzed here. These amplicons contained amplified genes not previously reported. Other amplified regions containg well-known oncogenes in GBMs were also detected at 7p12 (EGFR), 7q21 (CDK6), 4q12 (PDGFRA), and 12q13-15 (MDM2 and CDK4). In order to identify the putative target genes of the amplifications, and to determine the changes in gene expression levels associated with copy number change events, we carried out parallel gene expression profiling analyses using the same cDNA microarrays. We detected overexpression of the novel amplified genes SLA/LP and STIM2 (4p15), and TNFSF13B and COL4A2 (13q32-34). Some of the candidate target genes of amplification (EGFR, CDK6, MDM2, CDK4, and TNFSF13B) were tested in an independent set of 111 primary GBMs by using FISH and immunohistological assays. The novel candidate 13q-amplification target TNFSF13B was amplified in 8% of the tumors, and showed protein expression in 20% of the GBMs. CONCLUSION: This high-resolution analysis allowed us to propose novel candidate target genes such as STIM2 at 4p15, and TNFSF13B or COL4A2 at 13q32-34 that could potentially contribute to the pathogenesis of these tumors and which would require futher investigations. We showed that overexpression of the amplified genes could be attributable to gene dosage and speculate that deregulation of those genes could be important in the development and progression of GBM. Our findings highlight the important influence in GBM of signaling pathways such as the PI3K/AKT, consistent with the invasive features of this tumor

CD30 expression by bone marrow mast cells from different diagnostic variants of systemic mastocytosis

[Aims]: CD30 expression by bone marrow (BM) mast cells (MC) has been reported recently in systemic mastocytosis (SM) patients. The aim of this study was to investigate the potential diagnostic and prognostic value of CD30 expression in SM as assessed by multiparameter flow cytometry. [Methods and results]: A total of 163 consecutive BM samples corresponding to 142 SM patients and 21 non-mastocytosis cases were studied. CD30 was positive in most SM patients (80%), but in only one non-mastocytosis case (4.8%). When combined with CD25, CD30 contributed to an improved accuracy over that of CD25 alone (98% versus 93%) mainly because most (eight of nine) of the well-differentiated SM (WDSM), who lacked CD25, were CD30+. Similar levels of expression of CD30 were observed among all different subgroups of SM except mast cell leukaemia; among indolent SM (ISM) patients, no significant association was observed between the levels of CD30 expression and other clinical and biological features of the disease. [Conclusions]: The increased expression of CD30 associated with absence of CD25 contributes to the diagnosis of WDSM and its distinction from other subtypes of SM. By contrast, CD30 expression did not contribute either to prognostic stratification of ISM or to the differential diagnosis between ISM and aggressive SM cases.This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS) PI11/02399, PS09/00032 and RETICs RD09/0076/00133, RD09/0076/00074 and RD12/0036/0048 (FEDER) from the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain; Fundacion Sociosanitaria de Castilla-La Mancha (2010/008 y G-2010/C-002); Fundación Espanola de Mastocitosis (FEM 2010); and by a grant from Fundaçao para a Ciência e Tecnologia (FCT) of Portugal (SFRH/BD/22972/2005).Peer Reviewe

Clinical, immunophenotypic, and molecular characteristics of well-differentiated systemic mastocytosis

[Background]: Well-differentiated systemic mastocytosis (WDSM) is a rare variant of systemic mastocytosis (SM) characterized by bone marrow (BM) infiltration by mature-appearing mast cells (MCs) often lacking exon 17 KIT mutations. Because of its rarity, the clinical and biological features of WDSM remain poorly defined. [Objective]: We sought to determine the clinical, biological, and molecular features of a cohort of 33 patients with mastocytosis in the skin in association with BM infiltration by well-differentiated MCs and to establish potential diagnostic criteria for WDSM. Methods Thirty-three patients with mastocytosis in the skin plus BM aggregates of round, fully granulated MCs lacking strong CD25 and CD2 expression in association with clonal MC features were studied. [Results]: Our cohort of patients showed female predominance (female/male ratio, 4:1) and childhood onset of the disease (91%) with frequent familial aggregation (39%). Skin involvement was heterogeneous, including maculopapular (82%), nodular (6%), and diffuse cutaneous (12%) mastocytosis. KIT mutations were detected in only 10 (30%) of 33 patients, including the KIT D816V (n = 5), K509I (n = 3), N819Y (n = 1), and I817V (n = 1) mutations. BM MCs displayed a unique immunophenotypic pattern consisting of increased light scatter features, overexpression of cytoplasmic carboxypeptidase, and aberrant expression of CD30, together with absent (79%) or low (21%) positivity for CD25, CD2, or both. Despite only 9 (27%) of 33 patients fulfilling the World Health Organization criteria for SM, our findings allowed us to establish the systemic nature of the disease, which fit with the definition of WDSM. [Conclusions]: WDSM represents a rare clinically and molecularly heterogeneous variant of SM that requires unique diagnostic criteria to avoid a misdiagnosis of cutaneous mastocytosis per current World Health Organization criteria.Supported by grants from Asociación Española de Mastocitosis, Madrid, Spain (grant AEDM 2014); Instituto de Salud Carlos III, FEDER, Ministry of Economy and Competitivity, Madrid, Spain (grant PI11/02399); Fundación Ramón Areces, Madrid, Spain (grant CIVP16A1806); and Novartis Farmacéutica, S.A., Spain. I. Alvarez-Twose has received research support from Novartis Farmacéutica, S.A., Spain. A. García-Montero has received research support from Fundacion Ramon Areces (grant no. CIVP16A1806) and ISCIII Ministerio de Economia y Competitividad (grant no. PI11/02399). A. Orfao has received research support from Fundacion Ramon Areces (grant no. CIVP16A1806).Peer Reviewe

Nonaggressive systemic mastocytosis (SM) without skin lesions associated with insect-induced anaphylaxis shows unique features versus other indolent SM

Spanish Network on Mastocytosis (REMA): et al.[Background]: Indolent systemic mastocytosis (ISM) without skin lesions (ISMs-) shows a higher prevalence in males, lower serum baseline tryptase levels, and KIT mutation more frequently restricted to bone marrow (BM) mast cells (MCs) than ISM with skin lesions (ISMs+). Interestingly, in almost one-half of ISMs- patients, MC-mediator release episodes are triggered exclusively by insects. [Objective]: We aimed to determine the clinical and laboratory features of ISMs- associated with insect-induced anaphylaxis (insectISMs-) versus other patients with ISM. [Methods]: A total of 335 patients presenting with MC activation syndrome, including 143 insectISMs-, 72 ISMs- triggered by other factors (otherISMs-), 56 ISMs+, and 64 nonclonal MC activation syndrome, were studied. [Results]: Compared with otherISMs- and ISMs+ patients, insectISMs- cases showed marked male predominance (78% vs 53% and 46%; P <.001), a distinct pattern of MC-related symptoms, and significantly lower median serum baseline tryptase levels (22.4 vs 28.7 and 45.8 μg/L; P ≤.009). Moreover, insectISMs- less frequently presented BM MC aggregates (46% vs 70% and 81%; P ≤.001), and they systematically showed MC-restricted KIT mutation. [Conclusions]: ISMs- patients with anaphylaxis triggered exclusively by insects display clinical and laboratory features that are significantly different from other ISM cases, including other ISMs- and ISMs+ patients, suggesting that they represent a unique subgroup of ISM with a particularly low BM MC burden in the absence of adverse prognostic factors.Supported by Fondo de Investigaciones Sanitarias –FIS– of the Instituto de Salud Carlos III, Ministery of Economy and Competitivity, Madrid, Spain grant PS09/00032; Fundación Sociosanitaria de Castilla-La Mancha grants 2010/008 and G-2010/ C-002; Fundacióon Española de Mastocitosis grant FEM 2011; BioB-HVS is supported by grant RETICS (Redes Temáticas de Investigación Cooperativa en Salud) RD09/ 00760074 (Toledo, Spain); RTICC (Red Temática de Investigación Cooperativa en Cáncer) grants RD09/0076/00133, RD12/0036/0048; FIS; FEDER, Ministery of Economy and Competitivity, Madrid, Spain grant PI11/02399; and Fundación Ramón Areces, Madrid, Spain grant CIVP16A1806; and by Associazione Italiana Leucemie e Linfomi of Verona (AIL-Verona) and ASIMAS (Associazione Italiana Mastocitosi). L. Escribano has been supported by one or more grants from Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Fundacion Sociosanitaria de Castilla La Mancha, and from FEM. A. García-Montero has been supported by one or more grants from ISCIII. M. Mollejo and A. Orfao have been supported by one or more grants from Instituto de Salud Carlos III, Ministerio de Economía y Competitividad. L. Sánchez-Muñoz has been supported by one or more grants from Fundacion Sociosanitaria de Castilla La Mancha.Peer Reviewe

Complete response to gemtuzumab ozogamicin in a patient with refractory mast cell leukemia

Mast cell (MC) leukemia (MCL) is a subtype of systemic mastocytosis (SM) defined by the World Health Organization as ⩾ 20% of MCs in the bone marrow (BM) aspirate, with (leukemic variant) or without (aleukemic variant) ⩾ 10% of MCs in peripheral blood (PB). The European/American Consensus Group on Mastocytosis has recently proposed a new subclassification of MCL that distinguishes acute vs chronic MCL based on the presence vs absence of organ damage, respectively.Peer Reviewe

Simultaneous inhibition of pan-phosphatidylinositol-3-kinases and MEK as a potential therapeutic strategy in peripheral T-cell lymphomas

Obtained from Haematologica/the Hematology Journal website http://www.haematologica.orgPeripheral T-cell lymphomas are very aggressive hematologic malignancies for which there is no targeted therapy. New, rational approaches are necessary to improve the very poor outcome in these patients. Phosphatidylinositol- 3-kinase is one of the most important pathways in cell survival and proliferation. We hypothesized that phosphatidylinositol- 3-kinase inhibitors could be rationally selected drugs for treating peripheral T-cell lymphomas. Several phosphatidylinositol-3-kinase isoforms were inhibited genetically (using small interfering RNA) and pharmacologically (with CAL-101 and GDC-0941 compounds) in a panel of six peripheral and cutaneous T-cell lymphoma cell lines. Cell viability was measured by intracellular ATP content; apoptosis and cell cycle changes were checked by flow cytometry. Pharmacodynamic biomarkers were assessed by western blot. The PIK3CD gene, which encodes the δ isoform of phosphatidylinositol-3-kinase, was overexpressed in cell lines and primary samples, and correlated with survival pathways. However, neither genetic nor specific pharmacological inhibition of phosphatidylinositol-3-kinase δ affected cell survival. In contrast, the pan-phosphatidylinositol-3-kinase inhibitor GDC-0941 arrested all T-cell lymphoma cell lines in the G1 phase and induced apoptosis in a subset of them. We identified phospho-GSK3b and phospho-p70S6K as potential biomarkers of phosphatidylinositol-3-kinase inhibitors. Interestingly, an increase in ERK phosphorylation was observed in some GDC-0941-treated T-cell lymphoma cell lines, suggesting the presence of a combination of phosphatidylinositol-3-kinase and MEK inhibitors. A highly synergistic effect was found between the two inhibitors, with the combination enhancing cell cycle arrest at G0/G1 in all T-cell lymphoma cell lines, and reducing cell viability in primary tumor T cells ex vivo. These results suggest that the combined treatment of pan-phosphatidylinositol-3-kinase + MEK inhibitors could be more effective than single phosphatidylinositol-3-kinase inhibitor treatment, and therefore, that this combination could be of therapeutic value for treating peripheral and cutaneous T-cell lymphomas.This work was supported by grants from the Asociación Española Contra el Cáncer, Fondo de Investigaciones Sanitarias (PI051623, PI052800 and PI080856), RTICC (RD06/0020/0107) and Ministerio de Ciencia e Innovación (SAF2008-0387-1). EMS is supported by a grant from the Department of Education, Universities and Research of the Basque Government (BFI08.207). MSB is supported by a Contract Miguel Servet from Fondo de Investigaciones Sanitarias (CP11/00018

Chronic lymphocytic leukemia cells in lymph nodes show frequent NOTCH1 activation

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world. Pathogenic mechanisms involve multiple external events (such as microenvironmental and antigenic stimuli) and internal events (genetic and epigenetic alterations) that are associated with the transformation, progression and evolution of CLL. CLL is characterized by an accumulation of mature B cells in peripheral blood, bone marrow and lymphoid tissues. Extracellular stimuli play an important role in the development and maintenance of neoplastic cells. B-CLL cells proliferate and activate pathogenic signaling pathways in anatomical structures known as proliferation centers, which are usually more conspicuous in involved lymph nodes.1 Its clinical course is quite heterogeneous, whereby some patients progress rapidly and have short survival, whereas others have a more stable clinical course that may not need treatment for years.This work was supported by grants from the Ministerio de Economía y Competitividad (MINECO) (SAF2013-47416-R) Instituto de Salud Carlos III (ISCIII)- FEDER – MINECO- AES (CP11/00018, PI10/00621, RD012/0036/0060), and Asociación Española contra el Cancer (AECC). MS-B is supported by a Miguel Servet contract from ISCIII-FEDER (CP11/00018). Salary support to SG is provided by CP11/00018, from ISCIII-FEDER. JG-R is supported by a predoctoral grant from the Fundación Investigación Puerta de Hierro.S

Stratifying diffuse large B-cell lymphoma patients treated with chemoimmunotherapy: GCB/non-GCB by immunohistochemistry is still a robust and feasible marker

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous group of aggressive lymphomas that can be classified into three molecular subtypes by gene expression profiling (GEP): GCB, ABC and unclassified. Immunohistochemistry-based cell of origin (COO) classification, as a surrogate for GEP, using three available immunohistochemical algorithms was evaluated in TMA-arranged tissue samples from 297 patients with de novo DLBCL treated by chemoimmunotherapy (R-CHOP and R-CHOP-like regimens). Additionally, the prognostic impacts of MYC, BCL2, IRF4 and BCL6 abnormalities detected by FISH, the relationship between the immunohistochemical COO classification and the immunohistochemical expression of MYC, BCL2 and pSTAT3 proteins and clinical data were evaluated. In our series, non-GCB DLBCL patients had significantly worse progression-free survival (PFS) and overall survival (OS), as calculated using the Choi, Visco-Young and Hans algorithms, indicating that any of these algorithms would be appropriate for identifying patients who require alternative therapies to R-CHOP. Whilst MYC abnormalities had no impact on clinical outcome in the non-GCB subtype, those patients with isolated MYC rearrangements and a GCB-DLBCL phenotype had worse PFS and therefore might benefit from novel treatment approaches

Peripheral T-cell lymphoma: Molecular profiling recognizes subclasses and identifies prognostic markers

Peripheral T-cell lymphoma (PTCL) is a clinically aggressive disease, with a poor response to therapy and a low overall survival rate of approximately 30% after 5 years. We have analyzed a series of 105 cases with a diagnosis of PTCL using a customized NanoString platform (NanoString Technologies, Seattle, WA) that includes 208 genes associated with T-cell differentiation, oncogenes and tumor suppressor genes, deregulated pathways, and stromal cell subpopulations. A comparative analysis of the various histological types of PTCL (angioimmunoblastic T-cell lymphoma [AITL]; PTCL with T follicular helper [TFH] phenotype; PTCL not otherwise specified [NOS]) showed that specific sets of genes were associated with each of the diagnoses. These included TFH markers, cytotoxic markers, and genes whose expression was a surrogate for specific cellular subpopulations, including follicular dendritic cells, mast cells, and genes belonging to precise survival (NF-κB) and other pathways. Furthermore, the mutational profile was analyzed using a custom panel that targeted 62 genes in 76 cases distributed in AITL, PTCL-TFH, and PTCL-NOS. The main differences among the 3 nodal PTCL classes involved the RHOAG17V mutations (P < .0001), which were approximately twice as frequent in AITL (34.09%) as in PTCL-TFH (16.66%) cases but were not detected in PTCL-NOS. A multivariate analysis identified gene sets that allowed the series of cases to be stratified into different risk groups. This study supports and validates the current division of PTCL into these 3 categories, identifies sets of markers that can be used for a more precise diagnosis, and recognizes the expression of B-cell genes as an IPI-independent prognostic factor for AITL