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Dense (>98 th%) homogeneous mullite/Mo (32 vol.%) composites with two different Mo average grain sizes (1.4 and 3 μm) have been obtained at 1650°C in vacuum and in reducing condition. Depending on the Mo grain size and processing atmosphere, the K IC ranges from 4 to 7 MPa m 1/2 and σ f from 370 to 530 MPa. The MoO 2 –2SiO 2 ·3Al 2 O 3 –Mo system was found to be compatible in solid state, and a solid solution of ≈4 wt% of MoO 2 in mullite at 1650°C was detected. A solid state dewetting of MoO 2 from the surface of the Mo particle takes place during sintering. It was found that the absence of MoO 2 in the mullite/Mo composites by processing in reducing conditions increases the strength of the metal/ceramic interface and the plasticity of the Mo metal particles, thus strengthening the composite by a crack bridging mechanism. As a result, the K IC and the σ f values of the ceramic–metal composite were found to be ≈4 times and ≈2 times higher than the ones corresponding to the mullite matrix.

The possibility of fabricating continuous mullite/Mo FGMs with density closed to theoretical, by pressure slip casting of proof ethyl alcohol based slurries has been shown. Dense (> 98 th%) homogeneous mullite/Mo (32 vol%) composites with two Mo average grain sizes (1.5 and 3 μm) have been obtained at 1650°C in vacuum and under reducing conditions. Depending on the Mo grain size and processing atmosphere, the KIC ranged from 4 to 7 MPa m1 /2 and σf from 370 to 530 MPa. The MoO2-2SiO2 . 3Al2O3-Mo were found to be compatible in solid state and a solid solution of ~ 4 wt% of MoO2 in mullite at 1650°C was detected. A solid state dewetting of MoO2 from the surface of the Mo particle takes place during sintering. It was found that the absence of MoO2 in the mullite/Mo composites by processing in reducing conditions increases the plasticity of the metal particles strengthening the material by a crack bridging mechanism. Due to this fact, the KIC and the σf values of the metal/ceramic composite were found to be ~...

In the Funding Statement, a funding organization was incorrectly listed as providing one of the grants used in the research for the article. In addition, a funding organization and an additional grant were incorrectly omitted from the Funding Statement. The Funding Statement should read: "This work was supported by grants from the Spanish Fondo de Investigaciones Sanitarias-Fondos FEDER European Union (FIS PI08/0566 and FIS P12/02587) and Red de Investigacion Renal (REDinREN). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Mantle cell lymphoma (MCL) is considered a distinct type of B-cell lymphoma genetically characterized by the t(11;14) translocation and cyclin D1 overexpression. There is also a small subset of tumors negative for cyclin D1 expression that are morphologically and immunophenotypically indistinguishable from conventional MCL. Although in the last decades, the median overall survival of patients with MCL has improved significantly, it is still considered as one of the poorest prognoses diseases among B-cell lymphomas. Election of treatment for patients with MCL is complex due to the scarcity of solid evidence. Current available data shows that conventional chemotherapy does not yield satisfactory results as in other types of B-cell lymphomas. However, the role of other approaches such as autologous or allogenic stem cell transplantation, immunotherapy, the administration of consolidation or maintenance schedules, or the use of targeted therapies still lack clear indications. In view of this situation, the Spanish Group of Lymphomas/Autologous Bone Marrow Transplantation has conducted a series of reviews on different aspects of MCL, namely its diagnosis, prognosis, first-line and salvage treatment (both in young and elderly patients), new targeted therapies, and detection of minimal residual disease. On the basis of the available evidence, a series of recommendations have been issued with the intention of providing guidance to clinicians on the diagnosis, treatment, and monitoring of patients with MCL., The authors acknowledge the support of Pfizer Spain.

Background and Objectives Minimal residual disease (MRD) assessment by PCR in multiple myeloma (MM) has several shortcomings, including the lack of a suitable target. Kappa deleting element (KDE) rearrangements occur in virtually all Ig-lambda B-cell malignancies and in 1/3 of Ig-kappa are not affected by somatic hypermutation and, as in ALL, could be used as PCR targets. Methods We have first investigated the incidence, gene segment usage, and CDR3 composition of IGK-KDE rearrangements in 96 untreated myeloma patients. Second, we tested 16 KDE gene rearrangements as molecular targets for MRD assessment by RQ-PCR using a germline reverse primer and a germline Taqman probe in combination with allele-specific oligonucleotides (ASO) as forward primers. Results Monoclonal KDE rearrangements were amplified in 45% (43/96) of cases, monoallelic in 2/3 of them (29 cases), and biallellic in the remaining 14 cases. Overall, 88% of cases were successfully sequenced, KDE being equally frequently rearranged with VK and with intron-Recombination signal sequence (RSS). Median numbers of inserted and deleted nucleotides in the junctional region were one and five, respectively. Conclusions Using KDE rearrangements as additional PCR target for MRD assessment in MM improves the applicability of these studies in 9% of cases overall and in 20% of lambda cases. Its use in the latter subset could represent a significant advance.

There is barely any information about the prognostic significance of FLT3 expression and mutational status in cytogenetically distinct subgroups of acute lymphoblastic leukemia (ALL). We analyzed the presence of FLT3-tyrosine kinase domain (TKD) and FLT3-internal tandem duplication (ITD) mutations as well as FLT3 expression levels in 54 newly diagnosed patients with B-ALL (n=49) or T-ALL (n=5). All B/T-ALL samples tested negative for the presence of FLT3-TKD or FLT3-ITD. None of the T-ALL and E2A-PBX1+ B-ALL overexpressed FLT3. In contrast, mainly MLL-AF4+ B-ALL but also ETV6-RUNX1+, BCR-ABL+ or B-ALL displaying normal cytogenetics exhibited significantly higher FLT3 expression levels than normal bone marrow, supporting that aberrantly increased transcription of FLT3, rather than activating FLT3 mutations, contributes to the pathogenesis of these B-ALL. Using the median FLT3 expression as cut-off value we found that high-level FLT3 expression is associated with an extremely poor 1-year overall survival (OS; 0 vs 71%; P=0.002) and disease-free survival (DFS; 0 vs 43%; P=0.03) in MLL-AF4+ B-ALL but not in MLL-germline B-ALL. Cox regression analysis with OS/DFS as end points showed that age>14 years and high-level FLT3 expression were independent prognostic factors when all ALL patients were analyzed together. Importantly, when the MLL-AF4+ B-ALL subgroup was analyzed separately, high-level FLT3 expression was the only independent prognostic factor for OS and treatment outcome. These findings indicate that high FLT3 expression identifies MLL-AF4+ ALL patients at very high risk of treatment failure and poor survival, emphasizing the value of ongoing/future clinical trials for FLT3 inhibitors.

We have evaluated the use of CD138+ positively selected bone marrow samples to identify a molecular target for minimal residual disease assessment by polymerase chain reaction (PCR) in 25 untreated patients with multiple myeloma. A fraction of each sample was used for CD138+ selection, and the rest served as a reference control. VDJH, DJH, and Kde gene rearrangements were tested for amplification according to the BIOMED-2 Concerted Action. PCR products were directly sequenced in an automated ABI 3130 DNA sequencer using Big-Dye terminators. Within the CD138+ selected group, VDJH rearrangements were detected in all cases (100 %), DJH in 16 (64 %), and Kde in 18 (72 %) cases; whereas in the control samples, VDJH, DJH, and Kde rearrangements were detected in 19 (76 %), 11 (44 %), and 12 (48 %) cases, respectively. After sequencing, 24 (96 %) cases within the CD138+ group had a PCR target for MRD detection compared with 15 (60 %) cases in the control group. We conclude that the use of CD138+ positively selected bone marrow samples increases the applicability of minimal residual disease studies by PCR in patients with multiple myeloma.

All-trans retinoic acid (ATRA) induces complete remission in 64–100 % of patients with acute promyelocytic leukemia (APL), and is considered to be a safe agent. Pseudotumor cerebri is a neurological side effect of ATRA reported in pediatric patients, and which is characterized by raised cerebrospinal fluid pressure in the absence of any intracranial pathology or secondary causes of intracranial hypertension. Involvement of cranial nerves other than II and VI is very uncommon in idiopathic intracranial hypertension (IIH); peripheral facial nerve palsy is exceptional and has rarely been described in the context of treatment with ATRA. We describe the case of a 15-year-old female patient with APL who developed an IIH and involvement of cranial nerves (bilateral papilledema, left facial and right sixth nerves) after receiving induction therapy including ATRA. Viral infections and other causes of secondary cranial nerve lesions were excluded. Symptoms completely subsided with the temporary withdrawal of ATRA and did not recur after reintroducing the drug. To date, the patient has managed to receive the treatment as per protocol. In conclusion, we report an atypical presentation of IIH that merits consideration, especially with respect to young patients with APL receiving ATRA; our most important observation is that the drug could be safely reintroduced once the symptoms had resolved.

Background and objectives. PIM kinases (PIM1, PIM2, PIM3) are proteins known to be overexpressed in several hematological malignancies. In particular, in chronic lymphocytic leukemia (CLL) they are involved in cell survival, resistance to apoptosis (especially PIM2 and PIM3) and interactions with the microenvironment (PIM1). The aim of this study was dual: I) to evaluate the preclinical efficacy of PIM447, a pan PIM kinase inhibitor, in CLL and to study potential synergies with other drugs; and II) to evaluate the expression of PIM-kinases in different stages of the disease and correlate it with the prognosis and the sensitivity to the drug. Methods. Peripheral blood samples from untreated patients with different stages of the disease (monoclonal B lymphocytosis (MBL), stable CLL not requiring treatment (sCLL), and active CLL requiring treatment (aCLL)) were collected after informed consent. The ex vivo efficacy of PIM447 was analyzed by flow cytometry with annexin V in these samples. Moreover, PIM447 efficacy was also analyzed in two cell lines (MEC-1 and JVM-2) by MTT assay. Synergy with other drugs effective in CLL (bendamustine and fludarabine) was evluated with the calcusyn software. Protein levels of PIM Kinase proteins were evaluated by capillary electrophoresis immunoassay (WESTM ProteinSimple) in monoclonal B cells purified by CD19 selection with anti-CD19 magnetic microbeads and the autoMacs Cell separator (both from Miltenyi Biotec) from a subset of patients. Results. The pan PIM inhibitor, PIM447 was active in both cell lines tested, MEC-1 (IC50 5μM) and JVM2 (IC50 7μM), and also in monoclonal B cells from freshly isolated patients samples (sCLL=11; aCLL=5), with no difference in sensitivity between the different stages of the disease (IC50 of 4,8 μM and 4,7 μM for sCLL and aCLL respectively). There was a clear therapeutic window as treatment with PIM447 at doses toxic for monoclonal B cells, preserved T lymphocytes (figure 1) (median % of apoptosis for B cells and T lymphocytes respectively of 23 vs 20 at 5μM and 87 vs 35 at 10 μM). Moreover, PIM447 demonstrated to potentiate the activity of both bendamustine and fludarabine, being especially synergistic with this last one (combination index 0.1-0.6). A second objective was to analyze PIM2 protein expression by western blot in monoclonal B cells from these samples and correlate it with clinical and biological features. Up to now, it has been evaluated in 18 samples (MBL=4; sCLL=8; aCLL=6,). All of them expressed PIM-2. Expression levels of this protein were significantly higher in active CLL as compared with indolent stages of the disease (p=0,012). Patients with an unmutated IGHV status also displayed higher levels of PIM2 (p=0,01). Finally, samples with high PIM2 levels were slightly more resistant to PIM447 as compared with samples with lower protein levels (IC50 of 7,7 μM vs 5 μM, respectively). We are currently completing the analysis of the PIM2 levels of remaining samples and we are also measuring the levels of PIM1 protein, what will be available at the meeting. Conclusions: PIM-Kinase inhibition with PIM447 is effective in vitro in CLL cell lines and ex vivo in samples from patients. It synergizes with other agents especially fludarabine. PIM2 protein levels correlated with the clinical activity of CLL and with the mutational state of IGHV. Although all patients appear sensitive ex vivo to PIM447, further work is required to define PIM2 expression as a marker of sensitivity. Figure 1. Figure 1. Disclosures Ocio: Array BioPharma: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy; Mundipharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; MSD: Research Funding; Pharmamar: Consultancy, Research Funding; Jassen: Honoraria.

Here we perform whole-exome sequencing of samples from 105 individuals with chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults in Western countries. We found 1,246 somatic mutations potentially affecting gene function and identified 78 genes with predicted functional alterations in more than one tumor sample. Among these genes, SF3B1, encoding a subunit of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP), is somatically mutated in 9.7% of affected individuals. Further analysis in 279 individuals with CLL showed that SF3B1 mutations were associated with faster disease progression and poor overall survival. This work provides the first comprehensive catalog of somatic mutations in CLL with relevant clinical correlates and defines a large set of new genes that may drive the development of this common form of leukemia. The results reinforce the idea that targeting several well-known genetic pathways, including mRNA splicing, could be useful in the treatment of CLL and other malignancies.

A silver-hydroxyapatite nanocomposite has been obtained by a colloidal chemical route and subsequent reduction process in H2/Ar atmosphere at 350∘C. This material has been characterized by TEM, XRD, and UV-Visible spectroscopy, showing the silver nanoparticles (∼65 nm) supported onto the HA particles (∼130 nm) surface without a high degree of agglomeration. The bactericidal effect against common Gram-positive and Gram-negative bacteria has been also investigated. The results indicated a high antimicrobial activity for Staphylococcus aureus, Pneumococcus and Escherichia coli, so this material can be a promising antimicrobial biomaterial for implant and reconstructive surgery applications., This work was supported by Bioker Research S.L. and by the Spanish Ministry of Education and Science under Project CENIT-INTELIMPLANT (ref.: 20071210).M. Díaz has been supported by Spanish Ministry of Education and Science and CSIC under I3P Program financed by European Social Fund.

303 Mutations in the Toll-like receptor/ MYD88 pathway in young (£50 years) CLL patients Alejandra Martinez-Trillos, Magda Pinyol, Alba Navarro, Marta Aymerich, Pedro Jares, Manel Juan, Maria Rozman, Dolors Colomer, Julio Delgado, Eva Gine, Marcos Gonzalez-Diaz, Jesus M. Hernandez-Rivas, Enrique Colado, Consolacion Rayon, Angel R. Payer, Maria Jose Terol, Blanca Navarro, Victor Quesada, Xose S. Puente, Ciril Rozman, Carlos Lopez-Otin, Elias Campo, Armando Lopez-Guillermo, PhD MD, Neus Villamor Unitat d’Hematopatologia, Servei d’Anatomia Patologica, Hospital Clinic de Barcelona; IDIBAPS, Unitat de Genomica; Servei d’Inmunologia, Hospital Clinic de Barcelona; Unitat d’Hematopatologia, Servei d’Anatomia Patologica, Hospital Clinic de Barcelona; Unitat de Genomica, Unitat de Genomica, IDIBAPS; Servicio de Hematologia, Hospital Clinico Universitario, IBMCC, IBSAL, Centro de Investigacion del Cancer (USAL-CSIC), Universidad de Salamanca; Servicio de Hematologia, Hospital Universitario Central de Asturias; Servicio de Hematologia, Hospital Clinico de Valencia; Departamento de Bioquimica y Biologia Molecular, Universidad de Oviedo; Josep Carreras Leukemia Research Institute, Barcelona; Departamento de Bioquimica y Biologia Molecular, Universidad de Oviedo Table Main clinical and biological characteristics of 146 CLL patie Parameter Category Unmuta (n[12 Gender Male (%) 85 (6 Age (years), median (range) 44 (2 0 44/126 (3 Rai stage I-II 77/126 (6 III-IV 5/126 (4 Monoclonal component 7/127(6 b2microglobulin UNV 40/103 (3 IGHV Mutated 43/99 (4 CD38

APL is characterised by chromosomal rearrangements involving the gene encoding the myeloid transcription factor Retinoic Acid Receptor Alpha (RARα). While RARA is fused to PML in the majority of cases consequent upon the t(15;17)(q22;q21), significant insights can be gained into pathogenesis through analysis of APL involving alternative fusion partners. It is now apparent that the fusion partner has a critical bearing on disease biology, particularly with respect to sensitivity to molecularly targeted therapies i.e. all transretinoic acid (ATRA) and arsenic trioxide (ATO). While ATO sensitivity is restricted to PML-RARA+ APL, the fusion partner influences sensitivity to ATRA, with involvement of STAT5b or PLZF (ZBTB16) being associated with resistance and poorer clinical outcome. In the European Working Party study (Blood 2000;96:1297-1308) we previously established that ~10% of morphological APL cases lack the classic t(15;17). While PML is involved in the majority due to insertion events or more complex rearrangements, the molecular basis of disease in some patients has been unexplained. Analysis was undertaken in a normal karyotype APL (WBC 3.8 x 109/l, plt 7 x 109/l) presenting in a 37 year old man, PML-RARA negative by RT-PCR, with intact PML nuclear bodies on immunofluorescence. Due to the suggestive morphology, the patient was commenced on ATRA as a single agent (45mg/m2) pending further molecular investigation. ATRA induced maturation of the leukemic blasts, apparent in bone marrows taken at day 7 and day 16, associated with normalization of the platelet count (100 x 109/l by day 25). Diagnostic material was subject to gene expression profiling (TaqMan® Array Card, Life Technologies) and on unsupervised analysis clustered with PML-RARA+ cases, with a common signature associated with upregulation of HGF and FGF13 and downregulation of HOXA7, HOXA9 and MEIS1. 5’ RACE-PCR confirmed involvement of RARA and identified the fusion partner as IRF2BP2 (Interferon Regulatory Factor 2 Binding Protein 2), which is located at 1q42. The cDNA breakpoint involved fusion of exon 1 of IRF2BP2 to exon 3 of RARA leading to retention of the regions mediating binding to DNA, retinoic acid (ligand binding domain, LBD) and RXR. Chromosome 1 painting was undertaken, which appeared normal and a reciprocal RARA-IRF2BP2 fusion transcript was not detected, consistent with formation of IRF2BP2-RARA by cryptic insertion. Whole exome sequencing identified 17 non-synonymous exonic single nucleotide variants, three (with predicted functional impact) were in genes known to be recurrently mutated in AML (LRP1B, GATA2 and EZH2). The patient’s treatment response was tracked by real-time quantitative PCR (sensitivity 1 in 10e4), which showed persistent high level IRF2BP2-RARA fusion transcripts following single agent ATRA. The patient was commenced on the PETHEMA treatment schedule, achieved molecular remission (CRm) following the first course of chemotherapy and remains in ongoing CRm in the marrow 32 months from diagnosis. IRF2BP2 is a downstream target of p53 and repressor of the cell cycle regulator NFAT1; it is fused to CDX1 as a result of the t(1;5)(q42;q32) in mesenchymal chondrosarcoma, has been reported to be mutated in CLL and primary CNS lymphoma, but has not previously been implicated in myeloid malignancy. To investigate the biology of IRF2BP2-RARα, co-immunoprecipitation assays were performed in 293T cells using tagged constructs, which revealed that the fusion protein has the capacity to self-associate. In reporter assays, IRF2BP2-RARα behaved similarly to PML-RARα, inducing repression at retinoid response elements, which was overcome by 1μM ATRA. Full length IRF2BP2-RARA was cloned into the MSCV vector, expressed in lin- murine stem/progenitor cells and replating assays performed in three conditions (SCF, IL3 + IL6/ SCF, IL3, IL6 + G-CSF/ SCF, IL3, IL6, G-CSF + GM-CSF). Expression of IRF2BP2-RARA consistently induced large numbers of third round colonies under all 3 conditions; whereas, colony formation was completely abrogated in the presence of 1μM ATRA in accordance with the sensitivity to retinoid observed in vivo. In conclusion, these data extend the range of malignancies involving IRF2BP2, identifying a novel retinoid sensitive subtype of APL and provide further evidence that forced dimerization of RARα mediated by the fusion partner plays a critical role in leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Multiple myeloma (MM) pathogenesis has been explained for many years by the cancer biology dogma introduced by Peter Nowell: first, a single plasma cell would be immortalized by an error in the immunoglobulin genes rearrangement process; then, a progressive stepwise acquisition of somatic cell mutations would induce a sequential selection and domination by the fittest clone. In line with this idea of “myeloma stability”, SNP arrays studies in diagnostic-relapse paired samples have revealed the presence of common clonal characteristics. Biologically, the M-protein remains usually constant across MM evolution and further, the variable domain of the rearranged immunoglobulin heavy chain genes (or CDR3 region) has been used as a patient-specific myeloma fingerprint in minimal residual disease (MRD) studies. However, massive genome studies with Next Generation Sequencing (NGS) have challenged this concept, showing a significant intraclonal heterogeneity at diagnosis with the possible presence of several clonal progenitors or tumor-initiating cells. In this study, we have characterized and compared the CDR3 region in 52-paired samples from 26 MM patients aiming: 1) to assess mono-clonality in MM evolution through the analysis of the CDR3 sequence and, 2) to validate ASO RQ-PCR approaches for MRD in MM, based on the constancy and specificity of the CDR3 region. Samples were obtained at diagnosis and progression (19 pairs) or at 2 different timepoints of progressive disease (7 pairs). Median time between sampling was 2 years. M-protein subtype remained stable in all pairs but 1, associated with a light-chain escape phenomenon. All samples proceeded from bone marrow (BM) except for 2 pairs, composed by BM and extramedullary disease (spleen and testes). Two major cytogenetic changes were identified: increased 13q14 deletion (from 7 to 54%) in 1 pair and increased 17p (p53) deletion (from 5 to 87%) in a further one. Treatments administered between sampling included most of the current approaches used in MM (data not shown). Genomic DNA isolation, PCR amplification and sequencing were performed following conventional methods. Germline VH, DH and JH segments were identified by comparison with public databases. CDR3 region was first identified in all samples and then compared between the two samples in the 26 pairs: the sequence of nucleotides was constantly identical in each pair, including those associated with major cytogenetic changes, a light-chain escape, extramedullar vs. BM infiltration and relapsed (and therefore, treatment selected) vs. refractory disease. Therefore, we can first conclude that the main tumor clone in MM retains a specific signature across all stages of disease evolution that allows the identification of samples as evolutionary related. This major clone signature is not modified by clinical or biological changes in the disease nor under different treatment pressures and would thus identify disease relapse and progression. Our results have also a clear impact on the validity of molecular MRD techniques. The high rate of complete responses (up to 50-60%) currently achieved in MM has prompted the use of new techniques for disease assessment. Today, ASO RQ-PCR, based on the use of specific primers and probes complementary of the VDJH rearrangement, continues to be the most sensitive approach. One pitfall of this technique would be the potential instability of PCR targets over time, which would induce false negative results. In B-cell precursor ALL, this is estimated to happen in 30-40% of cases but has not been deeply evaluated in MM yet. With the present study, we can also conclude that the junction region of the VDJH rearrangement in MM constantly identifies the myeloma cells responsible for relapse and therefore can be used as a reliable target for MRD assessment by ASO RQ-PCR and more recently, by NGS methods. If the CDR3 region remains stable, the novel concept of clonal tiding in MM should not be interpreted as a poly- or oligoclonal but subclonal. In MM, tides can be subclonal, but the ocean remains monoclonal. Disclosures No relevant conflicts of interest to declare.

Obinutuzumab (GA101; G) is an anti-CD20 monoclonal antibody with activity in relapsed/refractory follicular lymphoma (FL) as a single agent and in combination with chemotherapy. G-chemotherapy induction followed by G-maintenance has not been evaluated for untreated FL. The open-label, randomized, phase Ib GAUDI study (NCT00825149) investigated safety and efficacy of G + CHOP (G-CHOP) or bendamustine (G-B) as first-line treatment for FL. We report data from patients (pts) who received G-maintenance after responding to G-chemotherapy induction. Pts with treatment-naïve CD20+ B-cell FL and ≥1 bi-dimensionally measurable lesion (CT scan; largest dimension >1.5cm) were allocated on a per-center basis to receive induction G IV 1000mg + standard CHOP (3-weekly, 6–8 cycles) or B IV 90mg/m2(4-weekly, 4–6 cycles). Induction responders received G-maintenance every 3 months for 2 years or until disease progression (PD). Pts completing maintenance were followed for a further 2 years, until PD or start of new anti-lymphoma therapy. Anti-infective prophylaxis was used at investigator discretion. The primary objective was safety. Secondary objectives included progression-free survival (PFS) and response rates. The overall safety population comprised 81 pts (G-B: n=41; G-CHOP: n=40). Baseline characteristics (age, sex, FLIPI status, bone marrow involvement, bulky disease [lesion ≥7cm], time from diagnosis) were balanced between arms. Median observation time from study start was 31 months (G-B) and 33 months (G-CHOP). The maintenance safety population comprised 72 pts (n=36 from each induction therapy arm). Most pts completed maintenance (G-B: 81%; G-CHOP: 72%). There were 17 discontinuations (24%; G-B: n=7; G-CHOP: n=10), due to an adverse event (AE)/intercurrent illness (n=9), insufficient therapeutic response (n=5), administrative/other (n=2) and death (n=1). During 2 years’ maintenance most pts had AEs: G-B, 100% (44% grade ≥3); G-CHOP, 78% (31% grade ≥3). The most common AE (all grades) was cough (G-B: 17%; G-CHOP: 11%). The grade ≥3 AEs mainly reflected infections and cytopenia. Six G-B pts (17%) experienced 7 grade ≥3 infections; 4 were considered treatment related (genital infection, oral herpes, pneumonia klebsiella, neutropenic infection). Five G-CHOP pts (14%) had one grade ≥3 infection each; 4 were considered treatment related (viral meningitis, respiratory tract infection [RTI], bacterial pneumonia [2 events]). Six G-B pts (17%) experienced 10 grade ≥3 cytopenia AEs; 7 were considered treatment related (anemia, febrile neutropenia, pancytopenia, neutropenia [2 events], thrombocytopenia [2 events]). No G-CHOP pt experienced a grade ≥3 cytopenia AE. AEs led to dose delays in 17% (G-B) and 6% (G-CHOP) of pts. Three pts (G-B: n=1; G-CHOP: n=2) had treatment-related AEs during, or within 24 hours of, an infusion (all grade 1–2). Two deaths (both G-CHOP) occurred during maintenance or maintenance follow-up; 1 due to PD and 1 due to a G-related AE (RTI leading to fatal lactic acidosis). At the end of maintenance, all pts with data available (G-B: n=41; G-CHOP: n=39) had experienced B-cell depletion (0.07x109cells/L). Median IgG levels remained within normal range during maintenance. In the overall safety population, complete response (CR) rate (based on CT scan rather than PET) as best overall response increased from end of induction (G-B: 37%; G-CHOP: 35%) to end of maintenance (G-B: 61%; G-CHOP: 70%). PFS rate at 32 months after first study drug was 92% (G-B) and 84% (G-CHOP). Median PFS was not reached; 10 pts (G-B: n=4; G-CHOP: n=6) had PD, including one transformation to diffuse large B-cell lymphoma. G-maintenance after G-chemotherapy induction was associated with a high CR rate in pts with previously untreated FL. Opportunistic infections occurred infrequently. Clinically relevant neutropenia was experienced by 14% of pts who received G-B induction but was not observed in G-CHOP pts. A phase III trial (GALLIUM) is investigating G versus rituximab in chemoimmunotherapy induction followed by immunotherapy maintenance in pts with untreated indolent non-Hodgkin lymphoma. Disclosures Off Label Use: Obinutuzumab is a type II CD20 monoclonal antibody which is licensed for use in combination with chlorambucil in untreated patients with CLL but is not currently approved for use in follicular lymphoma.. Grigg:Roche: Consultancy. Dreyling:Roche: Honoraria, Research Funding. Rule:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lei:Roche Products Ltd.: Employment. Wassner-Fritsch:Roche: Employment. Fingerle-Rowson:F. Hoffmann–La Roche: Employment. Marlton:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees.

A new pressureless hydrothermal method for the preparation of yttrium disilicate is presented. The obtained amorphous precursor was calcined at different temperatures to form the Y-, α-, β-, and γ-phases of Y2Si2O7, which have been characterized by DTA, XRD, TEM, and 29Si-MAS RMN and IR spectroscopy. It is shown that the proposed method allows the strictly controlled doping of yttrium disilicate using RE elements such as dysprosium (2.5 and 5 at. %). The luminescent properties, in terms of emission efficiency, of different polymorphs of Dy-doped Y2Si2O7 have been investigated by fluorescence measurements at room temperature. The results indicate that the β-phase doped with 2.5 at.% has the maximum efficiency. It is important to note that the efficiency of this phase is approximately 40% of that measured for the commercial phosphor Eu−Y2O3.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Background]: For years, the genetics of metastatic colorectal cancer (CRC) have been studied using a variety of techniques. However, most of the approaches employed so far have a relatively limited resolution which hampers detailed characterization of the common recurrent chromosomal breakpoints as well as the identification of small regions carrying genetic changes and the genes involved in them. [Methodology/Principal Findings]: Here we applied 500K SNP arrays to map the most common chromosomal lesions present at diagnosis in a series of 23 primary tumours from sporadic CRC patients who had developed liver metastasis. Overall our results confirm that the genetic profile of metastatic CRC is defined by imbalanced gains of chromosomes 7, 8q, 11q, 13q, 20q and X together with losses of the 1p, 8p, 17p and 18q chromosome regions. In addition, SNP-array studies allowed the identification of small (1.5 Mb) altered DNA sequences, many of which contain cancer genes known to be involved in CRC and the metastatic process. Detailed characterization of the breakpoint regions for the altered chromosomes showed four recurrent breakpoints at chromosomes 1p12, 8p12, 17p11.2 and 20p12.1; interestingly, the most frequently observed recurrent chromosomal breakpoint was localized at 17p11.2 and systematically targeted the FAM27L gene, whose role in CRC deserves further investigations. [Conclusions/Significance]: In summary, in the present study we provide a detailed map of the genetic abnormalities of primary tumours from metastatic CRC patients, which confirm and extend on previous observations as regards the identification of genes potentially involved in development of CRC and the metastatic process., This work has been partially supported by grants from the Consejeria de Sanidad, Junta de Castilla y Leon, Valladolid, Spain (SAN191/SA09/06 and SAN673/SA39/08), Fundacion Memoria de Don Samuel Solorzano Barruso, Salamanca, Spain, Caja de Burgos (Obra Social), Burgos, Spain, Grupo Excelencia de Castilla y Leon (GR37) and the RTICC from the Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia e Innovacion, Madrid, Spain (RD06/0020/0035-FEDER). JM Sayagués, M Gonzalez, ME Sarasquete and MC Chillon are supported by grants (CP05/00321, FI08/00721, CA08/00212 and CA/07/00077, respectively) from the ISCIII, Ministerio de Ciencia e Innovación, Madrid, Spain. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This paper is distributed under the terms of the Creative Commons Attribution-Non-Commercial-Share Alike licence.-- et al., Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution(1,2). Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes(3,4). The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer., This work was funded by the Spanish Ministry of Science and Innovation (MICINN) through the Instituto de Salud Carlos III (ISCIII) and Red Temática de Investigación del Cáncer (RTICC) del ISCIII. C.L.-O. is an Investigator of the Botin Foundation and D.T., of the ICREA program. We thank E. Santos for his support of this project, A. Carracedo and J. Benítez for genotyping studies, C. Fortuny for the supply of samples and N. Villahoz and M. C. Muro for their work in the coordination of the CLL-ICGC Consortium.

International audience; The International Cancer Genome Consortium (ICGC) was launched to coordinate large-scale cancer genome studies in tumors from 50 different cancer types and/or subtypes that are of clinical and societal importance across the globe. Systematic studies of over 25,000 cancer genomes at the genomic, epigenomic, and transcriptomic levels will reveal the repertoire of oncogenic mutations, uncover traces of the mutagenic influences, define clinically-relevant subtypes for prognosis and therapeutic management, and enable the development of new cancer therapies.

SummaryCurrently, molecular diagnosis of haemophilia A and B (HA and HB) highlights the excess risk-inhibitor development associated with specific mutations, and enables carrier testing of female relatives and prenatal or preimplantation genetic diagnosis. Molecular testing for HA also helps distinguish it from von Willebrand disease (VWD). Next-generation sequencing (NGS) allows simultaneous investigation of several complete genes, even though they may span very extensive regions. This study aimed to evaluate the usefulness of a molecular algorithm employing an NGS approach for sequencing the complete F8, F9 and VWF genes. The proposed algorithm includes the detection of inversions of introns 1 and 22, an NGS custom panel (the entire F8, F9 and VWF genes), and multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 102 samples (97 FVIII- and FIX-deficient patients, and five female carriers) were studied. IVS-22 screening identified 11 out of 20 severe HA patients and one female carrier. IVS-1 analysis did not reveal any alterations. The NGS approach gave positive results in 88 cases, allowing the differential diagnosis of mild/moderate HA and VWD in eight cases. MLPA confirmed one large exon deletion. Only one case did have no pathogenic variants. The proposed algorithm had an overall success rate of 99 %. In conclusion, our evaluation demonstrates that this algorithm can reliably identify pathogenic variants and diagnose patients with HA, HB or VWD.Supplementary Material to this article is available online at www.thrombosis-online.com.

To the Editor. —Bone marrow necrosis, which has been described in sickle cell disease, infectious diseases, and cancer,1-6is a disorder rarely diagnosed during life. In the November 1982Archives(1982;142:2223-2225), Pardoll et al reported a case of bone marrow necrosis associated with sickle cell disease. Reticuloendothelial scans showed widespread abnormalities consistent with marrow infarction; widespread microvascular obstruction by masses of sickle cells seems plausible as a mechanism of marrow necrosis.5Nevertheless, the bone marrow necrosis pathogenesis is unknown in neoplasia and infectious diseases. We report a case of bone marrow necrosis diagnosed antemortem in a patient with gastric metastatic adenocarcinoma, where postmortem pathologic studies may bring further knowledge about necrosis physiopathology. Report of a Case. —A 60-year-old man was admitted to the hospital after three weeks of fever, dyspnea, and bone pain in the lower part of the back. Physical examination showed a severely ill patient, pale with petechia on the limbs, and necrosis in his right toes.