ALBERT

Description: Diffuse large B-cell lymphomas (DLBCLs) can be divided into germinal-center B cell–like (GCB) and activated-B cell–like (ABC) subtypes by gene-expression profiling (GEP), with the latter showing a poorer outcome. Although this classification can be mimicked by different immunostaining algorithms, their reliability is the object of controversy. We constructed tissue microarrays with samples of 157 DLBCL patients homogeneously treated with immunochemotherapy to apply the following algorithms: Colomo (MUM1/IRF4, CD10, and BCL6 antigens), Hans (CD10, BCL6, and MUM1/IRF4), Muris (CD10 and MUM1/IRF4 plus BCL2), Choi (GCET1, MUM1/IRF4, CD10, FOXP1, and BCL6), and Tally (CD10, GCET1, MUM1/IRF4, FOXP1, and LMO2). GEP information was available in 62 cases. The proportion of misclassified cases by immunohistochemistry compared with GEP was higher when defining the GCB subset: 41%, 48%, 30%, 60%, and 40% for Colomo, Hans, Muris, Choi, and Tally, respectively. Whereas the GEP groups showed significantly different 5-year progression-free survival (76% vs 31% for GCB and activated DLBCL) and overall survival (80% vs 45%), none of the immunostaining algorithms was able to retain the prognostic impact of the groups (GCB vs non-GCB). In conclusion, stratification based on immunostaining algorithms should be used with caution in guiding therapy, even in clinical trials.

Description: The balance between actions of procoagulant and anticoagulant factors protects organisms from bleeding and thrombosis. Thus, antithrombin deficiency increases the risk of thrombosis, and complete quantitative deficiency results in intrauterine lethality. However, patients homozygous for L99F or R47C antithrombin mutations are viable. These mutations do not modify the folding or secretion of the protein, but abolish the glycosaminoglycan-induced activation of antithrombin by affecting the heparin-binding domain. We speculated that the natural β-glycoform of antithrombin might compensate for the effect of heparin-binding mutations. We purified α- and β-antithrombin glycoforms from plasma of 2 homozygous L99F patients. Heparin affinity chromatography and intrinsic fluorescence kinetic analyses demonstrated that the reduced heparin affinity of the α-L99F glycoform (KD, 107.9 ± 3nM) was restored in the β-L99F glycoform (KD, 53.9 ± 5nM) to values close to the activity of α-wild type (KD, 43.9 ± 0.4nM). Accordingly, the β-L99F glycoform was fully activated by heparin. Similar results were observed for recombinant R47C and P41L, other heparin-binding antithrombin mutants. In conclusion, we identified a new type of mosaicism associated with mutations causing heparin-binding defects in antithrombin. The presence of a fully functional β-glycoform together with the activity retained by these variants helps to explain the viability of homozygous and the milder thrombotic risk of heterozygous patients with these specific antithrombin mutations.

Description: Background FLT3, PIM1, PIM2 and CXCR4 are proteins implicated in the signal transduction of the regulation of proliferation, differentiation and survival of hematopoietic stem cells, at different levels. The impairing of these three processes, regulated by these molecules, constitutes the main hallmark of Acute Myeloid Leukemia (AML). The objectives of this work were to evaluate the expression level of the genes that codify such proteins in patients with AML; as well as correlate this level of expression with biological variables at diagnosis and survival. Methods peripheral blood or bone marrow samples from 31 healthy subject (HS) and 92 AML patients at diagnosis between 2004 and 2012 were studied. The median follow up was 14 months (69% of patients had died). We quantified the expression of FLT3, PIM1 and 2, and CXCR4 by real time PCR, employing primer pairs designed in our laboratory to amplify all known protein-coding transcripts; highlighting the presence of a 30.4% FLT3 ITD, 5.4% of other FLT3 mutations and 26% of NMP1 mutations. Results were then analyzed with the statistical package IBM“ SPSS” Statistics, v20 (IBM“, Nueva York). Results FLT3 was overexpressed in AML patients (FLT3 expression: controls 0.99 vs patients 36.97; p

Description: Abstract 2924 Background: Clonal composition and clone dynamic changes of neoplasms are a controversial issue, whose investigation is now facilitated by the development of massive parallel sequencing. Here we have analyzed the changes in the mutational spectrum associated with progression, treatment response and relapse in a multiple myeloma patient. We sequenced exomes for the primary quiescent-tumor, the progression and the relapsed samples. M&M: Patient and samples description Samples from asymptomatic, progression and relapse phases were compared by FISH and Whole exome massive parallel sequencing in a multiple myeloma patient carrying the t(4;14)(p16.3;q32) alteration. At relapse the cytogenetic study identified the presence of two major clones, 13q14 deletion and t(4;14)(p16.3;q32) in the 60% of the cells, and 17p13 deletion in the 12% of the cells. Whole exome sequencing was performed at CNAG (Barcelona, Spain) following standard protocols for high-throughput paired-end 76pb sequencing on the Illumina HiSeq2000 instruments (Illumina Inc., San Diego, CA). The variant calling was performed using an in house written software calling potential mutations showing a minimum independent multi-aligner evidence. Results: We performed whole exome sequencing on 3 tumor samples from the same patient: the first one at the time of diagnosis correspond to bone marrow infiltrated by 7% of plasma cells. The two additional samples, at progression and relapse, were done in CD138+ bone marrow cells, at this moment the percentage of infiltration was of 84% and 64% respectively. The germinal DNA from the same patient was used as reference. The mean coverage obtained for the four samples were 93x, with around 85% of bases with at least 20X coverage. After filtering, a total of 104 single nucleotide variations (SNV) were identified, some of them in more than one sample. The variations were classified into silent (25), missense (71), nonsense (6), and essential splice (2), according to their potential functional effect. In addition to t(4;14) and del13q14, progression and relapse samples shared 36 common SNVs. There were also some variants gained and/or loss in the different time points, suggesting the presence of at least five different clones, independent but related in their evolution. The two main clones were present in progression and relapse samples, but the ratio of the mutant alleles decreased in parallel to the decrement in the percentage of cells carrying on the described cytogenetic alterations Conclusions: There is a coupling between the cytogenetic and tumor sequence changes indicating that tumor at progression was composed by a dominant clone, together with multiple minor clones. Relapse after treatment was associated with multiple changes in the clone dynamics, progressive reduction of the main clone, emerging of new subclones and lost of minor clones. Dynamic changes along progression could be facilitated/induced by the therapy received. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2647 We have reported in B-NHL cell lines that the p38 MAPK was constitutively activated and was involved in the regulation of tumor cell resistance to cytotoxic drugs. Further, inhibition of this pathway reversed drug resistance. Based in these findings we hypothesized that the activation of the p38 MAPK pathway in patients with B-NHL may be associated with unresponsiveness to cytotoxic drug therapy. This study was designed to test this hypothesis. Eighty patients with Diffused Large B Cell Lymphoma (DLBCL) were used for analysis. Freshly derived tumor tissues from these patients were obtained from biopsies prior to any treatment. Tissue microarrays were prepared and examined by immunohistochemistry for the expression of both p38 MAPK and phosphorylated p38 MAPK (active). The antibodies were tested for specificity. The frequency of stained cells

Description: Abstract 3951 Aim: The aims of this study were on one hand, to evaluate the significance of achieving molecular response (MR) by fluorescent-PCR (F-PCR) of Ig genes in a prospective way, particularly in patients with singular cytogenetics' features. On the other hand, to compared MR with immunophenotypic response (IR) and complete remission by immunofixation (CR). Patients: 130 new MM patients who had achieved CR or VGPR were analyzed by PCR, multiparametric flow cytometry (MFC) and immunofixation at diagnosis, and after induction therapy or transplantation: 48 patients received conventional chemotherapy as induction therapy followed with ASCT and were included in the GEM2000 trial and the other 82 patients were enrolled in the GEM2005 clinical trials in which the induction and maintenance therapy were based on Bortezomib and/or Thalidomide. Methods: The molecular analysis of Immunoglobulin (Ig) gene rearrangements at diagnosis was carried out by fluorescent PCR in DNA from bone marrow samples according to the Biomed-2 protocols. Briefly three different multiplex PCRs: DHJ; Ig Kappa light chain (IGK) rearrangements K-VJ and Kappa deleting element (KDE). DHJ and light chain rearrangements detected at the diagnosis were used in the follow-up. The sensitivity of PCR was between 1/103 and 1/104 as Martínez P. et al previously published in BJH, 2008. Regarding MFC the following monoclonal antibody combinations (FITC/PE/PerCP-Cy5.5/APC) were used at the diagnosis to identify different aberrant plasma cells phenotypes which were used as patient-specific probes for Minimal Residual Disease. Results: 64 patients had negative F-PCR and 66 patients had positive F-PCR after induction therapy. The OS was 77.32% and 59.18%, respectively and the medians OS were 116 and 67 months for each group (P= 0.03). Regarding the PFS, 28 of the 64 F-PCR negative patients relapsed while there were 44 relapses in the group of positive F-PCR. The 5y PFS was 56.44% and 27.43% respectively; the medians PFS were 60 and 36 months respectively (P= 0.0005). The multivariate analysis showed that achieving molecular response by F-PCR had a independent prognostic value in PFS (P=0.003, HR 2.3). Interestingly amongst patients who achieved CR, F-PCR identified a population of patient with a better PFS, figure A (P= 0.04, HR 2.179).The applicability of the F-PCR was almost equivalent to that observed by MFC (91.5 vs. 92%). Nevertheless we found discordant results between these two response criteria. Approximately the 20% of patients who had achieved a MR no obtained IR. The opposite pattern also was observed (IR/noMR) in 10% of the patients. In order to see if the PCR showed differences in terms of survival in patients with high or low risk cytogenetics features at the diagnosis, we made two independent analysis: in one hand we analyzed 14 patients with high-risk cytogenetics [t(4;14), t(14;16) and/or del(17p)] and on the other hand 72 patients with standard-risk cytogenetics. In the former analysis, 4 of the 8 who had negative F-PCR relapsed after the induction therapy while the totality of the other six with positive F-PCR did. The medians PFS were 32 and 17 months respectively (P= 0.0002). In connection with the 72 patients with standard-risk cytogenetics, 13 of the 34 who had negative F-PCR relapsed while 24 of the 38 with positive F-PCR did. The medians PFS times were 75 and 38 months respectively, figure B (P= 0.01). Conclusion: F-PCR of IgH rearrangements is a simple, cheap and feasible method for evaluating response in MM patients after induction therapy or transplantation. Additionally achieving MR provides a similar prognostic value to IR in patients differentially treated, and particularly in patients with singular cytogenetics' features as well as in patients with CR by immunofixation. Disclosures: Paiva: Jansen-Cillag: Honoraria; Celgene: Honoraria. Rosiñol:Janssen: Honoraria; Celgene: Honoraria. Alegre:Janssen: Honoraria; Celgene: Honoraria. Blade:Centocor Ortho Biotech Research & Development: Research Funding. San Miguel:Janssen-Cilag: Honoraria; Celgene: Honoraria. Lahuerta:Celgene: Honoraria; Janssen: Honoraria.

Description: Human-induced changes in atmospheric composition are expected to affect primary productivity across terrestrial biomes. Recent changes in productivity have been observed in many forest ecosystems, but low-latitude upper tree line forests remain to be investigated. Here we use dendrochronological methods and isotopic analysis to examine changes in productivity, and their physiological basis, in Abies religiosa (Ar) and Pinus hartwegii (Ph) trees growing in high-elevation forests of central Mexico. Six sites were selected across a longitudinal transect (Transverse Volcanic Axis), from the Pacific Ocean toward the Gulf of Mexico, where mature dominant trees were sampled at altitudes ranging from 3200 to 4000m asl. A total of 60 Ar and 84 Ph trees were analyzed to describe changes in growth (annual-resolution) and isotopic composition (decadal-resolutions) since the early 1900s. Our results show an initial widespread increase in basal area increment (BAI) during the first half of the past century. However, BAI has decreased significantly since the 1950s with accentuated decline after the 1980s in both species and across sites. We found a consistent reduction in atmosphere to wood 13 C discrimination, resulting from increasing water use efficiency (20-60%), coinciding with rising atmospheric CO 2 . Changes in 13 C discrimination were not followed, however, by shifts in tree ring δ 18 O, indicating site- and species-specific differences in water source or uptake strategy. Our results indicate that CO 2 stimulation has not been enough to counteract warming induced drought stress, but other stressors, such as progressive nutrient limitation, could also have contributed to growth decline. Future studies should explore the distinct role of resource limitation (water vs . nutrients) in modulating the response of high-elevation ecosystems to atmospheric change. © 2013 Blackwell Publishing Ltd

Description: Large strike-slip faults in the eastern Betics are interpreted to have developed in a transcurrent setting in response to 4-6 mm/yr of Africa-Iberia NW-SE convergence. However, here we show that some of these faults are transfer faults accommodating heterogeneous late Miocene extension. The north–Cabrera dextral fault and other E–W to NE–SW strike-slip faults in the Sorbas basin were transfer faults produced under SW–NE extension. These faults together with related normal faults form the main boundaries of two sedimentary depocenters active between the Serravallian and the Tortonian. The older north Cabrera depocenter extended between the Serravallian and the early Tortonian (approx. 13.8 to 9 Ma), whilst the younger Gacía depocenter formed in response to late Tortonian extension (approx. 9 to 7.5 Ma). The latter formed to the west of the north Cabrera depocenter by a listric fan of normal faults with SW-directed transport that are linked by dextral and sinistral transfer fault segments. These faults root on a low-angle detachment cutting into the exhumed high-pressure Nevado–Filabride complex rocks at ~0.8 km depth. The present work reveals that 1) this extension was partially coeval with and kinematically linked to sinistral displacement along the Carboneras fault farther south in the Níjar basin; 2) this westward-directed extension produced elongated core-complexes and tilted-blocks to the north of the Carboneras fault and magmatic accretion upon thinned continental crust to the south, probably in response to slab tearing or detachment and associated edge delamination of the Iberian continental lithospheric mantle beneath the Betics.

Description: ABSTRACT Goodea gracilis is an endemic fish that only habitats in some water bodies of Central Mexico that are contaminated with cyanobacteria-producing microcystins (MC); however, a lack of information on this topic prevails. With the aim to generate the first approximation about the physiological changes elicited by cyanobacterium that produce MC congeners in this fish species, specimens born in the laboratory was exposed for 96 h to cell densities of 572.5, 1145, 2290, 4580, and 9160 × 10 6 cells of Microcystis aeruginosa strain LB85/L, and a set of novel endpoint related to hepatic gluconeogenesis (ADH/LDH) and pro-oxidant forces , H 2 O 2 ) in addition to biomarkers of oxidative damage and antioxidant response was evaluated in the liver. Results suggest that high inhibition of protein serine/threonine phosphatase (PP) may trigger many metabolic processes, such as those related to hepatic gluconeogenesis (ADH/LDH) and pro-oxidant , H 2 O 2 , TBARS, ROOH, RC O) as well as antioxidant (SOD, CAT, GPx) response to oxidative stress. Particularly, we observed that inhibition of LDH and PP, and H 2 O 2 increase and TBARS production were the key damages induced by high densities of M. aeruginosa . However, changes between aerobic and anaerobic metabolism related with ROS metabolism and ADH/LDH balance are apparently an acclimation of this fish species to exposure to cyanobacteria or their MCs. Fish species living in environments potentially contaminated with cyanobacteria or their MCs possess mechanisms of acclimation that allow them to offset the damage induced, even in the case of fish that have never been exposed to MCs. © 2014 Wiley Periodicals, Inc. Environ Toxicol, 2014.

Description: P2X7 is a purinergic receptor-channel; its activation by ATP elicits a broad set of cellular actions, from apoptosis to signals for survival. Here, P2X7 expression and function was studied in human ovarian carcinoma (OCA) cells, and biopsies from non-cancerous and cancer patients were analyzed by immunohistochemistry. Ovarian surface epithelium in healthy tissue expressed P2X7 at a high level that was maintained throughout the cancer. The cell lines SKOV-3 and CAOV-3 were used to investigate P2X7 functions in OCA. In SKOV-3 cells, selective stimulation of P2X7 by 2′(3′)-O-(4-benzoylbenzoyl) adenosine-5′-triphosphate (BzATP) induced a dose-dependent increase of intracellular Ca 2+ concentration ([Ca 2+ ] i ) but not cell death. Instead, BzATP increased the levels of phosphorylated ERK and AKT (pERK and pAKT), with an EC 50 of 44 ± 2 and 1.27 ± 0.5 μM, respectively; 10 μM BzATP evoked a maximum effect within 15 min that lasted for 120 min. Interestingly, basal levels of pERK and pAKT were decreased in the presence of apyrase in the medium, strongly suggesting an endogenous, ATP-mediated phenomenon. Accordingly: (i) mechanically stimulated cells generated a [Ca 2+ ] i increase that was abolished by apyrase; (ii) apyrase induced a decrease in culture viability, as measured by the MTS assay for mitochondrial activity; (iii) incubation with 10 μM AZ10606120, a specific P2X7 antagonist and transfection with the dominant negative P2X7 mutant E496A, both reduced cell viability to 70.1 ± 8.9% and to 76.5 ± 5%, respectively, of control cultures. These observations suggested that P2X7 activity was auto-induced through ATP efflux; this increased pERK and pAKT levels that generated a positive feedback on cell viability. © 2014 Wiley Periodicals, Inc.