ALBERT

Description: Imaging of diffractions is a challenge in seismic processing. Standard seismic processing is tuned to enhance reflections. Separation of diffracted from reflected events is frequently used to achieve an optimized image of diffractions. We present a method to effectively separate and image diffracted events in the time domain. The method is based on the common-reflection-surface-based diffraction stacking and the application of a diffraction-filter. The diffraction-filter uses kinematic wavefield attributes determined by the common-reflection-surface approach. After the separation of seismic events, poststack time-migration velocity analysis is applied to obtain migration velocities. The velocity analysis uses a semblance based method of diffraction traveltimes. The procedure is incorporated into the conventional common-reflection-surface workflow. We apply the procedure to 2D synthetic data. The application of the method to simple and complex synthetic data shows promising results.

Description: Time migration is an attractive tool to produce a subsurface image because it is faster and less sensitive to velocities errors than depth migration. However, a highly focused time image is only achievable with well-determined time-migration velocities. Therefore, a refinement of the initial time-migration velocities often is required. We introduced a new technique for prestack time migration, based on the common-migrated-reflector-element stack of common scatterpoint gathers, including an automatic update of time-migration velocities. The common scatterpoint gathers are generated using a new formulation of the double-square-root equation that is parametrized with the common-offset apex time. The common-migrated-reflector-element stack is a multiparameter stacking technique based on the Taylor expansion of traveltimes of time-migrated reflections in the paraxial vicinity of the image ray. Our 2D synthetic and field data examples demonstrated that the proposed method provides updated time-migration velocities that are more robust and have higher resolution compared with the initial time-migration velocities. The prestack time migration method also showed a clear improvement of the focusing of reflections for such geologic features as faults and salt structures.

Description: 〈span〉〈div〉ABSTRACT〈/div〉Assessment of the area of interest on tectonic overprint is a key aspect during various geoscientific exploration endeavors. Conventionally, 3D reflection imaging is used because it provides the highest resolution of the subsurface image. Tectonic features, e.g., faults or fractures, are imaged indirectly by means of discontinuities of the specular reflections. Specular reflections, however, are only a part of the backscattering wavefield. Geologic heterogeneities in the subsurface can act as scattering points or scattering edges, which both evoke diffracted waves. Thus, diffracted waves are a direct seismic response from subsurface heterogeneities, which have a size comparable with the prevailing wavelength or a curvature growing locally to infinity. We investigate a dedicated processing of diffracted waves, which allows direct imaging of such heterogeneities. The method is based on separation of reflections from diffractions with subsequent diffraction focusing. We apply a combination of frequency-wavenumber filtering and an adaptive-subtraction engine to isolate diffractions. Once the reflections are removed from a data set, diffracted waves are focused into their apexes yielding an image of scatter points, which can be interpreted as an indicator for the extent of the degree of inhomogeneities or can be used to map structural elements or fracture density. We use a modified coherence analysis as a focusing tool. Diffractions from the scattering points have a different phase response than diffractions from the edges. These differences can be distinguished and further guide the geologic interpretation.〈/span〉

Description: BACKGROUND: Loss of immune surveillance is critical in the pathogenesis of multiple myeloma (MM) and the progression from smoldering to symptomatic MM. To date, no clear efficacy signal has been observed with programmed-death 1 and programmed death ligand-1 inhibitors in patients with MM. General immune dysfunction in MM is well documented, but the evolving immune landscape in relapsed/refractory MM (RRMM) vs newly diagnosed MM (NDMM) is less well characterized. This study aimed to characterize immune profiles in peripheral blood and bone marrow from patients with NDMM and RRMM. METHODS: Peripheral blood samples were collected from 35 NDMM and 146 RRMM patients and 36 age-matched healthy volunteers (HVs). Cell surface and intracellular antigen staining using fluorochrome labeled antibodies was performed on a BD FACSCanto II flow cytometer. Bone marrow aspirates were collected from 26 NDMM and 73 RRMM patients, and the transcriptome was assessed by mRNA-Seq. RESULTS: In peripheral blood, T-cell populations differed between HVs and NDMM and RRMM patients. Absolute numbers of lymphocytes were higher in HVs than in NDMM and RRMM, regardless of the MM disease state. Absolute numbers of total CD4+ T cells and naïve CD4+ T cells were lower in RRMM patients, whereas CD4+ effector memory T cells as a proportion of total CD4+ T cells were increased in RRMM patients. Blood from RRMM patients also contained increased levels of proliferating CD4+ T cells, as evidenced by Ki67, ICOS, and HLA-DR, compared with blood from NDMM patients; HVs had values much closer to those from NDMM than from RRMM patients, suggesting a trend influenced by disease state or therapeutic intervention. In bone marrow, immunologic gene expression signatures were elevated in NDMM vs RRMM patients; the differences were similar to those in peripheral blood. Using limma to model the differential expression of all measured genes between NDMM and RRMM, we identified 367 genes that were elevated in NDMM patients vs 52 in RRMM patients. Gene set analyses using Molecular Signatures Database immunologic signatures (C7) applied to those 367 genes showed that naïve T-cell genes were increased in the bone marrow of NDMM vs RRMM patients. Gene set enrichment analysis with limma, using 489 gene sets from xCell representing 64 cell types and controlling for differences in tumor burden, indicated that macrophage, monocyte, and neutrophil genes were upregulated and T cells, particularly naïve CD4+ T cells, were downregulated in RRMM patients. Immunohistochemistry results from bone marrow biopsies showed increased programmed death-ligand 1 expression on tumor and infiltrating immune cells and increased CD8 infiltration into bone marrow in RRMM vs NDMM patients. Multiparameter immunofluorescence is underway to confirm these findings and further understand the tumor immune microenvironment in patient subsets. As expected, baseline RRMM immune cell populations depended on prior lines of therapy. Daratumumab-exposed RRMM patients had elevated total CD8+ T cells in peripheral blood but decreased CD38+, CD4+, and CD8+ T cells, as well as decreased total natural killer cells, compared with the daratumumab-naïve patients. Transcriptome analyses of bone marrow from daratumumab-exposed RRMM patients revealed increased T-cell gene expression signatures relative to marrow from daratumumab-naïve patients. Additionally, pomalidomide-exposed RRMM patients had increased activated CD4+ and CD8+ T cells vs pomalidomide-naïve patients. CONCLUSIONS: These data indicate that RRMM patients have peripheral blood and bone marrow environments with highly differentiated T-cell populations, whereas NDMM patients show elevated T-cell levels with proliferative capacity. Furthermore, the bone marrow of RRMM patients is enriched with neutrophils and macrophages; investigation is ongoing to determine if these cell types contribute to an immunosuppressive tumor microenvironment. Understanding immune system function based on disease progression, patient segments, and prior lines of therapy is imperative as treatment of MM improves, and it may inform the administration and sequence of next generation immunotherapeutics and identify predictive biomarkers for optimal treatment selection. Disclosures Pietz: Celgene Corporation: Employment. Tometsko:Celgene Corporation: Employment, Equity Ownership. Copeland:Celgene Corporation: Employment, Equity Ownership. Whalen:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Thompson:Celgene Corporation: Employment, Equity Ownership. Agarwal:Celgene Corporation: Employment, Equity Ownership. Foy:Celgene Corporation: Employment, Equity Ownership. Buchholz:Celgene Corporation: Employment. Komashko:Celgene Corporation: Employment. Dell'Aringa:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment.

Description: Polymorphonuclear leukocyte (PMN)–platelet interactions at sites of vascular damage contribute to local and systemic inflammation. We sought to determine the role of “outside-in” signaling by Src-family tyrosine kinases (SFKs) in the regulation of αMβ2-integrin–dependent PMN recruitment by activated platelets under (patho)physiologic conditions. Activation-dependent epitopes in β2 integrin were exposed at the contact sites between PMNs and platelets and were abolished by SFK inhibitors. PMNs from αMβ2−/−, hck−/−fgr−/−, and hck−/−fgr−/−lyn−/− mice had an impaired capacity to adhere with activated platelets in suspension. Phosphorylation of Pyk2 accompanied PMN adhesion to platelets and was blocked by inhibition as well as by genetic deletion of αMβ2 integrin and SFKs. A Pyk2 inhibitor reduced platelet-PMN adhesion, indicating that Pyk2 may be a downstream effector of SFKs. Analysis of PMN-platelet interactions under flow revealed that SFK signaling was required for αMβ2-mediated shear-resistant adhesion of PMNs to adherent platelets, but was dispensable for P-selectin–PSGL-1–mediated recruitment and rolling. Finally, SFK activity was required to support PMN accumulation along adherent platelets at the site of vascular injury, in vivo. These results definitely establish a role for SFKs in PMN recruitment by activated platelets and suggest novel targets to disrupt the pathophysiologic consequences of platelet-leukocyte interactions in vascular disease.

Description: We have developed a quantitative and sensitive flow cytometric method for the detection of human apoptotic lymphocytes that, unlike previously described assays, allows their identification in mixed populations of peripheral blood leukocytes as well as their immunophenotyping. Apoptotic lymphocytes are identified on the basis of peculiar light scatter changes, reflecting their smaller size and their modified nucleus/cytoplasm organization, and of the decreased expression of surface CD45 molecules. Based on these criteria, apoptotic lymphocytes generated by exposure to ionizing radiation can be easily distinguished from viable cells and from necrotic lymphocytes generated by treatment with antibody and complement. Using this assay, we reappraised the phenomenon of the in vitro apoptosis of lymphocytes from patients with human immunodeficiency virus (HIV) infection. Lymphocytes from HIV patients, unlike those from normal HIV-negative subjects, undergo apoptosis upon simple in vitro culture. We found that the percentages of lymphocytes undergoing apoptosis were significantly higher in patients with low CD4 cell counts (〈 400/microL) than in patients at earlier stages (〉 400 CD4 cells/microL). However, phenotypic analysis disclosed that apoptotic lymphocytes generated in these cultures were mostly CD8+ T cells and CD19+ B cells. Thus, in contrast to what has been previously suggested, the phenomenon of in vitro lymphocyte apoptosis might not be pathogenetically related to the depletion of CD4+ T cells in acquired immunodeficiency syndrome. Nevertheless, it might represent an useful marker of disease progression. Our assay allows the analysis of unfractionated peripheral blood leukocytes and thus the identification of apoptotic lymphocytes circulating in vivo. Apoptotic lymphocytes could indeed be detected in the circulation of a patient with cancer shortly after high-dose cytotoxic chemotherapy. By contrast, no apoptotic lymphocytes could be detected in vivo in patients with early or advanced HIV infection.

Description: Key Points This is the first trial to investigate PD-1 inhibitor, pembrolizumab, and an IMiD (pomalidomide) in MM with promising clinical efficacy. PD-L1 expression on myeloma cells and PD-1 on marrow infiltrating T lymphocytes are potential biomarkers for efficacy of PD-1 blockade.

Description: Destruction of immune cells in peripheral lymphoid tissues plays presumably a pivotal role in acquired immune deficiency syndrome pathogenesis. We found that cell suspensions obtained from lymph nodes of eight human immunodeficiency virus (HIV)-infected individuals contained variable proportions (2.1% to 18.3%, median 11.2%) of dead lymphocytes permeable to supravital dyes, represented by CD4+, CD8+, and B cells. The frequency of dead cells correlated directly (R = 0.847) with the amount of HIV provirus in the cell populations, and HIV provirus was enriched in the dead cell fractions. Similar proportions of dead cells were observed in cell suspensions from lymphadenopathic lymph nodes of HIV− donors, but not from small resting HIV− lymph nodes. Electron microscopic and flow cytometric analyses revealed that most dead cells from HIV+ lymph nodes lacked internucleosomal DNA fragmentation but displayed combined features of apoptosis and necrosis, eg, chromatin condensation and mitochondrial swelling. Cells with similar morphology were readily identified in lymph node tissue sections, and marked mitochondrial swelling could be occasionally observed in cells with otherwise normal morphology. Our findings have two major implications. One is that the in vivo cell death in HIV-infected lymph nodes occurs predominantly through a novel pathway, related to but distinct from classical apoptosis and characterised by early and severe mitochondrial damage. The second implication is that HIV-related lymphadenopathy is accompanied in vivo by massive destruction of uninfected lymph node cells. Comparable levels of cell death were observed in other inflammatory lymphadenopathies not related to HIV; however, the uniquely endless and generalized nature of HIV lymphadenopathy might render this “inflammatory” cell destruction a powerful pathogenetic mechanism, accounting for the progressive disruption and depletion of lymphoid tissues seen in HIV infection.

Description: Destruction of immune cells in peripheral lymphoid tissues plays presumably a pivotal role in acquired immune deficiency syndrome pathogenesis. We found that cell suspensions obtained from lymph nodes of eight human immunodeficiency virus (HIV)-infected individuals contained variable proportions (2.1% to 18.3%, median 11.2%) of dead lymphocytes permeable to supravital dyes, represented by CD4+, CD8+, and B cells. The frequency of dead cells correlated directly (R = 0.847) with the amount of HIV provirus in the cell populations, and HIV provirus was enriched in the dead cell fractions. Similar proportions of dead cells were observed in cell suspensions from lymphadenopathic lymph nodes of HIV− donors, but not from small resting HIV− lymph nodes. Electron microscopic and flow cytometric analyses revealed that most dead cells from HIV+ lymph nodes lacked internucleosomal DNA fragmentation but displayed combined features of apoptosis and necrosis, eg, chromatin condensation and mitochondrial swelling. Cells with similar morphology were readily identified in lymph node tissue sections, and marked mitochondrial swelling could be occasionally observed in cells with otherwise normal morphology. Our findings have two major implications. One is that the in vivo cell death in HIV-infected lymph nodes occurs predominantly through a novel pathway, related to but distinct from classical apoptosis and characterised by early and severe mitochondrial damage. The second implication is that HIV-related lymphadenopathy is accompanied in vivo by massive destruction of uninfected lymph node cells. Comparable levels of cell death were observed in other inflammatory lymphadenopathies not related to HIV; however, the uniquely endless and generalized nature of HIV lymphadenopathy might render this “inflammatory” cell destruction a powerful pathogenetic mechanism, accounting for the progressive disruption and depletion of lymphoid tissues seen in HIV infection.

Description: Abstract 4642 Background BARD1 (BRCA1-associated RING domain protein 1) was first described as a BRCA1 partner and tumour suppressor protein, muted in most cases of breast and ovarian cancer. BARD1 functions are BRCA1-dependent, requiring formation of a stable heterodimer through interaction of the RING finger domains. BRCA1-independent functions for BARD1 have recently been described, making it an interesting and important molecule for study. BARD1 is expressed in almost all human tissues, including haematological cells, testis and breast tissue, but it is over-expressed in leukaemias, sarcomas and testis cancer, implicating BARD1 in development of cancer. Different BARD1 isoforms are up-regulated in breast, ovarian and uterine cancers but markedly down-regulated or absent in healthy tissues. Therefore, the presence of these isoforms might be a risk factor or causal event in the cancer pathogenesis. We investigated the role of BARD1 isoforms in leukaemia, through the study of the epigenetic mechanisms involved in regulation of its expression and function. As such, we determined the expression of a specific BARD1 isoform in different human leukaemia cell lines the effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid, Vorinostat) of expression of BARD1. Results. Four human leukaemia cell lines (U937, NB4, K562 and HL60) express the BARD1 isoform of interest. Treatment of these cell lines with SAHA at 5 μ M resulted in decreased expression of this isoform (Fig. 1). Decreased expression of BARD1 by SAHA was also observed in in human breast cancer MCF7 cells, the usual model for BARD1 experiments and in the human neuroblastoma cell line Kelly (Fig. 2), but not in HeLa cells or an human epithelial carcinoma cell line (Fig. 2). The reduced expression of BARD1 was attributed to the action of 2 specific micro-RNAs (miRNAs), that directly bind its 3′untranslated region (UTR). SAHA-induced expression of miR-19a and miR-19b in primary human leukemia cells as demonstrated using miRNA microarray expression analysis and confirmed by Real-Time PCR (Fig. 3). These 2 miRNA were up-regulated after SAHA stimulation in human leukaemia cells. BARD1 is the predicted target for miR-19a and miR-19b based on miRBase database analyses. Transient transfection in NB4 cells with mimic miR-19a and mimic miR-19b confirmed expression of these miRNAs was associated with BARD1 down-regulation (Fig. 4 5.). The BARD1 3′UTR was cloned into a pGL3 vector with a downstream the luciferase gene reporting gene (Fig. 6). The plasmid was co-transfected in HeLa cells with each mimic miRNA and a luciferase assay was performed. We demonstrate that expression of miR-19a and miR-19b can directly bind BARD1 3′UTR, reducing luciferase activity in this system (Fig. 7), thereby confirming that BARD1 is a target of these miRNAs. These findings support our hypothesis that BARD1 is a promising target in leukemia and should be further investigated for its diagnostic and prognostic features. Disclosures: No relevant conflicts of interest to declare.