Gc Phenotypes Research Articles

Effects of CYP3A4 genetic polymorphisms on the pharmacokinetics and efficacy of perampanel in Chinese pediatric patients with epilepsy

ObjectiveThis study was the first to evaluate the effect of CYP3A4 gene polymorphisms on the plasma concentration and effectiveness of perampanel (PER) in Chinese pediatric patients with epilepsy. MethodsWe enrolled 102 patients for this investigation. The steady-state concentration was determined after patients maintained a consistent PER dosing regimen for at least 21 days. Plasma PER concentrations were measured using liquid chromatography–tandem mass spectrometry. Leftover samples from standard therapeutic drug monitoring were allocated for genotyping analysis. The primary measure of efficacy was the rate of seizure reduction with PER treatment at the final check-up. ResultsThe CYP3A4×10 GC phenotype exhibited the highest average plasma concentration of PER at 491.1 ± 328.1 ng/mL, in contrast to the CC phenotype at 334.0 ± 161.1 ng/mL. The incidence of adverse events was most prominent in the CYP3A4×1 G TT and CYP3A4×10 GC groups, with rates of 77.8 % (7 of 9 patients) and 50.0 % (46 of 92 patients), respectively. Moreover, the percentage of patients for whom PER was deemed ineffective was least in the CYP3A4×1 G TT and CYP3A4×10 CC groups, recorded at 11.1 % (1 of 9 patients) and 10.0 % (1 of 10 patients), respectively. There was a significant correlation between PER plasma concentration and either exposure or toxicity (both with p < 0.05). We suggest a plasma concentration range of 625–900 ng/mL as a suitable reference for PER in Chinese patients with epilepsy. ConclusionThe CYP3A4×10 gene's genetic polymorphisms influence plasma concentrations of PER in Chinese pediatric patients with epilepsy. Given that both efficacy and potential toxicity are closely tied to plasma PER levels, the CYP3A4 genetic phenotype should be factored in when prescribing PER to patients with epilepsy.

Mixed pituitary adenoma/pituitary neuroendocrine tumor-gangliocytoma: Immunohistochemical insights.

Mixed pituitary adenoma/PitNET-gangliocytomas (PA/PitNET-GC) have been reported in small series over the past 20 years; some had limited immunohistochemistry (IHC) data. We interrogated our experience over 20 years, focusing on patterns of the GC component and IHC results for anterior pituitary hormones, transcription factors, NFP, and CAM5.2. A search of cases from 2002 to 2023 yielded 20 cases: 7M:13F, ages 20-71 years; 17 macroadenomas, 1 microadenoma, 2 ectopic. GC was co-associated with 4 corticotroph, 2 densely granulated lactotroph, 5 mixed lactotroph-somatotroph, 1 immature PIT1-lineage tumor, and 8 sparsely granulated GH; the latter all had a minor lactotroph component. Patterns were: discrete nodular foci of GC (9/20), extensive GC differentiation often overshadowing the PA/PitNET (7/20), and intimate admixture of smaller bands of neuropil and individual metaplastic ganglion cells within PA/PitNET (4/20). NFP highlighted small cohesive regions of neuropil and identified greater axonal content, including individual axons within "pure" PA/PitNET areas, than appreciated on H&E. CAM5.2 IHC often revealed cells with neuronal morphologies to a greater extent than NFP and in different areas within the same tumor. These data suggest that the combined use of NFP and CAM5.2 IHC best reveals transition from PA to GC phenotype, with CAM5.2 positivity reflecting earlier stages of transformation.

Modelling the role of glucocorticoid receptor as mediator of endocrine responses to environmental challenge.

Glucocorticoid hormones (GCs) modulate acute 'stress' responses in vertebrates, exerting their actions across many physiological systems to help the organism face and overcome challenges. These actions take place via binding to the glucocorticoid receptor (GR), which determines not only the magnitude of the GC-mediated physiological response but also the negative feedback that downregulates GCs to restore homeostasis. Although GR function is assumed to determine GC regulation capacity, the associations between GR abundance and individuals' coping abilities remain cryptic. We developed a dynamic model fitted to empirical data to predict the effects of GR abundance on both plasma GC response patterns and the magnitude of GC-mediated physiological response. Individuals with higher GRs showed lower GC exposure, stronger physiological responses and greater capacity to adjust this response according to stressor intensity, which may be translated into more resilient and flexible GC phenotypes. Our results also show that among-individual variability in GR abundance challenges the detectability of the association between plasma GC measurements and physiological responses. Our approach provides mechanistic insights into the role of GRs in plasma GC measurements and function, which point at GR abundance fundamentally driving complex features of the GC regulation system in the face of environmental change. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

Clinical molecular subtyping reveals intrinsic mesenchymal reprogramming in gastric cancer cells

The mesenchymal cancer phenotype is known to be clinically related to treatment resistance and a poor prognosis. We identified gene signature-based molecular subtypes of gastric cancer (GC, n = 547) based on transcriptome data and validated their prognostic and predictive utility in multiple external cohorts. We subsequently examined their associations with tumor microenvironment (TME) features by employing cellular deconvolution methods and sequencing isolated GC populations. We further performed spatial transcriptomics analysis and immunohistochemistry, demonstrating the presence of GC cells in a partial epithelial-mesenchymal transition state. We performed network and pharmacogenomic database analyses to identify TGF-β signaling as a driver pathway and, thus, a therapeutic target. We further validated its expression in tumor cells in preclinical models and a single-cell dataset. Finally, we demonstrated that inhibition of TGF-β signaling negated mesenchymal/stem-like behavior and therapy resistance in GC cell lines and mouse xenograft models. In summary, we show that the mesenchymal GC phenotype could be driven by epithelial cancer cell-intrinsic TGF-β signaling and propose therapeutic strategies based on targeting the tumor-intrinsic mesenchymal reprogramming of medically intractable GC.

PTBP1 drives c-Myc-dependent gastric cancer progression and stemness

BackgroundGastric cancer (GC) tumorigenesis and treatment failure are caused by cancer stem cells. Polypyrimidine tract binding protein 1 (PTBP1) was shown to be involved in the development of embryonic stem cells and is now being considered as a therapeutic target for tumour progression and stem-cell characteristics.MethodsPTBP1 expression in GC samples was detected using tissue microarrays. Proliferation, colony formation, spheroid formation and stem-cell analysis were used to examine PTBP1’s role in tumorigenesis and stem-cell maintenance. In AGS and HGC-27 cells with or without PTBP1 deficiency, ubiquitin-related protein expression and co-precipitation assays were performed.ResultsWe identified that PTBP1 was aberrantly highly expressed and represented a novel prognostic factor in GC patients. PTBP1 maintained the tumorigenic activity and stem-cell characteristics of GC in vitro and in vivo. PTBP1 directly interacts with c-Myc and stabilises its protein levels by preventing its proteasomal degradation. This is mediated by upregulating the ubiquitin-specific proteases USP28 and limiting FBW7-mediated ubiquitination of c-Myc. Moreover, the depletion of PTBP1-caused tumour regression was significantly compromised by exogenous c-Myc expression.ConclusionsBy preserving the stability of c-Myc through the ubiquitin–proteasome pathway, the oncogene PTBP1 supports stem-cell-like phenotypes of GC and is involved in GC progression.

GC1f Vitamin D Binding Protein Isoform as a Marker of Severity in Autism Spectrum Disorders.

Autism spectrum disorders (ASD) are characterized by a wide spectrum of clinical, behavioral, and cognitive manifestations. It is, therefore, crucial to investigate possible biomarkers associated with specific ASD phenotypes. Ample literature suggests a possible role for vitamin D (VD) in influencing ASD clinical phenotypes. We analyzed three vitamin D binding protein gene (DBP) functional polymorphisms (rs2282679, rs7041, and rs4588), which are involved in the modulation of vitamin D serum concentration in 309 ASD children and 831 healthy controls. Frequency comparisons of single nucleotide polymorphisms (SNPs) alleles, genotypes, and GC isoforms (GC1f, G1s, and GC2)—generated by the combination of rs7041 and rs4588 alleles—were correlated with ASD diagnostic, behavioral, and functioning scales. The GC1f isoform was significantly more frequent in ASD compared with controls (18.6% vs. 14.5% pc = 0.02). Significantly higher scores for item 15 of the Childhood Autism Rating Scale (CARS) and lower ones for the Children’s Global Assessment Scale (CGAS) functioning scales were seen in ASD carrying the GC1f isoform. In GC phenotype analysis, a gradient of severity for overall CARS scores and CARS item 15 was observed, with scores decreasing according to the presence of GC1f-GC1f > GC1f-GC1s > GC1s-GC1s > GC1f-GC2 > GC2-GC2 isoforms. Similarly, lower CGAS scores were seen in carriers of the GC1f-GC1f isoform, whereas higher scores were present in those carrying GC2-GC2 (p = 0.028). This is the first study to evaluate possible relationships between GC variants and the different aspects of ASD in Italian ASD children. Results, although needing to be validated in ampler cohorts, suggest that the GC1f isoform could be a marker of severity in ASD that may be useful in establishing the intensity of therapeutic and rehabilitative protocols.

Single-Cell Multi-Omic and Spatial Analysis of Nodal B Cell Non-Hodgkin Lymphomas Reveals Plasticity in B Cell Maturation As a Driver of Intratumor Heterogeneity

Introduction Intratumor heterogeneity (ITH) is increasingly recognized as an important factor in the variability of B cell non-Hodgkin lymphoma (B-NHL) patient outcomes. It is well established that distinct nodal B cell non-Hodgkin lymphoma (NBNHL) entities originate from different stages of the humoral affinity maturation process, but drivers of ITH are not well understood. In this study, we aimed to investigate whether the maturation process is a driver of ITH. Methods 51 diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and non-malignant reactive lymph node (rLN) samples from distinct patients were sequenced with CITE-Seq (coupling single-cell RNA-seq with surface epitope profiling), highly multiplexed immunohistochemistry (IHC) with 57 proteins, and single-cell B cell receptor sequencing. Transcription factor activites were inferred from gene expression using the SCENIC package. (Fig 1a)Results Using the rLN samples (n = 8), a single-cell reference map of nodal B cell maturation states was constructed based on known B cell maturation states and their markers. This reference map was used to classify maturation states in malignant samples, which were verified and refined against their maturation marker profiles. Comparable to the non-malignant context in rLN samples, variability in maturation states was also observed for malignant cells in most NBNHL tumors. Although commonalities in composition existed between samples within entities, such as a dominant pre-GC mantle cell population in MCL and abundance of memory B cells in MZL, we observed high variability in maturation state proportions between these samples (Fig 1b). Several GCB and ABC DLBCL samples contained subpopulations of both GC and post-GC phenotypes. BCR-sequencing confirmed that different intra-tumor maturation states observed were monoclonal, and therefore arose from the same cell of origin. Common epigenetic drivers of the maturation processes, such as the transcription factors EZH2 and EGR1, were upregulated in subpopulations advanced in maturation. Known pathways common driving malignant transformation in NBNHL pathogenesis, BCL6 and BCL2, were upregulated in centroblasts and post-GC subpopulations respectively in tumors of the same entities. Conclusions Our findings reveal maturation is not halted during malignancy, but rather remains a plastic and divergent process. Ongoing maturation and malignant transformation processes are intertwined as the driving forces of intratumor heterogeneity, blurring the known boundaries of NBNHL entities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evolution of the Tumor Microenvironment throughout Progression and Transformation of EZH2 Mutant Follicular Lymphoma

The genetic hallmarks of follicular lymphomas include BCL2 translocations and somatic mutations of epigenetic modifier genes such as EZH2. Histologically, FLs typically feature a rich microenvironment, most notably featuring extensive follicular dendritic cell (FDC) networks with dendrites making extensive contact with lymphoma cells. In recent work we showed that the main effect of EZH2 gain-of-function mutations in GC B-cells is to enable them to become less dependent of T-cell help and strengthen their immune synapse formation with FDCs, which induces aberrant proliferation and survival of GC centrocytes and hence formation of a unique lymphoma-permissive immune niche. However, it is still unknown how interactions between EZH2 mutant GC B-cells and other immune cells change throughout the progression of the disease.To evaluate the evolution of the tumor microenvironment (TME) throughout EZH2 mutant lymphoma progression, we developed a genetically engineered mouse model designed for conditional expression of gain-of-function Ezh2 mutant and BCL2 in GC B-cells, Rosa26 LSL.BCL2.IRES.GFP;Ezh2 Y641F;Cγ1Cre (hereafter BCL2/Ezh2 Y641F), in which Cγ1-driven Cre excision of a STOP cassette in the Rosa26 locus leads to overexpression of BCL2 and GFP, and expression of the endogenous Ezh2 mutant Y641F. This mouse model develops low-grade follicular-like lymphoma, characterized by expanded follicles composed largely of centrocyte neoplastic GC B-cells and extensive FDC meshwork, along with presence of CD4 +, TFH and regulatory T cells (FOXP3 +), as depicted by multiparametric in situ imaging and multiparametric flow cytometry. At cytological level, cells are predominantly small with condensed chromatin, irregular nuclei, scant cytoplasm, and inconspicuous nucleoli, without sheets of large cells. Over time, these low grade FLs progress to advanced grade, characterized by disruption of follicle structures, expansion of centroblast-like large tumor cells and reduced CD4 + T cell infiltration and FDC meshwork.Furthermore, we have developed a murine transformed FL cell line by sequential passages of an original BCL2/Ezh2 Y641F low-grade FL into immunodeficient Rag1KO mice. We have transduced this BCL2/Ezh2 Y641F cell line with luciferase, and it successfully engrafted and homed to lymphoid organs when injected i.v. into immunocompetent C57BL6 mice. The cell line consists of high proliferative GC B-cells with multiple and irregular nuclei, open chromatin and prominent nucleoli that resemble DLBCL. The microenvironment of tumors developed in C57BL6 mice is characterized by disruption of follicle structures, severe reduction or absence of FDC meshwork, decreased CD4 +, CD8 + and Tregs, downregulation of MHC-I and MHC-II. Therefore, our mouse model also recapitulates progression and transformation stages.Since MYC translocations are frequent events that occur during histological transformation of FL, we modeled this more aggressive EZB MYC subtype of DLBCL. For that, we further crossed our BCL2/Ezh2 Y641F mice to Rosa26 LSL.MYC.IRES.hCD2. We generated cohorts of transplanted mice by injecting bone marrow cells from BCL2, BCL2/Ezh2 Y641F, BCL2/MYC, and BCL2/Ezh2 Y641F/MYC mice into lethally irradiated C57BL6 recipients. Recipient mice (n=5 per group) were immunized with SRBC to induce formation of GCs and sacrificed 5.5 months post bone marrow transplant. All genotypes showed expansion of FAS +CD38 -BCL2 GFP+ GC B-cells in spleen and lymph nodes; however, the proportion of GC B-cells in Ezh2 Y641F was significantly increased compared with Ezh2 WT, in both presence and absence of MYC. In contrast, FAS -CD38 +BCL2 GFP+ and CD138 +BCL2 GFP+ cells were significantly increased in MYC + mice and decreased in Ezh2 Y641F, suggesting that Ezh2 mutation is required to maintain the GC phenotype. The acquisition of Ezh2 Y641F in BCL2/MYC mice induced a reduction of CD4 + and CD8 + in the TME, and decreased TFH without changes in TFR proportions in GCs. Strikingly, FDC meshwork was significantly shrank in MYC + cases, and partially restored by acquisition of Ezh2 Y641F, indicating that MYC overexpression may contribute to acquire FDC independency for the survival of lymphoma cells.Our results indicate that progression of FL critically affects the TME and acquisition of additional mutations such as MYC alters the interactions between lymphoma cells and tumor supportive immune cells in TME. DisclosuresMelnick: Epizyme: Consultancy; Janssen: Research Funding; KDAC Therapeutics: Current holder of individual stocks in a privately-held company; Celgene Corporation: Consultancy; Constellation Pharmaceuticals: Consultancy; Astra Zeneca: Consultancy; Daiichi Sankyo: Consultancy; Exo Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sanofi US Services: Research Funding.

Treatment of High Risk DLBCL with DA-R-EPOCH in Those Unable to Tolerate Intensive Chemotherapy: Well-Tolerated and Effective, but Inadequate in Double/Triple Hit or Expressing DLBCL

Treatment of High Risk DLBCL with DA-R-EPOCH in Those Unable to Tolerate Intensive Chemotherapy: Well-Tolerated and Effective, but Inadequate in Double/Triple Hit or Expressing DLBCL

Global Transcriptional States of Follicular Lymphoma B Cells Highlight Distinct Groups of Tumor Identity Associated with Somatic Alterations and Tumor Microenvironment

Background: Follicular Lymphoma (FL) is the second most common non-Hodgkin lymphoma and presents with significant clinical, cellular, molecular, and genetic heterogeneity. Despite the name and defining microanatomic location, the genetic and molecular identity and pathogenesis of the FL tumor cell is largely uncharacterized. Prior clinical and molecular classifications of FL have been primarily driven by pathologic classification (Grade 1-3b), genetic classification (M7-FLIPI), or gene expression profiling (IR-1/2). Using a unique cohort of 93 FL tumors, we have explored the transcriptomic signature of purified FL B cells, along with their matched whole tumor, and identified unique molecular subsets which are defined by distinct pathway activation, immune content, and genomic signatures identified through whole exome sequencing (WES). Methods: Frozen tumor biopsies from 93 untreated FL (Grade 1-3b) patients enrolled in the University of Iowa/Mayo Clinic Lymphoma SPORE were used for the study. DNA was isolated from whole tumor cell suspensions and RNA was isolated from both whole tumor and B cell enriched cell suspensions. RNA sequencing (RNAseq) and WES was performed in the Mayo Clinic Genome Analysis Core. RNAseq and WES data were processed using the Mayo Clinic standard pipeline and novel driver genes were identified using 20/20+ driver analysis. Copy number variants were identified using GISTIC 2.0. NMF clustering and single sample gene set testing, for B cell lineage and tumor microenvironment (TME) signatures, was performed in R using the NMF and SingScore packages. Results: NMF consensus clustering of FL B cell RNAseq data identified two distinct subsets, C1 (n=32) and C2 (n=57). Clinically, C1 was associated with being FL grade 3 (p&amp;lt;0.001) and more aggressive therapy (e.g. immunochemotherapy) at diagnosis while other clinical factors were similar between the groups. To determine the biologic underpinnings that drove the NMF analysis, the top NMF metagenes were incorporated (n=389 for C1 and n=293 for C2) for GO analysis. Among those genes were AICDA, IRF4, and EZH2 in C1, and BCL2 in C2. Additionally, we identified significant gene set enrichment with NMF metagenes and calculated differential gene set expression comparing C1, C2, and normal B cells (n=5). Together, these analyses suggest C1 displays significantly (p&amp;lt;0.05) enhanced DNA replication and metabolic activity, whereas C2 displays significantly enhanced cytoskeleton rearrangement, repressed inflammatory cytokine signaling, and NF-kB activity. Based on recent scRNAseq data suggesting that FL derives from distinct germinal center B cells, the B lineage difference between C1 and C2 was explored. Both subgroups were heterogeneous for dark (DZ), intermediate (INT), light (LZ) zones, and plasmablast (PBL) gene signatures. When compared to benign B cell samples, C1 was enriched for a pre-PBL (p&amp;lt;0.01), and C2 for an INT phenotype related to a LZ signature (p&amp;lt;0.01). To define if a genomic signature was associated with C1 and C2, mutations and copy number variants (&amp;gt;5% frequency) were examined. TNFAIP3, TP53, and BCL6 alterations were enriched in C1 samples, whereas C2 associated with alterations in BCL2, KMT2D, CREBBP, REL, and MYC. Finally, B cell clusters were analyzed for TME signatures. C1 samples displayed significant enrichment of macrophage, cytotoxic cell, gamma-delta-Tcell, and endothelial cell TME elements (p&amp;lt;0.05) consistent with an inflamed/hot tumor. In contrast, C2 samples exhibited significantly lower signatures (p&amp;lt;0.05) in all entities except for the fibroblast signature, compared to C1 and normal, consistent with an immune desert phenotype. Conclusion: Our results suggest that B cells from FL patients display two distinct transcriptomic signatures. C1 identifies an immunologically active tumor, driven by TNFAIP3 alterations, with pre-PBL characteristics, DNA replication and repair, inflammatory cytokine secretion/signaling, and hyper-metabolic characteristics. C2 identifies an immunologically quiet tumor, driven by alterations in BCL2 and chromatin modifiers, with an intermediate GC phenotype, repressed cytokine signaling, and active cell cycle progression and cytoskeleton rearrangement. This study improves our understanding of the mechanisms driving FL tumors and motivates further investigation into the relationship between tumor intrinsic factors that may influence the TME. Disclosures Maurer: Morphosys: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Nanostring: Research Funding; Celgene / BMS: Research Funding. Ansell:ADC Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; AI Therapeutics: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squibb: Research Funding. Cerhan:NanoString: Research Funding; BMS/Celgene: Research Funding. Novak:Celgene/BMS: Research Funding.

MYC Associated and Double Protein Lymphoma: Subset Analysis of SWOG S9704

Background: Outside of Burkitt lymphoma (BL), MYC rearrangements confer negative prognostic significance, particularly when co-associated with BCL2 rearrangements. While “double hit lymphomas (DHL)”, occurring in 5-10% of diffuse large B cell lymphoma (DLBCL) patients (pts), have known inferior outcomes with R-CHOP (Savage Blood 2009; Barrans J Clin Oncol 2010), emerging reports suggest that “double protein lymphomas (DPL)” with immunohistochemical (IHC) overexpression of MYC and BCL2 also fare poorly with standard therapy. The frequency of DPL is reportedly higher than DHL and may account for 20-30% of DLBCL cases (Johnson J Clin Oncol 2012; Hu Blood 2013). The optimal management strategy for DHL and DPL is undefined, and there is limited data on the potential role of consolidative transplant. SWOG S9704 was a phase III randomized study of aggressive NHL treated with CHOP +/-R for 5 cycles and then either 3 additional cycles of CHOP +/-R for autologous stem cell transplant (ASCT) in first remission (Stiff N Engl J Med 2013), which gives a unique opportunity to evaluate DPL outcomes in a prospective dataset. Methods: Among 370 eligible pts, 260 had DLBCL or B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (BCLU); cases underwent morphologic review, and IHC analysis for MYC protein (>40% positive cells) was performed in 198 cases. BCL2 IHC (>30% positive cells), cell of origin (COO) classification (GC vs non-GC per Hans algorithm), and FISH studies for MYC were performed in all MYC IHC positive (+) cases with sufficient tissue. BCL2 FISH was also performed in cases positive for a MYC translocation via FISH. Clinical annotations were obtained through SWOG statistical center, and review of S9704 database. Results: There were 27 pts with MYC IHC+ among 198 available samples (13.6%), 19 of whom had concurrent BCL2 IHC+. Three of 6 evaluated pts had DHL via FISH (DHL group), and all were also DPL via IHC; thus, 16 pts had DPL without DHL. 8 patients with MYC IHC + had neither BCL2 positivity via FISH nor IHC (non-DHL, non-DPL group). For the 16 DPL pts (excluding 3 DHL pts), median (med) age was 59.9 y (range, 45-65y), 10 were male, 6 had bulky disease, all had elevated LDH (including 11 with LDH >2X ULN), and med AA IPI was 2. Among 11 evaluated pts for COO, 5 had GC whereas 6 had non-GC DLBCL phenotypes. All 3 DHL pts were female, and all had GC phenotype via Hans criteria. Among non-DHL/non-DPL pts (n=8), med age was 54.5y (range, 49-65y), 5 were male, 3 had bulky disease and all had elevated LDH. In terms of treatment, only 12 of 16 DPL pts were randomized (5 HCT, 7 no HCT), all 3 DHL pts were randomized (2 HCT) and 4 of 8 non-DHL/non-DPL pts were randomized. Reasons for non-randomization (n=8) were progression, lack of response or death. The med f/u for the entire cohort of 27 MYC IHC+ pts is 127 m (range, 94-147m); 21 pts have died (17 no HCT; 4 HCT groups). Med PFS for no HCT vs. HCT for all 27 MYC IHC + pts is 9m (95% CI: 5.0, 13.9) vs. not reached and 2-yr PFS is 16% (95% CI: 3.9%, 34.9%) vs. 63% (95% CI: 22.9%, 86.1%), respectively (Figure 1, intent-to-treat; Figure 2, randomized pts only). For the 16 DPL pts, 9 pts in the no HCT and 3 pts in the HCT group have progressed or died; med PFS for no HCT vs. HCT is 11m (95% CI: 6.7, 18.1) vs. 41m (95% CI: 7.1, NR), respectively, and 2-yr PFS is no HCT vs. HCT is 18% (95% CI: 2.9%, 44.2%) vs. 60% (95% CI: 12.6%, 88.2%), respectively Among 12 DPL pts actually randomized, 2-yr PFS was 29% for no HCT and 60% for HCT, but confidence intervals overlap substantially. All 3 DHL pts have progressed and died at a med of 6.5m. Conclusions: This is a subset analysis of the prospective S9704 trial; one of the largest data sets to address the question of upfront ASCT. True DHL were rare even in this high-risk cohort and had a dismal prognosis. The incidence of DPL was also less than reported in other series, and may reflect that adverse biologic features are more common in non-transplant, and thus older, populations. In an intent–to-treat analysis, there is a trend that MYC IHC+ and DPL patients consolidated with ASCT have improved outcomes; however, nearly one-third of MYC IHC+ pts were unable to be randomized due to early progression or death, suggesting that better induction regimens are needed. While these data are limited by small numbers, there seems to be a role for upfront ASCT in DPL which should be further explored. Support: CA32102, CA38926, CA21115 and in part by Bristol-Myers Squibb. Disclosures No relevant conflicts of interest to declare.

Intrinsic rescaling of granule cells restores pattern separation ability of a dentate gyrus network model during epileptic hyperexcitability.

The dentate gyrus (DG) is thought to enable efficient hippocampal memory acquisition via pattern separation. With patterns defined as spatiotemporally distributed action potential sequences, the principal DG output neurons (granule cells, GCs), presumably sparsen and separate similar input patterns from the perforant path (PP). In electrophysiological experiments, we have demonstrated that during temporal lobe epilepsy (TLE), GCs downscale their excitability by transcriptional upregulation of "leak" channels. Here we studied whether this cell type-specific intrinsic plasticity is in a position to homeostatically adjust DG network function. We modified an established conductance-based computer model of the DG network such that it realizes a spatiotemporal pattern separation task, and quantified its performance with and without the experimentally constrained leaky GC phenotype. Two proposed TLE seizure mechanisms were implemented in various degrees and combinations: recurrent GC excitation via mossy fiber sprouting and increased PP input. While increasing PP strength degraded pattern separation only gradually, already the slight elevation of sprouting drastically (non-linearly) impaired pattern separation. In most tested hyperexcitable networks, leaky GCs ameliorated pattern separation. However, in some sprouting situations with all-or-none seizure behavior, pattern separation was disabled with and without leaky GCs. In the mild sprouting (and PP increase) region of non-linear impairment, leaky GCs were particularly effective in restoring pattern separation performance. These results are compatible with the hypothesis that the experimentally observed intrinsic rescaling of GCs serves to maintain the physiological function of the DG network.

Computational analysis optimizes the flow cytometric evaluation for lymphoma

Although many clinical laboratories are adopting higher color flow cytometric assays, the approach to optimizing panel design and data analysis is often traditional and subjective. In order to address the question "What is the best flow cytometric strategy to reliably distinguish germinal center B-cell lymphoma (GC-L) from germinal center hyperplasia (GC-H)?" we applied a computational tool that identifies target populations correlated with a desired outcome, in this case diagnosis. Cases of GC-H and GC-L evaluated by flow cytometric immunophenotyping using CD45, CD20, kappa, lambda, CD19, CD5, CD10, CD38, were analyzed with flowType and RchyOptimyx to construct cellular hierarchies that best distinguished the two diagnostic groups. The population CD5-CD19+CD10+CD38- had the highest predictive power. Manual reanalysis confirmed significantly higher CD10+/CD38-B-cells in GC-L (median 12.44%, range 0.74-63.29, n = 52) than GC-H (median 0.24%, 0.03-4.49, n = 48, P = 0.0001), but was not entirely specific. Difficulties encountered using this computational approach included the presence of CD10+ granulocytes, continuously variable B-cell expression of CD38, more variable intensity antigen staining in GC-L and inability to assess the contribution of light chain restriction. Computational analysis with construction of cellular hierarchies related to diagnosis helped guide manual analysis of high dimensional flow cytometric data. This approach highlighted the diagnostic utility of CD38 expression in the evaluation of B-cells with a CD10+ GC phenotype. In contrast to computational analysis of non-neoplastic cell populations, evaluation of neoplastic cells must be able to take into consideration increased variability in antigen expression.

Computational analysis optimizes the flow cytometric evaluation for lymphoma.

Background: Although many clinical laboratories are adopting higher color flow cytometric assays, the approach to optimizing panel design and data analysis is often traditional and subjective. In order to address the question "What is the best flow cytometric strategy to reliably distinguish germinal center B-cell lymphoma (GC-L) from lymphoid hyperplasia (GC-H)?" we applied a computational tool that identifies target populations correlated with a desired outcome, in this case diagnosis. Design: Cases of GC-H and GC-L with a germinal center phenotype, evaluated by flow cytometric immunophenotyping using CD45, CD20, kappa, lambda, CD19, CD5, CD10, CD38, were analyzed with flowType and RchyOptimyx to construct cellular hierarchies that best distinguished the two diagnostic groups. Results: The population CD5-CD19+CD10+CD38- had the highest predictive power. Manual reanalysis confirmed significantly higher CD10+/CD38-B-cells in GC-L (median 12.44%, range 0.74 - 63.29, n=52) than GC-H (median 0.24%, 0.03 - 4.49, n=48, p=0.0001), but was not entirely specific. Difficulties encountered using this computational approach included the presence of CD10+ granulocytes, continuously variable B-cell expression of CD38, more variable intensity antigen staining in GC-L and inability to assess the contribution of light chain restriction. Conclusion: Computational analysis with construction of cellular hierarchies related to diagnosis helped guide manual analysis of high dimensional flow cytometric data. This approach highlighted the diagnostic utility of CD38 expression in the evaluation of B-cells with a CD10+ GC phenotype. In contrast to computational analysis of non-neoplastic cell populations, evaluation of neoplastic cells must be able to take into consideration increased variability in antigen expression. © 2013 Clinical Cytometry Society.

The association of cytokine gene polymorphism with reflux nephropathy

ObjectiveTo identify genetic risk factors for the progression of vesicoureteral reflux (VUR) to reflux nephropathy, we examined polymorphisms of multiple cytokine genes among VUR patients with or without renal scarring. MethodsA total of 238 VUR patients aged between 1 and 18 years with (n = 113) or without renal scarring (n = 125) were included. The presence of renal scarring was demonstrated by renal parenchymal examination using Technetium-99m dimercaptosuccinate scintigraphy. Sera of the patients were examined for tumor necrosis factor-alpha (TNF-α, −308), transforming growth factor-beta1 (TGF-β1, +869, +915), interleukin-6 (IL-6, −174), interleukin-10 (IL-10, −1082, −819, −592) and interferon-gamma (IFN-γ, +874) gene polymorphisms using the polymerase chain reaction sequence-specific primer method. ResultsAmong patients with renal scarring, frequencies for the T/T G/C and C/C G/C genotypes of TGF-β1 gene (p = 0.003), GCC/GCC genotype of IL-10 gene (p = 0.015), GC phenotype of IL-6 gene (p = 0.001) and T/T genotype of IFN-γ gene (p = 0.001) were higher compared to patients without renal scarring. Regarding the TNF-α gene, among patients with low grade VUR only, the G/G genotype was associated with an increased risk. ConclusionsCertain genotypes of cytokine gene polymorphisms seem to be associated with an increased or decreased susceptibility to reflux nephropathy, which may explain why only a proportion of VUR patients progress to reflux nephropathy. This information may aid in prediction of prognosis and implementing more aggressive management strategies at earlier stages. Further immunogenetic studies may identify novel targets for the management and prevention of the condition.

Comprehensive genomic meta-analysis identifies intra-tumoural stroma as a predictor of survival in patients with gastric cancer

ObjectiveGastric adenocarcinoma (gastric cancer, GC) is a major cause of global cancer mortality. Identifying molecular programmes contributing to GC patient survival may improve our understanding of GC pathogenesis, highlight new...

Prognostic significance of MYC, BCL2, and BCL6 rearrangements in patients with diffuse large B‐cell lymphoma treated with cyclophosphamide, doxorubicin, vincristine, and prednisone plus rituximab

Diffuse large B-cell lymphomas (DLBCLs) are a biologically heterogeneous group in which various gene alterations have been reported. The aim of this study was to investigate the frequency and prognostic impact of BCL2, BCL6, and MYC rearrangements in cyclophosphamide, doxorubicin, vincristine, and prednisone plus rituximab (R-CHOP)-treated DLBCL cases. Tissue microarrays were constructed from 239 cases of DLBCL, and the expressions of CD10, BCL6, MUM1/IRF4, and BCL2 were evaluated by immunohistochemistry. MYC, BCL2, and BCL6 rearrangements were investigated by interphase fluorescence in situ hybridization on tissue microarrays. Survival analysis was constructed from 145 R-CHOP-treated patients. MYC, BCL2, and BCL6 rearrangements were detected in 14 (6%), 36 (15%), and 69 (29%) of 239 DLBCL patients. Double or triple rearrangements were detected in 7 (3%) of 239 DLBCL cases. Of these, 4 had BCL2 and MYC, 2 had BCL6 and MYC, and 1 had BCL2, BCL6, and MYC rearrangements. The prognosis of these cases was extremely poor, with a median survival of 9 months. MYC rearrangement was associated with significantly worse overall survival (P = .01), especially for the cases with GC phenotype (P = .009). BCL6 rearrangement also predicted significantly shorter overall survival (P = .04), especially for the non-GC phenotype (P = .03). BCL2 rearrangement had no prognostic impact on outcome. International Prognostic Index (P = .004) and MYC rearrangement (P = .009) were independent poor prognostic factors. Analysis of MYC gene rearrangement along with BCL2 and BCL6 is critical in identifying high-risk patients with poor prognosis.

MYC+ Diffuse Large B Cell Lymphomas (DLBCL) Treated in Randomized Prospective Salvage Therapy, RICE or RDHAP Followed by BEAM Plus Autologous Stem Cell Transplantation (ASCT). A BioCORAL Report

Abstract 594▪FN2▪This icon denotes a clinically relevant abstract Background. Approximately 5% to 10% of DLBCLs harbor a MYC/8q24 oncogene rearrangement. We determined the prognostic significance of MYC+ DLBCL in a selected population of patients with relapsed/refractory DLBCL included in the CORAL and prospectively treated by RICE or RDHAP followed by BEAM and ASCT1. METHODS. Among the 396 patients included, 150 patients were analysed with break-apart FISH probes for MYC/8q24, BCL2/18q21, and BCL6/3q27. Correlations with clinical characteristics at diagnosis and at relapse, immunohistochemistry (IHC) for CD10, BCL6, MUM1, FOXP1, and BCL2 expression, cell of origin determined according to different IHC algorithms2-5 and by transcriptomic analysis, response to treatment and survivals were analysed considering presence or absence of MYC rearrangement. RESULTS. Twenty-four (16%) cases exhibited a MYC rearrangement, associated in 87.5% (n=21) of the cases with a BCL2/18q21 rearrangement (n=16), with a BCL6/3q27 rearrangement (n=4), and with both BCL2/18q21 and BCL6/3q27 rearrangements (n=4). Among the matched cases analysed (n=57), biological characteristics including tumor immunophenotype and breakpoints occurrence, were identical between primary and secondary biopsies. Presence of MYC+ rearrangement was significantly associated with CD10 expression (p= 0.0067) and a GC phenotype, based either on Hans algorithm (p<104) or on Alizadeh GC signature (p=0.0015). Patients with MYC+ DLBCL presented significantly with more advanced aaIPI (p =.03). Sixty-three percent of them presented a time to relapse < 12 months, compared to 51% in patients with MYC− DLBCL (p=0.2925). Before HDT+ASCT, CR and ORR (PR+CR) were observed in 56% and 63% in patients with MYC+ DLBCL, compared to 75% and 87% in patients with MYC− DLBCL (p=0.3442). Outcome of patients with MYC+ DLBCL was significantly worse than MYC− DLBCL, with a 4-y PFS at 22% vs 45% (p =.0105) and a 4-y OS at 33% vs 62% (p =.0192). Type of treatment, RDHAP or RICE did not have any impact on survival with a 4-y PFS at 23% vs 22% (p=NS) and a 4-y OS at 44% vs 28% (p=NS), respectively. No difference of outcome was observed between patients with simple hit and with complex hits MYC+ DLBCL. CONCLUSION. MYC rearrangement is an early event in DLBCL and is significantly associated with a GC phenotype. Patients with MYC+ DLBCL presented with a more advanced disease, and have a significant inferior prognosis than patients with MYC− DLBCLs. Their outcome seems not influenced by the proposed salvage therapy. Disclosures:No relevant conflicts of interest to declare.

Lymphoma recurrence 5 years or more following diffuse large B-cell lymphoma: Clinical characteristics and outcome

8562 Background: Diffuse large B-cell lymphoma (DLBCL) usually relapses early following treatment but some relapses happen 5 years or later. Few data exist regarding clinical characteristics and outcome of these patients (pts). Methods: We performed a retrospective analysis of all pts from two centers in Lyon/France between 1980–2003 who presented a biopsy proven relapse 5 years or later following diagnosis of DLBCL. All available biopsies were revised and immunohistochemistry (IHC) completed. Results: Among 1492 pts with DLBCL, 54 were eligible. Clinical characteristics at diagnosis were: median age 57 y; stage I-II 63% (34/54); IPI low/low intermediate 84% (41/49) and extranodal involvement (EN) 66% (35/53). IHC at diagnosis: CD20 100% (46/46), CD10 28% (10/36), bcl-6 53% (9/17), MUM1 48% (11/23), bcl-2 68% (19/28), germinal-center phenotype (GC) 57% (12/21) and non-GC 43% (9/21). 47/53 received CHOP/ACVBP-like regimens, 1 autologous transplantation (ASCT) and 1 rituximab. Median time from diagnosis to relapse was 7.4 years (5–20.5 years). 44 pts (81%) had DLBCL histology at time of relapse and 10 pts (19%) indolent histology. MUM1 expression at diagnosis was associated with DLBCL histology at relapse (p=0.037). Clinical characteristics at relapse were: median age 66 y; stage I-II 48% (26/54); 73% (31/43) with DLBCL at relapse had EN. 54% (15/28) with DLBCL at relapse had a GC phenotype and 46% (13/28) a non-GC phenotype. Treatment at relapse included rituximab in 21/54 and ASCT in 15/54 with 7 pts receiving both. Estimated 5-year event-free survival (EFS) and overall survival (OS) after relapse were 25% and 35% for all pts. Pts with DLBCL histology at relapse had an estimated 5-year EFS and OS of 18% and 28%. Pts with indolent histology had an estimated 5-year EFS and OS of 55% and 67%. Conclusions: Patients with DLBCL who present a late relapse usually had localized stage, favorable IPI and extranodal involvement at diagnosis. However, even if initial characteristics at time of first treatment were favorable, outcome of pts with DLBCL at time of relapse remains poor and aggressive treatment, such as ASCT, should be pursue whenever possible. Some patients relapsed with indolent histology and have a better outcome. No significant financial relationships to disclose.

The Intersection of Epstein-Barr Virus with the Germinal Center

The current model of Epstein-Barr virus (EBV) infection and persistence in vivo proposes that EBV uses the germinal center (the GC model) to establish a quiescent latent infection in otherwise-normal memory B cells. However, the evidence linking EBV-infected cells and the GC is only indirect and limited. Therefore, a key portion of the model, that EBV-infected cells physically reside and participate in GCs, has yet to be verified. Furthermore, recent experiments suggested that upon infection of GC cells the viral growth latency transcription program is dominant and GC functionality and phenotype are ablated, i.e., EBV infection is not consistent with GC function. In this study we show that in vivo, EBV-infected B cells in the tonsils retain expression of functional and phenotypic markers of GC cells, including bcl-6 and AID. Furthermore, these cells are physically located in the GC and express a restricted form of latency, the default latency program. Thus, the EBV default latency transcription program, unlike the growth latency program, is consistent with the retention of GC functionality in vivo. This work verifies key components of the GC model of EBV persistence and suggests that EBV and the GC can interact to produce the latently infected memory cells found in the periphery. Furthermore, it identifies latently infected GC B cells as a potential pathogenic nexus for the development of the EBV-positive, GC-associated lymphomas Hodgkin's disease and Burkitt's lymphoma.