Dual Translocation Research Articles

Optogenetic clustering and membrane translocation of the BcLOV4 photoreceptor

Optogenetic tools respond to light through one of a small number of behaviors including allosteric changes, dimerization, clustering, or membrane translocation. Here, we describe a new class of optogenetic actuator that simultaneously clusters and translocates to the plasma membrane in response to blue light. We demonstrate that dual translocation and clustering of the BcLOV4 photoreceptor can be harnessed for novel single-component optogenetic tools, including for control of the entire family of epidermal growth factor receptor (ErbB1-4) tyrosine kinases. We further find that clustering and membrane translocation are mechanistically linked. Stronger clustering increased the magnitude of translocation and downstream signaling, increased sensitivity to light by ~threefold-to-fourfold, and decreased the expression levels needed for strong signal activation. Thus light-induced clustering of BcLOV4 provides a strategy to generate a new class of optogenetic tools and to enhance existing ones.

Prospective Multi-Center Phase I/II Trial of Lenalidomide and Dose-Adjusted EPOCH-R in MYC-Associated B-Cell Lymphomas

Introduction Dual translocation of MYC and BCL2 or the dual overexpression of these proteins in patients with aggressive B-cell lymphomas, termed double-hit lymphoma (DHL) and double expressing lymphoma (DEL), respectively, have worse outcomes after standard chemoimmunotherapy with R-CHOP. DA-EPOCH-R has been used as a strategy to overcome chemoresistance. Lenalidomide (LEN) is an immunomodulatory agent with multiple anti-tumor properties including inhibition of the NF-kB pathway, which may enhance the treatment response in MYC-driven lymphomas. This phase I/II study evaluated the safety and efficacy of DA-EPOCH-R with LEN in DHL and DEL. The phase I portion has previously been reported with recommended phase II dose (RP2D) of LEN 15mg daily (day 1-14) per 21 day cycle with DA-EPOCH-R (Godfrey et al. Cancer 2019). Here we report the phase II results. Methods Adult patients with newly diagnosed DLBCL (stage II-IV) with dual MYC and BCL2 translocations (DHL) or with IHC ≥40% MYC and ≥70% BCL2 (DEL) were treated with LEN (15mg) daily on days 1-14 every 21 days with DA-EPOCH-R for 6 cycles. Patients with complete remission (CR) or partial remission (PR) received either a consolidative auto-SCT (if eligible) or maintenance LEN 10mg daily (days 1-14 every 21 days) for 12 cycles. The primary objective was to determine the 1- and 2-year progression free survival (PFS) with LEN plus DA-EPOCH-R. Secondary objectives included end-of-induction CR and overall response rate, as well as adverse events (AE) by CTCAE v4.0. Intention-to-treat analysis was performed, and response/survival data included phase I patients. Data lock was 7/20/22. Pre-specified primary evaluation of PFS was done with an optimal two-stage design and planned enrollment of at least 46 patients (up to 55). We hypothesized the addition of LEN to DA-EPOCH-R would increase 1-year PFS from 40% (null) to at least 60% (alternative) (α=0.05 and β=0.20). This study was IRB-approved and registered (NCT02213913). Results 54 patients with median age 65 (25-82) including 15 patients in the phase I portion (12 at RP2D or above) were enrolled and treated between 12/2014 and 1/2020. 48/54 (88.9%) were stage III or IV, and 27/54 (50.0%) had DHL. During induction treatment, 40/54 (74.1%) received all six cycles and 50/54 (92.6%) received at least four cycles. CNS prophylaxis was optional, and all 33/54 (61.1%) received IT methotrexate. The 1- and 2-year PFS were 83.3% (95% CI 70.4-91.0) and 77.3% (95% CI 63.5-86.5), respectively. The 1- and 2-year OS were 92.5% (95% CI 81.3-97.1) and 88.4% (95% CI 76.0-94.6). At median follow up 29.3 months, the median PFS and OS were not reached (Figure 1). The ORR after LEN and DA-EPOCH-R induction was 49/54 (90.7%) with CR of 46/54 (85.2%). Among responders, 31/49 (63.3%) received maintenance LEN with median 11 cycles (1-12), and 14/31 (45.2%) completed full maintenance. AE during induction was the most common reason for not receiving maintenance (9/18, 50%), and 9/31 (29.0%) stopped maintenance due to an AE. Only 1/49 (2.0%) received an auto-SCT. Of the patients with DHL (n=27), the ORR was 23/27 (85.2%) with CR of 22/27 (81.5%). For patients with DHL (median follow-up 29.2 months), the 1- and 2-year PFS were both 74.1% (95% CI 53.2-86.7), and the 1- and 2-year OS were 88.7% (95% CI 68.8-96.2) and 84.1% (95% CI 62.5-93.7). Median PFS and OS were not reached. For treatment-related toxicity, 43/54 (79.6%) experienced a grade 3-4 hematologic AE, and 19/54 (35.2%) had grade 3-4 neutropenic fever. 23/54 (42.6%) had any grade infection, and 12/54 (22.2%) were grade 3-4. 10/54 (18.5%) had a thromboembolic event, and 5/54 (9.3%) were grade 3. Peripheral sensory neuropathy was the most common non-hematologic AE with 38/54 (70.4%) any grade and 2/54 (3.7%) grade 3-4. Other common non-hematologic AEs included fatigue (66.7%), constipation (53.7%), and nausea (51.9%) with most being grade 1-2 and only 2-6% grade 3. Two patients (3.7%) had a therapy-related myeloid neoplasm at 6 months and 1.5 years after starting treatment, respectively. There was no treatment-related mortality. Conclusion LEN with DA-EPOCH-R for patients with DEL and DHL has a high response rate, encouraging survival, and met the primary endpoint for PFS. Considering that the vast majority of relapses are expected in the first 24 months after therapy, these results are promising and warrant further study. The specific benefit attributable to LEN needs to be tested in a randomized setting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular cytogenetic characterization of a novel wheat\u2013Psathyrostachys huashanica Keng T3DS-5NsL\u20225NsS and T5DL-3DS\u20223DL dual translocation line with powdery mildew resistance

BackgroundPsathyrostachys huashanica Keng (2n = 2x = 14, NsNs) carries many outstanding agronomic traits, therefore is a valuable resource for wheat genetic improvement. Wheat–P. huashanica translocation lines are important intermediate materials for wheat breeding and studying the functions of alien chromosomes. However, powdery mildew resistance in these translocation lines has not been reported previously.ResultsThis study developed a novel wheat–P. huashanica translocation line TR77 by selecting a F7 progeny from the cross between heptaploid hybrid H8911 (2n = 7x = 49, AABBDDNs) and durum wheat line Trs-372. Chromosome karyotype of 2n = 42 = 21II was observed in both mitotic and meiotic stages of TR77. Genomic in situ hybridization analysis identified two translocated chromosomes that paired normally at meiosis stage in TR77. Molecular marker analysis showed that part of chromosome 5D was replaced by part of alien chromosome fragment 5Ns. It meant replacement made part 5DL and part 5NsL·5NsS existed in wheat background, and then translocation happened between these chromosomes and wheat 3D chromosome. Fluorescence in situ hybridization demonstrated that TR77 carries dual translocations: T3DS-5NsL·5NsS and T5DL-3DS·3DL. Analysis using a 15 K-wheat-SNP chip confirmed that SNP genotypes on the 5D chromosome of TR77 matched well with these of P. huashanica, but poorly with common wheat line 7182. The translocation was physically located between 202.3 and 213.1 Mb in 5D. TR77 showed longer spikes, more kernels per spike, and much better powdery mildew resistance than its wheat parents: common wheat line 7182 and durum wheat line Trs-372.ConclusionsTR77 is a novel stable wheat–P. huashanica T3DS-5NsL·5NsS and T5DL-3DS·3DL dual translocation line and showed significant improved spike traits and resistance to powdery mildew compared to its parents, thus, it can be an useful germplasm for breeding disease resistance and studying the genetic mechanism of dual translocations.

Structural Basis of Lipid Scrambling and Ion Conduction by TMEM16 Scramblases

The plasma membrane of eukaryotic cells is asymmetric, with polar and charged lipids sequestered to the inner leaflet when the cell is at rest. Activation of phospholipid scramblases rapidly collapses this asymmetry and externalizes negatively charged phosphatidylserine lipids, activating extracellular signaling networks that control processes such as apoptosis, blood coagulation, membrane fusion and repair. The TMEM16 family is comprised of Ca2+‐dependent Cl− channels and phospholipid scramblases with non‐selective ion channel activity. Structural and functional analyses of TMEM16 scramblases identified a membrane‐exposed hydrophilic groove that serves as a dual translocation pathway for ions and lipids. The mechanisms underlying Ca2+‐dependent gating of TMEM16 scramblases/non‐selective channels, lipid permeation and the emergence of their ion conduction pore remain poorly understood. We used cryo‐electron microscopy to determine the structures of a fungal scramblase/non‐selective channel from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs in the presence and absence of Ca2+. These structures reveal that Ca2+ binding induces a global rearrangement of the transmembrane and cytosolic regions, resulting in opening of the lipid permeation pathway by way of rearrangements of TM4 and TM6. The structures of the scramblase/nanodisc complex in the presence and absence of Ca2+ show that the scramblases induce a pronounced distortion of the surrounding membrane: the outer leaflet is bent along the dimer axis of the protein and the membrane is thinned near the opened lipid translocation pathway thereby facilitating transbilayer lipid movement.Molecular dynamics simulations of a second closely related fungal TMEM16 scramblase, nhTMEM16, revealed that a hydrophobic lock between TM3 and TM4 is essential to maintain the open, lipid‐conductive conformation of the scramblase. MD simulations suggest that disruption of this lock favors the positioning of TM4 giving rise to a Ca2+‐bound conformation that is closed to the membrane and thus non‐conductive for lipids. Indeed, mutations at the TM3/TM4 lock severely impair lipid scrambling. Notably, these mutants retain ion channel activity, indicating that the dual activity scramblase/channel was converted into a channel only protein. The cryoEM structure of a channel‐only mutant reveal a continuous, protein‐delimited pore of sufficient size to allow ion permeation. Our results show that TM6 and TM4 are the gating elements of TMEM16 scramblases where TM6 movement is directly controlled by Ca2+‐binding and TM4 rearrangements are controlled by a hydrophobic lock between TM3 and TM4.Support or Funding InformationNIGMS 5R01 GM106717

The role of stem cell transplant for lymphoma in 2017.

The role of stem cell transplant for lymphoma in 2017.

Beyond RCHOP: A Blueprint for Diffuse Large B Cell Lymphoma Research.

Diffuse large B cell lymphoma (DLBCL) comprises multiple molecular and biological subtypes, resulting in a broad range of clinical outcomes. With standard chemoimmunotherapy, there remains an unacceptably high treatment failure rate in certain DLBCL subsets: activated B cell (ABC) DLBCL, double-hit lymphoma defined by the dual translocation of MYC and BCL2, dual protein-expressing lymphomas defined by the overexpression of MYC and BCL2, and older patients and those with central nervous system involvement. The main research challenges for DLBCL are to accurately identify molecular subsets and to determine if specific chemotherapy platforms and targeted agents offer differential benefit. The ultimate goal should be to maximize initial cure rates to improve long-term survival while minimizing toxicity. In particular, a frontline trial should focus on biologically defined risk groups not likely to be cured with cyclophosphamide, doxorubicin, vincristine, and prednisone plus rituximab (R-CHOP). An additional challenge is to develop effective and personalized strategies in the relapsed setting, for which there is no current standard other than autologous stem cell transplantation, which benefits a progressively smaller proportion of patients. Relapsed/refractory DLBCL is the ideal setting for testing novel agents and new biomarker tools and will require a national call for biopsies to optimize discovery in this setting. Accordingly, the development of tools with both prognostic and predictive utility and the individualized application of new therapies should be the main priorities. This report identifies clinical research priorities for critical areas of unmet need in this disease.

Impact of dual expression of MYC and BCL2 by immunohistochemistry on the risk of CNS relapse in DLBCL

AbstractDual expression of MYC and BCL2 by immunohistochemistry (IHC) is associated with poor outcome in diffuse large B-cell lymphoma (DLBCL). Dual translocation of MYC and BCL2, so-called “double-hit lymphoma,” has been associated with a high risk of central nervous system (CNS) relapse; however, the impact of dual expression of MYC and BCL2 (dual expressers) on the risk of CNS relapse remains unknown. Pretreatment formalin-fixed paraffin-embedded DLBCL biopsies derived from patients subsequently treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) were assembled on tissue microarrays from 2 studies and were evaluated for expression of MYC and BCL2 by IHC. In addition, cell of origin was determined by IHC and the Lymph2Cx gene expression assay in a subset of patients. We identified 428 patients who met the inclusion criteria. By the recently described CNS risk score (CNS–International Prognostic Index [CNS-IPI]), 34% were low risk (0 to 1), 45% were intermediate risk (2 to 3), and 21% were high risk (4 or greater). With a median follow-up of 6.8 years, the risk of CNS relapse was higher in dual expressers compared with non-dual expressers (2-year risk, 9.7% vs 2.2%; P = .001). Patients with activated B-cell or non–germinal center B-cell type DLBCL also had an increased risk of CNS relapse. However, in multivariate analysis, only dual expresser status and CNS-IPI were associated with CNS relapse. Dual expresser MYC+ BCL2+ DLBCL defines a group at high risk of CNS relapse, independent of CNS-IPI score and cell of origin. Dual expresser status may help to identify a high-risk group who should undergo CNS-directed evaluation and consideration of prophylactic strategies.

Abstract 5303: Correlative results from PRELUDE, a phase III study of enzastaurin (ENZA) vs placebo (PBO) in patients (pts) with high-risk diffuse large B-cell lymphoma (DLBCL) following a response to R-CHOP therapy

Abstract Background: Protein kinase Cβ (PKCβ) is critical for B-cell signaling and survival and overexpression of PKCβ in DLBCL is associated with inferior survival. ENZA, an oral serine/threonine kinase inhibitor, targets PKCβ. Here we report immunohistochemical (IHC) and fluorescence in-situ hybridization (FISH) correlative analyses. Methods: PRELUDE (NCT00332202) was a phase III, double-blind maintenance study of 758 pts with DLBCL who were randomized 2:1 to ENZA 500 mg daily (1125-mg loading dose) (n = 504) or PBO (n = 254), respectively, following CR to induction therapy with R-CHOP. The primary endpoint was disease-free survival (DFS). Overall survival (OS) and assessment of biomarkers specific to ENZA and DLBCL were secondary endpoints. Pre-treatment formalin-fixed, paraffin-embedded samples were assessed via IHC staining in a blinded manner (Eric Hsi) for cell of origin (COO) using Hans’ algorithm. In addition, IHC was performed for c-MYC, BCL2, BCL6, FOXP1, and MUM1 (10% increments/% tumor cells stained), markers relevant to ENZA, including PKCβ2. FISH was performed to identify translocations involving c-MYC, BCL2, and BCL6. Cox regression was used to determine statistical associations between efficacy outcomes and dichotomized markers, adjusting for treatment and additional baseline covariates. All tests of association were conducted at a 2-sided α = 0.05. Results: There was no difference in 4-year DFS (70% vs 71%) or OS (81% vs 82%) between ENZA and PBO arms, respectively. A total of 243 (32%) pts had ≥1 evaluable sample available for IHC and FISH. There was no difference in outcome for pts based on COO (GCB vs non-GCB). Independent of treatment, significant associations were observed for BCL2 (pre-specified cutpoint 20%) and MUM1 (cutpoint 30%) with OS, and FOXP1 (cutpoint 80%) by IHC with DFS (HR [95% CI]: 2.19 [1.01-4.73]), p = 0.031; 1.97 [1.04-3.71], p = 0.032; 1.74 [1.04-2.90], p = 0.032, respectively). Associations were not significant for other IHC markers, including c-MYC and BCL6. Low PKCβ2 (<50% expression) had numerically better (but not significant) DFS/OS vs high PKCβ2. Dual translocation lymphoma by FISH was identified in two pts (1.2%) each for c-MYC/BCL2 and c-MYC/BCL6, and one pt (0.6%) had a triple hit involving c-MYC/BCL2/BCL6. Conclusions: No difference in outcomes between treatment arms was observed for the trial. Independent of treatment, COO was not prognostic of outcomes. Pts with low PKCβ2 expression had numerically better DFS/OS compared with high PKCβ2 expression. Significant treatment-independent associations were observed for BCL2 and MUM1 with OS and for FOXP1 with DFS (low IHC expression corresponded to better outcomes). The small number of double hit and triple hit lymphomas seen in the study may reflect the enrollment of CR patients with more favorable biology. Citation Format: Eric D. Hsi, Kerry J. Savage, Sonali M. Smith, Fritz Offner, Scott P. Myrand, Thomas M. Habermann, Donald E. Thornton, Boris K. Lin, Tuan S. Nguyen, Oday Hamid, Michael Crump. Correlative results from PRELUDE, a phase III study of enzastaurin (ENZA) vs placebo (PBO) in patients (pts) with high-risk diffuse large B-cell lymphoma (DLBCL) following a response to R-CHOP therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5303. doi:10.1158/1538-7445.AM2015-5303

XIV. The pathology of transformation of indolent B cell lymphomas.

XIV. The pathology of transformation of indolent B cell lymphomas.

Clinicopathological Features and Prognosis of B-Cell Lymphoma, Unclassifiable, with Features Intermediate Between Diffuse Large B-Cell Lymphoma and Burkitt Lymphoma with t(14;18) and 8q24 Translocation in the Rituximab Era

Introduction: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of diseases in terms of morphology, immunohistochemistry and molecular features. The t(14;18)(q32;q21) translocation juxtaposes the BCL2 gene, normally located at 18q21, with the IGH locus on 14q32. The t(14;18)(q32;q21) occurs in about 80-90% of follicular lymphomas and about 25-30% of de novo DLBCLs. On the other hand, the t(8;14)(q24;q32) translocation juxtaposes the c-MYC protooncogene, which is located at 8q24, to the immunoglobulin heavy chain (IGH) gene, located at 14q32, resulting in deregulated expression of c-MYC. t(8;14)(q24;q32) has been found in 80-90% of Burkitt lymphoma cases and in 5-15% of DLBCL cases. B-cell lymphoma having both t(14;18) and 8q24, so called double hit lymphoma (DHL), is rare. The pathological diagnosis in most cases of DHL is B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (BCLU). Patients with DHL have elevated serum LDH levels, advanced stage, bone marrow involvement, extranodal involvement and the presence of B symptoms. In the present study, we evaluated the clinicopathological characteristics and prognoses of patients with BCLU with DHL.Patients and Methods: A total of 368 patients with DLBCL and BCLU were treated from 2007 to 2013. Chromosomal data were available in 195 of the 368 patients. Pathologic evaluation of the materials from each patient was performedat several central review meetings by six hematopathologists in the ALTSG pathology review board. Patients were treated with cyclophosphamide, vincristine, bleomycin, etoposide, doxorubicin, and prednisolone (CyclOBEAP) regimen or CHOP regimen. Rituximab was administered to all patients. The median follow-up period was 44 months (range, 12-61 months).Results: t(14;18) + 8q24 dual translocation was seen in 18 (9.2 %) of the 195 patients with DLBCL and BCLU. There were 12 males and 6 females, with a median age of 62 (range, 47-76) years. Stage III/IV was found in 56%, bone marrow infiltration was found in 39％, central nervous system (CNS) infiltration was found in 17％, and high risk of international prognostic index (IPI) was found in 67％. Among the 18 patients with the DHL, extranodal sites of disease were bone marrow (7 patients), CNS (3 patients), pleural effusion (5 patients), and gastrointestinal tract (3 patients). Furthermore, 8 patients had at least two extranodal localizations. Immunophenotyping analysis (CD20, CD5, CD10, BCL2, BCL6, MUM1, Ki-67) was performed and showed BCLU with a germinal center type in all cases. Ki-67 staining ranged from 30-90%. All lymphoma cells were positive for CD20 and negative for CD5. CD10, BCL2, BCL6, and MUM1 were positive in 89%, 75%, 88%, and 19%, respectively. The 4-year overall survival (OS) rate was 72% among the patients with dual translocation (n=18) and 75% among the patients in the other chromosomal abnormalities group (n=177). The 4-year progression-free survival (PFS) rate was 52% among the patients with the dual translocation and 71% among the patients in the other chromosomal abnormalities group. The 4-year OS rate of the stage I/II and stage III/IV groups was 100% and 47%, respectively (P=0.016). We next examined the survival curve of patients in whom data on serum LDH levels were available. The 4-year OS rates of the high (>2N) and low LDH groups (<2N) were 33 and 100%, respectively (p=0.0002). According to the IPI, the 4-year OS of patients with L or L-I risk and those with H-I or H risk was 100% and 50%, respectively (p=0.03).Conclusions: Among patients with DHL, there was one subgroup that had a good prognosis. We elucidated the clinicopathological condition of especially the subgroup of DTL with poor prognosis, and prognostic improvement of this disorder is expected in the future by considering a new treatment strategy for these subgroups. DisclosuresNiitsu:Chugai pharmaceutical: Research Funding. Takahashi:Chugai pharmaceutical: Research Funding. Kohri:Chugai pharmaceutical: Research Funding. Asou:Chugai pharmaceutical: Research Funding. Sakai:Chugai pharmaceutical: Research Funding. Miura:Chugai pharmaceutical: Research Funding. Okamoto:Chugai pharmaceutical: Research Funding.

Front-Line, Dose-Escalated Immunochemotherapy Is Associated with a Significant PFS (but not OS) Advantage in 401 Patients (Pts) with Double-Hit Lymphomas (DHL): A Systematic Review and Meta-Analysis

Introduction: “Double-hit lymphoma” (DHL), characterized by the presence of both c-Myc and BCL-2 rearrangements, is an aggressive B-cell non-Hodgkin’s lymphoma (NHL) that is clearly associated with an inferior prognosis compared to standard risk DLBCL. DHL pts do poorly when treated with R-CHOP and typically cannot be salvaged once relapsed. Studies examining the treatment of DHL pts have been small and there is a lack of consensus in the lymphoma community as to whether DHL pts benefit from dose-escalated chemotherapy regimens in the front-line setting. To address this question, we generated pooled, meta-analytic estimates of the effectiveness of dose-escalated approaches vs. standard dose immunochemotherapy in prolonging overall survival (OS) and progression-free survival (PFS) in newly diagnosed DHL pts.Methods: DHL was defined by the presence of c-Myc rearrangement (FISH) in combination with BCL2 rearrangement (FISH) in pts with DLBCL or transformed follicular lymphoma. Treatment regimens included dose intensive (DI; R-Hyper-CVAD, R-CODOX-M/IVAC), intermediate dose [Dose adjusted (DA)-R-EPOCH], or standard dose (R-CHOP). PubMed, Embase, and abstracts at 3 specific congresses from 1/2009 to 6/2014 were searched using terms “lymphoma” with “DHL”, “dual hit”, “dual translocation”, “double-hit”, or “myc” and “bcl2”. Randomized controlled trials and observational studies evaluating treatment of DHL were eligible for inclusion. Studies of ≤ 10 DHL pts were excluded. Data were collected from study authors or extracted from publications as the proportions of pts surviving at specific follow-up times or individual event times. Data were synthesized to estimate the hazard ratios of higher-dose treatments relative to R-CHOP using a Weibull proportional hazards model within a Bayesian meta-analysis framework.Results: The initial search yielded 996 hits and 57 congress abstracts. After independent review by three investigators, 11 studies examining 401 pts were included in the analysis. Pts were treated with either R-CHOP (n=180), DA-R-EPOCH (n=91), or DI regimen (n=130) in the front-line setting. There were no significant differences in baseline characteristics of pts between included studies or across treatment regimens (median age at diagnosis 60 years, median Ki-67 88%, 53% IPI ≥ 3). The estimated median (and 95% credible intervals [CrIs; the Bayesian analog to confidence intervals]) OS and PFS for the entire cohort were 27.8 (15.1-48.5) and 20.5 (10.9-36.0) months. No significant differences in OS (n=374) were observed among any of the treatments (estimated med OS: R-CHOP, DA-R-EPOCH and DI were 24.2, 37.5, and 29.5 months, respectively, Figure A). Estimated hazard ratios (HR) of OS were 0.77 (95% CrI: 0.50-1.11) for DA-R-EPOCH and 0.90 (95% CrI: 0.62-1.25) for DI relative to R-CHOP. The PFS (n=357) among pts receiving DA-R-EPOCH (HR 0.64, 95% CrI: 0.42-0.92) or DI (HR 0.75, 95% CrI: 0.51-1.05) was similar and DA-R-EPOCH demonstrated significant improvement over R-CHOP (estimated med PFS: R-CHOP, DA-R-EPOCH and DI of 13.9, 27.8, and 21.9, respectively, Figure B).Conclusions: In the largest study and first meta-analysis of treatment outcomes in DHL pts, we have demonstrated that dose-escalated chemotherapy was associated with a significant increase in PFS (particularly DA-R-EPOCH) which did not translate into an OS advantage. This suggests DHL is a disease of chemotherapy resistance. One can consider the use of dose-escalated front-line therapies as they can lead to a PFS advantage and that consolidation strategies should be developed to reduce the likelihood of relapse. These results highlight (1) outcomes remain poor for DHL pts regardless of the intensity of R-chemotherapy induction (2) the need for routine screening for MYC and BCL2 by FISH in DLBCL pts with high-risk features and (3) the need for prospective induction and consolidation strategies that incorporate our growing understanding of the biological basis of DHL such as combinations of novel targeted therapies and/or upfront consolidation with CAR-T-cells. [Display omitted] DisclosuresCohen:Pharmacyclics: Consultancy; Seattle Genetics: Consultancy; BMS: Research Funding; Janssen: Research Funding. Mato:Celgene: Honoraria, Research Funding.

The Impact of Concurrent MYC BCL2 Protein Expression on the Risk of Secondary Central Nervous System Relapse in Diffuse Large B-Cell Lymphoma (DLBCL)

Introduction: Recent studies have established that concurrent MYC and BCL2 protein expression by immunohistochemistry (IHC) identifies a subgroup of patients with diffuse large B-cell lymphoma (DLBCL) with a poor outcome. Classic dual translocation MYC/ BCL2, so called 'double hit’ disease, is associated with a high risk of central nervous system (CNS) relapse; however the impact of concurrent MYC and BCL2 protein expression on the risk of CNS relapse remains unknown. Further, robust biological markers that accurately predict the risk of CNS relapse in DLBCL would also be of value in clinical practice.Methods: Cases of pre-treatment formalin fixed paraffin embedded DLBCL in two tissue microarrays were independently scored by two expert hematopathologist (GWS and KLT or PF and AM) for expression of MYC (Epitomics Y69), BCL2 (Dako 124), CD10, BCL6 and MUM1 by IHC. MYC and BCL2 positivity were defined as ≥ 40% and ≥ 50% cells with staining, respectively, in accordance with previously established cutoffs (Johnson, JCO 2012; 30). Cases with discordant scores were reviewed by a third hematopathologist (RDG) to reach a consensus. Cell of origin (COO) was assigned according to the Hans IHC algorithm (Hans, Blood 103: 2004) as well as by the recently described gene expression profiling Lymph2Cx 20 gene assay based on NanoString technology (Scott, Blood 2014; 123) in the subset of patients with ≥ 40% tumor content. Patients treated with at least one cycle of R-CHOP chemotherapy with curative intent were included and those with established CNS disease at diagnosis were excluded.Results: 447 patients were identified with the following baseline clinical characteristics: Median age 65 y (16-92y); males n=280, 63%; performance status ≥ 2, n= 147, 33%; stage 3 or 4 disease n=242, 54%; elevated LDH n=219, 47%; EN > 1 n= 80, 17%. With a median follow-up of 6.75 years for living patients, the 3 year time to progression, progression-free and overall survival for all patients were 68%, 66%, and 73%, respectively. In total, 131 (29%) were MYC+BCL2+ and 316 (71%) were non-MYC+BCL2+. By COO assignment using the Hans algorithm (n=444), 192 were non-GCB (43%) and 252 were GCB (57%) and by the Lymph2Cx (n=308); 103 were ABC (33%), 172 were GCB (56%) and 33 (11%) were unclassifiable. The 2 year cumulative risk of CNS relapse for the whole cohort was 4.3%. The cumulative risk of CNS relapse was higher in cases that were MYC+BCL2+ (2 year risk 9.4% vs 2.4%, P=0.001) with similar results obtained if classic MYC+BCL2+ double hit cases are excluded. There were no cases of CNS relapse in cases MYC+ alone by IHC. By COO, patients with a non-GCB phenotype by the Hans algorithm had an increased risk of CNS relapse (2 year risk 6.9% vs 2.6%, P=0.03) and similarly, cases assigned as ABC DLBCL by the Lymph2Cx assay also identified a group with a higher risk of CNS relapse compared to GCB cases (9.5% vs 2.5%, P=0.03) (Figure 1). In Cox regression multivariate analysis including the COO (Hans), IPI group (0/1 vs 2/3 vs 4/5) and MYC/BCL2 IHC, only the IPI (HR 2.18, P=0.02) and MYC+BCL2+ IHC (HR=3.76, P=0.007) were associated with an increased risk of CNS relapse. Similar results were obtained using the Lymph2Cx COO designation. Within the IPI risk groups, MYC+BCL2+ status further stratified patients in the intermediate risk group (IPI 2 or 3, n=206) into a higher risk group (2 year CNS relapse 12.6%) and a low risk group (2 year CNS relapse 2.9%) (P=0.01). A similar trend was observed in the high IPI risk group (IPI 4 or 5, n=86, 2 year CNS relapse MYC+BCL2+ 17.2% vs 4.7%, P=.0.18) but it was not useful in the low IPI risk group (IP1 0 or 1 (n=155), 2 year CNS relapse 4% vs 1%, P=0.39) where the overall risk was low. Within the COO subgroups, MYC+BCL2+ status also defined a group at high cumulative risk of CNS relapse within the non-GCB subtype (12.9% vs 3%, P=0.001) and by the Lymph2Cx defined ABC subtype (16.9% vs 2.2%, P= 0.03) and a trend was observed for GCB defined by Lymph2Cx (6.6% vs 1.5%. P=.08) but not by Hans criteria (P=0.40).Conclusion: Concurrent expression of MYC and BCL2 protein in DLBCL defines a group of patients at high risk of CNS relapse, independent of the IPI and COO. MYC+BCL2+ status may help to further risk stratify patients in the intermediate and high IPI risk groups and within the ABC subtype to identify patients who should undergo additional diagnostic testing and in whom to explore the effectiveness of prophylactic CNS strategies. [Display omitted] DisclosuresSavage:F Hoffmann-La Roche: Other. Sehn:Roche: Research Funding. Connors:Seattle Genetics, Inc.: Research Funding; Roche: Research Funding. Gascoyne:Hoffman La-Roche: Research Funding.

Dual translocation pathways in smectic liquid crystals facilitated by molecular flexibility

We investigate translocation mechanisms in smectic A liquid crystals (LCs) by a realistic, coarse-grained model of a LC compound comprising a stiff azobenzene core with flexible tails. We observe that the molecules can permeate from one smectic layer to the next via two different mechanisms, with and without significant reorientation, the former being facilitated through transverse interlayer intermediates. This is possible due to the intrinsic flexibility of the molecules. The two processes lead to characteristic signatures in the Van Hove self-correlation function, which can also be observed experimentally.

Dual Translocation Is An Independent Poor Prognostic Factor in Diffuse Large B-Cell Lymphoma,

Abstract 3683 Background:Diffuse large B-cell lymphoma (DLBCL) is a heterogenous group of diseases, that are associated with variable survival outcomes depending on the presence of certain genetic and molecular features. Of particular interest is a subset known as the “double-hit” (DH) lymphoma. DH lymphoma (dual translocation) is defined by the presence of a chromosomal breakpoint affecting the MYC/8q24 locus with a second oncogene translocation, most commonly a BCL2 rearrangement, and less commonly involving BCL6 or CCND1 rearrangements. However, the incidence of DH lymphoma remains to be determined. These patients typically have poor prognostic factors, with a dismal outcome when treated with rituximab-CHOP (RCHOP) chemotherapy. The aim of this study was to identify any clinical defining characteristics in patients with DH lymphoma, and to compare their outcomes with that of DLBCL patients without dual translocation. Methods:202 newly diagnosed DLBCL patients, of whom 90% received rituximab based chemotherapy, were investigated using immunohistochemistry and fluorescence in situ hybridization (FISH), using breakapart FISH probes targeting BCL2, BCL6, MYC, CCND1 and IgH genes. The clinical characteristics and survival outcomes of patients who were identified with DH lymphoma were compared to those without the dual translocation. Results:Out of the 202 patients with DLBCL, we identified 10 cases (5%) with two or more concurrent translocations involving MYC and BCL2, or MYC and BCL6. Among the 10 patients with DH lymphoma, there were 6 patients with concurrent BCL2 and MYC translocations, 1 patient with BCL6 and MYC translocations and 3 patients with all 3 abnormalities. 7 of the 10 patients were male, with a median age of 68 years (42 – 84). Patients with DH lymphoma also presented with a significantly higher incidence of high-risk clinical features, including advanced stage disease, bulky disease, extranodal disease, bone marrow involvement and a high IPI score. Interestingly, the majority of patients with DH lymphoma expressed a germinal center (GC) phenotype (8 out of 9 patients) based on the Han’s criteria. These patients also demonstrated a significantly poorer overall survival (OS) when compared to patients without dual translocation (2 yr OS 33% vs 84%, p = < 0.001). On multivariate analysis, the presence of a dual translocation was found to be an independent poor prognostic factor for OS (hazard ratio 8.84, 95% CI 3.54 to 22.08). Other factors predictive of an inferior OS included age, stage, bone marrow involvement and patients treated without rituximab. Conclusions:Our findings showed that the presence of dual translocation is an independent poor prognostic factor in DLBCL. It was present in 5% of our cohort and was associated with more advanced disease. Patients with dual translocation also had a significantly poorer survival following treatment with standard chemotherapy such as RCHOP, even though most patients exhibited the GC phenotype. Therefore, the use of novel agents in combination with chemotherapy is an area that deserves further exploration in this type of lymphoma. Disclosures:No relevant conflicts of interest to declare.

Variant Philadelphia Chromosome Identified by Interphase Fluorescence In Situ Hybridization (FISH) without Evidence on G-banded Karyotyping and Metaphase FISH

A variant Philadelphia chromosome (Ph) is generated from translocation of one or more partner chromosomes in addition to chromosomes 9 and 22. We have described the cases of 2 patients bearing variant Ph detected by interphase FISH but not detected by G-banded karyotyping and metaphase FISH. FISH was performed using BCR/ABL dual color dual fusion translocation probes (Abbott Molecular, USA). A 52-year-old man was diagnosed with acute leukemia of mixed phenotype. G-banded karyotyping showed 46,XY,t(9;22)(q34;q11.2)[12]/47,idem,+der(22)t(9;22)[5]/46,XY[3]. Interphase FISH revealed nuc ish(ABL1,BCR) × 3(ABL1 con BCR × 2)[329/450]/(ABL1,BCR) × 4(ABL1 con BCR × 3)[5/450]/(AL1,BCR) × 3(ABL1 con BCR × 1)[44/450]. Metaphase FISH showed ish (9;22)(ABL1+,BCR1+;BCR+,ABL+)[22]/der(22)(BCR+,ABL1+)[3]. The other case was that of a 31-yr-old male patient diagnosed with CML in the blastic phase. G-banded karyotyping of all 20 metaphase cells showed 47,XYYc,dup(1)(q21q32),del(7)(p11.2),t(9;22)(q34;q11.2). Interphase FISH revealed nuc ish(ABL1,BCR) × 3(ABL1 con BCR × 2)[254/600]/(ABL1,BCR) × 3(ABL1 con BCR × 1)[191/600]. Metaphase FISH showed ish t(9;22)(ABL1+,BCR+;BCR+,ABL1+)[16]. These results suggest that typical t(9;22) and variant Ph may coexist in the same patient, and interphase FISH may facilitate the detection of the variant Ph that cannot be detected by G-banded karyotyping alone.

Follicular lymphoma transformation to dual translocated Burkitt-like lymphoma: improved disease control associated with radiation therapy

Dual translocated (or "dual hit") lymphomas are highly aggressive B cell neoplasms associated with an extremely poor prognosis. The optimal treatment for these lymphomas remains undefined. We present two cases of follicular lymphoma with transformation to Burkitt-like lymphoma. In both cases dual translocations involving both the bcl-2 and c-myc loci were present. Each patient underwent intensive induction immunochemotherapy followed by autologous stem cell transplantation and radiation therapy. The first patient received post-transplant mediastinal radiation and developed recurrence in multiple areas outside of the radiation field. The second patient received total body irradiation as part of the conditioning regimen, and is without recurrence 18 months after transplant, and 24 months after diagnosis of the dual translocated lymphoma. We review dual translocation B cell lymphoma in the setting of transformation from follicular lymphoma, and suggest a potential role for total body irradiation in the management of this highly aggressive non-Hodgkin lymphoma.

Cotranslocation of Methyl Parathion Hydrolase to the Periplasm and of Organophosphorus Hydrolase to the Cell Surface of Escherichia coli by the Tat Pathway and Ice Nucleation Protein Display System

A genetically engineered Escherichia coli strain coexpressing organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH) was constructed for the first time by cotransforming two compatible plasmids. Since these two enzymes have different substrate specificities, the coexpression strain showed a broader substrate range than strains expressing either one of the hydrolases. To reduce the mass transport limitation of organophosphates (OPs) across the cell membrane, MPH and OPH were simultaneously translocated to the periplasm and cell surface of E. coli, respectively, by employing the twin-arginine translocation (Tat) pathway and ice nucleation protein (INP) display system. The resulting recombinant strain showed sixfold-higher whole-cell activity than the control strain expressing cytosolic OP hydrolases. The correct localization of MPH and OPH was demonstrated by cell fractionation, immunoblotting, and enzyme activity assays. No growth inhibition was observed for the recombinant E. coli strain, and suspended cultures retained almost 100% of the activity over a period of 2 weeks. Owing to its high level of activity and superior stability, the recombinant E. coli strain could be employed as a whole-cell biocatalyst for detoxification of OPs. This strategy of utilizing dual translocation pathways should open up new avenues for cotranslocating multiple functional moieties to different extracytosolic compartments of a bacterial cell.

Clinical features and prognosis of de novo diffuse large B-cell lymphoma with t(14;18) and 8q24/c-MYC translocations

Diffuse large B-cell lymphoma (DLBCL) having both t(14;18) and 8q24 translocations is rare. We evaluated the clinical characteristics and prognoses of patients with DLBCL carrying both t(14;18) and 8q24 translocations. A total of 1972 patients with non-Hodgkin's lymphoma were treated in the Adult Lymphoma Treatment Study Group (ALTSG) from 1998 to 2007. Nineteen cases of de novo DLBCL with the dual translocation were identified. The dual translocation was observed in 19 of 394 patients with DLBCL (10 males and 9 females, with a median age of 61 years). The dual translocation was observed significantly more frequently among patients with high lactate dehydrogenase levels, B symptoms, bone marrow involvement and advanced stage. Immunophenotyping was performed and showed DLBCL with a germinal center type in the majority of cases. Progression-free survival and overall survival rates were significantly lower in patients with the dual translocation than in those with other translocation. DLBCL patients with concurrent t(14;18) and 8q24 translocations have very poor prognosis. Even if patients had a complete response to chemotherapy, they subsequently suffered early relapse. In this study, only a few patients received rituximab, and its usefulness could not be assessed. Future studies with larger numbers of patients are required.

Engineering of a monomeric fluorescent protein AsGFP499 and its applications in a dual translocation and transcription assay

The tetrameric green fluorescent protein AsGFP(499) from the sea anemone Anemonia sulcata was converted into a dimeric and monomeric protein by site-directed mutagenesis. The protein was engineered without prior knowledge of its crystal structure based on a sequence alignment of multiple proteins belonging to the GFP-family. Crucial residues for oligomerisation of AsGFP(499) were predicted and selected for mutation. By introduction of a single site mutation (S103K) the A/B subunit was disrupted whereas two substitutions were necessary to separate the A/C subunit (T159K/F173E). This method can be applied as a general predictive method for designing monomeric proteins from multimeric fluorescent proteins. The maturation temperature was optimised to 37 degrees C by a combination of Site-directed and random mutagenesis. In cell-based assays, the NFATc1A (nuclear factor of activated T-cells, subtype 1, isoform A)-AsGFP(499) chimera formed massive cytoplasmic aggregates in HeLa cells, which prevented the shuttling of NFATc1A into the nucleus and consequentially its transcriptional activity. In contrast, the cells expressing the NFATc1A in fusion with our engineered dimeric and monomeric fluorescent mutants were homogeneously distributed throughout the cytoplasm, making these stable cell lines functional in both translocation and transcriptonal assays. This new dual cellular assay will allow the screening and discovery of new drugs that target NFAT cellular processes.

The clinical presentation and prognosis of diffuse large B-cell lymphoma with t(14;18) and 8q24/c-MYC rearrangement

Diffuse large B-cell lymphomas (DLBCL) are common lymphomas that have been classified into three subgroups on the basis of their patterns of gene expression. The aim of this study was to characterize the clinical, biological, immunophenotypic and cytogenetic features of DLBCL with concurrent t(14;18) and 8q24/c-MYC rearrangement. Sixteen cases of DLBCL with the dual translocation were identified between 1998 and January 2006. The clinical features of these cases were examined and morphological, immunohistochemical, flow cytometric and cytogenetic analyses were performed. All patients had aggressive features: B symptoms (81%), ECOG performance status >2 (81%), elevated lactate dehydrogenase (100%), stage IV disease (100%) with at least one extra-nodal localization (bone marrow, blood and central nervous system involvement in 93%, 50% and 50%, respectively) and age-adjusted IPI score of 3 in 81%. Despite intensive chemotherapy regimens (including allogeneic transplants), all patients died of disease progression. Progression-free and overall survival was 4 and 5 months, respectively. Immunophenotyping analysis (CD20, CD10, Bcl-6, Mum-1, Bcl-2 CD138, MIB1, CD19, CD5, CD38 and sIg) was performed and showed DLBCL with a germinal center (GC) profile. Ki-67 staining ranged from 70 to 90%. All cases assessed by cytogenetics analysis [conventional cytogenetic and/or fluorescence in situ hybridization (FISH)] had a complex karyotype. In one case, we identified a 8q24/c-MYC translocation variant never reported in DLBCL before: t(8;9)(q24;p13) and t(14;18)(q32;q21). The BCL-6 rearrangement was investigated by FISH and found to rearranged in four cases. In conclusion, DLBCL with concurrent t(14;18) and 8q24/c-MYC rearrangement is a subgroup of GC-DLBCLwith poor outcome. It is worth searching for the coexistence of dual translocations in Bcl-2-positive DLBCL with unusual aggressive presentation.