bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel SAHA-Bendamustine Hybrid Induces Apoptosis of Leukemia Cells
Authors: Yu Jing , Qiu Shaowei , Ge Qiufu , Wang Ying , Wei Hui , Guo Dianwu , Chen Shuying , Liu Shuang , Rao Qing , Wang Jianxiang , Wang Min
Published: October 2019

Introduction Hybrid anticancer drugs are of great therapeutic interests as they can potentially overcome the flaws of conventional chemotherapy drugs and improve their efficacy. Histone deacetylase inhibitors (HDACi) and DNA damaging agents have showed synergistic effects in recent studies. In this study, we reported a novel hybrid NL-101 that combines chemo-active groups from suberoylanilide hydroxamic acid (SAHA) and bendamustine, the typical HDACi and alkylating agent respectively.The anticancer effect of NL-101 and its possible mechanisms were investigated in human leukemia cell lines and primary leukemia cells.

Methods MTT assay was performed to determine the proliferation of Kasumi-1 and NB4 cells treated with NL-101. Cell cycle distribution and apoptosis rate were detected by flow cytometry. Western-blot analysis was used to analyze the level of acetylated H3 as well as apoptotic-related proteins including γ-H2AX, PARP, caspase-3, Bax, Bcl-2 and Bcl-xL. Bone marrow mononuclear cells of AML patients were isolated by density gradient centrifugation. Wright staining and Western blot were performed to determine the inducing apoptosis effect.

Results NL-101 inhibited the proliferation of leukemia cell lines Kasumi-1 and NB4 cells with similar IC50 to that of SAHA. Cell cycle analysis indicated that NL-101 induced S phase arrest. As expected, apoptotic cell death was observed in response to NL-101 treatment. After treatment with 2 µmol/L NL-101 for 48 hours, the apoptosis rate of Kasumi-1 and NB4 cells were (60.19±12.01)% and (49.43±11.61)%, respectively. Western blot analysis showed that NL-101 exposure could induce the accumulation of acetylated Histone H3 and γ-H2AX as the biomarker of DNA double-strand breaks. Anti-apoptotic protein Bcl-xL involved in mitochondrial death pathway was also decreased. Moreover, NL-101 induced apoptosis with a low micromolar IC50 in various leukemia cell lines but not in nonmalignant cell line HEK293. The efficacy of NL-101 was also tested in human primary leukemia cells and all the treated samples exhibited apoptosis confirmed by the morphological examination and expression of apoptotic markers.

Conclusions The novel SAHA-bendamustine hybrid NL-101 inhibited the proliferation and induced apoptotic cell death of leukemia cell lines and primary leukemia cells. It presented the properties of both HDAC inhibition and DNA damaging. Down-regulation of Bcl-xL was also involved in the apoptosis induction. These results indicated that NL-101 might be a potential compound for the treatment of leukemia.

Disclosures

Wang: Bristol Myers Squibb: Consultancy; Novartis: Consultancy.
Aggravated Bone Density Decline after Symptomatic Osteonecrosis in Children with Acute Lymphoblastic Leukemia
Authors: den Hoed Marissa , Pluijm S.M.F. , Te Winkel Mariël L. , de Groot-Kruseman Hester A. , Fiocco Marta , Hoogerbrugge Peter M. , Leeuw J.a. , Bruin Marrie , van der Sluis Inge M. , Bresters Dorine , Lequin Maarten H. , Roos Jan C. , Veerman A. , Pieters Rob , van den Heuvel-Eibrink Marry M.
Published: October 2019

Background: Osteonecrosis (ON) and decline of bone mineral density (BMD) are serious side effects during and after treatment of childhood acute lymphoblastic leukemia (ALL). It is unknown whether ON and low BMD co-occur in the same patients, and whether these two osteogenic side effects can influence each other’s development during pediatric ALL treatment.

Methods: BMD and the incidence of symptomatic ON were prospectively assessed in 466 patients with ALL (4-18 years of age) treated according to the dexamethasone-based Dutch Child Oncology Group-ALL9 protocol. Symptomatic ON was defined as persistent pain in arms or legs not caused by vincristine administration, and confirmed by magnetic resonance imaging. Bone mineral density of the lumbar spine (BMDLS) (n=466) and of the total body (BMDTB) (n=106) were measured by dual X-ray absorptiometry at ALL diagnosis, after 32 weeks of treatment, at cessation of treatment (109 weeks) and 1 year after cessation of treatment. BMD was expressed as age-matched and gender-matched standard deviation scores (SDS; Z-score). Multivariate linear mixed models were adjusted for age at diagnosis.

Results: Thirty patients (6.4%) suffered from ON. At cessation of treatment, mean BMDLS was -1.28 SDS (SD: 1.27, n=332; p<0.01) and BMDTB was -0.74 SDS (SD: 1.29, n=65; p<0.01) lower in ALL patients compared to their healthy peers. At baseline, BMDLS and BMDTB did not differ between patients who developed or who did not develop ON (mean BMDLS ON+: -0.90 vs. ON-: -1.14, p=0.36; mean BMDTB ON+: 0.07 vs. ON-: 0.25 p=0.65). At cessation of treatment, patients with ON seem to have a trend for a lower mean BMDLS (ON+: -1.68 vs. ON-: -1.31, p=0.18) and they have a lower mean BMDTB (ON+: -1.91 vs. ON-: -0.59, p=0.01) than patients without ON. Multivariate analyses showed that BMDTB change during follow-up was significantly different for patients with ON than without ON (interaction group time, p=0.04). Between BMD measurements before and after the diagnosis, patients with ON seemed to have a more rapid decline of BMDTB than in patients of the same age without ON (mean BMDTB difference -1.13 vs. -0.62, p=0.10).

Conclusion: We conclude that symptomatic ON and low BMD during antileukemic treatment co-occur in pediatric ALL patients. BMD status at ALL diagnosis does not seem to precede ON. However, the development of ON seems to aggravate BMD decline during antileukemic treatment, most likely due to bone destruction and the advised physical immobilization.

Disclosures

No relevant conflicts of interest to declare.
The Negative Impact of Underweight and Weight Loss on Survival of Children with Acute Lymphoblastic Leukemia
Authors: den Hoed Marissa , Pluijm S.M.F. , de Groot-Kruseman Hester A. , Te Winkel Mariël L. , van den Akker Erica L.T. , Hoogerbrugge Peter M. , van den Berg Henk , Leeuw J.a. , Bruin Marrie , Bresters Dorine , Veerman A.J.P. , Fiocco Marta , Pieters Rob , van den Heuvel-Eibrink Marry M.
Published: October 2019

Background: Body mass index (BMI: kg/m2) and change in BMI during treatment might influence treatment outcome of pediatric patients with acute lymphoblastic leukemia (ALL). However, previous studies in pediatric acute lymphoblastic leukemia reported contradictory results. Therefore, we studiedthe influence of (change in) BMI on treatment outcome in pediatric ALL patients who were treated according to a dexamethasone-based protocol (Dutch Childhood Oncology Group [DCOG] ALL-9).

Patients and Methods: Data on body composition were prospectively collected from a cohort of newly diagnosed Dutch pediatric ALL patients (N=762, age 2-17 years), treated from 1997-2004. BMI at diagnosis was expressed as standard deviation scores (SDS) and categorized into underweight (≤–1.8SDS), or normal weight and overweight (>–1.8SDS). BMI loss was defined after 32 weeks of treatment. Dual X-ray absorptiometry scans were performed in a nested single center cohort (n=106) to assess the contribution of %fat and lean body mass to BMI. Multivariate analyses were corrected for age at diagnosis and risk treatment grpup.

Results: Multivariate analyses showed that patients with underweight (8%) had an increased risk of relapse (Hazard Ratio (HR) 1.79, 95% CI: [1.04-3.10], and a similar overall survival (HR 1.10 [0.57-2.10]). BMI loss during the first 32 weeks of treatment was associated with a decreased overall survival (HR 2.10 [1.14-3.87]), and a similar risk of relapse (HR 1.27 [0.70-2.30]) compared to patients without BMI loss. Dual X-ray absorptiometry revealed that BMI loss mainly consisted of a loss of lean body mass and gain in %fat.

Conclusion: Underweight at diagnosis is associated with an increased risk of relapse and a BMI loss early during treatment is associated with an increased mortality. This suggests that these patients might benefit from exercise interventions and a high-quality nutrient diet during therapy.

Disclosures

No relevant conflicts of interest to declare.
Acute Megakaryocytic Leukemia (AMegL) Is Associated with Inferior Outcomes Relative to Other Acute Myeloid Leukemia (AML) Morphologies
Authors: Giri Smith , Martin Michael G , Pathak Ranjan , Li Bojia , Prouet Philippe E
Published: October 2019

Introduction: Acute Megakaryocytic Leukemia (AMegL) is a rare subtype of Acute Myeloid Leukemia (AML) resulting from uncontrolled proliferation of megakaryoblasts. Due to its rarity it is unclear if AMegL morphology is independently associated with outcomes in AML.

Methods: We used the Surveillance, Epidemiology and End Results (SEER) 18 database to identify adult patients with AML diagnosed between 1991-2011 using the appropriate International Classification of Disease (ICD-O-3) histology codes. Cases of APL (M3) were excluded. Kaplan Meier Survival curves were generated to compare survival statistics between AMegL subtype and the rest of AML types. Multivariate analysis was done using Cox Linear Proportional Hazard Regression model. Statistical analysis was done using Statistical Package of Social Sciences (SPSS) version 21.0 (IBM Corporation, Armonk, NY).

Results: Of 45,564 cases of AML identified, 304 (0.7%) belonged to the AMegL subtype. The median ages were 69 (range 18-111), and 67.5 (range 18-92) years, respectively (p value 0.08); the proportion of males were 54.3% and 59.2% respectively (p value 0.09), the median year of diagnosis was 2004 in AMegL versus 2002 in other AML. Whites comprised 82.6% and 84.7% respectively. The 5 year overall survival rates were 17.5% and 10.6% respectively. Kaplan Meier Survival curves showed significantly inferior survival for AMegL relative to other AML cases (p value of log rank test 0.01). On multivariate analysis AMegL was as an independent predictor of increased mortality (adjusted RR 1.23, 95% CI 1.09-1.38, p value <0.01), after adjusting for age of diagnosis, sex, race and year of diagnosis. This remained significant after removing core binding factor leukemia cases from the analysis (RR 1.21; 95% CI 1.07-1.36, p value <0.01)

Conclusion: AMegL is a rare subtype of AML which is associated with significantly worse survival in comparison to other AML subtypes. We recommend that the National Comprehensive Cancer Network (NCCN) consider adding AMegL morphology to the list of adverse prognostic factors and as a reason to consider allogeneic stem cell transplantation.

Table 1: Multivariate Analysis showing the independent predictors of survival in the study population. Variable RR 95% CI of RR P value Lower Upper AML M7 type 1.229 1.092 1.383 0.001 Race - White - American Indian/Alaska Native - Asian or Pacific Islander - Black 1.0 1.166 0.957 1.112 0.994 0.919 1.070 1.366 0.996 1.156 0.06 0.03 <0.01 Age at diagnosis 1.035 1.034 1.036 <0.01 Year of Diagnosis 0.985 0.983 0.987 <0.01 Male Sex 1.040 1.019 1.061 <0.01

Disclosures

No relevant conflicts of interest to declare.
Allogeneic but Not Autologous Stem Cell Transplant Attenuates the Negative Prognostic Impact Dictated By Pretransplant MRD Positivity
Authors: Buccisano Francesco , Maurillo Luca , Del Principe Maria Ilaria , De Angelis Gottardo , Cerretti Raffaella , Ditto Concetta , Di Veroli Ambra , Picardi Alessandra , Cudillo Laura , Cefalo Mariagiovanna , Sarlo Chiara , Fedele Giorgia , Refrigeri Marco , Lo Coco Francesco , Arcese William , Amadori Sergio , Venditti Adriano
Published: October 2019

Multiparametric flow cytometry (MPFC) detection of minimal residual disease (MRD) represents a robust surrogate for the quality of complete remission (CR) and reliably predicts clinical outcome. In our experience, MRD detection provides prognostically relevant information when assessed at the post-consolidation time point. Ten years ago we demonstrated that the amount of MRD before autologous stem cell transplant (AuSCT) affected outcome. More recently, other authors have extended this observation to allogeneic stem cell transplant (ASCT) showing that pre-transplant MRD is a major determinant of prognosis regardless of graft-versus-leukemia (GVL) effect. The aim of our study was to evaluate, in an extended series of patients submitted to AuSCT or ASCT, the impact of a pre-transplantation MRD positive (MRDpos) or negative (MRDneg) status on overall survival (OS) and disease free survival (DFS). We analyzed 173 MRDpos and 53 MRDneg patients of whom 67 were submitted to AuSCT and 51 to ASCT. Eighty-two patients received no transplant because of age, poor performance status or insufficient stem cell harvest whereas 26, all in the MRDpos group, relapsed before transplant delivery. In the AuSCT group, before transplant, 32/67 (48%) were MRDneg and 35/67 (52%) MRDpos, with MRDneg group showing a superior OS (55% vs 20%, p=0.007). In the ASCT group, before transplant, 45/51 (88%) were MRDpos and 6/51 (12%) MRDneg. For 21 out of 51 (41%) sources of stem cells were matched unrelated donors (12) or haploidentical donors (9). In this subgroup, MRDpos and MRDneg patients shared a comparable 5-years OS (60% vs 56%, p=NS), with a 36% survival gain for those MRDpos who received ASCT as compared to AuSCT. Among MRDneg patients, no survival differences were demonstrated between those submitted to AuSCT or ASCT (55% vs 60%, p=NS). Such a lack of difference is likely due to the higher treatment related mortality (ASCT 3/6, 50% vs AuSCT 2/32, 6%, p=0.003) which counterbalanced the lower relapse rate in the ASCT group (ASCT 0/6, 0% vs AuSCT 11/32, 34%, p=NS). In conclusion, ASCT confers a significant survival advantage to MRDpos patients, attenuating the negative prognostic impact of pre-transplant MRD positivity. ASCT may expose MRDneg patients to an excess of toxicity suggesting that in these patients the allogeneic option should be postponed after a second remission. In MRDpos patients, AuSCT does not represent a valid therapeutic choice and ASCT, which should be timely delivered, also considering alternative sources of stem cells.

Disclosures

No relevant conflicts of interest to declare.
Defining the Primate T Cell Transcriptome during Graft Versus Host Disease: New Data Implicating the Hedgehog and Aurora Kinase Pathways in Pathogenesis and Prevention
Authors: Furlan Scott N. , Watkins Benjamin , Flynn Ryan P , Ramakrishnan Swetha , Singh Karnail , Giver Cynthia R. , Hanby Kelly , Stempora Linda , Garrett Aneesah P. , Ziegler Carly GK , Tharp Gregory , Bosinger Steven , Blazar Bruce R. , Waller Edmund K. , Kean Leslie S.
Published: October 2019

Graft versus host disease (GvHD) is the most common complication of hematopoietic stem cell transplant (HCT). However, our understanding of the molecular pathways that cause this disease remains incomplete, leading to inadequate targeted strategies for prevention and treatment. To address this, we determined the gene expression profile of non-human primate (NHP) T cells during active and partially controlled acute GvHD (aGvHD), in order to accomplish two goals: 1) uncover important genetic drivers of aGvHD and 2) identify novel, targetable pathways for optimal aGvHD prevention.

Utilizing microarray technology, we measured the gene expression profiles of flow cytometrically purified CD3+ T cells from NHP recipients of MHC partially-matched HCT in three treatment cohorts resulting in increasing degrees of survival: 1) no immunoprophylaxis (No Rx, MST = 7.5); 2) sirolimus monotherapy (MST = 17) tacrolimus-methotrexate (Tac-Mtx) dual prophylaxis (MST = 49). Arrays were performed on T cells purified on Day +14 post-transplant (unless terminal analysis occurred earlier due to severe disease). This comparison allowed us to determine the impact of both mTOR and calcineurin inhibition on the molecular pathways dysregulated during GvHD, and to determine which genes and pathways remained dysregulated despite prophylaxis.

Pathways identified by this strategy may contain new therapeutic targets unaffected by current immunoprophylactic approaches. We found that the transcriptional profile of donor T cells from HCT recipients with unprophylaxed GvHD was characterized by significant perturbation of pathways regulating T cell proliferation, effector function and cytokine synthesis (Figure 1a). By identifying pathways unaffected by sirolimus or tac-mtx therapy (Figure 1b), we discovered multiple potentially druggable targets not previously implicated in the pathophysiology of aGvHD. These targets prominently included the hedgehog and the aurora kinase A pathways. Utilizing a murine aGvHD model, we demonstrated that pharmacologic inhibition of these pathways could mitigate disease and improve survival (Figure 2a,b).

These data provide the first identification of the T cell transcriptome of primate acute GvHD and the hedgehog and aurora kinase A pathways as novel potential targets for prevention of this disease.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Haploidentical Second Allogeneic Hematopoietic Stem Cell Transplantation for the Treatment of Acute Leukemia Relapse after First Allo-HSCT: A Retrospective Registry Analysis of 60 Patients on Behalf of the German Cooperative Transplant Group
Authors: Christopeit Maximilian , Tischer Johanna , Bornhäuser Martin , Uharek Lutz , Pfrepper Christian , Behre Gerhard , Niederwieser Dietger , Kröger Nicolaus , Rösler Wolf , Klein Stefan A. , Beelen Dietrich , Hausmann Andreas , Bethge Wolfgang , Schmid Christoph
Published: October 2019

Background: Relapse of acute leukemia (AL) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is frequently treated with 2nd allo-HSCT. Donor change has not yielded a significantly different outcome over choosing the original HLA-identical donor (Christopeit et al., JCO 2013). Using a haploidentical donor at second allo-HSCT might represent a feasible option (Tischer et al., BMT 2014)

Study design and population: To define the role for second haploidentical allo-HSCT for AL relapsing after 1st allo-HSCT, a retrospective analysis was conducted 63 consecutive patients (female n=30, male n=33; AML n=51, ALL n=12) from 9 German centers were included. Median age was 40 years (range, 16-65). Grafts at 1st allo-HSCT were from matched related (32%), matched unrelated (33%), mismatch unrelated (18%), haploidentical donors (6%), and other donors, including cord blood (8%). Median duration of complete remission (CR) after 1st allo-HSCT was 414 days (range, 18-1633). Relapse was initially treated by cytoreductive chemotherapy in all cases; stage at start of conditioning for haploidentical second allo-HSCT was CR in 27%, active disease in 66% and not evaluated in 8%. Conditioning for second HSCT was myeloablative/reduced in 14%/86% To overcome the HLA barrier, 23 patients (36%) received ex vivo T-cell depletion (TCD), following either CD3/CD19 negative or CD34 positive selection. 4 patients received in vivo TCD only, two received no TCD at all, and 35 patients (55%) received high-dose cyclophosphamide post-transplant according to the Baltimore protocol.

Results: Neutrophil engraftment was achieved after a median of 12 days (range, 8-26). 50 patients (78%) achieved CR after 2nd haploidentical allo-HSCT, out of which 23 (46%) relapsed again. After a median follow-up of 425 days, 47 patents had died, 22 from leukemia, and 25 from treatment-related causes. Kaplan-Meier estimated overall survival at one and two years from haploidentical second HSCT was 41+/-6% and 19+/-6%.

Conclusions: Haploidentical second allo-HSCT is a promising approach to the treatment of AL relapse after first allogeneic transplant. OS rates at least comparable to alternative treatments were observed. Different strategies to overcome the HLA barrier seem feasible. This retrospective study was registered as NCT01997918 at clinicaltrials.gov.

Maximilian Christopeit and Johanna Tischer as well as Wolfgang Bethge and Christoph Schmid contributed equally to this work.

Disclosures

No relevant conflicts of interest to declare.
Phagocytic Function of Macrophages Is Impaired By Chronic Lymphocytic Leukemia and high–grade Lymphoma Progression and Can be Highly Effectively Restored for Chemo-Immunotherapy
Authors: Schwamb Janine , Reinart Nina , Vorholt Daniela , Ulas Thomas , Ristovska Vangica , Braun Christian J , Hemann Michael T , Schultze Joachim , Hallek Michael , Pallasch Christian P.
Published: October 2019

Macrophage polarity has recently been shown to play a pivotal role in progression and prognosis of human malignancies. In CLL the high dependence of the malignant cell to the tumor microenvironment has revealed macrophages as major mediators of leukemia cell survival. In contrast, macrophage activation also offers novel therapeutic strategies for leukemia cell targeting. Here we analyze the reciprocal relationship of leukemia cells and macrophages and the specific functional impact of phagocytosis in leukemia progression and therapy.

We employed the humanized hMB-Lymphoma and the Eµ-TCL1 CLL mouse model. Thioglycollate-induced macrophages, bone marrow derived murine macrophages and human monocyte derived macrophages were used for in vitroevaluation of phagocytosis either by bead based approaches or by ADCC.

Applying gene-expression profiling of macrophages in Eµ-TCL1 mice we could identify profound transcriptional alterations indicating a re-programming during leukemogenesis. Functional genomic analysis particularly revealed impaired phagocytic function induced during leukemia onset. In vivo and ex vivo phagocytosis assays of primary macrophages showed a significant reduced phagocytic activity during CLL progression. Human macrophages in co-culture with CLL cells in vitro compared to healthy B-cells similarly showed defects in phagocytosis. In the humanized leukemia model we similarly observed impaired phagocytosis resulting in resistance towards therapeutic antibody/macrophage mediated therapy. Resistance was actively induced by increasing leukemia cell infiltration in the bone marrow. Impaired macrophage function could be identified being mediated by secretory crosstalk such as release of PGE2 by leukemia cells and Cholecalciferol. We could restore phagocytic function by combination regimens involving therapeutic antibodies and chemotherapy. Specifically, an acute secretory activating phenotype (ASAP), releasing cytokines from leukemia cells induces macrophage infiltration and phagocytic activity in the bone marrow. Thus, malignant cells can be effectively targeted by modulation of macrophage polarization and function.

In conclusion, we have identified decreased phagocytic activity of macrophages as key functional aspect in leukemia and lymphoma associated macrophages. Inversely, enhancing phagocytosis rendered essential for the re-sensitization of refractory niches treatment towards monoclonal antibodies defined by macrophage polarity. Overall macrophage function represents a key therapeutic target in CLL and other B-cell malignancies.

Disclosures

No relevant conflicts of interest to declare.
In Vitro and in Vivo Imaging of Initial T-B Cell Interactions in the Setting of B Cell-Based Cancer Immunotherapy
Authors: Wennhold Kerstin , Klein-Gonzalez Nela , von Bergwelt-Baildon Michael , Shimabukuro-Vornhagen Alexander
Published: October 2019

In recent years, there has been a growing interest in the use of B cells for cellular immunotherapy, since B cell-based cancer vaccines have yielded promising results in preclinical animal models. Contrary to dendritic cells (DCs), we know little about the migration behavior of B cells in vivo. Therefore, we investigated the interactions between CD40-activated (CD40) B cells and cytotoxic T cells in vitro and the migration behavior of CD40B cells in vivo.

The dynamic interactions of human antigen-presenting cells and antigen-specific T cells were observed by time-lapse videomicroscopy. The migratory and chemoattractant potential of CD40B cells was analyzed by flow cytometry and standard transwell migration assays. GFP+ CD40B cells or CD40B cells isolated from Luciferase+mice were used for subsequent in vivo studies.

Murine CD40B cells show similar migratory and chemotactic characteristics compared to human CD40B cells. Upon CD40-activation, B cells upregulate the important molecules involved in lymh node homing (CD62L, CCR7/CDCR4), which are functional and induce chemotaxis of T cells in vitro. Striking differences were observed for interactions of human CD40B cells or DCs with T cells. Antigen-loaded CD40B cells differ from immature and mature DCs by displaying a rapid migratory pattern undergoing highly dynamic, short-lived (7.5 min) and sequential interactions with cognate T cells. In vivo, CD40B cells migrate to the spleen and the lymph nodes, where they enrich in the B cell zone before traveling to B cell/ T cell boundary close to the T cell zone.

CD40B cell interactions with T cells are dynamic and short-lived and thereby differ from DCs. Taken together, the migration behavior of CD40B cells and their interaction with T cells underline their potential as cellular adjuvant for cancer immunotherapy.

Disclosures

No relevant conflicts of interest to declare.
Conversion of Danger Signals into Cytokine Signals By Hematopoietic Stem and Progenitor Cells for Regulation of Stress-Induced Hematopoiesis
Authors: Zhao Jimmy L. , Ma Chao , O'Connell Ryan , Mehta Arnav , Wang Jun , Seet Christopher S. , Crooks Gay M. , Rao Dinesh S. , Heath James , Baltimore David
Published: October 2019

Mammals respond to infection by rapid production of innate immune cells. Hematopoietic stem and progenitor cells (HSPCs) have the ability to respond to pathogens directly through toll-like receptors. However, how the pathogen sensing ability of HSPCs may contribute to immune cell output remains unknown. Using a novel single-cell proteomic platform and mouse genetic models, we have shown that in response to toll-like receptor stimulation, short-term HSCs and multipotent progenitor cells produce copious amounts of diverse cytokines through NF-κB signaling. Interestingly, the cytokine production ability of HSPCs trumps mature immune cells in both magnitude and breadth. Among cytokines produced by HSPCs, IL-6 is a particularly important regulator of myeloid differentiation and HSPC proliferation. This is especially important in mediating rapid early myeloid cell recovery during neutropenia after chemotherapy or HSC transplant. This study has uncovered an important property of HSPCs that enables them to convert danger signals into versatile cytokine signals for the regulation of stress-induced hematopoiesis. To translate these findings into human hematopoietic system, we have recently developed a human cytokine chip and our preliminary analysis suggests that purified human bone marrow HSCs also have the capacity to secrete cytokines.

Disclosures

No relevant conflicts of interest to declare.
Concomitant Systemic and Neuro-Meningeal Non-Hodgkin’s Lymphoma: The Role of Consolidation with Intensive Chemotherapy and Autologous Stem Cell Transplantation. a Retrospective Study of 65 Cases
Authors: Damaj Gandhi , Ivanoff Sarah , Coso Diane , Ysebaert Loic , Choquet Sylvain , Houillier Caroline , Abarah Wajed , Marjanovic Zora , Garidi Reda , Diouf Momar , Gac anne Claire , Dupuis Jehan , Morschhauseur Franck , Ghesquieres Herve , Soussain Carole
Published: October 2019

Purpose

To determine the characteristics of Non-Hodgkin’s lymphoma (NHL) patients with concomitant systemic and central nervous system localization at diagnosis, as well as the impact of consolidation with high dose chemotherapy followed by hematopoietic stem cell rescue on outcome

Patients and Methods

Newly diagnosed NHL patients with concomitant systemic and cerebral or meningeal involvement at diagnosis have been included in this study. Patients were retrieved from the database of the participating centers of the LYSA and LOC study groups

Results

Sixty-five patients (37 males; 28 females) were included. Median age was 60 years (23-85). Histological subtype was mainly diffuse large B-cell lymphoma (n=54; 83%). The IPI was >2 in 43 (66%) patients. LDH level was elevated in 27 (55%) patients. Median number of extranodal positive sites was 2 (1-5) and bone marrow involvement was documented in 30 (46%) patients. CNS involvement was documented in 51 patients. Paravertebral and epidural compressive mass with (n=5) or without (n=2) CSF involvement were present. Five patients had both CNS and peripheral nervous system involvement. Anthracycline-based chemotherapy with high dose metothrexate with or without cytarabine was the most chemotherapy used. Autologous stem cell transplantation was performed in 21 patients in response. BEAM (n=9) or thiotepa-based (n=9) conditioning regimen was the most intensive chemotherapy used before autologous SCT

Post-chemotherapy ORR was 77%(CR69%;PR8%). 3-year overall survival (OS) and progression free survival (PFS) were 48±7% and 46±7% respectively. The consolidation strategy using high dose chemotherapy and autologous stem cell transplant positively impacted patient’s outcome. For the whole group as well as for patients ≤65 years, the 3-year OS and PFS were (77%vs29%;p=0.002) and (77%vs25%;p=0.001) and (75% vs 37%;p=0.002) and (75%vs32%;p=0.007) respectively.

In multivariate analysis and for the whole group of patients, the absence high dose therapy had a negative impact on 3-year OS and PFS [p=0.003;HR=5.05[1.76-14.49]) and PFS by [p=0.002;HR=5.46(1.91-15.26)] respectively. This is remained true for younger patients 65 years or less who had poorer 3-year OS [p=0.036;HR=3.41 (1.08-10.75)].

Conclusion

First line consolidative high dose therapy followed by autologous SCT in patients with systemic and neuro-meningeal NHL improve patient’s outcome

Disclosures

No relevant conflicts of interest to declare.
Ibrutinib Enhances the Anti-Tumor Immune Response Induced By Intratumoral Injection of a TLR9 Ligand
Authors: Sagiv-Barfi Idit , Kohrt Holbrook , Burckhardt Laura , Czerwinski Debra K , Levy Ron
Published: October 2019

We have designed a novel approach for treating tumors utilizing a combination of active immunotherapy and targeted kinase inhibition. We injected tumors with unmethylated CG-enriched oligodeoxynucleotides (CpG), an agonist for the toll like receptor 9 (TLR9). This in situ CpG injection resulted in local tumor eradication by NK cells and macrophages but on its own was not able to induce a systemic anti-tumor immune response. Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (Btk), a key enzyme in the signaling pathway downstream of B Cell receptor (BCR) is now known to also inhibit ITK, a key enzyme in the survival of Th2 T cells. By doing so, it can shift the balance to the more effective Th1 immune response.

In our murine model, the combination of in situ injection of CpG and systemic treatment with Ibrutinib resulted in eradication of tumor cells not only in the local site but also at distant sites. This anti-tumor immune response required both CD4 and CD8 T cells as the effect was lost in nude, scid and T cell depleted mice. Additionally, transferred T cells from animals previously treated with CpG and Ibrutinib protected na•ve mice from tumor challenge.

This immune-enhancing effect of Ibrutinib is unexpected. It reflects on the mechanism of action of the drug against B cell malignancies and it provides a novel way of enhancing anti-tumor immune responses against other types of cancer.

Figure 1 Figure 1.

A. six to eight weeks BALB/C mice were inoculated with 1x106 H11 cells s.c. into the right and left sides of their abdomen, tumor growth was monitored with a digital caliper. Therapy was started when tumors reached a size of 0.7-1cm in the largest diameter, CpG (100µg/injection) was given intratumorally to the left tumor every day in days 1-5, Ibrutinib (6mg/kg) was given daily, intraperitonealy (IP) on days 1-8. B. Growth curve of the right (non-treated) tumor C. Mice survival D. Mice at day 4 after treatment.

Disclosures

No relevant conflicts of interest to declare.
Cytoskeleton Regulator PAK4 Plays a Role in Growth and Survival of Myeloma with a Potential Therapeutic Intervention Using PAK4 Allosteric Modulators (PAMs)
Authors: Fulciniti Mariateresa , Bandi Rajya Lakshmi , Amodio Nicola , Cagnetta Antonia , Senapedis William , Baloglu Erkan , Anderson Kenneth C. , Munshi Nikhil C.
Published: October 2019

P21-activated serine/threonine kinase 4 (PAK4) is a major effector of Cdc42 and is essential for cytoskeleton reorganization. PAK4 is activated in cancer cells, promotes cell migration and anchorage-independent cell growth, and protects against apoptosis induction. With cellular migration playing a significant role in multiple myeloma (MM) cell growth and survival, we investigated the expression and subcellular localization of PAK4 in MM cells. We observed a high level of un-phosphorylated PAK4 in the cytoplasm and high levels of phosphorylated PAK4 in the nucleus. In a gain-of-function study, over-expression of PAK4-eGFP in PAK4-deficient MM cells (RPMI8226) significantly increased cell proliferation and survival. Conversely, in a loss-of-function study, conditional knock-down of PAK4 expression with TRIPZ-lentiviral vectors decreased MM cell proliferation and survival proportionally to the reduction in PAK4. With a significant impact of PAK4 on MM cell growth, we identified a class of orally bioavailable PAK4 allosteric modulators (PAMs; e.g. KPT-6604, -7189, -7657, -8752). We observed inhibition of MM cell growth and survival after treatment with PAMs even in the presence of bone marrow microenvironment. In addition, there is a significant correlation between PAK4 expression and the inhibition concentration (IC50s) of PAMs in proliferation assays. Moreover, inhibition of PAK4 induced receptor and mitochondrial-mediated apoptotic pathways via Caspase-3, -8, and -9 activation. PAMs had no significant effect on normal PBMCs, suggesting a favorable therapeutic index in MM treatment. Finally, in two murine models of human myeloma, orally bioavailable KPT-8752 given daily was able to inhibit tumor growth in vivo and prolong overall survival. In summary, PAK4 plays an important cellular and molecular function in myeloma and its inhibition with a new class of PAK4 allosteric modulators provides a novel therapeutic approach for the treatment of MM.

Disclosures

Senapedis: Karyopharm: Employment. Baloglu:Karyopharm: Employment. Anderson:Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy.
Effects of Monoclonal Gammopathy of Undetermined Significance (MGUS) on Outcomes after Solid Organ Transplant
Authors: Goebel Teresa E , Schiltz Nicholas K , Pillai Aiswarya Chandran , Caimi Paolo , Koroukian Siran M , Woodside Kenneth J , Campagnaro Erica L
Published: October 2019

Background:

MGUS is a precancerous condition which can progress to multiple myeloma (MM) or other hematologic malignancy. MGUS increases the risk of developing MM approximately 25-fold, and increases lifetime risk of infection, venous thromboembolism (VTE), and skeletal related events (SRE). Solid organ transplant requires immunosuppression, which is associated with overlapping risks, including hematologic malignancy such as post transplant lymphoproliferative disease (PTLD). Hypothesizing that MGUS would further increase risk of PTLD and other complications after transplant, we described risk of these outcomes in patients with MGUS prior to solid organ transplant (MGUS+) compared to those without MGUS (MGUS-).

Methods:

We used the 2005-2011 California State Inpatient, Emergency, and Ambulatory Databases. MGUS and complications were identified by ICD-9 diagnosis codes, and solid organ transplant by ICD-9 procedure codes. Patients with the ICD-9 diagnosis code 273.1 documented on or before the day of solid organ transplant surgery were defined as MGUS+. We used logistic regression to analyze complications in MGUS+ versus MGUS- transplant patients.

Results:

Of 24,358,669 patients, we identified 22,062 solid organ transplant patients. Transplant patients were 8.8% African American, 29.5% Hispanic, 43.4% White, 15.1% other, and 3.2% had data missing. 72 were MGUS+ prior to solid organ transplant. Median age of MGUS+ was 61.5 years versus 51 years for MGUS-. Outcomes are shown in table 1.

Table 1. Transplant Patients Outcome MGUS + N=72 MGUS – N=21,990 Odds Ratio (95% CI) PTLD 0 161 n/a MM ≤10 37 34.90 (12.11, 100.61) Lymphoma 0 193 n/a VTE 20 3,202 2.26(1.35, 3.79) SRE 18 2,320 2.83(1.66, 4.83) Infection 50 11,612 2.03(1.23, 3.36)

*Frequencies ≤10 are reported as such per the data use agreement.

Conclusions:

Compared to MGUS-, MGUS+ solid organ transplant patients had higher risk of VTE, SRE, and infection, but did not have higher risk of PTLD or other lymphomas. MGUS+ transplant patients are more likely to develop MM than MGUS-; however, this risk is similar to that historically attributed to MGUS, in patients who have not undergone transplant. These data show increased risk of certain complications in MGUS+ patients, and do not support screening for MGUS to assess risk of PTLD prior to solid organ transplant.

Disclosures

No relevant conflicts of interest to declare.
The Benefit of FVIII Measurement: Tailored Treatment with Obi-1, a Recombinant Porcine Sequence Factor VIII, in Subjects with Acquired Hemophilia a
Authors: Oladapo Abiola , Epstein Joshua , Novack Aaron , Farin Heinrich D.
Published: October 2019

Introduction: A significant unmet need in the management of acquired hemophilia A (AHA) is a replacement therapy that can be used without consideration of anti-human FVIII titer and allows for the ability to measure FVIII levels. The ability to measure FVIII levels during treatment: (1) identifies treatment response and leads to the ability to individually tailor treatment; (2) reduces treatment safety concerns; and (3) optimizes the utilization of therapy during treatment to minimize cost impact. OBI-1 is a recombinant porcine sequence FVIII developed to meet this need.

Objective: To determine the impact of FVIII monitoring on OBI-1’s utilization during the treatment phase in the OBI-1 Phase 2/3 trial in AHA.

Methods: A post-hoc analysis of the utilization data from the prospective, open label, Phase 2/3 study of OBI-1 in AHA was conducted. Subjects were administered an initial dose of 200U/kg of OBI-1 and subsequent doses were based on subject’s post-infusion FVIII activity levels and clinical assessments. The impact of FVIII monitoring on OBI-1’s utilization was evaluated by comparing: (A) the initial administered dose to the subsequent doses infused in the treatment period; and (B) the initial total administered dose within the first 24 hours of treatment to the doses infused in the subsequent 24-hour period(s) in the treatment period. The magnitude and the statistical significance of the dose adjustment were determined using non-parametric statistics (signed rank test).

Results: Of the 29 subjects enrolled in the study, 65.5% (n=19) were males and median (range) age was 70 (42-90) years. Twenty-eight of the enrolled subjects had AHA while presence of anti-human FVIII titer could not be confirmed in the twenty-ninth subject. Of the subjects with AHA, 27 had a serious bleeding episode while one was enrolled for hemostatic cover for a planned surgery. Two subjects (7.1%) successfully responded requiring only the initial administered dose (100U/kg; 200U/kg). Two additional subjects were successfully treated within the first 24 hours. After the initial dose, subsequent doses indicated a median (IQR) dose reduction of 41.2% (50%) [n=26, p<0.01]. After the first 24-hour period, subsequent 24-hour periods showed a median (IQR) dose reduction of 65.4% (32.5%) [n=24, p<0.0001].

Conclusion: Study data indicate a significant reduction in the treatment dose after the initial dose or subsequent daily dosing after the first 24 hours. The ability to measure and monitor FVIII activity levels allow for the tailoring of OBI-1’s treatment dose and regimen. This measurability likely has a significant impact on dosing decisions to control the bleed.

Disclosures

Oladapo: Baxter Healthcare Corporation: Employment. Epstein:Baxter Healthcare Corporation: Employment. Novack:Baxter Healthcare: Employment. Farin:Baxter Healthcare: Employment.
Granzyme B Is a Key Regulator of Plasmacytoid Dendritic Cell Immunogenicity
Authors: Jahrsdörfer Bernd , Beyer Thamara , Schrezenmeier Hubert , Debatin Klaus-Michael , Fabricius Dorit
Published: October 2019

Despite their low frequency of 0,1 - 0,5% in the peripheral blood, human plasmacytoid dendritic cells (pDC) are important modulators of adaptive immune responses and have meanwhile taken the step from the bench to promising initial tumor vaccination studies. PDC express high levels of the IL-3-receptor CD123, MHC class II and blood dendritic cell antigens (BDCA)-2 and -4. Apart from secretion of IFN-alpha and their capacity to rapidly initiate antigen-specific CD8+ T cell responses by cross-presentation, they produce large amounts of the serine protease granzyme B (GrB). GrB exhibits various non-cytotoxic functions including T cell regulation and the support of antigen processing and phagocytosis. We therefore hypothesized that pDC may use their GrB for both T cell regulation and for antigen uptake and processing. PDC were isolated using magnetically labeled antibodies against CD123, and cultured in the presence of various stimuli. Here, we demonstrate that pDC are potent producers of enzymatically active GrB, which reached maximal concentrations up to two logs higher than those produced by cytotoxic cells including CTL and NK cells. The strongest inducers of GrB in pDC were IL-3 and IL-10, whereas toll-like receptor (TLR)-7 or TLR-9 ligands such as CpG ODN or imiquimod as well as CD40 ligand suppressed GrB induction. We demonstrate that pDC-derived GrB can suppress effector T cell proliferation by GrB-mediated degradation of the T cell receptor zeta-chain. Moreover, we tested the uptake of fluorescently labeled antigenic material into pDC after lysis of tumor cells by freeze-thaw cycles and irradiation. These studies revealed a correlation between pDC GrB expression and antigen uptake, with GrBhigh pDC taking up significantly more antigenic material than GrBlow pDC. Our data support a novel concept, in which pDC acquire their maximal immunogenic potential in a two-step activation process: First, high levels of GrB are induced by IL-3 and IL-10, thereby supporting antigen processing and uptake into pDC, while preventing precocious activation of T cells. Subsequently, TLR agonists and CD40 ligand turn off GrB in pDC and initiate the transfer of MHC-antigen complexes and co-stimulatory molecules to the pDC surface, resulting in highly immunogenic pDC. Our data may be of translational relevance for the further development of novel DC vaccination strategies in tumor therapy.

Disclosures

No relevant conflicts of interest to declare.
Interleukin-7 Represses the Immunoregulatory Function of Human TCRαβ+ CD4- CD8- Double-Negative (DN) T Cells By Activating Akt/mTOR Signaling
Authors: Allgaeuer Andrea , Gerbitz Armin , Mackensen Andreas , Voelkl Simon
Published: October 2019

Regulatory T (Treg) cells have been shown to be involved in downregulating immune responses in autoimmunity, transplant rejection, and graft-versus-host disease (GvHD). Recently, a novel subset of TCRαβ+ CD4- CD8- (double negative, DN) T cells has been described to possess a very potent suppressive activity on allogeneic T-cell responses in both mice and humans. Of interest, clinical studies in patients after allogeneic stem cell transplantation (SCT) revealed an inverse correlation between the frequency of circulating DN T cells and the severity of GvHD. Interleukin (IL)-7 is required for survival and homeostasis of mature T-cells and has been shown to modulate the functional activity of Treg cells. Here we asked whether IL-7 affects the suppressive activity of human DN T cells. We found that human DN T cells isolated from peripheral blood lymphocytes do express the IL-7 receptor and are able to transduce IL-7 downstream signaling. Intriguingly, IL-7 significantly diminished the suppressive activity of DN T cells towards allogeneic CD4+ and CD8+ T cells. Thereby, IL-7 did not render the responder T cells insensitive to suppression. Instead, blocking of key signaling molecules revealed that IL-7 has a direct impact on the suppressive activity of DN T cells. Further analyses demonstrated that IL-7 activates Akt/mTOR signaling in DN T cells. Importantly, selective inhibition of the Akt or mTOR protein kinase was able to reverse the IL-7 effect thereby restoring the suppressive function of DN T cells, whereas inhibition of other central T-cell signaling pathways did not. Thus, DN T-cell mediated suppression is impaired by IL-7 due to crucial activation of the Akt/mTOR signaling cascade. These findings indicate that IL-7 and Akt/mTOR signaling are critical factors for the suppressive capacity of DN T cells. Targeting of these pathways by pharmacological agents may restore and/or enhance DN T-cell functionality in GvHD.

Disclosures

No relevant conflicts of interest to declare.
Platelets Protect Against Murine Pneumonia
Authors: Feng Shuju , Evans Scott , Wu Jessica , Afshar-Kharghan Vahid , Kroll Michael H.
Published: October 2019

Platelets have been called ancient granulocytes because they are capable of recognizing and destroying microbial invaders through their release of a variety of anti-microbial proteins. Experimental evidence indicates that this may involve direct platelet-microbial interactions or occur indirectly through platelet-leukocyte interactions, neutrophil histone-platelet interactions and/or platelet-directed neutrophil extracellular trap formation. To test the hypothesis that platelets contribute to innate lung immunity, we examined the clinical course, lung histopathology and pathogen burden of mice -/+ antibody-mediated platelet depletion who were subjected to aerosolized bacteria (Streptococcus pneumoniae or Pseudomonas aeruginosa). Mice (C57BL/6, 10 weeks) were injected by tail vein with a purified rat monoclonal antibody directed against mouse GpIbα (or control antibody) resulting in platelet depletion to ~ 20% at 3 hours that persisted for 48 hours. 30 minutes after GpIbα antibody injection mice were placed into a closed system for 60 minutes where they were exposed to 10 ml nebulized S. pneumoniae (2-6 x 1011 CFU/ml) or P. aeruginosa (1-4 x 1010 CFU/ml). All platelet depleted animals infected with S. pneumoniae died by day 4 after infection, whereas all platelet replete mice survived the infection. Infection with P. aeruginosa resulted in death of all animals, although the platelet replete mice lived longer than the platelet depleted mice. No platelet depleted sham-infected animals died. The lungs of animals were harvested, fixed and prepared for histopathology with hematoxylin and eosin staining; or harvested, homogenized and plated for bacterial CFU measurements. Whole lungs of platelet-depleted and infected mice were congested. Histopathology showed increased red cells in the venules of platelet-depleted S. pneumoniae-infected mice but no difference in air-space hemorrhage in comparison with platelet-depleted sham-infected lungs and platelet-replete S. pneumoniae-infected lungs. Platelet depletion resulted in ~ 5-fold increase in lung S. pneumoniae CFUs and ~ 10-fold increase in P. aeruginosa CFUs. These results indicate that platelets provide defense against pneumonia. The mechanism of this effect is not known. Experiments are ongoing to determine if it is a consequence of platelet-dependent hemostasis, platelet-leukocyte interactions and/or direct platelet-microbial interactions leading to the release of bactericidal compounds.

Disclosures

Kroll: Boerhinger-Ingelheim: Membership on an entity's Board of Directors or advisory committees; Aplagon Therapeutics: Membership on an entity's Board of Directors or advisory committees.