bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevention of Fetal/Neonatal Alloimmune Thrombocytopenia in a Preclinical Model By Monoclonal Anti-HPA-1a Antibody Prophylaxis: Structural and Functional Properties of Antibody Variant Isoforms
Authors: Mørtberg Trude Victoria , Zhi Huiying , Vidarsson Gestur , Foss Stian , Michaelsen Terje E , Skogen Bjørn , Stuge Tor Brynjar , Andersen Jan Terje , Newman Peter J. , Ahlen Maria Therese
Published: November 2021

Maternal alloantibodies to paternally inherited platelet antigens can cause fetal/neonatal alloimmune thrombocytopenia (FNAIT), rendering the fetus or newborn prone to bleeding and intracranial hemorrhage (ICH) with risk of lifelong disabilities or death. In Caucasians, the vast majority of cases are due to antibodies to the Human Platelet Antigen (HPA)-1a epitope on glycoprotein (GP)IIIa. To date, there are neither any means to prevent or reduce the risk of alloimmunization and subsequent FNAIT, nor safe and efficient treatment during affected pregnancies. Thus, a prophylactic regimen by administration of monoclonal antibodies to women at risk would be highly beneficial. Knowledge about the optimal functional design of prophylactic monoclonal antibodies for antenatal or post-natal use is however still limited. We have previously isolated and characterized a fully human anti-HPA-1a monoclonal antibody, mAb26.4. In the current study we have explored the prophylactic potential of this antibody by testing a panel of different IgG1 designs, including the wild-type mAb26.4, variants with modified Fc-region N-glycans, as well as an effector silent variant. We performed analyses of properties relevant for immunosuppression: in vitro Fc-receptor binding and capacity to induce phagocytosis, in vivo half-life measurements in humanized FcRn mice and platelet clearance in a recently developed transgenic mouse strain with a recreated HPA-1a epitope on murine GPIIIa. The prophylactic capacity by antibody-mediated immune suppression in vivo, were further tested in the FNAIT pre-clinical murine model, in which BALB/c females can be immunized by transfusion of HPA-1a-expressing platelets from the transgenic mice and where subsequent breeding of pre-immunized mice with transgenic males cause thrombocytopenia in off-springs mimicking FNAIT. By intravenous administrations of mAb26.4 variants prior to platelet transfusions, the mice generated no or low anti-platelet responses compared to control mice, and normal platelet counts in pups upon subsequent breeding. Our data thus successfully demonstrates efficient immunosuppression and prevention of FNAIT by anti-HPA-1a human monoclonal variants, providing further support for potential use in humans.

Disclosures

Skogen: Prophylix Pharma AS: Current holder of individual stocks in a privately-held company. Newman: Rallybio: Consultancy, Research Funding.
Flow Cytometry Based Identification of CD26+ Leukemic Stem Cells in the Peripheral Blood Has a Potential Role in the Rapid Diagnosis of Chronic Myeloid Leukemia
Authors: Sharma Praveen , Sachdeva Man Updesh Singh , Naseem Shano , Sreedharanunni Sreejesh , Das Reena , Malhotra Pankaj , Varma Neelam
Published: November 2021

Background: Demonstration of t(9;22)(BCR-ABL1) fusion is gold standard for the diagnosis of chronic myeloid leukemia (CML). We performed a flowcytometric assay to identify CD26+ CML leukemic stem cells (LSCs) for its value as a standalone diagnostic investigation for the diagnosis of CML and its utility for detection of residual disease in CML patients on therapy.

Methods: Patients of CML/ CML on follow-up were included and peripheral (PB) and/or bone marrow (BM) samples were utilized for flowcytometric analysis. PB and/or BM of patients with diseases other than CML were used as controls. Under 'lyse-wash-stain-wash' sequence, the sample was incubated with a pre-titrated custom-made antibody cocktail in a 'test' tube containing CD45, CD34, CD38 and CD26 mo-abs. Acquisition was carried out on BD FACS Canto II and analysis was done with Diva Software. Clinical data including demographic details, complete blood count and BM findings were also noted.

Results: A total of 104 samples (63 PB and 41 BM) from 64 patients [confirmed & treatment naïve CML (n=30), CML on follow-up (n=15), non-CML (n=19)] were tested. The median (range) time for reporting of PB/BM examination, molecular genetic studies and flow cytometry for CD26+ CML LSCs was 5 (3-11 days), 4 (3-6 days) and 1 (0-1 day) respectively. CD26+ LSCs were identified in all patients with a confirmed diagnosis of CML (Median=0.07%, range 0.002%-26.79%), and also in 8/15 patients of the follow-up group, who also reported persisting levels of BCR-ABL1. None of the patients in the non-CML group and follow-up CML patients with negative RT-PCR results showed the presence of CD26+ LSCs. Also, there was a strong correlation between CD26+ CML LSCs in the PB and BM (r=0.917).

Conclusion: Flow cytometric assessment for CD26+ LSCs is quick with reporting time of even less than an hour. Flow cytometric identification of CD26+ LSCs in the peripheral blood can be a cheap, rapid, robust and potential diagnostic tool for the diagnosis of CML compared to available testing methods. It is independent of BCR-ABL1 transcript type and its role in residual disease monitoring needs further investigation.

Disclosures

No relevant conflicts of interest to declare.
Expression of Paraoxonase-2 in Different Stages of Cutaneous T Cell Lymphoma: Is It a Potential Biomarker for Aggressiveness?
Authors: Morsia Erika , Rupoli Serena , Molinelli Elisa , Sartini Davide , Offidani Anna Maria , Emanuelli Monica , Salvolini Eleonora , Olivieri Attilio , Goteri Gaia
Published: November 2021

Introduction: Paraoxonase 2 (PON2) is an intracellular membrane protein that belong to the PON gene family. This enzyme exerting a crucial role against production of reactive oxygen species within mithochondrial respiratory chain. Some reports provided clinical evidence for a link between PON2 and different types of malignancies and recently a growing attention has been focused on exploring the role of PON2 in cancer. Moreover, in contrast to solid tumors, a low expression levels of PON2 protein were detected in hematological malignancies like acute myeloid leukemia and B-cell lymphomas. The aim of this study was to investigate the role of PON2 in the cutaneous T cells Lymphoma (CTCL).

Methods: We performed an immunohistochemistry analysis of PON2 protein expression at various stages CTCL. We analyzed PON2 protein using skin biopsies from stage I (n= 3) and stage II (n= 6) Mycosis Fungoides (MF) and stage III/IV (n= 5) erythrodermic MF/Sézary Syndrome (SS) and control (n=6) tissues. Values were expressed as percentage of PON2-positive staining cells, whereas the intensity of PON2 positivity was semi-quantitatively scored from negative to -/+/+++. Subsequent statistical analysis was carried out to explore the existence of correlations between intra-tumor enzyme level and clinical-pathological features at diagnosis.

Results: In our preliminary study, results showed PON2 down-expression in SS compared to controls. Among CTCL, we found significant differences in enzyme levels between MF and erythrodermic MF/SS; in fact, PON2 expression was higher in patients with not erythrodermic MF compared to patients with erythrodermic MF/SS. (Table I) Moreover, we found no significant differences in enzyme levels between MF patients and controls.

Conclusion: Our study is the first to demonstrate downregulation of PON2 intra-tumoral in SS patients while protein levels were higher in stage I and II MF patients. These findings seem to suggest that PON2 expression levels could be negatively related with tumor aggressiveness in CTCL: an interesting phenomenon where PON2 is upregulated in the early stages and downregulated in the late stages of CTCL. Further, in vitro studies are needed to clarify the mechanism related to PON2 expression.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Role of Daratumumab in Relapsed and Refractory Multiple Myeloma Patients: Meta-Analysis and Literature Review
Authors: Abodunrin Faith O , Tauseef Abubakar , Silberstein Peter
Published: November 2021

Introduction:

In 2015, U.S. Food and Drug Administration granted daratumumab approval, based on two phase II studies, as monotherapy (16 mg/kg in heavily treated patients) for MM patients who have received at least three prior lines of therapy, including a proteasome inhibitor and an immunomodulatory agent, or patient's double refractory to these agents [1]. Daratumumab is a human IgG1 monoclonal antibody targeting CD38, expressed at high levels on malignant cells in multiple myeloma (MM) [2].

METHODS AND METHODOLOGY:

This meta-analysis was conducted according to Cochrane Collaboration guidelines and reported as per PRISMA guidelines and its summary is given in Figure 1. Two authors (AA and MZ) performed a systematic literature search using databases such as MEDLINE (via PubMed), Embase, and Cochrane library using the medical search terms: "daratumumab, refractory, relapsed, multiple myeloma". Three of the totals of 679 articles reviewed were selected as having identical primary and secondary outcomes

Results:

A total of 3 studies including 1533 patients (849 in Daratumumab treatment group while 684 patients in control group) were included in the study (Figure I). Mean age was 65 years in both groups. 54.4% had a progression free survival in daratumumab group as compared to 8% in control. Overall response rate, very good partial response, and complete response favors treatment group while minimal and partial response outcome favored control group. Adverse effects were more common in treatment group as compared to treatment group.

Discussion:

In ELOQUENT-3 trial, role of Elotuzumab was studied in RRMM patients which showed ORR of 53% in treatment group in comparison to our study which showed ORR of 85.2% with significant p-value of <0.001 [3]. Similar kind of results were seen with Eloquent-2 trial which showed overall response rate of 79 % respectively [4].

References:

1. Overdijk MB, Jansen JH, Nederend M, Lammerts van Bueren JJ, Groen RW, Parren PW. The Therapeutic CD38 Monoclonal Antibody Daratumumab Induces Programmed Cell Death via Fcγ Receptor-Mediated Cross-Linking. 2016;197(3):807-13.

2. Dima D, Dower J, Comenzo RL, Varga C. Evaluating Daratumumab in the Treatment of Multiple Myeloma: Safety, Efficacy and Place in Therapy. Cancer management and research. 2020;12:7891-903.

3. Dimopoulos MA, Dytfeld D, Grosicki S, et al. Elotuzumab plus Pomalidomide and Dexamethasone for Multiple Myeloma. N Engl J Med. 2018 Nov 8;379(19):1811-1822.

4. Zhang T, Wang S, Lin T, Xie J, Zhao L, Liang Z, et al. Systematic review and meta-analysis of the efficacy and safety of novel monoclonal antibodies for treatment of relapsed/refractory multiple myeloma. Oncotarget. 2017;8(20):34001-17.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Platelet Mediated Monocyte/Macrophage Immune Training
Authors: Li Chen , Maurya Preeti , Nieves-Lopez Benjamin , Ture Sara , Morrell Craig N.
Published: November 2021

Platelets are essential mediators of vascular and immune homeostasis as well as mediators of thrombosis. Platelet functions in hemostasis and thrombosis have received great attention in both basic and clinical research, however, emerging research indicates platelets are also critical components of the immune system. Platelets have important roles in many inflammation-associated diseases including sepsis, atherosclerosis, and peripheral artery disease.

In recent years, much interest has focused on the acute outcomes of interactions between platelets and monocytes/macrophages in immune responses to bacterial infections, as several studies have demonstrated that platelets contribute to bacteria clearance and inflammation resolution by regulating monocyte/macrophage responses and their immune differentiation. However, macrophages are long-lived cells and past stimulation and immune interactions can change their responses to future stimuli - a concept termed 'innate immune memory'. Whether platelet interactions with monocytes/macrophages alters their inflammatory responses to secondary stimuli in a long-lasting manner remains unclear.

We have now found that platelet interactions with monocytes and macrophages as a first stimulus, changed their responses to secondary stimuli, such as lipopolysaccharide (LPS) and unmethylated cytosine-phosphate-guanine (CpG). We incubated monocytes or macrophages with control buffer or platelets overnight and then removed the platelets prior to the addition of LPS or CpG as secondary stimulation. LPS and CpG induced IL-6 and TNFα were reduced by platelet pre-incubation compared to platelet naïve macrophages. Despite reduced cytokine secretion, platelet pre-incubation increased monocyte and macrophage proliferation in response to secondary stimuli. Furthermore, the platelet mediated macrophage secondary stimuli phenotype was preserved for several days after platelet exposure indicating a genetic reprogramming mediated mechanism. Circulating monocytes from platelet deficient TPOR -/- mice and adult mice made platelet deficient also had higher Il6 expression and secrete more IL-6 in response to stimulation compared to monocytes from WT mice indicating in vivo platelet mediated monocyte immune-training. Furthermore, the genetic reprogramming may be dependent on a HIF1α dependent process, as platelet pre-incubation increased macrophage Hif1α expression, a known mediator of trained immunity.

Monocytes and macrophages, are 'immune plastic' and adapt to their environment in a functional manner. These novel data indicate that platelets 'reprogram' monocytes/macrophages and shape their responses to future stimulation.

Disclosures

No relevant conflicts of interest to declare.
Platelets up-Regulate Tumor Cell Programmed Death-Ligand 1 through Epidermal Growth Factor Receptor Signal Transduction
Authors: Guo Qiuchen , Malloy Michael W , Roweth Harvey G. , Italiano Joseph E. , Battinelli Elisabeth
Published: November 2021

Introduction: Programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) are important immune checkpoint proteins in cancer immunotherapy and targeted therapies against PD-L1 have significantly prolonged many patients' lives. Recently, high baseline platelet to lymphocyte ratio was reported to be associated with decreased patient response rate to immune checkpoint inhibition (ICI) therapies, including anti-PD-L1 therapy, suggesting the potential role of platelets in tumor immunity. Platelets express PD-L1 on their surface, and platelets binding to PD-L1 negative tumor cells can "decorate" tumor cells with PD-L1 and protect against T cell-mediated cytotoxicity. However, whether platelet can affect PD-L1 expression on tumor cells is still unknown.

Methods: In this study, we designed platelet-tumor cell co-culture systems to investigate whether direct or indirect exposure to platelets affects tumor cell PD-L1 surface expression. Considering platelets can be artificially activated by commonly used cell culture medium, the co-culture was performed in platelet resuspension buffer (HEPES, NaCl, KCl, MgCl2, NaHCO3, Glucose, pH7.4) supplied with fetal bovine serum and L-glutamine. After 24 hours of co-culture, platelets were washed out and fresh culturing medium was added to tumor cells and cultured for another 24 hours. At the end of the experiments, tumor cells were harvested and the PD-L1 expression analyzed by flow cytometry and RT-qPCR.

Results and discussion: Here we report that direct co-culture of platelets with either breast cancer cell line MDA-MB-468 or lung cancer cell line A549 increased tumor cell PD-L1 surface expression by up-regulating PD-L1 transcription. This platelet-induced tumor cell PD-L1 up-regulation can be partly reduced by pre-treating platelets with antiplatelet agents such as aspirin and ticagrelor, suggesting platelet activation contributes to platelet induced tumor cell PD-L1 up-regulation. The up-regulation of tumor cell PD-L1 by platelets was not due to abundant platelet cytokines such as C-C Motif Chemokine Ligand 5 (CCL5) and C-X-C motif chemokine 5 (CXCL5). However, both an epidermal growth factor (EGF) neutralizing antibody and cetuximab (EGFR neutralizing monoclonal antibody) decreased the platelet-induced increase in tumor cell PD-L1, suggesting that platelets initiate tumor cell PD-L1 transcription through the EGF signaling pathway. Our data indicate a novel function of platelets in tumor immunity and warrant further investigation to determine if targeting platelets offers a novel adjuvant approach to improve ICI therapy.

Disclosures

Italiano: Sierra Oncology: Consultancy; PlateletBio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Carrick Therapeutics: Consultancy.
The Prognostic Impact of Abnormal Protein Bands in Multiple Myeloma: A Systematic Review and Meta-Analysis
Authors: Low Soon Khai , Ho Matthew , Jamshed Saad
Published: November 2021

Introduction: The detection of abnormal protein bands (APB) different from the original monoclonal protein in patients with multiple myeloma (MM) who underwent stem cell transplantation and/or chemotherapy has been reported. These atypical serum immunofixation patterns may be monoclonal (also termed secondary monoclonal gammopathy of undetermined significance) or oligoclonal bands (OB). The prognostic significance of this phenomenon remains controversial. This systematic review and meta-analysis aimed to summarize and analyze the evidence for the association between APB with survival in MM patients.

Methods: A systematic search of PubMed, Cochrane, Google Scholar, Medline-Ovid, CINAHL, and ERIC, was conducted using relevant search terms. All studies were screened using predefined selection criteria and critically appraised for quality assessment following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guideline. All studies that described the presence of APB, defined as monoclonal spike with heavy or light chain immunoglobulin distinct from the original paraprotein at initial diagnosis of MM, and its associations with survival were included. Studies that reported multivariate adjusted hazard ratios (HR) were further included for meta-analysis. Random-effects model was used to synthesize the pooled HR estimate for the association of APB with survival. Cochran's Q statistics and I2 tests were used to evaluate statistical heterogeneity.

Results: A total of 24 out of 181 eligible studies were included for qualitative synthesis. Four studies (including 1115 patients) that reported HR estimates were included in the final meta-analysis. The random-effect summary HR for the progression-free survival among patients with APB was 0.44 (95% CI, 0.31-0.65) with no statistical heterogeneity (I2=0%, p=0.93). The pooled HR for the association with overall survival was 0.31 (95% CI, 0.14-0.66) with moderate statistical heterogeneity (I2=45%; p=0.16) for patients with APB. One study (Silva et al, 2017) only included patients with at least very good partial response while the other three studies reported similar findings of higher occurrence of APB with complete response (Tovar et al, 2013; Jo et al, 2014; Zou et al, 2014). These results are also consistent with the presumed association of APB with complete response as suggested in other studies included in qualitative review.

Conclusions: This study indicated a potential prognostic value of APB for favorable outcomes in the context of both overall and progression-free survival in MM patients after treatment. Further research is needed to evaluate the prognostic impact of the sole emergence of APB irrespective of treatment response.

Figure 1 Figure 1.

Disclosures

Jamshed: Takeda: Honoraria.
Recent Updates on Blinatumomab: A Bispecific T-Cell Engaging Antibody in Relapsed and Refractory B-Cell Acute Lymphoblastic Leukemia
Authors: Saleem Tabinda , Rafae Abdul , Wahab Ahsan , Ehsan Hamid , Neupane Karun , Rehman Mohammad Ebad Ur , Pachika Pranali , Ali Rabia , Ali Sundas , Anwer Faiz
Published: November 2021

Introduction: Blinatumomab is a bispecific T-cell engaging antibody that binds and allows CD3 cytotoxic T cells to recognize and eradicate CD-19 positive malignant B cells. Blinatumomab is currently under study for relapsed and refractory B-cell acute lymphoblastic leukemia (RR B-ALL).

Methods: A comprehensive literature search was conducted across various data sets, including PubMed, Cochrane, and Embase, and presented data in ASH and ASCO. A review of the most recent data is summarized in this abstract.

Results: Eight clinical trials are currently under various phases of the study evaluating blinatumomab in RR B-ALL. In the phase 1/2 trial by Locatelli et al., 110 patients treated with blinatumomab showed complete remission (CR) with <5% blast cells of 62.7% at a dose of 5-15 µg/m 2. In phase 1b trial by Horibe et al., nine Japanese children received blinatumomab at a dose of 5 µg/m 2/day for week 1, followed by (f/b) 15 µg/m 2/day for week 2-4 and showed an CR in 56% patients.

In a phase II study, Martinelli et al., treated 45 patients at a dose of 9 µg/day in week 1, f/b 28 µg/day; 36% of the patients achieved CR. In another phase II trial by Gokbuget et al., a 15 µg/day dose in 20 patients resulted CR in 50% pts. Topp et al. reported CR of 43% in 39 patients, and Stein et al. reported 45% CR in 64 patients treated with a similar regimen.

In phase III trials, Rambaldi et al., reported 36% CR in 119 patients and, Kantarjian et al. reported CR of 43.9% in 271 patients treated with blinatumomab at a dose of 9 µg/day during week 1 and 28 µg/d onwards for up to 3 and 6 cycles, respectively.

In all the trials, blinatumomab was given via 4-week continuous IV infusion followed by 2-week treatment-free interval per cycle. The most common grade 3 and 4 adverse effects are listed in the table 1.

Conclusion: Blinatumomab is showing promising results in RR B-ALL with a good side effect profile. However, the final results of these trials are awaited.

Figure 1 Figure 1.

Disclosures

Anwer: Janssen pharmaceutical: Honoraria, Research Funding; Allogene Therapeutics: Research Funding; BMS / Celgene: Honoraria, Research Funding; GlaxoSmithKline: Research Funding.
Limited Role of Antithrombin Deficiency in Hypercoagulopathy Associated with Nephrotic Syndrome
Authors: Abdelghani Eman , Waller Amanda P. , Wolfgang Katelyn J , Kerlin Bryce A.
Published: November 2021

Background: Nephrotic syndrome (NS) is associated with an acquired hypercoagulopathy that drives venous thromboembolic comorbidities. The molecular mechanisms underlying NS-associated hypercoagulopathy are incompletely understood, however previous studies proposed marked urinary loss of antithrombin (AT) as a primary etiology. We have previously demonstrated, in rodent NS models, that there is no correlation between AT antigen and thrombin generation, which we have previously used to characterize the hypercoagulopathy. The objective of this study is to examine the association between AT antigen, AT activity, and hypercoagulopathy along with NS disease severity markers (proteinuria and plasma albumin) in 2 cohorts that include both pediatric and adult NS patients.

Methods: Plasma samples were obtained from both cohorts as follows: (1) Samples were obtained on presentation from 147 adult and pediatric patients participating in the NEPTUNE prospective observational cohort study (2) Samples were obtained on presentation and after 7 weeks of glucocorticoid therapy (GC) in steroid sensitive and steroid resistant pediatric patients participating in the PNRC study. AT antigen and activity assays were performed using ELISA and chromogenic assays, respectively. Thrombin generation assay, plasma albumin, and urine protein-to-creatinine ratio were determined as previously described.

Results: There was no significant relationship between AT antigen or AT activity and thrombin generation parameters. AT antigen and activity also did not correlate with NS severity markers (plasma albumin and proteinuria) in the NEPTUNE cohort (Figure1). In PNRC pediatric cohort, no significant correlation was found between AT antigen and thrombin generation or other disease severity markers. However, AT activity was significantly associated with both plasma albumin and endogenous thrombin potential at presentation (Figure 2) and at follow-up. Interestingly, GC therapy significantly improved AT activity in steroid-sensitive but not steroid-resistant NS patients (Figure 3). A comprehensive literature review and meta-analysis was also performed which revealed that clinically significant AT deficiency (<0.7 IU/mL) is uncommon in both pediatric and adult NS patients.

Conclusion: AT antigen does not correlate with thrombin generation-defined hypercoagulopathy in adult or pediatric NS patients. AT activity does correlate with hypercoagulopathy in pediatric NS patients and improves significantly with successful remission inducing therapy. These data suggest that pediatric NS patients may have a qualitative, but not quantitative, AT deficiency that is responsive to therapy. The mechanism underlying this qualitative deficiency should be characterized in future studies.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Modulating Endothelial Cells with EGFL7 to Diminish aGVHD after Allogeneic Bone Marrow Transplantation in Mice
Authors: Guimond Martin , Moutuou Moutuaata , Goda Chinmayee , Sell Nathalie , Kaylan Sonu , Karunasiri Malith , Kulkarni Rohan SUDHIR , Goulard Marie , Kovovich Sofia , Naumann Eric , Ackaoui Antoire , Bigras Charles-Etienne , Daudelin Francis , Ruddich Alex , Garzon Ramiro , Ranganathan Parvathi , Dorrance Adrienne M.
Published: November 2021

Acute graft versus host (aGVHD) is the second cause of death after allogeneic-hematopoietic stem cell transplant (allo-HSCT) underscoring the need for novel therapies. Based on previous work that endothelial cell dysfunction is present in aGVHD and that epidermal growth factor-like domain 7 (EGFL7) plays a significant role in decreasing inflammation by repressing endothelial cell activation and T cell migration, we hypothesized that increasing EGFL7 levels after allo-HSCT will diminish the severity of aGVHD. Here, we show that treatment with recombinant EGFL7 (rEGFL7) decreases aGVHD severity and improves survival in recipient mice after allogeneic transplantation with respect to controls without affecting graft versus leukemia effect. Histopathology analysis revealed higher amount of leukocyte infiltration in both large intestine and liver of PBS group compared to rEGFL7-treated mice. Furthermore, damage to the gut was reduced in EGFL7 treated mice. Finally, we showed that rEGFL7 treatment results in higher thymocytes, T, B and dendritic cells in recipient mice after allo-HSCT. This study constitutes a proof of concept of the ability of rEGFL7 therapy to reduce GHVD severity and mortality after allo-HSCT.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
LC3-Associated Phagocytosis in Bone Marrow Macrophages Suppresses AML Progression through Mitochondrial DAMP Induced Sting Activation
Authors: Moore Jamie A , Mistry Jayna J , Hellmich Charlotte , Horton Rebecca H , Wojtowicz Edyta , Jibril Aisha , Wileman Tom , Jefferson Matthew , Beraza Naiara , Bowles Kristian M , Rushworth Stuart A
Published: November 2021

The bone marrow (BM) microenvironment regulates acute myeloid leukemia (AML) initiation, proliferation and chemotherapy resistance. Following cancer cell death, a growing body of evidence suggests an important role for uncleared apoptotic debris in regulating the immunologic response to, and growth of, solid tumors. LC3-associated phagocytosis (LAP) maintains tissue homeostasis by regulating immune responses, such as tumor immunity. Here we investigate the role of LAP in macrophage within the BM microenvironment of AML.

We find that depletion of BM macrophages via clodronate liposomes increased AML growth in-vivo. We show that LAP is an important pathway in BM macrophage to process dead and dying cells in the AML microenvironment. We used two syngeneic leukemia models (HOXA9/Meis1 and MN1) to investigate the role of LAP on AML proliferation. AML cells were injected into LAP deficient (Atg16L1 E230-) and wild-type (Atg16L1 E230+) mice. Targeted inhibition of LAP leads to accumulation of apoptotic cells (AC) and apoptotic bodies (AB) in the tumor microenvironment resulting in accelerated leukemia growth and decreased animal survival.

Mechanistically, we show, via cytokine arrays and gene analysis, that the phagocytosis of AML derived AB via LAP in BM macrophage resulted in STING pathway activation in the phagocytic cells. Furthermore, through inhibition of STING using H-151 STING inhibitor, we show that STING activation in vivo supressed leukemia growth. STING activation can lead to a type I IFN response and to recruitment of cytotoxic T-cells. We saw no increase in CD8 + T-cell numbers or activation, however, via ex vivo analysis found that STING activation is required for phagocytic functions in macrophages.

Next, we found that leukemic AB can induce a STING response in BM derived macrophages and that leukemic AB have increased mitochondria content that are processed by macrophages. Moreover, we identify that mitochondrial damage associated molecular patterns (DAMPs) from leukemic AB are processed by BM macrophages via LAP. Additionally, the depletion of mitochondrial DNA (mtDNA) in AML derived AB identified that the mtDNA from leukemic AB is responsible for the induction of STING signalling in BM macrophages.

In summary, we report that LAP in BM macrophage of apoptotic debris in the AML microenvironment suppresses leukemic growth, through mechanisms stimulated by AML apoptotic bodies which contain mtDNA in the BM microenvironment. This process is mediated by the activation of the STING pathway.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Ridd Is Required for the Prevention of Chronic Gvhd By Targeting IRE-1a/Xbp-1s Signaling
Authors: Choi Hee-Jin , Tang Chih-Hang Anthony , Tian Linlu , Wu Yongxia , Sofi Mohammed Hanief , Schutt Steven Douglas , Hu Chih-Chi Andrew , Yu Xue-Zhong
Published: November 2021

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is an effective therapeutic procedure to treat hematological malignancies. However, the benefit of allo-HCT is limited by a major complication, chronic graft-versus-host disease (cGVHD). Since transmembrane and secretory proteins are generated and modified in the endoplasmic reticulum (ER), the ER stress response is of great importance to secretory cells including B cells. By using conditional knock-out (KO) of XBP-1, IRE-1α or both specifically on B cells, we demonstrated that the IRE-1α/XBP-1s pathway, one of the major ER stress response mediators, plays a critical role in B cell pathogenicity on the induction of cGVHD in murine models of allo-HCT. Endoribonuclease activity of IRE-1α not only activates XBP-1s transcription factor by converting unspliced XBP-1 (XBP-1u) mRNA into spliced XBP-1 (XBP-1s) mRNA but also cleaves other ER-associated mRNAs through regulated IRE-1α-dependent decay (RIDD). Besides, it is known that ablation of XBP-1s production leads to unleashed activation of RIDD. Therefore, we hypothesized that RIDD plays an important role in B cells during cGVHD development. In this study, we found that B cells deficient for XBP-1s reduced ability to induce cGVHD, which however was reversed by inactivation of IRE-1α, highlighting the role of RIDD in controlling cGVHD (Fig. A). Activation of RIDD targets IgM mRNA of (Fig. B), a contributor to organ damage and fibrosis in cGVHD, which correlated with dysregulated expression of MHC II and costimulatory molecules such as CD86, CD40, and ICOSL in B cells (Fig. C). Alloreactive T cells need to be primed by APCs to initiate GVHD, and specifically, CD86 and CD40 mediated-costimulation from APCs has been demonstrated to play an essential role in eliciting cGVHD. We demonstrated that alloreactivity of T cells, especially CD4 T cells, can be recovered by suppressing RIDD in XBP-1s-deficient B cells (Fig. D). Since IRE-1α carrying a S729A mutation shows ablated RIDD activity without effect on splicing XBP-1 mRNA, we investigated the contribution of B cells from S729A knock-in mice to confirm the role of RIDD in B cells. We found that B cells from S729A mice increased GVHD severity (Fig. E). S729A B cells showed significant increases in IgM secretion (Fig. F), GC cell differentiation (Fig. G), and the expression levels of MHCII and co-stimulatory factors (Fig. H). In conclusion, these results provide a novel insight on how ER stress response regulates B cell activity after allo-HCT and suggest RIDD is an important mediator for reducing cGVHD pathogenesis.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Outcomes of High Grade B- Cell Lymphoma with MYC and BCL2 and/or BCL6 Rearrangements: An Atlantic Canadian Centre Perspective
Authors: Kelly Erica , Forward Nicholas Allen , Keating Mary-Margaret
Published: November 2021

Background: High Grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (also known as Double-hit lymphoma (DHL)) is an aggressive B cell Non-Hodgkin lymphoma which has recently been recognized by the WHO as a distinct clinical entity from Diffuse Large B cell lymphoma (DLBCL). Compared to patients with DLBCL, patients with DHL have poor outcomes with standard R-CHOP chemoimmunotherapy, and the optimal treatment regimen for this high-risk lymphoma is unknown. Due to clinical and genetic overlap with Burkitt lymphoma (which shares translocations involving MYC), treatment of DHL with more intensive Burkitt lymphoma protocols is a common approach. However, these regimens are associated with significant toxicity, and the superiority of these regimens over standard R-CHOP remains uncertain. At our institution, R-CODOX-M/IVAC has been the preferred regimen for fit younger patients with DHL.

Objective: To review the outcomes of patients with DHL treated with R-CODOX-M/IVAC at our institution.

Methods: In this retrospective single-centre study, patients with DHL receiving R-CODOX-M/IVAC were identified by cross-referencing provincial cancer care data and hospital pharmacy data. All adult patients with a diagnosis of DHL and who were treated with R-CODOX-M/IVAC between January 2009 and January 2019 were included in the analysis. The primary outcomes is complete response (CR) per standard lymphoma response criteria. Secondary outcomes include median progression-free survival (PFS), overall survival at 1 year, and median overall survival. Kaplan-Meier analysis was used for survival analyses.

Results: 14 patients with DHL and meeting the inclusion criteria were identified and included in the analysis. The complete response rate was 35.7%. With a median followup time of 26 months, the median PFS was 31.8 months, and the median OS was 54.3 months. The overall survival at 1 year was 77%.

Conclusions: A previous meta-analysis demonstrated inferior outcomes for patients with DHL receiving RCHOP, with a median PFS of only 12.1 months. The optimal treatment of these patients remains to be defined. Dose-adjusted regimens have been associated with a significant improvement in PFS compared to RCHOP (21.6 months versus 12.1 months). Previous retrospective data suggests R-CODOX-M/IVAC results in more favourable outcomes 2 year PFS of 41% and 2 year OS of 53% (Sun et al., Clinical Lymphoma, Myeloma & Leukemia, 2015). Our data, although limited, that R-CODOX-M/IVAC may be associated with improved outcomes compared to published results with RCHOP. The limitations of this study include its retrospective design, lack of matched controls receiving RCHOP, and its small sample size. Nevertheless, this study adds to the small body of available evidence on this difficult to treat clinical entity.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Iron Chelation Therapy in Low- to Intermediate- Risk Myelodysplastic Syndrome Patients: A Systematic Review and Meta-Analysis
Authors: Yang Sarah , Zhang Ming Chan , Li Allen , Leong Russell , Mbuagbaw Lawrence , Crowther Mark
Published: November 2021

Background

Transfused patients with low- to intermediate-risk myelodysplastic syndromes (MDS) are known to suffer from iron overload. Iron chelation therapy (ICT) may improve outcomes, however, it remains to be thoroughly investigated.

Methods

We conducted a systematic review and meta-analysis to assess the benefit of ICT in patients with MDS. MEDLINE, EMBASE, and Cochrane CENTRAL were searched for studies on ICT for low- to intermediate-risk MDS that reported adjusted hazard ratios (aHR) or overall survival (OS). Two reviewers independently screened titles and abstracts and subsequent full texts for eligibility. Studies were extracted for general demographics, AML progression rate, incidence of cardiac injury, and median OS or aHR. aHR risk ratios were calculated using a random-effects model meta-analysis. Risk of bias was assessed using the Cochrane Risk of Bias tool for randomized controlled trials (RCTs) and Newcastle-Ottawa scale for non-randomized studies.

Results

The initial search yielded 1177 citations, of which we included 11 observational studies (n = total patient number) and one RCT (n = total patient number) for analysis. The heterogeneity was moderate (I 2=57%; P=0.02). There was a significant reduction in risk of mortality in patients with iron overload and low- or intermediate-risk MDS treated with ICT (aHR 0.43; 95% CI 0.32-0.57; P < 0.00001) (figure 1). The median OS among patients receiving ICT and patients receiving no ICT was reported for nine studies. The median OS for patients receiving ICT was consistently longer than the median OS in the non-ICT group across all studies (figure 2).

Conclusion

Iron chelation therapy (ICT) is associated with a lower risk of mortality in low- to intermediate- risk myelodysplastic syndrome patients. Given the limited number of RCTs, more high-quality studies are required before ICT becomes a standard of care for this group of patients

Figure 1 Figure 1.

Disclosures

Crowther: Syneos Health: Honoraria; Precision Biologicals: Honoraria; Pfizer: Speakers Bureau; CSL Behring: Speakers Bureau; Bayer: Speakers Bureau; Hemostasis reference laboratories: Honoraria.
Prothrombotic Phenotype of a Protein S Mutant Lacking Anticoagulant Activity
Authors: Springborn Stephanie , Ghosh Sourav , Schulte Marie L , Rothlin Carla , Branchford Brian R
Published: November 2021

Protein S is a vitamin K-dependent protein that plays an important role in balancing pro- and anti-thrombotic responses to vascular injury. On one hand, it circulates bound to activated protein C, functioning as an anticoagulant complex that downregulates the activities of coagulation factors V and VIII. On the other hand, protein S is a ligand for platelet TYRO3 and MERTK, which, along with the third paralog AXL, constitute the TAM family of receptor tyrosine kinases that functions to potentiate the action of platelet-activating agonists, ultimately resulting in activation of the α IIbβ3 integrin. The relative importance of these two activities in vivo, however, is not known. To better understand the importance of the TYRO3/MERTK-stimulating ability of protein S and thus gain additional insight into its role in platelet activation, we used CRISPR/Cas9 technology to generate mice with a D136A mutant form of protein S that lacks the ability to bind protein C and function as an anticoagulant, while retaining its ability to bind platelet-activating TAM receptors - hereafter termed Pros1 D136A mice. Since homozygosity for this variant was embryonically lethal, all assays were carried out in wild-type animals and their heterozygous littermates comprising a wild-type Pros1 allele and an allele encoding Pros1 D136A. Though there was no significant difference between Pros1 D136A mice and their wild-type littermates in either a collagen/epinephrine-induced pulmonary embolism model or in tail vein bleeding times, platelets from Pros1 D136A mice accumulated at the injury site to a significantly greater degree following in vivo laser injury to the cremaster muscle microvasculature. Taken together with the embryonic phenotype of Pros1 D136A homozygous mice, these data support the notion that protein S can function to augment platelet responsiveness. Further studies on this and other ligands for the TAM family of receptor tyrosine kinases should provide additional insights into their roles in physiological platelet activation.

Disclosures

Branchford: Bio Products Laboratory: Honoraria; Novo Nordisk: Honoraria.
Bendamustine-Bortezomib-Dexamethasone in Heavily Pretreated Multiple Myeloma: Old PLUS New Drugs in NOVEL Agents' Era
Authors: Cerchione Claudio , Catalano Lucio , Nappi Davide , Pareto Anna Emanuele , Pane Fabrizio , Martinelli Giovanni
Published: November 2021

Bendamustine is an old bi-functional alkylating agent which has proved to be effective in relapsed, refractory and in new diagnosed Multiple Myeloma (MM). Thus, aiming to provide further insights in this field, also in novel agents'era, we present here a retrospective, real-life analysis of patients with relapsed/refractory MM (rrMM), who had received salvage therapy with bendamustine in combination with bortezomib and dexamethasone (BVD)

81 patients (44 M/37 F), with rrMM, median age at diagnosis 59.4 years (r. 36-82), median age at start of treatment 63.6 years (r.37-86) treated with several lines of treatments (median 6, r. 2-11), every refractory to all the drugs previously received (also Bortezomib), received BVD (B 90 mg/sqm days 1,2; V 1.3 mg/sqm days 1,4,8,11, D 20 mg days 1,2,4,5,8,9,11,12, Pegfilgrastim day +4) every 28 days, until progression.

All patients had previously received bortezomib-based and IMIDs-based treatments, and 32% (26/81) had also received radiotherapy. 69% (56/81) had undergone single or double autologous and three (2%) allogeneic stem cell transplant. All patients were relapsed and refractory to last therapies received before BVD.

Bendamustine was well tolerated, with grade 3-4 transfusion-dependent anemia in 56% (46/81) of patients, and 43% (35/81) grade 3-4 neutropenia (no ospedalization was required, no septic shocks were observed). No severe extrahematologic toxicity was observed, only grade 1 gastrointestinal side effect (nausea), treated by common antiemetic drugs.

According to IMWG, ORR was 63% (51/81: 7 CR, 18 VGPR, 15 PR, 11 MR) with 11 PD and 19 patients in SD, which can be considered as an impressive result in this subset of rrMM patients. In particular, for 11 patients, BVD was, after having achieved at least a PR, a bridge to second auSCT, and for two patients a bridge to alloSCT. Eight patients have surprisingly achieved a notable PR after failure of novel agents (i.e. Carfilzomib, Daratumumab and Pomalidomide).

Median time to response was 1.3 months (r.1-3), median OS from diagnosis was 67.3 months (r.6-151), median OS from start of Bendamustine was 9.6 months (r.2-36).

The triplet Bendamustine-Bortezomib-Dexamethasone has shown significant efficacy in a particularly severe setting of patients, relapsed and refractory to all available therapeutic resources, and, in particular cases, it could be considered as a bridge to a second autologous or allogenic SCT, also after failure of novel agents.

Disclosures

Martinelli: Abbvie: Consultancy; Jazz Pharmaceuticals: Consultancy; Daichii Sankyo: Consultancy; Celgene /BMS: Consultancy, Speakers Bureau; Roche: Consultancy; Pfizer: Consultancy, Speakers Bureau; Incyte: Consultancy; Stemline Therapeutics: Consultancy; Astellas: Consultancy, Speakers Bureau.
Immune Checkpoint Inhibitors (ICI) Enhance Cancer Associated Thrombosis (CAT) in Murine Model of Colon Carcinoma
Authors: Swaidani Shadi , Jun Shim Young , Rayman Patricia , Pavicic Paul G. , Diaz-Montero C. Marcela , Khorana Alok A , McCrae Keith R.
Published: November 2021

Abstract

Background:

Venous thromboembolism (VTE) is an important immune-related adverse event (irAE) associated with immune checkpoint inhibitors (ICI) cancer therapy. To better understand the pathogenesis of ICI-induced VTE during ICI treatment, we utilized a murine model of cancer associated thrombosis (CAT) to examine the impact of ICI treatment on the development of flow restriction-induced thrombosis.

Methods

A syngeneic colon carcinoma murine model (CT26) on BALB/c background was utilized to evaluate venous thrombosis following inferior vena cava (IVC) ligation. Non-tumor-bearing and tumor-bearing animals were treated with therapeutic doses of ICI: anti-programmed cell death receptor 1 antibody (anti-PD1) and cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA4) or isotype control antibodies. Mice underwent surgical treatment for IVC ligation followed by surgical retrieval of thrombus. Western blot analysis was performed on plasma cleaved high molecular weight kininogen (cHK), and citrullinated histone H3 (CitH3).

Results

Tumor-bearing mice undergoing IVC ligation after anti-PD1 and anti-CTLA4 infusion developed larger thrombi compared to mice treated with isotype control IgG. Thrombus weights in CT26 tumor-bearing mice treated with ICI (19.56±4.41 mg) were significantly increased compared to those in mice treated with isotype control IgG (14.67±2.76 mg) (P=0.043). The weight of thrombi in non-tumor-bearing mice was not affected by ICI treatment (9.25±2.22 mg with control IgG vs 9.33±1.15 mg with ICI). In addition, there was a significant increase in thrombus length in mice treated with ICI (9±1.02 mm vs 7.61±0.99 mm with IgG, P=0.039). Cleaved high molecular weight kininogen (cHK), an indicator of contact activation was increased in plasma pre-IVC occlusion from ICI-treated mice compared to IgG-treated mice (68% vs 38% in HK cleavage). Citrullinated histone H3 (CitH3), a NETosis marker, was elevated in plasma (and in thrombus) post-IVC occlusion from ICI-treated mice compared to that of IgG-treated mice.

Conclusions

ICI treatment of tumor-bearing mice undergoing flow restriction-induced thrombosis resulted in enhanced clot formation. Larger thrombi in ICI treated mice was accompanied with enhanced contact activation and NETosis marker CitH3.

Figure 1 Figure 1.

Disclosures

Khorana: Halozyme: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Anthos: Consultancy, Honoraria. McCrae: Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy; Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria.