bluuebunny/crossref_metadata_2025 huggingface dataset:
Bifunctional Immunoactive siRNAs as an Approach to Personalized AML Therapy
Authors: Meinl Hanna , Zeitlhöfler Marcus , Kocabey Samet , Liedl Tim , Hiddemann Wolfgang , Subklewe Marion , Endres Stefan , Rothenfußer Simon , Lichtenegger Felix S
Published: October 2019

The prognosis of acute myeloid leukemia (AML) is poor due to frequent relapse after initial remission. The development of new approaches to postremission therapy for elimination of minimal residual disease remains a major scientific and clinical challenge. We strive to combine two different innovative therapeutic concepts to develop a new specific and personalized treatment for AML. siRNAs are used to knock down either a gene that drives leukemogenesis due to genetic alterations in specific cases of AML (e.g., FLT3, NPM1) or a gene that is essential for the survival of the leukemic cells (e.g., BRD4, MCL1, PLK1). By adding a triphosphate modification to the 5’ end, the siRNA molecules additionally become ligands for the cytosolic pattern recognition receptor RIG-I (retinoic acid inducible gene I). Its activation mimics viral infection and leads to the production of inflammatory cytokines and induction of apoptosis in the target cell. We expect these bifunctional molecules to result in a decrease of viable AML cells and in the induction of an immune response similar to an active immunization.

This concept was successfully tested in vitro for several target genes in AML cell lines. We could demonstrate that the specific gene knockdown leads to inhibited proliferation, increased apoptosis and higher sensitivity to chemotherapeutic agents. Activation of RIG-I by triphosphate-modified RNA additionally stimulated an inflammatory response by the leukemic cells and increased the apoptosis rate.

A major hurdle for all siRNA-based anti-cancer strategies is the specific delivery of the RNA into tumor cells. In vivo liposomal transfection of siRNA molecules has been used in various tumor models, but generally results in ineffective and unspecific delivery. We are testing DNA-based nanoparticles coupled with molecules that target receptors specific for or overexpressed on AML cells. By coupling bifunctional siRNA molecules to these nanoparticles, they should be efficiently and selectively transported into the cytosol of AML cells.

Proof-of-concept in vivo studies in AML mouse models are in preparation. The long-term goal of this project is the development of a set of bifunctional siRNA molecules for the individualized treatment of AML.

Disclosures:

No relevant conflicts of interest to declare.
Locally Directed Therapy In Limited Stage Mantle Cell Lymphoma
Authors: Dalia Samir , Chavez Julio C , Bello Celeste M. , Chervenick Paul A. , Sokol Lubomir , Tao Jianguo , Sotomayor Eduardo M. , Shah Bijal D.
Published: October 2019

Introduction

Mantle-cell lymphoma (MCL) is a rare B-cell malignancy that usually presents in stage III-IV disease (classical) with poor outcomes even with aggressive therapy. Rarely, patients present with localized disease and the role of localized or systemic therapy remains a clinical choice.

Purpose

To assess outcomes in our patients with early stage MCL with a focus on treatment approaches.

Methods

Patients with early stage (Ann Arbor Stage I or II) between 1992 and 2012 were identified through our MCL database. Demographic , treatment, and outcomes data was collected for each patient. Cox regression was used to identify risk factors for progression and survival. Kaplan-Meier (KM) method was used to estimate median time to progression (TTP) and overall survival (OS), and comparisons were estimated using the log rank test. A p-value of <0.05 was considered significant.

Results

From our database of 300 patients with MCL, 34 were identified to have stage I or II disease (Table 1). 7 received local therapy, consisting of either radiation or resection. 20 received systemic therapy, including 1 who received only rituximab. 7 received a combination of local therapy and systemic therapy, including 2 who received rituximab with radiation.

With a median followup of 73.7 months, TTP was 22.8 months and OS was 83.8 months. MIPI could be determined for 56% of the cohort, however, was neither prognostic for TTP or OS. WBC was available for 74% of the cohort, and was prognostic for OS only [HR 1.46 (95%CI 1.02-2.08, p-value=0.04)]. None of the remaining MIPI covariates were prognostic. Ki67 was reported for only 8 patients limiting analysis as a continuous variable. Blastoid/Pleomorphic histology, alternatively, was prognostic for OS [HR 24.5 (95%CI 1.22-25.72, p-value=0.03)], but not TTP. Locally directed therapy was not associated with a decrease in OS or TTP, with some patients showing no evidence of relapse with extended followup (figure 1).

Conclusions

In our series patients with early stage MCL demonstrated a longer TTP and OS than those with extensive stage disease. The MIPI failed to stratify patients, likely related to small sample size. Aggressive histology may confer a worse prognosis, though, it remains unclear whether this can be abrogated by more intensive management, as all with blastoid/pleomorphic histology received some form of systemic therapy. Notably, as described by others, locally directed therapy did not compromise outcomes, with some showing extended TTP and OS.

Disclosures:

No relevant conflicts of interest to declare.
Cytogenetic Aberrations Characterize Mantle Cell Lymphoma With Bone Marrow Involvement and Provide The Most Important Prognostic Information
Authors: Yi Shuhua , Li Zengjun , An Gang , Li Chengwen , Zou Dehui , Qi Junyuan , Peihong Zhang , Qiu Lugui
Published: October 2019

Introduction

The clinical characteristics and cytogenetic aberrations of mantle cell lymphoma (MCL) is well described in the West, but not in China, and also not mentioned in patients with bone marrow involvement (BMI). The aim of this study was to characterize the clinical and cytogenetic feature of Chinese patients with MCL.

Methods

During the period July 2003 through November 2012, 50 MCL patients with BMI were diagnosed at our Hospital. Cytogenetic aberrations were detected by FISH on bone marrow cells, using a panel probes including Rb1, TP53, ATM and c-MYC.

Results

The median age of the 50 patients was 55.5 years at diagnosis, with 38 male patients (76%). Eighteen patients had B symptom, 36 patients with splenomegaly, while 4 patients had hepatomegaly. Twenty-four of the forty-five patients had elevated serum LDH. According to MIPI system, 13 patients (27.7%) were classified into medium risk group, while 34 patients (72.3%) in the high risk prognosis group. In aspect of the cytogenetic aberrations, eleven of forty-two patients (26.2%) had Rb-1 deletion, 7/39 patients (17.9%) with ATM deletion, 16/42 patients (38.1%) with TP53 deletion, while 15/37 patients (40.5%) had c-MYC abnormality, including amplification and translocation.

With a median follow-up of 21.5 months, the median estimated progression-free survival (PFS) was 15 months (95% CI 8.6-21.4), and the median estimated overall survival (OS) was 27 months (95% CI 17.5-36.5). Using the Kaplan-Meier method, the MIPI high risk group, deletion of Rb-1, ATM, TP53 and c-MYC abnormality were the adverse prognostic factors for PFS, while deletion of Rb-1, ATM, TP53 and c-MYC abnormality predicted the worse OS in the univariate analysis. All other clinical characteristics did not significantly influence the PFS and OS(p>0.05). TP53 deletion and c-MYC abnormality were the independent prognostic factors for both of PFS and OS in the multivariate analysis.

Conclusions

The outcome of MCL with BMI was poor. TP53 deletion and c-MYC abnormality were common in MCL with BMI and represented the worst factors for survival.

Disclosures:

No relevant conflicts of interest to declare.
Neutropenic and Non-Neutropenic Fever In Diffuse Large B-Cell Lymphoma Patients Treated With Rituximab-CHOP Chemotherapy
Authors: Park Silvia , Kim Won Seog , Kim Seok Jin , Hong Jung Yong
Published: October 2019

Introduction

Rituximab-CHOP (R-CHOP) is the standard chemotherapy in DLBCL patients. Febrile neutropenia (FN) is the major toxicity of this regimen, but we also observe non-neutropenic fever (NNF) during the recovery period after R-CHOP.

Method

Between September 20089 and January 2013, a total of 1142 lymphoma patients were registered in the prospective cohort. Among these, 397 DLBCL patients who had completed R-CHOP based chemo cycles as first line treatment were analyzed.

Results

Of the 397 patients, 160 (40.3%) and 39 (9.8%) experienced FN and NNF, and 24 (6.0%) exhibited both. Approximately 50% of the initial FN occurred after the 1st cycle whereas NNF was mainly observed around the 4th cycle (28.2%). Including multiple FN occurrences in 65 patients (40.9%), there were 276 FN events in 160 patients. Besides unexplained fever (47.8%), catheter related infection was predominated (14.2%) and Coagulase negative staphylococcus was the most common microorganism causing bacteremia (27.7%). Thirty nine patients with NNF exhibited a total of 42 episodes and atypical pneumonia comprised the highest proportion (54.8%). Of the identified organisms causing atypical pneumonia, PJP was most common (n=4), adenovirus (n=3), RSV (n=2), and CMV (n=1) came next.

Conclusion

FN and NNF during R-CHOP treatment showed different clinical manifestation regarding their timing of initial episode and causes.

Disclosures:

No relevant conflicts of interest to declare.
Interferon-Alpha With Or Without Rituximab Achieves a High Response Rate and Durable Responses In Relapsed FL: 17 years’ Experience In a Single Centre
Authors: Radesi-Sarghi Sinziana , Arbion Flavie , Dartigeas Caroline , Delain Martine , Benboubker Lotfi , Herault Olivier , Colombat Philippe , Gyan Emmanuel
Published: October 2019

Background

Maintenance interferon-alpha (IFNa) immunotherapy after induction chemotherapy prolongs progression-free survival (PFS) in untreated follicular lymphoma (FL). Little information is available about IFNa use in relapsed FL.

Objectives

To evaluate the benefit of IFNa as treatment for low-burden FL relapse.

Methods

In this single-centre retrospective study, we analysed all patients with molecular or clinical relapsed low- burden FL who received 3MIU IFNa three times a week alone (16 times) or in combination with 4-weekly rituximab (R) (11 times), without chemotherapy, according to the institution’s policy. All responses were evaluated according to the IWG 1999 criteria, except for molecular relapses which are described separately.

Results

20 patients (13 men, 7 women), treated for a total of 27 times with IFNa were identified, with a median follow-up of 85 months. At diagnosis, 17 patients had advanced III-IV stage and 5 of them a high tumour burden, according to GELA criteria. 5 patients were treated for molecular IGH-BCL2 relapses. The FLIPI score was intermediate or high risk in 37% of patients. The median age of patients at the first IFNa +/- R treatment was 51 years (range: 30-65). Patients had previously received a median of three lines of treatment (range: 1-6) and 59% had received anthracycline-based chemotherapy. In the 22 situations with measurable disease, the overall response rate (OR) was 70%, with 59% complete responses (CR). For patients with only detectable molecular disease (n=5), IFNa was used alone for a median duration of 15 months (range: 12- 21 months), MRD becoming negative in 4 patients. The only factor significantly correlated with maximum response was low FLIPI score calculated at study entry (p=0.02). Median PFS was 56.9 months, (95% CI: 0.8-113). The R-IFNa and IFNa subgroup outcomes did not statistically differ in response or PFS. The FLIPI score calculated at initiation of IFNa treatment was predictive of time to relapse (P=0.014). The overall survival at 5 years was 95 % (95%CI: 91-99): 100%  in the  IFNa group and 91% (95%CI: 82-100) in the R-IFNa group. Reported toxicity was low.

Conclusion

Interferon alpha with or without rituximab achieve high response rates in relapsed low burden follicular lymphoma. These results compare favorably with previous reports of the efficacy of R alone, and of R with IFNa in relapse. Further research is required to explore the role of IFNa in the management of FL.

Disclosures:

Gyan: Roche: Research Funding.
Resistance To Vorinostat In Hematological Malignancies May Involve Cytoprotective UPR and Correlates With Increased Sensitivity To Bortezomib-Induced Cell Death
Authors: Dupéré-Richer Daphné , Kinal Mena , Pettersson Filippa , Miller Wilson H.
Published: October 2019

Histone deacetylase inhibitors (HDACi) have shown promising activity against hematological malignancies in clinical trials and have led to the approval of vorinostat for the treatment of cutaneous T-cell lymphoma. However, as with many cancer therapies, de novoresistance is common and acquired resistance inevitably follows sensitivity. This issue is particularly difficult to resolve in HDACi therapy, as the mechanism of action is still unclear and may involve several components.

Our objective was to understand the molecular mechanisms underlying resistance to HDACi in order to design better combination strategies and to identify predictive biomarkers for response to HDACi therapy.

To gain insight into HDACi resistance, we developed vorinostat-resistant clones using a dose escalation protocol in the monocytic-like, histiocytic lymphoma cell line U937 and the diffuse large B-cell lymphoma SUDHL6. Indeed, resistant cells grow in 4 µM vorinostat without induction of cell death. Using a variety of targeted drugs, we evaluated the lethal dose (LD)50in the resistant cells versus their parental counterpart in order to screen for potential pathways involved in resistance to vorinostat.

We found that the vorinostat-resistant cells are cross-resistant to other HDACi but not all. Interestingly, the resistant cells exhibit increased sensitivity toward bortezomib, an inhibitor of the proteasome and to chloroquine, an inhibitor of autophagy. Both these drugs target protein processing, suggesting its importance in driving resistance to HDACi. We found that, in addition to elevated autophagy, vorinostat-resistant cells exhibit marks of ER stress, such as dilated ER when visualized by electron microscopy. Moreover, resistant cells have an increased protein synthesis rate and an accumulation of ubiquitinated proteins compared to their parental counterparts. Consistent with this, we observe activation of the UPR in the resistant cells.

We hypothesized that activation of UPR could be a mechanism of vorinostat resistance, because the UPR induces the upregulation of pro-survival genes and may induce autophagy. Understanding vorinostat resistance holds clinical relevance in terms of improving HDACi therapy and being able to identify subsets of patients who would benefit most from combination therapy, such as vorinostat with bortezomib.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation Of Large Granular Lymphocytes and Endothelial-Cell-Related Biomarkers In Patients With Chronic Myeloblastic Leukemia: Comparison Among 3 TKIs
Authors: Ito Tomoki , Uoshima Nobuhiko , Maeda Yasuhiro , Hotta Masaaki , Yoshomura Hideaki , Fujita Shinya , Satake Atsushi , Ishii Kazuyoshi , Urase Fumiaki , Hayashi Kunio , Nomura Shosaku
Published: October 2019

Background

Tyrosine kinase inhibitors (TKIs) currently represent the main therapy for chronic myeloblastic leukemia (CML). Although they are therapeutically effective, some TKI-related events such as pleural effusion and elevation of large granular lymphocytes (LGL) have been reported. In addition, these events itself may affect the therapeutic response to TKIs. In the present study, we measured the levels of some cytokines, chemokines, soluble factors and coagulation markers in patients with CML, and investigated the relationship between these markers and TKI-related events.

Methods

The subjects were 48 patients with CML. Blood samples were collected and levels of cytokines (interleukin (IL)-6, tumor necrosis factor-α, high-mobility group box 1), chemokines (monocyte chemotactic protein (MCP)-1, RANTES), soluble molecules (soluble vascular cell adhesion molecule-1, soluble E-selectin, angiopoietin-2, vascular endothelial growth factor, and soluble thrombomodulin) and coagulation markers (plasminogen activator inhibitor (PAI)-1, platelet-derived microparticles (PDMP)) were measured by ELISA.

Results

Levels of all markers except IL-6 and MCP-1 were significantly increased in patients with de novo CML. Level of LGL/lymphocyte was significantly higher in dasatinib-treated CML patients compared to other TKI-treated patients. LGL/lymphocyte and many biomarkers exhibit the time-dependent increase after dasatinib treatment. In addition, LGL/lymphocyte was already increasing in patients with pleural effusion before dasatinib treatment.

Conclusion

These results suggest that LGL and endothelial-cell-related factors play an important role in the pathophysiology of CML and its response to TKIs.

Disclosures:

No relevant conflicts of interest to declare.
Identification Of Mutations In Bcr-Abl Gene In Newly Diagnosed Indian Patients With Chronic Myelogenous Leukemia (CML)
Authors: Shyam Aggarwal , Verma Binuja , Vyas Bijal , Jauhri Mayank
Published: October 2019

Background & Objectives

Mutations in Bcr-Abl gene is responsible for resistance after Imatinib treatment in 30-60% of the cases. Currently the data does not support testing for these mutations in Imatinib naïve cases. However there is a data emerging that suggest that base line mutations in the Bcr-Abl gene do exist even before Imatinib is started, especially in patients with high SOKAL score. In India the failure rate of Imatinib is higher in comparison to western literature because patients are detected for CML chronic phase (CP) at a later stage. This study was designed to identify mutations in Abl-KD in Indian CML patients with no previous exposure to tyrosine kinase inhibitor.

Methods

This study targeted a cohort of CML-CP patients (n=47) without prior TKI exposure. KD mutations were analyzed by direct sequencing.

Results

A total of 47 samples were evaluated, out of which 2 samples were identified to have the same variation. This heterozygous missense variation (A206D) in the Abl1 gene was located on SH2 domain of Exon 4. The change was from C>A in codon 206, which caused a protein change from Alanine to Aspartic acid (p.A206D). By using the mutation taster software, this change was predicted to be disease causing with a high probability of .99 (∞1). One of the patients died within one year of diagnosis in spite of imatinib treatment.

Interpretation & conclusion

A novel mutation with putative pathogenicity was identified in the TKI-naïve Indian CML patients, supporting the concept that naturally occurring KD mutations are present in leukemic cell prior to drug exposure.

Disclosures:

No relevant conflicts of interest to declare.
Real World Analysis Of Duration Of TKI Treatment Among Patients With Chronic Myeloid Leukemia
Authors: Henk Henry J , Woloj Mabel , Shapiro Mark , Whiteley Jennifer
Published: October 2019

Background

Chronic myeloid leukemia (CML) accounts for 10-15% of all adult leukemias in the US. CML treatment has improved in the past few years with the introduction of imatinib in 2001, which has improved overall survival in patients with CML. Despite its success, there remains a subset of individuals who are either imatinib-resistant or imatinib-intolerant. With the approval of 2nd generation TKIs (dasatinib, nilotinib) beginning in 2006, approximately 40–50% of imatinib-resistant and imatinib-intolerant patients achieve complete cytogenetic response (CCyR) when switching TKI. Few studies have estimated duration of TKI use since the approval of 2nd generation TKIs. The objective of this study is to explore current treatment patterns of TKIs by line of therapy in the US.

Methods

U.S. health plan claims data from 01 January 2007 through 31 December 2011 were used to examine duration of TKI treatment by line of therapy for patients with CML aged 18 and older. Patients were required to have a prescription fill for a TKI and a CML diagnosis (ICD-9-CM code 205.1). Duration of TKI use was determined based on a gap in TKI coverage of 180 or more consecutive days following TKI initiation or switch to another TKI within the 180 day window. To account for censoring due to disenrollment for the health plan or end of the study period, median duration of 1st and 2nd line treatment is estimated via the Kaplan-Meier estimator.

Results

We identified 695 who started TKI treatment and had diagnosis for CML from 2007 through 2011. More than half (58%) were male; mean age was 55 years (SD 14 years). The most common first line TKI was imatinib (82%) with dasatinib and nilotinib use equally distributed (9%). Of patients starting a 2nd line TKI, dasatinib represented 59% and nilotinib 37% (Table).

Since 2007, the median duration of 1st line TKI was 39 months and 2nd line TKI was 22 months. Median duration of treatment for 1st line (p=0.4342) and 2nd line treatment (p=0.1762) were not significantly different by TKI (Figure). Among those who initiated 2nd line TKI, the majority were switched from imatinib to dasatinib or nilotinib (86%). Mean time to switch was 12 months and did not differ when switching from imatinib to dasatinib or nilotinib (p=0.7233).

Conclusions

Duration of TKI therapy is an informative measure as it is a proxy measure of treatment effectiveness as well as patient adherence, which is important to maintain optimal therapeutic outcomes. Among CML patients, we found median duration of treatment in both the 1st and 2nd line to be similar by TKI.

Disclosures:

Henk: OptumInsight: Employment. Woloj:Pfizer Inc.: Employment. Shapiro:Pfizer Inc.: Employment. Whiteley:Pfizer Inc.: Employment.
Erosion Of The Chronic Myeloid Leukemia Stem Cell Pool By PPARγ Agonists
Authors: Prost Stephane , Relouzat Francis , Spenchian Marc , Massonnet Gérald , Beressi Jean-Paul , Verhoeyen Els , Maneglier Benjamin , Castaigne Sylvie , Chomienne Christine , Chretien Stany , Rousselot Philippe , Leboulch Philippe
Published: October 2019

Whether cancer is maintained by a small number of stem cells or is composed of proliferating cells with approximate phenotypic equivalency is a central question in cancer biology. In the stem cell hypothesis, relapse after treatment may occur by failure to eradicate cancer stem cells. The chronic myeloid leukemia (CML) is quintessential to this hypothesis. CML is a myeloproliferative disorder that results from dysregulated tyrosine kinase activity of the fusion oncoprotein BCR-ABL. During the chronic phase, this sole genetic abnormality (chromosomal translocation Ph1+: t(9;22)(q34;q11)) at the stem cell level causes increased proliferation of myeloid cells without loss of their capacity to differentiate. Without treatment, most patients progress to the blast phase when additional oncogenic mutations result in a fatal acute leukemia made of proliferating immature cells. Imatinib mesylate and other tyrosine kinase inhibitors (TKIs) that target the kinase activity of BCR-ABL have improved patient survival dramatically. However, fewer than 10% of patients reach the stage of complete molecular response (CMR), defined when BCR-ABL transcripts become undetectable in blood cells3. Failure to reach CMR results from the inability of TKIs to eradicate quiescent CML leukemia stem cells (LCSs). Here we show that the residual CML LSC pool can be gradually purged by the glitazones, antidiabetic drugs that are agonists of peroxisome proliferator-activated receptor gamma (PPARg). We found that activation of PPARg by the glitazones decreases expression of STAT5 and its downstream targets HIF2α and CITED2, which are key guardians of CML LSC's quiescence and stemness. When pioglitazone was temporarily given to three CML patients in chronic residual disease in spite of continuous treatment with imatinib, all of them achieved sustained CMR, up to 4.7 years after withdrawal of pioglitazone. This suggests that clinically relevant cancer eradication may become a generally attainable goal by combination therapy that erodes the cancer stem cell pool.

Disclosures:

No relevant conflicts of interest to declare.
A Murine Model Of Hemorrhagic Shock/Resuscitation Demonstrates Transfusion Related Acute Lung Injury (TRALI) Induced By Stored Platelet Transfusions
Authors: McKenzie Christopher G.J. , Kim Michael , Safavian Dana , Rotstein Ori D , Semple John W.
Published: October 2019

Transfusion related acute lung injury (TRALI) is one of the leading causes of transfusion fatalities. There have been several animal models of TRALI developed including ex vivo lung models demonstrating the importance of human anti-neutrophil antibodies in TRALI and in vivo transfusion models showing how biological response modifiers can induce recipient lung damage. In addition, an in vivo antibody-mediated TRALI model has also been established and has shown close similarities with human TRALI. Most of the animal models have a two-hit paradigm where the first hit is delivered in the form of a noxious substance such as lipopolysaccharide and the second hit is a transfusion of either a blood product or an antibody. Here we describe a novel clinically relevant two-hit model where the first hit is hemorrhagic shock followed by a second hit in the form of a platelet transfusion. Severe combined immunodeficient (SCID) mice were anesthetized and had half of their total blood volume was removed via a carotid cannula to instigate hypovolemic shock. After one hour of shock, the mice were reperfused with either Ringer’s lactate solution or fresh or three day aged platelets. 34-1-2s, a monoclonal anti-mouse MHC class I antibody known to induce TRALI was used as a positive reperfusion control. Two hours following reperfusion, the mice were sacrificed and blood was obtained via cardiac puncture in order to measure serum levels of the neutrophil chemoattractant macrophage inflammatory protein-2 (MIP-2). Lungs were removed to determine the percentage of accumulated neutrophils and to measure edema. Compared with control mice reperfused with Ringer’s lactate solution or mice administered fresh platelets, serum MIP-2 levels in mice administered with three day aged platelets were significantly elevated and were comparable to those observed in mice infused with 34-1-2s. Pulmonary neutrophil accumulation in the mice transfused with 3 day stored platelets was also elevated to levels observed in 34-1-2s treated mice. Pulmonary edema as measured by lung Wet/Dry ratios was elevated in mice receiving the 3 day aged platelets. In vivo administration of a MIP-2 antagonist (SB225002, 4mg/kg) intraperitoneally immediately before reperfusion significantly reduced all TRALI symptoms. These results show that an animal model of hypovolemic shock exhibits TRALI upon reperfusion with stored platelets and is dependent on MIP-2 production. This clinically-relevant model will be valuable in studying potential therapeutic interventions to prevent TRALI following transfusions,

Disclosures:

No relevant conflicts of interest to declare.
Calcineurin Inhibitor-Free GVHD Prophylaxis with Post-Transplant Cyclophosphamide and Brief-Course Sirolimus Results in Low Rates of Non-Relapse Mortality and Chronic GVHD Following Matched Related and Unrelated Donor Peripheral Blood Stem Cell Transplantation (PBSCT)
Authors: Sanacore Melissa , Bashey Asad , Sizemore Connie A. , Holland H. Kent , Morris Lawrence E. , Brown Stacey , Zhang Xu , Solomon Scott R.
Published: October 2019

Calcineurin inhibitors (CNIs) form the foundation of current GVHD prophylaxis regimens. Although advances in immunosuppressive regimens have had a significant impact on the incidence and severity of acute GVHD, it is noteworthy that CNIs have had little impact on chronic GVHD. We hypothesized that a CNI-free regimen consisting of post-transplant cyclophosphamide (Cy) and brief-course sirolimus would decrease the risk of chronic GVHD and non-relapse mortality and improve outcomes following reduced intensity allogeneic PBSCT. Twenty-seven patients with high risk hematologic malignancies were enrolled in the study: median age 61 years (25-73). All patients had a 10/10 locus matched donor; MRD=18, MUD=9. Conditioning consisted of fludarabine 30mg/m2 on days -9 to -6, IV busulfan 130 mg/m2 on days -5 to -4, and Cy 14.5 mg/kg on days -3 and -2 followed by unmanipulated PBSCT. Post-grafting immunosuppression consisted of Cy 50 mg/kg/day on days 3 and 4 and sirolimus starting day +5 and completing day+90 in the absence of GVHD. Donor engraftment occurred in all patients with a median time to neutrophil and platelet recovery of 15 and 30 days, respectively. The median day +90 donor T cell and myeloid chimerism was 94% (40-100%) and 100% (11-100%) respectively. Three patients received donor lymphocyte infusions for incomplete donor T cell chimerism. The cumulative incidence of grade II-IV acute GVHD, grade III-IV acute GVHD, all chronic GVHD, and severe chronic GVHD was 41%, 15%, and 32%, and 12% respectively. Non-relapse mortality (NRM) and relapse incidence at 2 years was 4% and 17% respectively. With a median follow-up of 18 months, the estimated 2 year overall and disease-free survival was 71% and 80% respectively for the whole cohort, while it was 87% and 89%, respectively in the subgroup of 18 patients receiving MRD transplants. Good immune reconstitution was evidenced by low cytomegalovirus reactivation rates, occurring in only 4 of 19 at-risk patients (21%). Transplant-related toxicity included BK virus-associated cystitis in 33% of patients and a non-fatal hepatotoxicity syndrome in three patients consisting of transaminase elevation and ascites, with resolution following discontinuation of sirolimus. CNI-free GVHD prophylaxis with post-transplant Cy and brief-course sirolimus achieves consistent donor engraftment, low rates of GVHD and NRM, and excellent outcomes in recipients of HLA-identical donor allogeneic PBSCT.

Disclosures:

Off Label Use:fludarabine, cyclophosphamide, and sirolimus are not FDA-labeled for stem cell transplantation.
The Use Of Two Photon Microscopy To Image Vaso-Occulsion In Pulmonary Microvessels Of Living Mice With Sickle Cell Disease
Authors: Ralainirina Natacha , Sonderegger Ferron Lynn , Pei Hong , Chodaczek Grzegorz , Linden Joel
Published: October 2019

Although sickle cell anemia is initiated by red cell pathology, it is accompanied by an inflammatory immune response involving platelets and white blood cells that contribute to vaso-occlusvie episodes including painful vaso-occlusive crises (pVOC) and acute chest syndrome (ACS). In order to better understand the cellular and molecular bases of vaso-occlusion we are in the process of developing procedures to image microvessels in the lung, liver and spleen of living mice by 2-photon microscopy, a procedure that is based on excitation of a fluorophore by two photons simultaneously. The two-photon technique utilizes infrared light that efficiently penetrates tissues up to 200 microns with low phototoxicity allowing time-lapse imaging. Two-photon intravital microscopy can be used to study the behavior of intravascular cells during vaso-occlusive events.

Mice are prepared for lung intravital microscopy by the intraperitoneal injection of a mixture of ketamine and xylazine. Additional anesthesia is added during experimentation. The trachea is opened and the mouse intubated. The chest is opened to allow access to the left lobe of the lung through a window that is a few millimeters in diameter. PBS is applied to keep the lung moist. A custom built suction device is placed on the lung and covered with a cover glass at the same time pressure is exerted to seal the organ and the glass cover together. Throughout the procedure, the mouse is held at a temperature of 37°C. Once surgery is completed, a mixture of antibodies coupled to fluorophores is given by retro-orbital injection. In order to minimize photobleaching we used antibodies conjugated to Alexa Fluor 488, Alexa Fluor 555 or Alexa Fluor 647.

We are able to visualize and quantify interactions between red blood cells, white blood cells, and endothelial cells as well as the expression of adhesion molecules on endothelial cells in real time. During pVOC triggered by hypoxia, cell adhesion of neutrophils, lymphocytes and monocytes to the endothelium is observed that is associated with an increase in endothelial expression of ICAM-1 and V-CAM. We label endothelial cells with anti-CD31, lymphocytes with anti-CD45, monocytes with anti-Ly6C and neutrophils with anti-Ly6G. Platelets are labeled with anti-CD41 or anti-CD62P, NK cells with anti-NKp46, and macrophages with anti-F4/80 and anti-CD1d. We are able to quantify cell shape, rolling, adhesion and movement.

Our preliminary results demonstrate that it is possible in real time to image the sequence of events occurring during pulmonary vasoocclusion in sickle cell disease. In conclusion, intravital 2-photon microscopy holds great potential for enabling us to better understand inflammatory responses within the blood vessels of living SCD mice.

Disclosures:

No relevant conflicts of interest to declare.
Systems Biology and Epigenetic Mechanisms in Erythropoiesis
Authors: Socolovsky Merav
Published: October 2019

Irreversible rapid cellular decisions are often controlled by network motifs known as bistable switches. We identified a cell-cycle regulated bistable switch that controls activation of the erythroid transcriptional program during early S phase of the last generation of erythroid colony-forming-unit progenitors (CFUe). This switch drives a rapid, multi-layered commitment event that activates GATA-1 transcription, renders the cells dependent on erythropoietin, and brings about chromatin reconfiguration at erythroid gene loci. In addition, it triggers an unusual process of genome-wide DNA demethylation, the first known example of such a process in somatic cell development. Approximately 25 to 30 percent of all methylation marks are lost from essentially all genomic elements during erythroid terminal differentiation. The bistable switch activating erythroid transcription consists of two linked double-negative feedback interactions of the erythroid transcriptional repressor PU.1, which antagonizes both S phase progression, and the erythroid master transcriptional regulator GATA-1. During operation of the switch, a rapid S phase-dependent decline in PU.1 activates GATA-1 transcription. The dependence of this switch on S phase progression coincides with a dramatic change in the nature of S phase itself, which becomes shorter and 50 percent faster. The accelerated intra-S phase DNA synthesis rate is essential for the loss of genome-wide DNA methylation, which in turn is required for the rapid induction of erythroid genes.

Disclosures:

No relevant conflicts of interest to declare.
Novel Targeting Strategies for Leukemia-Initiating Cells in Myeloid Neoplasms
Authors: Sauvageau Guy , Pabst Caroline , Lemieux Sebastien , Hébert Josée , Krosl Jana
Published: October 2019

In this session we will present results from our chemo-genomic screens designed to categorize more accurately human acute myeloid leukemia (AML) subsets based on both their genetic make-up (RNA and exon Next Generation Sequencing) and their response to clinically-approved chemicals. The success of this project relies on our recent development of newly defined culture conditions that support the majority of leukemia stem cells in short-term cultures (Pabst et. al., submitted) and availability of a large collection of clinically annotated specimens (J.H., BCLQ). Integration of chemical and genetic data is possible through our novel bioinformatics tools developed for this purpose (Lemieux et. al., submitted). In addition to the generation of more accurate prognostic tools, these studies set the stage for drug repositioning and personalized medicine for human AML.

Disclosures:

No relevant conflicts of interest to declare.
ceRNAs and ceRNA Networks in Normal and Malignant Hematopoiesis and Their Therapeutic Implications
Authors: Pandolfi Pier Paolo
Published: October 2019

We will discuss exciting new data regarding the role of pseudogenes, long intergenic non-coding RNAs (lincRNAs) and miRNAs in the pathogenesis of leukemia and lymphoma, as also studied in vivo in the mouse, and their therapeutic implications. We will also focus on competing endogenous RNAs (ceRNAs), circular(circ)-ceRNAs and pseudo-ceRNAs and give important attention to how understanding their cross-talk and the ceRNA language will facilitate efforts to deconvolute ceRNA networks and their role in the pathogenesis of hematopoietic malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Platelets, Tumor Cell Invasiveness, and Metastasis
Authors: Hynes Richard O. , Begum Shahinoor , Labelle Myriam
Published: October 2019

Platelets have long been known to promote metastasis, and multiple mechanisms have been proposed to explain this phenomenon, including adhesion, coagulation, and protection against natural killer (NK) cells or turbulence. One mechanism that has been little explored is the possibility that platelets might secrete growth factors or provide other stimuli that could enhance the malignant properties of tumor cells. We have shown that pretreatment of carcinoma cells with platelets induces an EMT-like transformation in their properties in vitro and renders them much more metastatic after introduction into mice. TGF-β, produced by platelets and released on their activation is essential for both the in vitro and the in vivo effects. However, TGF-β alone is insufficient; platelet-tumor cell contact is also required and this contact activates NFkB signaling, which synergizes with the TGF-β signaling. Both signals are required for the enhancement of metastasis. In addition to enhancing migration and invasion in vitro, platelets enhance extravasation in vivo. Earlier work has shown that both P-selectin (expressed on platelets) and L-selectin (expressed on leukocytes) are essential for efficient metastasis, and aggregates of tumor cells, platelets, and leukocytes can be observed at sites of tumor cell arrest and extravasation. It has also been demonstrated by others that leukocytes can enhance extravasation and metastatic seeding. Therefore, we have been interested in the question of the relative roles of platelets and leukocytes in these processes. Which cell types are recruited at the sites of metastatic seeding? Does one cell type depend on another? Which cell types enhance metastasis? What roles do the platelets play in recruiting the other cell types? The involvement of platelets in enhancing metastasis also raises questions about the effects of platelets on circulating tumor cells (CTCs). Could platelets enhance the metastatic capacity of CTCs? Could it be the case that only those CTCs that are associated with platelets and/or leukocytes are functionally involved in seeding metastases? Such aggregates are not scored in most current assays for CTCs and will require new investigative approaches. Platelet participation in metastasis also raises the possibility of therapeutic interventions targeting platelet-specific targets and the paracrine interactions between them and other cells.

Disclosures:

No relevant conflicts of interest to declare.
Platelets and the Tumor Cell Microenvironment
Authors: Byzova Tatiana V. , Kerr Bethany A. , Feng Weiyi , McCabe Patrick
Published: October 2019

The link between tumors and the hemostatic system, first of all platelets, has been acknowledged by numerous studies. Platelets activation at the tumor microenvironment and subsequent intravascular clotting are often associated with cancer complications. Recent clinical studies demonstrated that platelet inhibitors substantially decrease risk of tumor metastasis and mortality in cancer patients, thereby emphasizing clinical importance of platelets. However, the precise function of platelets in cancer progression remains enigmatic. Growing tumors secrete a plethora of growth factors and cytokines (tumor secretome) aiming to change its micro- and macro-environment. These factors promote angiogenesis, induce inflammation, and even stimulate changes in bones prior to metastasis. Tumor secretome signals are likely transmitted through the circulation; however, these signals need to be protected from degradation or utilization prior to reaching their target. Due to their ability to intake and secrete back a wide spectrum of proteins, platelets could function as a mobile storage compartment for the tumor secretome. Using platelet infusion and depletion during tumor growth, we assessed the role of platelets in tumor growth and tumor-induced bone remodeling. We demonstrated that platelet infusion stimulates communications between bone marrow and the tumor, while platelet depletion had the opposite effects. Importantly, tumor-induced changes in bone structure were completely abolished in the absence of platelets. Using platelets from knockout mice with various platelet defects, we demonstrated that platelet secretion (but not aggregation) was crucial for their function in tumor progression. In particular, the function of platelet α-granules is essential for the pro-tumorigenic role of platelets. Analysis of the tumor secretome revealed that 77 percent of tumor-secreted factors are highly enriched within platelets over plasma. Several of these factors are essential mediators of bone remodeling. Thus, our data show that platelet α-granule sequestration and secretion represent a cellular mechanism mediating communication between the bone microenvironment and the primary tumor.

Disclosures:

No relevant conflicts of interest to declare.
Selecting T-Cell Subsets for Adoptive T-Cell Therapy to Optimize Potency and Persistence
Authors: Riddell Stanley , Turtle Cameron , Hudecek Michael , Sommermeyer Daniel , Jensen Michael C.
Published: October 2019

Adoptive T-cell therapy with tumor-reactive T cells is emerging as a highly effective strategy for eliminating even the most advanced chemotherapy refractory malignancies. Endogenous T cells specific for tumor-associated antigens can sometimes be isolated and expanded from the patient’s blood or tumor infiltrate, or more expeditiously can be engineered by gene transfer to express a T-cell receptor specific for a tumor associated MHC/peptide complex or a synthetic chimeric antigen receptor (CAR) specific for a tumor associated cell surface molecule. The remarkable regression of advanced acute lymphocytic leukemia and lymphoma in patients treated with T cells engineered to express CD19-specific CARs illustrates the potential for this approach to transform clinical care. Therapeutic activity is variable in individual patients, however, and this appears to correlate with the ability of transferred, tumor-reactive T cells to persist and proliferate in vivo, and to retain effector function. These attributes may reflect both the qualities of the T cells that are isolated or engineered for therapy, and the local tumor microenvironment that may contain regulatory T cells; cells that express ligands that engage inhibitor receptors on effector T cells or cytokines that inhibit effector T-cell proliferation. The CD4+ and CD8+ T cell pools in normal individuals contain a variety of naïve, memory, and regulatory T-cell subsets that differ in epigenetic, transcriptional, and functional properties. Because most clinical protocols have used polyclonal peripheral blood mononuclear cells as recipients for CAR gene transfer, the composition of T-cell products that are being administered is highly variable, particularly when the T cells are obtained from cancer patients that have received prior cytotoxic chemotherapy that can skew the phenotypic composition of the peripheral T-cell pool. As a consequence, transferring tumor-targeting receptors into polyclonal unselected cell populations provides poor control over the cellular composition of the final T-cell product, which may in part explain the marked differences in efficacy and toxicity that have been observed in the clinic, and may complicate regulatory approval of these novel therapies. Methods to derive T cells from distinct naïve and memory T-cell subsets have been developed, enabling the rapid production of therapeutic T cells of uniform composition. The results of preclinical studies that illustrate the improved potency of defined T-cell products that are engineered with tumor-specific CARs, and the clinical implementation of this approach in B-cell malignancies will be presented.

Disclosures:

Riddell: Cell Medica: Consultancy, Membership on an entity’s Board of Directors or advisory committees; ZetaRx: Consultancy.
Therapeutic Targeting of the Bcl2 Family
Authors: Letai Anthony , Davids Matthew S. , Chonghaile Triona Ni , Deng Jing , Patel Luv
Published: October 2019

Many, perhaps most, cancer chemotherapy agents kill cancer cells via the mitochondrial pathway of apoptosis that is controlled by the Bcl-2 family of proteins. Bcl-2 family proteins regulate commitment to cell death by controlling mitochondrial outer membrane permeabilization (MOMP). To better understand how cancer cells commit to apoptosis, and what drugs might make them commit to apoptosis, we have studied perturbing mitochondria with BH3 peptides that are derived from pro-death Bcl-2 family proteins. Using this provocative test, which we call BH3 profiling, we are able to measure how close a cell is to the threshold of apoptosis, a property we call “priming”. Priming corresponds to sensitivity to chemotherapy. Moreover, BH3 profiling can be used to detect dependence on Bcl-2 and Bcl-xL for survival, which predicts cytotoxic response to small molecule antagonists such as ABT-199 and ABT-263. In acute lymphoblastic leukemia, we find that dependence on either Bcl-2 or Bcl-xL varies from case to case, with very important consequences for sensitivity to ABT-199 and ABT-263. In chronic lymphocytic leukemia, ABT-199 has already demonstrated significant clinical activity that corresponds to its on-target activity in mitochondria in vitro. We have been testing how this in vitro mitochondrial activity in BH3 profiling assays might be translated into a useful clinical predictive biomarker. Finally, we can measure how short term incubation with many kinds of drugs, including targeted pathway inhibitors, can increase cancer cell priming, including for primary lymphoid malignancy cells. This short term increase in priming predicts subsequent cancer cell death, including in clinical treatment. We call this method “Dynamic BH3 Profiling” and are exploring how it might best be utilized in the clinic.

Disclosures:

Letai: Dana-Farber Cancer Institute: Patents & Royalties; AbbVie: Consultancy.