bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficacy and Safety of JWCAR029 in Adult Patients with Relapsed and Refractory B-Cell Non-Hodgkin Lymphoma
Authors: Yan Zixun , Wang Wen , Zheng Zhong , Hao Ming , Yang Su , Li James , Cheng Shu , Wang Li , Zhao Wei-li
Published: October 2019

Introduction

JWCAR029 is a novel CD19-directed 4-1BB stimulated chimeric antigen receptor T (CAR-T) cell type, which is different from JWCAR017 with independent production of CD4 and CD8 T cells and transfusion in non-fixed ratio. We conducted a single arm, open-label, dose escalation Phase I trial of JWCAR029 in relapsed and refractory B-cell non-Hodgkin lymphoma (NCT03355859).

Methods

From January to July 2018, 10 patients have been enrolled in this trial, including eight diffused large B cell lymphoma (DLBCL) and two MALT lymphoma, with median age of 47 years (range 32 to 59 years). All the patients received immunochemotherapy as induction and more than two lines of salvage treatment. Two patients received bridging chemotherapy after T-cell collection due to rapid tumor progression, followed by re-evaluation before CAR-T cell infusion. Lymphodepletion preconditioning was accomplished by fludarabine 25mg/m2/d and cyclophosphamide 250mg/m2/d on Day-4 to D-2, followed by CAR-T cell infusion on Day0. JWCAR029 was administrated as a single infusion in escalation dose levels, from 2.5×107 CAR-T cells (dose level 1, DL1) to 5.0×107 CAR-T cells (dose level 2, DL2) and to 1.0×108 CAR-T cells (dose level 3, DL3) according to mTPI-2 algorithm. Circulating blood count, serum biochemistry, and coagulation status were follow-up after infusion. Cytokines were assessed on a Luminex platform. Tumor evaluation was performed on Day 29 by PET-CT. PK data were detected by flow cytometry and real-time quantitative polymerase chain reaction system. All the adverse events were recorded. The study was approved by the Shanghai Rui Jin Hospital Review Board with informed consent obtained in accordance with the Declaration of Helsinki.

Results

The demographic characteristics of the patients were demonstrated in Table 1. Among six evaluable patients (3 of DL1 and 3 of DL2), the ORR was 100% on Day 29, including four complete remission and 2 partial remission. Cytokine release syndrome (CRS) was 100% in Gr 1, with main symptoms as fever (<39.0 degrees), fatigue, and muscle soreness. No neurotoxicity was observed. Four of the six patients with fever >38.0 degrees used prophylactic IL-6 Inhibitor (8mg/kg, ACTEMRA, two patients administered twice). No patients received steroids. The CRS showed no difference between dose level groups (p>0.99). Adverse effects included leukopenia (Gr 3-4: 83.3%, Gr 1-2: 16.7%), hypofibrinogenemia (Gr 1: 16.7%, Gr 2-4: 0%), liver dysfunction (Gr 1: 33.3%, Gr 2-4: 0%), elevated CRP (Gr 1: 83.3%, Gr 2-4: 0%), ferritin (Gr 1-2: 83.3%, Gr 2-4: 0%), or IL-6 (Gr 1-2:100%, Gr 3-4: 0%, Table 2).

Conclusion

Although long-term follow-up was needed, the preliminary data of six patients in this trial have demonstrated high response rates and safety of JWCAR029 in treating relapsed and refractory B-cell non-Hodgkin lymphoma.

Disclosures

Hao: JW Therapeutics: Employment, Equity Ownership.
A Successful Experience with Brentuximab Vedotin in Relapsing/Refractory Cutaneous T-Cell Lymphoma: Two Case Reports
Authors: Gorenkova Liliya , Kravchenko Sergey K. , Kovrigina Alla M. , Savchenko Valery G.
Published: October 2019

Primary cutaneous T-cell lymphomas are non-Hodgkin lymphomas of the skin defined by the presence of malignant T lymphocyte clonality.

Mycosis fungoides (MF) accounts for more than half of these lymphomas, while CD30+ lymphoproliferative skin diseases (primary cutaneous anaplastic lymphoma and lymphomatiod papulosis (LyP)) comprise 25% of these neoplasms, and rarely seen tumors including subtypes of primary cutaneous peripheral T cell lymphoma nonspecified (NOS).

A high expression of CD30 antigen on tumor cells is detected in primary cutaneous CD30+ lymphomas and transformed MF, whereas it may be observed in other entities, but a low level.

While most patients with cutaneous CD30+ lymphoproliferative disorders experience an indolent disease course with an excellent prognosis, up to 30% have progressive disease with 8% of patients succumbing to their cancer. Systemic immunomodulatory or chemotherapeutic agents are often used for patients with such advanced disease, with CD30 being an increasingly attractive therapeutic target.

Herein, we report two cases of a relapsing lymphomatiod papulosis and a refractory primary cutaneous peripheral T cell lymphoma NOS. Target therapy with brentuximab vedotin either as a single agent or combined with chemotherapy resulted in a stable long-term remission.

Patient 1, a man of 45 years of age, received treatment for Hodgkin lymphoma about 5 years ago. Already at that time, he noted self-regressing crops of pruritic papules or nodules on the face. In a year after chemotherapy had stopped, this skin elements recurrent with subsequent partial regression. LyP type D was identified on histological examination (picture 1).

Following several lines of therapy, frequent relapses were noted and a lesser quantity of lesions tended to resolve leaving scars and hyperpigmentation.

The complete remission was achieved after 6 cycles of brentuximab vedotin, at a follow-up period of 12 months no evidence of relapse were found (picture 2).

Patient 2, a 30-year aged man. First solitary skin lesions occurred 2.5 years ago. The patient received combination therapy with low doses of chemotherapeutic agents and subsequently had progression of the disease. The patient received more 3 lines of high-dose chemotherapy; however, the disease was rapidly progressive with increasing size of lesions and newly appearing tumor lesions in the skin (picture 3). The remission was achieved after a combination regimen which included Dexa Beam courses and brentuximab vedotin (picture 4). The matched allogeneic bone marrow transplantation is planned for the patient to consolidate the remission.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
The Role of DNAM-1/Tigit Axis of NK Cells in the Regulation of Alloreactive T Cell Responses and the Potential Mechanism
Authors: Sheng Lixia , Ouyang Guifang , Qitian MU , Huang He
Published: October 2019

Objective: Previous studies has showed the important role of NK cell in the regulation of alloreactive T cell response and negative regulation of GVHD. The paired receptors DNAM-1 and TIGIT, which bind the same ligands but have opposite effects on NK cell function, might present as potential targets for the GVHD treatment. This study is designed to explore the role of TIGIT/DNAM-1 balance of NK cells in the regulation of alloreactive T cell responses and the potential mechanism.

Methods: Antibodies blocking of TIGIT or DNAM-1, over-expression of TIGIT or DNAM-1 via lentiviral transduction and knockdown of TIGIT or DNAM-1 by lentiviral shRNA were used to manipulate the TIGIT/DNAM-1 balance on NK cell. Cytotoxicity assay using alloantigen activated T cells as targets were used to evaluate the regulating function of NK cell on alloreactive T cell responses. Western blot and small molecule inhibitors against PI3K were combined to investigate whether the PI3K-Akt-ERK signaling cascade is involved in the signal transduction process following TIGIT/DNAM-1-PVR engagement.

RESULTS: Blocking of DNAM-1 by an anti-DNAM-1 antibody and knockdown of DNAM-1 expression by lentiviral shRNA both resulted in deceased cytotoxicity of NK cells against alloantigen activated T cells, while over-expression of DNAM-1 via lentiviral transduction resulted in enhanced cytotoxicity. Blocking of TIGIT by an anti-TIGIT antibody and knockdown of TIGIT expression by lentiviral shRNA both resulted in increased cytotoxicity of NK cells against alloantigen activated T cells, while over-expression of TIGIT via lentiviral transduction resulted in decreased cytotoxicity. Increases in NK cytotoxicity against activated T cells through TIGIT knockdown could be overcome by blocking DNAM-1 signaling. Simultaneously, over-expression of DNAM-1 or knockdown of TIGIT expression resulted in an increase of the phosphorylation levels of Akt and ERK1/2 in NK cells after contacted with activated T cells, which could be overcome by pretreating NK cells with anti-DNAM-1 or PI3K small molecule inhibitor. Pretreating alloantigen activated T cells with anti-PVR also resulted in deceased cytotoxicity and Akt and ERK1/2 phosphorylation in DNAM-1 over-expression NK cells.

Conclusion: The paired receptor DNAM-1/TIGIT on the surface of NK cells compete the same PVR ligand on the surface of activated T cells and the DNAM-1/TIGIT axis is involved in the regulation of cytotoxicity of NK cells on alloantigen activated T cells through PI3K-Akt-ERK cascade phosphorylation. The DNAM-1/TIGIT expression balance may present as biomarkers for aGVHD and potential targets for aGVHD therapy.

Disclosures

No relevant conflicts of interest to declare.
IL-6 As a Potential Cell Released Marker of Prostate Cancer Cell Death
Authors: Gilazieva Zarema , Tezcan Gulcin , Garanina Ekaterina , Sakr Nawar , Arkhipova Svetlana , Vinogradov Lev , Rizvanov Albert , Khaiboullina Svetlana
Published: October 2019

Introduction: Interleukin 6 (IL-6) is proinflammatory cytokine which is produced by cell when Nod-like receptors (NLRs) are activated. Increased production of IL-6 was shown to promote tumor growth and metastasis.Therefore, it could be suggested that activation of NLRs could support malignancy. PC-3, prostate tumor derived cells, was shown to produce IL-6; however, little is known about the role of NLRs in regulation of cytokine secretion.

Method: PC-3 cells were seeded in 48 well plate cell culture plates and culturd in presence of VX765, Nigericin or Camptothesin. After 24 hours incubation, cell death was determined using Annexin V test. Additionally, IL-6 level was assessed in culture medium using Bio-Plex Pro™ Human Chemokine Panel (Biorad). Data was analyzed using One Way Anova, Post Hoc Tukey test, SPSS 20.

Results: Viability of PC3 cells was reduced when treated with Nigericin (37.74±0.33%) and Champtothesin (10.10±0.23%) as compared to untreated cells (P<0.01), while VX765 did not affect cell viability (4.78±0.28 %; P= 0.645). Nigerecin significatly increased IL-6 production in PC3 cells when compared to untreated cells. Interestingly, IL-6 levels in culture medium of PC3 cells treated with nigerecin was significantly higher than that in cells treated with VX765 and campthothesin (p< 0.001).

Conclusion: It appears that IL-6 can be produced by PC3 cells upon stmulation of NLRs. Since NLRs activation was associated with reduced cell vitality, we suggest that increased IL-6 level in culture medium could be the result of the cytokine release from dead cells.

This study was supported by RFBR Grant #18-34-01000 and Program of Competitive Growth of KFU.

Disclosures

No relevant conflicts of interest to declare.
Evaluation of Ocular Complications By Using Optical Coherence Tomography in Children with Sickle Cell Disease
Authors: Vatansever Esra , Vatansever Mustafa , Dinç Erdem , Temel Gulhan , Unal Selma
Published: October 2019

Purpose: This study was aimed to evaluate ocular abnormalities by using optical coherence tomography (OCT) in children with sickle cell disease (SCD) and to search the relationship between OCT and clinical findings.

Methods:The study included 108 eyes of 54 SCD patients as well as 110 eyes of 55 healthy subjects with no ocular pathology as a control group. A complete ophthalmologic examination was performed. Pupillary dilation was induced and macular sections were obtained with spectral domain OCT. Macular sections were evaluated according to the ETDRS map. Inner and outer retinal thicknesses were measured using the software in the device.

Results:Foveal splaying, temporal thinning of retinal thickness, and vascular tortuosity were found to be significantly higher in the patient group than the control group (p <0.0001 for all three parameters)(Figure-1).The foveal diameter (1592.39 ± 175.56 μ) was significantly increased in the patient group compared to the control group (1391.01 ± 175.56 μ) (p<0.0001) and the foveal depth was significantly decreased in the patient group (121.15 ± 26.83 μ) compared to the control group (146.1 ± 12.25 μ) (p<0.0001). The mean total retinal thickness was 253.53 ± 22.31 μ in the patient group and 261.03 ± 18.48 μ in the control group and the difference was statistically significant (p = 0.007). Likewise, central retinal thickness was significantly decreased in the patient group (219.35 ± 10.53 μ) compared to the control group (235.32 ± 12.51 μ) (p <0.0001).

Conclusions:Our study suggests that subclinical retinal involvement may occur in children with SCD. Foveal splaying, temporal thinning of retinal thickness, and vascular tortuosity are significant OCT findings in the children with SCD. It is seen that OCT is an important imaging method in addition to routine ophthalmologic examination to detecting ocular abnormalities in the early period and follow-up of this patient group.

Keywords :Sickle Cell Disease, Childhood, Optical Coherence Tomography

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
The Von Willebrand Factor Tyr2561 Allele Is a Gain-of-Function Variant and a Potential Risk Factor for Early Myocardial Infarction
Authors: Schneppenheim Reinhard , Hellermann Natalie , Brehm Maria Alexandra , Klemm Ulrike , Obser Tobias , Huck Volker , Schneider Stefan W , Denis Cecile V. , Tischer Alexander , Auton Matthew , Maerz Winfried , Xu Emma-Ruoqi , Wilmanns Matthias , Zotz Rainer B.
Published: October 2019

The frequent von Willebrand factor (VWF) variant p.Phe2561Tyr is located within the C4 domain, which also harbors the platelet GPIIb/IIIa-binding RGD sequence. To investigate its potential effect on hemostasis, we genotyped 865 patients with coronary artery disease (CAD), 915 with myocardial infarction (MI) and 417 controls (Ludwigshafen Risk and Cardiovascular Health Study (LURIC)) and performed functional studies of this variant. A univariate analysis of male and female carriers of the Tyr2561 allele ≤55 years revealed an elevated risk for repeated MI (Odds ratio: 2.53, 95% CI:1.07-5.98). The Odds ratio was even higher in females ≤55 years at a value of 5.93 (95% CI:1.12-31.24). Cone and plate aggregometry showed, that compared to Phe2561, Tyr2561 was associated with increased platelet aggregate size, both in probands' blood and with the recombinant variants. Inhibition of the VWF-GPIIb/IIIa interaction abrogated the effect. Microfluidic assays revealed that the critical shear rate for inducing aggregate formation was decreased to 50% by Tyr2561 compared to Phe2561. Differences in C-domain circular dichroism spectra resulting from Tyr2561 suggest an increased shear sensitivity of VWF, as a result of altered association of the C-domains that disrupts the normal dimer interface. In summary, our data emphasize the functional impact of the VWF C4-domain for VWF-mediated platelet aggregation in a GPIIb/IIIa-dependent manner and provide the first evidence that a functional variant of VWF plays a role in arterial thromboembolism.

Disclosures

Schneppenheim: SHIRE: Consultancy; CSL Behring: Consultancy.
Identification of a Novel CEBPE Enhancer Essential for Granulocytic Differentiation
Authors: Shyamsunder Pavithra , Shanmugasundaram Mahalakshmi , Mayakonda Anand , Teoh Weoi Woon , Han Lin , Lim Mei Chee , Fullwood Melissa , An Omer , Yang Henry , Jizhong Shi , Hossain Md.Zakhir , Madan Vikas , Koeffler H. Phillip
Published: October 2019

CEBPE is a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors essential for granulocytic differentiation. CEBPE is expressed in a stage-specific manner during myeloid differentiation and regulates transition from the promyelocyte to the myelocyte stage. It is essential for secondary and tertiary granule formation in granulocytes. We and others found germline mutations of the CEBPE gene in patients with neutrophil-specific granule deficiency. Their neutrophils display atypical bilobed nuclei, lack expression of granule proteins and these patients often have frequent bacterial infections. Cebpe knock-out mice resemble this clinical phenotype displaying a block in terminal differentiation and absence of secondary granule proteins.

Given the tissue specific expression of CEBPE, we were interested in identifying genomic regions and factors that could regulate its lineage specific expression. Our CEBPE ChIP-seq in murine bone marrow cells showed binding of CEBPE to a region 6kb upstream of Cebpe gene. Chromosome conformation capture-on-chip (4C-seq) demonstrated an interaction between this putative regulatory element (6kb upstream region) and the core promoter of Cebpe. Analysis of available DNase-seq data sets revealed that the region bound by CEBPE displayed an open chromatin only in myeloid lineage cells. Further examination revealed binding of a myriad of hematopoietic transcription factors to the +6kb enhancer in HPC-7 (hematopoietic progenitor cells) and in 416B (myeloid progenitor cells), indicating that this region/enhancer might regulate the expression of CEBPE.

Targeting of this region using dCas9-KRAB in murine 32D cells caused significant downregulation of RNA and protein levels of CEBPE compared to control cells. These targeted cells also exhibited impaired granulocytic differentiation with lower transcript levels of secondary granule proteins (Ltf and Ngp).

To investigate further the role of the +6kb enhancer region in myelopoiesis, mice were generated with deletion of this region using CRISPR/Cas9 technology. Germ line deletion of the +6kb enhancer resulted in reduced levels of CEBPE and its target genes, accompanied by a severe block in granulocytic differentiation and a complete absence of CD11b+/Gr1hi population. This phenotype is nearly identical to our Cebpe KO mice.

In summary, we have identified a novel enhancer crucial for regulating Cebpe, and required for normal granulocytic differentiation.

Disclosures

No relevant conflicts of interest to declare.
Multidimensional Study on the Prognostic Factors of Acute Myeloid Leukemia Patients with FLT3-ITD mutations Based on the Next-Generation Sequencing Platform
Authors: Zhang Yi , Zhao Bide , Wang Yungui , Wang Dongmei , Pan Hanzhang , Wang Chengcheng , Lou Feng , Cao Shanbo , Jin Jie
Published: October 2019

Purpose: This research aimed to investigate the features and effects of FLT3-ITD mutations in Chinese acute myeloid leukemia (AML) population, and to explore whether ITD variant allele frequency (VAF), number of mutations, insertionposition and length will have influence on the patients' overall survival (OS) and relapse-free survival (RFS).

Methods:A total of 101 newly diagnosed AML (non-APL) patients with FLT3-ITD mutations from December 2013 to March 2018 were recruited. Their bone marrow or peripheral blood samples were sequenced by NGS 185-gene platform, then the sequencing data were analyzed in combination with the patients' clinical information.

Results:The most common concomitant mutations in FLT3-ITD patients were NPM1, DNMT3A, TET2, IDH2, RUNX1, and WT1. The patients' median VAF is 0.344 (0.084-0.487). Among 101 patients, 80 (79.2%) carried 1 ITD mutation, 17 (16.8%) carried 2, 3 (3.0%) carried 3, and only 1 (1.0%) carried 4. In most patients (76/101) the ITD fragments were inserted in the JM region. And the median length of the insertion fragments was 54 (12-252) bp. According to the NCCN guidelines 2018, the cutoff value of FLT3-ITDmutation VAF between different stages of prognosis is 0.33. However, in our research, it was better to set 0.30 (p=0.0465 for OS; p=0.4929 for RFS), but not 0.33 (p=0.4270 for OS; p=0.3828 for RFS), as the cutoff value to tell differences of survival. Meanwhile, the numbers of carried ITD was significantly related to prognosis. The patients with single ITD mutation were found to have better clinical outcomes than those with multiple ITD mutations (p= 0.0031 for OS; p=0.0001 for RFS). Patients with ITD insertion in the hinge region of JM had a trend of poorer OS. The insertion length of ITD fragment correlated with the patients' RFS, but not OS. The longer the insertion was, the worse RFS of the patient showed (p=0.0323, p trend=0.0049).

Conclusion:The VAF, the number of mutations, and the insertion length of the FLT3-ITDmutations will significantly affect the prognosis of AML patients in Chinese population.

Disclosures

Jin: College of Medicine, Zhejiang University: Employment; The National Natural Science Foundation of China: Research Funding.
The Role of High Molecular Weight Kininogen in Lymphoma Microvascular Density and Growth
Authors: Erdemir Huseyin Halil , Shukla Meenal , McCrae Keith R.
Published: October 2019

Background: Non-Hodgkin lymphoma (NHL) is a common malignancy in childhood and adolescence. The prognosis of relapsed and refractory NHL remains poor, and new approaches to therapy are needed. High molecular weight kininogen (HK) is a central component of the kallikrein-kinin system. Previous work has demonstrated that in vitro, the kallikrein-cleaved form of HK induces apoptosis of proliferating endothelial cells. Prior studies performed in our laboratory have demonstrated the increased angiogenesis and tumor growth in the absence of HK in B16F10 melanoma and Lewis lung carcinoma models. However, the role of endogenous kininogen in modulating the growth and of hematologic malignancy is unknown. To elaborate the role of HK in in the development and progression of a murine lymphomas, we assessed growth and vascularization of T-cell lymphoma xenografts in wild type (WT) and kininogen deficient (mKNG1-/-) mice.

Methods: Murine T-cell lymphoma (EL-4) cells (1 x 10^6) were implanted into wild-type and mKNG1-/- mice. Tumor size was measured using calipers and tumor volume was calculated using the formula V = length x width^2 x 0.52. Fifteen days after cell implantation, tumors were harvested, processed and analyzed by immunofluorescent staining with anti-CD31 antibodies to determine microvascular density (MVD).

Results: EL-4 lymphomas grew more rapidly and to larger sizes in mKNG1-/- mice compared to wild-type mice, with significant differences apparent by day 11 after tumor implantation (p<0.001). By Day 15, the volume of tumors in mKNG1-/- mice was approximately ~2-fold greater than in wild-type mice (Figure 1A). Weights of EL-4 Lymphomas were determined after 15 days of implantation and found significantly different between two groups (Figure 1B). To assess whether the difference in growth rates of lymphomas in wild-type and mKNG1-/- correlated with differences in angiogenesis, the microvascular density within tumors from these animals was compared by counting vessels stained with anti-CD31 antibodies (Figure 1D). Compared to wild-type mice, lymphomas in kininogen deficient mice demonstrated significantly greater microvascular density when either random fields were assessed (Figure 1C and 1D, P < 0.001).

Conclusion: These findings suggest that HK is an important endogenous regulator of angiogenesis and tumor growth in this T- cell Lymphoma model. Although the role of kininogen has been described in solid tumor models, this study is the first to demonstrate that kininogen specifically angiogenesis in a hematopoietic tumor. Further work is needed to understand the mechanisms underlying these findings and inform anti-angiogenic therapies to increase the options for patients with hematopoietic malignancies such as NHL.

Disclosures

No relevant conflicts of interest to declare.
Comparision of the Clinical and Prognostic Characteristic in Adult and Pediatric AML1/ETO-Positive Acute Myeloid Leukemia
Authors: Yu Guopan , Zhou Jiaheng , Zhou Jinchan , Qiu Jiale , Yin Zhao , Yin Changxin , Xu Dan , Jiang Xuejie , Liu Qifa , Meng Fanyi
Published: October 2019

AML1/ETO-positive acute myeloid leukemia (AML) is a heterogeneous malignancy. Up until now, the difference between adult (>14-year old) and pediatric (≤14-year old) AML1/ETO+ AML remains unclear. In this retrospective multi-center study we analyzed 173 AML1/ETO+ AML patients including 98 adults and 75 kids, and found that AML-M2 phenotype was the most common morphology, especially higher in adult patients with an incidence of 92.9%, as compared with 82.7% (P=0.005) in pediatric patients. Furthermore, higher incidence of extramedulary leukemia (29.6% vs. 13.3%, P=0.001) and C-KIT mutation (21/67, 31.3% vs. 12/66, 18.2%, P=0.079) were observed in adult patients than pediatric patients. No significant difference in gender ratio, peripheral white blood cells count, immunology and cytogenetic abnormality including affiliated cytogenetic abnormalities and lose of sex chromosmoe between the two age groups. Among adult patients, idarubicin combined with cytarabine (IA) or daunorubicin combined with cytarabine (DA) was the major induction regimens, while FLAG (fludalabin,cytarabine and G-CSF) combined with idarubicin or DA combined with etoposid (DAE) was the main regimens in pediatric patients for one to two cycles. Not only the first-cycle complete remission (CR) rate (69/74, 93.2% vs. 56/95, 58.9%, P <0.001), but also the second-course cumulative CR rate (73/74, 98.6% vs. 82/95, 86.3%, P=0.004) in pediatric patients was much better than that in adult patients. After obtaining CR, standard dose cytarabine (SDAC)-based or medium dose cytarabine (MDAC)-based regimens were given to the adult patients as the major consolidation regimens, while pediatric patients received MDAC or homoharringtonine combined with cytarabine and etoposid (HAE). With a median of 23.5(2-126) months follow-up, cumulatively 35/87(40.2%) patients relapsed and 39/98(39.8%) cases died in adult group, which were much higher than that in pediatric patients, with the cumulative incidence of recurrence of 18/74(24.3%) (P=0.032) and cumulative death rate of 13/75(17.3%) (P=0.001). Survival analysis showed that EML [RFS: HR 3.20(1.63-6.28), P=0.001; OS: HR 2.92(1.55-5.51), P=0.001] and C-KIT mutation [RFS: HR 3.17(1.47-6.93), P=0.003; OS: HR 2.24(1.03-4.86), P=0.041] were the adverse factors for relapse free survival (RFS) and overall survival (OS). Nevertheless, neither these two factor were negative for the survival of pediatric patients. Taking together, significant difference in bone marrow morphology, incidence of EML and C-KIT mutation, also prognostic factors were observed between the adult and pediatric AML1/ETO+ AML patients. Intensive induction might be a main reason for higher CR rate and better survival in pediatric patients.

Disclosures

No relevant conflicts of interest to declare.
In Vitro Approach for the Identification of Exceptional Responders in Acute Myeloid Leukemia
Authors: O'Brien Eric , VanCauwenbergh Brett , Byerly Luke , Merk Alexander , Sarangdhar Mayur , Wunderlich Mark , Basu Mitali , Mizukawa Benjamin , Breese Erin H. , Seibel William , Perentesis John Peter
Published: October 2019

Each case of Acute Myeloid Leukemia (AML) represents a unique ecosystem. Despite recent advances in our understanding of the genetic landscape of AML, this information remains insufficient to accurately match patients with targeted therapies. While pediatric and adult AML share phenotypic similarities, pediatric AML represents a genetically distinct disease from adult AML, and will benefit from independent genomic studies and novel therapeutic strategies. Real-time ex-vivo functional screening can identify mechanisms underpinning drug response and diversity between tumors, aiding in patient stratification. We established an in vitro drug screening system that incorporates cytokine signaling to better model inflammation, stem cell function, and niche derived support of leukemic blasts. This approach provided insight into variability between patients who currently would be placed on the same therapeutic regimen.

Samples were acquired from 12 pediatric AML patients, after informed consent was obtained. Samples were enriched for blasts, and cultured in the presence of SCF, TPO, FLT3-L, IL-3, and IL-6 (KTF36). A panel of 38 drugs was selected from a larger screen of 1839 compounds done on commercially available hematological malignancy cell lines. Drugs included standard chemotherapy agents used in AML and drugs currently under clinical development. Cells were exposed to drugs for 72hrs. An MTS assay was performed and results reported as % of viable cells remaining, after normalization to vehicle control wells. Targeted DNA NGS sequencing of 406 genes, 31 introns, and RNA sequencing of 265 genes was performed for genetic characterization.

In vitro drug screening revealed variations in drug sensitivity between samples and revealed time ex-vivo and cytokine milieu to be important factors affecting response of the same cells to the same drugs. Engraftment of long term cultures into immunodeficient mice produced aggressive disease in all cases, indicating robust support of stem cell function via addition of KTF36 cytokines. When possible, clinical response to therapy was compared with in vitro response to the same drugs. This screening approach highlighted well established agents that showed significant activity in highly refractory disease providing rationale for further clinical trial development. An unsupervised clustering showed drug sensitivity primarily correlated with the presence of MLL-X fusion, NRAS/KRAS and PTPN11 alterations. Linear Regression with interaction effects showed drug sensitivity/resistance to be highly selective for single/signature of specific molecular alterations.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Does Race Impact Survival in Multiple Myeloma?
Authors: Kennedy-LeJeune Elizabeth , O'Neal Hollis , Miles Lauren , Hamer Diana , Cataldo Vince D.
Published: October 2019

Purpose:

Multiple myeloma (MM), a cancer involving abnormal antibody production from plasma cells, accounts for approximately 1.8% of all new cancer cases in the US. The International Staging System (ISS) for MM classifies patients with serum beta2 microglobulin < 3.5mg/l and serum albumin ≥3.5g/dl as having stage I disease, patients with serum beta2 microglobulin ≥ 5.5 mg/l as having stage III disease, and all others as having stage II disease. The median survival by stage at time of diagnosis is 62 months, 45 months, and 29 months, respectively, for stages I, II, and III.

The median age at time of diagnosis of MM is 69 years, with approximately 62% of all patients being at least 65 years old at time of diagnosis. There is a 2-fold increase in MM incidence amongst blacks as compared to whites in both the male and female population. This study was conducted to further characterize our MM patient population and to potentially identify racial and gender impact on mortality.

Methods:

A retrospective chart review of all patients in our geographic region diagnosed with MM between 2012 and 2017 was conducted using the Louisiana Tumor Registry Board database. Clinical data abstraction included epidemiology, laboratory, and pathologic data at time of diagnosis, and time to autologous stem cell transplant and death, if applicable. We performed chi-square analysis, linear regression, and survival analysis to compare survival outcomes between groups.

Results:

Chart review data was available for 184 of the 243 patients diagnosed with MM between 2012-2017, including 89 (47.6%) males and 98 females (52.4%), and 90 (48.1%) whites and 88 (47.1%) blacks. Females were significantly older than males (66.1 vs 62.6 years, p=0.037) at time of diagnosis yet whites were not significantly older compared to blacks (65.1 vs. 63.9 years, p=0.498). Among the 108 patients with documented stage of disease, 17 (15.7%) patients were diagnosed with stage I MM compared to 40 patients (37.0%) diagnosed at stage II and 51 (47.2%) diagnosed at stage III. With regards to stage, there was no significant difference between race or sex in our population. When adjusted for gender and age at diagnosis, race was a significant predictor of survival time with survival being 6.45 months shorter for black patients with MM compared to whites with the disease. (95% CI, 1.1 to 11.8; p = 0.019)

Conclusion:

Our data suggest that race exists as an independent predictor of shorter survival in our black patient population with MM. Additional studies investigating the biologic diversity of this disease are warranted to explain this racial disparity.

Disclosures

No relevant conflicts of interest to declare.
Osteoclast Stimulatory Transmembrane Protein (OCSTAMP) mRNA Levels and Clinical Variables in Persons with Plasma Cell Myeloma
Authors: Zhou Ya-Lan , Lu Jin , Gale Robert Peter , Yang Shen-Miao , Wu Li-Xin , Li Jin-Lan , Qin Ya-Zhen , Liu Yan-Rong , Jiang Hao , Jiang Qian , Zhang Xiaohui , Xu Lan-Ping , Liu Kai-Yan , Huang Xiao-Jun , Ruan Guorui
Published: October 2019

Background Bone disease is an important determinant of quality-of-life and survival of persons with plasma cell myeloma (PCM). Biomarkers of bone disease could be useful to predict risk and monitor therapy. In our prior analyses OCSTAMP (osteoclast stimulatory trans-membrane protein) mRNA levels were identified as increased above normals in persons with PCM. OCSTAMP encodes a membrane-anchored cell surface receptor promoting nucleation of osteoclasts involved in bone resorption and osteoclast differentiation.

Aims Measure levels of OCSTAMP mRNA in subjects with PCM and interrogate clinical associations.

Methods OCSTAMP mRNA levels were quantified by quantitative real-time polymerase chain reaction (RT-qPCR) in 224 bone marrow samples from 160 subjects with PCM including 160 newly-diagnosed; 55 in remission and 9 with recurrent PCM. Results were compared with 42 normals and data expressed as ratio of OSSTAMP mRNA/ABL mRNA. Associations with clinical variables were interrogated and comparisons analyzed using the Chi-square test.

Results OCSTAMP mRNA levels were significantly greater than normals in 111 subjects (69%, [95% confidence interval l[CI], 62-77%], P<0.001) with newly-diagnosed PCM, in 5 (56% [23,88%]; P=0.39) relapsing after initial therapy and in 12 in remission (22% [11-33%; P<0.001). mRNA levels in samples from newly-diagnosed subjects (median, 0.52%; range, 0-121%) were significantly higher than in samples from subjects in remission (0.01%; range, 0-4.7%) or normals (0.02%; range, 0-0.10%; both p<0.001). Levels in subjects in remission and normals were similar (p=0.85). Median OCSTAMP mRNA level was used to dichotomize subjects into low (median 0.06%; range, 0-0.51%) and high (3.8%; range, 0.54-121%) cohorts (P<0.001). Subjects in the high cohort were more likely to be in Durie/Salmon stage-3 (p=0.035), to have serum albumin concentrations <35g/L (p=0.036), have serum C-reactive protein (CRP) concentrations ≥8mg/L (p=0.03) and have ≥1 pathological bone fractures (p=0.002).

Conclusions OCSTAMP is highly transcribed in persons with newly-diagnosed and recurrent PCM compared with normals whereas persons in remission have similar levels to normals. OCSTAMP mRNA levels correlate with several clinical variables including Durie/Salmon stage, serum albumin C-reactive protein levels and likelihood of pathological bone fractures. OCSTAMP mRNA levels are potentially useful as a biomarker if our data are validated.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Rrx-001 Is a Phase 3 Small Molecule Dual Inhibitor of CD47 and Sirpα with Activity in Multiple Myeloma
Authors: Cabrales Pedro , Carter Corey , Oronsky Bryan , Reid Tony
Published: October 2019

Background:

CD47 binds to SIRPα on the surface of macrophages delivering a "do not eat" signal to suppress phagocytosis. To evade macrophage-mediated destruction, CD47 is frequently overexpressed in cancers, including multiple myeloma (MM). CD47 inhibition is an area of intense therapeutic interest with three anti-CD47 monoclonal antibodies (mAbs) currently in clinical trials. However, these mAbs have been associated with severe hematologic toxicities. RRx-001 is a minimally toxic small molecule that dually downregulates CD47 on tumor cells and SIRPα on macrophages and triggers tumor associated macrophage phagocytosis of tumor cells in vitro and in vivo. RRx-001 is entering Phase 3 trials for the treatment of multiple cancer indications.

Methods:

The effect of RRx-001 on the expression of CD47 and SIRPα on macrophages was evaluated with Western blotting and flow cytometry. An in vitro phagocytotic assay was used to determine whether RRx-001 promoted engulfment of multiple myeloma cells by macrophages. Transcriptional mRNA profiling in murine tumor associated macrophages (TAMs) was performed to analyze the cytokine profile of TAMs in the presence or absence of RRx-001. Finally, myeloma bearing mice were treated with RRx-001 in the presence or absence or absence of clodronate to determine the effect of macrophage depletion on RRx-001 anticancer activity.

Results:

RRx-001 was shown to downregulate CD47 and SIRPα expression on tumor cells and macrophages, respectively, and to promote the phagocytosis of multiple myeloma tumor cells. RRx-001 also stimulated the production of pro-inflammatory cytokines in TAMs. In tumor bearing mice, depletion of macrophages by clodronate reduced the antitumor effects of RRx-001.

Conclusions:

RRx-001 is a Phase 3-ready small molecule innate immune checkpoint inhibitor, which triggers tumor associated macrophage phagocytosis of high-expressing CD47 tumor cells. Dual downregulation of CD47 and SIRPα by RRx-001 results in TAM repolarization and phagocytosis of tumor cells. Depletion of macrophages by clodronate in tumor-bearing mice reduced the antitumor effects of RRx-001 and further suggests that the target of RRx-001 is the macrophage.

Disclosures

Cabrales: EpicentRx Inc: Research Funding. Carter:EpicentRx Inc: Employment. Oronsky:EpicentRx Inc: Employment. Reid:EpicentRx Inc: Employment.
Amino Bisphosphonates Are Associated with Decreased Mortality Compared to Non-Amino Bisphosphonates in the Treatment of Multiple Myeloma
Authors: Razavi Marianne , Mhaskar Rahul , Djulbegovic Benjamin
Published: October 2019

Introduction

Osteolytic bone lesion is a hallmark of multiple myeloma (MM). Bisphosphonates have been shown to be effective inhibitors of osteoclastic activity, leading to improvement of skeletal related morbidity in MM. Based on their chemical structure, bisphosphonates can be classified into two categories: amino bisphosphonates (ibandronate, pamidronate and zoledronate) and non-amino bisphosphonates (clodronate, etidronate). Although both categories are efficient for the management of myeloma related bone disease, their effect on mortality has been controversial. Some studies demonstrated beneficial effect associated with the administration of amino bisphosphonates, while others showed no such effect. We evaluated if amino bisphosphonates improve survival compared with non-amino bisphosphonates, when added to standard therapy for myeloma.

Methods

To address our objectives, we conducted a systematic review and network meta-analysis of all randomized controlled trials (RCT) estimating the effect of bisphosphonates in MM. We identified trials by searching electronic databases such as MEDLINE, CENTRAL and EMBASE. We compared the effect of amino bisphosphonates group to non-amino bisphosphonates group, versus the reference placebo group or no treatment. The primary outcome was overall mortality. We conducted a network meta-analysis under a random effects model. Pooled hazard ratios (HR) with 95% confidence intervals were estimated. To further evaluate the effect between the groups, a test for subgroup differences was applied. All analysis were conducted using Revman and R.

Results

Overall 14 studies were identified from 1982 to 2015, enrolling a total of 2706 patients: 5 studies tested bisphosphonates with non-amino group and 9 with amino group. The results from the network meta-analysis (Figure 1) showed a decrease in overall mortality for the amino bisphosphonates (HR 0.76, 95% CI 0.62 to 0.94) compared to the non-amino bisphosphonates (HR 1.06, 95% 0.87 to 1.3) and placebo. The statistical test for subgroup differences was significant (Q=4.69, p=0.03).

Conclusion

Based on the totality of randomized evidence, we showed for the first time that amino bisphosphonates compared to non-amino bisphosphonates versus placebo or no treatment reduce mortality in MM. The use of amino bisphosphonate should be further encouraged as anti-myeloma treatment and not solely as supportive treatment.

Disclosures

No relevant conflicts of interest to declare.
Loss of HDAC11 Promotes Myeloid-Derived Suppressor Cells Inhibition of T Cell Function in a Murine Lymphoma Microenvironment
Authors: Chen Jie , Cheng Fengdong , Wang Michael , Sahakian Eva , Powers John J. , Pinilla Ibarz Javier , Smith Mitchell R. , Sotomayor Eduardo
Published: October 2019

Myeloid-derived suppressor cells constitute a heterogeneous population of immature myeloid cells derived from bone marrow that negatively regulate both innate and adaptive immunity in the tumor microenvironment. Previous work in our lab had demonstrated that MDSCs lacking histone deacetylase 11 (HDAC11) displayed an increase in suppressive activity against IFN-γ producing CD8+ T cells. The upregulated suppressive activity of HDAC11KO MDSCs was associated with a more aggressive tumor growth pattern when compared with wild type control mice.

Mechanistically we have found that tumor infiltrated HDAC11KO MDSCs isolated from lymphoma-bearing mice displayed up-regulation of expression and enzymatic activity of arginase 1 and Nos2, two enzymes that are crucial in regulating MDSCs suppressive function, when compare with wild type MDSCs. However, both arginase activity and NO production were at a similar level in the wild type and HDAC11KO MDSCs isolated from spleens of the same lymphoma-bearing mice. This finding suggests that HDAC11KO MDSCs are more suppressive within the tumor microenvironment. Moreover, the aberrant enzymatic activities of Arg1 and Nos2 in HDAC11KO MDSCs correlate with over-expression of the lineage-specific transcription factor C/EBPβ, which has been previously shown to be essential for the differentiation of functional MDSCs. Furthermore, ChIP analysis confirmed that HDAC11 is recruited to the C/EBPβ gene promoter where exerts a negative regulatory effect upon gene transcription.

Unlike MDSC's in which absence of HDAC11 is associated with a suppressive phenotype, T-cell lacking HDAC11 are hyper-reactive and endowed with strong antitumor activity. To assess which phenotype will be the dominant one in vivo, we performed adoptive immune cell transfer experiments of both MDSC and/or T-cells from either wild type or HDAC11 KO mice into C57BL/6 lymphoma-bearing animals. The transfer of HDAC11KO MDSCs was able to eliminate, at least partially, the anti-tumor effect elicited by the HDAC11KO T cells in the lymphoma microenvironment

Taken together, we have uncovered a previously unknown role for HDAC11 as a transcriptional regulator of MDSCs phenotype and function in a murine lymphoma model. A better understanding of this novel role of HDAC11 in myeloid biology will lead to targeted epigenetic therapies to manipulate the suppressive effect of these immunoregulatory cells in vivo.

Disclosures

No relevant conflicts of interest to declare.
Description of Molecular Prognostic Markers in AML Uruguayan Patients
Authors: Zubillaga Maria Noel , Dell Oca Nicolas , Cappetta Mónica , Manrique Gonzalo , Elizondo Maria , Verónica Perez , Tort José
Published: October 2019

Acute myeloblastic leukemias (AML) are the most frequent acute leukemias in adults. Recent genomic and candidate gene studies identified novel recurrent somatic mutations in patients with AML with biological, clinical and therapeutic significance. In Uruguay, the characterization of AML is dependent on the integration of cytomorphology, immunophenotype, cytogenetics and molecular biology (mutations in FLT3, NPM1, CEBPA and cKIT). This allows stratification in prognostic risk groups and rationalization of therapeutic resources. However, with this molecular markers, complete stratification is not always succesful in patients with normal karyotype ( NK) .

In order to expand the AML genetic markers analysis in Uruguayan patients ( 3.000.0000 persons country ) ; DNA samples from 49 adult patients at the onset of the disease and 3 healthy controls were analyzed by next generation sequencing (NGS, Illumina) using a customized panel of 30 cancer associated genes. After excluding synonymous variants and those with a population frequency >1%, we ended with 493 variants in promoters and intron ends, and 173 in the 28 genes coding regions. Notably, all patients had at least one variant.

While none of the non-coding region variants were reported as pathogenic in COSMIC and/or ClinVar databases, 45 of those falling in coding regions were reported as pathogenic. The pathogenic variants were detected in 36/49 patients and 1 healthy control distributed in 16 genes. The most affected genes were TET2, NRAS, DNMT3, FLT3 and cKIT with 7, 6, 5, 4, 4 variants respectively.

This preliminar findings in this study, highlight the relevance of detecting gene variability underlying prognostic risk subgroups to deliver patient tailored clinical decision support. The correlation between the genetic abnormalities, associated with the clinicopahtological features and the epigenetic studies have prognostic significance, and are continued to be analyzed.

Disclosures

No relevant conflicts of interest to declare.
TALEN®-Based Gene Editing in Hematopoietic Stem Cells
Authors: Busser Brian W , Temburni Sonal , Boyne Alex , Juillerat Alexandre , Poirot Laurent , Duchateau Philippe
Published: October 2019

Autologous gene therapy in human hematopoietic stem cells (HSCs) has shown the potential to transform the treatment of genetic diseases of the blood and immune system. However, viral vector delivery has shown issues with insertional mutagenesis, unregulated transgene expression and durability. Genome editing via homology directed repair (HDR) in human HSCs can mitigate these issues by correcting and repairing the endogenous gene permitting precise spatio-temporal gene expression. Transcription activator-like effector nuclease (TALEN®) are a designer nuclease that enables the site-specific introduction of double-stranded breaks (DSBs) in the genome. Repair of these DSBs occurs largely through one of two pathways, non-homologous end joining (NHEJ) and HDR. NHEJ is an error-prone pathway that often results in insertions or deletions (indels) whereas HDR is a precise mechanism that uses a homologous piece of DNA (usually the homologous chromosome or sister chromatid) to accurately repair the lesion by recombination. However, HDR can be manipulated by supplying an exogenous DNA template with homology arms that start at the DSB as single-stranded oligonucleotides or as double-stranded DNA to introduce any genetic modifications encoded in the template DNA. Recent studies have shown efficient HDR in numerous cell types using adeno-associated virus (AAV) to deliver template DNA. Here we combined TALEN® with AAV containing a repair template to identify the conditions required to efficiently modify disease-relevant alleles in HSCs. We first describe a strategy to remove the nuclease target site in the template DNA even when the target site was located in regulatory sequences. Optimization of the transfection, transduction and culturing conditions of HSCs shows highly efficient allele modification using low doses of AAV and nuclease. Importantly, alleles more often show evidence of HDR as opposed to NHEJ. Further, colony forming unit assays and a xenograft model confirm that modified HSCs are multipotent. Lastly, we show that differentiated progeny of modified HSCs produce similar amounts of mRNA from the modified allele as wild-type unmodified alleles. In total, our results show that combining TALEN® with repair template delivery via AAV is a robust gene editing system for HSCs.

Disclosures

Busser: Cellectis: Employment, Patents & Royalties: Cellectis. Temburni:Cellectis: Employment, Patents & Royalties: Cellectis. Boyne:Cellectis, Inc: Employment, Patents & Royalties: Cellectis. Juillerat:Cellectis: Employment, Patents & Royalties: Cellectis. Poirot:Cellectis: Employment, Patents & Royalties: Cellectis. Duchateau:Cellectis: Employment, Patents & Royalties: Cellectis.