bluuebunny/crossref_metadata_2025 huggingface dataset:
Pathway-Directed High Throughput Drug Screen Identifies PI3K Inhibitors That Synergistically Potentiate Anti-Tumor Activity of HDAC Inhibitors in Mycosis Fungoides and Sezary Syndrome
Authors: Ai Weiyun , Yang Chen-Yen , Faraj Razan , Rakhshandhroo Taha , Afghani Shervin , Pincus Laura , Bandyopadhyay Sourav , McCormick Frank
Published: October 2019

Introduction and Objectives: Mycosis fungoides and Sezary syndrome (MF/SS) represent a group of heterogeneous diseases. Recent studies demonstrated dysregulation of several signaling pathways in MF/SS, including PI3K/AKT, JAK/STAT, RAS and NFkB pathways. We performed a high throughput drug screen to determine the potential of novel agents targeting these pathways for the treatment of MF/SS.

Materials and Methods: We compiled a libraryof 94 compounds targeting pathways known to be relevant in cancer biology. These included kinases involved in growth factor receptor signaling, HDACs, proteasome, DNA repair and regulators of apoptosis. The compounds were screened for anti-proliferative activity against four MF/SS cell lines in high throughput proliferation assays. Selected hits were further studied in xenograft models of MF/SS and in primary T cell lymphomas. Promising candidates from different classes were also tested in combination therapy assays using a matrix block method across dose gradients of each compound designed to detect synergistic activities.

Results: From the high throughput screen, we identified 14 compounds with anti-proliferative activity in MF/SS, including multiple inhibitors of the PI3K pathway. PI3K inhibitors emerged as preliminary hits in this screen and secondary validation assays confirmed the class effect of PI3K inhibitors. From this class, the PI3K inhibitor BKM120 was selected for in vivo studies. In a xenograft model of MF, BKM120 exhibited striking anti-tumor activity measured by a marked suppression of tumor growth and prolonged survival of tumor-bearing mice compared with vehicle control. In a search for even more effective combination therapies, we identified that BKM120 and the HDAC inhibitor class of compounds exhibit synergistic anti-proliferative effects in MF/SS tumor cells. Each of three HDAC inhibitors including LBH, Romidepsin and Vorinosat showed synergistic activity with BKM120, most evident at the GI50 concentrations of each drug, and apparent in both growth inhibition and apoptotic assays.

Conclusion: BKM120 is highly active in preclinical models of MF/SS. Furthermore, it synergistically potentiates the effect of HDAC inhibitors against MF/SS tumor cells. These are highly promising approaches for the treatment of MF/SS and warrant clinical investigation.

Disclosures

No relevant conflicts of interest to declare.
Increased Expression of Circulating Mir-22 in the Peripheral Blood of Patients with Myelodysplastic Syndromes Was Associated with Poorer Prognosis
Authors: Meng Yuesheng , Hua Fanli , Li Ying , Gao Song
Published: October 2019

The molecular pathogenesis of myelodysplastic syndromes (MDS) has not been completely elucidated. Deregulation of expression of some microRNA has been implicated in hematological disorders including MDS. An oncogenic role for miR-22 was recently suggested in MDS in which the expression of miR-22 was increased. However, there were corroborations showing that it could be a tumor suppressor in acute myeloid leukemia. In this study, we examined the expression levels of circulating miR-22 in the plasma of patients with MDS and evaluated its significance in clinical context. The diagnosis of MDS was made according to the WHO classifications and subtypes included 8 cases of RA, 14 cases of RCMD and 6 cases of RARS/RCMD-RS, 10 cases RAEB1 and 7 cases RAEB2. Twenty cases of healthy donors were enrolled as normal control. Written informed consent for sample collection was obtained from all subjects enrolled. EDTA-anticoagulated peripheral blood samples were collected and centrifuged. Circulating microRNAs were purified from the plasma with miRNeasy serum kit (Qiagen). Expression of miR-22 was reverse- transcripted and measured with stem-loop real time quantitative polymerase chain reaction assay. First of all, we proved that the expression of miR-22 was detectable in the plasma of patients with MDS as well as of healthy donors. Our result showed that the expression levels of circulating miR-22 in MDS patients were higher than that in healthy donors (medians 1.360 vs 1.000, P<0.05), among which 13 patients (37.1%) showed marked increase of miR-22 expression (>2 times). Due to the high heterogeneity of MDS and relatively smaller sample size, the subtypes were grouped into two major categories based on IPSS: the low-risk group (including RA, RCMD and RARS/RCMD-RS, totally 18 cases) and the high-risk group (RAEB1 and RAEB2, 17 cases in total). Relative levels of circulating miR-22 were higher in the high-risk group than in the low-risk group (medians= 1.081 vs 2.317, P<0.05). A follow-up study revealed an association of the expression levels of miR-22 with prognosis. Patients with increased expression of miR-22 had poorer clinical response (Chi-square analysis, P<0.05) and lower overall survival rate (Kaplan-Meier analysis, P<0.05). We compared the levels of miR-22 in the plasma with that of mononuclear cells of bone marrow in some MDS patients and found that the relative levels of miR-22 in the plasma and bone marrow cells were not directly co-related (P>0.05), indicating it could be an independent prognostic factor. In summary, this study confirmed an oncogenic presence of miR-22 in MDS. Given the fact that there is a lack of blastic cells in the bone marrow of patients with low-risk MDS but their plasma or serum is easy to obtain, the detection of circulating miR-22 may be of value as a molecular marker in the prognosis of MDS.

Disclosures

No relevant conflicts of interest to declare.
Brca1 Deficiency Causes Bone Marrow Failure and Spontaneous Hematologic Malignancies in Mice
Authors: Vasanthakumar Aparna , Arnovitz Stephen , Marquez Rafael , Lepore Janet , Rafidi George , Davis Elizabeth M. , Asom Anase , Weatherly Madison , Neistadt Barbara , Duszynski Robert , Vardiman James W. , Le Beau Michelle M. , Godley Lucy A. , Churpek Jane E.
Published: October 2019

Background: Fanconi anemia (FA) is caused by mutations in one of seventeen genes that make up the FA DNA double strand break (DSB) repair pathway. Recently, two individuals with biallelic germline BRCA1 mutations, each consisting of one null and one hypomorphic mutation, were identified and noted to have features consistent with FA, including congenital anomalies and increased chromosomal breakage of lymphocytes on exposure to diepoxybutane (Domchek et al. Cancer Discov. 2013 Apr; (4):399-405; and Sawyer et al. Cancer Discov. Epub. 2014 Dec 3.), adding BRCA1 as the newest FA gene. However, neither patient developed bone marrow failure (BMF), making the bone marrow effects of BRCA1 deficiency still unclear.

Methods: To test the hypothesis that Brca1 is also essential in hematopoiesis, we developed a conditional mouse model with Mx-1 Cre-mediated Brca1 deletion and examined the effects of Brca1 deficiency on hematopoiesis in this model.

Results: At baseline, Brca1-/- mice have macrocytic anemia and leukopenia. Further, by 6 months of age, 30% and 50% of the Brca1-/- mice develop spontaneous BMF or hematologic malignancies (HM), respectively. Brca1-/- mice develop a diverse range of HM, including T-cell lymphomas and acute myeloid leukemias, suggesting a defect in an early hematopoietic progenitor population. Methylcellulose colony forming assays also demonstrate a defect in progenitor cell function with Brca1-/- bone marrow cells forming fewer colonies (44.4±31.9) than Brca1+/+ cells (200.3±30.5, p=0.004) at baseline, and show FA-like hypersensitivity to the DNA cross-linking agent, Mitomycin C (MMC) (mean colony survival % at 10 nM MMC 40% versus 82% and at 50 nM 1% vs 56%). Spectral karyotyping of bone marrow cells from mice that developed BMF demonstrated chromatid exchanges and breaks. Similarly, multiple chromosomal translocations were seen in the myeloid leukemia cells, implicating genomic instability in the pathogenesis of these disorders.

Conclusions: Taken together, our results show that loss of Brca1 in murine bone marrow causes hematopoietic defects and MMC sensitivity similar to that seen in humans with FA, providing strong evidence that Brca1 is critical for normal hematopoiesis and that Brca1 is a bona fide FA gene. This novel mouse model provides the opportunity to gain functional insight into the key stage(s) of hematopoiesis that require Brca1 and the effects of Brca1 haploinsufficiency, as seen in humans, on hematopoiesis. Further, as nearly all of the single gene FA mouse models to-date have failed to recapitulate the bone marrow phenotype of human FA, this model will be critical for deepening our understanding of the pathogenesis of FA.

Disclosures

No relevant conflicts of interest to declare.
KIT D816V Mutation Positive Bone Marrow Mesenchymal Stem Cells in Indolent Systemic Mastocytosis Are Associated with Disease Progression
Authors: Garcia-Montero Andres C , Jara-Acevedo Maria , Alvarez-Twose Ivan , Teodosio Cristina , Sanchez-Muñoz Laura , Muñoz-Gonzalez Javier Ignacio , Mayado Andrea , Matito Almudena , Caldas Carolina , Morgado Jose M , Escribano Luis , Orfao Alberto
Published: October 2019

PURPOSE: Multilineageinvolvement of bone marrow (BM) hematopoiesis by the somatic KIT D816V mutation is present in a subset of adult indolent systemic mastocytosis (ISM) patients in association with a poorer prognosis. Here we investigated the potential involvement of BM mesenchymal stem cells (MSC) from ISM patients by the KIT D816V mutation and its potential impact on disease progression and outcome.

METHODS: The KIT D816V mutation was investigated in highly-purified BM MSC and other BM cell populations from 83 ISM patients followed for a median of 116 months. MC clonality was further evaluated in female patients by the pattern of inactivation of the X chromosome (XCIP).

RESULTS: KIT D816V-mutated MSC were detected in 22/83 (27%) ISM patients. All MSC-mutated patients had multilineage KIT mutation (100% vs. 30%, p=0.0001) and they more frequently showed involvement of lymphoid plus myeloid BM cells (59% vs 22%; P =.03) and a polyclonal XCIP of the KIT- mutated BM MC (64% vs 0%; P =0.01) vs other multilineage ISM cases. Moreover, presence of KIT D816V-mutated MSC was associated with more advanced disease features of ISM, a greater rate of disease progression (50% vs 17%; P =.04) and a shorter progression-free survival at 10, 20 and 30 years (P ≤.003).

CONCLUSION: Overall, these results support the notion that ISM patients with mutated MSC may have acquired the KIT mutation in a common pluripotent progenitor cell, prior to differentiation into MSC and hematopoietic precursor cells, before the X-chromosome inactivation process occurs. From a clinical point of view, acquisition of the KIT mutation in an earlier BM precursor cell confers a significantly greater risk for disease progression and a poorer outcome.

Disclosures

No relevant conflicts of interest to declare.
TOP2A Knockdown Resensitizes Carfilzomib-Resistant Hmcls to Carfilzomib
Authors: Reale Antonia , Khong Tiffany T. , Mithraprabhu Sridurga , Savvidou Ioanna , Ramachandran Malarmathy , Spencer Andrew
Published: October 2019

Introduction/background: Multiple myeloma (MM) remains incurable despite the introduction of novel therapeutic agents. Microarray-based technologies were adopted in our study to determine if a genetic signature associated with resistance to carfilzomib, a second-generation proteasome inhibitor already in use in clinical settings, could be identified.

Materials and Methods: 18 genetically heterogeneous human myeloma cell lines (HMCLs) were treated with carfilzomib and a cell viability profile was assessed categorizing the HMCLs as sensitive, intermediate or resistant to carfilzomib. Following categorization gene expression profiling was performed and validated with q-RT-PCR and knockdown assays.

Results: 29 genes were differentially regulated between the sensitive and resistant cell lines. Top genes based on intensity values and biological significance were: LOC731314, TSPAN13, APH1B, TSPYL5, COX7B2, PCSK1N, LRRC38, TCIRG1, TOP2A, ADM2, ITM2A, TSPAN13, STOM, UBE2C, SNHG8. Gene ontology (GO) enrichment analysis identified two pathways that were significantly different between the resistant and sensitive HMCLs; pathogenic escherichia coli infection (p=0.002) and lysosome (p=0.006). Eight GO terms were enriched: 4 related to biological processes and 4 related to cellular components.

TOP2A, an enzyme that controls and alters the topologic states of DNA during transcription and is involved in cell cycle and proliferation, was identified to be overexpressed in resistant HMCLs. It functions as the target for several anticancer agents and a variety of mutations in this gene have been associated with the development of drug resistance. TOP2A may be used as a predictive factor for patient selection for specific protocols or as independent prognostic marker in solid tumors. TOP2A was also overexpressed in the 'proliferation cluster' associated with greater proliferation rate and poor outcome in newly diagnosed MM patients. Suppression of TOP2A by siRNA in carfilzomib-resistant HMCLs significantly resensitised the cell lines to carfilzomib.

Conclusion: Our results suggest that TOP2A is overexpressed in carfilzomib-resistant HMCLs indicating a possible role as a predictive marker of response to carfilzomib in MM.

Disclosures

No relevant conflicts of interest to declare.
In Vivo Response to Cytarabine Chemotherapy Uncovers the Role of the Oxidative and Energetic Metabolism in the Chemoresistance of Human Primary AML Stem Cells
Authors: Aroua Nesrine , Sarry Jean-Emmanuel , De Toni Fabienne , Carson Robin , Vergez Francois , Saland Estelle , Sugita Mayumi , Carroll Martin , Recher Christian , Danet Gwenn
Published: October 2019

Resistant cells to conventional chemotherapy - which targets DNA synthesis - are thought to be rare and enriched in quiescent leukemic stem cells (LSCs) in acute myeloid leukemia (AML). In order to develop an improved understanding of chemotherapy resistance in AML, we analyzed the response to cytarabine (Ara-C) in our xenograft model with 20 primary AML patient specimens from two clinical sites. After demonstrating AML engraftment, mice are treated with Ara-C given IP daily for 5 days as a single agent at 60 mg/kg daily, which correlates with human dosing. In all mice treated with this regimen, there was a 4- to 46-fold cytoreductive effect at 1 week post-treatment. While in vivo Ara-C treatment induces changes in CD34pos CD38pos/neg phenotypes of 50% of AML-engrafted mice, this treatment does not induce any significant changes in cell cycle distribution and does not enrich in G0 quiescent cells in AML-engrafted mice. Surprisingly we observed that Ara-C induces a decrease or no change in the frequency of SL-ICs (control, 1:2,500-21,900 versus Ara-C, 1:4,500-34,200), suggesting that Ara-C would not spare LSCs in vivo and equally kills both cycling and quiescent cells. Furthermore, transcriptomic analysis of Ara-C residual leukemic cells (RLCs) from three different patient-derived xenografts confirmed that RLCs are not enriched in stem cell marker genes at 1 week post-treatment. However, our gene signature of the in vivo AML resistance is enriched in genes involved in inflammatory, immune and stress response. When tested in three independent AML cohorts (Verhaak et al. 2009; TGCA, 2011; Metzeler et al. 2011), this signature is associated with an unfavorable prognosis in patients treated with intensive chemotherapy. Finally we have shown that RLCs are PIMOpos cells with both high ROS content and active mitochondrial mass and membrane potential in AraC-treated xenografted mice compared to control mice. Accordingly, AML cells exhibiting high OXPHOS energetic phenotype are resistant to Ara-C chemotherapy in vivo while low OXPHOS AML cells are sensitive to AraC in NSG mice. Altogether these results suggest a novel model of cytarabine chemotherapy resistance in AML based on the oxidative and mitochondrial energy metabolism in vivo.

Disclosures

No relevant conflicts of interest to declare.
A Point-of-Care Platform for Hematopoietic Stem Cell Gene Therapy
Authors: Adair Jennifer E , Haworth Kevin G , Sauvageau Guy , Heimfeld Shelly , Hocum Jonah D , Trobridge Grant D. , Kiem Hans-Peter
Published: October 2019

Hematopoietic stem and progenitor cell (HSPC) gene therapy holds great promise but requires highly technical and dedicated facilities. Current state-of-the-art requires ex vivo HSPC gene transfer in dedicated Good Manufacturing Practices (GMP) facility infrastructure, limiting treatment to highly developed countries. A patient point-of-care strategy would therefore make HSPC gene therapy available to patients worldwide. We developed a short, semi-automated, mostly-closed platform for ex vivo isolation and lentivirus (LV) gene modification of CD34+ HSPCs from either bone marrow or mobilized peripheral blood sources using the CliniMACS Prodigy® (Figure 1). Experiments were performed with a biosafety cabinet and personal protective equipment to simulate anticipated conditions in clinical facilities of underdeveloped countries. A total of 7 custom programs were developed for bone marrow or mobilized peripheral blood CD34+ cell isolation and LV transduction. Addition of a pyrimidoindole derivative, UM 729, permitted efficient transduction of CD34+ HSPCs in <18 hours. Complete semi-automated production took <34 hours from collection to infusion of the gene modified cell product, requiring very little operator hands-on time. Autologous, LV gene-modified CD34+ HSPCs from two nonhuman primates produced using this platform engrafted and supported multilineage hematopoietic repopulation after myeloablative total body irradiation at 1020 cGy. Total cell doses achieved were 27 x 106 and 5.4 x 106 CD34+ cells/kg body weight, respectively. We observed stable, persistent and polyclonal gene marking in peripheral blood granulocytes up to 40% and lymphocytes up to 15% within 4 months after infusion. Neither animal displayed evidence for increased toxicity, including potential contamination from the cell products. We then validated processing of human bone marrow and mobilized apheresis products from healthy adult donors. We demonstrate efficient isolation of human CD34+ HSPCs and up to 60% transduction efficiency with a clinical-grade LV currently being tested in a phase I clinical trial for treatment of HIV-associated lymphoma. Products tested met current FDA approved specifications for infusion. Xenotransplantation of these products into immunodeficient mice resulted in polyclonal engraftment over 12 weeks. These data demonstrate preclinical safety and feasibility of a patient point-of-care strategy for ex vivo LV gene transfer into HSPCs. This platform represents a major advance in global portability of LV mediated HSPC gene therapy.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
An HSP90 Inhibitor, PU-H71, Antagonizes Stroma-Induced Pro-Survival Effects in CLL through Its Inhibition of Multi-Component B-Cell Receptor Signaling Pathway
Authors: Guo Ailin , Lu Pin , Zhen Chaojie , Chiosis Gabriela , Wang Yue Lynn
Published: October 2019

Background: Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B-cells in the hematopoietic system. The B-cell receptor (BCR) plays an essential role in the pathogenesis of CLL and many components of the BCR signaling pathway are known clients of HSP90. HSP90 is a highly conserved molecular chaperone that ensures the proper folding and stabilization of its client proteins. In this study, we investigated whether PU-H71 a novel purine-scaffold HSP90 Inhibitor, has anti-tumor activity in CLL by destabilizing BCR signaling pathway constituents.

Design: Fresh CLL cells were isolated and cultured ex vivo with or without stromal co-culture. Molecular and cellular events were studied in PU-H71-treated and control CLL cells.

Results: Immunoblotting revealed that a significantly higher amount of HSP90 is present in CLL cells than in peripheral blood mononuclear cells (PBMC), suggesting the chaperone is pathogenically relevant. We found that PU-H71 caused the death of CLL cells in a dose and time dependent manner while the viability of either PBMC or normal B lymphocytes were not affected. PU-H71 induced apoptosis resulting in CLL cell death as it caused mitochondrial cytochrome C release and a decrease in the abundance of several anti-apoptotic proteins. Interestingly, PU-H71 has the ability to counteract the pro-survival effects of the stroma and caused apoptosis in CLL cells co-cultured with stroma. To gain mechanistic insights into how PU-H71 acts, we examined the BCR signaling pathway. We found that the amounts of several key components of the pathway were reduced by PU-H71 treatment. This occurred even in the presence of stromal co-culture. The results suggest that PU-H71 antagonizes the function of HSP90 leading to the destabilization of the BCR signaling transducers. A chemical pull-down experiment revealed the co-existence of the BCR components and HSP90 in the same complex, suggesting these BCR constituents are indeed clients of HSP90 in CLL cells. Further, specific genetic knock-down of the signal transducers by siRNA confirmed their key roles in mediating the survival of CLL cells.

Conclusions: PU-H71 antagonizes stroma-induced pro-survival effects in CLL through its inhibition of the B-cell receptor signaling pathway. Our results suggest that PU-H71 may serve as a useful therapy against CLL and is worth further clinical development.

Disclosures

No relevant conflicts of interest to declare.
Risk of Early Mortality in Patients with Newly Diagnosed Multiple Myeloma
Authors: Liu Chia-Jen , Hsu Pei , Lin Ting-Wei , Gau Jyh-Pyng , Hsiao Liang-Tsai , Tzeng Cheng-Hwai , Chen Po-Min , Yu Yuan-Bin , Chiou Tzeon-Jye , Liu Yao-Chung , Liu Jing-Hwang
Published: October 2019

Background

The overall survival of patients with multiple myeloma has been improved greatly over the last two decades with the advances of treatment. Several studies reported that this improvement in survival has been ascribed to the broader use of novel drugs and autologous tandem transplantation. However, there were still a certain portion of myeloma patients died early after diagnosis. We therefore aim to investigate the risk factors of early mortality (death within 60 days after diagnosis) in patients with multiple myeloma.

Patients and Methods

We included in this study 451 consecutive patients with multiple myeloma, newly diagnosed at an Asian tertiary medical center between January 1, 2002 and April 30, 2015. A total 57 subjects who developed early mortality were identified. Risk factors for early mortality in myeloma patients were collected and analyzed.

Results

Compared with non-early mortality myeloma patients, early mortality patients had higher probability of being male, primary plasma cell leukemia, low platelet count, low serum albumin, high corrected serum calcium, high serum creatinine, high LDH, high serum β2-microglobulin, poor performance status, and high ISS stage. With multivariate analysis, we found that male (adjusted OR 2.93, 95% CI 1.17-7.31), serum albumin < 3.5g/dl (adjusted OR 2.76, 95% CI 1.17-6.52), corrected serum calcium ≥ 12mg/dl (adjusted OR 3.56, 95% CI 1.47-8.63) and LDH ≥ 250U/L (adjusted OR 3.30, 95% CI 1.62-6.74) were significant risk factors of early mortality. Pneumonia represented as the leading cause of early mortality in myeloma patients (n = 18, 31.5%), followed by renal failure (n = 7, 12.2%).

Conclusion

Early mortality rate is high (12.6%) in patients with multiple myeloma. Patients of male gender, low serum albumin, high corrected serum calcium and LDH are at risk of early mortality. More than one third myeloma patients (21 out of 57) who developed early mortality are died of infection. Identifying the risk group and providing prompt intervention, such as prophylaxis antibiotics, may reduce the incidence rate of early mortality and improve the life expectancy of myeloma patients.

Disclosures

No relevant conflicts of interest to declare.
A Bioinformatics Approach Revealed a Differential Expression of RKIP-Related Genes in Pre-Multiple Myeloma and Multiple Myeloma: Clinical Implications
Authors: Shvartsur Anna , Chatterjee Devasis , Chen Haiming , Berenson James , Garban Hermes , Bonavida Benjamin
Published: October 2019

Introduction: Multiple myeloma (MM) is a clonal plasma-cell neoplastic disorder arising from an indolent premalignant disease known as monoclonal gammopathy of undetermined significance (MGUS) that can evolve to asymptomatic pre-MM and later to symptomatic MM. MM is a biologically complex and a heterogeneous disease. The metastatic suppressor/anti-resistance factor, Raf-1 kinase inhibitor protein (RKIP), is poorly expressed in the majority of cancers. In contrast, inactive phosphorylated RKIP is overexpressed in MM and assisting in cell proliferation and resistance. Active RKIP inhibits the RAF/MEK/ERK1/2 and NF-κB pathways.

Rationale: A molecular identification of stage-specific pre-MM and MM biomarkers will support a more precise diagnosis/prognosis, an effective therapeutic regime, and will assist in the identification of novel therapeutic molecular targets.

Objective: To identify RKIP-related gene products in pre-MM and MM as novel biomarkers and therapeutic targets.

Hypothesis: We hypothesized that there would be differential molecular signatures between the pre-MM and MM disease-related to RKIP-regulated genes.

Methodology: We examined transcriptomic datasets on Oncomine platform (Life Technologies) for pre-MM and MM.

Results: Several gene products were found to be commonly overexpressed (e.g., RKIP, Bcl-2 and DR5); and underexpressed (e.g., Bcl-6 and TNFR-II) in both pre-MM and MM, suggesting a plausible distinctive general molecular signature of the disease. Importantly, a significant inverse correlation of differentially expressed pro-apoptotic genes was observed in pre-MM: overexpression of Fas and TNF -α and underexpression of YY1; versus expression of anti-apoptotic genes in MM: overexpression of YY1 and underexpression of Fas and TNF -α.

Conclusion and Future Directions: The findings suggested differential molecular signatures between pre-MM and MM. We are currently analyzing the relationship between RKIP and both overexpressed and underexpressed gene products to identify molecular pathways with cross-talks for the regulation of RKIP.

Clinical Implication: The proposed findings might lead to a more precise diagnosis/prognosis of MM and its disease stages, and can identify novel molecular therapeutic targets for pre-MM and MM.

Disclosures

No relevant conflicts of interest to declare.
Efficacy and Safety of Bortezomib Based Regimens Retreatment in Relapsed Multiple Myeloma Patients: Results from a Retrospective Study.
Authors: Wu Wenjun , Zheng Gaofeng , Han Xiaoyan , Zhao Yi , He Donghua , Yang Yang , He Jingsong , Zheng Weiyan , Sun Jie , Shi Jimin , Wei Guoqing , Huang He , Cai Zhen
Published: October 2019

Objective: To evaluate the efficacy and safety of bortezomib retreatment in relapsed multiple myeloma (MM) patients, who previously responded to bortezomib. Methods: This retrospective observational study included data from 45 patients and evaluated the efficacy and safety of bortezomib based retreatment in these patients, who had achieved at least a partial response (PR) on initial bortezomib therapy in our hospital from May 2006 to May 2013. Results: The overall response rate (ORR) was 71.2%, among them 9% patients achieved CR, 11.1% patients achieved very good partial response (VGPR), 51.1% patients achieved PR. All patients were divided into 3 groups according to the response of initial bortezomib therapy, including CR group, VGPR group and PR group. After bortezomib retreatment, the ORR of the 3 groups was 76.9%, 75% and 62.5%, respectively. According to the response of bortezomib retreatment, the patients were divided into 2 groups: group 1 who at least achieved PR, group 2 who showed no response. The median progression-free survival (PFS) after bortezomib retreatment for group 1 and 2 was 9( 95% confidence interval 7.947~10.051) and 10 (95% confidence interval 8.381∼11.619) months, respectively (P>0.05), while the median overall survival (OS) after bortezomib retreatment was 71 (95% confidence interval 66.694∼75.306)) and 37 (95% confidence interval 1-28) months, respectively (P<0.05). In patients with bortezomib retreatment had different degrees of adverse events (AE) , the most AE for grade 1~2. The most common grade ≥3 AE was thrombocytopenia, neutropenia and anemia. The incidence rate of grade ≥3 AE peripheral neuropathy bortezomib was 15%. Conclusion: Bortezomib based regimens retreatment was effective and tolerable in relapsed MM patients, who had achieved at least a partial response (PR) on initial therapy. The incidence rate of AE was not significantly increased.

Disclosures

No relevant conflicts of interest to declare.
CD38 Protects Tumor Cells from NK Lysis
Authors: Wang Anlai , Yang Guang , Song Zhili L , Adrian Francisco J
Published: October 2019

Expression of the type II glycoprotein CD38 in hematological malignancies is abnormally high compared to that in normal cells of hematopoietic origin. Across all malignancies, the highest CD38 expression levels are detected on the surface of multiple myeloma (MM) cells. The high CD38 level has prompted the development of anti-CD38 antibody-based therapies, like daratumumab and isatuximab (SAR650984), that are promising therapeutics for the treatment of patients with MM. Cellular functions associated with CD38 include co-receptor mediated signaling, cell adhesion and ecto-enzymatic conversion of NAD to modulate calcium signaling and adenosine synthesis; but other than allowing antibody-mediated engagement of innate immunity, the role of CD38 in MM is not fully understood.

In order to investigate a potential immune-protective role for CD38, we evaluated a panel of 15MM and 6 diffuse large B-cell lymphoma (DLBCL) cell lines with varying levels of CD38 for their susceptibility to NK-mediated lysis. Briefly, calcein AM pulsed target cells were incubated with NK-92.CD16 NK cells (E:T=3:1) for 1 hr and cell lysis measured as a function of the fluorescent calcein released into the supernatant. In an initial experiment, we observed that the percentage of cell lysis for all lines with >100,000 CD38s per cell was low, below 10%. The cell lines with <100,000 CD38s per cell were more susceptible to NKs, with a median percentage cell lysis of around 25%. The observed levels of cell lysis were not correlated with the levels of released IFNγ or TNFα.

The differential lytic activity against the target cells led to the hypothesis that CD38 might have a role in protecting the tumor cells from NK-mediated lysis. To investigate this, we first knocked down CD38 in SUDHL-8 cells using shRNA, decreasing the levels of CD38 from 246,000 to 51,000 per cell. This resulted in an increase in the percentage of lysed cells from 25% to more than 50%, likely reflecting a loss of protection against the NK cells. We next overexpressed CD38 in U266 cells, increasing the CD38 levels from 9,000 to 327,000 per cell. As a result, the percentage of lysis decreased from more than 60% to around 30%, indicating that U266 cells are now more resistant to the lysis by NK cells. The protection that CD38 seems to confer to CD38-overexpressing U266 cells is completely lost by addition of isatuximab which triggers CD16-mediated lysis (antibody dependent cellular toxicity, ADCC).

Whether the protective function of CD38 described here is a direct or an indirect protection mechanism is currently under investigation.

Disclosures

Off Label Use: isatuximab (SAR650984, anti-CD38 antibody). Song:Sanofi: Employment. Adrian:Sanofi: Employment.
Phase 2 Trial of Concurrent Monosialotetrahexosylganglioside in the Prophylactic Treatment of Bortezomib-Induced Peripheral Neuropathy in Patients of Multiple Myeloma
Authors: Wang Liang , Xia Zhongjun , Chen Xiaoqin
Published: October 2019

Backgrounds

Bortezomib is an important drug in the treatment of multiple myeloma (MM), and peripheral neuropathy (PN) is a significant dose-limiting toxicity of bortezomib. No effective prophylaxis has been defined for PN. Monosialotetrahexosylganglioside (GM), a nerve-protecting drug, is often used to promote growth of nerve and function restoration of damaged nerve. The role of GM in the prophylaxis of bortezomib-induced PN in MM patients has never been investigated.

Methods

A phase 2 clinical trial was conducted in newly diagnosed MM patients to evaluate the value of GM in the prophylaxis of bortezomib-induced PN. All eligible patients were treated with VD (bortezomib 1.3mg/㎡,subcutaneous injection, d1 ,8,15,22, and dexamethasone 40mg, po,d1 ,8,15,22, 4 weeks a cycle) or CyBorD (cyclophosphamide 300mg/㎡,po,d1 ,8,15, bortezomib 1.3mg/㎡,subcutaneous injection, d1 ,8,1 5,22, and dexamethasone 40mg, po,d1 ,8,15,22, 4 weeks a cycle) for at least 4 cycles. GM was used at a dosage of 100mg/day intravenously at d1 -2, 8-9, 15-16, 22-23. No other nerve-protective drugs or thalidomide-containing regimens were allowed. The primary endpoint was overall incidence rate of PN (the grade of PN was recorded according to CTCAE v3.0). The secondary endpoints included duration of PN, complete response rate after 4 cycles of treatment, 1-year PFS and OS rate. (This trial was registered in ClinicalTrial.gov, NCT02093910).

Results

From February 2014 to February 2015, 25 patients of newly diagnosed MM were enrolled. The median age was 55 years old (37-75), and male to female ratio was 19:6. 5 patients had ISS stage I disease, 6 patients with stage II, and the remaining 14 patients with stage III. All patients received a median of 4 cycles (range 2-9) of Bortezomibcontaining regimens. At the time of data analysis, 84% of patients had at least partial response, 48% had at least very good partial response, and 24% had complete response. 7 patients experienced PN after a median of 2 cycles (range 1-4) of treatment, resulting in the overall PN rate of 28%. Among these 7 patients, only 1 patient (4%) had grade 2 PN, leading to dose reduction of bortezomib, and all other patients had grade 1 PN. During treatment, 1 patient (4%) had grade 2 diarrhea, and another 1 patient (4%) had herpes zoster infection. The concurrent use of GM did not introduce new side effects and seemed not compromise the efficacy of bortezomib. At a median follow up time of 8 months, 1-year PFS rate and OS rate were speculated to be 69.8% and 100%, respectively.

Conclusions

The early-term analysis of this phase 2 trial found it feasible to concurrently use GM and bortezomib-containing regimens, and GM had the potential role of reducing bortezomib-induced PN rate and severity without compromising efficacy. This needs to be validated in future phase 3 randomized clinical trials.

Disclosures

No relevant conflicts of interest to declare.
The Impact of Driver Mutations of JAK2, Calr, or MPL in Patients with Myelofibrosis Undergoing Hemopoietic Stem Cell Transplantation
Authors: Bacigalupo Andrea , Pozzi Sarah , van Lint Maria Teresa , Bregante Stefania , Barosi Gianni , Pietra Daniela , Rumi Elisa , Milanesi Chiara , Cazzola Mario
Published: October 2019

Introduction. The vast majority of patients with primary myelofibrosis (PMF) have been shown to carry driver mutations of JAK2, CALR (calreticulin gene) or MPL ì, and these molecular leasions may impact the clinical course of the disease. In a recent paper (Blood 2014;124:1062) patients with CALR mutation, had a lower risk of developing anemia, thrombocytopenia, marked leukocytosis, thrombosis and showed better overall survival, than either JAK2 -mutant or triple-negative patients.

Aim of the study. The aim of the present study was to assess whether CALR exon 9 mutation, conferred a survival advantage in MF patients undergoing a hemopoietic stem cell transplant (HSCT).

Patients. All patients were selected for transplant by two authors (AB and GB) and grafted in Genova.

Patients are outlined in 2 groups: 15 with CALR mutation and 31 with wild-type CALR; the latter included 21 patients with JAK2 (V617F), 7 patients with MPL mutation and 3 patients triple negative. There were no significant differences, between the 2 groups in terms of DIPSS, spleen size, circulating CD34+ cell count, donor type, year of HSCT and conditioning regimens.

Mutation status. Granulocyte JAK2 (V617F) mutation status and mutant allele burden were assessed using a quantitative polymerase chain reaction (qPCR)-based allelic discrimination assay on a Rotor-Gene 6000 real-time analyzer (Qiagen), as previously described.1,2 Patients without JAK2 (V617F) were evaluated for MPL exon 10 mutations using a high-resolution melt (HRM) assay or Sanger sequencing.3,4 Patients with nonmutated JAK2 and MPL were studied for CALR exon 9 mutations as reported in our original paper or by Sanger sequencing, as described elsewhere.4

Results. With a median follow up of over 500 days for both groups, we could find no significant differences in CALR -mutated patients as compared to the remaining ones: transplant related mortality (26% vs 29%, respectively); relapse (20% vs 39%, p=0.2), relapse related death (20% vs 22%) and survival (47% vs 52%, p=0.7).

When dissecting the patients with wild-type CALR, we could indeed see a survival advantage for the small number of MPL -mutated patients (n=7): when these were compared against all the others (n=39), again with similar disease and transplant characteristics, survival was 86% vs 44% (p=0.04) and relapse 14% vs 36% (p=0.2). In multivariate analysis, DIPPS was the only predictor of survival.

Conclusions. These data suggests that CALR mutation does not confer a survival advantage in PMF patients undergoing an allogeneic HSCT. MPL mutation, on the other hand, though present in a very small number of patients, may be worth investigating further in a larger number of patients.

Disclosures

No relevant conflicts of interest to declare.
Reconstitution of Epstein-Barr Virus-Specific T Lymphocytes at the Early Stage of Allogeneic Stem Cell Transplantation
Authors: Lin Ren , Fan Zhiping , Zhao Ke , Jiang Qianli , Sun Jing , Liu Qifa
Published: October 2019

Backgroud:

Epstein-Barr virus (EBV) infection/reactivation is an common complication following allogeneic stem cell transplantation (allo-HSCT) which usually occure at the early stage after transplantation. EBV-specific cytotoxic T lymphocytes (EBV-CTLs) are considered to provide protection from EBV infection/reactivation and associated diseases in recipients of allo-HSCT. Herein, we evaluated the levels of EBV-CTLs early after transplantion and investigated the associaton between the reconstitution of EBV-CTLs and EBV infection/reactivation after allo-HSCT.

Methods:

Thirty patients undergoing allo-HSCT between November 2013 to September 2014 were enrolled in this study. Of these patients, 16 received HLA-matched donor and 14 received HLA-mismatched transplantation. Antithymocyteglobulin was given in 20 patients. EBV-CTLs were tested using Enzyme-linked immunospot assay (ELISPOT) presenting 9 peptides derived from 3 EBV proteins in +30, +90 and +180 days after transplantation.

Results:

EBV-CTLs, although low levels, were detected in 90% (27/30) patients in +30 days after HSCT. In most patients, EBV-CTLs levels in +90 days was significantly higher than +30 days and returned to normal levels within +180 days after HSCT. Thirteen patients developed EBV infection/reactivation within the median time of 48 (range, 23-110) after transplantation. EBV-CTLs levels had no significant difference among the patients with or without infection/reactivation in +30 days while the patients with EBV infection/reactivation had higher EBV-CTLs levels than those without in +90d after HSCT.

Conclusion:

Reconstitution of EBV-CTLs seems rapid in both HLA-matched and -mismatched donor transplantation. The association between EBV infection/reactivation and EBV-CTLs levels at monthly intervels after HSCT should be studied further.

Disclosures

No relevant conflicts of interest to declare.
The Relationship Between Some Cytokines and Graft Versus Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation in Thalassemia Major Patients
Authors: Wu Xuedong , Chen Libai , Xu Yanjun , Wen Jianyun , Ruan Yongsheng , He Yuelin , Li Chunfu
Published: October 2019

Background: Research has shown that the relationship between cytokines and graft versus host disease is close, but there are still a lot of cytokines and graft versus host disease relationships which are not clear. At the same time, thalassemia patients with graft versus host disease may exist in particularity.

Objects: To investigate the relationship between some cytokines and graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in β-thalassemia major patients.

Methods: The study included 16 patients who developed graft-versus-host disease (GVHD) after receiving hematopoietic stem cell transplantation in the Department of Pediatrics in Nanfang Hospital between the time period of 2011 July to 2015 March. We observed the dynamic variation of interleukin 6 (IL-6), interleukin 8(IL-8), interleukin 12(IL-12), tumor necrosis factor-α(TNF- α) and macrophage migration inhibitory factor(MIF) in 16 patients before onset of GVHD, when GVHD occurred, at fourth and seventh day after onset of GVHD, and when GVHD almost disappeared.

Results: There was a significant difference in serum levels of interleukin-6, interleukin-12, tumor necrosis factor-α, macrophage migration inhibitory factor at different times. The highest levels of different cytokines appeared when GVHD occurred, followed by those at seventh day after it occurred. There was also a significant difference in serum levels of interleukin-8 at different times, and the highest level appeared at fourth day after GVHD occurred. There is a significant positive correlation between IL-6 and IL-12,IL-12 and TNF-α,IL-12 and MIF,TNF- α and MIF when GVHD occurred. There is a significant positive correlation between IL-12 and TNF-α when GVHD occurred and on the fourth day . There is a significant positive correlation between IL-12 and TNF-α,IL-12 and MIF,TNF-α and MIF when GVHD appeared on the seventh day. There is a significant positive correlation between IL-6 and IL-12,IL-12 and TNF-α,IL-12 and MIF,TNF-α and MIF when GVHD almost disappeared.

Conclusion: The dynamic expression of IL-6,IL-8,IL-12,TNF-α and MIF can estimate the immune function of β-thalassemia major patients who develops graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, and can be used as the immunobiology indicators for the early diagnosis of graft-versus-host disease. IL-6, IL-8, IL-12, TNF-α and MIF may have correlation with each other in different time of graft versus host disease.

Disclosures

No relevant conflicts of interest to declare.
Complete Resolution of Extensive Chronic Graft-Versus-Host Disease with Ibrutinib
Authors: Scholoff Audrey , Obi Gloria , Baker Kelty R. , Carrum George , Kamble Rammurti T
Published: October 2019

We herein document a complete response of extensive chronic graft-versus-host disease (cGvHD) to a Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib. A 41 years old female with primary refractory MCL underwent mismatched unrelated donor (MMUD) allogeneic hematopoietic stem cell transplantation in December 2011 (conditioning with CY/TBI and alemtuzumab, graft=6.6x 106/kg CD-34+ cells, tacrolimus alone for GVHD prevention). Following engraftment on day 11, she developed grade III acute GvHD involving the skin and gut on day 17 of transplantation that persisted beyond 100 days post-transplant. Her cGvHD was treated with steroids, but remained active and extensive. Despite persistent cGvHD and 100% donor chimerism she relapsed in July 2012. Treatment with radiation and bendamustine with rituximab failed. By December 2013, the patient had extensive cGvHD manifesting as scleromatous skin thickening, oral ulcers and sclerosis of the buccal mucosa, ocular dryness and diarrhea, and was started on ibrutinib1 560 mg once daily for relapsed MCL. After 8 weeks of therapy, cGvHD had begun to improve. Oral steroids were reduced and ultimately stopped after 26 weeks of ibrutinib; after 30 weeks treatment all cGvHD manifestations resolved completely. A complete remission for MCL was documented at 8 weeks of ibrutinib initiation. Currently she continues to be on 560 mg daily ibrutinib without cGvHD exacerbation or MCL relapse for 22 weeks and 52 weeks, respectively. Chronic graft versus host disease (cGvHD) is mediated donor T cells. The role of B cells in the pathogenesis of cGvHD is increasingly recognized. Two murine studies have explored the role of ibrutinib in cGVHD-like syndromes, one in which there is T cell driven sclerodermatous cGvHD and a second in which there is Ab driven multiorgan system cGvHD that includes bronchiolitis obliterans (BO). Administration of ibrutinib decreased the incidence and severity of sclerodermatous, and improved pre-existing lesions and also improved pulmonary fibrosis and reduced BO. Animals lacking BTK and ITK did not develop cGVHD, indicating that these molecules are critical to cGVHD development. Our report provides the evidence that BTK inhibition led to complete resolution of cGvHD and supports exploration of its role in future clinical trials.

Disclosures

No relevant conflicts of interest to declare.
Pretransplantation Positron Emission Tomography Predicts Outcome Following Autologous Stem Cell Transplantation for Relapsed or Refractory Hodgkin Lymphoma
Authors: Dulery Remy , Marjanovic Zora , Aoudjane Malek , Corre Elise , Lemonnier Marie-Paule , Vekhoff Anne , Coppo Paul , Mohty Mohamad
Published: October 2019

Despite high-dose therapy followed by autologous stem cell transplantation (ASCT), roughly 50% of patients with relapsed or primary refractory Hodgkin lymphoma can be cured. To identify poor risk patients who could benefit from post-ASCT treatments, we carried out an analysis of 32 patients treated at Saint-Antoine Hospital. The study included 18 males and 14 females with a median age of 31.8 years (range, 17.5-64.8). Ann Arbor staging was I-II in 14 patients and III-IV in 18 patients. Only 2 patients did not have adverse prognosis factors at diagnosis. Sixteen patients had received more than 1 salvage therapy (ST) prior ASCT, among whom 4 patients had received Brentuximab-Vedotin. Patients were evaluated with computed tomography and fluorodeoxyglucose-positron emission tomography (PET) prior to ST and again before ASCT. Relapse had occurred less than 12 months after the 1st treatment in 17 patients. Twelve patients had never achieved complete remission before ASCT. Disease status at transplant was complete remission in 16 patients, partial response in 16 patients and progressive disease in 1 patient. TEP was positive in 4 patients.

After a median follow-up of 66,9 months (12.6-139.4), the cumulative incidence of relapse was 43.75%, the event-free survival (EFS) 50% and the overall survival (OS) 81.2%. PET status before ASCT clearly identified poor risk patients (5-year OS 25% and 89.3% for PET-positive and negative patients respectively, p=0.002) and was the only significant factor associated with OS by multivariate analysis [HR: 6.52; 95% CI 1.3-32.67] (p=0.023).

In conclusion, pre-ASCT PET status appears to be the main prognosis factor in patients with relapsed or refractory Hodgkin lymphoma. Studies evaluating new conditioning regimens, post-ASCT maintenance with Brentuximab-Vedotin or tandem allogeneic stem cell transplantation are warranted for pre-ASCT PET-positive patients.

Figure 1. Overall survival according to TEP status at transplant (log-rank P value) Figure 1. Overall survival according to TEP status at transplant (log-rank P value)

Disclosures

Coppo: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.