bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting HDAC6 Is a Novel Approach to Augment the Therapeutic Benefit of Bromodomain Inhibition in Multiple Myeloma
Authors: Carew Jennifer S. , Espitia Claudia M. , Zhao Weiguo , Kelly Kevin R. , Nawrocki Steffan T.
Published: October 2019

Multiple myeloma (MM) is a plasma cell disease that represents the second most common adult hematologic malignancy in the United States. Advances in the treatment of MM, including the development of proteasome inhibitors have improved clinical outcomes. However, many patients fail to respond to these newer agents or relapse after initial response. Constitutive activation of the MYC oncogene is a frequent pathogenic event in MM that drives disease progression making it an ideal therapeutic target. Recent studies suggest that inhibition of bromodomain and extra terminal (BET) protein family members including BRD4 decreases the expression of c-MYC and other key oncogenic factors such as BCL-2 and displays significant activity against MM. Here, we demonstrate that shRNA-mediated knockdown of BRD4 or treatment with the BRD4 antagonist JQ1 decreased the expression of c-MYC, BCL-2, and BCL-XL, diminished cell viability, disrupted clonogenic survival, and triggered apoptosis in a panel of MM cell lines. Subsequent assays conducted in MM cell lines confirmed a strong reduction in MYC levels and activity following treatment with JQ1 along with consistent increases in the expression of genes regulated by BRD4 including CDKN1A and HEXIM1. Interestingly, gene expression profiling assays revealed that the histone deacetylase HDAC6 was also highly significantly elevated in all MM cell lines and primary patient specimens treated with JQ1. Based on its ability to alter cellular survival capacity, we hypothesized that HDAC6 induction may reduce the anti-myeloma activity of JQ1. To test this hypothesis, we utilized shRNA-mediated knockdown of HDAC6, the pan-HDAC inhibitor vorinostat, and the HDAC6-selective inhibitor ACY-1215 (rocilinostat) and evaluated the impact of each on the anti-MM effects of JQ1. Abrogation of HDAC6 activity synergistically enhanced the activity of JQ1 in a panel of MM cell lines. These effects were also observed in primary CD138+ cells obtained from patients with MM in a manner that was not affected by prior treatment history. The increased efficacy of these therapeutic combinations was associated with apoptosis induction as evidenced by enhanced caspase-3 cleavage and further reductions in c-MYC expression. These data suggest that HDAC6 induction may represent a key mechanism that promotes drug resistance and/or limits the efficacy of bromodomain inhibitor therapy. Our collective findings indicate that abrogation of HDAC6 activity with ACY-1215 or vorinostat is a novel and promising approach to augment the efficacy of bromodomain inhibitors for MM that warrants further investigation.

Disclosures

No relevant conflicts of interest to declare.
Increased Levels of Thrombopoietin and IPF in Patients with Iron Deficiency Anemia
Authors: Kawasugi Kazuo , Yamamoto Tadashi , Shirafuji Naoki , Oka Yoko
Published: October 2019

Background

Thrombocytosis has been reported in about 50% of patients with iron deficiency anemia (IDA). However, the mechanism of the increase in the number of platelets is unknown. To investigate the relationship of endogenous thrombopoietin (TPO) and circulating platelet counts, we measured the plasma levels of TPO, immature platelet fraction (IPF) and erythropoietin ( EPO ) in 25 patients with IDA.

Patients and methods

Patients with IDA (newly diagnosed) were recruited from February 2013 through March 2014 in Teikyo University School of Medicine. TPO (Immuno-Biological Laboratory, Japan), was determined by an EILSA. IPF was measured on the Sysmex XE2100. EPO (Roch Diagnostic Japan) was determined by an EILSA.

Results and Conclusions

The number of platelets was increasing in the half patients with IDA in this study. Peripheral blood samples for measuring IPF were obtained from 300 healthy adult controls (160 women and 140 men) and 20 patients with IDA, consisting of 19 women and 6 men with age ranging from 16 to 75 years. The etiology of iron deficiency was gastrointestinal (GI) blood loss, menometrorrhagia, previous GI surgery, and low dietary iron intake. The mean absolute-IPF was significantly higher for IDA patients than for healthy control (8193±571 vs. 4254±193 p＜0.05). The endogenous levels of TPO were significantly elevated in patients with IDA (216.4±41 pg/ ml) as compared with normal controls (116.5±23 pg/ml). Also, The endogenous levels of EPO were significantly elevated in patients with IDA as compared with normal controls. There was a significant inverse correlation between serum Fe levels and platelet counts (r=0.551, p＜0.05). Also, a positive correlation (r=0.311, p＜0.05) between platelet counts and endogenous level of TPO was found. Moreover, there was a significant inverse correlation (r=-0.458, p＜0.05) between Hb and TPO. These results suggest that TPO is active regulator, in thrombopoiesis and megakaryocytopoiesis in patients with IDA. Also, it appears there is relationship between erythropoiesis and TPO.

Disclosures

No relevant conflicts of interest to declare.
Clinical Research on Hematological Malignancies Complicated with dic-a Single Center Report in China
Authors: Wei Qing , Niu Ting
Published: October 2019

Objective This study aimed to explore the most appropriate way of diagnosis and treatment among the patients with hematological malignancies complicated with disseminated intravascular coagulation (DIC) by evaluating the risk factors which would affect the prognosis and the efficiency of therapies.

Methods The data of 162 definitely-diagnosed patients in West China Hospital between 2009 and 2013 has been collected and analyzed in our retrospective study. We have summarized the information on clinical features, laboratory indicators and interventions, which has been statistically analyzed to evaluate the effect on mortality.

Results Elderly patients, AML patients accompanied with hyperleukocytosis, APL patients accompanied with differentiation syndrome combined deteriorated DIC symptoms, patients accompanied with infections, refractory/relapsed patients, patients with any of the following clinical symptoms such as gastrointestinal bleeding, intracranial hemorrhage, more than three sites of bleeding, acute renal dysfunction,acute respiratory dysfunction, acute left heart failure, MOF or shock, presented higher mortality (P<0.05).We found that there was difference in terms of mortality when we defined the cut-off value of PLT as 10×109/L (P<0.05). According to the ISTH criteria or Chinese guideline, the patients with abnormal PTAAPTTA AT-III, or ISTH score>6 had poorer prognosis. In our study, we tried to classify Fig < 1 g/L or > 4 g/L as a Fig abnormal group, the rest of the patients classified as a Fig normal group, then we found the abnormal Fig group had an increased mortality (P < 0.05). In addition, it had been found that patients who received standard chemotherapy had lower mortality (P < 0.05). With respect to non-APL patients, ones who took anti-fibrinolysis agents had similar mortality compared with the patients who didn't (P>0.05).

Conclusions Among the patients of hematological malignancies complicated with DIC, the patients with any of above clinical risk factors or abnormal laboratory indicators would have poorer prognosis. Moreover, the cut-off value of PLT should be dropped when we diagnose or evaluate this kind of patients’ prognosis. We put forward a new cut-off value of Fig as described above and it is valuable to evaluate the prognosis. The treatment of underlying diseases and substitute therapy act as basis of these patients, however, the adverse effects should be pay attention to as well as infections and acute heart failure. No benefit has been shown in the non-APL patients treated with anti-fibrinolytic agents.

Disclosures

No relevant conflicts of interest to declare.
Management of Major Bleeding Caused By Rivaroxaban and the Use of Desmopressin
Authors: Jajeh Ahmad
Published: October 2019

Rivaroxaban is a new anticoagulant that is substituted for Coumadin on a large scale in the treatment and prevention of Deep Vein Thrombosis DVT and Pulmonary Embolism PE. It is an oral agent that inhibits Factor Xa. The most attractive attribute of this new anticogulant is the lack of monitoring PT/INR. However, out of many cases put on Rivaroxaban a few reports of major and threatening bleed that could be fatal. Particularly, the the GI bleeding. Unfortunately, no set standard antidote or management is available when such catastrophic bleeds happen. This abstract present our experience with three major bleeding cases that presented with massive GI bleeding. Two are associated with peptic ulcer upon Upper GI endoscopy. Two males and one female age 60, 71 (males) and 71 (female). The first two patients were treated with Prothrombin complex product. The female patient presented with sever anemia of 4 grams of Hb with hematemesis and bright red blood per rectum. The Prothrombin complex product was not readly available . She was given multipe doses of Fresh Frozen Plasma FFP and multiple units of packed red blood cells. She was also given a product Profilnine which contains Factor II, IX and VII. Patient's coagulation profile of PTT, PT and Thrombin time were corrected. However, she continue to have bright blood per NG suction. Upon receiving D-DAVP Desmopressin 0.3 micrograms per Kg she stopped bleeding and EGD was done later with sclerosing treatment of gastric ulcer and ligation. Patient was given later a small dose of Prothrombine complex when was available since the last dose of Rivaroxaban was given less than 13 hours from her presentation to the hospital. All of the mentioned patients had prolongation of PT/INR/PTT at presentation. Thrombin time was monitored in all of them. All patients had survived the magor GI bleeding. D-DAVP were given to all of them.

In conclusion D-DAVP Desmopressin should be considered as an adjuvant drug in patient presentong with major GI bleeding secondary to Rivaroxaban.

Disclosures

No relevant conflicts of interest to declare.
The Prospective Use of Prothrombin Complex Concentrates in an Administrative Region of the Province of Quebec.
Authors: Bogaty Chloë , Breton Carolle , Laliberte Johanne , Marceau Daniele , Marceau Daniele
Published: October 2019

Introduction:

Prothrombin complex concentrates (PCCs) are used in the rapid reversal of warfarin and all other vitamin K deficiencies in patients with a major bleed or who require an urgent procedure. The Local Advisory Committee on Transfusion Medicine standardised a protocol for the use of PCCs, outlining the indications and contra-indications for their use.

Methods:

The charts of all patients having received PCCs between a two year period at the 4 hospital centers of region 12 were reviewed to evaluate the respect of guidelines, adherence to the protocol, and the clinical outcomes of patients.

Results:

From Nov. 2009 to Nov. 2011, 129 patients were treated with PCCs: 97 (75%) in the emergency department, 14 (11%) in the intensive care unit, 16 (12%) on medical or surgical floors, and 2 (1.5%) in the operating room. 116 prescriptions (90%) respected the guidelines; among the extra, 4 (3%) were for elective surgeries and 3 (2.3%) for an elevated INR without evidence of bleeding. One patient did not have an INR prior to administration, 10 (8%) did not have a repeat INR 30-minutes post-administration, and Vitamin K was omitted in 10 cases (8%). The prescription is based on weight: 120 (93%) received the correct dose, 3 (2.3%) received too high a dose, and 5 (3.8%) received too low a dose. The clinical outcome was favourable in 94 cases (73%), with rapid reversal of the INR and eventual discharge from hospital. 22 (17%) died during hospitalization and 10 (8%) were lost to follow-up due to transfer to a neurosurgical facility. The INR was less than 1.4 in 86% of cases 30-minutes post-administration. 6 (4.6%) thromboembolic complications occurred: two pulmonary embolisms, two deep vein thrombosis, one stroke, one disseminated intravascular coagulation. Five had resumed warfarin, but none had a therapeutic INR.

Conclusion:

Our review demonstrates that the majority of physicians correctly apply the recommended practice guidelines for the use of PCCs, that these are effective at rapidly reversing the INR, and are associated with favourable clinical outcomes in the majority of cases. However, thromboembolic events are frequent, emphasizing that anticoagulation therapy should be resumed quickly once clinically appropriate

Disclosures

No relevant conflicts of interest to declare.
Over-Expression of Heme Oxygenase-1 in Peripheral Blood Predicts the Progression and Relapse Risk of Chronic Myeloid leukemia
Authors: Wang Jishi , Wei Sixi , Wang Yating , Chai Qixiang , Fang Qin , Zhang Yaming , Lu Yinghao
Published: October 2019

Background There are limited eligible clinical markers at present to monitor the progress of chronic myeloid leukemia (CML). Heme oxygenase-1 (HO-1), as one of the most important oxidation-regulating enzymes in vivo, suggests the onset and progression of cancer when highly expressed. Furthermore, HO-1 level is related with the occurrence and development of hematological diseases. But the relationship between HO-1 expression and progression/relapse of CML has seldom been studied hitherto. This study aimed to investigate the relationship between them to find out a new molecular marker for prediction.

Methods A total of 60 peripheral blood and bone marrow (BM) samples from 25 CML patients in different phases were collected respectively to detect the expressions of HO-1 and bcr/abl using real-time PCR. Routine blood test was performed to detect the changes of leukocyte and platelet counts. The proportion of primitive cells in BM was detected by flow cytometry. The relationship between high HO-1 expression and CML progression and relapse was explored by the analysis of variance by Wilcoxon test and linear regression analysis. The diagnostic accuracy and cutoff values were determined by receiver operating characteristic curve.

Results Relative expression of HO-1 mRNA in CML patients peripheral blood was significantly higher than that of donors (P <0.0001), which were 0.57±3.78 and (1.417±1.125)×10–6, respectively. HO-1 expression level in CML patients was 0.061 5±0.062 4, which decreased to 0.009 4±0.006 7 upon CMoR, and remained remarkably higher 0.016 3±0.017 5 than that of normal donors (1.417±1.125)×10–6, P <0.001. When relapse occurred, HO-1 expression significantly increased from 0.020 6±0.021 0 to 3.852±10.285 in CMoR stage and undergoing relapse. According to progression of CML, HO-1 expression level in CML patients increased from CP (0.009 5±0.017 6) to AP (0.028 0±0.055 7) and then to BP (0.276 7± 0.447 0) . And there was a linear correlation between HO-1 expression and proportion of primitive CML cells. The diagnostic accuracies and cutoff values of HO-1 expression for CML-CP, CML-AP, and CML-BP were 1.0, 0.748, and 0.965, respectively, as well as 0.000 070, 0.001 917, and 0.020 696, respectively.

Conclusion HO-1 may be a potential molecular indicator for the progression and relapse of CML.

Disclosures

No relevant conflicts of interest to declare.
Synergistic Lethal Effects Between Gemcitabine and Arsenic Trioxide on Non-Hodgkin Lymphoma Cell Lines Is Associated with Modulation of PI3K/Akt Signaling Pathway
Authors: Zhang Mingwan , Xu Xiaojun , Ye Yongbin , Guo Ziwen , Bai Xiaochun , Wang Jing , Zhou Ruiqing , Qiu Dafa , He Huiqing , He Xin , Yi Wenfang , Weng Guangyang , Kunyuan Guo
Published: October 2019

OBJECTIVE: To explore the synersistic lethal effects of gemcitabine(GEM) with arsenic trioxide(ATO) on proliferation and apoptosis in the non-Hodgkin lymphoma cells ,and the major molecular signaling pathway involved in these effects.

METHODS: Human non-Hodgkin lymphoma cell lines Jurkat and Raji were used to determine the effects of GEM and ATO (alone or in combination) in vitro, as well as the PBMCs.Cell proliferation was measured by CCK-8 analysis. Cell cycle distribution and apoptosis were monitored by flow cytometry. The self-renewal and proliferating ability of cells were examined by methylcellblose conlony-forming units(CFU)assay.The activation of casepase-3, Bcl-2, and Akt was assessed by Western blot analysis.

RESULTS: ATO and GEM inhibited cell viability in a dose- and time-dependent manner, and induced cell cycle arrest and apoptosis.

The combination of ATO and GEM displays a synergistic or at least additive antitumor activity in vitro against cells than each single treatment. An obvious inhibition of NHL cells proliferation and an increased population of cells in apoptosis were observed in the combination treatment group, but not on PBMCs. ATO combined GEM at IC20 concentration did caused 50% inhibition of NHL cells proliferation. Afterwards, the rest of NHL cells were strongerly arrested in G0/G1 stage by ATO and GEM than each single treatment. Additionally, combination treatment also significantly reduced the ability of colony formation in NHL cells than treatment with either agent alone. Moreover, ATO blocked the Akt signaling pathway, subsequently inhibited Bcl-2 expression, reactivated cleaved caspase-3, while GEM did not. The combination group augmented these effects than each single agent.

CONCLUSIONS: ATO and GEM might be a potential candidate of the combination therapy for treatinghumanrefractory and relapsed malignant lymphoma, which also might be a salvage approach for patients who will be autologous transplantation in clinical trials; these effects were apparently associated with the modulation of PI3K/Akt signaling pathway.

Disclosures

No relevant conflicts of interest to declare.
Influence of MDR1 C3435T Genotype, Sokal Score and Hasford Score on the Overall Survival of Chronic Myelid Leukemia Patients Treated with Imatinib
Authors: Francis Jose , Biswajit Dubashi , Sundaram Rajan , Pradhan Suresh Chandra , Chandrasekaran Adithan
Published: October 2019

Aim: Imatinib is considered as the first-line drug of choice for the treatment of CML who is positive for Philadelphia chromosome. To correlate various prognostic scores (Sokal, Hasford and Eutos) and pharmacogenetic markers like ABCG2 421 C>A, SLC22A1 1386C>A, SLC22A1 1022C>T, SLC22A1 1222A>G, ABCB1 3435T>C, ABCB1 2677 G>T/A with the overall and progression-free survival in CML patients.

Methods: The patients who were started on imatinib from December 2009 to January 2014 at Regional Cancer Centre (RCC), JIPMER, Puducherry, India were recruited into the study. DNA was extracted from peripheral blood using phenol-chloroform method. The genotype status was identified using RT-PCR technique (AB7300, Applied Biosystems) using validated SNP genotyping kits. The influence of various factor on OS and PFS was determined using log-rank test (SPSS Ver 20.0).

Results: A total of 119 patients was identified as BCR/ABL positive and started on Imatinib treatment. Out of this 113 patients were in Chronic phase and six were in Accelerated phase. The median age of the patient group wad 39 years (18-74). The median overall survival and progression-free survival was found to be 25 months and 19 months respectively. The three yr overall survival was 94.5%. None of the prognostic scores or genotypes correlated with progression-free survival of the patients in this study cohort. The low-risk category of Sokal and Hasford groups had a statistically better overall survival compared to intermediate and high-risk groups (p value 0.030 and 0.014 using log-rank test analysis). The Mutant alleles for MDR1 C3435T had a better overall survival compared to other genotypes (p value <0.001 using log-rank test).

Conclusion: The Sokal score and Hasford score, not the Eutos score were better predictors of overall survival in our study. Out of the pharmacogenetic markers analyzed, MDR1 C3435T polymorphism had an influence on the overall survival.

Acknowledgments: The study was supported by the Science and Engineering Research Board (SERB) research grant (SB/FT/LS-147/2012).

Disclosures

No relevant conflicts of interest to declare.
Molecular Aberrations of Chronic Neutrophilic Leukemia: The CSF3R and SETBP1 Mutations
Authors: Cui Yajuan , Li Bing , Gale Robert Peter , Jiang Qian , Xu Zefeng , Qin Tiejun , Zhang Peihong , Zhang Yue , Xiao Zhijian
Published: October 2019

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by sustained elevated neutrophil levels with <10% immature cells according to WHO criteria. Recently, oncogenic mutations affecting the granulocyte-colony stimulating factor receptor (CSF3R) were reported to be highly prevalent in CNL. SET binding protein 1(SETBP1) mutations were also reported in some CNL cases. We reviewed clinical suspected 12 “CNL” patients and performed CSF3R and SETBP1 sequencing. All the 6 who met WHO criteria for CNL carried CSF3R T618I mutation. Four of the 6 also had SETBP1 mutations and a 5th had a CALR mutation (c.1154-1155insTTGTC). Two other who met these criteria but also had monoclonal gammopathy with uncertain significance (MGUS) had no CSF3R, SETBP1 or CALR mutation. The rest 4 carried infection or tumor and thus were excluded. We conclude including CSF3R and SETBP1 mutations into WHO criteria for CNL may improve diagnostic accuracy and may have therapeutic implications.

Disclosures

No relevant conflicts of interest to declare.
The Effects of CCL3 on Osteoblast Inhibition in Myeloma-Induced Bone Disease
Authors: Fu Rong , Zhao Sijie , Shao Zonghong
Published: October 2019

Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow. A common manifestation of the disease is bone destruction that caused by increased osteoclastic bone resorption and decreased bone formation. Suppression of osteoblast activity appears to persist even in patients enjoying long-term complete remission that without any detectable MM tumor cells. CCL3, also known as MIP-1α, is a pro-inflammatory and chemoline. To investigate if CCL3, a recently described osteoclast stimulatory factor,was possibly involved in the inhibition of osteoblast in MM-induced bone disease,osteoblast from BM of MM patients and healthy donors were cultured in vitro. Our results showed the proliferation and osteogenic potential of osteoblasts from MM patients were suppressed. MM-derived OBs expressed higher levels of CCR1 compared with normal controls. It’s an indication that CCL3 is able to influence OBs in MM bone disease (MBD).We cultured MM-derived OBs in osteogenic media and then stimulated them with CCL3 in the absence or presence of neutralizing antibody against CCL3. Our data suggested that CCL3 impaired matrix mineralization and suppressed osteocalcin (OCN).Gene expression analysis of osteoblastic transcription factors in CCL3-treated OBs revealed a Runx2 and Osterix downregulation. CCL3 antibody at least partially restored OBs activity with an upregulation of OCN levels and Runx2 and Osterix expression. These results showed that CCL3 may contribute to OB/OC imbalance by inhiting OBs’ dedifferentiation and function in MBD.

Disclosures

No relevant conflicts of interest to declare.
Impact of Novel Treatments on Multiple Myeloma Survival
Authors: Robinson David , Kaura Satyin , Kiely Daniel , Hussein Mohamad A. , Nersesyan Knar , Durie Brian G. M.
Published: October 2019

BACKGROUND: Multiple myeloma survival has significantly improved with the introduction of new therapeutic classes in the early 2000s. The magnitude of improvement in survival with better tolerated therapy is essentially transforming the disease from an acute condition, to a potentially manageable chronic disease . The impact of further advances in multiple myeloma therapy is not yet known but can be projected based on analysis of historical data and positive outcomes that are expected for future clinical trials.

METHODOLOGY: Analysis of data from the Surveillance, Epidemiology and End Results (SEER) were performed using SEERStat software. The first part of this analysis compares the median relative survival, based on cohort analysis, and five year relative survival, based on period analysis, for different time periods that coincided with the introduction of newer, better tolerated, more effective therapies for treating multiple myeloma. The second part of this analysis attempts to estimate future changes in survival, that may potentially result from additional disease modifying therapies planned to become available over the next 5-10 years. This will be accomplished through analysis of evidence-based metrics including extrapolation from early phase clinical trial data and validation with key opinion leaders.

RESULTS: Median, cumulative and 5 year relative survival of multiple myeloma have improved over the last decade primarily as a result of the introduction of newer therapies such as thalidomide, lenalidomide and bortezomib. Based on analysis of SEER data, median relative survival from the 1980s to 2000 showed modest changes, averaging approximately 30 months through the two decades. Thereafter, median survival increased to nearly 50 months by 2007 (FIGURE 1). Five year relative survival improved from about 30% in 1990 to over 46% in 2010 (FIGURE 2).

SUMMARY: This analysis highlights the dramatic improvements in multiple myeloma survival over the last three decades in the U.S., and demonstrates the transformative impact and value of novel therapies for multiple myeloma. The most substantial improvements were observed during the period between 2001 and 2007, when multiple myeloma median survival rates increased by 45%. This improvement has predominantly been driven by the introduction of novel classes of drugs and agents, including immunomodulatory and proteasome inhibitors. Over the next 10 years we expect to see a similar trend, where further advances in the treatment of multiple myeloma will yield additional novel therapies that could result in improvements of comparable or better magnitude.

FIGURE 1 FIGURE 1.

FIGURE 2 FIGURE 2.

Disclosures

Kaura: Celgene: Employment, Equity Ownership. Kiely:Celgene: Employment, Equity Ownership. Hussein:Celgene: Employment, Equity Ownership. Durie:Celgene: Advisory Board Celgene, IRC Onyx, DMC Millennium, IRC J&J Other.
Does AL Amyloidosis Have Unique Gene Expression Profile? Meta-Analysis Results
Authors: Kryukov Fedor , Kryukova Elena , Brozova Lucie , Pyszko Petr , Kufova Zuzana , Nemec Pavel , Kubiczkova-Besse Lenka , Jarkovsky Jiri , Hajek Roman
Published: October 2019

Background

Immunoglobulin light chain amyloidosis (AL amyloidosis) is a plasma cell dyscrasia characterized by deposition of amyloid fibrils in various organs and tissues, derived from monoclonal immunoglobulin light chains (LC), leading to organ dysfunction. Until now there is no systematic study of genomic expression of patients with AL amyloidosis compared to other monoclonal gammopathies.

Aim of this study was to reveal genomic expression differences between AL amyloidosis patients versus healthy donors (ND), monoclonal gammopathy of undetermined significance (MGUS), smouldering myeloma (SMM) and multiple myeloma (MM) patients.

Methods

Gene expression profiling data from GEO database (GSE6477, GSE24128) comprised of ND (n=15), MGUS (n=21), SMM (n=24), MM (n=69) and AL (n=16) patients were used. To ensure the comparability between arrays and eliminate the batch effect, quantile normalization was performed. To identify the AL patients´ gene expression profile which significantly differs in comparison with ND, MGUS, SMM or MM patients, we applied SAM algorithm for 6588 selected genes that fulfilled the condition having standard deviation above the median. Genes with fold change ≥1.5 or ≤0.5 and false discovery rate >0.001 were considered as significant. Prediction analysis of microarrays (PAM) using nearest shrunken centroids and cross validation assay was carried out to identify the smallest subsets of genes that were able to accurately predict classes.

Results

Comparison of AL patients vs. ND revealed 229 changed genes, AL vs. MGUS 126 genes, AL vs. SMM 338 genes and AL vs. MM 110 genes, respectively. Out of them, the most significantly changed 100 genes from each comparison were further analysed. Some of the genes occurred repeatedly, thus only 256 of genes from all comparisons were unique. Using PAM algorithm we found the genes that have the best predictive power for differentiation AL patients from ND, MGUS, SMM and from MM patients was as followed: RPS6, RPS14, RPS17, RPLA18A, TMEM66, EIF3L, CCDC72, GLTSCR2, NDUFS6, DUSP1, IGLJ3, IGHM, IGKV1-5IGKC///IGKV1-5///IGKV1D-8 and IGHA1///IGHA2///IGHD///IGHG1///IGHG3///IGHG4///IGHM/// IGHV3-23///IGHV4-31///IGHV4-59. Gene expression changes are presented on Figure 1.

Conclusion

Based on offered meta-analysis, 16 genes representing mostly ribosomal proteins and immunoglobulin regions allow us to detect specific aberrant clone responsible for pre-amyloid production and can serve as candidates for further detailed study.

Work was supported by grants IGA NT 12130, NT 14575 and NT 13190.

Figure 1. Gene expression for the genes which best discriminate AL patients from ND, MGUS, SMM and MM patients Figure 1. Gene expression for the genes which best discriminate AL patients from ND, MGUS, SMM and MM patients

Disclosures

Hajek: Merck: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Honoraria.
Value Research of T-Regs Cells in Peripheral Blood of Patients with Multiple Myeloma
Authors: Golenkov Anatoly , Mitina Tatyana , Chernykh Yuliya , Lutskaya Tatyana , Mitin Alexandr , Litvina Marina
Published: October 2019

In this study we have assessed FOXP3 and its isoforms expression by CD4+ T cells from peripheral blood of 37 patients with various forms of multiple myeloma (MM) and 15 healthy volunteers. Newly diagnosed MM was in 17 patients; resistant/relapsed MM was registered in 13 patients and 7 patients had remission after chemotherapy. Significant role of FOXP3 expression by CD4+ T lymphocytes in pathogenesis of MM was shown. Thus, increase of FOXP3-expressing cells percent among CD4+ T cells is registered in patients with newly diagnosed MM and in resistant/relapsed patients. The main contribution to this percent increment is made by FOXP3D2 isoform expressing cells. In addition to the percent increment the number of CD4+FOXP3+ T cells is also risen in patients with newly diagnosed MM. The level of FOXP3 expression by CD4+ T cells also reflects the effectiveness of antitumor therapy, considering normalization of CD4+FOXP3+ T cells number and percent in patients with disease remission. We also discuss the possible relationship between FOXP3 mRNA post-transcriptional modification, namely alternative splicing, with stable expression of FOXP3 in T-regs cells.

The level of an expression of T-regs cells can will be applied as criterion of efficiency of antitumor therapy.

Disclosures

No relevant conflicts of interest to declare.
Leukemia-Initiating Activity in T-ALL Requires Active Hif1a and Wnt Signaling
Authors: Giambra Vincenzo , Jenkins Catherine E , Lam Sonya H , Hoofd Catherine , Belmonte Miriam , Weng Andrew P
Published: October 2019

Prior work has shown that NOTCH1 is a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL) with activating NOTCH1 mutations occurring in over 50% of cases (Weng et al, Science 2004) and loss-of-function mutations in its negative regulator FBXW7 occurring in 8-15% of cases (O’Neil et al, J Exp Med 2007; Thompson et al, J Exp Med 2007). Subsequent work has shown that continued Notch signaling is required for maintenance of T-ALL leukemia stem cells (Armstrong et al, Blood 2009; Tatarek et al, Blood 2011; Giambra et al, Nat Med 2012).

Several lines of evidence have substantiated genetic interactions between the Notch and Wnt signaling pathways in various contexts, and Wnt signaling has been shown to play important roles in hematopoietic stem cell biology and also in hematopoietic cancers such as acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Luis et al, Leukemia 2012). To address what role if any Wnt signaling may play in T-ALL, we generated primary murine leukemias by viral transduction of bone marrow progenitors with activated NOTCH1, then delivered a fluorescent Wnt reporter construct (7TGP; Fuerer & Nusse, PLoS ONE 2010) by lentiviral transduction, and retransplanted the leukemias to interrogate Wnt signaling activity in vivo.

We report here that active Wnt signaling is restricted to minor subpopulations within bulk T-ALL tumors, and that these Wnt-active subsets are highly enriched for leukemia-initiating cell (LIC) activity. Moreover, using Ctnnb1loxP/loxP animals we show that inducible Cre-mediated deletion of β-catenin or enforced expression of a dominant-negative TCF construct severely compromises LIC activity. We also show that β-catenin levels are upregulated by hypoxia through Hif1a stabilization, and that deletion of Hif1a also severely compromises LIC activity. Interestingly, Wnt-active subsets are distributed diffusely throughout the marrow interstitial space suggesting that tumor infiltration induces formation of local hypoxic niches as opposed to taking up residence in pre-existing anatomic compartments with low oxygen tensions. Taken together, these results suggest a model in which hypoxic niches in vivo facilitate Hif1a-dependent accumulation of β-catenin which drives Wnt signaling and self-renewal of leukemia stem cells. Finally, we show using patient-derived xenografts that antagonism of Hif1a or Wnt signaling also compromises human LIC activity, suggesting that pharmacologic targeting of these pathways could have therapeutic application in patients with T-ALL.

Disclosures

No relevant conflicts of interest to declare.
High Serum Free Kappa Chains Are Frequently Missed By Serum Immunofixation
Authors: Sidana Surbhi , Rybicki Lisa , Reu Frederic J. , Daly Thomas
Published: October 2019

Background: Serum free light chains (sFLC) and immunofixation (IFE) analysis are used to detect monoclonal proteins. We noticed that some multiple myeloma (MM) patients (pts) had negative IFE results despite very high sFLC levels. This analysis was done to determine the frequency of this finding in a large cohort.

Methods: Following IRB approval, samples with simultaneous sIFE and sFLCs ordered from 1/2013 to 9/2013 were identified by querying our lab electronic database. Freelite (R) Human Kappa & Lambda Free kit (The Binding Site, Birmingham, UK) was used for sFLC and SPIFE® ImmunoFix-15 gels (Helena Laboratories, Beaumont, TX) for IFE. Clinical review was performed for a subset of patients with discrepant results between the two assays.

Results: 4404 samples from 2200 pts were identified with simultaneous sFLC and sIFE results. Overall 348 of 4404 (7.9%) samples had an abnormal sFLC ratio but a negative sIFE. Of 205 pts (457 samples) with involved serum free kappa above the expected IFE threshold (200 mg/L), 42 pts (103 samples) had negative sIFEs (20.5% pts; 22.5% samples) despite median free kappa of 556.6 mg/L (range 208.1 to 4954.4). This was much less common for involved free lambda. Only 4 of 107 pts with free lambda above 200mg/L had negative sIFEs (3.7%). Information of the nature of the plasma cell disorder was available on 40 of the 42 patients with free kappa > 200mg/L and negative IFE. In this group, 62.5% (n=25) had symptomatic MM, 12.5% (n=5) had asymptomatic MM, 5% (n=2) had light chain amyloidosis, 5% (n=2) had both amyloidosis and MM, and 15% (n=6) had MGUS or a not yet fully worked up plasma cell disorder.

Conclusions: Serum IFE, with a commonly used kit, missed about 20% of patients with free kappa above the expected IFE threshold, while free lambda was detected more reliably. Results suggest current criteria for complete remission which rely on immunofixation and bone marrow plasma cell infiltration may need to be revised for kappa light chain myeloma.

Disclosures

No relevant conflicts of interest to declare.
Reversibility of Renal Insufficiency in Patients with Newly Diagnosed Multiple Myeloma and the Role of different induction Regimens
Authors: Bao Li , Lu Jin , Huang Xiaojun
Published: October 2019

We retrospectively compared the impact of treatments based on thalidomide, bortezomib or tranditional regimen (VAD) on renal function recovery in a consecutive, unselected population of newly diagnosed myeloma patients presenting with renal failure. Between May 2005 and February 2014, 130 (24.3%) patients with newly diagnosed multiple myeloma and RI were treated upfront with VAD or a novel agent containing regimen and all received a similar supportive care in our single center. A complete recovery of renal function(renal complete response )was observed in 56.1% of patients treated with bortezomib, in 38.9% with thalidomide and in 28.6% with VAD (P=0.033), a significant improvement of renal function (≥renalPR (renal partial response)) was observed in 63.6% of patients treated with bortezomib, in 66.1% with thalidomide and in 42.9% with VAD (P=0.162). There has a significantly difference in the rates and quality of renal response among patients with different quality of myeloma response: renal CR rates for patients who achieved ≥VGPR, PR, MR or NR were 63.1% vs 46.8% vs 35.7% vs 25.8%, P<0.05, respectively. eGFR ≥30 ml/min and myeloma response higher than PR were independently associated with a higher probability of major renal response in the multivariate analysis. The median follow-up for all patients was 36.5 months and the median survival was 74 months. The median survival for patients of groups B,T and VAD was79 months,71 months and 39 months, respectively (P=0.007). Patients died within the first 2 months from initiation of therapy and the corresponding frequencies in groups B, T and VAD were 0%, 2.8% and 17.9%, respectively (P<0.05). In multivariate analyses on survival, myeloma response higher than PR and new agents-based therapy are identified independent prognostic factors. For newly diagnosed myeloma patients with RI, bortezomib or thalidomide based regimen should be selected promptly to achieve rapid effective myeloma rate and high rates of renal recovery.

Disclosures

No relevant conflicts of interest to declare.
The Cancer/Testis Antigens of SSX4, MAGE-A3, and CTAG2 Are Attractive Targets for Cancer Immunotherapy in Korean Patients with Multiple Myeloma
Authors: Choi Nu-Ri , Jung Sung-Hoon , Lee Hyun-Ju , Vo Manh-Cuong , Hoang My-Dung , Kim Hyeoung-Joon , Lee Je-Jung
Published: October 2019

Introduction: Cancer/testis antigens (CTAs) are an attractive target for cancer immunotherapy because of a tumor-restricted expression and remarkable immunogenicity. Several CTAs have been used as a source of tumor antigen in dendritic cell therapy against multiple myeloma (MM), but there was no report the CTAs in Asian patients with MM. In this study, we evaluate the expression of 10 CTAs on malignant plasma cells of bone marrow in 18 Korean patients with relapsed or refractory MM.

Materials and methods: Eighteen patients with relapsed or refractory MM were classified as four categories according to paraprotein subtypes: IgG (n=7), IgA (n=5), light chain-lambda (n=3), and light chain-kappa (n=3). The expression pattern of 10 CTAs, including NY-ESO-1, SSX2, SSX4, SSX5, MAGE-A3, MAGE-C1, MAGE-C2, BAGE2, CTAG2, and SPA7, was studied by real-time quantitative polymerase chain reaction in CD138+ cells of BM mononuclear cells (MNCs) obtained from MM patients. In addition, we compared it with expression pattern of CTAs in the MNCs from healthy normal donors and the CD138- cells of BM MNCs from MM patients as controls.

Results: In CD138+ cells of BM MNCs from the patients, five CTAs, including SSX2, SSX4, MAGE-A3, MAGEC2, and CTAG2, showed high frequency and overall 5.4 to 63.9 fold increase expression in the quantitative mRNA survey compared to MNCs from healthy donors and CD138- cells of BM MNCs from patients. Expression pattern of 5 CTAs was slightly different by paraprotein subtypes: IgA subtype - SSX4 (17.1 fold increase), MAGE-A3 (11.0 fold increase), and CTAG2 (5.9 fold increase); IgG subtype - CTAG2 (63.9 fold increase), SSX4 (40.2 fold increase), and MAGE-A3 (39.9 fold increase); lambda light chain subtype - CTAG2 (42.4 fold increase), SSX4 (29.0 fold increase), and MAGE-A3 (24.4 fold increase); kappa light chain subtype - SSX2 (6.4 fold increase), MAGE-C2 (6.2 fold increase), MAGE-A3 (5.4 fold increase), and SSX4 (5.4 fold increase).

Conclusion: This study suggests that three CTAs, such as SSX4, MAGE-A3, and CTAG2, highly expressed on malignant plasma cells are potentially promising targets for cancer immunotherapy in Korean patients with relapsed or refractory MM.

Disclosures

No relevant conflicts of interest to declare.
Colesevelam Hydrochloride for the Treatment of Lenalidomide Induced Diarrhea
Authors: Watson Melanie , Nooka Ajay K. , Gleason Charise , Valla Kelly , Kaufman Jonathan L. , Lonial Sagar
Published: October 2019

INTRODUCTION: Lenalidomide is an immunomodulatory drug used for both induction and maintenance in Multiple Myeloma patients. In the pooled multiple myeloma studies, 29.2% of patients reported diarrhea with lenalidomide and dexamethasone. Colesevelam hydrochloride, a drug used primarily for lipid-lowering, has shown efficacy when used for diarrhea in patients receiving lenalidomide in European studies.

METHOD: In our retrospective analysis, we have evaluated the response of twenty patients receiving lenalidomide and experiencing chronic diarrhea between October 29, 2013, and August 5, 2014. Patients were prescribed colesevelam hydrochloride, 1875mg orally twice daily. Patients were instructed to dose their lenalidomide and colesevelam separated by at least two hours. The objective is symptomatic improvement in lenalidomide induced diarrhea.

RESULTS: Twelve males and eight females on lenalidomide based therapy experiencing chronic diarrhea were prescribed colsevelam. Seventeen patients were receiving single agent lenalidomide and three patients were receiving combination therapies (elotuzumab/lenalidomide/dexamethasone:1 pt, lenalidomide/bortezomib/dexamethasone: 1 pt, and lenalidomide/cyclophosphamide/dexamethasone: 1 pt). Median age of the patients was 60 year (range: 44-77 yrs). Median prior lines of therapy were 1 (range: 1-6). Median time from initiation of lenalidomide to Colesevelam hydrochloride is 44 months (0-64 months). Eighty-five percent of patients have noted improvement and 30% experienced total resolution of symptoms. Median time from initiation of Colesevelam hydrochloride to symptomatic response is 4 weeks (range, 0-17). In one patient response was seen in less than a week.

CONCLUSION: Multiple Myeloma patients on lenalidomide therapy with chronic diarrhea noted improvement or total resolution of symptoms with the addition of colesevelam, thus allowing patients to stay on therapy, improving survival as well as quality of life.

Disclosures

Gleason: Celgene: Consultancy; Novartis: Consultancy. Lonial:Millennium: The Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding.
Cytotoxic T-Lymphocytes Recognize and Target Leukemic Stem Cells
Authors: Schneider Vanessa , Zhang Lu , Rojewski Markus , Bullinger Lars , Hofmann Susanne , Goetz Marlies , Doehner Konstanze , Doehner Hartmut , Buske Christian , Greiner Jochen
Published: October 2019

Leukemic stem cells (LSC) might be the source for leukemic disease self-renewal and account for disease relapse after treatment, which makes them a critical target for further therapeutic options. Treatment for acute myeloid leukemia (AML) patients became more effective, yet relapse rates are still high. Immunotherapeutic approaches might overcome the immune escape mechanisms of LSC and might be a therapeutic option. Leukemia-associated antigens (LAA) represent targets that are recognized by cytotoxic T-lymphocytes (CTL) and might be useful for specific immunotherapy.

We enriched the LSC containing fraction as CD34+CD38- cells (called LSC-EF) of 20 AML patients using FACS, analyzed expression patterns of LAA in LSC-EF and compared them to patterns in enriched HSC und bulk AML cells by microarray expression analysis and qRT-PCR. In immunoassays, we analysed the functional inhibition of LCS in colony forming assays (CFU) of AML patients by stimulated T cells.

We detected a high expression of several LAA in LSC-EF. PRAME (p = 0.0085), RHAMM (p = 0.03), WT1 (p = 0.04) and Proteinase 3 (p = 0.04) showed significant differential expression in LSC-EF compared to HSC. Survivin (p = 0.7), HAGE (p = 0.5), SSXIP2 (p = 0.4) and Aurorakinase A (p = 0.7) did not indicate a significant differential expression. BAGE does not seem to be an ideal candidate as it is significantly higher expressed on normal HSC (p = 0.0056). Further, we evaluated the expression differences of LSC-EF compared to leukemic bulk. PRAME, RHAMM and WT1 qualified as interesting potential targets as they are lower expressed on leukemic bulk than on LSC-EF. In contrast, Proteinase 3 indicates a higher expression on leukemic bulk but lower in LSC-EF. qRT-PCR confirmed our array data.

In immunoassays, T cells stimulated against LAA indicated a significant inhibition of CFUs in AML patient samples. PRAME, RHAMM and WT1 showed good immunogenic responses with CTL recognition rates in a range of 58-83%. PRAME hereby proved as most effective using two different patient samples, with a reduction of 83% colonies (p = 0.004). WT1 shows a significant reduction of 74% (p = 0.05) and RHAMM of 58% colonies (p = 0.2).

Taken together, several LAA are significantly expressed on the LSC-EF, whereas PRAME, RHAMM and WT1 seemed most promising as these LAA are differentially expressed compared to normal stem cells and stimulation of cytotoxic T cells against these LAAs inhibit LSC in CFU assays.

Disclosures

No relevant conflicts of interest to declare.