bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycobacterial Infections in Adult Recipients of Allogeneic Hematopoietic Stem Cell Transplantation: A Cohort Study in a High Endemic Area
Authors: Yao-Chung Liu , Wu Chi-Jung , Chien Sheng-Hsuan , Fan Nai-Wen , Hu Ming-Hung , Gau Jyh-Pyng , Liu Chia-Jen , Yu Yuan-Bin , Hsiao Liang-Tsai , Chiou Tzeon-Jye , Liu Jin-Hwang
Published: October 2019

Background

Mycobacterial infection is an important and potentially life-threatening complication of patients receiving organ transplantations. Notably, for the recipients of allogeneic hematopoietic stem cell transplantation (HSCT), there are few supporting results to explore post-transplant mycobacterial infections. Taiwan is a high endemic area of the infection. We therefore aim to investigate the incidence, risk factors, and survival of post-transplant mycobacterial infections, including mycobacterium tuberculosis (MTB) and nontuberculous mycobacterium (NTM).

Patients and Methods

We included in this study 422 adult patients undergoing allogeneic HSCT at an Asian tertiary medical center between January 2003 and December 2013. A total 26 subjects who developed post-transplant mycobacterial infections were identified. Risk factors, clinical features, and survival for post-transplant mycobacterial infections in recipients of HSCT were collected and analyzed.

Results

Post-transplant mycobacterial infections occurred in 26 (6.2%) of 422 HSCT patients. Two-year cumulative incidences in MTB and NTM were 1.4 % and 5.4%. In the multivariate analysis, being age > 45 years (adjusted HR 2.21, 95% CI 1.01-4.83)) and extensive chronic graft-versus-host disease (cGVHD) (adjusted HR 4.95, 95% CI 2.14-11.46) were identified as independent risk factors of post-transplant mycobacterial infections. There was a trend as a risk factors in relapsed patients (P=0.088). For patients with cGVHD, there was a significant difference of post-transplant survival between mycobacterial infections and none (median: 6.82 months versus not reached, P=0.036). Pneumonia with or without other infections contributed most to mortality (n = 11, 42.3%).

Conclusion

The 2-year cumulative incidence is higher in NTM (5.4%) than TB (1.4%) in recipients of allogeneic HSCT. Patients with age > 45 years, and extensive cGVHD are at risk of mycobacterial infections. Poor post-transplant survival in cGVHD patients developed mycobacterial infections and 42.3% patients in our study died of pneumonia. Once a high-risk group is identified, much effort is required to target new approaches for prevention, early detection and treatment of infections.

Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
Increase of Regulatory γδ T Cells Reduces the Incidence of Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Gao Li , Xuan Li , Wu Xiuli , Fan Zhiping , Huang Fen , Yi Zhangshan , Li Yangqiu , Liu Qifa
Published: October 2019

Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the primary means for treatment of hematologic and non-hematologic malignancies. Acute graft-versus-host disease (aGVHD) is a major complication associated with high morbidity and mortality after transplantation. Regulatory γδ T cells (γδ Tregs), as a novel subset of γδ T cells with Foxp3 expression and immunosuppressive effect, are found to effectively alleviate aGVHD in mice model. However, whether γδ Tregs involve in the occurrence of aGVHD in recipients of allo-HSCT remains unclear. Therefore, the aim of this study is to investigate the effect of γδ Tregs in the occurrence of aGVHD in recipients of allo-HSCT.

Methods The immunophenotyping of γδ Tregs was analyzed in peripheral blood from 24 aGVHD and 24 paired non-aGVHD patients (undergoing transplantation at the same time, basic data matched), using flow cytometry. The expression levels of immunoregulatory-associated molecules (Foxp3, CD25, CTLA-4, GITR, TLR8, RORc, STAT-1 and STAT-3) were analyzed in peripheral blood, using real-time RT-PCR with SYBR GreenⅠstaining. Liquichip technology was used to analyze the concentration of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10, IL-17 and tumour necrosis factor-α (TNF-α) in cultural supernatant.

Results The proportions of total γδ T and Vδ2 T cells in non-aGVHD patients were significantly higher than that in aGVHD patients (P=0.044, P=0.000), and the proportion of Vδ1 T cells was similar between the two groups (P=0.061). Compared with aGVHD patients, the proportions of Foxp3+γδ T, Foxp3+Vδ1 T, Foxp3+Vδ2 T cells and Tregs were increased in non-aGVHD patients (P=0.005, P=0.014, P=0.026 and P=0.000). There was a negative correlation between the incidence of aGVHD and the percentages of Foxp3+γδ T, Foxp3+Vδ1 T and Foxp3+Vδ2 T cells (P=0.014, rs=-0.477; P=0.001, rs=-0.610; P=0.040, rs=-0.405). The mRNA expression levels of Foxp3 and TLR8 genes were significantly increased in non-aGVHD patients compared with aGVHD patients (P=0.042, P=0.002). There were no significant difference in the expression levels of CD25, RORc, CTLA-4, GITR, STAT-1 and STAT-3 genes (P=0.234, P=0.194, P=0.056, P=0.524, P=0.142 and P=0.775). In addition, the concentrations of IL-10 and TNF-α were significantly increased in aGVHD patients (P=0.002, P=0.036), and the concentrations of IL-4, IL-17 and IFN-γ were similar between the two groups (P=0.379, P=0.414, P=0.170).

Conclusions Increase of γδ Tregs might reduce the incidence of aGVHD after allo-HSCT.

Disclosures

No relevant conflicts of interest to declare.
Does Cell Size Matter?: Utilizing Mean Cell Volume in Hospitalized Patients As a Screen to Determine Common Causes of Anemia Including Iron Deficiency Anemia, Vitamin B12 and Folate Deficiency
Authors: Gupta Manika , Copley Kathleen , Keeney Michael , Chin-Yee Ian
Published: October 2019

Background: The approach to anemia is traditionally based on the Mean Cell Volume. Based on this approach anemia is subdivided into microcytic, normocytic and macrocytic causes. This approach may not accurately discern common causes of anemia in hospitalized patients. Previous studies suggest the MCV may not be a sensitive measurement to differentiate iron deficiency anemia (IDA) and megaloblastic anemia due to vitamin B12 or folate deficiency.

Methods: In a retrospective, single-centre study at London Health Sciences Center, all adult patients (age 18 years or older) with confirmed IDA, vitamin B12 and folate deficiency and their associated MCV and RDW values at LHSC over a one year period were reviewed. IDA was defined as hemoglobin less than 115 g/l and ferritin less than 30 (M) and 10 (F). Vitamin B12 deficiency was defined as a value of less than 145.

Results: 1119 patients were identified with confirmed IDA, B12 or Folate deficiency. 894 patients had IDA of which 564 patients had low MCV (sensitivity 63.1%) and 797 patients had low MCV or high RDW (sensitivity 89.1%). Of the 96 patients with vitamin B12 deficiency anemia, 12 patients had high MCV (sensitivity 12.5%) and 70 patients had high MCV or high RDW (72.9%). Only one of 2244 patients who had RBC folate measured had an actual folate deficiency.

Conclusion: Our results confirm that a normal MCV does not exclude IDA or vitamin B12 deficiency. Clinicians need to be aware of the low sensitivity of the MCV as a screen. The sensitivity of MCV for IDA or vitamin B12 deficiency is improved with indices such as RDW. Folate deficiency is rare in North America and should not be routinely ordered for assessment of nutritional anemia.

Disclosures

No relevant conflicts of interest to declare.
Wait Times and Bed Capacity for Hematopoietic Cell Transplantation in Ontario: A System Dynamics Model
Authors: Wang Jonathan , Vahid Saba , Sherrie Hertz , Kouroukis C. Tom
Published: October 2019

Objectives: Cancer Care Ontario (CCO) is the provincial governmental organization responsible for planning hematopoietic cell transplantation (HCT) services in Ontario, Canada. The objective of this project is to develop a capacity planning model to investigate the effects on wait times of adding extra bed capacity for allogeneic transplant (ALLOHCT) in HCT centers.

Approach: A high-level process flow diagram was generated to understand patient flow at a 6-bed HCT unit within a hospital in Ontario and validated through consultation. This flow diagram was used to construct a system dynamics model to simulate patient flow. The model was parameterized with data from CCO, Discharge Abstract Database, and with hospital and clinical expert input. The effects at six months were projected for five scenarios: 1) current state; 2) increase bed capacity by 1 bed; or 3) increase bed capacity by 2 beds; 4) increasing patient demand by 20 patients per year; 5) combination of scenarios 3 and 4. Provincial clinical consensus established a benchmark wait time of 42 days for ALLOHCT from ready to transplant to the transplant date. In addition, the estimated number of beds required to reduce the wait times to the provincial benchmark within 1 year was calculated.

Results:The addition of 1 ALLOHCT bed resulted in a reduction of 22% and 11% to the ALLOHCT wait times and wait lists, respectively. The addition of 2 beds resulted in a reduction of 38% and 22% to the wait times and wait lists, respectively. If the demand increases by 20 patients per year, the addition of 2 beds resulted in a reduction of 16% in the wait times and while the wait list may experience a brief reduction, after 6 months, the wait list size will have increased by 9% as a result of the increased demand. In order to reduce the wait times to the provincial benchmark within 1 year, an additional 8 beds are needed.

Considerations: Concurrent planning for additional health human resources (physicians, nurses, etc…) needs to be done to ensure the additional beds are adequately staffed. This model also only considers the effects of adding beds within 1 year. There may be instances where bed space cannot be immediately opened and new capital is required. Additionally, the demand for ALLOHCT continues to increase, which in turn drives up the number of arrivals to the queue. A multi-year model will be built to account for timing of bed openings and increasing demand for ALLOHCT.

Conclusion:Using a system dynamics model, we are able to quantify the relationship between ALLOHCT bed capacity and wait times at an HCT center. This model can be used to estimate the ALLOHCT bed requirements for sites in other jurisdictions where ALLOHCT demand and wait time benchmarks are known.

Disclosures

Kouroukis: Janssen: Research Funding; Karyopharm: Research Funding.
Splenectomy Protects Humans from Red Cell Alloimmunization
Authors: Evers Dorothea , van der Bom Johanna G , Tijmensen Janneke , de Haas Masja , Middelburg Rutger A , de Vooght Karen , van de Kerkhof Daan , Visser Otto , Pequeriaux Nathalie , Hudig Fransisca , Zwaginga Jaap Jan
Published: October 2019

Background and aim

Being the primary organ that clears and processes ageing and damaged red cells, the spleen has been suggested to play a central role in red cell alloimmunization in mice experiments. We evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans.

Methods

We performed a nested case-control study among a source population of 24,063 patients who received their first and subsequent red cell transfusion in one of six participating hospitals in the Netherlands between 2005 and 2013. Cases were defined as patients who developed a first transfusion-induced clinically relevant red cell alloantibody. Each case was matched to two non-alloimmunized control patients, based on the number of (lifetime) transfusions and the hospital. Using multivariate logistic regression analyses, we evaluated the association between (a history of) splenectomy and the risk for alloimmunization.

Results

Among a source population of 24,063 patients, 505 patients (2.1%) developed red cell alloantibodies and were matched to 1,010 controls. Splenectomy had been performed in one case and 19 controls. In 12 patients, splenic injury was caused by severe trauma or complicated abdominal surgery. No patient underwent a splenectomy in the context of an autoimmune disease.

Using multivariate logistic regression analysis with conditioning on identified potential confounders, we found that splenectomized patients had a 20-fold reduced risk of alloimmunization as compared to patients lacking a history of splenectomy (adjusted RR 0.05, 95% confidence interval (CI) 0.01-0.55).

With the assumption that the red cell exposure patterns of the 19 splenectomized controls represented the red cell exposure pattern of all splenectomized patients within the source population we estimated that the total number patients with alloimmunization after splenectomy was 24; yielding a RR of 0.04 which confirms our logistic regression based RR.

Interpretation

In conclusion, in accordance with the findings in mice, our findings suggest that splenectomized patients are strongly protected from primary red cell alloimmunization.

Disclosures

No relevant conflicts of interest to declare.
Gut Colonization with Carbapenem Resistant Enterobactriaceae (CRE) Adversely Impacts the Outcome in Patients with Hematological Malignancies: Results of a Prospective Surveillance Study
Authors: Jaiswal Sarita Rani , Gupta Satyanker , Sherawat Amit , Vinod Seena , Jacob Hensey , Abraham Sajin V , Chaturvedi Anushree , Chakrabarti Suparno
Published: October 2019

CRE is the major concern in the patients suffering from hematological malignancy but the data on the impact of colonization in such patients is scarce. We conducted a prospective surveillance study of CRE on 225 patients of haematological malignancies (HM), who required hospital admission between October 2013 to January 2016. The median age of the cohort was 45 years(range,2-84). The cohort included a broad representation of diseases; AML (n=58), ALL(n=44), Lymphoma (n=76), Myeloma(n=22), CML(8) and Others (n=17). Rectal swabs were taken for cultures in all patients during first day of admission and repeated subsequently on weekly basis for continuous hospital stay or in subsequent admissions.47 (21%) patients were colonized with CRE on admission. This increased to 45% on prolonged hospital stay/ contact for more than 4 weeks with 72 (32%) patients being colonized overall. The highest number of patients colonized with CRE at diagnosis was those with AML (30%). The non-relapse mortality (NRM) of the cohort at 6 months was 10.3%. This was 14% in those with acute leukemia compared to 5.8% in others (p=0.02). Furthermore, this was highest in patients with AML (22.9% vs 6.1% in others; p=0.001). CRE related events accounted for 77% of mortality. NRM was 29.6% in those colonized with CRE during hospitalization compared to 10.5% in those colonized at admission with no NRM in those not colonized with CRE (p=0.0001). Amongst patients with AML, colonization with CRE during hospitalization was associated with a NRM of 54.9%(95%CI 32.4-67.4%) compared to those colonized at admission(11.8; 95%CI 4.0-19.6) and those not colonized(0%)(p=0.0001). This trend was true for patients without AML as well (27.3% vs 10.3% vs 0%; p=0.0001). On multivariate analysis, colonization with CRE during hospitalization was the most important risk factor for NRM in patients with hematological malignancies (HR-7.1; 95%CI 3.1-16.1). Our data demonstrates that a substantial proportion of patients with HM are colonized with CRE without prior hospitalisations but those with nosocomial colonization have the highest risk of mortality, particularly in those with AML.

Figure Figure.

Disclosures

No relevant conflicts of interest to declare.
Adenosine A2B Receptor Promotes Erythrocyte Oxygen Release to Counteract Tissue Hypoxia and Injury
Authors: Peng Zhangzhe , Luo Renna , Song Anren , Sun Kaiqi , Liu Hong , Zhang Yujin , Adebiyi Morayo G. , Xia Yang , Kellems Rodney E. , Huang Aji
Published: October 2019

Insufficient tissue oxygen (O2) availability (hypoxia) is commonly seen in patients with cardiovascular diseases (CVD), chronic kidney diseases (CKD), respiratory diseases, hemolytic disorders and certain cancers. Without interference, pathological hypoxia is dangerous because it promotes disease progression, organ damage and failure. Although erythrocyte is the only cell type responsible for delivering O2, its function in pathological tissue hypoxia remains largely unknown. Here using unbiased high throughput metabolomic profiling, we found that 2,3-biphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite enhancing O2 delivery, was significantly induced in the erythrocytes of mice infused with angiotensin II (Ang II), a potent vasopeptide commonly increased in the circulation of CVD and CKD patients to induce tissue hypoxia and injury via vasoconstriction. Mouse genetic studies demonstrated that Ang II induced local tissue accumulation of adenosine and that elevated adenosine-mediated erythrocyte A2B adenosine receptor (ADORA2B) activation was beneficial by inducing 2,3-BPG production, triggering O2 release to prevent multiple tissue hypoxia, inflammation and kidney damage. Mechanistically, we revealed that AMPK is an intracellular signaling molecular functions downstream of ADORA2B underlying elevated 2,3-BPG production by inducing BPG mutase activity. Finally, we translated our mouse study to human and confirmed that the levels of 2,3-BPG and P50 were elevated in erythrocytes of mild CKD patients compared to healthy individuals and further increased in severe CKD patients compared to mild CKD patients. Taken together, we demonstrate that erythrocyte ADORA2B plays a key protective role in Ang II-induced tissue damage by increasing 2, 3-BPG and oxygen release to counteract tissue hypoxia.

Disclosures

No relevant conflicts of interest to declare.
Haptoglobin and Hemopexin Infusion Efficiently Activates the Nrf2/HO-1 Axis and Inhibits Inflammation and Vaso-Occlusion in Murine Sickle Cell Disease
Authors: Belcher John D , Chen Chunsheng , Nguyen Julia , Abdulla Fuad , Nguyen Phong , Nguyen Hao , Zhang Ping , Brinkman Nathan , Vercellotti Gregory M
Published: October 2019

Free hemoglobin and hemin, released by red blood cells during intravascular hemolysis, promote vasculopathy, inflammation, thrombosis, and renal injury. Plasma haptoglobin and hemopexin tightly bind free hemoglobin and hemin, respectively, thwarting these clinical sequelae. In sickle cell disease (SCD), chronic hemolysis can deplete plasma haptoglobin and hemopexin in humans and mice. To explore mechanisms mediating this protection and provide a basis for supplementation in SCD patients, dorsal skin fold chambers were implanted onto Townes-SS mice and microvascular stasis (% non-flowing venules) was measured in response to a hemoglobin challenge. Human haptoglobin, hemopexin, or albumin was co-infused with hemoglobin or 1 hour after hemoglobin at equimolar concentrations. Sickle mice co-infused with hemoglobin/haptoglobin, hemoglobin/hemopexin or hemoglobin/haptoglobin/hemopexin had less stasis 1 to 4 hours after infusion, compared to albumin- and saline-treated mice (*p<0.01, Figure A). Haptoglobin, hemopexin, or haptoglobin/hemopexin given to Townes-SS mice 1 hour after hemoglobin, decreased stasis 2 and 3 hours after supplementation, while the venules of mice treated with albumin remained static (*p<.01, Figure B). Plasma hemoglobin and heme levels in Townes-SS mice were not different between treatment groups 3 and 4 hours after supplementation. Haptoglobin or hemopexin infusion increased hepatic Nrf2 and HO-1 and decreased pro-inflammatory NF-ĸB phospho-p65 expression relative to albumin 3 and 4 hours after supplementation (p<.05). The combination of haptoglobin/hemopexin was similar to either scavenger alone. Inhibition of the enhanced HO-1 activity afforded by haptoglobin or hemopexin with tin protoporphyrin blocked the stasis protection, confirming the critical cytoprotective role of HO-1. Haptoglobin and hemopexin, but not albumin, are cytoprotective in part by efficiently delivering heme to CD163 and CD91 and activating the Nrf2/HO-1 axis.

Figure Figure.

Disclosures

Belcher: CSL-Behring: Research Funding; Imara: Research Funding. Chen:Imara: Research Funding. Brinkman:CSL-Behring: Employment. Vercellotti:CSL-Behring: Research Funding; Imara: Research Funding.
Novel Lyn-Mediated Regulation of CLEC-2 Signaling By Gq-Coupled Receptors in Platelets
Authors: Badolia Rachit , Inamdar Vaishali , Manne Bhanukanth , Dangelmaier Carol , Kunapuli Satya P.
Published: October 2019

The CLEC-2 agonist, rhodocytin, elicits powerful platelet activation signals in conjunction with Src family kinases, Syk, and PLCg2. Previous reports have shown that rhodocytin-induced aggregation is dependent on secondary mediators. However, the role of secondary mediators in CLEC-2 signaling is not defined. In this study we report that CLEC-2-induced Syk activation is aspirin-sensitive and that TxA2 plays an important role in the most proximal events such as CLEC-2 phosphorylation and Syk activation. We also show that the activation of other GPCRs, such as the ADP receptors and PARs, can also potentiate CLEC-2 signaling. By using the Gq-inhibitor, UBO-QIC, or P2Y1/P2Y12 antagonists, we show that the Gq-signaling, but not G12/13 or Gi, is essential for GPCR-induced Syk phosphorylation downstream of CLEC-2. We further elucidated the signaling involved in Gq-mediated Syk phosphorylation and identified an important role for PKCs downstream of PLCb regulating SFK activation (Figure 1A). Using Lyn-knock out murine platelets we identified a potential role for Lyn downstream of the Gq-pathway in potentiating CLEC-2 signaling by using. We suggest that, at low concentration of CLEC-2 agonist, the unclustered CLEC-2 receptors are phosphorylated by the Gq-activated Lyn resulting in the potentiation of the initial CLEC-2 signal (Figure 1B). However, Gq receptors by themselves cannot phosphorylate CLEC-2 receptors and require minimal levels of ITAM-containing receptor stimulation in order to initiate unclustered CLEC-2 receptor phosphorylation. Together, these results provide evidence for a novel Lyn-mediated regulation of CLEC-2 signaling by Gq-coupled receptors thereby leading to potentiation of CLEC-2-induced signaling (Figure 1C).

Figure 1 A) Western blot showingeffect Gq-pathway inhibitors on CLEC-2 signaling. B) Effect of low concentration of CLEC-2 agonist in Lyn-knock out murine platelets. C) Model showing role of Gq-activated Lyn in CLEC-2 signaling. Figure 1. A) Western blot showingeffect Gq-pathway inhibitors on CLEC-2 signaling. B) Effect of low concentration of CLEC-2 agonist in Lyn-knock out murine platelets. C) Model showing role of Gq-activated Lyn in CLEC-2 signaling.

Disclosures

No relevant conflicts of interest to declare.
The Performance of the Original and Simplified Wells Scores in Combination with Age-Adjusted D-Dimer Testing in the Diagnostic Management of Pulmonary Embolism
Authors: Kraaijpoel Noémie , van Es Nick , Büller Harry R , Klok Frederikus A , Huisman Menno V , den Exter Paul L , Galipienzo Javier , Middeldorp Saskia , Bossuyt Patrick M
Published: October 2019

Background: Among patients with suspected pulmonary embolism (PE), imaging can be safely withheld in those with a 'PE unlikely' Wells score and a negative D-dimer. A simplification of the Wells score has been proposed to improve clinical applicability (Table 1), but its performance is less clear, in particular in combination with age-adjusted D-dimer testing.

Objectives: To compare the performance of the original and simplified Wells scores alone and in combination with age-adjusted D-dimer testing.

Methods: Individual patient data from 7,268 patients with clinically suspected PE enrolled in 6 prospective diagnostic management studies were used. The discriminatory performance, calibration, and diagnostic accuracy of the original and simplified Wells scores were evaluated. The efficiency and failure rate of both dichotomized scores combined with age-adjusted D-dimer testing were compared using a one-stage random effects meta-analysis. Efficiency was defined as the proportion of patients in whom PE could be considered excluded based on a 'PE unlikely' Wells score and a D-dimer below the age-adjusted treshold, defined as ≤500 µg/L in patients of 50 years or younger and the patient's age times 10 µg/L in those older than 50 years. The failure rate was defined as the proportion of patients subsequently diagnosed with symptomatic venous thromboembolism during 3-month follow-up.

Results: The discriminatory performance of the original and simplified Wells scores was comparable (c-statistic 0.73 [95% CI 0.72-0.75] vs. 0.72 [95% CI 0.70-0.73]). When combined with age-adjusted D-dimer testing, the original and simplified Wells rules had comparable efficiency (33% [95% CI 25-42%] vs 30% [95% CI 21-40%]) and failure rates (0.9% [95% CI 0.6-1.5%] vs. 0.8% [95% CI 0.5-1.3%]).

Conclusion: Among patients with suspected PE, the original and simplified Wells rules in combination with age-adjusted D-dimer testing have similar performance in ruling out the disease. Given its ease of use in clinical practice, the simplified Wells rule may be preferred.

Disclosures

Huisman: Boehringer Ingelheim Pharma GmbH & Co.KG: Other: Grant support; GlaxoSmithKline: Other: Grant support; Bayer HealthCare: Other: Grant support; Pfizer: Other: Grant support; Actelion: Other: Grant support.
Dual Activation of NRF2 in Multiple Myeloma and Bone Marrow Mesenchymal Stromal Cells Regulates Chemotherapy Resistance
Authors: Sun Yu , Zaitseva Lyubov , Shafat Manar S , Bowles Kristian M , Rushworth Stuart A
Published: October 2019

Background

The cornerstone treatments of multiple myeloma (MM) are proteasome inhibitors bortezomib (BZ) and carfilzomib (CFZ). However, MM still remains incurable for that MM cells rapidly develop resistance to chemotherapy. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathways have been shown to contribute to the malignant phenotypes of several cancers through effects on proliferation and drug sensitivity. NRF2 functions to rapidly change the sensitivity of the cells environment to oxidants and electrophiles by stimulating the transcriptional activation of drug metabolism and antioxidant genes. NRF2 is negatively regulated by proteasome degradation through its inhibitor KEAP1. The aim of this study was to determine if proteasome inhibitor induced NRF2 signalling orchestrates survival of MM in the bone marrow (BM) microenvironment.

Methods

To investigate the role of NRF2 in the MM microenvironment primary human MM and BM mesenchymal stromal cells (MSC) were obtained under UK ethical approval (LREC ref 07/H0310/146). NRF2 activity in MM and BM-MSC was measured by NRF2 protein expression, target genes expression and using promoter assays. Lentiviral mediated shRNA knockdown of NRF2 in the MM and BM-MSC. The NRF2 inhibitor, brusatol was used to verify the knockdown experiments.

Results

Results show that primary MM and MM cell lines have increased NRF2 activity in response to the proteasome inhibitors BZ and CFZ as measured by increased nuclear NRF2, increased NRF2 regulated genes and increased ARE activity in the promoter of heme oxygenase-1. Expression of basal NRF2 was high in the majority of primary MM cells and cell lines tested. Pharmacological inhibition and shRNA mediated knock-down of NRF2 showed a significant reduction in survival of MM cells, when treated alone and in combination with BZ or CFZ. Investigations also revealed that BM-MSC had increased NRF2 activity in response to BZ and CFZ. Moreover, knockdown of NRF2 in BM-MSC or pharmacological inhibition of NRF2 in BM-MSC/MM co-cultures reverses the protection conferred to MM by BM-MSC in response to BZ and CFZ.

Conclusion:

Here we show the first description of NRF2 driven cytoprotective responses in MM. We show that NRF2 in MM is activated by both BZ and CFZ which subsequently activates pro-survival mechanisms in response to proteasome inhibition. Furthermore, NRF2 is also activated in the BM microenvironment by BZ and CFZ, which also confers protection to MM. This highlights the importance of NRF2 in regulating MM drug resistance within the BM microenvironment through independent actions in both the tumour and the non-malignant BM-MSC which support it.

Disclosures

Rushworth: Infinity Pharmaceuticals: Research Funding.
Vitamin D Blocks CLL Cell-Mediated MDSC Induction
Authors: Mougiakakos Dimitrios , Bruns Heiko , Böttcher Martin , Qorraj Mirjeta , Fabri Mario , Dindorf Jochen , Busch Leonhard , Jitschin Regina , Mackensen Andreas
Published: October 2019

Chronic lymphocytic leukemia (CLL) is the leukemia with the highest incidence amongst adults. CLL-associated immune defects promote tumor immune escape and antagonize immune-based therapies. We and others have reported the accumulation of so-called myeloid-derived suppressor cells (MDSCs) in CLL. Accumulation of immunosuppressive CD14+HLA-DRlow monocytic MDSCs is associated with advanced disease and poor prognosis. The mechanisms re-polarizing CLL-monocytes remain unknown. Here, we describe that CLL-cell-derived exosomes elicit a phenotypical and functional skewing of regular monocytes towards MDSCs. However, pre-treating CLL-cells with vitamin D led to a loss of the exosomes' MDSC-promoting capability. In fact, higher vitamin 25D serum concentrations were linked to lower levels of circulating MDSCs in CLL. A plethora of bioactive molecules including microRNAs (miRs) is shuttled in exosomes. MiR-155 is found abundantly in CLL-exosomes and using antagomir against miR-155 prevented exosomal MDSC-induction. Furthermore, exosomes from patients with lower vitamin D levels contained more miR-155 copies. Accordingly, vitamin D application reduced miR-155 production in CLL-cells Overall, we identified that exosomal miR-155 transfer promotes MDSCs in CLL. Vitamin D interferes with this interplay and could thereby represent a mean for enhancing immune responses in CLL.

Disclosures

No relevant conflicts of interest to declare.
Transcriptional Repression of the LMO2 Oncogene By Ikaros in T-Cell Acute Lymphoblastic Leukemia
Authors: Ding Yali , Song Chunhua , Gowda Chandrika S. , Kapadia Malika , Payne Kimberly , Dovat Sinisa
Published: October 2019

LIM domain only protein 2 (LMO2) is a regulator of hematopoiesis and an oncogene that is overexpressed in a subset of T-cell acute lymphoblastic leukemia (T-ALL). Overexpression of LMO2 in T-ALL is associated with a poor prognosis. The mechanisms that regulate LMO2 expression in T-ALL are still unknown. Here, we present evidence that expression of LMO2 in T-ALL is regulated at the transcriptional level by Ikaros, a tumor suppressor protein whose deletion is associated with the development T-ALL. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in primary human acute lymphoblastic leukemia cells and in cell lines demonstrated Ikaros occupancy of the LMO2 promoter. Ikaros binding at the LMO2 promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL and B-ALL cells. The role of Ikaros in the regulation of LMO2 transcription in T-ALL was tested using gain-of-function and loss-of-function experiments. Ikaros knock-down with siRNA resulted in increased transcription of LMO2 in T-ALL. Overexpression of Ikaros in human T-ALL was associated with strongly reduced transcription of LMO2. In mice, T-ALL cells that are derived from Ikaros-knockout mice express high levels of LMO2. Transduction of these cells with Ikaros-containing retrovirus, results in a sharp reduction of LMO2 expression. Since Ikaros function in T-ALL is negatively regulated by the pro-oncogenic Casein Kinase II (CK2), we tested whether CK2 inhibition can enhance Ikaros-mediated transcriptional repression of LMO2. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor, resulted in reduced expression of LMO2 in primary human T-ALL. Inhibition of CK2 was associated with increased Ikaros binding at the LMO2 promoter. Ikaros knock-down restored high expression of LMO2 in T-ALL cells that were treated with CK2 inhibitors. These data show that Ikaros is a major regulator of LMO2 transcription in T-ALL and that CK2 inhibition requires Ikaros activity to repress LMO2 transcription. Increased Ikaros binding was associated with reduced histone H3K9ac and H3K4me3 marks at the LMO2 promoter suggesting that Ikaros regulates LMO2 transcription via chromatin remodeling. In conclusion, these results provide evidence that expression of the LMO2 oncogene is regulated by Ikaros and CK2 in T-ALL. Targeting CK2 with specific inhibitors has been used as a therapeutic strategy in a preclinical model of T-ALL. The presented data reveal a novel mechanism of therapeutic action for CK2 inhibitors - repression of LMO2 expression via Ikaros. These results provide a rationale for the use of CK2 inhibitors in T-ALL with LMO2 overexpression.

Disclosures

No relevant conflicts of interest to declare.
FOXM1, CDK6 and Rb Dependent Drug Resistance and Senescence in Myeloma
Authors: Gu Chunyan , Chen Ruini , Jing Xuefang , Janz Siegfried , Yang Ye
Published: October 2019

We recently reported that FOXM1 is a promising therapeutic target in multiple myeloma (MM), particularly for the subset of patients with high-risk disease.Because high-risk myeloma exhibits a strong predilection to early drug-resistant relapse following first-line therapy, we here decided to evaluate the role of FOXM1 in the acquisition of drug resistance by myeloma cells.

We analyzed gene expression profiles of 88 paired myeloma samples at baseline and relapse from the UAMS Total Therapy 2 cohort and found that FOXM1 mRNA levels were significant upregulated in relapsed myeloma and that this was associated with poor event-free and overall survival. Laboratory studies showed that enforced expression of FOXM1 in human myeloma cell lines (HMCLs) results in decreased sensitivity of cells to widely used myeloma drugs, such as bortezomib and doxorubicin. This was observed in vitro, in both bulk cell and soft-agar culture, and in vivo using xenografting in mice. Biochemical analysis of HMCLs revealed physical interaction of FOXM1 with CDK6 and Rb, key regulators of cell cycle progression and cellular senescence, respectively.Treatment with small-compound CDK6 inhibitor, inhibited myeloma growth, decreased clonogenicity of myeloma, and ameliorated FOXM1-dependent senescence. Genetic and pharmacological targeting of FOXM1 in myeloma cells using shRNA and thiostreptone respectively, led to growth arrest and senescence, while elevated expression of FOXM1 reversed these phenotypes.

In sum, our findings implicating the FOXM1-CDK6-Rb network in drug resistance and senescence of high-risk myeloma point to new treatment opportunities for this difficult-to-cure neoplasm.

Disclosures

No relevant conflicts of interest to declare.
Bone Marrow Derived Mesenchymal Stromal Cells Reduce the Incidence of Chronic Graft-Versus-Host Disease after Allogenetic Hematopoietic Stem Cell Transplantation
Authors: Zhao Ke , Fan Zhiping , Huang Fen , Wang Shunqing , Xiang Peng , Zhang Qi , Liu Qifa
Published: October 2019

Background Chronic graft-versus-host disease (cGVHD) is the common long-term complication and the leading cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT), which has a significant impact on patient quality of life. Currently, mesenchymal stromal cells (MSCs) have been considered as a promising therapy for treating refractory acute/chronic GVHD and engraftment failure (EF) or poor graft function (PGF), but the efficacy of MSCs in the prophylaxis of cGVHD is rarely reported. Methods Eighty-two patients who received MSCs treatment after allo-HSCT were enrolled in our study, including 45 patients with refractory aGVHD, 33 patients with EF or PGF and 4 patients with virus infection. In order to evaluate the influence of bone marrow (BM) derived MSCs for the incidence and severity of cGVHD, 308 consecutive patients who underwent allo-HSCT in the same period but without MSCs treatment were considered as the control group. And no statistical significance was found between the two groups for the demographic and transplant characteristics. MSCs were given at a median dose of 1×106 cells/kg once weekly. Results All eighty-two patients in MSC group received a median of 5 (range:3-12) doses of MSCs per patient. In MSC group, seventeen patients (20.7%) had cGVHD, including 14 patients with limited cGVHD and 3 patients with extensive cGVHD. In the control group, 140 patients (45.5%) had cGVHD, including 95 patients with limited and 45 extensive cGVHD. The 2-year cumulative incidence of cGVHD was 26.7% (95%CI: 16.1%-37.5%) in MSC group and 50.4% (95%CI: 37%-63.8%) in the control group (P=0.037). Furthermore, the extensive cGVHD (3/82) in the MSC group were also significantly lower than those (45/308) in the control group (P=0.021). In addition, at a median follow-up time of 189 (range: 112-1035) days post-transplantation, 4 patients relapsed in MSC group and 13 patients relapsed in the control group. The incidence of tumor relapse was not different between the two groups (P=0.629). No short-term toxic side effects were observed and other secondary tumor occurred after MSCs treatment. Conclusion MSCs derived from BM reduce the incidence and severity of cGVHD after allo-HSCT, but not increase the risk of tumour relapse.

Disclosures

No relevant conflicts of interest to declare.
Feasibility of Allogeneic Hematopoietic Stem Cell Transplant for High Risk FLT3-ITD Mutant Patients with Acute Myeloid Leukemia in CR1- a Real Word Analysis
Authors: Kansagra Ankit J. , Alkhateeb Hassan B , Hefazi Mehrdad , Yalniz Fevzi , Hogan William J , Litzow Mark R , Patnaik Mrinal M
Published: October 2019

Background:

Hematopoietic stem cell transplantation (HCT) remains the most effective postremission therapy for high-risk acute myeloid leukemia (AML). FMS-like tyrosine kinase3 (FLT3) mutations occur in about 30% of patients with cytogenetically normal AML. FLT3-ITD (internal tandem duplications) mutations are associated with short-lived remissions and poor outcomes. These adverse effects can be offset by allogeneic HCT in first complete remission (CR1). We carried out this study to get a real world perspective on feasibility of allogeneic HCT in CR1 for FLT3-ITD mutant AML patients.

Methods: The Mayo Clinic AML database was queried for all patients that had tested positive for the FLT3 ITD, from January 2003 to December 2014. 75 patients were identified, of which 28 (37%) underwent allogeneic HCT in CR1. 12 were excluded as they were transplant ineligible from an age standpoint, 6 patients actually had MDS and 2 were excluded for primary induction failure. Clinical and outcomes data were abstracted retrospectively.

Results:

29 patients were included in the final analysis, with a median age of 57 years (range 21-68), 38% males. Median time from diagnosis to last follow up was 339 days. 24(83%) had normal cytogenetics. 26(90%) received treatment with anthracycline + cytarabine based therapy, 3(10%) were enrolled on a clinical trial. 25(87%) patients went on to receive consolidation therapy. 22 (75%) of the patients relapsed after achieving a CR1, with a median time to relapse of 134 days (range 27-1710 days). Only 8 (26%) of the 22 patients were able to achieve a CR2 with salvage chemotherapy, of which 6 (75 %) successfully underwent allogeneic HCT.2 (25%) out of 8 patients received FLT3 inhibitor in 1st relapse. We then assessed the reasons why relapsed patients did not undergo HCT in CR1. The most common being; relapse during evaluation/work up for HCT in 8(27%) patients, 5 (17%) had comorbidities that precluded HCT, 3 (10%) were never referred for HCT due to personal preferences. At last follow up 19(65%) patients were dead.

Conclusion:

While allogeneic HCT is an effective consolidative strategy for FLT3 ITD mutant AML patients, in the real world, a large number are not transplanted in CR1 due to delays in donor search and transplant scheduling strategies resulting in disease relapse. Effective strategies to avoid delays in scheduling, rapid donor searches and the use of alternative donor sources are much needed for these patients.

Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
Schistocytosis in B12 Deficiency (B12D) Reduces the MCV and Raises the RDW in the Absence of Iron Deficiency: a Cause of Unnecessary Plasmapheresis (PP)
Authors: Savjani Maushmi , Draksharam Padma , Braverman Albert S
Published: October 2019

Background:

RBC size variation and microcytes in B12D are often ascribed, but rarely shown, to be due to co-existing Fe deficiency. Schistocytes (aka poikilocytes) were included in early descriptions of pernicious anemia (PA). As it also causes thrombocytopenia, B12D may be confused with thrombotic thrombocytopenic purpura (TTP). Because of the urgency of PP for TTP, it has sometimes been initial therapy for B12D with schistocytes.

Purpose:

Here we describe 3 PA patients, and review reports of 15 other severely anemic B12D cases, with schistocytes. As they are smaller than mature RBC, they may normalize the MCV, while raising the RDW. We wished to determine whether primary lab studies help to distinguish B12D from TTP.

Case Reports:

These patients presented with symptoms of severe anemia:

Ferritins were normal or high, haptoglobins <30, anti-intrinsic factor and parietal cell antibodies present, and complete response to B12 therapy occurred. Significant numbers of schistocytes were present in all, as was a distinctive population of minute, hypochromic schistocytes (MHS).

Review:

We found 15 reports of individual B12D patients with schistocytes, published between 2003 and 2015. All but 2 vegans had PA, 6 being anti-IF Ab+. Median & median ages were 52 & 53, [Hb]'s 5.9 & 6.0, MCV's 120 & 117 (3 normal), platelets 97 & 85, WBC 3200 & 3120, LDH's 4050 & 5860; reticulocyte levels were all low for severe anemia. Iron deficiency anemia was not found; ferritin and/or Fe/TIBC were reported normal in 4. Six legible blood smear microphotographs were included, confirming schistocytosis. Five patients had one or more PP before B12 deficiency was discovered. All 15 responded completely to B12. MHS were also observed on 4 of the 6 legible microphotographs, and noted by authors. For comparison, our survey of 36 reported TTP legible blood smears failed to detect MHC.

Conclusions:

Schistocytes are observed in some severely anemic B12D, may account for as much as 6.7% of RBC, and alter their indices. Schistocytosis and thrombocytopenia suggest TTP, and may lead to unnecessary PP. But such patients' low reticulocyte counts and very high LDH levels are not typical of TTP.

Table Table. Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
Correlation of Serum Ferritin Levels with Liver and Heart Mri T2 and Liver Iron Concentration in Beta Thalassemia Intermediate Patients: A Contemporary Issue
Authors: Karimi Mehran , Amirmoezi Fatemeh , Haghpanah Sezaneh , Ostad Seyed pouria , Lotfi Mehrzad , Sefidbakht Sepideh , Rezaian Shahed
Published: October 2019

Background: B-Thalassemia intermediate (B-TI) is a genetic disease that is milder than beta thalassemia major. The accumulation of iron in different organs causes tissue damage. The T2* magnetic resonance imaging (MRI) technique is currently the gold standard for iron load detection. However, it is expensive and needs an expert radiologist to report findings. Therefore, we conducted this study to determine an optimal cut-off value of ferritin in proportion to T2 MRI for early detection of cardiac and hepatic iron overload in patients with beta thalassemia intermediate.

Methods: This cross-sectional study was conducted on 108 patients with B-TI who referred to tertiary Hospital, Shiraz University of Medical Sciences, Shiraz, Iran. Serum ferritin, hepatic and cardiac T2 MRI were assessed. The ROC curve was used to determine the sensitivity and specificity of cut-off value.

Results: Serum ferritin levels showed a statistically significant negative correlation with T2 hepatic MRI (r= -0.290, P value=0.003) and positive correlation with LIC (r= 0.426, P value ˂ 0.001) in the patients with BTI. However, T2 cardiac MRI was not significantly correlated with serum ferritin levels (P value= 0.073).According to the analysis of ROC curves, the best cut-off value for ferritin to show early diagnosis of liver iron overload was 412 ng/ml. calculated sensitivities and specificities were 0.78 and 0.82 for T2 liver MRI and 0.76 and 0.86 for liver iron concentration (LIC) respectively.

Conclusion: Serum ferritin levels of 412 ng/ml might be considered as a cut-off point to evaluate hepatic iron overload before using expensive, not readily available T2 MRI. This level of serum ferritin (around 500 ng/ml) could be considered for starting iron chelation therapy in patients with B-TI in areas where T2 MRI is not available.

Disclosures

No relevant conflicts of interest to declare.
S1P Receptor 1-Mediatd Elevated IL-6 Signaling Underlies Chronic Inflammation and Multiple Tissue Damage in Sickle Cell Disease
Authors: Zhang Yujin , Zhao Shushan , Couturier Jacob , Kellems Rodney E. , Lewis Dorothy L. , Xia Yang
Published: October 2019

Objective-Sphingosine 1 phosphate (S1P) is a biolipid involved in chronic inflammation in multiple inflammatory disorders. Recent studies revealed that elevated S1P contributes to sickling in sickle cell disease (SCD), a devastating hemolytic genetic disorder associated with severe chronic inflammation. Here we evaluated the impact of S1P in chronic inflammation and underlying mechanisms in SCD.

Approach and Results-First, we demonstrated that interfering with S1P receptor signaling by FTY720, a FDA approved drug, significantly reduced systemic and local inflammation without anti-sickling effects. These findings led us to discover that IL-6 was highly elevated in the circulation and that increased S1P signaling via S1P receptors contributed to the induction of IL-6 in SCD mice. Genetic deletion of IL-6 in SCD significantly reduced local and systemic inflammation and multiple tissue damage and kidney dysfunction. Moreover, we determined that elevated IL-6 led to increased macrophage infiltration and elevated S1P1 gene expression in the macrophages of multiple tissues in SCD mice. Mechanistically, we reveled that S1P-S1P1 signaling reciprocally upregulated IL-6 gene expression in primary mouse macrophages in a JAK2-dependent manner.

Conclusion-Altogether, we revealed that elevated S1P signaling via S1P1-induced IL-6 is a key signaling network functioning as a malicious positive feed forward loop sustaining inflammation and promoting tissue damage in SCD. Our findings immediately highlight novel therapeutic possibilities.

Disclosures

No relevant conflicts of interest to declare.
Erythrocytes Retain "Hypoxic Purinergic Memory" for Faster Acclimatization upon Re-Ascent
Authors: Song Anren , Zhang Yujin , Liu Hong , Sun Kaiqi , Peng Zhangzhe , Wu Hongyu , Kellems Rodney E. , Roach Robert , Xia Yang
Published: October 2019

Improved and faster acclimatization to high altitude upon re-ascent is seen in humans. However, molecular basis for initial acclimatization and retention upon re-ascent to high altitude remains largely unknown. Here we report that plasma adenosine levels are induced by high altitude and retained at higher levels upon re-ascent in health lowlanders and that its elevation is positively associated with quicker acclimatization upon re-ascent. Unexpectedly, we found that, 1) erythrocyte equilibrative nucleoside transporter 1 (eENT1) is the major adenosine transporter in erythrocyte. 2) eENT1 protein level is downregulated in both humans at high altitude and mice under hypoxia. 3) Deletion of eENT1 allows rapid accumulation of plasma adenosine to offset hypoxic tissue damage in mice. Mechanistically, 4) we revealed that ADORA2B-mediated PKA-dependent phosphorylation, ubiquitinization and proteasomal degradation underlies hypoxia-mediated reduced eENT1 protein. Thus, the reduced eENT1 resulting from the initial hypoxia establishes erythrocyte "hypoxic purinergic memory" to promote quicker and higher accumulation of plasma adenosine and faster acclimatization upon re-ascent.

Disclosures

No relevant conflicts of interest to declare.