bluuebunny/crossref_metadata_2025 huggingface dataset:
SLC2A5 Overexpression in Childhood Philadelphia Chromosome Positive Acute Lymphoblastic Leukaemia
Authors: Zhao Pan , Zheng Yuhuan , Niu Ting
Published: October 2019

To study glycolysis/glycogenesis-related genes expression in childhood B cell acute lymphoblastic leukaemia (B-ALL), we performed a microarray-based analysis using published gene expression profiles. We found that gene SLC2A5, which encoded fructose transporter GLUT5 that facilitated cell fructose uptake, was up-regulated in Philadelphia chromosome positive ALL (Ph+ALL). Microarray-based analyses also suggested that SLC2A5 expression was significantly down-regulated in childhood B-ALL with t(1;19) or 11q23 mutation. High SLC2A5 expression was found in the patients who had recurrence within 3 years, early relapse, shortened complete remission duration, and positive minimal residue disease (MRD) status after treatment. The overexpression of SLC2A5 at both mRNA level and protein level in Ph+ALL was confirmed in a validation cohort of childhood B-ALL. We also validated the correlation of SLC2A5 expression and MRD status. In a mechanistic study using a human Ph+ALL cell line, we found that BCR-ABL kinase might regulate GLUT5 expression via c-myc. The tyrosine kinase inhibitors imatinib and dasatinib repressed GLUT5 expression and the cell uptake of fructose. Fructose protected the tumour cells from nutrition deficiency and drug-induced cell death. Overall, our findings showed that SLC2A5 was up-regulated in childhood Ph+ALL. The expression of SLC2A5 correlated with childhood B-ALL clinical factors, such as MRD status. Since TKI was able to inhibit GLUT5 expression, repression of fructose utility after TKI treatment contributes to TKI-induced Ph+ALL cytotoxicity. Targeting GLUT5 might be promising in B-ALL treatment, especially for Ph+ALL patients with high expression of SLC2A5.

Disclosures

No relevant conflicts of interest to declare.
Encouraging Outcome of Using Sibling HLA-Matched Fresh Cord Blood for Children β-Thalassemia Major Transplantation
Authors: Wen Jianyun , Chen Libai , Liu Sixi , He Yuelin , Ruan Yongsheng , Pei Fuyu , Feng Xiaoqin , Li Chunfu , Wu Xuedong
Published: October 2019

Background: Hematopoietic stem cell transplantation(HSCT) is currently the only curative treatment for thalassemia major (TM). Cord blood (CB) from a sibling has different characteristics from marrow and has potential advantages and disadvantages as a stem cell source.

Methods: We conducted a retrospective study of fresh cord blood transplantation (F-CBT) from the matched human leukocyte antigen (HLA)-identical sibling donors in 35 children (median age at transplantation: 4 years, range:1-7 years) with β-TM from June 2010 to December 2016. The conditioning protocol included intravenous busulfan, cyclophosphamide, fludarabine, and thiotepa.

Results:The median collected CB volume was 130ml (range: 79-209ml). The median infused total nucleated cell (TNC) dose was 9.38×107/kg (range: 2.73-18.91×107/kg). One patient had graft failure (GF) on +30day after F-CBT and one patient died from respiratory and heart failure that developed from a pulmonary infection. Of the 33 patients who had a successful engraftment, two patients developed grade II~III acute graft-versus-host disease(GVHD); and one with grade I extensive chronic GVHD was observed during the long-term follow-up period. The median time to neutrophil recovery was +27 days (range: +14 to +49days). The platelet and hemoglobin engraftment times were +33 days (range: +19 to +73 days) and +26 days (range: +11 to +74 days), respectively. All the patients were followed up by December 31, 2017; the median follow-up time was 45 months, and the estimated 5-year overall survival (OS) and TM-free survival (TFS) of F-CBT were 97.1% and 94.2%, respectively.

Conclusions: F-CBT from a matched HLA-identical sibling donor is an effective treatment option for β-TM in children with less GVHD.

Disclosures

No relevant conflicts of interest to declare.
Prognostic Value of Minimal Residual Disease before Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia
Authors: Pérez-López Olga , Caballero-Velázquez Teresa , Colado Enrique , Alonso Sara , González-Campos José , Prats-Martín Concepción , Rodríguez-Torres Nancy , Escamilla Gomez Virginia , Espigado Ildefonso , Pérez-Simón Jose Antonio
Published: October 2019

Introduction

Several studies have shown that the minimal residual disease (MRD) in acute myeloid leukemia (AML) patients has a prognostic value after induction and consolidation therapy. Nevertheless the relapse is the most important cause of treatment failure in these patients, although they achieved a negative MRD, and even after an allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nowadays, the value of the MRD before allogeneic BMT is still controversial.

Method

Multicentric study where we have studied correlative AML patients who went under an allo-HSCT in a situation of complete response, between 2012 and April'18. The MRD was analyzed by 8-coloured multiparametric flow cytometry, at least with 2 tubes per patient and 1,000,000 events per tube. We evaluated the prognostic value of the MRD before allo-HSCT.

Results

Between January'12 and April'18 we have gathered 90 allogeneic BMT in AML patients who were in CR, with a median age of 45 years old (17 - 66). The pre-HSCT situation was 1st complete remission (CR) in 75 patients and 2nd CR in 15. In 45 patients the conditioning regimen was myeoablative. In the group of patients (67) where we could know the risk group at diagnosis, the distribution was: low risk 18%, intermediate risk 59.7% and high risk 22.4%. The 46.7% of the donors were not related. In the last follow-up after allo-HSCT 24 patients have suffered a relapse (26.7%) and 41 (45.5%) have died (17 cases of mortality related to the transplant and 24 not related). In the global analysis the median follow-up of the overall survival (OS) was 37.5 months. Among the 90 patients, MRD was valuable in 86. Ten of 59 patients (16.9%) with negative MRD relapsed vs 12/27 (44.4%) with positive MRD, p= 0.016. If we consider only patients in 1st CR, 9/50 (18%) patients with negative MRD relapsed vs 10/22 (45.5%) with positive MRD, p= 0.02. This statistically significant difference does not exist if we consider only patients in 2nd CR. The median follow-up of OS and event free survival (EFS) was not reached in the negative MRD group and 571 days and 299 days in the positive MRD group. OS and EFS at 2 years after transplantation were 65% and 64% in the negative MRD group and 42% and 37% in the positive MRD group, p= 0.03 and p= 0.008 respectively (figure 1).

Conclusions

The detected MRD by 8-colour multiparametric flow cytometry previous an allo-HSCT in patients with AML in 1st CR is a prognostic factor in terms of relapse. Patients with a positive MRD before the allo-HSCT have a poorer OS and EFS than the patients with a negative MRD.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Risk Factors for Induction Related Mortality in Adults Patients with Acute Lymphoblastic Leukemia: Report from a Hispanic Population
Authors: Inclan-Alarcon Sergio I , Bourlon Christianne , Fierro-Angulo Oscar Manuel , Garcia-Ramos Jesus A , Riviello-Goya Santiago , Demichelis-Gómez Roberta
Published: October 2019

Introduction

Acute lymphoblastic leukemia (ALL) represents 20-30% of acute leukemia in adults. Higher incidence and inferior outcomes in Hispanic population have been described. In Latin Americans induction mortality (IM) is a major cause of death representing 20-50% vs.7-11% in developed countries. Our aim was to determine risk factors (RF) related to IM in ALL Hispanic adult patients.

Methods

We retrospectively analyzed clinical data of ≥18yo patients with ALL diagnosed and treated at our institution within 2009 and 2016.

Results

A total of 170 patients were included. Median age was 29 years (16-70), 64% were AYA, 96.8% had B-cell ALL, and 62.3% received Hyper-CVAD.

IM rate was 13.4%. In 64.1% IM was related to an infectious cause. The most frequent infection was pneumonia (39.8%). Gram-negative etiology was more prevalent (35.5% vs. 10.2%), however, IM rate was higher in gram-positive infections (26.3% vs .13.6%; p=.028).

RF related to IM in univariate analysis were: CNS involvement (OR4.6,95%IC2.8-9.5;p=<.001), tumor lysis syndrome (TLS) (OR 5.6, 95% CI 2.2-14.1; p=<.001), need for dialysis (OR 28.9, 95% CI 5.3-157.1; p=<.001), primary hypertension (OR 3.5, 95% CI 1.0-12.7; p=.052), shock status (OR 10.3, 95% CI 3.9-27.3; p=<.001), ECOG³2 (OR 1.9, 95% IC 1.1-3.4; p=.022), T-ALL (OR 2.2, 95% IC 1.1-4.3; p=.026), Hyper-CVAD (OR 1.9, 95% IC 1.1-3.8; p=0.51), ventilation assistance (OR 7.7, 95% IC 2.8-21; p=<.001), and vasopressor use (OR 7.6, 95% IC 2.8-20.6; p=<.001). In multivariate analysis TLS, need for dialysis and shock, kept statistical significance.

Conclusions

To our knowledge, this is the largest study that evaluates the impact over IM of biological, social, and economic factors in Hispanic adult patients with ALL. We identified factors not previously described such as hypertension and need for dialysis. Multicenter prospective studies most be urged to asses and validate these RF, and design a bedside prognostic score that can predict an increased risk of IM at ALL diagnosis.

Disclosures

No relevant conflicts of interest to declare.
STAT3 Inhibition Enables TLR9-Driven Differentiation of Cbfb/Myh11 acute Myeloid Leukemia Cells to Antigen-Presenting Cell Phenotype In Vivo
Authors: Kortylewski Marcin , Duttagupta Priyanka , SU Yu-Lin , Adamus Tomasz , Marcucci Guido
Published: October 2019

STAT3 transcription factor is persistently activated in cancer cells and in diverse tumor-associated immune cells being an important oncogene and an essential immune checkpoint regulator. It is a highly desirable but challenging therapeutic target for pharmacological intervention. We previously demonstrated that ligand for the intracellular receptor TLR9 (CpG oligonucleotide) allows for the uptake and cytoplasmic delivery of decoy oligodeoxynucleotides (dODNs) to inhibit transcriptional activity of STAT3 in a variety of human and mouse acute myeloid leukemia (AML) cells. Here, we demonstrate that CpG-STAT3dODN injected intravenously to mice engrafted with Cbfb/MYH11 AML, dramatically altered leukemic cell morphology, by reducing nucleus-cytoplasm ratio and increasing mitochondria size, and also resulting in metabolic shift from glycolysis to oxidative phosphorylation, resulting in increased mitochondrial ATP production. The transcriptional profiling of AML cells isolated from spleens of mice treated using CpG-STAT3dODN vs control CpG-scrODN or PBS, revealed the increased expression of genes regulating myeloid cell differentiation (Irf8, Cebpa, Gadd45a) and antigen-presentation (B2m, CIIta, IL-12a, Ifng), with decrease of leukemia-related regulators Runx1and Run1t1. Thein vivodifferentiation of leukemic cells was confirmed using flow cytometry by detecting appearance of the population of AML-derived CD11b+/MHCII+/CD86+myeloid cells after CpG-STAT3dODN but not control treatments. Furthermore, we confirmed that the differentiated AML cells have immunogenic effects, which drive expansion of Th1 effector T cell responses in Cbfb/MYH11 as well as C1498 AML models. Overall, our studies suggest that STAT3-inhibition can unleash immune-stimulatory potential downstream of TLR9 signaling transforming AMLcells into antigen-presenting cells. These findings support development of CpG-STAT3dODN strategy as a new bi-functional agent for AML immunotherapy.

This project described was supported by the National Cancer Institute of the National Institutes of Health under award number R01CA213131 to M.K.

Disclosures

Kortylewski: N/A: Patents & Royalties: I am an inventor on the US patent 9,976,147.
LILRB4 Signaling in Leukemia Cells Mediates T Cell Suppression and Tumor Infiltration
Authors: Deng Mi , Gui Xun , Kim Jaehyup , Zheng Junke , Zhang Ningyan , An Zhiqiang , Zhang Chengcheng
Published: October 2019

Immune checkpoint blockade therapy has been successful in treating some types of cancers but has not shown clinical benefits for treating leukemia. This result suggests that leukemia exploits unique escape mechanisms. Certain immune inhibitory receptors that are expressed by normal immune cells are also present on leukemia cells. It remains unknown whether these receptors can initiate immune-related primary signaling in tumor cells. Here we show that LILRB4, an ITIM-containing receptor and a monocytic leukemia marker, supports tumor cell infiltration into tissues and suppresses T cell activity via ApoE/LILRB4/SHP-2/uPAR/Arginase-1 signaling axis in acute myeloid leukemia (AML) cells. Blocking LILRB4 signaling using knockout and antagonistic antibody approaches eliminated AML development. Thus, LILRB4 orchestrates tumor invasive pathways in monocytic leukemia cells by creating an immune-suppressive microenvironment. LILRB4 represents an attractive target for treatment of monocytic AML.

Disclosures

Deng: Immune-Onc Therapeutics Inc.: Patents & Royalties. Gui:Immune-Onc Therapeutics Inc.: Patents & Royalties. Kim:Immune-Onc Therapeutics Inc.: Patents & Royalties. Zheng:Immune-Onc Therapeutics Inc.: Patents & Royalties. Zhang:Immune-Onc Therapeutics Inc.: Patents & Royalties. An:Immune-Onc Therapeutics Inc.: Patents & Royalties. Zhang:Immune-Onc Therapeutics Inc.: Patents & Royalties.
Thiotepa-Based Conditioning Regimen Compared to Non-Thiotepa Conditioning Regimen Prior to Allogeneic Stem Cell Transplantation in β Thalassemia Major: Impact on Survival
Authors: Tan Wanxia , He Yuelin , Feng Xiaoqin , Wu Xuedong , Li Chunfu , Jianyun Liao , Peng Zhiyong , Ruan Yongsheng , Hui Ge , Liu Xiaoting , Ren Yuqiong
Published: October 2019

This study is to investigate the survival impact of thiotepa as part of the conditioning therapy for β thalassemia major (TM) patients, undergoing HSCT from haploidentical and sibling cord blood transplant. We retrospectively analyzed 262 transplant patients and compared outcomes between Cyclophosphamide -fludarabinebusulfan - thiotepa (with TT group)and Cyclophosphamide fludarabine -busulfan(without TT group) regimens from July 2014 to July 2018. Transplantation was performed from 86 matched sibling (32.8%), 103 unrelated (39.3%) ,13 matched parent(5%), 41 haploidentical (15.6%), and 19 sibling cord blood (7.3%) donors, with a median follow-up of 27 months(1months-72months) and median age of 7 years old(2 ys-18ys). The overall survival (OS), thalassemia free survival (TFS), graft rejection (GR) and transplantation related mortality (TRM) among TT group and without TT group in matched sibling transplant patients was 97.6%, 97.6%, 0%, 2.4% and91.6%, 91.6%, 0%, 8.4% (P=0.355 in TFS), respectively; in unrelated transplant patients was 97.9%, 97.9%, 0%, 2.1% and 95.2%,93.3%, 1.9%, 4.8% (P=0.256 in TFS), respectively; in matched parent transplant was 90.9%, 90.9%, 0%, 9.1% and 100%. 100%, 0%, 0%;( P=0.546 in TFS), respectively; whereas, in haploidentical transplant was 100%, 96.9%, 3.1%, 0% and 88.9%, 77.8%, 12.5%, 11.1%(P=0.043 in TFS); and in sibling cord blood transplant was 100%, 100%, 0%, 0% and 100%, 62.3%, 29.9%, 0% (P=0.035 in TFS). Our data suggest that thiotepa-based conditioning regimen in TM is effective and safe and survival outcome of thiotepa-based regimen in haploidentical transplant and sibling cord blood transplant are encouraging. Thiotepa-based regimen may improve survival outcome in haploidentical transplant and sibling cord blood transplant.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Confirming the Presence of a Smoldering T and B PLL Associated with Improved 5 Year OS: Using the National Cancer Data Base
Authors: Parsons Benjamin , Peter Polewski , Go Ronald S. , Borgert Andrew , Gaddam Mamatha , Narayana Swapna
Published: October 2019

Background:

Prolymphocytic leukemia (PLL) is a rare cancer with incidence of 0.48/1,000,000. It is comprised of 2 subtypes, T or B cell. It generally is associated with a poor median OS of 1-3 years. However, some patients have an initial smoldering course not requiring treatment. Limited population data exists describing the outcomes of PLL. We studied PLL patients who met a definition of smoldering, not requiring treatment within 180 days of diagnosis, using the National Cancer Data Base (NCDB).

Methods:

Patient level data were obtained from the NCDB Participant User File. The NCDB collects hospital cancer registry data representing 70% of newly diagnosed cancer cases in the US population. We identified PLL patients (ICD-O: 9832 PLL, NOS, ICD-O: 9833 B-PLL, and ICD-O: 9834 T-PLL) diagnosed in 2004-2015 for demographics, disease and treatment characteristics. We included patients diagnosed from 2004-2013 in the overall survival (OS) analyses. Patients who received no treatment as part of their first course of treatment and who did not have a subsequent treatment within 180 days were considered smoldering PLL.

Results:

From 2004-2015, 1606 patients were diagnosed with PLL and 451(28%) were smoldering PLL. Of the 1606 patients, T-PLL represented 781(49%), B-PLL 480(30%), and PLL, NOS 345(21%). Of the 451 smoldering PLL patients observed as T-PLL represented 48%(215), B-PLL 29%(133), and PLL, NOS 23%(103). The median age at diagnosis was 69 [59 - 78] years. There was a male predominance with 60 % males. The median OS for the entire cohort was 20.6 months. The % 5yr overall survival (OS) for smoldering PLL was 40% vs 29%(for non-smoldering). Within the PLL subtypes PLL, NOS and B-PLL were associated with better 5yr OS 38.56% and 35.53%, compared to T-PLL 12.89%. Only 65(4%) patients underwent an early stem cell transplant(SCT).

Conclusions:

We identified a population of patients not needing treatment within 180 days of diagnosis representing smoldering PLL with a significantly improved OS. Additionally, PLL, NOS and B-PLL were associated with an improved OS. The use of early SCT is low in PLL.

Disclosures

No relevant conflicts of interest to declare.
The Efficacy and Safety of Super High-Dose Cytarabine Based Strategies in Childhood Acute Myeloid Leukemia Treatment
Authors: WU Zelin , Liang Ming , Zhang Hui , Wu Ziliang
Published: October 2019

Objective The aim of this study was to evaluate the effect of super high-dose cytarabine (SHDAC), a regimen first created by the author, on the five-year overall survival (OS) and event-free survival (EFS) of children with AML. Methods From July 2001 to April 2016, a total of 25 pediatric patients with newly diagnosed AML (exclueding secondary AML、APL and Down Syndrome AML) were enrolled. Patients received consolidate chemotherapy with the same regimen of high-dose cytarabine (3g/m2 every 12 hours by continuous infusion for 6 days) after remission induction chemotherapy. Then phase II consolidation therapy (also known as early intensive treatment) was performed according to the regimen of high-dose cytarabine (3g/m2 every 12 hours on days 1, 3, and 5 for four cycles, a total of 108g/m2) in 16 cases, while the other 9 cases received the treatment with another regimen (H3A7, M3A7 and VP16 combined with cytarabine alternately at intervals ranging from 2 to 6 months). The total treatment course spanned about three years, with regular post-treatment follow-up tracking for more than five years. Results Originally 26 cases were enrolled, but 1 case was not relieved after two courses of treatment and quitted therapy, so only 25 cases were enrolled finally. The total remission rate was 96%. There were 12 cases (48%) achieving complete remission within one course, 12 cases (48%) within two consecutive remission course and one case (48%) ,only one case(4%) achieving complete remission within with in five courses. The five-year OS and EFS of these 25 AML patients in this study were 77±0.98% and 77±1.22% respectively. Four patients (16%) died (1 treatment-related (4%), 3 relapse (12%) including 3 marrow relapse and 1 combined with testicular relapse). The average relapse time was 29.6 months and the median time was 14.8 months. Conclusion The application of super high-dose cytarabine (a total of 108g/m2 cytarabine, with a regimen of 3g/m2 every 12 hours by continuous infusion for 6 days as consolidation chemotherapy and 3g/m2 every 12 hours on days 1, 3, and 5 for four cycles as the early intensive treatment) is clinically safe and effective, with minimal hospitalization expense index (HEI). For patients with financial difficulties, especially those in developing countries, super high-dose cytarabine shows landslide advantages and practicality. Therefore, there should be more collaborative clinical applications as well as research upon related mechanisms.

Disclosures

No relevant conflicts of interest to declare.
AMP-activated protein kinase links acetyl-CoA homeostasis to BRD4 recruitment in acute myeloid leukemia
Authors: Jiang Yajian , Hu Tianyuan , Wang Tao , Shi Xiangguo , Kitano Ayumi , Eagle Kenneth , Hoegenauer Kevin A. , Konopleva Marina Y. , Lin Charles Y. , Young Nicolas L. , Nakada Daisuke
Published: October 2019

There is increasing evidence that the metabolic regulation of acute myeloid leukemia (AML) cell growth interacts with epigenetic pathways of gene expression and differentiation. Jiang et al link inhibition of glucose metabolism to epigenetic changes and altered transcriptional pathways in leukemic cells and demonstrate synergy between simultaneously targeting metabolism and chromatin modifiers in suppression of AML.
A common polymorphism in the annexin V Kozak sequence (−1C>T) increases translation efficiency and plasma levels of annexin V, and decreases the risk of myocardial infarction in young patients
Authors: González-Conejero Rocio , Corral Javier , Roldán Vanessa , Martı́nez Constantino , Marı́n Francisco , Rivera José , Iniesta Juan A. , Lozano Marı́a L. , Marco Pascual , Vicente Vicente
Published: October 2019

Annexin V has phospholipid-binding capacity and plays a potent antithrombotic role. Recently, a C to T transition has been described in the Kozak region of this gene, affecting the nucleotide preceding the initiation ATG codon. We have developed a simple method to detect this genetic change, showing by analysis of 580 Mediterranean white subjects that the −1C to T transition (−1C>T) is a common polymorphism (allele frequency, 0.121). This polymorphism is in linkage disequilibrium with a new C>G polymorphism located 27 bp downstream in intron 2. We show that −1C/C carriers presented significantly lower plasma levels of annexin V than −1C/T subjects (0.45 ± 0.20 ng/mL versus 0.73 ± 0.28 ng/mL, respectively;P = .02). In vitro transcription/translation experiments support that the −1T allele increases translation efficiency. The clinical relevance of the −1C>T change was investigated in consecutive patients with nontraumatic spontaneous intracranial hemorrhage (n = 225), deep venous thrombosis (n = 151), and coronary heart disease (n = 101). Finally, we also studied 166 survivors of an acute myocardial infarction occurring at age of 45 or less. This polymorphism seems to have a minor effect in bleeding disorders, but to play a protective role against early myocardial infarction, reducing by 2-fold the risk of developing the disease (P = .006; odds ratio, 0.51; 95% confidence interval, 0.30-0.85).
Human fetuses are able to mount an adultlike CD8 T-cell response
Authors: Hermann Emmanuel , Truyens Carine , Alonso-Vega Cristina , Even Jos , Rodriguez Patricia , Berthe Aurélie , Gonzalez-Merino Eric , Torrico Faustino , Carlier Yves
Published: October 2019

Fetal/neonatal immune responses generally are considered to be immature and weaker than that of adults. We have studied the cord-blood T cells of newborns congenitally infected with Trypanosoma cruzi, the protozoan agent of Chagas disease. Our data demonstrate a predominant activation of CD8 T cells expressing activation markers and armed to mediate effector functions. The analysis of the T-cell receptor beta chain variable repertoire shows the oligoclonal expansion of these T lymphocytes, indicating that activation was driven by parasite antigens. Indeed, we have detected parasite-specific CD8 T cells secreting interferon-γ after coincubation with live T cruzi. This response is enhanced in the presence of recombinant interleukin-15, which limits the T-cell spontaneous apoptosis. These findings point out that the fetal immune system is more competent than previously appreciated, since fetuses exposed to live pathogens are able to develop an adultlike immune CD8 T-cell response.
Rituximab treatment of refractory fludarabine-associated immune thrombocytopenia in chronic lymphocytic leukemia
Authors: Hegde Upendra P. , Wilson Wyndham H. , White Therese , Cheson Bruce D.
Published: October 2019

Fludarabine can exacerbate idiopathic thrombocytopenia (ITP) in chronic lymphocytic leukemia (CLL). We report 3 CLL patients with refractory fludarabine-associated ITP who responded to rituximab. The patients had Rai stages III, III, and IV disease. Before fludarabine treatment, the platelet counts were 141 000/μL, 118 000/μL, and 70 000/μL. ITP developed within week 1 of cycle 3 in 2 patients and within week 2 of cycle 1 in 1 patient. Platelet count nadirs were 4000/μL, 1000/μL, and 2000/μL, respectively, and did not respond to treatment with steroids or intravenous immunoglobulin. Rituximab therapy (375 mg/m2 per week for 4 weeks) was begun on days 18, 23, and 20 of ITP. Patient 1 achieved a platelet count of more than 50 000/μL at day 21 and more than 133 000/μL at day 28, patient 2 achieved a platelet count of more than 50 000/μL at day 4 and more than 150 000/μL at day 10, and patient 3 achieved a platelet count of more than 50 000/μL at day 5 and 72 000/μL at day 28 of rituximab therapy, with platelet response durations of 17+, 6+, and 6 months. These results suggest rituximab can rapidly reverse refractory fludarabine-associated ITP.
Generation of Tumor-Specific Cytotoxic T Lymphocytes in Multiple Myeloma Using Dendritic Cells Pulsed with Tumor-Derived Heat Shock Protein gp96.
Authors: Yi Qing , Qian Jianfei , Xie Jin , Wang Siqing , Freeman Muta E. , Barlogie Bart
Published: October 2019

To develop more effective immunotherapy strategies for patients with multiple myeloma (MM), it is important to identify novel tumor antigens. Heat shock protein gp96, a highly conserved glycoprotein, is a chaperon molecule that carries intracellularly processed peptides from proteins synthesized by the cells, including those of tumor-specific or -associated proteins. Recent studies in solid tumors have shown that tumor-derived gp96 is immunogenic and potent at stimulating the generation of tumor-specific cytotoxic T lymphocytes (CTLs). In this study, we examined whether myeloma-derived gp96 can be used as potent myeloma antigen. To generate myeloma-specific CTLs, immature dendritic cells (DCs), obtained from cultures of blood monocytes from HLA-A2+ healthy individuals or patients, were pulsed with gp96 protein purified from the myeloma cell line U266 (A2+), and induced maturation using the cytokine cocktail. Autologous T cells were then stimulated every two weeks with these DCs, and cytotoxicity was examined against gp96-pusled DCs, myeloma cell lines, and primary myeloma cells isolated from patients. After several cycles of in vitro stimulation, specific CTL lines were obtained, which consisted of both CD4+ and CD8+ T cells. These CTLs demonstrated not only specific cytotoxicity against gp96-pusled DCs and the cell line U266, but also significantly killed A2+ primary myeloma cells. No killing was observed against A2+ normal lymphocytes including B cells or A2− myeloma cell lines and primary myeloma cells from patients. Using the cold target inhibition assay, we showed that the same CTLs mediated the killing of both gp96-pulsed DCs and myeloma tumor cells. The cytotoxicity was MHC class I- and, to a lesser extent, class II-restricted, indicating that the gp96 contained both class I- and II-restricted tumor-derived peptides and that myeloma cells naturally present these peptides in the context of their surface MHC molecules. Upon antigen stimulation, these CTLs secreted predominantly interferon-g, detected by the ELISPOT assay and intracellular staining, indicating that they belong to the type-1 T-cell subsets. Furthermore, the CTLs lysed the target cells mainly through the perforin-mediated pathway because Concanamycin A but not Brefeldin-A, the selective inhibitors for perforin- or Fas-, respectively, mediated pathways, abrogated the cytolytic activity of the cells. These results therefore show that myeloma-derived gp96-specific CTLs are able to lyse myeloma tumor cells including primary myeloma plasma cells from patients and, thus, provide a rationale for gp96-based immunotherapy in MM.
Identification of a New Population of Human Cord Blood Cells with Lymphoid-Restricted Reconstituting Potential in Sublethally Irradiated Immunodeficient Mice.
Authors: Christ Oliver , Smith Clayton , Leung Karen , Hamilton Melisa , Eaves Connie J.
Published: October 2019

Throughout adult life, human hematopoiesis is sustained by the activity of a small compartment of pluripotent stem cells with extensive self-renewal potential. Available evidence suggests that these cells undergo a process of progressive lineage restriction similar to that described for murine hematopoietic cells, although many of the intermediate stages of human hematopoiesis have not yet been characterized. In human hematopoietic tissues, cells with short-term (<4 months) as well as long term (>4 months) repopulating activity (termed STRCs and LTRCs, respectively) are distinguished by their differential ability to engraft sublethally irradiated NOD/SCID-β2microglobulin null mice as well as their transient versus sustained output of differentiated cells. In previous studies, both a myeloid-restricted type of human STRC (STRC-M) and a type of STRC with lymphoid as well as myeloid potential (STRC-ML) have been identified. STRC-Ms are CD34+CD38+ and produce mainly erythroid progeny for the first 3–4 weeks post-transplant. In contrast, STRC-MLs are CD34+CD38− and produce progeny only between weeks 5 and 12 post-transplant which consist mainly of B-lymphoid cells plus some granulopoietic cells. We show here that both STRC-MLs and STRC-Ms are similarly distributed among lin- cord blood cells with intermediate to high levels of aldehyde dehydrogenase activity (ALDH-int/hi) as evidenced by staining with the fluorescent dye BAAA. In addition, BAAA-staining has allowed a previously undescribed primitive cell with low ALDH activity (ALDH-lo) and lymphoid-restricted repopulating activity to be identified. Assessment of NOD/SCID-β2microglobulin null mice transplanted with various subsets of cord blood cells further demonstrated that these “STRC-Ls” are CD38− and 10-fold more prevalent in the CD133+ subset of the low-density SSC-low ALDH-lo/neg population but, numerically, are equally distributed between the CD133+ and CD133− fractions because of the proportionately larger size of the CD133− subpopulation. Phenotype analysis of CD34+CD38− cord blood cells revealed a small and distinct ALDH-lo subset that expressed 10-fold higher levels of CD7 than any other CD34+CD38− cells. However, transplantation of this small CD7++ subset into NOD/SCID- β2microglobulin null mice revealed that they accounted for very few of the ALDH-lo STRC-Ls. The discovery of a CD38− ALDH-lo population of lymphoid-restricted human cells with in vivo reconstituting activity identifies a key step in the process of human hematopoietic cell lineage determination and the ability to prospectively isolate these progenitors separately from other types of short- and long-term repopulating cells present in normal human hematopoietic tissues should greatly facilitate future analysis of the mechanisms regulating their normal differentiation or malignant transformation.
Protein Signature of LMP1 Signaling in PTLDs Identifies and Mimics Inter/Perifollicular CD30+ EBV− B Blasts.
Authors: Shaknovich Rita , Basso Katia , Bhagat Govind , Alobeid Bachir , Cattoretti Giorgio
Published: October 2019

EBV-associated B-cell Post-Transpant Lymphoproliferative Disorders (PTLDs) represent a diverse group of lesions morphologically, in clinical presentation and behaviour, ranging from early reversible lesions to monomorphic aggressive lymphomas.

Polymorphic cases, which represent the focus of our analysis, contain a mixture of cells in various EBV latency stages, defined by EBNA1, EBNA2 and LMP1 immunostaining.

LMP1 is a key viral protein for cellular transformation and, analogously to CD40, engages TNF Receptor Associated Proteins and activates NF-kB and NF-kB-responsive genes.

We analyzed the protein signature of LMP1 in PTLDs and non-PTLD tonsils by double staining for LMP1, CD30, CD20, Pax5 and signaling molecules. A remarkably conserved set of proteins, associated with LMP1/CD40 signaling and NF-kB activation is expressed both in the EBV-infected lymphoid population in polymorphic PTLDs and in a normal B-cell subset(s) in reactive tonsils. These proteins include highly expressed CD30, JunB, nuclear cRel, TRAF-1, Bcl-XL, MUM1, CCL22 and downregulated BCL6 and CD10.

We observed that EBV infection, possibly through LMP1 and LMP2A signaling, results in varioius degrees of differentiation within the neoplastic clone. EBER+ terminally differentiated mucosa-associated IRTA-1+ marginal zone B-cells and CD138+ plasma cells were identified in most cases, including control post-transplant tonsils with no overt disease. We document for the first time in situ, in-vivo evidence of EBV latently infected post-Germinal Center B cells of marginal and plasma cell types in PTLDs. Polymorphic PTLD cases represent EBV-induced expansion of B cells, mimicking CD40L-like activated Peri/Interfollicular CD30+ normal B-cells.
Thromboelastometry in Patients Receiving Platelet Transfusion after Chemotherapy.
Authors: Dehmel Henning , Tiede Andreas , Horter Silvia , Ganser Arnold , von Depka Mario
Published: October 2019

Prophylactic platelet transfusion is frequently administered to reduce the risk of hemorrhage in patients undergoing chemotherapy for leukemia or cancer. A platelet count of 10 or 20x109 L−1 is often used as an occasion for platelet transfusion. In clinical studies, the bleeding risk was similar using either threshold (Rebulla, NEJM1997; 337:1870, Wand, Blood1998; 91:3601). Moreover, serious bleeding events were not related to the patients’ platelet count. Thromboelastometry with ROTEM® Whole-Blood Coagulation Analyzer (Pentapharm, Munich, Germany) is a method that may provide a better estimate of the risk of hemorrhage because it also depends on platelet function and plasma coagulation. We therefore performed a pilot study in 13 adult patients receiving prophylactic platelet transfusion after chemotherapy for leukemia or lymphoma. ROTEM® was performed using an ellagic acid based activating reagent (in-TEM®, Pentapharm). Clotting profiles were evaluated using the following thromboelastometric measures: clotting time (CT), clot formation time (CFT), and maximum clot firmness (MCF). The median platelet count before transfusion was 9x109 L−1 (range 1 to 20). CT was within the normal range in all patients (median 168 s, range 125–212, reference range 100–240). In contrast, a prolonged CFT (median 139 s, range 51–4526, reference range 30–110) and reduced MCF (median 34 mm, range 19–54, reference range 50–72) was recorded. Correlation between platelet count and CFT (R=−0.41, Spearman) or MCF (R=+0.48) was weak and not statistically significant. 15 min after platelet transfusion, there was an increase in platelet count (median 39x109 L−1, range 23–48) and a substantial improvement of CFT (median 99 s, range 44–332) and MCF (median 49 mm, range 40–64). Changes in thromboelastometric measures were due to platelet transfusion as the addition of cytochalasin D (fib-TEM®, Pentapharm) resulted in identical profiles before and after transfusion. Changes in platelet count correlated with changes in MCF (R=+0.73, P<0.01), but not CFT (R=−0.40, P=0.17). Comparing patients who had a maximum of one mild bleeding symptom (n=9) with patients who experienced one severe or at least two mild bleeding symptoms (n=4), there was no difference in platelet counts (median 10 vs. 9x109 L−1, p=0.77). In contrast, there was a trend towards a shorter CFT (median 111 vs. 388 s, P=0.09) and higher MCF (median 39 vs. 29.5 mm, P=0.09) in the group of non-bleeding patients. In summary, ROTEM® seems to be a sensitive method to detect hemostatic changes in patients with severe thrombocytopenia receiving platelet transfusion and to identify thrombocytopenic patients with an increased bleeding risk. Therefore, further studies to evaluate ROTEM® as a means to estimate the bleeding risk in thrombocytopenic patients are warranted.
Treatment with rhEPO Decreases the Need for Transfusion in Patients with βThalassemia.
Authors: Bachir Dora , Habibi Anoosha , Pissard Serge , Ourabah Zohra , Galacteros Frederic
Published: October 2019

The usage of human recombinant erythropoietin (rhEPO) in βthalassemia shows in many sudies, that not only it increases the synthesis of γ chains, but it also improves erythroid parameters. Among our population of thalassemic patients, eight patients with βthalassemia intermedia that were regulary transfused, were given escalating doses of rhEPO mostly darpoietin-α. Before receiving rhEPO, all patients had undergone splenectomy, also received iron chelation therapy, and folic acid supplementation. No patient received iron supplementation together with rhEPO.

With rhEPO, two patients were able to discontinue their requirements for transfusions. In the others, haemoglobin level was maintained above 8 g/dl. Four patients experienced bone pain which was successfully treated with hydroxyurea. All patients had improvement in their quality of life. This small series illustrates that rhEPO may be useful and cost effective in the long term (considering the risks of red blood cell transfusion) in some selected splenectomized transfusion-dependent patients with βthalassemia intermedia.

Results

Transfusion requirements (ml/kg/year) Patients Year of birth Sex Final doses of rhEPO (UI*kg/week) Follow-up (months) Before After Hydroxyurea * interruption of rhEPO because of participation to ICL 670 A 0107 protocol 1 1975 M 970 33 150 75 + 2 1973 F 1365 34 125 0 + 3 1965 M 820 33 130 0 + 4 1959 F 650 27 125 98 + 5 1979 F 715 15 210 160 * + 6 1978 M 1035 7 255 90 − 7 1987 F 450 3 135 80 − 8 1974 M 900 7 190 75 +
Impact of Plasmacytoid Dendritic Cell (PDC) Recovery on Outcome after Reduced-Intensity Conditioning (RIC) Allogeneic Stem Cell Transplantation (Allo-SCT).
Authors: Mohty Mohamad , Blaise Didier , Faucher Catherine , Bardou Valerie-Jeanne , Gastaut Jean-Albert , Viens Patrice , Olive Daniel , Gaugler Beatrice
Published: October 2019

Dendritic cells (DCs) are crucial in the induction of immune responses. Among DC subsets, the reconstitution of the natural type I-interferon-producing plasmacytoid DCs (PDC) has been proposed to play a major role in establishing immune competence, given the capacity of PDCs to efficiently expand either specific cytotoxic T lymphocytes, or to promote regulatory T cells, contributing to an impaired immune response. Therefore, we investigated the impact of circulating PDCs measured at the third month after RIC-allo-SCT, in 54 patients with hematological and non-hematological malignancies who received a RIC-allo-SCT from an HLA-identical sibling, in order to determine whether this could provide a convenient indicator for long term outcome. The median absolute count of PDCs measured at 3 months after RIC-allo-SCT was 0.725/μL (range, 0–23.2). In a multiple logistic regression analysis including all relevant parameters (demographic and graft characteristics, RIC regimens, CMV infections, and acute GVHD), only the absence of clinically significant grade II-IV acute GVHD was associated with an improved PDC recovery at 3 months (P=0.003; OR=6.4; 95%CI, 1.9–22). Being the major type I IFN-secreting cells, we also investigated whether PDCs recovered after allo-SCT are functional in response to viral stimulation. Patients experiencing grade 0-I acute GVHD could secrete significantly higher amounts of IFN-alpha as compared to patients with grade II–IV aGVHD (mean, 91 pg/ml vs. 0 pg/ml respectively; P=0.002), likely highlighting the deleterious impact of corticosteroids therapy on PDC function. The CD34+ stem cell dose and other lymphoid subsets infused with the allograft did not affect PDC recovery. Though PDC count could not predict death from progression or relapse, patients with a “high” PDC recovery profile had an improved overall survival (OS; P=0.03), in contrast to patients with a “low” PDC recovery profile who had an increased incidence of late transplant-related mortality (GVHD, infections) (P=0.03). In addition, the overall incidence of late infections (viral, fungal and bacterial) was significantly higher in the “low” PDC recovery group as compared to the “high” PDC recovery group (59% vs. 19%; P=0.002), illustrating the importance of PDCs in anti-infectious immune responses. In a multivariate analysis, only a “high” PDC count was significantly predictive of a decreased risk of death (P=0.04; RR=0.34; 95%CI, 0.12–0.96). The role and impact of rare immune effector cells would tend to be more evident in truly RIC and less toxic regimens. In this study, we could show that monitoring of PDCs may be useful for patients’ management (closer surveillance, infection prophylaxis…), and may have a significant impact on the probability of a favorable outcome in the context of RIC-allo-SCT.
Inhibitory Effects of Lactic Acid on Human Antigen-Specific CD8+ T-Cells.
Authors: Kreutz Marina , Fischer Karin , Hoffmann Petra , Volkl Simon , Edinger Matthias , Andreesen Reinhard , Mackensen Andreas
Published: October 2019

A characteristic feature of inflammatory lesions or tumor sites is local acidosis, which is attributed to the local increase in lactic acid production. We studied the effect of such an acidic environment on the immune functions of antigen-specific CD8+ T-cells by incubating the cells in the presence of various concentrations of lactic acid for up to 48h. CD8+ T-cells were isolated from healthy donors and expanded by weekly stimulation with autologous dendritic cells pulsed with a mutated HLA-A2-binding Melan-A (ELAGIGILTV) peptide. The obtained T cell population consisted of at least 90% CD8+ and about 60% Melan-A specific T cells, as determined by Melan-A multimer staining. Incubation of CD8+ T cells with up to 20mM lactic acid for 24h did not cause T-cell apoptosis or cell death as determined by combined annexin/propidium iodide staining. However, the proliferative capacity of CD8+ T cells, as determined by 3H-thymidine uptake, was strongly inhibited. Similar results were obtained when we determined cytokine production and cytotoxic activity of the cells after a 24h culture period in 5-20 mM lactic acid. Production of both, IL-2 and IFN-gamma was strongly diminished in comparison to untreated cells, as determined by intracellular staining after stimulation with PMA/ionomycin for 5h in the presence of monensin. Analysis of the antigen-specific cytolytic capacity revealed that CD8+ T cells pre-cultured with lactic acid were less effective in killing antigen-loaded T2 target cells as compared to untreated CD8+ T cells. In parallel, the intracellular contents of the cytotoxic effector molecules granzyme-B and perforin was diminished. Re-adjusting the pH of the medium to a physiological value of pH7.4 could partially revert the effect of lactic acid. Treatment of CD8+ T cells with sodium lactate instead of lactic acid had no inhibitory effect. We conclude, that lactic acid is an important modulator of CD8+ T-cell function and may contribute, together with other factors, to immune escape mechanisms in the tumor environment.
Effect of ADP and ATP Receptor Antagonism on Erythrocytosis-Induced Platelet Aggregate Formation under Dynamic Flow Conditions.
Authors: Peerschke Ellinor I.B. , Silver Richard T. , Weksler Babette , Savion Naphtali , Varon David
Published: October 2019

Red blood cells (RBC) contribute significantly to hemostasis and thrombosis under oscillatory flow conditions, and erythrocytosis has been associated with increased thrombotic risk. To capture dynamic effects of RBC on platelets, we used a recently described Cone and Plate (let) Analyzer (CPA), to evaluate the effect of hematocrit (Hct) on platelet function in whole blood under arterial flow conditions (1800 sec-1, 2 min, 25°C). Anticoagulated blood, reconstituted to varying hematocrits with autologous RBC, demonstrated a significant increase (30–50%, n= 12, p < 0.05) in adherent platelet aggregate size at Hct levels >45%. No significant effect on platelet adhesion was noted. Increases in aggregate size were not accompanied by significant platelet dense or alpha granule secretion, measured using 14C-serotonin release and Platelet Factor 4 ELISA assays, respectively. However, when cell-free supernatants (SNT) were prepared post shearing to investigate the presence of proaggregatory activity by adding SNT to fresh PRP (1:10 SNT: PRP ratio), and quantifying the formation of small platelet aggregates with a laser light scatter aggregometer, SNT derived from sheared high Hct samples contained nearly twice (1.75 + 0.16, n=5, p < 0.05) the activity of sheared normal Hct samples. The erythrocytosis-induced increase in platelet aggregate size was not affected by pretreatment of blood with 0.05 mM aspirin, but could be prevented completely by antagonism of P2Y1 (MRS2179, 200 μM), P2Y12 (2-methylthioadenosine 5′-monophosphate, 10–100 μM), or P2X1 (MRS2159, 200 μM), ADP and ATP receptors, respectively, as well as by converting exogenous ADP to ATP with a combination of creatine phosphate (2 mM) and creatine phosphokinase (20 U/ml). Whereas negligible platelet granule secretion was measured post shear, metabolic inhibition of RBC with 1% sodium azide or 0.25% glutaraldehyde fixation (2 h, 37°C) fully inhibited erythrocytosis-enhanced increases in platelet aggregate size. Thus, the adenine nucleotides contributing to erythrocytosis-enhanced platelet aggregate formation under physiologic shear appear to be derived predominantly from RBC. Taken together, the data suggest that ADP and ATP are both required for erythrocytosis-induced large platelet aggregate formation, but that the presence of ATP alone is not sufficient. The results may further suggest a role for direct ADP or ATP receptor blockade for the prevention of erythrocytosis-associated thrombotic complications.