bluuebunny/crossref_metadata_2025 huggingface dataset:
Remodeling Ca2+ Flux By ORP4L Is Essential for Leukemia Stem Cells (LSCs) Survival
Authors: Zhong Wenbin , Olkkonen Vesa , Bing Xu , Zhu Biying , Li Guoping , Li Jian , Yi Qing , Yan Daoguang
Published: October 2019

Acute myelogenous leukemia (AML) is one of the deadliest hematological malignancies and there is at present no efficient strategy to defeat it. New detailed insight into AML leukemia stem cells (LSCs) survival will facilitate the identification of targets for the development of new therapeutic approaches. Recent work has provided evidence that LSCs are defective in their ability to employ glycolysis, but are highly reliant on oxidative phosphorylation, and the maintenance of mitochondrial function is essential for LSCs survival. It is increasingly clear that Ca2+ released constitutively from endoplasmic reticulum (ER) is taken up by mitochondria to sustain optimal bioenergetics and cell survival. Here we report three striking findings: 1) oxysterol-binding protein (OSBP)-related protein 4 (ORP4L) is expressed in LSCs but not in normal hematopoietic stem cells (HSCs). 2) ORP4L is essential for LSC bioenergetics; It forms a complex with PLCβ3 and IP3 receptor 1 (ITPR1) to control Ca2+ release from the ER and subsequent cytosolic and mitochondrial parallel Ca2+ spike oscillations that sustain pyruvate dehydrogenase (PDH) activation and oxidative phosphorylation. 3) ORP4L inhibition eradicates LSCs in vitro and in vivo through impairment of Ca2+-dependent bioenergetics. These results suggest a novel role of ORP4L in governing Ca2+ release to sustain mitochondrial function and survival of LSCs and identify ORP4L as a putative new oncoprotein and therapeutic target for LSCs elimination.

Disclosures

No relevant conflicts of interest to declare.
Clinical Research and Genomic Analysis on Relapsed or Refractory Angioimmunoblastic T Cell Lymphoma with Chidamide Monotherapy
Authors: Wu Yu , Xu Juan , Tang Yuan , Jia Yongqian
Published: October 2019

BACKGROUND: This study was to evaluate the efficacy and safety of chidamide monotherapy, which is a new histone deacetylase inhibitor (HDAci) of the benzamide class, in relapsed or refractory Angioimmunoblastic T cell lymphoma (AITL), to investigate its genomic expression signatures as well as the mechanisms of chidamide's anti-lymphoma effect and its resistance.

METHODS: Two cases with relapsed or refractory AITL were treated with chidamide. We performed a repeated biopsy on relapsed lymphomas and did whole genome next-generation sequencing (NGS) testing on drug -resistant tumor samples.

RESULTS: The first patient is a 54-year-old man presented with stage Ⅳ AITL. He underwent autologous stem cell transplantation in his first complete remission (CR) but relapsed 7 months later. In view of resistance to multiple lines of chemotherapy and poor performance status, chidamide was adaministered orally at a standard dose of 30mg twice a week. Pulmonary lesions regressed quickly and a second CR was achieved. Adverse events included grade 2 cytopenias, diarrhea, and reversible QT interval prolongation. The disease free survival was 6 months and AITL relapsed again 4 months ago. He responded to low-dose chidamide combined with lenalidomide and dexamethasone and remains well. Formalin-fixed, paraffin-embeded tumor tissues collected at first relapse were tackled for whole-genome sequencing. The other patient is a 62-year-old woman who had stage Ⅲ AITL. Disease progressed after two cycles of combined chemotherapy, thereafter a standard dose of chidamide monotherapy was initiated. With well tolerability, an unconfirmed CR was achieved and lasted for nearly 3 months. She is still alive but remains refractory to various salvage therapeutics, including chemotherapies, arsenic trioxide, thalidomide, and pralatrexate. A rebiopsy was perferomed and the histopathological findings confirmed the relapse of AITL, associated with Epstein-barr virus infection. Fresh tumor tissues were sent for whole-genome sequencing. In both cases, NGS testing identified mutations of RHOA gene, epigenetic regulators TET2, IDH1, and DNMT3A, as well as CD28.

CONCLUSIONS: Chidamide, a low nanomolar inhibitor of HDAC1, 2, 3, and 10, was approved in China for the management of relapsed and/or refractory peripheral T cell lymphoma. It's reported that patients with AITL tended to have higher response rates and more durable responses to chidamide treatment. Our report showed single-agent chidamide is a reasonable approach to treat the formidable disease but seemed difficult to achieve a sustained remission. Various genetic, epigenetic, and immune alterations involve in the pathogensis of AITL, which provide targets for chidamide therapy. High-throughput sequencing approach is very helpful to clarify the mechanisms.

Disclosures

No relevant conflicts of interest to declare.
Kidney Damage in the Manifestation of Lymphoproliferative Diseases
Authors: Boldukyz Dzhumabaeva Tolgonbaevna , Ludmila Birjukova Semenovna , Hunan Julhakyan , Saliya Maryna , Ludmila Roshchina Sergeevna
Published: October 2019

Objectives:

Kidney lesions at lymphoproliferative diseases mainly described with disease progression or relapse. Renal involvement in the manifestation of lymphoproliferative diseases were observed rarely. Pathogenesis of renal damage is obscure.

Aim: To determine especially renal damage in the manifestation of lymphoproliferative diseases.

Subjects and methods. The study included 19 patients (13 males, and 6 females, age 63.3±8.8 years) with lymphoproliferative diseases accompanied by renal failure: chronic lymphocytic leukemia (n=12), marginal zone lymphoma (n=4), follicular lymphoma (n=1), Waldenstrom's macroglobulinemia (n=1), diffuse large B-cell lymphoma (n=1). Nephrotic syndrome was observed in 3 patients and renal failure in 18 ones. The average creatinine level was 330.9±152.3 μmol/l, glomerular filtration rate was 25.7±12.9 ml/min. The presence of monoclonal IgMk secretion (n=6), Bence Jones protein kappa (n=9), increased level of free light chains (n=4), cryoglobulin (n=4). The renal biopsy was studied by histological, immunohistochemical, immunofluorescence, and electron microscopy.

Results:Glomerulonephritises were revealed in 10 cases (52.6%): membranoproliferative glomerulonephritis (n=2), membranous (n=2), mesangiocapillary (n=3), fibrillary (n=1), immunotactoid glomerulonephritis (n=1), minimal-change disease (n=1). Neoplastic lymphoid infiltration of renal parenchyma was detected in 10 (52.6%) cases. The massive diffuse small cell lymphoid proliferation was determined in 1 case, focal infiltration in 9 ones. Neoplastic lymphoid infiltration was associated with glomerulonephritis in 3 cases and with carcinoma of the kidney in 4 cases. Large focal lymphoid proliferation was found in 1 case in a patient with diffuse large B-cell lymphoma. AL - amyloidosis was identified in 2 cases and the thrombotic microangiopathies in 2 ones.

Conclusion. Glomerular disease, lymphoid tumor infiltration, sometimes AL - amyloidosis, thrombotic microangiopathy were detected in the manifestation of lymphatic tumor. In all cases examined, there was a secretion of monoclonal immunoglobulin, protein Bence Jones, free light chains and cryoglobulin, pathogenetic value in the development of glomerulopathy should be clarified.

Disclosures

No relevant conflicts of interest to declare.
The Clinical Features and Prognosis of Lymphoplasmacytic Lymphoma/ Waldenström Macroglobulinemia: A Large Series Analysis from a Single Center
Authors: Xiong Wenjie , Yi Shuhua , Li Heng , Li Zengjun , Lv Rui , Liu Wei , Xu Yan , Zou Dehui , Hao Mu , Qiu Lugui
Published: October 2019

Background: Lymphoplasmacytic Lymphoma/Waldenströmmacroglobulinemia (LPL/WM) is a rare B-cell proliferative malignancy characterized by immunoglobulin M monoclonal gammopathy and /or bone marrow infiltration by lymphocyte, lymphoplasmacytic and/or plasma cells. The clinical features and prognosis of WM/LPL have less been investigated in Chinese patients. Moreover, few studies had reported the prognostic value of cytogenetic aberrations in Chinese WM/LPL patients.

Methods: We systematically reviewed the records of 233 LPL/WM patients, who had been diagnosed at institute of Hematology&Blood Disease Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College.

Results: The median age of 233 patients was 62 years (range 32-87) and the male to female ratio was 2.76 (171/62). The most common symptoms at diagnosis were fatigue (59.1%) and bleeding (11.2%). The patients in low, intermediate and higher risk group of the international prognostic scoring system for WM (ISSWM) occupied 14.9%, 32.6% and 52.6%, respectively. 90.4% and 16% patients showed an elevated serum β2-microglobulinand LDH. The percentages of hepatomegaly, splenomegaly, peripheral lymphadenopathy, B symptom were 14.9%, 34.5%, 43% and 29.3%, respectively. The degree of splenomegaly was always mild or moderate, only 19.1% patients with giant spleen. The karyotypes abnormalities were not common in LPL/WM patients. Only 8.9% (14/157) patients had abnormal chromosome karyotypes. The most common cytogenetic aberration detected by fluorescence in situ hybridization (FISH) were deletion of 17q13 (9/120, 7.5%), trisomy 12(5/58, 8.6%). Other aberrations include 13q14 deletion in 3 of 128 patients (2.3%), 11q22 deletion in2 of 90 patients (2.2%). Univariate analysis showed that age>62 years, leukocytopenia, thrombocytopenia, anemia, cytopenias ≥ 2, elevated LDH, abnormal chromosome karyotypes, P53 deletion and high risk of ISSWM were adverse risk factors for OS. But only clinical effect not reaching partial response (p=0.006), elevated LDH (p=0.049) and leukocytopenia (p=0.048) were the independent prognostic factors at multivariate analysis.

Conclusion: Distinct characteristics exist in WM in China compared with the Western country. Elevated LDH and leukocytopenia were the independent adviser factors for OS in this series patient.

Disclosures

No relevant conflicts of interest to declare.
Failure Pattern of Treatment of Hodgkin Lymphoma -- Retrospective from a Single Institution
Authors: Tan Tran-Der , Chiou Lun-Wei
Published: October 2019

Backgound:

Hodgkin lymphoma is a highly curable malignant disease by chemotherapy or chemoradiation. However, there is still small percentage of patients can not achieved complete remission or relapsed later then died of disease refractoriness. We investigated the treatment failure pattern in those relapsed and refractory patients.

Method:

This is a retrospective study of patients treated at our institute in ten years with ABVD +/- RT. For relapsed patients, the salvage chemotherapy includes DHAP, ICE, or GDP, and patients may undergo high dose chemotherapy plus hematopoietic stem cell transplantation when second remission achieved.

Results:

Between 2002 and 2012, we have treated 108 patients of Hodgkin lymphom and the ten-year overall survival was 92% with stage I and II (N=63) versus III and IV (N=45) were 100% and 80%, respectively. Twenty patients got disease relapsed after ABVD +/- RT with 6 patients died of disease refractoriess and 14 patients achieved second remission after salvage chemotherapy then underwent hematopoietic stem cell transplantation with 11 survived over ten years. In these 20 relapsed patients, the initial involved sites include lung (n=8), bone (n=5),bone marrow (n=5), liver (n=3), mediastinal bulky (n=2), nodal only (n=2).

Conclusion:

Stage IV with extramedullary involvement over lung, bone, bone marrow and liver are high risk for treatment failure and earlier intensive treatment is warranted.

Disclosures

No relevant conflicts of interest to declare.
Everolimus Is Active in Pretreated Hodgkin Lymphoma after Failure of Brentuximab Vedotin: Single Institute Experience
Authors: Dada Reyad , Yassin Fawwaz
Published: October 2019

Introduction: Patients with Hodgkin lymphoma (HL) relapsing after ASCT and those not eligible for myeloablative therapy can be salvaged with conventional chemotherapy or new novel agents such the immunotoxin brentuximab vedotin and the recently FDA approved anti-PD-1 inhibitor nivolumab. Other options include the mTOR inhibitor everolimus (1) for which, the data in literature is scarce. Moreover, there is no data on the efficacy of everolimus after failure of brentuximab vedotin.

Patients and methods: In this study, we retrospectively analyzed the outcome of patients with relapsed/ refractory HL treated with single agent everolimus at our center between July 2015 and July 2016. All patients were started on everolimus 10 mg daily. Response to treatment was assessed every 2 months. Primary endpoint was the response rate of everolimus as single agent in patients with relapsed/refractory HL.

Results: We identified 5 patients with heavily pretreated HL who received single agent everolimus. Mean age was 24.4 years (range, 20-30). Mean lines of previous treatment was 6.4 (range, 5-8). All patients failed brentuximab vedotin. Two patients failed previous autologous stem cell transplantation. Prior treatments included ABVD, radiation, ICE, ESHAP, miniBEAM, GVD, brentuximab vedotin, gemzar plus brentuximab vedotin, bendamustine, and ASCT.

Mean duration of everolimus treatment was 5 months (range 4-7). One patient showed complete metabolic remission after 5 months of treatment. This remarkable response was achieved after failure of 6 lines. Partial remission and stable disease each were documented in 40%. Grade 1 neutropenia was reported in 80%. Grade 2 anemia, thrombocytopenia, fatigue and stomatitis were observed in 60%, 80%, 20% and 20% respectively. Everolimus dose reduction (5 mg) was required in two cases (one patient had grade 2 thrombocytopenia and other one had grade 3 fatigue). The latter discontinued the treatment after 4 months and is still in complete remission 5 months after discontinuation. No pulmonary toxicity was observed.

Conclusion:The presented data suggest that everolimus is effective in heavily pretreated HL patients who failed conventional treatment including ASCT and new novel agents with 60% ORR. This result is the first of its kind showing efficacy of everolimus after failure of brentuximab vedotin.

Literature: Johnston PB et al. A Phase II trial of the oral mTOR inhibitor everolimus in relapsed Hodgkin lymphoma. Am J Hematol. 2010 May;85(5):320-4.

Disclosures

No relevant conflicts of interest to declare.
JQ1, a c-MYC Inhibitor Targets Hexokinase II and Enhances the Cytotoxic Effects of Chemotherapeutic Agents in Diffuse Large B-Cell Lymphoma (DLBCL)
Authors: Gu Juan J , Barth Matthew J. , Zhang Qunling , Mavis Cory , Hernandez-Ilizaliturri Francisco J.
Published: October 2019

Background: Results from thecollaborative trial in relapsed aggressive lymphoma (CORAL) study demonstrated a poor clinical among patients treated with second-line chemo-immunotherapy in the post-rituximab era, stressing the need to identify and/or optimize novel targeted agents. C-myc expression was found correlate with a poor clinical outcome in patients with newly diagnosed or relapsed/refractory DLBCL. Previously, we found that JQ1 overcomes rituximab-chemotherapy resistance in lymphoma pre-clinical models. JQ1 exhibited synergistic activity when combined with chemotherapy agents. We also demonstrated that hexokinase II was highly expressed in rituximab resistant cell lines (RRCL), and its expression was associated with a deregulation in the glucose metabolism and an increase in the apoptotic threshold leading to chemotherapy resistance. In our current work, we evaluated molecular studies dissecting the cellular pathways affected by JQ1.

Methods: A panel of rituximab-sensitive (RSCL) and RRCL cell lines were exposed to escalating doses of JQ1 (0-8μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism 6 software. Loss of mitochondrial membrane potential (∆ψm) following JQ1 exposure was assessed by DiOC6 staining. Changes in c-MYC, HKII, HKI, Voltage dependent anion channel protein (VDAC) and p21 expression levels were determined by western blotting. HKII was silencing using siRNA interference in RRCL (Raji-4RH) and changes in HKII, HKI, VDAC, c-MYC and p21 protein expression was determined. HKII knockdown or scramble Raji-4RH cells were exposed to different chemotherapy agents and JQ1; cell viability and mitochondrial membrane potential was accessed.

Result:JQ1 induced a dose-and time- dependent cell death in cell lines. In vitro exposure cells to JQ1 resulted in a loss of mitochondrial potential and induced G1 cell cycle arrest. In vitro exposure to JQ1 decreased c-MYC and HKII expression levels and induced of p21 expression. Complete silencing of HKII re-sensitized cell to chemotherapy (doxorubicin, vincristine and bortezomib). On the other hand, HKII knockout abrogated JQ1 anti-tumor activity.

Conclusion: Our data suggests that JQ1 had anti-tumor activity against rituximab-sensitive or resistant pre-clinical models. The inhibition of HKII following JQ1 exposure may contribute to lower the mitochondrial membrane potential and enhance the chemotherapeutic effect. Our finding highlights a better understanding in the molecular events triggered by JQ1 as a potential role in the treatment of B-cell malignancies.

Disclosures

No relevant conflicts of interest to declare.
PDGFR Inhibitor Imatinib May be a Potent Drug in Treating Essential Thrombocythemia
Authors: Li Xia Zhou , Mo Yang , Jie Yu Ye , Chong Beng H
Published: October 2019

Approximates 80% of essential thrombocythemia (ET) patients carrying JAK2, CALR, MPL mutations. However, there is still 20% of ET patients are negative for all three mutations. Our previous studies have demonstrated that platelet-derived growth factor (PDGF) and its receptor (PDGFR) has a potential effect in the regulation of hematopoiesis and megakaryopoiesis. Therefore, we speculated that PDGF/PDGFR may plays an important role in megakaryocyte/platelet related disease, such as ET. In this study, we found an increase of PDGF-BB and PDGFR expression in ET patients comparing to the normal persons. We then used the adjusted dosage of PDGF-BB (1.5ug/kg/d) to establish an ET mimic mouse model. We found that PDGF-BB can enhance platelet production in vivo significantly, while PDGFR inhibitor imatinib can block this effect. Bone marrow histology data also confirmed the megakaryopoiesis effect of PDGF, as shown by an increased number of megakaryocytes in PDGF-treated mouse. The addition of imatinib can block the effect, evident by a decrease number of megakaryocytes and an increase of apoptosis. Furthermore, we demonstrated that PDGF-BB activated theP-JAK2, the P-STAT3 and the P-AKT expression in the megakaryocytic cell line CHRF-288, while addition of imatinib can reduce the phosphorylation level of all three genes. Our results suggested that the overexpression of PDGF-BB and PDGFR may be important in the pathogenesis of ET, especially in the JAK2-mutation negative ET. The elevated PDGF-BB activates PDGFR with subsequently activation of the JAK2/ STAT3 and PI3K/AKT pathway. Imatinib may have an effect on treating ET via blocking PDGFR and the following JAK2/STAT3 and PI3K/AKT pathway. This study suggests that PDGF/PDGFR may be involved in the pathogenesis of essential thrombocythemia, and the expression of PDGF-BB and PDGFR is potential to be a diagnostic index of ET. Blockage of PDGFR by imatinib may be a new therapeutic target for ET.

Figure 1 Effect of PDGF-BB on Jak2/Stat3 and PI3K/Akt pathway of megakaryocytes. Figure 1. Effect of PDGF-BB on Jak2/Stat3 and PI3K/Akt pathway of megakaryocytes. Figure 2 The platelet numbers and the megakaryocytes of each group mice. Figure 2. The platelet numbers and the megakaryocytes of each group mice. Figure 3 Potential mechanisms for imatinib in treating essential thrombocythemia. Figure 3. Potential mechanisms for imatinib in treating essential thrombocythemia.

Disclosures

No relevant conflicts of interest to declare.
Azacitidine Use in a Low Income Country, Effectiveness of a 100mg Fixed Dose
Authors: Torres Jorge Carlos , Zapata Nidia , Cervera Eduardo , Sanchez Sergio , Aguilar Manuel
Published: October 2019

Azacitidine, a pyrimidine nucleoside analog of cytidine, causes hypomethilation of DNA. Currently FDA approved for treatment of low and intermediate MDS with complete responses around 50%. And Acute myeloid leukemia (AML) in the eldery

In the CALGB studies, the usual dose is 75mg/m2 in 28 day cycles, with dose modifications according to toxicity.

In low income countries such as Mexico, one course of Azacitidine is around 500 dollars, median income in Mexico is 4,910 PPP (purchasing power parity); vs 30,616 in the USA. So, azacitidine treatment is far from reach for most of the common population, particularly those who do not have insurance.

This is a retrospective observational study, of a compassionate use program of a fixed dose of Aza at 100mg. We analyzed data from patients that were treated with Aza between 2012 and 2016, and collected data in 2016. The aim of the study was to assess the effectivity of the fixed dose. For that purpose, we collected information from the physical and electronic file. We analyzed: Hemoglobin level before and after treatment, independence of transfusion, ANC recovery, number of courses, and overall survival. We conducted our research in a public institution in Mexico (Instituto Nacional de Cancerología) and a private institution (Medica Sur).

We included acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients, regardless of age, previous treatment and comorbidities (we included patients with renal failure, hypertension etc.)

We included 8 patients in our study, 6 males and 2 females, with a mean age of 69.9 years (49-87). We had 2 AML and 6 MDS.

We had 2 high risk AML and according to IPSS-R: 1 very low, 2 low, 1 intermediate, 1 high and 1 very high risk MDS. As for the Karyotype we had 1 complex KT, 4 normal KT and 1 Del 7q Del 5q +8.

All patients received at least one dose of Aza, with mean number of cycles of 4. We have a mean survival of 439 days (110-1385). 6/8 patients achieved transfusion independency within 3 doses of Aza. 6/8 patients achieved ANC but lost eventually lost response. 5/8 patients are alive in follow up. 3 patients died of infectious complications. 2 patients never achieved transfusion independence or ANC.

The information recovered suggests that a fixed dose of 100mg is as feasible as a higher dose, at least when no other treatment or higher dose can be administered. We still are analyzing the survival data in order to find other bad prognosis factor within this population.

Disclosures

No relevant conflicts of interest to declare.
Ibrutinib-Based Therapy in the Treatment of Relapsed and Refractory Chronic Lymphocytic Leukemia
Authors: Kuvshinov Alexey , Voloshin Sergei , Martynkevich Irina , Martynenko Ludmila , Garifullin Andrei , Kleina Elizaveta , Shuvaev Vasily
Published: October 2019

Introduction . The development of Bruton's tyrosine kinase inhibitors and their introduction into clinical practice represent a major advance in the treatment of chronic lymphocytic leukemia (CLL). Several studies in CLL have determined the achievement of minimal residual disease (MRD) negativity as an independent favorable prognostic factor, associated with superior progression-free survival (PFS) and overall survival. MRD status is the single best posttreatment predictor of long-term outcomes after treatment, regardless of the treatment protocol or the presence of other pre-existing prognostic indicators.

Aim.To estimate of response rate, PFS and MRD after ibrutinib-based therapy in the treatment patients with relapsed/refractory CLL.

M ethods.21 pts with relapsed/refractory CLL were included in the analysis. Stratification of patients into groups based on therapy. Group 1 (n = 14): 2nd and subsequent lines of rituximab-based chemotherapy (RB - 10, FCR - 4) and Group 2 (n = 12): ibrutinib-based therapy (420 mg daily oral Ibrutinib ± Chemoimmunotherapy). We have used NCI-IWCLL revised guidelines for treatment initiation and assessment of response. MRD was detected by multicolor flow cytometry of bone marrow in patients achieved a complete or partial remission: Group 1 - 7 pts, Group 2 - 5 pts.

Results. The median age was 60.5 years (45-83) in Group 1 and 62.5 years (49-82) in Group 2 and median previous lines of therapy was 1 (1-4) and 2 (1-4), respectively. Patients with unfavorable chromosomal abnormalities were detected in each group: Group 1 - 2 pts (combination del(11q) with del(13q)); Group 2 - 1 pts (del(17p)).

Overall response rate (ORR) in Group 1 was 71.4% (complete remission (CR) - 1 pt, partial remission (PR) - 9 pts, stable disease (SD) - 3 pts, progression disease - 1 pt). Group 2: ORR 91.7% (CR - 3 pts, PR - 8 pts; SD - 1 pt). Statistically significant differences in the frequency of ORR between groups were not detected (p>0.05).

MRD-negative remission rate was 40% (2/5, CR - 1 pt) in Group 2 compared to 14.3% (1/7 pts, CR) in Group 1 (p>0.05). Statistically significant differences in PFS were detected between Group 1 vs. Group 2 (p=0.0006). Median PFS in Group 2 has not been reached. Median PFS in Group 1 was 16.9 month.

Conclusion. Ibrutinib is highly effective at controlling disease, but best responses are typically partial remission, and patients must remain on treatment to maintain disease control. Evaluation of response and MRD after ibrutinib-containing therapy in the treatment of patients with relapsed/refractory chronic lymphocytic leukemia require further research.

Disclosures

Shuvaev: Pfizer: Honoraria; BMS: Honoraria; Novartis pharma: Honoraria.
Alterations in Circulating Lymphoid Populations after Post-Transplant Cyclophosphamide Administration in Haploidentical Hemopoietic Cell Transplantation
Authors: Shahin Kifah , Mattar Zamil , Bradstock Kenneth
Published: October 2019

High dose post-transplant cyclophosphamide (PTCy) administered early after stem cell infusion has facilitated the use of haploidentical related donors for allogeneic transplantation, but the precise immunosuppressive action of PTCy remains unclear. Using flow cytometry of peripheral blood leucocytes, we investigated changes in circulating lymphoid populations immediately before and after administration of PTCy in 10 patients undergoing haploidentical allogeneic transplantation after reduced intensity conditioning therapy. Compared to baseline levels prior to the first dose of PTCy, there were significant reductions in natural killer cell and monocyte numbers by Day 7 post-transplant, but no changes in T cell numbers. However, by Day 20 there was a significant reduction in CD8+ T effector cells, with significant increases in CD4+ T regulatory cells. The activation marker CD69 was detectable on CD8+ cells, particularly T effectors, at Day 3, but decreased significantly by the third week post-transplant. These changes were confirmed on donor T cells in 2 informative cases using HLA allotype-specific antibodies. Although other factors may have contributed, the reductions in numbers and levels of activation of effector T cells, and increases in regulatory T cells, are consistent with alterations in the immune response induced by PTCy after haploidentical transplant.

Disclosures

Bradstock: DendroCyte BioTech Ltd: Other: Laboratory IP contracted via ANZAC Research Institute to DendroCyte BioTech Ltd.
The Checkpoint for Agonist Selection Precedes Conventional Selection in Human Thymus
Authors: Verstichel Greet , Vermijlen David , Martens Liesbet , Goetgeluk Glenn , Saeys Yvan , Hilde Cheroutre , Vandekerckhove Bart
Published: October 2019

The thymus plays a central role in self-tolerance by preventing strongly self-reactive thymocytes from accumulating as naïve T cell receptor (TCR) αβ+ T cells in the periphery. The elimination of auto-reactive T cells from the naïve pool is in part mediated by deletion during conventional negative selection. Alternatively, self-reactive thymocytes can also be positively selected in response to strong TCR signals during agonist selection and functionally differentiate to innate TCRαβ + T cells such as the CD8αα+ double negative (DN) T cells. How thymocytes discriminate between these opposite outcomes remains unclear.

We identified a novel agonist-selected PD-1+ CD8αα+ subset of mature CD8+ T cells in human thymus. Using the same markers a similar population was also identified in cord blood at about the same frequency as TCRγδ+ cells. This population expresses high levels of Helios, indicative of strong TCR engagement, and displays an effector phenotype associated with agonist selection. Indeed, PD-1+CD8αα+ T cells exhibit innate production of IFN-γ and an elevated T-bet to Eomes ratio typical of effector CD8 T cells. These cells are CD62L-, CXCR3+ and Hobit high suggesting that these cells leave the thymus and home to the tissues. Interestingly, in vitro CD3/TCR stimulation of sorted early post-β-selection thymocyte blasts uniquely gives rise to this innate subset, whereas small CD4+CD8+ double positive precursors fail to survive strong TCR signals. The generation of the innate subset seems to arise also in vivo from early post-β-selection thymocyte blasts as these two populations have an identical TCRα repertoire: ex vivo isolated PD-1+CD8αα+ thymocytes are skewed for early 3' TRAV and 5' TRAJ rearrangements compared to conventional CD8 T cells. A similar skewing was found in early post-β-selection thymocyte blasts. As TCRα rearrangements are terminated by TCR engagement of agonist selection, this is strong evidence for a precursor progeny relationship.

Together, we conclude that human CD8αα+ T cells are preferentially selected by strong TCR engagement on a subset of progenitors that express a full TCRαβ early on, leading to the generation of a post-selection T cell population with innate functional capacity and a markedly distinct TCR repertoire. These findings uncover the heterogeneity among DP precursors in their potential to survive strong selection signals and suggests that the decision making in the thymus to divert immature thymocytes to the agonist selection pathway occurs early before conventional selection of DP cells. We propose that progression through the immature thymic developmental program influences the outcome of TCR engagement with early post-β-selection thymocytes triggered by strong TCR signals preferentially giving rise to innate CD8αα+ T cells in humans.

Disclosures

No relevant conflicts of interest to declare.
Tumor Associated Myeloid Cells and their Contribution to Tumor Outcome
Authors: Merad Miriam
Published: October 2019

My laboratory studies the mechanisms that control the development, homeostasis and function of tissue resident myeloid cells, which mostly consists of macrophages and dendritic cells (DC).

Macrophages are hematopoietic cells that perform tissue-specific functions critical for regulating and maintaining organ homeostasis. In contrast to most other hematopoietic cells, macrophages that reside in quiescent tissues originate from early hematopoietic precursors that take residence in tissues prior to birth. In this presentation, I will discuss how tissue cues control macrophage functional diversity to ensure tissue integrity. I will also discuss recent data from our laboratory showing that developmental diversity contributes to shaping macrophage functional specialization and that ontogenically distinct macrophages differently regulate tumor response to radiation therapy.

In contrast to macrophages, DC are constantly repopulated by DC restricted precursors that are recruited from the blood circulation. Our laboratory has contributed to the mapping of the regulatory network of DCs, and the identification of a lineage of DC, the CD103+ DC, which are specialized in the induction of CD8+ T-cell immunity. Here I will discuss recent data from the laboratory that revealed the critical role for tumor-associated CD103+ DC in tumor response to checkpoint blockade and argue for the need to study human CD103+ DC equivalent in clinical cancer immunotherapy.

Disclosures

No relevant conflicts of interest to declare.
Studies on Erythropoiesis
Authors: GOLDWASSER EUGENE , JACOBSON LEON O. , FRIED WALTER , PLZAK LOUIS F.
Published: October 2019

It has been shown that plasma from animals that have been injected with cobaltous chloride rapidly develops a high titer of erythropoietin.

The gross properities of the active material appear to be the same from cobalt-treated as from phenylhydrazine-treated animals.

Other metal ions and a complex ion have been studied as stimulants for erythropoietin formation; none was as effective as cobaltous ion with the exception of cobaltic hexamine.
Selenoid (Crescent) Bodies
Authors: CUADRA MANUEL
Published: October 2019

"Selenoid (Crescent) Bodies" are known in hematology as Cuerpos en Media Luna in Spanish and Corps en demi-lune in French.

An original method of staining selenoid bodies and one which is suitable for morphologic structural studies has been described. The method is also suitable for staining stromas of erythrocytes.

Selenoid bodies originate from erythrocytes and correspond to their stromas. Selenoid bodies can be produced in vitro by the spreading of blood on a slide. Two factors influence their formation: a mechanical factor by friction of the erythrocytes against the surface of the slide and a chemical essential factor represented by the lipids of the blood, increasing the fragility of the erythrocytes.

The number of selenoid bodies in any smear of blood is directly proportional to its contents in lipids, either from an exogenous source (alimentary lipemia) or from an endogenous origin.

Selenoid bodies have also been found in the dog, sheep, rabbit, guinea pig, rat and hen.
The Life Span of Red Cells in the Rat and the Mouse as Determined by Labeling with DFP32 in Vivo
Authors: VAN PUTTEN L. M. , Croon Fineke
Published: October 2019

The red cells of rats and mice were tagged in vivo by injection of diisopropylphosphorofluoridate (DFP), labeled with P32, and the disappearance of radioactivity from the circulating red cells was determined. From the data obtained, it is concluded that the disappearance of the red cells is linear with time and that the red cells in both rats and mice have a true life span without measurable random destruction.

The life span of the erythrocytes was found to be 60.0 ± 3.2 (S.D.) days in the rat and 40.7 ± 1.9 (S.D.) days in the mouse.

Especially in the mouse the determination of the red cell life span with DFP32 is a simple procedure.
Clonal diseases of large granular lymphocytes [see comments]
Authors: Loughran TP Jr
Published: October 2019

Three distinct clinical syndromes occur in patients with increased numbers of circulating LGL. Patients with T-LGL leukemia have clonal proliferations of CD3+ LGL typically associated with chronic neutropenia and autoimmune features. NK-LGL leukemia is characterized by clonal CD3- LGL proliferation with an acute clinical presentation marked by massive hepatosplenomegaly and systemic illness. However, most patients with increased numbers of CD3- LGL do not have clinical features of NK-LGL leukemia and have a chronic clinical course. X- linked gene analyses have supported a polyclonal LGL lymphocytosis in this syndrome. Further studies are needed to determine whether clonal progression can occur in these patients.
Control of programmed cell death in normal and leukemic cells: new implications for therapy
Authors: Sachs L , Lotem J
Published: October 2019

Programmed cell death (apoptosis) is a normal process by which cells are eliminated during normal embryonic development and in adult life. Disruption of this normal process resulting in illegitimate cell survival can cause developmental abnormalities and facilitate cancer development. Normal cells require certain viability factors and undergo programmed cell death when these factors are withdrawn. The viability factors are required throughout the differentiation process from immature to mature cells. Although many viability factors are also growth factors, viability and growth are separately regulated. Viability factors can have clinical value in decreasing the loss of normal cells including the loss that occurs after irradiation, exposure to other cytotoxic agents or virus infection including AIDS. There is no evidence that occurs after irradiation, exposure to other cytotoxic agents or virus infection including AIDS. There is no evidence that cancer cells are immortal. Programmed cell death can be induced in leukemic cells by removal of viability factors, by cytotoxic therapeutic agents, or by the tumor-suppressor gene wild-type p53. All these forms of induction of programmed cell death in leukemic cells can be suppressed by the same viability factors that suppress programmed cell death in normal cells. A tumor-promoting phorbol ester can also suppress this death program. The induction of programmed cell death can be enhanced by deregulated expression of the gene c-myc and suppressed by the gene bcl-2. Mutant p53 and bcl-2 suppress the enhancing effect on cell death of deregulated c-myc, and thus allow induction of cell proliferation and inhibition of differentiation which are other functions of deregulated c-myc. The suppression of cell death by mutant p53 and bcl-2 increases the probability of developing cancer. The suppression of programmed cell death in cancer cells by viability factors suggests that decreasing the level of these factors may increase the effectiveness of cytotoxic cancer therapy. Treatments that downregulate the expression or activity of mutant p53 and bcl-2 in cancer cells should also be useful for therapy.
L-selectin mediates neutrophil rolling in inflamed venules through sialyl LewisX-dependent and -independent recognition pathways
Authors: von Andrian UH , Chambers JD , Berg EL , Michie SA , Brown DA , Karolak D , Ramezani L , Berger EM , Arfors KE , Butcher EC
Published: October 2019

The glycoprotein (GP) L-selectin initiates adhesive interactions between leukocytes and endothelial cells (EC). It functions as a lymphocyte-lectin homing receptor recognizing carbohydrate determinants of the peripheral lymph node addressing on high endothelial venules. It also mediates neutrophil rolling, the earliest interaction of neutrophils with acutely inflamed venules. Neutrophil L-selectin presents sialyl-LewisX (sLe(X)) as a ligand to P- and E-selectin in vitro, and we have proposed that this is a major mechanism of L- selectin-mediated rolling in vivo. In contrast, the contribution of neutrophil L-selectin as a receptor protein recognizing one (or more) ligand(s) on inflamed EC is unclear. To address this question, an sLe(X)-negative murine pre-B cell line, L1–2, that can neither bind vascular selectins nor roll in inflamed rabbit venules, was transfected with human L-selectin cDNA. L-selectin expression in stable transfectants was sufficient to confer significant rolling in vivo. Rolling was unaffected by neuraminidase treatment but completely blocked by anti-L-selectin monoclonal antibody (MoAb) DREG-56. Thus, L- selectin can initiate leukocyte interactions with EC determinants potentially through recognition of endothelial carbohydrates. In contrast, when human neutrophils were tested, rolling was reduced, but not abolished, by MoAb DREG-56. Likewise, treatment with neuraminidase or anti-sLe(X) MoAbs decreased, but did not abrogate, neutrophil rolling, consistent with residual EC recognition via L-selectin. Combination of MoAb DREG-56 and neuraminidase resulted in almost complete loss of rolling, as did removal of glycosylated L-selectin by chymotrypsin. Together with the demonstrable rolling of L-selectin transfectants, our results support the concept of a bidirectional interaction between L-selectin bearing sLe(X) on neutrophils and activated EC in vivo. These findings also suggest that L-selectin may mediate rolling of lymphocytes that lack carbohydrate ligands for E- or P-selectin, although probably less efficiently than through bidirectional recognition.
Extended-cycle elutriation to adjust T-cell content in HLA-disparate bone marrow transplantation
Authors: Quinones RR , Gutierrez RH , Dinndorf PA , Gress RE , Ney AB , Taylor B , Karandish S , Carter CS , Luban NL , Reaman GH
Published: October 2019

We report the development of a double-cycle elutriation (DCE) technique separating 3 or greater logs of T cells from a stem-cell-enriched marrow fraction and the results of phase I T-cell depletion studies with HLA-disparate related bone marrow transplantation (BMT) donors in two patient groups. In group 1, 10 patients with refractory hematopoietic malignancies received combination chemotherapy, total body irradiation (TBI), and immunosuppression (pre- and post-BMT), and hematopoietic rescue with a marrow transplant, depleted of T cells by elutriation. Potentially to promote engraftment and a graft-versus- leukemia (GVL) effect, 0.5 to 0.75 x 10(5) T cells/kg were added back. All 10 patients engrafted. Five patients developed acute graft-versus- host disease (GVHD; four grade II, one grade III) and two subsequently developed chronic GVHD. Two patients have relapsed (median follow-up, 206 days; range, 46 to 1,035). Four patients died of BMT-related complications (three of infection, one of veno-occlusive disease [VOD]). Four patient are disease-free survivors (median follow-up, 960 days; range, 670 to 1,035). Group 2 included five infants, four with congenital lymphohematopoietic deficiencies and one with refractory acute lymphocytic leukemia (ALL). In these infants, busulfan and increased cyclophosphamide were substituted for TBI. Only the ALL patient received added T cells. Three patients engrafted: one has stable mixed chimerism, one relapsed with ALL, and one rejected the marrow. One patient had primary autologous recovery, while another failed to engraft. None developed GVHD. We conclude that, in this setting of HLA-disparate BMT with post-BMT antithymocyte globulin (ATG) and corticosteroids, DCE significantly depletes T cells from the marrow and that a defined number of T cells can be added without the occurrence of severe GVHD.
Hematopoietic precursors resistant to treatment with 4- hydroperoxycyclophosphamide: requirement for an interaction with marrow stroma in addition to hematopoietic growth factors for maximal generation of colony-forming activity
Authors: Rowley SD , Brashem-Stein C , Andrews R , Bernstein ID
Published: October 2019

We tested the ability of CD34+lin- precursor cells isolated from marrow after treatment with 4-hydroperoxycyclophosphamide (4HC) to generate colony-forming cells (CFC). In liquid cultures, recombinant human stem cell factor (SCF), in combination with interleukin-1 (IL-1), IL-3, IL- 6, granulocyte-macrophage colony-stimulating factor, or granulocyte colony-stimulating factor caused untreated, but not 4HC-treated, CD34+lin- cells to form CFC. However, generation of CFC from CD34+lin- cells treated with 60 micrograms/mL of 4HC was possible in the presence of an irradiated allogeneic stromal cell layer. This generation was increased when combinations of hematopoietic growth factors including SCF and IL-3 were added. Maximal generation of CFC was seen after 11 to 21 days of culture. At that time, generation of CFC from CD34+lin- 4HC- treated cells equalled that from untreated cells. The phenotype of these 4HC-resistant CD34+lin- precursors was also further defined as CD38-. These studies show that the generation of CFC from the 4HC- resistant, highly immature population of CD34+lin- cells requires an as yet undefined interaction with marrow stroma in addition to known hematopoietic growth factors.
Key functional role and lineage-specific expression of selected HOXB genes in purified hematopoietic progenitor differentiation
Authors: Giampaolo A , Sterpetti P , Bulgarini D , Samoggia P , Pelosi E , Valtieri M , Peschle C
Published: October 2019

Although it is well established that homeobox (HOX) genes play a key role in normal human embryogenesis, the expression and function of HOX genes in normal hematopoiesis is largely unknown. We have investigated by reverse transcriptase-polymerase chain reaction the mRNA expression of HOXB cluster genes (32 to 52 position in the cluster: from HOXB2 through B9) in 72% to 88% purified hematopoietic progenitor cells (HPCs) from adult peripheral blood induced in liquid suspension culture to gradual erythroid or granulopoietic (largely eosinophilic) differentiation and maturation by differential growth factor (GF) stimulus (ie, low-dose interleukin-3 [IL-3] and granulocyte-macrophage colony-stimulating factor [GM-CSF] and high-dose erythropoietin, or saturating amounts of IL-3/GM-CSF, respectively). Only B3 is expressed in quiescent HPCs. After GF treatment B3 expression is enhanced in the initial 24 hours and then through differentiation and maturation in erythroid and granulopoietic cultures. HOXB4 and B5 are induced at slightly later times and expressed through maturation in both lineages, whereas B6 is selectively induced in granulocytic differentiation. B2 is transiently expressed at low level in the granulopoietic pathway, whereas it is detected only in advanced stages of erythropoiesis: B7, B8, and B9 are essentially not detected. Functional studies were performed with antisense phosphorothioate oligomers to HOX mRNAs and included control analysis of the targeted mRNA. The results are strictly coherent with the HOX mRNA expression pattern: (1) anti-B3 oligomer (alpha-B3) treatment of purified HPCs induces a striking blockade of both erythroid and granulomonocytic colony formation (similarly, alpha-B3 treatment of K562 cell line causes a significant dose-related inhibition of cell proliferation); (2) alpha-B6 selectively and markedly inhibits granulomonocytic colony formation; (3) alpha-B4 and alpha-B5 cause a significant, less pronounced decrease of both colony types; (4) finally, alpha-B2 and alpha-B7, -B9 exert little and no effect, respectively. These studies provide novel evidence on the coordinate expression of selected HOXB cluster genes in erythropoiesis and granulopoiesis, particularly in the early stages of differentiation: B3 apparently functions as a master gene in early hematopoiesis, whereas B6 exerts a key selective function in the granulopoietic pathway.
Molecular basis for type 1 antithrombin deficiency: identification of two novel point mutations and evidence for a de novo splice site mutation
Authors: Jochmans K , Lissens W , Yin T , Michiels JJ , van der Luit L , Peerlinck K , De Waele M , Liebaers I
Published: October 2019

Inherited type 1 antithrombin (AT) deficiency is characterized by a reduction in both immunologically and functionally detectable protein. The disorder is associated with a high risk of thromboembolic disease. We have investigated the molecular basis of type 1 AT deficiency in three unrelated families. We have used the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis, followed by direct sequencing of the seven exons and the intron-exon junctions of the AT gene. Two novel point mutations were identified. A T to C single-base substitution was found in codon 421 in exon 6 (nucleotide position 13380), leading to an AT 421 isoleucine to threonine substitution. In another kindred, one of three Cs at nucleotide (nt) positions 5448 to 5450 in exon 3A (codon 151 or 152) was deleted, resulting in a frameshift mutation and predicting premature termination of protein translation at codon 251. In a third family, a previously reported G to A substitution, at nt position 9788 in intron 4, 14 bp in front of exon 5, was found. We have demonstrated the creation of a de novo exon 5 splice site by ectopic transcript analysis of lymphocyte mRNA. In all cases, the affected individuals were heterozygous for the mutation and no variant AT protein was detected.