bluuebunny/crossref_metadata_2025_split huggingface dataset:
Studies on Thrombopoiesis
Authors: IZAK G. , NELKEN D. , HERZOG MISS A. 
Published: October 2019

Abstract
Thrombocytopoiesis was studied by direct, continuous observation in tissue cultures of bone marrows taken from three patients with chronic idiopathic thrombocytopenic purpura and from normals following the addition of a potent anti-platelet serum to the culture media.
The process of platelet production was similar in these two conditions and followed the pattern observed in bone marrow cultures of healthy individuals. The breakdown of megakaryocytes of patients with idiopathic thrombocytopenic purpura and those treated with anti-platelet serum was greatly accelerated.
No morphologic evidence of injury to the megakaryocytes was present.
The platelets produced showed degenerative changes, they were agglutinated and underwent phagocytosis by the myeloid elements and reticulum cells were present in the cultures.
Congenital Amegakaryocytic Thrombocytopenia with Congenital Deformities and a Leukemoid Blood Picture in the Newborn
Authors: EMERY JOHN L. , GORDON R. R. , RENDLE-SHORT JOHN , VARADI S. , WARRACK A. J. N. 
Published: October 2019

Abstract
Two infants with absent radii are described. Both had hemorrhagic manifestations which occurred within 24 hours of birth, and from which they eventually died.
The most important hematologic feature was absence of megakaryocytes in the bone marrow.
Five cases showing a similar picture have been recorded, four as amegakaryocytic thrombocytopenia, and one as congenital leukemia.
It is suggested that these cases are related on the one hand to Fanconi’s anemia, and on the other to congenital leukemia.
Dithizone Staining of Myeloid Granules
Authors: MCNARY WILLIAM F. 
Published: October 2019

Abstract
A dithizone-acetone-water solution may be used to stain myeloid granules in blood smears and bone marrow preparations. It is necessary to lake the erythrocyte before staining in order to remove interfering substances. The results indicate that zinc, known to be present its leukocytes, is cytoplasmic and not associated with the myeloid granules. It is suggested that the method described may be of value to complement the peroxidase stain as it is rapid, simple, and utilizes solutions which may be stored for lengthy periods.
Variation in the Duration of Survival of Patients with Acute Leukemia
Authors: MACMAHON BRIAN , FORMAN DONALD 
Published: October 2019

Abstract
Survey of records of 34 Brooklyn hospitals and of selected hospitals elsewhere in New York City revealed records of 623 white residents of Brooklyn diagnosed as having acute leukemia during the period 1943-52. Diagnosis was on the basis of marrow biopsy or autopsy in 79 per cent. Date of death was known for 96 per cent.
Almost half of these patients died within one month of diagnosis, and three quarters before the end of the third month. Ten per cent survived for 6 months and 3 per cent for one year. The mean interval between diagnosis and death was 2.4 months.
The duration of survival after diagnosis was longer for the group of patients in whom the cell type was diagnosed as lymphocytic, than in those diagnosed as granulocytic. Percentage of patients surviving three months was also significantly higher in the lymphocytic than in the granulocytic group.
Average duration of survival was almost twice as long for patients in the first decade as for those in any subsequent age group. Survival was shorter for patients in whom the total white blood cell count taken at diagnosis was high.
Duration of survival after diagnosis increased with increasing duration of symptoms prior to diagnosis. This suggests another limitation to the use for prognostic purposes of the interval between onset of symptoms and death.
No association of survival with sex, religion or ABO blood group was detected.
Alternative Splicing and Expression of Class II Tubulin Beta (TUBB2B) Are Associated with Outcome in Diffuse Large B-Cell Lymphoma
Authors: Taskinen Minna , Koivula Satu , Chen Ping , Karjalainen-Lindsberg Marja-Liisa , Cervera Alejandra , Delabie Jan , Holte Harald , Hautaniemi Sampsa , Leppä Sirpa 
Published: October 2019

Abstract
Abstract 1557

Background:
The aim was to compare differentially expressed exons and splicing variants between diffuse large B-cell lymphoma (DLBCL) patients, who had relapsed or remained in remission after dose dense chemoimmunotherapy.

Design and methods:
We performed genome-wide exon array analysis from four DLBCL cell lines and 38 tumor tissues from young (<65 years) DLBCL patients with high-risk (aaIPI>1) disease. The patients were treated in a Nordic phase II protocol with six courses of R-CHOEP-14 followed by systemic central nervous system prophylaxis with one course of high dose methotrexate and high dose cytarabine. At the time of the analysis, median follow up was 34 months, predicted 3-year progression free survival (PFS) 78% and overall survival (OS) 79%. RNA for quantitative PCR validation was available from 20 patients. Two DLBCL cell lines and eight patient samples were further analyzed with high throughput RNA sequencing. In addition, microarray data set from 233 DLBCL patients treated with chemoimmunotherapy (Lymphoma/Leukemia Molecular Profiling Project (LLMPP)) was utilized for validation.

Results:
Differentially expressed exons between relapsed and non-relapsed patients were screened using criteria of p ≤ 0.05 and fold change ≥1.6 converting to 566 differentially expressed genes, of which 131 coded proteins. One of the identified genes with possible alternative splicing was TUBB2B, which encodes therapeutic target of taxanes and vinca alkaloids. The expression of TUBB2B, and specifically the expression of exon 3, was found to be suppressed in relapsed patients in comparison to patients remaining in remission. Differential expression of TUBB2B whole transcript and exon 3 was confirmed with RNAseq and quantitative PCR. Studies in lymphoma cell lines provided further support for the existence of different TUBB2B isoforms. According to Kaplan Meier estimates the patients with high (>median) expression levels of TUBB2B exon 3 had better 3-year PFS and lymphoma related OS rates than the patients with low expression levels (95% vs. 61%, p=0.015 for PFS, 100% vs. 75%, p=0.024 for OS). The prognostic significance of TUBB2B gene expression was validated in LLMPP data set (3-year OS 80% vs. 67%, p=0.040).

Conclusions:
The results provide evidence that differential expression and splicing of TUBB2B gene can discriminate the outcome of homogenously treated high risk DLBCL patients.

Disclosures:
No relevant conflicts of interest to declare.
Impact of ASXL1 Mutations On Outcome After Reduced Intensity Allograft in Patients with Myelofibrosis
Authors: Kröger Nicolaus , Koenecke Christian , Alchalby Haefaa , Zabelina Tatjana , Bollin Robin , Gehlhaar Marten , Dobbernack Vera , Kade Sofia , Huang Liu , Ganser Arnold , Ayuk Francis , Wolschke Christine , Thol Felicitas , Heuser Michael 
Published: October 2019

Abstract
Abstract 1740
Mutations in the additional sex combs like 1 gene (ASXL1) have been described recently in patients with myelodysplastic and myeloproliferative syndromes. While in MDS ASXL1 is associated with worse prognosis, the role in MPN is not well defined. The aim of this study was to investigate the prognostic impact of ASXL1 mutations in 136 patients with primary myelofibrosis or post ET/PV myelofibrosis who underwent allogeneic stem cell transplantation after reduced intensity conditioning.
Out of 136 patients sufficient genomic DNA prior transplantation was available for 103 patients. The median age of 48 female and 55 male patients was 59 years (range, 33–75y). After busulfan (n= 97) or treosulfan (n=6) based dose-reduced conditioning, patients received a stem cell graft from a related (n= 17) or unrelated donor (n=48). ASXL1 was sequenced by Sanger sequencing, and positive patients were confirmed by an independent sequence analysis.
Mutations in ASXL1 were found in 27 patients (26%), of whom 24 had frameshift mutations (23%). ASXL1 mutated and non mutated patients were equally distributed between patients with unrelated (19% pos/81% neg) and related (21% pos/79% neg) donors. ASXL1 mutations were found at the same frequency in Lille low risk (15%), intermediate risk (24%) and high risk (20%) patients.
After a median follow up of 41 months (range 26–62) the cumulative incidence of non- relapse mortality at 1 year and relapse at 5 years were 26% (95% CI 16–36) and 26% (95% CI 16–36), respectively, and the 5-year OS was 56% (95% CI 44–68).
Patients with ASXL1 mutation did not differ significantly to those without mutation in non relapse mortality (35% vs. 22% p=0.4), relapse incidence (24% vs. 27%, p=0.9) and regarding overall survival. Overall survival for ASXL1 mutated and wildtype patients was not significantly different (47% vs. 59%, p= 0.5) in patients with any ASXL1 mutation and in the subgroup of ASXL1 frameshift mutated patients (48% vs. 59%, p= 0.6). The rates of acute grade II-IV GvHD and of chronic GvHD were similar in ASXL1 mutated and wildtype patients (44% vs. 36%, p=0.5 and 52% vs. 46%, p=0.7, respectively). In summary, ASXL1 is one of the most frequently mutated genes in PMF patients. Patients with ASXL1 mutations have a similar outcome after dose reduced allogeneic HSCT as patients with wildtype ASXL1.

Disclosures:
No relevant conflicts of interest to declare.
Whole Genome Sequencing of Four CD34+-Derived iPSC Polycythemia Vera Clones From a Single Female
Authors: Tian Lifeng , Piterkova Lucie , Wang Linghua , Ye Zhaohui , Cheng Linzhao , Wheeler David A. , Hakonarson Hakon , Prchal Josef T 
Published: October 2019

Abstract
Abstract 1755
Polycythemia vera (PV) is an acquired clonal hematopoietic disorder characterized by the JAK2V617F somatic mutation, which, however, constitutes only a variable part of the PV clone. Even within the JAK2V617F subclone, heterozygous and homozygous JAK2V617F progenitors coexist. Further, family clustering of PV suggests the existence of predisposing germline mutations. The nature of these pre-JAK2V617Fsomatic and germline mutations has not yet been identified, and the search for these predisposing genetic lesions using marrow and blood cells is hampered by their genetic heterogeneity. In order to overcome this obstacle, we generated 5 inducible pluripotent stem cell lines (iPSC) from the same PV female with different JAK2 genomic configuration (4 from CD34+ blood cells, one from marrow mesenchymal cells having germ-line DNA configuration). Genomic DNA was isolated from the cell lines, followed by whole genome sequencing (WGS) at >20X depth, and whole exome sequencing (WES) at >130X depth.
We have developed a comprehensive WGS/WES pipeline for sequence alignment and variant calling. The major components are: a) Pre-processing and quality control of raw reads outputted from sequencing instruments; b) Align the read against the Human Genome Project reference using BWA (Burrows-Wheeler Aligner); c) Local realignment at indels using GATK (Genome Analysis Toolkit); d) Identify germline/somatic single nucleotide variants (SNVs), copy number variants (CNV) and structural variants (SV) using GATK, Varscan2, Breakdancer, CNVnator, and Pindel. The variant calls are intersected and merged. Calls detected by two or more algorithms are regarded as high confidence.
Two somatic mutations, rs1047840, and rs3795677 were identified by WGS and WES in the CD34+ cell derived iPSCs. rs1047840 (E589K) is a SNP in EXO1 gene that encodes a protein with 5' to 3' exonuclease activity as well as an RNase H activity. EXO1 is involved in DNA mismatch repair and homologous recombination. rs1047840 conferred increased risk of breast, lung, and gastric cancer. rs3795677 (R64H) is a SNP in FMN2 gene, which is required for microtubule-independent chromatin positioning during metaphase. Interestingly, these two mutations are all common SNVs, and they are located in a duplicated region on 1q43. Whole genome analysis is ongoing to characterize and validate the complex mixture of SVs in this region, identify potential translocations and determine the exact genomic breakpoints. The variants will be validated in non-iPSC cells from the same individual to rule out iPSC artifacts.
This work identifies somatic mutations in EXO1 and FMN2, and CNVs in 1q43 as the potential genetic defects involved in the early pathogenesis of PV. It also demonstrates the value of combining WGS and WES for the identification of somatic mutations.

Disclosures:
No relevant conflicts of interest to declare.
Ten Year Survival in Patients with Myeloma Is Characterized by the Persistence of Immunological Control Which Is Detected by Immunological Biomarkers
Authors: Brown Ross D , Suen Hayley , Bryant Christian E , Yang Shihong , Favaloro James , Gibson John , Ho P Joy , Fromm Phillip , Hart Derek NJ , Joshua Douglas E 
Published: October 2019

Abstract
Abstract 1818
There is clinical evidence for the presence of a limited degree of host-tumor control in patients with multiple myeloma (MM) but the exact mechanisms involved are not known. Patients who survive for more than ten years are likely to have the most active immunological host-tumour control and are an ideal cohort to study. We now add to our preliminary observations using a range of immunological biomarkers in the 29 of these patients who attend our clinic.
51% of MM patients (n=264) had expanded CD3+CD8+ TCRVβ+ CD57+T cell clones detected by TCRVβ analysis (Beckman, BetaMark). Clonality was confirmed by IgH CDR3 sequencing. These clones accounted for 14.3% (median) of the CD3 cells (range 4–49%). CFSE tracking demonstrated the anergic nature of these clonal T cells (median 6% proliferation) compared with other CD8 cells (70% proliferation) while Geneset analysis of mRNA microarrays (Affymetrix U133) identified that anergy was caused by upregulated RAS, CSK, TOB and suppressed ERK pathways. Unlike recent reports for T-LGL, microarray analysis suggested that there was no evidence of STAT3 upregulation in the MM T cell clones. Functional studies suggested that these non-proliferating clones have split anergy as interferon-γ production was normal. In contrast, all ten year survivors had expanded T cell clones and serial studies demonstrated a >8 year persistence of the same clone in 8 out of 12 patients studied. More importantly, unlike the other MM patients, the T cell clones in 19 out of 21 of the ten year survivors studied were not anergic. The Treg/Th17 ratio in the ten year survivors was significantly lower than other MM patients (median 1.9 vs 12.0; p<0.0002) and even lower than age matched normals (median 5.6; p<0.006). This difference in the ten year survivors was due to an increased absolute Th17 cell (p<0.005) number and a reduced absolute Tregnumber (p<0.05). MM patients had a reduced number of absolute 6-Sulfo-LacNAc (SLAN)-DCs, a major source of IL-12, compared to both age matched controls and long term survivors (p < 0.01). Allo SLAN-DCs stimulated a higher proliferative response by MM T cells than could be achieved with mononuclear preparations.
In conclusion, MM patients have expanded clones of cytotoxic T cells that exhibit split anergy and these cells are potential candidates for restoring immunological control of MM and other cancers. The normalised Treg/Th17 ratio suggests that the induced tolerance associated with increased Treg cell numbers is also absent in these patients. Our observations that the ten year MM survivors do not have anergy in their clonal T cells and have less Treg suppression offers a unique cohort for future studies.

Disclosures:
No relevant conflicts of interest to declare.
Bortezomib Induced Peripheral Neuropathy Is Related to Altered Level of Brain Derived Neurotrophic Factor in the Circulating Blood.
Authors: Azoulay David , Lavie David , Gatt Moshe E. , Surio Celia , Akaria Luiza , Perlman Riki , Braester Andrei , Yehuda Dina Ben 
Published: October 2019

Abstract
Abstract 2819
Peripheral Neuropathy (PN) is a major side effect and dose-limiting factor of Bortezomib treatment in multiple myeloma (MM). The exact mechanism underlying Bortezomib induced PN (BIPN) is unknown, and the risk factors which increase susceptibility for developing this syndrome have yet to be elucidated. Brain Derived Neurotrophic Factor (BDNF) is a nerve growth factor which is responsible for the development, maintenance, and repair of the peripheral nervous system. Aim: In this work we examined alterations in the levels of BDNF in circulating blood and searched for their relation with BIPN development.

Method:
24 patients with Multiple Myeloma receiving Bortezomib based regimen and 30 patients with other hematological malignancies (OHM) were examined. We used ELISA to quantify soluble BDNF (sBDNF) level in patient's platelets-poor plasma (PPP) and flow cytometry or western blotting to quantify BDNF in platelets. PN was assessed and graded according to the cancer common toxicity criteria index.

Results:
The level of sBDNF in MM patients PPP at diagnosis was similar to the level of sBDNF in patients with other hematological malignancies at diagnosis (3.86±2.47 ng/ml Mean ± SD in MM and 3.70±2.5 ng/ml in OHM). During treatment with Bortezomib the level of sBDNF was reduced only in the group of MM patients that developed BIPN (1.94±1.7 ng/ml P value<0.05) whereas the level of sBDNF in MM patients who did not develop BIPN remained equal to the level at diagnosis. Correspondingly, a negative correlation between the level of sBDNF and the severity of BIPN was observed. Analysis of platelets, the main cellular storage of BDNF in the peripheral blood, revealed a high incidence of BDNF-containing platelets as well as increased BDNF content within platelets of patients with BIPN as compared to patients without BIPN. No difference in the number of platelets between patients with BIPN and patients without BIPN was observed.

Conclusions:
Our results suggest that alterations in the circulating blood level of BDNF may play role in the pathophysiology of BIPN development. Detailed investigation regarding potential mechanisms by which BDNF is involved in BIPN is required. Furthermore, BDNF could serve as a useful biomarker for early detection of Bortezomib induced BIPN.

Disclosures:
No relevant conflicts of interest to declare.
BTK Inhibition Targets in Vivo CLL Proliferation Through Its Effects On B-Cell Receptor Signaling Activity.
Authors: Wang Y. Lynn , Cheng Shuhua , Ma Jiao , Guo Ailin , Lu Pin , Leonard John P. , Coleman Morton , Liu Momo , Buggy Joseph J. , Furman Richard R. 
Published: October 2019

Abstract
Abstract 2903

Purpose:
Bruton tyrosine kinase (BTK) is a component of the B-cell receptor signaling pathway. Ibrutinib (previously known as PCI-32765), a first in class, covalent BTK inhibitor, has demonstrated significant clinical activity against CLL in early clinical trials. Understanding the molecular mechanisms of action would shed light on CLL pathophysiology and provide additional opportunities for the development of new therapies.

Experimental Design:
The anti-tumor activity of ibrutinib in CLL has been investigated previously using either an ex vivo approach or a mouse model (Herman et.al, Blood. 2011;117:6287–96 and Ponader et.al, Blood. 2012;119:1182–9). In this study, we have chosen, instead, a patient-oriented in vivo approach by using samples from an ongoing phase 1b trial of ibrutinib (NCT01105247). We prospectively collected serial samples from CLL patients (n=14) before and at several time points after the initiation of therapy and analyzed them for cellular and molecular signaling events.

Results:
We demonstrated that levels of the phosphorylated BTK protein (p-BTK) in CLL cells from treatment-naïve patients were significantly higher than in normal B cells, explaining why CLL cells are more susceptible to BCR inhibition than normal B cells. Response assessments, performed at the end of cycle 2 (∼Day 56), demonstrated nodal responses in all patients by CT scan. Ex vivo apoptosis did occur but required high concentrations of ibrutinib (>500 nM). In addition, in vivo apoptosis was rarely observed in serial peripheral blood samples collected from treated patients. With these serial samples, we found that the population of Ki67+ cells were gradually decreased over a 28-day ibrutinib treatment course. Using a newly established co-culture system that induces CLL proliferation in vitro, the analysis of several parameters, including Ki-67 expression, cell growth and bromodeoxyuridine (BrdU) incorporation (shown in the figure), revealed that the proliferation of CLL cells was directly inhibited by ibrutinib (200 nM). Furthermore, activities of BTK and downstream signaling events, such as the phosphorylation of PLCg2, AKT and ERK, were all suppressed over time in ibrutinib-treated patients.

Conclusions:
With primarily an in vivo approach, we have demonstrated that the blockage of cell proliferation was a major effect of ibrutinib against leukemic CLL cells. Blocking cell proliferation via inhibition of BTK-mediated signaling concurs with clinical responses in ibrutinib-treated CLL patients.

Disclosures:
Leonard: Pharmacyclics Inc.: Consultancy, Honoraria. Buggy:Pharmacyclics: Employment, Equity Ownership.
Daratumumab, a Human CD38 Antibody Induces Apoptosis of Myeloma Tumor Cells Via Fc Receptor-Mediated Crosslinking.
Authors: Jansen J. H. Marco , Boross Peter , Overdijk Marije B. , van Bueren Jeroen J. Lammerts , Parren Paul W.H.I. , Leusen Jeanette H. W. 
Published: October 2019

Abstract
Abstract 2974
Daratumumab (DARA) is a human CD38 monoclonal antibody with broad-spectrum killing activity. DARA is in clinical development for multiple myeloma (MM) and has potential in other hematological tumors on which CD38 is expressed. In these clinical studies the adverse events have been manageable and marked reductions in paraprotein and bone marrow plasma cells have been observed. The killing activity of DARA on CD38-expressing tumor cells has previously been mainly attributed to complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).
In this study we explored whether DARA could also kill CD38-positive cells via induction of apoptosis after immobilization of DARA via Fc receptors (FcRs) in vitro. To this end, target cells were co-incubated in vitro with hFcgRI-expressing cells, lacking ADCC activity, in the absence or presence of DARA. Apoptosis was measured in flow cytometry for annexin-V/7AAD positivity. Treatment of the Burkitt's lymphoma cell line Ramos with DARA in combination with hFcgRI-expressing cells, led to a significant enhancement of annexin–V/7AAD positive cells (p<0.05 Dunn's multiple comparison test). Similar results were obtained with patient-derived multiple myeloma cell lines L363 and UM9, which are transduced with CD38 to obtain comparable expression levels to primary myeloma cells. This indicates that FcR-mediated crosslinking of DARA induces apoptosis which might contribute to the mechanism of action. We are currently performing experiments to investigate the contribution of DARA-mediated apoptosis in vivo in tumor growth inhibtion. Therefore, DARA efficacy is studied in a peritoneal syngeneic mouse model with EL4-CD38 cells, a mouse lymphoma cell line transfected with human CD38. The EL4-CD38 cells showed efficient apoptosis induction after FcR-mediated crosslinking of DARA in vitro. For this mouse model we will make use of NOTAM mice, which have a functionally inactive FcR associated gamma chain leading to cell membrane expression of all FcRs, but without signaling capacity (de Haij et al. Cancer Research 2010). Leucocytes of these mice are capable of antibody crosslinking via FcRs, but incapable of inducing cytotoxicity via ADCC.
In conclusion, in vitro data suggest that FcR-mediated crosslinking of DARA induces apoptosis of CD38 expressing tumor cells, and can thus be considered an additional mechanism of action for DARA.

Disclosures:
Jansen: Genmab BV: Research Funding. Overdijk:Genmab BV: Employment. Lammerts van Bueren:Genmab BV: Employment. Parren:Genmab BV: Employment.