bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment of Burkitt's Like Lymphoma: A Single Institution Case Series
Authors: Ranpura Vishal , Shah Shilpan S.
Published: October 2019

Abstract 4962

Background: Burkits's like (BL) lymphoma is a new pathologic entity with features partially resembling to Burkitt's lymphoma and diffuse large B cell lymphoma. The incidence of BL lymphoma is infrequent and may present with lymph node enlargement, involvement of bone marrow and peripheral blood. The natural history of BL lymphoma and its treatment remains unclear. Here we present a single institution case series of five patients diagnosed with BL lymphoma and their treatment. Methods: We searched the pathology reports of all patients diagnosed with lymphoma over the last one year. No inclusion or exclusion criteria were used. Results: We identified a total of five patients with diagnosis of BL lymphoma. All patients presented with lymphadenopathy and with no involvement of bone marrow, central nervous system or peripheral blood. Table 1 summarizes characteristics of all patients, their chemotherapy regimen and subsequent response to treatment. Conclusion: BL lymphoma is a new pathologic entity with low incidence. Treatment with DA-EPOCH and R-HyperCVAD has very good response rate. The data is limited by single institution case series and limited follow up time. Further studies are recommended to evaluate optimal chemotherapy regimen. Table 1: Characteristics, presentation, treatment and response in patients with BL lymphoma

No Age Sex LDH, Presentation Treatment Response Duration of response 1 68 F 386 Parotid and abdominal lymphadenopathy DA-R-EPOCH CR 16months 2^ 44 M 536 Neck, Axillary, media, abdomen pelvis and sacral mets R-HyperCVAD PR 3months 3* 66 F 310 Inguinal and pelvic lymphadenopathy R-HyperCVAD f/b Autologus BMT, CR 14months 4** 71 M 237 Parotid mass 3 cycles of DA-EPOCH f/b 3 cycles of R-CHOP CR 9months 5^ 52 M 186 Neck Lymphadenopathy R-HyperCVAD CR 3months DOX, DOXOL, and anthracenediones in soluble fractions of human myocardial strips after sequential DOX loading/clearance and anthracenedione treatment Notes: DA-REPOCH: Dose Adjusted Rituximab-Etoposide, Vincristine, Cyclophosphamide, Doxorubicine, Prednisone, R-HyperCVAD: Rituximab, hyperfractionated Cyclophosphamide, Vincristine, Doxorubicin, Dexamethasone, CR: complete remission, BMT: bone marrow transplantation, R-CHOP: rituximab, doxorubicin, cyclophosphamide, vincristine and prednisone. CNS: central nervous system * patient underwent bone marrow transplant because of relapse at current presentation. ** patient was changed from DA EPOCH to R-CHOP as patient was not able to tolerate EPOCH ^ Epatients are halfway through their treatment cycles and are actively getting treatment

Disclosures:

No relevant conflicts of interest to declare.
The Vitamin B12 Content of Human Liver Tissue and Its Nutritional Significance
Authors: SWENDSEID MARIAN E. , HVOLBOLL ELIZABETH , SCHICK GERALDINE , HALSTED JAMES A.
Published: October 2019

Range and average values are reported for the vitamin B12 content of liver tissue obtained at autopsy from 132 individuals. Th average value for all age groups was approximately 0.70 γ per Gm. of wet tissues. In cirrhosis the average value was 0.26 γ per Gm. It was concluded that there are no apparent differences in vitamin B12 stores in the various age groups. Also, the amount of vitamin stored is relatively large in terms of requirement and is probably sufficient to last for a period of several years.
Specific and Cross-reacting Antibodies in ABO Heterospecific Twin Pregnancy
Authors: ZUELZER WOLF W. , COHEN FLOSSIE , ROBINSON ABNER R. , BEATTIE KATHRYN
Published: October 2019

A "study in depth" is reported concerning the case of hemolytic disease of a group B infant born to a group O mother, whose group A twin was apparently unaffected. It was shown that the hemolytic disease of the affected infant was due to a specific anti-B antibody. The study included parallel examinations of the antibodies in the sera of the three individuals.

The specificity of the B-anti-B reaction was demonstrated in vitro and in vivo. The powerful anti-B antibody of the mother had no effect on the group A twin, in whose serum anti-B was present in large amounts.

In vitro, studied by the usual technics of cross absorption the maternal and the fetal anti-A and anti-B serum antibodies behaved as if strictly specific. By applying successive absorption, elution and neutralization techniques, however, it was possible to demonstrate additional cross-reacting antibodies in the maternal serum which could be separated from one another and from the specific anti-A and anti-B antibodies. From the erythrocytes of the normal group A twin such cross-reacting antibody could be eluted.

The cross-reacting anti-B antibody, isolated in pure form in eluates, could be shown to be loosely attached to group A erythrocytes without producing visible agglutination reactions while after elution from A cells it did visibly agglutinate group B cells. It could be eluted from A cells, absorbed by fresh A cells and reeluted while retaining its anti-B effect. It was neutralized by group B saliva only. A separate anti-A antibody with similar properties was eluted from B cells and specifically neutralized by group A saliva.

A partial affinity of these antibodies for heterologous erythrocytes but not for specific soluble substances was thus demonstrated. These findings support neither the linkage hypothesis of cross reactions between anti-A and anti-B nor the C-anti-C hypothesis of hemolytic disease. They are in keeping with the view that group O sera contain variable complexes of anti-A and anti-B antibodies, composed of multiple fractions with different partial specificities. It is suggested that the occurrence or non-occurrence of cross-reacting antibodies found in sera of group O mothers whose infants develop hemolytic disease is best explained on this basis. It is further stressed that the demonstration of an antibody in mother or child in ABO hemolytic disease does not necessarily indicate its pathologic significance.
Megaloblastic Anemia Following the Use of Primidone
Authors: NEWMAN M. J. D. , SUMNER D. W.
Published: October 2019

Two cases of atypical megaloblastic anemia are described in patients under treatment with Primidone.

It is argued that megaloblastic anemia may occur as a toxic effect of this drug as well as of Dilantin.
Studies on Thrombopoiesis
Authors: IZAK G. , NELKEN D. , HERZOG MISS A.
Published: October 2019

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

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

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

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 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 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 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 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 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 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 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.
Role of β7 Integrin in Hematopoietic Stem Cell Trafficking.
Authors: Murakami Jodi , Xu Baohui , Franco Christopher B. , Galli Stephen J. , Weissman Irving L. , Chen Ching-Cheng
Published: October 2019

Abstract 2992

β7 integrin (β7), a well studied integrin chain, associates with either the α4 or αE chains to form a heterodimer on the cell surface. While it plays a key role in the migratory activities of hematopoietic progenitors and mature effector cells in the periphery, the overall importance of β7 in adult hematopoiesis remains controversial. We have recently identified a small subpopulation of hematopoietic stem cells (HSCs) in the bone marrow (BM) that express β7 integrin at steady state. These β7+ HSCs are capable of long-term reconstitution of both the lymphoid and myeloid compartments, ruling out the possibility that β7 expression on HSCs constitutes a marker for a homogeneous lineage-restricted progenitor population. We also found that these β7+ HSCs have an inherent advantage over β7− HSCs in competitive reconstitution assays. Loss of β7 results in the retention of HSCs in the BM and reduced HSC reconstitution potential. Quantitative real-time PCR revealed that β7 deficient HSCs downregulated the chemokine receptor Cxcr4. Furthermore, blockade of the α4β7 integrin ligand, mucosal addressin cell adhesion molecule 1 (MAdCAM-1), exhibits a similar phenotype in HSC retention and competitive reconstitution. Interestingly, we discovered that β7 deficient mice have enhanced survival and faster hematopoietic recovery after 5-fluorouracil-induced myeloablative stress, indicating that the decreased engraftment of β7 deficient HSCs may not due to a functional defect, but rather is more likely caused by impaired homing capabilities. Taken together, these data strongly suggest that β7 integrin is essential for HSC homing during the transplantation process.

Disclosures:

No relevant conflicts of interest to declare.
Impact of Thymic Function On Chronic Graft-Versus-Host Disease (cGVHD).
Authors: Mahadeo Kris M. , Shah Ami J , Masinsin Bernadette , Kapoor Neena , Abdel-Azim Hisham , Parkman Robertson
Published: October 2019

Abstract 3052

The initial analysis of the impact of cGVHD on thymic function used TREC analysis and demonstrated a sustained decrease in thympoiesis in hematopoietic stem cell transplant (HSCT) recipients with a history of cGVHD (Blood 97:1458, 2011). We have reported that HSCT recipients, who had the clinical resolution of their cGVHD, had increases in their resting regulatory T lymphocytes (rTreg) as compared to HSCT recipients with active cGVHD [ASH 2011]. The increase in rTreg lymphocytes suggested that clinical resolution of cGVHD might be due to improved thymic function, which resulted in the increased production of rTreg lymphocytes.

To measure the thymic function of pediatric HSCT recipients with cGVHD, we determined the frequency of recent thymic emigrants (RTE; CD4+, CD45RA+, CD31+) in the whole CD4 T lymphocyte population and the CD4 regulatory T (Treg; CD127-, CD25+) and CD4 conventional T lymphocytes (Tcon; CD127+, CD25+) subpopulations of normal individuals, HSCT recipients without a history of cGVHD (n =16) and HSCT recipients with a history of cGVHD (n=20). The frequency of RTE in total, Treg, and Tcon lymphocytes of the HSCT recipients with cGVHD was decreased (P=0.001) compared to HSCT recipients without a history of cGVHD but was the same as normal individuals. Thus, pediatric HSCT recipients with cGVHD have thymic function that is similar to normal individuals, but have decreased thymic reserves compared to HSCT recipients without cGVHD.

However, since rTreg lymphocytes (CD4+, CD127-, CD25+, CD45RA+, FoxP3+) represent only 20% of total Treg lymphocytes, we specifically determined the thymic contribution to rTreg lymphocytes. The RTE content of rTreg lymphocytes was determined in HSCT recipients with active or resolved cGVHD. The RTE content of the rTreg lymphocytes (as a percentage of total CD4 T lymphocytes) was similar in HSCT patients with both active and resolved cGVHD. Thus, the increase in rTreg lymphocytes present in HSCT recipients with the clinical resolution of their cGVHD is not due to an increase in the frequency of RTE in their rTreg lymphocyte population. Therefore, the increase in rTreg lymphocytes, that is associated with the clinical resolution of cGVHD, may be due to either their decreased conversion to Th17 lymphocytes or their decreased apoptosis.

Disclosures:

No relevant conflicts of interest to declare.
A Simple and Predictive Index for the Evaluation of Risk Stratification Following Hematopoietic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia in Remission.
Authors: Huang Xiao Jun , Wang Yu
Published: October 2019

Abstract 3066

Background.

The impact of risk-related parameters has not been defined in transplant settings.

Design and methods.

We analyzed the data of 255 consecutive patients (median age: 26) with acute myeloid leukemia (AML) in their first or second remission (CR1 or CR2) after haploidentical hematopoietic stem cell transplantation (HSCT).

Results.

Three parameters were found to be predictive of outcome: response after induction therapy, WBC count at diagnosis, and cytogenetics. These three factors were combined to yield two risk groups. The 2-year cumulative incidences of relapse for patients at low and high risk were 8% and 36% (p = 0.001), respectively. The three-year probabilities of leukemia-free survival (LFS) for these two groups were 80% and 52% (p = 0.001), respectively. Multivariate analysis for relapse and for LFS showed that not achieving CR after 2 courses of therapy was the strongest independent prognostic factor (p=0.001, and p=0.019, respectively). In addition, in a subgroup of patients with quantification of minimal residual disease (MRD) at the time of HSCT, positive MRD at this time point was correlated with a poor outcome.

Conclusions.

Our results suggest that the index is simple and predictive, so it might be applied to assist with risk-directed post-transplant therapy for young patients with AML in CR who have received myeloablative haploidentical HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Anti-CD33 Chimeric Antigen Receptor Therapy For Acute Myeloid Leukemia
Authors: O'Hear Carol E. , Heiber Joshua , Geiger Terrence L.
Published: October 2019

CD33 is a protein expressed on most myeloid leukemia cells and committed myelomonocytic and erythroid progenitor cells, but not on other cell types. This makes it an attractive therapeutic target for acute myeloid leukemia (AML). CD33 positive cells can be specifically targeted for destruction through the use of a chimeric antigen receptor (CAR) expressed on cytotoxic T-lymphocytes (CTL). We have expressed a CD33-specific 41BB/CD3z second generation humanized chimeric antigen receptor (CAR) on human T cells by MSCV retroviral transduction, allowing for the generation of large numbers of CAR-modified CTLs. We assessed the activation and specific lytic ability of these CAR-modified T-cells by using murine and human CD33-transduced cell lines, as well as control CD33 negative lines. Our experiments show that anti-CD33 CAR CTLs specifically kill CD33-transduced 1498 and EL4 cells lines, with greater than 90% killing at 24 hours at a 1:1 effector to target ratio. These CTLs do not kill 1498 and EL4 cells that have not been transduced with CD33. In addition, anti-CD33 CTLs show significant (>90%) killing of multiple CD33 positive AML cell lines, including CHRF-288-11, HU-3, and UT-7, CMK, HL-60, Molm13, and Mv4-11. We have transduced a similar murine anti-CD33 CAR onto mouse CTLs. When these CTLs are injected into C57BL/6 mice with 1498 tumor cells transduced with CD33, the mice have a reduction in tumor burden compared with mice that have been injected with tumor cells and MSCV-negative control CTLs. This data suggests that our anti-CD33 CAR shows promise of therapeutic efficacy in the treatment of AML.

Disclosures:

No relevant conflicts of interest to declare.
Successful Engraftment of Hematopoietic Stem Cells into Immunocompetent Recipients Using Only Anti-CD117 Antibody and CD47 Blockade As Conditioning
Authors: Chhabra Akanksha , Ring Aaron M , Weiskopf Kipp , Weissman Irving L , Shizuru Judith A.
Published: October 2019

Bone marrow transplantation (BMT) is an efficacious therapy for many otherwise incurable hematologic malignancies and disorders that affect production of hematopoietic cells, including life-threatening anemias, and immunodeficiency syndromes. In a successful transplantation, clearance of bone-marrow niches must be achieved for donor hematopoietic stem cell (HSC) to engraft. Current methods to clear niche space rely on radiation and/or chemotherapy, which can impart toxic adverse effects that greatly limit the potential clinical utility of BMT. Thus, there is a major clinical need for safer conditioning regimens. To this end, a monoclonal antibody against CD117 (ACK2) has been shown to transiently deplete HSCs in immunocompromised mice and enable donor cell engraftment. However, this regimen has proved ineffective in immunocompetent animals. We hypothesized that HSC-depletion by this antibody may occur, in part, through Fc-mediated effector functions, such as antibody-dependent cellular phagocytosis (ADCP). We thus sought to enhance the ADCP activity of anti-CD117 antibodies by blocking the inhibitory CD47-SIRPα “don’t eat me” pathway. Consistent with previous reports, we found treatment of wild-type mice (C57BL/6) with ACK2 alone resulted in only modest and transient depletion of hematopoietic stem and progenitor cells (HSPCs). Strikingly, the addition of the CD47-antagonist CV1 (Weiskopf & Ring, 2013) in combination with ACK2 resulted in rapid and prolonged depletion of bone marrow HSPCs as well as clearance of the bone marrow niche. This depletion was accompanied by profound reductions in hematocrit and blood leukocyte counts not observed in mice treated with ACK2 alone. In support of the role of Fc receptors in the treatment regimen, depletion of HSCs was not observed in mice lacking functional Fc receptors, nor in animals treated with high-doses F(ab) fragments of ACK2. Finally, high levels of granulocyte chimerism (~70%) were achieved post-BMT in mice receiving the combination of ACK2 and CV1 as compared to no chimerism in animals treated with ACK2 alone (p-value <0.0001). Our results show that targeted biologic agents have the potential to replace the toxic preconditioning therapies that are currently clinically utilized. This can lead to safer preparative regimens, which will allow BMT to treat a broader patient population and larger spectrum of hematologic disorders.

Disclosures

Ring: Stanford University: PCT/US2013/021937 Patents & Royalties. Weiskopf:Stanford University: PCT/US2013/021937 Patents & Royalties. Weissman:Stanford University: PCT/US2013/021937 Patents & Royalties.
Metabolic Activity Distinguish Reserve and Primed HSCs
Authors: Li Zhenrui , He Xi , Paulson Ariel , Zhao Meng , Qian Pengxu , Tao Fang , Ding Wenxing , Dobrowsky Rick , Li Linheng
Published: October 2019

A population of reserve HSCs has been reported to be in a deep-quiescent (or dormant) state and function as a back-up population of HSCs to support life-time hematopoiesis (Li and Clevers, 2010; Wilson et al., 2008). Currently, characterization of reserve HSCs is mainly based on cell cycle quiescence, however, the metabolic state in reserve HSCs is yet to be defined. Here, we show that reserve HSCs maintain not only a quiescent state but also an overall low metabolic activity whereas the primed HSCs maintain still a quiescent state but primed at metabolic state. First, we used CD49b(Benveniste et al., 2010) to further separate conventional long-term (LT) HSCs (CD34-Flk2-Lineage-Sca-1+c-Kit+)(Yang et al., 2005) into CD49blo and CD49bhi subpopulations and confirmed their enrichment with previously identified dormant (Scl-H2B-GFP label retaining cells, LRCs), thus we termed CD49blo and CD49bhi subpopulations as candidates of reserve and primed HSCs. We then determined the cell cycle frequencies of reserve, primed, and ST-HSCs (Cd34+Flk2-LSK) respectively as once in 3 months, 3-4 weeks, and 3-4 days. Functionally, Reserve HSCs had on average 3.5-fold higher functional capacity compared with primed HSCs. RNA-seq analysis revealed that reserve HSC predominantly expressed a list of imprinting genes that associate with growth-restriction functions; primed HSCs expressed relatively-high number of genes involving in mitochondria fusion, organization, and function, while ST-HSCs expressed genes reflecting an active cycling state. Conducting metabolic assays, we found that reserve HSCs not only maintain quiescence but also maintain overall low metabolic activity in both glycolysis and mitochondrial capacity. In contrast, primed HSCs are in a quiescent state, but with their metabolic state primed as evidenced by an increased glycolytic activity and mitochondrial potential for subsequent active proliferating state in ST-HSCs. Intriguingly, our data suggests that the functionality of reserve HSCs correlates to metabolic state rather than cell cycle.

Disclosures

No relevant conflicts of interest to declare.
T-Cell Receptor Engagement Confers Resistance to Chemotherapy in T-Cell Lymphoproliferative Disorders
Authors: Wang Tianjiao , Polk Avery , Lu Ye , Wilcox Ryan A
Published: October 2019

Background: Chemotherapy resistance and primary refractory disease are common among patients with the more common peripheral T-cell lymphomas (TCL) treated with anthracycline-based chemotherapy. Antigen-receptor signaling activates a number of proliferation and survival pathways in normal lymphocytes. When activated in lymphomas, antigen-receptor signaling might confer resistance to chemotherapy. Until recently, the role of antigen-receptor signaling in B-cell malignancies was poorly understood. It is now appreciated that B-cell receptor (BCR) signaling is an important driver of B-cell lymphoma growth and survival, and represents an attractive therapeutic target in these lymphomas. In contrast, the potential role of T-cell receptor (TCR) signaling in T-cell lymphomagenesis is poorly understood. The observation that most (≈95%) TCLs express an intact TCR suggests that malignant T cells, like their B-cell counterparts, may benefit from antigen-receptor signaling.

Methods: TCL cell lines and primary patient specimens were subjected to TCR activation by CD3/CD28 beads. Gene expression profiling was performed by Affymetrix human gene 2.1 ST array at different time points (4, 8, 24 hours) following TCR engagement. Results were further validated by immunophenotyping by flow cytometry, western blot and cytokine measurement by enzyme-linked immunosorbent assay (ELISA). The proliferation and chemoresistance of the TCL cells following TCR engagement were determined by MTT assay.

Results: Microarray gene expression profiling of TCLs showed differential gene expression upon TCR engagement by CD3/CD28 beads at different time points. A total of 1274 genes were differentially expressed commonly in the patient specimens. Some of the differentially expressed genes were confirmed in independent samples by immunophenotyping (CD25, CD30, CD69, CD137, CCR4, GITR) and cytokine (IL2, IL13, IL10, IFNγ) release. Gene enrichment analysis of the common differentially expressed genes showed that pro-proliferation and pro-survival signaling pathways, including TCR (FDR: 0.05%), MAPK (FDR: 2.8%), NFκB (FDR: 0.1%) and cytokines (FDR: 0.03%) were enriched in TCR-engaged TCL cells. The activation of TCR and NFκB signaling in TCR-engaged TCLs was also confirmed by western blot and flow cytometry. More importantly, the proliferation of TCLs increased by 2-4 fold when cocultured with CD3/CD28 beads for 72 hours (p<0.01). In the presence of either vincristine or romidepsin, the viability of TCLs increased by 1.5-4 fold by CD3/CD28 bead treatment (p<0.01). TCR-dependent proliferation and chemoresistance in TCLs was significantly inhibited by agents targeting TCR and NFκB signaling pathways.

Conclusions: T-cell receptor dependent signaling pathways are rational therapeutic targets in the T-cell lymphomas.

Disclosures

No relevant conflicts of interest to declare.