bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular and Molecular Architecture of Hematopoietic Stem Cells and Progenitors in Genetic Models of Bone Marrow Failure
Authors: Heidemann Stephanie C. , Bursic Brian , Zandi Sasan , Li Hongbing , Klaassen Robert J , Abish Sharon , Rayar Meera , Breakey Vicky R. , Moshiri Houtan , Dhanraj Santhosh , de Borja Richard , Shlien Adam , Dick John E. , Dror Yigal , Abelson Sagi
Published: November 2019

Inherited bone marrow failure syndromes (IBMFSs) such as Fanconi Anemia (FA) and Shwachman-Diamond syndrome (SDS) feature progressive cytopenia and a risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Using deep immunophenotypic analysis of hematopoietic stem cells (HSCs) and early progenitors in bone marrow samples from 13 FA/SDS patients without MDS/AML and 6 healthy controls, we revealed relative survival of progenitors that phenotypically resembled granulocyte-monocyte progenitors (GMP) in the patients. In contrast, HSCs, multipotent progenitors, common myeloid progenitors and megakaryocyte-erythroid progenitors were remarkably reduced in FA/SDS patients compared to controls. Whole exome and targeted sequencing of GMP-like cells in leukemia-free patients revealed a prominently higher mutation load in FA/SDS patient samples than in healthy controls. Patient samples also manifested molecular changes that are characteristic of AML: increased G>A/C>T variants, decreased A>G/T>C variants, increased trinucleotide mutations at Xp(C>T)pT, decreased lower mutation rates at Xp(C>T)pG sites compared to other Xp(C>T)pX sites and enrichment for cancer signature 1. AML cells have previously been reported as commonly harboring this cancer signature. Potential pre-leukemic targets in the GMP-like cells from FA/SDS patients included SYNE1, DST, HUWE1, LRP2, NOTCH2 and TP53. Serial analysis of GMPs from a SDS patient, who progressed to AML revealed a gradual increase in mutational burden, enrichment of G>A/C>T signature and emergence of new clones. Interestingly, the molecular signature of marrow cells from two FA/SDS patients with AML was similar to that of FA/SDS without transformation. The predicted founding clones in SDS-AML harbored mutations in several genes including TP53, while in FA-AML the mutated genes included ARID1B and SFPQ. In conclusion, we described an architectural change in the hematopoietic hierarchy of FA/SDS with remarkable preservation of GMP-like populations harboring unique mutation signatures. GMP-like cells might represent a cellular reservoir for clonal evolution.

Disclosures

No relevant conflicts of interest to declare.
Classification and Prognosis of Central Nervous System Involvement in Erdheim-Chester Disease
Authors: Cohen-Aubart Fleur , Idbaih Ahmed , Galanaud Damien , Law-Ye Bruno , Emile Jean-François , Charlotte Frédéric , Donadieu Jean , Amoura Zahir , Haroche Julien
Published: November 2019

Background: Erdheim-Chester disease (ECD) is a non-Langerhans cell histiocytosis, characterized by the infiltration of tissues by foamy CD68+CD1a- histiocytes, with 1500 known-cases since 90 years. Mutations activating the MAPK pathway are found in more than 80% of ECD patients, mainly the BRAFV600E in 57 to 70%. Central nervous system (CNS) involvement during ECD lead to significant morbidity and mortality. We assessed CNS manifestations in a French cohort of 253 ECD patients. Since 2012, close to 200 patients worldwide with multi-systemic and refractory ECD (e.g. heart and/or CNS involvements) have benefited from BRAF and MEK inhibitors, which have proven highly beneficial.

Methods: CNS manifestations were determined by clinical examination and brain and/or spine magnetic resonance imaging (MRI). Targeted therapy efficacy was assessed using physician and radiologist global assessment.

Results: Ninety-seven (38%) among the 253 of the whole cohort of ECD patients had CNS involvement. CNS involvement was significantly associated with a younger age at diagnosis (mean 55.5 years) and at symptoms onset (mean 50.5 years), and also with the presence of the BRAFV600E mutation (in 77% of cases), xanthelasma (34%), and diabetes insipidus (36%). Median survival among patients with CNS involvement was significantly lower than in ECD patients without CNS involvement (124 months versus 146 months, p=0.03). Seventy-four CNS MRI were centrally reviewed, corresponding to 3 patterns: tumoral in 66%, degenerative in 50%, and vascular in 18%. Targeted therapy (BRAF and/or MEK inhibitors) was associated with improvement of symptoms in 43% of patients, and MRI improvement in 45%.

Conclusions: CNS manifestations are typically associated with a poor prognosis in ECD. Three distinct patterns may be recognized: tumoral, degenerative, and vascular. Targeted therapy leads to clinical and/or imaging improvement in almost 50% of patients.

OffLabel Disclosure:

Targeted therapies such as BRAF inhibitor (vemurafenib) and MEK inhibitor (cobimetinib) are used in severe histiocytosis in EU but are off label
Perception of Dentists and Dental Hygienists in Saudi Arabia about Bleeding with Dental Procedures in Patients Using Antithrombotic Medications
Authors: Alsheef Mohammed Abdullah , Aljohany Sarah , Gray Jenny , Zaidi Abdul Rehman Z. , Kullab Ghaydaa Juma
Published: November 2019

Background:

Antithrombotic medications are frequently used for the prevention of certain conditions such as venous thromboembolism (VTE). A major complication of treatment with antithrombotic is the risk of hemorrhage, especially after specific dental procedures.

Objectives:

This study was conducted to measure the knowledge of dentists and hygienists in Saudi Arabia about treating patients who are receiving new oral anticoagulants (NOACs) and determining if dentists and hygienists are treating patients according to international guidelines.

Methods:

Study design: An English language questionnaire was piloted and then revised and distributed amongst various dental institutions within Riyadh, Saudi Arabia.

Study Subjects: 302 participants (Dentists 94.7% and dental hygienists 5.3%) working within Saudi Arabia, 58.3 were males, and 41.7% were females. 30.5% of the subjects' experience was between 5 and 10 years.

Inclusion criteria: Dentists and hygienists who are practicing in Saudi Arabia during the defined study period and willing to participate.

Results:

It appears that there is still much confusion over-treating patients on antithrombotic medications, not only with the NOACs (i.e. Rivaroxaban, Dabigatran, Apixaban) but even with the more established medications, such as Warfarin and Aspirin; only approximately 30% of respondents were able to correctly respond to the appropriate method of treating patients receiving these medications. A large number of respondents stated that they did not know how to treat or would not treat patients on these antithrombotic medications (29.8% - 89.2%) which is very concerning, considering the large number of patients who are receiving antithrombotic medications.

Conclusion:

The familiarity of dentists and dental hygienists with the NOACs is very poor compared to the more established antithrombotic. Therefore, dental professionals need to be made more aware of the NOACs in order to facilitate proper and timely management.

Figure

Disclosures

No relevant conflicts of interest to declare.
JAK2V617F Hematopoietic Stem and Progenitors Are More Resistant to Chronic Blockage of IL-10 Signaling in a Murine Model of Myeloproliferative Neoplasms
Authors: Lai Hew Yeng , Trieu Tiffany , Han Tiffany , Brooks Stefan , Craver Brianna , Ramanathan Gajalakshmi , Fleischman Angela G.
Published: November 2019

Rationale:

Chronic inflammation is a key feature of Myeloproliferative Neoplasm (MPN), inflammation drives symptom burden and disease progression. We have found that monocytes from MPN patients persistently produce Tumor Necrosis Factor-alpha (TNF) following Toll-like receptor stimulation due to dampened Interleukin 10 (IL-10) receptor signaling which usually serves to negatively regulate TNF production (Lai et al, 2019). Chronic inflammation leads to hematopoietic stem cell (HSC) exhaustion, and IL-10 plays an important role in HSC self-renewal (Kang et al, 2007).

JAK2V617Fknock-in HSC do not have a selective advantage over wild-type cells in competitive repopulation assays, this suggests that JAK2V617FHSC gain a selective advantage only under specific circumstances. Here, we investigate whether blockade of IL-10 signaling allows JAK2V617F HSC to gain a selective advantage over wild-type cells. We hypothesize that blocking IL-10 signaling impairs wild-type but not JAK2V617Fmutant HSC and thereby allowing JAK2 V617Fmutant cells to expand.

Results:

We used competitive transplantation to assess the impact of blocking IL-10 signaling on the selective advantage of JAK2V617Fversus wild-type HSC. We transplanted equal numbers of whole bone marrow cells from wild-type (CD45.1) and JAK2 V617F(CD45.2) mice into lethally irritated recipients (CD45.1/2). At day 60 post transplantation we injected mice intraperitoneally with 0.1mg IL-10R blocking antibody (n=5) or PBS (n=4) weekly. The relative contribution of JAK2V617Fcells to donor derived peripheral blood leukocytes (n=4) decreased over time (Figure 1A) in the PBS group. In contrast, the relative contribution of JAK2V617Fcells increased after 120 days in the IL-10R blocking antibody group (Figure 1A).

Mice were sacrificed at 186 days post-transplant to assess spleen size and quantify hematopoietic stem and progenitor cells. Mice treated with IL-10R blocking antibody had larger spleens than the PBS group (Figure 1B). The LKS (linnegc-kit+Sca-1+), which contains hematopoietic stem and progenitor cells, was expanded in the mice treated with IL-10R blocking antibody (Figure 1C). The relative contribution of JAK2V617Fcells in the LKS compartment (Figure 1D) as well as whole bone marrow (Figure 1E) was higher in the IL-10R blocking antibody group as compared to the untreated group. Bone marrow from these mice were used for secondary transplants to functionally assess HSC fitness, results are forthcoming.

Conclusions:

In vivo blockade of IL-10R signaling increases the competitive ability of JAK2V617Fmutant cells. Spleen size is also augmented with IL-10R blockade, likely a reflection of an increase JAK2V617Fmutant cell burden. This suggests that defects in IL-10 signaling may be contributing to expansion of the mutant cells.

Disclosures

Fleischman: incyte: Speakers Bureau.
Glucocorticoids Regulate the Splicing Factor MBNL1, a Potential Control Point for B-Cell Specification
Authors: Pufall Miles A. , Fang Mimi , Bradley Robert K. , Guo-Liang Chew , Lanier Ross , Tasian Sarah K.
Published: November 2019

Glucocorticoids, such as dexamethasone and prednisone, are mainstays in the treatment of lymphoid malignancies and autoimmune diseases. To understand the mechanism of how glucocorticoids induce cell death, we integrated a comprehensive, genome-wide shRNA screen with differential gene expression analysis in B-cell acute lymphoblastic leukemia (B-ALL). In addition to activating pro-apoptosis genes and repressing anti-apoptosis genes, we found that repression of B-cell development genes contributes to dexamethasone-induced cell death in B-ALL. Similarly, elevated glucocorticoid levels brought on by stress in stroke victims causes lymphocytopenia by inducing of apoptosis in immature B-cells, but not hemopoietic stem cells or mature B-cells. Additionally, chronic stress has been associated with immune suppression and increased risk for autoimmune disease. Together these findings suggest that glucocorticoids play a normal role in B-cell development, and that elevated glucocorticoid levels in response to stress can alter that development.

A control point in this mechanism may be MBNL1. MBNL1 is a sequence-specific RNA binding factor that alters mRNA splicing, stability, and localization. In this work, we show that glucocorticoid suppression of MBNL1 contributes to induction of cell death in B-ALL. Deletion of MBNL1 by CRISPR/Cas9 in the B-ALL cell line NALM-6 induces accumulation and depletion of mRNA levels for hundreds of genes. A large number of B-cell specification genes, including CD19, CD79A, SPI1, and LEF1, are repressed upon MBNL1 deletion, whereas the most highly activated gene is CD34, which is a marker for the less mature lymphoid progenitor cells. Depletion of MBNL1 also changed the splicing of B-cell development genes, including quantitative depletion of LEF1 exon 6. These data suggest that depletion of MBNL1 induces de-differentiation of B-cell precursors. Consistent with this model, an analysis of gene expression in differentiating B-cells revealed a rapid elevation of MBNL1 expression during B-cell specification (after the lymphoid precursor stage), and a concomitant repression of CELF2, an RNA-binding splicing factor that has been shown to oppose MBNL1. We therefore propose that glucocorticoids play a physiological role in B-cell development by restraining B-cell specification through repression of key genes, including MBNL1.

Figure

Disclosures

Tasian: Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corportation: Research Funding.
Carcinocythemia with Microfilaria: Two Diverse Pathologies in One Case
Authors: Samal Swagatika , Mishra Shruti , Mohamedali Rasheeda , Kumar Kishore
Published: November 2019

Introduction:

Carcinocythemia means circulating tumor cells in peripheral blood. It is a very rare finding I routine practice. The first ever case was reported in 1869 by Ashworth, but it was not till 1976 that the terminology carcinocythemia was coined by Cary et al [1,2]. Only a handful of cases have been reported in literature [3]. We present one such rare case which had an additional finding of micro-filaria in it.

Case Report:

A 60-year-old male patient, hailing from Eastern Ghats of India, presented to the emergency with sudden onset paresis of bilateral lower limbs, hemoptysis, abdominal distention, and urine retention, constipation and persistent chest pain. On evaluation the power of bilateral lower limb was ¼.

He was a known case of Stage IV adenocarcinoma of lungs with bilateral adrenal metastasis. He had received four cycles of PEM + Carboplatin followed by 3 cycles of Carboplatin and Paclitaxel. Last dose of chemotherapy was administered fifteen days prior to presentation.

The patient was not responding to chemotherapy.

He was receiving blood transfusion.

The peripheral smear of the patient revealed circulating tumor cells along with micro-filaria. Cell block was prepared from the sample but unfortunately did not reveal much for special staining. The species of filaria was that of Wuchereria bankrofti. Interestingly, the patient lacked eosinophilia. The total count was 11,000/cumm; Hemoglobin: 8.2gm/dl and Platelet count was 2,10,000/cumm. The counter had flagged for atypical lymphocytes.

The patient deteriorated fast and expired within few hours of presentation. Thus, further evaluation was not possible.

Conclusion:

Carcinocythemia is a rare phenomenon but as a hemato-pathologist vigilance is mandated while evaluating peripheral blood smears to detect such extra-ordinary findings.

References:

1. Ashworth T. A case of cancer in which cells similar to those in the tumors were seen in the blood after death. Aust Med J. 1869;14:146-149.

2. Carey RW, Taft PD, Bennett JM, et al. Carcinocythemia (carcinoma cell leukemia). An acute leukemia-like picture due to metastatic car- cinoma cells. Am J Med. 1976;60(2):273-278.

3. Ronen S., Kroft S.H.,Olteanu H., Hosking P.R.,Harrington A.M.Carcinocythemia: A rare entity becoming more common? A 3-year, single institution series of seven cases and literature review. Int J Lab Hematol. 2019;41:69-79.

Figure

Disclosures

No relevant conflicts of interest to declare.
Near Universal Underestimation of the Risk of Recurrent Venous Thromboembolism (VTE)- an Institutional Survey of Thrombophilia
Authors: Pawar Tushar , Cuker Adam , Garrahy Ian , Donato Anthony , Stevenson Amber , Forman Daniel
Published: November 2019

Introduction: Current guidelines suggest consideration of indefinite anticoagulation for patients with unprovoked proximal deep vein thrombosis (DVT) and pulmonary embolism at low to intermediate risk for bleeding. In clinical practice, many patients are not treated with prolonged anticoagulation placing them at an increased risk for recurrent VTE. An underappreciation of the risk of recurrent thrombosis may result in the premature discontinuation of anticoagulation in some high-risk patients.

Methods: An online, anonymous questionnaire was sent to all physicians and advanced practice providers in a 711-bed academic community hospital inquiring about their practice, specialty, medical degree, years in practice, and basic questions regarding their comfort and frequency of prescribing anticoagulants. The questionnaire also asked the providers how they would manage ten various clinical scenarios related to anticoagulation management. The participants were graded and scores were calculated based upon percent correct. One question asked the clinicians to estimate the five-year risk of recurrent thrombosis off anticoagulation in a 70-year-old male after completing one year of therapeutic anticoagulation for an unprovoked proximal DVT. The choices given ranged from 1% to 30%. Data was de-identified, SPSS was used to perform t-test and descriptive analysis.

Results: Only 63 (15%) out of 420 respondents appreciated that there is approximately a 30% risk for recurrent VTE after stopping anticoagulation. A slight majority felt that the risk was 5% or less (55.9%). Approximately one-third (29.1%) estimated the risk to be 15%. In the context of practice setting, 22 (37%) from 109 (27%) in-patient providers answered the question correctly, compared to 11 (18%) from 97 (24%) of out-patient providers. In terms of experience, providers with an experience of >10 years (30, 50% correct from 162, 40% responses) did better compared to providers with <10 years of experience (30, 50% correct form 242, 60% responses). t-test results show that providers who answered correctly (30% recurrence), have significant better scores (mean 70.50), compared to those who answered it wrong (mean 57.01), with p-value<0.001.

Conclusion: Underestimation of the risk of recurrent VTE was pervasive in a large, academic healthcare system. It seems likely that this knowledge gap exists outside our institution, and it very likely contributes to premature termination of anticoagulation in some high-risk patients. Quality improvement efforts are needed to educate providers about VTE recurrence risk.

Disclosures

No relevant conflicts of interest to declare.
CNS Prophylaxis in Diffuse Large B-Cell Lymphoma & Its Outcomes- an Institutional Review
Authors: Vaddepally Raju , Adhami Angie , Siedlecki Charles , Kuriakose Philip
Published: November 2019

Background: Diffuse large B-cell lymphoma (DLBCL) has an approximately 5% risk of recurrence in the central nervous system, and carries a very poor prognosis. Prophylactic intrathecal chemotherapy (ITC) with agents such as Methotrexate (MTX) can mitigate the risk of recurrence and improve survival. We report a single-institution, retrospective analysis of the effect of ITC administration on outcomes in patients with DLBCL, based on CNS-IPI risk stratification.

Methods: We screened 325 DLBCL patients from our tumor registry- of these only 292 had clinical information to risk-stratify per IPI scoring- low-risk, n = 54, intermediate-risk, n = 198, and high-risk n = 40. These patients then further categorized based on the NCCN prognostic model to assess the risk of CNS disease (CNS-IPI) which includes age >60, LDH >normal, ECOG >1, Stage III or IV, Extranodal involvement >1 site, kidney or adrenal gland involvement with scores 0-1 low-risk, 2-3 intermediate-risk, and 4-6 or kidney or adrenal involvement as high-risk. The effect of ITC administration on outcomes such as overall survival (OS) at 1-year (yr) and 5-yr were calculated using univariate two-group comparisons.

Results: Demographic and clinical variables include- Age >60- 61% (n=201); mostly Caucasians- 71%(n=199); Germinal Center(GC) 25%(n=73), non-GC 74%(n=212); mean IPI score-2.8(n=196), R-CHOP-87%(n=233); and, ITC in 15%(n=45). Seven patients in low-risk patients received ITC for unclear reasons. Intermediate risk patients treated with ITC showed a trend towards 90% (n=26) 1yr OS, and 67%(n=8) 5-yr survival compared to who did not receive ITC (non-ITC) who had 84%(n=127) 1-yr, and 55%(n=52) 5-yr OS (p-value 0.57 and 0.43 respectively). High-risk patients treated with ITC showed a trend towards 100%(n=3) 1-yr, and 0% 5-yr OS compared to non-ITC who had 85%(n=23) 1-yr , and 29%(n=5) 5-yr OS (p-value 1.0 and 1.0 respectively).

Conclusions: Our results suggest that in DLBCL patients with the intermediate-risk group may benefit from intrathecal chemotherapy. Our study is limited owing to small sample size in each subset of CNS risk stratification- this limits the interpretation of the results, and should be interpreted cautiously given nonsignificant P-values. However, we believe these results can encourage further research to evaluate the effect of CNS prophylaxis in the intermediate-risk group of DLBCL patients.

Disclosures

Kuriakose: Alexion: Consultancy, Honoraria, Speakers Bureau; Bayer: Consultancy.
Astragalus Polysaccharide Promotes Hematopoiesis in an Irradiated Mouse Model and Reduces Apoptosis of Hematopoietic Cells
Authors: Xu Wan-huang , Li Liang , Yang Liuming , Xin Hongwu , Chong Beng H , Yang Mo
Published: November 2019

Background: Huangqi (the dried root of Radix Astragali) is a notable Chinese herb that has been used to promote hematopoiesis for centuries. Astragalus polysaccharide (ASPS), a bioactive constituent extracted from Radix Astragali, may play an essential role for hematopoiesis. The objective of this study is to investigate the hematopoietic effects of ASPS in mouse models and explore underlying mechanism, which provides scientific basis for future applications.

Methods: An irradiation-induced myelosuppressive mouse model and colony-forming unit (CFU) assays were used to determine the in vivo hematopoietic effects of ASPS. The anti-apoptotic effects of ASPS were also examined by using annexin V, caspase-3, and JC-1 assays with flow cytometry in HL-60 cells.

Results: In the myelosuppressed mouse model, ASPS enhanced the recovery of platelet and red blood cell at day 21, and white blood cell at day 14. ASPS also promoted the CFU formation of all lineages (MK, GM, E, GEMM and F). Morphological study of bone marrow revealed that as compared to the control group, tri-lineage hematopoiesis was preserved in the ASPS- and TPO-treated group. Overall cellularity (MTC/area) of ASPS-treated group was similar to that of TPO. Furthermore, in-vitro study showed that 100 μg/ml of ASPS had maximum effect on CFU formation. ASPS also reduced apoptosis via inhibiting mitochondrial/Caspase-3 signal pathway.

Conclusions: ASPS may promote hematopoiesis in irradiated myelosuppressive mice by enhancing hematopoietic proliferation and inhibiting apoptosis of hematopoietic cells.

Disclosures

No relevant conflicts of interest to declare.
Fibg390-396A Limits Metastatic Potential Via a Mechanism Independent of Fibrin-Myeloid Cell Interactions, but Prospectively through the Disruption of FXIII-Mediated Fibrin Crosslinking
Authors: Francisco Brenton J , Sharma Balkrishan , Rosenfeldt Leah A. , Mureb Duaa , Palumbo Joseph S.
Published: November 2019

Previous studies have shown that fibrin(ogen) supports metastasis. In order to better define the mechanisms coupling fibrin(ogen) to metastasis, we compared metastatic potential in immunocompetent mice carrying specific structure/function alterations in fibrinogen and controls. FibrinogenAEK mice, which have a germ-line mutation in the thrombin cleavage site that essentially "locks" fibrinogen in its soluble state, exhibited diminished metastatic potential relative to FibWT mice. Notably, previous studies have established that factor XIII is also a significant determinant of metastasis. Taken together, these studies suggest that stable fibrin polymer formation is important in the metastatic process. However, FibAEK retained significant metastatic potential relative to mice with complete fibrinogen deficiency, indicating that fibrinogen possesses prometastatic properties in the absence of polymer formation.

Fibrin(ogen) has also been shown to mediate inflammatory cell functions through direct engagement of leukocyte integrins independently of its role in platelet aggregation. Given previous studies demonstrating that myeloid cells are promote metastatic potential, we hypothesized that fibrin(ogen)-mediated leukocyte engagement represents one mechanism by which fibrinogen drives metastasis. Consistent with this view, mice expressing a mutant fibrinogen lacking the y chain binding motif for the leukocyte integrin αMβ2 (Fibγ390-396A) had diminished metastatic potential in both spontaneous and experimental metastasis assays. Furthermore, fate analyses revealed that Fibγ390-396A results in diminished survival of newly-formed metastatic foci. The early reduction in metastatic potential observed in Fibγ390-396A mice suggests that fibrin(ogen) promotes metastasis by recruiting myeloid cells to the early metastatic niche. In order to explore this hypothesis, we performed experimental metastasis assays in immunocompetent mice in which macrophages or neutrophils were specifically depleted using either clodronate or an anti-Ly6G antibody. In contrast to mice carrying the Fibγ390-396A mutation, depletion of macrophages or neutrophils had no significant impact on the early survival of metastatic foci. These studies suggest that expression Fibγ390-396A limits metastatic potential via a mechanism independent of interactions with myeloid cells.

In addition to disrupting fibrin-αMβ2 interactions, elimination of the γ chain 390-396a binding motif has also been shown to limit factor XIII binding to fibrinogen. Given the established importance of FXIII in metastasis, it is conceivable that even subtle alterations in the kinetics of fibrin cross-linking resulting from expression of Fibγ390-396A limits metastatic potential.

Disclosures

Palumbo: Ionis Pharmaceuticals: Research Funding.
Autologous Stem Cell Transplantation Outcomes in Patients with Myeloma and Renal Dysfunction
Authors: Monge Jorge , Jayabalan David S , Niesvizky Ruben , Coleman Morton , Flicker Kari , Solomon Robin S. , Rossi Adriana C. , Guo Jin
Published: November 2019

Background

Renal impairment (RI) is a common complication and a negative prognostic factor in patients with Multiple Myeloma (MM). Patients with severe renal dysfunction may not benefit from optimal therapy as they are often excluded from autologous stem cell transplantation (ASCT). The aim of this study was to analyze the outcome of MM patients with prior RI undergoing ASCT and to evaluate the effect of transplant on renal and hematologic response.

Methods

From 147 eligible MM patients who underwent ASCT between 2002 and 2019, 131 were included in this single-center retrospective cohort study. Renal function (eGFR in ml/min/1.73m2 by MDRD) was evaluated at the time of MM diagnosis and prior to ASCT, as well as 30 and 100 days after ASCT. Patients were classified by renal function: A) eGFR <30; B) eGFR 30-59; or C) eGFR >60 prior to ASCT. ANOVA was used for comparison of renal function at diagnosis, at transplant, 30 days post ASCT, and 100 days post ASCT. Log rank testing was used for overall survival (OS) analysis of group A, B and C

Results:

Patients with renal dysfunction at baseline had lower ORR than controls to induction therapy, 71 v 78%. ASCT provided deepening responses to both groups, ORR 83 v 86% respectively. No renal responses were seen with ASCT, whereas 16% of patients with renal dysfunction at diagnosis had renal response prior to transplant. Of note, only 51% of patients with renal dysfunction received conditioning with Mel200, compared with 94% of control group. Overall survival benefit is seen for patients with eGFR>60 over any level of dysfunction (p = 0.0079).

Conclusion

Myeloma patients with renal dysfunction are a high risk group, and require special attention. Rapid and effective induction therapy remains an important step in reversing renal damage. In patients who do not recover kidney function, autologous stem cell transplantation should be considered, providing depth of response benefit. The conditioning regimen used for these patients warrants further investigation.

Figure

Disclosures

Niesvizky: Takeda, Amgen, BMS, Janssen, Celgene: Consultancy, Research Funding. Coleman:Gilead, Bayer, Celgene: Consultancy, Research Funding, Speakers Bureau; Merck: Research Funding; Pharmacyclics: Speakers Bureau; Kite Pharmaceuticals: Equity Ownership. Rossi:BMS: Research Funding; Janssen, Celgene, Amgen: Consultancy.
Absence of Damaging Effects of Stem Cell Donation in Unrelated Donors Assessed By FISH and Gene Variance Screening
Authors: Nacheva Elisabeth P. , Ahyee Trudy , Navarette C , Addada Juanah , Pawson Rachel , Syzdlo Richard , Mackinnon Stephen , Shaw Bronwen E. , Madrigal J. Alejandro
Published: November 2019

Granulocyte-Colony-Stimulating factor (G-CSF) is currently the standard mobilizing agent for peripheral blood stem cell (PBSC) donation. While G-CSF use is associated with mild transient side-effects, concerns that it may trigger hematological malignancy were raised by a study of PBSC donors found to display epigenetic and genetic changes similar to those observed in leukemia patients. Although several studies have failed to reproduce the original data, possible long-term effects of G-CSF mobilization on chromosome integrity remain unclear. In the setting of a multi-center clinical trial we screened blood samples from PBSC donors for aberrations at cellular and gene level that are common in hematological malignancies using fluorescence in situ hybridization (FISH) and next generation sequencing (NGS) assays. Samples collected before, on the day of donation, 90 and 180 days after G-CSF admission confirmed the absence of short-term effects in 50 PBSC donors on both quiescent and dividing cells. The data obtained from interphase cells and chromosome preparations from 50 individuals tested 3 to 5 years after PBSC donation did not differ either from individuals post bone marrow (BM) donation over a similar time period or healthy persons. In an attempt to finally resolve the residual uncertainty surrounding the effects of the G-CSF administration we used next generation sequencing to search for cryptic genome aberrations that may remain undetected by FISH screening. The gene profile of samples from 47 PBSC donors assessed using a panel targeting 54 genes (TruSight Myeloid panel, Illumina) was found to be stable throughout the study period. These data conclusively demonstrate the absence of any detrimental effects on the genome integrity of lymphocytes caused by PBSC or BM donation.

Disclosures

Shaw: Therakos: Other: Speaker Engagement.
Enabling Gene-Edited, Regulatory-like, T Cells (edTreg) for Treatment of IPEX and Other Autoimmune Disorders
Authors: Honaker Yuchi , Sommer Karen , Dahl Noelle , Xiang Yufei , Lopez Christina , Scharffenberger Samuel , Christian Michelle , Torgerson Troy R. , Scharenberg Andrew M. , Rawlings David J.
Published: November 2019

IPEX (immunedysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome is a severe congenital autoimmune disorder in males resulting from hemizygous inheritance of a mutant FOXP3 allele. FOXP3 encodes a transcription factor that governs the development, maintenance, and function of regulatory T cells (Treg). We have developed a cell therapy strategy for treatment of IPEX using a gene-editing approach in which CRISPR/Cas9 RNPs are co-delivered with an AAV6 donor template designed to integrate into the FOXP3 locus an expression cassette containing the MND promoter driving expression of a functional FOXP3 cDNA and a surface LNGFR tag linked by a 2A ribosomal skip peptide. This approach enforces heterologous FOXP3 expression in IPEX CD4 effector T cells (Teff), while simultaneously eliminating expression of the endogenous FOXP3 allele. The resultant high level and stable expression of functional FOXP3 converts Teff to Treg-like cells with immunosuppressive activity. Using an optimized protocol, we obtained efficient HDR rates across multiple healthy donors. Edited cells were consistently enriched to >95% purity by a magnetic LNGFR antibody selection and expanded 50-fold in a week. Expression of FOXP3 cDNA in edited cells was sufficient to enforce Treg-like phenotypes including the up-regulation of Treg-associated markers (CD25, CTLA-4, and ICOS), and down-regulation of CD127 and inflammatory cytokines (IL2, IFNgamma, TNFalpha). Importantly, we demonstrate sustained in vivo suppressive activity of these edited Treg-like cells (edTreg) in a xeno-GvHD mouse model. edTreg (as well as expanded natural Treg) limited effector T cell expansion and tissue infiltration and significantly protected mice from xeno-GvHD induced by co-transferred autologous effector T cells. Along with preliminary data showing successful editing in CD4 T cells from IPEX patients, our data provide key pre-clinical proof-of-concept and safety data supporting use of edTreg in a clinical trial for IPEX and, potentially, for use in other autoimmune diseases.

Disclosures

Torgerson: Shire: Consultancy; CSL Behring: Consultancy; ADMA Biosciences: Consultancy; UCB: Consultancy. Scharenberg:Casebia Therapeutics LLc: Employment, Equity Ownership; Alpine Biosciences: Consultancy, Equity Ownership; Generation Bio: Equity Ownership.
Feasibility of Peripheral Blood Stem Cell Collection from Heterozygoussickle Cell Trait Donors
Authors: Foell Juergen , Morez Morad , Troeger Anja , Kleinschmidt Katharina , Pfirstinger Beatrix , Brosig Andreas , Burkhardt Ralph , Corbacioglu Selim , Ahrens Norbert
Published: November 2019

Introduction

Allogeneic haematopoietic stem cell transplantation (HSCT) is currently the only curative treatment for sickle cell disease (SCD) offered to patients with a fully matched sibling (MSD) or matched unrelated donor (MUD). With a MSD/MUD donor availability of below 20% a haploidentical HSCT from a family donor either with post-transplant cyclophosphamide (post-cy) or with a TCRαß/CD19 depleted graft (T-haplo) is an increasingly successful alternative for the majority of patients. In contrast to post-cy where mostly bone marrow is used, T-haploHSCT requires the generation of a G-CSF stimulated peripheral stem cell grafts. Almost uniformly haploidentical relatives of SCD patients are heterozygous carriers of SC trait. For these donors with a genetically heterozygous sickle trait, G-SCF represents a major safety concern with regard to triggering a sickle cell crisis. Therefore, the use of sickle trait donors is prohibited or not recommended in several countries, and guidelines are currently not available due to lack of evidence.

Methods

Haploidentical related donors with heterozygousSCD (group S, n=13) as well as healthy donors (group O, n=9) were stimulated with G-CSF. In all donors a similar mobilizations schedule using G-CSF was applied. All donors were applied 10µg/kg body weight (Neupogen®or Granocyte®) sub cutaneous daily with first apheresis on day 5 and day 6 when necessary. The amount of mobilized CD34+ cells were assessed after mobilization. In addition, the harvested stem cell preparations were analysed for CD34 content after apheresis and patients monitored for potential serious adverse effects (AE) during and after the apheresis.

Results

Mobilization was tolerated well with only mild and typical AE related to apheresis procedure such as citrate reactions, joint pain, ostealgia and headache, observed equally in both groups. In most of the donors (n=13/13 and 8/9), we were able to collect a sufficient amount of CD34+peripheral stem cells (mean of 16.3 and 8.2 x 10^6/kg in group S and O, respectively). We observed slight increases in LDH and reticulocyte counts in few donors also without significant differences between both groups. In particular, there were no severe neurologic side effects and no problems like sickle cell crisis despite mean pre-apheresis hemoglobin concentrations of 14.1 (range 11.9-17.5) g/dL in group S.

Conclusions

Collectively, these data indicate that in the absence of a MSD or MUD donor, haploidentical family donors with heterozygoussickle cell trait provide a safe and effective alternative for collection of peripheral CD34+stem cells for a T-haplo HSCT.

Disclosures

No relevant conflicts of interest to declare.
Megakaryocyte Ablation Ameliorates Bone Marrow Fibrosis By Reducing Myofibroblast Differentiation in a Mouse Model of Primary Myelofibrosis
Authors: Chung Yutein , Jin Huiyan , Trasorras Claire , Gonzalez Francina , Min Sang Hee
Published: November 2019

Primary Myelofibrosis (PMF) is a chronic myeloproliferative disorder characterized by excessive bone marrow (BM) fibrosis that can lead to ineffective hematopoiesis and reduced survival. PMF patients develop several megakaryocyte (MK) abnormalities including increased proliferation, abnormal morphology and upregulated expression of pro-fibrotic genes. Although several studies support the role of MK in BM fibrosis, the significance of MK in the development of BM fibrosis has not been demonstrated in vivo.

Here, we investigated the in vivo role of MK in the pathogenesis of BM fibrosis. First, we reproduced an established bone marrow transplantation (BMT) mouse model of PMF. To achieve this, lethally irradiated wild-type mice were transplanted with wild-type hematopoietic stem cells (HSC) transduced with either MPLW515L gene construct (MPLW515L BMT) or MIGR1 empty vector (MIGR1 BMT). Consistent with previous studies, starting at day 14 post-BMT, MPLW515L BMT mice gradually developed increased number of leukocytes and platelets, increased proliferation of abnormal megakaryocytes in the BM and spleen, hepatosplenomegaly with extra-medullary hematopoiesis, and reticulin fibrosis in the BM. Notably, robust fibrosis was also seen in the spleen and liver.

Next, to study the significance of MK in the development of BM fibrosis, we developed a MPLW515L BMT mouse model in which MK lineage could be selectively ablated. We accomplished this using PF4-Cre inducible diphtheria toxin receptor transgenic mice (iDTR+/-/PF4-Cre) as BMT donors. HSCs from iDTR+/-/PF4-Cre mice were transduced with MPLW515L or MIGR1 empty vector and transplanted as described above. Beginning day 14 post-BMT, recipient mice were injected with diphtheria toxin (DT) or water every 48 hours. At day 20 and day 29, DT injection significantly depleted MK in both MPLW515L BMT and MIGR1 BMT groups compared to water injection. Importantly, DT-injected MPLW515L BMT mice displayed attenuated fibrosis in the BM, spleen, and liver compared to water-injected MPLW515L BMT mice. In addition, DT treatment decreased the level of alpha smooth muscle actin in both BM and spleen of MPLW515L BMT mice, which suggests that MKs are critical for myofibroblast differentiation. MK ablation did not leukocytosis, thrombocytosis or hepatosplenomegaly. Together, our study show that successful MK ablation in vivo reduces fibrosis development in the BM, spleen, and liver of the MPLW515L mouse model of PMF.

In summary, these results support the essential role of MK in the pathogenesis of BM fibrosis in PMF. Further studies are underway to elucidate the mechanisms by which MK contribute to fibrosis in PMF.

Disclosures

No relevant conflicts of interest to declare.
Sequential CD19-22 CAR T therapy induces sustained remission in children with r/r B-ALL
Authors: Pan Jing , Zuo Shiyu , Deng Biping , Xu Xiuwen , Li Chuo , Zheng Qinlong , Ling Zhuojun , Song Weiliang , Xu Jinlong , Duan Jiajia , Wang Zelin , Yu Xinjian , Chang Alex H. , Feng Xiaoming , Tong Chunrong
Published: November 2019

Pan and colleagues report one of the first prospective evaluations of planned sequential chimeric antigen receptor (CAR) T-cell therapy targeting CD19 and then CD22 in a phase 1 trial, indicating acceptable toxicity and encouraging durable efficacy in pediatric patients with relapsed/refractory (r/r) acute lymphoblastic leukemia (ALL).
Bolder guppies do not have more mating partners, yet sire more offspring
Authors: Herdegen-Radwan Magdalena
Published: November 2019

Background

Intra-individual stable but inter-individually variable behaviours, i.e. personalities, are commonly reported across diverse animal groups, yet the reasons for their maintenance remain controversial. Therefore, studying fitness consequences of personality traits is necessary to discriminate between alternative explanations.

Results

Here, I measured boldness, a highly repeatable personality trait, and reproductive success in male guppies, Poecilia reticulata. I found that bolder males had higher reproductive success than their shyer conspecifics and they sired offspring with females who had larger clutches.

Conclusions

This result provides direct evidence for fitness consequences of boldness in the guppy. It suggests that the effect may be driven by bolder males mating with more fecund females.
MicroRNA-124-3p suppresses mouse lip mesenchymal cell proliferation through the regulation of genes associated with cleft lip in the mouse
Authors: Suzuki Akiko , Yoshioka Hiroki , Summakia Dima , Desai Neha G. , Jun Goo , Jia Peilin , Loose David S. , Ogata Kenichi , Gajera Mona V. , Zhao Zhongming , Iwata Junichi
Published: November 2019

Background

Cleft lip (CL), one of the most common congenital birth defects, shows considerable geographic and ethnic variation, with contribution of both genetic and environmental factors. Mouse genetic studies have identified several CL-associated genes. However, it remains elusive how these CL-associated genes are regulated and involved in CL. Environmental factors may regulate these genes at the post-transcriptional level through the regulation of non-coding microRNAs (miRNAs). In this study, we sought to identify miRNAs associated with CL in mice.

Results

Through a systematic literature review and a Mouse Genome Informatics (MGI) database search, we identified 55 genes that were associated with CL in mice. Subsequent bioinformatic analysis of these genes predicted that a total of 33 miRNAs target multiple CL-associated genes, with 20 CL-associated genes being potentially regulated by multiple miRNAs. To experimentally validate miRNA function in cell proliferation, we conducted cell proliferation/viability assays for the selected five candidate miRNAs (miR-124-3p, let-7a-5p, let-7b-5p, let-7c-5p, and let-7d-5p). Overexpression of miR-124-3p, but not of the others, inhibited cell proliferation through suppression of CL-associated genes in cultured mouse embryonic lip mesenchymal cells (MELM cells) isolated from the developing mouse lip region. By contrast, miR-124-3p knockdown had no effect on MELM cell proliferation. This miRNA-gene regulatory mechanism was mostly conserved in O9–1 cells, an established cranial neural crest cell line. Expression of miR-124-3p was low in the maxillary processes at E10.5, when lip mesenchymal cells proliferate, whereas it was greatly increased at later developmental stages, suggesting that miR-124-3p expression is suppressed during the proliferation phase in normal palate development.

Conclusions

Our findings indicate that upregulated miR-124-3p inhibits cell proliferation in cultured lip cells through suppression of CL-associated genes. These results will have a significant impact, not only on our knowledge about lip morphogenesis, but also on the development of clinical approaches for the diagnosis and prevention of CL.
Tuberculosis outbreaks among students in mainland China: a systematic review and meta-analysis
Authors: Bao Hongdan , Liu Kui , Wu Zikang , Wang Xiaomeng , Chai Chengliang , He Tieniu , Wang Wei , Wang Fei , Peng Ying , Chen Bin , Jiang Jianmin
Published: November 2019

Background

In recent years, tuberculosis outbreaks in schools have occurred more frequently in China than in other parts of the world, and have posed a public health threat to students and their families. This systematic review aimed to understand the epidemiological characteristics of tuberculosis (TB) outbreaks and analyze the factors associated with TB outbreaks in schools in China.

Methods

We conducted this systematic review following the standard procedures of the Cochrane Collaboration and the Preferred Reporting Items for Systematic Review and Meta-Analysis statement. The meta-analysis was performed with STATA using a random effects model.

Results

We included 107 studies involving 1795 student patients with TB in mainland China. The results of the systematic analysis indicated that TB outbreaks were more frequently reported in senior middle schools and in Eastern China. The outbreaks mainly occurred during the winter and spring, and the median outbreak duration was 4 months. The meta-analysis showed that the total attack rate and the class attack rate of tuberculosis outbreaks among students were 4.60% (95% CI 3.80 to 5.70%) and 22.70% (95% CI 19.20 to 27.00%), respectively. Subgroup analysis showed that outbreaks that occurred at universities or colleges had a relatively higher attack rate than those occurred in senior middle schools. The prevalence of latent tuberculosis infection (LTBI) among close contacts was 23.70% (95% CI 19.50 to 28.90%). The median case-finding interval was 2 months, and 47.40% of the index cases had a case-finding delay.

Conclusion

The results of our review indicated that school TB outbreaks were reported most frequently in senior middle schools in China. The attack rates of outbreaks at universities or colleges were higher than those in senior middle schools. The TB outbreaks in schools usually occurred over prolonged periods. The case-finding delay in the index cases must be reduced to prevent transmission in classes and schools. Effective surveillance and screening of presumptive TB cases in schools should be strengthened to reduce outbreaks in schools.