bluuebunny/crossref_metadata_2025 huggingface dataset:
The Survival of Transplant Ineligible Multiple Myeloma Patients Treated with Bortezomib-Based Chemotherapy Is Affected By the Patient Fitness
Authors: Lee Ji Hyun , Kim Sung-Hyun , Huh Seok Jae , Jo Jae-Cheol , Shin Ho-Jin , Lee Won-Sik , Lee Yoojin , Lee Ho Sup
Published: October 2019

Background

Frail patients who are in a poor perfomance status and have serious comorbidities or abnormal laboratory results comprise about one-third of the transplant ineligible multiple myeloma (MM). These patients, most of the cases, are excluded from the clinical studies. By far, the outcome of the real world data according to the patients' fitness in transplant ineligible MM patients is limited.

Method

Four hundred and fifteen patients with MM who have been treated with bortezomib, melphalan and prednisone (VMP) as a first-line treatment were retrospectively analyzed. 4 factors included into the frailty scores were Age, ECOG PS (Eastern cooperative group performance status), eGFR (estimated glomerular filtration rate), and number of comorbidities (heart disease, lung disease, liver disease, cerebrovascular disease, diabetes mellitus). These factors were di- or trichotomized by the reference of previously published papers and the meaningful cut-offs in our data. The points given to each variables were after the following criteria: Age < 70 (point 0), 70-79 (point 1), ≥ 80 (point 2); ECOG PS 0-2 (point 0), 3-4 (point 2); eGFR ≥ 60 mL/min/1.73 m2 (point 0), < 60 mL/min/1.73 m2 (point 1); comorbidity 0-1 (point 0) and ≥ 2 (point 1). Patients were further classified by frailty scores: fit (points 0-2), unfit (points 3-5) and frail (point 6).

Result

The median follow-up duration was 26.72 months (range 0.37 - 180.20 months). All of the factors including the Age ≥ 80, ECOG PS 3-4, eGFR < 60 mL/min/1.73 m2, and comorbidity ≥ 2 statistically significant for overall survival (OS) by univariate analysis. In multivariate analysis, ECOG PS 3-4 and eGFR < 60 mL/min/1.73 m2 remained statistically significant for OS and Age ≥ 80 was marginally significant for OS (Table 1). The response rates by the fit, unfit, and frail were as follows: 77.5% (148/191), 71.4% (55/77), and 0% (0/2) (P= 0.028). The 3-year overall survival rates by the frailty were 66%, 41%, and 0% in the fit, unfit or frail patients respectively and showed statistically significant difference (Figure 1 and Table 2, P= 0.000). The comparison of the frailty score with international staging system (ISS) and revised ISS (R-ISS) revealed statistical significance of all of the prognostic systems affecting OS by the univariate analyses but R-ISS and frailty score remained significantly affecting OS.

Conclusion

Unfit and frail patients showed a significant decrease in OS compared to fit patients. But there was no difference between the unfit and fit patients in terms of response rate in patients with transplant ineligible MM patients who have received bortezomib-containing regimen. Treatment of the unfit and frail patients needs more caution on toxicity management and appropriate dosing schedules to improve the survival outcome.

Disclosures

No relevant conflicts of interest to declare.
Domain-Focused CRISPR-Cas9 Screen Identifies the E3 Ubiquitin Ligase Substrate Adaptor Protein SPOP as a Novel Repressor of Fetal Hemoglobin
Authors: Lan Xianjiang , Khandros Eugene , Grevet Jeremy D. , Peslak Scott A. , Bhardwaj Saurabh , Keller Cheryl A. , Giardine Belinda , Garcia Ben A. , Hardison Ross C. , Shi Junwei , Blobel Gerd A.
Published: October 2019

Elevated levels of fetal hemoglobin (HbF) are beneficial to patients with sickle cell disease (SCD) and b-thalassemia. The identification of pharmacologically controllable pathways that regulate HbF levels remains an important goal in the treatment of these disorders. BCL11A and LRF (ZBTB7A) are the two major transcriptional repressors of g-globin (fetal beta-type globin). However, since transcription factors are challenging to inhibit with small molecules, we aimed to identify novel HbF regulators that might be amenable to control by pharmacologic means.

Using an improved protein-domain based CRISPR-Cas9 screen recently developed in our laboratory (Grevet et al., Science 361:6399, 285-290), we screened for new HbF regulators using a gRNAs library targeting chromatin associated proteins including BTB domain containing factors. We identified SPOP, a substrate adaptor of the CUL3 ubiquitin ligase complex, as a novel inhibitor of g-globin expression. Depletion of SPOP strongly elevated the mRNA and protein levels of g-globin in both HUDEP-2 and primary human CD34+ derived erythroid cells. The effects of SPOP loss were remarkably selective as evidenced by RNA-seq and whole cell mass spectrometry. Importantly, SPOP depletion did not appear to impair cell growth or differentiation. Mechanistically, we demonstrated that the repressive role of SPOP on g-globin levels is dependent on the CUL3 ubiquitin ligase complex. Additionally, forced expression of a dominant negative form of SPOP (Y87N) unable to bind to its substrates strongly induced HbF production. We also found that SPOP depletion did not measurably alter LRF levels and modestly reduced BCL11A levels. Overexpression of BCL11A did not restore g-globin silencing in SPOP depleted cells, indicating that SPOP works largely independently of these two factors. Finally, we tested whether the effects of SPOP depletion can be augmented through the use of experimental pharmacologic HbF inducers, such as pomalidomide. In proof of principle experiments we found that SPOP depletion and pomalidomide treatment induced HbF production to levels higher than either treatment alone.

In sum, our work uncovered SPOP as a new regulator of HbF and suggests targeting SPOP alone or in combination with other HbF inducers may provide a novel therapeutic strategy for the treatment of hemoglobinopathies such as sickle cell disease and b-thalassemia.

Disclosures

No relevant conflicts of interest to declare.
Increased SERUM VON Willebrand Levels in Chronic Lymphocytic Leukemia (CLL) Patients Are Related to a Shorter Time to Treatment
Authors: Tryfou Thommais , Papaioannou Paraskevi , Repousis Panagiotis , Gkioka Annita Ioanna , Sachanas Sotirios , Bitsani Aikaterini , Dimou Maria , Maltezas Dimitrios , Koudouna Aspasia , Kopsaftopoulou Anastasia , Chamoulkou Konstantina , Grafakos Ioannis , Kokori Styliani , Tsantes Argyrios , Pangalis Gerasimos , Panayiotidis Panayiotis , Kyrtsonis Marie-Christine
Published: October 2019

CLL is a frequent and usually indolent disease that require only follow-up in about 2/3 of patients while the rest 1/3 of patients are symptomatic, have a worse outcome and need to be treated. The role of angiogenesis in CLL prognosis remains unclear. It has been shown that CLL plasma promotes von Willebrand factor (vWF) secretion, and expression in human umbilical vein endothelial cell cultures (HUVECs) [1]. Therefore, we investigated whether serum vWF levels in CLL patients at diagnosis could provide prognostic information.

Patients and methods: 33 CLL patients were studied of whom 55%, 32% and 13% were Binet staged A, B and C respectively. 45% never required treatment while 55% required treatment either at the time of diagnosis or during their follow-up. The time to first treatment (TFT) was 34 months. Serum vWF was measured by ELISA (R&D Quantiquine, duo Set) in frozen sera drawn at patients' diagnosis and in 10 healthy individuals (HI). Measurements were performed in duplicate according to the manufacturer's instructions. Statistical analysis was performed by conventional methods, using the SPSS v.22 software. P values below 0,05 were considered statistically significant.

Results: Serum vWF levels ranged from 178,5 to 14353,5 pg/ml (median 1028,5) in patients and from 374,6 to 1141,3 pg/ml (median 528,5) in HI, the difference being statistically significant (p< 0,05). Patients with Serum vWF levels above 528,5 pg/ml (median normal value) had a significantly shorter TFT than the others (p=0,01) as shown in figure. Otherwise, serum vWF levels correlated only with hypogammaglobulinemia (p=0,04).

Conclusion: Serum vWF levels were increased in CLL patients. Importantly, we showed for the first time, to our knowledge, that higher vWF levels correlated with a shorter TFT, suggesting that increased vWF levels reflect active neoangiogenesis that in turn, contributes to a more aggressive disease behavior.

[1] Shahidi M et al, Induction of endothelial cell proliferation and von Willebrand factor expression and secretion by leukemic plasma of patients with chronic lymphocytic leukemia before and after inhibition of NF-κB. Blood Coagul Fibrinolysis. 2016 Sep;27(6):711-6.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Integrin Activity Reduces Immunogenic Cell Death By Inhibiting Cell Surface Presentation of ERp57 and Calreticulin
Authors: Liu Chi-Chao , Leclair Pascal , Pedari Foujan , Mahdis Monajemi , Sly Laura , Reid Gregor S. D. , Lim Chinten James
Published: October 2019

Calreticulin presentation on the cell surface is an important hallmark of immunogenic cell death (ICD), serving as the 'eat me' engulfment signal for professional phagocytes. ERp57 is an interacting and co-translocating protein with calreticulin, but unlike surface calreticulin, surface ERp57 is not known to be immunogenic. Since calreticulin interacts with α-integrins via the conserved GFFKR motif within the integrin cytoplasmic domain, we assessed whether integrin function can modulate cell surface calreticulin and ERp57 levels in ICD. When stimulated to engage integrin substrates, leukemic T-lymphoblasts treated with an ICD-inducer exhibited decreased surface calreticulin and ERp57 compared with cells under non-adherent conditions. The inhibitory effect on surface calreticulin and ERp57 was recapitulated for cells in suspension and treated with various agents that stimulate integrin activation, including Mn2+ and the LIBS antibodies 9EG7 and HUTS21. Similarly, cells expressing a mutant truncated α-integrin bearing only GFFKR as the cytosolic tail, also exhibited low surface calreticulin and ERp57 when treated with ICD-inducers under non-adherent conditions. Furthermore, integrin β1 null cells with overall reduced α-integrin expression exhibited enhanced surface calreticulin and ERp57, consistent with an inhibitory role for integrins in ICD. We generated calreticulin or ERp57 null strains by CRISPR-Cas9, and show that ICD-induced surface calreticulin is ERp57-dependent, while surface ERp57 is not calreticulin-dependent. Using permeabilization techniques that distinguish between cytosolic and ER staining, we found that ICD-inducers promoted the accumulation of cytosolic calreticulin and ERp57 that originated from the ER. In similar fashion, ER to cytosolic trafficking for calreticulin is ERp57-dependent, while for ERp57, it is not calreticulin-dependent. We also showed that integrin-mediated inhibition of surface calreticulin was due to reduced cytosolic to surface translocation coupled with normal ER-cytosolic release, suggesting that activated integrins acts to sequester calreticulin within the cytosol. T-lymphoblasts co-treated with an ICD-inducer and integrin activators exhibited reduced phagocytosis by macrophages when compared with treatment with only ICD-inducer. Our study reveals a previously uncharacterized function of integrins as negative regulators of immunogenic cell death by suppressing the presentation of cell surface calreticulin.

Disclosures

No relevant conflicts of interest to declare.
Cost-Effective Use of White Blood Cell Growth Factors in the Veterans Administration
Authors: Knopf Kevin , Hrureshky William , Love Bryan , Norris Leann , Bennett Charles L.
Published: October 2019

The use of granulocyte colony-stimulating (G-CSF) factors to avoid chemotherapy induced neutropenia is a major cost driver in clinical oncology. Short acting formulations, while less convenient, are 39% the cost of the longer formulation (Pegfilgrastim). We analyzed the use of G-CSF vs. Neulasta in the Veterans Administration (VA) health care system as currently practiced by clinical judgement and analyzed this economically. A survey of 23 sites within the VA on the relative frequency of use of filgrastim, Tbo-filgrastim, Filgrastim-sndz and Pegfilgrastim was conducted. Cost was estimated using 340B pricing.

A maximally cost-effecient strategy of 100% Tbo-filgrastim would result in a cost of $62,336 per 100 patient episodes. Tbo-filgrastim was the preferred treatment in 18 of 23 sites surveyed. Cost results ranged from a minimum of $62,336 (4 of 23 sites) to a maximum of $201,356 (2 of 23 sites) with a mean cost of $99,080. The VA was overall 73% efficient, and therefore highly cost-effective, in minimizing use of Pegfilgrasim.

As in Europe cost-efficiency favors the use of Tbo-filgrastim over Pegfilgrastim; further cost-saving opportunities exist with the following strategies : (1) avoid G-CSF use where there is no convincing evidence of efficacy (i.e. low risk patients, treatment of neutropenia rather than prophylaxis) (2) use daily filgrastim in lieu of pegfilgrastim whenever possible (3) use Tbo-filgrastrim rather than filgrastrimn (4) continue to examine strategies using 2 or 4 day courses of G-CSf as opposed to 8 days which would cut cost an additional 50 to 75%.

Survey data showed that uptake of biosimilar G-CSF in the VA system has been extremely rapid. All sites are using biosimilar GCSF for all new patients; 6 of 23 sites were comfortable shifting current patients on branded G-CSF to the biosimilar. Survey data shows an overwhelming use of filgrastrim compared to pegfilgrastim in the VA system, in contradistinction to the rest of the United States - resulting in an efficiency of 73% (i.e. only 27% of patients are being given pegfilgrastrim). This represents a great savings to the VA system which can be emulated in other sites in the United States. Switching to a Tbo-filgrastim, although heralded by some as cost savings, only engendered a cost savings of 2.2% - small compared to other clinical changes.

Disclosures

No relevant conflicts of interest to declare.
Symptom Duration Is Positively Correlated with Factor XIIIa Activity in Acute Pulmonary Embolism
Authors: Brailovsky Yevgeniy , Hoppensteadt Debra , Iqbal Omer , Simpson Kevin , Mcclane Nathan , Fareed Jawed , Darki Amir
Published: October 2019

Background

Acute pulmonary embolism (PE) patients are at variable risk of morbidity, mortality, and response to therapy. Patients often present at various time points from the symptom onset. Several factors may shed light into the state of endogenous thrombotic and fibrinolytic system at the time of presentation. Factor XIIIa plays a critical role in clot stabilization and may impact clot dissolution. Relation of Factor XIIIa activity and symptom duration is not known.

Methods

We prospectively collected blood samples from patients evaluated by Pulmonary Embolism Response Team at a tertiary care center. Blood was centrifuged, and plasma collected for analysis. We used ELISA method utilizing a commercially available kit from Hyphen, BioMed (Neuville-sur-Oise France), specific for measurement of Factor XIIIa, D-dimer, and Pro-TAFI antigens.

Baseline clinical characteristics were collected from electronic medical record. Symptom duration was gathered from patient subjective assessment. Additional workup included radiographic and echocardiographic evaluation.

We performed correlation analysis to test the association between symptom duration and Factor XIIIa activity, D-dimer, and Pro-TAFI antigen. Additionally we performed linear regression analyses to quantify the degree of association of symptom duration and Factor XIIIa activity.

Results

±±9.8, 8 patients were treated with catheter directed thrombolysis, while the rest were treated with anticoagulation alone. Symptom duration was positively correlated with Factor XIIIa activity (r2=0.227). More so, for every one day increase in symptom duration the Factor XIIIa activity was increased by 2.2%. (p=0.014). We demonstrated no correlation between symptom duration and D-Dimer (p=0.58) or symptom duration and Pro-TAFI antigen (p=0.84).

Conclusion

In patients with acute PE, symptom duration positively correlated with Factor XIIIa activity, for every one day increase in symptom duration the Factor XIIIa activity was increased by 2.2%. Future studies are needed to ascertain the impact of Factor XIIIa activity and clot dissolution as well as functional outcomes.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Second Transplants for Relapsed Acute Leukemia: Real World Experience from Argentina
Authors: Bet Lucia Antonella , Berro Mariano , Paganini Maria Ines , Basquiera Ana , Palmer Silvina , Garcia Juan Jose , Real Juan Jose , Martinez Rolon Juliana , Castro Martin , Kusminsky Gustavo
Published: October 2019

INTRODUCTION

Acute leukemia relapsing after allogeneic stem cell transplantation (AlloSCT) is usually associated with dismal prognosis. Treatment options in this population remain limited. We evaluated the outcomes of second AlloSCT in patients with acute leukemia relapsed after previous AlloSCT in centers from Grupo Argentino de Trasplante de Médula Ósea (GATMO)

METHODS AND RESULTS

We carried out a retrospective analysis in 21 adult patients who received a second transplant as a salvage treatment for relapsed acute leukemia. Main outcomes were overall survival (OS) and leukemia free survival (LFS). Variables evaluated were re-induction chemotherapy regimen, conditioning regimen, donor type, early immunosuppression tapering, and the use of maintenance treatment.

Mean age was 28 years old (range 16-53). Thirteen were under 30 years, 11 were women, 16 were acute myeloid leukemia. Median time from the first transplant to the relapse was 16,3 months (range 2 to 91 moths). Fourteen patients were MRD negative at the time of second AlloSCT (4 of them in aplasia) and 7 were either positive MRD or refractory disease. Almost all patients received a different re-induction chemotherapy regimen as well as a different conditioning compared to the first transplant. Sixteen out of 21 patients were transplanted with a different donor. Patients without graft versus host disease (GVHD) at day 100 started immunosuppression tapering. Considering the 16 patients alive/relapse free at day 100, half of them received maintenance therapy (6 azacitidine, one sorafenib and one dasatinib).

With a median follow up of 24 months, 1-year OS and LFS was 62% and 38%.

Age was a strong predictor of survival. There were no patients ≥30 years alive or relapse free beyond 6 months of the procedure, while LFS at 2 years was 58% (8/13) in patients younger than 30 years (p=0.0007). One year LFS was 60% for patients receiving any type of maintenance, compared to 33% of patients who did not receive maintenance (p=0.2). There were no differences in changing the donor or the pre-transplant status.

CONCLUSION

Second AlloSCT for relapsed acute leukemia is a potential curative option. Younger patients showed better outcomes. Patients relapsing after 6 months could have better prognosis. Maintenance treatment after second transplant should be evaluated, however prospective studies are needed in this scenario to draw better conclusions.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Single Cell RNA Sequencing of Serial Tumor and Blood Biopsies from Lymphoma Patients on an in Situ Vaccination Clinical Trial
Authors: Shree Tanaya , Sathe Anuja , Czerwinski Debra K. , Long Steven R. , Ji Hanlee , Levy Ronald
Published: October 2019

The critical determinants of effective antitumor immune responses, whether native or induced by therapy, remain poorly understood due to the complexity and plasticity of the immune system. To better profile and track these responses, we have employed the novel approach of performing single cell RNA sequencing for paired gene expression and immune repertoire analysis on tumor fine needle aspirates and peripheral blood of lymphoma patients undergoing immunotherapy on a clinical trial (NCT02927964).

In this in situ vaccination study, patients with low-grade lymphoma received local low-dose radiation and intratumoral SD-101 (a TLR9 agonist) to one site of disease, with systemic ibrutinib (a BTK inhibitor) added in the second week of treatment. Tumor fine needle aspirates and peripheral blood samples were obtained prior to treatment, at one week (prior to ibrutinib initiation) and at six weeks after treatment start. Single cell preparations were processed using 10X Genomics' single cell RNA transcription and library preparation protocol, followed by sequencing on the Illumina platform. Cells were sequenced to an average depth of 50,000 reads/cell for gene expression libraries and 5000 reads/cell for TCR sequencing. Identification of variable genes, principal component and/or canonical correlation analysis, graph-based clustering and differential expression analysis of single-cell gene expression data was performed using the Seurat algorithm. Single-cell TCR repertoires were analyzed using TCR-specific analysis software. This data is being integrated with data from multiparameter flow cytometry and functional immune assays for these same patients, as well as with their clinical outcomes.

Sequencing libraries have been prepared from 37 samples from 4 patients thus far. We have successfully generated single cell gene expression and TCR libraries from 3,000-10,000 cells from tumor fine needle aspirates and peripheral blood, with excellent sequencing quality metrics obtained. From detailed analyses of one patient's samples thus far, we have identified distinct immune populations in blood and tumor (Figure 1), including light-chain restricted B-cells, with good concordance with flow cytometry. Preliminary results show changes occurring in immune cell frequencies and phenotypes at the treated tumor site, at distant tumor sites and in the peripheral blood when samples from before and after treatment are compared. Sample collection, sequencing, and analysis are ongoing.

Deep profiling of serial biopsies during immunotherapy using single cell RNA sequencing promises to illuminate underlying cellular dynamics, and paired with clinical outcome data, determinants of response. Ultimately, this may provide a roadmap for successful translation of single-cell genomics into the clinic for treatment monitoring and response prediction.

Disclosures

No relevant conflicts of interest to declare.
Pharmacological Targeting of Osteoblast-Induced MDS and AML
Authors: Mosialou Ioanna , Galan-Diez Marta , Vandenberg Andrew , Ali Abdullah Mahmood , Raza Azra , Kousteni Stavroula
Published: October 2019

The bone marrow hematopoietic stem cell niche has emerged as a regulator and instigator of hematological myeloid malignancies. Activation of b-catenin signaling in osteoblasts, by deletion of exon3 or by inactivating mutations in APC, induces myelodysplasia (MDS), rapidly progressing to acute myeloid leukemia (AML) in mice. Activated b-catenin signaling is present in osteoblasts of one third of MDS and AML patients and it is the most active pathway in stromal cells of MDS patients suggesting that it may sustain dysplastic hematopoiesis and progression to MDS and AML in humans. Therefore, targeting this pathway may provide a new therapeutic approach for this subgroup of MDS/AML patients. To examine this hypothesis we searched for an FDA-approved compound with the ability to inhibit b-catenin signaling and identified all-trans-retinoic acid (ATRA) as a potential candidate. We show here that treatment of osteoblastic cells with ATRA inhibits the induction of b-catenin target genes by Wnt3a and abrogates nuclear localization of b-catenin, as shown by immunohistochemistry and western blot analysis. These effects are at least in part independent of its phosphorylation by GSK3b on exon3 and are mediated by the retinoic acid receptor a (RARa) since silencing RARa, but not RARb or RARg, in osteoblasts abolishes the inhibitory effect of ATRA on Wnt3a-induced b-catenin signaling. Similarly, treatment of leukemic mice expressing constitutively active b-catenin in their osteoblasts with ATRA, inhibited b-catenin signaling as measured by expression of its target genes Axin2 and Lef1 in bone. At the same time, ATRA improved anemia and thrombocytopenia, decreased the percentage of blasts in bone marrow and blood and prolonged overall survival in these leukemic mice as compared to vehicle-treated mice. The response to ATRA treatment of two MDS patients with active b-catenin in their osteoblasts, and not responding to other treatments, was monitored. The hematologic phenotype of the patients improved and disease status stabilized so that patients remain transfusion independent and disease-related symptom-free with a good quality of life thereafter. The improvement in disease status coincided with absence of b-catenin in the nucleus as flow cytometry analysis showed a high percentage of patient osteoblasts expressing activated b-catenin in pre-therapy samples but not in on-therapy samples. These results suggest that ATRA may find a new use in the treatment of the specific portion of MDS/AML patients with activated b-catenin in their osteoblasts.

Disclosures

Raza: Geoptix: Speakers Bureau; Onconova: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Kura Oncology: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Syros: Research Funding.
Pomalidomide-Dexamethasone in the Management of Heavily Pretreated Multiple Myeloma
Authors: Cerchione Claudio , Nappi Davide , Pareto Anna Emanuele , Pane Fabrizio , Catalano Lucio
Published: October 2019

Background:

Pomalidomide is a new generation IMID, with a very good compliance, thanks to oral administration, which can be used also in heavily pretreated patients, in a domestic setting.

Aims:

In this retrospective observational trial, It has been evaluated efficacy and tolerance of pomalidomide plus dexamethasone (PD) as salvage regimen in heavily pretreated patients with relapsed and refractory MM (rrMM), whose prognosis is particularly severe.

Methods:

33 patients (19 M/14 F), with rrMM, median age at diagnosis 69 years (r. 52-84), and median age at start of treatment 76 years (r.56-89) treated with several lines of treatments (median 7, r. 2-11), every refractory to all the drugs previously received (also Bortezomib, Thalidomide and Lenalidomide), received Pomalidomide-Dexamethasone (Pomalidomide 4 mg for 21 days, Dexamethasone 40 mg days 1,8,15,22, pegfilgrastim day +8) every 28 days, until progression.

ISS was equally distributed, and cytogenetic was evaluable in 14 patients, and in particular three del13q and one t(11;14) were present. All the patients had previously been treated with schedule containing bortezomib and IMIDs. 60% (20/33) of them had undergone at least to a single ASCT.

All patients were relapsed and refractory to last therapies received before PD.

Results:

Pomalidomide was well tolerated, with grade 3 anemia in 51% (17/33) of patients, 36.3% (12/33) grade 3 neutropenia (pegfilgrastim in primary prophylaxis was given, no hospitalization was required, no septic shocks were observed), 30.3% (10/33) grade 3-4 thrombocytopenia without hemorrhagic events and transfusion-dependence. No severe extra-hematologic toxicity was observed.

According to IMWG, ORR1 (≥PR) was 45.4% (15/33: 4 CR, 5 VGPR, 6 PR), but, considering that we are evaluating a cohort of heavily pretreated patients without any other alternative treatment, with really poor prognosis, another parameter should be considered, ORR2 (≥SD), considering stable disease as a successful result in progressive MM. ORR2 was 78.7% (26/33: 4 CR, 5 VGPR, 6 PR, 11 SD). These can be considered as impressive result in this subset of patients.

Oral treatment gives a really good compliance, in frail and unfit patients, and response, when present, is always really fast (median time to response: 2 months (r.1-6)), median OS from diagnosis was 92 months (range 21-234), median OS from start of pomalidomide was 9 months (range 1-25).

Conclusions:

Pomalidomide-dexamethasone has shown significant efficacy and a very good compliance, thanks to oral administration, in a particularly severe setting of heavily pretreated patients, relapsed and refractory to all available therapeutic resources.

Disclosures

No relevant conflicts of interest to declare.
Adverse Outcomes in Concurrent Sickle Cell Disease and Hematological Malignancies
Authors: Perimbeti Stuthi Pavani , Hou Kevin Ye , Ramanathan Sabarina , Woodard Adonas , Kyung Daniel , Crilley Pamela Ann , Topolsky David , Ward Kristine , Styler Michael
Published: October 2019

Introduction:

Case reports have suggested that there is an increased risk of hematological malignancies with sickle cell disease (SCD). We aimed to investigate the prevalence and mortality of select hematological malignancies, including: acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), polycythemia vera (PV), essential thrombocytopenia (ET), Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL), primary myelofibrosis (PMF) in patients hospitalized with SCD.

Methods:

We queried the Healthcare Cost and Utilization Project's Nationwide Inpatient Sample to identify the hospitalizations with SCD using ICD-9 codes 282.6, 282.60, 282.61, 282.62, 282.63, 282.64, 282.68, 282.69 from 1999 to 2014. Cases were then stratified based on the concurrent presence of the above hematological malignancies. The percentages of patients with each type of hematological malignancy were obtained using Chi-square analysis. In addition, we compared outcomes between patients with and without SCD who had hematological malignancies. Bivariate analysis for in-hospital mortality percentage was performed using Chi-square test. Multivariate analysis to evaluate the risk of death during hospitalization was performed using Cox proportional hazard regression with alpha set at 0.05.

Results:

There were 307,424 admissions (weighted=1,513,168) with SCD. Within these admissions, 0.04% (n=516) were associated with AML, 0.05% (n=720) with HL, 0.02% (n=47) with ALL, 0.07% (n=1,028) with NHL, 0.69% (n=10,654) with ET, 0.01% (n=20) with PMF and 0.01% (n=18) with PV. The hazard ratio for mortality (95% C.I.) with SCD compared to without SCD was 3.60 (1.24-4.67) for AML (p <0.001), 4.56 (2.78-6.78) for ET (p <0.001), 2.37 (1.41-5.67) for NHL (p=0.003), 1.5 (0.8-4.5) for HL (p=0.08), 1.1 (0.5-3.2) for ALL (p=0.04), 1.3 (1.1-3.2) for PMF (p= 0.03) and 1.7 (0.6-2.9) for PV (p=0.05).

Discussion:

The most frequently encountered hematological malignancy in patients with SCD was ET, followed by NHL and then HL. After controlling for multiple confounders including age, race, sex, comorbidities and socioeconomic status, the hazard of death during hospitalization with SCD, ET, AML, NHL and ALL are significantly higher compared to those without SCD.

While uncommonly encountered, concomitant hematological malignancy and SCD portends a significantly worse outcome, particularly in ET and AML. Potential explanations include iron overload from prior transfusions, increased infection risk due to asplenia and vascular damage from previous vaso-occlusive events. Newer advances in the management of SCD might improve subsequent outcomes in hematological malignancies.

Disclosures

No relevant conflicts of interest to declare.
The Role of LAMP5 in Innate Immune Signaling Is Critical for the Survival of MLL Leukemias
Authors: Gracia-Maldonado Gabriel , Clark Jason , Pierre Philippe , Mulloy James C. , Kumar Ashish R
Published: October 2019

Introduction

Chromosomal translocations involving the Mixed Lineage Leukemia (MLL, also called KMT2A) gene account for around 80% of the acute lymphoid leukemia (ALL) and 35%-50% of the acute myeloid leukemia (AML) diagnosed in infants, and are associated with poor outcomes. Despite improvements of conventional chemotherapies and treatments, patients with MLL-rearranged leukemia have weak response to treatment and poor prognosis. There is thus an urgent need to understand the molecular pathogenesis of these leukemias to develop more effective therapies. Gene expression studies show that MLL-leukemias uniquely display over-expression of Lysosome-associated Membrane Protein 5 (LAMP5) in both ALL and AML In normal hematopoiesis, LAMP5 expression is restricted to non-activated plasmacytoid dendritic cells (pDC), where it has been shown to be required for transport of TLR9 from VAMP3+/LAMP2+/LAMP1- endolysosomal vesicles to the LAMP1+ late lysosomal compartments, modulating the signaling and production of Type I interferon to TNF production. Here, we tested the hypothesis that LAMP5 is required for MLL leukemia wherein it modulates innate immune signaling.

Methods and Results

In previous studies, we found LAMP5 expression being restricted to MLL-fusion leukemia cell lines. Additionally, in an inducible model, LAMP5 expression directly correlated with that of the MLL-fusion protein MLL-AF9. . Knockdown on LAMP5 in MLL-fusion leukemia cells inhibited their propagation both in vitro and in vivo whereas the non-MLL leukemias were unaffected. To determine the mechanisms by which LAMP5 promotes growth in MLL leukemias, first we studied the localization of LAMP5 in these cells. Using immunofluorescent confocal microscopy we detected LAMP5 mainly in LAMP2+/LAMP1+ compartments. Additionally, LAMP5 co-localized with MYD88, a known scaffold protein required for IL1R/TLR signaling. Accordingly, LAMP5-knockdown abrogated TLR signaling as evidenced by reduced activation of JNK, MAPK p38, IRF7 and NFKB. Conversely, over-expression of LAMP5 in the non-MLL leukemia cell line Kasumi-1 led to increased activation of JNK, p38, IRF7 and NFKB, indicating that the innate-immune signaling pathway is regulated by LAMP5 expression in leukemias.r

Conclusions

Collectively, these results demonstrate that LAMP5 is required for the survival of MLL leukemia and that it plays an important role in the activation of the IL1/TLR signaling pathway. Overall, based on our results and the limited expression in normal hematopoiesis, we propose that LAMP5 could potentially serve as a therapeutic target with a wide therapeutic-window to treat MLL-leukemias.

Disclosures

No relevant conflicts of interest to declare.
Identification of GBA Gene Mutations in 19 Pakistani Unrelated Patients of Gaucher Disease
Authors: Naz Arshi , Abedin Qurat , Ahmed Shariq , Siddiqui Saima , Shamsi Tahir
Published: October 2019

Introduction: Gaucher disease (GD) is one of the lysosomal storage diseases that is rare and inherited autosomal recessively. There is insufficiency of glucocerebrosidase enzyme that leads to the build up of un-degraded substrates in white blood cells causing anemia, hepatosplenomegaly and skeletal disease. This enzyme deficiency is linked with the defect of its gene (GBA) that codes for this enzyme. Initial diagnosis is made by the estimation of glucocerebrosidase level in blood and confirmed by genetic analysis of GBA gene. To identify the mutations of GBA gene in Pakistani patients with GD from different regions of Pakistan.

Sampling & methodology: The sample and demographic data was collected in National Institute of Blood Disease and Bone marrow Transplantation after approval of IRB and written informed consent of patients. We collected total 19 blood samples, out of which 5 had Gaucher's disease, 10 samples were parents of the index cases and 4 were control. The methodology consisted of DNA extraction and quantification from peripheral blood. Genetic analysis of coding regions of GBA gene was done via gene amplification, gel electrophoresis and sequencing.

Result: Mutation was found in two out of five families that makes the prevalence of GBA gene mutation 40%. These were diagnosed on reduced enzyme levels and found to have L444P (c.1448T>C) mutation in homozygous form in 10th exon of GBA gene. The parents of that patient carried the same mutation in one allele. Rest of the patients who were diagnosed on bone marrow morphology showed no mutation in GBA gene.

Conclusion: Our results illustrate that GBA gene mutation was found in those patients who were diagnosed by the estimation of β-glucosidase enzyme levels rather than on bone marrow morphology. In our population, the mutation L444P was found, which is the most frequent gene mutation found in the world. Since this study is conducted in a small number of patients therefore it is recommended that large cohorts of patients should be evaluated in future for genetic mutations among Gaucher's patients in Pakistan

Key words: Gaucher disease, storage disorder, GBA gene

Disclosures

No relevant conflicts of interest to declare.
Effects of intravenous immunoglobulin on platelet count and antiplatelet antibody disposition in a rat model of immune thrombocytopenia
Authors: Hansen Ryan J. , Balthasar Joseph P.
Published: October 2019

Experiments were conducted to investigate the effects of intravenous immunoglobulin (IVIG) in a rat model of immune thrombocytopenia (ITP). Rats were pretreated with 0 to 2 g/kg IVIG and then challenged with an antiplatelet antibody (7E3, 8 mg/kg). IVIG effects on 7E3-induced thrombocytopenia and on 7E3 pharmacokinetics were determined. IVIG pretreatment led to significant changes in the degree and time-course of 7E3-induced thrombocytopenia (P = .031). Nadir percent platelet counts were 121% to 279% greater in animals treated with IVIG (0.4-2 g/kg) than in animals receiving 7E3 alone. IVIG treatment also led to dose-dependent increases in 7E3 clearance (P < .001), with more than 2-fold increases in 7E3 clearance seen following the highest dose of IVIG. In vitro experiments showed that IVIG effects on platelet count are not likely due to anti-idiotypic inhibition of 7E3-platelet binding and that IVIG did not directly bind to 7E3. Consequently, IVIG-7E3 binding cannot explain the increase of 7E3 clearance following IVIG treatment. We propose that the observed increase in 7E3 clearance with IVIG therapy is due to saturation of the FcRn salvage receptor for IgG. The importance of the effect of IVIG on 7E3 clearance to the prevention of thrombocytopenia in these animals is unclear at present; nonetheless, these data provide experimental support for a new mechanism of IVIG action in ITP (ie, IVIG-mediated increases in antiplatelet antibody elimination). This model of ITP will be useful for further investigations of IVIG mechanism of action and for development of new therapies for ITP.
A prospective study on the evolution of the T-cell repertoire in patients with Sézary syndrome treated by extracorporeal photopheresis
Authors: Ingen-Housz-Oro Saskia , Bussel Annette , Flageul Béatrice , Michel Laurence , Dubertret Louis , Kourilsky Philippe , Gachelin Gabriel , Bachelez Hervé , Musette Philippe
Published: October 2019

Sézary syndrome is a leukemic form of epidermotropic cutaneous T-cell lymphoma related to the malignant proliferation of clonal CD4+ T cells. Extracorporeal photochemotherapy may induce a transient improvement of the clinical signs, but its efficiency is discussed. To investigate the frequency of the T-cell clone in the peripheral blood of patients with Sézary syndrome and to monitor its evolution in patients treated using extracorporeal photopheresis or chemotherapy, we used the immunoscope technique. In one patient, we observed a decrease of the relative frequency of the clone from 15.6% to 0%, paralleling a complete remission of the clinical disease and a disappearance of the circulating Sézary cells. In the other cases, the evolution of the relative frequency paralleled the initial improvement of the clinical status and the absence of long-term efficiency in patients treated with extracorporeal photopheresis or chemotherapy. We observed a quick-acting direct cytotoxicity of the association 8MOP + UVA on the T-cell clone. The immunoscope technique appears to be an efficient tool to appreciate the amount of tumoral cells and to monitor the evolution of the clonal component in the Sézary syndrome.
Recombinant Factor VIIa Enhances Platelet Adhesion and Aggregation under Flow Conditions at Normal and Reduced Platelet Count.
Authors: Lisman Ton , Adelmeijer Jelle , Cauwenberghs Sandra , van Pampus Elisabeth C.M. , Heemskerk Johan W.M. , de Groot Philip G.
Published: October 2019

Recombinant factor VIIa (rFVIIa) has been developed for treatment of inhibitor-complicated hemophilia. Increasing clinical evidence shows that rFVIIa also appears safe and effective as pro-hemostatic agent in various other clinical disorders including patients with thrombocytopenia. Relatively little is known about the mechanism of action of rFVIIa in hemophilia and its various other potential indications. It is generally accepted that rFVIIa functions by enhancement of thrombin generation at the site of injury. It is, however, unknown if and how this affects platelet adhesion and aggregation. Previously, we have shown that rFVIIa-mediated thrombin generation substantially enhances deposition of platelets from patients with Glanzmann thrombasthenia (a qualitative or quantitative deficiency of the integrin αIIbβ3) to subendothelial proteins (Blood2004, 103: 1720). Here, we investigated the effect of rFVIIa-mediated thrombin generation on platelet adhesion and aggregation under flow conditions at normal and reduced platelet count. A mixture of washed platelet and red cells was perfused over collagen or fibrinogen in presence or absence of a thrombin-generating system consisting of purified coagulation factors rFVIIa, X, and prothrombin in the presence of calcium chloride. Addition of this thrombin-generating system enhanced platelet adhesion and aggregation to collagen and adhesion and spreading to fibrinogen at normal platelet count and at platelet numbers as low as 10.000/μl. rFVIIa-mediated thrombin generation enhanced the activation state of platelets adhered under flow as measured by intracellular calcium fluxes, and enhanced the exposure of procoagulant phospholipids as measured by annexin A5 binding. It was shown that FITC-labeled rFVIIa bound to collagen-adhered platelets under flow conditions in experiments in which whole blood from severely thrombocytopenic patients was used. We believe thrombin is generated by platelet-bound rFVIIa independent of tissue factor. Taken together, increased platelet adhesion and aggregation by rFVIIa-mediated thrombin formation may explain the therapeutic effects of rFVIIa in thrombocytopenic conditions and in patients with a normal platelet count by (1) enhancement of primary hemostasis and (2) enhancement of procoagulant surface leading to elevated fibrin formation. These platelet-promoting effects of rFVIIa might also, in part, explain the therapeutic effects of rFVIIa in other indications.
Rhesus Box Variation: Multiple RHD Deletion Events and Implications for Testing RHD Heterozygosity.
Authors: Flegel Willy A. , Moulds Joann M. , Wagner Franz F.
Published: October 2019

The RHD gene encoding the Rhesus blood group antigen D is flanked by two highly homologous DNA segments of about 9,000 bp, the upstream and downstream Rhesus boxes. In haplotypes with an RHD deletion, the fusion of the two Rhesus boxes within the 1,463 bp identity region generates the single hybrid Rhesus box. Its detection has been applied to determine RHD zygosity. For instance, testing for RHD heterozygosity is of clinical interest, because all children of an RHD homozygous father will be D positive and at risk of hemolytic disease of the newborn, if the mother carries an anti-D. Aberrant Rhesus boxes can confound this application and appear to be frequent among Africans. A comprehensive, systematic analysis was lacking. We sequenced about 5,850 bp of the upstream and downstream Rhesus boxes in 18 samples that were representative for all four D clusters and of the hybrid Rhesus box in 4 samples that were mistyped in assays for the hybrid Rhesus box. The known differences between upstream and downstream Rhesus boxes were in part restricted to subsets of RHD alleles. We detected 46 additional polymorphism, which represented single nucleotide substitutions, short insertions or deletions. Gene conversions were found in the upstream Rhesus boxes of RHDψ, DAU-1 and DAU-3 and in the downstream Rhesus boxes of Ccdes, weak D type 4.1, type 4.2 and DAU-0. All 6 different gene conversions started or ended in or near the identity region, which may have been instrumental for generating these gene conversions. Recombinations between haplotypes were likely in several alleles like DIII type 4. Four non-standard hybrid Rhesus boxes were suggestive of multiple independent RHD deletion events.

We found considerable variation of Rhesus box sequences, which explained the limitations for RHD heterozygosity testing that were previously observed in several laboratories. The general topology of the genomic Rhesus locus was however similar in all cases and consistent with our initial description. There was evidence for 5 probably independent RHD deletion events. Testing RHD heterozygosity in Africans is best based on quantitative PCR or amplification of the full length hybrid Rhesus box. Because in Europeans who harbored RHD alleles belonging to African D clusters the Rhesus box variation was also substantial, use of two complementary methods for hybrid Rhesus box detection may even be advisable in whites. We conclude that a long range PCR spanning the whole hybrid Rhesus box, while technically demanding, is not influenced by the kinds of aberrations observed in this study and may hence remain the standard for research purposes and quality assurance.
Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) Regulates Activated Complement C5a in Vivo.
Authors: Nishimura Toshihiko , Myles Timothy , Nagashima Mariko , Morser John , Pearl Ronald G. , Leung Lawrence L.K.
Published: October 2019

The latent plasma carboxypeptidase thrombin-activatable fibrinolysis inhibitor (TAFI) is activated by thrombin/thrombomodulin on the endothelial cell surface, and functions in dampening fibrinolysis. We recently showed that TAFIa efficiently inactivates bradykinin, C5a, and thrombin-cleaved osteopontin, suggesting that it may have a broad anti-inflammatory role. In this study, we examined the pulmonary inflammatory responses in a C5a-induced alveolitis model in wild-type (WT) and TAFI deficient mice. TAFI deficient mice had been backcrossed into a pure C57/B6 background. C5a was instilled into the trachea of anesthetized mice, which were then allowed to recover. Six hours later, bronchiole alveolar lavage (BAL) was performed and the extent of pulmonary inflammation determined. C5a caused a dose-dependent increase in cell counts (WBC/ul) in the BAL fluids in WT mice (saline control, 5.3±4.1 (mean±SD); C5a at 0.05 mg/ml, 28.7±8.5, C5a at 0.1 mg/ml, 60.4±23.8; n=6 for saline and low-dose C5a, n=12 for high dose C5a; p < 0.0005 and < 0.0001 for low-dose and high-dose C5a compared to saline control respectively). Significantly increased BAL cell counts in the TAFI deficient mice in response to C5a stimulation were noted (saline control, 3.3±3.9; low-dose C5a 70±11.2; high-dose C5a 159.6±58.7; n=6 for saline and low-dose C5a, n=12 for high dose C5a; p < 0.00001 and < 5E-0.6 respectively), representing ~2.4–2.6 fold increase in BAL cell counts in the TAFI deficient mice. Determinations of the wet/dried lung weight ratios, an index of pulmonary edema, showed similarly significant results (WT mice: 4.1±0.14, 4.25±0.1, 4.52±0.15 and TAFI-deficient mice: 4.27±0.41, 5.3±0.53, 5.77±0.62 for saline control, low- and high-dose C5a respectively). Our data indicate that in the absence of TAFI, the chemotactic and inflammatory effects of C5a in pulmonary inflammation were enhanced. We are in the process of testing whether E229K thrombin, which selectively activates protein C and TAFI, will ameliorate the C5a alveolitis response in WT mice. Our results support our thesis that the function of TAFIa is not restricted to fibrinolysis. It has a broad anti-inflammatory role and may serve as a homeostatic counterbalance to thrombin’s inflammatory functions.