bluuebunny/crossref_metadata_2025 huggingface dataset:
Critical Role for Apobec and Its Interacting Partners in Mediating Mutations and Cell Growth in Multiple Myeloma (MM)
Authors: Talluri Srikanth , Samur Mehmet Kemal , Shi Jialan , Prabhala Rao , Avet-Loiseau Hervé , Shammas Masood A. , Munshi Nikhil
Published: October 2019

The APOBEC family of cytidine deaminases include AID (activity induced deaminase) and 10 related APOBEC enzymes (A1,A2,A3A,A3B,A3C,A3D,A3F,A3G,A3H and A4). AID is well studied for its role in somatic hyper mutation and class switch recombination of immunoglobulin genes. APOBECs (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) have been shown to have roles in mRNA editing and in antiviral immunity. Recently, a causal role for the AID/APOBECs in inducing somatic mutations in myeloma has been proposed and we have previously published that APOBEC signature mutations as a frequent event in Myeloma. We have also observed that APOBEC-mediated mutations may account for mutations associated with progression of smoldering myeloma to MM.

We further investigated the role of APOBEC in genomic changes in MM and observed that APOBEC expression and activity is elevated in myeloma cell lines as well as patient samples. Using knockdown and over expression approaches, we showed that depletion of APOBECs in myeloma cell lines reduces genomic instability. Following APOBEC3G knock down we observed decreased DNA damage (by g-H2AX), decrease in acquisition of new copy number events over time, and reduced mutational load, strongly suggesting that inhibiting APOBECs could be a potential approach to reduce genome evolution in MM.

We next investigated the effect of APOBEC inhibition on myeloma cell proliferation. We observed that Sh-RNA-based APOBEC knock down in MM1S and H929 MM cell lines, led to significant inhibition of MM cell proliferation, and induction of apoptotic cell death. Associated with APOBEC knockdown, we also observed increased levels of Cyclin-dependent kinase inhibitor 1B (p27Kip1) at both RNA and protein level. By immunoprecipitation we found that APOBEC3G interacts and inhibits the RNA binding protein DEAD-END 1 (DND1), thereby preventing it from inhibiting miR-221-mediated targeting of p27 transcripts. Knockdown of DND1, or over-expression of miR-221 in APOBEC-depleted cells rescued the cell proliferation defects with concomitant decrease in p27 levels. These results show that APOBCs bind to and sequester DND1, leading to miR-221-mediated depletion of p27. In the absence of APOBEC, DND1 prevents the degradation of p27 mRNA, leading to elevated p27 levels and inhibition of cell cycle, suggesting a role for APOBECs in regulating MM cell proliferation that might be independent of its RNA/DNA mutator function. Taken together, these results indicate a significant functional role for APOBECs both in genome evolution as well as cell growth in myeloma and may constitute an important therapeutic target.

Disclosures

Munshi: OncoPep: Other: Board of director.
Survival Outcomes of Young Multiple Myeloma Patients: A Single Center Experience
Authors: Aytan Pelin , Yeral Mahmut , Gereklioglu Cigdem , Korur Aslı , Tanrıkulu Funda , Solmaz Soner , Buyukkurt Nurhilal , Kasar Mutlu , Asma Suheyl , Boga Can , Kozanoglu Ilknur , Ozdogu Hakan
Published: October 2019

Multiple myeloma (MM) is a disease of the elderly. It shows rapidly increasing incidence with increased age. Although MM is rare in young people, patients diagnosed before the age of 50 years account for 10% of the overall incidence and approximately 2% are diagnosed before 40 years of age. The aim of the study was to assess the clinical results of the young MM patients diagnosed at the age of ≤45 years.

All the MM patients who were ≤45 years old at the time of diagnosis and who were admitted to our clinic between January 2004 and December 2017 were retrospectively assessed. There was a total of 31 patients and 14 (45.2%) of them were females and 17 (54.8%) of them were males. The mean age was 40.4±3.8 years. The most frequent myeloma types were Ig G Kappa (n:12, 38.7%) and Kappa light chain (n: 10, 32.3%). The remaining types were Ig A lambda (n:4, 12.9%), Ig G lambda (n:2, 6.5%), Lambda light chain (n: 2, 6.5%) and non-secretory myeloma (n:1, 3.2%). Before transplantation 12 patients (38.7%) were in complete remission, 11 patients (35.5%) were in very good partial remission, 4 patients (12.9%) were in partial remission state and 4 patients (12.9%) had refractory disease. VAD + VCD protocol was given to 12 (38.7%) patients. 6 (19.8%) patients had VCD, 3 (9.7%) got VCD + VRD, 2 (6.5%) got VAD and 8 (25.8%) got other chemotherapy protocols. Transplantation was done in 26 patients (83.9%). Overall 9 patients (29%) died during the study period. Second or tandem transplantation was performed to 10 patients and 30% (n:3) of these re-transplanted patients died. The overall survival for these patients was estimated to be 70% (95% CI: 64.2-75.8).

In conclusion MM in patients younger than 45 years is rare. In these patients Ig G Kappa and Kappa Light chain types are the most frequent types. Heterogeneous of individual patients may exist within these young myeloma patients. For those who are expected to show a poor response to current treatments despite their young age, the efficacy of an alternative therapeutic strategy such as front line allo-HSTC should be further investigated.

Disclosures

No relevant conflicts of interest to declare.
Identification and Characterization of EBV Genome in NKT Cell Lymphoma
Authors: Su Yi , Yang Xi , Yi Hai , Fan Fangyi , Sun Haoping , Lai Sihan , Niu Ting
Published: October 2019

Background: Epstein-Barr virus (EBV) is closely related to (Natural Killer/T) NKT cell lymphoma. However, the genomic characteristics of EBV genome in NKT cell lymphoma are poorly understood.

Methods: We targeted the EBV gene sequence of 5 samples of NKT cell lymphoma tissue and identified the genomic variation of EBV in NKT cell lymphoma, and compared with other EBV strains by bioinformatics.

Results: The EBV genome was successfully retrieved from 5 samples and the variation was detected. We found a total of 3220 mutations in 5 cases of NKT cell lymphoma, including 2996 substitutions, 105 insertion and 119 deletion, of which 2103 of the mutations in the gene coding region, others were in the non-coding region. The non-synonymous mutations in 5 samples were 563, 586, 596, 667 and 578, respectively. Most of them were located in the exons of tegument, membrane glycoprotein, transcription factor and replication. Phylogenetic analysis of LMP1, EBNA1, EBNA2, EBNA3A, BZLF1, BLLF1 were performed with other EBV strains. The results showed that the LMP1 and BZLF1 genes of EBV in NKT cell lymphoma were different from those of other EBV strains, suggesting that LMP1 and BZLF1 were specific in NKT cell lymphoma. The comparison of EBNA2 and EBNA3A between EBV in NKT cell lymphoma and other strains suggested that the EBV in NKT cell lymphoma was type I EBV. The characteristics of EBNA1 in different strains have no correlation and need further investigation.

Conclusion: By targeting sequencing technology, we had shown that there were many variations of EBV in NKT cell lymphoma, including substitutions, insertions and deletions, and most of the variations were located in the gene coding region. The EBV in NKT cell lymphoma was type I EBV. LMP1 and BZLF1 in the EBV genome of NKT cell lymphoma were tumor specific and might serve as potential biomarkers for EBV genotyping.

Disclosures

No relevant conflicts of interest to declare.
Evidence for Role of Enzymatic Operations in Shared T-Cell Receptors
Authors: Woodcock Mark , Buntzman Adam , Vincent Benjamin G.
Published: October 2019

Background:

There exist shared T cell receptors (TCR) found in the circulating repertoires of healthy individuals. The exact mechanism for why some sequences are more commonly shared is unclear, but it has been hypothesized that the number of recombination permutations of V, D and J segments that produce a particular shared sequence is predictive (i.e. the "convergent recombination" hypothesis). An alternative hypothesis is that sharing is driven by decreased biochemical energy required to generate the shared sequences (i.e. the "enzyme proxy" hypothesis).

Methods:

Circulating T cells were collected from 100 healthy donors and TCR repertoires were profiled. Using a novel algorithm, the number of recombination events that could produce each TCR was calculated ("path count"). Sequence features associated with enzymatic operations - such as V and J region chewback and non-templated nucleotide addition - were calculated as well ("enzymatic" parameters). Multivariable regression was used to identify TCR sequence features associated with TCR clonotype sharing between individuals.

Results:

Nearly 38 million unique TCR sequences from the donor cohort were profiled. Based on regression coefficient analysis of significance, enzymatic parameters were overall negatively associated with TCR sharing (betas [-1.24x10-2 - 3.88x10-3], average -5.67x10-3, maximum p<0.001), while path count had a positive association (beta 1.73x10-2, p<0.001). When these parameters were used to predict sharing, a model using enzymatic parameters had similar performance to a model using only path count. The creation of a model with both enzymatic and path count parameters led to a significant improvement in predictive ability over both models (minimum delta LR 56950, p<0.001). The enzymatic variables and path count had a weak negative correlation (pearson correlation [-0.518 - -0.407]).

Conclusions:

Our findings indicate that TCR sharing is inversely related with increasing complexity of enzymatic operations required during VDJ recombination, and is predictive of receptor sharing between circulating T-cell repertoires in humans. The number of recombination paths is positively associated with TCR sharing, and carries predictive information indepenent of enzymatic operation. This suggests that shared TCRs are favored due to a combination of enzymatic kinetics as well as available recombination paths.

Disclosures

Buntzman: - An Autoimmune Research Fund: Membership on an entity's Board of Directors or advisory committees. Vincent:Merck: Research Funding.
A Reciprocal Interaction between Immune Reconstitution and Hemorrhagic Cystitis after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Xiao Min , Luo Xiaofeng , Ren Jinhua , Yang Xiaozhu , Zhu Haojie , Lin Kangni , Chen Minmin , Chen Shaozhen , Li Qian , Chen Zhizhe , Hu Jianda , Yang Ting
Published: October 2019

Background

Hemorrhagic cystitis (HC) is one of the serious complications of allogeneic hematopoietic stem cell transplants (allo-HSCT), with the potential to cause significant morbidity. However, the underlying mechanism of HC is not yet clear.

Objective

To explore the reciprocal interaction between immune reconstitution and HC after allo-HSCT.

Methods

101 patients undergoing allo-HSCT in our department during July, 2014 to April, 2017 were analyzed. JC virus(JCV), BK virus (BKV), cytomegalovirus (CMV) by quantitative real-time PCR were performed on all blood or urine samples post-HSCT. Non-specific T and B cell immune reconstitution were assessed by the expression of CD3, CD4, CD8, CD56, CD10, CD19, CD20 and CD22 with multiparametric flow cytometry, and specific T cell phenotype and cytokine production were determined by the expression of CD3, CD4, CD8, CD69, IL-2, IFN-gamma and TNF-alpha on PBMCs after the stimulation with one of four BKV peptides (tAg,Tag,VP-1,VP-2,VP-3).

Results

62 out of total 101 patients were male and 39 were female. The median age was 27 years (range, 1-54). 85 patients were diagnosed as malignancy hematologic disease (AML=44, ALL=26, MDS=4, CML=6, NHL=5), while 16 were non-malignancy hematologic disease (SAA = 14, fanconi=1, thalassemia=1). Through the cox regression analysis, the hazard ratio (HR) of HC (95% confidence interval) for BKV viruria was 4.866 (1.908-12.406), with a P-value of 0.001. Those with primary malignance disease also had higher odds of developing HC, with an HR of 5.101. Both of them were independent risk factors in multivariate analysis. There was no significant difference on the expression of CD3, CD4, CD8 and CD56 representative the non-specific T cell immune reconstitution between BK+ and BK- group, but the non-specific B cell immune reconstitution stained with CD19+, CD20+, CD22+ was significant delayed in BK+ group. Moreover, the specific T cell immune reconstitution performed in 6 cases were significant delayed in BK+ group as well compared to that in BK- group.

Conclusion

HC is correlated to BKV viruria and primary malignance disease in allo-HSCT. Poor specific immune reconstitution post-HSCT increased the risk of BK virus reactivation, and BK virus infection delayed the immune reconstitution after transplantation vise versa.

Disclosures

No relevant conflicts of interest to declare.
Potent and Timed Epigenetic Repression of PD-1 in Human T Cells
Authors: Pearl Jocelynn , Urnov Fyodor D , Stamatoyannopoulos John A
Published: October 2019

T cell exhaustion mediated by checkpoint genes - most prominently PD-1 - is believed to play a major role in limiting the efficacy of chimeric antigen receptor (CAR) T cell therapies of hematologic malignancies. Clinically durable responses to CAR-T therapy of B cell malignancies almost invariably occur in patients who achieve a complete remission by day 28 post infusion. Accumulating data indicate that the impact of T cell exhaustion is most prominent during the early post-infusion interval. PD-1 has recently been found to function as a tumor suppressor in T cells; as such permanent disablement of PD-1 expression via genetic knockout may thus increase long term adverse events from cell therapy.

We sought to develop an approach for increasing the potency and efficacy of CAR-T cell therapy of B cell and other hematologic malignancies by timed epigenetic abrogation of PD-1 expression that was limited to the critical early (21-28d) therapeutic interval. To accomplish this we engineered synthetic transcription factors coupled to a modified KRAB repressive domain that densely tiled the transcriptional regulatory regions of PD-1 at up to single-base resolution.

To identify TF-repressors that were both potent and specific, we transiently transduced each engineered TF-repressors separately into primary total CD3+, CD4+, or CD8+ human T-cells. These experiments revealed striking position dependence of TF-repressor activity, indicating that spatial and rotational positioning of the repressive domain was critical for function. We identified several TF-repressors that exhibited highly potent repression (>90%), and screened these for gene selectivity using transcriptome-wide deep RNA-seq. These studies yielded a TF-repressor with both high potency (>90%) and true single-gene (PD-1) specificity on a genomic scale.

Next we examined the durability of expression. Transduction of TF-repressor mRNA resulted in rapid expression and activity, with PD-1 repression observable beginning at 12 hours and peaking by 48 hours post transduction. Experiments with tagged TF-repressors revealed that substantially all translated protein had dissipated within 72 hours post transduction. Remarkably, however, potent PD-1 repression reliably persisted for >3 weeks post-transduction.

Collectively, our studies show the feasibility of generating highly potent and single-gene-selective epigenome editing reagents capable of reprogramming clinically relevant genes over a defined temporal interval. This approach should be broadly applicable to other key regulators of clinical response to produce genome-targeted medicines without incurring the genotoxic consequences of double-stranded DNA breaks.

Disclosures

No relevant conflicts of interest to declare.
LAMTOR2 Is Dispensable for T Cell Receptor-Mediated Signaling of Thymocytes but Controls Medullary Thymic Epithelial Cells
Authors: Wendeler Lina , Huber Lukas A , Klein Christoph , Kotlarz Daniel
Published: October 2019

Human LAMTOR2 deficiency is characterized by severe congenital neutropenia, growth failure, partial albinism, as well as B and T cell deficiencies (Bohn et al., Nat Med 2007). To determine the role of the endosomal adaptor LAMTOR2 in T cell development and homeostasis we used conditional knockout mouse models.

Mx1-Cre-driven knockout of Lamtor2 resulted in reduction of thymus weight and total thymocyte numbers. Immunophenotyping revealed an impaired T cell development characterized by a partial block at the double negative CD4-CD8- T cell precursor stage after 7 and 21 days of poly I:C injection that induced deletion of the Lamtor2 gene. Since Mx1-Cre-driven knockout does not allow a discrimination between T cell intrinsic and extrinsic effects, we next generated pre-TCRα-iCre conditional knockout mice. In contrast to Mx1-Cre-Lamtor2fl/fl mice, mice with T cell-specific knockout of Lamtor2 showed normal frequencies of total thymocytes and T cell progenitor subsets. Furthermore, LAMTOR2-deficient thymocytes exhibited normal TCR signaling (p-ERK, p-LAT, p-LCK, p-PLCγ, Nur77) and internalization of TCRβ upon stimulation with anti-CD3ε +/- anti-CD28, indicating that LAMTOR2 in T cells is dispensable for thymocyte development.

To assess whether T cell developmental defects in Mx1-Cre-Lamtor2fl/fl mice are caused by a dysfunctional thymic epithelium, we analyzed thymic epithelial cells (TECs) after 4 days of poly I:C injection by flow cytometry and detected a reduced ratio of CD45-EpCAM+UEA-1+Ly51- medullary TECs (mTECs) to CD45-EpCAM+UEA-1-Ly51+ cortical TECs in LAMTOR2-deficient mice. Further studies are underway to determine the role of LAMTOR2 in mTECs.

Taken together, our findings show that LAMTOR2 is not required for TCR-mediated signaling but plays a critical role in controlling mTEC homeostasis.

Disclosures

No relevant conflicts of interest to declare.
The Management of Cardiovascular Diseases in Patients with Hemophilia: The French Coche Registry
Authors: Guillet Benoit , Wibaut Benedicte , Falaise Céline , Lebreton Aurelien , Castet Sabine , Gautier Philippe , Claeyssens Ségolène , Schved Jean-François , Cayla Guillaume
Published: October 2019

Introduction: Over the last decades, life expectancy of patients with hemophilia (PWH) has significantly improved and therefore, age-related comorbidities like cardiovascular disease (CVD) are now common features. Using antithrombotic and antiplatelet agents, as needed for managing CVD, proves complex in PWH given that these drugs, along with the often required invasive procedures, interfere with hemostasis; general recommendations are not applicable as such.

Method: To collect data on current real-life practices in this patient population, the COCHE registry (COmorbidités Cardiovasculaires chez les patients HEmophiles) has been implemented back in July 2011. This French observational study sought to describe prospectively the management of CVD in PWH who require treatment with antiplatelet agents, antithrombotics, or both.

Results: By January 2018, this registry had prospectively included 67 PWH: 59 hemophilia A and 8 hemophilia B patients (47 mild, 10 moderate, and 10 severe), with a median age of 65±12.5 years. Overall, 52 patients were included for coronary artery disease (CAD) and 16 for atrial fibrillation (AF), with one patient presenting both CAD and AF. Among the 67 patients enrolled, 49 were receiving on-demand replacement therapy. Identified cardiovascular risk factors were arterial hypertension (n=44), overweight or obesity (n= 35), dyslipidemia (n=30; four tested positive for human immune deficiency virus [HIV]), smoking (n=17), family coronary disease history (n=11), non-insulin dependent diabetes mellitus (NIDDM) (n=11), and insulin dependent diabetes mellitus (IDDM) (n=3).

Conclusion: The proposals discussed herein are largely based on the experience we gained whilst managing PWH with CVD from the COCHE registry. To us, a multidisciplinary approach appears paramount.

Disclosures

Lebreton: Sobi: Consultancy, Research Funding.
Chromatin Organization By SATB1 Regulates HSP70 Induction in Early Erythropoiesis and Lost in Diamond Blackfan Anemia
Authors: Wilkes Mark C , Takasaki Kaoru , Youn Minyoung , Chae Hee-Don , Narla Anupama , Sakamoto Kathleen M.
Published: October 2019

Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome. The disease usually presents within the first year of life and is associated with anemia, congenital abnormalities, and an increased risk of developing cancer. The most prevalent mutations are found in the ribosomal protein RPS19, accounting for over 25% of cases but approximately 70% of DBA patients carry mutations in ribosomal genes. Downregulation of GATA1 has been attributed to the disease, but the upstream mechanisms leading to aberrant erythropoiesis is only beginning to be elucidated.

SATB1 is highly upregulated in lymphocytes (especially thymocytes) and steadily downregulated in all myeloid ineages during differentiation. However the more modest expression of SATB1 in early progenitors is required for self-renewal of HSCs and globin switching in early erythropoiesis. Using transcriptomics, we identified SATB1 expression is prematurely lost in RPS19-insufficient erythropoiesis and that over a third (16/42) of RPS19-sensitive early erythroid genes were rescued upon SATB1 re-expression. One of the most deregulated transcripts encode the chaperone HSP70.

GATA1 is an essential master regulator in erythroid differentiation and high GATA1 protein levels are required for efficient erythropoiesis. In DBA, GATA1 protein expression is drastically diminished. Two mechanisms contribute to this. At the translational level, GATA1 transcripts fail to be translated adequately due to ribosomal inefficiency at binding short, unstructured 5'UTRs. At the protein level,HSP70 loss leads to reduced GATA1 protein stability. During healthy erythropoiesis HSP70 genes are drastically upregulated (3.21, 4.18 and 1.37- fold) and this is lost in RPS19 insufficiency. Here we report that upregulation of HSP70 in healthy erythropoiesis requires SATB1. SATB1 binds to 3 sites within a 40kb region surrounding the HSP70 gene loci and recruits a distal predicted enhancer element to the proximal promoters via formation of two chromatin loops. During RPS19-insufficiency this chromatin organization is lost, but SATB1 re-expression restores chromatin looping and HSP70 expression, stabilizing GATA1 and promoting efficient erythropoiesis. As with HSP70, it is likely that permissive chromatin organization is essential for other RPS19-sensitive erythroid genes rescued by SATB1 re-expression.

Disclosures

No relevant conflicts of interest to declare.
Supplementation with Selenium As Prevention for the Development of Mucositis in Patients Undergoing HSCT. Results of a Pilot Study
Authors: Cantu Rodriguez Olga , Gomez-Almaguer David , Gutierrez Aguirre Cesar Homero , Mancias Consuelo , Tarín Arzaga Luz del Carmen , Gómez-De León Andrés , Herrera-Rojas Miguel , Garza-Ocañas Lourdes , Hawing Jose Angel
Published: October 2019

Introduction:

Numerous clinical trials have been conducted in order to reduce or prevent the appearance of mucositis in patients undergoing cancer treatment and some of these have appeared in the clinical guidelines as recommendations (Keefe,DM Cancer 2007). However, to date no molecule has been shown to prevent appearance of this kind of lesions. Selenium, a trace element in the organism, cofactor of multiple biological processes, has been used previously for mucositis chemoprevention in head and neck tumors. We conducted a study to know if selenium supplementation has an impact on the development of mucositis.

Objective:

We aim to know the impact of Selenium in patients undergoing hematopoietic stem cell transplantation, in the presentation and duration of mucositis comparing against placebo.

Methods: We performed a double blind clinical trial in a single center pilot study approved by our ethics committee. All patients candidates for autologous or allogeneic HSCT above 18 years were submitted for our study previous informed consent by our institution. Patients were randomized into one of two arms: a) selenium methionine, b) placebo.

Previously randomized, patients were administered supplementation with oral selenium methionine at a dose of 400mcg/day or placebo starting 7 days prior transplantion. We determined a baseline measurement of serum and urinary selenium. Subsequent measurements were made each week for a total of 28 days. Evaluations were made with validated SMAQ questionnaires which also included scales of mucositis severity, using the WHO mucositis scale. All of them performed by an observer blinded to what supplementation were being administered to each patient.

Results: We have recruited a total of 31 patients, of whom 16 were allogeneic and 15 autologous. We observed that 38.7% developed some degree of mucositis, most of them on the allogeneic arm (10 patients). Selenium consumption arm was associated with a median decrease of 32% (1.9 vs 2.8) in the WHO scale in the severity of mucositis compared to the placebo arm (p=0.046), however we didn't find difference in mucositis resolution time between groups (p=0.162). On the other hand, selenium supplementation was associated with shorter platelet engraftment time (p=0.041), without any significant difference in neutrophil engraftment time. With a follow up of 112 days, there are no difference about mortality or other analized variables.

Conclusions: Supplementation with selenium was associated with a slight decrease in the severity of mucositis in comparison with placebo. Without any relevant findings in relation to mortality or other variables analized.

Disclosures

Gomez-Almaguer: Novartis: Consultancy; AbbVie: Consultancy. Herrera-Rojas:Janssen: Employment.
Role of Bone Marrow Infiltration and POD 24 in Follicular Lymphoma
Authors: Rivas-Vera Silvia , Acosta-Maldonado Brenda Lizeth , Estrada-Martínez Lizeth Elisua
Published: October 2019

Follicular lymphoma (FL) is the most common indolent lymphoma with a median survival approaching 20 years. However, there is significant clinical heterogeneity with subsets of patients experiencing transformation, early recurrence or refractory disease. Some authors found that progression of disease within 24 months of diagnosis, in patients treated with chemoimmunotherapy (POD24), is associated with poor overall survival (OS).

OBJECTIVE

Evaluate the POD24 as an early clinical endpoint in FL and evaluate FL international prognostic index (FLIPI), and other baseline risk factors at diagnosis for overall survival and relapse.

METHODS

We conducted a retrospective and observational study in which 160 patients with follicular lymphoma who received R-CHOP at National Institute of Cancerology, Mexico from 2011 to 2017. We analyze with Kaplan Meier curves, log rank test and logistic regression model.

RESULTS

We analyze 160 patients, median of age was 53 years (26- 88), with a female : male ratio of 1.17:1. In this group: 86% had hemoglobin >12 mg/dL, LDH was normal in 62%, 1-4 nodal areas were affected in 64%, 56% of patients had high FLIPI score, 27% had B symptoms and we found bone marrow infiltration in 30% of cases; grade 2 Follicular lymphoma was the most common histological subtype (40.5%). Most of patients achieved complete response (81%), 12% partial response after the first line therapy. Only 13 patients (8%) presents relapse. Sixty four percent received maintenance. Only 13 of patients relapsed, 10 after 24 months and only 3 in first 24 months from diagnostic (POD 24). Fig. 1

Overall survival in our population was 89% to 8 years, the factors with statistical significance in the bivariate analysis were the more nodal regions affected (1-4), high FLIPI score and POD 24

POD 24, adjusted to FLIPI score, was an independent predictor of survival in regression analysis (p<0.041, HR:35 IC 95% 1.15-1060.21). The bone marrow infiltration at diagnosis was the only independent predictor for relapse (p<0.007, HR: 6.9, IC 95% 1.7- 28.2).

CONCLUSION:

In our study group, POD 24, was an important predictor of overall survival, and bone marrow infiltration at diagnosis was the only predictor factor for relapse.

Disclosures

No relevant conflicts of interest to declare.
Rituximab Combined with Donor Lymphocyte Infusion Cured Immunohemolysis after Refractory Hematopoietic Stem Cell Transplantation
Authors: Liao Jianyun , Peng Zhiyong , Liu Huaying , He Yuelin , Li Chunfu
Published: October 2019

Background

Immuno-hemolytic anemia is a severe complication after allogeneic hematopoietic stem cell transplantation (HSCT). Primary or secondary reaction between recipient lymphocytes and donor RBC antigens was considered as one of cause of post-transplantation immune-hemolysis (PTI). Different treatments can be administered according to the donor cell chimeric status and antibody titer of blood types of the donor in recipient.

Objective:

To find the ways of treatment of PTI after HSCT.

Methods:

A retrospective analysis about the complications of 707 patients underwent HSCT in our center from January 2010 to July 2017 was made. Of them, PI occurred in 28 patients with positive direct Coomb's test. By donor cell chimerism status, the 28 patients with PTI were divided into two groups. In group 1 (n=11), the patients presented full donor chimerism and received rituximab or/and steroid treatment only; In group 2 (n=17), the patients presented mixed chimerism and received rituximab and gradually decreasing immunosuppressive agents with/without, thereafter, donor lymphocyte infusion (DLI).

Results: A total of 28 patients was cured after treatment. Of 11 cases in group 1, five patients developed grade 1-2 acute GVHD and four of which developed pancytopenia. Of 17 patients with mixed chimerism (75-99.5%) in group 2, four had post-HSCT pure red blood cell aplasia and the major blood type incompatibility between the donor and recipients. In group 2, six patients with PTI were cured by rituximab and gradually decreasing immunosuppressive therapy only; 11 patients had no respond to the treatment of these treatments, and were cured after combined with DLI.

Conclusion:

When PTI occurred, chimerism status may help us to determine how to treat PTI. For patients with full donor cell chimerism, it may be fit to give rituximab and to reduce immunosuppressive agent gradually. DLI may be given to the patients with mixed chimerism and no responding to the combined therapy of rituximab and immunosuppressive agent reduction.

Disclosures

No relevant conflicts of interest to declare.
Observations from the First 1,000 Suspected aHUS Patients, Tested By a Rapid aHUS Genetic (NGS) Panel: Causative Genes Identified in a Subpopulation of Our Database
Authors: Anderson-Furgeson James , Kain Jamey , Venkataramani Sujatha , Lim Jooeun , Ero Mike , Lewis Bradley
Published: October 2019

Atypical Hemolytic Uremic Syndrome (aHUS) is a life-threatening, progressive genetic disease. aHUS results from uncontrolled activation of the complement system, which then targets endothelial cells, leading to thrombotic microangiopathy (TMA). The disease commonly progresses to kidney failure and death. Currently, aHUS is diagnosed by ruling out other diseases which cause TMA. Treatment is often performed with eculizumab, a terminal inhibitor of complement, which is expensive. Due to the severity of aHUS and the expense of treatment, rapid diagnosis of the disease is desirable. With a genetic sequencing assay for aHUS with a turnaround time of two days, we have gained comprehensive genetic understanding of a population of patients displaying acute clinical presentation of aHUS. The genes sequenced for the assay are CFH, MCP (CD46), CFI, C3, CFB, CFHR1, CFHR3, CFHR4, CFHR5, THBD, PLG, and DGKE, alongside deletion/duplication analysis for CFHR1-3 and CFHR1-4. Currently, over 1000 patients have been tested using this panel. We reviewed de-identified aggregate results for a subset of patients tested using this panel (416 patients). We tabulated the frequency of clear-cut causative genetic variants in each of the genes among the patients tested positive for aHUS. The most common variant among the positives tabulated is the large deletion in CFHR1, at 23%. 23% of positives have a variant in MCP, 15% in CFH, 10% in THBD, 9% in CFI, 6% in C3, 6% in PLG, 5% in CFB, 3% in CFHR5, and 1% in DGKE. Furthermore, we report the frequency of positive, equivocal and negative results, breakdown of rare variant types per gene, frequency of patients with multiple rare variants, frequency of novel variants in the genes sequenced, frequency of deletion-duplication events in CFHR1-3 and CFHR1-4, and the CFH and MCP/CD46 risk polymorphisms. These data may provide the largest overview to date of genetic variants in risk-factor genes in patients where an aHUS diagnosis is suspected.

Disclosures

No relevant conflicts of interest to declare.
High incidence of human herpesvirus 6 infection with a high viral load in cord blood stem cell transplant recipients
Authors: Sashihara Junji , Tanaka-Taya Keiko , Tanaka Shinya , Amo Kiyoko , Miyagawa Hiromi , Hosoi Gaku , Taniguchi Tomokuni , Fukui Takafumi , Kasuga Naoki , Aono Toshiya , Sako Masahiro , Hara Junichi , Yamanishi Koichi , Okada Shintaro
Published: October 2019

Human herpesvirus 6 (HHV-6) infection in recipients of cord blood stem cell transplants (CBSCTs) was estimated by semiquantitative and real-time quantitative polymerase chain reaction (PCR) and reverse-transcription PCR. Of the CBSCT recipients, 7 (70%) of 10 had active HHV-6 infection after transplantation, and all 7 were inferred from their age to have already had a primary infection. Because HHV-6 DNA is seldom detected in cord blood, these cases were considered likely to represent reactivation. In contrast, the 3 patients without HHV-6 infection were all believed to be naive regarding HHV-6 primary infection because of their age and the results of PCR assays given before the transplantation procedure. The incidence of HHV-6 infection after transplantation was significantly higher (P < .05) than after bone marrow (BM) transplantation and peripheral blood stem cell (PBSC) transplantation, when recipients without primary HHV-6 infection prior to transplantation were excluded (CBSCT, 100%; BMT/PBSCT, 56.3%). Real-time PCR revealed a higher level of viral DNA in the peripheral blood mononuclear cells from CBSCT recipients than from BMT/PBSCT recipients or patients with exanthem subitum (P < .05). HHV-6 mRNA of the U79/80gene was also detected by reverse-transcription PCR in all analyzed patients with HHV-6 infection. Its detection was correlated with the emergence of viral DNA in the plasma and symptoms such as fever and rash. Thus, HHV-6 infection was more frequent and the viral load was higher in CBSCT recipients with prior primary infection.
Liposomal Amphotericin B (L-AMB) Is Superior to Amphotericin B Deoxycholate (AmB-d) in Preventing Breakthrough Fungal Infections in Patients with Prolonged Neutropenia and Fever: Results of a Sub-Group Analysis of an Empirical Antifungal Therapy Trial.
Authors: Cordonnier Catherine , Walsh Thomas J. , Bresnik Mark
Published: October 2019

In a trial of empirical antifungal therapy in persistently febrile neutropenic patients, L-AMB was shown to be comparable in overall efficacy with significantly less nephrotoxicity and infusion-related reactions when compared to AmB-d. In addition, significantly fewer proven breakthrough invasive fungal infections (IFI) occurred in the L-AMB treatment group. (Walsh TJ, et al, NEJM1999; 340: 764–71). In order to better evaluate the maximum efficacy benefit of L-AMB in a higher risk patient subset, a sub-group analysis was performed on those patients with duration of neutropenia greater than or equal to the median duration for the entire study population. The results of this sub-group analysis are presented.

Methods: From the original data set, the median duration of neutropenia from the time of treatment initiation was determined. Clinical success (defined by a composite endpoint), frequency of proven breakthrough IFI and all cause mortality were reported by treatment group for patients with duration of neutropenia greater than or equal to the median. Investigator-reported proven breakthrough IFI were reviewed and verified by an independent, blinded expert.

Results: A total of 687 patients were enrolled in the trial. Median duration of neutropenia following initiation of antifungal therapy was 7 days (range 1–50). 392 patients had neutropenia >/= 7 days, (L-AMB=205), (AmB-d=187). Clinical success: L-AMB: 128/205 (62.4%); AmB-d: 112/187 (59.9%), and all cause mortality: L-AMB: 19/205 (9.3%); AmB-d: 24/187 (12.8%) were not significantly different. The frequency of proven breakthrough IFI favored L-AMB: L-AMB: 7/205 (3.4%); AmB-d: 18/187 (9.6%), p=0.01. Organisms isolated: L-AMB: 2 Candida spp (blood); 4 Aspergillus spp. (3 pneumonia, 1 wound); 1 Mucor sp (pneumonia). AmB-d: 9 Candida spp (8 blood, 1 pneumonia); 7 Aspergillus spp (4 pneumonia, 1 sinus, 1 pericarditis, 1 wound); 1 Cryptococcus albidus (blood); 1 unclassified mould (pneumonia). The difference in emergent proven IFI for the patients with neutropenia <7d trended in favor of L-AMB, but was not statistically significant: L-AMB 3/138 (2.2%); AmB-d 8/157 (5.1%).

Conclusions: Overall clinical success and all cause mortality remained equivalent for the 2 treatment arms in the subset of patients with prolonged neutropenia. However, L-AMB significantly reduced the frequency of proven breakthrough IFI in this high-risk patient population. These data support the efficacy of L-AMB as empirical therapy, particularly for persistently neutropenic patients at higher risk for invasive fungal infections.
In Vivo Downregulation of Bcl-2 by G3139 (G) Enhances Rituximab (R) Anti-Tumor Activity: Effect of Sequential Versus Concurrent G3139 Administration on Survival in a Lymphoma-Bearing SCID Mouse Model.
Authors: Ramanarayanan Jeyanthi , Hernandez-Ilizaliturri Francisco J. , Czuczman Myron S.
Published: October 2019

Bcl-2 over expression has been associated with poor prognosis and chemoresistance in various hematologic and solid tumor malignancies. G, an antisense oligonucleotide (ASO) targeting Bcl-2, downregulates Bcl-2 expression favoring apoptosis in cancer cells. Previously, we have demonstrated that G enhances R anti-tumor activity in various lymphoma pre-clinical models. Clinical trials are being conducted to evaluate the safety and efficacy of G3139 combined with R in relapsed/refractory NHL patients. However, the optimal sequence of administration of G and R is unknown. Our main objective was to determine the optimal anti-tumor schedule to administer G in combination with R. Six 8-week old SCID mice were inoculated via tail vein injection with Raji cells (1 x 106 on day 0). After five days, to allow for tumor engraftment, mice were assigned to receive placebo [P], G, R, R followed by G [R→G], G followed by R [G→R] or G preceding each dose of R [G+R]. In addition, other control groups consisted of lymphoma-bearing mice treated with G3622 (ASO control) [reverse sequence, RS], trastuzumab (isotype) [I] and R in combination with G3622 [R+RS]. Antibodies were administered vial tail vein injection at a dose of 10mg/kg/dose x 4 doses and ASO were administered intraperitoneally at 5mg/kg/dose x 8 doses. The end-point of the study was survival. Survival analysis was performed by Kaplan-Meier curves and p values calculated using log rank test. Differences in survival of lymphoma-bearing SCID mice were noted according to the schedule of G/R administration. Synergistic activity was observed in animals treated with [G+R]. A modest improvement in the mean survival was noted in animals treated with [G] (26 days) or [G→R] (28 days) when compared to control mice (20 days) (P < 0.001). Anti-tumor activity was observed in SCID mice treated with R (mean survival 34 days) and was improved by the sequential addition of G [R→G] (median survival 48 days, P = 0.038). Concomitant administration of G and R resulted in the highest anti-tumor activity as measured by the longest survival (mean 80 days, P < 0.001). Our data demonstrates differences in R/G anti-tumor activity depending on the schedule of administration of G and R. Sequential administration of R followed by G resulted in a better disease control than G followed by R. However, concurrent G and R administration was shown to be superior to sequential strategies in improving survival of lymphoma-bearing SCID mice. Our pre-clinical data suggest the importance of a concurrent administration of these agents in clinical trials whereby, down-regulation of Bcl-2 by G allows enhancement of rituximab-associated anti-tumor activity.
Herpes Simplex Virus Infection of Endothelial Cells Is Enhanced by Thrombin.
Authors: Sutherland Michael R. , Friedman Harvey M. , Pryzdial Edward L.
Published: October 2019

Herpes simplex virus type 1 (HSV1) and type 2 (HSV2) are highly prevalent and form life-long infections. While acute effects are not often critical, chronic infection has been linked to vascular disease. To better understand the underlying basis for the clinical correlations, our lab has demonstrated that HSV can initiate and sustain clot formation directly on the virus surface, bypassing the normal cellular requirements. This is facilitated by anionic phospholipid and tissue factor on HSV1 and HSV2, and the participation of at least HSV1- encoded glycoprotein C (gC) in factor VIIa-dependent factor X (FX) activation, emulating tissue factor. The evolution of the HSV surface as a procoagulant agent suggests that generation of thrombin is advantageous to the viral lifecycle. The current study therefore examines the hypothesis that thrombin enhances infection. Several purified HSV1 strains were evaluated, including the wild-type lab strain, MacIntyre, and the clinical isolate, NS. These viruses were compared to gC-deficient HSV1, ns-1, and the gC-restored strain, rns. As an example of HSV2, strain G, was also examined. ns-1 has been shown previously to generate less FXa per particle than the gC-containing viruses. To restrict in situ thrombin production, human umbilical vein endothelial cells were inoculated in serum-free culture media. Consistent with our hypothesis, standard plaque-forming assays revealed that co-incubation of the virus and cells with purified thrombin enhanced infection by up to 400%. The observed half-maximal concentration was approximately 20 nM thrombin for all viruses studied. As a demonstration of specificity, hirudin attenuated the effects of purified thrombin. The involvement of the protease activated receptor 1 (PAR1) in this mechanism was shown using the functional signaling analogue, thrombin receptor activating peptide (TRAP). In the absence of serum, TRAP resulted in increased infection comparable to that of purified thrombin. Approximately half the effect of TRAP was observed at 10uM, while a PAR4-activating peptide had no effect. Enabling the production of thrombin during the infection process, plaque-forming assays were also conducted with serum in the cell culture media. The presence of serum enhanced infectivity by as much as 20-fold. At a constant hirudin concentration of 5 U/ml, infection was attenuated by approximately 50% for the gC-deficient strain, ns-1, whereas the other viruses were only marginally or insignificantly affected, supporting a role for thrombin generation in situ and the participation of gC. Collectively, these data show that thrombin production by HSV enhances infection through a mechanism involving PAR1-mediated cell modulation.
Differential Binding of Plasminogen, Plasmin, and Angiostatin4.5 to Cell Surface β-Actin: Implications for Cancer-Mediated Angiogenesis.
Authors: Soff Gerald A. , Wang Hao , Doll Jennifer A. , Jiang Keyi , Cundiff Deborah L.
Published: October 2019

Angiostatin4.5 (AS4.5), the naturally occurring human angiostatin isoform, is the product of plasmin autoproteolysis and consists of kringles 1 to 4 and ~85% of kringle 5. An important question related to tumor angiogenesis is the mechanism by which tumor cells convert plasminogen to angiostatin locally and yet the angiostatin acts systemically. We recently reported that cancer cell surface globular β-actin mediates plasmin autoproteolysis to AS4.5. We now characterize the binding of plasminogen, plasmin and AS4.5 to globular β-actin. Plasminogen and plasmin bind comparably to immobilized globular β-actin, with a KD of approximately 140 nM. The binding is inhibited by ε-aminocaproic acid (EACA), a lysine analog, indicating the requirement for a lysine-kringle domain interaction. Using a series of peptides derived from β-actin in competitive binding studies, we show that the actin domain necessary for binding of plasminogen is within “GDEAQSKRGILTLKY”, (amino acids 55 to 69), which includes lys61 and lys68. Substitution of lys61 or Lys68 with arginine results in the loss of the peptide’s ability to block plasminogen binding, indicating that lys61 and Lys68 are necessary for plasminogen binding. Other actin peptides, including peptides with other lysines, did not inhibit the plasminogen-actin interaction. While plasminogen and plasmin bind to actin efficiently, AS4.5 did not bind, even at high concentrations (500 nM). Plasminogen, plasmin, and AS4.5 all contain kringles 1–4, however, kringle 5 is truncated in AS4.5, suggesting that the kringle 5 is the domain by which plasminogen and plasmin bind to actin. We thus tested pure recombinant kringle 5 for its ability to specifically bind actin. Isolated kringle 5 was shown to bind to actin supporting the hypothesis that intact kringle 5 is necessary for plasminogen and plasmin to bind to cell surface β-actin, and the truncated kringle 5 in AS4.5 results in its release from β-actin. We propose the following paradigm: Plasminogen binds cell surface globular β-actin, where uPA mediates activation to plasmin which remains bound to actin. Actin then mediates plasmin autoproteolysis, with an intra-kringle 5 cleavage, resulting in AS4.5 formation. With the truncation of kringle 5, the AS4.5 dissociates from the surface actin and enters the systemic circulation. These data may help explain why AS4.5 is formed locally, on cancer cell surfaces, and yet acts systemically.