bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evolution of Clinically Relevant Subclones during Chemotherapy Treatment of ALL As Determined By Single-Cell DNA and RNA Sequencing
Authors: Albertí Servera Llucia , Demeyer Sofie , Govaerts Inge , Gielen Olga , Segers Heidi , Uyttebroeck Anne , Boeckx Nancy , Maertens Johan A. , De Keersmaecker Kim , Cools Jan 
Published: November 2019

Acute lymphoblastic leukemia (ALL), which is the most common cancer in children, shows extensive genetic intra-tumoral heterogeneity. This heterogeneity might be the underlying reason for an incomplete response to treatment and for the development of relapse. In order to envision the clinical implementation of a refined risk-category strategy based on ALL subclonal composition, it is essential to first generate a reference single-cell map and accumulate evidence on how the subclonal composition affects the response to treatment. For that, we performed large-scale and integrative single-cell genome and transcriptome profiling of pediatric samples at diagnosis, during drug treatment and in case of relapse. We used the 10x Genomics platform for single-cell RNA-sequencing analysis (around 4000 cells per sample) and the Tapestri Platform (Mission Bio) for targeted single-cell DNA-sequencing (around 5000 cells per sample) of the most mutated genomic regions in ALL. For the later, we developed a custom panel that covers 305 ALL mutational hotspots across 110 genes.
We have determined a reference single-cell map of the cellular (based on the gene expression profile) and the clonal composition (based on the co-occurrence of mutations at each individual cell) for pediatric ALL at diagnosis (8 T-ALL and 10 B-ALL patients). We have also reconstructed the tumor phylogeny highlighting the order of mutational acquisition and the most likely pattern of evolution. Moreover, we have studied how T-ALL evolves during drug treatment at single-cell resolution in 4 patients, unraveling which are the most sensitive clones to the therapy, which are the most resistant ones and when relapse clones originated. Single-cell RNA-sequencing also provided information on how normal hematopoiesis recovers during chemotherapy treatment.
The results show that ALL is typically composed by a major clone and 5-10 smaller clones that have different sensitivities to the therapy. We have been able to detect minor clones (<1% of the cells) at diagnosis that are clinically relevant. These very rare clones are either not responding to the therapy (present at minimal residual disease) or form the relapsed leukemia. These findings could have clinical implications for improved risk-stratification methods based on individualized patient's molecular profiles.

Disclosures
Maertens: Gilead Sciences: Other: Grants, personal fees and non-financial support; Cidara: Other: Personal fees and non-financial support; Amplyx: Other: Personal fees and non-financial support; F2G: Other: Personal fees and non-financial support; Merck: Other: Personal fees and non-financial support; Pfizer: Other: Grant and personal fees; Astellas Pharma: Other: Personal fees and non-financial support.
Middle East Respiratory Syndrome (MERS) Infection in Hematological and Oncological Patients: A Case Series from a Tertiary Care Center in Saudi Arabia
Authors: Alaskar Ahmed , Bosaeed Mohammed , Rehan Hina , Mendoza May Anne , Alahmari Bader , Othman Adel , Alhejazi Ayman , Abuelgasim Khadega , Damlaj Moussab , Alzahrani Mohsen , Gmati Giamal , Salama Hind , Abolfotouh Mostafah , Shaheen Naila 
Published: November 2019

We present the largest to date of a case series of nine patients with hematological and oncological malignancies who were infected with Middle East Respiratory Syndrome Coronavirus (MERS-CoV). MERS-CoV is a novel beta-coronavirus with a high fatality rate in comorbid patients. The majority of MERS cases globally were reported from Saudi Arabia (1983 cases, including 745 related deaths with a case-fatality rate of 37.5%) according to the WHO update of February 2019. All were clinically stable before acquiring the virus.
Most of the cases had an active disease as relapse or refractory with three cases being neutropenic. The clinical presentation and radiological features of the patients were variable and inconsistent (Table 1). Diagnosis was confirmed with RT-PCR assays targeting upstream of the E gene and the open-reading frame gene 1a which had to be done repeatedly and required an average of 3 (with max. of 7) samples for a test to be positive (Table 2). All the patients developed respiratory failure, were admitted to the critical care unit (ICU) and required mechanical ventilation. The length of hospital stay ranged from 15 - 48, with an average of 24 days. Unfortunately, all nine patients died within days after admission to the ICU. In addition, the time from diagnosis to death has an average of 9 days ranging from 2-24 days, respectively.
In conclusion, MERS CoV infection in hematology/oncology patients has a very poor prognosis regardless of the status of the underlying disease. The clinical presentation is not distinctive and confirming the diagnosis requires numerous respiratory samples. Measures to prevent nosocomial outbreaks should include proper compliance with personal protection equipment by health-care workers when managing patients with suspected and confirmed MERS-CoV infection and prompt isolation of infected patients. Future research is required to enhance our understanding of the disease and to evaluate superior diagnostic and therapeutic options.

Disclosures
No relevant conflicts of interest to declare.
Lymphatic Control of the Tumor Immune Microenvironment
Authors: Swartz Melody A. 
Published: November 2019

Tumor engagement or activation of surrounding lymphatic vessels is well-known to correlate with tumor progression and metastasis in melanoma and many other cancers. We and others have identified several mechanisms by which the lymphatic growth factor VEGF-C and lymphangiogenesis can promote metastasis, including (i) increasing immune suppressive cell types and factors in the tumor microenvironment both directly and indirectly, (ii) inhibiting maturation of antigen-presenting cells and T cell activation, and (iii) driving changes in the stromal microenvironment that promote both cancer invasion and immune suppression. However, lymphatic activation also enhances communication with cells in the draining lymph node by antigen and cell transport, which may trigger the initiation of adaptive immune responses against the tumor. Under normal conditions, the potential anti-tumor effects are rendered 'dormant' by the pro-tumor immune suppression, and the tumor progresses. However, we are now observing that lymphangiogenic tumors are exceptionally responsive to immunotherapy, implying that the anti-tumor aspects can be unleashed when the overall balance of pro- and anti-tumor immune aspects is tipped enough towards the latter (e.g., upon tumor cell killing). On the mechanistic side, we are finding that 'lymphangiogenic potentiation' depends on tumor cell infiltration of both CD103+ dendritic cells and naïve T cells, driving local T cell education post-immunotherapy and antigen spreading. On the translational side, we are developing novel strategies to exploit lymphangiogenesis for cancer immunotherapy. Understanding the yin and yang of lymphatic activation in the tumor microenvironment and how it affects immunity may lead to exciting new translational strategies for cancer immunotherapy.

Disclosures
No relevant conflicts of interest to declare.
Synergistic Activity of Belantamab Mafodotin (anti-BCMA immuno-conjugate) with PF-03084014 (gamma-secretase inhibitor) in Bcma-Expressing Cancer Cell Lines
Authors: Eastman Stephen , Shelton Christopher , Gupta Ira , Krueger Julie , Blackwell Christina , Bojczuk Paul M 
Published: November 2019

Multiple myeloma (MM) is a plasma cell malignancy characterized by clonal proliferation of plasma cells within the bone marrow. B cell maturation antigen (BCMA) is a cell-surface receptor required for the survival of plasma cells and is also ubiquitously expressed on MM cells. Belantamab Mafodotin (GSK2857916) is a humanised monoclonal anti-BCMA antibody, which is afucosylated and conjugated to the microtubule-disrupting agent monomethyl auristatin-F (MMAF). Upon binding to BCMA on the cell surface, belatamab mafodotin is rapidly internalised and the cytotoxic moiety (cys-mcMMAF) is released, leading to direct cell death.
BCMA is directly shed from the plasma membrane by gamma-secretase, a type-I sheddase. In order to further enhance belantamab mafodotin activity, we sought to increase cell surface levels of BCMA by blocking shedding of BCMA with a gamma-secretase inhibitor (GSI). We then determined the effect on the activity of belantamab mafodotin by combining belantamab mafodotin with PF-03084014, a highly-selective GSI. In order to understand combination effects against immuno-conjugate activity, a 3-day proliferation assay on a panel of multiple myeloma and lymphoma cell lines with varying levels of BCMA expression was conducted. The assay showed a 50 to 3,000-fold EC50 shift in cell lines sensitive to belantamab mafodotin across multiple lymphoma cell types.
Antibody-dependent cellular cytotoxicity (ADCC) activity of belantamab mafodotin in combination with PF-03084014 was also examined. In a 24-hour ADCC Jurkat reporter assay, an EC50 shift across multiple BCMA-expressing cell lines was observed. Even cell lines with very low BCMA expression, such as Raji, showed a synergistic increase in ADCC activity in combination with PF-03084014. Cell lines that were non-responsive in the cell proliferation assay, showed activity in the ADCC assay, indicating low-expressing BCMA cell lines remain sensitive to belantamab mafodotin, alone and in combination with PF-03084014.
Synergistic effect from this preclinical work provided rationale to support clinical evaluation of belantamab mafodotin in combination with PF-03084014 in a planned clinical trial (DREAMM-5).

Disclosures
Eastman: GlaxoSmithKline: Employment, Equity Ownership. Shelton:GlaxoSmithKline: Employment, Equity Ownership. Gupta:GlaxoSmithKline: Employment, Equity Ownership. Krueger:GlaxoSmithKline: Employment, Equity Ownership. Blackwell:GlaxoSmithKline: Employment, Equity Ownership. Bojczuk:GlaxoSmithKline: Employment, Equity Ownership.
Low-Dose Triptolide Enhances Bromodomain Inhibitor JQ1-Induced Apoptosis of Acute Myeloid Leukemia Cells Via Down-Regulation of c-Myc Following RAS/MEK/ERK Inhibition
Authors: Xu Bing , Shi Yuanfei , Zhao Haijun , Qiu Yi 
Published: November 2019

The current therapy for acute myeloid leukemia (AML) remains greatly challengeable because most patients ultimately relapse due to resistant to current chemotherapies. Therefore, development of novel therapies using a variety of agents targeting varying pathways is needed to further improve patients' survival. Here we report that low dose triptolide (TPL), a natural product derived from plant Tripterygium wilfordii, could enhance bromodomain inhibitor JQ1-induced apoptosis of AML cell lines as well as primary cells from patients with de novo and relapsed or refractory (R/R) AML, while sparing their normal counterparts. Moreover, combination of TPL and JQ1 showed a more powerful activity to suppress AML cell growth in a xenograft model than single agent. Mechanistically, the synergetic effects of TPL and JQ1 were associated with impaired mitochondrial membrane potential, increased reactive oxygen species (ROS) production and imbalance of the Bcl2 family of pro-apoptotic and anti-apoptotic proteins, leading to caspase-dependent apoptosis. Meanwhile, RNAseq analysis revealed that ERK/MAPK signaling cascades are responsible for the enhanced activity of TPL in combination with JQ1 against AML. In summary, this study demonstrates that low-dose TPL synergizes with JQ1 to target AML through inhibiting ERK/MAPK signaling and mitochondrial apoptosis pathway, supporting a potential option of combination of TPL and JQ1 for AML treatment.

Disclosures
No relevant conflicts of interest to declare.
Noninvasive Microscopic Imaging Reveals Increased Leukocyte Adhesion and Rolling in Skin of Acute Graft-Versus-Host Disease Patients Compared to Post-Transplant Controls
Authors: Saknite Inga , Byrne Michael T , Jagasia Madan , Tkaczyk Eric R. 
Published: November 2019

BACKGROUND. The nature and kinetics of leukocyte migration are intimately connected to the pathophysiology of acute graft-versus-host disease (aGVHD). Real-time visualization of leukocyte-endothelial interactions in patients post hematopoietic transplantation (HCT) may augment clinical diagnosis and patient management. In this cross-sectional pilot study, we aimed to test the feasibility of parameters characteristic of leukocyte motion in skin capillaries as potential imaging biomarkers to detect aGVHD.
STUDY POPULATION. We enrolled 16 post-HCT patients: 8 patients with any organ aGVHD and 8 patients with no organ aGVHD on the day of the imaging. Diagnoses were retrospectively confirmed on day 100 by a transplant physician (MB), who was blinded to the results of the confocal imaging. 7 out of 8 aGVHD patients had only skin involvement: stage 1 (N=2 patients), stage 2 (N=2), and stage 3 (N=3) cutaneous aGVHD, whereas one patient had stage 1 gut aGVHD with no skin involvement. 6 out of 8 aGVHD group patients required systemic therapy with steroids.
METHODS. To noninvasively visualize leukocyte motion in the topmost capillaries of post-HCT patients' skin, we used a clinical reflectance confocal microscope (Vivascope 1500, Caliber I.D.). It enables real-time en face view of individual cells at 9 frames per second. We took twenty 30-second videos of different capillaries in the left volar forearm and left upper chest. We counted the number of adherent and rolling leukocytes per 10 minutes of videos per patient.
RESULTS. We found increased leukocyte rolling in the aGVHD group (average of 3 rolling leukocytes per patient), compared to post-HCT controls (average of 1 rolling leukocyte). Highest leukocyte rolling (>8 rolling leukocytes) was associated with increased all-cause mortality, but not predictive of transplant-related mortality (TRM). Similarly, we found increased leukocyte adhesion in the aGVHD group (average of 3 adherent leukocytes per patient), compared to post-HCT controls (average of 1 adherent leukocyte). Interestingly, we observed 7 adherent leukocytes in one patient who had isolated gut aGVHD and no skin involvement.
DISCUSSION. Our preliminary results show altered leukocyte-endothelial interactions in the skin capillaries of aGVHD patients, suggesting that confocal microscopy may be an important tool in augmenting the clinical diagnosis and managing post-HCT patients. Future studies should test whether there is a difference in the pattern of change in parameter values between patients who did and who did not develop aGVHD.
ACKNOWLEDGEMENT. This work was supported in part by Career Development Award Number IK2 CX001785 from the United Sates Department of Veterans Affairs Clinical Science R&D (CSRD) Service.

Disclosures
Byrne: Karyopharm: Research Funding. Jagasia:Janssen: Research Funding; Kadmon: Consultancy; Incyte: Consultancy. Tkaczyk:Incyte: Consultancy.
Monitoring of Recombinant Activated Coagulation Factor VII (rFVIIa) Therapy in Patients (pts) with Inherited Factor VII (FVII) Deficiency
Authors: Galstian Gennadii M. , Polevodova Olesya A. , Yakovleva Elena , Shchekina Antonina , Davydkin Igor , Kudlay Dmitriy 
Published: November 2019

Introduction and Objectives: Inherited FVII deficiency is a rare hemorrhagic disorder.Clinical bleeding does not correlate with the level of FVII plasma activity. FVII involves in the extrinsic coagulation pathway. ROTEM is point of care method. EXTEM is one of ROTEM screening tests. EXTEM analyses the extrinsic pathway of coagulation. Aim of the study was to evaluate hemostatic effect and safety of rFVIIa treatment in pts with inherited FVII deficiency.
Materials and Methods: The results of rFVIIa treatment were investigated in 4 pts (1 male, 3 females) with inherited FVII deficiency. Pts were treated with rFVIIa before gynecological (2), abdominal (1) and orthopedic surgery (1). Pts received 30 mcg/kg of rFVIIa (Koagil VII, Generium, Russia) before surgery. Plasma FVII activity, fibrinogen, INR, APTT, ROTEM and thromboelastography (TEG) parameters were investigated before rFVIIa administration, then in 15 minutes, in 2 hours, in 6 hours and in 12 hours.
Results: Before rFVIIa administration median plasma FVII activity was low, CT EXTEM was long and INR was greater than 3. TEG, APTT and fibrinogen were in normal ranges (table 1, fig. 1). Fifteen minutes after rFVIIa administration FVII:C activity increased, INR and CT EXTEM decreased. Hemostatic effect of rFVIIa remained during 12 hours. During this period CT EXTEM correlated with INR (r = 0.97, p<0.001). Other parameters did not change significantly. None patients had hemorrhagic complications.
Conclusion: CT EXTEM allowed to evaluate hemostatic effect of rFVIIa in operating room and good correlated with INR in pts with inherited FVII deficiency.

Disclosures
No relevant conflicts of interest to declare.
The Frequency of Calr and MPL Gene Mutations in Jak2 V617F - Positive Chronic Myeloproliferative Neoplasms in Russia
Authors: Makarik Tatiana V , Abdullaev Adhamjon O , Kulikov Sergei M. , Nikulina Elena E , Treglazova Svetlana A , Subortseva Irina N , Kovrigina Alla M. , Melikyan Anait L. , Sudarikov Andrey B , Savchenko Valery G. 
Published: November 2019

Background. Ph-negative chronic myeloproliferative neoplasms (MPNs) are characterized by proliferation of one or more myeloid cell lineages and include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Somatic Jak2, MPL and CALR gene mutations are responsible for more than 90% of NPM cases. These mutations affect sequential stages of prolipherative signal transduction and therefore after the emergence of one type of mutation another types basically should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases.
Aim. To evaluate frequencies of MPL and CALR mutations in Jak2 positive MPN cases for Russian cohort of patients.
Methods. Archival DNA samples from MPN patients followed up at the National Research Center for Hematology between 2014 and 2019 included into retrospective study. DNAs and RNAs were extracted from blood using reagent kit from Interlabservice (Russia). Jak2 V617F mutation was quantified by real-time PCR kit from Syntol (Russia) according to manufacturers instructions. CALR exon 9 deletions/insertions were analyzed by fragment analysis (sensitivity >= 3%). MPL W515L/K mutations were assessed by in-house allele specific PCR. All cases were tested for phi-negativity using BCR-ABl p210 PCR kit from Interlabservice (Russia).
Results. At least one of the mutations was found in 3863 cases. Jak2 V617F mutation - 3385 cases (87.6%); CALR insertion or deletion - 471 case (12.2%); MPLW515L/K mutation - 31 case (0.8%). We have found 28 cases (0.7%) with Jak2 and CALR mutations combined and 3 cases (0.1%) with Jak2 and MPL mutations in the cohort studied. Matched measures were obtained at least twice at different time points during the course of disease for these cases. No cases with simultaneous CALR and MPL mutations were detected. In 23 from 31 (74%) cases with combined mutations Jak2 V617F allele burden was lower than 3%. Among cases with combined mutations 5 were diagnosed with PV, 8 - with ET, 8 - with PMF and 10 with unclassified MPN. No correlations between diagnosis, mutation combination or allele burden were found.
Conclusions. Based on the data, obtained on retrospective DNA samples we cannot state whether combined mutations are present in different clones of myeloid cells or in one. Indirectly, the fact that more often mutations in CALR and MPL genes were found in the cases with a low Jak2 V617F allele burden may indicate that additional mutations occur in the "competing" cell clone. Further prospective studies with mutation monitoring over the therapy are required to assess the value of combined mutations for MPN pathogenesis.

Disclosures
No relevant conflicts of interest to declare.
Determining Postoperative Thrombocyte Reduction Cut Off to Predict 30-Day Mortality in Patients Undergo Coronary Artery Bypass Graft Surgery
Authors: Rinaldi Ikhwan , Mansjoer Arif 
Published: November 2019

Background
There are many factors associated with early mortality after CABG, including postoperative thrombocytopenia (Kertai, 2016). Many factors during CABG surgery, such as administration of heparin or cardio pulmonary bypass during surgery are related to thrombocyte count reduction (Hamid, Akhtar, Naqvi, & Ahsan, 2017; Arepally, 2017). However, it is possible for a post-CABG patient to suffer a significant thrombocyte reduction without reaching the thrombocytopenic state (thrombocyte count <150000/µL). Up to this time, there is still lack of study about association between thrombocyte reduction after surgery and 30-day mortality in patients undergo CABG. This study aim to determine cut off point for postoperative thrombocyte reduction as a predictor of 30-day mortality after CABG surgery.
Method
This is a retrospective cohort study using medical record of 263 adult patients who underwent CABG surgery in dr. Ciptomangunkusumo National Hospital on 2012-2015. Thrombocyte reduction was determined by substracting preoperative thrombocyte count from postoperative thrombocyte count. Receiver operating curve (ROC) analysis between percentage of thrombocyte reduction and 30-day mortality after surgery was done to obtain the sensitivity and specificity value of a particular degree of thrombocyte reduction. Cut off point was obtained from intersection between sensitivity and specificity value.
Result
Thirty-day mortality rate after CABG surgery in this study was 11.9%. Cut off point obtained from ROC analysis was 30% with area under the curve (AUC) 0.671. The sensitivity of this cut off point to predict early mortality after CABG surgery was 64.5%, while the specificity was 64.7%
Conclusion
Thrombocyte reduction more than or equal to 30% can be used as a predictor of 30-day mortality after CABG surgery.
Figure

Disclosures
No relevant conflicts of interest to declare.