bluuebunny/crossref_metadata_2025 huggingface dataset:
The Cost of Hospitalization for Thromboembolic Events in Patients with Colon or Lung Cancer
Authors: Borget Isabelle , Meyer Guy , Scotté Florian , Martelli Nicolas , Vainchtock Alexandre , Elalamy Ismail
Published: October 2019

Background: Venous Thromboembolic Event (VTE) is a common complication for cancer patients, leading to hospitalizations that increase the burden of cancer management. We evaluated the incidence and costs of VTE-related hospitalizations for patients with colon cancer (CC) or lung cancer (LC).

Methods: The French national hospital database (PMSI) was analyzed in order to identify patients whose colon cancer or lung cancer was diagnosed in 2010 and who were hospitalized for a VTE at least once during the following two years. The numbers of stays induced by a VTE, and the corresponding number of patients hospitalized, were determined using the disease-specific ICD-10 codes. Associated hospital costs were estimated from the perspective of the third-party payer, using the French official tariffs, on the 1,111 and 1,641 hospitalizations with VTE classified as primary or related diagnosis only (PD / RD) for colon and lung cancer patients, respectively.

Results: We identified 47,954 new patients with colon cancer and 39,454 new patients with lung cancer; in each group, 2,866 (6.0%) and 3,775 (9.6%), respectively, were hospitalized for a VTE or had a VTE during their hospital stay. During the 2 years of follow-up of these 6,641 patients, 1,707 (25.7%) were hospitalized for a VTE recurrence. In total, 4,104 stays for CC patients and 5,962 stays for LC patients were analyzed, including stays for recurrence.

In colon cancer patients, the mean cost per stay for a VTE classified as PD / RD amounted to 3,612 Euros and 3,457 Euros for first event and recurrence, respectively, and in lung cancer patients, to 3,674 Euros and 3,222 Euros for first event and recurrence, respectively. During the time of the study, mean hospitalization cost per patient who had at least one stay for recurrence was 5,441 Euros and 5,676 Euros in colon and lung cancer, respectively. Over a 2-year period, the total cost of hospital stays induced by VTEs classified as PD / RD reached 3.99 million Euros and 5.90 million Euros for colon and lung cancer, respectively, including a total amount of 1.49 million Euros for VTE recurrence.

Conclusion: In colon and lung cancer patients, VTE-related hospitalizations remain frequent and induce an elevated cost. This economic burden may be reduced by decreasing the occurrence of thromboembolic complications, using adequate prophylaxis and efficient management in this at-risk population.

Table: Cost of VTE-related hospital stays for patients with colon cancer or lung cancer 1st event Recurrence Global(1st event and recurrence) Colon cancer Mean cost/stay (Euros) 3,612 3,457 3,588 Total cost (Euros) 3,377,219 608,499 3,985,718 Lung cancer Mean cost/stay (Euros) 3,674 3,222 3,599 Total cost (Euros) 5,022,549 879,492 5,902,041

Disclosures

Borget: LEO Pharma: Honoraria. Meyer:LEO Pharma: Research Funding. Scotté:LEO Pharma: Honoraria. Martelli:LEO Pharma: Honoraria. Vainchtock:HEVA: Research Funding.
Acute Myeloid Leukemia-Derived Exosomes Transform Bone Marrow Niche into Leukemic Niche.
Authors: Kumar Bijender , Garcia Mayra , Weng Lihong , Lewis Xiaoman , Murakami Jodi , Hu Xingbin , McDonald Tinisha , DiGiusto David , Kumar Ashish , Bhatia Ravi , Chen Ching-Cheng
Published: October 2019

Increasing evidence suggests that leukemia cells take shelter in the bone marrow (BM) niche, where they hide from chemotherapy and continue to divide. As yet, how leukemia cells alter the BM niche to facilitate their growth and assist them in evading chemotherapy is unclear. In this study, we provide compelling evidences that acute myeloid leukemia (AML), through exosome secretion, transformed the BM niche to facilitate their own growth and suppress normal hematopoiesis.

Using AML xenograft and MLL-AF9 knock-in mouse model, we show that leukemia cells as well as AML-derived exosomes stimulate the growth of BM stromal progenitors and blocked the osteolineage development in our stromal compartment analysis. Histological analysis and micro-CT examination confirmed loss or thinning of the bone in both leukemia and leukemic exosome-treated animals. Expression of cell adhesion molecules (NCAM1, VCAM1, CD44, OPN & ICAM1) and factors important for angiogenesis (Angpt1, Angpt2 and VEGF) are upregulated, whereas genes important for HSC maintenance (CXCL12 and SCF), osteoblast (OCN, OSX, Notch3 and IGF1) and chondrocyte (ACAN, SOX9) development are suppressed.

While we observed increases in phenotypic LT-HSC in AML-derived exosomes treated mice, these mice show reduced multilineage reconstitution ability, increased cell cycle entry and higher sensitivity to myeloablative stress suggesting that HSCs from exosome-treated mice have lower stem cell activity than their counterparts from normal mice.In addition, leukemia-modified stroma cells exhibit marked reduction in ability to support normal HSCs. Pre-treatment of AML-derived exosome “prime” the animal for leukemia cell invasion and accelerate leukemia progression. Conversely,disruption of exosome secretion by targeting Rab27a in AML cells significantly delays leukemia progression. These data strongly support the notion that leukemia-modified niches favor leukemic cell proliferation and suppress normal hematopoiesis.

Disclosures

No relevant conflicts of interest to declare.
Tumor Suppressor Role of HIF-1alpha in Leukemia-Initiating Cells in Flt3-ITD-Induced Myeloproliferative Neoplasm
Authors: Velasco Talia , Cammenga Jörg
Published: October 2019

Hypoxia-induced signalling is a major regulator in normal and malignant hematopoiesis. The transcription factor HIF-1alpha plays a crucial role in the quiescence and self-renewal of hematopoietic stem cells as well as leukemia-initiating cells (LICs) of acute myeloid leukemia and chronic myeloid leukemia. Better understanding of the requirement of the molecular response to hypoxia in LICs could lead to new therapies targeting this pathway.

We have therefore investigated the effect of HIF-1alpha loss on the phenotype and biology of FLT3-ITD induced myeloproliferative neoplasm (MPN).

Using a combined transgenic mouse model (Mx1-Cre; Hif-1alphafl/fl; Flt3ITD/+) we showed that deletion of HIF-1alpha leads to a more severe MPN phenotype reflected by higher numbers of white blood cells and myeloid cells in peripheral blood, as well as a more severe splenomegaly. Loss of long-term hematopoietic stem cells (LT-HSCs: Lin- Sca1+ cKit+ CD48- CD150+) and increased number of lineage-restricted progenitors (Lin- Sca1+ cKit+ CD48+ CD150-) were the most pronounced effects on a cellular level upon the loss of HIF-1alpha. The proliferative effect of the HIF-1alpha loss was cell intrinsic and not at the expense of the ability of the LICs to self-renew because the disease was transplantable into secondary recipients recapitulating the same phenotype. Mice transplanted with FLT3-ITD induced MPN lacking HIF-1alpha succumbed to their disease (average survival of 35 weeks after transplant), while animals transplanted with MPN with wild-type HIF-1alpha suffered from MPN but did not die in the observation period of 60 weeks.

These findings are in contrary to what has been previously described for the role of HIF-1alpha in leukemia initiating cells and lead us to propose that HIF-1alpha could act as a tumor suppressor gene, inhibiting proliferation in myeloid malignancies. Our results provide evidence that targeting HIF-1alpha can lead to disease progression of MPN while not affecting self-renewal of LICs.

Disclosures

No relevant conflicts of interest to declare.
Pediatric Myelodysplastic Syndrome: Experience from a Single Center
Authors: Cetin Mualla , Unal Sule , Bayhan Turan , Aytac Selin , Kuskonmaz Baris , Tavil Betul , Uckan-Cetinkaya Duygu , Gumruk Fatma , Tuncer A. Murat
Published: October 2019

Pediatric myelodysplastic syndrome (MDS) is a group of heterogeneous clonal disorder with lesser frequency compared to adults. Additionally, there is much rare data in pediatric age group in relation to presentational findings and treatment.

The clinical and laboratory data, in addition to therapeutic interventions and outcomes of 47 patients in a single centre who were diagnosed between January 2001-May 2014 were summarized.

Median age of the study group was 2.8 years (0.1-16.8). The most common complaints at presentation included fever (27%), fatigue (17%), bleeding (15%), abdominal distention (13%), in addition to other rare presentational complaints including pallor, rash, vomiting, irritability, jaundice, stridor, abdominal pain and easy bruising. The underlying disorder was established as: neurofibromatosis in 5, Down syndrome in 3, secondary to prior chemotherapy in 2 (ALL and PNET), Fanconi anemia in 1, Jacobsen syndrome in 1, Klinefelter in 1. Final diagnosis was MDS in 22, JMML in 19, hypoplastic MDS in 4 and chemotherapy related MDS in 2. Median Hb, WBC, thrombocyte counts at presentation were 8.7 g/dl (4.1-12.7), 10.3x109/L (1.3-117) and 55x109/L (4-1515), respectively. Of the mutations studied related to MDS in 22 of the patients, k-ras positivity was the most common (23%). The most common cytogenetic abnormality was chromosome 7 related abnormalities (25%). Of the patients, 21 (45%) are alive and of these alive patients 62% are alive subsequent to hematopoietic stem cell transplantation.

The patients with pediatric MDS may present with various complaints and they may have underlying genetic diseases causing propensity for MDS. The survival is better among patients who underwent hematopoietic stem cell transplantation.

Disclosures

No relevant conflicts of interest to declare.
Comparison of IgA Heavy/Light Chain Immunoassays with Capillary Zone Electrophoresis and Total Immunoglobulin Measurements
Authors: Gartcheva Lidia , Petkova Violeta , Dimitrova Keranka , Petkova Veronika , Ganeva Penka , Guenova Margarita
Published: October 2019

Introduction: Capillary zone electrophoresis (CZE) and measurement of total immunoglobulins (total Ig) are standard techniques for the identification and quantification of monoclonal immunoglobulins (M-Ig). Heavy/light chain (HLC) pair analysis allows discrimination between Igκ and Igλ and as a result allows measurement of both the monoclonal involved and polyclonal uninvolved HLC pair. We compared measurement of M-Ig by CZE and total Ig to HLC levels.

Methods: 93 samples from 8 patients with IgA intact immunoglobulin multiple myeloma (MM) were analysed. M-Ig was measured using CZE Sebia Capillarys 2 system, total IgA (tIgA) and IgAκ and IgAλ HLC concentrations on a SPAPLUS analyser. IgA HLC levels were measured with Hevylite® and compared to published normal ranges (IgAκ (g/L): 0.57-2.08, IgAλ (g/L): 0.44-2.04, IgAκ/IgAλ: 0.78-1.94). Passing and Bablok fit analysis was used to determine correlation between the assays.

Results: Measurement of the involved HLC pair (iHLC) (median: 12.45g/L; range: 0.64-44.71g/L) compared well with CZE (n=34; median: 11.04g/L; range: 1.24-37.71g/L.; y= 1.2x + -2.65, R2= 0.94). Measurement of iHLC (median: 0.88. range: 0.05-21.55g/L) also compared well with tIgA measurement (n=65 median=1.44g/L; range: 0.227-21.11g/L, y=0.85x + -0.26, R2=0.98). Percent changes in iHLC concentrations from baseline through treatment compared well with CZE (n=28; y=1.59x + 0.15 R2=0.93) and tIgA (n=57; y=1.06x + 0.01, R2=0.96). Of the 34 samples with quantifiable M-protein by CZE, 32(94%) had an abnormal HLC ratio. The two discrepant samples were follow up samples from the same patient, where HLC normalised alongside total IgA entering the normal reference range. In addition, all 15 samples (15/65; 23%) where tIgA concentration was above the normal reference range all had abnormal HLC ratios. M-Ig was not detected by CZE in 48/82 (57%) samples, 46/48 of the samples (96%) had a normalised HLC ratio. In 38/65 (58%) samples, tIgA concentrations were within the normal reference range, and 34 (90%) had a normalised HLC ratio. HLC ratio for all patient samples normalised in subsequent samples following treatment.

Conclusion: The measurement of M-Ig is comparable between Hevylite® and both CZE and tIgA. The presented data indicate that Hevylite® is a more sensitive test for detecting residual disease and warrants prospective studies on larger cohorts of patients.

Acknowledgments:This work was partially supported by the National Science Fund (D02-35/2009).

Disclosures

Guenova: Novartis Pharma Sevices Bulgaria: Consultancy, Research Funding, Speakers Bureau; Roche Bulgaria: Consultancy, Research Funding, Speakers Bureau; Amgen Bulgaria: Consultancy, Research Funding, Speakers Bureau; Sanofi-Aventis Bulgaria: Consultancy, Research Funding, Speakers Bureau.
Evaluation of Anti-Neoplastic Effects of a New Hemostatic Agent Ankaferd Blood Stopper on Myeloma Cell Line and Plasmocytoma Development in Balb/c Mice: Results of the First in Vitro and in Vivo Study
Authors: Avcu Ferit , Guner Mustafa , Misirci Muammer , Elci Pinar , Safali Mukerrem , Goker Hakan , Haznedaroglu Ibrahim Celalettin
Published: October 2019

Ankaferd Blood Stopper (ABS), a unique traditional herbal mixture, has been used topically to stop bleeding for centuries in Anatolia. As well as ABS has been used as a blood-stopping agent, it may also have a considerable therapeutic benefit, because of its anti-infective, anti-neoplastic, and wound healing properties. The aim of this study is to investigate the anti-neoplastic effects of the ABS on myeloma cell line, in vitro and on the plasmocytoma development in Balb/c mice by intraperitoneal injection of pristane, in vivo. We therefore sought to evaluate the efficacy of ABS on MM cells and to study the modulation of cell-death pathways. The cytotoxicity of ABS against the MM cell lines (RPMI-8226, and ARH-77) was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-dye reduction assay. Responses to ABS by RPMI-8226 and ARH-77 cell lines were dose dependent but not time dependent. The IC50 values for RPMI 8226 and ARH 77 myeloma cell lines in 24h were 12,84 μL/mL and 13,86 μL/mL, respectively. Various cell-death characteristics such as caspase-3, Bcl-2, Bax were studied in response to ABS, but we couldn’t demonstrate specific features of apoptotic cell death, in vitro. We have also investigated the effect of the ABS on the pristane (2.6.10.14-tetramethylpentadecane)-induced plasmacytoma (PCT) development on six-week-old BALB/c mice. Three groups of mice were treated with intraperitoneal ABS (1 mg/kg, 0.5mg/kg, and 0,1mg/kg) per-week for eight weeks after pristane-induced PCT development. The study was stopped at twelfth week, the remaining mice were autopsied, and peritoneal tissues were examined histologically for PCTs. A database of different groups’ mice was analyzed using Kaplan-Meier and Cox regression statistics based on variables. Kaplan-Meier analysis revealed a difference of the survival of pristane-induced alone between the groups of pristane-induced plus ABS 1 mg/kg, 0.5mg/kg, and 0.1mg/kg. (Log-rank, p=0.016; p<0.001 and p<0.001; respectively). The present results indicate that direct anti-tumor effect of ABS on pristane-induced PCT and significantly increased survival. This hypothesis needs to now be further investigated in clinical trials.

Disclosures

No relevant conflicts of interest to declare.
A Phase II Trial of Chloroquine in Combination with Bortezomib and Cyclophosphamide in Patients with Relapsed and Refractory Multiple Myeloma
Authors: Montanari Francesca , Lu Meina , Marcus Sara , Saran Ankeeta , Malankar Aditya , Mazumder Amitabha
Published: October 2019

Background: preclinical data suggest that autophagy can serve as an adaptive mechanism facilitating tumor cell survival and resistance to therapy-induced apoptosis. Subsequently, inhibitors of autophagy are thought to enhance the efficacy of therapeutic strategies designed to induce tumor cell apoptosis. Based upon our preclinical results, we hypothesized that the addition of chloroquine, an autophagy inhibitor, to the combination of a proteasome inhibitor, and cyclophosphamide might result in a synergistic effect.

Patients and Methods: We designed a phase II trial of bortezomib, cyclophosphamide and chloroquine in patients with refractory myeloma (defined by > 25% increase in M-protein) who progressed on a combination of bortezomib and cyclophosphamide. Treatment consisted of 42 day cycles repeated until disease progression with bortezomib 1.3 mg/m2 IV on days 1, 4, 8, 11, 22, 25, 29 and 32; cyclophosphamide 50 mg orally twice a day; and chloroquine at 500 mg orally daily on days 1-14 and 22-35. The primary endpoint was to estimate clinical benefit (CR+VGPR+PR+SD) after 2 cycles.

Results: Between October 2011 and April 2013, 11 patients including 7 men and 4 women were enrolled and treated. The median age was 62 years (range 53-80). All patients were ISS stage II and III. Prior therapies for most patients included transplant, bortezomib, cyclophosphamide and lenalidomide. One patient withdrew after C1D1 and two patients progressed prior to completion of cycle 1. Of the 8 evaluable patients who completed at least 2 cycles, 3/8 achieved a partial response, 1/8 showed stable disease and 4/8 progressed with a clinical benefit rate of 4/10 (40%). Among the 3 patients who achieved a partial response, median duration of response was 4 months (250, 131 and 117 days respectively). The most common non-hematological adverse events (AEs) observed in >25% of patients (any Gr/Gr >3) and felt to be at least possibly related to the study drug were: fatigue (91%/18%); constipation (72%); myalgia, nausea (64% each); cough (55%), diarrhea, neuropathy (45% each); congestion (36%/9%); anorexia and dyspnea (27% each). The most commonly observed hematological AEs were neutropenia (45%/36%) and thrombocytopenia (55%/45%). Data from LC3 and beclin1 assays used to measure correlations of clinical response with in vitro autophagy inhibition will be presented.

Conclusions: this study suggests that the addition of chloroquine to bortezomib and cyclophosphamide is effective in overcoming proteasome inhibitor resistance in a significant fraction of heavily pretreated patients, with an acceptable toxicity profile.

Disclosures

No relevant conflicts of interest to declare.
Synergistic Antitumor Effects of Chimeric Antigen Receptor-Modified T Cells and Oncolytic Virotherapy
Authors: Wang Xingbing , Gottschalk Stephen , Song Xiao-Tong
Published: October 2019

Oncolytic vaccinia virus (VV) therapy has shown promise in preclinical models and in clinical studies. However their use has been limited by constraints on delivery and effective tumor targeting. The combination of VV therapy with Chimeric Antigen Receptor-modified T cells (CAR-T cells) might overcome these limitations, since CAR-T cells can direct VV to the tumors. In our previous studies, we have developed a novel T-cell Engager Armed Vaccinia Virus (TEA-VVs) strategy that expresses secretory bispecific antibodies that bind both to CD3 and a tumor cell surface antigen EphA2. We demonstrated that EphA2-TEA-VV displays significantly enhanced antitumor activity by inducing bystander killing of tumor cells that are not infected with virus, compared to non-modified VV. In this study, we aimed to evaluate the efficacy of combination therapy of HER2-CAR-T cells with EphA2-TEA-VV. This combinational therapy should exerts its anti-tumor activity through three mechanisms: i) HER2-CAR-T cells recognize and lyse HER2-positive tumor cells, ii) VV infect, replicate in, and lyse tumor cells, and iii) bi-specific antibody expressed by EphA2-TEA-VV directs T cells to recognize EphA2 and kill tumor cells, overcoming tumor heterogeneity. In our preliminary studies, we demonstrated that EphA2-TEA-VV can effectively infect and replicated in HER2-CAR-T cells. In coculture assays, preinfectin of HER2-CAR-T cells with EphA2-TEA-VV resulted in strongly enhanced killing of HER2+EphA2+ A549 tumor cells. These results illustrate the potential of combining CAR-T cells and TEA-VV for synergistic effects that more effectively treat cancer. 

Disclosures

No relevant conflicts of interest to declare.
Prevalence of Iron Deficiency and Iron Deficiency Anemia in Syrian Infants
Authors: Youssef Lama A , Albaroudi Issam N , Khodder Majed
Published: October 2019

Background: Iron deficiency (ID), with or without anemia, is the most common micronutrient malnutrition problem worldwide. Iron deficiency anemia (IDA) in infancy is linked to long-term motor and cognitive deficits. Infants and children in developing countries are particularly vulnerable to ID due to a negative iron balance resulting from increasing demands for growth that surpass dietary supplies. Recently, health reports have emerged on rising child malnutrition due to shortages of children’s foods and medicines in Syria. Nevertheless, factual data on prevalence are lacking.

Objective: To estimate the prevalence of ID and IDA in a cohort of non-displaced Syrian infants living in Damascus between November, 2011 and March, 2013.

Design/Methods: This was a prospective observational study conducted at the Children’s Hospital-based primary care clinic in Damascus. Information on type of feeding milk (maternal, iron-fortified formula or cow milk), and demographic characteristics was collected by face-to-face interviews with the parents of the study subjects. Hematological profile (complete blood count, reticulocytes and reticulocyte hemoglobin content (CHr)), and biochemical tests including serum ferritin and iron, and total iron-binding capacity were carried out. ID and IDA were diagnosed according to the 2010 American Academy of Pediatrics guidelines.

Results: Out of 155 infants assessed for eligibility, 135 infants met the inclusion criteria and were evaluated for iron deficiency. The average age was 12.4 ± 5.5 months (mean ± SD), and female to male ratio was 57:78. Only 31 (23%) of the study subjects were iron sufficient and not anemic, whereas 97 (72%) had ID, 75 (55.5%) had IDA, and 7 (5.2%) had non-iron deficiency anemia (thalassemia, hemolytic anemia, and macrocytic anemia). Prevalence of ID was statistically higher in males than females (85.33% vs. 62.26% respectively) (P=0.003). IDA was more prevalent in infants living in the countryside (76.25%) in comparison with their urban peers (53.84%) (p=0.04). Unexpectedly, ID and IDA were similarly prevalent regardless of the predominant type of milk fed to the infants (P >>0.05).

Conclusion: Our results unveil high prevalence of ID and IDA in Syrian infants between 2011 and 2013. Higher rates are expected in displaced infants and in remote regions of the country. These alarming findings call for immediate national and international collaborative efforts to provide iron supplementation and correct other existing malnutrition in Syrian children.

Disclosures

No relevant conflicts of interest to declare.
Palmitoyl Acyltransferase DHHC9 Is Required for Efficient Induction of Leukemia By Oncogenic NRAS
Authors: Liu Ping , Jiao Bo , Zhang Ruihong , Zhao Huanbin , Zhang Chun , Wu Min , Qiu Qingsong , Li Donghe , Zhao Xuemei , Ren Ruibao
Published: October 2019

Hyperactivation of RAS is common in human cancer, including hematological malignancies. Since RAS proteins are difficult to target, identification of alternative means to block RAS oncogenic signaling is critical for developing therapies against RAS-driven cancer. The biological activity of RAS proteins relies upon post-translational modifications (PTMs) that anchor RAS to cellular membranes. Among RAS PTMs, palmitoylation is required for the high-affinity plasma membrane binding of NRAS, HRAS and KRAS4A. NRAS mutations are common in human hematological malignancies. We have previously shown that palmitoylation is essential for NRAS leukemogenesis, suggesting that targeting RAS palmitoylation may be an effective therapy for RAS related cancers. In previous studies, we blocked NRAS palmitoylation by mutating the palmitoylation site in NRAS. Therapeutic intervention of RAS palmitoylation requires targeting enzymes that mediate the modification reaction. Protein S-palmitoylation is catalyzed by the DHHC family of palmitoyl acyltransferases (PATs). Thus far 24 mammalian PATs have been identified. It has been shown that DHHC9 (a 364-amino acid protein encoded by ZDHHC9) is the ortholog of yeast Ras2 PAT and constitutes a mammalian PAT with specificity for H- and NRAS in vitro. Increased expression of DHHC9 has been found in various cancers. In this study we investigated the role of DHHC9 in normal hematopoiesis and NRAS leukemogenesis in vivo. We found that DHHC9 is not the only PATs for RAS palmitoylation in vivo and the Zdhhc9 knockout mice were born and grew similarly as the wild type mice. The frequency of lineage-specific populations and hematopoietic stem cell phenotypes were also similar in mice with knockout alleles of Zdhhc9 as that of wild type mice. However, loss of DHHC9 prolonged the survival of mice with myeloproliferative disease or T-cell acute lymphoblastic leukemia evoked by oncogenic NRAS expressed either from the endogenous locus or from retroviral promoter. These results demonstrate that DHHC9 is dispensable for normal hematopoiesis, but plays an important role in the pathogenesis of NRAS-induced leukemias. The findings suggest that palmitoyl acyltransferase DHHC9 may serve as a safe and effective target for developing therapies against NRAS-related cancers.

Disclosures

No relevant conflicts of interest to declare.
Carma1 Is Important for T-ALL CNS Infiltration and Survival in an Animal Model of T-ALL
Authors: Oruganti Sreenivasa Rao , Halsey Christina , Krebsbach Savannah , Asperti-Boursin Francois , Patel Sumit , Matlawska-Wasowska Ksenia , Winter Stuart S. , Cannon Judy
Published: October 2019

Acute lymphoblastic leukemia (ALL) is one of the most common childhood cancer, and can arise from the T or B cell lineage (T-ALL or B-ALL respectively). T-ALL patients have almost 80% cure rate, however leukemic cell migration to the central nervous system (CNS) remains a major therapeutic challenge. Little is known about potential regulators of T-ALL cell migration to the CNS, and identifying new molecules that can drive T-ALL to the CNS may provide new targets for drug therapy to specifically block T-ALL CNS infiltration. Recently it has been shown that a chemokine receptor CCR7 is an essential regulator of T-ALL cell infiltration into the CNS. CCR7 is a homeostatic chemokine receptor expressed on T cells that controls the homing of cells to lymph nodes, including na•ve T cells, dendritic cells, and lymphoma cells. Aberrant CCR7 expression has also been associated with migration of gastric, breast and head and neck cancers. We recently identified the molecule CARMA1 as a signaling mediator that acts downstream of CCR7.

We now find that CARMA1 regulates CNS migration in T-ALL. We have established a xenograft model of T-ALL, where we can assess the engraftment of a T-ALL cell line into the NOD/SCID/IL2Rγ2 immunodeficient mice. Using luciferase expressing T-ALL cells lacking CARMA1, we imaged tumor load using the IVIS imaging system. Animals receiving T-ALL cells lacking CARMA1 show a significant delay in the timing of leukemia development as well as longer overall survival compared to animals receiving wild type T-ALL cells (n=25, P value < 0.0001). In addition, animals injected with CARMA1-knockdown T-ALL cells have lower tumor burden in the brain. In some cases, no tumor cells were found in the brains of animals receiving CARMA1-knockdown T-ALL cells. Using immunohistochemistry and flow cytometry, we also find lower tumor burden in liver and spleen of mice injected with CARMA1-knockdown T-ALL cells compared to WT cells. We confirmed the role of CARMA1 in a NOTCH1ΔE-induced T-ALL animal model. Similar to the xenograft model, we find that mice receiving CARMA1KO-Notch1ΔE survived longer than mice receiving with WT- Notch1ΔE. These results demonstrate for the first time that CARMA1 may play an important role in T-ALL induction and leukemic cell migration to the CNS.

Numerous studies have demonstrated the importance of CARMA1 in normal T cell function. We now find that CARMA1 is also involved in controlling the migration of T-ALL cells to the CNS as well as survival of animals with T-ALL. These results suggest that CARMA1 may provide a novel therapeutic target in T-ALL.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
T-Antigen Expression Causes Macrothrombocytopenia and Extensive Hemophagocytosis in Mice Lacking the Sialyltransferase ST3Gal-I Specifically in Megakaryocytes
Authors: Grozovsky Renata , Giannini Silvia , Ramsey Haley , Sola-Visner Martha , Hoffmeister Karin M
Published: October 2019

Changes in glycans expression have been associated with defects in blood platelet counts. However, the role of posttranslational modifications on platelet production is poorly understood. Six genes encoding sialyltransferases (ST)3Gal-I to -VI that form a2-3 sialic acid linkage have been identified in the mammalian genome, and deletion of St3gal1 and St3gal4 genes has been associated with macrothrombocytopenia in mice. We and others have shown previously that St3gal4-null platelets are cleared by the hepatic Ashwell-Morell receptor. Loss of ST3Gal-I activity has been associated with core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr expression, also known as tumor-associated or Thomsen-Friedenreich antigen (T antigen). We here investigated the detailed mechanisms of macrothrombocytopenia associated with St3gal1 deficiency by generating St3gal1loxP/PF4+ mice that lack ST3Gal-I specifically in the megakaryocyte (MK) lineage. Blood platelet counts were reduced by ~50% in St3gal1loxP/PF4+ mice, compared to control mice. Other blood cell counts were normal in St3gal1loxP/PF4+ mice. The clearance rate of St3gal1-null platelets was increased by ~15%, as determined by in vivo platelet biotinylation. Bone marrow MK numbers were normal in St3gal1loxP/PF4+ mice, compared to control mice, indicating that mechanisms other than clearance regulate circulating platelet counts in St3gal1loxP/PF4+ mice. Both St3gal1loxP/PF4+ platelets and bone marrow MKs had increased T antigen expression, as evidenced by flow cytometry using peanut agglutinin (PNA) binding. St3gal1loxP/PF4+ mice had increased bone marrow macrophage numbers, as evidenced by immunohistochemistry and flow cytometry using the macrophage marker F4/80. Macrophages in St3gal1loxP/PF4+ mice had increased expression of CD68 (macrosialin), as determined by immunohistochemistry and flow cytometry, indicative of an activated macrophage state. Consistently, St3gal1loxP/PF4+ bone marrow smears stained with May-Grunwald/Giemsa revealed increased hemophagocytosis. Macrophage ablation by in vivo injection of clodronate-encapsulated liposomes normalized blood platelet counts and size, and significantly reduced the numbers of activated macrophages in St3gal1loxP/PF4+ mice. Together, our data indicates that platelet production in the bone marrow is reliant on correct glycosylation on MK surface proteins and that the intimate interaction between MKs and macrophages play an important role in regulating platelet production and bone marrow homeostasis.

Disclosures

No relevant conflicts of interest to declare.
The Efficacy of Low-Dose Cytosine Arabinoside Therapy for Patients of Down Syndrome with Transient Abnormal Myelopoiesis
Authors: Kanzawa Takahiro , Suzuki Kyogo , Sakaguchi Hirotoshi , Yoshida Nao , Kato Koji
Published: October 2019

Transient abnormal myelopoiesis (TAM) occurs in about 10% of neonates with Down syndrome. While TAM is generally considered as self-limiting disease, substantial number of patients suffer from serious complications including liver fibrosis or pericardial effusion and eventually leads to fatal clinical outcomes. Low-dose cytosine arabinoside (CA) therapy has been reported to be effective for clinical symptoms of TAM patients, but it is still controversial as to which patient should be treated. We have retrospectively reviewed the clinical outcomes of TAM patients in our institution to confirm the efficacy as well as the safety of low-dose CA therapy by comparing the clinical outcomes before and after introduction of CA.

Before introduction of CA, we have experienced 20 TAM patients of 13 male and 7 female between September 1992 and November 2008. The median gestational age of those was 37w4d (range, 31w5d - 39w2d) , median peak value of white blood cell (WBC) count was 48×109/L (range, 19.8 - 399×109/L), and median peak direct bilirubin(DB) level was 0.9 mg/dL (range, 0.1 - 18.4 mg/dL), respectively. Sixteen of twenty patients (80%) had various complications including dyspnea, hepatosplenomegaly, pericardial effusion, and ascites.

Since 2009, we have introduced low dose CA (0.4 - 1.0mg/kg/day) for those with high WBC count (>100×109/L) until WBC count decrease to 10×109/L . Three patients received chemotherapy, but one patient with high WBC count did not, because consent was not obtained. The median gestational age of those was 36w4d (range, 33w6d - 39w2d) , median peak value of WBC count was 106×109/L (range, 101 - 267×109/L), and median peak DB level was 1.95mg/dL (range, 1.0 - 11.0 mg/dL), respectively. All of them had various complications including dyspnea, hepatosplenomegaly, pericardial effusion, liver fibrosis and ascites.

Before introduction of low dose CA therapy, five of six patients (83%) with high WBC count (>100×109/L) died of liver failure(4 of 5 patients) or AML, and one of 14 patients (7%) with low WBC count (<100×109/L) died of pneumonia at the age of eight without any relation to TAM. After introduction of CA, all three patients who received chemotherapy are alive without serious complications related to TAM or chemotherapy. Their blast cells in the peripheral blood disappeared promptly and liver dysfunction as well as biomarker of liver fibrosis is getting normalized. One patient who did not receive chemotherapy died of liver failure in spite of supportive care.

It is suggested that low dose CA therapy is safely conducted and is effective to improve the clinical outcomes of TAM patients with high WBC count. Prospective analysis is mandatory to confirm our findings by multi-institutional basis.

Disclosures

No relevant conflicts of interest to declare.
Role of Microbes in B-Cell Lymphopoiesis and Early Ig Repertoire Development
Authors: Wesemann Duane R. , Shrestha Akritee , Magee Jennifer , Chen Yuezhou , Silver Jared , Granato Alessandra
Published: October 2019

Progenitor (pro-) and precursor (pre-) B cells express the recombination-activating gene (RAG1/RAG2) endonuclease, which initiates the V(D)J recombination reaction that assembles Immunoglobulin heavy (IgH) and light (IgL) chain variable region exons from germline gene segments. Expression of productively assembled IgH μ chains and IgL (Igκ or Igλ) chains generates IgM molecules that form the B-cell antigen binding receptor (BCR) with a diverse primary repertoire of binding specificities. As the newly assembled and expressed IgM interacts with local antigens, RAG expression can continue, allowing continued Igκ V(D)J recombination that can replace the previously assembled VκJκ exon with one that generates a new specificity. This "receptor editing" process provides an antigen-mediated regulatory checkpoint that helps shape the pre-immune BCR repertoire. As the principal location of early B-cell development, the bone marrow antigenic environment is the main force affecting receptor editing. However, the extent to which self-antigens outside of the bone marrow — as well as environmental antigens — may shape the primary Ig repertoire is not known. Here we show that early B-cell development takes place in peripheral sites, including the small intestinal mucosa, in young mice. Cells in these sites co-express RAG with cytoplasmic Igκ and Igμ, indicating active receptor editing; and, correspondingly, they have significant differences in Vκ usage compared to RAG2-expressing bone marrow B-lineage cells and other RAG2-expressing B-lineage cells from other peripheral sites. In addition, the luminal microbial environment appears to also affect Igλ/Igκ ratios. We conclude that early B-cell development can occur in the periphery, including the intestinal mucosa, where commensal microbes may influence BCR diversification and gut pre-immune Ig repertoires.

Disclosures

No relevant conflicts of interest to declare.
Molecular Modification of the Microenvironment by the Tumor Clone
Authors: Ghobrial Irene M.
Published: October 2019

Multiple Myeloma (MM) is a disease of plasma cells with specific localization in the bone marrow. Recent studies have shown that MM is consistently preceded by Monoclonal Gammopathy of Undetermined Significance (MGUS). This has given rise to the concept of a myeloma precursor disease and raised questions about the biologic events leading to progression of these precursor states to symptomatic MM. The genomic complexity in MM was recently corroborated by next generation sequencing which revealed heterogeneity in tumors with tumor progression in a branching rather than in a linear manner, leading to substantial clonal diversity and coexistence of wide genetic heterogeneity. Although many factors regulating tumor progression are tumor cell autonomous, they are insufficient to induce progression and metastasis, and a permissive microenvironment is required for frank malignancy to emerge. Indeed, studies have shown that the tumor microenvironment is a key regulator in many steps of the invasion-metastasis cascade including tumor oncogenesis, egress, protection in the circulation, preparation of the metastatic niche, organ-specific homing, and the permissive role of the microenvironment in tumor colonization. These studies provide proof of bidirectional interactions that occur between tumor cells and the nearby microenvironment that are permissive for tumor initiation and progression, establishing a positive-feedback loop that may be self-amplifying. Although the bone marrow (BM) microenvironment is commonly referred to as the “non-tumor” entity, it has to be kept in mind that it is a complex network including a broad range of cells and factors that act as the "malignant niche" that is permissive for tumor growth in MM. Indeed, the BM microenvironment consists of three components: the cellular component (hematopoietic and non-hematopoietic cells, including mesenchymal stromal cells, osteoclasts, osteoblasts, endothelial cells, and immune cells such as T-regs, myeloid derived suppressor cells and plasmacytoid dendritic cells); the extracellular matrix component; and the soluble component (such as exosomes, cytokines, circulating free miRNA and growth factors). Multiple biological processes are affected by interactions between abnormal plasma cells and the BM microenvironment including homing to the BM, spread to secondary BM sites by the bloodstream, generation of paracrine factors, osteoclastogenesis, inhibition of osteogenesis, immune cell dysregulation, and angiogenesis lead to the progression of MM from early MGUS stages to overt metastatic myeloma.

Disclosures

Ghobrial: BMS: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Millennium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Off Label Use: CXCR4 inhibitors, SDF-1 inhibitors.
MYd88 Blockade Caused Reduction of PDL1 on Tumor Infiltrating MDSCs in a Murine Model of Melanoma
Authors: Forghani Parvin , Waller Edmund K
Published: October 2019

Introduction: Myeloid differentiation primary response gene 88 (Myd 88) is an important adaptor molecule for the activation of NADPH oxidase and regulation of arginase-1, which are responsible for the suppressive function of myeloid-derived suppressor cells (MDSCs). Blockade of Myd88 signaling induces antitumor effects in mice by skewing the immunosuppressive function of myeloid-derived suppressor cells. As the PD-L1/PD-1 axis has been characterized as a potent inhibitor of immune activation, particularly through inhibition of effector T cell function, we characterized the effect of Myd88 on checkpoint expression on tumor-infiltrating MDSC/T cells in a murine model melanoma.

Methods: Pathogen-free 8-10-week-old WT(B6-background) and Myd 88−/− mice that been backcrossed to a C57BL/6 genetic background were challenged with 1 × 106 B16 (F1) tumor cells s.c. On day 14, mice were sacrificed and spleen and tumors were removed and digested into single-cell suspensions, blocked with anti-FcR mAbs and analyzed for surface and intracellular staining by flow cytometry. We analyzed CD11b+/Gr-1+hi/int myeloid cells subsets and T cells in the blood, spleen and tumors of mice by flow cytomery.

Results: The growth of B16 melanoma tumor was significantly slower in Myd 88−/− mice compared with WT mice. No significant difference between two groups was found in the frequency of absolute number of MDSC subsets and expression of PDL1 check-point marker on spleen-derived MDSC subsets. In contrast CD4(+) and C8(+) T cells residing in spleens of Myd88(-/-) mice showed increased expression of TNF-α/IFN-α and GrZB compared with T cells from wild-type mice following short-term activation with PMA/iono. Of note, the frequencies and absolute numbers of infiltrating CD11b+/Gr1+ MDSC in tumor-bearing Myd 88−/− mice were lower than those in WT mice. Also we found that viable CD11b+/Gr1+ MDSC subsets from WT mice expressed higher level of PD-L1 compared with MDSCs from Myd 88−/− mice in concordance with the reduced expression of PD-1 on tumor infiltrating CD4+ T cells in Myd 88−/− mice. Collectively, the profile of PD-L1 and PD-1 expression in tumor microenvironments is favorably altered to enhance adaptive immune response in myd 88 KO vs WT mice harboring B16 melanoma.

Conclusion: The results of this study provide further evidence that blocking Myd 88 signaling increases anti tumor immunity against melanoma, and that the enhanced immunity can be explained, in part, by reduction of expression PDL1/PD1 immune checkpoint molecules. Considering the importance of tumor-infiltrating MDSCs in regulating anti tumor immunity in the tumor microenvironment, our findings could provide insight into the design of new therapeutics targeting Myd 88. Further experiments are needed to show how alteration in profile of PDL1 checkpoint expression on MDSCs influences anti-tumor T cell responses.

Disclosures

No relevant conflicts of interest to declare.
Tumor Cell Thrombin/PAR-1 Signaling Drives Pancreatic Ductal Adenocarcinoma Growth and Dissemination
Authors: Flick Matthew J. , Rewerts Cheryl , Cruz Carolina , Palumbo Joseph S. , Luyendyk James P. , Yang Yi , Konieczny Stephen F.
Published: October 2019

Pancreatic ductal adenocarcinoma (PDAC) accounts for ~85% of diagnosed pancreatic cancers and is among the most lethal malignancies. The 5-year survival rate for pancreatic cancer patients has improved only marginally in the last 40 years (3% → 7%), with effectively no change in survival profile for patients with metastatic disease (2%). High mortality is linked to the aggressive and invasive nature of the malignancy and poor efficacy of limited treatment options, which collectively highlight the need for novel treatment strategies. Notably, analyses of pancreatic cancer in patients and animal models have demonstrated that PDAC is associated with robust coagulation system activity. Previous work has shown that patient PDAC tumor cells often express high levels of tissue factor (TF) and protease-activated receptor (PAR)-1. To determine the potential contribution of tumor cell derived-TF and PAR-1 to PDAC growth and metastasis, a novel tumor cell line (termed KPC2) was derived from mice in which PDAC tumorigenesis was induced by activation of two established pancreatic cancer alleles, KrasG12D and Trp53R172H. In transplant studies, tumor growth and experimental metastasis were evaluated using KPC2 cells in which TF or PAR-1 expression was suppressed by shRNA knockdown. In addition, the interplay of tumor-derived TF and PAR-1 with host factors in promoting tumor growth and experimental metastasis were evaluated in mice with genetically imposed deficits in coagulation system components. TF knockdown (to ~10% of the parental line) in KPC2 cells resulted in a significant diminution of both primary tumor growth and experimental metastasis. This reduction appeared to be linked to thrombin activity as primary tumor growth and experimental metastasis of parental KPC2 cells were significantly reduced in fIIlow mice (which constitutively express 10% of normal prothrombin) relative to wild-type mice. PAR-1 knockout mice displayed similar KPC2 growth and experimental metastasis to wild-type animals indicating that stromal cell-derived PAR-1 was not significant determinant. In stark contrast, shRNA-mediated knockdown of PAR-1 in KPC2 (to ~10% of the parental line) cells resulted in significantly diminished tumor growth and experimental metastasis. Diminished tumor growth was linked to reduced expression of the macrophage chemokine MCP-1 and the metalloproteinase MMP9 by the tumor cells as well as reduced thrombin-stimulated ERK phosphorylation. Our results suggest that a major mechanism of PDAC growth and dissemination is through TF/thrombin-driven PAR-1 signaling on tumor cells.

Disclosures

No relevant conflicts of interest to declare.