bluuebunny/crossref_metadata_2025_split huggingface dataset:
Unique Effects of p53−/− Leukemic Cells On Mesenchymal Stromal Cell Gene Expression Profile in Vitro
Authors: Ling Xiaoyang , Chen Ye , Ruvolo Peter P. , Ruvolo Vivian , Wang Zhiqiang , Zhang Min , Shi Yuexi , Konopleva Marina , Davis Richard E , Andreeff Michael 
Published: October 2019

Abstract
Abstract 3468
Mesenchymal stromal cells (MSC) participate in the generation of the microenvironmental bone marrow niche which protects normal and leukemic stem cells from injuries, including chemotherapy. MSC produce numerous factors that aid in this function; however, little is known about how leukemic cells affect MSC. In this study, paired murine AML cells, MLL/ENL/FIT3-ITD/p53−/− and MLL/ENL/FIT3-ITD/p53wt, originally derived from C57BL/6 mice (Zuber et al. Genes & Dev. 2009), were co-cultured with MSC from the same strain. After 48 hrs, MSC were isolated by FACS sorting using CD45−/PDGFr+ as markers. Total RNA was profiled on Illumina WG6 mouse whole-genome bead arrays by standard procedures. The significance analysis of microarrays (SAM) method identified 429 differentially-expressed genes (DEG) whose expression in MSC differed significantly (false discovery rate, 10%) in co-cultures with p53−/− (C78) vs. p53wt (C147) leukemic cells. Differences in these DEG were highly consistent in replicates (Figure 1). The results demonstrate that: 1) p53 status (p53−/− vs. p53wt) of AML cells affects GEP patterns in co-cultured MSC. Comparison of the GEP in MSC co-cultured with p53−/− (78) or p53wt (147) (Fig 1) identified the following 5 genes that showed the most significant differences (up- or down-regulated): up-regulated: WNT16, WNT5, IGFBp5, GCNT1, ATP1B1; down-regulated: NOS2, DCN, CCL7, CCL2, CAR9, CCL4. These were selected for qPCR validation, and the results confirmed the array data. In addition, immunohistochemical staining showed that WNT16 was up-regulated in MSC co-cultured with p53wt leukemic cells. In addition, CXCL5 was found up-regulated in MSC co-cultured with p53−/− leukemic cells. These results were consistent with the GEP data. 2) Leukemic cells alter MSC Signaling proteins in vitro: Western blotting showed that Stat3, Akt, PTEN, CXCL5 and HIF-1α were up- regulated in MSC co-cultured with p53−/− leukemic cells as compared to p53wt leukemic cells (48 hrs). Additional analyses showed that the downstream targets of HIF-1α, VEGFa and VEGFc, but not VEGFb, were up-regulated. Taken together, these results suggest that 1) leukemic cells with different p53 genetic background co-cultured with normal MSC have profoundly differential effects on GEP of normal MSC; 2) MSC co-cultured with p53−/− leukemic cells resulted in increased levels of onco-proteins such as Akt and HIF-1α when compared to MSC co-cultured with p53wt leukemic cells. Results suggest, for the first time, that the genetics of leukemic cells determines gene expression in co-cultured MSC. In vivo experiments are in progress to provide in vivo evidence for the existence of a novel model of leukemia-stroma interactions where the genetics of the tumor cell impacts stromal cell biology.

Disclosures:
No relevant conflicts of interest to declare.
Expression of Human Leukocyte Antigen B61 Is Associated with Idiopathic Aplastic Anemia, Hepatitis-Associated Aplastic Anemia and Fulminant Hepatic Failure in Children
Authors: Takahashi Yoshiyuki , Ohara Akira , Kobayashi Ryoji , Yabe Hiromasa , Kikuchi Akira , Yagasaki Hiroshi , Morimoto Akira , Watanabe Ken-ichiro , Ito Etsuro , Kudo Kazuko , Fukuda Akinari , Sakamoto Seisuke , Kasahara Mureo , Nakazawa Atsuko , Kojima Seiji 
Published: October 2019

Abstract
Abstract 3487

Introduction:
Increasing evidence supports the notion that aplastic anemia (AA) defined as peripheral blood pancytopenia and bone marrow hypoplasia is an autoimmune disease mediated by T-cells attacking hematopoietic progenitor cells. Around 10% of acquired AA is associated with acute hepatitis, yet a causative virus has not been identified in most patients. Like other autoimmune disorders, adult AA is associated with human leukocyte antigen (HLA)-DR15. However, HLA-DR15 is not significantly associated in children with AA (Fuhrer et al. Pediatric Blood Cancer 2007 and Yoshida et al. Br J Haematol 2008). Human leukocyte antigen associations might differ between children and adults with AA. We therefore investigated the HLA types among patients with idiopathic aplastic anemia, hepatitis-associated aplastic anemia and fulminant hepatic failure who underwent liver transplantation.

Patients and methods:
We investigated data from 321 children (male, n = 202; female, n = 119; median age, 10 [range 0 – 18] years) with severe and very severe AA. All patients were enrolled in the AA-92 or AA-97 study from 1992 to 2010 and received immunosuppressive therapy (IST) using antithymocyte globulin and cyclosporine. Two hundred and sixty nine children had idiopathic AA and 52 had AA associated with hepatitis. The HLA alleles of 25 children with fulminant hepatic failure who underwent liver transplantation between 2006 and 2011 were characterized. Differences in all HLA alleles between the patients and healthy controls in the Japanese population were statistically analyzed at the phenotype level by χ2 and t tests using PRISM software.

Results:
The prevalence of HLA-B61 was significantly higher in idiopathic AA (21.9%, P < 0.0001), hepatitis associated AA (19.2%, P = 0.0089) and fulminant hepatic failure (20%, P = 0.0008) than in healthy Japanese controls (10.7%). No other HLA class I and II alleles including HLA-DR15 significantly differed between children with AA and healthy controls. Furthermore, HLA distribution did not significantly differ between patients who responded completely or partially to immunosuppressive therapy (IST) and non-responders.

Conclusions:
We did not uncover a significant association between AA and HLA-DR15 in children. Associations between HLA and disease might differ between children and adults with AA. HLA-B61 was positively associated with idiopathic aplastic anemia, hepatitis-associated aplastic anemia and fulminant hepatic failure in pediatric patients. These findings suggest that a common autoimmune mechanism is responsible for both hepatic- and bone marrow-related pathologies in children.

Disclosures:
No relevant conflicts of interest to declare.
Notch/HES1 Implicated As a Tumor Suppressor Mechanism in Human AML in Vivo
Authors: Golfman Leonard S , Kannan Sankaranarayanan , Hall Mandy A , Zweidler-McKay Patrick A 
Published: October 2019

Abstract
Abstract 3524

Background:
Although Notch signaling contributes to T cell leukemogenesis, the role of Notch in human AML is unclear. We and others have found that activation of Notch signaling inhibits AML growth and survival, e.g. a tumor suppressor like effect. However it is not known what the consequences of activating or inhibiting Notch signaling are in human AML in vivo.

Approach:
To determine whether Notch signaling would have growth inhibiting effects in vivo, we stably-transduced ML1 human AML cells with the constitutively-active forms of Notch1 and Notch2 (ICN1, ICN2), the common Notch target gene Hairy/Enhancer of Split 1 (HES1) or the pan-Notch inhibitor dominant-negative Mastermind-like (dnMAML) and performed in vivo competitive proliferation assays. Briefly, following transduction, each vector type were sorted to 50% GFP+ (containing the gene of interest) and GFP- (untransduced control cells). Groups of NSG mice were injected with these 50:50 mixtures of ML1 cells, and peripheral blood levels of GFP- and GFP+ cells were measured with flow cytometry for anti-human CD45 and GFP.

Results:
Peripheral blood engraftment of human CD45+ ML1 cells by week 5 was similar (2–5%) for ICN1, ICN2 and HES1 injected mice, but was significantly higher (23%) in dnMAML injected mice (Panel A). Similar to in vitro competitive proliferation assays, ICN1, ICN2 and HES1 all led to decreased relative numbers of GFP%+ cells, with 1%, 4% and 16% respectively (Panel B). Importantly, dnMAML had little effect on AML proliferation in vitro, however led to dramatic increases in GFP% as well as early morbidity and mortality due to increased leukemia burden, with 93% GFP+ (Panel B), demonstrating a selective advantage for dnMAML-expressing ML1 in vivo. When GFP+ (transduced) ML1 peripheral engraftment was directly compared to GFP- (parental CD45+) engraftment, the GFP- control cells had similar engraftment rates (2–5%) across groups of mice, while the GFP+ engraftment rates were significantly lower in ICN1, ICN2, and HES1 groups, but significantly higher in the dnMAML group (Panel C), demonstrating enhanced engraftment/proliferation in dnMAML-expressing cells.

Conclusions:
This suggests a previously unreported concept, namely that endogenous Notch ligands can inhibit human AML growth in vivo. This data supports the hypothesis that Notch behaves as a tumor suppressor in AML, and suggests the potential use of Notch agonists in human AML.

Disclosures:
No relevant conflicts of interest to declare.
Intensified Chemotherapy Regimen for Very High Risk Childhood B-Precursor Acute Lymphoblastic Leukemia (B-ALL): Results From Dana-Farber Cancer Institute (DFCI) ALL Consortium Protocol 05–01
Authors: Vrooman Lynda M. , Stevenson Kristen E. , Harris Marian , Neuberg Donna S. , Sallan Stephen E. , Silverman Lewis B. 
Published: October 2019

Abstract
Abstract 3563

Background:
High levels of minimal residual disease (MRD) at the end of 4-weeks of multiagent induction chemotherapy have been shown to be associated with a high risk of subsequent relapse in pediatric patients (pts) with B-ALL. Published reports indicated that pediatric B-ALL pts with high end-induction MRD had event-free survival (EFS) rates < 50% when treated with standard chemotherapy.[Zhou, Blood 2007; Borowitz Blood 2008] Pts with high-risk (HR) cytogenetic abnormalities, such as low hypodiploidy and MLL gene rearrangements (MLL-R) also have a high relapse risk. On DFCI ALL Consortium Protocol 05–01, we piloted an intensified regimen for B-ALL pts with high end-induction MRD and/or HR cytogenetics.

Methods:
Between 2005–2010, 482 evaluable pts aged 1–18 years with B-ALL were enrolled. Pts were initially classified as standard-risk (SR) or high-risk (HR) based on NCI age/WBC criteria. MRD was prospectively evaluated at the end of the 4-week induction phase via RQ-PCR analysis of IgH or TCR rearrangements. Results were reported as the ratio of copy numbers of target gene:GAPDH; a ratio >0.001 was considered high MRD. Pts with high MRD or HR cytogenetics (hypodiploidy with <45 chromosomes or MLL-R) were reclassified at the end of induction phase as very high risk (VHR), and received 2 additional chemotherapy cycles beginning at week 7 (cycle 1: cyclophosphamide, low-dose cytarabine, 6-MP; cycle 2: high-dose cytarabine, etoposide, dexamethasone, L-asparaginase), followed by the DFCI ALL Consortium HR consolidation phase, including 30 weeks of L-asparaginase and doxorubicin to a cumulative dose of 300 mg/m2. After consolidation, pts received a standard maintenance phase. Total duration of treatment was 25 months.

Results:
51 pts (11%) were classified as VHR, 25 of whom had been initially classified as SR and 26 as HR. 35 VHR pts had high end-induction MRD as the sole VHR criterion; 16 had HR cytogenetics. 9 pts relapsed (all marrow-involved) and 1 pt developed a secondary AML. There were no deaths in first remission. With median follow-up of 4.4 yrs, the 5-yr EFS (95% confidence interval) for all 51 VHR pts was 76% (60,87)[Figure 1] and 5-yr overall survival was 81% (60,92). The 5-yr EFS was 81% (59,90) for the 35 pts with high MRD.

Conclusion:
Intensification of chemotherapy (without stem cell transplant) resulted in a relatively favorable EFS in VHR B-ALL pts (defined by the presence of either high end-induction MRD or HR cytogenetics). More pts and longer follow-up will be necessary to confirm these promising results.

Disclosures:
No relevant conflicts of interest to declare.
Utility of Peripheral Blood BCR-ABL Mutation Analyses in Patients with Unexplained Neutrophilic Leukocytoses. Incidence of a Positive Test and Its Clinical Correlates
Authors: Dao Bao , Dowell Jonathan E , Illum Henrik , Shah Sachin , Sharma Anant 
Published: October 2019

Abstract
Abstract 4444

Background:
In patients with chronic unexplained neutrophilic leukocytoses (UNL), underlying Chronic Myeloid Leukemia (CML) is a concern. In these patients, peripheral blood BCR-ABL mutation analyses using fluorescent in situ hybridization (FISH) is often performed to investigate for CML. Diagnostic yield of this test, in patients presenting UNL and especially in those with mild leukocytoses is uncertain.

Objective:
To establish the overall incidence BCR-ABL mutation in patients with UNL using peripheral blood FISH analyses. To determine the incidence within subgroups based on severity of leukocytoses. To explore the association between a positive test and the presenting clinical and laboratory features.

Patients and Methods:
We performed a retrospective chart review (2000–2010) of adult patients referred to the hematology service at the Dallas Veterans Administration Medical Center for evaluation of UNL. All patients had undergone, peripheral blood BCR-ABL mutation analyses by FISH. Neutrophilic leukocytoses were defined as a white blood count (WBC) above 11 × 109/L along with an absolute neutrophil count (ANC) greater than 7 × 109/L. It was considered unexplained, if it persisted on repeat testing and the referring physician deemed its etiology as uncertain. The primary end point was incidence of BCR-ABL positivity in patients with UNL. Incidence of BCR-ABL positivity within subgroups based on degree of WBC elevation defined as mild (11–20 × 109/L), moderate (>20–50 × 109) or severe (>50 × 109) was also calculated. Univariate and multivariate regression analyses of various laboratory and clinical features was performed to identify any confounding variables.

Results:
The median time from discovery of leukocytoses to the performing the test was 1545 days (n=285). 26 (9.1%) patients were found to be positive for BCR-ABL (n= 286). Majority had mild leukocytoses (n=202). The incidence increased with the severity of leukocytoses. Mild = 4/202 (2%), moderate = 9/62 (14.5%), severe = 13/22 (59.1%). On univariate analyses, a positive test was associated with presence of an elevated LDH (OR=107 CI 14.17–809.99), myeloid left shift (OR=107 CI 14.17–809.99) and hepatosplenomegaly (OR 3.87 CI 1.57–9.54). On multivariate regression, the presence of a myeloid left shift was strongly associated with a positive test (OR=67.20 CI 8.36–540.36). Myeloid left shift was seen in 3/4 (75%) of patients with a mild leukocytoses and positive test for BCR-ABL,

Conclusion:
In patients with UNL, peripheral blood FISH for BCR-ABL mutation should be restricted to those with moderate to severe leukocytoses. Incidence of BCR-ABL is especially low (2%) in patients with mild leukocytoses (<20 × 109/L). In these patients BCR-ABL testing should be limited to those with an unexplained myeloid left shift.

Disclosures:
No relevant conflicts of interest to declare.
Impact of Disease Status On Outcomes of Allogeneic Hematopietic Stem Cell Transplantation for Refractory Acute Myeloid Leukemia
Authors: Zhou Qianlan , Tang XiaoWen , Qiu Huiying , Sun Aining , Ruan Changgeng , Depei Wu 
Published: October 2019

Abstract
Abstract 4470

Objective:
To evaluate the impact of disease status on outcomes of allogeneic hematopietic stem cell transplantation (allo-HSCT) for refractory acute myeloid leukemia (AML).

Methods:
32 patients with refractory AML received allo-HSCT after myeloablative conditioning regimen, including 17 patients not in remission (NR) and 15 patients in complete remission (CR) at the time of transplant. Treatment related adverse events, relapse rate and leukemia free survival (LFS) were compared in two groups.

Results:
There were no difference between two groups regarding patients gender, age, cytogenetic risk, donor type, stem cell resource, conditioning regimen, median number of CD34+ cells. 30 patients engrafted successfully. Only one patient failed to engraftment and another one died from veno-occlusive disease. Compared with CR group, NR group had a higher treatment related mortality (29.4% vs.13.3%,p=0.254), relapse rates (33.3% vs. 20.0% P=0.341),relative higher incidence of aGVHD (35.3% vs. 20.0%, p=0.287) and cGVHD (44.4% vs. 36.4%, p=0.217), but no significant difference was observed. 2-year LFS of the two groups were comparable (35.3% vs.40.0%,p=0.267). For the patient with refractory AML, univariate analysis showed that two factors, such as less than 35 years old and cGVHD, were benefit for overall survival.

Conclusion:
Regarding treatment related mortality, relapse rates, incidence of GVHD, there were no significant difference between the patients who achieved CR or NR prior to transplant. 2-year LFS of patients with CR and NR was comparable. In summary, our results showed that allo-HSCT is an appropriate treatment option for refractory AML patients.

Disclosures:
No relevant conflicts of interest to declare.
Extramedullary Relapse of Acute Leukemia After Allogeneic Hematopoietic Stem Cell Transplantation —Single Center Experience
Authors: Ge Lin , Tang XiaoWen , Ye Fan , Sun Aining , Ruan Changgeng , Depei Wu 
Published: October 2019

Abstract
Abstract 4473

Objective:
To investigate the incidence, risk factors and clinical outcome of extramedullary relapse (EMR) following allogeneic hematopoietic stem cell transplantation (allo- HSCT).

Methods:
We retrospectively studied the patients with AL (n =410) who underwent allo-HSCT at our center between 2001 and 2012 who developed either bone marrow (BM) or extramedullary (EM) relapse.

Results:
1. The incidence of overall relapse was 22% (90/410), with 68 cases of medullary relapse (16.6%), 13 cases of isolated extramedullary relapse (3.2%) and 13 cases of extramedullary relapse with concomitant bone marrow involvement (3.2%); 2. The 3-year overall survival (OS) of EMR and BMR was 14.1% and 13.8% respectively (p=0.027). Not only the 6-month overall survival (OS), but also the 12-month OS, the 24-month OS and the 36-month OS post relapse in patients with EMR was significantly better than that of BMR patients. Acute lymphoblastic leukemia (ALL) subtype (P=0.006) and complete remission status (P=0.047) at transplant yielded better 6-month survival in patients with EMR.; 3.Relapse sites included central nervous system (n=17), testicular (n=4), lymphatic (n=2), skin (n=2), bone (n= 1); 4.CNSL is the most common subtype of EMR. Univariate analysis showed that the factors associated with CNSL included ALL as the underlying diagnosis (relative risk [RR] = 2.727, p= 0.044) and sourse of peripheral blood stem cell (RR= 1.412, P= 0.018).

Conclusion:
CNSL is the most common subtype of EMR. Univariate analysis showed that ALL subtype and source of PBSC were associated with an increased risk of CNSL relapse. Patients with EMR have better overall survival compared with those with BMR.

Disclosures:
No relevant conflicts of interest to declare.
Pretransplant Parathyroid Hormone Level Has No Correlation with Platelet Recovery After Stem Cell
Authors: Kim Soo-Jeong , Kim Yundeok , Hyun Shin-Young , Jang Ji-Eun , Hwang Doh Yu , Cheong June-Won , Kim Jin Seok , Kim Yu Ri , Lee Hye Won , Min Yoo-Hong 
Published: October 2019

Abstract
Abstract 4495

Background
Stem cell fate is influenced by specialized microenvironments called as stem cell niche. Osteoblasts are considerd to play very important role in stem cell niche. They are activated by parathyroid hormone (PTH) or PTH related protein through PTH/PTHrP receptor and produce hematopoietic growth factors. In few large animal studies PTH treatment after hematopoietic stem cell transplantation (HSCT) improved post-transplant platelet count and CD4 lymphocyte count. Effect of PTH on cord blood stem cell transplantation to improve engraftment is under clinical trial. Therefore, authors investigated on correlation between pre-transplant serum PTH level and platelet recovery after allogeneic HSCT.

Methods
From January 2008 to December 2009, we measured pre-transplant serum PTH, ferritin of 28 patients who underwent allogeneic HSCT. Post-transplant platelet was checked at the point of post-transplant 14 days, 30 days, 60 days, 90 days and 180days. Surveillance of cytomegalovirus was done by weekly or biweekly CMV-PCR assay. Acute graft-versus-host disease (GVHD) was graded by IBMTR classification and chronic GVHD by NIH scoring system.

Results
Five patients received graft from partially matched donors and the others from fully matched donors, 15 from unrelated and 13 from related donors. Nineteen patients were in high risk at diagnosis. Nonmyeloablative conditioning was applied to 7 patients. Only 1 patient used bone marrow as stem cell source. Median dose of infused stem cell was 5.7×10∧6/kg (range 1.68 – 12.74). All patients were successfully engrafted. Acute GVHD more than grade 2 occurred in 15, and chronic GVHD more than moderate degree in 7. Five patients relapsed and 15 patients died of relapse or treatment related mortality. Pre-transplant serum PTH had no correlation with platelet count recovery. There was no correlation with pre-HSCT PTH and acute or chronic GVHD. However patient with lower PTH level had tendency to more frequent CMV reactivation (P=0.098).

Conclusion
Our hypothesis was that higher pre-transplant PTH would be related to better platelet recovery but failed to find correlation between two of them. There are various factors which affect engraftment and graft function. Role of PTH for engraftment should be evaluated in larger number of cases.

Disclosures:
No relevant conflicts of interest to declare.