bluuebunny/crossref_metadata_2025 huggingface dataset:
Genome-Wide Single-Nucleotide Polymorphism-Array Based Karyotyping Detects Clonal Aberrations, and Predicts the Risk of Imatinib Failure In Chronic Myeloid Leukemia.
Authors: Han Boram , Huh Jungwon , Yi Jun Ho , Lee Ha Yeon , Kim Jong-Won , Kim Hee-Jin , Kim Yeo-Kyeoung , Kim Hyeoung Joon , Moon Joon Ho , Sohn Sang Kyun , Jung Chul Won , Kim Dong Hwan (Dennis)
Published: October 2019

Abstract 3387

Background:

The current study investigated the diagnostic and clinical role of genome-wide, single nucleotide polymorphism arrays (SNP-A) which can detect microscopic copy number changes in chronic myeloid leukemia (CML).

Methods and Materials:

Genome-Wide Human SNP 6.0 Array (Affymetrix, CA, USA) was used for SNP-A karyotyping analysis in 132 CML patients. All patients were treated with imatinib and treatment outcomes were compared according to the presence of clonal aberrations (CAs) detected by SNP-A or additional cytogenetic abnormalities (ACAs) detected by metaphase cytogenetics (MCs).

Result:

Forty four clonal aberrations were identified in addition to t(9;22) (40 losses, 2 gains, 2 loss of heterozygosity [LOH]) that were not detected by MCs. The 9q34 deletions were found in 11% of cases, while 22q11.2 deletions were observed in 14% of cases. Nine cases harbored both 5'-ABL and 3'-BCR deletions adjacent to the t(9;22) breakpoint (7%). Copy number gains were identified at 8p and 9p, and losses at 2q, 7q, 8q, 11q, 13q, and 16p. Two cases with variant translocation showed additional deletions on the 3rd chromosome with involvement in variant translocations, which were missed by MC. We defined 3 commonly deleted regions (CDRs) on 9q34 and 22q11.2: CDR1 on 9q34 spanned approximately 162 kb between 9q34.11 and 9q34.12; CDR2 on 22q11.23 spanned 138 kb; CDR3 on 22q11.23 encompassed 102 kb.

When we compared treatment outcomes according to the combination of the presence of CAs by SNP-A and/or ACAs, the patients having both CAs and ACAs showed a higher risk of failure following imatinib therapy than those with either CAs/ACAs or none (p=0.015).

Conclusion:

The present study suggests that SNP-A analysis is a useful tool for detection of clonal aberrations in the CML genome, and could improve the prediction of prognosis in CML patients. In addition, our data suggest that SNP-A analysis is a useful tool to detect cryptic aberrations in CML genome, could improve the prediction of prognosis in CML patients and enables us to delineate CDRs on 9q34 and 22q11.2 which might be associated with CML leukemogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Hammersmith Score Is Able to Identify Chronic Myeloid Leukemia Patients with Poor Prognosis Before Treatment with Second-Generation TKIs.
Authors: Breccia Massimo , Latagliata Roberto , Stagno Fabio , Gozzini Antonella , Abruzzese Elisabetta , Rossi Antonella Russo , Santopietro Michelina , Cannella Laura , Sorà Federica , Vigneri Paolo , Trawinska Malgorzata , Sica Simona , Specchia Giorgina , Santini Valeria , Alimena Giuliana
Published: October 2019

Abstract 3409

A score aiming at early identification of CML patients showing sensitivity to second generation TKIs was proposed by the Hammersmith group. The score was created by analizing 80 patients and was based on 3 prognostic factors: previous cytogenetic response to imatinib, Sokal risk and recurrent neutropenia during imatinib. Subsequently, the score was validated in a small series of 28 patients. Aim of our study was to confirm the validity of this score and to establish its strength on a large group of CML patients resistant to imatinib and treated with second generation TKIs. One hundred twenty-seven patients were collected from 6 different Italian hematologic centers. There were 66 males and 61 females, median age 54 years (range 25–80). Twenty-seven patients received interferon before imatinib. Thirty patients had primary resistance, whereas 97 patients received second-generation TKI after acquired resistance to imatinib. The application of Hammersmith score was possible in 118 patients with available data: 52 patients were identified as good risk, 27 patients as intermediate risk and 38 patients as poor risk. The 1-year cumulative incidence of complete cytogenetic response (CCR) was 73% in good risk patients, 40% in intermediate risk patients and 23% in poor risk patients (p=0.0001). Similarly, the cumulative incidence of major molecular response (MMR) was 52% in good risk, 28% in intermediate risk and 13% in poor risk category (p=0.001). In the evaluation of event-free survival (EFS), events were considered loss of hematologic or cytogenetic response, disease progression, death for any cause, toxicity: the estimated 2-year event-free survival (EFS) was 89% in good risk, 70% in intermediate risk and 55% in poor risk group (p=0.0001). Progression-free survival (PFS) was defined as survival without evidence of accelerated or blastic phase: the estimated 2-year PFS was 97% in good risk, 93% in intermediate risk and 87% in poor risk category (p=0.05). Kaplan Meier estimated 2-year overall survival (OS) was 100% in the good risk, 93% in the intermediate risk and 82% in the poor risk category (p=0.001). In conclusion, as suggested by Milojkovic et al, some prognostic factors before starting second generation TKIs might predict cytogenetic response and outcome. As far as we known, the so-called Hammersmith score was not yet validated in large series of patients: we demonstrated that this score was able to discriminate patients at high risk of failure and consequent progression before treatment with second generation TKIs.

Disclosures:

No relevant conflicts of interest to declare.
Large Granular Lymphocyte Expansion Following Allogeneic Hematopoietic Stem Cell Transplantation Is Associated with the Presence of CMV Reactivation and Probably with Acute GvHD and Shows An Indolent Outcome.
Authors: Nann Sabine , Tzankov Alexander , Cantoni Nathan , Halter Jörg , Heim Dominik , Tsakiris Dimitrios , Arber Caroline , Buser Andreas , Gratwohl Alois , Tichelli Andre , Rovo Alicia
Published: October 2019

Abstract 3461

Expansion of CD3+ large granular lymphocytes (T-LGL) can be observed in situations such as viral infection, autoimmune disease, malignant neoplasm, and following allogeneic hematopoietic stem cell transplantation (HSCT). We sought to evaluate in patients treated with allogeneic HSCT incidence, characteristics, and clinical significance of persistent post-transplant T-LGL expansion. In this single center retrospective cohort study, we included all patients seen between January/08 and December/09 in our out-patient clinic for their regular follow-up. In patients with persistent lymphocytosis (>3 G/l for >3months) and an abnormal CD4/CD8 ratio, an extensive immunophenotyping was assessed; in case of an abnormal expansion of T-LGL cells a TCR gene rearrangement was performed. In 14 (7%) out of 215 evaluated patients a T-LGL expansion was diagnosed. Patients' characteristics with and without T-LGL expansion are summarized in Table 1. The median time between HSCT and diagnosis of lymphocytosis was 12 months (1-58). The median lymphocyte count was 4.24 G/l (3.0-26.5). The median duration of lymphocytosis was 29 (4-176) months. In 13/14 cases there was a CD3+/CD8+ immunophenotype, in 1 case was CD3+CD4+. In 5/14 patients a clonal TCR-gene rearrangement was observed. None of the patients presented neutropenia. Mild anemia was observed in 8/14 patients (57%), and thrombocytopenia in 2/14 patients; both changes were most probably not related with the T-LGL expansion. None of the patients had typical clinical signs of a T-LGL leukemia. In the univariate analysis acute GvHD and CMV reactivation were the only variables associated with T-LGL expansion, In the multivariate the relative risk (RR) of CMV reactivation was 5.063 (95%CI: 1.586–16.160; p=0.006) and the RR of acute GvHD grade 2–4 was 2.831 (95%CI: 0.831–9.648; p=0.096).

Conclusion:

we detected a T-LGL expansion in 7% of patients after HSCT. No symptoms or clinical signs related to T-LGL leukemia were observed. The T-LGL expansions, even when monoclonal, showed a chronic but indolent course. They have to be considered rather as an expression a chronic stimulation, triggered by causes such CMV reactivation or acute GvHD rather than as a malignant transformation. The question whether a T-LGL expansion plays a GvL role could not be answered in this study due to the small number of patients and the study design.

Disclosures:

No relevant conflicts of interest to declare.
Treatment of FLT3-ITD Positive AML Relapsing After Allogeneic Hematopoietic Stem Cell Transplant (HSCT) with Sorafenib.
Authors: Sharma Manish R , Ravandi Farhad , Chiattone Alexandre , Kebriaei Partow , Popat Uday , Giralt Sergio , Bashir Qaiser , Qazilbash Muzaffar H. , Nieto Yago , Yuen Carrie Hai-Lai , Parmar Simrit , Kantarjian Hagop M. , Cortes Jorge , Kadia Tapan , Borthakur Gautam , Champlin Richard E. , Lima Marcos De
Published: October 2019

Abstract 3471

AML with the internal tandem duplication of the Fms-Like Tyrosine kinase-3 gene (FLT3-ITD) has a poor prognosis, with high relapse rates. Sorafenib is an oral multikinase inhibitor of tyrosine kinases including Flt-3 with activity in AML. We used this drug to treat patients relapsing after allogeneic HSCT and herein present the results.

Patients and Methods.

We retrospectively evaluated 16 patients with FLT3-ITD AML that received sorafenib for at least a 7-day course either alone (n=8; 50%) or in combination (n=8; 50%) to treat AML relapsing at a median of 3 months (range, 1–7) after HSCT. Median age was 34 years (range, 20–603). Cytogenetics was diploid in 69%, high-risk in 19% and unknown in 12%. Treatment history prior to HSCT included sorafenib in 38% of the cases, and 30% were status post a 2nd HSCT. Donors were related (50%) or unrelated (50%). Seven patients (44%) received and failed other salvage chemotherapy prior to starting sorafenib (median=2 treatments; range, 1–5). At start of sorafenib salvage treatment, median WBC was 22.5 K/mm3 (range, 0.6–119), and median bone marrow blast was 58% (range, 12–88%), while 75% of the patients had circulating blasts. Sorafenib doses were as follows: 400 mg (n=3) or 800 mg daily (n=4) in combination to idarubicin/Ara-C, or 800 mg daily with 5-azacitidine (n=1); single agent: 800 mg (n=6) or 1200 mg (n=2) daily. Median duration of sorafenib single agent therapy was 39.5 days (range, 10–100), and in combination it was 7 days (range, 7–32). Complete remission (CR) rate was zero; 2 patients achieved a PR. Median reduction in bone marrow (n=9) and peripheral blood (n=12) blasts was 0% (range, 0–46), and 50% (range, 0–88), respectively. Two patients were ‘bridged’ to a 2nd HSCT, which led to CR durations of 53 and 106 days. Median survival after sorafenib initiation was 81 days and it was similar with single agent versus combination therapy (Figure 1 and 2). All patients have died.

Conclusions.

Treatment of overt AML relapse with sorafenib was unsuccessful as described here. It is possible that ‘pre-emptive’ treatment in the presence of minimal residual disease will be more effective, but this remains to be proven.

Disclosures:

No relevant conflicts of interest to declare.
Monitoring of Hematopoietic Chimerism by Real-Time Quantitative PCR of Single Nucleotide Polymorphisms In Samples with Low DNA Quantities.
Authors: Bach Christian , Tomova Elmira , Goldmann Katja , Lauer Birgit , Roesler Wolf , Mackensen Andreas , Winkler Julia , Spriewald Bernd M
Published: October 2019

Abstract 3491

Novel methods to quantify chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) based on single-nucleotide polymorphism (SNP) specific real-time quantitative polymerase chain reaction (qPCR) require high amounts of input DNA. The applicability of these methods in cases where DNA quantity is limiting, however, is currently unclear. Here, we demonstrate that SNP typing performed with just 5ng of input DNA still allowed for the distinction of positive (mean ct 28.05) and negative (ct > 36) signals. In addition, we confirm the high informative value of a set of 19 SNP markers, with 12 markers exceeding 20% informativity in our population (n=74). Of interest, a four-fold reduction of input DNA for qPCR standard curves did not alter PCR efficiencies. Most importantly, in 7 out of 16 allogeneic HSCT patients who experienced disease relapse, a retrospective analysis using the SNP-qPCR method revealed re-appearance of recipient chimerism earlier (mean 95 days) compared to previous results obtained by a short tandem repeat (STR) specific PCR. Taken together our data clearly demonstrate that SNP-qPCR is more sensitive compared to the widely used STR-PCR even with reduced amounts of input DNA. We therefore recommend that SNP-qPCR is preferable to STR-PCR for chimerism analysis in cases where DNA quantity is a limiting factor.

Disclosures:

No relevant conflicts of interest to declare.
Allogeneic Transplant In Children with Severe Aplastic Anemia – Excellent Outcomes with the Use of a Fludarabine Based Conditioning Regimen
Authors: George Biju , Mathews Vikram , Viswabandhya Auro , Abraham Aby , Kavitha Lakshmi M , Chandy Mammen , Srivastava Alok
Published: October 2019

Abstract 3519

There is limited data on the use of fludarabine based conditioning regimen in children with aplastic anemia though these regimens are increasingly being used in developing countries. Thirty four children (aged < 15 years) including 21 males and 13 females with a median age of 8 years (range: 2–15) underwent allogeneic transplant (HSCT) at our centre between 2004 and 2010 using HLA identical sibling or family donors. The median time from diagnosis to HSCT was 5 months (range: 2–96) and the median number of transfusions prior to HSCT was 10 (range: 3– 64). Five patients (14.7%) had failed treatment with Antithymocyte globulin (ATG) and 4 (11.4%) had an intracranial bleed in the 3 months prior to HSCT. Conditioning regimen consisted of Fludarabine 150 mg/m2 over 5 days and Cyclophosphamide 120 mg/kg over 2 days. Antithymocyte globulin (ATGAM) [10 mg/kg/day for 4 days] was used in 9 patients. Graft source consisted of either bone marrow [9 patients] or G-CSF stimulated peripheral blood stem cells (PBSC)[25 patients]. Graft versus host disease (GVHD) prophylaxis consisted of Cyclosporine and short course methotrexate. The median cell doses infused were 4.9 × 108 TNC/Kg for bone marrow and 8.5 × 108 MNC/Kg for PBSC. Thirty patients (88.2%) engrafted while 2 (5.8%) had primary graft failure. Two children died on Day 0 and Day +5 respectively due to infection. The median time to neutrophil engraftment was 14 days (range: 8–19) while the median time to platelet engraftment was 12 days (range: 7–24). Two patients (5.8%) developed veno-occlusive disease (VOD) of the liver while none developed hemorrhagic cystitis. Acute graft versus host disease (GVHD) was seen in 7 patients (23.3%) and was grade 1–2 in all patients. Chronic GVHD was seen in 31% of patients who could be evaluated and was limited in nature in a majority of patients. Of the 2 patients with primary graft failure, one was rescued with a second transplant while the second expired due to fungal pneumonia. The Day 100 mortality was 14.1%. Secondary graft failure was seen in 1 patient but was rescued with a second transplant using the same donor. At a median follow up of 30 months (range: 1 –67), 29 patients are alive for a 3 year OS of 85.1%. Fludarabine based conditioning regimens are associated with favorable outcomes in Indian children with aplastic anemia. These results are superior to an OS of 37% with the use of ATG and Cyclosporine in a similar population (George B et al PHO March 2010).

Disclosures:

No relevant conflicts of interest to declare.
Cysteinyl Leukotrienes Signal Via Overexpressed CysLT1, but Not CysLT2 Receptor In Chronic Lymphocytic Leukemia
Authors: Drost Adriana , Funk Mirjam , Norz Karoline , Zipfel Anne , Kanz Lothar , Mohle Robert
Published: October 2019

Abstract 3612

The G Protein-coupled receptors (GPCRs) CysLT1 and CysLT2 are both expressed in peripheral blood mononuclear cells (PBMNC). Their ligands are inflammatory mediators of the cysteinyl leukotriene (cysLT) family and contribute, together with ligands of other GPCRs such as chemokines, to migration and proliferation of a variety of cell types. In the present study, we demonstrate by real-time RT-PCR that CysLT1 mRNA is overexpressed in B-CLL cells compared to both normal PBMNC and normal CD19+ B cells, whereas only low levels of CysLT2 mRNA are present. GPCR-typical cellular responses such as intracellular calcium fluxes and actin polymerization, which were induced by the cysLT LTD4 in CLL cells in a dose-dependent manner, were suppressed by the CysLT1 antagonist MK571, the prototype of the lukast family of drugs used in asthma treatment (e.g., montelukast). However, MK571 and other lukasts also block the leukotriene transporter MRP. The fact that the CysLT1 antagonist LY171883, which has no effect on MRP, also abrogated the responses of CLL cells indicates that the effects of cysLTs are mediated solely by CysLT1. Moreover, also chemotaxis was induced by the cysLT LTD4 in B-CLL cells (optimum at low nanomolar concentrations) and could be blocked by MK571. We further observed inhibiting effects of MK571 on cysLT-induced Erk/MAP kinase phosphorylation in B-CLL cells, which suggests an involvement of CysLT1 in cell proliferation. Indeed, MK571 significantly induced apoptosis of CLL cells in vitro and reduced survival of cultured B-CLL cells. However, the concentrations for a significant reduction of cell viability were higher (1-10 μM) than the concentration required for efficient CysLT1 receptor inhibition (0.1 μM). Thus, the influence of MK571 on B-CLL cell survival may mainly be due to direct effects on apoptosis occuring at the higher dose level and/or blocking of the leukotriene transporter MRP. Accordingly, LY171883 and the combined CysLT1/2 antagonist Bay-u9773 did not induce CLL apoptosis. Our results suggest that CysLT1 is overexpressed in B-CLL cells and involved in cell trafficking, similar to the chemokine receptor CXCR4. Considering the redundancy among GPCRs and the limited effect of MK571 on cell survival, CysLT1 for its own represents a less attractive therapeutic target using the currently available CysLT1 antagonists of the lukast family. However, combined inhibition of several GPCRs highly expressed in CLL cells including cysLT1 and CXCR4 may constitute a promising therapeutic approach and should be further evaluated.

Disclosures:

No relevant conflicts of interest to declare.
B-Cell Lymphoma Cells Overexpressing BMI-1 Are Correlated with Drug Resistance through Enhanced Expression of Survivin and Are Effectively Eliminated by T Cells with Anti-CD38 Chimeric Receptor
Authors: Bhattacharyya Joyeeta , Mihara Keichiro , Ohtsubo Motoaki , Yasunaga Shin'ichiro , Takihara Yoshihiro , Hoshi Masaharu , Kimura Akiro
Published: October 2019

Abstract 3613

BMI-1 is essential for the self-renewal and proliferation of leukemic and hematopoietic stem and progenitor cells. Increased expression of BMI-1 is known to be an indicator for a poor prognosis in cancer patients. Analysis of the expression of BMI-1 and survivin in 6 patients with B-cell lymphoma (3 drug-resistant and 3 sensitive cases) showed that in the drug-resistant patients, high levels of BMI-1 and survivin were maintained even after drug administration in vitro. However, there observed was a down-regulation of both BMI-1 and survivin expression in the drug-sensitive patients. BMI-1 transduction induced the drug-resistance of two B-cell lymphoma cell lines, HT and RL, to the anti-cancer drugs etoposide and oxaliplatin, but not to irinotecan. The expression of survivin was clearly augmented in the cells transduced with BMI-1. Moreover, we detected sustained expression of survivin level in the presence of etoposide in the BMI-1-overexpressing cells. By contrast, the mock-transduced cells succumbed in the medium with anti-cancer drugs with an accompanying decrease in the expression of survivin as well as BMI-1. Survivin has been reportedly implicated in resistance to chemotherapeutic agents. Intriguingly, survivin mRNA levels in BMI-1-overexpressing cells were consistent with those in controls. Also, the level of survivin was enhanced by treatment with a proteasomal inhibitor, MG132, suggesting that overexpression of BMI-1 stabilized survivin expression post-translationally. We further showed that sh RNA-mediated knockdown of BMI-1 or survivin restored sensitivity to etoposide in the HT cells overexpressing BMI-1. Our findings suggest survivin as a potential target for BMI-1. Thus BMI-1, by acting as an upstream regulator, may control the expression of survivin, facilitating drug resistance in B-cell lymphoma. Next, we examined whether B-cell lymphoma cells overexpressing BMI-1 are abrogated by immunotherapy with T cells containing anti-CD38 chimeric receptor in vitro. Interestingly, these B-cell lymphoma cells were effectively eliminated by specific T cells against B-cell lymphoma cells bearing CD38. These results suggest that the immunotherapy is useful for treatment of patients with B-cell lymphoma cells overexpressing BMI-1, which are refractory to chemotherapeutic reagents. BMI-1 may be an important prognostic marker as well as a future therapeutic target in the treatment of drug-resistant lymphomas.

Disclosures:

No relevant conflicts of interest to declare.
TEL-AML1 Affects the Regulation of Cytoskeleton and Causes Alteration In Cellular Adhesive and Migratory Properties In An In Vitro Model of Pre-Leukemia.
Authors: Palmi Chiara , Fazio Grazia , Brunati Ilaria , Cazzaniga Valeria , André Valentina , Sozzani Silvano , Villa Antonello , Ford Anthony , Greaves Mel , Biondi Andrea , Cazzaniga Giovanni
Published: October 2019

Abstract 3624

Introduction:

The t(12;21) chromosome translocation generating TEL-AML1 chimeric fusion gene is a frequent initiating event in childhood leukaemia. Its impact is to generate a clone of covert, clinically silent pre-leukemic B cell progenitors. The leukemia arises only following second, post-natal hit/genetic events occurring years later. Moreover, relapse of leukemia is frequently arising from the pre-leukemic clone.

Aim

of our study is to investigate how TEL-AML1 expression can sustain this covert condition for many years. In a recent paper we described that the fusion gene rendered the B precursors resistant to the inhibitory activity of TGFbeta. Here we want to inquire into other factors that can explain the positive selection of the pre-leukemic clones over the normal counterpart. In particular, given the importance of the interaction with the microenvironment for survival signals for normal and leukemic stem cells, we question if the fusion gene causes changes in cellular adhesive and migratory properties.

Methods:

the study was performed by using two different models: i) a TEL-AML1 inducible expression system on the murine pro-B Ba/F3 cell line and ii) murine primary B lymphocytes (pre-BI cells) isolated from fetal liver, stably transduced with the pMIGR1-TEL-AML1-IRES-GFP construct. Gene expression assays were performed by using TaqMan (Applied Biosystems) and PCR Array technologies (SABioscences).

Results:

The expression of TEL-AML1 in Ba/F3 cell line causes over-expression of genes regulators of the cytoskeleton, specifically involved in cellular movement and in the regulation of actin dynamics. This gene expression alteration results in changes in the cellular morphology and phenotype: the cells acquire long extensions and several molecules involved in cell adhesion and migration are disregulated. Moreover, the TEL-AML1 positive cells present an increased ability to adhere to the ICAM1 substrate, but they also show a significant defect in the chemotactic response to CXCL12 in transwell migration assays in vitro, although the expression and the recycling of CXCR4 receptor are unaffected. This inability is not due to defects to migrate in general, as spontaneous motility is enhanced, but it is associated with a defect in CXCR4 signaling. In particular, CXCL12 calcium flux and ERK phosphorylation were inhibited. Those results have been confirmed in murine PreBI primary cells.

Conclusions:

in our murine models, TEL-AML1 affects the cytoscheleton regulation and causes alteration in cellular adhesive and migratory properties. We are now investigating how these alterations can give advantages to the pre-leukemic cells in the pathogenesis of TEL-AML1–expressing leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Epigenetic Silencing of a Novel Candidate Tumor Suppressor “miR” Predicts Poor Prognosis In High-Risk MDS and AML.
Authors: Treppendahl Marianne B. , Qiu Xiangning , Hother Christoffer , Andersen Mette K. , Kjeldsen Lars , Jones Peter , Liang Gangning , Gr∅nbæk Kirsten
Published: October 2019

Abstract 3629

Introduction:

A tumor suppressor has long been sought for in the commonly deleted region on chromosome 5q in myelodysplastic syndrome and acute myeloid leukaemia (MDS/AML). MicroRNAs (miRs) may have tumor suppressor function since they may repress the expression of proto-oncogenes through post-transcriptional regulation by binding to the 3′UTR of mRNA. It has recently been shown that several types of haematological malignancies have globally altered expression of miRs. DNA methylation cause targeted down regulation of individual tumor suppressor genes that may be directly involved in the pathogenesis of MDS/AML and we speculated whether epigenetic downregulation of miRs also plays a role in MDS/AML pathogenesis.

Methods:

Cell lines were treated with the hypomethylating agent 5-azacytidine. Upregulated miRs were identified by microarray (Exiqon) analysis and confirmed by RT qPCR analysis. Transcription start site was determined by 5′RACE and promoter methylation by methylation specific melting curve analysis. MiR expression and processing were analysed by qPCR, Northern blotting, and si-RNA mediated knock down of Drosha. Clinical data were collected from patient files.

Results:

We focused on one upregulated miR that locates to the commonly deleted region of AML/MDS. We found that the processing of this “miR” was independent of Drosha, and northern blotting showed that this was in fact a different type of non-coding RNA (ncRNA). The gene that encodes this ncRNA has a CpG island at the transcription start site. This CpG island is unmethylated in normal CD34+ cells, normal peripheral blood lymphocytes and methylated in three different myeloid cells lines (HL60, NB4, F-36P). Normal CD34+ cells and normal peripheral blood lymphocytes expressed the ncRNA, while no expression was seen in the cell lines. However, treatment by 5-azacytidine derepressed expression of this ncRNA. Bone marrow mononuclear cells from 101 high-risk MDS and AML patients, isolated at the time of diagnosis, were examined for methylation of the ncRNA promoter. Methylation was detected in 48% of the patients. Patients with no methylation have a significant better survival as compared to patients with methylation of the promotor.

Discussion:

This is the first example of targeted methylation of this type of RNA that has not previously been implicated in cancer. The preliminary results indicate that methylation of this ncRNA may be used as a prognostic marker for survival in high-risk MDS- and AML patients. We are currently investigating the function of this ncRNA by performing stable transfections into MDS/AML cell lines.

Disclosures:

Jones: Lilly: Consultancy; Millipore: Consultancy.
Downregulation of Protein Arginine Methyltransferase – 4 (PRMT4) Promotes MiR-223 Expression and Myeloid Differentiation.
Authors: Vu Ly P , Zhao Xinyang , Perna Fabiana , Nimer Stephen D
Published: October 2019

Abstract 3632

Protein arginine methyltransferase 4 is a Type I member of PRMT family, that catalyses the addition of a methyl-group to arginine residues of a wide range of proteins, including histones, transcription factors, and RNA binding proteins. PRMT4 has been shown to regulate gene expression through its interaction with various transcription factors and via methylation of numerous substrates. Although PRMT4 has been reported to play an important role in T cell development, lung development and adipocyte differentiation in mouse models, the function of PRMT4 during hematopoiesis has not been studied.

To investigate the function of PRMT4 in the hematopoietic system, we utilized human CD34+ haematopoietic stem/progenitor cells (HSPCs). We observed that PRMT4 protein level is markedly downregulated during the myeloid differentiation of CD34+ cells without a significant change in the mRNA level. We then utilized a loss of function approach, using short hairpin RNAs, and found that knockdown of PRMT4 leads to an acceleration of myeloid differentiation, with a concomitant loss of the clonogenic potential of the cells. Interestingly, knocking down PRMT4 results in upregulation of miR-223, a myeloid specific microRNA. We also found that, during the myeloid differentiation of CD34+ cells, miR-223 expression steadily increased. Using a microRNA target prediction program, we identified a binding site for miR-223 in the 3′-UTR region of PRMT4 and found that when we over-expressed miR-223 in CD34+ cells, PRMT4 protein expression decreased. To determine the importance of PRMT4 downregulation in myeloid differentiation, we expressed the PRMT4-ORF (that should not be regulated by microRNAs) in CD34+ cells. The forced expression of PRMT4, that lacks the 3′-UTR region, leads to a block in myeloid differentiation and the inability of cells to up-regulate miR-223 during differentiation. Taken together, these data indicate a regulatory loop between PRMT4 and miR-223 that controls the differentiation of CD34+ toward the myeloid lineage.

To examine how PRMT4 regulates transcription of miR-223, we examined the miR-223 locus and found a RUNX1 binding site in the promoter of pri-miR-223. We discovered that PRMT4 interacts with RUNX1 and methylates RUNX1 at a specific arginine residue. This results in the recruitment of several novel interacting partners, which appear to control the expression of miR-223. Thus our results indicate that PRMT4 regulate the transcription of miR-223 transcription via its effects on RUNX1. Our study demonstrates a novel function of PRMT4 in myeloid differentiation, through regulation of RUNX1 function and miR-223 expression.

Disclosures:

No relevant conflicts of interest to declare.
Genetic and Epigenetic Silencing of Mesoderm Specific Transcript (MEST) In Acute Myelogenous Leukemia.
Authors: Poetsch Anna R , Claus Rainer , Bullinger Lars , Witte Tania , LÃ¼bbert Michael , Rehli Michael , Döhner Konstanze , Plass Christoph
Published: October 2019

Abstract 3639

About 10 % of patients with either myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) present either with deletions on the long arm of chromosome 7 or monosomy 7. These chromosomal aberrations are associated with a poor prognosis. Following biallelic inactivation as proposed by Knudsen, a “second hit” of the remaining allele might be required for loss of gene function. Epigenetic silencing might display such a hit in particular since no additional genetic hits could be identified so far. The role of epigenetic regulation might be of special importance in patients with -7/7q-, since several studies have shown that patients harbouring a chromosome 7 abberrations do more benefit from a therapy with demethylating agents as compared to patients with other chromosomal changes.

To address the issue of epigenetic silencing in these AML cases we utilized DNA methylation profiling to identify the potential tumor suppressor genes on chromosome 7.

We used MBD2 based enrichment of methylated DNA from 4 AML patient samples with monosomy 7 and from 23 patient samples with with other chromosomal changes including normal karyotype AML. For validation we analyzed regional DNA methylation using quantitative MassArray technology on DNA from 115 del(7q) AML or monosomy 7 patients as well as normal karyotype patients (n=20) and CD34 + cells from healthy individuals (n=5).

We could identify the gene MEST to be silenced by hypermethylation (> 30 %) of a CpG island on the remaining allele in 20% of the patients with -7/7q- and 40% of patients with normal karyotype. Thus, preferentially in patients that do not have a deletion already.

MEST is an imprinted gene located on 7q32.2. However, silencing is correlated with hypermethylation of a CpG island located at an alternative promoter of MEST, independent of the methylation status of the imprinting control region.

In two patient samples with monosomy 7 and hypermethylation of MEST (> 80%), DNA methylation was erased after 5-Aza-2′-deoxycytidine (DAC) treatment to less than 10 %. In concordance, also AML cell lines with hypermethylation of the MEST locus loose methylation after sublethal DAC treatment which leads to a reexpression of the gene.

Thus, we hypothesize that MEST functions as a tumor suppressor in AML and is genetically as well as epigenetically silenced AML. Reactivation of MEST by demethylating treatment may contribute to the mechanism by which demethylating drugs display their therapeutic potential in leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Allogeneic Hematopoietic Cell Transplantation (AHCT) for Primary Cutaneous T Cell Lymphoma (CTCL): a Center for International Blood and Marrow Transplant Research (CIBMTR) Review
Authors: Lechowicz Mary Jo , Agovi Manza , Carreras Jeanette , Lazarus Hillard M. , Laport Ginna G. , Montoto Silvia , Maloney David G , Hari Parameswaran
Published: October 2019

Abstract 364

Patients (pts) with advanced stage CTCL have a poor prognosis with standard therapy whereas AHCT possibly is curative. Published experience with AHCT for CTCL is modest. We report the outcomes of a large cohort of CTCL pts receiving AHCT (N=133) reported to the CIBMTR from 2000–2009. The majority of pts 129 (97%) were diagnosed with Mycosis Fungoides or Sezary Syndrome. Only 8 (6%) received AHCT in a complete remission (CR) while 46 pts (35%) never achieved CR pre-AHCT. Most pts (83%) were transplanted beyond 12 months from diagnosis. Median (range) time from diagnosis to transplant is 29 (4-206) months. Peripheral blood was the main graft source (83% vs. 14% marrow and 3% cord blood). Donor types included: 66 HLA-identical siblings (50%), 56 unrelated donors (42%), 11 from other related donors (8%). Reduced intensity conditioning (RIC) was used in 64%. Recipients of RIC were on average older (median age 51 vs. 44 yr for myeloablative) and transplanted in later study years. Among the myeloablative regimens, cyclophosphamide with total body irradiation was the most common (38%) whereas among RIC, fludarabine in combination with busulfan, cyclophosphamide or melphalan was used in 50%. GVHD prophylaxis regimens varied. Overall mortality at 30 and 100 days was 7% and 16%, respectively. Overall survival at 6 mo and 2 yr was 75% (95% CI 67–82%) and 44% (95% CI 34–53%), respectively. Conditioning intensity was not different statistically for the overall survival of these pts (figure 1). Treatment related mortality (TRM) at 6 mo and 2 yr was 10% (95% CI 5–17%). Progression/relapse of CTCL was 44% (95% CI 34–54%) at 6 mo and 58% (95% CI 47–68%) at 2 yr. Progression free survival at 6 mo was 46% (95% CI 36–57%) and at 2 yr 32% (95% CI 23–42%). The incidence of grade II-IV acute GVHD was 36% (95% CI 22–50%) and of chronic GVHD 31% (95% CI 19–45%) in a subset of reported pts. Infection as a cause of death does not seem to be increased in CTCL transplanted pts. Conditioning intensity did not impact TRM or the risk of progression. This very large series of allogeneic HCT demonstrates feasibility in pts with advanced CTCL with a low TRM and long term progression free survival in approximately one third of pts. Progressive disease was the primary cause of treatment failure in this cohort of pts with advanced disease.

Figure 1. Figure 1.

Disclosures:

No relevant conflicts of interest to declare.
Cytokine-Induced Killer Cells Facilitate Immune Reconstitution After Allogeneic BMT In Mice.
Authors: Mase Shintaro , Nishimura Ryosei , Kuroda Rie , Maeba Hideaki , Naka Kazuhito , Araki Raita , Ikawa Yasuhiro , Koizumi Shoichi , Yachie Akihiro
Published: October 2019

Abstract 3719

Cytokine-induced killer (CIK) cells are ex vivo–expanded T lymphocytes expressing both natural killer (NK)– and T-cell markers. We have reported that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft versus host disease (GVHD) with retention of antitumor activity mediated by NKG2D, which is an activating receptor expressed on NK cells. With the purpose of potential application of CIK cells in a clinical hematopoietic stem cell transplantation, the problem we have to consider next is whether CIK cells could promote an engraftment and facilitate an immune reconstitution. To this end, lethally irradiated BALB/c mice were injected with minimal number of MHC incompatible C57BL/6 bone marrow (BM) cells alone, in which almost half of mice died because of graft failure, or with CIK concurrently. The mice receiving BM plus CIK cells survived with 93% without GVHD, demonstrating that CIK cells significantly promote an engraftment (P<0.05). In particular, recovery of CD3+ T-cells was significantly faster (p<0.05) in the mice receiving BM plus CIK cells than those receiving BM cells alone. Next we further evaluated whether CIK cells also promote an engraftment in the mice receiving non-myeloablative conditioning using low dose total body irradiation. As expected, CIK cells promoted an engraftment and favored immunoreconstitution, especially from the early time point after BMT. In conclusion, our study clearly demonstrated that CIK cells promote an engraftment and facilitate an immure reconstitution without GVHD. As recent studies demonstrated that sufficient number of CIK cells could be expanded even from washouts of cord blood units bags, infusion of CIK cells would be a potent strategy for preventing a graft-failure in clinical settings, especially after cord blood transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Augmented Regulatory T Cell Response After Photochemical Treatment Alleviates Acute Graft-Versus-Host Disease and Improves Survival.
Authors: Kanathezhath Bindu , Larkin Sandra K , Kaplan Julika , Walters Mark C. , Kuypers Frans
Published: October 2019

Abstract 3740

Graft-versus-Host disease (GVHD) causes significant morbidity and mortality after allogeneic transplantation. Regulatory T cells of both donor and host origin can limit GVHD. Acute GVHD early post transplantation is heralded by a cytokine storm induced by a dominant T helper type 1(Th1) response, which damages host tissues like skin, gut, liver and lungs. We hypothesized that co-transplantation of photochemically (S-59) treated T cells modulates T cell effecter subsets and their cytokine milieu, and thereby reduces acute GVHD. To this end, we transplanted whole bone marrow cells in a major histocompatibility complex (MHC) antigen mismatched murine model using marrow with T cells from C57BL/6J AKR (acute GVHD model), with and without S-59 treatment. We observed equivalent elevation of CD4+Th1 (IL-2 and IFN-g), cytotoxic CD8+ (FAS, IFN-g and TNF-a) and CD4+Th2 (IL-4, IL-5, IL-6, IL-13) cytokines in recipients of both groups during the first week after transplantation. While the Th1 cytokines persisted as long as 10 days after transplantation, there was a shift to a regulatory T cell (Treg) cytokine profile (Transforming growth factor b [TGF-b] and IL-10) in the group that received the S-59 treated T cells. TGF-b and IL-10 levels were higher in the peripheral blood and bone marrow of the study group compared to controls (table). This was accompanied by the appearance of FoxP3High CD4+ CD25+ Tregs in the spleen and CD4+ Th17 cytokine (IL-17) elevation in the thymic compartment of recipients that received S-59 treated T cells (mean-28.44pg/ml versus 1.45pg/ml, p=0.0059). In-vivo tracking of S-59 treated T cells demonstrated the disappearance of these cells in the peripheral blood, spleen, bone marrow and thymus by 48 hours after transplantation. Nonetheless, we noted that recipients of S-59 treated T cells had significantly less acute GVHD and better overall survival (p=0.0001). In summary, our experiments indicate that there is an initial dominance of inflammatory and CD4 Th1 cytokines immediately post transplantation. Co-transplantation of S-59 treated T cells shifts the effecter CD4 T cell profile to resemble a Treg phenotype. Despite the absence of circulating photochemically treated T cells, significant alterations in the recipient cytokine milieu persisted after transplantation. Thus, S-59 treated T cells appear to exert an important immunomodulatory effect to ameliorate GVHD and improve survival after MHC-mismatched allogeneic transplantation.

Blood (pg/ml) Bone marrow (pg/ml) Thymus (pg/ml) TGF-b [S59] 8482.7 * 7062.1 2926.9 TGF-b [control] 1.1 301.55 46.5 IL-10 [S59] 419.0 ** 174.0 30.4 IL-10 [control] <1 82.1 33.44 ρ value- * 0.0001 ** 0.0006

Disclosures:

No relevant conflicts of interest to declare.
Gpr171, a Putative P2Y-Like Receptor, Counteracts Monocyte Lineage Specification When Over-Expressed In Murine Hematopoietic Progenitors.
Authors: Rossi Lara , Lemoli Roberto M. , Goodell Margaret A.
Published: October 2019

Abstract 3758

Despite intense investigation, the molecular mechanisms underlying the multi-step process that leads from committed progenitors to terminally differentiated cells are still poorly understood. Due to the capability of responding to a variety of chemically different compounds, G protein-coupled receptors (GPRs) have been shown to play a pivotal role in regulating the interaction of hematopoietic cells with the surrounding microenvironment. A novel GPR gene, Gpr171, encodes an orphan receptor putatively related to the family of purinergic receptors (P2YRs) for extracellular nucleotides, a group of mediators previously shown to be involved in the regulation of HSCs. Gpr171 has been indicated as a putative, novel member of the family of P2Y receptors, but no information is currently available on the ligand responsible for its activation. Likewise, its functional role in biological systems remains completely unknown.

By sequence analysis, we reconstructed Gpr171 phylogenesis in mice. We explored the functional role of Gpr171 in the hematopoietic system by transducing Sca-1+ hematopoietic progenitor cells (HPCs) with MSCV bicistronic retroviral vectors (thus driving the enforced expression of Gpr171 along with GFP). Transduced HPCs were analyzed both in vitro and in vivo.

By phylogenetic analysis, we confirmed that Gpr171 is evolutionally related to members of a P2Y gene-cluster localized on mouse chromosome 3. As a first step towards unveiling a role for Gpr171, we investigated its expression profile and function in the murine hematopoietic system. As opposed to other P2YRs, Gpr171 emerged to be significantly repressed in cells undergoing myeloid differentiation. We therefore hypothesized that Gpr171 might be involved in repressing monocyte lineage-specification. Consistent with our assumption, we observed a decreased expression of myeloid markers upon induction of Gpr171 in hematopoietic progenitors (HPCs), as well as a dramatic reduction in the capability of generating myeloid colonies in vitro. Furthermore, mice transplanted with HPCs over-expressing Gpr171 display a significant reduction in the percentage of Mac-1+Gr-1− monocytes.

Our findings establish, for the first time, Gpr171 biological function and unveil its unique role in repressing monocyte differentiation. Gpr171 enforced expression induced a significant decrease in the proliferation rate of myeloid cells, as well as a reduction in the expression of monocyte-specific markers, both in vitro and in vivo. Our findings contribute to the delineation a peculiar evolutionary history for Gpr171 as compared to other members of the P2Y family, suggesting a rather unique role in counteracting myeloid differentiation.

Disclosures:

No relevant conflicts of interest to declare.
Olfactomedin 4 Down-Regulates Neutrophil Killing of Gram-Positive and Gram-Negative Bacteria.
Authors: Liu Wenli , Yan Ming , Liu Yueqin , Coleman William G , Rodgers Griffin P.
Published: October 2019

Abstract 3777

Introduction:

Olfactomedin 4 (OLFM4) is a member of olfactomedin-related glycoprotein family, which is specifically expressed in neutrophils and gastrointestinal tract. OLFM4 expression is upregulated in gastrointestinal cancer and inflammatory diseases such as chronic inflammatory bowel disease and Helicobacter pylori infection. It has been shown that OLFM4 is a target gene of NF-kB and Notch pathways and that OLFM4 down-regulates innate immunity to H. pylori infection. However, its potential biological functions in neutrophils still remain to be defined. The goal of this study is to determine whether OLFM4 is involved in the bactericidal activity of neutrophils using an OLFM4 deficient mouse model.

Results:

1. OLFM4 expression in neutrophils is upregulated in response to Staphylocococus aureus (Gram positive) and Escherichia coli (Gram negative) bacteria. 2. We have shown that neutrophils from OLFM4 deficient mice have increased intracellular killing of S. aureus and E. coli bacteria in vitro. 3. The OLFM4 deficient mice displayed enhanced bacterial clearance in vivo when the mice were challenged with intra-peritoneal injection of S. aureus and E. coli. 4. To elucidate the molecular mechanisms that mediate these effects, we performed a yeast 2-hybrid screen and found that OLFM4 interacts with cathepsin C (dipeptidyl peptidase I or DPPI), a lysosomal cysteine protease that has a degraditive function as an exopeptidase and is essential for activation of neutrophil granule-associated serine proteases including neutrophil elastase, cathepsin G and proteinase 3. The direct association of OLFM4 with cathepsin C was confirmed in human primary neutrophils. 4. We have demonstrated that OLFM4 is a direct substrate of DPPI and inhibits DPPI activity in transfected 293T cells. 5. The cathepsin C activity in neutrophils from OLFM4 deficient mice was significantly higher than that in neutrophils from wild-type littermate mice in the absence or presence of bacterial infection, suggesting that OLFM4 is an endogenous inhibitor of cathepsin C in neutrophils. 6. We have also demonstrated increased activities of neutrophil elastase, cathepsin G and proteinase 3 (whose processing and maturity require cathepsin C activity) in OLFM4 deficient neutrophils compared with wild-type neutrophils. 7. Activation of NADPH oxidase, myeloperoxidase (MPO) activity, and neutrophil phagocytosis were not altered in OLFM4 deficient neutrophils compared with wild-type neutrophils.

Conclusion:

These results suggest that OLFM4 is an important regulator of neutrophil bacterial killing activity via negative regulation of cathepsin C activity and its down stream granule-associated serine proteases.

Disclosures:

No relevant conflicts of interest to declare.