bluuebunny/crossref_metadata_2025 huggingface dataset:
First and Second High Dose Treatment Supported by Autologous Hematopoetic Cell Transplantation Rescue in Multiple Myeloma
Authors: Bozdag Sinem Civriz , Topcuoglu Pervin , Beksac Meral , Ilhan Osman , Ozcan Muhit , Gurman Gunhan , Arslan Onder
Published: October 2019

Abstract 4528

Purpose:

We aimed to evaluate the outcome of multiple myeloma patients underwent double autologus hematopoietic cell transplantation in our center.

Patient&Method:

We retrospectively analyzed 31 multiple myeloma patients (22F;9M) receiving two times high dose therapy supported by auto-HCT in Adult BMT Unit of Ankara University between 2005–2010. Median age was 52 years (34–66ys) prior to first auto-HCT. We evaluated the response rates of progression free survival (PFS) of both transplantation, respectively and also overall survival (OS) from the diagnosis.

Results:

The median time from diagnosis to the first transplantation was 11 months (5–68ms). The response rates were 87% before the first auto-HSCT [2 Complete response (CR),4 near-CR, 7 very good partial remission (VGPR), 14 PR) but four patients (11%) had refractory disease. The response of first auto-HCT was complete remission or partial response in 50% of the pts and 36% for stable disease. Only 4 pts was progressed at early period of 1st auto-HCT. The median time from the 1st to 2nd auto-HSCT was 16 months (3–64 ms). Seventy-seven percent patients sue to progressive disease responded completely or partially to one or two step treatment regimens before the second auto-HSCT. When evaluated the responses after the second auto-HCT, 14 (46%) patients were in CR or near-CR, 12 (38%) for stabile disease and 5 for refractory. PFS was estimated similar after both first and second auto-HCT (median 9 months vs 10 months, p=0.3) (Graphic 1). Five- year OS from the diagnosis was 75±10%.

Conclusion:

PFS of the myeloma pts was estimated similar median time after 1st&2nd auto-HSCT. Collection of adequate hematopoietic stem cells enough for more than one auto-HCT in myeloma patients can be appropriate. Although the majority of responses after the first AHSCT were partial responses, complete or near complete responses were dominant after the second AHSCT.Improvement in responses and the similarity of progression free survival can be explained by the contribution of the new antimyelom treatment options

Disclosures:

No relevant conflicts of interest to declare.
The Involvement of NF-κB Subunits in the Interaction Between CLL-B Cells and Bone Marrow Stromal Cells
Authors: Sun Aining , Feng Guo , Xu Jingjing , Wang Wenjuan , Depei Wu
Published: October 2019

Abstract 4590

Objective:

To investigate the function of NF-κB signaling pathway in the interaction between chronic lymphocytic leukemia B cells and bone marrow stromal cells.

Method:

The expression of NF-κB family members at mRNA and protein levels were examined by quantitative RT-PCR and Western bloting analyses individualy, and the differences between CLL-human bone marrow stromal cell (hBMSC) and non-CLL-hBMSC were investigated with the methods above. Cell death was measured by flow cytometry analysis after B-CLL cells were co-cultured with hBMSC and treated with proteasome inhibitors. The changes of NF-κB expression at the protein levels were examined by Western bloting after co-culture experiment.

Result:

The expression of NF-κB family members turned out to be heterogeneous at both mRNA and protein levels in B-CLL cells, and the members demonstrated a different κB-DNA binding activities. The mRNA expression of NF-κB family members in B-CLL cells was shown at a remarkable higher level than that of the controls. The relative mRNA expression of relA was 0.0214±0.012, whereas it was 0.0130±0.012 for the controls of CD19 positive cells. The relative mRNA expression of p50 and p52 were 66.0860±21.649 and 0.0208±0.011 respectively, and the corresponding expression of CD19 positive cells were 24.8440±9.749 and 0.0065±0.002. The differences were statistically significant. There was no notable difference between CLL-hBMSC and Non-CLL-hBMSC. hBMSC protected B-CLL cells against the proteasome inhibitors and facilitated the survival of B-CLL cells.

Conclusion:

NF-κB expression is heterogeneous in bone marrow B-CLL cells. There is no significant difference between CLL-hBMSC and non-CLL-hBMSC. hBMSC can protect the survival of B-CLL cells dependending on the endogenous NF-κB activity. hBMSC can increase the drug resistance of B-CLL cells to proteasome inhibitors.

Disclosures:

No relevant conflicts of interest to declare.
ZAP-70 Expression Measured by Quantitative PCR (QPCR) As a Prognostic Marker in Chronic Lymphocytic Leukaemia (CLL)
Authors: Mollee Peter , Banh Raymond H. , Cheung Catherine , Saal Russell , Chambers Lynne , Pelly Tennille , Gill Devinder S
Published: October 2019

Abstract 4602

Aim

ZAP-70 expression provides important prognostic information in CLL, however, routine flow cytometric assessment has been limited by difficulties in method standardisation. We aimed to test whether ZAP-70 expression measured by quantitative PCR was superior to flow cytometric assessment.

Methods

We determined ZAP-70 expression by QPCR on CD19 selected peripheral blood B-cells in 95 patients with CLL and compared this to ZAP-70 measured by flow cytometry. Utility as a predictor of IgVH mutation status and clinical outcome was compared along with other standard prognostic markers.

Results

95 patients were analysed: median age 62years, 62% male, Binet stage at diagnosis (A 84%, B 11%, C 5%). 46% progressed to require treatment at a median time to first treatment of 7.6 years. Median overall survival has not been reached at a median follow-up of 5 years. 72% were ZAP-70 positive (defined as >20%) by flow cytometry, 32% were CD38 positive and 41% had unmutated IgVH status. Median ZAP-70 by QPCR was 3,437 per 10,000 copies of ABL (range, 163 to 30,452). ZAP-70 by flow cytometry and QPCR were significantly correlated (Spearman’s correlation coefficient 0.52, p=0.002). Positive ZAP-70 by flow cytometry had a sensitivity of 100% and specificity of 56% for an unmutated IgVH status. Using a cut-off of 5000, ZAP-70 by QPCR had a sensitivity of 91% and specificity of 88% for an unmutated IgVH status.

Correcting for Binet stage, a shorter time to first treatment was predicted by higher ZAP-70 by QPCR (p=0.001), CD38 positivity (p=0.004) and unmutated IgVH status (p=0.003), but not ZAP-70 positivity by flow cytometry (p=0.09).

Conclusion

ZAP-70 expression determined by QPCR provides better prediction of IgVH mutation status and time to first treatment in patients with CLL than does ZAP-70 expression as measured by flow cytometry.

This research was supported by a grant from the Leukaemia Foundation of Australia.

Disclosures:

Banh: Leukaemia Foundation of Australia: Research Funding.
Title: Promoter Methylation of Selected Genes and Response to Azacitidine (AZ) Therapy in Patients with Non-BCR-ABL Myeloproliferative Disorders (MPDs)
Authors: Achille Nicholas , Michaelis Laura , Smith Scott E. , Germano Eliza , Zeleznik-Le Nancy J. , Nand Sucha
Published: October 2019

Abstract 4625

Background:

Gene silencing via methylation of CpG islands in the promoter regions of many genes but specifically of APAF1, p15INK4B, p16INK4A, RARB, and CDH1 appears to play a role in pathogenesis of myeloid malignancies. Azacitidine (AZ) causes demethylation by inhibiting DNA methyltransferase and has already been shown to be an effective therapy for myelodysplastic syndromes. The demethylation induced by AZ is detectable in about 48 hours and increases significantly after 5 days of therapy. After that, the effect tends to plateau.

Methods:

We initiated a Phase 2 study of patients with non-BCR-ABL MPDs to determine clinical response to AZ therapy and correlate it with promoter DNA methylation and gene re-expression. The protocol was approved by the institutional IRB. Patients received AZ 75mg/m2 s/c for days 1–7 and repeated every 28 days for a minimum of 4 cycles. Responders were allowed to continue treatment until disease progression. Pretreatment and D 7 peripheral blood samples were analyzed for promoter methylation status and expression of the 5 genes mentioned above. Bisulfite conversion of DNA was followed by quantitative PCR using primers specific for methylated or for unmethylated promoter regions. For gene re-expression analysis, quantitative RT-PCR was performed with RNA isolated from the same patient samples and the same time points as the DNA methylation analyses.

Results:

Seven patients were enrolled before the study closed due to lack of accrual. The diagnoses were: Myelofibrosis (MF) 4, essential thrombocythemia 1, unclassified MPD with dysplasia 2. One patient with MF and one with unclassified MPD responded, the latter with normalization of marrow karyotype. Both responses were accompanied by significant decrease in APAF1 promoter methylation and surprisingly, an increase in promoter methylation of RARB. In three of the non-responders, APAF1 methylation increased. In patients with decreased Apaf1 methylation, a statistically significant increase in mRNA expression was observed.

Conclusions:

Within its limitations, this small trial shows that the methylation status of selected genes, particularly of APAF1 and RARB (inversely) is associated with response to treatment with azacitidine in patients with MPDs. In non-responders, Apaf1 methylation appears to increase. A larger study will be necessary to confirm these preliminary observations.

Disclosures:

Smith: Seattle Genetics, Inc.: Research Funding; Cephalon: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Spectrum: Consultancy; GSK: Speakers Bureau. Nand:Celgene Corporation: Research Funding.
Methylation Status of the Collagen Prolyl-3 Hydroxylases (C-P3H) and C-P4H Genes in Multiple Myeloma: P3H2 Is Selectively Methylated
Authors: Hatzimichael Eleftheria , Dasoula Aggeliki , Lagos Konstantinos , Kartalou Georgianna , Dranitsaris George , Briasoulis Evangelos , Crook Tim
Published: October 2019

Abstract 4639

Background–Aim:

Prolyl hydroxylation is an important and the most common post-translational modification that affects the structure and function of collagen. Enzymes that participate in this process are the collagen prolyl-3 hydroxylases (C-P3H) and the C-P4H. We have previously showed that P3H3 is methylated in multiple solid tumour types, whereas methylation in P3H2 appears to be specific for breast cancer and multiple myeloma (MM). In this study we assessed the CpG methylation status of the P3H genes (P3H1, P3H2, P3H3) in a larger cohort of MM patients and for the first time the CpG methylation status of the P4H genes (P4HA1, P4HA2, P4HA3) and investigated for clinical relevance.

Patients and Methods:

Bone marrow aspirate samples from 47 MM patients (28 males, median age 66 years) were obtained at diagnosis. Methylation-specific PCR (MSP) was employed to study the CpG methylation in P3H1, P3H2, P3H3, P4HA1, P4HA2 and P4HA3 CpG islands. Genomic DNA was isolated and bisulphite modified using commercially available kits (QIAmp DNA mini kit, Qiagen and EZ DNA methylation kit, Zymo Research respectively). Control methylated and unmethylated genomic DNAs were included in each experiment. For the study of the P3H2 CpG methylation status we used 2 sets of independent primers, which map to different areas of P3H2 CpG island. Ten bone marrow samples from patients with border line thrombocytopenia that were proved to have no neoplasia served as negative controls. Logistic regression analyses were used to measure the association between gene methylation and age, b2 microglobulin, serum albumin, hypercalcemia, ISS stage, extramedullary disease, bone disease, anemia and renal failure (eGFR< 50 ml/min).

Results:

None of the six genes under study was methylated in the control bone marrow samples. Methylation in the CpG island of P3H2 was detected in 44 % of patients (95% CI 27.8–62.8%) No methylation was detected in the CpG islands of P3H1, P3H3, P4HA1, P4HA2 and P4HA3. Trends noted were that patients with methylated P3H2 were more likely to have ISS stage 3 (OR 2.2, p=0.1). Overall no significant association was found with any clinical parameter although the study might have been underpowered due to the small population size.

Conclusions:

P3H2 is methylated at high frequency in MM, whereas P3H1, P3H3, P4HA1, P4HA2, P4HA3 were unmethylated, implying a striking specificity for methylation in P3H2 in MM. These findings warrant further evaluation in a larger sample of patients in order to better define the prognostic and clinical utility of P3H2 methylation in MM.

Disclosures:

No relevant conflicts of interest to declare.
Epigenetic Inactivation Targets the Arginine Biosynthetic Pathway At Two Levels in Multiple Myeloma
Authors: Hatzimichael Eleftheria , Dasoula Aggeliki , Syed Nelofer , Szlosarek Peter , Dranitsaris George , Crook Tim , Briasoulis Evangelos
Published: October 2019

Abstract 4640

Background.

Arginine is critical for the growth of certain human cancers and is an intermediary in the synthesis of various bioactive molecules. Argininosuccinate synthetase-1 (ASS1) catalyses the rate-limiting step in arginine biosynthesis, the conversion of citruline to arginine. Argininosuccinate lyase (ASL) is an enzyme that catalyses the reversible breakdown of arginosuccinate producing arginine and fumerate. Down-regulation of ASS1 has been demonstrated in several solid cancers and has been shown to confer sensitivity to arginine deprivation (arginine auxotrophic tumors). Methylation-dependent silencing of the ASS1 promoter may account for ASS1 down-regulation and arginine auxotrophy. Arginine deprivation using arginine deiminase (ADI-PEG20) is currently considered as a novel therapeutic intervention for cancer, and early clinical trials have shown promising results in solid tumors. In this perspective, we investigated for the first time the methylation status of the ASS1 and ASL CpG islands in multiple myeloma (MM) and analyzed for clinical relevance.

Methods.

Genomic DNA was extracted from bone marrow aspirate samples from 46 MM patients (28 male, 18 female, median age 64 years) obtained at diagnosis. Methylation-specific PCR (MSP) was employed to study the methylation in the ASS1 and ASL CpG islands. DNA was isolated and bisulphite modified using commercially available kits (QIAmp DNA mini kit, Qiagen and EZ DNA methylation kit, Zymo Research respectively). Control methylated and unmethylated genomic DNAs were included in each experiment. Ten bone marrow samples, with no neoplastic cells, from patients with borderline thrombocytopenia served as negative controls. Logistic regression analysis was used to measure the association between gene methylation and sex, age, ISS stage, presence of extramedullary disease, renal failure (eGFR<50 ml/min) and bone disease.

Results.

Methylation in the CpG island of ASS1 was detected in 71.7% patients (95% CI 57–82%) and of ASL in 37% patients (95% CI 24–51%), while simultaneous methylation in both genes was present in 10 patients. None of the two genes were detectably methylated in the control group. Patients with ASS1 methylation were less likely to have bone disease (p=0.04, OR=0.22) and extramedullary disease (p=0.05, OR=0.22). There was no statistically significant difference in overall survival by methylation status of the studied genes.

Conclusions.

Arginine biosynthesis genes ASS1 and ASL are methylated in MM. Methylation of ASS1 was found to be more frequent and negatively associated with bone or extramedullary disease. Further evaluation of ASS1 and ASL methylation is warranted in MM in the perspective of expanding arginine deprivation trials in these patients

Disclosures:

No relevant conflicts of interest to declare.
Assessment Levels of Factor VIII and IX Inhibitors in Patients with Hemophilia in North West of Iran: What Is the Main Reason of Low Inhibitor Titer and Rate in Our Patients?
Authors: Dolatkhah Roya , Movasaghpour Akbari Ali Akbar , Asvadi Kermani Iraj , Sanaat Zohreh , Rezamand Azim , Nemati Fateme , Nikkhah Hosein
Published: October 2019

Abstract 4662

Introduction

Today, the development of inhibitors against Factor VIII (FVIII) and Factor IX (FIX) is seen as the most serious complication of haemophilia A and B therapy. Recent studies on the role of the causative haemophilic mutation, race and ethnicity, family history of inhibitors and the possible influence of HLA genotype in inhibitor formation have revealed new and exciting insights. That is challenging conventional thinking about inhibitor development risk and type of factor products, recombinant or plasma-derived. The use of recombinant factor concentrates has revolutionized the treatment of severe factor VIII and IX deficiency. One of the most important complications is the development of antibodies (Inhibitors).

Material and Methods

Ninety two patients with haemophilia A and 12 patients with Haemophilia B have been studied. Confirmatory tests including one stage FVIII and FIX assay has been performed using STA Deficient FVIII and FIX, an immune-depleted plasma intended in plasma by analyzers of the STA line suitable with these reagents (Diagnostica Stago,France).Presence of Factor VIII and IX inhibitors have been tested by Bethesda Assay. All of the patients use mostly plasma-derived factor products, and on-demand treatment.

Results

Among 92 haemophilia A patients, FVIII levels were between 0.14–14.40 IU/dl (mean 2.91 ± 2.62), FIX levels were between 0.17 to 4.37 IU/dl (mean 1.53 ± 1.38) in12 haemophilia B patients. PT activity was 68.7–134 (mean 101.05 ± 15.13), APTT was 28.90 – 102 (mean 60.66 ± 13.50). FVIII inhibitor levels were between 0–1.14 BU (mean 0.04 ± 0.20) in 5 severe Hemophilia A patients (5.45%) and FIX Inhibitor levels were between 0–0.65 BU (mean 0.10 ± 0.23) in 2 Hemophilia B patients.

Discussion

Alloantibodies (inhibitors) against FVIII or FIX represent a major complication in patient care because they render classical substitution therapy ineffective. Inhibitors occur at a frequency of 20–30% in severe and 3–13% with mild or moderate haemophilia A, and 3% in haemophilia B, respectively. An alternative pathomechanism may underlie inhibitor development in patients with mild hemophilia A. Although it has been reported that inhibitors in patients with mild haemophilia are related to periods of intensive treatment or surgery, this has never been properly studied in children with severe haemophilia.

The low inhibitor rate with Low Titers in our patients may be demonstrate the role of type of factor products, recombinant or plasma-derived, which in this study was mostly use of plasma-derived factor products, and on-demand treatment. Also detailed evaluation of major risk factors of development of Factor VIII and IX inhibitors in our patients is required.

Disclosures:

No relevant conflicts of interest to declare.
The Role of Helicobacter Pylori in Japanese Children with Chronic Idiopathic Thrombocytopenic Purpura
Authors: Nakadate Hisaya , Kitazawa Jun-Ichi , Horikoshi Yasuo , Nagasawa Masayuki , Imaizumi Masue , Kobayashi Naoaki , Kobayashi Masao , Takahashi Yukihiro , Fujisawa Kohji , Shirahata Akira , Bessho Fumio
Published: October 2019

Abstract 4680

Objective

Although many cases of adulthood chronic idiopathic thrombocytopenic purpura (cITP) achieving remission after eradication of H. pylori have been reported, little is known about childhood cITP. We conducted the prospective study to establish the prevalence and clinical relevance of eradication of H. pylori infection in children with cITP in Japan.

Methods

84 Japanese children with ciTP were entered this study from January 2006 to April 2007. The patients included 40 males and 44 females, with a median age of 13.4 years. For detection of H. pylori, 13 C-urea breathe test (UBT) and/or a stool antigen test were performed. Patients infected with H. pylori were treated according to our eradication protocol.

Results

8 of 84 patients (9.5 %) were positive for H. pylori infection. Six of eight patients, 4 males and 2 females with a median age of 14 years were received the therapy, all but one boy aged 10 years, were successfully eradicated (83.3 %). A transient improvement in platelet counts was identified in one patient; another 4 had no improvement during the 12 months of follow-up after completion of treatment.

Conclusion

The prevalence of H. pylori infection in Japanese children with cITP was 9.5 %. Although, we had demonstrated high successful eradication rate (83.3 %), we concluded that the eradication of H.pylori might ameliorate the clinical courses of cITP in a small subgroup of patients.

Disclosures:

No relevant conflicts of interest to declare.
Induction of Indoleamine 2,3-Dioxygenase Expression by Interferon-g in Human Mesenchymal Stem Cells Inhibits Graft Versus Host Disease
Authors: Lee Myoung Woo , Kim Dae Seong , Kim Hye Jin , Son Meong Hi , Lee Soo Hyun , Jung Hye Lim , Yoo Keon Hee , Sung Ki Woong , Koo Hong Hoe
Published: October 2019

Abstract 4698

Background:

It is important to overcome the limitations such as graft rejection and graft versus host disease (GvHD) in allogeneic hematopoietic stem cell transplantation. Mesenchymal stem cells (MSCs), which evoke only minimal immune reactivity, may have anti-inflammatory and immunomodulatory effects.

Purpose:

In this study, we aimed to identify the immunomodulatory properties of human MSCs and to elucidate the possible mechanism of their properties for clinical treatment of allogeneic conflicts using MSCs.

Materials & Methods:

We conducted a comparative analysis about the immunomodulatory properties of MSCs derived from adult human tissues, including bone marrow (BM), adipose tissues (AT), umbilical cord blood (CB), and cord Wharton's jelly (WJ), in vitro and in vivo models.

Results:

AT-MSCs, CB-MSCs, and WJ-MSCs effectively suppressed phytohemagglutinin (PHA)-induced T-cell proliferation as effectively as did BM-MSCs. Levels of interferon (IFN)-g secreted from activated T-cells increased over time, but these levels were significantly reduced when cocultured with each type of MSCs. In addition, expression of indoleamine 2,3-dioxygenase (IDO) increased in MSCs treated with IFN-γ via JAK/STAT1 signaling pathways. Treatment with anti-IFN-g antibodies, JAK1/2 inhibitor or STAT1 siRNA restored PHA-induced T-cell proliferation. Use of an antagonist, 1-methyl-L-tryptophan, also restored PHA-induced T-cell proliferation, suggesting that IDO contributes to IFN-g-induced immunosuppression in MSCs. Moreover, infusion of IFN-g-treated MSCs decreased symptoms for human peripheral blood-derived mononuclear cells-induced GvHD in NOD/SCID mice, which resulted in an increase of survival rate of in vivo GvHD model.

Conclusion:

These data indicate that IFN-γ produced by activated T-cells is correlated with induction of IDO expression in MSCs by IFN-γ receptor/JAK/STAT1 pathway, which resulted in suppression of T-cell proliferation. Our findings suggest that MSCs derived from BM, AT, CB, or WJ could be used for clinical treatment of allogeneic conflicts.

Disclosures:

No relevant conflicts of interest to declare.
Low Incidence and severity of Graft-Versus-host disease after outpatient allogeneic Peripheral Blood Stem Cell Transplantation employing a Reduced-Intensity Conditioning
Authors: Cantú-Rodríguez Olga , Flores-Jiménez Juan A. , Gutiérrez-Aguirre Cesar H , Ruiz-Arguelles Guillermo J. , López-Otero A , Mancías-Guerra Consuelo , Gonzalez-Llano Oscar , Treviño-Montemayor Oscar , Jaime-Pérez Jose C. , Martínez-Cabriales Silvia , Gomez-Almaguer David
Published: October 2019

Abstract 4707

Introduction.

Graft-versus-host disease (GVHD) is the most common complication of allogeneic hematopoietic cell transplantation (alloHCT) and may affect the transplant outcome. Its incidence is higher when the preparative regimen used is a non-myeloablative and the stem cell's source is peripheral blood after mobilization.

Objective.

Demonstrate the 10 years incidence of GVHD in 2 Mexican transplant centers using peripheral hematopoietic stem cell transplantation (PHSCT) in related donors after non-myeloablative conditioning.

Patients and methods.

Three hundred and four patients with hematological and non-hematological malignancies that underwent outpatient PHSCT after non-myeloablative conditioning between October 1998 and July 2008 were included. The age ranged between 1 and 71 years (median of 30.5). One hundred and eighty-five patients were men and 119 women. The median of cells CD34+ infused was 4.9 × 106/kg (0.23-17.70).They received cyclosporine 4mg/kg per day and intramuscular methotrexate 5mg/ m2 in days +1, +3, +5 and +11 for GVHD prophylaxis.

Results.

Two hundred thirty-nine (80%) patients were successfully engrafted. The conditioning regimen was delivered as an outpatient procedure in all individuals. One hundred and fifty-four patients (64%) developed acute and/or chronic GVHD. Sixty four patients (26.7%) developed acute GVHD, 50 (20.9%) developed chronic GVHD, and 40 (16.7%) with acute GVHD progressed to chronic. Twenty seven (26%) patients who developed acute GVHD were grade III or IV, and 30 (33.2%) of chronic GVHD patients presented in the extensive way. GVHD was the cause of dead in 40 patients; 24 of acute GVHD and 16 of chronic GVHD, even when immunosuppressive therapy (high dose steroids and rituximab) was used. Last death was 24 months ago; subsequently, alemtuzumab was included in GVHD treatment. The cumulative incidence of acute GVHD was 37.6%.

Conclusion.

The higher incidence of GVHD reported in other studies after PBSC transplant seems to decrease if non-myeloablative conditioning regimens are used. We suggest that the combination of a reduced-intensity conditioning transplant and the outpatient procedure is the responsible for the low incidence of GVHD in our patients.

Disclosures:

No relevant conflicts of interest to declare.
Establishing a New Zebrafish Model to Study Malignant Transformation in Myeloproliferative Disorders
Authors: Alghisi Elisa , Malagola Michele , Santoriello Cristina , Skert Cristina , Filì Carla , Bergonzi Cesare , Peli Annalisa , Turra Alessandro , Cancelli Valeria , Ribolla Rossella , Cattina Federica , Mione Marina , Russo Domenico
Published: October 2019

Abstract 4711

Background:

Myeloproliferative diseases (MPDs) are a group of hematological malignancies characterized by the abnormal increase of different blood cells in peripheral blood and in other hematological organs. The molecular pathways involved in disease pathogenesis and progression as well as in drug sensitivity and resistance are not fully elucidated yet. Given the physiological similarity to mammals, the zebrafish (ZF) could accelerate the study of the molecular basis of these diseases. In the last decade several human leukaemia associated oncogenes have been transiently over-expressed in ZF embryos in order to perturb the myelo-erythroid compartment, with the aim to create experimental models to discover new molecular pathways that can became potential targets for new chemical compounds.

Methodogy:

We took advantage of the binary Gal4/UAS system to express oncogenic human HRAS in the ZF hematopoietic compartment. We used the tg(MAZe) driver line, a specific transgenic line that allows random mosaic expression of Gal4-VP16 after heat shock(hs) treatment(1). This driver line was mated with a tg(UAS:eGFP-HRASV12)(2) responder line in order to mimic somatic events leading to human oncogene expression.

Results:

Surprisingly a single heat shock at 30hpf, (referred here as HRASV12hs) induces human HRASV12 expression in hematopoietic progenitors as judged by the expansion of the hematopoietic tissue. Histological analysis showed an increased number of monocytes/macrophages. Blood smear of HRASV12hs larvae showed the expansion of blasts and myeloid precursors. Quantitative analysis of gene expression highlights a remarkable increase of myelo-erythroid restricted genes associated with a slight increase of staminality markers (pu1, mpx, lmo2, mpl, CD41 and c-myb). Moreover fish raised to adulthood developed hyper-plastic kidney marrow, the site of definitive haematopoiesis, the equivalent of mammalian bone marrow.

Conclusions and Future Perspectives:

The combination of hyper-proliferation of blood cells associated with the expansion of the hematopoietic compartment that we found in this model reproduces the pathological features of human myeloproliferative disorders. This study shows that it is possible to drive the expression of leukemia associated oncogenes to the hematopoietic compartment in developing ZF thus reproducing some of the features of human blood malignancies in an in vivo model. Our Group is now testing different driver lines in order to induce oncogene expression in earliest hematopoietic progenitors and is focusing on different responder lines expressing human oncogenes involved in hematopoietic malignancies.

Disclosures:

No relevant conflicts of interest to declare.
CD80-IL2 Expressing Myeloma Cells for Immune Gene Therapy of Multiple Myeloma
Authors: Giunti Giulia , Malone David , Chan Lucas , David Darling , Kordasti Shahram Y , Barber Linda , David Oppenheim , Farzaneh Farzin , Schey Stephen
Published: October 2019

Abstract 4718

Improved experimental therapies are needed for Multiple Myeloma (MM). Despite major progress in treatment and initial induction of remission, myeloma remains an incurable disease. Although immunotherapy and, in particular, the employment of NK cells offers an approach of interest for the treatment of Multiple Myeloma (MM), recent studies have shown that myeloma cells utilise a number different mechanisms to impair NK and T cell functions. Important amongst these mechanisms is the reduced expression of CD80 in the sub-populations of PBMC isolated from myeloma patients.

We have previously demonstrated CD80/IL-2 mediated stimulation of NK and T cells isolated from AML patients (as measured by proliferation, cytokine release and target cell specific cytolytic activity). In the present study we have examined the ability of genetically modified MM cells engineered to express CD80 and IL-2 to stimulate NK cell functions. These studies confirm the ability of MM cells to suppress NK cell functions in healthy PBMC and show that in contrast to the unmodified MM cells, the CD80/IL-2 expressing MM cells are able to stimulate a moderate increase in NK and T cell numbers and a significant increase in the fraction of NK cells with activatory receptors (NKp44, NKp30, NKp46) and activation markers (CD69) on the cell surface of both NK and T cells. More importantly for potential therapeutic applications the stimulated NK cells show increased cytolytic activity against the unmodified MM cells.

This data suggest that CD80/IL-2 MM cells may be able to overcome the immune suppressive functions of unmodified MM cells and to stimulate NK, and T cell mediated responses against the unmodified MM cells. Therefore CD80/IL-2 expressing MM cells may provide a suitable cellular vaccine for NK cell stimulation and possibly the induction of broader ranging immunological responses against multiple myeloma cells.

Disclosures:

No relevant conflicts of interest to declare.
Dexamethasone Reduces Viability of Bone Marrow Derived Macrophages
Authors: Ohana Yasmin , Souroujon Miriam C , Mittelman Moshe , Neumann Drorit
Published: October 2019

Abstract 4732

Glucocorticoids are steroid hormones with pleotropic effects and are widely used in clinical practice. Glucocorticoids affect almost every cell of the immune system. Here, we show that dexamethasone (DEX), a synthetic glucocorticoid, decreases viability of naïve bone marrow-derived macrophages (BMDM) in a dose dependent manner. A 24-hour incubation with 0.1, 0.5 and 1 microM DEX reduced BMDM viability by 22%, 26% and 34%, respectively, compared with untreated cells. Annexin-PI staining suggests an apoptotic mechanism. Administration of DEX in the presence of lipopolysaccharide (LPS) protected the BMDM against DEX-mediated cell death.

In addition to its effect on cell viability, DEX also significantly reduced the percentage of BMDM expressing high levels of the differentiation and activation markers F4/80 and CD11b (5 fold decrease mediated by 1 microM DEX). Treatment of BMDM with 1 microM DEX also led to a ∼ 50% decrease in macrophage inflammatory protein 1 alpha (MIP-1-alpha) mRNA expression levels.

These findings may contribute to a better understanding of DEX effects on naïve and activated macrophages and may have clinical implications in various diseases, including infection, inflammation and neoplastic diseases.

Glucocorticoids are steroid hormones with pleotropic effects and are widely used in clinical practice. Glucocorticoids affect almost every cell of the immune system. Here, we show that dexamethasone (DEX), a synthetic glucocorticoid, decreases viability of naïve bone marrow-derived macrophages (BMDM) in a dose dependent manner. A 24-hour incubation with 0.1, 0.5 and 1 microM DEX reduced BMDM viability by 22%, 26% and 34%, respectively, compared with untreated cells. Annexin-PI staining suggests an apoptotic mechanism. Administration of DEX in the presence of lipopolysaccharide (LPS) protected the BMDM against DEX-mediated cell death.

In addition to its effect on cell viability, DEX also significantly reduced the percentage of BMDM expressing high levels of the differentiation and activation markers F4/80 and CD11b (5 fold decrease mediated by 1 microM DEX). Treatment of BMDM with 1 microM DEX also led to a ∼ 50% decrease in macrophage inflammatory protein 1 alpha (MIP-1-alpha) mRNA expression levels.

These findings may contribute to a better understanding of DEX effects on naïve and activated macrophages and may have clinical implications in various diseases, including infection, inflammation and neoplastic diseases.v

Disclosures:

No relevant conflicts of interest to declare.
Neutropenia Does Not Delay Lymphopoietic Regeneration in NODSCIDcγ−/− Mice
Authors: Bugl Stefanie , Wirths Stefan , Martin R Müller , Melanie Märklin , Wiesner Tina , Malenke Elke , Kopp Hans-Georg
Published: October 2019

Abstract 4831

Introduction:

Previously it was demonstrated that lymphopoiesis is rapidly established after transplantation of wild type stem cells into lymphopenic NODSCIDcγ−/− mice. These data were interpreted as evidence for an “empty” preformed lymphopoietic niche being replenished by lymphoid progenitors. We hypothesized that antibody-induced neutropenia might influence early post transplant fate decision to myeloid rather than lymphoid differentiation resulting in delayed lymphoid reconstitution.

Materials and Methods:

25,000 flow sorted CD45.2-expressing wild type Lin-/Sca1+/c-Kit+ (LSK) cells from C57BL/6 mice were transplanted into sublethally irradiated B-/T-/NK-cell deficient NODSCIDcγ−/− mice (CD45.1). Three groups of n = 7 mice received anti-Gr1 or anti-1A8 i.p. every 48 h to induce continuous antibody-mediated neutropenia vs. PBS as control. Blood was harvested at regular intervals to monitor the engraftment. After 16, 22, and 34 days, animals were sacrificed and underwent blood and bone marrow analysis.

Results:

Hematopoietic regeneration started with the emergence of donor-derived monocytes in all groups as well as neutrophils in the control group as early as 9 days after transplantation. On day 14, B cells were to be detected for the first time, followed by T lymphocytes approximately 20 days after transplantation. Besides the fact that neutrophils were undetectable in the antibody treated groups, the peripheral blood revealed no significant changes between the neutropenic mice and the control group at any point of time. At the bone marrow level, an increase of LSK and granulocyte-macrophage progenitors (GMPs) at the expense of megakaryocyte erythrocyte progenitor cells (MEPs) was found in neutropenic mice. Common lymphoid progenitors (CLPs), however, were not significantly different.

Conclusions:

The engraftment of wild type donor cells after hematopoietic stem cell transplantation into NODSCIDcγ−/− mice started with the production of monocytes and neutrophils. B-lymphocytes were detectable by day 14 after transplantation. The production of T-cells started around day 20. Continuous antibody-mediated neutropenia did not significantly delay lymphoid regeneration. Although the marrow of neutropenic mice displayed increased proliferation of granulocyte progenitors, CLPs were unchanged. We conclude that the detection of donor-derived lymphocytes in the host peripheral blood is a relatively early event after LSK transplantation. Moreover, antibody induced neutropenia is not sufficient to induce sustainable changes in early hematopoietic fate decisions on the bone marrow level.

Disclosures:

No relevant conflicts of interest to declare.
Hodgkin Lymphoma in Jordan; A Retrospective Analysis of 477 Cases in King Hussein Cancer Centre
Authors: Addasi Alaa
Published: October 2019

Abstract 4862

BACKGROUND

Jordan is a small country with an estimated mid year population in 2008 of 5 850 000, 3015000 of whom are males and 2 835 000 are females (male: female ratio 1.06: 1). (Department of Statistics Jordan, 2008).

About 12.7 % of the population is under 5 years old, and 37.3 % under 15 years old. 11%if the population are 15–19 year old, with a M:F ratio 1.06:1 as well) Only 3.3 % of the total population is above the age of 65 years old (sex ratio of 1.01 male per 1 female in this age group).

According to the Jordan Cancer Registry Report for 2008, Lymphoma is the fourth most common Cancer in the country. A total of (4606 ) new cases of cancer were recorded among Jordanians in the year 2008, 333 (7.2%) of whom had a diagnosis of lymphoma.111 (2.4%) were diagnosed as Hodgkin's lymphoma (HL), and 222 (4.8%) as Non Hodgkin Lymphoma(NHL).

OBJECTIVE

In this study, we aim to characterize some of the clinico-pathological features of Hodgkin lymphoma in Jordan by analyzing the data available for patients referred to King Hussein Cancer Center over a seven year period.

PATIENTS AND METHODS

A retrospective analysis was conducted of adults (>18 years) lymphoma patients referred to KHCC, between 1/1/2003 and 31/12/2010. Clinical features and histological subtypes were prospectively established for all patients registered in the Lymphoma Service Database. Pathology review and original paraffin block were mandated for all patients.

RESULTS

Over the 8 year period of 2003–2010,1329 lymphoma patients were referred to KHCC and registered in the Lymphoma Service Database, of whom 477 (35.9%) were diagnosed with Hodgkin's lymphoma. Among this group all 477 patients were adults 18 years or older (100%), as children are treated in a different department. The median age was 35 years, (with an age range of 18–77), and 5% of patients were above the age of 60. 290 (61 %) of the patients were males, 187 (39%) were females, with a male to female (M:F) ratio of 1.55:1.

276 (57.8%) of the HL cases had a diagnosis of nodular sclerosis Hodgkin lymphoma (HDNS), making it the most common histological subtype. 120(25.2%) had mixed cellularity Hodgkin lymphoma (HDMC), 9 (1.9%) had lymphocyte-rich Hodgkin lymphoma (HDLR), and 6 (1.2%) had lymphocyte-depleted Hodgkin (HDLD). Nodular lymphocyte predominance Hodgkin lymphoma (NLPHD) cases were 33, and constituted 6.9% of the HL cohort.

CONCLUSION

Hodgkin lymphoma appears to constitute a bigger share of the lymphoma burden in Jordan, as opposed to Europe and the US. Clinico-pathological features, however, appear to be closer to those described in Western countries, with similar incidence of HDNS, and HDMC subtypes, but possibly with less incidence of HDLR and HDLD subtypes.

Disclosures:

No relevant conflicts of interest to declare.
Trisomy 8 Remains An Intermediate Cytogenetic Risk Factor in Patients with Acute Myeloid Leukemia (AML) Treated with Stem Cell Transplantation
Authors: Walter Claudia Ulrike , Almhareb Fahed , Chaudhri Naeem A. , Zahrani Hazzaa Al , Bakr Mohammad , Ibrahim Khalid , Hussain Fazal , Mohamed Said , Rasheed Walid , Alsharif Fahad , Aljurf Mahmoud , Albitar Maher
Published: October 2019

Abstract 4900

Background:

Trisomy 8 is the most common chromosomal change in AML, as sole aberration in 5 % of cytogenetically abnormal cases and occurring together with other abnormalities in an additional 10% and little is known about the pathogenetic impact of trisomy 8. Most studies reported trisomy 8 in AML to be associated with intermediate risk. However, some reports have identified a poorer outcome than is usually seen in the intermediate group, considering trisomy 8 an adverse prognostic factor. More importantly, the clinical behavior and outcome of patients with trisomy 8 who are treated with hematopoietic stem cell transplantation (HSCT) is not known. We investigated the outcome in patients with trisomy 8 who were treated with HSCT.

Methods:

Bone marrow conventional cytogenetic and FISH data from 110 adult AML patients was reviewed and patients divided into three cytogenetic risk groups according to the European LeukemiaNet reporting system while separating patients with trisomy 8 into another, fourth, independent group. Patients with trisomy 8 as part of a complex karyotype (complex = 3 or more abnormalities) were excluded. We also compared outcome of patients with trisomy 8 to the intermediate-risk group alone and adverse-risk group alone, respectively. The AML cohort included 62 (56%) patients treated in their first remission (CR1) while most non-CR1 AML patients were treated with HSCT in CR2. The median age of all patients was 25 years (range: 14–58).

Results:

Patients with trisomy 8 were 9 (8%). Patients with trisomy 8 showed significantly longer overall survival (OS) (P=0.02) and event-free survival (EFS) (P=0.03) as compared to patients with adverse cytogenetics. The outcome of trisomy 8-positive patients was similar to the intermediate-risk group.

Conclusions:

This data suggests that trisomy 8 in AML is an intermediate-risk factor when patients are treated with HSCT. While we did not compare the outcome of HSCT with the outcome of trisomy 8-positive patients treated with intensive chemotherapy, most likely the cytogenetic risk of trisomy 8 for patients after HSCT is similar to their cytogenetic risk when treated with chemotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation of the Intra-LABORATORY Reproducibility of Percentage of Blast Counting In MYELODYSPLASIA
Authors: Wuilleme Soraya , Eveillard Marion , Barillet Sophie , Accard Françoise , Avet-Loiseau Hervé , Delaunay Jacques , Garand Richard
Published: October 2019

Abstract 4901

In the two International Prognostic Scoring Systems of Myelodysplastic Syndrom (MDS), the percentage of BLAST cells in the Bone Marrow is the most important parameter implicated in score: either directly in the IPSS (International Prognostic Scoring System) (1) or indirectly in the WPSS (Who classification-based prognostic scoring system) (2). In these two systems, as in the recommendations of the WHO 2001 (3), the morphological criteria defining blasts cells compared to promyelocyte cells are not specified. In 2005, the IWGM-MDS (International Working Group on Morphology of myelodysplastic syndrome) (4-6) has established morphological criteria defining Blasts cells.

Our objective in this study is to evaluate the reproducibility, among five observers of our laboratory, of the counting of the marrow blasts of 73 myelodysplasia. This study was conducted in several stages. 1st step was to test the implementation of the MDS-Foundation (www.mds-foundation.org/virtualmicroscopy) by 5 observers. The 2nd step was to perform correlation test of the selected cells from RAEB (selected and stained in our laboratory conditions), for the 5 observers. Finally Step 3 was to assess the correlation of blast percentage of 73 bone marrow smears from MDS patients, selected due to the presence of an excess of blast cells in the first reading on the bone aspiration.

Our results show that the correlation on counting blasts is generally satisfactory (percentage agreement: Test 1 = 86% and test 2 = 94%), while the concordance on the counting blasts of bone marow smears of 73 MDS patients and concordance on the WHO classification seems less satisfactory (agreement 3/5 observers. = 95% but agreed to 4–5/5 observers = 64%). These results can be explained partly by the inter-observer variability and by the variability of some parameters specific to smear marrow (poor quality smears, the staining and/or poverty of smears).

In conclusion, the evaluation of the blasts in the MDS must be achieved: (i) at least 500 cells counted (ii) by at least two different observers (iii) by a third observer in discordant case. Despite these recommendations, the assessment of the blasts in myelodysplasia is difficult to achieve in many cases.

Disclosures:

No relevant conflicts of interest to declare.
Bendamustine for Mantle Cell Lymphoma: Retrospective Analysis of the Spanish Experience
Authors: García-Noblejas Ana , Navarro Matilla Belén , Da Silva Rodriguez Carolina , De Oña Navarrete Raquel , Ramirez Sánchez María José , Nicolás Concepción , Pérez Fernández Ricardo , Sanchez Gonzalez Blanca , Domingo-Domènech Eva , Sanchez Blanco Jose Javier , Cannata-Ortiz Jimena , González-López Tomás José , Arranz Sáez Reyes
Published: October 2019

Abstract 4942

INTRODUCTION.

Patients with Mantle cell lymphoma (MCL) have an adverse outcome after relapse due to chemorefractory disease with conventional treatments. Bendamustine, a nitrogen mustard compound chemically related to the alkylating agents, has demonstrated high efficacy with a low toxicity profile in reported clinical trials.

AIM.

To analyze the Spanish experience in patients with relapsed/refractory MCL treated with Bendamustine.

METHODS.

Retrospective analysis of spanish experience in relapsed/refractory MCL treated with Bendamustine alone or in combination. This study has been approved by local ethical committees.

RESULTS.

Currently, there are 36 patients registered and 28 are available for this analysis. Patients'characteristics: 69% male, median age 65 years old (range 41–81), 87% ECOG≤ 1, 83% Ann Arbor stage IV, 37% high risk MIPI and 9% blastic variant. Previous regimens were CHOP or CHOP like ± R in 42.5%, HyperCVAD/MtxAraC ± R in 42.5%, R-CVP in 9% and other regimens in 6%. Median number of previous treatments were 2.6 (range 1–6), all patients had received prior Rituximab and 73% had chemosensitive disease to the last treatment. Bendamustine regimen was R-B (R-375mg/m2 D1, B-90 mg/m2 D1-2) in 78% patients, R-B with B-70 mg/m2 in 8%, B alone in 3%, R-B-Bortezomib in 3% and R-B plus consolidation (SCT, Y90Ibritumomab-tiuxetan) in 8%. Median number of cycles was 4.61 (range 1–7). G- CSF support was administered in 43% of cycles. Response: Overall response rate was 73%, with 43% CR & uCR and 30% PR. Survival: Median overall survival from diagnosis is 8,26 years (range: 1.6–11,6 years) without plateau. Median progression free survival (PFS) after Bendamustine treatment was 16 months (95% CI: 11.7–20.4), data that compares favourably with patients' PFS to previous therapy (12 months, 95% CI: 6.5–17.5). Median PFS for patients who achieved CR/uCR is 32.6 months (95% CI: 19.9–45.4) versus 11 months in patients with PR (95% CI: 3.9–18.8). With a median follow-up for surviving patients of 12 months since Bendamustine treatment, the estimated OS at 3 years is 47% (+ SD 14%). Toxicity: No treatment related mortality has been described so far. Over 152 cycles, only 10 hospitalizations due to febrile neutropenia were reported. No one case of lysis tumoral syndrome has been reported.

CONCLUSION.

Bendamustine plus Rituximab is a good rescue treatment in non selected pretreated patients with mantle cell lymphoma. CR rate and duration of response seem to reproduce in current clinical practice the good data reported in previous clinical trials and compares favourably with other available treatments.

Disclosures:

No relevant conflicts of interest to declare.