bluuebunny/crossref_metadata_2025_split huggingface dataset:
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
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
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
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
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
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
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
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
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.