bluuebunny/crossref_metadata_2025 huggingface dataset:
Cladribine (2-CdA) Effectiveness in Patients with Low Grade Non-Hodgkin’s Lymphoma - 7 year Polish Study Report.
Authors: Ceglarek Bernadeta B.
Published: October 2019

2-Chlorodeoxyadenosine (2-CdA, Cladribine) was used in patients with advanced, low-grade lymphomas (Lymphoplasmacytoid, Centroblastic/centrocytic, Centrocytic, Lymphocytic) resistant to conventional therapy. There was conducted a trial of 2-CdA group versus control group (patients treated different chemotherapy: Chlorambucil, COP, CHOP) in 85 patients. There were 47 men and 38 women with median age of 55 years were treated. Forty five patients with low-grade lymphomas were given 2-CdA at 0.1 mg/kg/d as a 7–5 day continuous infusion every 4–6 weeks. A total number of courses was different (2–7), median of three courses per patient, of 2-CdA were administered. All patients were evaluable for toxicity and for response.

The results of overall response for 2-CdA group and control group are in table 1.

Table 1- The results of overall response for 2-CdA group and control group

2-CdA Group Control Group P CR (%) 16.7 10.8 p>0.5 PR (%) 28.3 43.2 p>0.5 Overall (%) 45.0 54.0 p>0.5

The results of 2-CdA therapy are showed in tabel 2.

Table 2 - The results of 2-CdA therapy in low grade NHL patients.

2-CdA First line therapy II–III line therapy P Numbers of pts. 15.0 30.0 CR (%) 15.2 15.4 p>0.05 PR (%) 30.3 30.8 p>0.05 Overall (%) 45.5 46.2 p>0.05

The results of 2-CdA therapy LG NHL patients (I-6pts; II-7pts.; III-25pts.; IV-7 pts.) concerned with clinical stage are showed below:

Clinical Stage-CR (%)

I-50

II-42.9

III-12

IV-14.3

P>0.05

Clinical Stage-PR (%)

I-33.3

II-42.9

III-40.0

IV-28.6

P>0.05

Toxicity, in particular opportunistic infections (< or = grade 2, 35.6–48.4% in 2-CdA group v 31.6–64.7% in chemotherapy treated group; P>0.05) and myelosuppression (< or = grade 2 leucopenia, 31.1% in 2-CdA group v 25.0% in chemotherapy treated group, P>0.05 and thrombocytopenia 0–II grade WHO), were more frequent in 2-CdA group (II–III line therapy). The median response duration was 12 months (range, 3 to 44+). I observed less death numbers in 2-CdA first line patients than II–III line 2-CdA group (P=.00132).

Conclusions : There was no statistical significant difference in frequency overall response (CR+PR) between 2-CdA group and chemotherapy treated group.

2-CdA therapy as a consecutive line caused an increase in mortality risk in contrast to the first line therapy.

2-CdA therapy as II–III line therapy allowed to receive complete remission in 9.4% previously treated low grade non Hodkin’s lymphoma patients.
Study on Mechanisms of Telomerase Regulation in Arsenic Trioxide Inducing Apoptosis in Myelodysplasia Cell Line.
Authors: Tong Hongyan , Jin Jie , Xu Weilai , Qian Wenbin , Lin Maofang
Published: October 2019

The telomerase activity can be down regulated by arsenic trioxide (As2O3), which is regarded as an apoptotic induction agent, is confirmed in many kinds of tumor cells. To investigate the mechanisms of telomerase regulation and to explore the correlation of As2O3 inducing apoptosis and telomerase regulation in MUTZ-1 cells, which are established as a high-risk myelodysplasia Cell line that derived from a MDS patient (FAB subtype refractory anemia with excess of blasts), a quantitative assessment of the telomerase activity by TRAP-ELISA and detection of the expression levels of hTERT, TRF1 (TTAGGG repeat binding factor 1), TRF2 (TTAGGG repeat binding factor 2), bcl-2, bax mRNA were performed, together with the assessment of the apoptosis by means of translocation of phosphatidylserine (PS) through flow cytometry assay. The results indicated that a typical apoptotic cell group distribution of DNA content was represented in the MUTZ-1 cells after being exposed to As2O3 at the range of concentration from 1μmol/L to 8μmol/L in a dose-dependent manner (r=0.736, P<0.001) and time-dependent manner (r=0.674, p<0.05), and the telomerase activity was down-regulated in a time-dependent manner (r=−0.976,P=0.024), and the expression level of hTERT mRNA in MUTZ-1 cells was represented in a dose-dependent manner (r=−0.892,P=0.042) and time-dependent manner (r=−1.000,P=0.04), after the cells were treated by As2O3 at the dosage as above. It was showed that a significant correlation between the decreased telomerase activity and the increased percentage of apoptotic cells in the treated cells (r=0.938,P=0.018), and there was a strong relationship between the telomerase activity and the mRNA expression of hTERT gene (r=0.783,P=0.022). However, As2O3 has no obvious effect on the expression level of TRF1 mRNA and TRF2 mRNA, which were regarded as two telomere-binding proteins. Further findings indicated that the inhibition of telomerase activity in MUTZ-1 cells was accompanied with down-regulated mRNA expression of bcl-2 gene (densitometry readings: 0.255±0.017 vs 0.466±0.069, P<0.05) and decreased ration of bcl-2/bax (densitometry reading ratios: 0.890±0.083 vs 0.546±0.014, P<0.05) at the dosage of 4μmol/L for 24 hours. These observations suggest that the apoptosis induced by As2O3 on MUTZ- 1 cells might be mediated through the inhibition of telomerase activity regulated by expression of hTERT gene, which implies that may be one of the mechanisms of As2O3 inducing apoptosis in MUTZ-1 cells.
Lysophosphatidic Acid (LPA) Protects Primary Chronic Lymphocytic Leukemia (CLL) Cells from Apoptosis: Role for LPA as a Survival Factor in CLL.
Authors: Gibson Spencer B. , Hu Xiaojie , Haney Neil , Kabore Albert F. , Johnston James B.
Published: October 2019

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+/CD5+ lymphocytes, and this is achieved primarily through a block in apoptosis. The mechanisms underlying this defect are not fully understood. Several proteins have been shown to protect CLL cells from apoptosis and one of these is albumin that solublizes lipids in the plasma. A lipid found in plasma, lysophosphatidic acid (LPA), protects epithelial and fibroblast cells from apoptosis. We investigated whether LPA could be a survival factor in CLL. Herein, we demonstrate that LPA effectively protects the B cell line BJAB and the CLL-like cell line I-83 from etoposide, fludarabine, and chlorambucil-induced apoptosis. In primary CLL cells, plasma from either healthy or CLL patients significantly reduces spontaneous and drug-induced apoptosis. However, delipidation of the plasma reduces its protective effect. In addition, LPA protects primary CLL cells but not healthy lymphocytes from apoptosis. By western blotting, the LPA receptor 1 (LPA1) expression is increased in primary CLL cells compared to normal lymphocytes. Treatment of primary CLL cells with the LPA receptor antagonist diacylglycerol pyrophosphate (DGPP) reverses the protective effect of LPA against apoptosis. Over expression of the LPA1 receptor protects cells from apoptosis and downregulation of the receptor blocks LPA mediated protection against spontaneous apoptosis. The protective effect of LPA is inhibited by blocking activation of the PI-3K/AKT signaling pathway. These results indicate that LPA is a survival factor in primary CLL cells and that drugs targeting the LPA receptors might be an effective therapy for this disease.
Monoclonal Gammopathy of Undertermined Significance (MGUS): Clinical Predictors of Malignant Transformation in 434 Patients with a Long Follow-Up from a Single Institution.
Authors: Rosiñol L. , Montoto S. , Bladé J. , Cibeira M.T. , Rozman M. , Nadal E. , Giné E. , Aymerich M. , Esteve J. , Montserrat E.
Published: October 2019

Background: MGUS is a common disorder characterized by the presence of a small serum M-protein in individuals with no evidence of multiple myeloma (MM), Waldenström’s macroglobulinemia (WM) or primary amyloidosis (AL). Although about one fourth of these individuals will evolve into a malignant disease with a transformation rate of 1% per year, there are not well-established predictors of outcome.

Aim: To identify predictor features of malignant transformation in a large series of patients with MGUS and prolonged follow-up.

Patients and methods: Four hundred and thirty-four patients (200 M/234 F; median age 66 years) diagnosed with MGUS in a single institution from September 1970 to January 2001 with a minimum follow-up of one year were included in the study. All patients had an M-protein size < 30g/L. Bone marrow aspirates were reviewed independently by two of the authors and the proportion of bone marrow plasma cells (BMPC) were estimated from a 500 cell-count by each observer. The median follow-up was 5.2 years (range: 1–28.8 years) with 84 patients followed for more than 10 years.

Results: The type of M-protein was IgG in 67.2% of the cases, IgA in 18.8%, IgM in 11.9%, light chain in 1% and biclonal in 1%. The light chain was kappa type in 56.4% of the patients. The median M-protein size was 15.6 g/L (<10 g/L in 10.8%, 10–20 g/L in 61.7%, and > 20g/L in 27.4%). The median percentage of BMPC in 305 reviewed samples was 4.6% (range: 0.4–25). After a median follow-up of 5.2 years, 50 patients (11.5%) have evolved into a malignant monoclonal gammopathy (44 MM, 5 WM and 1 AL). The median time to progression was 5.4 yrs (range: 1.4 – 16.9). The risk of transformation was 15.4% (95% CI: 10.5–20.3) and 34% (95% CI 22.6–45.3) and 34% (95% CI: 22.6–45.3) at 10 and 20 years, respectively. The variables associated with a higher risk of transformation were IgA-type (p=0.003), kappa light chain (p=0.009), the amount of M-protein (<15 vs >15 g/L, p=0.005) and the percentage of BMPC (<5% vs >5%, p=0.007).

Conclusions: In this series of patients with MGUS, the type (i.e. IgA or kappa) and size of M-protein (>15g/L) as well as the percentage of bone marrow plasma cells (>5%) significantly predicted malignance transformation.
Fatty Acid Synthase Inhibitor Induces Apoptosis and Inhibits Proliferation in Multiple Myeloma Cells.
Authors: Wang Weiqin , Wu Dong
Published: October 2019

Objective: To investigate the effect of fatty acid synthase (FAS) inhibitor (cerulenin) in multiple myeloma cells and its mechanism.

Methods: Proliferatory inhibition rate of cerulenin in multiple myeloma cells U266 and RPMI8226 was assayed by MTT, apoptosis analysis and cell DNA cycle analysis by flow cytometry (FCM).

Results: Treated with 5ug/ml~640ug/ml of cerulenin for 24h, the proliferation of U266 and RPMI8226 cells were obviously inhibited with dosage related effect at the same concentration, the inhibition rate of human skin fibroblast are all lower than 28%. When U266 and RPMI8226 are treated for 12h with 20ug/ml of cerulenin the apoptosis rate revealed by FCM are 51.3% and 48.8% respectively. Cell DNA cycles analysis show the cell DNA is blocked in G1 phase and S phase cells decreased in both of U266 and RPMI8226.

Conclution: FAS cold induce MM cell apoptosis and inhibit MM cell proliferation. FAS might be a new potential target for MM therapy.
p38 alpha MAP Kinase Inhibitor Inhibits Human Myeloma Cell Growth in Vivo.
Authors: Reddy Mamatha , Ma Jing Y. , Kerr Irene , Henjarappa Nirupama , Navas Tony , Chakravarty Jit , Dugar Sundeep , Higgins Linda S. , Protter Andrew A. , Medicherla Satyanarayana
Published: October 2019

The p38 MAPK signaling pathway plays an important role in different pathological conditions and specific inhibitors of p38 alpha and beta MAPK block production of major inflammatory cytokines. Human multiple myeloma (MM) is an incurable neoplasm of B cells, and MM disease progression is affected by immunoregulatory elements such as IL-6 and other cytokines. Recent literature indicates that an important function of p38 MAP kinase is the generation of signals of critical value to the control of normal and malignant hematopoiesis by cytokines and growth factors. We therefore examined the effect of a p38 alpha MAP kinase specific inhibitor, SD-282, on human myeloma cell (RPMI8266) tumor growth in immunodeficient beige-nude-xid mouse xenograft plasmacytoma model. Treatment (90 mg/kg/bid by oral gavage) was initiated in mice with palpable tumor size (~200 mm3) as judged by tumor volume. In mice with palpable tumors, 21 days of SD-282 treatment significantly reduced tumor volume. When SD-282 treatment was initiated in mice with pronounced tumor size (~800 mm3) there remained a significant reduction in tumor volume. Histological assessment at the end of dosing from animals administered SD-282 or vehicle demonstrated a significant reduction in tumor volume and number of neoformed microvessels in the SD-282 treatment group. It also significantly reduced HSP27 and p38 expressions in tumor cells.

In conclusion, we report that the p38 inhibitor inhibits human myeloma cell growth in vivo via p38 MAP kinase signaling pathway. The current study provides strong in vivo evidence supporting use of p38 inhibitor therapy in patients with multiple myeloma
Rapid Clinical Scale Isolation of CD25hiCD4+ Regulatory T Cells.
Authors: Niemand Claudia , Conrads Carina , Siemer Ramona , Assenmacher Mario
Published: October 2019

Several publications during the last few years have reported CD25hiCD4+ regulatory T cells (Tregs) to prevent or to reverse disease in different mouse models of experimental autoimmune encephalomyelitis (EAE), colitis, graft rejection and graft-versus-host-disease (GvHD). As mouse and human Tregs share many phenotypical and functional characteristics, Tregs could provide a promising therapeutic approach for various human autoimmune diseases and pathological alloresponses. Here we have shown that Tregs can be isolated from leukapheresis harvests by CD25 enrichment using the CliniMACS technology (n=13). By this procedure we obtained 2.32x108 (± 1.12x108, range 0.71–4.42x108) cells out of 1010 mononuclear cells with a mean purity of 52.12% (± 12.11%, range 25.48–66.61%) for CD25hiCD4+ cells. Around 90% of enriched cells were CD25+CD4+. Among contaminating CD4− cells most cells were CD25+ which were further characterized by counterstaining to be mainly CD19+ B cells and a few CD8+, CD56+ or CD123+ cells. It is possible to deplete the CD19+ or CD8+ cells by using CD19 Microbeads or CD8 Microbeads respectively with the CliniMACS Instrument before CD25 enrichment. Combined depletion of different cells, e.g. CD19+ and CD8+ cells is conceivable. Isolated cells were phenotypically and functionally characterized. The majority of the CD25hiCD4+ T cells expressed glucocorticoid-induced tumor necrosis factor receptor (GITR), CD62L and CD45RO. In addition, isolated cells were able to suppress the proliferation and activation of cocultured conventional CD4+ cells after polyclonal stimulation with anti-CD3 antibody. We conclude that the large-scale isolation of CD25hiCD4+ regulatory T cells for clinical applications (e.g. therapy of autoimmune diseases, graft rejection or GvHD) is possible by using the CliniMACS CD25 Reagent and the CliniMACS Instrument.
Development of Functional Haemophilus Influenzae Type B Antibodies after Vaccination of Autologous Stem Cell Transplant Recipients.
Authors: Van der Velden Ankie M.T. , Claessen Anke M.E. , Van Velzen-Blad Heleen , Biesma Douwe H. , Rijkers Ger T.
Published: October 2019

The conditioning regiments for autologous stem cell transplantation (aSCT) lead to impairment of the immune system and susceptibility to infections. Although infectious complications are more prominent in allogeneic transplantation, it is also a major source of morbidity in patients undergoing aSCT. Therefore, vaccination of patients after stem cell transplantation has been recommended. We conducted a prospective follow-up study to determine quantitative and qualitative aspects of the humoral immune response to multiple vaccinations with conjugated Haemophilus influenzae type b (Hib) vaccine in autologous stem cell transplant recipients. Sixteen patients (twelve with multiple myeloma and four with non-Hodgkin’s lymphoma) received a conjugated Hib vaccine at 6, 8 and 14 months after transplantation. Antibody titres were determined by ELISA, 21 days after each vaccination. Adequate anti-Hib antibody responses (i.e. a four fold or greater increase in antibody levels in addition to a minimal titre of 50 U/ml corresponding to 18.8 μg/ml) were achieved in 7 (44%) of the patients after one vaccination. A second and third vaccination resulted in an adequate anti-Hib antibody response in 12 (75%) and 14 (88%) patients, respectively. Functionality of anti-Hib antibodies was assessed by measurement of antibody avidity. Repeated vaccination induced maturation of antibody avidity as demonstrated by increased resistance to sodium thiocyanate (NaSCN) treatment (Figure 1; the avidity index (AI) is defined as the molarity of NaSCN at which 50% of the amount of IgG antibodies bound to the coated antigen in the absence of NaSCN has been eluded. Mean AI plus range are shown after one, two and three vaccinations.). Subsequently, opsonizing activity of antibodies was determined by a phagocytosis assay. FITC-labelled Hib was incubated with serum and added to fresh polymorphonuclear cells (PMN). Mean fluorescence intensity (MFI) was measured by flowcytometry. Anti-Hib antibodies supported phagocytosis by PMN’s after multiple vaccinations (Figure 2; MFI at different time points is shown). In conclusion, multiple vaccinations with Hib-conjugate vaccine in autologous stem cell recipients result in high antibody response rates and functional maturation of antibodies.
The Use of Reduced Intensity Allogeneic Stem Cell Transplantation (alloSCT) for Recurrence after Autologous Stem Cell Transplantation (ASCT) in Hodgkin’s Disease (HD).
Authors: Krishnan Amrita Y. , Nademanee Auayporn , Fung Henry C. , Popplewell Leslie , Rodriguez Roberto , Kadhimi Zaid Al , Kirschbaum Mark , Kogut Neil , Raubitschek Andrew , Schriber Jeffrey , Zain Jasmine , Smith Eileen , Cheng Nigel , Qian Dajun , Molina Arturo , Forman Stephen J.
Published: October 2019

Autologous stem cell transplantation(ASCT) can cure up to 50% of patients with relapsed or refractory HD. However, for patients who relapse post ASCT or develop post transplant myelodsyplasia (t-MDS), standard chemotherapy options offer little chance of long-term disease free survival (DFS). The use of allogeneic stem cell (alloSCT) may provide a means of control of the HD through the immune mediated effects of an allogeneic approach and also be curative for concomitant t-MDS. While traditional alloSCT in HD patients was fraught with high treatment related mortality, a reduced intensity conditioning regimen may reduce upfront mortality while still providing long term DFS.

Between 11/00 and 1/04, fourteen patients underwent reduced intensity alloSCT at the City of Hope Cancer Center. Median age at alloSCT was 31years (range18–47). Median time from HD diagnosis to alloSCT was 36mos (range 3–123). Eleven patients had relapsed post ASCT and one post syngeneic transplant. One of the pts also had t- MDS. The median time from ASCT to alloSCT was 20 mos(range1–45). Ten pts received sibling alloSCT and four unrelated donor. The conditioning regimen consisted of Fludarabine 125mg/m2+ Melphalan 140mg/m2. All patients received a Cyclosporin(CSA) based graft versus host disease (GVHD) prophylaxis regimen; nine in conjunction with Mycophenolate Mofetil and three in conjunction with Methotrexate. Seven developed acute GVHD (3-GrIII-IV). Two patients died of GVHD. There was no other significant regimen related toxicity. Median length of followup for surviving patients is 55 mos. For eleven evaluable pts, one year OS and DFS are 77% (95%CI 49–100) and 58% (95%CI 25–91) respectively (figure 1). The pt with concomitant t-MDS also achieved a hematologic remission. In conclusion, reduced intensity alloSCT may provide a salvage for heavily pretreated patients with HD that have failed ASCT and who otherwise have a poor prognosis, and it can also provide a treatment for late secondary complications of transplantation such as t-MDS.
Molecular Relapse of Acute Lymphoblastic Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation Can Be Successfully Treated with Donor Lymphocyte Infusions.
Authors: Dominietto Alida , Raiola Anna Maria , Grasso Raffaella , Garuti Anna , di Grazia Carmen , Bregante Stefania , Gualandi Francesca , Ibatici Adalberto , Lamparelli Teresa , van Lint Maria Teresa , Frassoni Francesco , Bacigalupo Andrea
Published: October 2019

Background. Hematologic relapse is seen in 20–40% of adult acute lymphoblastic leukemia (ALL) patients undergoing an allogeneic hematopoietic stem cell transplant (HSCT) and is usually non responsive to donor lymphocyte infusions (DLI). This has suggested a lack of graft vs leukemia effect (GvL) in ALL patients. It is currently possible to monitor minimal residual disease (MRD) post-HSCT and this may allow to identify patients at high risk of hematological relapse.

Objectives. To monitor MRD in B-ALL patients post-HSCT and to treat MRD positive patients with DLI.

Patients and Methods. MRD was evaluated on bone marrow samples using nested polymerase chain reaction (PCR) and real time PCR (RT-PCR) assay to analyze respectively IgH VDJ and BCR-ABL gene rearrangement. Molecular positivity/relapse was defined with two consecutive positive PCR assays. MRD monitoring was performed in 28 patients grafted from an HLA-identical sibling (n=19), family mismatched related donor (n=2) or matched unrelated donor (n=7). Median follow-up was 47 months (range 5–163). Sixteen patients (57%) were in early disease phase at the time of transplant.

Results. We identified 3 groups of patients. A) 14 patients (50%) had no evidence of MRD after HSCT. B) 6 (21%) had a positive MRD and received escalating dose DLI, within 60 days and C) 8 (29%) had a positive MRD, but did not received DLI, because not available or because of an early hematological relapse. Median time from HSCT to molecular relapse was 139 days (range 46–1048). The median follow up was 1385 days (144 – 4877). Median number of infused CD3+ cells was 0.6x10^7/Kg of recipient body weight (range 0.01–7). Hematological relapse was seen in 0%, 0% and 88% respectively of group A,B,C (p=0.0001) and disease-free survival (DFS) was 100% , 67% , 12% (p=0.0001).

Conclusions. This study shows that (1) MRD monitoring of ALL patients post-transplant identifies patients at high risk of hematologic relapse and (2) that treatment with DLI, on the basis of MRD positivity, significantly reduces the risk of leukemia relapse and may improve DFS. These data also confirm the existence of a GvL effect, previously shown in this disease (Weiden et.al NEJM, 300, 1068; 1979).
Drug Resistance and In Vitro Selection of Human Hematopoietic Cells Transduced with Cytidine Deaminase.
Authors: Flasshove Michael , Rattmann Ina , Lehmberg Kai , Bardenheuer Walter , Sorg Ursula R. , Seeber Siegfried , Moritz Thomas
Published: October 2019

Retroviral gene transfer of human cytidine deaminase into murine hematopoietic cells has been shown to confer drug resistance against cytidine analogues such as araC (1-β-D-arabinofuranosylcytosine) and gemcitabine (2′,2′-difluorodeoxycytidine). Since data for primary human hematopoietic cells are lacking so far, we transduced human CD34+ enriched umbilical cord blood cells with the retroviral vector SF91-CDD-IRES-EGFP containing the cDNA of cytidine deaminase (CDD) and EGFP as a reporter gene. The transduced cells were tested in clonogenic assays for resistance against increasing concentrations of araC or gemcitabine. Gene transfer of SF91-CDD-IRES-EGFP resulted in a transduction efficiency of 28 ± 2% (n = 4). A significant higher survival of CFU-C in the presence of araC could be demonstrated as compared with the mock control expressing EGFP only. This protective effect was improved by FACS enrichment of EGFP+ cells prior to functional analysis, and was observed for a dose range of 30 – 500 nM araC. Gemcitabine resistance was also tested with cells enriched for EGFP expression. For CFU-C a significant higher survival of colonies at concentrations of 8 and 10nM could be shown. This protection was more pronounced for CFU-GM than for BFU-E. The IC50 value increased 3fold for CFU-GM but only 1.4fold for BFU-E and 1.8fold for total CFU-C in comparison to the mock control (n = 4). In vitro selection of CDD transduced progenitor cells was investigated by incubation in the presence of 30 – 100nM araC for 4 or 8 days. Before and after culture cells were analysed for EGFP expression and tested in clonogenic assays for araC resistance. Significant selection of CDD transduced cells as measured by EGFP expression was observed for all examined concentrations. After 4 days incubation with 60nM araC clonogenic assays revealed a significant selection of progenitor cells resistant to 100 – 500nM araC. Incubation of transduced cells in 6 – 15nM gemcitabine for 4 or 8 days likewise allowed successful in vitro selection. Transplantation of CDD transduced CD34+ cells in sublethally irradiated NOD/SCID mice resulted in up to 81% engraftment of human CD45+ cells and a rate of up to 4.6% of human CD45+/EGFP+ cells after 8 weeks. These data demonstrate that retroviral gene transfer of CDD protects human hematopoietic cells from the cytotoxic drugs araC and gemcitabine and allows selection of transduced cells. Successful engraftment of CDD transduced cells in NOD/SCID repopulating cells could be observed.
Impact of Extracorporeal Photochemotherapy on the Clinical and Economical Management of Patients Affected with GVHD.
Authors: Perotti Cesare , Isernia Paola , Del Fante Claudia , Viarengo Gianluca , Bressan Daniela , Santini Tiziana , Salvaneschi Laura
Published: October 2019

Extracorporeal photochemotherapy (ECP) is an effective, relatively new technique, FDA approved for cutaneous T-cell lymphoma, employed as second-third line treatment in patients affected with GVHD not or poorely responsive to standard immunosuppressive drugs. Both aGVHD and cGVHD are the major complications after stem cell transplantation (till to 50% and 80% of the patients, respectively). Corticosteroids, cyclosporine, micofenolate, tacrolimus and various experimental monoclonal antibodies (anti CD40 ligand, anti TNFα etc) are the drugs employed to control GVHD and are burdened with important short and long term side effects. Recently we revised our data about 102 pts (children and adults) treated by ECP in our Institution. The overall response was 75%, permitting to taper or suspend the immunosuppressive therapy (IST) in 67.6% of the pts. The economical and social impact (cost analysis and quality of life) of ECP vs standard IST was calculated basing on the National Price List and on Short Form Quality of Life (QoL) Scoring System. The cost of ECP comprehensive of the leukapheresis procedure, waste matherials and dedicated personnel was estimated as 684.51 Euro/procedure. On the other side, when the solely costs of the most common IST drugs (corticosteroids, cyclosporine) were considered for 5 months of treatment, an evident and obvious economical advantage emerged (120,3 E). On the contrary, when the costs of hospitalisation and day hospital regimen derived from the most common side effects related to the solely use of IST were included in our cost analysis studies, an economical advantage for long term ECP treatment (calculated on 16 procedures) was demonstrated (14952.16 E vs 17553 E). Moreover, when the “real life implication” calculated on the QoL parameters were considered, the advantages were more evident. In conclusion, the tie of respecting strictly an imposed program of budget calculated on the short period may exert an inhibitory effect in introducing new diagnostic or therapeutic procedures ignoring that the improvement or the cure of the patient has always a positive economical counterpart, expecially when the impact of a new technology is considered in a long term view.
User Preference for 3 Hemophilia Factor Reconstitution Methods.
Authors: Mannix Sally , Brewster Jessica , Rentz Anne M. , Lloyd Andrew , Butler Regina , Larson Peter J.
Published: October 2019

Introduction: Treatment of hemophilia includes the reconstitution of lyophilized FVIII concentrates with diluent followed by intravenous injection. Several reconstitution devices exist for infusion of concentrates. The Bio-Set® (Biodome, Issoire France) is a new needleless device for reconstituting a factor VIII concentrate, Kogenate® FS (Bayer Healthcare, Elkhart, IN).

Objective: Comparison of preferences for reconstitution methods by hemophilia patients, caregivers to hemophilia patients, and care providers.

Methods: A total of 35 hemophilia A patients who infuse themselves, 67 caregivers, and 59 infusion nurses were recruited following an IRB-approved protocol. Infusion method names were blinded to participants to prevent brand recognition. Reconstitution methods used in this study were Bio-Set®, a conventional 2 vial, transfer needle reconstitution method, and Baxject (Baxter Healthcare, Westlake Village CA). Participants viewed a video demonstrating the three methods, watched the interviewer demonstrate each method, and then practiced each method once. Each participant performed 1 timed round with each method and completed preference questionnaires about each method. The preference questionnaire was scored into domains for safety/worry and ease/confidence, in addition to a total preference score. Participants’ comments about the reconstitution methods were also recorded.

Results: Participants preferred the Bio-Set® in terms of worry/safety, ease/confidence, and overall preference. These results were consistent across participant group. Participants agreed on their preferences for the Bio-Set® in terms of safety from needlesticks, amount of waste produced, and convenience for traveling (all p<0.0001). Analysis of the qualitative data indicated that exposure to needles and product contamination were issues commonly noted. Safety and complexity of instructions were also common issues for participants.

Conclusions: Results of this study show that patients, caregivers, and nurse providers prefer the Bio-Set® reconstitution device over other methods for preparing factor concentrates for infusion.
Comparable Survival in Newly Diagnosed Multiple Myeloma (MM) after VAD Induction with High Dose Therapy Using Melphalan 140mg/M2 + TBI 12 Gy (MEL+TBI) Versus Standard Therapy with VBMCP and No Benefit from Interferon (IFN) Maintenance: Final Clinical Results of Intergroup Trial S9321 in the Context of IFM 90 and MRC VII Trials.
Authors: Crowley John , Fonseca Rafael , Greipp Phillip , Jason McCoy , Bart Barlogie
Published: October 2019

ECOG, CALGB and SWOG (coordinating group) enrolled 899 patients with newly diagnosed MM to receive VAD x 4 (813 were considered eligible), followed by randomization to PBSC-supported MEL-TBI (n=261) versus VBMCP (n=255), using CTX 4.5g/m2 + G-CSF for PBSC mobilization in all patients. Responders to VBMCP or MEL-TBI were randomized to IFN (n=121) or no maintenance (n=121). With a median follow-up of alive patients of 60 mos, median EFS and OS from 1st randomization were 25 and 62 mos for MEL-TBI and 21 and 53 mos for VBMCP (p=.14 for EFS, p=.87 for OS). EFS and OS from IFN randomization were also identical at 23/59 mos for IFN versus 18/74 mos for observation (p=.2/.6). 87 VBMCP failures received salvage autotransplants, with a post-relapse median survival of 24 mos which was similar to the 27 mos observed among the remaining 83 VBMCP failures managed with standard salvage therapies. When examined in the context of 2 other randomized trials (IFM 90, MRC VII), OS from baseline of S932, limited to patients <60 yr (as in IFM 90 + MRC VII), is intermediate between the high dose arms of IFM 90/MRC VII and the standard dose arms of these two trials (randomization at baseline) (Figure 1). OS from the time of randomization to high or standard dose therapy on S9321 (again restricted to age <60 yr) was similar to OS timed from transplant for the high dose arms of IFM 90 and MRC VII. (Figure 2).

Figure Figure

The comparable outcome after high- versus standard-dose therapies on S9321, interpreted in the context of 2 other published randomized clinical trials, may relate to (1) higher dose intensity with VBMCP than with other standard regimens or (2) a detrimental effect of TBI in S9321. We conclude that with the current follow-up MEL-TBI is no better than VAD/VBMCP with the option of salvage transplant and that IFN maintenance is of no benefit in this maintenance setting. Data from this trial will be provided to MRC to be included in their on-going meta-analyses of high-dose therapies and IFN maintenance.
In Vivo Fate Tracing Studies Using the SCL Stem Cell Enhancer: Embryonic Hematopoietic Stem Cells Significantly Contribute to Adult Hematopoiesis.
Authors: Gothert Joachim R. , Gustin Sonja E. , Hall Mark A. , Green Anthony R. , Gottgens Berthold , Izon David J. , Begley C. Glenn
Published: October 2019

Evidence for the direct lineage relationship between embryonic and adult hematopoietic stem cells (HSCs) in the mouse is primarily indirect. In order to study this relationship in a direct manner we expressed the tamoxifen-inducible Cre-ERT-recombinase under the control of the SCL-stem-cell-enhancer in transgenic mice (HSC-SCL-Cre-ERT). To determine functionality, HSC-SCL-Cre-ERT transgenics were bred with the Cre-reporter-mice ROSA26R and R26R-EYFP. Flow-cytometric and transplantation studies revealed tamoxifen-dependent recombination occurring in more than 90% of adult long-term HSCs, whereas the targeted proportion within mature progenitor populations was significantly lower. Moreover, the transgene was able to irreversibly tag embryonic HSCs on days 10 and 11 of gestation. These cells contributed to bone marrow hematopoiesis five months later. In order to investigate whether the de novo HSC-generation is completed during embryogenesis, HSC-SCL-Cre-ERT marked fetal liver cells were transplanted into adult recipients. Strikingly, the proportion of marked cells within the transplanted and the in vivo-remaining HSC-compartment was not different, implying that no further HSC-generation occurred during late fetal and neonatal stages of development. These data demonstrate for the first time the direct lineage relationship between mid-gestation embryonic and adult HSCs in the mouse. Additionally, the HSC-SCL-Cre-ERT mice will provide a valuable tool to achieve temporally controlled genetic manipulation of HSCs.
Expression Profile of Aging Hematopoietic Stem Cells.
Authors: Chambers Stuart M. , Shaw Chad A. , Goodell Margaret A.
Published: October 2019

A decline in stem cell function has been suggested to contribute to vertebrate aging. Several labs have documented a reduction in transplant efficiency and skewing in lineage contribution when murine bone marrow or hematopoietic stem cells (HSC) from old donors were transplanted into young recipients. Paradoxically, evidence from several labs including ours has shown that the percentage of phenotypically defined HSC in C57Bl/6 mice increases with age (Fig. 1). Within the serum of aged animals, systemic inflammatory markers such as C reactive protein in humans and IL-1 in mice have been shown to steadily increase with age, however it is unclear if systemic inflammation plays a role in these age-related HSC phenotypes. In order to investigate what might account for these functional changes in HSC aging, we have characterized gene expression in aged HSC using Affymetrix microarrays, examining expression profiles of HSC purified from C57Bl/6 mice that are 2-, 6-, 12-, 21-months old. Using polynomial regression over the time course, we have found more than 700 genes that are 2-fold up-regulated and more than 400 genes that are down-regulated with time. We used the Gene Ontology to categorize age-regulated genes, and have identified the category of ‘inflammatory response’ to be significantly enriched in genes that are up-regulated with age. Strikingly, several NF-Kb regulated genes, previously associated in other tissues with aging and inflammation (e.g. P-selectin, Cox-2, and ICAM-1) are up-regulated in aged HSC. The expression patterns of more than 15 genes, including clusterin, serum deprivation response, and growth hormone receptor, have been validated by quantitative real-time PCR. Furthermore, expression of several surface markers, including P-selectin, a protein that plays a role in inflammation, has been validated at the protein level by flow cytometry. In order to determine if the gene expression changes observed in aged HSC is caused by intrinsic or extrinsic factors, we have transplanted HSC from 21-month-old mice into young recipients and are assessing changes in their expression profile compared to young transplanted HSC. These data demonstrate a role for inflammation in HSC-aging, and may identify genes involved in stem cell transplant efficiency, lineage specification, and the onset of organismal aging.

Figure 1: Sca-1 enriched Side Population cells at the indicated ages (in months). The percentage of cells residing within the SP increases 9-fold with age. Figure 1:. Sca-1 enriched Side Population cells at the indicated ages (in months). The percentage of cells residing within the SP increases 9-fold with age.
Single 4-Dose Rituximab Treatment for Low-Tumor Burden Follicular Lymphoma (FL): Survival Analyses with a Follow-Up (F/Up) of at Least 5 Years.
Authors: Solal-Celigny Philippe , Salles Gilles Andre , Brousse Nicole , Franchi-Rezgui Patricia , Soubeyran Pierre , Delwail Vincent , Deconinck Eric , Haioun Corinne , Foussard Charles , Sebban Catherine , Tilly Herve , Milpied Noel-Jean , Boue Francois , Karsenti Jean-Michel , Lederlin Pierre , Najman Albert , Thieblemont Catherine , Morschhauser Franck , Berriot-Varoqueaux Nathalie , Bergougnoux Loic , Colombat Philippe
Published: October 2019

Rituximab alone may be given as initial treatment for FL either as a single treatment or as an induction treatment followed by a maintenance. In 2002, we have reported the response rates and progression-free survival (PFS) of 49 patients (pts) treated with 4 weekly 375 mg/m² doses of rituximab (Blood2002; 97: 101–6). All patients had FL and a low-tumor burden according to the GELF criteria. These pts have been followed during at least 5 years. According to the Follicular Lymphoma International Prognostic Index (Blood, in press). 22 pts were in the low-risk group (45%), 20 (41%) in the intermediate risk group and 7 (14%) in the poor risk group.

Best response rate was 80% with 49% CR/CRu and 31% PR, and response was maintained in 34% without further treatment after at least 5 years. Median F/Up was 60 months. The median PFS was 18 months. Among these 49 pts, only 3 pts died (2 from NHL, 1 from lung carcinoma).

Among the 32 pts who were bcl-2 positive in the blood and/or the bone marrow before treatment with rituximab, 10 (33%) became negative and 20 (67%) remained positive at d50. 6 (60%) relapsed among the former and 15 (75%) among the latter. Median PFS was 37 months for pts who became bcl-2 negative and 14 months for those who remained positive (p[log-rank test] = 0.10). Among the 4 patients who had information on molecular biology who did not relapse after a 5-year F/Up, 3 were bcl-2 negative and 1 was bcl-2 positive.

The long F/Up of these pts (i) confirms the median PFS of 18 months for all pts and the median relapse-free survival of 27 months for best responders (ii) shows that some patients (i.e. 28% of all pts and 34 % of responders) may have a relapse-free survival longer than 5 years after a single first line treatment with rituximab in monotherapy ; (iii) shows an excellent overall survival (only 3 death (6%) during the study) in these FL pts. These results confirm the relevance of on-going trials comparing a maintenance treatment with rituximab and treatment at the time of relapse.
Patients with Follicular Lymphoma, Grade 3, Have a Prolonged Relapse-Free Survival Following Aggressive Combination Chemotherapy.
Authors: Ganti Apar Kishor , Weisenburger Dennis D. , Smith Lynette M. , Hans Christine P. , Bociek R. Gregory , Bierman Philip J. , Vose Julie M. , Armitage James O. ,   The Nebraska Lymphoma Study Group
Published: October 2019

Clinical prognostic factors for grade 3 follicular lymphoma (FL3), using the new WHO classification, have not yet been clearly defined. Hence, we conducted a retrospective study to identify the outcome and clinical features predictive of survival in patients with FL3. Two hundred and two patients diagnosed with FL3 using the Berard criteria, who were staged and treated with various aggressive combination chemotherapy regimens containing either an anthracycline or mitoxantrone, are included in this study. After a median follow-up of 8.4 years (range, 0.5 – 19.5 years), 105 patients (52%) have died and 97 patients (48%) are alive at last contact. The estimated 10-yr event-free survival (EFS) and overall survival (OS) are 34% (95% CI: 26–41%) and 45% (95% CI: 37–53%), respectively. By multivariate analysis, older age (≥60 years; Figure 1) and a low hemoglobin (<12 gm/dL) were independent adverse predictors of OS (RR: 2.3, 95% CI: 1.5–3.7, p=0.0002; and RR: 1.9, 95% CI 1.2–3.0, p=0.0095, respectively). A low hemoglobin and advanced stage (III/IV) at presentation were independent adverse predictors of EFS (RR: 1.8, 95% CI: 1.2–2.8, p=0.0072; and RR: 1.7, 95% CI: 1.1–2.5, p=0.0096 respectively). When patients were compared based on age, there were no significant differences in the distribution of adverse prognostic factors among patients ≥60 years of age as compared to younger patients. There were also no differences in the frequency of relapse/progression (10-year rate of 50% in patients <60 years vs. 44% in patients ≥60 years, p=0.71) or lymphoma-specific/treatment-related deaths between the two age groups (10-year rate of 30% in patients <60 years vs. 35% in patients ≥60 years, p=0.17; Figure 2). Although younger patients (<60 years) had a significantly better OS and EFS as compared to older patients, there were no differences in the relapse/progression rates or lymphoma/treatment-related survival. Thus, aggressive combination chemotherapy led to durable remissions in 50–60% of patients with FL3.

Figure Figure Figure Figure
First Report of the Phase III North American Trial of Decitabine in Advanced Myelodysplastic Syndrome (MDS).
Authors: Saba Hussain , Rosenfeld Craig , Issa Jean-Pierre , DiPersio John , Raza Azra , Klimek Virginia , Slack James , de Castro Carlos , Mettinger Karl , Kantarjian Hagop
Published: October 2019

MDS is a disease of the elderly marked by dysplasia of hematopoetic cell lines resulting in cytopenias and AML progression. There is currently no treatment approved to alter the natural history of the disease. Aberrant methylation associated with cancer is a potential target for pharmacologic therapy, and DacogenTM (decitabine) for injection (SuperGen, Inc.), a cytosine analogue, indirectly depletes methylcytosine after incorporation into DNA with subsequent inactivation of DNA methyltransferases. We report the results of a Phase III trial of decitabine (DAC) vs. supportive care (Supp.Care) in adult MDS patients with IPSS Intermediate (Int)-1 (31%), Int-2 (44%) and high risk disease (26%). Secondary MDS (14%) and previously treated (27%) MDS patients were not excluded. Bone marrows were assessed by a blinded, independent pathologist. 170 patients (accrued from July 2001 through April 2003 at 23 centers) were randomized 1:1 to either Supp.Care or DAC (a 3 hr infusion of 15mg/m2/hr every 8 hrs on 3 consecutive days every 6 wks for up to 10 cycles. The groups were comparable for numerous risk factors, including time from diagnosis (median 29 weeks for DAC and 35 weeks for Supp.Care). Kaplan Meier (KM) curves for time to AML progression or death showed early and clinically meaningful separation (consistent with clinical benefit) in favor of DAC in all patients (intent to treat; ITT), Int-2/High Risk, and High Risk patients. Using the Cox proportional hazards model for the ITT population, the probability of progression to AML or death was 1.68-fold greater for Sup.Care than for DAC (p=0.023).

Median Time to AML or Death (Days)

MDS group (n) DAC Supp.Care p-value 1Protocol specified test. 2Preferred test for analysis of early separation of KM curves. n=89 n=81 Wilcoxon1,2 Log rank1 All Patients (170) 338 263 0.046 0.204 Int-2/High Risk (118) 334 189 0.005 0.040 High Risk (44) 260 79 0.001 0.006 Treatment naïve (124) 354 189 0.005 0.034

Figure Figure

Investigator reported response rate by International Working Group criteria was 25% (10% CR, 15% PR) for DAC vs. 0% for Supp.Care (p< 0.001), with responses equally distributed across baseline subgroups. Time to response was 100 days and median duration was estimated at >9 months. Responders (CR and PR) vs. non-responders had a median survival of 678 days vs. 406 days (p= 0.038 Wilcoxon). There were no treatment related deaths. As expected, grade 3–4 toxicity (including hematologic toxicity, febrile neutropenia) occurred in more DAC patients than in Supp.Care patients. Most patients tolerated treatment well. DAC appears a promising therapy for MDS with manageable toxicity. Final results of independently reviewed response rates and clinical benefit (transfusion independence; Quality of Life) will be presented.
Fixed-Dose, Weight-Adjusted, Unfractionated Heparin (UFH) Given Subcutaneously (sc) without Laboratory Monitoring for Acute Treatment of Venous Thromboembolism (VTE): Randomized Comparison with Low-Molecular-Weight-Heparin (LMWH).
Authors: Kearon Clive , Ginsberg Jeffrey J. , Julian Jim , Douketis James , Solymoss Susan , Ockelford Paul , Jackson Sharon , Turpie Graham , McCarty Donna , MacKinnon Betsy , Hirsh Jack , Gent Michael ,   (for the FIDO Investigators)
Published: October 2019

Background: Treatment of VTE with UFH is usually by intravenous (iv) infusion with adjustment of dose in response to the activated partial thromboplastin time (APTT). However, (1) there is no evidence that adjusting UFH in response to APTT results reduces recurrent VTE or bleeding if patients are receiving an appropriate dose of UFH, and (2) sc UFH might be preferable to iv UFH. We hypothesized that UFH is as effective and safe as LMWH when each is administered sc in weight-adjusted, fixed-doses.

Methods: We performed a multicentre, randomized, open-label, trial that compared fixed-dose UFH (first dose 333 u/kg sc, then 250 u/kg sc twice-daily) with fixed-dose LMWH (100 u/kg sc twice-daily) for initial treatment of acute VTE. Patients were followed for 3 months during which they received warfarin (target INR 2.0–3.0). Outcomes were adjudicated by a blinded committee.

Results: 703 patients who presented with acute DVT (81%) or PE (19%) were enrolled and received study drug (68% entirely as outpatients). Of the 352 LMWH patients, 74% received dalteparin and 26% received enoxaparin. Recurrent VTE occurred in 13 (3.8%) UFH and 12 (3.4%) LMWH patients in the 3 months (difference of 0.3%; 95% CI, −2.6 to 3.3%). Major bleeding occurred in 4 (1.1%) UFH and 5 (1.4%) LMWH patients in the first 10 days (difference of −0.3%; 95% CI, −2.3 to 1.7%), and a total of 6 (1.7%) UFH and 12 (3.4%) LMWH patients at 3 months. 18 (5.2%) UFH and 22 (6.3%) LMWH patients died.

Conclusion: Fixed-dose sc UFH is as effective and as safe as fixed-dose sc LMWH for treatment of acute VTE. Our findings: (1) support the use of fixed-dose sc UFH as a convenient and inexpensive alternative to LMWH; and (2) question the need for APTT monitoring of UFH therapy.