bluuebunny/crossref_metadata_2025 huggingface dataset:
Keratinocyte-Growth-Factor-Prophylaxis: Positive Effect on Intestinal Mucositis Following Autologous and Allogeneic Peripheral Blood Stem Cell Transplantation in Haemtological Malignancies: A Sequential Cohort Study.
Authors: Grothe Wilfried , Bauer Martin , Rupprecht Andreas , Christopeit Maximillian , Muetherig Anke , Weber Thomas , Theurich Sebastian , Becker Mike , Behre Gerhard
Published: October 2019

Introduction: Keratinocyte Growth factor (KGF) prophylaxis reduces the extent and duration of mucosal barrier injury following intensive chemotherapy. Following this prophylaxis a marked reduction of infections and consecutively the need for antibiotic treatment can be observed. The extent of oral mucositis (OM) is easely to access. The extent of instestinal musocitis (IM) is more difficult to determine: The serum level of Citrulline, an intermediate product of the uric acid cycle mainly synthesized in the small intestine, serves as a marker of small intestine injury. Aim of the present study was to determine the extent of OM and IM before and after the introduction of KGF-prophylaxis in their treatment.

METHODS: In 09/2006 KGF (60μg/kg body weight per day, for 3 days before conditioning therapy respectively after HSCT) was introduced at our institution on a compassionate use basis. Five months before the introduction and thereafter the extent of OM and IM in autologous and allogeneic HSCT was evaluated prospectively. Beside routine clinical and laboratory values including the daily oral mucositis score (DMS) and daily gut score (DGS) the plasma citrulline level was determined in week 1 (W1), week 2 (W2) and week 3 (W3) following HSCT. Moreover the dosage of therapeutic intravenous (i.v.) antibiotic treatment with meropeneme, piperacilline/combactam and teicoplanin in addition to antibiotic prophylaxis was noted. Pt with oropharyngeal radiotherapy were excluded from analysis.

RESULTS: Until 04/2007 36 pt were evaluated. 27 pt underwent autologous and 9 allogeneic HSCT. 13 of the autologous and 6 of the allogeneic transplanted pt received KGF. In pt with KGF clinically significant OM and moderate to severe IM were seen less often (W1: 1/19 pt vs. 3/17 pt and W2: 1/19 pt vs. 2/17 pt without KGF). These differences were statistically non significant. Citrulline serum levels (in μmol/l) were significantly higher in pt receiving KGF (W2: average 16.8 vs. 15.9 in pt without KGF (p = 0.02)). The therapeutic use of i.v. antibiotics was lower for pt receiving KGF (average of 10.5 days vs. 26.8 days for pt without KGF (p = 0.0001)). Subgroup analysis confirmed the described tendencies in autologous HSCT.

SUMMARY: Even in this small number of pt a positive effect of KGF on oral and intestinal mucositis was detectable. The reduced need for therapeutic i.v. antibiotics seen as a tendency in this study should be re-evaluated in a prospective pharmacoeconomic study to prove cost effectiveness of KGF treatment.
Patient Safety in the Context of Disease Outcome and Effectiveness of Therapy.
Authors: Van Hoef Marlies E.H.M.
Published: October 2019

Safety is more than pharmacovigilance. Safety is also optimization of disease outcome with acceptable side effects. Safety is in this context related to effectiveness. Risk management in treatment is levering the risks an benefits. The process is characterized by five steps, risk definition, risk assessment, risk control, risk communication and risk review. In risk definition terms as safety, efficacy and effectiveness will be explained and related to the treatment. The risks of therapeutic measurements are characterized by the five classes of the common terminology criteria. The benefits of a treatment by among others efficacy, quality of life and effectiveness. In order to assess the risks literature review, analysis of scientific results, evaluation of risks related to prognostic factors for disease outcome are to be considered. In risk control, risk reduction or minimization and acceptance of certain risks will need to be considered. The role of therapeutic measurements as well as the disease stage and timing of therapy will be addressed in the context of in risk control. In risk communication explanation of this balance and the various forementioned parameters is important to offer most optimal prediction of beneficial disease outcome and effectiveness of a treatment. We will introduce the concepts described hereabove and address that limited side effects may actually be very unsafe in the context of effectiveness of therapy.
Empirical Antifungal Therapy for Patients with Neutropenia and Persitent Fever - Systematic Review and Meta-Analysis.
Authors: Goldberg Elad , Gafter-Gvili Anat , Paul Mical , Robenshtok Eyal , Vidal Liat , Leibovici Leonard , Shpilberg Ofer
Published: October 2019

Background: Opportunistic invasive fungal infections (IFIs) are a major concern in the management of immunocompromised patients with hematological malignancies. The practice of administering antifungal therapy to neutropenic patients with persistent fever has become a standard care. Conflicting data exists concerning the efficacy of empirical antifungal therapy.

Objectives: This study aims to evaluate if empirical antifungal therapy reduces mortality and prevents invasive fungal infections (IFI).

Methods: Systematic review and meta-analysis including randomized controlled trials (RCTs) comparing empirical antifungal treatment with placebo or no intervention (control), or another regimen, in neutropenic patients with persistent fever. The Cochrane Library, MEDLINE, conference proceedings and references were searched until 2007. Outcomes assessed were: All-cause mortality, documented IFI, and adverse events. Relative risks (RR) with 95% confidence intervals (CIs) were estimated and pooled.

Results: Our search yielded 26 trials, 6 of which compared polyenes or azoles to control, and 20 compared between different regimens of polyenes, azoles or glucan synthesis inhibitors. Compared to control, there was no difference in all-cause mortality (RR 0.98; 95% CI 0.58–1.66, 5 trials, Fig.1). The risk for developing documented IFI was lower (RR 0.23; 95% CI 0.08–0.65, 4 trials, 4 events in the treatment group versus 18 in the control). When azoles (fluconazole, ketoconazole, itraconazole, voriconazole) were compared to polyenes (amphotericin B in 8 trials, liposomal ampho B in 1 trial) there was a trend in favor of azoles for decreased all-cause mortality (RR 0.89; 95% CI 0.73–1.09, 9 trials) and for decreased documented IFI (RR 0.70; 95% CI 0.47–1.04, 8 trials). Adverse events of any kind were less frequent in the azole group (RR 0.40; 95% CI 0.34–0.66, 5 trials), as were those which required discontinuation (RR 0.48; 95% CI 0.38–0.62, 7 trials).

Conclusions: Our review demonstrates that empirical antifungal therapy does not reduce mortality. Although it reduces IFIs, data are based on a small number of trials and events. The use of amphotericin B as empirical antifungal therapy seems unwarranted since it appears to be less effective than azoles, with no mortality benefit and an increased rate of side effects. Future trials should pursue a pre-emptive approach using improved diagnostic tools (such as galactomannan testing, high resolution CT), to identify the patients for whom antifungal treatment is warranted.

Figure Figure
Immunoglobulin Prophylaxis in Patients Undergoing Hematopoietic Stem Cell Transplantation - Systematic Review and Meta-Analysis.
Authors: Raanani Pia , Gafter-Gvili Anat , Paul Mical , Ben-Bassat Isaac , Leibovici Leonard , Ofer Shpilberg
Published: October 2019

Background: Many studies have evaluated the role of polyvalent immunoglobulins (IVIG) and CMV-hyperimmune IVIG (CMV-IVIG) prophylaxis in patients undergoing hematopoietic stem cell transplantation (HSCT), with inconsistent results and implications for practice.

Objectives: To evaluate the role of IVIG and CMV- IVIG prophylaxis in HSCT.

Methods: Systematic review and meta-analysis of randomized controlled trials comparing IVIG or CMV- IVIG with placebo or no intervention (control) for prophylaxis in HSCT. The Cochrane Library, MEDLINE, conference proceedings and references were searched until 2007. Primary outcome: all-cause-mortality ; Secondary outcomes: CMV infections, acute graft versus host disease (GVHD), interstitial pneumonitis (IP), veno-occlusive disease (VOD) and adverse events. Relative risks (RR) with 95% confidence intervals (CIs) were estimated and pooled.

Results: Nineteen trials met inclusion criteria. IVIG was compared to control in 10 trials (7 IVIG in allogeneic HSCT, 2 allogeneic/autologous HSCT and 1 autologous HSCT). When IVIG was compared to control, there was no difference in all-cause mortality (RR 1.03; 95% CI 0.89–1.18, 6 trials, Fig.). There was a reduction in the number of CMV infections (RR 0.70; 95% CI 0.50–0.99, 4 trials) and in the number of episodes of IP (RR 0.61; 95% CI 0.43–0.87, 6 trials). There was no significant difference in the number of episodes of acute GVHD (RR 0.93; 95% CI 0.83–1.05, 6 trials). The risk for VOD was increased (RR 2.71; 95% CI 1.06–6.94, 3 trials) as were adverse events in the IVIG group (RR 8.12; 95% CI 3.15–20.9, 5 trials). CMV-IVIG was compared to control in 7 trials, all allogeneic HSCT. There was no difference in all-cause mortality (RR 0.86; 95% CI 0.63–1.16, 4 trials). In addition, there were no differences in the risk for developing CMV infections (RR 1.07; 95% CI 0.86–1.33, 7 trials), acute GVHD (RR 1.02; 95% CI 0.72–1.44, 5 trials) or IP (0.95; 95% CI 0.58–1.56, 5 trials).

Conclusions: Our review demonstrates that IVIG prophylaxis for HSCT reduces the risk for CMV infections and IP without a significant influence on mortality. The beneficial effects should be weighed against the higher incidence of adverse events, VOD, and cost associated with the use of prophylactic IVIG in HSCT patients. Conversely, the use of CMV-IVIG was not associated with a change in any of the parameters.

Outcomes Summary Outcome IVIG vs. control CMV-IVIG vs. control All Cause Mortality RR 1.03; 95% CI 0.89–1.18 RR 0.86; 95% CI 0.63–1.16 CMV infections RR 0.70; 95% CI 0.50–0.99 RR 1.07; 95% CI 0.86–1.33 acute GVHD RR 0.93; 95% CI 0.83–1.05 RR 1.02; 95% CI 0.72–1.44 IP RR 0.61; 95% CI 0.43–0.87 RR 0.95; 95% CI 0.58–1.56 VOD RR 2.71; 95% CI 1.06–6.94 No data Adverse events RR 8.12; 95% CI 3.15–20.9 RR 7.0; 95% CI 0.38–129.34 Figure Figure
GVHD Prophylaxis with Cyclosporine + Mycophenolate Mofetil Improves Outcome Compared to Cyclosporine Alone in Patients Receiving Reduced Intensity Conditioning Hematopoietic Stem-Cell Transplantation from Unrelated Donor for Hematological Diseases.
Authors: Brissot-Davaine Eolia , Moreau Philippe , Ayari Sameh , Delaunay Jacques , Guillaume Thierry , Pelletier Jean-Baptiste , Harousseau Jean-Luc , Chevallier Patrice
Published: October 2019

Introduction: As observed in myeloablative allograft, incidence of acute and chronic GVHD remains a major problem in patients receiving RIC HSCT in term of TRM and quality of life. So far, the best GVHD prophylaxis has not been yet established in this settings, as it could depend on the type of donor or of the RIC regimen. Thus, GVHD incidence is expected to be higher in patients receiving transplantation from unrelated donor compared to related donor. Here we have compared two GVHD prophylaxis regimen (Cyclosporine (CsA) alone vs CsA + Mycophenolate mofetil (MMF)) in patients receiving RIC allograft from unrelated donor in order to demonstrate the favourable impact of a stronger immmunosupressive treatment in term of overall survival (OS) due to less GVHD incidence.

Patients and Methods: We have studied retrospectively 40 successive patients from our institution who received RIC allogeneic HSCT from unrelated donor for haematological malignancies. Two GVHD prophylaxis regimen were administrated: CsA alone in Group 1 (n=19) from 2000 to 2005 and CsA +MMF in Group 2 (n=21) from 2005. Indeed, the observation of high incidence of acute GVHD in the former group conducted us to modify our strategy of GVHD prophylaxis. Almost all patients received a Slavin’s like RIC regimen associating Fludarabine, Busulfan and ATG. The graft consisted of peripheral blood stem cells for all patients except 4 patients in Group 1 who received bone marrow and 3 patients in Group 2 who received unrelated cord blood stem cells. The 2 groups were comparable in term of sex, status at transplant (CR1 vs others) and age at transplant (median age 49 years in Group 1 and 54 years in Group 2, p=0.05). Median follow-up for survival patients were 35 months and 12 months in Group 1 and 2 respectively. All patients have given informed consent.

Results: OS was significantly higher in Group 2: median 300 days (range: 36–2620 days) in group 1 versus median not reached (range: 181–812 days) in Group 2. OS was 40% at 1 year in group 1 compared to 92% in Group 2, p=0.0004. This discrepancy was due to the observation of a trend for higher acute GVHD grade III–IV occurrence in group 1 compared to Group 2 (75% vs 41%, p=0.21) and significant more deaths due to acute GVHD in Group 1 compared to Group 2 (4 patients vs 1, p<0.05). No difference was observed in term of relapse when comparing the 2 groups.

Conclusion: With restriction due to the short follow-up in Group 2, we demonstrated here the favourable impact of the combination of CsA + MMF as GVHD prophylaxis in patients receiving RIC transplant from unrelated donor as we demonstrate a significant benefit in term of overall survival due to less acute GVHD.
Post Transplant Lymphoproliferative Disorders (PTLD): Prevalence, Clinical Features and Outcome.
Authors: Colorio Cecilia , Puente Dolores , Rossi Andrea , Jaimovich Gregorio , Tabares Maria , Requejo Alejandro , Bonini Nicolas , Milovic Vera , Pombo Gonzalo , Feldman Leonardo
Published: October 2019

PTLD is a life threatening complication developing in 1–5% of transplant (Tx) recipients. Epstein Barr (EBV), Cytomegalovirus (CMV) and immunosuppression (IS) were identified as risk factors. Aggressive evolution and therapy related toxicity generate poor prognosis. We retrospectively analyzed data from 18 PTLD patients (pts) diagnosed at our hospital between March 1994- June 2007. All of them were at least one graft recipient and were under IS.

RESULTS: Out of 1206 adult Tx recipients, 18 (1,5%) developed PTLD. Ninety-four percent were men, mean age 42,9 years (range: 18–72). Our study comprises 10 heart, 2 kidney, 2 lung, 1 heart-lung, 1 liver and 2 non related allogeneic bone marrow (BM) recipients. IS protocols included Cyclosporin A (16 pts), Azathioprine (8), Mycophenolate mofetil (7), Tacrolimus (4) and antitymocyte globulin (2). Pretx recipients serology for EBV and CMV were positive in 87 and 94% respectively. Sixty-six percent (12/18) disclosed late onset PTLD (median 49 months), all of them were solid organ (SO) recipients. BM recipients developed the disease within the first 6 months after Tx. Fifty percent presented extranodal involvement, 3/18 with BM infiltration and 3/18 showed lymphoma in the graft. Histologycal findings: 15/18 B cell lymphoma (8/15 large cell lymphoma), 1 peripheral T cell lymphoma, 1 early lesion infectious mononucleosis-like PTLD, 1 polymorphic hyperplasia PTLD. Immunophenotype: 89% were CD20+. Fifty percent of the group achieved response, 7/9 with complete response(CR). Three/9 pts obtained CR with reduction of IS alone.Treatment included Rituximab (63% pts), CHOP (56%) and radiotherapy (25%). Eleven/18 pts died, 4 due to disease progression, 6 by sepsis and 1 because of underlying hematological disease relapse.

CONCLUSIONS: In our experience, PTLD was an uncommon complication. The late onset of the disease was the most frequent form of presentation among SO recipients. B cell lymphoma was the main diagnosis (83%). Fifty percent (9/18) achieved response and 78% of them showed CR. Disease progression and sepsis were the most important causes of death.
Bone Marrow Transplant (BMT) for Acute Myeloid Leukemia (AML) Patients with Translocation (6; 9).
Authors: Haddad Abdo S. , Smith April , Bolwell Brain , Kalaycio Matt
Published: October 2019

Response and long term outcome to chemotherapy treatment in patient with AML may differ significantly depending on many factors identified at the time of diagnosis. Age, cytogenetics, and history of myelodysplasia are predictors of survival in patients undergoing induction chemotherapy. AML characterized by t (6;9) (p23; q34) - DEK/CAN fusion occurs only in 1–5% of AML cases. This translocation involves recurrent breakpoints in the DEK gene on chromosome 6p23 andin the CAN gene on chromosome 9q34. It predicts a poor outcome with chemotherapy alone and BMT is usually recommended as a result. However, the curative potential of BMT for t(6; 9) AML patients is unknown. We reviewed our experience treating AML with BMT. Five patients with t (6; 9) AML and one with high-risk myelodysplastic syndrome (MDS) underwent BMT following induction chemotherapy. The prep regimen was Busulfan/Cyclophosphamide in all but one patients and standard GVHD prophylaxis was used. No patient received a T-Cell depleted graft. The median follow up of survivors was 24 months (9–38 months). Our small series provides intriguing data suggesting the potential for cure of patients with AML and t (6; 9). While our data requires confirmation in a larger series of patients, our results provide a rationale to recommend BMT to these high risk patient with AML.

Table 1: Characteristics Pt. # Dx Date Age BMT Remission tatus GVHD Relapse A/D Months (Pt. #) Patient number, (Dx) Diagnosis, (BMT) bone marrow transplant date, (GVHD) graft versus host disease, (A/D) Alive/Dead from initial diagnosis, (CR) Complete remission, Matched unrelated donor, and (MSD) Matched sibling donor 1 10/04 37 MSD 6/05 CR1 Yes No A +34 2 06/04 36 MUD 10/06 CR2 NO No A +38 3 03/06 50 MUD 7/06 CR1 NO Yes D +13 4 09/05 20 MUD 12/05 CR1 NO No A +24 5 11/06 39 MSD 2/07 CR1 Yes No A +9 6 10/06 41 MSD 2/07 Refractory No No A +10
Study on Effect Hemopoiesis Reconstituted of Auto-Mesenchymal Stromal Cells or Allo-Mesenchymal Stromal Cells Transplantion.
Authors: Wang Chun Yan , Tan Huo , Huang Zhen Qian , Li Hai Ming , Liu Dan , Zheng Run Hui
Published: October 2019

Objective: To study effect hemopoiesis reconstituted of auto-mesenchymal stromal cells (MSCs) or allo-mesenchymal stromal cells transplantion.

Methods: MSCs were obtained from transplantion patient or embryo by culturing in Zhongshan university stem cell center. 2 patient(SLE, lymphoma) were transplant auto-MSCs before transplantion auto- haemopoietic stem cell(HSC), MSCs were 2.43×107/kg,2.76×107/kg, CD34+ cells were 2.13×104/kg,2.54×104/kg. 2 patient(CML, lymphoma) were transplant allo-MSCs(from embryo) after transplantion (+60 and +180), patient with CML was allo-HSCT, patient with lymphoma was auto-HSCT. MSCs were 2.23×105/kg. Rasults: 2 patients with combination transplantation auto-MSCs and auto-HSC were not adverse effect, neutrophil≥0.5 ×109/ L were +1 day and +10 day, platelet ≥20 ×109/ L were +1day and +8 day, granulocyte deficiency time were 0day and 7days. Two patients were complete remission(273 day and 124 day). Another two patient hemopoiesis delay (after transplantation +60 and +180) were transplanted allo-MSCs(from embryo), one patient with CML rised blood cell after transplantion, but 10 days later the patient relapsed, bone marrow showed acute transformation of chronic myelocytic leukemia. The patient died due to giving up trentment. The patient with lymphoma were transplanted allo-MSCs (from embryo), blood cells transient rised, but blood cells decreased after one week. Now the patient were complete remission (+300 day), but blood cells were lower. The two MSCs (from embryo) transplantation were failure.

Conclusion: It is important that how to chose the MScs transplantation time. Combination transplantation auto-MSCs and auto-HSC can promote hemopoiesis re-establish, especially to fit old age patient. If patient with hemopoiesis delay after transplantation were trantplanted allo-MSCs maybe further research. [ key word] MSCs, transplantation, haemopoietic stem cell, hemopoiesis re-establish
High-Dose Therapy Followed by Autologous HSCT in Patients with Multiple Myeloma. Results of a Retrospective Multicenter Analysis Conducted by The Polish Myeloma Group.
Authors: Knopinska-Posluszny Wanda M. , Hellmann Andrzej , Taszner Michal , Dmoszynska Anna , Jedrzejczak Wiktor , Holowiecki Jerzy , Walewski Jan , Warzocha Krzysztof , Skotnicki Aleksander , Lange Andrzej , Kuliczkowski Kazimierz , Sulek Kazimierz , Komarnicki M. , Robak Tadeusz
Published: October 2019

In recent years high-dose therapy with autoHSCT has become the treatment of choice for eligible patients with multiple myeloma. This disease is now one of the most common indications for autotransplantation. The aim of this study was the clinical assessment of patients who underwent autotransplantation of progenitor cells and an analysis of the results of conducted treatment. The Polish Myeloma Group collected results of auto HSCT from 12 transplantology centres in Poland conducted between 1995 and 2006. We analyzed retrospectively the prognostic influence of pre-transplant characteristics on response and survival in 498 patients. Virtually all the patients received peripheral blood stem cell support after conditioning with melphalan (95%). We evaluated the influence of age, type of myeloma, Durie-Salmon stage, presence of renal impairment, plasma cell infiltration, albumin and b2 microglobulin level at diagnosis, status prior to and post HSCT, time from diagnosis to HSCT on overall survival (OS) and progression-free survival (PFS) to define patients with better prognosis. 232 females and 266 males underwent auto HSCT, and these included 297 (59,6%) with IgG, 97 (19,5%) with IgA, 31 (6%) with B-J, 22 (4,4%) with non-secretory myeloma. Bone structure changes were ascertained in 355 patients (71%). Bone marrow involvement higher than 20% was found in 282 patients (56,6%) at diagnosis. A decreased level of albumin (<35g/l) was determined in 168 patients (33,7%), and b2microglobuline level above 3,5 mg/l in 124 patients (24,9%). Transplantation of progenitor cells was conducted as consolidation of first line treatment following chemotherapy according to VAD in the majority of patients (74,7%). This number increased to 246 (49,4%) following transplantation. Double transplantation was conducted in 132 patients (26,5%). Median OS and PFS obtained were 3272 (1391–4232) and 1158 (102–3767) days respectively. CR achieved before transplantation, normal renal function, albumin level above 35g/l, b2 mikroglobulin below 3,5mg/l and DS stage I, were associated with a longer OS and PFS (p<0,05). This retrospective, multicenter study confirms the efficacy and safety of autoHSCT in multiple myeloma patients. Additional confirmation is given of the increased rate of CR, and the significantly prolonged survival observed in complete responders. Taking the above into account the employment of new drugs, such as thalidomide or bortezomib, which allow the achievement of a higher percentage of remissions should in the future bring about an improvement of the efficacy of transplantation in multiple myeloma.
The Construction and Expression of Recombination Adenovirus Vector of G156A O6-methylguanine-DNA-methyltransferase.
Authors: Wang JiShi , Yang Yuan , Jiang XueJie , Li DongBo , Fang Qin
Published: October 2019

OBJECTIVE: To transfect G156A mutant MGMT gene into peripheral blood mononuclear cells(PBMCs), peripheral blood CD34+ cells, umbilical cord blood CD34+ cells and K562 cells through recombinant adenovirus vector and detect the expression of ΔMGMT in these cells, to study the protective effect of the ΔMGMT gene for bone marrow cells against alkylating agent.

METHODS: We obtained a full-length cDNA fragment encoding human O6-methylguanine-DNA-methytransferase (MGMT) gene from human liver tissue by RT-PCR. ΔMGMT cDNA was made by site-directed mutagenesis. Positive clones were identified by diagnostic restriction analysis and sequenced to confirm the presence of the desired mutation. ΔMGMT cDNA was released by digestion with XhoI and BamHI, purified and then ligated into adenovirus expression vector pAd5CMV-NpA. The recombinant adenovirus vectors pAd5CMV-ΔMGMT and pAd5CMV-EGFP were constructed and transfected into the packing cell lines HEK293 cells by liposome encapsulation method. The recombination adenvirus were propagated, purified, and titrated to 1x1012pfu/mL. PBMCs, peripheral blood CD34+ cells, umbilical cord blood CD34+ cells and K562 cells were incubated with a mixture containing adenovirus. Fluorescence microscopy was employed to test the transfection of recombinant adenovirus vectors.The expression of ΔMGMT gene was detected by Real-time PCR and Western Blot and contrasted to the cells which were transfected into the recombinant adenovirus including EGFP. 3-(4,5-dimethyhhiazol-2-yl)-2,5-dipheyhetrazolium bromide (MTT) and CFU-assays were used to detect the drug-resistance of the ΔMGMT transfected cells.

RESULTS: MGMT gene was cloned successfully and the mutant G156A MGMT genes were obtained. High titer recombinant adenovirus had been made. Real time-PCR showed that mutant drug resistance gene expressed strongly. Western-Blot verified that cells infected with Adv-EGFP displayed EGFP immunoreactivity and basal levels of MGMT immunoreactivity; in contrast, cells infected with Adv-ΔMGMT showed a strong ΔMGMT immunoreactivity. Fluorescence microscopy showed EGFP positive in control group. The mutant drug resistance gene target cells conferred 12.4 ∼ 18.6-fold stronger resistances to BCNU and O6-benzylguanine (BG) in MTT test than control group. CFU-assays showed the colony-forming units was more detected in transfected group compared with vacant vector group (P<0.05).

CONCLUSIONS: The results suggested that transduction of the mutant G156A MGMT into hematopoietic progenitor cells mediated by recombinant adenovirus vectors may lead to their selective resistance to the combined use of O6-benzylguanine and alkylating agents, and this study provided an experimental foundation for improving combination chemotherapy tolerance in clinical practice.
Prevention of Ph+ Acute Lymphoblastic Leukemia by Nanoparticulate Delivery of a DNA Vaccine in Syngeneic Mice.
Authors: Koechling Joachim , Rott Yvonne , Arndt Stefanie , Green Jordan , Anderson Daniel , Langer Robert , Stripecke Renata , Wittig Burghardt , Schmidt Manuel
Published: October 2019

In contrast to gene therapy of solid tumors, only a few preclinical studies exist about gene therapy of acute lymphoblastic leukemia. Previously, we showed that vaccination of mice with syngeneic BCR-ABLp185 expressing leukemia cell lines modified to express costimulatory molecules and cytokines induce a systemic immunity against wild type leukemia. However, the difficulties to culture and transfect human leukemia cells limit the clinical application of leukemia cell based vaccines. Thus, we evaluated the pre-immunization of mice with DNA based vaccines subsequently challenged by the cell line BM185. In order to avoid the limitations and serious side effects associated with viral vectors we used nonviral gene delivery methods. Minimalistic immunogenically defined gene expression (MIDGE) vectors encoding a BCR-ABLp185 fusion specific peptide or GM-CSF were used as DNA vaccine and double stem-loop immunomodulators (dSLIM), containing three CpG-motifs were used as immune adjuvant. We provide biometrical and CTL data that shows specific immunization and protection of mice which received the complete vaccine BCR-ABL/GM-CSF/dSLIM by GeneGun delivery of naked DNA. Mean tumor-free survival (p=0.019) and overall survival (p=0.008) were significantly longer compared to non-vaccinated mice. Furthermore we show that BCR-ABL specific sequences are required to prevent Ph+ acute lymphoblastic leukemia by DNA vaccination. However, survival rate was moderate and only 26.7% of vaccinated mice survived and remained tumor-free. While current nonviral genetic tumor vaccine systems are less effective than viral vaccines, in particular when tumor associated antigens are weakly immunogenic, microencapsulation of tumor-specific DNA with poly (β-amino esters) might be a promising, safe and efficient nonviral delivery method for genetic vaccines. These biodegradable cationic polymers can bind and condense DNA into nanoparticles, deliver large DNA payloads and show low toxicity. Therefore, we prepared DNA-polymer complexes of MIDGE-vectors and end-modified poly (butane diol diacrylate co amino pentanol) C32-117 or C32-118 which self-assemble into particles with effective diameters of 200 nm and zeta potentials of 9.8 mV in PBS. We compared the immunization of mice receiving the vaccine BCR-ABL/GM-CSF/dSLIM by intradermal injection as naked DNA or alternatively, complexed with C32-117 or C32-118. We present data about tumor growth, survival rate, tumor free and overall survival of vaccinated and control mice.
Lymphoma and Chromosomal Instability in Artemis Deficient Mice - A Challenge for Long Term Malignancy Free Survival and Choice of Optimal Conditioning Regimen in Artemis Gene Therapy Trials.
Authors: Hauer Julia , Benjelloun Fatine , Romana Serge , Lelorc’h Marc , Fischer Alain , Villartay de Jean-Pierre , Cavazzana-Calvo Marina
Published: October 2019

Correction of Artemis deficiency, which causes a T−/−B−/− negative and radiosensitive type of SCID, is subject of murine gene therapy trials as an alternative treatment protocol for Hematopoietic Stem Cell Transplantation (HSCT). A conditioning regimen consisting of irradiation or busulfan in mice is indispensable for a succesfull engraftment. Gene therapy related toxicity on the basis of vector integration and conditioning regimen has been observed previously. Herein we provide evidence that Artemis deficiency harbours an additional risk factor for the development of lymphoproliferative disease. We provide in vivo evidence, that non gene corrected Artemis mice are able to develop lymphoma facilitated by irradiation and/or aging. Artemis−/− mice were irradiated at 3 Gy. Lymphoma were detected in 2 out of 10 Artemis−/− mice at the age of 4 to 6 months. Lymphoproliferative disease were exclusively of T-cell origin and arose from the thymus carrying a highly CD4+ or CD4+/CD8+ phenotype. Additionally, increased chromosomal rearrangements were detected in bone marrow cells of irradiated Artemis−/− mice compared to non irradiated control mice. Karyograms of bone marrow cells of 3 Gy-irradiated, 4 months old mice were stained with the Spectral Karyotyping (SKY) method. Analysis of 5 out of 20 metaphases revealed chromosomal translocations whereas in two out of 5 abnormal metaphases chromosome 15 was involved in the translocations. On contrary in karyograms of 4 to 6 weeks old Artemis−/− Sca1+ cells, which were used for viral transduction twenty out of twenty metaphases showed no obvious rearrangement. Even stimulation with cytokines in the course of viral transduction (SCF, FLT3L, TPO, IL6 and IL11) for five days did not increase chromosomal instability. Here we provide evidence, that Artemis−/− mice have an additional increased risk of developing hematopoietic malignancies, due to chromosomal instability which is independent of gene therapy. Human cells might not harbour the same riskfactor. However chromosomal instability in human Artemis−/− cells needs to be analysed in more detail for the choice of an optimal conditioning regimen and long term malignancy free survival in clinical trials.
Drug Resistance Gene Therapy by Simultaneous Lentiviral Overexpression of MDR1 in Combination with the MGMT P140K Mutant in Human Hematopoietic Stem Cells.
Authors: Spier Isabel , Maier Patrick , Laufs Stephanie , Veldwijk Marlon R. , Wenz Frederik , Zeller W. Jens , Fruehauf Stefan
Published: October 2019

Background: In this study we cloned a vector based on a self-inactivating lentiviral backbone containing MDR1 (multidrug resistance 1 gene) connected by an ECMV-IRES-element with MGMT P140K (O6-BG-resistant mutant of O6-methylguanine-DNA methyltransferase). The chemoprotective potential of this HR′SIN-MDR-IRES-MGMT combination vector was compared to single vectors (HR′SIN-MDR, HR′SIN-MGMT).

Methods: HL60 and CD34+ cells were transduced with the various vectors. After chemotherapeutical treatment MTT assays were used to detect chemoresistance levels in HL60 cells, CD34+ cells were held in liquid culture under differentiation conditions and analysed by FACS for MDR1 expression.

Results: HL60 cells transduced with the combination vector showed significant chemoresistance to O6-BG/ACNU (IC50 13x higher compared to untransduced control), the IC50 of cells transduced with HR′SIN-MGMT was 35x higher. The IC50 of paclitaxel (MDR1 substrate) was 24x higher in cells transduced with HR’SIN-MDR and 25x higher with HR’SIN-MDR-IRES-MGMT compared to untransduced control. Combined exposure of cells to O6-BG/ACNU and paclitaxel showed a survival advantage of cells transduced with the combination vector (IC50 6.25x higher), for the single vectors the IC50 was 1.63x higher (MDR1) and 2.08x higher (MGMT) compared to untransduced control. Treatment of CD34+ cells with increasing concentrations of doxorubicin (up to 0.8 μM) resulted in a higher fraction of MDR1-positive cells either with HR′SIN-MDR (26.6x) or with HR′SIN-MDR-IRES-MGMT (30.6x) compared to untreated cells. After combination treatment (20μM O6-BG/16μM BCNU and 0.4μM doxorubicin) the fraction of MDR1-positive cells was higher for HR′SIN-MDR-IRES-MGMT (14x) than HR′SIN-MDR (8x) transduced cells.

Conclusion: The protective effect of the combination vector is comparable with the single vectors for monotherapy and superior for combined therapy. The combination vector presents simultaneous protective effects of two drug resistance genes, thus reducing the risk of insertional mutagenesis by one transduction process. Consequently our results might help to reduce myelotoxic side effects and increase the chemotherapeutic efficiency.
Efficient Clonal Gene Delivery into Human Embryonic Stem Cells Via Active Transgenesis Using PiggyBac Transposition.
Authors: Zhang Ting-Ting , Ulrey Clayton L. , Liu Shan-Run , Yang Rui , McConnell Sean C. , Huo Yongliang , Ryan Thomas M.
Published: October 2019

The regulation of any transgene integrated into patient derived ES cells (via nuclear transfer or reprogramming) needs to be studied in great detail prior to their differentiation and transplantation back into the patient. The ability to efficiently deliver transgenes and clonally isolate transduced human embryonic stem (hES) cells for further study should be a prerequisite for their general therapeutic use. In this study active transgenesis using the piggyBac (pB) transposon system has been used to efficiently deliver transgenes to hES cells. This transfection method utilizes transient expression of pB transposase (TPase) to efficiently insert transgenes flanked by pB inverted terminal repeats (ITRs) directly into the hES cell genome. In the two plasmid system, the pB TPase is produced in trans from the "Helper" plasmid and the transgene(s), inserted between pB ITRs, are provided in the "Donor" plasmid. In the single plasmid system both the transgene and the transposase are on the same DNA molecule, but only the transgene(s) is located between the ITRs. Amaxa nucleofection was used to efficiently deliver the supercoiled transposon plasmid(s) into hES cells. Here, we used the single plasmid pB system to deliver a GFP transgene to hES cells. Dozens of clonal lines of GFP transgenic human ES cells were produced in a single experiment. GFP fluorescent colonies were isolated, subcloned, and analyzed for transgene copy number. Chromosomal transgene integration sites were mapped by plasmid rescue. The pB preferred tetranucleotide target sequence, TTAA, was confirmed at each insertion site analyzed. The distribution of chromosomal insertion sites were analyzed for more than 50 mapped insertions. About 60% of the transposon insertions were within 5 kb of human RefSeq genes and 45% were located within transcription units. Additionally, we have used the two plasmid pB system to deliver a human β-globin transgene along with GFP into hES cells followed by clonal isolation. This method easily transferred more than 13 kb of exogenous DNA into the hES cells. Efficient pB-mediated active transgenesis and clonal isolation of transduced hES should enable a careful analysis of the regulation of specific transgenes during the directed differentiation of potential therapeutic hES cell subclones.
Impact of Anemia on Medical Resource Utilization and Hospitalization Cost in Women with Obstetrical Bleeding.
Authors: James Andra , Patel Snehal , Dinh Quinn
Published: October 2019

Objective: Anemia during pregnancy has been associated with adverse maternal and fetal outcomes. Although women with obstetrical bleeding are at increased risk for developing anemia, little is known about the prevalence and burden associated with anemia in hospitalized women with this condition. This study was conducted to estimate the prevalence, demographic characteristics, medical resource utilization, and hospitalization cost associated with anemia in hospitalized women with obstetrical bleeding in the U.S.

Methods: The Healthcare Cost and Utilization Project Nationwide Inpatient Sample (2003) was queried using ICD-9-CM codes to identify all pregnancy-related discharges as well as discharges with diagnosis codes for conditions associated with obstetrical bleeding. Descriptive statistics were used to evaluate demographic characteristics, medical resource utilization components, and hospitalization cost for two groups: cases with anemia and cases without anemia.

Results: Of the estimated 4,525,714 pregnancy-related discharges in the U.S. in 2003, more than 250,000 recorded diagnosis codes associated with obstetrical bleeding. Nearly 1 in 5 of these women had an anemia diagnosis. A diagnosis of anemia in hospitalized women with obstetrical bleeding was associated with a 9-fold increase in blood transfusion (p<.0001), 33% longer average length of stay (p<.0001), and 50% higher mean cost per hospitalization (p<.0001).

Conclusions: Anemia and blood transfusion are frequently observed in hospitalized women with obstetrical bleeding. To improve outcomes in these patients and alleviate the adverse impact of anemia on postpartum health status, greater provider awareness of the prevalence and burden of illness associated with anemia in hospitalized women with obstetrical bleeding is warranted.
Trend and Geographic Variations in the Incidence of Waldenstrom’s Macroglobulinemia in the United States over 17 Years from 1988 to 2004.
Authors: Wang Michael , Zhou Yuhong , Wang Haijun , Yi Qing , Zhang Yanxia , Alexanian Raymond , Kwak Larry , Du Xin
Published: October 2019

Background: Waldenstrom’s Macroglobulinemia (WM) is an uncommon subtype of B cell malignancy. The updated trend of its incidence in the United States has not been reported.

Objective: To better understand the incidence and epidemiological features of WM in the United States.

Methods: We conducted a population-based incidence study with 1463 patients (pts) with WM identified from the Surveillance, Epidemiology, and End Results (SEER) tumor registries over 17 years. From the SEER data, 78,558 pts of all ages diagnosed with non-Hodgkin’s lymphoma (NHL) were identified between 1988 and 2004 in 9 SEER areas. Of these pts, 1463 had pathologically confirmed WM, accounting for 2% of NHL. The incidence with 95% confidence intervals were generated from SEER*Stat Software and was age-adjusted to the U.S. year 2000 standard population. Annual Percent Change (APC)for the incidence was calculated and considered to be statistically significant if p value was less than 0.05. The crude 1-year survival rate was calculated as the ratio of the number of pts who survived over 1 year and the number of pts diagnosed with WM.

Results: Of the1463 pts with WM, median age at diagnosis was 73. Overall incidence rate was 0.37/100,000/year, which increased with age from 0.03 in pts aged <50 to 1.03 in pts aged 60–69 and 2.76 in pts aged 80 or older. The age-adjusted incidence did not change significantly from 0.34 in 1988 compared with 0.36 in 2004. Therefore was no significant Annual Percent Change (0.97%, p>0.05). Incidence of WM was higher in men (0.52) than in women (0.26) (P<0.001). There were substantial geographic variations (p<0.001) in the incidence of WM. The age-adjusted incidence was highest in Seattle (0.49) and lowest in Atlanta (0.19). The incidence was higher in Caucasians (0.40) than in African-Americans (0.17) and other races (0.20). Annual Percentage Change was 1.3% in Caucasians (p<0.05).

Conclusion: The overall incidence of Waldenstrom’s Macroglobulinemia did not change significantly from 1988 to 2004, and was significantly higher in male Caucasians. There were substantial geographical variations in the incidence of Waldenstrom’s Macroglobulinemia in the United States.
Thalidomide-Dexamethasone vs Interferon-α-Dexamethasone as Maintenance Treatment after ThaDD Induction for Multiple Myeloma (MM): Final Analysis of a Prospective, Randomized Study.
Authors: Offidani Massimo , Polloni Claudia , Corvatta Laura , Piersantelli Maria-Novella , Galieni Piero , Visani Giuseppe , Alesiani Francesco , Catarini Massimo , Brunori Marino , Burattini Maurizio , Centurioni Riccardo , Ferranti Mario , Giuliodori Luciano , Candela Marco , Mele Anna , Marconi Monica , Leoni Pietro
Published: October 2019

Maintenance therapy with Thalidomide in MM offer controversial results. De novo or relapsing MM patients presenting at least a minor response after ThaDD were randomized to receive Interferon 3 MU x 3/week or thalidomide 100 mg /d. Both groups were given also Dexamethasone 20 mg/d x 4 days every month. Actually, we have randomized 50 patients in both treatment arms. The two groups were matched for main prognostic factors and response. During maintenance, both the ID and TD regimens improved response obtained by induction in only 10% and 11% of patients, respectively (p=0.832). After a median 2-years maintenance follow-up for both arms, 30 ID patients (60%) relapsed vs 17 (33%) TD ones (p=0.009). Time-to-progression (TTP) was significantly higher in the TD group vs the ID one (p= 0.024). So that TTP amounted to 23% in the ID group vs the 44% in the TD one. In addition, 3-years overall survival (OS) was significantly better in the TD arm with a value of 67% vs 46% of ID (p = 0.030). Both treatment arms were overall fairly well tolerated: fever, anorexia, weight loss, fatigue, liver and heart function abnormalities and hematologic toxicities were significantly more frequent in the ID cohort whereas neurotoxicity was somewhat more frequent in the TD one. This turned into a rate of therapy dropouts rate significantly higher in the ID group than in the TD one (26% vs 8%; p=0.017). Anyway, estimated risk for treatment interruption due to side effects in a 3-years period with thalidomide was only 21% vs 44% for interferon (p =0.014). We concluded that, in MM patients responding to standard induction therapy, low-dose Thalidomide maintenance therapy is feasible even in the long run and offers a significantly longer control over residual disease when compared to standard maintenance regimen.
A Transgenic Mouse Model of Multiple Myeloma Shows Heterogeneity in Drug Response with Alterations Due to Selective Gene Modifications.
Authors: Gosse Mary A. , Van Ness Brian G.
Published: October 2019

Multiple myeloma (MM) is a type of cancer characterized by malignant plasma cells (PCs) in the bone marrow. One difficulty in treating patients is that there is no single genetic event or abnormality common to all patients, resulting in heterogeneity of genes expressed, tumor aggressiveness, and response to therapy. In addition, mouse models of myeloma are limited. However, deregulation of MYC and BCL-XL are both common in patients’ tumors. Our lab has previously shown that a transgenic mouse line targeting overexpression of c-Myc and Bcl-xL to PCs results in an early expansion of non-malignant PCs, which then develop into clonal malignancies in multiple tissue types. Tumors develop quickly (median 16 weeks) in all mice and these cells can be adoptively transferred to syngeneic recipient mice to form tumors in as little as 10 days. Our lab has analyzed cell surface markers and generated genetic profiles on these Bcl-xL/Myc tumor cells, which show genetic heterogeneity in B-cell, PC, and myeloma markers, similar to human MM. Additionally, these tumor cells can be grown in vitro. The purpose of this study was to evaluate different drug sensitivities of our Bcl-xL/Myc primary tumor lines, and to determine if the sensitivities of these Bcl-xL/Myc tumors are dependant on the deregulated expression of specific genes. After treating cells with varying concentrations of dexamethasone, melphalan, and doxorubicin for a number of days, it was discovered that different tumor lines from the Bcl-xL/Myc transgenic mouse line have a range of sensitivity, from highly resistant to extremely sensitive. Three of these cell lines show a dose-response to varying concentrations of dexamethasone. Additionally, one cell line shows evidence of IL-6 protection against dexamethasone-induced apoptosis. Next, Bcl-xL/Myc primary cell lines were treated with BH3I-2′, a Bcl-xL small molecule inhibitor, at varying concentrations. Cell lines overexpressing Bcl-xL were more resistant to BH3I-2′, compared to S194 control cells, which express Bcl-xL at normal levels. One of the Bcl-xL/Myc cell lines did not show an increase in apoptosis or decrease in cell viability, but maintained the same number of cells over four days, whereas in other cell lines, viability started to decrease after 12 hours. Bcl-xL/Myc cell lines were also treated with siRNA targeted against Ezh2 and Cyclin D1, D2 and D3 transcripts, which have been shown to be deregulated in human myeloma as well as our own cell lines. These tumor lines are being treated with dexamethasone, melphalan and doxorubicin to evaluate the cell lines’ new sensitivities to chemotherapy. In total, our results show the Bcl-xL/Myc transgenic mouse tumor model demonstrates genetic heterogeneity that may be associated with heterogeneity in tumor responses. This serves as a useful model for human myeloma progression and allows us to examine the role of selective gene regulation in therapeutic response.