bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical Outcomes of Patients with T Cell NHL Undergoing Allogeneic Stem Cell Transplant.
Authors: Zain Jasmine , Palmer J. , Tsai N. , Popplewell L. , Nadamanee A. , Krishnan A. , Nakamura R. , Rodriguez R. , Forman S. J. , Kirschbaum M.
Published: October 2019

Background: T cell NHL represent approximately 10–15% of all lymphomas. Pts with T cell NHL are often treated similarly to patient with B cell NHL, although the clinical outcomes of most patients with T- NHL tend to be worse with the exception of ALK positive anaplastic large cell lymphomas (ALCL). Updated classifications in recent years have recognized specific clinical and pathologic T cell entities with distinct clinical courses and this poses a challenge to the systemic study of these diseases. The exact role of allogenic transplant in T- cell NHL is unknown.

Methods: We looked at 45 pts who underwent an allogeneic HSCT for T cell lymphomas between Jan 2000 to Dec 2005. There were 18 males, 27 females. Median age at transplant was 32 (7–74). Histology was Mycosis fungoides /Sezary syndrome (SS/MF) n=10, T cell lymphobalstic leukemia (PTLL) n=16, PTCL including AILD and ALCL n=14, NKT cell n=5. Syngeneic donor 1, Sibs 31, MUD 13. Median time from diagnosis to transplant was 12.5 (3.6–88.3) mo. Source of stem cells BM 9, PBSCT 35, cord blood 1. Conditioning, fully ablative 29, reduced intensity 16. Median number of prior regimens MF/SS 5, PTLL 2, PTCL 3, NKTL 2 with only 2 pts with prior auto transplants. 18 pts were in remission at the time of transplant and 12 pts had induction failure.

Results: Median follow up from transplant was 45.3 mo(0.7–64.7) with a 55.6%OS. Incidence of GVHD was Acute n= 27 extensive chronic n=20. Cause of death was transplant related in 16 pts with only 3 pts dying of disease progression. The overall survival is 61.2 % at 1 year,55.4% at 2 years and 48.5% at 5 years with a 5 year progression free survival at 48.5%. Based on different histologies, the results are 50.0% overall survival at 1 year and 40.0% at 2 and 5 years for SS/MF, 55.6% at 1 and 2 years for PTLL, 70.1% at 1 and 2 years and 52.6% at 5 years for PTCL and 80% at 1 year for NKT shown in fig 1 and 2.

Conclusion: Allogenic transplant can result in long term survival for some patients with T cell NHL suggesting a graft vs lymphoma effect. Timing of transplant needs to be better defined. Most patients are heavily pretreated which may have resulted in more transplant related mortality with 11/20 pts dying of infection. Most patients did not have a prior auto transplant indicating early progression of disease after standard therapies defined for B cell malignancies.

Overall Survival by Histology  T-Cell Lymphoma with Allogenic Transplant  Sample Size: 45 patients Overall Survival by Histology  T-Cell Lymphoma with Allogenic Transplant  Sample Size: 45 patients Progression-Free Survival by Histology  T-Cell Lymphoma with Allogenic Transplant  Sample Size: 45 patients Progression-Free Survival by Histology  T-Cell Lymphoma with Allogenic Transplant  Sample Size: 45 patients
Persistent Centre Effect but Improvement of Leukemia-Free Survival of HLA Identical Hematopoietic Stem Cell Transplantation for Adults with AML in First CR in Europe over the Period 1987–2005 an Analysis on Behalf of the ALWP of EBMT.
Authors: Katsahian Sandrine , Labopin Myriam , Frassoni Francesco , Rocha Vanderson , Chevret Sylvie
Published: October 2019

Background. Among major European centres which had undertaken more than 30 bone-marrow transplantations(BMT) between Jan 1, 1987, and Dec 31, 1995, the outcome of allogenic BMT for acute myeloid leukemia (AML) in first complete remission has been shown to be influenced by the centre in which the procedure is done. To determine whether similar variation exists 10 years later, we analysed leukaemia free survival (LFS), relapse incidence (RI) and treatment related mortality (TRM) across the same 13 transplant centres.

Methods. Thirteen centres were included in the analysis which enrolled 515 patients (aged 16–55 years), between 1987 and 1995, for acute myeloid leukaemia in first complete remission and 455 patients were enrolled between 1996 and 2005. Patients were followed from the date of transplantation to the time of event (Relapse and/or deaths). Regression models taking into account centre effect was adapted to the outcome and adjusted for relevant covariates

Findings The overall results at 5 years were 57% (95% CI 54–61) for leukaemia-free survival (LFS), 22% (19–25) for relapse incidence (RI), and 21% (18–24) for treatment related mortality (TRM) with a range for centres of 41–67%, 13–41%, and 13–41%, respectively. LFS at 5 years before 1995 was significantly lower 54% than after 1995 62% (p=0.02). Relapse Incidence at 5 years before 1995 was not significantly lower 23% than after 1995 21% (p=0.34). Treatment-related mortality incidence at 5 years before 1995 was not significantly lower 25% than after 1995 21% (p=0.13). Analysis showed the centre effect to be highly significant for LFS and TRM, but not for RI.

Interpretation These findings show that the results of BMT have improved over time in Europe. At the same time this study confirms a significant centre-specific variation in the success of bone transplantation in Europe. This effect has persisted despite advances in transplantation, which have lead to improvements in patient outcomes (leukaemia free survival and Treatment Related Mortality). Further studies are needed to elucidate the causes of this variation, with the goal of developing strategies to minimize the centre effect and ensure the best possible outcomes for all transplant recipients.
The Use of Intravenous Compared with Oral Busulfan in High-Dose Chemotherapy and Autologous Stem Cell Transplantation (ASCT) for Lymphoma Is Associated with Improved Relapse-Free Survival.
Authors: Dean Robert , Pohlman Brad , Sweetenham John William , Sobecks Ronald , Kalaycio Matt , Andresen Steven , Rybicki Lisa , Lowe Christopher , Brown Stacey , Curtis Julie , Bolwell Brian
Published: October 2019

The efficacy of high-dose chemotherapy and ASCT for lymphomas is generally believed to be independent of the preparative regimen used, although toxicities may vary. Delayed toxicities after ASCT led us to investigate IV busulfan in older patients as a substitute for oral busulfan in a preparative regimen of busulfan/cyclophosphamide/etoposide (BuCyVP), based on data suggesting lower treatment-related mortality after ASCT with the IV formulation. We then queried whether other outcomes of ASCT for lymphomas were improved by substituting IV busulfan for oral busulfan. We retrospectively analyzed the outcomes of 308 patients over age 50 years who underwent ASCT for NHL (93%) or HL (7%) at the Cleveland Clinic from 1996 through 2/2006. Oral BuCyVP (“PO Bu”) was used in 261 patients and IV BuCyVP (“IV Bu”, 0.8 mg/kg IV x 14 doses given without monitoring or adjustment for serum Bu levels) in 47 patients. Differences in baseline characteristics were that the IV Bu group was more heterogeneous in the number of prior chemotherapy regimens (P=0.013), more often received chemotherapy for stem cell mobilization (P=0.026), had fewer NHL patients with a high-intermediate or high IPI score (P<0.001), and had fewer patients with stage III or IV disease (P=0.046). Follow up from ASCT was significantly longer in the PO Bu group (median 45.7 vs. 8.8 months). CD34+ cell doses and hematopoietic recovery were similar between groups.

Median overall survival was 43.6 months after ASCT with PO Bu but was not reached for the IV Bu group. Kaplan-Meier analysis demonstrated the unanticipated finding that IV Bu was associated with a lower rate of relapse (P=0.013), superior relapse-free survival (P=0.008), and a marginal improvement in overall survival (P=0.06).

Figure Figure

Univariate Cox models identified the route of busulfan administration and disease status at ASCT as significant predictors of relapse. Age at transplant, the number of prior regimens, and LDH at transplant were significant predictors of mortality. Only the route of busulfan administration significantly predicted RFS in univariate analysis. In a multivariable model, the route of busulfan administration was the strongest predictor of RFS (P=0.043). Age was also predictive of RFS (P=0.047). These preliminary results suggest that replacing PO Bu with IV Bu is associated with a reduced relapse rate and improved RFS following ASCT for lymphoma. In contrast to other reports, we observed these improvements without targeting Bu dosage according to serum levels. Further study is necessary to confirm these findings and to ascertain whether IV Bu will favorably influence overall survival in this setting.
Prospective, Randomized Comparison between Double Transplantation (T) with Melphalan (200 mg/m2) and Triple T with Intermediate Dose Melphalan (100 mg/m2) in Patients with Multiple Myeloma (An Interim Analysis).
Authors: Ludwig Heinz , Linkesch Werner , Kasparu Hedwig , Krieger Otto , Spicka Ivan , Gastl Gunther , Greil Richard , Drach Johannes , Thaler Josef , Kuhn Ingrid , Zojer Niklas , Hinke Axel
Published: October 2019

In this study we compare the efficacy and toxicity of double T with M 200 with triple T using M 100 in newly diagnosed pts with MM. 175 pts have as yet been enrolled and 135 have been randomized after VAD induction treatment and stem cell collection to either tandem or triple transplantation. Reasons for non-randomization were too early for T (n=22), PD after induction (n=5), early death (n=3), toxicity (n=3), pt. refusal (n=3), and others (n=4). Median age of the 135 pts randomized was 59 years (range: 27–70 years). Stage I: 8%, stage II: 17%, stage III: 75%, Paraprotein Isotypes: IgG: 55%, IgA: 25%, IgD: 2%, IgM: 1%, Light chain: 17%. Induction treatment: 3 cycles of VAD; stem cell priming: IEV (ifosfamide (2g/m2)-etoposide (150mg/m2), d1–3; epirubicin (50mg/m2), d1) and G-CSF (5mg/kg) from d4. Conditioning regimen: M 200 mg/m2 or M 100 mg/m2 plus G-CSF; day 5 until WBC >2000/ml. Pts with ≥ SD after T were subsequently randomized to interferon 3MU, TIW (Schering Plough) plus prednisone (25mg, po., TIW), or interferon alone. QoL was assessed with the EORTC QLQ C30 instrument. Chi2 test for trend was used for comparison of response and survival endpoints were estimated by the Kaplan-Meier product limit method. Response rates after VAD were CR: 11%, PR: 65%, SD: 22% and PD: 2%. Median time from start of VAD to start of T was 137 in the double and 139 days in the triple T arm. Response rates did not differ between pts treated with double or triple T (CR: 47% vs. 43%, PR: 47% vs. 47%, SD 2% vs. 6%, PD: 4% vs. 4%, p=0.60, respectively). Median PFS for both groups combined was 36 mos, but showed a tendency for shorter PFS in pts on triple T (23 mos. vs. 37.5 mos; HR 1.26, 95% CI: 0.72–2.23, p=0.21). Median of OS has not been reached yet, but did not differ between both groups. Hematological toxicity was similar in both groups. There was a tendency for more grade 2–4 mucositis and for grade 3–4 vomiting, nausea, and fatigue in the double T group. Global QoL (questions 29 and 30) was comparable between both groups, before (5 (2.5–6.5) vs. 5 (4.5–6)) and after double (5.8 (3–8.5) or triple T (6 (2.5–7)). In conclusion, triple T with intermediate dose M resulted in similar response rates, slightly shorter PFS but similar OS and was associated with less mucositis and slightly less vomiting, nausea and fatigue.

Progression-free survival by randomization group Progression-free survival by randomization group
Second Stem Cell Harvest from Unrelated Donors for Patients with Relapse or Graft Failure after First Transplantation: Patient Age >50 Years and Relapse of Malignant Disease Are Associated with Poor Outcome.
Authors: Platzbecker Uwe , Zapf Susanne , Theuser Cathrin , Schmidt Alexander , Rutt Claudia , Ehninger Gerhard , Bornhauser Martin
Published: October 2019

Introduction: Hematopoietic progenitor cells from unrelated donors are increasingly used for allogeneic stem cell transplantation (SCT). A second donation of peripheral blood stem cells (PBSC) or bone marrow is widely accepted in case of graft failure of relapse.

Patients and methods: We retrospectively investigated the outcome of seventy (n=70) patients with hematological malignancies (AML/MDS n=29, CML n=19, ALL n=17, other n=5) receiving a second SCT using PBSC (n=67) or bone marrow (n=3) from the same unrelated donors. Patients had a median age of 30 years (range 1–59) and the reason for a second donation of stem cells consisted of either relapse (n=37) or graft failure (n=33). Whereas in 21 patients no conditioning was used in the remaining patients it was busulfan or TBI-based in the majority of cases.

Results: After a median follow-up of 33 months (range 7.9–76.2) after 2nd SCT a total of 55 patients died due to treatment-related mortality (63%) or relapse (16%) resulting in a median overall survival (OS) for all patients of 3 months. The OS was significantly better in patients < 50 years (38 vs. 8 %, p=0.01) and in case of graft failure (37% vs. 7%, p=0.02). Time interval between SCT, HLA-disparity, disease type (AML/MDS vs. CML vs. ALL), infused CD34+ cells/kg or graft source at first transplant had no impact.

Conclusion: These data show the lack of efficacy of a second infusion of HSC from the same donor especially in older patients and in cases in which relapse of malignancy is the reason for second transplant. Given the currently unknown long-term effects of a repeated exposure to G-CSF, the factors mentioned above should be kept in mind whenever a request for a second HSC donation is considered.
Reduced-Intensity Allogeneic Transplantation after Failure of Autologous Transplantation: A Prospective Multi-Center CALGB Study.
Authors: Bashey Asad , Owzar Kouros , Johnson Jeffrey L. , Devine Steven M. , Shadduck Richard K. , Shea Thomas C. , Linker Charles
Published: October 2019

Relapse of malignancy (RM) remains the most important cause of treatment failure following high-dose chemotherapy and autologous hematopoietic transplantation (AHCT). Allogeneic transplantation (allo-HCT) can salvage some patients who relapse or develop MDS following AHCT. However, myeloablative allo-HCT has been associated with a prohibitive transplant-related mortality (TRM) in such patients. CALGB study 100002 prospectively assessed the safety of reduced-intensity allo-HCT (RICT) in patients who failed prior AHCT. Eligibility required the development of RM or MDS > 6 months following AHCT for a hematologic malignancy and an available HLA-identical sibling (MSD) or 9/10 locus matched related donor, or a 10/10 matched unrelated donor. Between Dec 02 and Mar 06, 82 patients were registered from 11 CALGB centers. Patient characteristics: median age = 51 (17–70); male-63%; diagnoses - NHL 30%, HD 26%, MM 18%, MDS 16%, AML 8%, CLL 2%; donor - MSD 43%, MUD 55%, 9/10 MRD 2%. Median time between AHCT and RICT was 2.6 yrs (0.6–5.7 yrs). Preparative regimen for patients with MSD was fludarabine 30 mg/m2/d x 5 d, busulfan (i.v.) 3.2 mg/kg/d x 2 d. For patients without MSD rabbit ATG (Thymoglobulin ®) 2.5mg/kg/d x 4 d was added. PBSC 2–8 x 106 CD34+ cells/kg was used as the graft. GVHD prophylaxis consisted of tacrolimus tapering at d +90 and methotrexate 5 mg/m2 x 3 doses for MSD patients. For non-MSD patients, tacrolimus was tapered starting d +180, a fourth dose of methotrexate and MMF (d 0 to d +60) were added. Primary endpoint was TRM at 6 months. For 56 patients with > 6 months post-RICT follow-up (median follow-up 1.3 yrs), 6 month TRM was 8.7% (MSD patients 4.2%, non-MSD patients 9.3%). Total cumulative TRM was 14.3% (MSD 16.7%, non-MSD 12.5%). Reported cumulative rates of II–IV and III–IV acute GVHD are 28.2% and 11.8% respectively. Chimerism studies were performed centrally on peripheral blood (PB) specimens. The percentage of patients from MSD patients who achieved full (> 90%) donor T-cell (CD3) chimerism in PB on day 30, 60, 90 and 180 post transplant are 15, 40, 47, 57 and 60% respectively. In contrast, for non-MSD patients the corresponding rates are 78, 68, 86 and 91 % respectively. Both MSD and non-MSD patients achieved full (>90%) donor chimerism in myeloid cells by d 30 in > 80% of patients. This study observed a <10% 6-month TRM for RICT following AHCT failure in the multi-center, co-operative group setting. Also, the use of a more potent GVHD prophylaxis regimen seems to enable TRM rates for non-MSD patients to approximate those seen for MSD patients. MSD patients achieve slower onset of full-donor chimerism in T-cells with this regimen. A successor study will assess means of increasing early T-cell chimerism in the MSD patients.
Beyond HLA: Impact of Immunoreactive Mismatches of Minor Histocompatibility Antigens on Probability of Relapse and Graft-Versus-Host Disease after Hematopoietic Stem Cell Transplantation from HLA-Matched Unrelated Donors.
Authors: Markiewicz Miroslaw , Siekiera Urszula , Wojnar Jerzy , Wieczorkiewicz Agata , Wylezol Iwona , Giebel Sebastian , Karolczyk Agnieszka , Holowiecki Jerzy
Published: October 2019

Factors contributing to relapses and GVHD following allogeneic Hematopoietic Stem Cell Transplantations (HSCT) from HLA-matched unrelated donors are not well established. We analyzed 35 patients (pts) transplanted from HLA 10/10 alleles (A, B, C, DRB1, DQB1) matched unrelated donors (URD) in the Dept. of Hematology and BMT, Katowice, Poland, with use of the same standard operating procedure from January 2004 until March 2006. Indication for HSCT was AML (13 pts), ALL (8), CML (7), MDS (2), PNH (3), CLL (1), myeloid sarcoma (1). Preparative regimen was Treosulfan+Fludarabine (23 pts), TBI+Cy (9) and Bu+Cy (3). Alleles encoding 11 minor Histocompatibility Antigens (mHA: HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, HwA-9, HwA-10, UGT2B17, HY) were analyzed for each donor-recipient pair with use of Dynal AllSet mHA typing kit and PCR-SSP method. Only immunogenic mHA mismatches were analyzed. Information on whether mHA mismatches might result in Host-versus-Graft or Graft-versus-Host responses was established with use of the minor Histocompatibility Knowledge Database of Leiden University Medical Center’s minor Histocompatibility Workshop.

Patients transplanted from donors with mHA mismatched in HVG direction had significantly higher probability of relapse (64+/− 8% versus 10+/− 9%, p=0.003) when compared to pairs without mHA HVG immonogenicity. Patients transplanted from donors with mHA mismatched in GVH direction had higher probability of aGVHD (88+/− 8% versus 53+/− 11%, p=0.14) and cGVHD (41+/− 16% versus 37+/− 17%, p=0.65) when compared to pairs without GVH immunogenicity. GVHD was also observed more often on day +100 (33% versus 6%, p=0.06) when mHA mismatch in GVH direction was present. Observed influence of HVG and GVH immonogenicity on probability of relapse and GVHD did not lead to significantly different survival in observed groups of pts. These results indicate that mHA immunogenic mismatches in HVG and GVH direction may be responsible for relapse and GVHD, respectively, in HSCT recipients from HLA-matched unrelated donors. The study is being continued to confirm these primary observations and to establish the associations of specific mHA mismatches with HSCT outcomes.

Impact of mHA mismatch on probability of relapse and GVHD Immunogenicity of mHA mismatches HVG no HVG p # of patients 18 17 Relapse at 1 year 64%+/−8 10%+/−9 0.003 Immunogenicity of mHA mismatches GVH no GVH p # of patients 16 19 aGVHD 88%+/− 8 53%+/−11 0.14 cGVHD at 1 year 41%+/−16 37%+/−17 0.65 Impact of mHA mismatch on probability of survival Immunogenicity of mHA mismatches HVG GVH only HVG only GVH no HVG no GVH p # of patients 9 9 7 10 Survival at 9 months 63%+/−17 57%+/−25 83%+/−15 69%+/−15 0.86
Allogeneic Cytokine-Induced Killer Cells Traffick to GVHD Target Tissue but Cause Less GVHD, While Retaining Tumor Cytotoxicity and Facilitating Bone Marrow Engraftment.
Authors: Nishimura Ryosei , Baker Jeanette , Beilhack Andreas , Negrin Robert
Published: October 2019

Cytokine-induced-killer cells (CIK), generated from splenocytes in mice and PBMC in human by the timed addition of IFN-γ, anti-CD3 MAbs and IL-2, express both T cell and NK cell markers and have cytotoxic activity against autologous and allogeneic tumors. We previously showed that adoptive transfer of allogeneic CIK in a murine model did not cause severe acute GVHD, leading to full survival. However the precise mechanism of reduced GVHD and retained GVT activity is not fully understood. To this end, we first evaluated the trafficking patterns of luciferase-transgenic CIK in a major MHC mismatched BMT model. In vivo bioluminescence imaging (BLI) showed that both CIK and fresh splenocytes homed to and proliferated initially in secondary lymphoid organs including the spleen, followed by infiltration of the gut and skin by day 7. All mice receiving fresh splenocytes died by day 10. Signal from gut mucosa in animals receiving allogeneic CIK cells was transient, however signals from other sites such as mesenteric lymph nodes were high and persistent. By day 21 allogeneic CIK reinfiltrated intestinal tissue without causing severe GVHD. To further investigate potential changes in the expression of surface molecules in vivo, we transplanted GFP positive fresh splenocytes and GFP positive CIK cells into major mismatched irradiated hosts, and then on day 7 analyzed activation markers, homing molecules, apoptosis related genes, and peripheral tolerance induction molecules such as CTLA-4 and PD-1 by FACS. No significant differences were noted between CIK and splenocytes. However we found that the cell division rate of CIK cells in vivo was much slower than those of splenocytes utilizing a CFSE based cell proliferation assay. This might lead to less GVHD in mice receiving CIK cells. Next we evaluated whether CIK retained tumor killing activity in vivo using A20 luciferase lymphoma cells into lethally irradiated MHC major mismatched receipients subcutaneously. We visualized the reduction of tumor by BLI. GFP positive CIK, which were extracted on day 7 following their transplantation into MHC-mismatched allogeneic recipients, retained tumor cytotoxicity in vitro. Finally, to study whether CIK facilitate BM engraftment, we quantified the absolute number of cells from the peripheral blood in MHC major-mismatched nonmyeloablative BMT. On day 30 mice receiving CIK showed rapid BM engraftment, especially in CD4 (p=0.0007) and CD8 (p<0.0001) subpopulations. In conclusion, allogeneic CIK homed to and expanded in GVHD target organs without causing acute GVHD, retained tumor killing activity and promoted BM engraftment.
Ex Vivo Gene Trasfer for Hemophilia A That Enhances Safety and Results in Sustained In Vivo Factor VIII Expression from Lentivirally-Engineered Endothelial Progenitors.
Authors: Matsui Hideto , Shibata Masaru , Brown Brian , Labelle Andrea , Hegadorn Carol , Andrews Chandler , Hebbel Robert P. , Galipeau Jacques , Hough Christine , Lillicrap David
Published: October 2019

Hemophilia is an excellent candidate disorder for the use of gene therapy as a treatment modality. However, significant obstacles have been encountered with systemic delivery of viral vectors that have prevented sustained expression of the therapeutic protein. Investigation of alternative gene therapy strategies for hemophilia that enhance safety and facilitate long-term, therapeutic levels of the transgene product is imperative. In this study, we evaluated an ex vivo gene therapy strategy for hemophilia A. Circulating endothelial cell progenitors (blood outgrowth endothelial cells - BOECs) were isolated from canine and mouse blood and transduced with a third generation self-inactivating lentiviral vector encoding the canine FVIII transgene under the transcriptional control of either the CMV promoter or an endothelial cell-specific regulatory element. Transduced BOECs were injected either intravenously (IV) or subcutaneously mixed with Matrigel (SC+Matrigel) into NOD/SCID mice. Canine FVIII antigen levels were assayed at weekly intervals using an Asserachrom VIII:Ag ELISA that detects canine FVIII against a background of normal murine FVIII levels in the NOD/SCID mice. The mean FVIII antigen levels in mice injected with BOECs at 3 weeks following treatment were 37.5 mU/mL and 105.8mU/mL, for IV and SC+Matrigel administration, respectively. These FVIII antigen levels were sustained up to 12 weeks at therapeutic levels (21.3mU/mL and 21.7mU/mL, for IV and SC+Matrigel administration respectively). To evaluate if the observed loss of FVIII expression by 12 weeks post-treatment resulted from transcriptional silencing of the viral promoter, the CMV promoter was replaced with the endothelial cell-specific thrombomodulin (TM) promoter and transduced BOECs were implanted SC with Matrigel. In contrast to results from the CMV-regulated transgene, sustained therapeutic levels of FVIII have been documented for the duration of the study with the TM-regulated construct (34.3 mU/mL at 3 weeks and 22.5 mU/mL at 20 weeks) Immunostaining at 18 weeks after SC implantation of the transduced BOECs, shows that these cells still express FVIII and von Willebrand Factor. Biodistribution analysis by flow cytometry and quantitative PCR demonstrated that SC-implanted BOECs were retained inside the scaffold and were not detected at any other anatomic site. These results indicate that genetically-modified endothelial progenitors implanted in a SC scaffold can provide sustained therapeutic levels of FVIII and are a promising safe delivery vehicle for gene therapy of hemophilia. Currently, these engineered cells have been implanted into immunocompetant mice and FVIII levels are being assessed.
Chimeric Immunoreceptor (T-Body) Targeting of Acute B-Cell Lymphoblastic Leukemia (B-ALL) through Lentivirus Engineering of Primary Human T Cells.
Authors: Milone Michael C. , Fish Jonathan , Carpenito Carmine , Lakal Mehdi , Ofori Ella , Danet-Desnoyers Gwenn , Riley James L. , Grupp Stephan A. , June Carl H.
Published: October 2019

T-bodies that combine the antigen recognition capability of single chain antibody molecules (scFv) with the signal transduction domains of activating receptors can be used to generate T cells with novel, engineered specificities independent of their endogenous TCR. We have developed a series of T-bodies with specificity towards CD19, a molecule with expression that appears restricted to normal and malignant B cells. These T-bodies contain the activating signals from the costimulatory T cell receptors, CD28 and/or 4-1BB, in tandem with the TCR-ζ signal transduction domain. Using lentiviral gene transfer and T cells expansion via bead-immobilized anti-CD3 and anti-CD28 antibodies, we can generate T cells 95% of which express the T-bodies. Lentiviral transduction combined with this efficient T cell expansion system has the potential to produce >1011 antigen-specific T cells without the need for a cell selection step. Interestingly, we have noted significant constitutive signaling effects of T-body overexpression on transduced T cells that may been overlooked previously due to lower transduction efficiency. Despite the observed constitutive effects, we show that lentiviral-engineered T cells expressing anti-CD19 T bodies efficiently kill CD19 expressing cell lines and primary B-ALL cells in an antigen-specific manner. T-body engineered T cells also produce cytokines in response to antigen triggering with different patterns of cytokine production by T cells engineered with different T-bodies. In particular, a T-body receptor containing the 4-1BB signal transduction domain combined with the TCR-ζ domain stimulates a >6-fold increase in IL-6 production compared with other receptor combinations in CD4+ T cells. In contrast, a CD28 and TCR-ζ domain containing T body uniquely enhances IL-2 (>3-fold) and TNF-α (> 7-fold) secretion by CD8+ T cells. These results indicate that the functional characteristics of our tested T-body signaling constructs are distinct and depend upon the particular receptor and T cell subset combination. We are also comparing the anti-tumor response generated by adoptively transferred T-body engineered T cells in leukemia bearing immunodeficient mice as a pre-clinical, therapeutic model. In addition to elucidating important functional differences of our lentiviral constructs, these studies indicate that an important variable in clinical trials will be optimizing the cytosolic signaling domain for the desired T cell subset. The present results suggest that distinct combinatorial signaling domains may be required for optimal function in CD4 and CD8 engineered T cells.
The Use of Nitrous Oxide To Treat Pain of Lumbar Punctures in Ambulatory Pediatric Leukemia and Lymphoma Patients.
Authors: Hargens Liesl , Slagerman Kathleen , Moertel Christopher , Moore Caron , Slomiany David , Messinger Yoav
Published: October 2019

Nitrous oxide is an anesthetic gas that has been used extensively to decrease pain during dental procedures. Recent publications report that nitrous oxide administration to pediatric patients is safe and effective in reducing pain and anxiety associated with a variety of medical procedures. We completed a randomized, crossover, pilot study in our outpatient clinic to compare the efficacy of nitrous oxide (N2O) to standard conscious sedation (SCS) in providing analgesia during lumbar puncture (LP) in children with leukemia and lymphoma.

Two nurses evaluated 12 subjects during a N2O LP and 10 subjects during a SCS LP (midazolam and either fentanyl or morphine). Inter-nurse reliability was not different for any measure. The mean time for clinic visit was statistically shorter for the N2O group, 34 minutes, compared to 57 minutes for SCS (p=0.010). The mean length of procedure was 9 minutes using N2O or SCS. The type of sedation did not affect the difficulty performing the LP. The incidence of side effects (nausea, vomiting, combative behavior, and unsteadiness when leaving for home) was similar in the N2O and SCS groups. Pain control was equivalent. The nurse, parent, and subject reported pain during the procedure as zero for 5/12 subjects with N2O and 3/10 subjects with SCS. Parents and subjects were very satisfied with N2O (parent=1.8, child=2.8) and SCS (parent=1.5, child=2.2) sedation (10-point Likert scale with 1=very satisfied, 10=very dissatisfied). After completion of both procedures, 8/12 subjects preferred N2O whereas 2/10 preferred SCS. All twelve subjects who received N2O returned to normal activity within an hour of leaving the clinic and 3/10 subjects who received SCS required longer than one hour.

Nitrous oxide is effective in reducing procedural pain with few side effects while decreasing time required for clinic visit and return to normal activity. Patient and parent satisfaction was high with more patients and families preferring N2O sedation to SCS.

Parent and Nurse Mean Pain Scores Before, During, and After Lumbar Puncture Using N2O and SCS (10-point Likert Scale) Pre-Procedure Pain During Procedure Pain Post-Procedure Pain Parent Nurse Parent Nurse Parent Nurse N2O 0.3 0 1.4 0.9 0.5 0.3 SCS 0.3 0.1 2.5 1.9 1.5 0.7
Cancer/Testis Genes in Multiple Myeloma: Expression Patterns and Vaccination Strategies Determined by Microarray Analysis.
Authors: Condomines Maud , Hose Dirk , Reme Thierry , Hundemer Michael , De Vos John , Baudard Marion , Moehler Thomas , Pantesco Veronique , Schved Jean-Francois , Rossi Jean-Francois , Goldschmidt Hartmut , Klein Bernard
Published: October 2019

Cancer-testis (CT) antigens are expressed in testis and malignant tumors, but rarely in non-gametogenic tissues. Due to this pattern, they represent attractive targets for cancer vaccination approaches. The aims of the present study were (1) to assess for the first time the expression of CT genes on a pangenomic basis in multiple myeloma (MM), (2) to provide selection strategies of CT antigens for clinical vaccination trials and (3) to assess the impact of CT gene expression on event-free survival. We report here the expression pattern of CT genes in purified MM cells (MMC) of 64 patients with newly-diagnosed MM, 12 patients with monoclonal gammopathy of unknown significance (MGUS), in normal plasma cell and B cell samples and in 20 MMC lines, using gene expression profiling (GEP). Out of 46 CT genes interrogated by the Affymetrix HG U133 Set arrays, 35 were expressed in MMC of at least one patient, according to the Affymetrix “present” call (frequency range: 2% – 66%). Of these, 24 CT genes were expressed in more than 5% of the MMC samples and 25 are located on chromosome X. MMC of 98% of the patients expressed at least one CT gene, 86% at least two, and 70% at least three CT genes. By using a set of 10 CT genes including KM-HN-1, MAGE-C1, MAGE-A3/6/12, MAGE-A5, MORC, DDX43, SPACA3, SSX-4, GAGE-1–8 and MAGE-C2, a combination of at least three CT genes - desirable to circumvent tumor escape mechanisms and immune tolerance - could be obtained in MMC of 67% of the patients. Thus, gene expression profiling can be used to select CT antigens as vaccination targets in individual patients. In a series of MMC from 111 patients treated with the same high-dose chemotherapy and autologous peripheral blood stem cell transplantation protocol and having a median two-year follow-up, we found that the expression of six CT genes, i.e. CTAG-1B, CTAG-2, MAGE-A1, MAGE-A2, MAGE-A3 and MAGE-A6 was associated with a shorter event-free survival (EFS). Furthermore, considering only the 25 CT genes encoded by chromosome X, a CT-Xhigh cluster comprising MMC of one third of the patients (35 of 111) could be defined using a binary hierarchical clustering based on Affymetrix call. Patients in the CT-Xhigh cluster had a shorter EFS (median 13 months) compared to patients in the CT-Xlow cluster (median 18 months, P = .003). The CT-Xhigh clsuter included more patients with a stage III disease (P = .004). These results confirm data from previous studies indicating that patients expressing some CT genes located on chromosome X have a poor outcome.
JNK Regulates DKK1 Expression in Multiple Myeloma Cells.
Authors: Colla Simona , Zhan Fenghuang , Yaccoby Shmuel , Epstein Joshua , Barlogie Bart , Shaughnessy John D.
Published: October 2019

Multiple myeloma (MM), a tumor of terminally differentiated plasma cells, depends on the bone marrow for growth and survival. MM is characterized by extensive bone loss and osteolytic lesions located at the sites of medullary plasmacytomas, suggesting that myeloma cells alter the biology of bone remodeling. Wnt signaling in osteoblasts is essential for the maintenance of osteoblast and osteoclast homeostasis and thus coupled bone turnover. MM plasma cells, but not those from healthy donors or MGUS, secrete Wnt signaling inhibitors DKK1, sFRP-2 and sFRP-3. The production of these factors contributes to osteolytic lesions by inhibiting Wnt signaling in the bone marrow milieu. It is currently not known how DKK1 expression is activated and regulated in MM. Here we report that DKK-1 expression is significantly lower in 45 myeloma cell lines (MMCL) and 5 secondary plasma cell leukemias/ pleural effusions compared to primary myeloma cells and down-regulated in all 9 primary myeloma cells with high DKK1 expression levels (Affymetrix Signal > 1,000) following co-culture with osteoclasts. This inhibition of DKK1 is associated with the simultaneous down-regulation of JUN, FOS, and FOSB, of which JUN has been shown to regulate DKK1 (Grotewald and Ruther, 2002). Ectopic expression of c-Jun in Jun negative MMCLs did not induce DKK1 expression, indicating that DKK1 transcription in this cell type is not solely dependent on c-Jun. DKK1 transcription increased 2- to 20-fold in myeloma cells from 21 of 42 primary MM following 48 hour of in-vivo exposure to thalidomide and 19 of 24 exposed to lenolidomide. The JNK-specific inhibitor SP600125 could significantly inhibit the up-regulation of DKK1 in 7 of 9 primary MM cells following in-vitro exposure to thalidomide or lenolidomide. Finally, treatment of MMCL and primary MM with the JNK activator Anisomycin resulted in up-regulation of DKK1 2- to 5-fold, but not in the presence of SP600125. These data indicate that DKK1 expression in MM cells is, in part, dependent on JNK signaling and suggests that JNK inhibitors may be an effective means of inhibiting both DKK1 expression and MM bone disease.
Upregulation of c-Jun Induces Cell Death Via Caspase-Triggered c-Abl Cleavage in Human Multiple Myeloma.
Authors: Podar Klaus , Raab Marc S. , Tonon Giovanni , Sattler Martin , Barila Daniela , Tai Yu-Tzu , Yasui Hiroshi , Raje Noopur , Depinho Ronald A. , Hideshima Teru , Chauhan Dharminder , Anderson Kenneth C.
Published: October 2019

The tyrophostin adaphostin (NSC680410) achieves remarkeable responses in patients with chronic myelocytic leukemia (CML), including Bcr/Abl-positive, Bcr/Abl-negative, and Bcr-Abl T315I mutant tumor cells resistant to both imatinib mesylate and second- generation BMS354825 and AMN107. In addition it also demonstrates cytotoxicity against chronic lymphcytic leukemia (CLL) and acute myelocytic leukemia (AML) cells. Several mechanisms have been proposed as a basis for its robust anti-tumor activity including generation and release of reactive oxygen species (ROS), cytochrome-c and apoptosis- inhibiting factor (AIF), caspase- cleavage, JNK activation, as well as inactivation of Raf-1, Stat3, and Stat5. As these adaphostin- targeted pathways are relevant to MM pathogenesis, we here sought to determine the potential molecular sequelae and therapeutic promise of adaphostin in MM. After demonstrating the anti- myeloma cytotoxicity of adaphostin, we carried out expression profiling of adaphostin- treated MM cells to identify its molecular targets. Surprisingly, c-Jun was the most upregulated gene even at the earliest point of analysis (2 hours). We also observed adaphostin- induced c-Abl cleavage in immunoblot analysis. Proteasome inhibitor bortezomib, but not melphalan or dexamethasone, induced similar effects, indicating unique agent- dependent mechanisms. Using caspase inhibitors as well as caspase- resistant mutants of c-Abl (TM-c-Abl and D565A-Abl), we then showed that c-Abl cleavage in MM cells requires caspase activity. Importantly, knockdown of c-Jun and c-Abl expression by siRNA confirms that adaphostin- induced c-Jun upregulation triggers downstream caspase- mediated c-Abl cleavage, inhibition of MM cell growth, and induction of apoptosis. Finally, our data suggest that this mechanism may not be restricted to MM, but may also be important in a broad range of malignancies including erythroleukemia and solid tumors.
PU.1 Is Downregulated in a Subset of Multiple Myeloma by the Methylation of Its -17 kb Upstream Regulatory Region and the Promoter.
Authors: Okuno Yutaka , Tatetsu Hiro , Ueno Shikiko , Hata Hiroyuki , Yamada Yasuhiro , Takeya Motohiro , Iino Tadafumi , Akashi Koichi , Tenen Daniel G. , Mitsuya Hiroaki
Published: October 2019

It has been reported that disruption of transcription factors critical for hematopoiesis, such as C/EBPa and AML1, is involved in leukemogenesis. PU.1 is a transcription factor important for both myeloid and lymphoid development. We reported that mice in which the levels of PU.1 were 20% of that of wild-type developed acute myeloid leukemia, T cell lymphoma, and a CLL-like disease. These findings strongly suggest that PU.1 has tumor suppressive activity in multiple hematopoietic lineages. Last year, we reported that PU.1 is downregulated in a majority of multiple myeloma cell lines and and freshly isolated CD138 positive myeloma cells from certain number of myeloma patients, and that tet-off inducible exogenous expression of PU.1 in PU.1 negative myeloma cell lines induced cell growth arrest and apoptosis. Based on their PU.1 expression levels, we divided the myeloma patients into two groups, namely PU.1 high and PU.1 low-to-negative, (cutoff index of 25th percentile of the PU.1 expression level distribution among all patients). The PU.1 low-to-negative patients had a significantly poorer prognosis than the PU.1 high patients. To elucidate the mechanisms of downregulation of PU.1, we performed sequence and epigenetic analysis of the promoter region and the -17 kb upstream region that is conserved among mammalians and important for proper expression of PU.1. There are no mutations in these regions of all five myeloma cell lines. In contrast, the -17 kb upstream region was highly methylated in 3 of 4 PU.1 negative myeloma cell lines, while the promoter region was also methylated to various levels in all five myeloma cell lines including one PU.1 positive cell line. These data suggested that the downregulation of PU.1 in myeloma cell lines might be dependent on the methylation of both regulatory regions of PU.1 gene, especially the -17 kb upstream region. We also evaluated the mechanisms of cell growth arrest and apoptosis of myeloma cell lines induced by PU.1. Among apoptosis-related genes, we identified that TRAIL was upregulated after PU.1 induction. To evaluate the effect of upregulation of TRAIL, we stably introduced siRNA for TRAIL into myeloma cell lines expressing PU.1, and we found that apoptosis of these cells was partially suppressed by siRNA for TRAIL, suggesting that apoptosis of myeloma cells induced by PU.1 might be at least partially due to TRAIL upregulation. We are currently performing DNA microarray analysis to compare the expression levels of genes between before and after PU.1 induction, in order to further elucidate the mechanisms of cell growth arrest and apoptosis.
Toward Defining the Functional Relevance of Wnt Signaling in Osteoclasts in Multiple Myeloma.
Authors: Qiang Ya-Wei , Chen Yu , Brown Nathan , Ojha Rohit P. , Rudikoff Stuart , Barlogie Bart , Shaughnessy John D.
Published: October 2019

Disruption of Wnt signaling is thought to play a central role in the development of myeloma bone disease. Although Wnt signaling in osteoblasts (OB) is essential for their differentiation and Wnt signaling in OB indirectly regulates osteoclast (OC) development through the regulation of both OPG and RANKL, little is known about the direct role of Wnt signaling in osteoclastogenesis. In this study, we sought to characterize the Wnt signaling pathway and its functional role in OC formation using the macrophage cell line Raw 264.7, which can be induced to differentiate into OC, as well as human primal progenitor osteoclast cells isolated from bone marrow of normal donors and patients with MM. We first analyzed the pattern of Wnt signaling components in these cells by RT-PCR and sequence analysis. High level of expression of Wnt receptors Frizzled (Fz) Fz2, 3, 4, 5, 7, 8 and 9, and co-receptors LRP5, and LRP6, and transcription factors TCF1, TCF3, and LEF1 was observed in 10 of 10 OC cells from MM patients and 4 of 4 OC from normal donors and Raw264.7. Wnt3a conditioned medium and recombinant Wnt3a induced increased levels of total and un-phosphorylated beta-catenin in all cells. Pretreatment of OC cells with sFRP1 decreased Wnt3a induced increased beta-catenin. Pretreatment with DKK1, a specific inhibitor of the canonical Wnt pathway, blocked Wnt3a induced stabilization of beta-catenin. Additionally, the GSK3beta inhibitor lithium chloride induced stabilization of beta-catenin in OC and Raw264.7 cells in dose-dependent fashion. Wnt-3a induced TCF/LEF transcriptional activity in Raw264.7 cells transfected with TOPflash compared with Raw264.7 transfected with FOPflash, which possess mutant sequence for binding beta-catenin. These results suggest that Wnt signaling functions in primal MM OC cells, as well as OC cell lines. Finally, we sought to determine the biological function of Wnt signaling in OC cells. The results showed that Wnt-3a did not affect the proliferation and survival of OC cells, nor increased formation of TRAP positive cells. Wnt3a did not synergize with RANKL and M-CSF in induction of OC differentiation. Additional biological functional assays are underway to determine the direct role of Wnt signaling on OC. Identification of the role of Wnt signaling in osteoclastogenesis may improve our understanding of the bone lytic process in multiple myeloma.
Inhibition of p38α MAPK Reduces Tumor Burden, Prevents the Development of Myeloma Bone Disease, and Increases Survival in the 5T2 and 5T33 Murine Models of Myeloma.
Authors: Vanderkerken Karin , Medicherla Satya , Coulton Les , Van Camp Benjamin , Protter Andy , Higgins Linda , Menu Eline , Croucher Peter
Published: October 2019

The bone microenvironment plays a critical role in supporting the growth and survival of myeloma cells and the development of osteolytic bone disease. Signalling through p38 α MAPK mediates synthesis of myeloma cell survival factors by stromal cells; whereas, inhibiting p38 α MAPK reduces myeloma cell proliferation and inhibits osteoclast formation in vitro. However, it is unclear whether p38 α MAPK inhibition will prevent the growth and survival of myeloma cells and the bone disease in vivo. The aim of this study was to determine whether SCIO-469, a selective p38 α MAPK inhibitor, would inhibit myeloma growth and prevent the development of bone disease in the 5TMM syngeneic models of myeloma.

Treatment of 5TMM cells, in vitro, with SCIO-469 resulted in a clear inhibition of p38 phosphorylation, as assessed by Western blotting and an inhibition up to 35% of stromal cell induced 5T33MM proliferation.

Injection of 5T2MM murine myeloma cells into C57Bl/KaLwRij mice resulted in the growth of myeloma in bone and the development of bone disease characterized by increased osteoclast surface (p<0.05), a reduction in cancellous bone (p<0.01) and the presence of osteolytic bone lesions on x-ray (p<0.01). Treatment of 5T2MM-bearing mice with SCIO-469 (150mg/kg in the diet, therapeutical treatment from paraprotein detection) resulted in a 42% decrease in serum paraprotein and prevented development of osteolytic lesions (p<0.01). Injection of 5T33MM cells into C57Bl/KaLwRij mice also resulted in the development of myeloma but not associated bone disease. Treatment of 5T33MM-bearing mice from the time of tumor cell injection with SCIO-469 resulted in a decrease in serum paraprotein (8.8+/−1.4g/dl to 0.04+/− 0.03g/dl, p<0.001) and a reduction in the proportion of tumor cells in the bone marrow (67 +/− 8.1% to 1.09 +/− 0.58%, p<0.001). Kaplan-Meier analysis demonstrated an increase in disease-free survival (veh=27.5 days vs 96 days, p<0.001) after treatment of the mice with SCIO-469.

These data demonstrate that targeting p38 α MAPK with SCIO-469 is associated with an anti-myeloma effect, which indirectly prevents the development of myeloma bone disease.