bluuebunny/crossref_metadata_2025 huggingface dataset:
Early Immunoglobulin Free Light Chain (FLC) Response Post Autologous Peripheral Blood Stem Cell Transplant Predicts for Hematologic Complete Response in Patients with Multiple Myeloma.
Authors: Dispenzieri Angela , Rajkumar S. Vincent , Plevak Matthew F. , Katzmann Jerry A. , Kyle Robert A. , Larson Dirk , Lacy Martha Q. , Clark Raynell , DeGoey Roberta , Hayman Suzanne R. , Kumar Shaji K. , Gertz Morie Abraham
Published: October 2019

Background: FLC is a valuable diagnostic tool to monitor response in patients with AL and oligosecretory MM. It is also prognostic in patients with MGUS. Its value in patients with non-oligosecretory myeloma has not been defined. Because of the short half-life of free-light chains, an early drop in free-light chain might be predictive for long term outcome post PBSCT.

Methods: Between 8/1/03 and 12/10/04, 45 patients with multiple myeloma and abnormal FLC ratios (less than 02.6 or greater 1.65), who were undergoing PBSCT consented to have waste serum from routine post-transplant daily blood draws collected and frozen for future FLC analysis. The immunoglobulin FLC assay (Binding Site, U.K.) was run on samples collected from day +1 until patients were dismissed from our transplant center. Hematologic response was coded according to standard IBMTR criteria at approximately 3 months status post transplant (BJH 1998). Two types of immunoglobulin FLC response were coded: 50% reduction (FLC50) and 90% reduction (FLC90) of the difference of the involved and uninvolved FLC (Leukemia 2006). Two time periods of FLC response were analyzed: early (day 7); and delayed (day 19, range 13 to 24). Endpoints analyzed were overall response (>=PR), complete response rate (CR), progression free survival (PFS), and overall survival (OS).

Results: The median age of the myeloma group was 59 years, and 53% were male. Thirty-three patients were monoclonal kappa, and 12 lambda. Forty-two percent were transplanted in response/plateau, 22% were primary refractory, and 36% relapsed disease. The overall hematologic response rate was 93% with a CR rate of 33%. Twenty-five patients have relapsed at a median of 14.2 months. Twelve patients have died, and median overall survival for the group has not been reached at a median follow-up of 24.6 months. Forty-nine and 58 percent of patients had early and delayed FLC90, respectively. Early FLC90, but not delayed FLC90, predicted for complete response (p<0.001). Neither early nor delayed FLC90 predicted for either PFS or OS. In addition, 89% and 96% of patients had an early and delayed FLC50, respectively. FLC50 at either time point did not predict for overall response, CR, PFS, or OS.

Conclusion: Immunoglobulin free light chain responses are seen within the first weeks post high dose chemotherapy with peripheral blood stem cell transplant. A 90% reduction in FLC seen within the first seven days predicts for complete response. However, this does not yet translate into prolonged PFS or OS, but with longer follow-up separation of the survival curves may be seen.
Toll-Like Receptors 2 and 4 Are Involved in the Induction of Graft-Versus-Host-Disease in Mice.
Authors: Nogai Axel , Heimesaat Markus M. , Thiele Marc , Bereswill Stefan , Thiel Eckhard , Goebel Ulf B. , Uharek Lutz
Published: October 2019

BACKGROUND: Intestinal Graft-versus-Host disease is a frequent and often lethal complication after allogenic stem cell transplantation. Since NOD2 polymorphisms have been recognized as potential triggers of severe intestinal GvHD in humans, we have developed murine transplantation models to investigate the role of different pattern recognition receptors (PRR) in GvHD and GvL. Here we report our results on the role of TLR2 and TLR4 for the induction of GvHD.

METHODS: Severity of GvHD in wildtype (wt) C57B/10 (H-2Db), TLR2−/−, TLR4−/−, and combined TLR2−/−TLR4−/− C57B/10 mice was investigated. Mice received treosulfan 2000 mg/kg from day -3 to -1 and cyclophosphamide 200 mg/kg day -1 prior to injection of 10×10^6 H-2Dd BM cells and 5×10^6 splenocytes (SC). Survival and GvHD score were assessed daily. Engraftment was determined every 2 weeks in pB and at the end of the experiments in bone marrow by flow cytometry. T cell alloreactivity in GvH direction was assessed by MLR using splenocytes as stimulators from PRR-deficient mice or wt as control and CFSE-staining as read-out. The relevance of PRR ligands for the enhancement of GvH alloreactivity was determined by addition of lipid A, lipopetides, or CpG.

RESULTS:

in vivo data: The transfer of 10×10^6 BMC + 5×10^6 SC induced a severe GvHD in all wt recipients, leading to death of 90% of the animals within 20 days. Recipient mice lacking either TLR2 or TLR4 showed only a slightly and not significantly decreased GvHD lethality. In recipients lacking both PPRs, i.e. TLR2 and TLR4, GvHD was generally milder and the majority (60%) of the animals survived until day 20 (p<0.05). However, the long term survival was not significantly improved. Differences in clinical severity of GvHD were confirmed histologically. In vitro data: Stimulation with cells from TLR2−/− and TLR4−/− mice resulted in a decreased alloreactivity in MLR. A median of 2% of Balb/c CD4+ T cells proliferated in response to C57B/10 stimulators. The addition of the TLR2 and TLR4 ligands lipopeptide, Lipid A and CpG significantly (p<0.05) increased the proliferation of CD4+ T cells in a specific manner more than twofold.

CONCLUSION: Our in vivo and in vitro data consistantly show that bacterial components are involved in triggering GvH alloreactivity via different types of PPRs. Binding of bacterial substances to TLR2 and TLR4 leads to activation of the immune system and subsequent induction of GvHD. Our data provide an experimental basis for the development of strategies for modulation of the intestinal gut flora by selective gut decontamination and/or probiotic regimens to prevent GvHD in humans.
Mechanism of Alemtuzumab Depletion of T Cells in Stem Cell Products.
Authors: Gulen Dumrul , Bociek Robert G. , Coskun Ugur , Devetten Marcel P. , Talmadge James E.
Published: October 2019

Alemtuzumab (a humanized murine monoclonal antibody [MAb] against CD52) is an effective drug for in vitro T cell purging of allogeneic stem cell products when a product is incubated with 10mg of antibody for 30 minutes prior to infusion. Recovery of CD34+ cells has been excellent and T cell purging with alemtuzumab has resulted in a decreased incidence of grade 2–4 GVHD and a reduced incidence of extensive GVHD and a low overall risk of graft failure. However, a higher incidence of cytomegalovirus (CMV) reactivation, respiratory and polyoma virus infections have been observed as has an increased relapse rate in patients with chronic myelogenous leukemia. In vitro purging, using alemtuzumab and infusion of unwashed products, has been associated with a reduced incidence of clinically significant GVHD, an increase in infectious complications and a possible increase in relapse rates. The present studies optimize the purging protocol and examine the mechanism of alemtuzumab purging to identify the role of antibody dependant cell mediated cytotoxicity (ADCC) versus antibody dependant complement mediated cytotoxicity (Ab–C’). Both ADCC and Ab–C’ mediated cytotoxicity were active, although ADCC was noticeably more effective. However, cellular viability was improved by incubation with 10% autologous serum. A two hour incubation (1–2×01e8 cells/ml) resulted in a 73% reduction in CD3+ cells, which was increased to 85% after a 16 hour co-incubation. In addition, natural killer (NK) and B cells were purged with alemtuzumab; as were lineage negative (Lin−)-HLA-DR+CD123+ dendritic cells (DCs) but not Lin-HLA-DR+CD11c+ DCs. Immature myeloid suppressor cells (IMSCs), which are Lin−DR−CD33+ or DR−CD14−CD11b+, have a low sensitivity to alemtuzumab cytotoxicity as did hematopoietic progenitor cells (CFU-c function or CD34+ number). This is an exciting observation as IMSCs have the potential to induce T cell tolerance. Optimal T cell purging, with retention of CD34+ hematopoietic stem cell number and activity, requires a 4–6 hr co-incubation of 2×10e8/ml hematopoietic stem cells, 100 ug/ml alemtuzumab in 10% autologous serum at 4C and removal of alemtuzumab by washing in Plasma-Lyte. Co-incubation at 4C and the presence of 10% autologous serum, was required to minimize the toxicity of a 24 hr co-incubation using a high frequency (1–2×10e8/ml) of hematopoietic cells. The selective depletion of T cells, with a retention of IMSCs, suggests that such a purging protocol has potential to not only reduce acute and chronic GVHD but to also induce major histocompatibility complex tolerance. However, our clinical studies need to be completed in order to assess the impact on leukemic relapse.
Interest of FDG-PET Scanning for the Monitoring of Immunotherapies for Follicular Lymphoma.
Authors: Baudard Marion , Comte Frederic , Conge Anne Marie , Mariano-Goulart Denis , Klein Bernard , Rossi Jean-Francois
Published: October 2019

Except in the subset of patients with localized disease, follicular lymphoma (FL), the most common type of indolent non-Hodgkin’s lymphoma, should still be regarded as an incurable malignancy with a relentless relapsing course. New therapeutic approaches have been developed including anti-tumor vaccination strategies with induced humoral and cellular immune responses revealed by sensitive biological analyses but no imaging of the lymphocyte activation developed so far. Because [18F]fluorodeoxyglucose (FDG) is sequestred in metabolically active cells, we prospectively investigated the use of FDG-PET (positron emission tomography) to kinetically monitor cell activation in 7 FL patients vaccinated by repeated intradermal (ID) and intravenous injections of tumor lysate-pulsed autologous dendritic cells (DC-lysate). Patients were administered 5 vaccines and all patients developed a positive delayed-type hypersensitivity reaction at the site of DC-lysate ID injections after several vaccines administration, testifying of the induction of a biological response. Among the 4 patients with measurable disease at time of vaccination, 2 complete remissions (CR) and 2 disease progressions were observed. Three patients with initial normal computed tomographies (CT) were still in CR 12 months after the last vaccine. PETs were performed before vaccination, 14 days and 1 month after the last vaccine. PET revealed initial malignant nodes infracentimetric on concomitant CT, and was very sensitive in documenting disease progression even in patients with no significant variations of CT findings. A transient marked increase in FDG uptake within malignant nodes was observed early after vaccine administration in a patient who achieved a CR (fig 1-3-4-5), while ranges of tracer uptake variations were less important with a continuous increase of FGD uptake, size and number of involved loci in non responding patients (fig 2: follow up of intensity of FDG uptake by 6 tumoral nodes in a non responding pt). In conclusion, FDG-PET is a non invasive imaging procedure which besides being useful for the staging and the evaluation of treatment efficacy in FL, might be of crucial interest to detect cell responses induced by immunotherapies.

Figure Figure
Induction of Polyclonal T-Cell Responses Against Cutaneous T-Cell Lymphomas by Autologous Dendritic Cells Transfected with Amplified Tumor RNA.
Authors: Ni Xiao , Richmond Heather , Shpall Elizabeth , Duvic Madeleine
Published: October 2019

The indolent course of cutaneous T-cell lymphoma (CTCL) patients makes them attractive candidates for immunotherapeutic strategies. However, tumor specific antigens are unknown in CTCL, and thus this is an obstacle to the development of CTCL vaccines. Recently, vaccinations with the tumor antigen repertoire has shown to be a superior strategy to elicit protective immunity for hematological malignancies since it does not require definition of tumor antigens or HLA haplotyping (Borrello IM, Sotomayor EM. Cancer Control, 9:138–151,2002). Studies have shown that dendritic cells (DC) transfected with tumor mRNA amplified from primary tumor cells can stimulate anti-tumor T cell responses (Boczkowski et al, Cancer Research, 60: 1028–1034,2000; Muller et al, Blood, 103: 1763–1769, 2004). In this study, we determined the efficacy of ex vivo induction of anti-CTCL T cell responses using autologous DCs transfected with tumor RNA amplified from CTCL cells. We established a protocol for amplification of whole CTCL mRNA. DCs were generated from CD14+monocytes of patients with CTCL, transfected with amplified tumor RNA (5μg/106), matured with IL-1β, TNF-α, IL-6, and PGE2, and co-cultured with autologous peripheral blood lymphocytes. Induction of CTCL-specific cytotoxic T lymphocytes (CTL), determined by IFN-γ and Granzyme B secretion, was measured by ELISPOT assays. As a result, DCs transfected with amplified tumor RNA induced a 1–3 fold increase in specific IFN-γ secreting and Granzyme B releasing CTLs, compared to control DCs that were not transfected, in 3 of 5 patients. There was only an induction of granzyme B releasing CTLs seen in the fourth patient. There was no induction of either IFN-γ secreting or granzyme B releasing CTLs in the fifth CTCL patient who had low CD8+ T cells in the peripheral blood. These data suggest that vaccinations using DCs transfected with amplified tumor RNA might be a potent new strategy in the treatment of CTCL. It provides the rationale for further investigation of a potentially clinically effective and broadly applicable treatment for CTCL patients, and has the advantage of not requiring large amounts of tumor cells or characterization of a specific relevant antigen in each patient.
Quantitative HLA Class I Expression and KIR-Mismatch between NK-Cell-Donor and Patient Predict NK Cell Mediated Lysis of Leukemic Cells from Children with Acute Lymphatic Leukemia.
Authors: Pfeiffer Matthias M. , Schumm Michael , Dietz Klaus , Feuchtinger Tobias , Handgretinger Rupert , Lang Peter J.
Published: October 2019

Relapses represent a major problem after transplantations in children with ALL. Natural Killer (NK) cells have been shown to exert remarkable Graft versus Leukemia effects after mismatched stem cell transplantation in myeloic leukemia, whereas the efficacy against lymphatic leukemia is still unclear. We therefore measured intensity of HLA class I expression on leukemic blasts by quantitative FACS analysis and investigated the impact of quantitative HLA class I expression, of several adhesion molecules and of KIR-mismatch on NK cell mediated lysis of the leukemic blasts from 21 pediatric patients with ALL. Expression of HLA class I molecules differed widely from patient to patient (range 5000–500000) and was reduced in comparison to B cells from healthy donors in 70% of cases. NK cells killed leukemic blasts very heterogeneously but a clear association between number of HLA class I molecules per cell and specific lysis (range 13–98%) was found (r2=0.68, p<0.0001). For the subgroup of leukemic blasts without KIR-mismatch, this association was even stronger (r2=0.98), whereas a weak association was found for the subgroup with KIR-mismatch, since most of these targets were lysed more efficiently than one could expect according to HLA class I expression alone. KIR-mismatch alone (t-test, p=0.45) as well as different patterns of adhesion molecules (ICAM1-3, LFA1/3) and CD95 had no significant influence. However, a multivariate model taking both HLA expression and KIR-ligand-mismatch into account, provided an even stronger association (r2=0.87 p<0.0001) for the whole group. Lysis was mainly dependent on HLA class I expression and in addition on KIR-mismatch for these leukemic cells. Assessment of HLA expression on leukemic blasts and KIR-receptor-ligand-mismatch between donor and recipient may be valuable to define patients who will benefit most from a NK mediated GvL effect.
Stimulation of Anti-Tumor Immunity Using Dendritic Cells Transduced with Fowl Pox Vector Expressing MUC-1 and Costimulatory Molecules (PANVAC-F).
Authors: Vasir Baldev , Lenahan Corrine , Rosenblatt Jacalyn , Bissonnette Adam , Wu Zekui , Kufe Donald , Avigan David
Published: October 2019

In pre-clinical models, vaccination with attenuated vaccinia and fowl pox virus expressing tumor antigens and costimulatory molecules (CD54/ICAM-1; CD58/LFA-1 and CD80/B7.1-TRICOM) potently stimulates anti-tumor immune responses. However, vaccine efficacy may be limited by intrinsic deficiencies of native dendritic cells (DC) populations in patients with malignancy that are required to process and present the virally introduced antigens. An alternative strategy involves the transduction of ex vivo generated activated DCs. We have examined the capacity of DCs transduced with a fowl pox vector expressing MUC-1, CEA, and TriCOM (PANVAC-F) to elicit antigen specific responses and expand activated as compared to regulatory T cell populations. Partially mature DCs were generated from leukopak preparations obtained from normal volunteers by culturing adherent peripheral blood mononuclear cells for 5 days with GM-CSF and IL-4. DCs were transduced with PANVAC-F vector and matured with either TNFa or the combination of PG-E2, TNFa, IL-6 and IL-1b. In 5 serial studies, transduction with PANVAC-F resulted in mean MUC1 expression in 64.6% (SEM: ±1.6) of cells with a mean fluorescent intensity (MFI) of 249.8 (SEM: ±45.7). In addition, transduced DCs also demonstrated high levels of expression of class II (99%; MFI:250), CD54 (98.4%;MFI:516), CD58 (98.8%;MFI:155) and CD80 (78.2%; MFI:115). Of note, transduced DCs demonstrated higher levels of the maturation marker CD83 (40.3%; SEM: ±1.6, n=3) as compared to untransduced DCs 19.7% (SEM: ±2.9) (p=0.02) suggesting that transduction enhanced DC maturation and activation. Transduced DCs matured with PGE2, TNFa, IL-6 and IL-1b as compared to TNFa alone demonstrated higher levels of CD83 and CCR7 and a more stable phenotype following withdrawal of cytokine support. To assess the ability of PANVAC-F to stimulate tumor antigen specific responses, the presence of T cells binding the MUC-1 specific tetramer was quantified following stimulation of autologous T cells derived from HLA*0201 healthy donors. An increase in CD8+ and MUC1+ cells were observed (8.3%) with stimulation with PANVAC-F transduced DCs as compared to untransduced DCs (2.5%). The capacity of DCs transduced with PANVAC-F to stimulate interferon gamma producing activated T cells, as compared to CD4+/CD25+/Foxp3+ regulatory T cell populations is being assessed. In summary, PANVAC-F transduced DCs stimulate expansion of antigen specific T cell populations suggesting their potential role as tumor vaccines for MUC-1/CEA expressing tumors. We are initiating a trial for patients with ovarian carcinoma in early (marker only) relapse in which patients will be randomized to undergo serial vaccination with PANVAC-V/PANVAC-F or DCs transduced with PANVAC-F. The capacity to generate anti-tumor immunity in vivo will be assessed as a primary endpoint.
Combination Therapy of Anti-Cancer Drug with FNIII14 Peptide of Fibronectin Effectively Overcome Cell Adhesion Mediated-Drug Resistance of Acute Myelogenous Leukemia.
Authors: Matsunaga Takuya , Fukai Fumio , Tanaka Maki , Kobune Masayoshi , Chiba Hiroki , Niitsu Yoshiro
Published: October 2019

We have previously reported that in acute myelogenous leukemia (AML), drug resistance is induced by the attachment of α4β1-integrin (very late antigen (VLA)-4) on leukemic cells (AML cells) to fibronectin (FN) on bone marrow (BM) stromal cells, and this interaction is crucial in BM minimal residual disease (MRD) and prognosis after chemotherapy (Matsunaga T et al, Nature Med2003, 9, 1158–1165). We also reported a successful overcome of drug resistance in human AML cells transplanted mice with the use of anti-VLA-4 antibody. However, a drawback of progressive multifocal leukoencephalopathy by anti-VLA-4 antibody administration was reported in multiple sclerosis and Chron’s disease patients. Therefore, in this study, we examined whether FNIII14, a 22-mer peptide derived from FN which was reportedly potent to block the interaction between FN and VLA-4, VLA-5 or αvβ3-integrin (Fukai F et al. Clin Cancer Res 2002, 8, 2455–2462), can overcome cell adhesion mediated drug resistance (CAM-DR) induced by VLA-4/FN interaction in AML cells. By in vitro culturing system, FNIII14 dose dependently inhibited adhesion of two AML cell lines (U937 cells, HL-60 cells) and fresh AML cells from patients to FN, and significantly circumvented CAM-DR of these AML cells to cytosine arabinoside (Ara C). The mechanism for this circumvention of CAM-DR involved conformational inactivation of β1-integrin as revealed by FACS through inside out inhibitory signal from complex FNIII14/its 50kDa receptor resulting in down regulation of FAK/Akt/Bcl-2 signal which is responsible for VLA-4/FN induced CAM-DR. In a mouse model of MRD, a 100% survival rate was achieved by combining FNIII14 and Ara C, in contrast to a slight prolongation of survival by Ara C alone. These results indicate that combination treatment of anticancer drugs and FNIII14 is the potent modality for eradication of MRD in BM, and should improve the prognosis of AML.
Multicenter Evaluation of Fungal Prophylaxis in Hematopoietic Stem Cell Transplantation.
Authors: Barron Michelle A. , Fishman Neil , Baillie G. Mark , Brake Helga
Published: October 2019

BACKGROUND: The University HealthSystem Consortium (UHC) conducted a benchmarking study to assess members’ compliance with published guidelines for prevention of fungal infections in hematopoietic stem cell transplant (HSCT) recipients.

METHODS: Adult HSCT patients were evaluated by retrospective chart review of cases discharged between 01/01/04 and 06/30/05. Data collected included demographics, use of prophylaxis (px), and outcomes. Patients were classified as high or low risk for fungal infection according to NCCN and CDC guidelines.

RESULTS: Thirteen UHC member hospitals submitted a total of 242 HSCT cases. Patient characteristics and administration of antifungal therapy are shown in Table 1. 57% (137/242) of patients were classified as high risk for fungal infection. Overall, 85% (205/242) of patients received antifungal px. Compliance with national guideline directed antifungal px was 54%. Of patients who did not receive px, 59% (22/37) were high-risk. 32% (7/22) of these patients required empiric antifungal therapy, 29% (2/7) had a confirmed fungal infection and 1 of these patients died. Of patients who received antifungal px, 44% (90/205) were classified as low risk. 6.7% (6/90) went on to require empiric therapy and 2 died. 84.4% of patients had fluconazole as at least one of their prophylactic agents, and 34% of patients received prophylactic antifungals that provided coverage for both Aspergillus and Candida species. 5.4% (13/242) of HSCT recipients died during the target hospitalization. 76.9% (10/13) of these patients were high risk. 61.5% (8/13) had signs/symptoms of a suspected or definitive fungal infection at the time of death (6 were high risk).

CONCLUSION: Most HSCT patients did not receive appropriate fungal px based on current guidelines. Failure represented both under and over utilization of px. Further study is required to explain poor adherence to current guidelines.

Table 1: Patient Characteristics by Degree of Risk High Risk (n=137) Low Risk (n=105) % (n) % (n) *ABX = broad spectrum antibiotics Chemotherapy prior to admission 53.3% (73) 61.9% (65) Radiation prior to admission 23.4% (32) 17.1% (18) Allogeneic Transplant 75.9% (104) 17.1% (18) Cytomegalovirus disease 11.7% (16) 6.7% (7) GVHD 15.3% (21) 1.0% (1) Mucositis 55.5% (76) 7.6% (8) Systemic corticosteroids 63.5% (87) 44.8% (47) Fever unresponsive to >4 days of ABX 19.0% (26) 12.4% (13) Neutropenia during antifungal therapy 39.4% (54) 27.6% (29) History of Aspergillus infection 3.6% (5) 0.0% (0) Antifungal therapy: Prophylaxis 83.9% (115) 85.7% (90) Failed prophylaxis 21.2% (29) 5.7% (6) Empiric therapy 25.4% (32) 10.5% (11) Definite treatment 6.6% (8) 0.0% (0)
Treatment of Steroid-Resistant Graft-Versus-Host Disease with Monoclonal Antibodies in Children.
Authors: Schechter Tal , Afzal Samina , Finkelstein Yaron , Koren Gideon , Doyle John , Dupuis Laura Lee
Published: October 2019

Acute graft-versus-host disease (aGVHD) carries a major risk of morbidity and mortality in patients undergoing hematopoietic stem cell transplantation (HSCT). The prognosis is poor if aGVHD does not respond to corticosteroid treatment. Recently, monoclonal antibodies such as daclizumab, a humanized monoclonal IgG1, and infliximab, a chimeric monoclonal antibody that binds the precursor of tumor-necrosis factor-alfa, have shown promising results in the treatment of corticosteroid-resistant aGVHD. The reported response rate to monoclonal antibody therapy in adults with aGVHD reaches as high as 67%. Data describing the efficacy of monoclonal antibodies in children with corticosteroid-resistant aGVHD are limited. We conducted a retrospective analysis to evaluate the efficacy of daclizumab and/or infliximab in children diagnosed with steroid-resistant aGVHD in the Hospital for Sick Children, Toronto, from July 2002 to December 2005. Corticosteroid-resistant aGVHD was defined as aGVHD which did not respond or worsened after a minimum of 5 days of corticosteroid therapy. Complete response was defined as full recovery without any signs of aGVHD; partial response was defined as improvement of aGVHD symptoms in at least one organ without worsening in other organs. Sixteen children were treated for aGVHD, thirteen of them had aGVHD grade 3 or 4. The organs involved were gut (n=6), skin (n=4), liver (n=2) and multi-organ involvement (n=4). Thirteen children were given daclizumab; one was treated with infliximab and 2 children with their combination. Fourteen children received a full course of monoclonal antibodies for aGVHD. An additional child died after the first dose (from multi-organ failure) and one child developed reactive arthritis attributed to daclizumab. Seven of the 14 children (50%) who completed treatment responded: 5 had complete response and 2 had a partial response. Nine out of the 16 children died during the study period: 8 due to Transplant Related Mortality (TRM) and 1 due to relapse; three children developed fatal fungal infection and one had fatal adenovirus infection during or shortly after monoclonal antibody treatment. Median length of follow up in the remaining 7 patients was 18 months. We conclude that monoclonal antibodies were effective in the treatment of children with corticosteroid-resistant acute GVHD. The risk for infection, mainly fungal, was high.
The Incidence and Risk Factors of the Euthyroid Sick Syndrome Soon after Allogeneic Hematopoietic Stem Cell Transplantation in Children.
Authors: Cho Bin , Lee Dae-Hyoung , Jeong Dae-Chul , Kwon Young-Joo , Jang Pil-Sang , Chung Nak-Gyun , Kim Hack-Ki , Hwang Hui Sung , Park Young-Shil , Kim Sun Young , Park Ji Kyoung
Published: October 2019

Purpose: In this study, we analyzed the short term change of thyroid function, incidence and risk factors of thyroid dysfunction soon after allogeneic hematopoietic stem cell transplantation(HSCT) in children.

Method: We enrolled 80 pediatric patients following allogeneic HSCT, at the Catholic HSCT center between January, 2004 and February, 2006. Serum TSH(thyroid stimulating hormone), total serum thyroxine, total serum triiodothyronine levels were systematically measured in 80 patients before and 1 month, 6 month and 12 month after HSCT.

Results: Thyroid function was statistically decreased at 1 month after HSCT(P < 0.001). Thyroid dysfunction at 1 month was observed in 43(54%) of 80 patients, 31(39%) of whom presented with euthyroid sick syndrome. Thyroid dysfunction was normalized within 1 year after HSCT(Table).

In univariate analysis, malignant disease and presence of acute GvHD(grade ≥II) were risk factors for euthyroid sick syndrome(P = 0.04, 0.01). In multivariate analysis, we could not detect an independent risk factor for euthyroid sick syndrome(P = 0.19, 0.06).

Conclusion: The present study suggests that the incidence of thyroid dysfunction is high after allogeneic HSCT. Therefore, regular monitoring of thyroid hormone levels after HSCT is required.

Short-term changes in thyroid function after HSCT Pre HSCT Post HSCT 1 month Post HSCT 6 month Post HSCT 12 month T3 (ng/dL) 161 ± 27 100 ± 35 123 ± 35 124 ± 35 T4 (μg/dL) 8.7 ± 1.6 6.5 ± 2.4 7.3 ± 1.8 7.7 ± 1.6 TSH (mIU/L) 2.9 ± 1.9 1.2 ± 1.3 2.4 ± 1.8 2.1 ± 1.3
Inflammatory Events in Early Posttransplantation Period Are Associated with the Relapse Kinetics of Acute Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Kim Sung-Yong , Min Chang-Ki , Lee Mi-Jin , Cho Byung-Sik , Eom Ki-Seong , Kim Yoo-Jin , Lee Seok , Kim Hee-Je , Cho Seok-Goo , Kim Dong-Wook , Lee Jong-Wook , Min Woo-Sung , Park Jong-Won , Kim Chun-Choo
Published: October 2019

Time to relapse of acute leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is variable. The relapse kinetics may be influenced by graft-versus-leukemic (GVL) reaction mediated by donor immune cells and inflammatory cytokines. We wondered if the inflammatory cytokines released during the occurrence of the early post-transplantation inflammatory events may affect GVL effect thus influencing the relapse kinetics. We retrospectively analyzed the correlation between early post-transplantation inflammatory events and relapse kinetics in 55 patients with acute leukemia who relapsed following allo-HSCT. Patients were divided into two groups, early and late-relapsed group according to the time to relapse from transplantation. Multivariate logistic regression analysis revealed that the incidences of conditioning associated diarrhea (CAD) ≥ 500ml/day and febrile episode (FE) were significantly lower in the early-relapsed group than in the late-relapsed group (P=0.046 and 0.014, respectively). Serum levels of C-reactive protein, a surrogate marker of inflammation, on day 21 after transplantation (CRP21) were significantly lower in the early-relapsed group than in the late-relapsed group (P=0.009). FE, CAD ≥ 500ml/day and CRP21 were independent predictable factors for relapse kinetics based on multivariate Cox regression analysis (P=0.037, 0.002 and 0.025; hazard ratio=0.360, 0.221 and 0.971, respectively). Our analysis indicated the inflammatory events in early post-transplantation period could delay the relapse of acute leukemia early after allo-HSCT presumably because GVL effect was enhanced with the cytokines released by the events.

Figure 1 Cumulative Incidences of relapse according to the presence of febrite episode (A) and conditioning regimens associated diarrhea ≥ 500mg/day (B) based on multivariate Cox regression analysis. The patients who had either febrite episode (FE) or server conditioning regimens associated diarrhea (CAD) ≥ 500mg/day in early posttransplantation period at longer relapse free survival compared with the patients who did not (P=0.035 and 0.002). Figure 1. Cumulative Incidences of relapse according to the presence of febrite episode (A) and conditioning regimens associated diarrhea ≥ 500mg/day (B) based on multivariate Cox regression analysis. The patients who had either febrite episode (FE) or server conditioning regimens associated diarrhea (CAD) ≥ 500mg/day in early posttransplantation period at longer relapse free survival compared with the patients who did not (P=0.035 and 0.002).
Role of Intensive Conditioning Regimens Followed by Allogeneic Stem Cell Transplantation in Patients with Acute Lymphoblastic Leukemia: A Long-Term Follow-Up.
Authors: Minami Jiro , Asai Osamu , Dobashi Nobuaki , Yano Shingo , Osawa Hiroshi , Takei Yutaka , Yahagi Yuichi , Takahara Shinobu , Ogasawara Yoji , Otsubo Hiroko , Yamaguchi Yuko , Saito Takeshi , Maruyama Dai , Kobayashi Tatsunosuke , Hoshi Yasutaka , Usui Noriko
Published: October 2019

Purpose: Acute lymphoblastic leukemia (ALL) remains a difficult disease to treat in adults and less than 40% of ALL patients could be alive at 5 years post-diagnosis. We investigated whether intensive conditioning regimens improve outcome of patients with ALL.

Methods: Retrospectively, we analyzed 24 patients (median age; 29 years) with ALL who underwent allogeneic transplantation in our institute between February 1991 and October 2003. The median follow-up time was 7.7 years. Sixteen patients were in first complete remission (CR1) and five patients were in second CR (CR2) and three patients did not attain CR (non-CR) at the time of transplantation. Five patients with Philadelphia (Ph) chromosome-positive ALL were included. Preparative regimens were cyclophosphamide (CY), total body irradiation (TBI) and etoposide based regimen (n=23) or CY, TBI, and cytosine arabinoside (n=1) followed by marrow (n=21) or peripheral blood (n=3) transplant from unrelated (n=7) or related donors (n=17).

Results: The 8-year overall survival (OS) rate and the 8-year disease-free survival rate for all cases were 48% and 39%, respectively. The 8-year OS of Ph negative and positive patients were 56% and 20%. The 8-year OS of CR1, CR2 and non-CR patients were 50%, 60% and 0%, respectively. Overall probability of the 8-year relapse rate was 47%. Treatment related mortality was 25%.

Conclusion: These results suggest that CY/TBI based intensive regimens are feasible for ALL patients and may be effective for Ph negative ALL patients in not only CR1 but also CR2.
In Multiple Myeloma (MM) CAMPATH-1 Antibodies “In the Bag” Reduce the Early Toxicity of Stem Cell Transplantation from Graft vs. Host Disease (GvHD) and Seem To Be Associated with Improved Long Term Outcome.
Authors: Novitzky Nicolas , Thomas Valda , Toit Cecile du
Published: October 2019

Introduction: Although allogeneic stem cell transplantation remains the only curative approach in patients with MM, its role remains controversial. Early high treatment related mortality, particularly from infections, GvHD and later, from disease recurrence remains substantial challenges. This study reviewed the outcome of consecutive patients who received T-cell depleted grafts from HLA identical siblings.

Patients and Methods: Patients with symptomatic MM (stage II, n= 4 & III, n=17) who had an HLA identical sibling were initially treated until response with anthracycline or steroid based combinations. Three patients had, in addition, undergone autografting. Individuals who received cytokine mobilized peripheral blood progenitor cell (PBPC) grafts were also prescribed therapeutic serum levels of cyclosporine for 90 days post transplantation. The objective of the study was to determine transplant related mortality OS and DFS.

Results: Twenty one patients with a median age of 44 (range 37– 56) years who had responded to chemotherapy (CR or VGPR) received stem cell enriched grafts from HLA identical siblings. Median performance status was 1 (0 – 2). Five individuals had significant organ dysfunction from effects of the disease. At presentation median albumin and β2MG blood levels were 30 g/L and 3.2 ng/mL, respectively. Myeloablative conditioning was radiotherapy (n= 12) or chemotherapy (n= 9) based. GvHD prophylaxis consisted of T-cell depletion with CAMPATH-1G (n=7) or H (n=14) antibodies “in the bag”. BM had all been treated with ex vivo with CAMPATH-1G (median 20 mg) while PBPC grafts in 14 patients were incubated with CAMPATH-1H (median 10 mg). Patients received a median of 23 ×104/kg CFU-GM in 0.87 BM mononuclear cells or 9.22 ×108/kg PBPC (CD34+: 4.46 ×106/kg). Median time to engraftment was 12 days. Two patients developed GvHD (grade 2) and 3 limited chronic form (1 progressed from acute). The 1 year non relapse mortality was 19%. Six patients suffered disease recurrence. One refused further therapy. Three of 5 responded to DLI and 2 remain in unsustained remission. Fatal events appeared lower in patients receiving chemotherapy based conditioning (mortality 11% vs. 50; p= 0.06), exposure of stem cells to CAMPATH-1H (15% vs. 62.5%; p= 0.04) and a lower median CAMPATH-1 dose given (p= 0.01). Cox analysis confirmed that lower CAMPATH-1 dose was associated with improved outcome. Fourteen (67%) patients survive at a median of 1101 days (range 385–5309) and 62% are disease free.

Conclusions: In this cohort of chemotherapy responsive patients with advanced myeloma, ex vivo T-cell depletion of allogeneic grafts was associated with good protection from GvHD and 19% 1-year transplant related mortality. Low grades of GvHD post transplantation were associated with a favourable impact in the long term survival.
Long Term Results of Allogeneic Stem Cell Transplant for CML in Pediatric Patients: A Study of 50 Cases Transplanted over 20 Years in a Single Institution.
Authors: Funke Vaneuza A.M. , Pettengil Claudia , Bonfim Carmem M.S. , Ruiz Jefferson , Bitencourt Marco A. , Setubal Daniela C. , Nunes Elenaide C. , Oliveira Michel M. , Azambuja Ana Paula , Lima Denise H. , Kojo Keiko T. , Datamanager Heliz Regina A. Neves , Neto Jose Zanis , Medeiros Carlos R. , Pasquini Ricardo
Published: October 2019

Introduction: Chronic myeloid leukemia (CML) accounts for 2–3% of the leukemias in childhood. The only potential curative treatment is allogeneic hematopoietic stem cell transplantation (HSCT), although promising results achieved with imatinib mesylate in adults substantiate its use as a therapeutic alternative for children. The aim of this study is to analyze the outcomes of HSCT in pediatric patients regarding overall survival (OS) and main causes of death.

Materials and methods: Retrospective analysis of children aged 1–17 years, diagnosed with CML who underwent HSCT in a single institution in Brazil between jan/1984 and aug/2005. Survival was estimated by Kaplan-Meier curves. Log Rank test was used for comparison of continuous variables.

Results: Fifty patients were assessed, 31 male and 19 female. Median age of 13,5 years (1–17). Forty one patients (82%) were in first chronic phase (CP1) and 9 in advanced phases. The interval between diagnosis and HSCT had a median time of 17,5 months (5–84). The source of stem cells was bone marrow in 44 patients (88%), umbilical cord blood in 5 (10%) and peripheral blood stem cell in 1 (2%). Thirty nine patients (78%) underwent related HSCT and 11 (22%) unrelated donor HSCT. Conditioning regimens: busulfan and cyclophosphamide in 35 patients (70%) and TBI containing regimens in 15 (30%). Complete engraftment occurred in 82% of the transplants. Acute (a) graft-versus-host-disease (GVHD) grades II–IV occurred in 44% of the patients, with 20% grade IV. Extensive chronic (c) GVHD occurred in 15/40 patients (38%). Fifteen patients (32%) relapsed after HSCT. Mortality in the study population was 48% and the main causes of death were: relapse in 6 patients (25%), a-GVHD in 6 (25%) and c-GVHD in 4 (17%). Estimated OS in 20 years was 50%, with a median survival of 1926 days. When analyzed separately, patients in CP1 who received related HSCT and immuneprophilaxis with three drugs (steroids, cyclosporine and methotrexate) had an estimated OS in 20 years of 70%.

Conclusions:

Long term follow up of these children with CML who underwent allogeneic HSCT demonstrate an OS of 50%, reaching 70% in low risk patients. Main causes of death were relapse, acute and chronic GVHD.
Autologous Non-Myeloablative Hematopoietic Stem Cell Transplantation for Refractory Systemic Vasculitis.
Authors: Statkute Laisvyde , Oyama Yu , Barr Walter G. , Satkus Jolita , Loh Yvonne , Verda Larissa , Burt Richard K.
Published: October 2019

Background

Current mortality rate in systemic vasculitis (SV) patients with the use of cytotoxic / immunosuppressive therapy is over 20 % at 5 years in some studies. For patients refractory to conventional therapy new strategies aimed at aggressive induction of remission and relapse prevention are being sought. We here report our single center experience in treating 4 patients with refractory SV employing non-myeloablative autologous hematopoietic stem cell transplantation (HSCT).

Patients and Methods

Four patients with refractory SV (2 - with neurovascular Behcet’s disease, 1 - with neurovascular Sjogren’s syndrome, and 1 - with Wegener’s granulomatosis) were involved in an IRB and FDA approved phase I clinical trial of high dose chemotherapy and autologous HSCT. Peripheral blood stem cells were mobilized with cyclophosphamide (Cy) 2g/m2 IV and granulocyte-colony stimulating factor 10 mcg/kg/day SQ and were CD34+ enriched. Conditioning regimen consisted of Cy 200 mg/kg and rabbit anti-thymocyte globulin 5.5 mg/kg IV. Disease activity and cumulative organ damage were assessed using Birmingham Vasculitis Activity Score (BVAS) and Vasculitis Damage Index (VDI), respectively. Primary and secondary endpoints were transplant-related toxicity, survival and change in BVAS and VDI after the transplant.

Results

All 4 patients tolerated HSCT well without transplant related mortality or any significant toxicity. At median follow up of 28 (range 22–35) months all patients are alive. Three patients (1 each with Behcet’s, Sjogren’s, and Wegener’s) entered a sustained remission at 6, 6 and 24 months after the transplant. Both, BVAS and VDI, markedly decreased within 6 months post-transplant. In a patient with WG C-ANCA levels became undetectable at 24 month evaluation corresponding to clinical and radiological improvement. All 3 patients who achieved remission discontinued immunosuppressive therapy at the time of transplant and have not required since. Two patients with pre-transplant steroid dependency discontinued prednisone at 6 and 14 months after HSCT. One patient with BD who is positive HLA-B51, now 22 months since HSCT, has never achieved remission and is still requiring immunosuppressive therapy.

Conclusion

We suggest autologous HSCT utilizing a non-myeloablative regimen is a safe and effective treatment for select patients with SV refractory to conventional immunosuppressive therapies. However, in patients with significant genetic predisposition, more aggressive approach such as allogeneic HSCT or cord blood transplantation might be considered.
Highly Efficient mRNA- and cDNA-Based Transient Gene Delivery into Human Progenitor Cells.
Authors: Greiner Jochen , Torzewski Jan , Ponsaerts Peter , Rojewski Markus T. , Kronawitter Desiree , Schrezenmeier Hubert , Hombach Vinzenz , Dohner Hartmut , Schmitt Michael , Wiesneth Markus , Zimmermann Oliver , Wiehe Juliane M.
Published: October 2019

The method of gene transfer into progenitor cells is critical as viral vector transduction involves the risk of tumor induction by non-specific genomic integration. Non-viral transfection systems often fail due to low transfection efficiency. However, gene transfer into human CD34+ hematopoietic progenitor (HPC) and mesenchymal stem cells (MSC) is an essential tool for in vitro- and in vivo-applications and therapeutic strategies such as tissue engineering and gene therapy. We recently reported an transient genetic labelling of human CD34+ HPC with deltaLNGFR-plasmid-DNA for in vivo application: Transient transfection was efficient for both, CD34+ HSC (41% ± 2%) and leukemia cell lines (55% ± 4.9%) using the method of nucleofection. Moreover, mature myeloid cells (CD66b+) derived from transfected human CD34+ HPC and leukemia cells maintained deltaLNGFR expression at a high percentage (70% ± 1.6% and 58% ± 2% respectively). In this work, we investigated labelling of CD34+ HPC with mRNA. Human CD34+ HPC and human MSC were transfected with in vitro-transcribed mRNA for deltaLNGFR, a marker gene approved for human in vivo-application, using nucleofection. EGFP was used as a control. 24h after nucleofection, FACS-analysis showed a higher transfection efficiency compared to plasmid transfected CD34+ HPC and MSC: A high transfection frequency was found for mRNA-transfected HPCs using deltaLNGFR (82.4±9.7%) and EGFP (88.7±2.6%). We found also a high transfection rate for MSC using the marker genes deltaLNGFR (92.4±3.6%) and EGFP (83.3±4.1%). Cell viability was not affected by mRNA-transfection. Moreover, differentiation assays of deltaLNGFR-selected MSC after transfection, showed that differentiation of MSC into mesenchymal cells like chondrocytes, adipocytes and osteoblasts was not affected by mRNA nucleofection. Taken together, mRNA based nucleofection is a powerful, highly efficient and non-toxic approach for transient labelling of human progenitor cells or, via transfection of selective proteins, for transient manipulation of stem cell function. It may thus be useful to transiently manipulate stem cell characteristics and combine principles of gene therapy and tissue engineering.
Therapeutic Efficacy of Engineered T Cells Targeting CD19 in a B-Cell Lymphoma Model.
Authors: Hawkins Robert E. , Gilham David E. , Thistlethwaite Fiona C. , Radford John A. , Cheadle Eleanor J.
Published: October 2019

The success of monoclonal antibodies in the treatment of certain cancers demonstrates that immune based therapies can work and are particularly effective in B cell malignancies. However, tumours can still avoid antibody mediate mechanisms of attack and there is currently no estalbished method of effectively recruiting T cells to harness their potential anti-tumour effects. We are exploring gene therapy approaches to endow T cells with antibody type specificity in order to more efficiently target and lyse tumours and thereby improve the overall immune therapy of cancer

T cells grafted with a CD19 specific receptor consisting of a CD19 scFv linked to human CD3zeta (CD19z) were tested for their potency against B cell lymphoma lines in vivo. T cells were engineered using retroviral vestors to possess a CD19 specific receptor which endows the T cells with specificity for B cell lymphoma. The vector incorporates a truncated hCD34 gene as a marker to facilitate assessment of transduced cells using as clinically applicable, non-immunogenic marker gene. Mice bearing B cell lymphoma were treated with a systemic infusion of targeted T cells with or without non-myeloablative chemotherapy. Human T cells targeting CD19 cured 40% of SCID/beige mice with 6 day established metastatic tumour but only in conjunction with a single dose of cyclophosphamide. Murine T cells expressing the CD19z receptor were also effective with cure of 24hr established s.c human CD19+ tumour in SCID/beige and immunocompetent mice. Pretreatment with cyclophosphamide did not affect T cell engraftment or efficacy in immuno-compromised animals but was necessary for T cell engraftment in immuno-competent animals.

These results which parallel the approach successfully used with tumour infiltrating lymhocytes in melanoma patients conclusively demonstrate that the combination of engineered T cells with “pre-conditioning” chemotherapy significantly impacts upon tumour growth in vivo and this evidence supports the development of phase I clinical trials targeting B cell lymphoma.