bluuebunny/crossref_metadata_2025 huggingface dataset:
Correcting Flow Cytometric Blast Counts for Blood Contamination in Bone Marrow Aspirates.
Authors: Loken Michael R. , Chu Sung-Chao , Fritschle Wayne K. , Li Dian-Kun , Wells Denise A.
Published: October 2019

An accurate blast count is pivotal in the diagnosis, classification and prognosis of patients with myelodysplasia. Blast counts in all previous classification schemes are based on morphologic assessment of marrow aspirates with a poor correlation to blast counts determined by flow cytometry. A significant problem in blast enumeration by flow cytometry is the variable hemodilution of the marrow during collection for flow cytometric analysis. Blast counts can vary depending on which aspirate tube is used for flow analysis, e.g., 2.4%, 1st 5ml tube; 0.62%, 2nd 5ml tube; 0.58% 3rd 5ml tube. Morphologists circumvent this problem by selecting a region for assessment close to a spicule with minimal blood dilution. Cell surface antigens can be used to distinguish mature cells found in blood as distinct from immature cells identified in marrow. CD16 intensity on neutrophils reaches a maximum at the band/segmented stage of development with a low coefficient of variation, thereby becoming a marker for mature myeloid forms. A simple method to distinguish immature from mature myeloid cells was developed to assess extent of blood contamination in marrow aspirates using a combination of CD16, CD13, and CD45. The average mature neutrophil content of a marrow was determined from phenotypically normal bone marrow biopsy specimens, assumed to have minimal blood contamination. The proportion of dimCD16 cells gated on the myeloid cells based on CD45 and right angle light scatter in 31 biopsy specimens was 82% (range 69–93, SD=6.2) (Figure 1). A value of 80% (rather than 82%) was used for the subsequent calculations to correct for the excess mature neutrophils found in an aspirate as compared to the biopsies (Corrected Blasts = [80 / % dim CD16 myeloid] x determined blast count). To test this hypothesis bone marrow aspirates were diluted with blood at different ratios to mimic blood marrow hemodilution. Blasts (defined as CD45 dim, low right angle light scatter, HLA-DR positive, CD11b negative) were determined for the various dilutions, then corrected based solely on the proportion of dim CD16 myeloid cells (Figure 2). A marrow from an MDS case was also diluted (1:5 v/v) with blood for comparison. The original marrow contained 80% dim CD16 myeloid cells with a blast count of 9.2%. After dilution, only 12% dim CD16 cells were detected with 1.1% blasts, however upon correction (6.67), the blast count was 7.3%, close to the original determination. This approach may provide for more standardization and consistency in the determination of blast counts in MDS marrow specimens using flow cytometric analysis.

Figure 1, CD16 of marrow myeloid cells. Figure 1,. CD16 of marrow myeloid cells. Figure 2, Uncorrected/Corrected Blast Count Figure 2,. Uncorrected/Corrected Blast Count
Lenalidomide after Allogeneic Stem Cell Transplantation in Multiple Myeloma.
Authors: Lehmann Friederike , Jean El-Cheikh , Zabelina Tatjana , Ayuk Francis , Wolschke Christine , Petersen Lars , Bacher Ulrike , Zander Axel R. , Blaise Didier , Mohty Mohamad , Nikolaus Kroeger
Published: October 2019

Background: Lenalidomide is an immunmodulatory drug, orally administered once daily, which is effective in relapsed multiple myeloma (MM). Here, we evaluated the efficacy and toxicity of lenalidomide in 16 MM patients with relapsed disease after allogeneic stem cell transplantation (allo-SCT).

Methods: Median age was 57 (range 37–68) years. The series included 5 females and 11 males. Prior to allo-SCT, all patients were heavily pretreated with multiple lines of chemotherapy, including high dose therapy with autologous stem cell support. Also, prior to lenalidomide treatment, other salvage therapies included donor lymphocyte infusions (DLI) in 93% of cases, thalidomide in 63%, and bortezomib in 88%. NCI-criteria were used to define toxicity. Lenalidomide was given 25 mg orally once daily on day 1–21 every 28 days. No prophylactic anticoagulation was used.

Results: The overall median number of completed cycles was 5 (range 1–10). Very good partial response (VGPR) was achieved in 13%, partial remission (PR) in 53%, and stable disease (SD) in 27% of the patients. During the follow up period, disease progression was observed in 50% of cases, and death in one patient. The median progression-free survival was 8 months, while the median overall survival was not yet reached. Hematotoxicity was the primarily and major encountered side effect (leukopenia: 6% grade 4, 19% grade 3, 19% grade 2, 38% grade 1; thrombopenia: 19% grade 3, 19% grade 2, 31% grade 1). This myelotoxicity led to dose reduction (usually 10 mg) in 44% of the patients. Infectious complications were observed in 25%. Non-hematological toxicity was also seen in 75% of cases (56% grade 1, 19% grade 2), consisting of cramps in the leg, constipation, symptomatic fatigue, nausea and progressing polyneuropathy (n=1). Thrombembolic complications (cerebral infarction) were observed only in one patient, who was receiving concomitant corticosteroid treatment for acute graft-versus-host disease (GvHD), but neurological symptoms resolved completely. GvHD of the skin under lenalidomide treatment was seen in 25% of cases (one grade 2, and 3 grades 1), with one case occurring shortly after an additional DLI.

Conclusions: Lenalidomide is effective in relapsed patients with MM after allo-SCT. Major toxicity is myelotoxicity, which required dose reduction in a majority of patients. With this background, a dose-finding study to determine the optimal lenalidomide dose as maintenance therapy after allo-SCT has already started.
CD26 Inhibition on CD34+ or Lineage− Human Umbilical Cord Blood Donor HSC/HPC Improves Long-Term Engraftment into NOD/SCID/Beta2null Immunodeficient Mice.
Authors: Christopherson Kent W. , Paganessi Laura A. , Napier Stephanie , Porecha Nehal K.
Published: October 2019

Given the tremendous need for and potential of umbilical cord blood to be utilized as a donor source for hematopoietic stem cell transplantation in adults, there is a strong push to overcome the constraints created by the limited volumes and subsequent limited hematopoietic stem and progenitor cell numbers available for hematopoietic stem cell transplantation from a single collection. We have previously described the use of CD26 inhibitor treatment of donor cells as a method to increase the transplant efficiency of mouse hematopoietic stem and progenitor cells into a mouse recipient (Christopherson, KW 2nd, et al, Science2004. 305:1000–1003). We therefore hypothesized that inhibition of CD26 on human donor cord blood prior to transplant would result in an increase in long-term engraftment potential. To test this hypothesis, we isolated CD34 enriched (CD34+) or lineage depleted (lin−) pooled cord blood cells, evaluated their level of CD26 expression and activity, and then tested their ability to engraft into the NOD/SCID/B2mnull immunodeficient mouse model of hematopoietic stem cell transplantation with or without prior CD26 inhibitor treatment. We observed that long-term engraftment into the recipient mouse bone marrow at twelve weeks post sub-lethal irradiation (350cGy), followed by transplantation of 1x105 pooled donor cells was 13.4±2.0% and 55.5±4.0% CD45+ human cells in the untreated, and CD26 inhibitor (Diprotin A) treated CD34+ donor cells respectively. We also observed engraftment levels in the mouse recipient bone marrow of 6.2±0.7% and 47.0±2.8% CD45+ human cells for untreated and CD26 inhibitor treated lin− cells respectively. These measurements represent approximately a 4-fold and 7.5-fold improvement in the engraftment of long-term repopulating cells resulting from CD26 inhibitor treatment of either CD34+ or lin− donor cells respectively. These pre-clinical results establish a basis on which to propose the use of CD26 inhibitor treatment of donor cord blood units prior to transplantation for the purpose of improving transplant efficiency and subsequently patient outcome.
Generation of Cytomegalovirus pp65-Specific T Cells from Seronegative Donors.
Authors: Stamer Mindy M. , Dunham Kimberly , Bao Lei , Lucas Kenneth G.
Published: October 2019

Cytomegalovirus (CMV) can cause significant disease in allogeneic stem cell transplant (SCT) recipients. Adoptive transfer of donor derived CMV-specific cytotoxic T lymphocytes (CTL) is currently used for prophylaxis and treatment of CMV infections in allogeneic transplant recipients, but this is only available for patients whose donors are CMV seropositive. We developed a method to prime and expand CMVpp65-specific T cells from the peripheral blood mononuclear cells (PBMC) of CMV seronegative donors. Monocyte-derived dendritic cells isolated from four CMV seronegative donors were pulsed with CMVpp65 peptides for two hours prior to T cell stimulation. CMVpp65 peptides consisted of 15 mers with an 11 amino acid overlap. T cells obtained from CMV seronegative donors were primed every seven days for three weeks using CMVpp65 peptide pulsed dendritic cells. Interleukin-7 and interleukin-12 were initially added to the T cell cultures to promote CTL differentiation. After ten days, interleukin-2 together with interleukin-7 and interleukin-12 were added at three day intervals. Intracellular cytokine staining (ICS) and 51Cr-release assays were used to measure the activity and specificity of CMVpp65 primed CMV seronegative donor T cells seven to ten days after the last stimulation. A four color ICS analysis revealed that CMVpp65-specific IFN-γ-producing CD4+ (Th1) T cells were expanded from two of the four seronegative donors; one of these two donors had CMVpp65-specific killing as measured by 51Cr-release assay, the other displayed non-specific killing. One of the other two donors exhibited CMVpp65 killing but lacked IFN-γ production. PBMC isolated from a CMV seronegative donor three weeks post CMV vaccination also exhibited CMV-specific killing after only one week in vitro stimulation, with no CMV specificity pre-vaccination. In conclusion CMV-specific effector cells can be expanded from some CMV seronegative donors using CMVpp65 peptide pulsed dendritic cells, interleukin-2, interleukin-7, and interlukin-12. These cells may have the potential to be used in adoptive cell therapy to prevent or treat CMV infections in SCT recipients from CMV seronegative donors. An alternative strategy for patients with persistent CMV infection may be to use CTL derived from a vaccinated donor. Analysis of CMVpp65-specific T cells from additional seronegative donors is in progress to further optimize ex vivo expansion.
IL-2- and IL-15-Activated Donor NK Cells Infusion Reduces GVHD and Prolongs Survival in Melanoma-Bearing Mice after MHC-Mismatched Hematopoietic Cell Transplantation.
Authors: Wu De Pei , Chen Guang Hua , Yang Ming Zhen , Tang Xiao Wen , Sun Ai-ning
Published: October 2019

The function of NK cells is regulated by a balance between signals transmitted via actvating and inhibitory receptors, including killer-immunoglobulin-like receptors (KIRs). Murine NK cells express subgroups of H-2-specific receptors of the C-type lectin superfamily, termed Ly49, that regulate their function. We investigated if alloreactive NK cells would reduce GVHD and mediate graft-versus-tumor effects in mice undergoing MHC-mismatched allogeneic HCT. Six-week-old female C57BL/6 mice were injected intravenously with 2×105 B16 melanoma cells via lateral tail vein. seven days following melanoma inoculation, melanoma-bearing mice were irradiated (1000cGy) with 6MV X—ray and transplanted with 5x106 bone-marrow cells and 10x106 splenocytes from MHC-mismatched BALB/c mice. One day following transplantation, recipient mice were given a single infusion of donor IL-2 and IL-15-activated NK cells. Recipients of alloreactive NK cells had a significant reduced clinical GVHD scores(p<0.01), less melanoma lung metastasis, and prolonged survival (p<0.01) compared to HCT recipients not receiving alloreactive NK cells. These data provide an in vivo evidence that a single infusion of alloreactive NK cells can reduce GVHD and prolong melanoma-bearing mice survival via graft-versus-tumor effects following MHC-mismatched allogeneic HCT.
Escape of Minor Histocompatibility Antigen-Restricted Anti-Leukemia Immunity Associated with Loss of Specific HLA-I Locus Expression in a Leukemia Patient after an Intensive Immunotherapy.
Authors: Chang Lung-Ji , Tong Chun-Rong , Tang Jih-Luh , Akatsuka Yoshiki , Han Shuhong , Liang Yin , Li Chi-Cheng , Dong Lujia , Wu Tong , Lu Dao-Pei
Published: October 2019

Differential graft versus host response after bone marrow transplantation (BMT) is key to leukemia patient survival. The development of specific graft versus leukemia response targeting tissue-specific minor histocompatibility antigens (mHAgs) is a novel therapeutic approach to treating hematopoietic malignancies. A pilot intensive immune cell therapy was applied to an acute myeloid leukemia (AML) patient who relapsed around day 43 after receiving unrelated allogeneic BMT with three allele disparities for HLA-A, C and DQB1. The patient received more than thirty infusions of dendritic cell-co-cultured donor-derived immune cells including cytotoxic T lymphocytes (CTL) generated against AML lysate-pulsed dendritic cells (DC), AML-DC fusion, and DC pulsed with HLA-A*2402-restricted ACC1Y mHAg epitope. The latter was expressed by recipient but not by donor hematopoietic cells. ACC1Y-peptide-major histocompatibility complex (MHC) multimer staining detected CTL in the patients peripheral blood at different time points during treatment. Four months after the first mHAg-CTL infusion, CTL-resistant AML cells were detected in relapsed pleural effusion and peripheral blood. Further analysis of the relapsed AML cells revealed complete loss of surface class I HLA-A*2402 and class II HLA molecules. Although the AML-specific immune cells were effective in killing the original AML in CTL assays in vitro, the late relapsed AML became resistant to the early AML-primed CTL. This result suggests that rapidly evolving AML cells may escape the intensive anti-leukemia allo-immune pressure including the mHAg-specific CTL through selective loss of A*2402 expression - the ACC1Y-restricted HLA locus. As the patient suffered ongoing multi-focal extramedullary relapse in muscles, sub-mucosal tissue, intestine, spinal cord, and brain, rare cancer cells resistant to mHAg-specific CTL could evolve quickly. Our study indicates that immune escape through diminished HLA expression, which resulted in lack of cancer cell killing by AML-specific immune cells, including the mHAg-restricted CTL, had developed during the therapy. Therefore, leukemia patients with high load of AML or multi-focal extramedullary relapse may not endure prolonged benefit from a single arm immune cell therapy.
The Generation of Potent Dendritic Cells Can Be Modulated by Interaction between Immature Dendritic Cells and Invariant NK Cells with the Presence of Stimulatory Cytokines and TLR Agonist.
Authors: Yang Deok-Hwan , Choi Bo-Hwa , Kang Hyun-Kyu , Kim Sang-Ki , Kim Yeo-Kyeoung , Kim Hyeoung-Joon , Lee Je-Jung
Published: October 2019

For the therapeutic DC-based immunotherapy, peripheral blood derived-monocytes are cultured with the presence of exogenous GM-CSF and IL-4 by in vitro amplification. However, the different cells of the innate and adaptive immune system interact with each other and affect the activation and maturation of DCs for subsequent T-cell priming in vivo. We investigated what kind way of cultivation was more effective for the induction of mature DCs (mDCs) in vitro, compared the coculture with invariant NK cells and iDCs under the stimulatory molecules to the coculture with iDCs added to the activated NK cells stimulated by same molecules.

Experimental Methods: To evaluate the NK-cell-mediated DC maturation, we divided two different combinations of the coculture of NK cells and iDCs. In the activated NK-cell-mediated DC maturation, isolated CD3-CD56+ NK cells were activated by IL-2 alone, combined IL-2 with TLR3 agonist (poly I:C) and combined IL-2 with TLR3 agonist and IFN-α. These activated NK cells were cocultured with iDCs and were differentiated into mDC. In the invariant NK-cell-mediated DC maturation, isolated CD3−CD56+ NK cells were cocultured with iDC, and then, the invariant NK cells and iDCs were stimulated simultaneously by same stimulatory methods. After harvesting the mDCs and their supernatants, the phenotype and functional capacities of mDCs were analyzed and IL-12p40 productions of mDCs were estimated by immunoassay.

Results: The expressions of several molecules on DCs and IL-12p40 productions by DCs were significantly higher under the stimulation of IL-2, TLR3 agonist and IFN-α than IL-2 alone or combined with IL-2 and TLR3 agonist in the invariant NK-cell-mediated DC maturation. However, the DC phenotypes and IL-12p40 productions in the activated NK-cell-mediated DC maturation did not show any differences to the same stimulatory methods. When comparing the functional capacities and IL-12p40 productions according to the way of the activated NK-cell-mediated and invariant NK-cell-mediated DC maturation, the functional capacities of DCs from the invariant NK-cell-mediated maturation were significantly higher than those of DC from the activated NK-cell-mediated maturation with the stimulation of IL-2, TLR3 agonist, and IFN-α. IL-12p40 productions were more improved from the invariant NK-cell-mediated maturation than the activated NK-cell-mediated maturation.

Conclusions: The invariant NK cells can promote the generation of DCs through the coculture with iDCs under the stimulation of IL-2, TLR3 agonist and IFN-α. However, the activated NK cells did not enhance the DC functions. This study emphasized the potential for manipulating the interaction between DC and NK cells in the generation of mDCs.

Figure Figure Figure Figure
Novel Nanoparticle-Based Approaches To Target B-Cell Malignancies.
Authors: Weaver James A. , Brannen Candice , DeSimone Joseph , Orlowski Robert Z.
Published: October 2019

Introduction: Current approaches to the management of B-cell malignancies include the use of systemic treatments with cytotoxic chemotherapeutics that are not targeted to the tumor cell itself. By improving the ability to deliver lethal agents to the malignant cell clone, such as by targeting the unique tumor idiotype, it is possible that the therapeutic index of our current armamentarium could be improved, thereby optimizing patient outcomes. Nanoparticles represent one attractive approach in this area, since their high surface to volume ratio provides a strong driving force for diffusion into the tumor microenvironment.

Methods: Studies were performed using Ramos and SUP-B8 human Burkitt’s lymphoma cell lines, as well as cell lines representative of other hematologic malignancies. Commercially available nanoparticles (Qdots; Invitrogen) as well as nanoparticles generated using PRINT (particle replication in non-wetting templates) technology, were used with idiotype and control peptides.

Results: A biotinylated peptide recognized by the SUP-B8 idiotype was bound to streptavidin-containing Qdots, as well as to 200 nm polymeric PRINT particles. Both of these nanoparticle types bound specifically to SUP-B8 Burkitt’s lymphoma cells, as determined by the induction of a fluourescence shift by flow cytometry. These same particles showed no detectable binding to Ramos Burkitt’s lymphoma cells, whose idiotype recognizes a distinct set of peptide sequences, nor to other malignant cell lines, such as RPMI 8226 and ANBL-6 multiple myeloma cells. Moreover, when these nanoparticles were coated with control biotinylated peptides, no binding was seen to any of these cell types, demonstrating the specificity of this approach. Studies utilizing a number of techniques, including fluorescence and electron microscopy, revealed that, after binding, these idiotype-specific particles were internalized by SUP-B8 cells, but not by control cells, or when control peptides were used. Studies in vivo are underway to determine if these targeted nanoparticles can be used for imaging, as well as for delivery of lethal cargoes such as doxorubicin.

Conclusion: Nanoparticles can be targeted to specific neoplastic B-cell clones through the use of peptides recognized by the surface idiotype, possibly thereby sparing toxic effects to other B-cell clones, as well as to non-lymphoid cells. The ability to generate nanoparticles of various chemistries, sizes, and porosity, as well as nanoparticles containing a wide array of cargoes, will allow these to be used as platforms for the future personalization of therapy of B-cell malignancies.
Plasmatic Levels of FLT-3 Ligand and SDF-1 and Expression of FLT-3 and CXCR4 in CD34+ Cells Population Prior and Post Mobilization of Peripheral Blood Stem Cell.
Authors: Cecyn Karin Z. , Schimieguel Dulce M. , Kimura Eliza Y. , Yamamoto Mihoko , Oliveira José Salvador R.
Published: October 2019

Introduction: Peripheral blood stem cells (PBSC) became the main source of cells for autologous and allogeneic marrow transplantation. FLT3L is capable to inducing multi-lineage hematopoietic cell differentiation in vivo and in vitro. It was postulated that an increase in FLT-3 levels correlated with a compensatory mechanism to boost the amount of PBSC. The stromal-derived- factor-1 (SDF-1) is a member of the family of α-chemokines that binds to the seven-span transmembrane G-protein-couple CXCR4. Mobilized human CD34+ progenitors cells express reduced levels of the SDF-1 receptor of CXCR4, which correlates with improved mobilization, suggesting the involvement of SDF-1/CXCR4 interactions in the trafficking of CD34+ cells from marrow to peripheral blood. Objective: To determine the behavior of the of FLT-3-L and SDF-1 plasma concentration, prior and post mobilization of PBSC. The cellular expression of the FLT-3 and CXCR4 in the CD34+ cell population in the bone marrow (BM) prior and post mobilization and in the apheresis product and to evaluate its association with the good and bad yield of CD34+cells.

Material and Methods: The plasma concentration of SDF-1 and FLT3-L was determined by ELISA, prior and after PBSC mobilization in 48 individuals, being 12 donors and 36 patients. The cellular expression of CXCR4 and FLT-3 in the CD34+ cell population, was estimated as the mean fluorescence intensity (MFI), in material of bone marrow (BM), prior and after mobilization and in the apheresis product. It was considered success of mobilization who had reached in the peripheral blood (PB), 15 or more CD34+cells and in the apheresis product ≥5x 106/Kg CD34+cells.

Results: Considering globally the total of individuals, it was observed a plasma concentration variation of FLT3L and SDF-1, prior and post mobilization of PBSC (p=0.001 and 0,012, respectively), even this, did not have influenced the good yield of CD34+ cells. When evaluated the cellular expression of the CXCR4, it was observed variations in the BM, prior and post mobilization of PBSC, for the myeloma group (p<0.001), however when the total of individuals was considered, the behavior was different. There was a variation between the measures prior and post mobilization, only to the success mobilization group(p=0.036). For the FLT-3, we find variations between BM mobilized and not mobilized for all studied groups, however it did not have relation with the yield of CD34+ cells.

Conclusion: Although, FLT-3L and SDF-1 prior and post mobilization showed variations in the plasmatic concentration in this study, the only variable that had association with the good yield of CD34+ cells was the cellular expression of the CXCR4.
Toxicity and Early Response after Single Daily Dose of Intravenous Busulfan and Melphalan as Conditioning Regimen for Autologous Stem Cell Transplantation in Patients with Multiple Myeloma.
Authors: Blanes Margarita , de la Rubia Javier , Lahuerta Juan J. , Gonzalez Juan D. , Ribas Paz , Solano Carlos , Sanz Miguel A.
Published: October 2019

Introduction: Conditioning regimen with oral busulfan (BU) and melphalan (MEL) for patients with multiple myeloma (MM) undergoing autologous transplant (ASCT) has been associated with a high incidence of liver complications, especially severe sinusoidal occlusive syndrome (SOS).

Material and methods: We conducted the current study to assess the toxicity profile and outcomes associated with ASCT after intravenous (iv) BU-based conditioning in 45 patients (24 M/21 F; median age 60 years, range 34–71) with MM. Front-line chemotherapy consisted of VBMCP/VBAD (31cases), VAD (8 cases), and other regimens (6 cases). At transplant, 8 patients were in complete response (2 with negative immunofixation) 24 in partial response, 13 with other responses (6 with minor response, 5 had progressive disease, 1 stable disease, and 1 patient no evaluated). Median interval from diagnosis to transplant was 11 months (range 5–80). Conditioning regimen consisted of iv BU (3.2 mg/kg in a single daily dose, days −5 to −3) and MEL (140 mg/m2, day −2). In 39 patients this was the first ASCT and the remaining 6 had relapsed after a previous ASCT conditioned with MEL-based regimens.

Results: Median number of CD34+ cells administered was 2.9 x10^6/kg (range 1–5.8). Hematopoietic recovery was fast with median (range) time to 0.5 PMN and 20 platelets x10^9/L of 12 (11–46) and 13 (9–64) days, respectively. The toxicity profile was favourable. Particularly, no case of SOS, commonly seen with the oral BU, has been reported. Mucositis was the non-hematopoietic toxicity most frequently seen (42 cases). Other toxicities observed were uncommon (gastrointestinal toxicity, 6 cases, increase of liver enzymes, 3 cases and mild cardiac failure, 1 case). Fever was observed in 38 patients: fever of unknown origin in 21 cases, microbiologically documented infection in 13 cases and clinically documented infection in the remaining 4 cases. Only 2 patients (4%) died due to transplant-related toxicity (one patient to pneumonia, day 51; the other patient to sepsis to Acinetobacter baumani, day 54). With a median follow-up of 18 months, 41 patients had been evaluated for response: 19 are in complete response (9 with negative immunofixation), 16 in partial response, 2 with minor response, 2 had progressive disease, and 2 transplant-related deaths. The 18 months actuarial overall and progression-free survival rates are 95±3% and 70±10%, respectively. Interestingly, in 19 patients significant improvement response was observed after transplant.

Conclusions: These preliminary results show that iv BU-containing regimen for MM is associated with an acceptable toxicity profile and with a high anti-myeloma effect.
Detection of Herpesvirus and Adenovirus Infections in Patients Undergoing Stem Cell Transplantation: Looking Below the Tip of the Iceberg with the Microarray Technology.
Authors: Ditzen Anette , Stichling Markus , Mueller Rene , Rutzka Anke , Hille Kristine , Illmer Thomas , Bornhaeuser Martin , Ehninger Gerhard , Jacobs Enno , Ehricht Ralf , Rohayem Jacques
Published: October 2019

Infections with herpesvirus and adenovirus is a major cause of morbidity and mortality in patients undergoing stem cell transplantation (SCT). In those patients, the frequency of herpesvirus and adenovirus co-infections as well as their contribution to the infectious burden remain so far unclear. In this study, we have postulated that co-infections with herpesviruses and adenoviruses are frequent in the 100 days following SCT, contributing significantly to the infectious burden. To address this issue, 615 blood samples collected from patients undergoing SCT (n=35) were retrospectively tested by quantitative real-time PCR for the presence of HSV 1/2, VZV, EBV, CMV, HHV6 A/B, and adenovirus. Infections with a single virus were detected in 46.1% (283/615) of the samples, whereas co-infections with two or three viruses were detected in 9.9% (61/615) and 2.1% (13/615) of the samples, respectively. Interestingly, about 50% of the patients (17/35) displayed a co-infection with two or more viruses, with [CMV+HHV6 A/B], and [EBV + HHV6 A/B +adenovirus] being most frequently detected, respectively. Comparison of viral loads indicated that the infectious burden increases proportionally to the number of viruses detected. Moreover, a DNA-microarray allowing simultaneous detection of HSV 1/2, VZV, EBV, CMV, HHV6 A/B, and adenovirus was developed. The assay displayed a high diagnostic accuracy, being an attractive device for routine assessment of the infectious burden in immunocompromised patients after SCT.
Bacterial, Viral and Fungal Infections in Patients after Allogeneic Stem Cell or Bone Marrow Transplantation - A Comparison between Patients with Related and Patients with HLA-Matched Non-Related Stem Cell Donors.
Authors: Rieger Christina T. , Tischer Johanna , Ostermann Helmut
Published: October 2019

Bacterial, viral and fungal pathogens frequently cause severe, life-threatening infections in immunocompromised patients after allogeneic stem cell transplantation (SCT). We investigated whether patients with related stem cell donors (group A) developed infections less frequently than patients with HLA-matched, non-related donors (group B). Fifty-nine consecutive patients treated at our transplantation unit between April 2004 and January 2005 were included into the analysis. We documented demographic and clinical characteristics at baseline, treatment, clinical course, microbiological examinations, clinical and radiological signs of infection and mortality. Of the total 59 patients analyzed, 22 received stem cells from related and 37 from HLA-matched non-related donors. Both groups were well balanced regarding age and weight. 50% of the patients in group A and 60% in group B were male. Most frequent diagnoses were acute myeloid leukemia (30 of 59 patients [50.8%]; group A: 68.2%; group B: 40.5%), multiple myeloma (15.2%), acute lymphoblastic leukemia (11.9%) and chronic myeloid leukemia (10.2%). Bone marrow was more often the stem cell source in group A (45.5%/ 10 patients) than in group B (10.8%/ 4 patients), peripheral stem cell transplantation respectively was predominant in the unrelated group (86.5%/ 32 patients) versus the family donor group (54.5%/ 12 patients), cord blood was used as unrelated stem cell source in1 patient (2.7%). Clinically documented infections occurred in 6% in group A and in 14% in group B. Pulmonary infiltrates were observed more frequently in group A (11 patients/ 50%) than in group B (16 patients/ 43.2%). The predominant findings were atypical infiltrates (total 16 patients), followed by signs of fungal (total 7 patients) and bacterial pulmonary infiltration (total 4 patients). Microbiologically documented infections were detected in all patients. The average number of pathogens was equal in both groups. Detected pathogens were HHV-6 (48 patients), coagulase-negative Staphylocci (17 patients), EBV (14 patients) and CMV (11 patients). Three fungal infections were detected by microbiological approaches in group A (2 × Candida albicans, 1 × Pitysporum ovale) compared to nine fungal infections in group B (5 × Candida albicans, 1 × Candida glabrata, 1 × Candida parapsilosis, 2 × Geotrichum capitatum). Two years after transplantation, 55.9% of patients were alive (group A: 68.2%; group B: 48.6%). Patients with AML had a two-year survival of 50% (group A: 53.3%; group B: 46.7%). In our study, we observed no clear relation between frequency of infection and donor type, yet there was a trend towards more invasive fungal infections in the unrelated group (13% group A vs. 24% group B).
Bloodstream Infection after Reduced-Intensity Umbilical Cord Blood Transplantation Versus Other Reduced-Intensity Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Takagi Shinsuke , Ishiwata Kazuya , Tsuji Masanori , Yamamoto Hisashi , Kato Daisuke , Matsuhashi Yoshiko , Kusumi Eiji , Seo Sachiko , Matsuno Naofumi , Matsumura Tomoko , Uchida Naoyuki , Masuoka Kazuhiro , Wake Atsushi , Miyakoshi Shigesaburo , Yoneyama Akiko , Matsuzaki Michio , Taniguchi Shuichi
Published: October 2019

Blood stream infection (BSI) is a major cause of transplant-related mortality (TRM) following allogeneic hematopoietic stem cell transplantation, and to overcome it, reduced-intensity preparative regimens were developed in recent years. However, little information has been reported on BSI after reduced-intensity cord blood transplantation (RI-CBT). To clarify the characteristics of BSI after RI-CBT, we compared the incidence of microbiologically documented BSI before day 100 between RI-CBT and reduced-intensity non-cord blood allogeneic hematopoietic stem cell transplant (RI-non-CBT) recipients in Toranomon hospital, Japan. RI-non-CBT group includes related bone marrow (rBM), related peripheral blood stem cell (rPBSC) and unrelated bone marrow (uBM) transplantation. We retrospectively reviewed the first events of BSI in 211 consecutive adult patients between Jan 2004 and July 2006. One hundred and fifteen patients received RI-CBT and 96 patients received RI-non-CBT (4 from rBM, 34 from rPBSC and 58 from uBM). The median ages of patients in both groups were 55 years. All of the preparative regimens were fludarabine-based and prophylaxis against GVHD was tacrolimus alone in most of the RI-CBT recipients and combination of calcinurin inhibitor and short-term methotrexate in most of the RI-non-CBT recipients. The median time to achieve neutrophil engraftment was delayed in RI-CBT group (day 20 vs. day15). The cumulative incidence of engraftment at day 60 was 73.0% in RI-CBT group versus 90.6% in RI-non-CBT group. The cumulative incidence of BSI was 39.3% at day 100 and RI-CBT group tended to have more BSI compared to RI-non-CBT group (46.1% vs. 31.3%, p=0.0122), particularly at the early points after transplantation. Median day of positive culture for bacteremia was earlier (day 9 vs. day 14) in RI-CBT group. In spite of reduced-intensity preparative regimen, RI-CBT in adults is associated with higher rates of BSI at early time points after transplantation.
Birth Order Does Not Affect Outcome in HLA-Identical Sibling Transplantation.
Authors: Copelan Edward , Sobecks Ronald , Dean Robert , Rybicki Lisa , Brown Stacey , Kalaycio Matt , Pohlman Brad , Sweetenham John William , Andresen Steven , Bolwell Brian
Published: October 2019

Fetomaternal and transmaternal sibling microchimerism might result in tolerance of hematopoietic stem-cell donors to recipients who were born before them. A single clinical study comparing first-born recipients and first-born donors provided evidence that birth order can have a significant impact on survival and on the incidence of acute graft-versus-host disease (GVHD) and relapse in HLA-identical sibling transplantation. In order to determine whether birth order might exert a consistent, biologically important effect, we compared survival and the incidence of acute GVHD, chronic GVHD, and relapse in recipients of HLA-identical transplants from sibling donors older than themselves to recipients with younger sibling donors. From 11/84 through 11/06, 410 first HLA-identical sibling transplants were performed. Patients were categorized by whether the sibling donor was older (n=187) or younger (n=223) than the recipient. As expected, the two groups differed with respect to patient age (P<0.001). The groups were similar with respect to gender, diagnosis, interval from diagnosis to transplant, extent of prior treatment, preparative regimen, stem-cell source, donor to patient gender, and CD34+ cell dose. Outcomes were estimated using the Kaplan-Meier Method and compared between the birth order groups using the log rank test. The incidence of acute GVHD, ≥ grade II acute GVHD, chronic GVHD, extensive chronic GVHD, relapse, survival, and disease-free survival were similar (0.39≤P≤0.85) between the two groups. The incidence of acute GVHD and survival in the two groups is shown below:

Figure Figure Figure Figure

After adjustment for differences between the two groups, multivariable assessment of sibling birth order was not prognostic for survival (P=.32). Only older (P=0.029) and male donor/female recipient (P=.017) were independent prognostic adverse factors. In this large study of the HLA-identical sibling transplantation, no impact of birth order on the incidence of GVHD, relapse incidence, or survival was detected. Although (because of different methodologies) not directly contradictory to a previous study of birth order, the present results suggest that further study of the impact of birth order on outcome of HLA-identical sibling transplantation is needed. Suggestions that birth order be integrated into donor-selection algorithms or that matched unrelated donors might be preferred to high-risk HLA identical siblings are premature.
Rituximab Reduces the Incidence of Acute Graft Versus Host Disease and Delays CD3+CD4+ Lymphocyte Recovery.
Authors: Christopeit Maximilian , Schuette Vincent , Behre Gerhard
Published: October 2019

The monoclonal antibody Rituximab has been successfully used to treat steroidrefractory chronic graft versus host disease (cGvH) after allogeneic stem cell transplantation (SCT). We hypothesized that Rituximab might reduce the incidence of aGvH. We found that patients conditioned with a regimen containing Rituximab experienced significantly less aGvH when compared to a regimen without Rituximab (23.1% versus 66.7%, p = 0.032). Furthermore did we find that the recovery of CD3+CD4+ lymphocytes was delayed following administration of Rituximab. As expected, patients were devoid of CD19+CD20+ B lymphocytes after administration of Rituximab; interestingly, both the recovery of CD3+CD8+ lymphocytes and CD16+CD56+ NK cells did not differ between patients receiving Rituximab and Rituximab-naíve patients. Furthermore, our data revealed a significant correlation of the transplants CD34+ HSC content with the occurrence of aGvH (p = 0.031) but only a trend towards a correlation of CD3+ cells with the occurrence of aGvH (p = 0.274). Taken together, our data establish a mechanism of how Rituximab might reduce the incidence of aGvH next to depleting host and perhaps donor antigen presenting cells. It remains to be elucidated whether this effect translates to a graft versus tumor effect and to survival as well.
ASBMT Risk Group and CD 34+ Dose Predicted for the Development of aGVHD in Allogeneic MRD Transplants When the Donors Who Received a Combined Mobilization and In Vivo TCD Regimen Using rhg-CFS and Dexamethasone.
Authors: Ashraf Khaleel K. , Tilden Arabella B. , Li Yufeng , Gupta Sameer , Lamb Lawrence S. , Salzman Donna E. , Lopez Richard , Vaughan William P.
Published: October 2019

We looked at factors affecting the development of acute and chronic GVHD in a cohort of 118 patients who underwent allogeneic matched sibling transplantation between 09/1998 to 05/2006 at the UAB BMT program. Donors were mobilized with rhg-CSF (10–16mcg/kg/day x4) and received dexamethasone 10mg/m2/day x3 days to effect in vivo T cell depletion (TCD). Total CD3 and CD34 cell dose were divided in to 4 quartiles (See table below). We then analyzed the influence of CD3+ and CD34+ dose quartiles on aGVHD, cGVHD, engraftment, TRM and relapse. (see table) Acute GVHD: Univariate Logistic regression indicated that the development of aGVHD is negatively associated with quartiles (q25–q50) interval of CD3 dose (p=0.0418). However this relationship was not independent of risk factor and CD34 dose. After using Multivariable Logistic regression analysis with adjustments for age, race and sex, we found that ASBMT risk factor and CD34 quartiles dose are strong predictors of aGVHD: patients who had high risk are about three times more likely to have aGVHD (OR=2.829, 95% CI 1.142–7.020, p=0.0250). The higher the CD34 dose also predisposed to higher incidence of aGVHD (p=0.0432). Median Survival: Patients who received the CD3 (P=0.00141) and CD34 (P=0.0270) in the q25–q50 quartile did better with improved median survival. Relapse and CD 3 quartiles: Using the - 2 log (LR) test shows the relapse free interval was longer if the CD3 dose is between q25 and q50 (p=0.0058); there was no relation between CD34 and relapse. TRM: Not associated with CD3 or CD 34 dose. WBC engraftment: Multivariable analysis indicated that the relationship is mainly an age effect. After adjusted for risk factor, GVHD status, CD3 or CD34 dose, the engraftment day is still significantly related to age at BMT (p=0.0252). No associations are observed for the relationship between engraftment day and GVHD risk. cGVHD: Neither CD34 dose (p=0.5339) nor CD3 (p=0.4209) dose were statistically associated with incidence and severity of cGVHD. To summarize, higher CD34 dose and higher risk ASBMT category predicted for higher incidence of aGVHD; neither CD3 nor CD 34 dose predicted for the development of cGVHD. Age was the single most important predictor of WBC engraftment.

CD3+ and CD34 Quartiles Quartile <25 25–50 50–75 >75 CD3 X10E8 <2.5 2.53–3.60 3.60–5.30 >5.30 CD34 X10E6 <4.92 4.92–7.79 7.79–11.2 >11.2
Cyclosporine Versus Cyclosporine/Mycophenolate Mofetil for GVHD Prophylaxis after Matched Related and Unrelated Minimal Intensity Allotransplantation.
Authors: Nelson Robert P. , Schwartz Jennifer E. , Yu Menggang , Robertson Michael , Haut Paul R. , Wood Lisa L. , Cox Elizabeth A. , Cornetta Kenneth , Abonour Rafat
Published: October 2019

The purpose of this study was to examine the efficacy of cyclosporine (CsA) alone versus CsA/mycophenolate mofetil (MMF) as GVHD prophylaxis in older patients who received a minimally-intensive allogeneic hematopoietic cell transplant (MIHCT) for the treatment of hematological malignancies (AML=26, MDS=15, NHL=8, CLL=5, MM=3, HD=4, CML=1, ALL=1). Patients (median age, 55 years; 41 males, 22 females) received HLA-matched related (n=33) or unrelated (n=30) peripheral blood hematopoietic cell infusions following immunosuppressive dosages of cyclophosphamide and fludarabine (Childs R, et al. Blood 94; 1999). The initial 28 consecutive patients received monitored CsA from day −1 through day +180 (if no GVHD). The next 35 subjects received CsA/MMF: CsA, day −1 through day +180 and MMF, 1000 mg twice daily, day +1 through day +60 (if no GVHD, then a taper). Patients were followed until death or at least 7 months. There were no differences between recipients of MRD and MUD transplants with respect to sex, age of the recipient, disease status, ABO matching, recipient CMV status or number of CMV-negative donor/recipient pairs. MUD donors were significantly younger and more likely to be CMV-negative. There was no difference in disease distribution between those who recieved CsA and those who recieved CsA/MMF prophylaxis. Follow-up ranged from 25–2241 days (median, 461 days for the CSA group; 449 days for the CSA/MMF group, 1290 days for survivors). All patients engrafted without growth factor support and no subjects experienced sinusoidal obstruction syndrome, mucositis or post-transplant lymphoproliferative disease. Thirty and 100-day transplant-related mortality for the cohort was 1.6% and 15.9% respectively. The incidence of Grade 2–4 aGVHD (n=63) was 49.2% (CsA, 57.1%, CsA/MMF, 42.9%, p=0.315). Grade 3 or 4 aGVHD occurred in 30% (CsA, 32.1%, CsA/MMF, 28.6%, p=0.788). Chronic extensive GVHD occurred in 53.8% (CsA, 61.5%, CsA/MMF, 46.2%, p=0.404) of at risk (survival greater than 100 days) recipients. The incidence of acute or chronic GVHD was not statistically different for MRD versus MUD recipients for either prophylaxis group (p=0.32 and 0.23, respectively). One-year, two-year and three-year overall Kaplan-Meier survival analyses were estimated to be 60.7%, 42.9% (CsA group) and 42.9%and 60%, 45.2% and 32.8% (CsA/MMF group, 0.708). Twenty-seven (42.9%) of the entire cohort are alive at a median of 1290 days post-transplant [CSA, 12/28, (42.9%); CsA/MMF, 15/35, (42.9%)]. Grade 3 or 4 aGVHD was associated with poor overall survival (p=0.0001) for the cohort. These findings provide evidence that MMF does not seem to provide substantial GVHD preventive or overall survival benefit when added to CsA after matched related/unrelated cyclophosphamide/fludarabine MIHCT in older patients with hematological malignancies.
Hematopoietic Stem Cell Gene Therapy for Wiskott-Aldrich Syndrome.
Authors: Boztug Kaan , Dewey Ricardo A. , Diez Ines Avedillo , Manfred Schmidt , Kerstin Schwarzwaelder , Jana Diestelhorst , Sonja Naundorf , Klaus Kuehlke , Irina Kondratenko , Laszlo Marodi , von Kalle Christof , Welte Karl , Klein Christoph
Published: October 2019

Wiskott Aldrich Syndrome (WAS) is a fatal primary immunodeficiency disorder caused by mutations in the cytoskeletal adaptor molecule WASP. We here report on our preliminary experience in two patients undergoing hematopoietic stem cell gene therapy upon retroviral WASP gene transfer. Both patients suffered from severe WAS and were treated with 8x10e6 and 7x10e6 /kg body weight transduced CD34 cells, respectively. Follow-up time is 9 and 10 months. Both patients show evidence of gene marking and WAS protein expression in multiple hematopoietic lineages. In particular, WASP-transgenic T and NK cells show a relative increase over time (up to 70% WASP-expressing cells). Both patients show a polyclonal pattern of hematopoiesis, as determined by LAM-PCR. No morphological or cytogenetic abnormalities were observed in the bone marrow. While one patient continues to suffer from autoimmune cytopenia, the other patient has had a marked clinical benefit. This interim analysis documents that hematopoietic gene therapy for WAS is feasible without undue short term toxicity. A longer follow-up period will be required to comprehensively evaluate the clinical and molecular outcome in these patients and to monitor for potential unwanted side-effects.