bluuebunny/crossref_metadata_2025 huggingface dataset:
Low Dose Thalidomide for the Treatment of Multiple Myeloma.
Authors: Bittencourt Rosane , Almeida Andreia D. , Friederich Joao R. , Fernande Flavo , Fogliatto Laura , Paz Alessandra , Astigarraga Claudia , Leugeur Denise , Silla Lucia
Published: April 2020

In the last two decades, we have seen a radical change in the therapy and prognosis of Multiple Myeloma (MM). Recent research on the role of the bone marrow microenvironment as integrant part of the biology of MM and the possible therapeutic interference at this level are leading to control and regression of the malignant plasma cell clone with a recognized clinical impact. Thalidomide, an old drug thought to interfere on the bone marrow microenvironment, is active not only for the treatment of refractory patients but also, as was recently shown, for induction and/or remission maintenance therapy. Most of the side effects (somnolence, constipation, fatigue, tremor, bradycardia, edema, and neuropathy) can be managed by reducing the dose and most patients need a dose reduction. The appropriate dose of thalidomide in myeloma is unknown. We treated thirty-five patients with MM with low-dose thalidomide (200mg or less). Twenty four patients were on maintenance therapy (13 after bone marrow transplantation, and 11 after chemotherapy with VCAP/VMCP); 5 patients were treated after relapse, 4 with refractory disease and 2 for remission induction as a first line therapy. Patients were treated at the Hematology and Bone Marrow Transplantation Service of the Hospital de Clínicas de Porto Alegre, RS, Brazil, from march/2001 to December/2003. The response as measured by hemoglobin level, immunoglobulin (M component) or urinary light chain concentration and bone marrow examination was evaluated before, 3, 6, and 12 months after the beginning of thalidomide.

Results: All patients are alive and well. Fifty one percent are on 100mg schedule; of the patients on maintenance therapy 90% are on complete remission or on a sustained plateau. A Wilcoxon ranks test for the immunoglobulin level before and after 6 months for the entire group showed p=0.001 (25–75%).

Conclusion: Low dose thalidomide is tolerable and effective.
Efficacy of Single-Agent Bortezomib Versus Thalidomide in Patients with Relapsed or Refractory Multiple Myeloma: A Systematic Review.
Authors: Prince Miles , Adena Michael , Smith Dell Kingsford , Hertel Judy
Published: April 2020

Aim: To perform a systematic review of the efficacy of monotherapy with bortezomib versus thalidomide in patients with relapsed or refractory multiple myeloma.

Methods: Published English literature from 1966 to June 2005 (MEDLINE, EMBASE, Cochrane library), publication reference lists, Janssen-Cilag Pty Ltd data-on-file, and abstracts from recent multiple myeloma conferences were reviewed. Prospective studies containing at least a single arm of any treatment group with n ≥ 30 and using continuing or variable thalidomide dosing were included. Studies adding dexamethasone for non-responders were excluded. Outcomes were analysed on an intent-to-treat basis. Statistical pooling was performed where possible for the following outcome measures: primary outcome of response rate, defined by a serum M-protein reduction ≥50% (A) and strict (e.g. EBMT) criteria (B), and for the secondary outcomes of overall survival and progression-free survival.

Results: One bortezomib (n=333, APEX, NEJM2005, 352; 2487–98) and 15 thalidomide (n=1007) studies were included. Patient baseline characteristics including age, gender, IgG:IgA, disease duration and β2M were well matched, except that 48% of bortezomib patients had received prior thalidomide. On an intent-to-treat basis, the overall estimate for response rate (A) was 53% for patients receiving bortezomib versus 32% for thalidomide (p<0.001, n=10 studies). For response rate (B) the estimate was 36% for patients receiving bortezomib versus 22% for thalidomide (p<0.001, n=4 studies). One-year survival was 81% for patients receiving bortezomib versus 67% for thalidomide (p<0.001, n=6 studies). Due to differences in disease monitoring and definitions of progression, it was not possible to compare results for progression-free survival.

Conclusion: In patients with relapsed or refractory multiple myeloma, bortezomib achieved significantly higher response rates and longer one-year survival than thalidomide, despite 48% of bortezomib-treated patients having received prior thalidomide.
Combination Chemotherapy Using Cyclophosphamide and High Dose Dexamethasone with or without Radiotherapy for the Treatment of Multiple Myeloma.
Authors: Kim Chul Soo , Lee Moon Hee , Kim Inho , Moon Yeonsook , Nahm Chung Hyun , Kim Hun Chung , Kim Woo Cheol , Loh John J.K.
Published: April 2020

Forty two people (men 22, women 20) diagnosed as Durie-Salmon stage IIIA or B multiple myeloma were treated with a combination chemotherapy consisting of cyclophosphamide at 400mg and dexamethasone at 40mg (CHD) regardless of body surface for 4 or 5 consecutive days every 3 or 4 weeks from August 1996 through July 2005. The treatment was repeated as long as the patient could tolerate or until disease progressed. The chemotherapy was given to 36 chemo-naïve patients as primary therapy and to 6 patients exposed to VAD (vincristine, doxorubicin, dexamethasone) as salvage therapy. Palliative loco-regional radiotherapy was also given to 20 of 44 patients. A total of 433 cycles (mean 10.3, range 2~28 per individual) of chemotherapy were given and the response evaluation was made in accordance to the criteria of Dr. Blade. SD (stable disease) was defined as reduction or increase of myeloma protein within 25%. Six of 36 chemo-naïve patients who did not respond to CHD received salvage therapy consisting of VAD or MP (melphalan plus prednisone). Eleven patients (26%) attained CR (complete remission) at a median of 7 cycles (range 4~10), 15 (36%) achieved PR (partial remission) at a median of 3 cycles (range 2~16), 12 (28%) stayed at SD, and 4 patients (10%) showed PD (progressive disease). The measurement of survival duration was started at the beginning of the CHD chemotherapy. The measurement was censored in 6 patients at the time of stem cell transplant, 5 autologous (one was disease free at 108 months after transplant), and 1 allogeneic. On a mean follow-up period of 15.1 months (range 3~59), 15 people remained alive at 3, 3, 3, 4, 6, 6, 8, 10, 14, 15, 16, 16, 16, 26, and 37 months. The mean response duration and OS (overall survival) of the patients who entered CR were 7 (range 2~18) and 31.3 (range 6+~59) months respectively. The correspondent durations of the patients in PR were 7.9 (range 2+~28) and 15 (range 3+~37+). The corresponding durations of the patients in SD were 8.3 (range 2~26) and 13.5 (3+~48). The mean OS duration of the patients in PD was 5.3 (range 3~8). The difference in OS durations between patients in PR and those in SD was not statistically meaningful. Five of 6 patients exposed to VAD responded to CHD, whereas one of 5 patients exposed to CHD did to VAD. Two of 4 patient exposed to CHD responded to MP. Adverse effect was minimal with non-fatal events including 5 febrile episodes of unknown origin, 3 cases of pneumonia, 2 cases of urinary tract infection, 1 case of peritonitis, 1 case of herpes zoster, and 1 case of intractable nausea during the 433 chemotherapy courses. There were 2 cases of fatal septicemia and 1 case of fatal pneumonia during the period. CHD combination chemotherapy seems an active and less toxic regimen in the treatment of multiple myeloma.
Low Dose Clofarabine Causes Significant Hematologic Toxicity in a Phase II Study of Relapsed or Refractory Multiple Myeloma.
Authors: Uy Geoffrey L. , Ruddell Alisa M. , Devine Steven M. , Tomasson Michael H. , DiPersio John F. , Vij Ravi
Published: April 2020

Clofarabine (Clolar™) is a purine nucleoside analog recently approved for the treatment of refractory pediatric ALL. The compound is structurally related to fludarabine and cladribine and has demonstrated activity in refractory adult and pediatric acute leukemias. In phase I studies of adults with clofarabine administered on days 1–5 of a 28 day cycle, the dose limiting toxicity (DLT) among patients with solid tumors and lymphoproliferative disorders was myelosuppression with a maximum tolerated dose (MTD) of 2 mg/m2 and 4 mg/m2 respectively. In contrast, for patients with acute leukemia the DLT was hepatotoxicity with the MTD of 40 mg/m2. Based on in vitro toxicity against myeloma cell lines and its activity in lymphoid malignancies, we initiated a phase II open-label study of low dose clofarabine in patients with relapsed or refractory multiple myeloma. This trial was designed using an optimal two-stage design (alpha =0.05 and power=0.9) with response rate as the primary endpoint. Nine patients will be enrolled in stage I. If one or more responses are observed, an additional 8 patients will be enrolled in stage II. Clofarabine 4mg/m2/day was given on days 1–5 of a 28 day cycle for up to 12 cycles or until disease progression. Eight patients have been enrolled to date. Baseline characteristics are as follows: median age 49 years (range 43–67), IgG (6 patients), IgA (1), light chain only (1). All had Stage IIIA disease and had previously undergone autologous transplants with a median of 3.5 (range 2–6) prior chemotherapy regimens. Hematologic toxicities were common in the first 6 patients treated. One patient developed Grade 3 thrombocytopenia (≤ 75,000/mm3), but all six had a >50% decrease in their platelet counts with recovery by day 28 of therapy. All six patients also developed neutropenia, Grade 3 (ANC ≤ 1000/mm3) in 4 patients and Grade 4 (ANC ≤ 500/mm3) in 2 patients. Interestingly, the timing of cytopenias was discordant between platelet and neutrophil counts. While platelet counts nadir on day 15, neutrophil counts nadir at day 25–28 of chemotherapy as platelet recovery is occurring. Dose delays of 1 to 2 weeks and dose reduction to 2 mg/m2/day have been required of all patients. Low dose clofarabine 4 mg/m2 x 5 days results in significant marrow suppression with a dose limiting toxicity of neutropenia. Alternative dosing schedules should be studied in future trials with clofarabine. Response data and toxicities for stage I will be complete and updated at the time of presentation.
In Vitro Generation of Highly-Purified Functional Invariant NKT Cells: A Strategy for Immunotherapy in Multiple Myeloma.
Authors: Song Weihua , van der Vliet Hans J.J. , Wang Ruojie , Prabhala Rao , Podar Klaus , Tai Yu-Tzu , Catley Laurence , Shammas Masood , Anderson Kenneth C. , Balk Steven P. , Exley Mark A. , Munshi Nikhil C.
Published: April 2020

Invariant NKT (iNKT) cells are characterized by the expression of invariant T-cell receptor encoded by Vα24-JαQ and NK receptors. iNKT cells recognize glycolipid antigens with CD1d restriction, and play an important immunoregulatory role in innate immunity. Through the production of IFN-γ, iNKT cells can contribute to immune surveillance in malignancy. However, in progressive multiple myeloma (MM), as well as in other advanced cancers, iNKT cells have a marked deficiency of ligand-dependent IFN-γ production. Thus, the development of effective iNKT cells is a novel strategy for the immunotherapy of MM. In this study, we report the establishment of highly purified primary iNKT cell lines from healthy donors and MM patients. iNKT cells derived from peripheral blood or bone marrow mononuclear cells were enriched with anti-TCRVα 24 mAb or anti-6B11 mAb and further expanded by several rounds of stimulation with α-GalCer-loaded dendritic cells. Phenotype analysis confirmed 95% purity in expanded iNKT cell lines. No significant phenotypic difference was observed in iNKT cells between healthy donors and MM patients. Most of iNKT cell lines are CD4+ cell lines (CD4+ cells > 90%, with less than 2% were CD8+ cells). Majority of iNKT cells expressed CD161 and CD28, whereas CD56 expression was at very low level. Following anti-CD3 or α-GalCer-loaded dendritic cells stimulation, iNKT cells showed strong proliferative activity as measured by 3H-TdR incorporation assay. These cells expressed high level of CD25 and produced high levels of IFN-γ as well as IL-2 as measured by ELISA assay. These results provide the preclinical feasibility of producing large volume of highly-enriched functional iNKT cells from myeloma patients and rationale to clinically evaluate the efficacy of adoptive transfer of iNKT cells in MM. Based on these results a clinical study is under development.
Thalidomide and Dexamethasone in Newly Diagnosed Multiple Myeloma.
Authors: McHugh Johnny , Keane Colm , Otridge Brian W. , Thornton Patrick , O’Gorman Peter
Published: April 2020

PURPOSE: Thalidomide and dexamethasone has been shown to have excellent antimyeloma activity in both relapsed and newly diagnosed patients. This combination offers a potential oral, less toxic alternative to standard regimens. This retrospective study evaluates the activity of the combination of thalidomide and dexamethasone in patients with newly diagnosed multiple myeloma.

METHODS: Thirty-two patients with newly diagnosed multiple myeloma were treated with thalidomide and dexamethasone. The median age of the patients was 69yrs (range 46 to 83). The median serum paraprotein level was 38g/l (range 2 to 76g/l). The median bone marrow plasma cell percentage was 50% (range 10% to 99%) The first sixteen patients were commenced on thalidomide at 200mg od with the dose increased by 200mg every two weeks if tolerated to a maximum of 800mg od. The most recent sixteen patients were commenced on thalidomide at 100mg od with the dose increased to a maximum of 200mg if tolerated. All patients received dexamethasone 10mg qds 4-days-on/4-days-off for the first four weeks and for a four day pulse every four weeks subsequently.

RESULTS: Twenty-two of the thirty-two patients (69%) showed a greater than 50% reduction in serum paraprotein and/or urinary Bence Jones protein (BJP) from baseline. From the lower thalidomide dose group thirteen out of sixteen patients(81%) showed a greater than 50% reduction in serum paraprotein and/or urinary BJP from baseline compared with nine out of sixteen (56%) in the higher thalidomide dose group. Thirteen patients (40%) showed a reduction in serum paraprotein and/or urinary BJP of greater than 90%, eight from the lower thalidomide dose group and five from the higher thalidomide dose group. Six of the patients failed to complete one month of treatment due to toxicities. They were all from the higher thalidomide dose group. Toxicities included drowsiness (eleven patients), constipation (six patients), rash (four patients) and peripheral neuropathy (four patients). None of the patients have suffered a venous thromboembolic event. Fewer toxicities were noted in the lower dose thalidomide group.

CONCLUSION: The combination of thalidomide and dexamethasone is safe and effective in the treatment of newly diagnosed multiple myeloma. There is less toxicity and no loss of efficacy with lower doses of thalidomide.
Distinct Gene Expression Pattern of CD34+ Stem and Progenitor Cells Mobilized by Pegfilgrastim after Cytotoxic Therapy in Multiple Myeloma in Comparison to G-CSF.
Authors: Bruns Ingmar , Steidl Ulrich , Kobbe Guido , Fenk Roland , Kliszewski Slawomir , Pechtel Sabrina , Haas Rainer , Kronenwett Ralf
Published: April 2020

Background: Current regimens for peripheral blood stem cell (PBSC) mobilization in patients with multiple myeloma are based on daily subcutaneous injections of G-CSF starting shortly after cytotoxic therapy. Recently a polyethylenglycole (PEG)-conjugated G-CSF (pegfilgrastim) has been introduced which has a substantially longer half-life than the original formula. Here, we compared the molecular phenotypes of CD34+ stem and progenitor cells mobilized by G-CSF with those mobilized by pegfilgrastim.

Study design and Methods: We examined immunomagnetically enriched CD34+ cells from leukapheresis products of 8 patients who received G-CSF and of 8 patients who were given pegfilgrastim using Affymetrix HG Focus GeneChips covering 8793 genes. The statistical scripting language ‘R’ was used for data analysis. Significantly differentially expressed genes were identified with the Significance Analysis of Microarrays (SAM) algorithm.

Results: Comparing CD34+ cells mobilized by G-CSF with pegfilgrastim-mobilized CD34+ cells 108 genes were differentially expressed (fold change 1.25 – 14.0, q- value 2.45–14.44%). 38 genes had a higher and 70 genes had a lower expression in CD34+ cells mobilized by G-CSF.

We found upregulation of genes characteristic for erythropoietic differentiation including haemoglobin chains and Erythroid Kruppel-like factor in G-CSF-mobilized CD34+ cells. Utilizing clonogenic assays we were able to functionally corroborate this finding as G-CSF-mobilized cells gave rise to a significantly higher number of burst-forming units erythroid (BFU-E) as compared to colony forming units granulocyte-macrophage (CFU-GM) (p=0.016). Cell cycle regulating genes were differentially expressed as well. Genes encoding for proteins that cause cell cycle arrest including human HTm4 were upregulated in G-CSF-mobilized cells, as opposed to an upregulation of cell cycle-promoting genes including Cyclin D2 and Hepatocyte Leukemia Factor (HLF) in pegfilgrastim-mobilized cells.

Moreover in pegfilgrastim-mobilized CD34+ cells we saw an upregulation of multiple genes involved in cellular immunogenicity like MHC class I and II antigens and genes encoding for proteins playing a role in antigen presentation.

Conclusion: Unconjugated G-CSF seems to be associated with an increased mobilisation of erythroid progenitors or an induction of erythropoiesis. Pegfilgrastim might result in mobilization of more immunogenic CD34+ cells. Unconjugated G-CSF and pegfilgrastim both seem to have an effect on cell cycle. Unconjugated G-CSF might rather induce cell cycle arrest and pegfilgrastim seems to lead to an increase of the cell cycle activity. This may be due to potentially different effects of continuously high serum levels of G-CSF maintained by pegfilgrastim and the pulsatile daily G-CSF injections on CD34+ cells.
Peripheral Blood Stem Cell Mobilization by G-CSF Alone and Engraftment Kinetics Following Autologous Transplantation in Children and Adolescents with Solid Tumor.
Authors: Watanabe Hiroyoshi , Watanabe Tsutomu , Suzuya Hiroko , Wakata Yoshifumi , Onishi Toshihiro , Nakagawa Ryuji , Okamoto Yasuhiro , Abe Takanori , Kawano Yoshifumi , Kagami Shoji , Takaue Yoichi
Published: April 2020

The purpose of this study was to examine the yield of PBSC, mobilized with G-CSF alone, and engraftment kinetics after autologous transplantation in pediatric cancer patients. In 55 patients (median age; 7 years, range 0–21) with various pediatric and adolescent solid tumors, PBSC were mobilized with G-CSF alone, and the yields of PBSC and engraftment following autologous PBSCT were evaluated retrospectively. Patients were categorized according to prior treatment; patients who had received less than 4 or 4 cycles of chemotherapy with/without local irradiation (Group 1: N= 21), patients who received more than 4 cycles of chemotherapy or 3 or more cycles of chemotherapy with extended irradiation (Group 2: N= 23), and patients who received high-dose chemotherapy with stem cell support (Group 3: N= 11). Ten microgram per kg of G-CSF was injected subcutaneously for mobilization when patients showed no influence of previous chemotherapy, and administration was continued for five days. The peaks of CD34+ cells and CFU-GM were observed on day 5 of G-CSF administration essentially in all patients. Aphereses were performed on days 5 and 6 of G-CSF treatment. Mobilization failure was observed in four patients in all groups. Compared with the results in patients mobilized by chemotherapy plus G-CSF (N=18), the progenitor cell yields were lower in those mobilized with G-CSF alone. However, there were no significant differences in WBC engraftment speed compared to the chemotherapy plus G-CSF mobilization group, although platelet recovery was delayed in patients with G-CSF alone, especially in patients in Group 3. The median time taken for ANC and platelet counts to reach 500 and 20K was 12 days (range 8–28) and 15 days (8–55), respectively, in all patients who were mobilized by G-CSF alone except for patients with progressive disease. In summary, mobilization with G-CSF alone can mobilize a sufficient number of CD34+ cells for successful autografting and sustained hematological reconstitution in pediatric patients with cancer, even in heavily pre-treated patients. Mobilization with G-CSF alone might offer some advantages, such as ease of determining a collection schedule without a daily determination of CD34+ cells in the blood, and the avoidance of neutropenic fever and additional transfusion.
Myeloid Progenitors Protect Against Radiation-Induced Intestinal Injury.
Authors: Brown Janice (Wes) , Shashidhar Sumana , Habtezion Aida , Burns-Guydish Stacy M. , Wang Sophia Y. , Domen Jos , Fong Tim , Contag Christopher H. , Omary M. Bishr
Published: April 2020

Gastrointestinal mucositis is a serious complication of intensive chemotherapy and radiotherapy (XRT) resulting in severe pain, impaired nutrition and an increased susceptibility to infection. We have previously reported that a single infusion of common myeloid and granulocyte-monocyte progenitors (CMP/GMP, c-kit+Sca-1+ CD34+ CD16/32+Lin-Thy1-) protects mice against lethal challenge with bacterial or fungal pathogens following chemotherapy or myeloablative radiation. Additionally, this protection is not HLA-restricted and correlates with the tissue myeloid content. We now present our findings on the effects of CMP/GMP infusion against radiation-induced injury in the gastrointestinal mucosa and liver. Following 950 cGy irradiation, Balb/c mice (H2d) received infusions of one of the following: [1] hematopoietic stem cells (HSC, c-kit+Thy1loLin-Sca-1+); [2] freshly isolated 10,000 CMP/20,000 GMP + HSC; or [3] 500,000 mixed myeloid progenitors (MP) expanded in vitro from HSC. In all cases, the HSC were MHC-matched (H2d), and CMP/GMP were MHC mis-matched (H2b). A cohort of mice served as XRT controls. Mice were sacrificed at d+6 or d+9 post-transplantation. Gross examination of the gastrointestinal tissues showed that mice in the XRT and HSC groups had severe hemorrhagic diarrhea in contrast to the mild to moderate diarrhea observed in the groups receiving either freshly isolated CMP/GMP or the expanded MP. At both time points, histologic examination of gastrointestinal tissue from the XRT controls revealed sub-mucosal bleeding in gastric and intestinal tissue; necrosis and ulceration in the stomach; crowding of villus-proximal epithelial cells in the small intestine; ulcerative areas and small foci of inflammation in the colon; and necrosis, portal tract damage, and dilated sinusoids/central veins in the liver. In comparison, the histologic changes were significantly less dramatic in the stomachs, colons, and livers of the mice that received either freshly isolated CMP/GMP or in vitro expanded MP. In a parallel experiment, irradiated mice were transplanted with bioluminescent CMP/GMP from transgenic FVB.luc+ mice (H2q) constitutively expressing firefly luciferase. Imaging of the live mice as well as harvested organs at d+6 and d+9 post-transplantation indicated the presence of CMP/GMP in the stomach and intestine, correlating with sites of attenuated XRT damage in our histologic analysis. These findings support the observation that repletion of the myeloid progenitor pool enhances the recovery of radiation-injured mucosa, and raises the possibility that similar applications may be considered in the human clinical setting.
Therapeutic Blockade of ICOS:ICOSL Interaction Alleviates Acute Graft Versus Host Disease.
Authors: Richter Guenther , Mollweide Andreas , Uhlmann Esther , Burdach Stefan
Published: April 2020

Inducible costimulator (ICOS) interaction with its ligand (ICOSL) is involved in several T cell effector functions. The abundant expression of ICOSL in a variety of target tissues of acute graft versus host disease (GVHD) provided the rationale to investigate its role in acute GVHD development. C57BL/6 mice were lethally irradiated and reconstituted with allogeneic spleen cells in the absence or presence of ICOSL-blocking reagents. Mice reconstituted with allogeneic spleen cells experienced 12–16% weight loss around day 4 after transplantation and died untreated of acute GVHD within 7–10 days after transplantation. Mice treated from day 3 after transplantation with an anti-ICOSL mAb gained weight again and survived for additional 18 days, although the treatment was already stopped at day 11 after transplantation. Such mice revealed less T cells in spleen at day 4 after transplantation with reduced effector activity. In contrast, the anti-ICOSL treatment starting from day 0 did not prevent GVHD. The difference between therapeutic (day 3) and prophylactic (day 0) anti-ICOSL treatment was independent of CD25+CD4+ regulatory T cells since their depletion did not abrogate the therapeutic effect of ICOSL blockade. Similarly, depletion of NK cells did not improve prophylactic anti-ICOSL treatment. However, our results clearly show that delayed, i.e. therapeutic blockade of ICOS:ICOSL interaction can interfere with acute GVHD development and alleviates disease significantly in a polyclonal T cell setting.
Use of Dendritic Cells Versus Peripheral Blood Lymphocytes To Segregate Alloreactive and Regulatory T Cells during Allogeneic Transplantation.
Authors: Katz Tami , Avivi Irit , Stroopinski Dina , Oliven Anna , Zuckerman Tsila , Karitzky Sandra , Moeneclaey Guy , Vasir Baldev , Rosenblatt Jacalyn , Lenahan Corrie , Rowe Jacob M. , Avigan David
Published: April 2020

A fundamental research objective in allogeneic transplantation is the selective depletion of alloreactive T cell populations that are responsible for GVHD while preserving mediators of anti-tumor immunity and immune reconstitution. Alloreactive T cells may be identified by their expression of activation markers, such as CD25, following stimulation by recipient antigen presenting cells. However, CD25 expression is also present on regulatory T cells, which suppress alloreactivity through the expression of inhibitory ligands such as CTLA-4 and GITR. In an effort to develop an optimal strategy to segregate these populations, we have examined the effect of conditions of allogeneic stimulation on the balance of alloreactive and regulatory T cell populations. Stimulation of lymphocytes with irradiated peripheral blood mononuclear cells results in a gradual increase in the percentage of CD4/CD25+ cells over 7 days of culture. Mean percentage of CD4/CD25 at day 3 and 6 was 3% and 11%, respectively. On day 3 of stimulation, these cells demonstrate a characteristic activated phenotype with low intensity of CD25 expression and absence of GITR and CTLA-4 expression. Following isolation by magnetic bead separation, CD25+ cells demonstrated high levels of proliferation in response to restimulation with allogeneic PBMC. In contrast, minimal proliferation was observed following stimulation of the CD25- fraction. On day 6 of culture increased intensity of CD25 expression was observed in a fraction of the stimulated population. A majority of this population now expressed GITR and CTLA-4, consistent with a regulatory phenotype. Lymphocytes stimulated with mature DC demonstrated a markedly different pattern. A marked rise in CD4+/CD25+ cells with high levels of CD25 expression was noted early in the culture period; Mean CD4+/CD25+ cells on day 3 of culture were 9%. These cells were uniformly positive for CTLA-4 and GITR. A further increase in the percentage of cells demonstrating a regulatory phenotype was noted on day 6 of culture. We have demonstrated that the balance of activated and regulatory cells is determined by the nature of the antigen presenting cell and the duration of T cell stimulation. Based on these findings, we plan to conduct a clinical trial in which patients will undergo allogeneic transplantation following the selective removal of alloreactive T cell populations.
CD34 Positive Cells Mobilized with Chemotherapy Plus Recombinant Human Growth Hormone (rhGH) and Recombinant Human Granulocyte Colony-Stimulating Factor (rhG-CSF) Allows Sustained Engraftment in Previously Non-Mobilizer Patients.
Authors: Almici Camillo , Verardi Rosanna , Ferremi Piero , Re Alessandro , Cattaneo Chiara , Ferrari Emilio , Braga Simona , Rossi Giuseppe , Marini Mirella
Published: April 2020

The availability of adequate amounts of peripheral blood progenitor cells (PBPC) is a prerequisite for the feasibility of high-dose therapy program in patients with neoplastic diseases. Due to prior chemio-radiotherapy or disease related factors, 20–30% of patients fails to mobilize sufficient amounts of PBPC. Therefore, non-mobilizer patients cannot complete their therapeutic program. The aims of the present study were: 1) to evaluate the combination rhGH+rhG-CSF after chemiotherapy as a mobilization regimen in previously non-mobilizers patients; 2) to assess safety and tolerability of rhGH+rhG-CSF treatment; 3) to evaluate hematopoietic recovery after reinfusion of rhGH+rhG-CSF mobilized PBPC. After informed consent, three patients (2 male, 1 female), that failed a previous mobilization cycle, were remobilized with chemotherapy plus rhGH (100μg/Kg/d, sc) and rhG-CSG (5μg/Kg/d, sc).

Case Disease TNC (x 108/Kg) CD34+ (x 106/Kg) CFC (x 106/Kg) LTC-IC (x 104/Kg) 1 LNH 8.5 7.6 5.68 15.9 2 LNH 18 6.2 3.57 5.0 3 MM 15 1.3 1.26 12.0

No specific side effects were noticed due to rhGH treatment. Reinfusionof PBPC after myeloablative therapy resulted in hematopoietic recovery with 16, 9, 11 days respectively to reach an absolute neutrophil count ≥ 500/μl and 24, 15, 15 days respectively to reach a platelet count ≥ 50000/μl. Hematopoietic engraftment is complete and sustained with a follow-up of 18, 11 and 9 months, respectively.

In conclusion our data confirm that the addition of rhGH to rhG-CSF allows mobilization and collection of PBPC in previously non-mobilizer patients, and therefore the programmed high-dose therapy program can be satisfactorily completed, with sustained hematopoietic recovery.
Merit of Initiating Peripheral Blood Stem Cell (PBSC) Collections at Low Level of Circulating CD34+ Cells.
Authors: Law Ping , Tan Lip Kun , Yasir Fathalha , Soh Teck Guan , Mah Joanna K.Y. , Li Jenny , Liu Te Chih , Chen Chien Shing
Published: April 2020

Most patients or donors undergoing leukapheresis (LP) for autologous or allogeneic PBSC transplantation requires multiple collections to achieve a sufficient CD34+ cell dose. LP is usually initiated when peripheral blood (PB) CD34+ reached a certain level, such as 20/μl. The aim of this retrospective analysis is to summarize our institutional experience of initiating LP at a low PB CD34+ cell level of 5/μl and investigate the merit of the practice. All patients or donors underwent LP (using Cobe Spectra or Baxter Amicus) processing 3 times the blood volume. A total of 170 LP procedures (118 autologous and 52 allogeneic) was performed in 74 adult patients or donors (> 40 kg) between Jan 2004 and May 2005. Autologous patients were mobilized with chemotherapy and G-CSF while allogeneic donors with G-CSF alone. A “good” LP product is defined as one having ≥ 1 x 106 CD34+ cells/kg so that a minimum dose of 3 x 106/kg can be achieved in 3 collections. Our result showed that each PBSC product contained 6.07 x 108 WBC/kg (median, range: 0.13–17.5) and 1.59 x 106 CD34+ cells/kg (0.14–24.9). Total CD34+ cells in PBSC products were correlated to PB CD34+ cell counts (r = 0.79, data not shown). As shown in Table 1, initiating LP at higher levels of PB CD34+ cell increased the proportion of good LP. Nevertheless, 76% of collections initiated at > 5 CD34+ cells/μl achieved good LP criterion. It is possible that the level of PB CD34+ cells was still increasing in many patients or donors after initiation of LP at the low level. However, some patients /donors still achieved minimim CD34+ cell dose when second LP day (Day 2) PB CD34+ cell level was lower than that of first LP day (Day 1) (Table 2). These patients /donors would likely NOT have been collected if higher levels of PB CD34+ cells were used as guideline for start of LP. Eleven patients /donors whose Day 2 CD34+ cell count was below that of Day 1 achieved minimum CD34+ cell dose when LP was initiated at < 20/μl. When LP was initiated at < 10/μl, four individuals achieved minimum dose. All 4 were autologous patients mobilized with chemotherapy and G-CSF (3 AML and 1 NHL). In conclusion, our results showed that initiating LP at low PB CD34+ cells can be helpful to some individuals. The guideline may be especially useful in those patients that can only be mobilized marginally.

Table 1: Initiating LP at Different Levels of PB CD34+ Cells PB CD34+ Cells # LP % Good LP > 5/μl 156 75.9 > 10/μl 119 87.4 > 20/μl 65 95.4 Table 2: Patient /Donor Achieving Dose and Levels of PB CD34+ Cells PB CD34+ Cells # Patients & Donors # Patients & Donors Achieving Dose # Patients & Donors that achieved dose when Day 2 PB CD34+ cells were lower than that of Day 1 < 20/μ l 46 35 (82.6%) 11 < 10/μ l 14 10 (71.4%) 4
Myeloablative Conditioning Is Well Tolerated by Older Patients Receiving T-Cell Depleted Grafts.
Authors: Novitzky Nicolas , Thomas Valda , Hale Geoff , Waldmann Herman
Published: April 2020

Introduction: Older age is associated with increased transplant related mortality from GvHD and organ dysfunction. Depletion of T-cells from stem cell grafts prevents GvHD and could allow more older patients to receive standard forms of myeloablative conditioning.

Patients and Methods: Adult patients with haematological malignancies received allogeneic grafts from HLA identical siblings. Conditioning was with radiation or chemotherapy based myeloablative schedules. PBPC grafts were incubated ex vivo with CAMPATH-1H (anti CD52) antibody and infused into patients. Patients were given therapeutic doses of cyclosporin until day 90 post transplantation. The survival of patients younger or older than the population median age was analysed to determine if age was a risk factor for outcome.

Results: 72 unselected patients aged from 15 to 59 years (median 44) received myeloablative conditioning followed by median of 2.64 (range 1.14–12.3) CD34+ cells exposed “ex vivo” to a median dose of 10 mg (range 7.5–50) CAMPATH-1H. There was one early death from sepsis and all the other patients engrafted. GvHD occurred in 11%. Death was procedure related in 12 (17%) and from relapse in 3. The median follow up is 539 (7–2316) days and 79% survive disease free. The diagnosis of acute leukemia and lymphoma was more common among younger and older individuals (p< 0.01) respectively. 75% of 33 younger individuals and 79% (p= 0.4) in the older cohort survive disease free, at a median follow up of 911 and 475 days (p= 0.4) respectively. Furthermore, when outcome of patients younger than 40 years, aged between 40 and 49 and older than 49 years were compared, there was again no difference. Cox analysis showed that survival was not associated with diagnosis of disease and that developing GvHD was the only adverse factor for survival (p< 0.04).

Conclusions: In patients receiving T-cell depleted grafts, as GvHD rates are low, myeloablative conditioning is well tolerated supporting the use of this transplantation modality up to the age of 59. This information is useful to select patients who would benefit most from reduced intensity conditioning schedules.
Potential Usefulness of Leukotriene B4 (LTB4) on Mobilization of Hematopoietic Progenitor Cells (HPC).
Authors: Mun Yeung-Chul , Lee Seung-Eun , Lee Kyung-Eun , Yoo Eun-Sun , Kang Eun Suk , Lee Sang min , Ahn Jee-Young , Bae Yun-Kyung , Kim Geun-Young , Lee Soon Nam , Chung Wha-Sun , Kim Min Kyoung , Hyun Myung Soo , Kim Jae-Hong , Seong Chu-Myong
Published: April 2020

The mechanisms on HPC mobilization seem to be multifactorial processes. Many cell adhesion molecules (VLA-4, ICAM-1, VCAM-1 etc), SDF-1/CXCR4, and proteases (ie, MMP-9) may be important players for this mobilization processes. However, finding more mechanisms on HPC mobilization is under intense scrutiny. So far, G-CSF is the best known cytokine for this purpose. Meanwhile, its limitation on using G-CSF is to take 4–6 days for the optimal mobilization at clinic and its side effects (ie; bone pain, high WBC counts) are not negligible. LTB4 is lipid mediator during the process of inflammation, having many roles (ie; inducer of chemotaxis, the production of nitric oxide, transepithelial migration of neutrophil). In present study, we focused on the roles of LTB4 on HPC mobilization and its feasibility on mobilization in vivo.

Samples were collected from the peripheral blood via heart puncture and the bone marrow on time dependent manner (1hr, 4hr, 6hr, 12hr, 24hr, 48hr) after LTB4 injection which was given via intravenously and the dose dependent manner of LTB4 (0.5μg, 1μg, 2μg, 3μg). These data were compared with two other groups after G-CSF injection and normal saline injection. Collected total nucleated cells were analyzed for Sca-1+/Lin- using flow cytometry and CFU studies. There were definite increase of Sca-1+/Lin- cells after 1μg of LTB4 injection group (TNC: 29.55(±0.92)×105/ml and HPC: 3.72(±0.09) %). Its control group showed as follows: TNC: 14.55(±0.21)×105/ml, HPC: 0.41(±0.04) %, (p<0.05). Mobilization was optimal only after 4hours of LTB4 injection (TNC: 36.25(±2.90)×105/ml & HPC: 3.50(±0.37) %). After 4hours of LTB4 injection, there was a obvious down pattern. In terms of quantity, more than 20 fold of HPC mobilization after one LTB4 injection was observed. There were no immediate toxicity in the cohort, though long term potential side effects are under investigations. In conclusion, we showed the rapid mobilization of HPC with LTB4 at only 4 hours post-injection. This finding with LTB4 could be significant in terms of shortening the optimal period of mobilization comparing to that by G-CSF, which takes at least 4–6 days by repetitive injection to reach the optimal timing for collection. The possible synergistic effects of G-CSF with LTB4 for larger quantity of HPC and the cellular and molecular mechanism(s) of mobilization by LTB4 are being investigated in our lab.
Effect of Storage Time and Temperature on Fresh Unprocessed Cord Blood.
Authors: Vowels Marcus R. , Stylianou Jessica , Mison Leigh
Published: April 2020

Studies on the optimal conditions to store fresh cord blood (CB) in order to optimise haematopoietic stem cell (HSC) recovery prior to processing and cryopreservation have produced conflicting data. In this study we investigate the effects of time and temperature on CB HSC after collection. We also investigate whether the process of cryopreservation and thawing aggravates stress created by storage prior to cryopreservation. 30 CB units were collected and transported to the laboratory within 5 hours of collection. An aliquot of each CB unit was tested for nucleated cell count (NCC), CD34 count, CD34 viability and CFU-GEMM, and then frozen with 10% DMSO. Each CB unit was then separated into three 10 ml portions and incubated at 3 different temperatures (4–8°C, 21–24°C and 29–31°C). After 24, 36 or 48 hours, samples were taken and tested. An aliquot of each of the portions was then cryopreserved. After a minimum of 1 week, the cryopreserved samples were thawed and tested. For fresh samples incubated for 24 hrs or 36 hrs there was no difference over time when CB was incubated at 4–8°C, whereas, at temperatures above 20°C, there was either a trend or significant decrease in CD34 and CFU colonies with time. For thawed samples, viable CD34 and CFU colonies were decreased below baseline at either one or both 24 hr and 36 hr time points. This was particularly apparent for CFU colony measurements for samples kept at 29–31°C. At 48 hrs, at each of the temperature ranges (4–8°C, 21–24°C and 29–31°C), CFU were significantly lower for fresh (21.1, 16.8 and 14.0; baseline 31.0) and thawed (9.3, 8.5 and 3.0; baseline 2.7) samples and viable CD34 were significantly lower for thawed (2.0, 2.46 and 1.8; baseline 2.7) samples respectively (p<0.001). In conclusion, the best survival /recovery was seen with CB stored at 4–8° C and for ≥ 36 hrs. Temperatures above room temperature (21–24°C) appeared detrimental. The data also indicate that this damage becomes more evident when tested after the CB has gone through the cryopreservation and thawing process, and is even more evident the higher the temperature and the greater the time of storage prior to cryopreservation. These results have implications for quality and safety of CB stored for clinical use.

Viable CD34 and CFU related to incubation time and temperature, tested pre- and post-cryopreservation. Viable CD34 cells x 10e6 CFU-GEMM /12,500 cells plated Storage temperature 4–8C° 21–24°C 29–31°C 4–8°C 21–24°C 29–31°C Baseline 3.4 3.4 3.4 20.9 20.9 20.9 Fresh 24 hrs 3.3 3.67 2.45 20.4 18.8 21.6 36 hrs 3.7 2.45 2.74 20.5 19.6 17.4 Frozen / Baseline 3.0 3.0 3.0 11.0 11.0 11.0 Thawed 24 hrs 2.7 2.8 2.5 11.8 11.8 6.1 36 hrs 2.5 2.84 2.1 10.6 10.1 4.7
Upregulation of MT1-MMP by Molecules Present in Leukaphereis Products Primes CD34+ Cell Homing.
Authors: Shirvaikar Neeta , Montano Jencet , Turner A.Robert , Ratajczak Mariusz Z. , Janowska-Wieczorek Anna
Published: April 2020

Hematopoietic stem/progenitor cells (HSPC) derived from mobilized peripheral blood engraft faster than those from bone marrow (BM) or cord blood (CB). We recently postulated that leukapheresis product supernatants (collected from G-CSF-mobilized patients) and their components (fibrinogen, fibronectin, thrombin, hyaluronic acid (HA) and C3 cleavage fragments) have a priming effect on HSPC homing by enhancing chemotactic responses to SDF-1 through increased incorporation of CXCR4 into membrane lipid rafts and stimulation of matrix metalloproteinase (proMMP-2 and proMMP-9) secretion in these cells (Blood2005; 105:40). As MT1-MMP has been shown to activate latent forms of MMP-2 and (indirectly) MMP-9 and is known to localize proteolytic activity at the leading edge of migrating cells, we decided to investigate MT1-MMPs’ role in the priming of HSPC homing. We observed that stimulation of BM and CB CD34+ cells with priming agents (HA, fibrinogen or thrombin) not only increased secretion of proMMP-2 and proMMP-9 (zymography) but also highly upregulated levels of MT1-MMP transcript (RT-PCR) and protein (Western blotting) in these cells. Moreover, trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), enhanced by priming agents, was inhibited by the potent MT1-MMP inhibitor epigallocatechin-3-gallate. Furthermore, priming agents (HA, fibrinogen and thrombin) increased levels of active MMP-2 in co-cultures of stromal cells (endothelial cells and BM fibroblasts) with CD34+ cells. To elucidate the mechanism of MT1-MMP upregulation by priming agents, we evaluated whether they affected MT1-MMP incorporation in ganglioside M1-enriched membrane lipid rafts in the same way as for CXCR4. We found that stimulation of hematopoietic and THP-1 cells with HA, fibrinogen and thrombin increased incorporation of MT1-MMP into membrane lipid rafts and these observations were confirmed using confocal microscopy. Hence it appears that MT1-MMP, like CXCR4, localizes in membrane lipid rafts, and this is enhanced by priming agents, leading to better homing of HSPC.
Higher Efficacy of Peripheral Blood Stem Cell Collection in Children Mobilized with High-Dose G-CSF.
Authors: Styczynski Jan , Debski Robert , Gornicka Hanna , Hulek Elzbieta , Wojtylak Pawel , Wysocki Mariusz
Published: April 2020

Objective: Analysis of efficacy of PBSC collection after mobilization by high-dose G-CSF in children and young adults, qualified for transplantation of autologous hematopoietic stem cells.

Patients and Methods: Total number of 21 patients (23 collection cycles) were included into the study, divided into 2 groups: group I of 10 patients (aged 16m-23y, weight 8.5–61kg) with solid tumors were mobilized with G-CSF (Neupogen) at dose 2x5 μg/kg bw/day (12 collection cycles); group II of 11 patients (aged 3–27y, weight 12.5–68kg) with solid tumors (n=6) or hematological malignancies (n=5), who were mobilized with G-CSF (Neupogen) at dose 2x12 μg/kg bw/day. PBSC collections were performed by COBE Spectra from the day when CD34 cell count exceeded 20/μl. Efficacy of procedure was determined by collection of total 2x106 CD34 cells/kg (minimal required cell dose), or 5x106 CD34 cells/kg (optimal cell dose).

Results: Group I: Peripheral blood CD34 cell count exceeded 20/μl in 7/12 (58%) patients at day 4 and 10/12 (83%) at day 6 of G-CSF administration. Total number of CD34 cells of 2x106 /kg kw and 5x106 /kg bw was obtained in 3/12 (25%) and 2/12 (17%) patients after first collection respectively; and in 8/12 (67%) and 4/12 (33%) after second collection, respectively. After 3 days of collection required number of cells was obtained in 9/12 (75%) and 6/12 (50%) patients, respectively. No patient reported side-effects related to G-CSF administration. Group II: Peripheral blood CD34 cell count exceeded 20/μl in 8/11 (73%) patients at day 4 and 11/11 at day 6 of G-CSF administration. Total number of CD34 cells of 2x106 /kg bw and 5x106 /kg bw was obtained in 9/11 (82%) and 3/11 (27%) patients after first collection respectively; and in 11/11 and 6/11 (55%) after second collection, respectively. After 3 days of collection, in all patients, but one, optimal number of cells was obtained. Two patients reported side-effects related to G-CSF administration (headache, bone pain). No differences were found between the rate of speed of PBSC mobilization in analyzed schemes of G-CSF administration, however group of patients mobilized with high-dose G-CSF, showed more efficient stem cell collection after 3 days of procedure (p=0.027; OR=14; 95%CI=1.1–402).

Conclusion: Mobilization of PBSC with high-dose G-CSF is a safe and effective method of hematopoietic stem cell collection, enabling high efficacy of this procedure.