bluuebunny/crossref_metadata_2025 huggingface dataset:
Higher Expression Levels of the Adhesion Molecules VLA-4 and ICAM-1 on Multiple Myeloma Cells after Chemotherapy Are Associated with Survival of a Multidrug Resistant Subpopulation in Vivo.
Authors: Schmidmaier Ralf , Mörsdorf Kerstin , Baumann Philipp , Emmerich Bertold , Meinhardt Gerold
Published: October 2019

Objectives: Primary drug resistance is a major problem in multiple myeloma (MM), an incurable disease of the bone marrow. Adhesion of multiple myeloma cells to bone marrow stromal cells (BMSC) has been shown to cause strong primary resistance. The adhesion molecules LFA-1 and VLA-4 are upregulated upon treatment with cytotoxic agents. Furthermore, we have shown that the corresponding ligands on HS-5 BMSCs, VCAM-1 and ICAM-1, are upregulated after incubation with melphalan, suggesting increase of adhesion mediated drug resistance after chemotherapy. In this context, the expression levels of important adhesion molecules on MM cells of consecutive MM patients before and after chemotherapy have been determined in this study.

Methods: The expression levels of VLA-1, VLA-4, VLA-5, LFA-1, VCAM, ICAM-1, CD138, CD38, and CD56 were determined on MM cell lines, HS-5 stromal cells, and primary myeloma cells of 20 consecutive patients by flow cytometry in comparison to isotype control. 9 patients had been pre-treated (mostly induction chemotherapy and high dose melphalan with stem cell rescue) and 11 patients had been at diagnosis without treatment. Interpatient comparison of treated and untreated patients was performed. Intrapatient analysis (before and after high dose chemotherapy) will be performed in the follow up.

Results: VLA-4 and ICAM-1 are upregulated after chemotherapy by 54% and 64%, respectively. Similar upregulation of CD38 could be observed (62%), whereas CD138 shows downregulation by about 50%. CD56, VCAM, and LFA-1 expression was not significantly altered after chemotherapy.

Conclusion: The adhesion molecules VLA-4 and ICAM-1, which are essential for MM-BMSC interaction, are upregulated after chemotherapy. This finding supports our preclinical data and the hypothesis, that adhered, primary drug resistant MM cells are selected by chemotherapy and herewith contribute to multidrug resistance in multiple myeloma.
Serum Soluble Syndecan-1 Levels Correlate with Erythropoietin and Inversely with Hemoglobin Levels in Untreated Anaemic Myeloma Patients: Possible Role of Syndecan-1 in the Mechanisms of Chronic Anaemia.
Authors: Kyrtsonis Marie-Christine , Vassilakopoulos Theodoros P. , Papadogiannis Argyro , Tzenou Tatiana , Dimopoulou Maria N. , Siakantaris Marina P. , Kokoris Styliani I. , Dimitriadou Evangelia , Angelopoulou Maria K. , Kalpadaki Christine , Pangalis Gerassimos A.
Published: October 2019

Impaired erythropoiesis with inappropriate erythropoietin (EPO) production is seen in about 40% of symptomatic multiple myeloma (MM) patients at diagnosis and worsens during terminal refractory disease. Up-regulation of proinflammatory and proapoptotic factors, namely TNF-alpha, INF-gamma, TGF-beta1, FAS-Ligand and TRAIL are involved in the pathogenesis of defective erythroid maturation. The involvement of these factors in the progression of MM has been studied and no correlation was found for TNF-alpha, INF-gamma and TGF-beta1, while it was shown that the up-regulation of FAS-L/TRAIL in MM cells contributes to MM aggressiveness. Increased soluble syndecan-1 (s-synd-1) serum levels have been shown to constitute an adverse prognostic factor for survival in MM. Furthermore at least 3 studies, including ours, also found a negative correlation of s-synd-1 levels with hemoglobin. The purpose of this study was to investigate the correlations between s-synd-1, hemoglobin (Hb), endogenous EPO levels and other parameters of disease in anaemic MM patients before any treatment including rHu-EPO administration. 41 MM patients (19 males and 22 females) with variable degree of anaemia (Hb <13.5 g/dl for men and <12.5 g/dlfor women) were studied. Median Hb was 10.5 g/dl (range 6.9–13.5); 32% had Hb below 10 g/dl. Median age was 68y. 7 patients had Durie and Salmon stage I, 21 stage II and 13 stage III. Serum EPO levels were determined by RIA and s-synd by ELISA (Diaclone Research, Besancon, France) in frozen sera of MM patients and 24 healthy individuals. Normal serum EPO levels range from 4.9 to 52 mU/ml in our laboratory. Median s-synd-1 was 36 ng/ml (9–75) in healthy individuals.The median value of EPO levels in MM patients was 40.1 mU/ml (7.2–313) and of s-synd-1 160 ng/ml (26–3500). Serum EPO levels correlated as expected with Hb (Spearman’s rho -0.223, p=0.14), creatinin (Spearman’s rho -0.32, p=0.04) but also with s-synd-1 (Spearman’s rho 0.33, p=0.03). Serum EPO did not correlate either with parameters reflecting disease mass such as beta2microglobulin (Spearman’s rho −0.19, p=0.90) and percentage of bone marrow infiltration (Spearman’s rho 0.118, p=0.48) or CRP (Spearman’s rho −0.189, p=0.29) that reflects proinflammatory cytokines’ activity. Our results in addition to the fact that s-synd-1 levels inversely correlate with Hb levels, suggest that s-synd-1 may be involved in the mechanisms leading to chronic anemia in multiple myeloma. Further studies are needed to confirm these findings.
The Clinical Characteristics and Therapeutic Status of Multiple Myeloma in China- a 23 Years Retrospective Analysis in One Single Center.
Authors: Qiu Lugui , Mai Yu-jie , Li Rei , Zou Dehui , Wang Yafei , Zhao Yao-zhong , Yang Renchi , Wang Jianxiang , Xiao Zhijian , Han Mingzhe , Qian Linsheng
Published: October 2019

The clinical characteristics and treatment results of the multiple myeloma patients admitted to our hospital from January 1980 to December 2002 were retrospectively analyzed and compared in order to put forward a feasible therapeutic strategy for MM in China.

Results:

1. Clinical characteristics (1) 432 cases with full follow-up statistics were analyzed. The median age was 57 years (20 to 80).The peak onset age were 50 to 60 years. The ratio of male to female was 2.35:1. (2) 81% patients had anemia(Hb<100 g/L) at diagnosis. The median proportion of tumor cells in the bone marrow was 0.38 (0.005–0.970). 72.1% with high β2-MG (>3mg/L), 60% of the patients had abnormities in X-rays. (3) M-protein peak was detected in 78.5% patients. IgG made up the majority in classification(48.1%), IgA (21.8%)and light-chain(20.5%)next to it. 144 patients were in stage IIIA(40%), and 87 cases in IIIB(24.1%)at first diagnosis. (4) The median survival duration was 27 months (1–137months).

2. Treatment results: 206 cases had integral therapeutic records in 432 patients. (1) 200 patients were administered with drugs therapy, and their 3-year and 5-year survival were 32.01% and 15.8% respectively. The OS, median survival, 3-year and 5-year survival in MP group(n=46) were 30.4%, 30 months, 21.96% and 13.2% respectively. 149 cases(72.3%)received multi-drug combined chemotherapy(CCT).The overall response was 50.3% and significant higher than that of MP group(P=0.01).While the median survival duration(30.5 months), 3-year and 5-year survival rates (35% and 16.7% respectively) were as same as MP group (P>0.05. (2). 38/200 cases (19%) were treated with interferon in combination with chemotherapy over 6 months. The overall response rates were 53.6%, and the median survival duration was 52 months, and presented an increased response(P=0.02)and a prolonged survival duration (P=0.0003) compared with the no interferon group(34.4%,27months respectively. (3)Thalidomide was administered to 29 cases(14.5%), including 5 who took it alone(2.5%). The overall response rates were 50.3%. (4) 6 patients received hematopoietic stem cell transplantation. 5 patients with autologous peripheral blood stem cell transplantation were alive averagely in 73±12.5 months by the end of the follow-up.

Conclusion:

1) MM patients in China had poor prognostic characteristics except of younger age compared with the westerns, especially most patients were diagnosed at III phase with significant lower survival.

2) CCT yielded higher response rates but no longer duration of survival benefit than MP when used as treatment for MM. INF-a and Thalidomide could benefit MM by combination with chemotherapy. Among patients who are <60 years old with good performance, HSC could prolong survival duration.
Campath-1H May Have Activity in the Treatment of Multiple Myeloma.
Authors: Gasparetto Cristina , Horwitz Mitchell E. , Gockerman Jon P. , de Castro Carlos M. , Moore Joseph O. , Smith Clay A. , Davis Patty , Lassiter Martha , Houser Lesa , Chao Nelson J. , Rizzieri David
Published: October 2019

Previously we have demonstrated that multiple myeloma cell lines and primary cells express CD52, the antigenic target of Campath-1H, and that they undergo apoptosis following treatment with alemtuzumab in-vitro (Gasparetto et al. ASH #3210,2002). Based on these observations, we initiated a clinical trial where patients with advanced, multiply relapsed MM were treated with subcutaneous (sc) alemtuzumab as a single agent. Nine patients (6 men/4 women, age range 45–69 yrs) were initially treated with the standard dose of 30mg sc three times per week for up to 12 weeks. All patients had at least 4 lines of prior therapy and 8/9 patients had undergone high dose chemotherapy followed by autologous peripheral blood stem cell transplant. Treatment related toxicities included pancytopenia in all patients with grade IV neutropenia in the first four patients, necessitating withdrawal from study. Subsequently, all patients were treated with G-CSF and aggressive transfusion support and no further patients were removed from study due to cytopenias. All patients were prophylaxed with Famvir, Fluconazole and Septra and no opportunistic infections were noted during treatment. Two patients developed acute renal insufficiency that reversed when alemtuzumab was discontinued. One patient completed the entire 12 weeks of treatment. One patient had a PR with a 40% reduction in M-protein after two months of treatment, which reversed once Campath-1H was discontinued. Pharmacokinetic studies of one patient demonstrated that it required 8–10 weeks of treatment using the sc protocol to achieve serum levels of alemtuzumab of 1ug/ml, the level considered to be tumoricidal in-vivo. Based on this observation, the treatment protocol was modified so that the initial week of escalating doses was given IV to achieve more rapid therapeutic levels. Following this, sc alemtuzumab was given as above. One patient with non-secretory multiple myeloma with relapsed disease following 5 prior lines of therapy has been treated with this modified protocol. Treatment with alemtuzumab and growth factor support in this patient has been well tolerated except for the development of RSV pneumonia. At 4 weeks, a PET scan demonstrated a significant overall decrease in metabolic activity in multiple bony areas consistent with a response to treatment. These initial results suggests that alemtuzumab is associated with significant toxicities in patients with advanced multiple myeloma including pancytopenia, infections and possibly renal insufficiency but all of these were reversible and have been minimized with aggressive prophylactic therapy. Alemtuzumab does appear to have modest activity against MM in this heavily pre-treated group suggesting that it should be explored in combination with other agents and at earlier stages of disease.
Human Cytomegalovirus Can Activate and Induce Apoptosis in Mesenchymal Stem Cells.
Authors: Wei Guoqing , Lin Maofang , Huang He , Cai Zhen
Published: October 2019

Objective Mesenchymal stem cells (MSCs) is a very important cell system in bone marrow stromal and human cytomegalovirus (CMV) infection is one of the most common complications following stem cells transplantation. Our studies were to investigate the CMV infection of MSCs as well as the apoptosis induction effects.

Methods MSCs were infected by 100,10,1 TCID50 of CMV. After 48h of culture with DMEM supplemented with 10% (v/v) fetal calf serum, MSCs shapes were observed. CMV particles and ultra structure in the cells were detected with transmission electron microscope. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene and GAPDH gene. Flow Cytometry was used to detect the apoptotic cells.

Results After the infection of CMV, shape of MSCs changed, turning from thin shuttle shape to round and thick ball shape, even escaping from wall. Cell shape changed more dominantly as the CMV titer increased. With transmission electron microscope, CMV particles and typical cellular apoptotic character could be seen in the cells. MSCs infected by CMV could express IE mRNA compare with the uninfected and there were more apoptotic and necrosis cells in the cells infected by CMV.

Conclusion CMV can not only infect MSCs and activate in the cells but also can induce apoptosis of the cells.
Hematopoietic Cell Differentiation from Common Marmoset (Callithrix jacchus) Embryonic Stem Cells by Their Genetic Manipulation Using the Third Generation Lentiviral Vector.
Authors: Kurita Ryo , Sasaki Erika , Hiroyama Takashi , Yokoo Tomoko , Nakazaki Yukoh , Izawa Kiyoko , Ishii Hajime , Tanioka Yoshikuni , Hanazawa Kisaburo , Hashiguchi Takao , Bai Yuan-Son , Soda Yasushi , Watanabe Sumiko , Asano Shigetaka , Tani Kenzaburo
Published: October 2019

Since the successful establishment of human embryonic stem (ES) cell lines in 1998, transplantation of differentiated ES cells to specific organ has been expected to complete its defective function. For the realistic medicine, the preclinical studies using animal model systems including non-human primates are essential. We have already demonstrated that non-human primates of common marmosets (CM) are suitable for the laboratory animal models for preclinical studies of hematopoietic stem cell therapy. In this study, we investigated the in vitro and in vivo differentiation of CM ES cells to hematopoietic cells by exogenous gene transfer methods in order to study the feasibility of future gene modified ES cell therapy. First, we tried various in vitro culture conditions including systems using embryoid bodies or co-culturing with stromal cells to induce hematopoietic cells, but the frequency of inducing hematopoietic cells was very low. The expression of CD45 and gata1 could not be detected in both conditions, suggesting that our culture conditions were incomplete for induction of hematopoietic cells from CM ES cells. Next we examined gene transduction methods by using VSV-G pseudotyped human immunodeficiency virus (HIV) vectors. We constructed the HIV vectors containing hematopoietic genes such as tal1/scl, gata1, gata2, hoxB4 and Lh2 genes under the EF1a promoter and transduced them into CM ES cells. Only in the case of tal1/scl overexpression, not other genes, hematopoietic induction from CM ES cells was dramatically increased and multi-lineage blood cells consisting of erythroid cells, granulocytes, macrophages and megakaryocytes, were confirmed by immunochemical and morphological analyses. Furthermore, RT-PCR results showed that several hematopoietic marker genes including CD34 were expressed higher in the tal1/scl overexpressed ES-derived cells. After the xenotransplantation of ES-derived cells into the immunodeficient mice, CM CD45+ cells and immature erythroids and megakaryocytic cells were observed only in the ES-tal1-injected mice, indicating that enforced expression of tal1/scl into ES cells led to highly efficient hematopoietic cell differentiation in vivo. Taken together, it was suggested that the transduction of exogenous tal1/scl cDNA into ES cells by HIV vector was the promising method for the efficient differentiation from CM ES cells to hematopoietic stem cells. Further examinations are required to determine the long-term hematopoietic reconstitute capacity and the safety of the tal1/scl transduced ES cells in marmoset for the purpose of developing new hematopoietic stem cell therapy.
Large Scale Analysis of Foamy Virus Vector Integration Sites in Human CD34+ Cells.
Authors: Trobridge Grant D. , Miller Daniel G. , Jacobs Michael A. , Allen James M. , Olson Erik , Kiem Hans-Peter , Kaul Rajinder , Russell David W.
Published: October 2019

The ability of retroviruses to efficiently integrate into the host cell’s genome has led to their use as gene delivery vehicles for gene therapy. However, integration in the genome can have adverse effects as observed in a gene therapy trial for X-linked SCID using an oncoretroviral vector. Recent studies have shown that an oncoretroviral vector integrated preferentially near transcription start sites and that a lentiviral vector integrated preferentially within genes. Foamy viruses are integrating retroviruses with many properties that distinguish them from onco- or lentiviruses, perhaps the most important characteristic for gene therapy being that they are non-pathogenic. We previously showed that foamy vectors efficiently transduce CD34+ SCID mouse-repopulating cells (SRCs) from human mobilized peripheral blood, demonstrating their potential for hematopoietic stem cell gene therapy. We present here the first large-scale analysis of foamy vector integration sites. Integration sites were determined by infecting human CD34+ cells or normal fibroblasts with a foamy vector carrying a bacterial origin of replication, then rescuing plasmids containing vector provirus-genomic junction sites in bacteria, and sequencing the foamy vector LTR-human genome junctions. Over 1900 unique integration sites in human CD34+ cells and 1000 unique sites in normal human fibroblasts were mapped using the human genome database. The foamy vector did not integrate preferentially into genes. The percentage of integration sites within Refseq genes in human CD34+ cells, human fibroblasts and randomly generated sites was 29, 23, and 32% respectively. Foamy vectors showed only a slight preference for integration within 1 kb 5′ or 3′ of Refseq transcription start sites. In summary, our data show that foamy vectors have a distinct integration site profile relative to oncoretroviral and lentiviral vectors. Future studies will be required to determine if the unique integration site preference of foamy vectors translates into a reduced risk for oncogenesis in gene therapy applications.
Adenovirus-Mediated PD-L1 Over-Expression Has Differential Effects on Allograft Survival in Murine Islet and Heart Transplant Models.
Authors: Gao Wenda , Yamashita Kenichiro , Sullivan Jennifer , Scaria Abraham , Strom Terry B. , Li Xian C.
Published: October 2019

Program death-1 (PD-1) is a negative regulator of the immune system. Blocking PD-1-mediated negative signaling accelerates autoimmune diseases, while engaging PD-1 with recombinant PD-L1Ig fusion protein potentiates the efficacy of co-stimulation blockade in prolonging allograft survival. However, soluble PD-L1Ig itself showed no graft-protecting effect, in contrast to its strong inhibition of T and B cell activation in vitro when applied in a plate-bound form. In this study, we tested the hypothesis that membrane-bound PD-L1 should prolong allograft survival due to its increased ability to crosslink PD-1 receptor. An adenovirus (Ad.PD-L1) was constructed to encode the full-length mPD-L1, followed by green fluorescent protein (GFP) gene linked by an IRES sequence. A control adenovirus (Ad.Ctrl) was similarly constructed that carries only the GFP gene. In islet transplant model, B6AF1 (H-2b/a) islets were infected with the adenoviruses, and then transplanted into C57BL/6 (H-2b) mice induced diabetic by streptozotocin. In heart transplant model, DBA/2 (H-2d) hearts were perfused with adenoviruses, and then transplanted into C57BL/6 mice. PD-L1 over-expression in islet did not prolong graft survival, but accelerated islet rejection (Ad.PD-L1: 9.0+/−3.5 days; Ad.Ctrl: 13.3+/−2.2 days). In contrast, infection with Ad.PD-L1 prolonged heart allograft survival (15.4+/−5.7 days) in C57BL/6 mice, which promptly rejected DBA/2 hearts (7.0+/−2.3 days, p<0.02). Thus, over-expression of membrane-bound PD-L1 has beneficial effect in an organ/tissue specific manner. Strategies other than direct expression of PD-L1 in the islet b cells need to be devised in order to utilize this negative pathway to prevent rejection.
Correlation between Hematopoietic Stem Cell Engraftment Assay Results and Bone Marrow Biopsy, Flow Cytometry, and Cytogenetics Findings.
Authors: Weir Edward G. , Murphy Kathleen , Batista Denise , Griffin Constance A. , Borowitz Michael J. , Ansari-Lari M. Ali
Published: October 2019

Hematopoietic stem cell transplantation following induction chemotherapy is an increasingly successful therapeutic option for patients with leukemia or lymphoma. The use of molecular assays in post-transplant patients has become the standard in evaluating these patients for evidence of engraftment or early recurrence of disease. The detection of residual host cells in the bone marrow (BM) or peripheral blood (PB) following stem cell transplantation often influences subsequent clinical management. The aim of our study is to determine the extent of correlation between the results of PCR-based stem cell engraftment (SCE) assays and BM biopsy (BMBx), multiparameter flow cytometry (FC) and cytogenetics findings in patients who have undergone stem cell transplantation as therapy for hematolymphoid malignancies. We retrospectively reviewed the results of 1103 serial SCE assays performed at The Johns Hopkins Hospital, and 596 of these had temporally corresponding BMBx, FC and/or cytogenetic analysis. Concordance between the results of SCE analysis and those of the latter assays was defined as the detection of similar host/donor compositions. While some cases demonstrated clear discordance between the results, a subset showed an equivocal correlation due to the unclear significance of <5% host DNA by SCE analysis. Of 318 SCE assays with concurrent BMBx, 167(52%) showed concordant results, 104(33%) showed discordant results, and 47(15%) demonstrated an equivocal correlation. Of 221 SCE assays with concurrent FC, 111(50%) showed concordant results, 73(33%) showed discordant results, and 37(17%) demonstrated an equivocal correlation. Additionally, SCE assays were performed on concurrent, paired BM and PB specimens in 168 patients. Concordant results were identified in 141(84%) pairs. Of the remaining 27 pairs, host DNA was detected in the PB of 16 cases in which the BM showed either donor only DNA (6 cases) or <5% host DNA (10 cases). Four cases showed <5% host DNA in the PB and chimeric DNA in the BM. However, donor only DNA was detected in the PB in 7 cases that demonstrated a chimeric BM. Lastly, concurrent SCE analysis and XY FISH analysis was identified in 28 cases. Concordance between these two assays was observed in 24 (86%) cases, whereas one (3%) case was discordant and 3 (11%) cases showed an equivocal correlation. In conclusion, both BMBx and FC show similar but weak correlations to SCE analysis. In contrast, XY FISH analysis demonstrates a strong correlation to SCE analysis. Furthermore, SCE analyses performed on paired PB and BM specimens show an overall good correlation. However, our data suggest that in a subset of cases, SCE analysis performed on PB may detect residual host DNA that is not detectable by SCE analysis of BM, possibly due to the heterogeneity of the marrow composition.
Selection of Cord Blood Units Based on CD34+ Cells for Storage in Cord Blood Bank.
Authors: Vandekerckhove Bart , Van haute Inge , Lootens Nele , De Buck Carine , Verdegem Linda , Van De Steene Viviane , De Smet Saskia , Craeye David
Published: October 2019

Over 170 000 cord blood units are stored worldwide. For the majority of patients in need of an allogeneic donor, a cord blood unit can be found which is identical or has less than 2 HLA mismatches. However, when partially mismatches cord blood units are used for transplantation, the number of CD34+ cells per kg body weight becomes important and should be higher than 1.7 105/kg (Blood2002;100:1611–1618). For this reason, it is important that only cord blood units with high CD34+ cell content are stored. Our standard selection criteria are volume above 40 mL and TNC of 10x108 or higher. Using these criteria, we routinely store 18% of collected units. The mean CD34+ cell content of stored units is 5.2 106 and TNC is 1.38x109. 94%, 52%, 12%, 4% of cord blood units can be used for patients weighing 10, 25, 50 and 75 kg respectively, based on the criterium of 1.7. 105 CD34+ cells/kg patient weight mentioned above.

To optimize CD34+ cell content in the stored units, we compared our present selection method based on TNC and volume (criterium 40/10/?) with a selection method based on volume, TNC and CD34 content. 131 samples with volumes above 40mL and TNC above 8x108 were analysed for CD34+ cell content before processing and subsequently processed in routine. Using this data set, we compared criterium 40/10/? with criteria 40/8/2 (volume:40mL, TNC:8x108, CD34 2x106). We found that both criteria resulted in an equal number of units acceptable for storage. CD34+ cell content increased from 4.5x106 for criterium 40/10/? to 4.8x106 for criterium 40/8/2 whereas TNC dropped from 14.6x109 to 14.1x109. 100%, 47%, 9% and 4 % (criterium 40/8/2) vs 88%, 43%, 9% and 4%(criteria 40/10/?) of cord blood is acceptable for patients weighing 10, 25, 50 and 75 kg resp., demonstrating that the 12% essentially unusable cord blood units are replaced by units which can be used for small children. In conclusion: by including CD34+ cell content in the selection criteria, CD34+ cell content increases so that 100% of the cord blood units can be used for children up to 10 kg. This measure however requires that CD34 measurement is performed on all units with TNC above 8x108 cells.
Comparison of Controlled vs Non-Controlled Rate Freezing of Umbilical Cord Blood Units.
Authors: Mrowiec Zbigniew R. , Fernandez-DeLeon Maryely , Marchioni Misty , Cieszynska Barbara
Published: October 2019

Controlled rate freezing is recognized as a method of choice for the cryopreservation of blood cells. DMSO, the cryoprotectant most commonly used for cryopreservation of cells, is well known to affect cell viability. It is therefore imperative to insure that cells are frozen without any appreciable delay after the addition of DMSO. Addition of cryoprotectant may take between 5–10 minutes per processed cord blood unit. In situations involving large scale processing, such as 5–10 cord blood units simultaneously, delay will occur, which could cause adverse affects on cell viability when controlled rate freezing is utilized

This study was designed to compare the effect of controlled with two non-controlled rate freezing methods on cord blood stem cell viability.

After adding DMSO to final concentration of 10%, each cord blood unit was split into two 25 ml volumes and enclosed in metal freezing canisters. The UCB units were covered by styrofoam sleeves and then placed directly into either a −80°C or a −140°C mechanical freezer. A corresponding portion of each unit was frozen utilizing a Forma Scientific, model 1010 controlled rate freezer.

Approximately 30 days after, matching units were thawed in a 37°C water bath and tested without washing.

Total Nucleated Cell count, Total CD34 count, 7AAD viability and colony forming units assay were performed on fresh and cryopreserved/thawed samples. Statistical pair analysis using student-t test was performed to indicate any statistically significant differences between each procedures.

(table1)

(table2)

Results indicate that all three methods produces similar cell viability as measured by total nucleated cell recovery, total CD34 counts, 7AAD viability and colony forming assays.

Analysis of fresh and frozen samples did not indicate that controlled rate freezing produces a better quality product after thawing compared to two uncontrolled freezing methods.

Controlled rate freezing vs −80oC dump method°

N=5 Fresh Sample Controlled Rate Freezing Non controlled Freezing at −80oC TNC (x10^6) 412.4 ±37.06 397.6±15.65 406.7±24.78 Total CD34+ (10^6) 0.23±0.08 0.19±0.08 p<0.05 0.19±0.07 p<0.05 Viability (%) 97.5±1.67 94.8±2.28 p<0.05 93.4±3.51 p<0.05 Total # of Colonies (x10^6) 0.27±0.09 0.21±0.10 p<0.05 0.20±0.08 p<0.05

Controlled rate freezing vs −140oC dump method

N=5 Fresh Sample Controlled Rate Freezing Non Controlled −140oC TNC (10^6) 365.4±100.87 372.1±102.18 365.9±104.9 Total CD34+ (x10^6) 0.28±0.08 0.023±0.05 p<0.05 0.23±0.05 p<0.05 Viability (5) 96.0±3.94 96.2±2.28 p<0.05 96.2±1.79 Total # of Colonies (10^6) 0.27±0.13 0.27±0.11 0.24±0.15
Long-Term Pre-Transplant Imatinib Can Improve the Outcome of Non-Myeloablative Stem Cell Transplantation without Increasing the Morbidity in Ph+ Chronic Myeloid Leukemia.
Authors: Kim Dong-Wook , Eon Ki-Seong , Kim Yoo-Jin , Kim Hee-Je , Min Chang-Ki , Lee Jong-Wook , Min Woo-Sung , Kim Chun-Choo
Published: October 2019

Background and Objectives: Although non-myeloablative preparative regimens are being increasingly used due to its reduced toxicity associated with allogeneic transplantation, post-transplant relapses are still problematic. The use of imatinib prior to transplant may potentially allow disease control before SCT and accordingly induce the better transplant outcome in CML patients.

Patients and Methods: The role of imatinib before a non-myeloablative stem cell transplantation (NST) was examined in 25 CML patients. Imatinib was pretreated for median 10.8 months. There were 13 men and 12 women with a median age of 35 (19 – 53) years. Of these, 15 patients were in accelerated phase (AP), and rest of them in chronic phase (CP) at the time of diagnosis.

Results: At the time of transplant, all but 1 patient were in complete cytogenetic response and 4 of them were in molecular remission. Normalized BCR-ABL transcript level decreased from 0.65 to 0.00163 during imatinib therapy. All the patients received fludarabine-based regimens. With a median follow-up of 6.9 months (range, 1.3 – 43 months), all the patients are alive and transplant-related mortality was not observed. After transplantation, 4 CP and 7 AP patients additionally achieved molecular remission. Acute graft versus host disease (GVHD) was observed in 4 (16%) of 25 patients, meanwhile chronic GVHD was observed in 10 (59%) of 17 evaluable patients.

Conclusions: This study suggests that the use of imatinib followed by NST can offer an advantage of reduced relapse without increased toxicity. Prospective randomized trial with a larger number of patients is remained to clarify these clinical observations.
Engraftment Syndrome after Reduced-Intensity Allogeneic Hematopoietic Stem Cell Transplantation: Impact of Graft-Versus-Host Disease Prophylaxis and T Cell Dose.
Authors: Dean Robert M. , Donley Kyle M. , Gea-Banacloche Juan , Steinberg Seth M. , Odom Jeanne , Krumlauf Michael , Castro Kathleen , Chinn Tracey , Fowler Daniel H. , Gress Ronald E. , Bishop Michael R.
Published: October 2019

Engraftment syndrome (ES), defined as noninfectious fever, rash, and noncardiogenic pulmonary edema occurring at the time of hematopoietic recovery after stem cell transplantation, may cause significant morbidity in the peri-transplant period. However, its association with GVHD is unclear, and it remains controversial whether T-cell alloreactivity, nonspecific neutrophil activation, or other factors contribute to the pathogenesis of this syndrome. To study this question, we retrospectively analyzed patients enrolled in 3 separate protocols of reduced-intensity allogeneic hematopoietic stem cell transplantation (RIST). Group A included patients with relapsed/refractory hematologic malignancies undergoing RIST with T-cell replete allografts and cyclosporine for GVHD prophylaxis (n=49). Group B consisted of patients with hematologic malignancies receiving T-cell replete allografts and cyclosporine/methotrexate for GVHD prophylaxis (n=20). Group C included patients with metastatic breast cancer undergoing RIST with T-cell depleted allografts (1 x 105 CD3+ cells/kg) and cyclosporine for GVHD prophylaxis (n=17). All patients received an identical reduced-intensity conditioning regimen with fludarabine and cyclophosphamide, followed by infusion of filgrastim-mobilized peripheral blood stem cells and post-transplantation filgrastim until neutrophil recovery. The incidences of ES and acute GVHD varied according to treatment group (ES: chi-squared test, p=0.066; acute GVHD: chi-squared test, p=NS):

Incidence of Engraftment Syndrome and Acute GVHD by Treatment Group

Group Graft Composition GVHD Prophylaxis Engraftment Syndrome Acute GVHD, Grades 2–4 A T-cell replete Cyclosporine 24/49 (49%) 32/49 (65%) B T-cell replete Cyclosporine/Methotrexate 5/20 (25%) 9/20 (45%) C T-cell depleted Cyclosporine 4/17 (24%) 8/17 (47%)

Logistic regression analysis identified hypoxemia requiring oxygen (OR 38.46, 95% CI 6.51 to 227.1; p=0.002), fever greater than 37 degrees C (OR 6.97, 95% CI 2.47 to 19.7; p<0.0001), and the maximum neutrophil count after engraftment (OR 1.07, 95% CI 1.02 to 1.125; p=0.0093) as factors strongly associated with steroid administration for ES. Subjects with acute GVHD displayed a modest trend toward increased frequency of ES (exact Cochran-Armitage trend test, p=0.103). The association of the intensity of GVHD prophylaxis and the maximum neutrophil count with the incidence of ES implies that both alloreactive T cells and donor granulocytes may play a role in the development of this complication after RIST. These results suggest that additional studies are warranted to explore the contribution of alloreactivity to the pathogenesis, prevention, and treatment of ES.
Prior Autologous Hematopoietic Cell Transplantation Confers an Adverse Prognosis for Recipients of Allogeneic Transplantation Following Nonmyeloablative Preparative Conditioning.
Authors: Schiller Gary J. , Emmanouilides Christos , Paquette Ron , Rosen Peter , Gansert Jennifer , Territo Mary C.
Published: October 2019

Allogeneic hematopoietic transplantation following nonmyeloablative preparative conditioning (also know as “mini-dose transplantation”) is frequently applied to patients who, on the basis of prior therapy, organ dysfunction, or co-morbid conditions may be considered ineligible for full, dose-intenstive treatment. This approach assumes that early regimen-related toxicity can be averted by the nonmyeloablative approach. We present the results obtained in a group of consecutive, unselected older adults with advanced hematologic neoplasia who underwent allogeneic transplantation from fully histocompatible siblings or unrelated donors after dose-reduced preparative conditioning in a single center. Thirty-four patients, median age 57 years (range 26–66) underwent allogeneic transplantation from siblings (14 patients) or unrelated donors (20 patients) following preparative conditioning consisting of chemotherapy only (typically fludarabine, cytarabine, and cyclophosphamide) or those drugs plus antithymocyte globulin, respectively. One recipient of a related allograft and one recipient of an unrelated allograft received preparative fludarabine/melphalan and busulfan/fludarabine/ATG, respectively. Diagnoses prior to transplantation included advanced acute leukemia and myelodysplasia (17 patients), nonHodgkin’s or Hodgkin’s lymphoma (11 patients), and CLL or Multiple Myeloma (6 patients). Allogeneic transplantation followed previous autologous transplantation in 17 patients. Time from previous autologous transplantation to nonmyeloablative allogeneic transplantation varied. Prior autograft conferred a significant adverse risk on recipients of allogeneic transplants following nonmyeloablative conditioning. Only two of 17 patients with a history of prior autograft enjoyed long-term survival as opposed to seven of 17 who had not undergone prior autologous transplant. Mortality before day + 100, typically attributed to treatment-related toxicities, occurred in 11 patients; nine of these patients had had prior autologous transplantation. Prior autologous transplantation typically was done for acute myelogenous leukemia in first complete remission and referral for allotransplant may have been an indicator of more aggressive disease biology. In conclusion, allogeneic transplantation following a nonmyeloablative preparative regimen may be associated with significant risk of early mortality in patients who undergo this treatment after prior autologous transplantation, especially for acute leukemia, suggesting that criteria for allogeneic transplantation after nonmyeloablative condition should take this risk into account. Further studies from multiple centers will be required to confirm this observation.
Antifungal Prophylaxis with Itraconazole in Patients with Acute Leukemia and after Stem Cell Transplantation (SZT): High Plasma Concentrations with a Loading Dose Strategy.
Authors: Sandherr Michael , Vogeser Michael , Hartig Carsten , Aulmann Christoph , Fuhrmann Stefanie , Wondzinski Linda , Oruzio Daniel , Schlimok Guenter
Published: October 2019

Invasive fungal infection (IFI) is a major complication in the treatment of leukemia. Itraconazole (ITR) is an azole antifungal that has activity against both Candida species and molds. Its prophylactic use in patients at high risk for IFI has been shown to reduce invasive mold infections, but the antifungal efficacy depends on a sufficient plasma concentration early in the course of prophylaxis. We tested a loading dose strategy to reach plasma levels above 500 μg/l by 48 hours.

ITR plasma concentrations from 30 patients (pts) in 51 treatment courses were sampled on day 3, 7 and 11 of prophylaxis which started one day after conclusion of chemotherapy. It consisted of 400 mg oral solution and 400 mg intravenous ITR on day 1+2 and continued with 400 mg oral solution daily until an ANC count > 500/μl, limiting toxicity or the use of empirical antifungal treatment. Plasma concentrations were measured by LC-MS/MS. Two pts received an allogeneic, 4 pts an autologous stem cell graft. Underlying diseases were 43 AML, 4 CML blast crisis, 3 Hodgkin’s disease and 1 Ph+ ALL. In 45 courses chemotherapy was based on Cytarabine plus an anthracycline.

Data from 51 courses were analyzed. Median time of neutropenia (ANC < 500/μl) was 11 days (4–25 days), median time of ITR was 14 days (4–28 days). Median ITR plasma concentrations were 902 μg/l (176–5149) on day 3, 604 μg/l (145–1945) on day 7 and 942 μg/l (191–2987) on day 11. The desirable plasma level of > 500 μg/l after 48 hours of ITR was achieved in 86% of courses and remained above that level in 64% on d 7 and 75% on d 11. No proven or probable IFI could be observed. In 15% of courses empiric antifungal treatment with Amphotericin B and Voriconazole was initiated because of persisting neutropenic fever. Colonization with Candida species was found in 32% of courses. Nausea and vomiting grade 1/2 CTC was observed 39%, diarrhea in 14% of courses.

Sufficient plasma concentrations 48 hours after starting antifungal prophylaxis with ITR can be reached with a loading dose combining oral solution and intravenous formulation. Toxicity is mild with only 10% of patients refusing oral solution. Our strategy suggests a high prophylactic efficacy in terms of preventing IFI in this high-risk group of patients.
Clinical Use of Caspofungin in Immunocompromised Children: A Multicenter Survey.
Authors: Lehrnbecher Thomas , Attarbaschi Andishe , Schuster Friedhelm , Herzog Nadine , Grigull Lorenz , Dworzak Michael , Beutel Karin , Laws Hans-Jürgen , Groll Andreas H.
Published: October 2019

Background : Caspofungin (CAS) is a novel echinocandin, which is approved for several antifungal indications in adults. Although the compound is not licensed in pediatric patients (pts), it is being used in children with refractory infections or intolerance to standard antifungal agents.

Methods: We conducted a multicenter retrospective survey to obtain data on clinical use, safety, and outcome in immunocompromised pediatric pts who received therapy with CAS.

Results : The survey identified 71 immunocompromised children and adolescents [mean age (range) 11.4 years (5 months –26 years)]. Out of the 29 females and 42 males, 53 suffered from hematological malignancies, 10 from bone marrow failure syndromes, 3 from solid tumors, 2 from congenital immunodeficiency, and 3 from non-malignant hematological disorders. Forty-two pts (59%) had undergone allogeneic blood stem cell transplantation, and 36 pts (51%) had an ANC of less than 500/μL at baseline. CAS was administered for proven (n=18), probable (15), and possible (20) invasive fungal infection, or as empirical antifungal treatment (18). All but one pt had received prior systemic antifungal therapy with amphotericin B (52) and/or triazoles (41). The 71 pts received CAS for a mean duration of 43.5 days (range, 2–218) as single agent (22) or in combination with other antifungal agents (49). The mean maintenance dosage of CAS was 1.2 mg/kg (range, 0.4–2.9) or 34.8 mg/msqu (range, 16.3–57.5). In none of the pts, CAS was discontinued prematurely due to clinical or laboratory adverse events. Clinical adverse events were described in 35 children (49%), mostly fever (28), nausea and vomiting (18), diarrhea (8), and headache (5). Increases in hepatic or renal function parameters were frequent in these pts that received multiple other therapeutic compounds. Whereas at the end of treatment (EOT), mean GPT and GOT were slightly elevated (from 41 at baseline to 61 U/L at EOT; p=0.002 and from 29 to 80 U/L, p=0.001, respectively), mean serum creatinine, bilirubin, and alkaline phosphatase values were not different from baseline. Complete responses, partial responses, or stabilization were observed in 4/8/3 of 18 evaluable pts with proven, in 4/2/3 of 13 pts with probable, and in 3/8/2 of 15 pts with possible invasive fungal infection. Of 16 evaluable pts who received CAS empirically therapy, 10 successfully completed therapy. Overall survival was 74% at EOT and 67% at three months post EOT (65 and 60 evaluable pts, respectively).

Conclusions: The data of this retrospective survey show that CAS displays acceptable safety and tolerance and may have useful antifungal efficacy for second line treatment of severely immunocompromised pediatric patients.
Anticipated Diagnosis of Adverse Events after Stem Cell Transplantation Using Highly Sensitive STR-PCR for Chimerism Quantification.
Authors: Nava Paola , Simon Ainhoa , Gonzalez-Pardo Gema , Jimenez Jose L. , Gonzalez-Rivera Milagros , Balsalobre Pascual , Carrion Rafael , Serrano David , Gomez-Pineda Alfonso , Diez-Martin Jose L. , Buño Ismael
Published: October 2019

Introduction: The current gold standard for chimerism quantification after allo-stem cell transplantation (SCT) is based on the polymerase chain reaction of DNA short tandem repeats (STR-PCR). This methodology is sex-independent, highly informative and quantitatively accurate. However, this method has a moderate sensitivity (1–3%).

Objective: To test the usefulness of increasing the assay sensitivity by 3 different approaches in order to detect recipient residual cells.

Material and Methods: Two sets of 20 samples, belonging to artificial mixtures of male and female peripheral blood (PB) cells in different proportions (% male: 100, 75, 50, 25, 10, 5, 3, 1, 0.1, 0) were prepared to test the assay sensitivity and reproducibility. Additionally 58 samples from 10 selected patients showing different clinical evolution post-SCT were retrospectively studied. Chimerism was analyzed on genomic DNA (purified QIAamp DNA blood kit, Qiagen) by multiplex STR-PCR (AmpFlSTR SGM Plus revealed on capillary electrophoresis in an ABI Prism 3100 automated DNA sequencer; Applied Biosystems). Three different approaches aimed to increase the sensitivity of STR-PCR were tested: (1) Increasing DNA concentration in the PCR (1x, 2.5x, 5x), (2) Increasing the concentration of the PCR product for the capillary electrophoresis (1x, 2.5x, 5x) and (3) Increasing the sample injection time into the capillary for the electrophoresis (10s, standard time, 30s and 50s).

Results: The standard STR-PCR showed a sensitivity of 1% in both sets of male-female PB artificial mixtures. Both approaches increasing DNA concentration in the PCR and the PCR product in the capillary electrophoresis did not achieved better sensitivity. Moreover, appropriate analysis of the samples was frequently hampered due to fluorescence saturation. However, longer injection times (30s, 50s) showed that currently underrepresented DNA fragments were conspicuously overestimated, giving a one-log increase in the sensitivity of the STR-PCR assay, which reached a 0.1%. This approach was used to re-analyze the 23 SCT patient samples previously identified as in complete chimerism by standard STR-PCR. The 0.1% sensitivity methodological approach allowed to detect residual/reappearing recipient cells in three of the 23 analyzed samples. Therefore this new approach would have allowed to anticipate the detection of graft rejection and disease relapse in 2/10 patients, 7 and 77 days respectively.

Conclusion: Long injection times (30s and 50s) in the capillary electrophoresis after STR-PCR result in a one log sensitivity increase (from 1% to 0.1%). This would allow earlier diagnosis of adverse events after SCT favoring a prompt introduction of therapeutic interventions.
Late Onset Hemorrhagic Cystitis (LOHC) Following Stem Cell Transplant (SCT): Risk Factors and Outcomes.
Authors: Janbay Nasser , Amin Albish , Visweshar N. , Ojha Rakesh , Mohsen Reyad , Randhawa Devinderdal , Stives S. , Benn H. , Manna Pradeep , Nath Rajneesh
Published: October 2019

Background. Hemorrhagic cystitis (HC) following stem cell transplant (SCT) is classified into two types, based on causes and time of appearance. Early-onset HC occurs within 48 hours of preparative regimen, and is thought to be a complication of high-dose chemotherapy containing preparative regimen. Late-onset HC occurs beyond 48 hours from the preparative regimen and has been reported to be associated with viral infections.

Goals. To establish risk factors for predicting LOHC and the impact on the outcomes following SCT.

Subjects and methods. We retrospectively analyzed the clinical records of 41 patients who underwent SCT from June 2002-June 2004, the median age 54 years (range 20–74), including 28 males and 13 females. The patients had the following diagnosis: AML/MDS=15, ALL=4, CML=2, NHD/HD=14, MM=4, solid tumor=1, and AA=1. Twenty-two patients received allogeneic transplants (matched sibling-10, matched unrelated-2, cord blood -10), and 19 patients underwent autologous SCT. Conditioning regimens included the following: BEAM=8, BU+CY±Thiotepa=17, TBI+CY±others=4, Melphalan±Fludara=12. Sixteen patients were considered to have poor risk disease (those beyond first complete remission or in relapse) and 25 patients had good risk disease. LOHC was defined as: RBCs in the urine without any trauma and persistence of >7 days. All patients who developed LO HC were tested for BK virus, CMV, and adenovirus in the urine and blood, which were measured by a real-time PCR assay.

Results. LOHC was detected in 11 patients (26.8%), 8 of which had BK viruria. median of 23 days (range 13–60 days), None of these patients were detected to have CMV or adenovirus. Nine patients (90%) that received cord blood SCT developed LO HC, while only two patients (6.4%) who did not have cord blood SCT developed LO HC (P=0.0001). Ten patients (58.8%) who had BU+CY±Thiotepa developed HC compared to one patient (4.2%) who did not have the same regimen and developed LO HC (P=0.0002). Nine patients (56.2%) who had poor risk disease developed LOHC, while two patients (8%) who had good risk disease developed LOHC (P=0.001). Eight patients (72.7%) who developed LOHC died compared to only six patients (20%) who did not develop LOHC and died (P=0.003). Among the 28 males, 10 patients developed LOHC, compared to only one female patient who developed LOHC (P=0.126).

Conclusion. Three predisposing factors were identified for the development of LOHC: cord blood transplant (P=0.0001), Busulphan use for pre-transplant conditioning (P=0.0002), and poor risk group characteristics (P=0.001). The mortality rate was significantly higher among patients who developed LOHC.