bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Stat3 Binding in the γ-Globin Promoter: The Role of DNA-Protein Interactions in Gene Silencing.
Authors: Yao Xiao , Cubillos Federico , Sangerman Jose , Pace Betty S.
Published: April 2020

We previously demonstrated that signal transducer and activator of transcription 3β (Stat3β) acts as a negative regulator of γ-globin gene expression. We speculate that Stat3β participates in the γ to β-globin switch recapitulated during erythroid maturation. Regulatory elements located between nucleotide −52 and +36 relative to the γ-globin cap site may define a silencing domain where Stat3β exerts its negative effect. We analyzed DNA-protein interactions in a putative Stat3 binding site at +9γ and an overlapping Stat3/GATA-1 consensus sequence at +26γ. The +9γ motif was required for Stat3β mediated γ-gene silencing in K562 cells and normal erythroid progenitors. Mutating the +26γ site disrupted both Stat3 and GATA-1 binding which was confirmed by electrophoretic mobility shift assay. We demonstrated for the first time direct interaction between Stat3 and GATA-1 using immunoprecipitation techniques and protein extracts isolated from K562 and mouse erythroleukemia cells. We speculate that developmental stage specific DNA-protein interactions in the −52 to +36 region are required for γ-gene silencing. To investigate this hypothesis, we tested the ability of Stat3β to bind in vivo using chromatin immunoprecipitation (ChIP) assay. K562 cells were treated with Interleukin-6 (100ng/ml) to activate Stat3β and then used for ChIP analysis with phosphorylated-Stat3β antibody. Specific PCR primers were designed to amplify the region between nucleotide −52 and +36 relative to the γ-globin cap site. We observed increased Stat3β binding to the γ-globin silencing domain in vivo after Interleukin-6 treatment. To compliment this approach, we modified our ChIP assay as follows. A trypsin digest step was added after cross-linking proteins to the γ-globin silencing domain in vitro. The samples will be analyzed by nanospray LC/MS/MS mass spectrometry to characterize the transcription factors that bind the silencing domain and interact with Stat3β in this region. Understanding the DNA-protein interactions required for γ-globin regulation will provide insights into mechanisms for globin gene silencing.
Prospective Study of Post-Transplant Microangiopathy in Allogeneic and Autologous Stem Cell Transplantation.
Authors: Zeigler Zella Rose , Shadduck R. , Lister John
Published: April 2020

Thrombotic thrombocytopenic purpura-hemolytic uremic syndromes (TTP-HUS) have been described as specific sequelae following stem cell transplantation (SCT).Twenty-two patients undergoing autologous SCT and thirty-three patients undergoing allogeneic stem cell transplantation were prospectively evaluated on days 0, 7, 14, 30, 60, 90, 120 following SCT for the development of Post-Transplant Microangiopathy (PTM) based upon a rising lactic dehydrogenase level and percent fragmented cells (FC). The protocols were approved by the Institutional Review Board of the Western Pennsylvania Hospital where the studies were performed. The grades of PTM were based upon the criteria described by Zeigler et al, Bone Marrow Transplant, 1995) as follows: Grade 0: Normal LDH level and normal percentage fragmented cells (0–1.2%), Grade 1: Increased Fragmented Cells (FC) with normal LDH level, Grade 2: Increased LDH level that is rising with 1.2–4.8% FC, Grade 3: Increased LDH level with 4.9–9.6% FC and Grade 4: Increased LDH level with >9.6% FC. The data is shown below in tabular form. These results indicate that almost all patients have increased fragmented cells following both autologous and allogeneic SCT. The median percent fragmented cells in both allogeneic and autologous SCT patients without evidence for PTM was 1.4% with a range of 0–5.2% Moreoeve the LDH level x ULN was 0.7 with a range of 0.5–1.8 x ULN in patients without evidence for PTM. The table below shows the data for the development of PTM in both allogeneic and autologous SCT patients with regard peak grade. Only two autologous patients had no increase in FC. The Grade of PTM was significantly lower in the autologous SCT patients compared to the allogeneic SCT patients. The table below lists the grades of PTM and the day of the peak Grade of PTM in both types of SCT patients. The Grades of PTM, peak LDH level and highest percent FC were higher in the allogeneic SCT patients. Eleven of twenty-two (50%) of the autologous SCT patients compared to thirty/thirty-three (91%) of the allogeneic SCT patients (p <0.001) had evidence for PTM (grades 2 to 4).

PTM Grade Autologous Allogeneic p Grade 0–1 (no PTM) 11/22 (50%) 3/33 (9%) <0.001 Grade 2 10/22 (45%) 10/33 (30%) NS Grade 3 1/22 (5%) 13 (39%) <0.01 Grade 4 0/22 (0%) 7/33 (21%) NS Day to Peak TM 31 (3–96) 26 (7–120) NS Peak LDH x ULN 1.1 (0.7–31.9) 2.5 (0.9–14.8) <0.001 Peak % FC 2.3 (0.5–4.9) 5.2 (1.9–19.2) <.001
Hepatic Veno-Occlusive Disease (VOD) after Hematopoietic Stem Cell Transplantation in Children: Retrospective Analysis in a Single Institution.
Authors: Kook Hoon , Kim Seok Joo , Sohn Kyoung Ran , Baik Hee Jo , Noh Ha Young , Cho Young Kook , Sung Jun Seung , Nam Ho Song , Hwang Tai Ju
Published: April 2020

Introduction: Hepatic VOD, a potential life-threatening complication, occurs early after stem cell transplantation (SCT). Reliable predictive factors, effective prophylaxis and treatments need to be established in case of severe VOD. To better understand clinical findings in children, we evaluated clinical and laboratory characteristics of hepatic VOD undergoing SCT in children. Methods: We retrospectively reviewed the medical records of all patients (n=116) receiving SCTs in Pediatric BMT unit between May, 1991 to June, 2004. The definition of VOD required two of the following three clinical findings before D+21 following SCT: hyperbilirubinemia (>2 mg/dL), ascites or sudden weight gain, and painful organomegaly. Ursodeoxycholic acid was started before conditioning. No routine heparin prophylaxis was used. Lipo-prostaglandin E1 (0.5 ng/kg/min) was used in most of unrelated transplants (20/23). Results: VOD developed in 11 patients (12.8%). The median age was 9.8 years (range, 2 to 13.9 years). Underlying diagnoses were ALL (n=3), AML (n=2), acute biphenotypic leukemia, severe aplastic anemia, neuroblastoma, and myelodysplastic syndrome (n=1, each). The median day of onset of VOD was D+9 (range, D-3 to D+19). VOD was classified as moderate in 5 (45%) and severe in 6 (55%) cases. At the median of D+12 (range, D-5 to D+56), total serum bilirubin elevated over 2 mg/dL, with the peak bilirubin level at D+18 (range, D-5 to D+59). Maximum level of serum bilirubin was 2.9 mg/dL (range, 2.1 to 9.2) in moderate VOD and 7.3 mg/dL (range, 2.0 to 24.2 mg/dL) in severe cases. Various treatment modalities have been applied. Successful control of VOD was possible with tissue plasminogen activator and heparin (2/5, 40%), ursodeoxycholic acid (2/5, 40%), N-acetylcysteine (3/5, 60%), and defibrotide (1/2, 50%). All of 5 patients with moderate VOD survived (range, 6+ to 67+ months). Five of 6 (83%) patients with severe VOD died of VOD before D+100 (range, D+7 to D+64). Conclusion: This retrospective study showed that the incidence of VOD in children was 12.8%, and the mortality of severe VOD is still unacceptably high, necessitating further development of effective treatment.
Comparison of G-CSF Administration Starting Day +5 Versus Based on White Blood Count Following Autologous Peripheral Blood Stem Cell Transplantation.
Authors: Craig Michael , Yao Yuan , Ericson Solveig
Published: April 2020

Granulocyte colony stimulating factor (G-CSF) is commonly used following autologous peripheral blood stem cell transplantation (PBSCT) to promote bone marrow recovery. However, the optimal timing of G-CSF in this setting is unknown. We randomized 23 patients undergoing autologous PBSCT for a variety of malignant disorders to G-CSF administration 5 μg/kg/day starting day +5 (Arm A) versus when white blood count (WBC) recovered to 0.2 x 109/L or 0.1 x 109/L for 2 consecutive days (Arm B). Results: All patients engrafted. The median time to absolute neutrophil count (ANC) > 1.0 x 109/L in Arms A and B was 12.1 vs. 12.8 days (p=0.77). There was no significant difference in number of days of temperature >38.3°C (4.3 vs. 4.4 days, p=0.71) or hospital stay (23.8 vs. 22.1 days, p=0.93) between Arms A and B respectively. The mean amount of G-CSF administered was 3210 μg/patient in Arm A vs. 2384 μg/patient in Arm B, a significant reduction (p=0.035). Conclusion: Waiting for early signs of engraftment after autologous PBSCT before starting G-CSF administration is associated with a decrease in amount of G-CSF administration by 25%, with no increase in days with fever, time to neutrophil recovery, or length of hospital stay.
Emergence of Mucormycosis in Onco-Hematology after Prolonged Use of Voriconazole: Attention Required.
Authors: Vigouroux Stephane , Morin Odile , Moreau Philippe , Mechinaud Francoise , Harousseau Jean-Luc , Milpied Noel
Published: April 2020

Voriconazole is a broad spectrum antifungal agent however ineffective against Mucor sp. It has proven to have a higher efficacy in the control of invasive aspergillosis when compared to amphotericin B. However, the prophylactic use of voriconazole has not yet been defined. Nevertheless, it has become an attractive option because of the broad spectrum, limited toxicity and possible oral administration of voriconazole. We report here four cases of mucormycosis (3 in 2003, 1 in 2004) that occured after prolonged use of voriconazole in hematologic patients. Patients 1 and 2 (23 and 34 years old, respectively) had relapsed acute myeloid leukemia, patient 3 (3 years old) had chronic myelomonocytic leukemia, and patient 4 (52 years old) had high-grade B cell lymphoma. All patients were severely immunosuppressed. Patient 1 had received a T cell depleted, haploidentical stem cell transplantation (SCT). Patients 2 and 3 had each received a matched unrelated allogeneic SCT complicated by subsequent acute and chronic graft versus host diseases. Patient 4 had been treated for 5 years with mycophenolate mofetil and steroids after a renal transplantation. All four patients received continuous treatment with the oral form of voriconazole for 7 to 30 weeks prior to the diagnosis of mucormycosis: three as primary prophylaxis and one for a bronchial aspergillosis. Mucormycosis was diagnosed 9 weeks after the lymphoma or between 27 and 33 weeks after SCT for the transplanted patients. Patients were subsequently treated with liposomal amphotericin B. Fever was the most common symptom, indicating an invasive infection in the 3 patients who died rapidly at 12, 13 and 45 days after the diagnosis. The surviving patient, an allogeneic SCT recipient, had a localized bronchial mucormycosis. No evidence of mucormycosis had been observed prior to the introduction of treatment with voriconazole beginning in October 2002. In addition, to date, mucormycosis has only been observed in patients that have had pre-exposure to voriconazole. Physicians should be mindful of the potential risk of invasive mucormycosis after prolonged use of voriconazole in immunocompromised hematologic patients. Moreover, the question of prophylactic use of voriconazole needs to be adressed further in randomized trials.
Outpatient High-Dose Chemotherapy and Autologous Hematopoietic Cell Transplantation for Multiple Myeloma Is Associated with a Low Infection Safety Profile.
Authors: Agha Mounzer E. , Yeager Andrew M. , Evans Carol , Borkowski Nikol , Scekeres Anne Marie , Miller Sarah , Tompkins Cheryl , Schwartz Rowena , Donnenberg Albert D. , Shogan Jeffrey
Published: April 2020

High-dose melphalan (MEL) and autologous hematopoietic cell transplantation (HCT) is now a standard therapy in patients (pts) with multiple myeloma (MM). This HCT procedure is sufficiently tolerable to be performed on either an outpatient (OP) or inpatient (IP) basis. We compared the infection profiles in 16 consecutive pts who underwent OP HCT and 31 consecutive pts who underwent IP HCT for MM over a 24-month period. The median age of each group was comparable (OP 58 yr, range 33–72 yr; IP 59 yr, range 32–74 yr). All pts received MEL 200 mg/m2 before HCT and anti-infective prophylaxis with oral acyclovir, fluconazole and levofloxacin. Empiric treatment of first neutropenic fever in the OP group was imipenem and vancomycin (VAN) and in the IP group was cefepime with addition of VAN for persistent fever and/or positive cultures with VAN-sensitive bacteria. Median time to neutrophils >0.5 x 109/L after HCT was 10 days in both groups (range 8–12 days for OP and 9–12 days for IP). Fever during neutropenia occurred in 7/16 (43.8%) of OP and 16/31 (51.6%) of IP HCT pts. Positive blood cultures (BCs) were identified in 3/7 (42.9%) of the febrile OP and 5/16 (31.3%) of the febrile IP HCT pts. Positive BCs in febrile OP HCT pts were coagulase-negative Staphylococcus (CNS) (2 pts) and Pseudomonas (1 pt). Positive BCs in febrile IP HSCT pts were CNS (2 pts), Streptococcus viridans (1 pt), Enterococcus (1 pt) and Bacillus (1 pt). One febrile OP HCT pt had cytomegalovirus pneumonia, and one afebrile IP HCT pt had a positive BC for CNS. Four febrile IP HCT pts with negative BCs had other infections, including cellulitis (2), facial abscess (1), and Clostridium difficile-associated colitis (CDAC) (1). Two other IP HCT pts (1 with fever and positive BC for CNS, 1 without fever) also had CDAC, for an overall incidence of CDAC of 3/31 (9.7%) after IP HCT. In contrast, no OP HCT pts had CDAC. Overall, the incidence of infections was 4/16 (25%) after OP HCT and 11/31 (35.5%) after IP HCT. We conclude that the infection profile of OP HCT compares favorably with that of IP HCT in pts with MM and is associated with a lower incidence of CDAC.
Clinical Significance of Positive Clostridium Difficile (CD) Toxin in the Stool of Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation (HSCT).
Authors: Terada Yoshiki , Onishi Yasushi , Kim Sung-Won , Kami Masahiro , Mori Shin-ichiro , Heike Yuji , Takaue Yoichi , Tanosaki Ryuji
Published: April 2020

CD infection is a common complication in immunocompromised patients, including those undergoing HSCT, with reported incidences ranging from 4% to 13%. However, there has been no previous detailed report on the clinical impact of positive CD toxin in patients after allogeneic HSCT with current supportive therapies. Therefore, we retrospectively reviewed the medical records of 422 patients who underwent allogeneic HSCT with a variety of preparative regimens at National Cancer Center Hospital from January 2000 to April 2004. CD toxin in stool samples was measured by the Latex particle agglutination method, and selected patients were examined further by endoscopy. Positive results with CD toxin were observed at least once in 51 patients, at a median time of 40 days (range, −1 to 212 days) following allogeneic HSCT. Most patients had severe watery diarrhea, along with other symptoms including fever, nausea, anorexia and abdominal cramping, which mimics pseudomembrane colitis. Twenty-seven of the 51 patients (53%) underwent endoscopic examination, and macroscopic findings were as follows; normal in 4 (15%), extensive edema in 11 (41%), and mucosal sloughing in 12 (44%), while none had pseudomembrane formation. Histological diagnosis revealed that 26 cases (96%) were compatible with GVHD, including 2 complicated with Cytomegalovirus colitis. Thirty-three patients (65%) were treated with oral vancomycin for a median of 14 days (2 to 46 days), while the remaining 18 were followed conservatively without any specific medication. Forty-four patients were evaluable for follow-up, and, regardless of management, all subsequently became negative for CD toxin. These findings suggest that positive CD toxin in the stool detected during the course of allogeneic HSCT may only reflect nonsignificant colonization or proliferation of CD in the gut, and hence may not necessarily imply clinically relevant pseudomembranous colitis which would require intense medical treatment. Additionally, in patients undergoing allogeneic HSCT, signs and symptoms of intestinal involvement may simply reflect concomitant GVHD per se or a pathology closely related to GVHD.
Full Donor Chimerism in Both Plasma and Mononuclear Cell after Haematopoietic Stem Cell Transplant (HSCT) at D28 Is Predictive of Acute Graft-Versus-Host Disease but Not Disease Relapse or Graft Failure.
Authors: Ma Shing Yan , Au Wing Yan , Lei Albert K.W. , Tse Eric , Chim Chor Sang , Liang Raymond , Kwong Yok Lam
Published: April 2020

Mononuclear cell mixed donor/recipient chimerism has been shown extensively to have high predictive value in disease relapse and graft rejection. Recently, donor DNA in plasma of transplant recipient has been suggested as a potential tool for post HSCT monitoring for disease relapse and graft rejection but its role has not been defined. Moreover, the impact of GVHD on plasma donor DNA remains unknown. In the current study, both the plasma and mononuclear cell donor chimerism of 40 patients post HSCT (mean age = 40; 35 myeloablative, 5 non-myeloablative) were analyzed at D28. Each patient was followed-up for one year or when disease relapse/graft rejection occurred. GVHD of grade II or above was also recorded to establish its correlation with the chimerism status. At D28, all patients were engrafted and alive. 5 (13%) patients had full donor chimerism in both plasma and mononuclear cell (Group A), 7 (18%) patients had full donor chimerism in the mononuclear cell only but not the plasma (Group B) and 28 (70%) patients failed to have full chimerism in both plasma and mononuclear cell (Group C). The sex, age, HLA-matching, donor relationship and ABO blood group matching had no impact on the chimerism status. None of the Group A patient had disease relapse/graft rejection at 1 year when compared to 13/35 (37%) of Group B and C patients (p=0.154, not significant).However, all the patients in Group A had acute GVHD which was significantly more than those in Group B and C (5/5 vs 12/35, p=0.009) and the acute GVHD would not alter the chimerism status. These results provide a foundation for further research on the potential role of plasma donor chimerism in predicting disease relapse/graft rejection and GVHD so that early intervention can be instituted.
Increased Risk for EBV-Lymphoma after Allogeneic Hsct in Adult Lymphoma Patients?A Retrospective Study of Reduced Compared to Myeloablative Conditioning.
Authors: Hagglund Hans G. , Brune Mats , Oberg Gunnar , Hagberg Hans , Theorin Niklas , Remberger Mats , Kimby Eva
Published: April 2020

The aim of this study was to compare the outcome of allogeneic HSCT after myeloablative versus reduced intensity conditioning (RIC) for lymphoma.

From January 1984 to June 2004, 87 patients underwent HSCT for lymphoma (including 41 patients with RIC, all grafted after May 1998). The diagnoses were 15 and 24 aggressive, 18 and 17 indolent and 8 and 5 HD lymphomas, in the RIC and myeloablative groups, respectively. There were 64 males and 23 females. The median age was 40 (16–61). The donors were 3 identical twins, 55 related and 29 unrelated donors. The conditioning was based on chemotherapy only in 32 and 8 and irradiation in combination with chemotherapy in 9 and 38 patients (p<0.001), in the RIC and myeloablative groups, respectively. Immunosuppression consisted of MTX and CsA in 68, CsA and MMF in 10, MTX or CsA in 4, T-cell depletion in 2 and 3 patients had no prophylaxis. In the RIC group the median age was significantly higher 49 (25–61) years than in the myeloablative group 38 (16–53) years (p<0.001). PBSC was used in 36 (88%) patients in the RIC group versus 17 (37%) (p<.001) in the myeloablative group.

The causes of deaths were 11 infections, 10 relapses, 6 EBV-lymphomas, 6 related to toxicity and 3 to GVHD and 1 renal cancer. Conclusions: Long term survival was seen in more than 50 % of patients with relapsing lymphoma after allogeneic HSCT. EBV lymphoma was the cause of death in 7% of the patients. The patient survival, TRM and relapse outcomes were similar after RIC and myeloablative conditioning but each subgroup is small. In the RIC group the patients were older, grafted in more advanced disease and the follow up shorter compared to the myeloablative group, therefore the results must be interpreted with care.

Results

RIC Myeloablative p GVHD (absolute incidence) Acute II-IV 43% 16% ns Chronic 32% 57% 0.06 Outcome (2-years probability) TRM 25% 39% ns Relapse 21% 16% ns Patient survival 61% 54% ns
Reduced Intensity Umbilical Cord Blood Transplantation(RI-UCBT) in Adult Patients with Graft Failure or Relapse after First Allogeneic Transplantation.
Authors: Masuoka Kazuhiro , Yuji Koichiro , Miura Yuji , Myojo Tomohiro , Kato Daisuke , Narimatsu Hiroto , Kusumi Eiji , Matsumura Tomoko , Wake Atsushi , Miyamoto Ko , Miyakoshi Shigesaburo , Yoneyama Akiko , Matsuzaki Michio , Ueyama Junichi , Morinaga Shinichi , Taniguchi Shuichi
Published: April 2020

Background: Graft failure, graft rejection, or disease relapse post allogeneic hematopoietic stem cell transplantation(HSCT) is life-threatening and serious complication that necessitate consideration for a second transplantation. Graft failure has been more frequently reported in patients with aplastic anemia with T-cell depletion of the graft, in cord blood transplants, or when unrelated or HLA-mismatched related donors were used. In second allogeneic stem cell transplant setting, current questions include suitable stem cell source, additional conditioning, immunosuppression, and the use of a different donor, still remain unknown. We performed a feasibility study of reduced intensity umbilical cord blood transplantation (RI-UCBT) in adult patients with graft failure or disease relapse after first allogeneic transplantation.

Patients and Methods: Nine patients (median age, 53 years; range 17–68) with advanced hematological diseases [AML (n=4), ALL (n=2), MDS (n=2), ATL (n=1)] who showed relapse or graft failure after first allogeneic transplantation underwent RI-UCBT with single cord blood unit at Toranomon Hospital between May 2003 and February 2004. Eight cases were in non-CR at transplant. A median time from first to second transplant was 226 days (range 31–475). The median number of mononuclear cells transfused was 2.3 x 107 /kg (range 1.8–3.5). HLA disparities were as follows; 5/6 in two cases, 4/6 in seven cases. Conditioning regimen mainly consisted of fludarabine 25 mg/m2 on days -7 to −3, melphalan 40mg/m2 (n=8) or busulfan 4mg/kg(n=1) day −3 to −2, and 4 Gy total body irradiation on day −1. Graft-versus-host disease (GVHD) prophylaxis was composed of ciclosporin alone (n=6) and tacrolimus alone (n=3).

Results: All of them achieved primary neutrophil engraftment (>500/μL) after a median of 22 days (range, 15–32) and achievement of donor T-cell chimerism was confirmed. Four cases developed grade II-IV GVHD, and one case developed limited chronic GVHD. Of all the nine cases received RI-UCBT, seven died from relapse (n=4), sepsis (n=2), and TMA (n=1). Two survived without relapse for +232 and +81 days, respectively.

Discussion: Although the number of patients is small and the follow-up period is short, our results corroborate that RI-UCBT for graft failure or relapse post first transplant is tolerated and may be worth considering for further evaluation. Neutrophil engraftment was achieved, however, disease relapse rate and treatment-related mortality is high. The management of disease status and development of ideal conditioning regimen will be the focus of future investigation.
Allogeneic and Syngeneic Hematopoietic Cell Transplants in Patients with AL-Amyloidosis: A Report from the European Group for Blood and Marrow Transplantation (EBMT).
Authors: Schoenland Stefan O. , Baccarani Michel , Campbell Andrew , Carreras Enric , Cordonnier Catherine , Grommisch Leanthe , Leblond Veronique , Littlewood Timothy J. , Verdonck Leo F. , Wood L. , Zachee Pierre , Zander Axel R. , Goldschmidt Hartmut , Niederwieser Dietger , Gahrton Goesta
Published: April 2020

AL amyloidosis is caused by a plasma cell dyscrasia in which clonal immunoglobulin light chains deposited in tissues leads to organ failure and death. Treatment with high dose melphalan and autologous PBSC rescue produces hematologic remissions in approximately 40% of evaluable patients and improvements in organ disease and quality of life. For a subgroup of patients, an allogeneic hematopoietic cell transplantation (HCT) may be useful. There is little experience with this procedure in patients with AL amyloidosis. We report the patients (pts) who were registered within the EBMT database. Eleven pts (median age 45 years, range 30–63; 3 female) with AL amyloidosis (n=10) or with multiple myeloma plus AL amyloidosis (n=1) underwent allogeneic (n=8) or syngeneic ( n=3) HCT during 1987–2002, 7 of them before 2000. Donors of allogeneic transplants were matched related siblings. Main organ manifestations were kidney, heart and liver. Indications for allogeneic HCT were young age, relapse after autologous HCT (n=2) and progressive or refractory disease. Conditioning regimen was ablative in 4 pts and reduced-intensity conditioning (RIC) was used in the other 4 pts. Three pts had T cell depletion as graft-versus-host-disease (GvHD) prophylaxis. Engraftment was evaluable in 7/8 pts, 2 pts rejected their grafts and 5 pts had durable engraftment. One out of 5 evaluable pts developed acute GvHD (grade IV), and 1 of 3 evaluable pts developed chronic GvHD (limited disease). Five pts (62,5%) died at a median of 51 (range 25 – 83) days after HCT, all before day +100. From 4 of these 5 pts causes of death are known: 1 pt died of a cerebral aspergillosis (day +54) and 3 pts died of amyloidosis. Three pts remain alive (median observation 27 months, range 12–79) after HCT. Best hematological responses after HCT in surviving pts were immunofixation-negative CR in 1 pt (with cGvHD), PR in 1 pt and SD in the third pt. One of the 3 pts with syngeneic HCT is alive about 60 months after the procedure, the other 2 pts. died of TRM day +7 and +18, respectively. In summary, a very high day +100 mortality was observed. Main problem was cardiac failure due to amyloidosis in the very early phase after HCT. Half of the pts survived RIC and ablative conditioning, respectively. We conclude that a very restrictive patient selection is necessary to decrease mortality as it has been shown for high-dose chemotherapy with autologous HCT. Whether RIC plays an more important role for this patient group in the future remains to be defined.
Hematopoietic Recovery after Allogeneic Transplantation Using G-CSF-Mobilized and Cryopreserved Peripheral Blood Stem Cells (PBSC).
Authors: Akahane Daigo , Onishi Yasushi , Won Sung , Kami Masahiro , Mori Shin-ichiro , Heike Yuji , Takaue Yoichi , Tanosaki Ryuji
Published: April 2020

Although PBSC is being used more often, the long-term reconstitution kinetics of donor-derived hematopoiesis have not yet been fully determined particularly in PBSC transplantation with a reduced-intensity regimen (RIST). This study focused on the long-term follow-up of hematopoiesis in recipients who underwent allogeneic transplantation with G-CSF-mobilized and cryopreserved PBSC from an HLA-matched related donor from June 1999 to January 2004. A total of 144 patients were analyzed ; 55 received conventional stem cell transplantation (CST) with a myeloablative regimen, whereas 89 received RIST. After PBSC were mobilized by 3 to 5 days of subcutaneous injection of G-CSF at a dose of 10μg/kg/day, leukapheresis was performed using a Spectra (Gambro) or AS104 (Fresenius) cell separator. Apheresed products were centrifuged (270 x g, 20 min) and depleted of platelets. Cells were then cryopreserved with 10% DMSO, 20% autologous plasma and RPMI 1640 using a programmed freezer, and stored in LiqN2 until use. All patients received G-CSF 5μg/kg/day starting from day 6 after transplantation. Hematological data were examined during recovery, at day 100, 1 year and 2 years. The median number of infused CD34+ cells was 3. 9 x 106/kg (range, 1.8–7.8) in CST and 3.5 x 106/kg (1.4–7.8) in RIST. The median number of days to ANC > 0.5 x 109/l and PLT > 50 x 109/l were, respectively, 13 (9–25) and 14 (7–83) in CST, and 11 (1–15) and 12 (1–44) in RIST. There was no engraftment failure, and no significant relationship was observed between the CD34+ cell dose (at least in the range of 2.0 x 106/kg) and the speed of engraftment in either group. Grade II to IV acute graft-versus-host disease (GVHD) and extensive chronic GVHD occurred in 23/55 cases (42%) and 12/43 (28%) in CST, and in 37/89 (42%) and 19/60 (32%) in RIST, respectively. Each blood component in peripheral blood in non-relapsed patients remained essentially within the normal range (WBC >3x109/l, Hb >10g/dl, PLT >1x1011/l) in 21/43 cases (49%) at day 100, in 25/26 (96%) at 1 year, and in 14/14 (100%) at 2 years in the CST group, whereas in the RIST group these values were 24/60 (40%) at day 100, 29/38 (76%) at 1 year, and 15/16 (94%) at 2 years, probably due to increased non-relapse complications including GVHD, etc. These results suggest that a CD34+ cell dose in PBSC of 2.0 x 106/kg before cryopreservation appears to be a good indication for stable long-term hematological recovery for both CST and RIST.
Factors Associated with Clinical Outcomes of 483 Allogeneic Peripheral Blood Stem Cell Transplants (PBSCT) in Brazil.
Authors: Vigorito Afonso C. , Aranha Francisco J.P. , Oliveira Gislaine B. , Eid Kátia A.B. , Lodi Fernanda M. , Azevedo Wellington M. , Colturato Vergílio , De Souza Mair P. , Barros José C. , Bittencourt Henrique , Silla Lúcia M.R. , Brandalise Silvia R. , Pontes Érica R. , Maiolino Angelo , Nucci Márcio , Ruiz Milton A. , Zanichelli Maria A. , De Souza Cármino A.
Published: April 2020

Uncertainty still exists with the effects of allogeneic PBSCT on the clinical outcomes of patients with hematological malignancies. Our aim was analyzed retrospectively the clinical outcomes of 483 patients who underwent an allo PBSCT in 9 Brazilians centers from May 1994 to February 2004. The analyzes included patients with hematological malignancies who underwent PBSCT from HLA identical siblings donors treated with G-CSF at a dose of 10mgr/kg/day, 5 days. Median age was 34ys old (2–57), advanced disease was present in 58%, conditioning without irradiation was 85%; GVHD prophylaxis with MTX/CsA was 91%; CD34+ median was 4.7X106/kg (0.51–71.6); the median follow-up for surviving patients was 797 days (8–3420); median day for neutrophils and platelets engraftment was 15 and 14, respectively; cumulative incidence (CI) for ³ 2 aGVHD was 38%; extensive cGVHD 63%; CI for transplant relate mortality (TRM) 59%; CI for relapse 37%; the estimates of OS and DFS at 9 ys are 33% and 42, respectively. The following factors were significantly associated with better outcomes for engraftment, aGVHD, cGVHD, OS, DFS, relapse, and TRM in univariate analyzes, respectively: neutrophils and platelets engraftment: CD34+ cell dose > 2.8X106/kg, GVHD prophylaxis other than MTX/CsA, and sex match other than female donor for male recipient; aGVHD: age<43ys old, and CD34 dose > 4.7X106/kg; cGVHD: age < 25ys, sex match other than female donor for male recipient, advanced disease and CD3+ dose < 170X106/kg; OS: early disease; DFS: early disease, CD34+ dose > 4.7X106/kg; relapse: age> 25ys, CD34+ cell dose > 2.8X106/kg, and early disease; TRM: early disease. All the results remained significant in multivariate analyzes, but CD34+ dose and platelet engraftment; age and CD34+ dose in aGVHD; age and CD3+ in cGVHD, and age in relapse. In our experience, sex match, CD34+ dose, GVHD prophylaxis may influence the engrafment, and sex match and disease phase the cGVHD. Furthermore, advanced disease had a negative impact on OS and TRM;. CD34+ higher dose and early disease were associated with better DFS and lower relapse.
B-Cell Depletion and Prevention of Hemolysis in Allogeneic Peripheral Blood Stem Cell Transplantation with Minor ABO Incompatibility.
Authors: Tavernier Emmanuelle , Thiebaut Anne , Nicolini Franck , Le Quoc Hung , Thomas Xavier , Chelghoum Youcef , Revesz Daniela , Hecquet Olivier , Dubost Philippe , Bourgeot Jean-Paul , Michallet Mauricette
Published: April 2020

Immune hemolysis is a severe complication of allogeneic peripheral blood stem cell (PBSC) transplantation with minor ABO-incompatibility, principally observed after conventional allogeneic PBSCT. The B-cell depletion of the PBSC graft is a prophylactic intervention which may decrease incidence and severity of this event. Eight patients who underwent allogeneic PBSCT in our institution between August 1999 and April 2004 were analysed. They all received a B-cell depleted transplantation performed because of minor ABO-incompatibility. There were 5 males and 3 females and the median age was 42.5 years (19–65). The pre-transplant diagnosis were solid tumours for 2 patients and haematological malignancies for 6. All received allogeneic PBSC recruited by G-CSF from 3 HLA identical unrelated and 5 HLA identical sibling donors. The median number of nucleated cells and CD34+ cells harvested were 7.75x108 /Kg (3.9–11) and 6x106/kg (4.3–8.7) and the median number of infused cells were 5.5x108 /Kg (3.4–8.71) and 5x 106/kg (3.1–8.33) respectively. Two patients received myeloablative and 6 patients reduced intensity conditioning regimens (RIC). As graft-versus-host-disease (GVHD) prophylaxis, cyclosporin with methotrexate was given after myeloablative conditioning. After RIC, cyclosporin alone (n=4) or cyclosporin associated to methotrexate (n=1) or cyclosporin associated to mycophenolate mofetil (n=1) were given. The ex-vivo manipulation consisted of a negative CD19 immuno-selection using the Isolex 300 I Baxter procedure. The median number of CD19+ cells (x106/kg) before and after ex-vivo manipulation was respectively 73 and 0.05, which represented a B-cell log depletion of 3.16. The ex-vivo manipulation nevertheless induced a nucleated cell depletion of 20% (from 7.75 x108 cells/kg to 5.52x108 cells/kg after B-cell depletion) and a CD34+ cell loss of 18.5% (from 6x106 cells/kg to 5x106 cells/kg). Engrafment was observed for all patients and median time to neutrophiles (>0.5 G/l) and platelets (>50 G/l) recovery were respectively 19 and 12 days. Acute GVHD occurred for 5 patients whereas 3 patients developed chronic GVHD. At last follow-up, 4 patients died, 3 due to progressive disease, one due to hepatic GVHD. None of these 8 patients developed any clinical severe hemolysis, except one patient who presented a biological hemolysis with the development of a positive antiglobulin test 3 weeks after transplantation, without any consequences. Three months after transplant and at the last follow-up, the 4 long-term alive patients showed a stable erythropoietic reconstitution with an ABO-RhD donor determination. These data indicate that B-cell depletion is an efficient and safe method which could be used in case of minor ABO incompatibility.
Reduced-Intensity Conditioning Regimen without Total Body Irradiation undergoing Umbilical Cord Blood Transplantation (non-TBI-RI-UCBT) Induces Successful Engraftment in Adult Patients with Advanced Hematological Malignancies.
Authors: Miyamoto Ko , Miyakoshi Shigesaburo , Kyo Aki , Yuji Koichiro , Kusumi Eiji , Sano Fumiaki , Taniguchi Shuichi , Nagosi Haruhisa
Published: April 2020

Purpose: In a phase I study to evaluate the feasibility and safety of non-TBI-RI-UCBT in patients with advanced hematological malignancies, who were ineligible for conventional allogeneic hematopoietic stem cell transplantation.

Patients and Methods: We treated a total of 6 patients (2 Hodgkin disease, 3 Follicular Lymphoma, 1 MDS overt leukemia). There were 4 female and 2 male, with a median age of 52.5 (46–62) years old. At the transplantation, all patients were in progressive disease (high-risk). Two patients with lymphoma had failed one or more preceding autologous transplants and a patient with overt leukemia had failed an allogeneic transplant. Backbone agent in the conditioning regimen was fludarabine, in combination with melphalan (n=4), or melphalan and thiotepa (n=2). Graft-versus-host disease (GVHD) prophylaxis incorporated cyclosporine alone (n=3), or FK506 alone (n=3). All of the patients received 4 of 6 HLA -antigen-matched umbilical cord blood transplantation. The median number of infused total nucleated cell dose and CD34 plus cell dose before freezing were 3.18 x 107 (2.77–4.7) /kg and 0,85 x 105 (0.57–1.91) /kg, respectively.

Results: All of the patients achieved primary neutrophil engraftment. The median time to reach 0.5 x 109/L neutrophils was 24 (13–31) days. Platelet counts above 20 x 109/L were achieved by 3 patients on median day of 40 (39–45) days. The median time to complete donor chimerism was 26.5 (16–35) days. Acute GVHD occurred in 2 of the 6 patients (grade I/II in 2). Chronic GVHD developed in none of the evaluable patients. Two of the 6 patients developed noninfectious fever before neutrophil engraftment. Three patients died of TRM (1 pneumonia, 1 thrombotic microangiopathy, 1 acute heart failure), and the other 2 patients died due to disease progression. At a median follow-up of 65.5 days (33–78), one of the 6 patients is currently alive in CR.

Conclusion: Although this study is preliminary with a small number of patients and short follow-up, our findings demonstrated the successful engraftment of non-TBI-RI-UCBT for adult patients with relapsed/refractory hematological malignancies. Prospective study to further evaluate the efficacy of non-TBI-RI-UCBT for hematological malignancies is warranted.
ENU Mutagenesis Screen Identifies a Novel Regulator of Platelet Production.
Authors: Stevenson William S. , Metcalf Donald , Hilton Douglas J. , Roberts Andrew W. , Alexander Warren S.
Published: April 2020

The genes that regulate variation in steady-state platelet count are largely unknown. We have attempted to identify novel genes that regulate platelet production by creating heritable models of thrombocytosis in mice by randomly inducing point mutations in the genome with chemical mutagenesis.

Mice with a high platelet count were identified in the third generation of an ENU mutagenesis screen and several different lines of thrombocytotic mice have been established. Subsequent breeding of one line has demonstrated that this mutation, called Plt2, is inherited in an autosomal recessive manner. Automated platelet counts for Plt2 homozygote mice were 1997±642x109/L (mean±2SD; n=51), 140% of those observed for wild-type mice (1487±372; n=56) and Plt2 heterozygote mice (1402±554; n=76). Analysis of Plt2 animals has shown increased numbers of megakaryocyte precursors (as measured by meg-CFC/5x105 cells to maximal cytokine stimulation) in the bone marrow (Plt2 40±20 compared to wild-type (WT) animals 28±8; n=7) and spleen (Plt2 16±6 compared to WT 8±3; n=7, p=0.01). Mature megakaryocytes were increased in frequency in bone marrow sections (Plt2 86.8±28.4 compared to WT 56.8±18.5) and demonstrate a similar maturation profile to control animals when their nuclear ploidy is measured by flow cytometry.

Reciprocal bone marrow transplantation experiments demonstrate that the thrombocytotic phenotype was observed in Plt2 homozygous recipients of either wild type or Plt2 homozygous bone marrow, but not observed in wild type recipients of Plt2 homozygous bone marrow. This data indicates that the Plt2 mutation is acting extrinsically on the haemopoietic system. Despite having an elevated platelet count, Plt2 mutant mice have inappropriately elevated serum thrombopoietin levels (Plt2 5420±2842; n=15) compared to controls (2947 ±2310; n=13; p<0.0001).

Mapping experiments have excluded both thrombopoietin and the thrombopoietin receptor, c-mpl, as the mutated gene. Further experiments will aim to map this mutation, which may be a novel regulator of thrombopoietin.
Rapid Cyclosporine Tapering and Isotretinoin Usage after Unrelated Donor Hematopoietic Stem Cell Transplantation in Juvenile Myelomonocytic Leukemia.
Authors: Yoo Keon Hee , Lee Soo Hyun , Sung Ki Woong , Koo Hong Hoe , Jung Hye Lim
Published: April 2020

Juvenile myelomonocytic leukemia (JMML) is a rare type of childhood leukemias, and allogeneic hematopoietic stem cell transplantation (HSCT) is known to be the only way to cure the disease. Unfortunately, relapse is still the most frequent cause of treatment failure after transplant in JMML. We investigated the feasibility of inducing graft versus leukemia (GVL) effect and the use of a differentiating agent even after unrelated HSCT in children with JMML. Seven consecutive patients with JMML underwent unrelated HSCT at a median age of 17 months. The sources of grafts were bone marrows (n=3) or HLA 1- or 2-antigen mismatched cord bloods (n=4). Only 3 of the 7 patients were in complete remission before transplantation. Intravenous busulfan, cyclophosphamide, and etoposide were used as preparative agents except in one who was conditioned with TBI-based regimen. Cyclosporine was used universally for GVHD prophylaxis with additional use of short-term methotrexate in bone marrow transplants and of methyl-prednisolone in cord blood transplants. Cyclosporine was tapered rapidly from around 1 month post-HSCT and isotretinoin (75–100 mg/m2/day) was used in selected patients who have any risk factors of relapse. Cyclosporine blood levels were 247.8±91.1, 146.6±104.2, and 88.8±52.6 ng/mL at 1, 2, and 3 months post-transplant, respectively. There was no grade 3 or 4 acute GVHD and only 2 patients developed grade 2 acute GVHD which was improved without additional treatment. Chronic GVHD was developed in 3 (1 limited, 2 extensive) of the evaluable 5 patents, which was all resolved after combined use of immune suppressive agents. Initial chimeric status analysis at 1 month revealed complete donor chimerism (CC) in 4 patients, mixed chimerism (MC) in 2 and autologous recovery (AR) in one. One of the patients with MC and the one with AR were in disease-free status. One patient whose chimeric status changed from CC to MC eventually relapsed. One patient with initial MC with residual disease turned to CC with complete remission. Another patient with initial MC but with no evidence of disease is on treatment with isotretinoin without relapse for 3 months even with persistent MC. The patient with AR relapsed early after transplant. Five patients are alive relapse-free and disease-free with a median follow-up of 16 months after transplant. The Kaplan-Meier probability of event-free survival was 66.7%. We suggest that GVL induction strategy with concomittant use of a differentiating agent might have a role to suppress leukemic relapse in JMML.
Pediatric Patients Transplanted with Positive Selected Stem Cells from Alternative Donors Have a Low Incidence of CMV Dnaemia.
Authors: Lang Peter , Hamprecht Klaus , Greil Johann , Bader Peter , Einsele Hermann , Neuheuser Friederike , Handgretinger Rupert , Niethammer Dietrich
Published: April 2020

Human cytomegalovirus (HCMV) remains a cause of serious infectious complications after allogeneic transplantation of hematopoietic stem cells, especially in recipients of T cell depleted grafts. Here, we investigated the incidence of CMV-DNAemia in a cohort of 100 pediatric patients with leukemias and nonmalignant diseases, who received megadoses of CD34+ or CD133+ selected (and therefore highly T-cell depleted) grafts from matched unrelated or mismatched related (haploidentical) donors. Graft versus host disease was minimized with this approach (acute GvHD grade III-IV 5%) but T-cell recovery was delayed in most patients.

All patients received prophylactic acyclovir. PCR screening for CMV was performed weekly from leukocytes and plasma and additional antiviral treatment was started in the case of positive findings. Cumulative incidence of CM-DNAemia at day 100 was 29%. Seropositive recipients (n=44) had a significantly higher incidence of CMV-DNAemia than seronegative recipients (n=56) (64% vs. 0.05%; p=0.0001). In contrast, the incidence was not influenced by the serostatus of the donors (patients with seropositve donors (n=41): 33%; patients with seronegative donors (n=59): 28%; p=0.6). D+R+ pairs were not superior to D-R+ pairs. CMV related over-all mortality was<10%. Conclusions: the incidence of CMVDNAemia was remarkably low in our transplanted patients, despite profound T-cell depletion. Donor seropositivity had no influence. An explanation for these observations may be, that only purified stem cells were transplanted (representing less than 1% of the total cell number of an unmanipulated graft) and therefore, patients received a minimal number of potentially CMV infected leukocytes.We suppose, that this effect may counterbalance the transiently impaired T-cell function in our patients and supports the use of CD34+ or CD133 selection in order to prevent GvHD.
Impact on Short and Long-Term Hematopoiesis of G-CSF Administered Posttransplant in Function of the Amounts of Peripheral Blood CD34+38− Cells Reinfused.
Authors: Henon Philippe R.G. , Sovalat Hanna , Ojeda-Uribe Mario , Arkam Yazid , Eisenmann Jean-Claude
Published: April 2020

We previously suggested that a reinfused threshold dose of CD34+38− cells = 5x104/kg b.w. better predict both short- and long-term engraftment after PBSCT than total CD34+ cells assessment, and should thus avoid unecessary postransplant (Tx) G-CSF administration. Therefore, we have further conducted a prospective study comparing postTx data from cancer patients undergoing autologous PBSCT and were administered or not G-CSF depending on the amount of CD34+38− cells reinfused. 48 patients (mean age 49y) were transplanted with, on average 2.5x104 CD34+38− cells/kg b.w. (range 1–49) and were consequently administered G-CSF 5 μg/kg daily from d5 to ANC = 109/1 (Group-I). 46 patients (mean age 50y) received an average of 20.5x104 CD34+38− cells/kg (range 5.5–162) without postTx G-CSF (Group-II). These 2 groups were compared and paired two by two with 2 groups of "historical" patients referred as controls : 11 patients (mean age 44y) had received, on average, 2.5x104 CD34+38− cells/kg b.w. (range 1.1–4.8) without G-CSF (Group-III); 29 patients (mean age 51y) received an average of 15.2x5x104 CD34+38− cells/kg b.w. (range 5.5–60) systematically associated with G-CSF for protocolar reasons (Group-IV). PostTx trilineage hematopoietic engraftment (up to 2 years), clinical and economical parameters were systematically recorded for each group of patients and statistically compared. PostTx ANC recovery occurred sooner, was faster and reached higher levels in the G-CSF groups (II and IV) compared to the others; platelets recovery kinetics was significantly faster in Group-III compared to the others; reticulocytic recovery was not statistically different whichever the group. When age, sex, disease, TBI did not significantly influence trilineage engraftment, a multiparametric study showed strong positive impacts of total CD34+ cells reinfused on ANC kinetics and of CD38− subset amounts on platelet kinetics, which was on the contrary slowered by G-CSF administration. Group-I patients received more transfusions, stayed longer hospitalized and costed more than those of the 3 other groups. Regarding long-term hematopoiesis, platelets and hemoglobin levels were globally higher in Group-III compared to the other groups, but still more dramatically compared to Group-IV from 1 to 9 months, which might be explained by differences in BM CD34+ and 38− subset differentiation.

In conclusion, if postTx G-CSF certainly accelerates ANC recovery, it seems to be to the detriment of short- an d median-term platelets and hemoglobin recovery, even in case of reinjection of CD34+38− cell doses = 5x104/kg b.w., which appears thus to be significantly discriminant for G-CSF administration decision.