scholarly journals Allo-HSCT from MUD/MRD As a Curative Treatment in Paroxysmal Nocturnal Hemoglobinuria

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1251-1251
Author(s):  
Miroslaw Markiewicz ◽  
Anna Koclega ◽  
Monika Dzierzak-Mietla ◽  
Patrycja Zielinska ◽  
Ewa Mendek-Czajkowska ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cyclosporine A ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Hemorrhagic Cystitis ◽  
Curative Treatment ◽  
Hematopoietic Stem ◽  
Donor Chimerism ◽  
Pnh Clone

Abstract Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal abnormality of the hematopoietic stem cell caused by somatic mutation in the phosphatidylinositol glycan-class A (PIG-A) gene located on the short arm of the X chromosome. Cells with lack phosphatidylinositol glycoproteins are more sensitive to complement-mediated lysis. Despite the efficient symptomatic treatment of hemolytic PNH with eculizumab, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment of the disease, although outcomes presented in the past were controversial. Material and methods: We report 32 allo-HSCTs: 31 from MUD and 1 from MRD performed for PNH in 2004-2014. Median age of recipients was 28 years (range 20-55) and donors 33(19-53), median time from diagnosis to allo-HSCT was 18(2-307) months. Median size of PNH clone was 80% granulocytes (0.41%-98%). Indication for allo-HSCT was aplastic/hypoplastic bone marrow (15 pts), overlapping MDS (2 pts), severe course of PNH with hemolytic crises and transfusion-dependency without access to eculizumab (15 pts). Additional risk factors were Budd-Chiari syndrome and hepatosplenomegaly (1 pt), history of renal insufficiency requiring hemodialyses (2 pts) and chronic hepatitis B (1 pt). The preparative regimen consisted of treosulfan 3x14 g/m2 plus fludarabine 5x30 mg/m2 (25 pts) or treosulfan 2x10 g/m2 plus cyclophosphamide 4x40 mg/kg (7 pts). Standard GVHD prophylaxis consisted of cyclosporine-A, methotrexate and pre-transplant ATG or thymoglobulin in MUD-HSCT. 2 pts instead cyclosporine-A received mycophenolate mofetil and tacrolimus. Source of cells was bone marrow (12 pts) or peripheral blood (20 pts) with median 7.7x10(8)NC/kg, 5.3x10(6)CD34+cells/kg, 24.2x10(6)CD3+cells/kg. Myeloablation was complete in all pts with median 9 days (6-13) of absolute agranulocytosis <0.1 G/l. Median number of transfused RBC and platelets units was 8.5(1-16) and 8(3-18). Results All pts engrafted, median counts of granulocytes 0.5 G/l, platelets 50 G/l and Hb 10 g/dl were achieved on days 17.5(13-33), 17.5(11-39) and 19.5(11-34). Acute GVHD grade I,II and III was present in 14, 6 and 1 pt, limited chronic GVHD in 11 pts. LDH decreased by 77%(5%-91%) in first 30 days indicating disappearance of hemolysis. 100% donor chimerism was achieved in all pts. In 1 patient donor chimerism decreased to 83% what was treated with donor lymphocytes infusion (DLI). 2 patients died, 1 previously hemodialysed pt died on day +102 in a consequence of nephrotoxicity complicating adenoviral/CMV hemorrhagic cystitis and second on day +56 because of severe pulmonary infection. Complications in survivors were FUO (7 pts), CMV reactivation (8), VOD (1), neurotoxicity (1), venal thrombosis (1), hemorrhagic cystitis (1) and mucositis (8). 30 pts (93.7%) are alive 42 months (1-85) post-transplant and are doing well without treatment. Complete disappearance of PNH clone was confirmed by flow cytometry in all surviving pts. Conclusions: Our results indicate, that allo-HSCT with treosulfan-based conditioning is effective and well tolerated curative therapy in PNH. Disclosures No relevant conflicts of interest to declare.

Allo-HCT from MUD/MRD for Paroxysmal Nocturnal Hemoglobinuria - 12 Years of Experience

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5886-5886 ◽  
Author(s):  
Miroslaw Markiewicz ◽  
Malwina Rybicka-Ramos ◽  
Monika Dzierzak-Mietla ◽  
Anna Koclega ◽  
Krzysztof Bialas ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cyclosporine A ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Conflicts Of Interest ◽  
Hemorrhagic Cystitis ◽  
Chronic Gvhd ◽  
Complete Disappearance ◽  
Hematopoietic Stem ◽  
Donor Chimerism ◽  
Pnh Clone

Abstract Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal abnormality of hematopoietic stem cell leading to lack of phosphatidylinositol glycoproteins, sensitizing cells to complement-mediated lysis. Despite the efficient symptomatic treatment of hemolytic PNH with eculizumab, allo-HCT is the only curative treatment for the disease, although outcomes presented in the past were controversial. Material and methods: We report 41 allo-HCTs: 37 from MUD and 4 from MRD performed for PNH in 2004-2016. Median age of recipients was 29(20-62) years and donors 30(19-53), median time from diagnosis to allo-HCT was 16(2-307) months. Median size of PNH clone was 80% granulocytes (0.5%-100%). Indication for allo-HCT was PNH with aplastic/hypoplastic bone marrow (19 pts), MDS (2 pts), overlapping MDS/aplasia (3 pts), severe course of PNH with hemolytic crises and transfusion-dependency without access to eculizumab (17 pts). Additional risk factors were Budd-Chiari syndrome and hepatosplenomegaly (1 pt), history of renal insufficiency requiring hemodialyses (2 pts), chronic hepatitis B (1 pt) and C (1 pt). The preparative regimen consisted of treosulfan 3x14 g/m2 plus fludarabine 5x30 mg/m2 (31 pts) or treosulfan 2x10 g/m2 plus cyclophosphamide 4x40 mg/kg (10 pts). Standard GVHD prophylaxis consisted of cyclosporine-A, methotrexate and pre-transplant ATG in MUD-HCT. 2 pts instead of cyclosporine-A received mycophenolate mofetil and tacrolimus. Source of cells was bone marrow (13 pts) or peripheral blood (28 pts) with median 6.3x108NC/kg, 5.7x106CD34+cells/kg, 24.7x107CD3+cells/kg. Myeloablation was complete in all pts with median 9(1-20) days of absolute agranulocytosis <0.1 G/l. Median number of transfused RBC and platelets units was 9(0-16) and 8(2-18). Results: All pts engrafted, median counts of granulocytes 0.5 G/l, platelets 50 G/l and Hb 10 g/dl were achieved on days 17.5(10-33), 16(9-39) and 19.5(11-34). Acute GVHD grade I,II and III was present in 16, 7 and 3 pt, limited and extensive chronic GVHD respectively in 11 and 3 pts. LDH decreased by 73%(5%-91%) in first 30 days indicating disappearance of hemolysis. 100% donor chimerism was achieved in all pts. In 1 patient donor chimerism decreased to 81% what was treated with donor lymphocytes infusion (DLI). 3 patients died, 1 previously hemodialysed pt died on day +102 due to nephrotoxicity complicating adenoviral/CMV hemorrhagic cystitis, two other SAA patients with PNH clone<10% died on days +56 due to severe pulmonary infection and +114 due to aGvHD-III and multi organ failure. Complications in survivors were FUO (10 pts), CMV reactivation (13), VOD (1), neurotoxicity (1), venal thrombosis (1), hemorrhagic cystitis (4) and mucositis (8). 38 pts (92.7%) are alive 4.2 (0.4-12) years post-transplant and are doing well without treatment. Complete disappearance of PNH clone was confirmed by flow cytometry in all surviving pts. Conclusions: Allo-HCT with treosulfan-based conditioning is effective and well tolerated curative therapy for PNH. Disclosures No relevant conflicts of interest to declare.

DNA Microarray Analysis of GPI-Negative Hematopoietic Stem Cells in Paroxysmal Nocturnal Hemoglobinuria Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1048-1048
Author(s):  
Kazuhiko Ikeda ◽  
Tsutomu Shichishima ◽  
Yoshihiro Yamashita ◽  
Yukio Maruyama ◽  
Hiroyuki Mano
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Data Set ◽  
Hematopoietic Stem ◽  
Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematological disorder which is manifested by complement-mediated hemolysis, venous thrombosis, and bone marrow failure. Deficiencies of glycosylphosphatidylinositol (GPI)-anchored proteins, due to mutations in the phosphatidylinositol glycan-class A (PIG-A) gene, contribute to complement-mediated hemolysis and affect all hematopoietic lineages in PNH. However, it is unclear how a PNH clone with a PIG-A gene mutation expands in bone marrow. Although some genes, including the Wilms’ tumor gene (Shichishima et al, Blood, 2002), the early growth response gene, anti-apoptosis genes, and the gene localized at breakpoints of chromosome 12, have been reported as candidate genes that may associate with proliferations of a GPI-negative PNH clone, previous studies were not intended for hematopoietic stem cell, indicating that the differences in gene expressions between GPI-negative PNH clones and GPI-positive cells from PNH patients remain unclear at the level of hematopoietic stem cell. To identify genes contributing to the expansion of a PNH clone, here we compared the gene expression profiles between GPI-negative and GPI-positive fractions among AC133-positive hematopoietic stem cells (HSCs). By using the FACSVantage (Becton Dickinson, San Jose, CA) cell sorting system, both of CD59+AC133+ and CD59− AC133+ cells were purified from bone marrow mononuclear cells obtained from 11 individuals with PNH. Total RNA was isolated from each specimen with the use of RNeasy Mini column (Qiagen, Valencia, CA). The mRNA fractions were amplified, and were used to generate biotin-labeled cDNAs by the Ovation Biotin system (NuGEN Technologies, San Carlos, CA). The resultant cDNAs were hybridized with a high-density oligonucleotide microarray (HGU133A; Affymetrix, Santa Clara, CA). A total of &gt;22,000 probe sets (corresponding to &gt;14,000 human genes) were assayed in each experiment, and thier expression intensities were analyzed by GeneSpring 7.0 software (Silicon Genetics, Redwood, CA). Comparison between CD59-negative and CD59-positive HSCs has identified a number of genes, expression level of which was statistically different (t-test, P &lt;0.001) between the two fractions. Interestingly, one of the CD59− -specific genes isolated in our data set turned out to encode a key component of the proteasome complex. On the other hand, a set of transcriptional factors were specifically silenced in the CD59− HSCs. These data indicate that affected CD59-negative stem cells have a specific molecular signature which is distinct from that for the differentiation level-matched normal HSCs. Our data should pave a way toward the molecular understanding of PNH.

Successful Hematopoietic Stem Cell Transplantation In a Child With Paroxysmal Nocturnal Hemoglobinuria

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5469-5469
Author(s):  
Gulsun Karasu ◽  
Yilmaz Ay ◽  
Sinan Akbayram ◽  
Suar Caki Kilic ◽  
Fügen Pekün ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Hemolytic Anemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Hematopoietic Stem ◽  
Matched Sibling ◽  
Pnh Clone

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder characterized by complement-mediated hemolysis, thrombosis, and bone marrow failure. The clinical manifestations of PNH are usually seen in adulthood and are very rarely reported in children. The experience with transplantation in the management of children with PNH is also very limited. Here in this paper, we report a child with PNHpresenting with an episode of hemolytic anemia who was treated successfully with hematopoietic stem cell transplantation (HSCT). Case Presentation An eleven-year-old patient presented with a history of fatigue. On physical examination, there was no lymphadenopathy, and liver and spleen were not palpable. Laboratory analysis revealed a hemoglobin of 5.7 g/dL, platelets of 124.000/mL, and white blood cells were 1420/ml. She had increased lactate dehydrogenase and reticulocyte count and decreased haptoglobulin level. Bone marrow biopsy showed erythroid hyperplasia and relative hypocellularity in myeloid cell lines. Chromosomal karyotyping of bone marrow cells was normal. To look for PNH, immunophenotyping was performed. Flow cytometric analysis showed a PNH clone within the RBCs, granulocytes and monocytes. These findings were consistent with a diagnosis of PNH and she was started on anti-complement therapy with eculizumab. Since matched sibling donor was available, she was referred to our center for transplantation. The conditioning regimen of HSCT consisted of fludarabine (40 mg/m2day -9 to -6) and busulphan (4 mg/kg, day -5 to -2) combined with anti-thymocyte globulin (ATG) (Fresenius, Munich, Germany) (5 mg/kg, day -2 to 0). GVHD prophylaxis consisted of metotrexate plus cyclosporin. The infusion of bone marrow stem cells contained 11.8x106 CD34+ cells/kg and 7.9 x108total nucleated cells/kg. The patient had no significant complications in the post-transplant period. Neutrophil and platelet engraftment occurred on posttransplant day 11 and day 18, respectively. She is alive and is doing well over 14 months following BMT. Recovery is complete with full donor chimerism and the eradication of PNH clone. Conclusion PNH can occur in children but is often misdiagnosed and mismanaged. Although with the advent of anti-complement therapy, pure hemolytic anemia is no longer a clear indication for HSCT in adult patient, it is not licensed for use in pediatrics. HSCT is the only curative option for patients with PNH and if suitable matched sibling donor is available, transplant should be considered. However, the experience with tranplantation is also very limited. This case is worth mentioning as it shows that busulphan, fludarabine and ATG can be safely and effectively used for conditioning in PNH in children. Disclosures: No relevant conflicts of interest to declare.

WT1 Transcripts Is a Powerful Leukemia Marker to Predict Early Relapse After Allogeneic Haematopoietic Stem Cell Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4488-4488
Author(s):  
Carlo Messina ◽  
Cristina Tresoldi ◽  
Maria Teresa Lupo Stanghellini ◽  
Alessandro Crotta ◽  
Stefania Girlanda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Expression Level ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Early Relapse ◽  
Expression Levels ◽  
Donor Chimerism ◽  
Before And After

Abstract Abstract 4488 Introduction Definition of leukemia remission requires the lack of disease markers at sub-microscopic level. This is highly important after a potentially curative approach as allogeneic hematopoietic stem cell transplant (HSCT), where early detection of relapse at molecular level may lead to modulation of immunosuppressive therapy or donor lymphocyte infusion (DLI). In AML various approaches has been used to define MRD, but still the majority of AML cases do not have a useful and sensitive MRD marker. Over-expression of Wilms' tumor gene 1 (WT1) in leukemic blasts has been reported in >80% of AML and >40% of MDS. Physiologic hematopoietic stem cell compartments also express WT1, however, a ‘malignant’ WT1 expression can be clearly distinguished based on quantitative detection methods such as semiquantitative RT-PCR. Quantification of WT1 expression levels can detect frequencies of leukemic cells in bone marrow (BM) and peripheral blood (PB) as low as 10-3 and 10-5, respectively. Therefore WT1 expression levels provide a new marker for leukemic blast cells regardless of the type of leukemia especially in the relevant percentage of patients that lacks a specific molecular marker of their disease and thus may be a useful marker MRD and may predict the relapse after allogeneic HSCT. Materials and methods We measured quantitative expression of WT1 at diagnosis, before and after allogeneic transplant monthly for the first six months and then every three months. The quantitative assessment of the WT1 transcript amount was performed by real-time quantitative polymerase chain reaction (RQ-PCR). Results Our study included 19 AML and 6 MDS pts who underwent allogeneic HSCT in our Institute between 12/2007 and 6/2009. Median age at diagnosis was 49 years (range 22-68). Bone marrow samples at diagnosis showed a WT1 median expression level of 5851.66 copies (range 77.98-31203.57). Fifteen pts (60%) had a specific cytogenetic marker that could allow MRD monitoring. At HSCT 17 pts (68%) were in CR, 5 (20%) had a refractory or relapsed disease, while 3 (12%) were transplanted upfront. Six pts (24%) received grafts from a matched sibling donors, 6 (24%) were transplanted from a matched unrelated donor (MUD), 10 (40%) from a familiar haploidentical donor and 3 (12%) received a cord blood unit. Myeloablative conditioning regimen consisted of Treosulfan 42 g/sqm, Fludarabine 150 mg/sqm, ALG 30 mg/kg and Rituximab 500 mg (last two drugs only for alternative donors). After HSCT a rapid decline of WT1 expression levels was observed in all pts that obtained or maintained a condition of CR. Two pts (8%) relapsed and both had an increase in WT1 expression before relapse. In the first relapsed pt, analysis of WT1 showed a dramatically increase between pre transplant level and day +28, while STR showed 100% donor chimerism. Relapse occurred on day +43, still on IST, with 69% of blast at AM evaluation and 40% donor chimerism. This pt was successfully reinduced with chemotherapy followed by allogeneic PBSC infusion without GvHD profilaxys obtaining a rapid reduction of WT1 (43.66). The pt developed GvHD that required a new IST, confirming a strong immune-surveillance of HSCT. The second pt was still on immunosuppressive treatment (IST) at the time of relapse (+136). WT1 in BM on day +30 was 30 cp and then increased gradually to 167 and 190 cp on day +67 and +102, respectively. Cytogenetic analysis and STR chimerism still showed a cCR with 100% donor chimerism. At relapse AM showed 10% blasts, WT1 expression level was 7447 cp with >95% donor chimerism. IST was discontinued and one month later WT1 expression level decreased to 1640 with >95% donor chimerism. In this situation we reasoned that DLI was the best available treatment. The total dose of donor T cell infused was 5 ×105 CD3/Kg. This procedure allowed an immune-mediated leukemia control with reduction of WT1 (1.58), cCR and 100% donor chimerism. Conclusions These data show that WT1 may be useful as a non-specific leukemia marker for monitoring MRD in AML and MDS after allogeneic HSCT and should enable the detection of early relapse allowing intervention at a more favourable stage than at overt relapse. We observed a complete concordance between WT1 expression levels and status of AML before and after allogeneic HSCT. Based on these results cases with increase of WT1 levels after HSCT and without GVHD may be candidate to immune intervention such as discontinuation of immunosuppression and/or DLI. Disclosures: Bonini: MolMed S.p.A.: Consultancy.

The Overview of the Short Term Curative Effects of HLA Haploidentical Hematopoietic Stem Cell Transplantation for Hematological Malignancies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5440-5440
Author(s):  
Jian Ouyang ◽  
Bing Chen ◽  
Yonggong Yang ◽  
Jingyan Xu ◽  
Xiaoyan Shao
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Hematological Malignancies ◽  
Mononuclear Cells ◽  
Hemorrhagic Cystitis ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Hematopoietic Reconstitution

Abstract From May 2003, 13 patients with refractory hematological malignancies received HLA haploidentical hematopoietic stem cell transplantations in out BMT center. 13 patients, including 9 male and 4 female, are with an average age of 31.4 years (range, 15 to 46). Among them, 4 cases were of accelerated phase of CML, 1 blast phase of CML, 1 polyleptic ANLL-M3, 1 ANLL-M2, 2 ANLL M4, 1 ANLL M6, 2 NHL, and 1 MDS-RA. Of the 13 donors, 8 were the mothers of the recipients, 4 were the siblings, and the rest was the son. All the donors were HLA haploidentical matched to the patients. 4 patients were conditioned with CY/TBI/Ara-C regimen, (Ara-C 3. 0g/m2, q12h, ×3 d; CTX45 mg/kg×2 d; TBI 5 Gy ×2 d, ATG5 mg/kg ×4 d)). 8 patients were conditioned with improved BU/CY regimen, (BU 4 mg/kg ×3 d, CTX 1. 8 g/m 2 ×2 d, Ara-C 2 g/m 2 × 1 d, Me-CCNU 250 mg/m 2 ×1 d, ATG5 mg/kg ×4 d) and 1 patient of MDS-RA was conditioned with nonmyeloablative regimen (Fludarabine 30 mg/m2/d × 5d, CTX 30 mg/kg/d x 2d, TBI 300 cGy d1. G-CSF was given to the donors at 250 mg /day for a continuous 5~7 days. On the 4th~8th day, their bone marrow was collected under epidural anesthesia. In the simple bone marrow transplantation, the amount of the bone marrow collected was 15~20 ml/kg recipient b.w. And BMT associated with HSCT was given to 9 of the patients. The average parameter of the mononuclear cells re-infused was 8.84×108/kg; while the CD34+ cells was 2.67×106/kg. The treatments of CsA, MTX, MMF, ATG, and anti-CD25 monoclonal antibody were given as prophylaxis for GVHD. All the 13 patients received standard supportive care, and got hematopoietic reconstitution. The mean time of engraftment with neutrophil count more than 1.0 x 109/L was 15 days and platelet count more than 20 x 109/L was 21 days. All the patients was tested with STR-PCR, and showed a genotype the same as the donor’s. 5 of the 13 patients suffered grade I~II acute GVHD. 1 patient suffered grade III dermo-GVHD 30 days after transplantation. The CMV-DNA of 3 patients turned out positive after the transplantation, 2 patients suffered grade I~II hemorrhagic cystitis. There were totally 2 deaths, one of which who was in the IV phase of NHL died due to recurrent disease after the transplantation, the other suffered graft rejection on the 28 day of the hematopoietic reconstitution, which complicated with centrum infection after the second transplantation. 11 recipients got CR after transplantation. 1 patient of the accelerated phase of CML relapsed on the +70 day and 1 M3 patient relapsed 18 months after transplantation, both of them died after ineffective therapy. One patient of accelerated phase of CML relapsed genetically 1 year after the transplantation, and then got remission after the administration of Glivec. Full donor-type engraftment was sustained successfully in these 8 recipients. The patients were followed up until June 2005, with a median follow-up time of 18 months (range, 12 to 25 months). Our initial results show that the conditioning regimens, either with or without radiotherapy, are able to transplant the haploidentical hematopoietic stem cell without first T lymphocytes depletion. If ATG, mycophenolate mofetil and anti-CD25 monoclonal antibody, etc. are applied, the incidence of severe aGVHD will be decreased. 8 of the 13 patients survived for more than one year after transplantation. It shows favorable short-term curative effects.

Eculizumab Treatment of Paroxysmal Nocturnal Hemoglobinuria Relapsing After Bone Marrow Transplant and Subsequent Clonal: Case Report

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5274-5274
Author(s):  
Andres L. Brodsky ◽  
Brenner Sabando Velez ◽  
Curutchet Ragusin
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Abdominal Pain ◽  
Case Report ◽  
Hematopoietic Stem Cell ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Clone Size ◽  
Hematopoietic Stem ◽  
Chronic Hemolysis ◽  
Terminal Complement

Abstract Abstract 5274 Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a chronic and life-threatening hematopoietic stem cell disorder characterized by deficiency of the GPI-anchored complement inhibitory proteins CD55 and CD59 on blood cells. The resulting uncontrolled complement activation is responsible for chronic hemolysis and can lead to serious clinical morbidities including thromboembolism (TE) and chronic kidney disease (CKD), which have been shown to increase risk of mortality. Patients may also experience debilitating quality-of-life (QoL) issues, including fatigue, shortness of breath, erectile dysfunction, and abdominal pain, attributed to chronic hemolysis and resultant nitric oxide scavenging by free hemoglobin. Although hematopoietic stem cell transplantation (HSCT) remains the only potentially curative option for PNH, the risk for substantial morbidities and mortality still exist. In patients with PNH undergoing HSCT, up to 45% die or develop acute or chronic graft-versus-host disease. Eculizumab, a first-in-class terminal complement inhibitor, is the only approved treatment for patients with PNH. By inhibiting terminal complement activity and chronic hemolysis, eculizumab reduces the incidence of TE, CKD, and transfusion requirements, improves anemia and QoL, and normalizes survival in patients with PNH. Aim: Report the benefits of eculizumab in a patient with PNH who relapsed after HSCT. Case Report: A 27-year-old woman presented in December 1993 with fever, diarrhea, hemoglobinuria, and acute renal failure requiring temporary hemodialysis (Table). She was subsequently diagnosed with PNH. In February 1995, allogeneic HSCT from an HLA-identical sibling donor was performed. In 2003, 8 years after successful engraftment, the patient relapsed and presented with hemoglobinuria, abdominal pain, corticosteroid dependence requiring 20 to 40 mg methylprednisone, and high transfusional requirements. In November 2007, she had a granulocyte clone size of 37.2% as determined by flow cytometry. In June 2009, she started eculizumab therapy which was associated with a rapid reduction in lactate dehydrogenase (LDH) from 4964U/L to 456U/L. She subsequently achieved resolution of asthenia, disabling fatigue, and abdominal pain, as well as transfusion independence and improvement in hemoglobin. In May 2010, she had a granulocyte clone size of 86.1% as determined by both CD55- and CD59-negative cells, and a granulocyte clone size of 98.7% as determined by CD16b-negative cells. Conclusions: The only potential cure for PNH—bone marrow transplantation—is associated with high risks of morbidity and mortality; therefore, for most patients the associated risks preclude this option. In this case study, we show that HSCT may not be curative in all patients and the PNH associated symptoms can arise after BMT. The potential benefits of eculizumab in this patient population should be considered in light of recent data that demonstrated normalized survival in PNH patients receiving long-term eculizumab treatment. Disclosures: Brodsky: Alexion Pharmaceuticals, Inc.: Consultancy, Speakers Bureau. Velez:Alexion Pharmaceuticals, Inc.: Consultancy.

Host CD4+CD25+FoxP3+ Regulatory T Cells Facilitate Donor Engraftment Without Impacting Graft Versus Host Disease Onset After Major-Mismatched Hematopoietic Stem Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1996-1996
Author(s):  
Antonio Pierini ◽  
Hidekazu Nishikii ◽  
Mareike Florek ◽  
Dennis B Leveson-Gower ◽  
Yuqiong Pan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cellular Therapy ◽  
Hematopoietic Stem ◽  
Graft Versus Host ◽  
Donor Chimerism

Abstract A major challenge following allogeneic hematopoietic stem cell transplantation (HCT) is to establish persistent engraftment of donor hematopoietic cells. Many strategies have been developed to permit engraftment involving high dose chemotherapy, serotherapy with anti-lymphocyte drugs or myeloablative irradiation resulting in highly toxic conditioning regimens. The introduction of less harmful therapies could result in less toxicity especially in the major mismatched setting and when reduced intensity conditioning is required. While recent studies have explored the mechanisms through which donor-type CD4+CD25+FoxP3+ regulatory T cells (Tregs) restrict the development of graft versus host and host versus graft reactions, less is known about the role of host-type Treg in the transplant setting. In syngeneic and minor mismatched HCT host Tregs comprise a major component of the Treg compartment in the first weeks after transplant. Moreover the transplant of in vitro primed host Tregs can improve donor engraftment in major mismatched models of HCT; therefore host Tregs could be one of the key controllers of the host versus graft reaction mediated by residual host CD4+ and CD8+ conventional T cells (Tcons), possibly influencing graft versus host disease (GvHD) onset and severity. In this study we investigated the role of host Treg after major mismatched HCT to understand their impact in graft facilitation and rejection and in GvHD induction and prevention. We investigated the mechanism through which this cell population works and we explored the feasibility and the effectiveness of host Treg adoptive transfer for cellular therapy in HCT animal models. Results CD4+CD25+FoxP3+ host Tregs persist for at least 28 days after total body irradiation (8 Gy) and transplantation of C57BL/6 (H-2b) T cell depleted bone marrow (TCD BM) into BALB/C (H-2d) mice. Host Treg could be found in spleen, lymph nodes and bone marrow with an increase in the Treg/CD4+ cell ratio. Moreover we observed that these residual host Tregs maintain their suppressive function in vitro if harvested 14 days after transplant and incubated with healthy mouse derived Tcons in a MLR. These results are even more relevant as transplanted mouse derived host Tcons lose their ability to proliferate confirming that host Tregs possess a numeric and functional advantage compared to residual host Tcons. Using FOXP3-DTR mice as hosts we observed that host Treg ablation results in reduced donor chimerism after major mismatched TCD BM transplant (p < 0.01, analysis performed 2 months after transplant). At the same time, the absence of host Tregs favors host CD4+ T cell persistence (p < 0.001) and delays B cell reconstitution (p < 0.001). Furthermore, we hypothesized that host Treg act as an immunological barrier for HSCs, providing a protective immunological niche. Confocal microscopic analysis of femurs performed at day 7 after TCD BM transplant confirmed that hypothesis showing host Tregs clustering in the epiphysis where donor hematopoietic stem cell (HSC) engraftment is mainly detectable. To strengthen these results and to provide a clinical translatable tool, we adoptively transferred 5x105/mouse highly purified unmanipulated host Tregs in a non myeloablative (TBI 5.5 Gy) major mismatched model of rejection. We found that the transferred host Tregs induce persistent full donor chimerism if injected together with a sublethal dose of donor Tcons (5x105/mouse, p=0.016) and transiently enhance donor chimerism in the first three weeks after transplant if injected with low dose interleukin-2 (IL-2, 50,000 IU bid for 7 days; p < 0.001) without impacting on GvHD incidence and lethality. The relatively low dose of injected Tregs, the possibility to stimulate and expand them in vivo with IL-2 and the safety of this model provide the first evidence of the feasibility of this clinical approach. Conclusion Our findings indicate that host Tregs facilitate bone marrow engraftment in major mismatched HCT models without impacting GvHD. Notably, our observations on the bone marrow environment after transplant strongly suggest that host Tregs can play a role in building the donor HSC cell niche. Finally host Treg adoptive transfer proved to be feasible and effective in animal models providing a new tool for cellular therapy and clinical translation. Disclosures: No relevant conflicts of interest to declare.

Monitoring of Bone Marrow Chimerism after Allogeneic Hematopoietic Stem Cell Transplantation – Evaluation of a qPCR Based Chimerism Assay

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3866-3866 ◽  
Author(s):  
Roland Reibke ◽  
Andrea Dick ◽  
Max Hubmann ◽  
Eva Hoster ◽  
Reinhard Henschler ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Early Detection ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Donor Chimerism

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) has become a valuable therapeutic option for malignant and non-malignant hematological diseases. Engraftment of donor cells is confirmed by repetitive testing for donor chimerism. Since the underlying malignant disease is host derived, the decrease of donor chimerism might precede or indicate the imminent relapse, enabling early intervention and presumably better outcome. STR-assay (Short-Tandem-Repeats-Assay) and XY-FISH (XY based Fluorescence in Situ Hybridization) respectively are routinely performed after HSCT. Furthermore CD34+ cell- chimerism and quantification of minimal residual disease in patients with informative markers might be used to detect early relapse. In this prospective, non-interventional study we evaluated the accuracy, reliability and feasibility of a qPCR based commercially available assay (Allele SEQR® Chimerism Assay, Abbott). In addition, the early detection of hematological relapse was analyzed as a clinical readout. Between May 2011 and January 2013 95 patients received allogeneic HSCT for MDS or acute leukemia (AML = 84) at our transplantation unit and were therefore included in our analysis. According to our local standard bone marrow samples were collected at standardized time points including days 30, 90, 180. The qPCR based Allele SEQR® Chimerism Assay consists of two parts: screening for discriminating, informative markers and subsequent actual quantification of the host DNA. For assay validation 68 patients were suitable. In all patient/donor pairs at least one informative marker could be found and quantitative results could be achieved. In 65/68 pairs (95.6%) even ≥ 2 markers could be identified, however, among 28 related donors three (10.7%) only revealed one discriminating marker. The sensitivity of the assay was proven by means of artificially spiked DNA samples mimicking an amount of 0.1%, 0.05% and 0.01% of “host“ DNA respectively. Testing was performed with 100ng/well patient DNA. The overall time needed for testing was less than four hours. We identified 61 patients with at least two samples in cytomorphological complete remission (CR) after allogeneic HSCT. The overall hematological relapse rate in our cohort was 23% until March 2014. To enable relapse prediction we quantified donor chimerism in CR samples. Scoring for suspicious results by the increase of host chimerism by at least one percent point, but not less than one third as compared to previous testing, was highly predictive for imminent relapse (overall relapse rate 86% vs. 15%, p=<0,001, Mantel-Byar Test). Median time from increased host chimerism to relapse was 68 days (25-201). Furthermore no patient relapsed within 120days without prior positive testing. The evaluated qPCR approach proved as a fast and highly sensitive tool for chimerism monitoring after HSCT. It may allow an early detection or exclusion of imminent relapse, enabling chimerism triggered therapy. In perspective we will validate this assay for peripheral blood samples, allowing more convenient and frequent testing. Disclosures Reibke: Abbott molecular Germany: Research Funding.

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