Facilitating cells as a venue to establish mixed chimerism and tolerance

Graft rejection and the toxicity associated with the use of non-specific immunosuppression remain the major limitations in pediatric solid organ transplantation. The induction of tolerance in transplant recipients is an elusive but achievable goal that will decrease the dependence on immunosuppressive agents. BMT is associated with a robust form of donor-specific transplantation tolerance. It achieves a state of chimerism, defined as the presence of donor marrow cells in the recipient. The two major toxicities in conventional bone marrow transplantation that have prevented its clinical application to induce tolerance are the toxicity of ablative conditioning and GVHD. Two forms of chimerism exist: full chimerism and mixed chimerism. In full chimerism, the hematopoietic system of the recipient is replaced by that of the donor following ablative conditioning. Full chimerism is associated with a relatively impaired immunocompetence for primary immune responses and an increased risk of GVHD. In addition, the 7-10% regimen-related mortality associated with ablation could not be accepted in solid organ allograft recipients. In mixed chimerism the donor hematopoietic system co-exists with that of the recipient. Mixed chimerism induces donor-specific tolerance and is associated with superior immunocompetence and a relative resistance to GVHD compared with full chimerism. Moreover, it can be achieved with partial conditioning, thereby reducing the regimen-related morbidity associated with myeloablation. Approaches to establish mixed chimerism using non-myeloablative-conditioning regimens have been aggressively pursued over the past decade. Mixed chimerism can be safely established with minimal conditioning, resulting in a significant reduction in risk compared with ablative conditioning. GVHD is the final hurdle that has prevented the widespread application of chimerism to induce tolerance. Donor T cells are the primary effector cells for GVHD. Although T cell depletion of the donor marrow avoids GVHD, it results in an increase in the rate of graft failure in MHC-disparate recipients. The dichotomy between GVHD and T cell depletion graft failure has recently been dissociated by the discovery of CD8+/TCR- graft FC. Purified HSC engraft readily in syngeneic recipients but not in MHC-disparate allogeneic recipients. The addition of small numbers of facilitating cells permits durable HSC engraftment in allogeneic recipients and avoids GVHD. Using FC to promote HSC engraftment following non-myeloablative conditioning could be a promising approach to establish tolerance in solid organ transplantation. This invited review focuses on recent developments in stem cell chimerism and tolerance that could bring the use of this approach to induce tolerance to solid organ transplantation one step closer to reality.     

Highly sensitive chimerism detection in blood is associated with increased risk of relapse after allogeneic hematopoietic cell transplantation in childhood leukemia

Analysis of chimerism in blood post‐HCT using STR‐PCR is routinely applied in parallel with quantification of MRD to predict relapse of leukemia. RQ‐PCR chimerism is 10‐ to 100‐fold more sensitive, but clinical studies in children are sparse. We analyzed IMC in blood samples following transplantation for acute lymphoblastic or myeloid leukemia in 56 children. IMC was defined as a minimum increase of (a) 0.1% or (b) 0.05% recipient DNA between two samples. The risk of relapse was higher in children with IMC of both 0.1% and 0.05% compared to children without IMC (HR 12.8 [95% CI: 3.9‐41.4; P < .0001] and 7.6 [95% CI: 2.2‐26.9; P < .01], respectively). The first IMC was detected at a median of 208 days prior to relapse. The 5‐year cumulative incidence of relapse for children with a single IMC was 45.5% (CI 12.3‐74.4) and 41.0% (14.2‐66.6) for IMC above 0.1% and 0.05%, respectively. However, in 47 and 38 children never attaining IMC > 0.1% and >0.05%, 10 and 8 children relapsed, respectively. In a landmark analysis, no association was found between IMC prior to 90 days post‐HCT and subsequent relapse by either classification of IMC and AUC for RQ‐PCR chimerism was 54.2% (95 CI 27.7‐ 84.8). Although limited by a retrospective design, these results indicate that monitoring of RQ‐PCR chimerism in peripheral blood may have a role in early detection of relapse in acute childhood leukemia .     

Stable mixed chimerism after hematopoietic stem cell transplantation in Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disease characterized by thrombocytopenia, eczema, impaired cellular and humoral immunity, and increased susceptibility to malignancy and autoimmunity. The only curative treatment for WAS is hematopoietic stem cell transplantation, especially in the presence of a matched sibling donor or matched unrelated donor. Here, we report the case of a 2.5-yr-old boy with WAS that resulted in mixed chimerism after having received bone marrow from his phenotypically identical grandfather. Although the patient has persistent thrombocytopenia (platelet counts 50-80 x 10(9)/L), he is currently alive and doing well at 36 months post-transplant and is free of any bleeding episodes.     

Evaluation of chimerism by quantitative PCR analysis of DNA polymorphism after allogeneic hematopoietic stem cell transplantation in a pediatric population with malignancies

Relapse remains the major pitfall to success for Allo-HSCT in children with malignancies. Ninety-one patients undergoing Allo-HSCT were retrospectively reviewed. Chimerism status was evaluated at days +30, +60, and +100 in PB. VNTR-PCR and STR-PCR were used for this purpose. Thirty-one patients recurred (34%) and none survived. Thirty-two remain alive in CR (35%). Patients who achieved a CC at those days had a significant higher RFS and OS than patients who did not. Twelve patients showing PMC had an increased risk of recurrence (p=0.02. OR 7.7). In the univariate analysis, the probability of death was higher in patients who were not in first CR before transplant (p=0.008.OR 2.09) and in those receiving cells not from PB (p=0.002.OR 2.03). In the multivariate analysis, the absence of CC at day +100 was associated with a higher probability of relapse (p=0.004. OR 10.8) and death (p=0.016. OR 9.3). Serial chimerism PCR-based analyses of polymorphic DNA markers can predict relapse. Patients with PMC are at the highest risk of recurrence. Patients receiving an Allo-HSCT in first CR from PB who achieve a CC at day +100 have a better outcome.     

The second mini-transplant for unstable mixed chimerism within the first 100 days after hematopoietic stem cell transplant in severe thalassemia

Allogeneic HSCT is the only curative treatment for severe thalassemia disease. MC occurs in one-third of these patients within the first two months after HSCT; this is a major risk factor of graft rejection, especially when RHCs are more than 25%. There is still no consensus for the management of MC, especially in the early phase of HSCT. The DLI has also been described in the treatment of MC following HSCT for hemoglobinopathies, but its success is still not guaranteed. The second HSCT has been an approach used in an attempt to cure patients who reject their graft. Concern about toxicity of conditioning regimen, the second HSCT is usually delayed for at least a year after the first HSCT. We would like to demonstrate the successful use of the second mini-allogeneic HSCT in hemoglobin E/β-thalassemia with evidence of unstable MC in the first 100 days after allogeneic HSCT to prevent further graft loss after allogeneic HSCT.     

Allogeneic hematopoietic stem cell transplantation in children with sickle cell disease

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents the only curative treatment for sickle cell disease (SCD), being successful in around 85-90% of patients. Mortality and long-term morbidity (including infertility, gonadal failure, and chronic graft-vs.-host disease) associated with conventional approaches curtail the number of patients who undergo allo-HSCT. Recently, it has been demonstrated that cord blood is as effective as and possibly safer than bone marrow in pediatric patients with SCD. Likewise, transplant strategies based on the use of reduced-intensity regimens and the induction of mixed chimerism have been explored to decrease allo-HSCT short- and long-term complications.     

Outcomes of pediatric identical living‐donor liver and hematopoietic stem cell transplantation

Chronic IS is associated with significant morbidity in transplant recipients. Moreover, IS does not prevent chronic graft failure frequently. Allograft immune tolerance in LT can be induced by complete donor chimerism through allogenic HSCT combined with identical LDLT. This approach may exempt patients from chronic lifelong IS. However, it is unclear whether its benefits justify its risks. Here, we present three cases from our institution and analyze seven additional reports of children treated with HSCT/LDLT, all receiving HSCT due to hemato‐oncological indications. In eight of 10 cases, donor macrochimerism resulted in allograft tolerance. Nine patients survived. One patient died due to fulminant ADV infection. Further complications were GvHD (n = 3) and bone marrow failure (n = 2). In conclusion, donor‐specific allograft tolerance can be achieved by identical‐donor HSCT/LDLT. However, at present, this approach should generally be limited to selected indications due to a potentially unfavorable risk–benefit ratio. Novel toxicity‐reduced conditioning protocols for HSCT/LDLT in the absence of malignant or non‐hepatic disease may prove to be a sufficiently safe approach for inducing graft tolerance in children receiving a LDLT in the future. This concept may reduce the burden of lifelong IS.     

Successful hematopoietic cell transplantation following cardiac transplantation in two pediatric patients

We report two patients who underwent cardiac transplantation at a young age and subsequently required a HCT for varied indications. Despite the challenges associated with HCT following cardiac transplant, including need for altered immunosuppression, toxicities related to chemotherapy exposure, and infection risks, both patients are currently alive and well. There is a paucity of such successful cases documented in the literature, and these cases highlight the critical importance of an experienced, multidisciplinary team when caring for this patient population.     

骨髓移植诱导大白兔特异性免疫耐受模型的建立

目的通过供体与受体的骨髓移植来诱导受体对供体的特异性免疫耐受。方法30只雄性和30只雌性新西兰大白兔分别作为供者和受者,随机配对,将30对大白兔再随机分为5组,即无处理对照组、单纯照射组、单纯骨髓移植(BMT)组、实验组(照射+BMT)和无关皮肤供体对照组,每组6对。受者给予7Gy的全身照射(TB I),24小时后耳缘静脉输注骨髓单个核细胞,5天后再次回输相同供者的骨髓单个核细胞,两次总的细胞数量为l~1.5×108/kg,在第二次回输细胞的同时,移植皮肤。观察皮肤移植存活时间、对第三方皮肤的排斥反应及对供体的单向混合淋巴细胞反应,并用多聚酶链式反应(PCR)检测嵌合体的形成。结果单纯照射和骨髓移植不能延长移植物的存活时间,也没有形成供受者混合嵌合;实验组皮肤移植物存活时间(45.83±3.97 d)比无处理对照组(10.83±0.75)、单纯照射组(12.5±1.0488)、单纯骨髓移植组(11.33±0.8165)、无关皮肤供体对照组(12.17±1.169)明显延长(P<0.01)。结论对大白兔模型,采用全身照射加大剂量骨髓移植的方法,可形成较长时间的混合嵌合,并获得一定程度的特异性免疫耐受。     

Liver transplantation in a child with liver failure due to chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation from the same unrelated living donor

Englert C, Ganschow R. Liver transplantation in a child with liver failure due to chronic graft‐versus‐host disease after allogeneic hematopoietic stem cell transplantation from the same unrelated living donor. Abstract: We report a case of a six‐yr‐old boy who developed chronic GVHD of the liver, intestines, and skin following allogeneic hematopoietic SCT. The boy received an allogeneic hematopoietic stem cell transplant at the age of two yr because of early recurrence of ALL. Chimerism analysis showed complete chimerism. In the following year, he developed GVHD despite adequate immunosuppressive therapy. Liver biopsy showed liver GVHD resulting in liver cirrhosis by the age of five yr. LTx was performed with a left liver lobe from the unrelated donor from whom the stem cells had been taken. Immunosuppressive therapy consisted of low‐dose steroids and low‐dose cyclosporine. The postoperative course was uneventful. Graft function was excellent, and we performed protocol biopsies at seven days and three wk as well as three, six, and nine months after transplantation; none of these showed any signs of rejection or GVHD. Immunosuppressive therapy was discontinued nine months after LTx. Three yr after transplantation, the boy is in good condition with normal graft function. To our knowledge, this is the first report on LTx following allogeneic hematopoietic SCT from the same unrelated living donor.     

Megakaryocyte chimerism after allogeneic stem cell transplantation in children

The aim of this study was to investigate the extent and the clinical implications of mixed chimerism in megakaryocytes after stem cell transplantation (SCT). Polymerase chain reaction analyzing allele length polymorphisms was used to determine the origin of immunomagnetically isolated megakaryocytes and leukocyte subpopulations after SCT in 13 children. Eleven were unselected consecutive cases while two were included due to known leukocyte mixed chimerism. Recipient DNA was detected in the megakaryocytes in six out of the 11 cases at levels between 1 and 100%. Coinciding mixed chimerism in the leukocyte populations was detected in two of the 11 cases. Of the two selected cases with known leukocyte mixed chimerism, two boys with aplastic anemia and Wiskott-Aldrich syndrome had 1-5 and 70% recipient megakaryocytes, respectively. Although the four relapses or deaths, within the 13 months of observation, were restricted to patients with multilineage or isolated megakaryocyte (n = 1) mixed chimerism, it was not possible to link any other apparent clinical problems, except a prolonged thrombocytopenia in one case, to the mixed chimerism in this limited study group.     

Haploidentical hematopoietic cell transplantation for disseminated Ewing sarcoma

We describe the case of a 13‐year‐old girl with multifocal disseminated Ewing sarcoma family of tumor (ESFT) who received a 5/8 human leukocyte antigen‐matched haploidentical hematopoietic cell transplantation to generate a graft‐versus‐tumor effect. The patient had grade 2 acute graft‐versus‐host disease (GVHD) of the skin and chronic GVHD nausea and abdominal pain that required prednisolone for 17 months, but has been free from ESFT for 3 years 10 months after therapy. The present case suggests a beneficial effect of haploidentical hematopoietic cell transplantation in disseminated ESFT.     

Successful treatment of disseminated mixed invasive fungal infection after hematopoietic stem cell transplantation for severe aplastic anemia

Concomitant infections are frequent and usually the causes of death in patients with severe AA. HSCT can restore hematopoiesis in AA, but it is usually life threatening when patients simultaneously have an IFI. Mixed IFIs have been reported on rare occasions. The exact diagnosis of IFIs is difficult because of low fungus culture rate, difficultly obtaining tissue specimens in severely immunocompromised patients or those with bleeding tendencies. Otherwise, treatment with anti-fungal drugs alone for DMIFI was always lethal in previous reports. Surgical resection is crucial for invasive zygomycosis, but severe pancytopenia and bleeding tendency make therapy difficult. Herein, we report that with a combination of aggressive anti-fungal drugs, HSCT, and surgery, we successfully treated a 10-yr-old boy with severe AA and pulmonary zygomycosis before HSCT and disseminated mixed invasive zygomycosis and aspergillosis after HSCT.     

Pediatric hematopoietic stem cell transplantation in China: Data and trends during 1998–2012

The success of treating a wide variety of pediatric diseases with HSCT, hematologic malignancies in particular, has resulted in an increased number of long‐term survivors. This study is the first large‐scale, multicentre report that describes the evolution of pediatric HSCTs in China during the period of 1998–2012. Of all 1052 patients, 266 cases were treated with autologous HSCs and 786 used allogeneic HSCs. The disease indications for HSCTs mainly included leukemias, lymphoma, solid tumors, and non‐malignant disorders. The total number of HSCTs, especially unrelated donor transplants, appeared to be increasing year by year. For patients with neuroblastoma, the therapeutic efficacy seemed to be poor, with a five‐yr OS and DFS rate of 34.5 ± 14.3% and 20.7 ± 9.6%, respectively. In contrast, the survival of patients with SAA was prominently improved, and their five‐yr OS and DFS rates were 82.8 ± 4% and 80.7 ± 4.1%, respectively. Patients who received cord blood transplants had a lower incidence of acute GVHD than that of PB and/or BM transplants from unrelated donors. This report offers us a valuable resource for evaluating the changes in HSCTs in China over the past 14 yr.