Mehmet Uzunel

Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells

Adult bone-marrow-derived mesenchymal stem cells are immunosuppressive and prolong the rejection of mismatched skin grafts in animals. We transplanted haploidentical mesenchymal stem cells in a patient with severe treatment-resistant grade IV acute graft-versus-host disease of the gut and liver. Clinical response was striking. The patient is now well after 1 year. We postulate that mesenchymal stem cells have a potent immunosuppressive effect in vivo.

Analysis of Tissues Following Mesenchymal Stromal Cell Therapy in Humans Indicates Limited Long-Term Engraftment and No Ectopic Tissue Formation†‡§

Mesenchymal stromal cells (MSCs) are explored as a novel treatment for a variety of medical conditions. Their fate after infusion is unclear, and long‐term safety regarding malignant transformation and ectopic tissue formation has not been addressed in patients. We examined autopsy material from 18 patients who had received human leukocyte antigen (HLA)‐mismatched MSCs, and 108 tissue samples from 15 patients were examined by PCR. No signs of ectopic tissue formation or malignant tumors of MSC‐donor origin were found on macroscopic or histological examination. MSC donor DNA was detected in one or several tissues including lungs, lymph nodes, and intestine in eight patients at levels from 1/100 to <1/1,000. Detection of MSC donor DNA was negatively correlated with time from infusion to sample collection, as DNA was detected from nine of 13 MSC infusions given within 50 days before sampling but from only two of eight infusions given earlier. There was no correlation between MSC engraftment and treatment response. We conclude that MSCs appear to mediate their function through a “hit and run” mechanism. The lack of sustained engraftment limits the long‐term risks of MSC therapy. STEM CELLS2012;30:1575–1578

Tissue repair using allogeneic mesenchymal stem cells for hemorrhagic cystitis, pneumomediastinum and perforated colon

Mesenchymal stem cells (MSC) possess anti-inflammatory properties and participate in tissue repair. We used MSC to heal therapy-induced tissue toxicity. Ten consecutive patients, treated with MSC due to tissue toxicity following allogeneic hematopoietic stem cell transplantation, (ASCT) were included. Their median age was 48 (13–64) years. Seven had hemorrhagic cystitis grades 2–5, two had pneumomediastinum and one had perforated colon and peritonitis. MSC donors were mainly third-party, HLA-mismatched (n=11), HLA-haploidentical (n=3) and, in two cases, the HLA-identical ASCT sibling donors. MSC were given intravenously, the median cell dose was 1.0 (range 0.7–2) × 106/kg. In five patients, the severe hemorrhagic cystitis cleared after MSC infusion. Gross hematuria disappeared after median 3 (1–14) days. Two patients had reduced transfusion requirements after MSC infusion, but died of multiorgan failure. In one of them, MSC donor DNA was demonstrated in the urinary bladder. In two patients, pneumomediastinum disappeared after MSC infusions. A patient with steroid-resistant graft-versus-host disease of the gut experienced perforated diverticulitis and peritonitis that was reversed twice by MSC. MSC is a novel treatment for therapy-induced tissue toxicity.

Low-intensity conditioning and hematopoietic stem cell transplantation in patients with renal and colon carcinoma

Summary:We have evaluated whether allogeneic hematopoietic stem cell transplantation (HSCT) could induce an antitumor effect in patients with metastatic solid tumors. A total of 12 HLA-identical siblings and 6 HLA-A-, -B- and –DRβ1-compatible unrelated grafts were used. Diagnoses were adenocarcinoma of kidney (n=10), colon (n=6), breast (n=1) and cholangiocarcinoma (n=1). Conditioning was fludarabine 30 mg/m2/day for 3 days and 2 Gy of total body irradiation. Recipients of unrelated HSCT were also given thymoglobuline and two additional days of fludarabine. The median CD34+ cell dose was 7.5×106/kg. Immunosuppression was mycophenolate mofetil and cyclosporin. Among all, 12 patients became complete donor chimeras within a median of 28, 29 and 65 days for B, myeloid and T cells, respectively. Two patients rejected the grafts, one developed marrow aplasia and three were mixed chimeras. The probability of grades II–IV acute graft-versus-host-disease (GVHD) was 57%. Regression of all tumor metastases was seen in one patient with colon carcinoma. Another patient with colon and two with renal carcinoma had regression of lung metastases, but progression of metastases in the liver and/or bone. Necrosis of lung metastasis was found in one further patient with renal carcinoma who died of graft-versus-host-disease (GVHD). In all, 10 patients died; four of transplant-related complications, one of trauma and five of progressive disease. Thus, progression was common after allogeneic HSCT in unselected patients with advanced solid tumors. However, the regression of some metastases associated with GVHD provides suggestive evidence that the GVHD effect may occur in renal and colon adenocarcinoma using reduced intensity conditioning.

Mesenchymal stem cells exert differential effects on alloantigen and virus-specific T-cell responses

Mesenchymal stem cells (MSCs) suppress alloantigen-induced T-cell functions in vitro and infusion of third-party MSCs seems to be a promising therapy for graft-versus-host disease (GVHD). Little is known about the specificity of immunosuppression by MSCs, in particular the effect on immunity to pathogens. We have studied how MSCs affect T-cell responses specific to Epstein-Barr virus (EBV) and cytomegalovirus (CMV). We found that EBV- and CMV-induced proliferation and interferon-gamma (IFN-gamma) production from peripheral blood mononuclear cells (PBMCs) was less affected by third-party MSCs than the response to alloantigen and that MSCs had no effect on expansion of EBV and CMV pentamer-specific T cells. Established EBV-specific cytotoxic T cells (CTL) or CMV-CTL cultured with MSCs retained the ability to proliferate and produce IFN-gamma in response to their cognate antigen and to kill virally infected targets. Finally, PBMCs from 2 patients who received MSCs for acute GVHD showed persistence of CMV-specific T cells and retained IFN-gamma response to CMV after MSC infusion. In summary, MSCs have little effect on T-cell responses to EBV and CMV, which contrasts to their strong immunosuppressive effects on alloreactive T cells. These data have major implications for immunotherapy of GVHD with MSCs and suggest that the effector functions of virus-specific T cells may be retained after MSC infusion.

Leukemia lineage-specific chimerism analysis is a sensitive predictor of relapse in patients with acute myeloid leukemia and myelodysplastic syndrome after allogeneic stem cell transplantation

One of the major complications after allogeneic stem cell transplantation (SCT) in patients with malignant disease is a high frequency of relapse. We have prospectively analyzed the clinical impact of recipient-derived chimeric cells in 30 patients with acute myeloid leukemia and myelodysplastic syndrome after SCT. In order to improve sensitivity and specificity, all samples were cell-separated by using immunomagnetic beads according to the patients leukemia phenotype, expressed at diagnosis or relapse before SCT. Twelve out of 30 patients experienced a clinical relapse after SCT. Median follow-up time for patients alive and without relapse (n = 15) was 30 (16–47) months. Mixed chimerism in peripheral blood (PB) and bone marrow (BM) ⩾1 month post SCT, in the leukemia-affected cell lineage, was detected in 14/30 patients. Ten of these 14 patients relapsed as compared to 2/16 with donor chimerism (DC) (P <0.01). All eight patients with MC in peripheral blood ⩾1 month after SCT relapsed vs 4/22 DC patients (P < 0.001). MC was detected a median of 66 (23–332) days before hematological relapse. No correlation was found between T cell MC and relapse. In this study, chimerism analysis showed a higher sensitivity and specificity vsmorphological examination. In conclusion, this study may provide a rational basis for treatment with adoptive immunotherapy at an earlier time after SCT than at present, in patients with AML and MDS, in order to treat recurrences of malignant disease.

Expression of Vascular Endothelial Growth Factor Receptor-2 or Tie-2 on Peripheral Blood Cells Defines Functionally Competent Cell Populations Capable of Reendothelialization

Background—Receptor tyrosine kinases that include vascular endothelial growth factor (VEGFR)-1, VEGFR-2, and Tie-2 regulate cardiovascular development and physiological and pathological angiogenesis. We were interested in the phenotypic and functional characterization of peripheral blood cells expressing these receptors and their therapeutic potential in vascular injury. Methods and Results—VEGFR-1+, VEGFR-2+, and Tie-2+ cells constituted ≈3.0±0.2%, 0.8±0.5%, and 2.0±0.3%, respectively, of the total population of mononuclear cells in blood. Phenotypic analysis demonstrated that all 3 cell populations mainly expressed markers of monocytic/macrophage lineage. Only VEGFR-2+ and Tie-2+ cells phenotypically, morphologically, and functionally differentiated to endothelial cells after culture, whereas VEGFR-1+ cells did not. None of the cell types proliferated in vitro. Only freshly isolated VEGFR-2+ or Tie-2+ cells but not VEGFR-2− or Tie-2− cell populations significantly contributed to efficient endothelialization of balloon-injured femoral arteries of nude mice. Furthermore, these cells also differentiated into &agr;-actin–positive smooth muscle cells. Administration of bromodeoxyuridine to animals transplanted with human endothelial progenitor cells showed that VEGFR-2+ and Tie-2+ cells proliferated in vivo. Conclusions—These data demonstrate that expression of VEGFR-2 and/or Tie-2 on peripheral blood cells defines functionally competent cell populations that proliferate in vivo and that contribute to reendothelialization. These findings may have implications for a cell-based approach in vascular diseases.

T cell mixed chimerism is significantly correlated to a decreased risk of acute graft-versus-host disease after allogeneic stem cell transplantation.

BACKGROUND It has been debated whether mixed chimerism (MC) is correlated to a decreased incidence of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). METHODS Between September 1996 and April 1999 we analyzed 102 patients for MC in the T-cell fraction post allogeneic SCT, using PCR amplification of variable numbers of tandem repeat (VNTR) loci. All samples, taken regularly post SCT, were cell separated using anti-CD3 immunomagnetic beads. RESULTS T-cell mixed chimerism was detected in 58 out of 102 patients (57%). Patient characteristics were comparable in the T-cell MC- and donor chimeric-group (DC). The median follow-up time for the MC group was 714 days (range 58 - 1248) as compared to 427 days (range 45 - 1042) for the DC group. Overall probability of acute GVHD grades II-IV was significantly higher in the DC group as compared to the MC group (52% vs. 5%, P<0.001). In multivariate analysis T-cell DC proved to be the most significant risk factor for acute GVHD grades II-IV. The cumulative incidence of relapse, among patients with malignant disease, did not show any statistical difference between the T-cell MC patients and the DC-group. There was a tendency for better overall survival in the T-cell MC group compared to the DC group (2 yrs; 73% vs. 54%, P=0.06). Among DC patients, 14/20 (70%) deaths were due to GVHD versus none in the MC-group(P<0.001). CONCLUSION T-cell mixed chimerism was significantly correlated to a decreased risk of moderate to severe acute GVHD and death by GVHD.

Improved Survival after Allogeneic Hematopoietic Stem Cell Transplantation in Recent Years. A Single-Center Study

We analyzed the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) over the past 2 decades. Between 1992 and 2009, 953 patients were treated with HSCT, mainly for a hematologic malignancy. They were divided according to 4 different time periods of treatment: 1992 to 1995, 1996 to 2000, 2001 to 2005, and 2006 to 2009. Over the years, many factors have changed considerably regarding patient age, diagnosis, disease stage, type of donor, stem cell source, genomic HLA typing, cell dose, type of conditioning, treatment of infections, use of granulocyte-colony stimulating factor (G-CSF), use of mesenchymal stem cells, use of cytotoxic T cells, and home care. When we compared the last period (2006-2009) with earlier periods, we found slower neutrophil engraftment, a higher incidence of acute graft-versus-host disease (aGVHD) of grades II-IV, and less chronic GVHD (cGHVD). The incidence of relapse was unchanged over the 4 periods (22%-25%). Overall survival (OS) and transplant-related mortality (TRM) improved significantly in the more recent periods, with the best results during the last period (2006-2009) and a 100-day TRM of 5.5%. This improvement was also apparent in a multivariate analysis. When correcting for differences between the 4 groups, the hazard ratio for mortality in the last period was 0.59 (95% confidence interval [CI]: 0.44-0.79; P < .001) and for TRM it was 0.63 (CI: 0.43-0.92; P = .02). This study shows that the combined efforts to improve outcome after HSCT have been very effective. Even though we now treat older patients with more advanced disease and use more alternative HLA nonidentical donors, OS and TRM have improved. The problem of relapse still has to be remedied. Thus, several different developments together have resulted in significantly lower TRM and improved survival after HSCT over the last few years.

Mixed chimerism in the B cell lineage is a rapid and sensitive indicator of minimal residual disease in bone marrow transplant recipients with pre-B cell acute lymphoblastic leukemia

One of the major problems after allogeneic bone marrow transplantation (BMT) is a high frequency of leukemia relapse. We have prospectively studied the presence of donor- and recipient-derived chimeric cells in bone marrow recipients with pre-B cell acute lymphoblastic leukemia (pre-B-ALL). The chimeric status of BMT recipients was compared to minimal residual disease (MRD) detection by analysis of immunoglobulin heavy chain (IgH) and T cell receptor (TcR) genes. Post-transplant blood and bone marrow samples from 12 patients with pre-B-ALL were studied. Five patients showed mixed chimerism (MC) in the CD19-positive cell fraction. Four of them have relapsed to date. The remaining patient with MC in the B cell lineage was also MRD positive in the same samples. All seven patients with donor chimerism in the B cell fraction remain in clinical remission (P = 0.01). In samples from all five patients having MC in the B cell lineage, the patient-specific IgH or TcR rearrangement was also detected. In three of four patients who relapsed, MC in the B cell lineage was seen more than 2.5 months prior to morphologically verified relapse. The results of this comparison suggest that routinely performed MC analysis of the affected cell lineage may facilitate post-BMT monitoring and rapid therapeutic decisions in transplanted patients with pre-B-ALL. Bone Marrow Transplantation (2000) 25, 843–851.