The Application of Sequential and Quantitative Analysis of Donor Chimerism in Adoptive Immunotherapy Following Allogeneic Stem Cell Transplantation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5118-5118
Author(s):  
Xiaowen Tang ◽  
De Pei Wu ◽  
Aining Sun ◽  
Ziling Zhu ◽  
Huirong Chang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Quantitative Analysis ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Adoptive Immunotherapy ◽  
Graft Rejection ◽  
Mixed Chimerism ◽  
Donor Chimerism ◽  
Time Point

Abstract Objective In order to investigate the value of sequential and quantitative analysis of chimerism to predict relapse or graft rejection after allogeneic stem cell transplantation, to determine the optional time point of adoptive immunotherapy and to estimate the efficacy of adoptive immunotherapy. Methods 70 patients who received HLA compatible allo-HSCT were evaluated. Peripheral blood and bone marrow were collected before and after transplantation in different period. Serial and quantitative analysis of donor chimerism (DC) both prior to and following adoptive immunotherapy were performed by multiplex PCR amplification of STR markers (STR-PCR) and capillary electrophoresis with fluorescence detection. Results 13 of 70 patients relapsed at median time of 6months post transplant (4/13 patients developed hematologic relapse and 9/13 patients appeared cytogenetic or molecular relapse respectively). 2 of 70 patients experienced graft rejection. Before the time of relapse or graft rejection, STR-PCR indicated the decreasing DC in all 15 patients, at levels ranging from 24.8%~86.2%. The declining value of donor chimerism (DC< 90%) was detected in four patients 26 days before relapse or graft rejection diagnosed clinically. Adoptive immunotherapy was used to treat 15 patients(9 CML, 5 AML, 1 MDS). A response to immunotherapy was achieved in 6 patients(6 CML) following withdrawal of CsA and in a further 4 patients(2 CML, 1 M2, 1 MDS) after additional donor lymphocyte infusion(DLI). In these patients, the value of DC increased with convertion to a predominant donor profile (>90%) or converted to stable full donor chimerism(FDC)(6 patients) or stable mixed chimerism(MC)(4 patients) shortly after immunotherapy. All these 10 patients who responsed to immunotherapy developed acute or chronic GVHD. While in the patients without response, the level of DC decreased persistently or declined after transient increasing. Three patients without response received second DLI but remain failed to therapy. Conclution The results demonstrate that the decreasing value of DC can identify the patients who have high risk of relapse or graft failure and can be used to guide adoptive immunotherapy implementation at early stage. The sequential and quantitative monitoring of DC has been shown to be a valuable tool to determine the optional time point of immunotherapy and to early evaluate the efficacy of treatment. Furthermore, it can present a rational basis for treatment intensification in patients who did not respond to first-line DLI treatment

Adoptive immunotherapy in canine mixed chimeras after nonmyeloablative hematopoietic cell transplantation

Blood ◽  
2000 ◽  
Vol 95 (10) ◽  
pp. 3262-3269
Author(s):  
George E. Georges ◽  
Rainer Storb ◽  
Jennifer D. Thompson ◽  
Cong Yu ◽  
Ted Gooley ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Adoptive Immunotherapy ◽  
Hematopoietic Cell Transplantation ◽  
Skin Grafting ◽  
Mixed Chimerism ◽  
Hematopoietic Stem ◽  
Donor Chimerism ◽  
Mixed Chimeras

Development of nontoxic and nonmyeloablative regimens for allogeneic hematopoietic stem-cell transplantation will decrease transplantation-related mortality caused by regimen-related toxic effects. In pursuit of this goal, a dog model of stable mixed hematopoietic chimerism was established in which leukocyte-antigen–identical litter mates are given sublethal total-body irradiation (2 Gy) before stem-cell transplantation and immunosuppression with mycophenolate mofetil and cyclosporine afterward. In the current study, we examined whether donor lymphocyte infusion (DLI) could be used as adoptive immunotherapy to convert mixed to complete donor chimerism. First, 8 mixed chimeras were given unmodified DLI between day 36 and day 414 after stem-cell transplantation. After a 10- to 47-week follow-up period, there were no significant changes in the percentage of donor engraftment. Next, we immunized the donor to the minor histocompatibility antigens (mHA) of the recipient by means of repeated skin grafting. Lymphocytes from the mHA-sensitized donor were infused between day 201 and day 651 after transplantation. All 8 recipients of mHA-sensitized DLI had conversion to greater than 98% donor chimerism within 2 to 12 weeks of the infusion. Complications from mHA-sensitized DLI included graft-versus-host disease in 2 dogs and marrow aplasia in 1. These results showed that the low-dose transplant regimen establishes immune tolerance, and mHA-sensitized DLI is required to break tolerance, thereby converting mixed to complete donor chimerism. We propose that mixed chimerism established after nonmyeloablative allogeneic stem-cell transplantation provides a platform for adoptive immunotherapy that has clinical potential in the treatment of patients with malignant diseases.

Adoptive immunotherapy in canine mixed chimeras after nonmyeloablative hematopoietic cell transplantation

Blood ◽  
2000 ◽  
Vol 95 (10) ◽  
pp. 3262-3269 ◽  
Author(s):  
George E. Georges ◽  
Rainer Storb ◽  
Jennifer D. Thompson ◽  
Cong Yu ◽  
Ted Gooley ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Adoptive Immunotherapy ◽  
Hematopoietic Cell Transplantation ◽  
Skin Grafting ◽  
Mixed Chimerism ◽  
Hematopoietic Stem ◽  
Donor Chimerism ◽  
Mixed Chimeras

Abstract Development of nontoxic and nonmyeloablative regimens for allogeneic hematopoietic stem-cell transplantation will decrease transplantation-related mortality caused by regimen-related toxic effects. In pursuit of this goal, a dog model of stable mixed hematopoietic chimerism was established in which leukocyte-antigen–identical litter mates are given sublethal total-body irradiation (2 Gy) before stem-cell transplantation and immunosuppression with mycophenolate mofetil and cyclosporine afterward. In the current study, we examined whether donor lymphocyte infusion (DLI) could be used as adoptive immunotherapy to convert mixed to complete donor chimerism. First, 8 mixed chimeras were given unmodified DLI between day 36 and day 414 after stem-cell transplantation. After a 10- to 47-week follow-up period, there were no significant changes in the percentage of donor engraftment. Next, we immunized the donor to the minor histocompatibility antigens (mHA) of the recipient by means of repeated skin grafting. Lymphocytes from the mHA-sensitized donor were infused between day 201 and day 651 after transplantation. All 8 recipients of mHA-sensitized DLI had conversion to greater than 98% donor chimerism within 2 to 12 weeks of the infusion. Complications from mHA-sensitized DLI included graft-versus-host disease in 2 dogs and marrow aplasia in 1. These results showed that the low-dose transplant regimen establishes immune tolerance, and mHA-sensitized DLI is required to break tolerance, thereby converting mixed to complete donor chimerism. We propose that mixed chimerism established after nonmyeloablative allogeneic stem-cell transplantation provides a platform for adoptive immunotherapy that has clinical potential in the treatment of patients with malignant diseases.

Increasing Mixed Chimerism Is an Important Prognostic Factor for Unfavorable Outcome in Children With Acute Lymphoblastic Leukemia After Allogeneic Stem-Cell Transplantation: Possible Role For Pre-Emptive Immunotherapy?

2004 ◽  
Vol 22 (9) ◽  
pp. 1696-1705 ◽  
Author(s):  
Peter Bader ◽  
Hermann Kreyenberg ◽  
Walter Hoelle ◽  
Gregor Dueckers ◽  
Rupert Handgretinger ◽  
...  
Keyword(s):  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Lymphoblastic Leukemia ◽  
Donor Lymphocyte Infusion ◽  
Mixed Chimerism ◽  
Acute Leukemias ◽  
Low Level

Purpose We recently reported that children with acute leukemias who show increasing mixed chimerism (MC) after allogeneic stem-cell transplantation have a significantly enhanced risk of relapse. Here we present the results of a prospective multicenter study to investigate (1) whether relapse of acute lymphoblastic leukemia (ALL) can be determined in advance by serial analysis of chimerism, and (2) if outcome can be influenced by withdrawal of immunosuppression and/or by low-dose donor lymphocyte infusion when increasing MC is detected. Patients and Methods Serial and quantitative analysis of chimerism was performed using a fluorescent-based short-tandem-repeat–polymerase chain reaction in 163 children with ALL. Results One hundred one patients revealed complete chimerism (CC) or low-level MC (CC/low-level MC); increasing MC was found in 46 patients; and decreasing MC, in 16 patients. Relapse was significantly more frequent in patients with increasing MC (26 of 46) than in patients with CC/low-level MC (eight of 101) or in patients with decreasing MC (0 of 16; P < .0001). The probability of 3-year event-free survival (EFS) was 54% for all patients, 66% for patients with CC/low-level MC (n = 101), 66% for patients with decreasing MC (n = 16), and 23% for patients with increasing MC (n = 46; P < .0001). Of the 46 patients with increasing MC, 31 received immunotherapy. This group had a significantly higher 3-year EFS estimate (37%) than the 15 patients who did not receive immunotherapy (0%; P < .001). Conclusion Serial analysis of chimerism reliably identifies patients at highest risk to relapse. The 3-year EFS of patients with increasing MC without immunotherapy was 0%, by which overt relapse could be prevented in a considerable group of patients.

Allogeneic stem cell transplantation for severe aplastic anemia: Graft rejection remains a problem

2009 ◽  
Vol 40 (1) ◽  
pp. 5-11 ◽  
Author(s):  
Şahika Zeynep Akı ◽  
Gülsan Türköz Sucak ◽  
Zübeyde Nur Özkurt ◽  
Zeynep Arzu Yeğin ◽  
Münci Yağcı ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Aplastic Anemia ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Graft Rejection ◽  
Severe Aplastic Anemia

Frequency and Relevance of CD10+CD19+ Hematogones after Allogeneic Stem Cell Transplantation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3280-3280
Author(s):  
Axel Nogai ◽  
Eckhard Thiel ◽  
Thomas Burmeister ◽  
Susanne Ganepola ◽  
Rita Lippoldt ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
B Cell ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Cell Recovery ◽  
Whitney Test ◽  
Donor Chimerism ◽  
Mann Whitney Test

Abstract Hematogones are B-lymphocyte precursors found in large frequencies after chemotherapies. In this study, the frequency of CD10+CD19+ hematogones was analysed routinely prior and post allogeneic stem cell transplantation and compared with moelculargenetic data for donor chimerism and for clonal translocations. Because of similarities in morphology and immunophenotype with frequent expression of CD19, CD10 and TdT they may undistinguishable from malignant B-cell lymphoblasts. As one example underlying the diagnostic difficulties, one patient with thrombopenia day +60 after allogeneic stem cell transplantation is presented. A relapse of Richter’s syndrome was suspected. Investigations of bone marrow specimens revealed a mixed chimerism, the frequency of cells coexpressing CD10 and CD19 was 28%. However, a CD19-sorted chimerism revealed an almost complete donor chimerism. Donor-lymphocytes were administered to improve graft function. Afterwards, donor chimerism reached 100% and platelets reached normal values, but hematogones continued to exceed 5% in the following specimens. As a result of such cases, bone marrow specimens after allogeneic stem cell transplantation were systematically analyzed for the presence of hematogones. METHODS: Hematogones were analyzed by routine 2-color flow cytometry. Cells coexpressing CD10 and CD19 with lymphocytic light scatter properties were regarded as hematogones. Percentage of cells was determined on the basis on total events. 133 patients undergoing allogeneic stem cell transplantation for AML, ALL, CLL, MM, NHL or aplastic anemia from 2003 to 2008 and surviving more than 60 days after transplantation were included in the analysis. During follow-up, bone marrow specimens were collected 1, 2, 3 and 12 months and in patients with suspected relapse. In total, 446 bone marrow specimens prior (186 specimen) and after (260 specimen) transplantation were collected and reevaluated for the frequencies of hematogones. RESULTS: The frequency of hematogones exceeded 5% in 8 of 186 specimens prior but in 62 of 260 specimens after transplantation (4.3% and 23.8%, respectively; p<0.001 Chi-Square). During follow-up, the median frequency of hematogones of patients in remission increased from 0.21% prior transplantion to 1.9% at two months after transplantation (range 0% to 13% and 0% to 32.0%, respectively; p=0.001 Mann-Whitney test) with no significant decrease of hematogones 3 and 12 months after transplantation. In contrast, the frequency of hematogones was significantly decreased (median 0.17%; range 0% to 16.7%; p=0.02 Mann-Whitney-test compared to day+60) in patients with relapses other than ALL. Patients with more than 5% hematogones at any time after transplantation were significantly younger (median 41 vs. 54 years; p=0.01 Mann-Whitney test) and received more often myeloablative conditioning therapies (p=0.016 Chi-square) compared to patients with less 5% hematogones. In contrast, the relapse rate or the overall survival, the underlying disease, source of stem cells, immunoglobulin levels prior and one year after transplantation as well as the number of transfused stem cells were not correlated with the maximal frequencies of hematogones. DISCUSSION: In this study, varying patterns of early B-cell recovery after allogeneic stem cell transplantation were found. Presence of large numbers of hematogones may be misinterpreted as a relapse in patients with B-cell malignancies. Presence of cells coexpressing CD10 and CD19 should be regarded with caution and always be interpreted with moleculargenetic data. The physiological and clinical effects of early B-cell recovery after allogeneic stem cell transplantation remain to be investigated in more detail.

Quantitative Analysis of Chimerism Using Insertion/Deletion Polymorphisms Is An Useful Predictor of Relapse After Allogeneic Stem Cell Transplantation for Acute Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3541-3541
Author(s):  
Nathalie Jacque ◽  
Nathalie Dhédin ◽  
Jean Louis Golmard ◽  
Madalina Uzunov ◽  
Stéphanie Nguyen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Quantitative Analysis ◽  
Stem Cell Transplantation ◽  
Acute Leukemia ◽  
Cell Transplantation ◽  
Tandem Repeat ◽  
Peripheral Blood ◽  
Hazard Ratio ◽  
Mixed Chimerism ◽  
Post Transplant

Abstract Abstract 3541 Poster Board III-478 Introduction Quantitative analysis of chimerism after allogeneic hematopoïetic stem cell transplantation (allo-HSCT) for acute leukemia is routinely used to monitor the kinetic of engrafment. Usually, chimerism is assessed by variable number tandem repeat (VNTR) or short tandem repeat (STR) amplification by polymerase chain reaction (PCR), which have a sensitivity of 1 to 5 %. Many studies have shown that these methods were not sensitive enough to predict relapse. Insertion/Deletion (InDel) polymorphism analysis by real-time quantitative PCR (InDel-QPCR) is a more sensitive method and thus it should be able to predict relapse earlier. Patients and methods We conducted a retrospective unicentric study including all consecutive patients transplanted for acute leukemia from May 2004 to January 2009 in the Pitié Salpétrière Hospital (Paris France). Seventy four patients (53 acute myeloblastic leukemia and 21 acute lymphoblastic leukemia) were included. Median age was 41 years (18-67). Conditionning regimen was myeloablative in 51 patients (69%). The donor was a HLA-identical sibling for 31 patients. The source of stem cell was bone marrow in 35 patients (47%), peripheral blood in 35 (47%) and umbilical cord blood in 4 (6%). Sixty four patients (86%) were in complete remission (CR) at the time of transplantation. InDel-QPCR was performed every month in peripheral blood samples with a reproducible sensitivity of 0.1% at least. An increasing mixed chimerism was defined as a one log increase between two successive chimerism assays when recipient rate was inferior to 0.1% and as any increase beyond this limit (0.1%). Results In this 74 patients population, overall survival was 65% at one year, with a median follow up of the survivors of 651days (129-1809). Thirty six patients developed an acute GVHD (2-4) and 21 a chronic GVHD. Eighteen patients (24%) presented a cytological relapse, at a median time of 203 days (63-1001) after transplant. In two patients, the quantification of recipient DNA rate was constantly superior to 1% at each time point post transplant and these patients presented an early relapse (at 3 and 4 months after transplant). In the 72 remaining patients, DNA rate was inferior to 1%: in 63 patients inferior to 0.1% and in 41 patients inferior to 0.01%. Among the 62 patients who presented an increasing mixed chimerism (IMC) at one point, 18 relapsed. Among the 24 patients who presented an IMC at two successive points, 16 relapsed. In univariate analysis, factors associated with higher risk of relapse were the status of disease at transplant (refractory disease+CR2 versus CR1, p=0.002, hazard ratio=9.54) and 2 successive IMC (p=0.0001, hazard ratio=8.74 (CI: 3.33-22.9). In multivariate analysis both factors remained significantly correlated with the incidence of relapse (status of disease p=0.003, hazard ratio=4.24(CI: 1.6-11.2) and two IMC p<0.0001, hazard ratio=9.20(CI: 3.44-24.57)). The median interval between the first IMC and the diagnosis of relapse was 45 days (7-154). At the time of the first IMC, the peripheral blood cell count was normal in all except one patient who had persistent post-transplant thrombocytopenia. Conclusion Analysis of chimerism by using In/Del polymorphism is a sensitive technique with a reproducible detection threshold inferior to 0.1%. The detection of two successive IMC is highly predictable of relapse in patients transplanted for acute leukemia. Therefore, the detection of an increasing chimerism should incite to perform a rapid control, in order to make a precocious diagnosis of relapse and to provide an early therapeutic intervention. This analysis could help to monitor minimal residual disease in post transplant patients without a more specific molecular marker of malignancy. Disclosures: No relevant conflicts of interest to declare.

Dynamics of Chimerism In Regulatory T Lymphocytes (Treg; CD4+/CD25+) After Allogeneic Stem Cell Transplantation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1328-1328
Author(s):  
Víctor Noriega ◽  
Carolina Martinez-Laperche ◽  
David Serrano ◽  
Gabriela Rodriguez-Macias ◽  
Mi Kwon ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Lymphocytes ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Conflicts Of Interest ◽  
Small Sample ◽  
Mixed Chimerism ◽  
Group 2

Abstract Abstract 1328 Introduction: CD4+CD25+ regulatory T-cells (Treg) play an important role in inducing and maintaining allogeneic tolerance and can inhibit graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). However, the dynamics of donor and recipient Treg cell populations after Allo-SCT has not been studied yet. Objective: To analyze the dynamics of chimerism in Tregs and compare it to that of T lymphocytes (TL; CD3+) and whole blood leukocytes (WBL). Material and Methods: The study includes 53 patients subjected to Allo-SCT (myeloablative and non-myeloablative). PB samples were obtained weekly during the first month and every 14 days after day +30 until complete chimerism (CC) was achieved, and at fixed time-points (+30, +60, +90, +180, +365) thereafter. TL and Tregs were purified from PB until CC was achieved using inmunomagnetic technology (Miltenyi Biotec; CD3+ Microbeads and CD4+/CD25+ Treg Isolation Kit, respectively). Chimerism analysis was performed by microsatellite PCR (STR-PCR; AmpFlSTR SGM Plus; Applied Biosystems) on genomic DNA obtained from WBL as well as on cell lysates obtained from purified TL and Tregs. Complete chimerism was considered in samples with percentage of recipient cells <1% (sensitivity of the STR-PCR) for WBL samples and <5% (minimum purity of 95% as estimated by flow cytometry) for purified cell lineages. Results: Median follow up for the whole cohort of patients was 338 days. CC was spontaneously achieved in WBL, TL and Tregs in 45/53 patients (85%) in a median time of 35.41, 38.8 and 42.5 days respectively. 5/3 patients (9%) suffered from graft failure/rejection showing mixed chimerism (MC) with increasing percentages of recipient cells both in WBL and leukocyte lineages. 3/53 patients (6%) maintained mixed chimerism (MC) in WBL and leukocyte lineages at day +180, with no signs of graft rejection or disease relapse. Analysis of the chimerism dynamics of those patients who spontaneously achieve CC revealed two different groups: Group 1 included 25/45 patients who achieved CC at the same time in WBL, TL and Tregs. Group 2 included 20/45 patients who achieved CC in WBL while maintaining MC in TL and Tregs. Interestingly enough, 9/20 patients from Group 2 maintained MC in Tregs 7–75 days after achieving CC in both WBL and TL (Figure 1). In a preliminary analysis, the small sample size precluded from obtaining statistically significant associations between the dynamics of chimerism in Tregs and the development of complications such as relapse or GVHD after SCT. Conclusions: To our knowledge, this is the first study dealing with Treg chimerism after SCT. We have shown it is feasible and can be performed on a routine basis together with standard lineage specific chimerism follow up. Although there is an association between chimerism dynamics in Tregs and TL, this is not absolute and a percentage of patients maintain residual Tregs of recipient origin after WTL and TL have become of complete donor origin. In this small cohort, Treg chimerism did not influence the development of post-SCT complications. Analysis of a larger and more homogeneous cohort would allow establishing the usefulness of Treg chimerism testing for the management of transplanted patients. Disclosures: No relevant conflicts of interest to declare.

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