Comparison study between automated and manual cell separator for fractionation of specific cells lineage for donor chimerism analysis

Abstract Abstract 3338 Poster Board III-226 Double umbilical cord blood transplantation (UCBT) results in higher engraftment rates as compared to single UCBT in adult patients. Sustained hematopoiesis is usually derived from a single cord blood unit (CBU). So far, the mechanism of predominance of a particular CBU is unresolved. Immunological rejection of one CBU has been proposed, as well as a selective growth advantage of one particular unit. Frequent serial chimerism studies in leukocyte subsets by use of HLA-specific monoclonal antibodies (mAbs) during the first month post transplant might contribute to unravel the mechanism of graft predominance. Methods. Seventeen consecutive patients (pts) with high risk hematological diseases received a double UCBT preceded by a non-myeloablative conditioning regimen (Cy 60 mg/kg/ Flu 160 mg/kg/ TBI 2×2 Gy). CBUs were selected by low resolution typing for HLA-A and –B loci and by allele typing for HLA-DRB1. The minimal required HLA-match grade was 4/6. If discriminating HLA mismatches between the 3 different parties were present, early analysis in leukocyte subpopulations was performed at day 11, 18, 25 and 32 post transplant by flowcytometry using lineage-specific (CD3, CD4, CD8, CD19, CD16/56, CD14, CD33) mAbs in combination with fluorochrome labeled HLA-antigen specific human mAbs. Results of day 32 were compared with routine chimerism analysis (VNTR) of peripheral blood TNC and T-cells. Results. Two pts were non-evaluable for engraftment due to early death and insufficient follow up, respectively. Donor engraftment in 15 pts occurred after a median of 30 days (range: 11-45). At 1 month post transplant, 9 pts showed complete single and 3 showed mixed chimerism by VNTR-analysis. In 3 patients, cells originating from both CBUs were present. Ultimately, complete single donor chimerism was established in 14 pts. Early chimerism studies with HLA-specific mAbs were performed in 8 pts. In all pts results at day 32 corresponded with chimerism analysis performed by VNTR. For 7 other pts no discriminating HLA-mAbs were available. Simultaneous 3-donor-origin detection of T-cells, monocytes and granulocytes based on HLA-disparities was possible in 4 pts. In 3 of them, all subsets showed a similar pattern: a clear predominance of a single CBU in all lineages as from day 11-18 onwards. Furthermore, the disappearing CBU was transiently detectable in these pts at day 11. In the fourth patient, T cells were of recipient origin at day 32 with predominance of a single CBU in all other subsets. The disappearing unit could be followed in another 4 pts, which revealed again a transient appearance of that unit in the various lineages at day 11. Although detectable in a total of 8 pts at day 11, all disappearing CBUs were completely lost within 18 days. The prefreeze TNC did not predict for the prevailing UCB, nor did the number of HLA-mismatches. Conclusions. These results show that double UCBT following a non-myeloablative regimen without ATG is associated with a rapid induction of complete single donor chimerism. The disappearing unit, although early detectable, was completely lost within 18 days in all patients, in whom it could be monitored by HLA-specific mAbs. We observed no disproportional increase of any leukocyte subset of the prevailing CBU nor of host leukocyte subset. This characteristic, uniform, early engraftment pattern in the presence of different HLA-disparities may rather argue for a difference in growth potential between both CBUs than an immunological rejection of the disappearing unit. Disclosures. No relevant conflicts of interest to declare.

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Real-Time Quantitative Y Chromosome Specific PCR vs. XY-FISH vs. Fluorescent STR-PCR for Chimerism Analysis after Allogeneic Sex-Mismatched PBSCT.

Blood ◽

10.1182/blood.v104.11.5047.5047 ◽

2004 ◽

Vol 104 (11) ◽

pp. 5047-5047

Author(s):

Ulrike Baak ◽

Olga Marinets ◽

Kathrin Rieger ◽

Igor Wolfgang Blau ◽

Lutz Uharek ◽

...

Keyword(s):

Complete Remission ◽

Real Time ◽

Y Chromosome ◽

Host Cells ◽

Fish Analysis ◽

Str Analysis ◽

Donor Chimerism ◽

Specific Pcr ◽

Highly Sensitive ◽

Chimerism Analysis

Abstract Chimerism analysis after allogeneic stem cell transplantation (SCT) is of major importance to monitor engraftment, graft rejection and disease relapse. Besides the routine application of short tandem repeat (STR)-based methods, XY-FISH comprises a well established method in sex-mismatched transplantation. While the sensitivity levels of these methods are sufficient for monitoring engraftment, a higher sensitivity seems desirable for detection of minimal residual disease (MRD). In this context highly sensitive quantitative Real-Time PCR (RQ-PCR) assays appear to be of great utility, among them a Real-Time quantitative Y chromosome-specific PCR (QYCS-PCR). QYCS-PCR allows chimerism analysis in male patients with female donors, accounting for about 25% of all transplantations. Here we have evaluated the clinical utility of this method compared to the routine methods of STR-PCR and XY-FISH in 36 samples of BM or PB derived from 11 patients at various time points after myeloablative (n=3) and nonmyeloablative (n=8) allogeneic PBSCT. Patients were transplanted for MM (5), AML (3), ALL (2) and NHL (1). Follow-up time ranged from 138 days to 1776 days post PBSCT. The majority of patients (n=8) showed a complete remission, whereas three patients experienced a relapse after PBSCT. Routine STR-analysis was performed applying 4 highly polymorphic markers in a multiplex PCR on an A.L.F. sequencer (sensitivity 5%). In the standardized XY-FISH analysis 200 nuclei were evaluated (sensitivity 1%). RQ-PCR was performed on a Rotor-Gene 3000 cycler. Serial dilutions of male mononuclear blood cells in female cells (100%, 50%, 25%, 10%, 1%, 0,1% and 0,01%) were prepared. The DFFRY gene and the HCK gene (control) were amplified in a duplex Real Time PCR (sensitivity 0,01%). Results of STR-analysis ranged from 40% to 100% donor chimerism. In the lower range of donor chimerism STR-PCR and XY-FISH showed a good correlation and a higher reproducibility than QYCS-PCR. In the majority of samples with complete chimerism in STR-PCR (n=20), compared to 13 samples with complete chimerism in XY-FISH, QYCS-PCR was able to detect residual host cells in 19 samples. Patients in complete remission showed a stable low level of persisting host cells (0,01-0,1%). In one patient with complete donor chimerism in STR-PCR and XY-FISH, QYCS-PCR was able to detect a rising level of host cells before clinical apparent relapse. Thus our data indicate that real-time quantitative Y chromosome-specific PCR based on the DFFRY gene allows highly sensitive and reliable detection of MRD in patients after sex-mismatched allogeneic transplantation. QYCS-PCR seems to be a valuable complementary tool complementing STR-PCR and XY-FISH. In some patients with complete donor chimerism in STR-PCR and XY-FISH analysis, it might allow earlier diagnosis of imminent relapse and offer more time for therapeutic intervention.

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Comparison Between Wilms' Tumor 1 (WT1) Expression Using a Standardized European Leukemia Net (ELN)-Certified Assay and Other Methods for Detection of Minimal Residual Disease (MRD) in MDS and AML Patients after Allogeneic Blood Stem Cell Transplantation

Blood ◽

10.1182/blood.v128.22.3447.3447 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3447-3447

Author(s):

Christina Rautenberg ◽

Sabrina Pechtel ◽

Barbara Hildebrandt ◽

Beate Betz ◽

Ariane Dienst ◽

...

Keyword(s):

Retrospective Analysis ◽

Research Funding ◽

Wilms Tumor ◽

Prospective Trial ◽

Real Life ◽

The Other ◽

Fish Analysis ◽

Donor Chimerism ◽

Chimerism Analysis ◽

Molecular Aberrations

Abstract Introduction An "ideal" marker to monitor MRD after allo-SCT should be informative in the majority of pts and facilitate the use of a method with high sensitivity and specificity in a standardized manner. In addition, to allow repeated monitoring in timely tight intervals but also ensuring patients' comfort such marker should ideally be measurable in peripheral blood (PB). Despite recent identification of several molecular aberrations AML and MDS, many of these do not fulfil the above-mentioned requirements, as they are present only in small patient groups, their potential instability during disease course, the absence of standardized assays and the need for BM as optimal sample source. A molecular marker which might provide these properties is the WT1 gene, as it is overexpressed in the majority of AML pts and in about 50% MDS pts and is measurable in PB by a standardized assay. To estimate its value after allo-SCT we compared serial WT1 measurement with other methods used to monitor MRD in a real-life situation. Patients and Methods For this retrospective analysis all AML and MDS pts who underwent allo-SCT at our center between 2012 and 2016 were screened for PB WT1 mRNA overexpression using the ELN certified Ipsogen® WT1 ProfileQuant® Kit. Pts with WT1 overexpression, as defined by an validated cut-off level of 50 copies/104 ABL copies, were routinely monitored after transplant. In addition, in all pts STR-based chimerism analysis was performed. In pts with chromosomal aberrations existing prior allo-SCT metaphase and FISH analysis was performed, while XY FISH was additionally conducted in pts with sex-mismatched donor-recipient constellation. Furthermore, pts with molecular markers were regularly monitored by NGS or qPCR. Results of WT1 monitoring were correlated with clinical course and compared with results obtained from the other methods. Results Of 104 screened pts 59 (57%) showed an WT1 overexpression at diagnosis and underwent an allo-SCT. This included 40 AML pts (WT1 overexpressed in 66%) and 19 MDS pts (WT1 overexpressed in 44%). Chimerism analysis was accessible in all 59 pts (100%), while 20 pts (34%) could also be monitored by XY-FISH. Additionally, in 40 pts (68%) cytogenetics and FISH were applicable, while 22 pts (37%) could be investigated by NGS or qPCR. Overall, in 5 pts MRD could be monitored by WT1 and chimerism only, while in 29 pts MRD could be monitored by 1, in 22 pts by 2 and in 3 pts by 3 additional methods. With a median follow up of 13 months (2 - 51) we analyzed a total of 472 WT1 samples reflecting a median of 8 samples per patient (2 - 19). One month after allo-SCT 57 pts (97%) showed complete remission and a rapid decrease of WT1 expression below threshold. Only 2 pts had persistant hematological disease with sustained WT1 overexpression. Twenty-four pts (41%) experienced hematologic (62%) or molecular (38%) relapse at a median of 126 d (28 - 938 ) after allo-SCT. In 20 (83%) of these at least one WT1 value wasabove the cut-off before relapse. Median time from first elevated WT1 to relapse was 2 weeks (0 - 27). Only 4 pts (17%) with molecular relapse showed WT1 expression below cut-off. In contrast, known molecular aberrations were found again in 63% and loss of complete donor-chimerism or a positive signal in XY-FISH analyses were only seen in 46% and 57% before relapse. Furthermore, cytogenetics or FISH detected known or new aberrations in 39% before relapse. From 35 pts remaining in CR for a median of 13 months, only 1 (3%) had a transient increase of WT1 expression above the cut-off, whereas WT1 levels of the other 34 pts persisted below. Three pts (9%) with ongoing remission showed a transient decrease of donor-chimerism, whereas even 31% of pts accessible for XY-FISH showed temporary conspicuous results. Conventional cytogenetics and FISH in pts with CR showed transient abnormalities in 18%, whereas in 14% with molecular aberrations these were temporary detectable. Conclusion Measurement of PB WT1 overexpression is an easy accessible method to monitor MRD after allo-SCT that can be employed by a standardized assay in the majority of AML and MDS pts independent from their individual leukemic genotype. Besides these advantages, the results from our real-life experience also suggest that WT1 overexpression allows sensitive detection of imminent relapse after allo-SCT. Taking into account the methodical restrictions of this retrospective analysis, our data requires confirmation in a prospective trial. Disclosures Gattermann: Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel, accomodation expenses, Research Funding. Kobbe:Jansen: Honoraria, Other: travel support; Celgene: Honoraria, Other: travel support, Research Funding.

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Donor Lymphocyte Infusion (DLI) for Mixed Chimerism 6 Months after T Cell Depleted Allogeneic Stem Cell Transplantation (TCD alloSCT) May Prevent Relapse.

Blood ◽

10.1182/blood.v108.11.3674.3674 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3674-3674

Author(s):

Erik W.A. Marijt ◽

Peter A. Von Dem Borne ◽

Renee M.Y. Barge ◽

Piette P. Deutz ◽

M. Floor Beaumont ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Acute Leukemia ◽

Relapse Rate ◽

Low Dose ◽

Mixed Chimerism ◽

Unrelated Donor ◽

Relapse Risk ◽

Donor Chimerism ◽

Chimerism Analysis

Abstract TCD alloSCT is associated with an increased relapse rate compared to non-TCD alloSCT. We and others found an association between the presence of mixed chimerism (MC) after transplantation (Tx) and an increased relapse risk. Since DLI for hematological relapse of acute leukemia cures only 20–30% of patients (pts), pre-emptive DLI might restore complete chimerism (CC) and decrease the relapse rate. Previously, we found that 50% of all relapses occurred within 6–7 months following Tx. We have demonstrated in pts with relapsed CML after TCD alloSCT that low dose DLI given 6–9 months after Tx was effective without inducing severe GVHD. Therefore, we evaluated whether low dose DLI administered 6–9 months after Tx for acute leukemia in pts with MC not suffering from GVHD could decrease the relapse rate without induction of severe GVHD. After conditioning with TBI and cyclophosfamide, 31 pts (AML 21, ALL 8, high risk MDS 2) were transplanted in CR with stem cells from an HLA-identical sibling donor (21), an HLA-matched unrelated donor (8), or a 1 or 2 antigen mismatched family donor (2). The graft was T cell depleted with 20 mg Campath ‘in the bag’. No additional GVHD prophylaxis was given. Bone marrow morphology, immunophenotyping, and chimerism analysis was performed at 3 and 6 months after Tx and at 1.5, 3, 6, 9, and 12 months after DLI. Six months after Tx 7 of 31 pts had relapsed, and 5 pts had died due to infections. In 1 of 19 remaining pts no chimerism analysis was performed, 16 pts were MC with a median of 2% (range 1–35) pt cells, and 2 pts were CC but later reverted to MC. Five of 16 MC pts suffered from GVHD prohibiting DLI and 1/16 MC pts did not receive DLI due to logistical problems, resulting in 12 eligible MC pts. A first DLI at a dose 3×10E6 CD3+ T cells/kg body weight (BW) was given at a median of 8.3 (range 6–22) months after Tx. The median follow-up after the first DLI was 10.5 (range 3–36) months. In 9 pts the percentage patient cells decreased after DLI (p<0.05, Wilcoxon rank test); in 6/9 pts donor chimerism increased to >99% showing the effectiveness of DLI to influence chimerism status. In 1 pt MC increased from 1 to 4% patient cells and in 2 pts MC remained stable. GVHD grade I developed in 1 pt and grade II in 3 pts after the first DLI. When no CC was obtained and no GVHD was present additional DLI was administered in escalating doses of 1×10E7 (1x), 3×10E7 (3x), and 1×10E8 T cells/kg BW (1x). No GVHD developed after subsequent escalated doses of DLI. Two MC pts who did not receive DLI due to GVHD at 6 months after Tx, relapsed within 9 months after Tx as did the patient in whom no chimerism was determined at 6 months. None of the 12 MC pts treated with DLI relapsed. In conclusion, our results indicate that low dose DLI projected to be administered 6–9 months after Tx increases the degree of donor chimerism in the majority of pts and may reduce the likelihood of relapse without severe GVHD. Since 7 of the 10 relapses occurred within the first 6 months after Tx, we aim to administer low dose DLI at 3–4 months after Tx to study its effect on chimerism and relapse risk in our next group of pts. Since the short interval between DLI and Tx will increase the likelihood of developing GVHD using unmodified DLI, also CD4 purified T cells will be administered.

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Persistence of Recipient-Derived Antiviral T cells Severely Influence Donor Chimerism Post Allogeneic HSCT,

Blood ◽

10.1182/blood.v118.21.3240.3240 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3240-3240

Author(s):

Sylvia Borchers ◽

Michael Stadler ◽

Susanne Luther ◽

Tina Ganzenmueller ◽

Brigitte Pabst ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cord Blood ◽

Cd8 T Cells ◽

T Cell Subsets ◽

Reduced Intensity Conditioning ◽

Donor Chimerism ◽

Conditioning Regimens ◽

Chimerism Analysis ◽

Cmv Reactivation

Abstract Abstract 3240 Analysis of donor chimerism is a well established technique to monitor engraftment and detect pending relapse in patients after allogeneic hematopoietic stem cell transplantation (HSCT). Over the last decade, use of unrelated and/or mismatched donors as well as alternative grafts like cord blood (CB) has increased, and, in addition reduced intensity conditioning regimens are widely applied. Thus, recipients are increasingly being exposed to both persistent mixed chimerism and infectious complications because of delayed immune reconstitution. Donor chimerism is analyzed routinely from peripheral blood cells in all recipients. In addition, in a prospective study to evaluate usefulness of subset chimerism, T cell chimerism is analyzed in selected patients since 2007. Reconstitution of CMV-specific CD8 T cells (CMV-CTL) is monitored by multimers (multimeric dye-labeled recombinant-MHC-I-peptide-complexes) since 2006 to evaluate CMV-specific immune reconstitution post HSCT. Using this method, HLA-restriction of the multimers enables detection of residual recipient CMV-CTLs in mismatched transplantations. Interestingly, we found that CMV reactivation is accompanied by a decline in donor chimerism in some patients and that recipient CMV-CTLs persisting post HSCT expand upon CMV reactivation. Table 1 summarizes first data of this analysis in patients transplanted between 2007 and 2011 in our centre. Table 1: Data from patients transplanted between 2007 and 2011 at MHH # underlying disease R/D gender Donor Conditioning regimen GvHD prophylaxis Graft 1 AML f/f MMUD Flamsa(TBI)/ATG CSA/MMF PBPC 2 AML f/f MMRD Flu/Melph/Thiotepa/ATG TCD PBPC 3 AML m/m MMUD Flamsa(TBI)/Thymo CSA/MMF PBPC 4 NHL m/f MUD Flu/Cy/Thymo CSA/MMF PBPC 5 ALL m/m MMUD TBI/Cy/ATG CSA/MMF PBPC 6 AA f/m hla-ident sibl. Flu/Cy/TBI/Thymo CSA/MTX BM 7 MDS m/m MMUD Flu/Cy/TBI/ATG CSA/MMF cord blood 8 NHL m/m MMUD Flu/Cy/ATG CSA/MTX PBPC # R/D CMV-serostatus aGvHD CMV reactivation (CMV-R) leukocyte chimerism decline post CMV-R T cell chimerism decline post CMV-R persisting recipient CMV-CTLs 1 R+D- yes 40, 61 yes yes 2 R+D- no 27, 90, 188 no yes 3 R+D+ yes no no yes 4 R+D+ no 18 yes yes yes 5 R+D- suspected 34, 90 no no yes 6 R+D- no 55 yes yes 7 R+D- yes 85, 113 yes yes yes* 8 R+D- no (HvG) 33 yes yes yes* * confirmed by chimerism analysis in enriched CMV-CTL Patient 7 received a double mismatched cord-blood graft. As expected the recipient-CMV-CTLs declined after HSCT and by day +50 post-HSCT no CMV-CTLs (A*0201-NLVP multimer) could be detected anymore. The patient had a CMV reactivation on day +85 as shown by pp65 antigenemia assay. On day +90, 72 CMV-CTLs/μl were detected, further increasing to over 200/μl by day +152. To further analyze the origin and functionality, CMV-CTLs detected on day +90 were enriched by MACS to a purity of 97% in the CD3+CD8+ T cells. Donor chimerism was only 4%. After reconstitution of autologous CMV-CTLs, the patient experienced an additional subclinical CMV reactivation on day +113, not requiring any treatment at this time. In patient 8 a subclinical CMV reactivation on day +33 led to proliferation of CMV-CTLs and HLA-A*24 restricted and -B*35 restricted CMV-CTLs rose from 0 cells/μl (A*24 0.05%, B*35 0.04% of CD3CD8 T-cells) to 1 cell/μl and 21 cells/μl (A*24 0.28%, B*35 4.05% of CD3CD8 T-cells), respectively. Donor chimerism decreased from 51% on day +33 to 0% by day +62. Chimerism analysis of T-cell subsets on day +62 and of CMV-CTLs on day +69 revealed a 0% donor chimerism in these subsets. We speculate that in this patient CMV reactivation led to an inflammatory environment, which might have promoted loss of the graft. Our data indicate that T cell-subset chimerism analyses may contribute to a better understanding of chimerism kinetics. Furthermore, recipient-derived CMV-CTLs may be able to control CMV reactivation, especially after reduced intensity conditioning but also after standard conditioning regimens (i.e. in patient 5), but can severely influence donor chimerism and thus might have negative effects as well. We are currently investigating donor chimerism in T cell subsets and CMV-CTL reconstitution to gain insight into the complex immune responses and reconstitution processes occurring after allogeneic HSCT or CB-SCT. Disclosures: No relevant conflicts of interest to declare.

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Monitoring of GRAFT Versus Leukemia Effect with CD34+ Specific DONOR Chimerism Analysis

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2013.12.464 ◽

2014 ◽

Vol 20 (2) ◽

pp. S276-S277

Author(s):

Maxim Norkin ◽

Christopher Ramin Cogle ◽

Helen Leather ◽

Amy Meacham ◽

Emma Rosenau ◽

...

Keyword(s):

Donor Chimerism ◽

Graft Versus Leukemia ◽

Chimerism Analysis ◽

Graft Versus Leukemia Effect

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Donor Lymphocyte Infusion in Patients with Thalassemia Major Who Have Mixed Chimerism Following Allogeneic Stem Cell Transplant

Blood ◽

10.1182/blood.v126.23.1965.1965 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1965-1965

Author(s):

Aby Abraham ◽

Eunice Sindhuvi ◽

Anu Korula ◽

Fouzia NA ◽

Alok Srivastava ◽

...

Keyword(s):

Stem Cell ◽

Graft Rejection ◽

Thalassemia Major ◽

Donor Lymphocyte Infusion ◽

Mixed Chimerism ◽

Cell Transplant ◽

Donor Chimerism ◽

Level Ii ◽

Chimerism Analysis ◽

Level I

Abstract Allogeneic stem cell transplantation (HSCT) is the only curative option for patients with thalassemia major (BTM). HSCT in BTM is associated with increased risk of graft rejection. Some patients do not achieve full donor chimerism after transplant despite hematopoietic engraftment. Early post transplant mixed chimerism (MC) is a known predictor of secondary graft rejection. Data is limited on the role of donor lymphocyte infusion (DLI) to prevent secondary graft rejection following detection of early MC in BTM. We report our experience with DLI in BTM patients who had developed progressive worsening early MC after HSCT. All patients with BTM who underwent HSCT at our centre between September 1994 and December 2013 were included in the analysis. During this period, 406 patients underwent HSCT for BTM. Median age was 8 years (range: 1 - 25) and 247 were males (60.8 %). Conditioning regimen was busulphan based in 274 (67.5%) or treosulfan based in 132 (32.5%). The graft source was BM in 305 (75.1%) and PBSC in 101 (24.9%) etc. Median CD34 stem cell dose was 9.24x106/kg (range: 2.1- 33). GVHD prophylaxis consisted of CSA and short course MTX. Patients who engrafted underwent chimerism analysis of peripheral blood at day 28 and between days 60-90. Subsequently chimerism analysis was done at the discretion of the treating physician. Chimerism analysis was done by qualitative and quantitative methods up to 2007 and by quantitative method from 2007. MC was defined by >5% recipient cells at any time point post transplant. The severity of MC was determined as per previously published criteria (level I= < 10%, level II = 10-25 %, level III = >25 %). In patients who developed MC, immunosuppression was tapered and stopped and chimerism monitored. Cyclosporine was tapered by approximately 30% every 2 weeks until there was stable MC or complete donor chimerism. If progressive loss of donor chimerism leading to level II or III MC occured on two consecutive occasions with a concurrent drop in hemoglobin despite stopping immunosuppression and in the absence of GVHD, DLI was given. Patients should have had at least 10% donor chimerism in order to have a DLI. There were 109 (41.6 %, out of 262 patients in whom chimerism was done) patients who developed MC. Of these, 35 (32.1%) developed progressively worsening MC and drop in hemoglobin despite tapering of immunosuppression (91% of these were within day 60). Of these 35 patients, 23 (65.7%) received DLI while 12 developed anemia requiring transfusion (4continued to be on blood transfusion and 8 underwent a second transplant). Among 23 patients who received DLI, 9 (39.1%) showed response (rise in Hb with CC or stable MC) while 14 (60.9%) failed to show any response. The median time to show response was 28 (8-192) days and the median time to peak response was 44 (23-442) days. The 14 patients who did not show any response developed anemia requiring transfusion (11 continued to be on blood transfusion while 3 underwent a second transplant). While 80% of those with level II chimerism responded to DLI, only 31.2% of those with level III chimerism showed a response (summarized in Table 1). DLI was well tolerated by the majority of the patients. However, 7 (30.4%) patients developed cytopenia. While 6 of these had level III chimerism at the time of DLI, 1 had level II chimerism. Two patients developed Grade IV cytopenia. Grade 2 acute GVHD developed in 3 (13%; liver=2, gut=1) which responded to immunosuppressive treatment. Chronic GVHD developed in 2 (8.7%: extensive=1). There was no mortality related to DLI. DLI helped close to 40% patients achieve either complete donor chimerism or stable MC who otherwise might have required a second stem cell transplant with minimal risk. There was a trend to suggest that early administration of DLI at level II MC was superior to DLI administered at level III. Table 1. Factors affecting the response to DLI n(%) Effect of DLI Response(CC+Stable MC) No response p Chimerism level I 0 0 0 II 5 4(80%) 1 III 16 5(31.2%) 11 0.07 MC 2 0 2 Lucarelli class I 1 1(100%) 0 II 8 3(37.5%) 5 III 14 5(35.7% ) 9 0.4 Conditioning Bu-Cy-ATG/ALG 12 4(33.3%) 8 Thio-Treo-Flu 11 5(45.5%) 6 0.7 Graft BM 17 5(29.4%) 12 PB 6 4(66.7%) 2 0.1 Time of 1st DLI <180 days 18 7(38.9%) 11 >180 days 5 2(40%) 3 1.0 Total 23 9(39.1%) 14 Disclosures No relevant conflicts of interest to declare.

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Double Umbilical Cord Blood Transplantation Preceded by a Reduced-Intensity Conditioning Regimen: Rapid Induction of Single Donor Chimerism and Highly Predictive Value of Early CD4+ T Cell and NK Cell Predominance

Blood ◽

10.1182/blood.v118.21.3026.3026 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3026-3026

Author(s):

Judith AE Somers ◽

Anneke Brand ◽

Bronno van der Holt ◽

Yvette van Hensbergen ◽

Kees Sintnicolaas ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Nk Cell ◽

Cord Blood Transplantation ◽

Conditioning Regimen ◽

Donor Chimerism ◽

Blood Transplantation ◽

Single Donor ◽

Chimerism Analysis ◽

Hla Mismatches

Abstract Abstract 3026 Background Double umbilical cord blood transplantation (UCBT) results in higher engraftment rates as compared to single UCBT in adult patients. Sustained hematopoiesis is usually derived from a single cord blood unit (CBU) after double UCBT. So far, the mechanism of predominance of a particular CBU is unresolved. In a prospective single-arm phase II study (HOVON-106) we monitored early engraftment kinetics to determine whether graft predominance after double UCBT is driven by specific leukocyte subpopulations. Methods 36 consecutive patients (pts) from 5 Dutch centers with high-risk hematological diseases received a double UCBT, preceded by a reduced-intensity conditioning regimen (Cy 60 mg/kg/ Flu 160 mg/m2/ TBI 2×2 Gy). CBUs were selected by intermediate resolution typing for HLA-A and -B loci and by high-resolution typing for HLA-DRB1. The minimal required HLA-match grade was 4/6. Chimerism analysis (STR-PCR) of unseparated peripheral blood (PB) and bone marrow (BM) cells was performed as from day +32 onwards. In addition, chimerism analysis in PB leukocyte subpopulations by flowcytometry using lineage-specific (CD45, CD3, CD4, CD8, CD19, CD16/56, CD14 and CD33) monoclonal antibodies (mAbs) in combination with human HLA-antigen specific mAbs (HLA-mAbs) was performed at day +11, +18, +25 and +32 if discriminating HLA-mismatches between recipient and CBUs were present. Day +32 flowcytometry results were compared to day +32 PB STR-PCR results. Results The median number of prefreeze total nucleated cells (TNC) per CBU was 2.3×107/kg (range: 1.5–5.5). Median numbers of post-thaw viable CD34+ cells, T-, B- and NK cells were 0.32 (range: 0–1.7), 4.4 (range: 0.4–36), 9.7 (range: 0.7–79) and 7.2 (range: 0.24–45) x105/kg, respectively. One pt was non-evaluable for engraftment due to insufficient follow up after early relapse. The cumulative incidence of neutrophil recovery (≥0.5×109/l) was 91% with a median time to neutrophil recovery of 33 days (range: 15–82). Primary graft failure occurred in 1 pt. Chimerism analysis, performed at day +32 by STR-PCR revealed single CBU predominance in all pts whereas residual non-engrafting CBU and recipient cells were detectable in only 3 and 7 pts, respectively. Simultaneous 3-donor-origin detection of leukocyte subpopulations by flowcytometry based on HLA disparities was possible in 12 pts. Flowcytometry using HLA-mAbs demonstrated single CBU predominance in various leukocyte subsets as from day +11 onwards in the majority of pts. Moreover, ultimate engraftment of a particular CBU was reliably predicted for by chimerism within the CD4+ (in 90% of pts) and NK cell (in 90% of pts) subsets at this early time point. In contrast, predominance of the engrafting CBU in monocytic en myeloid subsets was observed in only 70% and 33% of pts, respectively, at day +11. The numbers of CD8+ and B-cells were too low for analysis in the majority of pts. Predominance of the ultimate engrafting CBU was established in all subpopulations at day +18. Furthermore, the contribution of the non-engrafting CBU to the different leukocyte subsets was negligible or even absent as from day +18 onwards. Recipient hematopoiesis did not contribute to PB cell recovery either. The results of day +32 flowcytometry (CD45+ population) were similar to results of day +32 PB STR-PCR. The number of prefreeze TNC did not predict for the ultimately engrafting CBU, nor did the number of post-thaw CD34+, T, B or NK cells. Engraftment was not associated with the degree of HLA mismatches or presence of KIR ligand mismatches among recipient and CBUs. Conclusions These results show that single donor chimerism is rapidly established after double UCBT, preceded by a 4 Gy TBI-based conditioning regimen without ATG. In addition, our flowcytometry data suggest the occurrence of CBU predominance within 2 weeks post transplant, in the course of which both CD4+ and NK cell predominance at day +11 are highly predictive for ultimate single donor chimerism. That early engraftment pattern of leukocyte subsets might suggest a key role for either CD4+ T cells or NK cells in CBU predominance. Disclosures: Janssen: Novartis: Consultancy.

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Peripheral Blood CD34+ Specific Donor Chimerism Analysis As an Early Indicator of Disease Relapse After Allogeneic Hematopoietic Stem Cell Transplant in Patients with Myelodysplastic Syndromes and Acute Myelogenous Leukemia.

Blood ◽

10.1182/blood.v120.21.3074.3074 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3074-3074

Author(s):

Maxim Norkin ◽

Amy Meacham ◽

Mai H Ta ◽

Neil Benson ◽

Steven Goldstein ◽

...

Keyword(s):

Peripheral Blood ◽

Myelogenous Leukemia ◽

Cell Transplant ◽

Disease Relapse ◽

Str Analysis ◽

Hematopoietic Stem ◽

Donor Chimerism ◽

Cd34 Cells ◽

Acute Myelogenous ◽

Chimerism Analysis

Abstract Abstract 3074 Background: Disease relapse after allogeneic hematopoietic stem cell transplant (HCT) remains a major obstacle to treatment success and cure for patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). Early identification of patients at a high risk for disease relapse has significant practical implications allowing early therapeutic interventions. We evaluated a novel approach for an early identification of a post-transplant relapse in patients with MDS and AML. Methods: CD34+ specific donor chimerism analysis was obtained in 6 HCT recipients with CD34+ AML and MDS. Starting at day +30 post HCT CD34+ cells from peripheral blood were positively selected by immunomagnetic isolation and then underwent fluorescence-activated cell sorting. Purified CD34+ cells were subsequently evaluated for percentage of donor DNA contribution by short tandem repeat (STR) analysis. CD34+ specific donor chimerism analysis was repeated monthly until hematologic relapse or death occurred. Simultaneously, conventional donor chimerism analysis was measured in the subpopulations of peripheral blood cells. Results: CD34+ cells were isolated from peripheral blood with a very high purity >95% and in sufficient quantity for reliable STR analysis in all study patients. Out of 6 evaluated patients, 3 developed hematologic relapse confirmed by bone marrow evaluation. In each relapsed patient CD34+ specific chimerism dropped to <15% prior to the confirmation of hematologic relapse. However, conventional STR analysis in granulocytes, T-cells and B-cells remained >80% (Figure 1). Moreover, obvious and steady decline in CD34+ specific donor chimerism significantly preceded the time of hematologic relapse. In contrast, no decline in CD34+ chimerism was identified in 3 patients without hematologic relapse. (Figure 1). Conclusion: Though this is a preliminary study, the results suggest that CD34+ specific donor chimerism analysis is a useful technique for timely identification of hematologic relapse in MDS and AML patients after HCT and may provide significantly earlier relapse detection than conventional chimerism analysis. Disclosures: No relevant conflicts of interest to declare.

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