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1.
Ultrasensitive Chimerism Enhances Measurable Residual Disease Testing Post-Allogeneic Hematopoietic Cell Transplantation
Kanaan, S. B., Urselli, F., Radich, J. P., Nelson, J. L.
Blood advances. 2023
Abstract
Increasing mixed chimerism (reemerging recipient cells) after allogeneic hematopoietic cell transplantation (allo-HCT) can indicate relapse, the leading factor determining mortality in blood malignancies. Most clinical chimerism tests have limited sensitivity and are primarily designed to monitor engraftment. We developed a panel of qPCR assays using TaqMan chemistry capable of quantifying chimerism on the order of 1-in-a-million. At such analytic sensitivity, we hypothesized it could inform on relapse risk. As a proof-of-concept, we applied our panel on a retrospective cohort of acute leukemia patients with known outcomes post-allo-HCT. Recipient cells in bone marrow aspirates (BMA) remained detectable in 97.8% of tested samples. Absolute recipient chimerism proportions and rates at which these proportions increased in BMA in the first 540 days post-allo-HCT were associated with relapse. Detectable MRD (measurable residual disease) by flow cytometry in BMA post-allo-HCT showed limited correlation with relapse. This correlation noticeably strengthened when combined with increased recipient chimerism in BMA, demonstrating the ability of our ultrasensitive chimerism assay to augment MRD data. Our technology reveals an underappreciated usefulness of clinical chimerism. Used side-by-side with MRD assays, it promises to improve identification of patients with the highest risk of disease reoccurrence for a chance for early intervention.
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2.
STR typing of skin swabs from individuals after an allogeneic hematopoietic stem cell transplantation
von Máriássy, D., Reibke, R., Verbeek, M., Gätjens, B., Schiller, R., Anslinger, K.
International journal of legal medicine. 2023;137(1):227-236
Abstract
One of the pre-requisites for forensic DNA analysis is the fact that all nucleated cells of a person carry the same genetic information. However, this is not the case for individuals who have received an allogeneic hematopoietic stem cell or bone marrow transplantation, as all new cells formed by the bone marrow no longer show the genetic information of the recipient but that of the donor, while all other cells still carry the original information before transplantation. Thus, STR typing of a blood sample after successful transplantation yields a DNA profile that differs from the recipient's original profile and corresponds to the donor genotype instead. Evidence from a routine case suggests that transplanted individuals may show donor alleles in skin swabs, as well. In order to examine this issue more closely, various skin swabs from 28 patients who have received an allogeneic hematopoietic stem cell transplantation were examined in this study. Swabs from the right and left palm, the back of the hand, one of the two upper arms, and the neck were collected from each person. Ninety-one of the 140 resulting swabs delivered useful results. All of those samples showed mixtures of recipient and donor DNA with different mixture ratios and the proportions of donor and recipient alleles revealed inter- and intra-individual differences. Those results were discussed with respect to graft versus host disease.
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3.
Bone marrow CD34+ molecular chimerism as an early predictor of relapse after allogeneic stem cell transplantation in patients with acute myeloid leukemia
Malagola, M., Polverelli, N., Beghin, A., Bolda, F., Comini, M., Farina, M., Morello, E., Radici, V., Accorsi Buttini, E., Bernardi, S., et al
Frontiers in oncology. 2023;13:1133418
Abstract
BACKGROUND Minimal residual disease (MRD) monitoring is an important tool to optimally address post-transplant management of acute myeloid leukemia (AML) patients. METHODS We retrospectively analyzed the impact of bone marrow CD34+ molecular chimerism and WT1 on the outcome of a consecutive series of 168 AML patients submitted to allogeneic stem cell transplantation. RESULTS The cumulative incidence of relapse (CIR) was significantly lower in patients with donor chimerism on CD34+ cells ≥ 97.5% and WT1 < 213 copies/ABL x 10^4 both at 1(st) month (p=0.008 and p<0.001) and at 3(rd) month (p<0.001 for both). By combining chimerism and WT1 at 3(rd) month, 13 patients with chimerism < 97.5% or WT1 > 213 showed intermediate prognosis. 12 of these patients fell in this category because of molecular chimerism < 97.5% at a time-point in which WT1 was < 213. CONCLUSIONS Our results confirm that lineage-specific molecular chimerism and WT1 after allo-SCT (1(st) and 3(rd) month) are useful MRD markers. When considered together at 3(rd) month, CD34+ molecular chimerism could represent an earlier predictor of relapse compared to WT1. Further studies are necessary to confirm this preliminary observation.
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4.
Chimerism Testing by Next Generation Sequencing for Detection of Engraftment and Early Disease Relapse in Allogeneic Hematopoietic Cell Transplantation and an Overview of NGS Chimerism Studies
Liacini, A., Tripathi, G., McCollick, A., Gravante, C., Abdelmessieh, P., Shestovska, Y., Mathew, L., Geier, S.
International journal of molecular sciences. 2023;24(14)
Abstract
Chimerism monitoring after allogenic Hematopoietic Cell Transplantation (allo-HCT) is critical to determine how well donor cells have engrafted and to detect relapse for early therapeutic intervention. The aim of this study was to establish and detect mixed chimerism and minimal residual disease using Next Generation Sequencing (NGS) testing for the evaluation of engraftment and the detection of early relapse after allo-HCT. Our secondary aim was to compare the data with the existing laboratory method based on Short Tandem Repeat (STR) analysis. One hundred and seventy-four DNA specimens from 46 individuals were assessed using a commercially available kit for NGS, AlloSeq HCT NGS (CareDx), and the STR-PCR assay. The sensitivity, precision, and quantitative accuracy of the assay were determined using artificially created chimeric constructs. The accuracy and linearity of the assays were evaluated in 46 post-transplant HCT samples consisting of 28 levels of mixed chimerism, which ranged from 0.3-99.7%. There was a 100% correlation between NGS and STR-PCR chimerism methods. In addition, 100% accuracy was attained for the two external proficiency testing surveys (ASHI EMO). The limit of detection or sensitivity of the NGS assay in artificially made chimerism mixtures was 0.3%. We conducted a review of all NGS chimerism studies published online, including ours, and concluded that NGS-based chimerism analysis using the AlloSeq HCT assay is a sensitive and accurate method for donor-recipient chimerism quantification and minimal residual disease relapse detection in patients after allo-HCT compared to STR-PCR assay.
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5.
Posttransplant MRD and T-cell chimerism status predict outcomes in patients allografted with AML/MDS
Loke, J., McCarthy, N., Jackson, A. E., Siddique, S., Hodgkinson, A., Mason, J., Crawley, C. R., Gilleece, M., Peniket, A. J., Protheroe, R., et al
Blood advances. 2023
Abstract
Allogeneic stem-cell transplantation allows the delivery of curative graft-versus-leukemia (GVL) in patients with acute myeloid leukemia/myelodysplasia (AML/MDS). Surveillance of T-cell chimerism, measurable residual disease (MRD) and blast HLA-DR expression may inform whether GVL effectiveness is reduced. We report the prognostic impact of these biomarkers in patients allografted for AML/MDS. 187 patients from FIGARO, a randomized trial of reduced-intensity conditioning regimens in AML/MDS, were alive and relapse-free, at the first MRD timepoint and provided bone marrow for flow cytometric MRD monitoring and blood samples for T-cell chimerism analysis, requested to month+12. 29 (15.5%) patients had at least one MRD-positive result post-transplant. MRD-positivity was associated with reduced overall survival (OS) (HR:2.18, p=0.0028) as a time-varying Cox variable and remained significant irrespective of pre-transplant MRD status in multivariate analyses (p<0.001). 94 patients had sequential MRD with T-cell chimerism results at months+3/+6. Patients with full donor T-cell chimerism (FDTC) had an improved OS as compared with patients with mixed-donor T-cell chimerism (MDTC) (adjusted-HR=0.4, p=0.0019). In patients with MDTC (month+3 or +6), MRD-positivity was associated with decreased 2yr-OS (34.3% [95% CI:11.6-58.7] versus MRD-negative 71.4% [95% CI:52.2-84.0], p=0.001). In contrast, in the group with FDTC, MRD was infrequent and did not impact outcome. Amongst patients with post-transplant MRD-positivity, decreased HLA-DR expression on blasts significantly reduced OS, supporting this as a mechanism for GVL escape. Post-transplant MRD is an important predictor of outcome in patients allografted for AML/MDS and is most informative when combined with T-cell chimerism results, underlining the importance of a GVL effect in AML/MDS.
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6.
Immunosuppression Boost With Mycophenolate Mofetil for Mixed Chimerism in Thalassemia Transplants
Mehta, P., Singh, A., Halder, R., Verma, M., Agrawal, N., Ahmed, R., Bhurani, D.
Transplantation and cellular therapy. 2023;29(2):122.e1-122.e6
Abstract
Declining mixed chimerism (MC) portending impending graft failure is an undesirable outcome. However, for hemoglobinopathies in a stable state of MC, residual host cells persist without rejection in 30% to 40% of patients after hematopoietic stem cell transplantation (HSCT). Early detection and level of MC have been attributed to be significant in predicting the outcome of MC. Common clinical approach on MC is removal of immunosuppression. We retrospectively evaluated MC in transfusion dependent thalassemia patients who underwent HSCT in our institution between September 2013 and January 2022 to determine the outcome of MC on the basis of our approach of immunosuppression boost in comparison to conventional approach of immunosuppression tapering. Among 90 patients, 22 (24.4 %) had MC at some time point after transplantation with a median follow-up of 496 (67-1492) days. Immunosuppression withdrawal was done in 12 (54.5%) patients, whereas immunosuppression boost was given in 8 (36.3%) patients. In the immunosuppression withdrawal group, 2 (16.6%) patients evolved to complete chimerism, 5(41.6%) patients had persistent MC (PMC), whereas 5 (41.6%) patients had secondary rejection. All these rejections were at median of 186 (89-251) days after transplantation. In the immunosuppression boost group, all patients (n = 8) had PMC with no secondary rejection until median follow-up of 255(97-812) days after transplantation. We acknowledge that we need more experience with our unconventional approach of immunosuppression boost to obtain statistical significance in comparison to the conventional approach of tapering of immunosuppression.
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7.
Association of early donor chimerism status with survival outcomes in post allogeneic haematopoietic stem cell transplant patients of nonmalignant diseases
Sial, N., Ahmed, P., Wattoo, F. H., Ali, N.
JPMA. The Journal of the Pakistan Medical Association. 2022;72(3):464-470
Abstract
OBJECTIVE To highlight the association of early donor chimerism status at 2nd month with various survival outcomes. METHOD The retrospective study was conducted at the Armed Forces Bone Marrow Transplant Centre, Rawalpindi, Pakistan, and comprised patient data from January 2011 to July 2016. Data related to participants who underwent human leukocyte antigen-matched transplants for bone marrow failure syndrome and beta thalassemia major. Short tandem repeat-based polymerase chain reaction was used to assess donor chimerism status. Overall survival, disease-free survival, relapse-free survival, and graft versus host disease-free survival rates were noted. Data was analysed using SPSS 23. RESULTS Of the 106, 64(60.4%) had bone marrow failure syndrome and 42(39.6%) had beta thalassemia major. The overall median follow-up was 13.53 months (range: 1.81-62.73 months). Early donor chimerism status was associated with overall survival (p=0.02) and disease-free survival (p=0.01). Mixed donor chimerism was less hazardous in terms of overall survival (p=0.04) and disease-free survival (p=0.02). CONCLUSIONS Early mixed donor chimerism contributed to optimal survival in nonmalignant disease.
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8.
Utility of assessing CD3(+) cell chimerism within the first months after allogeneic hematopoietic stem-cell transplantation for acute myeloid leukemia
Bendjelloul, M., Usureau, C., Etancelin, P., Saidak, Z., Lebon, D., Garçon, L., Marolleau, J. P., Desoutter, J., Guillaume, N.
Hla. 2022;100(1):18-23
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Abstract
After allogeneic hematopoietic stem-cell transplantation (alloHSCT), the chimerism assay is used to monitor cell engraftment and quantify the respective proportions of donor/recipient cells in blood or bone-marrow samples. Here, we aimed to better assess the utility of determining CD3(+) cell chimerism within the first 6 months post alloHSCT. One hundred and thirty five patients diagnosed with acute myeloid leukemia were enrolled in this study. We observed significantly lower overall survival and relapse free survival for patients without full donor chimerism (<95%, <98%, <99%) in whole blood at Day 30, as well as at Day 90 after alloHSCT, than for patients with full donor chimerism. This outcome was not observed when assessing selected CD3(+) cells. However, at Day 90, patients with discordant whole blood versus selected CD3(+) cell chimerism showed both significantly lower overall survival and relapse free survival, giving an interest to assess selected cells chimerism.
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9.
Evaluation of a quantitative PCR-based method for chimerism analysis of Japanese donor/recipient pairs
Minakawa, K., Ono, S., Watanabe, M., Sato, Y., Suzuki, S., Odawara, S., Kawabata, K., Ueda, K., Nollet, K. E., Sano, H., et al
Scientific reports. 2022;12(1):21328
Abstract
Chimerism analysis is a surrogate indicator of graft rejection or relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Although short tandem repeat PCR (STR-PCR) is the usual method, limited sensitivity and technical variability are matters of concern. Quantitative PCR-based methods to detect single nucleotide polymorphisms (SNP-qPCR) are more sensitive, but their informativity and quantitative accuracy are highly variable. For accurate and sensitive chimerism analysis, a set of KMR kits (GenDx, Utrecht, Netherlands), based on detection of insertions/deletions (indels) by qPCR, have been developed. Here, we investigated informativity and validated the accuracy of KMR kits in Japanese donor/recipient pairs and virtual samples of DNA mixtures representative of Japanese genetic diversity. We found that at least one recipient-specific marker among 39 KMR-kit markers was informative in all of 65 Japanese donor/recipient pairs. Moreover, the percentage of recipient chimerism estimated by KMRtrack correlated well with ratios of mixed DNA in virtual samples and with the percentage of chimerism in HSCT recipients estimated by STR-PCR/in-house SNP-qPCR. Moreover, KMRtrack showed better sensitivity with high specificity when compared to STR-PCR to detect recipient chimerism. Chimerism analysis with KMR kits can be a standardized, sensitive, and highly informative method to evaluate the graft status of HSCT recipients.
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10.
The clinical application of SNP-based next-generation sequencing (SNP-NGS) for evaluation of chimerism and microchimerism after HLA-mismatched stem cell microtransplantation
Li, W., Xu, Y., Feng, Y., Zhou, H., Ma, X., Wu, D., Chen, S., Sun, A.
International journal of hematology. 2022
Abstract
Genetic diagnostic methods for evaluation of chimerism after HSCT, such as STR-PCR and XY-FISH, have limited sensitivity. When donor chimerism is in the micro range (< 1%), deviations in the accuracy of assessment are the most significant disadvantage of these common methods. We developed a highly sensitive method that applies SNPs based on NGS in order to explore the value of donor cell microchimerism in microtransplantation (MST). This improved SNP-NGS approach has higher sensitivity (0.01-0.05%) and only requires a small amount of DNA (8-200 ng). We retrospectively analyzed the clinical data of 48 patients with AML who received HLA-mismatched stem cell MST at our center to assess the impact of microchimerism on clinical prognosis. Patients whose duration of microchimerism was > 10.5 months (median) had a relapse rate of 26.1%, and had better 5-year LFS and OS (73.4% and 82.6%). In contrast, patients whose duration of microchimerism was < 10.5 months had a higher relapse rate (69.6%), and their 5-year LFS and OS were 30.4% and 43.5%. In conclusion, duration of donor chimerism is highly valuable for assessment of survival and prognosis in patients with AML who have received HLA-mismatched stem cell MST, especially the intermediate-risk group.