-
1.
Optimizing Donor Chimerism Threshold for Next Generation Sequencing Monitoring of Measurable Residual Disease Post-Allogeneic Stem Cell Transplant for Myeloid Neoplasms
Puzo, C. J., Tormey, C. A., Rinder, H. M., Siddon, A. J.
Transplantation and cellular therapy. 2023
Abstract
BACKGROUND Next-Generation Sequencing (NGS) is used to monitor genetically-measurable residual disease (gMRD) following allogeneic stem cell transplant (aSCT). It is unknown whether an upper limit of chimerism exists such that gMRD NGS testing can be safely forgone. METHODS We reviewed 61 AML and 24 MDS patients between 2016-2020 with at least 1 NGS panel before and after aSCT. Donor chimerism was quantified. Logistic regression characterized which factors predicted gMRD. Receiver operator curves (ROC) determined the optimal chimerism threshold for which gMRD would not be detected. Data from an additional 22 patients with follow-up NGS testing in 2022, was also analyzed to validate our proposed threshold. RESULTS Donor chimerism (OR= 0.38, 95% CI[0.10,0.62], p=0.02), as expected, was a significant predictor of gMRD. Age, gender, conditioning regimen, presence of a related donor, and diagnosis were not associated with gMRD. A chimerism threshold of 92.5% optimized sensitivity (97.7) and specificity (95.4) such that values >92.5% strongly predicted absence of gMRD (AUC= .986). The validation cohort demonstrated similar strongly predictive capability (AUC= .974) with appropriate sensitivity (100%) and specificity (90.9%). CONCLUSION NGS monitoring of gMRD is redundant at chimerism values greater than a more conservative threshold of 92.5% after stem cell transplant.
-
2.
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.
-
3.
Age Impacts Risk of Mixed Chimerism Following RIC HCT for Non-SCID Inborn Errors of Immunity
Fitch, T., Lane, A., McDonnell, J., Bleesing, J., Jordan, M., Kumar, A., Khandelwal, P., Khoury, R., Marsh, R., Chandra, S.
Transplantation and cellular therapy. 2023
Abstract
BACKGROUND Alemtuzumab, fludarabine and melphalan containing reduced intensity conditioning (RIC) is commonly used in patients undergoing allogeneic hematopoietic cell transplantation (HCT) for definitive treatment of high-risk inborn errors of immunity (IEI). Although survival is favorable, there is increased risk of mixed chimerism leading to secondary graft failure. OBJECTIVES Evaluate factors associated with risk of developing mixed chimerism, particularly the influence of age in patients undergoing allogeneic HCT for non-SCID IEI who received a uniform RIC regimen that included intermediate schedule alemtuzumab, fludarabine and melphalan. We hypothesized that age would impact incidence of mixed chimerism. STUDY DESIGN We retrospectively reviewed records of patients who underwent HCT for non-SCID IEIs with a uniform RIC regimen that included intermediate schedule alemtuzumab (1 mg/kg divided over days -14 to - 10), fludarabine (150 mg/m2 or 5 mg/kg if weight <10 kg divided over days -9 to -4), and melphalan (140 mg/m2 or 4.7 mg/kg if weight <10 kg on day-3) between 2010 and 2020 at our institution. Mixed chimerism was defined as <95% donor on 2 or more consecutive occasions on whole blood. RESULTS Ninety-three patients who underwent RIC-HCT for non-SCID IEI using intermediate schedule alemtuzumab, fludarabine, and melphalan were evaluated and categorized into 3 groups: <1, 1-5, and > 5 years of age. Forty-nine patients (52.7%) developed mixed chimerism at a median of 34 days post-HCT (range, 10-1396 days). Mixed chimerism developed in 88.9 % (n=16/18) for <1 year, 57.1% (n=20/35) for 1-5 years, and 35% (n=14/40) for those >5 years. Patients <5 years of age were significantly more likely to develop mixed chimerism (X(2) (3, N = 93) = 14.8, p =0.001). We observed a significantly increased cumulative incidence of developing mixed chimerism if < 1 year of age (p=0.0002). Competing risk regression analysis demonstrated an increase in odds of development of mixed chimerism for age <1 (OR 3.72, p = 0.006, 95% CI 1.46-19.46) compared to age >5 years and decrease in odds of mixed chimerism in patients who developed acute GVHD prior to any intervention (OR 0.24, p=0.005, 95% CI 0.09-0.65) There was no significant association between mixed chimerism and graft source, graft type, CD34+ or CD3+ cell dose, HLA match or underlying disease (HLH vs non-HLH). Additionally, the need for secondary intervention was evaluated; 27 patients (29.0%) required one or more secondary intervention(s) (DLI, CD34 Boost, or second HCT). Patients <1 year of age with mixed chimerism were significantly more likely to require secondary intervention for mixed chimerism than patients > 5 years (p =0.004). CONCLUSION Our study demonstrates that young age <5 years, especially age <1 year is associated with an increased risk of developing mixed chimerism in patients undergoing RIC-HCT for non-SCID IEI using intermediate schedule alemtuzumab, fludarabine, and melphalan. Our data suggest tailoring regimen intensity based on age to reduce the incidence of mixed chimerism. Children <5 years, particularly those <1 year of age, require a higher intensity regimen. Possible strategies include adding thiotepa or using a busulfan-based reduced toxicity regimen.
-
4.
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.
-
5.
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.
-
6.
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.
-
7.
Is Mixed Chimerism Post-allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Acute Lymphoid Leukemia a Prognostic Factor for Relapse?
Khan, S., AlSaif, Z., Siddiqui, K., AlSaedi, H., Al-Ahmari, A., Al-Jefri, A., Ghemlas, I., AlAnazi, A., Ayas, M.
Hematology/oncology and stem cell therapy. 2023;17(1):72-78
Abstract
Hematopoietic stem cell transplantation (HSCT) has been considered curative for children with high-risk acute leukemia (ALL), offering better survival. Short tandem repeat has been used as a marker of chimerism status after HSCT. The appearance of recipient cells >1% post-allogeneic stem cell transplant is defined as mixed chimerism (MC). Chimeric studies post-HSCT are dynamic. This study aimed to investigate the significance of recipient cells in post-HSCT pediatric ALL patients as a predictor of relapse of their primary disease. The rate of MC was 51.4% (19 out of 37 recipients). It was 48.6% (n = 18) during Day+100 and 12.9% (4 out of 31 recipients) during post-Day+100 follow-up until two years. No significant association was noted between MC and all grade overall acute graft-versus-host disease. A mortality rate of 35.1% (n = 13) and a median follow-up of 56.9 months (95% CI: 39.7-74.2) were observed for all but four (16.7%) of the survivors in remission. Regarding causes of death, transplant-related mortality was recorded in only 2 of 13 expired patients (15.4%); both succumbed to sepsis. No significant association was found between MC and primary causes of death. The cumulative probability of five-year overall survival and event-free survival was not found to be statistically significantly different for MC (≤1.0% vs. > 1.0%). In conclusion, our data did not show MC testing alone as an effective prognostic marker for detecting relapse; molecular and flow cytometric analyses should be considered in children with ALL post-HSCT for monitoring relapse.
-
8.
Peripheral blood CD34 donor chimerism has greater clinical utility than CD3 for detecting relapse after allogeneic stem cell transplantation for AML or MDS
Das, T. P., North, D., Fleming, S. A., Tan, J. L. C., Ivey, A., Cummings, N. J., Spencer, A., Patil, S. S., Widjaja, J. M. L., Swain, M. I., et al
Transplantation and cellular therapy. 2023
Abstract
BACKGROUND Monitoring donor chimerism (DC) may detect early relapse following allogeneic hematopoietic stem cell transplant for acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Most centers use unfractionated peripheral blood or T-cells to monitor DC although CD34(+) DC may be more predictive. Limited adoption of CD34(+) DC may be due to the lack of detailed, comparative studies. OBJECTIVE To address this knowledge gap, we compared peripheral blood CD34(+) and CD3(+) DC in 134 patients transplanted for AML or MDS. STUDY DESIGN In July 2011, the Alfred Hospital Bone Marrow Transplantation Service adopted routine monitoring of DC in the lineage-specific CD34(+) and CD3(+) cell subsets from peripheral blood at 1, 2, 3, 4, 6, 9 and 12-months post-transplant for AML or MDS. Immunologic interventions including rapid immune suppression withdrawal, azacytidine and donor lymphocyte infusions were pre-specified for CD34(+) DC ≤80%. RESULTS Overall, CD34(+) DC ≤80% detected 32 of 40 relapses (positive predictive value 68% and negative predictive value 91%) compared with 13 of 40 relapses for CD3(+) DC ≤80% (positive predictive value 52% and negative predictive value 75%). Receiver operator analysis showed superiority of CD34(+) DC, with the most value at Day 120. CD3(+) DC provided additional value in only 3 cases, preceding CD34(+) DC ≤80% by 1 month. We further show that the CD34(+) DC sample can be used to detect NPM1(mut), with the combination of CD34(+) DC ≤80% and NPM1(mut) identifying the highest risk of relapse. For patients in morphologic remission at the time of CD34(+) DC ≤80% (n=24), 13 patients (54%) responded to immunologic interventions (rapid withdrawal of immunosuppression, azacitidine or donor lymphocyte infusions) with recovery of CD34(+) DC >80% and 11 of these patients remained in complete remission for a median of 34 months (range 28-97 months). In contrast, the other 9 patients did not respond to the clinical intervention and relapsed within a median of 59 days after detecting CD34(+) DC ≤80%. The CD34(+) DC of responders (median 72%) was significantly higher than non-responders (56%) (p=0.015, Mann-Whitney U test). Overall, monitoring CD34(+) DC was considered clinically useful (early diagnosis of relapse enabling pre-emptive therapy or predicting low risk of relapse) in 107 of 125 (86%) evaluable patients. CONCLUSIONS We show that peripheral blood CD34(+) DC is feasible and superior to CD3(+) DC for predicting relapse. It also provides a source of DNA for MRD testing, which may further stratify risk of relapse. If validated by an independent cohort, our results suggest that CD34(+) should be used in preference to CD3(+) DC for detecting early relapse and guiding immunologic interventions following transplant for AML or MDS.
-
9.
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.
-
10.
One fits all: a highly sensitive combined ddPCR/pyrosequencing system for the quantification of microchimerism after hematopoietic and solid organ transplantation
Häuser, F., Mittler, J., Hantal, M. S., Greulich, L., Hermanns, M., Shrestha, A., Kriege, O., Falter, T., Immel, U. D., Herold, S., et al
Clinical chemistry and laboratory medicine. 2023
Abstract
OBJECTIVES A combined digital droplet PCR (ddPCR)/pyrosequencing assay system was developed that demonstrated advantages applicable to multiple qualitative and quantitative molecular genetic diagnostic applications. Data for characterizing this combined approach for hematologic stem cell transplantation (HSCT) and allele quantification from graft-derived cell-free (cf) DNA in solid organ transplantation (SOT) is presented. METHODS ddPCR and pyrosequencing assays targeting 32 SNPs/markers were established. ddPCR results from 72 gDNAs of 55 patients after allogeneic HSCT and 107 plasma-cfDNAs of 25 liver transplant recipients were compared with established methods/markers, i.e. short-tandem-repeat PCR and ALT, respectively. RESULTS The ddPCR results were in good agreement with the established marker. The limit of detection was 0.02 % minor allele fraction. The relationship between ddPCR and STR-PCR was linear with R(2)=0.98 allowing to transfer previously established clinical STR-PCR cut-offs to ddPCR; 50-fold higher sensitivity and a variation coefficient of <2 % enable the use of low DNA concentrations (e.g. pre-sorted cells). ddPCR detected liver allograft injury at least as sensitive as ALT suggesting that ddPCR is a reliable method to monitor the transplant integrity, especially when other biomarkers are lacking (e.g. kidney). CONCLUSIONS Combining pyrosequencing for genotyping and ddPCR for minor allele quantification enhances sensitivity and precision for the patient after HSCT and SOT. The assay is designed for maximum flexibility. It is expected to be suitable for other applications (sample tracking, prenatal diagnostics, etc.).