Assessment of chimerism by next generation sequencing: A comparison to STR/qPCR methods.

Chimerism analysis is used to evaluate patients after allogeneic hematopoietic stem cell transplant (allo-HSCT) for engraftment and minimal measurable residual disease (MRD) monitoring. A combination of short-tandem repeat (STR) and quantitative polymerase chain reaction (qPCR) was required to achieve both sensitivity and accuracy in the patients with various chimerism statuses. In this study, an insertion/deletion-based multiplex chimerism assay by next generation sequencing (NGS) was evaluated using 5 simulated unrelated donor-recipient combinations from 10 volunteers. Median number of informative markers detected was 8 (range = 5 - 11). The limit of quantitation (LoQ) was determined to be 0.1 % recipient. Assay sample number/batch was 10-20 and total assay time was 19-31 h (manual labor = 2.1 h). Additionally, 50 peripheral blood samples from 5 allo-HSCT recipients (related: N = 4; unrelated: N = 1) were tested by NGS and STR/qPCR. Median number of informative markers detected was 7 (range = 4 - 12). Results from both assays demonstrated a strong correlation (Y = 0.9875X + 0.333; R2 = 0.9852), no significant assay bias (difference mean - 0.08), and 100 % concordant detection of percent recipient increase ≥ 0.1 % (indicator of increased relapse risk). NGS-based chimerism assay can support all allo-HSCT for engraftment and MRD monitoring and simplify clinical laboratory workflow compared to STR/qPCR.

Assessment of chimerism and immunomodulation to prevent post-transplantation relapse in childhood acute myeloblastic leukemia: is it the right approach?

Relapse of acute myeloblastic leukemia (AML) after first allogenic hematopoietic stem-cell transplantation (allo-HSCT) is a fatal complication. Sixty-five children transplanted for AML were included in a prospective national study from June 2005 to July 2008 to explore the feasibility of preemptive immune modulation based on the monitoring of blood chimerism. Relapse occurred in 23 patients (35%). The median time between the last complete chimerism and relapse was 13.5 days (2-138). Prompt discontinuation of cyclosporin and the administration of donor lymphocyte infusions (DLIs) based on chimerism monitoring failed as a preemptive tool, either for detecting relapse or certifying long-term remission.

Multiplex STR panel for assessment of chimerism following hematopoietic stem cell transplantation (HSCT).

Short tandem repeat (STR) analysis is used in chimerism monitoring after allogeneic hematopoietic stem cell transplantation (HSCT) for patients with various hematologic malignancies. Commercial forensic STR kits often contain loci with huge differences in power of discrimination (PD) across populations, causing some loci to be less informative for chimerism analysis in certain populations. This study aimed to construct a new STR multiplex panel with highly informative loci for efficient chimerism analysis. Thirteen STR markers which exhibit high PD (> 0.9) in at least 80% of 50 populations globally were selected to form a new panel and used in STR analysis of 253 Malaysian subjects. Cumulative power of discrimination (CPD) and combined power of exclusion (CPE) were determined from 253 Malaysian individuals. Loci informativity was assessed and compared to the commercial AmpFLSTR Identifiler PCR Amplification kit in 14 donor-recipient pairs. The new panel had detected 202 unique alleles including five novel alleles from the 253 individuals with high CPD and CPE (> 0.99999999999999999 and > 0.999999997 respectively). All loci from the new panel in the donor-recipient pair analysis showed higher than 50% informativity, while five loci from the commercial kit demonstrated lower than 50% informativity. Four loci from the new panel ranked the highest informativity. A sequenced allelic ladder which consists of 202 unique alleles from the 253 subjects was also developed to ensure accurate allele designation. The new 13-loci STR panel, thus, could serve as an additional powerful, accurate, and highly informative panel for chimerism analysis for HSCT patients.

Chimerism as an important marker in post-transplant monitoring chimerism monitoring.

Cell chimerism determination is important for the monitoring of engraftment dynamics and for relapse prediction. Our cohort of 474 patients was divided into two groups according to the determination methods used over time, and by their chimerism status. A significant difference in survival was observed between mixed vs complete chimerism (P < 0.0001 vs P < 0.0002) in both patient groups, and also vs microchimerism (P = 0.0201) in the second group. Detection of mixed chimerism is thus a high-risk factor, and microchimerism is potentially a risk factor in the post-transplantation course. Methods with a high sensitivity for monitoring cell chimerism significantly improve the assessment of patients post-transplant, and they enable the identification of patients with high relapse risk. Supported by MH CZ-DRO (00023736, UHKT).

Ultra-Sensitive Droplet Digital PCR for the Assessment of Microchimerism in Cellular Therapies.

Current techniques to assess chimerism after hematopoietic stem cell transplantation (HSCT) are limited in both sensitivity and precision. These drawbacks are problematic in the context of cellular therapies that frequently result in microchimerism (donor chimerism <1%). We have developed a highly sensitive droplet digital PCR (ddPCR) assay using commercially available regents with good performance throughout the range of clinically relevant chimerism measurements, including microchimerism. We tested the assay using spiked samples of known donor-recipient ratios and in clinical samples from HSCT recipients and patients enrolled on clinical trials of microtransplantation and third-party virus-specific T cells (VSTs). The levels of detection and quantification of the assay were .008% and .023%, with high levels of precision with samples of DNA content ranging from 1 to 300 ng DNA. From the panel of 29 insertion-deletion probes multiple informative markers were found for each of 43 HSCT donor-recipient pairs. In the case of third-party cellular therapies in which there were 3 DNA contributors (recipient, HSCT donor, and T-cell donor), a marker to detect the cellular product in a background of recipient and donor cells was available for 11 of 12 cases (92%). Chimerism by ddPCR was able to quantify chimerism in HSCT recipients and comparison against standard STR analysis in 8 HSCT patients demonstrated similar results, with the advantage of fast turnaround time. Persistence of donor microchimerism in patients undergoing microtransplantation for acute myeloid leukemia was detectable for up to 57 days in peripheral blood and bone marrow. The presence of microtransplant product DNA in bone marrow T cells after cell sorting was seen in the 1 patient tested. In patients receiving third-party VSTs for treatment of refractory viral infections, VST donor DNA was detected at low levels in 7 of 9 cases. ddPCR offers advantages over currently available methods for assessment of chimerism in standard HSCT and cellular therapies.

Assessment of amiodarone-induced phospholipidosis in chimeric mice with a humanized liver.

It is important to consider susceptibility to drug-induced toxicity between animals and humans. Chimeric mice with a humanized liver are expected to predict hepatotoxicity in humans. Drug-induced phospholipidosis (DIPL), in which phospholipids accumulate, is a known entity. In this study, we examined whether chimeric mice can reveal species differences in DIPL. Changes in various phosphatidylcholine (PhC) molecules were investigated in the liver of chimeric mice after administering amiodarone, which induces phospholipidosis. Liquid chromatography-tandem mass spectrometry revealed that levels of PhCs tended to increase in the liver after administration of amiodarone. The liver of chimeric mice consists of human hepatocytes and residual mouse hepatocytes. We used imaging mass spectrometry (IMS) to evaluate the increase of PhCs in human and mouse hepatocytes after administration of amiodarone. IMS visualizes localization of endogenous and exogenous molecules in tissues. The IMS analysis suggested that the localized levels of several PhCs tended to be higher in the human hepatocytes than those in mouse hepatocytes, and PhC levels changed in response to amiodarone. Chimeric mice with a humanized liver will be useful to evaluate species differences in DIPL between mice and humans.

Risk assessment of relapse by lineage-specific monitoring of chimerism in children undergoing allogeneic stem cell transplantation for acute lymphoblastic leukemia.

UNLABELLED: Allogeneic hematopoietic stem cell transplantation is required as rescue therapy in about 20% of pediatric patients with acute lymphoblastic leukemia. However, the relapse rates are considerable, and relapse confers a poor outcome. Early assessment of the risk of relapse is therefore of paramount importance for the development of appropriate measures. We used the EuroChimerism approach to investigate the potential impact of lineage-specific chimerism testing for relapse-risk analysis in 162 pediatric patients with acute lymphoblastic leukemia after allogeneic stem cell transplantation in a multicenter study based on standardized transplantation protocols. Within a median observation time of 4.5 years, relapses have occurred in 41/162 patients at a median of 0.6 years after transplantation (range, 0.13-5.7 years). Prospective screening at defined consecutive time points revealed that reappearance of recipient-derived cells within the CD34(+) and CD8(+) cell subsets display the most significant association with the occurrence of relapses with hazard ratios of 5.2 (P=0.003) and 2.8 (P=0.008), respectively. The appearance of recipient cells after a period of pure donor chimerism in the CD34(+) and CD8(+) leukocyte subsets revealed dynamics indicative of a significantly elevated risk of relapse or imminent disease recurrence. Assessment of chimerism within these lineages can therefore provide complementary information for further diagnostic and, potentially, therapeutic purposes aiming at the prevention of overt relapse. This study was registered at clinical. TRIALS: gov with the number NC01423747.

Assessment of the use of immunosuppressants combined with cord blood for severe aplastic anemia.

BACKGROUND: The objective of this study was two-fold: 1) to investigate the changes of cytokines concentration in relation to severe aplastic anemia (SAA) when treated with immunosuppressants combined with cord blood (IS + CBI). and 2) to assess the curative effect of umbilical cord blood chimerism engraftment. METHODS: We selected 43 patients with SAA all treated with IS + CBI (newly diagnosed group). Among them, a total of 33 patients were treated effectively (effective group) while 10 cases were treated invalidly (invalid group). An additional 20 healthy individuals were selected as control (control group). The expression levels of IL-17, IL-22 and other cytokines in each group were detected by ELISA. The engraftment of cord blood stem cells was detected by using short tandem repeat-polymerase chain reaction (STR-PCR). RESULTS: 1. IL-17, IL-22 and other cytokines expressions in the newly diagnosed group were significantly higher than in the control group. 2. After six months, the levels in the effective group were significantly lower than pre-therapy levels (P < 0.05). The levels in the invalid group did not differ to those observed prior treatment. 3. After one and three months of treatment, a small amount of engraftment was found in the effective group. However, after six months, transplant rejection was observed in all patients. No effective engraftment was observed in the invalid group. CONCLUSION: 1) Th17 and Th22 producing cells in SAA patients significantly increased indicating a positive correlation between these biomarkers and the progression of SAA. 2) During the IS + CBI treatment the maintenance of a normal hematopoietic function depended on immunesup-pressants. Early umbilical cord blood chimerism engraftment may promote hematopoietic recovery.

Rapid single nucleotide polymorphism based method for hematopoietic chimerism analysis and monitoring using high-speed droplet allele-specific PCR and allele-specific quantitative PCR.

BACKGROUND: Chimerism analysis is important for the evaluation of engraftment and predicting relapse following hematopoietic stem cell transplantation (HSCT). We developed a chimerism analysis for single nucleotide polymorphisms (SNPs), including rapid screening of the discriminable donor/recipient alleles using droplet allele-specific PCR (droplet-AS-PCR) pre-HSCT and quantitation of recipient DNA using AS-quantitative PCR (AS-qPCR) following HSCT. METHODS: SNP genotyping of 20 donor/recipient pairs via droplet-AS-PCR and the evaluation of the informativity of 5 SNP markers for chimerism analysis were performed. Samples from six follow-up patients were analyzed to assess the chimerism via AS-qPCR. These results were compared with that determined by short tandem repeat PCR (STR-PCR). RESULTS: Droplet-AS-PCR could determine genotypes within 8min. The total informativity using all 5 loci was 95% (19/20). AS-qPCR provided the percentage of recipient DNA in all 6 follow-up patients without influence of the stutter peak or the amplification efficacy, which affected the STR-PCR results. CONCLUSION: The droplet-AS-PCR had an advantage over STR-PCR in terms of rapidity and simplicity for screening before HSCT. Furthermore, AS-qPCR had better accuracy than STR-PCR for quantification of recipient DNA following HSCT. The present chimerism assay compensates for the disadvantages of STR-PCR and is readily performable in clinical laboratories.

Monitoring of chimerism following allogeneic haematopoietic stem cell transplantation (HSCT): technical recommendations for the use of short tandem repeat (STR) based techniques, on behalf of the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping Chimerism Working Group.

Analysis of short tandem repeats (STR) is the predominant method for post-transplant monitoring of donor engraftment. It can enable early detection of disease relapse, level of engraftment and provide useful information on the graft-versus-host disease (GVHD)/graft-versus-tumour (GVT) effect, facilitating therapeutic intervention. Harmonization and standardization of techniques and result interpretation is essential to reduce the impact of laboratory variability on both clinical management and the results of multi-centre clinical trials. However, the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI) has highlighted significant issues inherent in STR testing that impact upon inter- and intra- laboratory variation. We present here consensus best practice guidelines and recommendations for STR chimerism testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 11 UK and Eire clinical laboratories. This document uses data obtained from the UK NEQAS LI Post-Stem Cell Transplant (SCT) Chimerism Monitoring Programme.

Tolerance--is it worth it?

We are entering an exciting time in the study of immunologic tolerance. Several cellular and molecular strategies have been developed that show promise in nonhuman transplant models and these approaches are just now appearing in clinical trials. Tolerance strategies that prevent immune rejection and obviate the need for immunosuppressive medications (with inherent risk of cancer, infection, and organ toxicity) would improve both graft and patient survival. Each tolerance protocol brings its own set of associated risks. As the results of these trials become available, we must continue to evaluate their successes and failures. The balance of these outcomes will help us answer the question: "Tolerance-Is it worth it?"

Assessment of the purity of isolated cell populations for lineage-specific chimerism monitoring post haematopoietic stem cell transplantation.

Following haematopoietic stem cell transplantation, monitoring the proportion of donor and recipient haematopoiesis in the patient (chimerism) is an influential tool in directing further treatment choices. Short tandem repeat (STR) analysis is a method of chimerism monitoring using DNA isolated from peripheral blood, bone marrow or specific isolated cell lineages such as CD3+ T cells. For lineage-specific STR analysis on cell populations isolated from peripheral blood, a qualitative estimation of the purity of each isolated population is essential for the correct interpretation of the test data. We describe a rapid, inexpensive method for the determination of purity using a simple flow cytometry method. The method described for assessing the purity of sorted CD3+ cells can be applied to any cell population isolated using the same technology. Data obtained were comparable to results from a commercial polymerase chain reaction (PCR)-based method for the assessment of purity (Non-T Genomic Detection Kit, Accumol, Calgary, AB, Canada) (P = 0.59). Of the 303 samples tested by flow cytometry, 290 (95.7%) exceeded 90% purity, and 215 (70.95%) were over 99% pure. There were some outlying samples, showing diversity between samples and the unpredictability of purity of isolated cell populations. This flow cytometry method can be easily assimilated into routine testing protocols, allowing purity assessment in multiple-sorted cell populations for lineage-specific chimerism monitoring using a single secondary antibody and giving results comparable to a PCR-based method. As purity of isolated cell lineages is affected by time after venepuncture and storage temperature, assessment of each sample is recommended to give a reliable indication of sample quality and confidence in the interpretation of the results.

Quantitative assessment of hematopoietic chimerism by quantitative real-time polymerase chain reaction of sequence polymorphism systems after hematopoietic stem cell transplantation.

BACKGROUND: Analysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by donor lymphocyte infusions. We developed a sensitive, reliable and rapid real-time PCR method based on sequence polymorphism systems to quantitatively assess the hematopoietic chimerism after HSCT. METHODS: A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time PCR in 101 HSCT patients with leukemia and other hematological diseases. The chimerism kinetics of bone marrow samples of 8 HSCT patients in remission and relapse situations were followed longitudinally. RESULTS: Recipient genotype discrimination was possible in 97.0% (98 of 101) with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Using serial dilutions of plasmids containing specific SP markers, the linear correlation (r) of 0.99, the slope between -3.2 and -3.7 and the sensitivity of 0.1% were proved reproducible. By this method, it was possible to very accurately detect autologous signals in the range from 0.1% to 30%. The accuracy of the method in the very important range of autologous signals below 5% was extraordinarily high (standard deviation <1.85%), which might significantly improve detection accuracy of changes in autologous signals early in the post-transplantation course of follow-up. The main advantage of the real-time PCR method over short tandem repeat PCR chimerism assays is the absence of PCR competition and plateau biases, with demonstrated greater sensitivity and linearity. Finally, we prospectively analyzed bone marrow samples of 8 patients who received allografts and presented the chimerism kinetics of remission and relapse situations that illustrated the sensitivity level and the promising clinical application of this method. CONCLUSION: This SP-based real-time PCR assay provides a rapid, sensitive, and accurate quantitative assessment of mixed chimerism that can be useful in predicting graft rejection and early relapse.

Assessment of chimeric mice with humanized liver as a tool for predicting circulating human metabolites.

The ability to predict circulating human metabolites of a candidate drug before first-in-man studies are carried out would provide a clear advantage in drug development. A recent report demonstrated that while in vitro studies using human liver preparations reliably predict primary human metabolites in plasma, the predictability of secondary metabolites, formed by multiple reactions, was low, with total success rates of < or =65%. Here, we assess the use of chimeric mice with humanized liver as an animal model for the prediction of human metabolism in vivo. Metabolism studies with debrisoquine and (S)-warfarin demonstrated significantly higher concentrations of their primary human abundant metabolites in serum or plasma in chimeric mice than in control mice. Humanized chimeric mice were also capable of producing human-specific metabolites of several in-house compounds which were generated through more than one metabolism reaction. This model is closer to in vivo human physiology and therefore appears to have an advantage over in vitro systems in predicting complex metabolites in human plasma. However, prediction of human metabolites failed for other compounds which were highly metabolized in mice. Although requiring careful consideration of compound suitability, this model represents a potential tool for predicting human metabolites in combination with conventional in vitro systems.

[A novel single nucleotide polymorphism-based method for quantitative assessment of chimerism after allogeneic stem cell transplantation.].

OBJECTIVE: To develop a novel single nucleotide polymorphism (SNP)-PCR based method for quantitative detection of chimerism after allogeneic haemopoietic stem cell transplantation (allo-HSCT), and to explore its feasibility, accuracy and superiority. METHODS: 18 SNP loci were sereened to identify informative markers for detecting chimerism in each donor/recipient pair before transplantation. Then the chimerism rate of each informative marker was analyzed by real-time quantitative PCR (RQ-PCR). The accuracy and sensitivity were verified by multiple proportion dilution and analogy chimerism compared with quantitative detection of short tandem repeat (STR)-PCR, fluorescence in situ hybridization (FISH) and fusion gene. RESULTS: (1) The average slope of the 17 time amplications of the internal control plasmid was -3.39, the average intercept was 39.97, correlation coefficients were more than 0.995, which was close to the theoretical level. The intra- and interassay variability was 0.50% and 1.1%, respectively, which were both in the allowed ranges. A linear correlation with artificial mixed chimerism is above 0.99 and a sensitivity of 0.01% proved reproducible. (2) At least one informative marker could be found in over 95% of 40 donor/recipient pairs. The results of the chimerisms derived from SNP-PCR were consistent with that from STR-PCR (96.7%), FISH and fusion gene analasis (P > 0.05); the quantitative results of special fusion gene transcripts were negtive in complete chimerism samples, and positive in mixed chimerism samples. CONCLUSIONS: This new assay which overcome the PCR competition and plateau biases of STR-PCR provides an accurate, reliable and rapid quantitative assessment of mixed chimerism after allo-transplantation. It is highly promising for of clinical application and may take the place of STR-PCR in the conventional chimerisim assessment.

The potential for tolerance in pediatric renal transplantation.

PURPOSE OF REVIEW: Immune tolerance has remained the Holy Grail in transplantation since the first kidney transplantation was performed over 50 years ago. Children present unique challenges in renal transplantation because of increased infectious risks and adolescent nonadherence to the intensive medication regimen necessary for allograft function. The current review presents recent studies and approaches toward achieving tolerance in renal transplantation and the prospects for pediatric patients. RECENT FINDINGS: A number of approaches have been used toward minimization of immunosuppression in pediatric patients, but none has yet achieved true operational tolerance with maintenance of allograft function of all immunosuppression. Recent work in adult renal transplant recipients using combined kidney-bone marrow transplantation by the induction of transient mixed chimerism has allowed for full withdrawal of immunosuppression with stable long-term renal function. SUMMARY: On the basis of recent adult studies, the prospect of immune tolerance in pediatric renal transplantation remains hopeful and may be ready for extension to the adolescent population.

Quantitative assessment of mixed chimerism in allogeneic stem cell transplant patients: a comparison of molecular genetic and cytogenetic approaches.

After allogeneic stem cell transplantation, dual donor and recipient populations may be present. Donor/recipient ratio changes over time may predict clinical outcome: accurate measurement of these changes are needed. Chimerism may be measured by XY-fluorescence in situ hybridization for donor/recipient sex mismatch, or polymerase chain reaction amplification of short tandem repeat loci with donor/recipient sex match. Patients were monitored by each method. Additionally, mononuclear cells from 2 sex-mismatched individuals were mixed and analyzed using both methods. Each gave concordant estimates of patient chimerism and discriminated cell population ratios in mixed blood. We conclude that cytogenetic and molecular methods give accurate donor chimerism estimates.

Whole-comparative genomic hybridization and "cell code" estimation: an application for assessment of cellular chimerism.

Whole-comparative genomic hybridization (W-CGH) allows one to identify copy number differences in highly repeated DNAs between two genomes. It allowed the identification of nuclear markers that can be used to distinguish cell populations from different individuals in a chimeric situation. We discuss the reliability of W-CGH accomplished with fluorescent in situ hybridization (FISH) and digital image analysis (DIA) to analyze the degree of chimerism in patients after allogeneic hematopoietic stem cell transplantation (HSCT).