Chimerism Analysis Research Articles

Cell-free DNA characteristics and chimerism analysis in patients after allogeneic cell transplantation

Cell-free DNA (cfDNA) isolated from plasma or serum has received increasing interest for diagnostic applications in pregnancy, solid tumors and solid organ transplantation. The reported clinical usefulness of cfDNA obtained from plasma or serum in patients undergoing allogeneic cell transplantation (alloHSCT) is scarce. ObjectiveTo analyze the potential clinical utility of cfDNA chimerism analysis after alloHSCT. Design and methodsA total of 196 samples obtained from 110 patients were investigated for their chimeric status both in peripheral blood and plasma using standard PCR for microsatellite amplification. Plasma DNA size distribution was analyzed using capillary electrophoresis. ResultsThe mean cfDNA concentration in the transplanted patients was 469ng/ml (range: 50–10,700ng/ml). The size range of almost 80% of the analyzed fragments was between 80 and 200bp. In 41 out of the 110 patients included in the study a mixture of donor and recipient plasma cfDNA was detected. There was a statistically significant difference in the percentage of plasma mixed chimerism between the patients without transplant related complications and the patients with either GvHD (p<0.05) or relapse (p<0.01). In those patients who showed improvement of GvHD also displayed a decrease in the observable percentage of recipient cfDNA during GvHD treatment. In patients without improvement or even with worsening of acute GvHD, stable or increasing levels of recipient cfDNA were detected. ConclusionscfDNA in combination with peripheral blood and bone marrow cell chimerism analysis might improve its utility in the clinic in particular in those patients with clinical complications after alloHSCT.

Étude du chimérisme après allogreffe de cellules hématopoïétiques : recommandations de la Société francophone de greffe de moelle et de thérapie cellulaire (SFGM-TC)

Chimerism analysis is an important step for the patient follow-up after hematopoietic stem cell transplantation. It is used to quantify the donor and the recipient part of a cell population issued from blood or bone marrow sample. In addition to hemogram, this technique is necessary to appreciate the quality of engraftment. The aim of this article is to propose some recommendation about methods, result analysis and therapeutic decision in hematopoietic stem cell transplantation for malignant or non-malignant diseases.

Development of lymphoma from the donor of haploidentical stem cell transplantation: A case report.

Post-transplantation lymphoproliferative disease (PTLD) is a serious complication following hematopoietic stem cell transplantation (HSCT). The majority of the cases develop during the first year after the transplantation and are associated with reactivation of the Epstein-Barr virus (EBV); the EBV-induced lymphoproliferation usually includes donor-derived B cells. We herein describe the case of a 28-year-old female patient who developed EBV-negative PTLD, namely diffuse large B-cell lymphoma (DLBCL), 6 months after receiving a haploidentical HSCT from her father. Chimerism analysis performed with XY fluorescence in situ hybridization revealed a B-cell PTLD originating from the donor. Unfortunately, the donor also developed DLBCL 380 days after donating progenitor cells, although he was hematologically normal at the time of donation. The present case demonstrated that disease transmitted from the donor may be a possible cause of PTLD.

P168 An algorithm to increase the informativeness of chimerism analysis in stem cell transplantation

Aim Chimerism analysis is an important tool for engraftment monitoring after hematopoietic stem cell transplantation (HSCT). It could have a highly predictive value if used appropriately. Therefore, we aim to develop a criteria for classification of patients according to their chimerism profile in order to detect earlier upcoming post-transplant complications. Methods In this retrospective study we have included 67 patients with hematologic malignancies. All patients were selected to be transplanted from HLA 10/10 compatible donor in order to decrease the role of HLA compatibility in post-HSCT period. The chimerism analysis was performed by a PCR-STR method. Donor cells ⩾99% were considered as a full donor chimerism (FDC). We used an intra-laboratory criteria to classify patients into four groups according to the dynamics of their donor chimerism (DC). The classification aimed to decrease the influence of the technical aspects of the STR platform, as well as its sensitivity, and increase its informativeness. Results Mortality (p = 0.004) and disease relapse (p Conclusions When individual drop-downs of the chimerism were taken into consideration, its monitoring seems to have a highly predictive value for disease relapse and mortality. Thus, regular monitoring of patients’ hematopoiesis with precise and sensitive methodology would greatly benefit the early detection of upcoming relapse or other life-threatening complication.

AB040. Preliminary study of chimerism detection in allogeneic hematopoietic stem cell transplantation using massively parallel sequencing

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is a common procedure for several hematologic disorders. The DNA chimerism detection of patient and donor of post-HSCT specimens is a crucial step for physician’s precision. Detection of patient and donor DNA chimerism has been relied on short tandem repeats (STRs) analysis using capillary electrophoresis (CE) platform as a gold standard. Recently, a massively parallel sequencing technology (MPS) has shown to improve sequencing performance with high throughput outcomes and with a large set of DNA markers. The objective of this study was to demonstrate the efficiency and performance of MPS towards chimerism analysis.

P162 Use of an automated chimerism calculator to increase speed, precision, and statistical analysis of results

Aim Chimerism testing is a valuable tool for determining the proportion of donor vs. recipient DNA in a sample. The commonly used polymerase chain reaction amplifications of short tandem repeats assay (Promega, Madison, WI) produces results for 21 loci, but only 9 loci are used in manual calculations. We sought to improve the tedious and time consuming process of manually calculating a patient’s percent chimerism by developing an automated chimerism calculator for rapid and accurate chimerism calculations based off of all 21 loci. Methods The calculator was developed using Microsoft Excel Macros and is able to report the percent donor chimerism +/− the margin of error. To determine whether precision is improved by using 21 loci, the results of a 21 loci analysis and a 9 loci analysis were compared in 111 patient samples. Additionally, each of the 21 loci was evaluated for its average deviation from the mean. Finally, 298 patient samples were analyzed by the program in order to determine if precision is equal at all chimerism percentages. Results When the same loci are analyzed, the chimerism calculator results are identical to the manual calculations, but completed in 10% of the time. The analysis of patient samples using 21 and 9 loci yielded, on average, chimerism percentages within 2.0% of each other. The average margin of error using 9 loci for analysis is 2.41% compared to 0.8% using all 21 loci. Among the 21 loci, the average deviation from the mean was found to range from 8.9% and 5.9%. Finally, we found that the average margin of error for patients between 90 and 100 percent donor chimerism is 0.19% while patients between 30 and 40 percent donor chimerism have 10.1% margin of error. Conclusions The chimerism calculator allows for routine use of all 21 STR loci with significantly reduced analysis time, 30% less margin of error, and an informative statistical analysis. In various laboratory settings, similar methods of computer assisted chimerism analysis can be easily implemented.

OR37 Persistence of T cells from the first donor after a second allogeneic stem cell transplantation

The monitoring of chimerism is a standard procedure to help assess engraftment and achievement of full donor lymphoid cells after allogeneic stem cell transplantation. T cell chimerism analysis is essential since T cells support the engraftment of hematopoietic stem cells and progenitor cells. TCRαβ T cells provide anti-tumor activity but can also cause graft vs. host disease (GVHD). In contrast, TCRγδ T cells have been found not to be associated with GVHD. Here we report a case in which residual T cells from the first donor were identified in a patient who received a second conditioning regiment and a subsequent second hematopoietic stem cell transplant. These cells from the first donor need to be closely monitored since they are TCRαβ T cells. A 10-year old male with high risk Acute Myeloid Leukemia received a 10/10 matched unrelated donor peripheral stem cell transplant (PSCT) with TCRαβ T cell depletion in May of 2015. He had been doing well until July 2016 when he relapsed. The patient underwent one dose of donor lymphocyte infusion in August 2016, with no improvement. He then received the second conditioning regimen and a KIR favorable maternal haplo-identical CD3/CD19 depleted PSCT on 11/9/16. The patient has done well overall post-transplant. He remains in remission with 100% donor engraftment without GVHD. Interestingly, his T cell engraftment analysis indicates he has T cell chimerism. 9% of his T cells are from the first unrelated donor and 91% are from the second donor. A subsequent analysis of TCRγδ T cell enriched engraftment showed that 100% of the TCRγδ T cells were from the second donor. This result suggests that the residual T cells identified from the first donor were most likely TCRαβ T cells. These results demonstrated that even after TCRαβ-depletion of the first graft and intensified immunosuppressive therapy conditioning regiment prior to the second transplant, CD3 + TCRαβ T cells from the first donor persisted. The patient is currently in remission. The expansion of the first donor T cells would strongly suggest a high risk of GVHD. Thus, it is clinically relevant to closely monitor these cells.

P072 Adaptation of NGS technology for engraftment monitoring in hematopoietic cell transplantation

Aim Increasingly, laboratories that support hematopoietic cell transplantation programs are implementing NGS technology for high resolution HLA genotyping. Extension of NGS to other laboratory services can provide efficiency in staffing and equipment usage. We have evaluated NGS of SNP and InDel polymorphisms for engraftment monitoring. Beyond workplace efficiencies, NGS of InDel polymorphism is not subject to stutter artifacts that can compromise results from the STR technology currently in wide use for chimerism analysis. Methods A panel of 49 polymorphisms on 20 chromosomes, comprising 37 SNPs on 18 chromosomes and 12 InDels on 10 chromosomes, was assembled. Initial amplification for the panel was reduced to 2 multiplexed PCRs with 5 ng of sample DNA per reaction. Total time for library preparation is 6 h, followed by a 7 h sequencing run on the MiSeq platform, with 1 h for chimerism analysis. Hands-on technologist time consists of just 3 of the 14 h time frame. Preliminary evaluation involved 4 sets of artificial mixtures of DNA from 2 unrelated individuals, with mixtures ranging from 10% to 0.1%, see Table. Results On average, 30 informative markers were identified between unrelated individuals. NGS achieved 0.5% sensitivity in all 4 sets of mixtures. The table shows the experimental values and standard deviation, SD, compared with the expected ratios among informative the polymorphisms. Informative InDel polymorphisms showed no false positive artifacts in unmixed specimens. Conclusions The results show that NGS technology provides informative, accurate and sensitive detection of a minority species in mixed specimens. With the 6 h library preparation and 7 h sequencing run, NGS offers rapid turnaround for result reporting. Additional testing is underway to assess the NGS technology in low cell count specimens and in mixtures of 3 or more unrelated individuals, intended to simulate retransplant and double cord donor transplant. Download : Download high-res image (233KB) Download : Download full-size image

P257 Automated isolation of lymphoid (CD3+, CD19+, CD56+) and myeloid (CD33/CD66B+, CD15+) subsets for use in lineage-specific chimerism analysis

Aim Lineage-specific chimerism analysis is used to monitor engraftment in different cell subsets following hematopoietic cell transplantation. Isolation of specific cell types can be time consuming, and so methods that automate the process are of great value to a busy chimerism laboratory. We have recently developed a method (EasySep™) to rapidly isolate different lymphoid (CD3+, CD19+, CD56+) and myeloid (CD33/CD66b+, CD15+) subsets from whole blood or buffy coat (BC). The aim of this study was to compare cell isolation performance for all 5 subsets between the automated RoboSep™-S and RoboSep™-16 instruments, which isolate 4 and 16 samples, respectively, and the manual silver “The Big Easy” EasySep™ magnet. Methods CD3+, CD19+, CD56+, myeloid and CD15+ cells were isolated from 0.9 mL BC samples from 5 different donors on each platform as per the manufacturer’s instructions. Following cell isolation, the percent purity by flow cytometry, the average number of cells isolated from the 0.9 mL BC sample, and the ug of DNA obtained from 2.5 × 10e5 isolated cells using QIAGEN spin columns were determined for each cell isolation platform (mean ± SD). Results Download high-res image (525KB) Download full-size image Conclusions No significant difference in cell purity, number of cells acquired, or ug of DNA obtained between the manual or automated isolation platforms was identified for any of the cell subsets analyzed (ANOVA). Thus use of the automated RoboSep™ platforms to isolate lymphoid and myeloid cell subsets will save time by increasing sample throughput up to 16-fold for busy chimerism testing laboratories.

T cell replete-haploidentical second hematopoietic stem cell transplantation for primary graft failure in pediatric patients with hematologic malignancies.

GF is one of the fatal complications of allogeneic HSCT. To rescue patients with primary GF, a second HSCT should be conducted as soon as possible, but the optimal donor source and technique have yet to be established. In this study, we retrospectively analyzed six children with hematologic malignancies who received TCR-haploidentical second HSCT for primary GF. The median interval between the prior HSCT and the second HSCT was 37.5days. All patients received fludarabine and ATG containing reduced-intensity re-conditioning before the second HSCT. All patients, except one who died early, achieved both neutrophil and Plt engraftment at a median time of 15 and 33days, respectively. Chimerism analysis showed that all engrafted patients achieved complete donor chimerism within 3weeks. Four patients developed acute GVHD, and three patients developed chronic GVHD. TRM occurred in two patients. Median follow-up of the four survivors was 6.8years, and all remained in sustained remission until the last follow-up. These results suggested that a TCR-haploidentical second HSCT for pediatric patients is feasible, and this approach may provide a potent option for children with primary GF.

Ivermectin activates GIRK channels in a PIP2 -dependent, Gβγ -independent manner and an amino acid residue at the slide helix governs the activation.

Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate-gated Cl- channel in parasites. It is known that IVM binds to the transmembrane domains (TMs) of several ligand-gated channels, such as Cys-loop receptors and P2X receptors. We found that the G-protein-gated inwardly rectifying K+ (GIRK) channel, especially GIRK2, is activated by IVM directly in a Gβγ -independent manner, but the activation is dependent on phosphatidylinositol-4,5-biphosphate (PIP2 ). We identified a critical amino acid residue of GIRK2 for activation by IVM, Ile82, located in the slide helix between the TM1 and the N-terminal cytoplasmic tail domain (CTD). The results demonstrate that the TM-CTD interface in GIRK channel, rather than the TMs, governs IVM-mediated activation and provide us with novel insights on the mode of action of IVM in ion channels. Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate-gated Cl- channel in parasites. It is also known that IVM binds to the transmembrane domains (TMs) of several ligand-gated channels, such as Cys-loop receptors and P2X receptors. In this study, we found that the G-protein-gated inwardly rectifying K+ (GIRK) channel is activated by IVM directly. Electrophysiological recordings in Xenopus oocytes revealed that IVM activates GIRK channel in a phosphatidylinositol-4,5-biphosphate (PIP2 )-dependent manner, and that the IVM-mediated GIRK activation is independent of Gβγ subunits. We found that IVM activates GIRK2 more efficiently than GIRK4. In cultured hippocampal neurons, we also observed that IVM activates native GIRK current. Chimeric and mutagenesis analyses identified an amino acid residue unique to GIRK2 among the GIRK family, Ile82, located in the slide helix between the TM1 and the N-terminal cytoplasmic tail domain (CTD), which is critical for the activation. The results demonstrate that the TM-CTD interface in GIRK channels, rather than the TMs, governs IVM-mediated activation. These findings provide us with novel insights on the mode of action of IVM in ion channels that could lead to identification of new pharmacophores which activate the GIRK channel.

Kinesin-1 promotes chondrocyte maintenance during skeletal morphogenesis.

During skeletal morphogenesis diverse mechanisms are used to support bone formation. This can be seen in the bones that require a cartilage template for their development. In mammals the cartilage template is removed, but in zebrafish the cartilage template persists and the bone mineralizes around the cartilage scaffold. Remodeling of unmineralized cartilage occurs via planar cell polarity (PCP) mediated cell rearrangements that contribute to lengthening of elements; however, the mechanisms that maintain the chondrocyte template that supports perichondral ossification remain unclear. We report double mutants disrupting two zebrafish kinesin-I genes (hereafter kif5Blof) that we generated using CRISPR/Cas9 mutagenesis. We show that zygotic Kif5Bs have a conserved function in maintaining muscle integrity, and are required for cartilage remodeling and maintenance during craniofacial morphogenesis by a PCP-distinct mechanism. Further, kif5Blof does not activate ER stress response genes, but instead disrupts lysosomal function, matrix secretion, and causes deregulated autophagic markers and eventual chondrocyte apoptosis. Ultrastructural and transplantation analysis reveal neighboring cells engulfing extruded kif5Blof chondrocytes. Initial cartilage specification is intact; however, during remodeling, kif5Blof chondrocytes die and the cartilage matrix devoid of hypertrophic chondrocytes remains and impedes normal ossification. Chimeric and mosaic analyses indicate that Kif5B functions cell-autonomously in secretion, nuclear position, cell elongation and maintenance of hypertrophic chondrocytes. Interestingly, large groups of wild-type cells can support elongation of neighboring mutant cells. Finally, mosaic expression of kif5Ba, but not kif5Aa in cartilage rescues the chondrocyte phenotype, further supporting a specific requirement for Kif5B. Cumulatively, we show essential Kif5B functions in promoting cartilage remodeling and chondrocyte maintenance during zebrafish craniofacial morphogenesis.

Characterization of High-Avidity Cytomegalovirus-Specific T Cells with Differential Tetramer Binding Coappearing after Allogeneic Stem Cell Transplantation.

CMV reactivation is a major complication after allogeneic stem cell transplantation (SCT). Immune reconstitution of CMV-specific CTLs (CMV-CTLs) is essential for virus control. During CMV-CTL monitoring using mutated HLA/CMV tetramers selectively detecting high-avidity T cells, we observed coappearance of CMV-CTLs with low (CMV tetlow CTLs) and high tetramer binding (CMV tethigh CTLs) in 53/115 CMV IgG+ patients stem cell transplanted from CMV IgG+ donors. However, the relevance of these coappearing differentially tetramer binding ("dual") CMV-CTLs was unclear. In this study, we investigated the kinetics, properties, and clinical impact of coappearing CMV tetlow and tethigh CTLs after allogeneic SCT. Patients with dual CMV-CTLs had more CMV tethigh than tetlow CTLs. Chimerism analysis of isolated CMV tetlow and tethigh CTLs revealed their exclusive donor origin. CMV tetlow and tethigh CTLs had an identical effector memory CD45RA-CCR7- and CD45RA+CCR7- T cell distribution, equal differentiation, senescence, and exhaustion marker expression and were negative for regulatory CD8+ T cell markers. Isolated CMV tetlow and tethigh CTLs were equally sensitive to CMV peptides in IFN-γ release and cytotoxicity assays. However, CMV tethigh CTLs proliferated more in response to low CMV peptide concentrations than tetlow CTLs. TCR repertoire analysis revealed that CMV tetlow and tethigh CTLs use different TCRs. Finally, dual CMV-CTLs were not associated with CMV antigenemia. In conclusion, these data show for the first time, to our knowledge, that both CMV tetlow and tethigh CTLs are functional effector T cells differing by proliferation, numbers in peripheral blood, and probably by their precursors without increasing the CMV reactivation risk after allogeneic SCT.

Abstract 4316: MCL-1 inhibition ameliorates the expansion of human leukemia in a dose dependent fashion

Abstract Myeloid cell leukemia-1 (MCL-1) is a prosurvival BCL-2 family member protein commonly overexpressed in cancer, including hematologic malignancies. The successful use of BH3 mimetics against other anti-apoptotic proteins has validated the potential of this line of therapy in hematologic malignancies. However, targeting BCL-2 family members, like MCL-1, with a small molecule inhibitor is challenging because these inhibitors mediate their effects through protein-protein interactions. Recently, we discovered potent (sub nM), selective small molecule MCL-1 inhibitors that exhibit cell-based activity and slow the growth of tumors in multiple mouse models. To determine tumor cell dependence on specific BCL2-family members, we employed BH3 profiling on a panel of myeloid tumor cells to reveal mitochondrial outer membrane permeablization (MOMP) in a cytochrome C release assay. Additionally, we determined growth inhibition of these cell lines in the presence of the potent MCL-1 inhibitor. These assays indicated MV-411 cells were dependent on MCL-1 and sensitive to MCL-1 inhibition with a GI50 &amp;lt;100nM. After cell line profiling, we began in vivo studies with the MCL-1 inhibitor in a systemic AML xenograft model. NSGS mice were sublethally irradiated and administered MV-411 cells intravenously. Engrafted mice received the MCL-1 inhibitor in 3 different doses (vehicle vs 10, 25 and 75mg/kg) daily via intraperitoneal injection. During treatment, the kinetics of MV-411 expansion was monitored via flow cytometry for the detection of human AML in the blood. At approximately 4 weeks after transplant, the vehicle mice became moribund, and all experimental groups were sacrificed for analysis of chimerism. Significant decreases in leukemic expansion were evident in the bone marrow (25 and 75mg/kg vs vehicle, P= .01, P &amp;lt;.001) and spleen (vehicle vs. 75mg/kg, P&amp;lt;.001) of treated mice in a dose-dependent fashion. MCL-1 blockade also eliminated splenomegaly in MCL-1 inhibitor treated mice. Successful use of the BCL-2 inhibitor, venetoclax, as a single agent and in combination with DNA methyltransferase inhibitors or low dose ara-C has proven the powerful role BH3 mimetics may play in AML therapy. However, mediation of resistance via alternative BCL-2 family antiapoptotic proteins remains a concern, and development of suitable inhibitors of MCL-1 and BCL-XL is important. These data show that we have developed a potent MCL-1 inhibitor, with in vivo efficacy in an AML mouse model. Further studies can determine the potential use of this drug to overcome resistance in the clinic. In addition, we will employ dynamic BH3 profiling of patient samples to determine the combination and sequencing of therapeutically applied BH3 mimetics. Citation Format: Haley E. Ramsey, Melissa A. Fischer, Taekyu Lee, Agnieszka E. Gorska, Pia Arrate, John Sensintaffer, Leah Hogdal, Edward Olejniczak, Stephen Fesik, Michael R. Savona. MCL-1 inhibition ameliorates the expansion of human leukemia in a dose dependent fashion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4316. doi:10.1158/1538-7445.AM2017-4316

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

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

Relevance of Chimerism Analysis After Allogeneic Stem Cell Transplantation

Hematopoietic stem cell transplantation is a potentially curative therapy for a range of malignant and non-malignant hematological diseases. Analysis of chimerism following allogeneic stem cell transplantation has been a routine method for the assessment of engraftment and early detection of graft failure. Lineage-specific chimerism monitoring is progressively used to specifically detect chimerism in one or more cell subsets, which may be undetected in assessment of the whole leukocyte population. The chimerism study in different leukocyte subpopulations increases sensitivity and specificity in the monitoring after transplantation, especially the analysis of T lymphocytes. All peripheral blood samples were separated into mononuclear cells and granulocytes by Ficoll density gradient centrifugation and T, B, and CD34+ was separated by immunomagnetic automatic cell separator. After DNA extraction, chimerism monitoring was performed using short tandem repeat by multiplex polymerase chain reaction followed by capillary electrophoresis. Quantification of chimerism was performed by determining the ratio of peak areas from donor and recipient informative short tandem repeat. Donor-recipient chimerism analysis in patients after allogeneic stem cell transplantation is a practical, feasible, and useful tool that predicts clinical outcomes and provides a guide for suitable therapeutic interventions.

Practical informativeness of short tandem repeat loci for chimerism analysis in hematopoietic stem cell transplantation

ObjectiveShort tandem repeat (STR) loci are most frequently used for chimerism analysis after hematopoietic stem cell transplantation (HSCT). The aim of this study was to evaluate the practical informativeness of STR chimerism by integrating theoretical and analytical points. MethodsTheoretical and practical informativess of 16 STR loci were evaluated from 1249 pairs of recipients and donors who were prepared for HSCT. ResultsTheoretical informativeness was influenced by genetic diversity including allele frequency and heterozygosity, and was higher in the unrelated HSCT group (90.5±5.3%) compared to the related HSCT group (66.2±4.4%). Practical informativeness was lower than theoretical (6.1±1.7%) because several STR loci were excluded due to stutter peaks and less reliable results, especially in type II-2 donor-recipient match pattern with no recipient-specific allele. We simulated an efficient STR combination for reliable chimerism analysis. Eight informative STR loci were required to analyze chimerism with at least one practically informative locus in the related HSCT group (D18S51, FGA, D2S1338, D13S317, D8S1179, D21S11, D16S539 and D7S820) while only three loci were needed in the unrelated group (D2S1338, FGA and D18S51). A minimum set of 2, 4 or 7 STR loci were required to provide at least 1, 3 or 5 practically informative loci in 95% of the unrelated HSCT group while 3, 8 or 12 loci were required in the related HSCT group. ConclusionWe deducted the practical informativeness of STR chimerism, identified the major influencing factors on the practical informativeness of each STR locus, and successfully simulated the efficient STR combination for reliable chimerism analysis.

Early mixed hematopoietic chimerism detection by digital droplet PCR in patients undergoing gender-mismatched hematopoietic stem cell transplantation.

Clinical decision making after allogeneic stem cell transplantation (HSCT) is partially based on hematopoietic chimerism analysis. Polymerase chain reaction amplification of polymorphic short tandem repeats (STR-PCR) is currently considered the gold standard for chimerism surveillance after transplantation. Nevertheless, this method has shown several limitations. Emerging technologies such as digital PCR (dPCR) has been applied to detect hematopoietic chimerism. Despite previous reports, the clinical usefulness of dPCR is unclear because the studies were performed in limited patient populations with short follow-ups. In order to compare hematopoietic chimerism detection time and rate, we analyzed 591 samples from 155 patients undergoing gender-mismatched HSCT using STR-PCR and dPCR. We also established the correlation between both methods in artificial DNA mixtures prepared in known proportions and in clinical samples. Depending on the artificial DNA mixture analyzed the correlation coefficient between both methods was 0.9946 and 0.9732. The limit of detection for dPCR was 0.01%. Of 157 samples with donor and recipient DNA, mixed chimerism (MC) was detected solely by dPCR in 66 samples. Within the group of patients relapsing after HSCT (n=32) MC was detected earlier in 15 of these patients with dPCR in comparison with STR-PCR. The mean time from MC detection to relapse was 155 days (range: 13-385 days) and 65 days (range: 0-203 days) for dPCR and STR-PCR, respectively. dPCR is a sensitive and accurate method for the quantification of hematopoietic chimerism allowing earlier MC detection compared to STR-PCR.

Donor chimerism and minimal residual disease monitoring in leukemia patients after allo-HSCT

In present research the comparative analysis of donor chimerism (DC) using different tests was performed to improve the diagnostic tool in patients with malignant hematological disorders after allo-HSCT. The RBC antigen typing, identification of ABO blood type and quantitative analysis of InDel-, STR-, Y-polymorphisms were carried out for detection of DC. In addition, the expression of well-known oncogenes and CD-markers for monitoring MRD was evaluated to predict relapse and clinical outcome. According to our research, the analysis of InDel polymorphism using AlleleSEQR-PCR is more sensitive test for estimation of DC as compared with other assays. Moreover, the sensitivity of AlleleSEQR-PCR may be increased after isolation of the CD34 cell population in bone marrow. Nevertheless, observation of high levels in DC (³95%) in some leukemia patients (ALL, Ph+, bcr-abl/p190+) during first 6 months after HSCT cannot exclude the possibility of relapse. Thus, the combined monitoring of both DC (InDel) and MRD (oncogenes, WT1 and CD-markers) is a more advisable and useful test in managing hematologic malignancies and predicting relapse risk after allo-HSCT.

Multiple modes of Lrp4 function in modulation of Wnt/β-catenin signaling during tooth development.

During development and homeostasis, precise control of Wnt/β-catenin signaling is in part achieved by secreted and membrane proteins that negatively control activity of the Wnt co-receptors Lrp5 and Lrp6. Lrp4 is related to Lrp5/6 and is implicated in modulation of Wnt/β-catenin signaling, presumably through its ability to bind to the Wise (Sostdc1)/sclerostin (Sost) family of Wnt antagonists. To gain insights into the molecular mechanisms of Lrp4 function in modulating Wnt signaling, we performed an array of genetic analyses in murine tooth development, where Lrp4 and Wise play important roles. We provide genetic evidence that Lrp4 mediates the Wnt inhibitory function of Wise and also modulates Wnt/β-catenin signaling independently of Wise. Chimeric receptor analyses raise the possibility that the Lrp4 extracellular domain interacts with Wnt ligands, as well as the Wnt antagonists. Diverse modes of Lrp4 function are supported by severe tooth phenotypes of mice carrying a human mutation known to abolish Lrp4 binding to Sost. Our data suggest a model whereby Lrp4 modulates Wnt/β-catenin signaling via interaction with Wnt ligands and antagonists in a context-dependent manner.