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Chimerism analysis after allogeneic hematopoietic stem cell transplantation serves to confirm engraftment, indicate relapse of hematologic malignancy, and attribute graft failure to either immune rejection or poor graft function. Short tandem repeat PCR (STR-PCR) is the prevailing method, followed by quantitative real-time PCR (qPCR), with detection limits of 1–5% and 0.1%, respectively. Chimerism assays using digital PCR or next-generation sequencing, both of which are more sensitive than STR-PCR, are increasingly used. Stable mixed chimerism is usually not associated with poor outcomes in non-malignant diseases, but recipient chimerism may foretell relapse of hematologic malignancies, so higher detection sensitivity may be beneficial in such cases. Thus, the need for and the type of intervention, e.g., immunosuppression regimen, donor lymphocyte infusion, and/or salvage second transplantation, should be guided by donor chimerism in the context of the feature and/or residual malignant cells of the disease to be treated. [ABSTRACT FROM AUTHOR]

Abstract A literature review does not provide information about the safety of autologous hematopoietic stem cell transplantation (HSCT) in a recipient who has previously received allogeneic HSCT. We treated a 69‐year‐old woman with diffuse large B cell lymphoma. The patient received autologous stem cell transplantation (ASCT) in the second complete remission of malignant lymphoma. The patient had undergone allogeneic hematopoietic SCT (allo‐HSCT) for chronic myeloid leukemia 20 years ago. Chronic myeloid leukemia had been in complete remission for the previous 20 years. Thus, the patient received autologous and allogenic HSCT 20 years apart. ASCT involves the patient receiving “self” hematopoietic cells from an allogeneic donor. In other words, this is immunologically the second allo‐HSCT. The allo‐HSCT 20 years ago was undergone by a related healthy brother, a human leukocyte antigen (HLA) 8/8 full matched donor. The conditioning regimen was reduced‐intensity consisting of fludarabine and busulfan. The patient did not experience acute or chronic graft‐versus‐host disease (GVHD) after allo‐HSCT. The second transplantation, ASCT was performed to the MEAM conditioning regimen. Engraftment was uneventful, and complete donor chimerism had been achieved even after ASCT. She suffered from an acute gastric mucosal lesion 52 days after ASCT. Pathological finding of gastric mucosa was nonspecific acute gastritis with significant neutrophil infiltration. Sex chromosome analysis of gastric mucosa demonstrated that mucosal cells had XX signals, whereas infiltrating neutrophils had XY signals. We speculated the patient onset of an acute gastric GVHD in this recipient after the second transplantation. This case remarked infiltration of neutrophils triggered GVHD reaction by resetting allogeneic immune reaction after the second transplantation. We describe a rare occurrence of GVHD reaction in a recipient of ASCT following allo‐HSCT.

Chimerism analysis after allogeneic hematopoietic stem cell transplantation serves to confirm engraftment, indicate relapse of hematologic malignancy, and attribute graft failure to either immune rejection or poor graft function. Short tandem repeat PCR (STR-PCR) is the prevailing method, followed by quantitative real-time PCR (qPCR), with detection limits of 1–5% and 0.1%, respectively. Chimerism assays using digital PCR or next-generation sequencing, both of which are more sensitive than STR-PCR, are increasingly used. Stable mixed chimerism is usually not associated with poor outcomes in non-malignant diseases, but recipient chimerism may foretell relapse of hematologic malignancies, so higher detection sensitivity may be beneficial in such cases. Thus, the need for and the type of intervention, e.g., immunosuppression regimen, donor lymphocyte infusion, and/or salvage second transplantation, should be guided by donor chimerism in the context of the feature and/or residual malignant cells of the disease to be treated.

Abstract Relapse is a major cause of treatment failure after hematopoietic stem cell transplantation (HSCT) for acute leukemia. Here, we report a monocentric retrospective study of all HSCTs for B cell acute lymphoblastic leukemia (ALL) performed during the years 2005–2021 (n = 138, including 51 children), aiming to identify the optimal use of lineage‐specific recipient‐donor chimerism analysis for prediction of relapse. In adults, relapse was associated with increased recipient chimerism in CD3+ bone marrow cells sampled at least 30 days before a relapse. Relapse could be predicted with a sensitivity of 73% and a specificity of 83%. Results were similar for children but with a higher recipient chimerism cutoff. Additionally, adults that had at least one chimerism value

Secondary malignancy of donor origin in the form of acute lymphoblastic leukemia (ALL), post-allogeneic hematopoietic stem cell transplant (HSCT) for beta thalassemia (BT) major, is exceedingly rare. A10 year old male child, the first and only product of non-consanguineous conception, was diagnosed with BT major at the age of 9 months when he had diarrhea and his parents (both of whom had thalassemia minor) noticed yellowing of the skin. Until the age of 10 years, he received regular blood transfusion and iron chelation, when the requirements got escalated and he consequently had to undergo myeloablative haploidentical HSCT from his mother. The post-transplant period was uneventful, and follow up with short tandem repeat chimerism analysis revealed complete donor chimerism on all occasions. Five years after the transplant, he developed fever with pancytopenia. Peripheral smear (PS) and bone marrow revealed blasts that were immunophenotypically precursor B-ALL. Cytogenetics revealed twenty diploid female metaphases with modal karyotype 46, XX[20], and again, complete donor chimerism was noted. Thus, a diagnosis of donor cell leukemia (DCL) was considered. Induction chemotherapy was initiated; however, the patient succumbed to systemic sepsis midway through induction therapy. No evidence of leukemia was noted in the patient's mother, who was followed up with PSs for 5 years. DCL has a poor prognosis. Greater understanding of the disease biology could allow for appropriate donor screening, notification and shielding the recipient from DCL and its grave consequences. [ABSTRACT FROM AUTHOR]

Currently there are no widely accepted guidelines for chimerism analysis testing in hematopoietic cell transplantation (HCT) patients. The objective of this review is to provide a practical guide to address key aspects of performing and utilizing chimerism testing results. In developing this guide, we conducted a survey of testing practices among laboratories that are accredited for performing engraftment monitoring/chimerism analysis by either the American Society for Histocompatibility & Immunogenetics (ASHI) and/or the European Federation of Immunogenetics (EFI). We interpreted the survey results in the light of pertinent literature as well as the experience in the laboratories of the authors. In recent years there has been significant advances in high throughput molecular methods such as next generation sequencing (NGS) as well as growing access to these technologies in histocompatibility and immunogenetics laboratories. These methods have the potential to improve the performance of chimerism testing in terms of sensitivity, availability of informative genetic markers that distinguish donors from recipients as well as cost. The results of the survey revealed a great deal of heterogeneity in chimerism testing practices among participating laboratories. The most consistent response indicated monitoring of engraftment within the first 30 days. These responses are reflective of published literature. Additional clinical indications included early detection of impending relapse as well as identification of cases of HLA-loss relapse. [ABSTRACT FROM AUTHOR]

A literature review does not provide information about the safety of autologous hematopoietic stem cell transplantation (HSCT) in a recipient who has previously received allogeneic HSCT. We treated a 69-year-old woman with diffuse large B cell lymphoma. The patient received autologous stem cell transplantation (ASCT) in the second complete remission of malignant lymphoma. The patient had undergone allogeneic hematopoietic SCT (allo-HSCT) for chronic myeloid leukemia 20 years ago. Chronic myeloid leukemia had been in complete remission for the previous 20 years. Thus, the patient received autologous and allogenic HSCT 20 years apart. ASCT involves the patient receiving "self" hematopoietic cells from an allogeneic donor. In other words, this is immunologically the second allo-HSCT. The allo-HSCT 20 years ago was undergone by a related healthy brother, a human leukocyte antigen (HLA) 8/8 full matched donor. The conditioning regimen was reduced-intensity consisting of fludarabine and busulfan. The patient did not experience acute or chronic graft-versus-host disease (GVHD) after allo-HSCT. The second transplantation, ASCT was performed to the MEAM conditioning regimen. Engraftment was uneventful, and complete donor chimerism had been achieved even after ASCT. She suffered from an acute gastric mucosal lesion 52 days after ASCT. Pathological finding of gastric mucosa was nonspecific acute gastritis with significant neutrophil infiltration. Sex chromosome analysis of gastric mucosa demonstrated that mucosal cells had XX signals, whereas infiltrating neutrophils had XY signals. We speculated the patient onset of an acute gastric GVHD in this recipient after the second transplantation. This case remarked infiltration of neutrophils triggered GVHD reaction by resetting allogeneic immune reaction after the second transplantation. We describe a rare occurrence of GVHD reaction in a recipient of ASCT following allo-HSCT., Competing Interests: The authors declare no conflict of interest., (© 2024 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

The monitoring of chimerism status in a hematopoietic stem cell transplantation patient is a crucial process and is performed periodically in a short time interval. A short tandem repeat marker is widely used for chimerism analysis due to its high discrimination power. However, the sensitivity of this approach was limited to 5% of a minor contributor and the interpretation is usually interrupted with PCR stochastic phenomena. Here, we developed an SNP panel for chimerism analysis using TaqMan technology. A set of SNPs was selected from Thai ancestry informative markers and open-access databases with proper criteria. We examined the 30 recipient-donor pairs that underwent HSCT and showed that the panel can provide an informative marker from 90% of all pairs. An early detection of artificial chimerism in post-HSCT samples was observed when compared with STR analysis. In addition, the detail of cases was discussed. [ABSTRACT FROM AUTHOR]

Background Donor factors have a variable correlation with cluster of differentiation (CD)34+ cell dose in allogeneic peripheral blood stem cell (PBSC) harvests. CD34+ cell dose affects the speed of hematopoietic recovery and percentage of donor chimerism in the recipient. Methods A total of 25 allogeneic PBSC transplants performed during a 3-year period were included. All donors underwent mobilization with filgrastim. Leukapheresis, flowcytometric CD34+ cell enumeration, and chimerism analysis were performed and correlated with recipient outcome. Results Besides age, all other donor parameters had a positive correlation with CD34+ cell count. Engraftment kinetics and chimerism had a positive correlation with the CD34+ yield of the PBSC product. Acute graft-vs-host disease (GVHD) was observed in patients with complete chimerism at day 30 after transplantation. Conclusion Adequate CD34+ cell yield happens in healthy donors, independent of donor demographic patterns with G-CSF only. A diverse population of donors can thus be approached for Matched Unrelated Donor (MUD) transplants. An accurate quantitative analysis of early donor chimerism in the recipient (at day 30) is an excellent tool for post-transplant monitoring for acute GvHD. [ABSTRACT FROM AUTHOR]

Chimerism analysis is a well-established method for monitoring the state of hematopoietic stem cell transplantation (HSCT) over time by analyzing peripheral blood or bone marrow samples of the recipient in several malignant and non-malignant hematologic diseases. From a clinical point of view, a continuous monitoring is fundamental for an effective early therapeutic intervention. This paper provides a comparative overview of the main molecular biology techniques which can be used to study chimerism after bone marrow transplantation, focusing on their advantages and disadvantages. According to the examined literature, short tandem repeats (STR) analysis through simple PCR coupled with capillary electrophoresis (STR-PCR) is the most powerful method which guarantees a high power of differentiation between different individuals. However, other methods such as real-time quantitative PCR (qPCR), digital PCR (dPCR), and next-generation sequencing (NGS) technology were developed to overcome the technical limits of STR-PCR. In particular, these other techniques guarantee a higher sensitivity, which allows for the detection of chimerism at an earlier stage, hence expanding the window for therapeutic intervention. After a comparative evaluation of the various techniques, it seems clear that STR-PCR still remains the gold standard option for chimerism study, even if it is likely that both dPCR and NGS could supplement or even replace the common methods of STR analysis.

Background: Recently molecular chimerism analysis after allogeneic hematopoietic stem cell transplantation (AHSCT) has become more important. The use of quantitative chimerism methods aims to assess the kinetics of engraftment to determine graft rejection and failure or relapse of the underlying disease after AHSCT. An accurate and sensitive determination of chimerism status is mandatory after AHSCT. This study aimed to compare two chimerism methods: Multiplex Short Tandem Repeat-Polymerase Chain Reaction (STR-PCR) and quantitative Real Time-PCR (qRT-PCR). Methods: Thirty-nine blood samples at +28 day were used to extract DNA. Most patients had been diagnosed with acute leukemia (74.3%) and other hematological diseases. For Multiplex STR-PCR method, PCR products were separated on an ABI 3130 Genetic Analyzer (Applied Biosystem, USA) and for qRT-PCR, an ABI 7500 (Applied Biosystem, USA) Plate System Real Time Analyzer was used to determine the quantification of chimerism percentage. Results: Of the 39 analyzed samples, 82% concordant chimerism results were detected for both STR-PCR and qRT-PCR methods. Ten mixed chimerisms (MC) were found by qRT-PCR whereas of only 3 MC cases were detected by STR-PCR. In the discordant group of 7 by qRT-PCR, we observed Acute Graft versus Host Disease (aGVHD). Three MC cases that were detected by both STR- and qRT-PCR methods died because of relapse. Conclusions: The quantitative chimerism method along with multiplex STR-PCR method is important for early detection of MC. qRT-PCR methods can be valuable options in the prevention of graft failure and assisting with fast and early treatment strategies for patients undergoing AHSCT. [ABSTRACT FROM AUTHOR]

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. Objective To analyze the potential clinical utility of cfDNA chimerism analysis after alloHSCT. Design and methods A 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. Results The mean cfDNA concentration in the transplanted patients was 469 ng/ml (range: 50–10,700 ng/ml). The size range of almost 80% of the analyzed fragments was between 80 and 200 bp. 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. Conclusions cfDNA 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. [ABSTRACT FROM AUTHOR]

The impact of persistent mixed chimerism ( MC) after haematopoietic stem cell transplantation ( HSCT) remains unclarified. We investigated the incidence of MC in peripheral blood beyond day +50 after HSCT and its impact on rejection, chronic graft-versus-host disease (c-GvHD) and relapse in 161 children receiving allogeneic HSCT for haematological malignancies. The 1-year incidence of late MC was 26%. Spontaneous conversion to complete donor chimerism ( CC) occurred in 43% of patients as compared to 62% after donor lymphocyte infusions. No graft rejection occurred. The 1-year incidence of c-GvHD was 20 ± 7% for MC, and 18 ± 4% for CC patients ( P = 0·734). The 3-year cumulative incidence of relapse ( CIR) according to chimerism status at days +50 and +100 was 22 ± 4% for CC patients vs. 22 ± 8% for MC patients (day +50; P = 0·935) and 21 ± 4% vs. 20 ± 7% (day +100; P = 0·907). Three-year CIRs in patients with persistent MC and patients with CC/limited MC were comparable (8 ± 7% vs. 19 ± 4%; P = 0·960). HSCT for acute leukaemia or myelodysplastic syndrome as secondary malignancies (hazard ratio ( HR) 4·7; P = 0·008), for AML ( HR 3·0; P = 0·02) and from mismatched donors ( HR 3·1; P = 0·03) were independent factors associated with relapse. Our data suggest that late MC neither protects from c-Gv HD nor does it reliably predict impending disease relapse. [ABSTRACT FROM AUTHOR]

Chimerism and clinical outcome data from 244 hematopoietic stem cell transplants in 218 children were retrospectively analyzed to assess their relevance for the detection of graft rejection and malignant relapse. Patients transplanted for a non-malignant disease had significantly higher proportions of residual recipient T cells in peripheral blood at one, three, and six months compared with patients transplanted for malignant disease. Recipient T-cell levels were below 50% at one month after transplantation in most patients (129 of 152 transplants). Graft rejection occurred more frequently in the group of patients with high levels of recipient cells at one month (10 graft rejections in the 23 patients with recipient T cells >50% at one month as compared to seven graft rejections occurred in 129 patients with recipient T cells <50% (p < 0.001). Multilineage chimerism data in 87 children with leukemia at one, three, and six months after transplantation were not correlated with subsequent relapse of malignant disease. In conclusion, early analysis of lineage-specific chimerism in peripheral blood can be used to identify patients who are at high risk of graft rejection. However, the efficacy of early chimerism analysis for predicting leukemia relapse was limited. [ABSTRACT FROM AUTHOR]

Little is known regarding the chimerism status after reduced-intensity conditioning transplantation when bone marrow is used as a stem cell source. We prospectively analyzed lineage-specific chimerism and retrospectively evaluated clinical outcomes in 80 adult patients who underwent unrelated donor bone marrow transplantation (URBMT) with fludarabine plus melphalan (FM) as the conditioning regimen. Mixed donor chimerism (MDC) was seen in 43 and 10 % of patients at days 14 and 28, respectively. Melphalan at ≤130 mg/m was associated with an increased incidence of MDC at day 28 ( P = 0.03). Patients with MDC at day 14 showed a marginally increased risk of primary graft failure and a marginally decreased risk of graft-versus-host disease. In multivariate analysis, MDC at day 14 was associated with higher overall mortality (hazard ratio (HR) = 2.1; 95 % confidence interval (CI), 1.1-4.2; P = 0.04) and relapse rate (HR = 3.0; 95 % CI, 1.2-7.5; P = 0.02), but not with non-relapse mortality (HR = 1.8; 95 % CI, 0.70-4.6; P = 0.23). Thus, the FM regimen yields prompt complete donor chimerism after URBMT, but the melphalan dose significantly impacts the kinetics of chimerism. Chimerism status evaluation at day 14 may be instrumental in predicting relapse after URBMT with the FM regimen. [ABSTRACT FROM AUTHOR]

Background Cytomegalovirus ( CMV)-specific CD8+ cytotoxic T lymphocytes ( CMV- CTLs) play a crucial role in preventing CMV disease. However, the actual in vivo dynamics of CMV- CTL clones after allogeneic hematopoietic stem cell transplantation (allo HCT) are still unclear. Methods Using a single-cell T-cell receptor repertoire analysis, we monitored clones and chimerism of CMV- CTLs in 3 CMV-seropositive allo HCT recipients from CMV-seronegative donors, with or without CMV reactivation. Results Nearly all of the CMV- CTLs during follow-up were CD45 RA− CCR7− effector memory/ CD45 RA+ CCR7− effector T cells, and were highly matured. In each case, the use of BV gene families was restricted, especially in BV5, 7, 28, and 29. Although no common predominant CMV- CTL clones were found, several shared motifs of complementarity-determining region-3 were identified among the 3 cases; QGA in all, TGE and TDT in Case 1 and Case 2, and RDRG in Case 2 and Case 3. In all cases, CMV- CTL clones that were detected for the first time after allo HCT persisted as the dominant clones. In Case 1, without CMV reactivation, recipient-derived CMV- CTLs exclusively persisted as a dominant clone, while all CMV- CTLs in the other 2 cases, with CMV reactivation, were donor derived. Conclusion Clone monitoring and chimerism analyses should help to further clarify novel aspects of immuno-reconstitution after allo HCT. [ABSTRACT FROM AUTHOR]

Relapse is a major cause of treatment failure after hematopoietic stem cell transplantation (HSCT) for acute leukemia. Here, we report a monocentric retrospective study of all HSCTs for B cell acute lymphoblastic leukemia (ALL) performed during the years 2005-2021 ( n = 138, including 51 children), aiming to identify the optimal use of lineage-specific recipient-donor chimerism analysis for prediction of relapse. In adults, relapse was associated with increased recipient chimerism in CD3 + bone marrow cells sampled at least 30 days before a relapse. Relapse could be predicted with a sensitivity of 73% and a specificity of 83%. Results were similar for children but with a higher recipient chimerism cutoff. Additionally, adults that had at least one chimerism value <0.12% in CD3 + peripheral blood cells within the first 60 days after HSCT had 89% probability of being relapse-free after 2-years compared to 64%. Results were similar for children but again necessitating a higher chimerism cutoff. These results suggest that high-sensitive lineage-specific chimerism analysis can be used for (1) early ALL relapse prediction by longitudinal chimerism monitoring in CD3 + bone marrow cells and (2) relapse risk stratification by analyzing CD3 + blood cells early post-HSCT., Competing Interests: Dan Hauzenberger is founder and co‐owner of Devyser AB. He is also part‐time employed by the company. Stephan Mielke has received speakers fee from Novartis and DNA Prime SA, has received travel support from and served in expert panel for Gilead/KITE, has received travel support and speakers fee from Celgene/BMS, has received research funding, travel support, and speakers fee from Kiadis Pharma, has served in expert panel for Bellicum, has received travel support and speakers fee from and has served in data safety monitoring board for Miltenyi, and has served in data safety monitoring board for Immunicum. The remaining authors have no conflict of interest to declare., (© 2022 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Abstract: The utility of routine chimerism analysis as a prognostic indicator of subsequent outcomes after allogeneic hematopoietic cell transplantation (HCT) with myeloablative conditioning regimens remains controversial. To address this controversy, routine chimerism test results at 2 to 6 months after HCT with myeloablative conditioning regimens were evaluated for association with subsequent risk of chronic graft-versus-host disease (GVHD), nonrelapse mortality (NRM), relapse, and overall mortality. Only 70 of 1304 patients (5%) had < 95% donor-derived cells in the marrow. Low donor chimerism in the marrow occurred more often in patients with low-risk diseases compared with those with higher-risk diseases and was significantly associated with a reduced risk of chronic GVHD. Among 673 patients evaluated, 164 (24%) had < 85% donor-derived T cells in the blood. Low donor T cell chimerism was more frequent in patients with low-risk diseases compared with those with higher-risk diseases, in those who received conditioning with busulfan compared with those who received conditioning with total body irradiation, and in those with lower-grade acute GVHD. Low donor T cell chimerism in the blood was significantly associated with a reduced risk of chronic GVHD but not with a reduced risk of relapse, NRM, or overall mortality. Routine testing of chimerism in the marrow and blood at 2 to 6 months after HCT with myeloablative conditioning regimens may be helpful in documenting engraftment in clinical trials, but provides only limited prognostic information in clinical practice. [Copyright &y& Elsevier]

Acute graft-versus-host disease (GvHD) is a rare complication after solid organ transplantation. We describe a 52-year-old female developing neutropenia and fever 48 days after single lung transplantation for chronic obstructive pulmonary disease. Bone marrow (BM) biopsy suggested drug-induced marrow failure, so immunosuppression was reduced. Five days later a maculopapular skin rash was observed, progressing to a generalized erythema with desquamation. Skin biopsy was suspectable for GvHD, so immunosuppression was re-initiated. PCR-based chimerism analysis of BM revealed 78% donor cells. Intensified immunosuppression resulted in temporary improvement, but BM aplasia recurred and the patient experienced severe GvHD of gut and liver. Despite extensive immunosuppression the patient died from multi-organ failure 99 days after transplantation. This report describes the occurrence of neutropenia as an early presenting sign of acute GvHD after lung transplantation. We therefore recommend incorporating GvHD in the differential diagnosis of neutropenia after solid organ transplantation, calling for early chimerism analyses. [ABSTRACT FROM AUTHOR]

Lineage-specific chimerism studies are commonly obtained at several time points after nonmyeloablative hematopoietic cell transplantation to assess the tempo and degree of engraftment, and to monitor graft rejection. For patients who receive myeloablative transplants, the value of frequent chimerism analyses using sensitive molecular techniques is less certain. In this study, a retrospective analysis was performed to assess the transplant outcome of 89 adult patients with ALL who had chimerism studies of unfractionated BM cells or peripheral blood subsets performed approximately 80 days after transplantation. These patients received unmanipulated, myeloablative transplants using either HLA-identical or HLA-mismatched, related or unrelated donor stem cells. Incomplete donor engraftment was present only in the CD3+ peripheral blood T cells in a small percentage of patients. There was no correlation of mixed chimerism with transplant outcome. Routine ‘day 80’ chimerism studies in this group of patients who receive intensive, myeloablative conditioning regimens are not recommended.Bone Marrow Transplantation (2008) 42, 271–274; doi:10.1038/bmt.2008.155; published online 26 May 2008 [ABSTRACT FROM AUTHOR]

Objective and Methods: Chimerism analysis has become a routine diagnostic procedure after haematopoietic allogeneic stem cell transplantation for early detection of relapse of disease or graft failure. Whereas some centres developed individual in-house short tandem repeat (STR) systems, others prefer commercial multiplex PCR systems. However, little is known about inter-assay variation, which could have a significant impact on treatment decision. We therefore compared two commercial multiplex PCR kits with our in-house STR system using different sample sources, such as peripheral blood (PB), bone marrow (BM) and specific leukocyte subsets. Results: Fifty samples of eighteen paediatric patients were analysed. For neither material, PB, BM and leukocyte subtypes, a significant difference between the STR systems tested was observed. Chimerism analyses of each single STR primer, which is component of both the in-house and the commercial STR system, did not reveal significant differences. Conclusion: Our analysis demonstrates that similar results can be obtained with both assays, even when using various sample sources. Further evaluation of different test systems will help to increase interlaboratory standardisation of chimerism analyses for early clinical intervention. [ABSTRACT FROM AUTHOR]

Both conventional chimerism analysis (CCA) and lineage-specific chimerism analysis (LCA) have potential pitfalls as diagnostic means for the detection of minimal residual disease after allogeneic hematopoietic cell transplantation (aHCT). Therefore, the present study examines the results of both methods in order to determine how predictive consecutive evaluations were, with respect to the risk that the patient would relapse during post-transplant follow-up and with respect to responsiveness to immunomodulatory treatment. A total of 168 individuals with acute myeloid leukemia (AML) (n=137) and myelo dysplastic syndrome (n=31) were investigated with CCA and LCA at mean intervals of 24 days (range: 11-116). The median follow-up after myeloablative aHCT was 22 months (range: 4-49). Of 168 patients, 65 experienced a clinical relapse after aHCT. CCA and LCA were comparatively sensitive and specific for relapse at the intervals of chimerism testing employed in this study. Of 32 patients, 10 who were offered donor lymphocyte infusions (DLI) treatment for increasing (n=29) or stable (n=3) mixed chimerism (MC) achieved at least transitory CC. The observation that all patients with increasing MC relapsed despite DLI treatment (54%) or withdrawal of immune suppression (24%) indicates that novel strategies to deal with rapidly evolving relapse in AML patients, such as shortening of chimerism monitoring intervals, need to be evaluated.Leukemia (2005) 19, 814-821. doi:10.1038/sj.leu.2403719 Published online 17 March 2005 [ABSTRACT FROM AUTHOR]

Abstract: A 52-year-old liver transplant recipient presented 8 months after transplantation with oral thrush, then 3 days later with oral ulcers and a diffuse rash, and 5 days later with an acutely reduced white blood cell count, rash, fever, and diarrhea. Bone marrow biopsy revealed severe aplasia. Although graft-versus-host disease (GVHD) was considered, the late onset of these symptoms was felt to render this etiology unlikely because GVHD usually occurs 2 to 6 weeks after transplantation. All potentially myelosuppressive medications were discontinued, and the patient was treated with high doses of hematopoietic growth factors. Because his symptoms continued, chimerism analysis was performed, which indicated that 96% of the peripheral blood mononuclear cells were of liver-donor origin. Ultimately, the patient underwent an allogeneic peripheral blood hematopoietic progenitor cell transplant from a human leukocyte antigen–identical brother, but he died 5 days after transplantation of overwhelming Candida kruseii infection. To our knowledge, this is the first chimerism-analysis–documented case of severe acute GVHD presenting so late after liver transplantation. It is of note that the patient had no known risks for GVHD in that he was relatively young and shared only one major human leukocyte antigen with his donor. Consideration should be given to GVHD as a cause of bone marrow aplasia at any time after organ transplantation. Storage of cell pellets from all transplant recipients and donors is highly recommended to facilitate the diagnostic evaluation. [Copyright &y& Elsevier]

Summary:Mast cell leukemia (MCL) is a rare form of aggressive mastocytosis with a reported median survival below 6 months. Casuistic reports suggest the effectiveness of allogeneic bone marrow transplantation (BMT) for MCL. However, these reports lack clear evidence for a graft-versus-mast-cell (GvMC) effect. We prospectively investigated the GvMC at different time points after allogeneic BMT and donor-lymphocyte infusions (DLI). Samples were gathered from a patient with MCL treated with allogeneic BMT from an unrelated HLA identical donor. Parameters for detection of a GvMC effect included flow cytometrical analysis of mast cell (MC) populations in peripheral blood and BM, BM smear and histology, chimerism analysis of flow cytometrically sorted BM CD117+/CD34-MC and testing for anti-mast cell reactivity of donor lymphocytes by interferon (IFN)-?ELISPOT. DLIs reduced MC from 5 to 0.5%. MC chimerism analysis demonstrated a complete recipient genotype after BMT, suggesting that the persistent mastocytosis was part of residual neoplastic disease. At 3.7 years after BMT, there is some evidence for relapse. In summary, BMT and DLIs attenuated the mastocytosis from an aggressive to an indolent form and may have improved the patients'prognosis. The in vitro data of our study indicate for the first time the existence of a GvMC effect.Bone Marrow Transplantation (2004) 34, 515-519. doi:10.1038/sj.bmt.1704627 Published online 26 July 2004 [ABSTRACT FROM AUTHOR]

Patients suffering from multiple endocrine neoplasia type 1 (MEN1) are predisposed to multiple endocrine tumors. The MEN1 gene product, menin, is expressed in many embryonic, as well as adult tissues, and interacts with several proteins in vitro and in vivo. However, the biological function of menin remains largely unknown. Here we show that disruption of the Men1 gene in mice causes embryonic lethality at E11.5–E13.5. The Men1 null mutant embryos appeared smaller in size, frequently with body haemorrhages and oedemas, and a substantial proportion of them showed disclosure of the neural tube. Histological analysis revealed an abnormal development of the nervous system and heart hypotrophy in some Men1 null embryos. Furthermore, Men1 null livers generally displayed an altered organization of the epithelial and hematopoietic compartments associated with enhanced apoptosis. Chimerism analysis of embryos generated by injection of Men1 null ES cells, showed that cells lacking menin do not seem to have a general cell-autonomous defect. However, primary Men1 null embryonic fibroblasts entered senescence earlier than their wild-type counterparts. Despite normal proliferation ability, Men1 null ES cells exhibited a deficiency to form embryoid bodies, suggesting an impaired differentiation capacity in these cells. The present study demonstrates that menin plays an important role in the embryonic development of multiple organs in addition to its proposed role in tumor suppression. [Copyright &y& Elsevier]

A patient with both a renal and pancreatic transplantation developed a diffuse large B-cell lymphoma, Epstein-Barr virus-related, 14 months after the surgical procedure. Tumor was confined to the transplanted organs: head of the pancreas and hilar lymph node of the transplanted kidney. Chimerism analysis demonstrated the tumor origin from donor lymphoid cells. Immunosuppression was discontinued and chemotherapy with cyclophosphamide, hydroxydaunomycin, vincristine, and prednisone (CHOP) was started. However, no response was observed after three courses of this regimen. Finally, a transplantectomy was carried out, followed by rituximab (anti-CD20 antibody), with the patient achieving a complete response (CR). Two years later the patient remains in CR. [ABSTRACT FROM AUTHOR]

Chimerism analysis is a well-established method for monitoring the state of hematopoietic stem cell transplantation (HSCT) over time by analyzing peripheral blood or bone marrow samples of the recipient in several malignant and non-malignant hematologic diseases. From a clinical point of view, a continuous monitoring is fundamental for an effective early therapeutic intervention. This paper provides a comparative overview of the main molecular biology techniques which can be used to study chimerism after bone marrow transplantation, focusing on their advantages and disadvantages. According to the examined literature, short tandem repeats (STR) analysis through simple PCR coupled with capillary electrophoresis (STR-PCR) is the most powerful method which guarantees a high power of differentiation between different individuals. However, other methods such as real-time quantitative PCR (qPCR), digital PCR (dPCR), and next-generation sequencing (NGS) technology were developed to overcome the technical limits of STR-PCR. In particular, these other techniques guarantee a higher sensitivity, which allows for the detection of chimerism at an earlier stage, hence expanding the window for therapeutic intervention. After a comparative evaluation of the various techniques, it seems clear that STR-PCR still remains the gold standard option for chimerism study, even if it is likely that both dPCR and NGS could supplement or even replace the common methods of STR analysis. [ABSTRACT FROM AUTHOR]