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Introduction: Degraders are an increasingly important sub-modality of small molecules as illustrated by an ever-expanding number of publications and clinical candidate molecules in human trials. Nevertheless, their preclinical optimization of ADME and PK/PD properties has remained challenging. Significant research efforts are being directed to elucidate the underlying principles and to derive rational optimization strategies. Areas covered: In this review, the authors summarize currently best practices in terms of in vitro assays and in vivo experiments. Furthermore, the authors collate and comment on the current understanding of optimal physicochemical characteristics and their impact on absorption, distribution, metabolism, and excretion properties, including the current knowledge of drug–rug interactions. Finally, the authors describe the pharmacokinetic prediction and Pharmacokinetic/Pharmacodynamic -concepts unique to degraders and how to best implement these in research projects. Expert opinion: Despite many recent advances in the field, continued research will further our understanding of rational design regarding degrader optimization. Machine-learning and computational approaches will become increasingly important once larger, more robust datasets become available. Furthermore, tissue-targeting approaches (particularly regarding the central nervous system will be increasingly studied to elucidate efficacious drug regimens that capitalize on the catalytic mode of action. Finally, additional specialized approaches (e.g. covalent degraders, LOVdegs) can further enrich the field and offer interesting alternative approaches. [ABSTRACT FROM AUTHOR]

Objective: Chimerism analysis is an important post-transplant assessment for allogeneic hematopoietic stem cell transplant (HCT) recipients. Although various chimerism analysis techniques are already established, they are limited in terms of sensitivity, versatility, and turnaround time. Our objective was to develop a digital droplet polymerase chain reaction (ddPCR) assay for chimerism analysis using ABO gene polymorphisms as markers. Methods: Our new chimerism analysis method utilizes ddPCR to assess the ABO gene polymorphisms that encode the ABO blood genotype. ABO genotypes were determined in blood samples from 15 HCT recipients using the O panel (rs8176719) and B panel (rs8176746 and rs8176747). Results: The two panels distinguished six ABO genotypes (AA, AO, BB, BO, AB, and OO). The results of chimerism analysis using ABO genotypes with ddPCR were compatible with those of established methods, such as SRY gene analysis and the use of short tandem repeat markers via standard PCR. Our method could distinguish chimerism in 77% of donor and recipient combinations in the Japanese population. Conclusions: We developed a sensitive and rapid chimerism analysis method for HCT using ABO gene polymorphisms in ddPCR. [ABSTRACT FROM AUTHOR]

Natural chimeras are prevalent in nature (> 10 phyla of protists, plants, invertebrates, and vertebrates), disrupting the conventional believe that genetically homogeneous entities are selected to prevent conflicts within an organism. Chimerism emerges as a significant ecological/evolutionary mechanism, shaping the life history characteristics of metazoans, and it develops in various forms, one of which is called 'microchimerism'. Furthermore, chimerism is a pivotal phenomenon, presenting complex biological and ecological expressions akin to a "double-edged sword", bypassing both innate and adaptive immune responses. Considering the proportionate contribution of chimeric partners and their spatial arrangements within chimeras, unveils six somatic states of chimerism (purged-chimerism, sectorial-chimerism, mosaic-chimerism, mixed-chimerism, microchimerism and multi-chimerism) and three states of germline chimerism (mixed-chimerism, male/female chimerism and parasitic germline chimerism). These diverse chimeric states are categorized into two distinct series of continua, namely 'somatic cell chimerism' and 'germline chimerism' scenarios where dynamic chimeric states transit into other states, and vice versa, within a specific continuum that relies on the concept of an endless 'Escherian stairwell' of chimerism states. Also, the same chimera may portray simultaneously, different chimeric states in various parts/organs. We further reviewed the evolutionary perspectives for chimerism, raising five commonly shared features of chimerism (multichimerism, ontogenic windows, reproductive chimerism, transmissible chimerism, germline hitchhiking) and 'costs' and 'benefits' accrued to chimerism, shared between invertebrates and vertebrates, including humans. We contest that 'microchimerism' lacks any quantitative definition, represents just a single facet in the multi-facet panorama of chimeric phenomena that demonstrate transitions over time into other states. All of the above carry evolutionary and clinical implications. [ABSTRACT FROM AUTHOR]

Despite its critical role in survival, many aspects of hematopoiesis remain unresolved. In the classical model of the hematopoietic program, quiescent hematopoietic stem cells (HSCs) sit at the top of the hematopoietic hierarchy, with the ability to self-renew and differentiate as needed. HSCs give rise to more proliferative progenitor cells, which possess multipotent potential, but have largely or completely lost self-renewal capabilities. Here, we have identified the tumor suppressor, Inhibitor of Growth 4 (ING4), as a critical regulator of multipotent progenitor (MPP) homeostasis. In the absence of ING4, we show that MPPs express a transcriptional program of hematopoietic activation, yet they remain quiescent with low levels of reactive oxygen species. Functionally, ING4-deficient MPPs are capable of robust regeneration following competitive bone marrow transplantation, resulting in substantially higher blood chimerism compared to wild-type MPPs. These data suggest ING4 deficiency promotes a poised state in MPPs, quiescent but transcriptionally primed for activation, and capable of converting the poised state into robust repopulation upon stress. Our model provides key tools for further identification and characterization of pathways that control quiescence and regeneration in MPPs. [ABSTRACT FROM AUTHOR]

Langerhans cell histiocytosis (LCH) is a disease caused by clonal expansion of CD1a+/CD207+ cells and is characterized by organ involvement and dysfunction. Treatment of LCH in children is risk-adapted, and multisystem LCH requires systemic therapy. Although systemic treatments such as chemotherapy and BRAF/MEK inhibitors have improved the cure rate of LCH, disease reactivation rates remain 30%, and eventually some patients progress to relapse-refractory LCH. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a promising salvage treatment strategy for children with relapse-refractory LCH. However, many questions such as the efficacy and indications of allo-HSCT, as well as suitable conditioning regimen are still undetermined for children with LCH. This review aimed to provide an update on advances in allo-HSCT for LCH in children, including indications, stem cell sources, conditioning regimens, chimerism, transplant-related complications, outcomes, and treatment of relapse. [ABSTRACT FROM AUTHOR]

Solid organ transplantation remains a life-saving treatment for patients worldwide. Unfortunately, the supply of donor organs cannot meet the current need, making the search for alternative sources even more essential. Xenotransplantation using sophisticated genetic engineering techniques to delete and overexpress specific genes in the donor animal has been investigated as a possible option. However, the use of exogenous tissue presents another host of obstacles, particularly regarding organ rejection. Given these limitations, interspecies blastocyst complementation in combination with precise gene knockouts presents a unique, promising pathway for the transplant organ shortage. In recent years, great advancements have been made in the field, with encouraging results in producing a donor-derived organ in a chimeric host. That said, one of the major barriers to successful interspecies chimerism is the mismatch in the developmental stages of the donor and the host cells in the chimeric embryo. Another major barrier to successful chimerism is the mismatch in the developmental speeds between the donor and host cells in the chimeric embryos. This review outlines 19 studies in which blastocyst complementation was used to generate solid organs. In particular, the genesis of the liver, lung, kidney, pancreas, heart, thyroid, thymus and parathyroids was investigated. Of the 19 studies, 7 included an interspecies model. Of the 7, one was completed using human donor cells in a pig host, and all others were rat–mouse chimeras. While very promising results have been demonstrated, with great advancements in the field, several challenges continue to persist. In particular, successful chimerism, organ generation and donor contribution, synchronized donor–host development, as well as ethical concerns regarding human–animal chimeras remain important aspects that will need to be addressed in future research. [ABSTRACT FROM AUTHOR]

Background and Clinical Significance: Ultrasonographic diagnosis of twin pregnancies has become routine, with chorionicity playing a crucial role in assessing associated risks. Traditionally, monochorionic (MC) twins were believed to derive from a single zygote, ensuring sex concordance. However, recent cases of dizygotic monochorionic (MCDZ) twins challenge this paradigm. In this paper, four cases of MCDZ twins with sex discordance are described. Case presentation: Case 1 and case 2 describe two spontaneous MC/diamniotic twin pregnancies in which sex discordance between twins was attributed to blood chimerism. Case 3 is about a MC/diamniotic twin pregnancy derived from a single blastocyst transfer after in vitro fertilization (IVF), and that was complicated by twin-to-twin transfusion syndrome, with zygosity testing confirming the dizygosity. Case 4 is a twin anemia polycythemia sequence diagnosed after birth in twins considered dichorionic during pregnancy (due to sex difference) and defined as monochorionic after placental examination. Conclusions: The prevalence of monochorionic dizygotic (MCDZ) twins remains uncertain, and many cases likely go unnoticed, particularly when twins are of the same sex. In twin pregnancies, determining chorionicity during the first-trimester ultrasound (US) is critical. Accurate identification of monochorionicity is essential for managing potential complications. Careful verification of sex concordance between twins is necessary. In cases of sex discordance, amniocentesis is required for karyotype evaluation and zygosity testing. [ABSTRACT FROM AUTHOR]

BACKGROUND Allogeneic hematopoietic stem-cell transplantation is the only curative treatment for myelofibrosis. Driver mutations are the pathophysiological hallmark of the disease, but the role of mutation clearance after transplantation is unclear. METHODS We used highly sensitive polymerase-chain-reaction technology to analyze the dynamics of driver mutations in peripheral-blood samples from 324 patients with myelofibrosis (73% with ]AK2 mutations, 23% with CALR mutations, and 4% with MPL mutations) who were undergoing transplantation after reduced-intensity conditioning. Mutations were detected before transplantation and at 30, 100, and 180 days after transplantation to measure clearance and its effect on relapse and cure. The two primary end points were relapse and disease-free survival. RESULTS At day 30 after transplantation, mutation clearance was found in 42% of the patients who had ]AK2 mutations, 73% of those who had CALR mutations, and 54% of those who had MPL mutations; the corresponding percentages at day 100 were 63%, 82%, and 100%. The cumulative incidence of relapse at 1 year was 6% (95% confidence interval [CI], 2 to 10) among patients with mutation clearance at day 30 after transplantation and 21% (95% CI, 15 to 27) among those without mutation clearance at day 30. Disease-free and overall survival at 6 years were 61% and 74%, respectively, among patients with mutation clearance at day 30 after transplantation and 41% and 60%, respectively, among those without mutation clearance at day 30. Mutation clearance at day 30 appeared to outperform traditional donor chimerism as a measure of response; it was independently associated with a reduced risk of relapse or progression (hazard ratio, 0.36; 95% CI, 0.21 to 0.61) and appeared to overcome differences in prognosis based on the type of driver mutation (JAK2 vs. MPL or CALR). CONCLUSIONS In patients with myelofibrosis, clearance of driver mutations at day 30 after transplantation appeared to influence relapse and survival, irrespective of the underlying driver mutation. [ABSTRACT FROM AUTHOR]

Introduction. The analysis of free circulating DNA (cfDNA) holds promise for molecular diagnostics, but its fragmentation and low concentration can complicate PCR analysis.Objective. To investigate the effect of target length on the amplification efficiency of Y-chromosome markers from cfDNA.Material and methods. Fifty cfDNA samples were obtained from 39 patients: patients after liver transplantation (n=19), patients with acute leukemia after allogeneic hematopoietic stem cell transplantation (n=10), and pregnant women (n=10). In addition, we prepared 16 chimeric samples by sequential dilution of male cfDNA into female cfDNA from healthy donors. We determined the proportion of male cfDNA using the Y-chromosome marker S02, which is 211 bp in length as suggested by M. Alizadeh et al. We also modified Alizadeh's primer design to obtain a DNA target with a length of 138 bp. The proportion of male cfDNA was also determined by fragment analysis using the amelogenin Y marker (84 bp) from the COrDIS Plus kit (Gordiz LLC, Russia).Results. In the three groups of patients, amplification of male cfDNA was more efficient when shorter DNA targets were used (p

Although graft‐versus‐host disease (GVHD) is a common complication of hematopoietic stem cell transplantation, it is rare after solid organ transplantation (SOT) or blood transfusion. We present a rare case of SOT‐derived and/or transfusion‐associated graft‐versus‐host disease (TA‐GVHD) in a 66‐year‐old man with interstitial lung disease who underwent bilateral lung transplantation (LT) from a 12‐year‐old female donor and required three units of packed red blood cells intraoperatively. He presented with signs and symptoms consistent with GVHD, and a bone marrow biopsy revealed an XX karyotype. He died 3 months after bilateral LT, and postmortem chimerism testing using next‐generation sequencing identified three sources of DNA within his bone marrow, including the recipient, the lung donor, and a third donor, thereby suggesting the presence of solid organ transplant graft‐versus‐host disease (SOT‐GVHD), TA‐GVHD, or a combination of both. [ABSTRACT FROM AUTHOR]

Background: Allo-HSCT is a curative therapy for patients with transfusion-dependent thalassemia (TDT). The high incidence of transplant-related complications is becoming an obstacle to safe and effective unrelated donor (URD) transplantation. Methods: In this retrospective study, we reported the survival outcomes and complications of transplantation in thalassemia patients using a novel regimen consisting of pre-transplantation immunosuppression (PTIS) and modified myeloablative conditioning based on intravenous busulfan, cyclophosphamide, fludarabine, and rabbit anti-human thymocyte immunoglobulin. Results: A total of 88 thalassemia patients received the novel conditioning regimen (NCR group), while 118 patients received the conventional conditioning regimen (CCR group). The median age at HSCT in the NCR group was older (7 years vs. 4 years, p < 0.05). No patient in the NCR group experienced primary graft failure, while the 3-year probabilities of OS and TFS were 96.6% and 93.2%, respectively. Even when the intensity of conditioning was reduced, OS (94.8% vs. 94.3%, p = 0.848) and TFS (89.8% vs. 92.5%, p = 0.663) in URD transplants in the NCR group were comparable to those in the CCR group, while the risk of autoimmune hemolytic anemia (AIHA) (0% vs. 15.1%) was lower. In addition, the NCR group had lower rates of mixed chimerism (7.1%). Conclusions: URD transplantation can achieve a comparable prognosis to matched sibling donor (MSD) transplantation with a lower incidence of AIHA due to PTIS and modified myeloablative conditioning regimen. [ABSTRACT FROM AUTHOR]

Myeloid chimerism better reflects donor stem cell engraftment than whole-blood chimerism in assessing graft function following allogeneic hematopoietic stem cell transplant (HCT). We describe our experience with 130 patients aged younger than 18 years, treated with allogeneic HCT using bone marrow or PBSC from HLA-matched donors for non-malignant diseases, whose pre-transplant conditioning therapy included alemtuzumab and who were monitored with lineage-specific chimerism after transplant. At 6 years post-transplant, overall survival (OS) was 91.1% and event-free survival (EFS) was 81.5%, with no grade III-IV acute GvHD or chronic GVHD observed. Recipient T-cells did not contribute to graft loss. Mixed T-cell chimerism (MC) did not affect EFS, and there was no connection between T-cell chimerism and myeloid chimerism in patients with MC or graft loss. MC significantly correlated with virus infection; more children with MC were CMV seropositive than those with complete chimerism (CC). Additionally, MC was more common in patients with CMV viramia post-transplant. CD8 T-cell reconstitution was affected by viral reactivation, including CMV, with CD8 T-cell counts higher in the MC group than in the CC group. Mixed T-cell chimerism is due to autologous, virus-specific, predominantly CD8, T-cell expansion, and is protective and not deleterious to the recipient. [ABSTRACT FROM AUTHOR]

Objectives To determine whether the information provided by short tandem repeat (STR) testing and bone marrow (BM) biopsy specimens following hematopoietic stem cell transplant (HSCT) provides redundant information, leading to test overutilization, without additional clinical benefit. Methods Cases with synchronous STR and flow cytometric immunophenotyping (FCI) testing, as part of the BM evaluation, were assessed for STR/FCI concordance. Results Of 1199 cases (410 patients), we found the overall concordance between STR and FCI was 93%, with most cases (1063) classified as STR–/FCI–. Of all discordant cases, 75 (6%) were STR+/FCI–, with only 5 (6.7%) cases best explained as identification of disease relapse. Eight cases were STR–/FCI+, representing relapsed/residual disease. Analysis of cases 1 year or more from transplant (54% of all cases) indicated only 9 (1.5%) were STR+/FCI–, and none uniquely identified relapse. Conclusions These data suggest that STR analysis performed 1 year or more post-HSCT does not identify unknown cases of relapse. Furthermore, while STR testing is critical for identifying graft failure/rejection within the first year posttransplant, FCI appears superior to STR at detecting late relapses with low-level disease. Therefore, STR testing from patients 1 year or more post-HSCT may be unnecessary, as BM biopsy evaluation is sufficient to identify disease relapse. [ABSTRACT FROM AUTHOR]

Simple Summary: Multiple myeloma (MM) is a blood cancer which classically has a prolonged course of remissions and relapses. Several new highly efficacious medications have been developed for MM in recent years, including some that utilize the body's own immune cells to control the cancer. Alongside the development of these new medications has been the evolution of tests to track small amounts of (measurable) residual disease (MRD). Several MRD tests can now find and track as little as 1 residual cancer cell in 1 million in certain cases. Having no detectable MRD has been independently associated with improved cancer control and longer survival—here, we review how these tests might be used as a companion to new immune therapies for MM. Several novel T-cell-based therapies have recently become available for multiple myeloma (MM). These T-cell redirecting therapies (TRTs) include chimeric antigen receptor T-cells (CAR-T) and bispecific antibodies (BiAbs). In both clinical trial and real-world data, these therapies have demonstrated high rates of deep clinical response, and some are now approved for second-line treatment for relapsed MM. The deep and sustained clinical responses these therapies are capable of inducing will require sophisticated response monitoring to provide meaningful information for patient care. Obtaining measurable residual disease (MRD) negativity has been validated as an independent positive prognostic marker for progression-free survival (PFS) and overall survival (OS) in both newly diagnosed and relapsed refractory patients with multiple myeloma. Assessment for MRD negativity was performed in all of the trials for FDA-approved TRT. Here, we summarize pertinent data for MRD assessment following TRT in MM and provide a rationale and structured framework for conducting MRD testing post TRT. [ABSTRACT FROM AUTHOR]

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Langerhans cell histiocytosis (LCH) is a disease caused by clonal expansion of CD1a+/CD207+ cells and is characterized by organ involvement and dysfunction. Treatment of LCH in children is risk-adapted, and multisystem LCH requires systemic therapy. Although systemic treatments such as chemotherapy and BRAF/MEK inhibitors have improved the cure rate of LCH, disease reactivation rates remain 30%, and eventually some patients progress to relapse-refractory LCH. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a promising salvage treatment strategy for children with relapse-refractory LCH. However, many questions such as the efficacy and indications of allo-HSCT, as well as suitable conditioning regimen are still undetermined for children with LCH. This review aimed to provide an update on advances in allo-HSCT for LCH in children, including indications, stem cell sources, conditioning regimens, chimerism, transplant-related complications, outcomes, and treatment of relapse.

Abstract Background Single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) are the predominant sources of information about the coding potential of uncultured microbial lineages, but their strengths and limitations remain poorly understood. Here, we performed a direct comparison of two previously published collections of thousands of SAGs and MAGs obtained from the same, global environment. Results We found that SAGs were less prone to chimerism and more accurately reflected the relative abundance and the pangenome content of microbial lineages inhabiting the epipelagic of the tropical and subtropical ocean, as compared to MAGs. SAGs were also better suited to link genome information with taxa discovered through 16S rRNA amplicon analyses. Meanwhile, MAGs had the advantage of more readily recovering genomes of rare lineages. Conclusions Our analyses revealed the relative strengths and weaknesses of the two most commonly used genome recovery approaches in environmental microbiology. These considerations, as well as the need for better tools for genome quality assessment, should be taken into account when designing studies and interpreting data that involve SAGs or MAGs. Video Abstract

The complete mitochondrial genome of Blue-fronted Redstart (Phoenicurus frontalis), GenBank accession number MT360379 (NC_053917), was published by Li and colleages in 2020. Here we show that this mitogenome is actually a chimera containing DNA fragments of both P. frontalis (15,518 bp, 92.5%) and Pink-rumped Rosefinch (Carpodacus waltoni eos, 1258 bp, 7.5%). This mitogenome has been re-used in at least three phylogenies. Our study confirms that mitogenomes are best verified with multiple gene trees, and that any anomalies should be investigated by direct comparison of sequences.

Haploidentical hematopoietic cell transplantation (haplo-HCT) is associated with an increased risk of allograft rejection. Here, we employed a major histocompatibility complex (MHC)-mismatched allogeneic HCT (allo-HCT) murine model to better understand the role of Gal-1 in immune tolerance. Transplanted mice were classified into either rejected or engrafted based on donor chimerism levels. We noted significantly higher frequencies of CD4+ T cells, CD8+ T cells, natural killer cells, IFN-g and TNF-a producing CD4+ T cells, and IFN-g producing dendritic cells and macrophages in rejected mice. Conversely, we found significantly increased frequencies of regulatory T cells (Tregs), predominantly Helios+, IL-10-producing CD4+ T cells, type 1 regulatory (Tr1) cells, and the proportion of Tr1+Gal-1+ cells in engrafted mice. Further, Gal- 1 specific blockade in Tregs reduced suppression of effector T cells in engrafted mice. Lastly, effector T cells from engrafted mice were more prone to undergo apoptosis. Collectively, we have shown that Gal-1 may favor HSC engraftment in an MHC-mismatched murine model. Our results demonstrate that Gal-1- expressing Tregs, especially at earlier time points post-transplant, are associated with inducing immune tolerance and stable mixed chimerism after HCT. [ABSTRACT FROM AUTHOR]

Background: Spontaneous remission of cancer is a rare and poorly understood phenomenon characterized by complete or partial remission of a malignancy in the absence of or with inadequate treatment. The underlying mechanism for such occurrences is poorly understood, however, immune mechanisms seem to play an important role in such cases. In recent years increasingly more data have become available in favor of the clinical benefit of low levels of chimerism in hematologic malignancies. One such instance of naturally occurring low-level chimerism is feto-maternal microchimerism which has been shown to influence cancer progression and, in some instances, to be a protective factor against malignancy. Case report: We report a case of a young female patient with aggressive primary mediastinal large B cell lymphoma refractory to two lines of chemo-immunotherapy achieving sustained complete metabolic remission of tumor while pregnant with twins. Results: A focus on feto-maternal microchimerism during and after pregnancy revealed transient levels of feto-maternal microchimerism in the peripheral blood of the patient as measured by quantifying the Y-chromosome-linked SRY gene. Conclusions: Microchimerism presents significant potential for enhancing our comprehension of disease mechanisms, uncovering novel therapeutic targets, and refining diagnostic and treatment approaches, especially concerning cancer. [ABSTRACT FROM AUTHOR]

Summary: Routine ABO blood group typing of apparently healthy individuals sporadically uncovers unexplained mixed‐field reactions. Such blood group discrepancies can either result from a haematopoiesis‐confined or body‐wide dispersed chimerism or mosaicism. Taking the distinct clinical consequences of these four different possibilities into account, we explored the responsible cause in nine affected individuals. Genotype analyses revealed that more than three‐quarters were chimaeras (two same‐sex females, four same‐sex males, one sex‐mismatched male), while two were mosaics. Short tandem repeat analyses of buccal swab, hair root and nail DNA suggested a body‐wide involvement in all instances. Moreover, genome‐wide array analyses unveiled that in both mosaic cases the causative genetic defect was a unique copy‐neutral loss of heterozygosity encompassing the entire long arm of chromosome 9. The practical transfusion‐ or transplantation‐associated consequences of such incidental discoveries are well known and therefore easily manageable. Far less appreciated is the fact that such findings also call attention to potential problems that directly ensue from their specific genetic make‐up. In case of chimerism, these are the appearance of seemingly implausible family relationships and pitfalls in forensic testing. In case of mosaicism, they concern with the necessity to delineate innocuous pre‐existent or age‐related from disease‐predisposing and disease‐indicating cell clones. [ABSTRACT FROM AUTHOR]

Background Allogeneic hematopoietic stem cell transplant for myeloid neoplasms with increased fibrosis is uncommon; morphologic features posttransplant can be concerning for persistent disease. Methods In this retrospective study, we identified 22 patients transplanted for myeloproliferative neoplasms or chronic myelomonocytic leukemia with fibrosis at our institution, and reviewed slides from pretransplant and posttransplant bone marrow biopsies. Clinical features and results of molecular, chimerism, and cytogenetic studies were retrieved from the medical record. Results Pretransplant bone marrow biopsies commonly exhibited hypercellularity, atypical megakaryocytes, and reticulin fibrosis. At day 100, 36% of biopsies had reticulin grade >MF1 and 33% of those tested had positive molecular studies, with no significant associations between day 100 marrow characteristics and molecular profile or peripheral count recovery times. In the 1 year posttransplant biopsies (n = 12), 7 of 9 had negative molecular studies; of these, none had reticulin grade >MF1, 1 had trichrome 1+, 2 had atypical megakaryocytes, and 1 was hypercellular. Conclusions Supporting recent literature, our study indicates that persistent day 100 reticulin fibrosis/collagen deposition does not show an association with day 100 molecular status. Our study additionally provides data for 12 patients with 1 year posttransplant marrow biopsies, with the majority of those lacking either increased fibrosis or molecular evidence of persistent disease. [ABSTRACT FROM AUTHOR]

Hemophagocytic Lymphohistiocytosis (HLH) is a rare disorder of immune dysregulation characterized by fever, cytopenias, and splenomegaly. Its primary form poses a therapeutic challenge due to its high fatality when left untreated. We retrospectively analyzed 28 patients who underwent related-donor allogeneic stem cell transplant for primary HLH from 2010 to 2021. Among them were 10 cases of familial HLH, 8 cases of Griscelli syndrome type 2, and 1 case each with PRF1 and STX11 mutations. All the patients underwent transplants with reduced-intensity or myeloablative conditioning and 26 of them achieved neutrophil engraftment at a median of day + 14. The donors were either fully matched (68%) or haploidentical (32%). With a median follow-up of 1 year, overall survival was 68% (n = 19) and disease-free survival was 64.4% (n = 18). OS was better in patients transplanted with a sibling donor (compared to parent donor), who achieved complete donor chimerism, and those transplanted early in the course of the disease (diagnosis to transplant duration less than 6 months). [ABSTRACT FROM AUTHOR]

Objectives: The Wilms' tumor gene 1 (WT1) plays a critical role in cell development and the regulation of essential genes involved in cell growth and metabolism. In the context of hematopoietic tumors, including acute myeloid leukemia (AML), WT1 has been identified as a potential marker for measurable residual disease (MRD) assessment. Relapse after allogeneic hematopoietic stem cell transplantation (allo-SCT) remains a significant challenge in AML treatment, highlighting the importance of MRD monitoring for risk stratification and treatment decisions. This study aimed to investigate the clinical significance of WT1 as a molecular marker for MRD and its correlation with chimerism in AML patients post-allo-SCT setting. Methods: We have included 58 patients with WT1-expression-positive acute myeloid leukemia (AML) who received allo-SCT in our center between 2016–2022. The exclusion criteria are as follows: not having WT1 polymerase chain reaction (PCR) measurement at diagnosis, not receiving allo-SCT, and not having a serial measurement of WT1 post-transplant. Pre- and post-transplant assessments were made with flow cytometry, WT1 PCR, and bone marrow morphological evaluations. Statistical analyses were carried out to explore correlations between WT1 levels, MRD markers, and chimerism post-transplantation. Results: We found that WT1 had a significant correlation with flow cytometry and bone marrow morphological evaluation, but not with chimerism. Interestingly, high WT1 expressors exhibited a more robust correlation with chimerism compared to the general cohort. The negative predictive value for post-allo-SCT relapse was 91.8% for the whole WT1 cohort; for high WT1 expressors, it was similar, at 87.5%. The negative predictive value for post-allo-SCT relapse was high for the whole WT1 cohort; for high WT1 expressors, it was similar. The WT1 MRD assay showed a high negative predictive value for post-allo-SCT relapse, consistent across both the entire cohort (91.8%) and high WT1 expressors (87.5%). Conclusions: WT1 expression levels may serve as a valuable ancillary marker in MRD assessment and relapse prediction post-allo-SCT in AML patients, particularly for those lacking specific fusion genes or mutations. However, further large-scale, controlled studies are needed to standardize WT1 MRD assays and establish clear guidelines for their clinical application. [ABSTRACT FROM AUTHOR]

BACKGROUND: Ischemia-reperfusion injury (IRI) is a phenomenon that affects transplant survival. The aim of our study was to examine the effects of IRI in isogenic and allogeneic muscle and skin transplantation models exposed to prolonged warm ischemia. METHODS: Forty-eight Lewis rats and 16 Brown-Norway rats were used to create four groups: Isogenic Inguinal Flap Transplantation (IST), Isogenic Gastrocnemius Muscle Flap Transplantation (IMT), Allogeneic Inguinal Flap Transplantation (AST), and Allogeneic Gastrocnemius Muscle Flap Transplantation (AMT). Malonyldialdehyde (MDA) and superoxide dismutase (SOD) levels were measured on postoperative days 1, 7, 21, 35, 63, 100, and 120 in all groups. Donor-specific chimerism (DSC) in peripheral blood was evaluated in the allogeneic groups on postoperative days 7, 21, 35, 63, 100, and 120. The microRNA-21 and microRNA-205 levels were evaluated on postoperative days 1, 7, and 120 in all groups. At the end of the study, a histopathological examination was performed. RESULTS: A statistically significant difference was found between the groups in terms of MDA and SOD levels. DSC was detected in the AMT group. A significant increase in microRNA-205 was observed, especially in the AMT group. There was no significant difference in the number of functional muscle units between the muscle transplantation groups. CONCLUSION: The presence of DSC in the AMT group and the lack of a significant difference in the number of functional muscle units in the IMT and AMT groups are noteworthy findings. [ABSTRACT FROM AUTHOR]

Vascularized composite allotransplantation (VCA) is the transplantation of multiple tissue types as a solution for devastating injuries. Despite the highly encouraging functional outcomes of VCA, the consequences of long-term immunosuppression remain the main obstacle in its application. In this review, we provide researchers and surgeons with a summary of the latest advances in the field of cell-based therapies for VCA tolerance. Four electronic databases were searched: PubMed, Scopus, Cumulative Index to Nursing and Allied Health Literature , and Web of Science. We used the Preferred Reporting Items for Systematic Reviews and Meta-Analysis as the basis of our organization. Hematopoietic stem cells prolonged VCA survival. A combination of immature dendritic cells and tacrolimus was superior to tacrolimus alone. T cell Ig domain and mucin domain modified mature dendritic cells increased VCA tolerance. Bone marrow-derived mesenchymal stem cells prolonged survival of VCAs. A combination of adipose-derived mesenchymal stem cells, cytotoxic T-lymphocyte antigen 4 immunoglobulin, and antilymphocyte serum significantly improved VCA tolerance. Ex-vivo allotransplant perfusion with recipient's bone marrow-derived mesenchymal stem cells increased VCA survival. Recipient's adipose-derived mesenchymal stem cells and systemic immunosuppression prolonged VCA survival more than any of those agents alone. Additionally, a combination of peripheral blood mononuclear cells shortly incubated in mitomycin and cyclosporine significantly improved VCA survival. Finally, a combination of donor recipient chimeric cells, anti-αβ-T cell receptor (TCR), and cyclosporine significantly prolonged VCA tolerance. Evidence from animal studies shows that cell-based therapies can prolong survival of VCAs. However, there remain many obstacles for these therapies, and they require rigorous clinical research given the rarity of the subjects and the complexity of the therapies. The major limitations of cell-based therapies include the need for conditioning with immunosuppressive drugs and radiation, causing significant toxicity. Safety concerns also persist as most research is on animal models. While completely replacing traditional immunosuppression with cell-based methods is unlikely soon, these therapies could reduce the need for high doses of immunosuppressants and improve VCA tolerance. [ABSTRACT FROM AUTHOR]

Citrus spp. are recalcitrant to in vitro shoot regeneration and we report an improved in planta protocol for genetic transformation of rough lemon that bypasses shoot regeneration in tissue culture. The features of the protocol were the use of an Agrobacterium suspension with an OD600 nm = 0.6–1.0 supplemented with 100 μg acetosyringone, gentle shaking of embryo axes pricked at shoot apical meristems (from 2-day-old germinating seeds) at 70 rpm during agro-infection, followed by growth and development of plantlets at 30 °C. PCR screening of 2-month-old T0 plants revealed the presence of an amplicon corresponding to the β-1,3-glucanase gene in the primary branches of 25 plants with a transformation efficiency of 7.74%. PCR analysis of the secondary branches of these plants after 18 months showed chimerism, i.e., the coexistence of transformed and untransformed branches in all 25 plants. Quantification of β-1,3-glucanase expression in the transformed secondary branches by qRT-PCR showed that plant number 32 had maximum (3.71-fold) relative transgene expression. The qRT-PCR analysis of all four tertiary branches arising from the transformed secondary branch of plant number 32 showed no significant differences in expression among themselves and from the transformed secondary branch, suggesting restoration of the transformed branches with uniform expression and dissociation of chimerism. Scanning electron microscopy examination of leaves from secondary and tertiary branches that uniformly expressed the transgene showed a smooth, waxy surface with non-significant variation in stomata, which had a narrow opening and a mean pore length of 4.22 ± 0.25–5.09 ± 0.36 µm. In contrast, the leaves of untransformed branch had a rough surface and a significantly large stomatal opening with a mean pore length of 7.82 ± 0.67 µm. The micro-morphological characteristics of the leaves confirmed the dissociation of chimerism in the transformed tertiary branches of plant number 32. The study demonstrates identification of chimerism after in planta transformation using PCR technique, and the novelty relates to monitoring dissociation of chimerism in transformed tertiary branches of T0 generation using qRT-PCR analysis and its corroboration by electron microscopy. The protocol for genetic transformation in plants described in the present study can be used for trait improvement by transgenesis. [ABSTRACT FROM AUTHOR]

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for various hematological, immunological and metabolic diseases, replacing the patient's hematopoietic system with donor-derived healthy hematopoietic stem cells. HSCT can be complicated by early and late events related to impaired immunological recovery such as prolonged hypogammaglobulinemia post- HSCT. We present a 16-year-old female patient with sickle-cell disease who underwent HSCT with stem cells from a human leukocyte antigen (HLA) class-II mismatched family donor.While cellular recoverywas good post-HSCT, the patient developed mixed chimerism and suffered from cervical lymphadenopathy, recurrent airway infections and cutaneous SLE. She presented with hypogammaglobulinemia and was started on immunoglobulin substitution therapy and antibiotic prophylaxis. B-cell phenotyping showed that she had increased transitional and naïve mature B cells, reduced memory B cells, and diminished marginal zone/natural effector cells. In-depth immunophenotyping and B-cell receptor repertoire sequencing ruled out an intrinsic B-cell defect by expression of activation-induced cytidine deaminase (AID), presence of somatic hypermutationsanddifferentiationintoIgG-andIgA-producingplasmacells in vitro. Immunohistochemistry and flow cytometry of lymph node tissue showed a clear block in terminal B-cell differentiation. Chimerism analysis of sorted lymph node populations showed that exclusively patient-derived B cells populated germinal centers, while only a minor fraction of follicular helper T cells was patient-derived. Given this discrepancy, we deduced that the HLA class-II disparity between patient and donor likely hinders terminal B-cell differentiation in the lymph node. This case highlights that studying disturbed cognate T-B interactions in the secondary lymphoid organs can provide unique insights when deciphering prolonged hypogammaglobulinemia post-HSCT. [ABSTRACT FROM AUTHOR]

Pre-treatment genotyping of four well-characterized toxicity risk-variants in the dihydropyrimidine dehydrogenase gene (DPYD) has been widely implemented in Europe to prevent serious adverse effects in cancer patients treated with fluoropyrimidines. Current genotyping practices are largely limited to selected commonly studied variants and are unable to determine phasing when more than one variant allele is detected. Recent evidence indicates that common DPYD variants modulate the functional impact of deleterious variants in a phase-dependent manner, where a cis- or a trans-configuration translates into different toxicity risks and dosing recommendations. DPYD is a large gene with 23 exons spanning nearly a mega-base of DNA, making it a challenging candidate for full-gene sequencing in the diagnostic setting. Herein, we present a time- and cost-efficient long-read sequencing approach for capturing the complete coding region of DPYD. We demonstrate that this method can reliably produce phased genotypes, overcoming a major limitation with current methods. This method was validated using 21 subjects, including two cancer patients, each of whom carried multiple DPYD variants. Genotype assignments showed complete concordance with conventional approaches. Furthermore, we demonstrate that the method is robust to technical challenges inherent in long-range sequencing of PCR products, including reference alignment bias and PCR chimerism. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome of Blue-fronted Redstart (Phoenicurus frontalis), GenBank accession number MT360379 (NC_053917), was published by Li and colleages in 2020. Here we show that this mitogenome is actually a chimera containing DNA fragments of both P. frontalis (15,518 bp, 92.5%) and Pink-rumped Rosefinch (Carpodacus waltoni eos, 1258 bp, 7.5%). This mitogenome has been re-used in at least three phylogenies. Our study confirms that mitogenomes are best verified with multiple gene trees, and that any anomalies should be investigated by direct comparison of sequences. [ABSTRACT FROM AUTHOR]

Donor cell leukemia (DCL) is a rare complication after allogeneic hematopoietic stem cell transplantation (HSCT) accounting for 0.1% of relapses and presenting as secondary leukemia of donor origin. Distinct in phenotype and cytogenetics from the original leukemia, DCL’s clinical challenge lies in its late onset. Its origin is affected by donor cell anomalies, transplant environment, and additional mutations. A 43-year-old woman, treated for early stage triple-negative breast cancer, developed mixed-phenotype acute leukemia (MPAL), 12 years later. Following induction chemotherapy, myeloablative conditioning, and allo-HSCT from her fully HLA-matched brother, she exhibited multiple cutaneous relapses of the original leukemia, subsequently evolving into DCL of the bone marrow. Cytogenetic analysis revealed a complex male karyotype in 20 out of 21 metaphases, however, still showing the MPAL phenotype. DCL diagnosis was confirmed by 90.5% XY in FISH analysis and the male karyotype. Declining further intensive chemotherapy including a second allo-HSCT, she was subsequently treated with repeated radiotherapy, palliative systemic therapies, and finally venetoclax and navitoclax but died seven months post-DCL diagnosis. This case underlines DCL’s complexity, characterized by unique genetics, further complicating diagnosis. It highlights the need for advanced diagnostic techniques for DCL identification and underscores the urgency for early detection and better prevention and treatment strategies.

AbstractA complete mitochondrial genome of Great Knot (Calidris tenuirostris), MK992912, was published by He and colleagues in 2020. Here we show that this mitogenome is actually a chimera containing DNA fragments of both C. tenuirostris (15,567 bp, 92.8%) and Pacific Golden Plover (Pluvialis fulva, 1208 bp, 7.2%). Detecting such errors is possible before publication if each sequenced fragment is separately analyzed phylogenetically before assembling the fragments into a single mitogenome. This mitogenome has been re-used in at least four phylogenies. The error is documented to avoid the perpetuation of erroneous sequence information in the literature.

Introduction: Parthenogenetic chimera is an extremely rare condition in human. Very few patients with parthenogenetic chimerism with XX/XY cells have been identified. Case Presentation: We report the clinical findings and molecular analysis of chimerism with a 46,XX/46,XY karyotype in a patient presenting idiopathic oligoasthenoteratozoospermia (OAT). To clarify the mechanism of chimera formation, short tandem repeat analysis using 21 loci was carried out. Quantitation of alleles in D6S1043, D12S391, fibrinogen alpha chain, and amelogenin revealed double paternal and one maternal genetic contribution to the patient, which is consistent with a parthenogenetic chimerism. The likely mechanism of chimerism formation was also discussed, followed by a literature review. Conclusion: This is the first documented case of parthenogenetic chimerism in an adult male with XX/XY cells presenting OAT. Improved cell sampling and more sensitive and specific detection methods are necessary to identify more patients with XX/XY chimerism for systematic studies on this condition in the future. Established Facts: Parthenogenetic chimera, resulting from endoreplication of the female pronucleus via mitotic divisions, is an extremely rare condition. Very few patients with parthenogenetic chimerism with XX/XY cells have been identified. The phenotypes of these patients vary from typical male genitalia to different degrees of ambiguous genitalia, and even to ovotesticular disorder of sex development. However, these patients' fertility ability in adulthood was not assessed since their conditions were usually discovered before childhood. [ABSTRACT FROM AUTHOR]

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]

Ascidians are marine filter-feeder chordates. Botrylloides ascidians possess diverse biological properties such as whole-body regeneration (WBR), hibernation/aestivation, blastogenesis, metamorphosis, and natural chimerism. However, the absence of distinctive morphological features often makes identification difficult. Botrylloides diegensis is an ascidian that has been misidentified in previous studies and is recorded in GenBank as Botrylloides leachii owing to the high morphological similarity between the sister species. More available sequences and strategies for identification would help resolve some of the confusion surrounding its ambiguous nature. We collected several Botrylloides samples from seven locations in Aotearoa New Zealand (Dunedin, Christchurch, Picton, Nelson, Whangateau, New Plymouth, and Invercargill) and barcoded the species based on Cytochrome Oxidase I, Histone 3, 18S, and 28S ribosomal RNA markers. Network and Bayesian trees confirmed the presence of three Botrylloides species: B. diegensis, Botrylloides jacksonianum, and Botrylloides affinis anceps. [ABSTRACT FROM AUTHOR]

Background: Head and neck cancer is the most common cancer in males and fifth most common in females in India. Inadequate screening programs and non-availability of local healthcare resources leads to late diagnosis and most cases present at an advanced stage. Surgical extirpation often results in complex, large defects. The concept of chimerism is useful in dealing with such extensive defects. Methods: Between July 2013 and May 2017, all patients who underwent primary reconstruction of head and neck defects following cancer extirpative surgery, with ALT-AMT chimera flap at a tertiary care cancer centre were included in this study. The patient data of age, sex, etiology, defect size, flap size, perforator configuration, anastomotic details, donor site closure, and complications was retrospectively collected as per the designated proforma from the hospital electronic record, departmental case record forms and the first author's personal logs, and analyzed. Results: Chimeric flaps based on the lateral circumflex femoral artery i.e. ALT plus AMT provide the desired qualities to address the complex defects. All thirteen patients had a large intraoral mucosal defect. Nine cases had a large extra oral skin defect. The average size of ALT was (112.5 cm2 area) and of AMT was (94.9 cm2 area). The combined area of ALT AMT was 28.5 X 9 = 256.5 cm2. All AMT pedicles were joining the ALT pedicle. 1 AMT was lost and all donor sites needed skin grafting. Conclusion: Chimeric ALT + AMT is a valuable option when complex, large, multidimensional and multicomponent defects need to be reconstructed. The advantage of utilising a single donor site and two independent flaps with a single microvascular anastomosis. Level of Evidence: Level IV, therapeutic study [ABSTRACT FROM AUTHOR]

Background: For many years, transplantation outcomes were uncertain and not hopeful, until histocompatibility testing spread. Common criteria for histocompatibility assays and communications' improvement allowed an efficient organ sharing system. The possibility of organ exchanges is closely linked to the importance of interlaboratory comparisons for histocompatibility and immunogenetics methods. The external proficiency testing (EPT) systems are the most powerful quality assurance tools. They help achieve harmonization of analyses, set a standard of performance, and a common interpretation. Methods: The external quality assurance program for diagnostic immunology laboratories (Garantía Externa de Calidad para Laboratorios de Inmunología Diagnóstica, GECLID) program nowadays runs 13 external quality assurance (EQA) histocompatibility and immunogenetics schemes, with the first of them from 2011 to date: serological and molecular: low- and high-resolution human leukocyte antigen (HLA), human platelet antigen (HPA), and killer inhibitory receptor (KIR) typing(HLA-B*27, HLA-B*57:01, and coeliac disease-related HLA), cell-dependent cytotoxicity (CDC) and flow cytometry (FC) crossmatches, anti-HLA and anti-HPA antibodies, and chimerism. Results: A total of 85 laboratories participated in this subprogram in the last 12years reporting over 1.69 M results: 1.46 M for anti-HLA and anti-HPA antibodies, 203.810 molecular typing data (HLA, HPA, and KIR genes), 2.372 for chimerism analyses, and 39.352 for crossmatches. Based on the European Federation for Immunogenetics (EFI) standards for EPT providers, the mean success rates ranged from 99.2% for molecular typing schemes and antibodies and 94.8% for chimerism, was 96.7% regarding crossmatches, and was 98.9% in serological typing. In 2022, 61.3% of the participating laboratories successfully passed every HLA EQA scheme, although 87.9% annual reports were satisfactory. Most penalties were due to nomenclature errors or misreporting of the risk associated to HLA and disease. Conclusion: This EQA confirms the reliability of HLA and immunogenetics assays in routine care. There is little heterogeneity of results of different assays used by participating laboratories, even when in-house assays are used. Reliability of test results is reasonably granted. [ABSTRACT FROM AUTHOR]

Chimerism-based strategies represent a pioneering concept which has led to groundbreaking advancements in regenerative medicine and transplantation. This new approach offers therapeutic potential for the treatment of various diseases, including inherited disorders. The ongoing studies on chimeric cells prompted the development of Dystrophin-Expressing Chimeric (DEC) cells which were introduced as a potential therapy for Duchenne Muscular Dystrophy (DMD). DMD is a genetic condition that leads to premature death in adolescent boys and remains incurable with current methods. DEC therapy, created via the fusion of human myoblasts derived from normal and DMD-affected donors, has proven to be safe and efficacious when tested in experimental models of DMD after systemic–intraosseous administration. These studies confirmed increased dystrophin expression, which correlated with functional and morphological improvements in DMD-affected muscles, including cardiac, respiratory, and skeletal muscles. Furthermore, the application of DEC therapy in a clinical study confirmed its long-term safety and efficacy in DMD patients. This review summarizes the development of chimeric cell technology tested in preclinical models and clinical studies, highlighting the potential of DEC therapy in muscle regeneration and repair, and introduces chimeric cell-based therapies as a promising, novel approach for muscle regeneration and the treatment of DMD and other neuromuscular disorders. [ABSTRACT FROM AUTHOR]

Chicken single‐chain fragment variable (IgY‐scFv) is a functional fragment and an emerging development in genetically engineered antibodies with a wide range of biomedical applications. However, scFvs have considerably shorter serum half‐life due to the absence of antibody Fc region compared with the full‐length antibody, and usually requires continuous intravenous administration for efficacy. A promising approach to overcome this limitation is to fuse scFv with immunoglobulin G (IgG) Fc region, for better recognition and mediation by the neonatal Fc receptor (FcRn) in the host. In this study, engineered mammalian ΔFc domains (CH2, CH3, and intact Fc region) were fused with anti‐canine parvovirus‐like particles avian IgY‐scFv to produce chimeric antibodies and expressed in the HEK293 cell expression system. The obtained scFv‐CH2, scFv‐CH3, and scFv‐Fc can bind with antigen specifically and dose‐dependently. Surface plasmon resonance investigation confirmed that scFv‐CH2, scFv‐CH3, and scFv‐Fc had different degrees of binding to FcRn, with scFv‐Fc showing the highest affinity. scFv‐Fc had a significantly longer half‐life in mice compared with the unfused scFv. The identified ΔFcs are promising for the development of engineered Fc‐based therapeutic antibodies and proteins with longer half‐lives. The avian IgY‐scFv‐mammalian IgG Fc region opens up new avenues for antibody engineering, and it is a novel strategy to enhance the rapid development and screening of functional antibodies in veterinary and human medicine. [ABSTRACT FROM AUTHOR]

Objective: To evaluate a PCR based method of Polyacrylamide gel electrophoresis of short tandem repeats and its quantification for detecting donor chimerism after haematopoietic stem cell transplantation in acute leukaemias. Methods: The descriptive study was conducted at Genetic Resource Centre (GRC) Lab Rawalpindi from Feb 2018 - Nov 2020. A total of twenty patients with acute leukaemias having undergone HSCT were selected and assessed for the analysis of chimerism status. DNA extraction from the whole blood was done by chelex method and short tandem repeats were amplified by using conventional STR-PCR assay. Electrophoresis was carried out and 6% Polyacrylamide gels were used for the resultant amplified DNA products and then followed by their densitometry. These patients had undergone HSCT from Pakistan Institute of Medical Science and Armed Forces Bone Marrow Transplant Centre. Results: The peaks in the PAGE densitometry represented the donor chimerism in all post transplant samples of the patients. Conclusion: Our study showed that densitometry of STR PCR PAGE is a useful and cheaper method for demonstration of donor chimerism in acute leukaemia patients having undergone HSCT. Hence this method can be a valuable option in the monitoring of chimerism status in these patients and therefore helps in preventing graft failure by fast and early treatment strategies for these patients. [ABSTRACT FROM AUTHOR]

Introduction: Chimerism is closely correlated with disease relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, chimerism rate is dynamic changes, and the sensitivity of different chimerism requires further research. Methods: To investigate the predictive value of distinct chimerism for relapse, we measured bone marrow (BM), peripheral blood (PB), and T-cell (isolated from BM) chimerism in 178 patients after allo-HSCT. Results: Receiver operating characteristic (ROC) curve showed that T-cell chimerism was more suitable to predict relapse after allo-HSCT compared with PB and BM chimerism. The cutoff value of T-cell chimerism for predicting relapse was 99.45%. Leukemia and myelodysplastic syndrome (MDS) relapse patients' T-cell chimerism was a gradual decline from 2 months to 9 months after allo-HSCT. Higher risk of relapse and death within 1 year after allo-HSCT. The Tcell chimerism rates in remission and relapse patients were 99.43% and 94.28% at 3 months after allo-HSCT (P = 0.009), 99.31% and 95.27% at 6 months after allo-HSCT (P = 0.013), and 99.26% and 91.32% at 9 months after allo-HSCT (P = 0.024), respectively. There was a significant difference (P = 0.036) for T-cell chimerism between early relapse (relapse within 9 months after allo-HSCT) and late relapse (relapse after 9 months after allo-HSCT) at 2 months after allo-HSCT. Every 1% increase in T-cell chimerism, the hazard ratio for disease relapse was 0.967 (95% CI: 0.948-0.987, P<0.001). Discussion: We recommend constant monitoring T-cell chimerism at 2, 3, 6, and 9 months after allo-HSCT to predict relapse. [ABSTRACT FROM AUTHOR]

Introduction: We investigated the potential role of HLA molecular mismatches (MM) in achieving stable chimerism, allowing for donor-specific tolerance in patients undergoing combined living donor kidney and hematopoietic stem cell transplantation (HSCT). Methods: All patients with available DNA samples (N=32) who participated in a phase 2 clinical trial (NCT00498160) where they received an HLA mismatched co-transplantation of living donor kidney and facilitating cell-enriched HSCT were included in this study. High-resolution HLA genotyping data were used to calculate HLA amino acid mismatches (AAMM), Eplet MM, three-dimensional electrostatic mismatch scores (EMS-3D), PIRCHE scores, HLA-DPB1 T-cell epitope group MM, HLA-B leader sequence MM, and KIR ligands MM between the donor and recipient in both directions. HLA MM were analyzed to test for correlation with the development of chimerism, graft vs. host disease (GvHD), de novo DSA, and graft rejection. Results: Follow-up time of this cohort was 6-13.5 years. Of the 32 patients, 26 developed high-level donor or mixed stable chimerism, followed by complete withdrawal of immunosuppression (IS) in 25 patients. The remaining six of the 32 patients had transient chimerism or no engraftment and were maintained on IS (On-IS). In host versus graft direction, a trend toward higher median number of HLA-DRB1 MM scores was seen in patients On-IS compared to patients with high-level donor/mixed chimerism, using any of the HLA MM modalities; however, initial statistical significance was observed only for the EMS-3D score (0.45 [IQR, 0.30-0.61] vs. 0.24 [IQR, 0.18-0.36], respectively; p=0.036), which was lost when applying the Bonferroni correction. No statistically significant differences between the two groups were observed for AAMM, EMS-3D, Eplet MM, and PIRCHE-II scores calculated in graft versus host direction. No associations were found between development of chimerism and GvHD and non-permissive HLA-DPB1 T-cell epitope group MM, HLA-B leader sequence, and KIR ligands MM. Conclusion: Our results suggest an association between HLA-DRB1 molecular mismatches and achieving stable chimerism, particularly when electrostatic quality of the mismatch is considered. The non-permissive HLA-DPB1 T-cell epitope group, HLA-B leader sequence, and KIR ligands MM do not predict chimerism and GvHD in this combined kidney/HSCT transplant patient cohort. Further work is needed to validate our findings. [ABSTRACT FROM AUTHOR]

Acute Myeloid Leukemia (AML) is the prototype of cancer genomics as it was the first published cancer genome. Large-scale next generation/massively parallel sequencing efforts have identified recurrent alterations that inform prognosis and have guided the development of targeted therapies. Despite changes in the frontline and relapsed standard of care stemming from the success of small molecules targeting FLT3, IDH1/2, and apoptotic pathways, allogeneic stem cell transplantation (alloHSCT) and the resulting graft-versus-leukemia (GVL) effect remains the only curative path for most patients. Advances in conditioning regimens, graft-vs-host disease prophylaxis, anti-infective agents, and supportive care have made this modality feasible, reducing transplant related mortality even among patients with advanced age or medical comorbidities. As such, relapse has emerged now as the most common cause of transplant failure. Relapse may occur after alloHSCT because residual disease clones persist after transplant, and develop immune escape from GVL, or such clones may proliferate rapidly early after alloHSCT, and outpace donor immune reconstitution, leading to relapse before any GVL effect could set in. To address this issue, genomically informed therapies are increasingly being incorporated into pre-transplant conditioning, or as post-transplant maintenance or pre-emptive therapy in the setting of mixed/falling donor chimerism or persistent detectable measurable residual disease (MRD). There is an urgent need to better understand how these emerging therapies modulate the two sides of the GVHD vs. GVL coin: 1) how molecularly or immunologically targeted therapies affect engraftment, GVHD potential, and function of the donor graft and 2) how these therapies affect the immunogenicity and sensitivity of leukemic clones to the GVL effect. By maximizing the synergistic action of molecularly targeted agents, immunomodulating agents, conventional chemotherapy, and the GVL effect, there is hope for improving outcomes for patients with this often-devastating disease. [ABSTRACT FROM AUTHOR]

After hematopoietic stem cell transplantation, chimerism assay is a useful approach to monitor the success of the transplant and to select the appropriate treatment strategy, such as donor leukocyte infusion or immunosuppressive drug dosage. Short tandem repeat PCR is the method that has been accepted as the gold standard for chimerism. However, it has not yet been sufficient to detect mixed chimerism in patients with minimal residual disease. Simultaneously, recent years have been marked by developing sensitive, high-throughput, and accurate molecular genetic assays. These novel methods have subsequently been adapted for the analysis of post-transplant chimerism. In this review, we discuss the technical features of both novel and conventional gold standard chimerism assays. We also discuss their advantages and disadvantages. [ABSTRACT FROM AUTHOR]

Individuals with 46,XX/XY chimerism can display a wide range of characteristics, varying from hermaphroditism to complete male or female, and can display sex chromosome chimerism in multiple tissues, including the gonads. The gonadal tissues of females contain both granulosa and germ cells. However, the specific sex chromosome composition of the granulosa and germ cells in 46,XX/XY chimeric female is currently unknown. Here, we reported a 30-year-old woman with secondary infertility who displayed a 46,XX/46,XY chimerism in the peripheral blood. FISH testing revealed varying degrees of XX/XY chimerism in multiple tissues of the female patient. Subsequently, the patient underwent preimplantation genetic testing (PGT) treatment, and 26 oocytes were retrieved. From the twenty-four biopsied mature oocytes, a total of 23 first polar bodies (PBs) and 10 second PBs were obtained. These PBs and two immature metaphase I (MI) oocytes only displayed X chromosome signals with no presence of the Y, suggesting that all oocytes in this chimeric female were of XX germ cell origin. On the other hand, granulosa cells obtained from individual follicles exhibited varied proportions of XX/XY cell types, and six follicles possessed 100% XX or XY granulosa cells. A total of 24 oocytes were successfully fertilized, and 12 developed into blastocysts, where 5 being XY and 5 were XX. Two blastocysts were transferred with one originating from an oocyte aspirated from a follicle containing 100% XY granulosa cells. This resulted in a twin pregnancy. Subsequent prenatal diagnosis confirmed normal male and female karyotypes. Ultimately, healthy boy–girl twins were delivered at full term. In summary, this 46,XX/XY chimerism with XX germ cells presented complete female, suggesting that germ cells may exert a significant influence on the sexual determination of an individual, which provide valuable insights into the intricate processes associated with sexual development and reproduction. [ABSTRACT FROM AUTHOR]