Pediatric Extracranial Germ Cell Tumors: Expression of microRNA.

BACKGROUND: Germ cell tumors (GCTs) may occur from the neonatal period to late adulthood, characterized by extensive clinical and pathologic heterogeneity. MicroRNAs are a family of small noncoding RNAs that regulate a wide array of biological processes including carcinogenesis. MicroRNAs may be used for many purposes in clinical diagnostics. Numerous studies have proven the diagnostic value of microRNA371-373 and microRNA302/367 expression in malignant GCT. The diagnostic value of microRNA375 is disputable, because while its value is confirmed by some research data, there are still others denying it. METHODS: The results of our own research on the relative expression of 10 microRNAs, including microRNA375, associated with GCT in the tumor tissues of 84 children and adolescents are presented. RESULTS: In our research, overexpression of microRNA 371-373, 302/367 detected in the group of malignant GCT subtypes. Statistically significant expression of microRNA375 have been defined not only in the group of malignant GCT subtypes, but also in the group of immature teratomas. Among malignant GCTs, high expression of microRNA375 is specific for yolk sac tumors. In the group of seminomas, embryonic carcinomas, and mature teratomas expression of microRNA375 was observed imperceptible, even so the results were statistically insignificant. CONCLUSION: Expression of microRNA 371-373, 302/367 is representative of malignant GCT subtypes. Statistically significant and high expression of microRNA375 attributable for yolk sac tumors and immature teratomas.

Genomic landscape and clonal evolution of acute myeloid leukemia with t(8;21): an international study on 331 patients.

Acute myeloid leukemia with t(8;21)(q22;q22) is characterized by considerable clinical and biological heterogeneity leading to relapse in up to 40% of patients. We sequenced coding regions or hotspot areas of 66 recurrently mutated genes in a cohort of 331 t(8;21) patients. At least 1 mutation, in addition to t(8;21), was identified in 95%, with a mean of 2.2 driver mutations per patient. Recurrent mutations occurred in genes related to RAS/RTK signaling (63.4%), epigenetic regulators (45%), cohesin complex (13.6%), MYC signaling (10.3%), and the spliceosome (7.9%). Our study identified mutations in previously unappreciated genes: GIGYF2, DHX15, and G2E3 Based on high mutant levels, pairwise precedence, and stability at relapse, epigenetic regulator mutations were likely to occur before signaling mutations. In 34% of RAS/RTKmutated patients, we identified multiple mutations in the same pathway. Deep sequencing (∼42 000×) of 126 mutations in 62 complete remission samples from 56 patients identified 16 persisting mutations in 12 patients, of whom 5 lacked RUNX1-RUNX1T1 in quantitative polymerase chain reaction analysis. KIT high mutations defined by a mutant level ≥25% were associated with inferior relapse-free survival (hazard ratio, 1.96; 95% confidence interval, 1.22-3.15; P = .005). Together with age and white blood cell counts, JAK2, FLT3-internal tandem duplicationhigh, and KIT high mutations were identified as significant prognostic factors for overall survival in multivariate analysis. Whole-exome sequencing was performed on 19 paired diagnosis, remission, and relapse trios. Exome-wide analysis showed an average of 16 mutations with signs of substantial clonal evolution. Based on the resemblance of diagnosis and relapse pairs, genetically stable (n = 13) and unstable (n = 6) subgroups could be identified.

Profiling of somatic mutations in acute myeloid leukemia with FLT3-ITD at diagnosis and relapse.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.

Multiple myeloma cells modify VEGF/IL-6 levels and osteogenic potential of bone marrow stromal cells via Notch/miR-223.

Bone marrow mesenchymal stromal cells (BMMSCs) represent a crucial component of multiple myeloma (MM) microenvironment supporting its progression and proliferation. Recently, microRNAs have become an important point of interest for research on micro-environmental interactions in MM with some evidence of tumor supportive roles in MM. In this study, we examined the role of miR-223 for MM support in BMMSCs of 56 patients with MM (MM-BMMSCs). miR-223 expression in MM-BMMSCs was reduced by the presence of MM cells in vitro in a cell-contact dependent manner compared to mono-cultured MM-BMMSCs. Co-cultivation of MM cells and MM-BMMSCs induced activation of notch amongst others via jagged-2/notch-2 leading to increased expression of Hes1, Hey2, or Hes5 in both cell types. Cultivation of MM-BMMSCs with increasing levels of recombinant jagged-2 reduced miR-223 and increased Hes1 levels in a concentration-dependent manner. Transient reduction of miR-223 levels increased VEGF and IL-6 expression and secretion by MM-BMMSCs. In addition, reduction of miR-223 degraded the osteogenic differentiation potential of MM-BMMSCs. Inhibition of notch signaling induced apoptosis in both MM cells and MM-BMMSCs. Furthermore, it increased miR-223 levels and reduced expression of VEGF and IL-6 by both cell types. These data provide first evidence that miR-223 participates in different MM supporting pathways in MM-BMMSCs inlcuding regulation of cytokine secretion and expression as well as osteogenic differentiation of MM-BMMSCs. More insights on the role of miR-223 in MM-BMMSCs and in cellular interactions between MM cells and MM-BMMSCs could provide starting points for a more efficient anti-myeloma treatment by targeting of notch signaling. © 2015 Wiley Periodicals, Inc.

Bone marrow T-cell infiltration during acute GVHD is associated with delayed B-cell recovery and function after HSCT.

B-cell immune dysfunction contributes to the risk of severe infections after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Delayed B-cell regeneration is found in patients with systemic graft-versus-host disease (GVHD) and is often accompanied by bone marrow (BM) suppression. Little is known about human BM GVHD. We analyzed the reconstitution kinetics of B-cell subsets in adult leukemic patients within 6 months after allo-HSCT. B-cell deficiency already existed before transplant and was aggravated after transplant. Onset of B-cell reconstitution characterized by transitional B-cell recovery occurred either early (months 2-3) or late (from month 6 on) and correlated highly positively with reverse transcription-polymerase chain reaction quantified numbers of κ-deleting recombination excision circles (KRECs). Delayed recovery was associated with systemic acute GVHD and full-intensity conditioning therapy. Histological analysis of BM trephines revealed increased T-cell infiltration in late recovering patients, which was associated with reduced numbers of osteoblasts. Functionally, late recovering patients displayed less pneumococcal polysaccharide-specific immunoglobin M-producing B cells on ex vivo B-cell activation than early recovering patients. Our results provide evidence for acute BM GVHD in allo-HSCT patients with infiltrating donor T cells and osteoblast destruction. This is associated with delayed B-cell reconstitution and impaired antibody response. Herein, KREC appears suitable to monitor BM B-cell output after transplant.

Multiple myeloma cells alter the senescence phenotype of bone marrow mesenchymal stromal cells under participation of the DLK1-DIO3 genomic region.

BACKGROUND: Alterations and senescence in bone marrow mesenchymal stromal cells of multiple myeloma patients (MM-BMMSCs) have become an important research focus. However the role of senescence in the pathophysiology of MM is not clear. METHODS: Correlation between senescence, cell cycle and microRNA expression of MM-BMMSCs (n = 89) was analyzed. Gene expression analysis, copy number analysis and methylation specific PCR were performed by Real-Time PCR. Furthermore, cyclin E1, cyclin D1, p16 and p21 genes were analyzed at the protein level using ELISA. Cell cycle and senescence were analyzed by FACS. MiRNA transfection was performed with miR-485-5p inhibitor and mimic followed by downstream analysis of senescence and cell cycle characteristics of MM-BMMSCs. Results were analyzed by Mann-Whitney U test, Wilcoxon signed-rank test and paired t-test depending on the experimental set up. RESULTS: MM-BMMSCs displayed increased senescence associated ß-galactosidase activity (SA-ßGalA), cell cycle arrest in S phase and overexpression of microRNAs. The overexpressed microRNAs miR-485-5p and miR-519d are located on DLK1-DIO3 and C19MC, respectively. Analyses revealed copy number accumulation and hypomethylation of both clusters. KMS12-PE myeloma cells decreased SA-ßGalA and influenced cell cycle characteristics of MM-BMMSCs. MiR-485-5p was significantly decreased in co-cultured MM-BMMSCs in connection with an increased methylation of DLK1-DIO3. Modification of miR-485-5p levels using microRNA mimic or inhibitor altered senescence and cell cycle characteristics of MM-BMMSCs. CONCLUSIONS: Here, we show for the first time that MM-BMMSCs have aberrant methylation and copy number of the DLK1-DIO3 and C19MC genomic region. Furthermore, this is the first study pointing that multiple myeloma cells in vitro reduce both the senescence phenotype of MM-BMMSCs and the expression of miR-223 and miR-485-5p. Thus, it is questionable whether senescence of MM-BMMSCs plays a pathological role in active multiple myeloma or is more important when cell interaction with myeloma cells is inhibited. Furthermore, we found that MiR-485-5p, which is located on the DLK1-DIO3 cluster, seems to participate in the regulation of senescence status and cell cycle characteristics of MM-BMMSCs. Thus, further exploration of the microRNAs of DLK1-DIO3 could provide further insights into the origin of the senescence state and its reversal in MM-BMMSCs.

Association between low uric acid levels and acute graft-versus-host disease.

Endogenous danger signals are increasingly recognized in the pathogenesis of graft-versus-host disease (GVHD). Uric acid (UA) is a known danger signal and is released from injured cells during conditioning for allogeneic hematopoietic stem cell transplantation (HSCT), but its role in GVHD is unclear. Here, we retrospectively analyze 228 consecutive patients with malignant diseases undergoing HSCT from 10/10-HLA-matched donors. Low UA levels at the time of HSCT (day 0) were significantly associated with acute GVHD grades II-IV in univariate (HR 0.836, p = 0.0072) and multivariate analyses (HR 0.815, p = 0.0047). There was no significant association between UA levels and overall survival, non-relapse mortality, and relapse. This is the first report demonstrating a negative association between UA levels and acute GVHD. Due to the easy assessment and established pharmaceutical modification of UA, our findings are potentially clinically relevant. Confirmation in independent cohorts and further investigations into underlying mechanisms, such as reduced antioxidative capacity in hypouricemia, are warranted.

Proteomic profiling reveals ACSS2 facilitating metabolic support in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by genomic aberrations in oncogenes, cytogenetic abnormalities, and an aberrant epigenetic landscape. Nearly 50% of AML cases will relapse with current treatment. A major source of therapy resistance is the interaction of mesenchymal stroma with leukemic cells resulting in therapeutic protection. We aimed to determine pro-survival/anti-apoptotic protein networks involved in the stroma protection of leukemic cells. Proteomic profiling of cultured primary AML (n = 14) with Hs5 stroma cell line uncovered an up-regulation of energy-favorable metabolic proteins. Next, we modulated stroma-induced drug resistance with an epigenetic drug library, resulting in reduced apoptosis with histone deacetylase inhibitor (HDACi) treatment versus other epigenetic modifying compounds. Quantitative phosphoproteomic probing of this effect further revealed a metabolic-enriched phosphoproteome including significant up-regulation of acetyl-coenzyme A synthetase (ACSS2, S30) in leukemia-stroma HDACi treated cocultures compared with untreated monocultures. Validating these findings, we show ACSS2 substrate, acetate, promotes leukemic proliferation, ACSS2 knockout in leukemia cells inhibits leukemic proliferation and ACSS2 knockout in the stroma impairs leukemic metabolic fitness. Finally, we identify ACSS1/ACSS2-high expression AML subtype correlating with poor overall survival. Collectively, this study uncovers the leukemia-stroma phosphoproteome emphasizing a role for ACSS2 in mediating AML growth and drug resistance.

Mesenchymal stromal cells of myelodysplastic syndrome and acute myeloid leukemia patients have distinct genetic abnormalities compared with leukemic blasts.

Mesenchymal stromal cells (MSCs) are an essential cell type of the hematopoietic microenvironment. Concerns have been raised about the possibility that MSCs undergo malignant transformation. Several studies, including one from our own group, have shown the presence of cytogenetic abnormalities in MSCs from leukemia patients. The aim of the present study was to compare genetic aberrations in hematopoietic cells (HCs) and MSCs of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients. Cytogenetic aberrations were detected in HCs from 25 of 51 AML patients (49%) and 16 of 43 MDS patients (37%). Mutations of the FLT3 and NPM1 genes were detected in leukemic blasts in 12 (23%) and 8 (16%) AML patients, respectively. Chromosomal aberrations in MSCs were detected in 15 of 94 MDS/AML patients (16%). No chromosomal abnormalities were identified in MSCs of 36 healthy subjects. We demonstrate herein that MSCs have distinct genetic abnormalities compared with leukemic blasts. We also analyzed the main characteristics of patients with MSCs carrying chromosomal aberrations. In view of these data, the genetic alterations in MSCs may constitute a particular mechanism of leukemogenesis.

Rabbit antithymocyte globulin (thymoglobulin) impairs the thymic output of both conventional and regulatory CD4+ T cells after allogeneic hematopoietic stem cell transplantation in adult patients.

Rabbit antithymocyte globulin-Genzyme™ is used to prevent graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Common disadvantages of treatment are infectious complications. The effects of rabbit antithymocyte globulin-Genzyme™ on thymic function have not been well-studied. Multicolor flow cytometry was used to analyze the kinetics of conventional and regulatory T cells in adult patients treated (n=12) or not treated (n=8) with rabbit antithymocyte globulin-Genzyme™ during the first 6 months after allogeneic hematopoietic stem cell transplantation. Patients treated with rabbit antithymocyte globulin-Genzyme™ had almost undetectable levels of recent thymic emigrants (CD45RA(+)CD31(+)) of both conventional and regulatory CD4T cells throughout the 6 months after allogeneic hematopoietic stem cell transplantation whereas CD4(+)CD45RA-memory T cells were less affected, but their levels were also significantly lower than in patients not treated with rabbit antithymocyte globulin-Genzyme™. In vitro, rabbit antithymocyte globulin-Genzyme™ induced apoptosis and cytolysis of human thymocytes, and its cytotoxic effects were greater than those of rabbit antithymocyte globulin-Fresenius™. Rabbit antithymocyte globulin-Genzyme™ in combination with a conditioning regimen strongly impairs thymic recovery of both conventional and regulatory CD4(+) T cells. The sustained depletion of conventional and regulatory CD4(+)T cells carries a high risk of both infections and graft-versus-host disease. Our data indicate that patients treated with rabbit antithymocyte globulin-Genzyme™ could benefit from thymus-protective therapies and that trials comparing this product with other rabbit antithymocyte globulin preparations or lymphocyte-depleting compounds would be informative.

AML with complex karyotype: extreme genomic complexity revealed by combined long-read sequencing and Hi-C technology.

Acute myeloid leukemia with complex karyotype (CK-AML) is associated with poor prognosis, which is only in part explained by underlying TP53 mutations. Especially in the presence of complex chromosomal rearrangements, such as chromothripsis, the outcome of CK-AML is dismal. However, this degree of complexity of genomic rearrangements contributes to the leukemogenic phenotype and treatment resistance of CK-AML remains largely unknown. Applying an integrative workflow for the detection of structural variants (SVs) based on Oxford Nanopore (ONT) genomic DNA long-read sequencing (gDNA-LRS) and high-throughput chromosome confirmation capture (Hi-C) in a well-defined cohort of CK-AML identified regions with an extreme density of SVs. These rearrangements consisted to a large degree of focal amplifications enriched in the proximity of mammalian-wide interspersed repeat elements, which often result in oncogenic fusion transcripts, such as USP7::MVD, or the deregulation of oncogenic driver genes as confirmed by RNA-seq and ONT direct complementary DNA sequencing. We termed this novel phenomenon chromocataclysm. Thus, our integrative SV detection workflow combing gDNA-LRS and Hi-C enables to unravel complex genomic rearrangements at a very high resolution in regions hard to analyze by conventional sequencing technology, thereby providing an important tool to identify novel important drivers underlying cancer with complex karyotypic changes.

Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation.

Infection with the human immunodeficiency virus type 1 (HIV-1) requires the presence of a CD4 receptor and a chemokine receptor, principally chemokine receptor 5 (CCR5). Homozygosity for a 32-bp deletion in the CCR5 allele provides resistance against HIV-1 acquisition. We transplanted stem cells from a donor who was homozygous for CCR5 delta32 in a patient with acute myeloid leukemia and HIV-1 infection. The patient remained without viral rebound 20 months after transplantation and discontinuation of antiretroviral therapy. This outcome demonstrates the critical role CCR5 plays in maintaining HIV-1 infection.

Relapse of NPM1-Mutated AML with Extramedullary Manifestation 17 Years after Allogeneic Hematopoietic Stem Cell Transplantation.

The majority of patients with acute myeloid leukemia (AML) with the NPM1 mutation achieve remission with intensive chemotherapy. However, many patients subsequently relapse, which occurs frequently within the first 2-3 years after therapy, while late relapse after more than 10 years is rare and can also represent secondary/therapy-associated AML without the NPM1 mutation. Here, we present a case of NPM1-mutated AML that developed medullary and extramedullary relapse 17 years after allogeneic stem cell transplantation, maintaining the NPM1 mutation and all other genetic alterations detected at first diagnosis. This exceptionally long latency between diagnosis and relapse of a genetically highly related leukemic clone implies the existence of therapy-resistant, persisting dormant leukemic stem cells in NPM1 mutant AML.

Intrathecal application of donor lymphocytes in leukemic meningeosis after allogeneic stem cell transplantation.

Extramedullary relapses after allogeneic stem cell transplantation, especially within the central nervous system (CNS), are not only difficult to treat but also associated with poor outcome. Although the graft-versus-leukemia (GvL) effect is nowadays accepted and well documented, it remains controversial whether one can make use of GvL effects in immunological-restricted areas ("sanctuary sites") like the central nervous system. Here, we present data of three hematological patients suffering from isolated CNS relapse of CML or AML after allogeneic stem cell transplantation. Patients received in addition to chemotherapy intrathecal infusions of donor lymphocytes by CD14 depletion of peripheral blood mononuclear cells from the correspondent allogeneic donor. Referring to an observation period of maximum 17 months no immediate or delayed side effects could be detected.

Polymorphisms in CXCR3 ligands predict early CXCL9 recovery and severe chronic GVHD.

Chronic graft-versus-host disease (cGVHD) is a major cause of mortality and morbidity after allogeneic stem cell transplantation (alloSCT). The individual risk of severe cGVHD remains difficult to predict and may involve CXCR3 ligands. This study investigated the role of single-nucleotide polymorphisms (SNPs) of CXCL4, CXCL9, CXCL10, and CXCL11, and their day +28 serum levels, in cGVHD pathogenesis. Eighteen CXCR3 and CXCL4, CXCL9-11 SNPs as well as peri-transplant CXCL9-11 serum levels were analyzed in 688 patients without (training cohort; n = 287) or with statin-based endothelial protection cohort (n = 401). Clinical outcomes were correlated to serum levels and SNP status. Significant polymorphisms were further analyzed by luciferase reporter assays. Findings were validated in an independent cohort (n = 202). A combined genetic risk comprising four CXCR3 ligand SNPs was significantly associated with increased risk of severe cGVHD in both training cohort (hazard ratio (HR) 2.48, 95% confidence interval (CI) 1.33-4.64, P = 0.004) and validation cohort (HR 2.95, 95% CI 1.56-5.58, P = 0.001). In reporter assays, significantly reduced suppressive effects of calcineurin inhibitors in constructs with variant alleles of rs884304 (P < 0.001) and rs884004 (P < 0.001) were observed. CXCL9 serum levels at day +28 after alloSCT correlated with both genetic risk and risk of severe cGVHD (HR 1.38, 95% CI 1.10-1.73, P = 0.006). This study identifies patients with high genetic risk to develop severe cGVHD.

Prognostic implications of mutations and expression of the Wilms tumor 1 (WT1) gene in adult acute T-lymphoblastic leukemia.

BACKGROUND: The role of the Wilms tumor 1 gene (WT1) in acute leukemias has been underscored by mutations found in acute myeloid leukemia identifying patients with inferior survival. Furthermore, aberrant expression of WT1 in acute myeloid leukemia was associated with an increased risk of relapse. No larger studies have performed a combined approach including WT1 mutation and expression analyses in acute T-lymphoblastic leukemia. DESIGN AND METHODS: We analyzed the WT1 mutations and the expression status in a total of 252 consecutive adult patients with newly diagnosed T-lymphoblastic leukemia, who were registered on the GMALL 06/99 and 07/03 protocols and had sufficient material available. The GMALL protocols included intensive chemotherapy as well as stem cell transplantation according to a risk-based model with indication for stem cell transplantation in first complete remission for early and mature T-lymphoblastic leukemia patients; patients with thymic T-lymphoblastic leukemia were allocated to a standard risk group and treated with intensive chemotherapy. RESULTS: Twenty of the 238 patients analyzed had WT1 mutations (WT1mut) in exon 7. WT1mut cases were characterized by immature features such as an early immunophenotype and higher WT1 expression. In thymic T-lymphoblastic leukemia, WT1mut patients had an inferior relapse-free survival compared to WT1 wild-type patients. T-lymphoblastic leukemia patients with aberrant WT1 expression (high or negative) showed a higher relapse rate and an inferior outcome compared to patients with intermediate WT1 expression. In the standard risk group of thymic T-lymphoblastic leukemia, aberrant WT1 expression was predictive for an inferior relapse-free survival as compared to patients with intermediate expression. In multivariate analysis, WT1 expression was of independent prognostic significance for relapse-free survival. CONCLUSIONS: WT1 mutations were associated with an inferior relapse-free survival in standard risk thymic T-lymphoblastic leukemia patients. Moreover, altered expression associated with inferior outcome also suggests a role of WT1 in T-lymphoblastic leukemia and the potential use of molecularly-based treatment stratification to improve outcome.

Long-Term Signs of T Cell and Myeloid Cell Activation After Intestinal Transplantation With Cellular Rejections Contributing to Further Increase of CD16+ Cell Subsets.

The intestine mediates a delicate balance between tolerogenic and inflammatory immune responses. The continuous pathogen encounter might also augment immune cell responses contributing to complications observed upon intestinal transplantation (ITx). We thus hypothesized that ITx patients show persistent signs of immune cell activation affecting both the adaptive and innate immune cell compartment. Information on the impact of intestinal grafts on immune cell composition, however, especially in the long-term is sparse. We here assessed activated and differentiated adaptive and innate immune subsets according to time, previous experience of cellular or antibody-mediated rejections or type of transplant after ITx applying multi-parametric flow cytometry, gene expression, serum cytokine and chemokine profiling. ITx patients showed an increase in CD16 expressing monocytes and myeloid dendritic cells (DCs) compared to healthy controls. This was even detectable in patients who were transplanted more than 10 years ago. Also, conventional CD4+ and CD8+ T cells showed persistent signs of activation counterbalanced by increased activated CCR4+ regulatory T cells. Patients with previous cellular rejections had even higher proportions of CD16+ monocytes and DCs, whereas transplanting higher donor mass with multi-visceral grafts was associated with increased T cell activation. The persistent inflammation and innate immune cell activation might contribute to unsatisfactory results after ITx.

Role of Donor Clonal Hematopoiesis in Allogeneic Hematopoietic Stem-Cell Transplantation.

PURPOSE: Clonal hematopoiesis of indeterminate potential (CHIP) occurs in the blood of approximately 20% of older persons. CHIP is linked to an increased risk of hematologic malignancies and of all-cause mortality; thus, the eligibility of stem-cell donors with CHIP is questionable. We comprehensively investigated how donor CHIP affects outcome of allogeneic hematopoietic stem-cell transplantation (HSCT). METHODS: We collected blood samples from 500 healthy, related HSCT donors (age ≥ 55 years) at the time of stem-cell donation for targeted sequencing with a 66-gene panel. The effect of donor CHIP was assessed on recipient outcomes, including graft-versus-host disease (GVHD), cumulative incidence of relapse/progression (CIR/P), and overall survival (OS). RESULTS: A total of 92 clonal mutations with a median variant allele frequency of 5.9% were identified in 80 (16.0%) of 500 donors. CHIP prevalence was higher in donors related to patients with myeloid compared with lymphoid malignancies (19.2% v 6.3%; P ≤ .001). In recipients allografted with donor CHIP, we found a high cumulative incidence of chronic GVHD (cGVHD; hazard ratio [HR], 1.73; 95% CI, 1.21 to 2.49; P = .003) and lower CIR/P (univariate: HR, 0.62; 95% CI, 0.40 to 0.97; P = .027; multivariate: HR, 0.63; 95% CI, 0.41 to 0.98; P = .042) but no effect on nonrelapse mortality. Serial quantification of 25 mutations showed engraftment of 24 of 25 clones and disproportionate expansion in half of them. Donor-cell leukemia was observed in two recipients. OS was not affected by donor CHIP status (HR, 0.88; 95% CI, 0.65 to 1.321; P = .434). CONCLUSION: Allogeneic HSCT from donors with CHIP seems safe and results in similar survival in the setting of older, related donors. Future studies in younger and unrelated donors are warranted to extend these results. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that donor CHIP might foster cGVHD development and reduce relapse/progression risk.

Chromosomal aberrations in bone marrow mesenchymal stroma cells from patients with myelodysplastic syndrome and acute myeloblastic leukemia.

OBJECTIVE: Bone marrow mesenchymal stroma cells (BMSC) are key components of the hematopoietic microenvironment. The question of whether BMSC from patients with hematological disorders have cytogenetic abnormalities is discussed controversially, some studies indicating that they are cytogenetically normal and others providing evidence of their aberrations. PATIENTS AND METHODS: We performed standard and molecular cytogenetic analyses of both hematopoietic cells and BMSC from 31 patients with myelodysplastic syndrome (MDS, n = 18) and acute myeloid leukemia (AML, n = 13) and 7 healthy individuals. Mononuclear cells were isolated from fresh bone marrow aspirates at the time of initial diagnosis for cytogenetic analysis of hematopoietic cells (HC) and selection of BMSC. RESULTS: Clonal cytogenetic aberrations were observed in HC from 8 (44%) MDS and 8 (61%) AML patients. Cytogenetic analyses of BMSC were successfully performed in 27 of the 31 cases. Structural chromosomal aberrations, including t(1;7), t(4;7), t(7;9), t(7;10), t(7;19), t(15;17), and others, were detectable in BMSC from 7 of 16 (44%) MDS and 6 of 11 (54%) AML patients. The breakpoints of chromosomes in BMSC were typical for leukemia aberrations. Two patients showed clonal chromosomal markers. CONCLUSIONS: BMSC from MDS and AML patients show chromosomal abnormalities. Although the majority of cytogenetic aberrations in BMSC were not clonal and differed from chromosomal markers in HC from the same individual, detection of typical chromosomal changes in BMSC suggests enhanced genetic susceptibility of these cells in MDS/AML. This may indicate potential involvement of BMSC in the pathophysiology of MDS/AML.

Single Nucleotide Polymorphisms in CD40L Predict Endothelial Complications and Mortality After Allogeneic Stem-Cell Transplantation.

Purpose Endothelial vulnerability is a potential risk factor for complications after allogeneic stem-cell transplantation (alloSCT). The CD40/CD40 ligand (CD40L) axis contributes to inflammatory diseases and is upregulated in endothelial cells upon activation, suggesting a role in alloSCT biology. Here, we studied single nucleotide polymorphisms (SNPs) in the CD40L gene in recipients of alloSCT. Patients and Methods Three CD40L SNPs (rs3092920, rs3092952, rs3092936) were analyzed for association with transplant-associated thrombotic microangiopathy, overall nonrelapse mortality (NRM), and NRM after acute graft-versus-host disease in 294 recipients of alloSCT without statin-based endothelial prophylaxis (SEP). The significant genotype was then put into perspective with established thrombomodulin ( THBD) gene polymorphisms. Findings were validated in an independent cohort without SEP and in an additional 344 patients who received SEP. Results The rs3092936 CC/CT genotype was associated with an increased risk of transplant-associated thrombotic microangiopathy ( P = .001), overall NRM ( P = .03), and NRM after acute graft-versus-host disease ( P = .01). The rs3092936 CC/CT genotype was largely mutually exclusive of high-risk THBD SNPs. Both CD40L and THBD SNPs predicted adverse overall survival (OS) and overall NRM to a similar extent in training cohort (OS, P = .04; NRM, P < .001) and validation cohort (OS, P = .01; NRM, P = .001) without SEP. In contrast, SEP completely abolished the influence of the high-risk CD40L and THBD SNPs ( P = .40). Conclusion An increased risk of endothelial complications can be predicted before alloSCT by genetic markers in the recipient's genome. The normalization of mortality risks in patients treated with SEP suggests a way of overcoming the negative effect of high-risk genotypes and warrants further clinical validation.