Blood

Allogeneic hematopoietic cell transplantation (allo-HCT) hinges on a delicate trade-off between graft-versus-tumor control and graft-versus-host disease (GvHD), mediated by donor T-cell recognition of antigens presented by recipient human leukocyte antigen (HLA) molecules. We hypothesized that, beyond allele-level matching, sequence divergence at peptide-binding grooves across donor and recipient HLA loci shapes these responses. To this end, we evaluated the effect of HLA evolutionary divergence (HED), a metric quantifying amino acid variability at HLA peptide-binding sites, on selected hematological malignancies in 4,695 patients undergoing allo-HCT from a 9/10 mismatched unrelated donor (MMUD), reported to the EBMT database. We examined (i) locus-specific recipient HED (HED-R) and (ii) "HED-mismatch" (HED-MM), capturing immunopeptidome divergence at the mismatched locus. While dichotomous mismatch status explained differences in survival and acute GvHD risk (with overall greater detriment for class I loci), HED metrics uncovered substantial within-mismatch heterogeneity. In DRB1 mismatched subgroup, HED-MM at this locus, independently predicted inferior relapse-free survival (RFS) with an attenuating time-dependent association, further modulated by cross-locus HED-R. In this subgroup, higher HED-R at HLA-A and HLA-C associated with increased risks of acute GvHD and non-relapse mortality, respectively. Among HLA-B-mismatched pairs, higher DRB1 HED-R associated with worse overall survival (OS) and RFS and higher relapse risk. In the HLA-A-mismatched subgroup, higher HED-R at HLA-A increased chronic GvHD risk. Collectively, HED-derived metrics complement conventional mismatch classification by capturing qualitative differences in donor-recipient immunopeptidome interactions and reveal a complex, non-linear interplay among alleles across mismatch subgroups that modulates the clinical impact of mismatching. KeypointsO_LIIn mismatched unrelated HCT, baseline risk varies across mismatch constellations, with class I mismatches more detrimental than class II. C_LIO_LIHED complements conventional HLA mismatch classification by capturing qualitative donor-recipient immunopeptidome interactions. C_LI

ObjectiveAutoimmune diseases (ADs) markedly elevate venous thromboembolism (VTE) risk, yet the shared genetic architecture and tissue-specific regulatory mechanisms of this "Autoimmune-Thrombotic Axis" remain poorly defined. We aimed to characterize the genomic landscape of immunothrombosis to identify causal links and therapeutic targets. Approach and ResultsWe integrated large-scale GWAS data for VTE and 16 ADs using a multi-omics framework, including pleiotropy scanning, local genetic correlation, and summary-based Mendelian randomization (SMR). We identified 21 Immunothrombotic Shared Loci (ISLs) and 274 pleiotropic genes enriched in complement and coagulation cascades. Mendelian randomization (MR) analysis revealed a robust causal effect of genetically predicted systemic lupus erythematosus (SLE) on VTE risk (OR = 1.018, 95% CI: 1.008-1.029, P = 0.0003). Mechanistically, IL6R and PLCL1 emerged as central mediators with distinct tissue-specific regulatory partitioning. Colocalization confirmed that shared genetic susceptibility is primarily driven by expression in arterial tissues (aorta and coronary) rather than exclusively in immune cells. Furthermore, the lead SNP rs4129267 was identified as a potential predictor for VTE in rheumatoid arthritis patients, and drug prioritization nominated TNF inhibitors as promising candidates for mitigating thrombotic burden. ConclusionThis study establishes the first genomic atlas of the autoimmune-thrombotic axis, demonstrating that vasculature-specific gene regulation drives immunothrombosis. These findings provide a biological basis for VTE risk stratification and suggest that genotype-guided therapy may optimize vascular outcomes in AD patients.

ObjectivePost-thrombotic syndrome (PTS), a common complication of deep vein thrombosis, lacks objective diagnostic biomarkers and its molecular mechanisms remain poorly understood. This study aimed to identify plasma biomarkers and clarify pathways using integrated multi-omics and machine learning. MethodsProteomic and metabolomic profiling of 75 PTS patients and 75 controls was performed. Differential expression analysis, pathway enrichment, and protein-metabolite network analysis were conducted. A multi-algorithm machine learning with 8 feature selection methods prioritized biomarkers. Validations and 14 models were assessed. Results1,104 proteins and 1,891 metabolites were differentially expressed. Citrate cycle and unsaturated fatty acid biosynthesis were enriched. Three proteins, namely DIP2B, KNG1, and SUCLG2, were consistently selected as core biomarkers. All of these proteins were significantly downregulated in PTS and externally validated. A random forest model utilizing these proteins achieved an accuracy of 97.7% in independent testing, with SUCLG2 being the most influential predictor. ConclusionThis study identifies a novel three - protein biomarker panel for the diagnosis of PTS and reveals an immunometabolic axis in the pathogenesis of PTS, which links inflammatory regulation with mitochondrial energy metabolism. These findings provide valuable insights into the development of diagnostic tools and targeted therapeutic approaches.

A role for substance P in promoting neurogenic inflammation and pain has been described in sickle cell disease (SCD). However its origin and contribution to SCD pathophysiology remain unclear. We measured substance P level in plasma from 225 patients with SCD and observed the highest concentrations during acute chest syndrome (ACS). Therefore, we tested the hypothesis that substance P may induce ACS. In transgenic sickle mice, unlike control mice, intravenous injection of substance P caused lethal crises with dose-dependent acute lung injuries. Activation of Fc{varepsilon}R1 with MAR-1 had similar effects, suggesting a role for mast cell or basophil activation and degranulation. Pretreatment of sickle mice with cromolyn, a stabilizer of mast cells and basophils, prevented lethal crisis and lung injuries induced by substance P injection. In SCD patients, blood cellular histamine levels and increased histidine decarboxylase activity were consistent with an involvement of circulating basophils. Flow cytometry analysis revealed higher basophil counts with increased activation and degranulation markers in patients compared with healthy controls. During vaso-occlusive crisis, absolute basophil counts tended to decrease, suggesting their recruitment outside the vascular compartment. The same results were observed in sickle mice after hypoxia-reoxygenation, intravenous hemin injection or substance P injection. Immunohistochemistry revealed the presence of mast cells and basophils in the lungs of sickle mice, but not in control mice, with further basophil recruitment and degranulation after intravenous substance P injection. In SCD patients, we observed extremely high levels of substance P in the sputum collected during ACS, consistently with mast cell and basophil degranulation in the lungs. In vitro, substance P was shown to be a potent chemoattractant for basophils via NK1R. Gene expression analysis on sorted circulating basophils from SCD patients revealed an increased expression of several chemokine receptors, including CCR3 and FPR1, which was confirmed by spectral flow cytometry and could contribute to the recruitment of basophils in the lungs. The two substance P receptors, NK1R and MRGPRX2, were also overexpressed, promoting the vicious cycle of substance P release and pain in SCD patients. Our results reveal a novel mechanism that contributes to the understanding of ACS pathogenesis and highlights the potential role of mast cells and basophils in SCD pathophysiology.

ObjectivesThe therapeutic efficacy of rituximab has reduced the discriminatory power of the International Prognostic Index (IPI) in diffuse large B-cell lymphoma (DLBCL), particularly within intermediate-risk categories. To address this "risk dilution," we aimed to develop and internally validate the AB-IPI (Albumin-BCL2 Refined Prognostic Index) using a hypothesis-driven approach that integrates tumor burden, host fitness, and tumor biology. MethodsThis multi-center retrospective study analyzed 289 patients with de novo DLBCL treated uniformly with R-CHOP immunochemotherapy. We combined the standard IPI with serum albumin < 3.6 g/dL (representing host fitness/rituximab pharmacokinetics) and BCL2 protein expression > 50% (representing tumor biology). The model was validated internally using bootstrapping with 1,000 resamples in accordance with TRIPOD Type 1b guidelines. This study adhered to the TRIPOD (Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis) statement for model development and internal validation (Type 1b). ResultsDuring the observation period, 115 death events were recorded. Multivariate Cox regression identified albumin < 3.6 g/dL (Hazard Ratio 2.62), IPI score > 2 (HR 2.13), and BCL2 > 50% (HR 1.72) as independent prognostic factors. The model maintained a robust Events Per Variable (EPV) ratio of 38.3. The AB-IPI stratified patients into four distinct risk groups with 5-year overall survival rates of 88.0% (Low), 76.1% (Intermediate-1), 45.0% (Intermediate-2), and 29.0% (High). The calibration plot demonstrated excellent agreement between predicted and observed probabilities, with a calibration slope of 0.98, indicating minimal optimism and robust risk estimation. Decision Curve Analysis (DCA) demonstrated that the AB-IPI provided a superior Net Benefit across a wide range of clinically relevant threshold probabilities. ConclusionsThe AB-IPI demonstrates superior clinical utility and calibration compared to the standard IPI. By identifying patients with compounded biological risks who are unlikely to be cured by R-CHOP alone, this score offers a practical framework for optimizing therapeutic strategies, such as the allocation of polatuzumab vedotin.

Iron deficiency (ID) is the most common nutritional deficiency worldwide, often caused by insufficient dietary intakes. Oral supplementation is one of the means to improve iron status. This study evaluated the efficacy and safety of two low-dose iron supplements - >Your< Iron Forte Capsules (YIFC) and Ferrous Sulfate Capsules (FSC) - in individuals with dietary ID. One hundred and one participants (mean age 30.6 years; 98% women) with low iron stores (mean serum ferritin 16.1 {micro}g/L) were randomized to receive either YIFC or FSC once daily for 12 weeks. Changes in blood indices and iron-related parameters were assessed at four and 12 weeks of intervention relative to baseline. The primary outcome was the change in hemoglobin (Hb) after 12 weeks. Eighty-seven participants completed the study. Both supplements significantly increased Hb at 12 weeks (YIFC: mean 6.52 g/L, p<0.001; FSC: mean 5.71 g/L, p<0.001). Product-related adverse events (AEs) were few (17% of all AEs) and of mild to moderate intensity only. One participant receiving FSC withdrew due to a probable product-related AE. The frequencies of product-related AEs were similar between study arms, however, statistically significantly more AEs judged to be definitely related to the product occurred in in the FSC arm. While product-related AEs were confined to the gastrointestinal tract in the YIFC arm, they affected multiple organ systems in the FSC arm. Supplementation with either YIFC or FSC proved as an effective, well-tolerated, and safe strategy for improving iron status in non-anemic dietary iron deficiency. In terms of the AE profile, supplementation with YIFC may offer advantages over supplementation with FSC.

BackgroundIn lung transplantation, de novo immunodominant donor-specific anti-HLA antibodies recognizing HLA-DQ antigens (dn-iDSA-DQ) are predominant and can induce chronic lung allograft dysfunction (CLAD). We previously developed a method to measure the active concentration of dn-iDSA-DQ. We aimed to determine whether this new quantitative biomarker is associated with transplantation outcomes. MethodsThis retrospective multicentre cohort study included 90 lung transplant recipients (LTRs) developing dn-iDSA-DQ, evidenced through single antigen flow beads (SAFB) follow-up. We measured the active concentration of dn-iDSA-DQ at the time of their first detection (T0) for all LTRs, and within the 2 years after DSA detection, whenever possible. SAFB dn-iDSA-DQ characteristics and clinical data were retrieved up to 5 years after DSA detection. ResultsWe tested 184 sera with SPR (n=90 at T0, n=94 within the 2 years after DSA detection), among which 63 (34.4%) had a quantifiable concentration of the dn-iDSA-DQ ([&ge;]0.3 nM). The median SAFB mean fluorescence intensity (MFI) of the dn-iDSA-DQ with a concentration [&ge;]0.3 nM was higher (p<0.0001), yet the correlation between SAFB MFI and active concentration was low (r=0.758, p<0.0001). In multivariate analysis, a concentration of the dn-iDSA-DQ [&ge;]0.3 nM at T0 was independently associated with a lower 2-year CLAD-free survival (HR 2.06, p=0.02). A concentration of the dn-iDSA-DQ [&ge;]0.3 nM within the 2 years from DSA detection was associated with a lower graft survival in univariate analysis. ConclusionsActive concentration of dn-iDSA-DQ appears as a valuable biomarker to identify pathogenic DSA at their first detection because of its association with CLAD.

TRAF1 is a pro-survival signaling adaptor that contributes to NF-{kappa}B activation downstream of a subset of TNFR superfamily members. TRAF1 is overexpressed in many cancers of mature B cells, including chronic lymphocytic leukemia (CLL). Previous studies have established that TRAF1 S146 is a target of phosphorylation by the kinase PKN1 and that PKN1 is required to prevent cellular inhibitor of apoptosis protein (cIAP)-dependent degradation of TRAF1 in the CD40 signaling complex. The kinase inhibitor OSST167 inhibits PKN1 in the nm range and its addition to primary CLL cells was shown to induce dose-dependent loss of TRAF1 and concomitant increases in activated caspase 3 and cell death. These studies identified PKN1 as a target for therapy of CLL. To identify more potent and specific PKN1 inhibitors for therapy of B cell cancers it is important to measure a direct target of PKN1, such as phospho-TRAF1. To this end, here we use overexpression of an S146A mutant of human TRAF1 in 293 cells to validate a recently generated phospho-TRAF1 S146-specific antibody and to confirm that this phosphorylation is lost upon treatment with OTSSP167. Using Cas/Crispr knockout in RAJI cells we also show that both PKN1 and the closely related family member PKN2 can phosphorylate TRAF1 S146. We further show that TRAF1 S146 is constitutively phosphorylated in primary human CLL cells, including those with p53 mutations and that this phosphorylation is sensitive to inhibition with OTSSP167. These findings provide support the development of more potent PKN1/2 inhibitors for CLL.

BackgroundIron metabolism disorder is highly prevalent before and after heart transplantation (HTx). The impact of pretransplant and posttransplant iron disorder on posttransplant outcomes is unclear. ObjectivePretransplant serum levels of key regulator proteins of iron metabolism (hepcidin, interleukin-6, erythroferrone) were tested for prediction of the composite outcome 1-year posttransplant all-cause mortality (ACM) or [&ge;]moderate acute cellular rejection (ACR). Furthermore, serum levels of these proteins were measured at 1-year posttransplant to explore their posttransplant course and association with ACR. ResultsIn a multicenter cohort including 276 consecutive HTx recipients, patients with or without outcome (n=118/158, respectively) did not differ for pretransplant demographics, mismatch of donor/recipient sex, mismatch of HLA epitopes, and hepcidin or interleukin-6 levels. However, pretransplant erythroferrone levels were higher (1.40 vs. 1.19 ng/mL; p=0.013) and hemoglobin levels were lower (124.5 vs. 127 g/L; p=0.004) among patients with the composite outcome. Pretransplant erythroferrone levels >2.25 ng/ml (4th-quartile) were significantly associated with the composite outcome in multivariable analysis (OR 2.17; 95% CI 1.19-3.94, p=0.011; reference: 1st-3rd quartiles). In adjusted predicted proportions analysis, the incidence of the composite outcome was higher in 4th-quartile patients when compared to 1-3rd -quartiles patients (58.0 vs. 37.7%; p=0.003). At 1-year posttransplant, 80.4% of patients with pretransplant erythroferrone levels >2.25 ng/ml remained high; 88.4% of patients with pretransplant erythroferrone levels [&le;]2.25 ng/ml had high levels posttransplant. In 1-year survivors with high erythroferrone levels and [&ge;]moderate ACR during the first postoperative year, the ratio of the opponent regulators of hepcidin gene expression, erythroferrone to interleukin-6, was higher when compared to those without ACR (1.18 vs. 0.41; p=0.016). Hepcidin levels were not different between these two subgroups indicating disproportionate ERFE increase. ConclusionHigh pretransplant erythroferrone levels predict the composite posttransplant outcome 1-year ACM and ACR. Disproportionately high posttransplant erythroferrone levels are related with [&ge;]moderate acute cellular rejection.

BackgroundAnthracyclines are central to childhood cancer therapy but predispose patients to cardiotoxicity leading to long-term cardiovascular risk. Endothelial injury and impaired repair contribute to this, yet pediatric data remain limited. ObjectiveTo longitudinally assess endothelial injury and repair in childhood cancer patients treated with anthracyclines by quantifying circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs). MethodsIn a single-centre retrospective cohort, children (<18 years) diagnosed with leukemia (n=35) or lymphoma (n=13) were studied at four timepoints: pre-treatment ("Pre"), [~]1-month- ("End"), 3 months- (3M), and 1 year- (1Y) post-treatment. Peripheral blood mononuclear cells were analyzed by flow cytometry to quantify CECs and EPCs, and EPC fate was assessed by p16 (senescence) and Annexin V (apoptosis). Cardiac injury biomarkers and left ventricular function were assessed at each timepoint. ResultsLongitudinal trends of CEC and EPC counts were similar between leukemia and lymphoma participants. CECs were highest at pre-treatment and declined significantly thereafter, though they remained marginally elevated during remission compared with healthy controls, indicating that endothelial damage had largely subsided following treatment. EPCs were also highest at pre-treatment and decreased to levels below healthy controls during remission, suggesting impaired baseline endothelial maintenance and repair. Furthermore, EPCs were predominantly senescent up to 1-year post-treatment. ConclusionsEndothelial injury resolves by treatment completion, but repair remains impaired during remission with EPC pools dominated by senescent cells. This suggests defective endothelial regeneration, rather than persistent injury, drives long-term cardiovascular complications and underscores the need to restore EPC viability and function in childhood cancer survivors.

PurposePrognosis in metastatic non-seminomatous germ cell tumors (mNSGCT) is currently guided by the IGCCCG classification, which incorporates tumor markers, organs involved with metastatic disease, and primary site but not histologic subtype. We aimed to evaluate whether specific histological components provide additional prognostic information in a large international mNSGCT cohort. Patient and MethodsWe analyzed clinical, pathologic, and outcome data from 662 patients with mNSGCT across multiple international centers. Cox regression and multivariable stepwise models were used to evaluate the impact of age, tumor histology, serum markers, primary site of disease, chemotherapy, IGCCCG, and post-chemotherapy surgery on overall survival. Analyses were performed using both complete-case and imputed datasets to account for missing values. ResultsThe presence of any percentage of embryonal carcinoma (EC) was independently associated with improved overall survival HR 0.603 (95% CI: 0.37-0.98, p=0.040), whereas yolk sac tumor (YST) predicted worse prognosis in complete-case analysis HR 2.27 (95% CI: 1.43 - 3.61 p = 0.001). Choriocarcinoma was also associated with a HR 1.58 (95% CI: 1.08 - 2.32 p= 0.019) adverse outcomes. IGCCCG risk classification remained a strong predictor of mortality HR up to 8.9 for Poor vs Good risk, (95% CI: 4.63 - 17.09 p < 0.001), but histologic components added significant independent prognostic value. Post-chemotherapy retroperitoneal lymph node dissection (RPLND) conferred a substantial survival benefit HR 0.44 (95% CI: 0.258 - 0.754 p=0.003). Interestingly, teratoma was not associated with mortality but was linked to younger age, testicular primaries, and higher likelihood of residual disease requiring surgery. ConclusionsHistological composition, particularly the presence of EC or YST, has a significant and independent impact on survival in mNSGCT, beyond established risk classifications. Integration of histological subtypes may enhance prognostic accuracy and guide individualized treatment strategies in advanced germ cell tumors.

BackgroundThe hyperpolymorphic nature and structural complexity of the human leukocyte antigen (HLA) genomic region present challenges for accurate and scalable typing across diverse sample types. While wholegenome sequencing (WGS) offers the opportunity to infer HLA genotypes without targeted enrichment, systematic benchmarks across sequencing platforms, biospecimens and coverage levels remain limited. ResultsWe assembled a multi-platform resource of WGS datasets derived from short-read (Illumina, MGI) and long-read (Oxford Nanopore Technologies R9 and R10) sequencing, spanning 29 biospecimens including cell lines, blood, buccal swab and saliva. We evaluated the performance of the HLA caller HLA*LA across 13 HLA genes, using a clinically validated assay as reference. WGSbased HLA genotyping achieved [~]95% accuracy across sequencing platforms, with Class I loci exhibiting higher accuracy than Class II. Crossplatform concordance was high, and performance remained consistent across Illumina, MGI and Oxford Nanopore chemistries. Analysis of blood, buccal swab and saliva samples showed that blood and buccal swabs supported accurate HLA inference, whereas saliva yielded reduced concordance. Downsampling experiments demonstrated that 15x coverage was sufficient to retain >95% accuracy at twofield resolution, with lower depths supporting lower-resolution typing. ConclusionsOur results demonstrate that WGS provides a robust, platformagnostic framework for accurate HLA genotyping across sample types and coverage levels. These benchmarks establish practical conditions for reliable HLA inference and underscore the utility of WGS for populationscale HLA analyses and future clinical applications.

Background & AimsPerihilar cholangiocarcinoma is an aggressive malignancy with clinical heterogeneity and poor long-term outcomes after resection. Current prognostic assessment relies mainly on anatomical staging and pathological features, which incompletely capture the entire postoperative risk. We aimed to determine whether integrative analysis of clinical, surgical, pathological and tumor genomic data could improve time-resolved, individualized recurrence-risk prediction after curative-intent resection. MethodsWe performed a multicenter retrospective study including patients undergoing curative-intent resection for perihilar cholangiocarcinoma in ten Spanish hospitals (2003-2023). Overall and disease-free survival were analyzed using Cox models. Outcome-agnostic clinical phenotypes were derived by unsupervised clustering of clinical and surgical features. Targeted tumor sequencing of cancer-associated hotspot regions and selected genes was performed. Prognostic models integrating clinical and genomic data were trained and evaluated in independent training/test sets using penalized and latent-component Cox frameworks, with time dependent discrimination. ResultsThe final cohort comprised 142 patients, with a median follow-up of 26.4 months. Recurrence occurred in 61.3% of patients, and 53.5% died during follow-up. Classical pathological factors were strongly associated with survival and recurrence. Unsupervised outcome-agnostic clustering identified three reproducible clinical phenotypes with markedly different recurrence patterns and survival, only partially explained by anatomical staging. Integrative clinical-genomic modelling further improved recurrence-risk prediction, achieving high discrimination in independent validation (time-dependent AUC [~]0.8). Moreover, the integrative model assigned higher risk over time to patients who relapsed. Patients combining unfavorable clinical phenotype with high genomic-derived risk exhibited a high probability of early recurrence. ConclusionsIntegrated clinical phenotyping and targeted genomic profiling substantially refine recurrence-risk stratification after resection of perihilar cholangiocarcinoma beyond anatomical staging alone. This provides a pragmatic framework for risk-adapted postoperative surveillance and therapeutic decision-making. Impact and ImplicationsThis study provides a data-driven framework integrating clinical, surgical and targeted genomic information to refine prognostic stratification after resection of perihilar cholangiocarcinoma, addressing the limitations of anatomy-based staging in capturing biological heterogeneity. The results are particularly relevant for clinicians managing postoperative surveillance and adjuvant strategies, as they identify patient subgroups with markedly different risks of early recurrence despite similar conventional staging. In practical terms, the combination of unsupervised clinical phenotyping and a targeted, biologically informed genomic panel could support risk-adapted follow-up intensity, selection for adjuvant or experimental therapies, and enrolment into clinical trials. While prospective validation is required before routine implementation, this approach offers a feasible and interpretable pathway toward precision postoperative management in a highly aggressive malignancy.

Allogeneic hematopoietic cell transplantation is the only curative option for many patients with acute myeloid leukemia (AML). In the current study, we designed and implemented a personalized assay, called v96, incorporating up to 96 mutations in 30 AML patients undergoing transplantation. The assay was performed on DNA derived in cells from the bone marrow as well as in cell-free plasma. All 30 (100%) of patients harbored molecular evidence of residual leukemia during remission that was detectable by the v96 assay, while only 6 (20%) had evidence of disease as assessed by conventional clinical assays. Furthermore, cell-free DNA from plasma proved to be more sensitive than DNA from cells of the bone marrow for identifying residual leukemia. The median number of mutants was 352-fold higher in plasma taken prior to transplantation for patients who relapsed compared to those who did not relapse. At two months post-transplantation, 27 of the 30 patients still harbored detectable leukemia as assessed by the v96 assay. Twenty-two of these patients had a subsequent decrease in leukemic burden assessed by the v96 assay, usually only after immunosuppression was discontinued and supporting a graft-versus-leukemia effect. These results document the feasibility of using a relatively large panel of carefully chosen mutations and a highly specific assay as non-invasive markers of therapeutic response in AML patients, minimizing the need for multiple bone marrow biopsies. STATEMENT OF SIGNIFICANCEWe report a blood test that tracks up to 96 patient-specific mutations and applied it to patients with AML who had undergone bone marrow transplantation. Using this test to evaluate cell-free plasma DNA, we found evidence of residual leukemia cells both during remission (prior to transplantation) in all patients, and two months following transplantation in 90% of patients. This test can mitigate the need for invasive bone marrow biopsies to follow patients with leukemia. Moreover, the test appears to be more accurate than standard assays for detecting residual leukemia, and has the potential to guide the timing of transplantation and subsequent therapeutic measures, thereby laying the foundation for future prospective studies.

T-cell lymphomas are often histologically indistinguishable from benign T-cell infiltrates. Clonality testing is frequently required for diagnosis. It lacks the spatial context and is slow and expensive, relying on complex, multiplexed PCR reactions, interpreted by experienced scientists or pathologists. We previously published details of a pair of highly specific monoclonal antibodies against the two alternatively used, but very similar, T-cell receptor {beta} constant regions, TCR{beta}1 and TCR{beta}2. We demonstrated the feasibility of immunohistochemical detection of TCR{beta}1 and TCR{beta}2 in formalin-fixed, paraffin-embedded (FFPE) tissue as a novel diagnostic strategy for T-cell lymphomas. Here we validate an improved pairing of TCR{beta}1/2 rabbit monoclonal antibodies, and demonstrate their utility for single and double immunostaining, including with a chimeric mouse anti-TCR{beta}2 antibody. Finally, we show that this staining is amenable to automated cell counting, permitting accurate calculation of the TCR{beta}2:TCR{beta}1 ratio.

BackgroundThe effects of Banff histological diagnoses on kidney transplant outcome have been well characterized. However, repeated observation of such histological injury across multiple biopsies in kidney transplant recipients remains insufficiently explored. MethodsIn an observational cohort (N=1819 transplantations with 5736 post-transplant biopsies, recurrent event survival models quantified transitions between diagnoses of T-cell mediated rejection (TCMR), antibody-mediated rejection (AMR), DSA-negative C4d-negative microvascular inflammation (MVIDSA-/C4d-), BK polyomavirus nephropathy (BKPyVAN), borderline TCMR (bTCMR), and probable AMR (pAMR), revealing patterns in the disease trajectories. In two observational cohorts (N=1818 transplantations with 5732 biopsies, N=853 transplantations with 975 biopsies), time-dependent cumulative covariates were constructed for TCMR, AMR, MVIDSA-/C4d- and BKPyVAN, enabling estimation of associations of repeated diagnoses with graft failure using multivariable cause-specific Cox models. ResultsThe incidence rate of a diagnosis was most strongly associated with earlier diagnosis of the same type, but associations between different types of diagnoses also occurred. The hazard of kidney graft failure was significantly increased by repeated observation of TCMR in multiple biopsies (HR 7.97, 95% CI 4.94 - 12.86), as well as by repeated AMR (HR 6.19, 95% CI 3.15 - 12.17), repeated MVIDSA-/C4d- (HR 4.53, 95% CI 2.15-9.54) and repeated BKPyVAN (HR 10.90, 95% CI 5.83 - 20.35). The hazard of graft failure was increased more after repeated diagnoses in transplants than after first diagnoses. The effects of repeated TCMR and repeated AMR remained significant even when observed in protocol biopsies in the absence of graft dysfunction. Repeated observation of BKPyVAN was the most detrimental of all diagnoses when observed in indication biopsies, but it was the least harmful when observed in protocol biopsies. ConclusionIncidence of Banff histological diagnoses appears to be affected by earlier diagnoses, especially those of the same type. These repeated observations of a specific diagnosis have an additional effect on the hazard of graft failure, underscoring a critical unmet need for adequate treatment strategies for these recurrent or persistent injury processes. Lay summaryIn two observational cohorts of 1819 and 750 kidney transplant recipients, kidney transplant biopsies were taken at multiple time points after transplantation. Based on the Banff classification for transplant pathology, various post-transplant diseases were diagnosed, often at more than one time point during follow-up. We assessed patterns in the occurrence of diagnoses over time, and related these diagnoses to survival of the kidney grafts using survival models with time-dependent cumulative diagnoses. We found that repeated observation of the same diagnosis was much more common than consecutive observations of different diagnoses. Repeated diagnoses of tissue injury also decreased kidney graft survival more compared to single diagnoses. This indicates that treatment options for patients with repeated or persistent diagnoses are currently inadequate and novel strategies are needed.

Anti-{beta}2-glycoprotein I (anti-{beta}2GPI) antibodies are central to the pathogenesis of antiphospholipid syndrome (APS), an autoimmune disease characterized by a strong predisposition to venous thromboembolism (VTE). In this study, we conducted a multi-ancestry genome-wide association study (GWAS) of quantitative total anti-{beta}2GPI levels in 5,969 participants enrolled in the Multi-Ethnic Study of Atherosclerosis (MESA) and identified a genome-wide significant association at the APOH locus. Paradoxically, genetically determined increases in anti-{beta}2GPI levels at this locus were associated with lower VTE risk. Fine-mapping and functional genomics prioritized the missense variant rs1801690 (W335S) in {beta}2GPI (apolipoprotein H, [APOH]) as the most likely causal variant. This variant has an allele frequency of 5-6% in European and East Asian ancestries but only 1% in African ancestries. Integrating prior experimental studies, molecular dynamics simulations and structure-based epitope prediction, we propose a dual-effect mechanism whereby W335S reduces thrombotic risk by disrupting phospholipid binding in Domain V, yet increases autoantibody production through conformational changes that enhance epitope exposure in Domains I and II. These findings mechanistically uncouple autoantibody formation from thrombotic risk in carriers of the W335S variant, and suggest that APOH genotype may represent a clinically relevant genetic biomarker with potential utility for thrombotic risk stratification in anti-{beta}2GPI-positive individuals.

AimsGestational diabetes mellitus (GDM) is the most common pregnancy-related medical complication. GDM is linked to aberrant immune responses in both mothers and offsprings, specifically, the subsequent development of inflammatory diseases. Whereas prior research has focused on specific immune cell subsets, a comprehensive overview of the impacts of GDM on maternal and fetal immune landscape is lacking. Here, we aim to comprehensively decipher how GDM modulates various immune cell populations in mothers and offsprings. MethodsA prospective, longitudinal case-control study was carried out. Maternal blood from GDM-affected (GDM, n=18) and non-GDM-affected (Ctrl, n=21) mothers were collected at ante-(36-38 weeks of gestation) and post-partum (6-8 weeks post-partum) timepoints. Cord blood from GDM (n=7) and Ctrl (n=11) pregnancies were collected upon C-section. They were analyzed with the state-of-the-art cytometry by time of flight (CyTOF) with a 40-marker panel. Additionally, a publicly available RNA-seq dataset for cord blood mononuclear cells was re-analyzed to confirm results from CyTOF experiments. ResultsCompared to Ctrl, GDM was associated with more activated maternal T cell subsets ante-partum, including increased CD45RO+ and Ki67+ CD4+ T cell populations, which reverted post-partum. GDM-affected maternal innate lymphoid cell (ILC) also exhibited increased granzyme B production ante-partum. On the other hand, in GDM-impacted cord blood, fetal T and B cells were more activated, displaying less naive and more effector phenotypes, further supported by RNA-seq analyses. ConclusionsOur comprehensive analyses revealed that GDM is linked to profound changes in the immune landscapes of the mothers (ante-/post-partum) and foetuses (at birth), casting novel insights towards GDM pathophysiology. Longitudinal immune profiling might be warranted for early detection and stratification of risk, and development of targeted interventions to prevent inflammatory disorders in GDM mothers and their offspring. Research in contextO_LIWhat is already known about this subject? O_LIThe maternal and intrauterine environments are important contributors to long-term health outcomes of mothers and offsprings. C_LIO_LISome maternal and fetal immunity changes have been observed in gestational diabetes mellitus (GDM)-affected pregnancies. C_LIO_LIGDM is associated with increased risk of later-life metabolic and inflammatory diseases in mothers as well as offsprings. C_LI C_LIO_LIWhat is the key question? O_LIWhat are the longitudinal alterations in maternal and fetal immune landscapes in GDM-affected pregnancies? C_LI C_LIO_LIWhat are the new findings? O_LIHigh-dimensional immune profiling provided the most comprehensive overview of alterations in maternal and fetal immune landscapes associated with GDM. C_LIO_LIGDM is associated with skewing of maternal CD4+ T cell and ILC towards activated phenotypes ante-partum. C_LIO_LIGDM is linked to more activated fetal T and B cell profiles. C_LI C_LIO_LIHow might this impact on clinical practice in the foreseeable future? O_LIUnderstanding the complex alterations in the maternal and fetal immune landscape in GDM-affected pregnancy provides insights into the long-term impacts of GDM on the mother and offspring. C_LI C_LI

Background and ObjectivePatients with upper urinary tract urothelial carcinoma (UTUC) undergoing radical surgery are at high risk of developing intravesical recurrences (IVR). The biology of IVR after surgery for UTUC is poorly understood, and urine markers to replace cystoscopic surveillance of the bladder are lacking. Here, we characterized the genomic landscape of UTUC and paired IVR to discover therapeutic targets and identify diagnostic markers for IVR. MethodsWe performed targeted next-generation DNA-sequencing of 571 genes in a cohort of 276 retrospectively and 138 prospectively enrolled UTUC patients who received radical surgery. Clonality and evolution were assessed in 79 paired UTUC-IVR cases. Key Findings and LimitationsMutations in TERT (72%) and FGFR3 (50%) were highly prevalent in UTUC, while mutations in KMT2C were associated with reduced risk of IVR. The mutually exclusive mutational profile of UTUC revealed five genomic subtypes with distinct clinicopathological and molecular characteristics, but none were associated with elevated IVR risk. Clonal evolution of paired UTUC-IVR occurred in 92% of cases via four evolutionary paths, with FGFR3 as a key driver in the largest path (36%). Additionally, hotspot mutations in the TERT promoter, and FGFR3 and HRAS genes were identified as potential markers for noninvasive surveillance by urine testing. Limitations include cohort heterogeneity and the selected gene-targeted sequencing approach. Conclusions and Clinical ImplicationsThe high FGFR3 mutation rate in UTUC and its association with IVR development support anti-FGFR targeted therapy to reduce IVR risk. The clonal relationship between UTUC and IVR underscores the potential for patient-friendly noninvasive urine tests for surveillance after radical surgery. SummaryUpper urinary tract urothelial carcinoma (UTUC) is a rare cancer with a high recurrence rate after surgery. We found that the FGFR3 gene is a potential therapeutic target to reduce the risk of recurrence, while recurrent mutations in TERT, FGFR3 and HRAS could serve as potential markers for noninvasive surveillance by urine testing after surgery for UTUC.

BackgroundPATZ1 fusion-positive central nervous system (CNS) tumors frequently harbor MN1::PATZ1 fusions as driver mutations, provisionally classified as a rare DNA methylation class of low-grade neuroepithelial tumors. Radiographically, they resemble pilocytic astrocytomas with tumor and cystic components, but their supratentorial cortex location and higher recurrence rates are distinguishing features. An intermediate clinical course, despite focal high-grade histopathology, underscores the need for longitudinal molecular and immune analyses to refine classification and standard therapy. Case SummaryA female pediatric patient presented with neurological symptoms, including headache and right upper extremity weakness. MRI revealed a large cystic lesion in the left frontal lobe, leading to a differential diagnosis of low-grade glioma and ependymoma. Genomic analysis identified an MN1::PATZ1 fusion. The tumor recurred after gross total resection prompting a second resection. Transcriptomic and histopathologic assessments identified multiglial lineage, and high-grade features closely related to adult glioblastoma alongside pro-inflammatory activity in the primary tumor. The recurrent tumor showed reduced malignancy, and oligodendroglioma-like features. Increased MHC gene expression, immune checkpoint receptors (PDCD1, CTLA4, TIGIT,TIM3), T cell regulators (CXCR6), and elevated macrophage frequency, coupled with reduced PD-L1 in the recurrent tumor, suggest a complex anti-tumor immune response constrained by T cell dysregulation. This case, along with two other MN1::PATZ1 fusion-positive tumors, identifies a distinct transcriptomic subtype separate from circumscribed astrocytic glioma, highlighting upregulation of growth factor receptor pathways, like PI3K/AKT, and immune dysfunction linked to recurrence. ConclusionLongitudinal multi-omics analyses of recurrent MN1::PATZ1 fusion-positive CNS tumors revealed tumor maturation, immune dysfunction, and potential therapeutic targets. Introductory ParagraphPATZ1 fusion-positive central nervous system (CNS) tumors are rare, predominantly pediatric and frequently recurrent neoplasms provisionally classified as neuroepithelial tumors. Their pronounced histopathological and clinical heterogeneity, along with limited immunological characterization complicates their treatment standardization. We report a new case of an MN1::PATZ1 fusion-positive CNS tumor with recurrence, highlighting its radiographic similarities to low-to-intermediate grade pediatric glioma. Longitudinal multi-omics analyses of this case, along with additional MN1::PATZ1 fusion-positive CNS tumors, however, delineates a transcriptome subtype resembling adult high-grade glioma, with activated oncogenic and pro-inflammatory programs. The recurrent tumor exhibits features of decreased malignancy and enhanced glial differentiation, phenotypically shifting towards oligodendroglioma, suggesting tumor maturation. This was accompanied by increased antigen presentation programs, indicating immune engagement, while increased immune checkpoint expression and microglia/macrophage frequency indicate T cell exhaustion and immunomodulation, respectively. This longitudinal study highlights potential therapeutic strategies targeting both the tumor and its immune environment in MN1::PATZ1 fusion-positive CNS tumors.