Acute myeloid leukemia (AML) is characterized by recurrent chromosomal abnormalities that form the basis of the European LeukemiaNet (ELN) risk classification and serve as essential determinants of prognosis and therapeutic decision-making. Conventional metaphase karyotyping remains the diagnostic gold standard for detecting these abnormalities; however, its utility is limited by longer turnaround times, often delaying critical clinical management. Here, we present a long-read sequencing-based (LRS) low-coverage whole genome sequencing (lcWGS) approach using Oxford Nanopore Technology as a rapid and scalable alternative for cytogenetic profiling. A total of 100 diagnostic AML samples were analyzed, comprising 50 retrospectively selected cases with known adverse-risk cytogenetics and 50 prospectively enrolled patients with clinically defined de novo AML. LcWGS demonstrated robust analytical performance, identifying chromosomal aberrations with 93% sensitivity, specificity, and overall accuracy, respectively. Complex karyotypes were reliably detected, with an area under the curve (AUC) of 0.971. Reproducibility was validated through replicate sequencing at two independent laboratories (R=0.99). LcWGS-derived estimates of clone size showed moderate correlation with conventional cytogenetic assessments (R=0.54). Patients with complex karyotypes identified by lcWGS exhibited significantly shorter overall and relapse-free survival, closely mirroring outcomes defined by conventional karyotyping and underscoring the value of lcWGS for risk stratification. Median turnaround time from sample receipt to bioinformatics interpretation was approximately 34 hours, enabling delivery of actionable karyotype results within 72 hours. These findings establish lcWGS as a rapid, reproducible, and accurate platform for detecting clinically relevant chromosomal abnormalities, addressing a critical need for timely risk stratification and treatment initiation in AML.

ABSTRACT:Core-binding factor acute myeloid leukemia (CBF-AML) is associated with KIT mutations and deregulated expression of KIT. We report results from the randomized, open-label, phase 3 trial of intensive chemotherapy with or without the multikinase inhibitor dasatinib in adult patients with CBF-AML. Patients received "3+7" induction therapy, followed by 4 cycles of high-dose cytarabine; in the investigational arm, patients received dasatinib 100 mg daily on days 8 to 21 in induction, and on days 6 to 28 in consolidation cycles, followed by 12-month single-agent dasatinib 100 mg daily. Primary end point was event-free survival (EFS). Secondary end points included overall survival, relapse-free survival, and cumulative incidence of relapse. A total of 202 patients were randomly assigned to the standard arm (n = 102) and to the dasatinib arm (n = 100). Median age was 49 years (range, 18-77); 94 patients had t(8;21), 108 had inv(16)/t(16;16); and 58 (28.7%) patients had a KIT comutation. There was no statistically significant difference in EFS (hazard ratio, 0.92; 95% confidence interval, 0.63-1.33; P = .66) or secondary end points between treatment arms. There was also no significant difference in EFS in subgroup analyses according to age, CBF-AML type, and KIT mutation status. The incidence of serious adverse events was higher in the investigational arm (64%) than in the standard arm (36%). In patients with CBF-AML, the addition of dasatinib to intensive chemotherapy failed to improve survival outcomes. The addition of dasatinib was associated with an increase in toxicity. This trial was registered at www.ClinicalTrials.gov as NCT02013648.

Eltrombopag combined with horse antithymocyte globulin and cyclosporine A (CSA) is the standard of care for patients with severe aplastic anemia who are not eligible for stem cell transplantation. No consensus exists on the optimal treatment of moderate aplastic anemia (MAA). The "Eltrombopag for MAA" (EMAA) trial is an investigator-initiated, prospective, randomized, placebo-controlled, double-blind, multicenter, phase III trial and compared eltrombopag+CSA (n=41) with placebo+CSA (n=44) as a first-line therapy for MAA patients with clinically significant cytopenia. In patients with evaluable response at week 24 (n=75), the overall response rate (ORR), including partial response (PR) and complete response (CR), was significantly higher in the eltrombopag+CSA arm (71.4%; 95% confidence interval [CI], 54.8%-83.8%) than in the placebo+CSA arm (42.5%; 95% CI, 28.5%-57.8%) (p=0.011, Fisher's exact test; primary endpoint). After response assessment at week 24 and unblinding, patients in the placebo arm without CR were switched to receive eltrombopag in addition to continued CSA. This group achieved an ORR of 73.5% by week 48 (p=0.009, McNemar test, comparing response at 24 vs. 48 weeks). Relapse rate and failure-free survival did not differ significantly among the arms. Eltrombopag was well tolerated without new safety concerns. Adding eltrombopag to CSA for first-line treatment of MAA significantly improves trilineage hematologic response at week 24 and addition of eltrombopag in patients without CR after 24 weeks of single-agent CSA treatment also significantly improves the ORR. The trial was registered at www.clinicaltrials.gov as #NCT02773225 and at European Union Drug Regulating Authorities Clincial Trials as EudraCT2014-000174-19.

The imaging-based assessment of response to treatment is essential for treatment planning and outcome prediction in oncology. Standardized classification systems enable reproducible follow-up assessments and improve comparability in the clinical practice and research. Depending on the tumor type and treatment approach different evaluation criteria and imaging modalities are applied. Metabolic information plays a central role in the assessment of hematological diseases and is gaining relevance in the assessment of solid tumors, which have previously been evaluated primarily based on their size. Additionally, the mechanism of action of the respective treatment should be considered, such as an expected size reduction after chemotherapy, altered contrast agent uptake after locoregional therapy or atypical response patterns, such as pseudoprogression after immunomodulatory therapy, which make repeated follow-up imaging mandatory. This article provides a structured overview of clinically relevant imaging-based response criteria and their specific applications.

Multiple myeloma treatment has experienced tremendous advances through chimeric antigen receptor (CAR) therapies directed to the B cell maturation antigen (BCMA), but remissions are usually transient. To mitigate the risk of BCMA immune escape, we aimed for a simultaneous targeting of BCMA together with the B cell-activating factor receptor (BAFF-R). Single-cell RNA sequencing discovered increased BAFF-R gene (TNFRSF13C) expression in relapsed and refractory multiple myeloma cases, and it emerged as prognostic marker for long-term complete responses. BAFF-R was expressed in plasma cells at earlier maturation stages compared with BCMA-positive plasma cell phenotypes. Bispecific BAFF-R/BCMA CARs endowed T cells with cytolytic efficacy against multiple myeloma cell lines and primary multiple myeloma cells. In vivo, the dual CAR compensated for BCMA downregulation when BAFF-R was expressed, preventing the evolution of antigen escape mutants that drive resistance to CAR T cell therapy. Our study proposes BAFF-R as a complementary target antigen suitable to eliminate malignant plasma cells with less advanced differentiation, lack of BCMA, and occurrence in dismal prognosis patients.

BACKGROUND:Distinguishing donor- vs. recipient-derived myelodysplastic neoplasm (MDS) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is challenging and has direct therapeutical implications. METHODS:Here, we took a translational approach that we used in addition to conventional diagnostic techniques to resolve the origin of MDS in a 38-year-old patient with acquired aplastic anemia and evolving MDS after first allo-HSCT. Specifically, we used single-cell transcriptional profiling to differentiate between donor- and recipient-derived bone marrow cells and established a strategy that additionally allows identification of cells carrying the MDS-associated U2AF1S34Y variant. RESULTS:The patient exhibited mixed donor chimerism combined with severely reduced erythropoiesis and dysplastic morphology within the granulocytic and megakaryocytic lineage along with the MDS-associated U2AF1S34Y mutation in the bone marrow. Single-cell transcriptional profiling together with targeted enrichment of the U2AF1S34Y-specific locus further revealed that, while the immune compartment was mainly populated by donor-derived cells, myelopoiesis was predominantly driven by the recipient. Additionally, concordant with recipient-derived MDS, we found that U2AF1S34Y-mutated cells were exclusively recipient derived with X but not Y chromosome-specific gene expression. CONCLUSION:Our study highlights the clinical potential of integrating high-resolution single-cell techniques to resolve complex cases for personalized treatment decisions. FUNDING:The study was funded by intramural resources of the Charité - Universitätsmedizin Berlin and the Berlin Institute of Health.

<p>Legends for Supplementary Figures S1-S6.</p>

Figure S1. Gating strategy to characterize and quantitate CD19+CD5+ leukemia Emu-Tcl1 cell frequencies and homeostatic chemokine receptor expression on splenic, LN, PB, and BM derived CD19+CD5+ gated Emu-Tcl1 tumor cells. This Figure complements Figure 1. Figure S2. Surface marker expression of murine Emu-Tcl1 leukemia cells is shown. Human primary B-CLL cells share characteristic expression of CXCR5. This Figure complements Figure 1. Figure S3. Enrichment plots of seven proliferation-related signatures are presented. Short term transfer of CXCR5+/+Emu-Tcl1 or CXCR5-/-Emu-Tcl1 tumor cells shows upregulation of the gene encoding the cell-cycle inhibitor p21 in CXCR5-deficient leukemia cells. Growth and activation delay of CXCR5-/- Emu-Tcl1 tumor cells can be reversed by direct contacts with stromal cells in vitro. This Figure complements Figure 2. Figure S4. (A) shows that CCR7-deficiency on leukemia cells does not affect their migratory behavior and complements Figure 3. (B) Ebi2-/-Emu-Tcl1 leukemia cells enter B cell follicles and the GC light zone area in a pattern comparable to Ebi2++Emu-Tcl1 leukemia cells, as presented in Figure 4, and (C) tumor growth of Ebi2-/-Emu-Tcl1 mice is unaltered compared to Wt Emu-Tcl1 mice. Figure S5. Human primary B-CLLs exhibit a characteristic FDC staining. This Figure complements Figure 4. Figure S6. Pertussis toxin (PTX) treatment induces apoptosis in Emu-Tcl leukemia cells co-cultured with FDC-HK stroma cells. This Figure complements Figure 4. Figure S7. Intravital application of anti-CD21/CD35 Fab fragments stains specifically GC light zone localized FDCs. These stainings complement Figure 5. Figure S8. Challenge of Rag2-/- mice with Emu-Tcl1 leukemia B cells induces FDC networks and a tumor cell-promoting cytokine and chemokine secretion. This Figure accompanies Figure 6. Figure S9. Abrogation of LTbetaR-signaling leads to disappearance of characteristic stromal elements within the B cell follicle; however, CD31+ blood vessels are not affected. In accordance with Figure 7, Ltalpha -/- Emu-Tcl1 mice exhibit substantially delayed leukemia growth and progression.

Polarization of leukemia-associated macrophages in spleen.

TOP 25 upregulated in Eμ-Tcl1 stroma by fold change.

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Neutrophil depletion does not affect the pro-inflammatory cytokine milieu in Eμ-Tcl1 leukemia B cells.

<p>Tumor-induced down- and upregulations of genes in the stroma.</p>

Supplemental Movie S2. Follicular motility of B cells. The Movie complements Figure 4J.

The extracellular matrix protein TGFBi is stronger expressed in tumor-bearing Eμ-Tcl1 mice.

Eμ-Tcl1 leukemia-associated splenic neutrophils exhibit a unique gene expression profile.

Supplementary Movie S1. Follicular motility of Emu-Tcl1 leukemia cells. The Movie complements Figure 4J.

Background: Phase II trials with 2nd or 3rd generation Tyrosine Kinase Inhibitors (TKI) with/without immunotherapy yield promising results in Ph+ ALL. However, Imatinib (IM) in combination with chemotherapy regimens of different intensities followed by allogeneic SCT is still the standard treatment for younger patients (pts) in many countries. Aims: GMALL Trial 08/2013 (NCT02881086) evaluates a concept with dose-reduced induction (ind) and intensive consolidation (CI) in combination with IM followed by SCT independent of MRD response in patients (pts) aged 18-55 years (yrs). Dose reduced conditioning (8 Gy TBI vs 12 Gy TBI) was recommended in pts older than 45 yrs. Methods: The 6 week ind included IM 600 mg/d together with low intensity chemotherapy (VCR, Dexa, PEG-ASP) and intrathecal prophylaxis. Bone marrow evaluation after 3 weeks separated ind I and II. Four doses of Rituximab were added if CD20 was >20%. C I (Dexa, VCR, HDMTX, HDAC, VP16) followed. Donor search was initiated in all pts at diagnosis. SCT was scheduled after C I; after an amendment, a TKI change was recommended if MRD >10-3 after CI. MRD was assessed by quantitative (RQ)-PCR of BCR::ABL in a central reference laboratory. Additional MRD assessment with IG/TR RQ-PCR was recommended. Results: 163 pts with a median age of 42 (18-55) yrs were evaluable. 64 (39%) pts had a WBC >=30,000/µL. CR rates after ind I, II and CI were 85%, 95% and 93%. Early death and failure rates were 5% and 1% after CI, respectively. Molecular complete remission (MolCR) rates, defined as PCR negativity for BCR::ABL1 transcripts with at least 10000 ABL1 copies, were 10% after ind I, 24% after ind II and 43% after CI. Low positive MRD (≤10-4) was observed in 8%, 19% and 20%, respectively, whereas the remaining pts experienced molecular failure (MolF) with MRD >10-4. 27/48 pts with MolF (59%) had MRD ≥10-3; 10/27 pts were switched to another TKI. Data are yet available from 8/10 pts. These pts. were switched to Dasatinib (n=7) or Ponatinib (n=1). Only 1/8 pts with available data had a molecular remission after 4 weeks of ponatinib. Overall survival (OS) at 1 yr was 82% and 74% at 3 yrs; remission duration (RD) was 92% and 89%, resp. OS at 3 yrs was 88%, 70% and 75% for pts aged 18-25, 26-45 and 46-55 yrs resp (p>0.05). WBC at diagnosis <> 30000/µL had no impact on 3y-OS of 73% vs 76% (p>0.05). 138 pts (85%) were transplanted in CR1 as scheduled after CI (Median time to SCT: 4 mo) with OS 86% and 81% at 1 and 3 yrs. OS was similar (86% vs 79% at 3 yrs, p>0.05) for matched sibling or matched unrelated SCT. The treatment-related mortality was 14%. OS after SCT was not impacted by status of BCR::ABL1 MRD after CI as defined above. Summary/Conclusion: Dose reduced induction including PEG-ASP in combination with IM was feasible and yielded high response rates leading to a molecular CR rate as high as 43% already after CI. A very high SCT realization rate (85%) was obtained at approximately 3 months from diagnosis likely contributing to the favorable overall outcome. It remains open to further trials whether BCR::ABL MRD at all has a prognostic impact in transplant-focused regimens. The role of alternative TKI and immunotherapies with the aim to reduce SCT indications will be evaluated in the upcoming GMALL EVOLVE trial. The trial was funded by Deutsche Krebshilfe.Keywords: Acute lymphoblastic leukemia, Treatment, Philadelphia chromosome

Neutrophil localization in human SMZL and benign spleen tissues.