Blood Cancer Journal

Mediastinal B-cell lymphomas are relatively frequent in young patients and include nodular sclerosis classic Hodgkin lymphoma (NSCHL), primary mediastinal large B-cell lymphoma (PMBL), and rarely, mediastinal gray zone lymphoma (MGZL). Occasional NSCHLs contain abundant Hodgkin/Reed Sternberg (HRS) cells that exhibit a syncytial growth pattern, which may create diagnostic challenges. Previous studies have demonstrated that PMBL can be distinguished from subtypes of diffuse large B-cell lymphoma (DLBCL) based on gene expression signatures using the Lymph3Cx gene expression profiling assay, which has been validated as a clinical test in our molecular diagnostics laboratory. Here, we demonstrate that syncytial variant NSCHL exhibits a PMBL-like gene expression profile using the Lymph3Cx gene expression profiling assay. It is critical for pathologists and oncologists to be aware of this potential diagnostic pitfall to avoid possible misdiagnosis.

Despite development of several effective therapies for multiple myeloma (MM), the prognosis of patients with partial deletion of chromosome 17 (del(17p)) and TP53 aberrations remains poor. By applying comprehensive multi-omics profiling analyses (whole exome and transcriptome sequencing plus proteomics) and functional ex vivo drug screening to samples from 167 patients with MM, we uncovered novel therapeutic vulnerabilities specific to TP53 mutated MM. Our findings revealed a distinct sensitivity profile to a range of inhibitors (mitotic, topoisomerase, HDAC, HSP90, IGF1R and PI3K/AKT/mTOR inhibitors) irrespective of 17p deletion status. Conversely, no increase in sensitivity was observed for monoallelic TP53 (del(17p) with WT TP53) when compared to WT TP53 samples, highlighting the remaining unmet clinical need. Notably, plicamycin, an RNA synthesis inhibitor linked to modulation of chromatin structure and increased transcription, emerged as particularly efficacious for TP53 mutated MM. The increased sensitivity correlated with higher protein expression of the drug targets: HDAC2, HSP90AA1 and multiple ribosomal subunits. Additionally, we observed increased RNA expression of G2M checkpoint, E2F targets and mTORC1 signaling in our cohort and the MMRF-CoMMpass (NCT01454297) study in TP53 mutated MM. Harmonization of multi-omics data with ex vivo drug screening results revealed that TP53 mutated MM is functionally distinct from MM with monoallelic TP53, and demonstrates that MM with mutated TP53, with and without del(17p), may be targetable by approved drugs. These results further indicate the need for regular monitoring by sequencing to identify these patients. KEY POINTSTP53 mutation in myeloma confers sensitivity to multiple compounds, including approved drugs, irrespective of del(17p) status. TP53 mutated myeloma links to higher expression of drug targets involved in cell proliferation, mRNA processing, and chromatin modulation.

Immunoglobulin light-chain amyloidosis (AL) has been reported to be associated with multiple myeloma (MM) in approximately 10-15% of cases, with the two conditions often coexisting. Understanding the interaction between these diseases is vital for improving patient outcome and developing targeted treatments. Our study investigates cellular heterogeneity in immunoglobulin light-chain amyloidosis (AL) and coexisting multiple myeloma (MM) or monoclonal gammopathy of undetermined significance (MGUS) (AL_MM) using single-cell RNA sequencing (scRNA-seq). A total of 21 bone marrow samples from 20 patients were analyzed, including 9 with AL_MM. We observed exclusive association of the 1q gain chromosomal aberration with the AL_MM group (p<0.01). The scRNA-seq data revealed 27 distinct cell clusters representing 14 unique cell types, with 6 clusters identified as plasma cell (PC) subpopulations. These subpopulations exhibited considerable inter- and intra-individual heterogeneity. Comparative analysis between the AL and AL_MM groups showed higher proportions of GMP, HSC, cDC2, and T cells in the AL group (p<0.05), suggesting a distinct tumor microenvironment. Differential gene expression analysis between the two groups identified 152 up-regulated and 134 down-regulated genes in AL_MM samples, with 28 of the up-regulated genes and 1 downregulated gene located on chromosome 1q. Furthermore, the application of inferCNV to the scRNA-seq data revealed amplifications in chromosome 4, 7, 11, 20, and 22 of AL samples. Our findings enhance the understanding of the molecular mechanisms underlying AL and its progression in patients with coexisting MM or MGUS. The study provides valuable insights into disease progression and cellular variability, supporting improved patient stratification and the development of targeted therapies for these complex hematological conditions. KEY POINTSO_LI1q gain is mainly restricted to amyloidosis patients with co-occurring sMM, MM, or MGUS. C_LIO_LIMultiple genes located on chromosome 1 are upregulated in plasma cells of patients with co-occurring AL and MM, potentially contributing to disease progression. C_LI

Multiple myeloma (MM) is consistently preceded by precursor conditions recognized clinically as monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM). We interrogate, for the first time, the whole genome sequence (WGS) profile of 18 MGUS and compare them with those from 14 SMMs and 80 MMs. We show that cases with a non-progressing, clinically stable myeloma precursor condition (n=15) are characterized by later initiation in the patients life and by the absence of myeloma defining genomic events including: chromothripsis, templated insertions, mutations in driver genes, aneuploidy, and canonical APOBEC mutational activity. This data provides evidence that WGS can be used to recognize two biologically and clinically distinct myeloma precursor entities that are either progressive or stable.

Richter transformation of Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) into classic Hodgkin lymphoma (CHL-RT) is rare and remains incompletely understood. Two histologic subtypes are recognized: type 1 (CLL/SLL with scattered Hodgkin/Reed-Sternberg (HRS) cells) and type 2 (HRS cells within a polymorphous inflammatory background). In this multi institutional study of 77 patients with CHL-RT (27 type 1 and 50 type 2), we characterized immune evasion markers, PD-L1/PD-L2 copy number alterations, tumor microenvironment, and performed targeted next-generation sequencing on 37 CLL/SLL samples. HRS cells in CHL-RT displayed immune evasion phenotypes similar to de novo CHL, though PD-L1 expression was lower in type 1 cases. PD-L1/PD-L2 gain/polysomy were frequent (83.3%). CLL/SLL with CHL-RT harbored increased mutations in XPO1, FBXW7, BIRC3, TRAF3, and HLA-A versus reference CLL/SLL. Similar mutational profiles, demographics, and survival outcomes support a biological continuum between type 1 and type 2 CHL-RT, with distinct genetic features in CLL/SLL predisposing to CHL transformation.

The epiCMIT (epigenetically-determined Cumulative MIToses) mitotic clock traces B-cell mitotic history via DNA methylation changes in heterochromatin and H3K27me3-containing chromatin. While high scores correlated with poor outcomes in CLL and MCL, its prognostic significance in SMZL remains unknown. Derived from 142 SMZL cases using DNA methylation microarrays, epiCMIT values were correlated with genomic, transcriptomic, and clinical data. EpiCMIT as a continuous variable was significantly higher in females (p=0.02), patients with IGHV1-2*04 allele usage (p<0001), intermediate IGHV somatic hypermutation load (97-99.9% identity, p=0.04), elevated mutational burden (25 vs. 17 mut/Mb, p=0.001), driver gene mutations [KLF2 (p<0.001), NOTCH2 (p<0.01), TP53 (p=0.01), KMT2D (p<0.001)], and del(7q) (p=0.01). Negative correlation between epiCMIT and telomere length (r=-0.29 p<0.001) supported the association between cumulated proliferation and telomere attrition. While univariate analysis highlighted epiCMIT as robust predictor of shorter treatment-free survival (TFS), multivariate analysis confirmed epiCMIT as an independent marker for shorter TFS. In summary, our matched multi-omic datasets facilitate the clinico-biological characterization of SMZL and introduces epiCMIT as a strong prognostic marker, identifying high-risk patients and predicting reduced treatment-free survival, hence providing a new tool for risk-adapted patient management.

Long-term follow-up of the CASSIOPEIA trial (NCT02541383) demonstrated superior progression-free survival (PFS) with daratumumab, both in combination with bortezomib, thalidomide, and dexamethasone during induction and consolidation, and during maintenance therapy, in transplant- eligible patients newly diagnosed with multiple myeloma (MM). However, outcomes among CASSIOPEIA patients remain heterogeneous across treatment groups. Measurable residual disease (MRD) is a strong indicator of the depth and duration of therapeutic response and is independently associated with both PFS and overall survival (OS), but it does not fully capture the biological diversity of MM. We performed a risk prediction analysis based on transcriptomic subgroups in CASSIOPEIA patients. A subset of 628 patients was characterized using RNA sequencing and consensus clustering identified five transcriptomic subtypes of MM. Long-term follow-up allowed the definition of three transcriptomic risk categories, with estimated 72-month PFS rates of 70%, 51%, and 27% for low, intermediate, and high-risk groups, respectively, among patients who received daratumumab in at least one treatment phase. In these patients, MRD negativity rates after consolidation and six months later were significantly higher in the low and high-risk groups compared with the intermediate-risk group. In the high-risk group, MRD status was not associated with PFS or OS. This suggests that, although daratumumab administered during both the induction/consolidation and maintenance phases improves the clinical outcomes of patients with activation of NSD2 or overexpressing members of the MAF family, highly aggressive minor clones may rapidly expand. These findings emphasize the need for novel therapeutic strategies in this high-risk population.

MALAT1 is a long non-coding RNA with oncogenic roles in cancer but poorly studied in indolent B-cell neoplasms. Here, MALAT1 expression was analyzed using RNA-seq, microarrays or qRT-PCR in primary samples from various clinico-biological subtypes of chronic lymphocytic leukemia (CLL, n=266) and follicular lymphoma (FL, n=61). In peripheral blood (PB) CLL samples, high MALAT1 expression was associated with a significantly shorter time to treatment, independently from other known prognostic factors, such as IGHV mutational status. Coding genes whose expression levels were associated with MALAT1 in CLL were predominantly related to oncogenic pathways stimulated in the lymph node (LN) microenvironment. Further analysis of MALAT1 expression by microarrays in paired CLL samples from PB/LN showed that its levels were maintained between both anatomical compartments, supporting that the clinical value of MALAT1 expression found in PB is mirroring expression differences already present in LN. Similarly, high MALAT1 expression in FL predicted for a shorter progression-free survival, and its correlated expressed genes were associated with pathways promoting FL pathogenesis. In summary, MALAT1 expression is related to pathophysiology and clinical behavior of indolent B-cell neoplasms. Particularly in CLL its levels could be a surrogate marker of the microenvironment stimulation and may contribute to refine the clinical management of these patients.

Non-ICANS neurotoxicities (NINTs) are serious, atypical toxicities associated with ciltacabtagene autoleucel, a commercial chimeric antigen receptor (CAR) T cell therapy approved for relapsed/refractory multiple myeloma. Risk factors contributing to the development of NINTs are poorly understood. In a cohort of 109 patients, we identify predisposing risk factors and propose strategies to mitigate NINTs. We show that high peak absolute lymphocyte count is a strong NINT predictor which directly correlates with flow cytometry-based peripheral blood CAR T cell quantitation. The observed CAR lymphocytosis was polyclonal with a bias towards CD4+ CAR T cells rich in memory marker expression. We then identified CAR lymphocytosis associated CD4+ CAR T cell populations which exhibited increased inflammatory pathway gene expression. Finally, we characterize NINT associated CD4+ CAR T cell populations which are potential therapeutic targets for future exploration. One Sentence SummaryCiltacabtagene autoleucel associated non-ICANS neurotoxicities are driven by high CD4+ CAR T cell expansion exhibiting memory marker expression and upregulated inflammatory gene signaling pathways.

Multiple myeloma (MM) is a hematological malignancy that, in many cases, is preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), pre-MM conditions with a lifelong risk of progression to symptomatic MM. Accurate diagnosis, prognostication, and adequate progress monitoring for these conditions require repeated invasive bone marrow biopsies. Additionally, current prognostic models for identifying higher-risk MGUS and SMM patients misclassify 20-30% of patients. Here, we apply Chromatin immunoprecipitation of cell-free chromatin (cfChIP-seq) from peripheral blood, which provides a genome-wide map of active promoters in the cells contributing to the circulating DNA, on a cohort of individuals with MGUS (n=65), SMM (n=27), patients with overt MM (n = 47), and healthy controls (n=82). We identified an increased representation of specific regions containing promoters specific to B-cells, plasma cells, and erythroblasts in MM blood samples. We used these regions to generate an MM score. This score is capable of diagnosing and staging the disease, and most importantly, identifying patients with a higher risk of 2-year progression (n = 18, p-value << 0.01). Our study highlights the potential of cfChIP-seq to contribute to differentiating plasma cell disease stages and monitoring progression non-invasively.

Background: Multiple myeloma (MM) progression is accompanied by remodeling of the bone marrow immune microenvironment. Local interactions among malignant plasma cells, stromal cells, myeloid cells, and immune cells not only support tumor cell survival, expansion, and immune escape, but are also closely associated with disease progression, therapeutic response, and clinical prognosis. Moreover, T cell exhaustion is a common T cells dysfunction in MM and limited efficacy of T cell-targeting therapies. However, the in situ organization and clinical significance of exhausted T cells in MM patients bone marrow remain insufficiently understood. Methods: In this study, we analyzed bone marrow Xenium 5K spatial transcriptomics data from control (Ctrl), monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SM), and MM samples. After canonical multi-sample integration and celltype annotation, we used Gaussian mixture model (GMM)-based spatial partitioning, and multilayer perceptron (MLP) machine learning for systematic characterization the T cell microenvironment in MM bone marrow. Results: Our results showed that exhaustion-like T cells increased during MM progression and formed spatially discrete T cell-enriched regions in the bone marrow, which we defined as exhaustion-like bone marrow T cell islands (eBM-TIs). These niches were mainly characterized by enhanced T cell-plasma cell communication associated with upregulated Galectin signaling. Pseudobulk analysis further showed enhanced IFN-related signaling in eBM-TIs, accompanied by upregulation of CXCR3 ligands such as CXCL9 and CXCL10, suggesting that the IFN-CXCL9/10 axis may contribute to T cell chemotaxis, maintenance of chronic inflammation, and formation of exhaustion-like states. By transferring spatial niche labels to scRNA-seq cohorts with available clinical staging information using MLP, we further found that the proportion of eBM-TI-like T cells was associated with higher disease risk and unfavorable prognostic outcomes. Conclusions: In summary, this study identifies eBM-TIs as a spatial niche in the MM bone marrow. These niches represent an important immune unit linking chronic inflammation, T cell exhaustion, and clinical risk, and may serve as a potential biomarker of MM disease progression.

Therapy-driven genomic changes in multiple myeloma (MM) remain poorly defined. We analyzed whole-genome sequencing (WGS) data from relapsed/refractory MM (rrMM, N=386) and identified regional 1p31.1-p12 (hereafter 1pCEN, a region proximal to the centromere) loss-of-heterozygosity (LOH) as the only enriched aberration showing strong therapy-associated clonal selection (clonal timing rank fold-change = 3.7, P<2.2x10-16). This event showed enriched co-occurrence with 1qGain (OR = 2.3 (1.5-3.8), P=2x10-4) forming a recurrent "double-hit" in rrMM. To validate the clonal selection process, we examined three longitudinal cohorts (180 patients, 390 samples) and confirmed clonal expansion of 1pCEN and consistent prevalence of the 1pCEN+1q double-hit (20-24%). Survival analyses demonstrated significantly reduced progression-free survival in rrMM patients with this double-hit compared with those without. Comparison with a large newly diagnosed MM (ndMM) cohort confirmed previously-described 1p32 LOH is the prognostic locus at baseline, whereas 1pCEN is therapy-selected and largely independent of the 1p32 locus. Thus, 1pCEN+1q represents a recurrent double-hit event that clonally emerges in rrMM, conferring selective advantage under drug exposure and is distinct from the ndMM high-risk markers defined by current consensus guidelines. These findings nominate 1pCEN as a new genomic biomarker in rrMM and 1pCEN+1q may help patient stratification for therapeutic monitoring. Key PointsA therapy-driven common genomic double-hit (1p31.1-p12 LOH with 1q gain) clonally emerges in relapsed/refractory myeloma.

Multiple myeloma (MM) is a cancer of malignant plasma cells in the bone marrow and extramedullary sites. We previously characterized a VQ model for human high-risk MM. Different VQ lines display distinct disease phenotypes and survivals, suggesting significant intra-model variation. Here, we use whole exome sequencing and copy number variation (CNV) analysis coupled with RNA-Seq to stratify VQ lines into corresponding clusters: Cluster I VQ cells carried recurrent amplification of chromosome (chr) 3 and displayed upregulation of growth pathways and high-risk myeloma gene signatures, whereas Cluster II cells had monosomy chr5 and overexpressed genes and pathways associated with positive response to bortezomib (Btz) treatment in human MM patients. Consistently, in sharp contrast to Cluster II VQ cells that showed short-term response to Btz, Cluster I VQ cells were de novo resistant to Btz in vivo. Our study highlights Cluster I VQ lines as highly representative of human high-risk MM subset.

Whole genome sequencing (WGS) of newly diagnosed multiple myeloma patients (NDMM) has shown recurrent structural variant (SV) involvement in distinct regions of the genome (i.e. hotspots) and causing recurrent copy number alterations. Together with canonical immunoglobulin translocations, these SVs are recognized as "recurrent SVs". More than half SVs were not involved in recurrent events. The significance of these "rare SVs" has not been previously examined. In this study, we utilize 752 WGS and 591 RNA-seq data from NDMM patients to determine the role of rare SVs in myeloma pathogenesis. 94% of patients harbored at least one rare SV event. Rare SVs showed an SV-class specific enrichment within genes and superenhancers associated with outlier gene expression. Furthermore, known myeloma driver genes recurrently impacted by point mutations were dysregulated by rare SVs. Overall, we demonstrate the association of rare SVs with aberrant gene expression supporting a driver role in myeloma pathogenesis. SIGNIFICANCECharacterization of multiple myeloma genome revealed that more than half structural variants are not involved in recurrent events. Here, we demonstrate that these rare SVs hold potential for myeloma pathogenesis through their gene expression impact. Rare SVs contribute to MM heterogeneity and have implications for development of individualized treatment.

Monoclonal antibodies targeting CD38 are a therapeutic mainstay in multiple myeloma (MM). While they have contributed to improved outcomes, most patients still experience disease relapse, and little is known about tumor-intrinsic mechanisms of resistance to these drugs. Antigen escape has been implicated as a mechanism of tumor cell evasion in immunotherapy. Yet, it is unknown whether MM cells can develop permanent resistance to anti-CD38 antibodies by acquiring genomic events leading to biallelic disruption of the CD38 gene locus. Here, by using whole genome and whole exome sequencing data from 701 newly diagnosed patients, 67 patients at relapse with naivety to anti-CD38 antibodies, and 50 patients collected at relapse following anti-CD38 antibodies. We report a loss of CD38 in 20% (10/50) of patients post-CD38 therapy, three of which exhibited a loss of both copies. Two of these cases showed convergent evolution where distinct subclones independently acquired similar advantageous variants. Functional studies on missense mutations involved in biallelic CD38 events revealed that two variants, L153H and C275Y, decreased binding affinity and antibody-dependent cellular cytotoxicity of the commercial antibodies Daratumumab and Isatuximab. However, a third mutation, R140G, conferred selective resistance to Daratumumab, while retaining sensitivity to Isatuximab. Clinically, patients with MM are often rechallenged with CD38 antibodies following disease progression and these data support a role for next generation sequencing to guide treatment selection.

ObjectiveTo evaluate the efficacy, safety, and prognostic factors of chimeric antigen receptor T-cell (CAR-T) therapy in patients with relapsed/refractory ileocecal region lymphoma and provide evidence for salvage therapy. MethodsA retrospective analysis was conducted on 21 ileocecal region lymphoma patients (IC group) and matched 23 non-ileocecal lymphoma patients (non-IC group) treated with CAR-T therapy between June 2014 and August 2024. Baseline characteristics, genetic mutations (next-generation sequencing), CAR-T cell kinetics (digital PCR), treatment response (WHO criteria), overall survival (OS), and progression-free survival (PFS) were assessed. Kaplan-Meier analysis, Cox regression, and Lasso models were used to identify prognostic factors. ResultsThe IC group showed significantly lower 3-month objective response rate (ORR) (55.56% versus 86.96%, P=0.036) and shorter median PFS (3.5 months versus 27 months, P=0.0003) compared to the non-IC group, but no OS difference (P=0.766). CAR-T cell expansion was higher in the IC group (CD19 Cmax: 67,290 versus 35,400, P=0.009), but persistence was similar. TP53 mutations (42.86% in IC group) showed no significant correlation with treatment response. Safety profiles (cytokine release syndrome [CRS] and immune effector cell-associated neurotoxicity syndrome[ICANS]) were comparable between groups (P>0.05). Multivariate analysis identified poor 3-month treatment response (HR=32.075, P=0.015) and higher pre-treatment lines (HR=8.6, P=0.036) as independent risk factors for PFS. Delayed B-cell recovery and low baseline lymphocyte counts were associated with worse OS. ConclusionCAR-T therapy is feasible and safe for relapsed/refractory ileocecal lymphoma, but short-term efficacy (ORR, PFS) is inferior to non-ileocecal patients. High CAR-T expansion did not improve long-term outcomes. Early response assessment (3 months), baseline lymphocyte levels, and B-cell recovery are critical prognostic indicators, suggesting the need for optimized timing and combination strategies to enhance efficacy.

High genomic complexity is linked to poor prognosis in chronic lymphocytic leukaemia (CLL), but its independent prognostic value remains uncertain amid emerging biomarkers. We analysed copy number alterations (CNA) in 495 treatment-naive patients from three randomized trials (CLL4, ADMIRE, ARCTIC), incorporating IGHV status, telomere length (TL), targeted sequencing, and DNA-methylation subtypes. Patients harboured low (LGC, 0-2 CNAs; n=334), intermediate (IGC, 3-4 CNAs; n=97), or high (HGC, [&ge;]5 CNAs; n=64) genomic complexity. U-CLL (81%, p<0.001) and short TL (61%, p<0.05) were enriched in HGC, and TL inversely correlated with CNA burden ({tau} = -0.147, p<0.001). 62% of HGC patients were n-CLL. TP53 dysfunction was associated with HGC (36%, p<0.001). Trisomy 12 and NOTCH1 mutations, were enriched in LGC (p<0.001). HGC predicted shorter progression-free and overall survival in all univariate models but only remained independently prognostic for OS only in CLL4 (HR=1.61, p=0.02). Of 64 HGC patients, 23 had TP53 dysfunction; 92% of TP53 wild-type cases had other high-risk features (TL-S, U-CLL, or n- CLL). HGC is associated with adverse outcomes but may reflect underlying biological risk rather than serve as an independent biomarker. Its interplay with telomere attrition, immunogenetics, and epigenetic subtype warrants further validation in targeted therapy-treated cohorts.

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.

BackgroundPatients with relapsed/refractory multiple myeloma (RRMM) and high-risk genetic abnormalities such as del(17p) and TP53 mutation have poor response to standard therapies and shorter survival compared to patients without these aberrations. Here, we investigated the activity and mechanism of action of peptide-drug conjugate melphalan flufenamide (melflufen) in TP53 wild type (TP53wt) and mutant (TP53mut) myeloma models and assessed the efficacy of melflufen in patients with del(17p) and/or TP53 mutation. MethodsWe evaluated melflufen activity ex vivo in 24 myeloma bone marrow (BM) samples and explored indicators of response from single cell RNA sequencing (scRNAseq) profiles. The efficacy of melflufen vs. control treatments was further investigated in TP53-/- and parental TP53wt myeloma cell lines. DNA damage, apoptosis kinetics, mitochondrial function, plus transcriptomic and metabolic data were analyzed to understand the mechanisms responsible for melflufen activity in the absence of p53. Patient outcome data from the OCEAN phase III clinical trial (NCT03151811), which investigated the clinical activity of melflufen in RRMM, were statistically analyzed to assess the impact of del(17p) and TP53 mutation on clinical response. ResultsBM plasma cell (PC) response to melflufen was independent of TP53 mutation status, with melflufen active in del(17p), TP53mut, and TP53wt samples. Differential analysis of scRNAseq data demonstrated that melflufen sensitive PCs had lower expression of p53 target genes and higher expression of genes associated with DNA damage repair and cell cycle checkpoints. Analysis of TP53-/- and TP53wt cell lines showed superior efficacy of melflufen in comparison to melphalan or cyclophosphamide. In the presence and absence of functional p53, melflufen robustly induced apoptosis, DNA damage, and mitochondrial dysfunction. In TP53-/- cells, melflufen treatment led to distinct changes in expression of genes associated with cell cycle checkpoint and apoptosis, which were not observed with melphalan treatment. Notably, post-hoc analysis of the OCEAN trial del(17p) patient population demonstrated favorable progression free survival in the del(17p) subgroup treated with melflufen plus dexamethasone compared to the pomalidomide plus dexamethasone arm. ConclusionsOur insights into the molecular mechanisms of melflufen activity in TP53mut myeloma support its clinical efficacy and application in the del(17p) and TP53mut patient population. Trial registrationNCT03151811, registration 2017-05-09.

Background CD19-directed CAR-T cell therapy can induce durable remissions in chronic lymphocytic leukemia (CLL), but response rates are lower than in other B-cell malignancies, in part because CLL is characterized by T-cell dysfunction, defective immune synapse formation, and impaired target-cell co-stimulation. Lenalidomide is an immunomodulatory drug with the potential to act on both sides of the CAR-T/CLL interface by improving T-cell fitness and modifying malignant B-cell susceptibility to immune engagement. Methods We are conducting an open-label, non-randomized phase I/II clinical trial VTB-CLL002 (ClinicalTrials.gov identifier: NCT06762431) evaluating the safety and efficacy of CD19 CAR-T cell therapy combined with concomitant lenalidomide in patients with relapsed or refractory CLL and small lymphocytic lymphoma followed by lenalidomide maintenance. The primary endpoint was safety. The secondary endpoint included overall response rate (ORR), complete response (CR), progression-free survival (PFS) and overall survival (OS). Results Twelve patients were treated. The median age was 60 years and the median number of prior lines of therapy was 2. All patients were BTK inhibitor-naive, and all had measurable disease at the time of infusion. CAR-T manufacturing was successful in all patients. All treated patients achieved complete remission, with a median time to response of 1 month. CAR T-cells expansion was observed in all patients, with a median peak expansion of 137 cells/L and a median time to peak expansion of 14 days. CAR T-cells remained detectable at the last follow-up in all patients, with persistence documented up to 24 months. At dose levels 2-3, eight of nine patients had ongoing MRD-negative responses at the time of analysis. Toxicity was clinically meaningful. Cytokine release syndrome (CRS) occurred in all patients, with severe CRS observed in 2 of 12 patients. ICANS occurred in 5 of 12 patients, including severe ICANS in 4 of 12 patients. One patient developed late grade 4 ICANS temporally associated with lenalidomide reintroduction and secondary CAR-T expansion. Early and late immune effector cell-associated hematotoxicity were common. In mechanistic studies, lenalidomide enhanced CAR-T proliferation and cytotoxicity, shifted CAR-T cells toward effector-associated phenotypes, reduced selected exhaustion markers during repeated antigen challenge, and increased IL-2 and IFN-{gamma} secretion. Lenalidomide also increased CAR-T/CLL conjugate formation and upregulated CD54/ICAM-1 on CLL target cells without broad induction of CD80, CD86, or CD40, consistent with improved adhesive target-cell engagement rather than classical co-stimulation. Transcriptomic profiling supported enhanced Th1/cytotoxic and T-cell activation-associated programs with lower T reg -associated genes in lenalidomide-treated CAR-T cells. Conclusions Lenalidomide-augmented CD19 CAR-T therapy demonstrated strong early clinical activity in relapsed/refractory CLL, characterized by deep responses, durable CAR-T persistence, and substantial incidence of immune effector-associated toxicities. These findings support further evaluation of lenalidomide as a rational CAR-T partner in CLL and suggest that its activity may involve both improved CAR-T fitness and enhanced target-cell engagement. Future studies should optimize lenalidomide timing and dosing to preserve response depth while reducing delayed immune-effector toxicity.