Blood Cancer Journal

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

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.

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.

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.

Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma characterized by heterogeneous clinical courses despite a common pathobiological initiating event. In this work we explore the genomic variants that characterize MCL and integrate transcriptomic data to comprehensively describe MCL biology. We performed whole exome sequencing (WES) on 28 tumor-normal pairs (lymph node and skin, respectively), as well as whole genome sequencing (WGS) and RNA sequencing on subsets of samples. We used established DNA and RNA analysis pipelines to detect single-nucleotide variants (SNV) and indels, structural variants, copy-number alterations, and RNA fusions. The canonical t(11;14)(q13;q32) CCND1::IGH translocation was detected in 8 of 10 WGS samples. Structural variant analysis additionally identified recurrent rearrangements involving KMT2A and PAFAH1B2. SNV and indel analyses revealed frequent mutations in ATM, TP53, CCND1, IGH, and NOTCH1. ATM exhibited diverse variant classes, including missense mutations, frameshift mutations, deletions, and duplications, while all detected NOTCH1 mutations were predicted loss-of-function frameshift variants. Copy-number analysis identified recurrent losses affecting DNA damage response genes, including TP53 and ATM, and recurrent gains involving transcriptional regulators and oncogenic signaling genes. Integrated pathway analysis demonstrated enrichment of transcriptional misregulation, DNA repair, PI3K/AKT signaling, and interleukin signaling pathways. We also identified recurrent alterations in candidate genes, including ASXL1, suggesting additional mechanisms of epigenetic dysregulation in MCL. Together, these findings provide a comprehensive description of somatic alterations in MCL and demonstrate that diverse genomic lesions converge on common pathways involved in genomic instability, transcriptional regulation, and tumor survival.

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.

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.

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.

Natural killer (NK) cell neoplasms are a diverse group of entities with often nonspecific clinical presentations, making immunophenotyping essential for diagnosis. Immunophenotyping by flow cytometry can identify clonal NK cell populations by detecting restricted expression patterns of NK cell receptors such as killer cell immunoglobulin-like receptors (KIRs). However, reactive NK cells may also demonstrate KIR restriction through expansion of self-KIR-expressing NK cells, leading to identification of NK clones of uncertain significance (NK-CUS). A well-described reactive NK subset, termed "adaptive" NK cells, arises in response to cytomegalovirus (CMV) infection or reactivation, often appears KIR-restricted, and is defined by coexpression of CD57 and the activating receptor NKG2C. Because CMV reactivation is common among patients undergoing evaluation for hematolymphoid malignancy, we hypothesized that NK-CUS may frequently correspond to this non-neoplastic adaptive NK cell subset. Here, we describe a flow cytometry panel for immunophenotypic characterization of cytotoxic lymphocytes that includes NKG2C, enabling detection of non-neoplastic adaptive NK cells. We show that NK-CUS frequently represent reactive NKG2C+ adaptive NK cells. We describe several cases that meet diagnostic criteria for NK-large granular lymphocytic leukemia (NK-LGLL) and demonstrate that the NK cell clones are non-neoplastic NKG2C+ adaptive NK cells arising in the setting of CMV viremia. Further, we show that NKG2C expression is uncommon by cytotoxic lymphocyte malignancies with recurrent molecular or cytogenetic abnormalities. Collectively, we demonstrate that NKG2C has a high specificity for reactive NK cell populations, and its inclusion in NK cell immunophenotyping panels is a useful strategy to more reliably distinguish between neoplastic and reactive NK cell populations.

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.

Mantle cell lymphoma (MCL) is one of the deadliest forms of Non-Hodgkins B-cell lymphoma. Typically, patients present with both overexpression of CyclinD1 and secondary mutations identified by genomic sequencing. Although MCL patients may initially respond to treatment, they eventually relapse and succumb to disease, highlighting the essential need to identify new targets for treatment. Here we performed proteomic profiling of healthy B cells and three different forms of B-cell malignancies, including MCL, to define the proteomic signature of MCL. We compared the proteome of each to MCL and identified 10 proteins that are specifically upregulated in MCL. Of these 10 proteins, seven of them show no transcriptional changes and have been overlooked by conventional RNA expression analysis. Further analysis of the proteomic signature reveals potential avenues for dual targeting in CAR T-cell therapy and provides guidance for personalized therapeutics based on protein expression. STATEMENT OF SIGNIFICANCEWe present a resource defining the protein landscape of MCL, CLL, and FL as compared to healthy b cells identified utilizing quantitative proteomics from primary patient samples. Applied to MCL, our results identify 10 proteins specifically upregulated in MCL that may prove to be therapeutic targets to treat the disease.

Flow cytometry can establish T cell clonality by detecting a restricted expression pattern of the T cell receptor (TCR) {beta} constant region (TRBC), expressed in association with CD3. However, T cell neoplasms frequently lose surface expression of the CD3/TCR complex, posing a challenge to demonstrating T cell lineage and clonality. To address this challenge, here we present a 12-color flow cytometry panel, called cytoTCR, to characterize cytoplasmic expression of CD3/TCR complex components. We apply cytoTCR to 38 patient specimens with immunophenotypically abnormal T cell populations, demonstrating this approach can efficiently establish T cell lineage and clonality in challenging T cell neoplasms that have lost surface CD3 expression. While we show that natural killer (NK)-lineage neoplasms can express cytoplasmic CD3 at similar levels to T cells, we show that absent expression of cytoplasmic TCR components by mature lymphocytes can help confirm NK cell lineage. We demonstrate that cytoTCR can detect cytoplasmic TRBC-restriction in challenging cases of null-phenotype anaplastic large cell lymphoma, which lack surface expression of pan-T cell antigens. In cases of T-lymphoblastic leukemia, cytoTCR shows that cytoplasmic TRBC expression matches the expected developmental stage of the leukemia. Finally, we use cytoTCR to characterize atypical cCD3-CD7- T cells in a patient with a history of T-lymphoblastic leukemia as well as recent CAR-T therapy, showing that this atypical population is polytypic and represents CAR-T product rather than residual disease. Our study presents a broadly applicable flow cytometric approach to simultaneously assess T cell lineage and clonality in suspected T lineage populations with absent surface CD3 expression.

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.

Diffuse Large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma in the Western world. First-line immunochemotherapy fails in approximately 30-40% of patients, with refractory and relapse patients presenting a dismal prognosis. Currently, these high-risk patients cannot be accurately identified at diagnosis. Using statistical modeling and machine learning approaches applied to large public DLBCL datasets, we identified a novel predictive signature based on the reactivation of eight normally silent tissue-dependent genes associated with survival. We then developed a multiplex RT-MLPseq based assay, compatible with formalin-fixed paraffin-embedded (FFPE) samples and transferable into routine clinical practice, enabling analysis of expression of these eight genes and validated their prognosis impact in an independent real-life cohort. This signature could be integrated with current prognostic indices and molecular classifications to improve patient stratification and guide treatment selection toward a personalized theragnostic approach, thereby enhancing management of non-responder patients. Data Sharing StatementFor access to original data, please contact: anouk.emadali@univ-grenoble-alpes.fr Key points- Ectopic activation of 8 tissue-specific genes defines a robust prognostic signature for survival stratification in DLBCL patients - A FFPE-compatible RT-MLPseq assay enables clinical use and improves risk stratification beyond IPI and COO, especially in high-risk patients

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.

Multiple myeloma (MM) is a plasma cell malignancy that frequently harbors activating mutations in NRAS and KRAS oncogenes. Previous clinical trials targeting the Ras/MAPK oncogenic pathway with MEK inhibitors (MEKi) were met with limited efficacy, and newer generation of Ras inhibitors (RASi) have not been specifically evaluated in MM patients. To investigate the vulnerabilities of Ras-mutant MM to targeted therapies, we examined the sensitivity of a panel of human MM cell lines to the RASi RMC-6236 (daraxonrasib) and the MEKi trametinib. Although Ras-mutant MM cells are responsive to oncogenic Ras signaling and are sensitive to RAS inhibition, their sensitivity to MEK inhibition is heterogeneous. Mechanistic studies revealed that c-Myc protein is destabilized by MEK inhibition only in MEKi-sensitive MM cells but not in MEKi-resistant cells, and pharmacological and genetic stabilization of c-Myc is sufficient to confer MEKi resistance. In contrast, Ras inhibition reduced c-Myc protein across all MM cell lines tested, regardless of their dependency on the MAPK pathway, and c-Myc expression was insufficient to promote RASi resistance. Together, these findings demonstrate that c-Myc protein stability differentiates the response of Ras-mutant MM cells to Ras and MEK inhibition, and suggest that direct targeting of the Ras oncoprotein, rather than its downstream MAPK pathway, may present a more effective strategy.

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.

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.