ABSTRACT Background The level of measurable residual disease (MRD) is one of the most important features correlating depths of response and long‐term outcomes in multiple myeloma (MM) and MRD evaluation is currently the gold standard tool for assessing treatment response. Nevertheless, reproducibility across laboratories is a major concern, as discrepancies among results make comparability impractical. Aims: herein, we report preliminary results from the “Italian MM‐MRD network” project. Patients & Methods MRD in bone marrow (BM) samples have been measured from newly diagnosed MM patients using next‐generation flow‐cytometry (NGF) or next‐generation sequencing (NGS) approaches in different laboratories. Results The NGF workgroup (7 laboratories) implemented the Euro‐Flow Standard‐Operating‐Protocol to reach minimum 1 × 10−5 sensitivity. The inter‐operator retrospective study (Stage 1) showed high inter‐center concordance in monoclonal plasma cells detection (ICC = 0.90, p < 0.001), whereas moderate concordance was observed in the inter‐laboratory correlation (Stage 2) in in‐vivo samples (ICC = 0.63, p < 0.001), reaching a median limit‐of‐detection (LOD) and limit‐of‐quantification (LOQ) of 8 × 10−6 and 2 × 10−5, respectively. Greater variability was also observed in the analysis of other BM cell populations. The NGS workgroup (4 laboratories) employed a targeted amplicon‐based approach to detect clonotypic IGH/IGK gene rearrangements in diagnostic samples, subsequently used to track MRD in mock samples. The experimental design was divided into three quality‐control (QC) rounds, focused on finding a shared strategy for clonotype identification (QC1: 100% concordance among centers), or quantifying MRD in mock samples (concordance: 81% [QC2]; 91% [QC3]). The 10−5‐sensitivity level was successfully reached in most of tested dilutions (QC2: 19/20 = 95%; QC3: 19/23 = 83%). Conclusion Overall, this pilot study provided preliminary data for MRD harmonization across Italian centers, paving the way for an expanded network, aiming at reducing variability, improving comparability, and enabling broader use of MRD‐monitoring in clinical practice.

Background/Objectives: FcγRIIIA presents a single nucleotide polymorphism at position 158 (V/F), which affects its binding affinity to the fragment crystallizable (Fc) of antibodies (Abs). In the presence of immune complexes, FcγRIIIA can mediate the inflammatory signaling, severity of bone disease, and osteoclastogenic activity. Based on this functional relevance, we hypothesized that the FcγRIIIA F158V polymorphism may influence the clinical presentation of multiple myeloma (MM). Methods: FcγRIIIA F158V genotyping was performed on genomic DNA extracted from peripheral blood samples of patients affected by MM or asymptomatic conditions named MGUS and SMM. We compared the allele frequency of FcγRIIIA-F158V polymorphism in 72 MM, 42 MGUS and 31 SMM and evaluated the association with clinical features and occurrence of high-risk chromosome abnormalities. Targeted NGS mutation analysis was performed on genomic DNA isolated from purified CD138+ bone marrow plasma cells (BMPCs) of 41 patients, to evaluate the association between somatic mutations and the FcγRIIIA F158V genotype. Results: the FcγRIIIA-158 V/V homozygous genotype was associated with high-risk cytogenetics, anemia, high beta-2 microglobulin levels, and more than 10 osteolytic lesions. V/V homozygous genotype was significantly associated with at least one mutation in RAS pathway genes (N-RAS, K-RAS or B-RAF). In the immune microenvironment, patients carrying the V/V homozygous genotype had a higher percentage of CD14+CD16++ non-conventional inflammatory monocytes than the V/F or FF genotype. Conclusions: Our study contributes to a better understanding of the interactions between genetic variants, tumor microenvironment, and therapeutic response in plasma cell dyscrasias, to identify molecular biomarkers for precision medicine in MM, MGUS and SMM.

Clonal hematopoiesis of indeterminate potential (CHIP) is a common age-related phenomenon. CHIP is prevalent in multiple myeloma (MM) patients, and several lines of investigations suggest it might be relevant for MM pathogenesis and clinical course. Phylogenetic studies indicate that CHIP and MM do not share a clonal origin. CHIP does not consistently affect survival in MM. However, CHIP has been associated with increased treatment-related toxicities. Recent single-cell RNA-sequencing data suggest that CHIP is associated with a more inflammatory and immunosuppressive tumor microenvironment (TME), characterized by dysfunctional myeloid and T cells. Furthermore, growing evidence highlights how anti-MM therapies such as alkylators and immunomodulatory drugs can favor the expansion of pre-existing mutant clones. Collectively, these data suggest a bidirectional interplay in which CHIP, acting as a modifier of the TME, may amplify inflammation driven by MM plasma cells and therapy, ultimately affecting MM progression and therapeutic response, while treatment pressure may reshape CHIP clonal architecture. Understanding CHIP dynamics in the context of MM treatment is therefore crucial to optimize therapeutic strategies, anticipate toxicities, and guide tailored approaches. This review summarizes current evidence supporting a translational and clinical impact of CHIP in MM.

Background: Multiple myeloma (MM) is always preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). While these precursor conditions are asymptomatic, they are not entirely benign and carry a lifelong risk of progression to MM. Unlike other cancers defined by pathology, malignant transformation from MGUS or SMM to MM has so far relied on demonstration of clinical end-organ damage or high tumor cell burden (i.e. SLiM criteria, SV Rajkumar Lancet Onc 2014), as morphology and cytogenetics cannot reliably distinguish them. Methods: To identify genomic features underlying clinical behavior in MGUS and SMM, a total of 374 patients with SMM (n= 290) or MGUS (n=84) with available whole exome (whole exome sequencing, WES, n=190) or whole genome sequencing (whole genome sequencing WGS, n=184) were included in the study. Overall, none of the patients had evidence of SLiM or CRAB criteria at the time of the biopsy. Based on the center of origin and availability of WES and WGS, 227 and 97 patients were used to form our study training and validation set. The study was designed in two parts: 1) defining a new classification for MM precursor conditions; 2) integrating genomics to existing prognostic score to improve accuracy in identifying patients at imminent risk of progression. Overall, 68 patients in the training set were either lost to follow-up or enrolled in a clinical trial and they were only included in the classification part of the study. Results: By leveraging the distribution of established MM-defining genomic events (Maura et al. JCO 2024) in the training set, we developed a workflow that differentiates MGUS and SMM into two genomically distinct entities: one with evidence of neoplastic transformation (i.e., genomic MM) and one without (i.e., genomic MGUS). Overall, 39% of MGUS and 91% of SMM cases had genomic evidence of neoplastic transformation in the training set (i.e., genomic MM). When we applied the same workflow and classification to the validation set, 46% and 83% of MGUS and SMM had evidence of neoplastic transformation, respectively. Importantly, none of the patients classified as genomic-MGUS progressed to MM in either the training or validation cohorts, with a median follow-up of 40 and 61 months, respectively. Moreover, all patients with IMWG 2/20/20 high risk SMM were classified as genomic MM in both the training and validation set. Interestingly, 5 out of 62 (8%) patients enrolled in early intervention trials for high risk SMM were genomically classified as genomic-MGUS. Overall, these data demonstrated that the MM genomic background and neoplastic transformation can be acquired very early in time, even before the SMM phase, in line with what observed in solid tumors. To avoid overestimating the prognostic impact of myeloma-defining genomic events—which are, by definition, enriched in the genomic-MM group compared to genomic-MGUS—we restricted our prognostic analysis to patients in whom transformation has already occurred (genomic-MM). In fact, distinct genomic drivers may predict malignant transformation but not necessarily the risk of imminent progression to MM once transformation has occurred. After running 5-fold cross validations corrected for IMWG 2/20/20, only presence of RAS mutations, genomic events involving MYC, presence of neoplastic copy number variant (CNV) signatures and APOBEC mutagenesis were predictive of early progression in the training set. Adding the presence of high-risk myeloma-defining genomic events to the IMWG 2/20/20 model (i.e., genomic-IMWG) significantly improves the accuracy of predicting MM progression in both the training (c-index 0.69 vs. 0.74) and validation sets (c-index 0.74 vs. 0.79), compared to the IMWG 2/20/20 alone. Conclusion: By leveraging comprehensive genomic profiling integrated with the clinical IMWG 2/20/20 risk score, we propose a novel classification and prognostication system for MM precursor conditions, which will better define patients who may benefit from early therapeutic intervention versus watch-and-wait monitoring.

Multiple myeloma (MM) is consistently preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). While these precursor conditions are asymptomatic, they are not entirely benign and carry a lifelong risk of progression to MM. Unlike other cancers defined by pathology, malignant transformation from MGUS or SMM to MM has so far relied on demonstration of clinical end-organ damage as morphology and cytogenetics cannot reliably distinguish them. In this study, using genomic data from 374 patients with MGUS or SMM (277 training, 97 validation), to our knowledge, we demonstrate for the first time the ability to identify malignant transformation in MGUS and SMM. We introduce the concept of genomic MM and genomic MGUS to differentiate the subsets of MGUS and SMM that are biologically malignant with genomic features indistinguishable from MM from the subset that is premalignant and unlikely to progress to malignancy. Importantly, we find that most SMM has biological features of malignant transformation indistinguishable from MM. As expected, this subset that we consider having genomic MM is associated with a high risk of progression to MM although some patients remained progression-free beyond 5 years. Conversely, 60% of MGUS and 10% of SMM have no evidence of malignant transformation (genomic MGUS), with no progression during follow-up. Integration of genomic features with the 2/20/20 International Myeloma Working Group model significantly improved the prediction of progression among genomic MM. These findings support the use of genomic criteria to refine the classification and the risk stratification in myeloma precursor conditions.

Multiple myeloma (MM) initiation is dictated by genomic events. However, its progression from asymptomatic stages to an aggressive disease that ultimately fails to respond to treatments is also dependent on changes of the tumor microenvironment (TME). Clonal hematopoiesis of indeterminate potential (CHIP) is a prevalent clonal condition of the hematopoietic stem cell whose presence is causally linked to a more inflamed microenvironment. Here, we demonstrate in 106 patients with MM that CHIP is frequently coexisting with MM at diagnosis, associates with a more advanced Revised International Staging System stage and higher age, and has a nonsignificant trend toward lower median hemoglobin. In our cohort, the 2 conditions do not share a clonal origin. Single-cell RNA sequencing in 16 patients with MM highlights significant TME changes when CHIP is present: decreased naive T cells, a proinflammatory TME, decreased antigen-presenting function by dendritic cells, and expression of exhaustion markers in CD8 cells. Inferred interactions between cell types in CHIP-positive TME suggested that especially monocytes, T cells, and clonal plasma cells may have a prominent role in mediating inflammation, immune evasion, and pro-survival signals in favor of MM cells. Altogether, our data reveal that, in the presence of CHIP, the TME of MM at diagnosis is significantly disrupted in line with what is usually found in more advanced disease, with potential translational implications. Our data highlight the relevance of this association and prompt for further studies on the modifier role of CHIP in the MM TME.

Multiple myeloma (MM) is characterized by genetic abnormalities in plasma cells, requiring precise genomic characterization for effective risk stratification and treatment. This study presents the Unique Molecular Assay (UMA) panel, a targeted DNA-sequencing approach designed to capture critical genomic aberrations in MM, including canonical immunoglobulin heavy chain translocations (t-IgH), copy number alterations (CNAs), and mutations in 82 genes. The UMA panel is the first MM sequencing panel validated against traditional methods like FISH and SNP-arrays across two laboratories for clinical-grade accuracy and reproducibility. The study included 150 patients whose DNA samples were analyzed using the UMA panel, achieving a median coverage of 233X with a requirement of ≥4 million reads per sample. The UMA panel demonstrated high concordance with FISH in detecting both CNAs and t-IgH, achieving a balanced accuracy >93%. Moreover, inter-laboratory validation confirmed the robustness and reliability of the panel on genomic alterations calls. Importantly, UMA panel enabled precise risk stratification based on the R2-ISS staging system, identifying high-risk features such as TP53 mutations and genome-wide CNAs. This comprehensive and cost-effective genomic profiling tool supports clinical decision-making and supports personalized treatment strategies in MM. The validated performance and scalability of the UMA panel suggest its potential to complement traditional diagnostic methods, offering detailed insights into the genomic landscape of MM.

Warm autoimmune hemolytic anemia (wAIHA) is a rare disorder caused by IgG autoantibodies targeting autologous erythrocytes. These antibodies bind specific epitopes on red blood cells, leading to extra-vascular hemolysis, primarily in the spleen. Standard treatment includes corticosteroids as first-line therapy and rituximab as second-line, with an efficacy exceeding 80%. However, responses are often transient, and relapse occurs in approximately 50% of patients within two years. The underlying mechanisms of relapse and progression to chronic disease remain poorly understood. To explore these aspects, we performed single-cell RNA sequencing (scRNA-seq) on bone marrow (BM) samples from n=9 wAIHA patients and n=4 healthy donors (HD) as part of the CYTOPAN protocol, approved by the local ethics committee and conducted in accordance with the Helsinki Declaration. Patient samples included five at diagnosis (under prednisone), three at remission (six months off therapy), and four at relapse following rituximab treatment. B-cell subpopulations were enriched in wAIHA patients compared to HD (15%, IQR 10–19 at diagnosis; 14%, IQR 10–15 at remission; versus 5%, IQR 3–5 in HD). Notably, mature B-cells were higher at diagnosis (72%, IQR 65–95 vs 42%, IQR 36–48 at remission and 37%, IQR 30–48 at relapse), whereas immature B-cells (pre-B and pro-B) were increased at remission and relapse (20%, IQR 17–22 and 28%, IQR 23–31 vs 19%, IQR 4–20 at diagnosis). Plasma cells (PC) were significantly more abundant at relapse. Longitudinal sampling of the three patients at remission further confirmed expansion of immature B-cell subsets. Functional analysis using GO and GSEA revealed stage-specific gene expression: immunoglobulin production, protein synthesis, and adaptive immune response genes were upregulated at diagnosis; cell-cycle and division-related genes were enriched at remission and relapse. These included pathways related to TNF-alpha signalling, estrogen/spermatogenesis, and E2F targets. Comparing remission to relapse highlighted an increased expression of TNF-alpha, IFN-alpha and gamma, apoptotic and hypoxic markers, KRAS, and inflammatory signals at relapse, while remission was marked by upregulation of microbial immunity and allograft rejection pathways, suggesting a more immature autoinflammatory profile at relapse. Marker gene analysis mirrored these findings, with upregulation of IGKV3D-11, IGHV3-74, IGHV3-7, CD27, IFNG-AS1, and FAM177B at diagnosis; and CD19, BTK, SYK, CD38, CD69, IGLL1, RAG1/2, and VPREB1/3 at remission/relapse. Inclusion of PCs reinforced the association with hypoxia, TNF-alpha, and glycolysis pathways at relapse, and confirmed absence of CD20 expression on these cells. Gene usage analysis, excluding IgM and IgD (consistently expressed across all disease stages), identified predominant usage of IGHG1 and IGHA1 constant regions and IGHV4-31 and IGHV4-34 variable genes at relapse. These preliminary findings suggest a shift toward an immature B-cell profile and accumulation of CD20-negative plasma cells in wAIHA patients at relapse post-rituximab, along with activation of genes involved in cell proliferation, TNF-alpha signaling, and CD38 expression, potentially revealing novel therapeutic targets.

ABSTRACT:Multiple myeloma (MM) is driven by clonal plasma cell (cPC)-intrinsic factors and changes in the tumor microenvironment (TME). To investigate whether residual polyclonal PCs (pPCs) are disrupted, single-cell (sc) RNA sequencing (scRNA-seq) and sc B-cell receptor analysis were applied in a cohort of 46 samples with PC dyscrasias and 21 healthy donors (HDs). Of 234 789 PCs, 64 432 were genotypically identified as pPCs with frequencies decreasing over different disease stages, from 23.66% in monoclonal gammopathy of undetermined significance to 3.23% in MMs (P = .00012). Both cPCs and pPCs had a comparable expression of typical lineage markers (ie, CD38 and CD138), whereas others were more variable (CD27 and ITGB7). Only cPCs overexpressed oncogenes (eg, CCND1/2 and NSD2), but CCND3 was often expressed in pPCs. B-cell maturation antigen was expressed on both pPCs and cPCs, whereas GPRC5D was mostly upregulated in cPCs with implications for on-target, off-tumor activity of targeted immunotherapies. In comparison with HDs, pPCs from patients showed upregulated autophagy and disrupted interaction with TME. Importantly, interferon-related pathways were significantly enriched in pPCs from patients vs HDs (adjusted P < .05) showing an inflamed phenotype affecting genotypically normal PCs. The function of pPCs was consequently affected and correlated with immunoparesis, driven by disrupted cellular interactions with TME. Leveraging our scRNA-seq data, we derived a "healthy PC signature" that could be applied to bulk transcriptomics from the CoMMpass data set and predicted significantly better progression-free survival and overall survival (log-rank P < .05 for both). Our findings show that genotypic sc identification of pPCs in PC dyscrasias has relevant pathogenic and clinical implications.

The combination of hypomethylating agents (HMA) with Venetoclax has become standard of care in patients with Acute Myeloid Leukemia (AML) unfit for intensive chemotherapy. Genetic and cellular resistance mechanisms, such as RAS and TP53 mutations as well as monocytic differentiation, were recently unveiled (Bataller et al. Blood Adv 2024). Nonetheless, a comprehensive understanding of the resistance mechanisms to HMA-Venetoclax is lacking. To gain further insights into cell-intrinsic and -extrinsic resistance mechanisms, we analyzed single-cell (sc) RNA-sequencing (RNA-seq) data coupled with TCR repertoire profiling from 10 longitudinal sample pairs from AML patients treated with HMA-Venetoclax. Bone marrow (BM) mononuclear cells from 10 AML patients were collected at diagnosis (TP1) and after the first treatment cycle (TP2). Median age at diagnosis was 70 years, 5 patients were considered at intermediate risk and 5 adverse as per the European LeukemiaNet 2022 classification. 5' sc-RNA-seq samples coupled with TCR profiling (10X Genomics) were analyzed with a bioinformatic pipeline based on Seurat, ScRepertoire and Bone Marrow Map packages in the R software. Mutational status was obtained by Next Generation Sequencing of bulk DNA at TP1. Six patients achieved Complete Response with or without complete count recovery (CR/CRi), 2 (20%) were refractory and 2 (20%) experienced early relapse. At TP1, 3 patients (30%) had TP53 abnormalities/complex karyotype; recurrently mutated genes included ASXL1 (30%), DNMT3A (30%) IDH1 (30%) NRAS (30%) and TET2 (30%). Non-responders were enriched in TP53 and RAS mutations. A total of 126.075 cells were analyzed to assess the role of BM architecture in treatment resistance. Of these, 39.697 cells were unbiasedly classified as Hematopoietic Stem Cells (HSC), Multipotent Progenitors (MPP), Granulocyte Monocyte Progenitors (GMP) and cells of monocytic derivation. Longitudinal differential expression analysis of leukemic cells, comparing non-responders to responders, unveiled the upregulation of: i) TNFa signaling via NF-KB in non-responders at TP2, shaped by the overexpression of JUNB and BCL2A1 in immature and monocytic cells respectively; ii) epithelial to mesenchymal transition (EMT), marked by PRSS2, LGALS1 and BASP1 upregulation in immature myeloid cells of non-responders at both timepoints. Furthermore, higher expression of an established leukemic stem cell score (LSC-17, Ng et al. Nature 2016) in the HSCs of non-responders was observed (p<0.001). Given the biological relevance of T-cells in AML, T cell phenotype and TCR repertoire were investigated to assess dynamic changes among T cells. A total of 45.539 T cells with paired TCR sequences were recovered. TCR clonotypes were labeled according to their relative frequency into small (<0.1%) medium (<1%), large (<10%) or hyperexpanded groups (>10%), identifying clonal diversity. From TP1 to TP2, responders showed a significant increase in CD4 naïve, CD8 central memory (CM) and effector memory (EM) subsets, combined with a significant increase in small TCR clonotypes (p<0.001), suggesting dynamic reshaping of TCR repertoire. Non-responders were also characterized by an increase of CD4 naïve cells, but decreased CM and EM cells from TP1 to TP2 (p<0.001). In this setting, no significant variations of TCR diversity were highlighted. Importantly, terminally exhausted CD8 T cells were observed only in patients who progressed during treatment. Finally, patients achieving long term response showed a persistent clonally expanded CD8 Terminally differentiated Effector Memory cell subset (TEMRA), characterized by GZMA/B, ZNF863 overexpression and FOS and GZMK downregulation, suggesting high cytotoxic activity involved in active immunosurveillance. This study provides novel findings associated with response in clonal and immune BM cells after treatment with HMA-Venetoclax in AML. In the context of TP53 and RAS mutation, EMT and inflammatory pathways shape the transcriptional profile of Venetoclax resistance: EMT emerges as a negative predictive factor while inflammatory pathways modulate the expression of Venetoclax targets at different cellular levels according to cell of origin. Increase in TCR diversity and persistence of highly cytotoxic TEMRA clones characterized long term responders while restricted TCR diversity and CD8 T cell exhaustion associated with early relapse.

Introduction The 2022 WHO and ICC classifications confirmed pre-fibrotic PMF (pre-PMF) as a myeloproliferative neoplasm subtype with distinct histopathological and clinical-biological parameters, for which specific prognostic models are still lacking. Aim To identify the most relevant clinical, histological, and molecular data at diagnosis as predictors of outcome in pre-PMF patients (pts) in the real-world setting. Methods The present multicenter, retrospective study included 762 pre-PMF pts who were diagnosed in 7 Italian hematological centers between January 2000 and June 2023 and were followed for a minimum of 6 months. Baseline clinical characteristics and outcome measures (death, thrombotic complications, fibrotic progression, and leukemic evolution) were assessed. Follow-up information was updated in December 2023. Results Median age of the pts included in this study was 62.8 years; 48.3% were male. All cases had no bone marrow fibrosis (MF-0, 28.6%) or only a slight increase in reticulin fibrosis (MF-1, 71.4%). A driver mutation was found in 92.8% of cases: JAK2V617F in 69.9%, CALR in 20.1%, and MPLW515L/K in 2.8%; 55 (7.2%) pts were triple-negative. After a median follow-up of 5.9 years, 168 (22.0%) pts died, 84 (11.0%) experienced fibrotic progression and 40 (5.2%) leukemic evolution. Thrombosis occurred before/at diagnosis in 137 (18.5%) and 64 (8.4%) pts, respectively, compared with 102 (13.4%) pts during follow-up. The 5-, 10-, and 15-year overall survival (OS) rate was 90.9%, 74.5% and 60.1%, respectively. In details, 5-, 10-, and 15-year cumulative incidence of thrombosis was 9.6%, 16.0% and 23.1%, respectively, compared with 4.9%, 12.8% and 20.6% for fibrotic progression, and 2.5%, 6.1% and 8.6% for leukemic evolution. In univariate analysis, factors associated with OS were female sex [Hazard Ratio (HR) 0.59, 95%CI 0.43-0.81], age at diagnosis (HR 2.66 per 10 years, 95%CI 2.25-3.15), previous thrombosis (HR 1.68, 95%CI 1.15-2.43), smoking habit, either former (HR 1.59, 95%CI 1.03-2.45) or current (HR 1.69, 95%CI 1.08-2.65), hypertension (HR 1.77, 95%CI 1.28-2.44) and diabetes (HR 2.02, 95%CI 1.37-2.98), hemoglobin (Hb) level (HR 0.81 per 1 mg/dL, 95%CI 0.75-0.86), white blood cell (WBC) (HR 1.06 per 1 x109/L, 95%CI 1.04-1.08) and platelet (PLT) count (HR 0.95 per 100 x109/L, 95%CI 0.90-1.00), peripheral blood (PB) blasts (HR 1.81 per 1%, 95%CI 1.33-2.48), LDH level (HR 1.08 per 100 IU/L, 95%CI 1.03-1.12), circulating CD34+ cells count (HR 1.07 per 10 x106/L, 95%CI 1.05-1.10), spleen diameter (HR 1.06 per cm below left costal margin, 95%CI 1.00-1.12), CALR mutations (HR 0.46, 95%CI 0.29-0.72), in particular type 1 (HR 0.35, 95%CI 0.19-0.65), presence of cytogenetic abnormalities (HR 1.81, 95%CI 1.15-2.84), and MF-1 reticulin fibrosis (HR 1.51, 95%CI 1.06-2.13). Moreover, we confirmed the prognostic relevance of IPSS, DIPSS, and DIPSS-plus (data not shown). The same was also for IPSET-Thrombosis [intermediate risk (IR) vs low risk (LR), HR 2.77, 95%CI 1.36-5.64; high risk (HR) vs LR, HR 5.18, 95%CI 2.79-9.64] and revised IPSET [LR vs very low risk (VLR), HR 3.66, 95%CI 1.23-10.87; IR vs VLR, HR 17.3, 95%CI 5.99-49.8; HR vs VLR, HR 17.1, 95%CI 6.28-46.8]. In multivariable analyses adjusted for gender and age at diagnosis, the following variables were confirmed to be associated with OS: diabetes (HR 1.70, 95%CI 1.15-2.51), Hb level (HR 0.83, 95%CI 0.77-0.89), WBC count (HR 1.05, 95%CI 1.03-1.07), PB blasts (HR 1.81, 95%CI 1.36-2.41), LDH level (HR 1.12, 95%CI 1.06-1.17), circulating CD34+ cells count (HR 1.06, 95%CI 1.04-1.09), spleen diameter (HR 1.16, 95%CI 1.09-1.24), CALR mutations (HR 0.54, 95%CI 0.34-0.85), in particular type 1 (HR 0.42, 95%CI 0.22-0.79), presence of cytogenetic abnormalities (HR 1.57, 95%CI 0.99-2.49), and MF-1 reticulin fibrosis (HR 1.54, 95%CI 1.08-2.19). Discussion In clinical practice, existing tools for overt PMF are often extended for prognosis and management guidance in pre-PMF. However, the daily concerns in pre-PMF management usually lie more in the prevention of thrombo-hemorrhagic episodes. Therefore, a risk stratification scheme based on clinical-pathological and molecular factors dedicated to pre-PMF would be needed, as it could help physicians not only with prognostication, but also in guiding management decisions. Updated analyses, including data on HMR mutations, will be presented.

IntroductionClonal hematopoiesis of indeterminate potential (CHIP), defined as the presence of somatic mutations in hematopoietic stem cells with a variant allele frequency (VAF) ≥2% in genes involved in hematologic cancers, has been linked to MASLD and severe liver disease.Aimto examine whether CHIP is associated with MASLD-related HCC and its major clinical determinants.Methodspatients with MASLD-related HCC (n=179), patients with advanced fibrosis without HCC (n=263), individuals with simple SLD (n=38) and healthy individuals (n=50) were enrolled. Age, sex, presence of type 2 diabetes (T2D), advanced liver fibrosis, AST and ALT levels were available.DNA was sequenced by the HiSeq 4000/NextSeq2000 platforms (Illumina). Somatic mutations were identified accepting a minimum variant coverage of 20, a minimum alternative allele count of 3 and a VAF between 0.02 and 0.46. Only somatic and variants associated to malignancy or CHIP were included.ResultsCHIP-defining lesions were identified in 92 out of 530 participants (17.3%).CHIP was found in 43 (24.0%) patients with HCC, 41 (15.6%) with advanced fibrosis without HCC, and in 8 (9.1%) without advanced fibrosis (p=0.006).CHIP prevalence was age-dependent, with a spike after the age of 65.The most frequently mutated gene was DNMT3A, followed by TET2, TP53 and ASXL1 (Figure 1).CHIP was associated to HCC independently of sex, diabetes, polygenic risk score of SLD and cirrhosis (OR 1.81, 95%CI 1.10-2.98; p=0.018) but association was lost when correcting for age.TET2 mutations were enriched in HCC and the association between HCC and TET2 remained significant when correcting for age, gender, diabetes, polygenic risk of SLD and cirrhosis (OR 4.35, 95%CI 1.18-16.01; p=0.029).Conclusionwe suggest a possible role of CHIP in the progression of SLD and TET2 mutations showed the strongest enrichment in HCC. Further studies are needed to clarify how CHIP can contribute to hepatic carcinogenesis.

Smoldering multiple myeloma (SMM) is an asymptomatic plasma cell (PC) neoplasm that may evolve with variable frequency into multiple myeloma (MM). SMM is initiated by chromosomal translocations involving the immunoglobulin heavy-chain locus or by hyperdiploidy and evolves through acquisition of additional genetic lesions. In this scenario, we aimed at establishing a reliable analysis pipeline to infer genomic lesions from transcriptomic analysis, by combining single-cell RNA sequencing (scRNA-seq) with B-cell receptor sequencing and copy number abnormality (CNA) analysis to identify clonal PCs at the genetic level along their specific transcriptional landscape. We profiled 20 465 bone marrow PCs derived from 5 patients with SMM/MM and unbiasedly identified clonal and polyclonal PCs. Hyperdiploidy, t(11;14), and t(6;14) were identified at the scRNA level by analysis of chimeric reads. Subclone functional analysis was improved by combining transcriptome with CNA analysis. As examples, we illustrate the different functional properties of a light-chain escape subclone in SMM and of different B-cell and PC subclones in a patient affected by W & auml;ldenstrom macroglobulinemia and SMM. Overall, our data provide a proof of principle for inference of clinically relevant genotypic data from scRNAseq, which in turn will refine functional annotation of the clonal architecture of PC dyscrasias.

Here, we reviewed clinical-morphological data and investigated mutational profiles by NGS in a single-center series of 28 consecutive patients admitted to our hospital between September 2011 and November 2021 for idiopathic hypereosinophilia (HE). Bone marrow (BM) morphology was evaluated in 22 patients: while in six subjects BM was unremarkable, in the remaining cases an increase in BM eosinophils was observed, together with a slight increase in BM fibrosis (MF-1) in 5/22 patients. A total of 4/28 patients had at least one genetic lesion by targeted NGS. In particular, the genes involved were: two each of TET2 and DNMT3A; and one each of JAK2V617F, ASXL1, PPM1D, and ZBTB33. Notably, JAK2V617F and TET2 mutations co-occurred, with the JAK2V617F-mutated sample also carrying TET2 lesions. Median VAF was 21%, with the exception of the oncodriver JAK2V617F, which showed a VAF > 50% in the reported case. Of note, of the four cases bearing lesions, 2/4 had multiple hits in different genes. While in recent years mutational analysis using NGS has proven to be able to differentiate clonal hematopoietic neoplasms from reactive processes in diagnostically difficult cases, we found somatic mutations in only 14.3% of patients who acceded to our hospital for idiopathic HE. More importantly, excluding the JAK2V617F-mutated case with an underlying MPN-Eo diagnosis, NGS was able to identify somatic mutations in only three cases, all older than 70 years. Consequently, the detection of these mutations in idiopathic HE patients should be interpreted with caution and only in the context of other supportive clinical-pathological findings.

Tables, figures, methods and references

Differential gene expression analysis of genes with comulative effect

Multiple Myeloma (MM) is driven by clonal plasma cell (PC)-intrinsic factors and changes in the tumorigenic microenvironment (TME). To investigate if residual polyclonal PCs (pPCs) are disrupted, single-cell (sc) RNAseq and sc B-cell receptor analysis were applied in a cohort of 46 samples with PC dyscrasias and 18 healthy donors (HDs). Out of n= 213,074 CD138pos PCs, 42,717 were genotypically identified as pPCs. Compared to HDs, we detected quantitative and qualitative differences in pPCs of patients showing immunoparesis, where we showed a pro-inflammatory status, driven by specific cellular interactions with TME. Finally, we derived a “hPC signature” that, once inferred in the CoMMpass dataset, was predictive of PFS and OS. Our findings show that genotypic, single-cell identification of pPCs in PC dyscrasias has relevant pathogenic and clinical implications.### Competing Interest StatementN.B. received honoraria for Amgen, GSK, Janssen, Jazz, Pfizer, Takeda. M.C.D.V. served as Advisory Board for Takeda, and on Speakers Bureau for Janssen and GSK. F.P. received honoraria during the last two years for lectures from Novartis, Bristol-Myers Squibb, Abbvie, GSK, Janssen, AOP Orphan and for advisory boards from Novartis, Bristol-Myers Squibb/ Celgene, GSK, Abbvie, AOP Orphan, Janssen, Karyiopharma, Kyowa Kirin and MEI, Sumitomo, Kartos. The remaining Authors declare no competing financial interest.