Smoldering Multiple Myeloma Patients Research Articles

Three-dimensional telomere profiling predicts risk of progression in smoldering multiple myeloma.

Smoldering multiple myeloma (SMM) is a precursor stage that precedes multiple myeloma (MM). SMM is heterogenous with nearly 40% of patients progressing to MM in the first 5 years. The high rate of progression of SMM patients highlights the need for early intervention, which underscores the importance of identifying SMM patients with the highest risk of progression. Several risk stratification models showed utility in identifying high-risk SMM patients; however, these systems showed limited sensitivity. To date, identifying high-risk SMM patients remains an important clinical need. In this study, we present the 3-dimensional telomere profiling as a structural biomarker capable of stratifying SMM patients as a function of genomic instability. Quantifying telomere dysfunction using the TeloView technology showed utility in risk stratification of cancer patients, particularly hematological malignancies. In this study, we analyzed 168 SMM patients. We report an AUC in ROC analysis of 0.8 using a subset of the patients as a training dataset. We then conducted a blind validation on a different cohort and demonstrated a positive predictive value of 85% and negative predictive value of 73%, with sensitivity and specificity of 83% and 76%, respectively. We examined the correlation between the TeloView prediction and the 20-2-20 scoring system, and cytogenetic abnormalities. We report a correlation of 53% with the 20-2-20 scores and over 60% correlation with cytogenetic abnormalities. The result of this study presents the telomere profiling as an effective biomarker able to stratify SMM patients to their respective risk groups with high sensitivity and specificity.

IS IT POSSIBLE TO PREDICT TUMOR PROGRESSION THROUGH GENOMIC CHARACTERIZATION OF MONOCLONAL GAMMOPATHY AND SMOLDERING MULTIPLE MYELOMA?

The study of monoclonal serum proteins has led to the generation of two major theories: one proposing that individuals who had monoclonal proteins without any symptoms or evidence of end-organ damage have a benign condition; the other one suggesting that some individuals with asymptomatic monoclonal proteins may progress to multiple myeloma and thus are affected by a monoclonal gammopathy of undetermined significance (MGUS). Longitudinal studies of subjects with MGUS have supported the second theory. Subsequent studies have characterized and defined the existence of another precursor of multiple myeloma, smoldering multiple myeloma (SMM), intermediate between MGUS and multiple myeloma. Primary molecular events, chromosome translocations, and chromosome number alterations resulting in hyperploidy, required for multiple myeloma development, are already observed in myeloma precursors. MGUS and SMM are heterogeneous conditions with the presence of tumors with distinct pathogenic phenotypes and clinical outcomes. The identification of MGUS and SMM patients with a molecularly defined high risk of progression to MM offers the unique opportunity of early intervention with a therapeutic approach on a low tumor burden.

The genomic profiling of high-risk smoldering myeloma patients treated with an intensive strategy unveils potential markers of resistance and progression

Smoldering multiple myeloma (SMM) precedes multiple myeloma (MM). The risk of progression of SMM patients is not uniform, thus different progression-risk models have been developed, although they are mainly based on clinical parameters. Recently, genomic predictors of progression have been defined for untreated SMM. However, the usefulness of such markers in the context of clinical trials evaluating upfront treatment in high-risk SMM (HR SMM) has not been explored yet, precluding the identification of baseline genomic alterations leading to drug resistance. For this reason, we carried out next-generation sequencing and fluorescent in-situ hybridization studies on 57 HR and ultra-high risk (UHR) SMM patients treated in the phase II GEM-CESAR clinical trial (NCT02415413). DIS3, FAM46C, and FGFR3 mutations, as well as t(4;14) and 1q alterations, were enriched in HR SMM. TRAF3 mutations were specifically associated with UHR SMM but identified cases with improved outcomes. Importantly, novel potential predictors of treatment resistance were identified: NRAS mutations and the co-occurrence of t(4;14) plus FGFR3 mutations were associated with an increased risk of biological progression. In conclusion, we have carried out for the first time a molecular characterization of HR SMM patients treated with an intensive regimen, identifying genomic predictors of poor outcomes in this setting.

Decreased neutrophil function in newly diagnosed multiple myeloma patients is restored with lenalidomide therapy.

Bacterial infections are common and a major cause of morbidity and mortality in multiple myeloma (MM). We have investigated the function of polymorphonuclear leukocyte (PMN), the immune system's first line of defense against bacteria, in peripheral blood (PB) and bone marrow (BM) samples from patients with newly diagnosed MM (NDMM), smoldering MM (SMM), monoclonal gammopathy of undetermined significance (MGUS) and healthy controls. Phagocytosis and oxidative burst in PMN cells from patients and healthy donors were investigated using PhagoTest and PhagoBurst assay. PMN from NDMM, SMM, and MGUS patients had reduced phagocytosis and oxidative burst ability compared with healthy controls. The dysfunction was most prominent in BM samples from MM, SMM, and MGUS patients. Importantly the reduced phagocytosis in MM patients was restored in patients on lenalidomide therapy. Consistently the ability of Escherichia coli stimulated oxidative burst in BM was reduced for the MM, SMM, and MGUS cohort in contrast to the healthy controls and the patients on lenalidomide treatment. Our results show that MM patients have neutrophil dysfunction that could contribute to susceptibility for bacterial infections and that lenalidomide therapy was associated with restored PMN function.

Abstract 6170: Risk stratification of smoldering multiple myeloma using fourier transform infrared spectroscopy of serum

Abstract Background: Smoldering multiple myeloma (SMM) is a state where patients have more than 10% clonal plasma cells or a serum monoclonal protein > 3g/dl but no evidence of end organ damage. Patients with SMM are at risk of developing active multiple myeloma (defined by the CRAB criteria). The best stratification system with respect to the risk of progression to active myeloma is the 20/2/20 (20% plasma cells, M protein >2.0 g/dl, and involved to uninvolved immunoglobulin free light chain ratio >20). The presence of ≥2 criteria predicts a 50 - 60% risk of progression within 2 years. Thus, many patients considered ‘high-risk’ may not progress in this time frame, and patients with ≤1 risk factor can progress to active myeloma. Therefore, improved risk stratification methods are needed to correctly identify patients at high risk of progression. We hypothesized that Fourier Transform InfraRed (FTIR) spectroscopic analysis of serum combined with artificial intelligence (AI) can correctly risk stratify patients with SMM. Methods: We identified 192 SMM patients: 96 who progressed and 96 who did not progress within 2 years of diagnosis. We used stored sera from the time of diagnosis to perform FTIR to determine differences in the spectra of patients who did/did not progress and train an AI algorithm. A randomized 10-fold cross-validation analysis was performed using 172 samples (86 from each group) for training and the remaining 20 for testing to assess the model across 10 independent iterations. Subsequently, we blindly tested the trained model against an independent set of serum samples (N=24) from patients who progressed ≤3 years of diagnosis. Receiver operator characteristic (ROC) analysis was used to assess the FTIR AI algorithm output compared to the clinical outcome data. Results: From the 96 patients who progressed, 48 had complete data for the 20/2/20 risk stratification. 11 patients (23%) had ≤1 of the criteria and so would have been considered at low likelihood to progress. Of the 96 who did not progress within 2 years, 49 had complete data for risk stratification. Of these, 27 (55.1%) met the high-risk criteria of the 20/2/20 classification and would have been predicted to progress within 2 years. FTIR/AI model correctly identified the patients at risk of progression (95% accuracy) with AUC of 0.96. Similarly, it was able to classify patients who did not progress within 2 years of diagnosis (94% accuracy) with an AUC of 0.96. FTIR/AI correctly predicted the patients at risk of progression within 2 years (90%) and 3 years (87.5%) in the blinded validation group with an AUC of 0.89 and 0.82, respectively. Conclusion: Analysis of serum in patients with newly diagnosed SMM with FTIR is able to correctly identify those at risk of progression within 2 or 3 years of diagnosis with an accuracy of ~90% in a blinded external validation study. FTIR may, therefore, be used to assess SMM patient risk before committing to potentially toxic therapy. Citation Format: David Dingli, Darlyna Khonkhammy, Caden Gunnarson, Grant Schlauderaff, Dan Q. Pham, Ali Khammanivong. Risk stratification of smoldering multiple myeloma using fourier transform infrared spectroscopy of serum [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6170.

Abstract 4807: Gaucher disease treatment reduces the progression of smoldering myeloma

Abstract Background: Gaucher disease (GD), a hereditary condition characterized by glucocerebrosidase deficiency, results in the impaired degradation of glucocerebroside (GL1) and its deacetylated form, lyso-GL1 (LGL1). This leads to the accumulation of these substrates primarily in the bone marrow (BM), liver, and spleen. Notably, individuals with GD face an increased risk of plasma cell dyscrasias (PCD) by approximately nine-fold. We present two compelling cases of the regression of smoldering multiple myeloma (SMM) following GD treatment, substantiated by biochemical evidence of lipid-reactive clonal paraproteins (PP) in both patients. Methods: Peripheral blood samples were obtained with IRB approval from patients with concurrent SMM and GD at the Icahn School of Medicine at Mount Sinai. Utilizing C-18 beads coupled with gly-sphingosine-coated liposomes and Sphingosine-1 phosphate bead liposome-based pulldown, we established the binding of glycolipids to serum PP. Results: In a 66-year-old male, hepatosplenomegaly and pancytopenia revealed compound heterozygous N370S/V394L mutations and reduced β-glucosidase activity, confirming Type 1 GD. Laboratory findings included substantial pancytopenia (WBC 2.5 k/µL, Hgb 12.1 g/dL, platelet 44 k/µL) and myeloma markers (M spike 1.58 g/dL, monoclonal IgA λ). A BM biopsy indicated 30% λ-restricted plasma cells. PET-CT showed hepatosplenomegaly without FDG avid lytic lesions, consistent with SMM. Imiglucerase enzyme therapy improved GD biomarkers, decreased hepatosplenomegaly, and blood count improvement (WBC 4.7 k/µL, Hgb 13.6 g/dL, platelet 66 k/µL). Notably, myeloma burden significantly reduced (M-spike 0.94 g/dL, IgA 1373 g/dL and free lambda 26.1 mg/L). Similarly, a 41-year-old female with mild fatigue, Hgb levels of 10.9 g/dL, a platelet count of 169 k/µL, M spike of 0.9 g/dL, IgG κappa on immunofixation, and IgG levels at 1730 mg/dL, indicating SMM. GBA analysis showed homozygous N370S mutation and reduced beta-glucosidase activity consistent with Type 1 GD. Substrate reduction therapy with eliglustat alleviated fatigue, reduced GD biomarkers, normalized cytopenias, and gradually decreased myeloma markers. Laboratory results indicated an M spike of 0.5 g/dL, IgG levels at 1295 mg/dL, and free kappa at 31.3 mg/L. To explore the interaction between clonal immunoglobulins (Ig) and lyso-lipids, we utilized Gly-sphingolipids (see methods) and demonstrated a robust interaction with IgA (patient 1) and IgG (patient 2) paraprotein by western blotting. Conclusion: Our experimental findings confirm specific clonal Ig binding in SMM and GD patients and demonstrate potent and timely benefits from addressing GD in managing SMM patients. This holds significance amid ongoing clinical trials exploring aggressive interventions for SMM, including bispecific and CAR-T therapies. Citation Format: Ravi Prakash Shukla, Anshuman Parekh, Violetta V Leshchenko, Kevin Barley, Deepa Vatti, Daniel Verina, Manisha Balwani, Samir Parekh. Gaucher disease treatment reduces the progression of smoldering myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4807.

Abstract 6848: Analysis of the bone microenvironment cells by single cell transcriptomics reveals altered features in multiple myeloma patients compared to pre-malignant monoclonal gammopathies

Abstract Multiple myeloma (MM) is a malignant plasma cell dyscrasia that can be preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). How the bone microenvironment (BME) changes and contributes to tumoral progression in MM remains unresolved. Published data on BME are mainly produced in vitro and few data are on MGUS/SMM patients. Up to date, no single-cell RNA sequencing database of the BME cells has been described in MM. The aim of this project was to characterize for the first time at single cell level, the BME in patients with newly diagnosed MM (MMD) and MGUS/SMM to identify alterations involved in tumoral progression. From 15 bone biopsies of MMD, MGUS and SMM patients, we depleted CD235a+, CD45+, CD31+, and CD138+ cells to enrich the rare BME non-hemopoietic cells. The CD45-CD31-CD235a-CD138- cells were analyzed by scRNAseq. Data were generated on Chromium 10X Genomics. Cellranger and Seurat pipeline in R software has been used. Cell identities were assigned by manual curation. The trajectory analysis has been made by Monocle3. Decouple R and FindMarkers were used to compare pathways, and differential gene expression (DGE) of the samples throughout the different conditions. A total of 35723 cells were profiled. We identified 13 BME cell type clusters: 7 mesenchymal stromal cells (MSCs) clusters (proliferating MSC, MSC DDK-OPG-, MSC DKK+, MSC RUNX2-, MSC OPG+, MSC Osteogenic, Perivascular MSC) and 6 osteoblasts (OBs) clusters (OB precursor, OB precursor LEPR+, OB BGLAP+, OB SPP1+, OB NFkB+, OB late). Psdeudotime analyses highlighted a complex trajectory of differentiation from more immature cell clusters (proliferating MSC) to late OB cluster, confirming the ability of the dataset to snapshot the in vivo complexity of BME and we highlighted an alteration of the MSCs clusters trajectories starting in SMM samples till a disruption in MM. Pathway analyses, in MMD compared to MGUS/SMM, revealed an upregulation of VEGF and PI3K activity in proliferating MSC, perivascular MSC and OB clusters. TRAIL pathway activity, known to suppress MM cell proliferation, was upregulated in MSC osteogenic and OB BGLAP+ in MGUS. Finally, DGE analyses revealed the modulation of different genes, many not yet described in MM. MGUS MSCs clusters showed an upregulation of FBLN1, SERPNF1 and MMD MSCs clusters dramatically downregulated WISP2 and upregulated SRGN, involved in MM cell proliferation. Interestingly, in SMM samples, we reported an upregulation of genes involved both in the promotion of osteoblastogenesis (SCARA3, SPON2) and inhibition (GREM1), likely underling the attempt to contrast the establishment of altered BME, characteristic of MM. Our approach is able to dissect the complex organization of the BME and to highlight at single cells level the alterations of the BME in patients with MM compared to MGUS and SMM. Citation Format: Paola Storti, Tommaso Torelli, Cristina Manferdini, Denise Toscani, Alessandro Guidi, Rosanna Vescovini, Nicolas Thomas Iannozzi, Benedetta Dalla Palma, Matteo Scita, Federica Librale, Stefania Ricci, Vincenzo Raimondi, Oxana Lungu, Giannalisa Todaro, Gabriella Sammarelli, Emanuela Aleo, Gina Lisignoli, Luca Agnelli, Nicola Giuliani. Analysis of the bone microenvironment cells by single cell transcriptomics reveals altered features in multiple myeloma patients compared to pre-malignant monoclonal gammopathies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6848.

Stromal alterations in patients with monoclonal gammopathy of undetermined significance, smoldering myeloma, and multiple myeloma

AbstractThe hallmark of multiple myeloma (MM) is a clonal plasma cell infiltration in the bone marrow accompanied by myelosuppression and osteolysis. Premalignant stages such as monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic stages such as smoldering myeloma (SMM) can progress to MM. Mesenchymal stromal cells (MSCs) are an integral component of the bone marrow microenvironment and play an important role in osteoblast differentiation and hematopoietic support. Although stromal alterations have been reported in MM contributing to hematopoietic insufficiency and osteolysis, it is not clear whether alterations in MSC already occur in MGUS or SMM. In this study, we analyzed MSCs from MGUS, SMM, and MM regarding their properties and functionality and performed messenger RNA sequencing to find underlying molecular signatures in different disease stages. A high number of senescent cells and a reduced osteogenic differentiation capacity and hematopoietic support were already present in MGUS MSC. As shown by RNA sequencing, there was a broad spectrum of differentially expressed genes including genes of the BMP/TGF-signaling pathway, detected already in MGUS and that clearly increases in patients with SMM and MM. Our data may help to block these signaling pathways in the future to hinder progression to MM.

Mode of progression in smoldering multiple myeloma: a study of 406 patients

The approach to patients with high-risk smoldering multiple myeloma (SMM) varies among clinicians; while some advocate early intervention, others reserve treatment at progression to multiple myeloma (MM). We aimed to describe the myeloma-defining events (MDEs) and clinical presentations leading to MM diagnosis among SMM patients seen at our institution. We included 406 patients diagnosed with SMM between 2013–2022, seen at Mayo Clinic, Rochester, MN. The 2018 Mayo 20/2/20 criteria were used for risk stratification. Median follow-up was 3.9 years. Among high-risk patients who did not receive treatment in the SMM phase (n = 71), 51 progressed by last follow-up; the MDEs included: bone lesions (37%), anemia (35%), hypercalcemia (8%), and renal failure (6%); 24% met MM criteria based on marrow plasmacytosis (≥60%) and/or free light chain ratio (>100); 45% had clinically significant MDEs (hypercalcemia, renal insufficiency, and/or bone lesions). MM diagnosis was made based on surveillance labs/imaging(45%), testing obtained due to provider suspicion for progression (14%), bone pain (20%), and hospitalization/ED presentations due to MM complications/symptoms (4%). The presentation was undocumented in 14%. A high proportion (45%) of patients with high-risk SMM on active surveillance develop end-organ damage at progression. About a quarter of patients who progress to MM are not diagnosed based on routine interval surveillance testing.

Kinetics and Biology of Circulating Tumor Cells (CTCs) and Measurable Residual Disease (MRD): Two Dynamic High-Risk Clones in Multiple Myeloma (MM)

BACKGROUND CTCs and MRD are two small clones associated with a high risk of progression in smoldering MM (SMM) and post treatment relapse in active MM. Despite growing knowledge about their clinical significance and biological features, these questions remain unanswered: 1) What is the prognostic value of CTC and MRD kinetics? 2) Is MM dissemination and on-treatment resistance driven by specific genomic alterations? 3) Are CTCs and MRD genetically related? AIM Analyze the kinetics and molecular features of CTCs and MRD in SMM and MM patients. METHODS CTC kinetics were investigated using NGF in 164 SMM patients with ≥3 assessments collected every 6 months in the iMMunocell study. MRD kinetics were investigated using NGF in 277 MM patients with ≥4 assessments in BM aspirates after induction, transplant, consolidation, and yearly during maintenance (GEM2012MENOS65-GEM2014MAIN trials). Unsupervised clustering based on CTC and MRD kinetics was performed using CONNECTOR. Molecular features of BM tumor cells at diagnosis were compared to paired CTCs in 94 SMM and MM patients and to paired MRD in 96 MM patients. In 10 cases, genomic data from tripartite BM tumor cells, CTCs, and MRD was available. Tumor samples were isolated based on patient-specific aberrant phenotypes using FACS. Whole exome and RNA-seq were performed in 343 and 217 samples, respectively. RESULTS Based on the assessment of CTCs in 754 PB samples, SMM patients clustered into 3 groups: sustained undetectable (n = 20), stable (n = 71) and evolving (n = 73) CTC levels. With a median follow-up of 2 years, the respective 2y progression-free rates were 100%, 94% and 81% ( P =.003). Risk stratification based on CTC kinetics (C-index 0.68) outperformed a single baseline assessment (C-index 0.61) and, along with the IMWG 20/2/20 model, had independent prognostic value (HR 3.4; P = .01). Patients with high risk cytogenetics showed more frequently evolving CTC levels. Studying genetic drivers of tumor egress, we found CTCs to closely resemble BM tumor cells (88% concordance at copy number [CNA] and somatic mutational [SNV] level). No recurrent cell-specific SNV or CNA was identified. CTC's transcriptional profile revealed 268 DEGs enriched in interferon alpha, gamma response and cell-adhesion pathways. Next, the prognostic value of MRD dynamics was analyzed according to 1,759 assessments, identifying 3 patient clusters: sustained undetectable (n = 100), stable (n = 104), and evolving (n = 73) MRD levels. Respective 6-year rates of PFS after consolidation were 90%, 75% and 5% ( P <.001), and of OS were 94%, 95% and 66% ( P<.001). Risk stratification based on MRD kinetics (C-index 0.82) outperformed a single MRD assessment before maintenance (C-index 0.62), the R-ISS (C-index 0.57) and R2-ISS (C-index 0.61) at diagnosis. MRD cells showed heterogeneous genomic evolution (60% median concordance) diverging from BM tumor cells at diagnosis due to de novo CNA and SNV that emerged at a subclonal level, particularly after transplant. After induction, MM driver mutations were detected or remained clonal in 13/19 (68%) MRD, whereas after transplant, this was true for only 5/13 (38%) MRD. No recurrent cell-specific SNV or CNA was identified. The transcriptional signature of MRD was treatment dependent, with common molecular features of on-treatment resistance found in all 96 patient-paired samples analyzed after various treatment stages. The 334 DEGs were commonly enriched in KRAS signaling hallmarks. Genomic profiles of tripartite BM tumor cells, CTCs and MRD from 10 MM patients confirmed a patient-specific mutational landscape with genomic heterogeneity in MRD. For instance, one patient exhibited BRAF & DIS3 mutations and del(13q) detected at baseline in BM tumor cells and CTCs, which persisted in MRD. After induction, de novo clonal SNVs, including a NEK11 mutation, emerged, followed by the appearance of 3 subclonal CNA after transplant, possibly contributing to resistance. CONCLUSIONS This is the largest dataset analyzing the kinetics and molecular features of CTCs and MRD in SMM and active MM. We showed superior prognostic value of CTC and MRD kinetics over single assessments. Tumor dissemination seems to be driven by transcriptional priming rather than acquired secondary genetic events, while on-treatment resistance is linked to genomic and transcriptional evolution, without recurrent genetic alterations and with common molecular signatures of resistance.

Immune Cells, Cytokines, and Gut Microbiota Landscape Along Monoclonal Gammopathy of Undetermined Significance (MGUS) to Multiple Myeloma (MM) Evolution

Introduction: Multiple myeloma (MM) is an incurable hematologic cancer characterized by abnormal plasma cell growth in the bone marrow (BM). It is preceded by two premalignant conditions: monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Understanding the transition from MGUS to MM is crucial for better risk stratification, early interventions, and targeted treatments. This study aims to gain insights into immunological changes and fecal microbiota variations along the MGUS to MM transition. Methods: To comprehensively evaluate the immune composition (myeloid/lymphoid T/B/NK subpopulations and immune checkpoint distribution) in both bone marrow (BM) and peripheral blood (PB), we designed six 10-color custom and two 8-color flow cytometry panels. We analyzed samples from 13 MGUS, 12 SMM, and 63 newly diagnosed MM patients. Additionally, we evaluated cytokine/chemokine abundance in BM and PB using a 48-plex Luminex plate on 72 samples from the same MGUS, SMM, and MM patients, along with 4 healthy donors (HD). Furthermore, we performed fecal microbiome profiling on samples from 3 MGUS, 6 SMM, and 9 MM patients to investigate potential relationships between bacterial composition and MM evolution. Statistical analysis was performed in R by using specific packages (such as FlowCT). Results: Through unsupervised analysis of T cells by flow cytometry, we identified a significant increase in circulating TEMRA CD8 T cells, mostly for CD57+ cells (mean, 7.5% for MGUS vs 20.1% for SMM vs 21.6% for MM; p<0.05) and a significant reduction in naïve CD8 T TIGIT+ cells (mean, 12.4% for MGUS vs 5.98% for SMM vs 4.16% for MM; p<0.001) and naïve CD8 T TIGIT+ TIM3+ (mean, 2.18% for MGUS vs 1.24% for SMM vs 1.17% for MM; p<0.001). Regarding CD4 T subsets, we observed a reduction in BM effector memory phenotype along disease evolution (mean, 7.07% for MGUS vs 4.54% for SMM vs 3.72% for MM; p<0.05). Interestingly, we confirmed the increase of BM CD4 T cells production of IL17 (mean: 6.7% for MGUS vs 22% for MM; p<0.05) in MM patients compared to those with MGUS. In the context of myeloid cell subsets, circulating non-classical monocytes expressing HLA-DR and CD11c (mean, 1.45% for MGUS vs 0.95% for SMM vs 0.69% for MM; p<0.05) as well as granulocytes (mean, 74.18% MGUS vs 73.86% SMM vs 65.59% MM; p<0.05) were found to be reduced along MM progression. No major differences were observed for other evaluated lineages. Regarding cytokines and chemokines, a concurrent decrease in GM-CSF, IFN-a2, IFN-g, IL-1b, IL-2, IL-2Ra, IL-3, IL-10, IL-13, and MCP-1/CCL2 levels within the BM plasma throughout MM progression was observed, thus supporting a global reduction in myeloid function as well as in T cell effector activity. Moreover, PDGF-BB, IFN-a2, IL-16, and IL-8 decrease was observed in PB plasma along MM progression. Lastly, while no differences were observed in term of alpha and beta diversity, we observed an overall increase in total abundance of fecal bacteria along MM evolution (maybe related to loss of myeloid/lymphoid control according to what previously described), with several bacterial genus significantly over-represented in MM patients, including Bariatricus, CAG-83, Alistipes, Bifidobacterium, Streptococcus, Ruminococcus D, Slackia A, and Coprococcus A, while others (such as Parabacteroides B) was found to be more represented in MGUS and SMM patients. The analysis of phyla abundance indicated Firmicutes D and Lactobacillales as more abundant in MM patients, while Actinobacteriota dominated in SMM patients, and Proteobacteria and Bacteroidota were more prominent in MGUS patients. Conclusions: Overall, this study demonstrated a progressive impairment of both myeloid and lymphoid compartment in term of cellular composition, cytokine production and release and control over gut microbiome. Recovering from T cell exhaustion and IL17 production remains a major challenge for these patients.

Stable MGUS and SMM Are Characterized By Distinct Senescent Features in the Pre-Malignant Plasma Cells and the Proximate Bone Microenvironment

Multiple Myeloma (MM) progresses from monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering MM (SMM). Despite similar primary genetic events as MM, MGUS and SMM PCs are non- or low-proliferative. Since oncogene activation and DNA damage drive senescence growth arrest to promote immune clearance of potentially malignant cells, we hypothesized that MGUS and SMM pre-malignant PCs exhibit senescence features. To test this, we performed gene set enrichment analysis (GSEA) of a published human PC gene array dataset (GSE5900) comparing normal, MGUS, and SMM PCs for custom senescence phenotyping gene sets (Senescence Up, SenUp; Senescent Cell Anti-Apoptosis Pathways, SCAPs; Senescence Growth Arrest, SenGA; Inflammatory Senescence Associated Secretory Phenotype, iSASP; Interferon SASP, IFN-SASP) versus biological aging gene sets (SenMayo; CellAge). MGUS and SMM PCs exhibited significant enrichment (NES>1.5, q<0.05) for senescence gene sets SenUp, SenGA, and SCAPs compared to normal PCs, with no enrichment for aging gene sets. MGUS PCs were also enriched for iSASP (NES=1.4, q=0.11), whereas SMM PCs were enriched for IFN-SASP (NES=2.3, q<0.05), characteristic of late-senescence. Thus, both MGUS and SMM PCs show enrichment of senescence gene sets compared to normal PCs with differential SASP profiles. Next, we evaluated MGUS, SMM, and MM patient PCs isolated by magnetic sorting for histological senescence features. PCs were immunostained for LMNB1 and HMGB1, and fluorescent in situ hybridized for α-satellite. Cellular senescence was defined as loss of ≥50% nuclear membrane LMNB1, loss of HMGB1, and ≥2 senescence-associated distensions of satellite (SADS) per nuclei. PCs from MGUS patients (N=12) exhibited increased percentage of senescent PCs (26.9±4.1%, Mean±SEM, p<0.0001) compared to SMM (11.0±1.9%, N=19, p<0.0001) and MM (11.6±1.5%, N=11, p<0.001). A subset analysis of SMM patients for which disease stability was known (≥2 years, N=6-8) showed no differences. These results demonstrate that the percentage of senescent PCs is increased in MGUS but not SMM PCs compared to proliferative disease. To assess PC senescence in the native bone marrow microenvironment (BMME), we evaluated trephine bone biopsies from MGUS and SMM patients that progressed or not to MM ≤10 years, as well as newly diagnosed MM. Biopsies were immunostained for CD138, LMNB1, and HMGB1. Whole biopsy images were analyzed using the artificial intelligence-based software HALO. Cells were classified as senescent if they were double negative and non-senescent if they were double positive for LMNB1 and HMGB1. The percentage of non-senescent PCs increased with progression to MM and positively correlated with total PC burden (R=0.52, p<0.0001), reflective of proliferative disease. Senescent PCs correlated with total senescent BMME (R=0.87, p<0.0001), consistent with a role for senescent cells to drive paracrine senescence. We performed proximity analysis to evaluate the cellular composition within 25um of senescent and non-senescent PCs. As expected, non-senescent PCs exhibited a significantly greater percentage of proximate non-senescent BMME in all groups (p<0.0001). In contrast, senescent PCs in stable MGUS (N=16) and SMM (N=14) were equally surrounded by senescent and non-senescent BMME. In progressing MGUS (N=21) and SMM (N=20), as well as MM (N=18), senescent PCs exhibited significantly reduced senescent BMME in their proximate environment (p<0.05). This suggests that senescent PCs in progressive disease, in contrast to stable disease, fail to drive local paracrine senescence responses. These data demonstrate that PCs from stable MGUS and SMM patients exhibit senescence features, with isolated PCs from stable MGUS showing increased senescence gene expression and histologic features. While stable SMM also exhibits senescence gene expression, they lose morphological features of senescence and show a preferential IFN SASP. Within the bone compartment, senescent PCs in stable MGUS and SMM exhibit the expected proximity to senescent BMME. In contrast, progressing MGUS and SMM, as well as NDMM, show a loss of proximate senescent BMME. Given the role of senescence to drive inflammation to clear potentially tumorigenic cells, these data support that failure to drive paracrine senescence may be key to the immune escape of pre-malignant cells in MGUS and SMM.

The 3D- Telomere Profiling Assay Identifies High Risk Smoldering Multiple Myeloma Patients with High Precision

Smoldering multiple myeloma (SMM) is an asymptomatic precursor stage of multiple myeloma (MM). According to the international Myeloma Working Group data (Blood Cancer J. 2020 Oct 16;10(10):102) the probability of progression to MM is 22% at 2 years, and 42% and 64% at 5 and 10 years, respectively. Thus, identification of patients with high risk of early progression to MM is crucial for optimal treatment management. Genomic instability is a sensitive indicator of disease progression in cancer. Telomere dysfunction is an early event in genomic instability. The 3-dimensional (3D) spatial profiling of telomeres using TeloView technology allows for quantification of telomere dysfunction and was shown to be instrumental in risk stratification of cancer patients generally, but particularly in selected hematological malignancies. Importantly, the utility of the 3D-telomere profiling was recently demonstrated in 2 clinical studies validating the utility of 3D telomere profiling as a structural biomarker to identify high risk SMM patients. The two studies achieved accuracy of >80% and specificity and sensitivity of over 80% & 76% respectively in a training dataset study followed by a blind validation (Kumar S.et al 2023. J Clin Oncol). To present the 3D telomere profiling assay as a reliable prognostic tool in the clinic, capable to precisely identify high risk SMM patients, it is important to demonstrate the repeatability and accuracy of the assay. In this study, we conducted a retrospective analytical validation including samples of 20 SMM patients with known progression outcome. The samples of each patient were blindly processed in triplets using the 3D-telomere assay, followed by the TeloView analysis. To eliminate any validation-bias the validation was conducted with uncontrolled variables. Samples were processed blindly by different operators on different dates using different instrumentation. We interrogated the repeatability and precision of the results of each patient analyzed on 2 levels. First, we calculated coefficient of variation (CV) of telomere predictors of each patient across the three runs; and secondly, we interrogated the repeatability of patient outcome using the scoring model developed and validated in (Kumar et al. 2023, J Clin Oncol) across the three runs of each patient. We set the acceptable concordance to 80% for CVs (CV <20%) and the acceptable repeatability of patient-outcome as predicted by the scoring model to 80% of the patients included in the study. We report CVs across all the telomere predictors, across the 3 runs of all patients ranging between 2.3 - 17.8. Furthermore, patient outcome as high/low risk of progression was consistent in 17 out of the 20 patients in all 3 runs (85%). The results of this study demonstrate the 3D-telomere profiling as an accurate and prognostic structural biomarker. The assay shows high level of precision, repeatability, and reliability, and presents a viable solution to accurately identify high risk SMM patients. This will potentially allow the treating physicians to make confident treatment decisions for high risk SMM patients based on the results of the 3D Telomere profiling assay.

Single-Cell RNA Sequencing of Circulating Tumor Cells in Precursor Myeloma Patients Reveals Mechanisms of Disease Dissemination

Introduction Multiple Myeloma (MM) is preceded by the precursors Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM), where some patients with precursor disease progress to active MM faster than others. BM biopsies are useful to monitor disease progression, however, they cannot be repeated often for continuous monitoring of tumor burden especially in the precursor disease management setting. Numerous studies have identified circulating tumor cell (CTC) levels in peripheral blood (PB) are a powerful biomarker of disease aggressiveness; CTC levels have associated with disease stage, PFS and OS in various trials and even outperformed BM to assess tumor burden in the SMM setting. Genomic profiling also demonstrated CTCs harbor the same copy number abnormalities, translocations and mutations as BM tumor cells, and CTCs can be serially monitored to detect the emergence of high-risk subclones in the blood. Further investigations of the molecular profile of both normal plasma cells (NPCs) and CTCs are needed to improve our understanding of myeloma pathogenesis and mechanisms of CTC dissemination across the MM disease continuum. Methods A cohort of 309 samples including CD138-enriched paired PB and BM aspirates and matched CD138- BM aspirates were collected from 103 individuals, including patients with MGUS (n=11), SMM (n=46), NDMM (n=17) and healthy donors (n=29). Malignant PCs from BM and PB underwent 5' single-cell RNA sequencing (scRNA-seq) and single-cell B-cell receptor sequencing (scBCR-seq) (10x Genomics), and CD138- BM immune cells underwent 5' scRNA-seq and single-cell T-cell receptor sequencing (scTCR-seq) to study immune alterations related the tumors' ability to circulate. To differentiate malignant from normal PCs, we used clonal V(D)J rearrangements. Differential expression (DE) and composition analyses were conducted using Wilcoxon's rank-sum tests. Results We successfully captured and profiled 774,647 BM tumor cells and 93,878 CTCs from the PB. The percent malignant plasma cells within the PB increased with disease stage, where NDMM patients had significantly more CTCs compared to MGUS patients (p=0.017) and low-risk SMM patients (p=0.0052). A median of 13.51%, 15.53%, and 18.34% CTCs were present from low (n=20), intermediate (n=10), and high-risk (n=16) SMM patients as defined by the International Myeloma Working Group's 2/20/20 criteria, suggesting sequencing-based CTC enumeration captures prognostically relevant differences in tumor burden. High expression of driver genes upregulated in patients with translocations, including CCND1, NSD2, and MAF, were detected in both BM tumor cells and CTCs in patients with t(11;14), t(4;14), and t(14;16) as identified by fluorescence in situ hybridization (FISH). In 5 patients with normal or inconclusive FISH results, we observed high levels of CCND2 and MAF in both BM and CTCs, indicating scRNA-seq can detect missed prognostically relevant cytogenetic abnormalities. A cytogenetic classifier model was developed to determine the accuracy of translocation calling using minimal numbers of CTCs, where the classifier's performance was able to consistently identify translocations downsampled to 50 CTCs. DE analysis of CTCs vs. BM tumor cells highlighted transcriptional similarity between BM and CTCs, validating their utility as a surrogate for analyzing BM tumor cells. DE analysis also revealed 8 genes significantly upregulated and 3 genes significantly downregulated in CTCs compared to BM tumor cells, providing novel insights into genes involved in PC circulatory potential. Pathway enrichment analysis revealed genes upregulated in CTCs were associated with epithelial mesenchymal transition, interferon response, and inflammation, consistent with CTC studies on MM patients, suggesting these pathways are dysregulated earlier in the disease continuum. Conclusions In the largest scRNA-seq study on CTCs to date, we demonstrate the utility of CTC-based molecular profiling for prognostication of patients with early-stage disease and provide novel insights into PC circulatory potential. Additional analyses are ongoing to gain further insight into intra-patient CTC heterogeneity and define high-risk disease CTC signatures that emerge throughout the MM disease continuum.

Psychological Impact in Individuals with Monoclonal Gammopathy of Undetermined Significance and Smoldering Multiple Myeloma

BACKGROUND: Monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) are medical conditions characterized by asymptomatic clonal plasma cell disorders that have the potential to develop into multiple myeloma (MM). The diagnosis of MGUS and/or SMM may be linked to psychological effects that can have a detrimental impact on patients' quality of life. The objective of our study was to evaluate the prevalence of adverse psychosocial effects among individuals with MGUS and/or SMM. METHODS: From August 2015 to February 2023, we conducted prospective interviews with all patients referred to the University of Arkansas for Medical Sciences who had newly diagnosed plasma cell dyscrasias, such as MGUS and SMM, before their consultation with the oncologist. Trained social workers performed an initial psychosocial evaluation and prepared a comprehensive narrative report for each patient referred during this period. Results: Our study consisted of 246 patients, with 115 having MGUS and 131 having SMM. The average age was 68 years, and 51.2% were male, with 80.5% identifying as white. Most patients (91%) were diagnosed incidentally through blood and/or urine tests, and 61.8% had a family history of cancer. Anxiety was found in 19.1% of patients, while 33.7% had a previous psychiatric disorder. Among those with a history of psychiatric disorders, 78.3% were taking medication, and 21.7% were receiving treatment from a psychiatrist/therapist. Anxiety or nervousness was reported by 22% of MGUS patients and 17% of SMM patients. About one third of patients had a history of psychiatric disorders, with 79% on medication and 26% seeing a psychiatrist/therapist. The presence of anxiety or a history of psychiatric disorders did not significantly differ between MGUS and SMM patients (p > 0.05). In our analysis, age, race, working status, and history of psychiatric disorders were significant risk factors for anxiety (p = 0.001, 0.033, 0.045, and <0.001, respectively). However, in the multivariable model, only race and history of psychiatric disorders remained significant (p = 0.037 and p = 0.006, respectively). White patients had 5 times higher odds of experiencing anxiety, while patients with a history of psychiatric disorders had 2.6 times higher odds (odds ratio: 5.1 and 2.7, respectively). The most common themes expressed by patients during interviews were 'fear, stress, and/or anxiety,' followed by 'emotional initial reaction' and ‘feeling upset.‘ Conclusion: In our study, about 20% of patients felt anxious about their diagnosis of MGUS or SMM, and around 33% reported previous psychiatric disorder. African American patients were less likely to feel anxious or nervous, while patients with a history of psychiatric disorders were more likely to feel anxious or nervous. Given that anxiety can negatively affect patients' well-being and quality of life, healthcare providers should prioritize comprehensive care that includes addressing and managing anxiety alongside the medical aspects of the disease.

Exploiting the Gut Microbiota to Boost Immunotherapy and Switch the Trajectory of Multiple Myeloma

Clonal proliferation of neoplastic plasma cells in the bone marrow (BM) and organ dysfunction characterizes multiple myeloma (MM), a treatable but incurable disease. Although asymptomatic smoldering MM (SMM) often precedes full blown MM, most patients affected by SMM are only offered active observation, which increases their frustration and anxiety. In transgenic Vk*MYC mice developing de novo MM, we found a direct link between gut microbiota composition and T helper 17 (Th17) lymphocytes, which migrated from the gut to the BM during asymptomatic MM (Early-MM), and favored the expansion of neoplastic plasma cells, thus fueling progression to symptomatic disease (Late-MM) [Calcinotto A. et al. Nat Commun (2018) 9(1):4832]. Similarly, in SMM patients, higher levels of BM Interleukin-17 (IL-17) predicted accelerated disease [Calcinotto A. et al. Nat Commun (2018) 9(1):4832]. Interestingly, Prevotella melaninogenica ( P.m.), a human commensal, limited MM aggressiveness in mice challenged with Vk*MYC-derived MM cells (t-Vk*MYC) by restraining Th17 cell expansion [Calcinotto et al. Nat Commun (2018) 9(1):4832]. Because the gut microbiota also contributes to the therapeutic efficacy of immune checkpoint blockade (ICB) and human neoplastic plasma cells express PD-L1, we hypothesized that modulation of the gut microbiota by P.m. could limit Th17 cell expansion in mice affected by Early-MM, thus fully exploiting the therapeutic potential of anti-PD-L1 against MM. Clinical trials combining anti-PD-1/PD-L1 antibodies, dexamethasone and immune modulatory drugs in MM were halted by US FDA because of unfavorable benefit-risk profile and life-threatening immune related adverse events (irAEs). Because IL-17 is involved in irAEs [Johnson et al. Cancer Immunol Res (2019) 7 (6): 860-865], we also hypothesized treatment with P.m. could restrain ICB-induced expansion of Th17 cells while unleashing CD8-mediated antitumor immunity. Early-MM Vk*MYC mice and t-Vk*MYC, the latter mimicking aggressive MM, were treated with P.m. and/or anti-PD-L1 and disease progression was monitored by paraprotein quantification in blood. Anemia provided clinical evidence of symptomatic disease. At sacrifice, gut, spleen and BM were analyzed by flow cytometry. In vitro assays were conducted to clarify the mechanism by which the gut microbiota influenced the induction of Th17 cells. To mimic cutaneous irAE, the skin of treated mice was exposed to imiquimod, which induces Th17-mediated inflammation [van der Fits et al. J Immunol (2009 )182(9): 5836-5845]. Body weight loss, skin inflammation, and inflammatory infiltrate into secondary lymphoid organs were taken as measure of imiquimod-induced toxicity. In Vk*MYC mice, all neoplastic plasma cells expressed PD-L1. Treatment of Early-MM Vk*MYC mice with anti-PD-L1 antibodies delayed progression to Late-MM, thus extending previous findings on the use of ICB in this model to anti-PD-L1 antibodies and Early-MM [Guillerey et al. J Clin Invest (2015 )125(5): 2077-2089]. Treatment with P.m. exerted a similar effect on disease progression, suggesting that a treatment as simple as taking a probiotic has an impact on MM evolution. When P.m. was combined with anti-PD-L1 antibodies, evolution to Late-MM was further delayed or even blocked. Single and combined treatments also reduced MM aggressiveness in t-Vk*MYC mice. Mechanistically, P.m. limited the expansion of gut-born Th17 cells and their accumulation in the BM of treated mice without dampening the antitumor cytotoxic response elicited by the ICB. Thus, only the combined treatment provided the most favorable CD8/Th17 and CD8/T regulatory cell ratios. Ex vivo, P.m.-conditioned BM DCs showed a limited propensity to produce Th17-polarizing cytokines. In vitro, stimulation of both human and mouse DCs with P.m. reduced polarization of naïve T cells toward a Th17 phenotype. Skin application of imiquimod in C57BL/6 mice induced psoriasis-like lesions and expansion of Th17 cells both in draining lymph nodes and spleen. The contextual administration of anti-PD-L1 antibodies worsened imiquimod-induced inflammation. An ongoing experiment will clarify if mice receiving P.m. and ICB experience reduced skin lesions. Taken together, our data support the development of microbiota-based strategies in combination with ICB to treat full-blown MM and to prevent progression of asymptomatic SMM to full-blown disease.

The Spop/BRD4/c-Myc Axis Modulates IMiDs Sensitivity in Multiple Myeloma

The development of novel agents including immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) has led to improved patient outcomes in multiple myeloma (MM); however, MM patients inevitably experience relapse and drug resistance. The expression level of CRBN, CRBN-binding proteins, and CRL4 CRBN ubiquitin ligase is considered to be associated with IMiDs resistance. Clinical studies have shown that MM patients can exhibit IMiDs resistance even without CRBN abnormalities, suggesting the molecular mechanisms of intrinsic resistance to IMiDs are not fully understood. To delineate the molecular mechanisms underlying IMiDs resistance, we performed genome-wide knockout (KO) screening in IMiDs-sensitive MM cells. For in vitro work, we used the MM cell lines MM1S, and H929. We used sgRNA to KO SPOP and BRD4 in MM cells. After performing a CRISPR KO screen in MM cells, we discovered that in addition to CRBN and its associated genes, sgRNAs targeting SPOP (speckle-type POZ protein, a speckled zinc finger protein) were highly enriched after treatment with Poma. Importantly, SPOP KO MM cell lines acquired significant resistance to Pom and Len treatment. To examine whether SPOP KO-induced IMiDs resistance was CRBN-pathway dependent, we assessed CRBN and its downstream interacting protein levels. SPOP KO showed no effect on CRBN expression; moreover, IMiDs can still trigger IKZF1 and IKZF3 degradation, associated with the downregulation of IRF4, suggesting that SPOP mediates sensitivity to IMiDs in a mechanism independent of CRBN-IKZF1/3 axis.Compared to plasma cells from normal donors, we found that the expression levels of SPOP were significantly decreased in MGUS patients, smoldering myeloma patients, newly diagnosed MM patients, and refractory relapsed MM patients (p-values: 2.4e-2, 2.0e-4, 9.9e-3, 3.17e-6, respectively). SPOP is the adaptor protein of the Cullin3-RING ubiquitin ligase complex. Therefore, the function of SPOP is determined by the characteristics of the substrates it recruits. We performed immunoprecipitation with overexpressed SPOP in MM cells, followed by mass spectrometry analysis, and identified BRD4 as a candidate protein binding to SPOP in MM, which was confirmed by Co-IP. KO of SPOP led to an upregulation of BRD4 protein levels in MM cells. As a downstream regulator of BRD4, the oncogene c-Myc showed a significant increase in protein expression in IMiDs-resistant MM patients. Furthermore, the KO of SPOP notably enhanced c-Myc expression in MM cells, while the KO of BRD4 in SPOP KO MM cells decreaed c-Myc expression and restored MM cell sensitivity to IMiD treatment. These findings suggest that SPOP modulates c-Myc expression through BRD4 in a CRBN-IKZF1/3 independent manner, thereby mediating MM cell sensitivity to IMiDs. BRD4 plays a significant role in the development and progression of various tumors. The BRD4 inhibitor JQ1 has been shown to exhibit anti-tumor effects in multiple types of cancer. We found that combination treatment with JQ-1 and IMiDs reversed IMiDs resistance of SPOP KO MM cell lines and primary MM cells from relapsed patients. Our data show that SPOP is a CRBN-independent modulator of IMiDs sensitivity by regulating the BRD4/c-Myc axis and provides the preclinical rationale for combining IMiDs with BRD4 inhibitors to overcome IMiDs resistance and improve patient outcomes.

CRIP1 Upregulated Along with chr1q-Gain Indicates Immune Dysregulation and Portends Poor Outcomes in Multiple Myeloma

Background: The gain of chromosome 1q (chr1q-gain), one of the frequently occurring copy number aberrations in multiple myeloma (MM), is associated with a poor prognosis. However, the specific high-risk genes involved and the underlying molecular mechanisms remain elusive. Method: In this study, we gathered a comprehensive dataset comprising bulk transcriptome data from 1271 multiple myeloma (MM) patients, including 413 patients from the GSE4581 dataset and 858 patients from the MMRF dataset. Additionally, we acquired single-cell RNA sequencing data from various disease stages, which included 12 MM patients, 7 patients with monoclonal gammopathy of undetermined significance (MGUS), 4 patients with amyloidosis (AL), 6 patients with smoldering multiple myeloma (SMM), and 11 healthy control donors from GSE117156. Cox regression and Kaplan-Meier curves were employed to identify and validate significant candidates associated with adverse prognosis among the overexpressed genes in patients with chr1q-gain. Furthermore, the expression profiles of the candidates in malignant plasma cells from MM patients compared to plasma cells in healthy donors were evaluated at single cell resolution. Gene Set Enrichment Analysis (GSEA) was conducted to uncover the pathways enriched in high-CRIP1 groups. To better understand the immune infiltration in MM, we utilized CIBERSORTx to infer the immune cell composition. Results: A total of 541 genes were found to be upregulated in multiple myeloma (MM) patients with chr1q-gain in the GSE4581 dataset, with a false discovery rate (FDR) of less than 0.05 and a log fold change (LogFC) greater than 0.5.We identified five genes (CRIP1, ANP32E, CKS1B, RBFA, TOP2A) associated with poor prognosis through univariate Cox and multivariate Cox regression analyses and successfully validated in MMRF dataset(n=858). Upon characterizing the expression of these candidate genes in malignant plasma cells at a single-cell resolution, we made an interesting observation: CRIP1 was uniquely expressed in multiple myeloma (MM) patients when compared to healthy donors, individuals with MGUS, SMM and AL patients. Moreover, the GSEA results showed significant enrichment of the “Anti-viral Mechanism by IFN-stimulated Genes” (Normalized Enrichment Score, NES=1.98, False Discovery Rate, FDR=0.017) and “Interferon Signaling” (NES=2.11, FDR<0.001) in CRIP1-low MM patients. These findings suggest that CRIP1 may play a role in the development of multiple myeloma by influencing the tumor immune microenvironment through the regulation of these pathways. Analysis of immune cell infiltration in MM showed the expression level of CRIP1 is negatively correlated with CD4 naïve T cells ( R=-0.24, P<0.001) and positively correlated with Monocytes ( R=0.19, P<0.001). Tumors with high CRIP1 expression showed increased infiltration of CD8 T cells, CD4 memory T cells, activated NK cells, M0 macrophages, and Eosinophils. Conclusions: The study showed that CRIP1 upregulated along with chr1q-gain was highly expressed in MM patients compared to healthy individuals and associated with immune dysregulation. The patients with higher CRIP1 expression have poorer survival. CRIP1 could serve as a potential therapeutic target and prognostic biomarker in multiple myeloma.

Immune Profiling and Responses of Smoldering Multiple Myeloma Patients Treated in a Phase Ib Study of Pvx-410 Vaccine Targeting XBP1/CD138/CS1 Antigens, and Citarinostat, a Histone Deacetylase Inhibitor (HDACi) with and without Lenalidomide

BACKGROUND AND SIGNIFICANCE Immunoparesis plays an important role in the progression of smoldering multiple myeloma (SMM). Therapies that augment the immune response may prevent or slow the progression to symptomatic multiple myeloma. We have previously shown that PVX-410, a multi-peptide cancer vaccine (OncoPep., Inc.) targeting XBP1/CD138/CS1, is immunogenic in SMM, as a single therapy and in combination with lenalidomide. Here, we investigated whether co-administration of PVX-410 with an oral HDAC inhibitor, citarinostat, +/- lenalidomide enhances vaccine specific immune response in patients with SMM. STUDY DESIGN AND METHODS This 2-cohort phase 1b multicenter study accrued 16 adults (≥18 years) with HLA-A2 + SMM and high risk of progression to active MM. Patients received citarinostat in combination with PVX-410 (double combination cohort) and with PVX-410 and lenalidomide (triple combination cohort). PVX-410 was given as six doses of 0.8 mg biweekly (weeks 0, 2, 4, 6, 8, and 10) via subcutaneous injection followed by a single dose of PVX-410 during follow-up visits at month 1, 2, 3, 6, and 9; 3 cycles of citarinostat (180 mg QD PO for 21 days every 28 days) and lenalidomide (25 mg on Days 1-21 every 28 days) were given to patients in the double and triple combination cohorts respectively in conjunction with the initial 6 doses of PVX-410. Patients were followed post-treatment for 12 months. Peripheral blood (PB) and bone marrow (BM) samples at baseline and month one after treatment were analyzed by single-cell RNA sequencing. T-cell receptor and B-cell receptor sequencing were also performed to investigate T cell and B-cell clonal expansion before and after treatment. Flow cytometric analyses of anti-MM functionality of the XBP1/CD138/CS1-specific CD8 + cytotoxic T lymphocytes (CTL) was performed at various timepoints from baseline up to 12-month post-treatment. The development of antigen-specific memory CD8 + CTL through upregulation of 41BB and production of Th1-type of cytokines (IFN-g, IL-2, TNF-a) were evaluated by flow cytometry. Deep whole genome sequencing was performed on subsets of CD138 + cells from BM isolates to corroborate our findings. RESULTS We screened 16 SMM patients; 15 (94%) received treatment (7 with the double and 8 with the triple combination). By the end of the 12-month post-treatment follow-up period no patients progressed to symptomatic myeloma in the double combination cohort; one patient in the triple combination cohort withdrew by month 3 for disease progression. Most patients receiving the double combination had stable disease (SD) as their best clinical response (n=5), one had partial response (PR) and one minimal response (MR). Triple combination therapy resulted in better clinical response including PR (n=2), MR (n=4) and SD (n=2). Single-cell transcriptomic analyses of 21 PB samples from 13 patients and 15 BM samples from 11 patients at baseline and month one after treatment is in progress. Immune monitoring studies on 125 samples from 15 different patients at various timepoints from baseline up to 12-month post-treatment are ongoing. Fourteen patients (100%) had at least one treatment-related adverse event (trAEs), mostly grade 1-2 in severity. The most common trAEs among patients receiving double vs triple combination were fatigue (71% vs 63%), injection site reactions (43% vs 50%), neutropenia (57% vs 38%), anemia (29% vs 38%), and diarrhea (0% vs 50%). There was one grade 3 trAE: specifically, a thromboembolic event in one patient who was receiving lenalidomide and despite aspirin prophylaxis, but who fully recovered. DISCUSSION The combination of PVX-410 with citarinostat, with and without lenalidomide is generally safe in this patient population. The objective clinical response was relatively modest given the short duration of therapy: longer follow up is needed to assess the impact on progression to active myeloma. PB and marrow transcriptome profiling along with the immunogenicity studies are in progress and will be presented. We will evaluate whether PB mononuclear cells have the potential to replace BM as a surrogate for genetic and immunological surveillance in SMM, as we anticipate that further understanding of the immune alterations among patients with SMM and the response to immunomodulatory therapy may provide insight on early intervention and prevention of progression to symptomatic disease.

PYGO2-MDR1 Axis in Multiple Myeloma Patients with 1q21 Amplification As Promising Target to Overcome Carfilzomib Resistance

Multiple myeloma (MM) is a hematological malignancy characterized by an accumulation of clonal plasma cells (PCs) in the bone marrow (BM). Gain and/or amplification of 1q21 (1q21+) is one of the most frequent secondary cytogenetic abnormalities present in MM patients and in smoldering MM (SMM). The incidence and copy number alteration (CNA) of the 1q21 region increase during the progression and relapse of the disease; in fact, additional copies of 1q21 can be detected in around 40% of newly diagnosed MM (NDMM) and 70% of relapse-refractory MM (RRMM). Recently, 1q21+ has been identified as an adverse prognostic factor in MM patients and its presence has been correlated with a shorter duration of the response in MM patients treated with carfilzomib (CFZ)-based regimens. Thus, the identification of possible target genes in 1q21 region is an emerging unmet medical need in MM patients. PYGOPUS2 (PYGO2) is a gene located in the 1q21 chromosomal region and it is a downstream target of β-catenin, and it has been shown to promote the transcription of gene target of the Wnt-signaling, forming a nuclear complex with β-catenin/BCL9. Different studies have reported that upregulation of PYGO2 is involved in tumorigenesis in breast and lung cancers, showing that overexpression of PYGO2 leads to drug resistance by promoting the activation of the multidrug resistance 1 polypeptide ( MDR1). The expression profile of PYGO2 in1q21+ MM patients and its possible role in CFZ resistance is still unknown and have been investigated in this study. Firstly, we purified CD138 + PCs from BM samples from 18 NDMM and from 11 SMM patients. Fluorescent in situ hybridization (FISH) analysis was performed on purified CD138 + PCs for all patients to access CNA in the region 1q21. A score indicating the number of 1q21 copies was calculated based on the FISH hybridization pattern of each patient. 14/29 (48%) patients presented CNA in the 1q21 region. The expression profile of all 29 samples was generated using GeneChip ClariomD Arrays (Affymetrix Inc., Santa Clara, CA, USA) . The sam package was used to identify differentially expressed genes between 1q21+ and control samples. Our analysis identified PYGO2 to be significantly upregulated in patients with 1q21+ when compared with controls (p= 0.0008). Additionally, we found a positive correlation between gene expression and the 1q21 copy number (p= <0.0001, r= 0.6738). Secondly, we evaluated PYGO2 mRNA expression levels in several human myeloma cell lines (HMCLs) with 1q21 CNA. Our results showed that PYGO2 is overexpressed in HMCLs with 1q21 CNA. Previous studies in solid tumor have shown that PYGO2 is upregulated in drug resistant cancer cells, and this upregulation results in overexpression of MDR1; indeed, we evaluated the mRNA expression of PYGO2 in CFZ-resistant HMCLs. Interestingly our results showed that both PYGO2 and MDR1 expression are upregulated in CFZ-R cells. We next examined the relationship between PYGO2 and MDR1. We generated a PYGO2-knockdown in JJN3 MM cell line using short hairpin RNA (shRNA) lentivectors. Knockdown of PYGO2 resulted in a significant decrease in expression PYGO2, a decrease in cell viability, and a reduction in MDR1 expression. In conclusion, our results show that PYGO2 expression is significantly upregulated in MM patients carrying 1q21+ and that its expression is correlated with 1q21 copy number. Moreover, we found that the PYGO2-MDR1 axis is involved in the CFZ resistance in HMCLs MM cells suggesting that PYGO2 could be a possible future druggable target in 1q21+ MM patients.