Multiple myeloma (MM) is a haematological malignancy of plasma cells characterized by substantial intraclonal genetic heterogeneity. Although therapeutic advances made in the past few years have led ...to improved outcomes and longer survival, MM remains largely incurable. Over the past decade, genomic analyses of patient samples have demonstrated that MM is not a single disease but rather a spectrum of haematological entities that all share similar clinical symptoms. Moreover, analyses of samples from monoclonal gammopathy of undetermined significance and smouldering MM have also shown the existence of genetic heterogeneity in precursor stages, in some cases remarkably similar to that of MM. This heterogeneity highlights the need for a greater dissection of underlying disease biology, especially the clonal diversity and molecular events underpinning MM at each stage to enable the stratification of individuals with a high risk of progression. Emerging single-cell sequencing technologies present a superlative solution to delineate the complexity of monoclonal gammopathy of undetermined significance, smouldering MM and MM. In this Review, we discuss how genomics has revealed novel insights into clonal evolution patterns of MM and provide examples from single-cell studies that are beginning to unravel the mutational and phenotypic characteristics of individual cells within the bone marrow tumour, immune microenvironment and peripheral blood. We also address future perspectives on clinical application, proposing that multi-omics single-cell profiling can guide early patient diagnosis, risk stratification and treatment strategies.
ABSTRACT
Triple‐negative breast cancers (TNBCs) account for ~25% of all invasive carcinomas and represent a large subset of aggressive, high‐grade tumors. Despite current research focused on ...understanding the genetic landscape of TNBCs, reliable prognostic and predictive biomarkers remain limited. Although dysregulated microRNAs (miRNAs) have emerged as key players in many cancer types, the role of miRNAs in TNBC disease progression is unclear. We performed miRNA profiling of 51 TNBCs by next‐generation sequencing to reveal differentially expressed miRNAs. A total of 228 miRNAs were identified. Three miRNAs (miR‐224–5p, miR‐375, and miR‐205–5p) separated the tumors based on basal status. Six miRNAs (high let‐7d‐3p, miR‐203b‐5p, and miR‐324–5p; low miR‐30a‐3p, miR‐30a‐5p, and miR‐199a‐5p) were significantly associated with decreased overall survival (OS) and 5 miRNAs (high let‐7d‐3p; low miR‐30a‐3p, miR‐30a‐5p, miR‐30c‐5p, and miR‐128–3p) with decreased relapse‐free survival (RFS). On multivariate analysis, high expression of let‐7d‐3p and low expression of miR‐30a were independent predictors of decreased OS and RFS. High expression of miR‐95–3p was significantly associated with decreased OS and RFS in patients treated with anthracycline‐based chemotherapy. Five miRNAs (let‐7d‐3p, miR‐30a‐3p, miR‐30c‐5p, miR‐128–3p, and miR‐95–3p) were validated by quantitative RT‐PCR. Our findings unveil novel prognostic and predictive miRNA targets for TNBC, including a miRNA signature that predicts patient response to anthracycline‐based chemotherapy. This may improve clinical management and/or lead to the development of novel therapies.—Turashvili, G., Lightbody, E. D., Tyryshkin, K., SenGupta, S. K., Elliott, B. E., Madarnas, Y., Ghaffari, A., Day, A., Nicol, C. J. B. Novel prognostic and predictive microRNA targets for triple‐negative breast cancer. FASEB J. 32, 5937–5954 (2018). www.fasebj.org
Patients with smoldering multiple myeloma (SMM) are observed until progression, but early treatment may improve outcomes. We conducted a phase II trial of elotuzumab, lenalidomide, and dexamethasone ...(EloLenDex) in patients with high-risk SMM and performed single-cell RNA and T cell receptor (TCR) sequencing on 149 bone marrow (BM) and peripheral blood (PB) samples from patients and healthy donors (HDs). We find that early treatment with EloLenDex is safe and effective and provide a comprehensive characterization of alterations in immune cell composition and TCR repertoire diversity in patients. We show that the similarity of a patient’s immune cell composition to that of HDs may have prognostic relevance at diagnosis and after treatment and that the abundance of granzyme K (GZMK)+ CD8+ effector memory T (TEM) cells may be associated with treatment response. Last, we uncover similarities between immune alterations observed in the BM and PB, suggesting that PB-based immune profiling may have diagnostic and prognostic utility.
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•EloLenDex is associated with 4-year PFS of 89% in patients with high-risk SMM•Immune reactivity, post-therapy immune normalization are associated with longer PFS•Higher abundance of GZMK+ cytotoxic T cells is associated with longer PFS•Blood-based immune profiling detects immune dysregulation associated with disease
Sklavenitis-Pistofidis et al. report results of a phase II trial of EloLenDex in patients with high-risk smoldering multiple myeloma and use single-cell RNA sequencing to identify biomarkers of outcomes. They show that immune cell composition affects progression-free survival and that blood-based immune profiling can detect immune alterations observed in the bone marrow.
Excessive proliferation and apoptosis‐resistance are hallmarks of cancer. Increased dynamin‐related protein 1 (Drp1)‐mediated mitochondrial fission is one of the mediators of this phenotype. ...Mitochondrial fission that accompanies the nuclear division is called mitotic fission and occurs when activated Drp1 binds partner proteins on the outer mitochondrial membrane. We examine the role of Drp1‐binding partners, mitochondrial dynamics protein of 49 and 51 kDa (MiD49 and MiD51), as drivers of cell proliferation and apoptosis‐resistance in non‐small cell lung cancer (NSCLC) and invasive breast carcinoma (IBC). We also evaluate whether inhibiting MiDs can be therapeutically exploited to regress cancer. We show that MiD levels are pathologically elevated in NSCLC and IBC by an epigenetic mechanism (decreased microRNA‐34a‐3p expression). MiDs silencing causes cell cycle arrest through (a) increased expression of cell cycle inhibitors, p27Kip1 and p21Waf1, (b) inhibition of Drp1, and (c) inhibition of the Akt‐mTOR‐p70S6K pathway. Silencing MiDs leads to mitochondrial fusion, cell cycle arrest, increased apoptosis, and tumor regression in a xenotransplant NSCLC model. There are positive correlations between MiD expression and tumor size and grade in breast cancer patients and inverse correlations with survival in NSCLC patients. The microRNA‐34a‐3p‐MiDs axis is important to cancer pathogenesis and constitutes a new therapeutic target.
BackgroundHigh-grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy characterized by resistance to chemotherapy and high rates of recurrence. HGSC tumors display a high ...prevalence of tumor suppressor gene loss. Given the type 1 interferon regulatory function of BRCA1 and PTENgenes and their associated contrasting T-cell infiltrated and non-infiltrated tumor immune microenvironment (TIME) states, respectively, in this study we investigated the potential of stimulator of interferon genes (STING) pathway activation in improving overall survival via enhancing chemotherapy response, specifically in tumors with PTEN deficiency.MethodsExpression of PTEN protein was evaluated in tissue microarrays generated using pretreatment tumors collected from a cohort of 110 patients with HGSC. Multiplex immunofluorescence staining was performed to determine spatial profiles and density of selected lymphoid and myeloid cells. In vivo studies using the syngeneic murine HGSC cell lines, ID8-Trp53–/–; Pten–/– and ID8-Trp53–/–; Brca1–/–, were conducted to characterize the TIME and response to carboplatin chemotherapy in combination with exogenous STING activation therapy.ResultsPatient tumors with absence of PTEN protein exhibited a significantly decreased disease specific survival and intraepithelial CD68+ macrophage infiltration as compared with intact PTEN expression. In vivo studies demonstrated that Pten-deficient ovarian cancer cells establish an immunosuppressed TIME characterized by increased proportions of M2-like macrophages, GR1+MDSCs in the ascites, and reduced effector CD8+ cytotoxic T-cell function compared with Brca1-deficient cells; further, tumors from mice injected with Pten-deficient ID8 cells exhibited an aggressive behavior due to suppressive macrophage dominance in the malignant ascites. In combination with chemotherapy, exogenous STING activation resulted in longer overall survival in mice injected with Pten-deficient ID8 cells, reprogrammed intraperitoneal M2-like macrophages derived from Pten-deficient ascites to M1-like phenotype and rescued CD8+ cytotoxic T-cell activation.ConclusionsThis study reveals the importance of considering the influence of cancer cell intrinsic genetic alterations on the TIME for therapeutic selection. We establish the rationale for the optimal incorporation of interferon activating therapies as a novel combination strategy in PTEN-deficient HGSC.
Multiple myeloma (MM) develops from well-defined precursor stages; however, invasive bone marrow (BM) biopsy limits screening and monitoring strategies for patients. We enumerated circulating tumor ...cells (CTC) from 261 patients (84 monoclonal gammopathy of undetermined significance, 155 smoldering multiple myeloma, and 22 MM), with neoplastic cells detected in 84%. We developed a novel approach, MinimuMM-seq, which enables the detection of translocations and copy-number abnormalities through whole-genome sequencing of highly pure CTCs. Application to CTCs in a cohort of 51 patients, 24 with paired BM, was able to detect 100% of clinically reported BM biopsy events and could replace molecular cytogenetics for diagnostic yield and risk classification. Longitudinal sampling of CTCs in 8 patients revealed major clones could be tracked in the blood, with clonal evolution and shifting dynamics of subclones over time. Our findings provide proof of concept that CTC detection and genomic profiling could be used clinically for monitoring and managing disease in MM.
In this study, we established an approach enabling the enumeration and sequencing of CTCs to replace standard molecular cytogenetics. CTCs harbored the same pathognomonic MM abnormalities as BM plasma cells. Longitudinal sampling of serial CTCs was able to track clonal dynamics over time and detect the emergence of high-risk genetic subclones. This article is highlighted in the In This Issue feature, p. 247.
Prevalence estimates for monoclonal gammopathy of undetermined significance (MGUS) are based on predominantly White study populations screened by serum protein electrophoresis supplemented with ...immunofixation electrophoresis. A prevalence of 3% is reported for MGUS in the general population of European ancestry aged 50 years or older. MGUS prevalence is two times higher in individuals of African descent or with a family history of conditions related to multiple myeloma. We aimed to evaluate the prevalence and clinical implications of monoclonal gammopathies in a high-risk US population screened by quantitative mass spectrometry.
We used quantitative matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and EXENT-iQ software to screen for and quantify monoclonal gammopathies in serum from 7622 individuals who consented to the PROMISE screening study between Feb 26, 2019, and Nov 4, 2021, and the Mass General Brigham Biobank (MGBB) between July 28, 2010, and July 1, 2021. M-protein concentrations at the monoclonal gammopathy of indeterminate potential (MGIP) level were confirmed by liquid chromatography mass spectrometry testing. 6305 (83%; 2211 from PROMISE, 4094 from MGBB) of 7622 participants in the cohorts were at high risk for developing a monoclonal gammopathy on the basis of Black race or a family history of haematological malignancies and fell within the eligible high-risk age range (30 years or older for PROMISE cohort and 18 years or older for MGBB cohort); those over 18 years were also eligible if they had two or more family members with a blood cancer (PROMISE cohort). Participants with a plasma cell malignancy diagnosed before screening were excluded. Longitudinal clinical data were available for MGBB participants with a median follow-up time from serum sample screening of 4·5 years (IQR 2·4-6·7). The PROMISE study is registered with ClinicalTrials.gov, NCT03689595.
The median age at time of screening was 56·0 years (IQR 46·8-64·1). 5013 (66%) of 7622 participants were female, 2570 (34%) male, and 39 (<1%) unknown. 2439 (32%) self-identified as Black, 4986 (65%) as White, 119 (2%) as other, and 78 (1%) unknown. Using serum protein electrophoresis with immunofixation electrophoresis, the MGUS prevalence was 6% (101 of 1714) in high-risk individuals aged 50 years or older. Using mass spectrometry, we observed a total prevalence of monoclonal gammopathies of 43% (1788 of 4207) in this group. We termed monoclonal gammopathies below the clinical immunofixation electrophoresis detection level (<0·2 g/L) MGIPs, to differentiate them from those with higher concentrations, termed mass-spectrometry MGUS, which had a 13% (592 of 4207) prevalence by mass spectrometry in high-risk individuals aged 50 years or older. MGIP was predominantly of immunoglobulin M isotype, and its prevalence increased with age (19% 488 of 2564 for individuals aged <50 years, 29% 1464 of 5058 for those aged ≥50 years, and 37% 347 of 946 for those aged ≥70 years). Mass-spectrometry MGUS prevalence increased with age (5% 127 of 2564 for individuals aged <50 years, 13% 678 of 5058 for those aged ≥50 years, and 18% 173 of 946 for those aged ≥70 years) and was higher in men (314 12% of 2570) compared with women (485 10% 5013; p=0·0002), whereas MGIP prevalence did not differ significantly by gender. In those aged 50 years or older, the prevalence of mass spectrometry was significantly higher in Black participants (224 17% of 1356) compared with the controls (p=0·0012) but not in those with family history (368 13% of 2851) compared with the controls (p=0·1008). Screen-detected monoclonal gammopathies correlated with increased all-cause mortality in MGBB participants (hazard ratio 1·55, 95% CI 1·16-2·08; p=0·0035). All monoclonal gammopathies were associated with an increased likelihood of comorbidities, including myocardial infarction (odds ratio 1·60, 95% CI 1·26-2·02; p=0·00016 for MGIP-high and 1·39, 1·07-1·80; p=0·015 for mass-spectrometry MGUS).
We detected a high prevalence of monoclonal gammopathies, including age-associated MGIP, and made more precise estimates of mass-spectrometry MGUS compared with conventional gel-based methods. The use of mass spectrometry also highlighted the potential hidden clinical significance of MGIP. Our study suggests the association of monoclonal gammopathies with a variety of clinical phenotypes and decreased overall survival.
Stand Up To Cancer Dream Team, the Multiple Myeloma Research Foundation, and National Institutes of Health.
This protocol details a staining technique optimized for immunophenotyping of human bone marrow immune populations using mass cytometry. The protocol accounts for the limitations of working with ...human bone marrow, such as reduced viability, low cell counts, and fragile cell pellets, to successfully acquire single viable cells ready for downstream analysis. This assay can be used to characterize the activation, exhaustion, and cytotoxicity of immune populations and ensure comprehensive immunophenotyping of human bone marrow clinical samples.
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•Staining protocol for assessment of immune cells in human bone marrow clinical samples•Validated mass cytometry panel for distinction of major immune populations•Insights on gating strategy for multiparameter mass cytometry data analysis
This protocol details a staining technique optimized for immunophenotyping of human bone marrow immune populations using mass cytometry. The protocol accounts for the limitations of working with human bone marrow, such as reduced viability, low cell counts, and fragile cell pellets, to successfully acquire single viable cells ready for downstream analysis. This assay can be used to characterize the activation, exhaustion, and cytotoxicity of immune populations and ensure comprehensive immunophenotyping of human bone marrow clinical samples.
Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor
(PPAR
) agonists that represent an effective class of insulin-sensitizing agents; however, clinical use is associated with ...weight gain and peripheral edema. To elucidate the role of PPAR
expression in endothelial cells (ECs) in these side effects, EC-targeted PPAR
knockout (
) mice were placed on a high-fat diet to promote PPAR
agonist-induced plasma volume expansion, and then treated with the TZD rosiglitazone. Compared with
-floxed wild-type control (
) mice,
treated with rosiglitazone are resistant to an increase in extracellular fluid, water content in epididymal and inguinal white adipose tissue, and plasma volume expansion. Interestingly, histologic assessment confirmed significant rosiglitazone-mediated capillary dilation within white adipose tissue of
mice, but not
mice. Analysis of ECs isolated from untreated mice in both strains suggested the involvement of changes in endothelial junction formation. Specifically, compared with cells from
mice,
cells had a 15-fold increase in focal adhesion kinase, critically important in EC focal adhesions, and >3-fold significant increase in vascular endothelial cadherin, the main component of focal adhesions. Together, these results indicate that rosiglitazone has direct effects on the endothelium via PPAR
activation and point toward a critical role for PPAR
in ECs during rosiglitazone-mediated plasma volume expansion.
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