Fine-needle aspiration biopsy and flow cytometry immunophenotyping of lymphoid and myeloproliferative disorders of the spleen.

BACKGROUND: Flow cytometry (FC) is a useful adjunct to fine-needle aspiration biopsy (FNAB) in the evaluation of lymphoproliferative disorders. The application of FC to FNAB of the spleen (sFNAB) is reported. METHODS: Flow cytometry was performed on 18 sFNAB collected over 3 years. The series comprised 10 cases of non- Hodgkin lymphomas (NHL), 2 cases insufficient for diagnosis, 2 cases of reactive hyperplasia (RH), and 4 cases of myeloid metaplasia (MM). FNAB was performed under ultrasound guidance using a 22-gauge needle. One or two passes were sufficient to prepare a conventional smear that was immediately evaluated to select the cases studied and to prepare a cell suspension for FC. The following fluoresceinated antibodies were used: CD3, CD19/kappa/lambda, FMC7/CD23/CD19, Bcl-2, and CD13/HLA-DR. In six cases, cytospins were also prepared for immunocytochemistry and were tested for CD20 (L26), CD45Ro, and kappa and lambda light chain expression. RESULTS: Flow cytometry contributed to the diagnosis of all cases of NHL by assessing light chain restriction. The specific subtype was also diagnosed by CD19/CD5 and CD 19/CD10 coexpression in two cases. Flow cytometry quantified the percentage of myeloid cells in MM cases and contributed to the cytologic diagnosis showing a polyclonal light chain expression in RH cases.Immunocytochemistry was effective and concordant in four cases. Patients tolerated the sFNAB well and no complications were reported. Cytologic and FC diagnoses were confirmed by follow-up and by histologic evaluation in cases in which splenectomy was performed for therapeutic purposes. CONCLUSION: Flow cytometry applied to sFNAB corroborates the cytologic diagnosis in lymphoid and myeloproliferative disorders of the spleen and allows therapeutic decisions avoiding splenectomy

miR-29b sensitizes multiple myeloma cells to bortezomib-induced apoptosis through the activation of a feedback loop with the transcription factor Sp1

MicroRNAs (miRNAs) with tumor-suppressor potential might have therapeutic applications in multiple myeloma (MM) through the modulation of still undiscovered molecular pathways. Here, we investigated the effects of enforced expression of miR-29b on the apoptotic occurrence in MM and highlighted its role in the context of a new transcriptional loop that is finely tuned by the proteasome inhibitor bortezomib. In details, in vitro growth inhibition and apoptosis of MM cells was induced by either transient expression of synthetic miR-29b or its stable lentivirus-enforced expression. We identified Sp1, a transcription factor endowed with oncogenic activity, as a negative regulator of miR-29b expression in MM cells. Since Sp1 expression and functions are regulated via the 26S proteasome, we investigated the effects of bortezomib on miR-29b-Sp1 loop, showing that miR-29b levels were indeed upregulated by the drug. At the same time, the bortezomib/miR-29b combination produced significant pro-apoptotic effects. We also demonstrated that the PI3K/AKT pathway plays a major role in the regulation of miR-29b-Sp1 loop and induction of apoptosis in MM cells. Finally, MM xenografts constitutively expressing miR-29b showed significant reduction of their tumorigenic potential. Our findings indicate that miR-29b is involved in a regulatory loop amenable of pharmacologic intervention and modulates the anti-MM activity of bortezomib in MM cells

Therapeutic vulnerability of multiple myeloma to MIR17PTi, a first-in-class inhibitor of pri-mir-17-92

The microRNA cluster miR-17-92 is oncogenic and represents a valuable therapeutic target in c-MYC (MYC)-driven malignancies. Here, we developed novel LNA gapmeR antisense oligonucleotides (ASOs) to induce RNase H-mediated degradation of MIR17HG primary transcripts and, consequently, to prevent biogenesis of miR-17-92 microRNAs (miR-17-92s). The leading LNA-ASO, named MIR17PTi, impaired proliferation of several cancer cell lines (n=48) established from both solid and hematologic tumors by on-target antisense activity, and more effectively as compared to miR-17-92s inhibitors. By focusing on multiple myeloma (MM), we found that MIR17PTi triggers apoptosis via impairment of homeostatic MYC/miR-17-92 feed-forward loops (FFLs) in patient-derived MM cells; and induced MYC-dependent synthetic lethality. We show that alteration of a BIM-centered FFL is instrumental for MIR17PTi to induce cytotoxicity in MM cells. MIR17PTi exerts strong in vivo anti-tumor activity in NOD-SCID mice bearing clinically relevant models of MM, with advantageous safety and pharmacokinetics profiles in non-human primates. Altogether, MIR17PTi is a novel pharmacological tool to be tested in early-phase clinical trials against MM and other MYC-driven malignancies

A targeted sequencing approach in multiple myeloma reveals a complex landscape of genomic lesions that has implications for prognosis

Background: Next-generation sequencing (NGS) studies have shown that mul- tiple myeloma is a heterogeneous disease with a complex subclonal architecture and few recurrently mutated genes. The analysis of smaller regions of interest in the genome (\u201ctargeted studies\u201d) allows interrogation of recurrent genomic events with reduces complexity of downstream analysis at a lower price. Aims: Here, we performed the largest targeted study to date in multiple myelo- ma to analyze gene mutations, deletions and amplifications, chromosomal copy number changes and immunoglobulin heavy chain locus (IGH) translo- cations and correlate results with biological and clinical features. Methods: We used Agilent SureSelect cRNA pull down baits to target: 246 genes implicated in myeloma or cancer in general in a mixed gene discovery/confirmation effort; 2538 single nucleotide polymorphisms to detect amplifications and deletions at the single-gene and chromosome level; the IGH locus to detect translocations. We sequenced unmatched DNA from CD138- purified plasma cells from 418 patients with multiple myeloma at diagnosis, with a median follow-up of 5.3 years. We sequenced at an average depth of 337x using Hiseq2000 machines (Illumina Inc.). We applied algorithms developed in- house to call genomic events, filtering out potential artifacts and germline vari- ants. We then ranker each event on its likelihood of being \u201concogenic\u201d based on clustering, recurrence and cross-reference with the COSMIC database. Results: We identified 2270 gene mutations in 412/418 patients, and of those 688 were oncogenic. 342 patients harbored at least one oncogenic mutation. 215/246 genes showed at lease one likely somatic mutation, but only 106 showed at least one oncogenic mutation. 63% of oncogenic mutations were accounted for by the top 9 driver genes previously identified (KRAS, NRAS, TP53, FAM46C, BRAF, DIS3, TRAF3, SP140, IRF4), implying our gene discov- ery effort did not identify novel mutated genes. We included deletion of tumor suppressors, amplification of oncogenes, chromosomal copy number changes and IGH translocations for a total of 76 variables, so that 413/418 patients showed at least one informative driver genomic event, (median 4/patient). We investigated pairwise associations between events and found significant corre- lations, such as TP53 mutations and del(17p), CYLD mutations and del(16), FAM46C mutations and del(1p), SF3B1 mutations and t(11;14). Hotspots muta- tions of IRF4 lysine p.123 showed an inverse correlation with a hyperdiploid karyotype and del(16) as opposed to other missense mutations scattered along the gene, which has pathogenic implications. Survival was negatively affected by the cumulative burden of lesions in an almost linear fashion, with median survival of 10.97 and 4.07 years in patients with =7 lesions respectively, and this was independent of the nature of the genomic events. Given the het- erogeneity and complex interplay of the variables we fitted a cox-proportional hazard model to predict survival. We found that mutations in TP53, amplifications of MYC, deletions of CYLD, amp(1q), del12p13.31 and del17p13 where the only significant events, all promoting shorter survival. In particular, TP53 muta- tions and deletions, often co-occurring, had an additive effect so that carriers of both showed a dismal survival of 17 months (Figure 1).Summary/Conclusions: Due to the complex genomic landscape in MM, a discovery effort still requires large studies to derive significant associations. We conclude that a targeted sequencing approach may provide prognostic models and give insights into myeloma biology

A high-risk, Double-Hit, group of newly diagnosed myeloma identified by genomic analysis

Patients with newly diagnosed multiple myeloma (NDMM) with high-risk disease are in need of new treatment strategies to improve the outcomes. Multiple clinical, cytogenetic, or gene expression features have been used to identify high-risk patients, each of which has significant weaknesses. Inclusion of molecular features into risk stratification could resolve the current challenges. In a genome-wide analysis of the largest set of molecular and clinical data established to date from NDMM, as part of the Myeloma Genome Project, we have defined DNA drivers of aggressive clinical behavior. Whole-genome and exome data from 1273 NDMM patients identified genetic factors that contribute significantly to progression free survival (PFS) and overall survival (OS) (cumulative R2 = 18.4% and 25.2%, respectively). Integrating DNA drivers and clinical data into a Cox model using 784 patients with ISS, age, PFS, OS, and genomic data, the model has a cumlative R2 of 34.3% for PFS and 46.5% for OS. A high-risk subgroup was defined by recursive partitioning using either a) bi-allelic TP53 inactivation or b) amplification (≥4 copies) of CKS1B (1q21) on the background of International Staging System III, comprising 6.1% of the population (median PFS = 15.4 months; OS = 20.7 months) that was validated in an independent dataset. Double-Hit patients have a dire prognosis despite modern therapies and should be considered for novel therapeutic approaches

Transcriptional Silencing of the Wnt-Antagonist DKK1 by Promoter Methylation Is Associated with Enhanced Wnt Signaling in Advanced Multiple Myeloma

The Wnt/β-catenin pathway plays a crucial role in the pathogenesis of various human cancers. In multiple myeloma (MM), aberrant auto-and/or paracrine activation of canonical Wnt signaling promotes proliferation and dissemination, while overexpression of the Wnt inhibitor Dickkopf1 (DKK1) by MM cells contributes to osteolytic bone disease by inhibiting osteoblast differentiation. Since DKK1 itself is a target of TCF/β-catenin mediated transcription, these findings suggest that DKK1 is part of a negative feedback loop in MM and may act as a tumor suppressor. In line with this hypothesis, we show here that DKK1 expression is low or undetectable in a subset of patients with advanced MM as well as in MM cell lines. This absence of DKK1 is correlated with enhanced Wnt pathway activation, evidenced by nuclear accumulation of β-catenin, which in turn can be antagonized by restoring DKK1 expression. Analysis of the DKK1 promoter revealed CpG island methylation in several MM cell lines as well as in MM cells from patients with advanced MM. Moreover, demethylation of the DKK1 promoter restores DKK1 expression, which results in inhibition of β-catenin/TCF-mediated gene transcription in MM lines. Taken together, our data identify aberrant methylation of the DKK1 promoter as a cause of DKK1 silencing in advanced stage MM, which may play an important role in the progression of MM by unleashing Wnt signaling

Targeting IL-17A in Multiple Myeloma: A Potential Novel Therapeutic Approach in Myeloma

We have previously demonstrated that interleukin-17A (IL-17) producing Th17 cells are significantly elevated in blood and bone marrow (BM) in multiple myeloma (MM) and IL-17A promotes MM cell growth via the expression of IL-17 receptor. In this study, we evaluated anti-human IL-17A human monoclonal antibody (mAb), AIN457 in MM. We observe significant inhibition of MM cell growth by AIN457 both in the presence and absence of BM stromal cells (BMSC). While IL-17A induces IL-6 production, AIN457 significantly down-regulated IL-6 production and MM cell-adhesion in MM-BMSC co-culture. AIN-457 also significantly inhibited osteoclast cell–differentiation. More importantly, in the SCIDhu model of human myeloma administration of AIN-457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth and reduced bone damage compared to isotype control mice. To understand the mechanism of action of anti-IL-17A mAb, we report here, that MM cells express IL-17A. We also observed that IL-17A knock-down inhibited MM cell growth and their ability to induce IL-6 production in co-cultures with BMSC. These pre-clinical observations suggest efficacy of AIN 457 in myeloma and provide the rationale for its clinical evaluation for anti-myeloma effects and for improvement of bone disease

Heterogeneity of genomic evolution and mutational profiles in multiple myeloma

Multiple myeloma is an incurable plasma cell malignancy with a complex and incompletely understood molecular pathogenesis. Here we use whole-exome sequencing, copy-number profiling and cytogenetics to analyse 84 myeloma samples. Most cases have a complex subclonal structure and show clusters of subclonal variants, including subclonal driver mutations. Serial sampling reveals diverse patterns of clonal evolution, including linear evolution, differential clonal response and branching evolution. Diverse processes contribute to the mutational repertoire, including kataegis and somatic hypermutation, and their relative contribution changes over time. We find heterogeneity of mutational spectrum across samples, with few recurrent genes. We identify new candidate genes, including truncations of SP140, LTB, ROBO1 and clustered missense mutations in EGR1. The myeloma genome is heterogeneous across the cohort, and exhibits diversity in clonal admixture and in dynamics of evolution, which may impact prognostic stratification, therapeutic approaches and assessment of disease response to treatment