Characterization of Complete Lncrnas Transcriptome Reveals Expression of Lncrnas As a Prognostic Factor and Linc-Smilo As a Potential Therapeutic Target in Multiple Myeloma

Deregulation of long non-coding RNAs (lncRNAs) is emerging as a common feature of different human tumors and their investigation may uncover novel biomarkers and oncogenic mechanisms. Previous studies have suggested that the alteration of some lncRNAs may play an important role in the pathogenesis of multiple myeloma (MM); however, the complete expression landscape of lncRNAs has not been elucidated. In the present work we characterized the lncRNAs transcriptome of MM and determined the potential involvement of lncRNAs in the pathogenesis and clinical behavior of MM. To characterize the MM transcriptome, we performed paired-end strand-specific RNA sequencing (ssRNA-seq) in 38 purified plasma cell (PC) samples from MM patients and in 3 bone marrow PCs (BMPCs) of healthy donors, as well as in distinct normal B-cell populations (Naïve, Centroblasts, Centrocytes, Memory and Tonsilar PCs). We identified 40,511 novel lncRNAs that were expressed, accounting for more than half of MM transcriptome (56%). This group of novel lncRNAs together with previously annotated lncRNAs comprised most (82%) of the MM transcriptome. We studied the transcriptional heterogeneity in MM samples and observed that lncRNAs showed a much more heterogeneous expression than coding genes, suggesting that these elements could contribute to the biological heterogeneity of the disease. Moreover, to determine differentially expressed genes, each MM patient was compared to normal BMPCs, detecting 19,886 lncRNAs deregulated (10,351 overexpressed and 9,535 downregulated) in more than 50% of patients. We then analyzed the transcriptional dynamics of MM considering the different stages of B-cell differentiation and focused on a group of 989 lncRNAs that were upregulated specifically in plasma cells from MM in comparison with the rest of B-cell stages (MM-specific lncRNAs). Next, we aimed to determine whether upregulation of MM-specific lncRNAs in MM was under epigenetic control so we analyzed the distribution of six histone modifications with non-overlapping functions (H3K4me3, H3K4me1, H3K27ac, H3K36me3, H3K27me3, and H3K9me3) of within the lncRNAs of interest by ChIP-seq in MM cases as compared to normal B cell subtypes. We detected 89 lncRNAs with de novo epigenomic activation. These data suggest an epigenetic rewiring in MM where the loci of most MM-specific lncRNAs are in an inactive state in normal cells and become active in MM. We focused on a specific lncRNA, LINC-SMILO, de novo epigenetically active and expressed in MM cells to determine whether upregulation of this lncRNA could play a role in the pathogenesis of the disease. Knockdown of LINC-SMILO in 3 different MM cell lines (MM.1S, MM.1R and KMS-11) using two different shRNAs, resulted in reduced proliferation and induction of apoptosis of myeloma cells. Using low input RNA-seq (MARS-seq), we found that inhibition of LINC-SMILO was associated with activation of ERVs (Endogenous retroviruses) and increase in interferon (IFN) induced genes and activation of IFN pathways, essential for MM cells survival. Finally, we aimed to determine whether the use of specific lncRNAs could improve the current prognostic stratification of MM patients using the IA11 release of CoMMpass data. We analyzed the prognostic value of lncRNAs using COX regression analysis and Backward elimination of Stepwise regression analysis, obtaining that the overexpression of the lncRNA PDLIM1P4 together with 1q amplification and 17p deletion stratified MM patients in three different risk groups (Figure 1). In summary, our study shows that the lncRNA transcriptome is widely altered in MM and suggests that some of the identified lncRNAs have marked prognostic influence and can be used as potential therapeutic targets for MM. Disclosures Paiva: Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Melnick:KDAc Therapeutics: Membership on an entity's Board of Directors or advisory committees; Constellation Pharmaceuticals: Consultancy; Epizyme: Consultancy; Janssenn: Research Funding.

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Clonal Analysis of IgM Cells in Multiple Myeloma Patients Suggests the Co-Existence of Normal and Malignant Arms of the Myeloma Clone.

Blood ◽

10.1182/blood.v104.11.1415.1415 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1415-1415

Author(s):

Brian J. Taylor ◽

Ming Ye ◽

Erin R. Strachan ◽

Tara M. Tiffinger ◽

Andrew R. Belch ◽

...

Keyword(s):

Multiple Myeloma ◽

B Cell ◽

Plasma Cells ◽

Somatic Hypermutation ◽

Immunomagnetic Separation ◽

B Cell Differentiation ◽

Reading Frame ◽

A Minor ◽

Pcr Targeting ◽

Insight Into

Abstract Analysis of immunoglobulin V genes, which undergo stepwise changes during B cell differentiation such as VDJ rearrangement, somatic hypermutation, and class switch recombination, provides insight into the point of transformation of B cell tumors. In Multiple Myeloma (MM), clonotypic VDJ sequences of malignant plasma cells are mutated, homogeneous, and associated with post-switch constant regions (either IgG or IgA, called the clinical isotype), suggesting the malignant arm of the MM clone arises from transformation events in the late stages of the germinal centre reaction. By contrast, the existence of clonotypic VDJ associated with pre-switch IgM is well established, and we have shown persistent clonotypic IgM is associated with advanced disease at diagnosis and poor survival in MM. Whether clonotypic IgM cells represent a malignant progenitor or a non-malignant population that parallels disease severity is unclear. To address these possibilities, we focused our analysis of clonotypic VDJ mutation profiles on IgM+ cells sorted by immunomagnetic separation from MM patient peripheral blood cells (PBMC). IgM clonotypic transcripts were amplified by hemi-nested RT-PCR targeting the CDR2-C mu constant region in IgM+ cells from 4/7 patients. These products were cloned, and 122, 28, 27, and 25 IgM clonotypic colonies were identified by specific CDR2/CDR3 PCR for patients 1–4 respectively. Each of these clones was sequenced, and mutations were identified by comparison with the closest germline V gene and tumor derived plasma cell VDJ sequences. An average mutation frequency of 0.005, significantly greater than the Taq error rate, was obtained for the 250–280 bp fragment downstream of CDR2, including the D-J-C mu region. Typically, MM clones were observed with 1–2 mutations in this region, many localizing to the D-J-C mu region. Small deletions that preserve reading frame were also observed in the D region of single clones of patients 1 and 4 respectively. The detection of intraclonal heterogeneity amongst clonotypic IgM cells may reflect a normal arm of the myeloma clone that co-exists with the post-switch malignant arm. In previous work examining bulk PBMC populations we had detected diversified clonotypic cells in the non-clinical isotype compartment of one patient, but, in accordance with studies performed by several other groups, were unable to detect diversified pre-switch counterparts. In this work we have focused on IgM+ MM B cells, a compartment of the MM clone that may remain driven by antigenic selection and undergo persistent clonal expansion. Our analysis gives insight into the nature of this proposed normal arm of the myeloma clone, revealing two coexisting subsets of pre-switch clonotypic IgM cells: a major set exhibiting homogeneity, identity with post-switch tumor VDJ, and questionable transformation status, and a minor clonally heterogeneous set which may represent the pre-malignant clone from which myeloma arose.

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First Description of B Cell Maturation Antigen Expression in Light Chain Amyloidosis

Blood ◽

10.1182/blood-2019-127332 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5452-5452

Author(s):

Susan Bal ◽

Allison Sigler ◽

Alexander Chan ◽

David J. Chung ◽

Ahmet Dogan ◽

...

Keyword(s):

Multiple Myeloma ◽

B Cell ◽

Board Of Directors ◽

Light Chain ◽

Research Funding ◽

Plasma Cells ◽

Staining Intensity ◽

Advisory Committees ◽

Light Chain Amyloidosis ◽

B Cell Maturation

Background B-cell maturation antigen (BCMA) is a transmembrane protein belonging to the tumor necrosis factor (TNF) superfamily involved in the regulation of B cell proliferation and survival as well as maturation/differentiation into plasma cells. In multiple myeloma cells, overexpression of BCMA has been shown to activate mitogen activated protein kinase pathways (AKT, ERK1/2, and NF-κB) and upregulates anti-apoptotic proteins (MCL1, BCL2, BCL-xL) resulting in cellular proliferation. Immunotherapeutic strategies targeting BCMA are showing great promise in heavily pre-treated refractory multiple myeloma. Light Chain Amyloidosis (AL) is a multisystem disorder of clonal plasma cells that results in the production of an abnormal light chain which misfolds and deposits in the organs leading to disruption of tissue architecture, cellular stress, dysfunction and eventually, death. The smaller burden and lower proliferative potential of the offending clonal plasma cells in amyloidosis may potentially lend itself favorably to immunotherapeutic strategies targeting BCMA. Given the efficacy of this approach in MM, the evaluation of BCMA expression on the surface of amyloidogenic plasma cells is warranted. Methods All patients diagnosed with Light chain Amyloidosis at Memorial Sloan Kettering Cancer Center, NY between January 1, 2012, and December 31, 2018, who had unstained bone marrow samples were identified. These unstained BM biopsy samples were prospectively stained for BCMA expression using Immunohistochemistry (IHC). We utilized a clinical-grade assay (clone D6; catalog sc-390147; company Santa-Cruz; monoclonal antibody; dilution 1:400) in a CLIA compliant setting. We scored the biopsies for BCMA expression, intensity, and site of staining. We also obtained their demographic details, staging, and cytogenetic information for the patients with available samples. Results During the queried period, 28 unstained samples were available for testing from the time of disease diagnosis. The median age of the population was 63 years (range 41-73). 64% of patients were male and consistent with the literature; a majority of patients (75%) had lambda-typic clonal plasma cells. Cytogenetic abnormalities using fluorescence in situ hybridization (FISH) were reviewed, t(11;14) was seen in 36% patients, and chromosome 1q and del 13q were each seen in 32% of patients. No patient had t(4;14) or del 17p. The median clonal PC burden in BM at diagnosis was 10% (range2-80%) and 36% had > 10% plasma cells. In clonal PCs, the median BCMA expression was 80% (range 20-100%). Only one patient had a staining intensity under 50% (20%). Membranous staining was noted in 82% of patients and a Golgi pattern in 11%. The median staining intensity was 2 (range 1-3). Of the patients with baseline diagnostic samples available for testing, six patients had additional unstained bone marrow samples for staining at the time of relapse. The majority of patients (83%) who relapsed had >10% plasma cells with a higher median plasma cell burden of 35% (range 10-80). The median BCMA expression was 65% (range 50-80) with no patient having <50% expression. The staining pattern was membranous in 50%, Golgi in 17%, and Golgi-membranous in 33%. At the time of relapse, the median clonal PC burden was 13% (range 5-30). BCMA expression continued to be present at the time of relapse with a median 75% (range 50-100) with predominantly membranous staining (83%). The median staining intensity in both diagnostic and relapsed tissue within the six samples studied was 1. Conclusions Our study represents the first description of BCMA expression on the surface of amyloidogenic plasma cells to our knowledge. BCMA is uniformly expressed by pathologic PCs in AL amyloidosis both at the time of diagnosis and relapse. Given the efficacy of BCMA directed therapy in multiple myeloma, further investigation of these agents in light-chain amyloidosis are warranted and may provide an effective therapeutic strategy in this devastating disease. Figure Disclosures Dogan: Corvus Pharmaceuticals: Consultancy; Celgene: Consultancy; Seattle Genetics: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Roche: Consultancy, Research Funding. Giralt:Takeda: Consultancy, Research Funding; Johnson & Johnson: Consultancy, Research Funding; Kite: Consultancy; Novartis: Consultancy; Actinium: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Miltenyi: Research Funding; Spectrum Pharmaceuticals: Consultancy. Hassoun:Novartis: Consultancy; Janssen: Research Funding; Celgene: Research Funding. Landau:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees; Caelum: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Oncogenic MAF in Co-Operation with IRF4 Confers Extensive Chromatin Re-Arrangement in Plasma Cells and Generates 'Neo-Enhancers' That Regulate Genes Critical for Myeloma Biology

Blood ◽

10.1182/blood-2019-122371 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3783-3783

Author(s):

Alexia Katsarou ◽

Nikolaos Trasanidis ◽

Jaime Alvarez-Benayas ◽

Foteini Papaleonidopoulou ◽

Keren Keren ◽

...

Keyword(s):

B Cell ◽

Research Funding ◽

Plasma Cells ◽

De Novo ◽

Gene Annotation ◽

Myeloma Cell ◽

Chromatin Accessibility ◽

Pathway Enrichment Analysis ◽

Rna Seq ◽

Motif Analysis

Overexpression of the transcription factor MAF, as a result of its juxtaposition to the IgH enhancer [MAF-translocated t(14;16)], is a myeloma-initiating event in 3-5% of patients with multiple myeloma (MM) and confers a poor prognosis. MAF is also overexpressed in another 40% of cases, often in co-operation with the oncogene MMSET. The mechanisms by which MAF overexpression impacts on the regulatory genome to generate the MAF-driven oncogenic transcriptome and its direct targets are not known. To address this, we employed a multi-layer -omics approach using primary myeloma plasma cells (PC) as well as myeloma cell lines (MMCL). First, we determined the chromatin accessibility and transcriptome profiles of MAF-translocated myeloma by performing ATAC-seq and RNA-seq, respectively, in purified bone marrow CD138+ PC from two patients with t(14;16) and three healthy donors. We identified 6,640 differentially accessible regions, 87% of which displayed enhanced chromatin accessibility in MAF samples compared to normal PC. Secondary analysis comparing this with ATAC-seq data from a set of 28 other MM samples, including hyperdiploid, MMSET and CCND1-translocated MM, revealed 33% of those regions to be MAF subgroup specific (1,949 regions), with the rest shared between MAF and other cytogenetic groups. Gene annotation and pathway enrichment analysis using GREAT confirmed overrepresentation of the MF myeloma patient signature, as previously identified in microarray datasets. RNA-seq detected significant upregulation of approximately 900 genes in MAF samples compared to normal counterparts, including MAF itself (top 4th hit) as well as its presumed targets (CCND2, ITGB7 and NUAK1). Next, we obtained the MAF cistrome using ChIP-seq in the MAF-translocated MMCL MM1.S and integrated it with the primary PC ATAC-seq data. This revealed that 31% (618/1,949) of the differentially accessible regions in MAF-translocated MM PC are also MAF-bound. Additional overlay with ENCODE ChromHMM epigenome map showed that 47% of MAF binding sites are on active enhancers and 42% on active promoters signifying potential direct regulation of the corresponding genes. Next, we superimposed the accessible and MAF-bound loci on the epigenomic landscapes of normal PC and other B-cell types using their corresponding ChromHMM maps (Blueprint consortium data). Interestingly, 56% (345/618) of the MAF-specific regions were not active in any stage of B cell development. This suggests that aberrant MAF overexpression and chromatin binding in PC is associated with de novo activation of these chromatin regions, over half of which (200/345; 58%) are enhancers; we termed these 'neo-enhancers'. Upon de novo motif analysis of MAF ChIP-seq in MAF-translocated JJN3 and MM1.S MMCL, we confirmed MAF as the first and, interestingly, IRF4 as the second top hit, suggesting a possible MAF-IRF4 functional interaction in myelomagenesis. Indeed, overlay of the accessible MAF-bound loci with IRF4 ChIP-seq data in MM1.S revealed 63% co-occupancy (including 62% of "neo-enhancers"), proposing a novel and extensive co-operative chromatin-based network between the two transcription factors. Final integration of the accessible MAF-bound regions with the paired transcriptomes of primary myeloma PC revealed a set 206 candidate enhancer-gene pairs. Strikingly, we identified two IRF4-cobound "neo-enhancers" linked to overexpression of TLR4 and CCR1, two genes known for their roles in myeloma cell proliferation and migration. We confirmed significant downregulation of both genes upon shRNA-mediated knockdown of MAF in the two MAF-translocated MMCL, MM1.S and JJN3, as well as the lethality of MAF depletion. Further, MAF overexpression in MAF-negative myeloma backgrounds led to transcriptional upregulation of these genes, further validating them as MAF targets. While CRISPR/Cas9i experiments targeting TLR4 are ongoing, preliminary results validated the functional role of the "neo-enhancer" in CCR1 gene expression. In conclusion, we demonstrate for the first time an extensive re-organisation of the PC chromatin conferred by oncogenic MAF in MM; we reveal its extensive co-operation with IRF4 in this process; we validate the directly MAF-regulated genes and functionally characterise neo-enhancers of key MAF-dependent genes that in addition to MAF itself are also critical for myeloma biology. Disclosures Hatjiharissi: Janssen: Honoraria. Caputo:GSK: Research Funding. Karadimitris:GSK: Research Funding.

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Phenotypic and functional analysis of B cell lines from patients with multiple myeloma

Blood ◽

10.1182/blood.v66.2.444.bloodjournal662444 ◽

1985 ◽

Vol 66 (2) ◽

pp. 444-446

Author(s):

M Goldstein ◽

J Hoxie ◽

D Zembryki ◽

D Matthews ◽

AI Levinson

Keyword(s):

Multiple Myeloma ◽

Functional Analysis ◽

Cell Differentiation ◽

Cell Growth ◽

B Cell ◽

Cell Lines ◽

Plasma Cells ◽

Plaque Assay ◽

Functional Markers ◽

B Cell Differentiation

We characterized phenotypic and functional properties of B cell lines obtained from patients with multiple myeloma to determine how well they conform to particular stages of B cell differentiation. This information is a prerequisite for using such lines as tools for studying B cell growth and the regulation thereof. Two lines, GM1312 and GM1500, expressed B1 and Ia, determinants on early B cells, but expressed little, if any, T10, a determinant expressed on plasma cells. By contrast, B1 and Ia were poorly expressed on two other lines, GM2132 and U266. T10 was expressed on GM2132 but not on U266. Using a reverse hemolytic plaque assay, we also assessed the numbers of cells actively secreting immunoglobulin (IgSCs) in such cultures to provide a functional marker of B cell differentiation. We observed consistently higher numbers of IgSCs in cultures of GM2132 than in GM1500 and GM1312. These phenotypic and functional markers were stable over several months. The data suggest that such cell lines represent early (GM1312, GM1500) and later stages (GM2132, U266) of B cell differentiation, although all lines were derived from patients with multiple myeloma.

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Multiple Myeloma Patients Treated at Academic Centers Have Improved Survival Outcomes

Blood ◽

10.1182/blood-2021-151899 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1971-1971

Author(s):

Victoria A. Vardell ◽

Daniel A. Ermann ◽

Srinivas K. Tantravahi ◽

Brian McClune ◽

Mary Nicole Steinbach ◽

...

Keyword(s):

Clinical Trial ◽

Multiple Myeloma ◽

Research Funding ◽

Stem Cell Transplant ◽

Cox Regression ◽

Autologous Stem Cell Transplant ◽

Cell Transplant ◽

Cox Regression Analysis ◽

Cancer Centers ◽

Facility Type

Abstract Background Treatment at academic cancer centers (ACs) has been associated with improved outcomes across hematologic malignancies, including acute myeloid leukemia and non-Hodgkin lymphoma. ACs offer the benefit of high treatment volume in addition to enrollment in clinical trials, involvement in post-graduate education, and expanded access to diagnostic and treatment related services. Though studies on multiple myeloma (MM) have demonstrated a survival benefit with treatment at both high-volume centers and at NCCN designated cancer centers, this is the largest study to date examining the benefit of academic centers. Methods The National Cancer Database was utilized to obtain data on patients diagnosed with MM between 2004-2017 for which data on treatment facility type was available. Using the Commission on Cancer facility categories, patients treated at ACs were compared to those treated at non-academic centers (NACs), including small and large volume community cancer centers. Demographic and treatment characteristics were compared between centers, with median overall survival (OS) assessed by Kaplan Meier. Cox regression analysis was used to asses the HR for OS by facility type, and adjusted on multivariate analysis for age, sex, race, insurance, time to treatment, and use of autologous transplant. Results Of the 179,769 MM patients available, 42.4% were treated at ACs (p<0.05). Patients treated at ACs were younger than those treated at NACs (mean age 64.7 years vs. 69.2 years, p<0.05) and patients > 75 years of age were more often treated at NACs (35.6% vs. 20.3%, p<0.05). ACs were more likely to treat Black and other minority patients, with Black patients representing 23.5% vs. 18%, and other minorities 5.6% vs. 3.6% of patients treated at ACs vs. NACs, respectively; all p<0.05. Academic centers were more likely to treat uninsured patients (6.3% vs. 4.1%), patients on Medicaid (7.9% vs. 5.5%) as well as privately insured patients (41.5% vs. 29.6%),( all p<0.05). The majority of Medicare patients were treated at NACs (60.8%), p<0.05. The time from diagnosis to treatment was longer at ACs, at 32.4 vs. 26.5 days (p<0.05), and patients were more likely to receive autologous stem cell transplant as first line treatment at ACs (6.3% vs 2.2%, p<0.05). While clinical trial data is limited in the NCDB, the majority of the 495 patients treated under a clinical trial were treated at ACs (393 vs 102, or 0.5% vs 0.1% of patients treated at ACs vs NACs, p<0.05). Median OS at ACs was significantly longer than at NACs, with median OS of 67.8 months (95% CI 66.89-68.79 months) compared to 38.6 months (95% CI 38.15-39.13 months) at NACs, p<0.05. One year OS was 75% vs 61%, 5-year OS 48% vs 33%, and 10-year OS of 22% vs 12% at ACs vs NACs, respectively; p<0.05. When adjusted for age, gender, race, insurance, time to treatment, and use of autologous transplant on Cox Regression analysis, the improvement in OS remained. Patients treated at AC had hazard ratio of 0.77 for all-cause mortality (95% CI 0.756-0.784) when referenced to NACs on multivariate analysis (p<0.05). Conclusion Patients with MM had significantly improved survival when treated at academic centers compared to all other facility types. The improvement in OS remained when controlled for available treatment and demographic features. Multiple factors, including specialized care, trial enrollment, and early access to autologous stem cell transplant may contribute to these improvements. Further investigations into the factors contributing to such disparities are required to standardize care and improve overall outcomes. Figure 1 Figure 1. Disclosures Tantravahi: CTI BioPharma: Research Funding; Novartis: Research Funding; BMS: Research Funding; Abbvie Inc.: Research Funding; Karyopharm Therapeutics Inc.: Consultancy, Honoraria, Research Funding. Sborov: SkylineDx: Consultancy; GlaxoSmithKline: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy.

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Onset of Regulatory B Cells Occurs at Initial Stage of B Cell Dysfunction in Multiple Myeloma

Blood ◽

10.1182/blood-2019-128703 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1780-1780 ◽

Cited By ~ 1

Author(s):

Zhongqing Zou ◽

Tingting Guo ◽

Jian Cui ◽

Li Zhang ◽

Ling Pan

Keyword(s):

Multiple Myeloma ◽

B Cells ◽

B Cell ◽

Natural Science ◽

Research Funding ◽

Mononuclear Cells ◽

Plasma Cells ◽

T Test ◽

High Dose ◽

Regulatory B Cells

Introduction :Multiple myeloma(MM) is caused by aggregation of clonal plasma cells, clinically presenting as evolvement in order from monoclonal gamma globulin disease (MGUS), smoldering MM (SMM), symptomatic MM to plasma cell leukemia. Regulatory B cells (Bregs), only a small immunosuppressive subgroup of B cells, have been recently identified in the setting of autoimmune diseases, immune thrombocytopenia (ITP) and gastric cancer . The role of Bregs in MM remains poorly defined. Here, the study was carried out on how Bregs correlate with evolution of MM, as well as how Bregs would be influenced by bortezomib, which is currently the first-line anti-MM agent. Methods :All patients met the International Myeloma Working Group (IMWG) Criteria for the Diagnosis of MM. Mononuclear cells (MNCs) were isolated from bone marrow(BM)but not peripheral blood(PB)at specified time points. Cell numbers were quantified by hemocytometer. The ratios of Bregs and B cells were delected by flow cytometry (FCM). Propidium iodide was widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic. The results were expressed as the mean ± SD. Comparisons between 2 groups were performed with Student's t-test. Multiple groups (≥3) were analyzed by one-way ANOVA, and paired groups were analyzed by two-way ANOVA or Student t test. Data were graphed and analyzed using GraphPad Prism 6.0. P < 0.05 was considered statistically significant. Results: The study firstly found that Bregs' ratio increased at very beginning stage of MM and headed for extinction during progression of MM. It showed that Bregs' ratios were 11.7 ± 6.3%, 11 ± 8.6%, 15.8 ± 6.8%, and 4.9 ± 2.1% at stage of MGUS (n=5), SMM (n=4), newly diagnosed MM (NDMM) (n=9), and relapsed or refractory MM (RRMM) (n=6), respectively (p<0.05). We then obtained mononuclear cells from NDMM samples. It revealed a positive correlation on both ratios and absolute numbers between Breg subgroup and B cell groups when B cells' ratio was higher than 5% of BMMNCs, whereas Bregs' ratio was rarely detected when B cells' ratio was lower than 5%. Subsequently, a retest was made to observe how Breg subgroup would be influenced by using bortezomib to target B-cell reservoir in MM. We added bortezomib of different concentrations to MNCs from NDMM samples within culture medium RPMI1640 (Ruikos Biotechnologies) for 24 hours (n=6). Bregs and B cells were totally killed when treated with high-dose bortezomib (n=3), while partially killed with low-dose bortezomib (n=3). To further explore how bortezomib could influence Bregs, another 6 samples were enrolled to demonstrate that the apoptotic absolute number of Bregs significantly increased after treatment of high-dose bortezomib (135615 ± 92085 v.s. 81132 ± 52908, P ≤ 0.05). Conclusions : Bregs were strongly entwined with the whole group of preserved B cell in MM. Bregs began to increase at very beginning stage of MM when B cells were preserved, accompanying with transition from MGUS to diagnostic MM. Bregs would substantially decreased while B-cell reservoir diminished during MM progression or by B cell targeted bortezomib. Disclosures Zou: the National Natural Science Foundation of China: Research Funding. Guo:the National Natural Science Foundation of China: Research Funding. Cui:the National Natural Science Foundation of China: Research Funding. Zhang:the National Natural Science Foundation of China: Research Funding. Pan:the National Natural Science Foundation of China: Research Funding.

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A Novel Risk Scoring System to Predict Survival in Soft Tissue-Related Extramedullary Multiple Myeloma

Blood ◽

10.1182/blood-2021-144837 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4749-4749

Author(s):

Beihui Huang ◽

Qing Wang ◽

Wuping Li ◽

Ying Zhao ◽

Juan Li

Keyword(s):

Multiple Myeloma ◽

Soft Tissue ◽

Plasma Cells ◽

Cox Regression ◽

Conflicts Of Interest ◽

Risk Group ◽

False Negative ◽

High Risk Group ◽

Risk Scores ◽

Cox Regression Analysis

Abstract Objective: Extramedullary multiple myeloma (EMM) is an aggressive subtype of multiple myeloma, especially those soft tissue-related extramedullary multiple myeloma (EM-S). Plasma cells often infiltrate in extramedullary tissues, but the proportion of plamas cells in bone marrow is not high, so FISH test often produces false negative results. Therefore, the R-ISS staging system cannot predict the really prognosis of this type of patients. A novel risk score is needed for these patients. Methos: A cohort of 73 patients with EM-S in four centers in China were included, excluding those only with bone related extramedullary multiple myeloma, primary or secondary plasma leukemia. We performed univariate and multivariate Cox regression analysis, generated risk scores to predict outcomes in EM-S MM and compared with R-ISS staging. Results: In the Cox multivariate model, high LDH, low count of platelet、central nervous system involved, abdominal viscera involved (including liver, spleen, pancreas, kidney) and Ki67≥70% were found to significantly and independently predict outcomes for EM-S. According to these parameters, the patients were divided into three groups: the low-risk group with a median TTP of 14.13 months, and the medium-risk group with a median TTP of 8.53 months. In the high-risk group, the median TTP was 2.73 months (p<0.05 between any two groups). This novel risk is better to predict prognosis of this group of patients than R-ISS stage, in which the TTP in R-ISS stage I was 20.33 months, R-ISS stage II 9.83 months, and R-ISS stage III 4.83 months (p=0.065). Conclusion: The novel risk stratification for EM-S may be may be superior to R-ISS for EM-S MM patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Feasibility and Safety of Drug-Coated Balloon-Only Angioplasty for De Novo Ostial Lesions of Left Anterior Descending Artery: Two Center’s Retrospective Study

10.21203/rs.3.rs-817229/v1 ◽

2021 ◽

Author(s):

Chuang Li ◽

Xuebo Ding ◽

Lefeng Wang ◽

Kuibao Li ◽

Xinchun Yang ◽

...

Keyword(s):

Risk Factors ◽

Regression Analysis ◽

Propensity Score ◽

Propensity Score Matching ◽

Cox Regression ◽

De Novo ◽

Cox Regression Analysis ◽

Left Circumflex Artery ◽

Left Anterior Descending Artery ◽

Drug Coated Balloon

Abstract Background:There is limited evidence of drug-coated balloon (DCB) only angioplasty in percutaneous treatment of complex de novo ostial coronary lesions. The major objective of our study is to explore the feasibility and test safety of this innovative approach in ostial lesions of left anterior descending artery (LAD). Methods:Patients treated with paclitaxel DCB or second-generation drug-eluting stent (DES) were retrospectively enrolled from two different large centers. The primary endpoints were defined as major adverse cardiovascular events (MACE) composed of cardiovascular death, target lesion revascularization (TLR), target vessel revascularization (TVR), and recurrent myocardial infarction related to target artery occlusion. Cox regression analysis were used to identify risk factors for MACE and propensity score matching is performed to minimize the selection bias.Results:A total of 53 patients were treated with paclitaxel DCB and 336 patients with DES in ostial lesions of LAD were recruited. In accordance with propensity score matching, 49 patients treated with DCB-only coordinated with 49 ones with the strategy of DES. After average follow-up time of 10 months, the rate of MACE trended to lower in DCB-only angioplasty treatment arm and triggered by post-procedure TLR (MACE: 6% vs. 4%, p=0.65; TLR: 2% vs. 4%, p=0.56). Cox regression analysis indicated that not DCB-only angioplasty was considered as an independent risk factor for adverse events after adjustment for cofound risk factors (HR: 1.748, p=0.48).Conclusions:Use of DCB-only approach in treatment of isolated ostial LAD disease could be an innovative and safe strategy without additional risk of aggressive progression of left circumflex artery.

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Ring Sideroblasts and SF3B1 Mutations in Myelodysplastic Syndromes (MDS): Are They Two Faces of the Same Coin? a Study on Behalf of the MDS Clinical Research Consortium (MDS CRC)

Blood ◽

10.1182/blood.v128.22.4321.4321 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4321-4321

Author(s):

Rami S. Komrokji ◽

John Barnard ◽

David P Steensma ◽

Amy E. DeZern ◽

Gail J. Roboz ◽

...

Keyword(s):

Regression Analysis ◽

Myelodysplastic Syndromes ◽

Research Funding ◽

Somatic Mutations ◽

Cox Regression ◽

Hazard Ratio ◽

Free Survival ◽

Cox Regression Analysis ◽

Mutational Status ◽

Hazard Ratios

Abstract Introduction Recurrent somatic mutations in SF3B1, a gene encoding a spliceosome component, have been identified in patients (pts) with myelodysplastic syndromes (MDS). SF3B1 mutations (MT) are more commonly detected in pts with ring sideroblast (RS) morphology and are associated with favorable outcome. The proposed 2016 World Health Organization (WHO) MDS classification categorizes pts with >5% RS and SF3B1 MT as MDS with RS, in contrast to prior WHO classifications which required ≥15% RS regardless of genotype. In this study, we explored the prognostic value of RS and SF3B1 MT and assessed the validity of the new proposal. Methods We identified 471 pts with MDS and known SF3B1 mutational status from MDS CRC institutions. RS were assessed as present or absent (RS +/-) based on bone marrow aspirate reports (n=157); in cases where quantitative data on RS% were available (n=41), pts were grouped in the 5-15% RS group or > 15% RS group. Survival was calculated from time of diagnosis. Cox regression analysis was used to estimate hazard ratios for overall survival (OS) and AML free survival (AFS), which was defined as time to death or AML transformation, respectively. Chi-squared and Wilcoxon tests were used to test for differences in categorical and continuous distributions, respectively. Results: Among 471 pts with known SF3B1 mutational status, 76 (16%) had MT. Pts with MT had lower-risk International Prognostic Scoring System (IPSS) scores compared to SF3B1 wild-type (WT; 79% vs 57%, p < .001). Among pts with MT, 50% had RS + compared to 19% RS + in the WT group (p < .001). MT were independently associated with better OS (HR 0.48, p= .001) and longer AFS (HR 0.5, p <.005) after adjusting for age and IPSS. We compared outcomes of four groups: WT/RS-, MT/RS-, WT/RS+, and MT/ RS +. Adjusting for age and IPSS, pts with MT/RS + had the best outcome, with hazard ratios for AFS of 4.2 for WT/RS- vs. MT/RS+ (p =.018), 4.1 for MT/RS- vs. MT/RS+ (p =.045), and 5.1 for WT/RS+ vs. MT/RS+ (p= .01). We compared 7 pts with 5-15% RS to 22 pts with >15% RS. Among patients with RS 5-15%, 4/7 pts (57%) were classified as MDS with excess blasts compared to 24% for those RS >15% (p=.09). Pts with 5-15% RS were more likely to be thrombocytopenic (5/7, 71%) compared to >15% RS (29%, p=.04). One patient (14%) with 5-15% RS had MT compared to 12 (55%) pts with > 15% RS, p= .06. In Cox regression analysis using the RS 5-15% group as the reference, the hazard ratio for RS > 15% for AFS was 0.26 (p = .034) and the hazard ratio for MT for AFS was 0.08 (p= .002). Conclusions SF3B1 somatic mutations in MDS are commonly associated with RS, better OS and longer AML-free survival. Patients with RS and MT had a significantly better outcome than those with either isolated RS or MT, or neither. These data support incorporation of SF3B1 mutation status into the WHO classification regardless of RS percent, though with differentiation for those with RS and MT. Disclosures Komrokji: Novartis: Consultancy, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy; Boehringer-Ingelheim: Research Funding. Roboz:Cellectis: Research Funding; Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy.

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Prognostic Factors in CD10 Negative Precursor B-Cell Acute Lymphoblastic Leukemia in Children: Data from Three Consecutive Trials ALL-BFM 86, 90, and 95.

Blood ◽

10.1182/blood.v104.11.1957.1957 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1957-1957

Author(s):

Anja Möricke ◽

Richard Ratei ◽

Wolf-Dieter Ludwig ◽

Jochen Harbott ◽

Arndt Borkhardt ◽

...

Keyword(s):

Regression Analysis ◽

Prognostic Factors ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Cox Regression ◽

Lymphoblastic Leukemia ◽

Cns Involvement ◽

Total N ◽

Cox Regression Analysis ◽

Cell Acute Lymphoblastic Leukemia

Abstract The overall unfavorable prognosis of CD10 negative (CD10−) precursor B-cell acute lymphoblastic leukemia (BCP-ALL) is well known. We analyzed 4473 pediatric patients (pts) <18 years (y) with BCP-ALL and immunophenotyping of CD10 enrolled from 1986 to 2000 in three consecutive ALL-BFM trials to explore prognostic factors in the CD10− subset. CD10 negativity was defined by CD10 expression in <20% of blasts. 233 pts (5.2%) were CD10−. In comparison to CD10 positive (CD10+) BCP-ALL pts, CD10− pts comprised more infants (age <1y 34% vs. 1%, p(X2)<0.001), more cases with hyperleukocytosis (WBC ≥100/nl 43% vs. 6%, p<0.001), more CNS involvement (CNS positive 10% vs. 2%; p<0.001) and an impaired treatment response (prednisone poor response (PPR) 22% vs. 5%, p<0.001; induction failure 6% vs. 1%, p<0.001). Estimated probability of 5 years event free survival (5y-pEFS) was significantly lower in pts with CD10− as compared to CD10+ BCP-ALL (49±3% vs 81±0.6%, p(log-rank)<0.001). Cox regression analysis including age, WBC, prednisone response (PR) and MLL/AF4 status as covariables revealed CD10 negativity as independent prognostic factor (RR 1.5, 95% confidence interval (CI) 1.1–2.1, p=0.01). Further analyses were performed within the CD10− group: 83% of infants and 60% of pts ≥1y were successfully analyzed for MLL/AF4. MLL/AF4 was detected in 55% of pts <1y and 27% of pts ≥1y. The well known risk factors for BCP-ALL (sex, age, WBC, CNS involvement, MLL/AF4 and PPR) also had prognostic impact within the CD10− group: n* 5y-pEFS* (%) SE (%) p (log-rank) *5 pts w/o reinduction were excluded sex female 109 55 5 0.022 male 119 40 5 age <1y 78 25 5 <0.001 ≥1y 150 62 4 WBC <100/nl 128 62 4 <0.001 ≥100/nl 100 33 5 CNS neg 181 54 4 0.011 pos 21 33 10 MLL/AF4 neg 95 53 5 0.001 pos 61 29 6 PR good 170 57 4 <0.001 poor 50 30 6 Out of a number of immunophenotypic markers, analyzed at different expression cut-off points, CD24 at missing or weak expression of <40% and CD65 at high expression of ≥40% were significantly correlated with unfavorable clinical characteristics and worse outcome within the CD10− group. Significant correlation with PR could only be demonstrated for expression of CD24, which is presumed to act as negative regulator in B-cell development through mediation of apoptosis. age<1y* WBC ≥100/nl* MLL/AF4 pos* PPR# pEFS§ n/total (%) n/total (%) n/total (%) n/total (%) % ±SE * all p(X2)<0.01, #CD24 p=0.01, CD65 n.s., §all p(log-rank]<0.001 CD24 <40% 37/77 (48) 52/77 (68) 33/56 (59) 24/73 (33) 32 ±5 CD24 ≥40% 34/122 (28) 35/122 (29) 18/76 (24) 20/119 (17) 59 ±5 CD65 <40% 56/180 (31) 67/180 (37) 39/120 (33) 39/175 (22) 30 ±7 CD65 ≥40% 22/44 (50) 32/44 (73) 21/34 (62) 10/41 (24) 54 ±4 Including age, WBC, PR and MLL/AF4 status as covariables, out of the analyzed markers only CD65 proved to be an independent prognostic factor in CD10− BCP-ALL (Cox regression analysis: RR 1.5, 95% CI 1.1–2.9, p=0.018). The identification of additional prognosis associated immunophenotypic markers may contribute to further refinement of treatment strategies for CD10− BCP-ALL pts.

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