scholarly journals Utx Insufficiency Cooperates with Braf V600E in the Induction of Myeloma in Mice

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1005-1005
Author(s):  
Ola Rizq ◽  
Naoya Mimura ◽  
Motohiko Oshima ◽  
Shuji Momose ◽  
Yaeko Nakajima-Takagi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Mouse Model ◽  
Plasma Cell ◽  
High Frequency ◽  
Research Funding ◽  
Plasma Cells ◽  
Braf V600e ◽  
Gene Sets ◽  
Plasma Cell Neoplasms

Abstract Introduction: Dysfunction of epigenetic pathways has been frequently implicated in hematological malignancies. In multiple myeloma (MM), EZH2, a methyltransferase that induces histone H3 lysine 27 trimethylation (H3K27me3), acts as an oncogene as evidenced by its overexpression, which was found to be positively correlated with disease progression (Kalushkova et al. PloS One. 2010). We have shown that inhibition of both EZH2 and its homolog EZH1 is effective in eradicating MM cells in vitro and in vivo (Rizq et al. Clin Cancer Res. 2017). In addition, inactivating somatic mutations in UTX/KDM6A, an X-linked histone demethylase that removes di- and tri-methyl groups from H3K27, are found in 3 - 10% of MM patients (van Haaften et al. Nat Genet. 2009 and Pawlyn et al. Clin Cancer Res. 2016), indicating a tumor suppressive role for UTX, which has yet to be delineated. Up till now, no mouse model has been generated to test Utx insufficiency in post germinal center (GC) B cells and plasma cells. On the other hand, an activating mutation V600E in the BRAF kinase gene is closely associated with aggressive disease features such as extramedullary disease and shorter overall survival in MM patients (Andrulis et al. Cancer Discov. 2013) and could accelerate induction of myeloma in mice. Methods: To investigate whether loss of Utx cooperates with Braf V600E in myelomagenesis in mice, we generated and analyzed mice with conditional knock-out allele of Utx and/or knock-in allele of Braf V600E combined with Cγ1-Cre allele, in which Cre is activated by immunization in post GC B cells. Results: Loss of Utx and Braf V600E synergistically induced post GC B-cell lymphoma and plasma cell neoplasms in mice and significantly shortened the survival of mice compared with control mice and either allele alone. Utx-/-Braf V600E females succumbed to death earlier than Utx-/YBraf V600Emales and Utx-/+Braf V600Efemales. Of note, plasma cell neoplasms developed at a high frequency in Utx-/YBraf V600Emales and Utx-/+Braf V600Efemales and, less frequently, in Utx-/-Braf V600E females. Mice with plasma cell neoplasms showed expansion of CD138+ plasma cells in bone marrow as well as spleen and/or lymph nodes, exhibiting extramedullary disease. Loss of Utx alleles and expression of Braf V600E were confirmed by genomic PCR of plasma cells. Importantly, the clonality of plasma cells was demonstrated by genomic PCR detecting rearrangements of immunoglobulin heavy and light chain genes. In addition, M protein was detected by serum protein electrophoresis (SPEP) at a high frequency. Notably, we were able to establish murine myeloma cell lines from moribund compound mice. These cells readily engrafted in the bone marrow of NOG mice after transplantation and caused myeloma-associated phenotypes including paraplegia in recipient mice. Interestingly, Utx-/-Braf V600E cells were sensitive to dual inhibition of EZH2 and EZH1 but not to specific inhibition of EZH2 in culture. They also showed decreased susceptibility to proteasome inhibitors when compared with human MM cell lines. To gain insight into the changes in the transcriptional landscape following Utx loss, we performed RNA sequencing (RNA seq) and then gene set enrichment analysis (GSEA). We found positive enrichment of gene sets related to Myc, implying that Myc is one of the main drivers of myelomagenesis in our mouse model. In addition, gene sets related to MM were significantly enriched following Utx loss. We are now working on ChIP sequencing (ChIP seq) of UTX-related histone modifications to evaluate the epigenetic impact of Utx loss on myelomagenesis. Conclusion: Utx insufficiency cooperates with Braf V600E in the induction of myeloma in mice. Our mouse model is a promising tool for understanding the role of epigenetic dysregulation in the pathogenesis of MM and evaluating novel anti-myeloma agents. Disclosures Okuno: Celgene: Research Funding. Tamaru:Nichirei Bioscience INC.: Research Funding; Takeda Pharmaceutical Company Limited: Speakers Bureau.

A Novel Role For Histone Deacetylase 11 (HDAC11) In Plasma Cell Differentation and Survival

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1907-1907
Author(s):  
Eva Sahakian ◽  
Jason B. Brayer ◽  
John Powers ◽  
Mark Meads ◽  
Allison Distler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
Plasma Cell ◽  
Research Funding ◽  
Plasma Cells ◽  
Myeloma Cells ◽  
Peripheral Blood Plasma

Abstract The role of HDACs in cellular biology, initially limited to their effects upon histones, is now appreciated to encompass more complex regulatory functions that are dependent on their tissue expression, cellular compartment distribution, and the stage of cellular differentiation. Recently, our group has demonstrated that the newest member of the HDAC family of enzymes, HDAC11, is an important regulator of IL-10 gene expression in myeloid cells (Villagra A Nat Immunol. 2009). The role of this specific HDAC in B-cell development and differentiation is however unknown. To answer this question, we have utilized a HDAC11 promoter-driven eGFP reporter transgenic mice (TgHDAC11-eGFP) which allows the monitoring of the dynamic changes in HDAC11 gene expression/promoter activity in B-cells at different maturation stages (Heinz, N Nat. Rev. Neuroscience 2001). First, common lymphoid progenitors are devoid of HDAC11 transcriptional activation as indicated by eGFP expression. In the bone marrow, expression of eGFP moderately increases in Pro-B-cells and transitions to the Pre- and Immature B-cells respectively. Expression of eGFP doubles in the B-1 stage of differentiation in the periphery. Of note, examination of both the bone marrow and peripheral blood plasma cell compartment demonstrated increased expression of eGFP/HDAC11 mRNA at the steady-state. These results were confirmed in plasma cells isolated from normal human subjects in which HDAC11 mRNA expression was demonstrated. Strikingly, analysis of primary human multiple myeloma cells demonstrated a significantly higher HDAC11 mRNA expression in malignant cells as compared to normal plasma cells. Similar results were observed in 4/5 myeloma cell lines suggesting that perhaps HDAC11 expression might provide survival advantage to malignant plasma cells. Support to this hypothesis was further provided by studies in HDAC11KO mice in which we observed a 50% decrease in plasma cells in both the bone marrow and peripheral blood plasma cell compartments relative to wild-type mice. Taken together, we have unveiled a previously unknown role for HDAC11 in plasma cell differentiation and survival. The additional demonstration that HDAC11 is overexpressed in primary human myeloma cells provide the framework for specifically targeting this HDAC in multiple myeloma. Disclosures: Alsina: Millennium: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Baz:Celgene Corporation: Research Funding; Millenium: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding.

Generation of a Novel, Multi-Stage, Progressive, and Transplantable Model of Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 327-327
Author(s):  
Takashi Asai ◽  
Silvia Menendez ◽  
Delphine Ndiaye-Lobry ◽  
Anthony R Deblasio ◽  
Kazunori Murata ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Animal Model ◽  
B Cells ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Class Switch Recombination ◽  
Class Switch ◽  
Multi Stage ◽  
Plasma Cell Neoplasms

Abstract Abstract 327 Multiple myeloma is characterized by the progressive expansion of monoclonal plasma cells in the bone marrow, which leads to the production of serum and/or urine monoclonal proteins and systemic complications including lytic bone lesions, renal abnormalities hypercalcemia, and infections. Although the treatment of multiple myeloma has vastly improved, multiple myeloma remains a generally incurable disease. Transgenic mouse models have been generated that develop plasma cell accumulations or myeloma, however these models are quite imperfect in mimicking the human disease. Quite serendipitously, we have generated a multi-stage, progressive, and transplantable mouse model of multiple myeloma, crossing a genetically modified mouse with aberrant class switch recombination with another modified mouse that has elevated DNA damage response signaling. We have reported that cells expressing the hypermorphic Rad50s allele show constitutive ATM activation, leading to cancer predisposition and aggressive hematopoietic failure in Rad50s/s mice. While deficiency of the transcription factor Mef/Elf4, which regulates the quiescence of hematopoietic stem/progenitor cells, can mitigate hematopoietic failure observed in Rad50s/s mice, we found that 70% of Mef−/−Rad50s/s mice more than 200 days old died from multiple myeloma, plasmacytoma, or plasma cell leukemia, confirmed by pathology, immunohistochemistry, flowcytometry (CD138/B220 profiles), and PCR analysis for VDJ recombination. Prior to the onset of the plasma cell neoplasms, the Mef−/−Rad50s/s mice show abnormal plasma cell accumulation in the peripheral blood and bone marrow, which worsens with age. As the mice age, they also develop progressive increases in g-globulin levels and decreases in serum albumin levels. Monoclonal protein peaks were frequently observed in the serum of mice older than 200 days, and in step with the progressive nature of these manifestations, anemia and lower bone mineral density becomes apparent as the mice further age. Overall, the median survival of the Mef−/−Rad50s/s mice is approximately 470 days. The plasma cell neoplasms derived from Mef−/−Rad50s/s mice can be transplanted into recipient mice and the onset of the transplanted disease is markedly accelerated, to approximately 4 weeks post transplantation. Thus, the transplanted neoplastic Mef−/−Rad50s/s plasma cells appear to be more aggressive than the original ones. Taken together, our findings suggest that the Mef−/−Rad50s/s animal model can recapitulate the spectrum and pace of human plasma cell neoplasms, including the progression from monoclonal gammopathy to multiple myeloma. Class switch recombination is facilitated in Mef−/−Rad50s/s B cells in vitro, compared with control, Mef−/−, and Rad50s/s B cells, thus the plasma cell neoplasms found in Mef−/−Rad50s/s mice may result from Rad50s-driven oncogenesis. This novel Mef−/−Rad50s/s myeloma animal model should be useful for the drug screening of novel anti-myeloma compounds, as well as defining the pathogenesis of multiple myeloma/plasma cell neoplasms. Disclosures: No relevant conflicts of interest to declare.

Humoral Immunity and Plasma Cell Changes in Patients Responding to CD19-Specific Chimeric Antigen Receptor (CAR)-Modified T-Cell Adoptive Immunotherapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1110-1110
Author(s):  
Vijay Bhoj ◽  
Michael C Milone ◽  
Carl H. June ◽  
David Porter ◽  
Stephan A. Grupp ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Humoral Immunity ◽  
Research Funding ◽  
Plasma Cells ◽  
Flow Cytometric ◽  
Antibody Titers

Abstract Introduction: T cells engineered to express chimeric antigen receptors (CARs) recognizing CD19 (CART19) can eliminate malignant cells in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). We and other groups have shown that persistent tumor eradication by CD19-specific T cell immunotherapy is accompanied by normal B-cell aplasia. It is assumed that responding patients cannot make new antibody responses post-successful CART19 treatment; however, the status of previously established humoral immunity in these patients is currently unknown. Understanding the consequence of successful CART19 therapy on established humoral immunity has implications for both the clinical management of CART19-treated patients as well as the potential application of this therapy to non-malignant diseases such as autoimmunity and transplantation. Methods: We performed a prospective, observational study of adult and pediatric patients with ALL and adults with relapsed/refractory CLL, who were enrolled in clinical trials of CART19 at our institution. Serum antibody titers to previously-generated vaccine or vaccine-related pathogens (Streptococcus pneumoniae, Tetanus toxoid, Hemophilus influenza type-B (HIB), Measles, Mumps, and Rubella) were determined along with a quantitative assessment of B-cell and plasma cell frequencies in blood and bone marrow aspirates. Specimens were collected during pre-established study assessments or additional time points when collected as required for clinical management. Due to the challenges of assessing plasma cells, multiple methods were employed for their quantification in fresh specimens including flow cytometry and immunohistochemistry (IHC). Flow cytometric assessment of plasma cells was performed on freshly obtained marrow samples. Only patients with at least 3 months of B-cell aplasia in the absence of regular intravenous immunoglobulin (IVIg) infusions were included in the study. Results: All patients had no evidence of leukemia or peripheral B cells post-CART19 infusion at the time of this study. Compared to pre-CART19 serum titers, antibodies to S. pneumoniae remained stable or increased in 9 of 12 patients despite lack of circulating B-cells. Antibody titers to Tetanus toxoid were stable or increased in 13 of 14 patients. Anti-HIB levels were stable or increased in 9 of 11 patients and antibodies to Measles, Mumps and Rubella were stable or increased in 12 of 13, 11 of 13, and 12 of 13 patients, respectively. Flow cytometric analysis of bone marrow aspirates after CART19 infusion revealed three patients with persistence of CD38+ CD138+ plasma cells (at 1, 3 and 9 months post infusion, respectively) despite a complete absence of peripheral CD19+ B cells. In 9 patients, CD20 and CD138 IHC analysis of bone marrow core biopsies revealed a decrease in plasma cell (ranges: 1-5% pre-CART19, 0-<1% post-CART19), consistent with our previously published data. Finally, in another subset of patients, neither B cells nor plasma cells were detectable by flow cytometry of aspirate material or IHC of core biopsies collected either pre- or post-CART19 treatment. Conclusions: The stable or increased titers of antibodies to previous vaccines are surprising and may, in part, reflect improved marrow function as a result of leukemia eradication. The demonstration of plasma cells in a subset of patients in the absence of detectable tumor or normal B cells provides strong evidence for the existence of a population of plasma cells that are resistant to lysis by CART19 cells. This is consistent with antibody titers to previously generated vaccine antigens, which remain stable despite effective CART19 treatment. The additional finding of a decrease in CD138+ cells in several patients by IHC suggests that some populations of plasma cells are either targeted directly by CART19 or have a short half-life (e.g. plasmablasts); CD138 is not sufficient to distinguish these populations. Overall, these results indicate that long-lived plasma cells are resistant to CART19, likely due to a loss of CD19 during plasma cell differentiation. Continued analysis of remaining plasma cells in the absence of ongoing B-cell maturation as a result of CART19 persistence may provide important information on turnover rates of these long-lived cells in humans. Disclosures Bhoj: Novartis: Research Funding. Milone:Novartis: Patents & Royalties, Research Funding. June:Novartis: Research Funding, Royalty income Patents & Royalties. Porter:Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. Lacey:Novartis: Research Funding. Mahnke:Novartis: Research Funding.

Should CD138 Immunohistochemistry be Standard Recommended Practice for Bone Marrow Evaluation of Plasma Cell Neoplasms?

2020 ◽  
Vol 154 (Supplement_1) ◽  
pp. S110-S110
Author(s):  
A Vijayanarayanan ◽  
K Inamdar ◽  
M Menon ◽  
P Kuriakose
Keyword(s):  
Bone Marrow ◽  
Plasma Cell ◽  
Linear Correlation ◽  
Plasma Cells ◽  
Pearson Correlation ◽  
Protein Electrophoresis ◽  
Pearson Correlation Coefficient ◽  
Cell Percentage ◽  
Significant Linear Correlation ◽  
Plasma Cell Neoplasms

Abstract Introduction/Objective Myeloma diagnosis by a pathologist requires 10% plasma cells (PC) or a biopsy proven plasmacytoma in addition to myeloma defining events. PC% &gt; 60% is a biomarker of malignancy under this definition. WHO allows for assesment of plasma cell percentage either by aspirate count or by CD138 immunohistochemistry (IHC). There is lack of consensus on aspirate smear adequacy for PC% estimation. Uneven distribution of plasma cells, hemodilution and/or patchy infiltration can lead to gross underestimation. We compared PC% by aspirate count and CD138 IHC and established corelation with serum protein electrophoresis (SPEP) values. Methods 67 myeloma cases were included after excluding cases with suboptimal or inadequate aspirate smears. Two hematopathologists evaluated the diagnostic marrow (therapy naive) for PC% by aspirate count and CD138 IHC on biopsy/clot section. Corresponding SPEP and Free light chain (FLC) values were obtained. Correlation coefficent was calculated using Pearson correlation coefficient (GraphPad Prism). Results The Ig subtypes included IgG (41/67) and IgA (17/67). 12 cases had available FLC values. Both average and median PC% by CD138 IHC was considerably higher (50%, 52%) compared to aspirate count (29%, 21%). However, PC% by aspirate smear count and CD138 IHC demonstrated a significant linear correlation (r=0.71, p60% by CD138 (and not by aspirate count). Conclusion CD138 IHC based PC% is consistently higher, nevertheless, statistically significant linear corelation is observed between aspirate count PC% and CD138 IHC. A significant linear correlation is observed between CD138 IHC and SPEP (IgG and IgA), however, no such correlation is observed with aspirate count. More cases were diagnosed as myeloma (11%) and higher propotion of cases (35%) had biomarker of malignancy i.e. PC% &gt;60% by CD138 IHC. Based on these findings, we propose estimation of PC% by CD138 immunostain be a recommended standard practice for better clinicopathologic and biologic correlation.

scholarly journals Complement receptors regulate differentiation of bone marrow plasma cell precursors expressing transcription factors Blimp-1 and XBP-1

2005 ◽  
Vol 201 (6) ◽  
pp. 993-1005 ◽  
Author(s):  
Dominique Gatto ◽  
Thomas Pfister ◽  
Andrea Jegerlehner ◽  
Stephen W. Martin ◽  
Manfred Kopf ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Cell Activation ◽  
Plasma Cells ◽  
Antibody Responses ◽  
Complement Receptors ◽  
Essentially Normal ◽  
Bone Marrow Plasma

Humoral immune responses are thought to be enhanced by complement-mediated recruitment of the CD21–CD19–CD81 coreceptor complex into the B cell antigen receptor (BCR) complex, which lowers the threshold of B cell activation and increases the survival and proliferative capacity of responding B cells. To investigate the role of the CD21–CD35 complement receptors in the generation of B cell memory, we analyzed the response against viral particles derived from the bacteriophage Qβ in mice deficient in CD21–CD35 (Cr2−/−). Despite highly efficient induction of early antibody responses and germinal center (GC) reactions to immunization with Qβ, Cr2−/− mice exhibited impaired antibody persistence paralleled by a strongly reduced development of bone marrow plasma cells. Surprisingly, antigen-specific memory B cells were essentially normal in these mice. In the absence of CD21-mediated costimulation, Qβ-specific post-GC B cells failed to induce the transcriptional regulators Blimp-1 and XBP-1 driving plasma cell differentiation, and the antiapoptotic protein Bcl-2, which resulted in failure to generate the precursor population of long-lived plasma cells residing in the bone marrow. These results suggest that complement receptors maintain antibody responses by delivery of differentiation and survival signals to precursors of bone marrow plasma cells.

Frequent Dysregulation of Fc Gamma Receptor IIb (Fc γRIIb) and Immunoreceptor Tyrosine-Based Inhibitory Motif (ITIM) Signaling Occurs in Multiple Myeloma (MM) Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2917-2917
Author(s):  
Jennifer Li ◽  
Andrew Leu ◽  
Mingjie Li ◽  
Ethan D Hobel ◽  
Kevin Delijani ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Critical Role ◽  
Rt Pcr ◽  
Binding Ability ◽  
Downstream Signaling

Abstract Abstract 2917 The inhibitory Fc receptor, Fc γRIIb, is expressed on plasma cells, controls their persistence in the bone marrow (BM) and their ability to produce serum Ig. Activation of Fc γRIIb leads to the phosphorylation of ITIM and recruitment of SH2-containing inositol 5'-phosphatase (SHIP) in plasma cells. Immunoreceptor tyrosine-based activation motif (ITAM) and ITIM provide the basis for two opposing signaling modules that duel for control of plasma cell activation. Fc γRIIb-mediated SHIP phosphorylation activates downstream ITAM or ITIM signaling. To determine whether multiple myeloma (MM) cells express Fc γRIIb, we performed immunohistochemical staining on bone marrow mononuclear cells from MM patients and controls. We found that not only CD20+ B cells expressed Fc γRIIb but more importantly CD138+ cells from MM patients also showed expression of this receptor. Next, we examined whether Fc γRIIb was present and expressed in CD138+ primary MM cells purified from fresh MM BM and the MM cell lines MM1s, RPMI8226, and U266 using PCR and RT-PCR on DNA and mRNA, respectively. We focused on the transmembrane domain of the Fc γRIIb gene with four primers from different parts of this domain since this portion plays a critical role in this receptor's function. The MM cell lines expressed different amounts of Fc γRIIb. Notably, we found that 17% (5/30) of MM patients showed absence of Fc γRIIb both using RT-PCR for mRNA and PCR for DNA. Moreover, use of these same primers on nonmalignant PBMCs from the MM patients also showed absence of this gene in the same five patients. As a result of these findings, we are currently sequencing Fc γRIIb in MM patients to determine if additional patients show mutational changes that affect the function of this receptor. We also further determined SHIP-1 phosphorylation using Western blot analysis since this protein mediates downstream signaling of Fc γRIIb. Following stimulation with Fc complexes, phosphorylation of SHIP-1 was markedly reduced in MM tumor cells compared to normal CD20+ B cells. Interestingly, the patients with missing Fc γRIIb expressed higher levels of SHIP-1 gene expression compared to patients with normal Fc γRIIb expression. We investigated the IgG-binding ability of MM patients (n=33) and normal donors (n=33) to Fc γRIIb. Each serum sample was incubated with cells from MHC1, a cell line that specifically expresses Fc γRIIb but not Fc γRI and Fc γRIIa. The results showed MM patients' serum IgG have much lower Fc γRIIb-binding ability than normal human IgG (P<0.05) by using both flow cytometric and immunofluorescence assays. Our findings suggest that the monoclonal protein produced by MM patients has a very low Fc γRIIb-binding ability and is incapable of signaling through the inhibitory ITIM pathway. Germline loss of Fc γRIIb in MM patients with variation in its expression level and its downstream signaling molecule SHIP and its phosphorylation as well as the inability of MM IgG to bind cells containing this receptor is a potential new mechanism that contributes to the uncontrolled growth of MM. Disclosures: Berenson: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Medtronic: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck: Research Funding; Genentech: Research Funding.

Detection and Monitoring of BRAF and NRAS Mutant Clones in Myeloma Patients By Digital PCR of Circulating DNA

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4196-4196 ◽  
Author(s):  
Even Holth Rustad ◽  
Hong Yan Dai ◽  
Eivind Coward ◽  
Kristine Misund ◽  
Anders Sundan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Plasma Cells ◽  
Digital Pcr ◽  
Braf V600e ◽  
M Protein ◽  
Bone Marrow Biopsies ◽  
Blood Samples ◽  
Sequential Samples ◽  
Clonal Development

Abstract Introduction Targeted mutation specific therapy is a promising approach in cancer therapy. However, an obstacle for this approach is the vast heterogeneity of the clonal composition and development. Tumor biopsies represent only a snapshot of the situation. Furthermore, monitoring of the clonal development is difficult because biopsies may not be representative for the whole tumor and availability of repeat biopsies is limited. To meet these difficulties we have established and optimized a method based on Digital PCR (dPCR) for analyses of circulating cell free (cf)DNA from sequential samples of serum and plasma from patients with multiple myeloma. Methods We investigated 19 patients for the BRAF V600E mutation. Nine were previously confirmed as mutation positive in bone marrow biopsies/purfied plasma cells by two independent methods (PCR/immunohistochemistry/whole exome sequencing) whereas 10 were mutation negative (Rustad et al Blood Cancer J 2015). Two patients with NRAS Q61K mutation detected in serial bone marrow samples were also included. In total, 67 serum and 21 EDTA-plasma samples were analyzed. Blood samples were taken, processed and frozen at -800 C within 1,5 hour. The samples were stored for a median of 5 years (range 0-23) before DNA isolation and analysis. Mutation detection by dPCR was performed using a droplet-based system and validated primer/probe-sets (BioRad). In-house validation and optimization of the assay was carried out using cancer cell lines OH2 and HT29 with NRAS Q61 and BRAF V600E mutations respectively. The limit of detection was 1-3 copies of mutated DNA per reaction and no false positives were detected. The threshold of positivity was set to 1 droplet per sample. Experiments were performed in accordance with the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines (Huggett et al Clin Chem 2013). Results BRAF or NRAS mutated cfDNA was detected in all patients with a confirmed mutation in tumor tissue, and in none of the mutation-negative controls (p = 0.000003, Fisher's exact test). When looking only at tumor tissue and blood samples obtained at the same time, mutation positivity was confirmed in the blood of 9/10 patients (p = 0.00012). Furthermore, there was a positive correlation between the percentage of mutated plasma cells in bone marrow biopsies and the concentration of mutated cfDNA (Spearman correlation R = 0.63, p = 0.025). Serial samples were analyzed from 5 patients and provided information about 3 different aspects: 1. Patients 1 (figure), 2 and 3, had large clones (50-100 %) of BRAF or NRAS mutated cells in diagnostic and relapse bone marrow samples. Mutated cfDNA correlated closely to M-protein levels in these patients as demonstrated in the figure. A corollary of the figure is that the BRAF mutated clone produces M protein and is sensitive to MP. 2. Patient 4 developed a pelvic extra medullary plasmacytoma with 75-100% BRAF mutation positive cells (immunohistochemistry), however, time-matched serum samples showed only a modest peak with 23 mutated copies/ml. 3. Patient 5 had a moderately sized BRAF V600E mutated clone of 50-75 % at diagnosis, which, according to serum levels, persisted through the disease course. However, two months prior to death, the patient rapidly deteriorated and became refractory to treatment. BM aspirate showed 95 % plasma cells with plasmablastic morphology. A serum sample contained > 600 ng/ml of cfDNA, 10-100 fold more than any other sample in our study, and was highly positive for BRAF V600E mutation (59 000 copies/ml). The patient clearly had expansion of an aggressive BRAF mutated clone that could easily be detected by serum analysis. Conclusions This study demonstrates that mutations such as BRAF V600E and NRAS Q61K can be reliably detected and monitored in sequential serum or plasma samples from myeloma patients. Quantitative mutation analysis compared to M protein in sequential samples provided significant information with clinical relevance. The great advantage of this approach is the easy access to blood samples compared to bone marrow aspirate/biopsy. This will facilitate studies of clonal development during treatment and detection of druggable mutations. Figure 1. Co-variation of M-protein and circulating BRAF V600E mutated DNA in patient 1. Figure 1. Co-variation of M-protein and circulating BRAF V600E mutated DNA in patient 1. Disclosures Waage: Celgene: Research Funding; Amgen: Research Funding; Janssen: Research Funding.

scholarly journals Evolution of the Tumor Microenvironment throughout Progression and Transformation of EZH2 Mutant Follicular Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 446-446
Author(s):  
Yusuke Isshiki ◽  
Dylan McNally ◽  
Ari M. Melnick ◽  
Wendy Béguelin
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cells ◽  
Tumor Microenvironment ◽  
Mouse Model ◽  
Cell Line ◽  
Immune Cells ◽  
Research Funding ◽  
Low Grade ◽  
Lymphoma Cells

Abstract The genetic hallmarks of follicular lymphomas include BCL2 translocations and somatic mutations of epigenetic modifier genes such as EZH2. Histologically, FLs typically feature a rich microenvironment, most notably featuring extensive follicular dendritic cell (FDC) networks with dendrites making extensive contact with lymphoma cells. In recent work we showed that the main effect of EZH2 gain-of-function mutations in GC B-cells is to enable them to become less dependent of T-cell help and strengthen their immune synapse formation with FDCs, which induces aberrant proliferation and survival of GC centrocytes and hence formation of a unique lymphoma-permissive immune niche. However, it is still unknown how interactions between EZH2 mutant GC B-cells and other immune cells change throughout the progression of the disease. To evaluate the evolution of the tumor microenvironment (TME) throughout EZH2 mutant lymphoma progression, we developed a genetically engineered mouse model designed for conditional expression of gain-of-function Ezh2 mutant and BCL2 in GC B-cells, Rosa26 LSL.BCL2.IRES.GFP;Ezh2 Y641F;Cγ1Cre (hereafter BCL2/Ezh2 Y641F), in which Cγ1-driven Cre excision of a STOP cassette in the Rosa26 locus leads to overexpression of BCL2 and GFP, and expression of the endogenous Ezh2 mutant Y641F. This mouse model develops low-grade follicular-like lymphoma, characterized by expanded follicles composed largely of centrocyte neoplastic GC B-cells and extensive FDC meshwork, along with presence of CD4 +, TFH and regulatory T cells (FOXP3 +), as depicted by multiparametric in situ imaging and multiparametric flow cytometry. At cytological level, cells are predominantly small with condensed chromatin, irregular nuclei, scant cytoplasm, and inconspicuous nucleoli, without sheets of large cells. Over time, these low grade FLs progress to advanced grade, characterized by disruption of follicle structures, expansion of centroblast-like large tumor cells and reduced CD4 + T cell infiltration and FDC meshwork. Furthermore, we have developed a murine transformed FL cell line by sequential passages of an original BCL2/Ezh2 Y641F low-grade FL into immunodeficient Rag1KO mice. We have transduced this BCL2/Ezh2 Y641F cell line with luciferase, and it successfully engrafted and homed to lymphoid organs when injected i.v. into immunocompetent C57BL6 mice. The cell line consists of high proliferative GC B-cells with multiple and irregular nuclei, open chromatin and prominent nucleoli that resemble DLBCL. The microenvironment of tumors developed in C57BL6 mice is characterized by disruption of follicle structures, severe reduction or absence of FDC meshwork, decreased CD4 +, CD8 + and Tregs, downregulation of MHC-I and MHC-II. Therefore, our mouse model also recapitulates progression and transformation stages. Since MYC translocations are frequent events that occur during histological transformation of FL, we modeled this more aggressive EZB MYC subtype of DLBCL. For that, we further crossed our BCL2/Ezh2 Y641F mice to Rosa26 LSL.MYC.IRES.hCD2. We generated cohorts of transplanted mice by injecting bone marrow cells from BCL2, BCL2/Ezh2 Y641F, BCL2/MYC, and BCL2/Ezh2 Y641F/MYC mice into lethally irradiated C57BL6 recipients. Recipient mice (n=5 per group) were immunized with SRBC to induce formation of GCs and sacrificed 5.5 months post bone marrow transplant. All genotypes showed expansion of FAS +CD38 -BCL2 GFP+ GC B-cells in spleen and lymph nodes; however, the proportion of GC B-cells in Ezh2 Y641F was significantly increased compared with Ezh2 WT, in both presence and absence of MYC. In contrast, FAS -CD38 +BCL2 GFP+ and CD138 +BCL2 GFP+ cells were significantly increased in MYC + mice and decreased in Ezh2 Y641F, suggesting that Ezh2 mutation is required to maintain the GC phenotype. The acquisition of Ezh2 Y641F in BCL2/MYC mice induced a reduction of CD4 + and CD8 + in the TME, and decreased TFH without changes in TFR proportions in GCs. Strikingly, FDC meshwork was significantly shrank in MYC + cases, and partially restored by acquisition of Ezh2 Y641F, indicating that MYC overexpression may contribute to acquire FDC independency for the survival of lymphoma cells. Our results indicate that progression of FL critically affects the TME and acquisition of additional mutations such as MYC alters the interactions between lymphoma cells and tumor supportive immune cells in TME. Disclosures Melnick: Epizyme: Consultancy; Janssen: Research Funding; KDAC Therapeutics: Current holder of individual stocks in a privately-held company; Celgene Corporation: Consultancy; Constellation Pharmaceuticals: Consultancy; Astra Zeneca: Consultancy; Daiichi Sankyo: Consultancy; Exo Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sanofi US Services: Research Funding.

AL Amyloidosis and Concomitant Myeloma: Time to Reconsider Assumptions

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4044-4044
Author(s):  
Wesley Witteles ◽  
Ronald Witteles ◽  
Michaela Liedtke ◽  
Sally Arai ◽  
Richard Lafayette ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Plasma Cell ◽  
Research Funding ◽  
Plasma Cells ◽  
Renal Involvement ◽  
World Health ◽  
Bone Lesions ◽  
Al Amyloidosis ◽  
Bone Marrow Biopsies

Abstract Abstract 4044 Background: Conventionally, multiple myeloma is believed to coexist in approximately 10% of AL amyloidosis patients. However, it is unclear whether this figure is too low based on current World Health Organization criteria. These criteria, mainly created to differentiate myeloma from monoclonal gammopathy of undetermined significance, include the presence of ≥ 10% plasma cells on a bone marrow biopsy or aspirate as being diagnostic of myeloma. Aims: To define the frequency and relevance of a concomitant diagnosis of myeloma in patients with AL amyloidosis. Methods: Records from consecutive patients with biopsy-proven AL amyloidosis treated at the Stanford University Amyloid Center were reviewed. Plasma cell percentages were determined by manual counts from bone marrow aspirate smears and by CD138 immunohistochemistry (IHC) performed on bone marrow core biopsies. Results: A total of 41 patients (median age 61 years, 32% female) were evaluated. The median number of organs involved with amyloidosis was 2 (range 1–4), with 28 patients (68%) having cardiac involvement, 22 patients (54%) having renal involvement, 15 patients (37%) having gastrointestinal involvement, 12 patients (29%) having soft tissue involvement, and 10 patients (24%) having nervous system involvement. All patients had bone marrow biopsies and aspirates performed at the time of amyloid diagnosis, with most undergoing both manual counts of plasma cells from aspirates and IHC from core biopsies. Based on conventional criteria, manual aspirate counts defined 15/28 (54%) patients as having myeloma, and IHC defined 26/31 (84%) patients as having myeloma (p=0.01). Only nine patients had a detectable serum paraprotein on immunofixation (median 1.1 g/dl, range 0.4–2.6). 81% of patients had an elevated serum free light chain (85% lambda), with a median level of 37.3 mg/dl (range 8.6–256 mg/dl). Compared to the frequency of elevated plasma cells, the prevalence of anemia (29%), hypercalcemia (14%), impaired kidney function (21%), and lytic lesions (7%) was low. After a median follow-up of 13 months (range 1–127 months), the one-year overall survival (74% vs. 58%) and three-year overall survival (50% vs. 50%) was not significantly different between patients with ≥10% plasma cells and patients with <10% plasma cells (p=NS). Discussion: As defined by bone marrow plasma cell involvement, a strikingly high percentage (84%) of AL amyloidosis patients would be considered to have concurrent myeloma. This figure is much higher than has been traditionally quoted in the literature, likely due to the utilization of newer methods of counting plasma cells. There was a low prevalence of myeloma-associated end-organ effects (hypercalcemia, anemia, renal insufficiency, lytic bone lesions), and a myeloma diagnosis had no impact on survival. Conclusion: In this cohort of AL amyloid patients, concomitant myeloma was present in the vast majority of patients using modern diagnostic techniques. The significance of this diagnosis appears to be minimal – calling into question whether the diagnostic criteria for myeloma should be redefined in this population. Disclosures: Witteles: Celgene: Research Funding. Liedtke:Celgene: Lecture fee, Research Funding. Schrier:Celgene: Research Funding.

scholarly journals High levels of circulating triiodothyronine induce plasma cell differentiation

2013 ◽  
Vol 220 (3) ◽  
pp. 305-317 ◽  
Author(s):  
Flavia Fonseca Bloise ◽  
Felipe Leite de Oliveira ◽  
Alberto Félix Nobrega ◽  
Rita Vasconcellos ◽  
Aline Cordeiro ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Cell Physiology ◽  
White Pulp ◽  
Plasma Cell Differentiation ◽  
Circulating Levels

The effects of hyperthyroidism on B-cell physiology are still poorly known. In this study, we evaluated the influence of high-circulating levels of 3,5,3′-triiodothyronine (T3) on bone marrow, blood, and spleen B-cell subsets, more specifically on B-cell differentiation into plasma cells, in C57BL/6 mice receiving daily injections of T3for 14 days. As analyzed by flow cytometry, T3-treated mice exhibited increased frequencies of pre-B and immature B-cells and decreased percentages of mature B-cells in the bone marrow, accompanied by an increased frequency of blood B-cells, splenic newly formed B-cells, and total CD19+B-cells. T3administration also promoted an increase in the size and cellularity of the spleen as well as in the white pulp areas of the organ, as evidenced by histological analyses. In addition, a decreased frequency of splenic B220+cells correlating with an increased percentage of CD138+plasma cells was observed in the spleen and bone marrow of T3-treated mice. Using enzyme-linked immunospot assay, an increased number of splenic immunoglobulin-secreting B-cells from T3-treated mice was detectedex vivo. Similar results were observed in mice immunized with hen egg lysozyme and aluminum adjuvant alone or together with treatment with T3. In conclusion, we provide evidence that high-circulating levels of T3stimulate plasmacytogenesis favoring an increase in plasma cells in the bone marrow, a long-lived plasma cell survival niche. These findings indicate that a stimulatory effect on plasma cell differentiation could occur in untreated patients with Graves' disease.

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