scholarly journals Characterization of an Acute Myeloid Leukemia Murine Model Driven By MLL/AF9: Effect of Retroviral Insertion Sites and Somatic Mutations on Gene Expression

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4329-4329
Author(s):  
Hui Mei Lee ◽  
Niloofar Zandvakili ◽  
Rhea Desai ◽  
Peter J. Browett ◽  
Purvi M Kakadia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Murine Model ◽  
Somatic Mutations ◽  
Fusion Gene ◽  
Advisory Committees ◽  
Leukemia Development ◽  
Insertion Sites ◽  
Secondary Leukemias

Abstract The MLL/AF9 fusion is found in approximately 30% of MLL-rearranged leukemias and has an intermediate prognosis. Genomically well-characterized murine leukemia models enable us to understand leukemogenesis. We generated a retroviral transduction murine bone marrow transplantation leukemia model (MBMTLM) using the MLL/AF9 fusion gene. Fifteen of 20 mice transplanted with syngeneic bone marrow transduced with a MLL/AF9 carrying retrovirus developed leukemia after a median latency of 149 days. Half a million leukemic bone marrow (LBM) cells from two of these primary leukemias, MA03-P and MA86-P, were transplanted into irradiated recipient mice to establish secondary leukemias, MA03-S (n=3) and MA86-S (n=4). Half a million LBM cells from these secondary leukemias were further transplanted into irradiated recipient mice to generate tertiary leukemias, MA03-T (n=3) and MA86-T (n=4). The latency of the leukemias shortened from 141 days in MA03-P to 18 and 22 days in MA03-S and MA03-T, respectively. Similarly, MA86-P had a latency of 98 days, and the latency was reduced to about 28 days in MA986-S and MA986-T. We used retroviral insertion sites (RISs) to track leukemia clones during serial transplantation. We identified 5 RISs in MA03-P. One RIS, RIS#1-03 at chromosome 7:4602500-4609499 accounted for 52.5% of the total RIS-related reads in MA03-P, while the other four RISs were each represented by fewer than 5% of the reads. Only RIS#1-03 was detected in all of the MA03 secondary and tertiary leukemias , indicating that the cells with RIS#1-03 were the dominant clone in MA03 leukemias. Two RISs were detected in MA86-P: RIS#1-86 at chromosome 19:41338500-41341999 and RIS#2-86 at chromosome 10:127106000-127109499 at 46.7% and 2.5%, respectively . RIS#1-986 was contained in the dominant clone as only this RIS was subsequently detected in the secondary and tertiary MA86 leukemias. The relatively long latency to leukemia development in our MLL/AF9 model was most likely due to the requirement of cooperating somatic mutations. We performed whole exome sequencing on DNA from LBM (n=15) and DNA from their corresponding germline (n=2). An average of 4.5 of single nucleotide variants (SNVs) and 11.4 indels affecting protein coding sequences were found in the MA03 family of leukemias (n=7) which, among others, mutated genes involved in tyrosine kinase pathways such as Epha5 and Pik3r1. We identified an average of 14.8 (SNVs) and 0.5 indels per exome in the MA86 leukemias (n=8). Transcription regulator (Brd1) and tumor suppressor genes (Stk11 and Trp53) were affected by somatic changes in the MA86 family. RNA sequencing was performed on LBM (n=15) and healthy bone marrow (HBM) (n=8). Principal component analysis (PCA) on the expression profiles showed that LBM samples clustered together. Differential gene expression analysis identified genes such as Six1, Eya1 and Bcor which had been reported in previous studies to be essential for leukemogenesis in MLL/AF9 murine model. We also observed downregulation of genes such as Gata2, Btg1, Ifitm1, which had been implicated in other types of leukemias. We next investigated the effect of the RISs and somatic mutations on gene expression. RIS#1-903 was in intron 1 of Ppp6r1. A reduction in fragments per kilobase of transcript per Million mapped reads (FPKM) of Ppp6r1 was observed in MA03 family leukemias compared to leukemias of the MA86 family which did not have RIS#1-03 and showed no difference to HBM samples (MA03: 87.71±1.5; MA86: 132.1±5.1; HBM: 77.56±1.7, p< 0.001). We then determined the expression of Tm9sf3 as it is located 600bp away from RIS#1-986. The FPKM of Tm9sf3 was significantly higher in LBM (both of MA903 and MA986 leukemias) than in HBM (LBM: 146.0±12.7; HBM: 64.66±2.8, p<0.001). In MA86 leukemias which all have RIS#1-86, the FPKM of Tm9sf was two fold higher than in MA03 leukemias without RIS#1-86 (MA86: 189.3±4.4; MA03:97.59±1.7, p < 0.001). In contrast, none of the somatic mutations had a significant effect on the expression of any of the mutated genes. In conclusion, we have established a MBMTLM driven by the MLL/AF9 fusion gene. This well-characterized model provides insights to further understand leukemia development and drug testing. Moreover, we demonstrated that RISs can have an impact on gene expression. Future work on whether Ppp6r1 and Tm9sf3 identified by our RIS analysis are drivers in MLL/AF9 leukemias is warranted. Disclosures Browett: MSD: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria.

Promoter-Wide Transcriptional Deregulation In Waldenstrom Macroglobulinemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3620-3620
Author(s):  
Yang Liu ◽  
Min Ni ◽  
Aldo M. Roccaro ◽  
Xavier Leleu ◽  
Yong Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Binding Sites ◽  
Research Funding ◽  
Enrichment Analysis ◽  
Promoter Regions ◽  
Advisory Committees ◽  
Pol Ii ◽  
Normal Controls

Abstract Abstract 3620 Introduction: Waldenstrom macroglobulinemia (WM) is a rare indolent non-Hodgkin lymphoma, characterized by bone marrow infiltration of clonal lymphoplasmacytic cells. Despite recent advances in understanding the pathogenesis of this disease, the molecular basis of WM etiology has not been clearly defined. We therefore performed genome-wide analysis of RNA polymerase II (pol II) binding sites and gene expression profiling in primary WM cells in order to comprehensively define the aberrant transcriptional regulation and related genes in WM. Methods: Primary CD19+ bone marrow derived WM cells and normal primary bone marrow were used. Genomic DNA was extracted using genome isolation kit (QIAGEN) after cross linking. All the DNA samples were sent for Chip assay and human promoter 1.0R array (Genepathway Inc.) which comprised of over 4.6 million probes tiled through over 25.500 human promoter regions. Each promoter region covers approximately 7.6kb upstream through 2.45kb downstream of the transcription start sites. For over 1,300 cancer associated genes, coverage of promoter regions was expanded to additional genomic content; for selected genes total coverage spans from 10kb upstream through 2.45kb downstream of transcription start sites. The published gene expression datasets (GDS2643) which included 10 CD19+ B cell from bone marrow of 10 WM patients and 8 normal controls was analyzed by d-chip software and normalized to normal control. The motif analysis was performed using Cistrome online tools from the Dana Farber Cancer Institute. The gene sets enrichment analysis (GSEA) was performed using GSEA online software from Broad institute. Results: A total of 13,546 high-confidence pol II sites were identified in WM samples and share a small percentage of overlap (11.5%) with the binding sites identified in normal controls. Combining the expression microarray data of WM patient samples and normal controls, we demonstrated a significant correlation between high levels of gene expression and enriched promoter binding of pol II. Notably, we also observed that the WM-unique pol II binding sites are localized in the promoters of 5,556 genes which are involved in important signaling pathways, such as Jak/STAT and MAPK pathways by applying gene set enrichment analysis (GSEA). Interestingly, we found that STAT, FOXO and IRF family binding sites motifs were enriched in the pol II-bound promoter region of IL-6 which plays a crucial role in cell proliferation and survival of WM cells. Moreover, the CpG island associated c-fos promoter was enriched for Pol II binding as compared to the normal control. Conclusion: The presence of increased Pol II binding and the identification of transcription factor motifs in the promoters of key oncogenes may lead to a better understanding of WM. Our findings suggest that altered transcriptional regulation may play an important role in the pathogenesis of WM. In addition, this study will provide novel insights into the molecular mechanism of WM etiology, and may lead to discovery of novel diagnostic molecular biomarkers and therapeutic targets for WM. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Deregulation of TNFRSF18 (GITR) Through Promoter CpG Island Methylation Induces Tumor Proliferation in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2424-2424
Author(s):  
Yang Liu ◽  
Yong Zhang ◽  
Phong Quang ◽  
Hai T Ngo ◽  
Feda Azab ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Advisory Committees ◽  
Brdu Assay

Abstract Abstract 2424 Introduction Tumor necrosis factor receptor super families (TNFRSFs) play an important role in activation of lymphocyte and cell apoptosis. However the function of TNFRSFs in multiple myeloma (MM) remains unknown. Loss of function mutation of Fas antigen (TNFRSF6) was identified in MM cells, thus suggesting the possible role of TNFRSFs in regulating MM pathogenesis. We therefore investigated the epigenetic mechanisms that may mediate inactivation of TNFRSFs and its functional role in MM. Methods Dchip software was utilized for analyzing gene expression dataset. DNA was extracted from both primary CD138+ MM plasma cells and MM cell lines using blood & tissue DNA isolation kit (Qiagen, Inc.). Expression of GITR in primary CD138+ plasma cells was detected by Imunohistochemistry (IHC) DNA methylation was analyzed by methylated DNA immunoprecipitation (Medip) assay and bisulfate sequencing. 5'azacytidine was used to demethylate genomic DNA. Gene expression was detected by qRT-PCR and confirmed at the protein level by flow cytometry and western-blot. Over-expression of GITR was obtained in MM1.S cells by using GITR recombinant plasmid and electroporation. Apoptosis was determined using Annexin/PI staining and flow cytometry analysis. Activation of apoptotic signaling was studied by western blot. Cell survival and proliferation were analyzed by MTT and BrdU assay, respectively. Recombinant GITR-lentivirus was obtained from the supernatant of culture medium after 72 hours transfection in 293 cells. GFP positive MM cells were sorted and analyzed by flow cytometry. In vivo effect of GITR on MM tumor growth was determined by injection of GITR over-expressing MM cells in null mice. Mice skull, femur and vertebrae were isolated after 4 weeks injection. Anti-human CD138+ mAb microbead was used to detect MM cells extracted from mice tissue by flow cytometry. Results Gene-expression profiling showed down-regulation of TNFRSFs, including TNFRSF11A, TNFRSF11B, TNFRSF8, TNFRSF10C, TNFRSF9, TNFRSF21, TNFRSF1B, TNFRSF1A and TNFRSF18, compared to normal plasma cells. Moreover, Our IHC results also showed that GITR expression was positive in primary CD138+ plasma cells from 9 normal bone marrow, but negative in 9 MM samples. Importantly, we found that low GITR expression significantly correlated with MM progression. Indeed, GITR gene levels were lower in smoldering and active MM patients compared to MGUS patients and normal donors. Promoter CpG island (CGI) methylation of GITR was indentified in 5 out of 7 MM primary bone marrow (BM)-derived CD138+ cells but not in normal BM-derived plasma cells. Bisulfate sequencing and Medip assay showed that methylation of GITR was significantly associated with GITR expression in 5 MM cell lines, including MM1.S, OPM1, U266, RPMI and INA6. Promoter CGI of GITR was highly methylated leading to complete silencing of GITR in MM1.S cell line. GITR expression was significantly up-regulated in MM cells upon treatment with the 5'azacytidine. MTT and BrdU assay revealed that the proliferation and survival of MM1.S cells was disrupted in the GITR over-expressing MM1.S cells, notably with inhibition of cell proliferation compared to control vector infected cells. Moreover induction of cytotoxicity in GITR over-expressing cells was confirmed by using GFP competition assay. GITR-induced apoptosis was supported by induction of caspase 8 and 3 cleavage. The inhibition of human CD138+ plasma cell growth in the bone marrow of SCID mice using a disseminated MM xenograft model was observed in the experimental group injected with GITR expressing cells compared to the control group after 4 weeks injection. Conclusion Our findings uncovered a novel epigenetic mechanism contributing to MM pathogenesis, showing the role of GITR methylation as a key regulator of MM cell survival. Disclosures: Roccaro: Roche:. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Baseline and on-Treatment Bone Marrow Microenvironments Predict Myeloma Patient Outcomes and Inform Potential Intervention Strategies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1882-1882 ◽  
Author(s):  
Samuel A Danziger ◽  
Mark McConnell ◽  
Jake Gockley ◽  
Mary Young ◽  
Adam Rosenthal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Expression Profiles ◽  
Cell Types ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Introduction The multiple myeloma (MM) tumor microenvironment (TME) strongly influences patient outcomes as evidenced by the success of immunomodulatory therapies. To develop precision immunotherapeutic approaches, it is essential to identify and enumerate TME cell types and understand their dynamics. Methods We estimated the population of immune and other non-tumor cell types during the course of MM treatment at a single institution using gene expression of paired CD138-selected bone marrow aspirates and whole bone marrow (WBM) core biopsies from 867 samples of 436 newly diagnosed MM patients collected at 5 time points: pre-treatment (N=354), post-induction (N=245), post-transplant (N=83), post-consolidation (N=51), and post-maintenance (N=134). Expression profiles from the aspirates were used to infer the transcriptome contribution of immune and stromal cells in the WBM array data. Unsupervised clustering of these non-tumor gene expression profiles across all time points was performed using the R package ConsensusClusterPlus with Bayesian Information Criterion (BIC) to select the number of clusters. Individual cell types in these TMEs were estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) augmented with 5 bone marrow- and myeloma-specific cell types. Results Our deconvolution approach accurately estimated percent tumor cells in the paired samples compared to estimates from microscopy and flow cytometry (PCC = 0.63, RMSE = 9.99%). TME clusters built on gene expression data from all 867 samples resulted in 5 unsupervised clusters covering 91% of samples. While the fraction of patients in each cluster changed during treatment, no new TME clusters emerged as treatment progressed. These clusters were associated with progression free survival (PFS) (p-Val = 0.020) and overall survival (OS) (p-Val = 0.067) when measured in pre-transplant samples. The most striking outcomes were represented by Cluster 5 (N = 106) characterized by a low innate to adaptive cell ratio and shortened patient survival (Figure 1, 2). This cluster had worse outcomes than others (estimated mean PFS = 58 months compared to 71+ months for other clusters, p-Val = 0.002; estimate mean OS = 105 months compared with 113+ months for other clusters, p-Val = 0.040). Compared to other immune clusters, the adaptive-skewed TME of Cluster 5 is characterized by low granulocyte populations and high antigen-presenting, CD8 T, and B cell populations. As might be expected, this cluster was also significantly enriched for ISS3 and GEP70 high risk patients, as well as Del1p, Del1q, t12;14, and t14:16. Importantly, this TME persisted even when the induction therapy significantly reduced the tumor load (Table 1). At post-induction, outcomes for the 69 / 245 patients in Cluster 5 remain significantly worse (estimate mean PFS = 56 months compared to 71+ months for other clusters, p-Val = 0.004; estimate mean OS = 100 months compared to 121+ months for other clusters, p-Val = 0.002). The analysis of on-treatment samples showed that the number of patients in Cluster 5 decreases from 30% before treatment to 12% after transplant, and of the 63 patients for whom we have both pre-treatment and post-transplant samples, 18/20 of the Cluster 5 patients moved into other immune clusters; 13 into Cluster 4. The non-5 clusters (with better PFS and OS overall) had higher amounts of granulocytes and lower amounts of CD8 T cells. Some clusters (1 and 4) had increased natural killer (NK) cells and decreased dendritic cells, while other clusters (2 and 3) had increased adipocytes and increases in M2 macrophages (Cluster 2) or NK cells (Cluster 3). Taken together, the gain of granulocytes and adipocytes was associated with improved outcome, while increases in the adaptive immune compartment was associated with poorer outcome. Conclusions We identified distinct clusters of patient TMEs from bulk transcriptome profiles by computationally estimating the CD138- fraction of TMEs. Our findings identified differential immune and stromal compositions in patient clusters with opposing clinical outcomes and tracked membership in those clusters during treatment. Adding this layer of TME to the analysis of myeloma patient baseline and on-treatment samples enables us to formulate biological hypotheses and may eventually guide therapeutic interventions to improve outcomes for patients. Disclosures Danziger: Celgene Corporation: Employment, Equity Ownership. McConnell:Celgene Corporation: Employment. Gockley:Celgene Corporation: Employment. Young:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Reiss:Celgene Corporation: Employment, Equity Ownership. Davies:MMRF: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; TRM Oncology: Honoraria; Abbvie: Consultancy; ASH: Honoraria; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria. Copeland:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Fitch:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Barlogie:Celgene: Consultancy, Research Funding; Dana Farber Cancer Institute: Other: travel stipend; Multiple Myeloma Research Foundation: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Millenium: Consultancy, Research Funding; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC. Trotter:Celgene Research SL (Spain), part of Celgene Corporation: Employment, Equity Ownership. Hershberg:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Dervan:Celgene Corporation: Employment, Equity Ownership. Ratushny:Celgene Corporation: Employment, Equity Ownership. Morgan:Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding.

scholarly journals Higher Expressions of PTH Receptor Type 1 and/or 2 in Bone Marrow Is Associated to Longer Survival in Newly Diagnosed Myeloma Patients Enrolled in Total Therapy 3

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3409-3409 ◽  
Author(s):  
Maurizio Zangari ◽  
Caleb K Stein ◽  
Shmuel Yaccoby ◽  
Donghoon Yoon ◽  
Christoph Heuck ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
P Value ◽  
Newly Diagnosed ◽  
Advisory Committees ◽  
Logrank Test ◽  
Total Therapy

Abstract Higher Expressions of PTH Receptor Type 1 and/or 2 in Bone Marrow is Associated to Longer Survival in Newly Diagnosed Myeloma Patients Enrolled in Total Therapy 3 INTRODUCTION: The Total Therapy 3 enrolled 303 newly diagnosed multiple myeloma patients at Myeloma Institute for Research and Therapy. Protocol included 2 cycles of VTD-PACE (bortezomib, thalidomide, and dexamethasone and 4-day continuous infusions of cisplatin, doxorubicin, cyclophosphamide, and etoposide) as induction and consolidation therapy after melphalan-based tandem transplantation, which is followed by 3 years of intended maintenance with VTD in year 1 and thalidomide/dexamethasone in years 2 and 3. As part of the protocol, gene expression profiling was performed from baseline bone marrow biopsy samples in 178 individuals. We have previously reported the clinical correlation between response to bortezomib and serum parathyroid hormone variations in myeloma patients as well as the interaction between receptor 1 and proteasome inhibitors function in cell line and myeloma mouse model. In this study we examine the PTH receptor 1 and 2 expression levels and their correlation to survival in total therapy 3 enrolled patients. METHOD: Gene expression profiling was performed using Affymetrix U133 plus 2.0 Microarrays (Santa Clara, CA) in baseline bone marrow biopsy samples from 178 patients enrolled on total therapy 3. Of these 178 patients, 108 were male. The overall median age of these patients was 59 years old at enrollment; 10 % of patients were considered to have high risk disease by 70 GEP model. Cox proportional hazards analysis was performed on the MAS5 normalized log 2 expression values of PTH1R and PTH2R using overall survival as the end point. Optimal dichotomous break points were found for PTH1R and PTH2R that corresponded to the maximum log rank test statistic from all cox proportional hazard models examined. To confirm PTH receptor expression in bone marrow, we performed real-time PCR using Taqman probes (PTH1R: Assay ID Hs00174895_m1 and PTH2R: Assay ID Hs00175044_m1) on subset of samples. RESULTS: Based on cox proportional hazards regression of PTH1R and PTH2R expression values, patients with higher PTH1R and PTH2R expression demonstrated better survival compared to lower expressing patients. PTH1R expression above optimal break point of 8.92 had a hazard ratio of 0.583 with a 95% confidence interval of (0.351, 0.969) and logrank test p-value of 0.035. PTH2R expression above optimal break point of 6.85 had a hazard ratio of 0.541 with a 95% confidence interval of (0.323, 0.905) and logrank test p-value of 0.018. Furthermore, the patients that were lower expressed in both PTH1R and PTH2R performed significantly poorer in outcome (n= 24 and median survival of 4.52 years logrank p-value+5.71e-05). Real-time PCR using Taqman probes was able to demonstrate relatively high levels of PTH1R and PTHR2 transcripts at bone marrow level. Figure 1 Figure 1. CONCLUSIONS: This is the first report indicating that PTH receptors type 1 and 2 gene expression levels are positively associated to overall survival in symptomatic multiple myeloma patients. Also we describe the presence of PTH2R at bone marrow level which function appear associated to myeloma control. These data confirm the correlation and close interaction between the survival of multiple myeloma patients and the parathyroid hormone axis. Disclosures Zangari: Norvartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Research Funding; Millennium: Research Funding. Heuck:Celgene: Honoraria; Foundation Medicine: Honoraria; Millennium: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. van Rhee:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Morgan:Celgene Corp: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Myeloma UK: Membership on an entity's Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity's Board of Directors or advisory committees; The Binding Site: Membership on an entity's Board of Directors or advisory committees; MMRF: Membership on an entity's Board of Directors or advisory committees.

Whole-Exome Sequencing In Myelodysplastic Syndromes With 5q- and Normal Karyotype

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1551-1551
Author(s):  
Vera Adema ◽  
Mar Mallo ◽  
Leonor Arenillas ◽  
María Díez-Campelo ◽  
Elisa Luño ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Exome Sequencing ◽  
Board Of Directors ◽  
Whole Exome Sequencing ◽  
Research Funding ◽  
Somatic Mutations ◽  
Normal Karyotype ◽  
Advisory Committees ◽  
Whole Exome ◽  
5Q Deletion

Abstract Introduction Myelodysplastic Syndromes (MDS) are a heterogeneous group of clonal myeloid stem cells disorders with high prevalence in the elderly characterized by inefficient hematopoiesis, peripheral blood (PB) cytopenias, and an increased risk of transformation to acute myeloid leukemia (AML). The karyotype is the clinical parameter with the strongest prognostic impact according the IPSS-R (Greenberg et al., 2012). The most frequent cytogenetic alteration is the chromosome 5q deletion (del[5q]) which as a single anomaly, confers a good prognosis and predicts an excellent response to lenalidomide. Whether other genetic abnormalities routinely cooperate with del(5q) is not known. Whole-exome sequencing (WES) is a powerful tool to identify somatic mutations in protein coding genes that might cooperate with del(5q). In order to better understand the genetic basis of MDS with del(5q), we performed whole-exome sequencing (assessing 334,378 exons) of tumor-normal paired samples from 21 MDS patients. Herein we describe the preliminary findings. The analysis is ongoing and the complete results will be presented in the meeting. Methods A total of 21 patients with MDS (16 with del(5q) as a sole abnormality, 3 with del(5q) and additional alterations and 2 with normal karyotype) were included in our study. We examined a total of 25 tumor samples (21 diagnostic bone marrow (BM) samples with matched CD3+ cells as a controls, additional BM samples from 3 patients during lenalidomide treatment and 1 bone marrow sample from a del(5q) patient after AML progression). DNA was extracted from BM samples and from isolated peripheral blood CD3+ cells (magnetic-activated cell sorting (MACS), MiltenyiBiotec GmbH, Germany). The purity of CD3+ cells was assessed by FC 500 flow cytometer (Beckman Coulter, Hialeah, Fl, USA). Only DNA that fulfilled quality controls required by WES were submitted. For each diagnostic sample, we performed Conventional G-banding cytogenetics and fluorescence in situ hybridization (FISH, to confirm or dismiss 5q deletions). Whole-exome targeted capture was carried out on 3 μg of genomic DNA, using the SureSelect Human Exome Kit 51Mb version 4 (Agilent Technologies, Inc., Santa Clara, CA, USA). The captured and amplified exome library was sequenced with 100 bp paired-end reads on an Illumina HiSeq2000. Whole-exome sequencing data were analyzed using an in-house bioinformatics pipeline as previously reported. Somatic mutations identified as alterations present in tumor but not in the matched CD3+ sample were validated by Sanger sequencing. Results In our preliminary analysis of WES from 12 patients (10 patients with 5q- and 2 patients with normal karyotype), a total of 249 non-silent somatic variant candidates were identified, of which 146 were confirmed as somatic mutations. Recurrent mutations were observed in three genes (ASXL1, NBPF10 and SF3B1) in 3 different patients. Seven genes (HRNR, JAK2, POTEG, MUC5B, PHLDA, TTN, ZNF717) were mutated in two patients. Mutations in several genes known to be mutated in MDS (ASXL1, JAK2, RUNX1, SF3B1, SRSF2 and TET2) were also identified. Patients with the 5q deletion had an average of 11 mutations whereas patients with normal karyotype had a higher mean (14.5). Mutated genes identified in both groups were HRNR, JAK2, MUC5B, NBPF10 and SF3B1. No mutations in TP53 were detected in this subset. Pathway analysis of the complete list of somatically mutated genes will be carried out once all 21 patients are analyzed. The four in-treatment samples will be examined from their matched diagnostic samples. Conclusions Whole-exome sequencing of largely del(5q) MDS patient samples identified both known and previously unreported somatic mutations. Analysis of additional samples will allow a more complete description of the genes and pathways that may cooperate with del(5q) in the development and progression of MDS. Acknowledgments Financial support: This work has been supported (in part) by a grant from Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02010); by Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER) (RD07/0020/2004; RD12/0036/0044); Acción COST BM0801: European Genomics and Epigenomics Study on MDS and AML; Sociedad Española de Hematología y Hemoterapia (SEHH) and MDS Celgene. Footnotes Rafael Bejar and Francesc Sole contributed equally. Disclosures: Díez-Campelo: Novartis and Celgene: Honoraria, Research Funding. Cañizo:Celgene Jansen-Cilag Arry Novartis: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Sanchez:Celgene: Honoraria, Research Funding. Bejar:Genoptix: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Solé:Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

scholarly journals Clonal Evolution in a Murine CALM-AF10 Leukemia: Evidence of Functional Heterogeneity of Leukemia Stem Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1158-1158
Author(s):  
Niloofar Zandvakili ◽  
Hui Mei Lee ◽  
Rhea Desai ◽  
Alyona Oryshchuk ◽  
Peter J. Browett ◽  
...  
Keyword(s):  
Stem Cells ◽  
Board Of Directors ◽  
Somatic Mutations ◽  
Clonal Evolution ◽  
Advisory Committees ◽  
Genetic Changes ◽  
Expression Levels ◽  
Long Latency ◽  
Marker Expression ◽  
Secondary Leukemias

Abstract Myeloid leukemia is caused by acquired genetic changes in haematopoietic stem cells. The combination of stepwise acquisition of genetic changes together with selection of the fittest clones results in great genetic and clonal heterogeneity. We used a CALM-AF10-driven retroviral transduction murine bone marrow transplantation leukemia model (MBMTLM) to study clonal hierarchy and clonal evolution starting with a primary leukaemia (Fig 1: Leu7) which developed after 131 days and had B220 marker expression on 4% of its cells. Limiting dilution assays (LDAs) showed that the leukemia stem cell (LSC) frequency of Leu7 was 1:2339 (95% confidence interval: 1:794-1:6885). Whole exome sequencing (WES) and analysis of the variant allele fraction of somatic mutations revealed that Leu7 was composed of a main clone (Fig 1: grey) with two subclones (blue and red). Half a million leukemic cells from Leu7 were transplanted into 4 sublethally irradiated recipients, which all developed secondary leukemias after a latency of 19 days (Leu7Sec1 to 4). All secondary leukemias showed similar B220 expression levels to Leu7, and all showed an expansion of the blue subclone. When again half a million cells each of one of the secondary leukemias (Leu7Sec2) were transplanted into 4 recipients, the expansion of the blue subclone continued, the red subclone vanished and, surprisingly, the proportion of B220 expressing cells increased to between 16 to 26%. LDAs showed that the LSC frequency of Leu7Sec2 had not changed. However, several of the leukemias from the LDAs had greatly varying latencies (27 to 193 days) and B220 marker expression (2 to 85%). Four of these tertiary LDA leukemias (Leu7Sec2Ter5 to 8), which each arose from a single LSC, were analysed more closely using WES. Leu7Sec2Ter5 showed a similar latency (27 days) and B220 expression levels like Leu7SecTer1 to 4 and also had the expansion of the blue subclone. Leu7Sec2Ter6 had a long latency of 69 days and a very low B220 expression. Leu7Sec2Ter6 was driven by a new, third subclone (pink), and both the blue and the red subclone disappeared. Very interestingly, Leu7Sec2Ter7 and Leu7Sec2Ter8 had a very long latency of 193 days, and showed an expansion of a subclone (green) of the red subclone. The B220 expression was high (37%) to very high (85%) in these two leukemias. Taken together, these observations paint an interesting picture with the blue subclone outcompeting the red subclone, as leukemias arising from the red subclone only appear after a long latency and in leukemias initiated by a single LSC, when there is no blue subclone LSC present. As the four leukemias (Leu7Sec2Ter5 to 8), which each were derived from a single LSC, showed striking differences in latency and surface marker expression, it can be concluded that this variation in phenotype is an intrinsic property of an individual LSCs most likely a consequence of the distinct combination of somatic mutations present in the individual LSCs. These observations also suggest that distinct LSCs with different properties might be present in a single human leukemia. Figure 1 Figure 1. Disclosures Browett: Janssen: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria.

scholarly journals Impact and Function of Somatic PHF6 Mutations in Myeloid Neoplasms

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3581-3581
Author(s):  
Brittney Dienes ◽  
Bartlomiej P Przychodzen ◽  
Michael Clemente ◽  
Wenyi Shen ◽  
Chantana Polprasert ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Board Of Directors ◽  
Somatic Mutations ◽  
Suppressor Gene ◽  
Advisory Committees ◽  
Nonsense Mutations ◽  
Dysmorphic Features ◽  
Normal Bone ◽  
Myeloid Neoplasms

Abstract Borjeson−Forssman−Lehmann syndrome (BFLS), a hereditary X-linked disorder characterized by mental retardation, truncal obesity, gynecomastia, hypogonadism and other dysmorphic features, is known to be caused by germline (GL) mutations of plant homeo domain finger protein 6 (PHF6). PHF6 is a highly conserved 41kDa protein showing ubiquitous expression in a variety of tissues, including bone marrow, CD34+ cells and blood leukocytes. Human PHF6 is located on chrXq26.2. Recently, rare somatic nonsense mutations and deletions have been detected in patients with T-ALL and AML and found in some T-ALL cell lines. Patients with BFLS with PHF6 mutations have been reported to develop leukemia, suggesting PHF6 mutations may predispose cancer. Although the actual function and molecular pathogenesis is unknown, PHF6 has been suggested to be a tumor suppressor gene involved in the control of myeloid development. In an index case of a young adult female patient with proliferative CMML with dysmorphic features, we have identified remarkable GL mosaicism for PHF6 mutation (p.K44fs), confirmed by deep sequencing of marrow, CD3+ cells and skin tissue. Subsequently, we screened patients with myeloid neoplasms by targeted multi-amplicon sequencing to determine the prevalence and distribution of PHF6 gene alterations. Sequencing results from 1072 cases were analyzed (728 by targeted deep sequencing and 344 by whole exome sequencing). In total, we identified 21 cases with PHF6 mutations, 13 of which were frameshift or nonsense mutations. Previously, PHF6 have been included in screening panels by Haferlach et al., (Leukemia 2014) and Papaemmanuil et al., (Blood 2013) and somatic mutations were found in 24/944 and 21/738 cases of MDS, respectively. These results along with ours suggest that PHF6 mutations are common driver events. The somatic nature of these defects was confirmed by analysis of non-clonal CD3+ lymphocytes, thus, PHF6 mutations occur at a frequency of 2.0% and are most frequently observed among patients with secondary AML (33%, P=.0021). Gender distribution showed a strong male predominance (76%), likely due to the location of PHF6 on chrX and indicating that retention of a single copy of PHF6 may be protective. SNP-array karyotyping showed that deletions of Xq, involving the PHF6 locus (Xq26), were present in about 1.2% of myeloid neoplasms and affect only female patients. As a family, plant homeo domain (PHD) finger genes are affected by mutations associated with various cancers. JARID1A, PHF23, NSD1 and NSD3 were described to serve as fusion partners with the NUP98 in a subset of AML cases. The most frequent chromosomal aberration observed in conjunction with PHF6 mutations was trisomy-8 (P=.08). The most commonly associated somatic mutations were in RUNX1 (N=7; P=.001), U2AF1 (N=5), ASXL1 (N=5), IDH1 (N=4), and DNMT3A (N=4). Interestingly, 6/7 cases with concomitant PHF6 and RUNX1 mutations showed a poor prognosis AML. Subsequent analysis of clonal architecture using variant allelic frequency calculations and serial samples for these cases suggested that PHF6 may function as a founder driver gene while RUNX1 mutations are acquired as secondary events. Recent studies proposed that PHF6 deficiency leads to impaired cell proliferation, cell cycle arrest at G2/M phase and an increase of DNA damage. To examine DNA damage and quantify double stranded breaks (DSBs) in primary cells from PHF6-mutants, those with wild-type (WT) PHF6 and normal bone marrow we used a flow cytometric anti-γH2AX assay, following induction of DNA damage with Camptothecin. As judged by greater percentages of anti-γH2AX labeled cells, DSBs were more common in mutant cases consistent with more DNA damage present in PHF6 mutant compared to WT MDS and normal bone marrow cells. In conclusion, our results indicate that PHF6 mutations are generally present in more aggressive types of myeloid neoplasms, frequently associated with RUNX1 mutations. Our functional in vitro studies along with recently published reports suggest an association of PHF6 deficiency with genomic instability and thereby provide a basis for a mutator phenotype conveyed by ancestral lesions, consistent with its role as a tumor suppressor gene. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen Corp: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity's Board of Directors or advisory committees.

Distinct Pathogenesis of Clonal Hematopoiesis Revealed By Single Cell RNA Sequencing Integrated with Highly Sensitive Genotyping Method

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-34
Author(s):  
Masahiro Marshall Nakagawa ◽  
Ryosaku Inagaki ◽  
Yutaka Kuroda ◽  
Yasuhito Nannya ◽  
Lanying Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Single Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Simultaneous Detection ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Single Cell Sequencing ◽  
Clonal Hematopoiesis

Background Recent evidence suggests that age-related clonal hematopoiesis (CH) might represent the earliest precursor of myeloid neoplasms. Although the exact mechanism of clonal selection that shapes CH is still to be elucidated, both cell intrinsic and non-cell intrinsic effects of mutations, including the interplay between mutated cells and the bone marrow environment, are thought to play important roles, which are best studied using single-cell sequencing analysis of both mutations and gene expression. Methods We performed single-cell sequencing of hematopoietic stem and progenitors (HSPCs) from BM of the 16 patients with CH along with 16 control patients without CH identified by screening otherwise healthy individuals who received hip joint replacement, using a novel platform that enables simultaneous detection of gene mutations and expression based on the Fluidigm C1-HT system. Sequence data were analyzed with Seurat (Stuart et al Cell 2019) with integration of genotyping information. Cells were clustered and each cluster was assigned by marker-gene expressions for major cell-types in HSPCs, including hematopoietic stem cell (HSC)-like and erythroid progenitors. Cells were grouped by their genotypes and pathway analysis were performed. Results In total, we identified 35 subjects who had CH-related mutations, including those affecting DNMT3A, TET2, ASXL1, SF3B1, PPM1D, IDH1, GNB1 and TP53, of which 11 had more than one CH-related mutation. Most of these mutations showed a low variant allele frequency (VAF) ≤ 0.05. However, clones having double mutations of DNMT3A/TET2 or those having biallelic TET2 mutations tended to show a higher VAF as high as 0.4, suggesting an enhanced clonal advantage for clones having multiple mutations. Using our novel single-cell platform, we analyzed 3,767 cells from control patients without CH and 1,474 mutated cells and 7,234 wild-type (WT) cells from patients with CH. By targeting both genomic DNA and RNA, we successfully obtained a sufficient number of single-cell reads for genes whose expression was too low to evaluate by only targeting RNA, such as TET2 and DNMT3A. Although some clones having a high-VAF mutation caused a skewed clustering to be detected as a CH clone, many clones with low-VAF mutations did not make distinct clusters, indicating the importance of genotyping at a single cell level to identify and characterize mutated cells. Simultaneous detection of genotype and expression allowed us to see the effect of CH-mutations on cell phenotype and differentiation. For example, cells having compound TET2/DNMT3A mutations were significantly enriched in the erythroid cluster, while another clone with double TET2 mutations were more enriched in the HSC-like cluster, compared to cells from individuals without CH (WTcont). These are in line with the previous findings of TET2/DNMT3A double knockout mice or TET2 knockout mice, respectively. In another case with an IDH1 mutation, IDH1-mutated (MUTIDH1) cells less contributed to the HSC-like fraction, showing an enhancement of cell proliferation-signature, compared to WT (WTIDH1) cells in the same patient. Strikingly, compared to WTcont cells, WTIDH1 cells were significantly enriched in the HSC-like fraction and showed an enhanced expression of cytokine-related pathway genes, which was in line with a finding seen in mouse cells treated with 2-hydroxy-glutalate, an mutant IDH-related oncometabolite. Similarly, when compared to WTcont cells, WT cells from patients with DNMT3A- (WTDNMT3A) or TET2- (WTTET2) mutated CH significantly showed an enhanced cell proliferation. HSC-like WTTET2 cells also showed aberrant IFN-response signatures compared to corresponding WTcont cells, which was confirmed in competitive transplantation of Tet2 heterozygous knockout (hKO) and WT cells in a mouse model; HSPCs of WT competitors transplanted with Tet2-hKO cells showed a significant enhancement of IFN-response signatures compared to those transplanted with WT cells. Intriguingly, monocytes of Tet2-hKO donors showed aberrant expression of S100a8/a9, which might contribute to the non-cell intrinsic effect of Tet2-hKO cells. Conclusions In CH, not only mutated cells but also surrounding WT cells show an aberrant gene expression phenotype, suggesting the presence of non-cell autonomous phenotype or an altered bone marrow environment that favors the positive selection of CH-clones. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Current Employment. Ogawa:Eisai Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Characterization of Philadelphia-Negative Myeloproliferative Neoplasms By the Bone Marrow Immune Microenvironment

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4593-4593
Author(s):  
Sung-Eun Lee ◽  
Seowon Choi ◽  
Gi June Min ◽  
Sung-Soo Park ◽  
Silvia Park ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
T Cell ◽  
Board Of Directors ◽  
Myeloproliferative Neoplasms ◽  
Cd8 T Cell ◽  
Fold Change ◽  
P Value ◽  
Immune Microenvironment ◽  
Advisory Committees

Abstract Backgound The Philadelphia-negative myeloproliferative neoplasms (MPNs) share similar molecular characteristics, in which the excessive myeloproliferations are driven by mutations in JAK2, CALR, MPL, and uncommon variants. More recently, the biological basis on acquisition of somatic mutations have been accumulated. However, symptoms, histomorphologic characteristics, and natural histories are different between MPN subsets. Although there are increasing evidences that inflammation have a key role in promoting MPN initiation and influencing disease evolution, characteristics of the bone marrow immune microenvironment between MPN subsets remains unclear exactly. Aims To characterize the bone marrow immune microenvironment of Philadelphia-negative MPNs, we carried out immune-related gene expression profiling of bone marrow aspirates (BMAs) from 33 MPN patients (6 PV, 6 ET, 6 early PMF, and 15 overt MF including 10 primary MF, 5 post-PV/ET MF) using nCounter Immunology Panel. Methods BMA samples collected at diagnosis using EDTA-coated tubes. Those samples were processed within 24 hours from collection to obtain mononuclear cells by density centrifugation using Ficoll-Paque. NanoString analysis using a 594-gene nCounter Immunology panel (Human v2 - nanoString) was performed on RNAs extracted from 33 MPN bone marrow aspirates. Results First, to investigate whether there are distinct gene expression signatures of immune cells between three subcategories of MPNs, we compared gene expression profiles (GEPs) between ET, PV, and overt PMF. Using a P-value cutoff of ≤0.05 and fold-change ≥ 2, 10 upregulated and 32 downregulated differentially expressed genes (DEGs) were identified in ET than PMF, and 9 upregulated and 11 downregulated DEGs were identified in PV than PMF, while we found no significant DEGs between ET versus PV, except seven genes. Second, we investigated differences in GEPs between early PMF and overt PMF. Thirty-two downregulated and 4 upregulated DEGs were identified in early PMF than overt PMF. Gene set analysis revealed that the expression of genes related to almost processes decreased in early PMF than overt PMF. Then, we questioned differences between PMF and post-PV/ET MF. Using a P-value cutoff of ≤0.05 and fold-change ≥ 2, 12 upregulated and 12 downregulated DEGs were identified. Next, we computed relative abundances of immune cell subpopulations, estimated based on expression counts from the entire panel of surveyed genes, and compared them between the subcategories of MPNs. The abundance measurement of exhausted CD8 + T cell genes were significantly lower in ET and PV, compared with overt PMF, suggesting T cell exhaustion was distinct in overt PMF, compared to ET and PV. Conclusions The results demonstrated that immune microenvironment signature was distinguishable in the subcategories of MPNs. In addition, inflammatory signature was enriched in the bone marrow of overt PMF and exhausted CD8 + T cell genes were distinct in overt PMT. Further investigation is warranted to determine the immunological factors critical for potential therapeutic targets to alleviate progress to myelofibrosis. Disclosures Kim: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AIMS Biosciense: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AML-Hub: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BL & H: Research Funding; BMS & Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Boryung Pharm Co.: Consultancy; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Handok: Consultancy, Honoraria; LG Chem: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria; Pintherapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Genzyme: Honoraria, Speakers Bureau; SL VaxiGen: Consultancy, Honoraria; VigenCell: Consultancy, Honoraria. Lee: Alexion, AstraZeneca Rare Disease: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Extracting Prognostic Molecular Information from PET-CT Imaging of Multiple Myeloma Using Radiomic Approaches

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1906-1906
Author(s):  
Christopher Wardell ◽  
Terri Lynn Alpe ◽  
Phil Farmer ◽  
Michael W Rutherford ◽  
Yan Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Somatic Mutations ◽  
Ct Scans ◽  
Focal Lesions ◽  
Advisory Committees ◽  
Pet Ct ◽  
Ngs Data

Abstract Introduction: Invasive bone marrow sampling is used in multiple myeloma (MM) diagnosis to obtain biological material, which can then be used to generate prognostically important genetic features. Physically sampling the bone marrow can be uncomfortable for the patient. Also, spatial heterogeneity is a common feature in MM, with multiple focal lesions (FLs) occurring throughout the skeleton, meaning a single sample from the iliac crest may be insufficient to capture intrapatient heterogeneity. An alternative strategy is to extract data directly from diagnostic positron emission tomography-computed tomography (PET-CT) scans of patients. These radiomic features can be used as a proxy from which to infer molecular and clinical phenotypes. Compared to physical sampling, there are several advantages, including rapid analysis, minimalizing patient discomfort, reduced cost and widespread availability of the required scanning equipment in hospitals. Methods: A series of 439 newly diagnosed MM patients were selected, all of which had diagnostic PET-CT scans. A radiologist examined these data and identified focal lesions in the axial skeleton of 136/439 (31%) patients. Focal lesions were manually segmented from the PET portion of the original DICOM data using a density-based thresholding method in 3DSlicer version 4.9.0. Pyradiomics version 1.3 was used to resample the voxels in the PET data to 4x4x4 mm and extract radiomic features from each FL. A combination of 10 filters and 7 feature classes were used and a total of 1679 radiomic features were generated per lesion. Radiomic features were a mixture of first order characteristics such as maximum intensity, shape characteristics and gray level matrix features. Hierarchical clustering was applied to the radiomic features, using the Pearson correlation between features as the distance metric and Ward's method for clustering. Next generation sequencing (NGS) data was available for samples from 58/136 (43%) patients with FLs in whole genome (WGS), whole exome (WES) or targeted panel (TP) modalities. The NGS data was used to detect translocations, copy number aberrations and somatic mutations. Results: There were 789 FLs identified in 136 patients, with each patient containing an average of 5.8 FLs. The median FL volume was 4350 mm3, with a median maximum 3D diameter of 29 mm. Hierarchical clustering across all FLs and radiomic features separated the FLs into 5 discrete clusters associated with various clinical and molecular features. However, clustering appeared to be independent of other classification systems based on gene expression profiling (GEP), including the UAMS classification system and GEP70 risk score. Clustering was also independent of the International Staging System (ISS) status suggesting that it can add additional prognostic information. Clusters also appeared to be independent of somatic mutations in genes previously reported as significantly mutated in MM. Patients commonly had FLs occurring in multiple clusters, suggesting that this method takes into account the heterogeneity between lesions in the same patient. Larger FLs were grouped primarily into two clusters consistent with them having distinct features that can be recognized by this approach. Looking across the different clusters distinct differences in clinical outcome were seen between the groups, with significant differences in both PFS (p=0.007) and overall survival (p=0.005), with worse prognosis being led by a cluster of smaller lesions. Conclusions: Radiomics provides a novel method to extract potentially important data from PET-CT scans which can define individual clusters that have different clinical, molecular and prognostic features. This can provide a novel non-invasive method to assess FLs based on both their physical and radiomic characteristics. Larger study sizes will be needed to confirm the differences in outcomes seen between groups. Disclosures Boyle: Celgene: Honoraria, Other: travel grants; Janssen: Honoraria, Other: travel grants; La Fondation de Frace: Research Funding; Abbvie: Honoraria; Amgen: Honoraria, Other: travel grants; Gilead: Honoraria, Other: travel grants; Takeda: Consultancy, Honoraria. Morgan:Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Research Funding; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Davies:TRM Oncology: Honoraria; MMRF: Honoraria; Abbvie: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria.

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