Whole-Exome Sequencing Identifies a Somatic Cell Mutation Signature That Predicts Relapse Risk and Survival Probability in Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 5-5
Author(s):  
Ehsan Malek ◽  
E. Ricky Chan ◽  
Daniel Qu ◽  
Jane Reese ◽  
Robert Fox ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Signaling Pathways ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Research Funding ◽  
Advisory Board ◽  
High Burden ◽  
Signature Genes ◽  
Whole Exome ◽  
Relapse Group

Introduction: Multiple myeloma (MM) is a plasma cell neoplasm associated with heterogeneous somatic alterations. Despite the development of novel anti-myeloma agents that have significantly prolonged patient survival, disease relapse remains a daunting problem. Our goal was to employ whole-exome sequencing (WES) to better describe the mutational landscape in MM beyond the tumor cell and identify genomic factors that might predict relapse. WES was performed using autograft samples obtained from MM patients that were then treated with high dose melphalan and autologous hematopoietic cell transplant (HCT). We identified a panel of genes that were most frequently mutated in all patients and then identified those genes mutated with greater frequency in patients that relapsed. A relapse burden signature was generated based upon the genes that were most frequently mutated genes in relapsed patients. Finally, the relapse burden signature was correlated with patient progression-free survival (PFS) and overall survival (OS) following autologous HCT. Methods. DNA was extracted from one ml of cryopreserved, mobilized hematopoietic cell product obtained from patients (N=93) that underwent HCT and was provided by the Case Comprehensive Cancer Center Hematopoietic Biorepository Core. Targeted sequencing was performed using the Tempus xE whole exome platform (Tempus, Chicago, IL). Variants were identified using a variant allele frequency (VAF) ≥0.1 for each sample. Variants were tabulated for each gene in each patient. Patients were grouped according to their relapse status; "No Relapse" (N=39) and "Relapse" (N=54) which corresponded to their post-HCT outcome. Relapse time was defined as time from transplant to event. Variants identified in each gene and patient group were counted and ranked. A relapse burden signature was defined and included twenty-two genes over-represented in the relapse group compared to the non-relapse group by > 10%. Genes in the relapse burden signature were subjected to gene set enrichment analysis (GSEA) and cross referenced against Gene Ontology (GO) categories. PFS and OS were defined as the time from transplant until the event of interest, with censoring at time of last follow up. Patients were regrouped according to their mutation burden in the relapse signature genes ("High burden" defined as >=six signature genes with variants) and their OS and PFS were analyzed with an R package (survival) to generate Kaplan-Meier curves and statistical significance based on a Chi-square test between low and high burden patients. Results: In total, 3,523 genes were identified as containing variants. Table 1 lists the top thirty genes that were identified and ranked based upon total number of mutations (mutational count) and most frequently mutated in relapsed and non-relapsed patients (sample count). We then identified those genes that were more frequently mutated by at least 10% in relapsed patients compared to non-relapsed patients (Fig. 1A). GSEA revealed that the relapse burden gene signature was associated with protein O-linked glycosylation, glycan processing, Golgi lumen and innate immune response activating cell surface receptor signaling pathways (Table 2). Interestingly, multiple mucin family members (Muc2, Muc3A, Muc12 and Muc19) were represented in the relapse burden signature. GO analysis indicated that the individual mucin genes were associated with the same signaling pathways that had been associated with the relapse burden signature by GSEA (Table 3). Importantly, a high relapse burden signature was correlated with a statistically significant reduction in both PFS and OS (Fig. 1B, C). Conclusion: Taken together, our results support the feasibility of WES to generate a relapse burden signature that predicts the risk of MM patients for relapse following HCT. Moreover, the mutational landscape associated with relapse, i.e. the specific genes mutated, has provided insights on the mechanisms of relapse. It is noteworthy that the relapse burden signature genes identified here were mutated at a much greater frequency than genes associated with clonal hematopoiesis of indeterminate potential (CHIP). The identification of patient subgroups at heightened risk of relapse can better guide treatment decisions. Future studies will be conducted to evaluate the effect of pathways identified here on myeloma cell survival and to validate actionable therapeutic targets. Disclosures Malek: Bluespark: Research Funding; Takeda: Other: Advisory board , Speakers Bureau; Medpacto: Research Funding; Janssen: Other: Advisory board, Speakers Bureau; Sanofi: Other: Advisory board; Clegene: Other: Advisory board , Speakers Bureau; Amgen: Honoraria; Cumberland: Research Funding. Caimi:Amgen: Other: Advisory Board; Bayer: Other: Advisory Board; Verastem: Other: Advisory Board; Kite pharmaceuticals: Other: Advisory Board; Celgene: Speakers Bureau; ADC therapeutics: Other: Advisory Board, Research Funding. de Lima:Celgene: Research Funding; BMS: Other: Personal Fees, advisory board; Incyte: Other: Personal Fees, advisory board; Kadmon: Other: Personal Fees, Advisory board; Pfizer: Other: Personal fees, advisory board, Research Funding.

scholarly journals Whole Exome Sequencing and Targeted Sequencing Reveal the Heterogeneity of Genomic Evolution and Mutational Profile in Smoldering Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 237-237
Author(s):  
Karma Salem ◽  
Jihye Park ◽  
Claudia Freymond ◽  
Marzia Capelletti ◽  
Daisy Huynh ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Research Funding ◽  
Plasma Cells ◽  
Targeted Sequencing ◽  
Target Coverage ◽  
Driver Genes ◽  
Whole Exome ◽  
Agilent Sureselect

Abstract Introduction: Recent data shows that multiple myeloma (MM) almost always arises from precursor states called Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM), but not all patients with MGUS or SMM develop MM. Risk factors of progression for SMM patients are largely based on tumor load as represented by an M-protein ≥ 3 g/dL, a free light chain (FLC) ratio outside the range of 0.125 to 8, and ≥ 10% plasma cells in the BM. However, the genetic lesions that underlie progression, the molecular factors that cause rapid versus slow progression, and the factors that distinguish the relatively indolent MGUS from SMM are not well known. Further, the genomic landscape of SMM is not well characterized. One potential factor is MYC overexpression. Bergsagel et al. have found that MYC levels increase when comparing MGUS, SMM and overt MM. Other frequently altered pathways in MM are NF-kB, MAPK and DNA damage. In addition, limited studies of paired SMM and MM samples show that in many cases, the aggressive subclones can already be detected, in small cell fractions, before overt MM develops. However, the cause of progression to MM is unclear, in large part because sequential genomic studies of MGUS/SMM progression have yet to be undertaken. To address these questions, in this study we examine clinically-annotated samples from patients with SMM. Methods: We performed whole exome sequencing (WES) (mean target coverage 50X/100X) on 49 germline-tumor matched samples from patients with SMM (DNA from bone marrow CD138+ plasma cells matched with germline DNA from peripheral blood mononuclear cells). Libraries were constructed using Agilent SureSelect XT2 library prep kit, and hybridized to Agilent's whole exome V5+UTR capture probes and then sequenced on HiSeq 2500 (Illumina). We also performed targeted deep sequencing using a custom enrichment bait set on 25 samples of progressor (n=12) and non-progressor (n=13) SMM samples. Libraries were also constructed with Agilent SureSelect XT2 library prep kit and enriched by hybridizing to an in-house designed customized target bait, then sequenced on HiSeq 2500. Sequencing data were analyzed using previously established analytic pipelines including MuTect, RecapSeg, GISTIC, MutSig, and ABSOLUTE. Results: The number of Somatic Single Nucleotide Variants (SSNVs) seen in SMM ranged from 1 to 98 nonsilent mutations with an average of 1.14 mutations/Mb, which is slightly lower than MM (1.6 mutations/Mb) from previous studies (p-value=0.05). This large and varying range of mutational load among samples suggests that SMM is likely a heterogenous entity where some patients are closer to MGUS and others closer to MM. We identified likely drivers in SMM in about ~32% of the samples, including mutations in MM candidate driver genes such as NRAS, KRAS and PTPN11(overall 36 events were present in COSMIC). SMM also had somatic CNAs in about ~50% of SMM samples, such as hyperdiploidy, gain of chromosome 1q, deletion of 13p and 17p, which match the hallmark chromosome changes seen in MM. Comparing deep targeted sequencing of 100 genes (mean target coverage 361X) in samples from 12 SMM patients who progressed to myeloma vs. 13 SMM patients who did not, we found non-synonymous mutations exclusive to progressors, suggesting that with more samples we may find genetic alterations that predict progression in SMM. Conclusion: This study demonstrates that WES and targeted sequencing of SMM patients can detect MM candidate driver genes as well as hallmark CNAs seen in MM patients. Further, there may be potential different mutational features between progressors and non-progressors. Thus, this approach can be used to identify genetic drivers of clonal progression from MGUS/SMM to MM that may present opportunities for early therapeutic intervention and prevention of disease progression. Disclosures Roccaro: Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:Takeda: Honoraria; Noxxon: Honoraria; Amgen: Honoraria; Novartis: Honoraria; BMS: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

Whole Exome Sequencing from Nine Independent Sites of Extraosseous Disease in a Single Patient with Relapsed Multiple Myeloma Show That Extramedullary Disease Arise through a Combination of Branched and Parallel Evolution

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2090-2090
Author(s):  
Heather Landau ◽  
Jun Fan ◽  
Sergio A. Giralt ◽  
Jonathan U. Peled ◽  
Hani Hassoun ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Exome Sequencing ◽  
Board Of Directors ◽  
Whole Exome Sequencing ◽  
Research Funding ◽  
Parallel Evolution ◽  
High Quality ◽  
Advisory Committees ◽  
Extramedullary Disease ◽  
Whole Exome

Abstract Introduction: Multiple myeloma (MM) is the second most common hematologic malignancy. Recent sequencing studies show evidence of massive genetic heterogeneity reflected in multiple parallel subclones already at diagnosis. Different subclones respond differently to given therapy. The role of treatment-driven subclonal skewing and intrinsic acquired mutations is poorly understood in the relapse setting. At relapse, the distribution of subclones throughout the bone marrow and at extramedullary sites is currently unknown. Methods: We performed a research autopsy on a single individual with IgG kappa MM who survived with multiple metastatic sites of disease for 10 years after diagnosis. Genomic DNA was extracted from histologically confirmed snap frozen normal tissue and nine independent sites of extraosseous disease. Whole exome sequencing (150X) was performed by the MSK Genomics Core. Data were filtered to remove germline polymorphisms and enrich for high quality somatic variants. Phylogenetics and subclonal analyses were performed using Treeomics and SCHISM. Results: The average on target coverage was 177X with 73% of bases covered a minimum of 100x. A total of 6348 somatic variants were initially identified. After filtering for high quality somatic variants, 1330 remained with 220 of these variants common to all MM samples and an average of 628 variants per sample. Annotation of high quality variants revealed potentially deleterious somatic mutations in NRAS, FAM46C, DYNC1, CREBBP, ATM, BIRC3, MGA, PPP6C and SMARCA4. Phylogenetics analyses (shown below) indicated the metastases arose through a combination of branched and parallel evolution, with the ATM mutation arising in one branch (one metastasis) that was distinct from a second clonal population containing the NRAS, FAM46C, BIRC3, CREBBP, DYNC1 and PPP6C mutations that were present in all eight other metastases. The MGA and SMARCA4 mutations further identified two additional subclones arising in from the NRAS/FAM46C mutant common ancestral clone. Clonality analyses independently supported this hierarchy by identifying NRAS and FAM46C as early clonal events and MGA and SMARCA4 as late events in the progression of this patient's disease. It also suggested a degree of subclonal mixing within each metastatic site. Conclusions: Using whole exome sequencing from nine independent sites of extraosseous disease in a single MM patient with relapse 10 years after initial diagnosis, we show that extramedullary disease arise through a combination of branched and parallel evolution. Two additional patients have also undergone research autopsy and results will be presented at the meeting. Figure Figure. Disclosures Landau: Prothena: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx/Amgen: Research Funding; Spectrum Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy. Hassoun:Binding Site: Research Funding; Novartis: Consultancy; Celgene: Research Funding; Takeda: Consultancy, Research Funding. Korde:Medscape: Honoraria. Landgren:Medscape Myeloma Program: Honoraria; Takeda: Honoraria; Amgen: Honoraria, Research Funding; BMS: Honoraria; Merck: Honoraria; Celgene: Honoraria, Research Funding.

scholarly journals Archetypes of AML Defined Using Whole Exome Sequencing and Clinical Characteristics in a Diverse Group of Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 597-597
Author(s):  
Christopher B. Benton ◽  
Koichi Takahashi ◽  
Prithviraj Bose ◽  
Feng Wang ◽  
Hsiang-Chun Chen ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Research Funding ◽  
De Novo ◽  
Gene Mutations ◽  
Advisory Board ◽  
Driver Mutations ◽  
Whole Exome ◽  
Clinical Scenarios

Abstract Background: Acute myeloid leukemia (AML) comprises a heterogeneous collection of morphologically related diseases. The identification and study of somatic mutations in AML using next-generation sequencing has led to insights into the pathogenesis of AML. Constellations of specific mutations may correlate with clinical phenotype and reveal the most relevant cooperating molecular pathways. Understanding AML biological pathways will contribute to more personalized, directed therapies and improve outcomes. Most sequencing efforts have focused on select groups of AML patients or select gene panels. Characterization of mutations identified by whole exome sequencing for AML patients from a variety of clinical scenarios has not been the subject of one study. Methods: Whole exome sequencing (WES) was performed, in addition to complete clinical molecular diagnostics, on leukemia samples from a diverse group of 141 consecutive AML patients who underwent complete clinical evaluation at a single quaternary leukemia care institution (MD Anderson Cancer Center, MDACC). The cohort included adults of all ages, treatment histories (treated/untreated), and ontogenies (de novo/secondary/therapy-related). To facilitate the identification of driver mutations from WES data, an automatic pipeline was developed to filter and annotate raw variants, and then select non-polymorphism, protein-coding changing mutations. A relational mutation/clinical database was created to analyze gene mutations and gene categories in the context of well-annotated mutations and complete clinical indices including treatments and outcomes. Results: We characterized the landscape of driver mutations in an unselected group of AML patients, and classified mutations by 9 predefined functional categories of known aberrant pathways implicated in AML (Figure 1). The frequency and distribution of gene mutations and gene categories were determined and compared to TCGA AML whole exome sequencing mutation data, derived from a de novo, untreated AML cohort. Forty-six separate genes/fusions were mutated in the MDACC cohort, including 326 mutations. The MDACC cohort included 23 cases (16%) with a TP53 mutation. Overall, a mean of 2.3+/-1.4 mutations-of-interest were identified per patient. For the MDACC dataset, the likelihood of co-occurrence and mutual exclusivity was determined for each gene (mutated in greater than 2 cases, n=34) and each gene category (n=9) versus every other gene, gene category, as well as each individual annotated clinical characteristic (n=30). 156 statistically significant co-occurring pairs and 71 statistically significant mutually exclusive pairs were found. We analyzed hierarchically clustered, strength-of-association correlograms to identify likely mutational frameworks with similarly associated clinical/molecular characteristics. We identified patterns of predominant genetic alterations that defined 4 distinct AML archetypes: 1) Transcription factor [TRNSXN] (16% of cases), 2) Nucleolar/Methylation/Signaling [NMS] (40%), 3) Spliceosome [SPLICE] (21%), and 4) TP53-mutated [TP53] (16%). Ten cases had no identifiable mutation-of-interest, compared to 4 cases without mutation in TCGA data. The defining mutational characteristics of each AML archetype, and select associated characteristics are shown in Table 1. Conclusions: Analysis of whole exome sequencing data along with clinical information from all-comer AML patients identifies archetypal classes of the disease, each with particular tendencies for specific mutational, clinical, and outcome characteristics. Our approach demonstrates that genomic characterization of mutations in a variety of diverse clinical scenarios has the potential to identify representative mechanisms of AML development across the spectrum of disease presentations. Disclosures Daver: Otsuka: Consultancy, Honoraria; Sunesis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Karyopharm: Honoraria, Research Funding; Ariad: Research Funding; Kiromic: Research Funding; BMS: Research Funding. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. DiNardo:Agios: Other: advisory board, Research Funding; Celgene: Research Funding; Novartis: Other: advisory board, Research Funding; Daiichi Sankyo: Other: advisory board, Research Funding; Abbvie: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.

P-047: Clonal evolution in multiple myeloma evaluated by Whole Exome Sequencing

2021 ◽  
Vol 21 ◽  
pp. S64
Author(s):  
Ritu Gupta ◽  
Gurvinder Kaur ◽  
Akanksha Farswan ◽  
Lingaraja Jena ◽  
Anubha Gupta ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Clonal Evolution ◽  
Whole Exome

scholarly journals Defining Clonal Plasticity Using Whole Exome Sequencing in Single Cells in an Index Case of amp1q21 Multiple Myeloma

2017 ◽  
Vol 17 (1) ◽  
pp. e108
Author(s):  
Surinder Sahota ◽  
Dean Bryant ◽  
Nicola Weston-Bell ◽  
Will Tapper ◽  
Arnold Bolomsky ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Index Case ◽  
Single Cells ◽  
Whole Exome

scholarly journals Whole-exome sequencing of cell-free DNA and circulating tumor cells in multiple myeloma

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
S. Manier ◽  
J. Park ◽  
M. Capelletti ◽  
M. Bustoros ◽  
S. S. Freeman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Exome Sequencing ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Whole Exome Sequencing ◽  
Cell Free Dna ◽  
Free Dna ◽  
Whole Exome

Whole Exome Sequencing of a Patient with Atypical Congenital Pure Red Cell Aplasia Has Enabled the Identification of a Novel Key Actor of Erythropoiesis That May be Involved in CDAI Physiopathology

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-4
Author(s):  
Elia Colin ◽  
Genevieve Courtois ◽  
Lydie Da Costa ◽  
Carine Lefevre ◽  
Michael Dussiot ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Homologous Recombination ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Research Funding ◽  
Pure Red Cell Aplasia ◽  
Red Cell ◽  
Whole Exome

Background: The development of next generation sequencing techniques has brought important insights into the molecular mechanisms of erythropoiesis and how these processes can be perturbed in human diseases. This strategy may be valuable in some hereditary erythroid disorders where a subset of patients does not carry any mutations in the supposed causal gene and for which transgenic mouse models do not recapitulate the phenotype, suggesting that additional genetic events may be involved in pathogenesis. Here, we report the case of an adult patient presenting with atypical pure red cell aplasia associated with facial dysmorphy and chronic leg ulcers. Whole exome sequencing revealed a heterozygous missense mutation (R725W) in the CDAN1 gene, which has been previously reported in congenital dyserythropoietic anemia type I (CDAI). However, this mutation was also detected in her healthy brother, suggesting that this event alone was not sufficient to explain her phenotype. According to this hypothesis, we found an additional germline heterozygous nonsense mutation (Q732X) in the MMS22L gene, which was not shared by her unaffected relatives. MMS22L is a protein involved in homologous recombination-dependent repair of stalled or collapsed replication forks. Additionally, MMS22L is able to bind newly synthesized soluble histones H3 and H4 and exhibits a histone chaperone activity. MMS22L loading onto ssDNA during homologous recombination is promoted by the histone chaperone ASF1. Interestingly, CDAN1 acts as a negative regulator of ASF1 by mediating its sequestration in the cytoplasm, which results in the blocking of histone delivery. Aims: As MMS22L has never been reported in erythropoiesis before, we aimed to investigate the role of MMS22L in human erythropoiesis. Based on the data summarized above, the purpose of this study was also to determine the effect of combined inactivation of MMS22L and CDAN1 on in vivo erythropoiesis, while exploring the functional cooperation between both proteins. Results: To decipher the role of MMS22L in human erythropoiesis, we assessed the consequences of complete MMS22L inactivation in human cord blood CD34+ progenitors as well as in CD36+ immature erythroblasts using shRNA lentiviruses. This resulted in a severe decrease of cell proliferation and differentiation due to G1 cell cycle arrest, with a slight increase of apoptosis. Interestingly, this phenotype was not observed when MMS22L was inactivated in the granulo-monocytic lineage, in which differentiation was maintained, suggesting that erythroid cells, that are highly proliferative, are more sensitive to MMS22L inactivation. To better understand the effect of combined CDAN1 and MMS22L haploinsufficiency observed in the proband, we used zebrafish as an in vivo model. Mms22l and cdan1 expression were simultaneously or separately downregulated by about 50% using antisens morpholino oligomers. 48 hours later, zebrafish embryos were stained with o-dianisidine to detect hemoglobin-containing cells. We found that combined knock-down of mms22l and cdan1 resulted in severe anemia, while knock-down of mms22l or cdan1 alone did not lead to any erythroid disorder. This experiment provides a proof-of-concept, indicating that the phenotype of the proband is indeed caused by the combination of both MMS22L and CDAN1 mutations. Finally, in order to decipher the cooperation between MMS22L and CDAN1 we used the human erythroid UT-7 cell line. We found that CDAN1 inactivation resulted in a severe decrease in MMS22L expression within the nucleus, suggesting that CDAN1 may regulate MMS22L expression or localization. We therefore wanted to confirm these results by assessing MMS22L expression in B-EBV cell lines established from two CDAI patients with CDAN1 compound heterozygous mutations. We found a great decrease in MMS22L expression within the nucleus of the CDAI patients' cells when compared to three control B-EBV cell lines. Based on these results, we suggest that impairment of MMS22L trafficking to the nucleus could be involved in CDA1 physiopathology. Conclusion: Through comprehensive genetic analysis of a single case with atypical congenital anemia, we demonstrated for the first time that MMS22L, a cell cycle regulator, is essential for the process of erythropoiesis. The crosstalk between MMS22L and CDAN1 is currently under investigation and could bring important new insights into the physiopathology of CDAI. Disclosures Hermine: Novartis: Research Funding; Alexion: Research Funding; AB Science: Consultancy, Current equity holder in publicly-traded company, Honoraria, Patents & Royalties, Research Funding; Celgene BMS: Consultancy, Research Funding; Roche: Consultancy.

Whole Exome Sequencing to Predict Response to Hypomethylating Agents in MDS

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1698-1698 ◽  
Author(s):  
Holleh D Husseinzadeh ◽  
Edward P Evans ◽  
Kenichi Yoshida ◽  
Hideki Makishima ◽  
Andres Jerez ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Research Funding ◽  
Somatic Mutations ◽  
Prolonged Exposure ◽  
Mutational Analysis ◽  
Outcome Analysis ◽  
Individual Treatment ◽  
Hypomethylating Agents ◽  
Whole Exome

Abstract Abstract 1698 Hypomethylating agents decitabine and azacitidine are standard treatments for myelodysplastic syndromes (MDS). In their use, one hopes to rectify cytopenias and prolong survival by retarding further disease progression. However, individual treatment responses vary from complete remission (CR) to complete refractoriness. In general, at least 4 cycles of therapy are administered prior to assessing response. Thus, patients may have prolonged exposure to ineffective therapy, suffering toxicities without clinical benefit, while alternative and potentially more effective treatments are delayed. Currently, there are no reliable phenotypic or mutational markers for predicting response to hypomethylating agents. Once whole exome sequencing (WES) became available for more routine analysis, we theorized that somatic mutational patterns may help identify patients who would most benefit from these drugs, thereby maximizing response rate by rational patient selection. To pursue this hypothesis, we screened a cohort of 168 patients with MDS who received either azacitidine or decitabine for the presence of somatic mutations. Only those who received sufficient therapy, i.e., completed at least 4 cycles, were selected for outcome analysis. Targeted Sanger sequencing, including a panel of up to 19 genes frequently affected by somatic mutations was performed. For a representative subset of 26 patients (this subset is expanding) of whom there were 15 responders and 11 non-responders, mutational analysis was performed by WES to select target genes for further analysis. WES utilizes paired DNA (tumor vs. CD3+ lymphocytes) to produce raw sequence reads aligned using Burrows-Wheeler Aligner (BWA). Variants are detected using the Broad Institute's Best Practice Variant Detection GATK toolkit. Median age was 68 years (range, 55–85), 50% were female, and MDS subtypes were as follows: RA/RCUD/RARS 13%, RCMD 16%, RAEB-1/2 20%, MDS/MPN & CMML-1/2 31%, and sAML 20%. Response was assessed using IWG 2006 criteria at 4 and 7 months after therapy initiation. Overall response was 48%; rate of CR (including marrow/cytogenetic CR) was 28%, any HI 20%, SD 22%, and no response 29%. The cohort was then dichotomized into “responders” and “non-responders,” with responders classified as those achieving CR or any HI. Baseline patient characteristics were similar between both groups, including average age at treatment initiation, disease subtypes, proportion of abnormal/complex karyotypes, and presence of common cytogenetic aberrations. Overall, the most frequently mutated genes include TET2/IDH1/IDH2, SRSF2, ASXL1, SF3B1, RUNX1, EZH2/EED/SUZ12, SETBP1, CBL, and PPIAF2. The highest rate of refractoriness was noted in mutants of TET2/IDH1/IDH2 (67%), SF3B1 (67%), U2AF1/2 (67%). We also identified several genes whose mutants were few but associated exclusively with refractory disease (100%), including KIT, ZRSR2, PRPF8, LUC7L2. We next applied a recursive partitioning algorithm to construct a decision tree for identifying the most pivotal mutations associated with response: we found mutant CBL and PPFIA2 to be strongly associated with response, whereas mutant U2AF1/2, SF3B1 and PRPF8 were strongly associated with refractoriness. Our final approach was to dichotomize the cohort by the presence/absence of each mutation/group of mutations, and response within mutant vs. wild type cases was compared. Among refractory cases, TET2/IDH1/IDH2 (26%) and SF3B1 (17%) were most frequently mutated; among responders, mutations in RUNX1 (19% vs. 4%]), CBL (14% vs. 0%), SRSF2 (23% vs. 9%), and SETBP1 (18% vs. 4%) were most frequent. When multiple genes were combined in “either-or” fashion, mutation in TET2, SF3B1, PRPF8, or LUCL71 was significantly associated with refractoriness (52%, p=.0287), whereas mutations of RUNX1, CBL, SRSF2, SETBP1, or PPFIA2 mutation was significantly associated with response (86%, p=.0001). Mutational patterns appear to predict response to standard hypomethylating agents. Identification of the most predictive genes could guide development of molecular maker-based selection of patients for hypomethylating agent therapy, but will require ongoing analysis and additional prospective testing for validation. Disclosures: Advani: Genzyme: Honoraria, Research Funding; Immunomedics: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

Driver Mutation in Waldenstrom's Macroglobullinemia and Their Clonal Heterogeneity during Progression and Relapse

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1092-1092 ◽  
Author(s):  
Jihye Park ◽  
Antonio Sacco ◽  
Salomon Manier ◽  
Karma Salem ◽  
Mira Mashood ◽  
...  
Keyword(s):  
New York ◽  
Disease Progression ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Copy Number ◽  
Research Funding ◽  
Targeted Sequencing ◽  
Whole Exome ◽  
Btk Inhibitor ◽  
Serial Samples

Abstract Background: Whole genome sequencing has recently shown the presence of recurrent mutations, such as MYD88, CXCR4 and ARID1A, in patients with Waldenstrom's Macroglobulinemia (WM) at diagnosis. Nevertheless, the contribution of each genomic aberration within the clonal evolution of the tumor during WM progression has not been reported. We therefore aimed to investigate whether sequential genomic events sustain WM disease progression by using whole exome sequencing (WES) and targeted sequencing of serial samples. In addition, we investigated if specific genomic alterations can change in response to treatment with samples before and after proteasome or BTK inhibitor treatments. Methods: We have sequenced 74 samples from 32 patients using WES or targeted sequencing technology. DNA was collected from bone marrow CD19-selected cells that were isolated from 6 patients with WM at different stages of disease (2-3 serial samples per patient, total 15 samples), and was subjected to library construction, followed by Agilent Sure-Select Human All Exon v2.0-based hybrid selection. Germline DNA was isolated from matched CD19-depleted peripheral blood samples. All libraries were sequenced with Illumina Hiseq 2500 instrument (New York Genome Center, Rockefeller University, New York, NY). Reads were aligned to GRCh37, and quality control, mutation calling, insertion and deletion identification, copy number variation detection, coverage calculations were accomplished via Firehose at Broad. Somatic Single Nucleotide Variations (SSNVs) were identified and annotated using MuTect and Oncotator, respectively. Insertions and deletions were detected using Strelka. In addition, the deep targeted sequencing was done with a customized bait set in 59 independent serial samples obtained from 26 patients who were treated with either proteasome inhibitor (Pi)- or BTK inhibitor (BTKi)-based therapies in 12 and 14 cases, respectively. Results: Whole exome sequencing, performed at a total average depth of 83X for germline and 85X for tumor samples, led to the identification of average of 14 non-silent mutations per sample (range 2-60). Base conversion signature showed dominant A>C/G transitions. Copy number analysis showed 6q deletion being the most prevalent one. MYD88 was the most recurrent somatic variants in WM patients (>93%), followed by several genes including IRS4, VCAN, CXCR4 and ALDH2, being detected in ~20-30% of the samples. Specifically, changes in the Cancer Cell Fraction (CCF) of these mutations, such as MYD88 and CXCR4, occurred in the serial samples depending on progression and response to therapy. The mutated genes mentioned above, including MYD88, CXCR4, LRG1 and VCAN, were also observed in targeted sequencing of 59 independent serial samples (mean target coverage 434X). CXCR4 was linked to disease progression exposed to Pi or BTK; but not detected in patients responding to therapies. MYD88 was present in patients with either progressive disease or PR/VGPRs. Specifically, different frequencies of MYD88 mutations were identified in patients with progression or PR/VGPR to BTKi-based therapies compared to Pi-based therapies. LRG1 was detected in a patient in response to Pi, while VCAN was observed in both patients showing progression or response to Pi regimens only. Conclusion: These findings reveal the occurrence of clonal variations in patients with WM during disease progression and response to therapies. MYD88 was confirmed to be the most prevalent somatic aberration, and was present in post-treatment samples of progressors and responders to Pi- or BTKi-based regimens. In contrast, mutations in CXCR4 were enriched in patients with WM progressing to either Pi or BTKi therapies. This study demonstrates that WES and targeted sequencing of serial samples of WM patients can detect clonal variations during disease progression. Disclosures Ghobrial: Celgene: Honoraria, Research Funding; Noxxon: Honoraria; Amgen: Honoraria; BMS: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria. Roccaro:Takeda Pharmaceutical Company Limited: Honoraria.

scholarly journals Mesenchymal Stromal Cells (MSCs) from Myelodysplastic Syndromes (MDS) Are Not Clonally Mutated I n Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1514-1514
Author(s):  
Johann-Christoph Jann ◽  
Maximilian Mossner ◽  
Vladimir Riabov ◽  
Eva Altrock ◽  
Nanni Schmitt ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Myelodysplastic Syndromes ◽  
Research Funding ◽  
Ex Vivo ◽  
Mutational Burden ◽  
Bm Niche ◽  
Whole Exome ◽  
Validation Experiments

Abstract Introduction There is increasing evidence for an active role of the bone marrow (BM) microenvironment in the pathogenesis of Myelodysplastic Syndromes (MDS). Genetically engineered murine models have shown that isolated mutations in the BM niche can disrupt the non-mutated hematopoietic compartment and induce MDS-like phenotypes. However, it is still unclear whether primary MDS in humans may possibly be associated with acquired mutations non-hematopoietic BM stroma cells. Although chromosomal aberrations and mutations have been described in in ex vivo expanded MSC cultures from MDS and AML patients, little validation has been performed to address whether such molecular lesions were not clonal outgrowths resulting from the strenuous and massively expansive cell culture procedures. Materials and Methods We performed whole exome sequencing on paired ex vivo expanded MSCs and native BM samples of n=98 MDS and associated myeloid neoplasia cases treated at the Department of Hematology and Oncology of the Medical Faculty Mannheim, Heidelberg University, Germany (median age 73 years, range 44-86). As controls, we included a cohort of n=28 samples from healthy subjects (median age 75 years, range 36-84). MSCs were expanded adherently on plastic dishes by seeding 5x10e6 mononuclear cells in StemMACS MSC Expansion Medium XF (Miltenyi Biotec) for a median of 34 days, (95% confidence interval 22-50d). Whole exome sequencing was carried out using Nextera DNA Flex Tagmentation kit (Illumina) with IDT xGene Research probe v1 at a median coverage at 88x with BM MNC as germline control accounting for possible LOH in the BM sample. Validation experiments were performed by deep re-sequencing of single CFU-F colonies (n=4 patients), sequencing of serial cultures (n=7 patients) and re-sequencing of primary sorted native bone marrow MSCs from n=9 patients. Results In the exome sequencing analyses of ex vivo expanded MSCs we discovered multiple recurrent mutations in MSCs of MDS patients including but not limited to genes such as ZFX (n=8/98) and RANK (n=5/98). MSCs from MDS patients displayed an overall higher mutational burden and increased replicative stress as determined by gH2AX and RPA staining, which correlated with the mutational burden and shorter telomeres as compared to healthy controls. The analysis of mutational signatures revealed that MDS MSCs were distinct compared to healthy MSCs. Furthermore, we found that MDS MSCs displayed increased senescence assessed by flow bGAL staining and associated inflammatory gene expression determined by IL6 qPCR/ELISA for n=32 cases. To investigate whether acquired mutations in MSCs were driven by the ex vivo expansion we performed individualized amplicon based deep re-sequencing of serial culture passages and different BM aspirations for n=7 patients as well as single colony re-sequencing in n=4 patient cases. Furthermore, we re-sequenced primary sorted CD45-,CD235a-,CD31+/-,CD271+/- BM cells of n=9 cases. All of these validation experiments indicated that the discovered mutations were associated with expansion in culture and but not present in clonally relevant cell populations in the primary BM in vivo. Discussion Together with previously published data of the BM niche of myeloid neoplasms, our results add to the notion that MSCs in MDS are molecularly and functionally altered. Nevertheless, our current comprehensive sequencing analyses leave little doubt that if acquired mutations in the stroma of MDS patients play a role in MDS disease initiation at all, then at such a low clonal and possibly locally confined level, that they are not detectable with currently feasible sample acquisition and methodology. In our current study, we discovered no evidence for acquired mutations in BM derived MSCs in MDS. Disclosures Schmitt: Affimed GmbH: Research Funding. Flach: Gilead: Current Employment. Hofmann: BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria. Nowak: Pharmaxis: Current holder of individual stocks in a privately-held company, Research Funding; Celgene: Honoraria; AbbVie: Other: Investigator on funded clinical trial; Tolero Pharma, Pharmaxis, Apogenix: Research Funding; Affimed: Research Funding; Takeda: Honoraria.

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