Mutational Patterns and Copy Number Changes at Diagnosis Are a Powerful Tool to Predict Outcome: Result of the Sequencing Study of 463 Newly Diagnosed Myeloma Trial Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 637-637 ◽  
Author(s):  
Eileen M Boyle ◽  
Brian A Walker ◽  
Christopher P Wardell ◽  
Alexander Murison ◽  
Lorenzo Melchor ◽  
...  
Keyword(s):  
Overall Survival ◽  
Targeted Therapy ◽  
High Risk ◽  
Personalized Medicine ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Copy Number ◽  
Whole Exome ◽  
Prognostic Groups ◽  
Mutation Group

Abstract Background: The main genetic features of myeloma identified so far have been the presence of balanced translocations at the immunoglobulin heavy chain (IGH) region and copy number abnormalities. Novel methodologies such as massively parallel sequencing have begun to describe the pattern of tumour acquired mutations detected at presentation but their biological and clinical relevance has not yet been fully established. Methods: Whole exome sequencing was performed on 463 presentation patients enrolled into the large UK, phase III, open label, Myeloma XI trial. DNA was extracted from germline DNA and CD138+ plasma cells and whole exome sequencing was performed using SureSelect (Agilent). In addition to capturing the exome, extra baits were added covering the IGH, IGK, IGL and MYC loci in order to determine the breakpoints associated with translocations in these genes. Tumour and germline DNA were sequenced to a median of 60x and data processed to generate copy number, acquired variants and translocation breakpoints in the tumour. Progression-free and overall survival was measured from initial randomization and median follow up for this analysis was 25 months. These combined data allow us to examine the effect of translocations on the mutational spectra in myeloma and determine any associations with progression-free or overall survival. Results: We identified 15 significantly mutated genes comprising IRF4, KRAS, NRAS, MAX, HIST1H1E, RB1, EGR1, TP53, TRAF3, FAM46C, DIS3, BRAF, LTB, CYLD and FGFR3. By analysing the correlation between mutations and cytogenetic events using a probabilistic approach, we describe the co-segregation of t(11;14) with CCND1 mutations (Corr 0.28,BF=1.5x106 (Bayes Factor)) and t(4;14) with FGFR3 (Corr=0.40, BF=1.12x1014) and PRKD2 mutations (Corr=0.23, BF=3507). The mutational spectrum is dominated by mutations in the RAS (43%) and NF-κB (17%) pathway, however they are prognostically neutral. We describe for the first time in myeloma mutations in genes such as CCND1 and DNA repair pathway alterations (TP53, ATM, ATR and ZFHX4 mutations) that are associated with a negative impact on survival in contrast to those in IRF4 and EGR1 that are associated with a favourable overall-survival. By combining these novel risk factors with the previously described adverse cytogenetic features and ISS we were able to demonstrate in a multivariate analysis the independent prognostic relevance of copy number and structural abnormalities (CNSA) such as del(17p), t(4;14), amp(1q), hyperdiploidy and MYC translocations and mutations in genes such as ATM/ATR, ZFHX4, TP53 and CCND1. We demonstrate that the more adverse features a patient had the worse his outcome was for both PFS (one lesion: HR=1.6, p=0.0012, 2 lesions HR=3.3, p<0.001, 3 lesions HR=15.2, p< 0.001) and for OS (one lesion: HR=2.01, p=0.0032, 2 lesions HR=4.79, p<0.001, 3 lesions HR=9.62, p< 0.001). When combined with ISS, we identified 3 prognostic groups (Group 1: ISS I/II with no CNSA or mutation, Group 2: ISS III with no CNSA or mutation or ISS I/II/III with one CNSA or mutation, Group 3: Two CNSA or mutation regardless of their ISS) thus identifying three distinct prognostic groups with a high risk population representing 13% of patients that both relapsed [median PFS 10.6 months (95% CI 8.7-17.9) versus 27.7 months (95% CI 25.99-31.1), p<0.001] and died prematurely [median overall survival 23.2 months (95% CI 18.2-35.3.) versus not reached, p<0.001] regardless of their ISS score. Finally, we have also identified a list of potentially actionable mutations for which targeted therapy already exists opening the way into personalized medicine in myeloma. Conclusion: We have refined our understanding of genetic events in myeloma and identified clinically relevant mutations that may be used to better stratify patients at presentation. Identifying high risk populations or patients that may benefit from targeted therapy may open the way into personalized medicine for myeloma. Disclosures Walker: Onyx Pharmaceuticals: Consultancy, Honoraria.

Whole-exome sequencing reveals potential germline and somatic mutations in 60 malignant ovarian germ cell tumors

2021 ◽  
Author(s):  
Juan Chen ◽  
Yan Li ◽  
Jianlei Wu ◽  
Yakun Liu ◽  
Shan Kang
Keyword(s):  
Germ Cell ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Peripheral Blood ◽  
Copy Number ◽  
Somatic Mutations ◽  
Germ Cell Tumors ◽  
Germline Mutations ◽  
Deletion Region ◽  
Whole Exome

Abstract Background Malignant ovarian germ cell tumors (MOGCTs) are rare and heterogeneous ovary tumors. We aimed to identify potential germline mutations and somatic mutations in MOGCTs by whole-exome sequencing. Methods The peripheral blood and tumor samples from these patients were used to identify germline mutations and somatic mutations, respectively. For those genes corresponding to copy number alterations (CNA) deletion and duplication region, functional annotation of was performed. Immunohistochemistry was performed to evaluate the expression of mutated genes corresponding to CNA deletion region. Results In peripheral blood, copy number loss and gain were mostly found in yolk sac tumors (YST). Moreover, POU5F1 was the most significant mutated gene with mutation frequency &gt; 10% in both CNA deletion and duplication region. In addition, strong cytoplasm staining of POU5F1 (corresponding to CNA deletion region) was found in 2 YST and nuclear staining in 2 dysgerminomas (DG) tumor samples. Genes corresponding to CNA deletion region were significantly enriched in the signaling pathway of regulating pluripotency of stem cells. In addition, genes corresponding to CNA duplication region were significantly enriched in the signaling pathways of RIG-I-like receptor, Toll-like receptor, NF-kappa B and Jak–STAT. KRT4, RPL14, PCSK6, PABPC3 and SARM1 mutations were detected in both peripheral blood and tumor samples. Conclusions Identification of potential germline mutations and somatic mutations in MOGCTs may provide a new field in understanding the genetic feature of the rare biological tumor type in the ovary.

scholarly journals Advancing Personalized Medicine Through the Application of Whole Exome Sequencing and Big Data Analytics

2019 ◽  
Vol 10 ◽  
Author(s):  
Pawel Suwinski ◽  
ChuangKee Ong ◽  
Maurice H. T. Ling ◽  
Yang Ming Poh ◽  
Asif M. Khan ◽  
...  
Keyword(s):  
Big Data ◽  
Personalized Medicine ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Data Analytics ◽  
Big Data Analytics ◽  
Whole Exome

scholarly journals RefCNV: Identification of Gene-Based Copy Number Variants Using Whole Exome Sequencing

2016 ◽  
Vol 15 ◽  
pp. CIN.S36612 ◽  
Author(s):  
Lun-Ching Chang ◽  
Biswajit Das ◽  
Chih-Jian Lih ◽  
Han Si ◽  
Corinne E. Camalier ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Copy Number ◽  
Copy Number Variants ◽  
Estimation Methods ◽  
Single Nucleotide Variants ◽  
False Positive Error ◽  
Reference Set ◽  
Genome Wide ◽  
Whole Exome

With rapid advances in DNA sequencing technologies, whole exome sequencing (WES) has become a popular approach for detecting somatic mutations in oncology studies. The initial intent of WES was to characterize single nucleotide variants, but it was observed that the number of sequencing reads that mapped to a genomic region correlated with the DNA copy number variants (CNVs). We propose a method RefCNV that uses a reference set to estimate the distribution of the coverage for each exon. The construction of the reference set includes an evaluation of the sources of variability in the coverage distribution. We observed that the processing steps had an impact on the coverage distribution. For each exon, we compared the observed coverage with the expected normal coverage. Thresholds for determining CNVs were selected to control the false-positive error rate. RefCNV prediction correlated significantly ( r = 0.96–0.86) with CNV measured by digital polymerase chain reaction for MET (7q31), EGFR (7p12), or ERBB2 (17q12) in 13 tumor cell lines. The genome-wide CNV analysis showed a good overall correlation (Spearman's coefficient = 0.82) between RefCNV estimation and publicly available CNV data in Cancer Cell Line Encyclopedia. RefCNV also showed better performance than three other CNV estimation methods in genome-wide CNV analysis.

Whole Exome Sequencing In Relapsed Pediatric T-ALL: Progression Into Relapse Is Characterized By An Increased Number Of Somatic Mutations and a Conservation Of Mutations In Leukemogenic Driver Genes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 228-228
Author(s):  
Joachim Kunz ◽  
Tobias Rausch ◽  
Obul R Bandapalli ◽  
Martina U. Muckenthaler ◽  
Adrian M Stuetz ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Initial Diagnosis ◽  
Driver Mutations ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Whole Exome ◽  
Recurrent Mutations

Abstract Acute precursor T-lymphoblastic leukemia (T-ALL) remains a serious challenge in pediatric oncology, because relapses carry a particularly poor prognosis with high rates of induction failure and death despite generally excellent treatment responses of the initial disease. It is critical, therefore, to understand the molecular evolution of pediatric T-ALL and to elucidate the mechanisms leading to T-ALL relapse and to understand the differences in treatment response between the two phases of the disease. We have thus subjected DNA from bone marrow samples obtained at the time of initial diagnosis, remission and relapse of 14 patients to whole exome sequencing (WES). Eleven patients suffered from early relapse (duration of remission 6-19 months) and 3 patients from late relapse (duration of remission 29-46 months).The Agilent SureSelect Target Enrichment Kit was used to capture human exons for deep sequencing. The captured fragments were sequenced as 100 bp paired reads using an Illumina HiSeq2000 sequencing instrument. All sequenced DNA reads were preprocessed using Trimmomatic (Lohse et al., Nucl. Acids Res., 2012) to clip adapter contaminations and to trim reads for low quality bases. The remaining reads greater than 36bp were mapped to build hg19 of the human reference genome with Stampy (Lunter & Goodson, Genome Res. 2011), using default parameters. Following such preprocessing, the number of mapped reads was >95% for all samples. Single-nucleotide variants (SNVs) were called using SAMtools mpileup (Li et al., Bioinformatics, 2009). The number of exonic SNVs varied between 23,741 and 31,418 per sample. To facilitate a fast classification and identification of candidate driver mutations, all identified coding SNVs were comprehensively annotated using the ANNOVAR framework (Wang et al., Nat. Rev. Genet., 2010). To identify possible somatic driver mutations, candidate SNVs were filtered for non-synonymous, stopgain or stoploss SNVs, requiring an SNV quality greater or equal to 50, and requiring absence of segmental duplications. Leukemia-specific mutations were identified by filtering against the corresponding remission sample and validated by Sanger sequencing of the genomic DNA following PCR amplification. We identified on average 9.3 somatic single nucleotide variants (SNV) and 0.6 insertions and deletions (indels) per patient sample at the time of initial diagnosis and 21.7 SNVs and 0.3 indels in relapse. On average, 6.3 SNVs were detected both at the time of initial diagnosis and in relapse. These SNVs were thus defined as leukemia specific. Further to SNVs, we have also estimated the frequency of copy number variations (CNV) at low resolution. Apart from the deletions resulting from T-cell receptor rearrangement, we identified on average for each patient 0.7 copy number gains and 2.2 copy number losses at the time of initial diagnosis and 0.5 copy number gains and 2.4 copy number losses in relapse. We detected 24/27 copy number alterations both in initial diagnosis and in relapse. The most common CNV detected was the CDKN2A/B deletion on chromosome 9p. Nine genes were recurrently mutated in 2 or more patients thus indicating the functional leukemogenic potential of these SNVs in T-ALL. These recurrent mutations included known oncogenes (Notch1), tumor suppressor genes (FBXW7, PHF6, WT1) and genes conferring drug resistance (NT5C2). In several patients one gene (such as Notch 1, PHF6, WT1) carried different mutations either at the time of initial diagnosis and or in relapse, indicating that the major leukemic clone had been eradicated by primary treatment, but that a minor clone had persisted and expanded during relapse. The types of mutations did not differ significantly between mutations that were either already present at diagnosis or those that were newly acquired in relapse, indicating that the treatment did not cause specific genomic damage. We will further characterize the clonal evolution of T-ALL into relapse by targeted re-sequencing at high depth of genes with either relapse specific or initial-disease specific mutations. In conclusion, T-ALL relapse differs from primary disease by a higher number of leukemogenic SNVs without gross genomic instability resulting in large CNVs. Disclosures: No relevant conflicts of interest to declare.

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.

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