scholarly journals Multiscale heterogeneity in gastric adenocarcinoma evolution is an obstacle to precision medicine

2020 ◽  
Author(s):  
Christoph Röcken ◽  
Anu Amallraja ◽  
Christine Halske ◽  
Luka Opasic ◽  
Arne Traulsen ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Copy Number ◽  
Validation Cohort ◽  
Intratumoral Heterogeneity ◽  
Discovery Cohort ◽  
Clinical Prognosis ◽  
Synonymous Mutations ◽  
Somatic Evolution ◽  
Whole Exome

Abstract Purpose: Cancer is a somatic evolutionary disease. Using multiregional whole exome sequencing, we tested the effect of somatic evolution on intratumoral heterogeneity and its putative clinical and biological implications in adenocarcinomas of the stomach and gastroesophageal junction (GC). Patients and Methods: The study comprised a prospective discovery cohort of 9 and a validation cohort of 463 GCs. Multiregional whole-exome sequencing was done using 48 tumor samples (range: 3-10 tumor samples/patient) of the discovery cohort.Results: In total, the discovery cohort harbored 16,537 non-synonymous mutations (mutations/sample: median n=159; mutations/patient: median n=369). Intratumoral heterogeneity of somatic mutations and copy number variants were present in all tumors of the discovery cohort. 53-91% of the non-synonymous mutations were not present in each patient’s sample; 399 genes harbored 2-4 different non-synonymous mutations in the same patient; 175 genes showed copy number variations, the majority being heterogeneous, including CD274 (PD-L1). Multi-sample tree-based analyses provided evidence for branched evolution being most complex in a microsatellite instable GC. The analysis of the mode of evolution showed a high degree of heterogeneity in deviation from neutrality within each tumor. Studies on the validation cohort showed that the subclonal loss of SMAD4 is an independent predictor for poor patient outcome in Caucasian patients.Conclusions: Neutral and non-neutral somatic evolution shape the mutational landscape in GC. It leads to complex spatial intratumoral heterogeneity and may have profound effects on patient management. It provides crucial information for an individualized understanding of clinical prognosis and therapeutic options in GC patients.

scholarly journals Multiscale heterogeneity in gastric adenocarcinoma evolution is an obstacle to precision medicine

2020 ◽  
Author(s):  
Christoph Röcken ◽  
Anu Amallraja ◽  
Christine Halske ◽  
Luka Opasic ◽  
Arne Traulsen ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Copy Number ◽  
Validation Cohort ◽  
Gastroesophageal Junction ◽  
Copy Number Variations ◽  
Intratumoral Heterogeneity ◽  
Discovery Cohort ◽  
Clinical Prognosis ◽  
Synonymous Mutations ◽  
Somatic Evolution

Abstract Background: Cancer is a somatic evolutionary disease, which adapts to environmental cues based on genetic constraints. Using multiregional exome sequencing, we tested the effect of somatic evolution on intratumoral heterogeneity and its putative clinical implications in adenocarcinomas of the stomach and gastroesophageal junction (GC). Methods: The study comprised a prospective discovery cohort of 9 and a validation cohort of 487 GCs. Multiregional whole-exome sequencing was done using 48 tumor samples (range: 3-10 tumor samples/patient) of the discovery cohort.Results: In total, the discovery cohort harbored 16,537 non-synonymous mutations (mutations/sample: median n=159; mutations/patient: median n=369). Intratumoral heterogeneity of somatic mutations and copy number variants were present in all tumors of the discovery cohort. 53-91% of the non-synonymous mutations were not present in each patient’s sample; 399 genes harbored 2-4 different non-synonymous mutations in the same patient; 175 genes showed copy number variations, the majority being heterogeneous, including CD274 (PD-L1). Phylogenetic analyses provided evidence for branched evolution being the most complex in a microsatellite instable GC. The analysis of the mode of evolution showed a high degree of heterogeneity in deviation from neutrality within each tumor. Studies on the validation cohort showed that the subclonal loss of SMAD4 is an independent predictor for poor patient outcome.Conclusions: Neutral and non-neutral somatic evolution shape the mutational landscape in GC. It leads to complex spatial intratumoral heterogeneity and may have profound effects on patient management. It provides crucial information for an individualized understanding of clinical prognosis and therapeutic options in GC patients.

scholarly journals Multiscale heterogeneity in gastric adenocarcinoma evolution is an obstacle to precision medicine

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Christoph Röcken ◽  
Anu Amallraja ◽  
Christine Halske ◽  
Luka Opasic ◽  
Arne Traulsen ◽  
...  
Keyword(s):  
Precision Medicine ◽  
Copy Number ◽  
Intratumoral Heterogeneity ◽  
Sample Analysis ◽  
Discovery Cohort ◽  
Synonymous Mutations ◽  
Somatic Evolution ◽  
Whole Exome ◽  
Evolutionary Trajectories ◽  
Node Metastases

Abstract Background Cancer is a somatic evolutionary disease and adenocarcinomas of the stomach and gastroesophageal junction (GC) may serve as a two-dimensional model of cancer expansion, in which tumor subclones are not evenly mixed during tumor progression but rather spatially separated and diversified. We hypothesize that precision medicine efforts are compromised when clinical decisions are based on a single-sample analysis, which ignores the mechanisms of cancer evolution and resulting intratumoral heterogeneity. Using multiregional whole-exome sequencing, we investigated the effect of somatic evolution on intratumoral heterogeneity aiming to shed light on the evolutionary biology of GC. Methods The study comprised a prospective discovery cohort of 9 and a validation cohort of 463 GCs. Multiregional whole-exome sequencing was performed using samples form 45 primary tumors and 3 lymph node metastases (range 3–10 tumor samples/patient) of the discovery cohort. Results In total, the discovery cohort harbored 16,537 non-synonymous mutations. Intratumoral heterogeneity of somatic mutations and copy number variants were present in all tumors of the discovery cohort. Of the non-synonymous mutations, 53–91% were not present in each patient’s sample; 399 genes harbored 2–4 different non-synonymous mutations in the same patient; 175 genes showed copy number variations, the majority being heterogeneous, including CD274 (PD-L1). Multi-sample tree-based analyses provided evidence for branched evolution being most complex in a microsatellite instable GC. The analysis of the mode of evolution showed a high degree of heterogeneity in deviation from neutrality within each tumor. We found evidence of parallel evolution and evolutionary trajectories: different mutations of SMAD4 aligned with different subclones and were found only in TP53 mutant GCs. Conclusions Neutral and non-neutral somatic evolution shape the mutational landscape in GC along its lateral expansions. It leads to complex spatial intratumoral heterogeneity, where lymph node metastases may stem from different areas of the primary tumor, synchronously. Our findings may have profound effects on future patient management. They illustrate the risk of mis-interpreting tumor genetics based on single-sample analysis and open new avenues for an evolutionary classification of GC, i.e., the discovery of distinct evolutionary trajectories which can be utilized for precision medicine.

scholarly journals Whole-exome sequencing reveals a novel homozygous mutation in the COQ8B gene associated with nephrotic syndrome

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mohd Fareed ◽  
Vikas Makkar ◽  
Ravi Angral ◽  
Mohammad Afzal ◽  
Gurdarshan Singh
Keyword(s):  
Nephrotic Syndrome ◽  
Disease Management ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Homozygous Mutation ◽  
Consanguineous Family ◽  
Clinical Prognosis ◽  
Whole Exome ◽  
Recurrent Mutations ◽  
Ckd Stage

AbstractNephrotic syndrome arising from monogenic mutations differs substantially from acquired ones in their clinical prognosis, progression, and disease management. Several pathogenic mutations in the COQ8B gene are known to cause nephrotic syndrome. Here, we used the whole-exome sequencing (WES) technology to decipher the genetic cause of nephrotic syndrome (CKD stage-V) in a large affected consanguineous family. Our study exposed a novel missense homozygous mutation NC_000019.9:g.41209497C > T; NM_024876.4:c.748G > A; NP_079152.3:p.(Asp250Asn) in the 9th exon of the COQ8B gene, co-segregated well with the disease phenotype. Our study provides the first insight into this homozygous condition, which has not been previously reported in 1000Genome, ClinVar, ExAC, and genomAD databases. In addition to the pathogenic COQ8B variant, the WES data also revealed some novel and recurrent mutations in the GLA, NUP107, COQ2, COQ6, COQ7 and COQ9 genes. The novel variants observed in this study have been submitted to the ClinVar database and are publicly available online with the accessions: SCV001451361.1, SCV001451725.1 and SCV001451724.1. Based on the patient's clinical history and genomic data with in silico validation, we conclude that pathogenic mutation in the COQ8B gene was causing kidney failure in an autosomal recessive manner. We recommend WES technology for genetic testing in such a consanguineous family to not only prevent the future generation, but early detection can help in disease management and therapeutic interventions.

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 > 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 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.

1265The results of whole exome sequencing in patients with bradyarrhythmias

EP Europace ◽  
2020 ◽  
Vol 22 (Supplement_1) ◽  
Author(s):  
E Polyakova ◽  
N Shcherbakova
Keyword(s):  
Family History ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Heart Diseases ◽  
Stress Test ◽  
Family Members ◽  
Pacemaker Implantation ◽  
Structural Heart Disease ◽  
Clinical Prognosis ◽  
Whole Exome

Abstract Introduction. Sick sinus syndrome (SSS) and atrioventricular block (AVB) are life-threatening cardiac arrhythmias, that sometimes can manifest itself with syncope and needs a pacemaker implantation even in children. Sometimes, SSS and AVB are accompanied by structural heart diseases such as septal defects, cardiomyopathies, but often the heart is structurally normal. Some genes associated with bradyarrhythmias are well known. At the same time, the etiology of the SSS is unidentified and may be genetic caused in 50% of patients with SSS. There are no studies on the prevalence of with bradyarrhythmia-associated mutations in children. The purpose of our work is to identify and study the types of mutations associated with SSS and AVB in children. Methods. We included in the study 15 patients (27% boys) with severe SSS and AVB, from the database of the Russian Pediatric Arrhythmia Center. 11 were the probands and 4 - family members.  Personal and family history, physical examination, including ECG, stress test, Holter monitoring, ECHO and other tests, and whole exome sequencing were made. The average age was 14.1 ± 4.5 (from 2 to 17). Results.  In 30% (5 pts) there was the combination of with bradyarrhythmias and structural heart disease. 7 pts (47%) had syncope, 4 pacemakers were implanted. 10 children (67%) had the genetic variants of genes associated with SSS and AVB: SCN5A, TNNI3K, KCNA5, TRPM4, ANK2 and others. Family history of cardiac diseases was positive in 5 probands; 2 probands had family members with implanted pacemakers. In 3 pts were likely pathogenic variants and in 7 pts - variants of unknown significance found. Conclusion.  We found the genetic cause of bradyarrhythmias in 67% of children. Further research and larger patient samples are required to study the prevalence of genetic types of and show the correlation of the genotype with the clinical prognosis. In addition, our work will enable practitioners to identify children from families with family forms of SSS, AVB and sudden cardiac death. Further research can help us determine the criteria for selecting children for genetic testing.

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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