scholarly journals Comprehensive Molecular Profiling of Idiopathic Erythrocytosis By Genetic Ancestry

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 945-945
Author(s):  
Caitlin O'Neill ◽  
Zarko Manojlovic ◽  
Ah-Reum Jeong ◽  
Yili Xu ◽  
Yuxin Jin ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Copy Number ◽  
Somatic Mutations ◽  
Chromatin Assembly ◽  
Whole Exome ◽  
Mutation Burden ◽  
History Of ◽  
Generation Sequencing ◽  
Idiopathic Erythrocytosis

Introduction: Idiopathic erythrocytosis (IE) is characterized by a persistently elevated hemoglobin, equivocal erythropoietin (EPO) levels, absence of janus kinase 2 (JAK2) mutations suggestive of polycythemia vera (PV) and no secondary cause. One study used a targeted 21 gene next-generation sequencing panel and identified novel variants in known erythrocytosis-related genes as well as novel genes associated with the oxygen-sensing pathway. However, expanded sequencing of blood and matched tissue samples in a large ethnically diverse group of IE patients has not been performed. Methods: All patients signed informed consent to participate in an observational study approved by the Institutional Review Board; they provide blood and buccal mucosa samples at study entry and at 24-month follow-up. Patients were enrolled if JAK2 testing and a complete work up for secondary causes was negative. They were required to have hemoglobin levels greater than 16 g/dL on two occasions or greater than 15 g/dL if undergoing phlebotomy. Our initial sequencing of 20 IE patients was performed utilizing high resolution whole-exome sequencing of circulating blood samples (disease) at a mean coverage of 390x and matched normal (buccal) samples at mean coverage of 300x. To stratify samples by genetic ancestry, we performed a population stratification principle component analysis (PCA) and STRUCTURE using Ancestry Informative Markers derived from 1K Genome Phase1_v3 Exome database. The primary in-silico analysis was performed on the baseline samples from treatment-naïve patients. The whole-exome data was generated in accordance to GATK's best practices with same filters applied as described by Exome Aggregation Consortium. The additional downstream in-silico paired analysis was performed using MutSig2.0 (Mutation Significance) algorithm to determine significant mutations and GISTIC (The Genomic Identification of Significant Targets in Cancer) to identify the significant copy number events, IPA (Ingenuity Pathway Analysis) to determine pathways along with other computational . Results: Median age at baseline was 52 years (range 35-71). Six patients (30%) were female and 14 (70%) were male. Median values and ranges for laboratory parameters at baseline were as follows: WBC 6.6 x 109/L (5-9.7), Hgb 17 g/dL (15.5-19.8), Plt 218 x 109/L (86-374), and EPO level 9.8 IU/L (2-14.3). Three patients had a personal history of malignancy, including 2 with lymphoma. Two patients had a family history of myeloid malignancy (chronic myeloid leukemia and PV). Our ancestry analysis of initial 20 patients with IE identified 6 patients with high European percent ancestry (EUR), 1 patient with high Asian percent ancestry (EAS) and 13 patients with high percent Ad Mixed ancestry (AMR). In our cohort, 60% (12/20) of patients had been also diagnosed with a liver disorder (11 with fatty liver, 1 with cirrhosis) that was not significantly different across populations. We identified, on average, 42 non-silent somatic mutations (not present in the buccal samples) in whole blood across our cohort with no statistical difference (p=0.671) in mutation burden between ancestry groups or between patients with and without liver disease. Age, gender, and ethnicity were not associated with mutation burden. Utilizing MutSig algorithm, we identified a novel candidate gene, CHAF1A, with high mutation prevalence of 30% in patients with IE. Further analysis of mutation landscape identified somatic nonsilent mutations in 25 known oncogenes which were present in at least 10% of patients. Our mutation signatures in IE identified a significant association with failure of double stranded DNA repair. Only one patient had a mutation in TET2. Further analysis of copy number indicated copy number loss in genes such as SETD3 and GSH associated with chromatin assembly which may suggest alterations in chromatin assembly and changes in the epigenome. Our analysis also identified a high number of 9p and 13q gains in patients with IE. Conclusion: In this study, we utilized high-resolution next generation sequencing in association with comprehensive clinical annotation to determine potential molecular drivers of IE in a multi-ethnic population. We identified somatic mutations in a subset of patients which may represent clonal hematopoiesis. Long term follow up of outcomes in this cohort may clarify the significance of these mutations in the pathogenesis of IE. Disclosures No relevant conflicts of interest to declare.

scholarly journals A simple method to estimate the in-house limit of detection for genetic mutations with low allele frequencies in whole-exome sequencing analysis by next-generation sequencing

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Takumi Miura ◽  
Satoshi Yasuda ◽  
Yoji Sato
Keyword(s):  
Next Generation Sequencing ◽  
Allele Frequency ◽  
Somatic Mutations ◽  
Limit Of Detection ◽  
Allele Frequencies ◽  
Genetic Mutations ◽  
Sequencing Data ◽  
Simple Method ◽  
Whole Exome ◽  
Generation Sequencing

Abstract Background Next-generation sequencing (NGS) has profoundly changed the approach to genetic/genomic research. Particularly, the clinical utility of NGS in detecting mutations associated with disease risk has contributed to the development of effective therapeutic strategies. Recently, comprehensive analysis of somatic genetic mutations by NGS has also been used as a new approach for controlling the quality of cell substrates for manufacturing biopharmaceuticals. However, the quality evaluation of cell substrates by NGS largely depends on the limit of detection (LOD) for rare somatic mutations. The purpose of this study was to develop a simple method for evaluating the ability of whole-exome sequencing (WES) by NGS to detect mutations with low allele frequency. To estimate the LOD of WES for low-frequency somatic mutations, we repeatedly and independently performed WES of a reference genomic DNA using the same NGS platform and assay design. LOD was defined as the allele frequency with a relative standard deviation (RSD) value of 30% and was estimated by a moving average curve of the relation between RSD and allele frequency. Results Allele frequencies of 20 mutations in the reference material that had been pre-validated by droplet digital PCR (ddPCR) were obtained from 5, 15, 30, or 40 G base pair (Gbp) sequencing data per run. There was a significant association between the allele frequencies measured by WES and those pre-validated by ddPCR, whose p-value decreased as the sequencing data size increased. By this method, the LOD of allele frequency in WES with the sequencing data of 15 Gbp or more was estimated to be between 5 and 10%. Conclusions For properly interpreting the WES data of somatic genetic mutations, it is necessary to have a cutoff threshold of low allele frequencies. The in-house LOD estimated by the simple method shown in this study provides a rationale for setting the cutoff.

Multifocal origin of occupational cholangiocarcinoma revealed by comparison of multilesion mutational profiles

2019 ◽  
Vol 41 (3) ◽  
pp. 368-376 ◽  
Author(s):  
Sachiyo Mimaki ◽  
Masahiko Watanabe ◽  
Masahiko Kinoshita ◽  
Riu Yamashita ◽  
Hiroshi Haeno ◽  
...  
Keyword(s):  
Bile Ducts ◽  
Somatic Mutations ◽  
Precancerous Lesions ◽  
Biliary Tree ◽  
Bile Duct Injuries ◽  
Whole Exome ◽  
Mutation Burden ◽  
Invasive Carcinomas ◽  
Occupational Cholangiocarcinoma

Abstract Recently identified occupational cholangiocarcinoma among printing workers is characterized by chronic bile duct injuries and precancerous or early cancerous lesions at multiple sites of the bile ducts. These observations suggested the potential multifocal carcinogenesis of the disease. We performed whole-exome analysis of multiple lesions, including the invasive carcinomas and precancerous lesions of four occupational cholangiocarcinoma cases. A much higher mutation burden was observed in both the invasive carcinomas (mean 76.3/Mb) and precancerous lesions (mean 71.8/Mb) than in non-occupational cholangiocarcinomas (mean 1.6/Mb). Most somatic mutations identified in 11 of 16 lesions did not overlap with each other. In contrast, a unique trinucleotide mutational signature of GpCpY to GpTpY was shared among the lesions. These results suggest that most of these lesions are multiclonal in origin and that common mutagenic processes, which may be induced by exposure to haloalkanes or their metabolites, generated somatic mutations at different sites of the bile ducts. A similarly high mutation rate had already been identified in the precancerous lesions, implying an increased potential for carcinogenesis throughout the biliary tree. These genomic features support the importance of ongoing close follow-up of the patients as a group at high risk of recurrence.

scholarly journals Clinical Implications of Copy Number Alteration Detection Using Panel-Based Next-Generation Sequencing Data in Myelodysplastic Syndrome

2020 ◽  
Author(s):  
Yoo-Jin Kim ◽  
SeungHyun Jung ◽  
Eun-Hye Hur ◽  
Eun-Ji Choi ◽  
Kyoo-Hyung Lee ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Myelodysplastic Syndrome ◽  
Somatic Mutation ◽  
Copy Number ◽  
Somatic Mutations ◽  
Clinical Implications ◽  
Next Generation ◽  
Clinical Variables ◽  
Ngs Data ◽  
Generation Sequencing

Abstract Background: Recent advancements in next-generation sequencing (NGS) technologies allow the simultaneous identification of targeted copy number alterations (CNAs) as well as somatic mutations using the same panel-based NGS data. We investigated whether CNAs detected by the targeted NGS data provided additional clinical implications, over somatic mutations, in myelodysplastic syndrome (MDS). Methods: Targeted deep sequencing of 28 well-known MDS-related genes was performed for 266 patients with MDS. Results: Overall, 215 (80.8%) patients were found to have at least one somatic mutation; 67 (25.2%) had at least one CNA; 227 (85.3%) had either a somatic mutation or CNA; 160 had somatic mutations without CNA; and 12 had CNA without somatic mutations. Considering the clinical variables and somatic mutations alone, multivariate analysis demonstrated that sex, revised International Prognostic Scoring System (IPSS-R) and NRAS and TP53 mutations were independent prognostic factors for overall survival. For AML-free survival, these factors were sex, IPSS-R, and mutations in NRAS, DNMT3A, and complex karyotype/TP53 mutations. When we consider clinical variables along with somatic mutations and CNAs, genetic alterations in TET2, LAMB4, U2AF1, and CBL showed additional significant impact on the survivals. Conclusions: Our study suggests that the concurrent detection of somatic mutations and targeted CNAs may provide clinically useful information for the prognosis of MDS patients.

Mutational landscape by targeted next-generation sequencing in EBV-associated lymphoepithelioma-like cholangiocarcinoma.

2019 ◽  
Vol 37 (4_suppl) ◽  
pp. 269-269
Author(s):  
Nai-Jung Chiang ◽  
Ming-Huang Chen ◽  
Li-Tzong Chen ◽  
Shan Yanshen
Keyword(s):  
Next Generation Sequencing ◽  
Peripheral Blood ◽  
Copy Number ◽  
Somatic Mutations ◽  
Tumor Tissue ◽  
Germline Mutations ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Generation Sequencing ◽  
Cancer Panel

269 Background: Lymphoepithelioma-like cholangiocarcinoma (LELCC) is a rare variant of intrahepatic cholangiocarcinoma, which is highly associated with EBV infection and abundant lymphoplasmacytic cell infiltration. However, there’s limited data of genetic background in LELCC. Therefore, we want to explore the mutation profiles of LELCC, as well as copy number variations. Methods: Five patients’ tumor tissues diagnosed as LELCC, with positive EBER expression were retrospectively collected and microscopically dissected. Of them, two patients’ peripheral blood mononuclear cell served as background of germline mutations. Targeted next-generation sequencing was performed using the ACTOnco Comprehensive Cancer Panel (Ion AmpliSeq Comprehensive Cancer Panel, Life Technologies) to target all coding exons of 409 cancer-related genes for analysis of tumor tissue and blood. PDL1 immunohistochemistry staining with 22C3 antibody pharmDx was applied. Results: All EBV-associated LELCC showed positive expression of PDL1 staining, with combined positive score from 5% to 30%. Both somatic and germline mutations can be detected in LELCC tissue because diffuse infiltration of lymphocytes over tumor. After adjusting for background germline mutation frequency from peripheral blood, only mutations with allele frequency less than or around 10% were considered as somatic mutations. Overall, 10 nonsynonymous somatic mutations were detected in 4 (80%) patients with a range of 1-5 mutations per sample. Mutations were identified including BARD1, EPHA5, MUC16, TNFAIP3, CD19, PTEN, TET1, RECQL4, CD79B, and KDM5A. Copy number changes were rare in this special population. Interesting, one patient who failed to gemcitabine plus cisplatin got partial response after anti-PD1 inhibitor treatment. Conclusions: LELCC is highly correlated with PDL1 expression in tumor and immune cells. It’s necessary to have both tumor tissue and peripheral blood for next-generation sequencing to identify truly somatic mutations in LELCC.

scholarly journals Next-Generation Sequencing Reveals Molecular Heterogeneity in Primary Testicular DLBCL

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1426-1426
Author(s):  
Shuozi Liu ◽  
Ping Yang ◽  
Jing Wang ◽  
Weilong Zhang ◽  
Hongmei Jing
Keyword(s):  
Next Generation Sequencing ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Somatic Mutations ◽  
Functional Enrichment ◽  
Venn Diagram ◽  
Next Generation ◽  
Pathogenic Genes ◽  
Whole Exome ◽  
Generation Sequencing

Abstract Background: Primary testicular lymphomas (PTLs) are extra-nodal large B-cell lymphomas (LBCLs) that respond poorly to current empirical treatment options. To date, no PTL whole-exome sequencing analysis based on the Chinese population has been reported. The main objective of this study was to explore the genomic landscape of PTDLBCL, correlating the results with clinical, histological, and immunogenetic features. Methods: We retrospectively analyzed the disease characteristics in a series of 20 patients (13 paired data and 7 unpaired data). In all cases, the diagnosis of PTL was made using appropriate diagnostic criteria for the 2008 WHO classification of lymphoid tumors with combinations of histologic, immunohistochemical, flow cytometric, and genetic evaluation. Medical records were reviewed for demographic and clinical data. We performed whole-exome next-generation sequencing (NGS) and mapped the mutational landscape. Based on the differential gene results, we performed KEGG and GO enrichment analysis, and five potential new pathogenic genes for DLBCL were obtained based on the Polyphen score. Results: A total of 20 DLBCL samples were collected. All samples were subjected to whole-exome sequencing (WES). A standard bioinformatics analysis process was used to identify somatic mutations in each sample, and an average of 1200 somatic mutations was identified, involving an average of 420 mutated genes. (Figure 1.) More than 85% of the somatic mutations were found in only 1 sample, indicating that somatic mutations are highly heterogeneous. In the matched 13 cases, we validated common gene mutations that have been reported, including PIM1 53.84% (7/13), CD79B 38.46% (5/13), CDKN2A 15.38% (2/13), and MYD88 15.38% (2/13). Venn diagram shows that 25% of the mutated genes were specific to the relapsed sample. GO analysis (Figure 2) shows the functional enrichment and differences between relapsed and non-relapsed samples. Non-relapsed samples are enriched in the herpes simplex virus 1 infection pathway. (Figure 3). Focusing on relapsed/non-relapsed samples, we identified 12 mutated genes specific to relapsed samples. Mutation Polyphen scores were calculated to assess mutation deleteriousness. Five potential new pathogenic genes for DLBCL were obtained. Conclusions: Based on whole-sequencing data, we validated the previously reported common genes for PTDLBCL. In addition, we performed GO and KEGG analysis according to the relapsed and non-relapsed groups. In addition, based on the difference in enrichment results, 12 significantly enriched mutated genes in relapsed DLBCL samples are obtained, and mutation Polyphen scores are calculated to assess mutation deleteriousness. And 5 potential new pathogenic genes of DLBCL are identified. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Genomic alterations caused by HPV integration in a cohort of Chinese endocervical adenocarcinomas

2021 ◽  
Author(s):  
Wenhui Li ◽  
Wanjun Lei ◽  
Xiaopei Chao ◽  
Xiaochen Song ◽  
Yalan Bi ◽  
...  
Keyword(s):  
Copy Number ◽  
Somatic Mutations ◽  
Hpv Infection ◽  
Copy Number Variations ◽  
Cervical Adenocarcinoma ◽  
Structural Variations ◽  
Driver Genes ◽  
Genetic Changes ◽  
Genomic Changes ◽  
Whole Exome

AbstractThe association between human papillomavirus (HPV) integration and relevant genomic changes in uterine cervical adenocarcinoma is poorly understood. This study is to depict the genomic mutational landscape in a cohort of 20 patients. HPV+ and HPV− groups were defined as patients with and without HPV integration in the host genome. The genetic changes between these two groups were described and compared by whole-genome sequencing (WGS) and whole-exome sequencing (WES). WGS identified 2916 copy number variations and 743 structural variations. WES identified 6113 somatic mutations, with a mutational burden of 2.4 mutations/Mb. Six genes were predicted as driver genes: PIK3CA, KRAS, TRAPPC12, NDN, GOLGA6L4 and BAIAP3. PIK3CA, NDN, GOLGA6L4, and BAIAP3 were recognized as significantly mutated genes (SMGs). HPV was detected in 95% (19/20) of patients with cervical adenocarcinoma, 7 of whom (36.8%) had HPV integration (HPV+ group). In total, 1036 genes with somatic mutations were confirmed in the HPV+ group, while 289 genes with somatic mutations were confirmed in the group without HPV integration (HPV− group); only 2.1% were shared between the two groups. In the HPV+ group, GOLGA6L4 and BAIAP3 were confirmed as SMGs, while PIK3CA, NDN, KRAS, FUT1, and GOLGA6L64 were identified in the HPV− group. ZDHHC3, PKD1P1, and TGIF2 showed copy number amplifications after HPV integration. In addition, the HPV+ group had significantly more neoantigens. HPV integration rather than HPV infection results in different genomic changes in cervical adenocarcinoma.

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