scholarly journals Next-generation sequencing of adrenocortical carcinoma reveals new routes to targeted therapies

2014 ◽  
Vol 67 (11) ◽  
pp. 968-973 ◽  
Author(s):  
J S Ross ◽  
K Wang ◽  
J V Rand ◽  
L Gay ◽  
M J Presta ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Adrenocortical Carcinoma ◽  
Single Case ◽  
Next Generation ◽  
Illumina Hiseq ◽  
Modest Improvement ◽  
Aggressive Form ◽  
Comprehensive Genomic Profiling ◽  
Cytotoxic Therapies ◽  
Generation Sequencing

AimsAdrenocortical carcinoma (ACC) carries a poor prognosis and current systemic cytotoxic therapies result in only modest improvement in overall survival. In this retrospective study, we performed a comprehensive genomic profiling of 29 consecutive ACC samples to identify potential targets of therapy not currently searched for in routine clinical practice.MethodsDNA from 29 ACC was sequenced to high, uniform coverage (Illumina HiSeq) and analysed for genomic alterations (GAs).ResultsAt least one GA was found in 22 (76%) ACC (mean 2.6 alterations per ACC). The most frequent GAs were in TP53 (34%), NF1 (14%), CDKN2A (14%), MEN1 (14%), CTNNB1 (10%) and ATM (10%). APC, CCND2, CDK4, DAXX, DNMT3A, KDM5C, LRP1B, MSH2 and RB1 were each altered in two cases (7%) and EGFR, ERBB4, KRAS, MDM2, NRAS, PDGFRB, PIK3CA, PTEN and PTCH1 were each altered in a single case (3%). In 17 (59%) of ACC, at least one GA was associated with an available therapeutic or a mechanism-based clinical trial.ConclusionsNext-generation sequencing can discover targets of therapy for relapsed and metastatic ACC and shows promise to improve outcomes for this aggressive form of cancer.

Next-generation sequencing yields promising targets in advanced cholangiocarcinoma (CCA).

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 209-209
Author(s):  
Rachna T. Shroff ◽  
Chaitanya Churi ◽  
Asif Rashid ◽  
Lopa Mishra ◽  
Mingxin Zuo ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Kras Mutation ◽  
Cancer Center ◽  
Next Generation ◽  
Illumina Hiseq ◽  
Md Anderson ◽  
Genetic Profiles ◽  
Next Generation Sequencing Ngs ◽  
Parp 1 ◽  
Generation Sequencing

209 Background: The incidence of CCA is rising and the clinical efficacy of systemic therapy is suboptimal. Next generation sequencing (NGS) technology offers potential for targeted therapeutics against genetically heterogenous solid tumors including cholangiocarcinoma (CCA). Methods: DNA was extracted from biopsy specimens of 61 patients (pts) with CCA seen at MD Anderson Cancer Center Houston, TX. DNA sequencing was performed for 3,769 exons of 236 cancer-related genes plus 47 introns from 19 genes to an average depth of 1000X using the Illumina HiSeq 2000 platform (performed by Foundation Medicine, Cambridge, MA). Results: 174genomic alterations (GA) were identified from 61 pt samples with an average of 2.85 GAs/pt (range 0-10). GAs identified were mutations (75%), amplifications (16%), loss/deletions (7%) and others (2%). 6 (10%) tumors showed no GAs. Most frequent GAs were TP53 (34%), KRAS (30%), ARID1A (15%), PBRM1 (11%), BAP1 (8%), ERBB2 (8%), FBXW7 (8%), SMAD4 (8%) and IDH1 (8%). ERBB2 GAs included 4 mutations and 1 amplification. KRAS mutation was associated with a statistically significant reduction in overall survival (OS). Mean OS in pts with KRAS mutation was 32 weeks vs. 63 weeks in KRAS wt (t = -2.126, p = 0.039). Ingenuity Pathway Analysis indicated disruption in cell cycle, proliferation, development, death and DNA repair pathways. Targetable signaling pathways from this study are described in the Table. Targetable GAs were noted in 62% of pts. These are potentially targetable by inhibitors to ERBB2, FGF, mTOR, MEK, BRAF and PARP-1. Conclusions: These data are the single largest compilation of NGS analysis on CCA pts and demonstrate the range of GAs that are eligible for investigational targeted therapies. These results can be used as a basis to develop personalized treatments for CCA pts based on individual genetic profiles. [Table: see text]

A novel homozygous frameshift mutation in the FUCA1 gene causes both severe and mild fucosidosis

2018 ◽  
Vol 71 (9) ◽  
pp. 821-824 ◽  
Author(s):  
Nasrollah Saleh-Gohari ◽  
Kolsoum Saeidi ◽  
Roya Zeighaminejad
Keyword(s):  
Next Generation Sequencing ◽  
Frameshift Mutation ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Gene Mutations ◽  
Next Generation ◽  
Lysosomal Storage ◽  
Illumina Hiseq ◽  
Almost All ◽  
Generation Sequencing

AimsFucosidosis is a rare autosomal recessive lysosomal storage disorder caused by α-L-fucosidase deficiency as a result of FUCA1 gene mutations. Here, we studied clinical features and the molecular basis of fucosidosis in a family from Iran, including two probands and nine family members.MethodsDNA sample of two probands were screened for gene defects using a next generation sequencing technique. The sequencing processes were performed on an Illumina Hiseq 4000 platform. Sequence reads were analysed using BWA-GATK.ResultsNext generation sequencing revealed a frameshift mutation caused by 2 bp deletion (c.837_838 delTG; p.Cys279) in the FUCA1 gene. The identified mutation was tested in all participants. Homozygous patients had almost all the complications associated with fucosidosis, while heterozygous carriers were unaffected.ConclusionsThe variant c.837_838 delTG; p.Cys279 has not been reported previously and is predicted to be pathogenic due to a premature stop codon.

scholarly journals Reliability of genomic variants across different next-generation sequencing platforms and bioinformatic processing pipelines

2020 ◽  
Author(s):  
Susanne Gerber ◽  
Stephan Weißbach ◽  
Stanislav Jur`Evic Sys ◽  
Charlotte Hewel ◽  
Hristo Todorov ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Allele Frequency ◽  
Low Complexity ◽  
Next Generation ◽  
Illumina Hiseq ◽  
Cross Sectional ◽  
Genomic Annotation ◽  
Complete Genomics ◽  
Sequencing Platforms ◽  
Generation Sequencing

Abstract Background Next Generation Sequencing (NGS) is the fundament of various studies providing insights into questions from biology and medicine. Nevertheless, integrating data from different experimental backgrounds can introduce strong biases. In order to methodically investigate the magnitude of systematic errors, we performed a cross-sectional observational study on a genomic cohort of 99 subjects each sequenced via (i) Illumina HiSeq X, (ii) Illumina HiSeq and (iii) Complete Genomics. Consequently, we systematically analyzed the heterogeneity between the sequencing cohorts with respect to genomic annotation and common filter criteria like minimum allele frequency (MAF). Results The number of detected variants/variant classes per individual was highly dependent on the sequencing technology. We observed a statistically significant overrepresentation of variants uniquely called by a single platform which indicates potential systematic biases. These variants were enriched in low complexity genomic regions and simple repeats. Furthermore, estimates of allele frequency were highly discrepant for a subset of variants in pairwise comparisons between different sequencing platforms. Applying common filters – such as MAF 5% and HWE- greatly reduced the heterogeneity between cohorts but still left discrepancies of several thousand variants after filtering.Conclusion We provide empirical evidence of systematic heterogeneity in variant calls between alternative experimental and data analysis setups. Our results highlight the potential benefit of reprocessing genomic data with harmonized pipelines when integrating data from different studies.

Molecular variations in uterine carcinosarcomas identify therapeutic opportunities

2020 ◽  
Vol 30 (4) ◽  
pp. 480-484
Author(s):  
Erin Crane ◽  
Wendel Naumann ◽  
David Tait ◽  
Robert Higgins ◽  
Thomas Herzog ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Protein Expression ◽  
Copy Number ◽  
Treatment Options ◽  
Therapeutic Targets ◽  
Mutational Load ◽  
Next Generation ◽  
Comprehensive Genomic Profiling ◽  
Poor Outcomes ◽  
Generation Sequencing

ObjectiveTo perform comprehensive genomic profiling on a large cohort of patients with uterine carcinosarcomas to identify potential therapeutic targets.MethodsMolecular profiling was conducted on 168 retrospectively de-identified patients with uterine carcinosarcomas using the Caris Life Sciences platform. Specimens were evaluated for aberrations in protein expression by immunohistochemistry, DNA sequence mutation using a 592-gene next generation sequencing panel, copy number amplification using next generation sequencing or in situ hybridization, and fusion events using NextGen RNA sequencing. Tumor mutational load and microsatellite instability were also evaluated.ResultsWe identified 168 patients with uterine carcinosarcoma; median age of the cohort was 67 years. The most common mutations were observed in the following genes: TP53 (86%), PIK3CA (34%), FBXW7 (23%), PTEN (18%), KRAS (16%), PPP2R1A (10%). Tumor mutational load was low to moderate in most cases (50% and 45%, respectively). HER2/neu (ERBB2) was amplified in 9% of tumors. Immunohistochemistry protein expression was elevated in TOP2A (95%), TS (80%), PTEN (76%), and TUBB3 (66%). Mismatch repair deficiency was rare (4%).ConclusionsMultiple somatic mutations and copy number alterations in genes that are therapeutic targets were identified in half of cases. Uterine carcinosarcomas represent an aggressive histology with limited treatment options and poor outcomes, and clinical trials are needed to validate new therapeutic targets.

Targeted Next Generation Sequencing of the Five Hemochromatosis Genes in Italian Patients with Iron Overload and Non-Diagnostic First Level Genetic Test: A Pilot Study

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4030-4030
Author(s):  
Sadaf Badar ◽  
Fabiana Busti ◽  
Giampiero Zamperin ◽  
Alberto Ferrarini ◽  
Paolo Bozzini ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Pilot Study ◽  
Iron Overload ◽  
Genetic Test ◽  
Conflicts Of Interest ◽  
Hereditary Hemochromatosis ◽  
Autosomal Recessive Disorder ◽  
Next Generation ◽  
Illumina Hiseq ◽  
Generation Sequencing

Abstract Background and Aim: Molecular diagnosis of HFE-related hereditary hemochromatosis (HH) is typically made by searching for the C282Y and H63D mutations (first level genetic test). However, in the Mediterranean area up to one third of patients with HH phenotype do not have the “diagnostic” genotypes (C282Y homozygosity, or C282Y/H63D compound heterozygosity). This pilot study was designed to develop a “second level” next generation sequencing (NGS)-based test for rapid and simultaneous analysis of the five HH genes (HFE, HFE2, HAMP, TFR2 and SLC40A1). Methodology: we studied 61 patients with relevant biochemical signs of iron overload (IO) and non-diagnostic first level genetic test suggesting a possible “non-HFE” HH. The five HH genes were captured by Halo-Plex™ technology, and then sequenced using aNGS platform (Illumina HiSeq 1000). Sequenced reads were aligned against human reference HG19 and analyzed by GoldenHelix™ software to annotate all the variants possibly involved in the disease. Results: In IO patients a large number of new non-synonymous variants (according to bioinformatics tools based on publicly available databases including the 1000-genomes project) were found. Many of them were relatively frequent and detected also in controls, thus being considered likely “non-pathogenic”, unless clearly enriched in patients. On the other hand, some rare variants (i.e. limited to a single or very few individuals), particularly in SCL40A1, TFR2, and HFE, were found exclusively in patients, and could be considered “potentially pathogenic”. Conclusions: The combination of the Halo-Plex™ approach with NGS platform and GoldenHelix™ algorithm appears a suitable approach for a better molecular characterization of patients with unexplained HH phenotype, and could represent a good option for second level genetic testing in referral centers. However, establishing the clinical relevance of NGS-detected “novel” genetic variants in a prevalently autosomal recessive disorder like HH remains a difficult task, requiring further functional studies and national/international collaborative efforts. Disclosures No relevant conflicts of interest to declare.

High concordance for MammaPrint 70 genes by RNA next generation sequencing.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 3065-3065
Author(s):  
Lorenza Mittempergher ◽  
Iris de Rink ◽  
Marja Nieuwland ◽  
Ron M Kerkhoven ◽  
Annuska Glas ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rna Sequencing ◽  
Pearson Correlation ◽  
Microarray Gene Expression Data ◽  
Good Prognosis ◽  
Rna Seq ◽  
Next Generation ◽  
Illumina Hiseq ◽  
Microarray Gene Expression ◽  
Generation Sequencing

3065 Background: The development of new biomarkers often requires fresh frozen (FF) samples. Recently we showed that microarray gene expression data generated from FFPE material are comparable to data extracted from the FF counterpart, including known signatures such as the 70-gene prognosis signature (Mittempergher L et al., 2011). As described by Luo et al (2010) RNA profiling using next generation sequencing (RNA-Seq) is now applicable to archival FFPE specimens. Methods: Technical performance and the comparison between the RNA-Seq 70-gene read-out and the MammaPrint test (Glas et al., 2006) is evaluated in a series of 15 patients (11/15 with matched FFPE/FF material). RNA-Seq was carried out using minor adjustments of the Illumina TruSeq RNA preparation method. RNA sequencing libraries were prepared starting from 100ng of total RNA. Next, the DSN (Duplex-Specific Nuclease) normalization process was used to remove ribosomal RNA and other abundant transcripts (Luo et al, 2010). The libraries were paired-end sequenced on the Illumina HiSeq 2000 instrument with multiplexing of 4 libraries per lane. The resulting sequences were mapped to the human reference genome (build 37) using TopHat 1.3.1(Trapnell et al., 2009). The HTSeq-count tool was used to generate the total number of uniquely mapped reads for each gene. Results: Between 14% and 45% of the total number of reads were assigned to protein-coding genes. The minimum coverage per 1000bp of CDS was 38 reads. The 70 MammaPrint genes were successfully mapped to the RNA-Seq transcripts. We calculated the Pearson correlation coefficient between the centroids of the original good prognosis template (van’t Veer et al., 2002) and the 70-gene read count determined by RNA-Seq of each sample. Predictions based on the 70-gene RNA-Seq data showed a high agreement with the actual MammaPrint test predictions (>90%), irrespective of whether the RNA-seq was performed on FF or FFPE tissue. Conclusions: New generation RNA-sequencing is a feasible technology to assess diagnostic signatures.

scholarly journals Verification of Underlying Genetic Cause in a Cohort of Russian Patients with Familial Hypercholesterolemia Using Targeted Next Generation Sequencing

2020 ◽  
Vol 7 (2) ◽  
pp. 16 ◽  
Author(s):  
Anna E. Semenova ◽  
Igor V. Sergienko ◽  
Diego García-Giustiniani ◽  
Lorenzo Monserrat ◽  
Anna B. Popova ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Total Cholesterol ◽  
Familial Hypercholesterolemia ◽  
Next Generation ◽  
Illumina Hiseq ◽  
Targeted Next Generation Sequencing ◽  
Cholesterol Levels ◽  
Pathogenic Mutations ◽  
Number Of Patients ◽  
Generation Sequencing

Russian patients with familial hypercholesterolemia (FH) were screened for pathogenic mutations using targeted next generation sequencing. Genetic testing was performed in 52 probands with definite or probable FH based on the Dutch lipid clinic network criteria (DLCN score ≥ 6). Blood samples were studied by massive parallel sequencing (Illumina HiSeq 1500 platform) using a custom capture library related to dyslipidemia and premature atherosclerosis. Mutations considered to be responsible for monogenic FH were identified in 48% of the probands: 24 with mutations in the LDLR gene and two with a mutation in the APOB gene. There were 22 pathogenic/likely pathogenic mutations in LDLR, eight of which have not been previously described in the literature. Four patients with a clinical picture of homozygous FH had two heterozygous LDLR mutations. Although mutation-negative patients had highly elevated total cholesterol and low-density lipoprotein cholesterol levels, only half of them had a family history of hypercholesterolemia. With respect to heterozygous FH, mutation-positive patients had higher maximum total cholesterol levels (p = 0.01), more severe carotid atherosclerotic lesions, and a higher percentage of premature peripheral artery disease (p = 0.03) than mutation-negative ones. However, the number of patients who suffered from myocardial infarction was similar between the two groups.

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