Clinical Validation of a Next-Generation Sequencing Screen for Mutational Hotspots in 46 Cancer-Related Genes

Singh, Rajesh R.; Patel, Keyur P.; Routbort, Mark J.; Reddy, Neelima G.; Barkoh, Bedia A.; Handal, Brian; Kanagal‐Shamanna, Rashmi; Greaves, Wesley O.; Medeiros, L. Jeffrey; Aldape, Kenneth; Luthra, Rajyalakshmi

doi:10.1016/j.jmoldx.2013.05.003

Cited by 295 publications

(248 citation statements)

References 23 publications

(19 reference statements)

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“…In addition to adequate depth of coverage, it was also imperative we generated sensitive and specific data; NGS can produce erroneous results secondary to chemistry sequencing errors, 29 formalin-fixation artifacts, 30 or suboptimal coverage, alignment, and/or variant calling. 31 We collected and tested samples from multiple clinically validated assays to ensure that we generated comparable results…”

Section: Detection Of Somatic Mutations In Ffpe Tumor Samplesmentioning

confidence: 99%

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies

Fisher

Zhang

Wang

et al. 2016

The Journal of Molecular Diagnostics

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We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated !500Â coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at !5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n Z 27), deletions (n Z 5), insertions (n Z 3), and multinucleotide variants (n Z 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/ 80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines. (J Mol Diagn 2016, 18: 299e315; http:// dx

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Section: Detection Of Somatic Mutations In Ffpe Tumor Samplesmentioning

confidence: 99%

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies

Fisher

Zhang

Wang

et al. 2016

The Journal of Molecular Diagnostics

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“…55,56 GNAQ mutation analysis was performed using DNA extracted from unstained formalin fixed, paraffin-embedded tissue sections. We tested for mutations in exon 5, codon 209 of the GNAQ gene using Sanger sequencing.…”

Section: Mutation Analysismentioning

confidence: 99%

Melanoma arising in association with blue nevus: a clinical and pathologic study of 24 cases and comprehensive review of the literature

et al. 2014

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Melanomas arising in association with blue nevi or mimicking cellular blue nevi comprise a relatively rare and heterogeneous group of melanomas. It remains controversial which prognostic indicators predictive of outcome in conventional cutaneous melanomas are applicable to this type of melanoma. Here, we describe the clinical and histopathologic features of 24 melanomas arising in association with blue nevi and correlate these with clinical outcome. The mean patient age was 49 years (range: 23-85) with a slight female predominance (15 females:9 males). The most common anatomic locations included the head and neck region (50%), the trunk (21%), and the buttock/sacrococcygeum (17%). Histologically, the tumors were typically situated in the mid to deep dermis with variable involvement of the subcutis, but uniformly lacked a prominent intraepithelial component. The mean tumor thickness (defined as either the standard Breslow thickness or, if not available due to the lack of orientation or lack of epidermis, the largest tumor dimension) was 20.9 mm (range: 0.6-130 mm). The mean mitotic figure count was 6.5/mm 2 (range: 1-30/mm 2 ). Perineural invasion was common (38%). Followup was available for 21 cases (median 2.1 years). The median overall survival, recurrence-free survival, time to local recurrence, and time to distant recurrence were 5.2, 0.7, 2.6, and 1.6 years, respectively. Logistic regression analyses demonstrated a significant association between tumor thickness and recurrence-free survival (hazard ratio ¼ 1.02 per mm; P ¼ 0.04) and reduced time to distant metastasis (hazard ratio ¼ 1.03 per mm; P ¼ 0.02) with a similar trend toward reduced time to local recurrence (hazard ratio ¼ 1.02 per mm; P ¼ 0.07). No other parameters (age, anatomic location, mitotic figures, lymphovascular or perineural invasion, or type of associated blue nevus) emerged as significant. In addition, we provide a comprehensive review of 109 cases of melanoma blue nevus type described in the English literature and summarize our findings in this context.

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“…The AmpliSeq Cancer Panel queries 739 known mutations in 46 well characterized cancer genes listed in the COSMIC database [18]. 200 ng of genomic DNA from each specimen was used to generate uniquely barcoded sequencing libraries.…”

Section: Ion Torrent Ampliseq Cancer Panelmentioning

confidence: 99%

Mutation and Transcriptional Profiling of Formalin-Fixed Paraffin Embedded Specimens as Companion Methods to Immunohistochemistry for Determining Therapeutic Targets in Oropharyngeal Squamous Cell Carcinoma (OPSCC): A Pilot of Proof of Principle

Saba

Wilson

Doho

et al. 2014

Head and Neck Pathol

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The role of molecular methods in the diagnosis of head and neck cancer is rapidly evolving and holds great potential for improving outcomes for all patients who suffer from this diverse group of malignancies. However, there is considerable debate as to the best clinical approaches, particularly for Next Generation Sequencing (NGS). The choices of NGS methods such as whole exome, whole genome, whole transcriptomes (RNA-Seq) or multiple gene resequencing panels, each have strengths and weakness based on data quality, the size of the data, the turnaround time for data analysis, and clinical actionability. There have also been a variety of gene expression signatures established from microarray studies that correlate with relapse and response to treatment, but none of these methods have been implemented as standard of care for oropharyngeal squamous cell carcinoma (OPSCC). Because many genomic methodologies are still far from the capabilities of most clinical laboratories, we chose to explore the use of a combination of off the shelf targeted mutation analysis and gene expression analysis methods to complement standard anatomical pathology methods. Specifically, we have used the Ion Torrent AmpliSeq cancer panel in combination with the NanoString nCounter Human Cancer Reference Kit on 8 formalin-fixed paraffin embedded (FFPE) OPSCC tumor specimens, (4) HPVpositive and (4) HPV-negative. Differential expression analysis between HPV-positive and negative groups showed that expression of several genes was highly likely to correlate with HPV status. For example, WNT1, PDGFA and OGG1 were all over-expressed in the positive group. Our results show the utility of these methods with routine FFPE clinical specimens to identify potential therapeutic targets which could be readily applied in a clinical trial setting for clinical laboratories lacking the instrumentation or bioinformatics infrastructure to support comprehensive genomics workflows. To the best of our knowledge, these preliminary experiments are among the earliest to combine both mutational and gene expression profiles using Ion Torrent and NanoString technologies. This reports serves as a proof of principle methodology in OPSCC.Electronic supplementary material The online version of this article

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Clinical Validation of a Next-Generation Sequencing Screen for Mutational Hotspots in 46 Cancer-Related Genes

Cited by 295 publications

References 23 publications

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies

Melanoma arising in association with blue nevus: a clinical and pathologic study of 24 cases and comprehensive review of the literature

Mutation and Transcriptional Profiling of Formalin-Fixed Paraffin Embedded Specimens as Companion Methods to Immunohistochemistry for Determining Therapeutic Targets in Oropharyngeal Squamous Cell Carcinoma (OPSCC): A Pilot of Proof of Principle

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