Oncomine comprehensive assay v3

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Oncomine Comprehensive Assay v3 is a targeted next-generation sequencing (NGS) panel designed to detect variants in a comprehensive set of genes associated with solid tumors. The assay covers over 500 unique genes and enables the detection of single nucleotide variants, small insertions and deletions, copy number variations, and select gene fusions. The assay is intended for use with the Ion Torrent platform.

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30 protocols using oncomine comprehensive assay v3

Comprehensive Cancer Mutation Profiling

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DNA was extracted from paraffin-embedded tissue blocks with a QIAamp Tissue KitTM (Qiagen, Hilden, Germany). Then, 10 ng DNA per tissue sample was provided for sequencing. The DNA library was created by multiplex polymerase chain reaction with the Ion AmpliSeq Cancer Hotspot Panel v2 (Thermo Fisher Scientific, Waltham, MA, USA), which covers the mutation hotspots of 50 genes. The panel includes driver mutations, oncogenes, and tumor suppressor genes. By mid-2018, the gene panel was expanded using the 161-gene next-generation sequencing panel of Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific, Waltham, MA, USA), which covers genetic alterations and gene fusions. The Ampliseq cancer hotspot panel was sequenced with an Ion PGM (Thermo Fisher Scientific, Waltham, MA, USA) and the Oncomine Comprehensive Assay v3 on an Ion S5 sequencer (Thermo Fisher Scientific, Waltham, MA, USA). The generated sequencing data were analyzed afterward with the help of the Ion Reporter Software (Thermo Scientific Fisher Scientific, Waltham, MA, USA). We referred to the BRCA Exchange, ClinVar, COSMIC, dbSNP, OMIM, and 1000 genomes for variant calling and classification.

Taghizadeh H., Mader R.M., Müllauer L., Fuereder T., Kautzky-Willer A, & Prager G.W. (2020). Outcome of Targeted Therapy Recommendations for Metastatic and Recurrent Head and Neck Cancers. Cancers, 12(11), 3381.

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Targeted Sequencing for Oncology

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DNA was extracted from paraffin-embedded tissue blocks with a QIAamp Tissue KitTM (Qiagen, Hilden, Germany) and 10 ng DNA per tissue sample was provided for sequencing. The DNA library was created by multiplex polymerase chain reaction with the 161-gene next-generation sequencing panel of Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific, Waltham, MA, USA). The panel includes driver mutations, oncogenes, tumor suppressor genes, and gene fusions. See supplementary information for complete list of the gene panel. The Oncomine Comprehensive Assay v3 was optimized for sequencing on an Ion Personal Genome Machine System (Thermo Fisher Scientific). The generated sequencing data were afterwards analyzed with the help of the Ion Reporter Software (Thermo Scientific Fisher). We referred to BRCA Exchange, ClinVar, COSMIC, dbSNP, OMIM and 1000 genomes for variant calling and classification. The variants were classified according to a five-tier system comprised of the modifiers pathogenic, likely pathogenic, uncertain significance, likely benign, or benign. This classification was based on the standards and guidelines for the interpretation of sequence variants of the American College of Medical Genetics and Genomics. The variants pathogenic and likely pathogenic were taken into consideration for the recommendation of targeted therapy.

Taghizadeh H., Müllauer L., Mader R.M., Füreder T, & Prager G.W. (2020). Molecularly guided treatment of metastatic parotid gland carcinoma in adults. Wiener Klinische Wochenschrift, 133(1), 32-40.

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Comprehensive Cancer Mutation Profiling

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DNA was extracted from paraffin-embedded tissue blocks with a QIAamp Tissue KitTM (Qiagen, Hilden, Germany), and 10 ng DNA per tissue sample was provided for sequencing. The DNA library was created by multiplex polymerase chain reaction (PCR) with the Ion AmpliSeq Cancer Hotspot Panel v2 (Thermo Fisher Scientific, Waltham, MA, USA), which covers mutation hotspots of 50 genes. The panel includes driver mutations, oncogenes, and tumor suppressor genes. By mid-2018, the gene panel was expanded using the 161-gene next-generation sequencing panel of Oncomine Comprehensive Assay v3 (Thermo Fisher Scientific), which covers genetic alterations and gene fusions (see supplemental information for complete list of the gene panel). The Ampliseq cancer hotspot panel was sequenced with an Ion PGM (Thermo Fisher Scientific) and the Oncomine Comprehensive Assay v3 on an Ion S5 sequencer (Thermo Fisher Scientific). The description of each mutation was presented according to the Human Genome Variation Society (HGVS).^{8 (link)}

Taghizadeh H., Müllauer L., Mader R.M., Schindl M, & Prager G.W. (2020). Applied precision medicine in metastatic pancreatic ductal adenocarcinoma. Therapeutic Advances in Medical Oncology, 12, 1758835920938611.

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Oncomine Comprehensive Assay for NSCLC

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Formalin‐fixed paraffin‐embedded samples were subjected to the Oncomine Comprehensive Assay v3 or Oncomine Dx Target Test (Thermo Fisher Scientific). The Oncomine Dx Target Test is a companion diagnostic test approved in Japan to evaluate patient tumor samples for multiple biomarkers associated with targeted therapies for NSCLC.

Hasegawa N., Kohsaka S., Kurokawa K., Shinno Y., Takeda Nakamura I., Ueno T., Kojima S., Kawazu M., Suehara Y., Ishijima M., Goto Y., Kojima Y., Yonemori K., Hayashi T., Saito T., Shukuya T., Takahashi F., Takahashi K, & Mano H. (2021). Highly sensitive fusion detection using plasma cell‐free RNA in non‐small‐cell lung cancers. Cancer Science, 112(10), 4393-4403.

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Profiling Oncogenic Mutations via NGS

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METex14 skipping mutations were identified through several methods including NGS of tissue using DNA- and RNA-based assays (TruSight Tumor 26 assay, Ilumina, San Diego CA; Oncomine Comprehensive Assay v3, Thermo Fischer, Waltham, MA; or FoundationOne CDx, Foundation Medicine, Cambridge, MA), or plasma-based NGS with commercially available assays (Guardant360 CDx, Redwood City, CA and FoundationOne LiquidCDx, Foundation Medicine, Cambridge, MA). Programmed death-ligand 1 (PD-L1) assessment was performed using the 22C3 pharmDx assay (Agilent Technologies, Dako, Carpinteria, CA) at all centers.

Lau S.C., Perdrizet K., Fung A.S., Mata D.G., Weiss J., Holzapfel N., Liu G., Bradbury P.A., Shepherd F.A., Sacher A.G., Feilotter H., Sheffield B., Hwang D., Tsao M.S., Cheng S., Cheema P, & Leighl N.B. (2023). Programmed Cell Death Protein 1 Inhibitors and MET Targeted Therapies in NSCLC With MET Exon 14 Skipping Mutations: Efficacy and Toxicity as Sequential Therapies. JTO Clinical and Research Reports, 4(10), 100562.

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FFPE DNA Extraction and Library Prep

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All laboratory work was performed according to the respective manufacturer’s protocol available online. The preparation of formalin-fixed, paraffin-embedded (FFPE) tissue samples was performed identically for all three workflows. The purification of DNA from the FFPE tissue samples was performed using the Maxwell^® RSC Instrument (Promega Corporation) with the Maxwell^® RSC FFPE Plus DNA Kit (Promega Corporation). Therefore, the tumor containing area of a tissue section or respectively the total area of a normal tissue section was scraped off the slide, placed into a 1.5 ml tube and centrifuged at maximum speed for 15 sec. 20 μl of 20 mg/ml Proteinase K and 180 μl Incubation Buffer was added. Overnight samples were heated at 70°C. After incubation the samples were mixed with 400 μl Lysis Buffer and transferred to the Maxwell^® RSC Cartridge. Further preparation and the instrument run were performed according to manufacturer’s protocol. The concentration of DNA was measured with the Qubit 4 Fluorometer (Invitrogen). For the library preparation, panel-specific amounts of DNA were used as input: 40 ng for the GeneRead™ QIAact BRCA UMI Panel (QIAGEN), 20 ng for the Oncomine™ Comprehensive Assay v3 (Thermo Fisher) and 20 ng for the AmpliSeq™ BRCA Panel (Illumina^®).

Ebner S., Winkelmann R., Martin S., Köllermann J., Wild P.J, & Demes M. (2022). Sequencing of BRCA1/2-alterations using NGS-based technology: annotation as a challenge. Oncotarget, 13, 464-475.

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Comprehensive Cancer Gene Panel Sequencing

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Up to five 10-μm-thick sections of formalin-fixed, paraffin-embedded tissue blocks were used for DNA and RNA sequencing analysis. Representative tumor areas were manually micro-dissected. DNA and RNA were extracted using the Invitrogen Recover All Total Nucleic Acid Isolation Kit (Invitrogen, Carlsbad, CA, USA). Massively parallel sequencing of cancer-related gene panels (Oncomine Comprehensive Assay v3, ThermoFisher Scientific Waltham, MA, USA) was performed using the Ion Chef and Ion Torrent sequencing platforms. Sequence data were aligned against the human reference genome (hg19 build). Bioinformatics data analysis was performed using Ion Reporter software (v.5.10.1.0) with the Oncomine Variants filter chain (v.5.6).

Jung H.J., Lee S.Y., Hong J.H, & Chun Y.K. (2020). Interobserver diagnostic reproducibility in advanced-stage endometrial carcinoma. Journal of Pathology and Translational Medicine, 55(1), 43-52.

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Genomic Profiling of Thyroid Cancers

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Surgically excised, formalin-fixed, routinely processed, paraffin-embedded tissue blocks of thyroid cancers were obtained from patients who had thyroidectomies performed by their local surgeons. Histologic sections were reviewed by an NCI pathologist (C-CL) who identified the carcinoma-containing areas that were then micro-dissected. Mutations were identified with a next-generation sequencing-based assay (Oncomine™ Comprehensive Assay v3; Thermo Fisher Scientific), which was performed and analyzed as described previously [18 (link)].

Kouatcheu S.D., Marko J., Tamura D., Khan S.G., Lee C.R., DiGiovanna J.J, & Kraemer K.H. (2020). Thyroid Nodules in Xeroderma Pigmentosum Patients: A Feature of Premature Aging. Journal of endocrinological investigation, 44(7), 1475-1482.

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Tumor Profiling Using NGS for Actionable Mutations

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To identify clinically actionable mutations targeted next-generation sequencing (NGS) was performed by VCU Pathology Molecular Diagnostics laboratory with clinically validated methodology used for diagnostic tumor profiling. PDX tumors were excised from mice, flash frozen, prepared into frozen sections using optimal cutting temperature (OTC) compound, and total nucleic acids extracted. NGS using the Oncomine Comprehensive Assay v3 (ThermoFisher, Waltham, MA, USA) was performed as previously described [25 (link)] to identify DNA mutations, DNA copy number variations, and RNA fusions across cancer-related genes.

Boyd D.C., Zboril E.K., Olex A.L., Leftwich T.J., Hairr N.S., Byers H.A., Valentine A.D., Altman J.E., Alzubi M.A., Grible J.M., Turner S.A., Ferreira-Gonzalez A., Dozmorov M.G, & Harrell J.C. (2023). Discovering Synergistic Compounds with BYL-719 in PI3K Overactivated Basal-like PDXs. Cancers, 15(5), 1582.

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Cancer Gene Panel Sequencing

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Before the library construction, a reverse transcription process was conducted to obtain cDNA from RNA. DNA and cDNA libraries were prepared using Oncomine™ Comprehensive Assay v3 (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. The panel covers 161 cancer driver genes, including kinase domain and DNA repair genes. The libraries concentrations were analysed using an Agilent 2100 Bioanalyzer. Sequencing was performed using the Ion Proton instrument with the Ion PI Hi-Q Sequencing 200 Kit (Thermo Fisher Scientific, Waltham, MA, USA).

Skubisz K., Januszkiewicz-Caulier J., Cybula P., Bakuła-Zalewska E., Goryca K., Paziewska A., Ambrożkiewicz F., Woliński K., Mikula M., Ostrowski J, & Dedecjus M. (2021). Higher EU-TIRADS-Score Correlated with BRAF V600E Positivity in the Early Stage of Papillary Thyroid Carcinoma. Journal of Clinical Medicine, 10(11), 2304.

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