Fusion-STAMP, FFPE

Molecular Oncology

The Solid Tumor Actionable Mutation Panel for Fusions (Fusion STAMP) is a next-generation sequencing (NGS) assay that detects potentially clinically actionable structural rearrangement events in cancers. The targeted sequencing approach and integrated bioinformatics workflow is optimized for sequencing of formalin fixed tumor tissue specimens. Fusion-STAMP is an NGS assay that detects chromosome rearrangement events in solid tumors and sarcomas that may guide diagnosis, prognosis, and therapeutic management. This test uses the FusionPlex Pan Solid Tumor v2 panel (Invitae) to detect gene rearrangements and select hotspot and splice variants in 137 genes relevant to solid tumors and sarcomas. AMP-based enrichment is used to optimize detection of fusions and partner genes with relatively low nucleic acid input from FFPE specimens. TNA is extracted from the sample, and cDNA is synthesized. The cDNA strands undergo end repair, adenylation, and ligation to half-functional universal adapters that include sample barcodes. This is followed by two rounds of nested PCR using gene-specific primers (GSP1 and GSP2) and a primer complementary to the universal adapter, allowing enrichment for target genes and identification of known and novel fusion partners. The target amplicon library is then quantitated and sequenced using the Illumina MiSeq system and analyzed using the Archer Analysis website/virtual machine.

The Stanford Actionable Mutation Panel for Fusions (Fusion-STAMP) is a targeted next generation sequencing method that detects potentially clinically actionable gene fusion events in cancer. The targeted sequencing approach and integrated bioinformatics workflow is optimized for sequencing of formalin fixed paraffin embedded tissue specimens. The workflow includes isolation of total RNA molecules, followed by efficient preparation of sequencing libraries and a target enrichment approach to capture mRNA transcript regions of interest for sequencing. The enrichment is done using custom designed libraries of capture oligonucleotides that target a specific set of expressed genomic regions. This panel fully targets the transcript isoforms of 43 genes which were selected based on their known impact as actionable targets of existing and emerging anti-cancer therapies, their prognostic features, and/or their utility as diagnostic cancer biomarkers. Pooled libraries are sequenced on an Illumina sequencing instrument.

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