Solid Tumor OncoPanel Hotspot and Fusion on RNA by NGS
Solid tumor, NGS, next generation sequencing, fusion, translocation, RNA, single nucleotide variant, SNV, hotspot
Molecular Diagnostics Laboratory
Clinical Indication and Relevance
The assay can identify molecular genomic alterations that may help provide a diagnosis and/or therapeutic information. Genomic alterations that can be identified include gene fusions, hotspot single nucleotide variants, small indels (<20bp), and a select number of oncogenic splicing variants.
The Solid Tumor HotSpot and Fusion OncoPanel is a custom-designed targeted RNA sequencing assay, created to detect gene fusions, exon skipping events, and select expressed hotspot mutations in solid tumors (see table 1 and 2 for gene lists). Selected gene targets represent recurrently altered genes expressed in solid tumors. Anchored Multiplex PCR chemistry is used for Archer FusionPlex library preparation with molecular barcoding from specimen RNA or total nucleic acid (TNA) targeting regions of interest in the Solid Tumor HotSpot and Fusion OncoPanel. After sequencing on the Illumina MiSeq, the Archer Analysis Bioinformatics Pipeline is used to detect relevant alterations, and uses the molecular barcodes for read deduplication and error correction.
Alterations are assessed as Tier I, Tier II, Tier III or Tier IV (benign or likely benign) based on professional guidelines (Li M et. al, 2017, PMID 27993330), and Tier I-III alterations are reported here after being assessed using public databases (such as COSMIC, OncoKB, Quiver Fusion Database, CIViC, and others).
TABLE 1: LIST OF GENES WITH HOTSPOTS TARGETED BY TEST
AKT1 BCOR BRAF CCND1 CIC CTNNB1
DICER1 EGFR ERBB2 FGFR1 FGFR2 FGFR3
GNA11 GNAQ GNAS H3F3A HIST1H3B HRAS
IDH1 IDH2 KIT KRAS MAP2K1 MED12
MET MTOR NFE2L2 NRAS NTRK1 NTRK3
PDGFRA PPP2R1A RAC1 RAF1 RET SF3B1
TABLE 2: LIST OF GENES INVOLVED IN FUSIONS TARGETED BY TEST
AKT1 AKT3 ALK AR ARHGAP26 ATRX
AXL BCAR4 BCL2 BCL2L1 BCOR BRAF
CAMTA1 CCND1 CD274 CDK6 CDKN2B-AS1 CIC
CTNNB1 DNAJB1 EGFR ERBB3 ERBB4 ERG
ESR1 ETV1 ETV4 ETV5 ETV6 EWSR1
FGFR1 FGFR2 FGFR3 FGFR4 FLI1 FOS
FOSB FOXA1 FOXO1 FUS GLI1 HMGA2
JAZF1 KIT MAML2 MAML3 MAST1 MAST2
MET MRTFB MN1 MYB MYBL1 NCOA1
NCOA2 NDRG1 NOTCH1 NOTCH2 NR4A3 NRG1
NTRK1 NTRK2 NTRK3 NUMB NUTM1 PAX3
PDGFD PDGFB PDGFRA PHF1 PIK3CA PLAG1
PPARG PRKCA RAF1 RELA RET ROS1
RSPO2 RSPO3 SLC45A3 SS18 SS18L1 STAT6
TAF15 TCF12 TCF7L1 TCF7L2 TERT TFE3
TFEB TFG THADA TMPRSS2 TP53 TRIO
USP6 VGLL2 YWHAE WWTR1 YAP1
Minimum Specimen Requirements
- Formalin Fixed Paraffin Embedded (FFPE) tissue; should not be more than 5 years old. Tumor percentage should be 40% in one area.
- Tissue blocks, including core needle biopsies. The Histology lab will cut 10 unstained slides at 5-10 microns and 1 H&E.
- Unstained FFPE tissue slides (10 slides at 5-10 microns thickness). And 1 H&E.
- Fine needle aspirate (FNA) processed as FFPE cell block (cytospin). The Histology lab will cut 10 unstained slides at 5-10 microns and 1 H&E.
- Cells grown in tissue culture or cells preserved by Cytogenetics. A corresponding H&E from the submitted sample is desired.
- Fresh or frozen tissue, at least 0.5 x 0.5 cm. Frozen tissue should be stored at -80C. An H&E slide is also required.
- Frozen tissue preserved in OCT (Optimal Cutting Temperature) and stored at -80C. An H&E slide is also required.
- Formalin-fixed paraffin embedded (FFPE) tissue blocks and slides can be delivered at controlled temperatures, not to exceed 30C.
- Fresh tissue samples should be delivered at room temperature in RPMI culture medium to the lab within 3 hours of collection, or snap frozen in liquid nitrogen at -70°C and packed in dry ice for delivery. Please do not allow frozen tissues to thaw.
- Tissue samples fixed in Zenker’s, B5, or Bouin’s fixatives
- Samples decalcified with strong acids
- Hematopathologic samples
- FFPE tissue scrolls are not desired, as they cannot be macrodissected. They require pathologist approval.
- Tumor percentage less than 40% on one area is not ideal. Tumor content between 5-40% in one area may be approved by a pathologist on a case-by-case basis.
- Frozen or fresh tissue samples or samples from cytogenetics must be accompanied by an H&E. In special situations, this may be approved by a pathologist.
Seven to 15 working days.View Requisition Sheet