Focus Panel

OVERVIEW

The AmpliSeq Focus Panel is an amplicon-based targeted resequencing assay designed to detect recurrent somatic single nucleotide variants (SNVs), small insertion and deletions (indels), copy number variants (CNVs) and fusions, putative fusions and other aberrant splicing events in genes that have clinical relevance to solid tumors.

INDICATIONS

Focus Panel testing is available for the following tumour types:

  • Lung Cancer  (Stage IIIB/IV non-small cell, non-neuroendocrine adenocarcinoma)
  • Lung Cancer (Treated, progression for EGFR T790M) – For tissue biopsies only
  • Low grade infiltrating glioma
  • Melanoma (Non-Resectable/Metastatic)

Additional indications that are funded for Focus Panel testing by Roche under the PREDiCTm study include:

  • Biliary Tract
  • Pancreatic
  • Sarcoma (NOS)
  • Salivary gland carcinoma
  • Head and Neck squamous cell carcinoma (HNSCC)
  • Thyroid carcinoma
  • Other indications (approval required)

Further information on the PREDiCTm study, including confirmation of eligibility for Focus Panel testing can be obtained by contacting Dr. Stephen Yip or Dr. Deepu Alex.

Focus Panel testing is also being funded by Novartis for the following indication:

  • Advanced-stage or metastatic hormone-receptor-positive, HER2-negative breast cancer that has progressed after treatment with hormonal therapy in postmenopausal women and men

The goal of this testing is to identify mutations in the PIK3CA gene, that may allow these patients to qualify for targeted therapy with the PIK3CA inhibitor Alpelisib (Piqray®).  Please contact Dr. Sean Young for inquiries and confirmation of patient eligibility.

TEST REQUIREMENTS

  1. Completed requisition form for the appropriate indication:
    • Currently funded indications (Lung, LGG, Melanoma) requisition
    • Indications funded under PREDiCTm by Roche requisition
    • Breast Cancer (Contact Dr. Sean Young or Dr. Stephen Chia for requisition)
  2. FFPE Tumour specimen (see Specimen Guidelines
    • minimum of 20% tumour content is required 

TURN-AROUND TIME

7-10 days (lung, melanoma) or 10-14 days (other indications) from receipt of specimen and completed, signed requisition form.

RESULTS REPORTING

  • Focus Panel reports include a list of variants classified into tiers of clinical significance: 
    • TIER I – VARIANTS OF STRONG CLINICAL SIGNIFICANCE 
    • TIER II – VARIANTS OF POTENTIAL CLINICAL SIGNIFICANCE 
    • TIER IIIA –VARIANTS OF UNCERTAIN CLINICAL SIGNIFICANCE 
    • TIER IIIB – VARIANTS OF UNCERTAIN FUNCTION 
  • Please see our Variant Classification Guidelines for additional details. 

GENES TARGETED

The Focus Panel interrogates for DNA alterations including SNVs, indels and CNVS in 39 genes (AKT1, ALK, AR, BRAF, CCND1, CDK4, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ESR1, FGFR1, FGFR2, FGFR3, FGFR4, GNA11, GNAQ, HRAS, IDH1, IDH2, JAK1, JAK2, JAK3, KIT, KRAS, MAP2K1, MAP2K2, MET, MTOR, MYC, NRAS, PDGFRA, PIK3CA, RAF1, RET, ROS1, SMO) while CNVs are interrogated in 8 additional genes (APC, BIRC2, BRCA1, CDK6, DCUN1D1, MED12, MYCN, NF1).  RNA alterations including recurrent gene fusions and aberrant splicing events involving 23 driver genes (ABL1, ALK, AKT3, AXL, BRAF, EGFR, ERBB2, ERG, ETV1, ETV4, ETV5, FGFR1, FGFR2, FGFR3, MET, NTRK1, NTRK2, NTRK3, PDGFRA, PPARG, RAF1, RET, ROS1) and putative fusions identified by a 3’/5’ imbalance in 4 driver genes (ALK, NTRK1, RET, ROS1).

Details for DNA Coverage including the genes and regions interrogated for SNVs/indels and/or CNVs can be found here.

Details for RNA Coverage including specific fusions detected can be found here.

METHODS

The AmpliSeq Focus Panel is an amplicon-based targeted resequencing assay designed to detect recurrent somatic DNA and RNA alterations in genes that are clinically relevant to solid tumors.  The test is performed in parallel on DNA and RNA samples obtained from the same tumor specimen using the Maxwell RSC FFPE TNA (total nucleic acids) extraction method.  Targeted DNA and RNA regions are amplified by multiplex PCR. Paired-end massively parallel sequencing of 150-bp fragments is performed with an illumina MiniSeq instrument.  DNA sequences are aligned and compared to reference genome hg19/GRCh37 to identify single nucleotide variants (SNVs), small insertions and deletions (indels) and copy number variants (CNVs).  RNA sequences are aligned to a custom reference to identify fusions, putative fusions and other aberrant splicing events.  Variants are interpreted and categorized based on their clinical impact using AMP/ASCO/CAP guidelines (PMID: 27993330) as follows: Tier I, variants with strong clinical significance (level A and B evidence); Tier II, variants with potential clinical significance (level C and D evidence); Tier IIIA, variants with uncertain clinical significance; Tier IIIB, variants with uncertain function and Tier IV, (likely) benign variants.