Detect thyroid and lung cancer-specific fusions and point mutations from RNA as part of Archer CTL comprehensive profiling

The Archer® FusionPlex® CTL Kit is a targeted NGS panel to detect gene fusions, SNV/indels, splicing and gene expression. This panel is expertly designed to cover relevant exons in 34 genes when used in combination with the VariantPlex™ CTL Panel.

For Research Use Only. Not for use in diagnostic procedures.

Highlights

  • Comprehensive fusion detection in lung and thyroid cancers
  • Confirmation of fusion events with expression imbalance
  • Expression profiling of relevant targets using molecular barcodes
  • Streamlined, lyophilized workflow mutation detection algorithms
  • Splice variant detection of EGFRvIII and MET exon 14 skipping

Expression imbalance provides secondary confirmation of fusion events

Functional gene fusions result in over-expression of one portion of the gene and lack of expression in the portion that has been translocated away. This results in an imbalance of expression levels within the gene of interest. Anchored Multiplex PCR uses molecular barcodes which enable the counting of unique molecules across a region to assess difference of expression levels. In the CTL panel, select targets are designed to enable the calculation of expression imbalance as a confirmatory method for detecting gene fusions.

Example of confirmatory testing with embedded expression imbalance

Figure 1 EML4-ALK Fusion Verification

Figure 1. This figure shows read statistics and visualization of the fusion EML4 exon 12 and ALK exon 20 in Archer Analysis (top) and sequencing reads spanning the breakpoint of ALK-EML4 (bottom). Hundreds of unique supporting reads span the exon-exon breakpoint.
» Click image to enlarge.


Figure 2 RNA Expression Imbalance

Figure 2. This was an NSCLC FFPE sample that was ambiguous by ALK break apart FISH testing. The ALK-EML4 fusion was called with 1,281 unique supporting reads. In addition, expression imbalance was observed with highly expressed region of ALK downstream of Exon 20 (red) in contrast to lack of expression upstream of Exon 20 (black).
» Click image to enlarge.


Gene Expression Profiling

Anchored Multiplex PCR enables robust expression profiling by using molecular barcodes to track each unique input molecule. After sequencing, the relative number of molecular barcodes in a targeted region can be counted up and normalized to determine expression levels. Two types of expression types are designed into the FusionPlex CTL panel, expression level and tissue expression. For expression level targets, the relative expression level and expression imbalance in the case of fusions can be determined by the MBC counts. For tissue expression targets, the sheer presence of these expression markers can indicate contamination or inaccurate sample collection. For example:

Data exhibit of PCA and Data exhibit of expression levels

Figure 3 Exhibit of PCA levels

Figure 3. Principal components extracted from per exon RNA expression in normal lung, thyroid, and adrenal inputs.
» Click image to enlarge.

Figure 4 Expression of Copy Number Variants

Figure 4. This figure shows detection of abnormal, driver expression events on relevant targets in three NSCLC FFPE samples. In the presence of BRAF V600A and EGFR L858R driver mutations, expression levels remained neutral. The ability to examine relative expression levels can aid in identifying driver events in the absence of a fusion or point mutation.
» Click image to enlarge.


FusionPlex CTL panel targets

Gene Accession Number Target Exon Direction Assay Type
AKT1 NM_005163 3 E17K Mutation
ALK NM_004304 19,(intron 19),20,21,22,23 5' Fusion
ALK NM_004304 N/A N/A Expression
ALK NM_004304 22,23,25 N/A Mutation
AXL NM_021913 18,19,20 3' Fusion
AXL NM_021913 5,11,15,17 5' Mutation
AXL NM_021913 N/A N/A Expression
BRAF NM_004333 11,15 N/A Mutation
BRAF NM_004333 7,8,9,10,11 5' Fusion
BRAF NM_004333 7,8,10 3' Fusion
BRAF NM_004333 N/A N/A Expression
CALC NM_001741 3,4 N/A Expression*
CCND1 NM_053056 1,2,3,4 5' Fusion
CCND1 NM_053056 1,2,4 3' Fusion
CCND1 NM_053056 N/A N/A Expression
CTNNB1 NM_001904 3 N/A Mutation
DDR2 NM_006182 17 N/A Mutation
EGFR NM_005228 8 (2-7 exon skipping event) 5' Splice Variant
EGFR NM_005228 18,19,20,21 N/A Mutation
EGFR NM_005228 N/A N/A Expression
ERBB2 NM_004448 20 N/A Mutation
FGFR1 NM_015850 2,8,9,10,17 5' Fusion
FGFR1 NM_015850 17 3' Fusion
FGFR1 NM_015850 N/A N/A Expression
FGFR2 NM_000141 2,8,9,10 5' Fusion
FGFR2 NM_000141 17 3' Fusion
FGFR2 NM_000141 N/A N/A Expression
FGFR3 NM_000142 17, (intron 17) 3' Fusion
FGFR3 NM_000142 8,9,10 5' Fusion
FGFR3 NM_000142 N/A N/A Expression
GNAS NM_000516 8,9 N/A Mutation
HRAS NM_005343 2,3 N/A Mutation
IDH1 NM_005896 4 N/A Mutation
IDH2 NM_002168 4 N/A Mutation
KRAS NM_004985 2,3,4 N/A Mutation
KRT20 NM_019010 2,5 N/A Expression*
KRT7 NM_005556 2,7 N/A Expression*
MAP2K1 NM_002755 2,3 N/A Mutation
MET NM_000245 2 3' Fusion
MET NM_000245 2,4,5,6,13,14,15,16,17,21 5' Fusion
MET NM_000245 Capture exon 14 skipping 5' Splice Variant
MET NM_000245 N/A N/A Expression
NRAS NM_002524 2,3 N/A Mutation
NRG1 NM_004495 1,2,3,6 5' Fusion
NTRK1 NM_002529 2,4,6,8,10,11,12,13 5' Fusion
NTRK1 NM_002529 N/A N/A Expression
NTRK2 NM_006180 5,7,9,11,12,13,14,15,16,17 5' Fusion
NTRK2 NM_006180 N/A N/A Expression
NTRK3 NM_002530 4,7,10,13,14,15,16 5' Fusion
NTRK3 NM_001007156 15 5' Fusion
NTRK3 NM_002530 N/A N/A Expression
PIK3CA NM_006218 10,21 N/A Mutation
PPARG NM_015869 1,2,3,5 5' Fusion
PPARG NM_015869 5 3' Fusion
PTH NM_000315 1,3 N/A Expression*
RAF1 NM_002880 4,5,6,7,9,10,11,12 5' Fusion
RET NM_020630 11,13,14,15,16 N/A Mutation
RET NM_020630 2,4,6,8,9,10,11,12,13 5' Fusion
RET NM_020630 N/A N/A Expression
ROS1 NM_002944 2,4,7,31,32,33,34,35,36,37 5' Fusion
ROS1 NM_002944 38 N/A Mutation
ROS1 NM_002944 N/A N/A Expression
SLC5A5 NM_000453 4,8 N/A Expression*
THADA NM_022065 24,25,26,27,28,29,30,36,37 3' Fusion
THADA NM_022065 N/A N/A Expression
TTF1 NM_007344 4,10 N/A Expression*
CHMP2A NM_014453 3,4 N/A Control
GPI NM_000175 15,16 N/A Control
RAB7A NM_004637 3,4 N/A Control
VCP NM_007126 14,15 N/A Control

*target is used to verify sample tissue origin


How to contact us

Address

2477 55th Street, Suite 202

Boulder, CO 80301

Phone

Phone: (877) 771 1093

Phone: (303) 357 9001

All content © 2017 ArcherDX, Inc.

For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.