Comprehensive thyroid and lung profiling from low-input FFPE

The Archer Comprehensive Thyroid and Lung (CTL) Assay is comprised of two complementary targeted NGS panels to detect gene fusions, expression, SNVs, CNVs, and indels from FFPE and FNA samples. CTL is expertly designed to utilize both DNA and RNA in the VariantPlex™ and FusionPlex™ CTL panels in parallel to cover relevant exons in 44 genes implicated in lung and thyroid cancers.

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

Features

  • Orthogonal verification - Confirm relevant mutation calls when using both panels in parallel
  • Parallel workflows - Libraries can be concurrently created from RNA and DNA in a single sample
  • In-house analysis - Comprehensive mutation detection for translational research is performed in-house, so there is no need for sample send-outs.



How it works

Archer CTL Parallel Workflow

Total nucleic acid (TNA) is purified from formalin-fixed, paraffin-embedded (FFPE) sample or a fine-needle aspirate (FNA). As little as 20ng TNA is then used with each panel to generate a targeted library from RNA (FusionPlex CTL) and DNA (VariantPlex CTL). The libraries are then pooled and sequenced, and the Archer Analysis bioinformatics platform analyzes the data to identify fusions, single nucleotide variants (SNVs) and insertions/deletions (indels) from the RNA and copy number variations (CNVs), SNVs and indels from the DNA in the sample.



Panel Targets

Gene Fusion SNV/Indel Splicing Expression SNV/Indel CNV
AKT1
ALK
AXL
BRAF
CALCA
CCND1
CTNNB1
DDR2
EGFR
EIF1AX
ERBB2
FGFR1
FGFR2
FGFR3
GNAS
HRAS
IDH1
IDH2
KIT
KRAS
KRT20
KRT7
MAP2K1
MDM2
MET
NRAS
NRG1
NTRK1
NTRK2
NTRK3
PDGFRA
PIK3CA
PPARG
PTEN
PTH
RAF1
RET
ROS1
STK11
SLC5A5
TERT
THADA
TP53
TSHR
TTF1



Complete lung cancer profiling means verification for all mutation types

The major types of lung cancer are non-small cell lung cancer (NSCLC) and small cell lung cancer, with NSCLC accounting for 85% of all lung cancer cases (1). NSCLC is driven by a variety of genetic mutation types, including SNVs, indels, CNVs and expression, and gene fusions. Figure 1 below shows the relative frequency of each type of mutation.

NSCLC mutation type frequency

Traditional molecular testing to detect these types of variants in NSCLC include morphology, immunohistochemistry (IHC), real time PCR (RT-PCR), PCR, pyrosequencing, fluorescence in situ hybridization (FISH), karyotyping, and array comparative genomic hybridization (aCGH). Each of these detection methods have cost, input, workflow, and accuracy challenges associated with them that makes comprehensive verification of each sample untenable. The advent of next-generation sequencing (NGS) has enabled nucleotide resolution results from which comprehensive and accurate tumor characterization is feasible, but certain library preparation techniques can result in lost information, noisy data, or have high input requirements. Using Archer CTL panels, labs can identify all relevant mutation types in NSCLC from low input FFPE samples.

Internal, Orthogonal Verification using CTL

Separate interrogation of somatic mutations in DNA and somatic mutations in RNA from the same sample has many inherent benefits. One powerful application is the internal, cross verification of fusions, CNVs, expressions, and SNVs enabled by AMP enrichment chemistry. Fusions can be verified by expression imbalance within the FusionPlex panel. CNVs and expression profiles between the two panels can verify or classify oncogenic mechanisms. SNVs in both RNA and DNA can confirm the presence of the mutation, expression and potential allelic imbalance. Concordance studies have never been so easy and powerful at nucleotide level resolution.

Internal orthogonal verification

Verify SNVs in both DNA and RNA

Overlapping hotspot coverage in FusionPlex and VariantPlex CTL panels enables SNV and indel cross-verification. Differences in allelic fraction detection can be attributed to RNA expression levels. In this case, the EGFR variant is driving the allelic imbalance.

Figure 1 NSCLC FFPE sample

Figure 1. This figure shows an NSCLC FFPE sample containing the EGFR L858R mutation at 22% AF in the DNA as detected in the VariantPlex library preparation (top). The mutation was also detected in the expressed RNA through the FusionPlex library preparation (bottom). Both CTL libraries were prepped from 50ng nucleic acid and sequenced to 1 million reads.
» Click image to enlarge.

Expression + CNV

Anchored MultiPlex PCR incorporates the ligation of molecular barcodes to unique input molecules prior to amplification. Counting and analyzing unique molecules allows for robust CNV detection in DNA and expression profiling in RNA. Comparing the two can verify results or classify oncogenic mechanism of the tumor.

Figure 2 Relative expression by gene

Figure 2. This figure shows relative expression by gene as detected by the FusionPlex CTL panel (top), and copy number variation (middle) as detected in the VariantPlex CTL panel in NSCLC FFPE samples. “Mutation” indicates the suspected driver mutation in the sample.
» Click image to enlarge.

Expression Imbalance for Fusion Confirmation

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 3 EML4-ALK Fusion Verification

Figure 3. 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 4 RNA Expression Imbalance

Figure 4. 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). Expression difference: p=0.01.
» Click image to enlarge.


References

  1. Herbst RS, Heymach JV, Lippman SM, Lung cancer. N. Engl. J. Med. 2008;359:1367–1380.
  2. Mazzaferri EL. Thyroid carcinoma: Papillary and follicular. In: Mazzaferri EL, Samaan N, eds. Endocrine Tumors. Cambridge: Blackwell Scientific Publications 1993:278-333.
  3. Kaplan, MM. Clinical evaluation and management of solitary thyroid nodules. In: Braverman, LE, Utiger RD, eds. Werner and Ingbar's The Thyroid: A Fundamental and Clinical Text, 9th ed. Philadelphia: Lippincott Williams & Wilkins; 2005:996-1010.
  4. Layfield LJ, Cibas ES, Gharib H, Mandel SJ. Thyroid aspiration cytology: current status. CA Cancer J Clin 2009;59:99-110. Available at: http://www.ncbi.nim.nih.gov/pubmed/19278960.
  5. Mazzaferri EL, Jhiang SM. Long-term impact of initial surgical and medical therapy on papillary and follicular thyroid cancer. Am J Med 1994;97:418-428. Available at: http://ncbi.nlm.nih.gov/pubmed/7977430

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.