Comprehensive Thyroid and Lung (CTL) kits enable parallel detection of copy number variants and expression levels by next-generation sequencing

By Laura Griffin, PhD on Tue Apr 12, 2016


  • Copy number variants (CNVs) are common oncogenic drivers in lung cancers, often encoding targetable proteins with unique drug sensitivities.
  • As CNVs can result in altered expression profiles of multiple genes through direct and indirect mechanisms, it is important to detect CNVs and expression levels across multiple genes.
  • Standard methods to detect CNVs are not amenable to multiplexing and cannot be easily coupled with assays to determine expression levels.
  • Next-generation sequencing (NGS) enables comprehensive profiling of CNVs and expression levels but exhibits poor detection sensitivity.
  • Anchored Multiplex PCR (AMP™) is a target enrichment strategy that greatly enhances NGS-based detection sensitivity.
  • Our data show that AMP enables sensitive NGS-based CNV detection and expression profiling of genes relevant in lung cancer (Figure 1, Figure 2).

CNVs in lung cancer

Copy number variants (CNVs) are frequently detected in lung cancers, driving approximately 24% of non-small cell lung cancers (NSCLCs) (1). CNVs arise from large-scale genomic amplifications and deletions, which can encompass multiple genes or only partial genes (2). CNVs often result in altered expression profiles due to altered gene dosages, but can also affect the expression of genes outside the amplified or deleted regions through indirect mechanisms (3). Furthermore, gene expression can become deregulated by mechanisms independent of CNVs. Therefore, in order to identify functional driver mutations it is necessary to simultaneously interrogate the copy number status as well as the expression level of multiple genes in a single sample.

EGFR overexpression has been detected in 40-80% of lung cancers (4) and is associated with poor prognosis in NSCLC (5). EGFR is targetable by clinically approved TKIs such as gefitinib and erlotinib, but amplified and mutated EGFR exhibit altered sensitivities to TKIs (6-10). However, resistance to TKIs frequently develops through the acquisition of resistance mutations in EGFR’s kinase domain as well as MET amplifications (11-15). Importantly, transcriptional upregulation of MET independent of gene amplification and EGFR drug resistance is also associated with an aggressive phenotype in multiple cancers, including NSCLC (16). MET is targetable by crizotinib, a dual ALK/MET tyrosine kinase inhibitor (17). Thus, determining the copy number status and expression levels of EGFR and MET in a single sample enables identification of targetable driver mutations in lung cancers.

CCND1 amplifications and overexpression have been reported in 5–30% and 18–76% of NSCLCs, respectively (18, 19). While CCND1 overexpression is associated with poor prognosis, the expressed protein, Cyclin D1, and it’s associated CDK proteins are targetable by CDK inhibitors that are currently undergoing clinical testing (20-24). CCND1 overexpression can be caused by many factors, including gene amplification and EGFR overexpression and stimulation (25). As EGFR activation can be inhibited by clinically approved drugs, determining the copy number and expression levels of both CCND1 and EGFR can reveal potential therapeutic targets.

Methods to detect CNVs

Standard techniques to determine copy number, including FISH and array comparative genomic hybridization (aCGH), are not amenable to multiplexing and cannot be easily coupled with assays to determine expression levels (26). Next-generation sequencing (NGS) of whole genomes and whole transcriptomes provides genome-wide copy number information and expression profiling (27-29). However, the comprehensive nature of these methods results in decreased sensitivity and increased cost, rendering them impractical for routine analysis of low input clinical samples types.

Anchored Multiplex PCR (AMP™) is a library preparation method for NGS that enriches for a panel of gene targets with both known and unknown mutations. Molecular barcoded adapters ligated to DNA fragments prior to PCR enable amplification of fragments as small as 50bp, thereby increasing read depth and coverage of target regions. Thus, AMP enhances NGS-based detection sensitivity and enables the detection of mutations from low input clinical sample types, such as formalin-fixed paraffin embedded (FFPE) specimens. Archer VariantPlex™ and FusionPlex™ assays use AMP to enrich for a panel of gene targets from DNA or RNA extracted from FFPE samples, respectively. We developed VariantPlex and FusionPlex Comprehensive Thyroid and Lung (CTL) kits to detect genetic aberrations in genes associated with thyroid and lung cancers. Therefore, comprehensive profiling with CTL kits provides a highly multiplexed, sensitive method to simultaneously detect CNVs and expression levels across multiple relevant genes.

MET CNV detection by FISH and VariantPlex CTL

To validate the ability of our VariantPlex CTL assay to enable detection of CNVs in low input FFPE samples, we acquired a NSCLC FFPE sample with a known MET amplification (Figure 1). Standard FISH testing using MET probes revealed MET amplifications in this sample (left panel). We then extracted genomic DNA from this sample, prepared a library using the VariantPlex CTL kit, and sequenced the library on an Illumina® instrument. Data analyzed using Archer Analysis showed a 24.5-fold amplification of the MET gene (right panel). These results show that MET CNV detected with VariantPlex CTL is consistent with MET FISH testing, the current standard method to determine copy number status of MET in FFPE samples.

MET amplification in an NSCLC FFPE sample is detected by FISH testing and by VariantPlex CTL-enabled NGS.
Figure 1. MET amplification in an NSCLC FFPE sample is detected by FISH testing and by VariantPlex CTL-enabled NGS. An NSCLC FFPE sample was subjected to FISH testing using MET probes (left) and AMP-based NGS using the VariantPlex CTL kit (right).

Comprehensive profiling of NSCLC FFPE samples with CTL kits

Next, we tested the ability our VariantPlex and FusionPlex CTL assays to enable parallel NGS-based detection of CNVs and expression levels (Figure 2). We extracted total nucleic acid from NSCLC FFPE samples and used the VariantPlex and FusionPlex CTL kits in parallel to prepare libraries for NGS. We identified samples with CCND1, EGFR, and MET amplifications, as well as a sample harboring an EGFR L858R driver point mutation and no detectable CNVs (bottom panel). Expression analysis supported these results, demonstrating increased expression of CCND1, EGFR, and MET genes that correlated with detected amplifications (top panel).

CTL kits enable parallel detection of CNVs and expression levels across relevant genes in NSCLC FFPE samples.
Figure 2. CTL kits enable parallel detection of CNVs (bottom) and expression levels (top) across relevant genes in NSCLC FFPE samples. Total nucleic acid was extracted from NSCLC FFPE samples containing indicated driver mutations (bottom grey bar). VariantPlex CTL kits were used to prepare libraries from genomic DNA and FusionPlex CTL kits were used to prepare libraries from RNA. Libraries were then sequenced on an Illumina® instrument, and data were analyzed using Archer Analysis. Copy numbers and expression levels were determined relative to the normal lung sample.

As described above, CCND1 overexpression can be caused by amplification of CCND1 as well as overexpression and stimulation of EGFR. Our data show amplification and overexpression of EGFR and CCND1 in the same sample, indicating a potential role for EGFR in CCND1 overexpression. In this case, further investigation would be necessary to determine the underlying mechanism(s) of CCND1 overexpression. We also observed a slight increase in CCND1 expression in the EGFR CNV-negative sample. This could be explained by the observed increase in CCND1 copy number, or by the EGFR L858R activating point mutation that renders EGFR constitutively active, which can also increase CCND1 expression. These results demonstrate the importance of interrogating multiple genes for copy number status and expression levels in the same sample. VariantPlex and FusionPlex CTL kits enable sensitive and comprehensive profiling of multiple genes relevant in lung cancers, which would not be possible with standard FISH testing.


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About Laura Griffin, PhD

Laura Griffin earned her PhD in Microbiology from the University of Colorado Denver, Anschutz Medical Campus. Her research focused on cancer virology, dissecting virus-host interactions during Human Papillomavirus infection. Laura is passionate about cancer research as well as effective education and communication of scientific ideas. Laura joined the ArcherDX team as Scientific Editor in January, 2016. In her spare time, Laura is a group fitness instructor and enjoys cycling, hiking, skiing, and snowshoeing in the Rocky Mountains of Colorado.

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