Profiling cancer gene mutations in clinical formalin-fixed, paraffin-embedded colorectal tumor specimens using targeted next-generation sequencing

Abstract

Purpose: The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology.

Methods: We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls.

Results: Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "false-positive" calls in clinically druggable oncogenes such as PIK3CA.

Conclusion: AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.

Keywords: AmpliSeq; Ion Torrent; Molecular diagnostics; Mutation; Next-generation sequencing; Precision oncology.

Figure 1.

Targeted NGS workflow: AmpliSeq and SimpliSeq. Abbreviations: emPCR, emulsion polymerase chain reaction; FFPE, formalin-fixed, paraffin-embedded; gDNA, genomic DNA; NGS, next-generation sequencing; PGM, Personal Genome Machine.

Figure 2.

Mutation detection using formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tumor specimens. Variants were detected by AmpliSeq (A) and SimpliSeq (B) in 44 FFPE genomic DNA samples. The red shade stands for variants with VF >5%; blue shade for variants with VF between 1% and 5%; and green shade for variants within a single gene containing multiple hotspot mutations, with VFs of both 1%–5% and >5%. Gray shade indicates no variants detected. The y-axis indicates the gene name, and the x-axis the identification of the matched pairings, i.e., the microdissected epithelial tumor and stroma tissues. False-negative variants in p53 and PTEN are marked with a circle (B). (C–E): Correlation of high-frequent variants (VF >5%) (C, D) and low-frequent variants (VF 1%–5%) (E) between AmpliSeq and SimpliSeq. Open circles within the marked area represent “true positive” and open circles on the x-axis represent “false positive.” Note that JAK2 was excluded from the above analysis and the outstanding “false-positive genes” (PIK3CA, NRAS, FGFR2) have been removed in (D, E). The percentage of bona fide variants (marked in red) was calculated based on SimpliSeq and AmpliSeq variant calls, and is provided in the upper right corner of each figure (C–E). Abbreviation: VF, variant frequency.

Figure 3.

Genetic landscape of variants detected in CRC tumor specimens. (A): The number of variants verified in each of the 22 CRC tumor specimens. (B): Intratumor molecular heterogeneity revealed by variants with different VF (13%–90%) in a single CRC tumor specimen. (C): Sequencing snapshot of the KRAS mutations in a single CRC tumor specimen. (D): Distribution of gene mutations in 22 CRC tumor specimens. Abbreviations: CRC, colorectal cancer; VF, variant frequency.

Figure 4.

Clinical application of AmpliSeq. (A): Implementing AmpliSeq NGS into routine clinical testing for precision oncology. (B): Orthogonal validation is recommended for variants with VF <5% and variants (VF >5%) with recurrent “false positives” (PIK3CA, NRAS, FGFR2, and JAK2). Abbreviations: gDNA, genomic DNA; NGS, next-generation sequencing; VF, variant frequency.