Ion ampliseq cancer hotspot panel

The Idylla™ ctEGFR mutation assay (Biocartis) has an estimated limit of detection (LOD) of 0.2% of allele frequency. This means that the Idylla™ ctEGFR system can detect mutations in a sample at an allele frequency of 0.2% or higher. The LOD for the Therascreen^® EGFR Plasma RGQ-PCR Kit (Qiagen) ranges between 0.15 and 0.2% allele frequency. The LOD for the Ion AmpliSeq Cancer Hotspot panel (ThermoFisher) is determined by the grade of detection sensitivity into the targeted sequence and the sequencing depth. With such an NGS-based system, commercial targeted panels can reliably have a limit of detection down to 0.01%, with a sequencing depth depending on the quality of the DNA isolated from the biological specimen. The LOD is typically lower in deeper sequencing runs, which provide more coverage of the targeted regions. The Ion AmpliSeq Cancer Hotspot panel has been designed to detect and accurately genotype most hotspot variants associated with many common cancer types, including lung adenocarcinoma. In addition to the sensitivity for the targeted sequences, the accuracy of the LOD for the Ion AmpliSeq Cancer Hotspot panel is also dependent on the quality of the sample. Poor-quality samples can reduce the accuracy of the LOD and may require additional sequencing depth or additional strategies to obtain an accurate result.

In the CRICKET study, baseline plasma samples were available for 25 patients and were analyzed using the next-generation sequencing Ion AmpliSeq Cancer Hotspot Panel (Thermo Fisher).^{7 (link)} For the CAVE trial, pretreatment plasma samples were collected for 67 patients and were analyzed using a reverse transcriptase–PCR test (IdyllaTM Biocartis platform).^{11 (link),20 (link)} In the CHRONOS trial, baseline liquid biopsy analysis was performed during the screening procedures. Overall, ctDNA from 52 patients was assessed using a ddPCR-based assay (Bio-Rad) for the identification of the most frequent KRAS, BRAF, and EGFR ECD alterations. Finally, for the 31 patients enrolled in the VELO trial who received rechallenge therapy, ctDNA was retrospectively evaluated using the IdyllaTM Biocartis assay.^{13 (link),14 (link),20 (link)}

DNA from the fresh-frozen pulverized tumor tissue was isolated using a DNEasy Blood and Tissue Kit (Qiagen, Venlo, The Netherlands). Targeted next-generation sequencing with a mean coverage of 500X coverage was performed on the Ion Torrent S5 system (ThermoFischer Scientific) using a custom next-generation sequencing (NGS) panel based on the Ion Ampliseq™ Cancer Hotspot Panel targeting mutational hotspots of 64 cancer-related genes. Variants with an allele frequency of at least 5% were reported. Variant call files were generated and analysis was performed using Alissa (Agilent Technologies Alissa Interpret v5.1.7).

Ten nanograms of DNA was used for preparing amplicon library using Ion AmpliSeq™ Library kit 2.0 (Ion Torrent; Thermo Fisher Scientific, Inc.) and Ion AmpliSeq™ Cancer hotspot panel (Ion Torrent; Thermo Fisher Scientific, Inc.), according to manufacturer’s instructions. This panel contains 207 primer pairs in a single tube and surveys hotspot regions of 50 oncogenes and tumor suppressor genes. The genes included in the panel were: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAS, GNAQ, HNF1A, HRAS, IDH1, JAK2, JAK3, IDH2, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, ST11, TP53 and VHL. Each library was barcoded using Ion Xpress™ Barcode Adapters kit (Ion Torrent; Thermo Fisher Scientific, Inc.).

Genomic DNA extraction was performed on paraffin-embedded tissue sections using the H&E-stained section as a guide and a cutoff of 60% tumor cellularity. DNA was amplified by multiplex PCR of targeted sequences in 50 genes using the Ion AmpliSeq Cancer Hotspot Panel v2 to generate an amplicon library. The genes included in this panel were ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11,GNAS,GNAQ, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1,MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL. The library was then clonally amplified by emulsion PCR, enriched and sequenced using the Ion AmpliSeq Cancer Hotspot Panel (v2, Thermo Fisher). The detection limit of this assay is 5% mutant alleles in a background of wild-type alleles. Reported variants from early cases were re-confirmed not to represent germline variants.

The genetic analysis of tumor specimens was performed by amplifying the extracted DNA (10 ng) using barcode adaptors (Ion Xpress Barcode Adapters 1–96 Kit, Life Technologies) with the Ion AmpliSeq Cancer Hotspot panel v.2 (Thermo Fisher), which contains 207 primer pairs and targets approximately 2800 hotspot mutations in the following 50 cancer‐related genes from the COSMIC database⁹: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAS, GNAQ, HNF1A, HRAS, IDH1, JAK2, JAK3, IDH2, KDR/VEGFR2, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, and VHL. Barcoded libraries were amplified using emulsion PCR on Ion Sphere particles, and sequencing was performed with an Ion Chef System and an Ion Proton Sequencer (Life Technologies) using an Ion PI Hi‐Q Chef Kit (Life Technologies). Research data obtained in this study are not shared.