Patients diagnosed with colorectal cancer were recruited with informed consent between July 2015 and June 2019 from the First Affiliated Hospital and the Second Affiliated Hospital of Wenzhou Medical University, China. Exclusion criteria include more than two pathological types, metastasis, treated with neoadjuvant chemotherapy or immunotherapy before surgery, or any cancer within the past 5 years. Tumor stages were determined according to the 8th edition of the American Joint Committee on Cancer (AJCC). The study was approved by the ethics committee of Wenzhou Medical University and its affiliated hospital.
Sample collection and DNA extraction
Resections or biopsies of primary solid tumors and adjacent tissues located 2-cm away from the tumor tissue, were taken immediately after surgery, snap frozen in liquid nitrogen, and stored at -80°C. DNA was extracted from tissue using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction, and stored at -20°C for further use. The DNA concentration was quantified by NanoDrop One (Thermo Fisher Scientific, Foster City, CA, USA).
FFPE tumor samples were analyzed by with hematoxylin-eosin (HE) staining to determine tumor purity. For each FFPE tissue, 20 slices of 10-μm-thick sections were used for DNA extraction with the QIAamp DNA FFPE Tissue kit (Qiagen). Concentration of DNA was determined by a Qubit 3.0 Fluorometer (Thermo Fisher Scientific).
CRC mutation panel design
The mutations were selected for either of the following three reasons: 1) present in at least two of the following three sources for CRC samples: TCGA (https://cancergenome.nih.gov/); International Cancer Genome Consortium (ICGC) database (https://icgc.org/); and publication by Muzny DM et al.; 2) potential resistance to CRC targeted therapy based on My Cancer Genome (http://www.mycancergenome.org), or 3) reported in a commercial cancer panel.
The CRC mutation panel was designed with Assay Designer software (MassARRAY Typer, Version 4.0, Agena Biosciences, San Diego, CA, USA), with primer sequences not overlapping known single nucleotide polymorphisms whenever possible.
CRC mutation detection and quantification by MALDI-TOF MS
CRC Mutation detection was optimized and performed by MassARRAY Analyzer 4 with CPM (Agena Biosciences). All primers were purchased from Integrated DNA Technologies (Integrated DNA Technologies, Coralville, IA, USA). All reagents were purchased from Agena Bioscience unless otherwise specified. Briefly, tissue genomic DNA was amplified by multiplex PCR. Shrimp alkaline phosphatase treatment was performed to inactivate surplus nucleotides. A primer extension reaction (iPLEX Pro) was performed with mass-modified terminator nucleotides, and the product was spotted on SpectroCHIP. Wild-type and mutant alleles were then discriminated by molecular weights determined by MassARRAY analyzer.
Allele calls were performed with MassARRAY Typer Analyzer software (Typer 4.0.26). Additionally, at least two investigators independently reviewed the mass spectra to further confirm the automated calls by the software. To estimate mutant allele frequency, the heights of raw spectral peaks corresponding to the wild-type and mutation allele were quantified. Mutation allele frequency was estimated by calculating mutant peak /(mutant peak + wild type peak).
Targeted NGS and data analysis
Genomic DNA was fragmented to an average size of 200 to 500 bp using Bioruptor Pico sonication device (Diagenode, Denville, NJ, USA). Sequencing libraries were prepared using the KAPA LTP Library Preparation Kit for Illumina (Kapa Biosystems, Wilmington, MA, USA) according to manufacturer's suggestions. Hybridization-based target enrichment was carried out with xGen Pan-Cancer Panel v2.4 (532 cancer-relevant genes), and xGen Lockdown Hybridization and Wash Reagents Kit (Integrated DNA Technologies). Captured libraries were amplified and purified using Agencourt AMPure XP Beads (Beckman Coulter, Atlanta, GA, USA). Concentrations and qualities of DNA libraries were analyzed by the Agilent High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA).
The libraries were paired-end sequenced on the Illumina HiSeq X platform (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Sequencing adapters and low quality bases were trimmed from raw sequencing reads using Trim Galore (v0.4.1; https:// github.com/FelixKrueger/TrimGalore). The filtered reads were then mapped to the reference Human Genome (hg38) using BWA-MEM (v0.7.12; https://github.com/lh3/bwa/tree/master/bwakit) with the default settings. The GATK (v18.104.22.168; https://software.broadinstitute.org/gatk/) was used for single nucleotide variation (SNV) identifications. SNV calls with at least 2.5% variant allele frequency (VAF) were retained, followed by annotation using ANNOVAR .
Mutation validation by capillary sequencing
A subset of samples were selected for Sanger sequencing to validate MS results. The capillary sequencing was performed with the BigDye Terminator Cycle Sequencing Kit and ABI 3730 Genetic Analyzer (Thermo Fisher Scientific).
Statistical analyses were performed with IBM SPSS Statistics 20.0. The χ2 test or Fisher exact test were used to compare baseline categorical variables, and the Kruskal-Wallis test was used to analyze the association between mutation and tumor size. The Cox proportional-hazards model of multivariate analysis was performed to analyze covariables, such as age, gender, clinical stage, location, differentiation, metastasis, treatment after surgery, and tumor size.