tissue sample
A total of 84 analytical samples were collected from surgical or biopsy specimens from 84 patients who underwent radical surgery for colorectal cancer at Saitama Medical University International Medical Center from January to December 2016. One case was excluded because the sample was too small. Therefore, we used metastatic lymph nodes instead of primary tumors. Double cancer was observed in 3 cases. In these cases, we selected the case with the greatest depth of tumor invasion or, if the depth was the same, the case with a lower degree of differentiation. Using hematoxylin and eosin-stained slides, tumor cells were located both visually and microscopically within the tissue specimen in consultation with a pathologist.
All patients underwent curative surgery followed by 5-FU-based adjuvant chemotherapy. Postoperative adjuvant chemotherapy consisted of a 5-FU: S-1-based regimen. capecitabine; tegafur-uracil and leucovorin calcium. Oxaliplatin combination therapy such as FOLFOX (5-FU, levofolinate, oxaliplatin), CAPOX (capecitabine and oxaliplatin), and SOX (S-1 and oxaliplatin). Oral uracil and tegafur plus leucovorin. Recurrence was defined as the date on which CRC recurrence was confirmed by imaging tests (computed tomography, magnetic resonance imaging, and positron emission tomography), endoscopy, or clinical examination. The follow-up period to monitor for recurrence was within 5 years after surgery. Clinical information was obtained by reviewing medical records and pathology reports (Table 1).
statistical analysis
Fisher’s exact test was performed to determine significant associations between genetic SNVs and cancer recurrence and non-recurrence (R package; https://bioconductor.org/packages/release/-bioc/html/edgeR.html). Logistic regression was used to test for confounders, Kaplan-Meier method was used to analyze overall survival, Student’s t test and Wilcoxon rank sum test were performed using JMP Pro 16 software (SAS Institute). was used to determine the mean value of the two groups. Inc., Cary, North Carolina, USA). All statistical tests are two-sided; p<0.05 was considered significant.
DNA extraction, quantification, and quality control
Samples from 84 patients were analyzed.Cancer areas were evaluated and recovered using previously reported methods [19]. Chromosomal DNA was isolated from formalin-fixed paraffin-embedded (FFPE) colorectal adenocarcinoma samples using the QIAamp DNA FFPE tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. DNA concentration was determined by measuring fluorescence using the Qubit dsDNA HS kit (Thermo Fisher Scientific, Waltham, MA, USA).
Targeted sequencing in our clinical CRC cases
We selected 50 autophagy-related genes and CRC-related genes and identified SNVs and insertions/deletions (INDELs) using targeted enrichment sequencing (see Additional file 1 : Table S1). . Several genes are required for autophagosome formation. They can be broadly divided into the following functional groups: three genes (PIK3R4, BECN1and ATG14Seven genes that contribute to the formation of the “Vps34 PI3 kinase complex” (MAP1LC3A, ATG3, ATG4A, ATG4B, ATG4C, ATG4Dand ATG7Five genes contributing to the “Atg8 binding system” (ATG5, ATG10, ATG12, ATG16L1and ATG16L2) Eight genes involved in the “Atg12 binding system” (ULK1, ATG13, RB1CC1, motor, raptor, Depter, AKT1S1and PTEN Genes required for the formation of “Atg1 protein kinase complex” (ATG9Aand ATG9B) important for “Atg9 and Atg2-Atg18 complexes” [20]. Furthermore, mitophagy is a selective mechanism responsible for autophagy-induced mitochondrial degradation and is involved in the metabolism of old mitochondria. 8 genes (pink 1, PRKN, BNIP3, BNIP3L, FUNDC1, opt-in, BCL2L13and Calcoco 2) contributes to “mitophagy receptors” [20].It was reported that class In colon models, induced autophagy proceeds through upregulation of the MEK/ERK pathway; classAnd autophagy contributes to the survival of CRC cells during starvation. 10 genes (class, NRAS, HRAS, United Arab Emirates, BRAF, RAF1, MAP2K1, map 2k2, map 1and map 3) Contributes to “RAS-MEK/ERK pathway” [21]. 6 genes (APC, CTNNB1, ERBB2, SMAD4, PIK3CAand TP53) Genes that are mutated in colorectal cancer were selected as colorectal cancer-related genes. [22]. The target regions were designed to be enriched in exonic regions and exon-intron junctions of all 50 genes (see Additional file 1: Table S1). The average percentile of target area covered was 98.49%.
Targeted capture and sequencing
A library of whole-genome sequences of all 50 known genes (see Additional file 1 : Table S1) was prepared using the HaloPlex Target Enrichment kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. Did. For each library, confirmation of enrichment and simple quantification of enriched target DNA was performed using the High Sensitivity D1000 Screen Tape System (Agilent Technologies). Pooled samples with different indices for multiplex sequencing were measured using a library quantification kit (Kapa Biosystems, Wilmington, MA, USA) to obtain molarity. High-throughput sequencing was performed on each pooled sample using 150 bp paired-end reads on a MiSeq or NextSeq platform (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.
Data analysis for next generation sequencing
Raw sequence read data passed FastQC quality check (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Read trimming with base quality was performed using FATX-toolkit v.0.0.14. [23]. Read alignment against the UCSC hg38 reference genome was performed using Burrows-Wheeler Aligner. [24].Unmappable reads were removed using SAMtools [25]. After filtering these reads, we used the Genome Analysis Toolkit (GATK) to perform local realignment and recalibration of the base quality score. We applied the GATK multiple sample calling protocol to detect SNVs and small INDELs. [26]. Coverage of the target area was estimated using GATK DepthOfCoverage. In this experiment, we used SelectVariants to select variants with “DP > 10” (coverage depth greater than 10 ×). Detected variants were annotated using ANNOVAR and pathogenicity was assessed using the ClinVar_20210501 database. [27].
Sanger sequence analysis
Sanger sequence analysis was performed to confirm the location of specific SNVs within the detected genes. PCR was performed using the PrimeSTAR Max DNA polymerase system (Takara Bio, Kusatsu, Japan). Thereafter, the PCR amplification product was extracted using QIAquick Gel Extraction Kit (QIAGEN). Reverse sequencing was performed according to the manufacturer’s instructions (BigDye; Applied Biosystems, Warrington, UK). Product sequencing was performed using an ABI 3500 automated DNA sequencer (Applied Biosystems).
Supplementary information
Target genes in selected clinical colorectal cancer cases (see Additional file 1: Table S1), correlations between all SNVs and recurrence rates (see Additional file 1: Table S2), and pathogenic SNVs and ClinVar-based Recurrence rate correlations (see Additional file 1: Table S2)File 1: Table S3) are described in Additional file 1.