Many studies failed to demonstrate benefit from the addition of targeted agents to current standard adjuvant FOLFOX chemotherapy in stage III colorectal cancer (CRC) patients. Intratumor heterogeneity may foster the resistant subclones and leads to cancer recurrence. Here, we built a cancer evolution model and applied machine learning analysis to identify potential therapeutic targets. Among 78 CRC cases, whole-genome (WGS) and deep targeted sequencing data generated from paired blood and primary tumor were used for phylogenetic tree reconstruction. Genetic alterations in the PI3K/ AKT, and RTK oncogenic signaling pathways were commonly detected in founding clones. The dominant subclones frequently exhibited dysregulations in the TP53, FBXW7/ NOTCH1 tumor suppression, and DNA repair pathways. Fourteen genetic mutations were simultaneously selected by random forest and LASSO methods. The logistic regression model had better accuracy (79%), precision (70%), and recall (65%) and area under the curve (AUC) (82%) for cancer recurrence prediction. Three genes, including MYO18A in the founding clone, FBXW7, and ATM in the dominant subclone, affected the prognosis were selected simultaneously by different feature sets. The in vitro studies, HCT-116 cells transfected with MYO18A siRNA demonstrated a significant reduction in cell migration activity by 20-40%. These results indicate that MYO18A plays a crucial role in the migration of human CRC cells. The cancer evolution model revealed the critical mutations in the founding and dominant subclones. They can be used to predict clinical outcomes and the development of novel therapeutic targets for stage III CRC.

Circulating cell-free DNA (cfDNA) analysis is an important tool for cancer monitoring. The patient-specific mutations identified in colorectal cancer (CRC) tissues are usually used to design the cfDNA analysis. Despite high specificity in predicting relapse, the sensitivity in most studies is around 40–50%. To improve this weakness, we designed a cfDNA panel according to the CRC genomic landscape and recurrent-specific mutations. The pathological variants in cfDNA samples from 60 CRC patients were studied by a next-generation sequencing (NGS) method incorporating the dual molecular barcode. Interestingly, patients in the disease positive group had a significantly higher cfDNA concentration than those in the disease negative group. Based on receiver operating characteristic analysis, the cfDNA concentration of 7 ng/mL was selected into the analytical workflow. The sensitivity in determining the disease status was 72.4%, which represented a considerable improvement on prior studies, and the specificity remained high at 80.6%. Compared to standard imaging and laboratory studies, earlier detection of residual disease and clinical benefits were shown on two cases by this cfDNA assay. We conclude this integrative framework of cfDNA analytical pipeline with a satisfactory sensitivity and specificity could be used in postoperative CRC surveillance.

Background Certain sequences of genomic mutations can lead to cancer formation and affect treatment outcomes and drug resistance. We constructed a cancer evolutionary tree using bulk-targeted deep sequencing to explore the impact of sequential and co-occurring somatic mutations on patients with stage III colorectal cancer (CRC). Methods A total of 108 stage III CRC patients from National Cheng Kung University Hospital (NCKUH) were recruited for this study between Jan. 2014 and Jan. 2019. Clinical information and tumor-targeted deep sequencing data were collected. Phylogenetic trees were reconstructed for evolutionary trajectories. We used a machine learning model for survival analysis. Results Six sequential somatic mutations stratified patients into seven subgroups based on survival. Patients carrying sequential germline followed by DNA damage response-related ATM or BRCA2 somatic mutations or non-TP53, APC somatic mutations had a better outcome than those without such mutations. The 4-year recurrence-free survival (RFS) probability was 88% in the low-risk group (G1) and 46% in the high-risk group (G2) (log-rank p-value 2e-05). The predictive efficacy by the area under the curve (AUC) was 0.73, 0.7, 0.797, and 0.88 at 2, 4, 6, and 8 years, respectively. The mutation status of mismatch repair (MMR) genes was not associated with RFS. Different genomic features were found between the groups. The orders of APC, KRAS and APC, BRCA2 sequential somatic mutations were associated with clinical outcomes. The occurrence of somatic mutations in BRCA2, such as TP53 somatic mutations, affected recurrence-free survival. Conclusions According to the evolution model, DNA damage response (DDR)-related ATM or BRCA2 somatic mutations are promising biomarkers for assessing the response of stage III CRC patients to oxaliplatin-based chemotherapy. The sequential order and co-occurring DDR somatic mutations are associated with recurrence-free survival.

The analysis of cell-free DNA (cfDNA) is rapidly emerging as a powerful approach to guide the clinical care of cancer patients. Several comprehensive cfDNA assays designed to detect mutations across several genes are now available. Here, we analyzed the use of a cfDNA panel in colorectal cancer (CRC) patients. Twenty-eight CRC patients with relapse or metastatic disease and 31 patients with no evidence of disease (NED) were enrolled. Genomic alterations in cfDNA were analyzed by the Oncomine™ Pan-Cancer Cell-Free Assay that detects hotspot mutations, small indels, copy number changes, and gene fusions across 52 genes. In the NED group, genomic alterations in cfDNA were detected in 12/31 patients (38.7%). The detection of alterations was more common in patients who were ≥60 years old, and the most common genomic alteration was a TP53 mutation. Fifty percent of the TP53 mutations were frequently or very frequently found in human cancers. Among 28 patients with relapse or metastatic disease, 22 (78.6%) had genomic alterations in cfDNA. The alterations were detected most frequently in TP53 (n = 10), followed by KRAS (n = 9). Actionable targets for CRC, including ERBB2 amplification and BRAF mutations, could be identified by this cfDNA assay. Compared with mutational profiling routinely analyzed using tumor samples, several additional targets with currently available therapies, including IDH1, IDH2, and PDGFRA mutations, were discovered. The cfDNA assay could identify potentially actionable targets for CRC. Identifying how to filter out cancer-like genomic alterations not derived from tumors remains a challenge.

In patients with unresectable metastatic disease confined to the liver, intra-arterial regional chemotherapy with implantable systems in an attractive option. Since April 1992, laparoscopic colorectal resections have been performed in our institution. Within this series of patients, three cases with bilateral liver metastasis from colon cancer were observed and underwent laparoscopic intra-arterial catheter implantation in the gastroduodenal artery for regional chemotherapy. In two patients the metastases were synchronous, and in both cases a laparoscopic colon resection was also performed, for tumors located in the cecum and in the sigmoid colon, respectively. The laparoscopic surgical technique for intra-arterial catheter implantation is described in detail. In this limited experience the procedure, from a purely technical point of view, was not considered difficult and was completed in 70 min on average. No complications were observed and the patient with metachronous liver metastasis was discharged on 3rd postoperative day.