Accurate detection of genomic alterations using high-throughput sequencing is an essential component of precision cancer medicine. We characterize the variant allele fractions (VAFs) of somatic single nucleotide variants and indels across 5095 clinical samples profiled using a custom panel, CancerSCAN. Our results demonstrate that a significant fraction of clinically actionable variants have low VAFs, often due to low tumor purity and treatment-induced mutations. The percentages of mutations under 5% VAF across hotspots in EGFR, KRAS, PIK3CA, and BRAF are 16%, 11%, 12%, and 10%, respectively, with 24% for EGFR T790M and 17% for PIK3CA E545. For clinical relevance, we describe two patients for whom targeted therapy achieved remission despite low VAF mutations. We also characterize the read depths necessary to achieve sensitivity and specificity comparable to current laboratory assays. These results show that capturing low VAF mutations at hotspots by sufficient sequencing coverage and carefully tuned algorithms is imperative for a clinical assay.

Tumors continuously evolve to maintain growth; secondary mutations facilitate this process, resulting in high tumor heterogeneity. In this study, we compared mutations in paired primary and metastatic colorectal cancer tumor samples to determine whether tumor heterogeneity can predict tumor metastasis. Somatic variations in 46 pairs of matched primary-liver metastatic tumors and 42 primary tumors without metastasis were analyzed by whole-exome sequencing. Tumor clonality was estimated from single-nucleotide and copy-number variations. The correlation between clinical parameters of patients and clonal heterogeneity in liver metastasis was evaluated. Tumor heterogeneity across colorectal cancer samples was highly variable; however, a high degree of tumor heterogeneity was associated with a worse disease-free survival. Highly heterogeneous primary colorectal cancer was correlated with a higher rate of liver metastasis. Recurrent somatic mutations in , and were frequently detected in highly heterogeneous colorectal cancer. The variant allele frequency of these mutations was high, while somatic mutations in other genes such as and were low. The number and distribution of primary colorectal cancer subclones were preserved in metastatic tumors. Heterogeneity of primary colorectal cancer tumors can predict the potential for liver metastasis and thus, clinical outcome of patients. .

Chemokine ligand 7 (CCL7) enhances cancer progression and metastasis via epithelial-mesenchymal transition (EMT). However, little is known about the molecular mechanism of CCL7-induced EMT signaling cascade in colon cancer. Thus, the objective of this study was to investigate CCL7-induced EMT signaling pathway and its role in the progression and metastasis of colon cancer. To demonstrate the effect of CCL7 on EMT induction, HCT116 and HT29 cells overexpressing CCL7 were generated. CCL7-induced EMT and its downstream signaling pathway were evaluated by both in vitro and in vivo experiments. In in vitro studies, CCL7 was found to interplay with CC chemokine receptor 3 (CCR3), resulting in enhanced cellular proliferation, invasion, and migration via ERK and JNK signaling pathway. To validate these findings, we established ectopic and orthotopic mouse models injected with CCL7-overexpressed cells. In ectopic mouse models, we observed that CCL7-overexpressed cells grew significantly faster than control cells. In orthotopic mouse models, we found that liver and lung metastasis developed only in mice injected with CCL7-overexpressed cells. This study is the first one focusing on the EMT cascade via CCL7-CCR3-ERK-JNK signaling axis in colon cancer. Our novel findings will improve our understanding on the mechanism of metastatic process and provide potential therapeutic strategies for preventing metastasis in colon cancer.