Intra-Tumor Heterogeneity of Colorectal Cancer Necessitates the Multi-Regional Sequencing for Comprehensive Mutational Profiling

Abstract

Background

The panorama and details of quantitative intratumor heterogeneity have not been fully investigated in colorectal cancer (CRC) patients with solitary lesion without distal metastasis, and its influences on sequencing interpretation and therapeutic strategies have not been explored.

Methods

Cancer tissues and matched blood from 70 sporadic CRC patients were collected and were divided into two cohorts. Four individual tissue biopsies were obtained from each of the 47 patients (multi-sample cohort). One random cancer tissue biopsy was obtained from each of the rest 23 patients (single-sample cohort). A 10 mL of blood was collected from all patients and the circulating cell-free DNA (cfDNA) was extracted. A 605-gene panel was used for targeted sequencing with tissue and paired blood.

Results

Mutational landscape revealed significantly higher mutational frequency in APC, CARD11 and CSMD3 in multi-sample cohort than single-sample cohort (P<0.05). The number of mutations and the ratio of trunk, shared and branch mutations showed extensive heterogeneity in multi-sample cohort, and the percentage of trunk mutations in major driver genes, including APC, TP53 and KRAS, was higher than 70%. A total of 929 mutations were detected in tissue/blood in multi-sample group, with 921(99.1%) from tissue and 472(50.8%) from blood (464 common mutations,49.9%). In contrast, 394 mutations were detected in tissue/blood in single-sample group, with 231 (58.6%) from tissue and 219 (55.6%) from blood (56 common mutations, 11.9%). The number of mutations of major driver genes detected in tissue was higher than that in blood in the multi-sample cohort, while it was similar in the single-sample group. Quantification analysis revealed differential correlation between tissue and blood VAF in trunk, shared and branch mutations. Meanwhile, VAF of trunk mutations was significantly higher than shared mutations and branch mutations. VAF exhibited significant differences among distinct stages, locations, differentiation and sex status.

Conclusion

Characteristic extensive heterogeneity was revealed for solitary CRC without distal metastasis. Multi-regional biopsy was necessary for comprehensive mutation detection in CRC.

Keywords:

• colorectal cancer
• multi-region sequencing
• intra-tumor heterogeneity
• circulating tumor DNA
• ctDNA
• APC
• KRAS
• TP53

Data Sharing Statement

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Please contact Dr Lele Song for dataset availability issues.

Ethics Statement

This study was approved by the Chinese PLA General Hospital Ethics Committee and conducted in accordance with the hospital’s guiding principles. Written informed consent and consent to publication were waived for the use of the clinical samples as retrospective samples were used in this study, and the fact that the study only revealed statistical data without disclosing any personal, clinical or sequencing data. Patient data were kept confidential, and the study was performed in compliance with the Declaration of Helsinki.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

Xinyi Liu, Mingyan Xu and Lele Song were employees of HaploX Biotechnology, who performed the NGS sequencing and data analysis in this study. Other authors declare no conflicts of interest in this study.