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

Abstract

Shaohua Guo,1,2,* Yumeng Ye,3,* Xinyi Liu,4 Yuan Gong,5 Mingyan Xu,4 Lele Song,4,6 Hongyi Liu1 1Department of General Surgery, The First Medical Center of the Chinese PLA General Hospital, Beijing, People’s Republic of China; 2Department of General Surgery, The Eighth Medical Center of the Chinese PLA General Hospital, Beijing, People’s Republic of China; 3Department of Experimental Pathology, Beijing Institute of Radiation Medicine, Beijing, People’s Republic of China; 4Department of Medical Division, HaploX Biotechnology, Shenzhen, People’s Republic of China; 5Department of Gastroenterology, The Second Medical Center of the Chinese PLA General Hospital, Beijing, People’s Republic of China; 6Department of Radiotherapy, The Eighth Medical Center of the Chinese PLA General Hospital, Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hongyi LiuDepartment of General Surgery, The First Medical Center of the Chinese PLA General Hospital, Beijing, People’s Republic of ChinaTel [phone]Email [email protected] SongDepartment of Radiotherapy, The Eighth Medical Center of the Chinese PLA General Hospital, Beijing, People’s Republic of ChinaTel [phone]Email [email protected]: 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

Keywords

• colorectal cancer
• multi-region sequencing
• intra-tumor heterogeneity
• circulating tumor dna
• ctdna
• apc
• kras
• tp53