Molecular characterization of circulating colorectal tumor cells defines genetic signatures for individualized cancer care

// Say Li Kong 1 , Xingliang Liu 1 , Nur-Afidah Mohamed Suhaimi 2 , Kenneth Jia Hao Koh 1 , Min Hu 2 , Daniel Yoke San Lee 2 , Igor Cima 2, 3, 4 , Wai Min Phyo 2, 5 , Esther Xing Wei Lee 2 , Joyce A. Tai 1 , Yu Miin Foong 2 , Jess Honganh Vo 2, 5 , Poh Koon Koh 2, 6 , Tong Zhang 1 , Jackie Y. Ying 2 , Bing Lim 1 , Min-Han Tan 2, 5, 6 and Axel M. Hillmer 1, 7 1 Genome Institute of Singapore, Singapore 138672, Singapore 2 Institute of Bioengineering and Nanotechnology, Singapore 138669, Singapore 3 German Cancer Consortium (DKTK), Essen/Dusseldorf, Heidelberg 69120, Germany 4 German Cancer Research Center (DKFZ), Heidelberg 69120, Germany 5 Lucence Diagnostics Pte Ltd, Singapore 159555, Singapore 6 Concord Cancer Hospital Singapore, Singapore 289891, Singapore 7 TRON-Translational Oncology at the University Medical Center of The Johannes Gutenberg University gGmbH, Mainz 55131, Germany Correspondence to: Say Li Kong, email: kongsl@gis.a-star.edu.sg Axel M. Hillmer, email: hillmer@gis.a-star.edu.sg Keywords: circulating tumor cells, colorectal cancer, targeted sequencing, druggable mutation, mutation signatures Received: October 12, 2016     Accepted: June 02, 2017     Published: July 10, 2017 ABSTRACT Studies on circulating tumor cells (CTCs) have largely focused on platform development and CTC enumeration rather than on the genomic characterization of CTCs. To address this, we performed targeted sequencing of CTCs of colorectal cancer patients and compared the mutations with the matched primary tumors. We collected preoperative blood and matched primary tumor samples from 48 colorectal cancer patients. CTCs were isolated using a label-free microfiltration device on a silicon microsieve. Upon whole genome amplification, we performed amplicon-based targeted sequencing on a panel of 39 druggable and frequently mutated genes on both CTCs and fresh-frozen tumor samples. We developed an analysis pipeline to minimize false-positive detection of somatic mutations in amplified DNA. In 60% of the CTC-enriched blood samples, we detected primary tumor matching mutations. We found a significant positive correlation between the allele frequencies of somatic mutations detected in CTCs and abnormal CEA serum level. Strikingly, we found driver mutations and amplifications in cancer and druggable genes such as APC, KRAS, TP53, ERBB3 , FBXW7 and ERBB2 . In addition, we found that CTCs carried mutation signatures that resembled the signatures of their primary tumors. Cumulatively, our study defined genetic signatures and somatic mutation frequency of colorectal CTCs. The identification of druggable mutations in CTCs of preoperative colorectal cancer patients could lead to more timely and focused therapeutic interventions.