Abstract 2906: Characterization of genetic intratumor heterogeneity of colorectal cancer and matching organoids
Authors: Arnadottir Sigrid S. , Jeppesen Maria , Lamy Philippe , Nordentoft Iver , Knudsen Michael , Vang Søren , Madsen Mogens R. , Thastrup Jacob , Thastrup Ole , Andersen Claus L. 
Published: July 2017

Abstract
Background: In the recent years it has become evident that intratumor heterogeneity of solid tumors, such as colorectal cancer (CRC), complicates development of efficient therapy strategies. A minor clone of the tumor might have the ability for treatment resistance and hence the ability for re-establishing the tumor. Organoid cultures derived from the patients’ tumors can be used for ex vivo drug screening prior to treatment of the patient. However this approach is only efficient if the organoids represent the heterogeneity of the tumor. In this study we aim to characterize the genetic intratumor heterogeneity of CRC by performing whole exome sequencing (WES) on multiple biopsies per tumor, and to see how well the heterogeneity is reflected in matching organoid cultures.
Methods: From five CRC patients, three biopsies were collected from separate areas of each tumor. Each biopsy was divided in two: one half was fresh frozen for DNA purification, and the other half was grown in vitro as organoid culture. When available, lymph node metastases (LNMs) were included. WES was performed using SeqCap EZ Exome v3.0, and Illumina NextSeq500. Matched germline DNA was used as reference to identify somatic mutations and copy number variations (CNVs) using Mutect2 and FACETS, respectively.
Results: Each tumor contained multiple mutations that were present in all biopsies and in the organoids as well, representing a common ancestral branch. However all tumor biopsies and organoids also contained private mutations. These constituted on average 10% of the mutations (range 3-18%) indicating spatial genetic heterogeneity in all tumors. Each organoid had private mutations not seen in the tumor area of origin and vice versa, indicating that each area contained multiple clones and only a subset was represented in the organoids. The extent of mutational differences between organoids and their area of origin was similar to the mutational differences between tumor areas. Implying that organoids do not reflect their local origin better than a single biopsy reflects the whole tumor. In one patient with LNMs it was observed that the mutational profile of the metastases resembled only one of the examined tumor areas. Surprisingly, not all the ancestral mutations found in the tumor biopsies were observed in the LNMs, suggesting that at least two clones co-existed in the area of origin and just one of these formed the metastases.
Conclusion: In the five patients studied; spatial genetic heterogeneity was observed, meaning that multiple biopsies are needed to picture the whole tumor. Genetic heterogeneity was also observed between primary tumor and metastases and our data support that the metastases were formed from a single cancer clone that did not dominate the primary tumor. From a genetic point of view, organoids do not seem to fully reflect the tumor area of origin and less so the whole tumor, indicating that care should be taken when using organoids as models of the primary tumor.
Citation Format: Sigrid S. Arnadottir, Maria Jeppesen, Philippe Lamy, Iver Nordentoft, Michael Knudsen, Søren Vang, Mogens R. Madsen, Jacob Thastrup, Ole Thastrup, Claus L. Andersen. Characterization of genetic intratumor heterogeneity of colorectal cancer and matching organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2906. doi:10.1158/1538-7445.AM2017-2906