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Somatic Ephrin Receptor Mutations Are Associated with Metastasis In Published OnlineFirst January 20, 2017; DOI: 10.1158/0008-5472.CAN-16-1921 Cancer Tumor and Stem Cell Biology Research Somatic Ephrin Receptor Mutations Are Associated with Metastasis in Primary Colorectal Cancer Lucy Mathot1, Snehangshu Kundu1, Viktor Ljungstrom€ 1, Jessica Svedlund2, Lotte Moens1, TomAdlerteg1, Elin Falk-Sorqvist€ 1,Veronica Rendo1, Claudia Bellomo3, Markus Mayrhofer4, Carme Cortina5, Magnus Sundstrom€ 1, Patrick Micke1, Johan Botling1, Anders Isaksson4, Aristidis Moustakas3, Eduard Batlle5, Helgi Birgisson6, Bengt Glimelius1, Mats Nilsson1,2, and Tobias Sjoblom€ 1 Abstract The contribution of somatic mutations to metastasis of colo- of mutant EPH receptors. To enable functional studies of EPHB1 rectal cancers is currently unknown. To find mutations involved in mutations, we demonstrated that DLD-1 colorectal cancer cells the colorectal cancer metastatic process, we performed deep expressing EPHB1 form aggregates upon coculture with ephrin mutational analysis of 676 genes in 107 stages II to IV primary B1 expressing cells. When mutations in the fibronectin type III colorectal cancer, of which half had metastasized. The mutation and kinase domains of EPHB1 were compared with wild-type prevalence in the ephrin (EPH) family of tyrosine kinase receptors EPHB1 in DLD-1 colorectal cancer cells, they decreased ephrin was 10-fold higher in primary tumors of metastatic colorectal B1–induced compartmentalization. These observations provide a than in nonmetastatic cases and preferentially occurred in stage III mechanistic link between EPHB receptor mutations and metas- and IV tumors. Mutational analyses in situ confirmed expression tasis in colorectal cancer. Cancer Res; 77(7); 1–11. Ó2017 AACR. Introduction colorectal cancer patients into those at a higher risk of devel- oping metastatic disease is of great clinical importance, as the Colorectal cancer develops due to a series of well-character- decision to give adjuvant therapy following resection of the ized mutations that arise in a particular order and affect both primary tumor is difficult (4). Many patients who may never oncogenes and tumor suppressor genes (TSG; ref. 1). The develop metastatic disease receive chemotherapy resulting in progression of colorectal cancer has been extensively studied unnecessary side effects, and a number of patients who would and characterized on a genetic level, due to the availability of benefit from elimination of micrometastases following radical biopsies from various stages in disease progression, that is, from resection remain unidentified. Discovery of mutations that can benign polyps to advanced carcinomas (2). However, while predict development of metastatic disease would help to reduce much is known about frequent mutations causing colorectal the number of patients who are overtreated, and could help to cancer, the genetic basis of metastasis is unclear (3). Stratifying increase the overall survival for higher risk patients. Although several features of cancer genomes have been associated with the dissemination of colorectal cancer, spe- fi 1Science for Life Laboratory, Department of Immunology, Genetics and Pathol- ci c gene mutations related to metastatic processes have yet to fi ogy, Uppsala University, Sweden. 2Science for Life Laboratory, Department of be con rmed. For example, loss of 1p36 has consistently been Biochemistry and Biophysics, Stockholm University, Solna, Sweden. 3Depart- associated with metastatic disease (5, 6) and mutations in the ment of Medical Biochemistry and Microbiology, Ludwig Cancer Research, tumor suppressor FBXW7 did not occur with distant metas- Science for Life Laboratory, Uppsala University, Sweden. 4Science for Life tases (7, 8). If metastasis-causing mutations arise late in Laboratory, Department of Medical Sciences, Uppsala University, Sweden. tumor development, they may be subclonal and therefore 5Oncology Programme, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain. 6Depart- present at a low frequency in the primary tumor (9). For this ment of Surgical Sciences, Colorectal Surgery, Uppsala University, Sweden. reason, and as the vast majority of genetic events known to contribute to colorectal cancer development are base-level Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). somatic mutations, we applied targeted deep sequencing to determine the mutational spectrum of pathways and systems L. Mathot and S. Kundu contributed equally to this work. associated with colorectal cancer development. Our aim was Corresponding Author: Tobias Sjoblom,€ Uppsala University, IGP, Dag Ham- to analyze tumor samples from metastatic and nonmetastatic € marskjold v 20, SE-751 85 Uppsala, Sweden. Phone: 46184715036; Fax: patients in order to identify less frequently mutated candidate 46184714808; E-mail: [email protected] cancer genes with high sensitivity to gain insight into the doi: 10.1158/0008-5472.CAN-16-1921 mutational spectrum of these tumors and discover novel Ó2017 American Association for Cancer Research. mechanisms of metastasis. www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst January 20, 2017; DOI: 10.1158/0008-5472.CAN-16-1921 Mathot et al. Table 1. Summary of patients and sequencing Instability phenotype CIN (n ¼ 83) MSI (n ¼ 24) Yes, at Yes, at Distant metastases No Yes, during follow-up diagnosis No Yes, during follow-up diagnosis Stage II III II III IV II III II III IV Cases 15 16 7 20 25 8 10 2 2 2 Postoperative adjuvant 11117 — 08 0 0 — therapy Median no. of mutations 10 9.5 9 9 8 83 69.5 81.5 87 128.5 per sample (range) (2–13) (6–16) (3–17) (1–36) (3–33) (7–122) (10–161) (61–102) (82–92) (87–170) Point mutations 111 146 55 181 221 330 530 99 112 169 InDels 21 12 10 25 30 201 228 64 62 88 NOTE: The protein coding regions of 676 genes in 107 T/N pairs, representing 100 colon cancer and 7 rectal cancer cases, were enriched and sequenced by Illumina sequencing to >1,000-fold average sequence depth followed by mutational analysis. Tumors were considered MSI if 1offive Bethesda markers showed instability. Materials and Methods tion coverage of the region of interest was required to be greater than 90% and the mean read depth >600-fold. Samples not Study design passing these criteria on the first attempt were resequenced and We aimed to collect 20 to 25 cases each of stages II and III sequencing data from both runs was merged. Samples not exceed- colorectal cancers with and without distant metastases, as well as ing >600-fold mean read depth thereafter were accepted if the 25 stage IV cases (6, 10). Fresh frozen tissue and whole blood mean read depth was greater than 250-fold (three T/N pairs). samples from 112 colorectal cancer patients [224 matched tumor/ normal (T/N) paired samples] were collected (Table 1). Sequenc- Statistical analyses ing libraries prepared from gDNA extracted from all patients Illumina sequencing adaptors were removed by cutAdapt ver- (tumor and matched normal tissue or blood) was sequenced on sion 0.9.5 (13) and the trimmed reads were subsequently aligned an Illumina HiSeq platform and a somatic mutation analysis was to the reference genome (hg19, March 2009 assembly) using performed. All data were included if the T/N pairs were correctly MosaikAligner version 2.1.33 allowing for a maximum of 5% matched, a read depth >250-fold was obtained for >90% of the mismatches in a read. A software for somatic mutation analysis in sequencing region of interest and the tumor was >40%. deep sequence datasets, ConfIdent (Adlerteg and colleagues; manuscript in preparation), was used for somatic mutation Patient samples detection. We assessed which genes were significantly mutated  m DNA was extracted from 196 frozen tissue samples (3 10- m- in CIN and MSI tumors using a combination of MutSigCV (14), thick sections) on a liquid handling workstation (Tecan Evo 150 number of nonsynonymous mutations per Mb and the nonsy- MCA LiHa RoMa; ref. 11). DNA from 28 EDTA whole blood nonymous to synonymous ratio (NS: S). We used the following samples was extracted using a QIAamp DNA Blood Midi Kit cut-offs for CIN tumors: Q-value of <0.1 for MutSigCV output, >1 fi (Qiagen). DNA was quanti ed using the Qubit HS dsDNA Kit mutation per Mb, and >2:1 NS: S ratio. Differences in the average (Invitrogen by Life Technologies). For the validation cohort of number of mutations and in mutation prevalence between met- BMPR2 metastatic colorectal cancer samples used for mutation astatic and nonmetastatic groups were assessed using Welch t test detection, DNA from 19 microsatellite unstable (MSI) FFPE and Fisher exact test. The Bonferroni method was used to correct tumor sections was extracted using the QIAamp DNA FFPE for multiple comparisons. Tissue Kit (Qiagen). In situ mutation analysis with padlock probes Tumor tissue purity, determination of genomic stability, Fresh frozen 4 mm sections of colon tumors with EPH receptor and T/N matching mutations were mounted on Superfrost Plus slides (ThermoFisher Affymetrix SNP 6.0 microarrays were used to assess tumor Scientific). Because of limited sample availability, a total of 15 purity and to confirm that samples had 40% TCC as initially EPH receptor mutations in 12 tumors were assessed by in situ assessed histologically. Microsatellite instability status was deter- mutation detection (two tumor sections contained two separate mined using MSI Analysis System, version 1.2 (ProMega) with sections, called 1 and 2). Probes specific for EPH receptor muta- 6 ng genomic DNA and analysis of five mononucleotide repeat tions were designed with one general and one specific detection Bethesda markers (BAT25, BAT26, NR-21, NR-24, and MONO- oligo sequence (Supplementary Table S1). Probe performance 27) on a 3130xl genetic analyzer (Applied Biosystems). T/N pair was first tested in T47D and U251 cell lines with known ephrin matching was performed using an MLGA based genotyping receptor mRNA expression levels (Supplementary Table S2).
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