Spatio-temporal mutation profiles of case-matched colorectal carcinomas and their metastases reveal unique de novo mutations in metachronous lung metastases by targeted next generation sequencing

Kovaleva et al. Molecular Cancer (2016) 15:63 DOI 10.1186/s12943-016-0549-8 RESEARCH Open Access Spatio-temporal mutation profiles of casematched colorectal carcinomas and their metastases reveal unique de novo mutations in metachronous lung metastases by targeted next generation sequencing Valentina Kovaleva1,2†, Anna-Lena Geissler1,2,3†, Lisa Lutz1, Ralph Fritsch4,5, Frank Makowiec5,6, Sebastian Wiesemann5,7, Ulrich T. Hopt5,6, Bernward Passlick5,7, Martin Werner1,2,5 and Silke Lassmann1,2,5,8* Abstract Background: Targeted next generation sequencing (tNGS) has become part of molecular pathology diagnostics for determining RAS mutation status in colorectal cancer (CRC) patients as predictive tool for decision on EGFR-targeted therapy. Here, we investigated mutation profiles of case-matched tissue specimens throughout the disease course of CRC, to further specify RAS-status dynamics and to identify de novo mutations associated with distant metastases. Methods: Case-matched formalin-fixed and paraffin-embedded (FFPE) resection specimens (n = 70; primary tumours, synchronous and/or metachronous liver and/or lung metastases) of 14 CRC cases were subjected to microdissection of normal colonic epithelial, primary and metastatic tumour cells, their DNA extraction and an adapted library protocol for limited DNA using the 48 gene TruSeq Amplicon Cancer PanelTM, MiSeq sequencing and data analyses (Illumina). Results: By tNGS primary tumours were RAS wildtype in 5/14 and mutated in 9/14 (8/9 KRAS exon 2; 1/9 NRAS Exon 3) of cases. RAS mutation status was maintained in case-matched metastases throughout the disease course, albeit with altered allele frequencies. Case-matched analyses further identified a maximum of three sequence variants (mainly in APC, KRAS, NRAS, TP53) shared by all tumour specimens throughout the disease course per individual case. In addition, further case-matched de novo mutations were detected in synchronous and/or metachronous liver and/or lung metastases (e.g. in APC, ATM, FBXW7, FGFR3, GNAQ, KIT, PIK3CA, PTEN, SMAD4, SMO, STK11, TP53, VHL). Moreover, several de novo mutations were more frequent in synchronous (e.g. ATM, KIT, PIK3CA, SMAD4) or metachronous (e.g. FBXW7, SMO, STK11) lung metastases. Finally, some de novo mutations occurred only in metachronous lung metastases (CDKN2A, FGFR2, GNAS, JAK3, SRC). (Continued on next page) * Correspondence: † Equal contributors 1 Institute for Surgical Pathology, Medical Center-Faculty of Medicine, University of Freiburg, Breisacherstr. 115A, 79106 Freiburg, Germany 2 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kovaleva et al. Molecular Cancer (2016) 15:63 Page 2 of 19 (Continued from previous page) Conclusion: Together, this study employs an adapted FFPE-based tNGS approach to confirm conservation of RAS mutation status in primary and metastatic tissue specimens of CRC patients. Moreover, it identifies genes preferentially mutated de novo in late disease stages of metachronous CRC lung metastases, several of which might be actionable by targeted therapies. Keywords: Colorectal cancer, Next generation sequencing, Metastases, FFPE Background Predictive molecular pathology mutation testing in selected genes is a routine diagnostic application in several epithelial tumour entities. This includes extended RAS testing (KRAS and NRAS exons 2,3,4) in advanced colorectal cancer (CRC) patients, which functions as well-established predictive biomarker for resistance to EGFR-targeted therapy (e.g. cetuximab or panitumumab) [1–5]. In addition, BRAF mutation testing is recommended for molecular grading of undifferentiated CRCs or as supportive tool for further molecular classification of microsatellite-instable CRCs [6]. Whilst dideoxy-Sanger sequencing, pyrosequencing or quantitative polymerase chain reaction-based assays were routinely used in the past, targeted next generation sequencing (tNGS) has by now evolved as a robust, time- and cost-efficient technique to accommodate the growing demand for mutation profiling in molecular pathology laboratories. Indeed, there are increasing numbers of reports on the general reliability and applicability of tNGS for mutation analyses of formalin-fixed and paraffin-embedded (FFPE) tissue specimens and/or the generation of tumour-entity specific tNGS gene panels, using different platforms and library preparation approaches [7–12]. In addition, several investigators compared primary colon and/or rectal tumours and metastases by NGS approaches in cohorts of 13 cases [13], 15 cases [14], 18 cases [15], 20 cases [16], 24 cases [17], 34 cases [18] or >400 cases [19]. These studies were based on fresh-frozen tissue specimens [15] or assessment of FFPE tissue specimens by amplicon-based semi-conductor NGS technology [13, 16, 17, 19], or evaluation of fresh-frozen tissue specimens with >70 % tumour cell content by whole exome sequencing [14, 18]. Reference to the otherwise mostly small tissue specimens and limited tumour cells of CRC liver or lung metastases in NGS performance and data interpretation is still sparse. Moreover, few of the previous studies focused on the individual clinicopathological and molecular characteristics of the investigated cases. One study [13] performed tNGS data analysis of 13 matched pairs of the primary tumour plus each one liver metastasis, reporting a 78 % match of mutations. In another study using semi-conductor NGS technology, mutations in for example APC, KRAS, FBXW7, PIK3CA, BRAF, SMAD4 were concordant between primary CRCs and their metastases, whereas 4 of 24 cases also showed de novo mutations in SYNE1, CTNNB1, TP53 and PTEN [17]. Similar findings were found in another study, which examined 17 paired primary and mainly synchronous metastatic CRC tissue specimens in a >400 CRC cohort [19]. In two whole exome sequencing based studies [14, 18], mutation profiles of key CRC associated genes were similar in primary and metastatic tumours by 53 % [14] and 57 % [18], with additional de novo mutations in metastases of 47 % [14] and 43 % [18]. No correlations of case-matched tumour specimens to clinico-pathological and molecular characteristics were made. Taken together, there is hence still a lack of studies which address the questi (...truncated)