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

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).
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.
Electronic supplementary material

The online version of this article (doi:10.1186/s12943-016-0549-8) contains supplementary material, which is available to authorized users

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Molecular Cancer. 15 (2016), 63, DOI 10.1186/s12943-016-0549-8, issn: 1476-4598 ; IN COPYRIGHT http://rightsstatements.org/page/InC/1.0 rs

Classification and Topics

• Keyword: Metastase ; Krebs ; Onkologie

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2016
• Creator: Kovaleva, Valentina ; Geißler, Anna-Lena ; Lutz, Lisa Magdalena ; Fritsch, Ralph ; Makowiec, Frank
• Contributor: Deutsches Krebsforschungszentrum ; Albert-Ludwigs-Universität Freiburg. Fakultät für Biologie ; Universitätsklinikum Freiburg. Institut für Klinische Pathologie ; DKTK German Cancer Consortium ; Tumorzentrum Freiburg - CCCF ; Universitätsklinikum Freiburg. Klinik für Innere Medizin I ; Albert-Ludwigs-Universität Freiburg. Centre for Biological Signalling Studies ; Klinik für Allgemein- und Viszeralchirurgie. Freiburg im Breisgau ; Klinik für Thoraxchirurgie ; Albert-Ludwigs-Universität Freiburg ; Albert-Ludwigs-Universität Freiburg. Medizinische Fakultät

Further information

• DOI: 10.1186/s12943-016-0549-8
• URN: urn:nbn:de:bsz:25-freidok-126287
• Rights: Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 14.08.2025, 10:46 AM CEST