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

Molecular Cancer

Research

Frank Makowiec¹  Ulrich T. Hopt¹  Bernward Passlick²  Sebastian Wiesemann²  Ralph Fritsch³  Lisa Lutz⁴  Valentina Kovaleva⁵  Martin Werner⁶  Silke Lassmann⁷  Anna-Lena Geissler⁸ 

[1]Comprehensive Cancer Center Freiburg, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany

[2]Department of General and Visceral Surgery, Medical Center- Faculty of Medicine, University of Freiburg, Freiburg, Germany

[3]Comprehensive Cancer Center Freiburg, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany

[4]Department of Thoracic Surgery, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany

[5]Department of Medicine I, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany

[6]Comprehensive Cancer Center Freiburg, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany

[7]Institute for Surgical Pathology, Medical Center-Faculty of Medicine, University of Freiburg, Breisacherstr. 115A, 79106, Freiburg, Germany

[8]Institute for Surgical Pathology, Medical Center-Faculty of Medicine, University of Freiburg, Breisacherstr. 115A, 79106, Freiburg, Germany

[9]German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany

[10]Institute for Surgical Pathology, Medical Center-Faculty of Medicine, University of Freiburg, Breisacherstr. 115A, 79106, Freiburg, Germany

[11]German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany

[12]Comprehensive Cancer Center Freiburg, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany

[13]Institute for Surgical Pathology, Medical Center-Faculty of Medicine, University of Freiburg, Breisacherstr. 115A, 79106, Freiburg, Germany

[14]German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany

[15]Comprehensive Cancer Center Freiburg, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany

[16]BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany

[17]Institute for Surgical Pathology, Medical Center-Faculty of Medicine, University of Freiburg, Breisacherstr. 115A, 79106, Freiburg, Germany

[18]German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany

[19]Faculty of Biology, University of Freiburg, Freiburg, Germany

关键词: Colorectal cancer;    Next generation sequencing;    Metastases;    FFPE;   

DOI  :  10.1186/s12943-016-0549-8

 received in 2016-07-05, accepted in 2016-10-06,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundTargeted 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.MethodsCase-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).ResultsBy 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).ConclusionTogether, 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.

【 授权许可】

CC BY   
© The Author(s). 2016