title: 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: 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.
  
  
  
  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).
  
  
  
  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).
  
  
  
  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.
authors.affiliation: Institute for Surgical Pathology, Medical Center-Faculty of Medicine, University of Freiburg, Breisacherstr. 115A, 79106, Freiburg, Germany.|German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Valentina
authors.initials: V
authors.lastname: Kovaleva
authors.affiliation: Institute for Surgical Pathology, Medical Center-Faculty of Medicine, University of Freiburg, Breisacherstr. 115A, 79106, Freiburg, Germany.|German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.|Faculty of Biology, University of Freiburg, Freiburg, Germany.
authors.forename: Anna-Lena
authors.initials: AL
authors.lastname: Geissler
authors.affiliation: Institute for Surgical Pathology, Medical Center-Faculty of Medicine, University of Freiburg, Breisacherstr. 115A, 79106, Freiburg, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Lutz
authors.affiliation: Department of Medicine I, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany.|Comprehensive Cancer Center Freiburg, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Ralph
authors.initials: R
authors.lastname: Fritsch
authors.affiliation: Comprehensive Cancer Center Freiburg, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany.|Department of General and Visceral Surgery, Medical Center- Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Makowiec
authors.affiliation: Comprehensive Cancer Center Freiburg, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany.|Department of Thoracic Surgery, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Wiesemann
authors.affiliation: Comprehensive Cancer Center Freiburg, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany.|Department of General and Visceral Surgery, Medical Center- Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Ulrich T
authors.initials: UT
authors.lastname: Hopt
authors.affiliation: Comprehensive Cancer Center Freiburg, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany.|Department of Thoracic Surgery, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Bernward
authors.initials: B
authors.lastname: Passlick
authors.affiliation: Institute for Surgical Pathology, Medical Center-Faculty of Medicine, University of Freiburg, Breisacherstr. 115A, 79106, Freiburg, Germany.|German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.|Comprehensive Cancer Center Freiburg, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Werner
authors.affiliation: Institute for Surgical Pathology, Medical Center-Faculty of Medicine, University of Freiburg, Breisacherstr. 115A, 79106, Freiburg, Germany. silke.lassmann@uniklinik-freiburg.de.|German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany. silke.lassmann@uniklinik-freiburg.de.|Comprehensive Cancer Center Freiburg, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg, Germany. silke.lassmann@uniklinik-freiburg.de.|BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany. silke.lassmann@uniklinik-freiburg.de.
authors.forename: Silke
authors.initials: S
authors.lastname: Lassmann
mesh_terms: D000328:Adult; D000368:Aged; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology*; D004252:DNA Mutational Analysis; D005260:Female; D015723:Gene Library; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000556:secondary; D008175:Lung Neoplasms / Q000556:secondary*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009367:Neoplasm Staging; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics
chemical_list: D016283:Proto-Oncogene Proteins p21(ras)
keywords: Colorectal cancer; FFPE; Metastases; Next generation sequencing