title: Heterogeneity of KRAS Mutation Status in Rectal Cancer.
abstract: BACKGROUND
  Anti-EGFR targeted therapy is of increasing importance in advanced colorectal cancer and prior KRAS mutation testing is mandatory for therapy. However, at which occasions this should be performed is still under debate. We aimed to assess in patients with locally advanced rectal cancer whether there is intra-specimen KRAS heterogeneity prior to and upon preoperative chemoradiotherapy (CRT), and if there are any changes in KRAS mutation status due to this intervention.
  
  
  METHODS
  KRAS mutation status analyses were performed in 199 tumor samples from 47 patients with rectal cancer. To evaluate the heterogeneity between different tumor areas within the same tumor prior to preoperative CRT, 114 biopsies from 34 patients (mean 3 biopsies per patient) were analyzed (pre-therapeutic intratumoral heterogeneity). For the assessment of heterogeneity after CRT residual tumor tissue (85 samples) from 12 patients (mean 4.2 tissue samples per patient) were analyzed (post-therapeutic intratumoral heterogeneity) and assessment of heterogeneity before and after CRT was evaluated in corresponding patient samples (interventional heterogeneity). Primer extension method (SNaPshot™) was used for initial KRAS mutation status testing for Codon 12, 13, 61, and 146. Discordant results by this method were reevaluated by using the FDA-approved KRAS Pyro Kit 24, V1 and the RAS Extension Pyro Kit 24, V1 Kit (therascreen® KRAS test).
  
  
  RESULTS
  For 20 (43%) out of the 47 patients, a KRAS mutation was detected. With 12 out of 20, the majority of these mutations affected codon 35. We did not obtained evidence that CRT results in changes of the KRAS mutation pattern. In addition, no intratumoral heterogeneity in the KRAS mutational status could be proven. This was true for both the biopsies prior to CRT and the resection specimens thereafter. The discrepancy observed in some samples when using the SNaPshot™ assay was due to insufficient sensitivity of this technique upon massive tumor regression by CRT as application of the therascreen® KRAS test revealed concordant results.
  
  
  CONCLUSIONS
  Our results indicate that the KRAS mutation status at the primary tumor site of rectal cancer is homogenous. Its assessment for therapeutic decisions is feasible in pre-therapeutic biopsies as well as in post-therapeutic resected specimens. The amount of viable tumor cells seems to be an important determinant for assay sensitivity and should thus be considered for selection of the analytical method.
authors.affiliation: Department of General, Visceral and Pediatric Surgery, University Medical Center Goettingen, Goettingen, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Jo
authors.affiliation: Department of Gastroenterology and Gastrointestinal Oncology, University Medical Center Goettingen, Goettingen, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: König
authors.affiliation: Department of Clinical Pharmacology, University Medical Center Goettingen, Goettingen, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Schirmer
authors.affiliation: Department of Pathology, University Medical Center Goettingen, Goettingen, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Kitz
authors.affiliation: Department of General, Visceral and Pediatric Surgery, University Medical Center Goettingen, Goettingen, Germany.
authors.forename: Lena-Christin
authors.initials: LC
authors.lastname: Conradi
authors.affiliation: Department of General, Visceral and Pediatric Surgery, University Medical Center Goettingen, Goettingen, Germany.
authors.forename: Azadeh
authors.initials: A
authors.lastname: Azizian
authors.affiliation: Department of General, Visceral and Pediatric Surgery, University Medical Center Goettingen, Goettingen, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Bernhardt
authors.affiliation: Radiotherapy and Radiology Munich, Munich, Germany.
authors.forename: Hendrik A
authors.initials: HA
authors.lastname: Wolff
authors.affiliation: Department of General, Visceral and Pediatric Surgery, University Medical Center Goettingen, Goettingen, Germany.
authors.forename: Marian
authors.initials: M
authors.lastname: Grade
authors.affiliation: Department of General, Visceral and Pediatric Surgery, University Medical Center Goettingen, Goettingen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Ghadimi
authors.affiliation: Department of Pathology, University Medical Center Goettingen, Goettingen, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Ströbel
authors.affiliation: Department of Pathology, University Medical Center Goettingen, Goettingen, Germany.
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Schildhaus
authors.affiliation: Department of General, Visceral and Pediatric Surgery, University Medical Center Goettingen, Goettingen, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Gaedcke
mesh_terms: D000230:Adenocarcinoma / Q000145:classification / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D059186:Chemoradiotherapy, Adjuvant; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000235:genetics*; D009367:Neoplasm Staging; D018365:Neoplasm, Residual / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D011379:Prognosis; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D012004:Rectal Neoplasms / Q000145:classification / Q000235:genetics* / Q000473:pathology / Q000628:therapy
chemical_list: C117307:KRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)