title: Heterogeneity of colorectal cancer (CRC) in reference to KRAS proto-oncogene utilizing WAVE technology.
abstract: BACKGROUND
  New drugs targeting specific genes required for unregulated growth and metastases have improved survival rates for patients with metastatic colorectal cancer. Resistance to monoclonal antibodies specific for the epidermal growth factor receptor (EGFR) has been attributed to the presence of activating point mutations in the proto-oncogene KRAS. The use of EGFR inhibitor monotherapy in patients that have KRAS wild type has produced response rates of only 10-20%. The molecular basis for clinical resistance remains poorly understood. We propose two possible explanations to explain these low response rates; 1) levels of resistant CRC cells carrying mutated KRAS are below the sensitivity of standard direct sequencing modalities (<5%) or 2) the standard practice of analyzing a single area within a heterogeneous tumor is a practice that can overlook areas with mutated KRAS.
  
  
  METHODS
  In a collaborative effort with the surgical and molecular pathology departments, 3 formalin fixed paraffin embedded tissue blocks of human CRC were obtained from the human tissue bank maintained by the Lifespan Pathology Department and/or the human tissue bank maintained by the Molecular Pathology Core of the COBRE for Cancer Research Development. The three specimens previously demonstrated KRAS mutations detected by the Applied Biosystems Kit. The Wave system 4500 (high performance ion-pairing liquid chromatography (IP-HPLC)) was utilized to evaluate tissue for the presence of KRAS proto-oncogene mutations at codons 12 and 13.
  
  
  RESULTS
  Initially, the sensitivity of WAVE technology was compared with direct sequencing by evaluating a dilutional series. WAVE detected mutant alleles at levels of 2.5% compared to 20% performed with standard direct sequencing. Samples from three patients were evaluated by WAVE technology. Eight samples from patient 1 were analyzed. In two of eight samples, no mutations were detected at concentrations as low as 5%. In one sample a mutation was noted by WAVE and not by direct sequencing. All four samples from patient 2 tested positive for Exon 12/13 mutations. Of the seven samples from patient 3, five were positive for Exon 12/13 mutations and two were negative for Exon 12/13 mutations.
  
  
  CONCLUSIONS
  In these studies the analysis of three patients' colorectal cancer tissues were analyzed utilizing the WAVE technology. Results demonstrated a greater degree of sensitivity in mutation detection when compared to standard sequencing. These studies also demonstrated heterogeneity of expression of KRAS mutations between areas of the tissue samples at a genomic level. The low clinical response rates to EGFR inhibition might be explained by the variation in mutation presence, which was dependent upon the region examined. The heterogeneity demonstrated in these studies provides another phenotypic variant that will impact clinical care.
authors.affiliation: Warren Alpert School of Medicine of Brown University, Providence, RI, USA. kperez@lifespan.org
authors.forename: K
authors.initials: K
authors.lastname: Perez
authors.forename: R
authors.initials: R
authors.lastname: Walsh
authors.forename: K
authors.initials: K
authors.lastname: Brilliant
authors.forename: L
authors.initials: L
authors.lastname: Noble
authors.forename: E
authors.initials: E
authors.lastname: Yakirevich
authors.forename: V
authors.initials: V
authors.lastname: Breese
authors.forename: C
authors.initials: C
authors.lastname: Jackson
authors.forename: D
authors.initials: D
authors.lastname: Chatterjee
authors.forename: V
authors.initials: V
authors.lastname: Pricolo
authors.forename: L
authors.initials: L
authors.lastname: Roth
authors.forename: N
authors.initials: N
authors.lastname: Shah
authors.forename: T
authors.initials: T
authors.lastname: Cataldo
authors.forename: H
authors.initials: H
authors.lastname: Safran
authors.forename: D
authors.initials: D
authors.lastname: Hixson
authors.forename: P
authors.initials: P
authors.lastname: Quesenberry
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D006801:Humans; D016612:Paraffin Embedding; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
keywords: Colorectal cancer; KRAS; WAVE system