title: Sanger sequencing in routine KRAS testing: a review of 1720 cases from a pathologist's perspective.
abstract: BACKGROUND
  Sanger sequencing (SS) of PCR products is still the most frequent method to test colorectal cancer for KRAS mutations in routine practice.
  
  
  METHODS
  An audit of SS on 1720 routine cases was carried out, taking into account age, gender, specimen type (resection vs biopsies), tumour site (primary vs metastasis), tumour stage, neoplastic cells abundance (>30% vs <30%) and fixation type (buffered formalin vs simple formalin). In a subset of 50 wild-type (WT) patients correlations between SS findings and response rate (RR), progression-free survival (PFS) and overall survival (OS) were also evaluated.
  
  
  RESULTS
  The tests were informative in 1691 cases (98.3%). Mutations were detected in 671 cases (39.6%). No significant differences in mutation rates were observed with respect to age (p=0.2), gender (p=0.2), specimen type (p=0.3) and formalin fixation (p=0.08). Conversely, KRAS mutant rate was higher in metastatic tissue (50% vs 39%, p=0.02), in samples with over 30% of neoplastic cells (43.4% vs 26.6%, p=0.02) and in tumours tested in stage IV (p=0.05). The RR of SS KRAS WT patients was 26% (one complete and 12 partial responses). The disease control rate (objective responses plus stable disease) was 56%. Median PFS was 4.4 months and median OS was 10.4 months.
  
  
  CONCLUSIONS
  Pathological criteria that make SS a more robust method for KRAS testing and treatment response prediction are neoplastic cell abundance, metastatic tissue sample and stage IV primary tumour.
authors.affiliation: Scienze Biomorfologiche e Funzionali, Universitá degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Umberto
authors.initials: U
authors.lastname: Malapelle
authors.forename: Claudio
authors.initials: C
authors.lastname: Bellevicine
authors.forename: Maria
authors.initials: M
authors.lastname: Salatiello
authors.forename: Caterina
authors.initials: C
authors.lastname: de Luca
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Rispo
authors.forename: Palmira
authors.initials: P
authors.lastname: Riccio
authors.forename: Lucianna
authors.initials: L
authors.lastname: Sparano
authors.forename: Alfonso
authors.initials: A
authors.lastname: De Stefano
authors.forename: Chiara
authors.initials: C
authors.lastname: Carlomagno
authors.forename: Francesco Maria
authors.initials: FM
authors.lastname: Maiello
authors.forename: Giulia
authors.initials: G
authors.lastname: Vita
authors.forename: Oscar
authors.initials: O
authors.lastname: Nappi
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Troncone
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D018572:Disease-Free Survival; D005260:Female; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000235:genetics*