title: Heterogeneity of KRAS, NRAS, BRAF and PIK3CA mutations in metastatic colorectal cancer and potential effects on therapy in the CAPRI GOIM trial.
abstract: BACKGROUND
  Evidence suggests that metastatic colorectal carcinoma (mCRC) has a high level of intratumor heterogeneity. We carried out a quantitative assessment of tumor heterogeneity for KRAS, NRAS, BRAF and PIK3CA mutations, in order to assess potential clinical implications.
  
  
  METHODS
  Tumor samples (n = 182) from the CAPRI-GOIM trial of first-line cetuximab + FOLFIRI in KRAS exon-2 wild-type mCRC patients were assessed by next-generation sequencing that allows quantitative assessment of mutant genes. Mutant allelic frequency was normalized for the neoplastic cell content and, assuming that somatic mutations usually affect one allele, the Heterogeneity Score (HS) was calculated by multiplying by 2 the frequency of mutant alleles in neoplastic cells. Therefore, HS virtually corresponds to the fraction of neoplastic cells carrying a specific mutation.
  
  
  RESULTS
  The KRAS HS ranged between 12 and 260 with mean value of 87.1 and median value of 84.4, suggesting that in most CRC, the majority of neoplastic cells carry mutant KRAS. Similar findings were observed for NRAS (HS range 35.5-146.7; mean 102.8; median 117.1). In contrast, in BRAF (HS range 17.1-120; mean 54.8; median 54.3) and PIK3CA (HS range 14.3-120; mean 59.5; median 47.3) mutant cases, only a fraction of neoplastic cells seem to carry the mutant allele. The response rate was 70% in KRAS mutant patients with an HS <33 (low KRAS; n = 10) and 45.7% in KRAS HS >33 patients (high KRAS; n = 35); median progression-free survival were 7.97 and 8.37 months, respectively. Low-KRAS tumors had a higher frequency of additional mutations in PIK3CA when compared with high-KRAS (6/10 versus 8/35).
  
  
  CONCLUSIONS
  KRAS and NRAS mutations are usually present in the majority of neoplastic cells, whereas BRAF and PIK3CA mutations often affect a limited fraction of transformed cells. Resistance to cetuximab in low-KRAS patients might be driven by the complex mutational profile rather than KRAS mutation load.
authors: Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori 'Fondazione Giovanni Pascale' IRCCS, Napoli Laboratory of Pharmacogenomics, Centro di Ricerche Oncologiche di Mercogliano (CROM)-Istituto Nazionale Tumori 'Fondazione Giovanni Pascale' IRCCS, Napoli nicnorm@yahoo.com n.normanno@istitutotumori.na.it.
authors: N
authors: N
authors: Normanno
authors: Laboratory of Pharmacogenomics, Centro di Ricerche Oncologiche di Mercogliano (CROM)-Istituto Nazionale Tumori 'Fondazione Giovanni Pascale' IRCCS, Napoli.
authors: A M
authors: AM
authors: Rachiglio
authors: Laboratory of Pharmacogenomics, Centro di Ricerche Oncologiche di Mercogliano (CROM)-Istituto Nazionale Tumori 'Fondazione Giovanni Pascale' IRCCS, Napoli.
authors: M
authors: M
authors: Lambiase
authors: Department of Clinical and Experimental Medicine 'F. Magrassi'-Medical Oncology, Seconda Università degli Studi di Napoli, Napoli.
authors: E
authors: E
authors: Martinelli
authors: Laboratory of Pharmacogenomics, Centro di Ricerche Oncologiche di Mercogliano (CROM)-Istituto Nazionale Tumori 'Fondazione Giovanni Pascale' IRCCS, Napoli.
authors: F
authors: F
authors: Fenizia
authors: Laboratory of Pharmacogenomics, Centro di Ricerche Oncologiche di Mercogliano (CROM)-Istituto Nazionale Tumori 'Fondazione Giovanni Pascale' IRCCS, Napoli.
authors: C
authors: C
authors: Esposito
authors: Laboratory of Pharmacogenomics, Centro di Ricerche Oncologiche di Mercogliano (CROM)-Istituto Nazionale Tumori 'Fondazione Giovanni Pascale' IRCCS, Napoli.
authors: C
authors: C
authors: Roma
authors: Department of Clinical and Experimental Medicine 'F. Magrassi'-Medical Oncology, Seconda Università degli Studi di Napoli, Napoli.
authors: T
authors: T
authors: Troiani
authors: GOIM, Gruppo Oncologico dell'Italia Meridionale, Bari.
authors: D
authors: D
authors: Rizzi
authors: Surgical Pathology Unit, Istituto Nazionale Tumori 'Fondazione Giovanni Pascale', IRCCS, Napoli.
authors: F
authors: F
authors: Tatangelo
authors: Surgical Pathology Unit, Istituto Nazionale Tumori 'Fondazione Giovanni Pascale', IRCCS, Napoli.
authors: G
authors: G
authors: Botti
authors: Medical Oncology Unit, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Foggia, Italy.
authors: E
authors: E
authors: Maiello
authors: GOIM, Gruppo Oncologico dell'Italia Meridionale, Bari.
authors: G
authors: G
authors: Colucci
authors: Department of Clinical and Experimental Medicine 'F. Magrassi'-Medical Oncology, Seconda Università degli Studi di Napoli, Napoli.
authors: F
authors: F
authors: Ciardiello
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D002166:Camptothecin / Q000031:analogs & derivatives / Q000627:therapeutic use; D002277:Carcinoma / Q000188:drug therapy / Q000235:genetics* / Q000556:secondary; D000068818:Cetuximab / Q000008:administration & dosage; D058534:Class I Phosphatidylinositol 3-Kinases; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005472:Fluorouracil / Q000627:therapeutic use; D020558:GTP Phosphohydrolases / Q000235:genetics; D005787:Gene Frequency; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D002955:Leucovorin / Q000627:therapeutic use; D008565:Membrane Proteins / Q000235:genetics; D009154:Mutation; D009944:Organoplatinum Compounds / Q000627:therapeutic use; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D016896:Treatment Outcome
chemical_list: C117307:KRAS protein, human; D008565:Membrane Proteins; D009944:Organoplatinum Compounds; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D000068818:Cetuximab; D002955:Leucovorin; D005472:Fluorouracil; D002166:Camptothecin
keywords: cetuximab; colorectal cancer; mutations; next-generation sequencing; tumor heterogeneity