Open Access Highly Accessed Research article

High resolution melting analysis of KRAS, BRAF and PIK3CA in KRAS exon 2 wild-type metastatic colorectal cancer

Joana G Guedes1, Isabel Veiga1, Patrícia Rocha1, Pedro Pinto1, Carla Pinto1, Manuela Pinheiro1, Ana Peixoto1, Maria Fragoso2, Ana Raimundo2, Paula Ferreira2, Manuela Machado2, Nuno Sousa2, Paula Lopes3, António Araújo4, Joana Macedo4, Fernando Alves5, Camila Coutinho6, Rui Henrique38, Lúcio L Santos7 and Manuel R Teixeira18*

Author Affiliations

1 Departments of Genetics, Portuguese Oncology Institute, Porto, Portugal

2 Departments of Oncology, Portuguese Oncology Institute, Porto, Portugal

3 Departments of Pathology, Portuguese Oncology Institute, Porto, Portugal

4 S. Sebastião Hospital, Santa Maria da Feira, Portugal

5 Trás-os-Montes e Alto Douro Hospital Center, Vila Real, Portugal

6 Alto Ave Hospital Center, Guimarães, Portugal

7 Departments of Surgery, Portuguese Oncology Institute, Porto, Portugal

8 Abel Salazar Biomedical Sciences Institute (ICBAS), University of Porto, Porto, Portugal

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BMC Cancer 2013, 13:169  doi:10.1186/1471-2407-13-169

Published: 1 April 2013



KRAS is an EGFR effector in the RAS/RAF/ERK cascade that is mutated in about 40% of metastatic colorectal cancer (mCRC). Activating mutations in codons 12 and 13 of the KRAS gene are the only established negative predictors of response to anti-EGFR therapy and patients whose tumors harbor such mutations are not candidates for therapy. However, 40 to 60% of wild-type cases do not respond to anti-EGFR therapy, suggesting the involvement of other genes that act downstream of EGFR in the RAS-RAF-MAPK and PI3K-AKT pathways or activating KRAS mutations at other locations of the gene.


DNA was obtained from a consecutive series of 201 mCRC cases (FFPE tissue), wild-type for KRAS exon 2 (codons 12 and 13). Mutational analysis of KRAS (exons 3 and 4), BRAF (exons 11 and 15), and PIK3CA (exons 9 and 20) was performed by high resolution melting (HRM) and positive cases were then sequenced.


One mutation was present in 23.4% (47/201) of the cases and 3.0% additional cases (6/201) had two concomitant mutations. A total of 53 cases showed 59 mutations, with the following distribution: 44.1% (26/59) in KRAS (13 in exon 3 and 13 in exon 4), 18.6% (11/59) in BRAF (two in exon 11 and nine in exon 15) and 37.3% (22/59) in PIK3CA (16 in exon 9 and six in exon 20). In total, 26.4% (53/201) of the cases had at least one mutation and the remaining 73.6% (148/201) were wild-type for all regions studied. Five of the mutations we report, four in KRAS and one in BRAF, have not previously been described in CRC. BRAF and PIK3CA mutations were more frequent in the colon than in the sigmoid or rectum: 20.8% vs. 1.6% vs. 0.0% (P=0.000) for BRAF and 23.4% vs. 12.1% vs. 5.4% (P=0.011) for PIK3CA mutations.


About one fourth of mCRC cases wild-type for KRAS codons 12 and 13 present other mutations either in KRAS, BRAF, or PIK3CA, many of which may explain the lack of response to anti-EGFR therapy observed in a significant proportion of these patients.