Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress
Clinical Collections
Advances in Alzheimer’s

At a glance: The STEP trials

A round-up of the STEP phase 3 clinical trials evaluating semaglutide for weight loss in people with overweight or obesity.
ADVANCED SEARCH
Log in
Top
BMC Cancer
Published in: BMC Cancer 1/2013

Open Access 01-12-2013 | Research article

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

Authors: Joana G Guedes, Isabel Veiga, Patrícia Rocha, Pedro Pinto, Carla Pinto, Manuela Pinheiro, Ana Peixoto, Maria Fragoso, Ana Raimundo, Paula Ferreira, Manuela Machado, Nuno Sousa, Paula Lopes, António Araújo, Joana Macedo, Fernando Alves, Camila Coutinho, Rui Henrique, Lúcio L Santos, Manuel R Teixeira

Published in: BMC Cancer | Issue 1/2013

Login to get access

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.
Appendix
Available only for authorised users
Literature
1.
Krause DS, Van Etten RA: Tyrosine kinases as targets for cancer therapy. N Engl J Med. 2005, 353 (2): 172-187. 10.1056/NEJMra044389.CrossRefPubMed
2.
Ciardiello F, Tortora G: EGFR antagonists in cancer treatment. N Engl J Med. 2009, 360 (15): 1579-CrossRef
3.
Vogelstein B, Kinzler KW: Cancer genes and the pathways they control. Nat Med. 2004, 10 (8): 789-799. 10.1038/nm1087.CrossRefPubMed
4.
Jass JR: Classification of colorectal cancer based on correlation of clinical, morphological and molecular features. Histopathology. 2007, 50 (1): 113-130. 10.1111/j.1365-2559.2006.02549.x.CrossRefPubMed
5.
Roberts PJ, Der CJ: Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene. 2007, 26 (22): 3291-3310. 10.1038/sj.onc.1210422.CrossRefPubMed
6.
Amado RG, Wolf M, Peeters M, Van Cutsem E, Siena S, Freeman DJ, Juan T, Sikorski R, Suggs S, Radinsky R: Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol. 2008, 26 (10): 1626-1634. 10.1200/JCO.2007.14.7116.CrossRefPubMed
7.
Van Cutsem E, Kohne CH, Hitre E, Zaluski J, Chien CRC, Makhson A, D'Haens G, Pinter T, Lim R, Bodoky G: Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med. 2009, 360 (14): 1408-1417. 10.1056/NEJMoa0805019.CrossRefPubMed
8.
Bokemeyer C, Bondarenko I, Makhson A, Hartmann JT, Aparicio J, de Braud F, Donea S, Ludwig H, Schuch G, Stroh C: Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer. J Clin Oncol. 2009, 27 (5): 663-671. 10.1200/JCO.2008.20.8397.CrossRefPubMed
9.
Karapetis CS, Khambata-Ford S, Jonker DJ, O'Callaghan CJ, Tu D, Tebbutt NC, Simes RJ, Chalchal H, Shapiro JD, Robitaille S: K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med. 2008, 359 (17): 1757-1765. 10.1056/NEJMoa0804385.CrossRefPubMed
10.
Tol J, Koopman M, Cats A, Rodenburg CJ, Creemers GJM, Schrama JG, Erdkamp FLG, Vos AH, van Groeningen CJ, Sinnige HAM: Chemotherapy, bevacizumab, and cetuximab in metastatic colorectal cancer. N Engl J Med. 2009, 360 (6): 563-572. 10.1056/NEJMoa0808268.CrossRefPubMed
11.
Hecht JR, Mitchell E, Chidiac T, Scroggin C, Hagenstad C, Spigel D, Marshall J, Cohn A, McCollum D, Stella P: A randomized phase IIIB trial of chemotherapy, bevacizumab, and panitumumab compared with chemotherapy and bevacizumab alone for metastatic colorectal cancer. J Clin Oncol. 2009, 27 (5): 672-680. 10.1200/JCO.2008.19.8135.CrossRefPubMed
12.
Di Fiore F, Blanchard F, Charbonnier F, Le Pessot F, Lamy A, Galais MP, Bastit L, Killian A, Sesboue R, Tuech JJ: Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy. Br J Cancer. 2007, 96: 1166-1169. 10.1038/sj.bjc.6603685.CrossRefPubMedPubMedCentral
13.
Lievre A, Bachet JB, Boige V, Cayre A, Le Corre D, Buc E, Ychou M, Bouche O, Landi B, Louvet C: KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol. 2008, 26 (3): 374-379. 10.1200/JCO.2007.12.5906.CrossRefPubMed
14.
De Roock W, Piessevaux H, De Schutter J, Janssens M, De Hertogh G, Personeni N, Biesmans B, Van Laethem JL, Peeters M, Humblet Y: KRAS wild-type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab. Ann Oncol. 2008, 19 (3): 508-515.CrossRefPubMed
15.
Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W: Mutations of the BRAF gene in human cancer. Nature. 2002, 417 (6892): 949-954. 10.1038/nature00766.CrossRefPubMed
16.
Wood LD, Parsons DW, Jones S, Lin J, Sjoblom T, Leary RJ, Shen D, Boca SM, Barber T, Ptak J: The genomic landscapes of human breast and colorectal cancers. Science. 2007, 318: 1108-1113. 10.1126/science.1145720.CrossRefPubMed
17.
Pinto P, Rocha P, Veiga I, Guedes J, Pinheiro M, Peixoto A, Pinto C, Fragoso M, Sanches E, Araujo A: Comparison of methodologies for KRAS mutation detection in metastatic colorectal cancer. Cancer Genet. 2011, 204 (8): 439-446. 10.1016/j.cancergen.2011.07.003.CrossRefPubMed
18.
Lungu O, Wright TC, Silverstein S: Typing of human papillomaviruses by polymerase chain reaction amplification with L1 consensus primers and RFLP analysis. Mol Cell Probes. 1992, 6 (2): 145-152. 10.1016/0890-8508(92)90059-7.CrossRefPubMed
19.
Pearson H, Stirling D: DNA extraction from tissue. Methods Mol Biol. 2003, 226: 33-34.PubMed
20.
Simi L, Pratesi N, Vignoli M, Sestini R, Cianchi F, Valanzano R, Nobili S, Mini E, Pazzagli M, Orlando C: High-resolution melting analysis for rapid detection of KRAS, BRAF, and PIK3CA gene mutations in colorectal cancer. Am J Clin Pathol. 2008, 130 (2): 247-253. 10.1309/LWDY1AXHXUULNVHQ.CrossRefPubMed
21.
Willmore-Payne C, Holden JA, Tripp S, Layfield LJ: Human malignant melanoma: detection of BRAF- and c-kit-activating mutations by high-resolution amplicon melting analysis. Hum Pathol. 2005, 36 (5): 486-493. 10.1016/j.humpath.2005.03.015.CrossRefPubMed
22.
Pichler M, Balic M, Stadelmeyer E, Ausch C, Wild M, Guelly C, Bauemhofer T, Samonigg H, Hoefler G, Dandachi N: Evaluation of high-resolution melting analysis as a diagnostic tool to detect the BRAF V600E mutation in colorectal tumors. J Mol Diagn. 2009, 11 (2): 140-147. 10.2353/jmoldx.2009.080100.CrossRefPubMedPubMedCentral
23.
24.
De Roock W, Claes B, Bernasconi D, De Schutter J, Biesmans B, Fountzilas G, Kalogeras KT, Kotoula V, Papamichael D, Laurent-Puig P: Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol. 2010, 11 (8): 753-762. 10.1016/S1470-2045(10)70130-3.CrossRefPubMed
25.
Loupakis F, Ruzzo A, Cremolini C, Vincenzi B, Salvatore L, Santini D, Masi G, Stasi I, Canestrari E, Rulli E: KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer. Br J Cancer. 2009, 101 (4): 715-721. 10.1038/sj.bjc.6605177.CrossRefPubMedPubMedCentral
26.
Okayama N, Nishioka M, Hazama S, Sakai K, Suehiro Y, Maekawa M, Sakamoto J, Iwamoto S, Kato T, Mishima H: The importance of evaluation of DNA amplificability in KRAS mutation testing with dideoxy sequencing using formalin-fixed and paraffin-embedded colorectal cancer tissues. Jpn J Clin Oncol. 2011, 41 (2): 165-171. 10.1093/jjco/hyq173.CrossRefPubMed
27.
Laurent-Puig P, Cayre A, Manceau G, Buc E, Bachet JB, Lecomte T, Rougier P, Lievre A, Landi B, Boige V: Analysis of PTEN, BRAF, and EGFR status in determining benefit from cetuximab therapy in wild-type KRAS metastatic colon cancer. J Clin Oncol. 2009, 27 (35): 5924-5930. 10.1200/JCO.2008.21.6796.CrossRefPubMed
28.
Di Nicolantonio F, Martini M, Molinari F, Sartore-Bianchi A, Arena S, Saletti P, De Dosso S, Mazzucchelli L, Frattini M, Siena S: Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J Clin Oncol. 2008, 26 (35): 5705-5712. 10.1200/JCO.2008.18.0786.CrossRefPubMed
29.
Moroni M, Veronese S, Benvenuti S, Marrapese G, Sartore-Bianchi A, Di Nicolantonio F, Gambacorta M, Siena S, Bardelli A: Gene copy number for epidermal growth factor receptor (EGFR) and clinical response to antiEGFR treatment in colorectal cancer: a cohort study. Lancet Oncol. 2005, 6 (5): 279-286. 10.1016/S1470-2045(05)70102-9.CrossRefPubMed
30.
Tol J, Dijkstra JR, Klomp M, Teerenstra S, Dommerholt M, Vink-Borger ME, van Cleef PH, van Krieken JH, Punt CJ, Nagtegaal ID: Markers for EGFR pathway activation as predictor of outcome in metastatic colorectal cancer patients treated with or without cetuximab. Eur J Cancer. 2010, 46 (11): 1997-2009. 10.1016/j.ejca.2010.03.036.CrossRefPubMed
31.
Barault L, Veyrie N, Jooste V, Lecorre D, Chapusot C, Ferraz JM, Lievre A, Cortet M, Bouvier AM, Rat P: Mutations in the RAS-MAPK, PI(3)K (phosphatidylinositol-3-OH kinase) signaling network correlate with poor survival in a population-based series of colon cancers. Int J Cancer. 2008, 122 (10): 2255-2259. 10.1002/ijc.23388.CrossRefPubMed
32.
Oliveira C, Pinto M, Duval A, Brennetot C, Domingo E, Espin E, Armengol M, Yamamoto H, Hamelin R, Seruca R: BRAF mutations characterize colon but not gastric cancer with mismatch repair deficiency. Oncogene. 2003, 22 (57): 9192-9196. 10.1038/sj.onc.1207061.CrossRefPubMed
33.
Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE: Tumorigenesis - RAF/RAS oncogenes and mismatch-repair status. Nature. 2002, 418 (6901): 934-10.1038/418934a.CrossRefPubMed
34.
Lurkin I, Stoehr R, Hurst CD, van Tilborg AA, Knowles MA, Hartmann A, Zwarthoff EC: Two multiplex assays that simultaneously identify 22 possible mutation sites in the KRAS, BRAF, NRAS and PIK3CA genes. PLoS One. 2010, 5 (1): e8802-10.1371/journal.pone.0008802.CrossRefPubMedPubMedCentral
35.
Velho S, Oliveira C, Ferreira A, Ferreira AC, Suriano G, Schwartz S, Duval A, Carneiro F, Machado JC, Hamelin R: The prevalence of PIK3CA mutations in gastric and colon cancer. Eur J Cancer. 2005, 41 (11): 1649-1654. 10.1016/j.ejca.2005.04.022.CrossRefPubMed
36.
Sartore-Bianchi A, Di Nicolantonio F, Nichelatti M, Molinari F, De Dosso S, Saletti P, Martini M, Cipani T, Marrapese G, Mazzucchelli L: Multi-determinants analysis of molecular alterations for predicting clinical benefit to EGFR-targeted monoclonal antibodies in colorectal cancer. PLoS One. 2009, 4 (10): e7287-10.1371/journal.pone.0007287.CrossRefPubMedPubMedCentral
37.
Scheffzek K, Ahmadian MR, Kabsch W, Wiesmuller L, Lautwein A, Schmitz F, Wittinghofer A: The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science. 1997, 277 (5324): 333-338. 10.1126/science.277.5324.333.CrossRefPubMed
38.
Buhrman G, Wink G, Mattos C: Transformation efficiency of RasQ61 mutants linked to structural features of the switch regions in the presence of Raf. Structure. 2007, 15 (12): 1618-1629. 10.1016/j.str.2007.10.011.CrossRefPubMedPubMedCentral
39.
Janakiraman M, Vakiani E, Zeng Z, Pratilas CA, Taylor BS, Chitale D, Halilovic E, Wilson M, Huberman K, Ricarte Filho JC: Genomic and biological characterization of exon 4 KRAS mutations in human cancer. Cancer Res. 2010, 70 (14): 5901-5911. 10.1158/0008-5472.CAN-10-0192.CrossRefPubMedPubMedCentral
40.
Bourne HR, Sanders DA, McCormick F: The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991, 349 (6305): 117-127. 10.1038/349117a0.CrossRefPubMed
41.
Wan PTC, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, Jones CM, Marshall CJ, Springer CJ, Barford D: Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004, 116 (6): 855-867. 10.1016/S0092-8674(04)00215-6.CrossRefPubMed
42.
Ikenoue T, Kanai F, Hikiba Y, Obata T, Tanaka Y, Imamura J, Ohta M, Jazag A, Guleng B, Tateishi K: Functional analysis of PIK3CA gene mutations in human colorectal cancer. Cancer Res. 2005, 65 (11): 4562-4567. 10.1158/0008-5472.CAN-04-4114.CrossRefPubMed
43.
Samuels Y, Wang ZH, Bardelli A, Silliman N, Ptak J, Szabo S, Yan H, Gazdar A, Powell DM, Riggins GJ: High frequency of mutations of the PIK3CA gene in human cancers. Science. 2004, 304 (5670): 554-10.1126/science.1096502.CrossRefPubMed
44.
Samuels Y, Diaz LA, Schmidt-Kittler O, Cummins JM, DeLong L, Cheong I, Rago C, Huso DL, Lengauer C, Kinzler KW: Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell. 2005, 7 (6): 561-573. 10.1016/j.ccr.2005.05.014.CrossRefPubMed
45.
Miled N, Yan Y, Hon WC, Perisic O, Zvelebil M, Inbar Y, Schneidman-Duhovny D, Wolfson HJ, Backer JM, Williams RL: Mechanism of two classes of cancer mutations in the phosphoinositide 3-kinase catalytic subunit. Science. 2007, 317 (5835): 239-242. 10.1126/science.1135394.CrossRefPubMed
46.
Gymnopoulos M, Elsliger MA, Vogt PK: Rare cancer-specific mutations in PIK3CA show gain of function. Proc Natl Acad Sci USA. 2007, 104 (13): 5569-5574. 10.1073/pnas.0701005104.CrossRefPubMedPubMedCentral
47.
Zhao L, Vogt PK: Helical domain and kinase domain mutations in p110 alpha of phosphatidylinositol 3-kinase induce gain of function by different mechanisms. Proc Natl Acad Sci USA. 2008, 105 (7): 2652-2657. 10.1073/pnas.0712169105.CrossRefPubMedPubMedCentral
48.
Kang SY, Bader AG, Vogt PK: Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic. Proc Natl Acad Sci USA. 2005, 102 (3): 802-807. 10.1073/pnas.0408864102.CrossRefPubMedPubMedCentral
49.
Bader AG, Kang SY, Zhao L, Vogt PK: Oncogenic PI3K deregulates transcription and translation. Nat Rev Cancer. 2005, 5 (12): 921-929. 10.1038/nrc1753.CrossRefPubMed
50.
Huang CH, Mandelker D, Schmidt-Kittler O, Samuels Y, Velculescu VE, Kinzler KW, Vogelstein B, Gabelli SB, Amzel LM: The structure of a human p110alpha/p85alpha complex elucidates the effects of oncogenic PI3Kalpha mutations. Science. 2007, 318 (5857): 1744-1748. 10.1126/science.1150799.CrossRefPubMed
51.
Mao C, Liao RY, Qiu LX, Wang XW, Ding H, Chen Q: BRAF V600E mutation and resistance to anti-EGFR monoclonal antibodies in patients with metastatic colorectal cancer: a meta-analysis. Mol Biol Rep. 2011, 38 (4): 2219-2223. 10.1007/s11033-010-0351-4.CrossRefPubMed
Metadata
Title
High resolution melting analysis of KRAS, BRAF and PIK3CA in KRASexon 2 wild-type metastatic colorectal cancer
Authors
Joana G Guedes
Isabel Veiga
Patrícia Rocha
Pedro Pinto
Carla Pinto
Manuela Pinheiro
Ana Peixoto
Maria Fragoso
Ana Raimundo
Paula Ferreira
Manuela Machado
Nuno Sousa
Paula Lopes
António Araújo
Joana Macedo
Fernando Alves
Camila Coutinho
Rui Henrique
Lúcio L Santos
Manuel R Teixeira
Publication date
01-12-2013
Publisher
BioMed Central
Published in
BMC Cancer / Issue 1/2013
Electronic ISSN: 1471-2407
DOI
https://doi.org/10.1186/1471-2407-13-169

Other articles of this Issue 1/2013

BMC Cancer 1/2013 Go to the issue

Research article

Pegfilgrastim prophylaxis is associated with a lower risk of hospitalization of cancer patients than filgrastim prophylaxis: a retrospective United States claims analysis of granulocyte colony-stimulating factors (G-CSF)

Research article

High expression of ezrin predicts poor prognosis in uterine cervical cancer

Research article

Genetic variations of E6 and long control region of human papillomavirus type 16 from patients with cervical lesion in Liaoning, China

Research article

The cysteinyl leukotriene 2 receptor contributes to all-transretinoic acid-induced differentiation of colon cancer cells

Research article

The novel myxofibrosarcoma cell line MUG-Myx1 expresses a tumourigenic stem-like cell population with high aldehyde dehydrogenase 1 activity

Research article

The prevalence of depression and anxiety among Chinese adults with cancer: a systematic review and meta-analysis
Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras
Prof. Fabrice Barlesi
Developed by: Springer Medicine
Image Credits