JCT  Vol.4 No.2 , April 2013
Mutation Status of KRAS, BRAF, PIK3CA and Expression Level of AREG and EREG Identify Responders to Cetuximab in a Large Panel of Patient Derived Colorectal Carcinoma Xenografts of All Four UICC Stages
ABSTRACT

To advance preclinical testing of novel targeted drugs in colorectal cancer (CRC) we established a panel of 133 mouse xenograft models from fresh tumor specimens of 239 patients with CRC of all four UICC stages. A subgroup of 67 xenograft models was treated with cetuximab, bevacizumab and oxaliplatin as single agents. Mutation status of KRAS (G12, G13, A146T), BRAF (V600E) and PIK3CA (E542K, E545K, H1047R) was assessed in all xenografts by allelespecific real-time PCR. KRAS codon 61 was assessed by conventional sequencing. AREG and EREG expression levels were analyzed by real-time PCR expression assays. In the treatment experiment we observed response rates of 27% (18/67) for cetuximab, 3% (2/67) for bevacizumab, and 6% (4/67) for oxaliplatin. Classification based on KRAS, BRAF and PIK3CA mutation status identified 15 of the responders (sensitivity 83%, confidence interval at p = 0.05 (CI): 59% - 96%), and 38 nonresponders (specificity 78%, CI: 63% - 88%). If any mutation except in KRAS codon 13 were considered, the classifier reached sensitivity of 94% and specificity of 69%. We improved specificity of the classifiers to 90% and 86% respectively by adding AREG and EREG RNA expression thresholds retrospectively. In patient-derived xenograft models, we found a predictive classifier for response to cetuximab that is more accurate than established biomarkers. We confirmed its potential performance in primary human tumors. For patients, the classifier’s sensitivity promises increased response rates and its specificity limits unnecessary toxicity. Given the scope of our xenograft models across all UICC stages, this applies not only to mCRC but also to the adjuvant setting of earlier stages. The xenograft collection allows to mimic randomized phase II trials and to test novel drugs effectively as single agents or in combinations. It also enables the development of highly accurate companion diagnostics as demonstrated by us for cetuximab.


Cite this paper
P. Pechańska, M. Becker, T. Mayr, B. Hinzmann, H. Adams, I. Klaman, K. Kretschmar, K. Kretschmar, K. Stecker, R. Mantke, R. Pauli, J. Pertschy, K. Hertel, K. Ridwelski, K. Hellwig, M. Pross, C. Radke, I. Fichtner, J. Hoffmann and A. Rosenthal, "Mutation Status of KRAS, BRAF, PIK3CA and Expression Level of AREG and EREG Identify Responders to Cetuximab in a Large Panel of Patient Derived Colorectal Carcinoma Xenografts of All Four UICC Stages," Journal of Cancer Therapy, Vol. 4 No. 2, 2013, pp. 678-693. doi: 10.4236/jct.2013.42083.
References
[1]   A. Jemal, F. Bray, M. M. Center, J. Ferlay, E. Ward and D. Forman, “Global Cancer Statistics,” Cancer Journal for Clinicians, Vol. 61, No. 2, 2011, pp. 69-90. doi:10.3322/caac.20107

[2]   B. Vicuna and A. B. Benson III, “Adjuvant Therapy for Stage II Colon Cancer: Prognostic and Predictive Markers,” Journal of the National Comprehensive Cancer Network, Vol. 5, No. 9, 2007, pp. 927-936.

[3]   J. B. O’Connell, M. A. Maggard and C. Y. Ko, “Colon Cancer Survival Rates with the New American Joint Committee on Cancer Sixth Edition Staging,” Journal of the National Cancer Institute, Vol. 96, No. 19, 2004, pp. 1420-1425.

[4]   D. Cunningham, Y. Humblet, S. Siena, D. Khayat, H. Bleiberg, A. Santoro, D. Bets, M. Mueser, A. Harstrick, C. Verslype, I. Chau and E. Van Cutsem, “Cetuximab Monotherapy and Cetuximab plus Irinotecan in IrinotecanRefractory Metastatic Colorectal Cancer,” The New England Journal of Medicine, Vol. 351, No. 4, 2004, pp. 337-345. doi:10.1056/NEJMoa033025

[5]   F. F. Kabbinavar, J. Schulz, M. McCleod, T. Patel, J. T. Hamm, J. R. Hecht, R. Mass, B. Perrou, B. Nelson and W. F. Novotny, “Addition of Bevacizumab to Bolus Fluorouracil and Leucovorin in First-Line Metastatic Colorectal Cancer: Results of a Randomized Phase II Trial,” Journal of Clinical Oncology, Vol. 23, No. 16, 2005, pp. 3697-3705. doi:10.1200/JCO.2005.05.112

[6]   L. B. Saltz, H. J. Lenz, H. L. Kindler, H. S. Hochster, S. Wadler, P. M. Hoff, N. E. Kemeny, E. M. Hollywood, M. Gonen, M. Quinones, M. Morse and H. X. Chen, “Randomized Phase II Trial of Cetuximab, Bevacizumab, and Irinotecan Compared with Cetuximab and Bevacizumab Alone in Irinotecan-Refractory Colorectal Cancer: The BOND-2 Study,” Journal of Clinical Oncology, Vol. 25, No. 29, 2007, pp. 4557-4561. doi:10.1200/JCO.2007.12.0949

[7]   L. B. Saltz, S. Clarke, E. Diaz-Rubio, W. Scheithauer, A. Figer, R. Wong, S. Koski, M. Lichinitser, T. S. Yang, F. Rivera, F. Couture, F. Sirzen and J. Cassidy, “Bevacizumab in Combination with Oxaliplatin-Based Chemotherapy as First-Line Therapy in Metastatic Colorectal Cancer: A Randomized Phase III Study,” Journal of Clinical Oncology, Vol. 26, No. 12, 2008, pp. 2013-2019. doi:10.1200/JCO.2007.14.9930

[8]   E. Van Cutsem, C. H. Kohne, E. Hitre, J. Zaluski, C. R. Chang Chien, A. Makhson, G. D’Haens, T. Pinter, R. Lim, G. Bodoky, J. K. Roh, G. Folprecht, P. Ruff, C. Stroh, S. Tejpar, M. Schlichting, J. Nippgen and P. Rougier, “Cetuximab and Chemotherapy as Initial Treatment for Metastatic Colorectal Cancer,” The New England Journal of Medicine, Vol. 360, No. 2009, pp. 1408-1417.

[9]   E. Van Cutsem, M. Peeters, S. Siena, Y. Humblet, A. Hendlisz, B. Neyns, J. L. Canon, J. L. Van Laethem, J. Maurel, G. Richardson, M. Wolf and R. G. Amado, “OpenLabel Phase III Trial of Panitumumab plus Best Supportive Care Compared with Best Supportive Care alone in Patients with Chemotherapy-Refractory Metastatic Colorectal Cancer,” Journal of Clinical Oncology, Vol. 25, No. 13, 2007, pp. 1658-1664.

[10]   L. B. Saltz, N. J. Meropol, P. J. Loehrer Sr., M. N. Needle, J. Kopit and R. J. Mayer, “Phase II Trial of Cetuximab in Patients with Refractory Colorectal Cancer That Expresses the Epidermal Growth Factor Receptor,” Journal of Clinical Oncology, Vol. 22, No. 7, 2004, pp. 1201-1208. doi:10.1200/JCO.2004.10.182

[11]   M. H. Cohen, J. Gootenberg, P. Keegan and R. Pazdur, “FDA Drug Approval Summary: Bevacizumab plus FOLFOX4 as Second-Line Treatment of Colorectal Cancer,” The Oncologist, Vol. 12, No. 3, 2007, pp. 356-361. doi:10.1634/theoncologist.12-3-356

[12]   K. Y. Chung, J. Shia, N. E. Kemeny, M. Shah, G. K. Schwartz, A. Tse, A. Hamilton, D. Pan, D. Schrag, L. Schwartz, D. S. Klimstra, D. Fridman, D. P. Kelsen and L. B. Saltz, “Cetuximab Shows Activity in Colorectal Cancer Patients with Tumors That Do Not Express the Epidermal Growth Factor Receptor by Immunohistochemistry,” Journal of Clinical Oncology, Vol. 23, No. 9, 2005, pp. 1803-1810. doi:10.1200/JCO.2005.08.037

[13]   T. D. Barber, B. Vogelstein, K. W. Kinzler and V. E. Velculescu, “Somatic Mutations of EGFR in Colorectal Cancers and Glioblastomas,” The New England Journal of Medicine, Vol. 351, No. 27, 2004, p. 2883. doi:10.1056/NEJM200412303512724

[14]   C. J. Allegra, J. M. Jessup, M. R. Somerfield, S. R. Hamilton, E. H. Hammond, D. F. Hayes, P. K. McAllister, R. F. Morton and R. L. Schilsky, “American Society of Clinical Oncology Provisional Clinical Opinion: Testing for KRAS Gene Mutations in Patients with Metastatic Colorectal Carcinoma to Predict Response to Anti-Epidermal Growth Factor Receptor Monoclonal Antibody Therapy,” Journal of Clinical Oncology, Vol. 27, No. 12, 2009, pp. 2091-2096. doi:10.1200/JCO.2009.21.9170

[15]   A. Bardelli and S. Siena, “Molecular Mechanisms of Resistance to Cetuximab and Panitumumab in Colorectal Cancer,” Journal of Clinical Oncology, Vol. 28, No. 7, 2010, pp. 1254-1261. doi:10.1200/JCO.2009.24.6116

[16]   W. De Roock, D. J. Jonker, F. Di Nicolantonio, A. Sartore-Bianchi, D. Tu, S. Siena, S. Lamba, S. Arena, M. Frattini, H. Piessevaux, E. Van Cutsem, C. J. O’Callaghan, S. Khambata-Ford, J. R. Zalcberg, J. Simes, C. S. Karapetis, A. Bardelli and S. Tejpar, “Association of KRAS p.G13D Mutation with Outcome in Patients with ChemotherapyRefractory Metastatic Colorectal Cancer Treated with Cetuximab,” JAMA, Vol. 304, No. 16, 2010, pp. 1812-1820. doi:10.1001/jama.2010.1535

[17]   F. Di Nicolantonio, M. Martini, F. Molinari, A. SartoreBianchi, S. Arena, P. Saletti, S. De Dosso, L. Mazzucchelli, M. Frattini, S. Siena and A. Bardelli, “Wild-Type BRAF Is Required for Response to Panitumumab or Cetuximab in Metastatic Colorectal Cancer,” Journal of Clinical Oncology, Vol. 26, No. 35, 2008, pp. 5705-5712. doi:10.1200/JCO.2008.18.0786

[18]   W. De Roock, V. De Vriendt, N. Normanno, F. Ciardiello and S. Tejpar, “KRAS, BRAF, PIK3CA, and PTEN Mutations: Implications for Targeted Therapies in Metastatic Colorectal Cancer,” The Lancet Oncology, Vol. 12, No. 6, 2011, pp. 594-603. doi:10.1016/S1470-2045(10)70209-6

[19]   H. Prenen, J. De Schutter, B. Jacobs, W. De Roock, B. Biesmans, B. Claes, D. Lambrechts, E. Van Cutsem and S. Tejpar, “PIK3CA Mutations Are Not a Major Determinant of Resistance to the Epidermal Growth Factor Receptor Inhibitor Cetuximab in Metastatic Colorectal Cancer,” Clinical Cancer Research, Vol. 15, No. 9, 2009, pp. 3184-3188. doi:10.1158/1078-0432.CCR-08-2961

[20]   A. Sartore-Bianchi, F. Di Nicolantonio, M. Nichelatti, F. Molinari, S. De Dosso, P. Saletti, M. Martini, T. Cipani, G. Marrapese, L. Mazzucchelli, S. Lamba, S. Veronese, M. Frattini, A. Bardelli and S. Siena, “Multi-Determinants Analysis of Molecular Alterations for Predicting Clinical Benefit to EGFR-Targeted Monoclonal Antibodies in Colorectal Cancer,” PLOS ONE, Vol. 4, No. 10, 2009, Article ID: e7287. doi:10.1371/journal.pone.0007287

[21]   W. De Roock, B. Claes, D. Bernasconi, J. De Schutter, B. Biesmans, G. Fountzilas, K. T. Kalogeras, V. Kotoula, D. Papamichael, P. Laurent-Puig, F. Penault-Llorca, P. Rougier, B. Vincenzi, D. Santini, G. Tonini, F. Cappuzzo, M. Frattini, F. Molinari, P. Saletti, S. De Dosso, M. Martini, A. Bardelli, S. Siena, A. Sartore-Bianchi, J. Tabernero, T. Macarulla, F. Di Fiore, A. O. Gangloff, F. Ciardiello, P. Pfeiffer, C. Qvortrup, T. P. Hansen, E. Van Cutsem, H. Piessevaux, D. Lambrechts, M. Delorenzi and S. Tejpar, “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,” The Lancet Oncology, Vol. 11, No. 8, 2010, pp. 753-762. doi:10.1016/S1470-2045(10)70130-3

[22]   T. S. Maughan, R. A. Adams, C. G. Smith, A. M. Meade, M. T. Seymour, R. H. Wilson, S. Idziaszczyk, R. Harris, D. Fisher, S. L. Kenny, E. Kay, J. K. Mitchell, A. Madi, B. Jasani, M. D. James, J. Bridgewater, M. J. Kennedy, B. Claes, D. Lambrechts, R. Kaplan and J. P. Cheadle, “Addition of Cetuximab to Oxaliplatin-Based First-Line Combination Chemotherapy for Treatment of Advanced Colorectal Cancer: Results of the Randomised Phase 3 MRC COIN Trial,” Lancet, Vol. 377, No. 9783, 2011, pp. 2103-2114. doi:10.1016/S0140-6736(11)60613-2

[23]   S. Khambata-Ford, C. R. Garrett, N. J. Meropol, M. Basik, C. T. Harbison, S. Wu, T. W. Wong, X. Huang, C. H. Takimoto, A. K. Godwin, B. R. Tan, S. S. Krishnamurthi, H. A. Burris III, E. A. Poplin, M. Hidalgo, J. Baselga, E. A. Clark and D. J. Mauro, “Expression of Epiregulin and Amphiregulin and K-Ras Mutation Status Predict Disease Control in Metastatic Colorectal Cancer Patients Treated with Cetuximab,” Journal of Clinical Oncology, Vol. 25, No. 22, 2007, pp. 3230-3237. doi:10.1200/JCO.2006.10.5437

[24]   J. B. Baker, D. Dutta, D. Watson, T. Maddala, B. M. Munneke, S. Shak, E. K. Rowinsky, L. A. Xu, C. T. Harbison, E. A. Clark, D. J. Mauro and S. Khambata-Ford, “Tumour Gene Expression Predicts Response to Cetuximab in Patients with KRAS Wild-Type Metastatic Colorectal Cancer,” British Journal of Cancer, Vol. 104, No. 3, 2011, pp. 488-495. doi:10.1038/sj.bjc.6606054

[25]   M. Jhawer, S. Goel, A. J. Wilson, C. Montagna, Y. H. Ling, D. S. Byun, S. Nasser, D. Arango, J. Shin, L. Klampfer, L. H. Augenlicht, R. Perez-Soler and J. M. Mariadason, “PIK3CA Mutation/PTEN Expression Status Predicts Response of Colon Cancer Cells to the Epidermal Growth Factor Receptor Inhibitor Cetuximab,” Cancer Research, Vol. 68, No. 6, 2008, pp. 1953-1961. doi:10.1158/0008-5472.CAN-07-5659

[26]   R. M. Goldberg, D. J. Sargent, S. N. Thibodeau, M. R. Mahoney, A. F. Shields, E. Chan, S. Gill, M. S. Kahlenberg, S. Nair and S. R. Alberts, “Adjuvant MfolFOX6 plus or minus Cetuximab (Cmab) in Patients (pts) with KRAS Mutant (m) Resected Stage III Colon Cancer (CC): NCCTG Intergroup Phase III Trial N0147,” 2010 ASCO Annual Meeting, 2010.

[27]   C. J. Allegra, G. Yothers, M. J. O’Connell, S. Sharif, N. J. Petrelli, L. H. Colangelo, J. N. Atkins, T. E. Seay, L. Fehrenbacher, R. M. Goldberg, S. O’Reilly, L. Chu, C. A. Azar, S. Lopa and N. Wolmark, “Phase III Trial Assessing Bevacizumab in Stages II and III Carcinoma of the Colon: Results of NSABP Protocol C-08,” Journal of Clinical Oncology, Vol. 29, No. 1, 2011, pp. 11-16. doi:10.1200/JCO.2010.30.0855

[28]   A. de Gramont, “AVANT: Results from a Randomized, Three-Arm Multinational Phase III Study to Investigate Bevacizumab with Either XELOX or FOLFOX4 versus FOLFOX4 alone as Adjuvant Treatment for Colon Cancer,” 2011 Gastrointestinal Cancers Symposium, 2011.

[29]   W. Talloen, D. A. Clevert, S. Hochreiter, D. Amaratunga, L. Bijnens, S. Kass and H. W. Gohlmann, “I/NI-calls for the Exclusion of Non-Informative Genes: A Highly Effective Filtering Tool for Microarray Data,” Bioinformatics, Vol. 23, No. 21, 2007, pp. 2897-2902. doi:10.1093/bioinformatics/btm478

[30]   G. Caponigro and W. R. Sellers, “Advances in the Preclinical Testing of Cancer Therapeutic Hypotheses,” Nature Reviews Drug Discovery, Vol. 10, No. 3, 2011, pp. 179-187. doi:10.1038/nrd3385

[31]   “The Catalogue of Somatic Mutations in Cancer (COSMIC),” http://cancer.sanger.ac.uk/ cancergenome/projects/cosmic

[32]   A. Bertotti, G. Migliardi, F. Galimi, F. Sassi, D. Torti, C. Isella, D. Corà, F. Di Nicolantonio, M. Buscarino, C. Petti, D. Ribero, N. Russolillo, A. Muratore, P. Massucco, A. Pisacane, L. Molinaro, E. Valtorta, A. Sartore-Bianchi, M. Risio, L. Capussotti, M. Gambacorta, S. Siena, E. Medico, A. Sapino, S. Marsoni, P. Comoglio, A. Bardelli and L. Trusolino, “A Molecularly Annotated Platform of PatientDerived Xenografts (‘Xenopatients’) Identifies HER2 as an Effective Therapeutic Target in Cetuximab-Resistant Colorectal Cancer,” Cancer Discovery, 2011, pp. 508-523.

[33]   Y. Becouarn and P. Rougier, “Clinical Efficacy of Oxaliplatin Monotherapy: Phase II Trials in Advanced Colorectal Cancer,” Seminars in Oncology, Vol. 25, No. 2, 1998, pp. 23-31.

[34]   E. Diaz-Rubio, J. Sastre, A. Zaniboni, R. Labianca, H. Cortes-Funes, F. de Braud, C. Boni, M. Benavides, G. Dallavalle and M. Homerin, “Oxaliplatin as Single Agent in Previously Untreated Colorectal Carcinoma Patients: A Phase II Multicentric Study,” Annals of Oncology, Vol. 9, No. 1, 1998, pp. 105-108. doi:10.1023/A:1008200825886

[35]   D. Machover, E. Diaz-Rubio, A. de Gramont, A. Schilf, J. J. Gastiaburu, S. Brienza, M. Itzhaki, G. Metzger, D. N’Daw, J. Vignoud, A. Abad, E. Francois, E. Gamelin, M. Marty, J. Sastre, J. F. Seitz and M. Ychou, “Two Consecutive Phase II Studies of Oxaliplatin (L-OHP) for Treatment of Patients with Advanced Colorectal Carcinoma Who Were Resistant to Previous Treatment with Fluoropyrimidines,” Annals of Oncology, Vol. 7, No. 1, 1996, pp. 95-98. doi:10.1093/oxfordjournals.annonc.a010489

[36]   M. L. Rothenberg, A. M. Oza, R. H. Bigelow, J. D. Berlin, J. L. Marshall, R. K. Ramanathan, L. L. Hart, S. Gupta, C. A. Garay, B. G. Burger, N. Le Bail and D. G. Haller, “Superiority of Oxaliplatin and Fluorouracil-Leucovorin Compared with Either Therapy Alone in Patients with Progressive Colorectal Cancer after Irinotecan and Fluorouracil-Leucovorin: Interim Results of a Phase III Trial,” Journal of Clinical Oncology, Vol. 21, No. 11, 2003, pp. 2059-2069. doi:10.1200/JCO.2003.11.126

[37]   M. H. Cohen, J. Gootenberg, P. Keegan and R. Pazdur, “FDA Drug Approval Summary: Bevacizumab plus FOLFOX4 as Second-Line Treatment of Colorectal Cancer,” The Oncologist, Vol. 12, No. 3, 2007, pp. 356-361. doi:10.1634/theoncologist.12-3-356

[38]   C. J. Allegra, G. Yothers, M. J. O’Connell, S. Sharif, N. J. Petrelli, L. H. Colangelo, J. N. Atkins, T. E. Seay, L. Fehrenbacher, R. M. Goldberg, S. O’Reilly, L. Chu, C. A. Azar, S. Lopa and N. Wolmark, “Phase III Trial Assessing Bevacizumab in Stages II and III Carcinoma of the Colon: Results of NSABP Protocol C-08,” Journal of Clinical Oncology, Vol. 29, No. 1, 2011, pp. 11-16. doi:10.1200/JCO.2010.30.0855

[39]   M. Peeters, T. J. Price, A. Cervantes, A. F. Sobrero, M. Ducreux, Y. Hotko, T. Andre, E. Chan, F. Lordick, C. J. Punt, A. H. Strickland, G. Wilson, T. E. Ciuleanu, L. Roman, E. Van Cutsem, V. Tzekova, S. Collins, K. S. Oliner, A. Rong and J. Gansert, “Randomized Phase III Study of Panitumumab with Fluorouracil, Leucovorin, and Irinotecan (FOLFIRI) Compared with FOLFIRI Alone as Second-Line Treatment in Patients with Metastatic Colorectal Cancer,” Journal of Clinical Oncology, Vol. 28, No. 31, 2010, pp. 4706-4713. doi:10.1200/JCO.2009.27.6055

[40]   S. Guerrero, I. Casanova, L. Farre, A. Mazo, G. Capella and R. Mangues, “K-Ras Codon 12 Mutation Induces Higher Level of Resistance to Apoptosis and Predisposition to Anchorage-Independent Growth than Codon 13 Mutation or Proto-Oncogene Overexpression,” Cancer Research, Vol. 60, No. 23, 2000, pp. 6750-6756.

[41]   H. Prenen, J. De Schutter, B. Jacobs, W. De Roock, B. Biesmans, B. Claes, D. Lambrechts, E. Van Cutsem and S. Tejpar, “PIK3CA Mutations Are Not a Major Determinant of Resistance to the Epidermal Growth Factor Receptor Inhibitor Cetuximab in Metastatic Colorectal Cancer,” Clinical Cancer Research, Vol. 15, No. 9, 2009, pp. 3184-3188. doi:10.1158/1078-0432.CCR-08-2961

[42]   Z. Saridaki, M. Tzardi, C. Papadaki, M. Sfakianaki, F. Pega, A. Kalikaki, E. Tsakalaki, M. Trypaki, I. Messaritakis, E. Stathopoulos, D. Mavroudis, V. Georgoulias and J. Souglakos, “Impact of KRAS, BRAF, PIK3CA Mutations, PTEN, AREG, EREG Expression and Skin Rash in ≥2nd Line Cetuximab-Based Therapy of Colorectal Cancer Patients,” PLoS One, Vol. 6, No. 1, 2011, Article ID: e15980. doi:10.1371/journal.pone.0015980

 
 
Top