Epidermal growth factor receptor immunohistochemistry: new opportunities in metastatic colorectal cancer

Journal of Translational Medicine

Peter W Hamilton³  Bharat Jasani²  Manuel Salto-Tellez³  Darragh G McArt³  Richard A Adams¹  Ryan A Hutchinson⁴ 

[1] Institute of Cancer and Genetics, Cardiff University School of Medicine, Institute of Medical Genetics Building, Heath Park, Cardiff CF14 4XN, UK;Department of Biomedical Sciences, Nazarbayev University School of Medicine, Astana 010000, Kazakhstan;Centre for Cancer Research and Cell Biology, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7AE, Northern Ireland, UK;Waring Laboratory, Department of Pathology, Centre for Translational Pathology, University of Melbourne, Parkville 3010, VIC, Australia

关键词: Localisation;    Image analysis;    Metastatic colorectal cancer;    Heterogeneity;    Personalised medicine;    Immunohistochemistry;    Epidermal growth factor receptor;   

Others  :  1221471
DOI  :  10.1186/s12967-015-0531-z

 received in 2015-01-24, accepted in 2015-05-12,  发布年份 2015

【 摘 要 】

The treatment of cancer is becoming more precise, targeting specific oncogenic drivers with targeted molecular therapies. The epidermal growth factor receptor has been found to be over-expressed in a multitude of solid tumours. Immunohistochemistry is widely used in the fields of diagnostic and personalised medicine to localise and visualise disease specific proteins. To date the clinical utility of epidermal growth factor receptor immunohistochemistry in determining monoclonal antibody efficacy has remained somewhat inconclusive. The lack of an agreed reproducible scoring criteria for epidermal growth factor receptor immunohistochemistry has, in various clinical trials yielded conflicting results as to the use of epidermal growth factor receptor immunohistochemistry assay as a companion diagnostic. This has resulted in this test being removed from the licence for the drug panitumumab and not performed in clinical practice for cetuximab. In this review we explore the reasons behind this with a particular emphasis on colorectal cancer, and to suggest a way of resolving the situation through improving the precision of epidermal growth factor receptor immunohistochemistry with quantitative image analysis of digitised images complemented with companion molecular morphological techniques such as in situ hybridisation and section based gene mutation analysis.

【 授权许可】

   
2015 Hutchinson et al.

【 图 表 】

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Figure2.

【 参考文献 】

• [1]Prahallad A, Sun C, Huang S, Di Nicolantonio F, Salazar R, Zecchin D et al.. Unresponsiveness of colon cancer to BRAF[V600E] inhibition through feedback activation of EGFR. Nature. 2012; 483(7387):100-103.
• [2]Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW et al.. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004; 350(21):2129-2139.
• [3]Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S et al.. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. 2007; 448(7153):561-566.
• [4]Corcoran RB, Ebi H, Turke AB, Coffee EM, Nishino M, Cogdill AP et al.. EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib. Cancer Discov. 2012; 2(3):227-235.
• [5]Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000; 100(1):57-70.
• [6]Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011; 144(5):646-674.
• [7]Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2001; 2(2):127-137.
• [8]Yarden Y. The EGFR family and its ligands in human cancer. Signalling mechanisms and therapeutic opportunities. Eur J Cancer. 2001; 37 Suppl 4:S3-S8.
• [9]Henriksen L, Grandal MV, Knudsen SL, van Deurs B, Grovdal LM. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands. PLoS One. 2013; 8(3):e58148.
• [10]McCourt CM, Boyle D, James J, Salto-Tellez M. Immunohistochemistry in the era of personalised medicine. J Clin Pathol. 2013; 66(1):58-61.
• [11]Anagnostou VK, Welsh AW, Giltnane JM, Siddiqui S, Liceaga C, Gustavson M et al.. Analytic variability in immunohistochemistry biomarker studies. Cancer Epidemiol Biomarkers Prev. 2010; 19(4):982-991.
• [12]Rivenbark AG, O’Connor SM, Coleman WB. Molecular and cellular heterogeneity in breast cancer: challenges for personalized medicine. Am J Pathol. 2013; 183(4):1113-1124.
• [13]Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA, Kinzler KW. Cancer genome landscapes. Science. 2013; 339(6127):1546-1558.
• [14]Burrell RA, McGranahan N, Bartek J, Swanton C. The causes and consequences of genetic heterogeneity in cancer evolution. Nature. 2013; 501(7467):338-345.
• [15]Aparicio S, Caldas C. The implications of clonal genome evolution for cancer medicine. N Engl J Med. 2013; 368(9):842-851.
• [16]Almendro V, Marusyk A, Polyak K. Cellular heterogeneity and molecular evolution in cancer. Annu Rev Pathol. 2013; 24(8):277-302.
• [17]de Bruin EC, Taylor TB, Swanton C. Intra-tumor heterogeneity: lessons from microbial evolution and clinical implications. Genome Med. 2013; 5(11):101.
• [18]Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E et al.. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012; 366(10):883-892.
• [19]Fisher R, Pusztai L, Swanton C. Cancer heterogeneity: implications for targeted therapeutics. Br J Cancer. 2013; 108(3):479-485.
• [20]Maughan TS, Adams RA, Smith CG, Meade AM, Seymour MT, Wilson RH et al.. 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. 2011; 377(9783):2103-2114.
• [21]Seymour MT, Maughan TS, Ledermann JA, Topham C, James R, Gwyther SJ et al.. Different strategies of sequential and combination chemotherapy for patients with poor prognosis advanced colorectal cancer [MRC FOCUS]: a randomised controlled trial. Lancet. 2007; 370(9582):143-152.
• [22]Fitzgibbons PL, Lazar AJ, Spencer S. Introducing new College of American Pathologists reporting templates for cancer biomarkers. Arch Pathol Lab Med. 2014; 138(2):157-158.
• [23]Idikio HA. Immunohistochemistry in diagnostic surgical pathology: contributions of protein life-cycle, use of evidence-based methods and data normalization on interpretation of immunohistochemical stains. Int J Clin Exp Pathol. 2009; 3(2):169-176.
• [24]Russnes HG, Navin N, Hicks J, Borresen-Dale AL. Insight into the heterogeneity of breast cancer through next-generation sequencing. J Clin Invest. 2011; 121(10):3810-3818.
• [25]Thomson TA, Hayes MM, Spinelli JJ, Hilland E, Sawrenko C, Phillips D et al.. HER-2/neu in breast cancer: interobserver variability and performance of immunohistochemistry with 4 antibodies compared with fluorescent in situ hybridization. Mod Pathol. 2001; 14(11):1079-1086.
• [26]Turashvili G, Leung S, Turbin D, Montgomery K, Gilks B, West R et al.. Inter-observer reproducibility of HER2 immunohistochemical assessment and concordance with fluorescent in situ hybridization [FISH]: pathologist assessment compared to quantitative image analysis. BMC Cancer. 2009; 9:165.
• [27]Gavrielides MA, Gallas BD, Lenz P, Badano A, Hewitt SM. Observer variability in the interpretation of HER2/neu immunohistochemical expression with unaided and computer-aided digital microscopy. Arch Pathol Lab Med. 2011; 135(2):233-242.
• [28]Atkins D, Reiffen KA, Tegtmeier CL, Winther H, Bonato MS, Storkel S. Immunohistochemical detection of EGFR in paraffin-embedded tumor tissues: variation in staining intensity due to choice of fixative and storage time of tissue sections. J Histochem Cytochem. 2004; 52(7):893-901.
• [29]Hewitt SM, Badve SS, True LD. Impact of preanalytic factors on the design and application of integral biomarkers for directing patient therapy. Clin Cancer Res. 2012; 18(6):1524-1530.
• [30]Neumeister VM, Parisi F, England AM, Siddiqui S, Anagnostou V, Zarrella E et al.. A tissue quality index: an intrinsic control for measurement of effects of preanalytical variables on FFPE tissue. Lab Invest. 2014; 94(4):467-474.
• [31]Press MF, Hung G, Godolphin W, Slamon DJ. Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression. Cancer Res. 1994; 54(10):2771-2777.
• [32]Tong LC, Nelson N, Tsourigiannis J, Mulligan AM. The effect of prolonged fixation on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast cancer: a prospective study. Am J Surg Pathol. 2011; 35(4):545-552.
• [33]Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ et al.. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 2007; 25(1):118-145.
• [34]Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH et al.. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol. 2013; 31(31):3997-4013.
• [35]Hammond ME, Hayes DF, Wolff AC, Mangu PB, Temin S. American society of clinical oncology/college of american pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Oncol Pract. 2010; 6(4):195-197.
• [36]Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol. 1998; 11(2):155-168.
• [37]Dowsett M, Nielsen TO, A’Hern R, Bartlett J, Coombes RC, Cuzick J et al.. Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group. J Natl Cancer Inst. 2011; 103(22):1656-1664.
• [38]Won JR, Gao D, Grant D, Cupples J, Rahemtulla A, Wolber R et al.. Variable performance of commercial epidermal growth factor receptor antibodies in detection of basal-like breast cancer. Histopathology. 2012; 61(3):518-519.
• [39]Ciardiello F, Tortora G. Epidermal growth factor receptor [EGFR] as a target in cancer therapy: understanding the role of receptor expression and other molecular determinants that could influence the response to anti-EGFR drugs. Eur J Cancer. 2003; 39(10):1348-1354.
• [40]Ciardiello F, Tortora G. EGFR antagonists in cancer treatment. N Engl J Med. 2008; 358(11):1160-1174.
• [41]Saltz LB, Meropol NJ, Loehrer PJS, Needle MN, Kopit J, Mayer RJ. Phase II trial of cetuximab in patients with refractory colorectal cancer that expresses the epidermal growth factor receptor. J Clin Oncol. 2004; 22(7):1201-1208.
• [42]Van Cutsem E, Peeters M, Siena S, Humblet Y, Hendlisz A, Neyns B et al.. Open-label phase III trial of panitumumab plus best supportive care compared with best supportive care alone in patients with chemotherapy-refractory metastatic colorectal cancer. J Clin Oncol. 2007; 25(13):1658-1664.
• [43]Pirker R, Pereira JR, von Pawel J, Krzakowski M, Ramlau R, Park K et al.. EGFR expression as a predictor of survival for first-line chemotherapy plus cetuximab in patients with advanced non-small-cell lung cancer: analysis of data from the phase 3 FLEX study. Lancet Oncol. 2012; 13(1):33-42.
• [44]You B, Chen EX (2011) Anti-EGFR monoclonal antibodies for treatment of colorectal cancers: development of cetuximab and panitumumab. J Clin Pharmacol
• [45]Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H, Santoro A et al.. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med. 2004; 351(4):337-345.
• [46]Amado RG, Wolf M, Peeters M, Van Cutsem E, Siena S, Freeman DJ et al.. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol. 2008; 26(10):1626-1634.
• [47]Lievre A, Bachet JB, Le Corre D, Boige V, Landi B, Emile JF et al.. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res. 2006; 66(8):3992-3995.
• [48]Lievre A, Bachet JB, Boige V, Cayre A, Le Corre D, Buc E et al.. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol. 2008; 26(3):374-379.
• [49]Di Fiore F, Blanchard F, Charbonnier F, Le Pessot F, Lamy A, Galais MP et al.. Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by cetuximab plus chemotherapy. Br J Cancer. 2007; 96(8):1166-1169.
• [50]Douillard JY, Oliner KS, Siena S, Tabernero J, Burkes R, Barugel M et al.. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med. 2013; 369(11):1023-1034.
• [51]Douillard JY, Siena S, Cassidy J, Tabernero J, Burkes R, Barugel M et al.. Randomized, phase III trial of panitumumab with infusional fluorouracil, leucovorin, and oxaliplatin [FOLFOX4] versus FOLFOX4 alone as first-line treatment in patients with previously untreated metastatic colorectal cancer: the PRIME study. J Clin Oncol. 2010; 28(31):4697-4705.
• [52]Bokemeyer C, Bondarenko I, Hartmann JT, de Braud F, Schuch G, Zubel A et al.. Efficacy according to biomarker status of cetuximab plus FOLFOX-4 as first-line treatment for metastatic colorectal cancer: the OPUS study. Ann Oncol. 2011; 22(7):1535-1546.
• [53]Stintzing S, Fischer von Weikersthal L, Decker T, Vehling-Kaiser U, Jager E, Heintges T et al.. FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab as first-line treatment for patients with metastatic colorectal cancer-subgroup analysis of patients with KRAS: mutated tumours in the randomised German AIO study KRK-0306. Ann Oncol. 2012; 23(7):1693-1699.
• [54]Bokemeyer C, Bondarenko I, Makhson A, Hartmann JT, Aparicio J, de Braud F et al.. 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.
• [55]Jonker DJ, O’Callaghan CJ, Karapetis CS, Zalcberg JR, Tu D, Au HJ et al.. Cetuximab for the treatment of colorectal cancer. N Engl J Med. 2007; 357(20):2040-2048.
• [56]Karapetis CS, Khambata-Ford S, Jonker DJ, O’Callaghan CJ, Tu D, Tebbutt NC et al.. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med. 2008; 359(17):1757-1765.
• [57]De Roock W, Claes B, Bernasconi D, De Schutter J, Biesmans B, Fountzilas G et al.. 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.
• [58]De Roock W, De Vriendt V, Normanno N, Ciardiello F, Tejpar S. KRAS, BRAF, PIK3CA, and PTEN mutations: implications for targeted therapies in metastatic colorectal cancer. Lancet Oncol. 2011; 12(6):594-603.
• [59]Vale CL, Tierney JF, Fisher D, Adams RA, Kaplan R, Maughan TS et al.. Does anti-EGFR therapy improve outcome in advanced colorectal cancer? A systematic review and meta-analysis. Cancer Treat Rev. 2012; 38(6):618-625.
• [60]Chung KY, Shia J, Kemeny NE, Shah M, Schwartz GK, Tse A et al.. Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol. 2005; 23(9):1803-1810.
• [61]Wierzbicki R, Jonker DJ, Moore MJ, Berry SR, Loehrer PJ, Youssoufian H et al.. A phase II, multicenter study of cetuximab monotherapy in patients with refractory, metastatic colorectal carcinoma with absent epidermal growth factor receptor immunostaining. Invest New Drugs. 2011; 29(1):167-174.
• [62]Cunningham MP, Essapen S, Thomas H, Green M, Lovell DP, Topham C et al.. Coexpression, prognostic significance and predictive value of EGFR, EGFRvIII and phosphorylated EGFR in colorectal cancer. Int J Oncol. 2005; 27(2):317-325.
• [63]Allo G, Bandarchi B, Yanagawa N, Wang A, Shih W, Xu J et al.. EGFR mutation-specific immunohistochemical antibodies in lung adenocarcinoma. Histopathology. 2014; 64(6):826-839.
• [64]Brabyn CJ, Kleine LP. EGF causes hyperproliferation and apoptosis in T51B cells: involvement of high and low affinity EGFR binding sites. Cell Signal. 1995; 7(2):139-150.
• [65]Braun S, Schlimok G, Heumos I, Schaller G, Riethdorf L, Riethmuller G et al.. ErbB2 overexpression on occult metastatic cells in bone marrow predicts poor clinical outcome of stage I-III breast cancer patients. Cancer Res. 2001; 61(5):1890-1895.
• [66]Ithimakin S, Day KC, Malik F, Zen Q, Dawsey SJ, Bersano-Begey TF et al.. HER2 drives luminal breast cancer stem cells in the absence of HER2 amplification: implications for efficacy of adjuvant trastuzumab. Cancer Res. 2013; 73(5):1635-1646.
• [67]Hirsch FR, Varella-Garcia M, Bunn PA, Di Maria MV, Veve R, Bremmes RM et al.. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol. 2003; 21(20):3798-3807.
• [68]Hirsch FR, Herbst RS, Olsen C, Chansky K, Crowley J, Kelly K et al.. Increased EGFR gene copy number detected by fluorescent in situ hybridization predicts outcome in non-small-cell lung cancer patients treated with cetuximab and chemotherapy. J Clin Oncol. 2008; 26(20):3351-3357.
• [69]Wulf MA, Bode B, Zimmermann D, Rufibach K, Weder W, Moch H et al.. Silver-enhanced in situ hybridization for determination of EGFR copy number alterations in non-small cell lung cancer. Am J Surg Pathol. 2012; 36(12):1801-1808.
• [70]Algars A, Lintunen M, Carpen O, Ristamaki R, Sundstrom J. EGFR gene copy number assessment from areas with highest EGFR expression predicts response to anti-EGFR therapy in colorectal cancer. Br J Cancer. 2011; 105(2):255-262.
• [71]Werner M, Chott A, Fabiano A, Battifora H. Effect of formalin tissue fixation and processing on immunohistochemistry. Am J Surg Pathol. 2000; 24(7):1016-1019.
• [72]Liotta LA, Espina V, Mehta AI, Calvert V, Rosenblatt K, Geho D et al.. Protein microarrays: meeting analytical challenges for clinical applications. Cancer Cell. 2003; 3(4):317-325.
• [73]Sartore-Bianchi A, Fieuws S, Veronese S, Moroni M, Personeni N, Frattini M et al.. Standardisation of EGFR FISH in colorectal cancer: results of an international interlaboratory reproducibility ring study. J Clin Pathol. 2012; 65(3):218-223.
• [74]Sartore-Bianchi A, Moroni M, Veronese S, Carnaghi C, Bajetta E, Luppi G et al.. Epidermal growth factor receptor gene copy number and clinical outcome of metastatic colorectal cancer treated with panitumumab. J Clin Oncol. 2007; 25(22):3238-3245.
• [75]Luber B, Deplazes J, Keller G, Walch A, Rauser S, Eichmann M et al.. Biomarker analysis of cetuximab plus oxaliplatin/leucovorin/5-fluorouracil in first-line metastatic gastric and oesophago-gastric junction cancer: results from a phase II trial of the Arbeitsgemeinschaft Internistische Onkologie [AIO]. BMC Cancer. 2011; 11:509.
• [76]Personeni N, Fieuws S, Piessevaux H, De Hertogh G, De Schutter J, Biesmans B et al.. Clinical usefulness of EGFR gene copy number as a predictive marker in colorectal cancer patients treated with cetuximab: a fluorescent in situ hybridization study. Clin Cancer Res. 2008; 14(18):5869-5876.
• [77]Mahipal A, Mcdonald MJ, Witkiewicz A, Carr BI. Cell membrane and cytoplasmic epidermal growth factor receptor expression in pancreatic ductal adenocarcinoma. Med Oncol. 2012; 29(1):134-139.
• [78]Pu YS, Huang CY, Kuo YZ, Kang WY, Liu GY, Huang AM et al.. Characterization of membranous and cytoplasmic EGFR expression in human normal renal cortex and renal cell carcinoma. J Biomed Sci. 2009; 16:82.
• [79]Ueda S, Ogata S, Tsuda H, Kawarabayashi N, Kimura M, Sugiura Y et al.. The correlation between cytoplasmic overexpression of epidermal growth factor receptor and tumor aggressiveness: poor prognosis in patients with pancreatic ductal adenocarcinoma. Pancreas. 2004; 29(1):e1-e8.
• [80]Spano JP, Fagard R, Soria JC, Rixe O, Khayat D, Milano G. Epidermal growth factor receptor signaling in colorectal cancer: preclinical data and therapeutic perspectives. Ann Oncol. 2005; 16(2):189-194.
• [81]Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A et al.. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 2001; 344(11):783-792.
• [82]Slamon D, Eiermann W, Robert N, Pienkowski T, Martin M, Press M et al.. Adjuvant trastuzumab in HER2-positive breast cancer. N Engl J Med. 2011; 365(14):1273-1283.
• [83]Mosesson Y, Mills GB, Yarden Y. Derailed endocytosis: an emerging feature of cancer. Nat Rev Cancer. 2008; 8(11):835-850.
• [84]Liao HJ, Carpenter G. Cetuximab/C225-induced intracellular trafficking of epidermal growth factor receptor. Cancer Res. 2009; 69(15):6179-6183.
• [85]Tomas A, Futter CE, Eden ER. EGF receptor trafficking: consequences for signaling and cancer. Trends Cell Biol. 2014; 24(1):26-34.
• [86]Piyathilake CJ, Frost AR, Manne U, Weiss H, Bell WC, Heimburger DC et al.. Differential expression of growth factors in squamous cell carcinoma and precancerous lesions of the lung. Clin Cancer Res. 2002; 8(3):734-744.
• [87]Einama T, Ueda S, Tsuda H, Ogasawara K, Hatsuse K, Matsubara O et al.. Membranous and cytoplasmic expression of epidermal growth factor receptor in metastatic pancreatic ductal adenocarcinoma. Exp Ther Med. 2012; 3(6):931-936.
• [88]De Craene B, Berx G. Regulatory networks defining EMT during cancer initiation and progression. Nat Rev Cancer. 2013; 13(2):97-110.
• [89]Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol. 2014; 15(3):178-196.
• [90]Mendoza MC, Er EE, Blenis J. The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation. Trends Biochem Sci. 2011; 36(6):320-328.
• [91]Vartanian S, Bentley C, Brauer MJ, Li L, Shirasawa S, Sasazuki T et al.. Identification of mutant K-Ras-dependent phenotypes using a panel of isogenic cell lines. J Biol Chem. 2013; 288(4):2403-2413.
• [92]Parsons BL, Myers MB. Personalized cancer treatment and the myth of KRAS wild-type colon tumors. Discov Med. 2013; 15(83):259-267.
• [93]Heinemann V, von Weikersthal LF, Decker T, Kiani A, Vehling-Kaiser U, Al-Batran SE et al.. FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab as first-line treatment for patients with metastatic colorectal cancer [FIRE-3]: a randomised, open-label, phase 3 trial. Lancet Oncol. 2014; 15(10):1065-1075.
• [94]Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M et al.. Genetic alterations during colorectal-tumor development. N Engl J Med. 1988; 319(9):525-532.
• [95]Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990; 61(5):759-767.
• [96]Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012; 487(7407):330-337.
• [97]Schreiber RD, Old LJ, Smyth MJ. Cancer immunoediting: integrating immunity’s roles in cancer suppression and promotion. Science. 2011; 331(6024):1565-1570.
• [98]Liotta LA, Kohn EC. The microenvironment of the tumour-host interface. Nature. 2001; 411(6835):375-379.
• [99]Emens LA, Silverstein SC, Khleif S, Marincola FM, Galon J. Toward integrative cancer immunotherapy: targeting the tumor microenvironment. J Transl Med. 2012; 10:70.
• [100]Galon J, Fridman WH, Pages F. The adaptive immunologic microenvironment in colorectal cancer: a novel perspective. Cancer Res. 2007; 67(5):1883-1886.
• [101]Mlecnik B, Tosolini M, Kirilovsky A, Berger A, Bindea G, Meatchi T et al.. Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction. J Clin Oncol. 2011; 29(6):610-618.
• [102]Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C et al.. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 2006; 313(5795):1960-1964.
• [103]Galon J, Pages F, Marincola FM, Thurin M, Trinchieri G, Fox BA et al.. The immune score as a new possible approach for the classification of cancer. J Transl Med. 2012; 10:1.
• [104]Galon J, Pages F, Marincola FM, Angell HK, Thurin M, Lugli A et al.. Cancer classification using the Immunoscore: a worldwide task force. J Transl Med. 2012; 10:205.
• [105]Galon J, Angell HK, Bedognetti D, Marincola FM. The continuum of cancer immunosurveillance: prognostic, predictive, and mechanistic signatures. Immunity. 2013; 39(1):11-26.
• [106]Angell H, Galon J. From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer. Curr Opin Immunol. 2013; 25(2):261-267.
• [107]Galon J, Mlecnik B, Bindea G, Angell HK, Berger A, Lagorce C et al.. Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours. J Pathol. 2014; 232(2):199-209.
• [108]Bindea G, Mlecnik B, Angell HK, Galon J. The immune landscape of human tumors: implications for cancer immunotherapy. Oncoimmunology. 2014; 3(1):e27456.
• [109]Hamilton PW, Bankhead P, Wang Y, Hutchinson R, Kieran D, McArt DG. Digital pathology and image analysis in tissue biomarker research. Methods. 2014; 70(1):59-73.
• [110]Van Schaeybroeck S, Allen WL, Turkington RC, Johnston PG. Implementing prognostic and predictive biomarkers in CRC clinical trials. Nat Rev Clin Oncol. 2011; 8(4):222-232.
• [111]Schneider MR, Wolf E. The epidermal growth factor receptor ligands at a glance. J Cell Physiol. 2009; 218(3):460-466.
• [112]Khambata-Ford S, Garrett CR, Meropol NJ, Basik M, Harbison CT, Wu S et al.. Expression of epiregulin and amphiregulin and K-ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab. J Clin Oncol. 2007; 25(22):3230-3237.
• [113]Jacobs B, De Roock W, Piessevaux H, Van Oirbeek R, Biesmans B, De Schutter J et al.. Amphiregulin and epiregulin mRNA expression in primary tumors predicts outcome in metastatic colorectal cancer treated with cetuximab. J Clin Oncol. 2009; 27(30):5068-5074.
• [114]Straussman R, Morikawa T, Shee K, Barzily-Rokni M, Qian ZR, Du J et al.. Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature. 2012; 487(7408):500-504.
• [115]Wilson TR, Fridlyand J, Yan Y, Penuel E, Burton L, Chan E et al.. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors. Nature. 2012; 487(7408):505-509.
• [116]Kuramochi H, Nakajima G, Kaneko Y, Nakamura A, Inoue Y, Yamamoto M et al.. Amphiregulin and epiregulin mRNA expression in primary colorectal cancer and corresponding liver metastases. BMC Cancer. 2012; 12:88.
• [117]Sunaga N, Kaira K, Imai H, Shimizu K, Nakano T, Shames DS et al.. Oncogenic KRAS-induced epiregulin overexpression contributes to aggressive phenotype and is a promising therapeutic target in non-small-cell lung cancer. Oncogene. 2013; 32(34):4034-4042.
• [118]Oda K, Matsuoka Y, Funahashi A, Kitano H. A comprehensive pathway map of epidermal growth factor receptor signaling. Mol Syst Biol. 2005; 1:2005.0010.
• [119]Mandell JW. Phosphorylation state-specific antibodies: applications in investigative and diagnostic pathology. Am J Pathol. 2003; 163(5):1687-1698.
• [120]Chafin D, Theiss A, Roberts E, Borlee G, Otter M, Baird GS. Rapid two-temperature formalin fixation. PLoS One. 2013; 8(1):e54138.
• [121]Theiss AP, Chafin D, Bauer DR, Grogan TM, Baird GS. Immunohistochemistry of colorectal cancer biomarker phosphorylation requires controlled tissue fixation. PLoS One. 2014; 9(11):e113608.
• [122]Phipps AI, Limburg PJ, Baron JA, Burnett-Hartman AN, Weisenberger DJ, Laird PW et al.. Association between molecular subtypes of colorectal cancer and patient survival. Gastroenterology. 2015; 148(1):77-87.e2.
• [123]Sadanandam A, Lyssiotis CA, Homicsko K, Collisson EA, Gibb WJ, Wullschleger S et al.. A colorectal cancer classification system that associates cellular phenotype and responses to therapy. Nat Med. 2013; 19(5):619-625.
• [124]Duncan JS, Whittle MC, Nakamura K, Abell AN, Midland AA, Zawistowski JS et al.. Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer. Cell. 2012; 149(2):307-321.
• [125]Ascierto PA, Kirkwood JM, Grob J-J, Simeone E, Grimaldi AM, Maio M et al.. The role of BRAF V600 mutation in melanoma. J Transl Med. 2012; 10:85.
• [126]Flaherty KT, Infante JR, Daud A, Gonzalez R, Kefford RF, Sosman J et al.. Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N Engl J Med. 2012; 367(18):1694-1703.
• [127]Larkin J, Ascierto PA, Dréno B, Atkinson V, Liszkay G, Maio M et al.. Combined vemurafenib and cobimetinib in BRAF-mutated melanoma. N Engl J Med. 2014; 371(20):1867-1876.
• [128]Polley MY, Leung SC, McShane LM, Gao D, Hugh JC, Mastropasqua MG et al.. An international Ki67 reproducibility study. J Natl Cancer Inst. 2013; 105(24):1897-1906.
• [129]Smits AJ, Kummer JA, de Bruin PC, Bol M, van den Tweel JG, Seldenrijk KA et al.. The estimation of tumor cell percentage for molecular testing by pathologists is not accurate. Mod Pathol. 2014; 27(2):168-174.
• [130]Viray H, Li K, Long TA, Vasalos P, Bridge JA, Jennings LJ et al.. A prospective, multi-institutional diagnostic trial to determine pathologist accuracy in estimation of percentage of malignant cells. Arch Pathol Lab Med. 2013; 137(11):1545-1549.
• [131]Shaw GL, Thomas BC, Dawson SN, Srivastava G, Vowler SL, Gnanapragasam VJ et al.. Identification of pathologically insignificant prostate cancer is not accurate in unscreened men. Br J Cancer. 2014; 110(10):2405-2411.
• [132]Foran DJ, Chen W, Yang L. Automated image interpretation computer-assisted diagnostics. Anal Cell Pathol [Amst]. 2011; 34(6):279-300.
• [133]Riley JS, Hutchinson R, McArt DG, Crawford N, Holohan C, Paul I et al.. Prognostic and therapeutic relevance of FLIP and procaspase-8 overexpression in non-small cell lung cancer. Cell Death Dis. 2013; 5(4):e951.
• [134]Petrovski S, Goldstein DB. Phenomics and the interpretation of personal genomes. Sci Transl Med. 2014; 6(254):254fs35.
• [135]Zemojtel T, Kohler S, Mackenroth L, Jager M, Hecht J, Krawitz P et al.. Effective diagnosis of genetic disease by computational phenotype analysis of the disease-associated genome. Sci Transl Med. 2014; 6(252):252ra123.
• [136]Lu JT, Campeau PM, Lee BH. Genotype-phenotype correlation—promiscuity in the era of next-generation sequencing. N Engl J Med. 2014; 371(7):593-596.
• [137]Zbuk KM, Eng C. Cancer phenomics: RET and PTEN as illustrative models. Nat Rev Cancer. 2007; 7(1):35-45.
• [138]Houle D, Govindaraju DR, Omholt S. Phenomics: the next challenge. Nat Rev Genet. 2010; 11(12):855-866.
• [139]Kelloff GJ, Sigman CC. Cancer biomarkers: selecting the right drug for the right patient. Nat Rev Drug Discov. 2012; 11(3):201-214.
• [140]Kent DM, Rothwell PM, Ioannidis JP, Altman DG, Hayward RA. Assessing and reporting heterogeneity in treatment effects in clinical trials: a proposal. Trials. 2010; 11:85.
• [141]Kleppe M, Levine RL. Tumor heterogeneity confounds and illuminates: assessing the implications. Nat Med. 2014; 20(4):342-344.
• [142]Cadioli A, Rossi G, Costantini M, Cavazza A, Migaldi M, Colby TV. Lung cancer histologic and immunohistochemical heterogeneity in the era of molecular therapies: analysis of 172 consecutive surgically resected, entirely sampled pulmonary carcinomas. Am J Surg Pathol. 2014; 38(4):502-509.
• [143]Baldus SE, Schaefer KL, Engers R, Hartleb D, Stoecklein NH, Gabbert HE. Prevalence and heterogeneity of KRAS, BRAF, and PIK3CA mutations in primary colorectal adenocarcinomas and their corresponding metastases. Clin Cancer Res. 2010; 16(3):790-799.
• [144]Xie T, Cho YB, Wang K, Huang D, Hong HK, Choi YL et al.. Patterns of somatic alterations between matched primary and metastatic colorectal tumors characterized by whole-genome sequencing. Genomics. 2014; 104(4):234-241.
• [145]Modjtahedi H, Essapen S. Epidermal growth factor receptor inhibitors in cancer treatment: advances, challenges and opportunities. Anticancer Drugs. 2009; 20(10):851-855.
• [146]Modjtahedi H, Khelwatty SA, Kirk RS, Seddon AM, Essapen S, Del Vecchio CA et al.. Immunohistochemical discrimination of wild-type EGFR from EGFRvIII in fixed tumour specimens using anti-EGFR mAbs ICR9 and ICR10. Br J Cancer. 2012; 106(5):883-888.
• [147]Fan QW, Cheng CK, Gustafson WC, Charron E, Zipper P, Wong RA et al.. EGFR phosphorylates tumor-derived EGFRvIII driving STAT3/5 and progression in glioblastoma. Cancer Cell. 2013; 24(4):438-449.
• [148]Bartlett JM, Campbell FM, Ibrahim M, O’Grady A, Kay E, Faulkes C et al.. A UK NEQAS ISH multicenter ring study using the Ventana HER2 dual-color ISH assay. Am J Clin Pathol. 2011; 135(1):157-162.
• [149]van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I, Hurlstone A et al.. The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell. 2002; 111(2):241-250.
• [150]Chakrabarty A, Sanchez V, Kuba MG, Rinehart C, Arteaga CL. Feedback upregulation of HER3 [ErbB3] expression and activity attenuates antitumor effect of PI3K inhibitors. Proc Natl Acad Sci USA. 2012; 109(8):2718-2723.
• [151]Laurent-Puig P, Manceau G, Zucman-Rossi J, Blons H. Dual blockade of epidermal growth factor receptor-induced pathways: a new avenue to treat metastatic colorectal cancer. J Clin Oncol. 2012; 30(13):1550-1552.