Crizotinib and Colorectal Cancer – A Couple to Be Tested?

Dr Stintzing is currently a postdoctoral fellow of the German Cancer Aid at the University of Southern California/Norris Comprehensive Cancer Center. Dr Stintzing’s research focus is on predictive and prognostic factors in the treatment of metastatic colorectal cancer.

Dr Lenz is the Associate Director for Clinical Research and Co-Leader of the Gastrointestinal Cancers Program at the University of Southern California/Norris Comprehensive Cancer Center. Dr Lenz is Professor of Medicine and Preventive Medicine, Section Head of GI Oncology in the Division of Medical Oncology and Co-Director of the Colorectal Center at the Keck School of Medicine of the University of Southern California.

Crizotinib, a multikinase inhibitor, has recently shown activity in the treatment of anaplastic lymphoma kinase (ALK) fusion gene positive non–small cell lung cancer (NSCLC)¹ and has therefore been approved for the treatment of ALK-positive NSCLC by the medical agencies in the United States and Europe.² Although only 3% to 5% of NSCLCs are EML4-ALK positive, this extended personalized treatment of lung cancer has changed clinical practice. Next to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib that are approved for the treatment of EGFR-mutated NSCLC,^3-5 crizotinib is another class of kinase inhibitor, being approved for a molecularly defined subset of lung cancer fragmenting NSCLC.

In metastatic colorectal cancer (mCRC), one of the most common tumors worldwide, kinase inhibitors targeting EGFR, vascular endothelial growth factor receptor (VEGFR), and others, with the exception of regorafenib, have failed to demonstrate clinical effectiveness, and therefore treatment strategies have been less successful. Whether this is a disease-specific issue or lack of patient selection is unknown.

This review will provide insights into molecularly defined subgroups and development of more effective therapies, with the focus on the pathways targeted by crizotinib in mCRC.

Crizotinib – Mechanism of Action – the NSCLC Story

Crizotinib (Xalkori) is an orally administered multikinase inhibitor that has been developed using a structure-based drug design program for inhibiting the hepatocyte growth factor receptor (c-Met)-signaling with a half maximal inhibitory concentration (IC50) of 5 to 20 nM.^6,7 Further evaluation revealed inhibition of cell proliferation in an ALK-positive large cell lymphoma cell with an IC50 of about 30 nM.⁷ In xenograft mouse models crizotinib was able to inhibit nucleophosmin-ALK (NPM-ALK) phosphorylation in a dose-dependent manner and induced apoptosis in tumorous tissue.⁸

Furthermore, inhibition of the oncogene ROS1 signaling has been described as a target of crizotinib.⁹ EML4-ALK fusion genes are found in about 5% of NSCLC. Because there is a negative association with smoking history in the subgroup of nonsmokers or patients with only a light smoking history, the EML4-ALK fusion gene is found in up to 10%.¹⁰ A phase 1 clinical trial established 250 mg bid as the recommended dosage for crizotinib. This trial has been extended, and patients bearing the EML4-ALK mutation have achieved an unexpected tumor response and clinical improvement. To reach the total of 119 patients, a cohort of about 1500 patients had to be screened for ALK fusion genes. This trial, conducted in heavily pretreated NSCLC patients bearing an EML4-ALK fusion, demonstrated an overall response rate of 60.8%, a progression-free survival (PFS) of 9.7 months, and a 1-year survival rate of 75%. The most common National Cancer Institute Common Terminology Criteria for Adverse Events grade 3/4 adverse events were neutropenia, elevated alanine aminotransferase, hypophosphatemia, and lymphopenia.¹ These data led to the approval of crizotinib in the treatment of EML4-ALK–positive NSCLC.²

Ongoing trials in lung cancer using crizotinib are testing the efficacy and safety of crizotinib in a randomized setting against standard chemotherapy with pemetrexed and platinum (NCT01154140)¹¹ or with other kinase inhibitors. Efficacy in other tumor entities is to be tested as well. Among those are anaplastic large cell lymphoma, glioma, and other advanced tumors if ALK fusion genes, ROS aberrations, or elevated c-Met expression is determined.¹¹

ALK Function – Fusion – Frequency

ALK is a member of the insulin receptor protein tyrosine kinases superfamily. A detailed review of structure and oncogenic activation has recently been published.¹² In short, ALK is involved in embryogenesis and is thought to be important for brain development,¹³ as ALK expression is seen during mouse embryogenesis. However, ALK knockout mice show no obvious abnormalities and have a normal lifespan.¹⁴ In adulthood, ALK is expressed in the brain, small intestine, testis, prostate, and colon,¹³ although the exact mechanism of action is not yet clear.¹⁵ Even the ligands to the ALK receptor in humans are still undiscovered, as ligands in animal models are lacking mammalian homologues. But the pathways to be activated by ALK signaling have been described using NPM-ALK fusion gene signaling in lymphoma cells. This fusion gene has the ability to activate ALK signaling by autophosphorylation. Among the pathways to be activated by ALK are the Ras/Raf/MEK/ERK1, JAK/STAT, PI3K/Akt, and PLc pathways, which lead to cell survival and cell division.^8,16 All these pathways are known to be important for oncogenesis and development of metastasis. The physiological signal transduction has not yet been elucidated. In addition to the NPM-ALK fusion gene, which drives 50% to 60% of the anaplastic large cell lymphoma cases,¹⁷ many other fusion genes with the ability to induce ALK signaling have been described. Although inflammatory myofibroblastic tumors have a reported frequency of 27%,⁷ clinically the most important ALK fusion gene in solid tumors is the EML4-ALK fusion gene in NSCLC. It can be found in a number of variants (currently 13 described variants) in about 5% of NSCLC patients.¹⁸ EML4-ALK fusion genes are able to activate Ras/ERK1/2, Akt, and JAK/STAT.¹⁹ As crizotinib has shown efficacy and therefore been approved for the treatment of NSCLC tumors harboring an ALK fusion gene, this has pushed the research efforts in the field of ALK fusion genes. During the past few years, in addition to NSCLC, ALK fusion genes have been found in many other solid tumors, including inflammatory myofibroblastic tumors,²⁰ breast cancer,²¹ esophageal squamous cell cancer,²² renal cell cancer,²³ and colorectal cancer (CRC).²¹ The frequency in adult solid tumors is less than 5% but is still undetermined in many tumors. Another tyrosine kinase targeted by crizotinib is c-Met.

Figure

c-Met Function

The MET proto-oncogene encodes for hepatocyte growth factor receptor (HGFR), also named c-Met, and has been described to be essential for embryonic development and can trigger invasive growth in cancer cells. In adulthood, c-Met signaling is important in many physiological processes such as wound healing, tissue regeneration, and morphogenic differentiation.²⁴ The activity of c-Met as a receptor tyrosine kinase is usually low in normal tissue but tends to be dysregulated in tumorous tissue.²⁵ Further experiments have shown that c-Met signaling by HGF facilitates cell proliferation, survival, differentiation, motility, and invasion,²⁴ all of which are important for oncogenesis. Somatic mutations of c-Met were described for the first time in hereditary papillary renal cell carcinomas in 2003²⁴ and have later been reported in other solid cancers such as gastric cancer, head and neck cancer, primary liver cancer, ovarian cancer, NSCLC, and thyroid cancer.²⁶ In addition to those activating mutations, higher levels of HGF leading to increased c-Met signaling have been described in many solid tumors, and overexpression of c-Met has been established in many solid tumors.²⁶ Among these are lung,²⁷ breast,²⁸ ovarian,²⁹ kidney,³⁰ colon,³⁰ gastric,³⁰ thyroid,³⁰ pancreas,³¹ and head and neck cancer.³² Higher c-Met expression was associated with poorer prognosis or advanced stage²⁶ in most of these solid tumors, making c-Met an interesting target for tumor growth inhibition.³³ Furthermore, analyses investigating NSCLC patients who have become resistant to anti-EGFR treatment with EGFR TKIs were able to establish c-Met amplification as one possible mechanism of anti-EGFR resistance.^34,35 Two possible bypass mechanisms have been proposed: 1) c-Met signaling might initiate downstream signaling via MAPK/PI3K/Akt independent of the EGFR, or 2) c-Met may be activated via ErbB crosstalk and therefore amplify its protumorigenic signaling.²⁶

Major downstream signaling pathways activated by c-Met include the PI3K/Akt pathway leading to proliferation and cell growth,³⁶ and the MAPK pathway.³⁷ Furthermore, receptor tyrosine kinase crosstalk, including EGFR, cell-cell adhesions, cell motility, and endocytosis, are influenced by c-Met signaling, emphasizing its potential role in cancer development.37 As c-Met signaling is known to have the ability to escape EGFR inhibition³⁸ in vitro, it would be of interest to biopsy CRC metastases that became resistant to anti-EGFR treatment to find a possible treatment option for c-Met inhibitors. A recent report showing clinical efficacy of crizotinib in 2 patients suffering from esophagogastric adenocarcinoma with c-Met amplification³⁹ does encourage testing this mechanism in CRC as well.

ROS1 Function

ROS1 is a receptor tyrosine kinase of the insulin receptor superfamily. Structurally similar to ALK, with which it shares a similarity of 48.92% in amino acid sequence,⁴⁰ the ligand is not yet known. It was first described in 1982,⁴¹ and its oncogenic potential was demonstrated in 198442 by transforming NIH3T3 cells to tumorous lesions in nude mice. Further investigations discovered ROS1 in glioblastoma cell lines.⁴³ In glioblastoma the fusion gene was called fusion in glioblastoma (FIG-ROS1).⁴⁴ Proving its importance in human solid tumors, ROS1 rearrangements have been described in NSCLC at a frequency of about 1.6%.45 In NSCLC, other fusion partners than in glioblastoma can be found. Different from FIG-ROS1, which is located in the Golgi apparatus,⁴³ CD74-ROS1,4^6,47 SLC34A2-ROS1,⁴⁶ SDC4-ROS1, TPM3-ROS1, EZR-ROS1, and LRIG3-ROS1,⁴⁸ which have been described in NSCLC patients, are all located on the cell surface. This might explain the difference in function and treatability. In gastrointestinal cancer, only the fusion genes FIG-ROS1⁴⁷ in cholangiocellular carcinoma and SLC34A2-ROS1⁴⁹ in gastric cancer have been described.

The signal pathways transduced by ROS1 are similar to those activated by ALK fusion genes. Most important is the activation of PI3K/Akt/mTOR50 and PLc pathways, leading to changes in differentiation and apoptosis and an increased translational capacity. The RAS/MAPK/MEK pathway does not seem to be activated by ROS1 signaling.⁵⁰ Interestingly, ROS1 is capable of phosphorylate cytoskeleton proteins and therefore interacts with alpha-, beta-, and delta-catenin,⁵¹ which may represent a possible interaction with the oncogenic potential of the beta-catenin-wnt pathway. Furthermore, ROS1 signaling can be inhibited by crizotinib.⁵²

Metastatic Colorectal Cancer

The treatment of mCRC has made notable progress during the past 10 years. Until recently, clinical effectiveness was based on chemotherapeutic combination regimens using 5-fluorouracil (5-FU) and leucovorin (folinic acid) in combination with either irinotecan (FOLFIRI regimen)⁵³ or oxaliplatin (FOLFOX regimen).⁵⁴ However, with the introduction of monoclonal antibodies to those combination therapies, median overall survival (OS) times of about 24 months have been reached.^55-59 Targets for those antibodies are either vascular endothelial growth factor (VEGF) with bevacizumab⁵⁹ or EGFR with cetuximab^56,57 and panitumumab.⁵⁵ Anti-EGFR antibodies do not have efficacy in the subset of patients with KRAS-mutated tumors and are therefore limited to the treatment of KRAS wild-type tumors. The activating KRAS mutation, with a frequency of about 40%, is the only accepted predictive factor in the treatment of CRC,⁶⁰ although it has become clear during the past few years that CRC is not 1 disease but many, defined by their individual genomic landscape.⁶¹ Along with the use of targeted therapy, the expansion of the chemotherapeutic regimen to FOL­FOXIRI has been successful in improving efficacy in randomized trials.⁶²

TKI Experience in mCRC
It has become evident that many CRCs are dependent on the activation of 3 signaling pathways that are in part overlapping and have the ability to crosstalk. RAS/RAF/MEK, PLCy/PIP2/IP3, and PI3K/AKT/mTOR signaling pathways have therefore become main targets of kinase inhibitor research. Furthermore, angiogenesis is an important mechanism in tumor growth and progression. Multiple inhibitors have been created⁶³ and are being tested alone or in combination. The Table lists ongoing phase 2 and phase 3 trials. The combination approach tries to increase efficacy by inhibiting 2 or more of these pathways at once to circumvent possible mechanisms of resistance. Although some of these inhibitors have shown promising results in phase 2 studies, most of them have failed to demonstrate activity in phase 3 trials. Therefore, the only approved multikinase inhibitor for the treatment of mCRC is regorafenib. Regorafenib, a multitarget angiogenesis inhibitor, has shown significantly increased survival in heavily pretreated CRC patients.⁶⁴ Some multikinase inhibitors like the VEGFR TKI cediranib⁶⁵ or the multitargeted angiogenesis inhibitor vatalanib⁶⁶ made it to phase 3 trial level but failed to demonstrate significant treatment advance. The reasons for that may well be that the whole patient population and not a molecular subset of “favorable” patients was treated. For example, vatalanib showed efficacy especially in the subset of patients presenting with a lactate dehydrogenase elevation.⁶⁶

Other interesting targets for kinase inhibitors in mCRC may be found in the EGFR-dependent pathway. Among those, the BRAF mutation with a frequency of about 4% is most likely a favorable target, as the BRAF inhibitor vemurafenib is already approved for the treatment of BRAF-mutated malignant melanoma.⁶⁷ Many phase 1/2 trials are currently testing a wide variety of TKIs in CRC.⁶³ The most important problem to be addressed is screening enough patients to define the patient cohort that most likely will benefit from those targeted therapies.

Table

The questions arise if crizotinib, a c-Met, ALK, and ROS1 inhibitor, would make sense in the treatment of mCRC; and if yes, which molecularly defined subset of patients would be most likely to benefit from crizotinib therapy?

Using immunohistochemistry (IHC), c-Met is overexpressed in 78% of primary CRC specimens, but only 8% express phospho-Met as a sign of activity.³⁰ c-Met gene amplification is known to be a marker for advanced CRC, and liver metastases tend to have higher c-Met expression than the primary tumor specimen.⁶⁸ Furthermore, c-Met has been described to be a negative prognostic marker in mCRC⁶⁹ and its ligand HGF to be a negative prognostic factor in Union for International Cancer Control stage III CRC.⁷⁰

Primary resistance to the anti-EGFR antibody cetuximab has been linked to c-Met activation in NSCLC71 and CRC cell lines.⁷² Using fluorescence in situ hybridization technique in a retrospective analysis of 85 chemotherapy-refractory CRC patients, primary resistance to cetuximab could not be attributed to c-Met overexpression in the primary tumor specimen.⁷³ This might well be due to a relatively rare occurrence of c-Met overexpression (7/76; 9.2%). This is supported by findings from a mouse xenograft model of mCRC in which no correlation between c-Met overexpression and therapeutic responsiveness to c-Met inhibitors could be established, but the transcriptional factor for c-Met, MACC1, was predictive for outcome.⁷⁴

In contrast, another group reported a possible influence of IHC-detected c-Met on PFS and OS in cetuximab-treated mCRC patients.⁷⁵

Because the relevance of c-Met in secondary resistance to anti-EGFR treatment in the clinical setting is still under discussion, a study investigating the evolution of the biology of CRC metastasis during anti- EGFR treatment is needed. Therefore, serial tumor biopsies and a comprehensive program to detect gene expression levels are essential.

ALK fusion genes being another target of crizotinib is a very rare event in CRC. The frequency of the EML4-ALK fusion gene is estimated to be 2.4%.²¹ Lately another ALK fusion gene (C2orf44-ALK) has been described by next-generation sequencing (NGS) screening of 40 CRC cases.⁷⁶ Additional ALK mutations (E17K mutation) have been found at a frequency of 6% in a cohort of 51 mCRC patients.⁷⁷ Therefore, there might be another group of CRC patients that could possibly benefit from an ALK inhibitor like crizotinib.

To date, no published data on the frequency and biological relevance of ROS1 in CRC are available.

Studies in mCRC currently testing the effect and safety of inhibitors of the c-Met signaling pathway include the following:

• Tivantinib (Daiichi Sankyo Inc, ARQ 197); a small molecule inhibiting the c-Met tyrosine kinase; it is being tested in a phase 1/2 setting with the combination of irinotecan and cetuximab in patients in whom frontline chemotherapy has failed (NCT01075048)¹¹
• LY2801653 (Eli Lilly); a small molecule inhibitor of c-Met being tested in a phase 1 setting in advanced cancers. It has shown antitumor activity in tumor xenografts.⁷⁸ CRC patients may be recruited (NCT01285037)¹¹
• Rilotumumab (Amgen, AMG 102); a fully human antibody that targets HGF and therefore inhibits c-Met signaling. It is being tested in a phase 1/2 setting alone and in combination with panitumumab in mCRC patients (NCT00788957)¹¹

As with many TKIs, development of resistance to crizotinib has been demonstrated in several in vitro studies. So far, the following mechanisms of resistance have been described: 1) cells undergo an oncogenic switch and become EGFR dependent,^79,80 2) cells express increased levels of MET and amplify KRAS and therefore overcome the inhibition by c-Met inhibitors and sustain high MAPK and PI3K/Akt signaling,⁸¹ and 3) recently a mutation of c-Met (Y1230H) has been described to overcome kinase inhibition.⁸²

Conclusion

ALK fusion genes are rare in mCRC, although with the NGS technique on its way this subgroup could grow as more ALK fusion genes may be identified. It is estimated that about 2.4% of all CRCs bear an ALK fusion gene, but so far the biological and clinical relevance have not been revealed. Also, ROS1 has not been described in CRC specimens. Both factors seem to be rare, and the screening for a trial targeting the ALK fusion gene in a randomized setting is difficult.

There are emerging data that c-Met expression levels are increased in pretreated CRC specimens. Furthermore, c-Met signaling has been shown to overcome anti-EGFR treatment and may therefore be an important factor of secondary resistance to anti-EGFR treatment. Although in vivo data on the mechanism on EGFR resistance are still missing, there are strong in vitro data suggesting this receptor tyrosine kinase crosstalk and oncogenic switch to maintain MAPK and PI3K/Akt signaling is important for a subgroup of CRC tumors. As this switch works vice versa, crizotinib in CRC should be combined with an anti-EGFR strategy to prevent crizotinib resistance. This concept of double inhibition (anti-EGFR and anti–c-Met) is currently being tested in several studies on the phase 1/2 level. Furthermore, a recent case report has been published showing that the combination of high-dose pemetrexed and crizotinib is tolerable and might expand the usage of crizotinib in NSCLC,⁸³ so a combination with chemotherapy seems to be an option to be tested in mCRC as well.

It might be clinically meaningful to biopsy CRC patients at the time of progression to evaluate c-Met expression levels repeatedly and treat the subgroup of patients with elevated c-Met expression levels in a randomized trial testing a combination of anti-EGFR antibody and crizotinib.

References
1. Camidge DR, Bang YJ, Kwak EL, et al. Activity and safety of crizotinib in patients with ALK-positive non-small-cell lung cancer: updated results from a phase 1 study. Lancet Oncol. 2012;13:1011-1019.
2. National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines). Non-Small Cell Lung Cancer. Version 2.2013. http://www.nccn.org/professionals/physician_gls/f_guide lines.asp#nscl.
3. Lynch TJ, Bell DW, Sordella R, 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;350:2129-2139.
4. Paez JG, Jänne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004;304:1497-1500.
5. Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A. 2004;101:13306-13311.
6. Ou SH. Crizotinib: a novel and first-in-class multitargeted tyrosine kinase inhibitor for the treatment of anaplastic lymphoma kinase rearranged non-small cell lung cancer and beyond. Drug Des Devel Ther. 2011;5:471-485.
7. Cui JJ, Tran-Dubé M, Shen H, et al. Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK). J Med Chem. 2011;54:6342-6363.
8. Christensen JG, Zou HY, Arango ME, et al. Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma. Mol Cancer Ther. 2007;6(12 Pt 1):3314-3322.
9. Chin LP, Soo RA, Soong, R, et al. Targeting ROS1 with anaplastic lymphoma kinase inhibitors: a promising therapeutic strategy for a newly defined molecular subset of non-small-cell lung cancer. J Thorac Oncol. 2012;7:1625-1630.
10. Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol. 2009;27:4247-4253.
11. www.clinicaltrials.gov. January 2013.
12. Roskoski R Jr. Anaplastic lymphoma kinase (ALK): structure, oncogenic activation, and pharmacological inhibition. Pharmacol Res. 2013;68:68-94.
13. Morris SW, Kirstein MN, Valentine MB, et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin’s lymphoma. Science. 1995;267:316-317.
14. Duyster J, Bai RY, Morris SW. Translocations involving anaplastic lymphoma kinase (ALK). Oncogene. 2001;20:5623-5637.
15. Iwahara T, Fujimoto J, Wen D, et al. Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system. Oncogene. 1997;14:439-449.
16. Chiarle R, Voena C, Ambrogio C, et al. The anaplastic lymphoma kinase in the pathogenesis of cancer. Nat Rev Cancer. 2008;8:11-23.
17. Morris SW, Kirstein MN, Valentine MB, et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin’s lymphoma. Science. 1994;263:1281-1284.
18. Sanders HR, Li HR, Bruey JM, et al. Exon scanning by reverse transcriptase-polymerase chain reaction for detection of known and novel EML4-ALK fusion variants in non-small cell lung cancer. Cancer Genet. 2011;204:45-52.
19. Palmer RH, Vernersson E, Grabbe C, et al. Anaplastic lymphoma kinase: signalling in development and disease. Biochem J. 2009;420:345-361.
20. Armstrong F, Duplantier MM, Trempat P, et al. Differential effects of X-ALK fusion proteins on proliferation, transformation, and invasion properties of NIH3T3 cells. Oncogene. 2004;23:6071-6082.
21. Lin E, Li L, Guan Y, et al. Exon array profiling detects EML4-ALK fusion in breast, colorectal, and non-small cell lung cancers. Mol Cancer Res. 2009;7:1466-1476.
22. Jazii FR, Najafi Z, Malekzadeh R, et al. Identification of squamous cell carcinoma associated proteins by proteomics and loss of beta tropomyosin expression in esophageal cancer. World J Gastroenterol. 2006;12:7104-7112.
23. Debelenko LV, Raimondi SC, Daw N, et al. Renal cell carcinoma with novel VCL-ALK fusion: new representative of ALK-associated tumor spectrum. Mod Pathol. 2011;24:430-442.
24. Birchmeier C, Birchmeier W, Gherardi E, et al. Met, metastasis, motility and more. Nat Rev Mol Cell Biol. 2003;4:915-925.
25. Jung KH, Park BH, Hong SS. Progress in cancer therapy targeting c-Met signaling pathway. Arch Pharm Res. 2012;35:595-604.
26. Sierra JR, Tsao MS. c-MET as a potential therapeutic target and biomarker in cancer. Ther Adv Med Oncol. 2011;3(1 suppl):S21-S35.
27. Wang NS, Liu C, Emond J, et al. Annulate lamellae in a large cell lung carcinoma cell line with high expression of tyrosine kinase receptor and proto-oncogenes. Ultrastruct Pathol. 1992;16:439-449.
28. Tuck AB, Park M, Sterns EE, et al. Coexpression of hepatocyte growth factor and receptor (Met) in human breast carcinoma. Am J Pathol. 1996;148:225-232.
29. Aune G, Lian AM, Tingulstad S, et al. Increased circulating hepatocyte growth factor (HGF): a marker of epithelial ovarian cancer and an indicator of poor prognosis. Gynecol Oncol. 2011;121:402-406.
30. Ma PC, Tretiakova MS, MacKinnon AC, et al. Expression and mutational analysis of MET in human solid cancers. Genes Chromosomes Cancer. 2008;47:1025-1037.
31. Furukawa T, Duguid WP, Kobari M, et al. Hepatocyte growth factor and Met receptor expression in human pancreatic carcinogenesis. Am J Pathol. 1995;147:889-895.
32. Liu N, Furukawa T, Kobari M, et al. Comparative phenotypic studies of duct epithelial cell lines derived from normal human pancreas and pancreatic carcinoma. Am J Pathol. 1998;153:263-269.
33. Trusolino L, Bertotti A, Comoglio PM. MET signalling: principles and functions in development, organ regeneration and cancer. Nat Rev Mol Cell Biol. 2010;11:834-848.
34. Turke AB, Zejnullahu K, Wu YL, et al. Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC. Cancer Cell. 2010;17:77-88.
35. Bean J, Brennan C, Shih JY, et al. MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci U S A. 2007;104:20932-20937.
36. Ponzetto C, Bardelli A, Zhen Z, et al. A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell. 1994;77:261-271.
37. Organ SL, Tong J, Taylor P, et al. Quantitative phospho-proteomic profiling of hepatocyte growth factor (HGF)-MET signaling in colorectal cancer. J Proteome Res. 2011;10:3200-3211.
38. Camp ER, Summy J, Bauer TW, et al. Molecular mechanisms of resistance to therapies targeting the epidermal growth factor receptor. Clin Cancer Res. 2005;11:397-405.
39. Lennerz JK, Kwak L, Ackerman A, et al. MET amplification identifies a small and aggressive subgroup of esophagogastric adenocarcinoma with evidence of responsiveness to crizotinib. J Clin Oncol. 2011;29:4803-4810.
40. Ou SH, Tan J, Yen Y, et al. ROS1 as a ‘druggable’ receptor tyrosine kinase: lessons learned from inhibiting the ALK pathway. Expert Rev Anticancer Ther. 2012;12:447-456.
41. Shibuya M, Hanafusa H, Balduzzi PC. Cellular sequences related to three new onc genes of avian sarcoma virus (fps, yes, and ros) and their expression in normal and transformed cells. J Virol. 1982;42:143-152.
42. Fasano O, Birnbaum D, Edlund L, et al. New human transforming genes detected by a tumorigenicity assay. Mol Cell B iol. 1984;4:1695-1705.
43. Birchmeier C, Sharma S, Wigler M. Expression and rearrangement of the ROS1 gene in human glioblastoma cells. Proc Natl Acad Sci U S A. 1987;84:9270-9274.
44. Charest A, Lane K, McMahon K, et al. Association of a novel PDZ domain-containing peripheral Golgi protein with the Q-SNARE (Q-soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor) protein syntaxin 6. J Biol Chem. 2001;276:29456-29465.
45. Rimkunas VM, Crosby KE, Li D, et al. Analysis of receptor tyrosine kinase ROS1-positive tumors in non-small cell lung cancer: identification of a FIG-ROS1 fusion. Clin Cancer Res. 2012;18:4449-4457.
46. Rikova K, Guo A, Zeng Q, et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell. 2007;131:1190-1203.
47. Gu TL, Deng X, Huang F, et al. Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma. PLoS One. 2011;6:e15640.
48. Takeuchi K, Soda M, Togashi Y, et al. RET, ROS1 and ALK fusions in lung cancer. Nat Med. 2012;18:378-381.
49. Lee J, Lee SE, Kang SY, et al. Identification of ROS1 rearrangement in gastric adenocarcinoma. Cancer. 2013;119:1627-1635.
50. Nguyen KT, Zong CS, Uttamsingh S, et al. The role of phosphatidyl­inositol 3-kinase, rho family GTPases, and STAT3 in Ros-induced cell transformation. J Biol Chem. 2002;277:11107-11115.
51. Zong CS, Chan JL, Yang SK, et al. Mutations of Ros differentially effecting signal transduction pathways leading to cell growth versus transformation. J Biol Chem. 1997;272:1500-1506.
52. Bergethon K, Shaw AT, Ou SH, et al. ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol. 2012;30:863-870.
53. Douillard JY, Cunningham D, Roth AD, et al. Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial. Lancet. 2000;355:1041-1047.
54. Andre T, Bensmaine MA, Louvet C, et al. Multicenter phase II study of bimonthly high-dose leucovorin, fluorouracil infusion, and oxaliplatin for metastatic colorectal cancer resistant to the same leucovorin and fluorouracil regimen. J Clin Oncol. 1999;17:3560-3568.
55. Peeters M, Price TJ, Cervantes A, et al. 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. J Clin Oncol. 2010;28:4706-4713.
56. Van Cutsem E, Köhne CH, Láng L, et al. Cetuximab plus irinotecan, fluorouracil, and leucovorin as first-line treatment for metastatic colorectal cancer: updated analysis of overall survival according to tumor KRAS and BRAF mutation status. J Clin Oncol. 2011;29:2011-2019.
57. Bokemeyer C, Bondarenko I, Makhson A, et al. Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer. J Clin Oncol. 2009;27:663-671.
58. Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med. 2004;350:2335-2342.
59. Saltz LB, Clarke S, Diaz-Rubio E, et al. Bevacizumab in combination with oxaliplatin-based chemotherapy as first-line therapy in metastatic colorectal cancer: a randomized phase III study. J Clin Oncol. 2008;26:2013-2019.
60. Di Fiore F, Blanchard F, Charbonnier F, et al. Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by cetuximab plus chemotherapy. Br J Cancer. 2007;96:1166-1169.
61. The Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487:330-337.
62. Loupakis F, Cremolini C, Masi G, et al. FOLFOXIRI plus bevacizumab (bev) versus FOLFIRI plus bev as first-line treatment of metastatic co­lorectal cancer (MCRC): results of the phase III randomized TRIBE trial. J Clin Oncol. 2012;30(suppl 34). Abstract 336.
63. Yu M, Grady WM. Therapeutic targeting of the phosphatidylinositol 3-kinase signaling pathway: novel targeted therapies and advances in the treatment of colorectal cancer. Therap Adv Gastroenterol. 2012;5:319-337.
64. Grothey A, Van Cutsem E, Sobrero A, et al. Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet. 2013;381:303-312.
65. Schmoll HJ, Cunningham D, Sobrero A, et al. Cediranib with mFOLFOX6 versus bevacizumab with mFOLFOX6 as first-line treatment for patients with advanced colorectal cancer: a double-blind, randomized phase III study (HORIZON III). J Clin Oncol. 2012;30:3588-3595.
66. Hecht JR, Trarbach T, Hainsworth JD, et al. Randomized, placebo-controlled, phase III study of first-line oxaliplatin-based chemotherapy plus PTK787/ZK 222584, an oral vascular endothelial growth factor receptor inhibitor, in patients with metastatic colorectal adenocarcinoma. J Clin Oncol. 2011;29:1997-2003.
67. Flaherty KT, Puzanov I, Kim KB, et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med. 2010;363:809-819.
68. Zeng ZS, Weiser MR, Kuntz E, et al. c-Met gene amplification is associated with advanced stage colorectal cancer and liver metastases. Cancer Lett. 2008;265:258-269.
69. Kammula US, Kuntz EJ, Francone TD, et al. Molecular co-expression of the c-Met oncogene and hepatocyte growth factor in primary colon cancer predicts tumor stage and clinical outcome. Cancer Lett. 2007;248:219-228.
70. Toiyama Y, Miki C, Inoue Y, et al. Serum hepatocyte growth factor as a prognostic marker for stage II or III colorectal cancer patients. Int J Cancer. 2009;125:1657-1662.
71. Krumbach R, Schüler J, Hofmann M, et al. Primary resistance to cetuximab in a panel of patient-derived tumour xenograft models: activation of MET as one mechanism for drug resistance. Eur J Cancer. 2011;47:1231-1243.
72. Liska D, Chen CT, Bachleitner-Hofmann T, et al. HGF rescues colorectal cancer cells from EGFR inhibition via MET activation. Clin Cancer Res. 2011;17:472-482.
73. Cappuzzo F, Varella-Garcia M, Finocchiaro G, et al. Primary resistance to cetuximab therapy in EGFR FISH-positive colorectal cancer patients. Br J Cancer. 2008;99:83-89.
74. Galimi F, Torti D, Sassi F, et al. Genetic and expression analysis of MET, MACC1, and HGF in metastatic colorectal cancer: response to met inhibition in patient xenografts and pathologic correlations. Clin Cancer Res. 2011;17:3146-3156.
75. Inno A, Di Salvatore M, Cenci T, et al. Is there a role for IGF1R and c-MET pathways in resistance to cetuximab in metastatic colorectal cancer? Clin Colorectal Cancer. 2011;10:325-332.
76. Lipson D, Capelletti M, Yelensky R, et al. Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies. Nat Med. 2012;18:382-384.
77. Carpten JD, Faber AL, Horn C, et al. A transforming mutation in the pleckstrin homology domain of AKT1 in cancer. Nature. 2007;448:439-444.
78. Yang WJ, Credille K, Gao H, et al. LY2801653, an orally available small molecule inhibitor of c-Met, demonstrated broad antitumor efficacy in patient derived xenograft models. Cancer Res. 2010;70(suppl). Abstract 3611.
79. Engelman JA, Zejnullahu K, Mitsudomi T, et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 2007;316:1039-1043.
80. Corso S, Ghiso E, Cepero V, et al. Activation of HER family members in gastric carcinoma cells mediates resistance to MET inhibition. Mol Cancer. 2010;9:121.
81. Cepero V, Sierra JR, Corso S, et al. MET and KRAS gene amplification mediates acquired resistance to MET tyrosine kinase inhibitors. Cancer Res. 2010;70:7580-7590.
82. Qi J, McTigue MA, Rogers A, et al. Multiple mutations and bypass mechanisms can contribute to development of acquired resistance to MET inhibitors. Cancer Res. 2011;71:1081-1091.
83. Gandhi L, Drappatz J, Ramaiya NH, et al. High-dose pemetrexed in combination with high-dose crizotinib for the treatment of refractory CNS metastases in ALK-rearranged non-small-cell lung cancer. J Thorac Oncol. 2013;8:e3-e5.