WO2015144184A1

WO2015144184A1 - Use of timp-1 as a biomarker in the egf-receptor inhibitor treatment of metastatic colorectal cancer

Info

Publication number: WO2015144184A1
Application number: PCT/DK2015/050069
Authority: WO
Inventors: Nils Aage Brünner; José Moreira; Ib Jarle Christensen; Line Schmidt TARPGAARD; Per PFEIFFER
Original assignee: University Of Copenhagen; Rigshospitalet; University Of Southern Denmark; The Region Of Southern Denmark
Priority date: 2014-03-26
Filing date: 2015-03-26
Publication date: 2015-10-01
Also published as: WO2015144184A8

Abstract

The present invention concerns the provision of biomarkers, in particular, TIMP-1 and RAS as biomarkers for the selection of patients in the treatment and prognosis of cancer, such as e.g. metastatic colorectal cancer (mCRC). The present invention also provides methods of patient selection, patient prognosis and methods of treatment of patients based on TIMP-1 levels in the blood, plasma or tumor tissue and the absence or presence of mutations in RAS, in particular KRAS, who are likely to benefit from treatment with an EGFR inhibitor alone or in combination with at least one chemotherapeutic agent. The present invention also provides for a method of treatment of m CRC in patients, which have been identified by the inventive method using the biomarker TIMP-1 and KRAS, who are likely to benefit from the treatment with the EGFR inhibitor cetuximab and at least one chemotherapeutic agent.

Description

Use of TIMP-1 as a biomarker in the EGF-Receptor inhibitor treatment of metastatic colorectal cancer

FIELD OF THE INVENTION The present invention concerns cancer biomarkers. In particular, the present invention concerns TIMP-1 as a biomarker for the selection of patients for treatment with EGF- Receptor inhibitors, methods of patient selection and methods of therapeutic treatment of mCRC.

BACKGROUND

Colorectal cancer (CRC) is the third most common cancer in the world, with nearly 1 .4 million new cases diagnosed in 2012. Despite optimized surgery and adjuvant chemotherapy, a large percentage of patients still experience recurrence of the disease.

Colorectal cancer usually develops slowly over a period of 10 to 15 years. The tumor typically begins as a non-cancerous polyp. A polyp is a growth of tissue that develops on the lining of the colon or rectum and it can subsequently become cancerous. Adenomatous polyps or adenomas, are the most likely to become cancers, though fewer than 10% of adenomas progress to cancer. Adenomas are common; an estimated one-third to one-half of all individuals will eventually develop one or more colorectal adenomas. Around one fifth of patients with CRC present with metastatic disease (mCRC) at time of diagnosis (synchronous disease), and up to 40% of primary CRC patients will develop metastases during the course of their disease, resulting in a relatively high overall mortality rate associated with CRC.

Incidence and death rates for colorectal cancer increase with age. Overall, 90% of new cases and 94% of deaths occur in individuals 50 years of age and older. The incidence rate of colorectal cancer is more than 15 times higher in adults older than 50 years of age than in adults 20 to 49 years of age.

Diagnosis of colorectal cancer via tumor biopsy is typically done during colonoscopy or sigmoidoscopy, depending on the location of the lesion. The extent of the disease is then usually determined by a CT scan of the chest, abdomen and pelvis. There are other potential imaging test such as PET and MRI which may be used in certain cases. Colorectal cancer staging is done based on the TNM system in which it is determined how much the initial tumor has spread, if and where lymph nodes are involved, and if and where metastases are found.

About 96% of colorectal cancers are adenocarcinomas, which evolve from glandular tissue. The great majority of these cancers arise from an adenomatous polyp, which is visible through a scope or on an x-ray-like image. The information on early detection in this document is most relevant to this type of cancer.

There are many known factors that increase or decrease the risk of colorectal cancer; some of these factors are modifiable and others are not. Modifiable risk factors that have been associated with an increased risk of colorectal cancer in epidemiologic studies include physical inactivity, obesity, high consumption of red or processed meats, smoking, and moderate-to-heavy alcohol consumption. A recent study found that about one-quarter of colorectal cancer cases could be avoided by following a healthy lifestyle, i.e., maintaining a healthy abdominal weight, being physically active at least 30 minutes per day, eating a healthy diet, not smoking, and not drinking excessive amounts of alcohol.

Nonmodifiable risk factors include a personal or family history of colorectal cancer or adenomatous polyps, and a personal history of chronic inflammatory bowel disease. People with a first-degree relative who has had colorectal cancer have 2 to 3 times greater risk of developing CRC compared to individuals with no family history; if the relative was diagnosed at a young age or if there is more than one affected relative, risk increases to 3 to 6 times that of the general population. About 20% of all colorectal cancer patients have a close relative who was diagnosed with the disease.

Alterations, such as gene mutations, gene amplifications, and protein over-expressions within the EGFR signaling cascade play a role in colorectal carcinogenesis. Over-expression of EGFR is seen in 60% to 80% of colorectal cancers, alluding to the potential utility of EGFR inhibition as a therapeutic option. Interestingly, EGFR expression does not appear to correlate with response to treatment in mCRC. Studies assessing EGFR amplification as a predictive marker have resulted in contradictory conclusions likely in part related to differences in fluorescence in situ hybridization assay methodology. Somatic mutations in EGFR are rarely, if ever, present in mCRC except as a possible mechanism of acquired resistance to EGFR inhibition.

EGFR mediates stimulation of cellular proliferation, survival, and motility and can be involved in the progression of cancer cells when EGFR is constitutively activated. Ligand binding, for example by EGF and TGF- , results in a conformational change of the EGFR and homo- or heterodimerization with other members of the HER receptor family with its family members HER2-4, in particular HER2. This will result in subsequent autophosphorylation of the cytoplasmic tyrosine kinase domain, which triggers downstream signaling events through the recruitment of adapter proteins such as e.g. SHC, GRB-2. There are two principle signaling pathways downstream of EGFR: (1 ) The ratsarcoma(Ras)/rapidly accelerated fibro-sarcoma (Raf)/mitogen-activated protein kinase kinase (MEK) pathway and (2) phosphatidylinositol-3- kinase(PI3K)/ protein kinaseB (AKT), which stimulates mitosis and inhibits apoptosis (Goffin and Zbuk, Clinical Therapeutics (2013), Vol. 35, No. 9: 1282-1303). The small GTPase KRAS, also known as V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog, encoded by the KRAS gene functions downstream of the EGFR and is involved in EGFR signaling in the KRAS-BRAF-MEK-ERK pathway. KRAS is a proto-oncogene encoding a small 21 -kD guanosine triphosphate (GTP)/guanosine diphosphate (GDP) binding protein involved in the regulation of the cellular response to many extracellular stimuli. KRAS belongs to the RAS protein family, which consists of four highly homologous enzymes (H-Ras, N-Ras and KRAS with its splice variants KRAS4A, KRAS4B) that are identical over the first 85 amino acids, 85% identical over the next 80 amino acids, and largely divergent within the C-terminal 24 amino acids, a domain that is referred to as the hypervariable region (HVR). After binding and activation by GTP, KRAS recruits the oncogene BRAF, which

phosphorylates MAP2K (mitogen-activated protein kinase kinase), thereby initiating MAPK signaling leading to the expression of the protein involved in cell proliferation, differentiation, and survival. KRAS is the mostly commonly mutated gene in this pathway, and 35%→45% patients with CRCs carry this mutation, which is an early event in colon tumorigenesis. H- RAS and N-RAS mutations are also found in CRCs, albeit at a lower frequency than KRAS mutations.

Each of the RAS isoforms can be locked into its GTP bound activated state via missense mutation, which typically involves amino acids in position 12, 13, or 61 . Mutant RAS proteins accumulate in the GTP-bound conformation due to defective intrinsic GTPase activity and/or resistance to inactivation by GTPase activating proteins (GAPs).

The most common mutations found in KRAS in CRC introduce amino acid substitutions at codons 12 and 13. KRAS mutations as the most common mutations found in CRC frequently induce glycine to valine substitutions at the catalytic sites of amino acids, which leads to the loss of GTPase activity and subsequent continuous binding of GTP to RAS (Yokota, Anti- Cancer Agents in Medicinal Chemistry, 2012, 12, 163-171 ). The substitution of other amino acids, usually aspartate and valine at codon 12 and aspartate at codon 13, results in the projection of larger amino acid side chains into the GDP/GTP binding pocket of the protein which interfere with GTP hydrolysis. As a result of those conformational and structural changes EGFR signaling becomes deregulated in response to the constitutive activation of KRAS protein (Herreros-Villanueva et al., Clinica Chimica Acta 431 (2014) 21 1-220).

This constitutive activation of KRAS results in the dysregulation of the downstream RAS-ERK signaling pathway independently of EGFR. Similarly, the kinase activity of the BRAF mutant protein is greatly elevated, which also constitutively stimulates downstream ERK activity independently of RAS and EGFR. Thus, the constitutive activation of KRAS or BRAF leads to EGFR-independent tumorigenicity in patients with CRC. Therefore, the oncogenic activation of the KRAS signaling pathway impairs the response of colorectal cancer cells to EGFR inhibitors such as for example cetuximab (Yokota, Anti-Cancer Agents in Medicinal

Chemistry (2012) 12: 163-171 ). However, anti-EGFR antibodies are able to block receptor signaling in tumors with wild-type KRAS. Various KRAS mutations in codon 12 were associated with different clinic-pathological features, for example mutations in KRAS codon 12, especially the C.35G N T (p.G12V) mutation, was associated with the highest frequency of mortality in CRC patients. It was also reported that the C.34G N C (p.G12R) and C.35G N T (p.G12V) mutations conferred more transforming ability than other KRAS mutations. Based on these findings, KRAS mutation testing has been attributed a major role in selecting patients for anti-EGFR antibody therapy.

Similarly, the kinase activity of the mutant protein BRAF may also result in constitutive stimulation downstream ERK activity and independently of RAS and EGFR. Thus, the constitutive activation of KRAS or BRAF mutation leads to EGFR-independent tumorigenicity in patients with CRC. Therefore, oncogenic activation of the RAS signaling pathway appears to impair the response of colorectal cancer cells to EGFR inhibitors, such as e.g. cetuximab.

Choice of first line treatment for patients with mCRC is based on tumour and patient related factors and molecular information for determination of individual treatment aim and thus treatment intensity. As a result of recent advances in the treatment of mCRC, median overall survival (OS) can now be as long as 30 months in selected patient groups and up to 70% of the patients will receive at least two lines of treatment.

First- and second-line chemotherapy with 5-fluorouracil (5-FU) and folinic acid (FA) in combination with either irinotecan (FOLFIRI) or oxaliplatin (FOLFOX) has been standard therapy for several years. These combinations are also being used together with either EGFR inhibitors or antiangiogenic drugs in patients with mCRC resulting in an improvement of median overall survival (OS) for mCRC patients to 20-22 months compared with 12 months with 5-FU/FA treatment alone.

Next to the treatment regimens FOLFIRI or FOLFOX, several other drugs are available for the treatment of mCRC singly or in combination with other chemotherapeutic agents, including for example the vascular endothelial growth factor (VEGF) antibody bevacizumab, EGFR antibodies cetuximab and panitumumab for RAS wildtype patients, the VEGF receptors 1 and 2 fusion protein aflibercept and the multitarget tyrosine kinase inhibitor regorafenib. Moreover, secondary resection and/or ablation e.g., by surgery or radiation treatment may contribute to long-term survival. Prognosis of mCRC depends on several patient-related factors, e.g. age of the patient, performance status as well as tumor-related biochemical factors, such as e.g spread of the disease, growth dynamics of the tumor, localization in the body as well as baseline values of carcino-embryonic antigen (CEA) or molecular factors, such as e.g. KRAS mutations. The consideration of these factors may be used to stratify cancer treatment, i.e. customizing it for a given patient considering the above factors. Thus, the determination of a patient's individual prognosis is therefore useful in the choice of treatment.

Despite the efforts which have been undertaken thus far in identifying predictive markers to allow for a stratification in the treatment of mCRC, only RAS mutations have been established, precluding treatment with EGFR antibodies (Stein et al, World J Gastroenterol 2014; 20(4): 899-907). Initially KRAS mutations in exon 2 (codon 12 and 13) have been found to be predictive for non-response to cetuximab or panitumumab. Although data are conflicting, KRAS codon G13D mutation, which is found in about 16% of KRAS mutated tumours, does not seem to preclude efficacy of cetuximab-based treatment in patients with KRAS mutations. However, according to clinical trial data, neither combining oxaliplatin with different fluoropyrimidine schedules and cetuximab, nor in panitumumab clinical trials available thus far, KRAS G13D mutated tumors seem to derive relevant benefit from anti- EGFR treatment with either cetuximab or panitumumab.

Other markers which have been used to stratify the mCRC patient population include the family of tissue inhibitor of metalloproteinases (TIMPs). TIMPs are endogenous inhibitors for matrix metalloproteinases (MMPs) that are responsible for remodeling the extracellular matrix (ECM) and involved in migration, invasion and metastasis of tumor cells. The TIMP family comprises TIMP-1 , -2, -3, and -4 as family members, of which each family members has its binding matrix metalloproteinase (MMP) partners, which regulate remodeling and turnover of the extracellular matrix (ECM) during normal development and pathological conditions. Among the TIMP members, TIMP-1 is the only protein that is glycosylated (Kim et al., BMB Rep. (2012) Nov;45(1 1 ):623-8).

TIMP-1 expression has been found to inversely correlate with the susceptibility to induction of apoptosis in various human Burkitt's lymphoma cell lines, in which forced TIMP-1 expression resulted in a reduced susceptibility to induction of apoptosis (Guedez et al, Blood 2001 , 102:2002-2010). Both TIMP-1 and TIMP-2 have also been reported to possess growth-promoting activity in epithelial and mesenchymal cells (Bertaux et al, J of Invest Dermatology 1991 ; 97:679-685, Hayakawa et al., FEBS Letters (1992)298:29-32).

Preclinical studies have provided evidence that TIMP-1 by binding to the CD63 tetraspanin receptor can activate the Akt-survival pathway. This pathway is also activated by EGFR, and it has been suggested that high TIMP-1 plasma or tumor cell levels will thereby render cancer cells insensitive to treatment with EGFR inhibitors such as e.g. cetuximab.

Recent studies were directed at examining a correlation between blood plasma levels of TIMP-1 in mCRC patients treated with a combinatorial chemotherapy of capecitabine and oxaliplatin (XELOX) as first line treatment. The results of this study indicated that high baseline TIMP-1 levels were associated with poor (shorter) overall survival (OS) (Frederiksen et al., Annals of Oncology (201 1 ) 22:369-375). A different study provided evidence that low baseline plasma levels of TIMP-1 in mCRC patients undergoing first line chemotherapy with 5-FU and irinothecan (FOLFIRI/FLIRI) were significantly associated with a better patient outcome (Sorensen et al., Clin Cancer Res (2007) 13: 41 17-4122).

A recent phase III trial, in which mCRC patients were administered the EGFR inhibitor cetuximab with continuous or intermittent fluorouracil, leucovorin and oxaliplatin (Nordic FLOX) in comparison to FLOX treatment alone as first-line treatment, examined the KRAS mutation status on treatment outcome. The results of the study indicated that KRAS status did not demonstrate any prognostic value that the administration of cetuximab adds a significant benefit to Nordic FLOX in first line treatment of mCRC (Tveit et al., J of Clin Oncol (2012) No:15, 1755-62).

Currently, the mutational status of the small GTPase RAS appears to be the only biomarker used to select patients with mCRC for EGFR inhibition. However, mCRC patients require further stratification to ascertain which of the patients derive the most benefit from a combined treatment with EGFR inhibitors and chemotherapy. Therefore, an unmet need for biomarkers still exists to select the most effective treatment for individual patients.

This need also extends to patients afflicted with other cancer types in which targeting EGFR is currently considered as a therapeutic treatment option including for example head and neck squamous cell carcinoma (HNSCC), prostate cancer or lung cancer. For example, the Bonner trial and EXTREME trial provided evidence that adding cetuximab to primary radiotherapy or chemotherapy increased overall survival in patients with locoregional advanced HNSCC or re-current and metastatic HNSCC. However, it remained unclear whether all patients benefited from the addition of the EGFR inhibitor cetuximab (Yakota, Int J. Clin. Oncol, 2014 Jan 21 ).

It is thus an object of the present invention to improve stratification of patients with cancer to EGFR inhibitor therapy with or without concomitant chemotherapy by providing additional predictive biomarkers.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that in cancer patients with EGFR expressing tumors, in which RAS is mutated, TIMP-1 levels as disclosed herein indicate patients that are likely to benefit from treatment in which an EGFR inhibitor administered. Accordingly, the present invention provides a method of identifying a cancer patient who is likely to benefit from treatment with an EGFR inhibitor comprising determining in vitro the levels of TIMP-1 of the patient and the absence or presence of a RAS mutation in a patient's tumor sample, wherein EGFR is expressed in the patient's tumor sample, and whereby the TIMP-1 levels and the presence of a RAS mutation in the patient's tumor sample indicate that the patient is likely to respond to a treatment with the EGFR inhibitor.

Accordingly, the present invention provides a method of identifying a cancer patient who is likely to benefit from treatment with an EGFR inhibitor comprising determining in vitro the levels of TIMP-1 of the patient; the absence or presence of a RAS mutation in a patient's tumor sample and the expression of EGFR in a patient's tumor sample, and whereby the TIMP-1 levels, the presence of a RAS mutation and EGFR expression in the patient's tumor sample indicate that the patient is likely to respond to a treatment with the EGFR inhibitor.

According to one embodiment, the present invention provides for a method of identifying a cancer patient who is likely to benefit from treatment with an EGFR inhibitor comprising determining in vitro the blood plasma levels of TIMP-1 of the patient, the absence or presence of a RAS mutation in a patient's tumor sample, wherein EGFR is expressed in the patient's tumor sample, and whereby the TIMP-1 blood plasma levels and the presence of a RAS mutation in the patient's tumor sample indicate that the patient is likely to respond to a treatment with the EGFR inhibitor. In one embodiment, the present invention provides a method of identifying a cancer patient who is likely to benefit from treatment with an EGFR inhibitor comprising determining in vitro TIMP-1 levels in a tumor sample from said patient, the absence or presence of a RAS mutation in said patient's tumor sample, wherein EGFR is expressed in the patient's tumor sample, and whereby the patient's TIMP-1 levels in said tumor sample and the presence of a RAS mutation in the tumor sample indicate that the patient is likely to respond to a treatment with the EGFR inhibitor.

According to one embodiment, the present invention provides a method of identifying a cancer patient who is likely to benefit from treatment with an EGFR inhibitor comprising determining in vitro TIMP-1 tumor tissue immunoreactivity in said patient's tumor tissue form said patient and the absence or presence of a RAS mutation in said patient's tumor sample, wherein EGFR is expressed in said patient's tumor sample and whereby the TIMP-1 tumor tissue immunoreactivity in said patient's tumor sample and the presence of a RAS mutation in said patient's tumor sample indicate that said patient is likely to respond to a treatment with an EGFR inhibitor.

According to one embodiment, the present invention pertains to predicting cancer patient response to EGFR-inhibitor treatment, which comprises the step of determining in vitro the patient's blood TIMP-1 levels, or the patient's blood plasma TIMP-1 levels, or the TIMP-1 levels in a tumor sample of said patient and determining the absence or presence of a RAS mutation in said patient's tumor sample expressing EGFR, wherein the plasma levels of TIMP-1 and the presence of a RAS mutation in said patient's tumor sample indicate an increased progression free survival (PFS) and/or overall survival (OS) of said patient when said patient is treated with an EGFR inhibitor.

In one embodiment, the present invention provides a method of identifying a patient non- responsive to a treatment with at least one EGFR inhibitor comprising determining in vitro the patient's blood TIMP-1 levels, or the patient's blood plasma TIMP-1 levels, the absence or presence of a RAS mutation in a tumor sample from said patient, wherein EGFR is expressed in said patient's tumor sample and whereby the blood or blood plasma levels of TIMP-1 are less than 250 ng/ml and the presence of a mutated KRAS in said patient's tumor sample indicate that said patient will not respond to a treatment with an EGFR inhibitor.

Accordingly, the EGFR inhibitor for use in the inventive method as disclosed herein in one embodiment is a monoclonal antibody, or a tyrosine kinase inhibitor.

Accordingly, the EGFR inhibitor for use in the inventive method as disclosed herein in one embodiment is one or more of cetuximab, panitumumab, erlotinib, gefitinib, afatinib, dacomitinib, neratinib, vandetanib, brivanib, tivantinib, crizotinib, XL-647, canertinib, pelitinib, PKI-166 or TAK-285.

More specifically, the EGFR inhibitor for use in the inventive method is in one embodiment one or more of cetuximab, panitumumab, erlotinib, gefitinib. According to the inventive method, the EGFR inhibitor is in one embodiment administered in combination with at least one additional chemotherapeutic agent, wherein the

chemotherapeutic agent in one embodiment is selected from the group consisting of capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, 6-mercaptopurine (6-MP), cladribine, clofarabine, 6-mercaptopurine (6-MP), cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, preferably a combination of 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA), or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx).

According to one embodiment, the cancer patient's TIMP-1 levels, preferably the blood plasma TIMP-1 levels in the inventive method are determined by means of an enzyme-linked immunosorbent assay (ELISA). Accordingly, the blood plasma levels according to the inventive method are in one embodiment at least about 250 ng/ml to about 1400 ng/ml, or at least about 250 ng/ml, 275 ng/ml, 300 ng/ml, 325 ng/ml, 350 ng/ml, 375 ng/ml, 400 ng/ml, 409 ng/ml, 425 ng/ml. Blood plasma levels of at least 250 ng/ml are in one embodiment indicative of the cancer patient being likely to benefit from treatment with an EGFR inhibitor. According to a more preferred embodiment, the RAS mutation in the method according to the present invention is an activating mutation. Accordingly, the RAS mutation in one

embodiment is a H-RAS, N-RAS or KRAS mutation.

In one embodiment, the N-RAS mutation according to the invention comprises at least one mutation, which is selected from the group consisting of G12C, G12D, G12R, G12S, G12A, G12V, G12R, G13C, G13R, G13A, G13D, G13V, G15W, G60E, Q61 P, Q61 L, Q61 R, Q61 K, Q61 H and Q61 E.

According to one embodiment, the H-RAS mutation according to the invention comprises at least one mutation selected from the group consisting of the amino acid substitutions G12R, G12V, G13C, G13R and Q61 R. More specifically, according to one embodiment, the KRAS mutation according to the invention comprises at least one mutation selected from the group consisting of the amino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G13C and G13D, G13R, G13S, Q61 K, Q61 L, Q61 P, Q61 R and A146V. Accordingly, the inventive method pertains to determining the presence or absence of a RAS mutation in one embodiment by amplifying RAS nucleic acid from a patient's tumor sample, suspected of harboring a mutation by means of PCR.

In one embodiment of the inventive method, the absence or presence of a RAS mutation in the patient's tumor is determined by amplifying RAS nucleic acid from said tumor and sequencing said amplified nucleic acid.

According to one embodiment of the inventive method, the EGFR expressed in the patient's tumor may be wild type EGFR or mutated EGFR.

In one embodiment of the present inventive method, the cancer is selected from the group consisting of colorectal cancer, metastatic colorectal cancer, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, non-small-cell lung cancer, melanoma, myeloid leukemia, juvenile myeloid leukemia, ovarian cancer, pancreas cancer, thyroid cancer, prostate cancer, and adenocarcinoma.

In one embodiment the cancer is colorectal cancer or metastatic colorectal cancer. In one embodiment the cancer is metastatic colorectal cancer. According to one embodiment, the present invention pertains to the use of the biomarkers RAS and TIMP-1 for predicting the pharmaceutical efficacy and/or clinical response of a combination comprising at least one EGFR inhibitor and at least one chemotherapeutic agent to be administered to a patient afflicted with cancer.

According to one embodiment, the present invention pertains to the use of the biomarkers KRAS and TIMP-1 for predicting the pharmaceutical efficacy and/or clinical response of a combination comprising at least one EGFR inhibitor and at least one chemotherapeutic agent to be administered to a patient afflicted with cancer.

According to a more specific embodiment, the at least one EGFR inhibitor according to the invention as defined above is selected from the group consisting of cetuximab,

panitumumab, erlotinib, gefitinib, afatinib, dacomitinib, neratinib, vandetanib, brivanib, tivantinib, crizotinib, XL-647, canertinib, pelitinib, PKI-166 and TAK-285.

More specifically, the present invention pertains to the use of the biomarkers KRAS and TIMP-1 for predicting the pharmaceutical efficacy and/or clinical response of a combination comprising the EGFR inhibitor cetuximab and at least one chemotherapeutic agent to be administered to a cancer patient.

According to a more specific embodiment, the use of the biomarkers KRAS and TIMP-1 according to the present invention comprises determining in vitro the absence or presence of a KRAS mutation, which is selected from the group consisting of the amino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G13C and G13D, G13R, G13S, Q61 K, Q61 L, Q61 P, Q61 R and A146V.

Accordingly, the use of the biomarkers TIMP-1 and KRAS according to the invention as disclosed above in one embodiment comprises determining in vitro the blood plasma TIMP-1 levels of the cancer patient.

Accordingly, the present invention pertains to the use of the biomarkers KRAS and TIMP-1 in a cancer patient, e.g. biliary tract cancer, bladder cancer, breast cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, non-small-cell lung cancer, melanoma, myeloid leukemia, juvenile myeloid leukemia, ovarian cancer, pancreas cancer, thyroid cancer, prostate cancer, adenocarcinoma, colorectal cancer or metastatic colorectal cancer to predict the pharmaceutical efficacy and/or clinical response of a combination comprising the EGFR inhibitor cetuximab and at least one chemotherapeutic agent to be administered to said cancer patient.

In a more specific embodiment, the at least one chemotherapeutic agent for use as defined above according to present invention is selected from the group consisting of 5- fluorouracil/folinic acid (5-FU/FA), capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, 6- mercaptopurine (6-MP), cladribine, clofarabine, 6-mercaptopurine (6-MP), cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, preferably a combination of 5-FU, leucovorin, 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5- FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx).

Accordingly, the present invention also provides for the use of the biomarkers KRAS and TIMP-1 as defined above, wherein cetuximab is used according to the present invention in a concentration of about 125 mg/m² to about 500 mg/m² body surface, preferably about 250 mg/m² to about 400 mg/m² body surface. Accordingly, cetuximab is administered according to the present invention every 5 days to 21 days, or every 7 days to 14 days, preferably every 5 days to 10 days, more preferably every 7 days or 14 days.

According to one embodiment, the present invention provides for a method of treating a patient with cancer, wherein the treatment comprises administering to a patient in need thereof a therapeutically effective amount of an EGFR inhibitor as defined above and at least one chemotherapeutic agent as defined above to a patient, if the patient is likely to benefit from the cancer treatment according to the inventive method as defined above, indicative of an increased progression free survival (PFS) and/or overall survival (OS). According to a more specific embodiment, the EGFR inhibitor for use in the method of treating a patient with cancer according to the invention is a monoclonal antibody or a tyrosine kinase inhibitor.

Accordingly, the EGFR inhibitor of the present invention for use as defined above in one embodiment is one or more of cetuximab, panitumumab, erlotinib, gefitinib, afatinib, dacomitinib, neratinib, vandetanib, brivanib, tivantinib, crizotinib, XL-647, canertinib, pelitinib, PKI-166 and TAK-285, preferably cetuximab.

In an even more preferred embodiment, according to the inventive method of treatment the EGFR inhibitor cetuximab is administered in a concentration of at least about 125 mg/m² to about 500 mg/m² body surface, preferably about 250 mg/m² to about 400 mg/m² body surface.

Accordingly, the blood plasma TIMP-1 levels in the patient treated according to the inventive method are in one embodiment at least about 250 ng/ml to about 1400 ng/ml, or at least about 300 ng/ml to about 1250 ng/ml, or at least about 250 ng/ml, 275 ng/ml, 300 ng/ml, 310 ng/ml, 325 ng/ml, 350 ng/ml, 375 ng/ml, 400 ng/ml, 409 ng/ml or 425 ng/ml. According to one embodiment, the present invention pertains to an EGFR inhibitor in combination with at least one chemotherapeutic agent for use in the treatment of cancer as defined above, wherein the tumor expresses a mutated KRAS and expresses EGFR as defined herein and whereby TIMP-1 levels are as defined herein.

According to a more specific embodiment, EGFR inhibitor in combination with at least one chemotherapeutic agent for use in the treatment of cancer according to the invention is a monoclonal antibody or a tyrosine kinase inhibitor, more specifically, the EGFR inhibitor is selected from the group consisting of cetuximab, panitumumab, erlotinib, gefitinib, afatinib, dacomitinib, neratinib, vandetanib, brivanib, tivantinib, crizotinib, XL-647, canertinib, pelitinib, PKI-166 and TAK-285. In a more preferred embodiment, the EGFR inhibitor in combination with at least one chemotherapeutic agent for use in the treatment of cancer according to the present invention as defined above is cetuximab. Accordingly, cetuximab may be administered in the inventive method of treatment in a concentration of about 125 mg/m² to about 500 mg/m² body surface, preferably of about 250 mg/m² to about 400 mg/m² body surface.

In a preferred embodiment, the present invention provides for an EGFR inhibitor in combination with at least one chemotherapeutic agent for use in the treatment of cancer as defined above, wherein the KRAS mutation is an activating mutation.

In a more preferred embodiment, the present invention pertains to an EGFR inhibitor in combination with at least one chemotherapeutic agent for use in the treatment of cancer as defined above, wherein the KRAS mutation is an activating mutation as defined above and whereby the cancer patient's blood plasma TIMP-1 levels are at least about 250 ng/ml, such as at least about 250 ng/ml to about 1400 ng/ml.

According to a more preferred embodiment, the EGFR inhibitor for use according to the invention is in one embodiment a monoclonal antibody or a tyrosine kinase inhibitor

Accordingly, the EGFR inihibitor in combination with at least one chemotherapeutic agent for use according to the invention is in one embodiment cetuximab.

Accordingly, the KRAS mutation in the inventive use of an EGFR inhibitor in combination with at least one chemotherapeutic agent as defined above comprises at least one mutation selected from the group consisting of the amino acid substitutions G12C, G12D, G12A,

G12V, G12S, G12F, G12R, G13C and G13D, G13R, G13S, Q61 K, Q61 L, Q61 P, Q61 R and A146V.

More specifically, the at least one chemotherapeutic agent for use in combination with an EGFR inhibitor according to the invention is selected from the group of chemotherapeutic agents as defined above.

Accordingly, the at least one chemotherapeutic agent for use in the treatment of cancer according to the invention in combination with the EGFR inhibitor cetuximab, in one embodiment is selected from the group consisting of 5-fluorouracil/folinic acid (5-FU/FA), capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, 6-mercaptopurine (6-MP), cladribine, clofarabine, 6-mercaptopurine (6-MP), cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or preferably from a combination of 5-FU and leucovorin, or a combination of 5-FU, leucovorin, and oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx). In a preferred embodiment, the present invention pertains to the EGFR inhibitor cetuximab in combination with 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin (FLOX) for use in the treatment of cancer, wherein the tumor expresses EGFR, a mutated KRAS as defined above and whereby the plasma TIMP1 levels in the patient are at least about 275 ng/ml to about 1400 ng/ml, preferably at least about 275 ng/ml, 300 ng/ml, 325 ng/ml, 350 ng/ml, 375 ng/ml, 400 ng/ml or at least about 425 ng/ml, or as defined herein according to the invention.

According to one embodiment, the present invention pertains to the EGFR inhibitor cetuximab in combination with 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin (FLOX) for use in the treatment of cancer, wherein the tumor expresses EGFR, a mutated KRAS as defined above and whereby the patient's tumor sample is TIMP-1 immunoreactive. In an even more preferred embodiment, the present invention provides for the EGFR inhibitor cetuximab in combination with 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin (FLOX) for use in the treatment of cancer as defined above, whereby the cancer is colorectal cancer and/or metastatic colorectal cancer.

DESCRIPTION OF THE DRAWINGS

Figure 1 : (A) Proposed molecular mechanisms of the EGFR signaling network induced by TIMP-1 .Non-therapeutic setting: the tumor cells will be driven by EGFR-1 signaling and the TIMP-1 signaling axis will only contribute modestly. (B) The predictive value of TIMP-1 ; the expression is under control of EGFR-1 -signaling, independently of the RAS/MAPK-axis, and TIMP-1 drives hyperproliferation of KRAS mutated cells. (C) Tumor cells are exposed to cetuximab: Expression of TIMP-1 will be inhibited, irrespective of KRAS status. This will not have a noticeable effect on KRAS wild-type tumor cells, but will abrogate the proliferative drive that TIMP-1 has on KRAS mutated tumor cells. (D) Potential mechanism of TIMP-1 mediated RAS signaling cascade activation, when EGFR signaling is inhibited by cetuximab. Figure 2: Nordic VII Study: Consort Diagram indicating sample size at each stage during the study. Assessed for Eligibility n=574571 ; n=5 excluded (1 mistaken includion, 1 consent withdrawn, 2 mis-diagnoses and 1 intercurrent death). Randomized n=566; n=14 excluded due to missing plasma samples. "Int.Flox" = intermittent Flox. Secondary analysis: Group A: KRAS wt n=46, KRAS mut n=23; Group B+C: KRAS wt n=101 , KRAS mut n=52. Figure 3: Kaplan-Meier estimates of survival probabilities for Progression Free Survival, PFS (A), and Overall Survival, OS (B) stratified by pre-treatment plasma TIMP-1 . Plasma TIMP-1 was categorized according to its fertile levels. P-value = test for trend.

Figure 4: Kaplan-Meier estimates of survival probabilities. (A) OS probabilities were estimated for patients treated, or not, with Cetuximab (+/- cetuximab) stratified by KRAS status (KRAS wt or mutant) and TIMP-1 level, below 201 ng/ml (first quartile) or above 409 ng/ml (third quartile). (B) The estimated survival probabilities based on the multivariable Cox regression model. The covariates are set to: CEA status (elevated), CRP (elevated), gender (male), age (70 years), multiple metastatic sites, good performance status, negative BRAF, treatment (cetuximab or not), KRAS status (wt or mutant), and TIMP-1 level equal to 201 ng/ml (first quartile) or equal to 409 ng/ml (third quartile).

Figure 5: Hazard ratio plot (solid line) with 95% CI (dotted lines) for OS comparing patients receiving FLOX + cetuximab versus those not treated with cetuximab as a function of the plasma TIMP-1 level and stratified by the KRAS mutational status. The estimates are based on the full multivariable model. Y-axis: Hazard Ration (95% CI), X-axis: TIMP-1 (ng/ml)

Figure 6: EGF induces TIMP-1 expression in CRC cells. CRC cell lines were serum- deprived for 24h prior to being stimulated with either 10 ng/mL or 50 ng/mL EGF for 24h and 48h. Controls, cultured with or without serum, were included in parallel. Immunoblotting of cell lysates was carried out using antibodies against P-Akt (Ser374), total Akt, TIMP-1 and p150^Glued (normalizing control). (A and B) Upper panels: immunoblots of HT-29 and HCT-15, respectively, lower panel: graph depicting pooled densitometry measurements of TIMP-1 levels relative to those of p150^Glued. Data points are presented as mean ± SEM of triplicate experiments. (C-E) graph depicting pooled densitometry measurements of TIMP-1 levels relative to those of p150^Glued.in immunoblots from DLD-1 , SW620, and Colo-205,

respectively.

Figure 7: TIMP-1 promotes malignant behavior of KRAS mutated cells. (A-D) TIMP-1 promotes colony formation in soft agar in /¾AS-mutated cells. Tumorsphere formation was quantified [in colony forming units (CFUs)] for DLD-1 clones bearing either a KRAS-mutated allele (KRAS G13D) or a wilt-type allele (KRAS wt) after 21 and 28 days of growth, in the presence of either rTIMP-1 (5ug/mL) or BSA (control). Visible colonies were counted by two independent observers. Data represents mean ± SEM (error bars), of triplicate experimenets. Significance was evaluated by two-way ANOVA with Sidak's multiple comparison post-test. Images were captured at 10 x magnification. (E) DLD-1 isogenic cell lines were serum- starved for 24 h before assessing the effect of rTIMP-1 (5ug/mL) on invasion in a 15-hour Boyden chamber invasion assay. Graph depicts the mean number of invaded cells of triplicate experiments. Invaded cells were counted by two independent observers.

Significance was determined by two-way ANOVA with Sidak's multiple comparison post-test.

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended items/claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Throughout this specification and the items/claims which follow, unless the context requires otherwise, the term "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step but not the exclusion of any other non-stated member, integer or step. The term "consist of" is a particular embodiment of the term "comprise", wherein any other non-stated member, integer or step is excluded. In the context of the present invention, the term "comprise" encompasses the term "consist of".

The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the items/claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of

SUBSTITUTE SHEET RULE 26 referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. The described objectives are solved by the present invention, preferably by the subject matter of the appended items/claims. More preferably, the present invention is solved according to a first embodiment by a method of identifying a cancer patient who is likely to benefit from treatment with an EGFR inhibitor, which comprises determining in vitro

a) the TIMP-1 blood levels of the patient,

b) the absence or presence of a RAS mutation in the patient's tumor sample, wherein EGFR is expressed in the patient's tumor sample, and whereby the plasma levels of TIMP-1 and the presence of a RAS mutation in the tumor sample indicate that the patient is likely to respond to a treatment with the EGFR inhibitor. Accordingly, the present invention provides a method for identifying a patient who is likely to benefit from treatment with an

EGFR inhibitor, which comprises determining in vitro TIMP-1 plasma levels of the patient and determining the presence or absence of a RAS mutation in the patient's tumor sample.

In one embodiment the present method of identifying a cancer patient who is likely to benefit from treatment with an EGFR inhibitor, further comprises determing EGFR expression in the patient's tumor sample.

The term "tumor sample" as used with the inventive method refers to a sample obtained from a patient. The tumor sample may be obtained from the patient by routine measures known to the person skilled in the art, such as biopsy (taken by aspiration or punctuation, excision or by any other surgical method leading to biopsy or resected cellular material). For those areas not easily reached surgical measures may be used by a surgeon to obtain the tumor sample for use in the inventive method. The process of obtaining a tumor sample from a patient does not form part of this invention. Accordingly, the term "tumor sample" as used within the context of the present invention, e.g in context of the inventive method of identifying a cancer patient who is likely to benefit from treatment with an EGFR inhibitor or e.g. in the inventive method of predicting cancer patient prognosis, or e.g. in the inventive method of identifying a patient non-responsive to a treatment with an EGFR inhibitor, refers to individual tumor cells, e.g. 1 - 10, or 1 -100 cells, or e.g. 10, 20, 30, 40, 50 - 100 cells, or 200, 300, 400, 500, 600, 700, 800, 900 or 1000, 2500, 5000, 10,000, 25,000, 50,000 cells or more, or parts of the tumor obtained by means of e.g. a biopsy, or may also refer to essentially all of the tumor, obtained from the patient. The term "tumor" as used in the present invention refers to an abnormal mass of tissue, e.g. a small group of cells, or a part of an organ, which contain or comprise neoplastic cells or have undergone neoplastic transformation. Accordingly, neoplasms and tumors may be e.g. benign, pre-malignant, or malignant. For example, tumors according to the invention in one embodiment includes neuroblastoma, intestine carcinoma such as rectum carcinoma, colon carcinoma, familiary adenomatous polyposis carcinoma and hereditary non-polyposis colorectal cancer, esophageal carcinoma, labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tong carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, medullary thyroidea carcinoma, papillary thyroidea carcinoma, renal carcinoma, kidney parenchym carcinoma, ovarian carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, pancreatic carcinoma, prostate carcinoma, testis carcinoma, breast carcinoma, urinary carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), adult T-cell leukemia lymphoma, hepatocellular carcinoma, gall bladder carcinoma, bronchial carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyo sarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma and plasmocytoma, preferably colorectal carcinoma and/or metastatic colorectal carcinoma. The term "cancer" as used in the present invention, e.g. in the inventive method as described above, refers to a cell that displays uncontrolled growth, invasion upon adjacent tissues, and often metastasis to other locations of the body. The cancer may be a sarcoma, lymphoma, leukemia, carcinoma, or blastoma. Accordingly, the cancer may be e.g. an epithelial cancer (carcinoma) of a vital organ, such as e.g. the pancreas, liver, lung and gut, or the cancer may be e.g. colorectal cancer, metastatic colorectal cancer, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, melanoma, myeloid leukemia, juvenile myeloid leukemia, ovarian cancer, pancreas cancer, thyroid cancer, prostate cancer, adenocarcinoma.

The term "overall survival" as used in the inventive method of determining cancer patient prognosis refers to the percentage of people in a study or treatment group who are still alive for a certain period of time after being diagnosed with cancer or after treatment was started for a disease, such as e.g. cancer. The term "progression-free survival" (PFS) as used within the context of the inventive method is used to describe the length of time during and after medication or treatment during which cancer does not worsen, e.g. the staging of the cancer according to the TNM system does not increase, e.g. from T1 to any of T2, T3 or T4. The term PFS may also be used in the present invention as a metric to study the health of a person afflicted with cancer to determine the effect of a new treatment, e.g. compound, antibody, chemotherapeutic agent, or treatment regimen. The time interval from the start of treatment to disease progression is typically defined as progression-free survival. It is a measure of the clinical benefit from therapy. PFS may also be used within the present invention as a metric to evaluate the cost effectiveness of a cancer treatment.

In one embodiment the cancer patient's TIMP-1 levels is the cancer patient's blood TIMP-1 levels. In one embodiment the cancer patient's blood TIMP-1 levels is selected from the group consisting of whole blood, plasma and serum. In a preferred embodiment the cancer patient's blood TIMP-1 levels is the patient's blood plasma TIMP-1 levels.

In one embodiment the cancer patient's TIMP-1 levels are pre-treatment TIMP-1 levels. Pre- treatment TIMP-1 levels may be levels before the cancer patient has received chemotherapy, or before the patient receives chemotherapy comprising at least one EGFR inhibitor as outlined herein.

Accordingly, the patient's blood TIMP-1 levels, or the patient's blood plasma TIMP-1 levels, or the TIMP-1 levels in a tumor sample of the patient in the inventive method may be determined by any technology known to the skilled person in the art, which allows the quantification of TIMP-1 in a patient's blood sample, or in a patient's blood plasma sample, or in a tumor sample from the patient. For example, blood TIMP-1 levels according to the invention may be determined in a blood sample from a cancer patient. The blood sample may be used immediately after the sample was obtained from the patient, e.g. the blood sample may be used to determine TIMP-1 levels therein, prior to centrifugation or blood clotting, or e.g. the blood sample may be allowed to clott for at least 30 minutes, after which the serum may be obtained by centrifugation at 1500 rcf for 15 min within two hours after blood collection. TIMP-1 levels may then e.g. be determined using an enzyme-linked immunosorbent assay (e.g. R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The patient's blood plasma TIMP-1 levels may e.g. be determined in a plasma sample from a patient, e.g. plasma may be obtained from a whole blood sample collected into commercially available anticoagulant-treated tubes e.g. EDTA-treated or citrate-treated tubes, heparinized tubes may also be used if the heparin used is endotoxin-free. Subsequently, cells may e.g. be removed from plasma by centrifugation for 10 to 15 minutes at 1 ,000-2,000 x g using a refrigerated centrifuge, the resulting plasma supernatant may then be used to determine the TIMP-1 levels therein, e.g. by means of an enzyme-linked immunosorbent assay (ELISA). For example, TIMP-1 levels may be determined by a sandwich immunoassay (ELISA) as disclosed in Holten-Andersen et al., Br J Cancer (1999) 80:495-503 for the quantitation of total plasma TIMP-1 levels, e.g. the immunoassay consists of a polyclonal anti-TIMP-1 antiserum raised in sheep for capture of antigen and a monoclonal anti-TIMP-1 lgG1

(MAC15) for detection of the bound antigen. This antibody recognizes both free and complexed TIMP-1 .

Alternatively, the plasma TIMP-1 levels, or the whole blood TIMP-1 levels according to the invention may be determined by determining free TIMP-1 in the plasma or whole blood sample of patients. The term "free" as used herein, refers to TIMP-1 that is not bound to a matrix metalloproteinases (MMPs). Thereto, the method as disclosed in Holten-Andersen et al Clin Chem. 2002 Aug;48(8):1305-13 may be employed, e.g. the immunoassay as disclosed above is carried out using the monoclonal antibody MAC19, which may for example be carried out as follows: 96-well microtiter plates (Maxisorp) may be coated overnight at 4 °C with an affinity purified sheep polyclonal anti-TIMP-1 antiserum (4mg/l; 100 μΙ/well). After blocking and washing a calibration curve for recombinant free TIMP-1 may e.g. be established by adding serial dilutions containing 12, 6, 3, 1 .5, 0.75, 0.375, and 0.1875 μ9/Ι (100 μΙ/well) to the plates; also, a 1 :26 dilution of a citrate plasma pool as a control and blank wells containing only sample dilution buffer may e.g. be included. Plasma samples may then e.g. be diluted 1 :26 in sample dilution buffer (20 μΙ_ of sample plus 500 μΙ_ of sample buffer; 100 μΙ/well) and added to the plates, which may then be incubated for e.g. 1 h at 30 °C. After binding of TIMP-1 , plates may for example be washed and incubated with 100 μΙ/well MAC19 (375 μο/Ι) for 1 h at 30 °C. After another round of washes, plates may e.g. be incubated for 1 h at 30 °C with the alkaline phosphatase conjugated rabbit anti-mouse antibody. After the final wash step, p-nitrophenyl phosphate substrate solution may for example be added (100 μΙ/well), and the plate may then be immediately read using kinetic rate measurements (mAU/min) in a plate reader at 405 nm for every 10 min over 60 min. An alkaline phosphatase conjugated rabbit anti-mouse antiserum (Dako, Glostrup, Denmark) is used as the final layer enabling the kinetic rate assay. Other examples of immunoassays for use in the inventive method may include e.g. direct ELISA using a labeled antibody recognizing an antigen immobilized on a solid support; or e.g. indirect ELISA using a labeled antibody recognizing a capture antibody forming complexes with an antigen immobilized on a solid support; or e.g. direct sandwich ELISA using a labeled antibody recognizing an antigen bound to a antibody immobilized on a solid support; and e.g. indirect sandwich ELISA, in which a captured antigen bound to an antibody immobilized on a solid support is detected by first adding an antigen-specific antibody, and then a secondary labeled antibody which binds the antigen-specific antibody. More preferably, however, the protein expression levels may be detected by e.g. sandwich ELISA as disclosed above, where a sample reacts with an antibody immobilized on a solid support, and the resulting antigen-antibody complexes are detected by adding a labeled antibody specific for the antigen, followed by enzymatic development, or by first adding an antigen-specific antibody and then a secondary labeled antibody which binds to the antigen-specific antibody, followed by enzymatic development. The blood plasma levels of TIMP-1 of the cancer patient, or the blood TIMP-1 levels of said patient, or the tumor TIMP-1 levels of said patient in any of the above embodiments of the inventive method, e.g. wherein TIMP-1 is used as a biomarker, are preferably determined prior to any treatment ("baseline" TIMP-1 levels) and may be determined as described herein.

According to the inventive method, TIMP-1 levels may also e.g. be determined in the tumor sample of the patient, e.g. TIMP-1 levels in the patient's tumor sample may be determined by ELISA, Western blotting, antibody array or immunohistochemistry. For example, the patient's tumor tissue may be homogenized by means of an ultrasonic homogenizer for e.g. 1 minute at a temperature of 4°C in a buffer comprising 0.01 M CaCI₂, 0.25% Triton 100 and optionally protease inhibitors. The homogenate may then e.g. be centrifuged for 30 min at 6000g. The resulting supernatant may then be subjected for further analyses, such as e.g. western blotting, ELISA, or antibody array analysis, such as e.g. antibody suspension bead arrays (Haggermark et al, Proteomics 2013, 13, 2256-2267). For example, anti-TIMP-1 antibodies, such as e.g. MAC 15 or MAC19, or VT1 ; VT2, VT4-VT8, may be immobilized onto magnetic beads (LuminexCorporation), e.g. the antibodies may be diluted to a concentration of 1 .6 _g/ml in a buffer comprising 2[N-Morpholino]-ethanesulphonic acid, pH 5.0 and may be incubated with the beads after activation of the carboxylic surface using 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide and N-hydroxysulphosuccinimide. In addition to the selected TIMP-1 protein target, one bead identity may e.g. be used for immobilization of an albumin-binding antibody (Dako), one bead identity e.g. for anti-human IgG (Jackson

ImmunoResearch), one for e.g. rabbit IgG (Bethyl) and one bead identity may e.g. be incubated with protein-free buffer. After incubation, the coupled beads may e.g. be washed and stored in a blocking reagent for ELISA (BRE, Roche) before all bead identities may be combined to create a bead array in suspension. The supernant as obtained above may then e.g. be diluted 1 :2 in PBS or PBS supplemented with 0.5% w/v bovine serum albumin (BSA, Sigma) and 0.1 % w/v rabbit IgG (Bethyl) (BIG buffer), using a liquid handler (SELMA, CyBio) and may be labeled at 4°C with a tenfold molar excess of biotin (NHS-PEG4-Biotin, Thermo Scientific) over total protein content. The labeling may e.g. be terminated after 2 h by by the addition of 1 MTris- HCI in a 250-fold molar excess over biotin and the reaction was allowed to occur for 20 min. A second dilution step of 1 :8 may be performed with a buffer composed of e.g. 0.5% w/v PVA, 0.8% (w/v) PVP and 0.1 % w/v casein (all Sigma-Aldrich)

supplemented with 10% v/v rabbit IgG, denoted PVXCas buffer. The samples may then be heat treated either at 56° or 72 ^<Ό for 30 min and cooled to 20 °C for 15 min in a thermocycler. Susequently, 45μΙ of sample may be incubated with 5μΙ of the bead array overnight at 20 °C under permanent rotation on a shaker. The bead-sample mixture may then subsequently be washed with 3 χ 100μΙ PBST (1 χ PBS, 0.05% Tween 20) using a wash station (BioTek EL406). Bound targets may be cross-linked to the immobilized antibodies by addition of e.g. 50μΙ 0.4% paraformaldehyde in PBS for 10 min, followed by a bead-washing step. For detection, 50 μΙ of a streptavidin conjugated fluorophore (R-phycoerythrin, Invitrogen) diluted to 0.5 μg/ml in PBST may be added and incubated for 20 min. Finally, the beads may be washed prior to addition of 100 μΙ PBST for measurement in a LX200 or FlexMap3D instrument (both Luminex Corporation) and e.g. for each bead identity, at least 50 events may be counted, whereby at least 34 events are required for reliable results and the median fluorescence intensity may be used as read-out of the assay. In the above assay, different standards of TIMP-1 may also be included in addition to the supernatants obtained from the patient's tumor samples for quantification purposes.

According to one embodiment, the inventive method of identifying a cancer patient who is likely to benefit from treatment with an EGFR inhibitor as disclosed above comprises determining in vitro the TIMP-1 immunoreactivity in the patient's tumor sample. Accordingly, the inventive method comprises determining TIMP-1 immunoreactivity in the patient's tumor sample by e.g. antibody suspension bead arrays as disclosed above, or e.g. by

immunohistochemistry (IHC). For example, the patient's tumor tissue may be embedded into paraffin and cut in 5μηι sections, which may then be subjected to dewaxing with xylene and rehydrated in ethanol/water dilution series and subsequent epitope retrieval by e.g. protelytic treatment with proteinase K, or heat-induced epitope retrieval for which different buffers may be used. For example, heat-induced epitope retrieval may be done in e.g. 10 mM citrate buffer, pH 6. The endogenous peroxidase activity in the sections may be quenched by immersion in 3% hydrogen peroxide for 5 min. The sections may then e.g. be incubated with at least one or more of the following antibodies to detect the TIMP-1 immunoreactivity in the patient's tumor sample according to the invention: MAC 15, MAC19, VT1 ; VT2, VT4, VT5, VT6, VT7 or VT8 (SSI Copenhagen, Denmark), for at least 30 min at room temperature, followed by visualization with e.g. DAB, e.g. for two periods of 3 minutes, with inbetween washes TBS, 0.05% tween-20, pH 7.6, after which the tumor tissue may subsequently be counterstained with e.g. hematoxylin. In the inventive method, TIMP-1 immunoreactivity may e.g. also be determined in a quantitatively. For example, TIMP-1 IHC may be done according to the method described in Barrow et al., J Clin Pathol 201 1 ; 64:208-214, utilizing one or more of the TIMP-1 antibodies as disclosed above, e.g. MAC 15, MAC19, or VT1 ; VT2, VT4, VT5, VT6, VT7, VT8. For example, formalin-fixed, paraffin-embedded (FFPE) sections (4- 8μηι) of the patient's tumor tissue may be mounted on coated slides and subjected to epitope retrieval using e.g. pressure heating in 0.001 M EDTA pH8.0 at 100 kPa for 6 min. The sections may then be subjected to quantum dot (QD) IHC staining, e.g. sections may be blocked for 20 min with 10% (v/v) normal goat serum (Vector Laboratories, Peterborough, UK) in TBS; washed in TBS wash and then blocked with avidin (Avidin/Biotin Blocking Kit, Vector Laboratories) for 15 min in Avidin; followed by a TBS wash; prior to block for 15 min in biotin (Avidin/Biotin Blocking Kit, Vector Laboratories). Sections may then be incubated with at least one primary antibody anti TIMP-1 antibody as disclosed above, diluted in 10% goat serum, followed by a wash in TBS and incubated with biotinylated goat antimouse IgG (product code BA-9200, Vector Laboratories) diluted 1 :150 (v/v) in 10% goat serum, followed by a TBS wash prior to incubation with streptavidin-coated Qdots (Invitrogen) diluted 1 :100 (v/v) in 10% (v/v) goat serum, followed by subsequent washes in TBS.

Alternatively, quantitative IHC to determine TIMP-1 levels in the patient's tumor tissue according to the invention may be done according to the method of Messersmith et al.

(Messersmith et al. Cancer Biology & Therapy (2005) 4:12, 1381 -1386), e.g. unstained 4 μηι sections may be deparaffinized and rehydrated in graded concentrations of alcohol by standard techniques prior to antigen retrieval in citrate buffer pH 6.0 for 30 minutes at l OCC.

The slides may then be cooled for 20 minutes before washing in 1 x TBST (Dako Corp.

Carpinteria, CA). All staining may be done using a DAKO Autostainer at room temperature.

Slides may then be incubated in 3% H₂0₂ for 10 minutes, followed by the appropriate dilution of at least one primary anti-TIMP-1 antibody as disclosed above for 60 minutes. If needed, antibodies may e.g. be diluted in Tris-HCI (0.2M, pH 7.5) (Quality Biological, Inc, Gaitersburg,

MD). Negative controls may be included in the study and be incubated for e.g. 60min with the antibody diluent solution (0.2M Tris-HCI, pH 7.5 from Quality Biological, Inc., Gaitersburg, MD). Staining may then be developed using e.g. the DAKO LSAB+ System (Dako Corp., Carpinteria, CA) with the following conditions: biotinylated link for 10 minutes (30 min for pEGFR), streptavidin for 10 min (30 min for pEGFR), and substrate-chromagen (DAB) Solution (DAKO Liquid DAB+ Substrate-Chromogen System) for 5 min. Slides may then be washed using 1 x TBST after incubation with each reagent and washed with dH₂0 following incubation with DAB. Analysis of the IHC treated patient's tumor tissue may be done using e.g. a computer controlled bright-field microscope coupled to a CCD camera capable of simultaneously detecting levels of hue (color), saturation (density) and luminosity (darkness), the data acquired therewith may then be used for further analysis, e.g. using ACIS software.

According to one embodiment, the present invention pertains to a method of identifying a patient non-responsive to a treatment with at least one EGFR inhibitor, which comprises determining in vitro a) the blood plasma levels of TIMP-1 of the patient, b) the absence or presence of a RAS mutation in a patient's tumor sample, wherein the EGFR is expressed in the patient's tumor sample and whereby the plasma levels of TIMP-1 are less than 250 ng/ml and the presence of a mutated RAS in the patient's tumor sample indicates that the patient will not respond to a treatment with an EGFR inhibitor. Accordingly, the TIMP-1 plasma levels according to the inventive method are in one embodiment 250 ng/ml or less, e.g. from about 50 ng/ml to about 245 ng/ml, or from about 100 ng/ml to about 230 ng/ml, or from about 125 ng/ml to about 225 ng/ml, or from about 150 ng/ml to about 200 ng/ml, or of about 55 ng/ml, 60 ng/ml, 65 ng/ml, 70 ng/ml, 75 ng/ml, 80 ng/ml, 85 ng/ml, 90 ng/ml, 95 ng/ml, 100 ng/ml to about 125 ng/ml, 130 ng/ml, 140 ng/ml, 150 ng/ml, 160 ng/ml, 170 ng/ml, 180 ng/ml, 190 ng/ml, 200 ng/ml, 212.5 ng/ml, 225 ng/ml, 230 ng/ml, 235 ng/ml, 240 ng/ml, 242.5 ng/ml, 245 ng/ml, 247.5 ng/ml in a cancer patient non-responsive to a treatment with at least one EGFR inhibitor. Accordingly, the TIMP-1 levels in the inventive method of identifying a patient non-responsive to a treatment with at least one EGFR inihibitor according to the invention may be determined as defined above, e.g. by means of an ELISA, or e.g. IHC, or e.g. by Western blotting, or e.g. by antibody array.

In one embodiment the TIMP-1 plasma levels in a cancer patient who is not likely to benefit from treatment with at least one EGFR inhibitor according to the inventive method are from about 50 ng/ml to about 55 ng/ml, such as from about 55 ng/ml to about 60 ng/ml, for example from about 60 ng/ml to about 65 ng/ml, such as from about 65 ng/ml to about 70 ng/ml, for example from about 70 ng/ml to about 75 ng/ml, such as from about 75 ng/ml to about 80 ng/ml, for example from about 80 ng/ml to about 85 ng/ml, such as from about 85 ng/ml to about 90 ng/ml, for example from about 90 ng/ml to about 95 ng/ml, such as from about 95 ng/ml to about 100 ng/ml, for example from about 100 ng/ml to about 1 10 ng/ml, such as from about 1 10 ng/ml to about 120 ng/ml, for example from about 120 ng/ml to about 130 ng/ml, such as from about 130 ng/ml to about 140 ng/ml, for example from about 140 ng/ml to about 150 ng/ml, such as from about 150 ng/ml to about 150 ng/ml, for example from about 150 ng/ml to about 160 ng/ml, such as from about 160 ng/ml to about 170 ng/ml, for example from about 170 ng/ml to about 180 ng/ml, such as from about 180 ng/ml to about 190 ng/ml, for example from about 190 ng/ml to about 200 ng/ml, such as from about 200 ng/ml to about 210 ng/ml, for example from about 210 ng/ml to about 220 ng/ml, such as from about 220 ng/ml to about 230 ng/ml, for example from about 230 ng/ml to about 240 ng/ml, such as from about 240 ng/ml to about 250 ng/ml.

The term "EGFR inhibitor" as used in the context of the inventive method of identifying a cancer patient who is likely to benefit from treatment with an EGFR inhibitor refers to any compound or substance that inhibits the biological activity of EGFR, e.g. dimerization upon ligand binding, or signal transduction events upon ligand binding.

According to one embodiment, the EGFR inhibitor for use in the present invention is a monoclonal antibody or a tyrosine kinase inhibitor, e.g. the EGFR inhibitor for use in the inventive method may be a monoclonal antibody, preferably a humanized or fully human monoclonal antibody, or a chimeric monoclonal antibody, which specifically binds to EGFR thereby preventing ligand binding to EGFR. The term "monoclonal antibody" as used in the inventive method refers to antibodies displaying a single binding specificity. The term "human monoclonal antibodies" refers to monoclonal antibodies which have variable and constant regions derived from human germline immunoglobulin sequences. The term "humanized" as used in the context of the present invention preferably refers to a monoclonal antibody in which the amino acid sequence is essentially identical to that of a human variant, despite the non-human origin of some of its complementarity determining region (CDR) segments responsible for the ability of the antibody to bind to its target antigen. The term "chimeric monoclonal antibody" as used in the present invention refers to a monoclonal antibody in which murine Fab fragments are spliced to human Fc.

According to the invention the EGFR inhibitor in one embodiment is a tyrosine kinase inhibitor. The term "tyrosine kinase inhibitor" as used in the above context of the inventive method of identifying a cancer patient who is likely to benefit from treatment with an EGFR inhibitor refers to all inhibitors of the tyrosine kinase activity of EGFR regardless of their mode of action. The term "monoclonal antibody" as used in the present invention refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies (mAb) for use in the present invention, are highly specific, being directed against a single antigenic site, e.g. they are directed against a single antigenic epitope of EGFR. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma (e.g. murine or human) method first described by Kohler et at., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). A "monoclonal antibody" may also be isolated from phage antibody libraries using the techniques described in Clackson et at., Nature, 352:624-628 (1991 ) and Marks et at., J. Mol. Biol., 222:581 -597 (1991 ).

According to one embodiment, the present invention provides for a method of predicting a cancer patient's response to EGFR-inhibitor treatment. The inventive method in one embodiment comprises the steps of determining in vitro the patient's blood TIMP-1 levels, such as the patient's blood plasma TIMP-1 levels, and/or the TIMP-1 levels in a tumor sample of the patient and determining the absence or presence of a RAS mutation in a patient's blood or tumor sample expressing EGFR, wherein the blood levels of TIMP-1 , or the blood plasma levels of TIMP-1 , or the TIMP-1 levels in a tumor sample of the patient and the presence of a RAS mutation in the patient's tumor sample indicate an increased progression free survival (PFS) and/or overall survival (OS) of the patient when the patient is treated with an EGFR inhibitor. According to one embodiment, the EGFR inhibitor for use in the present invention, e.g. for use in any of the above embodiments of the present invention, is a monoclonal antibody or a tyrosine kinase inhibitor.

According to a more specific embodiment, the EGFR inhibitor of the present invention is one or more of cetuximab, panitumumab, eriotinib, gefitinib, afatinib (BIBW2992), lapatinib, TAK- 285, CO-1686, neratinib, dacomitinib (PF299804), XL-647, vandetinib, canertinib (CI-1033), pelitinib (EKB-569), PKI-166 or TAK-285, neratinib, brivanib, tivantinib. Accordingly, the EGFR inhibitor of the present invention may e.g. be cetuximab, or panitumumab, or eriotinib, or gefitinib, or afatinib (BIBW2992), or lapatinib, or TAK-285, or CO-1686, or neratinib, or dacomitinib (PF299804), or XL-647, or vandetinib, or canertinib (CI-1033), or pelitinib (EKB- 569), or PKI-166 or TAK-285, or neratinib, or brivanib, or tivantinib, or e.g. cetuximab and eriotinib, or cetuximab and gefitinib, or cetuximab and afatinib, or cetuximab and dacomitinib, or cetuximab and lapatinib, or cetuximab and TAK-285, or cetuximab and CO-1686, or cetuximab and neratinib, or cetuximab and XL-647, or cetuximab and vandetinib, or cetuximab and canertinib, or cetuximab and pelitinib, or cetuximab and PKI-166, or e.g. panitumumab and eriotinib, or panitumumab and gefitinib, or panitumumab and afatinib, or panitumumab and dacomitinib, or panitumumab and lapatinib, or panitumumab and TAK- 285, or panitumumab and CO-1686, or panitumumab and neratinib, or panitumumab and XL-647, or panitumumab and vandetinib, or panitumumab and canertinib, or panitumumab and pelitinib, or panitumumab and PKI-166, or e.g. cetuximab, panitumumab and eriotinib, or cetuximab, panitumumab and gefitinib, or cetuximab, panitumumab and afatinib, or cetuximab, panitumumab and dacomitinib, or cetuximab, panitumumab and lapatinib, or cetuximab, panitumumab and TAK-285, or cetuximab, panitumumab and CO-1686, or cetuximab, panitumumab and neratinib, or cetuximab, panitumumab and XL-647, or cetuximab, panitumumab and vandetinib, or cetuximab, panitumumab and canertinib, or cetuximab, panitumumab and pelitinib, or cetuximab, panitumumab and PKI-166, or cetuximab, panitumumab and brivanib, or cetuximab, panitumumab and tivantinib, or e.g. cetuximab, eriotinib and gefitinib, or cetuximab, eriotinib and afatinib, or cetuximab, eriotinib and dacomitinib, or cetuximab, eriotinib and lapatinib, or cetuximab, eriotinib and TAK-285, or cetuximab, eriotinib and CO-1686, or cetuximab, eriotinib and neratinib, or cetuximab, eriotinib and XL-647, or cetuximab, eriotinib and vandetinib, or cetuximab, eriotinib and canertinib, or cetuximab, eriotinib and pelitinib, or cetuximab, eriotinib and PKI-166, or cetuximab, eriotinib and brivanib, or cetuximab, eriotinib and tivantinib, or e.g. panitumumab, eriotinib and gefitinib, or panitumumab, eriotinib and afatinib, or panitumumab, eriotinib and dacomitinib, or panitumumab, eriotinib and lapatinib, or panitumumab, eriotinib and TAK-285, or panitumumab, eriotinib and CO-1686, or panitumumab, eriotinib and neratinib, or panitumumab, eriotinib and XL-647, or panitumumab, eriotinib and vandetinib, or

panitumumab, eriotinib and canertinib, or panitumumab, eriotinib and pelitinib, or

panitumumab, eriotinib and PKI-166, or panitumumab, eriotinib and brivanib, or

panitumumab, eriotinib and tivantinib. In one embodiment, the above EGFR inhibitors according to the invention are combined with HER2 or HER3 inhibitors, which may be e.g. a monoclonal antibody or a tyrosine kinase inhibitor, e.g. the inhibitors may be one or more of trastuzumab, pertuzumab, U3-1287, or lapatinib, e.g. cetuximab may be combined with trastuzumab, or cetuximab may be combined with pertuzumab, or cetuximab may be combined with U3-1287, or e.g.

panitumumab may be combined with trastuzumab, or panitumumab may be combined with pertuzumab, or panitumumab may be combined with U3-1287, or e.g. cetuximab may be combined with trastuzumab, eriotinib, or e.g. cetuximab may be combined with trastuzumab, gefitinib, or e.g. cetuximab may be combined with trastuzumab, erlotinib, or e.g. cetuximab may be combined with trastuzumab, afatinib, or e.g. cetuximab may be combined with trastuzumab, lapatinib, or e.g. cetuximab may be combined with trastuzumab, TAK-285, or e.g. cetuximab may be combined with trastuzumab, CO-1686, or e.g. cetuximab may be combined with trastuzumab, neratinib, or e.g. cetuximab may be combined with trastuzumab, dacomitinib, or e.g. cetuximab may be combined with trastuzumab, erlotinib, XL-647, or e.g. cetuximab may be combined with trastuzumab, vandetinib, or e.g. cetuximab may be combined with trastuzumab, canertinib, or e.g. cetuximab may be combined with

trastuzumab, pelitinib, or e.g. cetuximab may be combined with trastuzumab, PKI-166, or e.g. cetuximab may be combined with trastuzumab, TAK-285, or e.g. cetuximab may be combined with trastuzumab, neratinib, or e.g. cetuximab may be combined with trastuzumab, brivanib, or e.g. cetuximab may be combined with trastuzumab, tivantinib, or e.g.

panitumumab may be combined with trastuzumab, erlotinib, or e.g. panitumumab may be combined with trastuzumab, gefitinib, or e.g. panitumumab may be combined with

trastuzumab, erlotinib, or e.g. panitumumab may be combined with trastuzumab, afatinib, or e.g. panitumumab may be combined with trastuzumab, lapatinib, or e.g. panitumumab may be combined with trastuzumab, TAK-285, or e.g. panitumumab may be combined with trastuzumab, CO-1686, or e.g. panitumumab may be combined with trastuzumab, neratinib, or e.g. panitumumab may be combined with trastuzumab, dacomitinib, or e.g. panitumumab may be combined with trastuzumab, erlotinib, XL-647, or e.g. panitumumab may be combined with trastuzumab, vandetinib, or e.g. panitumumab may be combined with trastuzumab, canertinib, or e.g. panitumumab may be combined with trastuzumab, pelitinib, or e.g. panitumumab may be combined with trastuzumab, PKI-166, or e.g. panitumumab may be combined with trastuzumab, TAK-285, or e.g. panitumumab may be combined with trastuzumab, neratinib, or e.g. panitumumab may be combined with trastuzumab, brivanib, or e.g. panitumumab may be combined with trastuzumab, tivantinib.

According to an even more specific embodiment, the EGFR inhibitor of the present invention is one or more of cetuximab, panitumumab, erlotinib, gefitinib. For example, the EGFR inhibitor according to the present invention may be cetuximab, panitumumab, erlotinib, gefitinib, or e.g. cetuximab and panitumumab, or e.g. cetuximab and erlotinib, or e.g.

cetuximab and gefitinib, or e.g. panitumumab and erlotinib, or e.g. panitumumab and gefitinib, or e.g. erlotinib and gefitinib, or e.g. cetuximab, panitumumab and erlotinib, or e.g. cetuximab, panitumumab and gefitinib.

According to one aspect of the present invention, the at least one chemotherapeutic agent, which is administered with the EGFR inhibitor in some of the embodiments of the inventive method as disclosed above is selected from the group consisting of capecitabine, 5-fluoro-2'- deoxyuiridine, irinotecan, 6-mercaptopurine (6-MP), cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, preferably a combination of 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA), or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a

combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx). The

chemotherapeutic agents may be provided as a single compound in combination with an EGFR inhibitor as disclosed above, or may e.g. be used in a combination of one, two, three or four chemotherapeutic agents in combination with one or more of the EGFR inhibitors as disclosed above, e.g. cetuximab, panitumumab, erlotinib or gefitinib.

Accordingly, in one embodiment at least one EGFR inhibitor, e.g. one, two, three or four EGFR inhibitors, may be administered with a combination of at least one chemotherapeutic agent as disclosed above, e.g. at least one EGFR inhibitor may be used with at least one chemotherapeutic agent as disclosed above, or least one EGFR inhibitor may be

administered in combination with two chemotherapeutic agents as disclosed above, or at least one EGFR inhibitor may be administered in combination with three chemotherapeutic agents as disclosed above, or at least one EGFR inhibitor may be administered in

combination with four chemotherapeutic agents as disclosed above, or two EGFR inhibitors may be administered in combination with one, two, three or four chemotherapeutic agents as disclosed above, or at least one, two or three EGFR inhibitors as disclosed above may be used in combination with at least one, two, three or four chemotherapeutic agents as disclosed above. Preferably, 5-FU, leucovorin, oxaliplatin and/or irinotecan may be combined with one or more of the EGFR inhibitors as disclosed above for as long as they provide the desired benefit to the patient. For example, cetuximab, panitumumab, erlotinib or gefitinib may be combined with 5-FU, leucovorin, oxaliplatin and/or irinotecan, e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin, or cetuximab may be combined with 5-FU, leucovorin, oxaliplatin and irinotecan, or panitumumab may be combined with 5-FU, leucovorin, oxaliplatin, or panitumumab may be combined with 5-FU, leucovorin, oxaliplatin and irinotecan, or erlotinib may be combined with 5-FU, leucovorin, oxaliplatin, or erlotinib may be combined with 5-FU, leucovorin, oxaliplatin and irinotecan, or gefinitib may be combined with 5-FU, leucovorin, oxaliplatin, or gefinitib may be combined with 5-FU, leucovorin, oxaliplatin and irinotecan. The EGFR inhibitors cetuximab, panitumumab, erlotinib or gefitinib as disclosed above in one embodiment may be used in combination with chemotherapeutic agents other than those disclosed above, e.g. cetuximab, panitumumab, erlotinib or gefitinib may be used in combination with one or more of afatinib (BIBW2992), dacomitinib (PF299804), neratinib, vandetanib, brivanib, tivantinib, crizotinib, XL-647, canertinib (CI-1033), pelitinib (EKB-569), PKI-166 or TAK-285, onartuzumab, rilotumumab, figitumumab, cixutumumab, ganitumab, lapatinib, or dovitinib. For example, cetuximab may be used in combination with afatinib, or in combination with dacomitinib, or in combination with neratinib, or in combination with vandetanib, or in combination with vandetanib, or in combination with brivanib, or in combination with crizotinib, onartuzumab, rilotumumab, figitumumab, cixutumumab, ganitumab, lapatinib, or in combination with TAK-285, dovitinib or e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin and afatinib or e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and afatinib, or e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin and dacomitinib or e.g. cetuximab may be combined with 5- FU, leucovorin, oxaliplatin, irinotecan and dacomitinib, or e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin and neratinib or e.g. cetuximab may be combined with 5- FU, leucovorin, oxaliplatin, irinotecan and neratinib, or e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin and vandetanib or e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and vandetanib, or e.g. cetuximab may be combined with 5- FU, leucovorin, oxaliplatin and brivanib or e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and brivanib, or e.g. cetuximab may be combined with 5- FU, leucovorin, oxaliplatin and tivantinib or e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and tivantinib, or e.g. cetuximab may be combined with 5- FU, leucovorin, oxaliplatin and crizotinib or e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and crizotinib, or e.g. cetuximab may be combined with 5- FU, leucovorin, oxaliplatin and crizotinib or e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and crizotinib, or e.g. cetuximab may be combined with 5- FU, leucovorin, oxaliplatin and dovitinib or e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and dovitinib, or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin and afatinib or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and afatinib, or e.g. panitumumab may be combined with 5- FU, leucovorin, oxaliplatin and dacomitinib or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and dacomitinib, or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin and neratinib or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and neratinib, or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin and vandetanib or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and vandetanib, or e.g panitumumab may be combined with 5-FU, leucovorin, oxaliplatin and brivanib or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and brivanib, or e.g panitumumab may be combined with 5-FU, leucovorin, oxaliplatin and tivantinib or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and tivantinib, or e.g panitumumab may be combined with 5-FU, leucovorin, oxaliplatin and crizotinib or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and crizotinib, or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin and dovitinib or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and dovitinib, or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin and afatinib or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and afatinib, or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin and dacomitinib or e.g. erlotinib may be combined with 5- FU, leucovorin, oxaliplatin, irinotecan and dacomitinib, or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin and neratinib or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and neratinib, or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin and vandetanib or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and vandetanib, or e.g. erlotinib may be combined with 5- FU, leucovorin, oxaliplatin and brivanib or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and brivanib, or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin and tivantinib or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and tivantinib, or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin and crizotinib or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and crizotinib, or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin and crizotinib or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and crizotinib, or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin and dovitinib or e.g. erlotinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and dovitinib, or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin and afatinib or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and afatinib, or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin and dacomitinib or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and dacomitinib, or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin and neratinib or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and neratinib, or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin and vandetanib or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and vandetanib, or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin and brivanib or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and brivanib, or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin and tivantinib or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and tivantinib, or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin and crizotinib or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and crizotinib, or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin and crizotinib or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and crizotinib, or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin and dovitinib or e.g. gefitinib may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and dovitinib, or e.g. XL-647 may be combined with 5-FU, leucovorin, oxaliplatin and dovitinib or e.g. XL-647 may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan, or e.g. canertinib (CI-1033) may be combined with 5-FU, leucovorin, oxaliplatin, or canertinib (Cl-1033)may be combined with 5-FU, leucovorin, oxaliplatin and irinotecan, or e.g. XL-647 may be combined with 5-FU, leucovorin, oxaliplatin, or XL-647 may be combined with 5-FU, leucovorin, oxaliplatin and irinotecan, or e.g. pelitinib may be combined with 5-FU, leucovorin, oxaliplatin, or pelitinib may be combined with 5-FU, leucovorin, oxaliplatin and irinotecan, or e.g. PKI-166 may be combined with 5-FU, leucovorin, oxaliplatin, or PKI-166 may be combined with 5-FU, leucovorin, oxaliplatin and irinotecan, or e.g. TAK-285 may be combined with 5-FU, leucovorin, oxaliplatin, or TAK-285 may be combined with 5-FU, leucovorin, oxaliplatin and irinotecan.

The EGFR inhibitors in combination with at least chemotherapeutic agent according to the invention as disclosed above may also e.g. be combined with an HER2 or HER3 inhibitor as disclosed above, e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and trastuzumab, or e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and pertuzumab, or e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and U3-1287, or e.g. cetuximab may be combined with 5- FU, leucovorin, oxaliplatin and pertuzumab, or e.g. cetuximab may be combined with 5-FU, leucovorin, oxaliplatin and U3-1287, or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and trastuzumab, or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and pertuzumab, or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin, irinotecan and U3-1287, or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin and pertuzumab, or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin and U3-1287, or e.g. panitumumab may be combined with 5-FU, leucovorin, oxaliplatin and trastuzumab, cetuximab may be combined with 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin and trastuzumab, or e.g. cetuximab may be combined with 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin and pertuzumab, or e.g. cetuximab may be combined with 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin and U3-1287, or e.g. panitumumab may be combined with 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin and trastuzumab, or e.g. panitumumab may be combined with 5- fluorouracil/folinic acid (5-FU/FA), oxaliplatin and pertuzumab, or e.g. panitumumab may be combined with 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin and U3-1287, or e.g.

cetuximab may be combined with Capecitabine, oxaliplatin and trastuzumab, or e.g.

cetuximab may be combined with Capecitabine, oxaliplatin and pertuzumab, or e.g.

cetuximab may be combined with Capecitabine, oxaliplatin and U3-1287, or e.g.

panitumumab may be combined with Capecitabine, oxaliplatin and trastuzumab, or e.g. panitumumab may be combined with Capecitabine, oxaliplatin and pertuzumab, or e.g. panitumumab may be combined with Capecitabine, oxaliplatin and U3-1287. According to a more specific embodiment, the EGFR inhibitor for use in the present invention as disclosed above is preferably one or more of cetuximab, panitumumab, erlotinib or gefinitinib. Accordingly, cetuximab may e.g. be used as disclosed above, or e.g.

panitumumab may be used as disclosed above, or e.g. erlotinib may be used as disclosed above, or e.g. gefinitinib may be used as disclosed above.

For example, according to the inventive method cetuximab may in one embodiment be administered in combination with 5-fluorouracil/folinic acid (5-FU/FA), or 5-FU, leucovorin, oxaliplatin (FOLFOX), or 5-FU, leucovorin, and irinotecan (FOLFIRI), or leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or with 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin (FLOX), or with Capecitabine and oxaliplatin (CapeOx). According to the invention, Panitumumab, may e.g. be administered in combination with 5-fluorouracil/folinic acid (5-FU/FA), or 5-FU, leucovorin, oxaliplatin (FOLFOX), or 5-FU, leucovorin, and irinotecan (FOLFIRI), or leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or with 5- fluorouracil/folinic acid (5-FU/FA), oxaliplatin (FLOX), or with Capecitabine and oxaliplatin (CapeOx).

According to the inventive method erlotinib may in one embodiment be combined with 5- fluorouracil/folinic acid (5-FU/FA), or 5-FU, leucovorin, oxaliplatin (FOLFOX), or 5-FU, leucovorin, and irinotecan (FOLFIRI), or leucovorin, 5-FU, oxaliplatin, and irinotecan

(FOLFOXIRI), or with 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin (FLOX), or with

Capecitabine and oxaliplatin (CapeOx). According to the inventive method gefitinib may e.g. be combined with 5-fluorouracil/folinic acid (5-FU/FA), or 5-FU, leucovorin, oxaliplatin (FOLFOX), or 5-FU, leucovorin, and irinotecan (FOLFIRI), or leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or with 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin (FLOX), or with Capecitabine and oxaliplatin (CapeOx). According to one or more of the embodiments of the inventive method as disclosed above cetuximab, panitumumab, erlotinib, gefitinib, afatinib, dacomitinib, neratinib, vandetanib, brivanib, tivantinib, crizotinib, XL-647, canertinib, pelitinib, PKI-166 or TAK-285 may also be combined with one or more of capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine;

leucovorin, cisplatin, carboplatin, oxaliplatin. Accordingly, cetuximab may be combined with one or more of e.g. capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or panitumumab may be combined with e.g. one or more of capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or erlotinib may be combined with e.g. one or more of capecitabine, 5-fluoro-2'- deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or gefinitib may be combined with e.g. one or more of capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or afatinib may be combined with e.g. one or more of capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or dacomitinib may be combined with e.g. one or more of capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine;

leucovorin, cisplatin, carboplatin, oxaliplatin, or neratinib may be combined with e.g. one or more of capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, cladribine, clofarabine, 6- mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or vandetanib may be combined with e.g. one or more of

capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or brivanib may be combined with e.g. one or more of capecitabine, 5-fluoro-2'- deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or tivantinib may be combined with e.g. one or more of capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or crizotinib may be combined with e.g. one or more of capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or XL-647 may be combined with e.g. one or more of capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or canertinib may be combined with e.g. one or more of capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or pelitinib, may be combined with e.g. one or more of capecitabine, 5-fluoro-2'- deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or PKI- 166 may be combined with e.g. one or more of capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, or TAK-285 may be combined with e.g. one or more of capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, cladribine, clofarabine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin.

According to one embodiment of the inventive method, the TIMP-1 levels, such as the blood TIMP-1 levels, preferably the blood plasma TIMP-1 levels of a cancer patient who is likely to benefit from treatment with at least one EGFR inhibitor are determined by means of an enzyme-linked immunosorbent assay (ELISA). For example, TIMP-1 levels may be determined by a sandwich immunoassay (ELISA) as disclosed above. The plasma TIMP-1 levels of a cancer patient who is likely to benefit from treatment with at least one EGFR inhibitor according to the present invention may also be determined by determining free TIMP-1 in the plasma of patients as disclosed above. The term "free" as used according to the invention refers to TIMP-1 that is not bound to a matrix metalloproteinases (MMPs).

The levels of TIMP-1 of the patient in any of the above embodiments of the inventive method according to the invention, e.g. wherein TIMP-1 is used as a biomarker, are preferably determined prior to any treatment ("baseline" TIMP-1 levels) and may be determined as described herein.

According to a more specific embodiment of the inventive method, the blood TIMP-1 levels in a cancer patient who is likely to benefit from treatment with at least one EGFR inhibitor , such as the blood plasma TIMP-1 levels, are at least least about 250 ng/ml to about 1400 ng/ml, or at least about 275 ng/ml to about 1300 ng/ml, or at least about 300 ng/ml to about 1250 ng/ml, or at least about 325 ng/ml to about 1200 ng/ml, or at least about 350 ng/ml to about 1 150 ng/ml, or at least about 375 ng/ml to about 1 100 ng/ml, or at least about 400 ng/ml to about 1050ng/ml, such as at least about 300 ng/ml to about 1350 ng/ml, or at least about 250 ng/ml , 255 ng/ml, 260 ng/ml, 265 ng/ml, 270 ng/ml, 275 ng/ml, 280 ng/ml, 285 ng/ml, 290 ng/ml, 295 ng/ml, 300 ng/ml, 305 ng/ml, 310ng/ml, 312 ng/ml, 315 ng/ml, 320 ng/ml, 325 ng/ml, 330 ng/ml, 335 ng/ml, 340 ng/ml, 345 ng/ml, 350 ng/ml, 375 ng/ml, 400 ng/ml, 409 ng/ml, 425 ng/ml, 430 ng/ml, 435 ng/ml, 440 ng/ml, 445 ng/ml, 450 ng/ml, 455 ng/ml, 460 ng/ml, 465 ng/ml, 470 ng/ml, 475 ng/ml, 480 ng/ml, 485 ng/ml, 490 ng/ml, 495 ng/ml or at least about 500 ng/ml. In one embodiment the TIMP-1 plasma levels in a cancer patient who is likely to benefit from treatment with at least one EGFR inhibitor according to the inventive method are from about 250 ng/ml to about 255 ng/ml, such as from about 255 ng/ml to about 260 ng/ml, for example from about 260 ng/ml to about 265 ng/ml, such as from about 265 ng/ml to about 270 ng/ml, for example from about 270 ng/ml to about 275 ng/ml, such as from about 275 ng/ml to about 280 ng/ml, for example from about 280 ng/ml to about 285 ng/ml, such as from about 285 ng/ml to about 290 ng/ml, for example from about 290 ng/ml to about 295 ng/ml, such as from about 295 ng/ml to about 300 ng/ml, for example from about 300 ng/ml to about 305 ng/ml, such as from about 305 ng/ml to about 310 ng/ml, for example from about 310 ng/ml to about 312 ng/ml, such as from about 312 ng/ml to about 315 ng/ml, for example from about 315 ng/ml to about 320 ng/ml, such as from about 320 ng/ml to about 325 ng/ml, for example from about 325 ng/ml to about 330 ng/ml, such as from about 330 ng/ml to about 335 ng/ml, for example from about 335 ng/ml to about 340 ng/ml, such as from about 340 ng/ml to about 345 ng/ml, for example from about 345 ng/ml to about 350 ng/ml, such as from about 350 ng/ml to about 360 ng/ml, for example from about 360 ng/ml to about 370 ng/ml, such as from about 370 ng/ml to about 380 ng/ml, for example from about 380 ng/ml to about 390 ng/ml, such as from about 390 ng/ml to about 400 ng/ml, for example from about 400 ng/ml to about 405 ng/ml, such as from about 405 ng/ml to about 410 ng/ml, for example from about 410 ng/ml to about 412 ng/ml, such as from about 412 ng/ml to about 415 ng/ml, for example from about 415 ng/ml to about 420 ng/ml, such as from about 420 ng/ml to about 425 ng/ml, for example from about 425 ng/ml to about 430 ng/ml, such as from about 430 ng/ml to about 435 ng/ml, for example from about 435 ng/ml to about 440 ng/ml, such as from about 440 ng/ml to about 445 ng/ml, for example from about 445 ng/ml to about 450 ng/ml, such as from about 450 ng/ml to about 460 ng/ml, for example from about 460 ng/ml to about 470 ng/ml, such as from about 470 ng/ml to about 480 ng/ml, for example from about 480 ng/ml to about 490 ng/ml, such as from about 490 ng/ml to about 500 ng/ml, for example from about 500 ng/ml to about 525 ng/ml, such as from about 525 ng/ml to about 550 ng/ml, for example from about 550 ng/ml to about 575 ng/ml, such as from about 575 ng/ml to about 600 ng/ml, for example from about 600 ng/ml to about 650 ng/ml, such as from about 650 ng/ml to about 700 ng/ml, for example from about 700 ng/ml to about 750 ng/ml, such as from about 750 ng/ml to about 800 ng/ml, for example from about 800 ng/ml to about 850 ng/ml, such as from about 850 ng/ml to about 900 ng/ml, for example from about 900 ng/ml to about 950 ng/ml, such as from about 950 ng/ml to about 1000 ng/ml, for example from about 1000 ng/ml to about 1 100 ng/ml, such as from about 1 100 ng/ml to about 1200 ng/ml, for example from about 1200 ng/ml to about 1300 ng/ml, such as from about 1300 ng/ml to about 1400 ng/ml. It is understood that the blood TIMP-1 levels in a cancer patient who is likely to benefit from treatment with at least one EGFR inhibitor according to the present invention, such as the blood plasma TIMP-1 levels, are in a preferred embodiment at least least about 250 ng/ml blood or blood plama. The receiver operating characteristic (ROC) curve is commonly used to evaluate a biomarker's ability for classifying disease or response status. The Youden Index (J), the maximum potential effectiveness of a biomarker, is a common summary measure of the ROC curve. According to one or more of the above embodiments of the present invention, e.g. in a method of identifying a cancer patient who is likely to benefit from treat with an EGFR inhibitor, or in a method of predicting cancer patient prognosis according to the invention, the RAS mutation is an activating mutation as disclosed above. Accordingly, the RAS mutation according to one or more of the above embodiments of the invention comprises as least one mutation in H-RAS, N-RAS, or KRAS as disclosed above, e.g. H-RAS comprises at least one mutation selected from the group comprising the amino acid substitutions G12R, G12V, G13C, G13R, Q61 R, or e.g. N-RAS comprises at least one mutation selected from the group comprising the amino acid substitutions G12C, G12D, G12R, G12S, G12A, G12V, G12R, G13C, G13R, G13A, G13D, G13V, G15W, G60E, Q61 P, Q61 L, Q61 R, Q61 K, Q61 H, Q61 E, or e.g. KRAS comprises at least one mutation selected from the group comprising the amino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G13C and G13D, G13R, G13S, Q61 K, Q61 L, Q61 P, Q61 R, A146V. For example determining the presence of a RAS mutation according to the inventive method may include e.g. determining the presence of H- RAS mutation G12R, or H-RAS mutation G12V, H-RAS mutation G13C, H-RAS mutation G13R, H-RAS mutation Q61 R, or N-RAS mutation G12C, or N-RAS mutation G12D, or N- RAS mutation G12R, or N-RAS mutation G12S, or N-RAS mutation G12A, or N-RAS mutation G12V, or N-RAS mutation G12R, or N-RAS mutation G13C, or N-RAS mutation G13R, or N-RAS mutation G13A, or N-RAS mutation G13D, or N-RAS mutation G13V, or N- RAS mutation G15W, or N-RAS mutation G60E, or N-RAS mutation Q61 P, or N-RAS mutation Q61 L, or N-RAS mutation Q61 R, or N-RAS mutation Q61 K, or N-RAS mutation Q61 H, or N-RAS mutation Q61 E, or e.g. KRAS mutation G12C, KRAS mutation G12D, KRAS mutation G12A, KRAS mutation G12V, KRAS mutation G12S, KRAS mutation G12F, KRAS mutation G12R, KRAS mutation G13C, KRAS mutation G13D, KRAS mutation G13R, KRAS mutation G13S, KRAS mutation Q61 K, KRAS mutation Q61 L, KRAS mutation Q61 P, KRAS mutation Q61 R, or KRAS mutation A146V. According to one embodiment of the inventive method of identifying a cancer patient who is likely to benefit from treatment with at least one EGFR inhibitor, the method comprises determining the presence or absence of a RAS mutation by amplifying RAS nucleic acid from a patient's tumor sample, suspected of harboring a mutation by means of PCR. For example, any PCR technology and primer pairs may be used, which are known to the person skilled in the art, such as e.g. those disclosed in Chang et al., Clinical Biochemistry 43 (2010), 296- 301 , e.g. a multiplex PCR may be used to amplify codons 12 and 13 of exon 2 and codon 61 of exon 3 of N-, H-, and KRAS genes with two pairs of universal primers for exons 2 and 3. For example, the following primers may be used:

SEQ ID NO exon primer sequence

SEQ ID NO:1 2 5'-CYKRBKDRMRATGACKGARTAYAARCTKGTGGT -

3'

SEQ ID NO:2 2 5'-ACCTCTATDGTKGGRTCRTATTC -3'

SEQ ID NO:3 3 5'-CAGGATTCYTACMGRAARCARGT -3'

SEQ ID NO:4 3 5 ' - TT K ATG G C AAA Y AC AC A V AG R A AG C -3'

As used herein, the letters are used according to the lUPAC notation, e.g. "Y" denotes pyrimidine, "K" denotes keto, e.g. G or C, "R" denotes purine, "B" C, G, or T, "D" denotes A, G, or T, "M" denotes A, C, "V" denotes A, C, or G. Following multiplex PCR amplification, the products may e.g. be purified to remove the primers and unincorporated deoxynucleotide triphosphates using PCR-M™ Clean Up System (Viogenebiotek Co., Sunnyvale, CA, USA). Purified DNA may then be semiquantified on a 1 % agarose gel in 0.5χ TBE and visualized by staining with ethidium bromide. The products may then e.g. be subjected to primer extension analysis using primers as disclosed in Chang et al., Clinical Biochemistry 43 (2010), 296-301 , e.g. such as those disclosed below: RAS Sequence

SEQ ID NO: 5 K 5 ' -AACTTGTGGTAGTTGGAGCT

SEQ ID NO: 6 K 5 ' -ACTGAATATAAACTTGTGGTAGTTGGAGCTG

SEQ ID NO: 7 K 5 ' -TGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGT

SEQ ID NO: 8 K 5 ' -GCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTG

SEQ ID NO: 9 K 5 ' -GCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGT

SEQ ID NO: 10 K 5 ' GGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGTC

SEQ ID NO: 11 K 5 ' -T₄₅ATTCTCGACACAGCAGGTCA

SEQ ID NO: 12 N 5 ' -AACTGGTGGTGGTTGGAGCA-3 '

SEQ ID NO: 13 N 5 ' -T₇AACTGGTGGTGGTTGGAGCAG-3 '

SEQ ID NO: 14 N 5 ' -T₁₄CAGTGCGCTTTTCCCAACAC-3 '

SEQ ID NO: 15 N 5 ' -T₂₂GTGGTGGTTGGAGCAGGTG-3 '

SEQ ID NO: 16 N 5 ' -T₂₉CTCATGGCACTGTACTCTTCTT-3 '

SEQ ID NO: 17 N 5 ' -T₃₆CTCATGGCACTGTACTCTTCT-3 '

SEQ ID NO: 18 N 5 ' -T₄₃CTCTCATGGCACTGTACTCTTC-3 '

SEQ ID NO: 19 H 5 ' -AGCTGGTGGTGGTGGGCGCC-3 '

SEQ ID NO:20 H 5 ' -T₇AGCTGGTGGTGGTGGGCGCCG-3 '

SEQ ID NO:21 H 5 ' -T₁₄TGGTGGTGGTGGGCGCCGGC-3 '

SEQ ID NO:22 H 5 ' -T₂₂GTGGTGGTGGGCGCCGGCG-3 '

SEQ ID NO:23 H 5 ' -T₂₉ACATCCTGGATACCGCCGGC-3 '

SEQ ID NO:24 H 5 ' -T₃₆ACATCCTGGATACCGCCGGCC-3 '

SEQ ID NO:25 H 5 ' -T₄₃CGCATGGCGCTGTACTCCTC-3 '

Various concentrations of probe for either codon 12, 13, or 61 may be employed (e.g. 0.03 - 0.6 μΜ) in the reactions containing 1 .5 μΙ of purified PCR products, as well as 4 μΙ of ABI PRISM SNaPshot Multiplex Kit (Applied Biosystems, Foster City, CA) containing AmpliTaq® DNA polymerase and fluorescently labeled dideoxynucleotide triphosphates (ddNTPs) (RGG-labeled dideoxyadenosine triphosphate, TAMRA-labeled dideoxycytidine triphosphate, ROX-labeled dideoxythymidine triphosphate, and R1 10-labeled dideoxyguanosine triphosphate). Each 10-μΙ mixture may then e.g. subjected to 25 single-base extension cycles consisting of a denaturing step at 96 °C for 10 s and primer annealing and extension at 55 °C for 35 s. After cycle extension, unincorporated fluorescent ddNTPs may then be incubated with 1 μΙ of shrimp alkaline phosphatase (United States Biochemical Co., Cleveland, USA) at 37 °C for 1 h, followed by enzyme deactivation at 75 °C for 15 min. The primer extension reaction products may then e.g. be resolved by automated capillary electrophoresis on a capillaryelectrophoresis platform, e.g. 14 μΙ of Hi-Di™ Formamide (Applied Biosystems) and 0.28 μΙ of GeneScan™- 120LIZ® Size Standard (Applied Biosystems) were added to 6 μΙ of primer extension products. All samples may then e.g. be analyzed on an ABI Prism 310 DNA Genetic Analyzer (Applied Biosystems) according to manufacturer's instructions using GeneScan™ 3.1 (Applied Biosystems).

According to one embodiment of the inventive method of identifying a cancer patient who is likely to benefit from treatment with at least one EGFR inhibitor, the method comprises determining the presence or absence of a RAS mutation by amplifying RAS nucleic acid from the patient's tumor sample and sequencing said amplified nucleic acid. Accordingly, RAS nucleic acid may be amplified using primers as disclosed above and sequenced. For example, H-RAS, N-RAS and KRAS nucleic acid may be amplified by PCR as disclosed above and subsequently subcloned using e.g. the TOPO TA Cloning Kit for sequencing (Invitrogen).

In the above inventive method, RAS nucleic acid may be obtained from the patient's tumor sample by any method known to the person skilled in the art. For example, any commercial kit may be used to isolate the genomic DNA, or mRNA from the patient's tumor sample, such as e.g. the Qlamp DNA mini kit, or RNeasy mini kit (Qiagen, Hilden, Germany). For example, if mRNA was isolated from the patient's tumor sample, cDNA synthesis may have to be carried out prior to the methods as disclosed herein, according to any known technology in the art.

For example, the nucleic acid to be isolated from the patients tumor may for example be one of genomic DNA, total RNA, mRNA or poly(A)+ mRNA. For example, if mRNA has been isolated from the the patient's tumor sample, the mRNA (total mRNA or poly(A)+ mRNA) may be used for cDNA synthesis according to well established technologies in prior art, such as those provided in commercial cDNA synthesis kits, e.g. Superscript® III First Strand

Synthesis Kit. The cDNA may then be further amplified by means of e.g. PCR and subsequently subjected to sequencing by e.g. Sanger sequencing or pyro-sequencing to determine the nucleotide sequence of e.g. codons 12 and 13 of the RAS gene, e.g. H-RAS, N-RAS or KRAS. Alternatively, the PCR product may e.g. also be subcloned into a TA TOPO cloning vector for sequencing. Other technologies than sequencing to determine the absence or presence of mutations in KRAS may be used in the inventive method such as e.g. Single Nucleotide Primer Extension (SNPE) (PLoS One. 2013 Aug 21 ;8(8):e72239).

According to one embodiment of the inventive method of identifying a cancer patient who is likely to benefit from treatment with at least one epidermal growth factor receptor (EGFR) inhibitor the EGFR expressed in the patient's tumor sample may be wildtype or mutated EGFR. As used in the context of the inventive method, "EGFR" or "wildtype EGFR" refers to the protein encoded by the mRNA having the sequence as provided by of GenBank

Accession NM 005228.3. EGFR is a member of the type 1 tyrosine kinase family of growth factor receptors, which play critical roles in cellular growth, differentiation, and survival.

Activation of these receptors typically occurs via specific ligand binding, resulting in hetero- or homodimerization between receptor family members of the HER family, e.g. HER2 -4, with subsequent autophosphorylation of the tyrosine kinase domain. This activation triggers a cascade of intracellular signaling pathways involved in both cellular proliferation (the ras/raf/MAP kinase pathway) and survival (the PI3 kinase/Akt pathway). The term "mutated" or "mutated EGFR" within the context of the present invention, e.g. in the inventive method of identifying a cancer patient who is likely to benefit from treatment with at least one EGFR inhibitor, refers to any mutation of the EGFR (e.g. amino acid substitution, insertion, deletion, or nucleotide polymorphisms (SNPs), chromosomal inversion) which alters its cellular function, e.g. a mutation may result in result in constitutive signaling of EGFR, resulting in a permanent activation of the receptor, or prolonged signaling of the receptor upon ligand binding. Examples of EGFR mutations include, but are not limited to, mutations on exons 18, 19, 20 and 21 of the EGFR gene. Accordingly, the most prevalent EGFR mutations are in- frame deletions of exon 19 (45%), followed by L858R substitution in exon 21 (41 %). Exon 18 mutations (G719A/C/S) account for -5% of the overall mutations. The exon 19 deletions, L858R in exon 21 , G719A/C/S in exon 18, the L861 Q and L861 R in exon 21 , are mutations that predict the probability of benefit from EGFR TKI therapy of adenocarcinomas. The insertion mutations in exon 20 (D770_N771 (insNPG), D770_N771 (insSVQ), D770_N771 (insG)) are the second most common. The mutated EGFR, which is expressed in the patient's tumor sample, may also harbor more than one mutation, e.g. one, two, three, four or more mutations. The term "EGFR expression" as used within the context of the present invention, e.g. in the context of the patient's tumor tissue, which expresses EGFR, refers to EGFR mRNA, but may also refer to EGFR protein encoded by the mRNA having the sequence as provided by GenBank Accession NM 005228.3, or protein fragments thereof, which are detectable by means of specific antibodies, such as e.g. cetuximab. According to a more specific embodiment, the inventive method of identifying a cancer patient who is likely to benefit from treatment with at least one EGFR inhibitor as defined above pertains to colorectal cancer, metastatic colorectal cancer, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, melanoma, myeloid leukemia, juvenile myeloid leukemia, ovarian cancer, pancreas cancer, thyroid cancer, prostate cancer or adenocarcinoma. Accordingly, the inventive method of identifying a cancer patient who is likely to benefit from treatment with at least one EGFR inhibitor comprises determining in vitro the blood plasma levels of TIMP-1 of the patient as defined above and the absence or presence of a KRAS mutation in a patient's tumor sample as defined above, wherein EGFR is expressed in the patient's tumor sample, and whereby the plasma levels of TIMP-1 and the presence of a RAS mutation, e.g. a H- RAS, or N-RAS, or KRAS mutation as defined above, indicate that the patient is likely to respond to a treatment with the EGFR inhibitor as defined above, or in combination with at least one chemotherapeutic agent as defined above.

According to one embodiment, the present invention pertains to the use of the biomarkers KRAS and TIMP-1 in predicting the pharmaceutical efficacy and/or clinical response of a combination comprising at least one EGFR inhibitor and at least one chemotherapeutic agent to be administered to a patient afflicted with cancer. Accordingly, KRAS and TIMP-1 may be used to predict the pharmaceutical efficacy and/or clinical response of a combination comprising at least one EGFR inhibitor as defined above and at least one chemotherapeutic agent as defined above, e.g. the biomarkers KRAS and TIMP-1 may be used according to the invention in predicting the pharmaceutical efficacy and/or clinical response of a patient afflicted with cancer who is administered at least one EGFR inhibitor as defined above in combination with at least one chemotherapeutic agent as defined above.

More specifically, the biomarkers KRAS and TIMP-1 may be used according to the invention in predicting the pharmaceutical efficacy and/or clinical response of a patient afflicted with cancer who is administered at least one EGFR inhibitor in combination with at least one chemotherapeutic agent as defined above, wherein the EGFR inhibitor is selected from the group comprising, panitumumab, erlotinib, gefinitinib, preferably, the at least one EGFR inhibitor according to the invention is cetuximab.

Accordingly, the biomarkers KRAS and TIMP-1 may be used in predicting the

pharmaceutical efficacy and/or clinical response of cetuximab in combination with at least one chemotherapeutic agent as defined above, e.g. the presence of at least one KRAS mutation in the patient's tumor sample as defined above, which is positive for EGFR as defined above whereby the patient's plasma TIMP-1 levels are as defined above, e.g. TIMP- 1 levels of at least about 250 ng/ml, 275 ng/ml, 300 ng/ml to about 600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml, 1000 ng/ml, 1 100 ng/ml, 1250 ng/ml, 1350 ng/ml, 1400 ng/ml, are indicative for a benefit of the treatment with the EGFR inhibitor cetuximab in combination with at least one chemotherapeutic agent as defined above. Accordingly, the use of the biomarkers KRAS and TIMP-1 according to the invention comprises determining in vitro the absence or presence of a KRAS mutation selected from the group consisting of the amino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G13C and G13D, G13R, G13S, Q61 K, Q61 L, Q61 P, Q61 R, A146V as disclosed above. For example, commercial PCR kits may be used, which are preferably FDA approved or approved be the respective relevant national regulatory health authority for use in the diagnosis of KRAS mutations. Commercial kits with and without FDA approval for in vitro diagnostic use include e.g. the "Signature®" kit (Asuragen), "TheraScreen®" kit (Qiagen), "EntroGen®" kit (EntroGen), "COBAS®" kit (Roche), "Mutector II™"kit (TrimGen) or the "StripAssay" (ViennaLab

Diagnostics). The inventive use of the biomarkers KRAS and TIMP-1 further comprises determining in vitro the plasma TIMP-1 levels as disclosed above, e.g. by means of an ELISA.

The at least one chemotherapeutic agent for use as defined above, is in one embodiment selected from the group consisting of 5-fluorouracil/folinic acid (5-FU/FA), capecitabine, 5- fluoro-2'-deoxyuiridine, irinotecan, 6-mercaptopurine (6-MP), cladribine, clofarabine, 6- mercaptopurine (6-MP), cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, preferably a combination of 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx).

More specifically, the EGFR inhibitor cetuximab may be used in the combination as defined above in a concentration of e.g. about 125 mg/m² to about 500 mg/m² body surface, or of about 250 mg/m² to about 450 mg/m² body surface, or of about 300 mg/m² to about 400 mg/m² body surface, or e.g. of about 250 mg/m² , 275 mg/m², 300 mg/m², 325 mg/m², 350 mg/m², 375 mg/m², 400 mg/m², 425 mg/m², 450 mg/m², 475 mg/m², or 500 mg/m² body surface, preferably about 250 mg/m² to about 400 mg/m² body surface, whereby the dosing of cetuximab may e.g. be computed according to the Dubois method, in which the body surface area of a subject (m²) is computed using the subject's body weight: m² = (wt kg⁰⁴²⁵ x height cm⁰⁷²⁵) x 0.007184. According to one specific embodiment, the inventive biomarkers KRAS and TIMP-1 may be used in predicting the pharmaceutical efficacy and/or clinical response of cetuximab in combination with at least one chemotherapeutic agent as defined above, whereby the at least one chemotherapeutic agent is administered e.g. every 7 days to 21 days, or 10 days to 14 days, or every 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, or preferably every 14 days.

More specifically, the inventive biomarkers KRAS and TIMP-1 may be used in predicting the pharmaceutical efficacy and/or clinical response of cetuximab in combination with at least one chemotherapeutic agent as defined above, whereby cetuximab is e.g. administered every 5 days to 21 days, or every 7 days to 14 days, or every 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, preferably every 5 days to 10 days, more preferably every 7 days or 14 days, e.g. in a dosing as defined above.

According to one embodiment, the present invention pertains to a method of treating a patient with cancer, wherein the treatment comprises administering a therapeutically effective amount of at least one EGFR inhibitor, and at least one chemotherapeutic agent to a patient in need thereof, if the patient is likely to benefit from the cancer treatment according as defined above indicative of an increased progression free survival (PFS) and/or overall survival (OS). The term "therapeutically effective amount" as used within the context of the method of treatment according to the invention means an amount of an EGFR inhibitor as disclosed above, e.g. at least one EGFR inhibitor as disclosed above, or in combination with at least one chemotherapeutic agent as disclosed above, effective for treating cancer as disclosed herein.

Accordingly, the present invention provides a method of treatment, which comprises administering a therapeutically effective amount of at least one EGFR inhibitor as defined above and at least one chemotherapeutic agent as defined above to a patient in need thereof, e.g. to a patient afflicted with cancer, such as e.g. colorectal cancer, metastatic colorectal cancer, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, melanoma, myeloid leukemia, juvenile myeloid leukemia, ovarian cancer, pancreas cancer, thyroid cancer, prostate cancer, adenocarcinoma, if the patient is likely to benefit from the cancer treatment according as defined above indicative of an increased progression free survival (PFS) and/or overall survival (OS), e.g. of an increased progression free survival (PFS) and overall survival (OS, or of an increased progression free survival (PFS), or of an increased overall survival (OS). A patient is likely to benefit from the method of treatment according to the invention, if the patient is identified by the inventive method according to one or more of the above embodiments of the present invention, e.g. if the patient's tumor sample expresses EGFR as defined above, a mutated KRAS as defined above and the patient's plasma TIMP-1 levels are as defined above, e.g. KRAS may comprise an activating mutation, such as one or more of the amino acid substitutions G12D, G12A, G12V, G12S, G12C, G12F, G12R, G13C and G13D and the plasma TIMP-1 levels are at least about 250 ng/ml, 260 ng/ml, 275 ng/ml, 300ng/ml, 325 ng/ml, 350 ng/ml, 375 ng/ml to about 600 ng/ml, 650 ng/ml, 700 ng/ml, 750 ng/ml, 800 ng/ml, 850 ng/ml, 900 ng/ml, 950 ng/ml, 1000 ng/ml, 1 100 ng/ml, 1200 ng/ml, 1300 ng/ml, or preferably at least about 300 ng/ml, 325 ng/ml, 350 ng/ml, 375 ng/ml, 400 ng/ml, 409 ng/ml, 425 ng/ml.

More specifically, the invention provides for a method of treatment, in which the EGFR inhibitor is a monoclonal antibody or a tyrosine kinase inhibitor. Accordingly, the EGFR inhibitor for use in the present invention may be one or more of cetuximab, panitumumab, erlotinib, gefitinib, afatinib, dacomitinib, neratinib, vandetanib, brivanib, tivantinib, crizotinib, XL-647, canertinib, pelitinib, PKI-166 or TAK-285, preferably cetuximab.

According to one embodiment, the method of treatment according to the invention comprises determining the presence or absence of a KRAS mutation as defined above by amplifying KRAS nucleic acid from a patient's tumor sample, suspected of harboring a mutation by means of PCR as defined above. More specifically, the presence or absence of a KRAS mutation may be determined by amplifying KRAS nucleic acid from the patient's tumor or tumor sample and sequencing the amplified nucleic acid as defined above. In the method of treatment according to the invention, the patient's tumor, which may result from e.g.

colorectal cancer, metastatic colorectal cancer, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, melanoma, myeloid leukemia, juvenile myeloid leukemia, ovarian cancer, pancreas cancer, thyroid cancer, prostate cancer, adenocarcinoma, expresses EGFR which may be wild type EGFR as defined above, or mutated EGFR as defined above.

In preferred embodiment, the present invention provides an EGFR inhibitor in combination with at least one chemotherapeutic agent for use in the treatment of cancer, wherein the tumor expresses EGFR and a mutated KRAS and whereby the blood TIMP-1 levels in the patient are least about 250 ng/ml to about 1400 ng/ml, or at least about 275 ng/ml to about 1300 ng/ml, or at least about 300 ng/ml to about 1250 ng/ml, or at least about 325 ng/ml to about 1200 ng/ml, or at least about 350 ng/ml to about 1 150 ng/ml, or at least about 375 ng/ml to about 1 100 ng/ml, or at least about 400 ng/ml to about 1050ng/ml, preferably of at least about 300 ng/ml to about 1350 ng/ml, or at least about 310 ng/ml, 325 ng/ml, 350 ng/ml, 375 ng/ml, 400 ng/ml, 409 ng/ml or 425 ng/ml.

In preferred embodiment, the present invention provides use of an EGFR inhibitor in combination with at least one chemotherapeutic agent for the manufacture of a medicament for the treatment of cancer, wherein the tumor expresses EGFR and a mutated KRAS and whereby the blood TIMP-1 levels in the patient are least about 250 ng/ml to about 1400 ng/ml, or at least about 275 ng/ml to about 1300 ng/ml, or at least about 300 ng/ml to about 1250 ng/ml, or at least about 325 ng/ml to about 1200 ng/ml, or at least about 350 ng/ml to about 1 150 ng/ml, or at least about 375 ng/ml to about 1 100 ng/ml, or at least about 400 ng/ml to about 1050ng/ml, preferably of at least about 300 ng/ml to about 1350 ng/ml, or at least about 310 ng/ml, 325 ng/ml, 350 ng/ml, 375 ng/ml, 400 ng/ml, 409 ng/ml or 425 ng/ml.

Accordingly, the present invention provides an EGFR inhibitor as defined above, e.g.

cetuximab, panitumumab, erlotinib, gefitinib, afatinib, dacomitinib, neratinib, vandetanib, brivanib, tivantinib, crizotinib, XL-647, canertinib, pelitinib, PKI-166 or TAK-285, in combination with at least one chemotherapeutic agent as defined above for use in the treatment of cancer, e.g. the at least one chemotherapeutic agent may be a combination of 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5- FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are at least as defined above, e.g. at least about 250 ng/ml, 275 ng/ml, 300 ng/ml, 325 ng/ml, 350 ng/ml to about 600 ng/ml, 650 ng/ml, 700 ng/ml, 750 ng/ml, 800 ng/ml, 850 ng/ml, 900 ng/ml, 1000 ng/ml, 1 100 ng/ml, 1200 ng/ml, 1300 ng/ml, or at least about 250 ng/ml to about 1400 ng/ml. In the method of treatment according to the invention, the at least one chemotherapeutic agent may be administered prior to, concomitant with or intermittent with the at least one EGFR inhibitor, e.g. the at least one chemotherapeutic agent may be administered prior to the administration of the EGFR inhibitor, such as e.g. cetuximab, or in combination with the EGFR inhibitor, e.g. on the same day or treatment cycle as the EGFR inhibitor is administered, or the at least one

chemotherapeutic agent may be administered intermittent to treatment with the at least one EGFR inhibitor, e.g. administered prior to and subsequently to administration of the at least one EGFR inhibitor. Accordingly, cetuximab may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above, or e.g. panitumumab may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of

Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above, or e.g. erlotinib may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above, or e.g. gefitinib may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5- FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above, or e.g. afatinib may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5- fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma

TIMP-1 levels are as defined above, or e.g. dacomitinib may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5- FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above, or e.g. neratinib may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin

(FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of

Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above, or e.g. vandetanib may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above, or e.g. brivanib may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5- FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above, or e.g. tivantinib may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5- fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above, or e.g. crizotinib may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5- FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above, or e.g. XL-647 may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin

Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above, or e.g. canertinib may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above, or e.g. pelitinib may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5- FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above, or e.g. PKI-166 may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5- fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above, or e.g. TAK-285 may e.g. be used in combination with 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5- FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), whereby the patient's plasma TIMP-1 levels are as defined above.

In a preferred embodiment, the present invention pertains to the use of cetuximab in combination with at least one chemotherapeutic agent as defined above for use in the treatment of cancer as defined above, wherein the tumor expresses EGFR, a mutated KRAS and whereby the plasma TIMP-1 levels in the patient are as defined above.

In a more preferred embodiment, the present invention pertains to the use of cetuximab in combination with at least one chemotherapeutic agent as defined above for use in the treatment of cancer as defined above, wherein the tumor expresses EGFR, a mutated KRAS as defined above, e.g. an activating mutation, which may comprise at least one of the amino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G13C and G13D, G13R, G13S, Q61 K, Q61 L, Q61 P, Q61 R, A146V and whereby the plasma TIMP-1 levels in the patient are as defined above.

More specifically, the present invention pertains to the EGFR inhibitor cetuximab in combination with at least one chemotherapeutic agent as defined above for use in the treatment of cancer as defined above, e.g. a combination of 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx), for use in the treatment of cancer as defined above, wherein the tumor expresses EGFR, a mutated KRAS as defined above, and whereby the plasma TIMP-1 levels in the patient are as defined above. Accordingly, cetuximab may be used e.g. in combination with capecitabine, or in combination with 5-fluoro-2'-deoxyuiridine, or in combination with irinotecan, or in

combination with 6-mercaptopurine (6-MP), or in combination with cladribine, or in combination with clofarabine, or in combination with cytarabine, or in combination with floxuridine, or in combination with fludarabine, or in combination with gemcitabine, or in combination with hydroxyurea, or in combination with methotrexate, or in combination with bleomycin, or in combination with paclitaxel, or in combination with chlorambucil, or in combination with mitoxantrone, or in combination with camptothecin, or in combination with topotecan, or in combination with teniposide, or in combination with colcemid, or in combination with colchicine, or in combination with pemetrexed, or in combination with pentostatin, or in combination with thioguanine; or in combination with leucovorin, or in combination with cisplatin, or in combination with carboplatin, or in combination with oxaliplatin, preferably cetuximab may be used in combination with e.g. 5-FU, leucovorin, or in combination with 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or in

combination with 5-FU, leucovorin, oxaliplatin (FOLFOX), or in combination with 5-FU, leucovorin, and irinotecan (FOLFIRI), or in combination with leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or in combination with Capecitabine and oxaliplatin (CapeOx), or any other conceivable combination of the above chemotherapeutic agents, for as long as the combination is therapeutically effective. As used herein, "therapeutically effective" means an amount of the at least one chemotherapeutic agent as defined herein that is sufficient to significantly induce a positive modification of a disease, e.g. cancer, as defined herein. At the same time, however, "therapeutically effective" refers to amounts of the above

chemotherapeutic agents that is small enough to avoid unnecessary side-effects and to permit a sensible relationship between advantage and risk.

In an even more preferred embodiment, the present invention pertains to use of the EGFR inhibitor cetuximab in combination with 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX) for use in the treatment of cancer as defined above, wherein the tumor expresses EGFR, a mutated KRAS as defined above whereby plasma TIMP-1 levels in the patient are e.g. at least about 250 ng/ml to about 1400 ng/ml, or 250 ng/ml, 255 ng/ml, 260 ng/ml, 265 ng/ml, 275 ng/ml, 300 ng/ml, 325 ng/ml, 350 ng/ml, 375 ng/ml, or of at least about 400 ng/ml to about 1400 ng/ml, or of at least about 300 ng/ml to about 800 ng/ml, or of at least about 325 ng/ml to about 700 ng/ml, or of at least about 350 ng/ml to about 900 ng/ml, preferably of at least about 275 ng/ml, 300 ng/ml, 325 ng/ml, 350 ng/ml, 375 ng/ml, 400 ng/ml, 409 ng/ml, or of at least about 425 ng/ml.

In one embodiment the present invention pertains to the EGFR inhibitor cetuximab in combination with 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX) for use in the treatment of cancer as defined above, wherein the patient's tumor expresses EGFR as defined above, a mutated KRAS as defined above and wherein the cancer patient's tumor is TIMP-1 immunoreactive, e.g. TIMP-1 may be detected in the patient's tumor as defined above, e.g. by Western blotting, ELISA, protein array, or preferably by immunohistochemistry as defined above. The term "immunoreactive" as used according to the invention refers to tissue, e.g. tumor tissue, to which anti-TIMP-1 antibodies, e.g. MAC15, MAC 19, or VT1 , VT2, VT4-VT8, specifically bind to, e.g. the antibody affinity is at least 10^"6M, 10^"7, 10^"8, or 10^" ⁹M. A given tissue may be immunoreactive with or without epitope retrieval as defined above, e.g. by boiling tissue sections in a citrate buffer as defined above, or by subjecting the tissue to a brief, e.g. for up to 5min proteinase K treatment at room temperature. In an even more preferred embodiment, the present invention pertains to the EGFR inhibitor cetuximab as defined above in combination with 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX) for use in the treatment of cancer, wherein the tumor expresses EGFR, a mutated KRAS as defined above, e.g. the mutated KRAS comprises at least one of the mutations G12C, G12D, G12A, G12V, G12S, G12F, G12R, G13C and G13D, G13R, G13S, Q61 K, Q61 L, Q61 P, Q61 R, A146V as defined above, whereby plasma TIMP-1 levels in the patient are e.g. at least about 250 ng/ml, 255 ng/ml, 260 ng/ml, 265 ng/ml, 270 ng/ml, 275 ng/ml, 300 ng/ml, 325 ng/ml, 350 ng/ml, 375 ng/ml, 400 ng/ml to about 1400 ng/ml, or at least about 300 ng/ml to about 800 ng/ml, or at least about 325 ng/ml to about 700 ng/ml, or at least about 350 ng/ml to about 900 ng/ml, preferably at least about 250 ng/ml, 275 ng/ml, 300 ng/ml, 325 ng/ml, 350 ng/ml, 375 ng/ml, 400 ng/ml, 409 ng/ml, or at least about 425 ng/ml, wherein the cancer is colorectal cancer and/or metastatic colorectal cancer.

Accordingly, the blood plasma levels of TIMP-1 of the patient in any of the above

embodiments of the present invention, e.g. wherein TIMP-1 and KRAS are used as biomarkers according to the invention as defined above to identify patients who are likely to benefit from a treatment with at least one EGFR inhibitor as disclosed above, or wherein the at least one EGFR inhibitor as disclosed above is used in combination with at least one chemotherapeutic agent as disclosed above, are preferably determined prior to any treatment ("baseline" TIMP-1 levels) and may be determined as described herein. ITEMS

1 . Method of identifying a cancer patient who is likely to benefit from treatment with an EGFR inhibitor comprising determining in vitro

a) the blood levels of TIMP-1 of the patient,

b) the absence or presence of a RAS mutation in a patient's tumor sample, wherein EGFR is expressed in the patient's tumor sample, and whereby the blood levels of TIMP-1 and the presence of a RAS mutation in the patient's tumor sample indicate that the patient is likely to respond to a treatment with an EGFR inhibitor.

2. Method according to item 1 comprising determing in vitro

a) the TIMP-1 blood plasma levels of the patient, b) the absence or presence of a RAS mutation in a patient's tumor sample,

wherein EGFR is expressed in the patient's tumor sample, and whereby the TIMP-1 blood plasma levels and the presence of a RAS mutation in the patient's tumor sample indicate that the patient is likely to respond to a treatment with an EGFR inhibitor.

3. Method according to item 1 comprising determing in vitro

a) the TIMP-1 levels in a tumor sample from said patient,

b) the absence or presence of a RAS mutation in said patient's tumor sample,

wherein EGFR is expressed in the patient's tumor sample, and whereby the TIMP-1 levels in said tumor sample from said patient and the presence of a RAS mutation in the tumor sample indicate that the patient is likely to respond to a treatment with an EGFR inhibitor.

4. Method according to item 3 comprising determing in vitro

a) TIMP-1 tumor tissue immunoreactivity in said patient's tumor tissue, b) the absence or presence of a RAS mutation in said patient's tumor sample,

wherein EGFR is expressed in the patient's tumor sample, and whereby the TIMP-1 tumor tissue immunoreactivity in said patient's tumor sample and the presence of a RAS mutation in the tumor sample indicate that the patient is likely to respond to a treatment with an EGFR inhibitor.

5. Method of predicting cancer patient response to EGFR-inhibitor treatment,

comprising the step of determining in vitro the patient's blood TIMP-1 levels, or the patient's blood plasma TIMP-1 levels, or the TIMP-1 levels in a tumor sample of said patient and determining the absence or presence of a RAS mutation in said patient's tumor sample expressing EGFR, wherein the plasma levels of TIMP-1 and the presence of a RAS mutation in said patient's tumor sample indicate an increased progression free survival (PFS) and/or overall survival (OS) of the patient when the patient is treated with an EGFR inhibitor.

Method of identifying a patient non-responsive to a treatment with at least one EGFR inhibitor comprising determining in vitro

a) the blood TIMP-1 levels of said patient, or the TIMP-1 blood plasma levels of said patient, or the TIMP-1 tumor tissue TIMP-1 levels in the patient's tumor,

b) the absence or presence of a RAS mutation in said patient's tumor sample, wherein EGFR is expressed in said patient's tumor sample and whereby TIMP-1 levels of less than 250 ng/ml and the presence of a mutated RAS in said patient's tumor sample indicate that the patient will not respond to a treatment with an EGFR inhibitor.

Method according to any one of items 1 -6, wherein the EGFR inhibitor is a monoclonal antibody or a tyrosine kinase inhibitor.

Method according to any one of items 1 -7, wherein the EGFR inhibitor is one or more of cetuximab, panitumumab, erlotinib, gefitinib, afatinib, dacomitinib, neratinib, vandetanib, brivanib, tivantinib, crizotinib, XL-647, canertinib, pelitinib, PKI-166 or TAK-285.

Method according to any one of items 1 -8, wherein the EGFR inhibitor is one or more of cetuximab, panitumumab, erlotinib or gefitinib.

Method according to any one of items 1 -9, wherein the EGFR inhibitor is

administered in combination with at least one chemotherapeutic agent, wherein the chemotherapeutic agent is selected from the group comprising capecitabine, 5- fluoro-2'-deoxyuiridine, irinotecan, 6-mercaptopurine (6-MP), cladribine, clofarabine,

6-mercaptopurine (6-MP), cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine,

pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, preferably a combination of 5-FU, leucovorin, or a combination of 5-fluorouracil/folinic acid (5-FU/FA), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of 5- fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx). Method according to any one of items 1 -10, wherein the cancer patient's TIMP-1 levels are determined by means of an ELISA, preferably in the cancer patient's blood plasma TIMP-1 levels are determined by means of an ELISA.

Method according to any one of items 1 -1 1 , wherein the TIMP-1 levels are at least about 250 ng/ml to about 1400 ng/ml, or at least about 275 ng/ml to about 1300 ng/ml, or at least about 300 ng/ml to about 1250 ng/ml, or at least about 325 ng/ml to about 1200 ng/ml, or at least about 350 ng/ml to about 1 150 ng/ml, or at least about 375 ng/ml to about 1 100 ng/ml, or at least about 400 ng/ml to about 1050ng/ml, preferably of at least about 300 ng/ml to about 1350 ng/ml, or at least about 275 ng/ml, 300 ng/ml, 325 ng/ml, 350 ng/ml, 375 ng/ml, 400 ng/ml, 409 ng/ml, 425 ng/ml. Method according to any one of items 1 -12, wherein the RAS mutation is an activating mutation.

Method according to item 13, wherein the RAS mutation is a H-RAS, N-RAS or KRAS mutation.

Method according to item 14, wherein the N-RAS mutation comprises at least one mutation selected from the group comprising the amino acid substitutions G12C, G12D, G12R, G12S, G12A, G12V, G12R, G13C, G13R, G13A, G13D, G13V,

G15W, G60E, Q61 P, Q61 L, Q61 R, Q61 K, Q61 H, Q61 E.

Method according to item 14, wherein the H-RAS mutation comprises at least one mutation selected from the group comprising the amino acid substitutions G12R, G12V, G13C, G13R, Q61 R.

Method according to any one of items 1 -14, wherein the KRAS mutation comprises at least one mutation selected from the group comprising the amino acid

substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G13C and G13D, G13R, G13S, Q61 K, Q61 L, Q61 P, Q61 R, A146V.

Method according to any one of items 1 -17, wherein the presence or absence of a RAS mutation is determined by amplifying RAS nucleic acid from a patient's tumor sample, suspected of harboring a mutation by means of PCR.

Method according to item 18, wherein the presence or absence of a RAS mutation is determined by amplifying RAS nucleic acid from said tumor and sequencing said amplified nucleic acid.

Method according to any one of items 1 -19, wherein the EGFR expressed in the patient sample is wild type EGFR.

Method according to any one of items 1 -20, wherein the EGFR expressed in the patient sample is mutated.

Method according to any one of items 1 -21 , wherein the cancer is colorectal cancer, metastatic colorectal cancer, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, melanoma, myeloid leukemia, juvenile myeloid leukemia, ovarian cancer, pancreas cancer, thyroid cancer, prostate cancer, adenocarcinoma.

Use of the biomarkers KRAS and TIMP-1 for predicting the pharmaceutical efficacy and/or clinical response of a combination comprising at least one EGFR inhibitor and at least one chemotherapeutic agent to be administered to a patient afflicted with cancer.

Use of the biomarkers KRAS and TIMP-1 according to item 23, wherein the at least one EGFR inhibitor is selected from the group comprising cetuximab, panitumumab, erlotinib, gefitinib, afatinib, dacomitinib, neratinib, vandetanib, brivanib, tivantinib, crizotinib, XL-647, canertinib, pelitinib, PKI-166 or TAK-285.

Use of the biomarkers KRAS and TIMP-1 according to item 23 or item 24, wherein the at least one EGFR inhibitor is cetuximab.

Use of the biomarkers KRAS and TIMP-1 according to any one of items 23-25, wherein the use comprises determining in vitro the absence or presence of at least one KRAS mutation selected from the group consisting of the amino acid

Use of the biomarkers KRAS and TIMP-1 according to any one of items 23-26, wherein the use comprises determining in vitro the blood plasma TIMP-1 levels of the cancer patient.

Use of the biomarkers KRAS and TIMP-1 according to any one of items 23-27, wherein the cancer is colorectal cancer, metastatic colorectal cancer, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, melanoma, myeloid leukemia, juvenile myeloid leukemia, ovarian cancer, pancreas cancer, thyroid cancer, prostate cancer, adenocarcinoma.

Use of the biomarkers KRAS and TIMP-1 according to any one of items 23-28, wherein the at least one chemotherapeutic agent is selected from the group comprising 5-fluorouracil/folinic acid (5-FU/FA), capecitabine, 5-fluoro-2'- deoxyuiridine, irinotecan, 6-mercaptopurine (6-MP), cladribine, clofarabine, 6- mercaptopurine (6-MP), cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, preferably a combination of 5-FU, leucovorin, or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan

(FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx).

30. Use of the biomarkers KRAS and TIMP-1 according to any one of items 23-29,

wherein cetuximab is administered in a concentration of about 125 mg/m² to about

500 mg/m² body surface, or of about 250 mg/m² to about 450 mg/m² body surface, or of about 300 mg/m² to about 400 mg/m² body surface, or of about 250 mg/m² , 275 mg/m², 300 mg/m², 325 mg/m², 350 mg/m², 375 mg/m², 400 mg/m², 425 mg/m², 450 mg/m², 475 mg/m², or 500 mg/m² body surface, preferably about 250 mg/m² to about 400 mg/m² body surface.

31 . Use according to any one of items 23-30, wherein the at least one chemotherapeutic agent is administered every 7 days to 21 days, or 10 days to 14 days, or every 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, preferably every 14 days.

32. Use according to any one of items 23-31 , wherein cetuximab is administered every 5 days to 21 days, or every 7 days to 14 days, or every 5, 6, 7, 8, 9, 10, 1 1 , 12, 13,

14, 15, 16, 17, 18, 19, 20 or 21 days, preferably every 5 days to 10 days, more preferably every 7 days or 14 days.

33. Method of treating a patient with cancer, wherein the treatment comprises

administering to a patient in need thereof a therapeutically effective amount of - an EGFR inhibitor, and

at least one chemotherapeutic agent,

if the patient is likely to benefit from the cancer treatment according to any one of items 1 -4, 7-22 indicative of an increased progression free survival (PFS) and/or overall survival (OS).

34. Method according to item 33, wherein the EGFR inhibitor is a monoclonal antibody or a tyrosine kinase inhibitor.

35. Method according to item 33 or item 34, wherein the EGFR inhibitor is one or more of cetuximab, panitumumab, erlotinib, gefitinib, afatinib, dacomitinib, neratinib, vandetanib, brivanib, tivantinib, crizotinib, XL-647, canertinib, pelitinib, PKI-166 or TAK-285.

36. Method according to item 35, wherein the EGFR inhibitor is cetuximab.

37. Method according to item 36, wherein the EGFR inhibitor cetuximab is administered in a concentration of at least about 125 mg/m² to about 500 mg/m² body surface, or of at least about 200 mg/m² to about 500 mg/m² body surface, or of at least about 250 mg/m² to about 450 mg/m² body surface, or of at least about 300 mg/m² to about

400 mg/m² body surface, or of at least about 250 mg/m² , 275 mg/m², 300 mg/m², 325 mg/m², 350 mg/m², 375 mg/m², 400 mg/m², 425 mg/m², 450 mg/m², 475 mg/m², or 500 mg/m² body surface, preferably about 250 mg/m² to about 400 mg/m² body surface.

38. EGFR inhibitor in combination with at least one chemotherapeutic agent for use in the treatment of cancer, wherein the tumor expresses EGFR, a mutated KRAS and whereby the plasma TIMP-1 levels in the patient are least about 250 ng/ml to about

1400 ng/ml, or at least about 275 ng/ml to about 1300 ng/ml, or at least about 300 ng/ml to about 1250 ng/ml, or at least about 325 ng/ml to about 1200 ng/ml, or at least about 350 ng/ml to about 1 150 ng/ml, or at least about 375 ng/ml to about 1 100 ng/ml, or at least about 400 ng/ml to about 1050ng/ml, preferably of at least about 300 ng/ml to about 1350 ng/ml, or at least about 310 ng/ml, 325 ng/ml, 350 ng/ml,

375 ng/ml, 400 ng/ml, 409 ng/ml or 425 ng/ml.

39. EGFR inhibitor in combination with at least one chemotherapeutic agent for use in the treatment of cancer according to item 38, wherein the EGFR inhibitor is a monoclonal antibody or a tyrosine kinase inhibitor.

40. EGFR inhibitor in combination with at least one chemotherapeutic agent for use in the treatment of cancer according to item 38 or item 39, wherein the EGFR inhibitor is selected from the group consisting of cetuximab, panitumumab, erlotinib, gefitinib, afatinib, dacomitinib, neratinib, vandetanib, brivanib, tivantinib, crizotinib, XL-647, canertinib, pelitinib, PKI-166 or TAK-285.

41 . Use according to item 40, wherein the EGFR inhibitor is cetuximab.

42. Use according to any one of items 38-41 , wherein the KRAS mutation is an

activating mutation.

43. Use according to any one of items 38-42, wherein the KRAS mutation comprises at least one mutation selected from the group consisting of the amino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G13C and G13D, G13R, G13S,

Q61 K, Q61 L, Q61 P, Q61 R, A146V.

44. Use according to any one of items 38-43, wherein the at least one chemotherapeutic agent is selected from the group comprising 5-fluorouracil/folinic acid (5-FU/FA), capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, 6-mercaptopurine (6-MP), cladribine, clofarabine, 6-mercaptopurine (6-MP), cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, preferably a combination of 5-FU, leucovorin, or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of Capecitabine and oxaliplatin (CapeOx).

EGFR inhibitor cetuximab in combination with 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin (FLOX) for use in the treatment of cancer, wherein the tumor expresses EGFR, a mutated KRAS and the patient's blood TIMP1 levels, or the patient's blood plasma TIMP-1 levels, or the tumor tissue TIMP-1 levels in the patient's tumor are at least about 250 ng/ml, 275 ng/ml to about 1400 ng/ml, or at least about 300 ng/ml to about 800 ng/ml, or at least about 325 ng/ml to about 700 ng/ml, or at least about 350 ng/ml to about 900 ng/ml, preferably at least about 250 ng/ml, 275 ng/ml, 300 ng/ml, 325 ng/ml, 350 ng/ml, 375 ng/ml, 400 ng/ml or at least about 425 ng/ml. EGFR inhibitor cetuximab in combination with 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin (FLOX) for use in the treatment of cancer according to item 45, wherein the tumor expresses EGFR, a mutated KRAS and wherein the patient's tumor tissue is TIMP-1 immunoreactive.

EGFR inhibitor cetuximab in combination with 5-fluorouracil/folinic acid (5-FU/FA), oxaliplatin (FLOX) for use in the treatment of cancer according to item 45 or item 46, wherein the cancer is colorectal cancer and/or metastatic colorectal cancer.

EXAMPLES

The following Examples are intended to further illustrate the invention. They are not intended to limit the subject matter or scope of the invention thereto.

Example 1 Patient characteristics In the NORDIC VII Study 566 patients with mCRC were included from 32 Nordic centers. Patients were randomized between; Nordic FLOX: 5-FU i.v. bolus 500 mg/m² and folinic acid 60 mg/m² day 1 -2, oxaliplatin 85 mg/m² day 1 every two week until progression (arm A); Nordic FLOX plus cetuximab (400 mg/m² day 1 , then 250 mg/m² weekly) until progression (arm B) or Nordic FLOX + cetuximab for 16 weeks, and weekly cetuximab as maintenance treatment until progression (arm C). Main inclusion criteria were: histologically confirmed mCRC (adenocarcinomas); age > 18 years and <75 years; WHO performance status (PS)≤ 2; no prior chemotherapy for mCRC, non-resectable and measurable disease according to the Response Evaluation Criteria in Solid Tumors (RECIST version 1 .0); last adjuvant chemotherapy≥ 6 months before inclusion; no previous oxaliplatin treatment; adequate haematological, renal and liver function.

The patients were treated until disease progression and followed until death or 30th April 2009. All patients provided written informed consent, and the study (including biomarker analyses) was approved by the Regional Ethics Committee (VEK ref. 20050053).

Biomarker analyses Total plasma TIMP-1 levels (free and in complex with matrix

metalloproteinase) were determined using the MAC15 antibody kinetic enzyme-linked immunosorbent assay (ELISA) as described (Holten-Andersen et al., Br J Cancer (1999) 80:495-503). Duplicate measurements were carried out and the mean values were used for statistical analysis. The mean intra-assay coefficient of variation (CV) was 5.1 % (range 1 .5%-9.8%) and the inter-assay CV was 6.7%. Statistical analysis The primary clinical endpoint for this biomarker study was OS determined as the time from randomization to treatment in NORDIC VII to time of death by any causes. The median follow-up time was 37 month (24-53 months). Cases in which patients were alive at this date were censored.

Secondary endpoint was PFS (primary endpoint of the NORDIC VII Study) defined as the time from randomization until objective disease progression. Descriptive statistics are presented as median levels and ranges. Analyses of measurements for PFS and OS were done using the Cox proportional hazards model. As the analyses performed comparing treatment arms did not reveal any substantial differences in terms of OS and PFS between the original study population and this subset of patients (please see below), we found it justified to pool arms B and C (i.e. + cetuximab treatment) for the statistical analyses. Thus, patients were stratified as receiving cetuximab or not, i.e. arm B and C versus arm A.

Survival probabilities for OS were estimated by the Kaplan-Meier method and tests for differences between strata were done using the log-rank statistic. Graphical presentation using Kaplan-Meier estimates of PFS and OS was shown grouping patients in tertiary TIMP- 1 levels. Multivariable analysis of PFS and OS was done using the Cox proportional hazard model. TIMP-1 concentrations in plasma were entered by the actual value on the log scale (base 2). Missing values for CEA (n = 27), CRP (n = 22), BRAF (n = 101 ) and KRAS (n = 62) were categorized separately and included in the final multivariable analysis. The final model included a three way interaction term (treatment with cetuximab +/- x KRAS mutational status x plasma TIMP-1 ).

The model was assessed using Schoenfeld and martingale residuals. In particular, the linearity assumption for plasma TIMP-1 on the log scale was evaluated using the supremum test for the cumulated martingales36. The results yielded P > .5 testing for the linearity of plasma TIMP-1 on the log scale. Ten-fold cross validation was performed in order to assess over-fitting37 showing almost similar results for the training and test sets (data not shown). P-values less than 5% were considered statistically significant. Statistical calculations were performed using SAS (version 9.2, SAS Institute, Cary, NC, USA) and R (R Core Team (2013). R: A language and environment for statistical computing. R statistical Computing. Vienna. Austria.)

RESULTS

Pre-treatment plasma TIMP-1 and demographic characteristics of the patients

The baseline demographic characteristics of the 426 patients with a pre-treatment plasma

TIMP-1 measurement are shown in Table 1 . The study populations are comparable and not different from the total intention to treat population of 566 patients. The median pre-treatment plasma TIMP-1 was 269 ng/mL (58 to 1318 ng/mL) with no differences between the two treatment groups (P = .97). The tumor cells were KRAS mutated in 147 patients (39%). There was no association between pre-treatment plasma TIMP-1 and gender, or number of metastatic sites. There were statistically significant associations between high pre-treatment plasma TIMP-1 and PS, location of the primary tumor, previous adjuvant chemotherapy, KRAS and BRAF status. The highest plasma TIMP-1 values were found in patient with low PS, primary tumor in colon, no adjuvant therapy, KRAS wild-type tumors and BRAF mutated tumors. (Table 1 ). Pre-treatment plasma TIMP-1 and associations to PFS and OS

Univariate Cox analyses including all patients showed that high pre-treatment plasma ΤΊΜΡ- 1 was significantly associated with shorter PFS (HR, 1 .22; 95% CI, 1 .07 to 1 .39; P = .003), and shorter OS (HR, 1 .55; 95% CI, 1 .33 to 1 .80; P < .0001 ) (Table 2). Kaplan-Meier estimates of survival probabilities for PFS and OS stratified by pre-treatment plasma TIMP-1 levels are shown in Figures 3 A and B.

Multivariate analysis (plasma TIMP-1 , KRAS and BRAF status, serum CRP, serum CEA, PS, number of metastatic sites, age, and gender) demonstrated that high plasma TIMP-1 was an independent biomarker of short OS (HR, 1 .35; 95% CI, 1 .12 to 1 .62; P = .016) while a significant interaction between plasma TIMP-1 and KRAS mutational status could not be demonstrated (P = .47). Plasma TIMP-1 was not significantly associated to PFS in this multivariate model (P = .82).

Pre-treatment plasma TIMP-1 and benefit from cetuximab treatment

Pre-treatment plasma TIMP-1 levels were not significantly different in non-responders versus responders (median 282 μg/\ vs. 260 μς/Ι; OR, 1 .16; 95% CI, .91 to 1 .49; P = .23).

Multivariate analysis showed a trend for an association between RR and pre-treatment plasma TIMP-1 (OR, 1 .34; 95% CI, .99 to 1 .82; P = .059). A significant interaction between RR, cetuximab treatment, KRAS mutational status, and TIMP-1 could not be shown (P = .48).

The results of the multivariate model for OS including pre-treatment plasma TIMP-1 , KRAS and BRAF status, age, gender, CRP, CEA, PS, number of metastatic sites and treatment +/- cetuximab are shown in Table 2. A significant 3-ways interaction between treatment +/- cetuximab, KRAS mutational status, and plasma TIMP-1 baseline level was demonstrated (P = .002). The HR for plasma TIMP-1 for patients with KRAS mutant tumors not treated with cetuximab was 4.45 (95% CI, 1 .73 to 1 1 .48) compared to 1 .04 (95% CI, .76 to 1 .42) if treated with cetuximab. A comparison of patients treated with cetuximab versus those not treated with cetuximab for the KRAS mutant subgroup showed a longer OS (HR, .48, 95% CI, .25 to .93) if the plasma TIMP-1 level was relatively high (3rd quartile), whereas the opposite was found for those with low levels of plasma TIMP-1 . There was no significant interaction between plasma TIMP-1 levels, treatment, and OS in patients with KRAS wild-type tumors (Table 2). The above described effects between plasma TIMP-1 levels, treatment and, OS are illustrated in Figure 5, indicating that patients with pre-treatment plasma TIMP-1 levels of about 250 ng/ml or greater and in which KRAS is mutated, show a significant benefit in terms of OS from the NORDIC FLOX + cetuximab treatment. Multivariate analysis of PFS could not demonstrate a similar significant association to plasma TIMP-1 (P = .058), however the analysis suggests a pattern similar to that seen for OS (Table 2).

Table 1 : Demographic and baseline clinical characteristics of the 423 patients with metastatic colorectal cancer included in the Nordic VII Study in whom pre-treatment pi TIMP-1 was measured.

Table 2: Univariate and multivariate Cox analyses of PFS and OS in the 426 patients with metastatic colorectal cancer (389 with progression, 285 deaths) included in the NORDIC VII Study according to pre-treatment plasma TIMP-1 and clinical parameters.

Progression-free Survival Overall Survival

Univariate Cox Multivariate Cox Univariate Cox Multivariate Cox analyses analyses analyses analyses

95% 95% 95% 95%

HR p-value HR P-value HR p-value HR P-value CI CI CI CI

0.27 0.25-

0.50¹ 0.48¹

0.93

0.91

Cetuxima 0.85 1.14² 0.85 1.08²

1.0 1.0 0.72- b Yes vs 0.68 b 0.48 a

5 0.80 9 1.63

No 1.29 1.33³ 1.40 2.16³

1.07- 1.27⁴ 1.63 0.83⁴

4.33

0.81 0.52-

2.19 1.03 0.87

1.84

0.63

0.76-

1.12

1.42

0.84⁵ 0.80 1.04⁵ 1.17- 2.15

Plasma 1.07 1.04⁶ 1.34 1.33 1.59⁶

1.2 1.5 <0.000

TI MP-1 0.003 b a

2 5 1 I.73- (log)* 1.39 2.15⁷ 1.04 1.80 4.45⁷

II.4 1.15⁸ 1.22⁸ 8

4.42

0.83- 0.81

1.79

1.62

0.84 0.82 0.86

Age per 0.9 0.9 0.88-

0.23 0.92 0.14 0.60 1.00 0.97 10 years 4 7 1.14

1.04 1.03 1.09

Gender,

0.88 0.74 0.74

1.0 0.9 0.90-

0.46 0.92 0.41 0.61 1.16 0.26

Female 8 4 1.49

1.32 1.13 1.19

vs. Male

BRAF 1.41 1.29 2.30

2.0 <0.000 <0.000 3.3 <0.000 3.10- <0.000 Mutant 1.73 4.74

0 1 1 1 1 7.23 1 vs. WT 2.82 2.33 4.77

0.74

1.46 0.78-

1.04⁹ 1.14⁹

1.66 1.29

2.37¹ 2.56¹

0 0 1.30-

KRAS 0.90 4.37 0.76 5.06

1.1 0.9

Mutant 0.30 b 0.88 a

2 1.34¹ 8 1.76¹

vs WT 1.39 1 0.97 1.27 1 1.16- 2.66

1.27¹ 1.85 0.67¹

2 2 0.33- 0.74 1.41

2.20 Metastati

1.13 1.13 1.22

c sites 1.4 1.5 1.20-

0.0026 1.43 0.0034 0.0006 1.60 0.0015 1 9 2.14

1.76 1.81 2.08

>1 vs 1

1.30 1.05 1.47

WHO PS 1.6 <0.000 1.8 <0.000 1.16-

1.33 0.020 1.52 0.0023 >1 vs O 1 1 8 1 1.98

1.99 1.68 2.39

Serum

CRP,

1.22 1.12 1.25

1.5 1.6 0.94-

0.0002 1.44 0.0046 0.0002 1.27 0.11

Elevated 0 1 1.71

1.85 1.86 2.05

vs.

normal

Serum

CEA,

1.24 1.03 1.38

1.6 1.9 1.19-

0.0004 1.39 0.034 0.0001 1.70 0.0038

Elevated 3 3 2.44

2.14 1.88 2.71

vs.

normal

HR = Hazard ratio. CI = Confidence interval.

^*Plasma TIMP-1 was included as a log transformed continuous variable (log base 2).

aP=0.004 for the interaction Treatment X KRAS X TIMP-1 .

bP=0.096 for the interaction Treatment X KRAS X TIMP-1 .

¹ HR for Cetuximab vs no cetuximab for KRAS mutant and TIMP-1 level at the 3^rd quartile (409 ng/ml).

²HR for Cetuximab vs no cetuximab for KRAS WT and TIMP-1 level at the 3^rd quartile (409 ng/ml).

3HR for Cetuximab vs no cetuximab for KRAS mutant and TIMP-1 level at the 1 ^st quartile (201 ng/ml).

⁴HR for Cetuximab vs no cetuximab for KRAS WT and TIMP-1 level at the 1 ^st quartile (201 ng/ml).

⁵HR for 2-fold difference TIMP-1 levels for mutant KRAS treated with Cetuximab.

6HR for 2-fold difference TIMP-1 levels for WT KRAS treated with Cetuximab.

⁷HR for 2-fold difference TIMP-1 levels for mutant KRAS not treated with Cetuximab.

⁸HR for 2-fold difference TIMP-1 levels for WT not treated with Cetuximab.

⁹HR for KRAS mutant vs WT receiving cetuximab and TIMP-1 level at the 3^rd quartile (409 ng/ml). ¹⁰HR for KRAS mutant vs WT not receiving cetuximab and TIMP-1 level at the 3^rd quartile (409 ng/ml).

¹¹ HR for KRAS mutant vs WT receiving cetuximab and TIMP-1 level at the 1 ^st quartile (201 ng/ml).

1²HR for KRAS mutant vs WT not receiving cetuximab and TIMP-1 level at the 1 ^st quartile (201 ng/ml).

Example 2

TIMP-1 plasma levels predict benefit from EGFR-inhibition therapy in KRAS-mutated metastatic colorectal cancer patients: results from the randomized, phase III NORDIC VII study.

ABSTRACT

It is now widely accepted that targeting the epidermal growth factor receptor (EGFR) can have efficacy in advanced colorectal cancer (CRC). What remains to be ascertained is which patients derive most benefit from inhibition of EGFR signaling. Tissue Inhibitor of

Metalloproteinases-1 (TIMP-1 ) is a pleiotropic factor that is predictive of survival outcome of CRC patients. Analysis of pre-treatment plasma samples of metastatic CRC patients (n=426) randomized to Nordic FLOX +/- cetuximab (NORDIC VII Study), showed TIMP-1 plasma levels to be associated with treatment outcome. Multivariate analysis demonstrated a significant interaction between plasma TIMP-1 , KRAS status, and treatment, with patients bearing KRAS mutated tumors and high TIMP-1 plasma level (>3rd quartile) showing a significantly longer overall survival when treated with cetuximab (HR 0.48; 95% CI, 0.25 to 0.93), as compared to patients not treated with cetuximab. Cell line analyses provided data consistent with a bimodal interaction between TIMP-1 and the EGFR/KRAS signaling axis, substantiating our clinical observation and providing a rationale for further clinical studies. Significance: Patients with KRAS mutated tumors and a high plasma TIMP-1 level showed benefit from EGFR-inhibition therapy. Based on these results, plasma TIMP-1 levels could be useful for selection of patients bearing KRAS mutated tumours that may derive benefit from EGFR-inhibition therapy.

INTRODUCTION

Different types of treatment are available for patients with advanced metastatic colorectal cancer (CRC), including targeted biological treatment, but there is a continued unmet need for effective, fast, point-of-care tests for predictive biomarkers in order to select the right treatment for individual patients. Epidermal growth factor receptor (EGFR) is recognized as a key player in CRC development and progression due to its effect on tumor-promoting processes such as cellular proliferation, survival, and motility. It is therefore not surprising that this receptor tyrosine kinase became a major therapeutic target, with several approved anti-cancer agents, such as cetuximab or panitumumab, currently used in the clinics.

Blockade of EGFR prevents ligand-induced activation of downstream effectors involved in intracellular signaling pathways, such as the RAS/RAF mitogen-activated protein kinase (MAPK) pathway, and phosphoinositide 3-kinase (PI3-K/Akt) pathway. Because activating mutations in downstream effectors, such as RAS, RAF, or PI3KCA, can result in persistent growth signaling, only RAS/RAF/PI3KCA wild-type patients are expected to derive benefit from anti-EGFR therapy. A comprehensive molecular characterization of human colon and rectal cancers, found that 55% of non-hypermutated tumours have alterations in KRAS, NRAS or BRAF, with a significant pattern of mutual exclusivity, stressing the importance of RAS testing in guiding treatment decisions with anti-EGFR targeted therapies in colorectal cancer patients. However, for yet unknown reasons a significant percentage of patients with KRAS mutated tumors may actually gain benefit from cetuximab treatment. Clearly, additional predictive biomarkers are needed to improve stratification of patients with mCRC to EGFR inhibitor therapies.

TIMP-1 is a 28kDa glycoprotein that can be found in the extracellular compartment in several tissues, and is present in various body fluids. TIMP-1 is one of four (TIMP-1 through 4) human natural endogenous inhibitors of matrix metalloproteinases (MMPs), a group of peptidases involved in degradation of the extracellular matrix. In addition to its function as inhibitor of MMPs, TIMP-1 can have tumor-promoting effects, including stimulation of cell proliferation, induction of anti-apoptotic signaling, and support of angiogenesis. Plasma TIMP-1 is elevated in patients with CRC and high plasma TIMP-1 levels are associated with poor prognosis in patients with primary or advanced CRC. TIMP-1 has also been implicated in resistance to various types of chemotherapy, and linked to thyroid carcinogenesis through an association with BRAF V600E signaling.

Based on previous work from our group, as well as from other groups, which has shown that TIMP-1 can be predictive of outcome in CRC and given that TIMP-1 can promote cell survival through the PI3-K/Akt signaling pathway, it was likely that TIMP-1 could influence response to anti-EGFR therapy. To address this hypothesis we performed a secondary analysis of the NORDIC VII study. This study was a three-arm, phase III prospective randomized clinical trial, of anti-EGFR therapy (cetuximab). Cetuximab is commonly used in combination with a chemotherapeutic backbone consisting of 5- uorouracil (5-FU) and either oxaliplatin or irinotecan. In patients with KRAS wild-type mCRC, addition of EGFR inhibitors to the conventional chemotherapy in first-line treatment increased the response rate (RR) by 7-15%. In the NORDIC VII study, no significant benefit of adding cetuximab to the Nordic FLOX regimen (bolus 5-FU and oxaliplatin) was found; neither in patients with KRAS wild- type, nor in patients with KRAS mutated tumors. Similar trials evaluating the efficacy of the FLOX regimen as first-line treatment of mCRC, have, for yet unknown reasons, been unsuccessful. On balance, the evidence indicates that when combined with cetuximab, only infusional 5-FU regimens are beneficial. In the present study, we tested interactions between plasma TIMP-1 levels and EGFR targeted treatment in the NORDIC VII Study (Nordic FLOX +/- cetuximab). Analysis of pre-treatment plasma samples (n=426) of metastatic CRC patients in the NORDIC VII Study showed TIMP-1 plasma levels to be associated with treatment outcome. Multivariate analysis demonstrated a significant interaction between plasma TIMP-1 , KRAS status, and treatment, with patients bearing KRAS mutated tumors and high TIMP-1 plasma level (>3rd quartile) showing a significantly longer overall survival when treated with cetuximab (HR 0.48; 95% CI, 0.25 to 0.93), as compared to patients with KRAS mutated tumors not treated with cetuximab. We show here that activated EGFR signaling induces significant increases in TIMP-1 expression in established CRC cell lines. Further, exogenous addition of human recombinant TIMP-1 (rTIMPI ) to CRC cells, promotes a more aggressive behavior, but only in RAS mutated cells.

MATERIALS AND METHODS

Cell lines and culture conditions

The colorectal cancer cell lines SW620 and Colo205 were purchased from the American Tissue Culture Collection (Rockville, MD, USA), while the HCT-15 and HT-29 were obtained from the NCI/Development Therapeutics Program. The DLD-1 cell line and matched pair of isogenic DLD-1 cell clones (KRAS G13D and KRAS wt) were a kind gift from Bert Vogelstein (Howard Hughes Medical Institute, The Johns Hopkins Medical Institution, USA). DLD-1 and derivative clones were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS); all other lines were cultured in RPMI-1640 medium supplemented with 10% FBS. All cells were maintained in cell culture flasks (TPP, Transadingen, Switzerland) at 37°C in a humidified, 5% C02 atmosphere under sterile conditions. All cell culture media were from Life Technologies (CA, USA), and plasticware was from Nunc (Thermo Fischer Scientific, USA). Cell identity was verified by short tandem repeat (STR) loci analysis at IdentiCell (Aarhus, Denmark). Cells were regularly tested for mycoplasma infection (Minerva Biolabs, Germany).

For EGF stimulation assays, cells were plated overnight, washed twice in PBS to remove serum remnants, and subsequently serum-starved in serum-free growth media for 24h, after which fresh serum-deprived medium containing human recombinant EGF (rEGF) (Sigma Aldrich, MO, USA) at 10 or 50 ng/mL was added and cells cultured for an additional 24h or 48h period. As controls, equivalent concentrations of rEGF solvent (acetic acid) or 10% FBS were added in each case, respectively. For TIMP-1 stimulation assays, DLD-1 isogenic cell lines (KRAS (wt/-) and (KRAS (-/G13D)) were plated for 48h, serum starved as previously described, for 4h, after which time human recombinant his6-tagged TIMP-1 (rTIMP-1 ) was added at a final concentration of 5μg/mL. Cells were incubated in the presence of rTIMP-1 for various time periods: Oh, 15min, 30min, 1 h, 2h, or 4h. As controls, equivalent

concentrations of bovine serum albumin (BSA) protein were added.

Western blot analysis

Whole cell extracts were obtained by lysis of 70-80% confluent cells. Briefly, cells were washed with ice-cold PBS before on-plate lysis with 250-500 μΙ_ M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, MA, USA) containing Pierce Protease and

Phosphatase Inhibitor Mini Tablets (Thermo Scientific, MA, USA). The lysates were centrifuged at 14000 x g for 10 min to remove cell debris and total protein concentrations of the samples were measured using a BCA protein Assay Kit (Novagen, CA, USA) according to manufacturer's instructions. For analysis, equivalent amounts of total protein (20 μg per well) were subjected to SDS-PAGE separation under reducing conditions. Proteins were blotted onto 0.2 μηι nitrocellulose membranes (Bio-Rad, CA, USA), and blocked in 5% milk or BSA (antibody-specific) in Tris-Buffered Saline and Tween 20 (TBS-T, 0.05 %) for 1 hour at room temperature. The membranes were incubated overnight with relevant primary antibodies diluted in 5% milk blocking solution (p150Glued, VT-7), or 5 % BSA (phospho- Akt(Ser374), Akt) in TBS-T at 4°C. After washing thrice for 10min in TBS-T, followed by detection of immune complexes with corresponding horseradish peroxidase-labeled species specific antibodies (Dako, Denmark), detection of immune complexes was done using the Amersham ECL-Select Western Blotting detection reagent (GE Healthcare Life Sciences, NJ, USA) or Clarity Western ECL Substrate (Bio-Rad, CA, USA) according to manufacturer's instruction and images were captured with a BioSpectrum Imaging system (Ultra-Violet Products, CA, USA). The anti-p150Glued antibody was from BD Biosciences (NJ, USA), the anti-TIMP-1 antibody was an in-house antibody (VT-7) previously described (40), and the anti-P-Akt antibody was from Cell Signaling Technologies (MA, USA).

In vitro invasion assay

In vitro invasive potential was determined using Corning ® BiocoatTM Matrigel® invasion chambers (VWR, Radnor, PA, USA). Briefly, cells were serum-starved for 24h, harvested by trypsin-EDTA treatment, and centrifuged to form a cell pellet (Sigma Aldrich, MO, USA). The cells were washed twice in serum-free medium, resuspended, and inoculated at a density of

onto an 8μηι pore Matrigel®-coated membrane. The inserts were inset in 24-well plates, with each well filled with 750 μΙ of 50 ng/mL rEGF medium, and subsequently incubated at 37°C for 14h. After incubation, non-invasive cells were carefully scraped off with a cotton swab and invasive cells were fixed in 100% methanol, stained with 10% Giemsa, and counted with a light microscope by two independent observers. All assays were performed in triplicate.

Soft agar clonogenic assay

To assess a potential differential biological effect of TIMP-1 on anchorage-independent growth in KRAS mutated cells, a double layer soft agar assay was performed. Monolayer cultures of DLD-1 isogenic cells (KRAS (wt/-) and (KRAS (-/G13D)) were prepared into single-cell suspensions using 0.01 % trypsin-EDTA. The cells were suspended in growth medium containing 0.18% low melting temperature agar (Sigma Aldrich, MO, USA) supplemented with either 5 μg/mL BSA or rTIMP-1 , seeded at a density of 1000 cells/750 μΙ_Λ/νβΙΙ on top of a solidified bottom layer of 0.75% agar in growth medium with 5.0 μg/mL rTIMP-1 or BSA (control) in 12-well plates. The following day 500 μΙ_ growth medium containing rTIMP-1 or BSA in corresponding concentrations was added. Each condition was set up in triplicate and three independent assays were performed. Visible colonies were counted independently by two different observers after 21 and 28 days. For each experiment multiple images were acquired and the size of the spheroids was estimated using Matlab software analysis of the images.

Patient characteristics

In the NORDIC VII Study 566 patients with mCRC were included from 32 Nordic centers. Patients were randomized between; Nordic FLOX: 5-FU i.v. bolus 500 mg/m2 and folinic acid 60 mg/m2 day 1 -2, oxaliplatin 85 mg/m2 day 1 every two week until progression (arm A); Nordic FLOX plus cetuximab (400 mg/m2 day 1 , then 250 mg/m2 weekly) until progression (arm B) or Nordic FLOX + cetuximab for 16 weeks, and weekly cetuximab as maintenance treatment until progression (arm C). Main inclusion criteria were: histologically confirmed mCRC (adenocarcinomas); age > 18 years and <75 years; WHO performance status (PS)≤ 2; no prior chemotherapy for mCRC, non-resectable and measurable disease according to the Response Evaluation Criteria in Solid Tumors (RECIST version 1 .0); last adjuvant chemotherapy≥ 6 months before inclusion; no previous oxaliplatin treatment; adequate haematological, renal and liver function.

The patients were treated until disease progression and followed until death or 30th April 2009. Pre-treatment plasma sample were available from 426 (75%) patients at baseline. The baseline demographic characteristics of the 426 patients with a pre-treatment plasma TIMP-1 measurement are shown in Table 1 . All patients provided written informed consent, and the study (including biomarker analyses) was approved by the Regional Ethics Committee (VEK ref. 20050053). Further details about the study have been published.

Biomarker analyses

Total plasma TIMP-1 levels (free and in complex with matrix metalloproteinase) were determined using a MAC15 antibody kinetic enzyme-linked immunosorbent assay (ELISA). Duplicate measurements were carried out and the mean values were used for statistical analysis. The mean intra-assay coefficient of variation (CV) was 5.1 % (range 1 .5%-9.8%) and the inter-assay CV was 6.7%.

Data on serum carcinoembryonic antigen (CEA), C-reactive protein (CRP), KRAS and BRAF mutational status of the tumor, WHO performance status (PS), and number of metastatic sites were retrieved from the original study report.

Statistical analysis

The primary clinical endpoint for this biomarker study was OS determined as the time from randomization to treatment in NORDIC VII to time of death by any causes. The median follow-up time was 37 month (24-53 months). Cases in which patients were alive at this date were censored. Secondary endpoint was PFS (primary endpoint of the NORDIC VII Study) defined as the time from randomization until objective disease progression. Descriptive statistics are presented as median levels and ranges. Analyses of measurements for PFS and OS were done using the Cox proportional hazards model. As the analyses performed comparing treatment arms did not reveal any substantial differences in terms of OS and PFS between the original study population and this subset of patients (please see below), we found it justified to pool arms B and C (i.e. + cetuximab treatment) for the statistical analyses. Thus, patients were stratified as receiving cetuximab or not, i.e. arm B and C versus arm A. Survival probabilities for OS were estimated by the Kaplan-Meier method and tests for differences between strata were done using the log-rank statistic. Graphical presentation using Kaplan-Meier estimates of PFS and OS was shown grouping patients in tertiary TIMP- 1 levels. Multivariable analysis of PFS and OS was done using the Cox proportional hazard model. TIMP-1 concentrations in plasma were entered by the actual value on the log scale (base 2). Missing values for CEA (n = 27), CRP (n = 22), BRAF (n = 101 ) and KRAS (n = 62) were categorized separately and included in the final multivariable analysis. The final model included a three way interaction term (treatment with cetuximab +/- x KRAS mutational status x plasma TIMP-1 ).

The model was assessed using Schoenfeld and Martingale residuals. In particular, the linearity assumption for plasma TIMP-1 on the log scale was evaluated using the supremum test for the cumulated martingales. The results yielded P > 0.05 testing for the linearity of plasma TIMP-1 on the log scale. Ten-fold cross validation performed in order to assess over- fitting showed almost similar results for the training and test sets (data not shown). P-values less than 5% were considered statistically significant. Statistical calculations were performed using SAS (version 9.2, SAS Institute, Cary, NC, USA) and R. The results of this project are reported in accordance with the REMARK guidelines.

RESULTS

Pre-treatment plasma TIMP-1 and associations to PFS and OS

We measured total plasma TIMP-1 levels (free and in complex with matrix metalloproteinase) in 426 samples that were available from the study, using an ELISA assay developed in- house. The two study populations (+/- cetuximab) were comparable and not different from the total intention to treat population of 566 patients. The median pre-treatment plasma TIMP-1 was 269 ng/mL (58 to 1318 ng/mL) with no differences between the two treatment groups (P = 0.97). Tumors were KRAS mutated in 147 patients (39%). There was no association between pre-treatment plasma TIMP-1 and gender, or number of metastatic sites. There were statistically significant associations between high pre-treatment plasma TIMP-1 and WHO PS, location of the primary tumor, previous adjuvant chemotherapy, KRAS and BRAF status. The highest plasma TIMP-1 values were found in patient with high WHO PS, primary tumor in colon, no adjuvant therapy, KRAS wild-type tumors and BRAF mutated tumors (Table 1 ). Univariate Cox analyses including all patients showed that high pre- treatment plasma TIMP-1 was significantly associated with shorter PFS (HR, 1 .22; 95% CI, 1 .07 to 1 .39; P = .003), and OS (HR, 1 .55; 95% CI, 1 .33 to 1 .80; P < .0001 ) (Table 2).

Kaplan-Meier estimates of survival probabilities for PFS and OS stratified by pre-treatment plasma TIMP-1 levels are shown in figs. 3A and 1 B, respectively. Multivariate analysis (plasma TIMP-1 , KRAS and BRAF status, serum CRP, serum CEA, PS, number of metastatic sites, age, and gender) demonstrated that high plasma TIMP-1 was an

independent biomarker of short OS (HR, 1 .35; 95% CI, 1 .12 to 1 .62; P = 0.016) while a significant interaction between plasma TIMP-1 and KRAS mutational status could not be demonstrated (P = 0.47). Plasma TIMP-1 was not significantly associated to PFS in this multivariate model (P = 0.82).

Pre-treatment plasma TIMP- 1 and benefit from cetuximab treatment

To determine if there was an association between TIMP-1 and response to cetuximab, we examined TIMP-1 plasma levels in patients responding, or not, to cetuximab. Pre-treatment plasma TIMP-1 levels were not significantly different in non-responders versus responders (median 282 μg/\ vs. 260 μς/Ι; OR, 1 .16; 95% CI, .91 to 1 .49; P = 0.23). Multivariate analysis showed a trend for an association between RR and pre-treatment plasma TIMP-1 (OR, 1 .34; 95% CI, .99 to 1 .82; P = 0.059). A significant interaction between RR, cetuximab treatment, KRAS mutational status, and TIMP-1 could not be shown (P = 0.48). The results of the multivariate model for OS including pre-treatment plasma TIMP-1 , KRAS and BRAF status, age, gender, CRP, CEA, PS, number of metastatic sites and treatment, or not, with cetuximab are shown in Table 2. A significant 3-ways interaction between treatment (+/- cetuximab), KRAS mutational status, and plasma TIMP-1 baseline level was demonstrated (P = 0.006). The HR for plasma TIMP-1 for patients with KRAS mutant tumors not treated with cetuximab was 4.45 (95% CI, 1 .73 to 1 1 .48) compared to 1 .04 (95% CI, .76 to 1 .42) if treated with cetuximab. A comparison of patients treated with cetuximab versus those not treated with cetuximab for the KRAS mutant subgroup showed a longer OS (HR, .48, 95% CI, .25 to .93) if the plasma TIMP-1 level was relatively high (3rd quartile), whereas the opposite was found for those with low levels of plasma TIMP-1 . There was no significant interaction between plasma TIMP-1 levels, treatment, and OS in patients with KRAS wild- type tumors (Table 2). The above described effects between plasma TIMP-1 levels, treatment and OS, are illustrated in figs. 4A and 2B, respectively. Multivariate analysis of PFS could not demonstrate a similar significant association to plasma TIMP-1 (P = 0.078), however the analysis suggests a pattern similar to that seen for OS (Table 2).

EGF induced TIMP- 1 expression in CRC cell lines

Our analysis of the NORDIC VII study demonstrated an association between high plasma TIMP-1 levels and benefit from adding cetuximab to the NordicFLOX regimen, but only in patients bearing KRAS-mutated tumors. To shed some light on the biological mechanisms underpinning this association, and address the possibility of a type 1 statistical error, we investigated the effect of EGFR signaling on TIMP-1 expression in cellular models of CRC. We analyzed five different CRC cell lines: SW620 (KRAS G12V, BRAF wt), Colo-205 (KRAS wt, BRAF V600E), HT-29 (KRAS wt, BRAF V600E), HCT-15 (KRAS G13D, BRAF wt), and DLD-1 (KRAS G13D, BRAF wt), for TIMP-1 expression upon stimulation with EGF, respectively. As shown in fig. 6, four of the five cell lines (Colo-205, HT-29, HCT-15, and DLD-1 , respectively) showed an increase, albeit to a varying degree, in TIMP-1 expression upon stimulation with EGF. This effect was dose-dependent, as stimulation with 50ng/ml_ EGF generally elicited relatively higher levels of TIMP-1 expression than 10ng/ml_ (fig. 6, compare 10ng/ml_ EGF with 50ng/ml_ EGF), and not associated with KRAS or BRAF status. In all cases phosphorylation of Akt was used as a measure of functional EGF signaling. In the cases of HCT-15 and HT-29 we found high basal levels of Akt phosphorylation even under serum-starvation growth conditions, consistent with the presence of activating mutations in BRAF and KRAS, respectively (figs. 6A and 3B). However, we could observe increased Akt phosphorylation after 24h of ligand stimulation of EGFR, indicating that the stimulatory potential of the signaling pathway was maintained, and thus the increase in TIMP-1 expression following EGF stimulation could be ascribed to stimuli transduced through the EGFR signaling axis in HCT-15 and HCT-29. Following 48h of EGF stimulation, we observed a continued dose-dependent EGF-induced increase in TIMP-1 expression (fig. 6A- C). Given the variable range in relative increases in TIMP-1 expression between the various cell lines in response to EGF stimulation, we analyzed EGFR expression in the five CRC cell lines used. We found that these cells lines had very different levels of EGFR expression, and that the levels of EGFR expression in the five cell lines were consistent with the effect of EGF on TIMP-1 expression we had observed. Thus, HT29 (strong EGFR expression), and HCT15 and DLD1 (moderate EGFR expression) cells, displayed very significant up- regulation of TIMP-1 stimulation upon exposure to EGF, whereas Colo-205 (weak EGFR expression), and SW620 (no detectable EGFR), showed very limited or no increase in TIMP- 1 expression, respectively (fig.6). Overall we could conclude that TIMP-1 expression is under regulation of the EGF-EGFR signaling axis in the studied CRC cell lines.

TIMP- 1 promotes colony formation in soft agar in KRAS-mutated cells only

In order to assess the effects of TIMP-1 on anchorage-independent growth, and to evaluate their dependency on KRAS mutational status, we performed a comparative soft agar colony formation analysis using a matched pair of isogenic DLD-1 cell clones (KRAS G13D and KRAS wt), in which either the wild-type or mutant KRAS allele has been disrupted. We found that in the DLD-1 cell clone with the wild-type KRAS allele (KRAS wt), the number and size of colony forming units (CFU) formed after 21 or 28 days incubation, respectively, were not affected by the continuous presence of TIMP-1 in the growth media (5 g/mL rTIMP-1 ) (fig. 7A, 21 days P > 0.999; 28 days P = 0.98). However, in the case of the DLD-1 clone with the KRAS-mutated allele (KRAS G13D), we observed an increase in both number, and size, of formed cell foci after 21 and 28 days incubation, respectively (fig. 7B, 21 days P = 0.04; 28 days P = 0.03). As expected, cells bearing the KRAS G13D mutated allele displayed increased ability to form colonies in soft agar, when compared to the clone with the KRAS wt allele (fig. 7B). The interaction between cell line and TIMP-1 exposure was non-significant at 21 days (P = 0.06), but it was significant after 28 days (P = 0.04).

TIMP-1 may enhance invasion in KRAS mutated cells only

To determine if TIMP-1 could promote invasion in a KRAS-dependent manner in CRC cells, we stimulated the matched pair of isogenic DLD-1 cell clones (KRAS G13D and KRAS wt) with exogenously added TIMP-1 (rTIMP-1 ; 5μg/mL) and compared the invasive potential of these cells to that of a control group (no added rTIMP-1 ), in a Boyden chamber invasion assay. We found that there was an interaction between addition of rTIMP-1 and KRAS mutational status (fig.7E). Whereas the DLD-1 cell clone bearing a wild-type KRAS allele (KRAS wt) showed no significant difference in invasive potential in the presence, or not, of TIMP-1 (fig. 7E; P = 0.999), KRAS mutated cells (KRAS G13D) responded to the presence of TIMP-1 in the growth medium (5.0 μg/mL rTIMP-1 ), becoming significantly more invasive (fig. 7E; KRAS G13D P = 0.0152). The invasive potential of DLD-1 KRAS mutated cells upon stimulation with TIMP-1 , compared with the BSA control, was significantly different to that of KRAS wt cells (P = 0.0328) (fig.7E). These results indicate that under the conditions of the assay, TIMP-1 promotes invasion only in KRAS mutated cells. DISCUSSION

Based on pre-clinical data from our laboratory, as well as published work from other groups, we hypothesized that high plasma TIMP-1 levels may interact with EGFR signaling and thereby affect the anti-tumor effects of EGFR inhibitors. To address this hypothesis we analyzed a potential association(s) between plasma TIMP-1 levels and clinical outcome in patients treated with a regimen based on an oxaliplatin backbone adding (or not) cetuximab (the NORDIC VII Study). The results, presented here, showed, an association between high plasma TIMP-1 levels and benefit from adding cetuximab treatment to the regimen in patients bearing KRAS mutated tumors (fig. 4 and Table 2). Thus, we have identified high plasma TIMP-1 levels as a novel predictive biomarker for cetuximab response in such KRAS mutated tumors (fig.4). In addition to the demonstration of a predictive effect of plasma TIMP- 1 for cetuximab treatment in KRAS mutated tumors, we were also able to confirm a strong prognostic effect of plasma TIMP-1 levels irrespective of treatment (fig.3 and Table 2). It should also be noted that the largest prognostic effect of plasma TIMP-1 was observed among KRAS mutated patients. Neither of the biological activities ascribed to TIMP-1 , be it the canonical MMP-dependent or the MMP-independent function, can explain the association we found between high plasma TIMP-1 levels and benefit from cetuximab treatment in KRAS mutated tumors. If anything, the PI3-K/Akt associated pro-survival effect should be deleterious for cetuximab treated patients, as well as independent of KRAS status. It was therefore conceivable that either we were dealing with a hitherto unknown function of TIMP- 1 , or more likely, that it was the expression of TIMP-1 , per se, that was important. We derived two assumptions from the data: first, given that plasma TIMP-1 levels were associated with benefit from cetuximab treatment in KRAS mutated tumors but not in KRAS wild-type tumors, one would expect TIMP-1 to have a different biological effect in KRAS- mutated cancer cells and in KRAS wild-type cancer cells. Second, as this effect was observed with cetuximab, it should be dependent on EGFR signaling but not on the

RAS/MEK signaling axis. We propose that the predictive value of TIMP-1 we observed is the outcome of two additive effects; first that TIMP-1 expression is under control of EGFR- signaling, independently of the RAS/MAPK-axis, and secondly that TIMP-1 potentiates an aggressive behavior in KRAS mutated cells but not KRAS wild-type cells. When tumor cells are exposed to cetuximab, expression of TIMP-1 will be inhibited, irrespective of KRAS status. This will not have a noticeable effect on KRAS wild-type tumor cells, but will abrogate the proliferative drive from TIMP-1 on KRAS mutated tumor cells (fig. 8), explaining the observation reported here. A corollary of this model is that blocking TIMP-1 may be an effective therapeutic strategy for patients bearing tumors with KRAS mutations.

These two levels of interaction are required to fully account for the predictive value of TIMP-1 specifically in cetuximab-treated KRAS-mutated patients and we addressed their validity employing CRC cellular models. We determined that TIMP-1 expression could be stimulated by exposing CRC cells to EGF ligand in a dose-dependent manner (fig. 6). This effect was directly related to expression levels of EGFR, supporting that the EGF-ligand/EGFR signaling axis plays an important regulatory role in TIMP-1 expression. We could also ascertain that TIMP-1 promoted colony formation and cell invasion in KRAS-mutated cells but not in KRAS wild-type cells (fig. 7), consistent with potentiation of aggressive behavior in KRAS mutated cells but not KRAS wild-type cells. This bimodal interaction between EGF-EGFR signaling and TIMP-1 expression on the one hand, and TIMP-1 mediated stimulation of cell invasion, specifically on KRAS-mutated cells, on the other hand, supports our clinical observation in the NORDIC VII study. The main question is then if these observations have any relevance in a clinical context. A number of studies provided evidence that have been deemed strong enough for the use of cetuximab and panitumumab to be restricted to patients with wild-type KRAS. However, there are a number of discordant findings, suggesting that there may be some patients with KRAS mutations that will derive benefit from anti-EGFR therapy and conversely some patients with wild-type KRAS will not. The NORDIC VII study itself, which was at the base of the data presented here, showed a trend towards an increased PFS for patients with KRAS muted tumors receiving cetuximab (median PFS of 7.8 versus 9.2 months), although this was not statistically significant (HR 0.71 ; P = .07). Clearly, there are a number of confounding factors, such as the exact nature of KRAS mutations (G13D or other), EGFR expression, first line therapy or metastatic treatment, chemotherapeutic backbone (irinotecan or oxaliplatin), or even administration regimen, which all seem to interplay and ultimately affect the outcome of anti-EGFR therapy in CRC patients. We provided evidence here confirming that plasma TIMP-1 is a prognostic biomarker in mCRC patients receiving oxaliplatin-containing treatment. Moreover, we were able to demonstrate a significant predictive effect of plasma TIMP-1 in relation to benefit from adding cetuximab to FLOX in patients with KRAS-mutated tumors. However, given that our analysis was conducted retrospectively, and the NORDIC VII trial was not specifically designed to assess the clinical activity of cetuximab in biomarker-specific subgroups, our results need to be validated in an independent cohort of patients with mCRC, and in patients with other cancer types where cetuximab (or another anti-EGFR therapy) could be a valid treatment option. Importantly, as an association between TIMP-1 expression and EGFR signaling has also been observed in other cellular contexts, and found to occur, at least under certain circumstances, via NF- B signaling, in a MEK-independent manner, it is probable that this is a general effect rather than a CRC-specific one. The same is true of the differential effect on KRAS-mutated cells, as it was recently shown that TIMP-1 induces hyperproliferation of KRAS(G12D)-transformed cells but not of KRAS wild-type pancreatic cells. Overall, our data provides a strong rationale to study the effect of plasma TIMP-1 as a predictive biomarker for cetuximab in a randomized study with a proper control group, which will allow for a separation of a prognostic and a predictive effect of plasma TIMP-1 .

Claims

1 . A method of identifying a cancer patient who is likely to benefit from treatment with an EGFR inhibitor comprising determining in vitro

a) the levels of TIMP-1 of the patient,

b) the absence or presence of a RAS mutation in the patient's tumor sample,

wherein EGFR is expressed in the patient's tumor sample, and whereby the levels of TIMP-1 of the patient and the presence of a RAS mutation in the patient's tumor sample indicate that the patient is likely to respond to a treatment with an EGFR inhibitor.

2. A method of predicting cancer patient response to EGFR-inhibitor treatment,

comprising the step of determining in vitro the patient's TIMP-1 levels and determining the absence or presence of a RAS mutation in said patient's tumor sample expressing EGFR, wherein the levels of TIMP-1 and the presence of a RAS mutation in said patient's tumor sample indicate an increased progression free survival (PFS) and/or overall survival (OS) of the patient when the patient is treated with an EGFR inhibitor.

3. A method of treating a cancer patient, said method comprising administering to a patient in need thereof a therapeutically effective amount of

an EGFR inhibitor, and

at least one chemotherapeutic agent,

if the patient is likely to benefit from the treatment evaluated by determining in vitro the patient's TIMP-1 levels and determining the absence or presence of a RAS mutation in said patient's tumor sample expressing EGFR, wherein the levels of TIMP-1 and the presence of a RAS mutation in said patient's tumor sample indicate an increased progression free survival (PFS) and/or overall survival (OS) of the patient when the patient is treated with an EGFR inhibitor.

4. The method according to any one of the preceding claims, wherein said levels of TIMP-1 of the patient is the blood levels of TIMP-1 of the patient.

5. The method according to claim 4, wherein said blood levels of TIMP-1 of the patient is the blood plasma levels of TIMP-1 .

6. The method according to any one of claims 1 -4, wherein said levels of TIMP-1 of the patient are the TIMP-1 levels in a tumor sample from said patient.

7. The method according to claim 6, wherein the TIMP-1 tumor tissue immunoreactivity in said patient's tumor sample is determined,

8. The method according to any one of the preceding claims, wherein the levels of TIMP-1 of the patient are pre-treatment TIMP-1 levels.

9. The method according to any one of the preceding claims, wherein TIMP-1 levels of at least 250 ng/ml and the presence of a mutated RAS in said patient's tumor sample indicate that the patient will respond to a treatment with an EGFR inhibitor.

10. The method according to any one of the preceding claims, wherein the TIMP-1 levels are at least about 250 ng/ml to about 1400 ng/ml, or at least about 275 ng/ml to about 1300 ng/ml, or at least about 300 ng/ml to about 1250 ng/ml, or at least about 325 ng/ml to about 1200 ng/ml, or at least about 350 ng/ml to about 1 150 ng/ml, or at least about 375 ng/ml to about 1 100 ng/ml, or at least about 400 ng/ml to about 1050ng/ml, such as at least about 300 ng/ml to about 1350 ng/ml, or at least about 250 ng/ml. 275 ng/ml, 300 ng/ml, 325 ng/ml, 350 ng/ml, 375 ng/ml, 400 ng/ml, 409 ng/ml or 425 ng/ml, which TIMP-1 levels indicate that the patient will respond to a treatment with an EGFR inhibitor.

1 1 . The method according to any one of the preceding claims, wherein TIMP-1 levels of less than 250 ng/ml and the presence of a mutated RAS in said patient's tumor sample indicate that the patient will not respond to a treatment with an EGFR inhibitor.

12. The method according to any one of the preceding claims, wherein the EGFR

inhibitor is one or more of a monoclonal antibody, a tyrosine kinase inhibitor, cetuximab, panitumumab, erlotinib, gefitinib, afatinib, dacomitinib, neratinib, vandetanib, brivanib, tivantinib, crizotinib, XL-647, canertinib, pelitinib, PKI-166 or TAK-285.

13. The method according to any one of the preceding claims, wherein the EGFR inhibitor is administered in combination with at least one chemotherapeutic agent, wherein the chemotherapeutic agent is selected from the group comprising capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, 6-mercaptopurine (6-MP), cladribine, clofarabine, 6-mercaptopurine (6-MP), cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, preferably a combination of 5-FU, leucovorin, or a

combination of 5-fluorouracil/folinic acid (5-FU/FA), or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or a combination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx).

14. The method according to any one of the preceding claims, wherein the RAS mutation is selected from one or more of

a. an activating mutation,

b. a H-RAS, N-RAS or KRAS mutation,

c. a N-RAS mutation comprising at least one mutation selected from the group consisting of the amino acid substitutions G12C, G12D, G12R, G12S, G12A, G12V, G12R, G13C, G13R, G13A, G13D, G13V, G15W, G60E, Q61 P, Q61 L, Q61 R, Q61 K, Q61 H and Q61 E,

d. a H-RAS mutation comprising at least one mutation selected from the group consisting of the amino acid substitutions G12R, G12V, G13C, G13R and Q61 R,

e. an activating KRAS mutation, and

f. a KRAS mutation comprising at least one mutation selected from the group consisting of the amino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G13C and G13D, G13R, G13S, Q61 K, Q61 L, Q61 P, Q61 R and A146V.

15. The method according to any one of the preceding claims, wherein the EGFR

expressed in the patient sample is wild type EGFR; or wherein the EGFR expressed in the patient sample is mutated.

16. The method according to any one of the preceding claims, wherein the cancer is selected from the group consisting of colorectal cancer, metastatic colorectal cancer, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, melanoma, myeloid leukemia, juvenile myeloid leukemia, ovarian cancer, pancreas cancer, thyroid cancer, prostate cancer and adenocarcinoma,

17. The method according to any one of the preceding claims, wherein the cancer is colorectal cancer or metastatic colorectal cancer.

18. The method according to claim 18, wherein the cancer patient's tumor expresses EGFR and a mutated KRAS and wherein the blood or blood plasma TIMP-1 levels in the cancer patient are at least about 250 ng/ml.

19. The EGFR inhibitor cetuximab in combination with 5-fluorouracil/folinic acid (5- FU/FA), oxaliplatin (FLOX) for use in the treatment of cancer, such as colorectal cancer, for example metastatic colorectal cancer, wherein

a. the cancer patient's tumor expresses EGFR and a mutated KRAS, and b. the cancer patient's blood TIMP1 levels are at least about 250 ng/ml or the cancer patient's tumor tissue is TIMP-1 immunoreactive.

20. Use of the biomarkers KRAS and TIMP-1 for predicting the pharmaceutical efficacy and/or clinical response of a combination comprising at least one EGFR inhibitor and at least one chemotherapeutic agent to be administered to a cancer patient.

21 . Use of the biomarkers KRAS and TIMP-1 according to claim 20, wherein

a. the at least one EGFR inhibitor is selected from the group comprising

cetuximab, panitumumab, erlotinib, gefitinib, afatinib, dacomitinib, neratinib, vandetanib, brivanib, tivantinib, crizotinib, XL-647, canertinib, pelitinib, PKI- 166 or TAK-285, and/or

b. the use comprises determining in vitro the absence or presence of at least one KRAS mutation selected from the group consisting of the amino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G13C and G13D, G13R, G13S, Q61 K, Q61 L, Q61 P, Q61 R and A146V, and/or c. the use comprises determining in vitro the TIMP-1 levels of the cancer

patient, such as the TIMP-1 blood levels or TIMP-1 tumor tissue

immunoreactivity, and/or the cancer is selected from the group consisting of colorectal cancer, metastatic colorectal cancer, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, endometrial cancer, kidney cancer, liver cancer, lung cancer, melanoma, myeloid leukemia, juvenile myeloid leukemia, ovarian cancer, pancreas cancer, thyroid cancer, prostate cancer and

adenocarcinoma, and/or

the at least one chemotherapeutic agent is selected from the group consisting of 5-fluorouracil/folinic acid (5-FU/FA), capecitabine, 5-fluoro-2'-deoxyuiridine, irinotecan, 6-mercaptopurine (6-MP), cladribine, clofarabine, 6- mercaptopurine (6-MP), cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel, chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide, colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin, cisplatin, carboplatin, oxaliplatin, preferably a combination of 5-FU, leucovorin, or a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or a combination of 5- FU, leucovorin, and irinotecan (FOLFIRI), or a combination of 5- fluorouracil/folinic acid (5-FU/FA) and oxaliplatin (FLOX), or a combination of leucovorin, 5-FU, oxaliplatin, and irinotecan (FOLFOXIRI), or a combination of Capecitabine and oxaliplatin (CapeOx).