WO2013190089A1

WO2013190089A1 - Molecular biomarkers for predicting outcome in lung cancer

Info

Publication number: WO2013190089A1
Application number: PCT/EP2013/062992
Authority: WO
Inventors: Rafael Rosell Costa; Miguel Tarón Roca
Original assignee: Pangaea Biotech, S.L.
Priority date: 2012-06-21
Filing date: 2013-06-21
Publication date: 2013-12-27

Abstract

The invention relates to methods for determining the survival of patients suffering EGFR mutation-positive lung cancer based on the determination of the levels of cell-free EGFR carrying said mutation. The invention also provides methods for typing or classifying patients based on the levels of free circulating EGFR carrying said mutation. The invention also provides computer programs and systems and kits for carrying out the above methods.

Description

MOLECULAR BIOMARKERS FOR PREDICTING OUTCOME IN LUNG

CANCER

FIELD OF THE INVENTION

The invention relates to the field of pharmacogenomics and, more in particular, to methods for predicting the survival of a lung cancer patient to a treatment with conventional chemotherapy or with an EGFR tyrosine kinase inhibitor-based chemotherapy, based on the detection of one sensitivity mutation in the EGFR gene towards an inhibitor of EGFR tyrosine kinase activity in a bio fluid of the patient.

BACKGROUND OF THE INVENTION

Non-small-cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancers, with 1.2 million new cases worldwide each year. NSCLC resulted in more than one million deaths worldwide in 2001 and is the leading cause of cancer-related mortality in both men and women (31% and 25%, respectively). The prognosis of advanced NSCLC is dismal. A recent Eastern Cooperative Oncology Group trial of 1155 patients showed no differences among the chemotherapies used: cisplatin/paclitaxel, cisplatin/gemcitabine, cisplatin/docetaxel and carboplatin/paclitaxel. Overall median time to progression was 3.6 months, and median survival was 7.9 months.

At diagnosis, patients with NSCLC can be divided into three groups that reflect both the extent of the disease and the treatment approach:

■ The first group of patients has tumors that are surgically resectable (generally stage I, stage II, and selected stage III tumors). This group has the best prognosis.

^■ The second group includes patients with either locally (T3-T4) and/or regionally (N2-N3) advanced lung cancer. Patients with unresectable or N2-N3 disease are treated with radiation therapy in combination with chemotherapy.

Selected patients with T3 or N2 disease can be treated effectively with surgical resection and either preoperative or postoperative chemotherapy or chemoradiation therapy.

■ The final group includes patients with distant metastases (Ml). This group can be treated with palliative radiation therapy or chemotherapy.

First-line treatment of NSCLC is carried out by platinum-based chemotherapy.

However, for the past several years, efforts have been focused on the development of targeted therapy direct against EGFR in non-small cell lung carcinoma (NSCLC). Several groups have independently identified frequent somatic mutations in the kinase domain of the EGFR gene in lung adenocarcinoma. These occur in 16% of lung adenocarcinoma specimens sequenced in the U.S. and 40% of those sequenced in Asia. Subsequent studies by multiple groups have now identified EGFR kinase domain mutations from many additional lung cancer patients. These mutations cluster in four groups, or regions; exon 19 deletions, exon 20 insertions, and point mutations at G719S and L858R. Deletions in exon 19 and the L858R mutation account for more than 90% of the total number of mutations found in lung adenocarcinoma. Thus far, the incidence of these kinase domain mutations is more common in adenocarcinomas than in lung cancers of other histological subtypes such as squamous cell carcinoma. These mutations are associated with sensitivity to specific inhibitors of the tyrosine kinase of EGFR, such as gefitinib (Iressa) and erlotinib (Tarceva), which has allowed the development of new protocols for first line treatment of NSCLC carrying these mutations with these tyrosine kinase inhibitors.

Erlotinib has been shown to improve progression-free survival compared with chemotherapy when given as first-line treatment for Asian patients with non-small-cell lung cancer (NSCLC) with activating EGFR mutations (Mitsudomi et al, Lancet Oncol 2010; 11 : 121-28). These results have been recently confirmed by Rosell et al. (Lancet Oncology, DOI: 10.1016/S1470-2045(11)70393-X) in non-Asian patients with advanced NSCLC by a prospective head-to-head phase 3 study comparing efficacy and safety of first-line erlotinib with platinum-based chemotherapy in patients. These results suggests a benefit in PFS with first-line erlotinib in a European population and confirm those improvements attained with EGFR targeted agents in Asian patients. However, there is still a need in the art for tools and methods useful for predicting survival of patients suffering lung cancer and, in particular, of patients suffering lung cancer carrying activating mutations in the EGFR gene. SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a method for predicting the outcome of a patient suffering lung cancer after treatment with chemotherapy, with an EGFR inhibitor or a combination thereof wherein said lung cancer carries at least an activating EGFR mutation, said method comprising determining in a bio fluid of said patient the levels of the cell-free EGFR gene carrying said at least one activating EGFR mutation wherein the detection of cell-free EGFR gene containing said mutation or the detection of an increased level thereof with respect to a reference value is indicative of a poor outcome of the patient or wherein an absence of detection of the cell- free EGFR gene containing said mutation or the detection of decreased levels thereof with respect to a reference value is indicative of a good outcome of the patient.

In a second aspect, the invention relates to a method of prognosing or classifying a subject with lung cancer positive for an activating EGFR mutation and treated with chemotherapy, with an EGFR inhibitor or a combination thereof said method comprising determining in a biofluid of said patient the level of cell-free EGFR gene carrying said EGFR mutation wherein the detection of cell-free EGFR gene containing said mutation or the detection of an increased level thereof with respect to a reference value is is used to prognose or classify the subject with lung cancer into a poor outcome group or wherein an absence of detection of the cell-free EGFR gene containing said mutation or the detection of decreased levels thereof with respect to a reference value is is used to prognose or classify the subject with lung cancer into a good outcome group.

In further aspects, the invention relates to a computer system that is provided with means for implementing the methods according to the invention, to a computer program comprising a programming code to execute the steps of the methods according to invention if carried out in a computer and to a computer-readable data medium comprising a computer program according to the invention in the form of a computer- readable programming code. In further aspects, the invention relates to a method for the further treatment of lung cancer in a subject and to a kit for for determining the outcome of a subject suffering from lung cancer.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Progression free survival in patients treated with chemotherapy according to EGFR mutations in cfDNA.

Figure 2: Progression free survival in patients treated with erlotinib according to EGFR mutations in cfDNA.

Figure 3: Overall survival according to EGFR mutations in cfDNA.

DETAILED DESCRIPTION OF THE INVENTION Method for predicting the survival of a lung cancer patient

The authors of the present invention have observed that, surprisingly, the level of cell-free EGFR carrying activating EGFR mutations in the serum of patients with non- small-cell lung cancer (NSCLC) carrying activating EGFR mutations can be used as a marker for outcome of the patients in response to conventional therapy or to therapy based on the use of inhibitors of the EGFR tyrosine kinase. This result is surprising because the levels of mutant EGFR in tissues does not provide significant differences in the survival of the two groups of patients. This result provides a useful prognostic marker in the clinical context.

Thus, in a first aspect, the invention relates to a method (first method of the invention) for predicting the outcome of a patient suffering lung cancer after treatment with chemotherapy, with an EGFR inhibitor or a combination thereof wherein said lung cancer carries at least an activating EGFR mutation, said method comprising determining in a biofluid of said patient the levels of the cell-free EGFR gene carrying said at least one activating EGFR mutation wherein the detection of cell-free EGFR gene containing said mutation or the detection of an increased level thereof with respect to a reference value is indicative of a poor outcome of the patient or wherein an absence of detection of the cell-free EGFR gene containing said mutation or the detection of decreased levels thereof with respect to a reference value is indicative of a good outcome of the patient.

The term "predicting", as used herein, refers to the determination of the likelihood that the patient will respond either favorably or unfavorably to a given therapy. Especially, the term "prediction", as used herein, relates to an individual assessment of any parameter that can be useful in determining the evolution of a patient. As will be understood by those skilled in the art, the prediction of the clinical response to the treatment with a biological drug , although preferred to be, need not be correct for 100% of the subjects to be diagnosed or evaluated. The term, however, requires that a statistically significant portion of subjects can be identified as having an increased probability of having a positive response. Whether a subject is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test, etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95%. The p-values are, preferably, 0.05, 0.02 or 0.01.

The term "outcome", as used herein, refers to any clinical observation or measurement relating to a patient's reaction to a therapy. Non-limiting examples of clinical outcomes include tumor response (TR), overall survival (OS), progression free survival (PFS), disease free survival, time to tumor recurrence (TTR), time to tumor progression (TTP), relative risk (RR), toxicity or side effect. "Overall Survival" (OS) intends a prolongation in life expectancy as compared to naive or untreated individuals or patients. "Progression free survival" (PFS) or "Time to Tumor Progression" (TTP) indicates the length of time during and after treatment that the cancer does not grow. Progression-free survival includes the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease. "Tumor Recurrence" as used herein and as defined by the National Cancer Institute is cancer that has recurred (come back), usually after a period of time during which the cancer could not be detected. The cancer may come back to the same place as the original (primary) tumor or to another place in the body. It is also called recurrent cancer. "Time to Tumor Recurrence" (TTR) is defined as the time from the date of diagnosis of the cancer to the date of first recurrence, death, or until last contact if the patient was free of any tumor recurrence at the time of last contact. If a patient had not recurred, then TTR was censored at the time of death or at the last follow-up. "Relative Risk" (RR), in statistics and mathematical epidemiology, refers to the risk of an event (or of developing a disease) relative to exposure. Relative risk is a ratio of the probability of the event occurring in the exposed group versus a non- exposed group.

In a preferred embodiment, the outcome of the patient is determined as progression- free survival or as survival.

The term "patient", as used herein, refers to all animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans, e.g., human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the patient is a male or female human of any age or race. In an embodiment the patient that has had lung cancer still is considered to have lung cancer.

The term "lung cancer" is meant to refer to any cancer of the lung and includes non-small cell lung carcinomas and small cell lung carcinomas. In a preferred embodiment, the methods of the invention are applicable to a subject suffering from NSCLC and/or that has suffered NSCLC. In a particular embodiment, the NSCLC is selected from squamous cell carcinoma of the lung, large cell carcinoma of the lung, and adenocarcinoma of the lung. Furthermore, the present method can also be applicable to a subject that has suffered or is suffering from any stage of NSCLC (stages 0, IA, IB, Ila, lib, Ilia, Illb o IV). In a preferred embodiment, the patient has had advanced lung cancer.

The term "lung cancer with an activating EGFR mutation", as used herein, refers to cancer wherein the EGFR gene carries one or more mutations in the tyrosine kinase domain which results in that the tyrosine kinase activity of EGFR is increased with respect to the wild type EGFR. These mutants are also characterised in that they show increased sensitivity to EGFR tyrosine kinase specific inhibitor such as erlotinib. Cancers carrying such a mutation can be identified by determining in a sample of the tumor the presence of the EGFR mutation. Suitable samples include, e.g., tumor biopsies which are excised from the tissue using any suitable method in the art. In particular, samples of a particular cell type, whether normal or diseased, may be micro- dissected using laser-capture micro-dissection ("LCM") techniques, as described in U.S. Pat. Nos. 5,843,657, 6,251,516 Bl, and 6,969,614 Bl, each of which is hereby incorporated by reference in its entirety. LCM allows for isolation of pure populations or subpopulations of the desired cell type, such as a diseased cell population or a normal cell population, or both from the same tissue sample. The cells of interest can be identified, e.g., by morphology, in situ immunohistochemistry, or fluorescent microscopy. By combining microscopy-based cell identification techniques with laser activation of the polymeric substrate to which the tissue sample is applied, very precise extraction of the desired cells is possible.

Methods for determining whether a given mutant is an activating EGFR mutation or confers sensitivity to a tyrosine kinase activity have been described in detail in the prior art and include, among others, a method as described in WO2006091889 based on the detection of the autophosphorylation capacity of EGFR as measured in cells over- expressing the variant EGFR in response to the treatment with a gefmtib (Iressa™) or panitumumab. In particular EGFR mutations, kinase or phosphorylation activity can be increased (e.g., by at least 5 percent, 10 percent, 15 percent, 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, or even 100 percent), as compared to wild type EGFR.

Activating EGFR mutations include one or more deletions, substitutions, or additions in the amino acid or nucleotide sequences of EGFR protein, or EGFR coding sequence. The activating EGFR mutant can also include one or more deletions, substitutions, or additions, or a fragment thereof, as long as the mutant retains or increases tyrosine kinase activity, compared to wild type EGFR. Particular EGFR mutants are described herein, where mutations are provided relative to the position of an amino acid in human EGFR, as described in the sequence provided in NCBI GenBank Reference Sequence: NP 005219.2. Exemplary EGFR mutations include, for example, mutations in exon 18, 19, 20 or 21. EGFR mutants showing an increased sensitivity to tyrosine kinase inhibitors include, without limitation, mutations at positions L858 in exon 21 such as L858R, L858P, L861Q or L861 point mutations in the activation loop (exon 21), in-frame deletion/insertion mutations in the ELREA sequence (exon 19) such as the E746-R748 deletion, the E746-A750 deletion, the E746-R748 deletion together with E749Q and A750P substitutions, del L747-E749 deletion combined with the A750P substitution, the L747S substitution in combination with the R748-P753 deletion, the L747-S752 deletion in combination with the E746V substitution, the L747- T751 deletion combined with an serine insertion, the AI insertion at positions M766- A767, the SVA insertion at positions S768-V769, or substitutions in at position 719 in the nucleotide binding loop (exon 18) such as G719A, G719C, G710S. Further activating mutations include, without limitation, mutations in the kinase domain, G719A, L858R, E746K, L747S, E749Q, A750P, A755V, V765M, S7681, L858P, E746-R748 del, R748-P753 del, M766-A767 Al ins, S768- V769 SVA ins, P772-H773 NS ins, 2402OC, 24820A, 2486T>C, 2491 G>C, 24940C, 251 0OT, 25390A, 25490T, 25630T, 2819T>C, 2482-2490 del, 2486-2503 del, 2544-2545 ins GCCATA, 2554-2555 ins CCAGCGTGG, or 2562-2563 ins AACTCC. Other examples of EGFR activating mutations are known in the art (see e.g., US Patent Publication No. 2005/0272083). In certain embodiments, the cell or cell line does not comprise a T790M mutation in the EGFR gene.

In a preferred embodiment, the patient shows at least a mutation conferring sensitivity to tyrosine kinase inhibitors. In a still more preferred embodiment, the patient shows a first mutation selected from the group of the L858R substitution and the ELREA deletion in exon 19.

The term "chemotherapy" as used herein refer to any treatment that reduces, prevents, mitigates, limits, and/or delays the growth of metastases or neoplasms, or kills neoplastic cells directly by necrosis or apoptosis of neoplasms or any other mechanism, or that can be otherwise used to reduce, prevent, mitigate, limit, and/or delay the growth of metastases or neoplasms in a subject with neoplastic disease. Suitable chemotherapeutic treatments include, without limitation, plant alkaloids, such as vincristine, vinblastine and etoposide; anthracycline antibiotics including doxorubicin, epirubicin, daunorubicin; fluorouracil; antibiotics including bleomycin, mitomycin, plicamycin, dactinomycin; topoisomerase inhibitors, such as camptothecin and its analogues; and platinum compounds, including cisplatin and its analogues, such as carboplatin. Other traditional chemotherapeutic agents suitable for use are known to those of skill in the art and include, asparaginase, busulfan, chlorambucil, cyclophosphamide, cytarabine, dacarbazine, estramustine phosphate sodium, floxuridine, fluorouracil (5-FU), hydroxyurea (hydroxycarbamide), ifosfamide, lornustine (CCNU), mechlorethamine HC1 (nitrogen mustard), melphalan, mercaptopurine, methotrexate (MTX), mitomycin, mitotane, mitsxantrone,, procarbazine, streptozocin,, thioguanine, thiotepa, amsacrine (m-AMSA), azacitidine,, hexamethylmeiamine (HMM),, mitoguazone (methyl-GAG; methyl giyoxal bis- guanyihydrazone; MGBG), semustine (methyl-CCNU), teniposide (VM-26) and vindesine sulfate.

In a preferred embodiment, the chemotherapy is a therapy with a platinum-based compound.

The term "platinum-based compound" (or drug)" as used herein refers to a compound comprising a heavy metal complex containing a central atom of platinum surrounded by organic and/or inorganic functionalities. Non-limiting examples of platinum-based drugs include cisplatin, carboplatin, oxilaplatin, nedaplatin, spiroplatin, iproplatin, satraplatin, pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogs thereof, and combinations thereof.

According to the present invention, an "EGFR inhibitor" is any agent that inhibits (blocks, reduces, antagonizes, decreases, reverses) the expression and/or biological activity of an epidermal growth factor receptor (EGFR), including any EGFR. Therefore, an inhibitor can include, but is not limited to, a product of drug/compound/peptide design or selection, an antibody or antigen binding fragment thereof, a protein, a peptide, a nucleic acid (including ribozymes, antisense, RNAi and aptamers), or any other agent that inhibits the expression and/or biological activity of an EGFR.

The terms "EGFR", "ErbBl" and "epidermal growth factor receptor" and are used interchangeably herein and refer to a tyrosine kinase which regulate signaling pathways and growth and survival of cells and which shows affinity for the EGF molecule. The ErbB family of receptors consists of four closely related subtypes: ErbBl (epidermal growth factor receptor [EGFR]), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4) and variants thereof (e.g. a deletion mutant EGFR as in Humphrey et al. (Proc. Natl. Acad. Sci. USA, 1990, 87:4207-4211). In a preferred embodiment, the EGFR is human. In a preferred embodiment, the EGFR inhibitor is an EGFR tyrosine kinase inhibitor. The type of EGFR tyrosine kinase inhibitor therapy for use according to the method of the present invention is not particularly limiting and may include any of the inhibitors mentioned above. In a preferred embodiment, the EGFR tyrosine kinase inhibitor is a dual EGFR inhibitor, a dual EGFR tyrosine kinase inhibitor or a EGFR tyrosine kinase inhibitor specific for EGFR carrying a resistance mutation.

The term "dual EGFR inhibitor", as used herein, refers to a composition which is capable of simultaneously inhibiting the tyrosine kinase activity of the intracellular domain of EGFR as well as its activation by the binding of the ligand to the extracellular domain. Illustrative and non- limitative example of such an inhibitor is, e.g. the composition comprising cetuximab (C225) as inhibitor of the extracellular domain and erlotinib (E) as inhibitor of the tyrosine kinase activity of the intracellular domain.

The term "dual EGFR tyrosine kinase inhibitor", as used herein, refers to a compound which is capable of simultaneously inhibiting EGFR and HER2 activity. Examples of such compounds include the EGFR and HER2 inhibitor CI- 1033 (formerly known as PD 183805; Pfizer); the EGFR and HER2 inhibitor GW-2016 (also known as GW-572016 or lapatinib ditosylate; GSK); the EGFR and JAK 2/3 inhibitor AG490 (a tyrphostin); the EGFR and HER2 inhibitor ARRY-334543 (Array BioPharma); BIBW- 2992, an irreversible dual EGFR HER2 kinase inhibitor (Boehringer Ingelheim Corp.) In a preferred embodiment, the EGFR inhibitor-based chemotherapy is an inhibitor of the EGFR tyrosine kinase. The expression "EGFR tyrosine kinase inhibitor", as used herein, relates to a chemical substance inhibiting "tyrosine kinase" which transfers a γ-phosphate group of ATP to a hydroxy group of a specific tyrosine in protein catalyzed by the tyrosine kinase domain of the receptor for epidermal growth factor (EGFR). Tyrosine kinase activity is measured by detecting phosphorylation of a protein. EGFR tyrosine kinase inhibitors are known in the art. For example, a tyrosine kinase inhibitor is identified by detecting a decrease the tyrosine mediated transfer phosphate from ATP to protein tyrosine residues.

The tyrosine kinase inhibitor is for example an erbB tyrosine kinase inhibitor. Alternatively the tyrosine kinase inhibitor is an EGFR tyrosine kinase inhibitor. The tyrosine kinase inhibitor is a reversible tyrosine kinase inhibitor. Alternatively the tyrosine kinase inhibitor is an irreversible tyrosine kinase inhibitor. Reversible tyrosine kinase inhibitors include for example, HKI-272, BIBW2992, EKB-569 or CL-387,785 or mimetics or derivatives thereof. Other tyrosine kinase inhibitors include those described in U.S. Pat. Nos. 6,384,051, 6,288,082 and US Application No. 20050059678, each of which is hereby incorporated by reference in their entirety.

EGFR tyrosine kinase inhibitors include, for example quinazoline EGFR kinase inhibitors, pyrido- pyrimidine EGFR kinase inhibitors, pyrimido-pyrimidine EGFR kinase inhibitors, pyrrolo- pyrimidine EGFR kinase inhibitors, pyrazolo -pyrimidine EGFR kinase inhibitors, phenylamino- pyrimidine EGFR kinase inhibitors, oxindole EGFR kinase inhibitors, indolocarbazole EGFR kinase inhibitors, phthalazine EGFR kinase inhibitors, isoflavone EGFR kinase inhibitors, quinalone EGFR kinase inhibitors, and tyrphostin EGFR kinase inhibitors, such as those described in the following patent publications, and all pharmaceutically acceptable salts and solvates of said EGFR kinase inhibitors: International Patent Publication Nos. WO 96/33980, WO 96/30347, WO 97/30034, WO 97/30044, WO 97/38994, WO 97/49688, WO 98/02434, WO 97/38983, WO 95/19774, WO 95/19970, WO 97/13771, WO 98/02437, WO 98/02438, WO 97/32881, WO 98/33798, WO 97/32880, WO 97/3288, WO 97/02266, WO 97/27199, WO 98/07726, WO 97/34895, WO 96/31510, WO 98/14449, WO 98/14450, WO 98/14451, WO 95/09847, WO 97/19065, WO 98/17662, WO 99/35146, WO 99/35132, WO 99/07701, and WO 92/20642; European Patent Application Nos. EP 520722, EP 566226, EP 787772, EP 837063, and EP 682027; U.S. Pat. Nos. 5,747,498, 5,789,427, 5,650,415, and 5,656,643; and German Patent Application No. DE 19629652. Additional non-limiting examples of low molecular weight EGFR kinase inhibitors include any of the EGFR tyrosine kinase inhibitors described in Traxler, P., 1998, Exp. Opin. Ther. Patents 8(12): 1599-1625.

Specific preferred examples of low molecular weight EGFR tyrosine kinase inhibitors that can be used according to the present invention include [6,7-bis(2- methoxyethoxy)-4-quinazolin-4-yl]- (3-ethynylphenyl)amine (also known as OSI-774, erlotinib, or TARCEVA (erlotinib HC1); OSI Pharmaceuticals/Genentech/Roche) (U.S. Pat. No. 5,747,498; International Patent Publication No. WO 01/34574, and Moyer, J. D. et al. (1997) Cancer Res. 57:4838-4848); CI- 1033 (formerly known as PD183805; Pfizer) (Sherwood et al, 1999, Proc. Am. Assoc. Cancer Res. 40:723); PD-158780 (Pfizer); AG-1478 (University of California); CGP-59326 (Novartis); PKI-166 (Novartis); EKB-569 (Wyeth); GW-2016 (also known as GW-572016 or lapatinib ditosylate; GSK); and gefitinib (also known as ZD 1839 or IRESSA.TM.; Astrazeneca) (Woodburn et al, 1997, Proc. Am. Assoc. Cancer Res. 38:633). A particularly preferred low molecular weight EGFR kinase inhibitor that can be used according to the present invention is [6,7-bis(2-methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl) amine (i.e. erlotinib), its hydrochloride salt (i.e. erlotinib HC1, TARCEVA), or other salt forms (e.g. erlotinib mesylate).

EGFR tyrosine kinase inhibitors also include, for example multi-kinase inhibitors that have activity on EGFR kinase, i.e. inhibitors that inhibit EGFR kinase and one or more additional kinases. Examples of such compounds include the EGFR and HER2 inhibitor CI- 1033 (formerly known as PD 183805; Pfizer); the EGFR and HER2 inhibitor GW-2016 (also known as GW- 572016 or lapatinib ditosylate; GSK); the EGFR and JAK 2/3 inhibitor AG490 (a tyrphostin); the EGFR and HER2 inhibitor ARRY-334543 (Array BioPharma); BIBW-2992, an irreversible dual EGFR/HER2 kinase inhibitor (Boehringer Ingelheim Corp.); the EGFR and HER2 inhibitor EKB-569 (Wyeth); the VEGF-R2 and EGFR inhibitor ZD6474 (also known as ZACTIMA ;AstraZeneca Pharmaceuticals), and the EGFR and HER2 inhibitor BMS-599626 (Bristol-Myers Squibb).

In another embodiment, an antisense strategy may be used to interfere with the kinase activity of a variant EGFR. This approach may, for instance, utilize antisense nucleic acids or ribozymes that block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme. For a general discussion of antisense technology, see, e.g., Antisense DNA and RNA, (Cold Spring Harbor Laboratory, D. Melton, ed., 1988).

Reversible short inhibition of variant EGFR gene transcription may also be useful. Such inhibition can be achieved by use of siRNAs. RNA interference (RNAi) technology prevents the expression of gene by using small RNA molecules such as small interfering RNAs (siRNAs). This technology in turn takes advantage of the fact that RNAi is a natural biological mechanism for silencing genes in most cells of many living organisms, from plants to insects to mammals (McManus et al., Nature Reviews Genetics, 2002, 3(10) p. 737). RNAi prevents a gene from producing a functional protein by ensuring that the molecule intermediate, the messenger RNA copy of the gene is destroyed. siRNAs can be used in a naked form and incorporated in a vector, as described below. One can further make use of aptamers to specifically inhibit variant EGFR gene transcription, see, for example, U.S. Patent 6,699,843. Aptamers useful in the present invention may be identified using the SELEX process. The methods of SELEX have been described in, for example, U. S. Patent Nos. 5,707,796, 5,763,177, 6,011,577, 5,580,737, 5,567,588, and 5,660,985.

An "antisense nucleic acid" or "antisense oligonucleotide" is a single stranded nucleic acid molecule, which, on hybridizing under cytoplasmic conditions with complementary bases in a RNA or DNA molecule, inhibits the latter's role. If the RNA is a messenger RNA transcript, the antisense nucleic acid is a counter-transcript or mRNA-interfering complementary nucleic acid. As presently used, "antisense" broadly includes RNA-RNA interactions, RNA- DNA interactions, ribozymes, RNAi, aptamers and Rnase-H mediated arrest.

Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these ribozymes, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1989, Science 245(4915) p. 276). Because they are sequence-specific, only mRNAs with particular sequences are inactivated.

Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (e.g., U.S. patent No 5,814,500; U.S. 5,811,234), or alternatively they can be prepared synthetically (e.g., u.s. patent No 5,780,607).

siRNAs have been described in Brummelkamp et al, Science 296; 550-

553,2002, Jaque et al ., Nature 418; 435-438, 2002, Elbashir S. M. et al. (2001) Nature, 411 : 494-498, McCaffrey et al. (2002), Nature, 418: 38-39; Xia H. et al. (2002), Nat. Biotech. 20: 1006-1010, Novina et al. (2002), Nat. Med. 8: 681-686, and U.S. Application No. 20030198627.

An important advantage of such a therapeutic strategy relative to the use of drugs such as gefitinib or erlotinib, which inhibit both the mutated receptor and the normal receptor, is that siRNA directed specifically against the mutated EGFR should not inhibit the wild-type EGFR. This is significant because it is generally believed that the "side effects" of gefitinib treatment, which include diarrhea and dermatitis, are a consequence of inhibition of EGFR in normal tissues that require its function.

In another embodiment, the compounds are antisense molecules specific for human sequences coding for an EGFR having at least one variance in its kinase domain. The administered therapeutic agent may be an antisense oligonucleotides, particularly synthetic oligonucleotides; having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA. The antisense sequence is complementary to the mRNA of the targeted EGFR genes, and inhibits expression of the targeted gene products (see e.g. Nyce et al. (1997) Nature 385:720). Antisense molecules inhibit gene expression by reducing the amount of mRNA available for translation, through activation of RNAse H or steric hindrance. One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences from a single targeted gene, or sequences that complement several different genes.

A preferred target gene is an EGFR with at least one nucleic acid variance in its kinase domain. Generally, the antisense sequence will have the same species of origin as the animal host.

Antisense molecules may be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the vector is introduced and expressed in the targeted cells. The transcriptional initiation will be oriented such that the antisense strand is produced as an RNA molecule. The anti-sense RNA hybridizes with the endogenous sense strand mRNA, thereby blocking expression of the targeted gene. The native transcriptional initiation region, or an exogenous transcriptional initiation region may be employed.

The promoter may be introduced by recombinant methods in vitro, or as the result of homologous integration of the sequence into a chromosome. Many strong promoters that are active in muscle cells are known in the art, including the β-actin promoter, SV40 early and late promoters, human cytomegalovirus promoter, retroviral LTRs, etc. Transcription vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences. Transcription cassettes maybe prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region. The transcription cassettes may be introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in cells, usually for a period of at least about one day, more usually for a period of at least about several days.

Aptamers are also useful. Aptamers are a promising new class of therapeutic oligonucleotides or peptides and are selected in vitro to specifically bind to a given target with high affinity, such as for example ligand receptors. Their binding characteristics are likely a reflection of the ability of oligonucleotides to form three dimensional structures held together by intramolecular nucleobase pairing. Aptamers are synthetic DNA, RNA or peptide sequences which may be normal and modified (e.g. peptide nucleic acid (PNA), thiophophorylated DNA, etc) that interact with a target protein, ligand (lipid, carbohydrate, metabolite, etc). In a further embodiment, RNA aptamers specific for a variant EGFR can be introduced into or expressed in a cell as a therapeutic.

Peptide nucleic acids (PNAs) are compounds that in certain respects are similar to oligonucleotides and their analogs and thus may mimic DNA and RNA. In PNA, the deoxyribose backbone of oligonucleotides has been replaced by a pseudo-peptide backbone (Nielsen et al. 1991 Science 254, 1457-1500). Each subunit, or monomer, has a naturally occurring or non-naturally occurring nucleobase attached to this backbone. One such backbone is constructed of repeating units of N(2-amino ethyl) glycine linked through amide bonds. PNA hybridises with complementary nucleic acids through Watson and Crick base pairing and helix fold. The Pseudo-peptide backbone provides superior hybridization properties (Egholm et al. Nature (1993) 365, 566-568), resistance to enzymatic degradation (Demidov et al. Biochem. Pharmacol. (1994) 48, 1310-1313) and access to a variety of chemical modifications (Nielsen and Haaima Chemical Society Reviews (1997) 73-78). PNAs specific for a variant EGFR can be introduced into or expressed in a cell as a therapeutic. PNAs have been described, for example, in U.S. Application No. 20040063906.

In a preferred embodiment, the EGFR inhibitor is a specific inhibitor of the tyrosine kinase activity of EGFR. In a still more preferred embodiment, the inhibitor is erlotinib or gefitinib. In a preferred embodiment, the method according to the invention allows predicting the survival of a patient after treatment with a combination of chemotherapy and treatment with an EGFR inhibitor. In a still more preferred embodiment, the combination of a chemotherapy and therapy with a therapy with an EGFR inhibitor comprises the sequential administration of chemotherapy and an EGFR inhibitor or of an EGFR inhibitor and chemotherapy. In one embodiment, the method allows predicting the survival of a patient which is treated first with chemotherapy and then with an EGFR inhibitor. In another embodiment, the method allows predicting the survival in a patient which is treated first with an EGFR inhibitor and then with chemotherapy.

In a first step, the method for predicting the outcome of a patient according to the invention comprises the determination in a biofluid of said patient of the levels of cell- free EGFR gene which contain said activating EGFR mutation.

The terms "cell-free DNA" and "circulating DNA", are used herein interchangeably to refer to free genomic DNA molecules that are not contained within any intact cells and can be obtained from any biofluid and, in particular, serum or plasma. It will be understood that the method of the present invention do not require the detection of the complete EGFR gene as cell-free DNA. Instead, the cell-free DNA is formed by a population of DNA molecules which are fragments of the EGFR gene which have a variable length and which contain the region of the EGFR gene wherein the activating mutation is found. Thus, the cell- free DNA refers to fragments of genomic DNA having at least 10 bp, 20 bp, 30 bp, 40, bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400, bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp, 6000 bp, 7000 bp, 8000 bp, 9000 bp, 10000 bp, 10000 bp, 20000 bp, 30000 bp, 40000 bp, 50000 bp, 60000 bp, 70000 bp, 80000 bp, 90000 bp, 100000 bp or more.

The term "biofluid" as used herein, relates to any fluid sample which can be obtained from the subject. Samples may be collected from a variety of sources from a mammal (e.g., a human), including a body fluid sample, blood, serum, plasma, sputum including saliva, plasma, nipple aspirants, synovial fluids, cerebrospinal fluids, sweat, urine, fecal matter, pancreatic fluid, trabecular fluid, cerebrospinal fluid, tears, bronchial lavage, swabbings, bronchial aspirants, semen, prostatic fluid, precervicular fluid, vaginal fluids, pre-ejaculate, etc. In a preferred embodiment, the biofluid is blood or serum.

In the practice of the invention biofluid such as blood is drawn by standard methods into a collection tube. In the case of blood, said tube preferably comprises siliconized glass, either without anticoagulant for preparation of serum or with EDTA, heparin, or similar anticoagulants, most preferably EDTA, for preparation of plasma. Plasma may optionally be subsequently converted to serum by incubation of the anticoagulated plasma with an equal volume of calcium chloride at 37°C for a brief period, most preferably for 1-3 minutes, until clotting takes place. The clot may then be pelleted by a brief centrifugation and the deproteinized plasma removed to another tube. Alternatively, the centrifugation may be omitted. Serum can also be obtained using clot activator tubes.

The term "level" as used herein in respect of cell-free EGFR refers to a numeric value that measures the amount or concentration of polynucleotides derived from the EGFR gene (fragments thereof) present in the sample. Since the cell- free DNA comprises a population of genomic fragments of variable length, the term "level" is to be understood as the concentration of genome fragments which comprise at least the mutation which is being studied.

The level of cell-free EGFR can be determined by any method suitable for determining the concentration of a specific gene in a sample. Alternatively, cell-free EGFR levels can be determined by measuring the levels of the corresponding mRNA carrying the activating EGFR mutation or by measuring the levels of the corresponding protein carrying the activating EGFR mutation. It can be desirable to confirm mutations in genomic DNA by analysis of transcripts and/or polypeptides, in order to ensure that the detected mutation is indeed expressed in the subject.

The term "nucleic acid" refers to a multimeric compound comprising nucleosides or nucleoside analogues which have nitrogenous heterocyclic bases, or base analogues, which are linked by phosphodiester bonds to form a polynucleotide such as DNA.

The term "DNA" refers to deoxyribonucleic acid. A DNA sequence is a deoxyribonucleic sequence. DNA is a long polymer of nucleotides and encodes the sequence of the amino acid residues in proteins using the genetic code. Mutations in genomic nucleic acid are advantageously detected by techniques based on mobility shift in amplified nucleic acid fragments. For instance, Chen et al. (Anal. Biochem., 1996, 239:61-9), describe the detection of single-base mutations by a competitive mobility shift assay. Moreover, assays based on the technique of Marcelino et al, BioTechniques 26: 1134-1148 (June 1999) are available commercially. In a preferred example, capillary heteroduplex analysis may be used to detect the presence of mutations based on mobility shift of duplex nucleic acids in capillary systems as a result of the presence of mismatches.

Generation of nucleic acids for analysis from samples generally requires nucleic acid amplification. Many amplification methods rely on an enzymatic chain reaction (such as a polymerase chain reaction, a ligase chain reaction, or a self-sustained sequence replication) or from the replication of all or part of the vector into which it has been cloned. Preferably, the amplification according to the invention is an exponential amplification, as exhibited by for example the polymerase chain reaction.

Many target and signal amplification methods have been described in the literature, for example, general reviews of these methods in Landegren, U., et al, Science, 1988, 242:229- 237 and Lewis, R., Genetic Engineering News 10: 1, 54-55 (1990). These amplification methods can be used in the methods of our invention, and include polymerase chain reaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligase hybridisation, Qbeta bacteriophage replicase, transcription-based amplification system (TAS), genomic amplification with transcript sequencing (GAWTS), nucleic acid sequence-based amplification (NASBA) and in situ hybridisation. Primers suitable for use in various amplification techniques can be prepared according to methods known in the art.

Once the nucleic acid has been amplified, a number of techniques are available for detection of single base pair mutations. One such technique is Single Stranded Conformational Polymorphism (SSCP). SCCP detection is based on the aberrant migration of single stranded mutated DNA compared to reference DNA during electrophoresis. Mutation produces conformational change in single stranded DNA, resulting in mobility shift. Fluorescent SCCP uses fluorescent-labelled primers to aid detection. Reference and mutant DNA are thus amplified using fluorescent labelled primers. The amplified DNA is denatured and snap-cooled to produce single stranded DNA molecules, which are examined by non-denaturing gel electrophoresis.

Chemical mismatch cleavage (CMC) is based on the recognition and cleavage of DNA mismatched base pairs by a combination of hydroxylamine, osmium tetroxide and piperidine. Thus, both reference DNA and mutant DNA are amplified with fluorescent labelled primers. The amplicons are hybridised and then subjected to cleavage using Osmium tetroxide, which binds to an mismatched T base, or Hydroxylamine, which binds to mismatched C base, followed by Piperidine which cleaves at the site of a modified base. Cleaved fragments are then detected by electrophoresis.

Techniques based on restriction fragment polymorphisms (RFLPs) can also be used. Although many single nucleotide polymorphisms (SNPs) do not permit conventional RFLP analysis, primer-induced restriction analysis PCR (PIRA-PCR) can be used to introduce restriction sites using PCR primers in a SNP-dependent manner. Primers for PIRA-PCR which introduce suitable restriction sites can be designed by computational analysis, for example as described in Xiaiyi et al. (2001) Bio informatics 17:838-839.

Furthermore, techniques based on WAVE analysis can be used (Methods Mol. Med. 2004; 108: 173-88). This system of DNA fragment analysis can be used to detect single nucleotide polymorphisms and is based on temperature-modulated liquid chromatography and a high-resolution matrix (Genet Test. 1997-98; 1 (3):201-6.)

Real-time PCR (also known as Quantitative PCR, Real-time Quantitative PCR, or RTQ- PCR) is a method of simultaneous DNA quantification and amplification (Expert Rev. Mol. Diagn. 2005(2):209-19). DNA is specifically amplified by polymerase chain reaction. After each round of amplification, the DNA is quantified. Common methods of quantification include the use of fluorescent dyes that intercalate with double-strand DNA and modified DNA oligonucleotides (called probes) that fluoresce when hybridised with a complementary DNA.

In a particular embodiment of the invention, the detecting step of the method of the invention is carried out by means of nucleic acid sequencing. Illustrative, non limitative, examples of nucleic acid sequencing methods are cycle sequencing (Sarkar et al, 1995, Nucleic Acids Res. 23: 1269-70) or direct dideoxynucleotide sequencing, in which some or the entire DNA of interest that has been harvested from the sample is used as a template for sequencing reactions. An oligonucleotide primer or set of primers specific to the gene or DNA of interest is used in standard sequencing reactions. Other methods of DNA sequencing, such as sequencing by hybridization, sequencing using a "chip" containing many oligonucleotides for hybridization (as, for example, those produced by Affymetrix Corp.; Ramsay et al, 1998, Nature Biotechnology 16: 40-44; Marshall et al, 1998, Nature Biotechnology 16: 27-31), sequencing by HPLC (DeDionisio et al, 1996, J Chromatogr A 735: 191-208), and modifications of DNA sequencing strategies such as multiplex allele-specific diagnostic assay (MASDA; Shuber et al, 1997, Hum. Molec. Genet. 6: 337-47), dideoxy fingerprinting (Sarkar et al, 1992, Genomics 13: 441-3;; Martincic et al, 1996, Oncogene 13: 2039-44), and fluorogenic probe-based PCR methods (such as Taqman; Perkin-Elmer Corp.; Heid et al, 1996, Genome Res. 6: 986-94) and cleavase-based methods may be used.

Alternatively, amplification can be carried out using primers that are appropriately labelled, and the amplified primer extension products can be detected using procedures and equipment for detection of the label. Preferably probes of this invention are labeled with at least one detectable moiety, wherein the detectable moiety or moieties are selected from the group consisting of: a conjugate, a branched detection system, a chromophore, a fluorophore, a spin label, a radioisotope, an enzyme, a hapten, an acridinium ester and a luminescent compound. As an illustrative, non limitative, example, in the method of the present invention the primers used can labelled with a fluorophore. More particularly, the reverse primer of the method of the present invention is labelled with the 6-FAM fluorophore at its 5 ' end. This fluorophore emits fluorescence with a peak wavelength of 522 nm. The PCR can be carried out using one of the primers labelled with, for example, either FAM, HEX, VIC or NED dyes.

In a preferred embodiment of the invention, the posterior detection and analysis of the DNA amplified with the method of the invention is carried out by the GeneScan technique as it is illustrated in EP2046985. Thus, as an illustrative, non limitative, example for carrying out the detecting step of the method of the invention, an aliquot of the PCR reaction (typically 1 μΐ) is added to 9 μΐ of formamide HI-DI and 0.25 μΐ of GeneScan marker -500 LIZ size standard. After denaturation, the sample is placed in the ABI 3130 Genetic Analyzer and capillary electrophoresis is carried out. The raw data is analysed using GeneScan software. This analysis is very important since the PCR products will be sized by extrapolation to an in-sample size standard. Using this technique inventors are able to detect in a very precise and accurate manner the mutation of interest.

In a particular embodiment of the invention, the biofluid, preferably serum or plasma, may be utilized directly for identification and quantification of the mutant DNA. In another particular embodiment, nucleic acid is extracted from the biofluid as an initial step of the invention. In such cases, the total DNA extracted from said samples would represent the working material suitable for subsequent amplification.

Once the sample has been obtained, amplification of nucleic acid is carried out. In a particular embodiment, the amplification of the DNA is carried out by means of PCR. The general principles and conditions for amplification and detection of nucleic acids, such as using PCR, are well known for the skilled person in the art. In particular, the Polymerase Chain Reaction (PCR) carried out by the method of the present invention uses appropriate and specific oligonucleotide primers or amplification oligonucleotides to specifically amplify the EGFR target sequences. The terms "oligonucleotide primers" or "amplification oligonucleotides" are herein used indistinguishably and refer to a polymeric nucleic acid having generally less than 1,000 residues, including those in a size range having a lower limit of about 2 to 5 residues and an upper limit of about 500 to 900 residues. In preferred embodiments, oligonucleotide primers are in a size range having a lower limit of about 5 to about 15 residues and an upper limit of about 100 to 200 residues. More preferably, oligonucleotide primers of the present invention are in a size range having a lower limit of about 10 to about 15 residues and an upper limit of about 17 to 100 residues. Although oligonucleotide primers may be purified from naturally occurring nucleic acids, they are generally synthesized using any of a variety of well known enzymatic or chemical methods. In a particular embodiment of the invention, such oligonucleotide primers enable the specific amplification of the DNA fragments corresponding to the deletion of specific nucleotides in the exon 19 at the EGFR gene.

Thus, in a particular embodiment, the method of the invention can be used for the detection of ELREA deletions at the exon 19. In a preferred embodiment, the present invention refers to a method for the detection of 9, 12, 15, 18, or 24 nucleotides deletions in the exon 19 at the EGFR gene. In another particular embodiment, the method of the invention can be used for the detection of the L858R mutation at the exon 21 of the EGFR gene.

The term "amplification oligonucleotide" refers to an oligonucleotide that hybridizes to a target nucleic acid, or its complement, and participates in a nucleic acid amplification reaction. Amplification oligonucleotides include primers and promoter- primers in which the 3' end of the oligonucleotide is extended enzymatically using another nucleic acid strand as the template. In some embodiments, an amplification oligonucleotide contains at least about 10 contiguous bases, and more preferably about 12 contiguous bases, that are complementary to a region of the target sequence (or its complementary strand). Target-binding bases are preferably at least about 80%, and more preferably about 90% to 100% complementary to the sequence to which it binds. An amplification oligonucleotide is preferably about 10 to about 60 bases long and may include modified nucleotides or base analogues.

The terms "amplify" or "amplification" refer to a procedure to produce multiple copies of a target nucleic acid sequence or its complement or fragments thereof (i.e., the amplified product may contain less than the complete target sequence). For example, fragments may be produced by amplifying a portion of the target nucleic acid by using an amplification oligonucleotide which hybridizes to, and initiates polymerization from, an internal position of the target nucleic acid. Known amplification methods include, for example, polymerase chain reaction (PCR) amplification, replicase-mediated amplification, ligase chain reaction (LCR) amplification, strand-displacement amplification (SDA) and transcription-associated or transcription-mediated amplification (TMA). PCR amplification uses DNA polymerase, primers for opposite strands and thermal cycling to synthesize multiple copies of DNA or cDNA. Replicase- mediated amplification uses QB-replicase to amplify RNA sequences. LCR amplification uses at least four different oligonucleotides to amplify complementary strands of a target by using cycles of hybridization, ligation, and denaturation. SDA uses a primer that contains a recognition site for a restriction endonuclease and an endonuclease that nicks one strand of a hemimodified DNA duplex that includes the target sequence, followed by a series of primer extension and strand displacement steps. An isothermal strand-displacement amplification method that does not rely on endonuclease nicking is also known. Transcription-associated or transcription-mediated amplification uses a primer that includes a promoter sequence and an R A polymerase specific for the promoter to produce multiple transcripts from a target sequence, thus amplifying the target sequence.

Preferred embodiments of the present invention amplify the EGFR target sequences using the present amplification oligonucleotides in a polymerase chain reaction (PCR) amplification. One skilled in the art will appreciate that these amplification oligonucleotides can readily be used in other methods of nucleic acid amplification that uses polymerase-mediated primer extension.

Methods for detecting mutations in the tyrosine kinase domain of the EGF receptor are known in the art, several corresponding diagnostic tools are approved by the FDA and commercially available, e.g. an assay for the detection of epidermal growth factor receptor mutations in patients with non-small cell lung cancer (Genzyme Corp.; see also Journal of Clinical Oncology, 2006 ASCO Annual Meeting Proceedings (Post-Meeting Edition). Vol 24, No 18S (June 20 Supplement), 2006: Abstract 10060). In a preferred embodiment, the mutations in EGFR are determined in serum samples as described in WO07039705 based on the use of specific Scorpion probes in combination with the Amplification Refractory Mutation System (ARMS) (Nucleic Acids Res., 1989, 17:2503-2516 and Nature Biotechnology, 1999, 17:804-807).

In a preferred embodiment, the levels of the EGFR gene carrying the activating mutation is measured by a method comprising the steps of

(i) amplifying the nucleic acid sequence corresponding to said specific region of the sensitivity mutation of the EGFR gene by means of PCR using a Peptide-Nucleic Acid probe, wherein said Peptide-Nucleic Acid probe is capable of specifically recognising and hybridising with the EGFR wild type sequence thereby inhibiting its amplification and

(ii) quantifying the level of the nucleic acid sequence of the at least one sensitivity mutation of the EGFR gene.

In a still more preferred embodiment, the Protein-Nucleic Acid probe which is capable of specifically recognising and hybridising with the EGFR wild type sequence thereby inhibiting its amplification has a sequence selected from the group consisting of SEQ ID NO:3 (for detecting ELREA deletions in exon 19) and SEQ ID NO: 10 (for detecting the L858R mutation in exon 21) such as it is described in WO08009740. In the amplifying step, the nucleic acid sequence corresponding to a specific region of the EGFR gene is amplified by means of PCR using a Protein-Nucleic Acid (PNA) probe. PNA probes are nucleic acid analogs in which the sugar phosphate backbone of a natural nucleic acid has been replaced by a synthetic peptide backbone, usually formed from N-(2-aminoethyl)-glycine units, resulting in an achiral and uncharged mimic. This new molecule is chemically stable and resistant to hydro lytic (enzymatic) cleavage and thus not expected to be degraded inside a living cell. Despite all these variations from natural nucleic acids, PNA is still capable of sequence-specific binding to DNA as well as RNA obeying the Watson-Crick hydrogen bonding rules. Its hybrid complexes exhibit extraordinary thermal stability and display unique ionic strength properties. In many applications, PNA probes are preferred to nucleic acid probes because, unlike nucleic acid/nucleic acid duplexes which are destabilized under conditions of low salt, PNA/nucleic acid duplexes are formed and remain stable under conditions of very low salt. Those of ordinary skill in the art of nucleic acid hybridization will recognize that factors commonly used to impose or control stringency of hybridization include formamide concentration (or other chemical denaturant reagent), salt concentration (i.e., ionic strength), hybridization temperature, detergent concentration, pH and the presence or absence of chaotropes. Optimal stringency for a probe/target sequence combination is often found by the well known technique of fixing several of the aforementioned stringency factors and then determining the effect of varying a single stringency factor. The same stringency factors can be modulated to thereby control the stringency of hybridization of a PNA to a nucleic acid, except that the hybridization of a PNA is fairly independent of ionic strength. Optimal stringency for an assay may be experimentally determined by examination of each stringency factor until the desired degree of discrimination is achieved.

PNA oligomers can be prepared following standard solid-phase synthesis protocols for peptides (Merrifield, B. 1986. Solid-phase synthesis. Science 232, 341- 347) using, for example, a (methyl-benzhydryl)amine polystyrene resin as the solid support. PNAs may contain a chimeric architecture, such as a PNA/DNA chimera, where a PNA oligomer is fused to a DNA oligomer.

Clinical samples contain DNA molecules with the wild-type allele in addition to DNA molecules with the mutant allele. So, under normal conditions, it is difficult to detect EGFR mutations (mutant allele) in a large background of wild-type EGFR genes (wild-type allele). In a particular case, the PNA probe utilized by the inventors is capable of specifically recognize and hybridize with the wild-type EGFR sequence. As an illustrative, non limitative example, the PNA probe to be used for carrying out the method of the present invention comprises the PNA probe described as the SEQ ID NO: 3 or SEQ ID NO: 10 in the Example accompanying the present invention. Such probe is added to the PCR reaction mix thus inhibiting amplification of the wild-type allele and favouring amplification of the mutant allele present in the sample, i.e. EGFR mutant, facilitating its posterior detection. Those of ordinary skill in the art will appreciate that a suitable PNA probe do not need to have exactly these probing nucleic acid sequences to be operative but often modified according to the particular assay conditions. For example, shorter PNA probes can be prepared by truncation of the nucleic acid sequence if the stability of the hybrid needs to be modified to thereby lower the Tm and/or adjust for stringency. Similarly, the nucleic acid sequence may be truncated at one end and extended at the other end as long as the discriminating nucleic acid sequence remains within the sequence of the PNA probe. Such variations of the probing nucleic acid sequences within the parameters described herein are considered to be embodiments of this invention.

In a second step, the first method of the invention involves correlating the detection/absence of detection or the levels of the EGFR gene carrying the activating mutation with a prediction of outcome. The correlation may indicate a poor outcome when cell- free EGFR gene containing said mutation is detected or wherein the levels of the EGFR gene carrying the activating mutation are increased levels with respect to a reference value. Alternatively, the correlation may indicate a good survival when cell- free EGFR gene containing said mutation is not detected or wherein the levels of the EGFR gene carrying the activating mutation are decreased levels with respect to a reference value.

The terms "detection" or "absence of detection" as used herein refers to the ability or inability to detect cell-free EGFR polynucleotides carrying the activating mutations using the methods provided herein.

The term "reference value", as used herein, refers to a laboratory value used as a reference for values/data obtained by laboratory examination of patients or samples collected from patients. The reference value or reference level can be an absolute value; a relative value; a value that has an upper and/or lower limit; a range of values; an average value; a median value, a mean value, or a value as compared to a particular control or baseline value. The reference value can be based on a large number of samples, such as from population of subjects of the chronological age matched group, or based on a pool of samples including or excluding the sample to be tested. In a preferred embodiment, the reference value is an absolute value. In another embodiment, the reference value is the level of mutated EGFR DNA in 2 pg/μΐ of diploid heterozygotic mutant genomic DNA or the level of concentration of mutated EGFR DNA in 1 pg/μΐ of haploid mutant genomic DNA. Thus, the skilled person will appreciate that the levels of the EGFR gene carrying the activating mutation in a bio fluid of patient can be considered as being increased if the signal corresponding to the mutated EGFR DNA detected in a given volume of biofluid of the patient is higher than the signal detected in a sample containing 2 pg/μΐ of genomic DNA wherein one of the copies of the EGFR gene contains the mutation under study when both the patient sample and the reference sample are analysed in parallel using the same technique. Alternatively, the levels of the EGFR gene carrying the activating mutation in a biofluid of patient can be considered as being decreased if the levels of mutated EGFR DNA detected in a given volume of biofluid of the patient are lower than the levels detected in a sample containing 2 pg/μΐ of genomic DNA wherein one of the copies of the EGFR gene contains the mutation under study when both the patient sample and the reference sample are analysed in parallel using the same technique. The genomic DNA used to generate the reference value is typically genomic DNA isolated from a tumor tissue carrying the mutation or genomic DNA from a cell line derived from lung cancer carrying said mutation such as, for example, cell lines carrying a deletion in exon 198 (e.g. the PC9 cell line as described by Koizumi et al, (Int. J. Cancer, 2005, 116: 36-44) or the HCC2279, the HCC827 or the H4006 cell lines) as well as cells carrying the L858R mutation (e.g. the H3255) or cells carrying the L858R and the T790M mutation (the H1975 cell line).

In other embodiments, the reference value is the level of mutated EGFR in a pool of samples from patients suffering from lung cancer with activating EGFR mutations. In another embodiment, mutated EGFR levels are determined in a population of subjects suffering lung cancer with activating EGFR mutations and the reference value is determined by the average or mean of the mutated EGFR levels in the population. In another embodiment, the reference value corresponds to the detection limit of the assay used for detecting mutated EGFR in the bio fluid.

In a particular embodiment, an increase in EGFR levels of at least 1.1 -fold, 1.5- fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90- fold, 100-fold or even more compared with the reference value is considered as "increased" expression. In a particular embodiment, a decrease in EGFR levels below the reference value of at least 0.9-fold, 0.75-fold, 0.2-fold, 0.1-fold, 0.05-fold, 0.025- fold, 0.02-fold, 0.01 -fold, 0.005-fold or even less compared with the reference value is considered as "decreased" expression.

In a preferred embodiment, the first method of the invention does not comprise the determination of any other marker, i.e. the level of EGFR gene carrying the activating mutation can be used on its own for predicting the outcome. The term "marker", as used herein, refers to any parameter which provides a correlation with a The term "marker" is considered to include clinical markers (e.g. ECOG performance status) as well as molecular markers. The term "molecular marker", "bio marker" or "biochemical marker" as used herein refers generally to a molecule, including a gene, protein, carbohydrate structure, or glycolipid, the expression of which in or on a mammalian tissue or cell can be detected by standard methods and which is associated quantitatively or qualitatively with the presence of a biological phenomenon (e.g. outcome of a patient suffering lung cancer or lung cancer carrying an activating EGFR mutation).

Method of prognosing or classifying a subject

The observation by the authors of the present invention that the level of mutant EGFR in a bio fluid of samples can be used as a marker for predicting survival in patients suffering EGFR-positive lung cancer provides a method for typing or classifying a patient into a poor survival or a good survival group. Thus, in another aspect, the invention relates to a method (second method of the invention) A method of prognosing or classifying a subject with lung cancer positive for an activating EGFR mutation and treated with chemotherapy, with an EGFR inhibitor or a combination thereof said method comprising determining in a bio fluid of said patient the level of cell-free EGFR gene carrying said EGFR mutation wherein the detection of cell-free EGFR gene containing said mutation or the detection of an increased level thereof with respect to a reference value is is used to prognose or classify the subject with lung cancer into a poor outcome group or wherein an absence of detection of the cell-free EGFR gene containing said mutation or the detection of decreased levels thereof with respect to a reference value is is used to prognose or classify the subject with lung cancer into a good outcome group.

The terms "prognosing", "classifying" and "typing", as used herein, mean categorizing a subject into a clinical outcome group, such as a poor survival group or a good survival group. In some embodiments, a subject is classified or prognosed according to whether the subject's risk score is above or below a control value. For example, prognosing or classifying comprises a method or process of determining whether an individual with NSCLC has a good or poor outcome, or grouping an individual with NSCLC into a good outcome group or a poor outcome group, based on whether the individual's calculated risk score is above or below the control value.

The terms "detection", "absence of detection" and "reference value" have been described in respect to the first method of the invention and are equally applicable to the second method of the invention. In a preferred embodiment, the reference value is an absolute value. In another embodiment, the reference value is the level or concentration of mutated EGFR DNA in 2 pg/μΐ of diploid heterozygotic mutant genomic DNA or the level of concentration of mutated EGFR DNA in 1 pg/μΐ of haploid mutant genomic DNA.

The term "good survival" as used herein refers to an increased chance of survival as compared to patients in the "poor survival" group. For example, the bio markers of the application can prognose or classify patients into a "good survival group". These patients are at a lower risk of death after surgery. In some embodiments, the patient is classified in a good survival group, and the patient does not receive chemotherapy.

The term "poor survival" as used herein refers to an increased risk of death as compared to patients in the "good survival" group. For example, gene signatures of the application can prognose or classify patients into a "poor survival group". These patients are at greater risk of death after surgery. In some embodiments, the patient is classified in a poor survival group, and the patient receives a chemotherapeutic regimen.

In a preferred embodiment, the patient has suffered advanced lung cancer. In a still more preferred embodiment, the lung cancer is Non Small Cell Lung Cancer.

In another preferred embodiment, the second method of the invention is carried out using serum or plasma as a biofluid. In a preferred embodiment, the biofluid sample is obtained prior to the treatment with chemotherapy.

In another embodiment, the second method of the invention is carried out in order to type or prognose a patient which has been treated with a combination of a chemotherapy and a therapy with an EGFR inhibitor. In another preferred embodiment, the combined treatment comprises the sequential administration of chemotherapy and an EGFR inhibitor or of an EGFR inhibitor and a chemotherapy.

Suitable chemotherapy compounds have been defined in the first method of the invention. In a preferred embodiment, the chemotherapy is a therapy with a platinum- based compound. Suitable platinum-based compounds have been mentioned in the context of the first method of the invention. In a preferred embodiment, the platinum- based compound is cisplatin or carboplatin.

Suitable EGFR inhibitors have been defined in the first method of the invention. In a preferred embodiment, the EGFR tyrosine kinase inhibitor is a dual EGFR inhibitor, a dual EGFR tyrosine kinase inhibitor or a EGFR tyrosine kinase inhibitor specific for EGFR carrying a resistance mutation. In a preferred embodiment, the EGFR inhibitor is a EGFR tyrosine kinase inhibitor. In a preferred embodiment, EGFR tyrosine kinase inhibitor is gefitinob or erlotinib.

In a preferred embodiment, the combination of a chemotherapy with a therapy with an EGFR inhibitor comprises the sequential administration of chemotherapy and an EGFR inhibitor or of an EGFR inhibitor and a chemotherapy.

In another preferred embodiment, the activating mutation in the EGFR gene is selected from L858R mutation and an (E)LREA deletion in exon 19 or a combination thereof.

In a preferred embodiment, the levels of EGFR carrying the activating mutation in the biofluid is measured by (i) amplifying the nucleic acid sequence corresponding to a region of the EGFR gene comprising the mutation of the EGFR gene by means of PCR using a Protein-Nucleic Acid probe, wherein said Protein-Nucleic Acid probe is capable of specifically recognising and hybridising with the EGFR wild type sequence thereby inhibiting its amplification and

In a preferred embodiment, the first method of the invention does not comprise the determination of any other marker, i.e. the level of EGFR gene carrying the activating mutation can be used on its own for predicting the outcome of the patients.

Computer systems and programs

In another aspect, the invention relates to a computer system that is provided with means for implementing the first or second method according to the invention. The computer system can include:

(a) at least one memory containing at least one computer program adapted to control the operation of the computer system to implement a method that includes: (i) receiving DNA methylation data e.g., the methylation profile of a CUP and the methylation profile of one or more primary tumors, (ii) determining the degree of identity between the methylation profile of the CUP and the methylation profile of the primary tumors and

(b) at least one processor for executing the computer program.

Another aspect of the present invention relates to a computer program for controlling a computer system to execute the steps according to the first, second or third method of the invention.

The present invention can be implemented on a stand-alone computer or as part of a networked computer system. In a stand-alone computer, all the software and data can reside on local memory devices, for example an optical disk or flash memory device can be used to store the computer software for implementing the invention as well as the data. In alternative embodiments, the software or the data or both can be accessed through a network connection to remote devices. In one networked computer system embodiment, the invention use a client -server environment over a public network, such as the internet or a private network to connect to data and resources stored in remote and/or centrally located locations. In this embodiment, a server including a web server can provide access, either open access, pay as you go or subscription based access to the information provided according to the invention. In a client server environment, a client computer executing a client software or program, such as a web browser, connects to the server over a network. The client software or web browser provides a user interface for a user of the invention to input data and information and receive access to data and information. The client software can be viewed on a local computer display or other output device and can allow the user to input information, such as by using a computer keyboard, mouse or other input device. The server executes one or more computer programs that enable the client software to input data, process data according to the invention and output data to the user, as well as provide access to local and remote computer resources. For example, the user interface can include a graphical user interface comprising an access element, such as a text box, that permits entry of data from the assay, e.g., the DNA methylation data levels or DNA gene expression levels of target genes of a reference pluripotent stem cell population and/or pluripotent stem cell population of interest, as well as a display element that can provide a graphical read out of the results of a comparison with a score card, or data sets transmitted to or made available by a processor following execution of the instructions encoded on a computer- readable medium.

A method for the further treatment of lung cancer In another aspect, the invention relates to A method for the further treatment of lung cancer in a subject in need thereof after treatment with chemotherapy, with an EGFR inhibitor, or a combination thereof, said method comprising;

(i) determining in a biofluid sample of said subject the level of cell-free EGFR gene carrying at least one activating EGFR mutation, and, if said level is higher than a reference value, (ii) administering an increased amount of said chemotherapy and/or said EGFR inhibitor to said subject, or administering an alternative anticancer therapeutic to said subject.

In a preferred embodiment, the reference value is 2 pg of cell-free mutated EGFR gene/μΐ of bio fluid.

In another preferred embodiment, the patient has advanced lung cancer. In yet another embodiment, the lung cancer is Non Small Cell Lung Cancer.

In another embodiment, the bio fluid is serum or plasma.

In another embodiment, the combination of a chemotherapy with a therapy with an EGFR inhibitor comprises the sequential administration of chemotherapy and an EGFR inhibitor or of an EGFR inhibitor and a chemotherapy. In another embodiment, the chemotherapy is a therapy with a platinum-based compound. In yet another embodiment, the platinum-based compound is cisplatin or carboplatin. In another embodiment, the EGFR inhibitor is a EGFR tyrosine kinase inhibitor. In yet another embodiment, the EGFR tyrosine kinase inhibitor is a dual EGFR inhibitor, a dual EGFR tyrosine kinase inhibitor or a EGFR tyrosine kinase inhibitor specific for EGFR carrying a resistance mutation. In yet another embodiment, the tyrosine- kinase inhibitor is erlotinib or gefitinib.

In another embodiment, the bio fluid sample is obtained prior to the treatment of the patient with chemotherapy, with an EGFR inhibitor or the combination thereof.

In another embodiment, the mutation in EGFR conferring sensitivity to an EGFR tyrosine kinase inhibitor is a L858R mutation, an (E)LREA deletion in exon 19 or a combination thereof.

In yet another embodiment, the levels of cell-free EGFR gene in the biofluid of the patient is measured by

(i) amplifying the nucleic acid sequence corresponding to a region of the EGFR gene comprising the mutation of the EGFR gene by means of PCR using a Protein-Nucleic Acid probe, wherein said Protein- Nucleic Acid probe is capable of specifically recognising and hybridising with the EGFR wild type sequence thereby inhibiting its amplification and (ii) quantifying the level of the amplified nucleic acid sequence of the at least one sensitivity mutation of the EGFR gene.

All other terms and expressions used to define the method of further treatment of lung cancer according to the invention have been explained in the previous methods of the invention and are equally applicable to the present method.

Kits of the invention

In another aspect, the invention relates to a kit for determining the outcome of a subject suffering from lung cancer after treatment with chemotherapy, with an EGFR inhibitor, or a combination thereof comprising: (a) means for quantifying in a biofluid sample of said subject the level of cell-free EGFR gene carrying at least one activating EGFR mutation; and (b) means for comparing the level quantified in (a) with a reference level.

In a preferred embodiment, the kit further comprises means for determining a therapy for further treating said lung cancer based on the comparison of the quantified expression level to the reference level.

In another aspect, the invention relates to a kit comprising: a) a reagent for quantifying in a biofluid sample of a subject suffering from lung cancer after treatment with chemotherapy, with an EGFR inhibitor, or a combination thereof, the level of cell- free EGFR gene carrying at least one activating EGFR mutation; and (b) one or more indices that have been predetermined to correlate levels of cell-free EGFR gene carrying at least one activating EGFR mutation in a biofluid sample to the outcome of the lung cancer treatment.

In another embodiment, the patient has advanced lung cancer. In yet another embodiment, the lung cancer is Non Small Cell Lung Cancer.

In another embodiment, the biofluid is serum or plasma.

In another embodiment, the combination of a chemotherapy with a therapy with an EGFR inhibitor comprises the sequential administration of chemotherapy and an EGFR inhibitor or of an EGFR inhibitor and a chemotherapy. In another embodiment, the chemotherapy is a therapy with a platinum-based compound. In yet another embodiment, the platinum-based compound is cisplatin or carboplatin.

In another embodiment, the EGFR inhibitor is a EGFR tyrosine kinase inhibitor. In yet another embodiment, the tyrosine kinase inhibitor is a dual EGFR inhibitor, a dual EGFR tyrosine kinase inhibitor or a EGFR tyrosine kinase inhibitor specific for EGFR carrying a resistance mutation. In a still more preferred embodiment, the kit according to claim 57 wherein EGFR tyrosine-kinase inhibitor is erlotinib or gefitinib.

All other terms and expressions used to define the kit according to the invention have been explained in the methods of the invention and are equally applicable to the present kit.

The invention is described by way of the following examples which are to be construed as merely illustrative and not limitative of the scope of the invention.

EXAMPLES

METHODS: MONITORING EGFR MUTATIONS IN SERUM Blood samples

Blood (15 mL) was collected from patients in three Vacutainer tubes (Becton Dickinson, Plymouth, UK), two for serum and one for plasma. Tubes were centrifuged twice at 2300 rpm for 10 min and the supernatant (serum or plasma) alliquoted. DNA was purified from 0.4 mL of serum or plasma by standard procedures, using the QIAamp DNA Blood Mini Kit (Qiagen), and resuspended in 20 of water. For each patient, DNA extraction and mutation analysis was performed per quadruplicate in two samples of serum and two samples of plasma. DNA from the cell line PC-9 was used as a mutated control for exon 19, and wild-type control for exons 20 and 21. DNA from the HI 975 cell line was used as a wild-type control for exon 19, and mutated control for exons 20 and 21. Nested length analysis of fluorescently labelled PCR products for EGFR deletions in exon 19

For the first PCR, primers were as follows: forward 5'- GTGCATCGCTGGTAACATCC-3 ' (SEQ ID NO: 1) and reverse 5'- TGTGGAGATGAGCAGGGTCT- 3' (SEQ ID NO: 2). Peptide Nucleic Acid (PNA): 5 '-AGATGTTGCTTCTCTTA-3 ' (SEQ ID NO: 3). The first PCR was performed in 25- μΐ volumes adding 2 μΐ of sample, 0.125 μΐ of Ecotaq Polymerase (Ecogen, Barcelona, Spain), 2,5 μΕ of PCR buffer xlO, 0,625 μΕ dNTPs (10 mM), 0,75 μΕ MgC12 (50 mM), 1.25 pmol of each primer (10 μΜ) and 12,5 μΐ_^ of 10 μΜ PNA probe. Amplification was as follows: 25 cycles of 30 seconds at 94°C, 30 seconds at 64°C, and 1 minute at 72°C (exons 19 and 21), or 35 cycles of 30 seconds at 94°C, 30 seconds at 58°C, and 1 minute at 72°C (exon 20).

For the length analysis, amplification was performed with the following primers: forward 5 '-ACTCTGGATCCCAGAAGGTGAG-3 ' (SEQ ID NO:4) and reverse 5 '- FAM-CC ACAC AGCAAAGCAGAAACTC-3 ' (SEQ ID NO: 5). Amplification (35 cycles) was done for 30 seconds at 94°C, 30 seconds at 58°C, and 1 minute at 72°C in 25-μ1 volumes adding 2 μΐ of sample, 0.1 μΐ of Ecotaq Polymerase (Ecogen, Barcelona, Spain), 2,5 μΕ of PCR buffer xlO, 0,625 μΕ dNTPs (10 mM), 1 μΕ MgC12 (50 mM), 1.25 pmol of each primer (10 μΜ) and 7,5 μΐ_^ of 10 mM PNA probe. One microliter of a 1/200 dilution of each PCR product was mixed with 0.5 μΐ of size standard (Applied Biosystems) and denatured in 9 μΐ formamide at 90°C for 5 minutes. Separation was done with a four-color laser-induced fluorescence capillary electrophoresis system (ABI Prism 3130 Genetic Analyzer, Applied Biosystems). The collected data were evaluated with the GeneScan Analysis Software (Applera, Norwalk, CT).

TaqMan assay for EGFR mutation in exons 20 (T790M) and 21 (L858R)

Primers and probes were as follows: exon 21 (forward primer, 5'- AACACCGCAGCATGTCAAGA-3 ' (SEQ ID NO: 6), reverse primer 5 '- TTCTCTTCCGCACCCAGC-3' (SEQ ID NO: 7); probes 5 '-FAM- CAGATTTTGGGCGGGCCAAAC-TAMRA-3 ' (SEQ ID NO: 8); and 5 '-VIC- TCACAGATTTTGGGCTGGCC AAAC-TAMRA-3 ' (SEQ ID NO: 9), PNA: AGTTTGGCCAGCCCA (SEQ ID NO: 10) and exon 20 (forward primer, 5'- AGGCAGCCGAAGGGCA-3 ' (SEQ ID NO: 11), reverse primer 5'- CCTCACCTCCACCGTGCA-3 ' (SEQ ID NO: 12); probes 5' VIC- CTCATCACGCAGCTCATG -MGB-3' (SEQ ID NO: 13); and 5 '-FAM- CTCATCATGCAGCTCATG - MGB-3' (SEQ ID NO: 14), PNA: TCATCACGCAGCTC (SEQ ID NO: 15)). Amplification was performed in 12.5-μ1 volumes using 1 of sample, 6.25 μΐ of Ampli Taq Gold PCR Master Mix (Applied Biosystems), 0.75 μΐ of each primer (10 μΜ), 0.25 μΐ, of probes (10 μΜ) and 0,625 μΐ of PNA (10 μΜ). Samples were submitted to 50 cycles of 15 seconds at 94°C and 1 minute at 60°C in an Applied Biosystems 7000 real-time cycler.

Calculations

For exon 19, a sample is considered positive (mutation detected) if a peak of mutated allele appears at least in one of the aliquots analyzed. The number of aliquots showing a mutated peak is recorded. In addition, another indicator is calculated as follows: area of the mutated peaks (in the four aliquots) / total area of the wt + mutated peaks (also in the four aliquots)

For exon 20, a sample is considered positive (mutation detected) if at least in one of the aliquots analyzed is positive.

RESULTS

EGFR mutations in cfDNA from serum were examined in the EURTAC (European Tarceva^® vs Chemotherapy) trial in European patients with advanced EGFR mutation-positive non-small-cell lung cancer. Median progression- free survival (PFS) in the erlotinib group was 9.7 months, in comparison with 5.2 months in the chemotherapy group (hazard ratio [HR] 0-37, 95% CI 0-25-0-54; p<0-0001). In the EURTAC study, the HR for patients with EGFR mutations detected in serum was 0.25 in favour of erlotinib. In the EURTAC study, the subgroup analysis of EGFR mutations in cfDNA indicated that the presence of EGFR mutations was an independent prognostic marker for PFS (HR 0-43, 95 CI 0-26-0-73; p=0-002). In the erlotinib group, median PFS was 12.6 months when EGFR mutations were not detected in cfDNA and 10.7 months when mutations were present in cfDNA (HR 048, 95% CI 0-22-0-97; p=0-04) (Figure 2). In the chemotherapy group, median PFS was 6 months when EGFR mutations were not found in cfDNA and 4.2 months when they were present in cfDNA (HR 0.48, 95% CI 0.22-0.95; p=0.035) (Figure 1). The presence of EGFR mutations in cfDNA in serum was also associated with shorter survival (HR 0.46, 95% CI 0.25-0-84; p=0.02) (Figure 3). Overall median survival was 26.8 months for patients without EGFR mutations detected in cfDNA and 15.5 months when EGFR mutations were present.

Table 1 : Multivariate analysis of PFS. No significant interaction between treatment and cfDNA EGFR mutation

Table 2: Multivariate analysis of survival. No interaction between treatment arm and cfDNA EGFR mutation.

Claims

1. A method for predicting the outcome of a patient suffering lung cancer after treatment with chemotherapy, with an EGFR inhibitor or a combination thereof wherein said lung cancer carries at least an activating EGFR mutation, said method comprising determining in a bio fluid of said patient the levels of the cell-free EGFR gene carrying said at least one activating EGFR mutation wherein

the detection of cell- free EGFR gene containing said mutation or the detection of an increased level thereof with respect to a reference value is indicative of a poor outcome of the patient

or wherein

an absence of detection of the cell-free EGFR gene containing said mutation or the detection of decreased levels thereof with respect to a reference value is indicative of a good outcome of the patient.

2. The method according to claim 1 wherein the reference value is 2 pg of cell- free mutated EGFR gene/μΐ of bio fluid.

3. The method according to according to claims 1 or 2 wherein the patient has advanced lung cancer.

4. The method according to any of claims 1 to 3 wherein the lung cancer is Non Small Cell Lung Cancer.

5. The method according to any of claims 1 to 3, wherein the bio fluid is serum or plasma.

6. The method according to any of claims 1 to 5 wherein the outcome is determined as the progression-free survival or as survival.

7. The method according to any of claims 1 to 6 wherein the combination of a chemotherapy with a therapy with an EGFR inhibitor comprises the sequential administration of chemotherapy and an EGFR inhibitor or of an EGFR inhibitor and a chemotherapy.

8. The method as defined in any of claims 1 to 7 wherein the chemotherapy is a therapy with a platinum-based compound.

9. The method as defined in claim 8 wherein the platinum-based compound is cisplatin or carboplatin.

10. The method according to any of claims 1 to 7 wherein the EGFR inhibitor is a EGFR tyrosine kinase inhibitor.

11. The method according to claim 10 wherein the EGFR tyrosine kinase inhibitor is a dual EGFR inhibitor, a dual EGFR tyrosine kinase inhibitor or a EGFR tyrosine kinase inhibitor specific for EGFR carrying a resistance mutation.

12. The method according to claim 11 wherein EGFR tyrosine-kinase inhibitor is erlotinib or gefitinib.

13. The method according to any of claims 1 to 12 wherein the biofluid sample is obtained prior to the treatment of the patient with chemotherapy, with an EGFR inhibitor or the combination thereof.

14. The method according to any of claims 1 to 13 wherein the mutation in EGFR conferring sensitivity to an EGFR tyrosine kinase inhibitor is a L858R mutation, an (E)LREA deletion in exon 19 or a combination thereof. 15. The method according to any of claims 1 to 14 wherein the levels of cell- free EGFR gene in the biofluid of the patient is measured by

(i) amplifying the nucleic acid sequence corresponding to a region of the EGFR gene comprising the mutation of the EGFR gene by means of

PCR using a Protein-Nucleic Acid probe, wherein said Protein- Nucleic Acid probe is capable of specifically recognising and hybridising with the EGFR wild type sequence thereby inhibiting its amplification and

quantifying the level of the amplified nucleic acid sequence of the at least one sensitivity mutation of the EGFR gene.

A method of prognosing or classifying a subject with lung cancer positive for an activating EGFR mutation and treated with chemotherapy, with an EGFR inhibitor or a combination thereof said method comprising determining in a biofluid of said patient the level of cell-free EGFR gene carrying said EGFR mutation

wherein

the detection of cell- free EGFR gene containing said mutation or the detection of an increased level thereof with respect to a reference value is is used to prognose or classify the subject with lung cancer into a poor outcome group or wherein

an absence of detection of the cell-free EGFR gene containing said mutation or the detection of decreased levels thereof with respect to a reference value is is used to prognose or classify the subject with lung cancer into a good outcome group.

17. The method according to claim 16 wherein the reference value is 2 pg of cell free mutated EGFR/μΙ of biofluid.

18. The method according to claims 16 or 17 wherein the patient has advanced lung cancer.

19. The method according to any of claims 16 to 18 wherein the lung cancer is Non Small Cell Lung Cancer.

20. The method according to any of claims 16 to 19, wherein the bio fluid is serum or plasma.

21. The method according to any of claims 16 to 20 wherein the combination of chemotherapy with a therapy with an EGFR inhibitor comprises the sequential administration of chemotherapy and an EGFR inhibitor or of an EGFR inhibitor and chemotherapy.

22. The method according to any of claims 16 to 21 wherein the chemotherapy is a therapy with a platinum-based compound.

23. The method according to claim 22 wherein the platinum-based compound is cisplatin or carboplatin.

24. The method according to any of claims 16 to 23 wherein the EGFR inhibitor is a EGFR tyrosine kinase inhibitor.

25. The method according to claim 24 wherein the EGFR tyrosine kinase inhibitor is a dual EGFR inhibitor, a dual EGFR tyrosine kinase inhibitor or a EGFR tyrosine kinase inhibitor specific for EGFR carrying a resistance mutation.

26. The method according to claim 25 wherein EGFR tyrosine-kinase inhibitor is erlotinib or gefitinib.

27. The method according to any of claims 16 to 26 wherein the biofluid sample is obtained prior to the treatment with chemotherapy.

28. The method according to any of claims 16 to 27 wherein the mutation in EGFR conferring sensitivity to an EGFR tyrosine kinase inhibitor is a L858R mutation, an (E)LREA deletion in exon 19 or a combination thereof.

29. The method according to any of the claims 16 to 28 wherein the level of cell- free EGFR gene carrying at least one sensitivity mutation of the EGFR in the bio fluid of the patient is measured by

30. A computer system that is provided with means for implementing the methods according to any of claims 1 to 29.

31. A computer program comprising a programming code to execute the steps of the methods according to any of claims 1 to 29 if carried out in a computer.

32. A computer-readable data medium comprising a computer program according to claim 31 in the form of a computer-readable programming code.

33. A method for the further treatment of lung cancer in a subject in need thereof after treatment with chemotherapy, with an EGFR inhibitor, or a combination thereof, said method comprising;

(i) determining in a biofluid sample of said subject the level of cell-free

EGFR gene carrying at least one activating EGFR mutation, and, if said level is higher than a reference value,

(ii) administering an increased amount of said chemotherapy and/or said EGFR inhibitor to said subject, or administering an alternative anti- cancer therapeutic to said subject.

34. The method according to claim 33 wherein the reference value is 2 pg of cell- free mutated EGFR gene/μΐ of biofluid.

35. The method according to according to claims 33 or 34 wherein the patient has advanced lung cancer.

36. The method according to any of claims 33 to 35 wherein the lung cancer is Non Small Cell Lung Cancer.

37. The method according to any of claims 33 to 36, wherein the bio fluid is serum or plasma.

38. The method according to any of claims 33 to 37 wherein the combination of a chemotherapy with a therapy with an EGFR inhibitor comprises the sequential administration of chemotherapy and an EGFR inhibitor or of an EGFR inhibitor and a chemotherapy.

39. The method as defined in any of claims 33 to 38 wherein the chemotherapy is a therapy with a platinum-based compound.

40. The method as defined in claim 39 wherein the platinum-based compound is cisplatin or carboplatin.

41. The method according to any of claims 33 to 40 wherein the EGFR inhibitor is a EGFR tyrosine kinase inhibitor.

42. The method according to claim 41 wherein the EGFR tyrosine kinase inhibitor is a dual EGFR inhibitor, a dual EGFR tyrosine kinase inhibitor or a EGFR tyrosine kinase inhibitor specific for EGFR carrying a resistance mutation.

43. The method according to claim 42 wherein EGFR tyrosine-kinase inhibitor is erlotinib or gefitinib.

The method according to any of claims 33 to 43 wherein the biofluid sample is obtained prior to the treatment of the patient with chemotherapy, with an EGFR inhibitor or the combination thereof.

The method according to any of claims 33 to 44 wherein the mutation in EGFR conferring sensitivity to an EGFR tyrosine kinase inhibitor is a L858R mutation, an (E)LREA deletion in exon 19 or a combination thereof.

46. The method according to any of claims 33 to 45 wherein the levels of cell-free EGFR gene in the biofluid of the patient is measured by

(i) amplifying the nucleic acid sequence corresponding to a region of the EGFR gene comprising the mutation of the EGFR gene by means of PCR using a Protein-Nucleic Acid probe, wherein said Protein- Nucleic Acid probe is capable of specifically recognising and hybridising with the EGFR wild type sequence thereby inhibiting its amplification and

(ii) quantifying the level of the amplified nucleic acid sequence of the at least one sensitivity mutation of the EGFR gene.

A kit for determining the outcome of a subject suffering from lung cancer after treatment with chemotherapy, with an EGFR inhibitor, or a combination thereof comprising: (a) means for quantifying in a biofluid sample of said subject the level of cell-free EGFR gene carrying at least one activating EGFR mutation; and (b) means for comparing the level quantified in (a) with a reference level.

48. The kit according to claim 47 wherein the kit further comprises means for determining a therapy for further treating said lung cancer based on the comparison of the quantified expression level to the reference level.

49. A kit comprising: a) a reagent for quantifying in a biofluid sample of a subject suffering from lung cancer after treatment with chemotherapy, with an EGFR inhibitor, or a combination thereof, the level of cell-free EGFR gene carrying at least one activating EGFR mutation; and (b) one or more indices that have been predetermined to correlate levels of cell- free EGFR gene carrying at least one activating EGFR mutation in a biofluid sample to the outcome of the lung cancer treatment.

50. The kit according to according to any of claims 47 to 49 wherein the patient has advanced lung cancer.

51. The kit according to any of claims 47 to 50 wherein the lung cancer is Non Small Cell Lung Cancer.

52. The kit according to any of claims 47 to 51, wherein the biofluid is serum or plasma.

53. The kit according to any of claims 47 to 52 wherein the combination of a chemotherapy with a therapy with an EGFR inhibitor comprises the sequential administration of chemotherapy and an EGFR inhibitor or of an EGFR inhibitor and a chemotherapy.

54. The kit as defined in any of claims 47 to 53 wherein the chemotherapy is a therapy with a platinum-based compound.

55. The kit as defined in claim 54 wherein the platinum-based compound is cisplatin or carboplatin.

56. The kit according to any of claims 47 to 55 wherein the EGFR inhibitor is a EGFR tyrosine kinase inhibitor.

57. The kit according to claim 56 wherein the EGFR tyrosine kinase inhibitor is a dual EGFR inhibitor, a dual EGFR tyrosine kinase inhibitor or a EGFR tyrosine kinase inhibitor specific for EGFR carrying a resistance mutation. The kit according to claim 57 wherein EGFR tyrosine-kinase inhibitor is erlotinib or gefitinib.

The kit according to any of claims 47 to 58 wherein the mutation in EGFR conferring sensitivity to an EGFR tyrosine kinase inhibitor is a L858R mutation, an (E)LREA deletion in exon 19 or a combination thereof.