MUTATIONS IN THE EXTRACELLULAR DOMAIN III OF EPIDERMAL GROWTH FACTOR RECEPTOR GENE

Abstract

The invention relates to new identified mutations in the epidermal growth factor receptor gene, leading to amino acidic changes which highly correlate with the resistance to a therapy regimen comprising cetuximab. The invention includes peptide sequences and primers to detect the mutations, as well as kits for predicting the response of a subject to a therapy regime comprising cetuximab. In particular, the invention is useful in the therapy regimen applicable to metastasic colorectal cancer.

Claims

1. A peptide sequence with a length from 17 to 100 amino acids and comprising the sequence SEQ ID NO: 13 X.sup.1SLKEISDGDVIIX.sup.4X.sup.5NX.sup.2, wherein X.sup.1 is selected from R and C; X.sup.4 is selected from S and L; X.sup.5 is selected from G and R; X.sup.2 is selected from K and T; and wherein at least one of X.sup.1, X.sup.4, X.sup.5 and X.sup.2 is, respectively, C, L, R or T.

2. The peptide sequence according to claim 1, with a length from 17 to 100 amino acids and comprising the sequence SEQ ID NO: 1 X.sup.1SLKEISDGDVIISGNX.sup.2, wherein: X.sup.1 is selected from R and C; X.sup.2 is selected from K and T; and wherein if X.sup.1 is C, then X.sup.2 is selected independently from K and T, and if X.sup.1 is R, then X.sup.2 is T.

3. The peptide according to claim 1, said peptide further comprising SEQ ID NO: 4 NLCYANTINWKKLFGTSGGKTKIIX.sup.3, wherein X.sup.3 is selected from S and R.

4. The peptide sequence according to claim 1, which comprises sequence SEQ ID NO: 5 X.sup.1SLKEISDGDVIISGNX.sup.2NLCYANTINWKKLFGTSGGKTKIIX.sup.3, wherein X.sup.1, X.sup.2 and X.sup.3 have the same meaning as in claims 1 and 2; and wherein if X.sup.1 is C, then X.sup.2 is selected independently from K and T, and if X.sup.1 is R, then X.sup.2 is T.

5. The peptide sequence according to claim 1, which comprises sequence SEQ ID NO: 14 X.sup.1SLKEISDGDVIIX.sup.4X.sup.5NX.sup.2NLCYANTINWKKLFGTSGGKTKIIX.sup.3, wherein X.sup.1, X.sup.2 and X.sup.3 , X.sup.4, and X.sup.5 have the same meaning as in claims 1, 2 and 3; and wherein at least one of X.sup.1, X.sup.4, X.sup.5 and X.sup.2 is, respectively, C, L, R or T.

6. An oligonucleotide comprising a sequence coding for SEQ ID NO: 1 or for SEQ ID NO: 13.

7. The oligonucleotide according to claim 6, further coding for SEQ ID NO: 4.

8. The oligonucleotide according to claim 6, comprising a sequence coding for SEQ ID NO: 5 or for SEQ ID NO: 14.

9. A set of primers consisting of SEQ ID Nos SEQ ID NOs: 6 (CAAAGTTTTCAGGGATACATTGTTTTT) and 7 (TTAAATGGGAATAGCCCTTCAATATT).

10. A kit which comprises a set of primers as defined in claim 9.

11. The kit according to claim 10, further comprising reagents for detecting mutations in the KRAS and/or PIK3CA, and/or BRAF genes, and/or additional mutations in EGFR gene.

12. An in vitro method of determining a therapy regimen for a subject, comprising: (a) use of a kit as defined in claim 10, wherein: (i) detection of a mutation in at least one gene selected from KRAS, PIK3CA, BRAF, and EGFR using the kit indicates that a therapy regimen comprising anti-EGFR monoclonal antibodies is not suitable; and (ii) absence of a mutation in KRAS, PIK3CA, BRAF, and EGFR using the kit indicates that a therapy regimen comprising anti-EGFR monoclonal antibodies is suitable; and (b) providing a suitable therapy regimen to the subject.

13. The method according to claim 12, wherein the therapy regimen comprising anti-EGFR monoclonal antibodies comprises cetuximab and/or panitumumab.

14. An in vitro method of determining the suitability of a therapy regimen comprising cetuximab and/or panitumumab for a subject, wherein the method comprises: (i) determining in a sample taken from the subject if mutations are present or absent in a fragment defined by SEQ ID NO: 12; and (ii) correlating the presence of any mutation identified in step (i) with resistance of the subject to the therapy regimen comprising cetuximab, or correlating the absence of mutations in step (i) with response of the subject to therapy regimen comprising panitumumab, and (iii) providing a suitable therapy regimen to the subject; wherein the determining step of step (i) is carried out using genotype methods and/or protein sequencing methods.

15. The in vitro method according to claim 14, wherein the sample in step (i) indicates that at least one of the following mutations are present or absent in SEQ ID NO: 12: (a) a change of an arginine to a cysteine at corresponding position 451 of the SEQ ID NO:2; (b) a change of a serine to a leucine at corresponding position 464 of the SEQ ID NO: 2; (c) a change of a glycine to an arginine at the corresponding position 465 of the SEQ ID NO: 2; and (d) a change of a lysine to a threonine at the corresponding position 467 of the SEQ ID NO: 2.

16. The in vitro method according to claim 14, wherein step (i) is performed with a set of primers as defined in claim 9.

17. The in vitro method according to claim 14, wherein step (i) further comprises determining the presence or absence of an arginine at position 492 of the amino acid sequence corresponding to SEQ ID NO: 2, and wherein in step (ii) the additional presence of the arginine identified in step (i) is correlated with resistance of the subject to the therapy regimen comprising cetuximab.

18. An in vitro method of identifying, in a sample taken from a subject, the presence or absence of a cysteine at position 451 of the amino acid sequence corresponding to SEQ ID NO: 2; and/or the presence or absence of a leucine at position 464 of the amino acid sequence corresponding to SEQ ID NO: 2; and/or the presence or absence of an arginine at position 465 of the amino acid sequence corresponding to SEQ ID NO: 2; and/or the presence or absence of a threonine at position 467 of the amino acid sequence corresponding to SEQ ID NO: 2, the method comprising determining the sequence of SEQ ID NO: 2, at least from position 450 to position 470,

19. The kit of claim 10, further comprising an oligonucleotide as defined in claim 6.

20. The in vitro method according to claim 14, wherein step (i) is performed with the kit of claim 10.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0074] FIG. 1 is the plot of two displays of sequencing results obtained by conventional Sanger sequencing (plot A) and Next Generation Sequencing (NGS) in a 454 GS Junior platform (Roche Applied Science, Mannheim, Germany) (plot B). It shows the acquisition of mutations in the EGFR ectodomain following treatment with cetuximab in two samples. (A) In patient #31, the post-treatment tumor sample had acquired an A.fwdarw.C substitution at nucleotide 1400 of EGFR gene that was not present in a pre-treatment biopsy, causing a substitution of a lysine to a threonine at amino acid 467 (K467T). (B) In patient #35, a C.fwdarw.T substitution at nucleotide 1351 of the EGFR gene was detected in the post-treatment sample, leading to a substitution of an arginine to a cysteine at amino acid 451 (R451C).

[0075] FIG. 2 is a Flow cytometry binding analysis of trypsinized NIH3T3 overexpressing wild-type EGFR (wt EGFR) and K467T EGFR mutant incubated with cetuximab (FIG. 2A) or panitumumab (FIG. 2B) as primary antibodies and using a secondary antibody conjugated with phycoerythrin directed against human IgG. C means counts; FL2H denotes the maximal signal intensity in the second channel of fluorescence detection with a band pass of 585±21 that is used to detect the phycoerythrin (PE) fluorescence; E means empty.

[0076] FIG. 3 is also a Flow cytometry binding analysis of trypsinized NIH3T3 overexpressing wild-type EGFR (wt EGFR) and S464L EGFR mutant incubated with cetuximab (FIG. 3A) or panitumumab (FIG. 3B) as primary antibodies and using a secondary antibody conjugated with phycoerythrin directed against human IgG. C means counts; FL2H denotes the maximal signal intensity in the second channel of fluorescence detection with a band pass of 585±21 that is used to detect the phycoerythrin (PE) fluorescence; E means empty.

[0077] FIG. 4 shows a Flow cytometry binding analysis of trypsinized NIH3T3 overexpressing wild-type EGFR (wt EGFR) and G465R EGFR mutant incubated with cetuximab (FIG. 4A) or panitumumab (FIG. 4B) as primary antibodies and using a secondary antibody conjugated with phycoerythrin directed against human IgG. C means counts; FL2H denotes the maximal signal intensity in the second channel of fluorescence detection with a band pass of 585±21 that is used to detect the phycoerythrin (PE) fluorescence; E means empty.

[0078] FIG. 5 shows a Flow cytometry binding analysis of trypsinized NIH3T3 overexpressing wild-type EGFR (wt EGFR) and R451C EGFR mutant incubated with cetuximab (FIG. 5A) or panitumumab (FIG. 5B) as primary antibodies and using a secondary antibody conjugated with phycoerythrin directed against human IgG. C means counts; FL2H denotes the maximal signal intensity in the second channel of fluorescence detection with a band pass of 585±21 that is used to detect the phycoerythrin (PE) fluorescence; E means empty.

DETAILED DESCRIPTION OF THE INVENTION

[0079] In general, the following words or phrases have the indicated definition when used in the description, examples and claims.

[0080] The term “therapy regimen” as used in the state of the art and also herein refers to any therapy intended to prevent, slow, arrest or reverse the growth of a precancerous lesion, cancer or a cancer metastasis. It includes chemotherapy, radiation therapy, immunotherapy, monoclonal antibody therapy or other methods.

[0081] By “response” is to be understood any kind of improvement either clinical or non-clinical selected from, but not limited to, measurable reduction in tumour size or evidence of disease or disease progression, stable disease, increase or elongation of progression of free survival or reduction in toxicity.

[0082] “Progression free survival” 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 partial response, as well as the amount of time patients have experienced stable disease.

[0083] “A complete response” to a therapy defines patients with valuable but non-measurable disease, whose tumour and all evidence of disease disappeared.

[0084] “A partial response” to a therapy defines patients with anything less than complete response.

[0085] “An anti-EGFR monoclonal antibody (anti-EGFR moAb)” relates to a monoclonal antibody and to a fragment thereof that are able to recognize epitopes in the EGFR sequence protein. Approved moAb which recognize different epitopes of EGFR are cetuximab and panitumumab, but other moAb could be used in the therapy regimen for facing cancer disclosed in the present invention. Suitable antibody fragments include F(ab), F(ab′), Fv and nanobodies, among others.

[0086] The expression “genotype methods” includes all those methodologies and processes suitable for determining the genotype or, which is the same for identifying the nucleotide in a given position. Examples of said methodologies encompass Sanger sequencing, pyrosequencing, allele-specific PCR, denaturing high pressure liquid chromatography (DHPLC), Allele Specific Primer Extension (ASPE), DNA biochips/microarrays and dynamic allele-specific hybridization (DASH).

[0087] For “protein sequencing methods” is to be understood any technique allowing to determine the amino acid sequence of a protein, as well as which conformation the protein adopts and the extent to which it is complexed with any non-peptide molecules. The determination of amino acid composition may be performed by hydrolysis or separation of the amino acids. Known technologies include the Sanger sequencing, Edman degradation and mass spectrometry.

[0088] If not indicated to the contrary, all sequences relating EGFR gene, mRNA variant and EGFR protein relate to the human one with the database accession numbers listed along the description. Also if not indicated to the contrary, oligonucleotide sequences are shown in the 5′-3′ direction, and peptide sequences are shown starting from the N-terminus amino acid ((also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) of the peptide, according to the convention for writing peptide sequences.

[0089] All the amino acid sequences, as well as of oligonucleotides may be synthesized following appropriate peptide or oligonucleotide chemical synthesis. Examples of peptide synthesis include solid-phase synthesis and liquid-phase synthesis, both processes coupling the carboxyl group or C-terminus of one amino acid to the amino group or N-terminus of another. Unintended reactions are avoided in solid-phase synthesis using protecting groups, such as 9-fluorenylmethyloxycarbonyl (Fmoc) and Tert-butyloxycarbonyl (t-Boc). Alternatively, the peptides may be obtained by DNA recombinant technologies. Oligonucleotides may be obtained by solid-phase synthesis using phosphoramidite method and phosphoramidite building blocks derived from protected 2′-deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, and U), or chemically modified nucleosides. Oligonucleotides may also be derived from DNA digestion with appropriate restriction enzymes.

[0090] As already explained above, prior art teachings show that mutations at domain III of EGFR may help to map critical points for the interaction of moAbs (cetuximab and/or panitumumab). Nonetheless, these data may serve to detect specific epitopes but they are not concluding in terms of resistance to treatment, since only specific amino acid exchanges encompass this information (that of resistance, either primary or secondary resistance). Particularly, acquired resistance to treatment is of great importance in order to modify the therapeutically approaches and avoid wasting time and efforts.

[0091] The present invention is based on novel mutations in the coding region of the EGFR gene. The novel mutations of the present invention are useful to predict the response to moAb-based therapy of a patient with mCRC. In particular, they are useful to predict primary resistance and the appearance of a secondary resistance.

[0092] As already indicated above, each of the disclosed nucleotide changes lead to the substitution to a cysteine at position 451 of SEQ ID NO: 2 (human EGFR protein), to a leucine at position 464 of SEQ ID NO: 2, to an arginine at position 465 of SEQ ID NO: 2, and to a threonine at position 467 of SEQ ID NO: 2. All these particular mutations are located in a fragment from amino acid 450 to amino acid 470 of this SEQ ID NO: 2, said fragment hereweith named SEQ ID NO: 12.

[0093] The peptide according to the invention, with a length from 17 to 100 amino acids and comprising the sequence SEQ ID NO: 1 includes any of the mutations R451C or K467T.

[0094] In a particular embodiment, this peptide is selected from the group consisting of: a sequence comprising SEQ ID NO: 1 wherein X.sup.1 is R and X.sup.2 is T; a sequence comprising SEQ ID NO: 1 wherein X.sup.1 is C and X.sup.2 is T; and a sequence comprising SEQ ID NO: 1 wherein X.sup.1 is C and X.sup.2 is K.

[0095] In a more particular embodiment, the peptide consists in SEQ ID NO: 1 and, more particularly that SEQ ID NO: 1 selected from the group consisting of SEQ ID NO: 1 wherein X.sup.1 is R and X.sup.2 is T; SEQ ID NO: 1 wherein X.sup.1 is C and X.sup.2 is T; and SEQ ID NO: 1 wherein X.sup.1 is C and X.sup.2 is K. These sequences are represented by SEQ ID NO: 8 (RSLKEISDGDVIISGNT), SEQ ID NO: 9 (CSLKEISDGDVIISGNT) and SEQ ID NO: 10 (CSLKEISDGDVIISGNK).

[0096] In a particular embodiment, the peptide with a length from 17 to 100 amino acids and comprising the sequence SEQ ID NO: 1, further comprises SEQ ID NO: 4

[0097] NLCYANTINWKKLFGTSGGKTKIIX.sup.3, wherein

[0098] X.sup.3 is selected from S and R.

[0099] The peptide comprising both SEQ ID NO: 1 and SEQ ID NO: 4 corresponds, in a particular embodiment, to a continuous amino acid sequence starting with SEQ ID NO: 1. This sequence has 42 amino acids and corresponds to a fragment of EGFR protein coded partially by exon 12 of the EGFR gene. It is represented by, or consists in SEQ ID NO: 5 (X.sup.1SLKEISDGDVIISGNX.sup.2NLCYANTINWKKLFGTSGGKTKIIX.sup.3)

[0100] In another particular embodiment, the peptide with a length from 17 to 100 amino acids and comprising the sequence SEQ ID NO: 13, further comprises SEQ ID NO: 4. This peptide comprising both SEQ ID NO: 13 and SEQ ID NO: 4 corresponds, in a particular embodiment, to a continuous amino acid sequence starting with SEQ ID NO: 13. It has 42 amino acids and corresponds to a fragment of EGFR protein coded partially by exon 12 of the EGFR gene. It is represented by, or consists in SEQ ID NO: 14 (X.sup.1SLKEISDGDVIIX.sup.4X.sup.5NX.sup.2NLCYANTINWKKLFGTSGGKTKIIX.sup.3)

[0101] Indeed, this SEQ ID NO: 5 includes any or all of the mutations R451C and K467T, and further it encompasses the option of including mutation S492R. Thus, X.sup.1, X.sup.2 and X.sup.3 have the same meaning as indicated above; and if X.sup.1 is C, then X.sup.2 is selected independently from K and T, and if X.sup.1 is R, then X.sup.2 is T.

[0102] Mutation S492R was firstly disclosed by the inventors in Montagut et al., (supra) as a key mutation for determining also resistance to moAb in cancers, including metastasic colorectal cancer.

[0103] Besides, SEQ ID NO: 14 includes any or all of the mutations R451C, S464L, G465R and K467T, and further it encompasses the option of including mutation S492R. Thus, X.sup.1, X.sup.2, X.sup.3, X.sup.4 and X.sup.5 have the same meaning as indicated above, but at least one of X.sup.1, X.sup.2, X.sup.4 or X.sup.5 is, respectively, C, L, R or T.

[0104] In another particular embodiment, the peptide sequence comprising SEQ ID NO: 1 or SEQ ID NO: 13 has a length from 17 to 50 amino acids. In another particular embodiment it has a length from 17 to 25 amino acids (that is 17, 18, 19, 20, 21, 22, 23, 24 or 25). In another most particular embodiment the peptide sequence has a length of 17 amino acids. In another particular embodiment it has a length of 21 amino acids, being any of SEQ ID NO: 1 or SEQ ID NO: 13 flanked in the N-terminal end by a leucine (L) and in the C-terminal end by the tripeptide N-Asparagine-Leucine-Cysteine-C (abbreviated NLC)

[0105] In addition, and as will be depicted in the examples below, the inventors also detected a new mutation leading to resistance to moAb in cancers, including metastasic colorectal cancer, namely a change of an isoleucine by a methionine at position 491 of SEQ ID NO: 2 (human EGFR protein). This mutation is herewith named I491M. The amino acid change I491M is the result of the nucleotide change A.fwdarw.G at nucleotide 1473 (also known herein as A1473G) of the mRNA variant 1 of the EGFR gene (Codon ATA is changed to ATG)

[0106] New mutations identified in the present invention are alternatives, but may also be used in combination to assure a proper therapy selection.

[0107] The invention encompasses oligonucleotides coding for SEQ ID NO: 1 or SEQ ID NO: 13. In the particular embodiment of an oligonucleotide coding for SEQ ID NO: 1 or SEQ ID NO: 13, optionally in combination with any embodiment above or below, said oligonucleotide further codes for SEQ ID NO: 4 and thus in another particular embodiment the oligonucleotide codes for SEQ ID NO: 5 or for SEQ ID NO: 14. Particular oligonucleotides are those consisting in nucleotide sequences coding for any of sequences SEQ ID NO: 8 to 10. These oligonucleotides, as above exposed, may be used as hybridization probes for detecting the mutations.

[0108] The kit according to the invention comprises, besides the set of primers disclosed above, oligonucleotide probes for detecting wild-type or mutated forms of EGFR gene coding for any of the mutations R451C and K467T. Examples of these probes for detecting mutated forms of EGFR gene, consists in oligonucleotides selected from those coding for any of SEQ ID NO: 1, 5, 8, 9 and 10.

[0109] The probes consisting in the oligonucleotides selected from those coding for any of SEQ ID NO: 1, 5, 8, 9 and 10 are nucleotide sequences comprising the several options of codon degeneracy in the corresponding mutation points.

[0110] Particular probes for the detection of the mutation R451C are those complementary to the mutated region of the EGFR, wherein the nucleotide changes resulting in the mutation R451C of the present invention is located, being either the coding or complementary region of the gene. Thus, they hybridize with a fragment of the nucleotide sequence carrying the mutation, and allows detecting the nucleotide change C.fwdarw.T at position 1351 disclosed above.

[0111] Particular probes for the detection of the mutation K467T are those complementary to the mutated region of the EGFR wherein the nucleotide changes resulting in the mutation K467T of the present invention is located, being either the coding or complementary region of the gene. Thus, it hybridizes with a fragment of the nucleotide sequence carrying the mutation, and allows detecting the nucleotide change A.fwdarw.C at position 1400 disclosed above.

[0112] Other particular oligonucleotide probes in the kit are for detecting wild-type or mutated forms of EGFR gene coding for any of the mutations S464L and G465R.

[0113] Particular probes for the detection of the mutation S464L are those complementary to the mutated region of the EGFR, wherein the nucleotide changes resulting in the mutation S464L of the present invention is located, being either the coding or complementary region of the gene. Thus, they hybridize with a fragment of the nucleotide sequence carrying the mutation, and allows detecting the nucleotide change C.fwdarw.T at position 1391 disclosed above.

[0114] Other particular probes for the detection of the mutation G465R are those complementary to the mutated region of the EGFR, wherein the nucleotide changes resulting in the mutation G465R of the present invention is located, being either the coding or complementary region of the gene. Thus, they hybridize with a fragment of the nucleotide sequence carrying the mutation, and allows detecting the nucleotide change G.fwdarw.A at position 1393 disclosed above.

[0115] For “nucleotide sequence carrying the mutation” is to be understood any of the coding or complementary DNA chains in the DNA genomic structure, as well as an mRNA chain which is going to be translated.

[0116] The kits of the invention may, optionally in combination with any embodiment above or below, further comprise additional reagents for detecting mutations in the KRAS and/or PIK3CA, and/or BRAF genes, and/or additional mutations in EGFR gene. These reagents include specific primers for detecting particular mutations in all these genes, and particularly mutations associated with resistance to a therapy regimen comprising cetuximab and/or panitumumab. Other reagents included in the kit relate to oligonucleotide probes that can hybridize either with the wild-type or mutated forms of all these genes.

[0117] Thus, in a particular embodiment, optionally in combination with any of the embodiments above or below, the kit comprises tools and means (reagents) to detect the mutations in KRAS selected from the group consisting of G12A; G12C; G12D; G12R; G12S; G12V; G13A; G13C, G13D; G13V as defined by Karapetis et al., “K-ras Mutations and Benefit from Cetuximab in Advanced Colorectal Cancer”, The New England Journal of Medicine—2008, Vol. 359, pp.: 1757-1765. All these mutations are placed on codons 12 and 13 of the protein sequence of K-ras identified with the GenBank accession number NP_004976.2 from Jul. 24, 2011 (named GTPase KRas isoform b precursor) and NP_203524.1 from Jul. 24, 2011 (named GTPase KRas isoform a precursor. In another preferred embodiment the kit comprises reagents to detect mutations in exons 9 and 20 of the PIK3CA gene that codifies for the PIK3CA protein with the GenBank accession number NP_006209.2 from Jul. 17, 2011; and/or the V600E mutation placed on codon 600 of the protein sequence of BRAF identified with the GenBank accession number NP_004324.2 from Jul. 24, 2011. In another preferred embodiment the kit comprises means (reagents) to detect mutation S492R in EGFR protein of SEQ ID NO: 2. In another preferred embodiment the kit comprises means (reagents) to detect mutation I491M in EGFR protein of SEQ ID NO: 2.

[0118] The kits of the invention are in particular for use in the prediction of the response of a subject to a therapy regimen comprising anti-EGFR monoclonal antibodies, in particular cetuximab and/or panitumumab. More particularly, the subject is affected with cancer, and the cancer is metastatic colorectal cancer.

[0119] The invention relates according to one aspect of the invention to an in vitro method of predicting the response of a subject therapy regimen comprising cetuximab and/or panitumumab, wherein the method comprises:

[0120] (i) determining in a sample taken from the subject and by means selected from the group consisting of genotype methods, and/or protein sequencing methods if mutations are present or absent in a fragment defined by SEQ ID NO: 12, which is a fragment from amino acid 450 to amino acid 470 of the consensus wild-type amino acid sequence of human EGFR of SEQ ID NO: 2;

[0121] and ii) correlating the presence of any mutation identified in step i) with resistance of the subject to the therapy regimen comprising cetuximab, or correlating the absence of mutations in step i) with response of the subject to therapy regimen comprising panitumumab.

[0122] In a particular embodiment of the in vitro method in step (i) there are determined within the SEQ ID NO: 12 if at least one of the following mutations are present or absent: a change of an arginine by a cysteine at corresponding position 451 of the SEQ ID NO:2; a serine by a leucine at corresponding position 464 of the SEQ ID NO: 2; a glycine by an arginine at the corresponding position 465 of the SEQ ID NO: 2; and of a lysine by a threonine at the corresponding position 467 of the SEQ ID NO: 2.

[0123] In another particular embodiment, the in vitro method of predicting the response of a subject therapy regimen comprising cetuximab and/or panitumumab comprises:

[0124] i) determining by means selected from the group consisting of genotype methods, and/or protein sequencing methods the presence or absence of at least one of the following amino acids:

[0125] a cysteine at position 451 of the amino acid sequence corresponding to SEQ ID NO: 2, and a threonine at position 467 of the amino acid sequence corresponding to SEQ ID NO: 2, in a sample taken from the subject; and ii) correlating the presence of any of the amino acids identified in step i) with resistance of the subject to the therapy regimen comprising cetuximab, or correlating the absence of all of these amino acids in step i) with response of the subject to therapy regimen comprising panitumumab.

[0126] This particular embodiment encompasses determining if SEQ ID NO: 1 is present in the sample of the subject, and further correlating in step ii) the presence of any of the mutations R451C and/or K467T with resistance of the subject to the therapy regimen comprising cetuximab, or correlating the absence of all of these amino acids in step i) with response of the subject to therapy regimen comprising panitumumab.

[0127] In a more particular embodiment of the method, optionally in combination with any embodiments above or below step i) encompasses determining if additionally SEQ ID NO: 4 is present in the sample of the subject. Thus, after determining if any of the mutations R451C and/or S464L and/or G465R and/or K467T is present, the method includes also determining if mutation S492R is present in EGFR protein.

[0128] Detection of mutation S492R relates to the particular embodiment of the in vitro method, optionally in combination with any of the embodiments below or above, wherein step i) further comprises determining the presence or absence of an arginine at position 492 of the amino acid sequence corresponding to SEQ ID NO: 2, and wherein in step ii) the additional presence of the arginine identified in step i) is correlated with resistance of the subject to the therapy regimen comprising cetuximab.

[0129] In another particular embodiment, optionally in combination with any embodiments above or below, the in vitro method of predicting the response of a subject therapy regimen comprising cetuximab and/or panitumumab, further comprises determining in step (i) the presence or absence of a methionine at position 491 of the amino acid sequence corresponding to SEQ ID NO: 2, and wherein in step ii) the additional presence of the methionine identified in step i) is correlated with resistance of the subject to the therapy regimen comprising cetuximab.

[0130] In another particular embodiment, optionally in combination with any of the embodiments above or below, step i) is performed with a set of primers consisting of SEQ ID NOs: 6 and 7.

[0131] Further to the amplification with the above mentioned primers, in a particular embodiment step i) is performed by genotype methods. In another most particular embodiment, optionally in combination with any embodiment above or below, said genotype method is selected from Sanger sequencing, pyrosequencing, droplet digital PCR (ddPCR), allele-specific PCR, denaturing high pressure liquid chromatography (DHPLC), Allele Specific Primer Extension (ASPE), DNA biochips/microarrays and dynamic allele-specific hybridization (DASH). In even a most particular embodiment, the genotype method is pyrosequencing.

[0132] Examples of pyrosequencing genotype methods include, among others, the next generation sequencing (NGS) methods known as 454 High though output pyrosequencing, sequencing by synthesis (Illumina), and Chain termination sequencing (Sanger sequencing).

[0133] Alternatively, step i) includes specific probes to detect wild-type or mutated points as genotyping method of the amplified regions. Particular probes are those oligonucleotides coding for SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. All these oligonucleotides are complementary to nucleotide sequences of mutated EGFR gene.

[0134] The in vitro method for predicting the response of a subject to a therapy regimen comprising cetuximab and/or panitumumab, is carried out in a sample comprising the tumour, in which the nucleotide changes in the EGFR gene of the present invention can be detected. In cases of mCRC, the sample can be used directly as obtained from the source or following a pre-treatment of the sample. The sample may additionally comprise normal tissue adjacent to said tumour. Accordingly, in case of mCRC the sample is selected from a primary colorectal cancer biopsy or a biopsy of a metastasis thereof. In other words, the sample may be a biopsy from colorectal cancer samples, including primary tumors and metastases. In a preferred embodiment, the metastasis is in the liver tissue.

[0135] Patients comprising any of the new identified mutations are likely to show response to a therapy regimen not comprising cetuximab as measured by any suitable clinical or sub-clinical increase or elongation in progression free survival.

[0136] In a preferred embodiment the therapy regimen is cetuximab alone or in combination with a chemotherapy regimen based on irinotecan, oxaliplatin and/or 5-fluorouracil (5-FU or 5FU). In a preferred embodiment the therapy regimen is panitumumab alone or in combination with a chemotherapy regimen based on irinotecan, oxaliplatin and/or 5-fluorouracil.

[0137] The invention further provides also methods for deciding and/or recommending a therapy regimen for subjects affected with cancer, preferably mCRC, comprising: i) determining by means selected from the group consisting of genotype methods, and/or protein sequencing methods the presence or absence of at least one of the following amino acids: a cysteine at position 451 of the amino acid sequence corresponding to SEQ ID NO: 2, and a threonine at position 467 of the amino acid sequence corresponding to SEQ ID NO: 2, in a sample taken from the subject; and ii) recommending the administration to said subject of an effective amount of cetuximab, or a composition thereof, if all the mutations are absent, or panitumumab, or a composition thereof, if at least one of the mutations is present.

[0138] The invention encompasses also an in vitro method for determining the acquired resistance to a therapy regimen comprising cetuximab, the method comprising, in a particular embodiment of this aspect:

[0139] i) determining by means selected from the group consisting of genotype methods, and/or protein sequencing methods, the presence or absence of at least one of the following amino acids:

[0140] a cysteine at position 451 of the amino acid sequence corresponding to SEQ ID NO: 2, and a threonine at position 467 of the amino acid sequence corresponding to SEQ ID NO: 2, in a sample taken from the subject; and

[0141] ii) correlating the presence of any of the amino acids identified in step i) with acquired resistance of the subject to the therapy regimen comprising cetuximab, or correlating the absence of all of these amino acids in step i) with response of the subject to therapy regimen comprising panitumumab.

[0142] As above exposed, the invention provides also an in vitro method of identifying, in a sample taken from a subject, the presence or absence of a cysteine at position 451 of the amino acid sequence corresponding to SEQ ID NO: 2; and/or the presence or absence of a threonine at position 467 of the amino acid sequence corresponding to SEQ ID NO: 2 by means selected from the group consisting of genotype methods, and/or protein sequencing methods. In a preferred embodiment the in vitro method of identifying the presence or absence of one or both of these mutations in SEQ ID NO: 2, further comprises identifying the presence or absence of an arginine at position 492 of SEQ ID NO: 2 by means selected from the group consisting of genotype methods, and/or protein sequencing methods.

[0143] In a particular embodiment, optionally in combination with any embodiment above or below, the method of identifying the presence or absence of a cysteine at position 451 of the amino acid sequence corresponding to SEQ ID NO: 2; and/or the presence or absence of a leucine at position 464 of the amino acid sequence corresponding to SEQ ID NO: 2; and/or the presence or absence of an arginine at position 465 of the amino acid sequence corresponding to SEQ ID NO: 2; and/or the presence or absence of a threonine at position 467 of the amino acid sequence corresponding to SEQ ID NO: 2, is carried out by determining the sequence of SEQ ID NO: 2 up to position 467 by means selected from the group consisting of genotype methods, and/or protein sequencing methods. In a preferred embodiment, the method is carried out by determining the sequence of SEQ ID NO: 2 from position 450 to 470 (SEQ ID NO: 12), and more preferably from 451 to position 467. For “determining a sequence up to a position” is to be understood that the sequencing is performed from oligonucleotide or amino acid from position 1 of said sequence to the position (nucleotide or amino acid) of interest (in this particular case, to amino acid 467 or to the nucleotide leading to this amino acid).

[0144] Throughout the description and claims the word “comprise” and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

EXAMPLES

Example 1

Tumor Samples and Patients

[0145] There was performed a proof-of-concept approach to study and characterize the presence of heterogeneous mutations emerging after cetuximab-based therapy in routine clinical practice. All mCRC consenting patients treated with anti-EGFR moAb at Parc de Salut Mar Biobank (MARBiobanc, Barcelona, Spain) Hospital del Mar institution between January 2010 and June 2013 were included in this study. In 34 patients the specimens were prospectively collected for this study and in 3 patients there were analyzed sequential biopsies taken in the past in the context of their routine clinical management. In the analysis, there were only included patients that had good quality paired pre- and post-treatment biopsies and that had acquired resistance to anti-EGFR based-therapy defined as progression disease following a) complete response or partial response or b) stable disease for more than 16 weeks (7-9). Response was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST)(Eisenhauer et al., “New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)”, Eur J Cancer 2009, Vol. 45(2):228-247). Tumoral biopsy obtained during the regular diagnosis procedure was used as the pre-treatment (initial) sample. In most cases this sample was obtained from the primary tumor during routine colonoscopy. A second initial biopsy from a metastatic site is not routine and was not performed unless necessary for pathologic diagnosis. The study included re-biopsy following treatment failure in patients that consented to this extra procedure. Re-biopsies at the time of progression were obtained from the most accessible lesion with less potential risk of related complications for the patient according to ethical considerations. Serum samples were collected before starting the cetuximab-based therapy and at the time of progression. When a mutation was detected in the post-treatment biopsy sample, the serum sample from that same patient was analyzed for that specific mutation. In this study, there are included nine cases (patients #21 to #28 and patient #36) that had been previously assessed for EGFR S492R, KRAS exon 2, BRAF V600E and PIK3CA mutations by direct sequencing and that in the current work were analyzed for the mutations reported above (R451C and K467T) using deep-sequencing technology. Biological samples were obtained from Parc de Salut Mar Biobank (MARBiobanc). This study was approved by the local Ethics Board (CEIC-2012/474111). All participating patients signed written informed consent.

[0146] For the KRAS, BRAF, NRAS, PIK3CA and EGFR sequencing, DNA extraction from tumoral samples was performed as previously described by Diaz et al. “The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers”, Nature-2012, Vol No. 486, pp.:537-40. Mutational analysis of KRAS (exons 2, 3 and 4), BRAF (exon15), NRAS (exons 2 and 3), PIK3CA (exons 9 and 20) and EGFR (exon 12, 13) was performed by Sanger sequencing using BigDye v3.1 (Applied Biosystems, Foster City, Calif.) following the manufacturer's instructions and analyzed on a 3500Dx Genetic Analyzer (Applied Biosystems). All cases were also screened by pyrosequencing using a Next Generation Sequencing (NGS) 454 GS Junior platform (Roche Applied Science, Mannheim, Germany). Moreover, processed and quality-filtered reads were analyzed using the GS Amplicon Variant Analyzer software version 2.5p1 (Roche). Mutations detected by NGS were confirmed by competitive allele-specific TaqMan® PCR (CAST-PCR, Applied Biosystems) when specific assays were available.

[0147] Primers for EGFR sequences were those disclosed above and defined by the set of primers consisting in SEQ ID NOs: 6 and 7. The pair of SEQ ID NO: 6 and SEQ ID NO: 7 served for amplifying entirely exon 12 that could contain mutations R451C and K467T, and some intron flanking regions. This sequence is represented by SEQ ID NO: 11:

TABLE-US-00001 caaagttttcagggatacattgtttttatattttcaccacatgattttt cttctctccaatgtagTGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAA CATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGAT GTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACT GGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAA CAGAGGTGAAAACAGCTGCAgtaagtcaccgctttctgtttagtttatg gagttggttctaatgggtcctttatttgtatttagaatattgaagggct attcccatttaa;
wherein underwritten nucleotides correspond to the sequences identical (for SEQ ID NO: 6) or complementary (for SEQ ID NO: 7) to the primers of the set, capital letters relate to exon 12 and non-capital are intron fragments.

[0148] Amplification was performed under the following conditions: 95° C. for 10 minutes; 40 cycles of 95° C., 1 minute, 60° C., 1′30″ and 72° C. 1 minute; and a final extension of 10 minutes at 72° C.

[0149] Besides, it was performed a Fluorescence in situ Hybridization (FISH). FISH was performed whenever there was sufficient remaining material following analysis of mutations. Amplification of EGFR was assessed by fluorescent in situ hybridization (FISH) using the LSI EGFR/CEP7 probe (Abbott Molecular Inc., DesPlaines, Ill.), as previously described (for example in document such as Salido et al., “Increased ALK gene copy number and amplification are frequent in non-small cell lung cancer”, J Thorac Oncol—2011, Vol. No. 6, pp.:21-7). KRAS amplification was analyzed using a dual colour FISH assay with KRAS/CEP12 probe (Abnova). Samples with a ratio KRAS/CEP12 greater than 3, in at least 10% of 50 analysed nuclei were scored. When the average number of chromosome 12 exceeded 2.5 or 4 per cell, the case was considered polysomic or high-polysomic respectively.

Example 2

Presence of R451C and K467T EGFR Mutations and Acquired Resistance to Cetuximab

[0150] As depicted in FIG. 1, which is a plot of two different displays of a Next Generation Sequencing (NGS) 454 GS Junior platform (Roche Applied Science, Mannheim, Germany), shows that some patients acquired mutations in EGFR ectodomain following treatment with cetuximab. FIG. 1(A) shows a patient #31, in which the post-treatment tumor sample had acquired an A.fwdarw.C substitution at nucleotide 1400 of EGFR gene that was not present in the pre-treatment biopsy, causing a substitution of a lysine to a threonine at amino acid 467 (K467T). The substitution is detected by the genotyping method and visualized (arrow) by means of a double pick (band or curve) in this position. Lower pick corresponded to the C nucleotide.

[0151] On the other side, in FIG. 1 (B) it is shown the display of the sequencing process (Read minus the Reference) from patient #35, in which a C.fwdarw.T substitution at nucleotide 1351 of the EGFR gene was detected in the post-treatment sample, leading to a substitution of a arginine to a cysteine at amino acid 451 (R451C). Substitution is also marked with an arrow and in this case the change is visualized by a negative value.

Example 3

Mutations in SEQ ID NO: 12 (Fragment from Amino Acid 450 to Amino Acid 470 of SEQ ID NO: 2) Involve Resistance to Cetuximab Treatment

[0152] 3A: EGFR Ectodomain Mutations and Acquired Resistance to Cetuximab in CRC Cell Models

[0153] It was previously reported that acquisition of resistance in CRC cells is associated with emergence of KRAS, BRAF and NRAS activating mutation. To discover additional mechanisms of resistance to EGFR blockade 5 CRC cell lines were exploited (DiFi, LIM1215, HCA-46, NCIH508, OXCO-2 and CCK81), which are highly sensitive to cetuximab. All these cell lines are wild type for KRAS, NRAS, BRAF and PIK3CA with the exception of NCIH508, which displays the p.E545K PIK3CA mutation. Altogether, these cell models recapitulate the molecular features of tumors from CRC patients likely to respond to anti EGFR therapies. For each line, at least five million cells were exposed continuously to cetuximab until resistant populations emerged. To define molecular mechanisms underlying acquisition of resistance, it was initially performed Sanger sequencing of genes involved in regulation of the EGFR signalling pathway (EGFR, KRAS, BRAF, NRAS, and PIK3CA). In accordance with previous reports, resistant populations often displayed KRAS, BRAF and NRAS mutations (See Misale et al. “Blockade of egfr and mek intercepts heterogeneous mechanisms of acquired resistance to anti-egfr therapies in colorectal cancer”, Sci Transl Med—2014; 6:224ra226). All of these alleles were detected in the resistant cells but not in the corresponding parental population from which they originated. Importantly, in several occasions multiple genetic alterations were concomitantly present in the resistant cell population indicating their polyclonal status. To assess the molecular features of individual clones it was therefore performed limited cell dilutions of LIM1215 and CCK81 as these cell lines are amenable to this procedure. Single clones were then subjected to Sanger sequencing for candidate genes (EGFR, KRAS, BRAF, NRAS, and PIK3CA). Notably, mutation profiling of clones identified three novel EGFR variants: S464L, G465R and I491M. Mutations S464L, G465R, together with mutations of Example 2 (R541C and K467T) are located in SEQ ID NO: 12 (a fragment defining part of the cetuximab binding epitope). Considering that the resistant derivatives are polyclonal, and in light of the limited sensitivity of the Sanger sequencing method, it was postulated that variants present in less than 20% of the cell populations might have remained undetected. To identify mutations present at low frequency it was employed droplet digital PCR (ddPCR) which is known to have a mutant/wild type sensitivity of 1:20000. ddPCR probes were designed and individually validated using control mutant DNA to detect EGFR variants previously identified in tumor biopsy or cell lines. This analysis unveiled the presence 3 new EGFR variants (S464L, G465R, and I491M) that were not detected by Sanger sequencing in resistant cell populations. ddPCR could not be performed in tissue samples because there was no sufficient material left. Overall, the mutational landscape of cell lines with acquired resistance to cetuximab, recapitulate the molecular profiles of tumors that relapsed upon cetuximab treatment.

[0154] ddPCR™ Supermix for Probes (Bio-Rad) using KRAS, NRAS, BRAF and EGFR assay (PrimePCR™ ddPCR™ Mutation Assay, Bio-Rad and custom designed). ddPCR was performed according to manufacturer's protocol and the results reported as percentage or fractional abundance of mutant DNA alleles to total (mutant plus wild type) DNA alleles. 8 to 10 μl of DNA template was added to 10 μl of ddPCR™ Supermix for Probes (Bio-Rad) and 2 μl of the primer/probe mixture. This 20 μl sample was added to 70 μl of Droplet Generation Oil for Probes (Bio-Rad) and used for droplet generation. Droplets were then thermal cycled with the following conditions: 5 minutes at 95° C., 40 cycles of 94° C. for 30 s, 55° C. for 1 minute followed by 98° C. for 10 minutes (Ramp Rate 2° C./sec). Samples were then transferred to a QX200™ Droplet Reader (Bio-Rad) for fluorescent measurement of FAM and HEX probes. Gating was performed based on positive and negative controls, and mutant populations were identified. Fractional Abundances of the mutant DNA in the wild-type DNA background were calculated for each sample using QuantaSoft software (Bio-Rad). Multiple replicates (minimum of four) were performed for each sample. ddPCR analysis of normal control gDNA from cell lines and no DNA template (water) controls were performed in parallel with all the samples, including again multiple replicates as a contamination-free control.

[0155] EGFR probes and primers sequences are available upon request.

[0156] Cell culture and generation of resistant cells utilized herein has already been previously described (see Misale S et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature. 2012; 486:532-6; Misale S, Arena Set al Blockade of EGFR and MEK intercepts heterogeneous mechanisms of acquired resistance to anti-EGFR therapies in colorectal cancer. Sci Transl Med. 2014; 6:224ra26). CCK81 cells were cultured in MEM medium (Invitrogen) supplemented with 5% FBS, 2 mM L-glutamine, antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) and grown in a 37° C. and 5% CO2 air incubator. CCK81 cetuximab-resistant derivatives were obtained by increasing the cetuximab dosage stepwise from 680 nM to 1.4 μM during the course of six months.

[0157] 3B: Presence of S464L, G465R and K467T EGFR Mutation and Resistance to Cetuximab

[0158] To establish whether the S464L, G465R and K467T EGFR mutations of the invention were responsible for the observed resistance to cetuximab, full-length wild-type EGFR and any of the S464L, G465R or K467T EGFR mutations were ectopically expressed in cultured NIH3T3 mouse embryonic fibroblast cell line that lack detectable endogenous EGFR expression.

[0159] EGFR was stimulated with its natural ligand EGF in the presence of cetuximab or panitumumab in transfected cells. Antibody binding was analyzed by flow cytometry using a secondary antibody to human IgG conjugated with phycoerythrin (PE). NIH 3T3 cells expressing the empty vector were used as a negative control (EMPTY). The percentage of cells binding to the antibody are shown in the two-dimensional dot plots of FIGS. 2-4. In this FIGS. 2-4, cell counts (C of “counts”, Y-axis) in the FL2H channel of the fluorescence detection are plotted for the assay with cetuximab (FIGS. 2A, 3A and 4A) and for the assay with panitumumab (FIGS. 2B, 3B and 4B).

[0160] In wild-type EGFR cells (EGFRWT in FIGS. 2-4A/B), both cetuximab and panitumumab inhibited EGFR activation, whereas in cells carrying the K467T mutation (EGFR_K467T in FIG. 2A/B), S464L (EGFR_S464L in FIG. 3A/B) and G465R (EGFR_G465R in FIG. 4A/B) panitumumab, but not cetuximab, effectively blocked EGF-induced EGFR activation. EMPTY is the negative control (EGFR non-expressing cells)

[0161] For the DNA constructs, the pLX301-EGFR WT construct, a generous gift from Dr. C. Sun and Prof R. Bernards (NKI, Amsterdam), was constructed from pLX301 (Addgene®). EGFR mutants containing the 4 point mutations (R451C, S464L, G465R, and K467T) were constructed using the QuikChange® II site-directed mutagenesis kits from Agilent Technologies with pLX301-EGFR WT plasmid as the template DNA. The presence of mutations was confirmed by DNA sequencing.

REFERENCES CITED IN THE APPLICATION

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