GB2488028A - mTOR mutants as biomarkers for mTOR inhibitor treatment - Google Patents

Abstract

A method of selection of a patient, who is a candidate for treatment with an mTOR inhibitor (mammalian target of rapamycin inhibitor), whereby to predict an increased or decreased likelihood of response to an mTOR inhibitor, wherein the amino acid at position 1977 of the mTOR protein is determined as being lysine or an adenine is determined as being present at position 5930 of the mTOR gene. The invention provides a method for determining the sequence of mTOR. The method also provides ARMS primers optimised for determining the sequence of mTOR. The invention also provides a diagnostic kit, comprising an ARMS primer. The use of mTOR inhibitors such as rapamycin (sirolimus) is claimed, particularly in cancer treatments.

Description

MTOR DIAGNOSTIC

The present invention relates to a method of selection of a patient, who is a candidate for treatment with an mTOR inhibitor drug, whereby to predict an increased or decreased likelihood of response to an mTOR inhibitor drug. The invention involves determining the presence or absence of a mutation within the sequence of mTOR. The method provides ARMS primers optimized for determining the sequence of mTOR, particularly at position 5930 in MTOR gene (according to SEQ ID NO: 1). The invention also provides a diagnostic kit, comprising an ARMS primer.

Growth factor/mitogenic activation of the phosphatidylinositol 3-kinase (PI3K)/AKT signalling pathway ultimately leads to the key cell cycle and growth control regulator mTOR, the mammalian target of rapamycin (alternatively referred to as FRAP (FKBP12 and rapamycin associated protein).

The PI3KIAKT signalling cascade is often dysregulated in cancers (Marone et at, Biochimica et Biophysica Acta -Proteins and Proteoinics, Volume 1784(1): 15 9-185, January 2008; LoPiccolo et at, Drug Resist Updat, 11, 32-50 (2008). In addition, a number of genetic disease have now been linked to the PI3KIAKT/mTOR pathway: Cowden syndrome, tuberous sclerosis, Peutz-Jeghers syndrome, and Birt-Hogg-Dubé syndrome are due to mutations or deletions of proteins in the PI3K1AKT/mTOR pathway (PTEN, TSC 1 and 2, LKB1 and folliculin, respectively) (LoPiccolo et at, Drug Resist Updat, 11, 32-50 (2008), Jozwiak Lancet Oncol. 2008 Jan; 9(1):73-9). Some of these genetic diseases usually develop as hamartomas which are very vascularised benign tumours. However, patients with Cowden syndrome, Peutz-Jeghers syndrome, and Birt-Hogg-Dubé syndrome have a significantly increased risk of cancer (breast and endometrial cancer in Cowden patients, gastrointestinal cancers in PJS patients, renal cancer in BHD patients). The PI3K!AKT/mTOR pathway is also implicated in a number of non malignant pathologies such as polycystic kidney disease (Masoumi et al., Drugs 67(17): 2495-5 10, 2007), chronic obstructive pulmanry disease (COPD) (Krymskaya BioD rugs 2007;21(2):85-95) and ocular conditions such as age related macular degeneration (AMD), glaucoma and uveitis.

The MTOR gene maps to human chromosome lp36.2 and encodes a 289 KDa protein.

The gene and protein sequences are available through NCBI Genbank databases with accession numbers NM_004958.3 (gene) and NP_004949.1 (protein). mTOR consists of a catalytic kinase domain, a FKBP12-rapamycin binding (FRB) domain, a putative auto-inhibitory domain (repressor domain') near the C-terminus and up to 20 tandemly repeated HEAT (Huntingtin, EF3, A subunit of PP2A and TOR) motifs at the N-terminus, as well as FRAP-ATM-TRRAP (FAT) and FAT C-terminus domains (Gingras eta!, Genes Dcv., 15, 807-826 (2001). HEAT motifs serve as protein-protein interaction parts, whereas FAT and FAT C-terminus domains participate in modulation of the catalytic kinase activity of mTOR (Guertin et a!, Trends MotMed., 11, 353-361(2005). The P13 K-related kinases (PIKKs), including TOR, Mecl/ATR, Tell/ATM, SMG-l, and DNA-PK have a catalytic domain related to P13K but are atypical Ser/Thr protein kinases.

mTOR (mammalian target of rapamycin) is a growth factor and nutrient-sensitive regulator of cell growth affecting a wide range of cellular functions including translation, transcription, mRINA turnover, protein stability, actin cytoskeleton reorganisation and autophagy (Guertin et a!, Cancer Ce!!, 12, 9-22 (2007). A growth factor such as insulin, after binding to its receptor, activates P13K via the protein IRS-i. P13K converts the substrate PIP2 into PIP3, activating downstream proteins PDK-i and AKT. mTOR is involved in two complexes: (i) mTORC1 containing mTOR, raptor, Gf3L and PRAS4O, is rapamycin-sensitive and activated by AKT and (ii) mTORC2 containing mTOR, rictor, PROTOR, GI3L and Sini is insensitive to rapamycin (Sarbassov eta!, Curr Blot 14:1296-302 (2004)). The three- dimensional (3D) structure of the fully assembled human mTORC1 was described using cryo-electron microscopy (Yip et a!,Mo!.CeH, 38, 768-774 (2010). The analysis reveals that mTORC1 is an obligate dimer with an overall rhomboid shape and a central cavity. The dimeric interfaces are formed by interlocking interactions between the mTOR and raptor subunits. Both the N-terminal and C-terminal of mTOR interact with raptor and PRAS4O is adjacent to raptor towards to N-terminal of mTOR.

mTORC 1-dependent phosphorylation of 56-kinase (p7056K) allows translation of ribosomal proteins via activation of its substrate ribosomal protein S6 (rpS6). mTORC 1 also phosphorylates the translation initiation factor 4E-BP 1 (PHAS-i), preventing its inhibitory binding to eIF4E and allowing the formation of an active eIF4F translation complex (Proud, Biochern.1 403:217-34 (2007)). p7056 kinase negatively regulates mTOR activation by phosphorylating the protein IRS-i which promotes its degradation by the proteosome. The mTORC2 complex directly phosphorylates and activates the upstream kinase AKT on serine 473. It also phosphorylates proteins involved in the cytoskeleton such as paxillin (Sarbassov eta!, Mo! CelL 22; 159-68 (2006), Jacinto and Hall, Nature Rev Mo! Cell Biol, 4, 117-126, (2005)). Finally, there is evidence that mTOR signalling regulates endothelial cell proliferation stimulated by vascular endothelial cell growth factor (VEGF), and partially controls VEGF synthesis through effects on the expression of hypoxia-inducible factor-la (HIF-I a) (Hudson et a!., Mo! Ce!] Biol, 2002, 22, 7004-70 14, Dancey, Exp OpEn Invest Drugs, 2005, 14, 313-328; Seeliger et al, Cancer Metastasis Rev. 2007 Dec;26(3-4):61 1-21).

Two main therapeutic strategies have been developed to antagonise mTOR activity -allosteric inhibitors of mTORC1 complex and direct inhibitors of mTOR kinase activity.

Rapamycin (sirolimus or RapamuneTM) and rapalogues (everolimus (RADOO 1 or CerticanTM), temsirolimus (CCI-779) and deforolimus (AP23 573) are allosteric inhibitors of mTORC1. For example, rapamycin binds to the FK506 binding protein, FKBP12.

Subsequently, the complex of FKBP12/Rapamycin binds to the FRB domain of mTOR within the mTORC1 complex, inhibiting its downstream signalling. Neither rapamycin nor FKBP12 can interact with mTOR independently of each other. In particular, rapamycin does not affect the kinase activity of mTOR but impairs the downstream signalling to p70 S6 Kinase and 4-EBP1. However, the inhibition of 4-EBP 1 is known to be only partial, effecting moderate protein translation. Rapamycin, potently inhibits proliferation or growth of normal cells (smooth muscle cells, T-cells) and tumour cells from rhabdomyosarcoma, neuroblastoma, glioblastoma and medulloblastoma, small cell lung cancer, osteosarcoma, pancreatic carcinoma and breast and prostate carcinoma (Faivre ci' a!, Nat.Rev.Drug Discov., 5, 67 1-688 (2006). Rapamycin has been approved and is in clinical use as an immunosuppressant, its prevention of organ rejection being successful and with fewer side effects than previous therapies (refs. 20, 21). Inhibition of mTOR by rapamycin and its analogues (RADOO1, Ccl- 779) is brought about by the prior interaction of the drug with the FK506 binding protein, FKBP12. Subsequently, the complex of FKBP12/rapamycin then binds to the FRB domain of mTOR and inhibits the downstream signalling from mTOR. Rapamycin has been approved as an immunosuppressant and for use in the prevention of organ rejection (reviewed in Neuhaus, et aL, Liver Transplantation, 7, 473-484 (2001); Woods and Marks, Ann Rev Mcd, 55, 169-178 (2004)). In addition, temsirolimus is approved for the treatment of mantle cell lymphoma, everolimus is approved for the treatment of renal cancer. Both agents along with deforolimus have a number of ongoing clinical trials. Despite their broad preclinical activity, rapamycin and analogues have shown limited clinical activity. Cloughesy (Cloughesy et a!, PLoS Med., 5, e8 (2008) showed that patients with PTEN negative tumours treated with rapamycin progressed on therapy. In particular, patients who had an increase in levels of pPRAS4O, a protein activated by AKT, in their tumours had a shorter time-to-progression on treatment. This demonstrated that feedback activation of AKT by relieving the negative feedback loop exerted by S6K was detrimental to rapamycin activity.

The second therapeutic strategy to inhibit mTOR activity is the direct inhibition of mTOR kinase activity. Tool compounds such as PP242, KU-0063794 and torin 1 (Feldman et al, PLoSBioL, 7, e38 (2009), (Thoreen et ai,.J.BiotChein., 284, 8023-8032 (2009), (Peterson et al, Cell, 137, 873-886 (2009), (Garcia-Martinez cit al,Biochein..J., 421, 29-42 (2009)) first showed that inhibition of mTOR kinase lead to inhibition of both mTORC 1 and mTORC2 complexes. More specifically, inhibition of4-EBP1 was more complete and associated with a greater inhibition of protein translation. In addition, inhibition of mTORC2 decreased SGK1 and AKT activity but also abrogated the feedback activation of AKT induced by the inhibition of S6K. Subsequently, molecules such as AZD8OSS (Chresta cit al, Cancer Res., 70, 288-298 (2010)) recapitulated these early findings and demonstrated a greater and broader antitumour activity compared to rapamycin.

Cancer cells adopt multiple strategies to become resistant to anticancer agents.

Kurmasheva et at. reviewed the main mechanisms of resistance to mTOR allosteric inhibitors (Kurmasheva cit ai,Br.J.Cancer, 95, 955-960 (2006)): -Rapamycin is a substrate for the drug transporter P-glycoprotein and overexpression of the transporter can lead to decreased sensitivity to rapamycin; -As 4-EBP1 is a main effector of mTORC 1, decreased expression of 4-EBP1 allowed cells to maintain cellular translation in presence of rapamycin (over-expression of eIF4E may have similar effects); -Proteins involved in cell cycle such as p27, Rb and p53 did alter the sensitivity to rapamycin; -Mutations in the FRB domain or mTOR or in FKBP12 could disrupt the interaction between FKBP 12 and mTOR and render the cells resistant to rapamycin (Dumont cit al, CelLIininunoL, 163, 70-79 (1995).

Mutations in the mTOR gene have been mostty observed in yeast to understand the mechanism of actions of mTOR inhibitiors (Dumont cit al, CelLimmunoL, 163, 70-79 (1995), (Ohne cit al, J.BiotChein., 283, 31861-31870 (2008). Sturgill et al. (Sturgill cit al, ACS Chem.BioL, 4, 999-1015 (2009) describe a model of mTOR catalytic domain with several residues between 2165 and 2356 expected to interact with ATP. The modeled catalytic region of human TOR is residues 1906 to 2526. Using S. cerevisiae and S. poinbe TOR as a model and aligning its structure to human mTOR, mutations previously reported in Sc TOR and corresponding to amino acids 2017 and 2419 in human TOR showed differential sensitivity to rapamycin or caffeine. However, mutations of the mTOR gene in cancer are a rare event: A survey of the Sanger database indicates that 18 mutations have been identified in the mTOR out of 918 tumours. No mutation was reported in breast cancer or at the 1977 position.

Cancer cells have developed multiple strategies to become resistant to mTOR inhibition but mutations appear to be infrequent suggesting that mTOR is critical for cell growth and survival.

The present invention permits the selection of a patient, who is a candidate for treatment with an mTOR inhibitor drug, in order to predict an increased or decreased likelihood of response to an mTOR inhibitor drug. It also permits monitoring a patient being treated with an mTOR inhibitor drug to determine whether or not over time the tumour acquires the T1977K substitution increasing likelihood of acquiring resistance to treatment with the mTOR inhibitor drug. According to one aspect of the invention there is provided a method of assessing the suitability of an individual for treatment with a compound that inhibits mTOR, the method comprising, a) in a tumour cell containing sample taken from the individual, determining the amino acid at position 1977 in mammalian target of rapamycin (mTOR) protein, or the nucleotide at position 5930 in MTOR gene, and assessing the suitability of an individual for treatment with a compound that inhibits mTOR by virtue of the amino acid or nucleotide present. In one embodiment, the presence of a threonine at position 1977 in the mTOR protein, or a cytosine at position 5930 of the MTOR gene is indicative of the suitability of the individual to treatment with the compound. In another embodiment, the presence of a lysine at position 1977 in the mTOR protein, or an adenine at position 5930 of the MTOR gene is indicative that the individual is unsuitabile for treatment with the compound.According to another aspect of the invention there is provided a method for determining whether or not a tumour cell or cell population is likely to be responsive or resistant to an mTOR inhibitor comprising, determining the amino acid at position 1977 (according to position in SEQ ID NO: 2) in mammalian target of rapamycin (mTOR) protein, or the nucleotide at position 5930 in MTOR gene (according to position in SEQ ID NO: 1) in the tumour cell(s), wherein the presence of a lysine at position 1977 in the mTOR protein, or an adenine at position 5930 of the MTOR gene is indicative that the tumour cell(s) is resistant to the mTOR inhibitor. The nucleotide at position 5930 in MTOR gene (according to position in SEQ ID NO: 1) in the tumour cell(s), need not be determined directly from the tumour cell(s), but could be carried out on a nucleic acid sample prepared from the tumour cell(s), such as an amplified (e.g. PCR amplified) nucleic acid sample.

According to another aspect of the invention there is provided a method for selecting a cancer patient for treatment with an mTOR inhibitor comprising determining the amino acid at position 1977 in mammalian target of rapamycin (mTOR) protein, or the nucleotide at position 5930 in MTOR gene, in a tumour sample obtained from the patient, and selecting the patient for treatment with an mTOR inhibitor if the mTOR protein in the tumour cells possess a threonine at position 1977 and! or nucleic acid encoding the MTOR gene in the tumour cells possess a cytosine at position 5930.

According to another aspect of the invention there is provided a method for selecting a patient for treatment with an mTOR inhibitor, the method comprising (i) providing a sample from a patient containing tumor-derived DNA or tumor cells; (ii) determining the nucleotide at position 5930 of the MTOR gene in the patient's tumour cells or tumour-derived DNA possesses, or the amino acid at position 1977 of the mTOR protein in the patient's tumour cells; and selecting a patient for treatment with an mTOR inhibitor based thereon. In particular, selecting the patient for treatment with an mTOR inhibitor if the mTOR protein in the tumour cells possess a threonine at position 1977 and! or nucleic acid encoding the MTOR gene in the tumour cells or tumor-derived DNA possess a cytosine at position 5930.

According to another aspect of the invention there is provided a method for predicting likelihood of response to treatment with an mTOR inhibitor in an individual diagnosed with cancer comprising determining the presence or absence of a mutation at position 5930 of the MTOR gene, in nucleic acid obtained from a biological sample from said individual, wherein the presence of a mutation indicates that the treatment will more likely to be ineffective in the individual that one without the mutation.

In one embodiment, the method comprises determining the sequence of MTOR gene in a sample obtained from the patient at position 5930 as defined in SEQ ID NO: 1. In one embodiment, the method comprises determining whether the sequence of MTOR gene in a sample obtained from the patient at position 5930, as defined in SEQ ID NO: 1, is cytosine, whereby to predict an increased likelihood of response to the mTOR inhibitor. In one embodiment, the method comprises determining whether the sequence of MTOR gene in a sample obtained from the patient at position 5930 as defined in SEQ ID NO: 1, is adenine. In one embodiment, the method comprises determining whether the sequence of MTOR gene in a sample obtained from the patient at position 5930, as defined in SEQ ID NO:1, is adenine, whereby to predict a decreased likelihood of response to the mTOR inhibitor.

According to another aspect of the invention there is provided a method for predicting the likelihood that a patient who is a candidate for treatment with an mTOR inhibitor will respond to said treatment, comprising determining the sequence of mTOR in a sample obtained from the patient at the following position as defined in SEQ ID NO: 2: position 1977, is threonine. In one embodiment, the method comprises determining whether the sequence of mTOR in a sample obtained from the patient at position 1977, as defined in SEQ ID NO:2, is threonine, whereby to predict an increased likelihood of response to the mTOR inhibitor. In one embodiment, the method comprises determining the sequence of mTOR in a sample obtained from the patient at position 1977 as defined in SEQ ID NO: 2. In one embodiment, the method comprises determining whether the sequence of mTOR in a sample obtained from the patient at position 1977, as defined in SEQ ID NO:2, is not threonine, whereby to predict a decreased likelihood of response to the mTOR inhibitor. In one particular embodiment, the method comprises determining whether the sequence of mTOR in a sample obtained from the patient at position 1977, as defined in SEQ ID NO:2, is lysine, whereby to predict a decreased likelihood of response to the mTOR inhibitor.

The methods of the invention are suitable for selecting patients for treatment with an mTOR inhibitor drug in whomever an mTOR inhibitor drug may work. mTOR inhibitor drugs have been proposed for use in a variety of tumour and non-tumour settings. The methods of the invention are particularly suitable for determining the suitability for treatment with an mTOR inhibitor drug in patients with cancer including, but not limited to, haematologic malignancies such as leukaemia, multiple myeloma, lymphomas such as Hodgkin's disease, non-Hodgkin's lymphomas (including mantle cell lymphoma), myelodysplastic syndromes and also solid tumours and their metastases such as breast cancer, lung cancer (non-small cell lung cancer, small cell lung, squamous cell carcinoma), endometrial cancer, tumours of the central nervous system such as gliomas, dysembryoplastic neuroepithelial tumor, glioblastoma multiforme, mixed gliomas, medulloblastoma, retinoblastoma, neuroblastoma, germinoma and teratoma; cancers of gastrointestinal tract such as gastric cancer, oesophagal cancer, hepatocellular (liver) carcinoma, cholangiocarcinomas, colon and rectal carcinomas, cancers of the small intestine, pancreatic cancers, cancers of the skin such as melanomas (in particular metastatic melanoma), thyroid cancers, cancers of the head and neck and salivary glands, prostate, testis, ovary, cervix, uterine, vulva, bladder, kidney cancers including renal cell carcinoma, clear cell and renal oncocytoma; squamous cell carcinomas, sarcomas such as osteosarcoma, chondrosarcoma, leiomyosarcoma, soft tissue sarcoma, Ewing's sarcoma, gastrointestinal stromal tumour (GIST), Kaposi sarcoma, pediatric cancers such as rhabdomyosarcomas, neuroblastomas.

In one embodiment the present invention is particularly suitable for use in patients with breast cancer, colorectal cancer, head and neck cancer, lung cancer, gastric cancer, prostate, haematological cancers, pancreatic cancer, renal cancer, neuroendocrine tumours, endometrial cancer and ovarian cancer.

The applicant has tested a variety of mTOR inhibitor compounds, with varying structures and mechanisms of action, and have found that the methods of the invention have broad application. As such it is predicted that the methods of the invention can be applied to any mTOR inhibitor. There are numerous scientific articles and patent filings describing novel mTOR inhibitor compounds, some mTOR inhibitor compounds such as sirolimus, temsirolimus and everolimus have received regulatory approval and many others are available from commercial sources. The person skilled in the art would therefore be able to identify an mTOR inhibitor for use in the present invention. Particularly suitable mTOR inhibitors include: rapamycin (sirolimus), azd8055 (compound 1 herein), BEZ-235, tetrandrine, INK- 128, NV-128, OST-027 (4-(4-amino-5-(7-methoxy-1 H-indol-2-yl)imidazo[5, 1-f][1,2,4]triazin- 7-yl)cyclohexanecarboxylic acid hydrochloride), Pp-242, deforolimus (MK8669), everolimus (RADOO 1), GSK-1059615, GSK-2 126458, PKI-5 87(PF-052 12384), GDC-0980, GDC-094 1 (2-(1 H-Indazol-4-yl)-6-[4-(methylsulfonyl)piperazin-1 -ylmethyl]-4-(4- morpholinyl)thieno[3,2-d]pyrimidine), KU-0063 794 (rel-5-[2-[(2R,6S)-2,6-dimethyl-4-morpholinyl]-4-(4-morpholinyl)pyrido[2, 3-d]pyrimidin-7-yl]-2-methoxybcnzenemethanol), Pt-lOS, BGT-226, PF-04691502, temsirolimus (CCI-779), WYE-354, AP23573, 3-[2,4-bis[(3 S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methyl-bcnzamide (see W02008/023 161), XL765(SAR245409), ridaforolimus, 401 (2-(4-Morpholinyl)-4H- pyrimido[2,l -a]isoquinolin-4-one) and niclosamide (5-Chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide).

The sample obtained from the patient may be any tumour tissue or any biological sample that contains material which originated from the tumour, for example a blood sample containing circulating tumour cells or DNA. In one embodiment the blood sample may be whole blood, plasma, serum or pelleted blood. In one embodiment a tumour sample is a tumour tissue sample. The tumour tissue sample may be a fixed or unfixed sample. In another embodiment the biological sample would have been obtained using a minimally invasive technique to obtain a small sample of tumour, or suspected tumour, from which to determine the mTOR sequence. In another embodiment the biological sample comprises either a single sample, which may be tested for any of the mutations as described hereinabove, or multiple samples, which may be tested for any of the mutations as described hereinabove.

In particular embodiments the tumour containing sample is a fresh tissue sample, a frozen tissue sample or a formalin-fixed paraffin-embedded tissue sample. In other embodiments the sample is a biofluid sample selected from: sputum, blood, plasma, serum, ascites fluid, urine, semen, prostatic fluid or cerebral fluid.

Generation of nucleic acids for analysis from samples generally requires nucleic acid amplification. Many amplification methods rely on enzymatic chain elongation (such as a polymerase chain reaction, ligase chain reaction, or a self-sustained sequence replication).

Preferably, the amplification according to the invention is or involves an exponential amplification, such as polymerase chain reaction (PCR).

The particular nucleotide at position 5930 of the MTOR gene can be determined by a variety of methods in the art. Particular methods include: sequencing, allele specific amplification (such as amplification refractory mutation system -ARMS), single strand conformation polymorphisms, polymerase chain reaction, restriction fragment length polymorphism (RFLP), WAVE analysis, assaying for mutant MTOR gene, denaturing gradient gel electrophoresis, high resolution melting or temperature gradient gel electrophoresis.

In one embodiment, the nucleotide at position 5930 in MTOR gene is determined by sequencing. In another embodiment, the nucleotide at position 5930 in MTOR gene is determined using a technique that involves polymerase chain reaction (PCR). In a further embodiment, the polymerase chain reaction uses an allele specific primer that detect the base at position 5930 of MTOR gene, as defined in SEQ ID NO: 1 In one embodiment of the invention there is provided a method as described hereinabove wherein the method for detecting a nucleic acid mutation in MTOR gene and thereby determining the sequence of MTOR gene, is selected from sequencing, WAVE analysis, restriction fragment length polymorphism (RFLP) and amplification reactions, such as amplification refractory mutation system (ARMS). ARMS is described in European Patent Publication No. 0332435, the contents of which are incorporated herein by reference, which discloses and claims a method for the selective amplification of template sequences which differ by as little as one base, which method is now commonly referred to as ARMS. RFLP is described by Zhong (Zhong et al. 2006 Clinica Chiinica Acta: 364, 205-208). In one embodiment of the invention there is provided a method as described hereinabove wherein the method for determining the sequence of MTOR gene in a sample obtained from a patient is the amplification refractory mutation system. In one embodiment ARMS may comprise use of an agarose gel, sequencing gel or real-time PCR. In one embodiment ARMS comprises use of real-time PCR. The ARMS assay may be multiplexed with a second PCR reaction that detects the presence of DNA in the reaction, thereby indicating successful PCR. TaqManTM technology may be used to detect the PCR products of both reactions using TaqManTM probes labeled with different fluorescent tags. The advantages of using ARMS rather than sequencing or RFLP to detect mutations are that ARMS is a quicker single step assay, less processing and data analysis is required, and ARMS can detect a mutation in a sample against a background of wild type polynucleotide.Amplification reactions are nucleic acid reactions which result in specific amplification of target nucleic acids over non-target nucleic acids.

The polymerase chain reaction (PCR) is a well known amplification reaction.

The term probe refers to single stranded sequence-specific oligonucleotides which have a sequence that is capable of hybridising to the target sequence of the allele to be detected.

The term primer refers to a single stranded DNA oligonucleotide sequence or specific primer capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied. The length and sequence of the primer must be such that they are able to prime the synthesis of extension products.

The term nucleic acid includes those polynucleotides capable of hybridising, under stringent hybridisation conditions, to the naturally occurring nucleic acids identified above, or the complement thereof Stringent hybridisation conditions' refers to an overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), Sx Denhardt's solution, 10% dextran sulphate, and 20 pg/mI denatured, sheared salmon sperm DNA, followed by washing the filters in 0.lx SSC at about 65°C.

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 242:229-237 (1988) 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.

Polymerase Chain Reaction (PCR) is a nucleic acid amplification method described inter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR consists of repeated cycles of DNA polymerase generated primer extension reactions. The target DNA is heat denatured and two oligonucleotides, which bracket the target sequence on opposite strands of the DNA to be amplified, are hybridised. These oligonucleotides become primers for use with DNA polymerase. The DNA is copied by primer extension to make a second copy of both strands.

By repeating the cycle of heat denaturation, primer hybridisation and extension, the target DNA can be amplified a million fold or more in about two to four hours. PCR is a molecular biology tool, which must be used in conjunction with a detection technique to determine the results of amplification. An advantage of PCR is that it increases sensitivity by amplifying the amount of target DNA by 1 million to 1 billion fold in approximately 4 hours. PCR can be used to amplify any known nucleic acid in a diagnostic context (Mok et aL, (1994), Gynaecologic Oncology, 52: 247-252).

Self-Sustained Sequence Replication (3SR) is a variation of TAS, which involves the isothermal amplification of a nucleic acid template via sequential rounds of reverse transcriptase (RT), polymerase and nuclease activities that are mediated by an enzyme cocktail and appropriate oligonucleotide primers (Guatelli et al. (1990) Proc. NaiL Acad. ScL USA 87:1874). Enzymatic degradation of the RNA of the RNA!DNA heteroduplex is used instead of heat denaturation. RiNase H and all other enzymes are added to the reaction and all steps occur at the same temperature and without further reagent additions. Following this process, amplifications of 106 to I o9 can been achieved in one hour at 42°C.

Ligation amplification reaction or ligation amplification system (LARILAS) uses DNA ligase and four oligonucleotides, two per target strand. This technique is described by Wu, D. Y. and Wallace, R. B. (1989) Genomics 4:560. The oligonucleotides hybridise to adjacent sequences on the target DNA and are joined by the ligase. The reaction is heat denatured and the cycle repeated.

QI Replicase In this technique, RNA replicase for the bacteriophage QJ3, which replicates single-stranded RNA, is used to amplify the target DNA, as described by Lizardi et al. (1988) Rio/Technology 6:1197. First, the target DNA is hybridised to a primer including a T7 promoter and a Q3 5' sequence region. Using this primer, reverse transcriptase generates a eDNA connecting the primer to its 5' end in the process. These two steps are similar to the TAS protocol. The resulting heteroduplex is heat denatured. Next, a second primer containing a Qf3 3' sequence region is used to initiate a second round of cDNA synthesis. This results in a double stranded DNA containing both 5' and 3' ends of the QI bacteriophage as well as an active T7 RNA polymerase binding site. T7 RNA polymerase then transcribes the double-stranded DNA into new RNA, which mimics the Qf3. After extensive washing to remove any unhybridised probe, the new RINA is eluted from the target and replicated by Qf3 replicase.

The latter reaction can create a 1 o fold amplification in approximately 20 minutes.

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 Conforruational 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 tetroxjde, 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.

Furthermore, techniques based on WAVE analysis can be used (Methods Mot Med. 2004;l08:173-88). 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):20 1-6.) Real-time PCR (also known as Quantitative PCR, Real-time Quantitative FCR, 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.

Specific primers known as Scorpion® primers can be used for a highly sensitive and rapid DNA amplification system. Such primers combine a probe with a specific target sequence in a single molecule, resuhing in a fluorescent detection system with unimolecular kinetics (Nuct Acids Res. 2000, 28:3752-376 1). This has an advantage over other fluorescent probe systems such as Molecular Beacons and TaqMan®, in that no separate probe is required to bind to the amplified target, making detection both faster and more efficient. A direct comparison of the three detection methods (Nucl. Acids Res 2000, 28:3752-376 1) indicates that Scorpions® perform better than intermolecular probing systems, particularly under rapid cycling conditions. The structure of one version of a Scorpion® primer is such that it is held in a hairpin loop conformation by complementary stem sequences of around six bases which flank a probe sequence specific for the target of interest (Nat Biotechnol. 1999, 17:804-807).

The stem also serves to position together a fluorescent reporter dye (attached to the 5'-end) in close proximity with a quencher molecule. In this conformation, no signal is produced. A PCR-blocker separates the hairpin loop from the primer sequence, which forms the 3'-end of the Scorpion®. The blocker prevents read-through, which would lead to unfolding of the hairpin loop in the absence of a specific target. During PCR, extension occurs as usual from the primer. After the subsequent denaturation and annealing steps, the hairpin loop unfolds and, if the correct product has been amplified, the probe sequence binds to the specific target sequence downstream of the primer on the newly synthesised strand. This new structure is thermodynamically more stable than the original hairpin loop. A fluorescent signal is now generated, since the fluorescent dye is no longer in close proximity to the quencher. The fluorescent signal is directly proportional to the amount of target DNA.

An alternative Scorpion® primer comprises a duplex of two complementary labelled oligonucleotides. One oligonucleotide of the duplex is labelled with a 5' end reporter dye and carries both the blocker non-coding nucleotide and PCR primer elements, while the other oligonucleotide is labelled with a 3' end quencher dye. The mechanism of action is then essentially the same as the Scorpion® hairpin primer described above: during real-time quantitative PCR, the 5' end reporter and 3' end quencher dyes are separated from each other leading to a significant increase in fluorescence emission.

Scorpions® can be used in combination with the Amplification Refractory Mutation System (ARMS) (Nuc!. Acids Res. 1989, 17:2503-2516, Nat. Biotechnol. 1999, 17:804-807) to enable single base mutations to be detected. Under the appropriate PCR conditions a single base mismatch located at the 3'-end of the primer is sufficient for preferential amplification of the perfectly matched allele (Newton et al., 1989), allowing the discrimination of closely related species. The basis of an amplification system using the primers described above is that oligonucleotides with a mismatched 3'-residue will not function as primers in the PCR under appropriate conditions. This amplification system allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis. It is simple and reliable and will clearly distinguish heterozygotes at a locus from homozygotes for either allele. ARMS does not require restriction enzyme digestion, allele-specific oligonucleotides as conventionally applied, or the sequence analysis of PCR products.

In one embodiment, the method of screening involves use of real time polymerase chain reaction (real time-PCR) with allele specific (ARMS) primers that detect single base mutations.

With respect to the base/nucleotide at position 5930 of MTOR gene (according to position in SEQ ID NO: 1), a cytosine is referred to herein as wild-type base or wild-type allele and something other than cytosine (in particular adenine) is referred to as mutant base or mutant allele.

In a further embodiment the method utilises a first primer pair to detect the wild type allele and a second primer pair is used to detect the mutant allele; and wherein one primer of each pair comprises: (a) a primer with a terminal 3' nucleotide that is allele specific for a particular mutation; and (b) possible additional mismatches at the 3' end of the primer.

Preferably, one primer in each pair as described above further comprises:-(a) a single molecule or nucleic acid duplex probe containing both a primer sequence and a further sequence specific for the target sequence; (b) a fluorescent reporter dye attached to the 5' end of the probe in close proximity with a quencher molecule within said single molecule or nucleic acid duplex; (c) one or more non-coding nucleotide residues at one end of said probe; (d) wherein said reporter dye and quencher molecule become separated during amplification of the target sequence.

The fluorescent probe system described above has the advantage that no separate probe is required to bind to the amplified target, making detection both faster and more efficient than other systems.

In one embodiment of the invention there is provided an ARMS method as described hereinabove wherein a first primer pair is used to detect the wild type allele and a second primer pair is used to detect the mutant allele; and wherein one primer of each pair comprises:- (a) a primer with a terminal 3' nucleotide that is allele specific for a particular mutation; and (b) possible additional mismatches at the 3' end of the primer.

In one scenario, one primer of each pair comprises:- (a) a single molecule or nucleic acid duplex probe containing both a primer sequence and a further sequence specific for the target sequence; (b) a fluorescent reporter dye attached to the 5' end of the probe in close proximity with a quencher molecule within said single molecule or nucleic acid duplex; (c) one or more non-coding nucleotide residues at one end of said probe; (d) wherein said reporter dye and quencher molecule become separated during amplification of the target sequence.

In one embodiment, the probe is a Scorpion® probe.

In one embodiment of the invention there is provided a method of determining the sequence of MTOR gene in a sample obtained from a patient comprising use of an ARMS primer capable of recognising the sequence of MTOR gene at position 5930 as shown in SEQ ID NO: 1. In one embodiment of the invention there is provided a method of determining the sequence of MTOR gene in a sample obtained from a patient comprising use of an ARIVIS primer and a companion primer optimized to amplify the region of a MTOR gene sequence comprising position 5930 as shown in SEQ ID NO: 1. The skilled person would understand that "optimized to amplify" comprises determining the most appropriate length and position of the forward primer and reverse primer. In one embodiment the ARMS primer capable of recognising the mutant base can be either of the forward or reverse primers. The forward reverse primers for use in the ARMS assay are optimized to amplify a region of less than 500 bases. In one embodiment the primers arc optimized to amplify a region of less than 250 bases. In one embodiment the primers are optimized to amplify a region of less than 200 bases. In one embodiment the primers are optimized to amplify a region of greater than 100 bases.

In one embodiment the ARMS forward primer is capable of recognising the sequence of MTOR gene at position 5930 as defined in SEQ ID NO: 1. In one embodiment the ARMS reverse primer is capable of recognising the sequence of MTOR gene at position 5930 as defined in SEQ ID NO: 1. By "recognising" in this context means specifically hybridising to and/or capable of facilitating primer extension therefrom. Either or the primers used in the ARMS assay may include locked nucleic acids to enhance or facilitate hybridisation to the substrate nucleic acid. Locked Nucleic Acid (LNA) oligonucleotides contain a methylene bridge connecting the 2'-oxygen of ribose with the 4'-carbon. This bridge resuhs in a locked 3 -endo conformation, reducing the conformational flexibility of the ribose and increasing the local organisation of the phosphate backbone. Braasch and Corey have reviewed the properties of LNA/DNA hybrids (Braasch and Corey, 2001, Chemistry & Biology 8,1-7).

Several studies have shown that primers comprising LNAs have improved affinities for complementary DNA sequences. Incorporation of a single LNA base can allow melting temperatures (Tm) to be raised by up to 41°C when compared to DNA:DNA complexes of the same length and sequence, and can also raise the Tm values by as much as 9.6°C. Braasch and Corey propose that inclusion of LNA bases will have the greatest effect on oligonucleotides shorter than 10 bases.

Implications of the use of LNA for the design of PCR primers have been reviewed (Latorra, Arar and Hurley, 2003, Molecular and Cellular Probes 17, 253-259). It was noted that firm primer design rules had not been established but that optimisation of LNA substitution in PCR primers was complex and depended on number, position and sequence context. Ugozolli et al (Ugozolli, Latorra, Pucket, Arar and Hamby, 2004, Analytical Biochemistry 324, 143-152) described the use of LNA probes to detect SNPs in real-time PCR using the 5' nuclease assay. Latorra et al (Latorra, Campbell, Wolter and Hurley, 2003, Human Mutation 22, 79-85) synthesised a series of primers containing LNA bases at the 3' terminus and at positions adjacent to the 3' terminus for use as allele specific primers.

Although priming from mismatched LNA sequences was reduced relative to DNA primers, optimisation of individual reactions was required.

In one embodiment the ARMS forward and/or reverse primer comprises a sequence in which one or more of the standard DNA bases have been substituted with a LNA base.

In one embodiment there is provided an ARIVIS probe capable of binding to the amplification product resulting from use of a pair of ARMS primer as described hereinabove in an ARMS assay. In one embodiment the ARMS probe comprises a sequence in which one or more of the standard DNA bases have been substituted with a LNA base. In one embodiment the ARMS probe comprises a Yakima YellowTM fluorescent tag on the 5' end.

In one embodiment the ARMS probe comprises a BHQTM quencher on the 3' end. The skilled person would recognise that the position at which the probe binds in the amplified product (and thus the sequence of the probe is complementary to) is restricted only by the boundaries imposed by the forward and reverse primers which determine the amplified product.

The Control probe can be used to confirm that the ARMS assay is working as intended and to confirm that there is DNA in the sample used in the ARMS assay. The skilled person would understand that the Control probe could be targeted to any chosen gene.

Table 1 ARMS Assay Primers and Probes The control gene is ul antitrypsin. Yakima Yellow and CyTM5 are fluorescent tags and BHQTM (Black Hole QuencherTM) and ElleQuencher are quenchers.

Emboldened bases indicate LNA bases where: E= A-LNA; L=C-LNA; P=G-LNA; and, ZT-

LNA Seq

Primer 5' Mod Primer Sequence 3' Mod ID NO ARMS forward primer ______ AGCCCCTTCCTGGTAGTCTCAAGC __________ 3

ARMS LNA

mutant Reverse Primer (1) _______ TCGTPGTAGECTTEGAAPCCACTZ __________ 4

ARMS LNA

mutant Reverse Primer (2) _______ TCGTPGTAGECTTEGAAPCCAGTZ __________ 5

ARMS LNA

mutant Reverse Primer (3) _______ TCGTPGTAGECTTEGAAPCCATTZ ___________ 6

ARMS LNA

mutant Reverse Primer (4) _______ TCGTPGTAGECTTEGAAPCCAATZ __________ 7 ARMS LNA Yakima probe YellowTM ACCAGCZCTTLCCCEACCCACA BHQTM 8 ARMS LNA Yakima probe Yel lowTM TCTZCTGZTTTTLCCTCAGGLCC BHQTM 9 Control primer AGGACACCGAGGAAGAGGACTT forward ________ ______________________________ ___________ 10 Control primer reverse _______ GGAATCACCTTCTGTCTTCATTT ___________ 11 Control LNA probe. pfM5 CTGCLTPAZGAGGGGAA ElleQuencher 12 In another aspect of the invention there is provided a method as described hereinabove wherein the method for determining the sequence of MTOR gene comprises determining the sequence of eDNA generated by reverse transcription of MTOR gene mRNA extracted from archival tumour sections or other clinical material. Extraction of RNA from formalin fixed tissue has been described in Bock et a!., 2001 Analytical Biochemistry: 295 116-117, procedures for extraction of RNA from non-fixed tissues, and protocols for generation of eDNA by reverse transcription, PCR amplification and sequencing are described in Sambrook, J. and Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.

In another aspect of the invention there is provided a method as described hereinabove wherein the method for determining the sequence of MTOR gene comprises amplification of individual exons of the MTOR gene, heteroduplex annealing of individual exons followed by digestion with Ce! I (as described in Crepin et al., 2006 Endocrinology: 36, 369-376; and Marsh et a!., 2001 Neoplasia: 3, 236-244).

Tn another aspect, the invention provides a mutant human FRAP po!ynucleotide comprising an adenine at position 5930, as defined in SEQ ID NO: 1, or a fragment thereof comprising at least 20 nucleic acid bases, such as at least 20, 25, 30, 35, 40, 45, 50, 75, 100 or more, provided that the fragment comprises position 5930 of SEQ ID NO: 1.

In a further aspect the invention provides a mutant human mTOR polypeptide comprising the following amino acid residue at the following position as defined in SEQ ID NO: 2: a lysine at position 1977, or a fragment thereof comprising at least 10 amino acid residues, such as at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 or more, provided that the fragment comprises position 1977 according to SEQ ID NO: 2.

In another aspect, there is provided a method for determining the sequence of mTOR in mRNA encoded by a mutant mTOR gene.

In another aspect of the invention there is provided a method as described herein wherein the method for determining the sequence of mTOR is selected from, for example, an immunohistochemistry-based assay which may use a slide from a single patient, or a tissue microarray (Mayr et al., 2006 American Journal of Clinical Pathology: 126, 10 1-109; Zheng et al., 2006 Anticancer Research: 26, 2353-2360) or application of an altemative proteomics methodology, which could comprise lysing cells, digesting the proteins, separating protein fragments on a gel, obtaining the peptide containing the mutated amino acid and analysing the peptide by mass spectrometry.

In another aspect the invention provides an antibody specific for a mutant human mTOR polypeptide as defined hereinabove.

A further aspect of the invention provides a diagnostic kit comprising a hybridisation or amplification primer capable of identifying the wild-type cytosine at position 5930 of the MTOR gene according to SEQ ID NO: 1, and a hybridisation or amplification primer capable of identifying the mutant adenine at position 5930 of the MTOR gene according to SEQ ID NO: 1, and optionally an ARMS companion primer, and optionally instructions for use.

A further aspect of the invention provides a diagnostic kit, comprising an ARMS forward or reverse primer capable of detecting a mutation in mTOR at position 5930, as defined in SEQ ID NO: 1, and optionally an ARMS companion primer, and optionally instructions for use. As described above, ARMS utilises a forward and reverse primer, one of which can discriminate between a mutant or wild-type base at the 3' end. Thus, depending on which primer (forward or reverse) binds at the location of the mutation to be detected, the other primer is referred to as the "companion" primer. In one embodiment of the invention there is provided a diagnostic kit, comprising an ARMS mutant primer comprising one or more LNA bases and capable of recognising the sequence of mTOR at position 5930, as defined in SEQ ID NO: 1, and optionally an ARMS companion primer, and optionally instructions for use. In one embodiment the diagnostic kit may be used in a method of predicting the likelihood that a patient, who is a candidate for treatment with an mTOR inhibitor, will respond to said treatment. Tn an alternative embodiment the diagnostic kit may be used in selecting a patient, who is a candidate for treatment with an mTOR inhibitor, for said treatment. In an alternative embodiment the diagnostic kit may be used to assess the suitability of a patient, who is a candidate for treatment with an mTOR inhibitor, for said treatment.

A further aspect of the invention provides a diagnostic kit, comprising an antibody specific for a mutant human mTOR polypeptide as defined hereinabove, and optionally instructions for use. In one embodiment the antibody is capable of binding to mTOR polypeptide comprising a lysine at position 1977, according to SEQ ID NO: 2. In another embodiment the antibody is capable of binding to mTOR polypeptide comprising a lysine at position 1977, according to SEQ ID NO: 2 in preference to a polypeptide comprising a threonine at position 1977, according to SEQ ID NO: 2.

In another embodiment the antibody is capable of binding to mTOR polypeptide comprising a threonine at position 1977, according to SEQ ID NO: 2. Tn one embodiment the diagnostic kit may be used in a method of predicting the likelihood that a patient, who is a candidate for treatment with an mTOR inhibitor, will respond to said treatment. In an alternative embodiment the diagnostic kit may be used in selecting a patient, who is a candidate for treatment with an mTOR inhibitor, for said treatment. In an alternative embodiment the diagnostic kit may be used to assess the suitability of a patient, who is a candidate for treatment with an mTOR inhibitor, for said treatment.

In a further aspect of the invention the ARMS primers and probes as described hereinabove may be used to determine the sequence of mTOR in a panel of cell lines expressing either the wild type or a mutant mTOR. Knowledge of whether the cell lines are expressing either wild type or mutant mTOR could be used in screening programmes to identify novel mTOR inhibitors with specificity for the mutant mTOR phenotype or novel inhibitors with activity against the phenotype associated with the wild type receptor. The availability of a panel of cell lines expressing mutant mTORs will assist in the definition of the signaling pathways activated through mTOR and may lead to the identification of additional targets for therapeutic intervention.

In a further aspect of the invention there is provided the use of a primer or a probe capable of recognising adenine or cytosine at position 5930 of MTOR gene, according to SEQ ID NO: 1, for predicting the response of a patient to treatment with an mTOR inhibitor.

In a further aspect there is provided the use of a primer or probe specific for position 5930 of MTOR gene, according to SEQ ID NO: 1, in the manufacture of a composition or kit for predicting the response of a patient to an mTOR inhibitor.

In a further aspect of the invention there is provided an oligonucleotide at least 12 nucleobases in length, such as at least 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 or more, identical or partly complementary to a sequence that has an adenine at position 5930 according to SEQ ID NO: 1. In a particular embodiment the oligonucleotide is less that 50 nucleobases. When the sequence is partly complementary to a sequence that has an adenine at position 5930 according to SEQ ID NO: 1, it must be capable of hybridising to said sequence so as to allow detection and/or strand elongation therefrom.

In another aspect the invention provides a method of preparing a personalised genomics profile for a patient comprising determining the sequence of mTOR in a sample obtained from the patient at the following position as defined in SEQ ID NO: 1: position 5930, and/or the following position as defined in SEQ ID NO: 2: position 1977, and creating a report summarising the data obtained by said analysis.

In a specific embodiment, the method as described hereinabove may be used to assess the pharmacogenetics of an mTOR inhibitor. Pharmaeogenetics is the study of genetic variation that gives rise to differing response to drugs. By determing the sequence of mTOR in a sample obtained from a patient and analysing the response of the patient to an mTOR inhibitor, the pharmacogenentics of the mTOR inhibitor can be elucidated.

In one embodiment the method for predicting the likelihood that a patient who is a candidate for treatment with an mTOR inhibitor will respond to said treatment, may be used to select a patient, or patient population, with a tumour for treatment with an mTOR inhibitor.

In one embodiment the method for predicting the likelihood that a patient who is a candidate for treatment with an mTOR inhibitor will respond to said treatment, may be used to predict the responsiveness of a patient, or patient population, with a tumour to treatment with an mTOR inhibitor.

The mTOR inhibitor/drug will be incorporated into a composition or formulation suitable for pharmaceutical administration to a subject in need thereof, by, for example, mixing the compound with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilisers, or other materials, as described herein.

The term "pharmaceutically acceptable" as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA); Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000 or Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.

Formulations suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

A tablet may be made by conventional means, e.g. compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g. povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmcthyl cellulose); fillers or dilucnts (e.g. lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc, silica); disintegrants (e.g. sodium starch glycolatc, cross-linked povidonc, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); and preservatives (e.g., methyl p-hydroxybenzoatc, propyl p-hydroxybcnzoatc, sorbic acid). Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

Formulations suitable for topical administration (e.g. transdermal, intranasal, ocular, buccal, and sublingual) may be formulated as an ointment, cream, suspension, lotion, powder, solution, past, gel, spray, aerosol, or oil. Alternatively, a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active compounds and optionally one or more excipients or diluents.

Formulations suitable for topical administration in the mouth include losenges comprising the active compound in a flavored basis, usually sucrose and aeaeia or tragacanth; pastilles comprising the active compound in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active compound in a suitable liquid carrier.

Formulations suitable for topical administration to the eye also include eye drops wherein the active compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active compound.

Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the active compound.

Formulations suitable for administration by inhalation include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.

Formulations suitable for topical administration via the skin include ointments, creams, and emulsions. When formulated in an ointment, the active compound may optionally be employed with either a paraffinic or a water-miscible ointment base.

Alternatively, the active compounds may be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-l,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof The topical formulations may desirably include a compound which enhances absorption or penetration of the active compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.

When formulated as a topical emulsion, the oily phase may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono-or dibasic ailcyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required.

Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active compound, such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 jig/mI, for example from about 10 ng/ml to about 1 jig/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs.

The size of the dose of each therapy which is required for the therapeutic or prophylactic treatment of a particular disease state will necessarily be varied depending on the host treated, the route of administration and the severity of the illness being treated.

Accordingly the optimum dosage may be determined by the practitioner who is treating any particular patient, and taking into consideration various factors known to modify the action of drugs including severity and type of disease, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. It may also be necessary or desirable to reduce the above-mentioned doses of the components of the combination treatments in order to reduce toxicity. Therapeutically effective dosages may be determined by either in vitro or in vivo methods.

The compositions described herein may be in a form suitable for oral administration, for example as a tablet or capsule, for nasal administration or administration by inhalation, for example as a powder or solution, for parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion) for example as a sterile solution, suspension or emulsion, for topical administration for example as an ointment or cream, for rectal administration for example as a suppository or the route of administration may be by direct injection into the tumour or by regional delivery or by local delivery.

Therapeutically effective dosages may be determined by either in vitro or in vivo methods. Calculating therapeutic drug dose is a complex task requiring consideration of medicine, pharmacokinetics and pharmacogcnetics. The therapeutic drug dose for a given patient will be determined by the attending physician, taking into consideration various factors known to modify the action of drugs including severity and type of disease, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. The mTOR inhibitor will however normally be administered to a warm-blooded animal so that a daily dose in the range, for example, 0.01mg/kg to 75mg/kg body weight is received, given, if required, in divided doses. The mTOR inhibitor may be administered orally such as in a tablet, cachet or capsule. The mTOR inhibitor may also be administered parenterally. In such cases lower doses will be used. Thus, for example, for intravenous administration, a dose in the range 0.01 mg/kg to 30mg/kg body weight will generally be used.

An mTOR inhibitor is an agent that inhibits the activity of mTOR complexes, such as allosteric inhibitors ofmTORCl (e.g. rapamycin and analogues) and mTOR kinase inhibitors (e.g. compound 1). The mTOR inhibitor may be an antibody or a small chemical molecule.

In one embodiment the mTOR inhibitor is an mTOR serine/threoninc kinase inhibitor. In one embodiment the mTOR inhibitor is an allostcric inhibitor and/or an mTOR tyrosinc kinase inhibitor.

In one embodiment the mTOR inhibitor is selected from or an antibody. In one embodiment the mTOR inhibitor is selected from the group consisting of: rapamycin (sirolimus), azd8OSS (compound 1 herein), BEZ-235, tetrandrine, TNK-128, NV-128, OST-027 (4-(4-amino-5-(7-methoxy-1H-indol-2-yl)imidazo[5,1-f][1,2,4]triazin-7-yl) cyclohexanecarboxylic acid hydrochloride), Pp-242, deforolimus (MK8669), everolimus (RADOO 1), GSK-1059615, GSK-2 126458, PKI-5 87(PF-052 12384), GDC-0980, GDC-094 1 (2-(1 H-Indazol-4-yl)-6-[4-(methylsulfonyl)piperazin-1 -ylmethyl]-4-(4- morpholinyl)thieno[3,2-d]pyrimidine), KU-0063 794 (rel-5-[2-[(2R,6S)-2,6-dimethyl-4-morpholinyl]-4-(4-morpholinyl)pyrido[2, 3-d]pyrimidin-7-yl]-2-mcthoxybenzenemethanol), P1-105, BGT-226, PF-04691502, temsirolimus (CCI-779), WYE-354, AP23573, 3-[2,4-bis[(3 S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methyl-benzamide (see W02008/023 161), XL765(SAR245409), ridaforolimus, 401 (2-(4-Morpholinyl)-4H- pyrimido[2,1-a]isoquinolin-4-onc) and niclosamide (5-Chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide). In one embodiment the mTOR inhibitor is selected from: sirolimus, everolimus, temsirolimus, 3-[2,4-bis[(3 S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7- yl]-N-methyl-benzamide and 1 -[5-[2,4-Bis[3(S)-methylmorpholin-4-yl]pyrido[2,3.- d]pyrimidin-7-yl]-2-methoxyphenyl]methanol. In one embodiment the mTOR inhibitor is 3- [2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl] -N-methyl-benzamide or 1 -[5-[2,4-Bis[3(S)-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-2- methoxyphenyl]methanol. In one embodiment the mTOR inhibitor is l-[5-[2,4-Bis[3(S)-methylmorpholin-4-yl]pyrido[2,3 -d]pyrimidin-7-yl] -2-methoxyphenyl] methanol An effective amount of an mTOR inhibitor will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient.

Accordingly, it is preferred for the therapist to titer the dosage and modi& the route of administration as required to obtain the optimal therapeutic effect. A typical daily or intermittent dosage, such as weekly, fortnightly or monthly, might range from about 0. 5mg to up to 300mg, 500mg, 1000mg or 1200 mg or more, depending on the factors mentioned above.

We contemplate that an mTOR inhibitor may be used as monotherapy or in combination with other drugs. The present invention is also useful in adjuvant, or as a first-line, therapy.

In one embodiment the methods of the present invention additionally comprises administration of an mTOR inhibitor to a patient selected for, or predicted to respond to treatment with an mTOR inhibitor according the methods described hereinabove.

In one embodiment the methods carried out on a patient's biological sample to determine the allele at position 5930 (according to SEQ ID NO: 1) or amino acid at position 1977 (according to SEQ ID NO: 2), further comprise administering an amount of an mTOR inhibitor to the patient identified as suitable for treatment with the drug. In a particular embodiment the mTOR inhibitor is administered to the patient after the determination step.

In a further aspect of the invention there is provided use of an mTOR inhibitor in preparation of a medicament for treating a patient, or a patient population, selected for, or predicted to respond to, treatment with an mTOR inhibitor according the methods described hereinabove.

In a further aspect of the invention there is provided a method of treating a patient, or a patient population, selected for, or predicted to have an increased likelihood of response to an mTOR inhibitor according to the method as described herein, comprising administering an mTOR inhibitor to said patient(s).

In a further aspect of the invention there is provided a method of treating a patient who is a candidate for treatment with an mTOR inhibitor comprising: (i) determining whether the sequence of mTOR in a sample obtained from the patient at the following position as defined in SEQ ID NO: 1: position 5930, is not adenine; or (ii) determining whether the sequence of mTOR in a sample obtained from the patient at the following position as defined in SEQ ID NO: 2: position 1977, is not lysine; and administering an effective amount of the mTOR inhibitor.

In a further aspect of the invention there is provided a method of treating a patient who is a candidate for treatment with an mTOR inhibitor drug comprising: (i) determining whether the sequence of mTOR in a sample obtained from the patient at position 5930, as defined in SEQ ID NO: 1, is not thymine; or (ii) determining whether the sequence of mTOR in a sample obtained from the patient at position 1977, as defined in SEQ ID NO: 2, is not methionine; and administering an effective amount of the mTOR inhibitor drug.

In a further aspect of the invention there is provided a method of treating a patient who is a candidate for treatment with an mTOR inhibitor drug, comprising: (i) determining whether the sequence of mTOR in a sample obtained from the patient at the following position as defined in SEQ ID NO: 1: position 5930, is cytosine; or (ii) determining whether the sequence of mTOR in a sample obtained from the patient at the following position as defined in SEQ ID NO: 2: position 1977, is threonine, and administering an effective amount of the mTOR inhibitor drug.

In a further aspect of the invention there is provided a method of treating a patient who is a candidate for treatment with an mTOR inhibitor drug comprising: (i) determining whether the sequence of mTOR in a sample obtained from the patient at position 5930, as defined in SEQ ID NO: 1, is cytosine; or (ii) determining whether the sequence of mTOR in a sample obtained from the patient at position 1977, as defined in SEQ ID NO: 2, is threonine; and administering an effective amount of the mTOR inhibitor drug.

In a further aspect of the invention there is provided a method of treating a patient suffering from cancer comprising determining whether or not the patient will respond favourably to an mTOR inhibitor according the methods of the invention described above, and administering an effective amount of an mTOR inhibitor to said patient if they are identified as likely to be responsive to treatment with an mTOR inhibitor.

In a further aspect of the invention there is provided use of an mTOR inhibitor in the manufacture of a medicament for the treatment of a patient identified as likely to be responsive to treatment with an mTOR inhibitor according to the methods described above.

In a further aspect of the invention there is provided use of an mTOR inhibitor in the manufacture of a medicament for the treatment of patient with cancer whose cancer cells have been determined to possess a threonine at position 1977 in the mTOR protein, or a cytosine at position 5930 of the MTOR gene according to any of the methods described herein.

In a further aspect of the invention there is provided an mTOR inhibitor for use in the treatment of a cancer patient whose cancer cells have been determined to possess a threonine at position 1977 in the mTOR protein, or a cytosine at position 5930 of the MTOR gene.

In a further aspect of the invention there is provided an mTOR inhibitor for use in the treatment of a cancer patient whose cancer cells possess a threonine at position 1977 in the mTOR protein, or a cytosine at position 5930 of the MTOR gene.

In a further aspect of the invention there is provided an mTOR inhibitor for use in the treatment of a cancer patient whose cancer cells have previously been identified as possessing a threonine at position 1977 in the mTOR protein, or a cytosine at position 5930 of the MTOR gene.

In a further aspect of the invention there is provided a method of treating a patient suffering from cancer comprising: providing a tumour cell containing sample from a patient; determining whether the MTOR gene or encoded protein is wild type or mutant; and administering to the patient an effective amount of an mTOR inhibitor if the tumour cells possess a wild type MTOR gene or encoded protein.

In one embodiment, the mTOR inhibitor is administered to a patient if the base/nucleotide at position 5930 of MTOR gene (according to position in SEQ ID NO: 1), is cytosine. In one embodiment, the mTOR inhibitor is administered to a patient if the amino acid at position 1977 of mTOR protein (according to position in SEQ ID NO: 2), is threonine.

In a further aspect of the invention there is provided use of an mTOR inhibitor to treat a cancer patient whose tumour cells have been identified as possessing a wild type mTOR protein.

In a further aspect of the invention there is provided an mTOR inhibitor for treating cancers with tumour cells identified as harbouring wild-type MTOR gene.

Examples

The invention is illustrated by the following non-limiting examples, in which Example 1 -Generation of cell line with acquired resistance to Compound 1 MCF7 and Calu-3 cells are breast and non small cell lung cancer cell lines, respectively. Both are sensitive to Compound 1 (1C50 determined by cell count -15 nM), with Compound 1 inducing cell death in both cell lines.

Compound 1 (AZD8O5 5) -(1 -[5-[2,4-Bis[3(S)-mcthylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl] -2-methoxyphenyl]methanol) is identified in Table 4 and is disclosed in MCF7 and Calu-3 cells were seeded into flasks in the presence of 45nM Compound 1 (MCF7/45 and Calu3/45) or 75nM Compound 1(MCF7/75 and Calu3/75). Control cells grown in absence of drug were cultured and passaged alongside the test flasks. (MCF7/0 and Calu3/0). When cells grown in presence of Compound 1 achieved an equivalent growth rate as the parental cells, cells growing in presence of 45 nM Compound 1 were either maintained in culture at this concentration (MCF7/45 and Calu3/45) or placed in the presence of increasing concentrations of Compound 1 up to 1 uM (MCF7/1000 and Calu3/1000) until the growth rate was equivalent to the parental cells. Cells grown in the presence of 75 nM Compound 1 were maintained in this condition ((MCF7/75 and Calu3/75).

The induced resistance was confirmed by growing the parental cell lines and resistant cell lines in presence of 1 RM Compound 1 and determining the TC5O of the cell lines in 2 independent experiments (see Table 2). The cell lines growing in 1 kM Compound 1 clearly show 1C50 values >1kM Compound 1.

Table 2 ICSO values for Compound 1 in sensitive and resistant cell lines MCF7/0 MCF7/1000 Calu3/0 Calu3/1000 Expt#1 23nM 5.2kM 268nM 10kM Expt#2 24 nM 4.5 wM 80 nM 2.3 kM Identification of mTOR mutation in MCF7 cells with acquired resistance to Compound 1: Genomic DNA from the 4 cell lines derived from MCF7 and the 4 cell lines derived from Calu3 was extracted (Qiagen DNeasy kit). Exons 2 to 58 of mTOR gene were sequenced. The sequencing and analysis was outsourced to Agencourt (Beckman Coulter Genomics).

A novel mutation T 1977K (found within exon 43) was detected in two of the MCF7 cell lines (see Table 3), the other resistant cell lines did not have this particular mutation. No other variants were observed in the coding regions sequenced.

Table 3 mTOR mutations Exon Exon43 Nucleotitle Change 5939A (TI NM_004958 (DS) Amino Acid Change TI 977K (ulu3,O - Calu3/45 - Calu3/75 - Calu3/1000 - MCF7/0 -MCF7/45 Homozygous mutation MCF7/75 -MCF7/1000 Heterozygous mutation Example 2 -Impact of resistance to Compound 1 on the sensitivity to other anticancer agents The sensitivity of the MCF7/1000 resistant cell line was determined against various compounds/drugs targeting the PI3K/mTOR pathway (see Table 4) and the Raf/MEKIERK pathway. Parental cells sensitive to rapamycin, PI3K/mTOR, P13K or AKT were found to be resistant to these agents (see Table 5). The parental cells were resistant to ErbB inhibitors such as gefitinib and lapatinib, as well as the IGF1R inhibitor NVP-AEW541. When tested on the resistant cells, these remained resistant. The parental cells were resistant to sorafenib and Compound 2 (Braf inhibitor) and resistant cells did not acquire greater sensitivity against these agents.

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-9' -[J-j7'g]oIozeiJTh/g' ]iAuoqd L99C 9OO OM --[jAuaqd(jAjnqopAoouiwy-)-J-__________________ c'-P hN9YflJ h C'II -)--[AqjowA-p,-uizeiodid(iAuojinsiAiuow) UV'O9tO9OO OM -H-9-(IA-fr-Iozepul-H U-g -one) )1Id qoojuauo o ii) oujiuouedoid[Auoqd[A--u!Iou!n b[a -g'j7]ozep!w!-T.N 9O99QQ OM 9CZa8-GIAN -9C-Table 5 Sensitivity of the parental and resistant MCF7 cells to other anticancer agents

____________________ _____________________ __________________

MCF7/0 MCF7/1000 Expt# 1 Expt#2 Expt# 1 Expt#2 Compound 1 0.023 13 0.02454 5.2 4.55 1 Rapamycin 0.048 0.0142 >10 >10 Bez235 0.0086 0.009656 1.18 1.234 Genentech P13K 0.2408 0.3208 4.48 >10 MK2206 4.104 300 Lapatinib 8.5 3.222 >10 5.057 Is Gefitinib 300 300 NVP-AEW541 1.421 1.728 >10 >10 Sorafenib 8.181 4.681 >10 >10 Compound 2 7.755 3.715 >10 >10 The strategy to develop cell lines with acquired resistance to Compound 1 was to subject the cells to test concentrations of Compound 1 which may induce a sub-optimal inhibition of the pathway and when resistance developed to strengthen the resistance mechanism by progressively increasing the concentration of Compound 1. In parallel, resistance could be developed using a higher concentration of Compound 1.

By aligning the mTOR gene sequence at the location of the T1977 amino acid in various species, it was observed that the T1977 amino acid is present in 40/42 species. Further, the PolyPhen tool (www.genetics.bwh.harvard.edu/pphl) was used to predict the impact of this variant on the protein structure. The query used PQS as a structure database. Parameters were set to be calculated on the first hit only. The difference in score was 1.711. The diagnostic was pre-computed. The calculation predicts that the T1977K variant is possibly damaging.

This suggests that nature strongly favours a threonine at position 1977 in mTOR.

Example 3 -Amplification Refractory Mutation System for detection of C5930A mutation in mTOR from Clinical Samples using ARMS LNA primers

Prophetic example.

An Amplification Refractory Mutation System assay (ARMS) may be used to detect the presence of a nucleotide base change in the mTOR gene compared to a background of normal DNA. Each ARMS assay is specific for a given mutation e.g. designed to detect a change from one base to another base at a given position. The assay is multiplexed with a second PCR reaction that detects the presence of DNA in the reaction, thereby indicating successful PCR. TaqManTM technology is used to detect the PCR products of both reactions using TaqManTM probes labelled with different fluorescent tags.

To undertake such an ARMS test, the DNA is extracted from the clinical sample as described in Example 1 and ARMS assay are carried out using the primers in Table 1.

A typical set of ARMS reactions would be carried out with about Sjil of genomic DNA containing varying proportions of mutant and wild type DNA and varying concentrations of input DNA in a total reaction volume of 25 p1 containing 1 Unit of Amplitaq gold DNA polymerase (N80080246, ABI) per reaction with final concentrations of 3.5 mlvi magnesium chloride, 200RM dNTPs (deoxyribonucleotide triphosphates) and 1.0 jiM of each ARMS mutant forward primer and ARMS reverse primer short in buffer (final buffer composition 15 mM Tris-HC1 Ph 8.3, 50 mM KCI). TaqManTM probes (Eurogentech). Cycle conditions: 95°C for 10 minutes followed by 40 cycles of 94°C for 45 seconds, 60°C for 45 seconds, 72°C for 1 minute in a Real Time PCR instrument (e.g. Stratagene Mx4000 or ABT 7900).

Claims (20)

1. Claims 1. A method for determining whether or not a tumour cell or cell population is likely to be responsive or resistant to an mTOR inhibitor comprising, determining the amino acid at position 1977 in mammalian target of rapamycin (mTOR) protein, or the nucleotide at position 5930 in MTOR gene (according to SEQ ID NO: 1) in the tumour cell(s), wherein the presence of a lysine at position 1977 in the mTOR protein (according to SEQ ID NO: 2), or an adenine at position 5930 of the MTOR gene is indicative that the tumour cell(s) is resistant to the mTOR inhibitor.
2. 2. The method as claimed in claim 1, wherein the tumour cell or cell population is taken from a cancer patient and the presence of a threonine at position 1977 in the mTOR protein, or a cytosine at position 5930 of the MTOR gene is indicative of the suitability of the individual to treatment with the compound.
3. 3. The method as claimed in claim 1 or 2, wherein the mTOR inhibitor is selected from the group consisting of: 1-[5-[2,4-Bis[3(S)-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl] -2-methoxyphenyl]methanol, rapamycin (sirolimus), BEZ-235, tetrandrine, INK-128, NV-128, OSI-027 (4-(4-amino-5-(7-methoxy-1 H-indol-2-yl)imidazo[5, 1-f][1,2,4]triazin-7-yt)cyclohexanecarboxytic acid hydrochloride), Pp-242, deforolimus (MK8669), everolimus (RADOO1), GSK-1059615, GSK-2126458, PKI-587(PF-05212384), GDC-0980, GDC-094 1 (2-(1 H-Indazol-4-yl)-6-[4-(methylsulfonyl)piperazin-1 -ylmethyl]-4- (4-morpholinyl)thieno[3,2-d]pyrimidine), KU-0063794 (rel-5-[2-[(2R,65)-2,6-dimethyl-4-morpholinyl]-4-(4-morpholinyl)pyrido[2, 3-d]pyrimidin-7-yl]-2-methoxybenzenemethanol), P1-105, BGT-226, PF-0469 1502, temsirolimus (CCI-779), WYE-354, AP23573, 3-[2,4-bis[(3 S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methyl-benzamide, XL765(SAR24S 409), ridaforolimus, 401 (2-(4-Morpholinyl)-4H-pyrimido[2, 1 -a]isoquinolin- 4-one) and niclosamide (5-Chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide).
4. 4. The method as claimed in claim 3, wherein the mTOR inhibitor is 1-[5-[2,4-Bis[3(S)-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl] -2-methoxyphenyl]methanol
5. 5. The method as claimed in any of the preceding claims, which method is used for selecting a cancer patient for treatment with an mTOR inhibitor.
6. 6. The method as claimed in any of the previous claims wherein the cancer is selected from the group consisting of: breast cancer, colorectal cancer, head and neck cancer, lung cancer, gastric cancer, prostate, haematological cancers, pancreatic cancer, renal cancer, neuroendocrine tumours, endometrial cancer and ovarian cancer.
7. 7. The method as claimed in any of the preceding claims, wherein the tumour containing sample is a solid tissue sample or a biofluid sample.
8. 8. The method as claimed in any of the preceding claims, wherein the solid tissue sample is selected from: a fresh tissue sample, a frozen tissue sample and a formalin-fixed paraffin-embedded tissue sample, and the biofluid sample is selected from: sputum, blood, plasma, serum, ascites fluid, urine, semen, prostatic fluid and cerebral fluid.
9. 9. The method as claimed in any of the preceding claims, wherein the nucleotide at position 5930 in MTOR gene (according to SEQ ID NO: 1) is determined by DNA sequencing, allele specific amplification, single strand conformation polymorphisms, polymerase chain reaction, restriction fragment length polymorphism (RFLP), WAVE analysis, assaying for mutant mTOR, denaturing gradient gel electrophoresis, high resolution melting or temperature gradient gel electrophoresis.
10. 10. The method according to any of the preceding claims wherein the nueleotide at position 5930 in MTOR gene (according to SEQ ID NO: 1) is determined by sequencing.
11. 11. The method according to any of the preceding claims wherein the nueleotide at position 5930 in MTOR gene (according to SEQ ID NO: 1) is determined using a technique that involves polymerase chain reaction (PCR).
12. 12. The method of any of the preceding claims, further comprising administering an amount of an mTOR inhibitor to the patient.
13. 13. A method of predicting likelihood of response to treatment with an mTOR inhibitor in an individual diagnosed with cancer comprising determining the amino acid at position 1977 (according to SEQ ID NO: 2) in mammalian target of rapamycin (mTOR) protein, or the nucleotide at position 5930 in MTOR gene (according to SEQ ID NO: 1) in a biological sample obtained from said individual, wherein the presence of a mutation at these locations indicates that the treatment will be more likely to be ineffective in the individual that one without the mutation.
14. 14. The method as claimed in claim 13, wherein if the MTOR gene possesses a cytosine at position 5930, or the mTOR protein posseses a threonine at position 1977 the patient is selected for treatment with an mTOR inhibitor.
15. 15. The method as claimed in claim 13, wherein if the MTOR gene possesses an adenine at position 5930, or the mTOR protein posseses a lysine at position 1977 the patient is not selected for, or is deselected from, treatment with an mTOR inhibitor.
16. 16. Use of an oligonucleotide primer capable of identifying the presence of an adenine or cytosine at position 5930 in MTOR gene for predicting the response of a patient to an mTOR inhibitor.
17. 17. An oligonucleotide capable of recognising the sequence of mTOR at position 5930,asdcfinedinSEQlDNO: 1.
18. 18. A diagnostic kit comprising a hybridisation or amplification primer capable of identifying the wild-type adeninc at position 5930 of MTOR gene, according to SEQ ID NO:, and a hybridisation or amplification primer capable of identifying the mutant cytosine at position 5930 of MTOR gene.
19. 19. A method of treating a patient suffering from cancer comprising determining whether or not the patient will respond favourably to an mTOR inhibitor according the method as claimed in any of claims 1 to 11 and 13 to 15, and administering an effective amount of an mTOR inhibitor to said patient if they are identified as likely to be responsive to treatment with an mTOR inhibitor.
20. 20. An mTOR inhibitor for use in the treatment of a patient suffering from cancer and whose cancer cells possess a threonine at position 1977 in the mTOR protein, or a cytosine at position 5930 of the MTOR gene.