Field of the Invention

The present invention relates to biomarkers for cancer, and in particular for cancers such as ovarian cancer, breast cancer, cervical cancer, endometrial cancer, lung cancer and prostate cancer. The invention specifically relates to the use of such biomarkers for predicting cancer prognosis (prognostic marker) or diagnosis (diagnostic marker) and response to treatment (predictive biomarker). Background to the Invention

RNA binding proteins (RBPs) regulate the decay kinetics and translational efficiency of mRNA transcripts by accelerating their degradation or prolonging their cytoplasmic half- life. In this way, the abundance of mRNAs and their encoded proteins can be altered in a manner that is independent from gene transcription. As RBPs are themselves activated by growth factors and cell signals, this tightly-regulated post-transcriptional mechanism enables the cell to rapidly adjust levels of protein expression in response to intrinsic and extracellular signals. In addition, RBPs can interact with up to thousands of mRNA transcripts, allowing the coordinated synthesis of multiple proteins involved in a single physiological function (termed an RNA operon). However, when the expression of an RBP is disrupted it can potentially disrupt cellular homeostasis and autonomously drive pathological processes by uncoupling the regulation of mRNA stability from cell signaling cues.

A protein recently identified as being a RBP is LARP1 (Castello et al Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149: 1393-1406).

LARP1 belongs to the La-related protein (LARP) family, three members of which: LARP1 , LARP3 (or genuine LA protein) and LARP7 have so far been implicated in cancer. An elevated expression of LARP1 has been shown to correlate with clinical outcome in hepatocellular carcinoma (Xie et al., Journal of Translational Medicine 11 : 272, 2013), LARP3 protein expression is increased in cervical cancer and higher levels have been shown to correlate with adverse outcome in lung cancer. In contrast, LARP7 is a potential tumor suppressor in gastric and cervical tumors.

LARP1 was first identified in Drosophila melanogaster, where it was shown to bind poly(A)-binding protein (PABP) and was required for embryonic development and fertility. Proteomic screens conducted in human embryonic cell lines have subsequently shown that LARP1 contributes to the stability and translation of 5TOP mRNAs (those bearing 5' terminal oligopyrimidine (5TOP) tracts) by interacting with them. 5TOP mRNAs are required for ribosome biogenesis and are regulated downstream of the mTOR

(mammalian target of rapamycin) complex 1 (mTORCI) kinase. Summary of the Invention

The present inventors have surprisingly found that LARP1 protein is present in the serum of cancer patients. LARP1 protein can therefore be used as an easily sampled peripheral biomarker of tumour LARP1 levels.

Accordingly, in a first aspect, the present invention provides a method of determining prognosis of cancer in a subject, comprising:

detecting the level of LARP1 protein in a serum or plasma sample from a subject; and comparing the level of LARP1 protein in said sample with the level of LARP1 protein in a control sample,

wherein an elevated level of LARP1 protein in said serum or plasma sample from said subject compared to the level of LARP1 protein in said control sample is indicative of poor prognosis of said cancer.

Detailed Description of the Invention

In a first aspect, the present invention relates to a method of determining prognosis of cancer. In other words, the first aspect of the invention relates to a method of determining the likely outcome or progression of cancer in a subject, for example the chances of survival of a subject with cancer over a particular period of time. The method of the first aspect of the invention can also be described as an assay. The method is typically carried out on a sample from a subject who has already been diagnosed with cancer. The present invention also relates to a method of diagnosing cancer. In other words, the invention relates to a method of identifying that a subject has cancer. Accordingly, in a second aspect the present invention provides a method of diagnosing cancer in a subject, comprising: detecting the level of LARP1 protein in a serum or plasma sample from a subject; and comparing the level of LARP1 protein in said sample with the level of LARP1 protein in a control sample,

wherein an elevated level of LARP1 protein in said serum or plasma sample from said subject compared to the level of LARP1 protein in said control sample indicates that said subject has cancer. The method of the second aspect of the invention can also be described as an assay. In the methods of the invention, the cancer is typically an epithelial cancer, for example ovarian cancer, cervical cancer, endometrial cancer, lung cancer (for example non-small cell lung cancer), prostate cancer, breast cancer or liver cancer (for example

hepatocellular carcinoma).

The methods of the invention involve detecting the level of LARP1 protein in a serum or plasma sample from a subject. Such a sample is typically derived from a sample of whole blood that has previously been obtained from a subject. A serum or plasma sample is then derived from the whole blood using an appropriate technique.

Serum is the liquid fraction of the blood that remains when a whole blood sample is allowed to clot. Accordingly, serum is obtained by allowing the whole blood sample to clot. This can be done, for example, by leaving the sample undisturbed at room

temperature for around 15-30 minutes. The serum can be obtained by removing the clot, for example by centrifuging the sample. This can be done, for example, at 1 ,000-2,000 x g for 10 minutes in a refrigerated centrifuge. The resulting supernatant is serum.

Plasma is produced when whole blood is treated with an anticoagulant. This can be done, for example, by collecting blood in tubes that are treated with an anticoagulant. Plasma can then be obtained by centrifugation. This can be done, for example, at 1 ,000-2,000 x g for 10 minutes in a refrigerated centrifuge. The resulting supernatant is plasma.

The methods of the invention are typically carried out on a sample that has previously been obtained from a subject. Thus, the taking of the sample does not typically form part of the methods of the invention and the methods of the invention are carried out on a sample that has been obtained from a subject. In some embodiments of the invention, however, the method also comprises taking the sample from the subject, for example by taking a blood sample. As used herein, the term "LARP1" means LARP1 protein. There are three putative protein isoforms encoded by LARP1 mRNA (NM_015315.4): isoform a (891 amino acids,

Gl_1 19582036), isoform b (1 158 amino acids, Gl_1 19582037) and isoform c (1019 amino acids, Gl_119582038). Figure 1 shows the nucleotide sequence of LARP1 mRNA (SEQ ID NO: 1) and the amino acid sequences of LARP1 isoforms a (SEQ ID NO: 2), b (SEQ ID NO: 3) and c (SEQ ID NO: 4). LARP1 can be detected in the serum or plasma sample using any suitable assay. A wide range of immunoassays are available to measure protein levels in a sample and these are typically based on antibody-antigen binding. For example, an enzyme-linked

immunosorbent assay (ELISA) such as a sandwich ELISA can be used. In a typical sandwich ELISA, capture antibodies are attached to a surface such as a microwell plate. In the present invention, the capture antibodies are specific to LARP1. Non-specific binding sites on the surface can then be blocked, for example using a blocking buffer. The serum or plasma sample is then added to the surface and any LARP1 in the sample binds to the capture antibodies. Unbound antigen (i.e. contaminants) can then be removed by washing the plate. A detection antibody is then added, which binds to bound LARP1. A secondary antibody is then added, which is linked to an enzyme and binds to the detection antibody. The enzyme to be used can be, for example, horseradish peroxidase (HRP), which catalyses the conversion of chromogenic substrates into coloured products and produces light when acting on chemiluminescent substrates. The plate can then be washed to remove any unbound antibody-enzyme conjugates. A substance containing the substrate of the enzyme is then added. If HRP is used as the enzyme various substrates can be used, including 3,3',5,5'-tetramethylbenzidine (TMB), 3,3'- diaminobenzidine (DAB), o-phenylenediamine dihydrochloride (OPD) and 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS), which are all chromogenic, or an enhanced chemiluminescent substrate (ECL). The subsequent reaction produces a detectable signal in the substrate. The detectable signal can be, for example, a colour change, fluorescence or electrochemiluminescence, depending on the substrate. The strength of the signal is indicative of the amount of the antigen, in this case LARP1. When the detectable signal is a colour change, a spectrometer is often used to give quantitative values for colour strength.

Non quantitative methods of detecting LARP1 in samples include Western blotting following sepharose-bead immunoprecipitation using anti-LARP1 antibodies. Anti-LARP1 antibodies are commercially available, for example from SDIX, LLC (Newark, DE).

Alternative methods of detecting LARP1 in samples include high performance liquid chromatography (HPLC) and other high-throughput techniques.

The methods of the present invention involve comparing the level of LARP1 protein in a serum or plasma sample from a subject with the level of LARP1 protein in a control sample. The control sample is typically a serum or plasma sample taken from a subject who is known not to be suffering from cancer, for example a healthy control subject. Alternatively, the control sample can be a serum or plasma sample taken from a subject with a benign tumour, for example a benign ovarian tumour. In some embodiments, the control sample has no LARP1 protein, or LARP1 protein is undetectable in the control sample.

In the method of the first and second aspect of the invention, an elevated level of LARP1 protein in the serum or plasma sample from the subject compared to the level of LARP1 protein in the control sample is indicative of poor prognosis of said cancer. In other words, the subject has a poor or low chance of survival, for example over a particular period of time. In other words, the subject is unlikely to survive for a particular period of time after the diagnosis of cancer is made. Without wishing to be limited by specific values, "poor prognosis" or "poor chance of survival" can mean, for example, that the subject has a 60% or lower, for example 50%, 40%, 30%, 20% or 10% chance of surviving for 100, 200, 400, 600, 800 or 1000 days after the date of diagnosis.

By "an elevated level of LARP1 protein" is meant a significantly higher level, in particular a statistically significantly higher level. Statistical tests known in the art can be used, for example the Wilcoxon signed rank sum test, the Mann-Whitney test or Cox Regression analyses. Statistical significance can be determined based on any suitable p-value, for example p<0.05, p<0.01 or pO.001.

Without wishing to be limited by specific values, an example of a typical elevated level of LARP1 protein in a sample is in the region of 750 to 1250 pg/ml, for example from 800 to 1200 pg/ml, from 900 to 1150 pg/ml, from 950 to 1100 pg/ml or around 1000 pg/ml. An example of a typical level of LARP1 protein in a control sample is in the region of 0 to 200 pg/ml, for example from 50 to 150 pg/ml or around 100 pg/ml. The subject is typically a human subject. However, the methods of the invention also find use in the field of veterinary medicine and can therefore be used to diagnose cancer in animal subjects, typically mammalian subjects, for example companion animals such as cats and dogs, or agricultural animals such as horses, cows and sheep. The present inventor hypothesises that plasma LARP1 is indicative of response to treatment with certain drugs. In particular, an elevated level of plasma LARP1 indicates that the subject does not respond to certain drugs.

Accordingly, in a third aspect, the present invention provides a method of determining whether a subject responds to a drug that acts on the PI3K AKT/mTOR pathway, comprising: detecting the level of LARP1 protein in a serum or plasma sample from a subject; and comparing the level of LARP1 protein in said sample with the level of LARP1 protein in a control sample,

wherein an elevated level of LARP1 protein in said serum or plasma sample from said subject compared to the level of LARP1 protein in said control sample indicates that the subject does not respond to said drug.

Conversely, if an elevated level of LARP1 protein is not found in said serum or plasma sample, this indicates that the subject does respond to a drug that acts on the

PI3K/AKT/mTOR pathway.

Accordingly, the third aspect of the invention extends to a method of determining whether a subject responds to a drug that acts on the PI3K/AKT/mTOR pathway, comprising: detecting the level of LARP1 protein in a serum or plasma sample from a subject; and comparing the level of LARP1 protein in said sample with the level of LARP1 protein in a control sample,

wherein a non-elevated level of LARP1 protein in said serum or plasma sample from said subject compared to the level of LARP1 protein in said control sample indicates that the subject does respond to said drug.

By a "non-elevated level of LARP1 protein" is meant that the level of LARP1 protein in the sample from the subject is comparable to the level of LARP1 protein in the control sample. Without wishing to be limited by specific values, an example of a typical level of LARP1 protein in a control sample is in the region of 0 to 200 pg/ml, for example from 50 to 150 pg/ml or around 100 pg/ml.

The third aspect of the invention relates to a method of determining whether a subject responds to a drug that acts on the PI3K/AKT/mTOR pathway. In other words, the third aspect of the invention relates to a method of determining that a subject responds to a particular drug or not and can therefore be used to decide whether to treat the subject with such a drug. Accordingly, this aspect of the invention provides an indication of a successful treatment protocol for a subject with cancer. The method of the third aspect of the invention can also be described as an assay. The method is typically carried out on a sample from a subject who has already been diagnosed with cancer. In some

embodiments, the method is carried out on a sample from a subject who is already being treated with one or more drugs that act on the PI3K AKT/mTOR pathway. The PI3K/AKT/mTOR pathway is one of the two main pathways involved in the regulation of translation. The PI3K pathway is activated in response to a change in nutrient and energy signals, resulting in phosphorylation and activation of the kinase AKT. AKT then phosphorylates the downstream effector kinase mTOR. mTOR forms two complexes, mTORCI and mTORC2, of which mTORCI is most influential for translation. mTORCI includes mTOR and the scaffold protein Raptor.

By "a drug that acts on the PI3K/AKT/mTOR pathway" is meant a drug that interacts with PI3K, AKT and/or mTOR or components interacting directly or indirectly with these proteins. Typically, the drug is an inhibitor of PI3K, AKT and/or mTOR.

Inhibitors of PI3K include wortmannin and its derivative demethoxyviridin, and LY294002.

Inhibitors of AKT include VQD-002, miltefosine and AZD5363.

Inhibitors of mTOR include rapamycin (sirolimus) and its derivatives (known as rapalogues), for example deferolimus, everolimus and temsirolimus, mTOR-KIs or multi- kinase inhibitors that have mTOR included as targets. For example, BEZ235 (or NVP- BEZ235) is a dual PI3K-mTOR inhibitor.

The method of the third aspect of the invention is typically carried out on a sample from a subject that has previously been treated with or is currently being treated with one or more drugs that act on the PI3K/AKT/mTOR pathway. In this aspect, the present invention relates to a method of monitoring whether a subject is responding to therapy with one or more drugs that act on the PI3K/AKT/mTOR pathway. In an alternative aspect, the subject is not being and has not been treated with one or more drugs that act on the PI3K/AKT/mTOR pathway. In this aspect, the present invention relates to a method of predicting whether a subject will respond to therapy with one or more drugs that act on the PI3K/AKT/mTOR pathway.

The method of the third aspect of the present invention gives an indication of whether a subject responds to a drug that acts on the PI3K/AKT/mTOR pathway, for example an inhibitor of PI3K, AKT and/or mTOR. This method can therefore also be used in combination with a method of treating cancer in a subject. The third aspect of the invention therefore also encompasses methods which comprise a further step of treating a subject. In these embodiments, a determination of whether or not a subject responds to a drug that acts on the PI3K/AKT/mTOR pathway is made using the method of the third aspect of the invention, and then the subject is treated (or further treated) with an appropriate drug.

If the method of the third aspect of the invention indicates that the subject does not respond to a drug that acts on the PI3K/AKT/mTOR pathway, the subject will need to be treated with an alternative drug. If the method of the third aspect of the invention indicates that the subject does respond to a drug that acts on the PI3K/AKT/mTOR pathway, the subject can be treated (or further treated) with a drug that acts on the PI3K/AKT/mTOR pathway.

Accordingly, in an embodiment the present invention provides a method of treating cancer in a subject in need thereof, comprising:

detecting the level of LARP1 protein in a serum or plasma sample from a subject; comparing the level of LARP1 protein in said sample with the level of LARP1 protein in a control sample;

identifying an elevated level of LARP1 protein in said serum or plasma sample from said subject compared to the level of LARP1 protein in said control sample; and

administering an anti-cancer therapy to said subject, wherein said anti-cancer therapy is not a drug that acts on the PI3K/AKT/mTOR pathway.

This embodiment also extends to an anti-cancer therapy for use in a method of treating cancer in a subject in need thereof, wherein the method comprises:

detecting the level of LARP1 protein in a serum or plasma sample from a subject; comparing the level of LARP1 protein in said sample with the level of LARP1 protein in a control sample;

identifying an elevated level of LARP1 protein in said serum or plasma sample from said subject compared to the level of LARP1 protein in said control sample; and

administering an anti-cancer therapy to said subject, wherein said anti-cancer therapy is not a drug that acts on the PI3K/AKT/mTOR pathway.

This embodiment also extends to the use of an anti-cancer drug in the manufacture of a medicament for the treatment of cancer in a subject in need thereof by a method comprising:

detecting the level of LARP1 protein in a serum or plasma sample from a subject; comparing the level of LARP1 protein in said sample with the level of LARP1 protein in a control sample; identifying an elevated level of LARP1 protein in said serum or plasma sample from said subject compared to the level of LARP1 protein in said control sample; and

administering an anti-cancer drug to said subject, wherein said anti-cancer drug is not a drug that acts on the PI3K/AKT/mTOR pathway.

The anti-cancer therapy that is not a drug that acts on the PI3K/AKT/mTOR pathway can be, for example, chemotherapy or radiotherapy or an anti-cancer drug. The anti-cancer drug can be, for example, a chemotherapeutic drug such as bleomycin, carboplatin, cisplatin, cyclophosphamide, dacarbazine, docetaxel, doxorubicin, etoposide, 5- fluorouracil, folinic acid, gemcitabine, irinotecan, oxaliplatin, paclitaxel, or a combination chemotherapeutic regimen such as AC (doxorubicin and cyclophosphamide), BEP (bleomycin, etoposide and platinum agent), Carbo/taxol (carboplatin and paclitaxel), FOLFIRINOX (5-flurouracil, folinic acid, irinotecan, oxaliplatin) or a chemotherapy agent or combination of agents given with a targeted therapy such as bevacizumab, or stem cell targeted therapy.

In another embodiment the present invention provides a method of treating cancer in a subject in need thereof, comprising:

detecting the level of LARP1 protein in a serum or plasma sample from a subject; comparing the level of LARP1 protein in said sample with the level of LARP1 protein in a control sample;

identifying a non-elevated level of LARP1 protein in said serum or plasma sample from said subject compared to the level of LARP1 protein in said control sample; and

administering a drug that acts on the PI3K/AKT/mTOR pathway to said subject.

This embodiment also extends to a drug that acts on the PI3K/AKT/mTOR pathway for use in a method of treating cancer in a subject in need thereof, wherein the method comprises:

detecting the level of LARP1 protein in a serum or plasma sample from a subject; comparing the level of LARP1 protein in said sample with the level of LARP1 protein in a control sample;

identifying a non-elevated level of LARP1 protein in said serum or plasma sample from said subject compared to the level of LARP1 protein in said control sample; and

administering a drug that acts on the PI3K/AKT/mTOR pathway to said subject. This embodiment also extends to the use of a drug that acts on the PI3K/AKT/mTOR pathway in the manufacture of a medicament for the treatment of cancer in a subject in need thereof by a method comprising:

detecting the level of LARP1 protein in a serum or plasma sample from a subject; comparing the level of LARP1 protein in said sample with the level of LARP1 protein in a control sample;

identifying a non-elevated level of LARP1 protein in said serum or plasma sample from said subject compared to the level of LARP1 protein in said control sample; and

administering a drug that acts on the PI3K/AKT/mTOR pathway to said subject.

The result of these methods is that the cancer in the subject is treated using the anticancer therapy or drug described herein. By "treated" is meant that the severity or symptoms of cancer are reduced, or eliminated altogether in the subject. For example, a tumour can be reduced in size or eliminated altogether using the anti-cancer therapy or drug.

The method of treatment can be of a human or animal subject and these aspects of the invention extend equally to uses in both human and veterinary medicine. The anti-cancer therapy or drug is preferably administered to a subject in a "therapeutically effective amount", this being sufficient to show benefit to the subject and/or to ameliorate, eliminate or prevent one or more symptoms of cancer. As used herein, "treatment" includes any regime that can benefit a human or a non-human animal, preferably a mammal. The treatment is typically administered to a subject or patient "in need thereof", i.e. a subject suffering from cancer.

In this embodiment, an anti-cancer drug can be administered to the subject by any appropriate route, for example by oral (including buccal and sublingual), nasal, topical (including transdermal) or parenteral (including subcutaneous, intramuscular, intravenous, intraperitoneal and intradermal) administration, depending on the type of anti-cancer drug used. The anti-cancer drug can be formulated using methods known in the art of pharmacy, for example by admixing the active ingredient with carrier(s) or excipient(s) under sterile conditions to form a pharmaceutical composition. Accordingly, in one embodiment the subject is administered a pharmaceutical composition comprising an anticancer drug and one or more carriers and/or excipients.

The anti-cancer therapy or drug can also be administered in combination with one or more other therapeutically active agents, for example one or more other anti-cancer drugs (for example in a combination chemotherapeutic regimen), anti-emetics or analgesics. Accordingly, the pharmaceutical composition for use in accordance with this aspect of the invention may also comprise one or more other therapeutically active agents in addition to an anti-cancer drug.

Dosages of the anti-cancer drug and/or pharmaceutical composition for use in the present invention can vary between wide limits, depending for example on the particular anticancer drug used, the age and disease stage of the patient, and a physician will ultimately determine appropriate dosages to be used.

The dosage can be repeated as often as appropriate. If side effects develop, the amount and/or frequency of the dosage can be reduced, in accordance with normal clinical practice.

The present invention is based on the findings that LARP1 protein is present in the serum of cancer patients, levels of plasma LARP1 correlate with outcome in various cancers and elevated plasma LARP1 correlates with poor response to treatment with certain drugs. These findings are unexpected as LARP1 would not be expected to be stable in serum and is the first example of a circulating RNA binding protein detectable in cancer patients. The present inventor has used these unexpected findings to arrive at the present invention.

Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.

The present invention will now be further described by way of reference to the following Examples which are present for the purposes of illustration only. In the Examples, reference is made to a number of Figures in which:

Figure 1 : A shows the nucleotide sequence of LARP1 mRNA (NM_015315.4). B shows the amino acid sequence of isoform a (891 amino acids, Gl_119582036). C shows the amino acid sequence of isoform b (1 158 amino acids, Gl_1 19582037). D shows the amino acid sequence of isoform c (1019 amino acids,

Gl_1 19582038).

Figure 2: An immuno-precipitation experiment showing elevated levels of LARP1 in the plasma of patients with ovarian cancer.

Figure 3 shows a protocol for producing tumorigenesis mouse models.

Figure 4 shows bioluminescence imaging of tumor load in the experiment described in Example 2. Example 1 - Identification of LARP1 as a circulating biomarker

Figure 2 is a Western blot following immuno-precipitation work showing detectable LARP1 in the plasma of a selection of patients with advanced ovarian cancer. LARP1 is detectable in plasma obtained from patients 2, 3 and 5; patients with 3B serous ovarian cancer, 1C endometrioid ovarian cancer and 3C serous ovarian cancer respectively. LARP1 was undetectable in samples 1 , 4 and 6; patients with 2A serous ovarian, unknown and 3C serous ovarian cancer. LARP1 was also negative in 1 benign and 2 control cases.

Conclusions:

LARP1 is an RNA-binding protein whose levels are detectable in the plasma of patients with ovarian cancers and theoretically with other epithelial cancers also. This makes LARP1 the first circulating RBP to be identified in patient serum. Example 2 - LARP1 is highly expressed in tumorigenesis mouse models

To model ovarian cancer, ovarian epithelial cells capable of undergoing spontaneous tumourigenesis and metastasis are implanted in syngeneic mice and tumor load is tracked by bioluminescence (Figure 4). Here we show (Figure 3) that there is high LARP1 expression in transformed cell lines before they are implanted. This supports a role for LARP1 in early tumorigenesis and thus as a diagnostic marker of cancer.