WO2012004565A1 - Cancer biomarker brf1 - Google Patents

Abstract

This invention relates to the transcription factor Brf1and its use as a biomarker in the diagnosis and prognosis of cancer.

Description

CANCER BIOMARKER BRF1

This invention relates to the identification of biomarkers for the screening, diagnosis and prognosis of cancers, such as prostate cancer .

Prostate cancer develops in the prostate, a gland that forms part of the male reproductive system and it is the most common cancer in men, excluding non-melanoma skin cancer.

Carcinoma of the prostate is generally a tumour of older men, and 95% of prostate carcinomas are adenocarcinomas. The rate at which the tumours grow varies but it tends to be slow growing. Patient survival is linked to the extent of the disease: if confined to the prostatic capsule, survival of over 5 years can be anticipated;

however, if cells metastasize from the prostate to other parts of the body, survival is typically 1 to 3 years.

Prostate cancer may cause pain, difficulty in urinating, problems during sexual intercourse, or erectile dysfunction. Other symptoms can potentially develop during later stages of the disease. Many factors, including age, genetics and diet, have been implicated in the development of prostate cancer.

Serum prostate specific antigen ("PSA") testing, along with digital rectal examination (DRE) is used clinically to screen men for prostate cancer. The lead time (time by which a cancer diagnosis is advanced) with PSA screening has been estimated to be, on average, -10 years. However, there is no proof as yet that prostate-specific antigen screening in asymptomatic men prevents prostate cancer deaths .

The major concern about prostate cancer screening is the potential for over-diagnosis of prostate cancers that would be unlikely to pose a threat for morbidity or mortality. The PSA "cut-point" above which further evaluation to rule out prostate cancer (by prostate biopsy) should be recommended is a controversial issue. In addition to prostate cancer, the prostate also frequently manifests benign enlargement (benign prostatic hyperplasia (BPH) and chronic or recurrent inflammation (prostatitis). Like prostate cancer, each of these conditions can elevate PSA.

Diagnosis of prostate cancer ultimately requires needle biopsy specimens and microscopic analysis of prostate tissue, which can be graded histologically, via the assignment of a Gleason grade or score (that predicts the behaviour of prostate cancer) .

Prostate cancers can be difficult to recognize and difficult to distinguish from other prostate abnormalities such as prostate intraepithelial neoplasia ("PIN") . Most experienced prostate pathologists use a combination of findings to make a diagnosis of prostate cancer based on needle biopsy, such as:

(a) basal epithelial cell markers, such as cytokeratins K5 and K14 - these can be used to help distinguish benign from

malignant glands in prostate tissue samples;

(b) immunohistochemical staining for AMACR, a prostate cancer biomarker discovered through cDNA microarray transcriptome

profiling, can aid in prostate cancer diagnosis;

However, neither of these immunohistochemistry reagents perfectly distinguishes prostate cancer: the absence of basal epithelial cell markers is not always diagnostic of prostate cancer, and AMACR expression is absent in some prostate cancers and present in PIN.

High-grade PIN is a likely precursor to prostate cancer and is a lesion characterized by the proliferation of malignant-appearing prostate epithelial cells within the confines of otherwise normal glandular structures. HG-PIN is identified in about 5% of men subjected to prostate biopsies. About 96 percent of all men diagnosed with prostate cancer survive at least five years, and 75 percent survive at least 10 years. Many men diagnosed will undergo watchful waiting regimes as opposed to active treatment. The most common treatment options for prostate cancer are surgery or hormonal therapy, depending on the staging at diagnosis. However, the percentage of cancer patients that relapse remains high (44-64%). Treatment is costly and harsh, and there is a need to identify the patients that would benefit from treatment.

There is an unmet need to develop diagnostic and prognostic markers that can identify those patients that are likely to progress at an early stage and to discriminate between low risk and high risk patients. This would avoid over-diagnosis and overtreatment of low risk men as well as avoid unnecessary use of anti-hormonal therapies which can lead to hormone-resistant PC (HRPC) .

The present inventors have recognised that expression of the transcription factor Brfl is closely associated with cancer, for example prostate cancer, and in particular with malignant cancers (e.g. prostate cancers with a Gleason score of 8 or more or a

Gleason grade of 3 or more) . Brfl may therefore be useful in screening, diagnosis and prognosis of cancers, such as prostate cancer .

One aspect of the invention provides a method of assessing an individual for cancer comprising;

providing a sample obtained from said individual, and;

determining the presence or amount of Brfl in one or more cells in said sample.

The presence of Brfl in the sample obtained from the individual may be indicative of said individual having or being at risk of having cancer. For example, the presence of Brfl in the sample may be indicative that the individual has cancer while the absence of one or more cells expressing Brfl in the sample may be indicative that the individual does not have cancer.

In some embodiments, expression of Brfl may be increased relative to controls, such as normal non-cancer cells.

The term cancer may include any type of solid cancer or malignant lymphoma and especially leukaemia, sarcomas, skin cancer, bladder cancer, breast cancer, uterus cancer, testicular cancer, ovary cancer, prostate cancer, lung cancer, colon and colorectal cancer, cervical cancer, liver cancer, head and neck cancer, oesophageal cancer, pancreas cancer, renal cancer, stomach cancer and cerebral cancer. Preferred cancers include prostate cancer, ovarian cancer, colon cancer and testicular cancer. In more preferred embodiments, a cancer as described herein is a prostate cancer.

Methods as described herein may be useful in distinguishing between cancer and non-cancer conditions. For example, prostate cancer and non-cancer conditions, such as prostate intraepithelial neoplasia and benign prostatic hyperplasia, may be distinguished.

Methods as described herein may be useful in distinguishing between benign cancer and malignant or aggressive cancer. For example, benign prostate cancer and malignant or aggressive prostate cancer may be distinguished. This may inform decisions about future treatment regimens .

The presence of one or more cells expressing Brfl in the sample obtained from the individual may be indicative of said individual having aggressive or malignant cancer, for example a prostate cancer with a Gleason score of 4, 5, 6, 7, 8 or more; or a Gleason grade of 3 or more.

An individual identified as having a malignant or aggressive cancer as described herein may be subjected to further treatment, for example by surgery or hormonal therapy, in accordance with standard medical practice. An individual identified as having a non- malignant or benign cancer may be subjected to mild or no further treatment and/or increased monitoring.

Brfl may therefore be useful as a stratification marker to identify patients who require increased monitoring or treatment.

The expression of Brfl may be useful in determining the prognosis of a cancer in an individual and the likely progress of the condition and the risk of morbidity and/or mortality. For example, the presence of one or more cells expressing Brfl in the sample may be indicative that the individual has a poor prognosis and a high risk of mortality and the absence of one or more cells expressing Brfl in the sample may be indicative that the individual has a good prognosis and a low risk of mortality.

An individual may be healthy or asymptomatic or may be suspected of or at risk of suffering from cancer, for example prostate cancer.

In other embodiments, an individual may be suffering from cancer and/or may have one or more symptoms of cancer, for example prostate cancer .

The individual may have been previously diagnosed as having cancer or suspected of having cancer by one or more diagnostic tests. For example, the individual may have been previously diagnosed as having prostate cancer or suspected of having prostate cancer by one or more prostate cancer tests, such as serum PSA testing; digital rectal examination (DRE) ; and/or immunohistochemical staining for AMACR or basal epithelial cell markers, such as cytokeratins K5 and K14. In some embodiments, the individual may be undergoing treatment for cancer and methods of the invention may be useful in determining the progress of the cancer treatment.

The Brfl biomarker described herein may be used in conjunction with known biomarkers. A method may further comprise determining the level, presence or expression of one or more other cancer markers. For example, a method of assessing prostate cancer may further comprise determining the level, presence or expression of one or more other prostate cancer markers, such as PSA, AMACR or basal epithelial cell markers, such as cytokeratins K5 and K14.

Methods and reagents for detecting these biomarkers are well known in the art.

A sample obtained from an individual preferably comprises one or more cells from the individual, preferably prostate cells. The sample may be a tissue sample, for example a biopsy from a cancerous tissue as described above, in particular a needle biopsy, or, in certain contexts, a non-cancerous tissue. The presence or amount of Brfl in one or more cells in the sample may be determined.

In other embodiments, a sample may be a sample of body fluid, such as saliva, cerebrospinal fluid, blood, urine or semen. For example, a sample of urine may be collected following digital rectal examination to dislodge cells into the urinary tract.

Brfl is a 90 kD polypeptide that is an essential component of the transcription factor TFIII B. It binds directly to RNA polymerase III and serves to recruit it to promoters. The TFIII B

transcription factor plays a central role in transcription

initiation by RNA polymerase III on genes encoding tRNA, 5S rRNA, and other small structural RNAs . Brfl (GenelD 2972) may have the amino acid sequence of Genbank accession numbers of NP_001510.2 GI : 22035556 (SEQ ID NO: 2) or NP_663718.1 GI : 22035558 (SEQ ID NO: 4) or may be a variant thereof. A variant Brfl polypeptide may comprise an amino acid sequence which has one or more allelic variations, for example, deletions, insertions or substitutions of one or more amino acids, relative to the wild-type Brfl sequence set out above. A variant polypeptide may have one, two, three, four or more mutations relative to the wild-type sequence. For example, a variant Brfl polypeptide may have at least 80%, at least 85%, at least 90%, at least 95% at least 98% or at least 99% sequence identity with a Brfl polypeptide sequence as set out above.

Brfl may be encoded by the nucleotide sequence of NM_001519.2 GI : 22035555 { SEQ ID NO: 1) or NM__145685.2 GI : 148833509 (SEQ ID NO:3) or a variant thereof. A variant Brfl nucleic acid may comprise a nucleotide sequence which has one or more allelic variations, for example, deletions, insertions or substitutions of one or more nucleotides, relative to the wild-type Brfl nucleotide sequence, as set out above. A variant nucleic acid may have one, two, three, four or more mutations relative to the wild-type sequence. For example, a variant nucleic acid may have at least 80%, at least 85%, at least 901, at least 95%, at least 98% or at least 99% sequence identity with a Brfl nucleotide sequence as set out above.

An allelic variant of a reference Brfl amino acid sequence of a biomarker may differ from a reference sequence by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20-30 or 30-50 amino acids. An allelic variant of a reference Brfl nucleotide sequence may differ from a reference nucleotide sequence of a biomarker by insertion, addition, substitution or deletion of 1 nucleotide, 2, 3, 4, 5-10, 10-20 20-30 or 30-50 nucleotides .

A method of assessing an individual for cancer according to the invention may thus comprise; providing a sample of cells obtained from the individual, and; determining the presence in the sample of one or more cells expressing Brfl and/or the number or proportion of cells in the sample of expressing Brfl. The presence, number or proportion of cells expressing Brfl may be indicative of said individual having or being at risk of having cancer. The presence, number or proportion of cells expressing Brfl may be indicative of the prognosis of the cancer in the individual i.e. the presence of one or more such cells may be indicative of that the cancer is aggressive or malignant cancer.

For example, a method of assessing an individual for prostate cancer may comprise; providing a sample of cells obtained from the individual, and; determining the presence in the sample of one or more cells expressing Brfl and/or the number or proportion of cells in the sample expressing Brfl. The presence, number or proportion of cells expressing Brfl may be indicative of said individual having or being at risk of having prostate cancer. The presence, amount or proportion of cells expressing Brfl may be indicative of the prognosis of the prostate cancer in the individual i.e. the presence, amount or proportion of cells expressing Brfl may be indicative that the prostate cancer is aggressive or malignant prostate cancer.

The expression of Brfl may be determined at the protein level by determining the presence or amount of Brfl polypeptide in the sample. For practical purposes, or at least commercial purposes bearing in mind cost and time, assessment of target protein expression at the protein level may be preferred over assessment at the nucleic acid level.

A method of determining the presence, absence or level of Brfl polypeptide in a sample from an individual, may include contacting the sample with a specific binding member which specifically binds to the Brfl polypeptide, and determining binding of the specific binding member to the sample. Binding of the specific binding member to the sample may be indicative of the presence or amount of Brfl within the sample.

An antibody or other binding member which specifically binds to an antigen such as Brfl may not show any significant binding to molecules other than the antigen. In some cases, an antibody may specifically bind to a particular epitope which is carried by a number of antigens, in which case the antibody will be able to bind to the various antigens carrying the epitope.

Samples to be subjected to a contact with a binding member in accordance with methods of the invention may be prepared using any available technique which allows binding of a specific binding molecule to the Brfl polypeptide. Various techniques are standard in the art.

Binding may be detected using any method or detectable label described herein. This, and any other binding detection method described herein, may be interpreted directly by the person performing the method, for instance, by visually observing a detectable label. Alternatively, this method, or any other binding detection method described herein, may produce a report in the form of an autoradiograph, photograph, computer printout, flow cytometry report, graph, chart, test tube, container or well containing the result, or any other visual or physical representation of a result of the method.

The amount of binding of the binding member to Brfl in the sample may be determined. Screening for Brfl binding and/or the

quantitation thereof may be useful, for instance, in screening patients for cancers and/or any other disease or disorder involving increased Brfl expression and/or activity.

A diagnostic or prognostic method may comprise (i) obtaining a tumour, tissue or fluid sample from a patient, (ii) exposing said tumour, tissue or fluid sample to one or more binding members which bind Brfl; and (iii) detecting the amount of binding of the binding member to the sample, as compared with a control sample, wherein an increased amount of binding as compared with controls may indicate increased or elevated Brfl expression in the sample.

The reactivities of a binding member such as an antibody on normal and test samples may be determined by any appropriate means.

Tagging with individual reporter molecules is one possibility. The reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals. The linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently . Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding binding molecule (e.g. antibody) and reporter molecule.

One favoured mode is by covalent linkage of each binding member with an individual fluorochrome, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable

fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine .

Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes that catalyse reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed. Further examples are horseradish peroxidase and chemiluminescence . The mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge. Many different assay formats suitable for detecting Brfl are known in the art, including non-competitive and competitive assays and immunoassays. Suitable approaches include immunohistochemical staining, immunocytochemical staining, Western Blotting,

immunofluorescence, enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies. All of these approaches are well known in the art. In some embodiments, an assay for Brfl in a sample may involve the use of a capture binding member with binds Brfl and a detection binding member which either binds to a different epitope on Brfl than the first binding member or which binds to the capture binding member. Suitable Brfl binding members may be produced using routine techniques or obtained from commercial sources (e.g. Novus

Biologicals Ltd, Cambridge UK; Abeam Ltd, UK) .

In some convenient assay formats, the capture binding member may be immobilised on a solid substrate and the detection binding member may be non-immobilised.

Preferred binding members for use in aspects of the present invention include antibodies and fragments or derivatives thereof ( ^antibody molecules' ) . Antibody molecules are well known in the art and are described in more detail below.

A binding member may be immobilised to the solid support in a number of different ways known in the art. For example, the binding member may be adsorbed directly to the solid support, e.g. through

electrostatic and/or hydrophobic interactions, such as in the case of plastic solid supports. Alternatively, the binding member may be covalently attached to the solid support. In this case, the solid support may be chemically modified to introduce or activate

functional chemical groups on the surface of the support, such as hydroxyl or amine groups, and the support crosslinked using

crosslinking agents such as gluteraldehyde, to facilitate covalent binding of the first member to the solid support. In a further alternative, the binding member may be indirectly attached to the solid support by a specific binding interaction, for example by an interaction between biotin and avidin, or by immobilising protein A or protein G to the solid support followed by specific binding to the binding member itself.

The capture binding member which binds Brfl may be immobilised at a solid support. The immobilised binding member may then be brought into contact with a sample of interest. If the sample contains Brfl, it will bind with the immobilised binding member. The detection binding member which binds Brfl is then added and allowed to bind. In other embodiments, the capture binding member is not immobilised and the detection binding member may be a binding member which binds Brfl at a different epitope from the capture binding member or a binding member which binds to the capture binding member. To allow detection, the second binding member may be labelled with a detectable label. Alternatively, binding of the second binding member may be determined using a third binding member, such as an antibody, which specifically binds to the second binding member and is labelled with a detectable label. For example, Brfl in the sample may be bound by the capture binding member, and immobilised at the support. Second binding member binds to the captured Brfl and is itself immobilised at the support. In some embodiments, labelled third binding member may bind to the second binding member and also be immobilised at the support. Brfl in the sample may be bound by the capture binding member. Second binding member binds to the captured Brfl or the capture antibody and is itself detected via a label or labelled third binding member binds to the second binding member and is detected. The third binding member may, for example, directly bind to the second binding member (e.g. an anti-IgG antibody) or may bind to an affinity tag which is linked or fused to the second binding member.

Suitable affinity tags include biotin or peptidyl sequences, such as MRGS(H)₆, DYKDDDDK (FLAG™), T7-, S- ( KETAAAKFERQHMDS ) , poly-Arg

(R5-6), poly-His (H2-10), poly-Cys (C4) poly-Phe ( Fll ) poly-Asp(D5- 16), Strept-tag II (WSHPQFEK) , c-myc (EQKLISEEDL) , Influenza-HA tag (Murray, P. J. et al (1995) Anal Biochem 229, 170-9), Glu-Glu-Phe tag (Stammers, D. K. et al (1991) FEBS Lett 283, 298-302), Tag.100 (Qiagen; 12 aa tag derived from mammalian MAP kinase 2), Cruz tag 09™ (MKAEFRRQESDR, Santa Cruz Biotechnology Inc.) and Cruz tag 22™ (MRDALDRLDRLA, Santa Cruz Biotechnology Inc.).

The amount of labelled second or third binding member bound directly or indirectly to the solid support is then measured, whereby the amount of labelled binding member detected is directly proportional to the amount of Brfl in the sample.

For example, a method of measuring Brfl in a sample may comprise; contacting the sample with a capture binding member with binds Brfl, and;

determining binding of said capture binding member to Brfl in the sample using a detection binding member which binds Brfl or a detection binding member which binds to the capture binding member. In other embodiments, the expression of Brfl may be determined at the nucleotide level by determining the presence or amount of Brfl nucleic acid, for example mRNA, encoding the Brfl polypeptide in the sample . Numerous methods are available for determining the presence or absence in a sample of a Brfl nucleic acid sequence and may for example include oligonucleotide probe binding, DNA amplification, for example by PCR, or microarray hybridisation. All of these approaches are well known in the art.

Another aspect of the present invention provides for a method of categorising a sample from an individual as (i) normal, (ii) potentially or actually pre-cancerous or cancerous, dysplastic, or neoplastic and benign or (iii) cancerous and malignant, the method including determining binding to a sample of a specific binding member directed against a Brfl polypeptide. The pattern or degree of binding may be compared with that for a known normal sample and/or a known abnormal sample.

A method may be used to characterise a cell, for example a cancer cell, as malignant, for example in the prognosis of a prostate cancer. Thus, binding of (e.g.) an anti- Brfl specific binding member to a sample provides for categorising the tissue from which the sample is derived as potentially or actually pre-cancerous or cancerous, benign, dysplastic or neoplastic or cancerous and potentially malignant and metastatic. The method may be used to pre screen samples before further analysis or testing. The method may also be useful for screening or analysis of samples previously tested using another prostate cancer diagnostic techniques.

A specific binding molecule which binds Brfl, for example an antibody molecule, may be provided in a kit, which may include instructions for use in accordance with a method of the invention and methods for interpreting and analyzing the data resulting from the performance of the assay. In the kit, the binding member may b labelled to allow its reactivity in a sample to be determined, e.g. as described further below. Further, the binding member may or may not be attached to a solid support. Such kits are provided as a further aspect of the invention. One or more other reagents may be included, such as labelling molecules, and so on (see below) . The reagents may include a second, different binding member which binds to the first specific binding member and is conjugated to a

detectable label (e.g., a fluorescent label, radioactive isotope or enzyme) . Antibody-based kits may also comprise beads for conducting an immunoprecipitation . Reagents may be provided within containers, which protect them from the external environment, such as a sealed vial. A kit may include one or more articles for providing or preparing the test sample itself, depending on the tissue of interest. A kit may include any combination of, or all of, a blocking agent to decrease non-specific staining, a storage buffer for preserving binding molecule activity during storage, staining buffer and/or washing buffer to be used during antibody staining, a positive control, a negative control and so on. Positive and negative controls may be used to validate the activity and correct usage of reagents employed in accordance with the invention and which may be provided in a kit. Controls may include samples, such as tissue sections, cells fixed on coverslips and so on, known to be either positive or negative for the presence of Brfl. The design and use of controls is standard and well within the routine

capabilities of those of ordinary skill in the art.

Methods of assessing a cancer condition as described herein may be employed before, during and/or after a course of prostate cancer treatment in order to determine the progress and/or effectiveness of the treatment; to determine the most appropriate treatment or level of monitoring for the individual.

Antibody molecules and other binding members that bind specifically to Brfl polypeptide may be useful both in the screening diagnosis and prognosis of prostate cancer. Antibodies may also be useful in the treatment of prostate cancer.

Antibodies that are specific for a Brfl polypeptide may be obtained using techniques that are standard in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep, monkey or bird such as chicken) with the protein or a fragment thereof, or a cell or virus that expresses the protein or fragment. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to Brfl. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, 1992, Nature 357: 80-82) . The production of specific monoclonal antibodies is also well established in the art.

As an alternative or supplement to immunising a mammal with a peptide, an antibody specific for a target may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous

bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047. The library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with the target or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest (or a fragment thereof) .

Antibodies may be modified in a number of ways. Indeed, unless context precludes otherwise, the term "antibody" should be construed as covering any specific binding substance having an antibody antigen-binding domain. Thus, this covers antibody fragments, derivatives, and functional equivalents, including any polypeptide comprising an immunoglobulin binding domain, whether natural or synthetic. Chimaeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimaeric antibodies are described in EP-A-0120694 and EP-A-0125023. Example antibody fragments, capable of binding an antigen or other binding partner are the Fab fragment consisting of the VL, VH, CI and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(ab')2 fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included. Recombinant expression of polypeptides, including antibody

molecules, is well-known in the art.

In some embodiments, an anti-Brfl antibody may be useful in the treatment of cancer, such as prostate cancer. In some therapeutic embodiments, an antibody molecule which is specific for Brfl may be conjugated or bound to a cytotoxic agent. Binding of the antibody molecule to the Brfl may be used to selectively target the cytotoxic agent to a cancer cells. Many suitable cytotoxic agents are known in the art.

Determination of binding to Brfl polypeptide in vivo may be used to identify localisations of cancer cells in the body. Labelled binding members against Brfl may be administered to an individual and binding within the body determined. When a radionucleotide such as Iodine-125, Indium-Ill, Thallium-201 or Technetium-99m is attached to an antibody or other binding members, and the antibody or other binding member localises preferentially in tumour rather than normal tissues, the presence of radiolabel in tumour tissue can be detected and quantitated using a gamma camera or scintigraphy. Radiolabelling with technetium-99m is described in Pak et al (1992), Nucl . Med. Biol. 19; 699-677. A review of cancer imaging with antibodies is provided by Goldenberg D.M., Int. J. of Biol. Markers 1992, 7; 183-188. The present invention of course extends to binding members directed against Brfl as disclosed, for use in any such in vivo method. As described above, Brfl has further been shown by the inventors to be associated with the malignancy in cancer, such as prostate cancer. Thus, methods of the invention may be useful in the

prognosis of an individual having cancer, such as prostate cancer.

Another aspect of the invention provides a method of determining the malignancy of a cancer in an individual, the method comprising,

determining the presence or absence of Brfl expression in a sample obtained from the individual.

For example, the presence or amount of Brfl polypeptide or nucleic acid may be determined in the sample. The presence of Brfl nucleic acid or polypeptide may be determined as described above.

Brfl expression may be indicative that the cancer is malignant (e.g. a prostate cancer having a Gleason grade of 3 or more and/or a Gleason score of 5, 6, 7, 8 or more). The absence of Brfl expression or the absence of Brfl above a threshold value relative to controls may be indicative that the cancer is benign (e.g. it has a Gleason score of 4 or less and/or a Gleason grade of less than 3) .

Another aspect of the invention provides a method for assessing the responsiveness of a cancer in an individual to treatment may comprise :

measuring the amount or expression of Brfl in samples obtained from the individual before and after said treatment, as described above,

wherein a decrease in the level of Brfl is indicative that the individual is responsive to the treatment.

The level or amount of Brfl may be measured in a first sample obtained from the individual before said administration as described above and in a second sample obtained from the individual after said administration. A difference, for example a decrease, between the level or amount of Brflin the first and second samples, may be indicative that the disease condition is responsive to said

treatment .

Another aspect of the invention provides a method for assessing the responsiveness of a cancer in an individual to treatment comprising:

(a) administering the cancer therapy to the individual; and,

(b) measuring the expression of Brfl in a sample obtained from the individual following said administration.

A reduction in the expression of Brfl following administration of the cancer therapy may be indicative of the efficacy of the therapy in the individual. A method for monitoring the treatment of cancer in individual may comprise :

(a) subjecting the individual to a regimen of cancer

treatment; and

(b) monitoring in samples obtained from the individual the level or amount of Brfl using a method described above during said treatment,

wherein a reduction in the level or amount of Brfl in samples obtained during the treatment is indicative that the regimen is effective for treating the cancer in the individual.

In the absence of sustained changes in the level or amount of Brfl in samples obtained from the patient during the treatment, the method may further comprise;

(c) altering the regimen of cancer treatment and subjecting the individual to the altered regimen;

(d) monitoring the level of Brfl in samples obtained from the individual using a method described herein, and

(e) repeating steps c) and d) until a sustained change in the level of Brfl is observed. wherein a change in the level or amount of Brfl which is sustained during the cancer treatment, for example a reduction, is indicative that the altered regimen is effective for treating the cancer in the individual.

In the presence of sustained changes in the level or amount of Brfl during the cancer treatment, the method may further comprise;

(c) altering the regimen of treatment and subjecting the individual to the altered regimen;

(d) monitoring the level of Brfl in samples obtained from the individual using a method described herein, and

(e) repeating steps c) and d) until a maximal change in the level of Brfl is observed.

wherein a maximal change in the level or amount of Brfl which is sustained during the treatment is indicative that the altered regimen is effective for treating the cancer in the individual.

Measurement of Brfl may also be useful in identifying a cohort of patients for clinical trials.

A method for identifying a cohort of patients may comprise:

(a) identifying a population of patients having cancer,

(b) measuring the amount of Brfl in samples obtained from the patients in the population as described above;

(c) identifying samples containing an amount of Brfl which is above or below a threshold value, and;

(d) identifying from the identified samples a cohort of patients . The identified cohort of patients may all have high or low levels of Brfl (i.e. levels above or below the threshold value).

Suitable treatments which may be monitored and/or assessed in a patient as described above are well known in the art and include cytotoxic regimes of drugs, such as topoisomerases (e.g. topotecan, irinotecan, rubitecan), alkylating agents (e.g. DTIC, temozolamide ) and platinum based drugs (e.g. carboplatin, cisplatin) and/or ionising radiation. The treatment of individuals using such agents and methods is well-known in the art.

Another aspect of the invention provides a method of treating cancer, such as prostate cancer, in an individual, the method comprising inhibiting the activity of Brfl polypeptide in one or more cells of said individual.

The activity or function of Brfl may be inhibited, for example, by administering an antagonist of Brfl to said individual. A suitable antagonist may be an antibody molecule, peptide or small organic molecule .

An antagonist which acts on Brfl may be specifically targeted to a tumour site using conventional techniques.

Suitable Brfl antagonists include antibody molecules as described above.

An alternative approach to inhibition employs regulation at the nucleic acid level to inhibit activity or function by down- regulating production of Brfl. The activity of Brfl polypeptide may be inhibited by reducing or abolishing expression of Brfl

polypeptide using anti-sense or RNAi technology.

Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of Brfl polypeptide so that its expression is reduced or completely or substantially completely prevented. In addition to targeting coding sequence, anti-sense techniques may be used to target control sequences of a gene, e.g. in the 5' flanking sequence, whereby the antisense oligonucleotides can interfere with expression control sequences. The construction of antisense sequences and their use is described for example in Peyman and Ulman, Chemical Reviews, 90:543-584, (1990) and Crooke, Ann. Rev. Pharmacol. Toxicol., 32:329-376, (1992).

Oligonucleotides may be generated in vitro or ex vivo for

administration or anti-sense RNA may be generated in vivo within cells in which down-regulation is desired. Thus, double-stranded DNA may be placed under the control of a promoter in a "reverse

orientation" such that transcription of the anti-sense strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the sense strand of the target gene. The complementary anti- sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein. Whether or not this is the actual mode of action is still uncertain. However, it is established fact that the technique works . The complete sequence corresponding to the coding sequence in reverse orientation need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding or flanking sequences of a gene to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A suitable fragment may have about 14-23 nucleotides, e.g. about 15, 16 or 17.

An alternative to anti-sense is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression; Angell & Baulcombe (1997) The EMBO Journal

16,12:3675-3684; and Voinnet & Baulcombe (1997) Nature 389: pg 553). Double stranded RNA (dsRNA) has been found to be even more effective in gene silencing than both sense or antisense strands alone (Fire

A. et al Nature, Vol 391, (1998)). dsRNA mediated silencing is gene specific and is often termed RNA interference (RNAi) . RNA interference is a two-step process. First, dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs (~2nt) . The siRNAs target the corresponding mRNA sequence

specifically for destruction (Zamore P.D. Nature Structural Biology, 8, 9, 746-750, (2001)

RNAi may be also be efficiently induced using chemically synthesized siRNA duplexes of the same structure with 3 '-overhang ends (Zamore PD et al. Cell, 101, 25-33, (2000)). Synthetic siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologeous genes in a wide range of mammalian cell lines

(Elbashir SM. et al. Nature, 411, 494-498, (2001))

Another possibility is that nucleic acid is used which on

transcription produces a ribozyme, able to cut nucleic acid at a specific site - thus also useful in influencing gene expression. Background references for ribozymes include Kashani-Sabet and

Scanlon, 1995, Cancer Gene Therapy, 2(3): 213-223, and Mercola and Cohen, 1995, Cancer Gene Therapy, 2(1), 47-59. Thus, an inhibitor of Brfl activity may comprise a nucleic acid molecule comprising all or part of the or wild type Brfl coding sequence or the complement thereof

Such a molecule may suppress the expression of the Brfl polypeptide and may comprise a sense or anti-sense Brfl coding sequence or may be a Brfl specific ribozyme, according to the type of suppression to be employed.

The type of suppression will also determine whether the molecule is double or single stranded and whether it is RNA or DNA.

Whether it is a polypeptide, peptide, nucleic acid molecule, small molecule or other pharmaceutically useful compound that is to be given to an individual, administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors .

A Brfl antagonist may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

Pharmaceutical compositions may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

The sequences of the database references recited in this

specification are also incorporated herein by reference in their entirety for all purposes.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures and tables described below.

Figure 1 shows immunohistochemical (ICH) analysis with a polyclonal antibody against Brfl on biopsy material from 149 patients with prostate cancer and 21 patients with benign prostate hyperplasia. Brfl levels are elevated significantly (P<0.001) in the cancer samples .

Figure 2 shows with a polyclonal antibody against Brfl on biopsy material from 21 patients with benign prostate hyperplasia and 149 patients with prostate cancer, split according to stage.

Figure 3 shows nuclear Brfl levels are predictive of survival in the 149 patients with prostate cancer analysed. Figure 4 shows immunohistochemical analysis of Brfl expression in normal prostate (upper) and a prostate cancer (lower) .

Figure 5 shows immunohistochemical analysis of Brfl expression in a prostate tissue section, where normal tissue (left) can be seen adjacent to tumour (right) .

Figure 6 shows immunohistochemical analysis of Brfl (lower) and Ki67 (upper) expression in a prostate cancer section (Gleason grade 4). Figure 7 shows immunohistochemical analysis of Brfl expression in sections of benign prostate hyperplasia (upper) and Gleason grade 5 prostate cancer (lower) .

Figure 8 shows immunohistochemical analysis of Brfl expression in sections of normal tissue (A and C) and tumour (B and D) from human testis (A and B) or ovary (C and D) .

Figure 9 shows immunohistochemical analysis of Brfl expression in colonic crypt cells before and 4 days after induction of colon carcinogenesis by cre-mediated deletion of the APC tumour

suppressor .

Experimental

A polyclonal antibody against Brfl was raised and used in immuno- histochemistry (IHC) on biopsy material of a cohort of 149 patients with prostate cancer and 21 with benign prostate hyperplasia (BPH) . The cohort of 149 patients had different grades of carcinoma and were all untreated. Brfl was observed to be significantly increased (P<0.001) in the cancer samples (Figure 1).

A breakdown of the 149 patients with carcinoma presented in Figure 1 is shown in Figure 2. Brfl elevation is seen in the earliest stages of cancer (Gleason grade 1-2) and does not change significantly as the disease progresses.

The survival curves were derived from the same 149 patients and are shown in Figure 3. Patients with low Brfl staining have a median survival of 3402 days, whereas the median is only 875 days for the patients with high Brfl staining. Ki67 and Brfl staining are not well correlated, showing that Brfl is not simply an indicator of proliferation .

These findings show that Brfl might be considered as a useful biomarker for prognosis and diagnosis and screening of prostate cancer .

Immunohistochemical analysis of Brfl expression was performed in normal prostate tissue and a prostate cancer. Brfl expression was found to be elevated in prostate cancer relative to normal prostate tissue (figure 4).

Immunohistochemical analysis of Brfl expression was performed in a prostate tissue section, where normal tissue (left) can be seen adjacent to tumour (right) . Brfl expression was found to be elevated in prostate cancer relative to normal prostate tissue (Figure 5) .

Immunohistochemical analysis of Brfl and Ki67 expression in a prostate cancer section (Gleason grade 4) was performed. Ki67 is a marker of cell proliferation (figure 6). It is clear that Brfl staining is not restricted to cells that stain positively for Ki67 (black) . This indicates that Brf1 elevation in cancers is not merely a consequence of cell proliferation. Brfl is therefore over- expressed in prostate cancers independently of cell proliferation

Immunohistochemical analysis of Brfl expression was performed on sections of benign prostate hyperplasia and Gleason grade 5 prostate cancer. Brfl expression was found to be elevated in malignant relative to benign prostate tissue (figure 7).

Immunohistochemical analysis of Brfl expression was performed in sections of normal tissue and tumour from human testis or ovary. Brfl staining was found to be elevated in the tumours relative to the normal tissue, indicating Brfl overexpression in the tumour tissue (figure 8). Elevated Brfl has also been detected in cases of human skin, cervical and endometrial cancer, as well as B cell lymphoma .

Immunohistochemical analysis of Brfl expression was performed in colonic crypt cells before (upper) and 4 days after (lower) induction of colon carcinogenesis by cre-mediated deletion of the APC tumour suppressor (figure 9). Brfl levels were found to be highly induced at a very early stage of carcinogenesis in mouse model of colon cancer initiation.

PCR analysis was performed on human prostate cancer cell lines (PC- 3) to detect Brfl expression. Brfl expression was detected in human prostate cancer cell lines following RT-PCR, but not in control cells . SEQUENCES

1 cggctgcgct caccggtagg ccccgctcgg gttccgccga agcccagccc ccgcaggtcg

61 cccctccgac gccggcgcgc cgcaagggag gccagctcgc tcgcagtggg gaggtcgcgg 121 ctccagtcct cgcgtccccg ccgtggtccc ggtgcctgtc ccatcccgcg ggcgcggccg 181 ttgcgggcgg cgcggcccgg gcgcgcgcgg cgaatctgcg gctgcgaatc ggctggagcg 241 gggcctcgcg agagcggcga ggcctgggcg gctgggcctg ggcgggcggc gcgggctgct 301 ccggaggctc gggtggcttg agagtcttgg gaggctccgg gcgctgcccg cgcggtcgcc 361 ggcatgacgg gccgcgtgtg ccgcggttgc ggcggcacgg acatcgagct ggacgcggcg 421 cgcggggacg cggtgtgcac cgcctgcggc tcagtgctgg aggacaacat catcgtgtcc 481 gaggtgcagt tcgtggagag cagcggcggc ggctcctcgg ccgtgggcca gttcgtgtcc 541 ctggacggtg ctggcaaaac cccgactctg ggtggcggct tccacgtgaa tctggggaag 601 gagtcgagag cgcagaccct gcagaatggg aggcgccaca tccaccacct ggggaaccag 661 ctgcagctga accagcactg cctggacacc gccttcaact tcttcaagat ggccgtgagc 721 aggcacctga cccgcggccg gaagatggcc cacgtgattg ctgcctgcct ctacctggtc 781 tgccgtacgg agggcacgcc gcacatgctc ctggacctca gcgacctgct ccaggtgaat 841 gtgtacgtgc ttggaaagac gtttcttctc ttggcaagag agctctgcat caatgcgccg 901 gccatagacc cgtgcctgta tattccacgc tttgcgcacc tgctggaatt cggggagaag 961 aaccacgagg tgtccatgac tgccctgagg ctcctacaga ggatgaagcg ggactggatg 1021 cacacaggcc ggcgcccctc gggcctctgc ggagcagcgc tcctggttgc agccagaatg 1081 catgacttca ggaggactgt gaaggaggtc atcagtgtgg tcaaagtgtg tgagtccacg 1141 ctgcggaaga ggctcacgga atttgaagac acccccacca gtcagttgac cattgatgag 1201 ttcatgaaga tcgacctgga ggaggagtgc gaccccccct cgtacacagc tgggcagagg 1261 aagctgcgga tgaagcagct tgaacaagtc ctgtcaaaaa aactggagga ggttgaaggt 1321 gaaatatcca gttaccagga tgcaattgag attgaactag aaaacagccg gccaaaggcc 1381 aaggggggcc tggccagcct ggcaaaagat ggctccaccg aggacaccgc gtccagcttg 1441 tgtggcgagg aggacacaga ggacgaggag ctggaagccg cggccagcca cctgaacaaa 1501 gacttatacc gggagctcct tggtggtgcc cccggcagct cggaagcagc aggaagcccc 1561 gagtggggcg gcagacctcc ggccctgggg tccctgctgg accccctccc cactgcagcc 1621 agcctgggca tctcagactc catccgcgaa tgcatctcct ctcagagcag cgaccccaaa 1681 gatgcttcag gagacggtga gctggacctc agtggcattg atgacctgga gattgacagg 1741 tacatcctga atgagtcgga agcccgcgtg aaggccgagc tgtggatgag ggagaacgcc 1801 gagtacctgc gggaacagag ggaaaaagaa gcaagaatag cgaaagagaa ggagctcggc 1861 atctacaagg aacacaagcc caagaagtct tgcaagcgac gggagccaat tcaggccagt 1921 accgccaggg aggccatcga gaagatgctg gagcagaaga agatctccag caagatcaat 1981 tatagcgtgc tccggggcct cagcagcgcc ggcgggggca gtccgcacag ggaggatgca 2041 cagcccgagc atagcgccag tgccaggaag ctgtcacgaa ggaggacgcc ggccagcaga 2101 agtggggctg accctgtgac cagtgtgggg aaaaggttga ggcctctggt gtctacgcag 2161 ccagcaaaga aggtggccac gggagaggct ttgctcccaa gctctcccac cctcggagct 2221 gagcctgcca ggccccaggc ggtgctggtg gagagcgggc ccgtgtcata ccacgccgac 2281 gaggaggctg acgaggagga gcctgacgag gaggacgggg agccctgcgt cagtgccctg 2341 cagatgatgg gcagcaacga ctatggctgt gatggcgatg aggacgacgg ctactgaagt 2401 gtggcctcca ggcaggtgat gtcctggcag ggggcctcgc gggtctcctc agcatcagac 2461 gggcttccag gaccgcagca ggcaggcccc agcgccgaga ctcctggtga caggtggcac 2521 ctgtcccaca gccctcgtcc catgtggaac ttaccattgg gattgtgttt ctattcagca 2581 agggaaaccg gaccaagcgt ctgcatgtgt gtgatcagat gtgggccggg tgtgtgcagg 2641 gctgggtccc gctgcctgcc gtcgactcat ccaaggaccc tccaaggctg gcagtgtggt 2701 gttgctacta ttaaggaaac aggcttgggg cagccccact gctggtccaa gtgtgtggag 2761 ggctgagtgt gctggccctg tgactcagga ccagctctgg agtctccagc ccaccctccg 2821 caccgtcccc tcctgagcag cactcggcgc cagcagcctc tgccagagtg gaagccagag 2881 ccctgcaggt gtccggcgca gccgtgggag ctgaggatct ggcacttgag aggcagcagc 2941 tccttgaagg tcctctgcct ccagctgtgg ccctgcatcc agatacctgc ctcgtccgag 3001 gcagacaccc ccacccctgc ctcctccaga cccccctccc cgctgcctgc accgcctgga 3061 gcagcatggg ggtcagaccc ctgctccagg gccacttgag ttgtgggccc aggagccctg 3121 cggctgccgg caggtgaact gagtgcccga cagctgagac cggcgcccac ccgtcctgag 3181 catagctctg taggcagtgc gggcatagcc tgcatagtgt cctggcgctg ggagttcccc 3241 gtggacagag ccagagggca gtggcgctcc ctgtcagagc tggatcaggc cccccatcga 3301 ggagggaggg cagacggagg cccgagagcc tccccaggcc tcttcgtggg aaggccccag 3361 taccactcgt aggaggtctc agctctggca tggctgcccc ggatgtggcc gagggggctt 3421 caccctgtgt ccttaggagg gggtggcctt gaggcagagc cgtgcctcac tgacccccag 3481 gggcctcatc ctccccatgg aatgggctgt atgtcctgcc ccaacttggc ccgcagcagg 3541 ccagaccccc ctacccccgc ccagagctca gtagccagcc tgggtcctgc cagggcttct 3601 cgagggcttg ggggaagaat agatttagta aagcaggaag atctgttgtt acttaacaga SEQ ID NO:l (NM 001519.2 GI:22035555)

1 mtgrvcrgcg gtdieldaar gdavctacgs vledniivse vqfvessggg ssavgqfvsl

61 dgagktptlg ggfhvnlgke sraqtlqngr rhihhlgnql qlnqhcldta fnffkmavsr

121 hltrgrkmah viaaclylvc rtegtphmll dlsdllqvnv yvlgktflll arelcinapa

181 idpclyiprf ahllefgekn hevsmtalrl lq mkrdwmh tgrrpsglcg aallvaaritih

241 dfrrtvkevi svvkvcestl rkrltefedt ptsqltidef mkidleeecd ppsytagqrk

301 Irmkqleqvl skkleevege issyqdaiei elensrpkak gglaslakdg stedtasslc

361 geedtedeel eaaashlnkd lyrellggap gsseaagspe wggrppalgs lldplptaas

421 lgisdsirec issqssdpkd asgdgeldls giddleidry ilnesearvk aelwmrenae

481 ylreqrekea riakekelgi ykehkpkksc krrepiqast areaiekmle qkkisskiny

541 svlrglssag ggsphredaq pehsasarkl srrrtpasrs gadpvtsvgk rlrplvstqp

601 akkvatgeal lpssptlgae parpqavlve sgpvsyhade eadeeepdee dgepcvsalq

661 mmgsndygcd gdeddgy

SEQ ID N0:2 NP 001510.2 GI:22035556

1 gtggcatggg gcactggctt tgctgatgag gagaccagac ttcagaggcc gtgtcacctg 61 gcctgggccg cacagctggt gcatgccaag ctcgcgtgca gacgaagctc cagaggcctg 121 gctgtcagga gggttcctcc ccgaggacac acacgcaacc tcctggaccg ccaggcttta 181 gacttttaaa acttccatat gcaggaaacc ccacaggacg cagccagcat ctcgggtcat 241 gggtgcacct gtcaccgcag tcctgggcta aacagtgaac aggagctctg tgtctctgtg 301 gtcgttacag ttgtagtgaa tgttccctgg gagagtcctc ttcagaagat atttgcggta 361 ttttcaacaa aggactctcc aggatggagg agcaactaca agtcaggaag gaaaaggctg 421 acaacccagt tttttaaaaa acgggcaaag gacgtgaaca gacaccatgc aatggcccag 481 agtgcacaaa tcatcatttc tcccaggaat ggggtgcccg agcccccagg cggctgaact 541 tgagacttgt cacgtagtgt tggtggaatg cagagcagct ggagatcacc tgctgcaggt 601 gggtgcgtaa ctccgcacgg ccaagccgtt tggaaagctg tagcatatgc atgagagtcc 661 ggaaagttcc gagagaaagt gtgtggatgc acccgccaag gacacaccca ccgaggccac 721 agctgcgatt tcatagtgtg ggaccccact ggcaaagaca cgctggctgc tgtggccgtg 781 gtttctgtcc cagccgtctg tttccataga aaaagacccg tgcctgtata ttccacgctt 841 tgcgcacctg ctggaattcg gggagaagaa ccacgaggtg tccatgactg ccctgaggct 901 cctacagagg atgaagcggg actggatgca cacaggccgg cgcccctcgg gcctctgcgg 961 agcagcgctc ctggttgcag ccagaatgca tgacttcagg aggactgtga aggaggtcat 1021 cagtgtggtc aaagtgtgtg agtccacgct gcggaagagg ctcacggaat ttgaagacac 1081 ccccaccagt cagttgacca ttgatgagtt catgaagatc gacctggagg aggagtgcga 1141 ccccccctcg tacacagctg ggcagaggaa gctgcggatg aagcagcttg aacaagtcct 1201 gtcaaaaaaa ctggaggagg ttgaaggtga aatatccagt taccaggatg caattgagat 1261 tgaactagaa aacagccggc caaaggccaa ggggggcctg gccagcctgg caaaagatgg 1321 ctccaccgag gacaccgcgt ccagcttgtg tggcgaggag gacacagagg acgaggagct 13S1 ggaagccgcg gccagccacc tgaacaaaga cttataccgg gagctccttg gtggtgcccc 1441 cggcagctcg gaagcagcag gaagccccga gtggggcggc agacctccgg ccctggggtc 1501 cctgctggac cccctcccca ctgcagccag cctgggcatc tcagactcca tccgcgaatg 1561 catctcctct cagagcagcg accccaaaga tgcttcagga gacggtgagc tggacctcag 1621 tggcattgat gacctggaga ttgacaggta catcctgaat gagtcggaag cccgcgtgaa 1681 ggccgagctg tggatgaggg agaacgccga gtacctgcgg gaacagaggg aaaaagaagc 1741 aagaatagcg aaagagaagg agctcggcat ctacaaggaa cacaagccca agaagtcttg 1801 caagcgacgg gagccaattc aggccagtac cgccagggag gccatcgaga agatgctgga 1861 gcagaagaag atctccagca agatcaatta tagcgtgctc cggggcctca gcagcgccgg 1921 cgggggcagt ccgcacaggg aggatgcaca gcccgagcat agcgccagtg ccaggaagct 1981 gtcacgaagg aggacgccgg ccagcagaag tggggctgac cctgtgacca gtgtggggaa 2041 aaggttgagg cctctggtgt ctacgcagcc agcaaagaag gtggccacgg gagaggcttt 2101 gctcccaagc tctcccaccc tcggagctga gcctgccagg ccccaggcgg tgctggtgga 2161 gagcgggccc gtgtcatacc acgccgacga ggaggctgac gaggaggagc ctgacgagga 2221 ggacggggag ccctgcgtca gtgccctgca gatgatgggc agcaacgact atggctgtga 2281 tggcgatgag gacgacggct actgaagtgt ggcctccagg caggtgatgt cotggcaggg 2341 ggcctcgcgg gtctcctcag catcagacgg gcttccagga ccgcagcagg caggccccag 2401 cgccgagact cctggtgaca ggtggcacct gtcccacagc cctcgtccca tgtggaactt 2461 accattggga ttgtgtttct attcagcaag ggaaaccgga ccaagcgtct gcatgtgtgt 2521 gatcagatgt gggccgggtg tgtgcagggc tgggtcccgc tgcctgccgt cgactcatcc 2581 aaggaccctc caaggctggc agtgtggtgt tgctactatt aaggaaacag gcttggggca 2641 gccccactgc tggtccaagt gtgtggaggg ctgagtgtgc tggccctgtg actcaggacc 2701 agctctggag tctccagccc accctccgca ccgtcccctc ctgagcagca ctcggcgcca 2761 gcagcctctg ccagagtgga agccagagcc ctgcaggtgt ccggcgcagc cgtgggagct 2821 gaggatctgg cacttgagag gcagcagctc cttgaaggtc ctctgcctcc agctgtggcc 2881 ctgcatccag atacctgcct cgtccgaggc agacaccccc acccctgcct cctccagacc 2941 cccctccccg ctgcctgcac cgcctggagc agcatggggg tcagacccct gctccagggc 3001 cacttgagtt gtgggcccag gagccctgcg gctgccggca ggtgaactga gtgcccgaca 3061 gctgagaccg gcgcccaccc gtcctgagca tagctctgta ggcagtgcgg gcatagcctg 3121 catagtgtcc tggcgctggg agttccccgt ggacagagcc agagggcagt ggcgctccct 3181 gtcagagctg gatcaggccc cccatcgagg agggagggca gacggaggcc cgagagcctc 3241 cccaggcctc ttcgtgggaa ggccccagta ccactcgtag gaggtctcag ctctggcatg 3301 gctgccccgg atgtggccga gggggcttca ccctgtgtcc ttaggagggg gtggccttga 3361 ggcagagccg tgcctcactg acccccaggg gcctcatcct ccccatggaa tgggctgtat 3421 gtcctgcccc aacttggccc gcagcaggcc agacccccct acccccgccc agagctcagt 3481 agccagcctg ggtcctgcca gggcttctcg agggcttggg ggaagaatag atttagtaaa 3541 qcaggaagat ctgttgttac ttaacagaaa aaaaaaaaaa aaaaa

SEQ ID NO:3 NM 145685.2 GI : 148833509 1 mtalrllqrm krdwmhtgrr psglcgaall vaarmhdfrr tvkevisvvk vcestlrkrl

61 tefedtptsq ltidefmkid leeecdppsy tagqrklrmk qleqvlskkl eevegeissy

121 qdaieielen srpkakggla slakdgsted tasslcgeed tedeeleaaa shlnkdlyre

181 llggapgsse aagspewggr ppalgslldp Iptaaslgis dsirecissq ssdpkdasgd

241 geldlsgidd leidryilne searvkaelw mrenaeylre qrekeariak ekelgiykeh

301 kpkksckrre piqastarea iekmleqkki sskinysvlr glssagggsp hredaqpehs

361 asarklsrrr tpasrsgadp vtsvgkrlrp Ivstqpakkv atgeallpss ptlgaeparp

421 qavlvesgpv syhadeeade eepdeedgep cvsalqmmgs ndygcdgded dgy

SEQ ID NO: 4 NP 663718.1 GI : 22035558

Claims

Claims :

1. A method of assessing an individual for cancer comprising; providing a sample obtained from said individual, and;

determining the presence, amount or expression of Brfl one or more cells in said sample.

2. A method according to claim 1 wherein the presence of Brfl in the sample obtained from the individual may be indicative of said individual having or being at risk of having cancer.

3. A method according to claim 1 wherein the presence of Brfl in the sample obtained from the individual may be indicative of said individual having malignant cancer. . A method according to any one of the preceding claims wherein the individual is asymptomatic and the presence of Brfl is diagnostic for cancer.

5. A method according to any one of the preceding claims wherein the individual has cancer and the presence of Brfl is prognostic for malignant or aggressive cancer.

6. A method according to any one of the preceding claims further comprising determining the presence of one or more of PSA, AMACR or cytokeratins K5 and K14 in the sample.

7. A method according to any one of the preceding claims wherein the sample is a needle biopsy.

8. A method according to any one of the preceding claims wherein the sample is a urine sample.

9. A method according to any one of the preceding claims wherein expression of Brfl is determined by determining the presence or amount of Brfl polypeptide in the sample. 10. A method according to claim 9 comprising contact the sample with an antibody molecule which specifically binds Brfl and determining the binding of the antibody molecule to the sample.

11. A method according to any one of the preceding claims wherein expression of Brfl is determined by determining the presence or amount of Brfl nucleic acid in the sample.

12. A method for assessing the responsiveness of a cancer in an individual to treatment comprising:

(a) administering the e cancer therapy to the individual; and,

(b) measuring the expression of Brfl in a sample obtained from the individual following said administration.

13. A method of treating cancer in an individual, the method comprising inhibiting the activity of Brfl polypeptide in one or more cells of said individual.

14. A method according to any one of claims 1 to 13 wherein the cancer is selected from prostate cancer, colorectal cancer, ovarian cancer and testicular cancer.