WO2018073382A1

WO2018073382A1 - Method for predicting prostate cancer metastasis

Info

Publication number: WO2018073382A1
Application number: PCT/EP2017/076786
Authority: WO
Inventors: Tianyu GUO; Lei Xu; Xueying Mao; Yong-Jie Lu
Original assignee: Queen Mary University of London
Current assignee: Queen Mary University of London
Priority date: 2016-10-19
Filing date: 2017-10-19
Publication date: 2018-04-26
Anticipated expiration: 2019-04-19
Also published as: GB201617723D0

Abstract

The present application relates to methods for predicting cancer metastasis using a combination of circulating tumour cells (CTCs) and prostate specific antigen (PSA). The present invention also provides methods of treatment of patients that have been diagnosed as being at risk of mestastasis according to a method of prognosis of the invention.

Description

METHOD FOR PREDICTING PROSTATE CANCER METASTASIS

The present application relates to methods for predicting cancer metastasis using a combination of circulating tumour cells (CTCs) and prostate specific antigen (PSA).

Prostate cancer is the most common cancer and the second most frequent cause of cancer-related death in European and North American men, with 35,000 new cases annually in the UK (according to Cancer Research UK). With the use of the PSA blood test to screen men for prostate cancer, small and organ-confined prostate cancers have been increasingly detected (Cuzick ef al. Lancet Oncol 2014; 15:e484-92 and Mulhem ef al. Am Fam Physician 2015; 92:683-8). With this improvement of early diagnosis in prostate cancer, many non-symptomatic indolent cancers are diagnosed. These cancers progress slowly and patients typically die of other causes (Cuzick et al. (2014) and Filson et al. CA Cancer J Clin 2015; 65:265-82). Therefore, the majority of prostate cancers confined within the prostate gland are indolent and most patients with localised cancer die with cancer, rather than as a result of cancer. However, if the disease develops into the metastatic stage, no treatment is currently available to save patients' lives (Lorente et al. Eur J Cancer 2014; 50:753-64). Hence, it is critical to predict which patients with localized cancer will develop or host an occult metastasis, the main cause of death from prostate cancer. Radionuclide bone-scan and computed tomography have been so far the gold standard to detect metastatic sites, but they are costly, time-consuming, and expose patients to radiation. Patients who are unlikely to have metastasis are better off avoiding these costly and potentially harmful procedures. Based on the observed relationship between PSA level and bone metastases (O'Sullivan ef al (2003) BJU Int 92, 685-689; Kosuda ef al (2002) Cancer 94, 964-972) recent National Comprehensive Cancer Network (NCCN) guidelines recommend bone scans to be carried out on patients with T1 stage and PSA > 20 ng/mL or T2 stage and PSA > 10 ng/mL (Mohler ef al (2016) J Natl Compr Cane Netw 14, 19-30).

Methods for predicting cancer metastasis based on patient blood samples which are better than PSA are therefore desirable. Such methods may include using CTCs. Current CTC technologies, such as CellSearch^® (the only FDA approved system), mainly rely on the capture and identification of those CTCs that express epithelial phenotype-specific markers epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK). However, CellSearch^® has previously been reported to detect fewer than two CTCs in many metastatic prostate cancer patients (Scher ef al. Lancet Oncol. 10, 233-239 (2009)). Furthermore, EpCAM antibody based CTC isolation methods may miss CTCs which have undergone epithelial to mesenchymal transition (EMT). This is an important limitation of CTC isolation methods since EMT is critical for cancer cell migration and invasion. A size and deformability based CTC isolation system, Parsortix™, has been optimized to capture CTCs based on the much larger size of tumor cells than the surrounding blood cells. Parsortix™ is described in EP2790020, which relates to methods and apparatus for segregating circulating tumor cells from blood cells in a whole blood sample. The efficiency of Parsortix™ in harvesting epithelial CTCs has been compared to CellSearch^® and IsoFlux, as well as its ability in isolation of CD45 negative (CD45-) circulating cells with mesenchymal properties (Xu ef al PLoS One 10, e0138032 (2015)). However, it is not presently possible to confirm the malignancy of CTCs whilst simultaneously confirming their identity using a marker based system.

Summary of the invention

To further investigate CTCs with mesenchymal features, the inventors have developed a novel technique to perform multiple rounds of fluorescence in situ hybridization (FISH) on the same slides after immunofluorescence staining in order to simultaneously identify epithelial and mesenchymal cell features and changes at multiple genomic regions. Use of this technique allows confirmation of the malignancy of CTCs with a mesenchymal phenotype. The role of CTCs with mesenchymal cell features in risk prediction has also been determined for either newly diagnosed or progressed prostate cancer patients.

The data in the present application show a strong association between PSA and metastasis, with a cut-off of 23 ng/mL as the best predictor. Although none of the analyses of CTCs alone outperformed PSA for metastatic correlation, a combined risk score (CRS) based on both PSA level and the amount of CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs (EMTing CTC count), significantly improved the metastasis prediction accuracy compared to PSA alone (92 % compared to 82%). This suggests that although PSA level correlates with CTC count, they are independent factors contributing to cancer metastasis, potentially by representing different aspects of tumour biology. This also represents an unexpected improvement over the currently available methods for predicting prostate cancer metastasis since the combination of two biomarkers which are associated typically either does not improve, or results in only a slight increase in prediction efficacy. Accordingly, the methods of the invention, especially involving a CRS based on both the amount of PSA and the amount of CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs may provide a marked advance in the clinical management of prostate cancer.

In a first aspect of the invention there is provided a method for predicting prostate cancer metastasis in a subject comprising measuring the amount of CTCs and the amount of PSA in one or more blood samples from the subject.

In a second aspect of the invention there is provided a method of treating cancer in a subject for whom cancer metastasis has been predicted according to a method of the first aspect of the invention. The method further comprises administering a therapeutic agent to the subject and/or adopting a therapeutic regimen. In a third aspect of the invention there is provided a combination of CTCs and PSA for use in the diagnosis of metastatic prostate cancer.

In a further aspect of the invention there is provided use of a combination of CTCs and PSA isolated from a subject in an ex vivo and/or in vitro method of predicting cancer metastasis.

In a further aspect of the invention there is provided use of a combination of CTCs and PSA isolated from a subject in an ex vivo and/or in vitro method of diagnosing metastatic cancer. In a still further aspect of the invention there is provided a stripping buffer comprising 1 % to 3 % sodium dodecyl sulfate (SDS), 0.055 M to 0.075 M Tris-HCI and 0.5 % to 1.1 % β-mercaptoethanol and wherein the stripping buffer has a pH from 6.6 to 7.0. The stripping buffer may comprise 2 % SDS, 0.0625 M Tris-HCI and 0.8 % β-mercaptoethanol, wherein the stripping buffer has a pH of 6.8 In a still further aspect of the invention there is provided a kit comprising specific binding molecules which bind to one or more of PSA, CD45, CK and/or vimentin (VIM).

Reference is made to a number of Figures as follows: Figure 1 - Representative images for different populations of detected cells in prostate cancer patients following five rounds of FISH in a CTC. (A) Immunofluorescence image for three types of circulating cells. Top row: One CK+/VIM-/CD45- cell (epithelial type, arrowed) adjacent to one CD45+ lymphocyte. Middle row: Two CK+/VIM+/CD45- cells (undergoing epithelial to mesenchymal transition (EMTing type), arrowed) adjacent to one CD45+ lymphocyte. Bottom row: Two CK-/VIM+/CD45- cells (mesenchymal type, arrowed) adjacent to one CD45+ lymphocyte. (B) Image following five rounds of FISH on one CTC post-immunostaining. (a): a CK-/VIM+/CD45- circulating cell, (b): first round of FISH: 6q16 (upper left arrow) and AR (remaining three arrows), (c): second round FISH for ERG rearrangement: RP1 1-476D17 (upper right arrow) and RP1 1-95121 (left most arrow); lower right arrow indicates substantially colocalised RP1 1-476D17 and RP1 1-95121. (d): third round FISH: C-MYC (upper left and lower right arrows) and NKX3.1 (middle arrow between upper left and lower right arrows), (e): fourth round FISH: RB1 (leftmost and rightmost arrows) and PTEN (top-centre and bottom-centre arrows), (f): fifth round FISH: CCND1 (arrows pointing towards top-left) and 16q22.1 (arrows pointing to top-right and bottom- left). FISH signals are indicated by arrows as described.

Figure 2 - CTC count correlation with cancer metastases. CTCs were categorized into epithelial CTCs, EMTing CTCs, mesenchymal CTCs, total CK+ CTCs, total VIM+ CTCs, and total CTCs. Figure 3 - Area under the ROC curve (AUC) of PSA alone and CRS for metastasis prediction.

Figure 4 - Box plots of CRS in patients without and with metastasis. Median (IQR: Q75- Q25%) value of CRS for patients without and with metastasis were 0.12 (0.21 1-0.076) and

1.023 (4.668-0.645). Five CRS values (12.324, 13.88, 18.888, 55.742 and 73.15) from patients with metastasis are not shown in the figure due to out of above scale. Wilcoxon rank-sum test showed significant difference in distribution (χ12 = 42.61 , p = 0.0001 , AUC = 0.921 (95% CI: 0.858-0.985)).

Detailed description of the invention

The present invention provides a method for predicting prostate cancer metastasis in a subject. The method comprises measuring the amount of CTCs and the amount of PSA in one or more samples from the subject. Usually, the sample is a blood sample. In some embodiments, if a metastasis and/or metastatic cancer is predicted, diagnosed or suspected, the methods of the invention may further comprise treating the patient for the cancer.

The invention also provides a combination of CTCs and PSA for use in the diagnosis of metastatic prostate cancer.

The invention also provides the use of a combination of CTCs and PSA isolated from a subject in an ex vivo and/or in vitro method of predicting prostate cancer metastasis. The invention also provides the use of a combination of CTCs and PSA isolated from a subject in an ex vivo and/or in vitro method of diagnosing metastatic prostate cancer.

As used herein the terms "measure" and "measuring" are used interchangeably with the terms "count" and "counting" in the context of measuring an amount or number of CTCs. The measuring may be manual (for example visually counting) or automated (for example detecting using a computer system and/or computer implemented method). The measuring may also be semi- automated (for example using a computer system and/or computer implemented method which requires essential inputs from a user or decisions made by a user). PSA

PSA may be measured accordingly to methods known in the art and/or the methods described herein. PSA measurement is typically by immunoassay. The immunoassay may be a two-site immunoenzymatic "sandwich" assay such as the Access Hybritech PSA assay (Beckman Coulter, Fullerton, CA.) The immunoassay may be a chemoluminscent, chromogenic or florescent assay. Accordingly, measuring the amount of PSA may comprise use of one or more anti-PSA antibodies. The anti-PSA antibodies may be raised in any suitable animal such as mouse, rabbit, rat, hamster, chicken or goat. The anti-PSA antibodies may be labelled. For example, the anti-PSA antibodies may be conjugated to an enzymatic label such as alkaline phosphatase or to paramagnetic particles.

Measuring the amount of PSA may also comprise use of a substrate. The substrate may be a chemolumiescent substrate, such as Lumi-Phos^** 530. Typically, the light production by reaction of the chemolumiescent substrate with the enzymatic label is proportional to the concentration of PSA in the sample. The amount of PSA in the sample may be determined by means of a multipoint calibration curve. Use of a calibration curve may comprise use of a weighted four parameter standard curve with a direct relationship of measured light produced (RLU) to concentration of PSA protein in the sample.

The amount of PSA may be expressed as a concentration. Typically the amount of PSA is expressed as a concentration in ng/mL. The amount of PSA typically refers to the serum concentration of PSA. Accordingly, the methods of the invention may comprise determining a serum PSA concentration. The amount of PSA may alternatively be expressed as, for example, a concentration in whole blood. The concentration in whole blood may be determined by direct measurement of other means. For example, the whole blood concentration of PSA may be proportionally adjusted from the serum concentration of PSA in a manner known to the person skilled in the art. In some embodiments, greater than or equal to a threshold amount of PSA is predictive of cancer metastasis in a subject. Alternatively, greater than a threshold amount of PSA may be predictive of cancer metastasis in a subject. The threshold amount may be a suitable value on which to establish whether a cancer has metastasised or will metastasise or is likely to metastasise. The threshold amount may be a predetermined amount of PSA in a blood sample which has been experimentally identified as providing a useful distinguishing point between subjects with high likelihood of metastasis and subjects with a low likelihood of metastasis. The data presented in the present application indicate that a serum PSA concentration of about 23 ng/mL may be a preferred threshold value where the different likelihoods of metastasis begin to become evident. Accordingly, in some embodiments, the threshold amount of PSA is about 23 ng/mL. Alternatively, in some embodiments, the threshold amount of PSA is about 22 ng/mL or about 24 ng/mL. Accordingly, the threshold amount of PSA may be from about 22 ng/mL to about 24 ng/mL.

Other threshold values may also be used, such as 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 ng/mL of PSA. The amount of PSA may be an average (e.g. median, mean, mode). Therefore, non-integer threshold values are also possible. In some embodiments, any integer or non-integer threshold value from 3 ng/mL to 2,000 ng/mL may be used.

The threshold amounts of PSA typically refer to the amount of PSA in blood serum. Accordingly, the methods of the invention may comprise determining a serum PSA concentration of at least a threshold concentration. The concentration is typically presented in ng/mL.

Circulating tumour cells (CTCs) The methods of the invention further comprise measuring the amount of CTCs in the blood sample. CTCs are cells that have been shed into the vasculature or lymphatics from a primary tumour or other cancer lesions and are carried around the body in the circulation. CTCs thus constitute seeds for the subsequent growth of additional tumours (metastases) in distant organs. Although defined as CTCs in the present application it will be apparent that the CTCs in a blood sample are not, strictly speaking, circulating once the blood sample has been taken. Cells defined herein as CTCs which possess any of the features described herein may simply be referred to as "cells characterised by [any of the above features of CTCs combined in any combination]".

CTCs may be have an epithelial phenotype (epithelial CTCs) a mesenchymal phenotype (mesenchymal CTCs) and may also be partially transitioned between epithelial and mesenchymal phenotypes (epithelial-mesenchymal CTCs, CTCs undergoing epithelial to mesenchymal transition (EMT) or EMTing CTCs). Accordingly, the CTCs may be partially transitioned from epithelial CTCs to mesenchymal CTCs. In some embodiments the CTCs are CD45 negative, CK positive and/or vimentin positive. Accordingly, the CTCs may be CD45 negative, CK positive and vimentin positive. Typically CTCs which are CD45 negative, CK positive and vimentin positive are partially transitioned from epithelial CTCs to mesenchymal CTCs. In some embodiments the CTCs are CD45 negative, CK negative and/or vimentin positive. Accordingly, the CTCs may be CD45 negative, CK negative and vimentin positive. Typically CTCs which are CD45 negative, CK negative and vimentin positive are mesenchymal CTCs.

In some embodiments the CTCs are CD45 negative, CK positive and/or vimentin negative. Accordingly, the CTCs may be CD45 negative, CK positive and vimentin negative. Typically CTCs which are CD45 negative, CK positive and vimentin negative are epithelial CTCs.

CD45 is expressed by leukocytes. CTCs may therefore be CD45 negative cells. Epithelial CTCs, CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs and mesenchymal CTCs may each be CD45 negative cells. CK is an epithelial marker which may be expressed by epithelial CTCs. CK is down-regulated during the epithelial to mesenchymal transition. Accordingly CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs may express CK. Mesenchymal CTCs typically do not express CK. Mesenchymal CTCs may therefore be CK negative cells.

Vimentin is a mesenchymal marker which may be expressed by mesenchymal CTCs. Vimentin is up-regulated during the epithelial to mesenchymal transition. Accordingly CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs may express vimentin. Epithelial CTCs typically do not express vimentin. Epithelial CTCs may therefore be vimentin negative cells.

In some embodiments, greater than or equal to a threshold amount of CTCs is predictive of cancer metastasis in a subject. Alternatively, greater than a threshold amount of CTCs may be predictive of cancer metastasis in a subject. The threshold amount may be a suitable value on which to establish whether a cancer has metastasised or will metastasise or is likely to metastasise. The threshold amount may be a predetermined amount of CTCs in a blood sample which has been experimentally identified as providing a useful distinguishing point between subjects with high likelihood of metastasis and subjects with a low likelihood of metastasis. The data presented in the present application indicate that the following amounts of CTCs per 7.5 mL of sample may be preferred threshold values where the different likelihoods of metastasis begin to become evident:

(a) 2 CTCs per 7.5 mL of sample, wherein the CTCs are CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs;

(b) 2 CTCs per 7.5 mL of sample, wherein the CTCs are mesenchymal CTCs;

(c) 3 CTCs per 7.5 mL of sample, wherein the CTCs are epithelial CTCs;

(d) 5 CTCs per 7.5 mL of sample, wherein the CTCs are CK positive CTCs;

(e) 2 CTCs per 7.5 mL of sample, wherein the CTCs are vimentin positive CTCs; and

(f) 7 CTCs per 7.5 mL of sample, wherein the CTCs are all CTCs. However, other threshold values may also be used, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 CTCs per 7.5 mL of sample. It will be appreciated that the threshold values may scale proportionately when the volume of the sample changes. For example, 2 CTCs per 7.5 mL of sample is equivalent to 4 CTCs per 15 mL of sample. Accordingly, in some embodiments, the threshold amount of CTCs per 7.5 mL of sample is selected from the group consisting of:

(b) 2 CTCs per 7.5 mL of sample, wherein the CTCs are mesenchymal CTCs;

(c) 3 CTCs per 7.5 mL of sample, wherein the CTCs are epithelial CTCs; (d) 5 CTCs per 7.5 mL of sample, wherein the CTCs are CK positive CTCs;

(f) 7 CTCs per 7.5 mL of sample, wherein the CTCs are all CTCs.

In some embodiments, the threshold amount of CTCs per 7.5 mL of sample is selected from the group consisting of:

(a) 2 CTCs per 7.5 mL of sample, wherein the CTCs comprise CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs;

(b) 2 CTCs per 7.5 mL of sample, wherein the CTCs comprise mesenchymal CTCs;

(c) 3 CTCs per 7.5 mL of sample, wherein the CTCs comprise epithelial CTCs;

(d) 5 CTCs per 7.5 mL of sample, wherein the CTCs comprise CK positive CTCs;

(e) 2 CTCs per 7.5 mL of sample, wherein the CTCs comprise vimentin positive CTCs; and

(f) 7 CTCs per 7.5 mL of sample, wherein the CTCs comprise all CTCs.

(a) 2 CTCs per 7.5 mL of sample, wherein the CTCs consist of CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs;

(b) 2 CTCs per 7.5 mL of sample, wherein the CTCs consist of mesenchymal CTCs;

(c) 3 CTCs per 7.5 mL of sample, wherein the CTCs consist of epithelial CTCs;

(d) 5 CTCs per 7.5 mL of sample, wherein the CTCs consist of CK positive CTCs;

(e) 2 CTCs per 7.5 mL of sample, wherein the CTCs consist of vimentin positive CTCs; and

(f) 7 CTCs per 7.5 mL of sample, wherein the CTCs consist of all CTCs.

In the embodiments above, as elsewhere, the epithelial CTCs, CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs and mesenchymal CTCs may be defined by the features of each phenotype described herein.

Accordingly, greater than 2, 3, 5 or 7 CTCs per 7.5 mL of sample may predict prostate cancer metastasis in a subject. Alternative values may apply, so that 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 CTCs per 7.5 mL of sample may predict prostate cancer metastasis in a subject.

The amount of CTCs per sample may be an average (e.g. median, mean, mode). Therefore, non- integer threshold values are also possible. In some embodiments, any integer or non-integer threshold value from 1 to 20 CTCs per 7.5 mL of sample may be used. Non-integer threshold values may also scale proportionately when the volume of the sample changes. For instance, 9.5 CTCs per 7.5 ml_ of sample is equivalent to 19 CTCs per 15 mL of sample and 10.5 CTCs per 7.5 mL of sample is equivalent to 21 CTCs per 15 mL of sample.

The step of measuring the amount of CTCs in a blood sample according to the method of the invention may involve measuring the amount of epithelial CTCs, mesenchymal CTCs and/or CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs.

In some embodiments, the methods of the invention further comprise comparing the amount of one type of CTCs to the amount of another type of CTCs to each other. The comparison of the amount of one type of CTCs to the amount of another type of CTCs may involve the amount of epithelial CTCs, mesenchymal CTCs and/or CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs. Typically, the comparison involves calculating the difference between the amount of one type of CTCs and the amount of another type of CTCs, for example by subtracting the amount of one type of CTCs from the amount of another type of CTCs. Alternatively, the difference may be calculated as the amount of one type of CTCs minus the square of the amount of another type of CTCs.

Alternatively, the difference between the amount of one type of CTCs and the amount of another type of CTCs may involve calculating the ratio or the fraction of one type of CTCs to the amount of another type of CTCs. Accordingly, it will be appreciated that as used herein the term "difference" may be substituted with the term "ratio", "fraction" or "fold difference".

Comparing the amount of CTCs and PSA to each other The method may comprise comparing the amount of PSA with the amount of CTCs. The comparison of the amount of PSA with the amount of CTCs may predict prostate cancer metastasis. The amount of PSA may be compared with the amount of any type of CTCs. The amount of PSA may therefore be compared with the CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs, mesenchymal CTCs, epithelial CTCs, all CK positive CTCs, all vimentin positive CTCs and any combination thereof. Accordingly the comparison may be between the amount of PSA and the total amount of CTCs (all CTCs). The data in the present application indicate that the optimal comparison for the purposes of predicting cancer metastasis is the comparison of the amount of PSA with the amount of CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs.

In some embodiments, the method comprises calculating a CRS using the amount of CTCs and the amount of PSA. The CRS may be calculated as described herein. For example, the amount of PSA (or "PSA score") and the amount of CTCs (or "CTC count") may be combined in a logistic regression. The outcome may be metastasis (yes, no). The coefficients obtained from this model may be used to compute the CRS. The data in the present application indicate that the CRS is optimally a combination of the amount of PSA with the amount of CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs.

In some embodiments, greater than or equal to a threshold CRS is predictive of cancer metastasis in a subject. Alternatively, greater than a threshold CRS may be predictive of cancer metastasis in a subject. The threshold amount may be a suitable value on which to establish whether a cancer has metastasised or will metastasise or is likely to metastasise. The threshold amount may be a predetermined CRS which has been experimentally identified as providing a useful distinguishing point between subjects with high likelihood of metastasis and subjects with a low likelihood of metastasis. The data presented in the present application indicate that a CRS threshold of about 0.357 may be a preferred threshold value where the different likelihoods of metastasis begin to become evident. Accordingly, in some embodiments, the threshold amount CRS is about 0.357.

However, other threshold values may also be used. In some embodiments, the threshold CRS is from about 0.2 to about 0.6. In some embodiments, the threshold CRS is from 0.27 to about 0.51. In some embodiments, the threshold CRS is about 0.276, about 0.357 or about 0.508. In some embodiments, the threshold CRS is from about 0.4 to about 0.5. In some embodiments, the threshold CRS is from about 0.35 to about 0.36. The above CRS thresholds may be particularly useful when the CRS is calculated as a combination of the amount of PSA with the amount of CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs. However, suitable CRS thresholds may also be calculated on the basis of the combination of the amount of PSA with the amount of any type of CTCs. Samples

The methods of the invention comprise measuring the amount of CTCs and the amount of PSA in one or more samples from a subject. The term "blood sample" is used herein to refer to blood which originates from a subject and/or which has been isolated from a subject. The blood sample is typically obtained from a vein. The blood sample may be obtained by venupuncture or using a fingerstick. Typically, the blood sample is acquired at the middle of phlebotomy after the collection for routine clinical blood test to avoid contamination with epithelial cells from the skin.

The term "blood sample" may denote any suitable sample of blood known to the person skilled in the art, such as whole blood, blood plasma and blood serum. Typically, the amount of CTCs is determined in a whole blood sample. Typically, the amount of PSA is determined in a blood serum sample. The blood serum may be separated by standard laboratory techniques known to the person skilled in the art. In alternative embodiments of the invention any sample may be used. The sample may be from tissue, cancer tissue, potential cancer tissue, prostate tissue, blood, urine, semen, prostatic secretions, needle aspirations or isolated cells. The isolated cells may be cells originating from a subject, from a blood sample, from prostate tissue, prostatic secretions, or isolated prostate cells.

The methods of the invention optionally comprise the step of obtaining a blood sample from the subject. In some embodiments, the method comprises obtaining a first blood sample from the subject and obtaining a second blood sample from the subject.

In some embodiments, the method comprises measuring the amount of PSA in a first sample and measuring the amount of CTCs in a second sample. Accordingly, in some embodiments the CTCs and PSA are not analysed in the same blood sample. The first and second samples may be derived from the same initial sample but processed separately. Alternatively, the first and second samples may be obtained at different time points and locations. The analysis of CTCs and PSA may be run separately or concurrently, as appropriate. Said comparison step may also detect the presence of particular types of cancer (for example localised prostate cancer, advanced prostate cancer, malignant prostate cancer, aggressive prostate cancer, CRPC or metastatic prostate cancer) and/or determine how extensive the therapeutic intervention should be for each patient. For example, the methods of the invention may detect prostate cancer that is likely to metastasise and distinguish this from prostate cancer that is less likely to metastasise. Alternatively, methods of the invention may detect aggressive prostate cancer and distinguish this from less-aggressive cancer.

Determining the severity of the therapeutic intervention required may be of particular importance in early stage prostate cancer, in order to balance risk with therapeutic benefit. Said comparison step may include comparing the amount of the PSA and/or CTCs with the amount of a biomarker in a control or reference sample.

The control biomarker may be a protein or a cell type, as appropriate. This control may be from the same sample, but be a biomarker different from the biomarker being assayed for predicting metastasis of prostate cancer or diagnosing metastatic prostate cancer (an internal reference).

The comparison step may therefore be with reference to the amount of other circulating proteins or cells within the same sample, which may include but are not restricted to red blood cells, platelets and white blood cells such as lymphocytes. Alternatively, the reference may be a biological sample taken from a healthy subject/individual. Alternatively still, the method may use reference data obtained from samples from the same patient at a previous point in time. In this way, the effectiveness of any treatment can be assessed and a prognosis for the patient determined. However, a control or reference sample is not always required. Accordingly, the invention also provides a method of predicting prostate cancer metastasis in a subject comprising measuring the amount of PSA in a first sample from the subject and measuring the amount of CTCs in a second sample from the subject.

In some embodiments, the method comprises enriching the sample for CTCs. The enriching may comprise selecting for, increasing the concentration of and/or isolating the CTCs. Accordingly, the enriching may be performed by isolating and/or counting the CTCs. The enriching, the isolating and/or the counting may be by size- and/or deformability-based sorting of the CTCs. The enriching may be performed according to any suitable method known to the skilled person, for example using Parsortix™ (as described in, for example EP2790020), Flowcytometry cell sorter, CellSearch^® and/or IsoFlux.

In some embodiments, the size- and/or deformability-based sorting of the CTCs excludes any cells with a dimension below about 10 μΐη. Therefore, the dimension of a CTC may be greater than 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 μιη. Size- and/or deformability-based separation modules and arrays are described below.

A sample (e.g. blood sample) can be enriched for analytes or cells of interest (e.g. CTCs) using one or more any methods known in the art (e.g. Guetta, EM et al. Stem Cells Dev, 13(1 ):93-9 (2004)) or described herein. The enrichment increases the concentration of cells of interest or ratio of cells of interest to other cells in the sample. For example, enrichment can increase concentration of an analyte or cell of interest, such as a CTC, by a factor of at least 2, 4, 6, 8, 10, 20, 50, 100, 200, 500, 1 ,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1 ,000,000, 2,000,000, 5,000,000, 10,000,000, 20,000,000, 50,000,000, 100,000,000, 200,000,000, 500,000,000, 1 ,000,000,000, 2,000,000,000, or 5,000,000,000 fold over its concentration in the original sample. Enrichment can also increase concentration of cells of interest in volume of cells / total volume of sample (removal of fluid). A fluid sample (e.g., a blood sample) of greater than 10, 15, 20, 50, or 100 mL total volume comprising components of interest, and it can be concentrated such that the component of interest into a concentrated solution of less than 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1 , 2, 3, 5, or 10 mL total volume.

Enrichment can occur using one or more types of separation modules. Several different modules are described herein, all of which can be fluidly coupled with one another in the series for enhanced performance. Enrichment may alternatively occur by selective lysis. Enrichment of cells of interest may occur using one or more size and deformability based separation modules. Examples of size- and/or deformability-based separation modules include filtration modules, sieves, matrixes, etc. Examples of size- and/or deformability-based separation modules contemplated by the present invention include those disclosed in International Publication No. WO 2004/1 13877. Other size based separation modules are disclosed in International Publication No. WO 2004/0144651.

A size- and/or deformability-based separation module comprises one or more arrays of obstacles forming a network of gaps. The obstacles are configured to direct particles as they flow through the array/network of gaps into different directions or outlets based on the particle's hydrodynamic size and deformability. For example, as a blood sample flows through an array of obstacles, nucleated cells or low deformability cells having a hydrodynamic size larger than a predetermined size, e.g., 8 microns, are directed to a first outlet located on the same side or the opposite side of the array of obstacles from the fluid flow inlet, while the enucleated cells or cells having a hydrodynamic size smaller than a predetermined size, e.g., 8 microns or high deformability cells with a predetermined size, e.g., 10 microns are directed to a second outlet also located on the opposite side of the array of obstacles from the fluid flow inlet. An array can be configured to separate cells smaller or larger than a predetermined size by adjusting the size of the gaps, obstacles, and offset in the period between each successive row of obstacles. For example, in some embodiments, obstacles or gaps between obstacles can be up to 10, 20, 50, 70, 100, 120, 150, 170, or 200 microns in length or about 2, 4, 6, 8, 9 or 10 microns in length. In some embodiments, an array for size- and/or deformability-based separation includes more than 100, 500, 1 ,000, 5,000, 10,000, 50,000 or 100,000 obstacles that are arranged into more than 10, 20, 50, 100, 200, 500, or 1000 rows. Preferably, obstacles in a first row of obstacles are offset from a previous (upstream) row of obstacles by up to 50% the period of the previous row of obstacles. In some embodiments, obstacles in a first row of obstacles are offset from a previous row of obstacles by up to 45, 40, 35, 30, 25, 20, 15 or 10% the period of the previous row of obstacles. Furthermore, the distance between a first row of obstacles and a second row of obstacles can be up to 10, 20, 50, 70, 100, 120, 150, 170 or 200 microns. A particular offset can be continuous (repeating for multiple rows) or non-continuous. In some embodiments, a separation module includes multiple discrete arrays of obstacles fluidly coupled such that they are in series with one another. Each array of obstacles has a continuous offset. But each subsequent (downstream) array of obstacles has an offset that is different from the previous (upstream) offset. Preferably, each subsequent array of obstacles has a smaller offset that the previous array of obstacles. This allows for a refinement in the separation process as cells migrate through the array of obstacles. Thus, a plurality of arrays can be fluidly coupled in series or in parallel, (e.g., more than 2, 4, 6, 8, 10, 20, 30, 40, 50). Fluidly coupling separation modules (e.g., arrays) in parallel allows for high-throughput analysis of the sample, such that at least 1 , 2, 5, 10, 20, 50, 100, 200, or 500 ml_ per hour flows through the enrichment modules or at least 1 , 5, 10, or 50 million cells per hour are sorted or flow through the device.

In an exemplary size- and/or deformability-based separation module, obstacles (which may be of any shape) are coupled to a flat substrate to form an array of gaps. A transparent cover or lid may be used to cover the array. The obstacles form a two-dimensional array with each successive row shifted horizontally with respect to the previous row of obstacles, where the array of obstacles directs component having a hydrodynamic size smaller than a predetermined size in a first direction and component having a hydrodynamic size larger that a predetermined size in a second direction. For enriching epithelial or circulating tumour cells from enucleated, the predetermined size of an array of obstacles can be get at 6-12 μιη or 6-8 μιη. For enriching CTCs from a mixed sample (e.g. a blood sample) the predetermined size of an array of obstacles can be between 6-12 μιη, 8-10 μιη or 9 μιη. The flow of sample into the array of obstacles can be aligned at a small angle (flow angle) with respect to a line-of-sight of the array. Optionally, the array is coupled to an infusion pump to perfuse the sample through the obstacles. The flow conditions of a size- and/or deformability-based separation module are such that cells are sorted by the array with minimal damage. This allows for downstream analysis of intact cells to be more efficient and reliable.

The size- and/or deformability-based separation module may be a Parsortix™ system. The Parsortix™ system is available commercially as Parsortix PR1 . The Parsortix™ system may be the optimised Parsortix™ system described in Xu et al (2015).

A size- and/or deformability-based separation module may comprise an array of obstacles configured to direct cells larger than a predetermined size to migrate along a line-of-sight within the array (e.g. towards a first outlet or bypass channel leading to a first outlet), while directing cells and analytes smaller than a predetermined size to migrate through the array of obstacles in a different direction than the larger cells (e.g. towards a second outlet).

A variety of enrichment protocols may be utilized, although gentle handling of the cells is preferred to reduce any mechanical damage to the cells or their DNA. This gentle handling may serve to preserve the small number of CTCs in the sample. Integrity of the cells being evaluated is an important feature to permit the distinction between the CTCs and other cells in the sample. In particular, the enrichment and separation of the CTCs using the arrays of obstacles produces gentle treatment which minimizes cellular damage and maximizes nucleic acid integrity permitting exceptional levels of separation and the ability to subsequently utilize various formats to very accurately analyse the genome of the cells which are present in the sample in extremely low numbers.

Enrichment of cells of interest (e.g. CTCs) occurs using one or more capture modules that selectively inhibit the mobility of one or more cells of interest. Preferably, a capture module is fluidly coupled downstream to a size- and/or deformability-based separation module. Capture modules can include a substrate having multiple obstacles that restrict the movement of cells or analytes greater than a predetermined size. Examples of capture modules that inhibit the migration of cells based on size are disclosed in U.S. Patent No. 5,837,1 15 and 6,692,952.

In some embodiments, a capture module includes a two dimensional array of obstacles that selectively filters or captures cells or analytes having a hydrodynamic size greater than a particular gap size (predetermined size), International Publication No. WO 2004/1 13877. In some cases a capture module captures analytes (e.g., cells of interest or not of interest) based on their affinity. For example, an affinity-based separation module that can capture cells or analytes can include an array of obstacles adapted for permitting sample flow through, but for the fact that the obstacles are covered with binding moieties that selectively bind one or more analytes (e.g., cell populations) of interest (e.g., CTCs) or analytes not-of-interest (e.g., white blood cells, red blood cells or epithelial cells). Arrays of obstacles adapted for separation by capture can include obstacles having one or more shapes and can be arranged in a uniform or non-uniform order. A two-dimensional array of obstacles may be staggered such that each subsequent row of obstacles is offset from the previous row of obstacles to increase the number of interactions between the analytes being sorted (separated) and the obstacles.

Binding moieties coupled to the obstacles can include e.g., proteins (e.g., ligands/receptors), nucleic acids having complementary counterparts in retained analytes, antibodies, etc. In some embodiments, an affinity-based separation module comprises a two-dimensional array of obstacles covered with one or more antibodies selected from the group consisting of: anti-PSA, anti-CD45, anti-CK and anti-vimentin. Examples of such affinity-based separation modules are described in International Publication No. WO 2004/029221.

A capture module may utilize a magnetic field to separate and/or enrich one or more analytes (cells) based on a magnetic property or magnetic potential in such analyte of interest or an analyte not of interest. For example, red blood cells which are slightly diamagnetic (repelled by magnetic field) in physiological conditions can be made paramagnetic (attributed by magnetic field) by deoxygenation of the haemoglobin into methaemoglobin. This magnetic property can be achieved through physical or chemical treatment of the red blood cells. Thus, a sample containing one or more red blood cells and one or more CTCs can be enriched for the CTCs by first inducing a magnetic property in the red blood cells and then separating the red blood cells from the CTCs by flowing the sample through a magnetic field (uniform or non-uniform).

For example, a blood sample can flow first through a size based separation module to remove enucleated cells and cellular components (e.g., analytes having a hydrodynamic size less than 6 μιη) based on size. Subsequently, the enriched nucleated cells and red blood cells are treated with a reagent, such as CO2, N2, or NaN02, that changes the magnetic property of the red blood cells' haemoglobin. The treated sample then flows through a magnetic field (e.g., a column coupled to an external magnet), such that the paramagnetic analytes (e.g., red blood cells) will be captured by the magnetic field while the CTCs and any other non-red blood cells will flow through the device to result in a sample enriched in CTCs. Additional examples of magnetic separation modules are described in US Application Serial No. 1 1/323,971 , filed December 29, 2005 entitled "Devices and Methods for Magnetic Enrichment of Cells and Other Particles" and US Application Serial No. 1 1/227,904, filed September 15, 2005, entitled "Devices and Methods for Enrichment and Alteration of Cells and Other Particles".

Subsequent enrichment steps can be used to separate the cells of interest (e.g. CTCs) from the other cell types such as nucleated red blood cells. A sample enriched by size- and/or deformability-based separation followed by affinity/magnetic separation may be further enriched for cells of interest using fluorescence activated cell sorting (FACS) or selective lysis of a subset of the cells.

When the analyte desired to be separated (e.g., PSA and/or CTCs) is not ferromagnetic or does not have a potential magnetic property, a magnetic particle (e.g., a bead) or compound (e.g., Fe³⁺) can be coupled to the analyte to give it a magnetic property. In some embodiments, a bead coupled to an antibody that selectively binds to an analyte of interest can be decorated with an antibody elected from the group of anti-PSA, anti-CK and anti-vimentin. In some embodiments a magnetic compound, such as Fe³⁺, can be couple to an antibody such as those described above. The magnetic particles or magnetic antibodies herein may be coupled to any one or more of the devices herein prior to contact with a sample or may be mixed with the sample prior to delivery of the sample to the device(s). Magnetic particles can also be used to decorate one or more analytes (cells of interest or not of interest) to increase the size prior to performing size- and/or deformability-based separation.

Magnetic fields used to separate analytes/cells in any of the embodiments herein can uniform or non-uniform as well as external or internal to the device. An external magnetic field is one whose source is outside a device herein (e.g., container, channel, obstacles). An internal magnetic field is one whose source is within a device. An example of an internal magnetic field is one where magnetic particles may be attached to obstacles present in the device (or manipulated to create obstacles) to increase surface area for analytes to interact with to increase the likelihood of binding. Analytes captured by a magnetic field can be released by demagnetizing the magnetic regions retaining the magnetic particles. For selective release of analytes from regions, the demagnetization can be limited to selected obstacles or regions. For example, the magnetic field can be designed to be electromagnetic, enabling turn-on and turn-off off the magnetic fields for each individual region or obstacle at will. In some cases, a fluid sample such as a blood sample is first flowed through one or more size- and/or deformability-based separation modules. Such modules may be fluidly connected in series and/or in parallel. For example a first outlet from a separation module can be fluidly coupled to a capture module. The separation module and capture module may be integrated such that a plurality of obstacles acts both to deflect certain analytes according to size and direct them in a path different than the direction of analyte(s) of interest, and also as a capture module to capture, retain, or bind certain analytes based on size, affinity, magnetism or other physical property.

In some embodiments, the enrichment steps performed have a specificity and/or sensitivity greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 99.1 , 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 99.95% The retention rate of the enrichment module(s) herein is such that > 10, 20, 30, 40, 50, 60, 70, 80, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 99.9 % of the analytes or cells of interest (e.g., PSA and/or CTCs) are retained. Simultaneously, the enrichment modules are configured to remove > 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 99.9 % of all unwanted analytes (e.g., red blood-platelet enriched cells) from a sample.

For example, the analytes of interest may be retained in an enriched solution that is less than 50, 40, 30, 20, 10, 9.0, 8.0, 7.0, 6.0, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, or 0.5 fold diluted from the original sample. Any or all of the enrichment steps may increase the concentration of the analyte of interest (PSA and/or CTCs), for example, by transferring them from the fluid sample to an enriched fluid sample (sometimes in a new fluid medium, such as a buffer).

Methods of detection In some embodiments, the presence or absence of PSA, CD45, CK and/or vimentin is determined by immunoassay, flow cytometry, immunofluorescence or immunohistochemistry. The immunoassay, flow cytometry, immunofluorescence or immunohistochemistry may be performed by any suitable method known in the art. In some cases, it is not necessary to isolate or enrich the CTCs and/or the PSA. The number of cells may simply be counted based on expression of any of the markers described herein. The counting may be automated. The counting may be performed by a computer programme.

In some embodiments, the methods further comprise measuring genomic alteration in the CTCs. The measuring genomic alteration may confirm the identity of the CTCs. For example, the measuring genomic alteration may identify one or more mutations of the CTCs and/or confirm the malignancy of the CTCs.

In some embodiments, the measuring genomic alteration comprises use of one or more nucleic acid molecule which hybridises under stringent conditions to a target genomic region. Accordingly, the nucleic acid molecule may be a primer or a probe. In some embodiments, the nucleic acid molecule detects a genomic alteration selected from the group consisting of polyploidy, a copy number gain, a copy number loss and a rearrangement event. For example, the one or more nucleic acid molecule detects a genomic alteration selected from the group consisting of AR gain, PTEN loss, ERG rearrangement, NKX3.1 loss, C-MYC gain, RB1 loss, CCND1 gain, 6q16 loss and 16q22.1 loss.

Accordingly, in some embodiments the target genomic region is selected from the group consisting of AR, PTEN, ERG, NXK3.1, C-MYC, RB1, CCND1, 6q16 and 16q22.1. However, the skilled person will understand that any suitable target genomic region may be selected.

In some embodiments, the PSA and/or CTCs are labelled. In some embodiments, the CTCs are labelled at one or more markers selected from the group consisting of CD45, CK and vimentin. PSA and/or the CTCs may be labelled by the binding of antibodies, antibody fragments and/or apatamers selected from the group consisting of anti-CD45, anti-CK and anti-vimentin. CTCs may also be labelled by a nucleic acid, such as a probe or a primer. Accordingly, the CTCs may be labelled by in situ hybridisation such as FISH.

In some embodiments, the FISH is repeated FISH. Repeated FISH is a procedure which involves repeatedly probing a single sample (for example a slide with fixed cells) according to an in situ hybridisation interspersed with a stripping step. Stripping may be as defined herein. FISH may be performed as described herein or using a known protocol. Repeated FISH generates genomic alteration information for multiple genomic regions in single cells. Repeated FISH can be used to confirm the malignancy of suspicious CTCs, uncovering the heterogeneity of cancer cells by analysing the differences in genomic alterations between individual cells and correlating genomic alterations with cellular features and different types of CTCs to understand mechanisms of metastases. Accordingly, the FISH may be on the same cells after immunofluorescence analysis. in situ hybridisation such as FISH and repeated FISH may be performed on cells after immunofluorescence analysis and stripping. The immunofluorescence analysis may, for example, be used to identify the cells as CTCs.

In some embodiments, the FISH or repeated FISH comprises stripping at 40 to 90 °C with a stripping buffer comprising 1 % to 3 % SDS, 0.055 M to 0.075 M Tris-HCI and 0.5 % to 1 .1 % β- mercaptoethanol and wherein the stripping buffer has a pH from 6.6 to 7.0. The stripping buffer may comprise 2 % SDS, 0.0625 M Tris-HCI and 0.8 % β-mercaptoethanol, wherein the stripping buffer has a pH of 6.8.

In some embodiments, the methods of the invention may further comprise comparing genomic alteration in the CTCs of the sample from the subject with genomic alteration in the CTCs of a control. The control may be a sample from a healthy individual. The comparison may be made by any suitable means known to the person skilled in the art. The comparison may assist in identifying one or more mutations and/or confirming that the CTCs are malignant.

Subjects

As used herein, the terms "subject" and "patient" are used interchangeably to refer to a human or a non-human mammal. The subject may be a companion non-human mammal (i.e. a pet, such as a dog, a cat, a guinea pig, or a non-human primate, such as a monkey or a chimpanzee), an agricultural farm animal mammal, e.g. an ungulate mammal (such as a horse, a cow, a pig, or a goat) or a laboratory non-human mammal (e.g., a mouse and a rat). The invention may find greatest application in connection with the treatment of male human subjects.

In any of the embodiments herein, the subject may be a human. The subject may be a subject undergoing treatment for cancer.

Metastasis

Metastasis of cancer cells is the process by which cancer cells from a malignant primary tumour invade the surrounding tissue and spread out into the body to seed secondary tumours. Secondary tumours are also capable of undergoing metastasis to spread further. Metastasis can also be characterised as the invasiveness potential of a cancer tumour. The invention can therefore be used to predict the invasiveness potential of prostate cancer in a subject.

Accordingly, the invention also relates to preventing or delaying the onset of metastatic disease in patients already diagnosed with a primary prostate tumor or with a proliferative lesion of the prostate. This may be achieved by administering an effective amount of a therapeutic agent or by adopting a therapeutic regimen after prediction of prostate cancer metastasis.

The phrase "predicting prostate cancer metastasis" refers to the process of predicting the likelihood of one or more secondary tumours being present in tissues besides the prostate in a patient. Accordingly, predicting prostate cancer metastasis may refer to determining the likelihood of an existing prostate cancer tumour seeding one or more secondary tumours in tissues excluding the prostate in the future. Alternatively, predicting prostate cancer metastasis may refer to determining the likelihood that an existing prostate cancer tumour will have already seeded one or more secondary tumours in tissues excluding the prostate. Accordingly, the invention also provides a method of detecting unknown, or "occult", secondary prostate tumours. The invention therefore also provides a method of diagnosing metastatic prostate cancer.

The phrase "predicting prostate cancer metastasis" does not refer to the ability to predict the likelihood of metastasis with 100% accuracy. Instead, the phrase "predicting prostate cancer metastasis" refers to an increased probability that a prostate cancer metastasis will occur; that is, that a prostate cancer metastasis is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition. Accordingly, the methods of the invention may further comprise a step of performing one or more scans and/or performing one or more biopsies to confirm metastasis and/or confirm the presence of one or more secondary tumours. The one or more biopsies may result in obtaining one or more biopsy samples. The scanning method and/or biopsy site may be selected on the basis of the tissues to which prostate cancer is known to metastasise. Prostate cancer is known to metastasise to bone, lymph nodes, the urethra, the bladder, the ureters, the rectum, the lungs and the liver. Therefore the scan may be a scan of bone, lymph nodes, urethra, bladder, ureters, rectum, lungs and/or liver. Accordingly, the biopsy site may be a bone, lymph nodes, the urethra, the bladder, the ureters, the rectum, the lungs and/or the liver. The most common site of prostate cancer metastasis is bone. Therefore the scan may be a bone scan. The scan may be a radionuclide bone scan and computed tomography (CT). The biopsy may be a bone marrow biopsy.

Since metastasis is the main cause of death from prostate cancer, predicting prostate cancer metastasis may also provide a determination of prognosis. A prognosis may be expressed as the amount of time a patient can be expected to survive. Alternatively, a prognosis may refer to the likelihood that the disease becomes latent or to the amount of time the disease can be expected to remain latent. Prognosis can be expressed in various ways; for example prognosis can be expressed as a percent chance that a patient will survive after one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, five years, ten years or the like. Alternatively, prognosis may be expressed as the number of months or years on average that a patient can expect to survive as a result of a condition or disease. The prognosis of a patient may be considered as an expression of relativism, with many factors affecting the ultimate outcome. For example, for patients with certain conditions, prognosis can be appropriately expressed as the likelihood that a condition may be treatable or curable, or the likelihood that a disease will go into remission, whereas for patients with more severe conditions prognosis may be more appropriately expressed as likelihood of survival for a specified period of time.

Accordingly, the invention also provides methods of determining prognosis of prostate cancer in a subject comprising measuring the amount of CTCs and PSA in one or more blood samples from the subject. Determining prognosis may also be based on whether a threshold amount of CTCs and/or PSA has been met and/or exceeded. Determining prognosis may also be based on whether a threshold value for a combined risk score (CRS) has been met and/or exceeded. Accordingly, the methods of the invention may comprise the step of determining prognosis according to the amount of the PSA, CTCs and/or the CRS in one or more samples; and/or the comparison of the amount of the PSA, CTCs and/or the CRS with a reference or control; whether a threshold amount of CTCs and/or PSA and/or CRS has been met and/or exceeded.

This method can also be used to determine a patient's suitability for treatment by determining the risk of the patient's cancer metastasising. However, methods of the invention indicate that the cancer is unlikely to metastasise, it may be decided that particular interventions (such as surgery) are not necessary, and the adverse side-effects of such treatment can be avoided.

Measuring the number of metastases may performed in any suitable manner known to the person skilled in the art. For example, measuring the number of metastases involve performing a scan and/or taking a biopsy. The biopsy may be a sample from the subject. The scanning method and/or sample collection site may be selected on the basis of the tissues to which the cancer is known to metastasise. For instance, prostate cancer is known to metastasise to bone, lymph nodes, the urethra, the bladder, the ureters, the rectum, the lungs and the liver. Therefore the scan for detecting prostate cancer metastases may be a scan of bone, lymph nodes, urethra, bladder, ureters, rectum, lungs and/or liver. Accordingly, the sample collection site for detecting prostate cancer metastases may be a bone, lymph nodes, the urethra, the bladder, the ureters, the rectum, the lungs and/or the liver. The most common site of prostate cancer metastasis is bone. Therefore the scan for detecting prostate cancer metastases may be a bone scan. When metastasis to bone is of interest, the scan may be a radionuclide bone scan and computed tomography (CT). The sample may be a bone marrow biopsy.

Cancers In some embodiments, the prostate cancer is localised prostate cancer, advanced prostate cancer, malignant prostate cancer, aggressive prostate cancer, castration resistant prostate cancer (CRPC) or metastatic prostate cancer.

Alternatively, the cancer may not be prostate cancer. For example, the cancer may be a carcinoma. The cancer may be selected from the group consisting of breast cancer, lung cancer, pancreatic cancer and colon cancer. The cancer may also be oesophageal or ovarian cancer. Where the cancer is not prostate cancer an alternative tissue specific biomarker to PSA may be used in combination with CTCs to predict cancer metastasis. The subject may be undergoing treatment for cancer. The subject may be undergoing treatment for cancer when the sample is obtained from the subject. The treatment may be any suitable treatment for the cancer affecting the subject, including any treatment described herein. In some embodiments, the cancer has progressed after the subject has undergone treatment for the cancer. A cancer which has progressed may be a cancer which has metastasised, a cancer where one or more tumours has increased in size and/or where the symptoms of the cancer are worsening or becoming less manageable for the patient.

The inventors have noted that the present invention is of particular relevance to prostate cancer, although it is applicable to other cancer types.

Prostate cancer can be classified according to The American Joint Committee on Cancer (AJCC) tumour-nodes-metastasis (TNM) staging system. The T score describes the size of the main (primary) tumour and whether it has grown outside the prostate and into nearby organs. The N score describes the spread to nearby (regional) lymph nodes. The M score indicates whether the cancer has metastasised (spread) to other organs of the body:

T1 tumours are too small to be seen on scans or felt during examination of the prostate - they may have been discovered by needle biopsy, after finding a raised PSA level. T2 tumours are completely inside the prostate gland and are divided into 3 smaller groups:

T2a - The tumour is in only half of one of the lobes of the prostate gland;

T2b - The tumour is in more than half of one of the lobes;

T2c - The tumour is in both lobes but is still inside the prostate gland. T3 tumours have broken through the capsule (covering) of the prostate gland- they are divided into 2 smaller groups:

T3a - The tumour has broken through the capsule (covering) of the prostate gland;

T3b - The tumour has spread into the seminal vesicles. T4 tumours have spread into other body organs nearby, such as the rectum (back passage), bladder, muscles or the sides of the pelvic cavity. Stage T3 and T4 tumours are referred to as locally advanced prostate cancer.

Lymph nodes are described as being 'positive' if they contain cancer cells. If a lymph node has cancer cells inside it, it is usually bigger than normal. The more cancer cells it contains, the bigger it will be:

NX The lymph nodes cannot be checked;

NO There are no cancer cells in lymph nodes close to the prostate;

N1 There are cancer cells present in lymph nodes.

M staging refers to metastases (cancer spread):

M0 - No cancer has spread outside the pelvis;

M1 - Cancer has spread outside the pelvis;

M1a - There are cancer cells in lymph nodes outside the pelvis;

M1 b - There are cancer cells in the bone; M1c - There are cancer cells in other places.

Prostate cancer can also be scored using the Gleason grading system, which uses a histological analysis to grade the progression of the disease. A grade of 1 to 5 is assigned to the cells under examination, and the two most common grades are added together to provide the overall Gleason score. Grade 1 closely resembles healthy tissue, including closely packed, well-formed glands, whereas grade 5 does not have any (or very few) recognisable glands. Scores of less than 6 and 6 have a good prognosis, whereas scores of more than 6 are classified as more aggressive. The Gleason score was refined in 2005 by the International Society of Urological Pathology and references herein refer to these scoring criteria (Epstein Jl, Allsbrook WC Jr, Amin MB, Egevad LL; ISUP Grading Committee. The 2005 International Society of Urological Pathology (ISUP) Consensus Conference on Gleason grading of prostatic carcinoma. Am J Surg Pathol 2005; 29(9): 1228-42). The Gleason score is detected in a biopsy, i.e. in the part of the tumour that has been sampled. A Gleason 6 prostate may have small foci of aggressive tumour that have not been sampled by the biopsy and therefore the Gleason is a guide. The lower the Gleason score the smaller the proportion of the patients will have aggressive cancer. Gleason score in a patient with prostate cancer can go down to 2, and up to 10. Because of the small proportion of low Gleasons that have aggressive cancer, the average survival is high, and average survival decreases as Gleason increases due to being reduced by those patients with aggressive cancer (i.e. there is a mixture of survival rates at each Gleason score).

The Gleason Score is the most widespread method of prostate cancer tissue grading used today. It is one determinant of a patient's specific risk of dying due to prostate cancer. Hence, once the diagnosis of prostate cancer is made on a biopsy, tumour grading, especially the Gleason score, is often then relied upon in considering options for therapy. However, the use of the Gleason score is limited by the invasive procedure of acquiring tissue samples which may cause psychological and physical burdens to patients.

The Gleason scoring system is based upon microscopic tumour patterns that are measured by a pathologist, based on a prostate biopsy. Several markers are observed, and then, additional ones are added for a final sum. (The "Gleason Score" and the "Gleason Sum" are same). The Gleason Score is the sum of the primary Gleason grade and the secondary Gleason grades.

When prostate cancer is present in the biopsy, the Gleason score is based upon the degree of loss of the normal glandular tissue architecture (i.e. shape, size and differentiation of the glands) as originally described and developed by Dr. Donald Gleason in 1974 (Gleason DF, and Mellinger GT, J Urol 1 1 1 :58-64, 1974).

The classic Gleason scoring diagram shows five basic tissue patterns that are technically referred to as tumour "grades". The subjective microscopic determination of this loss of normal glandular structure caused by the cancer is abstractly represented by a grade, a number ranging from 1 to 5, with 5 being the worst grade possible. The biopsy Gleason score is a sum of the primary grade (representing the majority of tumour) and a secondary grade (assigned to the minority of the tumour), and is a number ranging from 2 to 10. The higher the Gleason score, the more aggressive the tumour is likely to act and the worse the patient's prognosis.

Grade 1 : the cancerous tissue will closely resemble the normal tissue

Grade 2: tissue which still has well advanced structures, such as the glands; though they are also much larger and also the tissues are present amongst them.

Grade 3: tissue still has the recognizable glands; though, the cells are dimmer

Grade 4: the tissue has hardly any glands which are identifiable

Grade 5: there are no identifiable glands in the tissue

The Primary Gleason grade has to be greater than 50% of the total pattern seen (i.e. the pattern of the majority of the cancer observed). The Secondary Gleason grade has to be less than 50%, but at least 5%, of the pattern of the total cancer observed. The sum of the primary and secondary Gleason grades is shown as the Gleason score or sum (i.e. primary grade + secondary grade = GS; i.e. 4+3 or 3+4 = GS 7). Prostate cancers can also be staged according to how advanced they are. This is based on the TMN scoring as well as any other factors, such as the Gleason score and/or the PSA test. The staging can be defined as follows:

Stage I:

T1 , NO, M0, Gleason score 6 or less, PSA less than 10

OR

T2a, NO, M0, Gleason score 6 or less, PSA less than 10 Stage 11 A:

T1 , NO, M0, Gleason score of 7, PSA less than 20

OR

T1 , NO, M0, Gleason score of 6 or less, PSA at least 10 but less than 20:

OR

T2a or T2b, NO, M0, Gleason score of 7 or less, PSA less than 20

Stage MB:

T2c, NO, M0, any Gleason score, any PSA

OR

T1 or T2, NO, M0, any Gleason score, PSA of 20 or more

OR T1 or T2, NO, MO, Gleason score of 8 or higher, any PSA Stage III:

T3, NO, MO, any Gleason score, any PSA

Stage IV:

T4, NO, MO, any Gleason score, any PSA

OR

Any T, N1 , MO, any Gleason score, any PSA:

OR

Any T, any N, M1 , any Gleason score, any PSA

In the present invention, references herein to "aggressive prostate cancer" can refer to cancers having a Gleason score of more than 6, for example 7 or more or, in some cases, 8 or 9 or more. Aggressive prostate cancer may also be CRPC.

The invention may also be useful for detecting or diagnosing stage II to stage IV cancer or prostate cancer having a Gleason score of 6 or more, 7 or more or 8 or more or CRPC. It may be determined that a patient has aggressive prostate cancer by histological analysis. Alternatively (or additionally), a PSA level of more than 15, in particular more than 20 ng/ml may be indicative of aggressive prostate cancer, in particular in combination with higher Gleason scores. The level of risk (and hence aggressiveness) may be measured according to the National Institute for Health and Care Excellence guidelines on "Prostate Cancer: diagnosis and treatment" (document CG175 published January 2014), as follows:

Table 1

High-risk localised prostate cancer is also included in the definition of locally advanced prostate cancer. The above criteria were first descried by D'Amico ef a/., JAMA, 1998, 280:969-74. The present invention is useful in providing further information to the stratification shown in Table 1 and can provide a more reliable indication of which cancers will metastasise and therefore need treatment. In some embodiments, no metastases have been previously detected in the subject, the cancer is NO stage and/or the cancer is MO stage. Alternatively, the prostate cancer may be any stage described herein.

The methods of the invention may optionally comprise a step of comparing the amount of PSA and/or CTCs with a control or reference. The comparison with a control or reference may be to determine if cancer (for example prostate cancer such as localised prostate cancer, advanced prostate cancer, malignant prostate cancer, aggressive prostate cancer, CRPC or metastatic prostate cancer) is present or not. Accordingly, the prostate cancer may have been treated by hormone deprivation and the prostate cancer has one or more features selected from the group consisting of increased PSA concentration, a serum testosterone concentration of less than 50 ng/mL, a PSA rise of 25% or more from nadir and consecutive increases in PSA in measurements taken at least three weeks apart.

Alternatively, the subject may not have undergone treatment for cancer.

PSA may be measured accordingly to methods known in the art and/or the methods described herein.

In some embodiments of the invention, the analysis may be two fold; first, the presence of prostate cancer is determined, and second the likelihood of prostate cancer metastasis is predicted to decide what (if any) treatment should be given. The analysis may further comprise monitoring a patient's response to treatment.

Aggressive prostate cancer can be defined as a cancer that requires treatment to prevent, halt or reduce disease progression and potential further complications (such as metastases or metastatic progression). Ultimately, aggressive prostate cancer is prostate cancer that, if left untreated, will kill a patient. Hence the present invention is useful in diagnosing or detecting malignant or metastatic prostate cancer, or determining the prognosis for a patient by identifying patients at risk of malignant or metastatic prostate cancer. In some embodiments of the invention, "aggressive prostate cancer" may be determined by histological analysis. Aggressive prostate cancer may be CRPC. In some embodiments of the invention, the method of the invention may be combined with another test such as the PCA3 test to decrease the possibility of false positive or false negative results. Other tests may be a histological examination to determine the Gleason score, or an assessment of the stage of progression of the cancer.

In some embodiments, the prostate cancer has one or more advanced features selected from the group consisting of increased PSA concentration during treatment, a PSA concentration greater than or equal to 0.2 ng/mL, a primary Gleason score greater than or equal to 7 and greater than or equal to 5 metastases. The prostate cancer with one or more of the advanced features as defined herein may be a biochemical recurrence of prostate cancer after radical prostatectomy.

Following successful surgery (radical prostatectomy), PSA should be undetectable in blood samples (i.e. zero PSA, not 0-4 ng/dL) after about a month. However, some men will have a very low non-rising PSA after surgery, which can sometimes be related to normal prostate tissue left behind. This is uncommon, and referred to as benign regeneration. However, the most widely accepted definition of a cancer recurrence is a PSA > 0.2 ng/mL that has risen on at least two separate occasions at least two weeks apart and measured by the same lab. Accordingly, in some embodiments, increased PSA concentration during treatment comprises a PSA concentration greater than 0.2 ng/mL or has risen on at least two separate occasions at least two weeks apart.

In the post-radiation therapy setting, the most widely accepted definition a cancer recurrence is a PSA concentration that has risen from undetectable in at least three consecutive tests conducted at least two weeks apart and measured by the same lab. Some believe that failure after radiation is not clear until the PSA has risen 2 points above its lowest value after radiation. Accordingly, in some embodiments, increased PSA concentration during treatment comprises a PSA concentration that has risen in at least three consecutive tests conducted at least two weeks apart.

PSA velocity or PSA doubling time, both of which measure the rate at which PSA concentration increases, can be a very significant factor in determining is the aggressiveness of prostate cancer. Men with a shorter PSA doubling time or a more rapid PSA velocity after initial therapy tend to have more aggressive disease, and are therefore more likely to need more aggressive therapies. Likewise, men who have recurrence quickly after surgery (i.e. within 3 years) have a higher risk of aggressive disease. Methods of treatment

The invention also provides a method of treating cancer in a subject for whom metastasis has been predicted according to the invention, further comprising administering a therapeutic agent to the subject and/or adopting a therapeutic regimen. In some embodiments, the therapeutic regimen is selected from the group consisting of second line hormone therapy, hormone therapy, chemotherapy, radiotherapy, immunotherapy and bone- targeting therapy. As used herein, second line hormone therapy may refer to treatment with a therapeutic agent selected from the group consisting of anti-androgen, such as bicalutamide; anti-androgen withdrawal; corticosteroids such as dexamethasone, prednisolone, or hydrocortisone; triamcinolone; Ketoconazole; transdermal/oral oestrogen, such as Evorel or diethylstilbestrol. As used herein, hormone therapy may refer to treatment with a therapeutic agent selected from the group consisting of Abiraterone or Enzalutamide.

As used herein, chemotherapy may refer to treatment with a therapeutic agent selected from the group consisting of Docetaxel; Mitoxantrone; Paclitaxel; CL56 (Chlorambucil + lomustine); Estramustine (Estracyt); Melphalan; ECarboF (Epirubicin + Carboplatin + Folinic Acid + 5- Fluorouracil), ECarboX (Epirubicin + Carboplatin + Folinic Acid + Capecitabine) or Cabazitaxel.

As used herein, palliative radiotherapy may refer to radium 223 therapy (a type of internal radiotherapy treatment); local palliative external radiotherapy.

As used herein, immunotherapy may refer to Sipuleucel-T treatment.

As used herein, bone-targeting therapy may refer to drugs used to treat secondary bone cancer, such as Zoledronate, Clodronate, and Denosumab.

Accordingly, the therapeutic agent may be selected from the group consisting of an anti-androgen, such as bicalutamide; a corticosteroid such as dexamethasone, prednisolone, or hydrocortisone; triamcinolone; ketoconazole; transdermal or oral oestrogen, such as Evorel or diethylstilbestrol; Abiraterone; Enzalutamide; a chemotherapeutic agent such as Docetaxel, Mitoxantrone, Paclitaxel, CL56 (Chlorambucil + lomustine), Estramustine (Estracyt), Melphalan, ECarboF (Epirubicin + Carboplatin + Folinic Acid + 5-Fluorouracil), ECarboX (Epirubicin + Carboplatin + Folinic Acid + Capecitabine); or Cabazitaxel.

The therapeutic regimen and/or therapeutic agent may be selected on the basis of the prediction of metastasis, according to the method of the invention. For instance, a different therapeutic regimen and/or therapeutic agent may be selected if the likelihood of metastasis is high to if the likelihood of metastasis is low.

The methods of the invention may optionally comprise a step of comparing the amount of PSA, CTCs and/or the CRS with a control or reference. Accordingly, the invention provides a method for treatment of cancer comprising:

(a) administering a therapeutic agent and/or adopting a therapeutic regime to a patient,

(b) determining the amount of PSA and CTCs in a sample from the patient,

(c) comparing the amount of the PSA and/or CTCs in a sample to a reference or control, (d) continuing, modifying or discontinuing treatment on the basis of the comparison.

Optionally, the method of treatment of cancer further comprises a step of obtaining a sample from the patient. One or more samples may be obtained. For example, a first sample may be obtained prior to step (a) and a second sample may be obtained after step (a) and prior to step (b).

In another embodiment of the invention there is provided a method of treating or preventing cancer (for example prostate cancer such as localised prostate cancer, advanced prostate cancer, malignant prostate cancer, aggressive prostate cancer, castration resistant prostate cancer (CRPC) or metastatic prostate cancer), in a patient, comprising quantifying the amount of PSA and CTCs, in one or more blood samples obtained from a patient, comparing the values to a reference, and, if the detected values are greater than the reference, administering alternative treatment for the cancer. Methods of treating cancer may include administering secondary hormone therapy, new type of hormone therapy, chemotherapy and/or radiotherapy to the patient. The amount of PSA and/or CTCs may be quantified by any suitable means known to the skilled person, for example immunoassay, FISH, flow cytometry, immunofluorescence and/or immunohistochemistry.

The methods of treating cancer of the present invention are particularly useful in the treatment of aggressive prostate cancer, castration resistant prostate cancer (CRPC) or metastatic prostate cancer. In some embodiments, the methods of treatment are performed on patients who have been identified as having a particular amount of PSA and CTCs in one or more blood samples. Said amount is one that it is indicative of aggressive prostate cancer, castration resistant prostate cancer (CRPC) or metastatic prostate cancer.

In some embodiments of the invention, the methods may further comprise treating a patient for metastatic prostate cancer if metastatic prostate cancer is detected or suspected. If metastatic prostate cancer is detected or suspected based on the analysis of one or more blood samples, the presence of metastatic prostate cancer can be confirmed by, for example, detecting the presence and/or amount of one or more biomarkers in a sample of tissue or by a radionuclide bone-scan and computed tomography.

In some cases treatment for cancer involves resecting the tumour or other surgical techniques. For example, treatment for prostate cancer may comprise a radical prostatectomy, or bilateral orchiectomy. Treatment may alternatively or additionally involve treatment by chemotherapy and/or radiotherapy. Chemotherapeutic treatments include docetaxel and estramustine. Radiotherapeutic treatments include external beam radiotherapy, brachytherapy, or, as the case may be, prophylactic radiotherapy. Other treatments include abiraterone, enzalutamide, prednisolone, hormone therapy (including gonadorelin analogues such as buserelin, goserelin, histrelin, leuproelin and triptorelin and also including gonadotrophin-releasing hormone antagonists such as degarelix), anti-androgen treatment (such as androgen deprivation therapy using for example cyproterone acetate, flutamide, bicalutamide and abiraterone acetate), cryotherapy, high- intensity focused ultrasound, and/or administration of bisphosphonates and/or steroids. Palliative therapies include irradiation and strontium.

The methods of predicting prostate cancer metastasis, diagnosis of metastatic prostate cancer and treatment of prostate cancer, may further comprise detecting and/or identifying PSA and CTCs in one or more samples

The immunoassay, flow cytometry, immunofluorescence, immunohistochemistry and/or FISH may be performed by any suitable method and according to any method described herein. The specific binding molecules described herein may be used in the immunoassay, flow cytometry, immunofluorescence, immunohistochemistry and/or FISH. PSA is typically detected by a method comprising the binding of an anti-PSA antibody. The CTCs may be detected on the basis of any feature of CTCs defined herein, for example, CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs may be detected as cells which are CD45 negative, CK positive and vimentin positive. Malignant CTCs may be detected as positive for genomic alteration by FISH. Malignant CTCs that are partially transitioned from epithelial CTCs to mesenchymal CTCs may be detected as cells which are CD45 negative, CK positive and vimentin positive and which are positive for genomic alteration by FISH. Additional methods of the invention

The inventors foresee a number of further applications of the invention, including those described below. In a still further embodiment of the invention there is provided a method for determining the suitability of a patient for treatment for cancer (for example prostate cancer such as localised prostate cancer, advanced prostate cancer, malignant prostate cancer, aggressive prostate cancer, CRPC or metastatic prostate cancer), comprising detecting the amount of PSA and CTCs in one or more blood samples, optionally comparing the amount of PSA or CTCs with a control, and deciding whether or not to proceed with treatment for cancer is diagnosed or suspected, in particular if aggressive prostate cancer CRPC or metastatic prostate cancer is diagnosed or suspected.

There is also provided a method of monitoring a patient's response to therapy, comprising determining the amount of PSA and CTCs in one or more blood samples, obtained from a patient that has previously received therapy for prostate cancer (for example chemotherapy and/or radiotherapy). In some embodiments, the amount of PSA and/or CTCs is compared with the amount of PSA and/or CTCs obtained from a patient before receiving the therapy for prostate cancer. A decision can then be made on whether to continue the therapy or to try an alternative therapy based on the comparison of the PSA and/or CTCs.

Accordingly, the invention also provides a method for monitoring a patient's response to therapy comprising:

(a) determining the amount of PSA and optionally the amount of CTCs in a first sample obtained from a patient prior to administration of the therapy,

(b) determining the amount of CTCs and optionally the amount of PSA in a second sample obtained from a patient prior to administration of the therapy,

(c) determining the amount of PSA and optionally the amount of CTCs in the third sample obtained from a patient after administration of the therapy,

(d) determining the amount of CTCs and optionally the amount of PSA in the fourth sample obtained from a patient after administration of the therapy.

The method may optionally further comprise a step of administering the therapy (for example administering a therapeutic agent and/or adopting a therapeutic regime). The step of administering the therapy may be performed between steps (b) and (c) above.

In one embodiment, there is therefore provided a method comprising:

a) determining the amount of PSA and CTCs in one or more blood samples wherein the sample is obtained from a patient that has previously received therapy for prostate cancer; b) comparing the amount of PSA and/or CTCs determined in step a) with a previously determined amount of PSA and/or CTCs; and

c) maintaining, changing or withdrawing the therapy for prostate cancer.

The methods may comprise a prior step of administering the therapy for prostate cancer to the patient. In another embodiment, the method may also comprise a pre-step of determining the amount of PSA and/or CTCs in one or more blood samples obtained from the same patient prior to administration of the therapy. In step c), the therapy for prostate cancer may be maintained if an appropriate adjustment in the amount of PSA and/or CTCs is determined. For example, if there is a reduction in the amount of PSA and/or a reduction in the amount of CTCs, then treatment may be maintained. Alternatively, treatment may not need to be changed. If the amount of PSA and/or CTCs has altered sufficiently, for example back to what may be considered healthy or low-risk levels, then treatment for cancer may be withdrawn. Alternatively, it may be necessary to continue until therapy for a number of months or half a year until any signs of cancer disappear. If the amount of PSA and/or CTCs is unchanged or has worsened (for example there is an increase in the amount of CTCs and/or an increase in the amount of PSA, this may be indicative of a worsening of the patient's condition, and hence an alternative therapy for cancer may be attempted. In this way, drug candidates useful in the treatment of cancer (in particular aggressive cancer, such as aggressive prostate cancer and/or CRPC) can be screened.

The methods may also comprise a step of determining the risk and/or the rate of cancer progression. The methods may also comprise a step of administering a therapeutic and/or adopting a therapeutic regimen on the basis of the determination of the risk and/or the rate of cancer progression

In some embodiments of the invention, the methods are useful for individualising patient treatment, since the effect of different treatments can be easily monitored, for example by measuring PSA and/or CTCs in successive blood samples following treatment. The methods of the invention can also be used to predict the effectiveness of treatments, such as responses to hormone ablation therapy. Methods of the invention may therefore further comprise a step of detecting or determining the amount of a biomarker in a tissue sample. Typically the tissue sample will be a non-prostate tissue sample. For example, the tissue sample may be a bone marrow sample. The tissue sample may have been obtained previously from a patient, or the method may comprise a step of obtaining or providing said tissue sample. Analysis of tissue samples may also comprise a histological analysis, and any of the methods herein may be combined with a histological analysis to assist in the prediction of metastasis or the diagnosis of metastatic prostate cancer.

Methods of the invention may also further comprise a step performing a radionuclide bone-scan and computed tomography.

In another embodiment of the invention, there is provided a method identifying a drug useful for the treatment of prostate cancer (for example localised prostate cancer, advanced prostate cancer, malignant prostate cancer, aggressive prostate cancer, CRPC or metastatic prostate cancer), comprising:

(a) quantifying the amount of PSA and CTCs in one or more blood samples obtained from a patient;

(b) administering a candidate drug to the patient;

(c) quantifying the amount of PSA and CTCs in one or more blood samples obtained from the same patient at a point in time after administration of the candidate drug; and

(d) comparing the values determined in step (a) with the values determined in step (c), wherein an appropriate change in the amount of PSA and/or CTCs between the two samples identifies the drug candidate as a possible treatment for prostate cancer. For example, a decrease in the amount of PSA, or a decrease in the amount of CTCs may be indicative of the usefulness of the drug candidate in treatment of cancer. In some embodiments, the drug is a compound, an antibody or antibody fragment. Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, F(ab')2, Fv, scFv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to herein as "mAb".

In some embodiments, the methods of the invention further comprise measuring primary Gleason score, alkaline phosphatase (ALP) level and/or lactate dehydrogenase (LDH) level in one or more samples from the subject and/or measuring the number of metastases in the subject.

Stripping buffer

The invention also provides a stripping buffer comprising 1 % to 3 % SDS, 0.055 M to 0.075 M Tris- HCI and 0.5 % to 1.1 % β-mercaptoethanol wherein the stripping buffer has a pH from 6.6 to 7.0. In some embodiments, the stripping buffer comprises 2 % SDS, 0.0625 M Tris-HCI and 0.8 % β- mercaptoethanol wherein the stripping buffer has a pH of 6.8. The stripping buffer may for instance be useful in FISH following flow cytometry, immunofluorescence and/or immunohistochemistry protocols. In some embodiments, the stripping buffer is for use in predicting cancer metastasis and/or the diagnosis of metastatic cancer.

As used herein, "stripping" describes the removal of specific binding molecules such as primary and/or secondary antibodies. Accordingly, a stripping buffer is a composition that serves the function of removing specific binding molecules such as primary and/or secondary antibodies.

Kits

The invention also provides a kit comprising specific binding molecules which bind to one or more of PSA, CD45, CK and/or vimentin. In some embodiments, the specific binding molecules are antibodies, antibody fragments or aptamers. Accordingly, the kit may comprise anti-PSA, anti- CD45, anti-CK and/or anti-vimentin antibodies, antibody fragments or aptamers.

In some embodiments, the kit further comprises one or more nucleic acid molecule which hybridises under stringent conditions to a target genomic region. In some embodiments, the target genomic region is selected from the group consisting of AR, PTEN, ERG, NXK3.1, C-MYC, RB1, CCND1, 6q16 and 16q22.1. However, the skilled person will understand that any suitable target genomic region may be selected. In some embodiments, the kit further comprises a stripping buffer comprising 1 % to 3 % SDS, 0.055 M to 0.075 M Tris-HCI and 0.5 % to 1.1 % β-mercaptoethanol, wherein the stripping buffer has a pH from 6.6 to 7.0. In some embodiments, the stripping buffer comprises 2 % SDS, 0.0625 M Tris-HCI and 0.8 % β-mercaptoethanol wherein the stripping buffer has a pH of 6.8.

In some embodiments, the kit further comprises a nuclear dye. Any suitable nuclear dye known to the person skilled in the art may be used, such as DAPI, propidium iodide or a Hoescht nuclear dye such as Hoescht 3342. In an alternative embodiment, the kit may comprise one or more nucleic acid molecule which hybridises under stringent conditions to a target genomic region, as defined above. Alternatively, the kit may comprise a stripping buffer, as defined above. Any of the kit components defined above may be combined in any combination. Any of the kit components defined above may be combined in any combination. For example, the kit may comprise:

• One or more specific binding molecules, as defined herein,

• A stripping buffer, as defined herein,

• One or more nucleic acid molecules, as defined herein,

· A nuclear dye, as defined herein,

• One or more specific binding molecules and a stripping buffer,

• One or more specific binding molecules and one or more nucleic acid molecules,

• One or more specific binding molecules and a nuclear dye,

• A stripping buffer and one or more nucleic acid molecules,

· A stripping buffer and a nuclear dye,

• One or more nucleic acid molecules and a nuclear dye,

• One or more specific binding molecules, a stripping buffer and one or more nucleic acid molecules,

• One or more specific binding molecules, a stripping buffer and a nuclear dye,

· One or more specific binding molecules, one or more nucleic acid molecules and a nuclear dye,

• A stripping buffer, one or more nucleic acid molecules and a nuclear dye, or

• One or more specific binding molecules, a stripping buffer, one or more nucleic acid molecules and a nuclear dye.

For example, the kit may comprise:

• An anti-PSA specific binding molecule,

• an anti-CD45 specific binding molecule,

• an anti-CK specific binding molecule,

· an anti-vimentin specific binding molecule and optionally, • a nuclear dye, such as DAPI.

As used herein, a "kit" is a packaged combination optionally including instructions for use of the combination and/or other reactions and components for such use.

The specific binding molecule used in the invention can be a fragment of an antibody. It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. ef a/., Nature 341 :544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird ef a/., Science 242:423-426 (1988); Huston ef a/., PNAS USA 85:5879-5883 (1988)); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Hollinger ef a/., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)). Typically, the fragment is a Fab, F(ab')2 or Fv fragment or an scFv molecule. Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associated with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).

Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Hollinger & Winter, Current Opinion Biotechnol. 4:446- 449 (1993)), e.g. prepared chemically or from hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-id iotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described in Traunecker ef a/., EMBO Journal 10:3655-3659 (1991 ).

Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.

General definitions

As used herein the terms "number" or "concentration" may be used as an alternative to the term "amount". Accordingly, the methods of the invention may comprise a step of measuring the concentration of PSA and/or the number of CTCs in one or more blood samples. "About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

As used herein, the term "mutation" may refer to genomic copy number changes and/or changes in genomic structure.

As used herein, "nucleic acid(s)" or "nucleic acid molecule" generally refers to any ribonucleic acid or deoxyribonucleic acid, which may be unmodified or modified DNA or RNA. "Nucleic acids" include, without limitation, single- and double-stranded nucleic acids. As used herein, the term "nucleic acid(s)" also includes DNA as described above that contain one or more modified bases. Thus, DNA with backbones modified for stability or for other reasons are "nucleic acids". The term "nucleic acid(s)" as it is used herein embraces such chemically, enzymatically or metabolically modified forms of nucleic acids, as well as the chemical forms of DNA characteristic of viruses and cells, including for example, simple and complex cells.

As used herein, the terms "hybridizing to" and "hybridization" are interchangeably used with the term "specific for" and refer to the sequence-specific non-covalent binding interactions with a complementary nucleic acid, for example, interactions between a target nucleic acid sequence and a target specific nucleic acid primer or probe. In a preferred embodiment a nucleic acid, which hybridizes, is one which hybridizes with a selectivity of greater than 70 %, greater than 80 %, greater than 90 % and most preferably of 100 % (i.e. cross hybridization with other DNA species preferably occurs at less than 30 %, less than 20 %, less than 10 %). As would be understood to a person skilled in the art, a nucleic acid, which "hybridizes" to the DNA product of a genomic region of the invention, can be determined taking into account the length and composition.

As used herein, "stringent conditions for hybridization" are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1991. Stringent conditions may be defined as equivalent to hybridization in 6X sodium chloride/sodium citrate (SSC) at 45 °C, followed by a wash in 0.2 X SSC, 0.1 % SDS at 65 °C. Alternatively, stringent conditions may be defined as equivalent to hybridization in 50 % v/v formamide, 10 % w/v Dextran sulphate, 2X SSC at 37 °C, followed by a wash in 50 % formamide / 2x SSC at 42 °C.

The term "primer" as used herein refers to a nucleic acid, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e. in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and the method used. For example, for diagnostic applications, depending on the complexity of the target sequence, the nucleic acid primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

As used herein, the term "probe" means nucleic acid and analogues thereof and refers to a range of chemical species that recognise polynucleotide target sequences through hydrogen bonding interactions with the nucleotide bases of the target sequences. The probe or the target sequences may be single- or double-stranded DNA. A probe is at least 8 nucleotides in length and less than the length of a complete polynucleotide target sequence. A probe may be 10, 20, 30, 50, 75, 100, 150, 200, 250, 400, 500 and up to 10,000 nucleotides in length. Probes can include nucleic acids modified so as to have one or more tags which are detectable by fluorescence, chemiluminescence and the like ("labelled probe"). The labelled probe can also be modified so as to have both one or more detectable tags and one or more quencher molecules, for example Taqman® and Molecular Beacon® probes. The nucleic acid and analogues thereof may be DNA, or analogues of DNA, commonly referred to as antisense oligomers or antisense nucleic acid. Such DNA analogues comprise but are not limited to 2-O-alkyl sugar modifications, methylphosphonate, phosphorothiate, phosphorodithioate, formacetal, 3'-thioformacetal, sulfone, sulfamate, and nitroxide backbone modifications, and analogues wherein the base moieties have been modified. In addition, analogues of oligomers may be polymers in which the sugar moiety has been modified or replaced by another suitable moiety, resulting in polymers which include, but are not limited to, morpholino analogues and peptide nucleic acid (PNA) analogues (Egholm, ef a/., Peptide Nucleic Acids (PNA)-Oligonucleotide Analogues with an Achiral Peptide Backbone, (1992)). Preferred features for the second and subsequent aspects of the invention are as for the first aspect of the invention mutatis mutandis.

The present invention will now be described by way of reference to the following Examples and accompanying Drawings which are present for the purposes of illustration only and are not to be construed as being limiting on the invention.

Example 1 - Workflow of blood sample process for CTC & PSA analysis Sample collection and preparation:

A 7.5 mL of whole blood sample are drawn into EDTA Vacutainer tubes (Becton Dickinson and Company, Plymouth, UK) and acquired after getting discarding the initial 2-3 mL of blood to avoid contamination with epithelial cells from the skin. The blood samples are processed within 2 h after collection. Alternatively, they can be stored at room temperature or 4 °C and processed within 24 h of blood draw. If stored at 4 °C, the blood samples are warmed at 25-30 °C for 10-15 min before processing.

Sample isolation:

CTCs were isolated from peripheral blood mononuclear cells (PBMC) using Parsortix™ system. Details of the steps were shown below.

Whole blood Sample pre-processing, enriching mononuclear cell fraction

1. Fill 50 mL LeucoSep tube with 15.3 mL of Ficoll-Paque PLUS (GE Healthcare), and spin at 1 ,000g for 30 seconds at room temperature (RT).

2. Prepare 10 mL Ca⁺⁺ and Mg⁺⁺ free PBS (Sigma) to 50 mL conical tube for each 7.5 mL sample. Wet the pipette using PBS and add 1 mL of PBS to the LeucoSep tube to cover the frit.

3. Leave 0.3-0.5 mL of whole blood in EDTA vacutainer and gently transfer 7 mL of whole blood to the LeucoSep tube along the tube wall. Add 8.0 mL of PBS to the tube (to dilute sample and rinse remaining blood in the pipette and blood on the tube wall).

4. Centrifuge 15 min for fresh sample or 20 min for samples stored overnight at 1 ,000 g. Switch off brakes of centrifuge.

5. After spin, a layer of white cells can be seen just above the frit. Decant the supernatant containing cells from LeucoSep tube to a new 50 mL conical tube.

6. Rinse the same side wall of LeucoSep tube with 5 mL of PBS using 10mL pipette and add to the same 50 mL tube. Be careful not to suction the Ficoll-Paque PLUS through the frit; avoid pressing the pipette against the frit.

7. Repeat step 7 using the same 10 mL pipette.

8. Centrifuge the sample at 200 g for 8 minutes at RT with the braking setting to ON to collect the cell pellet.

9. Prepare 4.5 mL of isolation buffer to a new tube for each sample. 10. Use 25 ml_ pipette to gently aspirate off the supernatant as much as possible without disturbing the pellet. Use a P1000 pipette to remove the remaining if too much left close to the bottom (up to 0.5 mL might be left remaining).

1 1. Add 200 μΙ_ of isolation buffer to the pellet and gently tap the tube on the bench clockwise a few times to loosen the pellet. Tap patiently until the pellet is completely resuspended. Gently resuspend the cells with 200 μΙ_ low-binding tip and transfer to the EDTA vacutainer.

12. Rinse the residual cells in the 50 mL conical tube with another 1 mL isolation buffer and transfer to the same vacutainer. The final volume should be approximately 4.5-5 mL. CTC isolation using Parsortix™

1. Priming: Select the program PX2_P and perform according to instructions.

2. CTC isolation: select the program PX2_S26. Follow the instruction in the machine. During the isolation, shake the tube every 15 minutes to avoid the cells precipitation.

3. Tube cleaning: select the program PX2_CT2 after changing cleaning cassette. Put isolation cassette in original bag with orifice side facing up.

4. Harvest: put the isolation cassette back to the clamp and select the program PX2_H. Collect 200 μί cell suspension in 1.5 mL siliconised low retention tube.

5. Machine Clean: Select the program PX2_CT or PX2_C using cleaning cassette and PBS. Sample harvest and slide preparation:

200 \}L of CTC cell suspension is harvested into 1.5 mL Low-Retention microcentrifuge tube (Fisherbrand™), centrifuge at 1 ,000 g for 3 min, re-suspended in 10 buffer (0.075 M KCI) and transfer onto slide using Superslik™ surface and wide orifice pipette tips (VWR). The tube is rinsed with another 10 μί buffer and added to the drop on the slide. The slide is air-dried followed by fixation with acetone on ice for 20 min.

Immunofluorescence staining:

After blocking by 10% normal donkey serum for 10 min, cells for CTC analysis are incubated with PE-conjugated anti-CD45 (Clone: 5B1 , Miltenyi Biotec) for 15 min. Cells are then permeabilised with 0.1 % Triton X-100 for 5 min, stained with FITC-conjugated anti-CK (Clone: CK3-6H5, Miltenyi Biotec) and Alexa Fluor® 647-conjugated anti-VIM (Clone: EPR3776, abeam) for 30 min.

After the application of antibodies, slides are mounted in SlowFade® gold antifade mountant with DAPI. Enumeration is performed after the slide is scanned by Ariol image analysis system (Leica Microsystems (Gateshead) Ltd, UK), equipped with an Olympus BX61 microscope.

Criteria to identify CTCs:

Enumeration:

Epithelial CTCs are defined as those CK positive, VIM negative, CD45 negative, DAPI stained intact cells; EMTing CTCs are defined as those CK positive, VIM positive, CD45 negative, DAPI stained intact cells;

Mesenchymal CTCs are defined as those CK negative, VIM positive, CD45 negative, DAPI stained intact cells;

PSA Analysis:

The Access Hybritech PSA assay (Beckman Coulter, Fullerton, CA.) is a two-site immunoenzymatic "sandwich" assay. A sample is added to a reaction vessel with mouse monoclonal anti-PSA alkaline phosphatase conjugate, and paramagnetic particles coated with a second mouse monoclonal anti-PSA antibody. The PSA in the sample binds to the immobilized monoclonal anti-PSA on the solid phase while, at the same time, the monoclonal anti-PSA conjugate reacts with a different antigenic site on the sample PSA. Separation in a magnetic field and washing removes material not bound to the solid phase. A chemiluminescent substrate, Lumi- Phos^** 530 is added to the reaction vessel and light generated by the reaction is measured with a luminometer. The light production is proportional to the concentration of PSA in the sample. The amount of analyte in the sample is determined by means of a stored, multi-point calibration curve. PSA concentrations are calculated by using a calibration curve. This method utilizes a weighted four parameter standard curve with a direct relationship of measured light produced (RLU) to concentration of PSA protein in the serum sample. Serum results are expressed as ng/mL. Three levels of control are run for each test series. If, within a testing series, these controls do not conform to specifications as defined in the quality control manual, the entire series is invalidated. The steps of the PSA analysis method, including operating instructions, calculations and interpretation of results are as follows:

a. Preliminaries

1. Bring all controls and patient specimens to room temperature before use. Mix any specimens or controls that have been frozen. Centrifuge samples with particulate matter prior to testing.

2. Prime system (pipettor, dispense, and substrate) 4 times

3. Check reagent, substrate, wash buffer, and reaction vessel status. Load any needed supplies onto the instrument. Mix reagent pack contents by gently inverting pack several times before loading on the instrument. Do not invert open (punctured) packs— mix reagents by swirling gently.

b. Instrument Operation (see operator's manual for details).

1. Gently mix, uncap and load specimens into specimen racks, with the barcode in the open slot. Make sure there are no bubbles. Alternately, use the barcode wand to identify the specimens, and then load samples into the appropriate sample cups.

Load the racks onto the instrument.

2. Each day run a 1 :100 dilution of a "pool" of patient specimens to check for high dose hook. The pool result should not be higher than the highest patient result. Refer all questions to a supervisor. 3. Select the PSA-Hyb test. Note: if free PSA is also ordered, this test can be run on the same aliquot. Testing is done in singlicate.

4. The instrument automatically calculates all results. After testing is completed, results are printed and review by the technologist. Samples with results > 150 ng/mL are diluted off-line and repeated, and results are corrected for the dilution factor.

Samples with results < 0.1 ng/mL are repeated to confirm. Do not rerun samples that have sat on the Access for more than 60 minutes. Pour fresh aliquots before rerunning.

5. Remove specimens and controls. Return controls to the refrigerator and refreeze specimens.

6. Perform scheduled instrument maintenance (daily, weekly, and monthly) as outlined on the maintenance log. See the operator's manual for specific instructions.

Recording of Data

1. Specimen results are entered into the assay specific results table created from the send file corresponding to the specific sample box using Excel software (Microsoft Corporation, Redmond WA). A copy of this table is printed out and checked for accuracy of data entry.

2. Control results are entered to the Assay Specific QC/Levy-Jennings Table using the

Excel program. Compliance with the Westgard rules is evaluated. A copy of this table is printed out and checked for accuracy of data entry.

Example 2 - Detection of circulating cells with epithelial and mesenchymal features

Using an optimized CTC isolation and detection method (Xu ef al (2015)), blood samples from 81 prostate cancer patients (38 with untreated localized disease and 43 with CRPC) (Table 2 and Table 3) we analyzed to identify CK+/VIM-/CD45-, CK+/VIM+/CD45- and CK-/VIM+/CD45- circulating cells (Fig. 1A). Of the 38 patients with untreated localized disease, 18 (47%) patients had detectable CK+/VIM-/CD45- circulating cells, 7 (18%) patients had detectable CK+/VIM+/CD45- cells, and 21 (55%) patients had detectable CK-/VIM+/CD45- circulating cells. In the 43 with CRPC, 31 (72%), 30 (70%), and 38 (88%) patients had detectable CK+/VIM-/CD45-, CK+/VIM+/CD45-, and CK-/VIM+/CD45- circulating cells, respectively (Table 3). Of 81 patients studied, 58 (72%) patients had detectable CK+/CD45- cells regardless of VIM expression, while an additional 15 (19%) patients had no detectable CK+/CD45- cells but detectable CK-/VIM+/CD45- circulating cells. Table 2. Clinical characteristics and CTC cell count for all patients

Patient Primary PSA ALP LDH CRPC Prior treatment before sample Metastasis CK+/VIM- CK+/VIM+ CK-/VIM

ID Age GS (ng/mL) (U/L) (U/L) (Y/N) collection (Y/N) cell (n) cell (n) cell (n)

PC27 70 4+5 1574 54 367 Yes Dexamethasone Yes 3 0 9

PC29 78 4+3 736 46 391 Yes cabazitaxel Yes 4 3 19

PC17b 67 4+5 4626 1277 656 Yes Cisplatin, Etoposide Yes 0 2 6

PC33 60 5+5 39 337 366 Yes Dexamethasone Yes 0 0 5

PC32c 69 5+4 965 730 686 Yes Dexamethasone, Evorel Yes 33 20 53

PC36 80 4+5 260 76 407 Yes Docetaxel Yes 13 5 8

PC39 80 3+4 12 170 439 Yes Bicalutamide, Prostap Yes 2 5 2

PC40 58 4+4 22 n/a n/a No None None 2 26 5

PC22b 81 3+3 6000 1044 1 197 Yes Diethylstilbestrol Yes 4 10 0

PC41 69 4+5 39 312 756 Yes Bicalutamide, Degarelix Yes 5 13 1

PC42 84 n/a 1 19 75 421 Yes Diethylstilbestrol None 0 7 14

PC21 b 66 n/a 313 327 343 Yes Diethylstilbestrol Yes 3 4 2

PC43 63 4+3 16 104 448 Yes Dexamethasone None 9 1 4

PC44 83 3+5 633 46 391 Yes Diethylstilbestrol Yes 24 0 17

PC45 84 3+3 1 79 447 Yes Triamcinolone Yes 2 3 2

PC46 56 4+3 15 n/a n/a No None None 4 0 0

PC7d 60 4+5 693 79 489 Yes Triamcinolone Yes 20 4 5

PC47 76 4+4 2 66 282 Yes Dexamethasone Yes 7 1 2

PC49 88 4+3 1027 1339 774 Yes Dexamethasone Yes 1 0 13

PC50 76 n/a 160 62 375 Yes CL56 Yes 20 2 1

PC51 67 n/a 43 79 347 Yes Bicalutamide, Goserelin Yes 1 2 1 1

Diethylstilbestrol,

PC52 81 n/a 77 66 276 Yes Dexamethasone Yes 9 0 0

PC19b 73 n/a 40 93 329 Yes Enzalutamide Yes 1 2 8

PC54 86 5+3 29 61 354 Yes Dexamethasone Yes 18 10 2

PC56 62 5+5 9 181 413 Yes Prostatp Yes 3 3 6

PC58 80 n/a 5 83 409 Yes Cisplatin, Etoposide Yes 0 9 2

PC59 69 4+4 60 958 499 Yes Evorel, Triamcinolone Yes 23 2 0

PC57b 60 n/a 61 239 479 Yes Dexamethasone Yes 13 1 0

PC60 77 4+4 1 1 72 328 Yes Enzalutamide Yes 0 5 1 1

PC61 70 n/a 22 50 349 Yes ADT withdrawn Yes 1 2 7

PC16c 82 n/a 392 409 443 Yes Diethylstilbestrol Yes 2 1 1

PC63 74 n/a 550 766 509 Yes Dexamethasone, Evorel Yes 1 0 2

PC65 87 n/a 107 326 391 Yes ADT withdrawl Yes 4 2 5

PC66 58 3+3 5 n/a n/a No None None 0 0 2

PC69 67 5+5 4 131 374 Yes Bicalutamide, Leuprorelin Yes 3 4 3

PC68 73 4+3 5 82 407 Yes ADT withdrawn Yes 4 2 0

PC71 65 4+4 37 76 406 Yes Docetaxel Yes 0 3 4

PC73 67 3+4 12 n/a n/a No None None 0 0 1

Prostap, Stilboestrol,

PC72 74 4+5 23 203 324 Yes Dexamethasone Yes 0 0 1

PC75 63 3+3 5 n/a n/a No None None 1 0 1

PC77 66 3+3 12 n/a n/a No None None 0 0

PC80 62 4+5 26 n/a n/a No None None 5 0

PC81 69 3+3 4 n/a n/a No None None 0 0

PC82 66 3+3 6 n/a n/a No None None 1 0 5

PC83 76 3+4 10 n/a n/a No None None 0 0 0

PC84 56 3+3 45 n/a n/a No None None 0 6 4

PC85 69 3+3 8 n/a n/a No None None 0 0 0

PC92 34 3+3 4 n/a n/a No None None 0 0 0

PC93 75 4+3 8 n/a n/a No None None 4 0 0

PC96 73 3+4 5 n/a n/a No None None 2 0 0

PC 100 76 4+3 10 n/a n/a No None None 0 0 0

PC99 77 4+3 9 n/a n/a No None None 0 0 0

PC101 70 3+4 3 n/a n/a No None None 4 0 0

PC 103 71 3+4 9 n/a n/a No None None 2 0 0

PC 106 50 3+3 5 n/a n/a No None None 0 0 0

PC 107 73 4+3 7 n/a n/a No None None 0 0 3

PC 108 72 3+4 13 n/a n/a No None None 0 0 0

PC1 1 1 55 4+3 5 n/a n/a No None None 0 0 6

PC1 15 67 4+3 20 n/a n/a No None None 2 1 3

PC1 16 57 3+4 12 n/a n/a No None None 1 0 0

PC1 18 57 4+3 9 n/a n/a No None None 3 0 0

PC1 19 71 3+4 6 n/a n/a No None None 0 1 2

PC55 58 3+3 5 n/a n/a No None None 1 1 1

PC79 55 3+4 7 n/a n/a No None None 0 0 1

PC1 14 71 4+5 49 n/a n/a Yes Evorel, Dexamethasone Yes 2 0 6

PC 104 76 4+3 220 373 n/a Yes Prostap Yes 1 0 2

PC 105 55 4+5 52 81 393 Yes Bicalutamide, Goserelin Yes 0 0 4

PC 144 71 3+4 65 69 372 Yes Cyproterone, Prostap Yes 0 0 1

PC 125 83 n/a 324 135 341 Yes ADT withdrawn Yes 100 0 3

PC 122 78 3+4 14 n/a n/a No None None 0 0 0

PC 123 72 3+4 1 1 n/a n/a No None None 2 1 0

PC 124 69 4+4 20 n/a n/a No None None 2 0 1

PC 126 54 5+4 13 n/a n/a No None None 3 3 12

PC 127 66 3+4 367 62 317 Yes Bicalutamide None 0 0 13

PC 128 69 3+4 8 n/a n/a No None None 0 0 6

PC 129 84 4+5 189 73 472 Yes Bicalutamide Yes 0 1 4

PC 130 56 3+4 10 n/a n/a No None None 4 0 0

PC131 57 4+3 1 1 n/a n/a No None None 0 0 20

PC 132 61 >7 50 59 344 Yes Docetaxel Yes 0 1 6

PC 135 55 3+3 5 n/a n/a No None None 0 0 2

PC 136 73 3+4 8 n/a n/a No None None 3 0 15

GS: Gleason score; PSA: prostate specific antigen; ALP: alkaline phosphatase; LDH: lactate dehydrogenase; megakaryocytes: CK-/VIM-/CD45- cells with bi nuclei (see main text); ADT: androgen deprivation therapy; CL56: refers to chlorambucil + lomustine; n/a: data not availabl

Table 3. Summary of Clinical characteristics and CTC count for all patients

All patients, Patient with Patient with n = 81 untreated localized CRPC, disease, n = 38 n = 43

Age, y

Median (IQR) 69 (76-62) 66.5 (72-57) 73 (81-67)

PSA, ng/mL

Median (IQR) 15 (71-7.65) 8.91 (12.25-5.40) 61 (367-23)

Primary Gleason Score, n (%)

6 13 (16) 1 1 (29) 2 (5)

3+4 17 (21 ) 14 (37) 3 (7)

4+3 14 (17) 9 (24) 5 (12)

8-10 24 (30) 4 (10) 20 (46) n/a 13 (16) 0 (0) 13 (30)

Prior therapy, n (%)

No treatment 38 (47) 38 (100) 0 (0)

Systemic therapy 43 (53) 0 (0) 43 (100)

Metastasis, n (%)

No 41 (51 ) 38 (100) 3 (7)

Yes 40 (49) 0 (0) 40 (93)

CK+/VIM-/CD45- cell

Detectable patient number (%) 49 (60) 18 (47) 31 (72)

Cell number, median (IQR) 1 (4-0) 0 (2-0) 2 (9-0)

CK+/VIM+/CD45- cell

Detectable patient number (%) 37 (46) 7 (18) 30 (70)

Cell number, median (IQR) 0 (2-0) 0 (0-0) 2 (4-0)

CK-/VIM+/CD45- cell

Detectable patient number (%) 59 (73) 21 (55) 38 (88)

Cell number, median (IQR) 2 (6-0) 1 (4-0) 4 (8-2)

Total CTC

Detectable patient number (%) 73 (90) 30 (79) 43 (100)

Cell number, median (IQR) 6 (14-3) 3 (6-1 ) 1 1 (19-7)

IQR: interquartile range (Q75-Q25%); CRPC: castration-resistant prostate cancer; CK: cytokeratin;

VIM: Vimentin. Example 3 - Genetic evidence that CK-/VIM+/CD45- circulating cells are malignant cells with genomic alterations

Multiple FISH analysis after immunofluorescence staining was firstly tested using slides with lymphocytes spiked with PC3 cells. It was found that the immunofluorescence signals were completely removed by the stripping buffer, but not completely removed by 2XSSC buffer, fix solution or proteinase K digestion. The poly-lysine coating time length of the slides was further optimized to 45 minutes to best preserve cells for downstream FISH after signal striping. Less coating time freguently resulted in damaged or lost cells after signal striping. Using these optimized conditions, we detected clear nuclear morphology and FISH signals in up to the fifth round of FISH on cells after immunofluorescence (data not shown). The technigue was then successfully applied to 12 prostate cancer CTC cases for five rounds of FISH (Fig. 1 B) and one case for two rounds due to strong florescence background, to investigate the genetic alterations of nine genes. Changes of more than 30% of the genomic regions were detected in 68% of CK+/VIM-/CD45- cells, 57% of CK+/VIM+/CD45- cells and 54% of CK-/VIM+/CD45- cells, while only detected in 3.7% of CK-/VIM-/CD45+ lymphocytes and 7.8% of CK-/VIM-/CD45- cells (Table 4). The similar rate of genetic changes in the CK+/VIM-/CD45-, CK+/VIM+/CD45- and CK-/VIM+/CD45- circulating cells indicates that the majority of CK-/VIM+/CD45- cells were CTCs. While it is possible that small proportions of the above three circulating cell categories are of non-malignant origin, we considered all CK+/VIM-/CD45-, CK+/VIM+/CD45- and CK-/VIM+/CD45- cells for the correlation analysis between CTC numbers and clinical features, and categorized them as epithelial, EMTing or mesenchymal CTCs, respectively. When classifying cases as positive or negative for CTCs, the number of CK+/VIM-/CD45-, CK+/VIM+/CD45- and CK-/VIM+/CD45- circulating cells found in non- cancer healthy control cases were considered.

Table 4. Proportion of detected genetic changes in sub-populations of cells

0%^a <30% 30-49% 50-69% 70-99% 100%

Subtypes of CTCs n (%)^b n (%) n (%) n (%) n (%) n (%)

Epithelial (CK+/VIM-/CD45-), (n=25) 4 (16) 4 (16) 5 (20) 7 (28) 1 (4) 4 (16)

EMTing (CK+ VIM+/CD45-), (n=39) 1 1 (28) 6 (15) 9 (23) 8 (21 ) 2 (5) 3 (8)

Mesenchymal (CK-/VIM+/CD45-), (n=54) 8 (15) 17 (31 ) 17 (31 ) 9 (17) 2 (4) 1 (2)

Lymphocytes (CK- VIM-/CD45+), n=134 81 (60.4) 48 (35.8) 5 (3.7) 0 (0) 0 (0) 0 (0)

Negative cells (CK-/VIM-/CD45-), n=102 66 (64.7) 28 (27.5) 5 (4.9) 3 (2.9) 0 (0) 0 (0) ^aThe proportion of abnormal probes of the total countable probes in a cell (only with > 2 genomic region informative was included).

bThe number and percentage of cells in each category of detected circulating cells with certain freguency of genomic alterations. Example 4 - Association of CTC positivity with advanced clinical features in localized and metastatic prostate cancer

Analyzing blood samples from 12 healthy male donors, one, two and three CK-/VIM+/CD45- cells were detected respectively in three samples and none in the rest (median 0, range 0 to 3 cells per 7.5 mL). No CK+/VIM-/CD45- or CK+/VIM+/CD45- circulating cells were detected in any of the samples from healthy donors. Consequently, positive CTC cases were defined as those showing any CK+/VIM-/CD45-, any CK+/VIM+/CD45- or >3 CK-/VIM+/CD45- cells. Based on these criteria, 24 of 38 patients with untreated localized disease (63%) and 41 of 43 CRPC patients (95%) scored positive for CTCs. Excluding mesenchymal CTCs reduced CTC positivity to 20 (53%) in localized tumor patients and to 38 (88%) in CRPC patients. Upon investigation of the disparity of clinical features between CTC-score positive and negative patients in all 81 patients, high serum PSA level, high primary Gleason-sum score (GS) and metastatic status were significantly correlated with CTC- score positive patients. In the 38 untreated localized diseases, CTC-score positive cases were associated with high risk classification based on NCCN guideline version 1 , 2016 (Mohler ef al (2016) J Natl Compr Cane Netw 14, 19-30) (Table 5).

Table 5. Clinical features of CTC-score positive and negative patients

All prostate cancer patients

CTC negative patients CTC positive patients Xi² (P)

n=16 n=65

PSA ng/mL, median (IQR) 9.56 (12.75-5.73) 26 (174.5-8.35) 6.660 (0.0099) Primary GS, (n) 8.429 (0.0037)

< 7 6 7

= 7 9 22

> 7 1 23

n/a 0 13

Patients with untreated localized prostate cancer

CTC negative patients CTC positive patients

n=14 n=24

Risk classification, (n) 4.186 (0.0408) Low 5 4

Intermediate 9 14

High 0 6

All prostate cancer patients

CTC negative patients CTC positive patients P^*

n=16 n=65

Metastases, (n) 0.0015

No 14 27

Yes 2 38

IQR: interquartile range (Q75-Q25%). n/a: data not available; ^*: By Fisher's exact test Example 5 - EMTing CTC number was most significantly associated with presence of metastasis and high-risk localized tumor

When the numbers of different sub-populations of CTCs and cancer metastasis were correlated, the presence of metastases was significantly associated with higher number of any type of CTC and the association with EMTing CTCs was most significant (p = 0.0001 ), which was similar in significance when considering total CTCs (Fig. 2C, Table 6). When a receiver operating characteristic (ROC) curve was applied to compare PSA with CTCs in metastases prediction, EMTing CTC count (with an optimal cutoff point at > 2 cells) had an area under the ROC curve (AUC) of 0.755 compared to 0.823 for PSA (with an optimal cutoff point at > 23 ng/mL) (Table 7).

Table 7. ROC curves to predict metastases by CTC count and PSA level

Score Optimal Sensitivity Specificity AUC (95% CI) p value^*

Cut-point

PSA > 23 ng/mL 77.50% 90.24% 0.823 (0.720-0.927)

CRS > 0.357 92.50% 87.80% 0.921 (0.858-0.985) 0.0300

ALL CTC > 7 cells 78.05% 77.78% 0.795 (0.695-0.896) 0.9989

All CK+ CTC > 5 cells 57.50% 85.37% 0.770 (0.668-0.871 ) 0.9833

All VIM+ CTC > 2 cells 90.00% 53.66% 0.748 (0.636-0.859) 0.8655

EMTing CTC > 2 cells 57.50% 90.24% 0.755 (0.654-0.856) 0.9430

Epithelial CTC > 3 cells 50.00% 78.05% 0.696 (0.585-0.807) 0.4864

Mesenchymal CTC > 2 cells 75.00% 56.10% 0.668 (0.550-0.787) 0.1564

CTC-score Positive 95.00% 34.15% 0.646 (0.565-0.727) 0.0069

CRS: combined risk score; ^* Sidak adjusted p value using PSA as the standard Total CTC count (with an optimal cutoff point at > 7 cells) had a similar AUC to PSA (AUC: 0.795 Vs 0.823) (Table 7). PSA and EMTing CTC count were combined to create Combined Risk Score (CRS) as CRS = 0.012 x PSA + 0.1 15 x EMTing CTC count. The CRS (AUC = 0.921 ) was a significantly (p = 0.03) better predictor of metastasis than PSA score alone (Fig. 3). This combination with an optimal cutoff point of CRS > 0.357 resulted in a sensitivity of 92.5% and specificity of 87.8% compare to a sensitivity of 77.5% and specificity of 90.2% by PSA alone at an optimal cutoff point of PSA > 23 ng/mL. To achieve a sensitivity of 97.5%, CRS could simultaneously acguire a specificity of 80.49% while it was 0% by PSA alone (Table 8). Box plots of CRS in patients with/without metastasis are shown in Fig. 4. Table 8. Comparison of sensitivity and specificity to predict metastasis using PSA and CRS Score Cut-off Sensitivity Specificity

97.50% 0%

PSA

80.00% 87.8%

(ng/mL)

77.5% 90.24%

0.276 97.50% 80.49%

0.357 92.50% 87.80%

0.508 82.50% 90.24%

Materials and Methods Patients and blood samples

A total of 81 patients with written consent were recruited from December 2014 in St Bartholomew's Hospital, Barts Health NHS, London, UK, comprising 38 with untreated localized prostate cancer, and 43 with progressive CRPC (40 with metastasis) ready to commence an alternative treatment. Metastases were investigated by radionuclide bone scan and computed tomography (CT). Progressive diseases were defined with a minimum of two increasing PSA levels at least two weeks apart or by radiographic criteria new lesions by bone scan or as new or enlarged soft tissue by CT or magnetic resonance imaging. Blood specimens from 12 healthy donors were collected with signed Ethics committee approved consent form. 7.5ml_ of whole blood was donated from each participant for CTC enumeration. Blood samples were drawn into EDTA Vacutainer tubes (Becton Dickinson and Company, Plymouth, UK) and acquired at the middle of phlebotomy after the collection for routine clinical blood test to avoid contamination with epithelial cells from the skin. All blood samples were stored at room temperature and processed within 24 hours of blood draw. The clinical characteristics of the above 81 patients are detailed in Table 2 and Table 3. CTC harvest and fixation on slides

200 μΙ_ of CTC cell suspension isolated from 7.5 ml blood samples using Parsortix system was harvested into 1.5 mL Low-Retention microcentrifuge tube (FisherbrandTM), centrifuge at 1 ,000 g for 3 min, re-suspended in 10 μί buffer (0.075M KCI) and transfer onto slide using SuperslikTM surface and wide orifice pipette tips (VWR). The tube was rinsed with another 10 μί of buffer and added to the drop on the slide. The slide was air-dried followed by fixation with acetone on ice for 20 min.

Immunofluorescence staining

After blocking by 10% normal donkey serum for 10 min, cells for CTC analysis were incubated with PE-conjugated anti-CD45 (Clone: 5B1 , Miltenyi Biotec) for 15 min. Cells were then permeabilized with 0.1 % Triton X-100 for 5 min, stained with FITC-conjugated anti-CK (Clone: CK3-6H5, Miltenyi Biotec) and Alexa Fluora647-conjugated anti-VIM (Clone: EPR3776, abeam) for 30 min. Cells for megakaryocytes identification were incubated with anti-CD41 (Clone: M148, abeam) and anti-CD34 (Clone: H-140, Santa-cruz) for 1 h after permeabilized with 0.1 % Triton X-100 for 5 min. Alexa Fluora488 donkey anti-mouse and Alexa Fluora546 donkey anti-rabbit (Life technologies) secondary antibodies were then incubated with the cells for 20 minutes. After the application of antibodies, slides were mounted in SlowFade® gold antifade mountant with DAPI. Enumeration was performed after the slide was scanned by Ariol image analysis system (Leica Microsystems (Gateshead) Ltd, UK), equipped with an Olympus BX61 microscope.

Multiple FISH procedure

FISH analysis was performed for AR (RP1 1-479J1 ), PTEN (CTD-846G17), ERG (RP1 1-476D17 and RP1 1-95121 ), TMPRSS2 (RP1 1-535H1 1 ), NXK3.1 (RP1 1-213G6), C-MYC (RP1 1 -349C2), RB1 (RP1 1-305D15 and RP1 1-174110), CCND1 (RP1 1-278A17, RP1 1-599F23, RP1 1-681 H 17 and CTD-2009H2), 6q16 (RP1 1-639P13, RP1 1-25819, CTD-2281 M23 and CTD-2073M5), 16q22.1 (RP11-510M2), chromosome 1 centromere, RAF1 3' (RP1 1-64E16, RP1 1-136B7 and RP1 1- 738A2) and RAF1 5' (RP1 1-71514, RP1 1-764F12 and RP1 1-449E21 ). Chromosomel centromere, RAF1 3' and RAF1 5' were from Institute of Cancer Research. All other FISH probes were purchased from Life Technologies (UK). Probes were prepared as previously described (39). Probes for PTEN, RP1 1-95121 , TMPRSS2, NKX3.1, 6q16, 16q22.1 and RAF1 5' were labeled by Fluorescein-12-dUTP (Roche, IN, USA). Probes for ^R, RP1 1-476D17, C-MYC, RB1, CCND1 and RAF1 3' were labeled by Tetramethyl-rhodamine-5-dUTP (Roche, IN, USA). Before hybridization, slides were fixed in methanol: acetic acid (3:1 ) for 10 minutes, and then pretreated in 70% acetic acid in 10 minutes. Approximately 100 ng of each of the labeled clones were resuspended in hybridization buffer. The mixture was applied to the slide, denatured at 95°C for 10 minutes and incubated at 37°C overnight in a wet box. The slide was washed following the standard FISH method.

Two FISH probes were hybridized with the slide in each round of FISH. To apply new FISH probes, the FISH signal was removed in 70% formamide/2XSSC solution at 68°C for 4 minutes, followed by rinsing in 2XSSC and water. After being air dried, the slide was ready to hybridize with the new pair of FISH probes.

After mounting with antifade DAPI, the FISH signals were scanned by Ariol (Leica Microsystems (Gateshead) Ltd, UK). Statistics

Unless specifically noted, Wilcoxon rank-sum test was applied to assess the equality of CTCs between subgroups based on CTC-score. ROC curve analysis was used to test the ability of different subtypes CTCs as well as CRS to distinguish patients with metastasis. PSA score and EMTing CTC count were combined in a logistic regression. The outcome was metastasis (yes, no). The coefficients obtained from this model were used to compute CRS. Optimal cut-off point was calculated with an optimal corrected classified value to provide best available sensitivity and specificity. Rocgold function was used to independently test the equality of the ROC area of each method against the PSA as a gold standard curve. All statistical tests will be two sided and p- values of 0.05 will be accepted as statistically significant. No adjustment for multiple comparisons was performed. Statistical analyses were performed using Stata 13.0.

Claims

1. A method of predicting prostate cancer metastasis in a subject comprising measuring the amount of circulating tumour cells (CTCs) and the amount of prostate specific antigen (PSA) in one or more blood samples from the subject.

2. The method of claim 1 wherein the CTCs are selected from the group consisting of CTCs which are partially transitioned from epithelial CTCs to mesenchymal CTCs, mesenchymal CTCs, epithelial CTCs, all CK positive CTCs, all vimentin positive CTCs and all CTCs.

3. The method of claim 1 or claim 2, wherein the CTCs are partially transitioned from epithelial CTCs to mesenchymal CTCs.

4. The method of any one of claims 1 or 2 wherein the CTCs are CD45 negative, CK positive and/or vimentin positive.

5. The method of any one of claims 1 to 4 wherein the CTCs are CD45 negative, CK positive and vimentin positive.

6. The method any one of claims 1 or 2 wherein the CTCs are CD45 negative, CK negative and/or vimentin positive.

7. The method of any one of claim 1 , claim 2 or claim 6 wherein the CTCs are CD45 negative, CK negative and vimentin positive.

8. The method any one of claims 1 or 2 wherein the CTCs are CD45 negative, CK positive and/or vimentin negative.

9. The method of any one of claim 1 , claim 2 or claim 8 wherein the CTCs are CD45 negative, CK positive and vimentin negative.

10. The method of any preceding claim wherein greater than or equal to a threshold amount of CTCs is predictive of cancer metastasis in a subject.

11. The method of claim 10 wherein the threshold amount of CTCs per 7.5 mL of sample is selected from the group consisting of:

(b) 2 CTCs per 7.5 mL of sample, wherein the CTCs are mesenchymal CTCs;

(c) 3 CTCs per 7.5 mL of sample, wherein the CTCs are epithelial CTCs; (d) 5 CTCs per 7.5 ml. of sample, wherein the CTCs are CK positive CTCs;

(f) 7 CTCs per 7.5 mL of sample, wherein the CTCs are all CTCs.

12. The method of any preceding claim wherein greater than or equal to a threshold amount of PSA is predictive of cancer metastasis in a subject.

13. The method of claim 12 wherein the threshold amount of PSA is about 23 ng/mL.

14. The method of any preceding claim further comprising calculating a combined risk score (CRS) using the amount of CTCs and the amount of PSA.

15. The method of claim 14, wherein the CRS is calculated according to the following formula: CRS = (0.012 x PSA) + (0.1 15 x EMTing CTC count) wherein PSA is the concentration of prostate specific antigen in the blood sample in ng/ml, and wherein the EMTing CTC count is the number of circulating tumour cells (CTCs) that are CD45 negative, CK positive and vimentin positive.

16. The method of claim 14 or claim 15, wherein greater than or equal to a threshold CRS is predictive of cancer metastasis in a subject.

17. The method of claim 14 or claim 15 wherein the threshold CRS is from about 0.2 to about 0.6.

18. The method of any one of claims 14 to 17 wherein the threshold CRS is from about 0.27 to about 0.51.

19. The method of any one of claims 14 to 18 wherein the threshold CRS is about 0.276, about 0.357 or about 0.508.

20. The method of any one of claims 14 to 19 wherein the threshold CRS is from about 0.4 to about 0.5.

21. The method of any one of claims 14 to 20 wherein the threshold CRS is from about 0.35 to about 0.36.

22. The method of any one of claims 14 to 21 wherein the threshold CRS is about 0.357.

23. The method of any preceding claim, wherein the method comprises measuring the amount of circulating tumour cells (CTCs) and the concentration of prostate specific antigen (PSA) in one or more blood samples from the subject, wherein the combination of greater than or equal to 2 CTCs per 7.5 ml_ of sample and a concentration of greater than 23 ng/mL PSA is predictive of cancer metastasis in a subject, wherein the CTCs are CD45 negative, CK positive and vimentin positive.

24. The method of any preceding claim, wherein the method comprises measuring the amount of circulating tumour cells (CTCs) and the concentration of prostate specific antigen (PSA) in one or more blood samples from the subject, wherein the CTCs are CD45 negative, CK positive and vimentin positive, and wherein a combined risk score (CRS) greater than or equal to 0.357 is predictive of cancer metastasis, wherein the CRS is calculated according to the formula:

(0.012 x PSA concentration in the blood sample in ng/ml) + (0.1 15 x number of circulating tumour cells that are CD45 negative, CK positive and vimentin positive).

25. The method of any preceding claim further comprising enriching one or more samples for CTCs.

26. The method of claim 25 wherein the enriching is by isolating and/or counting the CTCs.

27. The method of any one of claims 25 to 26 wherein the enriching, the isolating and/or the counting is by size- and/or deformability-based sorting of the CTCs.

28. The method of claim 27 wherein the size-based sorting of the CTCs excludes any cells with a dimension below about 10 μιτι.

29. The method of any preceding claim wherein the subject is a human.

30. The method of any preceding claim wherein no metastases have been previously detected in the subject, the cancer is NO stage and/or the cancer is M0 stage.

31. The method of any preceding claim wherein the prostate cancer is localised prostate cancer, advanced prostate cancer, malignant prostate cancer, aggressive prostate cancer, castration resistant prostate cancer (CRPC) or metastatic prostate cancer.

32. The method of any preceding claim further comprising measuring primary Gleason score, alkaline phosphatase (ALP) level and/or lactate dehydrogenase (LDH) level in one or more samples from the subject.

33. The method of any preceding claim wherein the amount of PSA and/or CTCs is determined by immunoassay, flow cytometry, immunofluorescence or immunohistochemistry.

34. The method of any one of claims 4 to 33 wherein the presence or absence of CD45, CK and/or vimentin is determined by immunoassay, flow cytometry, immunofluorescence or immunohistochemistry.

35. The method of any preceding claim further comprising measuring genomic alteration in the CTCs.

36. The method of claim 35 wherein the measuring genomic alteration confirms the identity of the CTCs.

37. The method of any one of claim 35 to claim 36 wherein the measuring genomic alteration identifies one or more mutations of the CTCs and/or confirms the malignancy of the CTCs.

38. The method of any one of claim 35 to claim 37 further comprising comparing genomic alteration in the CTCs of one or more samples from the subject with genomic alteration in the CTCs of a control.

39. The method of claim 38 wherein the control is a sample from a healthy individual.

40. The method of any one of claim 35 to claim 39 wherein the measuring genomic alteration is by fluorescence in situ hybridization (FISH).

41. The method of claim 40 wherein the FISH is repeated FISH.

42. The method of any one of claim 40 to claim 43 wherein the FISH comprises stripping at 40 to 90 °C with a stripping buffer comprising 1 % to 3 % sodium dodecyl sulfate (SDS), 0.055 M to 0.075 M Tris-HCI and 0.5 % to 1.1 % β-mercaptoethanol and wherein the stripping buffer has a pH from 6.6 to 7.0.

43. The method of claim 42 wherein the stripping buffer comprises 2 % SDS, 0.0625 M Tris- HCI and 0.8 % β-mercaptoethanol and wherein the stripping buffer has a pH of 6.8.

44. The method of any preceding claim comprising taking a first blood sample from the subject and taking a second blood sample from the subject.

45. The method of any preceding claim wherein the amount of PSA is measured in a first sample and the amount of CTCs is measured in a second sample.

46. A method of treating cancer in a subject for whom metastasis has been predicted according to any preceding claim comprising administering a therapeutic agent to the subject and/or adopting a therapeutic regimen.

47. The method of claim 46 wherein the therapeutic regimen is selected from the group consisting of hormone therapy, second line hormone therapy, chemotherapy, radiotherapy, palliative radiotherapy, immunotherapy, and bone-targeting therapy, and/or the therapeutic agent is selected from the group consisting of an anti-androgen, such as bicalutamide; a corticosteroid such as dexamethasone, prednisolone, or hydrocortisone; triamcinolone; ketoconazole; transdermal or oral oestrogen, such as Evorel or diethylstilbestrol; Abiraterone; Enzalutamide; a

chemotherpapeutic agent such as Docetaxel, Estramustine, Mitoxantrone, Paclitaxel, CL56 (Chlorambucil + lomustine), Estramustine (Estracyt), Melphalan, ECarboF (Epirubicin + Carboplatin + Folinic Acid + 5-Fluorouracil), ECarboX (Epirubicin + Carboplatin + Folinic Acid + Capecitabine); or Cabazitaxel;

48. A combination of CTCs and PSA for use in the diagnosis of metastatic prostate cancer.

49. Use of a combination of CTCs and PSA isolated from a subject in an ex vivo and/or in vitro method of predicting prostate cancer metastasis.

50. Use of a combination of CTCs and PSA isolated from a subject in an ex vivo and/or in vitro method of diagnosing metastatic prostate cancer.

51. A stripping buffer comprising 1 % to 3 % SDS, 0.055 M to 0.075 M Tris-HCI and 0.5 % to 1.1 % β-mercaptoethanol wherein the stripping buffer has a pH from 6.6 to 7.0.

52. The stripping buffer of claim 51 wherein the stripping buffer comprises 2 % SDS, 0.0625 M Tris-HCI and 0.8 % β-mercaptoethanol wherein the stripping buffer has a pH of 6.8.

53. The stripping buffer according to any one of claim 51 to claim 52 for use in predicting cancer metastasis and/or the diagnosis of metastatic cancer.

54. A kit comprising specific binding molecules which bind to one or more of PSA, CD45, CK and vimentin.

55. The kit of claim 54 wherein the specific binding molecules are antibodies, antibody fragments or aptamers.

56. The kit of any one of claim 54 to claim 55 further comprising one or more nucleic acid molecule which hybridises under stringent conditions to a target genomic region.

57. The kit of any one of claim 54 to claim 56 further comprising a stripping buffer comprising 1 % to 3 % SDS, 0.055 M to 0.075 M Tris-HCI and 0.5 % to 1.1 % β-mercaptoethanol, wherein the stripping buffer has a pH from 6.6 to 7.0.

58. The kit of claim 57 wherein the stripping buffer comprises 2 % SDS, 0.0625 M Tris-HCI and 0.8 % β-mercaptoethanol, wherein the stripping buffer has a pH of 6.8.

59. The kit of any one of claim 54 to claim 58 further comprising instructions for use.