PRIORITY CLAIM

[001] This application claims priority to Australian Provisional Patent Application 2020903281 filed on 14 September 2020, the contents of which are hereby incorporated by reference.

FIELD

[002] The present disclosure relates to a marker associated with colorectal cancer and also associated with metastasis to the liver by colorectal cancer. In certain embodiments, the present disclosure relates to methods of assessing the risk of colorectal cancer or assessing the risk of metastasis by colorectal cancer. In other embodiments, the present disclosure relates to methods for treating colorectal cancer or metastasis of colorectal cancer using the level of marker, methods for diagnosis of colorectal cancer, methods for prognosis for time of disease-free survival from colorectal cancer, systems and software for assessing the risk of colorectal cancer or assessing the risk of metastasis to the liver, and products for determining the risk of colorectal cancer in a subject or determining the risk of metastasis of colorectal cancer to the liver.

BACKGROUND

[003] Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide, and therefore a critical healthcare priority. National screening programs for CRC, which have been introduced in more than 50 countries, increased the proportion of CRC patients who are diagnosed at an early disease stage. Indeed, the incidence of CRC in developing countries and in young adults is on the rise.

[004] Presently about 50% of patients with CRC are diagnosed with localized disease (stage I— II), nevertheless about 25% present at diagnosis with synchronous metastases in distant organs (stage IV). The liver is the most common site for distant metastases from cancers arising in the colon. Approximately 25% of CRC patients will develop secondary hepatic malignancy after resection of the primary tumour. For various reasons the majority of metastatic patients are not amenable to hepatic surgery. [005] Detection and appropriate staging of local recurrent disease, metachronous lesions, and distant metastases at an early, asymptomatic, and curable stage is an important goal. However, disease surveillance and management are challenging since there are no symptoms in the early stages of liver metastasis, and metastases are often unresponsive to available treatment options. Among primary CRC patients with stage I- III disease, approximately 30% will develop recurrent disease after initial treatment; whereas within secondary CRC patients with metastatic (stage IV) disease, up to 65% will relapse after treatment with curative intent.

[006] Risk prediction and early detection of disease can increase patient eligibility for a range of effective treatments, reduce morbidity, and improve overall survival. Accordingly, there is a need to be able to determine the risk of colorectal cancer in a subject and also a need to be able to determine the risk of metastasis of the cancer to the liver.

SUMMARY

[007] The present disclosure relates to a marker associated with colorectal cancer and metastasis to the liver by colorectal cancer, and to methods of assessing the risk of colorectal cancer or assessing the risk of metastasis by colorectal cancer. The present disclosure also relates to methods for treating colorectal cancer or metastasis of colorectal cancer using the level of marker, methods for diagnosis of colorectal cancer, methods for prognosis for time of disease-free survival from colorectal cancer, systems and software for assessing the risk of colorectal cancer or assessing the risk of metastasis to the liver, and products for determining the risk of colorectal cancer in a subject or determining the risk of metastasis of colorectal cancer to the liver.

[008] Certain embodiments of the present disclosure provide a method of assessing the risk of colorectal cancer in a subject, or assessing the risk of metastasis by colorectal cancer to the liver in a subject suffering from colorectal cancer, the method comprising:

(i) determining the level of circulating Secreted Frizzled-Related Protein 5 (SFRP5) in the subject; and

(ii) determining the risk of the colorectal cancer in the subject, or determining the risk of the metastasis to the liver in the subject suffering from colorectal cancer by the cancer, on the basis of the circulating level of Secreted Frizzled- Related Protein 5 so determined.

[009] Certain embodiments of the present disclosure provide a method of assessing the risk of colorectal cancer in a subject, the method comprising determining the risk on the basis of the circulating level of Secreted Frizzled-Related Protein 5 (SFRP5) in the subject.

[0010] Certain embodiments of the present disclosure provide a method of assessing risk of metastasis of colorectal cancer to the liver in a subject suffering from colorectal cancer, the method comprising determining the risk on the basis of the circulating level of Secreted Frizzled-Related Protein 5 (SFRP5) in the subject.

[0011] Certain embodiments of the present disclosure provide a method of diagnosis for colorectal cancer in a subject, the method comprising determining the level of circulating Secreted Frizzled-Related Protein 5 (SFRP5) in the subject and determining the likelihood of colorectal cancer in the subject on the basis of the circulating level of Secreted Frizzled-Related Protein 5 so determined.

[0012] Certain embodiments of the present disclosure provide a method of prognosis for time of disease-free survival of a subject suffering from colorectal cancer, the method comprising determining the level of circulating Secreted Frizzled-Related Protein 5 (SFRP5) in the subject and determining the time of survival for the subject on the basis of the of the circulating level of Secreted Frizzled-Related Protein 5 so determined.

[0013] Certain embodiments of the present disclosure provide use of Secreted Frizzled- Related Protein 5 (SFRP5) as a circulating marker for determining the presence of colorectal cancer in a subject or for determining risk of metastasis to the liver by a colorectal cancer in a subject.

[0014] Certain embodiments of the present disclosure provide a method of treating a subject for colorectal cancer, the method comprising: (i) determining the level of circulating Secreted Frizzled-Related Protein 5 (SFRP5) in the subject; and

(ii) treating the subject on the basis of the circulating level of Secreted Frizzled- Related Protein 5 so determined.

[0015] Certain embodiments of the present disclosure provide a method of treating a subject for metastasis to the liver by colorectal cancer, the method comprising:

(i) determining the level of circulating Secreted Frizzled-Related Protein 5 (SFRP5) in the subject; and

(ii) treating the subject on the basis of the circulating level of Secreted Frizzled- Related Protein 5 so determined.

[0016] Certain embodiments of the present disclosure provide a method of treating a subject for colorectal cancer, the method comprising treating the subject on the basis of an increased circulating level of Secreted Frizzled-Related Protein 5 in the subject.

[0017] Certain embodiments of the present disclosure provide a method of treating a subject for metastasis of a colorectal cancer to the subject, the method comprising treating the subject on the basis of a decreased circulating level of Secreted Frizzled-Related Protein 5 in the subject.

[0018] Certain embodiments of the present disclosure provide a system for assessing the risk of colorectal cancer in a subject, or for assessing the risk of tumour metastasis to the liver in a subject suffering from colorectal cancer, the system comprising a computer processor configured to receive information on the level of circulating SFRP5 in the subject and processing the received information to determine the risk of colorectal cancer in the subject or to determine the risk of metastasis of the colorectal cancer to the liver in the subject.

[0019] Certain embodiments of the present disclosure provide a computer-readable medium encoded with programming instructions executable by a computer processor to allow the computer processor to process information received on the level of circulating SFRP5 in a subject so as to determine the risk of colorectal cancer in the subject or to determine the risk of metastasis of colorectal cancer to the liver in the subject.

[0020] Certain embodiments of the present disclosure provide computer software encoded with programming instructions executable by a computer processor to allow the computer processor to process information received on the level of circulating SFRP5 in a subject so as to determine risk the risk of colorectal cancer in the subject or to determine the risk of metastasis of colorectal cancer to the liver in the subject.

[0021] Certain embodiments of the present disclosure provide a method of determining the risk of colorectal cancer in a subject, or determining the risk of metastasis of colorectal cancer to the liver in a subject suffering from colorectal cancer, the method comprising: transmitting information on the level of circulating SFRP5 in the subject to a computer processor; using the computer processor to process the information to determine the risk of colorectal cancer in a subject or to determine the risk of metastasis of colorectal cancer to the liver in a subject; and providing information on the risk so determined to a user.

[0022] Certain embodiments of the present disclosure provide a kit for performing a method as described herein.

[0023] Certain embodiments of the present disclosure provide a kit for determining the risk of colorectal cancer in a subject or for determining the risk of metastasis of colorectal cancer to the liver in a subject, the kit comprising:

(i) one or more reagents for processing a sample to allow detection of SFRP5; and

(ii) one or more reagents for detecting SFRP5; and optionally:

(a) one or more references, positive controls and/or negative controls; and/or

(b) instructions for determining the level of SFRP5; and/or

(c) information relating to the risk of metastasis to the liver by a cancer associated with the level of circulating SFRP5. [0024] Certain embodiments of the present disclosure provide a method of screening a subject for a colorectal cancer, the method comprising determining the level of secreted frizzled-related protein (SFRP5) in the subject and identifying a colorectal cancer in the subject on the basis of the level of SFRP5 in the subject, wherein an increased circulating level of SFRP5 is indicative of the presence of colorectal cancer in the subject.

[0025] Certain embodiments of the present disclosure provide a method of detecting colorectal cancer in a subject, the method comprising determining the level of secreted frizzled-related protein (SFRP5) in a gastrointestinal tissue sample from the subject and detecting a colorectal cancer in the subject on the basis of the level of SFRP5 in the subject.

[0026] Certain embodiments of the present disclosure provide use of secreted fizzled- related protein (SFRP5) as a diagnostic and/or prognostic marker for colorectal cancer.

[0027] Other embodiments are disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

[0028] Certain embodiments are illustrated by the following figures. It is to be understood that the following description is for the purpose of describing particular embodiments only, and is not intended to be limiting with respect to the description.

[0029] Figure 1 shows that circulating SFRP5 is elevated in early stage colorectal cancer and elevated SFRP5 is associated with improved disease-free survival. (A) Plasma concentration of SFRP5 was determined in patients that underwent a cholecystectomy (C, n = 30), surgical removal of bowel tissue for benign polyps or diverticulitis without pathological evidence of CRC (NC, n = 16), or with stage I (n = 32), stage II (n = 38), stage III (n = 40), or stage IV (n = 52) CRC. Data analysed using Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test. ***P < 0.001, *P < 0.05. (B) Disease-free survival (DFS) in patients with stage I-III CRC. Patients were dichotomized into low (< 28 ng/mL, n = 20) or high (> 28 ng/mL, n= 67) plasma SFRP5 concentration, and survival curves were compared using multi-variant Cox regression analysis. [0030] Figure 2 shows lower levels of circulating SFRP5 in patients diagnosed with vascular invasion or with liver metastasis. Patients diagnosed with vascular invasion (3 A, n=25) or with liver metastasis (3B, n=23) displayed significantly lower preoperative plasma SFRP5 concentration (P=0.002 and P=0.01, respectively). 3C. The Pearson’s correlation between SFRP5 levels and CRC patients’ age at operation (r=0.28).

DETAILED DESCRIPTION

[0031] The present disclosure is based on the demonstration that circulating SFRP5 is higher in patients with colorectal cancer, but is lower in CRC patients with either vascular invasion or liver metastasis. Low SFRP5 levels are associated with shorter disease-free survival. In addition, the expression of the SFRP5 transcript is lower in colorectal tumour tissue.

[0032] Certain embodiments of the present disclosure provide a method of assessing the risk of colorectal cancer in a subject, or assessing the risk of metastasis by colorectal cancer to the liver in a subject.

[0033] In certain embodiments of the present disclosure provide a method of assessing the risk of colorectal cancer in a subject, or assessing the risk of metastasis by colorectal cancer to the liver in a subject suffering from colorectal cancer, the method comprising:

(i) determining the level of circulating Secreted Frizzled-Related Protein 5 (SFRP5) in the subject; and

(ii) determining the risk of the colorectal cancer in the subject, or determining the risk of the metastasis to the liver in the subject suffering from colorectal cancer by the cancer, on the basis of the circulating level of Secreted Frizzled- Related Protein 5 so determined.

[0034] Secreted frizzled-related protein 5 (SFRP5) is a member of the SFRP family that contains a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzled proteins. SFRPs act as soluble modulators of Wnt signaling. The amino acid sequence of human SFRP5 is provided at UniProt accession number Q5T4F7-1. The HDNC accession number is HGNC: 10779. The term “Secreted frizzled-related Protein 5” or “SFRP5” as used herein also refers to a fragment or part of the protein, the circulating level of which reflects the circulating level of the full length protein.

[0035] In certain embodiments, the method is used for diagnostic purposes. In certain embodiments, the method is used for prognostic purposes.

[0036] In certain embodiments, the subject is a human subject suffering from, or susceptible to, colorectal cancer.

[0037] In certain embodiments, the level of circulating SFRP5 comprises the level of SFRP5 in blood, plasma or serum, or a processed fraction, component, or part of any of the aforementioned.

[0038] In certain embodiments, the level of circulating SFRP5 comprises the level of SFRP5 in plasma. In certain embodiments, the level of circulating SFRP5 comprises the level of plasma SFRP5.

[0039] Methods for processing blood, plasma and serum are known in the art. In certain embodiments, the method comprises determining the level of SFRP5 in a processed fraction, component or part of blood, plasma, or serum. It will be appreciated that methods for determining the level of circulating SFRP5 may require use of a processed fraction, component or part of blood, plasma or serum, such methods being known in the art.

[0040] Methods for determining the level of proteins in blood, plasma or serum as described, for example, in “Mosby's Manual of Diagnostic and Laboratory Tests” by Kathleen Deska Pagana, Timothy J. Pagana (2017), Elsevier Publishers.

[0041] In certain embodiments, the determining of the level of circulating SFRP5 comprises immunological detection. Immunological methods for detecting circulating are known in the art, for example in “The immunological Handbook” 4 ^th Edition by Rhys John, Chris Sheehan, Steve Binder and Jianwen He (2013), Elsevier Publishers. Antibodies to SFRP5, and kits utilising antibodies, are known in the art and are commercially available. [0042] In certain embodiments, the determining of the level of circulating SFRP5 comprises mass spectrometric detection. Mass spectrometric methods are known in the art, for example as described in Zhang et al (2007) Molecular & Cellular Proteomics 6.1: 64-71.

[0043] In certain embodiments, the method for determining the level of SFRP5 comprises immunological detection. In certain embodiments, the method of determining the level of SFRP5 comprises ELISA. ELISA kits for determining the level of human SFRP5 are commercially available.

[0044] In certain embodiments, an elevated or increased level of SFRP5 in the subject is indicative of an increased risk of suffering colorectal cancer.

[0045] In certain embodiments, a low or reduced level of SFRP5 in a subject suffering colorectal cancer is indicative of an increased risk of metastasis to the liver.

[0046] The level of the protein may be as compared to one or more of a known level, a reference sample, or one or more positive or negative controls.

[0047] In certain embodiments, a circulating level of 28 ng/ml or greater in the subject is indicative of an increased risk of colorectal cancer in the subject.

[0048] In certain embodiments, a circulating level of 30 ng/ml or greater, 40 ng/ml or 50 ng/ml or greater in the subject is indicative of an increased risk of colorectal cancer in the subject.

[0049] In certain embodiments, a circulating level of less than 25 ng/ml or less, 20 ng/ml or less, 15 ng/ml or less, or 10 ng/ml or less in the subject is indicative of a normal risk of colorectal cancer in the subject.

[0050] In certain embodiments, a circulating level of less than 25 ng/ml or less, 20 ng/ml or less, 15 ng/ml or less, or 10 ng/ml or less in the subject is indicative of a reduced normal risk of colorectal cancer in the subject. [0051] In certain embodiments, a circulating level of less than 28 ng/ml in a subject suffering from colorectal cancer is indicative of an increased risk of tumour metastasis.

[0052] In certain embodiments, the method comprises a computer assisted assessment of the level of SFRP5 to determine the risk of metastasis to the liver.

[0053] In certain embodiments, the computer assisted assessment comprises use of an algorithm. In certain embodiments, the algorithm provides the mathematical details of the relationship of the level of circulating SFRP5 to the risk of colorectal cancer or the risk of metastasis by colorectal cancer to the liver, and may also include the mathematical details of the influence of other clinical or biochemical factors. Algorithms may be developed by a person skilled in the art.

[0054] For example, a computer processor may comprise instructions that when executed by the processor cause the processor to compare data associated with the level of SFRP5 with the level of SFRP5 known to be indicative of the risk of colorectal cancer, or the risk of metastasis by colorectal cancer to the liver in a subject suffering from colorectal cancer, where the data associated with the level of SFRP5 (and any other relevant clinical, biochemical or other parameters) known to be indicative of the risk is held in computer readable memory.

[0055] In certain embodiments, the computer assisted assessment involves on-line interrogation of the computer processor with the level of SFRP5 (and any other clinical, biochemical or other factors) to determine the risk of colorectal cancer or metastasis to the liver.

[0056] In certain embodiments, the method is used for diagnostic purposes. In certain embodiments, the method is used for prognostic purposes. Research uses of the present disclosure are contemplated.

[0057] In certain embodiments, the method comprises assessing one or more clinical or biochemical characteristics of the subject and using the clinical characteristics in conjunction with the level of SFRP5 to determine the risk of colorectal cancer in the subject or the risk of metastasis to the liver in the subject suffering from colorectal cancer. [0058] In certain embodiments, the method comprises assessing one or more clinical and/or biochemical characteristics of the subject and using the clinical and/or biochemical characteristics in conjunction with the level of SFRP5 to determine the risk of metastasis to the liver by the cancer.

[0059] In certain embodiments, the one or more clinical or biochemical characteristics comprise one or more of liver function markers, liver fat content, cirrhosis of the liver, fibrosis of the liver, hepatitis, fatty liver disease, metabolic syndrome and type 2 diabetes.

[0060] In certain embodiments, the method further comprises detecting one or more liver disease markers and using the liver disease markers in conjunction with the level of SFRP5 to determine the risk of metastasis to the liver.

[0061] In certain embodiments, the method comprises using a computer processor to process data associated with the level of SFRP5 to assess the risk. Methods for assessing risk are known in the art.

[0062] In certain embodiments, the method comprises the use of a device to determine the level of SFRP5. For example, the method may comprise use of an ELISA reader or a mass spectrometer. In certain embodiments, the method comprises receiving data from the device and transferring the data to a computer processor.

[0063] In certain embodiments, the method comprises transferring the data/information over the internet to a computer processor.

[0064] In certain embodiments, the method comprises transferring data associated with the level of SFRP5 over the internet to a computer processor to generate a risk.

[0065] For example, web-based statistical software can be used to determine the risk and provide a probability of suffering from cancer and/or the risk of metastasis.

[0066] In certain embodiments, the method comprises processing the data/information to classify the risk in the subject as having an increased risk, a high risk, a moderate risk, a low risk, a normal risk or a decreased risk. [0067] Computer processors are known in the art. Method for sending and/or receiving data/information are known in the art.

[0068] In certain embodiments, the method comprises use of a system for assessing the risk using a computer processor. Systems utilising computer processors are known in the art.

[0069] In certain embodiments, the method is used to identify a subject suffering from colorectal cancer, for diagnosis of the presence of colorectal tumour in a subject, to identify a subject with an increased risk of metastasis of colorectal cancer in a subject, for prognosis of metastasis of colorectal cancer to the liver, for prognosis/prediction for time of disease-free survival and overall survival of a subject suffering from colorectal cancer, for selection of patients for treatment, for selection of patients into a clinical trial or for screening for colorectal cancer or liver metastasis.

[0070] In certain embodiments, the present disclosure provides a method of assessing the risk of colorectal cancer in a subject, the method comprising determining the risk on the basis of the circulating level of Secreted Frizzled-Related Protein 5 (SFRP5) in the subject.

[0071] In certain embodiments, a circulating level of 28 ng/ml or greater in the subject is indicative of an increased risk colorectal cancer in the subject.

[0072] In certain embodiments, a circulating level of 30 ng/ml or greater, 40 ng/ml or greater, 50 ng/ml or greater in the subject is indicative of an increased risk colorectal cancer in the subject.

[0073] In certain embodiments, a circulating level of less than 25 ng/ml or less, 20 ng/ml or less, 15 ng/ml or less, or 10 ng/ml or less in the subject is indicative of a normal risk of colorectal cancer in the subject.

[0074] In certain embodiments, a circulating level of less than 25 ng/ml or less, 20 ng/ml or less, 15 ng/ml or less, or 10 ng/ml or less in the subject is indicative of a reduced normal risk of colorectal cancer in the subject. [0075] In certain embodiments, a circulating level of less than 28 ng/ml is indicative of an increased risk of tumour metastasis in the subject suffering from colorectal cancer.

[0076] In certain embodiments, the present disclosure provides a method of determining risk of metastasis of colorectal cancer to the liver in a subject suffering from colorectal cancer, the method comprising determining the risk of on the basis of the circulating level of Secreted Frizzled-Related Protein 5 (SFRP5) in the subject.

[0077] In certain embodiments, a circulating level of less than 28 ng/ml is indicative of an increased risk of tumour metastasis in the subject suffering from colorectal cancer.

[0078] Certain embodiments of the present disclosure provide a method of diagnosis for colorectal cancer

[0079] In certain embodiments, the present disclosure provides a method of diagnosis for colorectal cancer in a subject, the method comprising determining the level of circulating Secreted Frizzled-Related Protein 5 (SFRP5) in the subject and determining the likelihood of colorectal cancer in the subject on the basis of the circulating level of Secreted Frizzled-Related Protein 5 so determined.

[0080] In certain embodiments, a circulating level of 28 ng/ml or greater in the subject is indicative of the presence of colorectal cancer in the subject.

[0081] In certain embodiments, a circulating kevel of 30 ng/ml or greater, 40 ng/ml or greater, or 50 ng/ml or greater is indicative of the presence of colorectal cancer in the subject.

[0082] In certain embodiments, a circulating level of less than 25 ng/ml or less, 20 ng/ml or less, 15 ng/ml or less, or 10 ng/ml or less in the subject is indicative of a normal risk of colorectal cancer in the subject.

[0083] In certain embodiments, a circulating level of less than 25 ng/ml or less, 20 ng/ml or less, 15 ng/ml or less, or 10 ng/ml or less in the subject is indicative of a reduced normal risk of colorectal cancer in the subject. [0084] Certain embodiments of the present disclosure provide a method of prognosis for time of disease-free survival of a subject suffering from colorectal cancer.

[0085] In certain embodiments, the present disclosure provides a method of prognosis for time of disease-free survival of a subject suffering from colorectal cancer, the method comprising determining the level of circulating Secreted Frizzled-Related Protein 5 (SFRP5) in the subject and determining the time of survival for the subject on the basis of the circulating level of Secreted Frizzled-Related Protein 5 so determined.

[0086] In certain embodiments, a circulating level of Secreted Frizzled-Related Protein 5 (SFRP5) of less than 28 ng/ml lower is associated with a shorter period of disease-free survival.

[0087] In certain embodiments, a circulating level of Secreted Frizzled-Related Protein 5 (SFRP5) of 28 ng/ml lower or greater is associated with a shorter period of disease-free survival.

[0088] Certain embodiments of the present disclosure provide use of Secreted Frizzled- Related Protein 5 (SFRP5) as a marker for determining the presence of colorectal cancer in a subject, or the likelihood of metastasis to the liver by a colorectal cancer in a subject.

[0089] Certain embodiments of the present disclosure provide a method of treating a subject for colorectal cancer based on the level of SFRP5 determined.

[0090] In certain embodiments, the present disclosure provides a method of treating a subject for colorectal cancer, the method comprising:

(i) determining the level of circulating Secreted Frizzled-Related Protein 5 (SFRP5) in the subject; and

(ii) treating the subject on the basis of the circulating level of Secreted Frizzled- Related Protein 5 so determined.

[0091] In certain embodiments, the subject is treated when the level of circulating SFRP5 in the subject is 28 ng/ml or more. [0092] In certain embodiments, the subject is treated when the circulating level of 30 ng/ml or greater, 40 ng/ml or greater, 50 ng/ml or greater.

[0093] Methods or treating colorectal cancer are known in the art, and include for example surgery, radiation therapy, chemotherapy, immunotherapy and targeted therapy.

[0094] Certain embodiments of the present disclosure provide a method of treating a subject for metastasis to the liver by colorectal cancer.

[0095] In certain embodiments, the present disclosure provide a method of treating a subject for metastasis to the liver by colorectal cancer, the method comprising:

(i) determining the level of circulating Secreted Frizzled-Related Protein 5 (SFRP5) in the subject; and

(ii) treating the subject on the basis of the circulating level of Secreted Frizzled- Related Protein 5 so determined.

[0096] In certain embodiments, the subject is treated when the level of circulating SFRP5 in the subject is less than 28 ng/ml.

[0097] Methods for treating liver metastasis from a colorectal cancer are known in the art and include, for example, surgical resection and chemotherapy.

[0098] In certain embodiments, the present disclosure provides a method of treating a subject for colorectal cancer, the method comprising treating the subject on the basis of an increased circulating level of Secreted Frizzled-Related Protein 5 in the subject.

[0099] In certain embodiments, the subject is treated when the level of circulating SFRP5 in the subject is 28 ng/ml or more.

[00100] In certain embodiments, the present disclosure provides a method of treating a subject for metastasis of a colorectal cancer to the subject, the method comprising treating the subject on the basis of a decreased circulating level of Secreted Frizzled-Related Protein 5 in the subject. [00101] In certain embodiments, the subject is treated when the level of circulating SFRP5 in the subject is less than 28 ng/ml.

[00102] Certain embodiments of the present disclosure provide a system for assessing the risk of colorectal cancer in a subject, or assessing the risk of tumour metastasis to the liver in a subject suffering from colorectal cancer.

[00103] In certain embodiments, the present disclosure provides a system for assessing the risk of colorectal cancer in a subject or assessing the risk of tumour metastasis to the liver in a subject suffering from colorectal cancer, the system comprising a computer processor configured to receive information on the level of circulating SFRP5 in the subject and processing the received information to determine the risk of colorectal cancer in the subject or the risk of metastasis of the colorectal cancer to the liver in the subject.

[00104] Systems utilising computer processors are known in the art. Examples are as described herein.

[00105] The system may further comprise devices such as an ELISA reader or a mass spectrograph, and software to determine the risk of colorectal cancer or the risk of metastasis of the colorectal cancer to the liver.

[00106] In certain embodiments, the present disclosure provides a system for assessing the risk of colorectal cancer in a subject or assessing the risk of tumour metastasis to the liver in a subject suffering from colorectal cancer using a computer processor configured to process a method as described herein.

[00107] The system may further comprise devices such as an ELISA reader or a mass spectrograph, and software to determine the risk of metastasis of the colorectal cancer to the liver.

[00108] Certain embodiments of the present disclosure provide a computer readable medium.

[00109] In certain embodiments, the present disclosure provides a computer-readable medium encoded with programming instructions executable by a computer processor to allow the computer processor to process information received on the level of circulating SFRP5 in a subject so as to determine the risk of colorectal cancer in the subject or the risk of metastasis of colorectal cancer to the liver in the subject.

[00110] Certain embodiments of the present disclosure provide a computer processor comprising a computer-readable medium as described herein.

[00111] Certain embodiments of the present disclosure provide computer software.

[00112] In certain embodiments, the present disclosure provides computer software encoded with programming instructions executable by a computer processor to allow the computer processor to process information received on the level of circulating SFRP5 in a subject so as to determine the risk of colorectal cancer in the subject or the risk of metastasis of colorectal cancer to the liver in the subject.

[00113] Software is as described herein.

[00114] In certain embodiments, the present disclosure provides a method of determining the risk of colorectal cancer in a subject or determining the risk of metastasis of colorectal cancer to the liver in a subject suffering from colorectal cancer, the method comprising: transmitting information on the level of circulating SFRP5 in the subject to a computer processor; using the computer processor to process the information to determine the risk of colorectal cancer in a subject or determining the risk of metastasis of colorectal cancer to the liver in a subject; and providing information on the risk so determined to a user.

[00115] In certain embodiments, the method comprises transmitting information via the internet to the computer processor.

[00116] In certain embodiments, the method comprises providing information on the risk via the internet to the user. [00117] Certain embodiments of the present disclosure provide a kit for performing a method as described herein.

[00118] Certain embodiments of the present disclosure provide a kit for determining the risk of colorectal cancer in a subject or determining the risk of metastasis of colorectal cancer to the liver in a subject, the kit comprising:

(i) one or more reagents for processing a sample to allow detection of SFRP5; and

(ii) one or more reagents for detecting SFRP5; and optionally:

(a) one or more references, positive controls and/or negative controls; and/or

(b) instructions for determining the level of SFRP5; and/or

(c) information relating to the risk of colorectal cancer and/or the risk of metastasis to the liver by a cancer associated with the level of circulating SFRP5.

[00119] For example, the kit may utilise a sandwich enzyme-linked immuno- sorbent assay (ELISA) technology. An antibody to SFRP5 may be used and be pre-coated onto a 96 or 384 well plate. Oher formats are contemplated. The kit may include standards, test samples, reagents for detection of the antibody, wash buffers, and reagents for quantifying the amount of SFRP5.

[00120] Certain embodiments of the present disclosure provide a method of screening a subject for a colorectal cancer.

[00121] In certain embodiments, the present disclosure provides a method of screening a subject for a colorectal cancer, the method comprising determining the level of secreted frizzled-related protein (SFRP5) in the subject and identifying a colorectal cancer in the subject on the basis of the level of SFRP5 in the subject.

[00122] In certain embodiments, an increased level of SFRP5 in the blood is indicative of the presence of colorectal cancer in the subject. In certain embodiments, an increased circulating level of SFRP5 is indicative of the presence of colorectal cancer in the subject. [00123] In certain embodiments, the method comprises determining the level of SFRP5 transcription in a gastrointestinal tissue sample

[00124] In certain embodiments, a decreased level of SFRP5 in the tissue is indicative that the tissue is cancerous.

[00125] In certain embodiments, the method is used to identify a colorectal cancer. In certain embodiments, the method is used to exclude the presence of a colorectal cancer.

[00126] In certain embodiments, the method comprises detecting SFRP5. Methods for detecting SFRP5 are known in the art and include, for example, immunological detection of SFRP5, or mass spectrometric detection of SFRP5.

[00127] In certain embodiments, the immunological detection comprises ELISA. For ELISA, typically the level of protein detected is compared to a standard curve of the concentration of the antigen (SRRP5) to the measured parameter, typically absorbance.

[00128] In certain embodiments, the method comprises obtaining a sample from the subject. In certain embodiments, the method comprises obtaining a sample from the subject and processing the sample to allow detection of SFRP5, for example by ELISA.

[00129] In certain embodiments, the sample is a blood, plasma or serum sample.

[00130] In certain embodiments, the sample is a tissue sample. In certain embodiments, the method comprises determining the level of SFRP5 in a tissue sample.

[00131] Methods for determining the level of a protein in a tissue sample include immunohistochemistry. Methods for assessing the level of a protein in a tissue sample are known in the art.

[00132] In certain embodiments, the sample comprises one or more of blood, plasma and serum. In certain embodiments, the method comprising determining the level of SFRP5 in blood, plasma or sera. [00133] Methods for obtaining blood, plasma, serum from a subject are known in the art. Methods for determining the level of proteins in blood, plasma or serum are known in the art, such as immunological detection and mass spectrometric detection. Immunological detection of proteins in blood, plasma or serum is known in the art, for example using ELISA or Western blotting. Mass spectrometric methods are known in the art, for example as described in Zhang et al (2007) Molecular & Cellular Proteomics 6.1: 64-71.

[00134] In certain embodiments, the method comprises determining the level of circulating SFRP5. In certain embodiments, the level of circulating SFRP5 comprises the level of SFRP5 in plasma. In certain embodiments, the level of circulating SFRP5 comprises the level of plasma SFRP5.

[00135] In certain embodiments, the method is used for diagnostic purposes. In certain embodiments, the method is used for prognostic purposes.

[00136] In certain embodiments, the present disclosure provides a method of screening a subject for a colorectal cancer, the method comprising determining the level of secreted frizzled-related protein (SFRP5) in the subject and identifying a colorectal cancer in the subject on the basis of the level of SFRP5 in the subject, wherein an increased circulating level of SFRP5 is indicative of the presence of colorectal cancer in the subject.

[00137] In certain embodiments, the present disclosure provides a method of screening a subject for a colorectal cancer, the method comprising determining the level of secreted frizzled-related protein (SFRP5) in a gastrointestinal sample from the subject and identifying a colorectal cancer in the subject on the basis of a decreased level of SFRP5 in the sample.

[00138] Certain embodiments of the present disclosure provide a method of detecting colorectal cancer in a subject

[00139] In certain embodiments, the method comprises determination of the level of circulating SFRP5 as described herein.

[00140] In certain embodiments, the method comprises determining the level of SFRP5 in a gastrointestinal sample. [00141] In certain embodiments, the method of detecting colorectal cancer in a subject, the method comprising determining the level of secreted frizzled-related protein (SFRP5) in a gastrointestinal tissue sample from the subject and detecting a colorectal cancer in the subject on the basis of the level of SFRP5 in the subject, wherein a decreased level of SFRP5 is indicative of colorectal cancer in the subject.

[00142] Methods for detecting SFRP5 include immunological detection, such as immunohistochemistry.

[00143] In certain embodiments, the method of detecting colorectal cancer in a subject, the method comprising determining the level of secreted frizzled-related protein (SFRP5) in a gastrointestinal tissue sample from the subject and detecting a colorectal cancer in the subject on the basis of the level of SFRP5 in the subject, wherein a decreased level of SFRP5 is indicative of colorectal cancer in the subject.

[00144] Certain embodiments of the present disclosure provide a method of detecting cancerous colorectal cells in a subject, the method comprising detecting cells in a gastrointestinal tissue having a decreased level of SFRP5.

[00145] Methods for determining the level of SFRP5 in tissue samples are as described herein.

[00146] In certain embodiments, the method is used for diagnostic or prognostic purposes. In certain embodiments, the method is used to inform a clinician as to whether to treat a subject. In certain embodiments, the method is used to inform a clinician as to how to treat a subject.

[00147] Certain embodiments of the present disclosure provide use of secreted fizzled- related protein (SFRP5) as a diagnostic and/or prognostic marker for colorectal cancer.

[00148] Certain embodiments of the present disclosure provide use of secreted fizzled- related protein (SFRP5) as a diagnostic and/or prognostic marker for assessing the risk of metastasis to the liver by a colorectal cancer.

[00149] The present disclosure is further described by the following examples. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.

EXAMPLE 1 - High preoperative levels of circulating SFRP5 predict better prognosis in colorectal cancer patients

[00150] ABSTRACT

[00151] Aim: To investigate the diagnostic and prognostic value of circulating SFRP5 (cSFRP5) in colorectal cancer (CRC). Patients & Methods: We evaluated preoperative cSFRP5 levels in CRC patients and controls (n=208). Results: We found significantly higher cSFRP5 levels in CRC patients compared to non-CRC controls (P<0.001). Notably, cSFRP5 levels are significantly lower in CRC patients with either vascular invasion (P=0.001) or liver metastasis (P=0.016). Accordingly, high cSFRP5 levels are associated with longer disease-free- survival in both univariate (P=0.024) and multivariable (P=0.015) analyses. Analysis of an independent TCGA tissue cohort revealed significantly lower SFRP5 RNA expression in CRC tumour tissue compared to adjacent normal mucosa (n=590 vs. 47; P<0.0001). Notably, the main source of SFRP5 is the digestive system, primarily the pancreas. Conclusions: Our findings confirm the role of cSFRP5 as physiologic tumour- suppressor and demonstrate its potential diagnostic and prognostic value in CRC.

[00152] Summary Points

• The role of circulating SFRP5 protein in colorectal cancer as well as its diagnostic/prognostic value remains unknown.

• The aim of this clinical study was to characterise the correlation between plasma SFRP5 levels and clinical parameters in CRC patients and to outline the expression, source and methylation state of SFRP5 in the human digestive system.

• We evaluated preoperative SFRP5 levels in patients undergoing cholecystectomy or surgical intervention for either stage I- IV CRC, diverticulitis or benign polyps, and analysed an independent tissues’ cohort from The Cancer Genome Atlas for SFRP5 gene expression/methylation.

• In our patient cohort, circulating SFRP5 is significantly higher in CRC patients compared to non-CRC controls.

• We also found that in our cohort circulating SFRP5 is lower in CRC patients with either vascular invasion or liver metastasis, accordingly low SFRP5 levels are associated with shorter disease-free survival.

• We also observed that the expression of SFRP5 transcript is significantly lower in colorectal tumour tissue compared to adjacent normal mucosa and that the main source of SFRP5 expression is within the digestive system, primarily the pancreas.

• Our findings suggest that SFRP5 production by normal gut mucosa could represent a physiological protective tumour-suppression mechanism and illustrate the potential value of cSFRP5 as diagnostic and prognostic marker in CRC.

[00153] BACKGROUND

[00154] The aim of this study was to determine the diagnostic and prognostic value of circulating SFRP5 in CRC patients. To this end, we investigated the correlation between the preoperative levels of SFRP5 and validated clinical-pathological parameters of CRC progression. In addition, we investigated the pattern of SFRP5 expression in human tissue.

[00155] METHODS

[00156] Patient plasma samples

[00157] Blood samples were obtained from patients operated on at Wakefield Hospital in Wellington New Zealand or at The Queen Elizabeth Hospital in Adelaide South Australia, with the approval of the New Zealand Central Regional Ethics Committee (CEN/11/09/056/ AM03) and the TQEH/LMH/MH Human Research Ethics Committee (HREC/14/TQEHLMH/164), respectively. The patient cohort included patients that had been diagnosed with CRC and had undergone primary tumor resection, or patients undergoing cholecystectomy or surgical intervention for either diverticulitis or benign polyps (non-CRC). All patients have fasted from the previous night until after blood sample collection. Exclusion criteria left-out people whose English language skills did not allow informed consent, pregnant women, children under 18 years old and people with an intellectual or mental impairment. Age, gender and all other clinical parameters were documented in a clinical database.

[00158] ELISA

[00159] The concentration of circulating SFRP5 protein in patient plasma samples was determined by ELISA according to the manufacturer’s instructions (Cloud-Clone Corp, Houston, USA). Briefly, 100 pL of SFRP5 standards and plasma samples were analyzed. The plates were read at 450 nm immediately using an iMark™ Microplate Reader (Bio Rad Inc.), and the concentration was determined according to the standard curve.

[00160] SFRP5 tissue expression analysis

[00161] RNA sequencing expression (median number of fragments per kilobase of exon per million reads, FPKM) and Infinium HumanMethylation450 BeadChip DNA methylation (beta-values) data were obtained from The Cancer Genome Atlas (TCGA) Research Network (http : //cancer genome . nih . go v/1 , as previously described (18). RNA sequencing expression data for normal tissues were obtained from The Genotype-Tissue Expression (GTEx) Project. The GTEx project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The data used for the analyses described in this manuscript were obtained from the GTEx Portal (http ://w w w . gtexportal . org/) on 03/03/19.

[00162] Statistical analysis

[00163] All statistical analyses were performed using Prism v7.0d for Mac OS X (GraphPad Software, Inc. La Jolla, CA, USA), unless otherwise stated. All tests were two-tailed and assessed at the 5% alpha level.

[00164] Differences between patient groups for age and SFRP5 plasma concentration or transcription expression were determined using Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test. Correlations between the concentration of plasma SFRP5 and different prognostic clinical-pathological were determined using Mann- Whitney test. Comparisons of SFRP5 expression between patient matched N and CRC were determined using Wilcoxon matched-pairs sign rank test. Comparisons of DNA methylation betavalues between N and CRC were determined using unpaired Welch’s t-test with Bonferroni correction for multiple comparisons. Correlations were determined using nonparametric Spearman r correlation.

[00165] Unadjusted and age-adjusted post hoc comparisons between groups for plasma SFRP5 concentration were determined using linear regression models. A log transformation was applied to the SFRP5 concentration prior to analysis in order to meet the assumptions of a linear regression model. The data were transformed back to a linear scale prior to reporting. As such, effects represent the ratio of two geometric means rather than the absolute differences between means. The analyses were completed using SAS v9.4 (SAS Institute Inc., Cary, NC, USA).

[00166] Patients were dichotomized into high or low plasma SFRP5 concentration using a concentration that produced the best separation. To choose the best separation, concentrations between the 20th to 80th percentiles were used to dichotomize all stage I- III patients (n = 87) into high or low concentration groups. Kaplan-Meier disease-free survival curves for the two groups were compared using the log-ranked (Mantel-Cox) test and the concentration cutoff yielding the lowest log-rank P value was selected. Univariate and multivariable survival analysis was performed using Cox regression using IBM SPSS version 24 for Mac OS X (IBM Inc., Armonk, NY, USA). [00167] RESULTS

[00168] Plasma SFRP5 concentration was elevated in early stage CRC patients

[00169] We evaluated SFRP5 levels in plasma samples from patients undergoing cholecystectomy (control) or surgical intervention for either stage I-IV CRC, or diverticulitis or benign polyps (non-CRC). In all cases, blood samples were collected from fasted patients prior to surgery, before administration of anesthesia. The concentration of SFRP5 in the plasma was determined in 208 patients, of which 30 underwent a cholecystectomy (C), 16 had surgical removal of the bowel for benign polyps or diverticulitis without pathological evidence of CRC (non-CRC), and 162 had either stage I (n = 32), stage II (n = 38), stage III (n = 40), or stage IV (n = 52) CRC (Figure 1A). Age was significantly associated with patient group (P = 0.0013), with cholecystectomy patients being significantly younger than either stage I (P = 0.0398), II (P = 0.0013) or III (P = 0.0024) CRC (Table SI). There were no other significant differences between patient groups but age. The median plasma SFRP5 concentration for all patients included in the study was 30.0 ng/mL (range 0.1 - 189.8 ng/mL). There was a statistically significant positive correlation between age and SFRP5 for all patients (Spearman r = 0.3820, 95% confidence interval (CI) 0.2557 to 0.4955, P < 0.0001). The plasma SFRP5 concentration was significantly associated with patient group (P < 0.0001) (Figure 1A, Table S2). There was no significant difference between the two control groups, cholecystectomy (C) and non-CRC (NC). Compared to the cholecystectomy group, plasma SFRP5 was significantly higher in CRC stage I (P = 0.0007), II (P = 0.0002) and III (P = 0.0004), but not stage IV (P = 0.0884) patients. Compared to the non- CRC group, SFRP5 was significantly higher in stage I (P = 0.0007), stage II (P = 0.0002), stage III (P = 0.0005), and stage IV (P = 0.0479). There were no statistically significant differences between stages I, II or III. However, stage IV patients had less circulating SFRP5 than either stage I (P = 0.0339), II (P = 0.0134) or III (P = 0.0225) CRC.

[00170] After adjustment for age, the association between patient group and plasma SFRP5 concentration remained significant (P = 0.002) (Table S2). There was no significant difference between the two control groups (P = 0.3063). Compared to patients in the cholecystectomy control group, the age adjusted plasma SFRP5 concentration was significantly higher in stage I (P = 0.0114), stage II (P = 0.0092) and stage III (P = 0.0135), but not stage IV (P = 0.3333). Compared to patients in the non-CRC group, the age adjusted concentration was significantly higher in stage I (P = 0.0015), stage II (P = 0.0012), and stage III (P = 0.0017), but not stage IV (P = 0.3063). There were no significant differences between stage I, II or III. The age adjusted SFRP5 concentration in stage IV was significantly lower than stage II (P = 0.0405) but was not significantly different to stage I (P = 0.0519) or stage III (P = 0.0602).

[00171] We analyzed the diagnostic performance of circulating SFRP5 concentration to discriminate between patients with and without CRC. The area under the receiver operating characteristic (ROC) curve comparing the combined patient control groups (C and NC) with stage I-IV CRC was 0.837 (95% CI 0.730 to 0.944; P < 0.0001; Figure IB). A threshold plasma SFRP5 concentration of 28 ng/mL was associated with 63% (95% CI 54% to 70%) sensitivity and 85% (72% to 92%) specificity. A threshold plasma SFRP5 concentration of 38 ng/mL was associated with 48% (40% to 56%) sensitivity and 93% (83% to 98%) specificity.

[00172] Elevated plasma SFRP5 level is predictive of better prognosis

[00173] We examined the correlation between the concentration of plasma SFRP5 and different prognostic clinical-pathological endpoints for the subset of CRC patients (n = 100) that had follow-up data (Table 1). Vascular invasion (P = 0.0011) and the presence or development of distant metastasis (P = 0.0163) were associated with significantly lower plasma SFRP5 level. No significant differences were observed for differences in gender, tumor site, differentiation, histological subtype, lymph node metastasis, perineural invasion or local recurrence. For the 87 patients with stage I-III CRC the median follow-up was 54 months (range 0.1 to 320 months). Survival analysis demonstrated that low plasma SFRP5 protein (defined as < 28 ng/ml) was associated with significantly shorter disease-free survival (DFS; Figure 1C; Table 2) with univariate (hazard ratio (HR) 3.672, 95% CI 1.183 to 11.50, P = 0.024) and multivariable analysis when the confounders stage and vascular invasion were taken into account (HR 2.385, 95% CI 1.181 to 4.816, P = 0.015). These findings indicate the prognostic value of SFRP5 in CRC.

[00174] Figure 1 shows circulating SFRP5 is elevated in early stage colorectal cancer and higher SFRP5 levels are associated with improved disease-free survival. (A) Plasma concentration of SFRP5 was determined in patients that underwent a cholecystectomy (C, n = 30), surgical removal of bowel tissue for benign polyps or diverticulitis without pathological evidence of CRC (NC, n = 16), or with stage I (n = 32), stage II (n = 38), stage III (n = 40), or stage IV (n = 52) CRC. Data analyzed using Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test. ***P < 0.001, *P < 0.05. (B) ROC curve for circulating SRFP5 concentration for the combined patient controls (C and NC, n = 46) and stage I-IV CRC (n = 162). Area under the curve (AUC) determined by ROC curve analysis. (C) Disease-free survival (DFS) in patients with stage I-III CRC. Patients were dichotomized into low (< 28 ng/mL, n = 20) or high (> 28 ng/mL, n= 67) plasma SFRP5 concentration, and survival curves were compared using Cox regression analysis.

[00175] Lower SFRP5 transcript expression in CRC tumors compared to adjacent normal mucosa

[00176] To investigate the source of the elevated plasma concentration of circulating SFRP5 we observed in CRC patients, we investigated SFRP5 transcript expression in normal and CRC tissues from the combined The Cancer Genome Atlas (TCGA) colon (TCGA-COAD) and rectal (TCGA-READ) datasets. The data for SFRP5 transcript expression levels, reported as FPKM (the median number of fragments per kilobase of exon per million reads), were available for 590 confirmed CRC patients, 47 of which had data from matched adjacent normal colonic mucosa as well. There was no difference in the median age of all 590 CRC patients (68 years, range 31 - 90) compared to the 47 patients with matched normal mucosa (73 years, range 40 - 90; P = 0.0585). Overall, the abundance of SFRP5 in CRC cells was low, with transcript detected in 260 of 590 CRC (44%) compared to all of the 47 normal mucosa. Expression of SFRP5 transcript was significantly lower in all CRC tissue (n=590) compared to adjacent normal mucosa (median FPKM 0, range 0 - 28.2, P < 0.0001; Figure 2A). Transcript expression level in patient-matched CRC (median 0, range 0 - 1.7) was significantly lower than in their normal mucosa (median 1.4, range 0.2 - 48.4 P < 0.0001; Figure 2B). Expression was lower in CRC tissue compared to the patient’s matched normal mucosa in 46 of the 47 (98 %) patients. Analysis of the TCGA datasets by disease stage revealed that compared to normal mucosa, expression of SFRP5 transcript in tumor cells was significantly lower in stages I (median 0, range 0 - 4.2, P < 0.0001), II (median 0, range 0 - 7.0, P < 0.0001), III (median 0, range 0 - 28.2, P < 0.0001), and IV (median 0.1, range 0 - 8.9, P < 0.0001) (Figure 2A). Expression was not significantly different between the stages I, II, III or IV. However, SFRP5 expression in normal mucosa was significantly higher in stage IV compared to combined stages I-III (median 0, range 0 - 28.2, P = 0.0144). Notably, analysis of tissue proportions demonstrated a statistically significant inverse correlation between SFRP5 expression and the percentage of tumor cells in the tissue (Spearman r = -0.11, 95% CI -0.19 to -0.03, P = 0.0078). Further analysis revealed SFRP5 level (FPKM > 0) was associated with a significant decrease in the percentage tumor cells (P = 0.0206). These data suggest that the source of SFRP5 is normal mucosa rather than tumor cells. There was no significant correlation with neither the percentage of necrosis, stromal cells, infiltrating neutrophils nor lymphocytes. However, there was a significant correlation between SFRP5 expression and the percentage of monocyte infiltration (Spearman r = 0.16, 95% CI 0.03 to 0.29, P = 0.0157), with SFRP5 expression (FPKM > 0) associated with a significant increase in infiltrating monocytes (P = 0.0142).

[00177] Having identified that SFRP5 transcript expression was decreased in cancer cells compared to normal mucosa, we examined if the reduced expression was associated with DNA methylation in the combined TCGA datasets. Infinium HumanMethylation450 BeadChip DNA methylation data were available for 317 of the 590 confirmed CRC patients (225 from TCGA-COAD and 92 from TCGA-READ), 32 of which had patient matched normal mucosa. Methylation data for SFRP5, expressed as beta-values, were available for 19 CpG probesets, of which 11 were located -1500 to -1 base pairs (bp) upstream of the transcription start site (TSS) and eight were in the gene body defined as +1 to +5248 bp downstream of the TSS. Methylation, expressed as average beta-value for each probeset, was significantly higher (hypermethylated) in CRC compared to normal mucosa (N) for all 14 probesets in the region between -464 and +766 bp from the TSS (Bonferroni adjusted P < 0.0001; Figure 2C). Differential methylation (deltaBeta), defined as a difference in average beta-values for N subtracted from CRC of > 0.2, was observed for 8 of 14 probesets, including 8 of 9 located within region between -169 and +672 bp of the TSS. There was a significant inverse correlation between SFRP5 promoter DNA methylation and gene expression (Spearman r = -0.23, 95% CI -0.33 to -0.11, P < 0.0001; Figure 2D) for CRC cancer cells. Together the above data indicate that the elevated levels of circulating SFRP5 we observed in CRC patients are unlikely to be due to secretion from CRC cancer cells. [00178] To assess the origin of circulating SFRP5, we investigated SFRP5 transcript expression in the major and relevant normal tissues obtained from The Genotype-Tissue Expression (GTEx) Project (Figure 2E). Transcript expression was significantly greater in pancreas compared to either colon or small intestine (P < 0.0001 for both), which in turn was greater than either stomach (P = 0.0002 and P = 0.0146 respectively), prostate (P < 0.0001 and P = 0.0005 respectively), liver or heart (P < 0.0001 for all). In contrast, expression was minimal in lung, adipose tissue and spleen. Together, these data raise the possibility that the elevated plasma SFRP5 in early stage CRC is due to elevated secretion from the pancreas and the non-cancerous gastrointestinal tissue, rather than from adipose tissue or immune cells.

[00179] Figure 2 shows SFRP5 expression and promoter DNA methylation in CRC patient-matched normal colonic mucosa and colorectal carcinoma cells. RNA-sequencing and DNA methylation data for patient tissues in the combined TCGA-COAD and TCGA- READ datasets were obtained from TCGA Research Network (http://cancergenome.nih.gov/). (A) SFRP5 transcript expression, reported as median number of fragments per kilobase of exon per million reads (FPKM). Data were available for 590 CRC and 47 normal mucosa (N), with stage I (n = 102), stage II (n = 211), stage III (n = 172), stage IV (n = 85) and stage not recorded (NR; n = 20). Data analyzed using Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test (****p < 0.0001). (B) Comparison of SFRP5 expression for patient matched N and CRC (n = 47). Data analyzed using Wilcoxon matched-pairs signed rank test. (C) Average beta-values for all available individual probe sets located in the region -5000 to +5000 base pairs (bp) from the SFRP5 transcription start site (TSS) for N (n = 32) and CRC (n = 317). Differential methylation (deltaBeta) was calculated by subtracting the average beta-value of N from CRC. Comparisons between N and CRC were considered statistically significant when the adjusted P value (adj. P) for the unpaired Welch’s t-test with Bonferroni correction for multiple comparisons was < 0.05 (*adj. P < 0.0001). (D) Correlation between SFRP5 expression and promoter DNA methylation. Average regional methylation was the average of the beta-values for the 14 probesets in the region between -464 and +766 bp from the TSS for an individual sample. Data analyzed using non-parametric Spearman r correlation. (E) Expression of SFRP5 transcript in normal tissues. RNA-sequencing data reported as median reads per kilobase per million mapped reads (RPKM), generated by the Genotype-Tissue Expression (GTEx) project [00180] DISCUSSION

[00181] Despite significant progress in our understanding of the pathogenesis of metastatic cancers, metastases are still unpreventable and difficult to treat. Due to the complexity of tumor progression, tumor composition, blood vessel structures, and drug resistance mechanisms, currently available therapies provide limited improvement of survival time and quality of life to metastatic patients. Thus, there is a critical medical need to develop new ways for risk prediction and prevention of liver metastasis in CRC patients.

[00182] The aim of this work was to determine the diagnostic and prognostic value of circulating SFRP5 as biomarker of metastatic progression in CRC. To the best of our knowledge this is the first demonstration that circulating SFRP5 levels display an inverse correlation with disease progression parameters in CRC patients. Our findings indicate that plasma SFRP5 levels are significantly higher in early stage (I to III) CRC patients compared to cholecystectomy and non-CRC (diverticulitis or benign polyps) patient controls. This observation requires validation in a larger patient cohort. In addition, patients diagnosed with vascular invasion or liver metastasis displayed lower plasma levels of SFRP5, and low plasma SFRP5 (< 28 ng/mL) was associated with shorter disease-free survival. These findings suggest that SFRP5 plays a protective role in CRC patients, possibly through maintenance of normal (metabolic and immunological) liver functions, in-line with its beneficial effects observed in other human pathological conditions (19-21).

[00183] Our clinical study has major limitations due to the retrospective nature of the analysis and small sample size, which does not allow any change of clinical practice. In addition, the level of circulating SFRP5 is result of complex biological and multifactorial processes, which could affect its value as diagnostic and prognostic biomarker in CRC. Indeed, as mentioned above SFRP5 has been associated with metabolic symptoms and with a proinflammatory state in visceral adipose tissue, in diabetes and obesity. Therefore, further validation of circulating SFRP5 as diagnostic/prognostic biomarker will require a prospective study including much larger CRC patient sample size. Abnormal epigenetic regulation of SFRPs has been detected in various human cancer types. The loss of SFRP gene expression results in activation of the Wnt pathway, which is a vital mechanism for various stages of tumor progression, including tumorigenesis, the epithelial- mesenchymal transition, metastasis, and drug resistance.

[00184] Previous studies showed that SFRP5 does not directly affect CCL2 and CXCL8 release from human adipocytes and skeletal muscle cells, respectively. Indeed, accumulating data suggests that the effects of SFRP5 in human cells are mediated through Wnt signaling modulation. WNT5A was shown to be involved in inflammatory processes and diseases. Other studies showed that SFRP5 production by gastric epithelial cells inhibits WNT5A-induced CCL2, but not CCL5 or CCL7, expression and macrophage chemotaxis, they therefore proposed that WNT5A overproduction and SFRP5 deficiency in gastric mucosa may together play an important role in gastric inflammation and carcinogenesis. Indeed, these studies have previously showed down regulation of local SFRP5 expression in primary gastric cancer tumors and in gastric cancer cell lines. Recent evidence suggests that different WNT5A mRNA isoforms play distinct roles in CRC prognosis. In addition, it was recently shown that antagonism of WNT5A by SFRP5 or by VEGF-A165b-neutralizing antibody could restore capillary growth and rescue the impaired angiogenesis phenotype in human adipose tissue. Therefore, it remains to be elucidated whether the protective role of SFRP5 is mediated primarily through Wnt signaling modulation or also by alternative mechanisms, and whether antagonism of a specific WNT5A downstream pathway is related to reduced risk of metastatic progression in CRC.

[00185] The source of the elevated circulating SFRP5 in CRC patients is largely unknown. Our analysis of an independent cohort of normal and cancerous colorectal tissues demonstrated that SFRP5 transcript expression was reduced in CRC tumor cells compared to normal colonic mucosa. The abundance of SFRP5 transcript in tumor cells was generally low, undetected in 330 of 590 CRC tumors, and SFRP5 expression inversely correlated with the percentage of tumor cells. Consistent with previous reports, reduced SFRP5 expression in CRC was associated with DNA hypermethylation of the promoter region. Together, these data reveal that the tumors are not the source of the elevated plasma SFRP5 in early stage CRC, suggesting that SFRP5 secretion could represent a response of the digestive system to CRC. Indeed, analysis of expression in normal tissues from the GTEx project demonstrated that SFRP5 transcript expression was abundantly expressed in the pancreas, followed by colon and small intestine, with minimal expression in adipose tissue, blood and spleen. These raise the possibility that the pancreas is the major source of elevated circulating SFRP5 observed in early stage CRC patients, rather than adipose tissue or immune cells. However, future studies are required to sustain this notion. Interestingly, we observed that SFRP5 transcript expression correlated with monocyte, but not lymphocyte or neutrophil infiltration (not depicted), suggesting SFRP5 can be involved in modulating local immune activity. This model represents a new mechanism by which tissue cells actively regulate immune activation and thus prevent the damage inflicted by chronic inflammation.

[00186] Our findings indicate that SFRP5 is a diagnostic and prognostic biomarker for the risk of liver metastasis in CRC patients. It would be of value to investigate in future studies whether circulating SFRP5 levels correlate with age in healthy individuals, and in patients with metabolic syndrome.

Table 1. Plasma SFRP5 concentration in CRC patients with follow-up data.

^tage IV compared to stage I-III NR, not recorded

NOS, not otherwise specified Table 2. Univariate and multivariable analysis of disease-free survival

Analysed as a continuous variable. Supplementary Table SI. Age for each patient group

Adjusted P-value for Kruskal- Wall is ANOVA with Dunn’s multiple comparisons test, and showing results for comparisons to the cholecystectomy group. All other comparisons were not significant.

Table S2. Unadjusted and age-adjusted post hoc comparisons between groups for plasma SFRP5 concentration

[00187] Although the present disclosure has been described with reference to particular embodiments, it will be appreciated that the disclosure may be embodied in many other forms. It will also be appreciated that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

[00188] Also, it is to be noted that, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context already dictates otherwise.

[00189] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[00190] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[00191] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations. [00192] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[00193] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

[00194] Future patent applications may be filed on the basis of the present application, for example by claiming priority from the present application, by claiming a divisional status and/or by claiming a continuation status. It is to be understood that the following claims are provided by way of example only, and are not intended to limit the scope of what may be claimed in any such future application. Nor should the claims be considered to limit the understanding of (or exclude other understandings of) the present disclosure. Features may be added to or omitted from the example claims at a later date.