WO2015172201A1

WO2015172201A1 - Biomarker of gastric cancer

Info

Publication number: WO2015172201A1
Application number: PCT/AU2015/050244
Authority: WO
Inventors: Alex BOUSSIOUTAS; Rita BUSUTTIL
Original assignee: Peter Maccallum Cancer Institute; The University Of Melbourne
Priority date: 2014-05-16
Filing date: 2015-05-15
Publication date: 2015-11-19

Abstract

The present disclosure relates generally to a biomarker of disease, more particularly a biomarker of gastric cancer, including agents, methods and protocols for the diagnosis, prognosis and treatment of gastric cancer, wherein the biomarker is secreted frizzled related protein 4 (SFRP4).

Description

BIOMARKER OF DISEASE Field of the Disclosure

[0001] The present disclosure relates generally to methods and protocols for the diagnosis, prognosis and treatment of gastric cancer.

Background of the Disclosure

[0002] Gastric cancer (GC) is the fourth most common cancer in the world and the second highest cause of cancer related deaths¹. At early stage, GC is usually asymptomatic and in countries lacking an established screening program, is often diagnosed at advanced stage. This late stage at diagnosis as well as the limited curative treatments available contributes to the high mortality rate of this disease. In most Western countries the 5-year survival rate is less than 20% . Survival is improved when there is a dedicated screening program allowing lesions in the stomach to be diagnosed early³' ⁴ and, in many cases, resected using minimally invasive endoscopic techniques. This has advantages for the patient and to the community in terms of productivity and health care costs. Current measures of prognosis that are routinely used for the management of patients with GC are somewhat limited.

[0003] Although there are a variety of prognostic measures⁵' ⁶ the best predictor of prognosis is the pathological TNM (Tumour, Node, Metastasis) stage and the AJCC/UICC staging system. TNM describes (i) the size and spread of the stomach tumour (T), (ii) whether cancer cells have spread to lymph nodes (N) and (iii) whether the cancer has spread to a different part of the body (M). TNM scores estimated by diagnostic imaging have limited ability to influence decisions of primary surgical procedure, degree of lymphadenectomy or the role of neo-adjuvant therapy.

[0004] Treatment for gastric cancer may include surgery, chemotherapy, and/or radiation therapy. Although new treatment regimes have been investigated, surgery (also referred to as resection) remains the most common treatment for gastric cancer, where part or all of the stomach is removed, as well as the surrounding lymph nodes for the purpose of removing all cancer and a margin of normal tissue. Depending on the extent of invasion and the location of the gastric tumour, resection may also include removal of part of the intestine or pancreas. However, surgical intervention by resection is only curative in less than 40% of patients, with the remaining cases presenting with recurrence, or the reappearance of gastric cancer. At present, there is no means of predicting recurrence in patients who have undergone resection of their gastric tumour.

[0005] Therefore, there is an urgent need to improve the survival from gastric cancer with potential methodologies including (a) improved diagnostics to identify persons with, or at risk of developing, gastric cancer, including recurrence, thus enabling stratification into appropriate therapy, and/or (b) improved therapeutics to treat and/or prevent gastric cancer.

Summary of the Disclosure

[0006] In one aspect of the present disclosure, there is provided a method of determining whether a subject has, or is at risk of developing, gastric cancer, the method comprising:

(a) measuring the expression of SFRP4 in a biological sample obtained from a subject; and

(b) comparing the level of expression of SFRP4 in the biological sample to a reference value;

wherein said comparison provides an indication as to whether or not the subject has, or is at risk of developing, gastric cancer.

[0007] In another aspect of the present disclosure, there is provided a method of stratifying a subject to a therapeutic regimen for treating or preventing gastric cancer, the method comprising the steps of:

(b) comparing the level of expression of SFRP4 in the biological sample to a reference value; (c) wherein said comparison provides an indication as to whether or not the subject has, or is at risk of developing, gastric cancer; and

(d) stratifying the subject identified in step (c) as having, or being at risk of developing, gastric cancer, to a therapeutic regimen for treating or preventing gastric cancer.

[0008] In an embodiment, the reference value is representative of a level of expression of SFRP4 in a population of healthy subjects or subjects that are not at risk of developing gastric cancer and wherein a level of expression of SFRP4 in the biological sample that is greater than the reference value is indicative of the subject having, or being at risk of developing, gastric cancer.

[0009] In another aspect of the present disclosure, there is provided a protocol for monitoring the efficacy of a therapeutic regimen for treating or preventing gastric cancer, the protocol comprising:

(a) obtaining from a subject having, or being at risk of developing, gastric cancer a first biological sample, wherein the first biological sample is obtained before or after commencement of treatment;

(b) obtaining from the same subject a second biological sample, wherein the second biological sample is obtained at a time point after commencement of treatment and after the first biological sample is obtained;

(c) measuring the expression of SFRP4 in the first and second biological samples; and

(d) comparing the level of expression of SFRP4 in the first biological sample with the level of expression of SFRP4 in the second biological sample;

wherein a change in the level of expression of SFRP4 between the first and second biological samples is indicative of whether the therapeutic regimen is effective or not.

[0010] In another aspect of the present disclosure, there is provided a kit comprising one or more reagents and/or devices for use in performing the methods and protocols disclosed herein. Brief Description of the Drawings

[0011] Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the following drawings.

[0012] Figure 1A shows the expression patterns that were positively correlated with T- stage as identified using K-means analysis. Eight patterns (K) of gene expression were chosen where every element on the 10.5K array was used.

[0013] Figure IB shows the expression profile of genes with patterns of expression similar to SFRP4 across various stages of gastric cancer progression (T1-T3). SFRP4 is denoted by *.

[0014] Figure 1C is a box plot of data from the Singapore cohort array showing increasing SFRP4 expression (mRNA) levels at more advanced T-stages (p=0.002754; Kruskal-Wallis test).

[0015] Figure ID is a box plot of data from the Affymetrix U133 plus 2 array showing increasing SFRP4 expression (mRNA) at more advanced T-stage (Kruskal-Wallis test; p= 0.0095).

[0016] Figure IE shows the percentage (%) area stained for SFRP4 by immunohistochemical analysis using an anti-SFRP4 antibody on gastric cancer tissue sections. The results indicate a positive correlation between immunohistochemistry staining by anti-SFRP4 antibody and T-stage. The staining was assessed semi- quantitatively on a scale of 0 to 3+. 0 = White bar; 1+ = Blue bar; 2+ = Red bar; 3+ = Black bar.

[0017] Figure IF shows photomicrographs of immunohistochemistry staining by anti- SFRP4 antibody on normal gastric tissue (Normal) and intestinal gastric cancer (IGC) tissue. The darker staining shown on the IGC tissue section is indicative of the overexpression of SFRP4 protein, as compared to the absence of staining in normal gastric tissue.

[0018] Figure 2 shows a Box plot of SFRP4 expression in gastric tissues (Figure 2A). 153 gastric tissues were run on Affymetrix U133 plus 2 arrays and stratified according to tissue type. Histologically normal gastric tissues (NN) exhibited significantly lower SFRP4 expression than other benign tissues (chronic gastritis (CG; p=0.001)) and intestinal metaplasia (IM; p=0.002). Highest levels of SFRP4 expression were observed in gastric tumour samples which are divided here into the 2 histological subtypes- intestinal (p=0.001) and diffuse gastric cancer (p=0.002). T-test was used for analysis. Figure 2B represents the results of laser capture microdissection (LCM), showing that SFRP4 mRNA was predominantly expressed in adjacent tumour stroma and epithelium.

[0019] Figure 3 shows Kaplan-Meier plots depicting progression free survival (i.e. period of survival between resection of gastric cancer and subsequent recurrence) versus T- stage. Figure 3A represents clinical data from the Peter MacCallum data set and Figure 3B represents clinical data from the Singapore data set. T-stage is classified on a scale of 1-4 with 1 being the least invasive and 4 being the most invasive; Black= Tl tumours, Red= T2 tumours and green=T3 and T4 tumours. Figures 3C and 3D depict Kaplan Meier plots with progression free survival used as an endpoint where clinical samples were classified as having either high or low SFRP4 expression (mRNA). For the Singapore data set (Figure 3C; p=0.01 , Peter MacCallum Cancer Centre data set; and Figure 3D; p=0.04, Singapore data set; logrank test).

[0020] Figure 4 shows SFRP4 protein levels in blood as determined by ELISA. Figures 4A shows that the concentration of SFRP4 protein in serum and plasma is not significantly different between normal healthy controls (Control) and patients with gastric cancer (before curative resection surgery). Figure 4B shows that the level of SFRP4 protein in serum is elevated in post-operative blood samples taken from patients who will eventually develop recurrence, whereas there is no change in the level of SFRP4 protein in serum in post-operative blood samples taken from patients who do not develop recurrence, even out to 36 months post-resection. A 1.15-fold change cut-off (depicted by the dotted line in Figure 4B) is capable of distinguishing patients who will recur from those who will not. Figure 4C shows plasma levels of SFRP4 protein (pg/mL) in pre-operative and postoperative plasma samples obtained at different time points in a cohort of 24 gastric cancer patients that underwent curative R0 resection. These patients were selected and matched for age, gender, stage, adjuvant therapy and type of resection. An elevation of SFRP4 levels in blood was noted as early as 4 weeks after curative resection, almost exclusively in those that recurred. All cases had curative surgery and were matched for age, sex, stage, resection and adjuvant therapy. (PFS = Progression free survival).

[0021] Figure 5A shows a receiver operator curve (ROC) analysis of recurrence in 69 gastric cancer (GC) patients analysing T stage (=), N stage (— ), SFRP4 protein levels (in plasma; PredictR (+)), and AJCC stage alone (#) and a logistic regression model of SFRP4 protein levels (in plasma) and AJCC stage combined (*) showing improvement in sensitivity and specificity for the prediction of recurrence. Figure 5B shows Area under ROC (AUC) of the different groups with 95% CI showing highest accuracy in the SFRP4 protein/ AJCC combination using a logistic regression model.

[0022] Figure 6 shows the level of CEA, CA19-9 and SFRP4 levels in plasma samples taken from 2 gastric cancer patients before resection (pre-op) and after resection (post-op; pre-recurrence and post-recurrence). The horizontal dotted lines represent the accepted threshold value for that particular test (note: the threshold value for SFRP4 is 1.15).

Detailed Description

[0023] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0024] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an agent" means one agent or more than one agent.

[0025] In the context of this specification, the term "about" is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result. Methods of diagnosis and prognosis

[0026] The present disclosure is predicated on the inventors' surprising findings that (i) SFRP4 expression is significantly greater in gastric cancer tissue as compared to the level of expression of SFRP4 in histologically normal or benign gastric tissue; (ii) SFRP4 expression is indicative of the histological nature of the gastric cancer (e.g., diffuse or intestinal); (iii) SFRP4 expression in gastric cancer tissue is increased, particularly at a more advanced T-stages of the disease; and (iv) elevated expression of SFRP4 predicts progression free survival and recurrence of gastric cancer in patients who have undergone resection. The present inventors have therefore shown, for the first time, that SFRP4 is a suitable diagnostic and prognostic indicator of gastric cancer.

[0027] Thus, in an aspect of the present disclosure, there is provided a method of determining whether a subject has, or is at risk of developing, gastric cancer, the method comprising:

Gastric cancer

[0028] Gastric cancer (also referred to as stomach cancer or gastric tumour) is a cancer arising from any part of the stomach, though originating from glandular epithelium of the gastric mucosa. Approximately 90% of all gastric cancers are characterised as adenocarcinomas, with the remaining are mostly lymphomas, carcinoids and/or stromal tumours. In its early stages, gastric cancer can be asymptomatic, producing no observable symptoms or nonspecific symptoms (i.e., symptoms that are not specific to gastric cancer). Symptoms typically arise occur when the cancer has reached an advanced stage, at which point it is likely to have metastasized to other parts of the body. It is for this reason that gastric cancer is often associated with relatively poor prognosis. Symptoms of gastric cancer include general discomfort or pain in the stomach area, vomiting, weight loss, difficulty swallowing, nausea, feeling full or bloated after a small meal and presence of blood in vomit and/or stools.

[0029] The presence of gastric cancer is typically confirmed by a gastroscopic exam, in which the stomach is visualized in situ via a fiber optic camera (endoscopy). Other tests that can be performed to confirm the presence of gastric cancer include, but are not limited to, an upper GI series (also referred to as barium roentgenogram) and computed tomography (CT scanning) of the abdomen. Thickening of the stomach wall of more than 1 cm which is focal, eccentric and enhancing will typically favor a diagnosis of malignancy. This can be confirmed by obtaining a biopsy sample of the gastric tumour and checking for the presence of cancerous cells. Various blood tests may also be performed, including a full blood count (FBC) to check for anemia and/or assessing a stool sample for the presence of blood.

[0030] Histologically, there are two major types of gastric adenocarcinoma (applying the Lauren classification): (i) intestinal type or (ii) diffuse type. Adenocarcinomas typically invade the gastric wall in an aggressive manner, infiltrating the muscularis mucosae, the submucosa, and the muscularis propria. Intestinal type adenocarcinoma tumour cells are typically characterised by irregular tubular structures, harbouring pluristratification, multiple lumens and reduced stroma. Depending on glandular architecture, cellular pleomorphism and mucosecretion, adenocarcinoma may present 3 degrees of differentiation: well, moderate and poorly differentiated. By contrast, a diffuse type adenocarcinoma (also referred to as mucinous, colloid, linitis plastica, leather-bottle stomach) is typically characterised by tumour cells that are discohesive and secrete mucus into the interstitium, producing pools of mucus/colloid (optically "empty" spaces) and is typically poorly differentiated.

[0031] If cancer cells are found in a tissue sample, an assessment is usually undertaken to determine the stage, or extent, of the disease, with respect to size and spread of the gastric cancer. The TNM system is often employed for this purpose, where (T) denotes the extent to which the gastric cancer has migrated through the stomach wall (invasion), (N) denotes the spread of gastric cancer cells to lymph nodes and (M) denotes the spread of gastric cancer cells to different parts of the body.

[0032] With respect to invasion (T), TX means the main tumour (primary) cannot be assessed, Tl means the tumour has started to grow into the wall of the stomach, Tl A means the tumour is within the inner layers of the stomach (the mucosa), TIB means the tumour has grown through the mucosa and into a layer of supportive tissue called the submucosa, T2 means the tumour has grown into the muscle layer of the stomach T3 means the tumour has grown into the outer lining of the stomach, T4 means the tumour has grown right through the outer lining of the stomach, T4A means the tumour has broken through the outer lining of the stomach wall and T4B means it has grown through the stomach wall and into other organs or body structures, such as the liver, oesophagus or abdominal wall.

[0033] With respect to (N), NX means that the lymph nodes cannot be assessed, NO means that there are no lymph nodes containing cancer cells and Nl, N2, N3A and N3B mean that there are cancer cells in one or more lymph nodes near to the stomach. With respect to (M), MX means that spread of the cancer cannot be assessed, M0 means the cancer has not spread to other organs and Ml means the cancer has spread to other parts of the body.

[0034] There are typically four clinical stages of gastric cancer:

[0035] Stage 0 - is the earliest stage of cancer and limited to the inner lining of the stomach. Also referred to as carcinoma in situ (CIS), it is a very early stage of gastric cancer. There are cancer cells in the stomach lining, but they are contained within the innermost layer of the lining. There is typically very little risk of any cancer cells having spread. These gastric cancers usually respond well to treatment by endoscopic mucosal resection when found very early (in routine screenings); otherwise by gastrectomy and lymphadenectomy without need for chemotherapy or radiation treatment;

[0036] Stage 1 - Penetration to the second or third layers of the stomach (Stage 1A) or to the second layer and nearby lymph nodes (Stage IB). Stage 1A means the cancer has grown no further than the lining of the stomach, with no cancer in the lymph nodes (Tl, NO, M0). Stage IB means either that the cancer is still within the stomach lining, but nearby lymph nodes contain cancer cells (Tl , Nl , MO) or there are no cancer cells in the lymph nodes, but the cancer has grown into the muscle of the stomach wall (T2, NO, MO).

[0037] Stage 2 - Penetration to the second layer and more distant lymph nodes, or the third layer and only nearby lymph nodes, or all four layers but not the lymph nodes. Stage 2A means that the cancer is still within the lining of the stomach, but between, for example, 3 and 6 nearby lymph nodes contain cancer cells (Tl , N2, M0) or the cancer has grown into the muscle layer of the stomach wall and is also in, for example, 1 or 2 nearby lymph nodes (T2, Nl, M0) or the cancer has grown into the outer layers of the stomach but there are no cancer cells in the lymph nodes (T3, NO, M0). Stage 2B means that the cancer is within the lining of the stomach wall but, for example, 7 or more lymph nodes contain cancer cells (Tl, N3, M0) or the cancer has grown into the muscle layer of the stomach and between, for example, 3 and 6 lymph nodes contain cancer cells (T2, N2, M0) or the cancer has grown into the outer layer of the stomach and is also in, for example, 1 or 2 nearby lymph nodes (T3, Nl, M0) or the cancer has grown through the outer lining but there are no cancer cells in nearby lymph nodes (T4A, NO, M0);

[0038] Stage 3 - Penetration to the third layer and more distant lymph nodes, or penetration to the fourth layer and either nearby tissues or nearby or more distant lymph nodes. Stage 3A means that the cancer has grown into the muscle layer of the stomach and, for example, 7 or more nearby lymph nodes contain cancer cells (T2, N3, M0) or into the outer lining of the stomach and between, for example, 3 and 6 nearby lymph nodes contain cancer cells (T3, N2, M0) or through the stomach wall and, for example, 1 to 2 nearby lymph nodes contain cancer cells (T4a, Nl , M0). In stage 3B, the cancer has grown into the outer lining of the stomach and more than, for example, 7 nearby lymph nodes contain cancer cells (T3, N3, M0) or right through the stomach wall and between, for example, 3 and 6 lymph nodes contain cancer (T4A, N2, M0) or right through the stomach wall into nearby tissues and organs, and the nearby lymph nodes may contain cancer (T4B, NO to 1, M0). In stage 3c, the cancer has grown right through the stomach wall and more than 7 nearby lymph nodes contain cancer (T4A, N3, M0) or grown right through the stomach wall into nearby tissues and organs, and the lymph nodes contain cancer (T4B, N2 to 3, M0); and [0039] Stage 4 - Cancer has spread to nearby tissues and more distant lymph nodes, or has metastasized to other organs further away from the stomach, such as the lungs, brain or bones (any T, any N, Ml). At this advanced stage, a cure is rarely possible, with the majority of patients entering palliative treatment to prolong life or improve symptoms.

[0040] Surgery remains the only real option as the treatment for patients diagnosed with gastric cancer. Gastric cancer has not been particularly sensitive to chemotherapeutic agents and, if used, has usually served to palliatively reduce the size of the tumour, relieve symptoms of the disease and/or increase survival time. Examples of chemotherapeutic agents that have been used to treat gastric cancer include fluorouracil (5-FU) or its analog capecitabine, carmustine, methyl-CCNU, adriamycin (doxorubicin), mitomycin C, cisplatin and taxotere, or combinations thereof. Studies have explored the benefit of chemotherapy before surgery (e.g., to shrink tumour size) or after surgery to destroy any residual cancer cells. Combination treatment with chemotherapy and radiation therapy has demonstrated some activity in selected post-surgical settings. Radiation therapy (also referred to as radiotherapy) uses high-energy electromagnetic radiation to damage cancer cells and stop them from proliferating. When used, it is generally in combination with surgery and chemotherapy, or used only with chemotherapy in cases where the individual is unable to undergo resection. Radiation therapy may also be used to relieve pain or blockage by shrinking the tumour during palliative care.

Secreted frizzled related protein 4

[0041] The term "secreted frizzled related protein 4", also referred to herein as "SFRP4", refers to the protein or polypeptide that is one of a family of secreted frizzled related proteins that contain a cysteine -rich domain homologous to the putative Wnt- binding site of Frizzled proteins. The term SFRP4 may also be used herein to refer to either or both of a SFRP4 polypeptide or a gene (polynucleotide) encoding an SFRP4 polypeptide, interchangeably. Those skilled in the art will recognize from the context of the disclosure whether the polypeptide or polynucleotide (gene) is the subject of the discussion.

[0042] There are at least eight members of the SFRP4 family, five of which have been identified in humans (SFRP41, SFRP42, SFRP43, SFRP4 and SFRP5)¹³. Structurally, the genes that encode these proteins are similar to Frizzled receptors, but lack the transmembrane domain. SFRP4 protein comprises 346 amino acids with a predicted molecular weight of 39.9 kDa and an actual molecular weight of approximately 50-55 kDa. The N-terminus contains a secretion signal peptide followed by a cysteine-rich domain of approximately 120 amino acids. The cysteine-rich domain is 30-50% identical to the extracellular putative Wnt-binding domain of frizzled (Fzd) receptors and is characterized by the presence of ten cysteine residues at conserved positions. These cysteines form a pattern of disulfide bridges. The C-terminal portion of SFRP protein is characterized by positively charged residues that appear to confer heparin-binding properties in some SFRP family members, namely SFRP1 and SFRP3, and contains a netrin-related motif (NTR) with six cysteine residues that most likely form three disulfide bridges. The six conserved cysteines in the N-terminus of SFRP4 share a similar spacing to SFRP3, whereas those of the other SFRP isoforms are different, indicating a disparity in disulfide bond formation. Uniquely, SFRP4 contains two additional cysteine residues.

[0043] SFRP4 is expressed in various normal tissues, including endometrium (e.g., stromal cells), ovary, kidney, heart, brain, mammary gland, cervix, pancreas, stomach, colon, lung, skeletal muscle, testis, eye, bone, prostate and liver. Since SFRPs share a similar cysteine-rich domain with the Fzd family of receptors, it has been suggested that they may act as soluble modulators that compete with Fzd to bind the Wnt ligands, thereby altering the Wnt signal. Reports have demonstrated that SFRP4 binds Wnt7a and perhaps Wnt3a.

[0044] The term SFRP4 includes vertebrate and non-vertebrate SFRP4. Suitable vertebrates that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates, rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g. , dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish. In an embodiment, the SFRP4 is a human SFRP4. [0045] In one embodiment, the human SFRP4 is a protein encoded by the mRNA sequences selected from the group consisting of those represented by GenBank accession numbers BC058911 , AK302357, AF026692, BC047684, AK303977 and DQ420628.

[0046] As used herein, the term "SFRP4" also includes homologs thereof. Thus, is an embodiment, elevated expression of a SFRP4 homolog in a post-operative blood sample of a patient following resection, as compared to the level of expression of the SFRP4 homolog in the blood of a healthy subject or a patient with gastric cancer prior to resection, is indicative of the risk of recurrence of gastric cancer in that patient. The term "homolog" typically refers to peptides with similar biological activity, although differ in amino acid sequence at one or more amino acid positions when the sequences are aligned. For example, the amino acid sequences of two homologous SFRP4 peptides may differ only by one amino acid residue within the aligned amino acid sequences of five to ten amino acids. Alternatively, two homologous SFRP4 peptides of ten to fifteen amino acids may differ by no more than two amino acid residues when aligned. Alternatively, two homologous SFRP4 peptides of fifteen to twenty or more amino acids can differ by up to three amino acid residues when aligned. Homologous SFRP4 peptides may also differ by up to approximately 5%, 10%, 20% or 25% of the amino acid residues when the amino acid sequences of the two peptide homologs are aligned.

[0047] Homologs of SFRP4 may be found in the same species (i.e., between two or more individuals of the same species), in related species and/or sub-species, or in different species. For example, for a human SFRP4, homologs include those found in non-human vertebrates and non-vertebrates. Suitable vertebrates that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates, rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g. , dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish. A preferred homolog is one found in a primate (e.g., a human, ape, monkey, chimpanzee). Alternatively, an SFRP4 homolog may be from the same species (e.g., human). [0048] Generally, homologs will have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a particular amino acid or nucleotide sequence, as determined, for example, by sequence alignment programs known in the art using default parameters (see, e.g., Needleman and Wiinsch, (1970, . Mol. Biol. 48: 444-453). In specific embodiments, the percent identity between nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. An non-limiting set of parameters (and the one that should be used unless otherwise specified) includes a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. In some embodiments, the percent identity or similarity between amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

Subject

[0049] The terms "subject," "individual" and "patient" are used interchangeably herein to refer to any subject to which the present disclosure may be applicable, particularly a vertebrate subject, and even more particularly a mammalian subject. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates, rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g. , dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish. In some embodiments, the subject is a primate (e.g., a human, ape, monkey, chimpanzee). In a preferred embodiment, the subject is a human. Biological sample

[0050] The biological sample can be any sample in which changes in the expression of SFRP4 reflect the presence of, or risk of developing, gastric cancer. Suitable biological samples could be determined by persons skilled in the art. For example, the level of expression of SFRP4 in a biological sample obtained from a first subject who has been diagnosed as having gastric cancer can be compared to a level of expression of SFRP4 in a biological sample obtained from substantially the same anatomical region of a second subject who is free of gastric cancer and/or has had no history of gastric cancer, wherein a higher level of expression of SFRP4 in the biological sample from the first subject as compared to the level of expression in the biological sample from the second subject is indicative that the biological sample is suitable for the purpose of diagnosing the presence or risk of developing gastric cancer, or of a subject's progression free survival, in accordance with the present disclosure.

[0051] In another illustrative example, the level of expression of SFRP4 in a biological sample obtained from a first subject who has been diagnosed as having gastric cancer can be compared to the level of expression of SFRP4 in a biological sample obtained from substantially the same anatomical region of a second subject who has also been diagnosed as having gastric cancer, albeit a different grade of gastric cancer; that is, a less or more advanced form of gastric cancer than the first subject, wherein a higher or lower level of expression of SFRP4 in the biological sample from the first subject as compared to the level of expression in the biological sample from the second subject is indicative that the biological sample is suitable for the purpose of diagnosing the presence or risk of developing gastric cancer, or of a subject's progression free survival, in accordance with the present disclosure.

[0052] A biological sample may include a sample that has been obtained, extracted, untreated, treated, diluted or concentrated from a subject. In some embodiments, the biological sample has not been extracted from the subject, particularly where the determination steps in accordance with the present invention (e.g., the expression of SFRP4) can be performed in situ. [0053] In some embodiments, the biological sample is a sample obtained from the subject that is reasonably expected to have, or be at risk of developing, gastric cancer. Non- limiting examples of biological samples include, but are not limited to, tissue, such as a biopsy sample or a resected tumour, bodily fluid (for example, blood, serum, plasma, saliva, urine, tears, peritoneal fluid, ascitic fluid, vaginal secretion, breast fluid, breast milk, lymph fluid, cerebrospinal fluid or mucosa secretion), umbilical cord blood, chorionic villi, amniotic fluid, an embryo, embryonic tissues, lymph fluid, cerebrospinal fluid, mucosa secretion, or other body exudate, fecal matter and one or more cells or extracts thereof that express SFRP4 (nucleic acid or protein), including subcellular structures obtained using protocols well established within the art. In some embodiments, the biological sample comprises blood, such as peripheral blood, or a fraction or extract thereof. The biological sample may comprise blood cells, such as mature, immature or developing leukocytes, including lymphocytes, polymorphonuclear leukocytes, neutrophils, monocytes, reticulocytes, basophils, coelomocytes, hemocytes, eosinophils, megakaryocytes, macrophages, dendritic cells, natural killer cells, or fraction of such cells (e.g. , a nucleic acid or protein fraction). In some embodiments disclosed herein, the biological sample is a whole blood sample. In some embodiments, the biological sample is a serum sample. In some embodiments, the biological sample comprises at least one gastric cancer cell. The biological sample may be derived from a gastric tumour or derived from a site that is distal (e.g., adjacent) the primary tumour.

[0054] The biological sample may be processed and analyzed for the purpose of determining the sample biomarker profile, in accordance with the present invention, almost immediately following collection (i.e., as a fresh sample), or it may be stored for subsequent analysis. If storage of the biological sample is desired or required, it would be understood by persons skilled in the art that it should ideally be stored under conditions that preserve the integrity of the biomarker of interest within the sample (e.g., at -80°C).

[0055] By "obtained" is meant to come into possession. Biological or reference samples so obtained include, for example, nucleic acid extracts or polypeptide extracts isolated or derived from a particular source. For instance, the extract may be isolated directly from a biological fluid or tissue of a subject. [0056] The present inventors have surprisingly found that elevated expression of

SFRP4 in a post-operative blood sample following resection can predict recurrence of gastric cancer in patients who have undergone resection. Hence, in an embodiment disclosed herein, the biological sample is a post-operative blood sample, taken after resection of a gastric tumour (i.e., post-resection). Thus, in another aspect of the present disclosure, there is provided a method of identifying a subject at risk of recurrence following resection, the method comprising the steps of:

(i) obtaining a post-operative blood sample from the subject following resection;

(ii) measuring the expression of SFRP4 in the post-operative blood sample; and

(iii) comparing the level of expression of SFRP4 in the post-operative blood sample to a reference value, wherein said comparison provides an indication as to whether or not the subject is at risk of recurrence, as herein described.

[0057] In another aspect disclosed herein, there is provided a method of identifying a subject at risk of recurrence following resection, the method comprising the steps of:

(i) measuring the expression of SFRP4 in the post-operative blood sample obtained from a subject following resection; and

(ii) comparing the level of expression of SFRP4 in the post-operative blood sample to a reference value, wherein said comparison provides an indication as to whether or not the subject is at risk of recurrence, as herein described.

[0058] A blood sample may be obtained, extracted, untreated, treated, diluted or concentrated from the subject. In some embodiments, the blood sample is obtained from the subject but does not undergo any further processing, such as dilution, extraction, treatment and the like. Non-limiting examples of suitable blood samples include serum and plasma. In an embodiment, the blood sample is plasma.

[0059] The blood sample may be processed and analyzed for the purpose of determining the expression of SFRP4 in accordance with the present disclosure, almost immediately following collection (i.e., as a fresh sample), or it may be stored for subsequent analysis. If storage of the blood sample is desired or required, it would be understood by persons skilled in the art that it should ideally be stored under conditions that assist to preserve the integrity of SFRP4 within the sample or at least of a substantial amount of SFRP4 within the sample (e.g., at -80°C).

[0060] The terms "obtain", "obtaining", "obtained" and the like, as used herein, are meant to come into possession. For example, a blood sample so obtained includes, for example, plasma or serum substantially separated from whole blood. In one embodiment, the blood sample is a sample selected from the group consisting of whole blood, plasma and serum. In another embodiment, the post-operative blood sample is plasma or serum.

[0061] The term "post-operative", as used herein, refers to any time point following surgery (i.e., following resection), although it will typically be a time point at which the level of expression of SFRP4 in the blood sample is expected to be elevated in blood of a sample population of patients who have gone on to develop recurrence as compared, for example, to the level of expression of SFRP4 in a blood sample from a healthy subject or to the level of expression of SFRP4 in a blood sample of patient with gastric cancer obtained prior to resection (i.e., pre-operative). In an embodiment, the post-operative blood sample is obtained from the subject at any time point from about 24 hours to about 24 months following resection, including, for example, at about 1 week, at about 2 weeks, at about 3 weeks, at about 4 weeks, at about 5 weeks, at about 6 weeks, at about 7 weeks, at about 8 weeks, at about 9 weeks, at about 10 weeks, at about 11 weeks, at about 4 months, at about 5 months, at about 6 months, at about 7 months, at about 8 months, at about 9 months, at about 10 months, at about 1 1 months, at about 12 months, at about 13 months, at about 14 months, at about 15 months, at about 16 months, at about 17 months, at about 18 months, at about 19 months, at about 20 months, at about 21 months, at about 22 months, at about 23 months or at about 24 months following resection, including at any other time point within the aforementioned ranges.

[0062] The present inventors have also surprisingly found that the level of expression of SFRP4 in a blood sample obtained from a patient that has gone on to develop recurrent gastric cancer is elevated in comparison to the level of expression of SFRP4 in a pre-operative blood sample from the same patient (i.e., prior to resection). Furthermore, the present inventors have surprisingly found that the level of expression of SFRP4 remains elevated at multiple time points following resection; for example, at about 1 week to about 3 months, at about 6 months to 2 years and at about 12 months to about 2 years following resection. Thus, in an embodiment, the post-operative blood sample is obtained from the subject from about 2 weeks to about 12 weeks following resection. In another embodiment, the post-operative blood sample is obtained from the subject from about 6 months to about 2 years following resection. In another embodiment, the post-operative blood sample is obtained from the subject from about 12 months to about 2 years following resection.

[0063] Surprisingly, the present inventors have also found that the level of expression of SFRP4 is independent of the presence of gastric cancer, as the level of expression of SFRP4 in blood remains elevated post-recurrence. Thus, in an embodiment, the methods disclosed herein comprise obtaining and analyzing post-operative blood samples from multiple time points following resection.

Measuring the expression of SFRP4

[0064] The term "expression" is used herein in its broadest context to denote a measurable presence of SFRP4, including the production of RNA message (gene expression), translation of RNA message into proteins or polypeptides (protein expression) and/or SFRP4 activity. For example, the term "SFRP4 expression" includes (i) the production of SFRP4 RNA message (i.e., SFRP4 gene expression), (ii) the translation of SFRP4 RNA message into SFRP4 protein and/or (iii) the transport of SFRP4 protein to the cell surface and/or (iv) the biological activity of SFRP4, as determined, for example, by changes in the level of expression of surrogate biomarkers of SFRP4 activity.

[0065] Suitable methods for measuring or analyzing the expression of SFRP4 would be known to persons skilled in the art. In some embodiments, it may be desirable to measure SFRP4 expression at the protein level. However, it will be understood that, in some instances, the SFRP4 can be a gene expression product such as transcript (e.g., mRNA) levels.

[0066] Methods of measuring expression products such as proteins and transcripts are known to persons skilled in the art, with some illustrative examples described below. In some embodiments, measuring the expression of SFRP4 comprises determining the level of SFRP4. As used herein the terms "level" and "amount" are used interchangeably herein to refer to a quantitative amount (e.g., weight or moles or number), a semi-quantitative amount, a relative amount (e.g., weight % or mole % within class or a ratio), a concentration, and the like. Thus, these terms encompasses absolute or relative amounts or concentrations of SFRP4 in a sample, including levels in a population of subjects represented as mean levels and standard deviations as shown in some of the Figures herein.

[0067] SFRP4 may be quantified or detected using any suitable technique, including, but not limited to, nucleic acid- and protein-based assays. In illustrative nucleic acid-based assays, nucleic acid is isolated from cells contained in a biological sample according to standard methodologies (Sambrook, et al., 1989, supra; and Ausubel et al., 1994, supra). The nucleic acid is typically fractionated (e.g., poly A⁺ RNA) or whole cell RNA. Where RNA is used as the subject of detection, it may be desired to convert the RNA to a complementary DNA. In some embodiments, the nucleic acid is amplified by a template-dependent nucleic acid amplification technique. A number of template dependent processes are available to amplify the SFRP4-encoding nucleotide sequences present in a given sample. An exemplary nucleic acid amplification technique is the polymerase chain reaction (referred to as PCR), which is described in detail in U.S. Pat. Nos. 4,683, 195, 4,683,202 and 4,800, 159, Ausubel et al. (supra), and in Innis et al , ("PCR Protocols", Academic Press, Inc., San Diego Calif., 1990). Briefly, in PCR, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the SFRP4 nucleotide sequence. An excess of deoxynucleotide triphosphates are added to a reaction mixture along with a DNA polymerase, e.g. , Taq polymerase. If a cognate SFRP4 nucleotide sequence is present in a sample, the primers will bind to the seqeunce and the polymerase will cause the primers to be extended along the sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the SFRP4 nucleotide sequence to form reaction products, excess primers will bind to the SFRP4 nucleotide sequence and to the reaction products and the process is repeated. A reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al , 1989, supra. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art.

[0068] In certain embodiments, the template-dependent amplification involves quantification of transcripts in real-time. For example, RNA or DNA may be quantified using the Real-Time PCR technique (Higuchi, 1992, et al , Biotechnology 10: 413-417). By determining the concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundance of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundance is only true in the linear range of the PCR reaction. The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. In specific embodiments, multiplexed, tandem PCR (MT-PCR) is employed, which uses a two-step process for gene expression profiling from small quantities of RNA or DNA, as described for example in US Pat. Appl. Pub. No. 20070190540. In the first step, RNA is converted into cDNA and amplified using multiplexed gene specific primers. In the second step each individual gene is quantitated by real time PCR.

[0069] In certain embodiments, target nucleic acids are quantified using blotting techniques, which are well known to those of skill in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provides different types of information, although cDNA blotting is analogous, in many aspects, to blotting or RNA species. Briefly, a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter. Subsequently, the blotted target is incubated with a probe (usually labelled) under conditions that promote denaturation and rehybridisation. Because the probe is designed to base pair with the target, the probe will bind a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above. Following detection/quantification, one may compare the results seen in a given subject with a control reaction or a statistically significant reference group or population of control subjects as defined herein. In this way, it is possible to correlate the amount of a biomarker nucleic acid detected with the likelihood that a subject is at risk of developing gastric cancer.

[0070] Also contemplated are biochip-based technologies such as those described by Hacia et al (1996, Nature Genetics 14: 441-447) and Shoemaker et al (1996, Nature Genetics 14: 450-456). Briefly, these techniques involve quantitative methods for analysing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ biochip technology to segregate target molecules as high-density arrays and screen these molecules on the basis of hybridization. See also Pease et al (1994, Proc. Natl Acad. Sci. U.S.A. 91 : 5022-5026); Fodor et al. (1991, Science 251: 767-773). Briefly, nucleic acid probes to SFRP4 nucleotide sequences are made and attached to biochips to be used in screening and diagnostic methods, as outlined herein. The nucleic acid probes attached to the biochip are designed to be substantially complementary to specific expressed SFRP4 nucleotide sequences, i.e., the target sequence (either the target sequence of the sample or to other probe sequences, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occur. This complementarity need not be perfect; there may be any number of base pair mismatches, which will interfere with hybridization between the target sequence and the nucleic acid probes of the present invention. However, if the number of mismatches is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. In certain embodiments, more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being desirable, are used to build in a redundancy for a particular target. The probes can be overlapping (i.e. have some sequence in common), or separate.

[0071] In an illustrative biochip analysis, oligonucleotide probes on the biochip are exposed to or contacted with a nucleic acid sample suspected of containing one or more biomarker polynucleotides under conditions favouring specific hybridization. Sample extracts of DNA or RNA, either single or double-stranded, may be prepared from fluid suspensions of biological materials, or by grinding biological materials, or following a cell lysis step which includes, but is not limited to, lysis effected by treatment with SDS (or other detergents), osmotic shock, guanidinium isothiocyanate and lysozyme. Suitable DNA, which may be used in the method of the invention, includes cDNA. Such DNA may be prepared by any one of a number of commonly used protocols as for example described in Ausubel, et al., 1994, supra, and Sambrook, et al., et al., 1989, supra.

[0072] Suitable RNA, which may be used in the method of the invention, includes messenger RNA, complementary RNA transcribed from DNA (cRNA) or genomic or subgenomic RNA. Such RNA may be prepared using standard protocols as for example described in the relevant sections of Ausubel, et al. 1994, supra and Sambrook, et al. 1989, supra).

[0073] cDNA may be fragmented, for example, by sonication or by treatment with restriction endonucleases. Suitably, cDNA is fragmented such that resultant DNA fragments are of a length greater than the length of the immobilized oligonucleotide probe(s) but small enough to allow rapid access thereto under suitable hybridization conditions. Alternatively, fragments of cDNA may be selected and amplified using a suitable nucleotide amplification technique, as described for example above, involving appropriate random or specific primers.

[0074] Usually the target biomarker polynucleotides are detectably labelled so that their hybridization to individual probes can be determined. The target polynucleotides are typically detectably labelled with a reporter molecule illustrative examples of which include chromogens, catalysts, enzymes, fluorochromes, chemiluminescent molecules, bioluminescent molecules, lanthanide ions (e.g., Eu³⁴), a radioisotope and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non- metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like. Illustrative labels of this type include large colloids, for example, metal colloids such as those from gold, selenium, silver, tin and titanium oxide. In some embodiments, in which an enzyme is used as a direct visual label, biotinylated bases are incorporated into a target polynucleotide.

[0075] The hybrid-forming step can be performed under suitable conditions for hybridizing oligonucleotide probes to test nucleic acid including DNA or RNA. In this regard, reference may be made, for example, to NUCLEIC ACID HYBRIDIZATION, A PRACTICAL APPROACH (Homes and Higgins, eds., IRL press, Washington D.C., 1985). In general, whether hybridization takes place is influenced by the length of the oligonucleotide probe and the polynucleotide sequence under test, the pH, the temperature, the concentration of mono- and divalent cations, the proportion of G and C nucleotides in the hybrid-forming region, the viscosity of the medium and the possible presence of denaturants. Such variables also influence the time required for hybridization. The preferred conditions will therefore depend upon the particular application. Such empirical conditions, however, can be routinely determined without undue experimentation.

[0076] After the hybrid-forming step, the probes are washed to remove any unbound nucleic acid with a hybridization buffer. This washing step leaves only bound target polynucleotides. The probes are then examined to identify which probes have hybridized to a target polynucleotide. The hybridization reactions are then detected to determine which of the probes has hybridized to a corresponding target sequence. Depending on the nature of the reporter molecule associated with a target polynucleotide, a signal may be instrumentally detected by irradiating a fluorescent label with light and detecting fluorescence in a fluorimeter; by providing for an enzyme system to produce a dye which could be detected using a spectrophotometer; or detection of a dye particle or a coloured colloidal metallic or non metallic particle using a reflectometer; in the case of using a radioactive label or chemiluminescent molecule employing a radiation counter or autoradiography. Accordingly, a detection means may be adapted to detect or scan light associated with the label which light may include fluorescent, luminescent, focussed beam or laser light. In such a case, a charge couple device (CCD) or a photocell can be used to scan for emission of light from a probe:target polynucleotide hybrid from each location in the micro-array and record the data directly in a digital computer. In some cases, electronic detection of the signal may not be necessary. For example, with enzymatically generated colour spots associated with nucleic acid array format, visual examination of the array will allow interpretation of the pattern on the array. In the case of a nucleic acid array, the detection means is suitably interfaced with pattern recognition software to convert the pattern of signals from the array into a plain language genetic profile. In certain embodiments, oligonucleotide probes specific for different biomarker polynucleotides are in the form of a nucleic acid array and detection of a signal generated from a reporter molecule on the array is performed using a 'chip reader'. A detection system that can be used by a 'chip reader' is described for example by Pirrung et al (U.S. Patent No. 5, 143,854). The chip reader will typically also incorporate some signal processing to determine whether the signal at a particular array position or feature is a true positive or maybe a spurious signal. Exemplary chip readers are described for example by Fodor et al (U.S. Patent No., 5,925,525). Alternatively, when the array is made using a mixture of individually addressable kinds of labelled microbeads, the reaction may be detected using flow cytometry.

[0077] In other illustrative embodiments, SFRP4 protein levels can be measured using protein-based assays known in the art. For example, an antibody-based technique may be employed to determine the level of SFRP4 in a sample, non-limiting examples of which include immunoassays, such as the enzyme-linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).

[0078] Protein-capture arrays that permit simultaneous detection and/or quantification of a large number of proteins may also be employed. For example, low- density protein arrays on filter membranes, such as the universal protein array system (Ge, 2000 Nucleic Acids Res. 28(2):e3) allow imaging of arrayed antigens using standard ELISA techniques and a scanning charge-coupled device (CCD) detector. Immuno-sensor arrays have also been developed that enable the simultaneous detection of clinical analytes. It is now possible using protein arrays, to profile protein expression in bodily fluids, such as in sera of healthy or diseased subjects, as well as in subjects pre- and post-treatment. Exemplary protein capture arrays include arrays comprising spatially addressed antigen- binding molecules, commonly referred to as antibody arrays, which can facilitate extensive parallel analysis of numerous proteins. Antibody arrays have been shown to have the required properties of specificity and acceptable background, and some are available commercially (e.g. , BD Biosciences, Clontech, BioRad and Sigma). Various methods for the preparation of antibody arrays have been reported (see, e.g. , Lopez et al., 2003 . Chromatogr. B 787: 19-27; Cahill, 2000 Trends in Biotechnology 7:47-51 ; U.S. Pat. App. Pub. 2002/0055186; U.S. Pat. App. Pub. 2003/0003599; PCT publication WO 03/062444; PCT publication WO 03/077851 ; PCT publication WO 02/59601 ; PCT publication WO 02/39120; PCT publication WO 01/79849; PCT publication WO 99/39210). Individual spatially distinct protein-capture agents are typically attached to a support surface, which is generally planar or contoured. Common physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads.

[0079] Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include colour coding for microbeads (e.g., available from Luminex, Bio-Rad and Nanomics Biosystems) and semiconductor nanocrystals (e.g., QDots™, available from Quantum Dots), and barcoding for beads (UltraPlex™, available from Smartbeads) and multimetal microrods (Nanobarcodes™ particles, available from Surromed). Beads can also be assembled into planar arrays on semiconductor chips (e.g., available from LEAPS technology and BioArray Solutions). Where particles are used, individual protein-capture agents (e.g., anti-SFRP4 antibodies or SFRP4-binding fragments thereof) are typically attached to an individual particle to provide the spatial definition or separation of the array. The particles may then be assayed separately, but in parallel, in a compartmentalized way, for example in the wells of a microtitre plate or in separate test tubes.

[0080] In an illustrative example, a protein sample, which is optionally fragmented to form peptide fragments (see, e.g., U.S. Pat. App. Pub. 2002/0055186), is delivered to a protein-capture array under conditions suitable for protein or peptide binding, and the array is washed to remove unbound or non-specifically bound components of the sample from the array. Next, the presence or amount of protein or peptide bound to each feature of the array is detected using a suitable detection system. The amount of protein bound to a feature of the array may be determined relative to the amount of a second protein bound to a second feature of the array. In certain embodiments, the amount of the second protein in the sample is already known or known to be invariant.

[0081] Another illustrative example of a protein-capture array is a Luminex -based multiplex assay, which is a bead-based multiplexing assay, where beads are internally dyed with fluorescent dyes to produce a specific spectral address. Biomolecules (such as an oligo or antibody) can be conjugated to the surface of beads to capture analytes of interest; that is, SFRP4 protein or a nucleic acid molecule encoding same. Flow cytometric or other suitable imaging technologies known to persons skilled in the art can then be used for characterization of the beads, as well as for detection of analyte presence. The Luminex technology enables are large number of proteins, genes or other gene expression products (e.g., 100 or more, 200 or more, 300 or more, 400 or more) to be detected using very small sample volume (e.g. , in a 96 or 384-well plate). In some embodiments, the protein-capture array is Bio-Plex Luminex- 100 Station (Bio-Rad).

[0082] In some embodiments, the level of SFRP4 can be normalized against a housekeeping biomarker. The term "housekeeping biomarker" refers to a biomarker or group of biomarkers (e.g., polynucleotides and/or polypeptides), which are typically found at a constant level in the cell type(s) being analysed and across the conditions being assessed. In some embodiments, the housekeeping biomarker is a "housekeeping gene." A "housekeeping gene" refers herein to a gene or group of genes which encode proteins whose activities are essential for the maintenance of cell function and which are typically found at a constant level in the cell type(s) being analysed and across the conditions being assessed.

[0083] It would be understood by those skilled in the art that the method of analyzing the expression of SFRP4 in a blood sample can be quantitative, semi-quantitative or qualitative in nature. For example, quantitative analyses will typically provide a concentration of SFRP4 in the sample within an appropriate error margin (e.g., mean +/- standard deviation). By contrast, semi-quantitative or qualitative analyses will typically provide an indication of the relative amount of SFRP4 in a sample. This may involve a comparison of an amount of SFRP4 protein in a first sample with an amount of SFRP4 protein in a second sample and making a determination as to the relative amount of SFRP4 protein between the first and second samples. In an embodiment, the methods and/or protocol disclosed herein comprise measuring the level of SFRP4 protein in the biological sample. SFRP4 protein levels may be measured using any suitable protein-based assays known in the art, illustrative examples of which are disclosed elsewhere herein.

[0084] The present inventors have also surprisingly found that the level of expression of SFRP4 protein is higher in gastric cancer tissue, as determined by immunohistochemistry, when compared to histologically normal or benign gastric tissue. Thus, in an embodiments, the methods and protocols disclosed herein comprise measuring the expression of SFRP4 protein by immunohistochemical techniques. Suitable immunohistochemical techniques would be familiar to persons skilled in the art, an example of which is disclosed herein with reference to the Examples.

[0085] It will also be understood that, in some instances, the methods and protocols disclosed herein comprise measuring the expression of an SFRP4 gene expression product in the biological sample, such as an SFRP4 transcript (e.g. , mRNA). Thus, in an embodiment, the methods and protocols disclosed herein comprise measuring the expression of a gene (nucleic acid molecule) that encodes SFRP4. Suitable nucleic acid- based techniques would be known to persons skilled in the art, illustrative examples of which are disclosed elsewhere herein.

[0086] In some embodiments, target nucleic acids are measured using blotting techniques, such as Southern blotting and Northern blotting.

[0087] As noted elsewhere herein, suitable methods for analyzing a sample for the expression of SFRP4 would be known to persons skilled in the art. In some embodiments, the expression of SFRP4 can be verified by observing, measuring and/or detecting an presence of SFRP4 at the protein level, for example, using an SFRP4-specific binding protein such as an anti-SFRP4 antibody or an SFRP4-binding fragment thereof. Suitable methods for observing, measuring and/or detecting the level of expression of SFRP4 at the protein level would be known to persons skilled in the art, illustrative examples of which include Western blot analysis and enzyme-linked immunosorbant assays.

[0088] In an embodiment, the methods disclosed herein comprise measuring SFRP4 protein; e.g., measuring the concentration of SFRP4 protein. In an embodiment, the level of SFRP protein is measured using an SFRP4 binding protein, such as an anti-SFRP4 antibody or an SFRP4-binding fragment thereof. As used herein, the phrase "SFRP4- binding fragment" means a portion of the referenced antibody molecule that retains the ability to bind specifically to or otherwise recognize the target molecule (i.e. , SFRP4). Examples of suitable fragments would be known to persons skilled in the art, illustrative examples of which include Fab, F(ab)₂, Fv and scFv fragments. Thus, in an embodiment disclosed herein, measuring the expression of SFRP4 in the post-operative blood sample comprises (i) exposing the post-operative blood sample to an SFRP4 binding agent under conditions that allow the SFRP4 binding agent to specifically bind to, and form a complex with, SFRP4 in the sample, and (ii) measuring the amount of complex formed, wherein the amount of complex is indicative of the amount of SFRP4 in the sample and, hence, indicative of whether or not the subject is at risk of recurrence. Suitable SFRP4 binding agents would be known to persons skilled in the art. Where the level of SFRP4 gene expression is being measured (e.g., SFRP4 mRNA levels), illustrative examples of suitable SFRP4 binding agents will include oligonucleotides, probes and primers. Where the level of SFRP4 protein is being measured, illustrative examples of suitable SFRP4 binding agents will include anti-SFRP4 antibodies or an SFRP4-binding fragments thereof, as described elsewhere herein.

[0089] In some embodiments, the level of expression of SFRP4 may be determined on the basis of the activity of SFRP4 in the sample. Thus, in an embodiment, a patient is identified as being at risk of recurrence where the level of activity of SFRP4 in a postoperative blood sample is greater than the level of activity of SFRP4 in the reference sample. As used herein, the term "activity" typically refers to the biological activity SFRP4; that is, a measurable biological response that in which SFRP4 plays a role. The activity of SFRP4 can be verified, for example, by observing, measuring and/or detecting changes in the expression of molecules implicated in a biological signalling pathway in which SFRP4 is a component, for example, in a suitable cell-based assay. Such molecules, also referred to herein as "surrogate markers", may be implicated in the biological signalling pathway upstream or downstream of SFRP4.

[0090] Examples of molecules implicated in SFRP4-mediated signalling would be known to persons skilled in the art, illustrative examples of which are those involved in Wnt7a activation (see, for example, Carmon and Loose, Mol. Cancer Res., 2008 6; 1017- 1028).

[0091] It would be understood by those skilled in the art that the method of analyzing the expression of SFRP4 in a blood sample can be quantitative, semi-quantitative or qualitative in nature. For example, quantitative analyses will typically provide a concentration of SFRP4 in the sample within an appropriate error margin (e.g., mean +/- standard deviation (SD) or standard error mean (SEM)). By contrast, semi-quantitative or qualitative analyses will typically provide an indication of the relative amount of SFRP4 in a sample. This may involve, for example, a comparison of an amount of SFRP4 protein in a first sample with an amount of SFRP4 protein in a second sample and making a determination as to the relative amount of SFRP4 protein between the first and second samples.

Reference Value

[0092] In some embodiments, the comparison step (i.e., to identify whether the subject has, or is at risk of developing, gastric cancer) is carried out in the absence of comparing the level of expression of SFRP4 in the biological sample to the level of expression in another biological sample. For example, the comparison may be carried out using a reference value; that is, a known or predetermined level of SFRP4 expression that is associated with, for example, the presence or absence of gastric cancer, a particular grade of gastric cancer, or a risk of developing gastric cancer, as described elsewhere herein.

[0093] The term "reference value" is referred to interchangeably herein as a "control value". In an illustrative example, the comparison may be carried out using a reference value that is representative of a known or predetermined level of SFRP4 expression in a reference sample or a plurality of reference samples, that is associated with, for example, the presence or absence of gastric cancer, or of a particular grade of gastric cancer. The term "reference sample" is also referred to herein as a "control sample". The reference value is typically a predetermined level of expression of SFRP4 that is representative of the level of expression in a particular cohort or population of subjects (e.g., normal healthy controls, subjects with gastric cancer, subjects with a history of gastric cancer, subjects who had no sign of gastric cancer at the time the reference sample was obtained but who have gone on to develop gastric cancer, etc). The reference value may be represented as an absolute number, or as a mean value (e.g., mean +/- standard deviation), such as when the reference value is derived from (i.e., representative of) a sample population of individuals.

[0094] The reference value may be equal to or not significantly different from the level of expression of SFRP4 in a sample population representative of patients with, or at known risk of developing, gastric cancer. Thus, a level of expression of SFRP4 in a sample from a test subject that is equal to, not significantly less than or greater than the reference value is indicative of the presence of gastric cancer in the test subject, or a risk of developing gastric cancer, including recurrence. Conversely, a level of expression of SFRP4 in a sample from a test subject that is significantly less than the reference value is indicative of the absence of gastric cancer in the test subject, or a low risk of developing gastric cancer, including recurrence.

[0095] The reference value may also be equal to or not significantly different from the level of expression of SFRP4 in a sample population representative of normal, healthy subjects or subjects who are otherwise free of gastric cancer. Thus, a level of expression of SFRP4 in a sample from a test subject that is lower than, equal to, or not significantly different from, the reference value is indicative of the absence of gastric cancer in the test subject, or a low risk of developing gastric cancer, including recurrence. Conversely, a level of expression of SFRP4 in a sample from a test subject that is significantly greater than the reference value is indicative of the presence of gastric cancer in the test subject, or a risk of developing gastric cancer, including recurrence. [0096] In some embodiments, the reference value can be a level of expression of

SFRP4 in a biological sample obtained from gastric tissue that has been characterised as (i) histologically normal (i.e., absent any signs of histological abnormalities) or (ii) benign (e.g., chronic gastritis or intestinal metaplasia). Thus, a level of expression of SFRP4 in a sample from a test subject that is lower than, equal to, or not significantly different from, the reference value is indicative of the absence of gastric cancer in the test subject, or a low risk of developing gastric cancer, including recurrence. Conversely, a level of expression of SFRP4 in a sample from a test subject that is significantly greater than the reference value is indicative of the presence of gastric cancer in the test subject, or a risk of developing gastric cancer, including recurrence. In an embodiment, the reference value is a level of expression of SFRP4 in a biological sample obtained from gastric tissue that has been characterised as histologically normal. In another embodiment, the reference value is a level of expression as measured in a sample of normal or benign gastric tissue obtained from the same subject.

[0097] Whilst persons skilled in the art would understand that using a reference value of SFRP4 expression that is derived from a sample population of individuals is likely to provide a more accurate representation of the level of expression in that particular population (e.g., for the purposes of the methods and protocols disclosed herein), in some embodiments, the reference value can be a level of expression of SFRP4 in a single biological sample.

[0098] In one embodiment, the reference value is a value that is representative of the level of expression of SFRP4 in a biological sample obtained from a healthy subject or a subject that is otherwise free of gastric cancer. Accordingly, a level of expression of SFRP4 in the biological sample from a test subject that is greater then, or not significantly different from (e.g., equal to), the reference value is indicative of the test subject having, or being at risk of developing, gastric cancer, including recurrence. Conversely, a level of expression of SFRP4 in a biological sample from a test subject that is less than the reference value is indicative of the absence of gastric cancer in the test subject, or a low risk of developing gastric cancer, including recurrence. [0099] Alternatively, or in addition, the reference value can be a value that is representative of the level of expression of SFRP4 in a biological sample obtained from a patient with gastric cancer or from a patient who had gone on to develop gastric cancer after the biological sample (i.e., the reference sample) was obtained. Accordingly, a level of expression of SFRP4 in the biological sample from a test subject that is greater than, or not significantly different from (e.g., equal to), the reference value is indicative of the test subject having, or being at risk of developing, gastric cancer, including recurrence. Conversely, a level of expression of SFRP4 in the biological sample from a test subject that is less than the reference value would be indicative of the test subject having, or being at risk of developing, gastric cancer, including recurrence.

[0100] The present inventors have also surprisingly found that the level of expression of SFRP4 is greater in diffuse gastric cancer tissue as compared to the level of expression of SFRP4 in intestinal gastric cancer tissue. Thus, the level of expression of SFRP4 in a biological sample may be used to predict whether a subject has a diffuse or intestinal form of gastric cancer. For example, where the reference value is representative of the level of expression of SFRP4 in a population of subjects diagnosed as having diffuse gastric cancer, then a level of expression of SFRP4 in a sample from a test subject that is significantly lower than the reference value is indicative of the likelihood that the subject has an intestinal form of gastric cancer. Conversely, where the reference value is representative of the level of expression of SFRP4 in a population of subjects diagnosed as having intestinal gastric cancer, then a level of expression of SFRP4 in a sample from a test subject that is significantly greater than the reference value is indicative of the likelihood that the subject has a diffuse form of gastric cancer.

[0101] In another embodiment, where the reference value is representative of the level of expression of SFRP4 in a population of subjects diagnosed as having diffuse gastric cancer, then a level of expression of SFRP4 in a sample from a test subject that is greater than, equal to or not significantly different from the reference value is indicative of the likelihood that the subject has a diffuse form of gastric cancer. Conversely, where the reference value is representative of the level of expression of SFRP4 in a population of subjects diagnosed as having intestinal gastric cancer, then a level of expression of SFRP4 in a sample from a test subject that is lower than, equal to or not significantly greater than the reference value is indicative of the likelihood that the subject has an intestinal form of gastric cancer.

[0102] The present inventors have also surprisingly found that the level of expression of SFRP4 at both the protein and gene expression (e.g., mRNA) level is increased at more advanced T-stages of gastric cancer. Thus, the level of expression of SFRP4 in a biological sample may be used as an indicator of the severity of disease or disease progression, as determined, for example, by T-stage. For instance, in some embodiments, the reference value is representative of the level of expression of SFRP4 in a population of subjects diagnosed as having a TO-stage or Tl -stage of gastric cancer, whereby a level of expression of SFRP4 in a sample from a test subject that is significantly greater than the reference value is indicative of the subject having a more advanced form of gastric cancer (e.g., a T1-, T2- or T3-stage cancer). Similarly, where the reference value is representative of the level of expression of SFRP4 in a population of subjects diagnosed as having a T2-stage or T3-stage of gastric cancer, then a level of expression of SFRP4 in a sample from a test subject that is significantly lower than the reference value is indicative of the subject having a less advanced form of gastric cancer (e.g., a TO-stage or Tl -stage cancer)

[0103] In other embodiments, where the reference value is representative of the level of expression of SFRP4 in a population of subjects diagnosed as having a TO-stage or Tl -stage of gastric cancer, a level of expression of SFRP4 in a sample from a test subject that is less than, equal to or not significantly different than the reference value is indicative of the subject has a TO- or Tl -stage cancer. Similarly, where the reference value is representative of the level of expression of SFRP4 in a population of subjects diagnosed as having a T2-stage or T3-stage of gastric cancer, then a level of expression of SFRP4 in a sample from a test subject that is significantly greater than, or not significantly different from (e.g., equal to), the reference value is indicative of the subject having T2- or T3-stage cancer. Recurrence and progression-free survival

[0104] As disclosed herein, the present inventors have surprisingly found that elevated expression of SFRP4 in a biological sample obtained following resection, as compared to the level of expression of SFRP4 in a biological sample obtained from of a healthy subject {i.e., a subject with no gastric cancer) or from a patient with gastric cancer prior to resection, can predict recurrence of gastric cancer in patients who have undergone resection. The present inventors have also found that a higher level of expression of SFRP4 in gastric cancer tissue is associated with poor prognosis, as determined by progression free survival, in patients who have undergone resection. The present inventors have therefore shown, for the first time, that SFRP4 is a suitable prognostic indicator of gastric cancer.

[0105] Thus, in an aspect disclosed herein, there is provided a method of determining whether a subject is at risk of recurrence, the method comprising:

wherein said comparison provides an indication as to whether or not the subject is at risk of recurrence.

[0106] In another aspect disclosed herein, there is provided a method of predicting progression free survival in a patient with gastric cancer, the method comprising:

wherein said comparison predicts progression free survival in the subject.

[0107] Resection, also referred to as radical gastrectomy or curative resection, remains the primary form of potentially curative treatment for gastric cancer. However, despite curative resection, recurrence of gastric cancer is common, affecting around 60% of patients. This is largely attributed to the fact that gastric cancer is often well advanced at the time of diagnosis. The rate of recurrence after curative resection typically varies, with studies reporting that more than 90% of patients relapse within 5 years after surgery, and 70% relapse within 2 years.

[0108] Recurrence of gastric cancer can be classified into several groups, including, but not limited to, (i) recurrence of gastric remnants, (ii) locoregional recurrence, (iii) peritoneal recurrence, (iii) liver metastasis, (iv) extrahepatic haematogenous metastasis, (v) lymph node metastasis and (vi) multiple metastasis. In clinical practice, recurrence is typically classified as locoregional (e.g., recurrence at the resection margin, within the lymph nodes or in the region of the resection, below the diaphragm and liver and above the pancreas and abdominal wound), peritoneal (recurrence in the abdominal cavity following intraperitoneal distribution including visceral metastasis and rectal shelf, pericholedochal, and periureteral infiltration) and haematogenous (recurrence has been defined as any metastatic lesion detected in the liver, lung, bone, ovary, spleen, testis, or other distant organs).

[0109] The two most common types of recurrence are haematogenous recurrence in relatively early-stage gastric cancer and peritoneal dissemination in advanced gastric cancer. Peritoneal recurrence accounts for about 50% of cases and usually occurs within 2 years after resection. Haematogenous recurrence accounts for about 40% of cases. By contrast, locoregional recurrence occurs in about 20% of cases.

[0110] There are many factors that are thought to influence recurrence of gastric cancer, including tumour size, location, therapeutic strategy, Borrmann classification, Lauren classification, lymph node metastasis, lymph node metastasis ratio, pN stage, lymphatic and/or blood vessel invasion, width of serosal changes, depth of invasion, and peritoneal cytology.

[0111] The TNM classification recommends the removal of at least 15 regional lymph nodes during resection for adequate pathological staging. The lymph node ratio (LNR) is the ratio between the number of metastatic nodes and the number of dissected nodes, and most studies consider 20% as the cutoff most frequently used. Tumour stage (such as depth of tumour invasion and lymph node metastasis) and Borrmann classification have generally been proven to be the main risk factors for recurrence of gastric cancer.

[0112] The Lauren classification groups gastric cancer into intestinal, diffuse, and mixed (unclassifiable) types. Earlier independent studies have reported that the rate of recurrence was 41 % in intestinal-type cases and 65% in diffuse-type cases. The incidence of locoregional, hematogenous, and peritoneal recurrence was 20%, 19%, and 9% in intestinal-type cases, respectively, and 27%, 16%, and 34% in diffuse-type cases, respectively. Compared with the intestinal type, the diffuse type showed a much greater predisposition to proliferate in the peritoneum. On the contrary, recurrence of intestinal- type tumours was predominantly found to be locoregional and hematogenous. However, the intestinal type metastasized primarily to the liver. The Borrmann classification has generally been considered to be an independent risk factor for recurrence of gastric cancer and Borrmann types III and IV occur more frequently in recurrent cancer.

[0113] Postoperative adjuvant chemotherapy has generally been accepted as a clinically effective method to reduce the recurrence rate in advanced gastric cancer. Preoperative therapy and surgical techniques may also affect the patterns of recurrence.

[0114] Many patients with gastric cancer typically present with advanced disease at initial diagnosis, which is one of the main contributory factors for the high rate of recurrence. Overall disease-specific survival of gastric cancer is generally poor. When recurrence occurs, the prognosis worsens, especially in patients with peritoneal recurrence. Nearly 50% of patients with potentially curable advanced gastric cancer will die of peritoneal recurrence and most patients will die within the first year of the diagnosis of recurrence, with mean survival time of about 8.7 (range, 2-66) months.

[0115] Patients with well-differentiated or moderately differentiated tumours have been shown to have a longer overall survival than those with poorly differentiated tumours (8 months). Multivariate analysis showed that tumour stage and gross characteristics were independent factors of the cumulative survival rate.

[0116] Lymph node metastasis is also an important prognostic indicator of recurrence after curative resection and is positively correlated with depth of tumour invasion. For example, lymph node-negative gastric cancer patients have a better prognosis than node-positive gastric cancer patients. Furthermore, recurrence in patients with lymph node-negative gastric cancer is typically less than that in patients with lymph node -positive gastric cancer.

[0117] The present inventors have surprisingly found that the level of expression of

SFRP4 protein in normal, healthy control subjects is not significantly different from the level of expression found in a pre-operative blood sample from patients with gastric cancer. Thus, in some embodiments, the level of expression of SFRP4 in the reference sample (also referred to herein as a "reference value", "reference level", "control value", control level" and the like) may be representative of a population of (i) normal (i.e., healthy) subjects, (ii) subjects with no history of gastric cancer or (iii) patients who have undergone resection but do not go on to develop recurrence within, for example, the first 3 years following resection. In some embodiments, the reference value is between about 20 ng/mL to about 150 ng/mL, more preferably between about 50 ng/mL to about 100 ng/mL, more preferably between about 50 ng/mL to about 80 ng/mL.

[0118] In an embodiment disclosed herein, the reference value is representative of a level of expression of SFRP4 in blood of a population of healthy subjects or representative of a level of expression of SFRP4 in blood of a population of patients with gastric cancer taken prior to resection and wherein a level of expression of SFRP4 in the post-operative blood sample that is greater than the reference value is indicative of a risk of recurrence.

[0119] In an embodiment, the reference value is representative of a pre-operative level of expression of SFRP4 in a population of subjects with, or at risk of developing, gastric cancer, wherein a level of expression of SFRP4 in the post-operative blood sample that is greater than the control level is indicative of a risk of recurrence. By "pre-operative" is meant a sample obtained before resection. In an embodiment, the reference value is the level of expression of SFRP4 in a sample that is obtained during or immediately following resection. In an embodiment, the reference value is the level of expression of SFRP4 in a sample that is obtained from the subject at any time point from about 24 hours prior to resection to about 24 hours following resection, from about 12 hours prior to resection to about 12 hours following resection, from about 6 hours prior to resection to about 6 hours following resection, from about 3 hours prior to resection to about 3 hours following resection, from about 2 hours prior to resection to about 2 hours following resection, from about 1 hour prior to resection to about 1 hour following resection, including at any other time point within the aforementioned ranges.

[0120] In an embodiment, the reference value is a level of expression of the SFRP4 in a pre-operative blood sample obtained from the same subject; that is, a level of expression in a blood sample obtained from the same subject at a time point prior to resection. Thus, in an embodiment, the method further comprises the step of obtaining a pre-operative blood sample from the subject before resection and comparing the level of expression of the SFRP4 in the post-operative blood sample with the level of expression of the SFRP4 in the pre-operative blood sample, wherein a level of expression of the SFRP4 in the post-operative blood sample that is greater than the level of expression in the preoperative blood sample is indicative of a risk of recurrent gastric cancer.

[0121] In some embodiments, the reference value may be representative of the level of expression of SFRP4 in blood of a population of patients who have undergone resection and have developed recurrence (also referred to interchangeably herein as a "positive reference value", "positive control value" and the like). In some embodiments, the positive reference value is at least about 1.1 -fold, preferably at least about 1.15-fold, higher than the level of expression of SFRP4 that is representative of a population of normal (i.e., healthy) subjects, of a population of subjects with no history of gastric cancer or of a population of patients who have undergone resection but do not go on to develop recurrence within, for example, the first 3 years following resection. Thus, in an embodiment disclosed herein, a subject is identified as being at risk of recurrence where the level of expression of SFRP4 in the post-operative blood sample from the subject is at least about 1.15-times greater than the level of expression of SFRP4 in the reference sample. In some embodiments, the positive reference value is from about 1.1 -fold to about 2-fold higher than the level of expression of SFRP4 that is representative of a population of normal (i.e., healthy) subjects, of a population of subjects with no history of gastric cancer or of a population of patients who have undergone resection but do not go on to develop recurrence within, for example, the first 3 years following resection. Risk

[0122] The term "risk", as used herein, denotes a subject's likelihood of developing gastric cancer, including recurrence following resection, based on the level of expression of SFRP4 in a biological sample, as determined for that subject. Accordingly, the terms "risk" and "likelihood" are used interchangeably herein, unless otherwise stated.

[0123] It would be apparent to persons skilled in the art that the risk of developing gastric cancer will vary, for example, from being at low, lower or decreased risk of developing gastric cancer to being at high, higher or increased risk of developing gastric cancer. By "low, lower or decreased risk" is meant that the subject is less likely to develop gastric cancer as compared to a subject determined to be a "high, higher or increased risk" subject. Conversely, a "high, higher or increased risk" subject is a subject who is more likely to develop cancer following resection as compared to a subject who is not at risk or a "low, lower or decreased risk" subject.

[0124] Likelihood is suitably based on mathematical modelling. An increased likelihood, for example, may be relative or absolute and may be expressed qualitatively or quantitatively. For instance, an increased risk may be expressed as simply determining the subject's level of expression of SFRP4 in a biological sample and placing the subject in a high or higher risk category, based upon the level of expression of SFRP4 that is representative of the level of expression of SFRP4 that corresponds to a high or higher risk of developing gastric cancer, as determined, for example, in biological samples of a population of patients who have developed gastric cancer. Alternatively, a numerical expression of the test subject's likelihood of developing gastric cancer may be determined based upon the level of expression of SFRP4 in the biological sample.

[0125] As used herein, the term "probability" refers to the probability of class membership for a sample as determined by a given mathematical model and is construed to be equivalent likelihood in this context. In some embodiments, likelihood is assessed by comparing the level of expression of SFRP4 to one or more preselected levels, also referred to herein as threshold or reference levels or values. Thresholds may be selected that provide an acceptable ability to predict recurrence. In illustrative examples, receiver operating characteristic (ROC) curves can be calculated by plotting the value of a variable versus its relative frequency in two populations in which a first population is considered at risk of recurrence following resection and a second population that is not considered to be at risk, or have a low risk, of recurrence following resection (called arbitrarily, for example, "healthy controls"). The second population may also be represented by patients who have undergone curative resection but do not present with recurrent gastric cancer over, for example, a 3 year period following the resection.

[0126] As disclosed herein, the greater the level of SFRP4 expression in the biological sample, the shorter the progression free survival. The term "progression free survival" is used to denote the time elapsed between treatment initiation (e.g., resection) and tumour progression or death. Progression free survival is often used in clinical trials as one way of measuring how well a particular treatment works. The determination of progression free survival need not be absolute (i.e., a specific time point between onset of treatment and the onset of disease progression or death). In some embodiments, the progression free survival may be approximate (e.g., 6-12 months, 1-2 years, etc).

[0127] As disclosed herein, a reference value can be used that is representative of the level of expression of SFRP4 in a biological sample obtained from a healthy subject or a subject that is otherwise free of gastric cancer. Thus, in an embodiment, a level of expression of SFRP4 in the biological sample from a test subject that is greater than the reference value is indicative of a poor prognosis; that is, of poor progression free survival. Conversely, a level of expression of SFRP4 in the biological sample from a test subject that is less than the reference value would be indicative of a progression free survival period that is longer than the progression free survival period of the patient from which the reference value was derived.

[0128] In another embodiment disclosed herein, the reference value can be a value that is representative of the level of expression of SFRP4 in a biological sample obtained from a patient with gastric cancer or from a patient who has undergone a resection of their gastric tumour but has subsequently developed recurrence. Thus, a level of expression of SFRP4 in the biological sample from a test subject that is greater than or not significantly different from (e.g., equal to) the reference value is indicative of a poor prognosis and a progression free survival that is shorter than or equal to the progression free survival period of the patient from which the reference value was derived. Conversely, a level of expression of SFRP4 in the biological sample from a test subject that is less than the reference value would be indicative of a progression free survival period that is longer than the progression free survival period of the patient from which the reference value was derived.

[0129] For any particular biomarker, such as SFRP4, a distribution of levels of expression for subjects who are at risk or are at no or at low risk of developing gastric cancer (including recurrence) may overlap. Under such conditions, a test may not absolutely distinguish a subject who is at higher risk of developing gastric cancer (e.g., recurrence) from a subject who is not at risk or at lower risk of developing gastric cancer with absolute (i.e., 100%) accuracy, and the area of overlap indicates where the test cannot distinguish the two subjects. A threshold can be selected, above which (or below which, depending on how a biomarker changes with risk) the test is considered to be "positive" and below which the test is considered to be "negative." The area under the ROC curve (AUC) provides the C-statistic, which is a measure of the probability that the perceived measurement will allow correct identification of a condition (see, e.g., Hanley et al., Radiology 143: 29-36 ( 1982)). In an embodiment disclosed herein, the threshold is between about 1.1 -fold and about 2-fold, preferably about 1.15-fold, higher than the level of expression of SFRP4 that is representative of a population of normal (i.e., healthy) subjects, of a population of subjects with no history of gastric cancer or of a population of patients who have undergone resection but do not go on to develop recurrence within, for example, the first 3 years following resection.

[0130] The level of risk, or a subject's likelihood of developing gastric cancer, including recurrence following resection, may be based on the level of expression of SFRP4 in a biological sample, as herein described, together with one or more additional risk factors, as determined for that subject. Suitable additional risk factors would be known to persons skilled in the art. Illustrative examples include those described by Pattison et al. (Predictors of outcome after surgery for gastric cancer in a Western cohort. ANZ Journal of Surgery. 2014; doi: 10.1 111/ans.12915), such as AJCC stage, Lauren classification and age at time of surgery.

[0131] For example, the present inventors have surprisingly found that the combination of SFRP4 expression and AJCC stage improves the sensitivity and specificity for the prediction of recurrence. Thus, in an embodiment, the methods disclosed herein further comprise using the subject's AJCC stage to determine whether the subject is at risk of recurrence and/or to predict progression free survival in that subject.

Therapeutic regimen

[0132] A subject who is identified as having, or being at risk of developing gastric cancer, including recurrence, can be stratified into a treatment group where an appropriate therapeutic regimen can be adopted or prescribed with a view to treating or preventing gastric cancer. Conversely, subjects identified as being free of gastric cancer, or being at no or low risk of developing gastric cancer, including recurrence, can be spared an otherwise taxing therapeutic regimen or, alternatively, a less aggressive therapeutic regimen (e.g., a lower dose of chemotherapeutic agent or radiation dose) can be adopted or prescribed. Thus, in an embodiment, the methods disclosed herein comprise the step of exposing (i.e., subjecting) the subject identified as having, or being at risk of developing gastric cancer, including recurrence, to a therapeutic regimen for treating or preventing gastric cancer.

[0133] In another aspect disclosed herein, there is provided a method of stratifying a subject to a therapeutic regimen for treating or preventing gastric cancer, the method comprising the steps of:

(c) wherein said comparison provides an indication as to whether or not the subject has, or is at risk of developing, gastric cancer; and (d) stratifying the subject identified in step (c) as having, or being at risk of developing, gastric cancer, to a therapeutic regimen for treating or preventing gastric cancer.

[0134] In another aspect of the present disclosure, there is provided a protocol for treating or preventing gastric cancer in a subject, the protocol comprising:

(a) identifying a subject at risk of recurrence in accordance with the methods disclosed herein; and

(b) subjecting the subject identified in step (a) as being at risk of recurrence to a therapeutic regimen for treating or preventing gastric cancer.

[0135] In another aspect disclosed herein, there is provided a method for treating or preventing gastric cancer in a subject, the protocol comprising:

(a) executing the steps of (i) measuring the expression of SFRP4 in a biological sample obtained from a subject, and (ii) comparing the level of expression of SFRP4 to a reference value, wherein said comparison provides an indication as to whether or not the subject has, or is at risk of developing, gastric cancer; and

(b) exposing a subject identified in (a) as having, or being at risk of developing, gastric cancer to an effective therapeutic regimen for treating or preventing gastric cancer.

[0136] The type of therapeutic regimen can be determined by persons skilled in the art and will typically depend on factors such as, but not limited to, the age, weight and general health of the subject. Another determinative factor may be the degree of risk of developing gastric cancer (including recurrence), as determined, for example, by the level of expression of SFRP4. For instance, for a subject identified as being at high or higher risk of developing gastric cancer, a more aggressive therapeutic regimen may be prescribed as compared, for example, for a subject who is deemed at low or lower risk of developing gastric cancer, as determined by the methods disclosed herein. Conversely, for a subject identified as being at low or lower risk of developing gastric cancer, a less aggressive therapeutic regimen may be prescribed as compared, for example, for a subject who is deemed at high or higher risk of developing gastric cancer, as determined, for example, by the methods disclosed herein. Similarly, for a subject identified as having a more advance stage of gastric cancer (as determined, e.g., by T-stage), a more aggressive therapeutic regimen may be prescribed as compared, for example, for a subject who has a less advanced stage of gastric cancer, as determined, for example, by the methods disclosed herein. Conversely, for a subject identified as having a less advance stage of gastric cancer (as determined, e.g., by T-stage), a less aggressive therapeutic regimen may be prescribed as compared, for example, for a subject who has a more advanced stage of gastric cancer.

[0137] Suitable therapeutic regimens would be known to persons skilled in the art, non- limiting examples of which include chemotherapeutic agents and/or radiotherapy. Therapeutic regimens will typically be designed by a medical practitioner or a team of medical practitioners, having regard, for example, to the age, weight, body mass index and general health of the subject, as noted elsewhere herein. Another determinative factor in the design of a suitable therapeutic regimen may be the degree of risk of recurrence, as determined, for example, by the level of expression of SFRP4. For instance, where the subject is determined to be at high risk of recurrence, a more aggressive therapeutic regimen may be prescribed as compared to a subject who is determined to be at no risk or low risk of recurrence.

[0138] In some embodiments, the therapeutic regimen comprises a combination of two or more treatment modalities (e.g., 2, 3 or more, 4 or more, 5 or more, 6 or more). Treatment modalities will typically be selected with a view to treating and/or preventing gastric cancer and/or gastric cancer recurrence.

[0139] As used herein the terms "treat", "treatment", "treating", "prevent", "preventing" and "prevention" refer to any and all uses which remedy a condition or symptom, prevent the establishment of a condition or disease, or otherwise prevent, hinder, retard, abrogate or reverse the onset or progression of a condition or disease or other undesirable symptoms in any way whatsoever. Thus, the terms "treating" and "preventing" and the like are to be considered in their broadest context. For example, treatment does not necessarily imply that a patient is treated until total recovery or cure. In conditions which display or a characterized by multiple symptoms, the treatment or prevention need not necessarily remedy, prevent, hinder, retard, or reverse all of said symptoms, but may prevent, hinder, retard, or reverse one or more of said symptoms. In the context of gastric cancer recurrence, the agents, uses, methods and protocols of the present disclosure that involve treatment or prevention may prevent, reduce, ameliorate or otherwise delay the recurrence of gastric cancer following resection, or of a highly undesirable event associated with gastric cancer recurrence or an irreversible outcome of gastric cancer recurrence, but may not of itself prevent recurrence of gastric cancer or an outcome associated therewith (e.g., a symptom associated with gastric cancer). Accordingly, treatment and/or prevention includes amelioration of the symptoms of gastric cancer recurrence or preventing or otherwise reducing the risk of recurrence (e.g., curative resection of the gastric tumour).

[0140] The term "inhibiting" and variations thereof, such as "inhibition" and "inhibits", as used herein, do not necessarily imply the complete inhibition of the specified event, activity or function. Rather, the inhibition may be to an extent, and/or for a time, sufficient to produce the desired effect. Inhibition may be prevention, retardation, reduction, abrogation or otherwise hindrance of an event, activity or function. Such inhibition may be in magnitude and/or be temporal in nature. In particular contexts, the terms "inhibit" and "prevent", and variations thereof may be used interchangeably.

[0141] In an embodiment disclosed herein, measuring the expression of SFRP4 in the biological sample comprises (i) exposing the biological sample to an SFRP4 binding agent under conditions that allow the SFRP4 binding agent to specifically bind to, and form a complex with, SFRP4 in the sample, and (ii) measuring the amount of complex formed, wherein the amount of complex is indicative of the amount of SFRP4 in the sample and, hence, indicative of whether or not the subject has, or is at risk of developing, gastric cancer. Suitable SFRP4 binding agents would be known to persons skilled in the art, as discussed elsewhere herein.

Monitoring Treatment

[0142] Without being bound by theory or a particular mode of practice, it also follows from the present disclosure that the methods disclosed herein can be used to monitor the efficacy of treatment of gastric cancer in a subject, whereby the level of expression of SFRP4 is determined (e.g., measured) in biological samples obtained from a subject at two or more separate time points, including before commencement of treatment, during the course of treatment and after cessation of treatment, to determine whether said treatment is effective, for example, in inhibiting the onset or progression of disease.

[0143] Thus, in another aspect disclosed herein, there is provided a protocol for monitoring the efficacy of a therapeutic regimen for treating or preventing gastric cancer, the protocol comprising:

[0144] In an embodiment, measuring the expression of SFRP4 in the first and second biological samples comprises (i) exposing the biological samples to an SFRP4 binding agent under conditions that allow the SFRP4 binding agent to specifically bind to, and form a complex with, SFRP4 in each of the samples, and (ii) measuring the amount of complex formed, wherein the amount of complex is indicative of the level of SFRP4 in the samples and, hence, indicative of whether the therapeutic regimen is effective or not. Suitable SFRP4 binding agents would be known to persons skilled in the art, as discussed elsewhere herein.

[0145] In an embodiment, a reduction in the level of expression of SFRP4 in the second biological sample as compared to the level of expression of SFRP4 in the first biological sample is indicative of an effective therapeutic regimen. Conversely, an increase in the level of expression of SFRP4 in the second biological sample as compared to the level of expression of SFRP4 in the first biological sample is indicative of an ineffective therapeutic regimen.

[0146] Where there has been no change or an increase in the level of expression of SFRP4 over time (indicative of ineffective treatment), the protocol may further comprises altering or otherwise modifying the therapeutic regimen with a view to providing a more efficacious or aggressive treatment. This may comprise administering to the subject additional doses of the same agent with which they are being treated or changing the dose and/or type of medication. Suitable therapeutic regimens will be known to persons skilled in the art, illustrative examples of which are disclosed elsewhere herein, including the use of chemotherapeutic agents and/or radiotherapy.

[0147] The manner in which the therapeutic regimen is to be altered or otherwise modified, if necessary, may depend on several factors, including the age, weight and general health of the subject. Another determinative factor may be the degree of risk of developing gastric cancer, as determined, for example, by the level of expression of SFRP4, or the stage of the gastric cancer. For instance, where the subject is determined to be at high or higher risk of developing gastric cancer, a more aggressive therapeutic regimen may be prescribed as compared to a subject who is determined to be at low or lower risk of developing gastric cancer. Conversely, where the subject is determined to be at low or lower risk of developing gastric cancer, a less aggressive therapeutic regimen may be prescribed as compared to a subject who is determined to be at high or higher risk of developing gastric cancer. In another illustrative example, where the subject is determined to have a more advanced form of gastric cancer (e.g., T2- or T3-stage), a more aggressive therapeutic regimen may be prescribed as compared to a subject who is determined to have a less advanced form of gastric cancer (e.g., TO- or Tl -stage). Conversely, where the subject is determined to have a less advanced form of gastric cancer (e.g., TO- or Tl -stage), a less aggressive therapeutic regimen (e.g., a lower dose or dosage regimen) may be prescribed as compared to a subject who is determined to have a more advanced form of gastric cancer (e.g., T2- or T3-stage).

[0148] The protocol for monitoring the efficacy of a therapeutic regimen, as disclosed herein, further enables determination of endpoints in pharmacotranslational studies. For example, clinical trials can take many months or even years to establish the pharmacological parameters for a medicament to be used in treating gastric cancer. Clinical trials can be expedited by selecting a therapeutic regimen (e.g., medicament and pharmaceutical parameters) that result in an SFRP4 expression profile that is associated with low or lower risk of gastric cancer, with a low or lower grade of gastric cancer or with a healthy state (e.g. , healthy condition). This may be determined, for example, by (1) providing a correlation of a level of expression of SFRP4 with the likelihood of having the healthy condition in order to obtain a reference value; and (2) determining the level of expression of SFRP4 in a sample from subject suspected of having or being at risk of developing gastric cancer, wherein a similarity of the subject's level of expression of SFRP4 after treatment to the reference value indicates the likelihood that the treatment regimen is effective for changing the health status of the subject to the desired health state (e.g. , healthy condition). This aspect of the present disclosure advantageously provides methods of monitoring the efficacy of a particular therapeutic regimen in a subject (for example, in the context of a clinical trial) already diagnosed as being at risk of developing gastric cancer.

Kits

[0149] In another aspect of the present disclosure there is provided a kit comprising one or more reagents and/or devices for use in performing the methods and/or protocols disclosed herein. The kits may contain reagents for analyzing the expression of SFRP4 in a biological sample in accordance with the methods and/or protocols as herein described.

[0150] Kits for carrying out the methods and/or protocols of the present disclosure may also include, in suitable container means, (i) a reagent for detecting SFRP4, (ii) a probe that comprises an anti-SFRP4 antibody (or SFRP4-binding fragment thereof) or a nucleic acid sequence that specifically binds to a nucleic acid molecule encoding SFRP4, (iii) a probe that comprises an antibody or antigen-binding fragment thereof or a nucleic acid sequence that is capable of measuring the expression of SFRP4, (iv) a label for detecting the presence of the antibody, antibody fragment or probe and/or (iv) instructions for how to determine the level of expression of SFRP4. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe and/or other container into which a first antibody specific for SFRP4 (or SFRP4-binding fragment thereof) or a first nucleic acid specific for the SFRP4-encoding nucleic acid molecule may be placed and/or suitably aliquoted. Where a second and/or third and/or additional component is provided, the kit will also generally contain a second, third and/or other additional container into which this component may be placed. Alternatively, a container may contain a mixture of more than one reagent, when required. The kits may also include means for containing the reagents (e.g. , nucleic acids, antibodies, polypeptides etc.) in close confinement for commercial sale. Such containers may include injection and/or blow-moulded plastic containers into which the desired vials are retained.

[0151] The kits may further comprise positive and negative controls, including a reference sample, as well as instructions for the use of kit components contained therein, in accordance with the methods and/or protocols disclosed herein.

[0152] All essential materials and reagents required for detecting and quantifying SFRP4 expression may be assembled together in a kit. The kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, blotting membranes, microtiter plates dilution buffers and the like. For example, a nucleic acid-based detection kit may include (i) an SFRP4-encoding polynucleotide (which may be used as a positive control), (ii) a primer or probe that specifically hybridizes to an SFRP4-encoding polynucleotide. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (Reverse Transcriptase, Taq, Sequenase™ DNA ligase etc. depending on the nucleic acid amplification technique employed), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits may also comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe. Alternatively, a protein- based detection kit may include (i) an SFRP4 polypeptide (which may be used as a positive control), (ii) an antibody that binds specifically to an SFRP4 protein, or an SFRP4-binding fragment thereof. The kit may also feature various devices (e.g. , one or more) and reagents (e.g. , one or more) for performing any one of the assays described herein; and/or printed instructions for using the kit to quantify the expression of a biomarker gene.

[0153] It will be appreciated that the above described terms and associated definitions are used for the purpose of explanation only and are not intended to be limiting.

[0154] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non- limiting examples.

[0155] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0156] The present disclosure is further described by reference to the following non- limiting examples.

Examples

Materials and Methods

( i) Patients and samples

[0157] Tumour samples were collected from 65 (cDNA arrays) or 99 (U133 plus 2 arrays) patients undergoing curative or palliative resection for gastric cancer (GC) in Melbourne or in China. Within 30 minutes of resection, samples were divided for storage in either liquid nitrogen (fresh frozen) or neutral buffered formalin.

[0158] Additional samples were taken from the cardia, fundus, body and antral mucosa of 1 1 patients at maximal distance from the tumour. These samples were pooled and used to extract a single batch of RNA to be used as a reference for all subsequent cDNA array experiments. Written informed consent was obtained from all patients prior to tissue collection.

[0159] Tables 1 and 2 summarize the characteristics for the samples utilised in the cDNA arrays and Affymetrix U133 plus 2 arrays, respectively. Table 1: cDNA array sample cohort

Parameter Number Parameter Number

Age (years) Pathology

Male 64 (32-83) Diffuse 23

Female 73.7 (47-85) Intestinal 35

Gender Mixed 6

Male 45 Adenosquamous 1

Female 20 Differentiation

Tumour location Undifferentiated 20

Antrum 14 Poor 20

GOJ 10 Moderate to poor 6

Greater curve-body 13 Moderate 13

Lesser curve-body 19 Well 4

Body unspecified 5 Other 2

Cardia 0 T Stage

Stomal 0 Tl 7

Chemo-radiotherapy T2 14

Adjuvant T3 44

No Adjuvant T4 0

Adjuvant AJCC Stage

ND IA 3

Paliative IB 9

No Paliative II 14

Paliative IIIA 22

ND IIIB 13

IV 3

Unspecified 1

Table 2: Affymetrix U133 plus 2 sample cohort

Parameter Number Parameter Number

Age (years) Pathology

Male 65.4 (32-83) Diffuse 39

Female 67 (33-86) Intestinal 50

Gender Mixed 10

Male 67 Differentiation

Female 32 Undifferentiated 29

Tumour location Poor 35

Antrum 20 Moderate 31

GOJ 18 Well 3

Carcinoma in

Greater curve-body 28 situ 1

Lesser curve-body 29 T Stage

Body unspecified 1 Tl 10

Cardia 2 T2 25

Stomal 1 T3 62

Chemo-radiotherapy T4 2

Adjuvant AJCC Stage

No Adjuvant 40 IA 5

Adjuvant 46 IB 14

ND 13 II 26

Paliative IIIA 23

No Paliative 21 IIIB 22

Paliative 24 IV 8

ND 54 Unspecified 1

[0160] Ethanol/formalin fixed tissue sections were paraffin embedded for histological use. 5μΜ sections of each sample was stained with haematoxylin and eosin (H&E) and classified independently by two pathologists. These results were compared to the pathology record prepared by the contributing institution. Any variations were further reviewed until a consensus was reached. Each specimen was scored for differentiation, Lauren classification, T-stage, percent tumour and inflammatory cells.

( ii) cDNA Microarray analysis

[0161] cDNA microarrays were run previously (see Boussioutas et al., 2003, Cancer Res., 63:2569-2577). Analysis and data visualisation was performed in Genespring (Silicon Genetics) for all microarray data analysis. All continuous variables were considered as parametric data and analysed with ANOVA using appropriate multiple testing correction factors such as Benjamini Hochberg false discovery rate (Benjamini and Hochberg, 1995; Controlling the false discovery rate: a practical and powerful approach to multiple testing. . Royal Stat. Soc, 57: 289-300) or Bonferroni correction (Overall and Doyle, 1996; False- positive error rates in routine application of repeated measurements ANOVA. Biopharm Stat. 6(1):69-81).

(Hi) Affymetrix U133 plus 2 arrays

[0162] Affymetrix U133 plus 2 arrays were run following the manufacturer's protocol using 5μg of RNA as starting material.

(iv) Immunohistochemistry

[0163] Tissue microarrays (TMA) were sectioned at 4μπι thickness and processed for immunostaining. Antibodies for SFRP4 (1 :250; provided by Lisa Horvarth (Garvan Institute of Medical Research), were used as the primary antibody. Staining was revealed with a horseradish peroxidise linked secondary antibody using the DAKO LSAB+ kit (DAKO), following the manufacturer's instructions.

(v) Cell culture

[0164] The human gastric cancer cell lines AGS (ATCC CRL-1739), SNU-1 (ATCC CRL- 5971) and N87 (ATCC CRL-5822) were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in DMEM (AGS) or RPMI (SNU- 1/N87) supplemented with 10% (w/v) fetal bovine serum, penicillin (100 U/ml) and streptomycin (lOOug/ml) (all from Invitrogen, Carlsbad, CA) and were maintained at 37°C in a humid incubator with 5% C0₂.

( vi) shRNA knockdown

[0165] A total of 3 shRNA clones were obtained for SFRP4 (VCFG) as well as a non- silencing control from the Open Biosystems pGIPz library. Clones were sequenced prior to use and the gene ID confirmed. shRNA-expressing lentiviral plasmids were transfected using Lenti-X packaging vectors into HEK293T cells (Open Biosystems). Viral containing media was collected and filtered. Virus was stored at -80°C until required. Target cells were seeded in 6-well plates and transduced when 60% confluent. Puromycin was used to select for positively transduced cells. Knockdown was confirmed at the mRNA and protein levels using real time PCR and Western blot.

( vii) Western blotting

[0166] Briefly, for each sample a total protein was run on a 12% sodium dodecyl sufate (SDS)/acrylamide gel. Proteins were transferred to a nylon membrane which was then blocked for 1 hour in (PBS/Tween, 5% milk powder). A polyclonal antibody for SFRP4 (R&D 1 : 10,000 dilution) and an anti-goat secondary (R&D) were applied. The human gene tubulin was used as an internal control.

(viii) SFRP4 ELISA

[0167] An SFRP4 enzyme-linked immunosorbent assay (ELISA) was performed using an ELISA kit for SFRP4 from USCNK Life Science Inc. following the manufacturers instructions. Serum or plasma samples were diluted 1:200 in phosphate buffered saline (PBS) before use.

(ix) Statistical analysis

[0168] All assays were performed in triplicate and found to be reproducible. Data is expressed as mean +/- standard error mean (SEM). Analyses was performed using Graphpad prism software.

Results

Example 1 - SFRP4 expression correlates with invasion

[0169] In order to identify key genes which may contribute to invasion, cDNA expression array data obtained for 65 tumours of known T-stage was interrogated (See Table 1 for clinical characteristics). As in clinical practice, T-stage was used as a surrogate for invasion of gastric cancer. Expression patterns that were positively correlated with T-stage (i.e., increasing expression with increments of T-stage) were identified using K-means analysis. Eight patterns (K) of gene expression were chosen where every element on the 10.5K array was used (see Figure 1A). [0170] The gene expression signatures selected served as models to enable selection of other genes highly correlated with each expression pattern. SFRP4 was the gene most strongly correlated with this pattern and was used to identify other genes with similar expression pattern (see Figure IB). 230 genes were discovered similar to SFRP4 expression with a correlation coefficient of 0.98 (see Table 3).

Example 2 - Validation on independent data sets/platforms

[0171] In addition to the cDNA arrays utilised in the initial discovery stage, a further 99 tumours were analysed using Affymetrix Human U133 plus 2 arrays (see Table 3). Although there is some sample overlap with the original cohort, the total number of samples within each T-stage has been increased. A box plot showing increasing SFRP4 expression (mRNA) levels at more advanced T-stages is represented in Figure 1C (p=0.002754; Kruskal-Wallis test). Due to the low number of T4 samples available, these samples were grouped with the T3 samples for the purpose of this analysis.

[0172] In order to further validate this finding, U133 plus 2 array expression data was obtained from another set of 178 gastric cancer samples originating from Singapore. T- stage information was available for 152 of these cases. SFRP4 expression (mRNA) data for these 152 samples are represented in Figure ID and also demonstrate increasing SFRP4 expression with more advanced T-stage (Kruskal-Wallis test; p= 0.0095).

Table 3: Top 60 genes similar to SFRP4

Common Genbank Product

SFRP4 AW089415

TFF3 NM_003226 trefoil factor 3 (intestinal)

v-kit Hardy-Zuckerman 4 feline sarcoma viral

KIT NM_000222 oncogene homolog precursor

IGFBP3 M31159 insulin-like growth factor binding protein 3

SYPL AI768845

EFEMP1 AI826799

ELA3A D00306 elastase 3, pancreatic (protease E)

decorin isoform a preproprotein; decorin isoform b precursor; decorin isoform c precursor; decorin isoform

DCN AF138300 d precursor; decorin isoform e precursor

GAS1 A W611727

SPARCL1 NM_004684 SPARC-like 1

FLNC NM_001458 gamma filamin

RAB23 NM_016277 Ras-related protein Rab-23

SFRP41 AI332407

THBS2 NM_003247 thrombospondin 2 precursor

LUM NM_002345 lumican

heat shock 27kDa protein family, member 7

HSPB7 NM_014424 (cardiovascular)

COMP NM_000095 cartilage oligomeric matrix protein precursor

ADH1C NM_000669 class I alcohol dehydrogenase, gamma subunit

S100A3 NM_002960 SI 00 calcium binding protein A3

SULF1 AW043713

ITGBL1 AI753143

FRZB U91903 frizzled-related protein

complement component 1 , q subcomponent, beta

C1QB NM_000491 polypeptide precursor

DACT1 NM_016651 DAPPER 1

ORM2 NM_000607 orosomucoid 1 precursor

SPON1 AB018305 spondin 1 , extracellular matrix protein

FAP U76833 fibroblast activation protein, alpha subunit

OGN NM_014057 osteoglycin preproprotein

FBLN5 NM_006329 fibulin 5 precursor

PLN M60411 phospholamban

AXL AI467916

BPHL NM_004332 biphenyl hydrolase-like

POLR2B BE614461

IGJ AV733266

CPZ BC006393 carboxypeptidase Z precursor

FMOD NM_002023 fibromodulin precursor

RAI17 AF070622 retinoic acid induced 17

DEFB1 U73945 defensin, beta 1, preproprotein Common Genbank Product

F2R BG026194

CPB1 NM_001871 pancreatic carboxypeptidase B 1 precursor

IGF1 AU144912

serine (or cysteine) proteinase inhibitor, clade A (alpha-

SERPINA1 NM_000295 1 antiproteinase, antitrypsin), member 1

TPM2 NM_003289 tropomyosin 2 (beta)

LIPF NM_004190 lipase, gastric

FGL2 NM_006682 fibrinogen-like 2

FBN1 AI264196

GYPC NM_002101 glycophorin C isoform 1 ; glycophorin C isoform 2

SSPN AL136756 sarcospan

ADH4 M15943 class II alcohol dehydrogenase 4 pi subunit

CYP1B 1 N21019

ELN AA479278

SPUVE NM_007173 protease, serine, 23 precursor

GARP NM_005512 glycoprotein A repetitions predominant precursor

TYRO protein tyrosine kinase binding protein isoform 1 precursor; TYRO protein tyrosine kinase binding

TYROBP NM_003332 protein isoform 2 precursor

LGALS1 NM_002305 beta-galactosidase binding lectin precursor

F13A1 NM_000129 coagulation factor XIII Al subunit precursor

TPM1 CA432736

DPYSL3 W72516

DPT NM_001937 dermatopontin precursor

Example 3 - SFRP4 expression increases with T-stage and cancer type

[0173] To assess the validity of this result at the protein level, immunohistochemistry was performed using several tissue microarrays (TMA) that included many of the cases in the original cohort. There was good concordance between the immunohistochemistry staining by anti-SFRP4 antibody and T-stage. The staining was assessed semi-quantitatively on a scale of 0 to 3+. Increasing SFRP4 protein expression was observed in T3 compared with Tl and T2 lesions (see Figure IE). SFRP4 shows overexpression in the majority of gastric cancers and is absent in normal gastric tissue (see Figure IF).

[0174] To examine any gastric cancer subtype- specific expression, the TMA analysis was stratified by gastric cancer subtype and the results showed higher proportion of 2+ and 3+ staining in diffuse gastric cancer. Staining was relatively weak, however, and this may be attributed to the secreted nature of SFRP4. The intestinal gastric cancer (IGC) samples that over-expressed SFRP4 showed a staining pattern consistent with a membranous/secreted protein (see Figure IF). This was more difficult to determine in the diffuse gastric cancer samples.

[0175] The Affymetrix U133 plus 2 array data were also stratified according to tissue type. As shown in Figure 2A, histologically normal gastric tissues (NN) exhibited significantly lower SFRP4 expression than other benign tissues (chronic gastritis (CG; p=0.001)) and intestinal metaplasia (IM; p=0.002). Highest levels of SFRP4 expression were observed in gastric tumour samples which are divided here into the 2 histological subtypes - Intestinal (p=0.001) and Diffuse gastric cancer (p=0.002). Laser capture microdissection (LCM) was also performed, the results showing that SFRP4 mRNA was predominantly expressed in adjacent tumour stroma and epithelium (see Figure 2B).

Example 4 - T-stage independently predicts progression free survival

[0176] In order to determine whether depth of invasion (T-stage) independently influenced the clinical outcome, the clinical data for the same samples used in the validation stage above were analysed. Kaplan-Meier plots depicting progression free survival (i.e. period of survival between resection of gastric cancer and subsequent recurrence) versus T-stage were generated (see Figures 3A and 3B). These data show that T-stage is able to predict progression free survival in both the Peter MacCallum Cancer Centre (Figure 3A; p=0.0047) and Singapore (Figure 3B; p=0.0558; logrank test) data sets, suggesting that gastric resection of T3- and T4-stage cancers confers a higher likelihood of recurrence than Tl- and T2-stage cancers.

Example 5 - Elevated SFRP4 expression predicts poor prognosis

[0177] Array-derived data were then used to determine whether SFRP4 expression levels can reliably predict prognosis. The Barcode method of analysis, which uses binary representation, was used to classify samples as having high or low SFRP4 expression. Clinical data from these samples were then used to generate a Kaplan Meier plot with progression free survival used as an endpoint. For the Singapore data set, all available arrayed samples were used for this analysis (n=178) since information regarding T-stage did not affect the expression of SFRP4. The results indicate that high SFRP4 expression levels (mRNA) were correlated with poor prognosis whilst patients harbouring tumours with low SFRP4 expression levels had a significantly lower risk of recurrence (p=0.01; Peter MacCallum Cancer Centre data set; Figure 3C) and p=0.04 (Singapore data set; Figure 3D; logrank test). Using a multivariate Cox proportional standards model, SFRP4 expression is independent of T-stage as a predictor of outcome.

Example 6 - Elevated SFRP4 expression predicts recurrence

[0178] Prospectively collected samples from control (non-cancer) and gastric cancer (GC) patients were used to determine whether SFRP4 can be used to diagnose patients with GC. A commercial ELISA kit was used to determine SFRP4 protein levels in serum and plasma samples from these individuals. Figure 4A shows that average SFRP4 protein levels are similar between both groups and, as such, a baseline SFRP4 protein level in blood is not a suitable diagnostic marker for GC. To determine whether SFRP4 protein levels are useful in identifying those patients at risk of developing recurrent disease, 11 patients with recurrent disease were identified and SFRP4 protein levels measured in plasma taken pre- operatively (baseline levels when SFRP4 levels are expected to be at their highest), postoperatively (first blood drawn post surgery), pre -recurrence (last blood drawn before clinical diagnosis of recurrence) and post recurrence (first blood drawn after clinical diagnosis of recurrence). As controls, 13 patients were identified who had undergone curative gastric resection and had no documented recurrence for at least 36 months postoperatively. Clinical characteristics of these patients are listed in Table 4.

Table 4: Clinical characteristics

Parameter Recurrence Non-recurrence

Age (years) 60.7 (33-83) 67.1 (55-78)

Gender

Male 8 10

Female 3 3

Tumour location Antrum 2 3

GOJ 4 1

Greater curve-body 3 2

Lesser curve-body 1 4

Cardia 1 3

H. pylori status

Positive 6 6

Negative 4 4

Unknown 1 3

Chemo-radiotherapy

Neo-Adjuvant

No Adjuvant 9 13

Adjuvant 2 0

Adjuvant

No Adjuvant 4 7

Adjuvant 7 6

Palliative

No Palliative 3 N/A

Palliative 8 N/A

Pathology

Diffuse 4 3

Intestinal 6 8

Mixed 1 0

Adenocarcinoma 0 1

Adenosquamous 0 1

Differentiation

Undifferentiated 1 2

Poor 6 6

Moderate 4 4

Well 0 1

T Stage

Tl 0 2

T2 3 4

T3 8 7

T4 0 0

AJCC Stage

IA 0 2

IB 1 2

II 3 5

IIIA 5 2

IIIB 1 2

IV 1 0

Surgery type

Proximal gastrectomy 3 1

Distal gastrectomy 4 6

Total gastrectomy 1 5 Oesophagogastrectomy 3 1

Margins

R0 11 12

Rl 0 1

Recurrence type

Local 1 0

Distant 5 0

Both 5 0

None 0 13

Time till recurrence 769(178-1304) N/A

[0179] Plasma SFRP4 protein levels were determined for all samples using a commercial ELISA-based kit (SEF878Hu- Cloud-Clone Corp. or USCN Life Science Inc, Cat#SEF878Hu). Each pre-operative blood sample was used as a baseline and SFRP4 protein levels for all subsequent blood samples were normalised to this sample. This analysis was performed individually for each patient and average values for all patients are plotted in Figure 4B. The data shown here indicate that SFRP4 levels remain constant for patients with no evidence of recurrence (n=13), whilst there is a significant increase in SFRP4 levels in patients who ultimately recur (n=l l), which was detectable as early as 7 days post-surgery. These levels continued to increase until clinical diagnosis of recurrence.

[0180] To validate potential utility of this test in a clinical setting, a 1.15 -fold change cutoff was set, which distinguished those patients who will recur from those who will not (see Figure 4B). SFRP4 levels in plasma were then measured from pre-operative and first postoperative blood samples from a further 13 recurrence and 11 non-recurrence patients. All samples with a fold-change equal to or above 1.15 were predicted to develop a recurrence and all those below this cut off were predicted to remain disease free. Based on these criteria, recurrence was correctly identified with a sensitivity of 76.92% and a specificity of

54.55%.

[0181] As also shown in Figure 4C, patients with recurrence (n=l l) had elevated circulating SFRP4 levels at the first postoperative blood test (range of time to test 1-3 months), as compared to patients who showed no evidence of recurrence as far as 36 months post-resection. SFRP4 levels remained elevated above baseline prior to clinical recurrence (pre -recurrence time range 6-24 months post-op). These data show that SFRP4 can be used as a predictor of recurrence as early as the first post-operative blood sample at 1 month post-op (p=0.003).

[0182] Logistic regression was also performed to determine if SFRP4 expression is predictive of recurrence, independent of stage. The results showed that SFRP4 had an Odds Ratio of 5.4 (p value = 0.02), which was independent of stage. This corresponds to a positive predictive probability for SFRP4 of 84%.

[0183] To assess the prediction accuracy based on SFRP4 alone and in addition to existing measures of recurrence risk, an ROC analysis was performed (Figure 5). This analysis used samples from 69 gastric cancer patients. Only the first post-operative blood sample was used to assess SFRP4 levels. As shown in Figures 5A and 5B, SFRP4 (PredictR) was able to predict recurrence, with a predictive accuracy (area under the curve; AUC) of 76% when used independently, with a 95% confidence interval of 65-88%. When SFRP4 levels were combined with AJCC stage, the AUC was 85%, with a 95% confidence interval of 76-94%. Thus, the combination of SFRP4 levels and AJCC stage was the most accurate predictor of recurrence. N stage had an accuracy of about 70% alone, but is limited due to issues of non- standardised nodal resections, making it less reliable when used alone.

[0184] When compared to similar biomarkers (CEA and CA19-9) that are commercially available and variably used in the diagnosis of gastric cancer, SFRP4 is the only marker that is elevated long before clinical recurrence. Figure 6 shows a comparison of CEA, CA19-9 and SFRP4 levels in plasma of two gastric cancer patients. The results show that SFRP4 is the only marker that rises before clinically detectable recurrence.

References

1. Parkin DM. International variation. Oncogene 2004;23:6329-40.

2. Hundahl SA, Phillips JL, Menck HR. The National Cancer Data Base Report on poor survival of U.S. gastric carcinoma patients treated with gastrectomy: Fifth Edition American Joint Committee on Cancer staging, proximal disease, and the "different disease" hypothesis. Cancer 2000;88:921-32.

3. Miki K, Fujishiro M, Kodashima S, Yahagi N. Long-term results of gastric cancer screening using the serum pepsinogen test method among an asymptomatic middle-aged Japanese population. Dig Endosc 2009;21:78-81.

4. Rosero-Bixby L, Sierra R. X-ray screening seems to reduce gastric cancer mortality by half in a community-controlled trial in Costa Rica. Br J Cancer 2007;97:837-43.

5. Monig S, Baldus SE, Collet PH, et al. Histological grading in gastric cancer by Goseki classification: correlation with histopathological subtypes and prognosis. Anticancer Res 2001;21:617-20.

Claims

1. A method of determining whether a subject has, or is at risk of developing, gastric cancer, the method comprising:

(a) measuring the expression of secreted frizzled related protein 4 (SFRP4) in a biological sample obtained from a subject; and

2. A method of stratifying a subject to a therapeutic regimen for treating or preventing gastric cancer, the method comprising the steps of:

(c) wherein said comparison provides an indication as to whether or not the subject has, or is at risk of developing, gastric cancer; and

3. The method of Claim 1 or Claim 2, wherein the reference value is representative of a level of expression of SFRP4 in a healthy subject, a subject that is otherwise free of gastric cancer, or a subject that is not considered at risk of developing gastric cancer, wherein a level of expression of SFRP4 in the biological sample that is greater than the reference value is indicative of the subject having, or being at risk of developing, gastric cancer.

4. The method of any one of Claims 1 to 3, wherein step (a) comprises measuring the expression of a gene that encodes SFRP4.

5. The method of any one of Claims 1 to 3, wherein step (a) comprises measuring the expression of SFRP4 protein.

6. The method of any one of Claims 1 to 5, wherein the biological sample is obtained from the subject after removal of a gastric tumour by resection and wherein said comparison provides an indication as to whether or not the subject is at risk of recurrence.

7. The method of any one of Claims 1 to 6, wherein the biological sample comprises a gastric cancer cell.

8. The method of any one of Claims 1 to 7, wherein the biological sample is a postoperative blood sample taken after resection of a gastric tumour.

9. The method of Claim 8, wherein the reference value is representative of a level of expression of SFRP4 in blood of a population of healthy subjects or representative of a level of expression of SFRP4 in blood of a population of patients with gastric cancer taken prior to resection, wherein a level of expression of SFRP4 in the post-operative blood sample that is greater than the reference value is indicative of a risk of recurrence.

10. The method of Claim 8 or Claim 9, wherein the reference value is a level of expression of SFRP4 in a pre-operative blood sample obtained from the same subject prior to resection of the gastric tumour.

11. The method of Claim 10, comprising the step of obtaining a pre-operative blood sample from the subject before resection and comparing the level of expression of the SFRP4 in the post-operative blood sample with the level of expression of the SFRP4 in the preoperative blood sample, wherein a level of expression of the SFRP4 in the postoperative blood sample that is greater than the level of expression in the pre-operative blood sample is indicative of a risk of recurrence.

12. The method of any one of Claims 8 to 11, wherein the subject is identified as being at risk of recurrence where the level of expression of SFRP4 in the post-operative blood sample from the subject is at least about 1.15-times greater than the level of expression of SFRP4 in the reference sample.

13. The method of any one of Claims 8 to 12, wherein the post-operative blood sample is obtained from the subject from about 1 week to about 2 years following resection.

14. The method of Claim 13, wherein the post-operative blood sample is obtained from the subject from about 2 week to about 3 months following resection.

15. The method of Claim 13, wherein the post-operative blood sample is obtained from the subject at about 1 month following resection.

16. The method of any one of Claims 8 to 15, wherein measuring the expression of SFRP4 in the biological sample comprises measuring the level of SFRP4 protein in the biological sample.

17. The method of any one of Claims 1 to 7, comprising the step of exposing the subject identified as having, or being at risk of developing, gastric cancer, to a therapeutic regimen for treating or preventing gastric cancer.

18. The method of any one of Claims 8 to 16, comprising the step of exposing the subject identified as being at risk of recurrence to a therapeutic regimen for treating or preventing gastric cancer.

19. The method of Claim 17 or Claim 18, wherein the therapeutic regimen comprises radiotherapy and/or the administration of a chemo therapeutic agent.

20. A protocol for monitoring the efficacy of a therapeutic regimen for treating or preventing gastric cancer, the protocol comprising:

(d) comparing the level of expression of SFRP4 in the first biological sample with the level of expression of SFRP4 in the second biological sample; wherein a change in the level of expression of SFRP4 between the first and second biological samples is indicative of whether the therapeutic regimen is effective or not.

21. The protocol of Claim 20, wherein a reduction in the level of expression of SFRP4 in the second biological sample as compared to the level of expression of SFRP4 in the first biological sample is indicative of an effective therapeutic regimen.

22. The protocol of Claim 20 or Claim 21, wherein the biological sample comprises gastric cancer tissue.

23. The protocol of any one of Claims 20 to 22, wherein step (c) comprises measuring the level of expression of a gene that encodes the SFRP4.

24. The protocol of any one of Claims 20 to 22, wherein step (c) comprises measuring the level of SFRP4 protein.

25. The protocol of any one of Claims 20 to 24, wherein the first and/or second biological sample is obtained from the subject after removal of a gastric tumour by resection.

26. The protocol of any one of Claims 20 to 25, wherein the first and/or second biological sample is a blood sample.

27. The protocol of any one of Claims 20 to 26, wherein where a change in the level of expression of SFRP4 between the first and second biological samples indicates that the therapeutic regimen is not effective in a subject, exposing the subject to a more aggressive therapeutic regimen for treating or preventing gastric cancer.

28. A kit comprising one or more reagents and/or devices for use in performing the method of any one of Claims 1 to 19 or the protocol of any one of Claims 20 to 27.

29. The method of any one of Claims 1 to 19 or the protocol of any one of Claims 20 to 27, wherein measuring the expression of SFRP4 in the sample comprises (i) exposing the sample to an SFRP4 binding agent under conditions that allow the SFRP4 binding agent to specifically bind to, and form a complex with, SFRP4 in the sample, and (ii) measuring the amount of complex formed.