GB2519830A - Methods for monitoring treatment response and relapse in ovarian cancer - Google Patents

Abstract

The invention relates to a method for monitoring the response to treatment in a patient undergoing ovarian cancer therapy comprising detecting the presence of a loss-of-function-related genetic alteration in the Pregnancy-Associated Plasma Protein A (PAPPA) gene or its regulatory or promoter sequences in a sample obtained from the patient, wherein the presence of a genetic alteration and downward change in the quantitative levels of the genetic alteration is indicative of response to therapy, and wherein no change or upward change in the quantitative levels of the genetic alteration is indicative of non-response to therapy. Also claimed are associated methods for assessing the occurrence of a relapse in patients previously treated with ovarian cancer therapy and methods for determining the likelihood of primary ovarian cancer spreading to another part of the body. Also claimed are associated methods detecting the presence and/or level of PAPPA expression or using the proportion of mitotic cells in metaphase or pro-metaphase in samples as a prognostic marker for ovarian cancer.

Description

METHODS FOR MONITORING TREATMENT RESPONSE AND RELAPSE

IN OVARIAN CANCER

Field of the Invention

This invention relates to the use of specific biological markers for staging ovarian cancer and for monitoring response to treatment, and detecting relapse after treatment, for ovarian cancer.

Background of the Invention

Neoplasms and cancer are abnormal growths of cells. Cancer cells rapidly reproduce despite restriction of space, nutrients shared by other cells, or signals sent from the body to stop reproduction. Cancer cells are often shaped differently from healthy cells, do not function properly, and can spread into many areas of the body. Abnormal growths of tissue, called tumours, are clusters of cells that are capable of growing and dividing uncontrollably. Tumours can be benign (noncancerous) or malignant (cancerous). Benign tumours tend to grow slowly and do not spread. Malignant tumours can grow rapidly, invade and destroy nearby normal tissues, and spread throughout the body. Malignant cancers can be both locally invasive and metastatic.

Ovarian cancer is a common form of gynecological cancer arising from the ovary or fallopian tube. More than 90% of ovarian cancers are classified as "epithelial ovarian cancer", arising from the surface (epithelium) of the ovary.

The risk of developing ovarian cancer appears to be affected by several factors, including age, genetics (including mutations in BRCA I and BRCA 2), conditions such as infertility and endomettiosis and use of post-menopausal estrogen replacement therapy.

Ovarian cancers readily metastasize by shedding cells into the naturally-occurring fluid within the peritoneal cavity. These cells can then implant on other abdominal structures including the uterus, urinary bladder, bowel and the lining of the bowel wall, forming new tumour growths and obstructions before cancer is even suspected.

Diagnosis of ovarian cancer usually involves physical examination, blood tests and trans-vaginal ultrasound. Diagnosis is confirmed with surgery to obtain biopsies. Ovarian cancer can be classified according to the International Federation of Obstetricians and Gynecologists (FIGO) Staging System. Early stage ovarian cancer (I/Il) is difficult to diagnose because the symptoms can be subtle and non-specific, therefore it is often not diagnosed until it spreads and advances to later stages (lll/IV).

Treatment for ovarian cancer can vary depending on the stage of progression of the cancer. Treatment usually involves surgery, chemotherapy with anti-proliferative drugs, such as taxanes or cisplatin, and sometimes radiotherapy, although radiation therapy is not effective for advanced stages.

However, the efficacy of chemotherapy can be reduced due to resistance or desensitization to chemotherapeutic drugs.

Ovarian cancer usually has a poor prognosis. The mortality rates are high because of a lack of any clear early detection or screening test, meaning that most cases are not diagnosed until they have reached advanced stages. More than 60% of women presenting with ovarian cancer have stage Ill or stage IV cancer, which has already spread beyond the ovaries. The five-year survival rate for all stages of ovarian cancer is 47%.

Pregnancy Associated Plasma Protein A (PAPPA) was identified in 1974 as one of four proteins of placental origin circulating at high concentrations in pregnant women, and later found clinical utility as a biomarker for Down's syndrome pregnancies. Its biological function remained an enigma for a quarter of a century until it was identified as a protease that regulates IGF bioavailability through cleavage of the inhibitory insulin-like growth factor binding protein-4 (IGFBP-4). Its role as a IGFBP-4 protease in a diverse range of cell types (e.g. fibroblasts, osteoblasts and vascular smooth muscle cells), together with a highly conserved amino acid sequence in vertebrates, indicated that PAPPA serves a basic function beyond placental physiology.

The localisation of the PAPPA gene on chromosome 9 has been shown by Callahan eta!. (Oncogene (2003) 22, p.590-601) as a region associated with loss of heterozygosity in ovarian cancer. This study showed that PAPPA loss was present in greater than 70% of the tumour samples tested.

US2005/0272034 (Conover et al) discloses that PAPPA activity is decreased in malignant ovarian cells due to a corresponding increase in expression of the precursor form of eosinophil major basic protein (proMBP), which circulates in complex with PAPPA and inhibits PAPPA carrying out is proteolytic function (cleavage of IGFBP-4). In contrast to this, another publication by the same group (Boldt and Conover; Endocrinology; (2011) 152 (4) pg1470- 1478) describes over-expression of PAPPA in ovarian cancer cells as a promoter of tumour growth in vivo. Therefore, the mechanisms by which PAPPA activity impacts upon tumour growth are not clearly understood in the art.

There is a need for improvements in methods for monitoring the effectiveness of treatment, monitoring for relapse and staging ovarian cancer.

Summary of the Invention

The present invention is based on the surprising finding that Pregnancy Associated Plasma Protein A (PAPPA) is required for normal progression through mitosis, and that PAPPA silencing is highly prevalent in invasive ovarian cancer and pre-invasive borderline tumours (termed atypia') predisposed to becoming invasive. The present invention provides an important understanding of the biological causes of ovarian cancer, and allows of ovarian cancer patients to be monitored in a more effective way.

The understanding that PAPPA is required for normal progression through mitosis, and that the loss of its expression or impaired functioning contributes significantly to the cancerous state, allows physicians to monitor patients' response to ovarian cancer therapies, and to detect relapse after treatment. PAPPA is also a useful biomarker for staging ovarian cancer, by detecting spread away from the site of the primary tumour to other parts of the body.

According to the first aspect of the invention, there is a method for monitoring the response to treatment in a patient undergoing ovarian cancer therapy, comprising detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences in a sample obtained from the patient, wherein the presence of a genetic alteration and downward change in its quantitative levels is indicative of response to therapy, and wherein no change or upward change in its quantitative level is indicative of non-response to therapy.

According to the second aspect of the invention, there is a method for monitoring the response to treatment in a patient undergoing ovarian cancer therapy, comprising detecting the presence and/or level of PAPPA in a sample obtained from the patient, wherein the absence of PAPPA, or the presence of PAPPA at a reduced level compared to a control is indicative of non-response to therapy.

According to the third aspect of the invention, there is a method for determining whether a patient, previously treated with ovarian cancer therapy, has suffered a relapse, comprising detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences in a biological sample obtained from the patient, wherein if a genetic alteration is identified, this is indicative of a relapse.

According to the fourth aspect of the invention, there is a method for determining whether a patient previously treated with ovarian cancer therapy, has suffered a relapse, comprising detecting the presence and/or level of PAPPA in a biological sample obtained from the patient, wherein the absence of PAPPA, or the presence of PAPPA at a reduced level compared to a control, is indicative of a relapse.

According to the fifth aspect of the invention, there is a method for determining whether a patient previously treated with ovarian cancer therapy has suffered a relapse, comprising identifying the proportion of mitotic cells in a tissue sample obtained from the patient that are in prophase or pro-metaphase and comparing to a pre-determined cut-off value, wherein if the proportion of cells in prophase or pro-metaphase is the same or greater than the cut-off value, this is indicative that the patient has suffered a relapse.

According to the sixth aspect of the invention, there is a method for determining whether a primary ovarian cancer in a patient has spread away from the site of the primary tumour to another part of the body, comprising detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences in a sample obtained from the patient, wherein the presence of a genetic alteration is indicative of spread.

According to the seventh aspect of the invention, there is a method for determining whether a primary ovarian cancer in a patient has spread away from the site of the primary tumour to another part of the body, comprising identifying the proportion of mitotic cells in a tissue sample obtained from the patient that are in prophase or pro-metaphase and comparing to a pre-determined cut-off value, wherein if the proportion of cells in a prophase or pro-metaphase is the same or greater than the cut-off value there is a risk that the cancer has spread, and wherein the tissue sample is from a site adjacent to or distant from that of the primary tumour.

According to the eighth aspect of the invention, there is a method for determining whether a primary ovarian cancer in a patient has spread away from the site of the primary tumour to another part of the body, comprising detecting the presence and/or level of PAPPA in a sample obtained from the patient, wherein the absence of PAPPA, or the presence of PAPPA at a reduced level compared to a control, is indicative of spread.

According to the ninth aspect of the invention, there is a method for predicting disease progression in a patient with invasive ovarian cancer, comprising identifying the proportion of mitotic cells in a tissue sample obtained from the patient that are in prophase or prometaphase and comparing to a pre-determined cut-off value, wherein if the proportion of cells in prophase or prometaphase is the same or greater than the cut-off, the prediction is reduced disease-free and overall survival.

According to the tenth aspect of the invention, there is a method for predicting disease progression in a patient with invasive ovarian cancer, comprising detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences in a sample obtained from the patient, wherein the presence of a genetic alteration is predictive of reduced disease-free and overall survival.

According to the eleventh aspect of the invention, there is a method for predicting disease progression in a patient with invasive ovarian cancer, comprising detecting the presence and/or level of PAPPA in a biological sample obtained from the patient, wherein the absence of PAPPA, or its presence at a reduced level compared to a control, predicts reduced disease-free and overall survival.

Description of the Figures

Figure 1 shows a multi-step progression model of ovarian tumourigenesis; Figure 2 shows mitotic phase distribution in human cancers. (A) shows representative images identifying distinct mitotic phases by phosphohistone H3 (H3SlOph) immunostaining in tissue sections of surgical biopsy specimens (1000x original magnification; scale bar 10 pm). (B) Pie charts showing the percentage of mitotic cells assigned to each mitotic phase in borderline ovarian cancer (n=21 patients), invasive ovarian cancer (n=154 patients), lung cancer (n=43 patients), bladder cancer (n=40 patients), colon cancer (n=59 patients) and lymphoma (n=48 patients). (C) shows photomicrographs of a representative case of ovarian cancer (400x magnification; scale bar 50 pm) which shows a high frequency of early mitotic figures (indicated by arrows) compared to lymphoma and bladder cancer (I000x magnification; scale bar 20 pm) as examples of the group of other cancers showing normal mitotic progression; Figure 3 is a receiver operating characteristic (ROC) curve for the prophase/prometaphase fraction, applying a minimum mitotic cell count of n=5.

Data shown are area under the curve (AUC). * indicates p < 0.0001 compared to a null diagnostics test; Figure 4 shows the distribution of the prophase/prometaphase fraction in borderline ovarian cancer, invasive ovarian cancer and other cancers (pooled). * indicates p c 0.0001 compared to other cancers (Mann-Whitney test); Figure 5 shows enrichment of early mitotic figures in ovarian cancer. Box plot shows the percentage of mitotic cells in prophase/prometaphase in a range of human cancers. Invasive ovarian carcinoma is characterised by a higher proportion of mitotic cells in prophase/prometaphase compared to other tumour types. Data are presented as the median (solid black line), interquartile range (boxed) and range (enclosed by dashed lines). Outlying cases are depicted as isolated points. * indicates p < 0.0001 compared to all other invasive samples (Pearson's test with continuity correction); Figure 6 shows that acquisition of the mitotic delay phenotype occurs early in multi-step ovarian tumour progression. (A) Bar chart showing the percentage of cases of other cancers (pooled; n=282), borderline ovarian carcinomas (n=21) and invasive ovarian carcinomas (n=154) exhibiting mitotic delay (p < 0.0001; Pearson's chi-square test). Cases are defined as delayed if the proportion of mitotic cells in prophase/prometaphase is at least one third.

This cut-point was chosen to allow the proportion of specimens in the combined group of other malignancies properly classified as non-delayed (96%) to be approximately equal to the proportion of ovarian cancer specimens properly classified as delayed (87%). Normal ovarian tissue samples did not contain the minimum number of mitotic cells required to make a statistically significant call for mitotic delay (n=5) and were therefore not included in this analysis. (B) Bar chart showing the percentage of mitotic cells in prophase/prometaphase in other cancers, borderline ovarian carcinomas and invasive ovarian carcinomas. (C) shows photomicrographs of representative cases of non-invasive borderline ovarian carcinoma immunostained for H3SlOph showing normal mitotic phase distribution (left panel) and a high proportion of niitotic cells in prophase/prometaphase (right panel) (400x magnification; scale bar 50 pm); Figure 7 shows that PAPPA is epigenetically silenced by promoter methylation in ovarian cancer. (A) shows a bar chart of the percentage of normal ovarian tissue (n=15); benign ovarian lesions (n=24), borderline ovarian lesions (n=30) and invasive ovarian carcinomas (n=174) with PAPPA promoter methylation as determined by MethyLight assay. The proportion of methylated cases was different between the sample types at p = 0.0029 (Pearson's chi-square test). (B) shows a bar chart of the percentage of cases expressing PAPPA protein as determined by immunohistochemistry (IHC). Positive PAPPA expression was defined as a score of 2 or more based on intensity and distribution. The proportion of cases expressing PAPPA was different between the sources at p c 0.0001 (Pearson's chi-square test). (C) shows photomicrographs of representative cases of borderline and invasive ovarian carcinomas with PAPPA expression in non-methylated cases and a lack of PAPPA expression in methylated cases (630x magnification; scale bar 20 pm); Figure 8 shows that PAPPA expression is inversely correlated with increasing tumour stage (p = 0.036; logistic regression with Wald approximation). Bar chart of percentage of stage I (n=51), stage 2 (n=22) and stage 3 (n=59) ovarian carcinomas expressing PAPPA protein as determined by immunohistochemistry. Positive PAPPA expression was defined as a score of 2 or more based on intensity and distribution; Figure 9 shows cell growth characteristics and PAPPA expression in ovarian cancer cell lines. (A) Representative phase-contrast image of Caov-3 cells (200x magnification). The population doubling time for this cell line is 50 hours which was calculated using a Countess1M automated cell counter. (B) Flow cytometry profile of untreated Caov-3 cells following P1 staining. Data were analysed using Multicycle AV software and is a summary of results obtained from three independent experiments. (C) Representative phase-contrast image of Ovcar-3 cells (200x magnification). The population doubling time for this cell line is 70 hours which was calculated using a CountessTM automated cell counter. (D) Flow cytometry profile of untreated Ovcar-3 cells following P1 staining. Data shown are representative of n=3. (F) MethyLight assays were performed on genomic DNA samples isolated from Caov-3 and Ovcar-3 cells.

PMR (percentage methylated reference gene) was obtained by dividing the PAPPA:COL2A1 ratio of a sample by the PAPPA:COL2A1 ratio of CpG methylated HeLa genomic DNA (control) and multiplied by 100. Data presented are the average ± standard error of mean (SEM) for n=3. * indicates p c 0.05 compared to Caov-3 (Student's unpaired t-test). (F) qRT-PCR was used to measure the relative levels of PAPPA mRNA in Caov-3 and Ovcar-3 cells.

PAPPA expression was normalised to the endogenous control GAPDH. Data shown are normalised to relative mRNA expression (RO) of Caov-3 ± RQ minimum and RQ maximum and are representative of n=3. * indicates p c 0.05 compared to Caov-3 (Student's unpaired t-test). (G) Western blot image of Caov-3 and Ovcar-3 crude cellular fractions probed with rabbit polyclonal anti-PAPPA antibody. -actin loading controls are shown in the lower panel. (H) The percentage of mitotic cells in prophase/prometaphase in Caov-3 and Ovcar-3 cells assessed by H3SlOph immunostaining. Data presented are the average ± SEM for n=3. * indicates p c 0.05 compared to Caov-3 (Student's unpaired t-test); Figure 10 shows that PAPPA knockdown in Caov-3 cells increases the number of cells accumulating in early mitosis and renders Caov-3 cells more invasive. (A) qRT-PCR analysis of Caov-3 cells transfected with negative control 51RNA or PAPPA silencer validated 5IRNA. Data were normalised to the endogenous control GAPDH for determination of relative mRNA expression (RO) ± RO minimum and RO maximum, and are representative of results obtained from two independent experiments. * indicates p c 0.05 compared to negative control 5iRNA (Student's unpaired t-test). (B) Western blot image of Caov-3 cells transfected with negative control siRNA or PAPPA siRNA. The membrane was probed with rabbit polyclonal anti-PAPPA antibody. -actin loading controls are shown in the lower panel. (C) Representative images (200x magnification) of Caov-3 cells transfected with negative control 5iRNA or PAPPA 5iRNA, immunostained with H3SlOph antibody and assigned to distinct mitotic phases as indicated by key. (D) The percentage of mitotic cells in prophase/prometaphase in negative control 5iRNA or PAPPA 5iRNA transfected Caov-3 cells. Data presented are the average ± SEM, of n=5. * indicates p < 0.05 compared to negative control siRNA (Student's unpaired t-test). (F) Number of invading Caov-3 cells as determined from stained membrane insert of a Boyden chamber treated with negative control 5iRNA compared to PAPPA 5iRNA transfected cells. Data shown are the average ± SEM for n=3. * indicates p c 0.05 compared to negative control 5iRNA (Student's unpaired t-test); Figure 11 shows that PAPPA overexpression in Ovcar-3 cells decreases the number of cells accumulating in early mitosis. (A) qRT-PCR analysis of Ovcar-3 cells transfected with negative control (empty plasmid) or PAPPA expressing plasmid. Data were normalised to the endogenous control GAPDH.

Data shown are normalised to relative mRNA expression (RO) of negative control ± RQ minimum and RQ maximum and are representative of n=3. * indicates p c 0.05 compared to negative control (Student's unpaired t-test). (B) Western blot image of Ovcar-3 cells transfected with negative control or PAPPA expressing plasmid. The membrane was probed with rabbit polyclonal anti-PAPPA antibody. -actin loading controls are shown in the lower panel. (C) Representative images (200x magnification) of Ovcar-3 cells transfected with negative control or PAPPA expressing plasmid, immunostained with H3SlOph antibody, and assigned to distinct mitotic phases as indicated by key. (D) The percentage of total mitotic cells in prophase/prometaphase in negative control or PAPPA cDNA transfected Ovcar-3 cells. Data shown are the average ± SEM for n=4. * indicates p < 0.05 compared to negative control (Student's unpaired t-test); Figure 12 shows that the PAPPA promoter is methylated in circulating tumour DNA isolated from ovarian cancer patients, whereas no methylation is detectable in healthy controls. (A) shows a representative amplification plot of a MethyLight assay for methylated PAPPA promoter using DNA purified from plasma of ovarian cancer patients (n=3) compared to a healthy control. The relative fluorescence (Rn) of one representative sample well is plotted against the number of POP amplification cycles. (B) shows a MethyLight assay amplification of CoI2A1 DNA that was used as a reference to estimate the DNA amount isolated from each sample; Figure 13 shows the percentage of ovarian cancer cases (n=5) exhibiting PAPPA promoter hypermethylation in circulating tumour DNA isolated from ovarian cancer patients compared to normal controls. Positive PAPPA methylation was defined as a PMR >50. AMP was obtained by dividing the PAPPA:COL2AI ratio of a sample by the PAPPA:COL2AI ratio of CpG methylated HeLa genomic control DNA and multiplied by 100; and Figure 14 shows the distribution of the detectable amount of PAPPA promoter methylation between ovarian cancer samples. The distribution of the Ct values of the the MethyLight assay against the methylated PAPPA promoter are shown, comparing control plasma samples (n=3, solid bars) and all tested ovarian-cancer related samples isolated from full blood (n=5, unfilled bars). The x-axis denotes the minimum of the Ct value range, while the y-axis denotes the frequency of samples in the range. In cases with no detectable amplification, the Ct is displayed as N/A.

Detailed Description of the Invention

US2005/0272034 discloses proMBP as a marker for ovarian neoplasia and describes how increased proMBP expression correlates with decreased PAPPA activity in malignant ovarian cells. Therefore the authors of US200510272034 propose reduced proteolytic activity of secreted PAPPA as a marker of ovarian neoplasia. This observation of decreased proteolytic activity of PAPPA in malignant ovarian cells is due to the pro-MBP regulatory loop, i.e. increased expression of pro-MBP results in increased formation of the pro-MBP/PAPPA complex, which prevents PAPPA from carrying out its proteolytic function of cleaving IGFBP-4, hence a corresponding decrease in measured PAPPA activity. Therefore, in the working model described by the authors of US200510272034, PAPPA activity is reduced solely as a direct consequence of increased levels of circulating proMBP.

In contrast, the present invention is based on a very different understanding of the role of PAPPA in ovarian cancer. The present inventors have identified that endogenous PAPPA levels and/or functional activity of PAPPA protein is supressed due to genetic alterations within the PAPPA gene, causing ovarian cells to become temporarily stalled in mitosis. This has implications for the development and progression of ovarian cancer.

The following definitions apply to terms used throughout this description and in relation to any of the aspects of the invention described herein.

The terms "patient" and "subject" are used interchangeably herein and refer to any female animal (e.g. mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents and the like, which is to be the recipient of the diagnosis. Preferably, the subject or patient is a human female.

The terms "drug" and "agent" are used interchangeably herein, and refer to a chemical or biological substance that exerts an effect on a biological system.

The present invention can utilise naturally-occurring or synthetic nucleic acids. The methods described herein can involve the synthesis of cDNA from mRNA in a test sample or the amplification of naturally-occurring nucleic acids.

Portions of cDNA corresponding to genes or gene fragments of interest can be amplified, and the product detected.

As used herein, "symptoms of ovarian cancer" include, but are not limited to, loss of appetite, indigestion, nausea, excessive gas and a bloated feeling, unexplained weight gain or an increased waist size, swelling in the abdomen associated with shortness of breath, pain in the lower abdomen, changes in bowel or bladder habits, including constipation, diarrhoea or needing to pass urine more often, lower back pain, pain during sex and abnormal vaginal bleeding.

As used herein, the term "a sample" includes biological samples obtained from a patient or subject, which may comprise a tissue sample obtained from the ovary, preferably from the surface epithelium, or fallopian tube, fluid samples including ascites, peritoneal fluid or washings, ovarian cyst fluid and blood/blood components.

Tissue samples may be taken from a primary ovarian tumour site in the ovaries of fallopian tubes or, in certain aspects of the invention, may be taken from a site that is adjacent to or distant form the primary tumour site. AS used herein "adjacent" refers to a site that is next to, but does not include, the primary (or original) tumour. "Distant" refers to a site that is not part of, or adjacent to, the primary tumour site, such as a different tissue or organ within the body. Tumour cells may spread from the primary tumour site to distant sites via the blood stream or lymphatic system.

Examples of suitable tissue samples include formalin-fixed paraffin-embedded (FFPE) biopsy and/or resection specimens. These comprise protein, DNA, mRNA and/or miRNA, other cellular and extracellular matter and tumour cells (if present in the subject). In the context of the present invention, tissue samples can be used to determine the PAPPA status of the subject (i.e. determine PAPPA expression/activity levels and presence of loss-of-function genetic alterations) and to identify the mitotic delay phenotype by analysing mitotic phase distribution in dividing cells within the tissue. Methods for taking a sample from a patient (biopsy) are conventional and will be apparent to the skilled person.

Ascites, peritoneal fluid or peritoneal washings and ovarian cyst fluid can comprise free DNA, mRNA and/or miRNA and tumour cells (if present in the subject). These samples can be used to investigate the PAPPA status of a subject. The skilled person will be familiar with standard techniques which are suitable for obtaining these samples from a subject.

As used herein, the term "blood sample" includes blood components, including plasma and serum. Circulating free DNA, mRNA, miRNA and, if present, circulating tumour cells, can be extracted from the blood sample and used to determine the PAPPA status of the subject. The skilled person will be familiar with standard phlebotomy techniques which are suitable for obtaining a blood sample from a subject.

The terms "cancer/cancerous" and "neoplasm/neoplastic" refer to or describe the physiological condition in mammals in which a population of cells are characterised by unregulated cell growth.

The terms "cancer cell" and "tumour cell" are grammatical equivalents referring to the total population of cells derived from a tumour or a pre-cancerous lesion. The terms "tumour" and "neoplasm" are used interchangeably herein and refer to any mass of tissue that results from excessive cell growth, proliferation and/or survival, either benign (noncancerous) or malignant (cancerous), including pre-cancerous lesions.

Some of the methods described herein involve establishing whether PAPPA is present in a sample at reduced levels compared to a control.

However, it is also envisaged that PAPPA protein may be present at or near to normal levels, but the expressed protein is inactive or active at reduced levels.

Accordingly, all aspects of the invention that involve establishing the presence and/or level of PAPPA in a sample also encompass monitoring the activity of PAPPA. PAPPA activity may be reduced by greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 90% compared to a control. Therefore, all references herein to determining whether PAPPA is present (or is present at a reduced level) compared to a control, encompasses the functional activity of PAPPA. Methods for detecting PAPPA protein and PAPPA activity are described in detail below.

The methods of the invention described herein are carried out ex vivo.

For the avoidance of doubt, the term "ox vivo" has its usual meaning in the art, referring to methods that are carried out in or on tissue in an artificial environment outside the body of the patient from whom the tissue sample has been obtained.

The term "mitosis" has its usual meaning in the art. Mitosis is the process by which a eukaryotic cell separates the chromosomes in its nucleus into two identical sets, in two separate nuclei. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle. The process of mitosis is characterised into stages corresponding to the completion of one set of activities and the start of the next. These stages are prophase, prometaphase, metaphase, anaphase and telophase.

The term "prophase" has its usual meaning in the art. Prophase refers to the stage where the chromatin in the nucleus becomes tightly coiled, condensing into discrete chromosomes.

The term "prometaphase" has its usual meaning in the art. During prometaphase the nuclear membrane disintegrates and microtubules invade the nuclear space.

The term "proliferating cells" refers to cycling cells that are actively dividing and progressing though interphase (G1, S and G2 phases) or M phase (mitosis) of the cell cycle.

The term "invasive cancer" refers to metastatic cancer, i.e. a primary ovarian lesion or tumour that has spread beyond the epithelium in which it developed, for example into the peritoneal cavity and has invaded and/or is growing in surrounding tissues such as uterus, urinary bladder, bowel and the lining of the bowel wall, forming new tumour growths and obstructions. Other routes of invasion of ovarian cancer include transcoelomic spread, which refers to the spread of a malignancy into body cavities via seeding the surface of the peritoneal, pleural, pericardial, or subarachnoid spaces. Figure 1 shows the multistep model of ovarian tumourigenesis and distinguishes between normal, benign, borderline and malignant tumours. Invasive cancer is also referred to as infiltrating cancer or malignant cancer. These terms are intended to include all primary invasive surface epithelial ovarian tumours, including adenocarcinomas (such as endometrioid tumour, serous cystadenocarcoinoma, papilliary, mucinous cystadenocarcinoma, clear-cell ovarian tumour, mucinous adenocarcinoma, papilliary serious cystadenocarcinoma), squamous cell carcinomas (epidermoid), germ cell tumours (such as teratoma and dysgerminoma) and other carcinomas such as sex cord-stromal tumours.

The terms "immunoassay", "immuno-detection" and immunological assay" are used interchangeably herein and refer to antibody-based techniques for identifying the presence of or levels of a protein in a sample.

The term "antibody" refers to an immunoglobulin which specifically recognises an epitope on a target as determined by the binding characteristics of the immunoglobulin variable domains of the heavy and light chains (VHS and VLS), more specifically the complementarity-determining regions (CDR5). Many potential antibody forms are known in the art, which may include, but are not limited to, a plurality of intact monoclonal antibodies or polyclonal mixtures comprising intact monoclonal antibodies, antibody fragments (for example F2b, Fab', and F1 fragments, linear antibodies single chain antibodies and multi-specific antibodies comprising antibody fragments), single chain variable fragments (scFS), multi-specific antibodies, chimeric antibodies, humanised antibodies and fusion proteins comprising the domains necessary for the recognition of a given epitope on a target. Preferably, references to antibodies in the context of the present invention refer to monoclonal antibodies. Antibodies may also be conjugated to various reporter moieties for a diagnostic effect, including but not limited to radionuclides, fluorophores or dyes.

The term "epitope" refers to the portion of a target which is specifically recognised by a given antibody. In instances where the antigen is a protein, the epitope may be formed from either a contiguous or non-contiguous number of amino acids (linear' or conformation' epitopes respectively), whereby in the case of the latter, residues comprising the epitope are brought together in the three-dimensional fold of the polypeptide. An epitope typically comprises, but is not limited to, 3-10 amino acids in specific positions and orientations with respect to one another. Techniques known in the art for determining the epitope recognised by an antibody (specifically whether or not an epitope comprises a given residue) include but are not limited to, site-directed mutagenesis or the use of suitable homologous proteins to the target protein, in combination with techniques for determining specific recognition or lack thereof, as exemplified below. By way of example and not limitation, an epitope may be determined as comprising a given residue by comparative analysis with a control comprising specific recognition of the native (non-substituted) target protein by said antibody; wherein diminished binding and/or lack of specific recognition by said antibody when compared with said control identifies a given residue as forming part of an epitope. Furthermore, structural analyses of antibody-target protein complexes via x-ray crystallography and/or nuclear magnetic resonance (NMR) spectroscopy, or suitable derivatives thereof, may also be used to determine the residues which constitute an epitope.

The term specifically recognises", in the context of antibody-epitope interactions, refers to an interaction wherein the antibody and epitope associate more frequently or rapidly, or with greater duration or affinity, or with any combination of the above, than when either antibody or epitope is substituted for an alternative substance, for example an unrelated protein. Generally, but not necessarily, reference to binding means specific recognition. Techniques known in the art for determining the specific recognition of a target by a monoclonal antibody or lack thereof include but are not limited to, FAGS analysis, immunocytochemical staining, immunohistochemistry, western blotting/dot blotting, ELISA, affinity chromatography. By way of example and not limitation, specific recognition, or lack thereof, may be determined by comparative analysis with a control comprising the use of an antibody which is known in the art to specifically recognise said target and/or a control comprising the absence of, or minimal, specific recognition of said target (for example wherein the control comprises the use of a non-specific antibody). Said comparative analysis may be either qualitative or quantitative. It is understood, however, that an antibody or binding moiety which demonstrates exclusive specific recognition of a given target is said to have higher specificity for said target when compared with an antibody which, for example, specifically recognises both the target and a homologous protein.

As used herein, "ovarian cancer therapies" includes the following: * Demethylation agents, including decitabine (5-aza-2'-deoxycytidine), farazabine, azaytidine (5-azacytidine), histone deacetylase inhibitors (such as hydroxamic acids (e.g. trichostatin A)), cyclic tetrapeptides (e.g. trapoxin B), depsipeptides, benzamides, electrophilic ketones, aliphatic acid compounds (e.g. phenylbutyrate and vaiproic acid), hydroxamic acids (e.g. vorinostat, belinostat and panobinostat), benzamides and phenylbutyrates.

Chemotherapeutic drugs, including: taxanes, platinum-containing chemotherapeutic agents, ymca alkaloids, alkylating antineoplastic agents, nucleoside analogue drugs, topoisomerase I/Il inhibitors, podophyllotoxins, camptothecins, folate antimetabolites, anthracyclines, tetrahydroisoquinoline alkaloids, PLK1 inhibitors and aurora kinase inhibitors. More specifically, the chemotherapeutic drug is preferably selected from one or more of the drugs paclitaxel (TaxolTM, AbraxaneTM), paclitaxel poliglumex, carboplatin (ParaplatinTTM), docetaxel (TaxotereTTM), doxorubicin (DoxilTM CaelyxTM, MyocetTM), gemcitabine (GemzarTM), cisplatin (PlatinTTM), topotecan (HycamtinTM), oxaliplatin (EloxatinTM), fluorouracil, leucovorin-modulated 5-fluorouracil, podofilox (CondyloxTM), etoposide (EtopophosTM), vinorelbine (NavelbineTTM), ifosfamide (lfexTM), pemetrexed (AlimtaTM), trabectedin (YondelisTM), tirapazamine, vintafolide, karenitecan and sapacitabine.

* Biological agents * Nanoparticles * Radiation therapy As used herein, the term biological agent" has its usual meaning in the art and refers to any therapeutic entity derived from a biological or biotechnological source or process, including modified derivatives thereof, or one chemically synthesised to be equivalent to a product from said source, process or derivative thereof Suitable biological agents include a peptide, a protein, an oligonucleotide, a polysaccharide, a cell-based product, a plant or animal extract, a recombinant protein, an antibody of any suitable type, including a monoclonal, a polyclonal antibody, a humanised antibody, a chimeric antibody, and an antibody fragment, an anti-cancer vaccine, blood or blood components (including erythrocytes, leukocytes, plasma and serum), a pro-drug, or combinations thereof. If the biological agent is an antibody, it will have specificity for a proliferating ovarian cancer cell. The antibody may itself be the therapeutic agent, or may be a carrier, bringing a therapeutic agent, such as a chemotherapeutic agent, to the site of action. In another example, the antibody may be conjugated to a radionuclide, to deliver the radionuclide to the site of proliferating ovarian cancer cells where it can exert its beneficial effect. An example of a suitable biologic drug for use in the treatment of ovarian cancer is farletuzumab (MORAb-003), which is a monoclonal antibody that acts against proliferating cells inS phase of the cell cycle by targeting folate receptor-alpha.

As used herein, the term "nanoparticle" refers to a particle having at least one dimension of nanometer (10-s meters) scale. Generally, but not necessarily, the term refers to a polymer sphere or spheroid having one dimension of less than or equal to about S000nm, including, 5, 10, 15, 20, 30, 50, 100, 200, 250, 300, 350, 400, 500, 750 and 1000 nm. The term includes nanoparticles comprising a number of layers and/or regions of different polymers and/or adsorbed agents. Nanoparticles are used in cancer therapy as drug delivery systems. Typically, the nanoparticles are liposomal and/or polymer-drug conjugates, able to target selectively proliferating cancer cells. Nanoparticles can be linked to biological molecules which can act as address tags, to direct the nanoparticles to specific sites within the body and within the nucleus. Common address tags are monoclonal antibodies, aptamers, streptavidin or peptides.

Nanoparticles useful in the therapeutic regimen of the invention may carry anti-proliferative agents (e.g. doxorubicin, DoxilTM).

As used herein, the term "radiation therapy" (also known as radiation oncology or radiotherapy) has its usual meaning in the art and refers to the medical use of ionizing radiation as part of a cancer treatment regimen to kill malignant cells that are progressing through the cell cycle (i.e. in any phase of the cell cycle). The radiation therapy may be internal or external radiotherapy.

External radiotherapy involves targeting doses (or "fractions") of high-energy beams of radiation, either X-rays or gamma rays, to the tumour. Internal radiotherapy involves positioning the source of radioactivity inside the body close to the tumour. This can be achieved in two ways: by brachytherapy or by radioisotope therapy. Brachytherapy involves placing a solid source of radiation next to a tumour to give a high dose of radiotherapy. Brachytherapy is often used to treat gynaecological cancers by placing the radiation source inside the vagina during the treatment. Radioisotope therapy involves administration of a radioactive substance, a radioisotope, either as an intravenous injection, or as an oral capsule or liquid.

The present invention has identified that suppression of endogenous Pregnancy Associated Plasma Protein A (PAPPA) levels is implicated in the development of malignant ovarian cancer. The inventors have shown that in ovarian tissue PAPPA is required for normal progression through mitosis. The endogenous suppression of PAPPA levels and/or functional activity of PAPPA cause an early mitotic delay phenotype, with cycling cells temporarily stalling in prophase/prometaphase.

Mitotic delay due to PAPPA suppression in ovarian cancer cells appears at first glance to be disadvantageous to tumour growth. However a major biological advantage is conferred to the mitotically delayed, neoplastic ovarian cell through the associated increase in acquiring invasive capacity. In ovarian cancer specimens, mitotic delay due to PAPPA silencing can be detected in virtually all cases of invasive cancer and also in a proportion of non-invasive borderline lesions. The gain in invasive capacity as a consequence of PAPPA loss therefore occurs early in multi-step ovarian tumour progression during the transition from non-invasive to invasive cancer. Detection of PAPPA deregulation and mitotic delay in clinical biopsy specimens offers a significant advance of the staging, management and prognostication of ovarian cancer.

Therefore, the present invention is useful in the context of: 1) Monitoring/determining the efficacy of a ovarian cancer treatment 2) Detecting a relapse after treatment 3) Staging of ovarian cancer 4) Prediction of disease progression of invasive ovarian cancer 1. Monitoring efficacy of treatment The present invention can be used to monitor the response to a treatment given for ovarian cancer.

As described above, conventional treatments for ovarian cancer include: * Surgery * Radiation therapy * Chemotherapy * Targeted therapy The present invention provides a way of measuring the success of the therapy and monitoring progress during therapy.

According to the first aspect of the invention, there is a method for monitoring the response to treatment in a patient undergoing ovarian cancer therapy, comprising detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences in a sample obtained from the patient, wherein the presence of a genetic alteration and downward change in its quantitative levels is indicative of response to therapy, whereas no change or upward change in its quantitative level is indicative of non-response to therapy.

The quantitative level of PAPPA genetic alterations in a patient's sample is tested at intervals during the therapeutic treatment, using a technique that generates a quantitative readout (e.g. PCR methods such as Digital PCR and Cold PCR). If the tumour cells are responding to the treatment, results with decreasing signal strength will be generated (i.e. a downward change in the quantitative level of PAPPA genetic alterations). If the tumour cells are resistant to the chosen therapy and continue to divide, then the signal strength will increase (i.e. an upward change in the quantitative level of PAPPA genetic alterations). If the quantitative level of PAPPA genetic alterations stays the same during the therapeutic treatment then this is indicative of stable disease.

Monitoring for genetic alteration is also useful in monitoring the response to therapy. If there is no change in the level of the mutant gene this indicates non-response. If there is a gradual decrease in the level of the mutant gene, then the patient's tumour is responding to treatment. If the mutant gene cannot be detected any longer it indicates that the patient is in remission.

In a preferred embodiment, the loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences is one or more of methylation, deletion, loss of heterozygosity, a point mutation, insertion, translocation or chromosomal breakage.

According to the second aspect of the invention, there is a method for monitoring the response to treatment in a patient undergoing ovarian cancer therapy, comprising detecting the presence and/or level of PAPPA in a sample obtained from the patient, wherein the absence of PAPPA, or the presence of PAPPA at a reduced level compared to a control is indicative of non-response to therapy.

Preferably the control is the patient's baseline PAPPA levels determined from a sample taken from the patient prior to treatment commencing.

Monitoring for the presence of PAPPA, or PAPPA activity, will aid in the monitoring of the effectiveness of therapy, as the PAPPA levels may initially increase at the start of therapy, but, if the patient is non-responsive to therapy, the PAPPA levels may decrease. A rise in levels of PAPPA compared with the patient's baseline control sample and subsequent maintenance of PAPPA levels at those of or similar to a healthy control will indicate a successful response to treatment.

In a preferred embodiment, both of the above methods are carried out.

Preferably these methods will be carried out on one or more blood samples taken from the patient undergoing therapy.

As used herein, the phrase patient undergoing ovarian cancer therapy" includes both patients who have begun, but not yet completed, a course of therapy, in which case the method is carried out during therapy, and patients who have completed the course of therapy, in which case the method is carried out following therapy. In the latter instance, the methods of the invention are useful for indicating or confirming that a patient is in remission. The patient may be being treated for primary or metastatic ovarian cancer.

The therapy may include any ovarian cancer therapy indicated herein.

2. Relapse The present invention may also be used to monitor a patient who is in complete or partial remission following treatment for primary or metastatic ovarian cancer, to determine whether there is a relapse or progressive disease.

The present invention can therefore be used to provide early warning of relapse.

The present invention is also useful in this context to help in the treatment of relapse or progressive disease.

As used herein, the term "relapse" has its usual meaning in the art, and refers to cancer returning, either in the ovary or at another site (i.e. metastatic cancer) following complete remission.

The term "complete remission" (also known as "pathologic remission") refers to the situation where no tumour cells are detectable following surgical removal of the cancerous tissue and chemotherapy.

"Partial remission" refers to a situation where the tumour has shrunk in size following chemotherapy but subsequent surgery to remove the remaining tumour is not possible (for example, due to the location of the tumour). This results in a stable disease state, where the tumour is present but not growing.

The term "progressive disease" refers to renewed tumour growth following a period of stable disease in a patient in partial remission.

According to this third aspect of the invention, there is a method for determining whether a patient, previously treated with ovarian cancer therapy, has suffered a relapse, comprising detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences in a biological sample obtained from the patient, wherein if a genetic alteration is identified, this is indicative of a relapse.

In a preferred embodiment, the loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences is one or more of methylation, deletion, loss of heterozygosity, a point mutation, insertion, translocation or chromosomal breakage.

According to a further fourth aspect, there is a method for determining whether a patient previously treated with ovarian cancer therapy, has suffered a relapse, comprising detecting the presence and/or level of PAPPA in a biological sample obtained from the patient. wherein the absence of PAPPA, or the presence of PAPPA at a reduced level compared to a control, is indicative of a relapse.

Preferably the control is the patient's baseline PAPPA levels determined from a sample taken from the patient following completion of treatment.

In the methods of the third and fourth aspects, the sample from the patient is typically blood, ascites, peritoneal fluid/washings and tissue, and is preferably a blood sample.

According to a further fifth aspect, there is a method for determining whether a patient previously treated with ovarian cancer therapy has suffered a relapse, comprising identifying the proportion of mitotic cells in a biological sample obtained from the patient that are in prophase or pro-metaphase and comparing to a pre-determined cut-off value, wherein if the proportion of cells in prophase or pro-metaphase is the same or greater than the cut-off value there is a risk that the patient has suffered a relapse.

In the method of the fifth aspect of the invention, the sample may be tissue or ascites, and is preferably a tissue sample, such as ovarian, fallopian tube tissue or a different tissue that the cancer appears to have spread to.

In these aspects of the invention relating to relapse monitoring, the patient will have been previously treated for ovarian cancer. For the avoidance of doubt, the phrase "previously treated" means that the patient will have been diagnosed with ovarian cancer and will have completed the prescribed treatment for the ovarian cancer and entered complete or partial remission before the methods of any of the third to fifth aspects of the invention are carried out.

Any of the three aspects mentioned above may be combined with each other, or with other conventional techniques such as imaging, in order to confirm whether relapse or progressive disease has occurred.

3. Staging After ovarian cancer has been diagnosed, tests are done to find out if the cancer cells have spread within the ovarian tissue or to other tissues. Usually, surgery is needed before staging can be complete. The surgeon takes many samples of tissue from the pelvis and abdomen to look for cancer.

Tests for staging include: * CT scan: Doctors often use CT scans to make pictures of organs and tissues in the pelvis or abdomen. An x-ray machine linked to a computer takes several pictures. Contrast material may be given by mouth or by injection into your arm or hand. The contrast material helps the organs or tissues show up more clearly. Abdominal fluid or a tumour may show up on the CT scan.

* Chest x-ray: X-rays of the chest can show tumours or fluid.

* Barium enema x-ray: The doctor may order a series of x-rays of the lower intestine. An enema is given with a barium solution. The barium outlines the intestine on the x-rays. Areas blocked by cancer may show up on the x-rays.

* Colonoscopy: The doctor inserts a long, lighted tube into the rectum and colon. This exam can help tell if cancer has spread to the colon or rectum.

These are the stages of ovarian cancer: * Stage I: Cancer cells are found in one or both ovaries. Cancer cells may be found on the surface of the ovaries or in fluid collected from the abdomen.

* Stage II: Cancer cells have spread from one or both ovaries to other tissues in the pelvis. Cancer cells are found on the fallopian tubes, the uterus, or other tissues in the pelvis. Cancer cells may be found in fluid collected from the abdomen.

* Stage Ill: Cancer cells have spread to tissues outside the pelvis or to the regional lymph nodes. Cancer cells may be found on the outside of the liver.

* Stage IV: Cancer cells have spread to tissues outside the abdomen and pelvis. Cancer cells may be found inside the liver, in the lungs, or in other organs.

The present invention can be used separately, or as an additional test complementing conventional staging techniques, to stage the cancer, and determine whether a primary ovarian cancer tumour has spread into the blood stream or to another tissue site.

According to the sixth aspect of the invention, there is a method for determining whether a primary ovarian cancer in a patient has spread away from the site of the primary tumour to another part of the body, comprising detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences in a sample obtained from the patient, wherein the presence of a genetic alteration is indicative of spread.

The patient sample will typically be blood, peritoneal fluid/washings, ascites and/or a tissue sample, and is preferably a blood sample.

In a preferred embodiment, the loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences is one or more of methylation, deletion, loss of heterozygosity, a point mutation, insertion, translocation or chromosomal breakage.

According to a seventh aspect, there is a method for determining whether a primary ovarian cancer in a patient has spread away from the site of the primary tumour to another part of the body, comprising identifying the proportion of mitotic cells in a biological sample obtained from the patient that are in prophase or pro-metaphase and comparing to a pre-determined cut-off value, wherein if the proportion of cells in a prophase or pro-metaphase is the same or greater than the cut-off value there is a risk that the cancer has spread.

The sample may be a tissue sample or ascites. The tissue sample is obtained from a site other than the primary tumour site; this may be from a site surrounding (i.e. adjacent to) the primary tumour site, or may be from a different site that is distant from (i.e. not at or adjacent to) the primary tumour site, e.g. colon, uterus or liver tissue.

According to an eighth aspect, there is a method for determining whether a primary ovarian cancer in a patient has spread away from the site of the primary tumour to another part of the body, comprising detecting the presence and/or level of PAPPA in a sample obtained from the patient, wherein the absence of PAPPA, or the presence of PAPPA at a reduced level compared to a control, is indicative of spread. Preferably control" refers to PAPPA levels measured in a sample from a healthy individual.

The sample will typically be blood, peritoneal fluid/washings, ascites and/or a tissue sample, and is preferably a blood sample.

For the avoidance of doubt, the term "spread" refers to a primary ovarian lesion or tumour that has spread beyond the epithelium in which it developed, for example into the peritoneal cavity, and has invaded and/or is growing in surrounding tissues such as uterus, urinary bladder, bowel and the lining of the bowel wall, forming new tumour growths and obstructions. Other routes of invasion of ovarian cancer include transcoelomic spread, which refers to the spread of a malignancy into body cavities via seeding the surface of the peritoneal, pleural, pericardial, or subarachnoid spaces.

In the above aspects relating to staging, the patient will have been diagnosed with ovarian cancer. Other tests may be performed on the patient to aid in the staging of the cancer. For example, one or more of chest x-ray, CT (CAT) scan, colonoscopy, may have been performed on the patient.

The results of the staging diagnosis can help in the prognosis (prediction of outcome) and in determining what further therapeutic intervention should be given.

The methods and techniques for carrying out the above aspects are as disclosed.

4. Prognostication of disease outcome The methods of the present invention may also be used to predict disease progression in a patient who has been diagnosed with invasive ovarian cancer.

Accordingly this aspect of the invention provides a method for predicting disease progression in a patient who has been diagnosed with invasive ovarian cancer, comprising identifying the proportion of mitotic cells in a tissue sample obtained from the patient that are in prophase or prometaphase and comparing to a pre-determined cut-off value, wherein if the proportion of cells in prophase or prometaphase is the same or greater than the cut-off value, the prediction is reduced disease-free and overall survival.

In a related aspect of the invention, a method for predicting disease progression in a patient who has been diagnosed with invasive ovarian cancer comprises detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences in a sample obtained from the patient, wherein the presence of a genetic alteration predicts reduced disease-free and overall survival.

Another related aspect provides a method for predicting disease progression in a patient who has been diagnosed with invasive ovarian cancer, comprising detecting the presence and/or level of PAPPA in a biological sample obtained from the patient, wherein the absence of PAPPA, or its presence at a reduced level compared to a control, is predictive of reduced disease-free and overall survival.

The term "control" may refer to PAPPA levels measured in a sample from a healthy individual, or may refer to the patient's baseline PAPPA levels determined from a sample taken from the patient prior to or during treatment.

Certain aspects of the present invention relate to an ex vivo method for determining whether ovarian cells are stalled in mitosis, by identifying a delayed mitotic phenotype. Identification of this phenotype comprises identifying the proportion of mitotic cells in a biological sample obtained from a patient that are in prophase or prometaphase and comparing to a pre-determined cut-off value.

The pre-determined cut-off value is at least 30% and preferably at least 33% or more. At least five, preferably at least ten, twenty or fifty of the cells within the sample must be undergoing mitosis. If at least 30% of these at least five mitotic cells are identified as being in prophase or prometaphase then the sample is deemed to have a delayed mitotic phenotype.

For example, if five of the cells within the sample are identified as being in mitosis, at least two of these cells must be in prophase/prometaphase in order for the mitotic delay phenotype to be identified. More preferably the proportion of mitotic cells in prophase/prometaphase in a sample from a patient having the delayed mitotic phenotype is greater than 33%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more. Typically, a "control" value for untransformed proliferating cells undergoing mitosis and not displaying the mitotic delay phenotype would be approximately 10-25%, e.g. 24% cells in prophase/prometaphase.

If fewer than five cells in a tissue sample are undergoing mitosis then the analysis of the proportion of mitotic cells that are in prophase/prometaphase will not be sufficiently significant to enable the delayed mitotic phenotype to be identified according to the methods of the invention. Therefore, the methods require there to be at least five mitotic cells in the sample being analysed at the time of analysis.

For methods of the present invention involving determination of the mitotic delay phenotype the biological sample is preferably an ovarian or fallopian tube tissue sample. Alternatively, in aspects of the invention relating to staging or relapse monitoring, the tissue sample may be from a different tissue type or organ that the ovarian cancer appears to have spread to. Tissue biopsies may be taken form a site that is adjacent to or distant from the site of the primary ovarian tumour.

Detection of whether the cells in a tissue sample are in prophase or prometaphase can be carried out using techniques conventional in the art. For example, immuno-detection techniques using specific antibodies may be used to characterise the mitotic phase of a cell. Immunohistochemistry (IHC) is an immuno-detection technique and refers to the process of detecting antigens in cells of a tissue section by visualising an antibody-antigen interaction. This can be achieved by tagging an antibody with a reporter moiety, preferably a visual reporter such as a fluorophore (termed immunofluorescence") or by conjugating an antibody to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction that can be detected and observed.

H3310 phosphorylation (H3SlOph) is a mitosis-specific modification essential for the onset of mitosis; the phosphorylation of the serine 10 of Histone H3 is important for chromosome condensation. Antibodies specific for H3SlOph are commercially available (e.g. Millipore and Active Motif) as are kits for carrying out mitotic assays.

Alternative markers of mitosis, such as phospho-specific nuclear laminin are also available commercially and may be utilised in the methods of the invention. Other methods for assessing and characterising the different phases of mitosis, such as the use of specialist stains and visual morphological analysis, are well known in the art, as will be appreciated by the skilled person.

The analysis can be carried out to provide a "snapshot" of the mitotic phase distribution in dividing cells in a tissue sample. In this way, a mitotic phase distribution analysis is obtained which is then used to characterise the proportion of mitotic cells that are in prophase or prometaphase.

The methods of the invention that involve identifying a delayed mitotic phenotype may further comprise detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene, or its regulatory or promoter sequences, in a sample obtained from the patient, according to the methods described below. Furthermore, additionally or alternatively, such methods may further comprise detecting the presence and/or level of PAPPA in a sample obtained from the patient, as described below.

Other aspects of the invention comprise detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene, or its regulatory or promoter sequences, in a sample obtained from the patient. The loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences is one or more of a point mutation, deletion, insertion, translocation, chromosomal breakage, loss of heterozygosity (LOH) or methylation. In order for a single genetic alteration or combination of two or more genetic alterations to be classified as a loss-of-function-related genetic alteration, the alteration must result in loss-of-functional PAPPA protein. In aspects of the invention that relate to detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene the sample may be selected from blood, ascites, peritoneal fluid or washings, and tissue, preferably ovarian or fallopian tube tissue. Alternatively, in aspects of the invention relating to staging or relapse monitoring, the tissue sample may be from a different tissue type or organ that the ovarian cancer appears to have spread to. Tissue biopsies may be taken from a site that is adjacent to or distant from (i.e. not adjacent to) the site of the primary ovarian tumour.

It is preferred that for aspects of the invention involving detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene, or its regulatory or promoter sequences the sample is a blood sample.

Additionally, the methods of the invention that involve detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene may further comprise detecting the presence and/or level of PAPPA in a sample obtained from the patient, and comparing to a control of reference value.

An absence or reduction in PAPPA is indicated if PAPPA is not present or is present at a level less than 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20% compared to the control. For example, if PAPPA is present at a reduced level compared to a control it may be present at an amount between 40%-80% of that of the control. Most preferably, PAPPA will be present at a level between 50%- 70%, e.g. approx. 60% compared to that of a control.

For methods of the invention that involve detecting the presence and/or level of PAPPA in a sample obtained from the patient, and comparing to a control of reference value the sample is preferably ovarian or fallopian tube tissue. The control is preferably a corresponding sample obtained from a population of untransformed proliferating ovarian cells, or it may be a reference value.

The presence of PAPPA is preferably identified using a PAPPA-specific antibody.

The presence of PAPPA may be identified using a PAPPA-specific antibody or a probe for the PAPPA gene, mRNA or a specific PAPPA mutation.

The present inventors have identified that one cause of PAPPA suppression in ovarian cancer cells (or pre-cancerous cells) is due to epigenetic changes, i.e. methylation of its DNA, primarily the PAPPA promoter region. DNA methylation, caused primarily by covalent addition of methyl groups to cytosine within CpG dinucleotides, occurs primarily in promoter regions of genes due to the large proportion of CpG islands found there. Hypermethylation results in transcriptional silencing.

Detecting the presence or absence of cancer by determining the methylation state of specific genes is known (but not in the context of PAPPA), and conventional methods for doing this may be adapted for use in the present invention. For example, methylation-specific PCR (MSP) has been used to determine the methylation status of specific genes. This technique, referred to also as MethyLight is described in Eads et al, Nucleic Acids Res. 2000; 28(8), and Widschwendler et at, Cancer Res., 2004; 64:3807-381 3, the content of each of which is incorporated herein by reference. Alternative methods include Combined Bisulphate Restriction Analyses, Methylation-sensitive Single Nucleotide Primer Extension and the use of CpG island microarrays.

Commercially available kits for the study of DNA methylation are available. The invention makes use of conventional methods for determining the methylation state of the PAPPA gene or its regulatory promoter sequences.

MethyLight is a high-throughput quantitative methylation assay that utilises fluorescence-based real-time PCR (TaqMan®) technology that requires no further manipulations after the PCR step. MethyLight is a highly sensitive assay, capable of detecting methylated alleles in the presence of a 10,000-fold excess of unmethylated alleles. The assay is also highly quantitative and can very accurately determine the relative prevalence of a particular pattern of DNA methylation using very small amounts of template DNA.

According to the present invention, MethyLight can be used to determine the methylation state of the PAPPA gene or regulatory or promoter regions in a sample of genomic DNA obtained from the patient. Determination of the methylation state of the PAPPA gene may comprise the following steps: Genomic DNA is extracted from the ovarian tissue sample and treated with sodium bisulfite to convert unmethylated cytosines to uracil residues (methylated residues are protected); ii. Primers and probes designed specifically for bisulfite-modified DNA, such as those detailed in Table 2 below, are used to amplify the bisulfite-targeted DNA sample. The primer/probe sets used include a methylated set specific for the PAPPA gene and a set specific for a reference gene (COL2A1) Di. The data are analysed and Ct values are calculated, for example by using ABI Step One Plus software; and iv. The percentage of fully methylated PAPPA molecules at the specific locus is calculated by dividing the PAPPA:COL2A1 ratio of a sample by the PAPPA:COL2A1 ratio of a positive control sample (for example, Sssl treated HeLa genomic DNA) and multiplying by 100.

Since MethyLight reactions are specific to bisulfite converted DNA, the generation of false positive results is precluded.

Although DNA methylation (hypermethylation) is one cause of PAPPA suppression, there may be other causes. For example, the PAPPA gene (or its regulatory sequences) may be mutated leading to transcriptional silencing. Point mutations, deletions, loss of heterozygosity and translocations may all cause the PAPPA gene to lose transcriptional activity. Without being bound by theory, it is believed that approximately 40% of PAPPA suppression in ovarian tissue is caused by epigenetic changes including hypermethylation. It is believed that the major cause of PAPPA suppression in ovarian tissue is due to loss-of-function genetic changes including point mutations, insertions, deletions, translocation, chromosomal breakage and loss of heterozygosity.

Point mutations are genetic changes where the mutation of a single DNA base to another base can lead to a non4unctional protein. These point mutations can be further classified into nonsense mutations, where the mutant gene brings the protein synthesis to a premature halt; missense mutations, where the altered codon results in the insertion of an incorrect amino acid into the protein; and frame-shift mutations, where the loss or gain of one or more nucleotides causes the codons to be misread, resulting in non-functional proteins. There are a variety of methods available for the detection of point mutations in molecular diagnostics. The choice of the method to be used depends on the specimen being analysed, how reliable the method is, whether the mutations to be detected are known before analysis and the ratio between wild-type and mutant alleles.

Denaturing gradient gel electrophoresis is a suitable technique for mutation detection, particularly for point mutations. A prolonged (48hr) proteinase K digestion method or DNA easy kit (Qiagen) can be used to extract genomic DNA. Double stranded DNA (POR fragments of 1kb) can be generated by multiplex PCR reaction covering the whole of the PAPPA coding region. In order to increase the efficiency of detection GO clamps can be attached to one of the POR primers. The DNA can then be subjected to increasing concentrations of a denaturing agent like urea or formamide in a gel electrophoresis set up. With increasing concentrations of denaturing agent domains in the DNA will dissociate according to their melting temperature (Tm).

DNA hybrids of 1kb usually contain about 3-4 domains, each of which would melt at a distinct temperature. Dissociation of strands in such domains results in the decrease of electrophoretic mobility, and a lbp difference is sufficient to change the Tm. Base mismatches in the heteroduplices lead to a significant destabilisation of domains resulting in differences in Tm between homoduplex and heteroduplex molecules. The homo and heteroduplices will be detected by silver staining after gel electrophoresis. This method offers the advantage that 100% of point mutations can be detected when heteroduplices are generated from sense and antisense strands (Cotton RG, Current methods of mutation detection, Mutat Res 1993; 285: 125-44).

Alternative methods available for the detection of point mutations include P0k-single stranded conformation polymorphism, heteroduplex analysis, protein truncation test, RNASE A cleavage method, chemical/enzyme mismatch cleavage, allele specific oligonucleotide hybridisation on DNA chips, allele specific P0k with a blocking reagent (to suppress amplification of wild-type allele) followed by real time P0k, direct sequencing of P0k products, pyrosequencing and next generation sequencing systems.

Other loss-of-function genetic changes that contribute significantly to PAPPA suppression in ovarian tissue are chromosome changes including gene deletions/insertions, wherein a gene or a cluster of genes is lost from, or inserted into in a chromosome, and translocations, which involve breakage and/or exchange of chromosomal fragments.

The technique of pyrosequencing can be used for detection of insertions, deletions, frame-shift mutations. Pyrosequencing is based on the sequencing-by-synthesis principle. In this method a single-stranded P0R/RT-PCR fragment is used as a template for the reaction. During the process of DNA replication after nucleotide incorporation, released PPi (inorganic phosphate) is converted to light by an enzymatic cascade; ATP sulfurylase which converts PPi to ATP in the presence of APS. This ATP would further drive the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light, which can be detected by a COD sensor and is visible as a peak in the pyrogram (Ronaghi, M., Uhlen, M., and Nyren, P, A sequencing method based on real-time pyrophosphate, Science; 1998b 281: 363-365). The light signal generated is linearly proportional to the nucleotides incorporated.

A prolonged (48hr) proteinase K digestion method or DNA easy kit (Qiagen) can be used to extract genomic DNA from the patient tissue sample.

P0k and sequencing primers for the PAPPA gene can be designed for use in pyrosequencing. P0k products can be bound to streptavidin-sepharose, purified washed and denatured using NaoH solution and washed again. Then the pyrosequencing primer can be annealed to the single-stranded P0k product and the reaction carried out on, for example, a Pyromark ID system (Qiagen) according to the manufacturers instructions.

Other methods available for detecting insertionsI deletions and frame-shift mutations are big dye terminator sequencing, next generation sequencing systems and heteroduplex analysis using capillary/microchip based electrophoresis.

Another major contributor to PAPPA suppression in ovarian tissue is loss of heterozygosity. All cells contain two copies of somatic genes -one copy inherited from each parent. If a cell develops a mutation in one of the alleles of a tumour suppressor gene like PAPPA, loss of the remaining allele (termed "loss of heterozygosity" or [OH") can initiate tumourigenesis.

Loss of heterozygosity (LOH) can be measured using various techniques, including the following: Southern blotting Tumour and reference control DNA is isolated and digested with restriction enzymes. Digested DNA is subjected to Southern blot. 32P-labelled DNA probes are designed to bind to restriction fragment length polymorphisms (RF[P). DNA probes are hybridised to the blot and then visualised by autoradiography. The advantage of Southern blotting is the frequency of false positives or negatives is lower than other techniques. The disadvantages are that this technique is low throughput and the amount of DNA required. If DNA is from FEPE tissues the fragmented nature of this DNA may inhibit the ability to be digested by restriction enzymes. Also, newer technologies described below have replaced radioactive labels with fluorescent labels.

PCR-based microsatellite analysis Primers are designed to amplify the microsatellite loci. For example, suitable primers for the amplification of KLF4 gene mapping within the FRA9E CFS regions are: 5' CAGAGGAG000AAGCCAAAGAG 3' (SEQ ID NO. 1) and 5' CACAGCCGTCCCAGTCACAGT 3' (SEQ ID NO. 2). One primer from each pair is labelled with T4 kinase and y [32P]ATP. PCR amplification is performed using DNA, labelled primer and unlabelled primer. PCR samples are separated by formamide and urea polyacrylamide gel electrophoresis. After fixing and drying, the gel is exposed to X-ray film. Bands are analysed by laser densitometry and the imbalance is defined as the ratio of allele intensities in the tumour sample relative to the ratio of alleles in normal DNA. The advantage of this technique is that it can analyse DNA from fresh tissue, FFPE tissue, and cells. However, DNA from FFPE tissue may be degraded and therefore too shod to be amplified by PCR. The disadvantage of this technique is the potential for false positives due to mis-priming. The technique is labour intensive and requires radioactive labels. This technique is usually carried out together with ORT-POR for e.g for 16 genes in the FRA9E site lost in ovarian cancers. PAPPA is one of the 16 genes found in this site.

Fluorescence in situ hybridisation (FISH) Samples from FFPE, frozen tissue sections, or cells are fixed and permeabilised. Oligonucleotide pairs are hybridised to target RNA. The use of multiple fluorescent-labelled probes enables multiplexing of targets. Samples are viewed under a fluorescence microscope. Alternatively, to detect DNA a metaphase spread from cells is generated and repetitive DNA sequences are blocked. A probe is designed to hybridise to the target and tagged with fluorophores, targets for antibodies, or with biotin. Samples are viewed under a fluorescence microscope. The advantage of FISH is that it detects balanced translocations. Cells are individually analysed and therefore a heterogeneous population or contaminating normal cells would not affect the analysis. The disadvantages of FISH are that it is labour intensive and in order to target DNA, and metaphase spreads need to be generated from cells.

Comparative genomic hybridisation (CGH) CGH technology can detect DNA copy number changes across the entire genome by comparing the hybridisation intensity of the subject samples compared to a reference sample. The DNA is fluorescently labelled and mixed with unlabelled human cot-I DNA to supress repetitive sequences. The DNA mix is hybridised to a normal metaphase spread. The ratio of subject and reference binding is determined by epifluorescence microscopy. The resolution is quite low; to detect a single copy loss the region must be at least 5-10 Mb and amplification changes less than 1 Mb are undetectable. There have been advances to improve the resolution by using array CGH (aCGH) instead of metaphase spreads. The aCGH uses similar technology to microarrays. High resolution aCGH are able to detect structural variations of a resolution of 200 bp.

However, aCGH has proved difficult to use with DNA from FFPE. Also, aCGH can require large quantities of subject DNA.

SNP Microarray Various SNP microarray technologies are available commercially, e.g. from Affymetrix and Illumina, and can be used in the present invention. The Affymetrix technology uses the same basic principles as DNA microarray: solid surface DNA capture, DNA hybridisation and fluorescence microscopy. The Illumina platform uses oligonucleotide bound BeadArray in micro-wells on either fibre optic bundles or planar silica slides. Normal and tumour DNA is fluorescently labelled and bound to predesigned SNA arrays. For each SNA, four to six probes are used. The fluorescent intensity of DNA bound to perfect match probes against mismatch probes is analysed. SNP microarray platforms can be utilised for high-throughput LOH detection. Microarrays can be used for DNA or RNA-analysis applications. The accuracy of SNA calls are based upon the model used to analyse the data including the different definitions of LOH thresholds. The frequently used Hidden Markov Model-based approaches (Green et al., BMC Cancer; 2010, 10:195) allow detection of [OH from hemizygous deletion of single alleles, but cannot detect copy-number neutral [OH.

Next generation sequencing Next generation sequencing technology is moving to replace SNP-arrays for the detection of LOH. Whole genome sequencing, targeted resequencing of a gene of interest, or exome sequencing of a gene of interest can be applied to detect SNPs. A range of tumour DNA, or tumour DNA compared to reference DNA can be sequenced by synthesis or by ligation technology. The DNA sequence is read by fluorescence or a change is pH, depending on the instrument used. The advantages of next generation sequencing are that it is high throughput, fast and has a low cost per base. The disadvantages are that the whole gene of interest may not be covered due to repetitive sequences or high GO content. The accuracy of SNP calls are based upon the model used to analyse the data including the different definitions of LOH thresholds.

Alternative methods of the invention require determining the presence (or absence) or the level of PAPPA in a patient's sample. This can be carried out by determining protein levels, or by studying the expression level of the gene coding for the protein. As used herein the term "expression level" refers to the amount of the specified protein (or mRNA coding for the protein) in the ovarian tissue sample. The expression level is then compared to that of a control. The control may be a tissue sample of a person that is known to not have cancer or may be a reference value. It will be apparent to the skilled person that comparing expression levels of a control and the test sample will allow a decision to be made as to whether the expression level in the test sample and control are similar or different and therefore whether the patient has or is at risk of invasive ovarian cancer.

Methods of measuring the level of expression of a protein in a biological sample are well known in the art and any suitable method may be used. Protein or nucleic acid from the sample may be analysed to determine the expression level, and examples of suitable methods include semi-quantitative methods such as in situ hybridisation (ISH) fluorescence and in situ hybridisation (FISH), and variants of these methods for detecting mRNA levels in tissue or cell preparations, Northern blotting, and quantitative POR reactions. The use of Northern blotting techniques or quantitative FOR to detect gene expression levels is well known in the art. Kits for quantitative PCR-based gene expression analysis are commercially available, for example the Quantitect system manufactured by Qiagen. Simultaneous analysis of expression levels in multiple samples using a hybridisation-based nucleic acid array system is well known in the art and is also within the scope of the invention. Mutation-specific POR may also be used, as will be appreciated by the skilled person.

Immunohistochemical detection of a mitotic marker (such as H3SlOph) can be carried out by preparing formalin-fixed, paraffin-embedded ovarian tissue sections are prepared and mounted on SuperFrost®Plus charged slides.

Following heat-mediated epitope retrieval, endogenous peroxidase activity is quenched and the sections are incubated with a first antibody (suitable H3SlOph antibodies are available, for example, from Millipore) which specifically recognises mitotic markers (such as phosphorylated H3SIO) within mitotic cells.

The section is then further incubated with a polymer-linked secondary antibody and peroxidase which enables a chromogenic signal to develop following addition with DAB, thereby allowing binding of the first antibody to the mitotic marker to be detected visually. The immunohistochemical procedure described above can be fully automated using commercially available immunostainers.

Analysis of mitotic phase distribution in a patent's sample or cultured cell line can be carried out using at least two consecutive serial sections from each sample and at least two cytospin preparations for each cell line or body fluid (e.g. aspirate) immunolabelled as described above and five to twenty high power fields (400x magnification) image captured and a minimum of 5 mitotic cells for each sample used to determine the mitotic phase distribution. All mitotic cells within the captured fields can be classified based on their chromosomal morphology as prophase/prometaphase, metaphase, anaphase and telophase, according to classical morphological criteria. A population of cells is classified as delayed' if at least 30% of mitotic cells reside in prophase/prometaphase.

PAPPA levels in an ovarian tissue sample can be determined using conventional immunological detection techniques, using conventional anti-PAPPA antibodies. The antibody having specificity for PAPPA, or a secondary antibody that binds to such an antibody, can be detectably-labelled. Suitable labels include, without limitation, radionuclides (e.g. 1251, 1311 35s, 3H, 32P or 140), fluorophores (e.g. Fluorescein, FITC or rhodamine), luminescent moieties (e.g. Qdot nanoparticles supplied by Quantum Dot Corporation, Palo Alto Calif) or enzymes (e.g. alkaline phosphatase or horse radish peroxidase).

Immunological assays for detecting PAPPA can be performed in a variety of assay formats, including sandwich assays e.g. (ELISA), competition assays (competitive RIA), bridge immunoassays, immunohistochemistry (IHC) and immunocytochemistry (ICC). Methods for detecting PAPPA include contacting a patient sample with an antibody that binds to PAPPA and detecting binding. An antibody having specificity for PAPPA can be immobilised on a support material using conventional methods. Binding of PAPPA to the antibody on the support can be detected using surface plasmon resonance (Biacore Int, Sweden). Anti- PAPPA antibodies are available commercially (e.g. HPA001667 from Sigma-Aldrich, MAI -46425 (5H9) from Thermo Scientific, 0A5A03208 from Aviva Systems Biology and A0230 from Dako). The immuno-detection of PAPPA is also disclosed in US 6172198, the content of which incorporated herein by reference.

Immunohistochemical detection of PAPPA within an ovarian or fallopian tube tissue sample can be carried out by preparing formalin-fixed, paraffin-embedded ovarian tissue sections mounted on SuperFrost-'-+ charged slides.

Following epitope retrieval by proteolytic digestion, endogenous peroxidase activity is quenched and the sections are incubated with a first anti-PAPPA antibody (available, for example, from DAKO). The section is then further incubated with a polymer-linked secondary antibody and peroxidase which enables a chromogenic signal to develop following addition with DAB, thereby allowing binding of the first antibody to the PAPPA protein to be detected visually. The immunohistochemical procedure described above can be fully automated using commercially available immunostainers. I mmunocytochemistry (ICC) can be used to detect PAPPA in cells contained in a fluid sample, such as peritoneal fluid or washings, orascites.

Preferred testing methodologies for different analytes are summarised in

Table 1.

Table I

Analyte Raw material Testing Methodology Ascites Tumour cells * FCR/ Methylation-specific FCR cyst fluid * Next generation (high throughput) Free DNA, mRNA or sequencing Peritoneal fluid or miRNA washings * ELISA * ICC/Immunofluorescence Smear * FISH/CISH * Mass spectrometry * Micro-chip-based immunomagnetic detection Blood, Circulating tumour cells * PCR/ Methylation specific PCR Plasma, Circulating free DNA, * Next generation (high throughput) Serum mRNA or miRNA sequencing * ELISA * ICC/Immunolluorescence * FISH/CISH * Mass spectrometry * Nanoparticle-based immunomagnetic detection FFPE biopsy, Protein * IHC Resection DNA * FISH/CISH specimen RNA * PCR/ Methylation specific PCR * Next generation (high throughput) sequencing * Mass spectrometry PAPPA protein expression can be classified using conventional methods, for example, a combined score for membrane and cytoplasmic staining intensity and staining distribution can be evaluated using the following scoring system: intensity of positive signal is scored as follows: negative (0), no staining is observed; weakly positive (1+), a faint/barely perceptible membrane/cytoplasmic staining is detected; moderately positive (2+), weak staining is detected; strongly positive (3+), strong membrane/cytoplasmic staining is detected. Distribution of staining is scored as follows: focal positivity of less than 10% of cells (1), positivity between 10-50% of cells (2), positivity over 50% of cells (3). Scores for intensity and distribution are combined to give a minimum score of 2 and a maximum score of 6.

For analysis of a relatively small number of PAPPA proteins, a quantitative immunoassay such as a Western blot or ELISA can be used to detect the amount of protein (and therefore level of expression) in a ovarian tissue sample. Semi-quantitative methods such as IHC and ICC can also be used.

To analyse a larger number of samples simultaneously, a protein array may be used. Protein arrays are well known in the art and function in a similar way to nucleic acid arrays, primarily using known immobilised proteins (probes) to "capture" a protein of interest. A protein array contains a plurality of immobilised probe proteins. The array contains probe proteins with affinity for PAPPA.

Alternatively, 20 Gel Electrophoresis can be used to analyse simultaneously the expression level of PAPPA. This method is well known in the art; a sample containing a large number of proteins are typically separated in a first dimension by isoelectric focusing and in a second dimension by size. Each protein resides at a unique location (a "spot") on the resulting gel. The amount of protein in each spot, and therefore the level of expression, can be determined using a number of techniques. An example of a suitable technique is silver-staining the gel followed by scanning with a Bio-rad FX scanner and computer aided analysis using MELANIE 3.0 software (GeneBio). Alternatively, Difference Gel Electrophoresis (DIGE) may be used to quantify the expression level (see Von Eggeling etal; Int. J. Mol Med. 2001 Oct; 8(4):373-7.

As explained above, all references herein to determining the presence/level of PAPPA also encompass determining the functional activity of PAPPA. PAPPA activity can be measured using conventional techniques. For example, PAPPA activity can be determined by examining IGFBP-4 proteolytic activity in a sample. Methods for detecting PAPPA activity are disclosed in US patent publication No. 2005/0272034, the content of which is incorporated herein by reference. Alternatively, loss of PAPPA activity may also be determined by mutation-specific POR analysis.

In one embodiment, PAPPA activity may be detected by screening for proteolytic cleavage of its substrate IGFBP-4 using immunoblotting. PAPPA secreted into the medium can be detected by incubating the media samples in a buffer, such as 50mM Tris (pH 7.5) supplemented with IGFBP-4. Samples can then be incubated (for example, at 3T0 for 4hrs) and the proteolytic products detected by immunoblotting using available commercial antibodies against IGFBP-4 protein.

Alternatively, PAPPA activity can be detected by using an ELISA (Enzyme linked immunosorbent assay), wherein specific antibodies against PAPPA are immobilised in the well of a microtitre plate. After washing away unbound protein the activity of PAPPA can be measured using a synthetic substrate which liberates a coloured product only if the primary specific reaction between PAPPA and its antibody has occurred and the bound PAPPA is active.

The colour developed is quantified spectrophotometrically using a microplate reader.

Alternatively, the interaction between PAPPA and its substrate IGFBP-4 can also be assessed using Biacore (Surface plasmon resonance technology) or Fluorescence polarisation assay. These methods offer the advantage of being very sensitive and specific and can easily be adapted to develop a high-throughput assay.

As a control for the above described methods a mutant PAPPA protein (E483Q), which is proteolytically inactive, may be used.

The content of all publications referred to herein is incorporated by reference.

The invention is described with reference to the accompanying drawings, by the following non-limiting examples.

EXAMPLE I -Mitotic delay phenotype and PAPPA expression levels as markers of ovarian cancer Physical units Throughout this example section, all references to hours, minutes, seconds, milliseconds and millivolts are abbreviated as hrs, mm, s, ms and my, respectively.

Tissue specimens Formalin-fixed, paraffin-embedded (FFPE) tissue resection specimens and tissue microarrays (TMA) were obtained from commercial sources (Tissue Solutions, Glasgow, UK, and Insight Biotechnology, Wembley, UK, respectively).

Samples included invasive ovarian carcinoma (n=188); borderline ovarian cancer (n=38); benign ovarian cancer (n=25); normal ovarian epithelium (n=30); colon adenocarcinoma (n=59); transitional cell carcinoma of the bladder (n=40); penile squamous cell carcinoma (n=33); gastric adenocarcinoma (n=30); malignant melanoma (n=29); small cell lung cancer (n=43); and non-Hodgkin lymphoma (n=48). Histological specimens had been reviewed by three independent qualified pathologists at diagnosis and assessed for histological subtype and nuclear grade according to World Health Organization (WHO) criteria.

Chemicals Chemicals were purchased from Sigma Aldrich (Dorset, UK) unless otherwise stated.

Cell culture Caov-3 cells (ATCC® HTB75TM) were cultured in DMEM (Life Technologies; Cat. No. 31966-021) supplemented with 10% EBS (Life Technologies; Lot #41Q822OK) and GIutaMAXTM (Life Technologies; Cat. No. 35050-038). Ovcar- 3 cells (ATCC® HTB161TM) were cultured in RPMI 1640 (LGC Standards; Cat.

No.30-2001) supplemented with 20% FBS (Life Technologies) and 10 pg/mI bovine insulin (Sigma Aldrich, Cat. No. 10516). Cells were cultured at 37°C with 5% CO2 and »= 95% humidity. Cells were harvested following incubation with TrypLETM Express (Life Technologies; Cat.No. 126005-010). Cell density and viability were determined by trypan blue exclusion using a Countess® Automated Cell Counter (Life Technologies; Mod No. C10281). The population doubling time was calculated by PDT = (t2 -ti) / 3.32 x (log n2 -log nl), where t is the sampling time and n is the cell density at the time of sampling.

Photomicrographs shown were taken using a Panasonic digital camera fitted to a Leica IL-LED microscope. Where indicated, Caov-3 cells were treated with 100 ng/ml IGFR blocking antibody (Millipore; Lot #30080) for 24 hrs, 100 ng/ml IGF-1 (Life Technologies; Lot #73159414A) for 48 hrs or 5 nM paclitaxel (Taxol® Sigma Aldrich; Lot #091M1781V) for 24 hrs. Ovcar-3 cells, where indicated, were treated with 100 ng/ml IGF-1 for 48 hrs or 25 nM paclitaxel for 24 hrs.

PAPPA overexpression Full length PAPPA cDNA (NM-002581.3) cloned into pCMV6-XL5 vector available commercially from Origene was used for this experiment. PAPPA cDNA (20 rig) was transfected into 2 x 106 Ovcar-3 cells using the Neon® Transfection System (Life Technologies; Model No. MPK5000) according to the manufacturer's recommendations with the following optimised settings: two pulses of 1050 my, 30 ms. 48 hrs post-transfection PAPPA expression was determined by qRT-PCR, immunoblotting and the phenotype established by mitotic phase distribution analysis.

RNA interference Knock-down of PAPPA expression was carried out using PAPPA Silencer® Validated 5iRNA (Life Technologies; Cat.No. 4390817). Non-targeting 5iRNA Silencer® Select negative control (Life Technologies; Cat. No. 439-0843) was used as a negative control. 100 nM siRNA was transfected into 2 x 106 Caov-3 cells using the Neon® Transfection System according to the manufacturers recommendations with the following optimised settings: two pulses of 1150 my, ms. Cells were incubated for 48 hrs and silencing of PAPPA was assessed by qRT-PCR, immunoblotting and the phenotype established by mitotic phase distribution analysis.

Cell cycle analysis Cells that reached 60-70% confluency were harvested by treatment with TrypLETM Express. The culture medium supernatant was pooled with the detached cells and centrifuged for 3 mm at 194 x g (this step was included to ensure retention of all mitotic cells and avoid loss through mitotic shake off). The cell pellet was washed and resuspended in PBS to give a concentration of 2 x 106 cells/mI. The resuspended cells were transferred to a Coulter Flow cytometry tube (Beckman Counter) and fixed by drop wise addition of 1.5 ml ice-cold 100% ethanol whilst vortexing. The cells were placed on ice for 30 mm to fix. Following ethanol fixation, the cells were pelleted by centrifugation for 5 mm at 194 x g.

The supernatant was carefully removed and the cells washed with PBS (added drop-wise whilst vortexing). The cells were finally resuspended in 300 p1 DNA Prep P1 solution (Beckman Coulter; Cat. No. 6607055) and incubated for 10 mm at room temperature in the dark prior to cell cycle analysis on the Navios Flow Cytometer (Beckman Coulter; Ser. No. AN36127). Data presented were analysed using the Multicycle-AV software (Phoenix Flow Systems V328).

Cytospin preparation Cells that had reached 60-70% confluency were harvested by treatment with TrypLETM Express. The culture medium supernatant was pooled with the detached cells and centrifuged for 3 mm at 194 x g (this step was included to ensure retention of all mitotic cells and avoid loss through mitotic shake off). The cell pellet was washed and resuspended in PBS to give a concentration of 0.5 x 106 cells/mI. The resuspended cells were cytospun onto Leica Snow Coat glass slides (Leica; Cat. No. 3808100GE) for 5 mm at 60 x g using a Cytospin 4 cytocentrifuge (Thermo Scientific; Mod. No. A78300101). Cytospins were fixed in 10% neutral buffered formalin for 10 mm at room temperature. Slides were either processed immediately or were stored overnight at 4°C in 10% neutral buffered formalin.

Immunohistochemistry Section deparaffinisation, antigen retrieval and immunostaining were performed using the Leica Bond-Ill Autostainer and Bond Polymer Refine Detection kit (Leica; Cat. No. D59800), according to the manufacturer's instructions. Heat-mediated antigen retrieval at pH 6.0 for 30 mm was used for both H3SlOph and PAPPA antigens. Primary antibodies were applied for 40 mm at the following dilutions: PAPPA (DAKO; Lot #00061479) at 1/200; H3SlOph (Millipore; Lot #2066052) at 1/2000. Cytospin preparations were immunostained using the same protocol without the deparaffinisation step. Incubation without primary antibody was used as a negative control and sections of tonsil and placenta were used as positive controls for H3SlOph and PAPPA antibodies, respectively.

Mitotic phase distribution analysis Two consecutive serial sections from each FFPE tissue sample and two cytospin preparations for each cell line were immunostained with H3SlOph. Five to 20 high power fields (400x magnification) were image-captured using a Leica DM2500 microscope, Leica DFC295 digital camera and Leica Application Suite software V4.1. A minimum of five mitotic cells for each sample were used to determine the mitotic phase distribution. All mitotic cells within the captured fields were classified based on their chromosomal morphology as prophase/prometaphase, metaphase, anaphase and telophase according to established criteria. Mitotic cells were classified as prophase/prometaphase if chromosome condensation was evident with or without the nuclear envelope intact (prophase or prometaphase, respectively). Metaphase was defined if the sister chromatids showed alignment at the metaphase plate in the centre of the spindle. Anaphase was defined if the sister chromatids were separated into two distinct sets migrating to opposite poles. Telophase was defined by the complete segregation of the sister chromatids and the reformation of the nuclear envelope around each set of chromosomes. A population of cells was classified as delayed' if at least a third of mitotic cells resided in prophase/prometaphase.

Laser capture microdissection Laser-capture microdissection was performed on a Leica LMD6500 microdissection microscope V7.4.1, using the Leica Application Suite software V4.2, a 355 nm ultraviolet (UV-A) Nd: YAG solid-state laser, and UVI 5x0.12 Microdissection objective (Leica). Formalin-fixed, paraffin-embedded sections (Tissue Solutions, Glasgow, UK) or tissue microarray cores (Insight Biotechnology, Wembley, UK) were cut at 5 pm thickness using a rotary microtome and mounted on PET-Membrane 1.4pm slides (Leica; Cat. No. 11505151). Sections were baked in a 37°C oven for 2 hrs and then deparaffinised in xylene, rehydrated through graded alcohols to water, stained with Mayer's haematoxylin for 10 s, washed in tap water and then air-dried for 20 mm. Using the Leica Application Suite software V4.2, regions of interest were selected with the drawing tool, cut out by the laser and collected by gravity into the cap of a system-mounted 0.2 ml PCR tube (Greiner Bio-One) containing 50 p1 of DNA extraction buffer [PCR buffer (Amplitaq; Cat. No. Y02028) supplemented with 1.5 mM Mg012, 0.5% Tween 20 and 0.32 mg/mI Proteinase K (Ambion; Cat.No. AM2546)]. The samples were vortexed, pulse centrifuged, and incubated at 56'C in a Veriti Thermocycler for a total of 48 hrs with 0.48 p1 of (20 mg/mi stock) Proteinase K added after 24 hrs of incubation. After 48 hrs incubation, samples were heated to 99t for 15 mm to inactivate Proteinase K, then cooled to 4'C for 10 mm, and stored at -20'C for future use. DNA concentration was determined using Nanovue Plus Spectrophotometer (GE Healthcare; Mod. No. 28956058).

MethyLight assay Genomic DNA was extracted from cells using the QlAamp DNA kit (Qiagen; Cat.No. 51304) according to the manufacturers protocol. DNA isolated was quantified using NanoVue Plus spectrophotometer. For each reaction 400-500 ng of genomic DNA from cell lines or from tissue (using laser capture microdissection) was bisulfite-modified using the EZ DNA Methylation-Gold Kit (Zymo Research; Cat. No. D5006) according to the manufacturers instructions.

Bisulfite-modified DNA was stored at -80°C until required. CpG methylated HeLa genomic DNA (New England Biolabs; Cat. No. N4007S) was used as positive control. Two sets of primers and probes were designed for bisulfite-modified DNA: a methylated set for PAPPA and collagen 2A1 to normalise for input DNA.

Real time PCR reactions were carried out using the TaqMan® Universal PCR Master Mix [No AmpErase® UNG (Life technologies; Cat. No. 00058004345-01)] with 20 ng DNA, 0.3 pM probe and 0.9 pM of both forward and reverse primer.

The reactions were carried out on a StepOne Plus Real Time PCR system (Life Technologies; Mod. No. 272006346). The cycling conditions were: 95°C (10 mm), followed by 50 cycles of 95°C (15 s), 60°C (1 mm). The results of the PCR reaction were analysed (DDC1 and RQ calculations) using the StepOne software V2.2. The percentage of methylated PAPPA was calculated by dividing the PAPPA: COL2AI ratio of the sample by the PAPPA: COL2A1 ratio of the CpG methylated HeLa genomic DNA and multiplying by 100. The abbreviation PMR (percentage methylated reference gene) was used to indicate this measurement.

Primers used for this study are shown in Table 2.

Table 2

Gene Forward primer Reverse primer Probe sequence 5'-3' sequence 5'-3' sequence 5'-3' PAPPA GCGTGTTTGTGCGAG CGCCTTCCGAATATACC 6-FAM-

AGTTGT CATT TCGCCCGAATATCTCTACGCCGCT-

(SEQ ID NO. 3) (SEQ ID NO.4) BHQ-1 (SEQ ID NO. 5) COL2A1 TCTAACAATTATAAAC GGGAAGATGGGATAGAA 6-FAM-

TCCAACCACCAA GGGAATAT CCTTCATTCTAACCCAATACCTATC

(SEQ ID NO. 6) (SEQ ID NO. 7) CCACCTCTAM-BHQ-1 (SEQ ID NO. 8) qRT-PCR analysis Total cellular RNA was isolated using the Ambion PureLink RNA Mini kit (Life Technologies; Cat. No. 12183018A), according to the manufacturer's instructions. qRT-PCR reactions were carried out using the TaqMan® RNA-to-CTTM 1-Step kit (Life Technologies; Cat. No. 4392938) according to the manufacturer's instructions. PCR reactions were carried out in StepOne Plus Real Time PCR system (Life Technologies). 400 ng template RNA, final primer concentration of 900 nM and final probe concentration of 250 nM was used in each individual PCR reaction. The probes used in this study were: PAPPA (assay ID Hs01032307_ml Life Technologies) which spans exons 21-22 and GAPDH (assay ID Hs03929097_gl Life Technologies) which locates to exon boundary 9 of transcript variant 1 and exon boundary 8 of transcript variant 2.

The cycling conditions were: 48°C (15 mm), 95°C (10 mm) followed by 40 cycles of 95°C (15 s), 60°C (1 mm). The results of the qRT-PCR reaction were analysed (DDCT and RQ calculations) using the StepOne software V2.2.

Immunoblofting Cells were lysed in crude cellular extraction buffer [10 mM HEPES pH 7.8, 10 mM KCI, 1.5 mM MgCI2, 0.34 M glucose, 10% Triton X-100 and lx Complete Protease Inhibitor cocktail (Roche; Cat. No. 04693132001)]. After incubation on ice for 10 mm the sample was centrifuged for 5 mm at 1,301 x g at 4°C. The supernatant containing the crude cellular extract was retained for further analysis. The protein concentration was determined using the Bradford protein assay kit (Pierce; Cat. No. 1856209) according to the manufacturer's protocol.

20-30 pg of crude cellular fractions and MagicMarkTM (Life Technologies; Cat.

No. LC5602) were separated using Novex® 4-20% Tris-Glycine SDS PAGE (Life Technologies; Cat. No. EC6028). Proteins were transferred from the polyacrylamide gel onto PVDF membrane using the iBlot® dry electroblotting system (Life Technologies; Mod. No. iBlo?). The membrane was blocked for 60 mm in PBS supplemented with 10% milk. The membrane was further probed with rabbit polyclonal anti-PAPPA antibody. This step was carried out overnight at 4°C with gentle agitation. After incubation with the primary antibody, the membrane was washed five times for 10 mm with PBS. HRP conjugated secondary goat anti-rabbit antibody (Dako; Cat. No. P0448) in PBS with 10% milk was added to the membrane and incubated for 60mm at room temperature.

Equal volumes of reagent A and B from ECLSeIectTM kit (GE Healthcare; Cat.

No. RPN2235) were added to the membrane and incubated for 1 mm at room temperature. Images were captured using the GeneGnome chemiluminescent detection system (Syngene; Mod. No. 75000). The membrane was reprobed with anti-p actin antibody (Sigma Aldrich; Lot #121M4846) to ensure equal loading of total protein in each lane.

Analysis of PAPPA protein expression Each FEPE tissue specimen was immunostained for PAPPA and digitally scanned at 200x magnification using the Leica SCN400 scanner. The entire section was viewed using the Leica SlidePath Gateway LAN software V2.0 and given a combined score for intensity and distribution of PAPPA staining. Intensity of positive signal was scored from 0-3+ (0 being negative for PAPPA and 3+ showing the highest staining intensity). Distribution of PAPPA staining was scored from 0-3 (0 being negative for PAPPA, 1 for focal positivity of less than 10% of cells, 2 if between 10-50% of cells were positive and 3 if over 50% of cells showed PAPPA positive staining). Positive PAPPA expression was defined as a combined score of 2 or above.

Invasion assay FBS (10%) mediated cell migration through an extracellular matrix was measured by Boyden chamber assay [(BD BiocoatTM Matrigel invasion chamber) BD Biosciences; Cat. No. 354480)] following the manufacturer's instructions.

Briefly, Caov-3 cells were transfected with PAPPA 5iRNA or control 5iRNA (negative control) as described above. Caov-3 cells were treated with IGFR blocking antibody or untreated as described previously. Ovcar-3 cells were treated with IGF-1 or untreated as described previously. Prior to transfections or treatments the cells were serum starved for 24 hrs. Caov-3 cells were collected in DMEM medium containing 5% BSA, counted and 0.5 x i05 cells were added to each invasion assay chamber. Ovcar-3 cells were collected in RPMI medium containing 5% BSA, counted and 2 x io5 cells were added to each invasion assay chamber. After incubation for 48 hrs the invasion chamber inserts were washed with PBS, fixed in 4% formaldehyde for 5 mm, stained with 0.01% crystal violet or haematoxylin and cells from random areas on the filters were counted.

Statistical analysis The proportion of mitotic cells in prophase/prometaphase was calculated for each specimen of premalignant and malignant tissue. Receiver Operating Characteristic (ROC) curves for differentiating ovarian cancer from other malignancies (pooled) using the proportion of cells in prophase/prometaphase were constructed for various minimum numbers of mitotic cells. It was clear that the ROC curve was not compromised by letting the minimum requirement be as low as five mitotic cells and was significantly better than a null diagnostic test (p <0.0001) (Figure 3). The p value was obtained by calculating the area under the curve (AUC) using the trapezoidal method and the result interpreted as a Mann-Whitney statistic with Wald approximation. In the interest of using as many specimens as possible, the evaluability threshold for analysis of mitotic delay was set to at least five mitotic cells observed per specimen. A specimen was declared delayed' if at least one third of its mitotic cells were in prophase/prometaphase. This requirement was derived by balancing the sensitivity and specificity associated with distinguishing ovarian cancer from other malignancies: 96% of evaluable ovarian cancer specimens had at least one third of their mitotic cells in prophase/prometaphase, while 87% of evaluable other malignancies had less than one third of their mitotic cells in prophase/prometaphase (p < 0.0001) (Figure 4). The proportion of evaluable specimens with mitotic delay was compared between sources of specimen (ovarian cancer, other malignancies) using Pearson's chi-squared test with Yates's continuity correction. The median proportion of mitotic cells in prophase/prometaphase was compared between sources of specimen using the Mann-Whitney test. All significance probabilities reported are two-sided. The association of mitotic delay with tumour differentiation and tumour stage was assessed using a logistic regression test with stagS grade as the continuous variable and Wald approximation. In vitro cell line data were statistically analysed using the Student's unpaired t-test. Results that were statistically significant at p < 0.05 are indicated by an asterisk symbol.

RESU LTS

Ovarian cancer is specifically enriched in early mitotic figures In evaluable tissue specimens from seven common human tumour types, including lung, colon, bladder, penile, melanoma, gastric and lymphatic cancer (n=282 patients), the majority of mitotic cells were in metaphase (Figure 2 and Figure 5). In marked contrast, the inventors found a strong prophase/prometaphase enrichment in 96% (148 out of 154 patients) of ovarian cancers compared with only 13% for the combined group of other malignancies (Figure 5 and Figure 6A). Cases were defined as displaying an early mitotic delay if at least one third of mitotic cells was in prophase/prometaphase. A minimum count of 5 mitotic cells was shown to be specific by ROC curve analysis (AUC: 0.9759, Figure 3). The cut-point of one third was chosen to allow the proportion of specimens in the combined group of other malignancies properly classified as non-delayed (87%) to be approximately equal to the proportion of ovarian cancer specimens properly classified as delayed (96%) (see Figure 4). The mean proportion of mitotic ovarian cancer cells in prophase/prometaphase was 55% (median 55%) compared with 24% (median 24%) in the other malignancies (Figure 2B-C, Figure 5 and Figure 6B), indicating that early mitotic delay is a hallmark of ovarian cancer. Importantly, the inventors found that the mitotic delay phenotype could be detected already in 86% (18 out of 21 patients) of non-invasive borderline lesions, in which the mean proportion of mitotic cells in prophase/prometaphase was 49% (median 48%) (Figure 2B and Figure 60). Thus, the inventors identified an unexpectedly high frequency of early mitotic figures (prophase/prometaphase) in nearly all tested ovarian cancers, revealing a formerly unrecognised delay in mitotic progression in this tumour type.

PAPPA loss is linked to mitotic delay in ovarian cancer Since promoter methylation represents a common mechanism for loss of tumour suppressor genes during cancer development, the inventors hypothesised that epigenetic silencing of PAPPA could be linked to the mitotic delay phenotype, which proved to be the case in ovarian cancer. MethyLight assays (Widschwendter, M. et al. Cancer research (2004) 64, 3807-3813) showed that PAPPA is strongly hypermethylated in the 5' regulatory region of the gene in invasive ovarian cancer and in non-invasive borderline ovarian cancer (Figure 7A). Forty% (70 out of 174 patients) of invasive ovarian cancers and 57% (17 out of 30 patients) of borderline ovarian cancers showed PAPPA hyperniethylation (defined as PMR>1; percentage methylated reference gene).

In contrast, PAPPA was non-methylated in 93% (14 out of 15 patients) of normal ovarian samples and 79% (19 out of 24 patients) of benign ovarian lesions (Figure 7A; note that 14 cases of invasive ovarian cancer, 8 cases of borderline ovarian cancer, 1 benign ovarian case and 15 cases of normal ovary were not available for MethyLight analysis due to poor preservation of DNA). This made PAPPA a strong candidate to explain the prophase/prometaphase delay found in ovarian cancer. To test if PAPPA promoter methylation indeed caused gene silencing, the inventors used a commercially available anti-PAPPA antibody (DAKO) for immunostaining of FFPE tissue sections. Immunostaining profiles showed that indeed 89% (58 out of 65 patients) of borderline and invasive ovarian cancers with methylated PAPPA promoter and exhibiting the mitotic delay phenotype were not expressing PAPPA protein (Figure 73 and 70).

Loss of PAPPA expression is linked to tumour progression Immunostaining profiles showed PAPPA protein expression in 52% (13 out of 25 patients) of benign ovarian lesions, 31% (11 out of 36 patients) of borderline ovarian carcinomas and 11% (20 out of 184 patients) of invasive ovarian carcinomas (Figure 7B-C). This indicates a significant trend showing a link between loss of PAPPA protein and tumour progression. PAPPA expression also inversely correlated with increasing tumour stage (Figure 8). The inventors showed that 16% (8 out of 51 patients) of stage 1 ovarian carcinomas expressed PAPPA, compared to 9% (2 out of 22 patients) of stage 2 and 3% (2 out of 59 patients) of stage 3 carcinomas (Figure 8). This trend was significant and indicates that PAPPA loss correlates with progression of ovarian cancer.

PAPPA epigenetic silencing is linked to an increase in the proportion of ovarian cancer cells showing mitotic delay In order to corroborate the hypothesis that PAPPA loss is associated with mitotic delay in ovarian cancer an experimental in vitro model was required. Therefore it was investigated whether in ovarian cancer cell lines a link exists between PAPPA loss and the mitotic delay phenotype. Ovarian cancer cell lines Caov-3 and Ovcar-3 (Figure 9A-D) were chosen for detailed study after determining PAPPA mRNA and protein levels (Figure 9F-G). Mitotic phase distribution was determined by morphological analysis of H3SlOph immunostaining. The Caov-3 cell line has no detectable methylation of the PAPPA promoter and expresses PAPPA at mRNA and protein level (Figure 9E-G). H3SlOph immunostaining demonstrated some mitotic delay (-A7% of the total mitotic cells in prophase/prometaphase) for this cell line (Figure 9H). In keeping with the inventors' in vivo findings, a cell line (Ovcar-3) with hypermethylated PAPPA promoter did not express PAPPA protein (Figure 9E-G) and exhibited a strong mitotic delay phenotype with -76% of mitotic cells in prophase/prometaphase (Figure 9H).

PAPPA loss renders ovarian cancer cells more invasive Next the inventors asked what biological advantage might be conferred to the neoplastic ovarian cell through perturbation of early mitotic progression. To address this question the inventors looked for any correlation between the mitotic delay phenotype and standard clinico-pathological features determined for each ovarian cancer specimen during routine clinical investigation (n=154 evaluable patients). This analysis did not reveal any linkage between mitotic delay and age of patient, tumour differentiation (grade), or morphological subtype (serous, endometrioid, mucinous, clear cell). The presence of the mitotic delay phenotype in nearly all invasive ovarian cancer specimens studied (96%, 148 out of 154 evaluable patients) raises the possibility that this mitotic defect might be linked to the acquisition of invasiveness. To test this hypothesis, the inventors induced the mitotic delay phenotype in Caov-3 cells by 5iRNA mediated F'APPA knockdown and measured the invasiveness of the manipulated cells in Matrigel-coated Boyden chamber assays. Relative to control siRNA, transfection of Caov-3 cells with PAPPA targeting siRNA resulted in significantly decreased PAPPA mRNA and protein expression (Figure bA-B).

Consistent with the PAPPA knockdown, Caov-3 showed a significant increase in the proportion of mitotically delayed cells (Figure IOC-D). Notably, experimental perturbation of mitotic progression in this cell model was associated with a significant increase in the number of cells invading through the matrix of a Boyden chamber (Figure bE). These results show that delayed progression through mitosis resulting from PAPPA down-regulation increases the invasive capacity of ovarian cancer cells.

PAPPA overexpression reverses the mitotic delay phenotype in ovarian cancer cells Since loss of PAPPA expression led to mitotic delay, it was hypothesised that PAPPA overexpression in a PAPPA negative cell line would reverse the mitotic delay phenotype. PAPPA was significantly overexpressed in Ovcar-3 cells compared to control transfections (Figure 1 lA-B). Analysis of mitotic phase distribution in PAPPA overexpressing cells confirmed there was a decrease in the proportion of mitotic cells in prophase/prometaphase (Figure 11C-D). In summary, the results in ovarian cancer cell lines demonstrate that PAPPA is required for progression through mitosis and that PAPPA downregulation through epigenetic silencing (Ovcar-3 cells) or experimentally by 5iRNA knockdown (Caov-3 cells) causes mitotic delay.

EXAMPLE 2 -Methylation as a molecular signature for ovarian cancer in liquid biopsies Materials Plasma samples from ovarian cancer and normal control plasma were obtained from a commercial source (Tissue Solutions, Glasgow, UK). Samples used in this study include healthy female control samples (n=3) and samples obtained from ovarian cancer patients (n=5). Sample characteristics are listed in Table 3.

All use of, or transfer of, blood or blood products was performed in accordance with Human Tissue Authority (HTA) Guidelines.

Table 3

Sample ID code Type Stage Grade Description

control Ni Normal female N/A N/A Human plasma control N2 Normal female N/A N/A Human plasma control N3 Normal female N/A N/A Human plasma Ovarian Endodermal Sinus OC1 Ill B 3 Human plasma cancer Tumour Ovarian Endodermal Sinus 0C2 IA 3 Human plasma cancer Tumour Ovarian Papillary Serous 0C3 IA 2 Human plasma cancer Adenocarcinoma Ovarian Serous 0C4 Ill B 3 Human plasma cancer Adenocarcinoma Ovarian Papillary Serous OCS I c 3 Human plasma cancer Cystadenocarcinoma Methods DNA isolation using the Norgen Plasma Circulating DNA kit Prior to DNA isolation, plasma samples were centrifuged at 16,000 xg for 5 mm to remove remaining cells and debris. Circulating genomic DNA was isolated from all plasma samples using the Norgen Plasma/Serum Circulating DNA Isolation Mini Kit (Geneflow, Cat no. P4-01 24, Lot no. 583601) according to the manufacturer's instructions. Circulating DNA was isolated from plasma in two preparation runs of 400 p1 per sample. The final elution step was performed with 50 p1 elution butter.

Bisulfite Modification DNA isolated from plasma was bisulfite modified using the EZ-DNA Methylation Gold Kit (Zymo Research, Cat no D5006, Lot no. ZRC1 75490) according to the manufacturer's instructions. CpG methylated HeLa genomic DNA (New England Biolabs, Cat no. 40075, Lot no. 51301) was treated in parallel as a reference for total and PAPPA-promoter methylated DNA. 100 i.iL CT conversion reagent was added to 50 pL of DNA isolation eluate. Samples were incubated at 98°C for 10 mm, then at 64°C for 2.5 hours on a MJ Mini 48-Well Personal Thermal Cycler (Bio-Rad, Cat no. FTC-i 148). Samples were processed, purified on a spin column and eluted. Bisulfite-modified DNA was analysed by MethyLight assay or stored at -80°C until required.

MethyLight assay PCR Two sets of primers and probes were designed for bisulfite-modified DNA: a methylated set for PAPPA and a set for collagen 2A1 (COL2AI) to normalise for input DNA. These were the same primer/probe sets shown in Table 2.

q-PCR reactions were carried out using the TaqMan® Universal PCR Master Mix [No AmpErase® UNG (Life technologies, Cat no. 4324018, Lot no. S 12230)] with 2 p1 of the eluted DNA, or H20 as negative control, 0.3 pM probe and 0.9 pM of both forward and reverse primer (shown below). The reactions were carried out in duplicate on a StepOne Plus Real Time PCR system (Life Technologies, Model No. 272006346). The cycling conditions were: 95°C (10 mm), followed by 60 cycles of 95°C (15 s), 6000(1 mm). The results of the PCR reaction were analysed (Ct calculations) using StepOne software V2.3 (Figure 14). The percentage of methylated PAPPA was calculated by dividing the PAPPA: COL2AI ratio of the sample by the PAPPA: COL2A1 ratio of the CpG methylated HeLa genomic DNA and multiplying by 100. The abbreviation PMR (percentage methylated reference gene) was used to indicate this measurement.

RESU LTS

Methylation of the PAPPA promoter can be specifically detected in circulating DNA from plasma of ovarian cancer patients Previous studies carried out by the inventors indicate that epigenetic silencing of PAPPA by promoter hypermethylation is a feature of ovarian cancer. In keeping with this finding, PAPPA promoter hypermethylation was detected in invasive ovarian cancer tumour tissues compared to normal ovarian tissue specimens.

Based on these findings, the inventors hypothesized that circulating tumour DNA carrying PAPPA promoter hypermethylation as a genetic alteration can be detected in plasma samples from ovarian cancer patients.

Figure 12A shows a representative MethyLight amplification plot using DNA purified from plasma of three ovarian cancer samples and one healthy control subject. PAPPA promoter methylation was detected in plasma of ovarian cancer samples, but not in healthy control samples. Figure 12B demonstrates, using the amplitication ot the endogenous control gene COL2A1, that DNA could be purified from all samples.

Figure 13 shows the percentage of plasma samples from ovarian cancer patients and healthy temale controls, in which PAPPA promoter methylation was detectable above the amplification threshold by MethyLight assay. The percentage methylated reference (PMR) was obtained by dividing the PAPPA:COL2A1 ratio of a sample by the PAPPA:COL2A1 ratio of CpG methylated HeLa genomic DNA (control) and multiplied by 100. Circulating DNA extracted from plasma of ovarian cancer samples showed that hypermethylation of the PAPPA promoter (PMR>50) was detectable in 60% of the samples (3 out of 5), compared to none of the control samples (0 out of 3).

Discussion Promoter methylation represents a common mechanism for loss of tumour suppressor genes during cancer development. The inventors previously established that epigenetic silencing of PAPPA is causally connected with ovarian cancer tumorigenesis. Previous studies showed that the PAPPA gene is strongly hypermethylated in the 5' regulatory region of the gene in invasive ovarian cancer and correlating with a loss of PAPPA protein expression. Using DNA extracted from formalin fixed paraffin embedded tissue blocks it was shown that 40% (70 out of 174 patients) of invasive ovarian cancers, showed PAPPA hypermethylation. In contrast, PAPPA was unmethylated in 93% (14 out of 15 cases) of normal ovarian samples. In this study, the inventors claim that an enhanced level of DNA containing methylation of the PAPPA promoter can be detected in the blood of ovarian cancer patients using a MethyLight assay.

Compared to tissue biopsies, blood samples (liquid biopsies) can be easily obtained, and are therefore a suitable sample type for cancer screening.

Circulating DNA is found in the plasma of healthy individuals in the range of 1-ng/ml and can be increased in cancer patients due to lysis of circulating tumour cell, or enhanced cell lysis in tumour regions. Circulating tumour DNA has been tested for tumour specific epigenetic changes such as DNA methylation (Board et al, Biomarker Insights (2), 2007). Notably, DNA methylation in the plasma has been described to correlate to the same epigenetic alterations found in the tumour biopsies (Skvortsova et al, Br J Cancer 94, 2006).

The method described here can be used as an aid in staging patients previously diagnosed with ovarian cancer. The method can be used to assess invasion of ovarian cancer cells into neighbouring tissue or spread to distant sites (staging) by monitoring the level of loss-of-function related genetic alterations in PAPPA.

The method described here can also be used for monitoring the response to treatment in a patient undergoing ovarian cancer therapy, comprising detecting the presence of a loss-of-function related genetic alteration in the PAPPA gene or its regulatory or promoter sequences in a sample obtained from a patient wherein if the genetic alteration is present but its quantitative level decreases during treatment, this is indicative of response to therapy, whereas no change or an upward change in its quantitative level is indicative of non-response to therapy. Moreover, this method can also determine if a patient previously subjected to ovarian cancer therapy has suffered a relapse. The method described in this patent can also be used for blood-based screening of asymptomatic subjects wherein the presence of loss-of-function related genetic alteration suggests an increased risk of ovarian cancer.

Taken together, the results presented here show that PAPPA promoter methylation can be used as a molecular signature for ovarian cancer screening in asymptomatic subjects, staging in invasive ovarian cancer patients, monitoring efficacy to treatment regimens and relapse monitoring in invasive ovarian cancer patients. The invention described in this patent may allow more accurate staging of ovarian cancer and effective monitoring of treatment response and may detect early relapse before it is detected by the standard tests in the clinic.

Claims (24)

1. CLAIMS1. A method for monitoring the response to treatment in a patient undergoing ovarian cancer therapy, comprising detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences in a sample obtained from the patient, wherein the presence of a genetic alteration and downward change in its quantitative levels is indicative of response to therapy, and wherein no change or upward change in its quantitative level is indicative of non-response to therapy.
2. 2. A method according to any of claim 1, wherein the loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences is one or more of methylation, deletion, loss of heterozygosity, a point mutation, insertion, translocation or chromosomal breakage.
3. 3. A method for monitoring the response to treatment in a patient undergoing ovarian cancer therapy, comprising detecting the presence and/or level of PAPPA in a sample obtained from the patient, wherein the absence of PAPPA, or the presence of PAPPA at a reduced level compared to a control is indicative of non-response to therapy.
4. 4. A method according to any of claims 1 to 3, wherein the sample is blood, ascites or peritoneal fluid/washings.
5. 5. A method for determining whether a patient, previously treated with ovarian cancer therapy, has suffered a relapse, comprising detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences in a biological sample obtained from the patient, wherein if a genetic alteration is identified, this is indicative of a relapse.
6. 6. A method according to claim 5, wherein the loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences is one or more of methylation, deletion, loss of heterozygosity, a point mutation, insertion, translocation or chromosomal breakage.
7. 7. A method for determining whether a patient previously treated with ovarian cancer therapy, has suffered a relapse, comprising detecting the presence and/or level of PAPPA in a biological sample obtained from the patient, wherein the absence of PAPPA, or the presence of PAPPA at a reduced level compared to a control, is indicative of a relapse.
8. 8. A method according to any of claims 5 to 7, wherein the sample is blood, ascites, tissue or peritoneal fluid/washings.
9. 9. A method for determining whether a patient previously treated with ovarian cancer therapy has suffered a relapse, comprising identifying the proportion of mitotic cells in a tissue sample obtained from the patient that are in prophase or pro-metaphase and comparing to a pre-determined cut-off value, wherein if the proportion of cells in prophase or pro-metaphase is the same or greater than the cut-off value, this is indicative that the patient has suffered a relapse.
10. 10. A method for determining whether a primary ovarian cancer in a patient has spread away from the site of the primary tumour to another part of the body, comprising detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences in a sample obtained from the patient, wherein the presence of a genetic alteration is indicative of spread.
11. 11. A method according to claim 10, wherein the loss-of4unction-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences is one or more of methylation, deletion, loss of heterozygosity, a point mutation, insertion, translocation or chromosomal breakage.
12. 12. A method according to claim 11, wherein the sample is a blood sample, ascites, peritoneal fluid/washings or tissue sample adjacent to or distant from that of the primary tumour.
13. 13. A method for determining whether a primary ovarian cancer in a patient has spread away from the site of the primary tumour to another part of the body, comprising identifying the proportion of mitotic cells in a tissue sample obtained from the patient that are in prophase or pro-metaphase and comparing to a pre-determined cut-off value, wherein if the proportion of cells in a prophase or pro-metaphase is the same or greater than the cut-off value there is a risk that the cancer has spread, and wherein the tissue sample is from a site adjacent to or distant from that of the primary tumour.
14. 14. A method for determining whether a primary ovarian cancer in a patient has spread away from the site of the primary tumour to another part of the body, comprising detecting the presence and/or level of PAPPA in a sample obtained from the patient, wherein the absence of PAPPA, or the presence of PAPPA at a reduced level compared to a control, is indicative of spread.
15. 15. A method according to claim 14, wherein the sample is a blood sample, ascites, peritoneal fluid/washings or tissue sample adjacent to or distant from that of the primary tumour.
16. 16. A method for predicting disease progression in a patient with invasive ovarian cancer, comprising identifying the proportion of mitotic cells in a tissue sample obtained from the patient that are in prophase or prometaphase and comparing to a pre-determined cut-off value, wherein if the proportion of cells in prophase or prometaphase is the same or greater than the cut-off, the prediction is reduced disease-free and overall survival.
17. 17. A method for predicting disease progression in a patient with invasive ovarian cancer, comprising detecting the presence of a loss-of-function-related genetic alteration in the PAPPA gene or its regulatory or promoter sequences in a sample obtained from the patient, wherein the presence of a genetic alteration is predictive of reduced disease-free and overall survival.
18. 18. A method for predicting disease progression in a patient with invasive ovarian cancer, comprising detecting the presence and/or level of PAPPA in a biological sample obtained from the patient, wherein the absence of PAPPA, or its presence at a reduced level compared to a control, predicts reduced disease-tree and overall survival.
19. 19. A method according to any of claims 17 or 18, wherein the sample is blood, ascites, tissues or peritoneal fluid/washings.
20. 20. A method according to any of claims 3, 7, 14 and 18, wherein the presence of PAPPA is identified using a PAPPA-specific antibody or a probe for the PAPPA gene, mRNA or a specific PAPPA mutation.
21. 21. A method according to any of claims 9, 13 and 16, wherein the cut-off value is at least 30% of the mitotic cells in the sample.
22. 22. A method according to any of claims 9, 13, 16 and 21 wherein at least five of the cells in the sample are in mitosis.
23. 23. A method according to any of claims 9, 13, 16, 21 and 23 wherein the proportion of cells that are in prophase or pro-metaphase is determined using immuno-detection.
24. 24. A method according to claim 23, wherein immuno-detection is carried out using an H3SlOph antibody.