WO2015114350A1 - Biomarker - Google Patents

Abstract

In one aspect, the present invention provides a method for detecting pancreatic cancer, cholangiocarcinoma, ovarian cancer or bladder cancer in a subject, the method comprising (a) determining a level of proline-rich protein 11 (PRRll) or PRRll expression in a sample obtained from the subject; and (b) comparing the level of PRRll or PRRll expression in the sample to a control level; wherein an increased level of PRRll or PRRll expression in the sample compared to the control level is indicative of the presence of pancreatic cancer, cholangiocarcinoma, ovarian cancer or bladder cancer in the subject.

Description

BIOMARKER

FIELD OF THE INVENTION

The present invention relates to the field of cancer diagnosis and treatment. In particular, the invention relates to a novel biomarker of pancreatic cancer and cholangiocarcinoma, as well as of ovarian cancer, bladder cancer and lung cancer, and to methods of treating such conditions following detection of the biomarker in a sample from a subject.

BACKGROUND TO THE INVENTION

Pancreatic cancer has minimal survival rates compared to a number of other more treatable cancers. For instance, typical survival is 7-12 months following diagnosis, with less than 20% of patients alive at 1 year, and less than 5% alive at 5 years (see e.g. Everhart 2009, Gastroenterology 136:1 134-1 1449). This is mainly due to very late diagnosis. Because of this, typically only 20% of newly diagnosed patients are suitable for surgery. Early diagnosis is thus critical and defining an 'at risk' group for screening is required; family history and risk factors such as chronic pancreatitis, diabetes, alcoholism & smoking can be used to define at risk groups. If diagnosed early enough for surgical intervention, five-year survival rates are 20-50%.

There is therefore a need for the establishment of new methods for early diagnosis, prognostic stratification and differential diagnosis of pancreatic cancer. Advances in these areas are pivotal to improve the prognosis of this malignancy, since timely surgical resection of early stage tumors is currently the only effective means of treatment of this disease.

Early symptoms of pancreatic cancer are rare, uncharacteristic and often present as pancreatitis. Thus, pancreatic cancers are commonly diagnosed in an advanced stage of the disease. Currently used diagnostic methods include imaging techniques such as endoscopic ultrasound (EUS), spiral computer tomography (CT), magnetic resonance cholangiopancreatography ( RCP) or endoscopic retrograde cholangiopancreatography (ERCP) (Dewitt 2006, Gastroenterol. Hepatol. (4):717-25). Unfortunately, the resolution of these technologies for detecting neoplastic lesions within the pancreas is in the range of 3-10 mm. Thus, they are not able to detect pancreatic neoplasia at a curable stage.

The serum concentration of conventional tumor markers such as CA19-9 is increased in a subset of pancreatic cancer patients (Fry 2008, Langenbecks Arch Surg. (393): 883-90). However, so far all available markers lack sensitivity and tumor specificity. Thus, new approaches are urgently needed to increase the diagnostic sensitivity towards the detection of pancreatic cancer, as well as prognostic subgroups of advanced tumors.

Cholangiocarcinoma is a very rare (1-2 cases per 100,000) type of cancer which occurs in the bile ducts. Similarly to pancreatic cancer, cholangiocarcinoma is often detected late and thus has a very poor prognosis. Non-resectable disease has a typical survival time of less than 6 months, while resectable disease has a 5 year survival rate of 20-50%. IMP3 is a biomarker typically considered to be the gold standard used in biliary brushes followed by cytology. IMP3 is very specific but lacks sensitivity in detecting all cases of cholangiocarcinoma.

Ovarian cancer occurs at a rate of approximately 20 cases per 100,000 women per year in Europe. The incidence of this cancer increases with age, particularly after the menopause. Genetic factors are known to play a role in some cases, since ovarian cancer in close relatives is a significant risk factor for development of the disease. Early diagnosis significantly improves survival rates, however diagnosis is often in the later stages of disease. At this late stage patients typically present with pelvic or abdominal pain, urinary frequency or urgency, increased abdominal size or bloating. This diagnosis may be confirmed by a pelvic examination, transvaginal ultrasonography and detection of carbohydrate antigen 125 (CA125) in the tumour tissue.

Accordingly, there is still a need for new biomarkers of pancreatic cancer and cholangiocarcinoma. In addition, there is a need for new biomarkers for further cancers, such as ovarian cancer, bladder cancer and lung cancer. In particular, there is a need for biomarkers which are sensitive, specific and capable of detecting these conditions in at-risk subjects at an early stage. Such biomarkers are capable of providing significant benefits by enabling successful intervention, including by surgery, in a greater number of patients and thereby improving survival rates.

SUMMARY OF THE INVENTION

Accordingly, in one aspect the present invention provides a method for detecting pancreatic cancer or cholangiocarcinoma in a subject, the method comprising (a) determining a level of proline-rich protein 11 (PRR11) or PRR11 expression in a sample obtained from the subject; and (b) comparing the level of PRR11 Protein or PRR11 expression in the sample to a control level; wherein an increased level of PRR11 or PRR11 expression in the sample compared to the control level is indicative of the presence of pancreatic cancer or cholangiocarcinoma in the subject.

In another aspect the present invention provides a method for detecting ovarian cancer or bladder cancer or lung cancer in a subject, the method comprising (a) determining a level of proline-rich protein 11 (PRR11) or PRR11 expression in a sample obtained from the subject; and (b) comparing the level of PRRll or PRRll expression in the sample to a control level; wherein an increased level of PRRll or PRRll expression in the sample compared to the control level is indicative of the presence of ovarian cancer, bladder cancer or lung cancer in the subject.

In one embodiment, PRRll is detected using an antibody which binds specifically to PRRll. Preferably the level of PRRll in the sample is determined by immunohistochemistry or an enzyme- linked immunosorbent assay (ELISA).

In another embidoiment, the level of PRRll expression is determined by analysing the level of PRRll mRNA in the sample obtained from the subject.

In one embodiment, the sample comprises a pancreatic biopsy sample or a pancreatic cyst drainage sample. In an alternative embodiment, the sample comprises a bile duct biopsy sample or a biliary brushing sample. In a further embodiment, the sample comprises an ovarian biopsy sample. In another embodiment, the sample comprises a bladder biopsy sample or urine sample. In another embodiment, the sample comprises a lung biopsy sample.

In one embodiment, the subject is suspected to be suffering from pancreatic cancer or cholangiocarcinoma. In another embodiment, the subject is suspected to be suffering from ovarian cancer bladder cancer or lung cancer.

In an alternative embodiment, the subject is at elevated risk of developing pancreatic cancer or cholangiocarcinoma. For instance, the subject may have a family history of pancreatic cancer or cholangiocarcinoma, or a genetic predisposition for developing pancreatic cancer or cholangiocarcinoma. In another embodiment, the subject is suffering from (chronic) pancreatitis, has a family history of (chronic) pancreatitis, and/or has a genetic predisposition for developing (chronic) pancreatitis. Preferably the method is capable of distinguishing pancreatic cancer from pancreatitis in the subject.

In another embodiment, the subject is at elevated risk of developing ovarian cancer, bladder cancer or lung cancer. For instance, the subject may have a family history of ovarian cancer, bladder cancer or lung cancer, or a genetic predisposition for developing ovarian cancer, bladder cancer or lung cancer.

In one embodiment, the control level comprises a level of PRRll or PRRll expression in a control sample from a subject not suffering from pancreatic cancer or cholangiocarcinoma. In another embodiment, the control level comprises a level of PRRll or PRRll expression in a control sample from a subject not suffering from ovarian cancer, bladder cancer or lung cancer. In one embodiment, the method further comprises determining a level of CA 19-9 or CA 19-9 expression in a (e.g. serum or plasma) sample obtained from the subject, and comparing the level of CA 19-9 or CA 19-9 expression in the sample to a control level; wherein an increased level of CA 19- 9 or CA 19-9 expression in the sample compared to the control level is indicative of the presence of pancreatic cancer in the subject.

In another embodiment, the method further comprises determining a level of IMP3 or IMP3 expression in a sample obtained from the subject, and comparing the level of IMP3 or IMP3 expression in the sample to a control level; wherein an increased level of IMP3 or IMP3 expression in the sample compared to the control level is indicative of the presence of cholangiocarcinoma in the subject.

In another embodiment, the method further comprises determining a level of CA125 or CA125 expression in a sample obtained from the subject, and comparing the level of CA125 or CA125 expression in the sample to a control level; wherein an increased level of CA125 or CA125 expression in the sample compared to the control level is indicative of the presence of ovarian cancer in the subject.

The sample may be for example a blood, plasma, serum or urine sample from the subject.

In a further aspect, the invention provides a method for treating pancreatic cancer or cholangiocarcinoma in a subject, comprising a) detecting pancreatic cancer or cholangiocarcinoma in the subject by a method as defined above; and b) treating the subject for pancreatic cancer or cholangiocarcinoma if the level of PRR11 or PRR11 expression is increased in the sample from the subject compared to the control level.

In a further aspect, the invention provides a method for treating ovarian cancer, bladder cancer or lung cancer in a subject, comprising a) detecting ovarian cancer, bladder cancer or lung cancer in the subject by a method as defined above; and b) treating the subject for ovarian cancer, bladder cancer or lung cancer if the level of PRR11 or PRR11 expression is increased in the sample from the subject compared to the control level.

In one embodiment, the treatment comprises surgery, chemotherapy and/or radiotherapy and/or treatment with a pathway inhibitor. Preferably the treatment comprises pancreatectomy. In another embodiment, the chemotherapy comprises administration of gemcitabine and/or oxaliplatin to the subject. Treatment with a pathway inhibitor may involve treatment with an inhibitor, such as a small molecular weight inhibitor which targets specific pathways. The inhibitor may, for example, be a VEGF or EGFR inhibitor.

In a further aspect, the invention provides a chemotherapeutic agent for use in treating pancreatic cancer or cholangiocarcinoma in a subject, wherein the subject has an elevated level of PRRll expression compared to a control subject not suffering from pancreatic cancer or cholangiocarcinoma.

In a further aspect, the invention provides a chemotherapeutic agent for use in treating ovarian cancer, bladder cancer or lung cancer in a subject, wherein the subject has an elevated level of PRRll expression compared to a control subject not suffering from ovarian cancer, bladder cancer or lung cancer.

In one embodiment, PRRll levels or expression are elevated in a pancreatic biopsy or bile duct biopsy sample derived from the subject. Preferably pancreatic cancer or cholangiocarcinoma has been detected in the subject by a method as defined above. In another preferred embodiment, the chemotherapeutic agent comprises gemcitabine and/or oxaliplatin.

In another embodiment, PRRll levels or expression are elevated in an ovarian biopsy or lung biopsy sample derived from the subject. Preferably ovarian cancer, bladder cancer or lung cancer has been detected in the subject by a method as defined above.

In another aspect, the invention provides a method for predicting progression of bladder cancer in a subject, the method comprising (a) determining a level of proline-rich protein 11 (PRRll) or PRRll expression in a sample obtained from the subject; and (b) comparing the level of PRRll or PRRll expression in the sample to a control level; wherein an increased level of PRRll or PRRll expression in the sample compared to the control level is indicative of progression of bladder cancer in the subject.

In one embodiment, an increased level of PRRll or PRRll expression in the sample compared to the control level is indicative of decreased recurrence-free survival time in the subject.

In another aspect, the invention provides a method for stratification of patients with pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, based on PRRll level and/or PRRll expression, which method comprises the following steps:

(a) determining a level of PRRll or PRRll expression in a sample obtained from the patient; and (b) comparing the level of PRRll or PRRll expression in the sample to a control level; wherein an high level of PRRll or PRRll expression in the sample compared to the control level indicates that the patient has a high risk of relapse and similar level of PRRll or PRRll expression in the sample compared to the control level indicates that the patient has a low risk of relapse.

The method may be for stratifying bladder cancer patients.

In the method, "high risk" patients may be subsequently assigned to a more frequent monitoring program and "low risk" patients may be subsequently assigned to a less frequent monitoring program.

The more frequent monitoring program may involve monitoring at least once a year, and the less frequent monitoring program may involve monitoring at intervals of more than one year. For example, the more frequent monitoring program may involve monitoring every 3-6 months, and the less frequent monitoring program may involve monitoring every 3-6 years.

Monitoring may involve cystoscopy and/or sample analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: IHC was developed for PRRll. PRRll was expressed in tumour glands (stained brown, blue nuclei) but not adjacent normal glands (black arrowheads) or chronic pancreatitis (red arrowheads). A. Expression of PRRll in tumour glands. B. Tumour and normal glands. C. Tumour and normal pancreatitis.

Figure 2: IHC for PRRll was performed on a pancreatic tissue microarray (TMA) comprised of 6mm cores. Where more than one pathology existed in a single core, both were scored. PPV - positive predictive value, NPV - negative predictive value. Sensitivity - 95%, specificity - 98%; PPV - 99% NPV - 94%.

Figure 3: PRRll IHC of cholangiocarcinoma (brown). Reactive epithelia are highlighted with red arrowheads, cholangiocarcinoma is highlighted with black arrowheads.

Figure 4: IHC for PRRll was performed on an ovarian tissue microarray (TMA). Data shows the percentage of ovarian tissue samples showing particular levels of staining for PRRll: none (no staining), weak (staining was inconsistent and/or weak), moderate (appreciable staining) or strong (very intense staining). Upper panel: all benign vs all tumour samples. Lower panel: individual benign and tumour types. PPV - positive predictive value, NPV - negative predictive value. Figure 5: IHC for PRRll was performed on a lung tissue microarray (T A). Data shows the percentage of lung tissue samples showing particular levels of staining for PRRll: none (no staining), weak (staining was inconsistent and/or weak), moderate (appreciable staining) or strong (very intense staining). Upper panel: all normal vs all tumour samples. Lower panel: normal and individual tumour types. PPV - positive predictive value, NPV - negative predictive value.

Figure 6: Immunohistochemical detection of PRRll in ovarian and lung cancers.

Figure 7: IHC for PRRll was performed on a bladder tissue microarray (TMA). Upper panels: Data shows the percentage of bladder tissue samples showing particular levels of staining for PRRll at various stages of the disease. None (no staining), weak (staining was inconsistent and/or weak), moderate (appreciable staining) or strong (very intense staining). Lower panel: Kaplan-Meier survival estimates showing recurrence-free survival over time for subjects with bladder cancer showing low or high expression of PRRll.

Figure 8: PRRll expression is restricted in normal tissue, a) PRRll expression was analysed by staining of the FDA 999c normal tissue micro array, b) 3 or 4 cores of normal tissue were analysed for each indicated tissue and representative images of no staining (-), weak (+), moderate (++) and strong (+++).

DETAILED DESCRIPTION OF THE INVENTION

In embodiments of the present invention, pancreatic cancer or cholangiocarcinoma may be detected in a subject using PRRll as a biomarker. In further embodiments, ovarian cancer, bladder cancer or lung cancer may also be detected using PRRll as a biomarker. Thus it has surprisingly been found that PRRll is a sensitive and specific biomarker of particular cancers. This advantageously allows the early detection of such conditions, thereby enabling early intervention and improved therapeutic outcomes.

Detecting pancreatic cancer

In one aspect, the present invention provides a method for detecting pancreatic cancer in a subject. By "detecting pancreatic cancer" it is typically meant that the method may be used to determine whether a subject is suffering from pancreatic cancer. Thus in particular embodiments, the method may be used, for diagnosing pancreatic cancer; screening a patient population for the presence of pancreatic cancer; and/or monitoring progression of pancreatic cancer in a subject. By "pancreatic cancer" as used herein it is typically meant a cancer which is derived from pancreatic cells. In one embodiment, the pancreatic cancer is pancreatic adenocarcinoma. The symptoms accompanying pancreatic cancer are known in the art.

Detecting cholangiocarcinoma

In another aspect, the present invention provides a method for detecting cholangiocarcinoma in a subject. By "detecting cholangiocarcinoma" it is typically meant that the method may be used to determine whether a subject is suffering from cholangiocarcinoma. Thus in particular embodiments, the method may be used, for diagnosing cholangiocarcinoma; screening a patient population for the presence of cholangiocarcinoma; and/or monitoring progression of cholangiocarcinoma in a subject.

By "cholangiocarcinoma" as used herein it is typically meant a cancer which originates in the bile ducts, e.g. in epithelial cells (or cells showing characteristics of epithelial differentiation) in the bile ducts. In one embodiment, the bile duct cancer is an adenocarcinoma.

Detecting ovarian cancer

In another aspect, the present invention provides a method for detecting ovarian cancer in a subject. By "detecting ovarian cancer" it is typically meant that the method may be used to determine whether a subject is suffering from ovarian cancer. Thus in particular embodiments, the method may be used for diagnosing ovarian cancer; screening a patient population for the presence of ovarian cancer; and/or monitoring progression of ovarian cancer in a subject.

By "ovarian cancer" as used herein it is typically meant a cancer which is derived from cells of the ovary. The symptoms accompanying ovarian cancer are known in the art. In particular embodiments, the ovarian cancer comprises endometrioid adenocarcinoma, mucinous cystadenoma, serous cystadenocarcinoma, stromal sarcoma or undifferentiated carcinoma. More preferably the ovarian cancer comprises serous cystadenocarcinoma or stromal sarcoma.

Detecting bladder cancer

In another aspect, the present invention provides a method for detecting bladder cancer in a subject. By "detecting bladder cancer" it is typically meant that the method may be used to determine whether a subject is suffering from bladder cancer. Thus in particular embodiments, the method may be used for diagnosing bladder cancer; screening a patient population for the presence of bladder cancer; and/or monitoring progression of bladder cancer in a subject. By "bladder cancer" as used herein it is typically meant a cancer which is derived from cells of the bladder. The symptoms accompanying bladder cancer are known in the art.

Detecting lung cancer

In another aspect, the present invention provides a method for detecting lung cancer in a subject. By "detecting lung cancer" it is typically meant that the method may be used to determine whether a subject is suffering from lung cancer. Thus in particular embodiments, the method may be used for diagnosing lung cancer; screening a patient population for the presence of lung cancer; and/or monitoring progression of lung cancer in a subject. Preferably the lung cancer comprises adenocarcinoma or squamous cell carcinoma.

By "lung cancer" as used herein it is typically meant a cancer which is derived from cells of the lung. The symptoms accompanying lung cancer are known in the art.

The method as referred to in accordance with the present invention includes a method which essentially consists of the aforementioned steps or a method which includes further steps. However, it is to be understood that the method, in a preferred embodiment, is a method carried out ex vivo, i.e. not practised on the human or animal body. The method, preferably, can be assisted by automation.

Diagnosis and monitoring

The term "diagnosing" as used herein refers to assessing whether a subject suffers from pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer, or not. The term includes individual diagnosis of pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer or their symptoms as well as continuous monitoring of a patient. Monitoring, i.e. diagnosing the presence or absence of pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer or the accompanying symptoms at various time points, includes monitoring of patients known to suffer from pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer as well as monitoring of subjects known to be at risk of developing pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer. Furthermore, monitoring can also be used to determine whether a patient is treated successfully or whether the symptoms thereof can be ameliorated over time by a certain therapy.

In some embodiments, the method may be used to distinguish pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer from a different condition showing similar symptoms and/or clinical signs. For instance, in one embodiment the method may be used to distinguish pancreatic cancer from chronic pancreatitis. In another embodiment, the method may be used to distinguish ovarian cancer from e.g. irritable bowel syndrome (IBS) or premenstrual syndrome (PMS).

Prognostic applications

In further aspects, PR 11 may be used as a biomarker in prognostic applications, e.g. for providing an indication of disease status, risk of progression, rate of progression and/or clinical outcome (e.g. probability of survival). Accordingly, the invention provides methods for predicting progression of a cancer (e.g. bladder cancer) comprising detecting PRR11 as described herein. In one embodiment, the method may be used to predict a probability of survival of the subject, for instance within a particular time period (e.g. within 3 months, 6 months, 1 year, 2 years, 5 years, 10 years or more). In a preferred embodiment, the method is used to predict a recurrence-free survival time of the subject, for instance in bladder cancer. In another embodiment, the method may be used to predict severity of the disease, e.g. the degree to which the cancer is advanced or the rate at which the cancer is predicted to advance in the future. In general, such prognostic methods may be performed in a similar manner to the methods described herein for detecting cancer, e.g. on the same types of subjects, using similar biomarker detection methods, control levlels and on the same types of sample. Typically an increased level of PRR11 expression is indicative of a negative prognosis, e.g. an increased risk of progression of the cancer, an increased rate of progression, and/or a decreased recurrence-free survival time.

The prognostic method may be used for patient stratification, in order to define a given patient as "high-risk" or "low-risk" for example in terms of likelihood of relapse. High-risk and low-risk patients may be recruited on to separate monitoring programs: the high-risk patients receiving more frequent monitoring than the low-risk patients. Monitoring may, for example involve periodic cystoscopy or sample analysis. Such subsequent monitoring may or may not include investigating the level of PRR11 or PRR11 expression.

Subjects

The term "subject" as used herein relates to animals and, preferably, to mammals. More preferably, the subject is a primate and, most preferably, a human.

In one embodiment, the subject is suspected to be suffering from pancreatic cancer or cholangiocarcinoma, i.e. the subject may already show one or more symptoms associated with the disease. Symptoms of pancreatic cancer and cholangiocarcinoma include, for example, persistent abdominal pain, upper back pain, jaundice (e.g. yellow skin/eyes, dark urine) and unexplained weight loss. In another embodiment, the subject may be suspected to be suffering from pancreatitis, i.e. the method may be applied in order to distinguish between pancreatitis and pancreatic cancer in the subject.

Alternatively, the subject may be at increased risk of developing pancreatic cancer or cholangiocarcinoma. For instance, the subject may have one or more relatives suffering from pancreatic cancer or cholangiocarcinoma, or a defined genetic predisposition for developing pancreatic cancer or cholangiocarcinoma. Alternatively the subject may be suffering from (chronic) pancreatitis, or may have one or more relatives suffering from (chronic) pancreatitis, and/or has a defined genetic predisposition for developing (chronic) pancreatitis. Further risk factors which may be present in the subject include diabetes, alchoholism and smoking.

In another embodiment, the subject is suspected to be suffering from ovarian cancer, i.e. the subject may already show one or more symptoms associated with the disease. Symptoms of ovarian cancer include, for example, persistent abdominal pain and bloating; pelvic, abdominal and/or back pain; increased urinary frequency or urgency; and eating difficulties and/or nausea. In another embodiment, the subject may be suspected to be suffering from another abdominal condition having one or more of the above symptoms, i.e. the method may be applied in order to distinguish between ovarian cancer and other abdominal conditions associated with the above symptoms.

Alternatively, the subject may be at increased risk of developing ovarian cancer. For instance, the subject may have one or more relatives suffering from ovarian cancer, or a defined genetic predisposition for developing ovarian cancer. For instance, the subject may have one or more mutations in the BRCA1 or BRCA2 genes which are associated with elevated ovarian cancer risk. Further risk factors which may be present in the subject include a history of breast cancer, infertility, hormone replacement therapy (HRT), obesity, endometriosis, dietary factors and smoking.

In another embodiment, the subject is suspected to be suffering from bladder cancer, i.e. the subject may already show one or more symptoms associated with the disease. Symptoms of bladder cancer include, for example, hematuria (blood in urine), pain during urination, or frequent urination. In another embodiment, the subject may be suspected to be suffering from e.g. a different condition having one or more of the above symptoms. For instance, the method may be applied in order to distinguish between bladder cancer and a condition having similar symptoms (e.g. hematuria), such as prostate infections, over-active bladder, cystitis, bladder or ureteric stones, kidney disease, and vascular malformations. Alternatively, the subject may be at increased risk of developing bladder cancer. For instance, the subject may have one or more relatives suffering from bladder cancer, or a defined genetic predisposition for developing bladder cancer. Smoking is one factor which is indicative of an increased risk of developing bladder cancer.

In another embodiment, the subject is suspected to be suffering from lung cancer, i.e. the subject may already show one or more symptoms associated with the disease. Symptoms of lung cancer include, for example, respiratory symptoms such as coughing, wheezing or shortness of breath; weight loss, fever or fatigue; and chest pain or difficulty swallowing. In another embodiment, the subject may be suspected to be suffering from e.g. a respiratory condition having one or more of the above symptoms. For instance, the method may be applied in order to distinguish between lung cancer and a respiratory condition such as tuberculosis, pneumonia, chronic obstructive pulmonary disease (COPD) or emphysema, bronchitis or lung infection.

Alternatively, the subject may be at increased risk of developing lung cancer. For instance, the subject may have one or more relatives suffering from lung cancer, or a defined genetic predisposition for developing lung cancer. Smoking is a particular risk factor which is indicative of an increased risk of developing lung cancer.

Sample

In general the term "sample" as used herein refers to samples from body fluids (e.g. blood, plasma, serum or urine) or samples derived, e.g., by surgical excision, aspiration, biliary brush or biopsy, from cells, tissues or organs, in particular from the pancreas, bile ducts, ovary, bladder or lung. In a preferred embodiment, the sample is a biopsy sample from the pancreas or bile duct. In another embodiment, the sample is a biopsy sample from the ovary, bladder or lung.

In another preferred embodiment, the sample is a urine sample, e.g. in the case where the method is used to detect bladder cancer. In bladder cancer, tumour cells normally originate from the lining of the bladder which is in direct contract of the urine. Hence the mechanistic pressures excreted during the voiding on the bladder results in tumour cells or cell fragments being released into the urine as well as general shedding into the bladder cavity prior to voiding. The urine is therefore an ideal sample for detection of bladder cancer, by methods such as ELISA.

Techniques for obtaining the aforementioned different types of biological samples are well known in the art. For instance, pancreatic biopsy samples may be obtained as described in e.g. Goldin SB et al., J Gastrointest Surg. (2007); ll(6):783-90; Cohen SJ et al., Curr. Treat. Options Oncol. (2000) 1:375-386 and Iglesias-Garcia J et al., World J Gastroenterol (2007) 13(2): 289-293. Ovarian biopsy samples may be obtained using methods as described in e.g. Fayez et al., Obstet Gynecol. (1976) 48(4):397-402; Portuondo et al., J Reprod Med. (1982) 27(2):67-72; and May et al., J Obstet Gynaecol. (2011) 31(6):535-8. Suitable samples may be obtained, for example, by fine needle aspiration biopsy, endoscopic brushings, laparoscopy and endoscopic ultrasound guided biopsies.

Fine needle aspiration may also be referred to as percutaneous needle biopsy and involves insertion of a needle into the affected organ under imaging guidance, capturing some tissue.

Endoscopic brushing techniques include a procedure referred to as endoscopic retrograde cholangiopancreatography (ERCP). In this method, a flexible tube comprising an endoscope is advanced from the mouth to the small intestine, near the pancreas. Biopsy tissue can be collected with a brush, using the images available from the endoscope.

Laparoscopy is a surgical procedure that uses several small incisions to collect tissue for biopsy. This method can also be used to determine if pancreatic cancer has spread further within the abdomen.

In endoscopic ultrasound techniques, an endoscope is advanced near the pancreas in a procedure similar to ERCP. An ultrasound probe on the endoscope locates the potentially cancerous tissue, and a needle on the endoscope is used to obtain a biopsy sample.

Preferably the sample is a solid tissue sample (e.g. a biopsy sample) from a subject suspected of suffering from pancreatic cancer or cholangiocarcinoma. In another preferred embodiment, the sample is a solid tissue sample (e.g. a biopsy sample) from a subject suspected of suffering from ovarian cancer, bladder cancer or lung cancer. Thus the tissue sample may comprise neoplastic tissue. In one embodiment, the sample comprises a tissue section, such as a fresh, frozen or paraffin-embedded tissue section, typically from a suspected diseased tissue or organ (e.g. pancreas, bile duct, ovary, bladder or lung). By "section" of a tissue sample is meant a single part or piece of a tissue sample, e.g. a thin slice of tissue or cells cut from a tissue sample. Multiple sections of tissue samples may be taken and subjected to analysis according to the present invention, e.g. on a tissue microarray as disclosed in US 2003/0215936. Typically the section is suitable for analysis by microscopy, e.g. visible light or fluorescent microscopy. The section may, for example, be placed on a solid support such as a microscope slide. Methods for preparing tissue samples for microscopic analysis are well known in the art.

In one embodiment, the tissue sample is fixed and embedded in paraffin or the like. The tissue sample may be fixed (i.e. preserved) by conventional methodology. One of skill in the art will appreciate that the choice of a fixative is determined by the purpose for which the tissue is to be histologically stained or otherwise analyzed. The length of fixation depends upon the size of the tissue sample and the fixative used. By way of example, neutral buffered formalin or paraformaldehyde may be used to fix a tissue sample.

Generally, the tissue sample is first fixed and is then dehydrated through an ascending series of alcohols, infiltrated and embedded with paraffin or other sectioning media so that the tissue sample may be sectioned. Alternatively, the tissue may be sectioned and then the sections fixed. The tissue sample may be embedded and processed in paraffin by conventional methodology. Once the tissue sample is embedded, the sample may be sectioned by a microtome or the like. Typically the sections may range from about 3 to 20, e.g. 5-10 microns in thickness. Once sectioned, the sections may be attached to slides by several standard methods. Examples of slide adhesives include, but are not limited to, silane, gelatin, poly-L-lysine and the like. By way of example, the paraffin embedded sections may be attached to positively charged slides and/or slides coated with poly-L-lysine.

If paraffin has been used as the embedding material, the tissue sections are generally deparaffinized and rehydrated to water. The tissue sections may be deparaffinized by several conventional standard methodologies. For example, xylenes and a gradually descending series of alcohols may be used. Alternatively, commercially available deparaffinizing non-organic agents may be used. After deparaffinization, the sections mounted on slides may be stained with one or more morphological stains (counterstains) for evaluation, if required. Generally, the section is stained with one or more dyes each of which distinctly stains different cellular components, for example, a xanthine dye, a thiazine dye or methylene blue. Typically the counterstain is a nuclear stain, in order to facilitate the identification and/or counting of individual cells. Staining may be optimized for a given tissue by increasing or decreasing the length of time the slides remain in the dye.

Proline-rich protein 11 (PRRll)

The amino acid sequence of human PRRll is disclosed in NCBI RefSeq database accession no. NP_060774.2. A nucleotide sequence encoding human PRRll is disclosed in NCBI RefSeq database accession no. NM_018304.3. Amino acid and nucleotide sequences corresponding to PRRll in other mammalian species may be found in similar publicly-available databases or by identifying sequences showing homology to the above human sequences.

Determining biomarker levels

In the present method, levels of PRRll are determined in an obtained sample from the subject. The amount of PRRll in the sample may be measured by any suitable method. For example, methods for detecting protein biomarkers may include the use of an antibody, aptamer, capture molecule, receptor, or fragment thereof which selectively binds to the protein. Antibodies which bind to PRRll are known or may be produced by methods known in the art, including immunization of an animal and collection of serum (to produce polyclonal antibodies) or spleen cells (to produce hybridomas by fusion with immortalised cell lines leading to monoclonal antibodies). The amino acid sequence of PRRll is known and available from publicly-accessible databases, and can be used to generate suitable immunogens for antibody production. Detection molecules such as antibodies may optionally be bound to a solid support such as, for example, a plastic surface or beads or in an array. Suitable test formats for detecting protein levels include, but are not limited to, an immunoassay such as an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Western blotting and immunoprecipitation.ln one embodiment, the method may comprise a step of staining the sample with a reagent that labels PRRll. By this it is meant that the reagent enables PRRll to be detected, for instance by binding to PRRll and providing a detectable signal. Typically the reagent used in this step is selective or specific for PRRll, in contrast to the morphological stain discussed above, thereby providing a stain which is uniquely indicative of the presence of PRRll. Thus "staining" refers to any step which renders PRRll detectable, particularly a histological method which renders PRRll detectable by microscopic techniques, such as those using visible or fluorescent light. One or more reagents may be used in combination in this step in order to detect PRRll, e.g. a first reagent may bind specifically to PRRll and a second reagent may bind to the first reagent and provide the detectable signal.

In a preferred embodiment, PRRll is detected in the sample by immunohistochemistry (IHC). IHC may be performed in combination with morphological staining as discussed in the preceding section (either prior to, but preferably thereafter). In IHC, PRRll is detected by an antibody which binds specifically to PRRll.

The antibody may be a monoclonal antibody, polyclonal antibody, multispecific antibody (e.g., bispecific antibody), or fragment thereof provided that it specifically binds to PRRll. Antibodies may be obtained by standard techniques comprising immunizing an animal with a target antigen and isolating the antibody from serum. Monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example. The antibody may also be a chimeric or humanized antibody.

Two general methods of IHC are available; direct and indirect assays. According to the first assay, binding of antibody to the target antigen is determined directly. This direct assay uses a labelled reagent, such as a fluorescent tag or an enzyme-labelled primary antibody, which can be visualized without further antibody interaction.

In a typical indirect assay, unconjugated primary antibody binds to the antigen and then a labelled secondary antibody binds to the primary antibody. Where the secondary antibody is conjugated to an enzymatic label, a chromagenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.

The primary and/or secondary antibody used for immunohistochemistry typically will be labeled with a detectable moiety. Numerous labels are available, including radioisotopes, colloidal gold particles, fluorescent labels and various enzyme-substrate labels. Fluorescent labels include, but are not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, Lissamine, umbelliferone, phycocrytherin and phycocyanin, and/or derivatives of any one or more of the above. The fluorescent labels can be conjugated to the antibody using known techniques.

Various enzyme-substrate labels are available, e.g. as disclosed in US 4,275,149. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be detected microscopically, e.g. under visible light. For example, the enzyme may catalyze a colour change in a substrate, or may alter the fluorescence or chemiluminescence of the substrate. Examples of enzymatic labels include luciferases (e.g. firefly luciferase and bacterial luciferase; US 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are well known.

Horseradish peroxidase may be visualised with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g. orthophenylene diamine (OPD) or 3,3',5,5'- tetramethyl benzidine hydrochloride (TMB). Alkaline phosphatase (AP) may be detected with para- nitrophenyl phosphate as chromogenic substrate. β-D-galactosidase (β-Gal) may be detected with the chromogenic substrate p-nitrophenyl-P-D-galactoside or fluorogenic substrate 4- methylumbelliferyl^-D-galactoside.

In an embodiment of the present invention, the immunohistochemistry step may be performed as follows. Following an optional blocking step, the tissue section is exposed to primary antibody for a sufficient period of time and under suitable conditions such that the primary antibody binds to P R11 in the tissue sample. Appropriate conditions for achieving this can be determined by routine experimentation. The tissue sample is then exposed to a secondary antibody which binds specifically to the primary antibody (e.g. the primary antibody is a mouse monoclonal antibody and secondary antibody is a rat anti-mouse IgG polyclonal antibody). PRR11 can then be visualised by applying to the sample a chromogenic substrate for an enzyme conjugated to the secondary antibody.

Specimens thus prepared may be mounted and coverslipped. The stained sample is now ready to be imaged for subsequent analysis. Typically the image is obtained under magnification, for instance using a microscope. In one embodiment the image is obtained by an automated image acquisition system, e.g. an apparatus capable of automatic scanning of prepared microscope slides. The imaging system may additionally be capable of automated image analysis. Suitable automated imaging systems are disclosed in, for example, US 6,718,053, US 7,233,340 and US 6,466,690.

The level of PRR11 in the sample may be assessed, for example, by visual scoring of the stained sample by a trained observer or by an automated image analysis system as described above. Typically such automated systems are capable of quantifying levels of an analyte in a histological sample by detecting a characteristic stain colour (corresponding to the chromogenic substrate used) in a microscopic image of the sample.

Typically the method comprises a step of detecting stained regions within the image. Pixels in the image corresponding to staining associated with PRR11 may be identified by colour transformation methods, for instance as disclosed in US 6,553,135 and US 6,404,916. In such methods, stained objects of interest may be identified by recognising the distinctive colour associated with the stain. The method may comprise transforming pixels of the image to a different colour space, and applying a threshold value to suppress background staining. For instance, a ratio of two of the RGB signal values may be formed to provide a means for discriminating colour information. A particular stain may be discriminated from background by the presence of a minimum value for a particular signal ratio. For instance pixels corresponding to a predominantly red stain may be identified by a ratio of red divided by blue (R/B) which is greater than a minimum value.

The transformed image may be further analysed to determine the presence of structures of interest, e.g. positively stained cell surfaces, by grouping together pixels in close proximity and having the same colour. Edge detection techniques may be applied to discriminate the cell membrane from other structures. In some embodiments cells may be identified, for example, by identifying nuclei stained with a counterstain. Antibodies

The detection methods described herein preferably use one or more antibodies which bind to P R11. Suitable antibodies are known or may be generated using known techniques.

Antibodies comprise immunoglobulin molecules. Immunoglobulin molecules are in the broadest sense members of the immunoglobulin superfamily, a family of polypeptides comprising the immunoglobulin fold characteristic of antibody molecules, which contains two β sheets and, usually, a conserved disulphide bond. Antibodies, as used herein, refers to complete antibodies or antibody fragments capable of binding to PRR11, and including Fv, ScFv, F(ab') and F(ab')₂, monoclonal and polyclonal antibodies, engineered antibodies including chimeric, CDR-grafted and humanised antibodies, and artificially selected antibodies produced using phage display or alternative techniques.

Antibodies may be obtained from animal serum, or, in the case of monoclonal antibodies or fragments thereof, produced in cell culture. Recombinant DNA technology may be used to produce the antibodies according to established procedure, in bacterial, yeast, insect or preferably mammalian cell culture. The selected cell culture system preferably secretes the antibody product.

Growing of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenished by a mammalian serum, for example foetal calf serum, or trace elements and growth sustaining supplements, for example feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid, or the like. The culture medium may be serum-free or animal-produce free, such as a chemically defined medium, in order to minimise animal derived contamination. Multiplication of host cells which are bacterial cells or yeast cells is likewise carried out in suitable culture media known in the art, for example for bacteria in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT, or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.

Insect cells may be cultured in serum free medium, which is cheaper and safer compared to serum containing medium. Recombinant baculovirus may be used as an expression vector, and the construct used to transfect a host cell line, which may be any of a number of lepidopteran cell lines, in particular Spodoptera frugiperda Sf9, as known in the art. Reviews of expression of recombinant proteins in insect host cells are provided by Altmann et al. (1999), GlycoconjJ 1999, 16, 109-23 and Kost and Condreay (1999), Curr Opin Biotechnol, 10, 428-33. In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies. Techniques for bacterial cell, yeast, insect and mammalian cell cultivation are known in the art and include homogeneous suspension culture, for example in an airlift reactor or in a continuous stirrer reactor, or immobilised or entrapped cell culture, for example in hollow fibres, microcapsules, on agarose microbeads or ceramic cartridges.

Large quantities of the desired antibodies can also be obtained by multiplying mammalian cells in vivo. For this purpose, hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody-producing tumours. Optionally, the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection. After one to three weeks, the antibodies are isolated from the body fluids of those mammals. For example, hybridoma cells obtained by fusion of suitable myeloma cells with antibody-producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example, Kohler and Milstein, (1975) Nature 256:495-497; US 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor, incorporated herein by reference. Techniques for the preparation of recombinant antibody molecules is described in the above references and also in, for example, EP 0623679; EP 0368684 and EP 0436597, which are incorporated herein by reference.

The cell culture supernatants are screened for the desired antibodies, preferentially by immunofluorescent staining of cells expressing the desired target by immunoblotting, by an enzyme immunoassay, for example a sandwich assay or a dot-assay, or a radioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culture supernatants or in the ascitic fluid may be concentrated, for example by precipitation with ammonium sulphate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like. If necessary and/or desired, the antibodies are purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or immunoaffinity chromatography, for example affinity chromatography with the a protein containing a target or with Protein-A. Antibodies generated according to the foregoing procedures may be cloned by isolation of nucleic acid from cells, according to standard procedures. Usefully, nucleic acids variable domains of the antibodies may be isolated and used to construct antibody fragments, such as scFv.

The methods described here may employ recombinant nucleic acids comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies. By definition such nucleic acids comprise coding single stranded nucleic acids, double stranded nucleic acids consisting of the coding nucleic acids and of complementary nucleic acids thereto, or these complementary (single stranded) nucleic acids themselves.

Antibodies may moreover be generated by mutagenesis of antibody genes to produce artificial repertoires of antibodies. This technique allows the preparation of antibody libraries; antibody libraries are also available commercially. Hence, artificial repertoires of immunoglobulins, preferably artificial ScFv repertoires, can be used as an immunoglobulin source.

Isolated or cloned antibodies may be linked to other molecules, for example nucleic acid or protein association means by chemical coupling, using protocols known in the art (for example, Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor, and Maniatis, T., Fritsch, E. F. and Sambrook, J. (1991), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press). Such methods may be used to produce labelled antibodies or to immobilize the antibody on a solid phase.

In some embodiments the antibody may be labelled. Typically a labelled antibody is capable of producing a detectable signal. The signal may be, for example, the generation of an enzymatic activity, such as protease activity, transcriptional activity or luminescence inducing activity. In one embodiment, the signal is emission or absorption of electromagnetic radiation, for example, light. The signal may be, for example, a colour change which takes place when the labelled antibody is present.

Methods of conjugating visible or fluorescent labels to various entities, including peptides, polypeptides and antibodies, are well known in the art. In certain embodiments, it may be desirable to include spacing means between the antibody and the label. The spacing means may comprise linkers or spacers which are polymers of differing lengths (the length of which may be controlled by controlling the degree of polymerisation). Numerous spacers and linkers are known in the art, and the skilled person will know how to choose and use these, depending on the application. The skilled person will also know what spacer length to use. Comparison to control

In embodiments of the present invention, the level of PRRll in the sample is compared to a control level. The control level may be, for example, a predetermined measurement of a level of PRRll which is present in a sample from a normal subject, i.e. a subject who is not suffering from pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer. In some embodiments, the control level may be derived from a subject (or a population of subjects) who is suffering from a condition such as (chronic) pancreatitis. The control level may, for example, be based on a mean or median level of the biomarker in a control population of subjects, e.g. 5, 10, 100, 1000 or more subjects (who may either be age- and/or gender-matched or unmatched to the test subject) who show no symptoms of pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer.

The control level may be determined using corresponding methods to the determination of PRRll levels in the test sample, e.g. using one or more samples taken from a control population of subjects. For instance, in some embodiments PRRll levels in control samples may be determined in parallel assays to the test samples. In alternative embodiments, the control level may have been previously determined, or may be calculated or extrapolated, without having to perform a corresponding determination on a control sample with respect to each test sample obtained.

For instance, where the level of PRRll is determined by immunohistochemistry the control sample may comprise a tissue section (e.g. from a pancreatic, bile duct, ovarian, bladder or lung biopsy) obtained from a subject known not to be suffering from pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer. Alternatively, the control sample may be obtained from the same (test) subject, but from a region of pancreas, bile duct, ovary, bladder or lung tissue which is not cancerous. Typically the control sample is processed for immunochemistry in a similar manner to the test sample, except that the anti-PRRll antibody is not applied to the control sample. Thus the control sample may be fixed, embedded in paraffin and counterstained as for the test sample, as well as being contacted with any secondary antibody and/or chromogenic agents used.

In embodiments of the present invention, an increased level of PRRll in the test sample compared to the control level is indicative of the presence of pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer in the subject. Preferably the level of PRRll in the test sample differs by at least 1%, 5%, at least 10%, at least 20%, at least 30%, or at least 50% compared to the control level.

Further biomarkers and diagnostic tools The present method may be used in combination with existing biomarkers, techniques and imaging tools suitable for the detection and diagnosis of pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer. For instance, the method may be used in combination with ultrasound (including endoscopic ultrasound), computer tomography, magnetic resonance, laparoscopy and other imaging techniques.

In the case of pancreatic cancer, the method may, for example, be used in combination with the detection of CA 19-9 (cancer antigen 19-9 or carbohydrate antigen 19-9, also known as sialylated Lewis (a) antigen) as a serum biomarker. Thus in one embodiment, the method further comprises determining a level of CA 19-9 in a serum (or plasma) sample obtained from the subject, and comparing the level of CA 19-9 to a control level. Methods for detecting CA 19-9 are known in the art. For instance, CA 19-9 may be detected using specific antibodies, e.g. as described in Proteomics (2012) 12(13):2212-20.

The control level may, for example, correspond to a level of CA 19-9 in serum of a normal subject not suffering from (pancreatic) cancer. Typically an increased level of CA19-9 compared to the control level is indicative of the presence of pancreatic cancer. For instance, a level of CA 19-9 in serum greater than about 37 U/mL may be considered to be an elevated level. Thus in some embodiments, an increased level of both PRR11 and CA 19-9 may be used to diagnose the presence of pancreatic cancer.

In the case of cholangiocarcinoma, the method may, for example, be used in combination with the detection of IMP3 (Insulin-like growth factor-ll mRNA-binding protein 3, U3 small nucleolar ribonucleoprotein). Thus in one embodiment, the method further comprises determining a level of IMP3 in a sample obtained from the subject, and comparing the level of IMP3 to a control level. Methods for detecting IMP3 are known in the art. For instance, IMP3 may be detected using immunohistochemistry of biopsy samples, e.g. as described in Int J Surg. (2013); 11(1):85-91. Amino acid and nucleotide sequences corresponding to IMP3 are disclosed in NCBI RefSeq database accession numbers NP_060755.1 and NM_018285.3 respectively.

The control level may, for example, correspond to a level of IMP3 in a normal subject not suffering from cancer (particularly cholangiocarcinoma). Typically an increased level of IMP3 compared to the control level is indicative of the presence of cholangiocarcinoma. Thus in some embodiments, an increased level of both PRR11 and IMP3 in the sample from the subject may be used to diagnose the presence of cholangiocarcinoma. In the case of ovarian cancer, the method may, for example, be used in combination with the detection of CA125 (cancer or carbohydrate antigen 125, also known as mucin 16 or MUC16) as a serum biomarker. Thus in one embodiment, the method further comprises determining a level of CA125 in a serum (or plasma) sample obtained from the subject, and comparing the level of CA125 to a control level. Methods for detecting CA125 are known in the art.

The control level may, for example, correspond to a level of CA125 in serum of a normal subject not suffering from (ovarian) cancer. Typically an increased level of CA125 compared to the control level is indicative of the presence of ovarian cancer. Thus in some embodiments, an increased level of both PRRll and CA125 may be used to diagnose the presence of ovarian cancer.

Determining PRR expression

Expression of PRRll mRNA may be determined by any method known to one skilled in the art. For example, levels of PRRll mRNA may be determined by quantitative RT-PCR, digital PCR, next generation sequencing or northern blotting. Each of these methods is well known to one skilled in the art. For example, expression of PRRll mRNA may be determined using an array, gene chip or gene set comprising one or more polynucleotides capable of specifically hybridising mRNA. Next generation sequencing and/or polynucleotides capable of specifically hybridising to PRRll may be used to detect deletions or mutations in the genes encoding PRRll protein.

Treating pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer

In one embodiment, the method described above may be used in order to select a treatment protocol for an individual subject. For instance, based on whether levels of PRRll are elevated in the subject, the subject may be treated for pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer, or alternatively for a different condition such as chronic pancreatitis.

In one embodiment, if levels of PRRll (and optionally one or more further biomarkers such as e.g. CA 19-9 or IMP3) in the sample from the subject are increased compared to the control level, the subject is treated for pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer. Typically such treatment may comprise surgery, chemotherapy and/or radiotherapy.

In one embodiment, the treatment for pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lung cancer comprises surgery. Typically surgery comprises a surgical intervention to remove (resect) the cancerous tissue, i.e. at least a portion of the pancreas and/or bile ducts. For instance, in particular embodiments the tail of the pancreas or alternatively the head of the pancreas may be removed, in some cases together with the duodenum. Thus the treatment may comprise a full or partial pancreatectomy. Localized cancers of the pancreas may also be resected using minimally invasive (laparoscopic) approaches. In the case of cholangiocarcinoma, the surgical treatment may comprise removal of all or part of the bile duct, in some cases together with parts of the liver, stomach, duodenum, pancreas, gall bladder and/or surrounding lymph nodes. Surgical treatment for ovarian cancer may comprise removal of one (unilateral oophorectomy) or both ovaries (bilateral oophorectomy), the fallopian tubes (salpingectomy), and the uterus (hysterectomy). For some very early tumors (stage 1, low grade or low-risk disease), only the involved ovary and fallopian tube may be removed (unilateral salpingo-oophorectomy).ln another embodiment, the treatment comprises chemotherapy, either alone or in combination with surgery. Chemotherapy typically refers to treatment with drugs or chemical compounds that target cancer cells. Chemotherapy may involve administration of a chemotherapeutic compound, which may have a cytotoxic or cytostatic effect, or which may induce a cyto-protective autophagy response in the cell. The chemotherapeutic agent may be an agent that induces apoptosis, such as p53-dependent apoptosis, or that induces cell cycle arrest, including p53-dependent cell cycle arrest, in a cell that is abnormally proliferating or cancerous. Commonly used chemotherapeutic agents include DNA damaging agents and genotoxic agents that can activate p53-dependent apoptosis or p53- dependent cell cycle arrest in a proliferating cell.

Suitable chemotherapeutic agents include an anthracycline, an alkylating agent, an alkyl sulfonate, an aziridine, an ethylenimine, a methylmelamine, a nitrogen mustard, a nitrosourea, an antimetabolite, a folic acid analogue, a purine analogue, a pyrimidine analogue, a podophyllotoxin, or a platinum-containing agent. For instance, the chemotherapy may comprise administration of tamoxifen or a related taxane (e.g. paclitaxel or docetaxel), gemcitabine, carboplatin, oxaliplatin, cisplatin, erlotinib, adriamycin, 5-fluorouracil, etoposide, camptothecin, topotecan, liposomal doxorubicin, or a derivative or analog thereof. Preferably the chemotherapy comprises administration of gemcitabine and/or oxaliplatin.

In a further embodiment, the treatment may comprise radiotherapy, either alone or in combination with radiotherapy or surgery. Protocols for performing radiotherapy in pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder and lung cancer are well known to a skilled person.

Pharmaceutical formulations

A chemotherapeutic agent may be formulated and administered to a subject in any suitable composition for the treatment of pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer and/or lung cancer. In particular embodiments, an effective amount of the chemotherapeutic agent is administered to the subject. In this context, the term "effective amount" means an amount effective, at dosages and for periods of time necessary to achieve the desired result, for example, to treat the specific cancer.

The chemotherapeutic agent may be administered to a subject using a variety of techniques. For example, the agent may be administered systemically, which includes by injection including intramuscularly or intravenously, orally, sublingually, transdermal^, subcutaneously, internasally. Alternatively, the agent may be administered directly at a site at which the cancer is located, e.g. by injection to the site, or surgical implantation, for example at a site of a tumour.

The concentration and amount of the chemotherapeutic agent to be administered will typically vary, depending on the cancer, the type of cell associated with the cancer, the type of agent that is administered, the mode of administration, and the age and health of the subject.

The chemotherapeutic agent may be formulated in a pharmaceutical composition together with a pharmaceutically acceptable carrier, excipient or diluent. The compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives and various compatible carriers. For instance the chemotherapeutic agent may be formulated in a physiological buffer solution.

The proportion and identity of the pharmaceutically acceptable carrier, excipient or diluent may be determined by the chosen route of administration, compatibility with live cells, and standard pharmaceutical practice. Generally, the pharmaceutical composition will be formulated with components that will not significantly impair the biological properties of the agent. Suitable carriers, excipients and diluents are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).

Kits

In further embodiments, the present invention provides a kit suitable for performing the method as described above. In particular, the kit may comprise reagents suitable for detecting PRR11, e.g. in a biopsy or urine sample. Typically the reagents may comprise antibodies, aptamers, receptors or fragments thereof which bind specifically to PRR11.

Such kits may optionally further comprise one or more additional components, e.g. reagents suitable for performing an ELISA assay using antibodies which bind specifically to PRR11. For instance, the kits may comprise capture and detection antibodies for PRR11, secondary antibodies, detection reagents, solid phases (e.g.reaction plates or beads), standards (e.g. known concentrations of each biomarker in the form of recombinant proteins) as well as buffers suitable for performing any of step of an ELISA method. The kits may further comprise vials, containers and other packaging materials for storing the above reagents, as well as instructions for performing a method as defined herein.

The invention will now be described by way of example only with respect to the following specific embodiments.

EXAMPLES

Materials and methods

All tissue for this study was either provided by Addenbrookes Hospital Tissue Bank or purchased from Stretton Scientific or Insight Biotechnology. All immunohistochemistry (IHC) was performed using a Bondmax Autostainer. PRR11 was stained using 1.5M Tris EDTA, pH8.0 for antigen recovery and rabbit anti-PRRll antibody (1:150, Atlas Antibodies) diluted in a buffer containing 300mM Tris buffered saline, 1% donkey serum (Sigma Aldrich) and 0.05% Tween. Nuclei were counterstained with haematoxylin and slides coverslipped using DPX. All slides were imaged using an Aperio scanner at 20x magnification.

Tissue was scored as none (no staining), weak (staining was inconsistent and/or weak), moderate (appreciable staining) or strong (very intense staining). Sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) were calculated where possible. All grouped p- values (n=>3) were calculated using a Kruskal-Wallis test. All pairwise comparisons were completed using a Mann-Whitney test. A p-value of <0.05 was classed as significant.

Immunohistochemical staining for PRR11 in pancreatic tumour tissue is highly specific and sensitive.

Immunohistochemistry using an anti-PRRll antibody was performed on pancreatic tissue biopsy samples (Fig. 1). Patients suffering from chronic pancreatitis have a high risk of developing pancreatic cancer, therefore a useful biomarker must distinguish between the two. PRR11 shows clear differentiation. The specificity and sensitivity of PRR11 to the presence of tumour in tissue is highly significant (see Fig. 2).

PRR11 is more sensitive than the current gold standard - combining both could improve diagnosis

Immunohistochemistry using an anti-PRRll antibody was performed on cholangiocarcinoma biopsy samples. Both dysplastic areas and cancer are strongly stained by PRR11, but there is also some weak staining of reactive tissue (see Fig. 3). As shown in Table 1 below, IHC for PRRll and IMP3 was scored on multiple slides from nine patients with cholangiocarcinoma. Results are expressed as a percentage of the possible positive or negative scores. Scores were given for reactive/inflammatory epithelia, dysplasia and cholangiocarcinoma. PPV - positive predictive value, NPV - negative predictive value.

Table 1

PRRll is more sensitive than IMP3; IMP3 is more specific. If used in tandem, IMP3 and PRRll could have greater clinical utility than if used separately.

PRRll as a biomarker in other cancers

Immunohistochemistry using an anti-PRRll antibody was performed on tissue biopsy samples from a number of further cancer types. PRRll is expressed at low levels in most normal tissues but is over-expressed in a number of tumour tissue types (see Table 2).

Table 2

Tissue Cancer type Normal tissue score Tumour tissue score

Oesophagus Squamous cell carcinoma - 7-

Liver Hepatocellular carcinoma - -1-

Thyroid Papillary carcinoma - -/-

Soft tissue Lymphoma - -/-

Tongue Squamous cell carcinoma - 2+/3+

Skin Squamous cell carcinoma 1+ 2+/3+

Lung Squamous cell carcinoma - 2+/2+

Lung Adenocarcinoma - -/- Colon Adenocarcinoma - 7-

Colon Adenocarcinoma - -/-

Stomach Adenocarcinoma - -/-

Stomach Signet ring cell carcinoma - -/-

Kidney Renal cell carcinoma - -/-

Kidney Renal cell carcinoma Missing -/-

Ovary Yolk sack tumour - -/-

Uterus Adenocarcinoma 1+ 2+/1+

Breast Ductal carcinoma - 7-

Breast Ductal carcinoma - -/-

Testis Seminoma - 7-

Prostate Adenocarcinoma 2+ 7-

Pancreas Adenocarcinoma - 2+/3+

Thymus Thymoma 2+ -/1+

As shown in Figure 4, PRRll is also a sensitive and specific biomarker of ovarian cancer. In particular, PRRll expression is elevated especially in endometrioid adenocarcinoma, mucinous cystadenoma, serous cystadenocarcinoma, stromal sarcoma or undifferentiated carcinoma. Cases of serous cystadenocarcinoma and stromal sarcoma show particularly strong PRRll expression compared to normal tissue.

Figure 5 shows that PRRll is a sensitive and specific biomarker of lung cancer. A high proportion of cases of adenocarcinoma and squamous cell carcinoma show elevated expression of PRRll compared to normal lung tissue. In cases of squamous cell carcinoma, PRRll expression is particularly strong.

Figure 7 shows that PRRll is expressed at high levels in bladder cancer. PRRll is expressed at various stages of the disease, and a high proportion of bladder cancer tissue samples show strong PRRll expression. Subjects showing high levels of expression of PRRll have decreased recurrence- free survival time compared to subjects showing low levels of expression of PRRll.

Figure 8 shows that PRRll expression is restricted in normal tissue. Conclusions

• Early diagnosis is key for treatment and survival of both pancreatic cancer and cholangiocarcinoma.

• PR 11 is a strong marker for the presence of tumour, with a low false-positive rate.

• Diagnostic potential is seen in several further, specific cancer types, including ovarian cancer, bladder cancer and lung cancer - PRR11 appears to highlight a loss of differentiation.

• Defining an at-risk population is crucial to allow earlier screening and the identification of treatable patients using biomarkers such as PRR11.

All references described herein are incorporated by reference. Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification. Further advantages and features of the present invention are apparent from the figures and non-limiting examples.

Claims

1. A method for detecting pancreatic cancer or cholangiocarcinoma in a subject, the method comprising:

(a) determining a level of proline-rich protein 11 (PRRll) or PRRll expression in a sample obtained from the subject; and

(b) comparing the level of PRRll or PRRll expression in the sample to a control level;

wherein an increased level of PRRll or PRRll expression in the sample compared to the control level is indicative of the presence of pancreatic cancer or cholangiocarcinoma in the subject.

2. A method for detecting ovarian cancer or bladder cancer in a subject, the method comprising:

(a) determining a level of proline-rich protein 11 (PRRll) or PRRll expression in a sample obtained from the subject; and

(b) comparing the level of PRRll or PRRll expression in the sample to a control level;

wherein an increased level of PRRll or PRRll expression in the sample compared to the control level is indicative of the presence of ovarian cancer or bladder cancer in the subject.

3. A method according to claim 1 or claim 2, wherein PRRll is detected using an antibody which binds specifically to PRRll.

4. A method according to any preceding claim, wherein the level of PRRll in the sample is determined by immunohistochemistry or an enzyme-linked immunosorbent assay (ELISA).

5. A method according to claim 1 or 2, wherein the level of PRRll expression is determined by analysing the level of PRRll mRNA in the sample obtained from the subject.

6. A method according to any of claims 1, 3, 4 or 5, wherein the sample comprises a pancreatic biopsy sample.

7. A method according to any of claims 1, 3, 4 or 5 wherein the sample comprises a bile duct or biliary brush biopsy sample.

8. A method according to any preceding claim, wherein the subject is suspected to be suffering from pancreatic cancer, cholangiocarcinoma, ovarian cancer or bladder cancer cancer.

9. A method according to any preceding claim, wherein the subject is at elevated risk of developing pancreatic cancer, cholangiocarcinoma, ovarian cancer or bladder cancer cancer.

10. A method according to claim 9, wherein the subject has a family history of pancreatic cancer, cholangiocarcinoma, ovarian cancer orbladder cancer, or a genetic predisposition for developing pancreatic cancer, cholangiocarcinoma, ovarian cancer or bladder cancer.

11. A method according to claim 9 or claim 10, wherein the subject is suffering from pancreatitis, has a family history of pancreatitis, and/or has a genetic predisposition for developing pancreatitis.

12. A method according to any preceding claim, wherein the method is capable of distinguishing pancreatic cancer from pancreatitis in the subject.

13. A method according to any preceding claim, wherein the control level comprises a level of PR 11 or PRR11 expression in a control sample from a subject not suffering from pancreatic cancer, cholangiocarcinoma, ovarian cancer or bladder cancer.

14. A method according to any preceding claim, further comprising determining a level of CA 19-9 in a sample obtained from the subject, and comparing the level of CA 19-9 in the sample to a control level; wherein an increased level of CA 19-9 in the sample compared to the control level is indicative of the presence of pancreatic cancer in the subject.

15. A method according to any preceding claim, further comprising determining a level of IMP3 in a sample obtained from the subject, and comparing the level of IMP3 in the sample to a control level; wherein an increased level of IMP3 in the sample compared to the control level is indicative of the presence of cholangiocarcinoma in the subject.

16. A method according to any preceding claim, further comprising determining a level of CA125 in a sample obtained from the subject, and comparing the level of CA125 in the sample to a control level; wherein an increased level of CA125 in the sample compared to the control level is indicative of the presence of ovarian cancer in the subject.

17. A method according to any preceding claim, wherein the sample is a blood, plasma, serum, or urine sample from the subject.

18. A method according to any of claims 2 to 5 or 8 to 16, wherein the sample comprises an ovarian biopsy sample.

19. A method according to any of claims 2 to 5 or 8 to 18, wherein the ovarian cancer comprises serous cystadenocarcinoma or stromal sarcoma.

20. A method according to any of claims 2 to 5 or 7 to 16, wherein the sample comprises a bladder biopsy sample or a urine sample.

21. A method for treating pancreatic cancer, cholangiocarcinoma, ovarian cancer or bladder cancer in a subject, comprising:

a) detecting pancreatic cancer, cholangiocarcinoma, ovarian cancer or bladder cancer in the subject by a method as defined in any preceding claim; and b) treating the subject for pancreatic cancer, cholangiocarcinoma, ovarian cancer or bladder cancer if the level of PRRll or PRRll expression is increased in the sample from the subject compared to the control level.

22. A method according to claim 21, wherein the treatment comprises surgery and/or chemotherapy and/or radiotherapy and/or treatment with a pathway inhibitor

23. A method according to claim 22, wherein the treatment comprises pancreatectomy.

24. A method according to claim 22 or claim 23, wherein the chemotherapy comprises administration of gemcitabine and/or oxaliplatin to the subject.

25. A chemotherapeutic agent for use in treating pancreatic cancer, cholangiocarcinoma, ovarian cancer or bladder cancer in a subject, wherein the subject has an elevated level of PRRll expression compared to a control subject not suffering from pancreatic cancer, cholangiocarcinoma, ovarian cancer or bladder cancer.

26. A chemotherapeutic agent for use according to claim 25, wherein PRRll levels are elevated in a pancreatic biopsy, bile duct biopsy, ovarian biopsy or bladder biopsy sample derived from the subject.

27. A chemotherapeutic agent for use according to claim 25 or claim 26, wherein pancreatic cancer, cholangiocarcinoma, ovarian cancer or bladder cancer has been detected in the subject by a method as defined in any of claims 1 to 20.

28. A chemotherapeutic agent for use according to any of claims 25 to 27, wherein the chemotherapeutic agent comprises gemcitabine and/or oxaliplatin.

29. A method for predicting progression of bladder cancer in a subject, the method comprising:

(a) determining a level of proline-rich protein 11 (PRRll) or PRRll expression in a sample obtained from the subject; and

(b) comparing the level of PRRll or PRRll expression in the sample to a control level;

wherein an increased level of PRRll or PRRll expression in the sample compared to the control level is indicative of progression of bladder cancer in the subject.

30. A method according to claim 28, wherein an increased level of PRRll or PRRll expression in the sample compared to the control level is indicative of decreased recurrence-free survival time in the subject.

31. A method for stratification of bladder cancer patients, based on PRRll level and/or PRRll expression, which method comprises the following steps:

(a) determining a level of proline-rich protein 11 (PRRll) or PRRll expression in a sample obtained from the patient; and

(b) comparing the level of PRRll or PRRll expression in the sample to a control level; wherein an high level of PRR11 or PRR11 expression in the sample compared to the control level indicates that the patient has a high risk of relapse and similar level of PRR11 or PRR11 expression in the sample compared to the control level indicates that the patient has a low risk of relapse.

32. A method according to claim 31, wherein "high risk" patients are subsequently assigned to a more frequent monitoring program and "low risk" patients are subsequently assigned to a less frequent monitoring program.

33. A method according to claim 32, wherein the more frequent monitoring program involves monitoring at least more than once a year, and the less frequent monitoring program involves monitoring at intervals one year or more.

34. A method according to claim 33, wherein the more frequent monitoring program involves monitoring every 3-6 months, and the less frequent monitoring program involves monitoring every 3-6 years.

35. A method according to any of claims 32 to 34, wherein monitoring involves cystoscopy and/or sample analysis.