RU2785737C2 - Chromogranin a as marker of bladder cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to prediction of bladder cancer. A method for prediction of non-neuroendocrine bladder cancer in an individual includes determination of a level of CgA in a sample of individual’s body fluid. If the level of CgA is higher than a predetermined threshold, then it is an indicator of a high risk of death related to malignant tumor after surgical treatment, and/or treatment using a drug, and/or radiotherapy. In a patient subjected to transurethral resection of the bladder, a twenty-month survival rate after treatment is 87% for patients with low preoperational CgA concentration and 70% for patients with a high preoperational level of CgA. In a patient subjected to radical cystectomy, a twenty-month survival rate after treatment is 76% for patients with low preoperational CgA concentration and from 33% for patients with a high preoperational level of CgA. The level of CgA is considered compared to a control level or the predetermined threshold, where the latter is from 100 ng/mg to 147 ng/ml, and serum samples of healthy individuals without malignant tumor in the history are used as control.

EFFECT: invention provides prediction of bladder cancer.

6 cl, 3 dwg, 10 tbl, 3 ex

Description

The technical field to which the invention belongs

[0001] The present invention relates to the field of clinical diagnostics. In particular, the invention relates to the prediction, risk assessment, risk stratification, monitoring and/or control of bladder cancer therapy based on chromogranin A (CgA) as a marker.

State of the art for the invention

[0002] Bladder cancer (bladder cancer) is the most common malignant neoplasm of the urinary tract. Considering newly diagnosed cases and its high recurrence rate, bladder cancer is one of the most common malignancies worldwide (Chamie et al. 2011, Goodison, Rosser and Urquidi 2013). Western countries are more affected, and men have a 3-4 times higher risk of developing compared to women (Burger et al. 2013a).

Based on the cells that become cancerous, different types of bladder cancer can be distinguished. The most common type of bladder cancer is transitional cell carcinoma (90%), followed by squamous cell carcinoma and adenocarcinoma (2-5%). Less than 1% of bladder cancer arises from neuroendocrine cells (Bertaccini et al. 2008, Pompas-Veganzones, Gonzalez-Peramato and Sanchez-Carbayo 2014). At diagnosis, approximately 70-75% of patients have superficial bladder cancer (stages Ta-T1), also known as non-muscle invasive bladder cancer (NMIBC). 25-30% of patients have muscle-invasive bladder cancer (MIBC, stages T2-T4) and/or metastatic bladder cancer (Clark et al. 2013, Mossanen and Gore 2014).

The recurrence and/or progression rate of bladder cancer is extremely high, where 50-70% of NMIBC patients may relapse, progress, or develop new disease within 5-7 years of treatment (Clark et al. 2013), and 10% - 30% of these relapses are invasive (Chamie et al. 2011, Goodison et al. 2013, Witjes et al. 2014). Approximately half (43%) of MIBC cases had non-invasive tumors at diagnosis that progressed despite organ-sparing treatment (EAU Guidelines on Muscle-invasive and Metastatic Bladder Cancer 2014).

[0003] Currently, a key element in the diagnosis and surveillance of bladder cancer is histopathological examination of biopsy specimens of bladder urothelium during cystoscopy/transurethral resection of a bladder tumor (TURBT). White light cystoscopy is a useful diagnostic tool in the hands of medical professionals, but has major limitations. Indeed, she often underestimates the stages of tumors, which can lead to undertreatment (Cauberg Evelyne, de la Rosette and de Reijke 2011). It is also moderately sensitive, expensive and invasive. Some extremely rare complications such as perforation due to the obturator reflex, bleeding, TUR syndrome, urinary tract obstruction, perforation (extra- or intraperitoneal), and infections may occur during this procedure.

Natural urine cytology (VUC) is also a widely used method for diagnosing and monitoring NMIBC, with high specificity but low sensitivity in well-differentiated (low-grade) tumors. Its accuracy ranges from only 20 to 40% for early stage tumors (AUA Guidelines 2007-2014, Goodison et al., 2013). It has shown better accuracy for high grade tumors and in situ carcinoma in particular. This property makes it an additional tool for transurethral resection (see below). Cystoscopy and VUC allow primary diagnosis and initial assessment of signs of tumors, including number, type, grade, and stage assessed.

Computed tomography (CT) and magnetic resonance imaging (MRI) can also be used to evaluate the primary tumor. However, in up to 40% of cases, they underestimate disease and can only discriminate between stages of Ta-T3a tumors with limitations (accuracy ranges from 55-92%), even though better accuracy in distinguishing invasive from non-invasive has been published (Maurer et al. 2013 ).

Assessment of metastasis status, and especially the condition of the nodes, is a key point in the prognostic evaluation of bladder cancer. Metastasis to the lymph nodes increases from a low incidence of 5–10% for non-muscle invasive bladder tumors to 15–20% for superficial muscle invasive tumors, up to 25–30% for deep muscle invasive tumors, and up to >40% for non-muscle invasive tumors. tumors (Shariat et al. 2012). Diagnosis of distant or regional metastatic bladder cancer is largely based on a variety of imaging modalities, including radiography, ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI). Ultrasound is the simplest, most noninvasive, and cost effective, but depends on the skill of the operator. CT and NMR are also non-invasive, and although the sensitivity of these methods has recently been improved, their effectiveness remains insufficient.

If the initial diagnosis is positive for bladder cancer, regardless of metastatic status, transurethral resection of the bladder tumor (TURBT) is performed. This therapeutic and diagnostic method is the initial therapeutic stage, characterized by endoscopic ablation of the entire tumor. It confirms the diagnosis and allows a more accurate assessment of the stage of the tumor, especially the depth of invasion of the bladder wall. However, TURBT is associated with a significant risk of underestimation, especially for T1 tumors (Babjuk 2009).

Moreover, due to incomplete resection, recurrent tumor growth can be observed within the first year (Kamat et al. 2013, Babjuk 2009). For this reason, repeated TURBT or maximum TURBT is strongly recommended in guidelines (Brausi et al. 2011).

[0004] While the course of the disease in patients with NMIBC can often be safely managed by "fine" TURBT with or without additional therapeutic measures (e.g., immunotherapy, chemotherapy), radical cystectomy (RCE) with bilateral removal of pelvic lymph nodes (PLND) has become the standard of care for patients with MIBC (Clark et al. 2013, Witjes et al. 2014). Some neoadjuvant chemotherapeutic agents have shown a survival advantage (Sharma, Ksheersagar and Sharma 2009) and are recommended for selected patients. RCE is also recommended for highly malignant and/or refractory NMIBCs and carcinomas in situ (CIS). Many radical surgery patients undergo urinary diversion or neobladder reconstruction. National Comprehensive Cancer Network (NCCN) guidelines recommend that patients without lymph node involvement at highest pathological risk may be treated with additional adjuvant radiotherapy with radiosensitization therapy or chemotherapy. However, these additional therapies may exacerbate toxicity and comorbidities (Clark et al. 2013).

After RCE alone in the total bladder cancer (BCa) group (all stages and grades), 5-year overall survival, disease-free survival, and cancer-specific survival were only 57%, 48%, and 67%, respectively, with late and local recurrences 37% and 6%, respectively (Yafi et al. 2011).

The survival benefit of standard pelvic lymph node removal (PLND) for MIBC cases has been shown to a large extent, and PLND is now an integral part of the RC recommended by consensus in the guidelines. However, its advantage in a surgical monotherapy modality is modest, and lymphadenectomy (LND) is often considered more diagnostic/prognostic (may provide nodal status) than therapeutic (Skinner and Sagalowsky 2014). Indeed, regional LND is a necessary step in staging due to the sensitivity limitations of current imaging modalities. Despite advances in surgical technique, imaging, perioperative disease management, and therapies, approximately 50% of patients develop metastases and die from bladder cancer (Stein JP, Skinner DG 2006).

Thus, neoadjuvant/adjuvant chemotherapeutic agents or radiotherapy and PLND or extended LND are contemplated (Skinner and Sagalowsky 2014) to improve the survival of high-risk patients. However, due to increased toxicity, risk of comorbidities, and cost, the benefits of these major interventions must be carefully weighed against changes in quality of life and potential complications, including bleeding, nerve injury, lymphocystosis, or thrombi in the extremities (Scarpato et al. 2015), and assign them only to cases with the highest risk.

One of the challenges for this type of advanced cancer is answering the question: which treatment is for which patient?

[0005] After RCE with or without LND, helical CT is the preferred imaging modality for identifying lung, lymph node, and liver metastasis (ICUD-EAU Bladder cancer Edition 2012), and EAU guidelines recommend scanning every 3-4 months abdomen, upper urinary tract (UUT), pelvis, and radiography or chest CT (Sharma et al. 2009) during the first year, every 6 months until the third year, and after this period, monitoring through annual imaging as clinically indicated. For RCE with cutaneous diversion (UD), a urethral flush cytology every six to 12 months is also recommended (Sharma et al. 2009). The frequency and methods of follow-up after radical cystectomy are not strictly consistent between guidelines, however, they differ only slightly.

[0006] Distant recurrence usually occurs within 24 months, where higher stage and lymph node status are the most important risk factors. Recurrence in the pelvis usually occurs (in 5-15% of patients) within 6-18 months after surgery, again depending on the initial stage and the condition of the lymph nodes. Secondary urethral tumors are rare, occurring 1–3 years after cystectomy, and have a poor survival rate. Upper urinary tract recurrence is rare and usually occurs late (28–49 months after cystectomy) (Witjes et al. 2014). Finally, some late recurrences may occur after more than 10 years, in which case patients also require very long follow-up after RCE.

[0007] Due to the prognostic heterogeneity of both NMIBC and MIBC, guidelines for the treatment of bladder cancer emphasize the importance and unmet need for better prognosis (EAU guidelines). Risk tables are available for NMIBC to improve treatment decisions and/or follow-up (Vedder et al. 2014). However, the European Organization for Research and Treatment of Cancer (EORTC) scoring system recommended in the EAU and AUA guidelines is rarely used in daily practice.

A number of recent publications have shown that these models are not very reliable. For example, Vedder et al. published that the scoring system can reasonably predict progression but not recurrence (Vedder et al. 2014).

An overestimation of recurrence and progression on the EORTC table is also shown in NMIBC, also in the subgroup after BCG treatment (Fernandez-Gomez et al., 2011).

Finally, Xylinas et al. showed that the EORTC scoring system has poor discrimination for both recurrence and progression, and highlighted the need to improve current prognostic tools (Xylinas et al. 2013).

To date, there is no table or scoring system for specifically assessing MIBC prognosis such as to guide treatment choice. The presence of regional lymph node metastases is the strongest predictor of tumor recurrence and disease-specific survival (DSS) in patients for MIBC (Skinner and Sagalowsky 2014). However, approximately half of superficial tumors may have micrometastases undetectable by imaging tools, while only 25% of radical surgery patients have lymph node (LN) metastases at the time of surgery (Svatek et al. 2010, EAU Guidelines on Muscle-invasive and Metastatic Bladder Cancer 2014).

Even though disease-specific survival (DSS) ranges from 85% to 90% after radical cystectomy for CIS, early radical cystectomy can be considered overtreatment in approximately 50% of patients (Burger et al. 2013b) .

[0008] Bladder cancer is a heterogeneous neoplasia requiring a large-scale treatment algorithm (sequential chemical or biological treatment, radiotherapy and surgical interventions) and extensive long-term follow-up. Tumors with similar histology may have different clinical developments, and these nuances are critical for proper disease management. Its complexity leads to a direct and indirect economic burden and makes it the most costly malignancy in terms of lifetime cost of care per patient (Brausi 2013, Chamie et al. 2011, Goodison et al. 2013, Mossanen and Gore 2014). In 2010, the cost of care for BCa was approximately $4 billion in the US (Mossanen and Gore 2014) and €3 billion in 2012 in the European Union (EU), including hospital care itself as the main cost component (counting 58%) . The total economic burden was €5 billion in the EU (Leal et al. 2015). In the US, the estimated lifetime cost of disease management for an individual with BCa ranges from $96,000 to $280,000 (Hansel et al. 2013). The medical costs associated with a diagnosis of MIBC are approximately $150,000, however, due to the long clinical course of the disease in the early stages, its prevalence relative to MIBC, and its procedurally oriented follow-up, the economic burden of NMIBC is generally considered to be greater (Svatek et al. 2014).

Underestimation of tumor stage is a major limitation of current diagnostic and prognostic tools. Moreover, to date, there are no reliable tools for predicting the progression of NMIBC to MIBC, nor for predicting the outcome of MIBC.

[0009] The use of biomarkers to predict or stratify risk for patients with bladder cancer can help the physician initiate appropriate treatment and reduce the costs associated with patient follow-up.

Some commercially determined urine markers have received FDA approval for the diagnosis and/or follow-up of bladder cancer but have not entered clinical practice. In contrast, no blood or urine predictive markers are currently endorsed by the National Institutes of Health or recommended in any guidelines. This is inconsistent with the BCA disease management strategy based on global risk.

[0010] In a recent large meta-analysis (Schmitz-Drager et al. 2015), the efficacy of most commercially available markers (BTA Stat®, NMP22, including BladderCheck® and FISH Urovysion™) was compared with VUC. He confirms that these molecular markers have much better sensitivity, especially for high-risk tumors (pT1G3, CIS) than urine cytology, despite no demonstrated predictive use. However, he also confirms that the lower specificity and reproducibility of such markers was a major limitation. In addition, these markers had no predictive value.

[0011] Chromogranin A (CgA) is a glycoprotein commonly expressed on neuroendocrine (NE) cells. CgA is a constituent of the secretory granules of most peptide-producing endocrine cells (Chuang and Liao 2003). It is physiologically released by exocytosis and can be detected in the blood. When a tumor develops in neuroendocrine tissue, it becomes the main source of circulating CgA. CgA secretion in the bladder is associated with rare cases of differentiated neuroendocrine tumors, which are known to have a poor prognosis (Alijo Serrano et al. 2007, Bertaccini et al. 2008). Neuroendocrine tissue markers, such as CgA, are often used to distinguish between neuroendocrine carcinoma (NEC) and transitional cell carcinoma (TCC), and are used to confirm the diagnosis of NEC and typically for small cell bladder cancer (SCBC) (Bertaccini et al. 2008, Cerulli et al. 2012, Iczkowski et al. 1999) or paraganglioma (Bagchi et al. 2015, Feng et al. 2013).

However, CgA has been shown to be expressed in neuroendocrine bladder cancer (NEBC) tissue, but to date, no study has evaluated the predictive effect of tissue and serum CgA levels for either neuroendocrine or urothelial BCAs.

Moreover, the presence of both small cells and transitional cells also did not predict outcomes (Chuang and Liao 2003).

Chuang et al. showed that CgA was expressed in 4 out of 10 SCBC tissue samples but found no association with patient prognosis. However, due to the small number of cases and the heterogeneous cohort, this study has limited reliability (Chuang and Liao 2003).

CgA has not been shown to be associated with survival in large and small cell neuroendocrine carcinoma of the bladder. In one study, TNM classification remained the only predictor of survival (Alijo-Serrano, 2007). The authors did not consider CgA to be useful for prognostic purposes. In addition, Soukup et al. recently found that urinary CgA was not associated with either the presence of primary NMIBC or cancer recurrence (Soukup et al. 2015). However, Bertaccini et al. found that 1 month after cystoprostatectomy for small cell urothelial carcinoma (SCUC), serum CgA levels decreased 10-fold compared to preoperative levels, but neither preoperative prognosis nor follow-up value was assessed (Bertaccini et al. 2008).

[0012] Matrix metalloproteinase-7 (MMP7) has been described as an independent marker in serum and urine for lymph node metastases (Gunes et al. 2013, Jager et al. 2013, Szarvas et al. 2010, Szarvas et al. 2011b), and as an independent predictive marker for BCa before surgery (Svatek et al. 2010, Szarvas et al. 2010, Szarvas et al. 2011a). High levels of MMP-7 have also been described as an independent risk factor for poor survival in specific types of metastatic bladder cancer (El Demery et al. 2014). See also WO 2007/144144 A1.

[0013] The present invention is based on the unexpected finding that chromogranin A has a high prognostic value in non-neuroendocrine bladder cancer. Thus, it can help overcome the above disadvantages of prior art diagnostic tools for bladder cancer.

The essence of the invention

[0014] The present invention relates to the use of chromogranin A (CgA) as a marker (particularly a prognostic marker) for bladder cancer, in particular non-neuroendocrine bladder cancer, and preferably urothelial carcinoma. In particular, CgA can be used as a marker in an in vitro assay to predict, assess risk, risk stratify, monitor and/or control therapy for bladder cancer, in particular non-neuroendocrine bladder cancer and preferably urothelial carcinoma.

[0015] The invention further relates to a method for predicting, assessing risk, risk stratifying, monitoring and/or controlling therapy for bladder cancer, in particular non-neuroendocrine bladder cancer and preferably urothelial carcinoma, in a subject, including determining the level of CgA and optionally, MMP7 in a bodily fluid sample of said subject. The present invention further relates to the use of diagnostic kits containing one or more CgA-specific antibodies for predicting, risk assessment, risk stratification, monitoring and/or therapy control of bladder cancer, in particular non-neuroendocrine bladder cancer, and preferably , urothelial carcinoma, in a subject. The invention also relates to methods for treating bladder cancer, in particular non-neuroendocrine bladder cancer and preferably urothelial carcinoma, in a subject, in which the level of CgA in a sample from said subject is determined.

Description of drawings

Figure 1: Serum CgA concentration for controls and cases

Figure 2: Predictive value of CgA and MMP-7 levels and their combination in patients undergoing surgical treatment (TURBT or RCE) (Kaplan-Meier curves with log-rank tests). DSS: disease-specific survival.

Figure 3: Predictive value of CgA and MMP7 levels and their combinations in a subgroup of patients treated with RCE (Kaplan-Meier curves with log-rank tests)

Detailed description of the invention

[0016] The present invention relates to the use of chromogranin A (CgA) as a marker for bladder cancer (in particular, non-neuroendocrine bladder cancer and preferably urothelial carcinoma), in particular as a prognostic marker for bladder cancer, in particular, for non-neuroendocrine bladder cancer, preferably urothelial carcinoma. Thus, CgA can be used as a marker in an in vitro assay to predict, assess risk, risk stratify, monitor and/or control therapy for bladder cancer, in particular non-neuroendocrine bladder cancer, preferably urothelial carcinoma. In other words, CgA proved to be a good biomarker for assessing disease severity in patients with bladder cancer, in particular in patients with non-neuroendocrine bladder cancer, preferably in patients with urothelial carcinoma. CgA can thus also be used in the management of the disease of patients ("patient management") with bladder cancer, in particular non-neuroendocrine bladder cancer and preferably urothelial carcinoma.

[0017] The present invention relates to predictive methods for subjects with bladder cancer (in particular, subjects with non-neuroendocrine bladder cancer, preferably subjects with urothelial carcinoma), including the step of determining the level of CgA (and optionally, MMP7) in a sample of physiological fluid specified subject. The invention particularly relates to a method for predicting, risk assessment, risk stratification, monitoring and/or control of therapy for bladder cancer (in particular, non-neuroendocrine bladder cancer, preferably urothelial carcinoma) in a subject, comprising the step of determining the level of CgA in the sample bodily fluid of said subject. Optionally, the level of MMP7 is further determined in the same or a different sample from the specified subject. The level of CgA (and optionally MMP7, as the case may be) in a sample from a subject is, in one aspect, indicative of the severity and aggressiveness of the bladder cancer (particularly non-neuroendocrine bladder cancer, preferably urothelial carcinoma) and/or outcome for the subject. An increased level of CgA (and optionally MMP7, as the case may be) in a sample from a subject, compared to a control level or a predetermined threshold, is indicative of a poor outcome for the subject. For example, the level of CgA in a sample from a subject is indicative of the subject's overall survival, or disease-specific survival of the subject, or progression-free survival of the subject.

[0018] In some embodiments, first determine the level of CgA in a sample from the subject and then perform additional tests if the level of CgA is above a predetermined threshold in another sample from the same subject, for example, then determine the level of MMP7 to refine or confirm the initial prediction based on at the CGA level. In other more preferred embodiments, first determine the level of MMP7 in a sample from the subject and then perform additional tests if the level of MMP7 is above a predetermined threshold in another sample from the same subject, for example, then determine the level of CgA to refine or confirm the initial prediction, based on MMP7 level. Thus, MMP7 and CgA levels can be used together to refine an initial prediction based on only one of these two markers.

[0019] The term "bladder cancer" (also referred to as "bladder cancer") in the context of the present invention preferably refers to bladder cancer of non-neuroendocrine origin. Thus, preferably, "bladder cancer" is not a neuroendocrine tumor (NET). The term "neuroendocrine" refers to neural or endocrine influences, and in particular to the interaction between the nervous and endocrine systems. In particular, the term "neuroendocrine" refers to cells that release a hormone into the blood in response to a nerve stimulus. "Neuroendocrine malignancies", therefore, are malignant neoplasias arising from cells of the endocrine (hormonal) and nervous systems. Neuroendocrine cancer of the bladder includes, for example, small cell carcinoma (SCC), carcinoid tumor, and large cell neuroendocrine carcinoma (LCNEC).

Thus, "bladder cancer" as used herein preferably refers to bladder cancer not arising from cells of the endocrine (hormonal) and nervous systems. Thus, preferably in the present description, the bladder cancer is not a small cell carcinoma, is not a carcinoid tumor, and is not a large cell neuroendocrine carcinoma. Bladder cancer in the context of the present invention is preferably selected from the group consisting of transitional cell carcinoma (TCC) (i.e. urothelial carcinoma (UC) of the bladder, also known as urothelial cell carcinoma (UCC)), squamous cell carcinoma and adenocarcinoma, more preferably, bladder cancer in the context of the present invention is a transitional cell carcinoma. The term "urothelial" specifically refers to urothelial carcinoma, denoting TCC of the urinary system. Thus, urothelial carcinoma in the context of the present invention means bladder urothelial carcinoma. Transitional cell carcinoma (=urothelial carcinoma) can be superficial bladder cancer (ie non-muscle invasive bladder cancer (NMIBC)) or muscle invasive bladder cancer (MIBC). The NMIBC may, for example, be papillary carcinoma or squamous carcinoma (eg, carcinoma in situ (CIS)). Generally, bladder urothelial carcinoma can, for example, also be classified as micropapillary, nested, plasmacytoid, sarcomatoid, or other variants, or it can have a mixed histology. Micropapillary or nested variants of urothelial carcinoma are particularly aggressive.

These bladder cancers can, for example, be classified according to the UICC/AJCC (International Union Against Cancer/American Joint Committee on Cancer) TNM chart and graded according to grade according to the WHO 1973-2004 classifications.

[0020] A subject in the context of the present invention suffers from non-neuroendocrine bladder cancer and/or is diagnosed with non-neuroendocrine bladder cancer prior to said CgA level determination. Preferably, in the present description, the subject does not have neuroendocrine bladder cancer (ie does not suffer from neuroendocrine bladder cancer). Thus, preferably, the subject is not diagnosed with neuroendocrine bladder cancer.

[0021] The term "subject" as used herein refers to a living human or non-human animal, preferably a mammal, most preferably a human. The subject is preferably a patient. The term "patient" as used herein refers to a living human or non-human animal (most preferably a human) who is receiving medical care or who is about to receive medical care due to a disease, in particular non-neuroendocrine cancer of the urinary tract. bubble. This includes individuals without established disease who are examined for signs of pathology. Thus, the methods and assays described herein are applicable to both human disease and veterinary disease.

[0022] The term "level" in the context of the present invention refers to the concentration (preferably expressed as mass/volume; wt./vol.; for example, as "ng/ml") of the marker (for example, CgA and/or MMP7) obtained from a sample from a subject, such as a patient with bladder cancer.

[0023] "Diagnosis" in the context of the present invention refers to the recognition and (early) detection of a disease or clinical condition in a subject and may also include differential diagnosis. Also, an assessment of the severity of a disease or clinical or histopathological condition may, in specific embodiments, be included in the term "diagnosis".

"Prognosis" refers to predicting the outcome or specific risk for a subject suffering from a particular disease or clinical condition, as used herein, not neuroendocrine bladder cancer. This may include an assessment of the chance of recovery or the chance of a poor outcome for said subject.

[0024] "Monitoring" or "monitoring therapy" refers to tracking an already diagnosed disease, disorder, complication or risk, for example, to analyze the progression of a disease (in the present description: not neuroendocrine bladder cancer) or the effect of a particular treatment on the progression of a disease or disorder . According to the present invention, the term "risk stratification" refers to the grouping of subjects into different risk groups according to their predictions for the future. Risk stratification also refers to stratification for taking preventive and/or therapeutic measures.

[0025] The term "managing the course of a patient's disease" in the context of the present invention refers to:

• the decision to enter a hospital or intensive care unit,

• the decision to transfer the patient to a specialized hospital or to a specialized department of a hospital,

• assessment for early discharge from the intensive care unit or hospital,

• allocation of resources (eg staff of physicians and/or nurses, diagnostic, therapeutic, surgical facilities).

[0026] The term "assessing the severity of the disease" refers to assessing the disease status of a patient, including tumor histopathology, metastatic status and disease progression, the likelihood of adverse events (including death), the likelihood of high costs per hospitalized patient, and the likelihood of prolonged hospitalization.

[0027] The term "outcome" in the context of the present invention refers to the severity of the disease, for example, the state of health of the patient after a certain time, for example, after 3 days, 5 days, 10 days, 14 days, 20 days, 3 weeks, 4 weeks , 30 days, 45 days, 60 days, 90 days, 3 months, 6 months, 1 year, 2 years or 5 years. Outcome can be expressed as a rate (percentage) in a population (eg, study or treatment group) or as a rate for an individual (ie, probability for a particular subject or patient). Thus, "overall outcome" can be expressed as "overall survival", which is the percentage of people in a group (for example, in the study group or in the treatment group) who are still alive for a specific period of time, after having they are diagnosed with a disease, or they are being treated for a disease, such as, in the present case, non-neuroendocrine bladder cancer. Overall survival is often quoted as a five-year survival rate, which is the percentage of people in a study or treatment group who are alive five years after they are diagnosed or have been treated for a disease. Thus, "overall outcome" or "overall survival" for a particular subject or patient is the probability of surviving over a particular period of time, i.e. it refers to the life expectancy of said subject or patient.

"Disease-specific survival" can be expressed as a "disease-specific survival rate", which is the percentage of people in a population (eg, study or treatment group) who do not die from a specific disease in a given time period. The time period usually begins at the time of diagnosis or treatment and ends at the time of death. Patients who died from causes other than the disease under investigation (in the present description: non-neuroendocrine bladder cancer) are not included in this measurement. Thus, "disease-specific" survival or outcome for an individual subject or patient means the probability of not dying from non-neuroendocrine bladder cancer in a given time period.

"Progression-free survival" is the length of time during and after treatment for a disease, such as non-neuroendocrine bladder cancer, that a subject or patient lives with the disease without getting worse. "Progression" may, for example, be the appearance of a new tumor (at a primary or distant site) ("relapse") or an increase in size, histological stage, grade, or symptom, particularly after a given curative or palliative treatment. Progression can, for example, be detected radiographically or biochemically. For example, in the case of NMIBC, progression may refer to progression from stage Ta to T1, to T2, to T3, to T4; or from stage G1/2 to G3, or from stage LN0 to LN+, or from stage M0 to M+. For non-metastatic MIBC, progression may refer to postoperative recurrence, progression from LN0 to LN+, or from M0 to M+. Disease-specific death can also be considered as progression. In metastatic MIBC, progression can, for example, be detected radiographically, for example according to the RECIST criteria.

In some aspects, poor outcome is, for example, increased risk of reduced life expectancy, increased risk of progression, increased risk of cancer-related death, and/or increased risk of recurrence after surgery and/or drug treatment, and/or radiotherapy.

[0028] The methods and uses of the present invention can be used in various situations and at various stages of the disease, for example, before intervention or after intervention. The terms "pre-intervention", "performed before the intervention" and "before the intervention" in the present description refer to the time before the start of the intervention for the treatment of non-neuroendocrine bladder cancer. An “intervention” is any medical intervention used to modify a health outcome. This definition includes drug administration, surgical procedures, device use, behavior modification treatments, care modification, and the like. Preferably, the sample is taken after the patient is admitted to the hospital or before the diagnosis of non-neuroendocrine bladder cancer is confirmed.

For example, in one aspect, the subject is diagnosed with non-neuroendocrine bladder cancer and has not (to date) been subjected to surgical treatment for said non-neuroendocrine bladder cancer. In such a case, the methods of the invention can be used to predict outcome or to select an appropriate treatment (eg, type of surgery).

The terms "post-intervention", "post-intervention" and "post-intervention" refer to the time after the intervention or treatment was started.

For example, in one aspect, the subject has already undergone surgical treatment of said non-neuroendocrine bladder cancer, such as radical cystectomy of said non-neuroendocrine bladder cancer. In such a case, the methods of the invention can be used to predict outcome and/or to select an appropriate follow-up treatment, and/or to monitor the subject (eg, disease progression). Thus, the level of CgA (and any other additional marker, including MMP7, depending on the situation) can be used for postoperative control. The term "postoperative control" in the context of the present invention refers to the monitoring of the specified subject after a surgical procedure in the specified subject.

[0029] "Prediction" in the context of the present invention, in one case, refers to predicting a complication, progression, or symptom before other symptoms or markers become apparent or change significantly. In the context of the present invention, terms such as "predictive value" refer to the statistical significance of a specific, defined measurement result. Thus, an increase in predictive value or predictive power in the context of the present invention means that the probability of a correct diagnosis, prognosis, stratification, or the like, based on a particular value determined from measuring the level of a particular marker in a sample, is increased.

[0030] As described herein above, the level of CgA in a sample from a specified subject (as well as the levels of any other additional markers such as MMP7) can be compared to a control level or a predetermined threshold. These reference levels or predefined thresholds may be absolute values or relative values.

Depending on the specific application, the control level may be the CgA level in the earliest sample from the same subject, for example, the control level may be the CgA level in a sample taken from the same subject prior to surgery (e.g., RCE), and a certain level may be derived from a sample taken after surgical treatment (eg, RCE). The same applies for other markers that can be further defined, such as MMP7.

The predetermined threshold for the level of CgA can, for example, be selected in the range from 100 ng/ml to 431 ng/ml, preferably in the range from 103 ng/ml to 191 ng/ml, more preferably from 130 ng/ml to 160 ng/ml ml, and most preferably 147 ng/ml. The choice of threshold for a particular application may be based, for example, on the desired specificity and/or selectivity of the assay; see below.

[0031] As described in the present description above, in addition to the level of CgA, the level of other markers, in particular biomarkers, can be determined in the same subject, preferably in the same sample. One marker that has proven to be of particular value along with CgA is matrix metalloproteinase-7 (MMP7). Thus, in the context of the present invention, preferably, additionally, the level of matrix metalloproteinase 7 (MMP7) is determined in the specified sample from the specified subject or in another sample from the specified subject, and the level of MMP7 in the sample from the subject is an indicator of the severity of non-neuroendocrine bladder cancer and/ or outcome for the subject. An increased level of MMP7 in a sample from a subject compared to a control level or a predetermined threshold may be indicative of a poor outcome for the subject, in particular an increased risk of reduced life expectancy, an increased risk of progression, an increased risk of cancer-related death, and/or an increased risk of recurrence after surgical treatment and/or drug treatment and/or radiotherapy. The level of MMP7 in the same sample or in a different sample from a subject may, in one aspect, be indicative of the subject's overall survival, or disease-specific survival of the subject, or progression-free survival of the subject. The predetermined threshold for the MMP7 level may, for example, be selected in the range of 4.4 to 21 ng/mL, preferably in the range of 5.4 to 10.1 ng/mL, more preferably in the range of 6 to 9 ng/mL. ml. Most preferably, the predetermined threshold for the MMP7 level is 7.75 ng/mL. The choice of threshold for a particular application may be based. for example, the desired specificity and/or selectivity of the assay; see below.

[0032] In a specific embodiment of the methods of the present invention,

(i) a CgA level in said sample above a predetermined threshold for CgA is indicative of a high risk of cancer-related death after surgical treatment (eg, TURBT, partial cystectomy (PCE), RCE, RCE in combination with regional or extended LND, metastasectomy), or treatments using drugs (chemotherapy, targeted therapy, immunotherapy) or radiotherapy, and

(ii) the CgA level in said sample is above a predetermined CgA threshold and the MMP7 level in said sample is above a predetermined MMP7 threshold is indicative of a very high risk of cancer-related death and shorter life expectancy after surgical treatment (e.g., TURBT, PCE, RCE , RCE in combination with regional or extended LND, metastasectomy), or drug treatments (chemotherapy, targeted therapy, immunotherapy), or radiotherapy.

[0033] Using the methods of the present invention, the medical professional is able to select the most appropriate treatment based on the patient's perceived risk. As described, the invention relates to a post-operative, non-invasive, inexpensive and easy-to-handle instrument for monitoring treatment efficacy and recurrence in bladder cancer, in particular non-neuroendocrine bladder cancer and preferably urothelial carcinoma. For example, the level of CgA and optionally, MMP7, can be determined at regular intervals during follow-up, and the control level can be the level determined prior to said surgical treatment and/or at the initial stage of follow-up.

[0034] In one specific aspect, preoperative level (preferably serum level) of CgA is used as a marker for overall and disease-specific survival in patients after surgery (eg, RCE). In a specific aspect of the invention, MMP7 is used as an additional marker for poor disease-specific survival in patients treated with RCE. MMP7 can also be used as an additional marker for muscle invasive tumor and/or metastasis.

[0035] As discussed, in addition to determining the level of CgA, you can additionally determine the level of other markers, in particular, biomarkers (including MMP7), and other clinical parameters for the subject. Such clinical parameter(s) for a subject may, for example, be selected from the group consisting of histological tumor subtype, metastasis status (lymph node and distant), lymphadenopathy, smoking or tobacco use, age, sex, family history, ethnicity, weight body mass index (BMI), cystoscopic data, urine cytology (VUC), ultrasound, CT, MRI and TURBT scans, and blood pressure. In a specific embodiment, at least one clinical parameter selected from the group including age, gender, systolic blood pressure, diastolic blood pressure, antihypertensive treatment, history of urinary tract disease and treatment, history of stroke, wheezing, index body weight, heart rate, temperature, presence of diabetes and current smoking habit.

[0036] In addition, the level of a marker selected from the group consisting of complement factor H-related protein and complement factor H, nuclear matrix protein BLCA-4, survivin (BIRC5, EPR-1), cytokeratin 8 (CK8), cytokeratin 18 ( CK18), cytokeratin 20 (CK20), CEA protein and bladder tumor cell-associated mucins, changes on chromosomes 3, 7, 17 and 9p21, CEA protein (CEA), CYFRA 21-1 (CK19), carbonic anhydrase, neurological sensor protein (NSE), c-reactive protein (CRP), nuclear mitotic apparatus protein 22 (NMP22), alkaline phosphatase, matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 3 (MMP-3), matrix metalloproteinase 9 (MMP-9), matrix metalloproteinase 10 (MMP-10), matrix metalloproteinase 13 (MMP-13), matrix metalloproteinase 26 (MMP-26), tissue inhibitor of metalloproteinases 1 (TIMP-1), tissue inhibitor of metalloproteinases 2 (TIMP-2), tissue inhibitor of metalloproteinases 3 (TIMP- 3), tissue inhibitor of metalloproteinases 4 (TIMP-4), alpha-1 antitrypsin, vascular endothelial growth factor (VEGF), placental growth factor (PLGF), vascular endothelial growth factor receptor 1 (VEGFR-1), soluble factor receptor 1 vascular endothelial growth factor receptor 2 (VEGFR-2), soluble vascular endothelial growth factor receptor 3 (sVEGFR-3), vascular endothelial growth factor A (VEGFA), endothelial growth factor Vascular C (VEGFC), insulin-like growth factor-binding protein 1 (IGFBP-1), insulin-like growth factor-binding protein 2 (IGFBP-2), insulin-like growth factor-binding protein 3 (IGFBP-3), insulin-like growth factor-binding protein 4 (IGFBP -4), insulin-like growth factor-binding protein 5 (IGFBP-5), insulin-like growth factor-binding protein 6 (IGFBP-6), transforming growth factor β (TGF-β), insulin-like growth factor (IGF), insulin-like growth factor receptor ( IGFR), in sulin-like growth factor 1 (IGF-1), insulin-like growth factor 1 receptor (IGF1R), endothelial growth factor (EGF), endothelial growth factor receptor (EGFR), proheparin-binding EGF-like growth factor (proHB-EGF), insulin-like factor mRNA-binding growth 2 protein 3 (IGFBP3), mRNA-binding insulin-like growth factor 2 protein 7 (IGFBP7), angiostatin, endostatin, plasminogen (PLG), aquaporin 1 (AQP-1), perilipin 2 (PLIN-2), human chorionic gonadotropin (hCG ), androgen receptor (AR), estrogen receptor (ER), prostate-specific antigen (PSA), free prostate-specific antigen (free PSA), total prostate-specific antigen antigen (total PSA), tumor necrosis factor α (TNFα), E-cadherin, elastin , fibronectin, collagen and vitronectin, can be determined in the specified sample or another sample from the subject. Among them, preferred additional markers are selected from complement factor H-related protein and complement factor H, nuclear matrix protein BLCA-4, cytokeratin 8 (CK8), cytokeratin 18 (CK18), CEA protein and bladder tumor cell associated mucins, changes in chromosomes 3, 7, 17 and 9p21, CEA protein (CEA), nuclear mitotic apparatus protein 22 (NMP22), alkaline phosphatase, tissue inhibitor of metalloproteinases 1 (TIMP-1) and tissue inhibitor of metalloproteinases 2 (TIMP-2).

[0037] The term "biomarker" (biological marker) refers to measurable and quantifiable biological parameters (for example, the concentration of a specific enzyme, the concentration of a specific hormone, the distribution of the phenotype of a specific gene in a population, the presence of biological substances) that serve as indicators of health-related and physiology of assessments such as disease risk, psychiatric disorders, environmental exposure and its effects, disease diagnosis, metabolic processes, drug dependence and substance abuse, pregnancy, cell line development, epidemiological studies, etc. In addition, a biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. A biomarker can be measured in a biological sample (e.g., whole blood, serum, plasma, urine), it can be a record of readings obtained from an individual (blood pressure, ECG, or ECG Holter monitoring), or it can be an imaging test (an echocardiogram or CT scan) (Vasan et al. 2006, Circulation 113:2335-2362). Biomarkers can indicate many characteristics of a health condition or disease, including the level or type of exposure to an environmental factor, genetic predisposition, genetic responses to exposures, biomarkers of subclinical or clinical disease, or measures of response to therapy. Thus, a simplified way of understanding biomarkers is to understand as indicators a sign of a disease (risk factor or biomarker), disease state (preclinical or clinical), or rate of disease development (progression). Accordingly, biomarkers can be classified as antecedent biomarkers (identifying the risk of developing a disease), screening biomarkers (screening for subclinical disease), diagnostic biomarkers (recognizing overt disease), grade-determining biomarkers (categorizing disease severity), or prognostic biomarkers (predicting the future course of a disease, including relapse and response to therapy, and monitoring the effectiveness of therapy). Biomarkers can also serve as surrogate endpoints. A surrogate endpoint is a point that can be used as an outcome in clinical trials to evaluate the safety and efficacy of medicines, instead of measuring the actual outcome of interest. The underlying principle is that changes in the surrogate endpoint follow closely with changes in the outcome of interest. Surrogate endpoints have the advantage that they can be obtained in a shorter time frame and at lower cost than endpoints such as morbidity and mortality, which require large clinical trials to evaluate. The added value of surrogate endpoints includes the fact that they are closer to the intervention/intervention of interest and can be more easily causally linked to it than more distant clinical events. An important disadvantage of surrogate endpoints is that if the clinical outcome of interest is influenced by multiple clinical factors (in addition to the surrogate endpoint), residual confounding may reduce the validity of the surrogate endpoint. It was suggested that the validity of a surrogate endpoint is greater if it can explain at least 50% of the effect of the treatment or intervention on the outcome of interest. For example, the biomarker may be a protein, peptide, or nucleic acid molecule.

The National Institutes of Health (NIH) defines a biomarker as a biological marker that is objectively measured and evaluated as an indicator of a normal biological process, a pathogenic process, or pharmacological responses to therapeutic interventions (Danesh et al. Clin Pharmacol Ther 2001. 169:416-468).

Some of the markers (particularly biomarkers) are "cancer markers", ie. they are markers associated with the diagnosis and/or prognosis of a malignant tumor, in the present description: not neuroendocrine bladder cancer.

[0038] The methods of the present invention may include the initial step of obtaining a sample of physiological fluid from the specified subject. The term "sample", as used in the context of the present invention, refers to a sample of physiological fluid obtained for the purposes of diagnosis, prediction or evaluation of a subject of interest, such as a patient. Preferred test samples include (whole) blood, serum, plasma and urine. Some test samples are more simply analyzed after a fractionation or purification procedure, such as separation of whole blood into serum or plasma components. Thus, in a preferred embodiment of the invention, the sample is selected from the group consisting of a (whole) blood sample, a serum sample, a plasma sample and a urine sample or an extract of any of the above samples. Preferably, the sample is a whole blood sample, most preferably a serum sample or a plasma sample. As used herein, "whole blood sample" means an unprocessed or substantially unprocessed blood sample. When appropriate, the sample may need to be homogenized or solvent extracted prior to use in the present invention in order to obtain a liquid sample. The liquid sample thus may be a solution or a suspension. Liquid samples may be subjected to one or more pre-treatments prior to use in the present invention. Such pretreatments include, but are not limited to, dilution, filtration, centrifugation, concentration, sedimentation, precipitation, or dialysis. Pre-treatments may also include the addition of chemical or biochemical agents to the solution, such as acids, bases, buffers, salts, solvents, reactive dyes, detergents, emulsifiers, or chelators.

"Plasma" in the context of the present invention is actually a cell-free supernatant of blood containing an anticoagulant obtained after centrifugation. Exemplary anticoagulants include calcium ion binding compounds such as EDTA or citrate and thrombin inhibitors such as heparinates or hirudin. Cell-free plasma can be obtained by centrifuging anticoagulant-exposed blood (eg, citrate, EDTA, or heparin-exposed blood) for at least 15 minutes at 2000-3000 g. Thus, it is preferred that plasma samples used in the context of the present invention are subjected to centrifugation at more than 1500 g for 30 minutes, preferably at least 2000 g for at least 30 minutes, more preferably at least at 3000 g for at least 20 minutes, most preferably at least 3000 g for at least 30 minutes.

"Serum" in the context of the present invention is the undiluted, extracellular portion of the blood after adequate clotting has been completed. Clotting is usually completed after 30 minutes. Serum can be obtained by centrifuging the coagulated sample for at least 10 minutes at a minimum speed of 1500 g. Thus, it is preferred that serum samples used in the context of the present invention are centrifuged at at least 1500 g for at least 10 minutes, preferably for at least 15 minutes, more preferably for at least 20 min. Most preferably, the serum sample is centrifuged at at least 3000 g for at least 20 minutes.

[0039] The level of CgA can be determined using appropriate assays. "Analysis" or "diagnostic analysis" can refer to any type used in the field of diagnostics. Such an assay may be based on the binding of the analyte to be detected to one or more binding probes with a certain affinity. With regard to the interaction between binding molecules and target molecules or molecules of interest, the affinity constant preferably exceeds 10 ⁸ M ^-1 .

[0040] Preferred detection methods include immunoassays in various formats, such as, for example, radioimmunoassay (RIA), chemiluminescent and fluorescent immunoassays, enzyme-linked immunosorbent assays (ELISA), Luminex bead arrays, protein microarray assays, rapid assay formats, or point-of-care (PoC) assays, such as, for example, immunochromatographic strip tests; and automated immunoassay systems, such as, for example, the BRAHMS KRYPTOR system. Particularly preferred are PoC test formats; for example, see St. John & Price, Clin Biochem Rev. 2014 Aug; 35(3): 155-167. In addition, mass spectrometry techniques can be used to detect and quantify CgA, MMP7 and/or additional biomarkers, for example selective response quantification (qSRM) can be used. For mass spectrometric measurements, chemical derivatization is usually performed prior to analysis and quantification of the target protein.

[0041] Immunoassays can be homogeneous or heterogeneous assays, competitive or non-competitive assays. In a particularly preferred embodiment, the assay is in the form of a sandwich assay, which is a non-competitive immunoassay in which the molecule to be detected and/or quantified binds to a first antibody and a second antibody. The first antibody may be associated with a solid phase, such as a bead, the surface of a well or other container, a chip, or a strip, and the second antibody is an antibody that is labeled, such as with a dye, a radioactive isotope, or a reactive or catalytically active group. The amount of labeled antibody bound to the analyte is then measured in an appropriate manner. The general composition and methods involved in "sandwich assays" are well established and known to those skilled in the art ((The Immunoassay Handbook, Ed. David Wild, Elsevier LTD, Oxford; 3rd ed. (May 2005), ISBN-13: 978 -0080445267; Hultschig C et al., Curr Opin Chem Biol 2006 Feb;10(1):4-10, the contents of which are incorporated herein by reference).

[0042] In a particularly preferred embodiment, the assay comprises two binding molecules, preferably antibodies, both of which are present in the form of dispersions in a liquid reaction mixture, where the first labeling component is attached to the first binding molecule, where the specified first labeling component is part of a labeling system based on quenching or enhancing fluorescence or chemiluminescence, and the second labeling component of said labeling system is attached to a second binding molecule such that upon binding of both binding molecules to the analyte, a measurable signal is generated allowing detection of the formed sandwich complexes in the solution containing the sample.

Even more preferably, said labeling system comprises rare earth metal cryptates or rare earth metal chelates in combination with a fluorescent dye or a chemiluminescent dye, in particular a cyanine type dye.

[0043] In the context of the methods of the present invention, the level of CgA and/or MMP7 is preferably determined using an immunoassay. Suitable immunoassays and antibodies for the detection of CgA are described, for example, in WO 2015/158701 A1 and Popovici et al. (2014. Clin Biochem 47: 87-91) (the contents of which are hereby incorporated by reference). For example, you can use the analysis of chromogranin A B·R·A·H·M·S KRYPTOR (B·R·A·H·M·S GmbH, Hennigsdorf, Germany). Additional commercial assays for the detection of chromogranin A are available and can be used: the Cis-Bio ELISA assay (Cisbio Bioassays, Codolet, France) uses two monoclonal antibodies targeting epitopes corresponding to amino acids 145-197 and 219-234, the DAKO assay ELISA (Dako Denmark A/S, Glostrup, Denmark) uses a rabbit polyclonal antibody targeted against the 23 kDa C-terminal fragment, the Euro-Diagnostica NEOLISA™ sandwich ELISA (Euro Diagnostica AB, Malmö, Sweden) uses two monoclonal antibodies, targeted against epitopes corresponding to amino acids 236-251 and 264-279 (see also WO 2011/135035 A1 and WO 99/58980 A1).

Suitable immunoassays for the detection of MMP7 are described, for example, in WO 2007/144144 A1 (the contents of which are thus incorporated by reference).

Commercially available assays for MMP7 include: Human Total MMP-7 Quantikine ELISA Kit or Human Magnetic Luminex Screening Assay or Human Luminex Screening Assay or Human MMP-7 Activity Assay Human MMP-7 Luminex Performance Assay, or Human MMP-7 Magnetic Luminex Performance Assay, or Human Total MMP-7 DuoSet ELISA, or Human MMP Premixed Luminex Performance Assay Kit, or Human Proteome Profiler Kit Protease Array Kit (R&D Systems Minneapolis, MN 55413, USA), ELISA kit for human total MMP-7 Human Total MMP-7 ELISA Kit (Aviva Systems Biology Corporation, San Diego, CA 92121, USA), ELISA kit for MMP7 (human ) MMP7 (Human) ELISA Kit (Abnova, Taipei 114, Taiwan), Human MMP7/Matrilysin ELISA Kit (Sandwich ELISA) (LifeSpan Biosciences, Seattle, Washington 98121, USA), on MMP7 Human ELISA Kit (Abcam, Cambridge, United Kingdom), MMP-7 SensoLyte® 490 MMP-7 Assay Kit *Fluorimetric* (ANASPEC, Fremont, CA 94555, USA), Bio- Plex Pro™ for Human MMP and TIMP (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.), Matrix Metalloproteinase 7 (MMP7) ELISA Kit ELISA Kit for Matrix Metalloproteinase 7 (MMP7) (Wuhan USCN Business Co., Ltd ., Wuhan City, China).

[0044] The CgA detection immunoassays described in WO 2015/158701 A1 are preferred in the context of the present invention. They are based on the use of two antibodies against CgA (sandwich immunoassay). The (first and/or second) antibodies or antigen-binding fragments or derivatives thereof from an immunoassay method as described in WO 2015/158701 A1 may, for example, be polyclonal antibodies, monoclonal antibodies or genetically modified monoclonal antibodies. Said first antibody is specific for an epitope in the CgA sequence (SEQ ID NO:1), preferably in the sequence spanning amino acids 124-144 of SEQ ID NO:1. The first antibody is preferably a monoclonal antibody. Said second antibody is specific for an epitope in the CgA sequence (SEQ ID NO:1), preferably in the sequence spanning amino acids 280-301 of SEQ ID NO:1. The second antibody is preferably a monoclonal antibody. In a particular immunoassay, the first antibody is specific for an epitope in the CgA sequence (SEQ ID NO:1) spanning amino acid residues 124-144 and the second antibody is specific for an epitope in the CgA sequence (SEQ ID NO:1) spanning amino acid residues 280 -301. The first and second antibodies are preferably monoclonal antibodies. The first antibody, or an antigen-binding fragment or derivative thereof, can, for example, be produced in a 537/H2 hybridoma cell line deposited as DSM ACC3231. The antibody produced in the 537/H2 hybridoma cell line specifically binds to amino acid residues 124-144 of the CgA sequence (SEQ ID NO:1). The second antibody, or antigen-binding fragment or derivative thereof, can, for example, be produced in the 541/E2 hybridoma cell line deposited as DSM ACC3232. The antibody produced in the 541/E2 hybridoma cell line specifically binds to amino acid residues 280-301 of the CgA sequence (SEQ ID NO:1). In a specific embodiment of the immunoassay, the first antibody is generated in a 537/H2 hybridoma cell line deposited as DSM ACC3231 and the second antibody is generated in a 541/E2 hybridoma cell line deposited as DSM ACC3232.

[0045] Biomarkers such as CgA may be fragmented into shorter peptides or proteins. Thus, in some cases also fragments of CgA or other peptide biomarkers such as MMP7 can be detected and their level in the sample can be determined. The term "fragment" refers to smaller proteins or peptides that can be obtained from larger proteins or peptides, which thus contain a partial sequence of a larger protein or peptide. These fragments can be obtained from larger proteins or peptides by saponification of one or more of their peptide bonds. Said peptide fragments are preferably at least about 15 to about 20 amino acids in length, more preferably at least about 25 to about 45 amino acids in length.

[0046] Measured levels of markers (CgA, MMP7, and others, as appropriate) can be correlated with a specific diagnosis and/or prognosis, for example, using a specific mathematical algorithm. In the context of the present invention, "algorithm" or "mathematical algorithm" refers to the use of a mathematical or statistical method or model used to compare a particular measured value with values in a reference population to stratify said measured value. This may, for example, be the median of the level of a particular molecule in an ensemble of predetermined samples, which means that the measured level of said molecule is compared to the mathematical median of the level of said molecule in a given number of samples. The number of samples used to determine the median is not particularly limited, but should be sufficient to ensure that the median is statistically significant. The number of samples used to determine the median may even increase over time as further measurement values for clinical samples are added to increase the statistical significance of the median. Preferably, the number of samples is chosen such that the median is statistically significant. Thus, said median is used as a reference value, whereby the measured level of the aforementioned molecule can be statistically correlated with a specific physiological state, for example, a predisposition to an adverse outcome for the patient, depending on the relative level above or below the median, and the degree of deviation of the measured value from specified median. Instead of the median, other statistical methods, such as quantile determinations (e.g., quartiles or percentiles), or mathematical models, preferably Cox regression, can be used similarly to the above description to obtain the above reference value and/or otherwise determine the significance of the measured value in relation to the physiological status of the subject from which the sample was obtained. These mathematical or statistical methods or models are well known to the person skilled in the art and their use in the context of medical applications is well established.

[0047] The term "correlated" or "correlation" as used herein in relation to the use of a diagnostic and prognostic marker(s) refers to comparing the presence or level of a marker(s) in a patient with its presence or level in individuals known to suffering from, or known to be at risk for, the condition. The level of the marker in a sample from a patient can be compared to a level known to be associated with a specific diagnosis. The level of the marker in the sample is said to be correlated with the diagnosis; that is, one of skill in the art can use the level of the marker to determine if a patient is suffering from a particular type of disease and take appropriate responses. Alternatively, the level of the marker in the sample can be compared to the level of the marker known to be associated with a good outcome (eg, absence of disease, etc.). In preferred embodiments, the bar of marker levels correlates with a global probability or a particular outcome.

[0048] In other embodiments, a positive likelihood ratio, a negative likelihood ratio, an odds ratio, or a hazard ratio is used as an indicator of a test's ability to predict risk or diagnose a disease. In the case of a likelihood ratio of a positive study result, a value of 1 indicates that a positive result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the affected group; and a value less than 1 indicates that a positive result is more likely in the control group. In the case of a likelihood ratio of a negative test result, a value of 1 indicates that a negative result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a negative result is more likely in the test group; and a value less than 1 indicates that a negative result is more likely in the control group. In particular preferred embodiments, markers and/or marker panels are selected, preferably having a positive or negative likelihood ratio of at least about 1.5 or more or about 0.67 or less, more preferably at least about 2 or more or about 0.5 or less, even more preferably at least about 5 or more or about 0.2 or less, even more preferably at least about 10 or more or about 0.1 or less, and most preferably , at least about 20 or more, or about 0.05 or less. The term "approximately" in this context refers to +/- 5% of a given measurement.

[0049] In the case of an odds ratio, a value of 1 indicates that a positive result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the affected group; and a value less than 1 indicates that a positive result is more likely in the control group. In specific preferred embodiments, markers and/or marker panels are selected, preferably having an odds ratio of at least about 2 or more, or about 0.5 or less, more preferably at least about 3 or more, or about 0.33 or less, even more preferably at least about 4 or more, or about 0.25 or less, even more preferably at least about 5 or more, or about 0.2 or less, and most preferably at least about 10 or more, or about 0.1 or less. The term "approximately" in this context refers to +/- 5% of a given measurement.

[0050] In the case of a hazard ratio, a value of 1 indicates that the relative risk of an endpoint (eg, death) is equal in both the "diseased" and "control" groups; a value greater than 1 indicates that the risk is higher in the affected group; and a value less than 1 indicates that the risk is higher in the control group. In specific preferred embodiments, markers and/or marker panels are selected, preferably having a hazard ratio of at least about 1.1 or more, or about 0.91 or less, more preferably at least about 1.25 or more, or about 0.8 or less, even more preferably at least about 1.5 or more, or about 0.67 or less, even more preferably at least about 2 or more, or about 0.5 or less, and most preferably at least about 2.5 or more, or about 0.4 or less. The term "approximately" in this context refers to +/- 5% of a given measurement.

[0051] The sensitivity and specificity of a diagnostic and/or predictive test depends on more than just the analytical "quality" of the test, they also depend on determining what constitutes an abnormal result. In practice, operating characteristic curves (ROC curves) are typically calculated by plotting the value of a variable against its relative frequency in "normal" (i.e. apparently healthy) and "diseased" populations ( i.e., in patients suffering from bladder cancer, in particular non-neuroendocrine bladder cancer and preferably urothelial carcinoma). For any particular marker, the distribution of marker levels for subjects with and without disease is likely to overlap. Under such conditions, the test does not absolutely distinguish normal from disease with 100% accuracy, and the area of overlap indicates where the test cannot distinguish normal from disease. A threshold is chosen above which (or below which, depending on how the marker changes with disease) a test is considered abnormal, and below which a test is considered normal. The area under the ROC curve is a measure of the likelihood that the observed measurement can allow correct identification of the condition. ROC curves can be used even when test results do not necessarily give an accurate count. As long as you can rank the results, you can get the ROC curve. For example, test results for "diseased" samples can be ranked according to grade (eg, 1=low, 2=normal, and 3=high). This ranking can be correlated with the results in the "normal" population, and get the ROC curve. These methods are well known in the art (see, for example, Hanley et al. 1982. Radiology 143: 29-36). Preferably, ROC curves produce an AUC greater than about 0.5, more preferably greater than about 0.7, even more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably, greater than about 0.9. The term "approximately" in this context refers to +/- 5% of a given measurement.

[0052] The horizontal axis of the ROC curve represents (1-specificity), which increases with the proportion of false positives. The vertical axis of the curve represents sensitivity, which increases with the proportion of true positives. Thus, for a particular cut-off threshold chosen, a value (1-specificity) can be determined and an appropriate sensitivity can be obtained. The area under the ROC curve is a measure of the likelihood that the measured level of a marker may allow the correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.

[0053] In specific embodiments, markers and/or marker panels are selected to have at least about 70% sensitivity, more preferably at least about 80% sensitivity, even more preferably at least about 85% sensitivity, even more preferably at least about 90% sensitivity, and most preferably at least about 95% sensitivity, combined with at least about 70% specificity, more preferably at least about 80% specificity, even more preferably, at least about 85% specificity, even more preferably at least about 90% specificity, and most preferably at least about 95% specificity. In particularly preferred embodiments, both sensitivity and specificity are at least about 75%, more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90 %, and most preferably at least about 95%. The term "approximately" in this context refers to +/- 5% of a given measurement.

[0054] Appropriate threshold levels for "stratification of subjects" into different groups (categories) should be determined for each specific combination of CgA level, additional markers (such as MMP7) and/or parameters, drugs and diseases. This can, for example, be done by grouping a reference population of patients according to their CgA level into specific quantiles, eg quartiles, quintiles, or even according to suitable percentiles. For each of the quantiles or groups above and below certain percentiles, hazard ratios can be calculated comparing the risk of a poor outcome, i.e. an "adverse effect", for example, in terms of survival rate, between those patients who were administered a particular drug and those who were not. In this scenario, a hazard ratio (HR) greater than 1 indicates a higher risk of adverse outcome for treated patients than for untreated patients. An HR below 1 indicates beneficial effects of a particular treatment in a group of patients. An HR of about 1 (eg +/- 0.1) indicates no increase in risk, but also no benefit from the drug for a particular patient population. By comparing the HR between specific patient quantiles with each other and with the HR of the general patient population, it is possible to identify those patient quantiles that are at increased risk and those that benefit from the drug, and thus stratify the subjects according to the present invention.

[0055] The term "therapeutic treatment stratification" or "therapy stratification" in the context of the present invention refers to the evaluation of an appropriate therapeutic treatment for a specified patient. Subclassification refers to an additional definition of a diagnosis according to subclasses of the diagnosed disease, disorder, complication, or risk, such as a definition according to mild and severe disease. The term "monitoring therapy" in the context of the present invention refers to the control and/or adjustment of therapeutic treatment of the specified patient.

[0056] The present invention also relates to methods for treating non-neuroendocrine bladder cancer, wherein CgA levels (and optionally, additional markers and/or clinical parameters such as MMP7 levels) are determined to determine the most appropriate treatment. For example, a patient having a relatively poor prognosis according to a certain elevated CgA level may benefit from more aggressive treatment, such as partial cystectomy instead of TURBT or triple bladder sparing, RCE instead of TURBT or triple bladder sparing treatment, or partial cystectomy, RCE in combination with PLND instead of RCE, extended or super-expanded LND instead of PLND, adjuvant chemotherapy or radiotherapy instead of one surgery, additional metastasectomy. This high-risk patient may also be selected for clinical trials testing new drugs.

[0057] In addition, the present invention relates to the use of kits containing antibodies specific for CgA, and optionally antibodies specific for MMP7, for predicting, risk assessment, risk stratification, monitoring and/or control of bladder cancer therapy, in particular , non neuroendocrine bladder cancer and preferably urothelial carcinoma. Kits may also contain additional components such as buffers and instructions for use. A preferred CgA detection kit is described in WO 2015/158701 A1 and contains

(i) a first antibody or antigen-binding fragment or derivative thereof that is specific for CgA or a fragment thereof; and

(ii) a second antibody or antigen-binding fragment or derivative thereof that is specific for CgA or a fragment thereof.

The first and second antibodies of the set are preferably specific for the same CgA fragment or fragments. For example, (i) the first antibody or antigen-binding fragment or derivative thereof is specific for an epitope contained within the sequence of SEQ ID NO:1; and/or (ii) the second antibody, or antigen-binding fragment or derivative thereof, is specific for an epitope contained within the sequence of SEQ ID NO:1. Preferably, the first antibody of the kit is an anti-CgA monoclonal antibody generated in a 537/H2 hybridoma cell line deposited as DSM ACC3231 and/or the second antibody is an anti-CgA monoclonal antibody generated in a 541/E2 hybridoma cell line deposited as DSM ACC3232.

The kit may contain one or more antibodies for detecting MMP7. For example, the set (in addition) contains

(i) a first antibody or antigen-binding fragment or derivative thereof that is specific for MMP7 or a fragment thereof; and

(ii) a second antibody or antigen-binding fragment or derivative thereof that is specific for MMP7 or a fragment thereof.

The first and second antibodies of the set are preferably specific for the same fragment or fragments of MMP7. For example, (i) the first antibody or antigen-binding fragment or derivative thereof is specific for an epitope contained within the sequence of SEQ ID NO:2; and/or (ii) the second antibody, or antigen-binding fragment or derivative thereof, is specific for an epitope contained within the sequence of SEQ ID NO:2.

[0058] The present invention relates in preferred aspects to:

1. The use of chromogranin A (CgA) as a prognostic marker for bladder cancer.

2. Use according to aspect 1, wherein the bladder cancer is non-neuroendocrine bladder cancer.

3. The use according to aspect 1 wherein the bladder cancer is selected from the group consisting of bladder urothelial carcinoma, bladder squamous cell carcinoma and bladder adenocarcinoma, preferably wherein the bladder cancer is bladder urothelial carcinoma.

4. Use according to aspect 3, wherein the bladder cancer is non-muscle invasive bladder cancer (NMBC) or muscle invasive bladder cancer (MIBC).

5. The use according to aspects 1-4 wherein CgA is used as a marker in an in vitro assay for predictive, risk assessment, risk stratification, monitoring and/or control of bladder cancer therapy.

6. A method for predicting, assessing risk, risk stratifying, monitoring and/or controlling therapy for bladder cancer in a subject, comprising the step of determining the level of CgA in a sample of body fluid of said subject.

7. The method of aspect 6 wherein the bladder cancer is non-neuroendocrine bladder cancer.

8. The method of claim 5 or 6 wherein the bladder cancer is selected from the group consisting of bladder urothelial carcinoma, bladder squamous cell carcinoma and bladder adenocarcinoma, preferably wherein the bladder cancer is bladder urothelial carcinoma.

9. The method of aspect 8 wherein the bladder cancer is non-muscle invasive bladder cancer (NMBC) or muscle invasive bladder cancer (MIBC).

10. The method according to aspects 6-9, wherein the level of CgA in a sample from a subject is indicative of bladder cancer severity and/or outcome for the subject.

11. The method of aspect 10 wherein an increased level of CgA in a sample from a subject compared to a control level or a predetermined threshold is indicative of poor outcome for the subject.

12. The method of aspect 10, wherein the level of CgA in the sample from the subject is indicative of the subject's overall survival, or the subject's disease-specific survival, or the subject's progression-free survival.

13. The method of aspect 12, wherein the poor outcome is an increased risk of reduced life expectancy, an increased risk of progression, an increased risk of cancer-related death, and/or an increased risk of recurrence after surgery and/or drug treatment, and/or radiotherapy.

14. The method according to any one of aspects 6-13, wherein the subject is diagnosed with bladder cancer and wherein the subject has not undergone surgical treatment for said bladder cancer.

15. The method according to any one of aspects 6-13, wherein the subject has undergone surgical treatment of said bladder cancer.

16. The method of aspect 15 wherein the subject has undergone a radical cystectomy of said bladder cancer.

17. The method according to any one of aspects 11-16, wherein the predetermined threshold for CgA level is selected in the range of 100 ng/mL to 431 ng/mL, preferably in the range of 103 ng/mL to 191 ng/mL, more preferably , from 130 ng/ml to 160 ng/ml, and most preferably, the threshold is 147 ng/ml.

18. The method of any one of aspects 6-17, wherein further, the level of matrix metalloproteinase 7 (MMP7) is determined in said sample from said subject, and wherein the level of MMP7 in the sample from the subject is indicative of bladder cancer severity and/or outcome for the subject .

19. The method of aspect 18 wherein an increased level of MMP7 in a sample from a subject compared to a control level or a predetermined threshold is indicative of a poor outcome for the subject, in particular an increased risk of reduced life expectancy, an increased risk of progression, an increased risk of associated cancer death and/or increased risk of recurrence after surgery and/or drug treatment and/or radiotherapy.

20. The method of aspect 18, wherein the level of MMP7 in the sample from the subject is indicative of the subject's overall survival, or the subject's disease-specific survival, or the subject's progression-free survival.

21. The method according to aspect 19, wherein the predetermined threshold for the MMP7 level is selected in the range of 4.4 to 21 ng/mL, preferably in the range of 5.4 to 10.1 ng/mL, more preferably in the range of 6 to 9 ng/ml, most preferably, the predetermined threshold for the MMP7 level is 7.75 ng/ml.

22. The method in accordance with any of aspects 11-21, where

(i) a CgA level in said sample above a predetermined threshold for CgA is indicative of a high risk of cancer-related death following surgery and/or drug treatment and/or radiotherapy, and

(ii) the CgA level in said sample is above a predetermined CgA threshold and the MMP7 level in said sample or another sample from the same subject is above a predetermined MMP7 threshold is indicative of a very high risk of cancer-related death and shorter life expectancy after surgical treatment, and /or drug treatment and/or radiotherapy.

23. The method according to any one of aspects 11-22, wherein the CgA level and optionally MMP7 is determined at regular intervals during follow-up, and where the control level is the appropriate level determined prior to said surgical treatment and/or at the initial stage follow-up.

24. The method according to any one of aspects 6-23, further comprising determining a clinical parameter for the subject selected from the group consisting of tumor histological subtype, metastasis status (lymph node and distant), lymphadenopathy, smoking or tobacco use, age, gender, family history, ethnicity, body weight, body mass index (BMI), cystoscopy, urine cytology (VUC), ultrasound, CT, MRI, and TURBT scans, and blood pressure.

25. The method according to any of aspects 6-24 further comprising determining the level of a marker selected from the group consisting of complement factor H related protein and complement factor H, nuclear matrix protein BLCA-4, cytokeratin 8 (CK8), cytokeratin 18 (CK18), CEA protein and bladder tumor cell-associated mucins, changes on chromosomes 3, 7, 17 and 9p21, CEA protein (CEA), nuclear mitotic apparatus protein 22 (NMP22), alkaline phosphatase, tissue inhibitor of metalloproteinases 1 (TIMP -1) and tissue inhibitor of metalloproteinases 2 (TIMP-2) in the specified sample or another sample from the subject.

26. The method according to any one of aspects 6-25, wherein the sample is from a bodily fluid selected from whole blood, serum, plasma, and urine.

27. The method according to any one of aspects 6-26, wherein the level of CgA and/or MMP7 is determined using an immunoassay.

28. The method of aspect 27 wherein the immunoassay is selected from the group consisting of radioimmunoassay (RIA), chemiluminescent and fluorescent immunoassay, enzyme immunoassay (EIA), ELISA, Luminex bead array assay, protein microarray assay and test on immunochromatographic strips.

29. The method of aspect 28 wherein the immunoassay is a sandwich immunoassay using first and second antibodies specific for CgA.

30. The method according to aspect 29, wherein one of the antibodies is labeled and the other antibody is solid phase bound or may be selectively bound to the solid phase.

31. The method of immunoassay according to aspect 30, wherein the first and second antibodies are present in the form of dispersions in a liquid reaction mixture, and where the first labeling component, which is part of a labeling system based on quenching or enhancing fluorescence or chemiluminescence, is associated with the first antibody, and the second labeling component of said labeling system is coupled to the second antibody so that after binding of both antibodies to CgA, a measurable signal is generated allowing detection of the resulting sandwich complexes in the measured solution.

32. An immunoassay method according to aspect 31, wherein the labeling system comprises rare earth metal cryptates or chelates in combination with a fluorescent dye or a chemiluminescent dye, in particular a cyanine type dye.

33. A method of using chromogranin A (CgA) levels in a subject's body fluid sample as a prognostic marker for bladder cancer.

Cited links

[0059] The entire contents of the references cited in the present description are hereby incorporated by reference.

Alijo Serrano, F., N. Sanchez-Mora, J. Angel Arranz, C. Hernandez & E. Alvarez-Fernandez (2007) Large cell and small cell neuroendocrine bladder carcinoma: an immunohistochemical and outcome study in a single institution. Am J Clin Pathol, 128, 733-9.

Babjuk, M. (2009) Transurethral Resection of Non-muscle-invasive Bladder Cancer. European Urology Supplements, 8, 542-548.

Bagchi, A., K. Dushaj, A. Shrestha, A. L. Leytin, S. A. Bhuiyan, F. Radparvar, S. Topchik, S. S. Tuli, P. Kim & S. Bakshi (2015) Urinary bladder paraganglioma presenting as micturition-induced palpitations, dyspnea, and angina. Am J Case Rep, 16, 283-6.

Bertaccini, A., D. Marchiori, A. Cricca, M. Garofalo, C. Giovannini, F. Manferrari, T. G. Gerace, R. Pernetti & G. Martorana (2008) Neuroendocrine carcinoma of the urinary bladder: case report and review of the literature. Anticancer Res, 28, 1369-72.

Brausi, M., J. A. Witjes, D. Lamm, R. Persad, J. Palou, M. Colombel, R. Buckley, M. Soloway, H. Akaza & A. Bohle (2011) A review of current guidelines and best practice recommendations for the management of nonmuscle invasive bladder cancer by the International Bladder Cancer Group. J Urol, 186, 2158-67.

Brausi, M. A. (2013) Primary prevention and early detection of bladder cancer: two main goals for urologists. Eur Urol, 63, 242-3.

Burger, M., J. W. Catto, G. Dalbagni, H. B. Grossman, H. Herr, P. Karakiewicz, W. Kassouf, L. A. Kiemeney, C. La Vecchia, S. Shariat & Y. Lotan (2013a) Epidemiology and risk factors of urothelial bladder cancer. Eur Urol, 63, 234-41.

Burger, M., W. Oosterlinck, B. Konety, S. Chang, S. Gudjonsson, R. Pruthi, M. Soloway, E. Solsona, P. Sved, M. Babjuk, M. A. Brausi, C. Cheng, E. Comperat, C. Dinney, W. Otto, J. Shah, J. Thurof, J. A. Witjes & C. International Consultation on Urologic Disease-European Association of Urology Consultation on Bladder (2013b) ICUD-EAU International Consultation on Bladder Cancer 2012: Non -muscle-invasive urothelial carcinoma of the bladder. Eur Urol, 63, 36-44.

Cauberg Evelyne, C. C., J. J. de la Rosette & T. M. de Reijke (2011) Emerging optical techniques in advanced cystoscopy for bladder cancer diagnosis: A review of the current literature. Indian J Urol, 27, 245-51.

Cerulli, C., G. M. Busetto, G. Antonini, R. Giovannone, M. Di Placido, G. Soda, E. De Berardinis & V. Gentile (2012) Primary metastatic neuroendocrine small cell bladder cancer: a case report and literature review . Urol Int, 88, 365-9.

Chamie, K., C. S. Saigal, J. Lai, J. M. Hanley, C. M. Setodji, B. R. Konety, M. S. Litwin & P. Urologic Diseases in America (2011) Compliance with guidelines for patients with bladder cancer: variation in the delivery of care. Cancer, 117, 5392-401.

Chuang, C. K. & S. K. Liao (2003) A retrospective immunohistochemical and clinicopathological study of small cell carcinomas of the urinary tract. Chang Gung Med J, 26, 26-33.

Clark, P. E., N. Agarwal, M. C. Biagioli, M. A. Eisenberger, R. E. Greenberg, H. W. Herr, B. A. Inman, D. A. Kuban, T. M. Kuzel, S. M. Lele, J. Michalski, L. C. Pagliaro, S. K. Pal, A. Patterson, E. R. Plimack, K. S. Pohar, M. P. Porter, J. P. Richie, W. J. Sexton, W. U. Shipley, E. J. Small, P. E. Spiess, D. L. Trump, G. Wile, T. G. Wilson, M. Dwyer & M. Ho (2013) Bladder cancer. J Natl Compr Canc Netw, 11, 446-75.

El Demery, M., G. Demirdjian-Sarkissian, S. Thezenas, W. Jacot, Y. Laghzali, B. Darbouret, S. Culine, X. Rebillard & P.-J. Lamy (2014) Serum Matrix Metalloproteinase-7 is an independent prognostic biomarker in advanced bladder cancer. Clinical and Translational Medicine, 3.

Feng, N., X. Li, H. D. Gao, Z. L. Liu, L. J. Shi & W. Z. Liu (2013) Urinary bladder malignant paraganglioma with vertebral metastasis: a case report with literature review. Chin J Cancer, 32, 624-8.

Fernandez-Gomez, J., R. Madero, E. Solsona, M. Unda, L. Martinez-Pineiro, A. Ojea, J. Portillo, M. Montesinos, M. Gonzalez, C. Pertusa, J. Rodriguez-Molina , JE Camacho, M. Rabadan, A. Astobieta, S. Isorna, P. Muntanola, A. Gimeno, M. Blas, JA Martinez-Pineiro & O. Club Urologico Espanol de Tratamiento (2011) The EORTC tables overestimate the risk of recurrence and progression in patients with non-muscle-invasive bladder cancer treated with bacillus Calmette-Guerin: external validation of the EORTC risk tables. Eur Urol, 60, 423-30.

Goodison, S., C. J. Rosser & V. Urquidi (2013) Bladder cancer detection and monitoring: assessment of urine- and blood-based marker tests. Mol Diagn Ther, 17, 71-84.

Gunes, M., A. S. Kemik, N. Pirincci, I. Gecit, K. Taken, M. B. Yuksel, M. Kaba & R. Eryilmaz (2013) Preoperative levels of matrix metalloproteinase-7 and -9 and tissue inhibitor of matrix metalloproteinase- 1 relation to pathologic parameters in bladder carcinoma patients. Asian Pac J Cancer Prev, 14, 873-6.

Hansel, D. E., J. S. Miller, M. S. Cookson & S. S. Chang (2013) Challenges in the Pathology of Non-Muscle-invasive Bladder Cancer: A Dialogue Between the Urologic Surgeon and the Pathologist. Urology.

Iczkowski, K. A., J. H. Shanks, W. C. Allsbrook, A. Lopez-Beltran, C. G. Pantazis, T. R. Collins, R. W. Wetherington & D. G. Bostwick (1999) Small cell carcinoma of the urinary bladder is differentiated from urothelial carcinoma by chromogranin expression, absence of CD44 variant 6 expression, a unique pattern of cytokeratin expression, and more intense gamma-enolase expression. Histopathology, 35, 150-6.

Jager, T., S. Tschirdewahn, F. Vom Dorp, G. Piechotta, H. Rubben & T. Szarvas (2013) [Siliconchiptechnology-based MMP-7 analysis in urine: An option for preoperative identification of lymph node metastasis in bladder cancer]. Urologie A, 52, 853-8.

Kamat, A. M., P. K. Hegarty, J. R. Gee, P. E. Clark, R. S. Svatek, N. Hegarty, S. F. Shariat, E. Xylinas, B. J. Schmitz-Drager, Y. Lotan, L. C. Jenkins, M. Droller, B. W. van Rhijn, P. I. Karakiewicz & C. International Consultation on Urologic Disease-European Association of Urology Consultation on Bladder (2013) ICUD-EAU International Consultation on Bladder Cancer 2012: Screening, diagnosis, and molecular markers. Eur Urol, 63, 4-15.

Leal, J., R. Luengo-Fernandez, R. Sullivan & J. A. Witjes (2015) Economic Burden of Bladder Cancer Across the European Union. EUR Urol.

Maurer, T., T. Horn, M. Heck, J. Gschwend, M. Eiber & A. Beer (2013) Current Staging Procedures in Urinary Bladder Cancer. Diagnostics, 3, 315-324.

Mossanen, M. & J. L. Gore (2014) The burden of bladder cancer care: direct and indirect costs. Curr Opin Urol.

Pompas-Veganzones, N., P. Gonzalez-Peramato & M. Sanchez-Carbayo (2014) The neuroendocrine component in bladder tumors. Curr Med Chem, 21, 1117-28.

Scarpato, K. R., A. K. Morgans & K. A. Moses (2015) Optimal management of muscle-invasive bladder cancer - a review. Res Rep Urol, 7, 143-51.

Schmitz-Drager, B. J., M. Droller, V. B. Lokeshwar, Y. Lotan, M. A. Hudson, B. W. van Rhijn, M. J. Marberger, Y. Fradet, G. P. Hemstreet, P. U. Malmstrom, O. Ogawa, P. I. Karakiewicz & S. F. Shariat (2015) Molecular markers for bladder cancer screening, early diagnosis, and surveillance: the WHO/ICUD consensus. Urol Int, 94, 1-24.

Shariat, S. F., B. Ehdaie, M. Rink, E. K. Cha, R. S. Svatek, T. F. Chromecki, H. Fajkovic, G. Novara, S. G. David, S. Daneshmand, Y. Fradet, Y. Lotan, A. I. Sagalowsky, T. Clozel , P. J. Bastian, W. Kassouf, H. M. Fritsche, M. Burger, J. I. Izawa, D. Tilki, F. Abdollah, F. K. Chun, G. Sonpavde, P. I. Karakiewicz, D. S. Scherr & M. Gonen (2012) Clinical nodal staging scores for bladder cancer: a proposal for preoperative risk assessment. Eur Urol, 61, 237-42.

Sharma, S., P. Ksheersagar & P. Sharma (2009) Diagnosis and treatment of bladder cancer. Am Fam Physician, 80, 717-23.

Skinner, E. C. & A. I. Sagalowsky (2014) Is extended lymphadenectomy of beneficial therapeutic value for T2 urothelial cancer? J Urol, 191, 1206-8.

Soukup, V., M. Kalousova, O. Capoun, R. Sobotka, Z. Breyl, M. Pesl, T. Zima & T. Hanus (2015) Panel of Urinary Diagnostic Markers for Non-Invasive Detection of Primary and Recurrent Urothelial Urinary Bladder Carcinoma. Urol Int, 95, 56-64.

Svatek, R. S., B. K. Hollenbeck, S. Holmang, R. Lee, S. P. Kim, A. Stenzl & Y. Lotan (2014) The Economics of Bladder Cancer: Costs and Considerations of Caring for This Disease. EUR Urol.

Svatek, R. S., J. B. Shah, J. Xing, D. Chang, J. Lin, D. J. McConkey, X. Wu & C. P. Dinney (2010) A multiplexed, particle-based flow cytometric assay identified plasma matrix metalloproteinase-7 to be associated with cancer-related death among patients with bladder cancer. Cancer, 116, 4513-9.

Szarvas, T., M. Becker, F. vom Dorp, C. Gethmann, M. Totsch, A. Bankfalvi, K. W. Schmid, I. Romics, H. Rubben & S. Ergun (2010) Matrix metalloproteinase-7 as a marker of metastasis and predictor of poor survival in bladder cancer. Cancer Sci, 101, 1300-8.

Szarvas, T., T. Jager, M. Becker, S. Tschirdewahn, C. Niedworok, I. Kovalszky, H. Rubben, S. Ergun & F. vom Dorp (2011a) Validation of circulating MMP-7 level as an independent prognostic marker of poor survival in urinary bladder cancer. Pathol Oncol Res, 17, 325-32.

Szarvas, T., B. B. Singer, M. Becker, F. Vom Dorp, T. Jager, A. Szendroi, P. Riesz, I. Romics, H. Rubben & S. Ergun (2011b) Urinary matrix metalloproteinase-7 level is associated with the presence of metastasis in bladder cancer. BJU Int, 107, 1069-73.

Vedder, M. M., M. Marquez, E. W. de Bekker-Grob, M. L. Calle, L. Dyrskjot, M. Kogevinas, U. Segersten, P. U. Malmstrom, F. Algaba, W. Beukers, T. F. Orntoft, E. Zwarthoff, F. X. Real, N. Malats & E. W. Steyerberg (2014) Risk prediction scores for recurrence and progression of non-muscle invasive bladder cancer: an international validation in primary tumors. PLoS One, 9, e96849.

Witjes, J. A., E. Comperat, N. C. Cowan, M. De Santis, G. Gakis, T. Lebret, M. J. Ribal, A. G. Van der Heijden & A. Sherif (2014) EAU guidelines on muscle-invasive and metastatic bladder cancer: summary of the 2013 guidelines. Eur Urol, 65, 778-92.

Xylinas, E., M. Kent, L. Kluth, A. Pycha, E. Comploj, R. S. Svatek, Y. Lotan, Q. D. Trinh, P. I. Karakiewicz, S. Holmang, D. S. Scherr, M. Zerbib, A. J. Vickers & S. F. Shariat (2013) Accuracy of the EORTC risk tables and of the CUETO scoring model to predict outcomes in non-muscle-invasive urothelial carcinoma of the bladder. Br J Cancer, 109, 1460-6.

Yafi, F. A., A. G. Aprikian, J. L. Chin, Y. Fradet, J. Izawa, E. Estey, A. Fairey, R. Rendon, I. Cagiannos, L. Lacombe, J. B. Lattouf, D. Bell, D. Drachenberg & W Kassouf (2011) Contemporary outcomes of 2287 patients with bladder cancer who were treated with radical cystectomy: a Canadian multicentre experience. BJU Int, 108, 539-45.

Sequences

Sequence 1 (SEQ ID NO:1): Human chromogranin A (CgA) without signal peptide (UniProt accession number P10645):

1 11 21 31 41

LPVNSPMNKG DTEVMKCIVE VISDTLSKPS PMPVSQECFE TLRGDERILS

51 61 71 81 91

ILRHQNLLKE LQDLALQGAK ERAHQQKKHS GFEDELSEVL ENQSSQAELK

101 111 121 131 141

EAVEEPSSKD VMEKREDSKE AEKSGEATDG ARPQALPEPM QESKAEGNNQ

151 161 171 181 191

APGEEEEEEE EATNTHPPAS LPSQKYPGPQ AEGDSEGLSQ GLVDREKGLS

201 211 221 231 241

AEPGWQAKRE EEEEEEEEAE AGEEAVPEEE GPTVVLNPHP SLGYKEIRKG

251 261 271 281 291

ESRSEALAVD GAGKPGAEEA QDPEGKGEQE HSQQKEEEEE MAVVPQGLFR

301 311 321 331 341

GGKSGELEQE EERLSKEWED SKRWSKMDQL AKELTAEKRL EGQEEEEDNR

351 361 371 381 391

DSSMKLSFRA RAYGFRGPGP QLRRGWRPSS REDSLEAGLP LQVRGYPEEK

401 411 421 431

KEEEGSANRR PEDQELESLS AIEAELEKVA HQLQALRRG

Sequence 2 (SEQ ID NO:2): Human matrix metalloproteinase 7 (MMP7) (UniProt accession number P09237)

1 11 21 31 41

MRTVLCAVC LLPGSLALPL PQEAGGMSEL QWEQAQDYLK RFYLYDSETK

51 61 71 81 91

NANSLEAKLK EMQKFFGLPI TGMLNSRVIE IMQKPRCGVP DVAEYSLFPN

101 111 121 131 141

SPKWTSKVVT YRIVSYTRDL PHITVDRLVS KALNMWGKEI PLHFRKVVWG

151 161 171 181 191

TADIMIGFAR GAHGDSYPFD GPGNTLAHAF APGTGLGGDA HFDEDERWTD

201 211 221 231 241

GSSLGINFLY AATHELGHSL GMGHSSDPNA VMYPTYGNGD PQNFKLSQDD

251 261

IKGIQKLYGK RSNSRKK

Examples

Example 1 : Detection of chromogranin A and MMP7 in patients with bladder cancer

Study design

[0060] Serum samples were collected from 188 patients undergoing bladder cancer (BCa) surgery: transurethral resection of the bladder (TURB) or radical cystectomy (RCE). Patients were enrolled at the Department of Urology of the University Hospital Duisburg-Essen between August 2008 and November 2011. Enrollment criteria for the study were a histopathological diagnosis of transitional cell carcinoma of the bladder, no history of another tumor, no chemotherapy prior to surgery, availability of a serum sample, and tracking potential. Neuroendocrine bladder cancer is not diagnosed in the population. Histological grade and T-stage were classified according to the 1973 and 2004 WHO classification and the 2009 TNM classification, respectively. Serum samples from 97 healthy individuals with no history of malignancy were used as controls.

[0061] The study was conducted in accordance with the ethical standards of the Declaration of Helsinki and approved by the hospital ethics board. Samples were centrifuged at 1500 rpm for 15 minutes, immediately aliquoted and stored at -80° C. until analysis.

The clinical characteristics of the patients are presented in Table 1.

[0062] The endpoints of this study were overall and cancer-specific survival. The cause of death was obtained from death certificates. The median follow-up for all patients was 24 months, and the median follow-up for survivors was 31 months. Fifty-six of 188 patients died during the follow-up period, 39 of them due to BCa.

Chromogranin A detection

[0063] Chromogranin A levels were measured in a fully automated B.R.A.H.M.S KRYPTOR® device (Thermo Scientific B.R.A.H.M.S GmbH, Hennigsdorf/Berlin, Germany) using an advanced homogeneous fluorescence sandwich immunoassay marketed under CgA II #839.050 (patent application WO 2015/ 158701 A1).

[0064] This system uses time-resolved cryptate enhanced emission (TRACE®) sensitive technology based on non-radiative energy transfer between a donor (europium cryptate) and an acceptor (XL665). Two mouse monoclonal antibodies are used in the assay. The LK2H10 antibody epitope is located between amino acids 280-301, and the PHE5 monoclonal antibody epitope is between amino acids 124-144. The coefficient of variation (CV) between runs is ≤ 12% and the intra-run CV for the Kryptor assay is ≤ 7%. The functional sensitivity of the assay is 13.7 ng/mL and the limit of quantitation is 13.71 ng/mL.

MMP7 detection

[0065] MMP-7 levels were measured in a fully automated B.R.A.H.M.S KRYPTOR® device (Thermo Scientific B.R.A.H.M.S GmbH, Hennigsdorf/Berlin, Germany) using a prototype advanced homogeneous fluorescence sandwich immunoassay (not yet marketed).

[0066] This system uses time-resolved cryptate enhanced emission (TRACE®) sensitive technology based on non-radiative energy transfer between a donor (europium cryptate) and an acceptor (XL665). The assay uses a goat polyclonal antibody and a mouse monoclonal antibody against human MMP7 labeled with europium cryptate -EuC- (Cis Bio International, Bagnols/Cèze, France) and Alexa Fluor 647 (Molecular Probes - Life technologies, Eugene, USA) as donor , respectively. Epitopes of polyclonal and monoclonal antibodies are not described. Recombinant MMP-7 (R&D Systems Europe) diluted in neonatal calf serum (Trina Bioreactives AG, Nänikon, Switzerland) was used as calibrator. The coefficient of variation (CV) between runs is ≤ 11% and the intra-run CV for the Kryptor assay is ≤ 9%. The functional sensitivity of the assay is 1.95 ng/mL

measurements

[0067] Single measurements were performed automatically by incubating 50 μl of each patient sample with 50 μl of each conjugated antibody solution at 37° C. for 29 minutes. To assess reproducibility and repeatability, 2 and 3 quality control (QC) samples were measured in duplicate for each run of CgA and MMP7 assays, respectively.

Statistical analysis

[0068] The lack of a normal distribution of serum concentration data (controlled by the Shapiro-Wilk test) indicates the use of a non-parametric two-tailed Wilcoxon rank sum test (Mann-Whitney test) for independent group comparisons.

For univariate and multivariate analysis, patients were subdivided into low and high concentration groups with respect to their CgA and MMP7 levels. Cut-off thresholds were determined to maximize predictive value, which corresponds to 147 ng/ml for CGA (80th percentile) and 7.75 ng/ml for ^MMP7 (66th ^percentile ). One-way analysis of disease-specific survival was performed using both the Kaplan-Meier log-rank test and one-way Cox analysis. Cox proportional hazards regression model was used for multivariate analysis. In all tests, a p value of at least 0.05 was considered significant. All statistical analyzes were performed using the SPSS software package, version 21.0 (Chicago, IL, USA).

Example 2 Preoperative Predictive Value of CgA in a Population Treated Using Surgery (Study from Example 1)

Comparison of serum CgA levels between tumor and control samples

[0069] CgA concentrations were significantly increased in serum samples from tumor patients compared to controls (median 3.9 ng/mL versus 29.4 ng/mL, respectively, P < 0.0001, Figure 1).

CgA concentration and clinicopathological parameters

[0070] In tumor patients, CgA concentrations were significantly higher in older patients and in men (p=0.026 and p=0.009, respectively, Table 2). CgA concentrations correlated with stages and grades of malignancy, but not with metastasis (Table 2).

Predictive value of CgA levels in a population treated with surgery

[0071] Univariate Analysis: The results of the univariate analysis are listed in Table 3 and Figure 2. Patient age did not affect overall or disease-specific survival, in contrast to gender, which contributed to disease-specific survival (HR=0.447, CI 0.247 -0.918, p=0.027). Stage, grade, and metastasis of tumors are significant predictors of overall and disease-specific survival (P≤0.001, Table 3). Serum CgA is also a strong predictor of overall and disease-specific survival. High serum CgA was significantly associated with poor overall and disease-specific survival in patients treated with surgery (HR=2.553, 95% CI, 1.406-4.566, p=0.002 and HR=2.295, 95% CI, 1.106- 4.764, p=0.026, respectively, Table 3; and p=0.021, Figure 2). Moreover, CgA was also a significant predictor of overall survival when adjusted as a continuous variable (HR=1.001, 95% CI, 1.000-1.002, p=0.024).

[0072] Multivariate analysis: The results of multivariate analysis are presented in Table 4. Multivariate analysis showed that high serum CgA concentration is independent of stage, grade and metastasis as a predictor of overall and disease-specific survival (HR=3.424, 95% CI, 1.856-6.319, p<0.001 and HR=3.629; 95% CI, 1.692-7.784; P=0.001, respectively, Table 4).

The 20-month post-treatment survival rate was 87% in patients with low preoperative CgA levels compared to 70% in patients with high preoperative CgA levels (Figure 2).

Patients with high preoperative CgA concentrations are at high risk of bladder cancer-related and all-cause mortality, and may benefit from more aggressive or experimental treatment, as described in section [0054].

Preoperative predictive value of CgA and MMP7 levels in the population treated with surgery

[0073] Comparison of serum MMP7 levels between tumor and control samples:

Serum MMP7 concentrations were measured in the same population. MMP7 concentrations were significantly increased in serum samples from tumor patients compared to controls (median 2.9 ng/mL versus 4.4 ng/mL, respectively, P < 0.001).

MMP7 concentration and clinicopathological parameters

[0074] In tumor patients, MMP7 concentrations were significantly higher in older patients (p < 0.001, Table 5), but not in males. MMP7 concentrations were associated with the presence of muscle-invasive tumor and metastasis (p=0.008, p=0.05, respectively, Table 5), but not with the grade of malignancy.

Predictive value of MMP7 levels in a population treated with surgery

[0075] One-way analysis: The results of one-way analysis are presented in Table 3 and Figure 2.

Serum MMP7 levels are a significant predictor of overall and disease-specific survival. High serum MMP7 was significantly associated with poor overall and disease-specific survival in patients treated with surgery (HR=3.764, 95% CI, 1.983-7.148, p<0.001 and HR=3.905, 95% CI, 2.291- 6.655, p<0.001, respectively, Table 3; and p<0.001, Figure 2).

[0076] Multivariate analysis: Multivariate analysis showed that high serum concentration of MMP7 is independent of stage, grade, and metastasis as a predictor of overall and disease-specific survival (HR=2.750, 95% CI 1.557-4.855, p<0.001 and HR =2.324; 95% CI, 1.187-4.548; P=0.014, respectively, Table 6).

The twenty-month survival rate was 89% in patients with low preoperative MMP7 concentrations compared to 59% in patients with high preoperative concentrations (Figure 2).

Predictive value of combined CgA and MMP7 levels in a population treated with surgery

[0077] Multivariate analysis showed that high concentration of MMP7 and high concentration of CgA in serum are stage, grade and metastasis independent predictor of overall and disease-specific survival (table 9). High serum CgA concentrations in combination with high MMP7 concentrations were associated with poor disease-specific survival in patients treated with surgery (p < 0.001, figure 2).

The 20-month post-treatment survival rate was over 90% in patients with low preoperative CgA and MMP7 levels, compared to 71% in patients with elevated serum levels of one marker and 50% with elevated levels of both CgA and MMP7 (Figure 2).

Patients with high preoperative CgA and MMP7 concentrations are at high risk of bladder cancer-related and overall mortality, and may benefit from more aggressive or experimental treatment, as described in section [0054].

Example 3 : Preoperative Predictive Value of CgA/MMP7 in an RCE Population (Study from Example 1)

[0078] Risk stratification for patients treated by radical cystectomy is of particular interest. Thus, the authors of the present invention also analyzed the predictive value of CgA levels, focusing solely on this group.

Predictive value of CgA levels in treated patients RCE

[0079] High serum CgA was a strong independent predictor of overall and disease-specific survival (HR=2.405, 95 CI 1.000-5.784, p=0.050 and HR=4.003, 95% CI 1.491-10.748, p=0.006, respectively, table 7). High CgA was significantly associated with poor disease-specific survival in patients treated with radical cystectomy (p=0.008, figure 3).

The 20-month survival rate after radical cystectomy was 76% in patients in whom a low preoperative CgA concentration was indicated, compared with 33% in patients in whom a high preoperative concentration was indicated.

Patients with high preoperative CgA concentrations are at high risk of bladder cancer-related and overall mortality, and may benefit from more aggressive or experimental treatment, as described in section [0054].

Predictive value of MMP7 levels in patients treated with RCE

[0080] High serum MMP7 was a strong independent predictor of overall and disease-specific survival (HR=2.456, 95% CI 1.294-4.662, p=0.006 and HR=2.195, 95% CI 1.057-4.560, p=0.035 respectively, table 8). High concentration of MMP7 was significantly associated with poor disease-specific survival in patients treated with radical cystectomy (p=0.028, figure 3)

[0081] The twenty-month survival rate after radical cystectomy was 78% in patients showing low preoperative MMP7 levels compared to 54% in patients with high preoperative MMP7 levels (Figure 3).

Predictive value of combined CgA and MMP7 levels in patients treated with RCE

[0082] Multivariate analysis showed that high concentration of MMP7 and high concentration of CgA in serum are stage, grade and metastasis independent predictor of overall and disease-specific survival (table 10). High serum CgA in combination with high MMP7 was significantly associated with poor disease-specific survival in patients treated with radical cystectomy ( p =0.001, Figure 3).

The 20-month survival rate after radical cystectomy was 79% in patients with low preoperative CgA and MMP7 levels, compared with 47% in patients with elevated serum levels of one marker and 20% with elevated levels of both CgA and MMP7.

Patients with high preoperative CgA and MMP7 concentrations are at high risk of bladder cancer-related and overall mortality, and may benefit from more aggressive or experimental treatment, as described in section [0056].

tables

Table 1: Clinical characteristics of patients

Variables cases Control population Size 188 97 Age (median and range) 71 (21-90) 63 (52-79) Gender (male/female) 149/39 56/41 Classification Non-invasive (cis-Ta-T1) 108 NA Invasive (T2-T4) 80 NA Stage Cis eight NA Ta 81 NA T1 nineteen NA T2 28 NA T3 27 NA T4 25 NA Grade of malignancy G1 37 NA G2 93 NA G3 58 NA Low grade (G1-2) 130 NA High grade (G3) 58 NA Metastases No 156 NA Yes 32 NA Surgical intervention TURB 101 NA RCE 87 NA

NA: not applicable

Table 2: Association of CgA concentration and clinicopathological parameters of the tumor population

Variables n Median (range) CgA concentration P Age ≤65 51 48.9 (0-1682.4) 0.026 >65 137 66.5 (19.4-1786.5) Floor Male 149 66.2 (0-1786.5) 0.009 female 39 45.1 (21.4-996.7) Stage Non-invasive (cis-Ta-T1) 108 65.8 (14.6-1682.4) 0.045 Invasive (T2-T4) 80 52.4 (19.4-1786.5) Grade of malignancy Low grade (G1-2) 130 69.6 (21.2-1786.5) <0.001 High grade (G3) 58 46.4(14.6-996.7) Metastases No 156 65.1(14.6-1786.5) NS Yes 32 47.9 (21.4-479.4)

Table 3: Association of CgA levels, MMP7 levels, and clinicopathological parameters with patient prognosis (univariate analysis)

Variables Overall survival disease-specific survival HR 95%CI P HR 95%CI P Age ≤65 reference reference >65 1.727 0.892-3.343 NS 1.951 0.860-4.427 NS Floor female reference reference Male 0.660 0.369-1.179 NS 0.447 0.247-0.918 0.027 Stage Non-invasive (Ta) reference reference Invasive (T1-T4) 3.705 2.093-6.558 <0.001 5.449 2.583- <0.001 Grade of malignancy Low grade reference reference High degree of malignancy 2.489 1.470-4.212 0.001 2.014 1.547-5.490 0.001 Metastases No reference reference Yes 5.523 3.213-9.492 <0.001 7.125 3.757- <0.001 Serum CgA level low concentration reference reference High concentration 2.553 1.406-4.566 0.002 2.295 1.106-4.764 0.026 Serum MMP7 low concentration reference reference High concentration 3.764 1.983-7.148 <0.001 3.905 2.291 -6.655 <0.001

Table 4: Association of CgA levels and clinicopathological parameters with patient prognosis (multivariate analysis)

Overall survival disease-specific survival Variables HR 95%CI p HR 95%CI p Whole population Stage (T1-T4) 1.822 0.888-3.742 0.102 3.134 1.223-8.033 0.017 Grade of malignancy (G3) 1.184 0.595-2.356 0.630 1.074 0.479-2.410 0.862 Metastasis (yes) 4.129 2.026-8.414 <0.001 4.736 2.084-10.760 <0.001 Serum CGA (high concentration) 3.424 1.856-6.319 <0.001 3.629 1.692-7.784 0.001

Table 5: Association of MMP7 concentration with clinicopathological parameters of patients

Variables n Median (range) concentration of MMP7 P Age ≤65 51 3.5 (1.6-83.4) <0.001 >65 137 4.9 (1.4-72.5) Floor Male 149 4.4 (1.6-72.5) NS female 39 4.7 (1.4-83.4) Stage Non-invasive (cis-Ta-T1) 108 4.1 (1.4-20.8) 0.008 Invasive (T2-T4) 80 5.3 (1.6-83.4) Grade of malignancy Low grade (G1-2) 130 4.4 (1.4-28.5) NS High grade (G3) 58 4.4 (1.6-83.4) Metastases No 156 4.4 (1.4-83.4) 0.05 Yes 32 5.7 (1.6-72.5)

Table 6: Association of MMP7 levels and clinicopathological parameters with patient prognosis (multivariate analysis)

Overall survival disease-specific survival Variables HR 95%CI P HR 95%CI P Whole population Stage (T1-T4) 2.274 1.120-4.620 0.023 3.647 1.504-8.846 0.004 Grade of malignancy (G3) 1.405 0.758-2.603 0.280 1.349 0.657-2.768 0.415 Serum MMP7 (high concentration) 2,750 1.557-4.855 <0.001 2.324 1.187-4.548 0.014

Table 7: Association of CgA levels and clinicopathological parameters with patient prognosis in the RCE-treated population (multivariate analysis)

Overall survival disease-specific survival Variables HR 95%CI P HR 95%CI P Patients treated with RCE Stage (T1-T4) 3.508 1.466-8.397 0.005 4.581 1.623-12.932 0.004 Grade of malignancy (G3) 1.244 0.581-2.666 0.574 1.107 0.464-2.6643 0.819 Metastasis (yes) 2.521 1.218-5.218 0.013 2.991 1.282-6.981 0.011 Serum CGA (high concentration) 2.405 1,000-5,784 0.050 4.003 1.491-10.748 0.006

Table 8: Association of MMP7 levels and clinicopathological parameters with patient prognosis in the RCE-treated population (multivariate analysis)

Overall survival disease-specific survival Variables HR 95%CI P HR 95%CI P Patients treated with RCE Stage (T1-T4) 3.458 1.561-7.661 0.002 3.901 1.567-9.712 0.003 Grade of malignancy (G3) 1.756 0.863-3.573 0.280 1.601 0.722-3.553 0.247 Serum MMP7 (high concentration) 2.456 1.294-4.662 0.006 2.195 1.057-4.560 0.035

Table 9: Association of MMP7 and CgA levels, and clinicopathological parameters with patient prognosis (multivariate analysis)

Overall survival disease-specific survival Variables HR 95%CI P HR 95%CI P Whole population Stage (T1-T4) 1.488 0.698-3.083 0.313 2.668 1.020-6.980 0.046 Grade of malignancy (G3) 1.391 0.704-2.749 0.343 1.235 0.552-2.760 0.608 Metastasis (yes) 3.543 1.765-7.114 <0.001 4.170 1.859-9.354 0.001 Serum CGA (high concentration) 3.164 1.681-5.955 <0.001 3.485 1.598-7.603 0.012 Serum MMP7 (high concentration) 2.324 1.323-4.082 0.003 1.959 1.005-3.817 0.048 Stage (T1-T4) 1.572 0.774-3.192 0.210 2.676 1.056-6.783 0.038 Grade of malignancy (G3) 1.378 0.690-2.749 0.363 1.324 0.576-3.046 0.509 Metastasis (yes) 4.036 2.003-8.131 <0.001 4,564 2.037-10.228 <0.001 Both CGA/MMP7 are high 6.062 2.782-13.206 <0.001 7.382 2.789-19.539 <0.001

Table 10: Association of MMP7 and CgA levels and clinicopathological parameters with patient prognosis in the RCE-treated population (multivariate analysis)

Overall survival disease-specific survival Variables HR 95%CI P HR 95%CI P Patients treated with RCE Stage (T1-T4) 3.629 1.496-8.805 0.004 4.777 1.665-13.703 0.004 Grade of malignancy (G3) 1.583 0.720-3.478 0.253 1.407 0.582-3.520 0.466 Metastasis (yes) 2.088 0.984-4.389 0.052 2.509 1.050-5.997 0.038 Serum CGA (high concentration) 2.713 1.110-6.634 0.029 4.556 1.652-12.583 0.003 Serum MMP7 (high concentration) 2.149 1.090-4.237 0.027 1.892 0.848-4.218 0.119 Stage (T1-T4) 3.557 1,490-8,493 0.004 4.697 1.661-13.286 0.004 Grade of malignancy (G3) 1.462 0.670-3.191 0.340 1.422 0.571-3.544 0.449 Metastasis (yes) 2.503 1.222-5.128 0.012 3.031 1.328-6.916 0.008 Both CGA/MMP7 are high 4,878 1.539-15.463 0.007 9.059 2.170-37.820 0.003

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SEQUENCE LIST

<110> Cézanne S.A.S.

<120> Chromogranin A as a marker of bladder cancer

<130> Z1039 PCT BLN

<150>EP 16 15 9474.2

<151> 2016-03-09

<160> 2

<170> BiSSAP 1.3

<210> 1

<211> 439

<212> PRT

<213> Homo sapiens

<220>

<223> Human chromogranin A (CgA) without signal peptide

<400> 1

Leu Pro Val Asn Ser Pro Met Asn Lys Gly Asp Thr Glu Val Met Lys

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Cys Ile Val Glu Val Ile Ser Asp Thr Leu Ser Lys Pro Ser Pro Met

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Pro Val Ser Gln Glu Cys Phe Glu Thr Leu Arg Gly Asp Glu Arg Ile

35 40 45

Leu Ser Ile Leu Arg His Gln Asn Leu Leu Lys Glu Leu Gln Asp Leu

50 55 60

Ala Leu Gln Gly Ala Lys Glu Arg Ala His Gln Gln Lys Lys His Ser

65 70 75 80

Gly Phe Glu Asp Glu Leu Ser Glu Val Leu Glu Asn Gln Ser Ser Gln

85 90 95

Ala Glu Leu Lys Glu Ala Val Glu Glu Pro Ser Ser Lys Asp Val Met

100 105 110

Glu Lys Arg Glu Asp Ser Lys Glu Ala Glu Lys Ser Gly Glu Ala Thr

115 120 125

Asp Gly Ala Arg Pro Gln Ala Leu Pro Glu Pro Met Gln Glu Ser Lys

130 135 140

Ala Glu Gly Asn Asn Gln Ala Pro Gly Glu Glu Glu Glu Glu Glu Glu

145 150 155 160

Glu Ala Thr Asn Thr His Pro Pro Ala Ser Leu Pro Ser Gln Lys Tyr

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Pro Gly Pro Gln Ala Glu Gly Asp Ser Glu Gly Leu Ser Gln Gly Leu

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Val Asp Arg Glu Lys Gly Leu Ser Ala Glu Pro Gly Trp Gln Ala Lys

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Arg Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Ala Glu Ala Gly Glu Glu

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Ala Val Pro Glu Glu Glu Gly Pro Thr Val Val Leu Asn Pro His Pro

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Ser Leu Gly Tyr Lys Glu Ile Arg Lys Gly Glu Ser Arg Ser Glu Ala

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Leu Ala Val Asp Gly Ala Gly Lys Pro Gly Ala Glu Glu Ala Gln Asp

260 265 270

Pro Glu Gly Lys Gly Glu Gln Glu His Ser Gln Gln Lys Glu Glu Glu

275 280 285

Glu Glu Met Ala Val Val Pro Gln Gly Leu Phe Arg Gly Gly Lys Ser

290 295 300

Gly Glu Leu Glu Gln Glu Glu Glu Arg Leu Ser Lys Glu Trp Glu Asp

305 310 315 320

Ser Lys Arg Trp Ser Lys Met Asp Gln Leu Ala Lys Glu Leu Thr Ala

325 330 335

Glu Lys Arg Leu Glu Gly Gln Glu Glu Glu Glu Asp Asn Arg Asp Ser

340 345 350

Ser Met Lys Leu Ser Phe Arg Ala Arg Ala Tyr Gly Phe Arg Gly Pro

355 360 365

Gly Pro Gln Leu Arg Arg Gly Trp Arg Pro Ser Ser Arg Glu Asp Ser

370 375 380

Leu Glu Ala Gly Leu Pro Leu Gln Val Arg Gly Tyr Pro Glu Glu Lys

385 390 395 400

Lys Glu Glu Glu Gly Ser Ala Asn Arg Arg Pro Glu Asp Gln Glu Leu

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Glu Ser Leu Ser Ala Ile Glu Ala Glu Leu Glu Lys Val Ala His Gln

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Leu Gln Ala Leu Arg Arg Gly

435

<210> 2

<211> 267

<212> PRT

<213> Homo sapiens

<220>

<223> Human matrix metalloproteinase 7 (MMP7)

<400> 2

Met Arg Leu Thr Val Leu Cys Ala Val Cys Leu Leu Pro Gly Ser Leu

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Ala Leu Pro Leu Pro Gln Glu Ala Gly Gly Met Ser Glu Leu Gln Trp

20 25 30

Glu Gln Ala Gln Asp Tyr Leu Lys Arg Phe Tyr Leu Tyr Asp Ser Glu

35 40 45

Thr Lys Asn Ala Asn Ser Leu Glu Ala Lys Leu Lys Glu Met Gln Lys

50 55 60

Phe Phe Gly Leu Pro Ile Thr Gly Met Leu Asn Ser Arg Val Ile Glu

65 70 75 80

Ile Met Gln Lys Pro Arg Cys Gly Val Pro Asp Val Ala Glu Tyr Ser

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Leu Phe Pro Asn Ser Pro Lys Trp Thr Ser Lys Val Val Thr Tyr Arg

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Ile Val Ser Tyr Thr Arg Asp Leu Pro His Ile Thr Val Asp Arg Leu

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Val Ser Lys Ala Leu Asn Met Trp Gly Lys Glu Ile Pro Leu His Phe

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Arg Lys Val Val Trp Gly Thr Ala Asp Ile Met Ile Gly Phe Ala Arg

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Gly Ala His Gly Asp Ser Tyr Pro Phe Asp Gly Pro Gly Asn Thr Leu

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Ala His Ala Phe Ala Pro Gly Thr Gly Leu Gly Gly Asp Ala His Phe

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Asp Glu Asp Glu Arg Trp Thr Asp Gly Ser Ser Leu Gly Ile Asn Phe

195 200 205

Leu Tyr Ala Ala Thr His Glu Leu Gly His Ser Leu Gly Met Gly His

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Ser Ser Asp Pro Asn Ala Val Met Tyr Pro Thr Tyr Gly Asn Gly Asp

225 230 235 240

Pro Gln Asn Phe Lys Leu Ser Gln Asp Asp Ile Lys Gly Ile Gln Lys

245 250 255

Leu Tyr Gly Lys Arg Ser Asn Ser Arg Lys Lys

260 265

<---

Claims (11)

1. A method for predicting bladder cancer in an individual, wherein the bladder cancer is non-neuroendocrine bladder cancer, wherein the method comprises the step of determining the level of CgA in the body fluid sample of said individual, and thus predicting: (i) if the CgA level in the patient's body fluid sample is above a predetermined threshold for CgA, then this is indicative of a high risk of cancer-related death after surgical treatment and/or drug treatment and/or radiotherapy, and (ii) in a patient undergoing transurethral resection of the bladder, the twenty-month survival rate after treatment is 87% in patients with low preoperative CgA and 70% in patients with high preoperative CgA, or (iii) in a radical cystectomy patient, the 20-month post-treatment survival rate is 76% in patients with low preoperative CgA and 33% in patients with high preoperative CgA; wherein the level of CgA in a sample from a patient is compared to a control level or a predetermined threshold, where the predetermined threshold for CgA is from 100 ng/mg to 147 ng/ml, and serum samples from healthy individuals without a history of malignancy are used as controls. 2. The method of claim 1, wherein the CgA level in the individual's sample is indicative of the individual's overall survival, or the individual's disease-specific survival, or the individual's progression-free survival. 3. The method of claim 1 or 2, wherein the poor prognosis is an increased risk of reduced life expectancy, an increased risk of progression, an increased risk of death from cancer, and/or an increased risk of recurrence after surgery and/or drug treatment, and/ or radiotherapy. 4. The method of claim 3, wherein the predetermined CgA level threshold is selected from 103 ng/mL to 191 ng/mL, more preferably 130 ng/mL to 160 ng/mL, and most preferably the threshold is 147 ng/mL. 5. The method of any one of claims 1 to 4, further comprising determining the level of matrix metalloproteinase 7 (MMP7) in said sample of the individual, and wherein the level of MMP7 in the individual's sample is indicative of bladder cancer severity and/or outcome in the individual. 6. The method according to claim 5, where the level of CgA and optionally MMP7 is determined at regular intervals during follow-up and where the control level is the corresponding level determined before said surgical treatment and/or drug treatment and/or radiotherapy, and/or at the initial stage of follow-up.

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