Chromogranin A as marker of bladder cancer

Abstract

The invention relates to chromogranin A as a marker of bladder cancer. The present invention relates to the use of chromogranin A (CgA) as a marker, in particular a prognostic marker, of bladder cancer, in particular non-neuroendocrine bladder cancer, and preferably urothelium cancer. In particular, CgA can be used as a marker in an in vitro assay for the prognosis, risk assessment, risk stratification, monitoring and / or treatment control of bladder cancer, particularly non-neuroendocrine bladder cancer, preferably urothelium cancer. The invention also relates to a method for the prognosis, risk assessment, risk stratification, monitoring and / or treatment control of bladder cancer, in particular non-neuroendocrine bladder cancer, preferably urothelium cancer, in a subject, comprising the step of determining the level of CgA and optionally MMP7 in a sample of a bodily fluid in said subject.

Description

[0001] This application is a divisional application of Chinese patent application No. 201780015679.3 entitled "Chromogranin A as a marker of bladder cancer". The original application was PCT international application PCT / EP2017 / 055279 filed on March 7, 2017, which entered the Chinese national phase on September 6, 2018. Technical Field

[0002] This invention belongs to the field of clinical diagnostics. In particular, this invention relates to the prognosis, risk assessment, risk stratification, monitoring, and / or treatment control of bladder cancer based on chromogranin A (CgA) as a biomarker. Background Technology

[0003] Bladder cancer (urinary bladder cancer) is the most common malignant tumor of the urinary tract. Considering newly diagnosed cases and its high recurrence rate, bladder cancer is one of the most common cancers in the world (Chamie et al. 2011, Goodison, Rosser, and Urquidi 2013). It is more prevalent in Western countries, and men are at 3 to 4 times higher risk of developing the disease compared to women (Burger et al. 2013a).

[0004] Different types of bladder cancer can be distinguished based on the cells that become cancerous. The most common type of bladder cancer is transitional cell carcinoma (90%), followed by squamous cell carcinoma and adenocarcinoma (2 to 5%). Less than 1% of bladder cancers originate from neuroendocrine cells (Bertaccini et al. 2008, Pompas-Veganzones, Gonzalez-Peramato, and Sanchez-Carbayo 2014). At diagnosis, approximately 70 to 75% of patients have superficial bladder carcinoma (stages Ta-T1), also known as non-muscle invasive bladder cancer (NMIBC). 25 to 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).

[0005] The recurrence and / or progression rate of bladder cancer is very high; 50 to 70% of patients with non-invasive bladder cancer (NMIBC) experience disease recurrence, progression, or new development within 5 to 7 years after treatment (Clark et al., 2013), of which 10% to 30% are invasive (Chamie et al., 2011; Goodison et al., 2013; Witjes et al., 2014). Approximately half (43%) of MIBC cases have non-invasive tumors at diagnosis, which progress despite organ-preserving therapy (EAU Guidelines on Muscle-invasive and Metastatic Bladder Cancer, 2014).

[0006] Currently, the diagnosis and monitoring of bladder cancer are based on histopathological examination of bladder urothelial tissue obtained from a biopsy during cystoscopy / transurethral resection of bladder tumor (TURBT). White light cystoscopy is a useful diagnostic tool for clinicians, but it is highly limited. In fact, it often understages tumors, which can lead to inadequate treatment (Cauberg, Evelyne, dela Rosette, and de Reijke 2011). It is also moderately sensitive, expensive, and invasive. A number of extremely rare complications can occur during the procedure, such as obturator reflex perforation, bleeding, TUR syndrome, urinary tract obstruction, perforation (extraperitoneal or intraperitoneal), and infection.

[0007] Voided Urine Cytology (VUC) is also a widely used method for the diagnosis and monitoring of non-MIBC, exhibiting high specificity but lower sensitivity in well-differentiated tumors (low-grade). Accuracy for early-stage tumors is only 20% to 40% (AUA Guidelines 2007–2014, Goodison et al., 2013). It shows high accuracy for high-grade tumors, particularly carcinoma in situ. This capability makes it a complementary tool to transurethral resection (see below). Cystoscopy and VUC allow for initial diagnosis and assessment of tumor characteristics, including number, type, grade, and estimated stage.

[0008] Computed tomography (CT) and magnetic resonance imaging (MRI) can also be used for the assessment of primary tumors. However, in up to 40% of cases, they underestimate the disease and can only slightly differentiate tumor stages Ta to T3a (accuracy 55 to 92%), although good accuracy in differentiating between invasive and non-invasive tumors has been reported (Maurer et al. 2013).

[0009] Assessing metastatic status, particularly lymph node status, is crucial for prognostic evaluation of bladder cancer. Lymph node metastasis increases from a low proportion of 5-10% in non-muscle-invasive bladder tumors to 15-20% in superficial muscle-invasive tumors, 25-30% in deep muscle-invasive tumors, and >40% in extravesical tumors (Shariat et al., 2012). Diagnosis of distant or regional metastatic bladder cancer relies primarily on various imaging techniques, including radiography, ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI). Ultrasound is the simplest, most non-invasive, and cost-effective, but it is dependent on operator skill. CT and MRI are also non-invasive, although their sensitivity has recently improved, their performance remains insufficient.

[0010] If the initial diagnosis is positive for bladder cancer without considering metastasis, transurethral resection of bladder tumor (TURBT) is performed. This diagnostic approach is the initial treatment step, characterized by endoscopic ablation of the entire tumor. It confirms the diagnosis and allows for more accurate assessment of the tumor stage, particularly the depth of invasion into the bladder wall. However, TURBT is associated with a significant risk of underestimation, especially for T1 tumors (Babjuk 2009).

[0011] Furthermore, due to incomplete resection, recurrent tumor growth can be observed during the first year (Kamat et al. 2013, Babjuk 2009). Therefore, guidelines strongly recommend repeat TURBT or maximum TURBT (Brausi et al. 2011).

[0012] Although patients with non-MIBC can often be safely managed with a “perfect” TURBT with or without other treatments (e.g., immunotherapy, chemotherapy), radical cystectomy (RCE) with bilateral pelvic lymph node dissection (PLND) has become the standard of care for MIBC patients (Clark et al. 2013, Witjes et al. 2014). Some neoadjuvant chemotherapy treatments have shown survival benefits (Sharma, Ksheersagar, and Sharma 2009) and are recommended for selected patients. RCE is also recommended for advanced and / or refractory NMIBC and carcinoma in situ (CIS). Many patients undergoing radical surgery undergo urinary diversion or neobladder reconstruction. The National Cancer Comprehensive Network (NCCN) guidelines recommend that lymph node-negative patients with the highest pathologically estimated risk may receive adjuvant additional radiation therapy and radiosensitization or chemotherapy. However, these additional treatments can increase toxicity and comorbidity (Clark et al., 2013).

[0013] In whole bladder cancer (BCa) (all stages and grades), the 5-year overall survival, recurrence-free survival and cancer-specific survival after RCE alone were only 57%, 48% and 67%, respectively, with distant and local recurrence rates of 37% and 6%, respectively (Yafi et al., 2011).

[0014] The benefit of standard pelvic lymph node dissection (PLND) in MIBC cases for patient survival is largely established, and PLND is now a major part of the recommended renal replacement therapy (RC) in guidelines. However, its benefit as a single surgical treatment strategy is moderate, and lymph node dissection (LND) is generally considered more diagnostic / prognostic (providing lymph node status) than therapeutic (Skinner and Sagalowsky 2014). In fact, regional LND is a necessary step in staging due to the limitations of current imaging techniques' sensitivity. Despite advances in surgical techniques, imaging, perioperative management, and treatment, approximately 50% of patients develop metastases and die from bladder cancer (Stein JP, Skinner DG 2006).

[0015] Then, neoadjuvant / adjuvant chemotherapy or radiotherapy and PLND or extended LND (Skinner and Sagalowsky 2014) are considered to improve survival in high-risk patients. However, due to increased toxicity, comorbidity risks, and costs, the benefits of these intensive interventions must be perfectly balanced against changes in quality of life and potential complications (including bleeding, nerve damage, lymphoceles, or extremity thrombus) (Scarpato et al., 2015) and are committed to the highest-risk cases.

[0016] One of the challenges of treating this advanced malignant tumor is answering the question: which patient should receive which treatment?

[0017] Following RCE with or without LND, spiral CT represents the preferred imaging modality for identifying lung, lymph node, and liver metastases (ICUD-EAU Bladder Cancer Edition 2012), and the EAU guidelines recommend 3 to 4 monthly abdominal, upper urinary tract (UUT), pelvic, and chest X-rays or CT scans during the first year (Sharma et al., 2009), 6 monthly until the third year, and annual monitoring thereafter as a clinical indication. For RCE with urinary diversion (UD), urethral flushing cytology every 6 to 12 months is also recommended (Sharma et al., 2009). The frequency and methods of follow-up after radical cystectomy are not strictly consistent across guidelines, but vary only slightly.

[0018] Distant recurrence typically occurs within 24 months, with higher stage and lymph node status being the most significant risk factors. Pelvic recurrence commonly occurs within 6 to 18 months post-surgery (5 to 15% of patients), depending on the initial stage and lymph node status. Secondary urethral tumors are rare, occurring 1 to 3 years after cystectomy, and have low survival rates. Upper urinary tract recurrence is rare and usually occurs late (28 to 49 months after cystectomy) (Witjes et al., 2014). Finally, some late recurrences may occur more than 10 years later, requiring very long-term monitoring after RCE.

[0019] Due to the prognostic heterogeneity between NMIBC and MIBC, bladder cancer guidelines emphasize the importance of better prognosis and unmet needs (EAU guidelines). For NMIBC, risk tables can be used 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 by the EAU and AUA guidelines is rarely used in daily life.

[0020] Recent publications have demonstrated that these models are not robust. For example, Vedder et al. disclosed that the scoring system can reasonably predict progression but not relapse (Vedder et al., 2014).

[0021] The overestimation of the risk of relapse and progression in the EORTC table has also been confirmed in NMIBC and in the subgroup of BCG treatment (Fernandez-Gomez et al., 2011).

[0022] Finally, Xylinas et al. demonstrated that the EORTC scoring system has low discrimination between relapse and progression, and emphasized the need to improve existing predictive tools (Xylinas et al., 2013).

[0023] To date, there are no specific tables or scoring systems to assess the prognosis of MIBC to guide treatment selection. The presence of regional lymph node metastases is the strongest predictor of tumor recurrence and disease-specific survival (DSS) in MIBC patients (Skinner and Sagalowsky, 2014). However, approximately half of superficial tumors may have micrometastases that cannot be detected by imaging tools, and only 25% of patients undergoing radical surgery have lymph node (LN) metastases at the time of surgery (Svatek et al., 2010, EAU Guidelines for Muscle Invasive and Metastatic Bladder Cancer 2014).

[0024] Although the disease-specific survival (DSS) rate after radical cystectomy for CIS is 85% to 90%, early radical cystectomy can be considered overtreatment in approximately 50% of patients (Burger et al., 2013b).

[0025] Bladder cancer is a heterogeneous tumor requiring large-scale treatment algorithms (sequential chemotherapy or biotherapy, radiation therapy, and surgical intervention) and extensive long-term monitoring. Tumors with similar histology can exhibit different clinical behaviors, and these nuances are crucial for proper management. This complexity results in both direct and indirect economic burdens, making it the most expensive malignancy in terms of lifetime healthcare costs per patient (Brausi 2013, Chamie et al. 2011, Goodison et al. 2013, Mossanen and Gore 2014). In 2010, healthcare costs for BCa were approximately $4 billion in the United States (Mossanen and Gore 2014), and in 2012 in the European Union (EU), they were €3 billion, including direct hospitalization as the major cost component (58%). The global economic burden in the EU was €5 billion (Leal et al. 2015). In the United States, the estimated cost of lifetime personal management of 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, but the economic burden of NMIBC is generally considered higher due to the long clinical course of early-stage disease, its prevalence relative to MIBC, and its procedurally oriented surveillance (Svatek et al. 2014).

[0026] Tumor underestimation is a major limitation of current diagnostic and predictive tools. Furthermore, to date, there are no reliable tools to predict the progression of NMIBC to MIBC, nor to predict the outcome of MIBC.

[0027] Biomarkers used for prognosis or risk stratification in bladder cancer patients can help doctors initiate appropriate treatment and reduce costs associated with patient monitoring.

[0028] Several commercially available urinary biomarkers have been FDA approved for the diagnosis and / or follow-up of bladder cancer, but have not penetrated into clinical practice. In contrast, there are currently no blood or urine prognostic biomarkers approved by national health agencies or recommended by any guidelines. This is inconsistent with BCA's global risk management strategy.

[0029] A recent large meta-analysis (Schmitz-Drager et al., 2015) compared the performance of the most commonly used commercially available biomarkers (BTA Stat®, NMP22 including BladderCheck®, and FISH Urovysion™) with VUC. These molecular biomarkers demonstrated better sensitivity than urine cytology, particularly for high-risk tumors (pT1G3, CIS), although their prognostic application was not confirmed. However, the lower specificity and reproducibility of these biomarkers were also confirmed as major limitations. Furthermore, these biomarkers lacked prognostic value.

[0030] Chromogranin A (CgA) is a glycoprotein normally expressed in neuroendocrine (NE) cells. CgA is a component of secretory granules in most peptide-producing endocrine cells (Chuang and Liao, 2003). It is physiologically released via exocytosis and can be detected in the blood. When tumors develop in neuroendocrine tissues, it becomes a major source of circulating CgA. CgA secretion in the bladder has been associated with rare cases of neuroendocrine-differentiated tumors known to have poor prognosis (Alijo Serrano et al., 2007; Bertaccini et al., 2008). Neuroendocrine tissue markers such as CgA are commonly used to differentiate neuroendocrine carcinoma (NEC) from transitional cell carcinoma (TCC), and to confirm the diagnosis of NEC. They are also commonly used 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).

[0031] However, while CgA has been shown to be expressed in the tissues of neuroendocrine bladder cancer (NEBC), no studies have to date assessed the prognostic impact of tissue and serum CgA levels on neuroendocrine and urothelial BCA.

[0032] Furthermore, the presence of both small cells and transitional cells does not predict the outcome (Chuang and Liao, 2003).

[0033] Chuang et al. demonstrated that CgA was expressed in 4 out of 10 SCBC tissue samples, but found no correlation with patient prognosis. However, the reliability of this study was limited due to the low number of cases and heterogeneous cohorts (Chuang and Liao 2003).

[0034] CgA has not shown any relationship with survival in large cell and small cell neuroendocrine bladder cancer. In one study, the only prognostic factor for survival remained TNM classification (Alijo-Serrano, 2007). The authors did not consider CgA useful for prognosis. Furthermore, Soukup et al. recently found that urinary CgA was not associated with the presence of primary NMIBC or with cancer recurrence (Soukup et al., 2015). However, Bertaccini et al. found that serum CgA levels were 10-fold lower than preoperative levels one month after cystoprostatectomy for small cell urothelial carcinoma (SCUC), but preoperative prognosis and follow-up values ​​were not assessed (Bertaccini et al., 2008).

[0035] Matrix metalloproteinase-7 (MMP7) has been described as an independent biomarker for lymph node metastasis in serum and urine (Gunes et al. 2013, Jager et al. 2013, Szarvas et al. 2010, Szarvas et al. 2011b), and also as an independent prognostic biomarker before BCa surgery (Svatek et al. 2010, Szarvas et al. 2010, Szarvas et al. 2011a). High MMP-7 levels have also been described as an independent risk factor for poor survival in certain metastatic bladder cancers (El Demery et al. 2014). See also WO 2007 / 144144 A1.

[0036] This invention is based on the surprising discovery that chromogranin A has high prognostic value in non-neuroendocrine bladder cancer. Therefore, it can help overcome the aforementioned shortcomings of existing diagnostic tools for bladder cancer. Summary of the Invention

[0037] This invention relates to the use of chromogranin A (CgA) as a biomarker (particularly a prognostic biomarker) for bladder cancer, particularly non-neuroendocrine bladder cancer, and preferably urothelial carcinoma. Specifically, CgA can be used as a biomarker in in vitro assays for the prognosis, risk assessment, risk stratification, monitoring, and / or treatment control of bladder cancer, particularly non-neuroendocrine bladder cancer, and preferably urothelial carcinoma.

[0038] The present invention also relates to methods for the prognosis, risk assessment, risk stratification, monitoring, and / or treatment control of bladder cancer, particularly non-neuroendocrine bladder cancer, and preferably urothelial carcinoma, in subjects, comprising the step of determining the levels of CgA and optionally MMP7 in a body fluid sample of the subject. The present invention further relates to the use of diagnostic kits comprising one or more CgA-specific antibodies for the prognosis, risk assessment, risk stratification, monitoring, and / or treatment control of bladder cancer, particularly non-neuroendocrine bladder cancer, and preferably urothelial carcinoma, in subjects. The present invention also relates to methods for treating bladder cancer, particularly non-neuroendocrine bladder cancer, and preferably urothelial carcinoma, in subjects, wherein the CgA level in a sample from the subject is determined. Attached Figure Description

[0039] Figure 1 Serum CgA concentrations in controls and cases

[0040] Figure 2 Prognostic value of CgA and MMP-7 levels and their combinations in patients undergoing surgical treatment (TURBT or RCE) (Kaplan-Meier curves with log-rank test). DSS: Disease-specific survival.

[0041] Figure 3 Prognostic value of CgA and MMP7 levels and their combinations in subgroups of patients treated with RCE (Kaplan-Meier curves with log-rank test). Detailed Implementation Plan

[0042] This invention relates to the use of chromogranin A (CgA) as a biomarker for bladder cancer (particularly non-neuroendocrine bladder cancer, and preferably urothelial carcinoma), particularly as a prognostic biomarker for bladder cancer, especially non-neuroendocrine bladder cancer (preferably urothelial carcinoma). Therefore, CgA can be used as a biomarker in in vitro assays for the prognosis, risk assessment, risk stratification, monitoring, and / or treatment control of bladder cancer, particularly non-neuroendocrine bladder cancer, preferably urothelial carcinoma. In other words, CgA has been shown to be a good biomarker for assessing disease severity in patients with bladder cancer, particularly in patients with non-neuroendocrine bladder cancer, preferably urothelial carcinoma. Therefore, CgA can also be used for the management of patients with bladder cancer (particularly non-neuroendocrine bladder cancer, preferably urothelial carcinoma) (“patient management”).

[0043] This invention relates to a method for the prognosis of subjects with bladder cancer (particularly subjects with non-neuroendocrine bladder cancer, preferably urothelial carcinoma), comprising the step of determining the level of CgA (and optionally MMP7) in a body fluid sample of the subject. The invention particularly relates to a method for the prognosis, risk assessment, risk stratification, monitoring, and / or treatment control of bladder cancer (particularly non-neuroendocrine bladder cancer, preferably urothelial carcinoma) in subjects, comprising the step of determining the level of CgA in a body fluid sample of the subject. Optionally, the level of MMP7 is also determined in the same sample or another sample of the subject. The level of CgA (and optionally MMP7, as appropriate) in the sample from the subject indicates, in one respect, the severity and aggressiveness and / or outcome of the subject's bladder cancer (particularly non-neuroendocrine bladder cancer, preferably urothelial carcinoma). Elevated levels of CgA (and optionally MMP7, as appropriate) in the sample from the subject, compared to control levels or a predetermined threshold, indicate an adverse outcome for the subject. For example, the level of CgA in the sample from the subject indicates overall survival of the subject, disease-specific survival of the subject, or progression-free survival of the subject.

[0044] In some embodiments, the CgA level in the sample is first determined, and if the CgA level is higher than a predetermined threshold, further testing is then performed in another sample of the same subject, such as subsequently determining the Mg7 level, to improve or confirm the initial prognosis based on the CgA level. In other, more preferred embodiments, the MMP7 level in the sample of the subject is first determined, and if the MMP7 level is higher than a predetermined threshold, further testing is then performed in another sample of the same subject, such as subsequently determining the CgA level, to improve or confirm the initial prognosis based on the MMP7 level. Therefore, MMP7 and CgA levels can be used together to improve an initial prognosis based solely on one of these two markers.

[0045] In the context of this invention, the term "bladder cancer" (also referred to as "bladder cancer") preferably refers to bladder cancer of non-neuroendocrine origin. Therefore, preferably, "bladder cancer" is not a neuroendocrine tumor (NET). The term "neuroendocrine" relates to neural or endocrine effects, particularly to the interaction between the neural and endocrine systems. Specifically, the term "neuroendocrine" relates to cells that release hormones into the bloodstream in response to neural stimulation. Thus, "neuroendocrine carcinoma" is a malignant tumor arising from endocrine (hormonal) and nervous system cells. Neuroendocrine bladder cancers include, for example, small cell carcinoma (SCC), carcinoid, and large cell neuroendocrine carcinoma (LCNEC).

[0046] Therefore, the term "bladder cancer" as used herein preferably refers to bladder cancer that is not generated by cells of the endocrine (hormonal) and nervous systems. Therefore, preferably, the bladder cancer referred to herein... noSmall cell carcinoma, not carcinoid tumor, and not large cell neuroendocrine carcinoma. In the context of this invention, bladder cancer is preferably selected from transitional cell carcinoma (TCC) (i.e., urothelial carcinoma of the bladder (UC), also known as urothelial cell carcinoma (UCC)), squamous cell carcinoma, and adenocarcinoma; more preferably, bladder cancer in the context of this invention is transitional cell carcinoma. The term "urothelial" specifically refers to urothelial carcinoma, i.e., TCC of the urinary system. Therefore, in the context of this invention, urothelial carcinoma refers to urothelial carcinoma of the bladder. Transitional cell carcinoma (= urothelial carcinoma) can be superficial bladder cancer (i.e., non-muscle-invasive bladder cancer (NMIBC)) or muscle-invasive bladder cancer (MIBC). NMIBC can be, for example, papillary carcinoma or squamous carcinoma (e.g., carcinoma in situ (CIS)). Generally, urothelial carcinoma of the bladder can be classified, for example, as microcapillary, nested, plasmacytic, sarcomatoid, or other variants, or can have mixed histology. Microcapillary or nested variants of urothelial carcinoma are particularly aggressive.

[0047] For example, these bladder cancers can be classified according to the UICC / AJCC (Union for international cancer control / American Joint Committee on Cancer) TNM table and graded according to the WHO 1973-2004 classification.

[0048] Prior to the determination of CgA levels, the subject in this invention context had and / or was diagnosed with non-neuroendocrine bladder cancer. Preferably, in this context, the subject did not (i.e. had) neuroendocrine bladder cancer. Therefore, preferably, the subject was not diagnosed with neuroendocrine bladder cancer.

[0049] The term "subject" as used herein refers to a living human or non-human animal, preferably a mammal, and most preferably a human. The subject is preferably a patient. As used herein, "patient" refers to a living human or non-human animal (most preferably a human) who is receiving or should receive medical care due to a disease, particularly non-neuroendocrine bladder cancer. This includes individuals with undetermined diseases undergoing pathological examination. Therefore, the methods and assays described herein are applicable to both human and veterinary diseases.

[0050] In the context of this invention, the term “level” refers to the concentration (preferably expressed as weight / volume; w / v; e.g., “ng / mL”) of a marker (e.g., CgA and / or MMP7) in a sample taken from a subject (e.g., a bladder cancer patient).

[0051] In the context of this invention, "diagnosis" refers to the identification and (early) detection of a disease or clinical condition in the subject, and may also include differential diagnosis. In some embodiments, an assessment of the severity of the disease or a clinical or histopathological condition may also be included in the term "diagnosis".

[0052] "Prognosis" refers to predicting the outcome or specific risk for a subject with a particular disease or clinical condition (in this case, non-neuroendocrine bladder cancer). This can include estimating the likelihood of recovery or the probability of an adverse outcome for the subject.

[0053] "Monitoring" or "treatment monitoring" involves tracking a diagnosed disease, condition, complication, or risk, such as analyzing the progression of a disease (here: non-neuroendocrine bladder cancer) or the impact of specific treatments on the progression of a disease or condition. In this invention, the term "risk stratification" refers to grouping subjects into different risk groups based on their further prognosis. Risk stratification also involves stratification based on the application of preventative and / or therapeutic measures.

[0054] In the context of this invention, the term "patient management" refers to:

[0055] • The decision to admit the patient to a hospital or intensive care unit.

[0056] • The decision was made to relocate the patient to a specialist hospital or specialist hospital unit.

[0057] • Assess early exit from the intensive care unit or hospital.

[0058] • Resource allocation (e.g., doctors and / or nurses, diagnosis, treatment, surgery).

[0059] The term “assessment of disease severity” involves evaluating a patient’s disease status, including the histopathology of the tumor, the metastatic status and progression of the disease, the likelihood of adverse events (including death), the likelihood of high hospitalization costs, and the likelihood of long-term hospitalization.

[0060] In the context of this invention, the term "outcome" refers to the degree of disease severity, such as the patient's health status after a specified period of time, such as 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. An outcome can be expressed as a ratio (percentage) in a population (e.g., a study or treatment group) or an individual ratio (i.e., the probability of a particular subject or patient). Thus, "overall outcome" can be expressed as "overall survival rate," which is the percentage of people who remain alive in a group (e.g., the study or treatment group) for a period of time after being diagnosed with a disease (e.g., non-neuroendocrine bladder cancer in this case) or starting treatment for a disease. Overall survival rate is typically expressed as a five-year survival rate, which is the percentage of people who survive in the study or treatment group five years after diagnosis or the start of treatment. Therefore, "overall outcome" or "overall survival" is a probability of survival for a particular subject or patient over a specific period of time, i.e., it is related to the life expectancy of said subject or patient.

[0061] "Disease-specific survival," also known as "disease-specific survival rate," is the percentage of people in a population (e.g., a study or treatment group) who do not die from a specific disease within a given time period. This time period typically begins at diagnosis or the start of treatment and ends at death. Patients who die from causes other than the disease under study (here: non-neuroendocrine bladder cancer) are not included in this measurement. Therefore, "disease-specific" survival or outcome means the likelihood that an individual subject or patient will not die from non-neuroendocrine bladder cancer within a given time period.

[0062] "Progression-free survival" is the length of time a subject or patient has had disease but has not progressed during and after treatment for disease (e.g., non-neuroendocrine bladder cancer). "Progression" is, for example, the appearance of any new tumor (at the primary or distant site) ("recurrence") or an improvement in size, histological stage, grade, or symptoms, especially after given curative or palliative treatment. Progression can be detected by radiography or biochemistry, for example. In the case of NMIBC, for example, progression can 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 can refer to postoperative recurrence, from stage LN0 to LN+, or from stage M0 to M+. Disease-specific death can also be considered progression. In metastatic MIBC, progression can be detected by, for example, radiography, or according to RECIST criteria.

[0063] In some respects, adverse outcomes include 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 following surgical and / or medical and / or radiation therapy.

[0064] The methods and uses of this invention can be applied to different conditions and stages of disease, for example, before or after intervention. The terms "before intervention," "before intervention," and "before intervention" as used herein refer to the time preceding an intervention for the treatment of non-neuroendocrine bladder cancer. "Intervention" means any medical intervention intended to alter health outcomes. This definition includes medication administration, surgery, device application, behavioral therapy, changes in the course of care, etc. Preferably, sampling is performed upon patient admission or before a diagnosis of non-neuroendocrine bladder cancer is confirmed.

[0065] For example, in one aspect, the subject has been diagnosed with non-neuroendocrine bladder cancer and (to date) has not undergone surgical treatment for said non-neuroendocrine bladder cancer. In this case, the method of the present invention can be used to predict outcomes or to select appropriate treatment (e.g., surgical selection).

[0066] The terms "post-intervention," "after intervention," and "after intervention" refer to the time elapsed since the start of the intervention or treatment.

[0067] For example, in one aspect, the subject has undergone surgical treatment for the non-neuroendocrine bladder cancer, such as radical cystectomy for the non-neuroendocrine bladder cancer. In this case, the method of the present invention can be used to predict outcomes and / or to select appropriate follow-up treatment and / or to monitor the subject (e.g., disease progression). Therefore, the level of CgA (and any other additional markers, including MMP7, as appropriate) can be used for postoperative control. In the context of the present invention, the term "postoperative control" refers to monitoring the subject after the subject's surgery.

[0068] In one instance, within the context of this invention, "prediction" refers to predicting complications, progression, or symptoms before other symptoms or biomarkers become apparent or have changed significantly. Within the context of this invention, terms such as "predictive value" refer to the statistical significance of a specific measured outcome. Therefore, within the context of this invention, an increased predictive value or predictive power implies an increased probability of correctly diagnosing, prognosing, stratifying, etc., based on a value determined by measuring the level of a biomarker in a sample.

[0069] As outlined above, the CgA level (and any other additional marker, such as MMP7 level) in a sample of the subject can be compared to a control level or a predetermined threshold. These control levels or predetermined thresholds can be absolute or relative values.

[0070] Depending on the specific application, the control level can be the CgA level from an earlier sample of the same subject. For example, the control level could be the CgA level from a sample taken from the same subject before surgical treatment (e.g., RCE), and the measured level could be from a sample collected after surgical treatment (e.g., RCE). This also applies to other biomarkers that can be determined separately, such as MMP7.

[0071] The predetermined threshold for CgA levels can be selected, for example, from 100 ng / mL to 431 ng / mL, preferably from 103 ng / mL to 191 ng / mL, more preferably from 130 ng / mL to 160 ng / mL, and most preferably from 147 ng / mL. The selection of the threshold for a particular application can be based, for example, on the desired specificity and / or selectivity of the assay; see below.

[0072] As outlined above, in addition to CgA levels, other biomarkers, particularly biomarkers, can be determined from the same subject, preferably from the same sample. One such biomarker that has been shown to have specific value in conjunction with CgA is matrix metalloproteinase-7 (MMP7). Therefore, in the context of this invention, it is preferable to additionally determine the level of matrix metalloproteinase-7 (MMP7) in the sample of the subject or another sample from the subject, and the level of MMP7 in the sample from the subject indicates the severity and / or outcome of the subject's non-neuroendocrine bladder cancer. Elevated MMP7 levels in the sample from the subject, compared to control levels or predetermined thresholds, can indicate adverse outcomes for the subject, particularly an increased risk of reduced life expectancy, increased risk of progression, increased risk of cancer-related death, and / or increased risk of recurrence following surgical and / or medical and / or radiotherapy. In one aspect, the MMP7 level in the same sample or another sample from the subject can indicate the subject's overall survival, disease-specific survival, or progression-free survival. The predetermined threshold for MMP7 levels can be selected, for example, from 4.4 to 21 ng / mL, preferably from 5.4 to 10.1 ng / mL, more preferably from 6 to 9 ng / mL. Most preferably, the predetermined threshold for MMP7 levels is 7.75 ng / mL. The selection of the threshold for a particular application can be based, for example, on the desired specificity and / or selectivity of the assay; see below.

[0073] In one specific embodiment of the method of the present invention,

[0074] (i) CgA levels above a predetermined CgA threshold in the sample indicate a high risk of cancer-related death following surgical treatment (e.g., TURBT, partial cystectomy (PCE), RCE, RCE combined with regional or extended LND, metastatic resection), or drug treatment (chemotherapy, targeted therapy, immunotherapy) or radiation therapy, and

[0075] (ii) CgA levels in the sample that are above a predetermined CgA threshold and MMP7 levels above a predetermined MMP7 threshold in the samples indicate a very high risk of cancer-related death and shorter lifespan following surgical treatment (e.g., TURBT, PCE, RCE, RCE combined with regional or extended LND, metastatic resection), drug treatment (chemotherapy, targeted therapy, immunotherapy), or radiation therapy.

[0076] Using the method of this invention, clinicians can select the most appropriate treatment based on the determined patient risk. As outlined, this invention provides a postoperative, non-invasive, inexpensive, and easy-to-use tool for monitoring treatment effectiveness and recurrence in bladder cancer, particularly non-neuroendocrine bladder cancer and preferably urothelial carcinoma. For example, levels of CgA and optionally MMP7 can be measured at regular intervals during follow-up, and control levels can be corresponding levels measured before said surgical treatment and / or at the initial step of follow-up.

[0077] In one particular aspect, preoperative CgA levels (preferably serum levels) are used as a biomarker for overall and disease-specific survival in patients after surgery (e.g., RCE). In another particular aspect of the invention, MMP7 is used as an additional biomarker for poor disease-specific survival in patients treated with RCE. MMP7 can also be used as an additional biomarker for invasive muscle tumors and / or metastases.

[0078] As discussed, in addition to measuring CgA levels, other biomarkers, particularly biomarkers (including MMP7), and other clinical parameters can be determined in subjects. Such clinical parameters may be selected, for example, from tumor histological subtype, metastatic status (lymph nodes and distal), lymphadenopathy, smoking or tobacco use, age, sex, family history, race, weight, body mass index (BMI), cystoscopy report, urine cytology (VUC), ultrasound, CT scan, MRI, and TURBT, and blood pressure. In one specific implementation, at least one additional clinical parameter selected from the group comprising: age, sex, systolic blood pressure, diastolic blood pressure, antihypertensive treatment, urinary tract disease and treatment history, history of stroke, wheezing, body mass index, heart rate, body temperature, presence of diabetes, and current smoking habit.

[0079] In addition, levels of markers selected from the following can be determined in the sample or another sample from the subject: complement factor H-related protein and complement factor H, nuclear matrix protein BLCA-4, survival proteins (BIRC5, EPR-1), cytokeratin 8 (CK8), cytokeratin 18 (CK18), cytokeratin 20 (CK20), CEA protein and bladder tumor cell-associated mucin, alterations in chromosomes 3, 7, 17, and 9p21, CEA protein (CEA), CYFRA. 21-1 (CK19), carbonic anhydrase, neurosensory protein (NSE), C-reactive protein (CRP), nuclear mitogen 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 metalloproteinase 1 (TIMP-1), tissue inhibitor of metalloproteinase 2 (TIMP-2), tissue inhibitor of metalloproteinase 3 (TIMP-3), tissue inhibitor of metalloproteinase 4 (TIMP-4), α-1 antitrypsin, vascular endothelial growth factor (VEGF), placental growth factor (PLGF), vascular endothelial growth factor receptor 1 (VEGFR-1), soluble vascular endothelial growth factor receptor 1 (sVEGFR-1), sfLT-1), vascular endothelial growth factor receptor 2 (VEGFR-2), soluble vascular endothelial growth factor receptor 3 (sVEGFR-3), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor 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), insulin-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 growth factor 2 mRNA-binding protein 3 (IGFBP3), insulin-like growth factor 2mRNA-binding 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 (total PSA), tumor necrosis factor α (TNFα), E-cadherin, elastin, fibronectin, collagen, and hyalin. Among them, the 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 mucin, alterations in chromosomes 3, 7, 17 and 9p21, CEA protein (CEA), nuclear mitogen 22 (NMP22), alkaline phosphatase, tissue inhibitor of metalloproteinases 1 (TIMP-1) and tissue inhibitor of metalloproteinases 2 (TIMP-2).

[0080] The term "biomarker" (biological marker) refers to measurable and quantifiable biological parameters (e.g., specific enzyme concentrations, specific hormone concentrations, specific gene phenotypic distributions in a population, the presence of biological substances) that serve as indicators in health and physiologically relevant assessments such as disease risk, mental illness, environmental exposure and its effects, disease diagnosis, metabolic processes, drug abuse, pregnancy, cell line development, epidemiological studies, etc. Furthermore, a biomarker is defined as a characteristic that is objectively measured and assessed as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions. Biomarkers can be measured on biological samples (e.g., whole blood, serum, plasma, urine), which can be records obtained from humans (blood pressure, ECG, or Holter monitoring), or they can be imaging tests (echocardiography or CT scan) (Vasan et al. 2006, Circulation 113:2335-2362). Biomarkers can indicate a variety of health or disease characteristics, including the level or type of exposure to environmental factors, genetic susceptibility, genetic response to exposure, biomarkers of subclinical or clinical disease, or indicators of response to treatment. Therefore, a simple way to think of biomarkers is as indicators of disease characteristics (risk factors or risk biomarkers), disease status (preclinical or clinical), or disease rate (progression). Thus, biomarkers can be classified as antecedent biomarkers (identifying the risk of disease onset), screening biomarkers (screening for subclinical diseases), diagnostic biomarkers (identifying obvious diseases), staging biomarkers (classifying disease severity), or prognostic biomarkers (predicting future disease progression, including relapse and response to treatment, and monitoring treatment efficacy). Biomarkers can also serve as surrogate endpoints. A surrogate endpoint is an endpoint that can be used as a clinical trial outcome to assess the safety and efficacy of treatment, rather than measuring the true outcome of interest. The basic principle is that changes in the surrogate endpoint are closely correlated with changes in the outcome of interest. Usufruct endpoints offer several advantages: they can be collected over a shorter timeframe and at a lower cost than endpoints such as morbidity and mortality, which require extensive clinical trials for evaluation. Other values ​​of surrogate endpoints include their closer proximity to the exposure / intervention of interest and their potential for stronger causal associations compared to more distant clinical events. A significant drawback of surrogate endpoints is that residual confounding can reduce their validity if the clinical outcome of interest is influenced by numerous factors (in addition to the surrogate endpoint itself). It has been suggested that surrogate endpoints are more effective if they explain at least 50% of the impact of the exposure or intervention on the outcome of interest. For example, biomarkers can be proteins, peptides, or nucleic acid molecules.

[0081] The National Institutes of Health (NIH) defines biomarkers as biological markers that are objectively measured and assessed as indicators of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions (Danesh et al. Clin Pharmacol Ther 2001. 169:416-46).

[0082] Some biomarkers (especially biomarkers) are “cancer biomarkers,” meaning they are biomarkers that are associated with the diagnosis and / or prognosis of cancer (in this article: non-neuroendocrine bladder cancer).

[0083] The method of the present invention may include an initial step of providing a body fluid sample of the subject. In the context of this invention, the term "sample" refers to a body fluid sample obtained for the diagnosis, prognosis, or evaluation of a subject of interest (e.g., a patient). Preferred test samples include (whole) blood, serum, plasma, and urine. Some test samples are easier to analyze after grading or purification procedures, such as separating whole blood into serum or plasma components. Therefore, in a preferred embodiment of the invention, the sample is selected from (whole) blood samples, serum samples, plasma samples, and urine samples, or extracts of any of the above samples. Preferably, the sample is a whole blood sample, most preferably a serum or plasma sample. As used herein, "whole blood sample" refers to an unprocessed or substantially unprocessed blood sample. Where appropriate, the sample may need to be homogenized or extracted with a solvent to obtain a liquid sample prior to use in the present invention. Therefore, the liquid sample may be a solution or a suspension. The liquid sample may undergo one or more pretreatments prior to use in the present invention. Such pretreatments include, but are not limited to, dilution, filtration, centrifugation, concentration, sedimentation, precipitation, or dialysis. Pretreatment may also include the addition of chemical or biological substances to the solution, such as acids, bases, buffers, salts, solvents, reactive dyes, detergents, emulsifiers, or chelating agents.

[0084] In the context of this invention, "plasma" is a nearly cell-free supernatant of blood containing an anticoagulant obtained after centrifugation. Exemplary anticoagulants include calcium-binding compounds such as EDTA or citrate and thrombin inhibitors such as heparin or hirudin. Cell-free plasma can be obtained by centrifuging anticoagulant (e.g., citrate, EDTA, or heparinized blood) at 2000 to 3000 g for at least 15 minutes. Therefore, plasma samples used in the context of this invention have preferably been centrifuged at more than 1500 g for 30 minutes, preferably at at least 2000 g for at least 30 minutes, more preferably at at least 3000 g for at least 20 minutes, and most preferably at at least 3000 g for at least 30 minutes.

[0085] In the context of this invention, "serum" refers to the undiluted extracellular portion of blood after sufficient coagulation. Coagulation typically completes after 30 minutes. Serum can be obtained by centrifuging the coagulated sample at a minimum speed of 1500 g for at least 10 minutes. Therefore, it is preferred that the serum sample used in the context of this invention has been centrifuged at at least 1500 g for at least 10 minutes, preferably at least 15 minutes, more preferably at least 20 minutes. Most preferably, the serum sample is centrifuged at at least 3000 g for at least 20 minutes.

[0086] CgA levels can be determined using appropriate assays. "Assay" or "diagnostic assay" can be of any type used in the diagnostic field. Such assays can be based on the binding of the analyte to be detected to one or more capture probes with a certain affinity. Regarding the interaction between the capture molecule and the target molecule or molecule of interest, the affinity constant is preferably greater than 10. 8 M -1 .

[0087] Preferred detection methods include various forms of immunoassays, such as radioimmunoassay (RIA), chemiluminescence and fluorescence immunoassays, enzyme-linked immunosorbent assays (ELISA), Luminex-based bead arrays, protein microarray assays, rapid tests, or point-of-care (PoC) forms, such as immunochromatographic strip assays and automated immunoassay systems, such as the BRAHMS KRYPTOR system. PoC assays are particularly preferred; see, for example, St. John & Price, ClinBiochem Rev. 2014 Aug; 35(3): 155–167. Furthermore, mass spectrometry can be used to detect and quantify CgA, MMP7, and / or other biomarkers, for example, using quantitative selective reaction monitoring (qSRM). For mass spectrometry measurements, chemical derivatization is typically performed before analysis and quantification of the target protein.

[0088] Immunoassays can be homogeneous or heterogeneous, competitive or non-competitive. 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 is bound to a first antibody and a second antibody. The first antibody may be bound to a solid phase (e.g., the surface of a bead, pore or other container, chip or strip), and the second antibody is an antibody labeled with, for example, a dye, a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by appropriate methods. General compositions and procedures involving “sandwich assays” are well determined and are known to those skilled in the art (The Immunoassay Handbook, edited by David Wild, Elsevier LTD, Oxford; 3rd edition (May 2005), ISBN-13: 978-0080445267; Hultschig C et al., Curr Opin Chem Biol. 2006 Feb;10(1):4-10, incorporated herein by reference).

[0089] In a particularly preferred embodiment, the assay comprises two capture molecules, preferably antibodies, both present as dispersions in a liquid reaction mixture, wherein a first labeled component is linked to a first capture molecule, wherein the first labeled component is part of a labeling system based on fluorescence-quenching or chemiluminescence-quenching or amplification, and a second labeled component of the labeling system is linked to a second capture molecule, such that when the two capture molecules bind to the analyte, a measurable signal is generated that allows detection of the sandwich complex formed in a solution containing the sample.

[0090] Even more preferably, the labeling system comprises rare earth cryptate or rare earth chelate in combination with fluorescent dyes or chemiluminescent dyes (especially anthocyanin dyes).

[0091] In the context of the method of the present invention, immunoassays are preferably used to determine the levels of CgA and / or MMP7. Suitable immunoassays and antibodies for detecting CgA are described, for example, in WO 2015 / 158701A1 and Popovici et al. (2014. ClinBiochem 47:87-91), which are incorporated herein by reference. For example, the B•R•A•H•M•S Chromogranin A KRYPTOR assay can be used (B•R•A•H•M•S GmbH, Hennigsdorf, Germany). Other commercially available assays for detecting chromogranin A are available and usable: the Cis-Bio ELISA assay (Cisbio Bioassays, Codolet, France) using two monoclonal antibodies targeting epitopes corresponding to amino acids 145-197 and 219-234; the DAKO ELISA assay (Dako Denmark A / S, Glostrup, Denmark) using a rabbit polyclonal antibody targeting the 23 kDa C-terminal fragment; and the Euro-Diagnostica NEOLISA. TM The sandwich ELISA assay (Euro Diagnostica AB, Malmö, Sweden) uses two monoclonal antibodies targeting epitopes corresponding to amino acids 236-251 and 264-279 (see also WO2011 / 135035 A1 and WO 99 / 58980 A1).

[0092] Suitable immunoassays for detecting MMP7 are described, for example, in WO 2007 / 144144 A1 (which is incorporated herein by reference).

[0093] Commercially available MMP7 assays include: Human Total MMP-7 Quantikine ELISA Kit, or Human Magnetic Luminex Screening Assay, or Human Luminex Screening Assay, or 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 Proteomics Analyzer Human Protease Array Kit (R&D Systems Minneapolis, MN 55413, USA), Human Total MMP-7 ELISA Kit (Aviva Systems Biology Corporation, San Diego, CA 92121, USA), MMP 7 (Human) ELISA Kit (Abnova, Taipei 114, Taiwan, China), and Human MMP7 / Matrilysin ELISA Kit (Sandwich ELISA) (LifeSpan). Biosciences, Seattle, Washington 98121, USA; MMP7 Human ELISA Kit (Abcam, Cambridge, United Kingdom); SensoLyte® 490 MMP-7 Assay Kit*Fluorescence* (ANASPEC, Fremont, CA 94555, USA); Bio-Plex Pro™ Human MMP and TIMP Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA); Matrix Metalloproteinase 7 (MMP7) ELISA Kit (Wuhan USCN Business Co., Ltd., Wuhan City, China).

[0094] In the context of this invention, the immunoassay for detecting CgA described in WO 2015 / 158701 A1 is preferred. It is based on the use of two antibodies against CgA (sandwich immunoassay). The (first and / or second) antibodies or their antigen-binding fragments or derivatives of the immunoassay method described in WO 2015 / 158701 A1 can be, for example, polyclonal antibodies, monoclonal antibodies, or genetically modified monoclonal antibodies. The first antibody is specific for an epitope in the CgA sequence (SEQ ID NO:1), preferably spanning amino acids 124 to 144 of SEQ ID NO:1. The first antibody is preferably a monoclonal antibody. The second antibody is specific for an epitope in the CgA sequence (SEQ ID NO:1), preferably spanning amino acids 280 to 301 of SEQ ID NO:1. The second antibody is preferably a monoclonal antibody. In a specific immunoassay, the first antibody is specific for an epitope in the CgA sequence (SEQ ID NO:1) spanning amino acid residues 124 to 144, and the second antibody is specific for an epitope in the CgA sequence (SEQ ID NO:1) spanning amino acid residues 280 to 301. Both the first and second antibodies are preferably monoclonal antibodies. The first antibody or its antigen-binding fragment or derivative can be produced, for example, by hybridoma cell line 537 / H2, which is deposited as DSM ACC3231. The antibody produced by hybridoma cell line 537 / H2 specifically binds to amino acid residues 124 to 144 of the CgA sequence (SEQ ID NO:1). The second antibody or its antigen-binding fragment or derivative can be produced, for example, by hybridoma cell line 541 / E2, which is deposited as DSM ACC3232. The antibody produced by hybridoma cell line 541 / E2 specifically binds to amino acid residues 280 to 301 of the CgA sequence (SEQ ID NO:1). In one particular embodiment of the immunoassay, the first antibody is produced by hybridoma cell line 537 / H2, which is preserved as DSM ACC3231, and the second antibody is produced by hybridoma cell line 541 / E2, which is preserved as DSM ACC3232.

[0095] Biomarkers such as CgA can be fragmented into shorter peptides or proteins. Therefore, in some cases, fragments of CgA or other peptide biomarkers (e.g., MMP7) can also be detected, and their levels in a sample can be determined. The term "fragment" refers to a smaller protein or peptide that can be derived from a larger protein or peptide, and thus contains a portion of the sequence of the larger protein or peptide. The fragment is derived from the larger protein or peptide by saponifying one or more peptide bonds. The peptide fragment is preferably at least about 15 to about 20 amino acids in length, more preferably at least about 25 to about 45 amino acids.

[0096] The levels of measured biomarkers (CgA, MMP7, and other biomarkers as appropriate) can be correlated with certain diagnoses and / or prognoses, for example, using specific mathematical algorithms. In the context of this invention, "algorithm" or "mathematical algorithm" refers to the use of mathematical or statistical methods or models to compare a measurement with values ​​in a reference population in order to stratify the measurement. This could be, for example, the median level of an entity in a predetermined sample set, meaning comparing the measurement level of that entity with the mathematical median level of that entity in a given number of samples. There is no particular limitation on the number of samples used to determine the median, but it should be sufficient to ensure the statistical significance of the median. The number of samples used to determine the median can even increase over time to improve the statistical significance of the median due to the addition of additional measurements from clinical samples. Preferably, the sample size is chosen to ensure the statistical significance of the median. Thus, by using the median as a reference, the measurement level of the aforementioned entity can be statistically correlated with some physiological state, for example, a tendency for adverse patient outcomes, depending on the relative level above or below the median and the degree of deviation of the measurement from that median. Other statistical methods, such as determining quantiles (e.g., quartiles or percentiles) or mathematical models, preferably Cox regression, can be used in place of the median to obtain the aforementioned reference values ​​and / or, in other cases, to determine the significance of the measurements relative to the physiological state of a given subject from which samples have been obtained. The mathematical or statistical methods or models described are well known to those skilled in the art and their application in the context of medical applications has been well established.

[0097] As used herein in the context of the use of reference diagnostic and prognostic biomarkers, the term "correlated" means comparing the presence or level of a biomarker in a patient to its presence or amount in a person known to have a particular condition or known to be at risk of a particular condition. Biomarker levels in a patient sample can be compared to levels known to be associated with a particular diagnosis. The biomarker levels in the sample are believed to be associated with a diagnosis; that is, a technician can use the biomarker levels to determine whether a patient has a particular type of disease and respond accordingly. Alternatively, biomarker levels in a sample can be compared to levels of biomarkers known to be associated with a favorable outcome (e.g., no disease, etc.). In a preferred embodiment, the levels of a set of biomarkers are associated with an overall probability or a specific outcome.

[0098] In other embodiments, positive likelihood ratio, negative likelihood ratio, odds ratio, or hazard ratio are used as measures of the trial's ability to predict risk or diagnose disease. In the case of positive likelihood ratio, a value of 1 indicates that a positive outcome is equally likely in both the "disease" and "control" groups; a value greater than 1 indicates that a positive outcome is more likely in the disease group; and a value less than 1 indicates that a positive outcome is more likely in the control group. In the case of negative likelihood ratio, a value of 1 indicates that a negative outcome is equally likely in both the "disease" and "control" groups; a value greater than 1 indicates that a negative outcome is more likely in the experimental group; and a value less than 1 indicates that a negative outcome is more likely in the control group. In some preferred embodiments, biomarkers and / or groups of biomarkers are preferably selected to show positive or negative likelihood ratios of at least about 1.5 or higher or about 0.67 or lower, more preferably at least about 2 or higher or about 0.5 or lower, more preferably at least about 5 or higher or about 0.2 or lower, more preferably at least about 10 or higher or about 0.1 or lower, and most preferably at least about 20 or higher or about 0.05 or lower. In this context, the term "about" refers to + / - 5% of a given measurement.

[0099] In the case of odds ratios, a value of 1 indicates that a positive outcome is equally likely in both the "disease" and "control" groups; a value greater than 1 indicates that a positive outcome is more likely in the disease group; and a value less than 1 indicates that a positive outcome is more likely in the control group. In some preferred embodiments, biomarkers and / or biomarker groups are preferably selected to show the following odds ratios: at least about 2 or higher or about 0.5 or lower, more preferably at least about 3 or higher or about 0.33 or lower, more preferably at least about 4 or higher or about 0.25 or lower, more preferably at least about 5 or higher or about 0.2 or lower, and most preferably at least about 10 or higher or about 0.1 or lower. In this context, the term "about" means + / - 5% of a given measurement.

[0100] In the case of hazard ratios, a value of 1 indicates that the relative risks at the endpoints (e.g., death) are equal in the "disease" and "control" groups; a value greater than 1 indicates a higher risk in the disease group; and a value less than 1 indicates a higher risk in the control group. In some preferred embodiments, biomarkers and / or groups of biomarkers are preferably selected to show hazard ratios of at least about 1.1 or higher or about 0.91 or lower, more preferably at least about 1.25 or higher or about 0.8 or lower, more preferably at least about 1.5 or higher or about 0.67 or lower, more preferably at least about 2 or higher or about 0.5 or lower, and most preferably at least about 2.5 or higher or about 0.4 or lower. In this context, the term "about" refers to + / - 5% of a given measurement.

[0101] The sensitivity and specificity of diagnostic and / or prognostic tests depend not only on the analytical “quality” of the test but also on the definition of what constitutes an abnormal outcome. In practice, receiver operating characteristic (ROC) curves are typically calculated by plotting the relationship between the value of a variable and its relative frequency in the “normal” (i.e., superficially healthy) and “disease” groups (i.e., patients with bladder cancer, particularly non-neuroendocrine bladder cancer, preferably urothelial carcinoma). For any given biomarker, the distribution of biomarker levels in subjects with or without the disease may overlap. Under such conditions, the test cannot perfectly distinguish between normal and disease with 100% accuracy; the overlapping area indicates that the test cannot differentiate between normal and disease. A threshold is chosen above which the test is considered abnormal (or below which, depending on how the biomarker varies with the disease); below which the test is considered normal. The area under the ROC curve is a measure of the probability that the perceived measurement allows for the correct identification of the disease. ROC curves can be used even when the test outcome may not provide an accurate number. ROC curves can be created whenever the outcomes can be arranged. For example, the test outcomes of “disease” samples can be ranked according to severity (e.g., 1 = low, 2 = normal, 3 = high). This ranking can be correlated with the outcomes in the “normal” population, and an ROC curve can be created. These methods are well known in the art (see, for example, Hanley et al., 1982, Radiology 143: 29-36). Preferably, the outcome of the ROC curve is an AUC greater than about 0.5, more preferably greater than about 0.7, more preferably greater than about 0.8, more preferably greater than about 0.85, and most preferably greater than about 0.9. In this context, the term “about” refers to + / - 5% of a given measurement.

[0102] The horizontal axis of the ROC curve represents (1-specificity), which increases with the false positive rate. The vertical axis of the curve represents sensitivity, which increases with the true positive rate. Therefore, for a specific cutoff value, the value of (1-specificity) can be determined, and the corresponding sensitivity can be obtained. The area under the ROC curve is a measure of the probability that a marker level allows for the correct identification of a disease or condition. Therefore, the area under the ROC curve can be used to determine the validity of the test.

[0103] In some embodiments, markers and / or groups of markers are selected to exhibit a combination of at least about 70% sensitivity, more preferably at least about 80% sensitivity, more preferably at least about 85% sensitivity, more preferably at least about 90% sensitivity, and most preferably at least about 95% sensitivity with at least about 70% specificity, more preferably at least about 80% specificity, more preferably at least about 85% specificity, 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%, more preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95%. In this context, the term "about" refers to + / - 5% for a given measurement.

[0104] For each specific combination of CgA levels, other biomarkers (e.g., MMP7) and / or parameters, drugs, and diseases, an appropriate threshold level must be determined to “stratify” subjects into different groups (categories). This can be done, for example, by grouping a reference population of patients into certain quantiles based on their CgA levels, such as quartiles, quintiles, or even appropriate percentiles. For example, in terms of survival, for each quantile or group above and below certain percentiles, a hazard ratio (HR) can be calculated by comparing the risk of adverse outcomes (i.e., “adverse effects”) between patients who have received treatment with a certain drug and those who have not. In this case, a HR greater than 1 indicates a higher risk of adverse outcomes for patients who have received treatment than for those who have not. An HR less than 1 indicates a beneficial effect of a certain treatment in a patient group. An HR of approximately 1 (e.g., + / - 0.1) indicates no increased risk for a particular patient group, but also no benefit from the drug. By comparing the HRs between certain quantiles of patients with each other and with the HR of the total patient population, quantiles of patients with high risk and those who benefit from the drug can be determined, thereby stratifying subjects according to the invention.

[0105] In the context of this invention, the term "stratification of therapeutic treatment" or "treatment stratification" refers to the evaluation of appropriate therapeutic treatment for the patient. Subclassification involves further defining the diagnosis based on different subcategories of the diagnosed disease, disorder, complication, or risk, for example, based on the severity or form of the disease. In the context of this invention, the term "treatment monitoring" refers to controlling and / or adjusting the patient's therapeutic treatment.

[0106] This invention also relates to a method for treating non-neuroendocrine bladder cancer, wherein the level of CgA (and optionally other biomarkers and / or clinical parameters such as the level of MMP7) is determined to identify the most appropriate treatment. For example, based on the determined CgA level, patients with a relatively poor prognosis may benefit from more aggressive treatment, such as partial cystectomy instead of TURBT or triple bladder preservation, RCE instead of TURBT or triple bladder preservation or partial cystectomy, RCE combined with PLND instead of RCE, prolonged or ultra-prolonged LND instead of PLND, adjuvant chemotherapy or radiotherapy instead of surgery alone, and additional metastatic tumor resection. These high-risk patients may also be eligible for clinical trials to test new drugs.

[0107] Furthermore, this invention relates to a kit comprising a CgA-specific antibody and optionally an MMP7-specific antibody for the use in the prognosis, risk assessment, risk stratification, monitoring, and / or treatment control of bladder cancer, particularly non-neuroendocrine bladder cancer, preferably urothelial carcinoma. The kit may also contain other components such as buffers and instructions for use. A preferred kit for detecting CgA is described and comprises in WO 2015 / 158701 A1:

[0108] (i) A first antibody or antigen-binding fragment or derivative thereof that is specific to CgA or a fragment thereof; and

[0109] (ii) A second antibody or its antigen-binding fragment or derivative that is specific to CgA or its fragments.

[0110] The first and second antibodies in the kit are preferably specific to the same CgA fragment. For example, (i) the first antibody or its antigen-binding fragment or derivative is specific to an epitope contained in the sequence SEQ ID NO:1; and / or (ii) the second antibody or its antigen-binding fragment or derivative is specific to an epitope contained in the sequence SEQ ID NO:1. Preferably, the first antibody in the kit is a monoclonal anti-CgA antibody produced as hybridoma cell line 537 / H2 deposited as DSM ACC3231, and / or the second antibody is a monoclonal anti-CgA antibody produced as hybridoma cell line 541 / E2 deposited as DSM ACC3232.

[0111] This kit may contain one or more antibodies for detecting MMP7. For example, the kit (additionally) contains...

[0112] (i) A first antibody or antigen-binding fragment or derivative thereof that is specific to MMP7 or a fragment thereof; and

[0113] (ii) A second antibody or its antigen-binding fragment or derivative that is specific to MMP7 or a fragment thereof.

[0114] The first and second antibodies in this kit are preferably specific to the same MMP7 fragment. For example, (i) the first antibody or its antigen-binding fragment or derivative is specific to the epitope contained in the sequence SEQ ID NO:2; and / or (ii) the second antibody or its antigen-binding fragment or derivative is specific to the epitope contained in the sequence SEQ ID NO:2.

[0115] In a preferred aspect, the present invention relates to:

[0116] 1. The use of chromogranin A (CgA) as a prognostic marker for bladder cancer.

[0117] 2. The use according to the first aspect, wherein the bladder cancer is a non-neuroendocrine bladder cancer.

[0118] 3. The use according to the first aspect, wherein the bladder cancer is selected from bladder urothelial carcinoma, bladder squamous cell carcinoma and bladder adenocarcinoma, preferably wherein the bladder cancer is bladder urothelial carcinoma.

[0119] 4. According to the use described in aspect 3, the bladder cancer is non-muscle-invasive bladder cancer (NMBC) or muscle-invasive bladder cancer (MIBC).

[0120] 5. The use as described in aspects 1 to 4, wherein CgA is used as a biomarker in in vitro assays for prognosis, risk assessment, risk stratification, monitoring and / or treatment control of bladder cancer.

[0121] 6. A method for prognosis, risk assessment, risk stratification, monitoring, and / or treatment control of bladder cancer in subjects, comprising the step of determining the level of CgA in a body fluid sample of the subjects.

[0122] 7. The method according to aspect 6, wherein the bladder cancer is a non-neuroendocrine bladder cancer.

[0123] 8. The method according to claims 5 to 6, wherein the bladder cancer is selected from bladder urothelial carcinoma, bladder squamous cell carcinoma and bladder adenocarcinoma, preferably wherein the bladder cancer is bladder urothelial carcinoma.

[0124] 9. The method according to aspect 8, wherein the bladder cancer is non-muscle-invasive bladder cancer (NMBC) or muscle-invasive bladder cancer (MIBC).

[0125] 10. The method according to aspects 6 to 9, wherein the CgA level in the sample from the subject indicates the severity and / or outcome of the subject's bladder cancer.

[0126] 11. The method according to aspect 10, wherein an elevated CgA level in the sample from the subject, compared to a control level or a predetermined threshold, indicates an adverse outcome for the subject.

[0127] 12. The method according to aspect 10, wherein the CgA level in the sample from the subject indicates the overall survival of the subject or the disease-specific survival of the subject or the progression-free survival of the subject.

[0128] 13. The method according to aspect 12, wherein the adverse 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 following surgical treatment and / or medical treatment and / or radiation therapy.

[0129] 14. The method according to any one of aspects 6 to 13, wherein the subject has been diagnosed with bladder cancer and wherein the subject has not undergone surgical treatment for the bladder cancer.

[0130] 15. The method according to any one of aspects 6 to 13, wherein the subject has undergone surgical treatment for the bladder cancer.

[0131] 16. The method according to aspect 15, wherein the subject has undergone radiation cystectomy for the bladder cancer.

[0132] 17. The method according to any one of aspects 11 to 16, wherein the predetermined threshold of the CgA level is selected from 100 ng / mL to 431 ng / mL, preferably from 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.

[0133] 18. The method according to any one of aspects 6 to 17, wherein the level of matrix metalloproteinase 7 (MMP7) is further determined in the sample of the subject, and wherein the level of MMP7 from the sample of the subject indicates the severity and / or outcome of the subject's bladder cancer.

[0134] 19. The method according to aspect 18, wherein an elevated MMP7 level in the sample from the subject, compared to a control level or a predetermined threshold, indicates an adverse outcome for the subject, particularly an elevated risk of reduced life expectancy, an elevated risk of progression, an elevated risk of cancer-related death, and / or an elevated risk of recurrence following surgical and / or medical and / or radiotherapy.

[0135] 20. The method according to aspect 18, wherein the MMP7 level in the sample from the subject indicates the overall survival of the subject or the disease-specific survival of the subject or the progression-free survival of the subject.

[0136] 21. The method according to aspect 19, wherein the predetermined threshold for the MMP7 level is selected from 4.4 to 21 ng / mL, preferably 5.4 to 10.1 ng / mL, more preferably 6 to 9 ng / mL, and most preferably the predetermined threshold for the MMP7 level is 7.75 ng / mL.

[0137] 22. The method according to any one of aspects 11 to 21, wherein

[0138] (i) CgA levels above a predetermined CgA threshold in the sample indicate a high risk of cancer-related death following surgical and / or pharmacological and / or radiotherapy, and

[0139] (ii) CgA levels in the sample that are above a predetermined CgA threshold and An MMP7 level above a predetermined MMP7 threshold in the sample or another sample from the same subject indicates a very high risk of cancer-related death and shorter lifespan following surgical and / or medical and / or radiation therapy.

[0140] 23. The method according to any one of aspects 11 to 22, wherein the levels of CgA and optionally MMP7 are determined at regular time intervals during follow-up, and wherein the control level is a corresponding level determined before said surgical treatment and / or at the initial step of the follow-up.

[0141] 24. The method according to any one of aspects 6 to 23, wherein clinical parameters selected from the following subjects are further determined: tumor histological subtype, metastatic status (lymph nodes and distal), lymphadenopathy, smoking or tobacco use, age, sex, family history, race, weight, body mass index (BMI), cystoscopy report, urine cytology (VUC), ultrasound examination, CT scan, MRI and TURBT, and blood pressure.

[0142] 25. The method according to any one of aspects 6 to 24, wherein the level of a marker selected from the following is further determined in the sample, or in another sample determining the object: 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 mucin, alterations in chromosomes 3, 7, 17 and 9p21, CEA protein (CEA), nuclear mitogen 22 (NMP22), alkaline phosphatase, tissue inhibitor of metalloproteinases 1 (TIMP-1) and tissue inhibitor of metalloproteinases 2 (TIMP-2).

[0143] 26. The method according to any one of aspects 6 to 25, wherein the sample is derived from a body fluid selected from whole blood, serum, plasma and urine.

[0144] 27. The method according to any one of aspects 6 to 26, wherein an immunoassay is used to determine the levels of CgA and / or MMP7.

[0145] 28. The method of aspect 27, wherein the immunoassay is selected from radioimmunoassay (RIA), chemiluminescent immunoassay and fluorescence immunoassay, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), Luminex-based bead array assay, protein microarray assay and immunochromatographic strip assay.

[0146] 29. The method of aspect 28, wherein the immunoassay is a sandwich immunoassay using CgA-specific first and second antibodies.

[0147] 30. The method according to aspect 29, characterized in that one of the antibodies is labeled, and the other antibody binds to or is selectively bound to the solid.

[0148] 31. The immunoassay method according to aspect 30, wherein first and second antibodies are dispersed in a liquid reaction mixture, and wherein a first labeling component, as part of a labeling system based on fluorescence or chemiluminescence elimination or amplification, binds to the first antibody, and a second labeling component of the labeling system binds to the second antibody, such that after both antibodies bind to CgA, a measurable signal is generated that allows the resulting sandwich complex to be detected in a measurement solution.

[0149] 32. The immunoassay method according to aspect 31, characterized in that the labeling system comprises a combination of rare earth cavitates or chelates with fluorescent or chemiluminescent dyes (especially anthocyanin dyes).

[0150] 33. A method for using chromogranin A (CgA) levels in bodily fluid samples of subjects as a prognostic marker for bladder cancer.

[0151] References cited

[0152] All references cited in this article are included in their entirety through citation.

[0153]

[0154]

[0155]

[0156]

[0157]

[0158] sequence

[0159] Sequence 1 (SEQ ID NO:1): Human chromogranin A (CgA) without signal peptide (UniProt accession number P10645):

[0160]

[0161]

[0162] Sequence 2 (SEQ ID NO:2): Human matrix metalloproteinase 7 (MMP7) (UniProt accession number P09237)

[0163]

[0164] Example

[0165] Example 1 Detection of human chromogranin A and MMP7 in bladder cancer patients

[0166] Experimental Design

[0167] Serum samples were collected from 188 patients who underwent surgical treatment for bladder cancer (BCa): transurethral resection of cystectomy (TURB) or radical cystectomy (RCE). Patients were enrolled in the Department of Urology at Duisburg-Essen University Hospital between August 2008 and November 2011. Enrollment criteria were histopathological diagnosis of transitional cell carcinoma of the bladder, absence of other tumor history, absence of preoperative chemotherapy, availability of sufficient serum samples, and likelihood of follow-up. No neuroendocrine bladder cancer was diagnosed in the population. Histological grading and T staging were performed according to the WHO 1973 and 2004 and 2009 TNM classifications. Serum samples from 97 healthy individuals with no history of cancer were used as controls.

[0168] The study was conducted in accordance with the ethical standards of the Declaration of Helsinki and approved by the hospital's ethics committee. Samples were centrifuged at 1500 rpm for 15 minutes, immediately aliquoted, and kept at -80°C until analysis.

[0169] The patients' clinical characteristics are shown in Table 1.

[0170] The endpoints of this study were overall-specific survival and cancer-specific survival. Cause of death was obtained from death certificates. The median follow-up time for all patients was 24 months, and for survivors, it was 31 months. Of the 188 patients, 56 died during follow-up, of whom 39 were related to BCa.

[0171] Detection of chromogranin A

[0172] Chromogranin A levels were measured using a fully automated BRAHMS KRYPTOR® instrument (Thermo Scientific B.RAHMS GmbH, Hennigsdorf / Berlin, Germany) based on the advanced homogeneous sandwich fluorescence immunoassay commercially available according to reference CgA II #839.050 (patent application WO 2015 / 158701 A1).

[0173] This system utilizes sensitive time-resolved amplified cavitary emission (TRACE®) technology based on nonradiative energy transfer between a donor (europium cavitary compound) and an acceptor (XL665). The assay employs two mouse monoclonal antibodies: the LK2H10 antibody with epitopes located between amino acid sequences 280 and 301, and the PHE5 monoclonal antibody with epitopes located between amino acid sequences 124 and 144. The inter-assay coefficient of variation (CV) is ≤12%, and the intra-assay CV for the Kryptor assay is ≤7%. The sensitivity of the functional assay is 13.7 ng / mL, and the limit of quantitation is 13.71 ng / mL.

[0174] MMP7 detection

[0175] MMP-7 levels were measured using an advanced homogeneous sandwich prototype fluorescence immunoassay (not yet commercially available) with a fully automated BRAHMSKRYPTOR® instrument (Thermo Scientific BRAHMS GmbH, Hennigsdorf / Berlin, Germany).

[0176] This system utilizes sensitive time-resolved amplified cavitary emission (TRACE®) technology based on nonradiative energy transfer between a donor (europium cavitary compound) and an acceptor (XL665). The assay uses a goat polyclonal anti-human MMP7 antibody and a mouse monoclonal anti-human MMP7 antibody labeled with europium cavitary compound-EuC- (Cis BioInternational, Bagnols / Cèze, France) and Alexa Fluor 647 (Molecular Probes – Lifetechnologies, Eugene, USA), respectively. Epitopes of the polyclonal and monoclonal antibodies are not described. Recombinant MMP-7 (R&D Systems Europe) diluted in newborn calf serum (Trina Bioreactives AG, Nänikon, Switzerland) was used as a calibrator. Inter-assay coefficient of variation (CV) ≤11%, and intra-assay CV ≤9% for the Kryptor assay. The sensitivity of the functional assay is 1.95 ng / ml.

[0177] Measurement

[0178] A single measurement was performed automatically by incubating 50 μL of each patient's sample with 50 μL of each conjugated antibody solution at 37°C for 29 minutes. To evaluate reproducibility and repeatability, two and three quality control (QC) samples were measured in duplicate for each run of the CgA and MMP7 assays, respectively.

[0179] Statistical analysis

[0180] The lack of serum concentration data and the normal distribution (controlled by the Shapiro-Wilk test) suggest using the nonparametric two-sided Wilcoxon rank-sum test (Mann-Whitney test) for independent group comparisons.

[0181] For univariate and multivariate analyses, patients were subdivided into low-concentration and high-concentration groups based on their CgA and MMP7 levels. Cutoff values ​​were determined to maximize prognostic values, corresponding to 147 ng / mL (80th percentile) for CgA and 7.75 ng / mL (66th percentile) for MMP7. Univariate disease-specific survival analyses were performed using both the Kaplan-Meier log-rank test and univariate Cox analysis. For multivariate analyses, a Cox proportional hazards regression model was used. A p-value of at least 0.05 was considered significant in all tests. All statistical analyses were performed using SPSS software package, version 21.0 (Chicago, IL, USA).

[0182] Example 2The preoperative prognostic value of CgA in surgical patients (Example 1 study)

[0183] Comparison of serum CgA levels between tumor and control samples

[0184] Compared with the control group, the serum CgA concentration in tumor patients was significantly elevated (median values ​​were 3.9 ng / mL and 29.4 ng / mL, respectively, P<0.0001). Figure 1 ).

[0185] CgA concentration and clinicopathological parameters

[0186] In cancer patients, CgA concentrations were significantly higher in older patients and in men (p=0.026 and p=0.009, respectively, Table 2). CgA concentrations were associated with stage and grade but not with metastasis (Table 2).

[0187] The prognostic value of CgA levels in surgical patients

[0188] Univariate analysis: The outcomes of the univariate analysis are listed in Table 3 and Figure 2 In contrast to sex, which had an impact on disease-specific survival (HR=0.447, CI 0.247–0.918, p=0.027), patient age did not affect overall or disease-specific survival. Tumor stage, grade, and metastasis were significant predictors of both overall and disease-specific survival (P≤0.001, Table 3). Serum CgA levels were also strong predictors of both overall and disease-specific survival. High serum CgA concentrations were significantly associated with poor overall and disease-specific survival in patients undergoing 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, Table 3, respectively); and p=0.021, respectively. Figure 2 Furthermore, when considered as a continuous variable, CgA is also an important predictor of overall survival (HR=1.001, 95% CI, 1.000–1.002, p=0.024).

[0189] Multivariate analysis: The results of the multivariate analysis are shown in Table 4. Multivariate analysis showed that high serum CgA concentration was an independent predictor of stage, grade, and metastasis of overall survival 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, Table 4, respectively).

[0190] The 20-month survival rate after treatment was 87% in patients with low preoperative CgA concentrations, compared to 70% in patients with high preoperative CgA levels. Figure 2 ).

[0191] Patients with high preoperative CgA concentrations have a high risk of bladder cancer-related death and all-cause mortality and may benefit from more aggressive or experimental treatments, as described in part

[0054] .

[0192] Preoperative prognostic value of CgA and MMP7 in surgical patients

[0193] Comparison of serum MMP7 levels between tumor and control samples:

[0194] Serum MMP7 concentrations were measured in the same population. Compared with the control group, serum MMP7 concentrations were significantly higher in tumor patients (median values ​​were 2.9 ng / mL and 4.4 ng / mL, respectively, P < 0.001).

[0195] MMP7 concentration and clinicopathological parameters

[0196] In cancer patients, MMP7 concentrations were significantly higher in older, non-male patients (p < 0.001, Table 5). MMP7 concentrations were associated with the presence of invasive muscle tumors and metastases (p = 0.008 and p = 0.05, respectively, Table 5), but were not graded.

[0197] The prognostic value of MMP7 levels in surgical patients

[0198] Univariate analysis: The results of the univariate analysis are shown in Table 3 and Figure 2 middle.

[0199] Serum MMP7 levels were significant predictors of both overall survival and disease-specific survival. High serum MMP7 concentrations were significantly associated with poor overall survival and disease-specific survival in patients undergoing surgical treatment (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, Table 3, respectively); and p < 0.001, respectively. Figure 2 ).

[0200] Multivariate analysis: Multivariate analysis showed that high serum MMP7 concentration was an independent predictor of stage, grade and metastasis of overall survival 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, Table 6).

[0201] The 20-month survival rate was 89% in patients with low preoperative MMP7, compared to 59% in patients with high preoperative concentrations. Figure 2 ).

[0202] The prognostic value of combined CgA and MMP7 levels in surgical patients

[0203] Multivariate analysis showed that high serum concentrations of MMP7 and CgA were independent predictors of stage, grade, and metastasis for both overall survival and disease-specific survival (Table 9). High serum concentrations of CgA, in conjunction with high MMP7 concentrations, were associated with poor disease-specific survival in patients undergoing surgical treatment (p < 0.001). Figure 2 ).

[0204] In patients with low preoperative CgA and MMP7, the 20-month survival rate after treatment exceeded 90%, compared to 71% in patients with one elevated serum marker and 50% in patients with both elevated CgA and MMP7. Figure 2 ).

[0205] Patients with high preoperative CgA and MMP7 concentrations have a high risk of bladder cancer-related and all-cause mortality and may benefit from more aggressive or experimental treatments, as described in part

[0054] .

[0206] Example 3 Preoperative prognostic value of CCE / MMP7 in patients undergoing RCE treatment (Example 1 study)

[0207] Risk stratification is particularly interesting for patients treated with radical cystectomy. Therefore, we also analyzed the prognostic significance of CgA levels in this group in isolation.

[0208] The prognostic value of CgA levels in patients undergoing RCE treatment

[0209] High serum CgA concentrations were strong independent predictors of both overall survival 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 concentrations were significantly associated with poor disease-specific survival in patients undergoing radical cystectomy (p = 0.008). Figure 3 ).

[0210] The 20-month survival rate after radical cystectomy was 76% in patients with low preoperative CgA concentrations, compared to 33% in patients with high preoperative CgA concentrations.

[0211] Patients with high preoperative CgA concentrations have a high risk of bladder cancer-related and all-cause mortality and may benefit from more aggressive or experimental treatments, as described in part

[0054] .

[0212] The prognostic value of MMP7 levels in patients undergoing renal replacement therapy (RCE)

[0213] High serum MMP7 concentrations were strong independent predictors of both overall survival 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 MMP7 concentrations were significantly associated with poor disease-specific survival in patients undergoing radical cystectomy (p = 0.028). Figure 3 )

[0214] The 20-month survival rate after radical cystectomy was 78% in patients with low preoperative MMP7 levels, compared to 54% in patients with high preoperative MMP7 levels. Figure 3 ).

[0215] The prognostic value of combined CgA and MMP7 levels in patients undergoing recurrent cerebral chemoembolization (RCE).

[0216] Multivariate analysis showed that high serum MMP7 and high CgA concentrations were independent predictors of stage, grade, and metastasis for both overall survival and disease-specific survival (Table 10). The combination of high serum CgA concentration and high MMP7 concentration was significantly associated with poor disease-specific survival in patients undergoing radical cystectomy (p = 0.001). Figure 3 ).

[0217] The 20-month survival rate after radical cystectomy was 79% in patients with low preoperative CgA and MMP7 levels, compared to 47% in patients with one elevated serum marker and 20% in patients with both elevated CgA and MMP7 levels.

[0218] Patients with high preoperative CgA and MMP7 concentrations have a high risk of bladder cancer-related and all-cause mortality and may benefit from more aggressive or experimental treatments, as described in part

[0056] .

[0219] sheet

[0220] Table 1: Clinical characteristics of patients

[0221]

[0222] Table 2: Association between CgA concentration and clinicopathological parameters in tumor patients

[0223]

[0224] Table 3: Association between CgA levels, MMP7 levels, clinicopathological parameters, and patient prognosis (univariate analysis)

[0225]

[0226] Table 4: Association between CgA levels and clinicopathological parameters and patient prognosis (multivariate analysis)

[0227]

[0228] Table 5: Association between MMP7 concentration and clinicopathological parameters in patients

[0229]

[0230] Table 6: Association between MMP7 levels and clinicopathological parameters and patient prognosis (multivariate analysis)

[0231]

[0232] Table 7: Association between CgA levels and clinicopathological parameters and patient prognosis in the RCE treatment population (multivariate analysis)

[0233]

[0234] Table 8: Association between MMP7 levels and clinicopathological parameters and patient prognosis in the RCE treatment population (multivariate analysis)

[0235]

[0236] Table 9: Relationship between MMP7 and CgA levels, clinicopathological parameters, and patient prognosis (multivariate analysis)

[0237]

[0238] Table 10: Association between MMP7 and CgA levels and clinicopathological parameters and patient prognosis in the RCE treatment population (multivariate analysis)

[0239]

[0240] The technical solutions corresponding to the original claims of the parent application are hereby incorporated in this specification:

[0241] 1. The use of chromogranin A (CgA) as a prognostic marker for bladder cancer.

[0242] 2. A method for prognosis, risk assessment, risk stratification, monitoring, and / or treatment control of bladder cancer in subjects, comprising the step of determining the level of CgA in a body fluid sample of the subjects.

[0243] 3. The use as described in Item 1 or the method as described in Item 2, wherein the bladder cancer is a non-neuroendocrine bladder cancer.

[0244] 4. The use according to item 1 or the method according to item 2, wherein the bladder cancer is selected from bladder urothelial carcinoma, bladder squamous cell carcinoma and bladder adenocarcinoma, preferably wherein the bladder cancer is bladder urothelial carcinoma.

[0245] 5. The method according to item 3 or 4, wherein the CgA level in a sample from the subject indicates the severity and / or outcome of the subject's bladder cancer.

[0246] 6. The method according to Project 5, wherein an elevated CgA level in a sample from the subject, compared to a control level or a predetermined threshold, indicates an adverse outcome for the subject.

[0247] 7. The method according to Project 5, wherein the CgA level in a sample from the subject indicates the overall survival of the subject, or the disease-specific survival of the subject, or the progression-free survival of the subject.

[0248] 8. The method according to Project 6, wherein the adverse 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 following surgical treatment and / or medical treatment and / or radiation therapy.

[0249] 9. The method according to any one of items 2 to 8, wherein the predetermined threshold for the CgA level is selected from 100 ng / mL to 431 ng / mL, preferably 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.

[0250] 10. The method of any one of items 2 to 9, wherein the level of matrix metalloproteinase 7 (MMP7) is further determined in the sample of the subject, and wherein the level of MMP7 from the sample of the subject indicates the severity and / or outcome of the subject's bladder cancer.

[0251] 11. Based on the method in item 9 or 10, where

[0252] (i) CgA levels above a predetermined CgA threshold in the sample indicate a high risk of cancer-related death following surgical and / or pharmacological and / or radiotherapy, and

[0253] (ii) A CgA level above a predetermined CgA threshold in the sample and an MMP7 level above a predetermined MMP7 threshold in the sample or another sample from the same subject indicate a very high risk of cancer-related death and shorter lifespan following surgical treatment and / or drug treatment and / or radiation therapy.

[0254] 12. The method according to any one of items 6 to 11, wherein the levels of CgA and optionally MMP7 are determined at regular time intervals during follow-up, and wherein the control levels are the respective levels determined before and / or at the initial step of the follow-up.

[0255] 13. The method according to Project 4, wherein the bladder cancer is non-muscle-invasive bladder cancer (NMBC) or muscle-invasive bladder cancer (MIBC).

[0256] 14. The method according to any one of items 2 to 13, wherein the sample is derived from a body fluid selected from whole blood, serum, plasma and urine.

[0257] 15. The method of any one of items 2 to 14, wherein the CgA level is determined using an immunoassay.

Claims

1. Use of a chromogranin A (CgA) detection reagent in the preparation of a diagnostic kit for the prognosis of non-neuroendocrine bladder cancer in subjects, wherein the prognosis identifies subjects who will benefit from more aggressive treatment, the method comprising: a) Determine the CgA level in the body fluid sample of the subject. b) Compare the determined CgA level in the sample with a control level or a predetermined threshold. The elevated CgA level in the sample, compared to the control level or a predetermined threshold, indicates that the subject will benefit from more aggressive treatment.

2. The use according to claim 1, wherein the more aggressive treatment is selected from: partial cystectomy instead of transurethral bladder tumor resection (TURBT), triple bladder preservation instead of TURBT, radical cystectomy (RCE) instead of TURBT, RCE combined with pelvic lymph node dissection (PLND) instead of RCE, extended or ultra-extended LND instead of PLND, adjuvant chemotherapy or radiotherapy instead of surgery alone, or additional metastatic tumor resection.

3. The use according to claim 1 or 2, wherein the elevated CgA level in the sample indicates 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 following surgical and / or medical and / or radiotherapy, thereby indicating benefit from more aggressive treatment.

4. The use according to claim 1 or 2, wherein the bladder cancer is selected from bladder urothelial carcinoma, bladder squamous cell carcinoma and bladder adenocarcinoma, preferably wherein the bladder cancer is bladder urothelial carcinoma.

5. The use according to claim 1, wherein the predetermined threshold for the CgA level is selected from 100 ng / mL to 431 ng / mL, preferably from 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.

6. The use according to claim 1 or 2, wherein the sample is derived from a body fluid selected from whole blood, serum, plasma and urine, wherein the body fluid is preferably serum.

7. The use according to claim 1, wherein the level of matrix metalloproteinase 7 (MMP7) is further determined in the sample of the subject, and wherein the level of MMP7 from the sample of the subject indicates the severity and / or outcome of the subject's bladder cancer.

8. The use according to claim 5 or 7, wherein (i) CgA levels above a predetermined CgA threshold in the sample indicate a high risk of cancer-related death following surgical and / or pharmacological and / or radiotherapy, and (ii) A CgA level above a predetermined CgA threshold in the sample and an MMP7 level above a predetermined MMP7 threshold in the sample or another sample from the same subject indicate a very high risk of cancer-related death and shorter lifespan following surgical treatment and / or drug treatment and / or radiation therapy.

9. The use according to claim 7, wherein the levels of CgA and optionally MMP7 are determined at regular time intervals during follow-up, and wherein the control levels are the respective levels determined before and / or at the initial step of the follow-up.

10. The use according to claim 8, wherein the levels of CgA and optionally MMP7 are determined at regular time intervals during follow-up, and wherein the control levels are the respective levels determined before and / or at the initial step of the follow-up.

11. The use according to claim 4, wherein the bladder cancer is non-muscle-invasive bladder cancer (NMBC) or muscle-invasive bladder cancer (MIBC).

12. The use according to claim 1 or 2, wherein the CgA level is determined using an immunoassay.

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