# A Serum Metabolomic Signature for the Detection and Grading of Bladder Cancer
## 1 Introduction  
Bladder cancer (BC) was the third most frequent cancer in Europe in 2018 (excluding skin cancers), with an estimated incidence in the same year of about 197,100 new cases and a mortality rate of 3.4% for both sexes. The incidence in men is 3-4 times higher than in women [1]. The highest incidence occurs in people over 60 years old [2]. In addition to age, other risk factors for the development of BC include cigarette smoking, chronic exposure to potential carcinogens (such as industrial chemicals: aromatic amines and nitrosamines), family history of concordant cancers [3; 4], pelvic radiation, pharmacological therapy with cyclophosphamide and thiazolinediones, chronic infections [5] and long-term consumption of arsenic-contaminated or chlorinated water [3; 4].  
Bladder cancer usually begins in transitional cells belonging to the urothel, i.e., the inner lining of the bladder. This transitional cell carcinoma (TCC), or urothelial carcinoma, is the most frequent type [6]. Starting from the urothel, the cancer can spread into the bladder wall and reach adjacent tissues and organs [7]. Based on the degree of invasion, BC is classified into NMIC (non-muscle-invasive cancer) which is confined to the mucosa and lamina propria of the bladder, and therefore does not invade the muscle (stages Ts, Ta, T1) and MIC (muscle-invasive cancer) which extends into the muscle (stage T2), into the perivescical fat layer (stage T3), adjacent tissues (stage T4), or ultimately metastas to distant organs (stage M1) [6]. BC can also be classified according to the cellular morphology and differentiation: well-differentiated (G1), moderately differentiated (G2) and poorly differentiated (G3) tumor. Tumor grade is an important factor that can affect prognosis and treatment options because G1 and G2 (classified as low-grade tumors) show better prognosis and are less likely to become invasive, while G3 (high grade tumors) are more aggressive [6]. In most patients with BC (approximately 85%), the initial symptom of the disease is hematuria which is usually intermittent and painless. In a lower percentage of cases (approximately 20%), the symptomatology consists of bladder irritability: urination frequency, urgency, and dysuria [5]. In patients with more advanced stages, other symptoms may be flank pain secondary to lymphadenopathy, ureter obstruction, and less specific symptoms such as weight loss, fatigue, and anorexia [8]. About 80% of newly diagnosed cases are classified as NMIC. Compared to MIC, these tumors have a better prognosis and consequently a significantly higher 5-year survival rate (\(\sim\)90% for NMIC and \(<\)50% for MIC) [3; 8]. However, patients with NMIC require a lifetime of follow-up by cystoscopy because the recurrence rate is very high. Approximately 50-70% of NMICs recur during the first two years after diagnosis with a 10-20% risk to progress invasively within 5 years [3; 9]. Prognosis and treatment of BC depend on various factors such as: stage, grade, number and size of tumor(s), whether this is an initial tumor or a recurrence, patient age, and general health conditions. Generally, NMICs are treated locally by transurethral resection (TUR), often accompanied by intravesical therapy with chemotherapeutic agents or immunotherapy [9]. However, patients with MIC need a radical cystectomy, generally followed by fairly aggressive systemic therapies [10]. Nevertheless, the 5-year survival rate for MIC remains about 50-70% [10; 11]. Biochemical differences between MIC and NMIC as well as among MICs, are hard to describe due to significant heterogeneity. Despite abundant studies describing these differences, it was not until 2020 that a molecular classification, based on transcriptomics profiling for MIC was proposed [12]. Herein, we report the results of a metabolomics-based profiling of bladder cancer to elucidate the metabolic changes generated by bladder cancer, stratifying the disease by stage and grade. We describe a potential non-invasive approach for BC screening and stratification.