Discovery of Volatile Biomarkers for Bladder Cancer Detection and Staging through Urine Metabolomics  
[PERSON]  
1 UCIBIO/REQUIMUTE, Department of Biological Sciences, Laboratory of Toxicology, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal, [EMAIL_ADDRESS] (F.A. ); [EMAIL_ADDRESS] (A.R.L. ); [EMAIL_ADDRESS] (M.d.L.B. )2 Cancer Biology & Epigenetics Group-Research Centre, Portuguese Oncology Institute of Porto (CI-IPOP), 4200-072 Porto, Portugal; [EMAIL_ADDRESS] (C.C.-M.); [EMAIL_ADDRESS] (M.C.M. ); [EMAIL_ADDRESS] (C.J. ); mmhenrique@icabs.upt (R.H.)3 Department of Pathology, Portuguese Oncology Institute of Porto, 42 strategies, including trans-urethral resection, complemented with intravesical immunotherapy or chemotherapy, whereas MIBC patients are treated with radical cystectomy plus neoadjuvant or adjuvant chemotherapy or, eventually, immunotherapy [4]. Hematuria (gross or microscopic) is the most common clinical manifestation of BC, occurring in 80-90% of patients [4]. Current standard diagnostic methods include cystoscopy, urinary cytology and imaging techniques (computed tomography (CT) and magnetic resonance imaging (MRI)) [5]. Cystoscopy is an invasive procedure which directly visualizes the bladder lining for presence of a tumor, with overall reported sensitivity and specificity of 68-100% and 57-97%, respectively [6]. This procedure can lead to urethral injury, urinary tract infection and hematuria. Urinary cytology, which detects cancer cells in urine, is a non-invasive method particularly useful as a complement to cystoscopy, with sensitivity and specificity for BC detection ranging 13-86% and 73-100%, respectively [6]. Besides the very low sensitivity for detection of low-grade tumors, this technique is operator dependent and can be hindered by low cellular yield, urinary tract infection and other comorbidities [5]. CT and MRI are mainly used for staging and evaluation for distant metastases, but concern remains regarding their ability to detect flat urothelial lesions, and accurately perform local-staging and nodal detection [7]. Urinary biomarkers represent an attractive alternative or adjunct to current BC standard diagnosis. An ideal BC biomarker should be non-invasive, objective, easy to interpret and have high sensitivity and specificity [8]. There are some urine tests currently approved by Food and Drug Administration (FDA) based on altered proteins [e.g., nuclear matrix protein 22 (NMP-22), complement factor H-related protein (BTA TRACK)] or chromosomal aberrations (e.g., UroVysionTM) [6; 9]. However, these tests have not replaced the current standard diagnostic methods as they are affected by hematuria, infection or inflammation leading to false-positive results [9]. In the era of precision medicine, multiple levels of molecular profiling (e.g., genome, epigenome, transcriptome, proteome, metabolome) of BC have been investigated for accurate diagnosis, prognostication, and prediction of treatment response [9]. In particular, the volatile fraction of the urine metabolome has shown very promising results for diagnosis of prostate and gastrointestinal cancers, as recently reviewed [10]. However, only one study has explored the urinary volatilome for identification of potential biomarkers for BC [11]. This study reported 89% sensitivity and 66% specificity, with overall 83% accuracy and area under the curve (AUC) of 0.868, for discrimination of BC (\(n=72\)) from cancer-free controls (\(n=46\)) based on the alterations in the levels of several ketones, aldehydes, acids and monoterenes. Herein, we aimed to investigate the performance of urinary volatilome in discriminating patients diagnosed with BC from cancer-free controls, based on the analysis of urine by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Because BC is more often diagnosed in men than women, urine samples from BC and control groups were gender matched.