## 3 Discussion  
The investigation of candidate volatile biomarkers for urological cancers has become of great interest over the last years due to a study by [PERSON] et al. which showed that dogs were able to detect BC in urine samples by sniffing [22]. Based on this observation, several researchers have focused on the application of electronic noses, i.e., devices that mimic the human olfactory system, for analysis of volatile metabolites present in urine headspace of cancer patients and cancer-free individuals [23]. Studies performed in urine of BC patients have demonstrated that electronic noses using different technologies can detect BC with sensitivity ranging from 60 to 93% and specificity from 67 to 93% [24; 25; 26]. However, electronic noses are based on the differentiation of odor fingerprints instead of chemical composition, not allowing for the identification of specific biomarkers. Thus, we aimed to identify volatile biomarkers of BC based on the analysis of urine headspace by HS-SPME-GC-MS. Our results unveiled a set of seventeen discriminant volatiles, including several alkanes, aldehydes, aromatic hydrocarbons, heterocyclic compounds, ketones and monoter, able to detect BC with an AUC of 0.851, 70% sensitivity, 89% specificity and 80% accuracy. Comparing with FDA-approved urinary biomarkers, this set of discriminant volatiles  
Figure 3: Heatmap illustrating the levels of urinary volatile metabolites changing between NMIBC (stage Ta/Ts and T1) and MIBC (stages \(\geq\)T2) patients. Columns correspond to each sample group, while rows correspond to the mean normalized peak area of each discriminant volatile metabolite colored from minimum (\(-\)1, dark blue) to maximum (1, dark red). revealed better overall sensitivity and specificity than NMP-22 (69% and 77%, respectively) and BTA TRACK (65% and 74%, respectively) [27]. In contrast with these FDA-approved biomarkers, the set of seventeen volatiles did not show an increase in sensitivity with higher tumor stage, unveiling higher sensitivity for detection of stage T1 (94% sensitivity and 80% sensitivity). This fact may be explained by the distinct urinary volatile profiles found between NMIBC stages (Ta/Ts vs. T1) and NMIBC vs. MIBC (stages \(\geq\)T2), with the latter characterized by higher levels of several alkanes (2,4-dimethylheptane, 4-methyloctane and decane) and aromatic compounds (1,2,3-trimethylbenzene, 1,2,4-trimethylbenzene and 1,2,4,5-tetramethylbenzene), and lower levels of 2,5-dimethylbenzaldehyde. The altered volatile metabolites may also provide new insights into the metabolic dysregulations occurring in BC, as suggested in Table 3 and Tables S3-S5. Several branched alkanes were found increased in urine of BC patients, namely 2-methylnonane, 2,4-dimethylheptane, 2,6-dimethyllnonane and 4-methyloctane. The potential endogenous origin of branched alkanes has been attributed by some researchers to peroxidation of polyunsaturated fatty acids [28], although this hypothesis has been challenged by the fact that there are no branched polyunsaturated fatty acids in the human body [29]. Despite the lack of information regarding the biochemical pathways, 2,4-dimethylheptane and 4-methyloctane were found increased in the headspace of a human non-small cell lung cancer cell culture suggesting a cellular origin [30]. Four aldehydes (2-furaldehyde, 2-methylbutanal, formaldehyde and hexanal) were found statistically decreased in urine of BC patients. The biological origin of 2-furaldehyde is still unknown. The decrease in hexanal and 2-methylbutanal levels may be attributed to oxidation by aldehyde dehydrogenase (ALDH) to yield carboxylic acids or a decrease in aldehyde formation through lipid peroxidation [14]. Hexanal has also been found decreased in urine of patients diagnosed with other cancer types (e.g., colorectal cancer, prostate cancer, leukemia and lymphoma) [31; 32; 33; 34; 35; 36; 37; 38; 39], whereas 2-methylbutanal was found decreased in patients diagnosed with head and neck cancer [37].