# A Serum Metabolomic Signature for the Detection and Grading of Bladder Cancer
## 4 Discussion  
Here we report the diagnostic performance of three machine learning ensemble models derived from the statistical comparison of the serum metabolomic fingerprints of subjects with and without BC. Detailed comparison of many hundreds of serum metabolites illustrates biochemical differences in patients on the basis of presence or absence of BC and, among the BC cases, by grade (high grade vs. low grade), showing an accuracy of 99% and 80%, respectively. The same approach was not able to discriminate between patients based on muscle invasiveness of the cancer. Results presented herein indicate that several serum metabolites and metabolic pathways are associated with BC. Analysis of the network of metabolic pathways that connect molecules, combined with a powerful machine learning algorithm, allows group separation and offers a new method to noninvasively screen for BC, as well as provide new biochemical insights based on the affected metabolomic pathways. The BC metabolome was characterized by lower levels of glucose, citric acid, and glutamine. On the contrary, lactic acid, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate increased in BC patients. The resultant low glucose and high lactic acid due to cancer metabolism has been established since 1920 [29]. The Nobel laureate [PERSON] was the first researcher who speculated about the advantages of anaerobic glycolysis for cancer due to the higher speed of this metabolism, and the low oxygen availability during rapid cancer growth [30]. Furthermore, relatively recent evidence indicates that bypassing the tricarboxylic acid cycle (TCA) is an efficient strategy for cancer cells which effectively mitigates ROS production and reduces cancer cell apoptosis [31]. Reduction of different glycolytic enzymes have been reported in tumors, including bladder cancer [32; 33; 34]. This results in unfettered energy production for tumor cells. Many cancer cells are able to subvert the feedback mechanisms that normally allosterically inhibit rate-controlling enzymes in glycolysis. In a normal cell, phosphofructokinase (PFK) is inhibited by ATP, limiting the glycolytic pathway when the cell is rich in energy. In cancer cells, high glucose concentrations increase the synthesis of fructose 2,6-bisphosphate from fructose 6-phosphate by 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases. Fructose 2,6-bisphosphate can override ATP-mediated PFK inhibition via the Warburg effect, whereby, increased expression of glucose transporters [35] as well as high activity of hexokinase [36] increases the concentration of fructose 2,6-bisphosphate which allosterically activates PFK. In addition, the specific PFK isozymes overexpressed in cancer cells are less sensitive to the ATP inhibition [37]. As a result of this poor ATP sensitivity, both dihydroxyacetone phosphate and 3-phosphoglyceraldehyde were up-regulated as reported in our metabolomics signature (Figure 5). Due to the Warburg effect, glycolysis does not proceed via the TCA, while pyruvate is shunted through lactate production aided by increasing lactate dehydrogenase (LDH) expression. LDH was considered a poor prognostic marker in BC because it reflects both high cytolysis levels and the high shunting rate of pyruvate [38]. These characteristic cancer biochemical pathways are represented by the serum metabolome described herein. Moreover, the elevated levels of phenylpyruvate and phenylacetate we report could be the result of the increased expression of LDH. The high reported levels of lactic acid in our metabolomics signature could also reflect a more sophisticated cancer metabolic pathway. Lactic acid could also affect the T-cells adjacent to the cancer. The activation of these cancer-associated immune cells depends on anaerobic glycolytic metabolism, leading to high production and excretion of lactic acid. The increased extracellular levels of lactic acid contrast that produced by the T-cells, in an against-gradient force [39; 40; 31] which slows the T-cell activation.