## 4 Discussion  
The primary option for the treatment of colorectal cancer is surgery. Adjuvant chemotherapy is strongly indicated in stage III disease and in stage II patients at high risk of relapse. Whereas, in low-risk stage II disease decision-making around adjuvant chemotherapy must be carefully evaluated. At present, postoperative surveillance via clinical, radiological and biomarkers examination often cannot identify early metastatic disease and/or postoperative micrometastatic residual disease. Based on these considerations, especially in stage II disease, improved risk-stratification tools are required to identify those patients who are most likely to benefit from adjuvant chemotherapy and need to be followed up more closely after surgery to timely detect systemic recurrence. On the other hand, accurate stratification instruments could prevent low-risk patients from unnecessary treatment and possible mild-to-severe adverse reactions. The analysis described in the present research article shows for the first time, to the best of our knowledge, the metabolomic variations among preoperative and postoperative NMR-based serum fingerprint of CRC patients. Furthermore, metabolomics as novel approach for risk-stratification in CRC setting was evaluated by studying differences between pre- and postoperative serum samples of each patient enrolled. With this innovative approach, each patient in the study population acts as his/her own control, thus eliminating noise from interindividual variability. Our data demonstrate that metabolomics profiles are influenced by the presence or absence of the cancerous mass. Indeed, the mPLS models calculated using each of the three NMR spectra acquired (namely, CPMG, NOESY, and DIFFUSION) show high discrimination accuracies (range 82-85%). This evidence poses an important question in terms of future study design, since sample collection when the tumor was still in place or after resection can significantly impact on metabolomic data. From the univariate analysis, it emerges that after surgery, there is a significant increase of pyruvate, HDL cholesterol, HDL phospholipids, HDL Apo-A1, and HDL Apo-A2. Moreover, we observed, postoperative, a significant decrement of acetone, 3-hydroxybutyrate, LDL-Chol/HDL-Chol ratio, and Apo-A1/Apo-B100 ratio. These data point to a relevant rewiring of the metabolic pathways associated to lipoproteins, ketone bodies, and energy metabolism. Depletion of pyruvate and increase of ketone bodies has been observed in sera of metastatic CRC patients with respect to healthy controls, and this evidence has been associated with an altered energy metabolism, probably reflecting an increased gluconeogenesis and fatty acid oxidation [31]. It is interesting to note that in our dataset, these three metabolites show trend inversions after surgery. Our data show an increase of several HDL-Chol and a decrease of LDL-Chol lipoprotein-related parameters post-surgery. This may be explained by the fact that, after cancer resection, an improvement in the inflammatory status of the gut is achieved, allowing for an improved lipid metabolism and lipid assimilation in the absence of the tumor. Strikingly, despite the low number of recurrence events registered, it is peculiar that the difference in HDL-Chol is particularly marked in relapsed patients and is coupled with a decrease of VLDL-Chol. It has been observed that in colorectal cancerous tissue, the levels of cholesterol and triglycerides were reduced and HDL-Cholesterol level increased, indicating that CRC development destroys the physiological balance of lipids and lipoproteins, leading to lipid metabolic disorders [40]. Preclinical and clinical studies have already investigated the role of cholesterol in CRC progression; however, a clear understanding of the molecular mechanism linking these two entities is still lacking [40; 41].