## 2 Results
### Fecal Metabolomic Profile  
In parallel, responders and non-responders were compared in terms of their baseline fecal metabolomic profiles. Similar to the GM data, PCoA of beta diversity based on Euclidean distances revealed significant segregation based on therapeutic response (\(p=0.05\), Adonis) (Figure 2A), with non-responders also showing significantly higher within-group variance (\(p<0.001\), Wilcoxon test) (Figure 2B). The discriminating metabolites between groups are reported in Table 3 (\(p\leq 0.05\)). Notably, butyric acid and its derivatives (i.e., methyl and propyl esters) were significantly over-represented in the non-responders (\(p\leq 0.02\)). \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Characteristic** & **Overall** & **Responders** & **Non-Responders** & **\(p\) Value** \\  & **(n = 31)** & **(n = 18)** & **(n = 13)** & **\(p\) Value** \\ \hline Physical activity, n (\%) b & & & & 0.1516 \\ \hline Low & 14 (45) & 6 (43) & 8 (57) & \\ Moderate & 11 (36) & 9 (82) & 2 (18) & \\ High & 6 (19) & 3 (50) & 3 (50) & \\ \hline Fiber: EPIC FFQ c & & & & 1.000 \\ \hline \(<\)25 g/die & 17 (65) & 12 (71) & 5 (29) & \\ \(\geq\)25 g/die & 9 (35) & 6 (67) & 3 (33) & \\ \hline Italian Mediterranean Index c & & & & 0.0838 \\ \hline
0–3 & 11 (42) & 10 (91) & 1 (9) & \\
4–11 & 15 (58) & 8 (53) & 7 (47) & \\ \hline BMI, body mass index; CT, computed tomography; FFM, fat-free mass; FPQ, food frequency questionnaire; IMAT, intramuscular adipose tissue; SAT, subcutaneous adipose tissue; SD, standard deviation; SM, skeletal muscle; TAT, total adipose tissue; VAT, visceral adipose tissue; VATSAT, the ratio of visceral to subcutaneous adipose tissue. \({}^{\mathrm{a}}\) Data are missing for six patients. b Assessed using the International Physical Activity Questionnaire. c Data are missing for five patients. & \\ \hline \hline \end{tabular}  
* Data are missing for six patients. b Assessed using the International Physical Activity Questionnaire. c Data are missing for five patients.  
\end{table} 
Table 2: \(Cont.\)Figure 1: Baseline gut microbiota in advanced melanoma patients in relation to therapeutic responses. (**A**) PCaA based on the Jaccard similarity index between the gut microbiota profiles of responders and non-responders. Ellipses include a 95% confidence area based on the standard error of the weighted average of sample coordinates. Significant separation between groups was found (\(p\) = 0.034, Adonis). (**B**) Boxplots showing the relative abundance distribution of genera differentially represented between groups. Wilcoxon test, * for \(p\) < 0.05, ** for \(p\) < 0.01. For _Phuscolarctobacterium_, only a non-significant trend of \(p\) = 0.06 was found. \begin{table}
\begin{tabular}{c c c c} \hline
**Metabolite** & **Non-Responders** & **Responders** & \(p\)**Value** \\ \hline Butanoic acid, methyl ester & 0.47 \(\pm\) 0.1 & 0.013 \(\pm\) 0.0063 & 0.002 \\
1-Hexanol, 2-ethyl- & 0.034 \(\pm\) 0.01 & 0.15 \(\pm\) 0.015 & 0.003 \\
2-Heptano, 6-methyl- & 0.031 \(\pm\) 0.0045 & 0.0069 \(\pm\) 0.0025 & 0.006 \\ Cyclohexancellor acid, ethyl ester & 0.41 \(\pm\) 0.12 & 0.0082 \(\pm\) 0.0029 & 0.007 \\ Butanoic acid & 25 \(\pm\) 4.3 & 5.7 \(\pm\) 0.63 & 0.02 \\ Butanoic acid, propyl ester & 1.3 \(\pm\) 0.36 & 0.1 \(\pm\) 0.035 & 0.02 \\
2H-Indol-2-one, 1,3-dihydro- & 0.11 \(\pm\) 0.037 & 0.12 \(\pm\) 0.011 & 0.02 \\
2-Hexanone & 0.076 \(\pm\) 0.022 & 0.18 \(\pm\) 0.021 & 0.04 \\
5,9-Undecadien-2-one, 6,10-dimethyl- & 0.13 \(\pm\) 0.024 & 0.34 \(\pm\) 0.047 & 0.04 \\ Anethole & 0.14 \(\pm\) 0.05 & 0.86 \(\pm\) 0.26 & 0.05 \\ Propanoic acid, ethyl ester & 0.12 \(\pm\) 0.037 & 0.007 \(\pm\) 0.0035 & 0.05 \\ \hline \end{tabular}
\end{table} 
Table 3: Fecal metabolites differentially represented at baseline in advanced melanoma patients in relation to therapeutic response.