## 2 Material and Methods  
### Patients and Healthy Donors  
In this prospective study, 20 patients with a histologic diagnosis of NSCLC, UC, RCC, or squamous cell carcinoma of head and neck (SCCHN) who were at the starting point of their treatment with a PD-1 inhibitor, either nivolumab or pembrolizumab, were enrolled. The study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization for Good Clinical Practice standards of care and was approved by the University Hospital of Patras Ethics Committee. A written informed consent was provided from the participants before their enrolment. Our patients' median age was 58 years (range 43-80), all of whom were Caucasians, and the male-to-female ratio was 14 to 6. The cancer diagnosis was as follows: fourteen patients were diagnosed with non-small-cell lung cancer (NSCLC), four with urothelial carcinoma (UC), one with renal cell carcinoma (RCC), and one with head-neck cancer of squamous cells (SCCHN). Patients' demographics are shown in Table 1. All study subjects included in the study were patients who needed treatment with monotherapy with immune checkpoint inhibitors as a second- or third-line treatment option. All patients were enrolled sequentially at recruitment and were not selected based on their cancer type. More patients included had NSCLC, since this type of cancer is more common in our Oncology department. None of the patients had past medical history of autoimmune disease. None of the patients were treated with corticosteroids at least 6 months before study enrolment [47]. Additionally, eight age- and sex-matched healthy donors with no history of malignancy or autoimmune disease were enrolled, representing the control subject group.  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{2}{*}{**Number**} & \multicolumn{2}{c}{**Patient or**} & \multirow{2}{*}{**Gender**} & \multirow{2}{*}{**Age**} & \multirow{2}{*}{**Malignancy**} & \multirow{2}{*}{**CPI**} & \multicolumn{2}{c}{**Line of**} & \multicolumn{2}{c}{**Timepoint of Last**} & \multirow{2}{*}{**Outcome**} & \multicolumn{2}{c}{**irAEs/**} \\  & & & & & & **Treatment** & & & **Evaluation** & & **Autoimmunity** \\ \hline
1 \(\dagger\) & Pt & M & 62 & Uca & pembrolizumab & 2 nd & 2 & PD & - \\ \hline
2 \(\dagger\) & Pt & M & 65 & NSCLC & nivolumab & 2 nd & 2 & PD & - \\ \hline
3 \(\dagger\) & Pt & F & 47 & NSCLC & nivolumab & 3 rd & 4 & PD & - \\ \hline
4 \(\dagger\) & Pt & M & 72 & NSCLC & nivolumab & 2 nd & 2 & PD & - \\ \hline
5 & Pt & M & 70 & NSCLC & nivolumab & 2 nd & 2 & R & - \\ \hline
6 \(\dagger\) & Pt & M & 54 & NSCLC & nivolumab & 2 nd & 3 & PD & - \\ \hline
7 & Pt & M & 65 & Uca & pembrolizumab & 2 nd & 4 & R & - \\ \hline
8 \(\dagger\) & Pt & M & 75 & Uca & pembrolizumab & 2 nd & 1 & PD & - \\ \hline \hline \end{tabular}
\end{table}
Table 1: Demographics of study subjects. Patients’ (Pt) and healthy donors’ (HD) demographics are shown in the table along with evaluation of response during the first 6 months of CPI treatment (irAEs: immune-related adverse events, PD: progressive disease, R: response; complete response; partial response; stable disease, M: male, F: female, \(\dagger\): indicates deceased patients, ANA: antinuclear antibody).