This is a retrospective observational historical-matched cohort study using clinical data from six hospitals of the Santeon hospital network.
The Santeon hospital network consists of seven large teaching hospitals and covers more than 11% of all the hospitalizations in the Netherlands. The Netherlands Cancer Registry (NCR) was used for identifying all patients diagnosed with NSCLC and for obtaining information on the date of diagnosis and the vital status. Individual patients are assigned a unique anonymous identifier, which enables them to be tracked in the Santeon Farmadatabase (SFD). The information of the SFD was used for collection of detailed information about the systemic treatments.  Finally, the patients’ medical records were used to complement the database with detailed information about the clinical and demographic characteristics, the use of concomitant medication and the treatment response. All data were gathered and stored at a Research Electronic Data Capture database (REDcap). 
Patients diagnosed with stage IV NSCLC between 1st January 2015 to 1st January 2019 and who started first-, second- or third-line immunotherapy before the 1st of January 2020 were assigned to the immunotherapy group. We matched every patient in the immunotherapy group to a patient with stage IV NSCLC who received conventional chemotherapy in the pre-immunotherapy era, and has been diagnosed before 1st January 2015 (see publication of Cramer et al for more details about this cohort) . Patients were (1:1) matched on gender, age groups (< 50 year, 50–60 year, 61–70 year, 71–80 year, > 80 year) and line of treatment (first, second or third).
The patient characteristics and demographics were collected manually from the patients’ medical records, including age, gender, body mass index (BMI), Eastern Cooperative Oncology Group-Performance status (ECOG-PS), the histology subtype, brain metastases and lines of systemic treatments. First-line treatment (1L) was defined as the initial systemic therapy used in the treatment of NSCLC. Second-line and third-line treatment (2L and 3L) were defined as the therapy given after discontinuation due to disease progression or completion of first or second-line treatment, respectively. Additionally, oncogenic driver mutation status (e.g. EGFR, ALK, ROS-1) and PD-L1 expression were collected if available.
Patient records were reviewed to collect information on the use of concomitant medication potentially affecting the microbiome  . The medication classes collected were antibiotics, proton-pump inhibitors, metformin, antidiabetics and opioids. Exposure was defined if any information on the use of these drugs was reported by the physician in the medical health record within a timeframe of one month before until one month after the start of the systemic therapy.
OS was defined as the time from the start of systemic therapy to death. Patients still alive at the end of follow-up on 1 January 2020 were censored at this date.
Statistical Software (SPSS version 26 for Windows: IBM) was used for statistical analysis. Categorical and continuous variables were summarized using descriptive statistics. To compare the immunotherapy and chemotherapy group, we used chi-squared tests (categorical variables) and independent t- test (continuous variables). The potential impact of concomitant medication on OS was analyzed through multivariable cox regression analyses. Possible factors associated with OS were first identified using a univariable analysis. All univariate predictors with a p-value ≤ 0.15 and three other relevant variables - type of treatment, the ECOG-PS and the use of antibiotics - were used to construct the multivariable model. In the final models, backward selection was applied to eliminate non-significant variables (p-value ≤ 0.10). Finally, the models were examined for the existence of effect modification by statistical testing of an interaction term between concomitant drugs of interest and the type of treatment (chemotherapy or immunotherapy). In order to investigate the difference between the lines of treatment, an exploratory analysis was performed for 1L patients and for 2L + 3L patients using the same approach described above. Survival curves using the Kaplan-Meier method were constructed to visualize - where considered of relevance- contrast between the use of concomitant medication and the type of treatment (chemotherapy or immunotherapy).
All methods were carried out in accordance with relevant guidelines and regulations. Our ethics committee - the Santeon institutional review board - approved the study (SDB 2019-013) and waived informed consent. The need for informed consent was waved because of anonymous data handling and the retrospective nature of the study. The study was performed in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.