Differences of T-cell subtypes and TMB between extracranial and intracranial samples were calculated by subtracting the results of the extracranial samples from the results of the matched intracranial sample of the same patient. Therefore, a positive difference indicated a higher value in extracranial samples and a negative difference a higher value in intracranial samples. The median difference was used as a cut-off to explore survival correlation.

Data normality was tested using the Shapiro–Wilk test. Differences between two groups were calculated by unpaired, two-sided Mann–Whitney U tests and differences between more than two groups were calculated by Kruskal–Wallis tests.

In order to obtain information on the association of the inflammatory microenvironment in BM and the brain-metastatic-free survival, we used Cox regression models to receive hazard ratios for the differences from extracranial to intracranial TIL subsets and TMB. Brain-metastatic-free survival was defined as time from diagnosis of primary tumor to radiological diagnosis of BM.

A two-sided P value of <0.05 was considered to indicate statistical significance. Due to the hypothesis-generating approach of the current paper, no correction for multiple testing was applied.9

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