Fig. 4: Dynamic changes in the mutational spectra in response to immunotherapy.

a, Prevalence of mutations per megabase (Mb, y axes) of 500 cancer-related genes in pretherapeutic diagnostic biopsies (left) and resected tumors (right) of two exemplary patients without (001-R-010) and with response (002-R-052) to study therapy. The specific mutations (nucleotide exchanges from C to A (C>A), G (C>G) or T (C>T), from T to A (T>A), C (T>C) or G (T>G), complex nucleotide replacements (complex) or multiple nucleotide variants (MNV)) are color-coded from dark blue to yellow. The minimal VAFs are depicted on the x axes. b, Subclonal dynamics between pretherapeutic biopsies and resected tumors of 14 patients; each line depicts an individual patient. Left, pathological regression (percentage reduction of viable tumor cells) following immunotherapy. Center, estimated total number of subclones in the resected tumor. Right, fraction of subclones enriched (‘fraction gained’) and depleted (‘fraction lost’) in the resected tumors. Fractions are visualized by color (with yellow for high, purple for low), and bubble size (large for high, small for low, no bubble for zero). c, Selection of genomically encoded putative resistance mechanisms in one of 43 patients with pretreatment and posttreatment tumor specimens for genomic analyses. Left, representative microphotographs of the pretherapeutic diagnostic tumor biopsy stained with H&E and with an anti-PD-L1 primary antibody. DNA sequencing of the tumor biopsy revealed pathogenic mutations of KRAS and TP53 and amplification of the CD274 (PD-L1)-encoding gene. Center, low magnification image of a H&E-stained section of the resected tumor showing massive necrosis, but a residual region of vital tumor cells on the left-hand margin. Right, high magnification photomicrographs representing the transition zone from necrotic tumor to residual viable tumor cells stained with H&E and with an anti-CD8 primary antibody demonstrating tumor-infiltrating T lymphocytes. DNA sequencing of the resected tumor confirmed the presence of pathogenic mutations of KRAS and TP53 and amplification of the CD274 (PD-L1)-encoding gene. In addition, copy number gain of MYC and a pathogenic IDH1 mutation were newly detected. A complete list of patients with enrichment of genomically encoded putative resistance mechanisms in resected tumors is presented in Supplementary Fig. 3.