Patients

A male in his late 60s with advanced melanoma underwent surgical resection and received anti–PD-1 mAb at Yamanashi University Hospital. The metastatic lesion was resected en bloc and found to be comprised of two lesions separated by connective tissue from which LN1 and LN2 samples were separately obtained (Fig. 1A). Each tumor-infiltrating lymphocytes (TIL) and DNA extracted from resected samples were used for WES and single-cell sequencing. A male in his middle 60s with advanced gastric cancer received anti–PD-1 mAb as third-line therapy at National Cancer Center Hospital East (Supplementary Fig. S1). Autopsy samples were used for WES and TCR sequencing. We obtained written informed consent for analyses. In addition, clinical data of various cancer patients who received PD-1 blockade monotherapies without any cytotoxic chemotherapies were analyzed to evaluate mixed responses in this study (Supplementary Tables S1–4). The protocols for these studies were approved by the appropriate institutional review board and ethics committees at the Yamanashi University Hospital, Chiba University Hospital, Shinshu University Hospital, Saitama Medical University International Medical Center, Okayama University Hospital, National Cancer Center Hospital East, and Chiba Cancer Center. This study was conducted in accordance with the Declaration of Helsinki.

There were different tumor cell clones between LN1 and LN2. A, CT and the pathology of a male in his late 60s. Before the initiation of treatment, LN1 and LN2 were located next to each other (arrows), which were subsequently resected surgically. Computed tomography (top) and HE staining (bottom) are presented. Scale bars, 2 mm. B, IHC. FFPE sections (3 μm) from LN1 and LN2 were stained. Representative CD8 and PD-1 double staining and the quantified summary are presented. Scale bars, 50 μm. C, Representative driver gene alterations. Extracted DNA from LN1 and LN2 were sequenced and representative driver gene alterations are presented. CN, copy number. D, Clonal evolution of tumor cells. The cellular prevalence of clones carrying individual nonsynonymous mutations in LN1 and LN2 was determined using PyClone. The determined cellular prevalence was used as input, and the phylogenetic relationships of clones were inferred with LICHeE. The means and SDs are shown. A t test was used to calculate statistical significance in B. **, P < 0.01.