PMC10328386_01
Comprehensive analyses of the UTUC lesions from the patient. (A) PET-CT scan performed before surgery for the patient indicating malignant lesions involving left upper-middle ureter and surrounding lymph nodes attaching aorta and psoas major. (B) Surgical specimens of left kidney, ureter, and resected lymph nodes. (C) Pathologic analysis for the primary tumor and staining of markers for tumor subtype, proliferation, EMT, and immune status (in order from top to bottom row). Strongly positive: CK7, CK20, ki-67, p53, EGFR/HER-1, E-cadherin, and PD-L1; mildly positive: HER-2; negative: CK5/6, CK14, PTEN, CD44, N-cadherin, vimentin, and ZEB1; infiltrating lymphocytes: 15% PD-1+, 50% CD3+, 40% CD4+, and 10% CD8+. (D) Transcriptome subtype classification for the patient's primary tumor using TCGA classifier based on RNA sequencing data. Left panel: principal component analysis to visualize separation among subtypes (luminal, luminal-papillary, luminal-infiltrated, basal-squamous, and neuronal) of TCGA UC cohort, and this patient clustered with the luminal-infiltrated subset; right panel: cosine similarity measure classifying the current case into the luminal-infiltrated subtype. (E) Bar charts for GO (left) and KEGG (right) categories significantly enriched in TP53-mutant cases of TCGA-UC dataset. (F) Bar charts for GO (left) and KEGG (right) categories significantly enriched in ERBB2-mutant cases of TCGA-UC dataset. CK, cytokeratin; EGFR/HER, human epidermal growth factor receptor; FDR, false discovery rate; GO, Gene Ontology; HE, hematoxylin-eosin; KEGG, Kyoto Encyclopedia of Genes and Genomes; PD-1, programmed death-1; PD-L1, programmed death-1 ligand; PTEN, phosphatase and tensin homolog; TCGA, The Cancer Genome Atlas; ZEB1, zinc finger E-box-binding homeobox-1.
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Surrounding hypermetabolic lymph nodes extended to paraaortic region, and no distant metastasis was found ( Figure 1A ). Retroperitoneoscopic RNU, regional lymphadenectomy, and cystoscopy were planned and performed, whereas a few paraaortic metastatic lesions were not successfully removed because of deep location and surrounding adhesion ( Figure 1B ). Immunohistochemical (IHC) staining was performed for representative markers of subtypes (cytokeratins), tumor growth (i.e., ki-67 and p53), epithelial-mesenchymal transition (EMT) (i.e., cadherins and vimentin) and immune status (PD-L1 and lymphocyte markers) ( Figure 1C ). UC subset of The Cancer Genome Atlas (TCGA) database was employed to deeply understand the features of this patient. Using TCGA classifier for UC, his lesion was clustered with luminal-infiltrated subtype based on RNA sequencing data ( Figure 1D ). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggested that highly expressed genes in TP53-mutant tumors were significantly associated with biological processes and pathways regarding assembly and repair of DNA or chromatin, i.e., DNA replication, DNA damage response, and mismatch repair ( Figure 1E ;  Supplementary Figure S1 ). Among ERBB2-mutant UC cases, GSEA showed that immune-activating categories [i.e., response to Interferon gamma (INF-gamma), natural killer (NK)-cell activation, and T helper type 1 (Th1)- and T helper type 2 (Th2)-cell differentiation] were highly enriched ( Figure 1F ;  Supplementary Figure S2 ).
Longitudinal surveillance of the patient's treatment course. (A) Preoperative PET-CT and serial MRI scans for monitoring the treatment response of lymphatic metastases at different follow-up time points. (B) Dynamic changes of serum tumor markers, systemic inflammatory indices and ctDNA parameters during the treatment. Postoperative (month 1.5) levels of CEA, CYFRA21-1, and CCF were decreased as compared with baseline values corresponding with surgical resection of primary tumor and most of the lymphatic metastases, whereas increased levels of CA72-4, CA15-3, bTMB, and mVAF were supported by rapid progression of residual metastatic lymph nodes as indicated by MRI scans. Durable response to ICT was supported by constantly ascending NK-cell count and descending trends of all other indices, especially Treg count, bTMB, and mVAF. (C) GEP scores of tumors with different immune status in IMvigor210 cohort with metastatic UC. (D) Overall survival curves for high-GEP and low-GEP subgroups of the anti-PD-1-treated IMvigor210 cohort. bTMB, blood tumor mutation burden; CA, carbohydrate antigen; CBP, carboplatin; CCF, cancer cell fraction; CEA, carcinogenic embryonic antigen; CYFRA21-1, cytokeratin-19 fragments; GEM, gemcitabine; GEP, T-cell-inflamed gene expression profile; ICI, immune checkpoint inhibitor; mVAF, mean variant allele frequency; NK, natural killer; NLR, neutrophil-to-lymphocyte ratio; PLR, platelet-to-lymphocyte ratio; Treg, regulatory T cell.
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Among ERBB2-mutant UC cases, GSEA showed that immune-activating categories [i.e., response to Interferon gamma (INF-gamma), natural killer (NK)-cell activation, and T helper type 1 (Th1)- and T helper type 2 (Th2)-cell differentiation] were highly enriched ( Figure 1F ;  Supplementary Figure S2 ). Magnetic resonance imaging (MRI) showed a significant enlargement of residual lymphatic metastases at six weeks after surgery ( Figure 2A ). Further ctDNA analyses showed a decrease in the cancer cell fraction (CCF) but remarkable increases in blood TMB (bTMB) and mean variant allele frequency (mVAF), which was calculated as the mean of VAFs of somatic mutated genes for each specimen ( Figure 2B ). GEP score in prediction of response to ICI agents. GEP score of this patient was high at 0.14, predicting active immunity and favorable response to PD-1 blockage ( Figures 2C, D ). Meanwhile, ctDNA analyses confirmed sustained decreases in CCF, bTMB, and mVAF ( Figure 2 ).