case_images: PMC10570427_01
case_images: (A) The level of beta-HCG during the treatment. (B) Computed tomography images of the lesions during the treatment.
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case_images: The tumor markers examination showed a significant increase in serum beta-HCG level of 6080.2 IU/L ( Figure 1A ) (normal range<5.0 IU/L). C-reactive protein, 52.3 mg/L (normal range<3 mg/L). CT of the abdomen revealed gallbladder cancer with invasion of the adjacent liver and multiple metastases in the liver ( Figure 1B ). No tumorous lesion, suggestive of a primary site, was identified in organs of the reproductive system by radiography ( Supplementary Figure 1 ). Contrast-enhanced CT was performed regularly, and the results suggested that the size of metastases and the extent of invasion were smaller than before ( Figure 1B ). To date, there was no evidence of tumor recurrence ( Figure 1B ) and the serum beta-HCG level remained in the normal range ( Figure 1A ). As shown in  Figure 3B , MYC targets v1, mitotic spindle, DNA repair, PI3K/AKT/mTOR signaling, and unfolded protein response of gene sets were expressed in relatively higher levels in the present case than gallbladder adenocarcinoma without HCG.
case_images: Pathological findings. (A) The metastatic lesion shows poorly differentiated carcinoma with large-scale necrosis (H&E x200). (B) Almost all cancer cells show a positivity for HCG (x200). (C) Cancer cells show a strong positivity correlation for PD-L1 (x400). (D) The resected gallbladder shows no tumor cells with infiltration of lymphocytes and plasma cells (left: x40; right: x200). (E) Immunofluorescence staining of CD4 (green) in combination with CD3 (red), CD20 (pink), and FOXP3 (rose red) (x40). (F) Immunofluorescence staining of CD68 (green) in combination with CD8 (red), CD163 (pink), and CD56 (rose red) (x40).
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case_images: Histopathological analysis of liver metastases showed poorly differentiated carcinoma with large-scale necrosis ( Figure 2A ). Immunohistochemical detection of various markers was further performed on the tumoral tissue and revealed strong positivity for HCG in most tumor cells ( Figure 2B ). The infiltration of large amounts of lymphocytes and plasma cells was observed in the specimen ( Figure 2D ). Immunofluorescence staining showed positivity for CD4, CD8, and CD68 ( Figures 2E, F ). Immunostaining showed tumor cells were positive for PD-L1 (Tumor cell Proportion Score, TPS >= 50%) ( Figure 2C ). As shown in  Figure 3B , MYC targets v1, mitotic spindle, DNA repair, PI3K/AKT/mTOR signaling, and unfolded protein response of gene sets were expressed in relatively higher levels in the present case than gallbladder adenocarcinoma without HCG.
case_images: Immune microenvironment of GBC producing HCG. (A) Volcanic map for differentially expressed genes. (Red dots: significantly upregulated genes. Blue dots: significantly downregulated genes. Grey dots: non-differentially expressed genes.) (B) Heatmap showing the relative expression levels of genes in GBC with ectopic HCG and gallbladder adenocarcinoma without HCG. (C) Heatmap showing the relative abundances of infiltrating immune cells in GBC with ectopic HCG and gallbladder adenocarcinoma without HCG. (D) The proportions of infiltrating immune cells in GBC with ectopic HCG and gallbladder adenocarcinoma without HCG. (P5: GBC with ectopic HCG, P1-P4: gallbladder adenocarcinoma without HCG).
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case_images: TIME features between GBC producing HCG and those without HCG, RNA-seq was performed, which refers to the use of high-through sequencing for cDNA sequencing to comprehensively obtain almost all transcripts of tissues of a tumor. The scatter plot was shown to demonstrate the variation of gene expression between two types of tumors ( Figure 3A ). As shown in  Figure 3B , MYC targets v1, mitotic spindle, DNA repair, PI3K/AKT/mTOR signaling, and unfolded protein response of gene sets were expressed in relatively higher levels in the present case than gallbladder adenocarcinoma without HCG. T cell gamma delta (4.5%), eosinophil (4.2%), and M1 macrophage (14.3%) than common gallbladder adenocarcinoma without HCG ( Figure 3C ). The dominant immune cell types in our case were macrophage and CD4+ T cells ( Figure 3D ).