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case_images: Treatment history and initial biological findings of the patient. (A). Timeline of treatment and radiological findings. Hematoxylin-eosin(HE) staining before treatment showed large tumor cells. The patient received the pemetrexed and cisplatin plus Keytruda (AP+K) regimen, and tumor shrinkage and partial response was observed. Thereafter, progression occurred within a short time during immune-maintenance therapy, and the tumor recurred and metastasized soon after surgical treatment. HE staining of the primary tumor foci and the 7th station of mediastinal lymph node metastases after surgery showed many tumor cells without immunotherapy-related pathological reactions. After one cycle of the second-line docetaxel and cisplatin plus Keytruda (DC+K) regimen, postoperative RNA-based NGS of tumor tissue showed CD47-MET fusion. The patient received savolitinib treatment and complete imaging remission was achieved. Unfortunately, in October 2023, the patient experienced recurrence and progression. Subsequent biopsy of the lung metastases revealed adenocarcinoma characterized by CD47-MET fusions, MET-ATP13A5 fusions, and a MET p.D1228H mutation. Consequently, the patient was initiated on a third-line therapy comprising a combination of albumin-bound paclitaxel, carboplatin, and bevacizumab (TC+B). The red arrows show the tumor regions. (B) PD-L1 immunohistochemistry (IHC) results of tumor tissues before treatment and after surgery. PD- L1 expression was both positive before and after treatment, and the PD-L1 [22C3] TPS in both cases was approximately 5%.
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case_images: Figure 1B ). Following four cycles of chemotherapy combined with pembrolizumab immunotherapy, a partial response was achieved. However, the lesion exhibited slow growth during the immunotherapy maintenance phase ( Figure 1A ). Table A1 ). Tumor mutation burden was 2.99 Muts/Mb while PD-L1 TPS was 5% in postoperative tumor tissues ( Figure 1B ). Unfortunately, new nodes in the right temporal lobe, intrapulmonary and mediastinal, showed suspicious metastases 1 month after surgery ( Figure 1A ). The lung and head lesions shrank and disappeared in 4 months ( Figure 1A ). Within 7 months, blood carcinoembryonic antigen (CEA) decreased to become normal, achieving complete remission ( Supplementary 3 Figure A1 ). Unfortunately, the patient was found to have significantly elevated CEA in October 2023, and chest CT suggested recurrence of the operative stump with new metastases in the mediastinal lymph nodes, both intrapulmonary, right pleura, and chest wall ( Figure 1A ). Following two treatment cycles, the lymph node and bilateral lung metastases exhibited complete regression, accompanied by a normalization of CEA levels ( Figure 1A ).
case_images: Biological examination of MET fusion. (A) Sequence diagram and structural schematic of MET fusion and MET p.D1228H mutation. Exon 5 of the CD47 gene and exon 15 of the MET gene were fused. The fusion gene retains the 5' end of CD47 and the 3' end of MET gene, which are thought to form a functional fusion protein at the RNA level. The fusion gene contains the regulatory dimerization domain (RDD) of the CD47 gene and the intact TKD of the MET gene. RDD drives the constitutive activation of the TKD, which theoretically produces kinase proteins that activate the downstream signaling pathway of MET
  (A1, 2). However, the MET p.D1228H mutation, which is in the kinase domain of the MET gene, confers resistance to TKIs (A3). Nevertheless, the fusion event between ATP13A5 and MET occurs within the promoter region and is not anticipated to result in the formation of a novel fusion protein (4A). (B) Results of MET protein IHC assay and FISH assay. (B1) Tumor showing invasive growth and composed of solid (~ 70%), cribriform (20%), and acinar (10%) architecture, with a scattered signet ring cell carcinoma component. (B2) Met protein IHC assay showing c-Met protein expression exhibiting a moderate to strong positive staining pattern in the cell membrane and is homogeneous among morphological variants. (B3)
  MET break-apart signals in 65% of tumor cells, demonstrating C-MET gene translocation. (B4) No heterogenous break-apart percentage in different areas of the histological variants. A normal MET/CEP7 ratio (1.6) and no copy number gain of MET gene (2.4 copies per cell) is present based on counting 50 tumor cells within the tumor area, suggesting no C-MET gene amplification.
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case_images: To identify any potential driver gene mutations, we performed RNA-based NGS (Burning Rock Biotech company of Chengdu China, OncoRNA assay) on the postoperative specimen with detection of a rare mutation of CD47-MET fusion ( Figures 2A1, A2 ). B2 ). Fluorescence in the situ hybridization (FISH) assay validated the C-MET translocation ( Figure 2B3 ) and no copy number gain of MET, suggesting no C-MET amplification ( Figure 2B4 ). Because the benefits of chemotherapy and immunotherapy are limited, savolitinib, a new MET-TKI, was prescribed. A repeat puncture biopsy of lung metastases showed adenocarcinoma, while DNA NGS (GenePlus company of Chengdu China, Gene+Seq2000, panel of 1021 genes) combined with RNA NGS (GenePlus company of Chengdu China, tumor-related RNA gene test platform) suggested CD47-MET fusion with MET p.D1228H mutation (55.1%) ( Figure 2A3 ). Additionally, the RNA NGS analysis revealed a novel MET-ATP13A5 fusion event that was not predicted to result in a functional protein product ( Figure 2A4 ).
case_images: Results of multiplex immunofluorescence detection of tumor microenvironment. (A-C) In general, CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), and M2-type tumor-associated macrophage (TAM) cells (CD68+CD163+); Treg cells (CD3+CD4+FOXP3+); and NK cells (CD16+CD56+) had a significantly lower percentage of infiltration in the tumor area than in the tumor stroma. CD4+ T cells, CD8+ T cells, M2 type TAM cells, PD-L1, and TIM3 had heterogeneous distribution in different tumor areas; (D), respectively, in HE images, mIF bright field, and mIF fluorescence maps delineating ROI 1 and ROI 2; (E1), the infiltration density of CD4+ T cells, CD8+ T cells, and Treg cells within ROI 2 was significantly higher than that in ROI 1. Expression of PD-L1 and TIM3 within ROI 2 was significantly higher than that in ROI 1; (E2) CD4+ T cells, CD8+ T cells, Treg cells, and M2 TAM infiltrated the peripheral stroma region more significantly than the tumor region in ROI 1, but the distribution was reversed in ROI 2.
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case_images: CD47-MET fusion and immunogenic resistance, we performed mIF (multiple immunofluorescence) assays on the surgical resection specimens. Compared to paraneoplastic tissue, the infiltration ratio of CD4+ T, CD8+ T, and M2-type tumor-associated macrophages (TAM), T-regulatory (Treg), and NK cells was relatively low in tumor tissue, whereas the infiltration of immune cells in different tumor regions presented with significant heterogeneity ( Figures 3A-C ). We identified two tumor regions with different degrees of immune cell infiltration in the mIF images:region of interest (ROI) 1 and ROI 2 ( Figure 3D ), and then we compared various immune cells' infiltration densities in the two regions. T, and Treg cells, excluding M2-TAMs, within ROI 2 were considerably higher than those of ROI 1, as were the positive expression rates of PD-L1 and TIM3 ( Figure 3E1 ;  Supplementary 4 Tables B1-B6 ). To further explore the immune microenvironment of the tumor, we analyzed the densities of various immune cells within and outside the boundaries of ROIs 1 and 2 at distances of 0-200, 201-400, 401-800, and 801-1000 microm. The infiltration degree of CD4+ T, CD8+ T, and Treg cells, and M2-TAMs were substantially higher in the peripheral stroma than in the tumor area in ROI 1. However, these cells were significantly higher in the tumor area than in the peripheral stroma in ROI 2 ( Figure 3E2 ;  Supplementary 4 Tables B7-B9 ).