PMC10470069_01
(A) Baseline PET-CT scan of the patient. SUVmax (LN10) = 17.3, SUVmax (LN7) = 5.6 (B) Hematoxylin and eosin staining of pretreatment specimens. (C) Immunohistochemical detection of ALK protein expression in pretreatment specimens. Tissue paraffin sections were blocked and incubated with primary antibody against ALK (1:50, DAKO North America, United States) overnight at 4 C. PBS was substituted for the antibody as a negative control. Cytoplasmic staining was considered ALK positive.
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F1
Positron emission tomography-computed tomography (PET-CT) and brain magnetic resonance imaging showed no signs of distant metastases (Figure 1A). A biopsy of the mass in the left lower lung basal segmental opening was performed under bronchoscopy, and the pathological findings indicated lung adenocarcinoma (Figure 1B). Next-generation sequencing of specimens obtained by bronchoscopy identified an EML4-ALK fusion mutation which is inherently a translocation fusion caused by inversion within the chromosome arm, and subsequently, an immunohistochemistry test indicated high expression of the ALK protein in the tumor tissue samples of this patient (Figure 1C). Although the patient's subcarinal region lymph nodes (LN7) did not change significantly during treatment (Supplementary Figure S1A), the patient's hilar region lymph nodes (LN10) became large again at the end of eight cycles of chemotherapy (Figure 2A, Supplementary Figure S1B). However, at this time, serum levels of tumor markers, especially CEA, had fallen to lower levels (Supplementary Figure S1C). Furthermore, the scores for the intra group were higher than those for the margin group, with Z7 having the highest TIS score and Z2 having the lowest TIS-related gene expression (Supplementary Figure S1D). The expression levels of immune checkpoint molecules at each sampling were explored further. The expression level of cytotoxic T lymphocyte antigen 4 (CTLA-4) was the highest in Z6 compared with the other samples, and the expression levels of several immune checkpoint markers (PDL2, TIM3, and 4-1BB) were higher in Z2 than in the other samples (Supplementary Figure S1E).
(A) CT scans of the patient at each stage of treatment. (B) Hematoxylin and eosin staining of the treated specimen. Blue arrows point to necrotic areas, yellow arrows point to atypical hyperplastic cells, and green arrows point to infiltrating lymphocytes.
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F2
Although the patient's subcarinal region lymph nodes (LN7) did not change significantly during treatment (Supplementary Figure S1A), the patient's hilar region lymph nodes (LN10) became large again at the end of eight cycles of chemotherapy (Figure 2A, Supplementary Figure S1B). CT showed that the lesion diameter was 42.4 mm 4 months later, with no significant change from the diameter at the end of chemotherapy (Figure 2A). The pathological findings of the postoperative lung lobe specimens indicated extensive necrosis in the tumor area with no residual tumor cells and massive chronic inflammatory cell infiltration around the tumor area, confirming the preoperative effectiveness of alectinib (Figure 2B).
(A) Sampling point location. (B) Clustering analysis of 770 gene expressions. (C) Venn diagram showing differentially expressed genes (DEGs) common to each tumor tissue sampling site and paracancerous tissue. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEGs. (E) Gene Ontology (GO) analysis of DEGs. (F) Evaluation of immune cell infiltration. (G) Heat map of the expression of immune-related signatures.
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F3
Multi-point tumor specimen sampling was performed (Figure 3A) with consent from the patient and her family. We found that there was no significant clustering at the four sampling sites, and some differences were seen between sampling locations (Figure 3B). Further, we identified differentially expressed genes (DEGs) between each tumor tissue sampling site and paracancer normal tissue (Figure 3C), and the 68 DEGs common to each combination were subjected to Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The KEGG analysis showed that the DEGs were mainly enriched in signaling pathways such as cytokine-cytokine receptor interaction and the chemokine signaling pathway (Figure 3D). GO analysis showed that DEGs were mainly related to granulocyte migration, monocyte chemotaxis, granulocyte chemotaxis, neutrophil migration, neutrophil chemotaxis, and positive regulation of T cells (Figure 3E). The results showed that Z6 tissues had the highest infiltration percentage of B cells and regulatory T cells (Treg), Z6 and Z7 tissues had higher percentages of CD8+ T cells and DCs, and Z2 tissues had the highest percentage of macrophages (Figure 3F). Treg, ARG1, and NOS2 (Figure 3G).