Fig 1: Representative immunohistochemical staining images for high-grade soft-tissue sarcoma. Brown-stained cells were considered to have positive staining; they were mostly immune cells, including lymphocytes and macrophages. To analyze the results, the CPS was determined. (A) Immunostaining for HLA-DRB4 (CPS<1) was negative in a sample from the good prognosis group. (B) Immunostaining for HLA-DRB4 (CPS=1) revealed positive staining in a sample from the poor prognosis group. (C) Immunostaining for HLA-DQA1 (CPS<1) was negative in a sample from the poor prognosis group. (D) Immunostaining for HLA-DQA1 (CPS=1) was positive in a sample from the good prognosis group. (E) Immunostaining for HLA-DQB1 (CPS<1) was negative in a sample from the poor prognosis group. (F) Immunostaining for HLA-DQB1 (CPS=1) was positive in a sample from the good prognosis group. Magnification, x200. CPS, combined positive score; HLA, human leukocyte antigen.
Fig 2: Kaplan-Meier survival analysis for expression of HLA-DRB4, HLA-DQA1 and HLA-DQB1 detected via immunohistochemistry. (A) Survival curves by HLA-DRB4 expression indicated no significant association with prognosis (P=0.489). (B) HLA-DQA1 expression was significantly associated with an increase in the survival rate (P=0.028). (C) Survival curves by HLA-DRB1 expression indicated no significant association with prognosis (P=0.500). Blue line, CPS-negative expression group; green line, CPS-positive expression group. CPS, combined positive score; HLA, human leukocyte antigen. Patient follow-up was conducted from the date of surgery until final follow up date identified in medical records; the date of death or the last visit to the hospital. The follow-up period ranged from 4 to 122 months (mean, 68.3 months). CPS, combined positive score; HLA, human leukocyte antigen.
Fig 3: Heat map generated from mRNA expression data reflecting 13 representative genes that were differentially expressed between the S and D prognostic groups. Red color indicates highly expressed genes. Groups: S, good prognosis; D, poor prognosis. HLA-DRB4, human leukocyte antigen; NCAM1, neural cell adhesion molecule; CD36; CD3, FOS, FCER2, Fc fragment of IgE receptor II; DOCK9, dedicator of cytokines 9; HLA-DQA1, HLA-DQB1, LAG3, lymphocyte antivation gene 3; FOXP3, forkhead box P3; BIRC5, baculoviral IAP repeat containing 5; DUSP4, dual specificity phosphatase 4.
Fig 4: Clinical validation of HLA class II expression based on TKI response(A) Volcano plot for pseudo-bulk-based DEGs between the resistant and sensitive groups. Red and blue dots represent upregulated genes in the resistant and sensitive groups, respectively.(B) The resistant group’s enriched gene ontology terms were illustrated in red, whereas the sensitive group’s enriched gene ontology terms were shown in blue.(C) Comparison of HLA class ? (HLA-DPB1, HLA-DQA1, and HLA-DRB1) expression between the TKI resistant (LCPE.R) and sensitive (LCPE.S) groups (Wilcoxon rank-sum test).(D) Boxplot for the comparison of JAK-STAT score between the resistant and sensitive groups (p = 0.032, Wilcoxon rank-sum test).(E) IHC staining of MHC class II in the two resistant (top) and two sensitive patient samples (bottom). 100× magnification and 200× partial magnification with a lattice length of 125 µm.(F) Boxplot for the comparison of IHC scores based on MHC class ? (HLA-DPB1, HLA-DQA1, and HLA-DRB1) expression between the resistant and sensitive groups.(G) Survival plot showing PFS dependence on high and low HLA-DPB1 (HR 2.837, 95% CI 1.333-6.041), HLA-DQA1 (HR 2.219, 95% CI 1.036-4.749), and HLA-DRB1 (HR 2.417, 95% CI 1.123-5.199) subtype expression.(H) Flow cytometry analysis of interferon (IFN)-? producing MPE cells from the two EGFR-TKI resistant (left) and two sensitive patient samples (right). TKI, tyrosine kinase inhibitor; DEGs, differentially expressed genes; IHC, immunohistochemistry; PFS, progression-free survival.
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