Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are currently the first-line standard of care for patients with non-small cell lung cancer (NSCLC) that harbor EGFR mutations. Nevertheless, resistance to EGFR-TKIs is inevitable. In recent years, although immune checkpoint inhibitors (ICIs) have significantly shifted the treatment paradigm in advanced NSCLC without driver mutation, clinical benefits of these agents are limited in patients with EGFR-mutated NSCLC. Compared with wild-type tumors, tumors with EGFR mutations show more heterogeneity in the expression level of programmed cell death ligand 1 (PD-L1), tumor mutational burden (TMB), and other tumor microenvironment (TME) characteristics. Whether ICIs are suitable for NSCLC patients with EGFR mutations is still worth exploring. In this review, we summarized the clinical data with regard to the efficacy of ICIs in patients with EGFR-mutated NSCLC and deciphered the unique TME in EGFR-mutated NSCLC.
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Humans ; Carcinoma, Non-Small-Cell Lung/genetics* ; Lung Neoplasms/genetics* ; ErbB Receptors/metabolism* ; Immunotherapy ; Mutation ; B7-H1 Antigen/genetics* ; Protein Kinase Inhibitors/pharmacology* ; Tumor Microenvironment

Objective: To accurately screen non-small cell lung cancer (NSCLC) patients with KRAS G12C mutation and to evaluate their clinicopathological features, prognostic factors and current treatment status. Methods: A total of 19 410 NSCLC cases diagnosed at the Department of Pathology of Shanghai Chest Hospital, Shanghai, China from January 2018 to September 2021 were retrospectively reviewed, and the cases with KRAS gene mutation detected by next-generation sequencing were included. The clinicopathological and genetic mutation data of these cases were collected and analyzed. Results: A total of 1 633 (8.4%) NSCLC patients carried a KRAS gene mutation, among whom G12C was the most frequent (468 cases, 28.7%) mutant subtype. The mutation was more commonly found in males (414/468, 88.5%), patients with a history of smoking (308/468, 65.8%), and patients with a pathological type of invasive adenocarcinoma (231/468, 49.4%). The most common co-mutated genes in KRAS G12C mutant NSCLC were TP53 (52.4%, 245/468), STK11 (18.6%, 87/468) and ATM (13.2%, 62/468). The proportion of PD-L1 expression (≥1%) in KRAS G12C mutant NSCLC was significantly higher than that in patients without G12C mutation [64.3% (90/140) vs. 56.1% (193/344), P=0.014]. Immune checkpoint inhibitors (ICIs) treatment significantly prolonged progression-free survival (PFS) in NSCLC patients (10.0 months vs. 5.0 months, P=0.011). However, combination of chemotherapy and ICIs with anti-angiogenesis inhibitors or multi-target inhibitors did not significantly improve PFS in patients with KRAS G12C mutant NSCLC (P>0.05). Patients with KRAS G12C mutation NSCLC treated with ICIs and KRAS G12C patients with TP53 mutation had significantly longer median PFS than those with STK11 mutation (9.0 months vs. 4.3 months, P=0.012). Conclusions: Patients with KRAS G12C mutant NSCLC have relatively higher levels of PD-L1 expression and can benefit from ICIs treatment. The feasibility of chemotherapy, ICIs therapy and their combination needs further investigation.
Humans ; Male ; B7-H1 Antigen/genetics* ; Carcinoma, Non-Small-Cell Lung/pathology* ; China ; Lung Neoplasms/pathology* ; Mutation ; Proto-Oncogene Proteins p21(ras)/genetics* ; Retrospective Studies ; Female

Objective: To investigate the clinicopathological features, treatment and prognosis of patients with RET fusion positive non-small cell lung cancer (NSCLC). Methods: A total of 1 089 NSCLCs were retrieved at Affiliated Hospital of Jiangnan University from August 2018 to April 2020. In all cases, multiple gene fusion detection kits (fluorescent PCR method) were used to detect the gene status of RET, EGFR, ALK, ROS1, KRAS, BRAF and HER2; and immunohistochemical method was used to detect the expression of PD-L1 and mismatch repair related proteins. The correlation between RET-fusion and patients' age, gender, smoking history, tumor stage, grade, pathologic type, and PD-L1, mismatch repair related protein expression was analyzed. Results: There were 22 cases (2.02%) detected with RET fusion-positive in 1 089 NSCLC patients, in which 11 males and 11 females; and the median age was 63.5 years. There were 20 adenocarcinomas, including 11 acinar predominant adenocarcinoma (APA), five solid predominant adenocarcinoma (SPA) and four lepidic predominant adenocarcinoma (LPA); There were one case each of squamous cell carcinoma (non-keratinizing type) and sarcomatoid carcinoma (pleomorphic carcinoma). There were 6 and 16 patients with RET fusion-positive who were in stage Ⅰ-Ⅱ and Ⅲ-Ⅳ respectively, and 16 cases with lymph node metastasis, 11 cases with distant metastasis. Among RET fusion-positive cases, one was detected with HER2 co-mutation. The tumor proportion score of PD-L1≥1% in patients with RET fusion positive lung cancer was 54.5% (12/22). Defects in mismatch repair protein expression were not found in patients with RET fusion positive NSCLC. Four patients with RET fusions positive (two cases of APA and two cases of SPA) received pratinib-targeted therapy, and two showed benefits from this targeted therapy. Conclusions: The histological subtypes of RET fusions positive NSCLC are more likely to be APA or SPA. RET fusion-positive NSCLC patients are associated with advanced clinical stage, lymph node metastases, and they may benefit from targeted therapy with RET-specific inhibitors.
Male ; Female ; Humans ; Middle Aged ; Carcinoma, Non-Small-Cell Lung/pathology* ; Lung Neoplasms/pathology* ; B7-H1 Antigen/genetics* ; Protein-Tyrosine Kinases/genetics* ; Proto-Oncogene Proteins c-ret/metabolism* ; Proto-Oncogene Proteins/genetics* ; Adenocarcinoma/pathology* ; Carcinoma, Squamous Cell/genetics* ; Mutation

Objective: To investigate and elucidate the clinicopathological and prognostic characteristics of SMARCA4-deficient non-small cell lung cancer. Methods: The clinicopathological and prognostic data were collected in 127 patients with SMARCA4-deficient non-small cell lung cancer diagnosed in Shanghai Pulmonary Hospital, Shanghai, China from January 2020 to March 2022. The variation and expression of biomarkers related to treatment were retrospectively reviewed. Results: One hundred and twenty-seven patients were eligible for enrollment. Among them 120 patients (94.5%) were male and 7 cases (5.5%) were female, while the average age was 63 years (range 42-80 years). There were 41 cases (32.3%) of stage Ⅰ cancer, 23 cases (18.1%) of stage Ⅱ, 31 cases (24.4%) of stage Ⅲ and 32 cases (25.2%) of stage Ⅳ. SMARCA4 expression detected by immunohistochemistry was completely absent in 117 cases (92.1%) and partially absent in 10 cases (7.9%). PD-L1 immunohistochemical analyses were performed on 107 cases. PD-L1 was negative, weakly positive and strongly positive in 49.5% (53/107), 26.2% (28/107) and 24.3% (26/107) of the cases, respectively. Twenty-one cases showed gene alterations (21/104, 20.2%). The KRAS gene alternation (n=10) was most common. Mutant-type SMARCA4-deficient non-small cell lung cancer was more commonly detected in females, and was associated with positive lymph nodes and advanced clinical stage (P<0.01). Univariate survival analysis showed that advanced clinical stage was a poor prognosis factor, and vascular invasion was a poor predictor of progression-free survival in patients with surgical resection. Conclusions: SMARCA4-deficient non-small cell lung cancer is a rare tumor with poor prognosis, and often occurs in elderly male patients. However, SMARCA4-deficient non-small cell lung cancers with gene mutations are often seen in female patients. Vascular invasion is a prognostic factor for disease progression or recurrence in patients with resectable tumor. Early detection and access to treatment are important for improving patient survivals.
Humans ; Male ; Female ; Aged ; Adult ; Middle Aged ; Aged, 80 and over ; Carcinoma, Non-Small-Cell Lung/pathology* ; B7-H1 Antigen/metabolism* ; Lung Neoplasms/pathology* ; Retrospective Studies ; China ; Prognosis ; Biomarkers, Tumor/analysis* ; DNA Helicases/genetics* ; Nuclear Proteins/genetics* ; Transcription Factors/genetics*

Objective To explore the effect of formononetin on immunity of mice with transplanted H22 hepatocarcinoma. Methods Male C57BL/6 mice were subcutaneously inoculated with H22 cells (4×10⁵) to establish a tumor-bearing mouse model. The mice were treated with formononetin [10 mg/(kg.d)] or [50 mg/(kg.d)] for 28 days, and then the tumor inhibition rate was calculated. Carrilizumab was used as a positive control drug. The expressions of CD8, granzyme B and forkbox transcription factor 3 (FOXP3) in HCC tissues were analyzed by immunohistochemical staining. The mRNA and protein expression of programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) in HCC tissues were detected by real-time PCR or Western blot analysis, respectively. The serum levels of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) were detected by ELISA. Results Formononetin increased the tumor inhibition rate and the positive rate of CD8 and granzyme B staining in tumor-bearing mice. There was no significant difference in the positive rate of FOXP3 staining in tumor tissues of mice in each group. Formononetin decreased the levels of IL-10 and TGF-β in serum of tumor-bearing mice, and decreased the relative expression of mRNA and protein of PD-1 and PD-L1 in tumor tissue of tumor-bearing mice. Conclusion Formononetin can activate CD8⁺ T cells and reduce the release of immunosuppressive factors in regulatory T cells by blocking PD-1/PD-L1 pathway and play an antitumor role.
Male ; Animals ; Mice ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/genetics* ; Interleukin-10/genetics* ; B7-H1 Antigen ; Granzymes/genetics* ; Programmed Cell Death 1 Receptor/metabolism* ; CD8-Positive T-Lymphocytes/metabolism* ; Mice, Inbred C57BL ; Transforming Growth Factor beta/genetics* ; RNA, Messenger/metabolism* ; Forkhead Transcription Factors/genetics* ; Cell Line, Tumor

Objective: PD-1/PD-L1 immune checkpoint treatment is effective for some triple-negative breast cancer populations with PD-L1 expression, but the response rate is still not satisfactory. This study aims to explore the mechanism of drug resistance to breast cancer anti-PD-1 therapies and the strategies for overcoming the resistance to PD-1therapies. Methods: By constructing a human triple-negative breast cancer drug-resistant cell line called BT-549R5 and a mouse breast cancer drug-resistant cell line called 4T1R3, and applying the whole-gene shRNA library screening, candidate drug resistance-associated molecules were obtained and verified by cytological experiments. The expression of Tyro3, Axl and MerTK of the TAM family in the 4T1R3 group was tested using the Western blot method. The down-regulation of CDK9 on the effect of T cells killing the BT-549R5 cells was observed through T cell killing tests, while the down-regulation of Tyro3 and CDK9 on the effect of anti-PD-1 therapies for transplanted breast tumors was observed in mouse tumor formation experiments. Results: The cell lines and animal models of breast cancer resistant to PD-1 treatment were successfully constructed. Tyro3, Axl and MerTK were highly expressed in 4T1R3 cells. Whole genome sequencing showed that Tyro3 and CDK9 were highly expressed in BT-549R5 cells. T cell killing experiment showed that the survival rate of BT-549R5 cells in the CDK9 down-regulated group and the control group decreased gradually with the increase of T cells, but the survival rate of BT-549R5 cells in the CDK9 down-regulated group decreased rapidly. Tumor formation experiment in mice showed that under anti-PD-1 treatment, the transplanted tumor in the 4T1R3 cell group grew rapidly compared with the 4T1 cell group (P<0.05), and the tumor volume of the 4T1R3 group was larger than that of the 4T1 group on Day 20. Nevertheless, the tumor growth rates in the CDK9-knockdown 4T1R3 cell group and the Tyro3-knockdown 4T1R3 cell group were similar to that of the 4T1 cell group, and the tumor volumes at day 20 were signiference lower than that of 4T1R3 cell group(P<0.05). Conclusions: Tyro3 and CDK9 are associated with the drug resistance to anti-PD-1 therapies for breast cancer. Inhibiting the expression of Tyro3 and CDK9 can reverse the drug resistance to breast cancer treatment.
Humans ; Animals ; Mice ; c-Mer Tyrosine Kinase/metabolism* ; Receptor Protein-Tyrosine Kinases/genetics* ; Axl Receptor Tyrosine Kinase ; Proto-Oncogene Proteins/metabolism* ; B7-H1 Antigen/genetics* ; Triple Negative Breast Neoplasms/genetics* ; Drug Resistance, Neoplasm ; Biomarkers ; Cell Line, Tumor ; Cyclin-Dependent Kinase 9

Humans ; B7-H1 Antigen ; Neoplasms/genetics*

Objective: PD-1/PD-L1 immune checkpoint treatment is effective for some triple-negative breast cancer populations with PD-L1 expression, but the response rate is still not satisfactory. This study aims to explore the mechanism of drug resistance to breast cancer anti-PD-1 therapies and the strategies for overcoming the resistance to PD-1therapies. Methods: By constructing a human triple-negative breast cancer drug-resistant cell line called BT-549R5 and a mouse breast cancer drug-resistant cell line called 4T1R3, and applying the whole-gene shRNA library screening, candidate drug resistance-associated molecules were obtained and verified by cytological experiments. The expression of Tyro3, Axl and MerTK of the TAM family in the 4T1R3 group was tested using the Western blot method. The down-regulation of CDK9 on the effect of T cells killing the BT-549R5 cells was observed through T cell killing tests, while the down-regulation of Tyro3 and CDK9 on the effect of anti-PD-1 therapies for transplanted breast tumors was observed in mouse tumor formation experiments. Results: The cell lines and animal models of breast cancer resistant to PD-1 treatment were successfully constructed. Tyro3, Axl and MerTK were highly expressed in 4T1R3 cells. Whole genome sequencing showed that Tyro3 and CDK9 were highly expressed in BT-549R5 cells. T cell killing experiment showed that the survival rate of BT-549R5 cells in the CDK9 down-regulated group and the control group decreased gradually with the increase of T cells, but the survival rate of BT-549R5 cells in the CDK9 down-regulated group decreased rapidly. Tumor formation experiment in mice showed that under anti-PD-1 treatment, the transplanted tumor in the 4T1R3 cell group grew rapidly compared with the 4T1 cell group (P<0.05), and the tumor volume of the 4T1R3 group was larger than that of the 4T1 group on Day 20. Nevertheless, the tumor growth rates in the CDK9-knockdown 4T1R3 cell group and the Tyro3-knockdown 4T1R3 cell group were similar to that of the 4T1 cell group, and the tumor volumes at day 20 were signiference lower than that of 4T1R3 cell group(P<0.05). Conclusions: Tyro3 and CDK9 are associated with the drug resistance to anti-PD-1 therapies for breast cancer. Inhibiting the expression of Tyro3 and CDK9 can reverse the drug resistance to breast cancer treatment.
Humans ; Animals ; Mice ; c-Mer Tyrosine Kinase/metabolism* ; Receptor Protein-Tyrosine Kinases/genetics* ; Axl Receptor Tyrosine Kinase ; Proto-Oncogene Proteins/metabolism* ; B7-H1 Antigen/genetics* ; Triple Negative Breast Neoplasms/genetics* ; Drug Resistance, Neoplasm ; Biomarkers ; Cell Line, Tumor ; Cyclin-Dependent Kinase 9

Objective: To investigate the expression of programmed death ligand-1 (PD-L1, SP142) and PD-L1 (22C3) in triple-negative breast cancer (TNBC), and analyze their correlation with the clinicopathological factors and prognosis. Methods: The clinicopathologic data of 259 patients with TNBC treated in Cancer Hospital from August 2010 to December 2013 were collected. Whole section of surgical tissue samples were collected to conduct PD-L1 (SP142) and PD-L1 (22C3) immunohistochemical (IHC) staining. The PD-L1 expression in tumor cells and tumor infiltrating immune cells were visually assessed respectively, the relationship between PD-L1 expression and clinicopathologic characterizes were analyzed. Univariable and multivariable Cox proportional hazards regression models were used to test the correlations between PD-L1 expression and disease-free survival (DFS) and overall survival (OS). Results: The positive rates of SP142 (immune cell score, ICs≥1%) and 22C3 (combined positive score, CPS≥1) were 42.1%(109/259) and 41.3%(107/259) in TNBC tissues, respectively, with a total coincidence rate of 82.3%. The Kappa value of positive expression cases was 0.571 and the distribution difference of SP142 and 22C3 positive expression cases was statistically significant (P<0.001). The PD-L1 positive patients were less likely to have vascular invasion (P<0.05), but with higher histological grade and Ki-67 proliferation index (P<0.05). The recurrence/metastasis cases(8) of the patients with positive PD-L1 (SP142) was significantly lower than that of patients with negative PD-L1(SP142, 27, P=0.016). The positive expression of PD-L1 (SP142) patients were longer DFS (P=0.019). The OS of patients with positive PD-L1 (SP142) were longer than those with negative PD-L1 (SP142), but without significance (P=0.116). The positive expression of PD-L1 (22C3) was marginally associated with DFS and OS of patients (P>0.05). Conclusions: The expression of PD-L1 (22C3) is different from that of PD-L1 (SP142) in TNBC, and the two antibodies can't be interchangeable for each other in clinical tests. PD-L1 (SP142) status is an independent prognostic factor of DFS in TNBC. The DFS is significantly prolonged in patients with positive expression of PD-L1 (SP142).
B7-H1 Antigen/genetics* ; Humans ; Immunohistochemistry ; Prognosis ; Triple Negative Breast Neoplasms/pathology*

OBJECTIVE: To investigate the potential inhibitory effect of interference with PD-L1 on B cell lymphoma in mice. METHODS: Three shRNA vectors for mouse CD274 (PD-L1) were constructed and transiently transfected into 293T cells. RT-qPCR was used to validate the interference efficiency of CD274. The shRNA vector that interfere efficiently with CD274 expression was packaged by using lentivirus packaging system to generate shRNA lentivirus, and then transfected into A20 lymphoma cell line. The methyl thiazol terazolium (MTT) assay was used to detect proliferation after 48 h culture of CD274-sh A20 cells. Meanwhile, BALB/c mice were hypodermically infected with CD274-sh A20 cells. Infected mice were observed daily and assessed to visualize tumor by in vivo fluorescence imaging. RESULTS: The proliferation rate of CD274-sh A20 cells in vitro was significantly lower than that of A20 cells (P<0.05). The tumor size detected by in vivo fluorescence imaging showed a significant reduce in tumor bearing mice with CD274-sh compared with other tumor bearing mice. And the weight and size of tumor in CD274-sh group were also significantly reduced compared with other group (P<0.05). Moreover, the survival time of tumor bearing mice in CD274-sh group was longer than that of the PD-L1 high expression group. CONCLUSION PD-L1 plays an important role in the incidence and the progression of lymphoma, and the shRNA-based PD-L1 knockdown can inhibit cell proliferation of A20 cells and partly suppress tumor growth.
Animals ; B7-H1 Antigen/metabolism* ; Cell Line, Tumor ; Humans ; Lymphoma ; Lymphoma, B-Cell ; Mice ; Mice, Inbred BALB C ; RNA, Small Interfering/genetics*