The impact of EGFR mutations on the incidence and survival of stages I to III NSCLC patients with subsequent brain metastasis

<div><p>Previous studies have demonstrated the association between EGFR mutations and distant metastasis. However, the association for subsequent brain metastasis (BM) in stages I-III non-small cell lung cancer (NSCLC) patients remains inconclusive. We conducted a retrospective analysis to clarify the impact of EGFR mutations on the incidence of BM and associated survival in patients with stage I-III NSCLC. A total of 491 patients screened for EGFR mutations were retrospectively enrolled. Brain MRI or CT was used to detect the BM. Cumulative incidence of subsequent BM and overall survival (OS) after diagnosis of BM were estimated by the Kaplan-Meier method and compared using log-rank test. We performed Cox proportional hazard regression for predictors of subsequent BM and determinants of OS after BM. The cumulative incidence of BM seemed higher in patients harboring EGFR mutations than those without EGFR mutations although it did not reach statistical significance (hazard ratio [HR] = 1.75, 95% confidence interval [CI] = 0.73~1.81). After adjusting possible confounders, including age, smoking, stage, and tumor size, EGFR mutation became one of the predictors for subsequent BM (HR = 1.89, 95% CI = 1.12~3.17, <i>p</i> = 0.017). Though there was no statistical difference in survival after BM between patients with EGFR mutations and wild-type EGFR (median survival: 17.8 vs. 12.2 months, HR = 0.79, 95% CI = 0.45–1.40), patients with EGFR 19 deletion (Del) tended to have a longer survival after BM than the non-EGFR 19 Del group (median survival: 29.4 vs. 14.3 months, HR 0.58, 95% CI = 0.32–1.09, <i>p</i> = 0.089). In conclusion, our data suggested EGFR mutation to be one of the predictors for subsequent BM in stage I-III patients. Given the small sample size, more studies are warranted to corroborate our results.</p></div

Cumulative incidence of brain metastasis (BM) in EGFR mutant versus wild-type patients.

<p>Cumulative incidence of brain metastasis (BM) in EGFR mutant versus wild-type patients.</p

Kaplan-Meier curve for overall survival in patients with mutant EGFR mutation versus those with wild type EGFR after the diagnosis of brain metastasis.

<p>Kaplan-Meier curve for overall survival in patients with mutant EGFR mutation versus those with wild type EGFR after the diagnosis of brain metastasis.</p

Basic characteristics.

<p>Basic characteristics.</p

Cox proportional hazard regression models for predictors of subsequent BM.

<p>Cox proportional hazard regression models for predictors of subsequent BM.</p

Summary of studies examining the association between EGFR mutations and brain metastasis in patients with stages I to III NSCLC.

<p>Summary of studies examining the association between EGFR mutations and brain metastasis in patients with stages I to III NSCLC.</p

Additional file 1 of Rab37 mediates trafficking and membrane presentation of PD-1 to sustain T cell exhaustion in lung cancer

Additional file 1: Table S1. The plasmids and their characteristics used in the current study. Table S2. Antibodies and their reaction conditions used in the current study. Table S3. Characteristics of NSCLC patients and normal individuals for ex vivo assays in the current study. Figure S1. The expression of PD-1 and Rab37 exhibited positive correlation in T cells. Figure S2. Rab37 mediates PD-1 membrane trafficking. Figure S3. Glycosylation on PD-1 promotes protein stability and transport to the PM. Figure S4. Rab37-mediated PD-1 PM presentation reduces T cell function. Figure S5. The serum biochemical parameters and major organ histology in LLC tumor-bearing Rab37 KO and WT mice. Figure S6. The relationship between Rab37 expression and population of PD-1/TIM3/CD8 cells derived from PBMCs treated with CD3 and CD28 antibodies

Upregulation of Tissue Factor by Activated Stat3 Contributes to Malignant Pleural Effusion Generation via Enhancing Tumor Metastasis and Vascular Permeability in Lung Adenocarcinoma

<div><p>Malignant pleural effusion (MPE) is a poor prognostic sign for patients with lung cancer. Tissue factor (TF) is a coagulation factor that participates in angiogenesis and vascular permeability and is abundant in MPE. We previously demonstrated that autocrine IL-6-activated Stat3 contributes to tumor metastasis and upregulation of VEGF, resulting in the generation of MPE in lung adenocarcinoma. In this study, we found IL-6-triggered Stat3 activation also induces TF expression. By using pharmacologic inhibitors, it was shown that JAK2 kinase, but not Src kinase, contributed to autocrine IL-6-induced TF expression. Inhibition of Stat3 activation by dominant negative Stat3 (S3D) in lung adenocarcinoma suppressed TF-induced coagulation, anchorage-independent growth <i>in vitro</i>, and tumor growth <i>in vivo</i>. Consistently, knockdown of TF expression by siRNA resulted in a reduction of anchorage-independent growth of lung adenocarcinoma cells. Inhibition of TF expression also decreased the adhesion ability of cancer cells in normal lung tissues. In the nude mouse model, both lung metastasis and MPE generation were decreased when PC14PE6/AS2-siTF cells (TF expression was silenced) were intravenously injected. PC14PE6/AS2-siTF cells also produced less malignant ascites through inhibition of vascular permeability. In summary, we showed that TF expression plays a pivotal role in the pathogenesis of MPE generation via regulating of tumor metastasis and vascular permeability in lung adenocarcinoma bearing activated Stat3.</p> </div

Blockage of Stat3 activation decreased TF activity and tumor formation <i>in vivo</i>.

<p>(A) Before injecting the cells into nude mice, Vec(1), Vec(2), S3C(1) and S3C(2) cells were harvested and the expression levels of TF and Stat3 were evaluated by Western blot analysis. (B) The same cells as (A) were harvested and TF activity assay were performed to measure the ability to initiate the coagulation cascade. (C) Nude mice were subcutaneously injected with 1 × 10⁶ cells of the vector control clone Vec(1) or dominant-negative Stat3 clones, S3D (1) and S3D(2). Tumor volume was measured weekly. The values of tumor volume represent an average of five mice for each group. *Significantly different from vector controls (P < 0.05) at indicated times (D).</p

Stat3 activation increased TF expression on cell surface in PC14PE6/AS2 cells.

<p>Cell lysates of PC14PE6/AS2-Vec(1), S3C(1), and S3C(2) (A) and cell lysates of Vec(1), S3D(1), and S3D(2) (B) were harvested and subjected to Western blot analysis to detect the expression levels of Stat3 and TF protein using various antibodies as indicated. (C) Vec (1), Vec(2), S3D(1), and S3D(2) cells were seeded on chamber slides for 24 h. To visualize cell surface TF expression, the cells were fixed without permeabilization and then stained with anti-TF antibody. Images were collected by confocal microscopy. (D) Vec(1), Vec(2), S3D(1), and S3D(2) cells were seeded on a 100 mm dish for 24 h. Cells were then fixed and stained with anti-TF antibody. Signals were analyzed by flow cytometry.</p