Shaodong Hong

Upregulation of PD-L1 by EGFR activation mediates the immune escape in EGFR-driven NSCLC: Implication for optional immune targeted therapy for NSCLC patients with EGFR mutation

Introduction: Epidermal growth factor receptor (EGFR) mutation status was reported to be associated with programmed death-ligand 1 (PD-L1) expression. However, the molecular mechanism of PD-L1 regulation by EGFR activation and the potential clinical significance of blocking PD-1/PD-L1 in EGFR-mutant non–small-cell lung cancer (NSCLC) treated with EGFR tyrosine kinase inhibitors (TKIs) were largely unknown. Methods: Western blot, real-time polymerase chain reaction, immunofluorescence, and flow cytometry were employed to explore the association between PD-L1 and EGFR activation. Then, we used EGFR-TKIs and downstream pathways inhibitors to clarify the detailed signaling pathway involved in PD-L1 regulation. Cell apoptosis, viability, and enzyme-linked immunosorbent assay test were used to study the immune suppression by EGFR activation and immune reactivation by EGFR-TKIs and/or PD-1 blocking in tumor cells and human peripheral blood mononuclear cells coculture system. Results: We found that EGFR activation by EGF stimulation, exon-19 deletions, and L858R mutation could induce PD-L1 expression. EGFR activation upregulated PD-L1 through p-ERK1/2/p-c-Jun but not through p-AKT/p-S6 pathway. PD-L1 mediated by EGFR activation could induce the apoptosis of T cells through PD-L1/PD-1 axis in tumor cells and peripheral blood mononuclear cells coculture system. Inhibiting EGFR by EGFR-TKIs could free the inhibition of T cells and enhance the production of interferon-&ggr;. Synergistic tumor cell killing effects were not observed with EGFR-TKIs and anti-PD-1 antibody combination treatment in coculture system. Conclusions: Our results imply that EGFR-TKIs could not only directly inhibit tumor cell viability but also indirectly enhance antitumor immunity through the downregulation of PD-L1. Anti-PD-1/PD-L1 antibodies could be an optional therapy for EGFR-TKI sensitive patients, especially for EGFR-TKIs resistant NSCLC patients with EGFR mutation. Combination of EGFR-TKIs and anti-PD-1/PD-L1 antibodies treatment in NSCLC is not supported by the current study but warrant more studies to move into clinical practice.

Patients with Exon 19 Deletion Were Associated with Longer Progression-Free Survival Compared to Those with L858R Mutation after First-Line EGFR-TKIs for Advanced Non-Small Cell Lung Cancer: A Meta-Analysis

Backgrounds It has been extensively proved that the efficacy of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) is superior to that of cytotoxic chemotherapy in advanced non-small cell lung cancer (NSCLC) patients harboring sensitive EGFR mutations. However, the question of whether the efficacy of EGFR-TKIs differs between exon 19 deletion and exon 21 L858R mutation has not been yet statistically answered. Methods Subgroup data on hazard ratio (HR) for progression-free survival (PFS) of correlative studies were extracted and synthesized based on random-effect model. Comparison of outcomes between specific mutations was estimated through indirect and direct methods, respectively. Results A total of 13 studies of advanced NSCLC patients with either 19 or 21 exon alteration receiving first-line EGFR-TKIs were included. Based on the data from six clinical trials for indirect meta-analysis, the pooled HRTKI/chemotherapy for PFS were 0.28 (95% CI 0.20–0.38, P<0.001) in patients with 19 exon deletion and 0.47 (95% CI 0.35–0.64, P<0.001) in those with exon 21 L858R mutation. Indirect comparison revealed that the patients with exon 19 deletion had longer PFS than those with exon 21 L858R mutation (HR19 exon deletion/exon 21 L858R mutation  = 0.59, 95% CI 0.38–0.92; P = 0.019). Additionally, direct meta-analysis showed similar result (HR19 exon deletion/exon 21 L858R mutation  = 0.75, 95% CI 0.65 to 0.85; P<0.001) by incorporating another seven studies. Conclusions For advanced NSCLC patients, exon 19 deletion might be associated with longer PFS compared to L858 mutation at exon 21 after first-line EGFR-TKIs.

Gemcitabine plus cisplatin versus fluorouracil plus cisplatin in recurrent or metastatic nasopharyngeal carcinoma: a multicentre, randomised, open-label, phase 3 trial

BACKGROUND Outcomes are poor for patients with recurrent or metastatic nasopharyngeal carcinoma and no well established first-line chemotherapy is available for the disease. We compared the efficacy and safety of gemcitabine plus cisplatin versus fluorouracil plus cisplatin in patients with recurrent or metastatic nasopharyngeal carcinoma. METHODS In this multicentre, randomised, open-label, phase 3 trial, patients with recurrent or metastatic nasopharyngeal carcinoma were recruited from 22 hospitals in China. Key inclusion criteria were Eastern Cooperative Oncology Group performance status of 0 or 1, adequate organ function, and measurable lesions according to Response Evaluation Criteria in Solid Tumors version 1.1. Patients were randomly assigned in a 1:1 ratio to receive either gemcitabine (1 g/m2 intravenously on days 1 and 8) and cisplatin (80 mg/m2 intravenously on day 1), or fluorouracil (4 g/m2 in continuous intravenous infusion over 96 h) and cisplatin (80 mg/m2 on day 1 given intravenously) once every 3 weeks for a maximum of six cycles. The randomisation was done centrally via an interactive phone response system using block randomisation with a size of six. The primary endpoint was progression-free survival assessed by the independent image committee in the intention-to-treat population. Safety analyses were done in patients who received at least one cycle of study drug. This study is ongoing and is registered with ClinicalTrials.gov, number NCT01528618. FINDINGS Between Feb 20, 2012, and Oct 30, 2015, 362 patients were randomly assigned to a group (181 to the gemcitabine [plus cisplatin] group and 181 to the fluorouracil [plus cisplatin] group). Median follow-up time for progression-free survival was 19·4 months (IQR 12·1-35·6). The median progression-free survival was 7·0 months (4·4-10·9) in the gemcitabine group and 5·6 months (3·0-7·0) in the fluorouracil group (hazard ratio [HR] 0·55 [95% CI 0·44-0·68]; p<0·0001). A total of 180 patients in the gemcitabine group and 173 patients in the fluorouracil group were included in the safety analysis. Significantly different treatment-related grade 3 or 4 adverse events between the gemcitabine and fluorouracil groups were leucopenia (52 [29%] vs 15 [9%]; <0·0001), neutropenia (41 [23%] vs 23 [13%]; p=0·0251), thrombocytopenia (24 [13%] vs three [2%]; p=0·0007), and mucosal inflammation (0 vs 25 [14%]; <0·0001). Serious treatment-related adverse events occurred in seven (4%) patients in the gemcitabine group and ten (6%) in the fluorouracil group. Six (3%) patients in the gemcitabine group and 14 (8%) patients in the fluorouracil group discontinued treatment because of drug-related adverse events. No treatment-related deaths occurred in either group. INTERPRETATION Gemcitabine plus cisplatin prolongs progression-free survival in patients with recurrent or metastatic nasopharyngeal carcinoma. The results establish gemcitabine plus cisplatin as the standard first-line treatment option for this population. FUNDING Sun Yat-Sen University Clinical Research 5010 Programme, Chinese National Natural Science Foundation project (grant numbers 81372502 and 81201917), the National High Technology Research and Development Program of China (863 program numbers 2012AA02A501 and 2012AA02A502), and the Natural Science Foundation of Guangdong (grant number S2013010016564).

Ratio of C-Reactive Protein/Albumin is An Inflammatory Prognostic Score for Predicting Overall Survival of Patients with Small-cell Lung Cancer

Recent studies have indicated that the C-reactive protein/ albumin (CRP/Alb) ratio is associated with clinical outcomes in patients with hepatocellular carcinoma (HCC). We examined the prognostic value of this ratio in patients with small-cell lung cancer (SCLC). In this retrospective study, a total of 367 eligible SCLC patients were analyzed and the correlation between the pretreatment CRP/Alb ratio and overall survival (OS) was investigated. The optimal cutoff level of CRP/Alb ratio was at 0.441. A low and high CRP/Alb ratio was assigned to 65.1% and 34.9% of patients, respectively. The median OS of patients with a high CRP/Alb ratio was worse than those in the low group (13.70 vs 18.90 months HR, 1.34; p = 0.005). Disease stage (p < 0.001), performance status (PS) (p < 0.001) and pretreatment LDH (p < 0.001) were also significant predictors of OS. Multivariate analyses showed that the CRP/Alb ratio is an independent prognostic factor (p = 0.025). This study demonstrated that the CRP/Alb ratio could independently predict OS in patients with SCLC, and had comparable prognostic value to other known prognostic markers. Therefore, the CRP/Alb ratio could have prognostic value and be a measurable biomarker in patients with SCLC.

Upregulation of PD-L1 by EML4-ALK fusion protein mediates the immune escape in ALK positive NSCLC: Implication for optional anti-PD-1/PD-L1 immune therapy for ALK-TKIs sensitive and resistant NSCLC patients

ABSTRACT Driver mutations were reported to upregulate programmed death-ligand 1 (PD-L1) expression. However, how PD-L1 expression and immune function was affected by ALK-TKIs and anti-PD-1/PD-L1 treatment in ALK positive non-small-cell lung cancer (NSCLC) remains poorly understood. In the present study, western-blot, real-time PCR, flow cytometry and immunofluorescence were employed to explore how PD-L1 was regulated by ALK fusion protein. ALK-TKIs and relevant inhibitors were used to identify the downstream signaling pathways involved in PD-L1 regulation. Cell apoptosis, viability and Elisa test were used to study the immune suppression by ALK activation and immune reactivation by ALK-TKIs and/or PD-1 blocking in tumor cells and DC-CIK cells co-culture system. We found that PD-L1 expression was associated with EGFR mutations and ALK fusion genes in NSCLC cell lines. Over-expression of ALK fusion protein increased PD-L1 expression. PD-L1 mediated by ALK fusion protein increased the apoptosis of T cells in tumor cells and DC-CIK cells co-culture system. Inhibiting ALK by sensitive TKIs could enhance the production of IFNγ. Anti-PD-1 antibody was effective in both crizotinib sensitive and resistant NSCLC cells. Synergistic tumor killing effects were not observed with ALK-TKIs and anti-PD-1 antibody combination in co-culture system. ALK-TKIs not only directly inhibited tumor viability but also indirectly enhanced the antitumor immunity via the downregulation of PD-L1. Anti-PD-1/PD-L1 antibodies could be an optional therapy for crizotinib sensitive, especially crizotinib resistant NSCLC patients with ALK fusion gene. Combination of ALK-TKIs and anti-PD-1/PD-L1 antibodies treatment for ALK positive NSCLC warrants more data before moving into clinical practice.

KRAS mutation-induced upregulation of PD-L1 mediates immune escape in human lung adenocarcinoma

It was reported that PD-L1 expression was correlated with genetic alterations. Whether PD-L1 was regulated by mutant Kirsten rat sarcoma viral oncogene homolog (KRAS) in non-small-cell lung cancer (NSCLC) and the underlying molecular mechanism were largely unknown. In this study, we investigated the correlation between PD-L1 expression and KRAS mutation and the functional significance of PD-1/PD-L1 blockade in KRAS-mutant lung adenocarcinoma. We found that PD-L1 expression was associated with KRAS mutation both in the human lung adenocarcinoma cell lines and tissues. PD-L1 was up-regulated by KRAS mutation through p-ERK but not p-AKT signaling. We also found that KRAS-mediated up-regulation of PD-L1 induced the apoptosis of CD3-positive T cells which was reversed by anti-PD-1 antibody (Pembrolizumab) or ERK inhibitor. PD-1 blocker or ERK inhibitor could recover the anti-tumor immunity of T cells and decrease the survival rates of KRAS-mutant NSCLC cells in co-culture system in vitro. However, Pembrolizumab combined with ERK inhibitor did not show synergistic effect on killing tumor cells in co-culture system. Our study demonstrated that KRAS mutation could induce PD-L1 expression through p-ERK signaling in lung adenocarcinoma. Blockade of PD-1/PD-L1 pathway may be a promising therapeutic strategy for human KRAS-mutant lung adenocarcinoma.

Multi-Targeted Antiangiogenic Tyrosine Kinase Inhibitors in Advanced Non-Small Cell Lung Cancer: Meta-Analyses of 20 Randomized Controlled Trials and Subgroup Analyses

Background Multi-targeted antiangiogenic tyrosine kinase inhibitors (MATKIs) have been studied in many randomized controlled trials (RCTs) for treatment of advanced non-small cell lung cancer (NSCLC). We seek to summarize the most up-to-date evidences and perform a timely meta-analysis. Methods Electronic databases were searched for eligible studies. We defined the experimental arm as MATKI-containing group and the control arm as MATKI-free group. The extracted data on objective response rates (ORR), disease control rates (DCR), progression-free survival (PFS) and overall survival (OS) were pooled. Subgroup and sensitivity analyses were conducted. Results Twenty phase II/III RCTs that involved a total of 10834 participants were included. Overall, MATKI-containing group was associated with significant superior ORR (OR 1.29, 95% CI 1.08 to 1.55, P = 0.006) and prolonged PFS (HR 0.83, 0.78 to 0.90, P = 0.005) compared to the MATKI-free group. However, no significant improvements in DCR (OR 1.08, 1.00 to 1.17, P = 0.054) or OS (HR 0.97, 0.93 to 1.01, P = 0.106) were observed. Subgroup analyses showed that the benefits were predominantly presented in pooled results of studies enrolling previously-treated patients, studies not limiting to enroll non-squamous NSCLC, and studies using MATKIs in combination with the control regimens as experimental therapies. Conclusions This up-to-date meta-analysis showed that MATKIs did increase ORR and prolong PFS, with no significant improvement in DCR and OS. The advantages of MATKIs were most prominent in patients who received a MATKI in combination with standard treatments and in patients who had previously experienced chemotherapy. We suggest further discussion as to the inclusion criteria of future studies on MATKIs regarding histology.

Development and External Validation of Nomograms for Predicting Survival in Nasopharyngeal Carcinoma Patients after Definitive Radiotherapy.

The distant metastasis free survival (DMFS) and overall survival (OS) differ significantly among individuals even within the same clinical stages. The purpose of this retrospective study was to build nomograms incorporating plasma EBV DNA for predicting DMFS and OS of nasopharyngeal carcinoma (NPC) patients after definitive radiotherapy. A total of 1168 non-metastatic NPC patients from two institutions were included to develop the nomograms. Seven and six independent prognostic factors were identified to build the nomograms for OS and DMFS, respectively. The models were externally validated by a separate cohort of 756 NPC patients from the third institutions. For predicting OS, the c-index of the nomogram was significantly better than that of the TNM staging system (Training cohort, P = 0.005; validation cohort, P = 0.03). The c-index of nomogram for DMFS in the training and validation set were both higher than that of TNM classification with marginal significance (P = 0.048 and P = 0.057, respectively). The probability of 1-, 3-, and 5-year OS and DMFS showed optimal agreement between nomogram prediction and actual observation. The proposed stratification of risk groups based on the nomograms allowed significant distinction between Kaplan-Meier curves for survival outcomes. The prognostic nomograms could better stratify patients into different risk groups.

PD-L1 is remarkably over-expressed in EBV-associated pulmonary lymphoepithelioma-like carcinoma and related to poor disease-free survival

Backgroud Programmed cell death-ligand 1 (PD-L1) and driver mutations are commonly seen in non-small-cell lung cancer (NSCLC). However, the prevelance of PD-L1 over-expression and its prognostic value in Epstein–Barr virus (EBV) associated pulmonary lymphoepithelioma-like carcinoma (LELC) remains poorly understood. Methods A total of 214 NSCLC patients and 113 surgically treated pulmonary LELC patients were included. Paraffin-embedded tumor sections were stained with PD-L1 antibody. Correlations between PD-L1 expression and clinicopathological features as well as survival outcomes were analyzed. Results The frequency of PD-L1 over-expression in NSCLC was 51.4%. No significant association was observed between common driver mutations and PD-L1 over-expression. Remakably, the positive rate of PD-L1 in pulmonary LELC was 74.3%. High PD-L1 expression was associated with impaired diseas-free survival (DFS) compared with low PD-L1 expression (p = 0.008). Multivariate analysis shows that PD-L1 expression level, N stage and M stage were independent prognostic factors for DFS. N stage and M stage but not PD-L1 expression level were significantly associated with overall survival (OS). Conclusions PD-L1 over-expression was not related to common driver mutations in NSCLC. Pulmonary LELC have remarkably high incidence of PD-L1 expression. PD-L1 was a negative prognostic factor for DFS in surgically resected pulmonary LELC. These findings may provide a rationale for immunotarget therapy in this virus-associated lung cancer.

Low Prognostic Nutritional Index (PNI) Predicts Unfavorable Distant Metastasis-Free Survival in Nasopharyngeal Carcinoma: A Propensity Score-Matched Analysis

Background Poor nutritional status is associated with progression and advanced disease in patients with cancer. The prognostic nutritional index (PNI) may represent a simple method of assessing host immunonutritional status. This study was designed to investigate the prognostic value of the PNI for distant metastasis-free survival (DMFS) in patients with nasopharyngeal carcinoma (NPC). Methods A training cohort of 1,168 patients with non-metastatic NPC from two institutions was retrospectively analyzed. The optimal PNI cutoff value for DMFS was identified using the online tool “Cutoff Finder”. DMFS was analyzed using stratified and adjusted analysis. Propensity score-matched analysis was performed to balance baseline characteristics between the high and low PNI groups. Subsequently, the prognostic value of the PNI for DMFS was validated in an external validation cohort of 756 patients with NPC. The area under the receiver operating characteristics curve (AUC) was calculated to compare the discriminatory ability of different prognostic scores. Results The optimal PNI cutoff value was determined to be 51. Low PNI was significantly associated with poorer DMFS than high PNI in univariate analysis (P<0.001) as well as multivariate analysis (P<0.001) before propensity score matching. In subgroup analyses, PNI could also stratify different risks of distant metastases. Propensity score-matched analyses confirmed the prognostic value of PNI, excluding other interpretations and selection bias. In the external validation cohort, patients with high PNI also had significantly lower risk of distant metastases than those with low PNI (Hazards Ratios, 0.487; P<0.001). The PNI consistently showed a higher AUC value at 1-year (0.780), 3-year (0.793) and 5-year (0.812) in comparison with other prognostic scores. Conclusion PNI, an inexpensive and easily assessable inflammatory index, could aid clinicians in developing individualized treatment and follow-up strategies for patients with non-metastatic NPC.