Si-Yang Liu

Potential Predictive Value of TP53 and KRAS Mutation Status for Response to PD-1 Blockade Immunotherapy in Lung Adenocarcinoma

Purpose: Although clinical studies have shown promise for targeting programmed cell death protein-1 (PD-1) and ligand (PD-L1) signaling in non–small cell lung cancer (NSCLC), the factors that predict which subtype patients will be responsive to checkpoint blockade are not fully understood. Experimental Design: We performed an integrated analysis on the multiple-dimensional data types including genomic, transcriptomic, proteomic, and clinical data from cohorts of lung adenocarcinoma public (discovery set) and internal (validation set) database and immunotherapeutic patients. Gene set enrichment analysis (GSEA) was used to determine potentially relevant gene expression signatures between specific subgroups. Results: We observed that TP53 mutation significantly increased expression of immune checkpoints and activated T-effector and interferon-γ signature. More importantly, the TP53/KRAS comutated subgroup manifested exclusive increased expression of PD-L1 and a highest proportion of PD-L1+/CD8A+. Meanwhile, TP53- or KRAS-mutated tumors showed prominently increased mutation burden and specifically enriched in the transversion-high (TH) cohort. Further analysis focused on the potential molecular mechanism revealed that TP53 or KRAS mutation altered a group of genes involved in cell-cycle regulating, DNA replication and damage repair. Finally, immunotherapeutic analysis from public clinical trial and prospective observation in our center were further confirmed that TP53 or KRAS mutation patients, especially those with co-occurring TP53/KRAS mutations, showed remarkable clinical benefit to PD-1 inhibitors. Conclusions: This work provides evidence that TP53 and KRAS mutation in lung adenocarcinoma may be served as a pair of potential predictive factors in guiding anti–PD-1/PD-L1 immunotherapy. Clin Cancer Res; 23(12); 3012–24. ©2016 AACR.

The Unique Characteristics of MET Exon 14 Mutation in Chinese Patients with NSCLC

Introduction: Predictive biomarkers of mesenchymal‐to‐epithelial transition factor (MET)‐targeted therapy remain elusive. Since the discovery of the MNNG HOS Transforming gene (MET) exon 14 mutation, it has been found to have the best potential to become one precise biomarker for MET‐targeted therapy. Here, we present the unique characteristics of MET exon 14 mutations in Chinese patients with NSCLC. Methods: A total of 1296 patients with NSCLC were screened for MET exon 14 mutations. Next‐generation sequencing was performed on the DNA of 968 patients and Sanger sequencing was conducted on complementary DNA of the other 328 patients. Immunohistochemical analysis and fluorescence in situ hybridization were also performed on all specimens. Results: Twelve patients had MET exon 14 mutations. These accounted for only 0.9% of adenocarcinoma. Thus, the mutations were present at less than half the frequency of their occurrence in Western patients (0.9% versus 3% in Chinese and white patients, respectively, χ2 = 15.1, p < 0.001). Samples from six patients with MET exon 14 mutations were analyzed using immunohistochemical analysis and fluorescence in situ hybridization. We found no significant relationships among the mutation, MET amplification, and MET overexpression. In two patients who received crizotinib, only one patient (who exhibited MET amplification) experienced a partial response; the progression‐free survival was 9 months. However, it remains unclear whether the sensitivity of this patient to crizotinib was conferred by the MET exon 14 mutation per se or by MET amplification. In the other patient with concomitant MET exon 14 skipping and KRAS G12D mutation, the disease progressed in only 1 month. Conclusions: MET exon 14 mutation per se may not be sufficiently robust for use in defining a subset of NSCLCs. Further research on MET exon 14 mutations, MET amplification, and MET overexpression is required. Maybe a panel of biomarkers will be necessary in the future.

Acquired MET Y1248H and D1246N mutations mediate resistance to MET inhibitors in non-small cell lung cancer

Purpose: MET amplification, responsible for 20% of acquired resistance to EGFR tyrosine kinase inhibitor (TKI) in patients with advanced non–small cell lung cancer (NSCLC), presents an attractive target. Numerous studies have conferred susceptibility of MET mutations and focal amplification to targeted MET-TKIs. However, the mechanism underlying MET-TKIs–induced resistance remains elusive. Experimental Design: We conducted a cohort of 12 patients with advanced NSCLC who developed resistance to a combinatorial therapy consisting of gefitinib and a type I MET-TKI. We performed capture-based targeted ultra-deep sequencing on serial tumor biopsies and plasmas ctDNA samples to detect and quantify genetic alterations. Results: We identified 2 newly acquired MET mutations, Y1248H and D1246N, in 2 patients and further confirmed their resistance against type I MET-TKIs in silco, in vitro, and in vivo. Interestingly, NIH3T3 cells harboring either mutation exhibited responses to type II MET-TKIs, suggesting sequential use of MET-TKIs may offer a more durable response. In addition, we also discovered that EGFR amplification may act as an alternative MET-TKI resistance mechanism. Conclusions: Our study provides insight into the diversity of mechanisms underlying MET-TKI–induced resistance and highlights the potential of sequential use of MET-TKIs. Clin Cancer Res; 23(16); 4929–37. ©2017 AACR.

EGFR mutation correlates with uninflamed phenotype and weak immunogenicity, causing impaired response to PD-1 blockade in non-small cell lung cancer

ABSTRACT Patients with EGFR mutations showed unfavorable response to programmed cell death-1 (PD-1) blockade immunotherapy in non-small cell lung cancer (NSCLC). Yet the underlying association between EGFR mutation and immune resistance remains largely unclear. We performed an integrated analysis of PD-ligand 1(PD-L1)/CD8 expression and mutation profile based on the repository database and resected early-stage NSCLC in Guangdong Lung Cancer Institute (GLCI). Meanwhile, 2 pool-analyses were set to clarify the correlation between EGFR mutation and PD-L1 expression, and the association of EGFR status with response to anti-PD-1/L1 therapy. Pool-analysis of 15 public studies suggested that patients with EGFR mutations had decreased PD-L1 expression (odds ratio: 1.79, 95% CI: 1.10–2.93; P = 0.02). Analysis of The Cancer Genome Atlas (TCGA) and the GCLI cohort confirmed the inverse correlation between EGFR mutation and PD-L1 expression. Furthermore, patients with EGFR mutation showed a lack of T-cell infiltration and shrinking proportion of PD-L1+/CD8+ TIL (P = 0.034). Importantly, patients with EGFR mutations, especially the sensitive subtype, showed a significantly decreased mutation burden, based on analysis of the discovery and validation sets. Finally, a pool-analysis of 4 randomized control trials confirmed that patients with EGFR mutation did not benefit from PD-1/L1 inhibitors (Hazard ratio [HR] = 1.09, P = 0.51) while patients with EGFR wild-type did (HR = 0.73, P < 0.00001). This study provided evidence of a correlation between EGFR mutations and an uninflamed tumor microenvironment with immunological tolerance and weak immunogenicity, which caused an inferior response to PD-1 blockade in NSCLCs.

The coexistence of MET over-expression and an EGFR T790M mutation is related to acquired resistance to EGFR tyrosine kinase inhibitors in advanced non-small cell lung cancer.

MET overexpression and the EGFR T790M mutation are both associated with acquired resistance (AR) to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in advanced non-small cell lung cancer (NSCLC). We characterized the frequency, underlying molecular mechanisms, and subsequent treatment for AR in MET overexpressing NSCLC patients with or without the T790M mutation. The study participants were 207 patients with advanced NSCLC and AR to EGFR-TKIs. The percentages of MET-, T790M- and MET/T790M-positive patients were 20.3% (42/207), 34.8% (72/207) and 6.8% (14/207), respectively. The disease control rate was 100% (5/5) for five patients with MET overexpression who received EGFR-TKIs plus a MET inhibitor. Among the MET/T790M-positive patients, seven received EGFR-TKIs plus a MET inhibitor and four received a T790M inhibitor, but no response was observed. The median post-progression survival (PPS) was 14.1, 24.5, and 10.7 months for MET-overexpressing, T790M-positive and MET/T790M-positive patients, respectively (P=0.044). c-Met, p-Met, ERBB3, and p-ERBB3 were highly expressed in MET-positive and MET/T790M-positive patients, but were poorly expressed in T790M-positive patients. EGFR, p-EGFR, AKT, p-AKT, MAPK, and p-MAPK were highly expressed in all three groups. These results suggest that MET/T790M-positive patients are at higher risk of AR to EGFR-TKIs, and have a worse PPS than patients with only MET overexpression or the T790M mutation alone. Clinical trials are needed to determine the best treatment for patients with both MET overexpression and the EGFR T790M mutation.

Ongoing clinical trials of PD-1 and PD-L1 inhibitors for lung cancer in China

Compared to chemotherapy, promising results have been obtained by blocking the PD-1 pathway using antibodies that inhibit programmed cell death protein 1 (PD-1) or programmed cell death protein ligand 1 (PD-L1). Furthermore, global researchers and doctors are exploring how to optimize this immunotherapy in 270 clinical studies. However, Chinese clinical trials of these agents remain in the early stages. We summarize the ongoing international and domestic clinical trials using PD-1 and PD-L1 inhibitors to treat lung cancer. This information can help researchers better understand the active and approved clinical trials in China, as well as the ongoing research regarding PD-1 and PD-L1 inhibitors.

Genetic and Immune Profiles of Solid Predominant Lung Adenocarcinoma Reveal Potential Immunotherapeutic Strategies

Introduction Subtype classification of lung adenocarcinoma (LUAD) divides different survivals and therapeutic vulnerabilities; however, little is known about the disease’s underlying molecular mechanism. This study sought to determine the genetic and immune profiles of histologic subtypes and identify the evidence for adjuvant immunotherapy. Methods We performed an integrated analysis of multidimensional data from a discovery set consisting of cohorts of The Cancer Genome Atlas and the Broad Institute data set from the LUAD public database and a validation set from the Guangdong Lung Cancer Institute. Immunohistochemical staining was carried out to determine the expression of the proteins programmed cell death 1 ligand (PD‐L1) and CD8. Results Patients with solid predominant LUAD showed poor disease‐free survival and a high frequency of relapse/metastasis compared with those with the nonsolid subtype of LUAD. The solid subtype tended to occur more frequently in those with a history of smoking. Solid predominant LUAD exclusively showed increased expression of PD‐L1 and a high proportion of dual positive PD‐L1– and tumor‐infiltrating lymphocytes. Meanwhile, a notable increase in the tumor mutation burden and higher frequency of GC>TA transversions were specifically identified in tumors of the solid subtype. Furthermore, the solid subtype of tumor displayed an active cytotoxic immune signature and increased incidence of genetic mutations related to immunogenicity. Conclusion Solid predominant LUAD was identified as a subtype with adaptive immune resistance, higher cytotoxic activity, and enhanced immunogenicity. These findings suggest that patients with solid predominant LUAD may represent a potential selective group that will benefit from adjuvant programmed cell death 1 blockade immunotherapy.

Comparing overall survival between first generation EGFR-TKIs and chemotherapy in lung cancer patients with Del19/L858R

Objective Combined overall survival (OS) analysis of Lux-Lung 3 and Lux-Lung 6 demonstrated that patients with epidermal growth factor receptor (EGFR) exon 19 deletions (Del19) would benefit from first-line second generation EGFR tyrosine kinase inhibitors (TKIs) afatinib but not for those with L858R. This study was to investigate the survival difference between first-line first generation EGFR-TKIs and chemotherapy in patients with either Del19 or L858R, and to directly compare OS in these two mutation groups. Methods Eligibles were all prospective and retrospective studies comparing EGFR-TKIs with conventional chemotherapy or receiving single agent EGFR-TKIs and demonstrating survival analysis based on mutation types. The primary outcome was OS measured as pooled hazard ratios (HRs). All measures were pooled using randomeffects models and 95% confidential interval (95% CI) was calculated. Results A total of 14 studies incorporating 1,706 patients with either Del19 or L858R were included. Enrolling patients with Del19 or L858R in randomized controlled trials (RCTs), first-line first generation EGFR-TKIs were associated with no OS benefit, compared with chemotherapy (pooled HRTKI/Chemo for Del19: 0.82, 95% CI: 0.64-1.06, P = 0.14; pooled HRTKI/Chemo for L858R: 1.15, 95% CI: 0.85-1.56, P = 0.38). Direct comparison of Del19 with L858R receiving with first-line first generation EGFR-TKIs demonstrated no significant survival difference (pooled HR19/21: 0.88, 95% CI: 0.67-1.16, P = 0.37). Conclusions Among patients with advanced non-small cell lung cancer (NSCLC) harboring Del19 and L858R, first-line first generation EGFR-TKIs demonstrated no survival benefit comparing with chemotherapy. Direct comparison between Del19 and L858R revealed no significant survival difference after first-line first generation EGFR-TKIs.

Mixed Responses to Systemic Therapy Revealed Potential Genetic Heterogeneity and Poor Survival in Patients with Non-Small Cell Lung Cancer.

BACKGROUND A subset of patients with non-small cell lung cancer (NSCLC) fosters mixed responses (MRs) to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) or chemotherapy. However, little is known about the clinical and molecular features or the prognostic significance and potential mechanisms. METHODS The records of 246 consecutive patients with NSCLC receiving single-line chemotherapy or TKI treatment and who were assessed by baseline and interim positron emission tomography/computed tomography scans were collected retrospectively. The clinicopathological correlations of the MR were analyzed, and a multivariate analysis was performed to explore the prognostic significance of MR. RESULTS The overall incidence of MR to systemic therapy was 21.5% (53/246) and predominated in patients with stage IIIB-IV, EGFR mutations and those who received TKI therapy (p < .05). Subgroup analyses based on MR classification (efficacious versus inefficacious) showed significant differences in subsequent treatment between the two groups (p < .001) and preferable progression-free survival (PFS) and overall survival (OS) in the efficacious MR group. Multivariate analyses demonstrated that the presence of MR was an independent unfavorable prognostic factor for PFS (hazard ratio [HR], 1.474; 95% confidence interval [CI], 1.018-2.134; p = .040) and OS (HR, 1.849; 95% CI, 1.190-2.871; p = .006) in patients with NSCLC. Induced by former systemic therapy, there were more T790M (18%), concomitant EGFR mutations (15%), and changes to EGFR wild type (19%) in the MR group among patients with EGFR mutations, which indicated higher incidence of genetic heterogeneity. CONCLUSION MR was not a rare event in patients with NSCLC and tended to occur in those with advanced lung adenocarcinoma treated with a TKI. MR may result from genetic heterogeneity and is an unfavorable prognostic factor for survival. Further studies are imperative to explore subsequent treatment strategies. The Oncologist 2017;22:61-69Implications for Practice: Tumor heterogeneity tends to produce mixed responses (MR) to systemic therapy, including TKI and chemotherapy; however, the clinical significance and potential mechanisms are not fully understood, and the subsequent treatment after MR is also a clinical concern. The present study systemically assessed patients by PET/CT and differentiated MR and therapies. The study identified a relatively high incidence of MR in patients with advanced NSCLC, particularly those treated with targeted therapies. An MR may be an unfavorable prognostic factor and originate from genetic heterogeneity. Further studies are imperative to explore subsequent treatment strategies.

Clinical relevance of PD-L1 expression and CD8+ T cells infiltration in patients with EGFR-mutated and ALK-rearranged lung cancer

OBJECTIVES EGFR-mutated or ALK-rearranged non-small cell lung cancer (NSCLC) often showed unfavorable clinical benefit to checkpoint inhibitors (CPIs). However, few reports exist with integrated analysis, to interpret the underlying mechanism of poor response to PD-1/PD-L1 inhibitors. We have retrospectively analyzed the tumor microenvironment (TME) based on tumor PD-L1 expression and CD8+ T cells infiltration in patients with EGFR mutations and ALK rearrangements, and the prognostic value of TME subtypes on overall survival (OS). MATERIALS AND METHODS Tumor samples from 715 patients with lung cancer were retrospectively collected at Guangdong Lung Cancer Institute. Tumoral PD-L1 expression (N = 715) and CD8+ T cells infiltration (N = 658) was determined by immunohistochemistry (IHC), based on which TME was categorized into four different subtypes: PD-L1+/CD8+, PD-L1-/CD8+, PD-L1+/CD8-, PD-L1-/CD8-. Proportion of four TME subtypes was determined, and overall survival with PD-L1 expression and TME was analyzed. RESULTS In patients with EGFR mutations or ALK rearrangements, proportion of PD-L1+/CD8+ tumors was the lowest (5.0%, 17/342), and that of PD-L1-/CD8- tumors was the highest (63.5%, 217/342). In patients with wild-type EGFR and ALK, 14.2% (45/316) tumors were PD-L1+/CD8+ and 50.3% (159/316) tumors were PD-L1-/CD8- (P < 0.001). Median OS of EGFR-mutated or ALK-rearranged lung cancer was 78.6 months in PD-L1 positive group and 93.4 months in PD-L1 negative group (HR 0.47, 95%CI 0.23-0.76, P = 0.005). PD-L1+/CD8+ group exhibited the shortest OS, with 44.3 months, but is likely to respond to CPIs. The PD-L1-/CD8+ group exhibited the longest OS but is unlikely to respond to CPIs. CONCLUSION Patients with EGFR mutations or ALK rearrangements exhibited lower PD-L1 and CD8 co-expression level in TME, which could be responsible for poor response to CPIs. PD-L1 and CD8 co-expression in EGFR-mutated or ALK-rearranged lung cancer is a biomarker for poor prognosis with shorter OS.