Prognostic prediction of lung adenocarcinoma by integrative analysis of RHOH expression and methylation.

BACKGROUND AND OBJECTIVE: The development of epigenetics holds great promise for diagnosis and treatment of lung adenocarcinoma (LUAD). The purpose of this work was to analyze the correlation between Ras Homolog Gene Family Member H (RHOH) expression and methylation in patients with LUAD and its association with survival. METHODS: Data related to gene expression, DNA methylation, and clinical features of LUAD from The Cancer Genome Atlas (TCGA) database were analyzed. A total of 50 patients were included in verification group. The methylation level of RHOH in verification group was detected by bisulfite amplicon sequencing. RESULTS: The RHOH methylation level in TCGA cohort was significantly and negatively correlated with its expression level (Cor = -0.5, p = 2.687e-33). Patients with hypermethylation and low expression of RHOH had significantly worse prognosis than patients with hypomethylation and low expression of RHOH (TCGA: p = 0.004; validation cohort: p = 0.006, HR: 4.740, 95% CI: 1.567-14.340). CONCLUSION: Our research revealed that RHOH may prove to be a new potential prognostic predictor for LUAD patients.

A systematic dissection of the epigenomic heterogeneity of lung adenocarcinoma reveals two different subclasses with distinct prognosis and core regulatory networks.

BACKGROUND: Lung adenocarcinoma (LUAD) is a highly malignant and heterogeneous tumor that involves various oncogenic genetic alterations. Epigenetic processes play important roles in lung cancer development. However, the variation in enhancer and super-enhancer landscapes of LUAD patients remains largely unknown. To provide an in-depth understanding of the epigenomic heterogeneity of LUAD, we investigate the H3K27ac histone modification profiles of tumors and adjacent normal lung tissues from 42 LUAD patients and explore the role of epigenetic alterations in LUAD progression. RESULTS: A high intertumoral epigenetic heterogeneity is observed across the LUAD H3K27ac profiles. We quantitatively model the intertumoral variability of H3K27ac levels at proximal gene promoters and distal enhancers and propose a new epigenetic classification of LUAD patients. Our classification defines two LUAD subgroups which are highly related to histological subtypes. Group II patients have significantly worse prognosis than group I, which is further confirmed in the public TCGA-LUAD cohort. Differential RNA-seq analysis between group I and group II groups reveals that those genes upregulated in group II group tend to promote cell proliferation and induce cell de-differentiation. We construct the gene co-expression networks and identify group-specific core regulators. Most of these core regulators are linked with group-specific regulatory elements, such as super-enhancers. We further show that CLU is regulated by 3 group I-specific core regulators and works as a novel tumor suppressor in LUAD. CONCLUSIONS: Our study systematically characterizes the epigenetic alterations during LUAD progression and provides a new classification model that is helpful for predicting patient prognosis.

EGFR-mutant lung adenocarcinoma harboring co-mutational tumor suppressor genes predicts poor prognosis.

INTRODUCTION: EGFR mutations occur most frequently in patients with lung adenocarcinoma in East Asia. However, the prognostic and therapeutic impact of co-mutational status of EGFR and tumor suppressor genes is not fully understood. This study aims to provide a deeper understanding of lung adenocarcinoma patients with co-mutation of EGFR and tumor suppressor genes. METHODS: From November 2009 to May 2016, 675 patients with lung adenocarcinoma who underwent complete surgery were included in this study. Samples were collected and pathologically examined. Whole-exome sequencing was performed on 197 samples, while direct sequencing of major driver genes, including EGFR, KRAS, ERBB2 and BRAF and Ion-torrent targeted sequencing of tumor suppressor genes, including TP53, KEAP1, MGA, NF1, RB1, SMARCA4 and STK11, were performed on 478 samples. Tumor mutational burden was calculated and survival analyses were performed. RESULTS: The frequency of EGFR and TP53 mutation was 409 (60.6%) and 215 (31.9%), respectively. Co-mutation of EGFR and TP53 occured in 151 patients (22.4%), while co-mutation of EGFR and at least one tumor suppressor gene occured in 184 patients (27.3%). Compared with patients with only EGFR mutations, patients with co-mutations of EGFR and TP53 had a higher tumor mutational burden (p = 0.007) and worse recurrence-free survival (p = 0.010), while patients with co-mutations of EGFR and at least one tumor suppressor gene had a higher tumor mutational burden (p = 0.007), worse recurrence-free survival (p = 0.016) and worse overall survival (p = 0.018). CONCLUSIONS: Lung adenocarcinoma patients harboring EGFR and co-mutational tumor suppressor genes should be regarded as a unique subgroup.

The CD24+ cell subset promotes invasion and metastasis in human osteosarcoma.

BACKGROUND: Osteosarcoma is the most common primary aggressive bone tumor affecting children and young adolescents. Metastases are often resistant to conventional chemotherapy and mean short-term survival. Development of valuable diagnostic indicators and targeting agents will have important implications for clinical diagnosis by the identification and characterization of molecules that contribute to its aggressive behavior. METHODS: We examined differential expression levels of common stem cell markers in osteosarcoma parental and sphere cells. In addition, we further analyzed the changes of candidate common stem cell markers before and after in vitro chemotherapy of osteosarcoma cells. The biological functions of CD24+ subpopulation in osteosarcoma such as proliferation, migration, invasion, tumorigenesis and metastasis were systematically investigated, and the correlations of CD24 levels with prognosis in patients with osteosarcoma were analyzed. FINDINGS: CD24+ Cells presented characteristics of TICs and resist drug-induced apoptosis. The prevention of tumor formation and metastasis by CD24 knockdown highlights the potential of CD24 as a therapeutic target for osteosarcoma. Moreover, the levels of CD24 in osteosarcoma samples were significantly correlated with the prognosis of patients. INTERPRETATION: CD24+ cell subset played an important role in osteosarcoma invasion and metastasis. FUNDING: National Natural Science Foundation of China (No.81772857); Shanghai Science and Technology Commission (18140902000); Shanghai Municipal Health Commission (2017ZZ01017; 17411950301).

Genomic and immune profiling of pre-invasive lung adenocarcinoma.

Adenocarcinoma in situ and minimally invasive adenocarcinoma are the pre-invasive forms of lung adenocarcinoma. The genomic and immune profiles of these lesions are poorly understood. Here we report exome and transcriptome sequencing of 98 lung adenocarcinoma precursor lesions and 99 invasive adenocarcinomas. We have identified EGFR, RBM10, BRAF, ERBB2, TP53, KRAS, MAP2K1 and MET as significantly mutated genes in the pre/minimally invasive group. Classes of genome alterations that increase in frequency during the progression to malignancy are revealed. These include mutations in TP53, arm-level copy number alterations, and HLA loss of heterozygosity. Immune infiltration is correlated with copy number alterations of chromosome arm 6p, suggesting a link between arm-level events and the tumor immune environment.

FGB and FGG derived from plasma exosomes as potential biomarkers to distinguish benign from malignant pulmonary nodules.

Previous proteomic analysis (label-free) of plasma exosomes revealed that the expression of FGG and FGB was significantly higher in the malignant pulmonary nodules group, compared to the benign pulmonary nodules group. The present study was performed to evaluate the role of plasma exosomal proteins FGB and FGG in the diagnosis of benign and malignant pulmonary nodules. We examined the expression levels of FGB and FGG in plasma exosomes from 63 patients before surgery. Postoperative pathological diagnosis confirmed that 43 cases were malignant and 20 cases were benign. The ROC curve was used to describe the sensitivity, specificity, area under the curve (AUC) of the biomarker and the corresponding 95% confidence interval. We confirmed that the expression levels of FGB and FGG were higher in the plasma exosomes of malignant group than in the benign group. The sensitivity and AUC of FGB combined with FGG detection to determine the nature of pulmonary nodules are superior to single FGB or FGG detection. FGB and FGG might represent novel and sensitive biomarker to distinguish benign from malignant pulmonary nodules.

tRNA-based prognostic score in predicting survival outcomes of lung adenocarcinomas.

As the most abundant noncoding RNA in cells, tRNA plays an important role in tumorigenesis and development. The report of tRNA on the pathogenesis of lung adenocarcinoma is rare. It is of great clinical significance to explore the relationship between tRNA expression and prognosis of lung adenocarcinoma. The expression level of tRNAs in lung adenocarcinoma tissues and paracarcinoma tissues was detected using a tRNA RT-qPCR array. A total of 104 lung adenocarcinomas were included in the analysis of the correlation between candidate tRNAs expression and prognosis. A tRNA-based prognostic model was constructed and validated using Cox proportional hazards regression. A nomogram was built to help clinicians develop treatment strategies. We screened a series of differentially expressed tRNAs between lung adenocarcinoma tissues and paracarcinoma tissues. Among these tRNAs, tRNAAsnATT , tRNAIleAAT , tRNALeuTAA , mt-tRNATrpTCA , mt-tRNALeuTAA , tRNAProAGG , tRNALysCTT-1 and tRNALeuAAG were associated with the clinicopathological characteristics of lung adenocarcinoma. tRNALysCTT-1 , mt-tRNASerGCT and tRNATyrATA were associated with cancer-specific survival. We constructed a prognostic model for lung adenocarcinoma using specific tRNA expression levels as reference factors. Multivariate analyses showed that tRNA-based prognostic score was a significant and important prognostic factor. The prognostic model based on the tRNAs expression signatures can help predict the prognosis of patients with lung adenocarcinoma.

Proteomic analysis of plasma exosomes to differentiate malignant from benign pulmonary nodules.

BACKGROUND: It is difficult to distinguish benign pulmonary nodules (PNs) from malignant PNs by conventional examination. Therefore, novel biomarkers that can identify the nature of PNs are needed. Exosomes have recently been identified as an attractive alternative approach since tumor-specific molecules can be found in exosomes isolated from biological fluids. METHODS: Plasma exosomes were extracted via the exoEasy reagent method. The major proteins from plasma exosomes in patients with PNs were identified via labelfree analysis and screened for differentially expressed proteins. A GO classification analysis and KEGG pathway analysis were performed on plasma exosomal protein from patients with benign and malignant PNs. RESULTS: Western blot confirmed that protein expression of CD63 and CD9 could be detected in the exosome extract. Via a search of the human Uniprot database, 736 plasma exosome proteins from patients with PNs were detected using high-confidence peptides. There were 33 differentially expressed proteins in the benign and malignant PNs. Of these, 12 proteins were only expressed in the benign PNs group, while 9 proteins were only expressed in the malignant PNs group. We further obtained important information on signaling pathways and nodal proteins related to differential benign and malignant PNs via bioinformatic analysis methods such as GO, KEGG, and String. CONCLUSIONS: This study provides a new perspective on the identification of novel detection strategies for benign and malignant PNs. We hope our findings can provide clues for the identification of benign and malignant PNs.

Clinical Significance of Complex Glandular Patterns in Lung Adenocarcinoma: Clinicopathologic and Molecular Study in a Large Series of Cases.

OBJECTIVES: To explore whether complex glandular patterns (CGPs) have a potential role in the clinical management of patients with lung adenocarcinoma. METHODS: We included 356 patients with lung adenocarcinoma with available clinicopathologic information, gene mutations, and clinical outcomes for analysis. RESULTS: We identified 54 (15.2%) CGP-predominant cases. The CGPs were associated with ALK rearrangement and HER2 mutation. Survival analysis showed that the clinical outcome of CGP-predominant patients was worse than that for acinar-predominant patients (overall survival [OS], 66.4 vs 90.3 months, P < .01; recurrence-free survival [RFS], 50.1 vs 73.1 months, P = .022) but was comparable with solid-predominant subtype tumors (OS, 66.4 vs 67.8 months, P = .558; RFS, 50.1 vs 41.3 months, P = .258). In particular, the coexistence of the cribriform and fused gland pattern was associated with the poorest survival, with a death risk increased by 2.25-fold (hazard ratio, 3.25; 95% confidence interval, 1.35-7.86, P = .009). CONCLUSIONS: Our results provide new insight into the potential role of CGPs in clinical management and will be beneficial for treatment decision making in patients with lung adenocarcinoma.

ALCAM+ stromal cells: role in giant cell tumor of bone progression.

Giant cell tumor of bone(GCTB) is a special benign tumor with variable aggressiveness and recurrence rate. Increasing evidences suggest that a subset of cells called cancer stem cells (CSCs) are present as cancer-initiating cells in a range of malignant tumors. However, the role of CSCs in benign tumor such as GCTB remains unknown, and the connection between the presence of CSCs and biological characteristics of GCTB is unclear. To investigate this issue, we screened a panel of markers of normal stem cells and CSCs and found ALCAM+ stromal cells possessed characteristics of stem-like cells. Subsequently a series of experiments such cell proliferation, migration and invasion assays were performed to investigate the biological characteristics of ALCAM+ stromal cells in vivo and in vitro. The clinical significance of ALCAM expression were further evaluated using Kaplan-Meier analyses. The ALCAM+ GCTB cells showed the stem cell properties of self renewal and had the capacity to differentiate in vitro. The ALCAM+ GCTB cells showed increased resistance for chemotherapy- or radiation-induced cell death. ALCAM knockdown reduced stem/progenitor characteristics in GCTB Cells. Furthermore, ALCAM expression was associated with outcome in GCTB patients. Our work demonstrates for the first time ALCAM+ tumorigenic sub-population within stromal GCTB cells and may represent a potential therapeutic target in aggressive and recurrent GCTBs.

Super enhancer associated RAI14 is a new potential biomarker in lung adenocarcinoma.

PURPOSE: Tyrosine kinase inhibitors (TKIs) are widely used to treat lung adenocarcinoma patients with EGFR mutations or ALK-fusions. However, patients with wild-type genes or TKIs-resistant mutations lack effective therapeutic targets. Extensive studies reveal that super enhancer (SE), a large cis-regulatory element, is associated with key oncogenes in a variety of cancers. By comparing the effect of SE on lung adenocarcinoma cell lines with normal cell line, this work attempts to find new biomarkers and potential therapeutic targets for lung adenocarcinoma. EXPERIMENTAL DESIGN: Chromatin Immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of H3K27ac (acetylation on lysine 27 of histone 3) was performed in lung adenocarcinoma cell lines SPC-A1 and SCH-1153. The differences in SE distribution were then analyzed among SPC-A1, SCH-1153, A549 and normal human lung fibroblasts (NHLF) to identify SE-associated oncogenes. The expression of SE-associated oncogenes was then detected by RNA-seq and further verified in 71 patients by real-time PCR. RESULTS: SE associated with many new oncogenes in lung adenocarcinoma, among which, RAI14 was up-regulated in A549 and 31 of 71 patients. High expression of RAI14 could inhibit cell proliferation, indicating its potential as a new biomarker for lung adenocarcinoma.