In vivo CRISPR screening unveils histone demethylase UTX as an important epigenetic regulator in lung tumorigenesis.

Lung cancer is the leading cause of cancer-related death worldwide. Inactivation of tumor suppressor genes (TSGs) promotes lung cancer malignant progression. Here, we take advantage of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated somatic gene knockout in a KrasG12D/+ mouse model to identify bona fide TSGs. From individual knockout of 55 potential TSGs, we identify five genes, including Utx, Ptip, Acp5, Acacb, and Clu, whose knockout significantly promotes lung tumorigenesis. These candidate genes are frequently down-regulated in human lung cancer specimens and significantly associated with survival in patients with lung cancer. Through crossing the conditional Utx knockout allele to the KrasG12D/+ mouse model, we further find that Utx deletion dramatically promotes lung cancer progression. The tumor-promotive effect of Utx knockout in vivo is mainly mediated through an increase of the EZH2 level, which up-regulates the H3K27me3 level. Moreover, the Utx-knockout lung tumors are preferentially sensitive to EZH2 inhibitor treatment. Collectively, our study provides a systematic screening of TSGs in vivo and identifies UTX as an important epigenetic regulator in lung tumorigenesis.

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.

Frequency and clinical significance of NF1 mutation in lung adenocarcinomas from East Asian patients.

NF1 is a tumor suppressor gene that negatively regulates Ras signaling. NF1 deficiency plays an important role in carcinogenesis. To investigate the frequency and clinical significance of NF1 mutation, we examined mutation status of NF1, TP53, LKB1 and RB1 in 704 surgically resected lung adenocarcinomas from East Asian patients using semiconductor-based Ion Torrent sequencing platform. Common driver events, including mutations in EGFR, KRAS, HER2, BRAF, MET, and fusions affecting ALK, RET and ROS1, were also concurrently detected. The correlation between NF1 mutations and clinicomolecular features of patients was further evaluated. Among 704 patients, 42 NF1 mutations were found in 33 patients (33/704, 4.7%), including 14 patients harboring EGFR/NF1 comutations (14/33, 42.4%). Comparing with EGFR-mutant patients, patients harboring NF1 mutations were closely associated with solid component subtype (p = 0.028). Comparing with KRAS mutations, NF1 mutations were found more common in female and never smokers (p = 0.003 and p = 0.004, respectively). Kaplan-Meier survival analysis revealed that patients harboring NF1 mutation had similar disease-free survival (DFS) and overall survival (OS) with patients with KRAS mutation. Although frequently overlapped with EGFR mutation, patients harboring NF1 mutation had significantly shorter DFS (p = 0.019) and OS (p = 0.004) than patients with EGFR mutation. During follow-up, one female patient with EGFR exon 19 deletion and NF1 Q1815X comutation showed poor response to EGFR TKIs (Gefitinib and Osimertinib) after disease relapse. In conclusion, NF1 mutations define a unique molecular and clinicopathologic subtype of lung adenocarcinoma. Examination of NF1 mutation may contribute to molecular subtyping and therapeutic intervention of 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.

The lymph node status and histologic subtypes influenced the effect of postoperative radiotherapy on patients with N2 positive IIIA non-small cell lung cancer.

BACKGROUND AND OBJECTIVE: To investigate the role of postoperative radiotherapy (PORT) in IIIA-N2 non-small cell lung cancer (NSCLC) patients and subgroups which derived benefit from PORT. METHODS: A total of 576 patients with pathological IIIA-N2 NSCLC, who underwent complete resection, were identified. Propensity score matching (PSM) methods were used to balance the patients' characteristics between two groups. Overall survival (OS) and relapse-free survival (RFS) were compared between PORT and non-PORT patients. RESULTS: On multivariable analysis, improved OS remained correlated with younger age, single N2 station involvement, less positive lymph nodes, and chemotherapy. After PSM, 121 PROT patients and 242 non-PORT patients were matched. PORT was not associated improved patients' OS (P = 0.735) or RFS ( P = 0.483). For patients who underwent postoperative chemotherapy (POCT), PORT could improve OS in single N2 station involved patients (HR: 0.572, 95%CI: 0.312 to 1.05, P = 0.040). Patients with papillary predominant adenocarcinoma also benefited from PORT with an increase in OS (HR: 0.350, 95%CI: 0.126 to 0.972, P = 0.033). CONCLUSIONS: For patients with completely resected IIIA-N2 NSCLC, mediastinal lymph node metastasis and histologic subtypes could influence the effect of PORT. Single N2 station involvement and papillary predominant subtype were predictors of benefit from PORT.

Genomic alterations dissection revealed MUC4 mutation as a potential driver in lung adenocarcinoma local recurrence.

Background: Lung adenocarcinoma (LUAD) is the most common histological type of lung cancer, of which genomic alterations play a major role in tumorigenesis. The prognosis of LUAD has been improved these years but nearly half of the patients still develop recurrence even after radical resection. The underlying mechanism driving LUAD recurrence especially genomic alterations is complicated and worth exploring. Methods: Forty-one primary tumors and 43 recurrent tumors were collected from 41 LUAD patients who received surgery resection after recurrence. Whole exon sequencing (WES) was performed to make genomic landscapes. WES data were aligned to genome and further analyzed for somatic mutation, copy number variation and structure variation. MutsigCV was used to identify significantly mutated genes and recurrence specific genes. Results: Significantly mutated genes including EGFR, MUC4 and TP53 were identified in primary and recurrent tumors. Some were found to be more specifically mutated in recurrent tumors, such as the MUC17, KRAS and ZNF families. In recurrent tumors, ErbB signaling pathway, MAPK pathway and cell cycle pathway were highly activated, which maybe the mechanism driving recurrence. The adjuvant therapy would affect tumor evolution and molecular features during recurrence. MUC4 was highly mutated in this study cohort, and it was a potential driver gene in LUAD recurrence by activating ErbB signaling pathway as a ligand of ERBB2. Conclusions: Genomic alteration landscape was changing during LUAD recurrence to construct a more suitable environment for the survival of tumor cells. Several potential driver mutations and targets during LUAD recurrence were identified, such as MUC4, and more investigation was needed to verify the specific functions and roles.

Identified optimal candidates for pulmonary resection in octogenarians with non-small cell lung cancer: a web-based predictive model.

Background: A survival benefit from pulmonary resection was observed in octogenarians with non-small cell lung cancer (NSCLC). Meanwhile, the identification of patients who can indeed benefit can be difficult. Therefore, we aimed to establish a web-based predictive model to identify optimal candidates for pulmonary resection. Methods: Octogenarians with NSCLC in Surveillance, Epidemiology and End Results (SEER) database were enrolled and split into the surgery and non-surgery groups based on whether they received pulmonary resection. Propensity-score matching (PSM) was utilized to eliminate the imbalance. Independent prognostic factors were identified. Patients in the surgery group who lived longer than the median cancer-specific survival (CSS) time of the non-surgery group were assumed to benefit from the surgery. The surgery group was further divided into the beneficial group and the non-beneficial group based on the median CSS time of the non-surgery group. Among the surgery group, a nomogram was established through a logistic regression model. Results: A total of 14,264 eligible patients were extracted, with 4,475 (31.37%) patients receiving pulmonary resection. Surgery was an independent favorable factor of prognosis after PSM (median CSS time: 58 vs. 14 months, P<0.001). A total of 750 (70.4%) patients lived longer than 14 months (beneficial group) in the surgery group. Factors including age, gender, race, histologic type, differentiation grade, and tumor-node-metastasis (TNM) stage were used to formulate the web-based nomogram. The precise discrimination and predictive capability of the model were validated through receiver operating characteristic curves, calibration plots, and decision curve analyses. Conclusions: A web-based predicted model was constructed to distinguish specific patients who can indeed benefit from pulmonary resection among octogenarians with NSCLC.

HyperChIP: identification of hypervariable signals across ChIP-seq or ATAC-seq samples.

Identifying genomic regions with hypervariable ChIP-seq or ATAC-seq signals across given samples is essential for large-scale epigenetic studies. In particular, the hypervariable regions across tumors from different patients indicate their heterogeneity and can contribute to revealing potential cancer subtypes and the associated epigenetic markers. We present HyperChIP as the first complete statistical tool for the task. HyperChIP uses scaled variances that account for the mean-variance dependence to rank genomic regions, and it increases the statistical power by diminishing the influence of true hypervariable regions on model fitting. A pan-cancer case study illustrates the practical utility of HyperChIP.

Comparison of perioperative and survival outcomes between sublobar resection and lobectomy of patients who underwent a second pulmonary resection.

BACKGROUND: Repeat pulmonary resection is widely accepted in clinical practice. This study aimed to compare sublobar resection (segmentectomy or wedge resection) with lobectomy in the treatment of patients who underwent a second pulmonary resection. METHODS: This study retrospectively included patients who underwent lobectomy or sublobar resection for second pulmonary resection. 1:1 propensity score matching (PSM) was performed to balance selection bias. Clinicopathological features, perioperative and survival outcomes of lobectomy and sublobar resection were compared. RESULTS: A total of 308 patients who underwent second pulmonary resection were identified: 71 (23.1%) who underwent lobectomy and 237 (76.9%) who underwent sublobar resection. After PSM, 58 patients for each group were selected with well-balanced clinicopathological characteristics. In patients who underwent sublobar resection, significantly shorter chest tube duration (days) (median, 4 vs. 2, p < 0.001) and postoperative hospital stay (days) (median, 6 vs. 4, p < 0.001) were observed. There was no significant difference in overall survival between these two groups after the second and first surgery (p = 0.65, p = 0.98), respectively. Subgroup analysis according to the type of the first resection showed consistent results. CONCLUSIONS: Sublobar resection may be considered as an alternative option for second pulmonary resection due to its perioperative advantages and similar survival outcomes compared with lobectomy.

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.

Is flexible bronchoscopy necessary in the preoperative workup of patients with peripheral cT1N0 subsolid lung cancer? -a prospective multi-center cohort study.

BACKGROUND: Necessity of flexible bronchoscopy (FB) examination as a routine preoperative work-up for peripheral clinical T1N0 subsolid lung cancer was unknown. METHODS: This was a prospective, multi-center clinical trial (NCT03591445). Patients with peripheral GGO nodules (GGNs) who were candidates for surgical resection were enrolled. FB examination was performed preoperatively. Surgical plan could be changed if any aberrant histologic and anatomic findings were detected by FB examination. Primary endpoint was the rate that surgical plan was changed by positive FB findings. Secondary endpoints were rate of positive FB findings and rate of procedural complications. RESULTS: Six hundred and fifteen patients with peripheral subsolid nodules detected by thoracic CT were enrolled. There were 187 (30.4%) male and 428 (69.6%) female patients, mean age was 54.85±10.41 y (range, 26-78). 262 (42.6%) patients had pure GGNs and 353 (57.4%) patients had part-solid nodules. Mean size of nodules was 13.87±6.37 mm (range, 5-30). FB examinations confirmed one (0.16%) adenocarcinoma, seven (1.14%) bronchial variations, one (0.16%) segmental bronchostenosis, one (0.16%) segmental bronchial occlusion and one (0.16%) bronchial inflammation. No complications of FB examinations occurred. 568 (92.35%) thoracoscopic and 47 (7.65%) open surgeries were performed. No established surgical plan was changed by positive FB findings. Final pathologies revealed 26 (4.2%) adenocarcinoma in situ (AIS), 240 (39%) minimal invasive adenocarcinomas (MIAs), 343 (55.8%) invasive adenocarcinomas (IADs), one (0.2%) adenosquamous cell carcinoma, one (0.2%) squamous cell carcinoma, two (0.3%) atypical adenoid hyperplasia and two (0.3%) inflammations. CONCLUSIONS: FB examination was unnecessary in the preoperative assessment of peripheral clinical T1N0 subsolid lung cancer.

Integrated analysis of optical mapping and whole-genome sequencing reveals intratumoral genetic heterogeneity in metastatic lung squamous cell carcinoma.

BACKGROUND: Intratumoral heterogeneity is a crucial factor to the outcome of patients and resistance to therapies, in which structural variants play an indispensable but undiscovered role. METHODS: We performed an integrated analysis of optical mapping and whole-genome sequencing on a primary tumor (PT) and matched metastases including lymph node metastasis (LNM) and tumor thrombus in the pulmonary vein (TPV). Single nucleotide variants, indels and structural variants were analyzed to reveal intratumoral genetic heterogeneity among tumor cells in different sites. RESULTS: Our results demonstrated there were less nonsynonymous somatic variants shared with PT in LNM than in TPV, while there were more structural variants shared with PT in LNM than in TPV. More private variants and its affected genes associated with tumorigenesis and progression were identified in TPV than in LNM. It should be noticed that optical mapping detected an average of 77.1% (74.5-78.5%) large structural variants (>5,000 bp) not detected by whole-genome sequencing and identified several structural variants private to metastases. CONCLUSIONS: Our study does demonstrate structural variants, especially large structural variants play a crucial role in intratumoral genetic heterogeneity and optical mapping could make up for the deficiency of whole-genome sequencing to identify structural variants.

Outcomes comparison between neoadjuvant chemotherapy and adjuvant chemotherapy in stage IIIA non-small cell lung cancer patients.

BACKGROUND: A neoadjuvant chemotherapy (NCT) is a feasible second-option other than an adjuvant chemotherapy (ACT); however, no definite conclusions have been drawn about whether or not a NCT is associated with better clinical outcomes for IIIA non-small cell lung cancer (NSCLC) patients. METHODS: We reviewed 68 clinical IIIA NSCLC patients who received preoperative chemotherapy (NCT group), and 535 pathological IIIA NSCLC patients who received ACT after surgery (ACT group). After a 1:1 propensity score matching (PSM), we compared the relapse-free survival (RFS) and overall survival (OS) rates as the long-term clinical outcomes, and hospital stay, surgery duration, postoperative complications as the short-term clinical outcomes. To evaluate the predictive value of the NCT response, we also assessed the response evaluation criteria in solid tumors (RECIST) response to NCT. RESULTS: There was no significant difference in RFS or OS between the NCT group and ACT group (RFS: P=0.1138; OS: P=0.4234). On multivariate analysis, large cell lung carcinoma (P=0.0264), bilobectomy (P=0.0039) and clinical N2 stage (P=0.0309) were independent predictive factors of a worse OS. Short-term clinical outcomes including the hospital stay and postoperative complications had no statistically distinct difference between the ACT and NCT groups. Meanwhile, the OS of the partial response (PR) patients group was better than the stable disease/progressive disease (SD/PD) (P=0.0205) and ACT (P=0.0442) group, but none of the clinical features we tested was found to be a predictive factor for a PR response. CONCLUSIONS: There was a non-significant difference between the long-term and short-term clinical outcomes of both NCT and ACT. The OS of PR patients was better than SD/PD and ACT, indicating that NCT response acts as a predictor for a higher long-term survival rate.

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.

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.

Survival following segmentectomy or lobectomy in elderly patients with early-stage lung cancer.

PURPOSE: To determine the survival following segmentectomy versus lobectomy in elderly patients with early-stage non-small cell lung cancer (NSCLC). METHODS: We identified 12324 elderly (≥ 70 years) patients with stage I ≤ 3 cm NSCLC in the Surveillance, Epidemiology and End Results (SEER) database. Propensity score methods were used to balance baseline characteristics of patients undergoing segmentectomy or lobectomy. Overall survival (OS) and lung cancer-specific survival (LCSS) of patients treated with segmentectomy versus lobectomy were compared in Cox regression models after adjusting, stratifying or matching patients based on propensity scores. RESULTS: Cox models adjusting, stratifying or matching propensity scores all showed that patients treated with segmentectomy had significantly worse OS and LCSS compared to lobectomy. Subgroup analysis of patients with tumors ≤ 2cm, aged ≥ 75 years, or had ≥ 7 lymph nodes examined also revealed survival advantage associated with lobectomy. CONCLUSION: Elder age alone could not justify the application of segmentectomy in early-stage lung cancer. Prospective randomized trials are warranted to validate our results.