MicroRNAs (miRNAs) are small, noncoding RNAs that can contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies have also linked different sets of miRNAs to metastasis through either the promotion or suppression of this malignant process. Interestingly, epigenetic silencing of miRNAs with tumor suppressor features by CpG island hypermethylation is also emerging as a common hallmark of human tumors. Thus, we wondered whether there was a miRNA hypermethylation profile characteristic of human metastasis. We used a pharmacological and genomic approach to reveal this aberrant epigenetic silencing program by treating lymph node metastatic cancer cells with a DNA demethylating agent followed by hybridization to an expression microarray. Among the miRNAs that were reactivated upon drug treatment, miR-148a, miR-34b/c, and miR-9 were found to undergo specific hypermethylationassociated silencing in cancer cells compared with normal tissues. The reintroduction of miR-148a and miR-34b/c in cancer cells with epigenetic inactivation inhibited their motility, reduced tumor growth, and inhibited metastasis formation in xenograft models, with an associated down-regulation of the miRNA oncogenic target genes, such as C-MYC, E2F3, CDK6, and TGIF2. Most important, the involvement of miR-148a, miR-34b/c, and miR-9 hypermethylation in metastasis formation was also suggested in human primary malignancies (n ‫؍‬ 207) because it was significantly associated with the appearance of lymph node metastasis. Our findings indicate that DNA methylation-associated silencing of tumor suppressor miRNAs contributes to the development of human cancer metastasis.

Mechanisms of acquired resistance to immune checkpoint inhibitors (ICIs) are poorly understood. We leveraged a collection of 14 ICI-resistant lung cancer samples to investigate whether alterations in genes encoding HLA Class I antigen processing and presentation machinery (APM) components or interferon signaling play a role in acquired resistance to PD-1 or PD-L1 antagonistic antibodies. Recurrent mutations or copy number changes were not detected in our cohort. In one case, we found acquired homozygous loss of B2M that caused lack of cell surface HLA class I expression in the tumor and a matched patient-derived xenograft (PDX). Downregulation of B2M was also found in two additional PDXs established from ICI-resistant tumors. CRISPR-mediated knock-out of B2m in an immunocompetent lung cancer mouse model conferred resistance to PD-1 blockade in vivo proving its role in resistance to ICIs. These results indicate that HLA Class I APM disruption can mediate escape from ICIs in lung cancer.

Rationale: Identification of risk factors for lung cancer can help in selecting patients who may benefit the most from smoking cessation interventions, early detection, or chemoprevention. Objective: To evaluate whether the presence of emphysema on low-radiation-dose CT (LDCT) of the chest is an independent risk factor for lung cancer. Methods: The study used data from a prospective cohort of 1,166 former and current smokers participating in a lung cancer screening study. All individuals underwent a baseline LDCT and spirometry followed by yearly repeat LDCT studies. The incidence density of lung cancer among patients with and without emphysema on LDCT was estimated. Stratified and multiple regression analyses were used to assess whether emphysema is an independent risk factor for lung cancer after adjusting for age, gender, smoking history, and the presence of airway obstruction on spirometry. Abbreviations: CI ϭ confidence interval; IQR ϭ interquartile range; LDCT ϭ low-radiation-dose CT; RR ϭ risk ratio L ung cancer is the deadliest malignancy in the world among men and women.

The complement system contributes to various immune and inflammatory diseases, including cancer. In this study we investigated the capacity of lung cancer cells to activate complement, and characterized the consequences of complement activation on tumor progression. We focused our study on the production and role of the anaphylatoxin C5a, a potent immune mediator generated after complement activation. We first measured the capacity of lung cancer cell lines to deposit C5 and release C5a. C5 deposition, after incubation with normal human serum, was higher in lung cancer cell lines than in non-malignant bronchial epithelial cells. Interestingly, lung malignant cells produced complement C5a even in the absence of serum. We also found a significant increase of C5a in plasma from patients with non-small cell lung cancer, suggesting that the local production of C5a is followed by its systemic diffusion. The contribution of C5a to lung cancer growth in vivo was evaluated in the Lewis lung cancer model. Syngeneic tumors of 3LL cells grew slower in mice treated with an antagonist of the C5a receptor. C5a did not modify 3LL cell proliferation in vitro but induced endothelial cell chemotaxis and blood-vessels formation. C5a also contributed to the immunosuppressive microenvironment required for tumor growth. In particular, blockade of C5a receptor significantly reduced myeloid-derived suppressor cells and immunomodulators ARG1, CTLA-4, IL6, IL10, LAG3 and PDL1 (B7H1). In conclusion, lung cancer cells have the capacity to generate C5a, a molecule that creates a favorable tumor microenvironment for lung cancer progression.

The present review is an update of the research and development efforts regarding the use of molecular biomarkers in the lung cancer screening setting. The two main unmet clinical needs, namely, the refinement of risk to improve the selection of individuals undergoing screening and the characterization of undetermined nodules found during the computed tomography-based screening process are the object of the biomarkers described in the present review. We first propose some principles to optimize lung cancer biomarker discovery projects. Then, we summarize the discovery and developmental status of currently promising molecular candidates, such as autoantibodies, complement fragments, microRNAs, circulating tumor DNA, DNA methylation, blood protein profiling, or RNA airway or nasal signatures. We also mention other emerging biomarkers or new technologies to follow, such as exhaled breath biomarkers, metabolomics, sputum cell imaging, genetic predisposition studies, and the integration of nextgeneration sequencing into study of circulating DNA. We also underline the importance of integrating different

Components of the SWI/SNF chromatin-remodeling complex, such as INI1, are inactivated in human cancer and, thus, act as tumor suppressors. Here we screened for mutations the entire coding sequence of BRG1 (SMARCA4), which encodes the ATPase of the complex, in 59 lung cancer cell lines of the most common histopathological types. Mutations were detected in 24% of the cancer cell lines, many of them in cells commonly used for lung cancer research. All mutations were homozygous and most predicted truncated proteins. The alterations were significantly more frequent in the non-small-cell lung cancer (NSCLC) type (13/37, 35%) as compared to the small-cell lung cancer (SCLC) type (1/19, 5%) (P<0.05; Fisher's Exact test) and BRG1 was the fourth most frequently altered gene in NSCLC cell lines. BRG1 mutations coexisted with mutations/deletions at KRAS, LKB1, NRAS, P16, and P53. However, alterations at BRG1 always occurred in the absence of MYC amplification, suggesting a common role in lung cancer development. In conclusion, our data strongly support that BRG1 is a bona fide tumor suppressor and a major factor in lung tumorigenesis.

A B S T R A C T PurposeNon-small-cell lung cancer (NSCLC) is a tumor in which only small improvements in clinical outcome have been achieved. The issue is critical for stage I patients for whom there are no available biomarkers that indicate which high-risk patients should receive adjuvant chemotherapy. We aimed to find DNA methylation markers that could be helpful in this regard. Patients and MethodsA DNA methylation microarray that analyzes 450,000 CpG sites was used to study tumoral DNA obtained from 444 patients with NSCLC that included 237 stage I tumors. The prognostic DNA methylation markers were validated by a single-methylation pyrosequencing assay in an independent cohort of 143 patients with stage I NSCLC. ResultsUnsupervised clustering of the 10,000 most variable DNA methylation sites in the discovery cohort identified patients with high-risk stage I NSCLC who had shorter relapse-free survival (RFS; hazard ratio [HR], 2.35; 95% CI, 1.29 to 4.28; P ϭ .004). The study in the validation cohort of the significant methylated sites from the discovery cohort found that hypermethylation of five genes was significantly associated with shorter RFS in stage I NSCLC: HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9. A signature based on the number of hypermethylated events distinguished patients with high-and low-risk stage I NSCLC (HR, 3.24; 95% CI, 1.61 to 6.54; P ϭ .001). ConclusionThe DNA methylation signature of NSCLC affects the outcome of stage I patients, and it can be practically determined by user-friendly polymerase chain reaction assays. The analysis of the best DNA methylation biomarkers improved prognostic accuracy beyond standard staging.

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