Tumor-Intrinsic Nuclear ß-Catenin Associates with an Immune Ignorance Phenotype and a Poorer Prognosis in Head and Neck Squamous Cell Carcinomas.

Activation of WNT/ß-catenin signaling has been associated with a non-T-cell-inflamed tumor microenvironment (TME) in several cancers. The aim of this work was to investigate the relationship between ß-catenin signaling and TME inflammation in head and neck squamous cell carcinomas (HNSCCs). Membrane and nuclear ß-catenin expression, PD-L1 expression, and CD8+ tumor-infiltrating lymphocyte (TIL) density were jointly evaluated by immunohistochemistry in a series of 372 HPV-negative HNSCCs. Membrane ß-catenin levels decreased in carcinomas compared to the normal epithelium. Positive nuclear ß-catenin was detected in 50 tumors (14.3%) and was significantly associated with a low CD8+ TIL density (168 cells/mm2 versus 293 cells/mm2 in nuclear-ß-catenin-negative cases; p = 0.01) and a tendency for a lower expression of PD-L1, resulting in association with a noninflamed TME (i.e., type II, immunological ignorance). Multivariate Cox analysis further demonstrated that low infiltration by CD8+ TILs (HR = 1.6, 95% CI = 1.19-2.14, p = 0.002) and nuclear ß-catenin expression (HR = 1.47, 95% CI = 1.01-2.16, p = 0.04) were both independently associated with a poorer disease-specific survival. In conclusion, tumor-intrinsic nuclear ß-catenin activation is associated with a non-inflamed TME phenotype and a poorer prognosis, thereby suggesting a possible implication as an immune exclusion mechanism for a subset of HNSCC patients.

PD-L1 expression correlates with tumor-infiltrating lymphocytes and better prognosis in patients with HPV-negative head and neck squamous cell carcinomas.

INTRODUCTION: The importance of immune tumor microenvironment in the prognosis of patients with head and neck squamous carcinomas (HNSCC) is increasingly recognized. We analyzed the prognostic relevance of PD-L1 and PD-1 expressions in relation to the infiltration by CD8+ and FOXP3+ tumor-infiltrating lymphocytes (TILs). METHODS: Samples from 372 surgically treated HPV-negative HNSCC patients were evaluated by immunohistochemistry for PD-L1 expression [both tumor proportion score (TPS) and combined proportion score (CPS)], PD-1 expression in immune cells, and density of infiltrating CD8+ and FOXP3+ TILs. PD-L1 expression and CD8+ TIL density were combined to establish the type of tumor microenvironment. RESULTS: 29.5% cases exhibited PD-L1 TPS positivity (≥ 1%), whereas PD-L1 CPS positivity (≥ 1%) was observed in 40% cases. 47.5% cases showed positive PD-1 expression (≥ 1%). PD-L1 and PD-1 positivity correlated with a high density of both CD8+ and FOXP3+ TILs. In univariate analysis, PD-L1 TPS positivity (P = 0.026), PD-L1 CPS positivity (P = 0.004), high density of CD8+ TIL (P = 0.001), and high density of FOXP3+ TIL (P = 0.004) were associated with a better disease-specific survival (DSS). However, in multivariate analysis, only high density of CD8+ TIL was associated with a better DSS (P = 0.002). The type of tumor microenvironment correlated with DSS (P = .008), with the better DSS observed in cases with type I (PD-L1 CPS positivity and high density of CD8+ TIL). CONCLUSIONS: High infiltration by CD8+ TIL is associated with better survival outcomes. Positive PD-L1 expression correlates with a high infiltration by TILs, explaining its association with better prognosis.

YES1 Drives Lung Cancer Growth and Progression and Predicts Sensitivity to Dasatinib.

Rationale: The characterization of new genetic alterations is essential to assign effective personalized therapies in non-small cell lung cancer (NSCLC). Furthermore, finding stratification biomarkers is essential for successful personalized therapies. Molecular alterations of YES1, a member of the SRC (proto-oncogene tyrosine-protein kinase Src) family kinases (SFKs), can be found in a significant subset of patients with lung cancer.Objectives: To evaluate YES1 (v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1) genetic alteration as a therapeutic target and predictive biomarker of response to dasatinib in NSCLC.Methods: Functional significance was evaluated by in vivo models of NSCLC and metastasis and patient-derived xenografts. The efficacy of pharmacological and genetic (CRISPR [clustered regularly interspaced short palindromic repeats]/Cas9 [CRISPR-associated protein 9]) YES1 abrogation was also evaluated. In vitro functional assays for signaling, survival, and invasion were also performed. The association between YES1 alterations and prognosis was evaluated in clinical samples.Measurements and Main Results: We demonstrated that YES1 is essential for NSCLC carcinogenesis. Furthermore, YES1 overexpression induced metastatic spread in preclinical in vivo models. YES1 genetic depletion by CRISPR/Cas9 technology significantly reduced tumor growth and metastasis. YES1 effects were mainly driven by mTOR (mammalian target of rapamycin) signaling. Interestingly, cell lines and patient-derived xenograft models with YES1 gene amplifications presented a high sensitivity to dasatinib, an SFK inhibitor, pointing out YES1 status as a stratification biomarker for dasatinib response. Moreover, high YES1 protein expression was an independent predictor for poor prognosis in patients with lung cancer.Conclusions: YES1 is a promising therapeutic target in lung cancer. Our results provide support for the clinical evaluation of dasatinib treatment in a selected subset of patients using YES1 status as predictive biomarker for therapy.

5 protein-based signature for resectable lung squamous cell carcinoma improves the prognostic performance of the TNM staging.

INTRODUCTION: Prognostic biomarkers have been very elusive in the lung squamous cell carcinoma (SCC) and none is currently being used in the clinical setting. We aimed to identify and validate the clinical utility of a protein-based prognostic signature to stratify patients with early lung SCC according to their risk of recurrence or death. METHODS: Patients were staged following the new International Association for the Study of Lung Cancer (IASLC) staging criteria (eighth edition, 2018). Three independent retrospective cohorts of 117, 96 and 105 patients with lung SCC were analysed to develop and validate a prognostic signature based on immunohistochemistry for five proteins. RESULTS: We identified a five protein-based signature whose prognostic index (PI) was an independent and significant predictor of disease-free survival (DFS) (p<0.001; HR=4.06, 95% CI 2.18 to 7.56) and overall survival (OS) (p=0.004; HR=2.38, 95% CI 1.32 to 4.31). The prognostic capability of PI was confirmed in an external multi-institutional cohort for DFS (p=0.042; HR=2.01, 95% CI 1.03 to 3.94) and for OS (p=0.031; HR=2.29, 95% CI 1.08 to 4.86). Moreover, PI added complementary information to the newly established IASLC TNM 8th edition staging system. A combined prognostic model including both molecular and anatomical (TNM) criteria improved the risk stratification in both cohorts (p<0.05). CONCLUSION: We have identified and validated a clinically feasible protein-based prognostic model that complements the updated TNM system allowing more accurate risk stratification. This signature may be used as an advantageous tool to improve the clinical management of the patients, allowing the reduction of lung SCC mortality through a more accurate knowledge of the patient's potential outcome.

Ruthenium counterstaining for imaging mass cytometry.

Imaging mass cytometry is a novel imaging modality that enables simultaneous antibody-based detection of >40 epitopes and molecules in tissue sections at subcellular resolution by the use of isotopically pure metal tags. Essential for any imaging approach in which antigen detection is performed is counterstaining, which reveals the overall structure of the tissue. Counterstaining is necessary because antigens of interest are often present in only a small subset of cells, and the rest of the tissue structures are not visible. As most biological tissues are nearly transparent or non-fluorescent, chromogenic reagents such as haematoxylin (for immunohistochemistry) or fluorescent dyes such as 4',6-diamidino-2-phenylindole (which stains nuclei for epifluorescence and confocal microscopy) are utilized. Here, we describe a metal-based counterstain for imaging mass cytometry based on simple oxidation and subsequent covalent binding of the tissue components to ruthenium tetroxide (RuO4 ). RuO4 counterstaining reveals general tissue structure both in areas with high cell content and in stromal areas with low cellularity and fibrous or hyaline material in a manner analogous to haematoxylin in immunohistochemical counterstaining or eosin or other anionic dyes in conventional histology. Our new counterstain approach is applicable to any metal-based imaging technique, and will facilitate the adaptation of imaging mass cytometry for routine applications in clinical and research laboratories. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A novel protein-based prognostic signature improves risk stratification to guide clinical management in early-stage lung adenocarcinoma patients.

Each of the pathological stages (I-IIIa) of surgically resected non-small-cell lung cancer has hidden biological heterogeneity, manifested as heterogeneous outcomes within each stage. Thus, the finding of robust and precise molecular classifiers with which to assess individual patient risk is an unmet medical need. Here, we identified and validated the clinical utility of a new prognostic signature based on three proteins (BRCA1, QKI, and SLC2A1) to stratify early-stage lung adenocarcinoma patients according to their risk of recurrence or death. Patients were staged according to the new International Association for the Study of Lung Cancer (IASLC) staging criteria (8th edition, 2018). A test cohort (n = 239) was used to assess the value of this new prognostic index (PI) based on the three proteins. The prognostic signature was developed by Cox regression with the use of stringent statistical criteria (TRIPOD: Transparent reporting of a multivariable prediction model for individual prognosis or diagnosis). The model resulted in a highly significant predictor of 5-year outcome for disease-free survival (p < 0.001) and overall survival (p < 0.001). The prognostic ability of the model was externally validated in an independent multi-institutional cohort of patients (n = 114, p = 0.021). We also demonstrated that this molecular classifier adds relevant information to the gold standard TNM-based pathological staging, with a highly significant improvement of the likelihood ratio. We subsequently developed a combined PI including both the molecular and the pathological data that improved the risk stratification in both cohorts (p ≤ 0.001). Moreover, the signature may help to select stage I-IIA patients who might benefit from adjuvant chemotherapy. In summary, this protein-based signature accurately identifies those patients with a high risk of recurrence and death, and adds further prognostic information to the TNM-based clinical staging, even when the new IASLC 8th edition staging criteria are applied. More importantly, it may be a valuable tool for selecting patients for adjuvant therapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Blockade of the Complement C5a/C5aR1 Axis Impairs Lung Cancer Bone Metastasis by CXCL16-mediated Effects.

RATIONALE: C5aR1 (CD88), a receptor for complement anaphylatoxin C5a, is a potent immune mediator. Its impact on malignant growth and dissemination of non-small cell lung cancer cells is poorly understood. OBJECTIVES: To investigate the contribution of the C5a/C5aR1 axis to the malignant phenotype of non-small cell lung cancer cells, particularly in skeletal colonization, a preferential lung metastasis site. METHODS: Association between C5aR1 expression and clinical outcome was assessed in silico and validated by immunohistochemistry. Functional significance was evaluated by lentiviral gene silencing and ligand l-aptamer inhibition in in vivo models of lung cancer bone metastasis. In vitro functional assays for signaling, migration, invasion, metalloprotease activity, and osteoclastogenesis were also performed. MEASUREMENTS AND MAIN RESULTS: High levels of C5aR1 in human lung tumors were significantly associated with shorter recurrence-free survival, overall survival, and bone metastasis. Silencing of C5aR1 in lung cancer cells led to a substantial reduction in skeletal metastatic burden and osteolysis in in vivo models. Furthermore, metalloproteolytic, migratory, and invasive tumor cell activities were modulated in vitro by C5aR1 stimulation or gene silencing. l-Aptamer blockade or C5aR1 silencing significantly reduced the osseous metastatic activity of lung cancer cells in vivo. This effect was associated with decreased osteoclastogenic activity in vitro and was rescued by the exogenous addition of the chemokine CXCL16. CONCLUSIONS: Disruption of C5aR1 signaling in lung cancer cells abrogates their tumor-associated osteoclastogenic activity, impairing osseous colonization. This study unveils the role played by the C5a/C5aR1 axis in lung cancer dissemination and supports its potential use as a novel therapeutic target.

Comparison of RNA-seq and microarray platforms for splice event detection using a cross-platform algorithm.

BACKGROUND: RNA-seq is a reference technology for determining alternative splicing at genome-wide level. Exon arrays remain widely used for the analysis of gene expression, but show poor validation rate with regard to splicing events. Commercial arrays that include probes within exon junctions have been developed in order to overcome this problem. We compare the performance of RNA-seq (Illumina HiSeq) and junction arrays (Affymetrix Human Transcriptome array) for the analysis of transcript splicing events. Three different breast cancer cell lines were treated with CX-4945, a drug that severely affects splicing. To enable a direct comparison of the two platforms, we adapted EventPointer, an algorithm that detects and labels alternative splicing events using junction arrays, to work also on RNA-seq data. Common results and discrepancies between the technologies were validated and/or resolved by over 200 PCR experiments. RESULTS: As might be expected, RNA-seq appears superior in cases where the technologies disagree and is able to discover novel splicing events beyond the limitations of physical probe-sets. We observe a high degree of coherence between the two technologies, however, with correlation of EventPointer results over 0.90. Through decimation, the detection power of the junction arrays is equivalent to RNA-seq with up to 60 million reads. CONCLUSIONS: Our results suggest, therefore, that exon-junction arrays are a viable alternative to RNA-seq for detection of alternative splicing events when focusing on well-described transcriptional regions.

The oncogenic RNA-binding protein SRSF1 regulates LIG1 in non-small cell lung cancer.

In recent years, the relevance of RNA metabolism has been increasingly recognized in a variety of diseases. Modifications in the levels of RNA-binding proteins elicit changes in the expression of cancer-related genes. Here we evaluate whether SRSF1 regulates the expression of DNA repair genes, and whether this regulation has a relevant role in lung carcinogenesis. An in silico analysis was performed to evaluate the association between the expression of SRSF1 and DNA repair genes. In vitro functional analyses were conducted in SRSF1 or DNA ligase 1 (LIG1)-downregulated non-small cell lung cancer (NSCLC) cell lines. In addition, the prognostic value of LIG1 was evaluated in NSCLC patients by immunohistochemistry. We found a significant correlation between the DNA repair gene LIG1 and SRSF1 in NSCLC cell lines. Moreover, SRSF1 binds to LIG1 mRNA and regulates its expression by increasing its mRNA stability and enhancing its translation in an mTOR-dependent manner. Furthermore, siRNA-mediated LIG1 inhibition reduced proliferation and increased apoptosis of NSCLC cells. Finally, the expression of LIG1 was an independent prognostic factor for NSCLC, as confirmed in a series of 210 patients. These results show that LIG1 is regulated by the oncoprotein SRSF1 and plays a relevant role in lung cancer cell proliferation and progression. LIG1 is associated with poor prognosis in non-small lung cancer patients.

Phenotypic and metabolic features of mouse diaphragm and gastrocnemius muscles in chronic lung carcinogenesis: influence of underlying emphysema.

BACKGROUND: Muscle wasting negatively impacts the progress of chronic diseases such as lung cancer (LC) and emphysema, which are in turn interrelated. OBJECTIVES: We hypothesized that muscle atrophy and body weight loss may develop in an experimental mouse model of lung carcinogenesis, that the profile of alterations in muscle fiber phenotype (fiber type composition and morphometry, muscle structural alterations, and nuclear apoptosis), and in muscle metabolism are similar in both respiratory and limb muscles of the tumor-bearing mice, and that the presence of underlying emphysema may influence those events. METHODS: Diaphragm and gastrocnemius muscles of mice with urethane-induced lung cancer (LC-U) with and without elastase-induced emphysema (E-U) and non-exposed controls (N = 8/group) were studied: fiber type composition, morphometry, muscle abnormalities, apoptotic nuclei (immunohistochemistry), and proteolytic and autophagy markers (immunoblotting) at 20- and 35-week exposure times. In the latter cohort, structural contractile proteins, creatine kinase (CK), peroxisome proliferator-activated receptor (PPAR) expression, oxidative stress, and inflammation were also measured. Body and muscle weights were quantified (baseline, during follow-up, and sacrifice). RESULTS: Compared to controls, in U and E-U mice, whole body, diaphragm and gastrocnemius weights were reduced. Additionally, both in diaphragm and gastrocnemius, muscle fiber cross-sectional areas were smaller, structural abnormalities, autophagy and apoptotic nuclei were increased, while levels of actin, myosin, CK, PPARs, and antioxidants were decreased, and muscle proteolytic markers did not vary among groups. CONCLUSIONS: In this model of lung carcinogenesis with and without emphysema, reduced body weight gain and muscle atrophy were observed in respiratory and limb muscles of mice after 20- and 35-week exposure times most likely through increased nuclear apoptosis and autophagy. Underlying emphysema induced a larger reduction in the size of slow- and fast-twitch fibers in the diaphragm of U and E-U mice probably as a result of the greater inspiratory burden imposed onto this muscle.

Improving selection criteria for lung cancer screening. The potential role of emphysema.

RATIONALE: Lung cancer (LC) screening using low-dose chest computed tomography is now recommended in several guidelines using the National Lung Screening Trial (NLST) entry criteria (age, 55-74; ≥30 pack-years; tobacco cessation within the previous 15 yr for former smokers). Concerns exist about their lack of sensitivity. OBJECTIVES: To evaluate the performance of NLST criteria in two different LC screening studies from Europe and the United States, and to explore the effect of using emphysema as a complementary criterion. METHODS: Participants from the Pamplona International Early Lung Action Detection Program (P-IELCAP; n = 3,061) and the Pittsburgh Lung Screening Study (PLuSS; n = 3,638) were considered. LC cumulative frequencies, incidence densities, and annual detection rates were calculated in three hypothetical cohorts, including subjects who met NLST criteria alone, those with computed tomography-detected emphysema, and those who met NLST criteria and/or had emphysema. MEASUREMENTS AND MAIN RESULTS: Thirty-six percent and 59% of P-IELCAP and PLuSS participants, respectively, met NLST criteria. Among these, higher LC incidence densities and detection rates were observed. However, applying NLST criteria to our original cohorts would miss as many as 39% of all LC. Annual screening of subjects meeting either NLST criteria or having emphysema detected most cancers (88% and 95% of incident LC of P-IELCAP and PLuSS, respectively) despite reducing the number of screened participants by as much as 52%. CONCLUSIONS: LC screening based solely on NLST criteria could miss a significant number of LC cases. Combining NLST criteria and emphysema to select screening candidates results in higher LC detection rates and a lower number of cancers missed.

Combined clinical and genomic signatures for the prognosis of early stage non-small cell lung cancer based on gene copy number alterations.

BACKGROUND: The development of a more refined prognostic methodology for early non-small cell lung cancer (NSCLC) is an unmet clinical need. An accurate prognostic tool might help to select patients at early stages for adjuvant therapies. RESULTS: A new integrated bioinformatics searching strategy, that combines gene copy number alterations and expression, together with clinical parameters was applied to derive two prognostic genomic signatures. The proposed methodology combines data from patients with and without clinical data with a priori information on the ability of a gene to be a prognostic marker. Two initial candidate sets of 513 and 150 genes for lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), respectively, were generated by identifying genes which have both: a) significant correlation between copy number and gene expression, and b) significant prognostic value at the gene expression level in external databases. From these candidates, two panels of 7 (ADC) and 5 (SCC) genes were further identified via semi-supervised learning. These panels, together with clinical data (stage, age and sex), were used to construct the ADC and SCC hazard scores combining clinical and genomic data. The signatures were validated in two independent datasets (n = 73 for ADC, n = 97 for SCC), confirming that the prognostic value of both clinical-genomic models is robust, statistically significant (P = 0.008 for ADC and P = 0.019 for SCC) and outperforms both the clinical models (P = 0.060 for ADC and P = 0.121 for SCC) and the genomic models applied separately (P = 0.350 for ADC and P = 0.269 for SCC). CONCLUSION: The present work provides a methodology to generate a robust signature using copy number data that can be potentially used to any cancer. Using it, we found new prognostic scores based on tumor DNA that, jointly with clinical information, are able to predict overall survival (OS) in patients with early-stage ADC and SCC.

New syngeneic inflammatory-related lung cancer metastatic model harboring double KRAS/WWOX alterations.

New mouse models with specific drivers of genetic alterations are needed for preclinical studies. Herein, we created and characterized at the genetic level a new syngeneic model for lung cancer and metastasis in Balb-c mice. Tumor cell lines were obtained from a silica-mediated airway chronic inflammation that promotes tumorigenesis when combined with low doses of N-nitrosodimethylamine, a tobacco smoke carcinogen. Orthotopic transplantation of these cells induced lung adenocarcinomas, and their intracardiac injection led to prominent colonization of various organs (bone, lung, liver and brain). Driver gene alterations included a mutation in the codon 12 of KRAS (G-A transition), accompanied by a homozygous deletion of the WW domain-containing oxidoreductase (WWOX) gene. The mutant form of WWOX lacked exons 5-8 and displayed reduced protein expression level and activity. WWOX gene restoration decreased the in vitro and in vivo tumorigenicity, confirming the tumor suppressor function of this gene in this particular model. Interestingly, we found that cells displayed remarkable sphere formation ability with expression of specific lung cancer stem cell markers. Study of non-small-cell lung cancer patient cohorts demonstrated a deletion of WWOX in 30% of cases, with significant reduction in protein levels as compared to normal tissues. Overall, our new syngeneic mouse model provides a most valuable tool to study lung cancer metastasis in balb-c mice background and highlights the importance of WWOX deletion in lung carcinogenesis.

Anaphylatoxin C5a creates a favorable microenvironment for lung cancer progression.

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 nonmalignant bronchial epithelial cells. Notably, 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, IL-6, IL-10, 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.

Elevated expression of complement factor I in lung cancer cells associates with shorter survival-Potentially via non-canonical mechanism.

While numerous membrane-bound complement inhibitors protect the body's cells from innate immunity's autoaggression, soluble inhibitors like complement factor I (FI) are rarely produced outside the liver. Previously, we reported the expression of FI in non-small cell lung cancer (NSCLC) cell lines. Now, we assessed the content of FI in cancer biopsies from lung cancer patients and associated the results with clinicopathological characteristics and clinical outcomes. Immunohistochemical staining intensity did not correlate with age, smoking status, tumor size, stage, differentiation grade, and T cell infiltrates, but was associated with progression-free survival (PFS), overall survival (OS) and disease-specific survival (DSS). Multivariate Cox analysis of low vs. high FI content revealed HR 0.55, 95 % CI 0.32-0.95, p=0.031 for PFS, HR 0.51, 95 % CI 0.25-1.02, p=0.055 for OS, and HR 0.32, 95 % CI 0.12-0.84, p=0.021 for DSS. Unfavorable prognosis might stem from the non-canonical role of FI, as the staining pattern did not correlate with C4d - the product of FI-supported degradation of active complement component C4b. To elucidate that, we engineered three human NSCLC cell lines naturally expressing FI with CRISPR/Cas9 technology, and compared the transcriptome of FI-deficient and FI-sufficient clones in each cell line. RNA sequencing revealed differentially expressed genes engaged in intracellular signaling pathways controlling proliferation, apoptosis, and responsiveness to growth factors. Moreover, in vitro colony-formation assays showed that FI-deficient cells formed smaller foci than FI-sufficient NSCLC cells, but their size increased when purified FI protein was added to the medium. We postulate that a non-canonical activity of FI influences cellular physiology and contributes to the poor prognosis of lung cancer patients.

Oxidative Damage and Telomere Length as Markers of Lung Cancer Development among Chronic Obstructive Pulmonary Disease (COPD) Smokers.

Lung cancer (LC) constitutes an important cause of death among patients with Chronic Obstructive Pulmonary Disease (COPD). Both diseases may share pathobiological mechanisms related to oxidative damage and cellular senescence. In this study, the potential value of leucocyte telomere length, a hallmark of aging, and 8-OHdG concentrations, indicative of oxidative DNA damage, as risk biomarkers of LC was evaluated in COPD patients three years prior to LC diagnosis. Relative telomere length measured using qPCR and serum levels of 8-OHdG were determined at the baseline in 99 COPD smokers (33 with LC and 66 age-matched COPD without LC as controls). Of these, 21 COPD with LC and 42 controls had the biomarkers measured 3 years before. Single nucleotide variants (SNVs) in TERT, RTEL, and NAF1 genes were also determined. COPD cases were evaluated, which showed greater telomere length (p < 0.001) and increased serum 8-OHdG levels (p = 0.004) three years prior to LC diagnosis compared to the controls. This relationship was confirmed at the time of LC diagnosis. No significant association was found between the studied SNVs in cases vs. controls. In conclusion, this preliminary study shows that longer leucocyte telomere length and increased 8-OHdG serum levels can be useful as early biomarkers of the risk for future lung cancer development among COPD patients.

Lung Cancer Screening in People With COPD: The Pamplona-IELCAP Experience.

INTRODUCTION: The Global Initiative for Obstructive Lung Disease (GOLD) recommends lung cancer screening for patients with Chronic Obstructive Pulmonary Disease (COPD), but data is lacking regarding results of screening in this high-risk population. The main goal of the present work is to explore if lung cancer screening with Low Dose Chest Tomography (LDCT) in people with COPD, allows lung cancer (LC) diagnosis in early stages with survival compatible with curative state. METHODS: This is a post hoc exploratory analysis. Pamplona International Early Lung Cancer Action Program (P-IELCAP) participants with a GOLD defined obstructive pattern (post bronchodilator FEV1/FVC<0.70) were selected for analysis. The characteristics of those who developed LC and their survival are described. A Cox proportional analysis explored the factors associated with LC diagnosis. RESULTS: Eight hundred and sixty-five patients (77% male, 93% in spirometric GOLD stage 1+2) were followed for 102±63 months. LC prevalence was 2.6% at baseline, with an annual LC diagnosis rate of 0.68%. Early-stage tumors predominated (74%) with a median survival (25-75th percentiles) of 139 (76-185) months. Cumulative tobacco exposure, FEV1%, and emphysema were the main predictors of an LC diagnosis. Eight (11%) patients with COPD had a second LC, most of them in early stage (92%), and 6 (8%) had recurrence. Median survival (25-75th percentiles) in these patients was 168 (108-191) months. CONCLUSIONS: Lung cancer screening of selected high-risk participants with COPD allowed the LC diagnosis in early stages with survival compatible with curative state.

Whole exome sequencing and machine learning germline analysis of individuals presenting with extreme phenotypes of high and low risk of developing tobacco-associated lung adenocarcinoma.

BACKGROUND: Tobacco is the main risk factor for developing lung cancer. Yet, while some heavy smokers develop lung cancer at a young age, other heavy smokers never develop it, even at an advanced age, suggesting a remarkable variability in the individual susceptibility to the carcinogenic effects of tobacco. We characterized the germline profile of subjects presenting these extreme phenotypes with Whole Exome Sequencing (WES) and Machine Learning (ML). METHODS: We sequenced germline DNA from heavy smokers who either developed lung adenocarcinoma at an early age (extreme cases) or who did not develop lung cancer at an advanced age (extreme controls), selected from databases including over 6600 subjects. We selected individual coding genetic variants and variant-rich genes showing a significantly different distribution between extreme cases and controls. We validated the results from our discovery cohort, in which we analysed by WES extreme cases and controls presenting similar phenotypes. We developed ML models using both cohorts. FINDINGS: Mean age for extreme cases and controls was 50.7 and 79.1 years respectively, and mean tobacco consumption was 34.6 and 62.3 pack-years. We validated 16 individual variants and 33 variant-rich genes. The gene harbouring the most validated variants was HLA-A in extreme controls (4 variants in the discovery cohort, p = 3.46E-07; and 4 in the validation cohort, p = 1.67E-06). We trained ML models using as input the 16 individual variants in the discovery cohort and tested them on the validation cohort, obtaining an accuracy of 76.5% and an AUC-ROC of 83.6%. Functions of validated genes included candidate oncogenes, tumour-suppressors, DNA repair, HLA-mediated antigen presentation and regulation of proliferation, apoptosis, inflammation and immune response. INTERPRETATION: Individuals presenting extreme phenotypes of high and low risk of developing tobacco-associated lung adenocarcinoma show different germline profiles. Our strategy may allow the identification of high-risk subjects and the development of new therapeutic approaches. FUNDING: See a detailed list of funding bodies in the Acknowledgements section at the end of the manuscript.

Phosphorylated tubulin adaptor protein CRMP-2 as prognostic marker and candidate therapeutic target for NSCLC.

Collapsin response mediator protein-2 (CRMP-2) is the first described and most studied member of a family of proteins that mediate the addition of tubulin dimers to the growing microtubule. CRMPs have mainly been studied in the nervous system, but recently, they have been described in other tissues where they participate in vesicle transport, migration and mitosis. In this work, we aimed at studying the role of CRMP-2 in lung cancer cell division. We first explored the expression of CRMP-2 and phosphorylated (Thr 514) CRMP-2 in 91 samples obtained from patients with localized nonsmall cell lung cancer. We observed a significant correlation between high levels of nuclear phosphorylated CRMP-2 and poor prognosis in those patients. Interestingly, this association was only positive for untreated patients. To provide a mechanistic explanation to these findings, we used in vitro models to analyze the role of CRMP-2 and its phosphorylated forms in cell division. Thus, we observed by confocal microscopy and immunoprecipitation assays that CRMP-2 differentially colocalizes with the mitotic spindle during cell division. The use of phosphodefective or phosphomimetic mutants of CRMP-2 allowed us to prove that anomalies in the phosphorylation status of CRMP-2 result in changes in the mitotic tempo, and increments in the number of multinucleated cells. Finally, here we demonstrate that CRMP-2 phosphorylation impairment, or silencing induces p53 expression and promotes apoptosis through caspase 3 activation. These results pointed to CRMP-2 phosphorylation as a prognostic marker and potential new target to be explored in cancer therapy.

Receptor of activated protein C promotes metastasis and correlates with clinical outcome in lung adenocarcinoma.

RATIONALE: Efficient metastasis requires survival and adaptation of tumor cells to stringent conditions imposed by the extracellular milieu. Identification of critical survival signaling pathways in tumor cells might unveil novel targets relevant in disease progression. OBJECTIVES: To investigate the contribution of activated protein C (APC) and its receptor (endothelial protein C receptor [EPCR]) in animal models of lung cancer metastasis and in patients with lung adenocarcinoma. METHODS: Signaling pathway triggered by APC/EPCR and its relevance in apoptosis was studied in vitro. Functional significance was assessed by silencing and blocking antibodies in several in vivo models of lung cancer metastasis in athymic nude Foxn1(nu) mice. We examined EPCR levels using a microarray dataset of 107 patients. Immunohistochemical analysis was performed in an independent cohort of 295 patients with lung adenocarcinoma. MEASUREMENTS AND MAIN RESULTS: The effects of APC binding to EPCR rapidly triggered Akt and extracellular signal-regulated kinase signaling pathways, leading to attenuated in vitro apoptosis. In vivo, silencing of EPCR expression or blocking APC/EPCR interaction reduced infiltration in the target organ, resulting in impaired prometastatic activity. Moreover, overexpression of EPCR induced an increased metastatic activity to target organs. Analysis of clinical samples showed a robust association between high EPCR levels and poor prognosis, particularly in stage I patients. CONCLUSIONS: EPCR and its ligand APC promote cell survival that contributes to tumor cell endurance to stress favoring prometastatic activity of lung adenocarcinoma. EPCR/APC is a novel target of relevance in the clinical outcome of early-stage lung cancer.