Differentially methylated regions within lung cancer risk loci are enriched in deregulated enhancers.

Genome-wide association studies (GWAS) have identified SNPs linked with lung cancer risk. Our aim was to discover the genes, non-coding RNAs, and regulatory elements within GWAS-identified risk regions that are deregulated in non-small cell lung carcinoma (NSCLC) to identify novel, clinically targetable genes and mechanisms in carcinogenesis. A targeted bisulphite-sequencing approach was used to comprehensively investigate DNA methylation changes occurring within lung cancer risk regions in 17 NSCLC and adjacent normal tissue pairs. We report differences in differentially methylated regions between adenocarcinoma and squamous cell carcinoma. Among the minimal regions found to be differentially methylated in at least 50% of the patients, 7 candidates were replicated in 2 independent cohorts (n = 27 and n = 87) and the potential of 6 as methylation-dependent regulatory elements was confirmed by functional assays. This study contributes to understanding the pathways implicated in lung cancer initiation and progression, and provides new potential targets for cancer treatment.

Lung cancer mortality reduction by LDCT screening-Results from the randomized German LUSI trial.

In 2011, the U.S. National Lung Cancer Screening Trial (NLST) reported a 20% reduction of lung cancer mortality after regular screening by low-dose computed tomography (LDCT), as compared to X-ray screening. The introduction of lung cancer screening programs in Europe awaits confirmation of these first findings from European trials that started in parallel with the NLST. The German Lung cancer Screening Intervention (LUSI) is a randomized trial among 4,052 long-term smokers, 50-69 years of age, recruited from the general population, comparing five annual rounds of LDCT screening (screening arm; n = 2,029 participants) with a control arm (n = 2,023) followed by annual postal questionnaire inquiries. Data on lung cancer incidence and mortality and vital status were collected from hospitals or office-based physicians, cancer registries, population registers and health offices. Over an average observation time of 8.8 years after randomization, the hazard ratio for lung cancer mortality was 0.74 (95% CI: 0.46-1.19; p = 0.21) among men and women combined. Modeling by sex, however showed a statistically significant reduction in lung cancer mortality among women (HR = 0.31 [95% CI: 0.10-0.96], p = 0.04), but not among men (HR = 0.94 [95% CI: 0.54-1.61], p = 0.81) screened by LDCT (pheterogeneity = 0.09). Findings from LUSI are in line with those from other trials, including NLST, that suggest a stronger reduction of lung cancer mortality after LDCT screening among women as compared to men. This heterogeneity could be the result of different relative counts of lung tumor subtypes occurring in men and women.

Perioperative course and quality of life in a prospective randomized multicenter phase III trial, comparing standard lobectomy versus anatomical segmentectomy in patients with non-small cell lung cancer up to 2 cm, stage IA (7th edition of TNM staging system).

OBJECTIVES: For early stage non-small cell lung cancer (NSCLC) retrospective data of functionally compromised patients undergoing segmentectomy showed equal outcomes for perioperative complications and quality of life (QoL) compared with lobectomy patients. However no prospectively randomized data comparing patients eligible for both procedures are available. MATERIALS AND METHODS: We conducted a prospective, randomized, multicenter phase III trial and investigated perioperative complications and QoL in patients with NSCLC stage IA (7th edition) undergoing segmentectomy versus lobectomy. The EORTC Questionnaire Core-30 (QLQ C-30) supplemented by thirteen-item lung cancer-specific module (LC13) was assessed before surgery, at discharge, 6 weeks, 3, 6 and 12 months post-surgery. RESULTS: 108 patients with verified or suspected NSCLC up to 2 cm diameter were enrolled, whereby 54 were assigned to lobectomy and 54 to segmentectomy. Due to nodal disease, tumor size and surgical reasons estimated during the operation, eight patients of the segmentectomy group received a lobectomy. In hospital and 90 days mortality was 0% in both groups. Perioperative complications were observed in 6 (11.3%) patients after segmentectomy and in 8 patients (14.8%) after lobectomy (p = 0.563), while the 90-day morbidity were 17% and 25.9% (9 and 14 patients), respectively (p = 0.452). Twelve months after surgery, there was a significant deterioration to the baselines of physical (p < 0.001) and cognitive functioning (p = 0.025), dyspnea (p < 0.001) and fatigue (p = 0.003) in the lobectomy group. Dyspnea showed a faster recovery in the segmentectomy compared to lobectomy group with statistical significance (p = 0.016 after 12 months). CONCLUSION: In patients with early-stage NSCLC, segmentectomy is associated with a statistically not significant lower perioperative morbidity and appears to provide a superior recovery in QoL compared with lobectomy patients.

Procedure-Specific Risk Prediction for Recurrence in Patients Undergoing Lobectomy or Sublobar Resection for Small (≤2 cm) Lung Adenocarcinoma: An International Cohort Analysis.

INTRODUCTION: This work was performed to develop and validate procedure-specific risk prediction for recurrence following resection for early-stage lung adenocarcinoma (ADC) and investigate risk prediction utility in identifying patients who may benefit from adjuvant chemotherapy (ACT). METHODS: In patients who underwent resection for small (≤2 cm) lung ADC (lobectomy, 557; sublobar resection, 352), an association between clinicopathologic variables and risk of recurrence was assessed by a competing risks approach. Procedure-specific risk prediction was developed based on multivariable regression for recurrence. External validation was conducted using cohorts (N = 708) from Japan, Taiwan, and Germany. The accuracy of risk prediction was measured using a concordance index. We applied the lobectomy risk prediction approach to a propensity score-matched cohort of patients with stage II-III disease (n = 316, after matching) with or without ACT and compared lung cancer-specific survival between groups among low- or high-risk scores. RESULTS: Micropapillary pattern, solid pattern, lymphovascular invasion, and necrosis were involved in the risk prediction following lobectomy, and micropapillary pattern, spread through air spaces, lymphovascular invasion, and necrosis following sublobar resection. Both internal and external validation showed good discrimination (concordance index in lobectomy and sublobar resection: internal, 0.77 and 0.75, respectively; and external, 0.73 and 0.79, respectively). In the stage II-III propensity score-matched cohort, among high-risk patients, ACT significantly reduced the risk of lung cancer-specific death (subhazard ratio 0.43, p = 0.001), but not among low-risk patients. CONCLUSIONS: Procedure-specific risk prediction for patients with resected small lung ADC can be used to better prognosticate and stratify patients for further interventions.

Molecular signatures in IASLC/ATS/ERS classified growth patterns of lung adenocarcinoma.

BACKGROUND: The current classification of human lung adenocarcinoma defines five different histological growth patterns within the group of conventional invasive adenocarcinomas. The five growth patterns are characterised by their typical architecture, but also by variable tumor biological behaviour. AIMS: The aim of this study was to identify specific gene signatures of the five adenocarcinoma growth patterns defined by the joint IASLC/ATS/ERS working group. METHODS: Total RNA from microdissected adenocarcinoma tissue samples of ten lepidic, ten acinar, ten solid, nine papillary, and nine micropapillary tumor portions was isolated and prepared for gene expression analysis. Differential expression of genes was determined using the R package "LIMMA". The overall significance of each signature was assessed via global test. Gene ontology statistics were analysed using GOstat. For immunohistochemical validation, tissue specimens from 20 tumors with solid and 20 tumors with lepidic growth pattern were used. RESULTS: Microarray analyses between the growth patterns resulted in numerous differentially expressed genes between the solid architecture and other patterns. The comparison of transcriptomic activity in the solid and lepidic patterns revealed 705 up- and 110 downregulated non-redundant genes. The pattern-specific protein expression of Inositol-1,4,5-trisphosphate-kinase-A (ITPKA) and angiogenin by immunohistochemistry confirmed the RNA levels. The strongest differences in protein expression between the two patterns were shown for ITPKA (p = 0.02) and angiogenin (p = 0.113). CONCLUSIONS: In this study growth pattern-specific gene signatures in pulmonary adenocarcinoma were identified and distinct transcriptomic differences between lung adenocarcinoma growth patterns were defined. The study provides valuable new information about pulmonary adenocarcinoma and allows a better assessment of the five adenocarcinoma subgroups.

The combination of the blood based tumor biomarkers cytokeratin 19 fragments (CYFRA 21-1) and carcinoembryonic antigen (CEA) as a potential predictor of benefit from adjuvant chemotherapy in early stage squamous cell carcinoma of the lung (SCC).

OBJECTIVES: To determine whether the tumor biomarkers cytokeratin 19 fragment (CYFRA 21-1) and carcinoembryonic antigen (CEA), which are prognostic in early-stage non-small cell lung cancer (NSCLC), can predict which patients benefit from adjuvant chemotherapy (CTx). MATERIALS AND METHODS: Serum samples were collected preoperatively from patients with NSCLC who underwent resection. Samples were retrospectively analyzed for CYFRA 21-1 and CEA via electrochemiluminescence immunoassay. Recurrence-free survival (RFS) was compared for patients who received adjuvant CTx versus surgery alone, stratified based on the following prognostic classifications: (1) tumor stage (pT1-2/N0 [stage I] or pT3/N0 or pT1-2/N1 [stage II]), (2) biomarker-based risk score, (3) clinical characteristics. Absolute 2-year RFS rates were calculated via Kaplan-Meier estimations; statistical significance level: 0.05. RESULTS: 227 patients were included (stage I: 69%; male: 67%; median age 65 years); 70 received adjuvant CTx. Median duration of sample collection was 58.8 months. All high-risk patients (by all three prognostic classifications) who received adjuvant CTx had a longer RFS versus those who received surgery alone. In patients with squamous cell carcinoma (SCC) classified as high risk by all three prognostic classifications, there was a benefit from adjuvant CTx versus surgery alone (tumor stage hazard ratio [HR] 4.9, p = 0.004; biomarker levels HR 9.4, p = 0.002; clinical characteristics HR 9.0, p = 0.003). None of the prognostic classifications were able to predict a benefit from adjuvant CTx in patients with adenocarcinoma. CONCLUSION: Baseline CYFRA 21-1 and CEA levels may provide further information to help clinicians decide which patients with SCC should receive adjuvant CTx. Further evaluation of these biomarkers is warranted.

Circulating and Tumor Myeloid-derived Suppressor Cells in Resectable Non-Small Cell Lung Cancer.

RATIONALE: Myeloid-derived suppressor cell (MDSC) expansion has been found to play a role in disease progression in patients with cancer. However, the characteristics of MDSCs in lung cancer are poorly understood. OBJECTIVES: We prospectively investigated MDSCs and inflammatory factors in tumor and peripheral blood samples from patients with resectable non-small cell lung cancer and studied their correlations with the disease prognosis. METHODS: A complex analysis of MDSC subsets and inflammatory mediators was performed using flow cytometry and a Bio-Plex assay. MEASUREMENTS AND MAIN RESULTS: A significant increase in the frequency of circulating monocytic (M)-MDSCs was observed in the patients with non-small cell lung cancer compared with the healthy donors (HDs). Moreover, the frequencies of M- and polymorphonuclear (PMN)-MDSCs were higher in tumors than in the peripheral blood of the same patients. This accumulation was associated with elevated concentrations of inflammatory mediators involved in MDSC migration to and activation in the tumor microenvironment. An analysis of the MDSC immunosuppressive pattern showed increased programmed death-ligand 1 expression on circulating cells from patients compared with HDs. Tumor PMN-MDSCs displayed higher programmed death-ligand 1 expression levels than the same cells in the peripheral blood. The frequency of CCR5 (C-C chemokine receptor 5) expression on circulating M-MDSCs was significantly higher in the patients than in the HDs. Clinical data analysis revealed negative correlations between recurrence-free survival and the frequencies of PMN-MDSCs and CCR5+ M-MDSCs in the circulation but not in tumors. CONCLUSIONS: Our findings suggest that the level of MDSCs in the peripheral blood but not in tumor tissues predicts recurrence after surgery.

[Implementation of Hyperthermic Intrathoracic Chemotherapy (HITHOC) in Germany]. / Anwendung der hyperthermen intrathorakalen Chemotherapie (HITHOC) in Deutschland.

INTRODUCTION: For several years, hyperthermic intrathoracic chemotherapy (HITHOC) has been performed in a few departments for thoracic surgery in a multimodality treatment regime in addition to surgical cytoreduction. Specific data about HITHOC in Germany are still lacking. METHODS: Survey in written form to all departments of thoracic surgery in Germany. The objective is the evaluation of HITHOC with respect to number, indications, technique, perioperative protection measure and complications. RESULTS: A total of 116 departments of thoracic surgery were contacted, with a return rate of 43% (n = 50). HITHOC was not performed in 33 departments, due to lack of resources or experience (n = 17), missing efficacy of the procedure (n = 8) and fear of excessive complication rates (n = 3). Since 2008, a total of 343 HITHOC procedures have been performed in 17 departments. Eight departments have their own perfusion machine, whereas the remaining departments borrow the perfusion machine. Indications were malignant pleural mesothelioma in all departments (n = 17), thymoma with pleural spread (n = 11) and secondary pleural carcinosis (n = 7). The HITHOC was performed in nearly all departments after closing the chest (n = 16), with a temperature of 42 °C (n = 12) and for 60 minutes (n = 15). Cisplatin was always used, either alone (n = 9) or in combination (n = 8). In all the participating departments, the aims of the HITHOC were improvement in local tumor control and prolonged recurrence-free and overall survival. Relevant HITHOC-associated complications were low. CONCLUSIONS: HITHOC is performed in at least 17 departments of thoracic surgery in Germany, and is widely standardised with protective measures and a low rate of complications. The aims of the HITHOC are improvement in local tumor control in pleural malignancies combined with prolonged overall survival and better quality of life.

Evaluation of NGS and RT-PCR Methods for ALK Rearrangement in European NSCLC Patients: Results from the European Thoracic Oncology Platform Lungscape Project.

INTRODUCTION: The reported prevalence of ALK receptor tyrosine kinase gene (ALK) rearrangement in NSCLC ranges from 2% to 7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proved to be a reproducible and sensitive technique. Reverse-transcriptase polymerase chain reaction (RT-PCR) has also been advocated, and most recently, the advent of targeted next-generation sequencing (NGS) for ALK and other fusions has become possible. This study compares anaplastic lymphoma kinase (ALK) evaluation with all four techniques in resected NSCLC from the large European Thoracic Oncology Platform Lungscape cohort. METHODS: A total of 96 cases from the European Thoracic Oncology Platform Lungscape iBiobank, with any ALK immunoreactivity were examined by FISH, central RT-PCR, and NGS. An H-score higher than 120 defines IHC positivity. RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers covered the most frequent ALK translocations. For NGS, the Oncomine Solid Tumour Fusion Transcript Kit (Thermo Fisher Scientific, Waltham, MA) was used. The concordance was assessed using the Cohen κ coefficient (two-sided α ≤ 5%). RESULTS: NGS provided results for 77 of the 95 cases tested (81.1%), whereas RT-PCR provided results for 77 of 96 (80.2%). Concordance occurred in 55 cases of the 60 cases tested with all four methods (43 ALK negative and 12 ALK positive). Using ALK copositivity for IHC and FISH as the criterion standard, we derived a sensitivity for RT-PCR/NGS of 70.0%/85.0%, with a specificity of 87.1%/79.0%. When either RT-PCR or NGS was combined with IHC, the sensitivity remained the same, whereas the specificity increased to 88.7% and 83.9% respectively. CONCLUSION: NGS evaluation with the Oncomine Solid Tumour Fusion transcript kit and RT-PCR proved to have high sensitivity and specificity, advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be assessed by using two techniques and a third one in discordant cases. We therefore propose a customizable testing algorithm. These findings significantly influence existing testing paradigms and have clear clinical and economic impact.

FOXM1 activates AGR2 and causes progression of lung adenomas into invasive mucinous adenocarcinomas.

Lung cancer remains one of the most prominent public health challenges, accounting for the highest incidence and mortality among all human cancers. While pulmonary invasive mucinous adenocarcinoma (PIMA) is one of the most aggressive types of non-small cell lung cancer, transcriptional drivers of PIMA remain poorly understood. In the present study, we found that Forkhead box M1 transcription factor (FOXM1) is highly expressed in human PIMAs and associated with increased extracellular mucin deposition and the loss of NKX2.1. To examine consequences of FOXM1 expression in tumor cells in vivo, we employed an inducible, transgenic mouse model to express an activated FOXM1 transcript in urethane-induced benign lung adenomas. FOXM1 accelerated tumor growth, induced progression from benign adenomas to invasive, metastatic adenocarcinomas, and induced SOX2, a marker of poorly differentiated tumor cells. Adenocarcinomas in FOXM1 transgenic mice expressed increased MUC5B and MUC5AC, and reduced NKX2.1, which are characteristics of mucinous adenocarcinomas. Expression of FOXM1 in KrasG12D transgenic mice increased the mucinous phenotype in KrasG12D-driven lung tumors. Anterior Gradient 2 (AGR2), an oncogene critical for intracellular processing and packaging of mucins, was increased in mouse and human PIMAs and was associated with FOXM1. FOXM1 directly bound to and transcriptionally activated human AGR2 gene promoter via the -257/-247 bp region. Finally, using orthotopic xenografts we demonstrated that inhibition of either FOXM1 or AGR2 in human PIMAs inhibited mucinous characteristics, and reduced tumor growth and invasion. Altogether, FOXM1 is necessary and sufficient to induce mucinous phenotypes in lung tumor cells in vivo.

C-reactive protein-albumin ratio is an independent prognostic predictor of tumor recurrence in stage IIIA-N2 lung adenocarcinoma patients.

OBJECTIVE: To systematically evaluate the prognostic value of nutrition/inflammation-based markers for recurrence-free survival (RFS) in pN2-stage IIIA lung adenocarcinoma patients. MATERIALS AND METHODS: Data from 156 patients who had pathologically confirmed pN2-stage IIIA primary lung adenocarcinoma and received complete surgical resection from 2010 to 2014 were retrospectively analyzed. The data for Glasgow prognostic score (GPS), modified GPS (mGPS), high-sensitivity mGPS, C-reactive protein/albumin ratio (CAR), neutrophil/lymphocyte ratio, platelet/lymphocyte ratio, and prognostic nutritional index were analyzed. Univariate and multivariate Cox proportional-hazards regression analyses were used to identify the prognostic factors associated with RFS. RESULTS: The optimal cutoff value for the CAR was set at 0.6. A significant correlation was found between the CAR and RFS (P=0.001) by univariate analysis. Multivariate analysis between RFS and the factors selected from univariate analysis showed that ECOG performance status, pneumonectomy, multi-level N2, and high CAR were independent predictors of RFS. CONCLUSION: The CAR was the best prognostic marker to predict tumor recurrence in pN2-stage IIIA lung adenocarcinoma patients among the 7 nutrition/inflammation-based markers. The preoperative CAR may identify patients with a high risk of postoperative tumor recurrence.

Functional T cells targeting tumor-associated antigens are predictive for recurrence-free survival of patients with radically operated non-small cell lung cancer.

In this prospective study, we examined postoperative follow-up and preoperative IFN-γ T cell responses against 14 non-small cell lung cancer (NSCLC)-associated antigens in the blood of 51 patients with NSCLC, 7 patients with benign pulmonary tumors, and 10 tumor-free patients by enzyme-linked immunospot assay. The phenotype and function of T cells specific for tumor-associated antigens (TAAs) in the blood or tumor tissue of 9 NSCLC patients were characterized in detail using TNF-α, IL-2, and IFN-γ cytokine capture assays. We found that circulating TAA-specific T cells were significantly enriched in NSCLC compared with tumor-free patients. The most frequently recognized TAAs were Aurora kinase A, HER2/neu, NY-ESO-1, and p53. TNF-α was the most abundant cytokine secreted by TAA-specific T cells in the blood as well as by in situ-activated tumor-infiltrating lymphocytes, most of which were effector memory cells. The absence of TAA-reactive T cells identified patients at higher risk of tumor recurrence, irrespective of tumor stage (OR = 8.76, 95% CI: 1.57-34.79, p = 0.008). We conclude that pre-existing TAA-reactive circulating T cells are a strong independent prognostic factor for recurrence-free survival. These data may help discriminating high-risk from low-risk patients, improving prognostication, and redirecting adjuvant therapy. Our findings suggest the therapeutic relevance of Aurora kinase A, HER2/neu, NY-ESO-1, and p53 as targets for immunotherapy. This study is registered on Clinicaltrials.gov with trial identification number: NCT02515760.

Low-dose photon irradiation induces invasiveness through the SDF-1α/CXCR4 pathway in malignant mesothelioma cells.

BACKGROUND: Low-dose photon irradiation has repeatedly been suspected to increase a risk of promoting local recurrence of disease or even systemic dissemination. The purpose of this study was to investigate the motility of malignant pleural mesothelioma (MPM) cell lines after low-doses of photon irradiation and to elucidate the mechanism of the detected phenotype. METHODS: H28 and H226 MPM cells were examined in clonogenic survival experiments and migration assays with and without various doses of photon and carbon ion irradiation. C-X-C chemokine receptor type 4 (CXCR4), SDF-1α, ß1 integrin, α3 integrin, and α5 integrin expressions were analyzed by quantitative FACS analysis, ELISA and western blots. Apoptosis was assessed via Annexin-V-staining. RESULTS: The migration of MPM cells was stimulated by both fetal bovine serum and by stromal cell-derived factor 1α (SDF-1α). Low doses of photon irradiation (1 Gy and 2 Gy) suppressed clonogenicity, but promoted migration of both H28 and H226 cells through the SDF-1α/CXCR4 pathway. Hypermigration was inhibited by the administration of CXCR4 antagonist, AMD3100. In contrast, corresponding doses of carbon ion irradiation (0.3 Gy and 1 Gy) suppressed clonogenicity, but did not promote MPM cell migration. CONCLUSION: Our findings suggest that the co-administration of photon irradiation and the CXCR4-antagonist AMD3100 or the use of carbon ions instead of photons may be possible solutions to reduce the risk of locoregional tumor recurrence after radiotherapy for MPM.

SMARCA4 and SMARCA2 deficiency in non-small cell lung cancer: immunohistochemical survey of 316 consecutive specimens.

The chromatin remodeling switch sucrose nonfermentable (SWI/SNF) complex has been increasingly implicated in the pathogenesis and dedifferentiation of neoplasms from several organs with prognostic and potential therapeutic implications. We herein investigated the expression of the SWI/SNF complex catalytic subunits SMARCA4 (BRG1) and SMARCA2 (BRM) in 316 consecutive non-small cell lung cancer (NSCLC) specimens on tissue microarrays (171 adenocarcinomas [ADCAs], 130 squamous cell carcinomas [SCCs], 9 adenosquamous carcinomas, and 6 large cell carcinomas) excluding undifferentiated/giant cell or rhabdoid carcinomas. Complete loss of SMARCA4 was observed in 8 (5.5%) of 146 evaluable pulmonary ADCAs and 6 (5.2%) of 115 evaluable pulmonary SCCs, whereas 9 (6.4%) of 140 ADCAs and 2 (1.7%) of 117 SCCs showed SMARCA2 loss. Two of 6 large cell carcinomas were SMARCA2 deficient. Concurrent loss of both markers was observed in 4 cases (2 ADCAs and 2 SCCs). Of 15 ADCAs with loss of either or both markers, 12 (80%) were TTF1 negative. In conclusion, SMARCA4 and SMARCA2 deficiency is observed in 5.1% and 4.8% of NSCLC, respectively. SMARCB1 expression was intact in all cases. The presence of differentiated histology (glandular or squamous) is a novel aspect among SWI/SNF-deficient carcinomas which in other organs generally are associated with undifferentiated/rhabdoid morphology. The predominance of TTF1 negativity among SWI/SNF-deficient pulmonary ADCA (80%) underlines the need to include these 2 markers in the evaluation of TTF1-negative ADCA of putative pulmonary origin. Given the recently documented potential of SMARCA4 loss as a predictor of chemosensitivity to platinum-based chemotherapy in NSCLC, recognition of the clinicopathological features of SMARCA4-deficient NSCLC in routine surgical pathology practice is recommended.

AURKA, DLGAP5, TPX2, KIF11 and CKAP5: Five specific mitosis-associated genes correlate with poor prognosis for non-small cell lung cancer patients.

The growth of a tumor depends to a certain extent on an increase in mitotic events. Key steps during mitosis are the regulated assembly of the spindle apparatus and the separation of the sister chromatids. The microtubule-associated protein Aurora kinase A phosphorylates DLGAP5 in order to correctly segregate the chromatids. Its activity and recruitment to the spindle apparatus is regulated by TPX2. KIF11 and CKAP5 control the correct arrangement of the microtubules and prevent their degradation. In the present study, we investigated the role of these five molecules in non-small cell lung cancer (NSCLC). We analyzed the expression of the five genes in a large cohort of NSCLC patients (n=362) by quantitative real-time PCR. Each of the genes was highly overexpressed in the tumor tissues compared to corresponding normal lung tissue. The correlation of the expression of the individual genes depended on the histology. An increased expression of AURKA, DLGAP5, TPX2, KIF11 and CKAP5 was associated with poor overall survival (P=0.001-0.065). AURKA was a significant prognostic marker using multivariate analyses (P=0.006). Immunofluorescence studies demonstrated that the five mitosis-associated proteins co-localized with the spindle apparatus during cell division. Taken together, our data demonstrate that the expression of the mitosis-associated genes AURKA, DLGAP5, TPX2, KIF11 and CKAP5 is associated with the prognosis of NSCLC patients.

A new classification method for MALDI imaging mass spectrometry data acquired on formalin-fixed paraffin-embedded tissue samples.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) shows a high potential for applications in histopathological diagnosis, and in particular for supporting tumor typing and subtyping. The development of such applications requires the extraction of spectral fingerprints that are relevant for the given tissue and the identification of biomarkers associated with these spectral patterns. We propose a novel data analysis method based on the extraction of characteristic spectral patterns (CSPs) that allow automated generation of classification models for spectral data. Formalin-fixed paraffin embedded (FFPE) tissue samples from N=445 patients assembled on 12 tissue microarrays were analyzed. The method was applied to discriminate primary lung and pancreatic cancer, as well as adenocarcinoma and squamous cell carcinoma of the lung. A classification accuracy of 100% and 82.8%, resp., could be achieved on core level, assessed by cross-validation. The method outperformed the more conventional classification method based on the extraction of individual m/z values in the first application, while achieving a comparable accuracy in the second. LC-MS/MS peptide identification demonstrated that the spectral features present in selected CSPs correspond to peptides relevant for the respective classification. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Typing of colon and lung adenocarcinoma by high throughput imaging mass spectrometry.

In advanced tumor stages, diagnosis is frequently made from metastatic tumor tissue. In some cases, the identification of the tumor of origin may be difficult by histology alone. In this setting, immunohistochemical and molecular biological methods are often required. In a subset of tumors definite diagnosis cannot be achieved. Thus, additional new diagnostic methods are required for precise tumor subtyping. Mass spectrometric methods are of special interest for the discrimination of different tumor types. We investigated whether it is possible to discern adenocarcinomas of colon and lung using high-throughput imaging mass spectrometry on formalin-fixed paraffin-embedded tissue microarrays. 101 primary adenocarcinoma of the colon and 91 primary adenocarcinoma of the lung were used to train a Linear Discriminant Analysis model. Results were validated on an independent set of 116 colonic and 75 lung adenocarcinomas. In the validation cohort 109 of 116 patients with colonic and 67 of 75 patients with lung adenocarcinomas were correctly classified. The ability to define proteomic profiles capable to discern different tumor types promises a valuable tool in cancer diagnostics and might complement current approaches. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Glycodelin is a potential novel follow-up biomarker for malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor with a short survival time arising from the mesothelial cells of the pleura. Soluble mesothelin-related peptide (SMRP), osteopontin or EFEMP1 (Fibulin-3) are well described biomarkers for malignant mesothelioma with moderate sensitivity and specificity. In this study, we characterized the expression of glycodelin, a marker for risk pregnancy, in MPM by RNA and protein analyses and investigated its potential as a MPM biomarker. We were able to detect glycodelin in the serum of MPM patients. Compared to benign lung diseases, the serum levels were significant increased. Patients with high glycodelin serum levels revealed a worse overall survival. The glycodelin serum levels correlated with the tumor response to treatment. A comparison of SMRP and glycodelin serum measurement in a large patient cohort demonstrated that the detection of both soluble factors can increase the reliable diagnostic of MPM. Glycodelin was highly expressed in MPM tumors. Analyses of a tissue micro array indicated that the immunomodulatory form glycodelin A was expressed in MPM and correlated with the survival of the patients. Altogether, glycodelin seems to be a new potential biomarker for the aggressive malignant pleural mesothelioma.

A Comprehensive MicroRNA Expression Profile of Liver and Lung Metastases of Colorectal Cancer with Their Corresponding Host Tissue and Its Prognostic Impact on Survival.

MicroRNAs are small non-coding RNAs with a length of 18-25 nucleotides. They can regulate tumor invasion and metastasis by changing the expression and translation of their target mRNAs. Their expression is substantially altered in colorectal cancer cells as well as in the adjacent tumor-associated stroma. Both of these compartments have a mutual influence on tumor progression. In the development of metastases, cancer cells initially interact with the host tissue. Therefore, compartment-specific expression signatures of these three locations-tumor, associated stroma, and host tissue-can provide new insights into the complex tumor biology of colorectal cancer. Frozen tissue samples of colorectal liver (n = 25) and lung metastases (n = 24) were laser microdissected to separate tumor cells and the adjacent tumor-associated stroma cells. Additionally, normal lung and liver tissue was collected from the same patients. We performed a microarray analysis in four randomly selected liver metastases and four randomly selected lung metastases, analyzing a total of 939 human miRNAs. miRNAs with a significant change >2-fold between the tumor, tumor stroma, and host tissue were analyzed in all samples using RT-qPCR (11 miRNAs) and correlated with the clinical data. We found a differential expression of several miRNAs between the tumor, the tumor-associated stroma, and the host tissue compartment. When comparing liver and lung metastases, miR-194 showed a 1.5-fold; miR-125, miR-127, and miR-192 showed a 2.5-fold; miR-19 and miR-215 a 3-fold; miR-145, miR-199-3, and miR-429 a 5-fold; miR-21 a 7-fold; and, finally, miR-199-5 a 12.5-fold downregulation in liver metastases compared to lung metastases. Furthermore miR-19, miR-125, miR-127, miR-192, miR-194, miR-199-5, and miR-215 showed a significant upregulation in the normal liver tissue compared to the normal lung tissue. Univariate analysis identified an association of poor survival with the expression of miR-125 (p = 0.05), miR-127 (p = 0.001), miR-145 (p = 0.005), miR-192 (p = 0.015), miR-194 (0.003), miR-199-5 (p = 0.008), miR-215 (p < 0.001), and miR-429 (p = 0.03) in the host liver tissue of the liver metastases. Colorectal liver and lung metastases have a unique miRNA expression profile. miRNA expression in the host tissue of colorectal liver metastases seems to be able to influence tumor progression and survival. These findings can be used in the development of tailored therapies.

Genetic Risk Can Be Decreased: Quitting Smoking Decreases and Delays Lung Cancer for Smokers With High and Low CHRNA5 Risk Genotypes - A Meta-Analysis.

BACKGROUND: Recent meta-analyses show that individuals with high risk variants in CHRNA5 on chromosome 15q25 are likely to develop lung cancer earlier than those with low-risk genotypes. The same high-risk genetic variants also predict nicotine dependence and delayed smoking cessation. It is unclear whether smoking cessation confers the same benefits in terms of lung cancer risk reduction for those who possess CHRNA5 risk variants versus those who do not. METHODS: Meta-analyses examined the association between smoking cessation and lung cancer risk in 15 studies of individuals with European ancestry who possessed varying rs16969968 genotypes (N=12,690 ever smokers, including 6988 cases of lung cancer and 5702 controls) in the International Lung Cancer Consortium. RESULTS: Smoking cessation (former vs. current smokers) was associated with a lower likelihood of lung cancer (OR=0.48, 95%CI=0.30-0.75, p=0.0015). Among lung cancer patients, smoking cessation was associated with a 7-year delay in median age of lung cancer diagnosis (HR=0.68, 95%CI=0.61-0.77, p=4.9∗10-10). The CHRNA5 rs16969968 risk genotype (AA) was associated with increased risk and earlier diagnosis for lung cancer, but the beneficial effects of smoking cessation were very similar in those with and without the risk genotype. CONCLUSION: We demonstrate that quitting smoking is highly beneficial in reducing lung cancer risks for smokers regardless of their CHRNA5 rs16969968 genetic risk status. Smokers with high-risk CHRNA5 genotypes, on average, can largely eliminate their elevated genetic risk for lung cancer by quitting smoking- cutting their risk of lung cancer in half and delaying its onset by 7years for those who develop it. These results: 1) underscore the potential value of smoking cessation for all smokers, 2) suggest that CHRNA5 rs16969968 genotype affects lung cancer diagnosis through its effects on smoking, and 3) have potential value for framing preventive interventions for those who smoke.