Combination of calretinin, MALAT1, and GAS5 as a potential prognostic biomarker to predict disease progression in surgically treated mesothelioma patients.

BACKGROUND: The role of cytoreductive surgery for epithelioid pleural mesothelioma within a multimodal treatment approach remains controversial. Carefully selected patients benefit from cytoreductive surgery and adjuvant chemotherapy, but there is no established biomarker to predict tumor recurrence or progression during the course of the disease. The aim of this study was to identify potential biomarkers to predict therapeutic response in terms of progression-free survival. METHODS: Between 03/2014 and 08/2022, preoperative blood samples were collected from 76 patients with epithelioid pleural mesothelioma who underwent cytoreductive surgery as part of a multimodal treatment approach. Identification of potential biomarkers was performed by determination of mesothelin and calretinin, as well as specific long non-coding RNAs and microRNAs. Receiver operating characteristic analysis, Kaplan-Meier survival analysis, and Cox regression were used to assess the association between biomarker concentrations and patient recurrence status and survival. RESULTS: MALAT1, GAS5, and calretinin showed statistically significant increased biomarker levels in patients with recurrence in contrast to recurrence-free patients after surgical treatment (p < 0.0001, p = 0.0190, and p = 0.0068, respectively). The combination of the three biomarkers resulted in a sensitivity of 68 % and a specificity of 89 %. CONCLUSION: MALAT1, GAS5, and calretinin could be potential biomarkers for the prediction of tumor recurrence, improving the benefit from multimodal treatment including cytoreductive surgery.

The pregnancy-associated protein glycodelin as a potential sex-specific target for resistance to immunotherapy in non-small cell lung cancer.

Lung cancer has been shown to be targetable by novel immunotherapies which reactivate the immune system and enable tumor cell killing. However, treatment failure and resistance to these therapies is common. Consideration of sex as a factor influencing therapy resistance is still rare. We hypothesize that the success of the treatment is impaired by the presence of the immunosuppressive pregnancy-associated glycoprotein glycodelin that is expressed in patients with non-small-cell lung cancer (NSCLC). We demonstrate that the glycan pattern of NSCLC-derived glycodelin detected by a lectin-based enrichment assay highly resembles amniotic fluid-derived glycodelin A, which is known to have immunosuppressive properties. NSCLC-derived glycodelin interacts with immune cells in vitro and regulates the expression of genes associated with inflammatory and tumor microenvironment pathways. In tumor microarray samples of patients, high glycodelin staining in tumor areas results in an impaired overall survival of female patients. Moreover, glycodelin colocalizes to tumor infiltrating CD8+ T cells and pro-tumorigenic M2 macrophages. High serum concentrations of glycodelin prior to immunotherapy are associated with a poor progression-free survival (p < 0.001) of female patients receiving PD-(L)1 inhibitors. In summary, our findings suggest that glycodelin not only is a promising immunological biomarker for early identification of female patients that do not benefit from the costly immunotherapy, but also represents a promising immunotherapeutic target in NSCLC to improve therapeutic options in lung cancer.

PITX2 as a Sensitive and Specific Marker of Midgut Neuroendocrine Tumors: Results from a Cohort of 1157 Primary Neuroendocrine Neoplasms.

As neuroendocrine tumors (NETs) often present as metastatic lesions, immunohistochemical assignment to a site of origin is one of the most important tasks in their pathologic assessment. Because a fraction of NETs eludes the typical expression profiles of their primary localization, additional sensitive and specific markers are required to improve diagnostic certainty. We investigated the expression of the transcription factor Pituitary Homeobox 2 (PITX2) in a large-scale cohort of 909 NET and 248 neuroendocrine carcinomas (NEC) according to the immunoreactive score (IRS) and correlated PITX2 expression groups with general tumor groups and primary localization. PITX2 expression (all expression groups) was highly sensitive (98.1%) for midgut-derived NET, but not perfectly specific, as non-midgut NET (especially pulmonary/duodenal) were quite frequently weak or moderately positive. The specificity rose to 99.5% for a midgut origin of NET if only a strong PITX2 expression was considered, which was found in only 0.5% (one pancreatic/one pulmonary) of non-midgut NET. In metastases of midgut-derived NET, PITX2 was expressed in all cases (87.5% strong, 12.5% moderate), whereas CDX2 was negative or only weakly expressed in 31.3% of the metastases. In NEC, a fraction of cases (14%) showed a weak or moderate PITX2 expression, which was not associated with a specific tumor localization. Our study independently validates PITX2 as a very sensitive and specific immunohistochemical marker of midgut-derived NET in a very large collective of neuroendocrine neoplasms. Therefore, our data argue toward implementation into diagnostic panels applied for NET as a firstline midgut marker.

CYFRA 21-1, CA 125 and CEA provide additional prognostic value in NSCLC patients with stable disease at first CT scan.

BACKGROUND: Serum tumor markers (STM) may complement imaging and provide additional clinical information for patients with non-small cell lung cancer (NSCLC). OBJECTIVE: To determine whether STMs can predict outcomes in patients with stable disease (SD) after initial treatment. METHODS: This single-center, prospective, observational trial enrolled 395 patients with stage III/IV treatment-naïve NSCLC; of which 263 patients were included in this analysis. Computed Tomography (CT) scans were performed and STMs measured before and after initial treatment (two cycles of chemotherapy and/or an immune checkpoint inhibitor or tyrosine kinase inhibitor); analyses were based on CT and STM measurements obtained at first CT performed after cycle 2 only PFS and OS were analyzed by Kaplan-Meier curves and Cox-proportional hazard models. RESULTS: When patients with SD (n = 100) were split into high- and low-risk groups based on CYFRA 21-1, CEA and CA 125 measurements using an optimized cut-off, a 4-fold increase risk of progression or death was estimated for high- vs low-risk SD patients (PFS, HR 4.17; OS, 3.99; both p < 0.0001). Outcomes were similar between patients with high-risk SD or progressive disease (n = 35) (OS, HR 1.17) and between patients with low-risk SD or partial response (n = 128) (PFS, HR 0.98; OS, 1.14). CONCLUSIONS: STMs can provide further guidance in patients with indeterminate CT responses by separating them into high- and low-risk groups for future PFS and OS events.

Prognostic value of tumor markers ProGRP, NSE and CYFRA 21-1 in patients with small cell lung cancer and chemotherapy-induced remission.

BACKGROUND: Despite successful response to first line therapy, patients with small-cell lung cancer (SCLC) often suffer from early relapses and disease progression. OBJECTIVE: To investigate the relevance of serum tumor markers for estimation of prognosis at several time points during the course of disease. METHODS: In a prospective, single-center study, serial assessments of progastrin-releasing peptide (ProGRP), neuron-specific enolase (NSE), cytokeratin-19 fragments (CYFRA 21-1) and carcino-embryogenic antigen (CEA) were performed during and after chemotherapy in 232 SCLC patients, and correlated with therapy response and overall survival (OS). RESULTS: ProGRP, NSE and CYFRA 21-1 levels decreased quickly after the first chemotherapy cycle and correlated well with the radiological response. Either as single markers or in combination they provided valuable prognostic information regarding OS at all timepoints investigated: prior to first-line therapy, after two treatment cycles in patients with successful response to first-line therapy, and prior to the start of second-line therapy. Furthermore, they were useful for continuous monitoring during and after therapy and often indicated progressive disease several months ahead of radiological changes. CONCLUSIONS: The results indicate the great potential of ProGRP, NSE and CYFRA 21-1 for estimating prognosis and monitoring of SCLC patients throughout the course of the disease.

Preliminary results from the EMoLung clinical study showing early lung cancer detection by the LC score.

BACKGROUND: Lung cancer (LC) causes more deaths worldwide than any other cancer type. Despite advances in therapeutic strategies, the fatality rate of LC cases remains high (95%) since the majority of patients are diagnosed at late stages when patient prognosis is poor. Analysis of the International Association for the Study of Lung Cancer (IASLC) database indicates that early diagnosis is significantly associated with favorable outcome. However, since symptoms of LC at early stages are unspecific and resemble those of benign pathologies, current diagnostic approaches are mostly initiated at advanced LC stages. METHODS: We developed a LC diagnosis test based on the analysis of distinct RNA isoforms expressed from the GATA6 and NKX2-1 gene loci, which are detected in exhaled breath condensates (EBCs). Levels of these transcript isoforms in EBCs were combined to calculate a diagnostic score (the LC score). In the present study, we aimed to confirm the applicability of the LC score for the diagnosis of early stage LC under clinical settings. Thus, we evaluated EBCs from patients with early stage, resectable non-small cell lung cancer (NSCLC), who were prospectively enrolled in the EMoLung study at three sites in Germany. RESULTS: LC score-based classification of EBCs confirmed its performance under clinical conditions, achieving a sensitivity of 95.7%, 91.3% and 84.6% for LC detection at stages I, II and III, respectively. CONCLUSIONS: The LC score is an accurate and non-invasive option for early LC diagnosis and a valuable complement to LC screening procedures based on computed tomography.

Early Detection of Lung Cancer Using Small RNAs.

INTRODUCTION: Lung cancer remains the deadliest cancer in the world, and lung cancer survival is heavily dependent on tumor stage at the time of detection. Low-dose computed tomography screening can reduce mortality; however, annual screening is limited by low adherence in the United States of America and still not broadly implemented in Europe. As a result, less than 10% of lung cancers are detected through existing programs. Thus, there is a great need for additional screening tests, such as a blood test, that could be deployed in the primary care setting. METHODS: We prospectively recruited 1384 individuals meeting the National Lung Screening Trial demographic eligibility criteria for lung cancer and collected stabilized whole blood to enable the pipetting-free collection of material, thus minimizing preanalytical noise. Ultra-deep small RNA sequencing (20 million reads per sample) was performed with the addition of a method to remove highly abundant erythroid RNAs, and thus open bandwidth for the detection of less abundant species originating from the plasma or the immune cellular compartment. We used 100 random data splits to train and evaluate an ensemble of logistic regression classifiers using small RNA expression of 943 individuals, discovered an 18-small RNA feature consensus signature (miLung), and validated this signature in an independent cohort (441 individuals). Blood cell sorting and tumor tissue sequencing were performed to deconvolve small RNAs into their source of origin. RESULTS: We generated diagnostic models and report a median receiver-operating characteristic area under the curve of 0.86 (95% confidence interval [CI]: 0.84-0.86) in the discovery cohort and generalized performance of 0.83 in the validation cohort. Diagnostic performance increased in a stage-dependent manner ranging from 0.73 (95% CI: 0.71-0.76) for stage I to 0.90 (95% CI: 0.89-0.90) for stage IV in the discovery cohort and from 0.76 to 0.86 in the validation cohort. We identified a tumor-shed, plasma-bound ribosomal RNA fragment of the L1 stalk as a dominant predictor of lung cancer. The fragment is decreased after surgery with curative intent. In additional experiments, results of dried blood spot collection and sequencing revealed that small RNA analysis could potentially be conducted through home sampling. CONCLUSIONS: These data suggest the potential of a small RNA-based blood test as a viable alternative to low-dose computed tomography screening for early detection of smoking-associated lung cancer.

Prognostic Impact of the Immune-Cell Infiltrate in N1-Positive Non-Small-Cell Lung Cancer.

INTRODUCTION: The tumoral immune milieu plays a crucial role for the development of non-small-cell lung cancer (NSCLC) and may influence individual prognosis. We analyzed the predictive role of immune cell infiltrates after curative lung cancer surgery. MATERIALS AND METHODS: The tumoral immune-cell infiltrate from 174 patients with pN1 NSCLC and adjuvant chemotherapy was characterized using immunofluorescence staining. The density and distribution of specific immune cells in tumor center (TU), invasive front (IF) and normal tissue (NORM) were correlated with clinical parameters and survival data. RESULTS: Tumor specific survival (TSS) of all patients was 69.9% at 5 years. The density of tumor infiltrating lymphocytes (TIL) was higher in TU and IF than in NORM. High TIL density in TU (low vs. high: 62.0% vs. 86.7%; p = .011) and the presence of cytotoxic T-Lymphocytes (CTLs) in TU and IF were associated with improved TSS (positive vs. negative: 90.6% vs. 64.7% p = .024). High TIL-density correlated with programmed death-ligand 1 expression levels ≥50% (p < .001). Multivariate analysis identified accumulation of TIL (p = .016) and low Treg density (p = .003) in TU as negative prognostic predictors in squamous cell carcinoma (p = .025), whereas M1-like tumor- associated macrophages (p = .019) and high programmed death-ligand 1 status (p = .038) were associated with better survival in adenocarcinoma. CONCLUSION: The assessment of specific intratumoral immune cells may serve as a prognostic predictor in pN1 NSCLC. However differences were observed related to adenocarcinoma or squamous cell carcinoma histology. Prospective assessment of the immune-cell infiltrate and further clarification of its prognostic relevance could assist patient selection for upcoming perioperative immunotherapies.

Corrigendum: Deep learning for the detection of anatomical tissue structures and neoplasms of the skin on scanned histopathological tissue sections.

[This corrects the article DOI: 10.3389/fonc.2022.1022967.].

Lung Adenocarcinoma Diagnosed at a Younger Age Is Associated with Advanced Stage, Female Sex, and Ever-Smoker Status, in Patients Treated with Lung Resection.

To date, the factors which affect the age at diagnosis of lung adenocarcinoma are not fully understood. In our study, we examined the relationships of age at diagnosis with smoking, pathological stage, sex, and year of diagnosis in a discovery (n = 1694) and validation (n = 1384) series of lung adenocarcinoma patients who had undergone pulmonary resection at hospitals in the Milan area and at Thoraxklinik (Heidelberg), respectively. In the discovery series, younger age at diagnosis was associated with ever-smoker status (OR = 1.5, p = 0.0035) and advanced stage (taking stage I as reference: stage III OR = 1.4, p = 0.0067; stage IV OR = 1.7, p = 0.0080), whereas older age at diagnosis was associated with male sex (OR = 0.57, p < 0.001). Analysis in the validation series confirmed the ever versus never smokers' association (OR = 2.9, p < 0.001), the association with highest stages (stage III versus stage I OR = 1.4, p = 0.0066; stage IV versus stage I OR = 2.0, p = 0.0022), and the male versus female sex association (OR = 0.78, p = 0.032). These data suggest the role of smoking in affecting the natural history of the disease. Moreover, aggressive tumours seem to have shorter latency from initiation to clinical detection. Finally, younger age at diagnosis is associated with the female sex, suggesting that hormonal status of young women confers risk to develop lung adenocarcinoma. Overall, this study provided novel findings on the mechanisms underlying age at diagnosis of lung adenocarcinoma.

High-resolution transcriptomic and epigenetic profiling identifies novel regulators of COPD.

Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering its environmental cause, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) across COPD stages. We show that the epigenetic landscape is changed early in COPD, with DNA methylation changes occurring predominantly in regulatory regions. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Data integration identified 110 candidate regulators of disease phenotypes that were linked to fibroblast repair processes using phenotypic screens. Our study provides high-resolution multi-omic maps of HLFs across COPD stages. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic lung diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients.

Comparison of the sensitivity of different criteria to select lung cancer patients for screening in a cohort of German patients.

INTRODUCTION: Trials of CT-based screening for lung cancer have shown a mortality advantage for screening in North America and Europe. Before introducing a nationwide lung cancer screening program in Germany, it is important to assess the criteria used in international trials in the German population. METHODS: We used data from 3623 lung cancer patients from the data warehouse of the German Center for Lung Research (DZL). We compared the sensitivity of the following lung cancer screening criteria overall and stratified by age and histology: the National Lung Screening Trial (NLST), the Danish Lung Cancer Screening Trial (DLCST), the 2013 and 2021 US Preventive Services Task Force (USPSTF), and an adapted version of the Prostate, Lung, Colorectal, and Ovarian no race model (adapted PLCOm2012) with 6-year risk thresholds of 1.0%/6 year and 1.7%/6 year. RESULTS: Overall, the adapted PLCOm2012 model (1%/6 years), selected the highest proportion of lung cancer patients for screening (72.4%), followed by the 2021 USPSTF (70.0%), the adapted PLCOm2012 (1.7%/6 year) (57.4%), the 2013 USPTF (57.0%), DLCST criteria (48.7%), and the NLST (48.5%). The adapted PLCOm2012 risk model (1.0%/6 year) had the highest sensitivity for all histological types except for small-cell and large-cell carcinomas (non-significant), whereas the 2021 USPTF selected a higher proportion of patients. The sensitivity levels were higher in males than in females. CONCLUSION: Using a risk-based selection score resulted in higher sensitivities compared to criteria using dichotomized age and smoking history. However, gender disparities were apparent in all studied eligibility criteria. In light of increasing lung cancer incidences in women, all selection criteria should be reviewed for ways to close this gender gap, especially when implementing a large-scale lung cancer screening program.

Longitudinal monitoring of cell-free DNA methylation in ALK-positive non-small cell lung cancer patients.

BACKGROUND: DNA methylation (5-mC) signals in cell-free DNA (cfDNA) of cancer patients represent promising biomarkers for minimally invasive tumor detection. The high abundance of cancer-associated 5-mC alterations permits parallel and highly sensitive assessment of multiple 5-mC biomarkers. Here, we performed genome-wide 5-mC profiling in the plasma of metastatic ALK-rearranged non-small cell lung cancer (NSCLC) patients receiving tyrosine kinase inhibitor therapy. We established a strategy to identify ALK-specific 5-mC changes from cfDNA and demonstrated the suitability of the identified markers for cancer detection, prognosis, and therapy monitoring. METHODS: Longitudinal plasma samples (n = 79) of 21 ALK-positive NSCLC patients and 13 healthy donors were collected alongside 15 ALK-positive tumor tissue and 10 healthy lung tissue specimens. All plasma and tissue samples were analyzed by cell-free DNA methylation immunoprecipitation sequencing to generate genome-wide 5-mC profiles. Information on genomic alterations (i.e., somatic mutations/fusions and copy number alterations) determined in matched plasma samples was available from previous studies. RESULTS: We devised a strategy that identified tumor-specific 5-mC biomarkers by reducing 5-mC background signals derived from hematopoietic cells. This was followed by differential methylation analysis (cases vs. controls) and biomarker validation using 5-mC profiles of ALK-positive tumor tissues. The resulting 245 differentially methylated regions were enriched for lung adenocarcinoma-specific 5-mC patterns in TCGA data and indicated transcriptional repression of several genes described to be silenced in NSCLC (e.g., PCDH10, TBX2, CDO1, and HOXA9). Additionally, 5-mC-based tumor DNA (5-mC score) was highly correlated with other genomic alterations in cell-free DNA (Spearman, ρ > 0.6), while samples with high 5-mC scores showed significantly shorter overall survival (log-rank p = 0.025). Longitudinal 5-mC scores reflected radiologic disease assessments and were significantly elevated at disease progression compared to the therapy start (p = 0.0023). In 7 out of 8 instances, rising 5-mC scores preceded imaging-based evaluation of disease progression. CONCLUSION: We demonstrated a strategy to identify 5-mC biomarkers from the plasma of cancer patients and integrated them into a quantitative measure of cancer-associated 5-mC alterations. Using longitudinal plasma samples of ALK-positive NSCLC patients, we highlighted the suitability of cfDNA methylation for prognosis and therapy monitoring.

Deep learning for the detection of anatomical tissue structures and neoplasms of the skin on scanned histopathological tissue sections.

Basal cell carcinoma (BCC), squamous cell carcinoma (SqCC) and melanoma are among the most common cancer types. Correct diagnosis based on histological evaluation after biopsy or excision is paramount for adequate therapy stratification. Deep learning on histological slides has been suggested to complement and improve routine diagnostics, but publicly available curated and annotated data and usable models trained to distinguish common skin tumors are rare and often lack heterogeneous non-tumor categories. A total of 16 classes from 386 cases were manually annotated on scanned histological slides, 129,364 100 x 100 µm (~395 x 395 px) image tiles were extracted and split into a training, validation and test set. An EfficientV2 neuronal network was trained and optimized to classify image categories. Cross entropy loss, balanced accuracy and Matthews correlation coefficient were used for model evaluation. Image and patient data were assessed with confusion matrices. Application of the model to an external set of whole slides facilitated localization of melanoma and non-tumor tissue. Automated differentiation of BCC, SqCC, melanoma, naevi and non-tumor tissue structures was possible, and a high diagnostic accuracy was achieved in the validation (98%) and test (97%) set. In summary, we provide a curated dataset including the most common neoplasms of the skin and various anatomical compartments to enable researchers to train, validate and improve deep learning models. Automated classification of skin tumors by deep learning techniques is possible with high accuracy, facilitates tumor localization and has the potential to support and improve routine diagnostics.

Multimodal therapy of epithelioid pleural mesothelioma: improved survival by changing the surgical treatment approach.

Background: The exact role and type of surgery for malignant pleural mesothelioma (MPM) remains controversial. This study aimed at analyzing a 20-year single center perioperative experience in MPM surgery at our high-volume thoracic surgery center and comparing the overall survival after trimodal extrapleural pneumonectomy (EPP) and extended pleurectomy and decortication combined with hyperthermic intrathoracic chemoperfusion (EPD/HITOC) and adjuvant chemotherapy with that after chemotherapy (CTx) alone. Methods: Patients with epithelioid MPM treated with neoadjuvant chemotherapy, EPP and adjuvant radiotherapy within a trimodal concept or EPD/HITOC in combination with adjuvant chemotherapy between 2001 and 2018 were included in this retrospective analysis. Surgical cohorts were compared to patients treated with standard chemotherapy. Results: Overall, 182 patients (69 EPP, 57 EPD/HITOC, 56 CTx) were analyzed. Due to occupational exposure to asbestos for most of the patients, 154 patients (84.6%) were male. The patients in the surgical cohorts were significantly younger than those in the CTx cohort. There was no significant difference between the proportion of patient age and side. The median overall survival of the EPD/HITOC cohort with 38.1 months was significantly longer than that of the EPP and CTx cohorts (24.0 and 15.8 months). Better survival was significantly associated with an ECOG 0 performance status, age below 70 years, and negative lymph node status. In the multivariate analysis, EPD/HITOC was significantly associated with improved overall survival. Perioperative morbidity was lower in the EPD/HITOC group than in the EPP cohort. Conclusions: EPD/HITOC is feasible and safe for localized epithelioid pleural mesothelioma. Changing the surgical approach to a less radical lung-sparing technique may improve overall survival compared to trimodal EPP.

Lung Adenocarcinoma Cell Sensitivity to Chemotherapies: A Spotlight on Lipid Droplets and SREBF1 Gene.

To explore the relationship between cancer cell SREBF1 expression, lipid droplets (LDs) formation, and the sensitivity to chemotherapies, we cultured lung adenocarcinoma cells H1299 (with LD) and H1563 (without LD) in a serum-free basal medium (BM) or neutrophil degranulation products containing medium (NDM), and tested cell responses to cisplatin and etoposide. By using the DESeq2 Bioconductor package, we detected 674 differentially expressed genes (DEGs) associated with NDM/BM differences between two cell lines, many of these genes were associated with the regulation of sterol and cholesterol biosynthesis processes. Specifically, SREBF1 markedly declined in both cell lines cultured in NDM or when treated with chemotherapeutics. Despite the latter, H1563 exhibited LD formation and resistance to etoposide, but not to cisplatin. Although H1299 cells preserved LDs, these cells were similarly sensitive to both drugs. In a cohort of 292 patients with non-small-cell lung cancer, a lower SREBF1 expression in tumors than in adjacent nontumor tissue correlated with overall better survival, specifically in patients with adenocarcinoma at stage I. Our findings imply that a direct correlation between SREBF1 and LD accumulation can be lost due to the changes in cancer cell environment and/or chemotherapy. The role of LDs in lung cancer development and response to therapies remains to be examined in more detail.

Cellular dissociation grading on biopsies of pulmonary squamous cell carcinoma provides prognostic information across all stages and is congruent with resection specimen grading.

Grading of squamous cell carcinomas (SCCs) based on tumour budding and cell nest size has been termed cellular dissociation grading (CDG) and was suggested as a robust outcome predictor when assessed in biopsies and resections of various extrapulmonary SCCs. In pulmonary SCC (pSCC), this has so far been shown only for resected cancers. As most lung cancers are inoperable, it is of utmost importance to clarify whether the prognostic impact of CDG is retained in the biopsy setting. Two independent pSCC biopsy cohorts from Munich (n = 134, non-resected) and Heidelberg (n = 135, resected) were assessed. Tumour budding and cell nest size measures were assembled into the three-tiered CDG system (G1-G3). Data were correlated with clinicopathological parameters and overall- (OS), disease-specific- (DSS), and disease-free survival (DFS). Interobserver variability and concordance between biopsy and resection specimen were also investigated. CDG was highly congruent between biopsy and resection specimens (κ = 0.77, p < 0.001). In both pSCC cohorts, biopsy-derived CDG strongly impacted on OS, DSS, and DFS (e.g. DFS: p < 0.001). In multivariate survival analyses, CDG remained a stage independent predictor of survival in both cohorts (DFS: p < 0.001 respectively; hazard ratio Munich cohort: CDG-G2: 4.31, CDG-G3; 5.14; Heidelberg cohort: CDG-G2: 5.87, CDG-G3: 9.07). Interobserver agreement for CDG was almost perfect (κ = 0.84, p < 0.001). We conclude that assessment of CDG based on tumour budding and cell nest size is feasible on pSCC biopsies and harbours stage independent prognostic information in resectable as well as non-resectable pSCC. Integration of this grading approach into clinicopathological routine should be considered.

Identification of NHLRC1 as a Novel AKT Activator from a Lung Cancer Epigenome-Wide Association Study (EWAS).

Changes in DNA methylation identified by epigenome-wide association studies (EWAS) have been recently linked to increased lung cancer risk. However, the cellular effects of these differentially methylated positions (DMPs) are often unclear. Therefore, we investigated top differentially methylated positions identified from an EWAS study. This included a putative regulatory region of NHLRC1. Hypomethylation of this gene was recently linked with decreased survival rates in lung cancer patients. HumanMethylation450 BeadChip array (450K) analysis was performed on 66 lung cancer case-control pairs from the European Prospective Investigation into Cancer and Nutrition Heidelberg lung cancer EWAS (EPIC HD) cohort. DMPs identified in these pre-diagnostic blood samples were then investigated for differential DNA methylation in lung tumor versus adjacent normal lung tissue from The Cancer Genome Atlas (TCGA) and replicated in two independent lung tumor versus adjacent normal tissue replication sets with MassARRAY. The EPIC HD top hypermethylated DMP cg06646708 was found to be a hypomethylated region in multiple data sets of lung tumor versus adjacent normal tissue. Hypomethylation within this region caused increased mRNA transcription of the closest gene NHLRC1 in lung tumors. In functional assays, we demonstrate attenuated proliferation, viability, migration, and invasion upon NHLRC1 knock-down in lung cancer cells. Furthermore, diminished AKT phosphorylation at serine 473 causing expression of pro-apoptotic AKT-repressed genes was detected in these knock-down experiments. In conclusion, this study demonstrates the powerful potential for discovery of novel functional mechanisms in oncogenesis based on EWAS DNA methylation data. NHLRC1 holds promise as a new prognostic biomarker for lung cancer survival and prognosis, as well as a target for novel treatment strategies in lung cancer patients.

Cross-ancestry genome-wide meta-analysis of 61,047 cases and 947,237 controls identifies new susceptibility loci contributing to lung cancer.

To identify new susceptibility loci to lung cancer among diverse populations, we performed cross-ancestry genome-wide association studies in European, East Asian and African populations and discovered five loci that have not been previously reported. We replicated 26 signals and identified 10 new lead associations from previously reported loci. Rare-variant associations tended to be specific to populations, but even common-variant associations influencing smoking behavior, such as those with CHRNA5 and CYP2A6, showed population specificity. Fine-mapping and expression quantitative trait locus colocalization nominated several candidate variants and susceptibility genes such as IRF4 and FUBP1. DNA damage assays of prioritized genes in lung fibroblasts indicated that a subset of these genes, including the pleiotropic gene IRF4, potentially exert effects by promoting endogenous DNA damage.

Intratumoral Heterogeneity and Immune Modulation in Lung Adenocarcinoma in Female Smokers and Never Smokers.

SIGNIFICANCE: Single-cell analysis of healthy lung tissue and lung cancer reveals distinct tumor cell populations, including cells with differential immune modulating capacity between smokers and never smokers, which could guide future therapeutic strategies.