EBUS-TBNA in Extrathoracic Malignancies: Diagnostic and Prognostic Implications.

PURPOSE: In patients with extrathoracic malignancies (EM) the role of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for the assessment of abnormal mediastinal lymph nodes (MLN) is controversial. The aim of this study was to assess the diagnostic yield and prognostic significance of EBUS-TBNA in these patients. METHODS: Retrospective analysis of patients with EM and abnormal MLN detected by Computed Tomography (CT) and/or Positron Emission Tomography (PET). RESULTS: A total of 161 patients with EM and abnormal MLN were included (93 males, 58%). The most common EM was melanoma (19%) and gastrointestinal cancer (17%). Assessed lymph nodes were mediastinal in 70% of cases and hilar in 30%. The most frequently sampled lymph nodes were subcarinal (45%) and lower right paratracheal (21%). Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of EBUS-TBNA for the diagnosis of malignancy were 88%, 100%, 100% and 87%, respectively. These values were similar regardless the type of EM except for head and neck tumors where the NPV was particularly low (67%). The diagnosis of neoplastic involvement by EBUS-TBNA implied a worse prognosis in terms of overall survival (p < 0.02) and cancer-specific survival (p < 0.001). CONCLUSIONS: In patients with EM and abnormal MLN, EBUS-TBNA has a high diagnostic yield. However, the NPV decrease in patients with head and neck tumors. Neoplastic MLN detected by EBUS-TBNA has prognostic implications in these patients.

Characterization of the MicroRNA Cargo of Extracellular Vesicles Isolated from a Pulmonary Tumor-Draining Vein Identifies miR-203a-3p as a Relapse Biomarker for Resected Non-Small Cell Lung Cancer.

In resected non-small cell lung cancer (NSCLC), post-surgical recurrence occurs in around 40% of patients, highlighting the necessity to identify relapse biomarkers. An analysis of the extracellular vesicle (EV) cargo from a pulmonary tumor-draining vein (TDV) can grant biomarker identification. We studied the pulmonary TDV EV-miRNAome to identify relapse biomarkers in a two-phase study (screening and validation). In the screening phase, a 17-miRNA relapse signature was identified in 18 selected patients by small RNAseq. The most expressed miRNA from the signature (EV-miR-203a-3p) was chosen for further validation. Pulmonary TDV EV-miR-203a-3p was studied by qRT-PCR in a validation cohort of 70 patients, where it was found to be upregulated in relapsed patients (p = 0.0194) and in patients with cancer spread to nearby lymph nodes (N+ patients) (p = 0.0396). The ROC curve analysis showed that TDV EV-miR-203a-3p was able to predict relapses with a sensitivity of 88% (AUC: 0.67; p = 0.022). Moreover, patients with high TDV EV-miR-203a-3p had a shorter time to relapse than patients with low levels (43.6 vs. 97.6 months; p = 0.00703). The multivariate analysis showed that EV-miR-203a-3p was an independent, predictive and prognostic post-surgical relapse biomarker. In conclusion, pulmonary TDV EV-miR-203a-3p is a promising new relapse biomarker for resected NSCLC patients.

Tumour markers with clinically controlled cut-offs for suspected cancer.

BACKGROUND: Due to insufficient scientific evidence, panels of tumour markers (TMs) are currently not recommended for use in suspected cancer. However, recent well-designed studies have revealed a potential clinical value in lung cancer. We analysed the diagnostic accuracy of a panel of 11 circulating TMs with clinically controlled thresholds in the differentiation of cancer from nonmalignant diseases. METHODS: We prospectively recruited 4776 consecutive patients presenting with focal or nonspecific symptoms suggestive of cancer who underwent testing for 11 serum TMs before diagnosis was known. The study abided by 2015 STARD guidelines. Tumour markers included, among others, carbohydrate antigen 19-9, carcinoembryonic antigen, alpha-fetoprotein, squamous cell carcinoma-associated antigen, prostate-specific antigen (males), neuron-specific enolase, progastrin-releasing peptide and carbohydrate antigen 125. Thresholds were adjusted for the presence of kidney failure, liver disease, effusions and dermatological disorders. Results showing ≥1 TMs with concentrations above threshold were considered positive. RESULTS: Benign diseases were diagnosed in 3281 (68.7%) patients and cancer in 1495 (31.3%), with epithelial cancers in 1214 (77% at stage IV). When applying criteria for controlled thresholds, overall specificity was 98%. Overall sensitivity of the panel in epithelial cancers was 72.2%, positive predictive value 93% and negative predictive value 90.5%. The area under the receiver operating characteristic curve was 0.920 (95% confidence interval, 0.902-0.924). CONCLUSIONS: By using clinically controlled cut-offs, the combined panel demonstrated an excellent ability to discriminate epithelial cancers from nonmalignant diseases. However, its use in clinical practice would need formal validation through a multicentre controlled trial assessing a panel-guided strategy vs. standard diagnosis.

A continuous responder algorithm to optimize clinical management of small-cell lung cancer with progastrin-releasing peptide as a simple blood test.

This study aimed to investigate whether changes in progastrin-releasing peptide (ProGRP) levels correlate with treatment response and can be used to optimize clinical management of patients with small-cell lung cancer. Patients with small-cell lung cancer (any stage) receiving chemotherapy were eligible. ProGRP was measured in serum/plasma at baseline and after each chemotherapy cycle using the Elecsys® ProGRP assay (Roche Diagnostics). Treatment response was assessed by computed tomography scan. The primary objective was to examine whether changes in ProGRP levels correlated with computed tomography scan results after two cycles of chemotherapy. The prognostic value of ProGRP among patients receiving first-line chemotherapy was also assessed. Overall, 261 patients from six centers were eligible. Among patients with elevated baseline ProGRP (>100 pg/mL), a ProGRP decline after Cycle 2 was associated with nonprogression (area under the curve: 84%; 95% confidence interval: 72.8-95.1; n = 141). ProGRP changes from baseline to end of Cycle 1 were predictive of response, as determined by computed tomography scan 3 weeks later (area under the curve: 87%; 95% confidence interval: 74.1-99.2; n = 137). This was enhanced by repeat measurements, with a 92% area under the curve (95% confidence interval: 85.3-97.8) among patients with ProGRP data after both Cycles 1 and 2 (n = 123); if a patient experienced a ≥25% decline in ProGRP after Cycle 1, and ProGRP remained stable or decreased after Cycle 2, the probability of finding progression on the interim computed tomography scan at the end of Cycle 2 was almost zero (sensitivity: 100%, specificity: 71%). Both ProGRP levels at baseline and at the end of first-line chemotherapy were prognostic; the latter provided a moderately improved hazard ratio of 2.43 (95% confidence interval: 1.33-4.46; n = 110) versus 1.87 (95% confidence interval: 1.04-3.37; n = 216). In summary, for patients with small-cell lung cancer and elevated baseline ProGRP levels, ProGRP may be a simple, reliable, and repeatable tool for monitoring response to chemotherapy and provide valuable prognostic information.

Clinical significance of long non-coding RNA HOTTIP in early-stage non-small-cell lung cancer.

BACKGROUND: HOTTIP, a long non-coding RNA located in the HOXA cluster, plays a role in the patterning of tissues with mesodermal components, including the lung. Overexpression of HOXA genes, including HOTTIP, has been associated with a more aggressive phenotype in several cancers. However, the prognostic impact of HOTTIP has not yet been explored in non-small-cell lung cancer (NSCLC). We have correlated HOTTIP expression with time to relapse (TTR) and overall survival (OS) in early-stage NSCLC patients. METHODS: Ninety-nine early-stage NSCLC patients who underwent surgical resection in our center from June 2007 to November 2013 were included in the study. Mean age was 66; 77.8% were males; 73.7% had stage I disease; and 55.5% had adenocarcinoma. A validation data set comprised stage I-II patients from The Cancer Genome Atlas (TCGA) Research Network. RESULTS: HOTTIP was expressed in all tumor samples and was overexpressed in squamous cell carcinoma (p = 0.007) and in smokers (p = 0.018). Patients with high levels of HOTTIP had shorter TTR (78.3 vs 58 months; p = 0.048) and shorter OS (81.2 vs 61 months; p = 0.023) than those with low levels. In the multivariate analysis, HOTTIP emerged as an independent prognostic marker for TTR (OR: 2.05, 95%CI: 1-4.2; p = 0.05), and for OS (OR: 2.31, 95%CI: 1.04-5.1; p = 0.04). HOTTIP was validated as a prognostic marker for OS in the TCGA adenocarcinoma cohort (p = 0.025). Moreover, we identified a 1203-mRNA and a 61-miRNA signature that correlated with HOTTIP expression. CONCLUSIONS: The lncRNA HOTTIP can be considered a prognostic biomarker in early-stage NSCLC.

Assessment of a Combined Panel of Six Serum Tumor Markers for Lung Cancer.

RATIONALE: We have previously identified six serum tumor markers (TMs) (carcinoembryonic antigen, carbohydrate antigen 15.3, squamous cell carcinoma-associated antigen, cytokeratin-19 fragment, neuron-specific enolase, and pro-gastrin-releasing peptide) related to the presence of lung cancer (LC). OBJECTIVES: To validate their individual performance in an independent cohort, and to explore if their combined assessment (≥1 abnormal TM value) is a more accurate marker for LC presence. METHODS: We determined these six TMs in 3,144 consecutive individuals referred to our institution by their primary care physician because of the clinical suspicion of LC. MEASUREMENTS AND MAIN RESULTS: LC was excluded in 1,316 individuals and confirmed in 1,828 patients (1,563 with non-small cell LC and 265 with small cell LC). This study validated the previously reported performance of each individual TM. We also showed that their combined assessment (≥1 abnormal TM) had a better sensitivity, specificity, negative predictive value, and positive predictive value (88.5, 82, 83.7, and 87.3%, respectively) than each TM considered individually and that it increased the diagnostic performance (area under the curve) of a clinical model that included tumor size, age, and smoking status. In patients with radiographic nodules less than 3 cm, the negative predictive value of the TM panel was 71.8%, hence providing some support for a more conservative diagnostic approach. Finally we identified two TMs (neuron-specific enolase and pro-gastrin-releasing peptide) that differentiate the risk of non-small cell LC from that of small cell LC. CONCLUSIONS: The combined assessment of a panel of six serum TMs is a more accurate marker for LC presence than these same TMs considered individually. The potential of these TMs in the diagnostic and screening settings deserves further research.

NKX2-1 expression as a prognostic marker in early-stage non-small-cell lung cancer.

BACKGROUND: NKX2-1, a key molecule in lung development, is highly expressed in non-small cell lung cancer (NSCLC), particularly in lung adenocarcinoma (ADK), where it is a diagnostic marker. Studies of the prognostic role of NKX2-1 in NSCLC have reported contradictory findings. Two microRNAs (miRNAs) have been associated with NKX2-1: miR-365, which targets NKX2-1; and miR-33a, which is downstream of NKX2-1. We have examined the effect of NKX2-1, miR-365 and miR-33a on survival in a cohort of early-stage NSCLC patients and in sub-groups of patients classified according to the mutational status of TP53, KRAS, and EGFR. METHODS: mRNA and miRNA expression was determined using TaqMan assays in 110 early-stage NSCLC patients. TP53, KRAS, and EGFR mutations were assessed by Sanger sequencing. RESULTS: NKX2-1 expression was upregulated in never-smokers (P = 0.017), ADK (P < 0.0001) and patients with wild-type TP53 (P = 0.001). A negative correlation between NKX2-1 and miR-365 expression was found (ρ = -0.287; P = 0.003) but there was no correlation between NKX2-1 and miR-33a expression. Overall survival (OS) was longer in patients with high expression of NKX2-1 than in those with low expression (80.8 vs 61.2 months (P = 0.035), while a trend towards longer OS was observed in patients with low miR-365 levels (P = 0.07). The impact of NKX2-1 on OS and DFS was higher in patients with neither TP53 nor KRAS mutations. Higher expression of NKX2-1 was related to higher OS (77.6 vs 54 months; P = 0.017) and DFS (74.6 vs 57.7 months; P = 0.006) compared to low expression. The association between NKX2-1 and OS and DFS was strengthened when the analysis was limited to patients with stage I disease (P = 0.005 and P=0.003 respectively). CONCLUSIONS: NKX2-1 expression impacts prognosis in early-stage NSCLC patients, particularly in those with neither TP53 nor KRAS mutations.

Low miR-145 and high miR-367 are associated with unfavourable prognosis in resected nonsmall cell lung cancer.

The transcription factors SRY-related HMG box (SOX)2 and octamer-binding transcription factor (OCT)4 regulate the expression of the miR-302-367 cluster. miR-145 regulates SOX2 and OCT4 translation and p53 regulates miR-145 expression. We analysed the expression of the miR-302-367 cluster and miR-145 and the mutational status of p53 in resected nonsmall cell lung cancer (NSCLC) patients and correlated results with time to relapse (TTR). Tumour and paired normal tissue samples were obtained from 70 NSCLC patients. MicroRNA expression was assessed with TaqMan MicroRNA Assays. p53 exons 5 to 8 were sequenced. miR-145 was downregulated (p<0.0001) and miR-367 was upregulated (p<0.0001) in tumour compared with normal tissue. Mean TTR was 18.4 months for patients with low miR-145 levels and 28.2 months for those with high levels (p=0.015). Mean TTR was 29.1 months for patients with low miR-367 levels and 23.4 months for those with high levels (p=0.048). TTR was shorter for patients with both unfavourable variables (p=0.009). Low miR-145 expression (p=0.049), the combination of unfavourable microRNA levels (p=0.02) and the combination of low miR-145 with p53 mutations (p=0.011) were independent markers of shorter TTR. In conclusion, miR-145 and miR-367 expression could be novel markers for relapse in surgically treated NSCLC. p53 may play a role in modulating miR-145 expression in NSCLC.

Integral mediastinal staging in patients with NON-SMALL cell lung cancer and risk factors for occult N2 disease.

BACKGROUND: In patients with non-small cell lung cancer (NSCLC), the presence of abnormal hiliar lymph nodes (clinical N1; cN1), central tumor location and/or tumor size (diameter >3 cm) increases the risk of occult mediastinal metastasis (OMM). This study investigates prospectively the diagnostic value of an integral mediastinal staging (IMS) strategy that combines EndoBronchial Ultrasound-TransBronchial Needle Aspiration (EBUS-TBNA) and Video-Assisted Mediastinoscopy (VAM) in patients with NSCLC at risk of OMM. METHODS: Patients with NSCLC and radiologically normal mediastinum assessed non-invasively by positron emission tomography and computed tomography of the chest (PET-CT), and OMM risk factors (cN1, central tumor and/or >3 cm) underwent EBUS-TBNA followed by VAM if the former was negative. Those with negative IMS underwent resection surgery of the tumor. RESULTS: EBUS-TBNA identified OMM in 2 out of the 49 patients evaluated (4%) and VAM in 1 of the 47 patients with negative EBUS (2%). Two patients with a negative IMS had OMM at surgery. Overall, the prevalence of OMM was 10%. EBUS-TBNA has a sensitivity of 40%, a negative predictive value (NPV) of 93.6%, and negative likelihood ratio of 0.60 (95%CI:0.30-1.16). The risk of not diagnosing OMM after EBUS was 6% and after IMS was 4.4%. CONCLUSION: Integral mediastinal staging in patients with NSCLC and clinical risk factors for OMM, does not seem to provide added diagnostic value to that of EBUS-TBNA, except perhaps in patients with cN1 disease who deserve further research.

Single nucleotide polymorphisms in tobacco metabolism and DNA repair genes and prognosis in resected non-small-cell lung cancer.

BACKGROUND: If tobacco-related carcinogens are not inactivated or extruded from the cell, they can damage the DNA. Single nucleotide polymorphisms (SNPs) in genes involved in tobacco metabolism, DNA repair, and multidrug resistance have been related to lung cancer susceptibility. We examined 13 SNPs in 10 of these genes and correlated the results with time to progression (TTP) and overall survival (OS) in 71 smoker or former smoker patients with resected non-small-cell lung cancer (NSCLC). MATERIALS AND METHODS: DNA was obtained from paraffin-embedded tumor. SNP analysis of the candidate genes was performed by allelic discrimination assay. Log-rank test, Kaplan-Meier plots, and Cox multivariate analysis were used to evaluate the association of TTP and survival with the SNPs evaluated. RESULTS: Patients with wild-type (wt) XPC rs2228001, wt CYP2C8 rs10509681, or non-wt NAT2 rs1799930 had a longer TTP. Patients with wt ERCC1 showed a nonsignificant trend towards longer TTP. No other relation between SNPs and TTP were observed. Patients harboring at least two unfavorable genotypes in these four genes had a shorter TTP and OS than patients with either one or no unfavorable genotypes. In the multivariate analysis, non-wt XPC rs2228001 and the presence of at least two unfavorable genotypes emerged as independent markers for shorter TTP. CONCLUSIONS: SNPs in tobacco metabolism and DNA repair genes may influence the clinical outcome of resected NSCLC.

Prognostic implications of miR-16 expression levels in resected non-small-cell lung cancer.

BACKGROUND: MicroRNAs are novel regulators of gene expression that are linked to the main oncogene networks, including the p53 pathway. p53 regulates the maturation process of miR-16 and miR-143. We analyzed the role as prognostic markers of miR-16 and miR-143 in 70 non-small-cell lung cancer (NSCLC) patients. METHODS: MicroRNAs were analyzed by TaqMan MicroRNA assays. Disease-free survival (DFS) and overall survival (OS) were examined using Kaplan-Meier curves with log-rank tests and the Cox proportional hazard model. RESULTS: When patients were classified in three groups according to their miR-16 expression levels, those with normal levels had the best outcome while those with high levels had the worst. DFS was 22.4 months for patients with high levels, 71.8 months for those with normal levels, and 55.8 months for those with low levels (P = 0.05). OS was 23.9 months for patients with high levels, 97.6 months for those with normal levels, and 63.5 months for those with low levels (P < 0.001). In the multivariate analyses, high miR-16 levels emerged as an independent prognostic factor for poor DFS (P = 0.001) and OS (<0.001). CONCLUSIONS: Our results provide the first hints that miR-16 levels in tumor samples may be a prognostic marker in NSCLC.

Monitoring HOTTIP levels on extracellular vesicles for predicting recurrence in surgical non-small cell lung cancer patients.

In resected non-small cell lung cancer (NSCLC), postsurgical recurrence is the major factor affecting long-term survival. The identification of biomarkers in extracellular vesicles (EV) obtained from serial blood samples after surgery could enhance early detection of relapse and improve NSCLC outcome. Since EV cargo contains long non-coding RNAs (lncRNAs), we aimed to analyze whether the oncogenic lncRNA HOTTIP, which higher expression in tumor tissue was related to worse outcome in NSCLC, could be detected in EV from NSCLC patients and serve as recurrence biomarker. After purification of EVs by ultracentrifugation in 52 serial samples from 18 NSCLC patients, RNA was isolated and HOTTIP was quantified by Real time PCR. We observed that patients that relapsed after surgery displayed increased postsurgical EV HOTTIP levels in comparison with presurgical levels. In the relapsed patients with several samples available between surgery and relapse, we observed an increment in the EV HOTTIP levels when approaching to relapse, which indicated its potential utility for monitoring disease recurrence. When we focused in EV HOTTIP levels in the first post-surgical sample, we observed that the detection of an increment of the expression levels in comparison to presurgical sample, predicted recurrence with high sensitivity (85.7%) and specificity (90.9%) and that patients had shorter time to relapse and shorter overall survival. In conclusion, our pilot study showed that EV HOTTIP is a potential biomarker for monitoring disease recurrence after surgery in NSCLC.

Role of the epithelial-mesenchymal transition-related circular RNA, circ-10720, in non-small-cell lung cancer.

BACKGROUND: Circular RNAs (circRNAs) are non-coding RNAs with a circular structure that have recently emerged as important regulators of tumorogenesis. Recently, several circRNAS, including circ-10720 have been related to epithelial-mesenchymal transition (EMT) process. In the present study, we have analyzed the role of circ-10720 in non-small-cell lung cancer (NSCLC) and studied its prognostic relevance in resected stage I-IIIa NSCLC patients. METHODS: Circ-10720 expression was analyzed using a custom TaqMan assay in four NSCLC cell lines (HCC44, A549, H23 and H1299) and in the normal immortalized lung cell line BEAS2B. Silencing of circ-10720 was performed using two custom siRNAs which were transfected using lipofectamine 2000. Protein levels were evaluated by Western blot and immunofluorescence. Wound healing and invasion assays were performed to evaluate the impact the circRNA on cell motility. Apoptosis was analyzed by evaluation of Caspase 3-7 activity and proliferation by MTS assay. Moreover, the expression levels of the circRNA were studied in 119 resected NSCLC patients. The expression in tumor tissue was correlated with the main clinicopathological characteristics and with time to relapse (TTR). RESULTS: Circ-10720 was overexpressed in HCC44 and A549 and underexpressed in H23 and H1299 NSCLC cell lines in comparison to BEAS2B normal immortalized lung cell line. CircRNA knockdown in the two circ-10720 overexpressing cell lines was associated with a decrease of Vimentin (VIM) and an increase of E-cadherin (CDH1) protein levels, loss of mesenchymal phenotype, and a significant reduction of migration and invasion capacity. After silencing circ-10720, the apoptosis rate increased and the proliferation was significantly reduced. Furthermore, circ-10720 was upregulated in tumor vs. normal tissue from 119 resected NSCLC patients. In the group of patients not receiving adjuvant treatment, those with high levels of circ-10720 had a shorter TTR than those with low levels and emerged as an independent prognostic value in the multivariate analysis. In tumor tissue, circ-10720 levels positively correlated with the EMT gene Twist1 levels. CONCLUSIONS: Circ-10720 regulates EMT, apoptosis and proliferation and acts as a biomarker of relapse in NSCLC.

EBUS-TBNA Cytological Samples for Comprehensive Molecular Testing in Non-Small Cell Lung Cancer.

Clinical guidelines promote the identification of several targetable biomarkers to drive treatment decisions in advanced non-small cell lung cancer (NSCLC), but half of all patients do not have a viable biopsy. Specimens from endobronchial-ultrasound transbronchial needle aspiration (EBUS-TBNA) are an alternative source of material for the initial diagnosis of NSCLC, however their usefulness for a complete molecular characterization remains controversial. EBUS-TBNA samples were prospectively tested for several biomarkers by next-generation sequencing (NGS), nCounter, and immunohistochemistry (PD-L1). The primary objectives were to assess the sensitivity of EBUS-TBNA samples for a comprehensive molecular characterization and to compare its performance to the reference standard of biopsy samples. Seventy-two EBUS-TBNA procedures were performed, and 42 NSCLC patients were diagnosed. Among all cytological samples, 92.9% were successfully genotyped by NGS, 95.2% by nCounter, and 100% by immunohistochemistry. There were 29 paired biopsy samples; 79.3% samples had enough tumor material for genomic genotyping, and 96.6% for PD-L1 immunohistochemistry. A good concordance was found between both sources of material: 88.9% for PD-L1, 100% for NGS and nCounter. EBUS-TBNA is a feasible alternative source of material for NSCLC genotyping and allows the identification of patient candidates for personalized therapies with high concordance when compared with biopsy.

Is invasive mediastinal staging necessary in intermediate risk patients with negative PET/CT?

BACKGROUND: Tumor involvement of mediastinal lymph nodes is of high importance in non-small cell lung cancer (NSCLC). Invasive mediastinal staging is recommended in selected patients without evidence of mediastinal involvement on staging by imaging. In the present study we aimed to evaluate the effectiveness of invasive mediastinal staging in reducing pN2, its impact on survival and the risk factors for occult pN2. METHODS: Patients with NSCLC tumors larger than 3 cm, central tumors or cN1 cases treated in our institution between 2013 and 2018 were prospectively included in the study. Incidence of pN2 and overall survival was compared among invasively staged (IS) and non-invasively staged groups (NIS). Multivariate analysis was performed to identify risk factors of pN2. RESULTS: A total of 201 patients were included in the study, 79 (39.3%) of whom were not invasively staged (NIS group) and 122 (60.7%) were invasively staged (IS group). Incidence of cN1 and mean PET/CT uptake was different among both groups. Prevalence of pN2 was similar in both groups (7.6% in NIS vs. 12.6% in IS; P>0.05). Median survival in IS-pN2 patients was 11 months longer than in NIS-pN2 group (33.6 vs. 22.5 months; P=0.245). cN1 emerged as the only a risk factor for pN2. CONCLUSIONS: Invasive staging does not reduce the incidence of pN2. However, this finding could be biased because in our series cN1 patients were more often staged and cN1 has been detected as a risk factor for pN2. In addition patient better selection after invasive staging might have an impact on overall survival. To conclude, invasive mediastinal staging in intermediate risk patients for positive mediastinal nodes is justified.

Long Non-Coding RNA NANCI/NKX2-1 Duplex Impacts Prognosis in Stage I Non-Small-Cell Lung Cancer.

BACKGROUND: NANCI, an intergenic long non-coding RNA (lncRNA) is essential for buffering NKX2-1 expression during embryonic development and in adult tissue. We analyzed NANCI and NKX2-1 in human lung embryonic samples and adult lung tissues and evaluated their potential as prognostic markers in stage I non-small cell lung cancer (NSCLC). METHODS AND RESULTS: NANCI and NKX2-1 expression was assessed by TaqMan assays in 18 human embryonic samples from 8 to 13 weeks, 59 non-tumoral (NT) lung tissue samples, and 98 stage I NSCLC tumor samples. NANCI and NKX2-1 expression in embryonic and NSCLC samples were downregulated in comparison to adult NT tissue. Patients with low expression of NANCI had shorter disease-free survival (DFS) and overall survival (OS) than those with high levels (47.6 vs 69.3 months, P=0.032 and 57.7 vs 77.6 months, P=0.021, respectively). When the expression levels of NANCI and NKX2-1 were evaluated in combination, four groups were identified (high NANCI/high NKX2-1, low NANCI/high NKX2-1, high NANCI/low NKX2-1 and low NANCI/low NKX2-1) with differential impact on DFS (P=0.042) and OS (P=0.024). Interestingly, the high NANCI/high NKX2-1 duplex group had longer DFS and OS than the other three groups (71.25 vs 46.3 months, P=0.009 and 81.3 vs 56.1 months, P=0.004, respectively). In the multivariate analysis, the high NANCI/high NKX2-1 duplex was identified as an independent prognostic factor for longer DFS (HR 0.346, 95% CI, 0.169-0.709; P=0.004) and OS (HR 0.309, 95% CI, 0.121-0.786; P=0.014). CONCLUSIONS: NANCI and the NANCI-NKX2-1 duplex impacts prognosis in stage I NSCLC patients.

Extracellular Vesicle lincRNA-p21 Expression in Tumor-Draining Pulmonary Vein Defines Prognosis in NSCLC and Modulates Endothelial Cell Behavior.

Hypoxia-induced upregulation of lincRNA-p21 in tumor tissue was previously shown by our group to be related to poor prognosis in resected non-small cell lung cancer (NSCLC) patients. In the present study, we have evaluated the presence of lincRNA-p21 in extracellular vesicles (EVs) from NSCLC patients and assessed its potential as a prognostic biomarker. High EV lincRNA-p21 levels in blood from the tumor-draining vein were associated with shorter time to relapse and shorter overall survival. Moreover, the multivariate analysis identified high lincRNA-p21 levels as an independent prognostic marker. In addition, lincRNA-p21 was overexpressed in H23 and HCC44 NSCLC cell lines and their derived EVs under hypoxic conditions. Functional assays using human umbilical vein endothelial cells (HUVECs) showed that tumor-derived EVs enriched in lincRNA-p21 affected endothelial cells by promoting tube formation and enhancing tumor cell adhesion to endothelial cells. Additionally, the analysis of selected EV microRNAs related to angiogenesis and metastasis showed that the microRNAs correlated with EV lincRNA-p21 levels in both patients and cell lines. Finally, EV co-culture with HUVEC cells increased the expression of microRNAs and genes related to endothelial cell activation. In conclusion, EV lincRNA-p21 acts as a novel prognosis marker in resected NSCLC patients, promoting angiogenesis and metastasis.

miR-34a as a prognostic marker of relapse in surgically resected non-small-cell lung cancer.

MicroRNAs (miRNAs) have been identified as promising prognostic markers in non-small-cell lung cancer (NSCLC) since they play an important role in oncogenesis. The miR-34 family is composed of three miRNAs (miR-34a, miR-34b and miR-34c) that are part of the p53 network and whose expression is directly induced by p53 in response to DNA damage or oncogenic stress. We have analyzed the impact of miR-34 expression on relapse and overall survival in surgically resected NSCLC patients. For this purpose, we used stem-loop reverse transcription-polymerase chain reaction to analyze the expression of the miR-34 family in paired tumor and normal tissue from 70 surgically resected NSCLC patients who received no postsurgical treatment until relapse. In addition, in patients with sufficient tumor tissue, we assessed p53 mutations and the methylation status of the MIRN34A gene promoter region and correlated these findings with miR-34a expression. Molecular findings were correlated with relapse and overall survival. The miR-34 family was downregulated in tumor compared with normal tissue, and low levels of miR-34a expression were correlated with a high probability of relapse (P = 0.04). A relation was also found between MIRN34A methylation and miR-34a expression (P = 0.008). Patients with both p53 mutations and low miR-34a levels had the highest probability of relapse (P = 0.001). In the multivariate analysis, miR-34a expression emerged as an independent prognostic marker for relapse. In summary, we have identified miR-34a as a novel prognostic marker in NSCLC patients, providing a potential mechanism for estimating a patient's risk of disease recurrence and a useful tool to help guide treatment decisions.

Usefulness of serum tumor markers, including progastrin-releasing peptide, in patients with lung cancer: correlation with histology.

BACKGROUND: Tumor markers have been extensively studied in lung cancer, reporting some relationship to the histology, but their clinical utility is not clear. METHODS: ProGRP, CEA, SCC, CA 125, CYFRA 21-1 and NSE were studied prospectively in 155 patients with unconfirmed suspicion of lung cancer and in 647 patients with lung cancer: 182 squamous, 205 adenocarcinomas, 19 large-cell lung cancer (LCLC), 175 small-cell lung cancer (SCLC) and 66 unspecific non-small-cell lung cancer (NSCLC). RESULTS: Abnormal tumor marker serum levels were found in less than 5.3% of the patients with benign diseases, excluding CA 125 (21.3%). Tumor markers were related to histological type and tumor extension with significantly higher CEA (p <0.01) and CA 125 (p <0.007) serum levels in adenocarcinomas, SCC (p <0.0001) and CYFRA 21-1 (p <0.008) in squamous tumors and ProGRP (p <0.0001) and NSE (p <0.0001) in SCLC. Tumor markers may be useful in the histological differentiation of NSCLC and SCLC. Patients with SCC serum levels >2 ng/ml were always NSCLC, while those with SCC <2 ng/ml and ProGRP >100 pg/ml and NSE >35 ng/ml were all SCLC patients. The sensitivity was 76.7 and 79.5%, specificity was 97.2 and 99.6%, with a positive predictive value of 98.6 and 98.6% and a negative predictive value of 60.7 and 92.9% in the differentiation of NSCLC and SCLC, respectively. CONCLUSIONS: Tumor marker determination in patients with suspicious signs of lung cancer suggests, in a few hours, the histological diagnosis in the majority of lung cancer patients.

MicroRNAs expressed during lung cancer development are expressed in human pseudoglandular lung embryogenesis.

BACKGROUND/AIMS: MicroRNAs (miRNAs) play a role during mouse embryonic development and are also important in carcinogenesis. In order to investigate whether there are similar patterns of miRNA expression levels in pseudoglandular human embryonic lung and in human lung tumors, we have analyzed 18 miRNAs (the let-7 family, the miR-17-92 cluster, miR-221 and miR-222) in human embryonic lung samples and in paired lung tumor and normal lung tissue samples and correlated the results with clinicopathological characteristics. METHODS: RNA was obtained from 12 human embryonic lung samples, 33 lung tumor samples and 33 paired normal lung samples. miRNAs were assessed by quantitative real-time PCR. RESULTS: Members of the let-7 family were downregulated and members of the miR-17-92 cluster and miR-221 were overexpressed both in embryonic lung tissue and in lung tumors. Low levels of let-7c were associated with absence of metastases (p = 0.015), early-stage non-small cell lung cancer (NSCLC, p = 0.05), and smokers (p = 0.009). High levels of miR-106a were associated with small-cell lung cancer (p = 0.031), and high levels of miR-19a with advanced NSCLC (p = 0.008). CONCLUSION: Our study lends support to the model of cancer as an alteration of normal development, as many miRNAs were similarly expressed in early human lung development and stage I-II of lung cancer development.