A signature of five 7-methylguanosine-related genes is a prognostic marker for lung squamous cell carcinoma.

Background: N7-methylguanosine (m7G) is an important posttranscriptional modification affecting mRNA and tRNA functions and stability. The genes regulating the m7G process have been previously found involved in the carcinogenesis process. We aimed to analyze the role of m7G-related genes as potential prognostic markers for lung squamous cell carcinoma (LSCC). Methods: Twenty-nine m7G-related genes were selected for the analysis in the LSCC cohort of the Cancer Genome Atlas (TCGA). Univariate, multivariate, and Kaplan-Meier analyses were used to evaluate the predictive value of risk model developed with m7G signature for overall survival (OS).. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of differentially expressed genes (DEGs) were performed for high- and low-risk LSCC groups. Results: We identified 17 differentially expressed m7G methylation-related genes in LSCC versus normal tissues. The expression of five m7G-related genes (EIF3D, LSM1, NCBP2, NUDT10, and NUDT11) was identified as an independent prognostic marker for OS in LSCC patients. A risk model with these five m7G-related genes predicted 2-, and 3-year survival rates of 0.623 and 0.626, respectively. The risk score significantly correlated with OS: LSCC patients with a higher risk score had shorter OS (P<0.01) and it was associated with lower immune response (P<0.01). Conclusions: We developed a novel m7G-related gene signature that can be of great utility to predict the prognosis for patients with LSCC.

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.

Screening for Prognostic microRNAs Associated with Treatment Failure in Diffuse Large B Cell Lymphoma.

Diffuse large B cell lymphoma (DLBCL) treatment with R-CHOP regimen produces 5-year progression-free survival and overall survival of around 60-70%. Our objective was to discover prognostic biomarkers allowing early detection of the remaining 30-40% with poor long-term outcome. For this purpose, we applied a novel strategy: from a cohort of DLBCL patients, treated with standard therapy, a discovery group of 12 patients with poor prognosis (advanced stage III-IV, R-IPI > 2) was formed, consisting of six chemoresistant (refractory/early relapse < 12 months) and six chemosensitive (complete remission > 3 years) subjects. By using microarray assays, the most differentially expressed miRNAs were defined as an initial set of prognostic miRNA candidates. Their expression was then analyzed in a validation cohort of 68 patients and the three miRNAs with the most significant impact on event-free and overall survival were selected. In the DLBCL cell line U-2932 the transfection with miR-1244 and miR-193b-5p, but not miR-1231, blocked the effect of CHOP on cell viability. A subsequent gene set enrichment analysis in patients revealed the implication of the first two miRNAs in cell cycle control and chemoresistance-related pathways, whereas the last one was involved in immunological processes. In conclusion, this novel strategy identified three promising prognostic markers for DLBCL patients at high risk of failure with standard therapy.

Human embryonic mesenchymal lung-conditioned medium promotes differentiation to myofibroblast and loss of stemness phenotype in lung adenocarcinoma cell lines.

BACKGROUND: When genes responsible for normal embryonic development are abnormally expressed in adults, it can lead to tumor development. This can suggest that the same mechanism that controls embryonic differentiation can also control tumor differentiation. We hypothesize that the malignant phenotype of lung cancer cells could acquire benign characteristics when in contact with an embryonic lung microenvironment. We cultured two lung cancer cell lines in embryonic lung mesenchyme-conditioned medium and evaluated morphological, functional and molecular changes. METHODS: The human embryonic mesenchymal lung-conditioned medium (hEML-CM) was obtained by culturing lung cells from embryos in the pseudoglandular stage of development. The NSCLC cell lines A549 and H1299 we cultured in the hEML-CM and in a tumor-conditioned medium. Morphological changes were analyzed with optical and transmission electron microscopy. To evaluate the functional effect of conditioned medium in tumor cells, we analyzed cell proliferation, migration, colony formation capacity in 2D and 3D and in vivo tumor growth capacity. The expression of the pluripotency genes OSKM, the adenocarcinoma marker NKX2-1, the lung surfactant proteins SFTP, the myofibroblast marker MYH and DNMT3A/3B was analyzed with qRT-PCR and the presence of the myofibroblast markers vimentin and α-SMA with immunofluorescence. Transcriptomic analysis was performed using Affymetrix arrays. RESULTS: The A549 and H1299 cells cultured in hEML-CM lost their epithelial morphology, acquired mesodermal characteristics, and decreased proliferation, migration, and colony formation capacity in 2D and 3D, as well as reduced its capacity to growth in vivo. The expression of OSKM, NKX2-1 and SFTP decreased, while that of DNMT3A/3B, vimentin, α-SMA and MYH increased. Distant matrix analysis based on transcriptomic profile showed that conditioned cells were closer to myoblast and human lung fibroblast than to normal epithelial immortalized lung cells. A total of 1631 for A549 and 866 for H1299 differentially expressed genes between control and conditioned cells were identified. CONCLUSIONS: To the best of our knowledge, this is the first study to report that stimuli from the embryonic lung can modulate the malignant phenotype of lung cancer cells, control their growth capacity and activate their differentiation into myofibroblasts. These findings could lead to new strategies for lung cancer management.

Octogenarians may benefit from stage-specific small cell lung cancer treatment.

BACKGROUND: Our study investigates treatment profiles in octogenarian patients with small cell lung cancer (SCLC) and assesses each treatment's role in a stage-specific manner. METHODS: Patient data from individuals with SCLC aged 80 years and older between 1988 and 2015 in the Surveillance, Epidemiology, and End Results Program (SEER) database were extracted. Cancer-specific survival (CSS) between patients with no treatment and different treatment groups were compared by the Kaplan-Meier method, with stratifications by stage. Cox Proportional Hazard model further identified independent prognostic factors. RESULTS: A total of 7,290 patients were included in this study. Notably, 3,358 (46.1%) patients did not receive active treatment. Compared with the no active treatment group, the CSS of patients who received treatment was significantly improved (median 6 vs. 0 months, P<0.001) and further validated in stage subgroups. Chemotherapy combined with local therapy was associated with the best CSS in regional and distant disease stages, with the hazard ratios (HR) and 95% confidence intervals (CI) being 0.30 (0.26-0.34) and 0.27 (0.25-0.30), respectively. Local therapy only appeared to confer better oncological outcomes (HR =0.33; 95% CI: 0.25-0.42) than chemotherapy only (HR =0.37; 95% CI: 0.29-0.47) in the localized disease stage. CONCLUSIONS: Although nearly half of octogenarians with SCLC did not receive active treatment in the real clinical setting, these patients may benefit from treatment. Chemotherapy combined with local therapy may provide the best treatment choice in octogenarians with advanced SCLC, while local therapy appears to play a more critical role in treating those with early-stage disease.

LncRNA Quantification from Extracellular Vesicles Isolated from Blood Plasma or Conditioned Media.

During the last years, the study of extracellular vesicles (EVs) and its cargo has gained interest in the scientific media. EVs have been found in all biofluids and it is postulated that all cells are capable to secrete a wide variety of these vesicles, which play a key role in different cell-to-cell communication processes as well as in the microenvironment modulation. In the EV cargo, DNA, protein, and RNA molecules can be found, including long noncoding RNAs (lncRNAs). Several authors consider the study of EV lncRNAs an ideal source of biomarkers due to the easy sampling of EVs in different biofluids and the high specificity of the lncRNA expression pattern.In the present chapter, a detailed explanation of the EV isolation workflow followed by RNA isolation and lncRNA gene expression study is provided for two sample sources: blood plasma and cell culture conditioned media. EVs from both plasma samples and cell cultured media are isolated using sequential ultracentrifugation method (UC), which has been reported as one of the best methods available to date in terms of purity. UC is followed by RNA extraction based on the combination of phenol/guanidine-based lysis of samples with silica-membrane-based purification of total RNA. LncRNA quantification is performed by qRT-PCR. This chapter includes detailed discussion on lncRNA quantification using hydrolysis probes, recommended housekeeping genes and evaluation of methods for comparing lncRNA levels between EVs and its parental cells. In summary, we describe here the main steps for a successful isolation of the EVs-lncRNA cargo, paying attention to how overcome the different challenges found in the experimental procedure and in the data analysis of lncRNA expression from this source.

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.

Lincp21-RNA as Predictive Response Marker for Preoperative Chemoradiotherapy in Rectal Cancer.

Preoperative chemoradiotherapy (CRT) is a standard treatment for locally advanced rectal cancer (RC) patients, but its use in non-responders can be associated with increased toxicities and resection delay. LincRNA-p21 is a long non-coding RNA involved in the p53 pathway and angiogenesis regulation. We aimed to study whether lincRNA-p21 expression levels can act as a predictive biomarker for neoadjuvant CRT response. We analyzed RNAs from pretreatment biopsies from 70 RC patients treated with preoperative CRT. Pathological response was classified according to the tumor regression grade (TRG) Dworak classification. LincRNA-p21 expression was determined by RTqPCR. The results showed that lincRNA-p21 was upregulated in stage III tumors (p = 0.007) and in tumors with the worst response regarding TRG (p = 0.027) and downstaging (p = 0.016). ROC curve analysis showed that lincRNA-p21 expression had the capacity to distinguish a complete response from others (AUC:0.696; p = 0.014). LincRNA-p21 was shown as an independent marker of preoperative CRT response (p = 0.047) and for time to relapse (TTR) (p = 0.048). In conclusion, lincRNA-p21 is a marker of advanced disease, worse response to neoadjuvant CRT, and shorter TTR in locally advanced RC patients. The study of lincRNA-p21 may be of value in the individualization of pre-operative CRT in RC.

Deciphering miRNA-Target Relationships to Understand miRNA-Mediated Carcinogenesis.

We now accept that the non-coding part of the genome is essential for fine-tuning most cellular functions, and that its deregulation drives carcinogenesis [...].

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.

circFBXW7 attenuates malignant progression in lung adenocarcinoma by sponging miR-942-5p.

BACKGROUND: As a type of non-coding RNA, circular RNAs (circRNAs) are considered to be functional molecules associated with human cancers. An increasing number of circRNAs have been verified in malignant progression in a number of cancers. The circRNA, circFBXW7, has been proven to play an important role in tumor proliferation and metastasis. However, whether circFBXW7 influences progression in lung adenocarcinoma (LUAD) remains unclear. METHODS: Quantitative real-time reverse transcriptase PCR (qRT-PCR) was used to verify circFBXW7 in LUAD cell lines and LUAD tissues. Kaplan-Meier analysis was then used to compare the disease-free survival (DFS) and overall survival (OS) of these LUAD patients. The biological function of circFBXW7 was examined by overexpression and knockdown of circFBXW7 using MTT assay, EdU assay, wound-healing assay, and Transwell in vitro assays. To explore the mechanism of the circFBXW7, RNA pull-down assay, dual luciferase reporter assay, and RNA immunoprecipitation (RIP) assay were employed to examine the interaction between circFBXW7 and miR-942-5p. Western blot was used to study the fundamental proteins associated with the epithelial-mesenchymal transition (EMT) pathway. In vivo studies with BALB/c nude mice subcutaneously injected with cells stably overexpressing circFBXW7 were performed to further validate the in vitro results. RESULTS: circFBXW7 was downregulated in LUAD cell lines and tissues, and LUAD patients with lower levels had shorter DFS and OS. The in vitro study showed that circFBXW7 overexpression inhibited proliferation and migration of A549 and HCC2279 cell lines. These results were confirmed by circFBXW7 knockdown, which showed the reverse effect. The in vivo model showed that the circRNA levels influenced the tumor growth. Finally, we determined that circFBXW7 target miRNA-942-5p which regulates the EMT gene BARX2. The modulation of circFBXW7 levels produced significant changes in EMT genes in vitro and in vivo. CONCLUSIONS: Our findings showed that circFBXW7 inhibits proliferation and migration by controlling the miR-942-5p/BARX2 axis in LUAD cell lines and its levels correlates with patient survival suggesting that regulating circFBXW7 could have therapeutic value in treating LUAD patients.

The DNA damage repair-related gene PKMYT1 is a potential biomarker in various malignancies.

BACKGROUND: Protein kinase membrane associated tyrosine/threonine 1 (PKMYT1) regulates cell cycle and is a part of DNA damage repair (DDR)-related signaling. Recent studies have identified a role for PKMYT1 in tumor immunity and DDR. Thus, we initiated this study aiming to characterize the molecular and immunological portrait of PKMYT1 in cancer. METHODS: Transcriptomic data extrapolated from Genotype-Tissue Expression (GTEx), The Cancer Genome Atlas (TCGA), and Cancer Cell Line Encyclopedia (CCLE) datasets were used to determine the mRNA expression levels of PKMYT1. PKMYT1 mRNA expression status was correlated with patients' prognosis as well as immune neoantigens, and immune checkpoints in 34 different tumors. The Tumor Immune Estimation Resource (TIMER) dataset was used to analyze immune infiltrating scores. RESULTS: PKMYT1 mRNA is differentially expressed in common tumors and high expression levels of PKMYT1 mRNA is associated with poor prognosis except for malignant thymoma (THYM). In addition, PKMYT1 mRNA expression was correlated with tumor-infiltrating immune cells particularly in lung squamous cell carcinoma, esophageal carcinoma, THYM, and lung adenocarcinoma. An upregulation of immune checkpoints and neoantigens was observed in tumors with a high PKMYT1 mRNA expression. Data from gene set enrichment analysis (GSEA) revealed that PKMYT1 is involved in tumor immunogenicity, metabolism, and cell cycle progression. CONCLUSIONS: PKMYT1 is differentially expressed in various cancers and exerts an important effect on tumor immunity and progression. The PKMYT1 gene holds the potential as a new potential biomarker. Therefore, further studies are clearly needed to elaborate our findings.

Long non-coding RNA NANCI/NKX2-1 duplex impacts prognosis in stage I non-small-cell lung cancer / La combinación de NANCI/NKX2-1 de ARN no codificante de cadena larga afecta al pronóstico del cáncer de pulmón de células no pequeñas en estadio I

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

CONTEXTO GLOBAL: NANCI, un ARN intergénico largo no codificante (lncRNA) es esencial para regular la expresión de NKX2-1 durante el desarrollo embrionario y en el tejido adulto. Analizamos la expresión de NANCI y NKX2-1 en muestras embrionarias de pulmón humano y tejidos pulmonares adultos, y evaluamos su potencial como marcadores pronósticos en el cáncer de pulmón de células no pequeñas (CPCNP) en estadio I. MÉTODOS Y RESULTADOS: La expresión de NANCI y NKX2-1 se evaluó mediante ensayos TaqMan® en 18 muestras embrionarias humanas de 8 a 13 semanas, 59 muestras de tejido pulmonar no tumoral (NT) y 98 muestras de tumor de CPCNP en estadio i. La expresión de NANCI y NKX2-1 en muestras embrionarias y NSCLC se encontraba reducida en comparación con el tejido NT adulto. Los pacientes con baja expresión de NANCI tuvieron una supervivencia libre de enfermedad (SLE) y una supervivencia general (SG) más cortas que aquellos con niveles altos (47,6 frente a 69,3 meses; p = 0,032 y 57,7 frente a 77,6 meses; p = 0,021, respectivamente). Cuando se evaluaron los niveles de expresión de NANCI y NKX2-1 combinados se identificaron 4 grupos (NANCI alto/NKX2-1 alto, NANCI bajo/NKX2-1 alto, NANCI alto/NKX2-1 bajo y NANCI bajo/NKX2-1 bajo) con impacto variable en la SLE (p = 0,042) y la SG (p = 0,024). Curiosamente, la combinación de NANCI alto/NKX2-1 alto presentó unas SLE y SG más largas que los otros 3 grupos (71,25 frente a 46,3 meses; p = 0,009 y 81,3 frente a 56,1 meses; p = 0,004, respectivamente). En el análisis multivariante, la combinación de NANCI alto/NKX2-1 alto se identificó como un factor de pronóstico independiente para una SLE más larga (HR: 0,346; IC 95%: 0,169-0,709; p = 0,004), al igual que la SG (HR: 0,309; IC 95%: 0,121-0,786; p = 0,014). CONCLUSIONES: NANCI y la combinación de NANCI-NKX2-1 afecta al pronóstico de los pacientes con CPCNP en estadio i

LincRNA-p21 Levels Relates to Survival and Post-Operative Radiotherapy Benefit in Rectal Cancer Patients.

LincRNA-p21 is a long non-coding RNA involved in the p53 pathway and angiogenesis regulation that acts as prognostic marker in several tumors. In the present study, we aimed to analyze the clinical value of lincRNA-p21 in 177 resected stage I-III colorectal cancer (CRC) patients. Tumor and normal paired tissue and plasma samples from tumor-draining mesenteric veins and paired peripheral veins were analyzed. LincRNA-p21 expression was determined by RTqPCR and correlated with disease-free (DFS) and overall survival (OS). LincRNA-p21 was downregulated in tumor versus normal tissue (p = 0.0012). CRC patients with high lincRNA-p21 expression had shorter DFS (p = 0.0372) and shorter OS (p = 0.0465). Of note, the major prognostic impact was observed in the subset of rectal cancer patients where patients with high lincRNA-p21 levels had worse DFS (p = 0.0226) and OS (p = 0.0457). Interestingly, rectal cancer patients with high lincRNA-p21 benefited from post-operative chemoradiotherapy, as indicated by a longer OS in the group of high lincRNA-p21 patients receiving post-operative chemoradiotherapy (p = 0.04). Finally, patients with high lincRNA-p21 levels in mesenteric vein (MV) had shorter OS (p = 0.0329). LincRNA-p21 is a marker of advanced disease and worse outcome in CRC. Moreover, rectal cancer patients with high lincRNA-p21 levels could benefit from post-operative chemoradiotherapy, and plasmatic-lincRNA-p21 is a promising liquid biopsy biomarker.

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.

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.

KRAS mutations by digital PCR in circulating tumor cells isolated from the mesenteric vein are associated with residual disease and overall survival in resected colorectal cancer patients.

PURPOSE: In colorectal cancer (CRC), circulating tumor cells (CTCs) are released into the mesenteric veins (MV). We chose to determine whether KRAS mutations detected in CTCs from blood obtained at the time of surgery could be a marker of survival. METHODS: From 52 surgically resected CRC patients who later relapsed, samples of tumor tissue, normal tissue, and blood from the peripheral vein (PV) and MV were obtained from each patient at the time of surgery. KRAS mutations were assessed by Sanger sequencing and digital PCR (DGPCR) in tissue samples and by DGPCR in CTCs. Mutant KRAS copy number was assessed in CTCs. Results were correlated with overall survival (OS). RESULTS: Sanger sequencing detected KRAS mutations in ten tumor samples (19.2%), while DGPCR detected mutations in 30 (58%). Mutations were detected in CTCs in 21 MV samples (40.4%) and 18 PV samples (34.6%). Patients with G13D mutations in CTCs from the MV had shorter OS than those with G12D mutations (28.1 vs 54.6 months; p = 0.025). Patients with a high mutant KRAS copy number in CTCs had shorter OS than those with a low mutant KRAS copy number (MV: 20.5 vs 43.7 months; p = 0.002; PV: 15.1 vs 38.2 months; p = 0.027). CONCLUSION: DGPCR is more efficient than Sanger sequencing for detecting KRAS mutations. KRAS G13D mutations and high mutant KRAS copy number are associated with shorter OS. The analysis of KRAS mutations in CTCs from blood obtained at the time of surgery can identify patients with a higher risk of relapse.

Microscopic analysis and microstructural characterization of the organic and inorganic components of dairy fouling during the cleaning process.

This study evaluated the organic residues of milk fouling using fluorescence and confocal laser scanning microscopy. The inorganic content was analyzed with energy-dispersive X-ray spectroscopy, complemented with inductively coupled plasma optical emission spectrometry. These techniques were applied to evaluate milk fouling cleanliness using an alkaline product and an enzymatic formulation based on protease and amylase. The results showed that the efficiency of enzymatic cleaning was 87.1% when it was evaluated at 55°C for 30 min, and with a medium of pH 8.5. No difference was found from the efficacy in eliminating dairy fouling observed for the chemical cleaning (86.9%). The fluorescence microscopy proved useful for determining the organic solid components in the outer layer of the dairy fouling. The fouling spatial disposition in 3 dimensions, obtained by confocal laser scanning microscopy, showed that it was formed of 51.3% sugars, 9.3% fats, and 39.4% proteins, with the enzymatic cleaning of these compounds being homogeneous, compared with chemical cleaning. The protein and lipid contents were in the surface layer, whereas sugars were located in the innermost part that contributes to the Maillard reaction during fouling formation. After enzymatic cleaning, the reduction in the concentration of Ca and P was 71.61 and 74.67%, respectively, compared with fouling intact. Thus, enzymatic cleaning, without the accumulation of Na from chemical cleaning, leaves 1.5 times less mineral than chemical cleaning. Knowing the content and structure of fouling in the industry helps to formulate better products to achieve proper levels of cleanliness. Additionally, studying the cleaning residues helps to avoid problems of cross-contamination between batches or subsequent microbial growths (biofilms) on surfaces with residues.