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.

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.

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.

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.

The tibiocalcaneal bundle of the deltoid ligament - Prevalence and variations.

INTRODUCTION: A precise understanding of the anatomy of the multiple bundles of the deltoid ankle ligament might have clinical impact. The most relevant deltoid anatomical series report a variable frequency of the tibiocalcaneal ligament, possibly the most important bundle to be reconstructed in medial ankle insufficiency. Our purpose was to access the deltoid's tibiocalcaneal ligament morphology in a large anatomical study as well as to perform a historical literature review on the reasons for its variable prevalence. MATERIALS AND METHODS: Forty-three ankle specimen were dissected to describe the prevalence of superficial and deep deltoid bundles, with special attention to the tibiocalcaneal ligament and its variants. RESULTS: All ankles had distinct deep and superficial bundles. In all 43 ankles the tibionavicular and tibiospring ligaments were clearly identified. The superficial posterior tibiotalar ligament was identified in 38 ankles (88%). The deep anterior tibiotalar bundle was identified in 35 ankles (81%). The deep posterior tibiotalar bundle was identified in all ankles. The tibiocalcaneal ligament was identified in 33 ankles (77%). In ten ankles there wasn't a direct bundle between the tibia and the sustentaculum tali. In all of these, however, we found some fibers spanning the gap between the tibiospring ligament and the sustentaculum tali. CONCLUSION: The tibiocalcaneal ligament is present in most specimens. In those in which we could not identify a direct bundle between the tibia and the calcareous we found a variant of the tibiospring ligament that connects to the sustentaculum tali.

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.

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.

Exosome Analysis in Tumor-Draining Pulmonary Vein Identifies NSCLC Patients with Higher Risk of Relapse after Curative Surgery.

Since tumor-draining pulmonary vein blood (PV) is enriched in tumor-secreted products, we hypothesized that it would also be enriched in tumor-derived exosomes, which would be important in the metastasis process. We characterized exosomes from PV of 61 resected non-small cell lung cancer (NSCLC) patients to evaluate its potential as relapse biomarkers. Exosomes were characterized using transmission electron microscopy, western blot and nanoparticle tracking analysis and we examined time to relapse (TTR) and overall survival (OS). Differences between PV and peripheral vein were found. PV was enriched in smaller exosomes than the paired peripheral vein (p = 0.01). Moreover, PV exosome size mode was able to identify relapsed patients (Area under the curve [AUC] = 0.781; 95%CI: 0.6641⁻0.8978), in whom exosome size was smaller (<112 nm; p < 0.001). The combination of PV exosome size and N (lymph node involvement) showed an AUC of 0.89 (95%CI: 0.80⁻0.97). Moreover, smaller PV exosome size was associated with shorter TTR (28.3 months vs. not reached, p < 0.001) and OS (43.9 months vs. not reached, p = 0.009). Multivariate analyses identified PV exosome size and stage as independent prognostic markers for TTR and OS. PV exosome size is a promising relapse biomarker after surgery that can add valuable information to clinical variables.

miR-21, miR-99b and miR-375 combination as predictive response signature for preoperative chemoradiotherapy in rectal cancer.

INTRODUCTION: Preoperative chemoradiotherapy (CRT) is a standard treatment for locally advanced rectal cancer patients. Despite the benefits of CRT, its use in non-responder patients can be associated with increased toxicities and surgical resection delay. The identification of CRT response biomarkers, such as microRNAs, could improve the management of these patients. We have studied the microRNA expression in pretreatment endoscopy biopsies from rectal cancer patients treated with CRT to identify potential microRNAs able to predict CRT response and clinical outcome of these patients. MATERIAL AND METHODS: RNA from pretreatment endoscopy biopsies from 96 rectal cancer patients treated with preoperative CRT were studied. Pathological response was graded according to the tumor regression grade (TRG) Dworak classification. In the screening phase, 377 miRNAs were studied in 12 patients with extreme responses (TRG0-1 vs TRG4). The potential role as predictive biomarkers for CRT response, disease-free survival (DFS) and overall survival (OS) of the miRNAs identified in the screening phase were validated in the whole cohort. RESULTS: In the screening phase, an 8-miRNAs CRT-response signature was identified: let-7b, let-7e, miR-21, miR-99b, miR-183, miR-328, miR-375 and miR-483-5p. In the validation phase, miR-21, miR-99b and miR-375 emerged as CRT response-related miRNAs while miR-328 and let-7e emerged as prognostic markers for DFS and OS. Interestingly, ROC curve analysis showed that the combination of miR-21, miR-99b and miR-375 had the best capacity to distinguish patients with maximum response (TRG4) from others. CONCLUSIONS: miR-21, miR-99b and miR-375 could add valuable information for individualizing treatment in locally advanced rectal cancer patients.

Prognostic Impact of miR-200 Family Members in Plasma and Exosomes from Tumor-Draining versus Peripheral Veins of Colon Cancer Patients.

OBJECTIVE: To evaluate the prognostic potential of expression levels of miR-200 family members (miR-200a, miR-200b, miR-200c, miR-429, miR-141) in plasma and exosomes from the tumor-draining vein (mesenteric vein [MV]) and peripheral vein (PV) of colon cancer (CC) patients. METHODS: We analyzed the expression of miR-200 family members in matched samples of MV and PV plasma from 50 resected patients with CC and correlated our findings with overall survival (OS). We also examined the content of these microRNAs in MV and PV exosomes. RESULTS: Expression levels were higher in MV than in PV (miR-200a, p < 0.001; miR-200b, p < 0.001; miR-429, p = 0.01; miR-200c, p = 0.05; miR-141, p = 0.05). Low levels of both miR-200c and miR-141 in MV plasma were associated with longer OS (p = 0.02). This association was maintained for the MV exosome cargo of miR-200c and miR-141 (p = 0.02). CONCLUSION: Our findings provide the first indication that expression levels of miR-200c and miR-141 in MV plasma can identify CC patients with poor prognosis. In addition, our results lend further support to the premise that tumor-draining veins constitute a better source of biomarkers than do PVs.

Rotational Instability after Anterior Talofibular and Calcaneofibular Ligament Section: The Experimental Basis for the Ankle Pivot Test.

The clinical diagnosis of the anterior talofibular ligament (ATFL) rupture is based on the findings from the medical history and the anterior drawer test, a maneuver that allegedly pushes the talus and rearfoot anteriorly, although with great variability in its sensitivity. We consider that an ATFL rupture is best evaluated by a rotational vector (i.e., a pivot test) owing to the uncompromised medial ligaments that will block any pure anterior translation of the talus underneath the tibia. We idealized a constrained ankle cadaver model that only allows talar movements in the axial plane. Our hypothesis was that progressive sectioning of the lateral ankle ligaments in this model would cause a progressive and significant angular laxity in internal rotation. Our results showed 3.67 degrees ± 1.2 degrees of talus rotational laxity in the intact ankle, 9.6 degrees ± 3.2 degrees after ATFL sectioning, and 13.43 degrees ± 3.2 degrees after ATFL and calcaneofibular ligament sectioning, indicating almost threefold increase in internal talocrural rotation after single ATFL sectioning and an almost fourfold increase after double (ATFL and calcaneofibular ligament) sectioning. We consider this evidence of rotational ankle laxity to be a major step in defining the correct movement to diagnose an ATFL rupture and propose a new term to avoid further inconsistencies and variability, "the pivot test."

Proteomic Analysis of Liquid Biopsy from Tumor-Draining Vein Indicates that High Expression of Exosomal ECM1 Is Associated with Relapse in Stage I-III Colon Cancer.

BACKGROUND: The analysis of exosomes in blood obtained from the tumor-draining mesenteric vein (MV) can identify tumor biomarkers before they reach target organs and form the premetastatic niche where circulating tumor cells can anchor. Our group has recently shown that microRNAs in plasma from the MV-but not the peripheral vein (PV)-have been related to liver metastases in colon cancer (CC) patients. Here we examine the exosomal protein cargo in plasma from the MV and paired PV in 31 CC patients. PATIENTS AND METHODS: The study included patients who were initially diagnosed with stage I-III CC and 10 healthy controls. Exosomes from the MV and PV of all patients and controls were isolated by ultracentrifugation and confirmed by cryogenic transmission electron microscopy. High-throughput proteomic analysis by mass spectrometry was used to identify expression levels of exosomal proteins. Findings were confirmed by Western blot. RESULTS: Exosomal ECM1 protein was more highly expressed in patients than in controls and was 13.55 times higher in MV from relapsed than relapse-free patients. High exosomal ECM1 expression was associated with liver metastases. Patients with high exosomal ECM1 expression in MV-but not PV-plasma had shorter time to relapse than those with low ECM1 expression (P = .04). CONCLUSION: High levels of exosomal ECM1 protein can identify CC patients with a higher risk of relapse. The analysis of exosomes isolated from the tumor-draining MV is a promising method for the identification of biomarkers before they reach the target organ.

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.

Epigenetic regulation mechanisms of microRNA expression.

MicroRNAs (miRNAs) are single-stranded RNAs of 18-25 nucleotides that regulate gene expression at the post-transcriptional level. They are involved in many physiological and pathological processes, including cell proliferation, apoptosis, development and carcinogenesis. Because of the central role of miRNAs in the regulation of gene expression, their expression needs to be tightly controlled. Here, we summarize the different mechanisms of epigenetic regulation of miRNAs, with a particular focus on DNA methylation and histone modification.