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.

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.

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.

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."

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.

Non-Coding RNAs in Hodgkin Lymphoma.

MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression by binding to the 3'-UTR of their target genes, can act as oncogenes or tumor suppressors. Recently, other types of non-coding RNAs-piwiRNAs and long non-coding RNAs-have also been identified. Hodgkin lymphoma (HL) is a B cell origin disease characterized by the presence of only 1% of tumor cells, known as Hodgkin and Reed-Stenberg (HRS) cells, which interact with the microenvironment to evade apoptosis. Several studies have reported specific miRNA signatures that can differentiate HL lymph nodes from reactive lymph nodes, identify histologic groups within classical HL, and distinguish HRS cells from germinal center B cells. Moreover, some signatures are associated with survival or response to chemotherapy. Most of the miRNAs in the signatures regulate genes related to apoptosis, cell cycle arrest, or signaling pathways. Here we review findings on miRNAs in HL, as well as on other non-coding RNAs.

Talonavicular joint arthroscopic portals: A cadaveric study of feasibility and safety.

BACKGROUND: The objectives of the study were to evaluate the safety of hypothetical arthroscopic portals from talonavicular joint and to evaluate their reproducibility and enforceability. METHODS: 19 cadaveric feet were marked and four arthroscopic portals were made (medial, dorsomedial, dorsolateral and lateral). The specimens were dissected in layers and the distances between neurovascular structures and the trocars were measured. RESULTS: Medial and dorsomedial portals were in average 8.3 and 8.7, respectively, to the saphenous vein and nerve. Dorsolateral portal was in average 8.1mm to the deep peroneal nerve and dorsalis pedis artery, and 9.1mm to the medial dorsal cutaneous branch of the superficial peroneal nerve. Lateral portal was in average 12.3mm to the intermediate dorsal cutaneous branch of the superficial peroneal nerve. CONCLUSION: Tested portals shown to have a good safety margin for the foot neurovascular deep dorsal structures and an acceptable safety margin for the superficial neurovascular structures.

Role of miR-200 family members in survival of colorectal cancer patients treated with fluoropyrimidines.

BACKGROUND AND OBJECTIVES: Surgery is the standard treatment for colorectal cancer (CRC), and adjuvant chemotherapy has been shown to be effective in stage III but less so in stage II. We have analyzed the expression of the miR-200 family in tissue samples from resected CRC patients and correlated our findings with survival to adjuvant treatment with fluoropyrimidines. METHODS: Tumor tissue samples were obtained from 127 surgically resected patients with stage I-III CRC. miRNA detection was performed using TaqMan MicroRNA assays. RESULTS: High levels of miR-200a and miR-200c were associated with longer overall survival, while high levels of miR-429 correlated with longer overall and disease-free survival (DFS). In the subgroup of 56 patients treated with fluoropyrimidines and in the smaller subgroup of 32 stage II patients treated with fluoropyrimidines, those with high levels of miR-200a, miR-200c, miR-141, or miR-429 had significantly longer overall and DFS. Low miR-429 levels were identified as an independent prognostic marker. High levels of miR-429 combined with 5-fluorouracil inhibited cell invasion in LOVO cells. CONCLUSIONS: miR-200a, miR-200c, miR-141, and miR-429 expression levels may identify CRC patients, including those with stage II disease, who are most likely to benefit from adjuvant chemotherapy.

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.

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.

Prevention of chemotherapy-induced left ventricular dysfunction with enalapril and carvedilol: rationale and design of the OVERCOME trial.

BACKGROUND: The current treatment of hematologic malignancies includes diverse potentially cardiotoxic chemotherapy agents, including high-dose myeloablative regimens used in autologous hematopoietic stem cell transplantation (HSCT). Many of these treatments could induce left ventricular dysfunction (LVD), and limit their efficacy. Angiotensin-converting enzime inhibitors and beta-blockers prevent LVD and prolong survival after infarction, and recent animal and pilot clinical studies suggest that they can prevent the development of chemotherapy-induced cardiac toxicity. METHODS: This is a prevention, parallel-assignment, randomized, controlled, clinical efficacy study. Ninety patients recently diagnosed of acute leukemia or undergoing autologous HSCT and with normal LV ejection fraction will be randomized to enalapril and carvedilol or to the control group. Echocardiogram and a cardiac magnetic resonance imaging studies will be performed at baseline and 6-9 months after randomization. The primary efficacy endpoint is the change from baseline in LV ejection fraction. Secondary endpoints include the assessment of LV volumes and diastolic function, and the incidence of death, heart failure, or LVD. CONCLUSIONS: The OVERCOME study will be the first clinical trial to test the preventive efficacy on LVD of combined treatment with enalapril and carvedilol administered to patients with hematologic malignancies submitted to current treatment with intensive chemotherapy.

Regulation of JAK2 by miR-135a: prognostic impact in classic Hodgkin lymphoma.

The behavior of classic Hodgkin lymphoma (cHL) is determined by both the intrinsic features of the tumor cells and the characteristics of the microenvironment, making the analysis of entire lymph nodes an effective approach to understanding the disease. We examined the influence of our previously reported 25-microRNA signature for cHL on clinical outcome in 89 homogeneously treated cHL patients with a median follow-up of 80 months. Patients with low miR-135a expression had a higher probability of relapse (P = .04) and a shorter disease-free survival (P = .02). Functional analysis of cHL cell lines showed that mature miR-135a levels increased after pre-miR-135a transfection, causing apoptosis and decreased cell growth. Target analysis showed a direct regulation by miR-135a of JAK2, a cytoplasmic tyrosine kinase involved in a specific subset of cytokine receptor signaling pathways. miR-135a-mediated JAK2 down-regulation led to decreased mRNA and protein levels of the antiapoptotic gene Bcl-xL, suggesting a role for Bcl-xL in miR-135a/JAK2-mediated apoptosis. Our findings confirm the critical role of miR-135a in the survival of cHL cells and in the prognosis of cHL patients, indicating that novel treatment approaches targeting miR-135a may potentially benefit these patients.

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.

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.

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.

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.