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.

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.

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.

Critical pathways are shared between embryogenesis and cancer; but while the first is an organized and well-regulated process, the last is characterized by entropy. Several works have evaluated the potential of the embryonic microenvironment for controlling tumor development, showing that embryonic stem cell conditioned media are able to reduce metastatic phenotype of cancer cells. In this context, we have previously observed that human embryonic mesenchymal lung cells-conditioned medium (hEML-CM) are able to induce differentiation in tumor cells. Using this model, we aimed to identify relevant genes involved in the embryonic control of tumorogenesis in non-small cell lung cancer (NSCLC) and to evaluate their utility as prognostic biomarkers. A549 cell line were cultured 48 hours in hEML-CM from human embryonic lungs of 10 weeks of development or control medium. Then, RNA was extracted and expression analysis was performed using Affymetrix HT Clariom™ S human microarray. After bioinformatic analysis, candidate genes were studied by qRT-PCR in a cohort of 91 early-stage NSCLC patients: mean age 69 years; 64.84% male; 48.35% stage I; 37.4% received adjuvant treatment; mean follow up 57 months; and 36 patients relapsed. The endpoints analyzed were disease free-survival (DFS) and overall survival (OS). A549 cells cultured with hEML-CM showed evidence of cell differentiation including morphological and molecular changes. Moreover, the metastatic potential of these cells was significantly reduced (lower proliferation and migration capacity). The analysis of the expression profile identified 7 genes previously linked to cancer, including DIO2 (p=0.003), which has been previously related to thyroid and brain development. In NSCLC patients DIO2 was upregulated in tumor tissue (p=0.002) and its expression was higher in smoking patients (p=0.035). Moreover, high levels of DIO2 were associated with shorter DFS (58.2 months vs. not reached; p=0.043) and shorter OS (76.3 months vs. not reached; p=0.024). The study of embryonic genes involved in tumorogenesis allowed the identification of DIO2, a new promising prognostic biomarker for early-stage NSCLC patients.

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.

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.

e15534 Background: Preoperative chemoradiotherapy (CRT) is a standard treatment for locally advanced rectal cancer (RC) patients (pts). Despite the benefits of CRT, its use in non-responder pts can be associated with increased toxicities and resection delay. The identification of CRT response biomarkers, such as long non-coding RNAs (lncRNA), could improve the management of these pts. LincRNA-p21 is a lncRNA involved in the p53 pathway and angiogenesis regulation that acts as prognostic marker in several tumors. We aim to study lincRNA-p21 expression in pretreatment samples from RC pts treated with CRT to evaluate whether lincRNA-p21 can act as a predictive biomarker for CRT response. Methods: RNA from pretreatment endoscopy biopsies from 58 RC pts treated with preoperative CRT at Mutua Terrassa University Hospital were analyzed. Pathological response was classified according to the tumor regression grade (TRG) Dworak classification. LincRNA-p21 expression was determined by RTqPCR and was correlated with CRT response and, time to relapse (TTR), disease-free survival (DFS) and overall survival (OS). Results: Samples from 58 pts were analyzed. Most of them males (n = 40, 69%). Median age at diagnosis of 67 (44-82) years. Fifty (86.2%) pts reported stage III and, 42 (72.4%) pts assessed TRG 0-3 with a 70.7% of downstaging reported. LincRNA-p21 was upregulated in stage III tumors (p < 0.0001) and also in tumors with worst response to CRT regarding TRG, TGR0-3 (TRG0-3, n = 42 vs TRG4, n = 16, p = 0.019). In this line, the ROC curve analysis showed that lincRNA-p21 expression had capacity to distinguish pts with maximum response (TRG4) from others (AUC:0.7; p = 0.02). The binary logistic regression analysis validated its independence as response biomarker. LincRNA-p21 levels correlated with pts relapse after surgery. When the pts were classified in 3 groups, high, medium and low, according to lincRNA-p21 levels, RC pts with highest lincRNA-p21 expression had the shortest TTR. TTR for pts with high levels was 74.5 months (m) (95% CI:31-117), while it was 114 m (95% CI:95-133) for those with Medium levels and 123 m (95% CI:111-134) for those with low levels (p = 0.047). Conclusions: LincRNA-p21 is a marker of advanced disease and worse response to CRT. LincRNA-p21 may add valuable information for individualizing pre-operative CRT in locally advanced RC pts.

Potential curative surgery is the preferential therapeutic option for early-stage non-small cell lung cancer (NSCLC). The outcome of these patients it is still very unfavorable, and it is believed that early detection and treatment of recurrence could improve their survival. Patient surveillance after surgery is mostly based in imaging techniques and few circulating biomarkers have been identified. Long non-coding RNAs (lncRNAs) and their analysis in exosomes have recently emerged as promising blood-based biomarkers.

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.

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.

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.

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. (C) 2020 Published by Elsevier Ltd on behalf of European Foot and Ankle Society.

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.

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.

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. 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. 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. The lncRNA HOTTIP can be considered a prognostic biomarker in early-stage NSCLC.

Background In resected non-small cell lung cancer (NSCLC) considerable recurrence rates has been reported (30%). Tumor secretes small extracellular vesicles known as exosomes, which plays a role in the metastasis process. Pulmonary tumor-draining vein blood has proved to be better source than peripheral vein to analyze the prognosis impact of exosomes (Navarro A, et al. Cancers 2019). LincRNA-p21 is a long non-coding RNA which upregulation in tumor tissue induced by hypoxia was related to poor prognosis in NSCLC (Castellano JJ. et al. JTO 2016). We aimed to evaluate the presence of lincRNA-p21 in exosomes and its potential as prognosis biomarker in resected NSCLC patients. Methods H23 and H1299 NSCLC cell lines were cultured in exosome-free medium 48h under normoxic/hypoxic conditions. LincRNA-p21 was inhibited using a siRNA and quantified using a TaqMan assay. Exosomes were purified by ultracentrifugation and characterized by electron microscopy, western blot and Nanosight. LincRNA-p21 was analyzed in exosomes from pulmonary tumor-draining vein, obtained during surgery, from 54 NSCLC patients. Results LincRNA-p21 was present in exosomes showing 18 times more representation than in their origin cells. Under hypoxic conditions exosomal lincRNA-p21 was upregulated in both cell lines (p Conclusions Tumor exosomes carry LincRNA-p21, which increases under hypoxia, and its analysis in tumor-draining vein arises as a promising prognosis marker. Legal entity responsible for the study The authors. Funding SAF2017-88606-P (AEI/FEDER, UE). Disclosure All authors have declared no conflicts of interest.

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.