Metformin as an Alternative Radiosensitizing Agent to 5-Fluorouracil During Neoadjuvant Treatment for Rectal Cancer.

BACKGROUND: Neoadjuvant chemoradiation for locally advanced rectal cancer combining 5-fluorouracil with radiation increases tumor regression compared with radiation alone. However, it occurs at the cost of significant treatment-related toxicity. Patients with rectal cancer using metformin have been associated with improved response to radiotherapy. OBJECTIVE: The purpose of this study was to evaluate the radiosensitizing effects of metformin in vitro and in vivo and compare it with a standard combination of radiation/5-fluorouracil. DESIGN: Colorectal cancer cell lines SW480, HT29, and HCT116 were used as models. Cell viability was compared under treatments with radiation, radiation/5-fluorouracil, metformin, radiation/metformin, and radiation/5-fluorouracil/metformin. Nude mice were injected subcutaneously with SW480 cells and treated for 1 week with radiation/5-fluorouracil, metformin, radiation/metformin, or radiation/5-fluorouracil/metformin. Tumor volume was evaluated for 4 weeks after treatment completion. The phosphorylation status of key proteins of the PI3K/Akt/mTOR pathway was determined by immunoblots. SETTINGS: This was an experimental study conducted in vitro and in vivo. PATIENTS: Animal models/cell lines were used. MAIN OUTCOME MEASURES: The end point was to investigate how metformin compares with 5-fluorouracil as a radiosensitizer. RESULTS: All cell lines significantly decreased cell viability after treatment with radiation/metformin when compared with radiation alone. Radiation/metformin was superior to radiation/5-fluorouracil in SW480 (37% vs 74%; p < 0.001). In HT29 and in HCT116, radiation/metformin was inferior to radiation/5-fluorouracil (40.0% vs 13.8%, p < 0.001 and 40.0% vs 7.0%, p < 0.001), mainly because of increased 5-fluorouracil toxicity (≤20% of cell viability). In vivo assays indicated that radiation/metformin treatment was comparable with radiation/5-fluorouracil (557 vs 398 mm; p > 0.05) and that the addition of metformin to the standard radiation/5-fluorouracil did not improve tumor response (349 mm; p > 0.05). Metformin exerted strong PI3K/Akt/mTOR pathway inactivation effects after 24-hour exposure (increasing pAMPK, p < 0.01; decreasing pAkt, p < 0.01; and pS6, p <0.05). LIMITATIONS: In vitro and in vivo chemoradiation regimens cannot be directly translated to human delivery methods. CONCLUSIONS: Metformin enhances tumor response to radiation in vitro and in vivo. Metformin is an attractive alternative radiosensitizing agent to be considered in future studies/trials. See Video Abstract at http://links.lww.com/DCR/B219. LA METFORMINA COMO AGENTE RADIOSENSIBILIZADOR ALTERNATIVO A 5FU DURANTE EL TRATAMIENTO NEOADYUVANTE PARA CÁNCER DE RECTO: La quimiorradiación neoadyuvante para el cáncer de recto localmente avanzado que combina 5FU con radiación aumenta la regresión tumoral en comparación con la radiación sola. Sin embargo, se produce a costa de una toxicidad significativa relacionada con el tratamiento. Los pacientes con cáncer de recto que usan metformina se han asociado con una mejor respuesta a la radioterapia.Evaluar los efectos radiosensibilizantes de metformina in vitro e in vivo y compararlo con la combinación estándar de radiación / 5FU.Se usaron como modelos las líneas celulares de cáncer colorrectal SW480, HT29 y HCT116. La viabilidad celular se comparó en tratamientos con radiación, radiación / 5FU, metformina, radiación / metformina y radiación / 5FU / metformina. A los ratones desnudos se les inyectó por vía subcutánea células SW480 y fueron tratados durante una semana con radiación / 5FU, metformina, radiación / metformina o radiación / 5FU / metformina. El volumen tumoral se evaluó durante 4 semanas después de la finalización del tratamiento. El estado de fosforilación de las proteínas clave de la vía PI3K / Akt / mTOR se determinó mediante inmunotransferencias.Estudio experimental in vitro e in vivo.Modelo animal / líneas celulares.El punto final fue investigar cómo la metformina se compara con 5FU como un radiosensibilizador.Todas las líneas celulares disminuyeron significativamente la viabilidad celular después del tratamiento con radiación / metformina en comparación con la radiación sola. La radiación / metformina fue superior a la radiación / 5FU en SW480 (37% frente a 74%; p <0,001). En el HT29 y el HCT116 la radiación / metformina fue inferior a la radiación / 5FU (40% vs 13.8%, p <0.001 y 40% vs 7%, p <0.001; respectivamente), debido principalmente al aumento de la toxicidad de 5FU (≤20% de la célula viabilidad). Los ensayos in vivo indicaron que el tratamiento con radiación / metformina era comparable a la radiación / 5FU (557 vs 398 mm, p > 0.05), y que la adición de metformina a la radiación estándar / 5FU no mejoró la respuesta tumoral (349 mm, p > 0.05). La metformina ejerció fuertes efectos de inactivación de la vía PI3K / Akt / mTOR después de 24 horas de exposición (aumentando pAMPK p < 0.01, disminuyendo pAkt, p < 0.01; y pS6, p < 0.05).Los regímenes de CRT in vitro e in vivo no se pueden traducir directamente a los métodos de entrega en humanos.La metformina mejora la respuesta tumoral a la radiación in vitro e in vivo. La metformina es un agente alternativo de radiosensibilización atractivo para ser considerado en futuros estudios / ensayos. Consulte Video Resumen en http://links.lww.com/DCR/B219. (Traducción-Dr Gonzalo Hagerman).

Exosomes from patients with septic shock convey miRNAs related to inflammation and cell cycle regulation: new signaling pathways in sepsis?

BACKGROUND: Exosomes isolated from plasma of patients with sepsis may induce vascular apoptosis and myocardial dysfunction by mechanisms related to inflammation and oxidative stress. Despite previous studies demonstrating that these vesicles contain genetic material related to cellular communication, their molecular cargo during sepsis is relatively unknown. In this study, we evaluated the presence of microRNAs (miRNAs) and messenger RNAs (mRNAs) related to inflammatory response and redox metabolism in exosomes of patients with septic shock. METHODS: Blood samples were collected from 24 patients with septic shock at ICU admission and after 7 days of treatment. Twelve healthy volunteers were used as control subjects. Exosomes were isolated by ultracentrifugation, and their miRNA and mRNA content was evaluated by qRT-PCR array. RESULTS: As compared with healthy volunteers, exosomes from patients with sepsis had significant changes in 65 exosomal miRNAs. Twenty-eight miRNAs were differentially expressed, both at enrollment and after 7 days, with similar kinetics (18 miRNAs upregulated and 10 downregulated). At enrollment, 35 differentially expressed miRNAs clustered patients with sepsis according to survival. The pathways enriched by the miRNAs of patients with sepsis compared with control subjects were related mostly to inflammatory response. The comparison of miRNAs from patients with sepsis according to hospital survival demonstrated pathways related mostly to cell cycle regulation. At enrollment, sepsis was associated with significant increases in the expression of mRNAs related to redox metabolism (myeloperoxidase, 64-fold; PRDX3, 2.6-fold; SOD2, 2.2-fold) and redox-responsive genes (FOXM1, 21-fold; SELS, 16-fold; GLRX2, 3.4-fold). The expression of myeloperoxidase mRNA remained elevated after 7 days (65-fold). CONCLUSIONS: Exosomes from patients with septic shock convey miRNAs and mRNAs related to pathogenic pathways, including inflammatory response, oxidative stress, and cell cycle regulation. Exosomes may represent a novel mechanism for intercellular communication during sepsis.

A straightforward assay to evaluate DNA integrity and optimize next-generation sequencing for clinical diagnosis in oncology.

Next generation sequencing (NGS) has become an informative tool to guide cancer treatment and conduce a personalized approach in oncology. The biopsy collected for pathologic analysis is usually stored as formalin-fixed paraffin-embedded (FFPE) blocks and then availed for molecular diagnostic, resulting in DNA molecules that are invariably fragmented and chemically modified. In an attempt to improve NGS based diagnostics in oncology we developed a straightforward DNA integrity assessment assay based on qPCR, defining clear parameters to whether NGS sequencing results is accurate or when it should be analyzed with caution. We performed DNA extraction from 12 tumor samples from diverse tissues and accessed DNA integrity by straightforward qPCR assays. In order to perform a cancer panel NGS sequencing, DNA library preparation was performed using RNA capture baits. Reads were aligned to the reference human genome and mutation calls were further validated by Sanger sequencing. Results obtained by the DNA integrity assays correlated to the efficiency of the pre-capture library preparation in up to 0.94 (Pearson's test). Moreover, sequencing results showed that poor integrity DNA leads to high rates of false positive mutation calls, specially C:G>T:A and C:G>A:T. Poor quality FFPE DNA samples are prone to generating false positive mutation calls. These are especially perilous in cases in which subclonal populations are expected, such as in advance disease, since it could lead clinicians to erroneous conclusions and equivocated conduct.

MicroRNA Expression Signature Is Altered in the Cardiac Remodeling Induced by High Fat Diets.

Recent studies have revealed the involvement of microRNAs (miRNAs) in the control of cardiac hypertrophy and myocardial function. In addition, several reports have demonstrated that high fat (HF) diet induces cardiac hypertrophy and remodeling. In the current study, we investigated the effect of diets containing different percentages of fat on the cardiac miRNA expression signature. To address this question, male C57Bl/6 mice were fed with a low fat (LF) diet or two HF diets, containing 45 kcal% fat (HF45%) and 60 kcal% fat (HF60%) for 10 and 20 weeks. HF60% diet promoted an increase on body weight, fasting glycemia, insulin, leptin, total cholesterol, triglycerides, and induced glucose intolerance. HF feeding promoted cardiac remodeling, as evidenced by increased cardiomyocyte transverse diameter and interstitial fibrosis. RNA sequencing analysis demonstrated that HF feeding induced distinct miRNA expression patterns in the heart. HF45% diet for 10 and 20 weeks changed the abundance of 64 and 26 miRNAs in the heart, respectively. On the other hand, HF60% diet for 10 and 20 weeks altered the abundance of 27 and 88 miRNAs in the heart, respectively. Bioinformatics analysis indicated that insulin signaling pathway was overrepresented in response to HF diet. An inverse correlation was observed between cardiac levels of GLUT4 and miRNA-29c. Similarly, we found an inverse correlation between expression of GSK3ß and the expression of miRNA-21a-3p, miRNA-29c-3p, miRNA-144-3p, and miRNA-195a-3p. In addition, miRNA-1 overexpression prevented cardiomyocyte hypertrophy. Taken together, our results revealed differentially expressed miRNA signatures in the heart in response to different HF diets. J. Cell. Physiol. 231: 1771-1783, 2016. © 2015 Wiley Periodicals, Inc.

Melatonin-induced temporal up-regulation of gene expression related to ubiquitin/proteasome system (UPS) in the human malaria parasite Plasmodium falciparum.

There is an increasing understanding that melatonin and the ubiquitin/ proteasome system (UPS) interact to regulate multiple cellular functions. Post-translational modifications such as ubiquitination are important modulators of signaling processes, cell cycle and many other cellular functions. Previously, we reported a melatonin-induced upregulation of gene expression related to ubiquitin/proteasome system (UPS) in Plasmodium falciparum, the human malaria parasite, and that P. falciparum protein kinase 7 influences this process. This implies a role of melatonin, an indolamine, in modulating intraerythrocytic development of the parasite. In this report we demonstrate by qPCR analysis, that melatonin induces gene upregulation in nine out of fourteen genes of the UPS, consisting of the same set of genes previously reported, between 4 to 5 h after melatonin treatment. We demonstrate that melatonin causes a temporally controlled gene expression of UPS members.

The somatic mutation profile of estrogen receptor-positive HER2-negative metastatic breast cancer in Brazilian patients.

Background: Breast cancer is the leading cause of cancer death among women worldwide. Studies about the genomic landscape of metastatic breast cancer (MBC) have predominantly originated from developed nations. There are still limited data on the molecular epidemiology of MBC in low- and middle-income countries. This study aims to evaluate the prevalence of mutations in the PI3K-AKT pathway and other actionable drivers in estrogen receptor (ER)+/HER2- MBC among Brazilian patients treated at a large institution representative of the nation's demographic diversity. Methods: We conducted a retrospective observational study using laboratory data (OC Precision Medicine). Our study included tumor samples from patients with ER+/HER2- MBC who underwent routine tumor testing from 2020 to 2023 and originated from several Brazilian centers within the Oncoclinicas network. Two distinct next-generation sequencing (NGS) assays were used: GS Focus (23 genes, covering PIK3CA, AKT1, ESR1, ERBB2, BRCA1, BRCA2, PALB2, TP53, but not PTEN) or GS 180 (180 genes, including PTEN, tumor mutation burden [TMB] and microsatellite instability [MSI]). Results: Evaluation of tumor samples from 328 patients was undertaken, mostly (75.6%) with GS Focus. Of these, 69% were primary tumors, while 31% were metastatic lesions. The prevalence of mutations in the PI3K-AKT pathway was 39.3% (95% confidence interval, 33% to 43%), distributed as 37.5% in PIK3CA and 1.8% in AKT1. Stratification by age revealed a higher incidence of mutations in this pathway among patients over 50 (44.5% vs 29.1%, p=0.01). Among the PIK3CA mutations, 78% were canonical (included in the alpelisib companion diagnostic non-NGS test), while the remaining 22% were characterized as non-canonical mutations (identifiable only by NGS test). ESR1 mutations were detected in 6.1%, exhibiting a higher frequency in metastatic samples (15.1% vs 1.3%, p=0.003). Additionally, mutations in BRCA1, BRCA2, or PALB2 were identified in 3.9% of cases, while mutations in ERBB2 were found in 2.1%. No PTEN mutations were detected, nor were TMB high or MSI cases. Conclusion: We describe the genomic landscape of Brazilian patients with ER+/HER2- MBC, in which the somatic mutation profile is comparable to what is described in the literature globally. These data are important for developing precision medicine strategies in this scenario, as well as for health systems management and research initiatives.

The structurally related auxin and melatonin tryptophan-derivatives and their roles in Arabidopsis thaliana and in the human malaria parasite Plasmodium falciparum.

Indole compounds are involved in a range of functions in many organisms. In the human malaria parasite Plasmodium falciparum, melatonin and other tryptophan derivatives are able to modulate its intraerythrocytic cycle, increasing the schizont population as well as parasitemia, likely through ubiquitin-proteasome system (UPS) gene regulation. In plants, melatonin regulates root development, in a similar way to that described for indoleacetic acid, suggesting that melatonin and indoleacetic acid could co-participate in some physiological processes due to structural similarities. In the present work, we evaluate whether the chemical structure similarity found in indoleacetic acid and melatonin can lead to similar effects in Arabidopsis thaliana lateral root formation and P. falciparum cell cycle modulation, as well as in the UPS of gene regulation, by qRT-PCR. Our data show that P. falciparum is not able to respond to indoleacetic acid either in the modulation of the intraerythrocytic cycle or in the gene regulation mediated by the UPS as observed for melatonin. The similarities of these indole compounds are not sufficient to confer synergistic functions in P. falciparum cell cycle modulation, but could interplay in A. thaliana lateral root formation.

Ubiquitin proteasome system and the atypical kinase PfPK7 are involved in melatonin signaling in Plasmodium falciparum.

We previously reported that melatonin modulates the Plasmodium falciparum erythrocytic cycle by increasing schizont stage population as well as diminishing ring stage population. In addition, the importance of calcium and cAMP in melatonin signaling pathway in P. falciparum was also demonstrated. Nevertheless, the molecular effectors of the indoleamine signaling pathway remain elusive. We now demonstrate by real-time PCR that melatonin treatment up-regulates genes related to ubiquitin/proteasome system (UPS) components and that luzindole, a melatonin receptor antagonist, inhibits UPS transcription modulation. We also show that protein kinase PfPK7, a P. falciparum orphan kinase, plays a crucial role in the melatonin transduction pathway, since following melatonin treatment of P. falciparum parasites where pfpk7 gene is disrupted (pfpk7(-) parasites) (i) the ratio of asexual stages remain unchanged, (ii) the increase in cytoplasmatic calcium in response to melatonin was strongly diminished and (iii) up-regulation of UPS genes did not occur. The wild-type melatonin-induced alterations in cell cycle features, calcium rise and UPS gene transcription were restored by re-introduction of a functional copy of the pfpk7 gene in the pfpk7(-) parasites.

Molecular Signature Expands the Landscape of Driver Negative Thyroid Cancers.

Thyroid cancer is the most common endocrine malignancy. However, the cytological diagnosis of follicular thyroid carcinoma (FTC), Hürthle cell carcinoma (HCC), and follicular variant of papillary thyroid carcinoma (FVPTC) and their benign counterparts is a challenge for preoperative diagnosis. Nearly 20-30% of biopsied thyroid nodules are classified as having indeterminate risk of malignancy and incur costs to the health care system. Based on that, 120 patients were screened for the main driver mutations previously described in thyroid cancer. Subsequently, 14 mutation-negative cases that are the main source of diagnostic errors (FTC, HCC, or FVPTC) underwent RNA-Sequencing analysis. Somatic variants in candidate driver genes (ECD, NUP98,LRP1B, NCOR1, ATM, SOS1, and SPOP) and fusions were described. NCOR1 and SPOP variants underwent validation. Moreover, expression profiling of driver-negative samples was compared to 16 BRAF V600E, RAS, or PAX8-PPARg positive samples. Negative samples were separated in two clusters, following the expression pattern of the RAS/PAX8-PPARg or BRAF V600E positive samples. Both negative groups showed distinct BRS, ERK, and TDS scores, tumor mutation burden, signaling pathways and immune cell profile. Altogether, here we report novel gene variants and describe cancer-related pathways that might impact preoperative diagnosis and provide insights into thyroid tumor biology.

Molecular machinery of signal transduction and cell cycle regulation in Plasmodium.

The regulation of the Plasmodium cell cycle is not understood. Although the Plasmodium falciparum genome is completely sequenced, about 60% of the predicted proteins share little or no sequence similarity with other eukaryotes. This feature impairs the identification of important proteins participating in the regulation of the cell cycle. There are several open questions that concern cell cycle progression in malaria parasites, including the mechanism by which multiple nuclear divisions is controlled and how the cell cycle is managed in all phases of their complex life cycle. Cell cycle synchrony of the parasite population within the host, as well as the circadian rhythm of proliferation, are striking features of some Plasmodium species, the molecular basis of which remains to be elucidated. In this review we discuss the role of indole-related molecules as signals that modulate the cell cycle in Plasmodium and other eukaryotes, and we also consider the possible role of kinases in the signal transduction and in the responses it triggers.

Histopathological Characterization and Whole Exome Sequencing of Ectopic Thyroid: Fetal Architecture in a Functional Ectopic Gland from Adult Patient.

Ectopic thyroid results from a migration defect of the developing gland during embryogenesis causing congenital hypothyroidism. But it has also been detected in asymptomatic individuals. This study aimed to investigate the histopathological, functional, and genetic features of human ectopic thyroids. Six samples were histologically examined, and the expression of the specific thyroid proteins was assessed by immunohistochemistry. Two samples were submitted to whole exome sequencing. An oropharynx sample showed immature fetal architecture tissue with clusters or cords of oval thyrocytes and small follicles; one sample exhibited a normal thyroid pattern while four showed colloid goiter. All ectopic thyroids expressed the specific thyroid genes and T4 at similar locations to those observed in normal thyroid. No somatic mutations associated with ectopic thyroid were found. This is the first immature thyroid fetal tissue observed in an ectopic thyroid due to the arrest of structural differentiation early in the colloid stage of development that proved able to synthesize thyroid hormone but not to respond to TSH. Despite the ability of all ectopic thyroids to synthetize specific thyroid proteins and T4, at some point in life, it may be insufficient to support body growth leading to hypothyroidism, as observed in some of the patients.

E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer.

BACKGROUND: Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. METHODS: We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. RESULTS: We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. CONCLUSIONS: We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

The use of personalized biomarkers and liquid biopsies to monitor treatment response and disease recurrence in locally advanced rectal cancer after neoadjuvant chemoradiation.

Neoadjuvant chemoradiotherapy (nCRT) followed by surgery is the mainstay treatment for locally advanced rectal cancer. Variable degrees of tumor regression are observed after nCRT and alternative treatment strategies, including close surveillance without immediate surgery, have been investigated to spare patients with complete tumor regression from potentially adverse outcomes of radical surgery. However, clinical and radiological assessment of response does not allow accurate identification of patients with complete response. In addition, surveillance for recurrence is similarly important for these patients, as early detection of recurrence allows salvage resections and adjuvant interventions. We report the use of liquid biopsies and personalized biomarkers for monitoring treatment response to nCRT and detecting residual disease and recurrence in patients with rectal cancer. We sequenced the whole-genome of four rectal tumors to identify patient-specific chromosomal rearrangements that were used to monitor circulating tumor DNA (ctDNA) in liquid biopsies collected at diagnosis and during nCRT and follow-up. We compared ctDNA levels to clinical, radiological and pathological response to nCRT. Our results indicate that personalized biomarkers and liquid biopsies may not be sensitive for the detection of microscopic residual disease. However, it can be efficiently used to monitor treatment response to nCRT and detect disease recurrence, preceding increases in CEA levels and radiological diagnosis. Similar good results were observed when assessing tumor response to systemic therapy and disease progression. Our study supports the use of personalized biomarkers and liquid biopsies to tailor the management of rectal cancer patients, however, replication in a larger cohort is necessary to introduce this strategy into clinical practice.

Comprehensive evaluation of the effectiveness of gene expression signatures to predict complete response to neoadjuvant chemoradiotherapy and guide surgical intervention in rectal cancer.

Neoadjuvant chemoradiotherapy (nCRT) may lead to complete tumor regression in rectal cancer patients. Prediction of complete response to nCRT may allow a personalized management of rectal cancer and spare patients from unnecessary radical total mesorectal excision with or without sphincter preservation. To identify a gene expression signature capable of predicting complete pathological response (pCR) to nCRT, we performed a gene expression analysis in 25 pretreatment biopsies from patients who underwent 5FU-based nCRT using RNA-Seq. A supervised learning algorithm was used to identify expression signatures capable of predicting pCR, and the predictive value of these signatures was validated using independent samples. We also evaluated the utility of previously published signatures in predicting complete response in our cohort. We identified 27 differentially expressed genes between patients with pCR and patients with incomplete responses to nCRT. Predictive gene signatures using subsets of these 27 differentially expressed genes peaked at 81.8% accuracy. However, signatures with the highest sensitivity showed poor specificity, and vice-versa, when applied in an independent set of patients. Testing previously published signatures on our cohort also showed poor predictive value. Our results indicate that currently available predictive signatures are highly dependent on the sample set from which they are derived, and their accuracy is not superior to current imaging and clinical parameters used to assess response to nCRT and guide surgical intervention.

Overexpression of miR-21-5p as a predictive marker for complete tumor regression to neoadjuvant chemoradiotherapy in rectal cancer patients.

BACKGROUND: Neoadjuvant chemoradiotherapy (nCRT) followed by radical surgery is the preferred treatment strategy for locally advanced rectal cancer. However, complete tumor regression is observed in a significant proportion of patients after nCRT, making them ideal candidates for alternative treatment strategies to this considerably morbid procedure. Identification of such patients based on clinical findings (complete clinical response - cCR) is difficult mainly because it relies on subjective clinical and imaging studies. Our goal was to identify biomarkers capable of predicting complete response to nCRT. METHODS: We analyzed miRNA expression profile using deep sequencing in rectal tumor biopsies prior to nCRT. Differential expression was investigated by EdgeR for a training (n = 27) and a validation (n = 16) set of patients to identify miRNAs associated with treatment response (complete vs. incomplete). In vitro experiments with two cancer cell lines were also performed in order to evaluate the possible role of miRNAs on response to nCRT. RESULTS: We found 4 miRNAs differentially expressed between complete and incomplete responders to nCRT. In addition, validation was performed using an independent group of patients and miR-21-5p was confirmed as being overexpressed in complete responders. Overall sensitivity and specificity of miR-21-5p expression in predicting complete response to nCRT was 78% and 86% respectively. Interestingly, in a subset of patients with cCR followed by early local recurrence, the expression level of miR-21-5p was considerably low, similarly to incomplete responders. We also found SATB1, a miR-21-5p target gene and known multidrug resistance gene, whose expression was inversely correlated with miR-21-5p expression. Finally, we performed functional experiments and showed that miR-21-5p and SATB1 may be directly involved with poor response to nCRT in rectal cancer patients. CONCLUSIONS: This study suggests miR-21-5p as a promising predictive biomarker, which should aid in the selection of patients with cCR to nCRT that potentially could be spared from radical surgery.

Mutational analysis of genes coding for cell surface proteins in colorectal cancer cell lines reveal novel altered pathways, druggable mutations and mutated epitopes for targeted therapy.

We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of "general" immune checkpoint drugs in this subset of patients.