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Targeting wild-type epidermal growth factor receptor (EGFR) using tyrosine kinase inhibitors (TKIs) never achieved its purported success in cancers such as head and neck squamous cell carcinoma, which are largely EGFR-dependent. We had previously shown that exceptional responders to TKIs have a genetic aberration that results in overexpression of an EGFR splice variant, isoform D (IsoD). IsoD lacks an integral transmembrane and kinase domain and is secreted in extracellular vesicles (EVs) in TKI-sensitive patient-derived cultures. Remarkably, the exquisite sensitivity to TKIs could be transferred to TKI-resistant tumor cells, and IsoD protein in the EV is necessary and sufficient to transfer the phenotype in vitro and in vivo across multiple models and drugs. This drug response requires an intact endocytic mechanism, binding to full-length EGFR, and signaling through Src-phosphorylation within the endosomal compartment. We propose a therapeutic strategy using EVs containing EGFR IsoD as a co-drug to expand the use of TKI therapy to EGFR-driven cancers.
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Glioblastoma (GBM) is the most aggressive and common primary malignant brain tumor with limited available therapeutic approaches. Despite improvements in therapeutic options for GBM patients, efforts to develop new successful strategies remain as major unmet medical needs. Based on the cytotoxic properties of aporphine compounds, we evaluated the biological effect of 12 compounds obtained through total synthesis of ( ±)-apomorphine hydrochloride (APO) against GBM cells. The compounds 2,2,2-trifluoro-1-(1-methylene-3,4-dihydroisoquinolin-2(1H)-yl)ethenone (A5) and ( ±)-1-(10,11-dimethoxy-6a,7-dihydro-4H-dibenzo[de,g]quinolin-6(5H)-yl)ethenone (C1) reduced the viability of GBM cells, with 50% inhibitory concentration ranging from 18 to 48 µM in patient-derived GBM cultures. Our data show that APO, A5 or C1 modulate the expression of DNA damage and apoptotic markers, impair 3D-gliomasphere growth and reduce the expression of stemness markers. Potential activity and protein targets of A5, C1 or APO were predicted in silico based on PASS and SEA software. Dopamine receptors (DRD1 and 5), CYP2B6, CYP2C9 and ABCB1, whose transcripts were differentially expressed in the GBM cells, were among the potential A5 or C1 target proteins. Docking analyses (HQSAR and 3D-QSAR) were performed to characterize possible interactions of ABCB1 and CYP2C9 with the compounds. Notably, A5 or C1 treatment, but not temozolomide (TMZ), reduced significantly the levels of extracellular ATP, suggesting ABCB1 negative regulation, which was correlated with stronger cytotoxicity induced by the combination of TMZ with A5 or C1 on GBM cells. Hence, our data reveal a potential therapeutic application of A5 and C1 as cytotoxic agents against GBM cells and predicted molecular networks that can be further exploited to characterize the pharmacological effects of these isoquinoline-containing substances.
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Temozolomida , Humanos , Temozolomida/farmacologíaRESUMEN
Evaluate the biological action of valproic acid in the acetylation of histones and in the methylation of tumor suppressor genes via oral rinse in patients with a previous history of head and neck squamous cell carcinoma (HNSCC). Forty-two active or former smokers were included in this randomized, double-blind, placebo-controlled trial. Oral rinse samples were collected prior to treatment with valproic acid or placebo and after 90 days of treatment. The methylation status of five tumor suppressor genes and histone acetylation were evaluated by pyrosequencing and ELISA techniques, respectively. Differences between the 90-day and baseline oral rinse acetylation and methylation results were analyzed by comparing groups. Thirty-four patients were considered for analysis. The mean percentage adherence in the valproic and placebo groups was 93.4 and 93.0, respectively (p = 0.718). There was no statistically significant difference between groups when comparing the medians of the histone acetylation ratio and the methylation ratio for most of the studied genes. A significant reduction in the DCC methylation pattern was observed in the valproic group (p = 0.023). The use of valproic acid was safe and accompanied by good therapeutic adherence. DCC methylation was lower in the valproic acid group than in the placebo group.
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Acetilación/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Ácido Valproico/uso terapéutico , Método Doble Ciego , Femenino , Neoplasias de Cabeza y Cuello/metabolismo , Histonas/metabolismo , Humanos , Masculino , Estudios Prospectivos , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismoRESUMEN
INTRODUCTION: Multiple myeloma (MM) remains an incurable disease, and patient survival requires a better understanding of this malignancy's molecular aspects. Heparanase (HPSE) is highly expressed in aggressive MM cells and related to tumor growth, metastasis, and bortezomib (BTZ) resistance. Thus, targeting HPSE seems to be a promising approach for MM treatment, and because microRNAs (miRNAs) have emerged as potential regulators of HPSE expression, the use of extracellular vesicles (EVs) can allow the efficient delivery of therapeutic miRNAs. METHODS: We used prediction algorithms to identify potential miRNAs that regulate negatively HPSE expression. RT-qPCR was performed to assess miRNAs and HPSE expression in MM lines (U266 and RPMI-8226). Synthetic miRNA mimics were electroporated in MM cells to understand the miRNA contribution in HPSE expression, glycosaminoglycans (GAGs) profile, cell proliferation, and cell death induced by BTZ. EVs derived from HEK293T cells were engineered with miRNAs to evaluate their therapeutic potential combined with BTZ. RESULTS: It revealed a direct association between BTZ sensitivity, HPSE, and miR-1252-5p expressions. Moreover, overexpression of miR-1252-5p significantly reduced HPSE expression and HPSE enzymatic activity in MM cells. The higher level of miR-1252-5p was correlated with a reduction of cell viability and higher sensitivity to BTZ. Further, EVs carrying miR-1252-5p increased MM cells' sensitivity to BTZ treatment. CONCLUSION: These results showed that miR-1252-5p could negatively regulate HPSE in MM, indicating the use of EVs carrying miR-1252-5p as a potential novel BTZ sensitization approach in MM cells.
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The content of tumor-derived extracellular vesicles (EVs) can regulate the tumor microenvironment and functionally acts in favor of cancer aggressiveness. To better elucidate the role of EVs in the interplay between immune system and tumor microenvironment, the purpose of this study was to analyze the effect of head and neck squamous cells carcinoma (HNSCC)-derived EVs on the modulation of inflammasomes - mediators of pyroptosis and secretion of inflammatory factors by macrophages. Our results showed that macrophages treated with the Vesicular Secretome Fraction (VSF) isolated from patient-derived HNSCC presented a reduction in the secretion of mature IL-1ß and caspase-1 without affecting cell viability. An analysis of the protein content of HNSCC-derived VSF by antibody array revealed that some of the most expressed proteins share a correlation with Transforming Growth Factor-beta (TGF-ß) activity. Since TGF-ß is related to the inhibition of the NF-kB-related pathways, including those required for the priming phase of the inflammasomes, we sought to evalute the interference of the VSF in the induction of inflammasome components. In fact, HNSCC-derived VSF inhibited the induction of pro-IL-1ß and pro-caspase-1 proteins and NLRP3 gene expression during the priming phase of inflammasome activation. Thus, our findings contribute to a better understanding of how tumor-derived EVs modulate inflammatory response by demonstrating their role in inhibiting NLRP3 inflammasomes.
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Purpose Among alkaloids, abundant secondary metabolites in plants, aporphines constitute a class of compounds with interesting biological activities, including anticancer effects. The present study evaluated the anticancer activities of 14 substances, including four aporphine derivatives acquired through the biomonitoring of (±)-apomorphine hydrochloride total synthesis from 2-phenethylamine and 3,4-dimethoxybenzaldehyde against head and neck squamous cell carcinoma (HNSCC). Methods The cytotoxic effects of compounds against a panel of HNSCC cell lines were determined by PrestoBlue cell viability assay, while the genotoxicity of substances was evaluated by micronucleus test. Cell death was detected by flow cytometry (Annexin V/7AAD) and western blot analysis was used to detect the presence of cleaved Caspase-3 molecules. Results The aporphine and isoquinoline derivatives APO, C1, and A5 significantly reduced HNSCC cell viability and promoted DNA damages in these cells. Further, by activating the Caspase-3 pathway, these substances were able to induce apoptosis. Conclusion Our results revealed that APO, C1, and A5 exhibit cytotoxic effects in HNSCC cells. The mechanisms of action appear to be partly via the generation of DNA damages and apoptosis induction through Caspase-3 pathway activation. This study provides preclinical data that suggest a potential therapeutic role for APO, C1, and A5 against head and neck cancer cells.
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Antineoplásicos Fitogénicos/farmacología , Aporfinas/química , Neoplasias de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Antineoplásicos Fitogénicos/química , Apoptosis , Aporfinas/farmacología , Caspasa 3/metabolismo , Proliferación Celular , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Células Tumorales CultivadasRESUMEN
Management of locally advanced head and neck squamous cell carcinoma (HNSCC) requires a multi-prong approach comprising surgery, radiation and/or chemotherapy, yet outcomes are limited. This is largely due to a paucity of biomarkers that can predict response to specific treatment modalities. Here, we evaluated TGFß3 protein levels in extracellular vesicles (EVs) released by HNSCC cells as a predictor for response to chemoradiation therapy (CRT). To this end, specific EV-fractions were isolated from cell lines or HNSCC patient plasma, and TGFß3 protein was quantified. In patients treated with CRT, TGFß3 levels were found to be significantly higher in plasma EV-fractions or non-responders compared with responders. High levels of TGFß3 levels in Annexin V-EVs were associated with the worst progression-free survival. In vitro experiments demonstrated that TGFß3 silencing sensitized HNSCC cells to cytotoxic therapies, and this phenotype could be rescued by treatment with exogenous. In addition, specific EV-fractions shed by cisplatin-resistant cells were sufficient to transfer the resistant phenotype to sensitive cells through activation of TGFß-signaling pathway. Therefore, our data show that TGFß3 transmitted through EV plays a significant role in response to cytotoxic therapy, which can be exploited as a potential biomarker for CRT response in HNSCC patients treated with curative intent.
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Biomarcadores de Tumor/sangre , Vesículas Extracelulares/metabolismo , Neoplasias de Cabeza y Cuello/terapia , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Factor de Crecimiento Transformador beta3/sangre , Adulto , Anciano , Quimioradioterapia/métodos , Cisplatino/administración & dosificación , Resistencia a Antineoplásicos/fisiología , Femenino , Neoplasias de Cabeza y Cuello/sangre , Humanos , Masculino , Persona de Mediana Edad , Paclitaxel/administración & dosificación , Tolerancia a Radiación/fisiología , Carcinoma de Células Escamosas de Cabeza y Cuello/sangreRESUMEN
Multiple myeloma (MM) is an incurable disease; a better understanding of the molecular aspects of this hematological malignancy could contribute to the development of new treatment strategies and help to improve the survival rates of patients with MM. Previously, the methylation status of the deleted in colorectal cancer (DCC) gene was correlated with the survival rate of patients with MM, thus the main goal of this study was to understand DCC contribution to MM tumorigenesis, and to assess the impact of DCC inhibition in the MM response to treatment with bortezomib. Our results demonstrated that hypermethylation of the DCC promoter inhibits gene expression, and DCC silencing is significantly correlated with a reduction in cell viability and an increase in cell death induced by bortezomib. In conclusion, our results suggested that hypermethylation is an important mechanism of DCC expression regulation in MM and that the absence of DCC contributes to the enhanced sensitivity to treatment with bortezomib.
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Bortezomib/farmacología , Receptor DCC/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Receptor DCC/antagonistas & inhibidores , Receptor DCC/genética , Metilación de ADN/efectos de los fármacos , Humanos , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
BACKGROUND: There is a paucity of plasma-based biomarkers that prospectively segregate the outcome of patients with head and neck squamous-cell carcinoma (HNSCC) treated with chemoradiation therapy (CRT). Plasma extracellular vesicles (EVs) might be an alternative source for discovery of new specific markers present in patients with HNSCC, which could help to re-direct patients to appropriate curative therapies without delay. METHODS: In order to identify new markers in plasma compartments, Cholerae toxin B chain (CTB) and Annexin V (AV) were used to isolate EVs from pooled plasma samples from patients with locally advanced HNSCC who responded (CR, n = 6) or presented incomplete response (NR, n = 6) to CRT. The crude plasma and EVs cargo were screened by antibody array. RESULTS: Of the 370 polypeptides detected, 119 proteins were specific to NR patients while 38 were exclusive of the CR subjects. The Gene Set Enrichment Analysis (GSEA) and Search Tool for the Retrieval of Interacting Genes (STRING) database analysis indicated that the content of circulating plasma EVs might have a relevant function for the tumor intercellular communication in the HNSCC patients. CONCLUSION: This study provides a list of potential markers present in plasma compartments that might contribute to the development of tools for prediction and assessment of CRT response and potentially guide therapeutic decisions in this context.
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Biomarcadores de Tumor/sangre , Carcinoma de Células Escamosas/patología , Quimioradioterapia/métodos , Vesículas Extracelulares/metabolismo , Neoplasias de Cabeza y Cuello/patología , Adulto , Anciano , Carcinoma de Células Escamosas/sangre , Carcinoma de Células Escamosas/terapia , Ensayos Clínicos Fase II como Asunto , Vesículas Extracelulares/patología , Femenino , Estudios de Seguimiento , Neoplasias de Cabeza y Cuello/sangre , Neoplasias de Cabeza y Cuello/terapia , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Análisis por Matrices de Proteínas , Mapas de Interacción de ProteínasRESUMEN
BACKGROUND: There is a paucity of plasma-based biomarkers that predict outcome in patients with head and neck squamous cell carcinoma (HNSCC) treated with chemoradiation therapy (CRT). Here, we evaluate the prognostic potential of plasma Melanoma-Antigen Recognized by T-cells 1 (MLANA) in this setting. METHODS: MLANA expression in HNSCC lines were evaluated by reverse transcription polymerase chain reaction, whereas plasma levels were quantified using ELISA in 48 patients with locally advanced HNSCC undergoing a phase 2 trial with CRT. RESULTS: MLANA is expressed at variable levels in a panel of HNSCC lines. In plasma, levels were elevated in patients with tumor relapse compared to those without (P < .004); 73.9% of the patients expressing high plasma MLANA levels progressed with recurrent disease (P = .020). Multivariate analysis showed that plasma MLANA levels and tumor resectability were independent prognostic factors for progression free survival. CONCLUSION: Plasma MLANA expression appears to be an effective noninvasive biomarker for outcomes in patients treated with CRT, and could potentially guide therapeutic decisions in this context.
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Quimioradioterapia , Neoplasias de Cabeza y Cuello/sangre , Antígeno MART-1/sangre , Carcinoma de Células Escamosas de Cabeza y Cuello/sangre , Adulto , Anciano , Biomarcadores de Tumor/sangre , Línea Celular Tumoral , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Quimioterapia de Inducción , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Análisis Multivariante , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/terapiaRESUMEN
Glioblastoma (GBM) is an incurable disease ranked among the deadliest solid cancers worldwide. A better understanding on the molecular aspects of this malignancy could contribute to the development of new treatment strategies and help to improve survival rates. Previously, our group had shown that GBM patients expressing the cancer/testis antigen Opa Interacting Protein 5 (OIP5) present a longer survival period than the OIP5-negative group. The main goal of this study was to evaluate the OIP5 contribution to GBM tumorigenesis and assess the role of OIP5 in GBM cell response to lomustine, an alkylating agent used in the treatment of this malignancy. So, the effect of OIP5 knockdown was evaluated in A172 and T98G GBM cell lines. Our results demonstrated that downregulation of the OIP5 stimulates glioma cell viability and inhibits cell death-induced necrosis prompted by lomustine. In conclusion, our data shows that OIP5 expression in GBM cells seems to be able to enhance lomustine cytotoxic effects, reinforcing that this gene is a potential therapeutic target and putative molecular biomarker for treatment response in GBM.
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Neoplasias Encefálicas/metabolismo , Proteínas Cromosómicas no Histona/genética , Resistencia a Antineoplásicos/genética , Glioblastoma/metabolismo , Antineoplásicos Alquilantes/farmacología , Neoplasias Encefálicas/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , Humanos , Lomustina/farmacologíaRESUMEN
Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analyzed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined 4 CCA clusters-fluke-positive CCAs (clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations; conversely, fluke-negative CCAs (clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3' untranslated region deletion as a mechanism of FGFR2 upregulation. Integration of noncoding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation of H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores-mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Our results exemplify how genetics, epigenetics, and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer.Significance: Integrated whole-genome and epigenomic analysis of CCA on an international scale identifies new CCA driver genes, noncoding promoter mutations, and structural variants. CCA molecular landscapes differ radically by etiology, underscoring how distinct cancer subtypes in the same organ may arise through different extrinsic and intrinsic carcinogenic processes. Cancer Discov; 7(10); 1116-35. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1047.
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Neoplasias de los Conductos Biliares/genética , Colangiocarcinoma/genética , Epigenómica/métodos , Estudio de Asociación del Genoma Completo/métodos , Islas de CpG , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Receptor ErbB-2/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: Carcinoma of the oral tongue (OTSCC) is the most common malignancy of the oral cavity, characterized by frequent recurrence and poor survival. The last three decades has witnessed a change in the OTSCC epidemiological profile, with increasing incidence in younger patients, females and never-smokers. Here, we sought to characterize the OTSCC genomic landscape and to determine factors that may delineate the genetic basis of this disease, inform prognosis and identify targets for therapeutic intervention. METHODS: Seventy-eight cases were subjected to whole-exome (n = 18) and targeted deep sequencing (n = 60). RESULTS: While the most common mutation was in TP53, the OTSCC genetic landscape differed from previously described cohorts of patients with head and neck tumors: OTSCCs demonstrated frequent mutations in DST and RNF213, while alterations in CDKN2A and NOTCH1 were significantly less frequent. Despite a lack of previously reported NOTCH1 mutations, integrated analysis showed enrichments of alterations affecting Notch signaling in OTSCC. Importantly, these Notch pathway alterations were prognostic on multivariate analyses. A high proportion of OTSCCs also presented with alterations in drug targetable and chromatin remodeling genes. Patients harboring mutations in actionable pathways were more likely to succumb from recurrent disease compared with those who did not, suggesting that the former should be considered for treatment with targeted compounds in future trials. CONCLUSIONS: Our study defines the Asian OTSCC mutational landscape, highlighting the key role of Notch signaling in oral tongue tumorigenesis. We also observed somatic mutations in multiple therapeutically relevant genes, which may represent candidate drug targets in this highly lethal tumor type.
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Carcinoma de Células Escamosas/genética , Neoplasias de la Lengua/genética , Adulto , Anciano , Anciano de 80 o más Años , Pueblo Asiatico/genética , Cromatina/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Pronóstico , Receptores Notch/genética , Análisis de Secuencia de ADN , Singapur , Adulto JovenRESUMEN
PURPOSE: Definitive radiation therapy is the mainstay of treatment for early stage laryngeal squamous cell carcinoma (LSCC). However, up to 30% of the patients do not respond to radiotherapy. Unfortunately, we are unable to predict which tumors are likely to respond to radiation, and which will be resistant and persist. Therefore, the development of novel markers to predict response to radiotherapy is urgently needed. This study was designed to evaluate the expression pattern of microRNAs (miRNAs) in LSCC in order to identify markers capable of segregating radioresistant and radiosensitive tumors and to investigate the relationship between the expression of these miRNAs and the prognosis of LSCC. METHODS: The expression profile of 667 miRNAs was determined in an initial screening of nine early-stage LSCC samples (5 radioresistant and 4 radiosensitive) using TaqMan Low-Density Array (TLDA). Real-time polymerase chain reactions were performed to validate the expression of selected miRNAs in an expanded LSCC cohort (20 radioresistant and 14 radiosensitive). The miRNA expression level was scored as high or low based on the median of the expression in the LSCC samples. RESULTS: A comprehensive miRNA expression profiling enabled the identification of four miRNAs (miR-296-5p miR-452, miR-183* and miR-200c) differentially expressed in radioresistant LSCC. Moreover, the analysis of additional 34 LSCC samples, confirmed the expression of miR-296-5p as significantly related to radioresistance (p = 0.002) as well as an association of this marker with recurrence (p = 0.025) in early stage laryngeal cancer. CONCLUSIONS: This study indicates that miR-296-5p expression is associated with resistance to radiotherapy and tumor recurrence in early stage LSCC, showing the feasibility of this marker as a novel prognostic factor for this malignance. Furthermore, miR-296-5p expression could be helpful in the identification of tumors resistant to radiotherapy; thus aiding the clinicians in the choice of the best therapeutic scheme to be used in each case.
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Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/radioterapia , MicroARNs/genética , Tolerancia a Radiación/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Perfilación de la Expresión Génica , Humanos , Neoplasias Laríngeas/patología , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias de Células Escamosas/genética , Neoplasias de Células Escamosas/patología , Neoplasias de Células Escamosas/radioterapia , Pronóstico , Análisis de SupervivenciaRESUMEN
BACKGROUND: The presence of metastatic disease in cervical lymph nodes of head and neck squamous cell carcinoma (HNSCC) patients is a very important determinant in therapy choice and prognosis, with great impact in overall survival. Frequently, routine lymph node staging cannot detect occult metastases and the post-surgical histologic evaluation of resected lymph nodes is not sensitive in detecting small metastatic deposits. Molecular markers based on tissue-specific microRNA expression are alternative accurate diagnostic markers. Herein, we evaluated the feasibility of using the expression of microRNAs to detect metastatic cells in formalin-fixed paraffin-embedded (FFPE) lymph nodes and in fine-needle aspiration (FNA) biopsies of HNSCC patients. METHODS: An initial screening compared the expression of 667 microRNAs in a discovery set comprised by metastatic and non-metastatic lymph nodes from HNSCC patients. The most differentially expressed microRNAs were validated by qRT-PCR in two independent cohorts: i) 48 FFPE lymph node samples, and ii) 113 FNA lymph node biopsies. The accuracy of the markers in identifying metastatic samples was assessed through the analysis of sensitivity, specificity, accuracy, negative predictive value, positive predictive value, and area under the curve values. RESULTS: Seven microRNAs highly expressed in metastatic lymph nodes from the discovery set were validated in FFPE lymph node samples. MiR-203 and miR-205 identified all metastatic samples, regardless of the size of the metastatic deposit. Additionally, these markers also showed high accuracy when FNA samples were examined. CONCLUSIONS: The high accuracy of miR-203 and miR-205 warrant these microRNAs as diagnostic markers of neck metastases in HNSCC. These can be evaluated in entire lymph nodes and in FNA biopsies collected at different time-points such as pre-treatment samples, intraoperative sentinel node biopsy, and during patient follow-up. These markers can be useful in a clinical setting in the management of HNSCC patients from initial disease staging and therapy planning to patient surveillance.
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Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/patología , Metástasis Linfática/diagnóstico , MicroARNs , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Femenino , Neoplasias de Cabeza y Cuello/genética , Humanos , Ganglios Linfáticos/patología , Metástasis Linfática/genética , Masculino , Persona de Mediana Edad , Pronóstico , Sensibilidad y Especificidad , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
Despite significant advances in the treatment of head and neck squamous cell carcinoma (HNSCC), the survival rate has not changed in the last decades. Therefore, the development of novel therapeutic strategies is pursued. Cancer-testis antigens (CTA) are strong immunogenic proteins with a tumor-restricted expression pattern, and are considered ideal targets for tumor-specific immunotherapeutic approaches. In this study, using an in silico approach, we selected, among 139 previously described CTA, candidates to be evaluated in 89 HNSCC and 20 normal mucosa samples. SPANX-CD (71.9%), MAGEB2 (44.9%), MAGEA1 (44.9%), MAGEB6 (32.6%), and CXORF48 (27.0%) were found frequently expressed in HNSCC, and over 85% of the tumors expressed at least one of these five CTAs. The mRNA positivity of CXORF48, MAGEB6, and CRISP2 presented significant associations with recognized clinical features for poor outcome. Furthermore, MAGEA3/6 positivity was associated with significantly better disease-free survival (DFS, P = 0.014), and the expression of this antigen was shown to be an independent prognostic factor for tumor recurrence. In conclusion, one of five selected CTAs is expressed in at least 85% of the HNSCCs, suggesting a possible usage as target for immunotherapeutic approaches, and the mRNA-positivity for MAGEA3/6 is shown to be an independent marker for DFS.
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Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Neoplasias de Cabeza y Cuello/genética , Proteínas de Neoplasias/genética , Recurrencia Local de Neoplasia/diagnóstico , Recurrencia Local de Neoplasia/genética , Testículo/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/metabolismo , Estudios de Casos y Controles , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Membrana Mucosa/metabolismo , ARN Mensajero/genética , Estudios Retrospectivos , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
BACKGROUND: Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. The purposes of this study were to determine the methylation profile of exfoliated tumors cells collected from patients with head and neck squamous cell carcinoma (HNSCC) and to evaluate its prognostic significance. METHODS: The methylation profile and level of a 20-gene panel were evaluated by quantitative methylation-specific polymerase chain reaction (qMSP) in exfoliated tumor cell samples from 96 patients with HNSCC. RESULTS: CCNA1 (60.4%), DCC (54.2%), and TIMP3 (35.4%) were frequently methylated in these samples. Patients with exfoliated tumors cells positive for DCC methylation showed a trend toward a lower local recurrence-free survival. CONCLUSION: These findings indicate that a low invasive method could be used to access the methylation profile of exfoliated cells from patients with HNSCC. Moreover, our data provide evidence that hypermethylation of DCC could be useful as prognostic indicator for this malignancy.
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Carcinoma de Células Escamosas/genética , Metilación de ADN , Genes Supresores de Tumor , Neoplasias de Cabeza y Cuello/genética , Anciano , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Estudios de Cohortes , Ciclina A1/genética , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Genes DCC/genética , Neoplasias de Cabeza y Cuello/mortalidad , Neoplasias de Cabeza y Cuello/patología , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , Medición de Riesgo , Carcinoma de Células Escamosas de Cabeza y Cuello , Estadísticas no Paramétricas , Tasa de Supervivencia , Inhibidor Tisular de Metaloproteinasa-3/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: Hypermethylation in the promoter regions is associated with the suppression of gene expression and has been considered a potential molecular marker for several tumor types, including head and neck squamous cell carcinomas (HNSCC). METHODS: To evaluate the gene hypermethylation profile as a prognostic marker, this retrospective study used a QMSP approach to determine the methylation status of 19 genes in 70 HNSCC patients. RESULTS: The methylation profile analysis of primary HNSCC revealed that genes CCNA1, DAPK, MGMT, TIMP3 and SFRP1 were frequently hypermethylated, with high specificity and sensitivity. TIMP3 and CCNA1 hypermethylation was significantly associated with lower rates of second primary tumor-free survival (p = 0.007 and p = 0.001; log-rank test, respectively). CONCLUSION: This study, for the first time, presents CCNA1 and TIMP3 hypermethylation as a helpful tool to identify HNSCC subjects at risk of developing second primary carcinomas.
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Carcinoma de Células Escamosas/genética , Ciclina A1/genética , Metilación de ADN , Neoplasias de Cabeza y Cuello/genética , Neoplasias Primarias Secundarias/genética , Inhibidor Tisular de Metaloproteinasa-3/genética , Carcinoma de Células Escamosas/epidemiología , Femenino , Neoplasias de Cabeza y Cuello/epidemiología , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Neoplasias Primarias Secundarias/epidemiología , Reacción en Cadena de la Polimerasa , Estudios RetrospectivosRESUMEN
Mutations in JAK-STAT signaling pathway genes have been associated with the development of various hematological tumors, but have not been investigated in head and neck tumors, and the PIK3CA, BRAF and KRAS genes have been described in a few cases of head and neck squamous cell carcinoma (HNSCC). In the present study, we determined the mutation status in members of the MAPK, PI3K-AKT and JAK-STAT pathways in HNSCC. Mutations in the KRAS, BRAF, PIK3CA, JAK1 and JAK2 genes were evaluated in 94 HNSCCs by direct DNA sequencing analysis using cDNA synthesized from RNA extracted from patient tumor cells. All patients evaluated had wild-type KRAS, BRAF and PIK3CA genes. Furthermore, although some known polymorphisms have been found in JAK1 genes (rs45598436, rs17127063, rs2230587, rs3737139, rs2230588 and rs12129819) and JAK2 (rs10429491, rs2230723, rs2230724 and rs41316003), no mutation could be detected. Our data indicate that mutations in these kinase genes seem to be rare events in HNSCC.