Safety of Allogeneic Adipose Tissue-Derived Mesenchymal Stem Cells for the Treatment of Complex Perianal Fistulas Not Associated With Crohn's Disease: A Phase I Clinical Trial.

BACKGROUND: Anal fistula treatment aims to eradicate the fistula, preserve the sphincter, prevent recurrence, and allow an early return to daily activities for the patient. Because of the difficulty of achieving these goals, stem cell-based therapy has emerged for the treatment of complex perianal fistula with promising results. OBJECTIVE: The objective of this study was to evaluate the safety of allogeneic mesenchymal stem cells in the treatment of complex anal fistula in patients without Crohn's disease. DESIGN: This was a prospective nonrandomized phase I clinical trial. SETTINGS: This study was conducted at a second-level hospital. PATIENTS: Twenty consecutive patients diagnosed with a complex fistula were included. INTERVENTIONS: All patients received 40 × 106 allogeneic mesenchymal stem cells. In patients with 2 tracts, 20 × 106 stem cells were applied on each tract. MAIN OUTCOME MEASURES: The patients were discharged 24 hours after the procedure and were evaluated at 1, 2, 4, 8, 16, and 24 weeks after the application. The long-term follow-up was performed 1 year after the procedure. RESULTS: The procedure was performed in a total of 20 patients from October 1, 2016, to October 31, 2017; 1 patient was eliminated from the final data analysis. No adverse effects were reported within the first 24 hours, and all the patients were discharged asymptomatic. Three patients (15%) presented with perianal abscess. In 1 patient, the abscess appeared at the fourth week, and, in the other 2 patients, the abscess was diagnosed at week 8. Complete closure was achieved in 13 (69%) patients. LIMITATIONS: This was a nonrandomized controlled trial. CONCLUSION: The use of allogeneic mesenchymal stem cells as a treatment is a safe option for the management of complex perianal fistula not associated with Crohn's disease. See Video Abstract at http://links.lww.com/DCR/B443. SEGURIDAD DE LAS CLULAS MADRE MESENQUIMALES ALOGNICAS DERIVADAS DEL TEJIDO ADIPOSO PARA EL TRATAMIENTO DE FSTULAS PERIANALES COMPLEJAS NO ASOCIADAS CON LA ENFERMEDAD DE CROHN ENSAYO CLNICO DE FASE I: ANTECEDENTES:El tratamiento de la fístula anal tiene como objetivo erradicar la fístula, preservar el esfínter, prevenir la recurrencia y permitir un retorno temprano a las actividades diarias del paciente. Debido a la dificultad de alcanzar estos objetivos, ha surgido una terapia basada en células madre para el tratamiento de la fístula perianal compleja con resultados prometedores.OBJETIVO:El objetivo de este estudio fue evaluar la seguridad de las células madre mesenquimales alogénicas en el tratamiento de la fístula anal compleja en pacientes sin enfermedad de Crohn.DISEÑO:Este fue un ensayo clínico prospectivo no aleatorizado de fase I.AMBIENTE:Este estudio se realizó en un hospital de segundo nivel.PACIENTES:Veinte pacientes consecutivos diagnosticados de fístula compleja.INTERVENCIONES:Todos los pacientes recibieron 40 x 106 células madre mesenquimales alogénicas, en pacientes con dos tractos, se aplicaron 20 x 106 células madre en cada tracto.PRINCIPALES MEDIDAS DE RESULTADO:Los pacientes fueron dados de alta 24 horas después del procedimiento y fueron evaluados 1, 2, 4, 8, 16, 24 semanas después de la aplicación. El seguimiento a largo plazo se realizó un año después del procedimiento.RESULTADOS:El procedimiento se realizó en un total de 20 pacientes desde el 1 de octubre de 2016 al 31 de octubre de 2017; un paciente fue eliminado del análisis de datos final. No se informaron efectos adversos en las primeras 24 horas, todos los pacientes fueron dados de alta asintomáticos. Tres pacientes (15%) presentaron absceso perianal. En un paciente, el absceso apareció a la cuarta semana y en los otros dos pacientes el absceso se diagnosticó en la octava semana. El cierre completo se logró en 13 (69%) de los pacientes.LIMITACIONES:Este fue un ensayo controlado no aleatorio.CONCLUSIÓN:El uso de células madre mesenquimales alogénicas como tratamiento es una opción segura para el manejo de la fístula perianal compleja no asociada con la enfermedad de Crohn. Consulte Video Resumen en http://links.lww.com/DCR/B443.

Matrix metalloproteinases 7, 8, 12, 13 gene haplotypes associated with colorectal cancer in a Mexican population.

BACKGROUND: Matrix metalloproteinases (MMPs) are involved in tumor invasion and progression in colorectal cancer (CRC). Variants rs11568818, rs11225395, rs2276109 and rs2252070 have been associated with this neoplasm. OBJECTIVE: To evaluate MMPs 7, 8, 12, and 13 haplotypes and their association with CRC. MATERIAL AND METHODS: One-hundred and four patients and 112 healthy individuals were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP). For the association analysis, odds ratio and confidence interval values were calculated. Haplotype and linkage disequilibrium (LD) analysis was performed with Arlequin software, v3.5. RESULTS: LD was present between rs2276109 and rs2252070. Haplotypes rs11568818(A)-rs11225395(T)-rs2276109(A)-rs2252070(A) and rs11568818(A)-rs11225395(C)-rs2276109(A)-rs2252070(G) were associated with CRC risk, and haplotypes rs11568818(G)-rs11225395(C)-rs2276109(A)-rs2252070(A) and rs11568818(A)-rs11225395(T)-rs2276109(A)-rs2252070(G), with protection. CONCLUSION: Variants rs2276109 and rs2252070 showed genetic linkage. Two haplotypes were associated with the development of CRC (ATAA and ACAG) and two were associated with protection (GCAA and ATAG). This study represents the first report on variants rs11225395 and rs2276109 frequency in a Mexican population.

ANTECEDENTES: Las metaloproteinasas (MMP) se involucran en invasión y progresión tumoral en cáncer colorrectal (CCR). Las variantes rs11568818, rs11225395, rs2276109 y rs2252070 se han asociado con esta neoplasia. OBJETIVO: Evaluar haplotipos de las MMP 7, 8, 12, y 13 y su asociación con CCR. MATERIAL Y MÉTODOS: Se genotipificaron 104 pacientes y 112 individuos sanos mediante reacción en cadena de la polimerasa con análisis del polimorfismo de los fragmentos de restricción (PCR-RFLP). Para el análisis de asociación fueron calculados valores de odds ratio e intervalo de confianza. El análisis de haplotipos y desequilibrio de ligamiento (LD) se realizó con el software Arlequin v3.5. RESULTADOS: Se presentó LD entre rs2276109 y rs2252070. Los haplotipos rs11568818(A)-rs11225395(T)-rs2276109(A)-rs2252070(A) y rs11568818(A)-rs11225395(C)-rs2276109(A)-rs2252070(G) se asociaron con riesgo de CCR y los haplotipos rs11568818(G)-rs11225395(C)-rs2276109(A)-rs2252070(A) y rs11568818(A)-rs11225395(T)-rs2276109(A)-rs2252070(G) con protección. CONCLUSIÓN: Las variantes rs2276109 y rs2252070 mostraron ligamiento génico. Dos haplotipos fueron asociados con el desarrollo de CCR (ATAA y ACAG) y dos fueron asociados con protección (GCAA y ATAG). Este estudio representa el primer reporte de frecuencias de las variantes rs11225395 y rs2276109 en población mexicana.

High frequency of MLH1 promoter methylation mediated by gender and age in colorectal tumors from Mexican patients.

INTRODUCTION: Several genes determine the development of colorectal cancer (CRC), such as MLH1, which encodes a protein that participates in DNA repair. MLH1 hypermethylation has been associated with gene silencing. OBJECTIVE: To analyze the methylation of five regions of MLH1 CpG island in colorectal tumors from Mexican patients. MATERIALS AND METHODS: One hundred and one tumor tissue samples were obtained from Mexican patients with CRC who provided informed consent. DNA was subjected to bisulfite conversion. Methylation of all five regions of the CpG island was evaluated using methylation-specific PCR. RESULTS: The frequency of methylation in Mexican patients with CRC was 25%. Regions A and B methylation was the main observed pattern (60%). Female patients showed a higher frequency of methylation (71%; OR 3.085; CI: 1.85-8.03; p = 0.02), and out of total methylated samples, 80% corresponded to individuals older than 45 years (p < 0.05). CONCLUSION: We calculated a methylation frequency for the MLH1 gene of 25% in Mexican patients with CRC, with this being the first report for this population. Female patients and patients older than 45 years showed a higher frequency of methylation.

INTRODUCCIÓN: Varios genes determinan el desarrollo de cáncer colorrectal (CCR), como MLH1, el cual codifica una proteína que participa en la reparación del ADN. La hipermetilación de MLH1 ha sido asociado con silenciamiento génico. OBJETIVO: Analizar la metilación de cinco regiones de la isla CpG de MLH1 en tumores colorrectales de pacientes mexicanos. MATERIALES Y MÉTODOS: Se obtuvieron 101 muestras de tejido tumoral de pacientes mexicanos con CCR, quienes proporcionaron su consentimiento informado. El ADN fue sometido a conversión por bisulfito. La metilación de las cinco regiones de la isla CpG fue evaluada utilizando PCR específica para metilación. RESULTADOS: La frecuencia de metilación en pacientes mexicanos con CCR fue del 25%. La metilación de las regiones A y B fue el principal patrón observado (60%). Las pacientes de sexo femenino mostraron una mayor frecuencia de metilación (71%) (odds ratio: 3.085; intervalo de confianza; 1.85-8.03; p = 0.02); y del total de muestras metiladas, el 80% fueron individuos mayores de 45 años (p < 0.05). CONCLUSIÓN: Calculamos una frecuencia de metilación para el gen MLH1 del 25% en pacientes mexicanos con CCR, siendo el primer reporte para esta población. Pacientes de sexo femenino y pacientes mayores de 45 años mostraron una mayor frecuencia de metilación.

Expression profile of NF-κB regulated genes in sporadic colorectal cancer patients.

Colorectal cancer (CRC) is the fourth leading worldwide cause of cancer-associated mortalities. Nuclear factor-κB (NF-κB) is a transcriptional regulator of multiple genes associated with CRC. Tumor tissue were compared with normal adjacent mucosa from 30 sporadic patients with CRC were investigated. A total of 8 non-CRC patients were analyzed as a control group. In the present study, the protein expression of NF-κB/p65 was detected by immunohistochemistry, and the gene expression profiles of cyclin D1 (CCND1), prostaglandin-endoperoxide synthase 2, vascular endothelial growth factor A, matrix metallopeptidase 9, BCL2 apoptosis regulator (BCL2), BCL2 like 1, nitric oxide synthase 2, tumor necrosis factor and arachidonate lipoxygenase were detected by reverse transcription-quantitative polymerase chain reaction. NF-κB/p65 and genes expression profiles were classified according to tumor-node-metastasis (TNM) clinicopathological parameters, followed by statistical analysis. Higher protein expression of NF-κB/p65 in the cytoplasm of tumor tissues compared with adjacent normal mucosa was reported; this increment was positively associated with all clinicopathological parameters, except for tumor localization site. The selected genes demonstrated a diverse associative pattern when analyzed with clinicopathological parameters. CCND1 was positively associated with all TNM parameters and BCL2 was negatively associated with all TNM parameters, thus indicating their importance as strong molecular biomarkers for CRC. According to these results, not all selected genes regulated by NF-κB/p65 show increased expression during CRC development, whereas the transcription factor did. The present study suggests that NF-κB/p65 overexpression is necessary for CRC establishment and progression, but its transcriptional activity is not sufficient to regulate all target genes in CRC. NF-κB/p65 and the gene expression profiles reported in the present study may be therapeutically useful. Considering the heterogeneity of the disease, the particular evaluation of these molecules may allow for the selection of proper diagnosis, treatment and follow-up for patients with sporadic CRC.

Novel Mutations in MLH1 and MSH2 Genes in Mexican Patients with Lynch Syndrome.

Background. Lynch Syndrome (LS) is characterized by germline mutations in the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2. This syndrome is inherited in an autosomal dominant pattern and is characterized by early onset colorectal cancer (CRC) and extracolonic tumors. The aim of this study was to identify mutations in MMR genes in three Mexican patients with LS. Methods. Immunohistochemical analysis was performed as a prescreening method to identify absent protein expression. PCR, Denaturing High Performance Liquid Chromatography (dHPLC), and Sanger sequencing complemented the analysis. Results. Two samples showed the absence of nuclear staining for MLH1 and one sample showed loss of nuclear staining for MSH2. The mutations found in MLH1 gene were c.2103+1G>C in intron 18 and compound heterozygous mutants c.1852_1854delAAG (p.K618del) and c.1852_1853delinsGC (p.K618A) in exon 16. In the MSH2 gene, we identified mutation c.638dupT (p.L213fs) in exon 3. Conclusions. This is the first report of mutations in MMR genes in Mexican patients with LS and these appear to be novel.

Association of LEP and ADIPOQ common variants with colorectal cancer in Mexican patients.

Leptin and adiponectin are cytokines produced by adipose tissue with opposite effects on tumor growth: the former stimulate whereas the latter inhibit it. The objective was to analyze the association of LEP A19G and ADIPOQ+45 T/G and +276 G/T polymorphisms in Mexican patients with colorectal cancer (CRC). 68 unrelated patients with CRC (study group) and 102 blood donors (control group); all subjects were Mestizos from western Mexico. The polymorphisms were established by PCR-RFLP on DNA samples obtained from peripheral blood. The LEP A19G polymorphism showed significant differences between CRC patients and control group (p= 0.01 for G/A genotype and p= 0.02 for the recessive model G/G +G/A); yet, in the analysis stratified by gender, this difference remained significant only in males. The ADIPOQ polymorphisms did not shown any significant differences. Our results suggest that the A19G LEP polymorphism is associated with CRC in Mexican patients.