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(1) Background: Intravesical mitomycin-C (MMC) combined with hyperthermia is increasingly used in non-muscle invasive bladder cancer (NMIBC), especially in the context of a relative BCG shortage. We aim to determine real-world data on the long-term treatment outcomes of adjunct hyperthermic intravesical chemotherapy (HIVEC) with MMC and a COMBAT® bladder recirculation system (BRS), (2) Methods: A prospective observational trial was performed on patients with NMIBC treated with HIVEC using BRS in nine academic institutions in Spain between 2012–2020 (HIVEC-E). Treatment effectiveness (recurrence, progression and overall mortality) was evaluated in patients treated with HIVEC MMC 40mg in the adjuvant setting, with baseline data and a clinical follow-up, that comprise the Full Analysis Set (FAS). Safety, according to the number and severity of adverse effects (AEs), was evaluated in the safety (SAF) population, composed by patients with at least one adjunct HIVEC MMC instillation, (3) Results: The FAS population (n = 502) received a median number of 8.78 ± 3.28 (range 1–20) HIVEC MMC instillations. The median follow-up duration was 24.5 ± 16.5 (range 1–81) months. Its distribution, based on EAU risk stratification, was 297 (59.2%) for intermediate and 205 (40.8%) for high-risk. The figures for five-year recurrence-free and progression-free survival were 50.37% (53.3% for intermediate and 47.14% for high-risk) and 89.83% (94.02% for intermediate and 84.23% for high-risk), respectively. A multivariate analysis identified recurrent tumors (HR 1.83), the duration of adjuvant HIVEC therapy &lt, 4 months (HR 1.72) and that high-risk group (HR 1.47) were at an increased risk of recurrence. Independent factors of progression were high-risk (HR 3.89), recurrent tumors (HR 3.32) and the induction of HIVEC therapy without maintenance (HR 2.37). The overall survival was determined by patient age at diagnosis (HR 3.36) and the treatment duration (HR 1.82). The SAF population (n = 592) revealed 406 (68.58%) patients without AEs and 186 (31.42%) with at least one AE: 170 (28.72%) of grade 1–2 and 16 (2.7%) of grade 3–4. The most frequent AEs were dysuria (10%), pain (7.1%), urgency (5.7%), skin rash (4.9%), spasms (3.7%) and hematuria (3.6%), (4) Conclusions: HIVEC using BRS is efficacious and well tolerated. A longer treatment duration, its use in naïve patients and the intermediate-risk disease are independent determinants of success. Furthermore, a monthly maintenance of adjunct MMC HIVEC diminishes the progression rate of NMIBC.

Resumen Introduccion El Blastocystis hominis (B. hominis) es un protozoo comunmente encontrado en el tracto gastrointestinal. Existen dudas sobre su significado clinico. El metronidazol (MTZ) es el tratamiento aconsejado de primera linea. Material y metodos Se realizo una revision retrospectiva entre 2011 y 2012. Se seleccionaron de forma aleatoria 151 de 383 muestras positivas para B. hominis . Los criterios de inclusion fueron: clinica sugestiva, indicacion de tratamiento y realizacion de control microbiologico. Se realizo una revision sistematica de los estudios que evaluan el efecto de MTZ sobre la infestacion por B. hominis . Resultados Cuarenta y seis pacientes cumplian criterios de inclusion (el 64% eran mujeres; edad, 44,2 ± 2 anos). Se utilizo MTZ en 39 pacientes, de los cuales 31 obtuvieron respuesta clinica (79,5%) pero solo 15 respuesta microbiologica (48,4%). No se aprecio una relacion dosis-efecto. Veinte pacientes sin respuesta microbiologica inicial recibieron una segunda tanda de tratamiento (MTZ, cotrimoxazol, paramomicina, otros), con una respuesta microbiologica del 70%. De forma global, se consiguio la curacion de B. hominis en un 72% (IC95%: 57-83%). De 54 tratamientos asociados a respuesta clinica, se produjo respuesta microbiologica en 31 (57%); mientras que de los 12 que se siguieron de ausencia de respuesta clinica solo se observo la curacion microbiologica en 2 (17%) (p = 0,022). La tasa de erradicacion en la revision sistematica oscilo entre 0 y 100%. Conclusiones Parece existir relacion entre la respuesta clinica y microbiologica al tratamiento de B. hominis . En nuestro entorno geografico la respuesta microbiologica al tratamiento con MTZ es insuficiente. La revision sistematica muestra que la respuesta a MTZ es muy variable.

// Clara Esteban-Jurado 1 , David Gimenez-Zaragoza 2 , Jenifer Munoz 1 , Sebastia Franch-Exposito 1 , Miriam Alvarez-Barona 3 , Teresa Ocana 1 , Miriam Cuatrecasas 4 , Sabela Carballal 1 , Maria Lopez-Ceron 1 , Maria Marti-Solano 5 , Marcos Diaz-Gay 1 , Tom van Wezel 6 , Antoni Castells 1 , Luis Bujanda 7 , Judith Balmana 8 , Victoria Gonzalo 9 , Gemma Llort 10 , Clara Ruiz-Ponte 3 , Joaquin Cubiella 11 , Francesc Balaguer 1 , Rosa Aligue 2 , Sergi Castellvi-Bel 1 1 Gastroenterology Department, Hospital Clinic, Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Catalonia, Spain 2 Biomedical Sciences Department, School of Medicine, University de Barcelona, Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia, Spain 3 Galician Public Foundation of Genomic Medicine (FPGMX), Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), Genomics Medicine Group, Hospital Clinico, Santiago de Compostela, University of Santiago de Compostela, Galicia, Spain 4 Department of Pathology, Hospital Clinic, Biobanc Clinic-IDIBAPS, Barcelona, Catalonia, Spain 5 Department of Pharmaceutical Chemistry, Philipps-University Marburg, Marburg, Germany 6 Leiden University Medical Center (LUMC), Leiden, Netherlands 7 Gastroenterology Department, Hospital Donostia–Instituto Biodonostia, Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas (CIBEREHD), Basque Country University (UPV/EHU), San Sebastian, Spain 8 High Risk and Cancer Prevention Unit, Medical Oncology Department, University Hospital Vall d’Hebron and Vall d’Hebron Institute of Oncology, Barcelona, Spain 9 Gastroenterology Department, Hospital Universitari Mutua de Terrassa, Terrassa, Barcelona, Spain 10 Clinical Oncology Department, Corporacio Parc Tauli, Sabadell, Barcelona, Spain 11 Gastroenterology Department, Complexo Hospitalario Universitario de Ourense, Instituto de Investigacion Biomedica Ourense, Pontevedra y Vigo, Ourense, Spain Correspondence to: Sergi Castellvi-Bel, email: sbel@clinic.cat Keywords: colorectal neoplasm, colorectal adenoma, genetic predisposition to disease, POLE, POLD1 Received: January 04, 2017 Accepted: February 18, 2017 Published: March 01, 2017 ABSTRACT Germline mutations in POLE and POLD1 have been shown to cause predisposition to colorectal multiple polyposis and a wide range of neoplasms, early-onset colorectal cancer being the most prevalent. In order to find additional mutations affecting the proofreading activity of these polymerases, we sequenced its exonuclease domain in 155 patients with multiple polyps or an early-onset colorectal cancer phenotype without alterations in the known hereditary colorectal cancer genes. Interestingly, none of the previously reported mutations in POLE and POLD1 were found. On the other hand, among the genetic variants detected, only two of them stood out as putative pathogenic in the POLE gene, c.1359 + 46del71 and c.1420G > A (p.Val474Ile). The first variant, detected in two families, was not proven to alter correct RNA splicing. Contrarily, c.1420G > A (p.Val474Ile) was detected in one early-onset colorectal cancer patient and located right next to the exonuclease domain. The pathogenicity of this change was suggested by its rarity and bioinformatics predictions, and it was further indicated by functional assays in Schizosaccharomyces pombe . This is the first study to functionally analyze a POLE genetic variant outside the exonuclease domain and widens the spectrum of genetic changes in this DNA polymerase that could lead to colorectal cancer predisposition.

Background Serrated polyposis syndrome (SPS) has been associated with an increased risk of colorectal cancer (CRC). Accordingly, intensive surveillance with annual colonoscopy is advised. The aim of this multicenter study was to describe the risk of advanced lesions in SPS patients undergoing surveillance, and to identify risk factors that could guide the prevention strategy. Methods From March 2013 to April 2015, 296 patients who fulfilled criteria I and/or III for SPS were retrospectively recruited at 18 centers. We selected patients in whom successful clearing colonoscopy had been performed and who underwent subsequent endoscopic surveillance. Advanced neoplasia was defined as CRC, advanced adenoma, or advanced serrated lesion that were ≥ 10 mm and/or with dysplasia. Cumulative incidence of advanced neoplasia was calculated and independent predictors of advanced neoplasia development were identified. Results In 152 SPS patients a total of 315 surveillance colonoscopies were performed (median 2, range 1 – 7). The 3-year cumulative incidence of CRC and advanced neoplasia were 3.1 % (95 % confidence interval [CI] 0 – 6.9) and 42.0 % (95 %CI 32.4 – 51.7), respectively. Fulfilling both I + III criteria and the presence of advanced serrated lesions at baseline colonoscopy were independent predictors of advanced neoplasia development (odds ratio [OR] 1.85, 95 %CI 1.03 – 3.33, P = 0.04 and OR 2.62, 95 %CI 1.18 – 5.81, P = 0.02, respectively). During follow-up, nine patients (5.9 %) were referred for surgery for invasive CRC (n = 4, 2.6 %) or because of polyp burden (n = 5, 3.3 %). After total colectomy, 17.9 % patients developed advanced neoplasia in the retained rectum. Conclusions Patients with SPS have a substantial risk of developing advanced neoplasia under endoscopic surveillance, whereas CRC incidence is low. Personalized endoscopic surveillance based on polyp burden and advanced serrated histology could help to optimize prevention in patients with SPS.

Objective Serrated polyposis syndrome (SPS) is associated with an increased colorectal cancer (CRC) risk, although the magnitude of the risk remains uncertain. Whereas intensive endoscopic surveillance for CRC prevention is advised, predictors that identify patients who have high CRC risk remain unknown. We performed a multicentre nationwide study aimed at describing the CRC risk in patients with SPS and identifying clinicopathological predictors independently associated with CRC. Design From March 2013 through September 2014, patients with SPS were retrospectively recruited at 18 Spanish centres. Data were collected from medical, endoscopy and histopathology reports. Multivariate logistic regression was performed to identify CRC risk factors. Results In 296 patients with SPS with a median follow-up time of 45 months (IQR 26–79.7), a median of 26 (IQR 18.2–40.7) serrated polyps and 3 (IQR 1–6) adenomas per patient were detected. Forty-seven patients (15.8%) developed CRC at a mean age of 53.9±12.8, and 4 out of 47 (8.5%) tumours were detected during surveillance (cumulative CRC incidence 1.9%). Patients with >2 sessile serrated adenomas/polyps (SSA/Ps) proximal to splenic flexure and ≥1 proximal SSA/P with high-grade dysplasia were independent CRC risk factors (incremental OR=2, 95% CI 1.22 to 3.24, p=0.006). Patients with no risk factors showed a 55% decrease in CRC risk (OR=0.45, 95% CI 0.24 to 0.86, p=0.01). Conclusions Patients with SPS have an increased risk of CRC, although lower than previously published. Close colonoscopy surveillance in experienced centres show a low risk of developing CRC (1.9% in 5 years). Specific polyp features (SSA/P histology, proximal location and presence of high-grade dysplasia) should be used to guide clinical management.

The development of genotyping technologies has allowed for wider screening for inherited causes of variable outcomes following drug administration. We have performed a genome-wide association study (GWAS) on 221 colorectal cancer (CRC) patients that had been treated with 5-fluorouracil (5-FU), either alone or in combination with oxaliplatin (FOLFOX). A validation set of 791 patients was also studied. Seven SNPs (rs16857540, rs2465403, rs10876844, rs10784749, rs17626122, rs7325568 and rs4243761) showed evidence of association (pooled P-values 0.020, 9.426E-03, 0.010, 0.017, 0.042, 2.302E-04, 2.803E-03) with adverse drug reactions (ADRs). This is the first study to explore the genetic basis of inter-individual variation in toxicity responses to the administration of 5-FU or FOLFOX in CRC patients on a genome-wide scale. © 2013 Macmillan Publishers Limited. All rights reserved 1470-269X/13.

Introduction Blastocystis hominis (B. hominis) is a protozoan commonly found in the gastrointestinal tract. There are doubts about its clinical significance. Metronidazole (MTZ) is the recommended first-line treatment. Materials and methods A retrospective review was carried out between 2011 and 2012. A total of 151 samples were randomly selected from 383 samples positive for B. hominis . Inclusion criteria were: suggestive symptoms, treatment indication and microbiological follow-up. A systematic review was performed of all studies that evaluated the effect of MTZ on B. hominis infection. Results Forty-six patients met the inclusion criteria (64% women; age, 44.2 ± 2 years). MTZ was used in 39 patients, 31 of whom obtained a clinical response (79.5%) but only 15 a microbiological response (48.4%). No dose-effect relationship was observed. Twenty patients with no initial microbiological response received a second round of treatment (MTZ, cotrimoxazole, paramomycin, others), with a microbiological response in 70%. Overall, B. hominis was cured in 72% (95% CI: 57–83%). Of 54 treatments associated with a clinical response, a microbiological response occurred in 31 (57%), while in the remaining 12 with no clinical response, microbiological cure was observed in only 2 (17%) ( P = .022). The eradication rate in the systematic review varied between 0% and 100%. Conclusions There seems to be a relationship between the clinical and microbiological response to B. hominis treatment. The microbiological response to MTZ treatment is insufficient in our geographical setting. The systematic review shows that the response to MTZ is very variable.

Colorectal cancer (CRC) risk associated with germline monoallelic MUTYH mutations remains controversial, although a slightly increased risk for this disease has been suggested. MUTYH and MSH6 proteins act in cooperation during the DNA repair process. Based on this interaction, it was hypothesized that the combination of heterozygote germline mutations in both genes could result in an increased CRC risk. To further clarify the interaction between MUTYH and MSH6, we analyzed the prevalence of MSH6 mutations in a cohort of CRC patients and controls previously tested for MUTYH mutations: CRC patients with and without a monoallelic MUTYH mutation (group I, n = 26; group II, n = 50, respectively), and healthy carriers with a monoallelic MUTYH mutation (group III, n = 21). In group I, we found three patients (11.5%) with MSH6 mutations, a missense mutation (p.R635G), a change in the 3′UTR region (c.*4098A > C) and a nonsense mutation (p.Q982X). In group II and III, no mutations were detected. In CRC patients, MSH6 mutations were more frequently found in MUTYH mutation carriers than in noncarriers (11.5% vs. 0%, P = 0.037). CRC patients carrying monoallelic MUTYH mutations harbor more frequently concomitant MSH6 mutations than patients without them, thus suggesting that both genes could act cooperatively and confer together an increased CRC risk.

The pathogenesis of cancer includes both genetic and epigenetic mechanisms. The term > refers to any alteration of gene expression, potentially hereditary, which is not accompanied by a modification in the DNA sequence. The effects of epigenetic changes include alteration of DNA transcription, aberrant activation of specific genes, predisposition of genetic instability through alteration of the control of chromosome replication and silencing of the genes implicated in cancer initiation and progression. Among the various epigenetic alterations that lead to altered gene expression, methylation is the main epigenomic mechanism implicated in cancer, whether through a phenomenon of overall hypomethylation or hypermethylation localized in the promoters of specific genes.

Colorectal cancer (CRC) is the second most common cancer worldwide and the second leading cause of cancer mortality in Western countries. The lifetime risk of CRC in the average-risk person—one aged above 50 years and without a personal or family history of this neoplasm—is 5% to 6%. This risk may be as high as 20% if first-or second-degree relatives have CRC and reaches a lifetime risk of 80% to 100% in hereditary syndromes. Knowledge of the genetic basis for CRC has been applied to genetic testing, genotypic-phenotypic correlations, and provision of a rationale for chemopreventive and therapeutic approaches. This article reviews the most relevant recent advances in the genetics of polyposis and nonpolyposis colorectal syndromes, emphasizing their clinical application in gene testing.

ARLTS1 was recently identified in chromosome 13q14 as a tumor suppressor gene of the ADP-ribosylation factor family with pro-apoptotic characteristics. Additionally, one of its genetic variants (W149X) was hypothesized to be a polymorphism associated with familial cancer. We performed a large case-control association study within the EPICOLON project aimed at evaluating the sporadic and familial colorectal cancer (CRC) risk associated with ARLTS1 genetic variants. Whereas P131L and W149X did not seem to affect CRC risk, C148R did show, for the first time in CRC, statistically significant differences between cases and controls [odds ratio (OR) = 1.45, 95% confidence interval (95% CI) = 1.13-1.86, P = 0.003], sporadic cases and controls (OR = 1.59, 95% CI = 1.13-2.23, P = 0.007) and familial cases and controls (OR = 1.55, 95% CI = 1.10-2.19, P = 0.01) in agreement with a hypothetical moderate increase of the cancer risk linked to the C148R ARLTS1 variant, both in sporadic and familial CRC cases.

Resumen Introduccion Los pacientes con adenomas avanzados (AA) presentan un mayor riesgo de desarrollar neoplasias colorrectales metacronicas, por lo que se recomienda realizar un control mas exhaustivo. La displasia de alto grado (DAG) es el marcador principal de transformacion carcinomatosa. Sin embargo, se desconoce el valor predictivo de esta para el desarrollo de neoplasia colorrectal avanzada en pacientes con AA. Objetivo Evaluar si la DAG supone un riesgo adicional al resto de caracteristicas que definen el AA (patron glandular velloso o tamano ≥ 1 cm) para desarrollar un cancer colorrectal (CCR) o un AA tras la reseccion endoscopica. Metodos Se registraron los pacientes con AA resecados endoscopicamente entre enero de 1996 y diciembre de 1997. Se excluyeron los que tenian antecedentes de CCR, enfermedad inflamatoria intestinal, historia de poliposis adenomatosa familiar o CCR no asociado a poliposis o ausencia de vigilancia endoscopica. Se evaluo el desarrollo de CCR o AA durante el seguimiento. Resultados Se clasifico a 71 pacientes incluidos en 2 grupos segun la presencia (n = 49) o ausencia (n = 22) de DAG en el adenoma basal. La probabilidad de desarrollar CCR o AA (rango logaritmico, p = 0,47; indice de Breslow, p = 0,58) o AA con DAG (rango logaritmico, p = 0,47; indice de Breslow, p = 0,53) durante el seguimiento fue similar en ambos grupos. El numero de polipos (p = 0,67), adenomas (p = 0,73), AA (p = 0,93) o AA con DAG (p = 0,88) metacronicos fue similar. Conclusion La DAG no supone un riesgo adicional para el desarrollo de CCR ni AA metacronico en los pacientes con AA. Por tanto, no seria necesario modificar el intervalo de seguimiento propuesto para el conjunto de los AA.

Background & Aims: Whereas it has conclusively been demonstrated that biallelic MutY human homolog (MYH) mutations confer a significant risk for colorectal cancer (CRC), the influence of monoallelic mutations remains controversial. Characterization of MYH-associated CRC is critical to identify individuals who might benefit from preventive strategies. This prospective, multicenter, case-control, population-based study was aimed at (1) establishing the CRC risk associated with specific germline MYH mutations and (2) devising a set of clinical criteria to identify MYH carriers among newly diagnosed CRC. Methods: Genotyping for Y165C and G382D was performed by TaqMan technology. Single-stranded conformation polymorphism analysis was performed in heterozygotes to screen for mutations in the entire gene. All individuals were re-screened for any additional pathogenic variant. Results: Biallelic and monoallelic MYH mutations were found in 8 (0.7%) and 19 (1.7%) of 1116 CRC patients, respectively. None of the 934 control subjects carried biallelic mutations, whereas 22 (2.3%) of them were monoallelic carriers. In a meta-analysis including all previous case-control studies, monoallelic MYH carriers were not at increased risk for CRC (odds ratio, 1.11; 95% confidence interval, 0.90–1.37), although a significant association was found with the Y165C mutation in either homozygotes or heterozygotes (odds ratio, 1.67; 95% confidence interval, 1.17–2.40). Furthermore, presence of more than 15 synchronous colorectal adenomas or CRC diagnosed before the age of 50 years was the most effective set of criteria for the identification of biallelic MYH mutation carriers. Conclusions: This study proposes the first set of clinical criteria designed to identify CRC patients with biallelic MYH mutations, and it argues against an increased risk for monoallelic carriers.

BACKGROUND: Colorectal cancer (CRC) is the second cause of cancer-related death in the Western world. Much of the CRC genetic risk remains unidentified and may be attributable to a large number of common, low-penetrance genetic variants. Genetic linkage studies in CRC families have reported additional association with regions 9q22-31, 3q21-24, 7q31, 11q, 14q and 22q. There are several plausible candidate genes for CRC susceptibility within the aforementioned linkage regions including PTCH1, XPA and TGFBR1 in 9q22-31, and EPHB1 and MRAS in 3q21-q24. METHODS: CRC cases and matched controls were from EPICOLON, a prospective, multicentre, nationwide Spanish initiative, composed of two independent phases. Phase 1 corresponded to 515 CRC cases and 515 controls, whereas phase 2 consisted of 901 CRC cases and 909 controls. Genotyping was performed for 172 single-nucleotide polymorphisms (SNPs) in 84 genes located within regions 9q22-31 and 3q21-q24. RESULTS: None of the 172 SNPs analysed in our study could be formally associated with CRC risk. However, rs1444601 (TOPBP1) and rs13088006 (CDV3) in region 3q22 showed interesting results and may have an effect on CRC risk. CONCLUSIONS: TOPBP1 and CDV3 genetic variants on region 3q22 may modulate CRC risk. Further validation and meta-analysis should be undertaken in larger CRC cohorts. British Journal of Cancer (2011) 105, 870-875. doi: 10.1038/bjc.2011.296 www.bjcancer.com Published online 2 August 2011, We are sincerely grateful to all patients participating in this study who were recruited in 25 (EPICOLON 1) and 14 (EPICOLON 2) Spanish hospitals as part of the EPICOLON project. We are also grateful to the Spanish National Genotyping Center (CEGEN-ISCIII)-USC and UPF nodes, and the Genome Analysis Platform of the CIC-BioGUNE. The work was carried out (in part) at the Esther Koplowitz Centre, Barcelona. SC-B, CF-R and MDG are supported by contracts from the Fondo de Investigacion Sanitaria (CP 03-0070 to SC-B, CM10/00084 to MDG, and PS09/02368 to CF-R). JM is supported by a contract from the CIBERehd. Both CIBERehd and CIBERER are funded by the Instituto de Salud Carlos III. This work was supported by grants from the Fondo de Investigacion Sanitaria/FEDER (06/1384, 08/0024, 08/1276, PS09/02368, 10/00918), Instituto de Salud Carlos III (Accion Transversal de Cancer), Xunta de Galicia (07PXIB9101209PR), Ministerio de Ciencia e Innovacion (SAF2010-19273), Asociacion Espanola contra el Cancer (Fundacion Cientifica y Junta de Barcelona), Fundacio Olga Torres (SCB and CRP) and FP7 CHIBCHA Consortium (LC-C, ACar and SC-B).

Purpose: Early-onset colorectal cancer (CRC) is suggestive of a hereditary predisposition. Lynch syndrome is the most frequent CRC hereditary cause. The MUTYH gene has also been related to hereditary CRC. A systematic characterization of these two diseases has not been reported previously in this population. Experimental Design: We studied a retrospectively collected series of 140 patients ≤50 years old diagnosed with nonpolyposis CRC. Demographic, clinical, and familial features were obtained. Mismatch repair (MMR) deficiency was determined by microsatellite instability (MSI) analysis, and immunostaining for MLH1, MSH2, MSH6, and PMS2 proteins. Germline MMR mutations were evaluated in all MMR-deficient cases. Tumor samples with loss of MLH1 or MSH2 protein expression were analyzed for somatic methylation. Germline MUTYH mutations were evaluated in all cases. BRAF V600E and KRAS somatic mutational status was also determined. Results: Fifteen tumors (11.4%) were MSI, and 20 (14.3%) showed loss of protein expression (7 for MLH1/PMS2, 2 for isolated MLH1, 3 for MSH2/MSH6, 7 for isolated MSH6, and 1 for MSH6/PMS2). We identified 11 (7.8%) germline MMR mutations, 4 in MLH1, 1 in MSH2, and 6 in MSH6. Methylation analysis revealed one case with somatic MLH1 methylation. Biallelic MUTYH mutations were detected in four (2.8%) cases. KRAS and BRAF V600E mutations were present in 39 (27.9%) and 5 (3.6%) cases, respectively. Conclusions: Loss of MSH6 expression is the predominant cause of MMR deficiency in early-onset CRC. Our findings prompt the inclusion of MSH6 and MUTYH screening as part of the genetic counseling of these patients and their relatives. Clin Cancer Res; 16(22); 5402–13. ©2010 AACR.

BackgroundColorectal cancer (CRC) multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect.Methodology/principal findingsWe examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals) and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2), RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008) and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047) as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006). Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17), SFRP1 (r = 0.83, 0.06), HPP1 (r = 0.64, p = 0.17), 3OST2 (r = 0.83, p = 0.06) and GATA4 (r = 0.6, p = 0.24). Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant difference in any evaluated gene.ConclusionsThese results provide a proof-of-concept that gene promoter methylation is associated with tumor multiplicity. This underlying epigenetic defect may have noteworthy implications in the prevention of patients with sporadic CRC.

Background & Aims Ten common low-penetrant genetic variants have been consistently associated with colorectal cancer (CRC) risk; little is known about the correlation between these variants and CRC phenotype. Characterization of such a correlation would improve CRC management and prevention programs. We assessed the association between these genetic variants and CRC phenotype in patients and modeled pairwise combinations to detect epistasis. Methods The validation population corresponded to a prospective, multicenter, population-based cohort (EPICOLON I) of 1096 patients with newly diagnosed CRC. The replication set was an independent, prospective, multicenter Spanish cohort (EPICOLON II) of 895 patients with newly diagnosed CRC. For individual single nucleotide polymorphism (SNP) association analyses, a multivariate method using logistic regression was applied in EPICOLON I and subsequently prospectively validated in EPICOLON II. Interactions between SNPs were assessed using the likelihood ratio test. Results Validated results confirmed that the C allele on 8q23.3 (rs16892766) was significantly associated with advanced-stage tumors (odds ratio [OR], 1.48; 95% confidence interval [CI], 1.15–1.90; P value=4.9 × 10 −3 ). The G allele on 8q24.21 (rs6983267) was more common in patients with a familial history of CRC (OR, 2.02; 95% CI, 1.35–3.03; P value=3.9 × 10 −4 ). The combination of rs6983267 on 8q24.21 and rs9929218 on 16q22.2 was associated with a history of colorectal adenoma (carriers of GG and AA, respectively; OR, 2.28; 95% CI, 1.32–3.93; P = 5.0 × 10 −4 ). Conclusions CRC susceptibility variants at 8q23.3, 8q24.21, and 16q22.2 appear to be associated with cancer phenotype. These findings might be used to develop screening and surveillance strategies.

In this paper we describe the history of lithiasis and the development of its treatment. We discuss the steps that led to the birth of endoscopic surgery and extracorporeal shockwave lithotripsy.We reviewed books and writings of History of Urology and Endourology.To know a little of the history and treatment of lithiasis, the contributions of Spanish urologists, as well as the instruments that made it possible.

Background Inflammatory bowel disease is a premalignant condition for developing colorectal cancer. Since a correlation has been suggested between telomere length, chromosomal instability and neoplastic transformation in this setting, we sought to investigate whether telomerase expression in colorectal mucosa may constitute a biomarker for malignant transformation in patients with inflammatory bowel disease. Patients and methods Forty-seven patients with inflammatory bowel disease with and without cancer or dysplasia were evaluated for human telomerase reverse transcriptase hTERT immunostaining in paraffin-embedded, formalin-fixed colorectal tissues. In addition, hTERT mRNA expression was assessed in fresh frozen specimens from a second set of 35 patients with inflammatory bowel disease at high or low risk for neoplastic transformation. Results Five out of 10 patients (50%) with colorectal cancer or high-grade dysplasia exhibited hTERT immunochemical detection in adjacent, non-transformed colonic mucosa. However, this phenomenon was also observed in non-affected mucosa of patients with either long-standing (13 out of 19 patients; 68%) or short duration (13 out of 18 patients; 72%) disease without cancer or dysplasia. On the other hand, hTERT mRNA expression in non-affected colorectal mucosa from patients at high risk for neoplastic transformation due to long-standing disease was higher than in those at low risk (7.42±6.43 vs. 2.87±1.47, respectively; p =0.006). Conclusions Whereas hTERT immunostaining provides equivocal results, the observation that patients at high risk for colorectal cancer because of long-standing inflammatory bowel disease overexpress hTERT mRNA in non-affected colorectal mucosa suggests its potential usefulness as a biomarker of the risk of malignant transformation.

En este trabajo se realiza una descripcion historica de los descubrimientos sobre la litiasis y la evolucion de su tratamiento. Se exponen los antecedentes que llevan al nacimiento de la endourologia y de la litotricia por ondas de choque. METODOS Hemos revisado los libros y las publicaciones de la historia de la urologia y de la endourologia. RESULTADOS Conocer la historia y el tratamiento de la litiasis, las aportaciones de los urologos espanoles asi como el desarrollo de los materiales que han contribuido a conseguirlo. CONCLUSIONES Conocer la historia y el tratamiento de la litiasis, las aportaciones de los urologos espanoles asi como el desarrollo de los materiales que han contribuido a conseguirlo.

The objective of this work is to analyze the treatment of staghorn calculi at our Department of Urology. We have to know the recent development of endoscopic surgery (percutaneous renal surgery) and external shock wave lithotripsy.We reviewed the surgical management of staghorn calculi during the period between 1987 and 2004.Percutaneous renal surgery was performed successfully in 24.1% of the cases. Persisting residual fragments appeared in 75.9% and were treated by ESWL or second endoscopic surgery.Percutaneous renal surgery may be considered the technique of choice to treat staghorn calculi. Endoscopic surgery has good results and little complications with low morbidity. In other cases the treatment is combined therapy, percutaneous renal surgery and ESWL.

The goal of this work is to evaluate the usefulness of NMP22 BladderChek in the diagnosis and follow-up of bladder cancer, comparing it with cystoscopy and urine cytology.Group 1: 109 asymptomatic patients on follow up for bladder cancer underwent cystoscopy, cytology and NMP22 BladderChek. Group 2:15 patients with history of hematuria underwent cystoscopy and NMP22 BladderChek.Group 1: 9 patients had tumor relapse. Sensitivity was 25% for NMP22 test, 50% for citology and 100% for cystoscopy Specificity was 91.1%, 94.1% and 95% respectively. Group 2: 12 patients had bladder cancer. The sensitivity was of 83.3% for NMP22 BladderChek and 100% for cystoscopy. The specificity was of 100% for NMP22 BladderChek and 66.7% for cystoscopy.The low sensitivity of NMP22 Bladder-Chek invalidates it as alternative method to cystoscopy in the follow-up of bladder cancer. But it can be recommended for screening in patients without history of bladder cancer but with an increased risk (smokers, patients with dysuria and hematuria).

Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer, is the most common form of hereditary colorectal cancer. It is characterized by early onset of colorectal cancer and other extracolonic-associated malignancies. This disorder is inherited in an autosomal dominant pattern and is due to a mutation in one of the DNA mismatch repair genes. Although clinical and molecular understanding of the syndrome has progressed dramatically in the last decade, diagnosis of the syndrome is still a clinical challenge. This review summarizes the main features of the syndrome and provides an update of its management.

Objetivo: Evaluar la utilidad del NMP22 BladderChek para el diagnóstico y el seguimiento del tumor vesical comparándolo con la citología y la cistoscopia. Métodos: Grupo 1 formado por 109 pacientes asintomáticos en seguimiento por cáncer de vejiga a los que realizamos citología, cistoscopia y el test NMP22. Grupo 2 constituido por 15 pacientes que consultan por hematuria. Se realiza cistoscopia y el test NMP22. Resultados: Grupo1, 9 pacientes presentaron recidiva vesical. La sensibilidad fue del 25% para el NMP22 BladderChek, del 50% para la citología y del 100% para la cistoscopia. La especificidad fue del 91.1% para el NMP22, del 94.1% para la citología y del 95% para la cistoscopia. Grupo 2, de los 15 pacientes 12 (80%) presentaron tumoración vesical. La sensibilidad del NMP22 fue del 83.3% y del 100% para la cistoscopia. La especificidad fue del 100% para el NMP22 y del 66.7% para la cistoscopia. Conclusiones: El NMP22 BladderChek no nos permite distanciar la realización de las cistoscopias en el seguimiento de los tumores vesicales, ya que presenta una baja sensibilidad. Recomendamos sin embargo su uso como screening de tumor vesical en pacientes de riesgo (aquellos que presentan hematuria, disuria y son fumadores).

The pathogenesis of cancer includes both genetic and epigenetic mechanisms. The termepigeneticrefers to any alteration of gene expression, potentially hereditary, which is not accompanied by a modification in the DNA sequence. The effects of epigenetic changes include alteration of DNA transcription, aberrant activation of specific genes, predisposition of genetic instability through alteration of the control of chromosome replication and silencing of the genes implicated in cancer initiation and progression. Among the various epigenetic alterations that lead to altered gene expression, methylation is the main epigenomic mechanism implicated in cancer, whether through a phenomenon of overall hypomethylation or hypermethylation localized in the promoters of specific genes.

Objetivo: Analizar los resultados del tratamiento de la litiasis coraliforme en nuestro servicio atendiendo a los avances tecnológicos que se han producido en los últimos años destacando el papel fundamental que supone el desarrollo de la cirugía endoscópica y de la litotricia extracorpórea por ondas de choque. Métodos: Hemos revisado todos los pacientes tratados de litiasis coraliforme en nuestro servicio entre los años 1987 y 2004. Resultados: Se consiguió la extracción completa con un sólo acto quirúrgico mediante nefrolitotomía percutánea en un 24.1%. En el 75.9% restante se realizó un tratamiento combinado mediante litotricia extracorpórea con ondas de choque o una segunda nefrolitotomía percutánea. Conclusiones: La endourología ha revolucionado el tratamiento de la litiasis coraliforme. Es eficaz sola y en combinación con la LEOC para el manejo de esta patología con un número reducido de complicaciones y una alta tolerabilidad por parte de los pacientes.

To show the beginning of spinal and epidural anesthesia in our country and the contributions of Spanish urologists.We reviewed books and writings of History of Medicine, Urology and Anesthesia and Doctoral thesis about spinal and epidural anesthesia.In the 20th century, surgeons also gave the anesthetic drugs to the patients. Spinal and epidural anesthesia were used for the first time in 1900. A lot of Spanish urologists like F Rusca Doménech, J.M. Batrina, M. Barragán Bonet, R. Lozano Monzón, L. Guedea Calvo, Gil Vernet, Fidel Pagés Miravé, V Sagarra Lascurain, Gómez Ulla, etc, did research, writings in scientific journals and Doctoral thesis about anesthesia.

Objetivo: Conocer como se desarrollaron en nuestro país las técnicas de la raquianestesia y la anestesia epidural, y cuales fueron las aportaciones de los urólogos españoles. Métodos: Hemos revisado los libros de Historia de la Medicina, de la Urología y de Anestesia, las publicaciones periódicas y las Tesis Doctorales que sobre este tema se realizaron en la época. Resultados: En buena parte del siglo XX aquellos que se dedicaban a la cirugía administraban también la anestesia. Es a partir del año 1900 cuando comienzan a desarrollarse la raquianestesia y la anestesia epidural siendo numerosas y fundamentales las aportaciones de los urólogos españoles como F. Rusca Doménech, J.M. Batrina, M. Barragán Bonet, R. Lozano Monzón, L. Guedea Calvo, Gil Vernet, Fidel Pagés Miravé, V. Sagarra Lascuraín, Gómez Ulla, etc. realizando publicaciones en revistas de prestigio, comunicaciones en congresos y tesis doctorales sobre la anestesia raquídea y epidural.

En un estudio poblacional previo en Espana (Eur J Gastroenterol Hepatol 2004; 16:39–45), el diagnostico clinico de CCHNP y otras formas familiares de CCR se observo en un 2,5% y 27%, respectivamente. Mientras el CCHNP es una enfermedad bien caracterizada, existen pocos datos sobre las caracteristicas de los pacientes con CCR familiar no sindromico. Objetivo Comparar las caracteristicas epidemiologicas del CCHNP respecto a otras formas familiares de CCR en nuestro medio. Metodos Estudio prospectivo, multicentrico de ambito nacional (EPICOLON-II). Se registraron todos los pacientes con CCR diagnosticado de novo atendidos en el periodo de Junio 2006 a Diciembre 2007 en hospitales de ambito nacional. Se recogieron de forma sistematica las caracteristicas demograficas, clinicas, relativas al tumor del probando, asi como una detallada historia familiar Resultados Los resultados corresponden a los primeros 699 casos de CCR de 10 hospitales con una historia familiar completa. El diagnostico clinico de CCHNP se establecio en 9 (1,3%) pacientes en base a los criterios de Amsterdam II. La comparacion entre los pacientes que cumplian los criterios de Amsterdam I o II no mostro diferencia alguna en relacion a las caracteristicas demograficas, clinicas o en relacion al tumor. Un total de 189 (26,8%) pacientes cumplian los criterios revisados de Bethesda. Todos los CCHNP cumplian al menos uno de los criterios de Bethesda. Del total de pacientes, 145 (20,7%) presentaban una historia familiar de CCR y 543 (78%) pacientes no presentaban ninguna historia familiar de CCR, representando el grupo de CCR esporadico. Estos grupos se diferenciaban en la localizacion colon derecho del tumor (p Conclusion Estos datos clinico-epidemiologicos aportan informacion adicional en la caracterizacion del CCHNP y otras formas de CCR familiar en Espana. De forma peculiar, la localizacion del CCR y de lesiones sincronicas adenomatosas pueden sugerir la existencia de una forma familiar de CCR.

Introduccion La multicentricidad en el cancer colorrectal (CCR) se ha relacionado principalmente con los sindromes hereditarios poliposicos y no poliposicos. Sin embargo, hay escasa evidencia de la implicacion de otros factores moleculares en la multicentricidad del CCR, sobre todo de la metilacion genica aberrante. Objetivo Evaluar la posible implicacion de la hipermetilacion genica en la multicentricidad del CCR, determinando el grado de metilacion en la region promotora de genes supresores de tumor implicados en el CCR, como prueba de la existencia de un posible defecto de campo o “field defect” subyacente. Pacientes y metodos Se incluyeron 82 pacientes, 41 con CCR sincronico o metacronico y 41 con CCR unico, apareados por edad y sexo, unos pertenecientes al proyecto Epicolon I y otros recogidos de forma prospectiva en el Servicio de Endoscopia del Hospital Clinic. Los pacientes con sindromes poliposicos, sindrome de Lynch o enfermedad inflamatoria intestinal fueron excluidos. Se extrajo DNA a partir de tejido congelado de mucosa tumoral y mucosa aparentemente sana correspondiente de cada individuo. Tras realizar un primer tratamiento bisulfito, se cuantifico la presencia de metilacion en la region promotora de 7 genes supresores de tumor: MGMT1 (region promotora), MGMT2 (region potenciadora), p16, sFRP1, HPP1, 3OST2, RASSF1A y GATA-4 mediante PCR a tiempo real (tecnica “Methylight”). El analisis de los datos fue realizado de forma cuantitativa y cualitativa mediante analisis univariante y multivariante no parametricos, utilizando como punto de corte el valor de PMR de 4, relacionado con la silenciacion del gen afecto. Resultados El grado de metilacion de todos los genes estudiados fue mayor en el tumor de los pacientes con CCR multiple que en el de los pacientes con CCR unico, siendo esta diferencia estadisticamente significativa para los genes MGMT1 (p=0,026), MGMT2 (p=0,001) y RASSF1A (p=0,01), y permaneciendo significativa tras ajustar por edad y sexo como posibles factores de confusion. El analisis cualitativo de los datos (punto de corte PMR=4) tambien revelo una mayor frecuencia de hipermetilacion en el tumor de los pacientes con CCR multiple comparado con el tumor de aquellos con CCR unico por lo que respecta a MGMT2 (40% vs. 14%, respectivamente; p=0,007), RASSF1A (17% vs. 0%, respectivamente; p=0,006) y GATA-4 (81% vs. 61% respectivamente; p=0,04), permaneciendo significativo tras ajustar por edad y sexo. De forma similar, el grado de metilacion en la mucosa normal correspondiente al tumor de los pacientes con CCR multiple fue mayor que en la mucosa normal de los pacientes con CCR unico en MGMT1, RASSF1A y p16, aunque no alcanzo significacion estadistica. Conclusion Los resultados de este estudio indican que la hipermetilacion genica se asocia a la multicentricidad en el CCR. Este potencial defecto de campo podria tener notables implicaciones en la vigilancia de los pacientes con formas no hereditarias de CCR.

Introduccion El cancer colorrectal (CCR) de debut precoz (⩽50 anos) es sugestivo de predisposicion hereditaria. El sindrome de Lynch, causado por mutaciones germinales en los genes de reparacion del DNA ( MLH1 , MSH2 , MSH6 y PMS2 ), es la forma mas frecuente de CCR hereditario. Sin embargo, unicamente un pequeno subgrupo de pacientes con CCR de debut precoz corresponde a esta patologia. Por ello, es fundamental identificar las bases moleculares del CCR en esta poblacion y asi poder establecer estrategias preventivas adecuadas. Objetivos Evaluar las caracteristicas clinicas, histologicas y moleculares de los pacientes con CCR de debut precoz, y analizar la prevalencia de alteracion del sistema de reparacion del DNA en esta poblacion. Pacientes y metodos Se seleccionaron de forma retrospectiva todos los pacientes con diagnostico de CCR a una edad ⩽50 anos que fueron tratados quirurgicamente en 2 centros espanoles entre los anos 95-07. Se recogieron datos demograficos, clinicos y familiares. Se realizo extraccion de DNA de tejido normal y tumoral parafinado de cada paciente. Se analizo inestabilidad de microsatelites (MSI) con el panel de mononucleotidos (BAT25, BAT26, NR21, NR24 y MONO27). Se definio inestabilidad alta (MSI-H) como >2/5 marcadores inestables y estabilidad (MSS) como ⩽2 marcadores inestables. La expresion de las proteinas MLH1, MSH2 y MSH6 fue valorada mediante inmunohistoquimica. BRAFV600E y los hotspots del exon 2 de KRAS se analizaron mediante sondas TaqMan alelo-especificas y secuenciacion directa, respectivamente. Resultados Se incluyeron 83 pacientes con una edad media de 44 anos (rango 23–50); 46 (56%) hombres. El 86% de los tumores estaban localizados en colon izquierdo, incluyendo 39% en el recto. Nueve tumores (11%) mostraron MSI-H y 13 (16%) perdida de expresion proteica (4 para MLH1, 2 para MSH2/MSH6 y 7 para MSH6 de forma aislada). Globalmente, 13 (16%) pacientes mostraron alteracion del sistema de reparacion del DNA, definida por perdida de expresion proteica y/o MSI-H. Todos los tumores con perdida de MLH1 o MSH2 mostraron MSI-H y solo 4 de 7 (60%) de los tumores con perdida aislada de MSH6 mostraron MSI-H. Ademas, se detecto la presencia de mutaciones en KRAS y BRAF en 16 (19,5%) y 3 (3,7%) casos, respectivamente. Las mutaciones de BRAF no se asociaron con perdida de expresion de MLH1. Conclusiones La alteracion del sistema de reparacion del DNA justifica menos del 20% de los casos de CCR de debut precoz. Esta cohorte muestra un perfil molecular de alteracion del sistema de reparacion diferente, caracterizado por perdida aislada de MSH6, baja sensibilidad del analisis de MSI con marcadores mononucleotidos, y baja frecuencia de mutaciones en la via RAS/RAF/ERK. Estas observaciones sugieren una alta frecuencia de mutaciones germinales en MSH6 y cuestionan la realizacion de MSI con el panel de mononucleotidos como metodo de cribado en esta cohorte.

Background Colorectal cancer (CRC) is the second leading cause of cancer death in developed countries. Familial aggregation in CRC is also important outside syndromic forms and, in this case, a polygenic model with several common low-penetrance alleles contributing to CRC genetic predisposition could be hypothesized. Mucins and GALNTs (N-acetylgalactosaminyltransferase) are interesting candidates for CRC genetic susceptibility and have not been previously evaluated. We present results for ten genetic variants linked to CRC risk in previous studies (previously identified category) and 18 selected variants from the mucin gene family in a case-control association study from the Spanish EPICOLON consortium. Methods CRC cases and matched controls were from EPICOLON, a prospective, multicenter, nationwide Spanish initiative, comprised of two independent stages. Stage 1 corresponded to 515 CRC cases and 515 controls, whereas stage 2 consisted of 901 CRC cases and 909 controls. Also, an independent cohort of 549 CRC cases and 599 controls outside EPICOLON was available for additional replication. Genotyping was performed for ten previously identified SNPs in ADH1C, APC, CCDN1, IL6, IL8, IRS1, MTHFR, PPARG, VDR and ARL11, and 18 selected variants in the mucin gene family. Results None of the 28 SNPs analyzed in our study was found to be associated with CRC risk. Although four SNPs were significant with a P-value < 0.05 in EPICOLON stage 1 [rs698 in ADH1C (OR = 1.63, 95% CI = 1.06-2.50, P-value = 0.02, recessive), rs1800795 in IL6 (OR = 1.62, 95% CI = 1.10-2.37, P-value = 0.01, recessive), rs3803185 in ARL11 (OR = 1.58, 95% CI = 1.17-2.15, P-value = 0.007, codominant), and rs2102302 in GALNTL2 (OR = 1.20, 95% CI = 1.00-1.44, P-value = 0.04, log-additive 0, 1, 2 alleles], only rs3803185 achieved statistical significance in EPICOLON stage 2 (OR = 1.34, 95% CI = 1.06-1.69, P-value = 0.01, recessive). In the joint analysis for both stages, results were only significant for rs3803185 (OR = 1.12, 95% CI = 1.00-1.25, P-value = 0.04, log-additive 0, 1, 2 alleles) and borderline significant for rs698 and rs2102302. The rs3803185 variant was not significantly associated with CRC risk in an external cohort (MCC-Spain), but it still showed some borderline significance in the pooled analysis of both cohorts (OR = 1.08, 95% CI = 0.98-1.18, P-value = 0.09, log-additive 0, 1, 2 alleles). Conclusions ARL11, ADH1C, GALNTL2 and IL6 genetic variants may have an effect on CRC risk. Further validation and meta-analyses should be undertaken in larger CRC studies.

Lynch syndrome, also known as hereditary non-polyposis colorectal cancer (HNPCC), is the most common form of hereditary colorectal cancer. Although great advances in the understanding of its molecular basis have taken place in the last decade, optimal selection of individuals for HNPCC genetic testing remains controversial. This is especially relevant since colonoscopy has been proven effective for reducing colorectal cancer incidence and mortality in individuals at-risk for this disorder. In this manuscript, we summarize the most significant contributions to this important issue that have appeared in the last few years.