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PDF file - 58K, Effect of Spn shRNA on human CCR tumor cell lines

PDF file - 77K, Hypermethylation analysis of Spn promoter in human CCR tumor samples

PDF file - 49K, Table 1: Clinical and pathologic features of patients in Cohort 1 (Stage II-III CRC); Table 2: Clinical and pathologic features of patients in Cohort 2 (stage IV CRC)

Purpose: The genomic region 17q21 is frequently associated with microsatellite instability and LOH in cancer, including gastric and colorectal carcinomas. This region contains several putative tumor suppressor genes, including Brca1, NM23, prohibitin, and spinophilin (Spn, PPP1R9B, neurabin II). The scaffold protein Spn is one of the regulatory subunits of phosphatase-1 (PP1) that targets PP1 to distinct subcellular locations and couples PP1 to its target. Thus, Spn may alter cell-cycle progression via the regulation of the phosphorylation status of the retinoblastoma protein, a direct target of PP1. Therefore, we analyzed whether Spn levels were reduced in colorectal carcinomas and whether Spn levels correlated with prognosis or response to therapy.Experimental Design: By means of immunohistochemistry or quantitative PCR, we studied the levels of Spn in stages II, III, and IV colorectal carcinoma tumors and correlated to other clinicopathologic features as well as prognosis or response to therapy.Results: Spn was lost in a percentage of human gastric, small intestine, and colorectal carcinomas. In patients with colorectal carcinoma, tumoral Spn downregulation correlated with a more aggressive histologic phenotype (poorer tumor differentiation and higher proliferative Ki67 index). Consistent with this observation, lower Spn protein expression levels were associated with faster relapse and poorer survival in patients with stage III colorectal carcinoma, particularly among those receiving adjuvant fluoropyrimidine therapy. We validated this result in an independent cohort of patients with metastatic colorectal carcinoma treated with standard chemotherapy. Although patients that achieved an objective tumor response exhibited Spn levels similar to nontumoral tissue, nonresponding patients showed a significant reduction in Spn mRNA levels.Conclusions: Our data suggest that Spn downregulation contributes to a more aggressive biologic behavior, induces chemoresistance, and is associated with a poorer survival in patients with advanced stages of colorectal carcinoma. Clin Cancer Res; 19(14); 3925–35. ©2013 AACR.

[Background]: The treatment of celiac disease (CD) is a lifelong gluten-free diet (GFD). The current methods for monitoring GFD conformance, such as a dietary questionnaire or serology tests, may be inaccurate in detecting dietary transgressions, and duodenal biopsies are invasive, expensive, and not a routine monitoring technique., [Objectives]: Our aim was to determine the clinical usefulness of urine gluten immunogenic peptides (GIP) as a biomarker monitoring GFD adherence in celiac patients and to evaluate the concordance of the results with the degree of mucosal damage., [Methods]: A prospective observational study was conducted involving 22 de novo CD patients, 77 celiac patients consuming a GFD, and 13 nonceliac subjects. On 3 d of the week, urine samples were collected and the GIP concentrations were tested. Simultaneously, anti-tissue transglutaminase antibodies, questionnaire results, clinical manifestations, and histological findings were analyzed., [Results]: Approximately 24% (18 of 76) of the celiac patients consuming a GFD exhibited Marsh II–III mucosal damage. Among this population, 94% (17 of 18) had detectable urine GIP; however, between 60% and 80% were asymptomatic and exhibited negative serology and appropriate GFD adherence based on the questionnaire. In contrast, 97% (31 of 32) of the celiac patients without duodenal damage had no detectable GIP. These results demonstrated the high sensitivity (94%) and negative predictive value (97%) of GIP measurements in relation to duodenal biopsy findings. In the de novo CD-diagnosed cohort, 82% (18 of 22) of patients had measurable amounts of GIP in the urine., [Conclusions]: Determining GIP concentrations in several urine samples may be an especially convenient approach to assess recent gluten exposure in celiac patients and appears to accurately predict the absence of histological lesions. The introduction of GIP testing as an assessment technique for GFD adherence may help in ascertaining dietary compliance and to target the most suitable intervention during follow-up., Supported by Ministerio de Economía, Industria y Competitividad and FEDER funds grant SAF2017-83700-R (to CS) and Junta de Andalucía grant PI-0427-2017 (to ÁP).

Canonical prefoldin is a protein cochaperone composed of six different subunits (PFDN1 to 6). PFDN1 overexpression promotes epithelial–mesenchymal transition (EMT) and increases the growth of xenograft lung cancer (LC) cell lines. We investigated whether this putative involvement of canonical PFDN in LC translates into the clinic. First, the mRNA expression of 518 non-small cell LC (NSCLC) cases from The Cancer Genome Atlas (TCGA) database was evaluated. Patients with PFDN1 overexpression had lower overall survival (OS; 45 vs. 86 months; p = 0.034). We then assessed the impact of PFDN expression on outcome in 58 NSCLC patients with available tumor tissue samples. PFDN1, 3, and 5 overexpression were found in 38% (n = 22), 53% (n = 31), and 41% (n = 24) of tumor samples. PFDN1, 3, and 5 overexpression were significantly associated with lower OS, lower disease-free survival (DFS), and lower distant metastasis-free survival (DMFS) for PFDN1 and 3 with a trend for PFDN5. In multivariate analysis, PFDN5 retained significance for OS (hazard ratio (HR) 2.56; p = 0.007) and PFDN1 for DFS (HR 2.53; p = 0.010) and marginally for DMFS (HR 2.32; p = 0.053). Our results indicate that protein response markers, such as PFDN1, 3, and 5, may complement mRNA signatures and be useful for determining the most appropriate therapy for NSCLC patients., This work was co-funded by the Spanish Ministry of Economy and Competitiveness (BFU2017-85420-R to JCR and BFU2016-7772-C3-1-P to SC) and by the Andalusian Government (BIO-271) and the University of Seville (US-1256285 to SC) in cooperation with the European Regional Development Fund.

Human epidermal growth factor receptor 2 (HER2) dysregulation is associated with tumorigenesis in gastric/gastroesophageal junction cancer; however, the number of patients with HER2-positive disease is unclear, possibly due to differing scoring criteria/assays. Data are also lacking for early disease. We aimed to assess the HER2-positivity rate using approved testing criteria in a large, real-life multinational population. HER2-positivity was defined as an immunohistochemistry staining score of 3+, or immunohistochemistry 2+ and HER2 amplification detected by in situ hybridization. A total of 4949 patients were enrolled and results showed that 14.2% of 4920 samples with immunohistochemistry results were HER2-positive. HER2-positivity was significantly higher in males (16.1% vs. 9.6% in females), in gastroesophageal versus stomach tumors (22.1% vs. 12.9%), in biopsy versus surgical samples (18.3% vs. 13.0%), in intestinal tumor subtypes versus diffuse (21.5% vs. 4.8%) and mixed types (21.5% vs. 8.5%) (P

[Background] Conflicting data exist on the role of pulmonary dendritic cells (DCs) and their maturation in patients with chronic obstructive pulmonary disease (COPD). Herein, we investigated whether disease severity and smoking status could affect the distribution and maturation of DCs in lung tissues of patients undergoing elective pneumectomy or lobectomy for suspected primary lung cancer., [Materials and Methods] A total of 75 consecutive patients were included. Spirometry testing was used to identify COPD. Lung parenchyma sections anatomically distant from the primary lesion were examined. We used flow cytometry to identify different DCs subtypes—including BDCA1-positive myeloid DCs (mDCs), BDCA3-positive mDCs, and plasmacytoid DCs (pDCs)—and determine their maturation markers (CD40, CD80, CD83, and CD86) in all participants. We also identified follicular DCs (fDCs), Langerhans DCs (LDCs), and pDCs in 42 patients by immunohistochemistry., [Results] COPD was diagnosed in 43 patients (16 current smokers and 27 former smokers), whereas the remaining 32 subjects were classified as non-COPD (11 current smokers, 13 former smokers, and 8 never smokers). The number and maturation of DCs did not differ significantly between COPD and non-COPD patients. However, the results of flow cytometry indicated that maturation markers CD40 and CD83 of BDCA1-positive mDCs were significantly decreased in smokers than in non-smokers (P = 0.023 and 0.013, respectively). Immunohistochemistry also revealed a lower number of LDCs in COPD patients than in non-COPD subjects., [Conclusions] Cigarette smoke, rather than airflow limitation, is the main determinant of impaired DCs maturation in the lung., This study was supported by a grant from the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Fondo de Investigación Sanitaria (project PS09/00826).

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License.-- et al., The paired box transcription factor Pax8 is critical for development of the eye, thyroid gland as well as the urinary and reproductive organs. In adult, Pax8 overexpression is associated with kidney, ovarian and thyroid tumors and has emerged as a specific marker for these cancers. Recently, Pax8 expression was also reported in human pancreatic islets and in neuroendocrine tumors, identifying Pax8 as a novel member of the Pax family expressed in the pancreas. Herein, we sought to provide a comprehensive analysis of Pax8 expression during pancreogenesis and in adult islets. Immunohistochemical analysis using the most employed Pax8 polyclonal antibody revealed strong nuclear staining in the developing mouse pancreas and in mature human and mouse islets. Astonishingly, Pax8 mRNA in mouse islets was undetectable while human islets exhibited low levels. These discrepancies raised the possibility of antibody cross-reactivity. This premise was confirmed by demonstrating that the polyclonal Pax8 antibody also recognized the islet-enriched Pax6 protein both by Western blotting and immunohistochemistry. Thus, in islets polyclonal Pax8 staining corresponds mainly to Pax6. In order to circumvent this caveat, a novel Pax8 monoclonal antibody was used to re-evaluate whether Pax8 was indeed expressed in islets. Surprisingly, Pax8 was not detected in neither the developing pancreas or in mature islets. Reappraisal of pancreatic neuroendocrine tumors using this Pax8 monoclonal antibody exhibited no immunostaining as compared to the Pax8 polyclonal antibody. In conclusion, Pax8 is not expressed in the pancreas and cast doubts on the value of Pax8 as a pancreatic neuroendocrine tumor marker., This work was supported by grants from the Fundacion Progreso y Salud (to B.R.G.), the Spanish Ministry of Science and Innovation, Instituto de Salud Carlos III (PI10/00871 to B.R.G) and the Juvenile Diabetes Research Foundation (9-2004-384 to E.C.I.T Plan Nacional de I+D+I 2008-2011, ISCIII (PI08/0018) co-funded by Fondos FEDER (to A.R.) and Consejería de Salud, Junta de Andalucía (PI0008 to A.R.).

To determine whether the different tumour necrosis factor alpha (TNF-alpha) promoter gene polymorphisms are involved in the development of steatosis in chronic hepatitis C.One hundred and thirty patients (89 men and 41 women; mean age 42.5 +/- 12.3 years) with chronic hepatitis C were included. Insulin resistance was measured according to the Homeostasis model assessment (HOMA IR). Serum leptin levels were also obtained and the body mass index and fat mass were calculated. Liver biopsy was carried out in all the patients, and steatosis was measured as one of four stages (0 to 3): stage 0, no steatosis; stage 1,25% of hepatocytes with steatosis; stage 2, 25-50%; and stage 3,50%. DNA samples were obtained in order to describe the polymorphisms at the TNF-alpha promoter gene position.Fifty-nine of the 130 (45.38%) patients had different degrees of steatosis, while 71/130 (54.62%) were not steatosic. Six of the 59 (10.2%) patients with steatosis presented mutations at the -238 position of the TNF-alpha promoter region, while 5/71 (7.0%) patients without steatosis also showed mutations at this position (P=NS). Seventeen of the 59 (28.8%) steatosic patients showed a mutation at the -308 position, while 16/71 (22.5%) without steatosis also had this mutation (P=NS). Insulin resistance, beta cells reserve, insulin and leptin levels showed no differences between patients with or without mutations at the promoter region of the TNF-alpha gene.TNF-alpha mutations do not seem to play any role in the development of steatosis in chronic hepatitis C.

[Purpose+ The genomic region 17q21 is frequently associated with microsatellite instability and LOH in cancer, including gastric and colorectal carcinomas. This region contains several putative tumor suppressor genes, including Brca1, NM23, prohibitin, and spinophilin (Spn, PPP1R9B, neurabin II). The scaffold protein Spn is one of the regulatory subunits of phosphatase-1 (PP1) that targets PP1 to distinct subcellular locations and couples PP1 to its target. Thus, Spn may alter cell-cycle progression via the regulation of the phosphorylation status of the retinoblastoma protein, a direct target of PP1. Therefore, we analyzed whether Spn levels were reduced in colorectal carcinomas and whether Spn levels correlated with prognosis or response to therapy., [Experimental Design] By means of immunohistochemistry or quantitative PCR, we studied the levels of Spn in stages II, III, and IV colorectal carcinoma tumors and correlated to other clinicopathologic features as well as prognosis or response to therapy., [Results] Spn was lost in a percentage of human gastric, small intestine, and colorectal carcinomas. In patients with colorectal carcinoma, tumoral Spn downregulation correlated with a more aggressive histologic phenotype (poorer tumor differentiation and higher proliferative Ki67 index). Consistent with this observation, lower Spn protein expression levels were associated with faster relapse and poorer survival in patients with stage III colorectal carcinoma, particularly among those receiving adjuvant fluoropyrimidine therapy. We validated this result in an independent cohort of patients with metastatic colorectal carcinoma treated with standard chemotherapy. Although patients that achieved an objective tumor response exhibited Spn levels similar to nontumoral tissue, nonresponding patients showed a significant reduction in Spn mRNA levels., [Conclusions] Our data suggest that Spn downregulation contributes to a more aggressive biologic behavior, induces chemoresistance, and is associated with a poorer survival in patients with advanced stages of colorectal carcinoma. © 2013 American Association for Cancer Research., This work was supported by grants from the Spanish Ministry of Science and Innovation (SAF2009-08605), Fondo de Investigacion Sanitaria (PI12/00137), Consejeria de Ciencia e Innovacion, and Consejeria de Salud of the Junta de Andalucia (CTS-6844 and PI-0142). A. Carnero's laboratory is also funded by a fellowship from Fundacion Oncologica FERO. P. Estevez-Garcia and I. Lopez-Calderero are supported by Rio Ortega Fellowships and S. Molina-Pinelo is supported by a Sara Borrell fellowship. R. Garcia-Carbonero is funded by the Instituto de Salud Carlos III, Ministerio de Sanidad, Spain (PI 10.02164).

Objectives: To elucidate the role of epithelial-mesenchymal transition markers in gastroenteropancreatic neuroendocrine tumors (GEP NETs) and the potential usefulness in their clinical management. Methods: One hundred ten GEP NET paraffin-embedded samples were immunohistochemically analyzed for E-cadherin, N-cadherin, β-catenin, vimentin, Snail1, Snail2, Twist, and Foxc2 protein expression. Results: The 5-year survival rate was reduced for those patients showing high Snail1 protein levels, a cytoplasmic E-cadherin pattern, reduced N-cadherin expression, and loss of E-cadherin/β-catenin adhesion complex integrity at the cell membrane. Interestingly, high β-catenin expression was useful in identifying a grade 1 NET subgroup with a favorable clinical course. Importantly, it also helped to discriminate small-cell vs large-cell grade 3 neuroendocrine carcinomas. Conclusions: β-Catenin and N-cadherin immunohistochemical detection might be a useful tool in the differential diagnosis of small-cell vs large-cell G3 neuroendocrine carcinomas. High Snail1 and Foxc2 expression is associated with the invasion and metastatic spread of GEP NETs.

One of the most challenging areas in pancreatic endocrine tumor (PET) biology is the identification of specific biomarkers that can morphologically and histochemically distinguish well-differentiated PETs from other types of neuroendocrine tumors (NETs). Novel markers could improve diagnosis accuracy and could consequently lead to a more appropriate and tailored treatment and therefore to a better prognosis.

Background and aims: The solute carrier family 11 member 1 (SLC11A1) gene (formerly Nramp1) encodes for the protein solute carrier family 11, member 1. It affects susceptibility and clinical outcome of autoimmune and infectious diseases. We investigated the possible role of the functional polymorphism located in the promoter region of SLC11A1 and tumour necrosis factor (TNF) genes in the progression of fibrosis in chronic hepatitis C. Methods: A total of 242 Caucasian Spanish patients with biopsy proven chronic hepatitis C and 194 healthy control subjects were genotyped for SLC11A1 and TNF promoter polymorphisms. Results: No significant differences in the distribution of frequencies among patient and control groups were observed. The SCL11A1 homozygous 2/2 genotype was rarely detected among patients showing advanced fibrosis (2/82; 2.4%) but was highly represented in those with mild fibrosis (29/160; 18.1%; odds ratio (OR) 8.85 (95% confidence interval (CI) 1.9–55.2, p c = 0.002). In patients carrying allele 3 of SLC11A1, the presence of −238 TNF A/G was associated with advanced fibrosis (14/26 (53.8%) v 68/216 (31.4%); OR 2.53 (95% CI 1.03–6.23); p = 0.02). Conclusions: SLC11A1 gene promoter polymorphism could influence fibrosis progression in chronic hepatitis C in that the homozygous genotype 2/2 exerts a protective effect against cirrhosis development. Also, the combination of TNF −238 A/G and the presence of allele 3 is conducive to progression to pre-cirrhotic or cirrhotic stages of the disease.

Mujer de 42 anos, estudiada en el Servicio de Digestivo por un cuadro de rectorragia en relacion con la menstruacion y con el cambio en el habito intestinal (estrenimiento). Se le realizo colonoscopia, apreciandose una estenosis rectosigmoidea, sin afectacion de la mucosa, que impedia el paso del endoscopio. La tomografia computarizada abdominal (fig. 1), mostro una lesion infiltrativa que afectaba a la pared anterior de la union rectosigmoidea, con configuracion «en abanico» (flechas negras pequenas), preservando la pared contralateral, asociado a una masa en el fondo de saco de Douglas (flecha blanca), y un pediculo de conexion entre ambas (flecha negra grande). Se le realizo sigmoidectomia y estudio histologico con inmunohistoquimica CK7 que mostro la existencia de glandulas endometriales.

e19105 Background: The anaplastic lymphoma kinase (ALK) fusion gene is a key oncogenic driver in a subset of patients with advanced non small cell lung cancer (NSCLC). ALK rearragement is detected in 2-7% of NSCLC patients. Fluorescence in situ hybridization (FISH) is the recommended method of detection. The clinical features of lung cancer that harbors ALK include light- or never-smokers, younger age, adenocarcinomas with acinar pattern or signet ring adenocarcinoma, and a lack of EGFR or KRASmutations. Crizotinib has shown an important benefit in terms of overall response rate (ORR) and progression free survival (PFS) in the 2nd/3rd line setting. Treatment related adverse events include gastrointestinal and visual disorders. Methods: Retrospective analysis of the clinico-pathological profile of ALK NSCLC patients between May 2011 and December 2012 in our region. Results: 10 cases (6,7%) were identified from 150 screened patients with adenocarcinoma histology and EGFR wild-type status. 7 (70%) patients were women. Median age at diagnosis was 62 years old (36-77). 9 (90%) patients were light-or-never smokers. All tumors were adenocarcinomas with EGFR wild-type status: Acinar growth pattern was detected in 4 cases (40%); 4 (40%) patients showed mucous cells and previous described signet-ring cells were detected in the last 2 (20%) cases. Only 5 (50%) patients received crizotinib therapy: 2 patients were treated during 1st line with partial response, 1 patient on 2nd line with stable disease and 2 patients received treatment in 3rdline with partial response. Only one patient required a dose reduction due to a grade 3 nausea and mucositis. Conclusions: Most of the patients with ALK rearragements in our serie have clinical and pathological characteristics to previously described. More women and older population were showed. In stead of the small sample size, pathological pattern based on acinar growth and mucous or signet-ring cells in adenocarcinomas with no EGFR mutation should guide the ALK screening. ORR and toxicity profile confirmed Crizotinib benefit as soon as ALK status was detected.