Lymph Node Tumor Burden Correlates With Tumor Budding and Poorly Differentiated Clusters: A New Prognostic Factor in Colorectal Carcinoma?

Introduction: Molecular lymph node (LN) staging in early colorectal cancer (CRC) has demonstrated to be more precise than conventional histopathology pN staging. Tumor budding (TB) and poorly differentiated clusters (PDCs) are associated with LN metastases, recurrences, and lower survival in CRC. We evaluated the correlation between the total tumor load (TTL) in LNs from CRC surgical specimens with patient outcome, TB, and PDC. Methods: In this retrospective multicentre study, 5,931 LNs from 342 stage I-III CRC were analyzed by both hematoxylin and eosin and molecular detection of tumor cytokeratin 19 mRNA by one-step nucleic acid amplification. TB and PDC were evaluated by hematoxylin and eosin and cytokeratin 19 immunohistochemistry. Results: One-step nucleic acid was positive in 38.3% patients (n = 131). Tumor Budding was low in 45% cases, intermediate in 25%, and high in 30%. Poorly Differentiated Clusters were low-grade G1 in 53%, G2 in 32%, and G3 in 15%. TB and PDC correlated with TTL, high-grade, lymphovascular and perineural invasion, pT, pN and stage (P < 0.001). TB, PDC, and TTL ≥ 6,000 copies/µL were associated with worse overall survival (P = 0.002, P = 0.013, and P = 0.046) and disease-free survival (P < 0.001). Discussion: The implementation of more sensitive molecular methods to assess LN status is a promising alternative approach to pN staging, which could be integrated to other factors to help risk stratification and management of patients with early-stage CRC. This study demonstrates the correlation of the amount of LN tumor burden with TB and PDCs. TTL is related to the outcome and could be used as a new prognostic factor in CRC (see Visual Abstract, Supplementary Digital Content 2, http://links.lww.com/CTG/A512).This work was supported by the Instituto de Salud Carlos III (grants PI17/01304 to J.C and M.C; grant PI16/00766 to F.B., and a Miguel Servet grant to J.C, cofunded by the European Regional Development Fund (ERDF) (CPII18/00026), through the Plan Estatal de Investigacion Científica y Tecnica y de Innovación, and Agencia de Gestiò d’Ajuts Universitaris i de Recerca (2017SGR653 and 2017SGR1035). CIBERehd is funded by the Instituto de Salud Carlos III. We also acknowledge the support of the CERCA Programme/Generalitat de Catalunya, and the Xarxa de Bancs de Tumors de Catalunya (XBTC)

Turismo LGTB. Unha aproximación ao caso de Galicia

Este traballo aborda unha tipoloxía turística que está en auxe na actualidade: o turismo LGTB (lesbianas, gays, transexuais e bisexuais). Inicialmente, preténdese identificar os desexos, necesidades e expectativas concretas do mercado turístico homosexual, analizando os estudos existentes para, poste-riormente, analizar a situación de Galicia en relación coa oferta e demanda existente mediante unha análi-se cualitativa xunto a un estudo exploratorio de carácter cuantitativo que complementa o primeiro. Unha vez analizados eses aspectos, este traballo pretende determinar e expoñer as directrices básicas a seguir por parte dos diferentes axentes turísticos que queiran atraer o colectivo LGTB. 

Expression of Epithelial and Mesenchymal Markers in Plasmatic Extracellular Vesicles as a Diagnostic Tool for Neoplastic Processes

Tumor-derived extracellular vesicles (TD-EVs) have active roles as cancer hallmark enablers. EVs RNA of epithelial and stromal cells carry information that facilitates the communication processes that contribute to oncological progression, so the objective of this work was to validate by RT-PCR the presence of epithelial (KRT19; CEA) and stromal (COL1A2; COL11A1) markers in RNA of plasmatic EVs in healthy and diverse-malignancy patients for the development of a non-invasive cancer diagnosis system using liquid biopsy. Ten asymptomatic controls and 20 cancer patients were included in the study, and results showed that the isolated plasmatic EVs by scanning transmission electron microscopy (STEM) andBiomedical Research Institute A Coru&ntilde;a nanoparticle tracking analysis (NTA) contained most exosome structures with also a considerable percentage of microvesicles. No differences were found in concentration and size distribution between the two cohorts of patients, but significant gene expression in epithelial and mesenchymal markers between healthy donors and patients with active oncological disease was shown. Results of quantitative RT-PCR are solid and reliable for KRT19, COL1A2, and COL11A1, so the analysis of RNA extracted from TD-EVs could be a correct approach to develop a diagnostic tool in oncological processes

The NMR structure of human obestatin in membrane-like environments: insights into the structure-bioactivity relationship of obestatin.

The quest for therapeutic applications of obestatin involves, as a first step, the determination of its 3D solution structure and the relationship between this structure and the biological activity of obestatin. On this basis, we have employed a combination of circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, and modeling techniques to determine the solution structure of human obestatin (1). Other analogues, including human non-amidated obestatin (2) and the fragment peptides (6-23)-obestatin (3), (11-23)-obestatin (4), and (16-23)-obestatin (5) have also been scrutinized. These studies have been performed in a micellar environment to mimic the cell membrane (sodium dodecyl sulfate, SDS). Furthermore, structural-activity relationship studies have been performed by assessing the in vitro proliferative capabilities of these peptides in the human retinal pigmented epithelial cell line ARPE-19 (ERK1/2 and Akt phosphorylation, Ki67 expression, and cellular proliferation). Our findings emphasize the importance of both the primary structure (composition and size) and particular segments of the obestatin molecule that posses significant α-helical characteristics. Additionally, details of a species-specific role for obestatin have also been hypothesized by comparing human and mouse obestatins (1 and 6, respectively) at both the structural and bioactivity levels

Structural statistics for the ensemble of the best 20 structures of human obestatin (1), its fragments and mouse obestatin (6).

<p>[a] The final CYANA target function value was computed for the structures calculated using CYANA.</p><p>[b] Average values of the 20 final energy-minimized CYANA conformers.</p><p>[c] Calculated using PROCHECK-NMR.</p><p>[d] Atomic differences are given as the average RMS difference of the mean coordinate structure (mean).</p

Analysis of the expression and functionality of GPR39 in ARPE-19 cells.

<p>(A) Immunocytochemical detection of GPR39 in ARPE-19 cells (objective magnification of 20x). (B) The effect of siRNA depletion of GPR39 on pAkt(S473) and pERK1/2(T202/Y204) in ARPE-19 cells after human obestatin treatment (<b>1</b>, 100 nM, 10 min). The ARPE-19 cells were transfected with GPR39 siRNA prior to obestatin <b>1</b> treatment. Equal amounts of protein in each sample were used to assess the expression of GPR39 by western blotting. The GPR39 level was expressed as the fold change relative to the control siRNA-transfected cells (mean ± SE). The protein expression was normalized relative to actin. The data are expressed as the mean ± SE. The asterisk (*) denotes <i>P</i><0.05 when comparing the treated control siRNA group with the control siRNA group; the dagger (#) denotes <i>P</i><0.05 when comparing the GPR39 siRNA group with the control siRNA group.</p

Immunocytochemical analysis of the Ki67 expression and BrdU incorporation in ARPE-19 cells.

<p>Immunocytochemical analysis of the Ki67 expression in ARPE-19 cells after 24 h of proliferation. A) Control. B) 10% FBS (v/v). C) 100 nM human obestatin (<b>1</b>). D) 100 nM human non-amidated obestatin (<b>2</b>). E) 100 nM human (6–23)-obestatin (<b>3</b>). F) 100 nM human (11–23)-obestatin (<b>4</b>). G) 100 nM human (16–23)-obestatin (<b>5</b>). H) 100 nM mouse obestatin (<b>6</b>). The magnification was 20x. I) Quantification of the immunocytochemical expression of Ki67 in ARPE-19 cells after treatment with 10% FBS (v/v; 100±2), 100 nM human obestatin (<b>1</b>; 84±1%), 100 nM human non-amidated obestatin (<b>2</b>; 33±2%), 100 nM human (6–23)-obestatin (<b>3</b>; 56±3%), 100 nM human (11–23)-obestatin (<b>4</b>; 67±3%), 100 nM human (16–23)-obestatin (<b>5</b>; 50±2%) and 100 nM mouse obestatin (<b>6</b>; 32±2%). The expression of Ki67 was expressed as the fold change relative to the expression level in FBS-treated cells in the positive control (mean ± SE). J) BrdU incorporation in ARPE-19 cells after treatment with 10% FBS (v/v; 100±1), 100 nM human obestatin (<b>1</b>; 84±4%), 100 nM human non-amidated obestatin (<b>2</b>; 39±2%), 100 nM human (6–23)-obestatin (<b>3</b>; 30±1%), 100 nM human (11–23)-obestatin (<b>4</b>; 52±3%), 100 nM human (16–23)-obestatin (<b>5</b>; 34±3%) and 100 nM mouse obestatin (<b>6</b>; 35±1%). The BrdU incorporation was expressed as the fold change relative to the level in FBS-treated cells in the positive control (mean ± SE). The data are expressed as the mean ± SE. The asterisk (*) denotes <i>P</i><0.05 when comparing the peptide-treated ARPE-19 cells groups with the human obestatin (<b>1</b>)-treated group.</p