Alba Díaz

Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells

Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs.

Acid ceramidase as a therapeutic target in metastatic prostate cancer

Acid ceramidase (AC) catalyzes the hydrolysis of ceramide into sphingosine, in turn a substrate of sphingosine kinases that catalyze its conversion into the mitogenic sphingosine-1-phosphate. AC is expressed at high levels in several tumor types and has been proposed as a cancer therapeutic target. Using a model derived from PC-3 prostate cancer cells, the highly tumorigenic, metastatic, and chemoresistant clone PC-3/Mc expressed higher levels of the AC ASAH1 than the nonmetastatic clone PC-3/S. Stable knockdown of ASAH1 in PC-3/Mc cells caused an accumulation of ceramides, inhibition of clonogenic potential, increased requirement for growth factors, and inhibition of tumorigenesis and lung metastases. We developed de novo ASAH1 inhibitors, which also caused a dose-dependent accumulation of ceramides in PC-3/Mc cells and inhibited their growth and clonogenicity. Finally, immunohistochemical analysis of primary prostate cancer samples showed that higher levels of ASAH1 were associated with more advanced stages of this neoplasia. These observations confirm ASAH1 as a therapeutic target in advanced and chemoresistant forms of prostate cancer and suggest that our new potent and specific AC inhibitors could act by counteracting critical growth properties of these highly aggressive tumor cells.

High-risk human papillomavirus is transcriptionally active in a subset of sinonasal squamous cell carcinomas.

It has been reported that high-risk human papillomavirus (HPV) is a causative agent of a subgroup of oropharyngeal carcinomas. In these tumors, the presence of the transcriptionally active HPV has been proved through the identification of HPV E6 or E7 messenger RNA (mRNA) transcripts. The aim of the study was to assess the HPV-active transcription in a series of sinonasal carcinomas, in correlation with the HPV DNA identification and the p16 immunohistochemistry. Seventy patients with squamous cell carcinomas of the sinonasal tract were included in the survey. The main clinicopathological characteristics were recorded. All tumors were investigated for HPV through the HPV DNA detection by PCR, using the SPF10 primers and by in situ hybridization, using the high-risk GenPoint probe (Dako, Glostrup, Denmark). HPV16 E7 mRNA transcripts detection was performed by RT-PCR in 27 cases. The immunostaining for p16 was performed in all cases. Fourteen carcinomas (20%) were positive for high-risk HPV by PCR: 13 HPV16 and one HPV35. In situ hybridization showed a dotted nuclear positivity in all these cases. HPV16 E7 mRNA was detected in seven tumors harboring HPV16; in the remaining HPV-positive cases, RNA did not reach the quality for analysis. Strong, diffuse positivity for p16 was observed only in the HPV-positive cases. The 14 HPV-positive squamous cell carcinomas were non-keratinizing or scarcely keratinizing tumors. No significant differences were found in terms of gender, age, or staging at diagnosis between HPV-positive and HPV-negative tumors. However, differences in disease-free survival and overall survival between both groups of patients were significant (P=0.004 and P=0.028, respectively). In conclusion, we have shown that HPV is the etiological agent of a subset of sinonasal carcinomas demonstrating the transcriptionally active HPV in these tumors. Immunostaining for p16 can be used as a surrogate marker to identify these tumors.

Factors associated with collagen deposition in lymphoid tissue in long-term treated HIV-infected patients.

Objective:The factors associated with fibrosis in lymphoid tissue in long-term treated HIV-infected patients and their correlation with immune reconstitution were assessed. Methods:Tonsillar biopsies were performed in seven antiretroviral-naive patients and 29 successfully treated patients (median time on treatment, 61 months). Twenty patients received protease inhibitors-sparing regimens and nine protease inhibitor-containing regimens. Five tonsillar resections of HIV-negative individuals were used as controls. Lymphoid tissue architecture, collagen deposition (fibrosis) and the mean interfollicular CD4+ cell count per μm2 were assessed. Results:Naive and long-term treated HIV-infected patients had a higher proportion of fibrosis than did HIV-uninfected persons (P < 0.001). Patients with greater collagen deposition showed lower levels of CD4+ cells in lymphoid tissue (P = 0.03) and smaller increase in peripheral CD4+ T cells (r = −0.40, P = 0.05). The factors independently associated with fibrosis in lymphoid tissue were age (P < 0.0001), treated patients with detectable lymphoid tissue viral load when compared with patients with undetectable lymphoid tissue viral load (median 5 vs. 12%, respectively, P = 0.017) and patients receiving a protease inhibitor-sparing vs. a protease inhibitor-containing regimen (median 8 vs. 2.5%, respectively, P = 0.04). Conclusion:Fibrosis in lymphoid tissue was associated with a poor reconstitution of CD4+ T cells and long-term antiretroviral therapy did not reverse this abnormality. HIV infection, older age, a detectable level of lymphoid tissue viral load in treated patients and protease inhibitor-sparing regimens seem to favour fibrosis in lymphoid tissue.

Integrative microRNA profiling in alcoholic hepatitis reveals a role for microRNA-182 in liver injury and inflammation

Objective MicroRNAs (miRNAs) are well-known regulators of disease pathogenesis and have great potential as biomarkers and therapeutic targets. We aimed at profiling miRNAs in alcoholic hepatitis (AH) and identifying miRNAs potentially involved in liver injury. Design MiRNA profiling was performed in liver samples from patients with AH, alcohol liver disease, non-alcoholic steatohepatitis, HCV disease and normal liver tissue. Expression of miRNAs was assessed in liver and serum from patients with AH and animal models. Mimic and decoy miR-182 were used in vitro and in vivo to evaluate miR-182s biological functions. Results MiRNA expression profile in liver was highly altered in AH and distinctive from alcohol-induced cirrhotic livers. Moreover, we identified a set of 18 miRNAs predominantly expressed in AH as compared with other chronic liver conditions. Integrative miRNA-mRNA functional analysis revealed the association of AH-altered miRNAs with nuclear receptors, IGF-1 signalling and cholestasis. Interestingly, miR-182 was the most highly expressed miRNA in AH, which correlated with degree of ductular reaction, disease severity and short-term mortality. MiR-182 mimic induced an upregulation of inflammatory mediators in biliary cells. At experimental level, miR-182 was increased in biliary cells in mice fed with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet but not upregulated by alcohol intake or fibrosis. Inhibition of miR-182 in DDC-fed mice reduced liver damage, bile acid accumulation and inflammatory response. Conclusions AH is characterised by a deregulated miRNA profile, including miR-182, which is associated with disease severity and liver injury. These results highlight the potential of miRNAs as therapeutic targets and biomarkers in AH.

Pigmented spindle cell nevus: clues for differentiating it from spindle cell malignant melanoma. A comprehensive survey including clinicopathologic, immunohistochemical, and FISH studies.

Pigmented spindle cell nevus (PSCN), also known as Reed nevus, is a distinctive melanocytic tumor that can show worrisome clinical and histologic features mimicking a malignant melanoma. From a series of 46 pigmented spindle cell melanocytic lesions, including 22 PSCN and 24 spindle cell malignant melanomas (SCMMs), we collected clinical and histopathologic characteristics and evaluated cell cycle and apoptosis regulators by immunohistochemistry. Moreover, fluorescence in situ hybridization (FISH) using probes targeting 6p25 (RREB1), 11q13 (CCND1), 6q23 (MYB), and centromere 6 was performed. PSCN presented in younger people, frequently in women, and were small lesions under 7 mm in diameter affecting the lower limbs, whereas SCMMs arose more frequently in the trunk, upper limbs, and head and neck region. Histologically, symmetry, good lateral demarcation, and uniformity of cellular nests were significantly differential features of PSCN, whereas pagetoid and adnexal spread were frequently seen in both tumors. Immunohistochemical markers that significantly differed from melanomas were Ki-67, cyclin D1, and survivin. FISH was positive in 1 of 15 PSCN and was negative in 4 of 15 SCMMs. These results correlated to a sensitivity of 73% and a specificity of 93%. In conclusion, in the evaluation of pigmented spindle cell melanocytic tumors, the integration of clinical and histologic assessment is essential. However, ancillary techniques such as proliferation antigen Ki-67, cyclin D1, survivin, and FISH can be useful as adjunctive tools.

Portal pressure and liver stiffness measurements in the prediction of fibrosis regression after SVR in recurrent hepatitis C.

Sustained virological response (SVR) improves survival in post‐liver transplant (LT) recurrent hepatitis C. However, the impact of SVR on fibrosis regression is not well defined. In addition, the performance of noninvasive methods to evaluate the presence of fibrosis and portal hypertension (PH) post‐SVR has been scarcely evaluated. We aimed to investigate the degree of fibrosis regression (decrease ≥1 METAVIR stage) after‐SVR and its associated factors in recurrent hepatitis C, as well as the diagnostic capacity of noninvasive methods in the assessment of liver fibrosis and PH after viral clearance. We evaluated 112 hepatitis C virus–infected LT recipients who achieved SVR between 2001 and 2015. A liver biopsy was performed before treatment and 12 months post‐SVR. Hepatic venous pressure gradient (HVPG), liver stiffness measurement (LSM), and Enhanced Liver Fibrosis (ELF) score were also determined at the same time points. Sixty‐seven percent of the cohort presented fibrosis regression: 43% in recipients with cirrhosis and 72%‐85% in the remaining stages (P = 0.002). HVPG, LSM, and ELF significantly decreased post‐SVR. Liver function significantly improved, and survival was significantly better in patients achieving fibrosis regression. Baseline HVPG and LSM as well as decompensations before therapy were independent predictors of fibrosis regression. One year post‐SVR, LSM had a high diagnostic accuracy to discard the presence of advanced fibrosis (AF) and clinically significant PH (AUROC, 0.902 and 0.888). Conclusion: In conclusion, SVR post‐LT induces fibrosis regression in most patients, leading to significant clinical benefits. Pretreatment HVPG and LSM are significant determinants of the likelihood of fibrosis regression. Finally, LSM accurately predicts the presence of AF and PH 1 year after SVR and thus can be used to determine monitoring strategies. (Hepatology 2018;67:1683‐1694).

Lymphoid Tissue Collagen Deposition in HIV-Infected Patients Correlates With the Imbalance Between Matrix Metalloproteinases and Their Inhibitors

We studied the lymphoid tissue biopsies of 20 patients with chronic human immunodeficiency virus (HIV) infection by analyzing collagen deposition, CD4+ cell number, and gene expression of metalloproteinases (MMPs; MMP-2, MMP-9) and tissue inhibitors of MMPs (TIMPs; TIMP-1, TIMP-2). HIV-infected patients had significantly increased collagen deposition (P = .001), fewer CD4+ T cells (P = .05), and decreased MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios (P = .01), compared with HIV-negative control patients. Moreover, we found a significant negative correlation between collagen deposition and the MMP-9/TIMP-1 ratio (ρ = -0.50; P = .047). To our knowledge, this is the first time that MMP/TIMP imbalance has been correlated with lymphoid tissue collagen deposition and incomplete immune recovery in HIV-infected patients, even after long-term antiretroviral treatment.

TERT and AURKA Gene Copy Number Gains Enhance the Detection of Acral Lentiginous Melanomas by Fluorescence in Situ Hybridization

The study of specific chromosomal loci through fluorescence in situ hybridization (FISH) is useful in differential diagnosis of melanocytic tumors. However, sensitivity rates vary, probably because of molecular heterogeneity. Acral lentiginous melanomas are characterized by copy number gains of small genomic regions, including CCND1, TERT, and AURKA. In a series of 58 acral melanocytic lesions, we explored the value of a four-color FISH probe, used in addition to determining MYC gene status, and assessed the potential diagnostic usefulness of newly developed probes targeting TERT and AURKA. Moreover, we tested CCND1, TERT, and AURKA protein expression by immunohistochemistry. The four-color FISH probe detected 85.3% of melanomas and 29.4% of TERT and AURKA copy number gains. Sensitivity was 97% (confidence interval 95%, 82.9% to 99.8%) for the combined results of all probes. No MYC copy number gains were detected. No nevi showed aberrations. Immunohistochemistry revealed a higher percentage of cells positive for CCND1, TERT, and AURKA protein in melanomas than in nevi (P ≤ 0.001). A significant correlation between gene copy number gain and protein expression was found for CCND1 (P = 0.015). Our results indicate that addition of specific FISH probes to the current probe could improve sensitivity for the diagnosis of acral melanomas. Further studies in larger numbers of cases are needed to validate these results.

High Prevalence of Liver Fibrosis Among European Adults With Unknown Liver Disease: A Population-Based Study

Background & Aims: Liver fibrosis is the main determinant of long‐term outcome in chronic liver diseases. Little is known about the prevalence of liver fibrosis in the general population. The aim of the study was to investigate the prevalence of liver fibrosis in the general adult population with unknown liver disease. Methods: This was a population‐based, cross‐sectional study performed in the Barcelona metropolitan area. Subjects aged 18 to 75 years old were identified randomly from citizens included in the primary health care registry. Of 4866 subjects invited, 3076 participated (63.2%). Liver fibrosis was estimated by measuring liver stiffness (LS) with transient elastography (TE). Liver histology was assessed in 92 subjects with increased LS. Results: Prevalence estimates of increased LS (≥6.8, ≥8.0, and ≥9.0 kPa) were 9.0%, 5.8%, and 3.6%, respectively. The etiology of liver disease was mainly nonalcoholic fatty liver disease (NAFLD), followed by alcohol risk consumption (consumption of ≥21 standard drinking units/wk in men and ≥14 standard drinking units/wk in women). Factors independently associated with increased LS were male sex, abdominal obesity, type 2 diabetes, serum glucose, high‐density lipoprotein, and triglyceride levels. Subjects without risk factors for NAFLD or without alcohol risk consumption had a very low prevalence of increased LS. The best cut‐off value of LS for significant liver fibrosis (F2–F4) was 9.2 kPa, with high sensitivity and specificity. TE was more accurate than alanine aminotransferase, NAFLD fibrosis score, or Fibrosis 4. An algorithm for screening for liver fibrosis using TE in the community setting is proposed. Conclusions: These findings show a high prevalence of silent liver disease with advanced fibrosis mainly related to NAFLD in adult European subjects without known liver disease. An LS value less than 9.2 kPa predicts the absence of significant liver fibrosis with high accuracy and could be used for screening purposes.