[Show abstract][Hide abstract] ABSTRACT: Background:
Gastric cancer (GC) is a deadly malignancy worldwide. In the past, it has been shown that cellular signaling pathway alterations play a crucial role in the development of GC. In particular, deregulation of the PI3K/AKT/mTOR pathway seems to affect multiple GC functions including growth, proliferation, metabolism, motility and angiogenesis. Targeting alterations in this pathway by microRNAs (miRNAs) represents a potential therapeutic strategy, especially in inhibitor-resistant tumors. The objective of this study was to evaluate the expression of 3 pre-selected miRNAs, miR-101-2, miR-125b-2 and miR-451a, in a series of primary GC tissues and matched non-GC tissues and in several GC-derived cell lines, and to subsequently evaluate the functional role of these miRNAs.
Twenty-five primary GC samples, 25 matched non-GC samples and 3 GC-derived cell lines, i.e., AGS, MKN28 and MKN45, were included in this study. miRNA and target gene expression levels were assessed by quantitative RT-PCR and western blotting, respectively. Subsequently, cell viability, clone formation, cell death, migration and invasion assays were performed on AGS cells.
miR-101-2, miR-125b-2 and miR-451a were found to be down-regulated in the primary GC tissues and the GC-derived cell lines tested. MiRNA mimic transfections significantly reduced cell viability and colony formation, increased cell death and reduced cell migration and invasion in AGS cells. We also found that exogenous expression of miR-101-2, miR-125b-2 and miR-451a decreased the expression of their putative targets MTOR, PIK3CB and TSC1, respectively.
Our expression analyses and in vitro functional assays suggest that miR-101-2, miR-125b-2 and miR-451a act as potential tumor suppressors in primary GCs as well as in GC-derived AGS cells.
[Show abstract][Hide abstract] ABSTRACT: Overexpression of Short and Raji variants of Cellular FLICE-like inhibitory protein (c-FLIP) is capable of inhibiting apoptosis, while the function of the Long isoform depends of c-FLIPL concentration in cells. The aim of this study was to determine the effects of c-FLIPL knockdown in cervical cell lines. SiHa, C-4I and C-33A cervical cancer cell lines were analyzed. c-FLIPL level expression was determined by quantitative real-time PCR and western blotting. c-FLIPL was transiently downregulated by siRNA. The effects of knockdown of c-FLIPL on cell viability, proliferation and apoptosis were assessed by comparing with scrambled siRNA-transfected cells. SiHa and C-4I c-FLIPL knockdown cells showed increased viability compared with scrambled siRNA-transfected cells (P<0.05), while C-33A cells did not show significant differences. Ki-67 and PCNA immunocytochemistry was performed to evaluate proliferation on these cervical cancer cell lines. SiHa cells with c-FLIPL knockdown showed elevated expression of Ki-67 protein compared with their scrambled counterparts (P<0.0001), while C-33A c-FLIPL knockdown cells showed a significantly lower in PCNA expression (P<0.01) compared with control. All three c-FLIP-transfected cell lines showed a higher level of apoptosis compared with their scrambled controls. Our results suggest that c-FLIPL could have effects in proliferation and apoptosis in cervical cancer cell lines.
Full-text · Article · Jun 2015 · International Journal of Morphology
[Show abstract][Hide abstract] ABSTRACT: Castleman's disease is an uncommon lymphoproliferative disorder which can be unicentric or multicentric. Hialine vascular variant is the most common pathologic form, which is usually unicentric and presenting as mediastinal tumors. We report a 31-year-old female with a history of retrosternal pain. A chest CAT sean showed a tumor in the posterior mediastinum. The patient was operated and the tumor excised. The pathology report showed a Castleman's disease. No other tumors were found in the patient, who had a favorable evolution.
No preview · Article · Jun 2014 · Revista medica de Chile
[Show abstract][Hide abstract] ABSTRACT: Signaling pathway alterations are important in the development of gastric cancer (GC). Deregulation of the PI3K/AKT/mTOR pathway plays a crucial role in the regulation of multiple cellular functions including cell growth, proliferation, metabolism, and angiogenesis. Our goal was to assess expression of proteins involved in the PI3K/AKT/mTOR pathway by immunohistochemistry (IHC) in tumor and nontumor gastric mucosa from patients with advanced GC. We evaluated 71 tumor and 71 nontumor gastric mucosa samples from advanced GC patients, selected from Hernán Henríquez Aravena Hospital (Temuco, Chile). The targets studied were PI3K, AKT, p-AKT, PTEN, mTOR, p-mTOR, P70S6K1, p-P70S6K1, 4E-BP1, p-4E-BP1, eIF4E, and p-eIF4E. Expression data were correlated with clinicomorphological data. Descriptive and analytical statistics were used (95 % confidence interval, p < 0.05). For survival analyses, the Kaplan-Meier method and the log-rank test were used. PI3K, AKT, p-AKT, p-mTOR, p-4E-BP1, P70S6K1, p-P70S6K1, eIF-4E, and p-eIF-4E proteins were significantly overexpressed in tumor tissue. Conversely, PTEN was underexpressed in tumor tissue, notably in pT3-pT4 tumors (p = 0.02) and tumors with lymph node metastases (p < 0.001). P70S6K1 expression was associated with pT3-pT4 tumors (p = 0.03). Moreover, PI3K (p = 0.004), AKT (p = 0.01), p-AKT (p = 0.01), P70S6K1 (p = 0.04), p-P70S6K1 (p = 0.001), and eIF-4E (p = 0.004) were overexpressed in tumors with lymph node metastases. Low expression of 4E-BP1 was associated with poor overall survival (p = 0.03). Our results suggest that the PI3K/AKT/mTOR pathway is activated in GC, with overexpression in tumor tissue of most of the studied proteins (total and phosphorylated). These might be considered as target for specific targeted therapy in GC.
Full-text · Article · May 2014 · Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin
[Show abstract][Hide abstract] ABSTRACT: Cervical cancer is a major health concern among women in Latin America due to its high incidence and mortality. Therefore, the discovery of molecular markers for cervical cancer screening and triage is imperative. The aim of this study was to use a genome wide methylation approach to identify novel methylation biomarkers in cervical cancer. DNA from normal cervical mucosa and cervical cancer tissue samples from Chile was enriched with Methylated DNA Immunoprecipitation (MeDIP), hybridized to oligonucleotide methylation microarray and analyzed with a stringent bioinformatics pipeline to identify differentially methylated regions (DMRs) as candidate biomarkers. Quantitative Methylation Specific PCR (qMSP) was used to study promoter methylation of candidate DMRs in clinical samples from two independent cohorts. HPV detection and genotyping were performed by Reverse Line Blot analysis. Bioinformatics analysis revealed GGTLA4, FKBP6, ZNF516, SAP130, and INTS1 to be differentially methylated in cancer and normal tissues in the Discovery cohort. In the Validation cohort FKBP6 promoter methylation had 73% sensitivity and 80% specificity (AUC = 0.80). ZNF516 promoter methylation was the best biomarker, with both sensitivity and specificity of 90% (AUC = 0.92), results subsequently found in a Prevalence cohort. Together, ZNF516 and FKBP6 exhibited a sensitivity of 84% and specificity of 81%, when considering both cohorts. Our genome wide DNA methylation assessment approach (MeDIP-chip) successfully identified novel biomarkers that differentiate between cervical cancer and normal samples. These biomarkers need to be further explored in case-control and prospective cohorts to validate them as cervical cancer biomarkers.
Full-text · Article · Nov 2013 · Epigenetics: official journal of the DNA Methylation Society
[Show abstract][Hide abstract] ABSTRACT: Gallbladder cancer (GBC) is a highly fatal disease with poor prognosis and few therapeutic alternatives. The molecular mechanisms involved in the pathogenesis of GBC remain poorly understood. The vascular endothelial growth factor A (VEGF-A) is a potent proangiogenic agent involved in the carcinogenesis of many human tumors and is an attractive target for cancer therapy. We characterized VEGF-A expression in advanced GBC and its relation to clinicopathologic features. VEGF-A expression was examined by immunohistochemistry in tissue microarrays containing 224 advanced gallbladder carcinomas and 39 chronic cholecystitis. The cases were classified as low or high expression to evaluate the association of VEGF-A expression level with clinicopathologic variables. The Kaplan-Meier method was used to estimate survival as a function of time, and survival differences were analyzed by the log-rank test. High expression of VEGF-A was observed in 81% (183/224) of tumors and 5.1% (2/39) of chronic cholecystitis (P<0.0001). The VEGF-A expression had a significant relationship with histologic grade and TNM stage (P<0.05). Moreover, 5-year survival analysis indicated that high expression of VEGF-A is associated with a poor prognosis in patients with advanced GBC (P=0.0116). Our results indicate that VEGF-A is highly expressed in GBC and correlates with poor prognosis, suggesting that VEGF-A expression could be used as a biomarker for predicting malignant behavior and for identifying a subset of patients who may benefit from anti-VEGF-A therapies.
Full-text · Article · Oct 2013 · Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry
[Show abstract][Hide abstract] ABSTRACT: Gallbladder carcinoma is a highly malignant tumor and a public health problem in some parts of the world. It is characterized by a poor prognosis and its resistance to radio and chemotherapy. There is an urgent need to develop novel therapeutic alternatives for the treatment of gallbladder carcinoma. The mammalian target of the rapamycin (mTOR) signaling pathway is activated in about 50% of human malignancies, and its role in gallbladder carcinoma has previously been suggested. In the present study, we investigated the phosphorylation status of the mTOR substrate p70S6K in preneoplastic and neoplastic gallbladder tissues and evaluated the effect of three mTOR inhibitors on cell growth and migration in gallbladder carcinoma cell lines.
Immunohistochemical staining of phospho-p70S6K was analyzed in 181 gallbladder carcinoma cases, classified according to lesion type as dysplasia, early carcinoma, or advanced carcinoma. Protein expression of AKT/mTOR members was also evaluated in eight gallbladder carcinoma cell lines by Western blot analysis. We selected two gallbladder carcinoma cell lines (G415 and TGBC-2TKB) to evaluate the effect of rapamycin, RAD001, and AZD8055 on cell viability, cell migration, and protein expression.
Our results showed that phospho-p70S6K is highly expressed in dysplasia (66.7%, 12/18), early cancer (84.6%, 22/26), and advanced cancer (88.3%, 121/137). No statistical correlation was observed between phospho-p70S6K status and any clinical or pathological features, including age, gender, ethnicity, wall infiltration level, or histological differentiation (P < 0.05). In vitro treatment with rapamycin, RAD001, and AZD8055 reduced cell growth, cell migration, and phospho-p70S6K expression significantly in G-415 and TGBC-2TKB cancer cells (P < 0.001).
Our findings confirm the upregulation of this signaling pathway in gallbladder carcinoma and provide a rationale for the potential use of mTOR inhibitors as a therapeutic strategy for human gallbladder carcinoma.
Full-text · Article · Oct 2013 · OncoTargets and Therapy
[Show abstract][Hide abstract] ABSTRACT: The general impression about gallbladder carcinomas is that they are uniformly fatal; however, for the early forms, an entirely different picture indicating a very good prognosis is evolving from the high-incidence regions. We subjected 190 early gallbladder carcinomas (EGBC), defined as carcinomas confined to and above the tunica muscularis (AJCC's Tis, T1a, and T1b), and identified in cholecystectomy specimens sampled entirely according to an established protocol, to detailed analysis. Average patient age was 57.9 years (29-95). In more than half of the cases (114/190; 60 %), the tumor was inapparent by gross examination. In 81 cases (42.6 %), carcinomatous epithelium abutted the muscularis, whereas 57.4 % (n = 109) were qualified as intramucosal with no overt contiguity with muscularis. Intraepithelial extension into Rokitansky-Aschoff sinuses (RAS) was found in 34 cases (17.8 %). At the time of data analysis, 171 patients (99 %) were alive. Overall actuarial survival was 92.3 % at 5 years and 90.4 % at 10 years. The 5- and 10-year actuarial survival rates of the intramucosal group (93.2 and 92.1 %, respectively) were not statistically different from that of the muscle-abutting group (89.7 % and 88.2 % ; p = 0.334). Patients with RAS involvement had a significantly shorter survival than those without (p < 0.001). Of the 33 patients with RAS involvement, 13 (39 %) died of disease, whereas only 6 of the 154 patients (4 %) without RAS involvement died of disease. Disease-related mortality in these cases occurred relatively late (median 48 months). EGBC has a very good prognosis with a 90 % 10-year survival rate. It is seen on average in patients almost a decade younger than those with advanced cancers. RAS involvement is an independent prognostic factor, and additional surgery may have to be considered for such cases. Occasional recurrences are encountered several years later, which suggests a field-effect phenomenon and warrants long-term follow-up.
Full-text · Article · Sep 2013 · Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin
[Show abstract][Hide abstract] ABSTRACT: Context.-Advanced gallbladder carcinoma (GBC) is a highly fatal disease with poor prognosis and few therapeutic alternatives. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a central role in cell growth and homeostasis. Its regulation is frequently altered in various tumors and is an attractive target for cancer therapy; however, its status in GBC remains unclear. Objective.-To characterize immunohistochemical expression and prognostic significance of phospho-mTOR in advanced gallbladder carcinoma. Design.-Phospho-mTOR expression was examined by immunohistochemistry in tissue microarrays containing 128 advanced GBCs and 99 cases of chronic cholecystitis, which were divided into 2 groups according to the presence or absence of metaplasia. To evaluate the association of the level of phospho-mTOR expression with clinical variables and patient survival, the advanced GBCs were classified as having low or high expression. Statistical analysis was performed by using a significance level of P < .05, and Kaplan-Meier curves were constructed for survival analysis. Results.-Immunostaining for phospho-mTOR was positive in 82 of 128 tumors (64.1%) and in 24% of chronic cholecystitis cases (16% nonmetaplasia and 32% with metaplasia) (P < .001). Survival analysis indicated that a high phospho-mTOR immunohistochemical expression was associated with poorer prognosis in patients with advanced GBC (P = .02). Conclusions.-Metaplasia is a common finding in chronic cholecystitis and is considered a precursor lesion of dysplasia. Our results suggest that the activation of mTOR occurs very early during the development of GBC, contributing to the carcinogenesis process. Phospho-mTOR expression is correlated with poor survival, supporting the potential of mTOR for targeted therapy.
Full-text · Article · Apr 2013 · Archives of pathology & laboratory medicine
[Show abstract][Hide abstract] ABSTRACT: Cervical cancer is a leading cause of cancer deaths in women worldwide and infection by high-risk human papillomavirus types is a precursor event. The cellular FLICE-like inhibitory protein (c-FLIP) has been found to be overexpressed in several types of cancers and could be associated with cervical cancer progression because of its ability to inhibit the apoptotic process. To detect c-FLIP expression in cervical cancer, an immunohistochemical staining was performed, using tissue microarrays, on a series of 536 archival biopsy samples, including normal cervical tissues, low-grade and high-grade squamous intraepithelial lesions, and squamous cervical carcinomas. The epithelium in the normal cervix and low-grade squamous intraepithelial lesions mainly stained negatively for c-FLIP, whereas high-grade intraepithelial lesions and cancer samples showed an elevated expression of c-FLIP. A direct association was observed between the increasing grade of the lesion and the intensity of c-FLIP staining, in which the frequency of intense c-FLIP expression increased from 12.5% in the normal tissue to 82.1% in the cervical cancer tissue. An increased expression of c-FLIP may be an important factor in the progression of cervical cancer. This finding could aid in identifying patients with preneoplastic lesions at greater risk of developing cervical cancer. c-FLIP expression in cervical tissue may be a potential cervical cancer progression marker.
Full-text · Article · Mar 2013 · International journal of gynecological pathology: official journal of the International Society of Gynecological Pathologists
[Show abstract][Hide abstract] ABSTRACT: Context:
Gallbladder cancer (GBC) is an aggressive neoplasia associated with late diagnosis, unsatisfactory treatment, and poor prognosis. Molecular mechanisms involved in GBC pathogenesis remain poorly understood. Connective tissue growth factor (CTGF) is thought to play a role in the pathologic processes and is overexpressed in several human cancers, including GBC. No information is available about CTGF expression in early stages of gallbladder carcinogenesis. Objective.- To evaluate the expression level of CTGF in benign and malignant lesions of gallbladder and its correlation with clinicopathologic features and GBC prognosis.
Connective tissue growth factor protein was examined by immunohistochemistry on tissue microarrays containing tissue samples of chronic cholecystitis (n = 51), dysplasia (n = 15), and GBC (n = 169). The samples were scored according to intensity of staining as low/absent and high CTGF expressers. Statistical analysis was performed using the χ(2) test or Fisher exact probability test with a significance level of P < .05. Survival analysis was assessed by the Kaplan-Meier method and the log-rank test.
Connective tissue growth factor expression showed a progressive increase from chronic cholecystitis to dysplasia and then to early and advanced carcinoma. Immunohistochemical expression (score ≥2) was significantly higher in advanced tumors, in comparison with chronic cholecystitis (P < .001) and dysplasia (P = .03). High levels of CTGF expression correlated with better survival (P = .04).
Our results suggest a role for CTGF in GBC progression and a positive association with better prognosis. In addition, they underscore the importance of considering the involvement of inflammation on GBC development.
Full-text · Article · Feb 2013 · Archives of pathology & laboratory medicine
[Show abstract][Hide abstract] ABSTRACT: Gallbladder cancer is an infrequent neoplasia with noticeable geographical variations in its incidence around the world. In Chile, it is the main cause of death owing to cancer in women over 40 years old, with mortality rates up to 16.5 per 100,000 cases. The prognosis is poor with few therapeutic options; in advanced cases there is only a 10% survival at 5 years.
Several studies mention the possible role of DNA methylation in gallbladder carcinogenesis. This epigenetic modification affects tumor suppressor genes involved in regulation pathways, cell cycle control, cell adhesion and extracellular matrix degradation, in a sequential and cumulative way. Determining DNA methylation patterns would allow them to be used as biomarkers for the early detection, diagnosis, prognosis and/or therapeutic selection in gallbladder cancer.
[Show abstract][Hide abstract] ABSTRACT: The bronchogenic cyst is a remanent of the development of the tracheobronchial tree. Alterations in cell migration during development cause the formation of cyst lining by respiratory epithelium. They are usually located in the thorax in lung and mediastinum. The definite diagnosis is realized with the pathology report. This is an uncommon pathological condition and the extra- thoracic forms are very unusual. We present two cases, one of which is of sublingual location and the other of subcutaneous location at the scapular site.
[Show abstract][Hide abstract] ABSTRACT: We describe a clinicopathologically distinct subtype of cholecystitis, the extensively calcific version of which has been presented in the clinical literature as "porcelain gallbladder (PG)." This cholecystitis, which we propose to refer to as hyalinizing cholecystitis (HC), is characterized by dense, paucicellular hyaline fibrosis transforming the gallbladder (GB) wall into a relatively thin and uniform band. The process diffusely effaces most of the normal structures of GB, and some cases show calcifications. To determine the clinicopathologic associations of HC, we systematically analyzed 4231 cholecystectomies (606 of which had carcinoma) histopathologically, in addition to a targeted search in our databases. Ninety-six cases of HC were identified (1.6% of cholecystectomies). Patients with HC were a decade older than ordinary cholecystitis patients (56 vs. 47; P<0.001), suggesting that HC may be a long-term complication of chronic injury in some patients. Calcifications of variable amounts and degrees were identified in two thirds of the cases. In addition, 10 cases showed diffuse marked calcifications and were considered separately as "complete porcelain" GB. Thirty-eight HC cases had carcinoma with a calculated frequency of 15% and an odds ratio of cancer risk of 4.6. Only 42% of the invasive cases were associated with calcifications; none of the 10 diffusely calcific cases had carcinoma. HC-related carcinomas were challenging diagnostically. They did not form distinct masses or any significant thickening (mean thickness, 2.6 vs. 4.0 mm in ordinary adenocarcinomas; P<0.002). Microscopically, they had widely scattered and bland-appearing glands embedded in the thin band of hyaline stroma of HC, commonly showing a disappearing lining, leaving behind the granular, necrotic intraluminal debris (regression) with or without calcifications, which could be the only sign of cancer in some sections. The morphologic features that allowed the recognition of these glands as malignant included their longitudinal axis parallel to the surface, their irregular contours, clear cytoplasm with distinct borders, nuclear irregularities, and washed-off chromatin. Surface epithelium, if preserved (and it was not in most cases), typically showed carcinoma in situ of either denuding or micropapillary types. HC-associated carcinomas, with a median survival of 7 months, appeared to have a clinical course at least as aggressive as that of regular carcinomas (median survival 12 months; P=0.02). In conclusion, HC is a distinct clinicopathologic entity, which is often associated with carcinoma, and the carcinomas arising from this group are often very subtle and prone to misdiagnosis microscopically. As HC is typically devoid of epithelium, any glandular elements on the wall of HC should be regarded as a suspect for carcinoma. This study also confirms recent findings in the radiology literature-it is not the complete (diffusely calcific) PG that is associated with cancer. Instead, a distinct, histopathologically defined form of cholecystitis, HC with minimal or no calcifications (incomplete PG), is associated with invasive carcinoma. Thus, imaging protocols ought to focus on the correlates of HC rather than fixating on calcifications. Further studies into the pathogenesis of this process and its mechanisms of progression to carcinoma are warranted.
Full-text · Article · Jun 2011 · The American journal of surgical pathology
[Show abstract][Hide abstract] ABSTRACT: The information in the literature on squamous cell and adenosquamous carcinomas of the gallbladder is highly limited. In this study, 606 resected invasive gallbladder carcinoma cases were analyzed. Squamous differentiation was identified in 41 cases (7%). Those without any identifiable glandular-type invasive component were classified as pure squamous cell carcinomas (8 cases) and those with the squamous component constituting 25–99% of the tumors were classified as adenosquamous carcinomas (26 cases) and included into the analysis. The remaining 7 that had <25% squamous component were classified as adenocarcinoma with focal squamous change and excluded. The clinicopathological characteristics of adenosquamous carcinoma/squamous cell carcinomas were documented and contrasted with that of ordinary gallbladder adenocarcinomas. The average patient age was 65 years (range 26–81); female/male ratio, 3.8. In only 13%, there was a preoperative clinical suspicion of malignancy. Grossly, 58% presented as thickening and hardening of the wall and 6% were polypoid. In 12%, mucosa adjacent to the tumor revealed squamous metaplasia. All pure squamous cell carcinomas had prominent keratinization. Giant cells and tumor-infiltrating eosinophils were observed in 29 and 51% of the squamous cell carcinomas/adenosquamous carcinomas versus 10% (P=0.02) and 6% (P=0.001) in gallbladder adenocarcinomas, respectively. All but three cases had ‘advanced’ (pT2 and above) carcinomas. Follow-up was available in 31 patients: 25 died of disease (median=5 months, range 0–20), and 6 were alive (median=64 months, range 5–112.5). The survival of patients with squamous cell carcinomas/adenosquamous carcinomas was significantly worse than that of gallbladder adenocarcinomas (P=0.003), and this adverse prognosis persisted when compared with stage-matched advanced gallbladder adenocarcinoma cases (median=11.4 months, P=0.01). In conclusion, squamous differentiation was noted in 7% of gallbladder carcinomas. The incidence of adenosquamous carcinoma (defined as 25–99% of the tumor being squamous) was 4%, and that of pure squamous cell carcinoma (without any documented invasive glandular component) was 1%. Pure squamous cell carcinomas often showed prominent keratinization. The overall prognosis of adenosquamous carcinoma/squamous cell carcinoma appears to be even worse than that of ordinary adenocarcinomas. Most patients died within a few months; however, those few who were alive beyond 2 years in this cohort experienced long-term survival.Keywords: adenosquamous; carcinoma; gallbladder; squamous