Positive Cytoplasmic Staining Research Articles

Downregulation of nuclear and cytoplasmic Chibby is associated with advanced cervical cancer.

Chibby has been identified as a putative tumor suppressor and antagonist to β-catenin, thereby controlling the Wnt signaling pathway. Chibby is typically downregulated in numerous types of cancer and may be associated with tumorigenesis. The present study aimed at clarifying the following: i) Whether Chibby antagonizes β-catenin in cervical cancer; ii) whether Chibby and β-catenin mRNA expression is associated with cancer progression; and iii) whether Chibby and β-catenin expression may be used as a biomarker. A total of 87 paraffin-embedded cervical sections with distinct cervical intraepithelial neoplasia (CIN) stages (chronic cervicitis, CIN 1, CIN 2, CIN 3 and invasive squamous cell carcinoma) were collected between June 2004 and October 2012 The mRNA expression level of Chibby and β-catenin was determined using the polymerase chain reaction. Protein expression and cellular localization of Chibby and β-catenin were determined using immunohistochemistry. Chibby and β-catenin were analyzed for possible association with the progression of cervical cancer. Chibby mRNA expression and the Chibby/β-catenin ratio were identified to be downregulated in invasive tumors. Positive cytoplasmic and nuclear staining for Chibby was associated with CIN staging and decreased as the CIN stage increased. In addition, the cytoplasmic and membrane intensity of β-catenin was associated with invasive tumors, in which a significantly increased level of protein expression was detected. Chibby may be a tumor suppressor in cervical cancer, since the dysregulation of Chibby expression is associated with tumorigenesis in cervical cancer. Chibby and β-catenin expression together may potentially to a biomarker for disease progression in cervical cancer.

High-resolution melting and immunohistochemical analysis efficiently detects mutually exclusive genetic alterations of adamantinomatous and papillary craniopharyngiomas.

Craniopharyngioma consists of adamantinomatous and papillary subtypes. Recent genetic analysis has demonstrated that the two subtypes are different, not only in clinicopathological features, but also in molecular oncogenesis. Papillary craniopharyngioma (pCP) is characterized by a BRAF mutation, the V600E (Val 600 Glu) mutation. Adamantinomatous craniopharyngioma (aCP) can be distinguished by frequent β-catenin gene (CTNNB1) mutations. Although these genetic alterations can be a diagnostic molecular marker, the precise frequency of these mutations in clinical specimens remains unknown. In this study, we first evaluated BRAF V600E and CTNNB1 mutations in four and 14 cases of pCP and aCP, respectively, using high-resolution melting analysis followed by Sanger sequencing. The results showed that 100% (4/4) of pCP cases had BRAF V600E mutations, while 78% (11/14) of the aCP cases had CTNNB1 mutations, with these genetic alterations being subtype-specific and mutually exclusive. Second, we evaluated BRAF V600E and CTNNB1 mutations by immunohistochemical analysis (IHC). All pCP cases showed positive cytoplasmic staining with the BRAF V600E-mutant antibody (VE-1), whereas 86% (12/14) of aCP cases showed positive cytoplasmic and nuclear staining for CTNNB1, suggesting a CTNNB1 mutation. Only one case of wild-type CTNNB1 on the DNA analysis showed immunopositivity on IHC. We did not detect a coexistence of BRAF V600E and CTNNB1 mutations in any single tumor, which indicated that these genetic alterations were mutually exclusive. We also report our modified IHC protocol for VE-1 staining, and present the possibility that BRAF V600E mutations can be used as a diagnostic marker of pCP in the differentiation of Rathke cleft cyst with squamous metaplasia.

Expression of cytokeratin-19 (CK19) in the classical subtype of papillary thyroid carcinoma: the experience of one center in the Silesian region.

Cytokeratin-19 (CK19) is recognized as a reliable tumor marker of papillary thyroid carcinoma (PTC). The general prognosis in the classical subtype of PTC (CSTPTC) remains favorable, but some cases can be very aggressive. The aim of this study was to evaluate the localization and intensity of immunohistochemical CK19 expression in CSTPTC and its associations with the clinical and pathological characteristics of patients with CSTPTC in the Silesian region. All the available clinical and histopathological data for 149 patients with CSTPTC from the Silesian region were retrospectively analyzed. The group consisted of 135 (90.6%) women and 14 (9.4%) men (mean age and SD: 52.3 ± 15.0). All these patients with CSTPTC underwent surgery at the same center between 2008 and 2013; the follow-up period was 24 to 90 months (mean and SD: 47 ± 20). In 142 (95.3%) of the patients with CSTPTC, positive cytoplasmic staining of CK19 was found. A higher expression of CK19 was observed in the group of patients without the recurrence of the disease (p = 0.015). CK19 expression was not associated with age, gender, tumor focality, disease stage, tumor size (pT), lymph node involvement (pN), or distant metastases (pM). Decreased CK19 expression in CSTPTC cases with relapse suggests that it plays a role in the carcinoma progression of CSTPTC. The association between lower CK19 expression and patients' unfavorable postoperative course could suggest its possible role as a marker of CSTPTC poor prognosis.

Changes in endogenous sulfur dioxide pathway in angiotensin II- induced myocardial hypertrophy in mice

Objective To explore the changes in the endogenous sulfur dioxide (SO2) pathway in the myocardial hypertrophy induced by the angiotensin Ⅱ(Ang Ⅱ) in mice. Methods Fourteen healthy C57BL mice, 9 weeks old, were randomly divided into control group(n=7) and Ang Ⅱ group(n=7), and capsule osmotic pump with pre-loaded 9 g/L saline and Ang Ⅱ was implanted into the back of each mouse subcutaneously.After 2 weeks, the mice were executed.The heart weight/body weight (HW/BW) and the left heart weight/full heart weight (LVW/HW) of the mice were measured.The microstructure of the cardiac myocyte was observed by hematoxylin-eosin (HE) staining under the microscope.The expression of myocardial alpha myosin heavy chain (α-MHC) was detected by immunohistochemistry and Western blot methods.SO2 enzymes aspartate aminotransferase 1 (AAT1) and AAT2 protein expression were detected by Western blot method.Myocardial SO2 content and AAT activity were measured by high performance liquid chromatography with fluorimetric detection and colometric method. Results Compared with control group, the HW/BW and LVW/HW in mice of Ang Ⅱ group were significantly increased(all P<0.01), the cardiac myocytes were hypertrophy, and α-MHC positive staining in the cytoplasm of myocardium was weakened.Moreover, Western blot data showed that α-MHC protein expression in heart tissue of Ang Ⅱ-treated mice was decreased significantly (all P<0.05). Simultaneously, the data showed that AAT2 protein expression, SO2 content and AAT activity in heart tissue of Ang Ⅱ-treated mice were also decreased markedly[(1.093±0.131) μmol/g protein vs.(0.737±0.233) μmol/g protein, P<0.05; (7.979±1.317) U/mg protein vs.(6.470±0.516) U/mg protein, P<0.01]. Furthermore, there was a negative correlation between LVW/HW and cardiac SO2 content in heart tissue (r=-0.56, P<0.05). Conclusions Myocardial endogenous SO2/AAT2 pathway is down-regulated in the development of myocardial hypertrophy induced by Ang Ⅱ in mice. Key words: Myocardial hypertrophy; Sulfur dioxide; Angiotensin Ⅱ

Cervical small cell neuroendocrine tumor mutation profiles via whole exome sequencing.

Cervical small cell neuroendocrine tumors (CSCNETs) are rare, aggressive neuroendocrine tumors (NETs). Reliable diagnostic and prognostic CSCNET markers are lacking, making diagnosis and prognosis prediction difficult, and treatment strategies limited. Here we provide mutation profiles for five tumor-normal paired CSCNETs using whole exome sequencing (WES). We expanded our assessment of frequently mutated genes to include publicly available data from 55 small intestine neuroendocrine tumors, 10 pancreatic neuroendocrine tumors, 42 small cell lung cancers, six NET cell lines, and 188 cervical cancers, along with our five CSCNETs. We identified 1,968 somatic mutations, including 1,710 missense, 106 nonsense, 144 splice site, 4 lncRNA, 3 nonstop, and 1 start codon mutation. We assigned functions to the 114 most frequently mutated genes based on gene ontology. ATRX, ERBB4, and genes in the Akt/mTOR pathway were most frequently mutated. Positive cytoplasmic ERBB4 immunohistochemical staining was detected in all CSCNET tumors tested, but not in adjacent normal tissues. To our knowledge, this study is the first to utilize WES in matched CSCNET and normal tissues to identify somatic mutations. Further studies will improve our understanding of how ATRX and ERBB4 mutations and AKT/mTOR signaling promote CSCNET tumorigenesis, and may be leveraged in novel anti-cancer treatment strategies.

Expression of stem cell markers in oral cavity and oropharynx squamous cell carcinoma

The aim of this study was to analyze the expression of CD24, CD44, CD133, ALDH1, CD29 (integrin-β1), and Ki-67 in squamous cell carcinoma of the oral cavity and oropharynx. Fifty-two tumors and 21 metastatic lymph nodes were evaluated by using immunohistochemistry. Seven of 52 cases (13.5%) showed positive cytoplasmic staining of aldehyde dehydrogenase 1; integrin-β1 was expressed in 45 of 50 cases (90%); 30 of 52 cases (57.7%) had positive membranous staining of CD44; CD24 was expressed in 44 of 50 cases (88%); and three of 52 cases (5.8%) stained positively for membranous CD133. Median proliferation rate, measured by Ki-67, was 37.1% for tumors. Five-year cancer-specific survival rates for the CD44-negative and CD44-positive groups were 74% and 38%, respectively, although this difference did not reach statistical significance (P=.052). Our study demonstrated the expression of putative stem cell markers in squamous cell carcinoma of the oral cavity and oropharynx, with participation of CD44-positive cells in association with poor survival outcome.

Leucine zipper, down regulated in cancer-1 gene expression in prostate cancer.

Numerous genetic alterations have been implicated in the development of prostate cancer (PCa). DNA and protein microarrays have enabled the identification of genes associated with apoptosis, which is important in PCa development. Despite the molecular mechanisms are not entirely understood, inhibition of apoptosis is a critical pathophysiological factor that contributes to the onset and progression of PCa. Leucine zipper, down-regulated in cancer 1 (LDOC-1) is a known regulator of the nuclear factor (NF)-mediated pathway of apoptosis through the inhibition of NF-κB. The present study investigated the expression of the LDOC-1 gene in LNCaP, PC-3, PNT1A and PNT2 prostate cell lines by reverse transcription-quantitative polymerase chain reaction. In addition LDOC-1 protein expression in normal prostate tissues and PCa was studied by immunohistochemistry. LDOC-1 messenger RNA resulted overexpressed in LNCaP and PC-3 PCa cell lines compared with the two normal prostate cell lines PNT1A and PNT2. The results of immunohistochemistry demonstrated a positive cytoplasmic LDOC-1 staining in all PCa and normal prostate samples, whereas no nuclear staining was observed in any sample. Furthermore, a more intense signal was evidenced in PCa samples. LDOC-1 gene overexpression in PCa suggests an activity of LDOC-1 in PCa cell lines.

Abstract 1792: Phospho-IGF-1R/IR provides independent prognostic information in primary breast cancer but is not associated with prognosis in endocrine-treated patients

Abstract Introduction The insulin-like growth factor (IGF) system is implicated in several cancers including breast cancer. Phosphorylated IGF type I receptor/insulin receptor (pIGF-1R/IR) indicates active IGF signaling. The signaling network may interfere with several treatment mechanisms and be involved in endocrine resistance. However, the prognostic value of pIGF-1R/IR in breast cancer is unclear. Aim and methods In order to elucidate the significance of activated IGF-IR/IR in breast cancer, the level of receptor phosphorylation was evaluated by immunohistochemistry on invasive breast cancer tissue microarrays from 984 primary breast cancer patients included in a population-based cohort in Sweden. Patients were followed for up to 11 years, the median follow-up for patients still at risk was five years. Phospho-IGF-1R/IR in relation to prognosis was analyzed using Cox regression, adjusted for age, invasive tumor size, axillary lymph node involvement, histological grade, estrogen receptor (ER) status, and body mass index. Results Phospho-IGF-1R/IR status was available for 902 patients (91.7%). Any positive cytoplasmic or membranous staining for pIGF-1R/IR was present in 749 patients (83.0%). pIGF-1R/IR+ was associated with ER+ OR (1.76; 95% CI 1.09-2.83). During follow-up, 112 patients had a first breast cancer event. Patients with pIGF-1R/IR positive tumors had lower risk for any breast cancer event compared to patients without pIGF-1R/IR (Log Rank P = 0.016), adjusted HR (0.60; 95% CI 0.40-0.91). In patients with ER+ tumors (n = 792, 87.9%), pIGF-1R/IR was evaluated in relation to prognosis and endocrine treatment. In patients who had not received endocrine treatment, pIGF-1R/IR was associated with significantly lower risk for any breast cancer event adjusted HR (0.37; 95% CI 0.15-0.92). In contrast, pIGF-1R/IR was not associated with prognosis in endocrine-treated patients adjusted HR (0.90; 95% CI 0.50-1.63). Conclusions These results indicate that IGF-1R/IR activation status may add independent prognostic information in primary breast cancer. In contrast to previous studies, pIGF-1R/IR+ was associated with better prognosis overall in this cohort. However, pIGF-1R/IR+ was not associated with prognosis in endocrine-treated patients. Taken together, these results are in line with the negative results from clinical trials with IGF-1R inhibitors in breast cancer patients, and highlight the complexity of the IGF-system. Citation Format: Sofie Björner, Ann Rosendahl, Maria Simonsson, Andrea Markkula, Karin Jirström, Signe Borgquist, Carsten Rose, Christian Ingvar, Helena Jernström. Phospho-IGF-1R/IR provides independent prognostic information in primary breast cancer but is not associated with prognosis in endocrine-treated patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1792.

Intracellular location of BRCA2 protein expression and prostate cancer progression in the Swedish Watchful Waiting Cohort.

Prostate cancer patients with inherited BRCA2 mutations have a survival disadvantage. However, it is unknown whether progression is associated with BRCA2 protein expression in diagnostic prostate cancer tissue, among men without inherited mutations. We conducted a nested case-control study within the Swedish Watchful Waiting cohort. The case group included all 71 patients who died from prostate cancer within 5 years from diagnosis and controls were all patients (n = 165) who lived at least 7 years after diagnosis. Tissue microarrays were stained using antibodies for C- and N-terminal domains of the BRCA2 protein. Location (nuclear, cytoplasmic and membranous) and magnitude (intensity and percentage) of expression were assessed. Logistic regression models produced odds ratios (OR) and 95% confidence intervals (CI) adjusted for age, year of diagnosis and Gleason score. Positive BRCA2 staining at the cell membrane was associated with reduced risk of death within 5 years (N-terminal: OR = 0.47, 95% CI = 0.21-1.04, P = 0.06; C-terminal: OR = 0.41, 95% CI = 0.18-0.91, P = 0.03) and low Gleason scores (P = 0.006). Positive cytoplasmic C-terminal staining was associated with higher Gleason scores and increased lethality (OR = 3.61, 95% CI = 1.61-8.07, P = 0.002). BRCA2 protein expression at the cell membrane and lack of C-terminal expression in the cytoplasm were associated with a reduced risk of rapidly fatal prostate cancer. BRCA2 protein expression in prostate cancer tissue may have independent prognostic value. The potential biological significance of BRCA2 expression at the cell membrane warrants further investigation.

Folic acid restores endothelial function in ACTH-induced hypertension

Hypertension is associated with increased oxidative stress and vascular endothelium dysfunction. The aim was to study the effect of folic acid (FA) on hypertension, blood nitric oxide (NO), homocysteine (HCY), malondialdehyde (MDA) and reduced glutathione (GSH); aortic tissue glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD); and vascular endothelial function in adrenocorticotrophic hormone (ACTH)-induced hypertension rats. Rats were treated with saline or FA alone (0.04g/L/day orally, control), or subcutaneous ACTH-induced hypertension (0.2mg/kg/day, ACTH) groups. Treated FA groups were started before (Folic–ACTH, prevention) and during (ACTH–Folic, reversal) ACTH administrations. Systolic blood pressure (SBP), thymus/body weight ratio, blood urea, creatinine, NO, HCY, MDA and GSH; aortic endothelium-dependent vasodilator (EDD) in response to acetylcholine (ACh), aortic tissue extract for CAT, GPx, and SOD activity; and histopathological changes of aorta and kidney were assessed. Saline or FA alone did not change SBP (P>0.05). FA, in prevention study, significantly decreased SBP, increased serum NO and GSH, enhanced relaxation response (EDD%) to 1×10−4M ACh; increased aortic tissue GPx, CAT and SOD activity, also revealed nearly normal endothelial cell layer and moderately positive cytoplasmic staining for CD34+ expression versus ACTH-treated rats (P<0.05). In contrast, FA, in reversal study, did not show significant changes in most of the measured parameters as ACTH-treated group (P>0.05). FA can be used as an adjuvant therapy for prevention and treatment of ACTH-induced hypertension. The protective role of FA in ACTH-induced hypertension could be attributed via decreasing HCY, MDA (oxidative stress); increasing NO, GSH, GPx, CAT, SOD activity (antioxidants); and restoring endothelial dysfunction.

Strong expression of polypeptide N-acetylgalactosaminyltransferase 3 independently predicts shortened disease-free survival in patients with early stage oral squamous cell carcinoma.

The polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts) family of enzymes regulates the critical initial steps of mucin-type O-glycosylation. Among GalNAc-Ts that may significantly influence cancer biology, thus affecting cell differentiation, adhesion, invasion, and/or metastasis, GalNAc-T3 exhibits a high expression in several human cancers, closely associated with tumor progression and a poor prognosis. However, the expression pattern of GalNAc-T3 in oral squamous cell carcinoma (OSCC) remains obscure. Since postoperative recurrence of even early stage OSCC (ESOSCC) occurs at an early phase, significantly affecting their clinical course and worse outcome, the identification of clinically significant accurate biomarkers is needed. Therefore, we investigated the correlation between the immunohistochemical GalNAc-T3 expression and various clinicopathological characteristics and recurrence using 110 paraffin-embedded tumor samples obtained from patients with surgically resected ESOSCC (T1–2N0). Recurrence was recognized in 37 of 110 (33.6 %) patients. The GalNAc-T3 expression was considered to be strongly positive when 20 % or more of the cancer cells showed positive cytoplasmic staining. Consequently, a strong expression of GalNAc-T3 was observed in 40 patients (36.4 %), showing a close relationship to poor differentiation, the presence of lymphatic and vascular invasion, and recurrence. Univariate and multivariate analyses further demonstrated that the patients with a strong GalNAc-T3+ status had markedly lower disease-free survival (DFS) rates, especially within the first 2 years postoperatively. Therefore, GalNAc-T3 might play a role in the pathogenesis of ESOSCC recurrence, and its immunohistochemical detection potentially predicts a shorter DFS and may be a useful parameter for providing clinical management against ESOSCC in the early postoperative phase.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-015-3928-7) contains supplementary material, which is available to authorized users.

The Effect and Mechanism of Celecoxib in Hypoxia-Induced Survivin Up-Regulation in HUVECs

Background/Aims: To investigate the roles of hypoxia-inducible factor 1α (HIF-1α), cyclooxygenase-2 (Cox-2) and its product, Prostaglandin E2 (PGE₂), in the mechanisms underlying hypoxia-induced survivin expression in human umbilical vein endothelial cells (HUVECs) and to examine the effect of celecoxib, a selective Cox-2 inhibitor, on survivin expression. Methods: HUVECs were exposed to a normal (95% O₂) or hypoxic (3% O₂) environment for 24 hrs. We observed the localized expression of survivin, Cox-2 and HIF-1α in HUVECs using immunocytochemistry and detected the inhibitory effects of celecoxib on the growth of HUVECs using an MTT assay. mRNA and protein levels of Cox-2, HIF-1α and survivin were determined by real-time PCR and Western blot analysis under hypoxic conditions for 0, 6, 12, or 24 hrs. The time course changes of HIF-1α and survivin protein expression induced by cobalt chloride (CoCl₂) were studied using Western blot analysis. We then treated HUVECs under hypoxia for 24 hrs with celecoxib (a Cox-2 selective inhibitor), genistein (a HIF-1α inhibitor) or exogenous PGE₂ to further investigate the changes in hypoxia-induced survivin expression. Results: Following 24 hrs of hypoxic treatment, cells exhibited strongly positive survivin, HIF-1α and Cox-2 cytoplasmic staining. Celecoxib (65 μM) effectively inhibited cell proliferation under hypoxic conditions. The protein and mRNA levels of Cox-2, HIF-1α and survivin were increased under hypoxia. The patterns of HIF-1α and survivin expression induced by CoCl₂ were similar to those induced by exposure to hypoxia. Genistein partially blocked survivin expression. Celecoxib reversed the hypoxia-induced survivin expression, whereas the addition of PGE₂ partially restored this effect. Conclusions: Hypoxia-induced survivin expression in HUVECs may be mediated by dual interdependent mechanisms directly involving HIF-1α and indirectly involving the Cox-2/PGE₂ pathways. Celecoxib may offset hypoxia-induced survivin expression.

Expression of the p53 target Wig-1 is associated with HPV status and patient survival in cervical carcinoma.

The p53 target gene WIG-1 (ZMAT3) is located in chromosomal region 3q26, that is frequently amplified in human tumors, including cervical cancer. We have examined the status of WIG-1 and the encoded Wig-1 protein in cervical carcinoma cell lines and tumor tissue samples. Our analysis of eight cervical cancer lines (Ca Ski, ME-180, MS751, SiHa, SW756, C-4I, C-33A, and HT-3) by spectral karyotype, comparative genomic hybridization and Southern blotting revealed WIG-1 is not the primary target for chromosome 3 gains. However, WIG-1/Wig-1 were readily expressed and WIG-1 mRNA expression was higher in the two HPV-negative cervical cell lines (C33-A, HT-3) than in HPV-positive lines. We then assessed Wig-1 expression by immunohistochemistry in 38 cervical tumor samples. We found higher nuclear Wig-1 expression levels in HPV-negative compared to HPV positive cases (p = 0.002) and in adenocarcinomas as compared to squamous cell lesions (p<0.0001). Cases with moderate nuclear Wig-1 staining and positive cytoplasmic Wig-1 staining showed longer survival than patients with strong nuclear and negative cytoplasmic staining (p = 0.042). Nuclear Wig-1 expression levels were positively associated with age at diagnosis (p = 0.023) and histologic grade (p = 0.034). These results are consistent with a growth-promoting and/or anti-cell death function of nuclear Wig-1 and suggest that Wig-1 expression can serve as a prognostic marker in cervical carcinoma.

Nuclear expression of the deubiquitinase CYLD is associated with improved survival in human hepatocellular carcinoma.

Background & AimsThe deubiquitinase CYLD removes (K-63)-linked polyubiquitin chains from proteins involved in NF-κB, Wnt/ß-catenin and Bcl-3 signaling. Reduced CYLD expression has been reported in different tumor entities, including hepatocellular carcinoma (HCC). Furthermore, loss of CYLD has been shown to contribute to HCC development in knockout animal models. This study aimed to assess subcellular CYLD expression in tumor tissues and its prognostic significance in HCC patients undergoing liver resection or liver transplantation.MethodsSubcellular localization of CYLD was assessed by immunohistochemistry in tumor tissues of 95 HCC patients undergoing liver resection or transplantation. Positive nuclear CYLD staining was defined as an immunhistochemical (IHC) score ≥3. Positive cytoplasmic CYLD staining was defined as an IHC score ≥6. The relationship with clinicopathological parameters was investigated. Cell culture experiments were performed to analyze subcellular CYLD expression in vitro.ResultsCytoplasmic CYLD expression was observed in 57 out of 95 (60%) HCC specimens (cyt°CYLD+). Nuclear CYLD staining was positive in 52 out of 95 specimens (55%, nucCYLD+). 13 out of 52 nucCYLD+ patients (25%) showed a lack of cytoplasmic CYLD expression. nucCYLD+ was associated with prolonged overall survival in patients after resection or liver transplantation (P = 0.007). 5-year overall survival rates were 63% in nucCYLD+ vs. 26% in nucCYLD- patients. Nuclear CYLD staining strongly correlated with tumor grading (P<0.001) and Ki67 positivity (P = 0.005). nucCYLD+ did not prove to be an independent prognostic parameter. In vitro, Huh7, Hep3B and HepG2 showed reduced CYLD levels compared to the non-malignant liver cell line THLE-2. Induction of CYLD expression by doxorubicin treatment led to increased cytoplasmic and nuclear expression of CYLD.ConclusionsExpression of nuclear CYLD is a novel prognostic factor for improved survival in patients with HCC undergoing liver resection or transplantation.

Prognosis significance of HER2 status and TACC1 expression in patients with gastric carcinoma.

HER2 amplification and/or expression occurs in gastric carcinoma (GC), but the role of HER2 in the prognosis of GC remains unclear. The dysregulation of transforming acidic coiled coil 1 (TACC1), a downstream gene of HER2, is thought to be involved in the development of GC. The aim of this study was to investigate the role and relationship of HER2 and TACC1 in GC. The expression of HER2 and TACC1 was analyzed using immunohistochemistry on 129 primary resected GC patients, and HER2 amplification was additionally determined by FISH. The data on clinicopathological features and relevant prognostic factors in these patients were analyzed. The expression (3+, 2+ and 1+) and the amplification of HER2 was observed in 57 cases (44.2 %) and 25 cases (19.4 %), respectively, and the correlation between HER2 expression and amplification was strong (p < 0.001). According to the FDA criteria, 24 cases (18.6 %) would have been considered as HER2 positive. A total 62 (48.1 %) GC tissues showed positive cytoplasmic staining of TACC1. There was a significant and positive association between TACC1 and HER2. HER2 positive was significantly associated with TNM stage (p = 0.019), and TACC1 expression was significantly associated with lymph node metastasis (p = 0.004) and TNM stage (p = 0.004). TNM stage, TACC1 expression and co-positive of both HER2 and TACC1 were independent prognostic factors. TACC1 expression is an independent prognostic indicator of GC. The correlation between TACC1 expression and HER2-positive status indicated a possible synergistic regulation of the two molecules and co-positive of both HER2 and TACC1 maybe a more valuable prognostic marker.

Immunohistochemical expression of SFRP1 and SFRP3 proteins in normal and malignant reproductive tissues of rats and humans.

Secreted frizzled-related proteins 1 and 3 (SFRP1 and SFRP3) act as Wnt signaling pathway antagonists and play an important role in embryonic development and carcinogenesis. The aim of the present study was to analyze immunohistochemically the distribution of 2 SFRP family proteins, SFRP1 and SFRP3, in an experimental rat model, in normal and intrauterine growth-restricted (IUGR) human placentas, and in a subset of the corresponding human trophoblastic tumors (pure choriocarcinomas and mixed germ cell tumors with choriocarcinoma component). In rats, expression of both SFRP1 and SFRP3 was pronounced in the perimetrium and myometrium, whereas decidual cells showed only occasional positive cytoplasmic staining. The most prominent expression of both proteins was found in blood vessel endothelial cells. Stereological variable of volume density (Vv, mm) showed statistically higher expression of SFRP1 and SFRP3 in human IUGR placentas than in normal pregnancy placentas (P<0.0001). Compared with adjacent normal/benign tissues, reduced expression of SFRP1 and SFRP3 was observed in human trophoblastic tumors (58.5% and 31.25%, respectively), although none of the examined tumors exhibited complete loss of either protein. Our study indicates that increased expression of both SFRP1 and SFRP3 may contribute to the pathogenesis of IUGR placental dysfunction, whereas the loss of these proteins may be involved in the development of human trophoblastic tumors.

Abstract 1849: High nuclear expression of the p53 target Wig-1 is associated with poor prognosis in cervical carcinoma

Abstract Cervical cancer is the third most common cancer in women worldwide, and is the most common cancer in Eastern Africa, South-Central Asia and Melanesia. Persistent HPV infection is an important feature of cervical cancer. The central role of chromosome 3q for progression from low-grade dysplastic lesions to higher grades and to invasive carcinomas has been well established. Gain of 3q can occur in morphologically normal cytology of women who developed cervical carcinomas after only a short latency. The Wig-1 gene (wt p53-induced gene 1) is located at the frequently gained chromosomal region 3q. We examined Wig-1 in eight cervical cancer cell lines (Ca Ski, ME-180, MS751, SiHa, SW 756, C-4I, C-33A, and HT-3) by FISH, Southern, Northern, qRT-PCR, and Western blotting. We did not detect any Wig-1 gene alterations in these cell lines. C33-A and HT-3 with strong Wig-1 mRNA expression were HPV negative while the HPV positive cells only exhibited low / moderate Wig-1 expression by Northern blotting. However, no association was found between Wig-1 protein expression and HPV infection. We also assessed Wig-1 expression in a series of 38 cervical tumors, including both squamous cell carcinoma (SCC) and adenocarcinoma (ADCA), by immunohistochemistry. Interestingly, patients who showed moderate nuclear Wig-1 expression levels and positive cytoplasmic Wig-1 staining in their tumors had significantly better prognosis than patients with high nuclear Wig-1 expression and no cytoplasmic staining (Log Rank P value = 0.042). Moreover, higher nuclear Wig-1 expression levels were associated with HPV negative cervical tumors (P=0.003). These results suggest that a marker like Wig-1 could have considerable impact on prediction of the prognosis in women with cervical cancer. Our previous results indicate that Wig-1 acts as a survival factor. Thus, high levels of nuclear Wig-1 may promote tumor growth and survival. The exact role of Wig-1 at the molecular level during cervical carcinoma development needs further investigation. Citation Format: Lidi Xu, Susanne Muller, Mikael Lerner, Svetlana Lagercrantz, Dan Grandér, Keng-Ling Wallin, Klas G. Wiman, Sonia Andersson, Catharina Larsson. High nuclear expression of the p53 target Wig-1 is associated with poor prognosis in cervical carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1849. doi:10.1158/1538-7445.AM2014-1849

Expression and functions of galectin-7 in ovarian cancer.

There is a critical need to develop effective new strategies for diagnosis and treatment of ovarian cancer. In the present work, we investigated the expression of galectin-7 (gal-7) in epithelial ovarian cancer (EOC) cells and studied its functional relevance. Immunohistochemical analysis of gal-7 expression in tissue microarrays showed that while gal-7 was not detected in normal ovarian tissues, positive cytoplasmic staining of gal-7 was detected in epithelial cells in all EOC histological subtypes but was more frequent in high grade tumors and metastatic samples. Gal-7 expression correlated with a significant difference in the overall survival of patients with ovarian serous cystadenocarcinoma. Furthermore, using human EOC cell lines, we found that gal-7 expression was induced by mutant p53. Mechanistically, Matrigel invasion assays and live cell imaging showed that gal-7 increased the invasive behavior of ovarian cancer cells by inducing MMP-9 and increasing cell motility. EOC cells can also secrete gal-7. Recombinant human gal-7 kills Jurkat T cells and human peripheral T cells, suggesting that gal-7 also has immunosuppressive properties. Taken together, our study validates the clinical significance of gal-7 overexpression in ovarian cancer and provides a rationale for targeting gal-7 to improve the outcome of patients with this disease.

Serum anti-osteopontin autoantibody as a novel diagnostic and prognostic biomarker in patients with hepatocellular carcinoma.

Osteopontin (OPN) is a secreted phosphorylated and glycosylated protein, which plays an important role in carcinogenesis and metastasis. In hepatocellular carcinoma (HCC), OPN is being investigated either as a therapeutic target gene or as a biomarker for diagnosis. Yet, the role of the anti-OPN autoantibody in HCC remains unclear. In the present study, the level of serum anti-OPN autoantibody in HCC was analyzed by enzyme-linked immunosorbent assay. Immunohistochemistry (IHC) was also performed to analyze protein expression profiles and the prognostic significance of OPN in HCC. In this study, the prevalence and titer of anti-OPN autoantibodies in HCC were significantly higher than these values in normal human serum (NHS) (P=0.001, P=0.000, respectively). When both α-fetoprotein and the autoantibody against OPN were used simultaneously as diagnostic biomarkers, the sensitivity was up to 65%. In IHC, 59 of the 83 (65.6%) HCC specimens expressed OPN with cytoplasmic positive staining. The overall survival (OS) of HCC patients with OPN-positive tumors was 28.81 months compared to 39.37 months for HCC patients with OPN-negative tumors (P<0.01). Furthermore, multivariate analysis showed that OPN overexpression was the strongest independent adverse prognostic factor for OS (P=0.02). Taken together, our data indicate that the anti-OPN autoantibody may be a supplementary serological biomarker for HCC, and is correlated with poor prognosis in HCC patients.

Pheochromocytoma-paraganglioma due to germline SDHB mutation in children are common

Introduction Pheochromoctyoma-paraganglioma syndrome (PHEO-PGL) due to SDH germline mutation is easily identified by loss of SDHB by immunohistochemistry. Pham et al . showed a 16% (5/30) prevalence of SDH mutation in a retrospective review of the Mayo clinic involving thirty patients (1975-2005). Aim Ascertain the prevalence of PHEO-PGL due to SDHB germline mutations in a local paediatric population by using IHC. Method 14 patients were ascertained from the pathology database with pheochromocytoma and or paraganglioma, SDHA and SDHB IHC was performed on FFPE blocks. Results Three patients had SDHB germline mutations, three had VHL mutation and eight had no genetic studies performed. Of the 14 patients, four had multiple tumours and of these, three showed loss of SDHB staining. All three patients with loss of SDHB by IHC had genetically confirmed germline mutations of SDHB. A novel observation was that three patients with VHL syndrome showed a characteristic weak granular cytoplasmic staining, a pattern distinct from SDHB loss and normal controls. Discussion (1) We found the prevalence of SDH germline mutations was 28% in children with PHEO-PGL, which is higher than historically identified. (2) In patients with multiple tumours the prevalence was 75% (3/4 patients). (3) We found a distinctive weak granular positive cytoplasmic SDHB staining pattern in tumours with VHL germline mutations.