EphA2 expression is associated with aggressive features in ovarian carcinoma.

PURPOSE: EphA2 (epithelial cell kinase) is a transmembrane receptor tyrosine kinase that has been implicated in oncogenesis. There are no published data regarding the role of EphA2 in ovarian carcinoma, which is the focus of the present study. EXPERIMENTAL DESIGN: Nontransformed (HIO-180) and ovarian cancer (EG, 222, SKOV3, and A2780-PAR) cell lines were evaluated for EphA2 by Western blot analysis. Five benign ovarian masses, 10 ovarian tumors of low malignant potential, and 79 invasive ovarian carcinomas were also evaluated for EphA2 expression by immunohistochemistry. All samples were scored in a blinded fashion. Univariate and multivariate analyses were used to determine significant associations between EphA2 expression and clinicopathological variables. RESULTS: By Western blot analysis, EG, 222, and SKOV3 cell lines overexpressed EphA2, whereas A2780-PAR and HIO-180 had low to absent EphA2 expression. All of the benign tumors had low or absent EphA2 expression. Among the invasive ovarian carcinomas examined (mean age of patients was 59.2 years), 60 (75.9%) tumors overexpressed EphA2 and the other 19 tumors had negative or minimal EphA2 expression. There was no association of EphA2 overexpression with ascites, likelihood of nodal positivity, pathological subtype, and optimum surgical cytoreduction (residual tumor <1 cm). However, EphA2 overexpression was significantly associated with higher tumor grade (P = 0.02) and advanced stage of disease (P = 0.001). The median survival for patients with tumor EphA2 overexpression was significantly shorter (median, 3.1 years; P = 0.004); the median survival for patients with low or absent EphA2 tumor expression was at least 12 years and has not yet been reached. In multivariate analysis using the Cox proportional hazards model, only volume of residual disease (P < 0.04) and EphA2 overexpression (P < 0.01) were significant and independent predictors of survival. CONCLUSIONS: EphA2 overexpression is predictive of aggressive ovarian cancer behavior and may be an important therapeutic target.     

Bcl-2 expression in cholangiocellular carcinoma is inversely correlated with biologically aggressive phenotypes

bcl-2 is known to play a crucial role in modulating carcinoma progression as well as in inhibiting apoptosis. However, its expression and clinical significance for cholangiocellular carcinoma (CCC) remains unclear. In the present study, we immunohistochemically investigated bcl-2 expression in 41 CCC. Thirteen cases (31.7%) were classified as bcl-2 positive, because more than 10% of the carcinoma cells expressed bcl-2. The expression of bcl-2 was inversely related to lymph node metastasis, vascular invasion, perineural invasion, the Ki-67 labeling index, aberrant p53 expression and the incidence of apoptotic cells. Furthermore, well or moderately differentiated carcinoma more frequently expressed bcl-2. These results suggest that downregulation of bcl-2 expression is strongly linked to highly biologically aggressive phenotypes of CCC.     

Lipocalin 2 expression is associated with aggressive features of endometrial cancer

ABSTRACT: BACKGROUND: Increased expression of lipocalin 2 (LCN2) has been observed in several cancers. The aim of the present study was to investigate LCN2 in endometrial cancer in relation to clinicopathologic phenotype, angiogenesis, markers of epithelial-mesenchymal transition (EMT), and patient survival. METHODS: Immunohistochemical staining was performed using a human LCN2 antibody on a population-based series of endometrial cancer patients collected in Hordaland County (Norway) during 1981-1990 (n = 256). Patients were followed from the time of primary surgery until death or last follow-up in 2007. The median follow-up time for survivors was 17 years. Gene expression data from a prospectively collected endometrial cancer series (n = 76) and a publicly available endometrial cancer series (n = 111) was used for gene correlation studies. RESULTS: Expression of LCN2 protein, found in 49% of the cases, was associated with nonendometrioid histologic type (p = 0.001), nuclear grade 3 (p = 0.001), >50% solid tumor growth (p = 0.001), ER and PR negativity (p = 0.028 and 0.006), and positive EZH2 expression (p < 0.001). LCN2 expression was significantly associated with expression of VEGF-A (p = 0.021), although not with other angiogenesis markers examined (vascular proliferation index, glomeruloid microvascular proliferation, VEGF-C, VEGF-D or bFGF2 expression). Further, LCN2 was not associated with several EMT-related markers (Ecadherin, N-cadherin, P-cadherin, beta-catenin), nor with vascular invasion (tumor cells invading lymphatic or blood vessels). Notably, LCN2 was significantly associated with distant tumor recurrences, as well as with the S100A family of metastasis related genes. Patients with tumors showing no LCN2 expression had the best outcome with 81% 5-year survival, compared to 73% for intermediate and 38% for the small subgroup with strong LCN2 staining (p = 0.007). In multivariate analysis, LCN2 expression was an independent prognostic factor in addition to histologic grade and FIGO stage. CONCLUSION: Increased LCN2 expression is associated with aggressive features and poor prognosis in endometrial cancer.     

Decreased expression of antioxidant enzymes is associated with aggressive features in ependymomas

The purpose of this study was to investigate the relationship between antioxidant enzyme expression and clinicopathological features in 67 ependymal tumors. We included into the analysis antioxidant enzymes (AOEs) and related proteins, such as, manganese superoxide dismutase (MnSOD), gammaglutamylcysteine synthetase catalytic and regulatory subunits (GLCL-C and GLCL-R), thioredoxin (Trx) and thioredoxin reductase (TrxR). Their expression was studied in 46 primary (10 grade I, 30 grade II and 6 grade III) and 21 recurrent (3 grade I, 12 grade II and 6 grade III) tumors. Immunoreactivity for MnSOD was found in 87%, GLCL-C in 74%, GLCL-R in 89%, Trx in 72%, TrxR in 54%, of primary tumors. Lower GLCL-C and GLCL-R expression was associated with higher tumor grade (P = 0.047 and 0.049, respectively). MnSOD, GLCL-C and TrxR expressions were significantly higher in tumors located in the spinal cord compared to those in the brain (P = 0.044, 0.046 and 0.004, respectively). In the primary tumors Trx-positivity was found to correlate significantly with patient survival. In univariate survival analysis patients whose tumors did not express Trx had shorter survival (P = 0.045) and there was even more significant association (P = 0.011) when only adults were included in the analysis (in the total material median follow-up time of Trx-positive tumors was 9.7 years and of Trx-negative 5.4 years). The results indicate that AOEs have several biological functions in ependymal tumors. Trx had important prognostic value: all adults with Trx-positive tumors were alive at follow-up (median 7.8 years).     

COX-2 expression is associated with an aggressive phenotype in ductal carcinoma in situ

Cyclooxygenase type-2 (COX-2) is overexpressed in malignant tumours including breast cancers, though the mechanism of upregulation is unclear. This study aimed to determine COX-2 expression in ductal carcinoma in situ (DCIS) in comparison to invasive breast cancer (IBC) and normal breast, and also to investigate the relationship of COX-2 expression with HER-2 expression, oestrogen receptor (ER), tumour grade and cellular proliferation (Ki67) in DCIS. Cyclooxygenase type-2, HER-2, ER and Ki67 expression were determined by immunohistochemistry on paraffin tissue sections of DCIS (n=187), IBC (n=65) and normal breast reduction tissue (n=60). Cyclooxygenase type-2 expression in DCIS (67%, P<0.001) and IBC (63%, P<0.001) was significantly greater than in normal breast (23%). There was no difference in COX-2 expression level between DCIS and IBC (P=0.87) or between normal breast from reduction mammoplasty tissue and normal breast ducts around DCIS (22%, P=0.29). In DCIS, COX-2 expression was associated with higher cellular proliferation rates (P<0.0001), nuclear grade (P=0.003), with ER negativity (P=0.003) and with HER-2 positivity (P<0.0001). Cyclooxygenase type-2 expression is upregulated in in situ breast cancer and is associated with surrogate markers of an aggressive DCIS phenotype including nonoestrogen-regulated signalling pathways. Cyclooxygenase type-2 inhibition may potentially prevent the development of ER-positive and ER-negative breast cancers.     

Deletion at 13q21 is associated with aggressive prostate cancers

Previous cytogenetic and molecular genetic analyses suggest that the q21 band of chromosome 13 harbors a tumor suppressor gene(s) involved in prostatic carcinogenesis. The precise genetic location, however, has not been defined. In this study, we examined prostate cancer specimens and cell lines/xenograft for genetic deletions at 13q21, using the methods of tissue microdissection and duplex PCR. Deletions at 13q21 were detected in 13 of 147 (9%) prostate cancer samples. Deletion of the same region was also detected in the LNCaP cell line and the PC-82 xenograft of prostate cancer. The overlapping region of deletion in LNCaP and PC-82 spans 3.1 cM or 2.9 cR, which is equivalent to 1-3 Mb. The endothelin receptor B gene, a possible tumor suppressor gene at 13q21, was not located in the region of deletion. Among the 13 prostate neoplasms with deletion at 13q21, 5 were metastases, and 7 were poorly differentiated primary tumors. The only primary tumor that was not poorly differentiated but had deletion occurred in one of the youngest patients (49 years) at diagnosis. These results provide evidence that 13q21 may harbor an unidentified gene(s) whose inactivation occurs in some aggressive carcinomas of the prostate. In addition, this study provides a framework for the cloning and identification of the 13q21 gene(s).     

Cytoplasmic accumulation of α-catenin is associated with aggressive features in laryngeal squamous-cell carcinoma

Aberrations in the function of α-catenin (α-cat), the anchoring protein of E-cadherin, are believed to cause dysfunction of the cadherin–catenin complex, leading to disturbed cell–cell adhesion. It has been suggested that expression of α-cat in human tumours might be a better indicator of aggressive phenotype than expression of E-cadherin. The value of α-cat as a prognostic marker in laryngeal squamous cell carcinoma (LSCC) is unclear. To determine the potential prognostic significance of α-cat, paraffin-embedded samples from 159 patients with invasive carcinoma left in the section and with long-term follow-up were evaluated immuno-histochemically for α-cat expression, and the results were related to histopathological grade, tumour stage and survival. Two patterns of staining were observed: pure membranous staining (57%) and membranous staining with cytoplasmic involvement (43%). Cytoplasmic involvement of α-cat was associated with dedifferentiation, advanced tumour stage and nodal status. In addition, supra-glottic tumours showed more often cytoplasmic involvement of α-cat than glottic tumours. Patients with cytoplasmic involvement appeared to have a trend towards poor overall survival, though without statistical significance. These results suggest that cytoplasmic involvement of α-cat is associated with aggressive behaviour and metastatic phenotype of LSCC. Int. J. Cancer (Pred. Oncol.) 79:546–550, 1998.© 1998 Wiley-Liss, Inc.     

Reduced expression of vasohibin-1 is associated with clinicopathological features in renal cell carcinoma

Vasohibin-1(VASH1) has recently been isolated as a novel negative feedback inhibitor of angiogenesis. Several studies have demonstrated that VASH1 plays important roles in tumor angiogenesis but the role of this angiogenic inhibitor in renal cell carcinoma (RCC) has not been elucidated until now. In this study, we investigated the expression pattern of VASH1 and the association with clinicopathological features in RCC. Expression of VASH1, hypoxia-inducible factor-1?? (HIF-1??), and microvessel density (MVD, labeled by CD34) was assessed by immunohistochemistry in 46 RCC specimens and 20 adjacent nontumorous renal tissues (ANRTs). Correlation between vasohibin-1 and HIF-1??, MVD, and clinicopathological features was then investigated. In RCC, VASH1 was expressed mainly in the cytoplasm and membrane of tumor cells and partly in vascular endothelial cells. In ANRT, it was mainly expressed in the cytoplasm and membrane of renal tubular epithelial cells and partly in vascular endothelial cells and glomerular mesangial cells. The expression level of VASH1 in RCC tissue was significantly lower than that in ANRT and was significantly reduced with the increased degree of malignancy in RCC tissues. In addition, a significantly negative correlation was noted between VASH1 expression and HIF-1?? expression and a significantly negative correlation was noted between VASH1 expression and MVD in RCC. Therefore, VASH1 expression is reduced and it associates with clinicopathological features in RCC. Based on our findings and the knowledge of other angiogenesis inhibitors, we postulate that VASH1 would potentially be a biomarker and a candidate for molecular targeted therapy for patients with RCC in the future.     

Overexpression of thymidylate synthase (TYMS) is associated with aggressive tumor features and early PSA recurrence in prostate cancer

Thymidylate synthase (TYMS) plays a role in DNA synthesis and is a target for 5-fluorouracil. In this study TYMS was analyzed by immunohistochemistry on a tissue microarray containing 11,152 prostate cancers. TYMS expression was higher in neoplastic than in normal prostate epithelium and was detectable in 72.9% of 10,223 interpretable cancers. It was considered strong in 21.9%, moderate in 33.4% and weak in 17.6% of tumors. TYMS overexpression was associated with deletions at 5q21 (p < 0.0001), 6q15 (p < 0.0001) and 3p13 (p = 0.0083) and gradually increased with the total number of these deletions present in the respective cancer sample (p < 0.0001). TYMS expression was unrelated to PTEN deletions (p = 0.9535) but tightly linked to high Gleason grade, advanced pathological tumor stage and early PSA recurrence (p < 0.0001). The prognostic value of TYMS was independent from the ERG status and deletions at 3p13, 5q21, and 6q15. In multivariate analyses the prognostic role of TYMS expression was independent of Gleason grade, pT stage, preoperative PSA, pN stage, or resection margins. TYMS expression analysis might result in clinically useful information in prostate cancer. The striking link to some but not all chromosomal aberrations might suggest a mechanistical link with specific types of DNA damage.     

Atypical localization of membrane type 1-matrix metalloproteinase in the nucleus is associated with aggressive features of hepatocellular carcinoma

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a versatile proteinase and recent studies indicated it could be internalized. Our earlier study found that it is overexpressed in hepatocellular carcinoma (HCC) and could promote intrahepatic metastasis. The present study was conducted to examine its subcellular localization and its clinicopathological significance in HCC after curative partial hepatectomy. Localization of MT1-MMP in 101 pairs of HCCs and their adjacent liver tissues, and 8 normal liver tissues was examined by the immunohistochemical method. MT1-MMP protein was localized at membrane and cytoplasm of hepatocytes in the normal and tumor adjacent liver tissues. In contrast, the HCCs were highly heterogeneous with variable degrees of membrane, cytoplasmic, and even nuclear staining. Interestingly, patients with presence of nuclear MT1-MMP were associated with poor overall survival (log-rank test, P=0.043) and large tumor size (>5 cm) (Fisher's exact test, P=0.031). Subcellular distribution was further demonstrated by Western blotting and immunofluorescence with Hep3B stable transfectant overexpressing MT1-MMP. Western blot analyses of subcellular fractions confirmed a differential partitioning of various post-translationally modified MT1-MMP in these fractions. Different antibodies corroborated the presence of MT1-MMP in the nuclear fraction. Concomitant nuclear presence of MMP2 with MT1-MMP further indicated its potential involvement in the nuclear functions. MT1-MMP co-localized with caveolin-1 at the perinuclear region, suggesting nuclear translocation of MT1-MMP via caveolae-mediated endocytosis. In summary, the association of nuclear MT1-MMP with aggressive tumor features including poor prognosis and large tumor expands its functional repertoire and further indicates a new functional role of MMPs within nuclei of tumor cells.     

S100A7 (psoriasin) expression is associated with aggressive features and alteration of Jab1 in ductal carcinoma in situ of the breast

Introduction  

The S100A7 (psoriasin) gene is highly expressed in ductal carcinoma in situ (DCIS) of the breast and can be downregulated in invasive carcinoma. Persistent S100A7 expression in invasive carcinoma is associated with a worse prognosis, and this effect may be mediated in part through interaction with the multifunctional cell signaling protein Jab1.     

Decreased adiponectin level is associated with aggressive phenotype of tongue squamous cell carcinoma

Circulating adiponectin levels are inversely associated with risk of various obesity‐related cancers. However, the effect of adiponectin on carcinogenesis and progression of tongue squamous cell carcinoma (TSCC) remains unknown. We measured serum adiponectin levels in 59 patients with TSCC and 50 healthy controls. Expression of adiponectin and its receptors in paired tumor and paracancerous specimens were determined by immunohistochemical staining (n = 37) and western blot (n = 30), respectively. Serum adiponectin level was lower in patients than in controls (5.0 ± 2.4 vs 8.4 ± 3.5 μg/mL, P < 0.01), and was inversely associated with histological grade and lymph node metastasis but not tumor size. Local adiponectin levels in tumor tissue gradually decreased as tumor‐node‐metastasis stage increased, while the expression of adiponectin receptors was unchanged. In addition, serum adiponectin levels in the TSCC patients without metabolic and cardiovascular diseases, or without smoking and drinking habits, were still lower than in controls. Furthermore, adiponectin inhibited the migration, but not proliferation, of SCC15 cells in vitro. These results indicate that a decreased adiponectin level is associated with risk of TSCC. Hypoadiponectinemia might be used as a biomarker to predict an aggressive phenotype of TSCC.     

Survivin expression is significantly linked to the dedifferentiation of thyroid carcinoma

Survivin is a novel member of the apoptosis protein inhibitors, but according to previous reports, it is also significantly linked to cell proliferating activity. In this study, we investigated the expression of survivin in thyroid neoplasms. Survivin was only occasionally expressed in normal follicular cells, whereas in follicular and papillary carcinomas, about 20% of cases were positive for survivin. The incidence was significantly higher in advanced stage papillary carcinoma (p=0.0080) and papillary and follicular carcinomas with poorly differentiated lesions (p=0.0150). In anaplastic carcinoma, survivin positivity was observed in 84% of the cases, which was in significantly higher incidence than in papillary or follicular carcinoma (p<0.0001). These results suggest that survivin is strongly related to the dedifferentiation of thyroid carcinoma.     

Low adipocyte hepatocellular carcinoma is associated with aggressive cancer biology and with worse survival

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, and non-alcoholic fatty liver disease is strongly associated with its development. To explore the role of adipocytes in HCC, we investigated intratumoral adipocytes, also known as cancer-associated adipocytes (CAA). Based on our prior breast cancer findings, we hypothesized that low intratumoral adipocytes would be associated with aggressive cancer biology, worse tumor microenvironment (TME), and clinical outcomes. The Cancer Genome Atlas (TCGA) was used and validated by the Gene Expression Omnibus (GEO) cohort. xCell algorithm was used to quantify intratumoral adipocytes and top 90% were defined as adipocyte high (AH) and bottom 10% as adipocyte low (AL). We found that AL-HCC was significantly associated with worse disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS). AL-HCC were higher-grade, had high MKI67 expression, enriched cell proliferation-related gene sets, and had increased altered fraction, aneuploidy, and homologous recombination defects. Also, anti-cancer immune cells, CD8, Th1, and M1 cells, as well as pro-cancer Th2 cells were increased in AL-HCC. Micro-RNAs miR-122 (associated with cholesterol metabolism) and miR-885 (associated with liver pathologies) were significantly increased in the AL TME. In conclusion, we found that AL-HCC has worse patient outcomes and is biologically more aggressive with enhanced cell proliferation. Our findings take initial steps to clarify the role of adipocytes in HCC.     

Down-regulation of CD9 expression during prostate carcinoma progression is associated with CD9 mRNA modifications.

PURPOSE: Cluster-of-differentiation antigen 9 (CD9) protein, a member of the tetraspanin family, has been implicated in carcinogenesis of various human tumors. Although decreased expression of the CD82 tetraspanin protein, a close CD9 relative, is associated with prostate cancer progression, CD9 expression has not been analyzed in this malignancy. EXPERIMENTAL DESIGN: CD9 expression in human prostatic adenocarcinoma was analyzed by immunohistochemistry on 167 primary tumors and 88 lymph node or bone metastases. CD9 cDNA was sequenced from two human prostate cancer cell lines, prostatic adenocarcinoma, high-grade prostatic intraepithelial neoplasia (PIN), and normal prostatic tissues. RESULTS: Although CD9 was detected in the epithelium of normal prostatic tissues, reduced or loss of CD9 expression within neoplastic cells was observed in 24% of 107 clinically localized primary adenocarcinomas, 85% of 60 clinically advanced primary adenocarcinomas, 85% of 65 lymph node metastases, and 65% of 23 bone metastases. Difference in CD9 expression between clinically localized and advanced diseases was highly significant (P < 1 x 10(-7)). Whereas there was no alteration of CD9 cDNA in normal tissues, all PC-3-derived cell lines, one PIN, and four prostatic adenocarcinomas harbored deletions in their CD9 cDNAs. Recurring CD9 point mutations were also found in PC-3M-LN4 cells, one PIN, and seven prostatic adenocarcinomas. CONCLUSIONS: CD9 expression is significantly reduced and even lost during prostate cancer progression. Moreover, deletions and mutations of the CD9 mRNA may be associated with loss of protein expression observed in tumor cells. Our data suggest that CD9 inactivation may play an important role in prostate cancer progression.     

Increased C-CRK proto-oncogene expression is associated with an aggressive phenotype in lung adenocarcinomas

The C-CRK gene, cellular homolog of the avian v-crk oncogene, encodes two alternatively spliced adaptor signaling proteins, CRKI (28 kDa) and CRKII (40 kDa). Both CRKI and CRKII have been shown to activate kinase signaling and anchorage-independent growth in vitro and CRKI transformed cells readily form tumors in nude mice. Affymetrix oligonucleotide arrays were used to analyse 86 lung adenocarcinomas and 10 uninvolved lung tissues. C-CRK mRNA expression was increased in more advanced (stage III versus stage I), larger (T(2-4) versus T(1)), and poorly differentiated tumors and in tumors from patients demonstrating poor survival (P=0.00034). An overlapping series of 93 lung adenocarcinomas (64 stage I and 29 stage III) and 10 uninvolved lung specimens were measured for quantitative differences in CRKI and CRKII protein levels using 2-D PAGE. CRK protein spots were identified using mass spectrometry and 2-D Western blotting. A significant increase in levels of the CRKI oncoprotein and the phosphorylated isoform of CRKII was observed in tumors (P<0.05). No difference in protein level was evident between stages. Concordant with mRNA expression, CRKI and CRKII were increased in poorly differentiated tumors (P<0.05). CRK immunohistochemical analysis of tumor tissue arrays using the same tumor series also demonstrated increased abundance of nuclear and cytoplasmic CRK in more proliferative tumors (P<0.05). This study provides the first quantitative analysis of discrete CRKI and CRKII protein isoforms in human lung tumors and provides evidence that the C-CRK proto-oncogene may foment a more aggressive phenotype in lung cancers.     

Loss of p63 and cytokeratin 5/6 expression is associated with more aggressive tumors in endometrial carcinoma patients

p63 and cytokeratin (CK) 5/6 are markers of basal and squamous differentiation in several normal epithelia and human tumors and are also suggested to be markers of progenitor or stem cells in certain stratified epithelia. In endometrial carcinoma, there is very limited information about the expression pattern of p63 or CK5/6 and no prognostic information. The aim of our study was to examine whether the expression of these markers was associated with a certain tumor phenotype in terms of other biomarkers, clinicopathologic characteristics and patient prognosis. Immunohistochemical expression of p63 and CK5/6 was examined using tissue microarrays (TMAs) in a large population-based series of 276 endometrial carcinomas with long and complete follow-up. Selected cases of normal and hyperplastic endometrium were examined for comparison (n = 15). Absence of p63 expression (70%) was significantly associated with nonendometrioid carcinomas, high histologic grade (FIGO), higher mitotic count and tumor cell proliferation by Ki-67, microsatellite instability (MSI) and loss of hMSH6 expression. A tendency toward reduced patient survival was also seen (p = 0.098). Presence of CK5/6 expression was more frequent in endometrioid tumors with squamous differentiation, while loss of CK5/6 expression (54%) was significantly associated with high FIGO stage, reduced beta-catenin expression, MSI and reduced patient survival (p = 0.0001); the latter was also found within the endometrioid subgroup (p = 0.0004). Multivariate survival analysis revealed that loss of CK5/6 expression had an independent prognostic impact in addition to well-known prognostic variables. Expression of both markers was increased in simple hyperplasia compared with normal endometrium. In complex hyperplasia, p63 expression was also increased, whereas CK5/6 was positive in areas with squamous differentiation only. Thus, loss of p63 or CK5/6 was associated with features of aggressive tumors, and lack of CK5/6 was significantly associated with reduced survival in multivariate analysis.