Tissue microarray-based analysis shows phospho-beta-catenin expression in malignant melanoma is associated with poor outcome

Beyond depth of invasion, there are very few prognostic markers to predict outcome in melanoma. It has been shown recently that the beta-catenin oncogene is mutated or shows altered subcellular localization suggesting that activation of beta-catenin mediated signaling plays a role in oncogenesis. We hypothesize that assessment of activated beta-catenin, as detected by a phospho-specific antibody, may be useful to predict outcome in melanoma. We use immuno-histochemical analysis of beta-catenin and phospho-beta-catenin, first to verify the specificity of the phospho-beta-catenin antibody and then to assay expression in a tissue microarray-based study. The subcellular localization of beta-catenin is membranous in some cases and cytoplasmic and nuclear in others. We validate the specificity of a ser33/37/thr41 phospho-beta-catenin antibody in transfected cells and show that the expression is almost exclusively localized to the nucleus in both cultured cells and human tissue. Evaluation of both total and phospho-beta-catenin antibodies showed that cytoplasmic/nuclear staining was more common in primary lesions, whereas nuclear phospho-beta-catenin was more common in metastatic lesions. High levels of nuclear phospho-beta-catenin are associated with significantly worse overall survival (51% vs. 25% overall survival at 5 years, p = 0.046). These results suggest that phospho-specific antibodies to beta-catenin define a unique subset of cases and that monitoring of phospho-beta-catenin expression may be useful for assessing prognosis in malignant melanoma.     

Quantitative in situ analysis of beta-catenin expression in breast cancer shows decreased expression is associated with poor outcome

The role of beta-catenin in breast cancer and its prognostic value is controversial. The prognostic value had been assessed previously in a series of nonquantitative immunohistochemical studies with conflicting results. In efforts to clarify the relationship between beta-catenin protein expression and breast cancer prognosis, we have assessed a retrospective 600 case cohort of breast cancer tumors from the Yale Pathology archives on tissue microarrays. They were assessed using automated quantitative analysis (AQUA) with a series of array-embedded cell lines for which the beta-catenin concentration was standardized by an ELISA assay. The expression levels of the standard clinical markers HER2, estrogen receptor (ER), progesterone receptor (PR), and Ki-67 were also assessed on the same cohort. X-tile software was used to select optimal protein concentration cutpoints and to evaluate the outcome using a training set and a validation set. We found that low-level expression of membranous beta-catenin is associated with significantly worse outcome (38% versus 76%, 10-year survival, validation set log-rank P = 0.0016). Multivariate analysis of this marker, assessed in a proportional hazards model with tumor size, age, node status, nuclear grade, ER, PR, HER2, and Ki-67, is still highly significant with a hazard ratio of 6.8 (P < 0.0001, 95% confidence interval, 3.1-15.1). These results suggest that loss of beta-catenin expression at the membrane, as assessed by objective quantitative analysis methods, may be useful as a prognostic marker or may be part of a useful algorithm for prognosis in breast cancer.     

ATG16L2 overexpression is associated with a good prognosis in colorectal cancer

BackgroundColorectal cancer (CRC) is a highly aggressive, high-incidence malignancy. Several biomarkers associated with the prognosis and metastasis of CRC have been identified. Our study aimed to evaluate the value of ATG16L2 protein as a new biomarker to predict the prognosis of patients with CRC.MethodsOne hundred and fifty-two pairs of paraffin-embedded tissue samples and 19 fresh tissue samples were collected from the Department of Pathology of Renji Hospital, Shanghai Jiao Tong University School of Medicine. All the patients had undergone surgery in the hospital’s Department of Gastrointestinal Surgery between 2013 and 2014. The samples were arranged on two tissue microarrays of normal (n=152) and tumor (n=152) tissue. The tissues were immunostained and graded as low (<50%) or high (≥50%) according to the proportion of ATG16L2-positive cells. An overexpression plasmid was constructed and transfected into RKO cells, and the cell proliferation and migration ability were detected. Finally, Flag-ATG16L2 RKO cells subcutaneous injection into the skin of BALB/c nude mice to determine the effects of ATG16L2 on the growth of subcutaneously transplanted tumors.ResultsATG16L2 expression was negatively correlated with lymph node metastasis (P<0.05) and tumor-node-metastasis stage (P<0.05). High ATG16L2 expression in tumor tissues was related to a good prognosis, with patients with a high expression of ATG16L2 displaying longer overall survival. In vitro, overexpression of ATG16L2 in a CRC cell line RKO cell led to a decrease in cell proliferation but had no obvious influence on cell migration. In vivo, the mice in the Flag-NC (as control) group exhibited faster tumor growth than those in the experiment group.ConclusionsATG16L2 expression is positively associated with patient prognosis in CRC. Further, ATG16L2 can negatively affect CRC cell proliferation in vitro and in vivo.     

Prognostic and diagnostic significance of beta-catenin nuclear immunostaining in colorectal cancer.

In the present study, we investigated the prognostic and diagnostic significance of beta-catenin nuclear immunostaining in 60 specimens of normal colorectal tissue; 180 specimens of colorectal polyps, adenomas, and carcinomas; and 40 specimens from patients with the simultaneous occurrence of polyps, adenomas, and carcinomas. Additional specimens from 59 patients with colorectal carcinoma and 14 patients with adenoma who subsequently developed carcinoma were examined for possible survival study. Immunohistochemical staining showed that the occurrence of nuclear beta-catenin correlated with the sequential stages in colorectal carcinogenesis, in which positive staining was observed in 0% of normal tissues, 8% of polyps, 92% of adenomas, and 100% of carcinomas. High immunohistochemical scores in colorectal carcinoma were significantly associated with lymph node metastasis and poor survival. Adenomas associated with synchronous or metachronous carcinomas showed significantly higher levels of nuclear beta-catenin compared with adenomas without associated carcinomas. Nuclear translocation of beta-catenin was rare or absent in other types of cytokeratin 20 positive adenocarcinomas examined (99 cases). Thus, it was positive in only 7% of colonic mucinous adenocarcinomas, 3% of pancreatic adenocarcinomas, 8% of ovarian mucinous cystadenocarcinomas, and 0% of gastric adenocarcinomas. However, 100% of primary and metastatic colorectal adenocarcinomas were positive for nuclear staining for beta-catenin. Thus, nuclear staining for beta-catenin may serve as an additional parameter to help distinguish colorectal adenocarcinomas from adenocarcinomas of other tissue sites. Collectively, the present large-scale study has clearly addressed the clinical significance of beta-catenin nuclear translocation with respect to tumor progression, survival, and differential diagnosis.     

High ALDH2 expression is associated with better prognosis in patients with gastric cancer

The relationship among alcohol, acetaldehyde, and gastric cancer (GC) is a very interesting research direction. Although many studies have focused on the correlation between ALDH2 polymorphism and GC, ALDH2 expression in GC and its relationship with the prognosis of GC patients remain to be fully understood. To explore these, 455 GC cases were included in this study. The relationships of ALDH2 expression with patients’ survival and clinicopathological characteristics were assessed. The immune infiltration characteristics of ALDH2 in GC were also analyzed. Furthermore, the gene regulatory network and functional pathways of ALDH2 in GC were investigated. We found that high expression of ALDH2 was associated with better prognosis in GC patients. GC patients with high ALDH2 expression had a lower degree of pathological malignancy, consistent with our hypothesis that ALDH2 may play as a tumor suppressor role in GC. Mechanistically, ALDH2 may cooperate with genes such as C5orf32, TSPAN8 and RILP to inhibit GC progression via regulating multiple signaling pathways and chemical carcinogenesis. Therefore, our study suggested that ALDH2, an important variant gene in Asians, might serve as a prognostic marker and a potential therapeutic target for patients with GC.     

DBC1 is over-expressed and associated with poor prognosis in colorectal cancer

Background

Deleted in breast cancer 1 (DBC1) was initially cloned from a region homozygously deleted in breast cancers, but its role in colorectal cancer remains unknown. The present study aims to examine the expression level of DBC1 and assess its prognostic value in human colorectal cancer.

Methods

Immunohistochemical staining was performed to detect the expression level of DBC1 in a series of 186 colorectal cancer patients. Immunohistochemical staining results were analyzed and compared statistically with various clinicopathological characters and overall survival.

Results

Compared with the corresponding non-tumor tissues, a higher expression level of DBC1 was detected in colorectal cancer (P < 0.01). Tissue microarray analysis revealed that DBC1 expression is significantly associated with tumor histological grade, TNM stage and metastatic status (P < 0.01). Importantly, Kaplan–Meier analysis showed that DBC1 expression is associated with shorter overall survival (P < 0.01). Univariate Cox regression suggested that DBC1 expression, poorly differentiation status and the presence of lymph node metastasis predict shorter overall survival in colorectal cancer (P < 0.05). Multivariate Cox regression analysis indicated that DBC1 acts as an independent prognostic factor in colorectal cancer (P < 0.01).

Conclusions

These results suggest that DBC1 is over-expressed in colorectal cancer and that it might serve as a predictor for selecting patients at high risk of poor prognosis.     

Tissue microarray analysis of EGFR gene amplification and gain in Bulgarian patients with colorectal cancer

Colorectal cancer is one of the most common neoplastic diseases and a leading cause of cancer-related deaths. Overexpression of epidermal growth factor receptor (EGFR) is commonly associated with this cancer. The aim of our study was to determine the frequency of epidermal growth factor receptor gene amplification and gain in a large number of colorectal carcinomas, arranged in a tissue microarray, in order to assess their role in colorectal cancer development. A tissue microarray of 498 patients with colorectal tumors was constructed, and 239 samples for EGFR copy number changes were successfully analyzed by fluorescence in situ hybridization. No amplification of EGFR was detected in our cohort of patients with colorectal tumors, and the EGFR gene was upregulated in only 2 tumors (0.84%). Therefore, the development of colon cancer in patients cannot be explained by copy number changes of the EGFR gene.     

Automated quantitative analysis of DCC tumor suppressor protein in ovarian cancer tissue microarray shows association with beta-catenin levels and outcome in patients with epithelial ovarian cancer.

BACKGROUND: The deleted in colorectal cancer (DCC) protein, the product of DCC tumor suppressor gene, is frequently altered in cancer. Preclinical data demonstrate that DCC regulates beta-catenin levels. Here, we sought to determine the association of DCC with beta-catenin protein levels, clinicopathological parameters and patient outcome in ovarian cancer using a method of in situ compartmentalized protein analysis. METHODS: A tissue array composed of 150 advanced-stage ovarian cancers, treated with surgical debulking and platinum-paclitaxel (Taxol) combination chemotherapy, was constructed. For evaluation of protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis (AQUA). RESULTS: One hundred and twelve patients (74%) had sufficient tissue for AQUA. The median follow-up time for the entire cohort was 33 months. Patients with low nuclear DCC expression had a 3-year progression-free survival (PFS) rate of 0% compared with 33% of those with high DCC expression (P = 0.0067). In multivariate analysis, low nuclear DCC expression level retained its prognostic significance for PFS. Between DCC and beta-catenin, a significant relationship was found, where tumors with low DCC had low beta-catenin and vice versa (P = 0.003). CONCLUSIONS: Low nuclear DCC levels predict for poor patient outcome in epithelial ovarian cancer. DCC may exert its antitumor function, in part, through regulation of beta-catenin levels.     

NEAT expression is associated with tumor recurrence and unfavorable prognosis in colorectal cancer

Long noncoding RNAs (lncRNAs) have recently been identified to be involved in various diseases including cancer. NEAT1 is a recently identified lncRNA with its function largely unknown in human malignancy. In the present study, we investigated NEAT1 expression in 239 cases of clinical colorectal cancer specimens and matched normal tissues. Statistical methods were utilized to analyze the association of NEAT1 with clinical features, disease-free and overall survival of patients. Results showed that NEAT1 expression in colorectal cancer was up-regulated in 72.0% (172/239) cases compared with corresponding normal counterparts, and related to tumor differentiation, invasion, metastasis and TNM stage. Kaplan-Meier analysis proved that NEAT1 was associated with both disease-free survival and overall survival of patients with colorectal cancer that patients with high NEAT1 expression tend to have unfavorable outcome. Moreover, cox’s proportional hazards analysis showed that high NEAT1 expression was an independent prognostic marker of poor outcome. These results provided the first evidence that the expression of NEAT1 in colorectal cancer may play an oncogenic role in colorectal cancer differentiation, invasion and metastasis. It also proved that NEAT1 may serve as an indicator of tumor recurrence and prognosis of colorectal cancer.     

Lower levels of nuclear beta-catenin predict for a poorer prognosis in localized prostate cancer

Beta-catenin in its role as a nuclear signaling molecule has been implicated in prostate carcinogenesis primarily through modulation of androgen receptor activity. We defined the pattern of beta-catenin protein expression in the nuclei of normal, hyperplastic and malignant human prostate tissue and determined whether differences in expression were associated with disease progression and prognosis. Five normal prostates, 26 benign prostatic hypertrophy specimens, 232 radical prostatectomy specimens from patients with clinically localized prostate cancer (PC) and 20 cases of advanced PC were assessed for beta-catenin expression using immunohistochemistry. Nuclear beta-catenin expression in localized PC was significantly lower than that in benign prostate tissue (p < 0.001) and significantly higher than that in advanced PC tissue (p < 0.001). In addition, lower levels of nuclear beta-catenin expression (< 10% of cancer cells) predicted for a shorter biochemical relapse-free survival in patients with localized PC (p = 0.008) and were inversely correlated with preoperative prostate-specific antigen (PSA) levels (p = 0.01). Analysis of the low-risk subgroup of patients with preoperative PSA levels < 10 ng/ml demonstrated that lower levels of nuclear beta-catenin expression (< 10% of cancer cells) again predicted for a poorer prognosis (p = 0.006). In conclusion, lower levels of nuclear beta-catenin expression are found in malignant compared to benign prostate tissue. In addition, lower nuclear beta-catenin expression is associated with a poorer prognosis in localized PC, in particular, in the low-risk subgroup of patients with preoperative PSA levels < 10 ng/ml. Thus, the level of nuclear beta-catenin expression may be of clinical utility as a preoperative prognostic marker in low-risk localized PC.     

High expression of MMP-9 is associated with better prognosis in extrahepatic bile duct cancer patients

Purpose

To evaluate the prognostic value of matrix metalloproteinase-9 (MMP-9) in patients with extrahepatic bile duct (EHBD) cancer undergoing surgical resection and adjuvant radiotherapy.

Novel reversible selective inhibitor of nuclear export shows that CRM1 is a target in colorectal cancer cells

Colorectal cancer arises via a multistep carcinogenic process and the deregulation of multiple pathways. Thus, the simultaneous targeting of multiple pathways may be a promising therapeutic approach for colorectal treatment. CRM1 is an attractive cancer drug target, because it can regulate multiple pathways and tumor suppressor proteins. In this study, we investigated the anti-tumor activity of a novel reversible CRM1 inhibitor S109 in colorectal cancer. Our data demonstrate that S109 inhibits proliferation and induces cell cycle arrest in colorectal cancer cells. Mechanistically, we demonstrate that the activity of S109 is associated with the nuclear retention of major tumor suppress proteins. Furthermore, the Cys528 mutation of CRM1 prevented the ability of S109 to block nuclear export and inhibit the proliferation of colorectal cancer cells. Interestingly, S109 decreased the CRM1 protein level via proteasomal pathway. We argue that reversible CRM1 inhibitors but not irreversible inhibitors can induce the degradation of CRM1, because the dissociation of reversible inhibitors of CRM1 changes the conformation of CRM1. Taken together, these findings demonstrate that CRM1 is a valid target for the treatment of colorectal cancer and provide a basis for the development of S109 therapies for colorectal cancer.     

HLA‐DR expression is associated with better prognosis in sporadic Australian clinicopathological Stage C colorectal cancers

Predicting patient outcome for colorectal carcinoma (CRC) with lymph node but not distant metastases remains challenging. Various prognostic markers have been identified including microsatellite instability (MSI) and possibly expression of the MHC Class II protein, HLA‐DR. About 15% of sporadic CRC exhibits MSI associated with methylation of the DNA mismatch repair gene hMLH1 promoter. In addition, a significant proportion of unselected CRC demonstrates expression of HLA‐DR. We sought to examine the relationship between HLA‐DR expression, MSI status and prognosis in sporadic Australian Clinicopathological (ACP) Stage C CRC. Two hundred seventy consecutive patients with sporadic ACP Stage C CRC were treated at Concord Repatriation General Hospital between 1986 and 1992. None of these patients received adjuvant chemotherapy and all were followed for a minimum of 5 years or until death. DNA was extracted from paraffin sections and MSI status determined by PCR. HLA‐DR expression was determined immunohistochemically using an antibody against the HLA‐DR α chain. MSI status could be assigned in 235 cases: 176 CRCs (74.9%) were microsatellite stable, whereas 23 (9.8%) had high levels of MSI (MSI‐H) and 36 (15.3%) had low levels of MSI (MSI‐L). HLA‐DR expression by CRC cells was seen in 148 (60.1%) cases and correlated with the presence of tumor‐infiltrating lymphocytes (p = 0.0005) and peritumoral lymphocytes (p = 0.003), but not other clinicopathological features or MSI status. HLA‐DR‐positive CRCs were strongly associated with better patient outcome (p < 0.0001). © 2009 UICC