Jan-Patrik Wiksten

MYC-Dependent Regulation and Prognostic Role of CIP2A in Gastric Cancer

BACKGROUND Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified human oncoprotein that stabilizes the c-Myc (MYC) protein. However, the clinical relevance of CIP2A to human cancers had not been demonstrated, but the mechanism of its regulation and its clinical role in cancer were completely unknown. METHODS Tissue microarrays consisting of 223 gastric adenocarcinoma specimens were evaluated for the presence of CIP2A using immunohistochemistry, and the association of CIP2A expression with survival was assessed using Kaplan-Meier analysis. The effects of MYC and CIP2A on each others expression and on cell proliferation were investigated in several gastric cancer cell lines using small interfering RNAs to CIP2A and MYC and immunoblotting. To further evaluate the role of MYC in CIP2A regulation, an inhibitor of MYC dimerization, 10058-F4, and an inducible MycER model were used. RESULTS Expression of CIP2A protein was associated with reduced overall survival for gastric cancer patients with tumors 5 cm or smaller, with a 10-year overall survival in the CIP2A-immunopositive group of 8.1% as compared with 37.6% in the CIP2A-negative group (difference = 29.5%, 95% confidence interval = 12.5% to 46.5%, P = .001). In gastric cancer cell lines, CIP2A depletion led to decreased proliferation and anchorage-independent growth of the cells, as well as to reduced stability and expression of MYC protein. Interestingly, MYC depletion led to reduced expression of CIP2A mRNA and protein. Moreover, experiments with an MYC inhibitor and activator suggested that MYC directly promotes CIP2A gene expression. Finally, CIP2A and MYC immunopositivities were associated in gastric cancer specimens (P = .021). CONCLUSIONS CIP2A immunopositivity is a predictor of survival for some subgroups of gastric cancer patients. CIP2A and MYC appear to be regulated in a positive feedback loop, wherein they promote each others expression and gastric cancer cell proliferation.

Cyclooxygenase-2 Is an Independent Prognostic Factor in Gastric Cancer and Its Expression Is Regulated by the Messenger RNA Stability Factor HuR

Purpose: Cyclooxygenase-2 (COX-2) promotes carcinogenesis and its expression associates with clinicopathologic characteristics in gastric cancer. HuR is an mRNA binding protein that controls the stability of certain transcripts including COX-2. We evaluated the prognostic significance of COX-2 and HuR expressions in gastric cancer and whether there exists a link between HuR and COX-2 expressions. Experimental Design: The study included 342 consecutive patients with histologically confirmed gastric adenocarcinoma, of whom 321 patients had tissue specimens available for COX-2 and 316 for HuR immunohistochemistry. Specimens were stained by COX-2– and HuR-specific monoclonal antibodies and scored by two independent observers. Correlation to clinical data and survival was assessed. TMK-1 gastric adenocarcinoma cells were treated with small interfering RNA against HuR and expressions of HuR and COX-2 were detected by immunofluorescence and Western blot analysis. Results: Patients with low COX-2 expression had a cumulative 5-year survival of 53% and those with high COX-2 expression had 16% (P < 0.0001). In multivariate analysis, COX-2 was an independent prognostic factor (P = 0.003). Cytoplasmic HuR expression was associated with high COX-2 expression (P < 0.0001) and with reduced survival (P = 0.004) whereas nuclear positivity for HuR was not. When TMK-1 cells were treated with HuR small interfering RNA, expressions of HuR and COX-2 were reduced. Conclusions: High COX-2 is an independent prognostic factor in gastric cancer. Cytoplasmic expression of HuR associates with high COX-2 expression and with reduced survival, and tissue culture experiments show that HuR can regulate expression of COX-2 in gastric cancer cells.

Epithelial and stromal syndecan-1 expression as predictor of outcome in patients with gastric cancer.

The prognostic value of the immunohistochemical expression of epithelial and stromal syndecan‐1 was evaluated in 296 patients with gastric carcinoma. Formalin‐fixed, paraffin‐embedded specimens of gastric adenocarcinomas were stained with mouse monoclonal antibody B‐B4 against human syndecan‐1. Loss of immunoreactivity (≤60% of cancer cells stained) was observed in 197 (67%) patients. Stromal immunoreactivity was observed in 28 (9%) patients. Loss of epithelial syndecan‐1 immunoreactivity correlated with a higher stage of disease (stages II–IV), tumour location in the upper third of the stomach, nodal metastases (N1 or N2), positive stromal syndecan‐1 staining, deep tumour penetration (to subserosa or deeper = T2–T4), larger tumour size (≥5 cm) and intestinal type of cancer. No correlation between epithelial syndecan‐1 immunoreactivity and age, gender, distant metastases, grade of differentiation or Borrmann classification was observed. Positive stromal syndecan‐1 immunoreactivity correlated with decreased epithelial syndecan‐1 expression, intestinal type of cancer and Borrmann type I. Patients with low epithelial syndecan‐1 expression in cancer cells had worse overall survival than patients with strong epithelial syndecan‐1 staining (p = 0.0012). Stromal syndecan‐1‐positive patients had a worse outcome than patients with syndecan‐1‐negative stroma (p = 0.0193). In Cox multivariate analysis, stromal syndecan‐1 immunoreactivity was a prognostic factor independent of TNM stage, surgery for cure and tumour size. Thus, the immunohistochemical expression of syndecan‐1 might be a predictor of outcome in patients with gastric adenocarcinoma.

Distal Pulse Palpation: Is It Reliable?

The aim of this study was to evaluate the reliability of distal pulse palpation. The dorsalis pedis and the tibialis posterior arteries of 25 patients with suspected lower limb arterial disease were independently palpated by three vascular surgeons and three medical students in the outpatient clinic and by two vascular nurses and one physician in the vascular laboratory. The palpation findings were compared to the ankle/brachial index (ABI). Palpable and unpalpable pulses were best separated with ABI 0.76 as the cutoff point. The degree of misdiagnosis was unacceptably high, with an underdiagnosis of more than 30%. The agreement was highest (kappa 0. 68, good) among the vascular laboratory personnel in the peaceful vascular laboratory and lowest (kappa 0.38, fair) among the vascular surgeons in the busy outpatient clinic. The poor agreement and the high proportion of misdiagnosis obtained in the outpatient clinic argue against the use of pulse palpation as a single diagnostic method. Palpable pulses with low ABIs clearly state the need for more objective measurements whenever ischemia is suspected. Yet, by carefully palpating both pedal arteries under good, nonhurried conditions the reproducibility and accuracy of pulse palpation can be tolerable.

MMP-7 overexpression is an independent prognostic marker in gastric cancer

To enable cancer to invade and to metastasize, the surrounding stroma must be degraded. Matrix metalloproteinase-7 (MMP-7) is capable of degrading many extracellular matrix proteins and cellular adhesions, is overexpressed in many malignancies, and plays a role in tumour progression. The purpose of this study was to evaluate the association between MMP-7 tissue expression and patients’ prognosis in gastric cancer. From 264 patients who underwent surgery for gastric cancer, surgical specimens were collected on tissue array blocks and stained by immunohistochemistry for MMP-7. In 27 (10.2%) of the specimens, immunopositivity was found as high, in 50 (18.9%) as moderate and in 51 (19.3%) as weak. In 136 cases (51.5%), the immunopositivity was negative. A statistically significant correlation appeared between high MMP-7 expression and poor survival. In conclusion, our results suggest that MMP-7 expression may prove helpful in evaluating gastric cancer prognosis.

MMP-2 but not MMP-9 associated with COX-2 and survival in gastric cancer

Background and aim: Matrix metalloproteinases (MMPs) MMP-2 and MMP-9 can degrade type IV collagen of extracellular matrix and basal membranes. As cyclo-oxygenase-2 (COX-2) has been shown to activate MMPs, creating one of the COX-2-promoted pathways of tumour growth and metastasis, the prognostic role of MMP-2 and MMP-9 in gastric cancer was assessed and their association with COX-2 expression was evaluated. Materials and methods: Samples were collected from 342 consecutive patients operated on for gastric cancer, of which 315 were acceptable for MMP-2, MMP-9 and COX-2 immunohistochemistry. Specimens were stained with specific antibodies, evaluated and categorised by two interpreters, and then correlated with clinical data and survival. Results: Epithelial MMP-2 immunoreactivity was associated with male sex, high stage, advanced penetration depth, non-curative surgery, high COX-2 expression and poor survival. Stromal MMP-2 expression correlated with high stage, intestinal type and non-curative surgery whereas MMP-9 correlated only with intestinal type. Stage, intent of surgery and COX-2 were independent prognostic factors. Conclusions: Epithelial MMP-2 expression in gastric cancer is associated with aggressive forms, COX-2 and poor survival, although MMP-2 was not an independent prognostic factor. In gastric cancer tumour growth is apparently induced by COX-2, and invasion is mediated by MMP-2.

COX-2 is associated with proliferation and apoptosis markers and serves as an independent prognostic factor in gastric cancer

Cyclooxygenase-2 (COX-2) is an important factor in gastric carcinogenesis, and COX-2 expression in gastric cancer patients correlates with prognosis. We have now studied the impact of COX-2 in comparison to six other tissue tumor markers, DNA index, and S-phase fraction (SPF) in a large series of gastric cancer specimens. From 342 consecutive patients, 337 archival tissue specimens were available for immunohistochemistry of COX-2, HuR, cyclin A, MMP-2, p53, p21, and Ki-67 and 313 for analysis of DNA index and S-phase fraction by flow cytometry. Associations between factors were assessed by chi-square test and survival analysis by the Kaplan–Meier method and Cox model. A significant association emerged between of COX-2 and p53 (p < 0.0001), Ki-67 (p = 0.013), DNA ploidy (p < 0.0001), and SPF (p < 0.0001). In an extended multivariate analysis, COX-2 and p53 expression were independent prognostic factors for poor survival, in addition to high stage and non-curative surgery. In gastric cancer, COX-2 expression associated with markers for apoptosis and proliferation, and furthermore, it was confirmed that COX-2 and p53 are strong prognostic indicators.

Prognostic significance of cyclin A in gastric cancer

High level of cyclin A promotes carcinogenesis, and overexpression of cyclin A has been associated with poor prognosis of cancer patients. We validated the prognostic role of cyclin A in gastric cancer and evaluated its correlation with expression of an mRNA stability factor HuR. From 342 consecutive histologically confirmed gastric cancer patients were obtained 325 representative tissue specimens for cyclin A and 316 for HuR immunohistochemistry. Specimens were stained by cyclin A and HuR specific monoclonal antibodies. Nuclear immunostaining detected in ≥≥≥≥5% of the tumor cells was considered the cut‐off for cyclin A positivity. Positive HuR immunoreactivity was scored as nuclear or cytoplasmic. Associations between scores, clinicopathological factors and survival were calculated by the χ2‐test, Fishers exact test, Kaplan‐Meier test and Cox model. Cyclin A detected in the nuclei of cancer cells was positive in 55% (179 of 325) of the specimens; 40% (127 of 316) of the specimens had cytoplasmic and 88% (279of 316) nuclear immunoreactivity of HuR. Cyclin A expression was an independent prognostic factor for poor survival. Cyclin A immunoreactivity was associated with old age, high stage, proximal location of the tumor, intestinal type, noncurative resection, advanced penetration depth and with nodal metastases but not distant metastases. Furthermore, cyclin A expression was associated with cytoplasmic HuR expression, whereas no association with nuclear HuR was evident. Cyclin A is an independent prognostic factor in gastric cancer, and one mechanism for its overexpression may depend on cytoplasmic localization of HuR.

Decreased xanthine oxidoreductase is a predictor of poor prognosis in early‐stage gastric cancer

Background: Xanthine oxidoreductase (XOR) is a key enzyme in the degradation of DNA, RNA and high-energy phosphates. About half of the patients with breast cancer have a decrease in XOR expression. Patients with breast cancer with unfavourable prognosis are independently identified by the loss of XOR. Aim: To assess the clinical relevance of XOR expression in gastric cancer. Methods: XOR levels were studied by immunohistochemistry in tissue microarray specimens of 337 patients with gastric cancer and the relation between XOR expression and a series of clinicopathological variables, as well as disease-specific survival, was assessed. Results: XOR was moderately decreased in 41% and was undetectable in another 14% of the tumours compared with the corresponding normal tissue. Decreased XOR was associated with advanced stage, deep tumour penetration, diffusely spread tumour location, positive lymph node status, large tumour size, non-curative disease, cellular aneuploidy, high S-phase fraction and high cyclooxygenase-2 expression, but not with p53 expression or Borrmann classification. Down regulation of XOR was associated with unfavourable outcome, and the cumulative 5-year gastric cancer-specific survival in patients with strong XOR expression was 47%, compared with 22% in those with moderate to negative expression (p<0.001). This was also true in patients with stage I–II (p = 0.01) and lymph node-negative (p = 0.02) disease, as well as in patients with smaller (⩽5 cm) tumours (p = 0.02). Conclusion: XOR expression in gastric cancer may be a new marker for a more aggressive gastric cancer biology, similar to that previously reported for breast cancer.

Epithelial Syndecan-1 Expression Is Associated with Stage and Grade in Colorectal Cancer

Objectives: Loss of epithelial heparan sulfate proteoglycan syndecan-1 has been associated with a more aggressive behavior in various cancer forms, but the prognostic significance of syndecan-1 expression in colorectal cancer is unclear. The aim of this study was to evaluate the prognostic value of immunohistochemical syndecan-1 expression in a series of 237 patients with colorectal cancer. Methods: Paraffin-embedded formalin-fixed specimens were stained with a syndecan-1-specific monoclonal antibody, and both the epithelial and stromal expression were analyzed. Results: Epithelial expression of syndecan-1 was seen in 222 tumors (94%), and it was associated with low stage of disease (p = 0.002) and low histological differentiation grade (p = 0.048). The cumulative 5-year survival of patients with weak and strong syndecan-1 expression was 49 and 54 %, respectively (p = 0.234). Syndecan-1 stromal immunoreactivity was observed in 138 tumors (58%), but lacked prognostic significance. Staining pattern and distribution can be viewed from digitized representative microscope slides (virtual slides) at http://www.webmicroscope.net/supplements/syndecan. Conclusions: The results are in line with previous reports in that low epithelial syndecan-1 expression was associated with a higher histological grade and a more advanced clinical stage of the patients. This study shows that syndecan-1 is expressed also in stromal tissue of colorectal cancer, but it does not support the proposed role of stromal syndecan-1 expression as a marker of poor clinical outcome.