Type I gamma phosphatidylinositol phosphate kinase modulates adherens junction and E-cadherin trafficking via a direct interaction with mu 1B adaptin.

Assembly of E-cadherin-based adherens junctions (AJ) is obligatory for establishment of polarized epithelia and plays a key role in repressing the invasiveness of many carcinomas. Here we show that type Igamma phosphatidylinositol phosphate kinase (PIPKIgamma) directly binds to E-cadherin and modulates E-cadherin trafficking. PIPKIgamma also interacts with the mu subunits of clathrin adaptor protein (AP) complexes and acts as a signalling scaffold that links AP complexes to E-cadherin. Depletion of PIPKIgamma or disruption of PIPKIgamma binding to either E-cadherin or AP complexes results in defects in E-cadherin transport and blocks AJ assembly. An E-cadherin germline mutation that loses PIPKIgamma binding and shows disrupted basolateral membrane targeting no longer forms AJs and leads to hereditary gastric cancers. These combined results reveal a novel mechanism where PIPKIgamma serves as both a scaffold, which links E-cadherin to AP complexes and the trafficking machinery, and a regulator of trafficking events via the spatial generation of phosphatidylinositol-4,5-bisphosphate.

Type I gamma phosphatidylinositol phosphate kinase modulates invasion and proliferation and its expression correlates with poor prognosis in breast cancer.

INTRODUCTION: The loss of E-cadherin based cell-cell contacts and tumor cell migration to the vasculature and lymphatic system are hallmarks of metastasis of epithelial cancers. Type I gamma phosphatidylinositol phosphate kinase (PIPKIgamma), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PI4,5P2) a lipid messenger and precursor to many additional second messengers, was found to regulate E-cadherin cell-cell contacts and growth factor-stimulated directional cell migration, indicating that PIPKIgamma regulates key steps in metastasis. Here, we assess the expression of PIPKIgamma in breast cancers and have shown that expression correlated with disease progression and outcome. METHODS: Using a tissue microarray, we analyzed 438 breast carcinomas for the levels of PIPKIgamma and investigated the correlation of PIPKIgamma expression with patient survival via Kaplan-Meier survival analysis. Moreover, via knockdown of the expression of PIPKIgamma in cultured breast cancer cells with siRNA, the roles of PIPKIgamma in breast cancer migration, invasion, and proliferation were examined. RESULTS: Tissue microarray data shows that approximately 18% of the cohort immunostained showed high expression of PIPKIgamma. The Kaplan-Meier survival analysis revealed a significant inverse correlation between strong PIPKIgamma expression and overall patient survival. Expression of PIPKIgamma correlated positively with epidermal growth factor receptor (EGFR) expression, which regulates breast cancer progression and metastasis. In cultured breast cancer cells, PIPKIgamma is required for growth factor stimulated migration, invasion, and proliferation of cells. CONCLUSIONS: The results reveal a significant correlation between PIPKIgamma expression and the progression of breast cancer. This is consistent with PIPKIgamma 's role in breast cancer cell migration, invasion, and proliferation.

A panel of antibodies to determine site of origin and malignancy in smooth muscle tumors.

Leiomyosarcomas are malignant smooth muscle tumors that occur most commonly in the gynecologic tract and soft tissue. There are different diagnostic criteria of malignancy for smooth muscle tumors arising at gynecologic and soft tissue sites and they may be managed differently but determining the primary site of a smooth muscle tumor can be difficult in some cases. In addition, the distinction between malignant and benign gynecologic tract smooth muscle tumors on morphologic grounds can be challenging. Using a series of tissue microarrays that contain 245 cases of leiomyosarcomas (102 gynecologic) with survival data, and 49 cases of uterine leiomyoma, we examined the ability of selected immune-markers (estrogen receptor (ER) and WT1) to distinguish between leiomyosarcomas of gynecologic and nongynecologic origin. In addition, we examined whether immunostains for p16, p53 and Ki-67 could distinguish between malignant and benign gynecologic smooth muscle tumors. ER nuclear positivity was observed in 3 and 50% of the nongynecologic and gynecologic leiomyosarcomas, respectively (P<0.001). Nuclear WT1 positivity was seen in 0 and 8% of the nongynecologic and gynecologic leiomyosarcomas, respectively (P<0.001). 87% of primary gynecologic leiomyosarcomas and 2% of uterine leiomyomas showed diffuse (>or=50% of cells) p16 staining (P<0.001). 23% of gynecologic leiomyosarcomas showed p53 immunopositivity (>or=50% of cells) whereas none of the leiomyomas were positive for p53 (P<0.001). 65% of the gynecologic leiomyosarcomas and 0% of the leiomyomas exhibited >10% Ki-67 proliferation index (P<0.001). Diffuse p16 and p53 immunopositivity and high Ki-67 proliferation index, singly or in combination, yielded an overall sensitivity of 92% and specificity of 98% for distinguishing between gynecologic leiomyosarcomas and leiomyomas and can be used as indicators of malignancy for gynecologic smooth muscle tumors. Although ER positivity can be used to support the gynecologic origin of a leiomyosarcomas, nuclear WT1 immunostaining is of little use.

Stromal mast cells in invasive breast cancer are a marker of favourable prognosis: a study of 4,444 cases.

PURPOSE: We have previously demonstrated in a pilot study of 348 invasive breast cancers that mast cell (MC) infiltrates within primary breast cancers are associated with a good prognosis. Our aim was to verify this finding in a larger cohort of invasive breast cancer patients and examine the relationship between the presence of MCs and other clinical and pathological features. EXPERIMENTAL DESIGN: Clinically annotated tissue microarrays (TMAs) containing 4,444 cases were constructed and stained with c-Kit (CD-117) using standard immunoperoxidase techniques to identify and quantify MCs. For statistical analysis, we applied a split-sample validation technique. Breast cancer specific survival was analyzed by Kaplan-Meier [KM] method and log rank test was used to compare survival curves. RESULTS: Survival analysis by KM method showed that the presence of stromal MCs was a favourable prognostic factor in the training set (P = 0.001), and the validation set group (P = 0.006). X-tile plot generated to define the optimal number of MCs showed that the presence of any number of stromal MCs predicted good prognosis. Multivariate analysis showed that the MC effect in the training set (Hazard ratio [HR] = 0.804, 95% Confidence interval [CI], 0.653-0.991, P = 0.041) and validation set analysis (HR = 0.846, 95% CI, 0.683-1.049, P = 0.128) was independent of age, tumor grade, tumor size, lymph node, ER and Her2 status. CONCLUSIONS: This study concludes that stromal MC infiltration in invasive breast cancer is an independent good prognostic marker and reiterates the critical role of local inflammatory responses in breast cancer progression.

Longitudinal outcome study of sessile serrated adenomas of the colorectum: an increased risk for subsequent right-sided colorectal carcinoma.

Sessile serrated adenomas (SSAs) are associated with colorectal carcinomas (CRCs) that demonstrate high microsatellite instability (MSI-H). Currently, SSAs are managed clinically in a similar fashion to adenomatous polyps (APs). We studied the natural history of SSA by analyzing the outcome of previously undiagnosed SSAs and comparing it with that of hyperplastic polyps (HPs) and APs. All colorectal polyps diagnosed between 1980 and 2001 as HP were selected for study. Polyps identified as possible SSAs were reviewed by 3 pathologists, and the diagnosis was confirmed. Clinical follow-up was obtained for each SSA patient and matched with control HP and AP patients. In total, 1402 colorectal polyps diagnosed as HP were examined and 81 polyps in 55 patients (5.8%) were rediagnosed as SSA. Of these, 40 SSA patients had no previous history of either CRC or AP with high-grade dysplasia (HGD). Of these 40 patients, 5 developed subsequent CRCs and 1 developed AP with HGD. The incidence of subsequent CRCs was significantly higher in SSA patients than in control patients with HP (12.5% vs. 1.8%) and AP (12.5% vs. 1.8%). All of the subsequent CRCs or APs with HGD developed in the proximal colon. Four of the 5 CRCs demonstrated a high microsatellite instability phenotype. We conclude that SSAs are high-risk lesions, with 15% of the SSA patients developing subsequent CRCs or APs with HGD. This incidence is higher than that of the control HP and AP patients, and would support close endoscopic follow-up in patients harboring SSAs.

Automated quantitative analysis of estrogen receptor expression in breast carcinoma does not differ from expert pathologist scoring: a tissue microarray study of 3,484 cases.

BACKGROUND: Estrogen receptor (ER) expression is routinely assessed by immunohistochemistry (IHC) in breast carcinoma. Our study compares visual scoring of ER in invasive breast cancer by histopathologists to quantitation of staining using a fully automated system. MATERIALS AND METHODS: A tissue microarray was constructed from 4,049 cases (3,484 included in analysis) of invasive breast carcinoma linked to treatment and outcome information. Slides were scored independently by two pathologists and scores were dichotomised, with ER positivity recognized at a cut-off of >1% positive nuclei. The slides were scanned and analyzed with an Ariol automated system. RESULTS: Using data dichotomised as ER positive or negative, both visual and automated scores were highly consistent: there was excellent concordance between two pathologists (kappa = 0.918 (95%CI: 0.903-0.932)) and between two Ariol machines (kappa = 0.913 (95%CI: 0.897-0.928)). The prognostic significance of ER positivity was similar whether determined by pathologist or automated scoring for both the entire patient cohort and subsets of patients treated with tamoxifen alone or receiving no systemic adjuvant therapy. The optimal cut point for the automated scores using breast cancer disease-specific survival as an endpoint was >0.4% positive nuclei. The concordance between dextran-coated charcoal ER biochemical assay data and automated scores (kappa = 0.728 (95%CI: 0.69-0.75); 0.74 (95%CI: 0.71-0.77)) was similar to the concordance between biochemical assay and pathologist scores (kappa = 0.72 (95%CI: 0.70-0.75; 0.70 (95%CI: 0.67-0.72)). CONCLUSION: Fully automated quantitation of ER immunostaining yields results that do not differ from human scoring against both biochemical assay and patient outcome gold standards.

Raft-dependent endocytosis of autocrine motility factor is phosphatidylinositol 3-kinase-dependent in breast carcinoma cells.

Autocrine motility factor (AMF) is internalized via a receptor-mediated, dynamin-dependent, cholesterol-sensitive raft pathway to the smooth endoplasmic reticulum that is negatively regulated by caveolin-1. Expression of AMF and its receptor (AMFR) is associated with tumor progression and malignancy; however, the extent to which the raft-dependent uptake of AMF is tumor cell-specific has yet to be addressed. By Western blot and cell surface fluorescence-activated cell sorter (FACS) analysis, AMFR expression is increased in tumorigenic MCF7 and metastatic MDA-231 and MDA-435 breast cancer cell lines relative to dysplastic MCF10A mammary epithelial cells. AMF uptake, determined by FACS measurement of protease-insensitive internalized fluorescein-conjugated AMF, was increased in MCF7 and MDA-435 cells relative to MCF-10A and caveolin-1-expressing MDA-231 cells. Uptake of fluorescein-conjugated AMF was dynamin-dependent, methyl-beta-cyclodextrin- and genistein-sensitive, reduced upon overexpression of caveolin-1 in MDA-435 cells, and increased upon short hairpin RNA reduction of caveolin-1 in MDA-231 cells. Tissue microarray analysis of invasive primary human breast carcinomas showed that AMFR expression had no impact on survival but did correlate significantly with expression of phospho-Akt. Phospho-Akt expression was increased in AMF-internalizing MCF7 and MDA-435 breast carcinoma cells. AMF uptake in these cells was reduced by phosphatidylinositol 3-kinase inhibition but not by regulators of macropinocytosis such as amiloride, phorbol ester, or actin cytoskeleton disruption by cytochalasin D. The raft-dependent endocytosis of AMF therefore follows a distinct phosphatidylinositol 3-kinase-dependent pathway that is up-regulated in more aggressive tumor cells.

Expression of protein elongation factor eEF1A2 predicts favorable outcome in breast cancer.

Breast cancer is the most common malignancy among North American women. The identification of factors that predict outcome is key to individualized disease management and to our understanding of breast oncogenesis. We have analyzed mRNA expression of protein elongation factor eEF1A2 in two independent breast tumor populations of size n = 345 and n = 88, respectively. We find that eEF1A2 mRNA is expressed at a low level in normal breast epithelium but is detectably expressed in approximately 50-60% of primary human breast tumors. We have derived an eEF1A2-specific antibody and measured eEF1A2 protein expression in a sample of 438 primary breast tumors annotated with 20-year survival data. We find that high levels of eEF1A2 protein are detected in 60% of primary breast tumors independent of HER-2 protein expression, tumor size, lymph node status, and estrogen receptor (ER) expression. Importantly, we find that high eEF1A2 is a significant predictor of outcome. Women whose tumor has high eEF1A2 protein expression have an increased probability of 20-year survival compared to those women whose tumor does not express substantial eEF1A2. In addition, eEF1A2 protein expression predicts increased survival probability in those breast cancer patients whose tumor is HER-2 negative or who have lymph node involvement.

MDM2 protein expression is a negative prognostic marker in breast carcinoma.

The protein encoded by the MDM2 oncogene inhibits the function of p53, leading to increased cell growth, avoidance of apoptosis, tolerance of genetic instability, and resistance to chemotherapy. The present study was performed to evaluate the relationship between MDM2 protein expression and survival in breast carcinoma. Two series of cases were used in this study: the first to identify the cutoff to be used in the interpretation of MDM2 immunostaining and perform preliminary survival analysis, and a second, independent series, to validate the findings from the first series and to perform multivariate analysis. For both series, archival sections of tissue microarrays were stained with anti-MDM2 antibody (NeoMarkers, Fremont, CA, USA) and MDM2 staining intensity was scored semiquantitatively. In the first series, 49 of 362 (14%) interpretable cases were positive for MDM2 expression, with 35 (10%) showing weak positivity and 14 (4%) strong positivity. Patients with MDM2-positive tumours had a significantly worse disease-specific survival than patients with MDM2-negative tumours (P=0.0022, 10-year DSS 61% (95% CI: 45-73) vs 73% (95% CI: 67-77)). No significant difference in survival was observed between patients with strongly and weakly MDM2-positive tumours (P=0.3). Accordingly, in the independent validation series weak and strong MDM2 positivity were combined and considered to be MDM2 positive. MDM2 expression was seen in 230/1747 (13%) interpretable cases in this series, with a significant difference (P<0.0001) in DSS between MDM2-negative and MDM2-positive cases (10 year DSS 58% (95% CI: 51-64) vs 73% (95% CI: 70-75)). MDM2 was an independent prognostic marker (HR=1.35, P=0.02) in a Cox regression model including MDM2 expression, tumour grade, nodal status, ER status and tumour size. Immunohistochemical studies of MDM2 in more than 2000 breast carcinomas show that MDM2 is an independent negative prognostic marker.

Nuclear beta-catenin in mesenchymal tumors.

Beta-catenin is a crucial part of the Wnt and E-cadherin signalling pathways, which are involved in tumorigenesis. Dysregulation of these pathways allow beta-catenin to accumulate and translocate to the nucleus, where it may activate oncogenes. Such nuclear accumulation can be detected by immunohistochemistry, which may be useful in diagnosis. Although the role of beta-catenin has been established in various types of carcinomas, relatively little is known about its status in mesenchymal tumors. A number of studies suggest that beta-catenin dysregulation is important in desmoid-type fibromatosis, as well as in synovial sarcoma. We wished to determine whether nuclear beta-catenin expression is specific to and sensitive for particular bone and soft-tissue tumors, including sporadic desmoid-type fibromatosis. We studied the nuclear expression of beta-catenin using tissue microarrays in a comprehensive range of bone and soft-tissue tumor types. A total of 549 cases were included in our panel. Nuclear immunohistochemical staining was determined to be either high level (>25% of cells), low level (0-25%) or none. High-level nuclear beta-catenin staining was seen in a very limited subset of tumor types, including desmoid-type fibromatosis (71% of cases), solitary fibrous tumor (40%), endometrial stromal sarcoma (40%) and synovial sarcoma (28%). Although occasional cases of fibrosarcoma, clear cell sarcoma and carcinosarcoma had high-level staining, no high-level nuclear beta-catenin expression was seen in any of 381 fibrohistocytic, muscular, adipocytic, chondroid or osseous tumor cases representing 42 diagnostic categories. All primary immunostain tissue microarray images are made publicly accessible in a searchable database. High-level nuclear beta-catenin staining serves as a useful diagnostic tool, as it is specific to a small subset of mesenchymal tumors.

TMA-Combiner, a simple software tool to permit analysis of replicate cores on tissue microarrays.

We have previously published a suite of software tools that facilitates the reformulation of tissue microarray (TMA) data so that it may be analyzed using techniques originally devised for analysis of cDNA microarray data. However, current microarray data often feature multiple scores for a given tissue sample and antibody combination. Furthermore, an efficient and systematic method for combining scores that takes into account the differing staining properties of tissue epitopes has not been described. We thus present the TMA-Combiner, a new Microsoft Excel-based macro that permits analysis of data for which tissues may have two or more scores per antibody, and permits combination of data from multiple different tissue microarrays. It accomplishes this by rendering one score per tissue per antibody from two or more scores, using one of multiple user-selectable combination rules developed to account for the differing staining properties of tissue epitopes. This greatly facilitates analysis of tissue microarrays, particularly for users with large repositories of data, and may facilitate discovery of biological trends and help refine diagnostic accuracy of tissue markers in clinical samples.