Dominic Fong

EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis.

Aims Epithelial cell adhesion molecule (EpCAM) is a cell surface protein with oncogenic features that is expressed on healthy human epithelia and corresponding malignant tumours. EpCAM expression frequently correlates with more aggressive tumour behaviour and new EpCAM-specific therapeutic agents have recently been approved for clinical use in patients with cancer. However, no consensus exists on how and when to evaluate EpCAM expression in patients with cancer. Material and methods EpCAM expression was assessed by a well-established immunohistochemical staining protocol in 2291 primary tumour tissues and in 108 metastases using the EpCAM-specific antibody clone VU1D9. A total immunostaining score was calculated as the product of a proportion score and an intensity score. Four expression subgroups (no, weak, moderate and intense) were defined. As described previously, the term ‘EpCAM overexpression’ was reserved for tissues showing a total immunostaining score >4. Results EpCAM was highly expressed in most tumours of gastrointestinal origin and in some carcinomas of the genitourinary tract. However, hepatocellular carcinomas, clear cell renal cell cancer, urothelial cancer and squamous cell cancers were frequently EpCAM negative. EpCAM expression in breast cancer depended on the histological subtype; lobular histology usually showed no or weak expression. Most metastases were EpCAM positive and they frequently reflected the expression phenotype of the primary tumour. Conclusion EpCAM expression was detected on adenocarcinomas of various primary sites. If EpCAM-specific antibodies are intended to be used in patients with cancer, we recommend prior immunohistochemical evaluation of EpCAM expression, particularly in patients with renal cell cancer, hepatocellular carcinoma, urothelial carcinoma, breast cancer and squamous cell carcinomas.

Overexpression of Epithelial Cell Adhesion Molecule Antigen in Gallbladder Carcinoma Is an Independent Marker for Poor Survival

Purpose: Gallbladder carcinoma is an aggressive type of cancer that is difficult to cure by conventional procedures. There thus is a need to identify novel molecular markers for the assessment of prognosis and as potential therapeutic targets. This retrospective study was designed to investigate the prognostic significance of epithelial cell adhesion molecule (Ep-CAM) overexpression in human gallbladder carcinoma. Experimental Design: Ep-CAM expression was examined immunohistochemically on paraffin-embedded tissue specimens from 99 patients who underwent surgical treatment for gallbladder carcinoma in the period between August 1988 and May 1999. Results: Ep-CAM overexpression was found in 63 (63.6%) of the tumor samples. Kaplan-Meier curves showed that Ep-CAM overexpression was significantly related to decreased overall survival (P < 0.01). Overall survival gradually worsened with increasing Ep-CAM scores. Notably, in the subgroup of pT1 tumors (n = 17), patients without Ep-CAM overexpression had a 5-year overall survival rate of 100% compared with 38% (P = 0.01) for patients with Ep-CAM-overexpressing tumors. By univariate analysis, no correlation was found with conventional clinicopathological parameters. Multivariate analysis, including Ep-CAM expression, pT stage, tumor grade, and resection margin involvement, showed that Ep-CAM overexpression was an independent prognostic marker in gallbladder carcinoma (P = 0.03; relative risk, 1.8). Conclusions: These results demonstrate for the first time that Ep-CAM overexpression is an independent prognostic marker in gallbladder carcinoma and that its prognostic impact should be validated prospectively. Furthermore, the Ep-CAM antigen represents an attractive target for specific therapies with monoclonal antibodies or specific vaccines in patients with Ep-CAM-overexpressing gallbladder carcinoma.

TROP2: a novel prognostic marker in squamous cell carcinoma of the oral cavity

Squamous cell carcinoma is by far the most common type of cancer of the oral cavity, representing more than 90% of all oral cancers. Despite refinement of surgical techniques and adjuvant therapies, the prognosis for patients with oral squamous cell carcinoma remains poor. Identification of prognostic factors related to tumor biology might improve this assessment. Recently, the human trophoblast cell-surface antigen TROP2 was found to be highly expressed in colorectal cancer, correlating with aggressiveness and poor prognosis. Thus, the aim of this study was to investigate TROP2 expression and its prognostic impact in oral squamous cell carcinoma patients. TROP2 expression was examined by immunohistochemistry in a series of 90 patients on a tissue microarray of paraffin-embedded specimens. Survival was calculated using Kaplan–Meier estimates. Parameters found to be of prognostic significance in univariate analysis were verified in a multivariate Cox regression model. TROP2 overexpression was observed in 52 (58%) of the tumor samples. Kaplan–Meier curves showed that TROP2 overexpression was significantly associated with decreased overall survival (P<0.01). Overall survival gradually worsened with increasing TROP2 scores. By univariate analyses, no correlation with conventional clinicopathological features was found. Multivariate Cox regression analysis revealed TROP2 overexpression to be an independent factor predictive of poor disease outcome (P<0.01). These results demonstrate that TROP2 overexpression is an independent prognostic marker in patients with oral squamous cell carcinoma. TROP2 overexpression was detectable in 58% of the tumor samples, indicating it to be a potential novel therapeutic target in squamous cell carcinoma of the oral cavity.

Regulatory T-cells in the graft and the risk of acute graft-versus-host disease after allogeneic stem cell transplantation

Background. FOXP3+ regulatory T-cells (Treg) are important regulators of allo-reactivity and may therefore represent an important predictor for the risk of graft versus-host disease (GVHD) after allogeneic stem cell transplantation. Methods. To determine the clinical significance of Treg-content in stem cell grafts, we analyzed 58 human leukocyte antigen (HLA)-identical sibling donors (34 patients received myeloablative and 24 patients reduced intense conditioning regimens) and correlated the Treg frequency with clinical outcome after stem cell transplantation (SCT). Results. A mean value of 9.1×106 CD4+FOXP3+ Treg per kg body weight (bw) of the recipient was transplanted (ranging from 0.7 to 33.7×106 Treg/kg bw). Graft content of Treg correlated with mononuclear cells and CD3+ T-cells. Patients receiving low numbers of Treg (Treglow) after myeloablative conditioning for SCT had a significantly increased cumulative incidence of 76% for acute GVHD when compared with 23% for individuals receiving high numbers of Treg (Treghigh). This observation, however, was not made in patients after reduced intense conditioning-SCT. Notably, relapse rate was not significantly different between Treglow and Treghigh patients in either patient group and overall survival was even increased in Treghigh patients after myeloablative SCT. Finally, low Treg graft levels represent an independent prognostic factor in multivariate analysis for the appearance of acute GHVD. Conclusion. Donor-derived Treg might be of particular significance for the development of acute GVHD after myeloablative SCT using HLA-identical sibling donors.

Ep-CAM expression in pancreatic and ampullary carcinomas: frequency and prognostic relevance

Aims: Pancreatic adenocarcinoma is an aggressive gastrointestinal malignancy with only a few long-term survivors even after radical surgery. Patients with ampullary cancer have a better prognosis but adjuvant therapy needs further improvement. Epithelial cell adhesion molecule (Ep-CAM) is strongly expressed in a variety of epithelial cancers and represents a promising target for immunological tumour therapy. Thus, the aim of this study was to investigate Ep-CAM expression and its potential prognostic impact in pancreatic and ampullary carcinomas. Methods: Ep-CAM expression was investigated retrospectively by immunohistochemistry in paraffin-embedded primary tumour tissue samples from a series of consecutive patients with pancreatic (n = 153) and ampullary cancer (n = 34). Results: Ep-CAM overexpression was observed in 85 of 153 pancreatic cancer specimens (56%) and in 29 of 34 ampullary cancer samples (85%). Overall, Ep-CAM failed to be an independent prognostic marker. However, subgroup analyses showed that Ep-CAM overexpression correlated with shorter overall survival among patients with ampullary cancer and advanced stage pancreatic cancer. In the latter subgroup, survival gradually worsened with increasing Ep-CAM scores. Furthermore, in ampullary cancer, Ep-CAM overexpression was found to correlate with tumour stage. Conclusions: Ep-CAM overexpression was detectable in the majority of cases with pancreatic and ampullary cancer. Therefore, Ep-CAM represents an attractive target for immune-based therapeutic interventions in these tumour entities. However, the prognostic value of Ep-CAM overexpression remains undetermined.

Hodgkin's disease variant of Richter's syndrome in chronic lymphocytic leukaemia patients previously treated with fludarabine

The transformation of chronic lymphocytic leukaemia (CLL) into large‐cell lymphoma (Richters syndrome, RS) is a well‐documented phenomenon. Only rarely does CLL transform into Hodgkins lymphoma (HL). To further analyse the clinico‐pathological and genetic findings in the HL variant of RS, we performed a single‐institution study in four patients, who developed HL within a mean of 107 months after diagnosis of CLL. All were treated with fludarabine. Three cases were Epstein–Barr virus (EBV)‐associated mixed cellularity (MC) HL, the fourth was nodular sclerosis (NS) HL without EBV association. The sites involved by HL included supra‐ and infradiaphragmal lymph nodes and the tonsils; stage IV disease was also documented. All patients presented with CLL treatment‐resistant lymphadenopathies and B‐symptoms. In two of the MC cases, molecular analysis performed on CLL samples and microdissected Hodgkin and Reed–Sternberg cells (HRSC) suggested a clonal relationship, while in NS no indication of a clonal relationship was detected. In summary, HL can occur in CLL patients at any site, up to 17 years after initial diagnosis, especially after treatment with fludarabine. The majority present with B‐symptoms and CLL treatment‐resistant lymphadenopathy, are of the MC type, clonally related to CLL and might be triggered by an EBV infection.

TROP2 expression as prognostic marker for gastric carcinoma

Background: In gastric cancer the recurrence rate is unacceptably high, even after R0 resection and (neo)adjuvant chemotherapy. Therefore, there is an urgent need for identification of predictive and/or prognostic biomarkers to select high-risk patients who might benefit from additional therapies. Expression of TROP2 has been shown to be associated with tumour aggressiveness and poor prognosis in patients with various epithelial cancers. Aims: To investigate TROP2 expression in gastric cancer and its correlation with clinicopathological features and disease outcome. Methods: Expression of TROP2 was investigated by immunohistochemistry of tumour specimens from 104 patients who underwent resection for gastric cancer. Parameters found to be of prognostic significance in univariate analysis were verified in a multivariate Cox regression model. Results: TROP2 was found to be overexpressed in 58 (56%) tumour samples. Significantly higher expression of TROP2 could be detected in intestinal-type carcinomas (p = 0.03). In intestinal-type gastric cancer, TROP2 overexpression was significantly correlated with shorter disease-free survival (DFS) (p = 0.03). Among the total group, TROP2 overexpression was predictive for poor disease-free (p<0.01) and overall (p = 0.03) survival in lymph node positive patients. Multivariate Cox regression analysis revealed TROP2 overexpression to be an independent prognostic marker for poor DFS in the subgroup of patients with intestinal-type gastric cancer irrespective of lymph node involvement. Conclusion: Results show that TROP2 is an independent prognostic marker for disease recurrence in intestinal type gastric cancer. Due to its wide distribution TROP2 may become an attractive therapeutic target in a subgroup of patients with gastric cancer.

Effects of EpCAM overexpression on human breast cancer cell lines

BackgroundRecently, EpCAM has attracted major interest as a target for antibody- and vaccine-based cancer immunotherapies. In breast cancer, the EpCAM antigen is overexpressed in 30-40% of all cases and this increased expression correlates with poor prognosis. The use of EpCAM-specific monoclonal antibodies is a promising treatment approach in these patients.MethodsIn order to explore molecular changes following EpCAM overexpression, we investigated changes of the transcriptome upon EpCAM gene expression in commercially available human breast cancer cells lines Hs578T and MDA-MB-231. To assess cell proliferation, a tetrazolium salt based assay was performed. A TCF/LEF Reporter Kit was used to measure the transcriptional activity of the Wnt/β-catenin pathway. To evaluate the accumulation of β-catenin in the nucleus, a subcellular fractionation assay was performed.ResultsFor the first time we could show that expression profiling data of EpCAM transfected cell lines Hs578TEpCAM and MDA-MB-231EpCAM indicate an association of EpCAM overexpression with the downregulation of the Wnt signaling inhibitors SFRP1 and TCF7L2. Confirmation of increased Wnt signaling was provided by a TCF/LEF reporter kit and by the finding of the nuclear accumulation of ß-catenin for MDA-MB-231EpCAM but not Hs578TEpCAM cells. In Hs578T cells, an increase of proliferation and chemosensitivity to Docetaxel was associated with EpCAM overexpression.ConclusionsThese data show a cell type dependent modification of Wnt signaling components after EpCAM overexpression in breast cancer cell lines, which results in marginal functional changes. Further investigations on the interaction of EpCAM with SFRP1 and TCF7L2 and on additional factors, which may be causal for changes upon EpCAM overexpression, will help to characterize unique molecular properties of EpCAM-positive breast cancer cells.

Dkk-3 expression in the tumor endothelium: a novel prognostic marker of pancreatic adenocarcinomas.

Dkk‐3 is proposed to be a new specific marker for tumor endothelial cells. Here we analyzed the clinical relevance of Dkk‐3 expression in pancreas adenocarcinomas and determined its role on endothelial cell growth in vitro. Microvessel density in tumor samples was immunohistochemically determined using Dkk‐3 and CD31 as endothelial cell markers, respectively. Based on the median microvessel density as a cut‐off point, patients were categorized into high and low microvessel density groups and a correlation with survival and clinical parameters was assessed. Moreover, the role of Dkk‐3 expression on chemosensitivity of endothelial cells was analyzed. In contrast to CD31 staining, Dkk‐3‐positive vessels were found only in tumor tissue and Dkk‐3 microvessel density significantly correlated negative with tumor grading. In survival analysis the median survival time was 7 months for patients with Dkk‐3 low, and 15 months for Dkk‐3 high microvessel density (P = 0.0013). Subset analysis of patients receiving gemcitabine therapy showed that overall survival was significantly decreased in Dkk‐3 low tumors than in high tumors (P = 0.009). In Cox regression Dkk‐3 emerged as a significant independent parameter (P = 0.024). Dkk‐3 overexpression in endothelial cells resulted in significantly enhanced growth inhibition after 5‐fluorouracil or gemcitabine treatment compared to control endothelial cells and cancer cell lines. Dkk‐3 low microvessel density was associated with tumor progression and worse clinical outcome. Overexpression of Dkk‐3 enhanced endothelial cell growth inhibition to chemotherapeutic drugs. Therefore, we suggest that Dkk‐3 high microvessel density may help to select patients who may benefit from chemotherapy. (Cancer Sci 2009)

Loss of membranous expression of the intracellular domain of EpCAM is a frequent event and predicts poor survival in patients with pancreatic cancer

Epithelial cell adhesion molecule (EpCAM) is a widely used immunohistochemical marker for epithelial human malignancies. Antibodies to target EpCAM are usually directed against its ectodomain (EpEX), but do not detect the intracellular domain (EpICD). The aim of this study was to compare membranous EpEX versus EpICD expression by immunohistochemistry.