Marleen J.E.M. Gosens

Circumferential Margin Involvement Is the Crucial Prognostic Factor after Multimodality Treatment in Patients with Locally Advanced Rectal Carcinoma

Purpose: After preoperative (radio)chemotherapy, histologic determinants for prognostification have changed. It is unclear which variables, including assessment of tumor regression, are the best indicators for local recurrence and survival. Experimental Design: A series of 201 patients with locally advanced rectal cancer (cT3/T4, M0) presenting with an involved or at least threatened circumferential margin (CRM) on preoperative imaging (<2 mm) were evaluated using standard histopathologic variables and four different histologic regression systems. All patients received neoadjuvant radiochemotherapy or radiotherapy. The prognostic value of all factors was tested with univariate survival analysis of time to local recurrence and overall survival. Results: Local recurrence occurred in only 8% of the patients with a free CRM compared with 43% in case of CRM involvement (P < 0.0001). None of the four regression systems were associated with prognosis, not even when corrected for CRM status. However, we did observe a higher degree of tumor regression after radiochemotherapy compared with radiotherapy (P < 0.001). Absence of tumor regression was associated with increasing invasion depth and a positive CRM (P = 0.02 and 0.03, respectively). Conclusions: Assessment of CRM involvement is the most important pathologic variable after radiochemotherapy. Although tumor regression increases the chance on a free CRM, in cases with positive resection margins prognosis is poor irrespective of the degree of therapy-induced regression.

Loss of membranous Ep-CAM in budding colorectal carcinoma cells

Tumor budding is a histological feature that reflects loss of adhesion of tumor cells and is associated with locoregional metastasis of colorectal carcinoma. Although nuclear localization of β-catenin is associated with tumor budding, the molecular mechanism remains largely elusive. In this study, we hypothesize that the epithelial cell adhesion molecule (Ep-CAM) is involved in tumor budding. In order to address this question, we performed immunohistochemistry on Ep-CAM using three different antibodies (monoclonal antibodies Ber-ep4 and 311-1K1 and a polyclonal antibody) and a double staining on β-catenin and Ep-CAM. In addition, Ep-CAM mRNA was monitored with mRNA in situ hybridization. Subsequently, we determined the effect of Ep-CAM staining patterns on tumor spread in rectal cancer. In contrast to the tumor mass, budding cells of colorectal carcinoma displayed lack of membranous but highly increased cytoplasmic Ep-CAM staining and nuclear translocation of β-catenin. mRNA in situ hybridization suggested no differences in Ep-CAM expression between the invasive front and the tumor mass. Importantly, reduced Ep-CAM staining at the invasive margin of rectal tumor specimens (n=133) correlated significantly with tumor budding, tumor grade and an increased risk of local recurrence (P=0.001, P=0.04 and P=0.03, respectively). These data demonstrate abnormal processing of Ep-CAM at the invasive margin of colorectal carcinomas. Our observations indicate that loss of membranous Ep-CAM is associated with nuclear β-catenin localization and suggest that this contributes to reduced cell–cell adhesions, increased migratory potential and tumor budding.

Combinations of Tumor and Treatment Parameters Are More Discriminative for Prognosis Than the Present TNM System in Rectal Cancer

Iris D. Nagtegaal and Marleen J.E.M. Gosens, Department of Pathology, Radboud University Medical Center, Nijmegen, the Netherlands Corrie A.M. Marijnen, Department of Radiotherapy, Netherlands Cancer Institute, Amsterdam, the Netherlands Harm J. Rutten, Department of Surgery, Catharina Hospital, Eindhoven, the Netherlands Cornelis J.H. van de Velde, Department of Surgery, Leiden University Medical Center, Leiden, the Netherlands J. Han J.M. van Krieken, Department of Pathology, Radboud University Medical Center, Nijmegen, the Netherlands

Signet ring cell differentiation in mucinous colorectal carcinoma

Approximately 10% of all colorectal carcinomas are mucinous carcinomas, characterized by extracellular mucin. Occasionally, mucin accumulates intracellularly in these tumours, causing signet ring cell differentiation. We hypothesized that signet ring cells arise from a separate genetic pathway. In this study the molecular background of signet ring cell differentiation was investigated by analysing genetic changes, changes in the expression of adhesion molecules, and mucin content. Furthermore, its clinical relevance was addressed.

Cyclooxygenase 2 Expression in Rectal Cancer Is of Prognostic Significance in Patients Receiving Preoperative Radiotherapy

Purpose: To determine the effect of cyclooxygenase (COX)-2 expression on clinical behavior in irradiated and nonirradiated rectal carcinomas. Experimental Design: Tumor samples were collected from 1,231 patients of the Dutch TME trial, in which rectal cancer patients were treated with standardized surgery and randomized for preoperative short-term (5 × 5 Gy) radiotherapy or no preoperative radiotherapy. Tissue microarrays were constructed from primary tumor material, and COX-2 expression was assessed by immunohistochemistry. Tumor cell apoptosis was determined by M30 immunostaining. Results: A high level of COX-2 expression after radiotherapy was associated with low levels of tumor cell apoptosis (P = 0.001). COX-2 expression had no significant effect on patient survival or tumor recurrence in nonirradiated tumors. However, in patients receiving preoperative radiotherapy, high level of COX-2 expression was associated with higher incidence of distant recurrences [P = 0.003; hazard ratio (HR), 1.7; 95% confidence interval (95% CI), 1.2-2.5] and shorter disease-free survival (P = 0.002; HR, 1.8; 95% CI, 1.2-2.5) and overall survival (P = 0.009; HR, 1.5; 95% CI, 1.1-2.0), independent of patient age, tumor stage, tumor location, or the presence of tumor cells in the circumferential resection margin. Conclusions: A high level of COX-2 expression after preoperative radiotherapy in resection specimens is associated with apoptosis resistance, high distant recurrence rates, and a poor prognosis in rectal cancer.

Preoperative radiochemotherapy is successful also in patients with locally advanced rectal cancer who have intrinsically high apoptotic tumours

BACKGROUND Not all patients with locally advanced rectal cancer (LARC) respond equally to neo-adjuvant radiochemotherapy (RCT). Patients with highly apoptotic less advanced rectal cancers do not benefit from short-term radiotherapy. This study investigates whether this is also the case in the setting of RCT for LARC. PATIENTS AND METHODS Tissue microarrays were constructed of biopsy and resection specimens of 201 LARC patients. Apoptosis (M30) and several apoptosis-regulating proteins [p53, Bcl-2, Bax, cyclooxygenase-2 (Cox-2) and mamma serine protease inhibitor (maspin)] were studied with immunohistochemistry. Subsequently, predictive values for local recurrence (LR), overall survival (OS) and histological tumour regression were analysed. RESULTS Apoptotic levels, quantified as the number of apoptotic cells/mm(2) tumour epithelium, were higher in posttherapy tissues compared with biopsies (P < 0.001). Biopsies from clinical T4 stage tumours demonstrated significantly higher levels of apoptosis than clinical T3 stage tumours (P = 0.020). Therapy-induced apoptosis was higher when the interval between the last day of irradiation and surgery increased (P < 0.001, correlation coefficient = 0.355). Pre- and posttherapy apoptosis, p53, Bcl-2, Bax and Cox-2 were not associated with LR, OS or tumour regression. Intense pretherapy cytoplasmatic staining of maspin indicated a higher risk on LR (P = 0.009) only. CONCLUSION Combined RCT is also successful in highly apoptotic tumours and is therefore independent of intrinsic apoptosis.