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More than 50% of patients with Dukes C colorectal cancer have disease recurrence and die within 5 years after surgical removal of their primary tumor. It is currently not possible to distinguish patients with good and bad prognosis. SMAD4 is an important tumor suppressor gene that mediates transforming growth factor-h superfamily signaling and is located in chromosome 18q21, a region with frequent genetic losses in these tumors. Allelic imbalance in 18q has been linked to poor prognosis in a subset of colorectal cancer patients. Therefore, we generated a tissue microarray containing triplicate tumor samples from 86 Dukes C patients and used immunohistochemistry to assess the relative expression level of SMAD4 and its value as a prognostic marker. In addition, SMAD4 was screened for mutations and two polymorphic microsatellite markers were used to assess the presence of allelic imbalance in these tumors. Patients with tumors expressing high SMAD4 levels had significantly better overall (P < 0.025) and disease-free (P < 0.013) survival than patients with low levels. This identifies SMAD4 as a prognostic marker for Dukes C colorectal cancer. Although all tumors with absent SMAD4 staining showed allelic imbalance in 18q21, tumors with 18q21 allelic imbalance as a group showed no difference in SMAD4 levels compared with tumors without allelic imbalance, suggesting that additional mechanisms of SMAD4 down-regulation exist. In addition, although SMAD4 mutations were found in five tumors, they were not associated with shorter survival. In conclusion, the level of expression of SMAD4 was found to be a more sensitive marker than 18q21 allelic imbalance and SMAD4 mutations, which were of no prognostic significance for these patients.One of the most common cytogenetic abnormalities in colorectal tumors is the loss of genetic material in chromosome 18q. This is believed to be indicative of an important tumor suppressor gene in this genomic location (1, 2). The identity of the gene targeted by these deletions in 18q has remained elusive and several genes, including SMAD2 and DCC (deleted in colorectal carcinoma), have been proposed (3 -5). Recently, SMAD4, a key transducer of transforming growth factor-h (TGF-h) superfamily signaling that is located in chromosome 18q21 and regulates cell proliferation, differentiation, and apoptosis (6 -8), has attracted considerable attention due to several findings. SMAD4 mutations have recently been shown to be associated with the occurrence of juvenile polyposis, an autosomal dominant syndrome predisposing to hamartomatous polyps and colorectal cancer (9, 10). In addition, frequent somatic mutations have been found in human colorectal tumors in several studies, further suggesting an important role for this gene in colorectal carcinogenesis (11,12). Moreover, animal studies have shown that SMAD4 inactivation is involved in the malignant transformation of gastrointestinal adenomas (13) and a reduction in SMAD4 mRNA levels has been observed during tumor progression (14).A significan...

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The receptor tyrosine kinase EPHB2 has recently been shown to be a direct transcriptional target of TCF/B-catenin. Premalignant lesions of the colon express high levels of EPHB2 but the expression of this kinase is reduced or lost in most colorectal carcinomas. In addition, inactivation of EPHB2 has been shown to accelerate tumorigenesis initiated by APC mutation in the colon and rectum. In this study, we investigated the molecular mechanisms responsible for the inactivation of EPHB2 in colorectal tumors. We show here the presence of mutations in repetitive sequences in exon 17 of EPHB2 in 6 of 29 adenomas with microsatellite instability (MSI), and 101 of 246 MSI carcinomas (21% and 41%, respectively). Moreover, we found EPHB2 promoter hypermethylation in 54 of the 101 colorectal tumors studied (53%). Importantly, EPHB2 expression was restored after treatment of EPHB2-methylated colon cancer cells with the DNA methyltransferase inhibitor 5-aza-2V-deoxycytidine. In conclusion, in this study, we elucidate the molecular mechanisms of inactivation of EPHB2 and show for the first time the high incidence of frameshift mutations in MSI colorectal tumors and aberrant methylation of the regulatory sequences of this important tumor suppressor gene. (Cancer Res 2005; 65(22): 10170-3)

We have recently reported that low tumor levels of SMAD4, a key mediator of transforming growth factor-h superfamily signaling, can predict the probability of recurrence in patients with Dukes C colorectal cancer who had surgery as the only form of treatment. However, standard treatment for Dukes C colorectal cancer patients currently involves the administration of 5-fluorouracil (5-FU)b ased adjuvant chemotherapy after surgery. Approximately 30% to 40% of these patients present with recurrence and die within 5 years, and there is great need for markers capable of predicting poor prognosis after the combined surgery/adjuvant treatment. In this study, we evaluate the prognostic value of SMAD4 in patients treated with surgery and 5-FU-based adjuvant therapy. We used immunohistochemistry and quantitative real-time reverse transcription-PCR to measure the levels of SMAD4 protein and mRNA expression in the primary tumors and a number of lymph node metastases from a series of 75 Dukes C colorectal cancer patients with at least 6 years of follow-up. Patients with tumors expressing low levels of SMAD4 protein or mRNA showed significantly shorted disease-free and overall survival than patients with high tumor levels of SMAD4. The median survival of patients with low SMAD4 protein or mRNA tumor levels was 1.4 and 1.2 years, respectively, whereas patients with high SMAD4 tumor level had a median survival of >9.3 years. In addition, the protein and mRNA levels of SMAD4 in lymph node metastases was significantly lower than in primary tumors (P = 0.006). In contrast, allelic imbalance in chromosome18q21was of no prognostic significance in these patients. In conclusion, low SMAD4 tumor levels identified a subset of patients with poor prognosis following surgery and 5-FU-based adjuvant therapy; therefore, these patients could be good candidates to receive combined treatment with additional chemotherapeutic agents such as CPT-11and/or oxaliplatin.Approximately 50% of the patients diagnosed with Dukes C colorectal cancer have disease recurrence and die within 5 years of surgical removal of their primary tumor. Given the relatively high risk of recurrence of these patients, 5-fluorouracil (5-FU) -based adjuvant chemotherapy is routinely given to the great majority of patients with Dukes C colorectal cancer. Although this aggressive chemotherapeutic treatment prevents disease recurrence in 10% to 20% of the patients, f30% to 40% of the patients will not respond to this treatment and will succumb to their disease (1, 2). Patients unlikely to respond to the standard 5-FU-based adjuvant therapy are good candidates to receive additional treatment. The platinum compound oxaliplatin and the camptothecin derivative CPT-11 have been shown to be effective chemotherapeutic agents that can be used alone or in combination with 5-FU (3 -6). The availability of these newer chemotherapeutic agents in addition to more experimental treatment options, such as cyclooxygenase-2 or epidermal growth factor receptor inhibitors (7,8), highlights ...