Maspin, a member of the Serpin protease inhibitor family, is overexpressed in poorly differentiated colorectal tumors and more frequently found in tumors with microsatellite instability. Immunohistochemical nuclear Maspin staining is predominantly seen in tumor cells at the invasion front of such cancers, suggesting that this molecule is associated with local tumor cell infiltration and aggressiveness. In a retrospective study, we studied nuclear Maspin expression as a potential prognostic tool in a total of 172 primary stage III colon cancers by immunohistochemistry. Of those 172 patients, 76 were treated by surgery only, and 96 patients received additional adjuvant 5-fluorouracil (5-FU) based chemotherapy. Nuclear Maspin expression was an independent adverse prognostic factor for overall survival in our patient cohort (hazard ratio 2.08; 95% CI, 1.13-3.81; p 5 0.018). However, patients with primary tumors expressing Maspin in the nucleus showed a significant treatment benefit from 5-FU chemotherapy (hazard ratio 0.384; 95% CI, 0.188-0.784; p 5 0.009) compared to adjuvantly treated patients whose tumors did not express this molecule. Nuclear Maspin expression is highly predictive of 5-FU chemotherapy response in patients with advanced stage colon cancer. Patients with negative immunhistochemical Maspin expression do not benefit from 5-FU treatment and may be candidates for an alternative (non-5-FU based) adjuvant therapy regime. ' 2005 Wiley-Liss, Inc.Key words: colon cancer; Maspin; prediction; 5-flourouracil; chemotherapy Adjuvant 5-fluorouracil (5-FU) chemotherapy is the current standard therapy after surgical resection of stage III colon carcinoma, with an overall 5-year survival of only 50% in affected patients. It is estimated that up to 40% of patients would be cured by surgery alone, and only 10% of patients benefit from 5-FU chemotherapy.
Promoter hypermethylation occurs in various tumors and leads to silencing of tumor-relevant genes. Thus, promoter methylation analysis (MA) has been established as an important tool in cancer research and diagnostics. Here we present MethyQESD (methylation-quantification of endonuclease-resistant DNA) as a fast, easy, precise and reliable method for quantitative MA without the need of bisulfite-treatment or fluorescent probes. Though MethyQESD principally works with any gene promoter we established MethyQESD for the mismatch repair gene MLH1 and tested its utility to differentiate between sporadic microsatellite unstable (MSI-H) colorectal cancer and hereditary nonpolyposis colorectal cancer (HNPCC) by quantitative MLH1 MA. We investigated formalin-fixed and paraffin-embedded tissue samples from a previously published, well-characterized tumor collective comprising 25 HNPCC, 14 sporadic MSI-H CRC and 16 sporadic microsatellite stable (MSS) CRC. We found a high accuracy of MethyQESD by spiking experiments with dilution series of methylated (SW48 cancer cell line) and unmethylated (blood) DNA (Pearson's r ¼ 0.9997 (proximal MLH1 promoter region), r ¼ 0.9976 (distal MLH1 promoter region)). MethyQESD and conventional quantitative MA using of 96 formalinfixed and paraffin-embedded CRC showed a high degree of concordance of both methods (Pearson's r ¼ 0.885). HNPCC tumors showed either null MLH1 methylation or a significantly lower degree of MLH1 methylation than sporadic MSI-H CRC (Po0.001). MLH1 methylation was negative in all MSS tumors. Receiver operating characteristic (ROC) curve analyses defined a cutoff value of 16.5% MLH1 methylation for specific and sensitive identification of sporadic MSI-H CRC (area under ROC curve: 1.000; asymptotic significance: Po0.001). Thus, quantitative MLH1 MA by MethyQESD provides a simple, fast and valuable tool to identify HNPCC candidates. Furthermore, MethyQESD works reliably with formalin-fixed paraffin-embedded tissue and simplifies DNA MA both for research and diagnostic purposes.
Relatives of index patients are not sufficiently informed about the importance of predictive testing and the re-commended surveillance guidelines. An insufficient implementation of Lynch syndrome specific aspects of the S3-guideline can be assumed. For an improved implementation barriers of physicians' adherence must be systemically analyzed. It is essential for these high-risk families to establish and enforce awareness in order to create intensified surveillance.
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