CTCs detection by CellSearch is a highly reproducible method that correlates with stage but not with other clinical and morphological variables in patients with colorectal cancer. Colon cancer tumor cells are detectable in all stages. Further studies are warranted.

The p16INK4a gene, localized within chromosome 9p21, has been identified as a cyclin-dependent kinase inhibitor and may negatively regulate the cell cycle acting as a tumor suppressor. Genetic alterations involving the 9p21 region are common in human cancers. A consecutive series of 64 untreated patients (median of follow up 53 months) undergoing surgical resection for locally advanced laryngeal squamous-cell carcinomas (LSCCs) has been studied prospectively. Our purpose was to investigate p16 alterations (9p21 allelic loss, hypermethylation and point mutations) and their possible association with clinico-pathological data and flow cytometric variables (DNA-ploidy and S-phase fraction (SPF)), and to determine the possible prognostic role of this gene in these tumors. PCR-based techniques were used for investigating 9p21 loss of heterozygosity (LOH) and methylation promoter status of the p16 gene. p16 mutations were detected by PCR-SSCP (single strand conformation polymorphism) and sequencing. 9p21 LOH was detected in 16/62 (26%) informative tumors, point mutations in 5% (3/64) and hypermethylation in 9% (6/64) of the cases. p16 alterations were significantly associated with high SPF and DNA-aneuploidy. By univariate analysis, poor histologic differentiation, stage IV, DNA-aneuploidy and p16 point mutations proved to be significantly related to quicker relapse, whereas these same factors, and in addition high SPF, 9p21 LOH and any p16 alterations were significantly related to shorter overall survival. By Cox proportional hazards analysis only histologic grade (G3) and p16 point mutations were independently related to both disease relapse and death. Our study has identified p16 point mutations as important biomolecular indicators in LSCCs.

The relationship between molecular abnormalities and neoplasm has been extensively reviewed and there is strong evidence that abnormalities of p53 gene represent the most common molecular change in human cancer. Such abnormalities can be detected in a number of ways. Several prior studies have revealed p53 protein expression via immunohistochemistry (IHC) in 42Ð69% of colorectal cancers (Scott et al, 1991;Auvinen et al, 1994). However, the prognostic value of this protein remains to be defined, probably due to variability of detection and retrieval systems (Wynford-Thomas, 1994). To be clinically useful, prognostic markers should be accessible to analysis with simple and reproducible procedures appropriate for routine use. We have adapted the recently developed luminometric immunoassay (LIA) (Borg et al, 1995), to quantitate p53 protein in archival colorectal cytosols.The purpose of the present study was: (1) to evaluate the relationship between p53 overexpression and clinicopathological data and (2) to assess the value of p53 as a biological marker of prognosis within each TNM class in a series of patients resected for colorectal cancer with a long follow-up. PATIENTS AND METHODS PatientsA series of 111 patients underwent surgical resection for primary colorectal adenocarcinoma at the II Department of Surgery, University Hospital ÔSan CarlosÕ, Madrid, between 1990 and 1992. Each patient is regularly followed up at 6-monthly intervals for a minimum of 5 years. Cases in which resections have been performed for metachronous carcinoma, carcinoma arising in familial adenomatous polyposis and ulcerative colitis are excluded. None of the patients had received preoperative radiotherapy or chemotherapy. Since 1992, stage III patients younger than 70 received adjuvant chemotherapy with 5-fluorouracil (5-FU) and leucovorin according to the prevailing protocol (four patients). Clinical staging was done on the basis of the TNM classification. Survival time was calculated from the date of surgery to the date of death or last follow-up, with times censored for patients dying of causes unrelated to colorectal cancer and those surviving. Median follow-up was 5 years. Tissue specimensSections from the colorectal adenocarcinoma and normal mucosa at the proximal/distal resection margins were obtained at surgical resection. These specimens were stored in liquid nitrogen and, prior to being pulverized in the frozen state and homogenized for the preparation of cytosols for protein measurement, cryostat sections were evaluated; all tumour samples used contained more than 80% tumour cells. Luminometric assayThe LIA is based on a combination of two monoclonal antibodies, Ab 1801 and DO 1, which detect both wild-type and mutant p53 protein in a sandwich-type assay. The Ab1801, which is immobilized onto a solid phase, is used for catching. Ab DO 1, labelled with a chemiluminescent compound (ABEI), is used for detection. The immunoassay was performed by incubating either 100 µl of p53 standard, controls or tumour cytosols, together with 10...

There is a subgroup of carcinomas which develop via the MSI pathway that carry an alteration of the BRAF gene. This alteration confers a poorer outcome on these patients within the total group of patients with MSI who have a better prognosis. This hypothesis should be further investigated in a larger study population due to the low incidence of BRAF mutations in colorectal cancer.

Background: As the enzyme telomerase extends the life of the cell through its ability to lengthen telomeres, its activity in different types of tumor has been evaluated as a possible factor involved in tumorigenesis. The aim of this study was to assess the prognostic significance of telomerase activity in patients with colorectal carcinoma. Patients and Methods: Telomerase activity was determined in 103 patients undergoing surgery for colorectal cancer between 2001 and 2003. Telomerase activity was determined by an enzyme-linked immunoassay based on the amplification of telomeric repeat sequences (TRAP assay). Results: 90% of our study population showed telomerase activity. Telomerase activity was related to tumor stage and site: a lower proportion of patients with stage A tumors showed telomerase activity compared to more advanced stages; and more patients with colon than with rectal carcinomas were telomerase positive. Multivariate analysis revealed that by adjusting for tumor stage, telomerase activity could be used to predict the risk of death or recurrence (p < 0.001). Conclusions: Activation of telomerase seems to be a frequent event related to the stage of the tumor in colorectal tumorigenesis. Our findings suggest that telomerase activity can predict a greater risk of death or recurrence, irrespective of the more conventional prognostic factors.