Prospective study of intratumoral microvessel density, p53 expression and survival in colorectal cancer

Adjuvant treatment of patients with colorectal cancer is hampered by a lack of reliable prognostic factors in addition to the clinicopathological staging system. A poorly defined but considerable fraction of Astler–Coller stage B patients will experience tumour recurrence, and some of the stage C patients will probably survive for a prolonged time after surgery without adjuvant treatment. Assessing parameters related to tumour angiogenesis has provided valuable prognostic information in different tumour types. The formation of new microvessels is part of the malignant phenotype in the majority of tumours. Alterations in tumour-suppressor genes, such as the p53 gene, or oncogenes, such as the ras gene, have been found to be responsible for changing the local balance of pro- and antiangiogenic factors in favour of the former. In this prospective study, intratumoral microvessel density (IMD) was assessed by immunostaining tissue sections for CD31 and counting individual microvessels in selected and highly vascular regions in specimens of 145 colorectal cancer patients. p53 protein overexpression was semiquantitatively determined after immunohistochemistry. In both uni- and multivariate analysis, high IMD was significantly associated with shorter survival in the patients undergoing surgery with curative intent (Astler–Coller stages A–C). p53 added prognostic power to IMD, both in Astler–Coller stage B and stage C patients. An association between IMD and mode of metastasis was also noted. High IMD was strongly associated with the incidence of haematogenous metastasis during follow-up, but not with the presence of lymphogenic metastasis observed at surgery. This study confirms the results of previous retrospective analyses of IMD and survival in colorectal cancer and warrants a clinical validation by randomizing stage B tumour patients with high IMD and p53 overexpression between adjuvant treatment or not. © 1999 Cancer Research Campaig

Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study

BACKGROUND: The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. METHODS: In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. RESULTS: Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. CONCLUSION: CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency

Novel KRAS Gene Mutations in Sporadic Colorectal Cancer

In this article, we report 7 novel KRAS gene mutations discovered while retrospectively studying the prevalence and pattern of KRAS mutations in cancerous tissue obtained from 56 Saudi sporadic colorectal cancer patients from the Eastern Province.Genomic DNA was extracted from formalin-fixed, paraffin-embedded cancerous and noncancerous colorectal tissues. Successful and specific PCR products were then bi-directionally sequenced to detect exon 4 mutations while Mutector II Detection Kits were used for identifying mutations in codons 12, 13 and 61. The functional impact of the novel mutations was assessed using bioinformatics tools and molecular modeling.KRAS gene mutations were detected in the cancer tissue of 24 cases (42.85%). Of these, 11 had exon 4 mutations (19.64%). They harbored 8 different mutations all of which except two altered the KRAS protein amino acid sequence and all except one were novel as revealed by COSMIC database. The detected novel mutations were found to be somatic. One mutation is predicted to be benign. The remaining mutations are predicted to cause substantial changes in the protein structure. Of these, the Q150X nonsense mutation is the second truncating mutation to be reported in colorectal cancer in the literature.Our discovery of novel exon 4 KRAS mutations that are, so far, unique to Saudi colorectal cancer patients may be attributed to environmental factors and/or racial/ethnic variations due to genetic differences. Alternatively, it may be related to paucity of clinical studies on mutations other than those in codons 12, 13, 61 and 146. Further KRAS testing on a large number of patients of various ethnicities, particularly beyond the most common hotspot alleles in exons 2 and 3 is needed to assess the prevalence and explore the exact prognostic and predictive significance of the discovered novel mutations as well as their possible role in colorectal carcinogenesis

IMMUNOHISTOCHEMICAL STUDY OF HEPATIC-FIBROSIS INDUCED IN RATS BY MULTIPLE GALACTOSAMINE INJECTIONS

Multiple injections of D-galactosamine induce liver fibrosis and cirrhosis in rats. The purpose of this immunopathological study was to correlate inflammation and hepatic extracellular matrix remodeling after repeated administration of galactosamine. Rats were given 10, 20, 30, 40, 60, 80, 100 and 140 intraperitoneal injections of D-galactosamine (500 mg/kg body wt, three times weekly). Controls received injections of saline solution. Cryostat sections of lives tissue obtained on biopsy or autopsy were immunostained with a panel of monoclonal and polyclonal monospecific antibodies reactive with macrophages, T and B lymphocytes, desmin, the extracellular matrix components fibronectin; laminin; collagen types I, III and IV; and the fibronectin receptor alpha 5 beta 1. Total RNA was extracted and Northern-blot analysis was performed with a specific cDNA probe for rat collagen type III. Spotty liver cell necrosis and mild portal and parenchymal inflammation was seen after 10 injections of galactosamine. After 20 to 40 injections, expansion of portal tracts, prominent bile ductular proliferation and gradual formation of fibrous septa were encountered with the development of cirrhosis at later intervals. These progressive histological changes were Paralleled by a gradual increase of desmin-positive cells in developing septa with deposition of fibronectin; collagen types I, III and IV; and Iaminin. Northern-blot analysis showed that this accumulation of extracellular matrix was not accompanied by increase of mRNA for collagen type III. In conclusion, repetitive administration of galactosamine causes progressive liver disease with prominent bile ductule proliferation and development of fibrous septa. These pathological alterations bear some resemblance to the morphological changes in chronic biliary disease in human beings