Clinical significance of stromal apoptosis in colorectal cancer

BackgroundEpithelial and stromal cells play an important role in the development of colorectal cancer (CRC). We aimed to determine the prognostic significance of both epithelial and stromal cell apoptosis in CRC.MethodsTotal apoptosis was determined by caspase-3 activity measurements in protein homogenates of CRC specimens and adjacent normal mucosa of 211 CRC patients. Epithelial apoptosis was determined by an ELISA specific for a caspase-3-degraded cytokeratin 18 product, the M30 antigen. Stromal apoptosis was determined from the ratio between total and epithelial apoptosis.ResultsEpithelial and stromal apoptosis, as well as total apoptosis, were significantly higher in CRC compared with corresponding adjacent normal mucosa. Low total tumour apoptosis (< or = median caspase-3 activity) was associated with a significantly worse disease recurrence (hazard ratio (HR), 95% confidence interval (95% CI): 1.77 (1.05-3.01)), independent of clinocopathological parameters. Epithelial apoptosis was not associated with clinical outcome. In contrast, low stromal apoptosis (< or = median caspase-3/M30) was found to be an independent prognostic factor for overall survival, disease-free survival and disease recurrence, with HRs (95% CI) of 1.66 (1.17-2.35), 1.62 (1.15-2.29) and 1.69 (1.01-2.85), respectively.InterpretationStromal apoptosis, in contrast to epithelial apoptosis, is an important factor with respect to survival and disease-recurrence in CRC

Poorer outcome in stromal HIF-2α- and CA9-positive colorectal adenocarcinomas is associated with wild-type TP53 but not with BNIP3 promoter hypermethylation or apoptosis

Stromal expression of hypoxia inducible factor 2α (HIF-2α) and carbonic anhydrase 9 (CA9) are associated with a poorer prognosis in colorectal cancer (CRC). Tumour cell death, regulated by a hypoxic stromal microenvironment, could be of importance in this respect. Therefore, we correlated apoptosis, TP53 mutational status and BNIP3 promoter hypermethylation of CRC cells with HIF-2α- and CA9-related poor outcome. In a series of 195 CRCs, TP53 mutations in exons 5–8 were analysed by direct sequencing, and promoter hypermethylation of BNIP3 was determined by methylation-specific PCR. Expressions of HIF-2α, CA9, p53, BNIP3 and M30 were analysed immunohistochemically. Poorer survival of HIF-2α and CA9 stromal-positive CRCs was associated with wild-type TP53 (P=0.001 and P=0.0391), but not with BNIP3 methylation. Furthermore, apoptotic levels were independent of the TP53 status, but lower in unmethylated BNIP3 CRCs (P=0.004). It appears that wild-type TP53 in CRC cells favours the progression of tumours expressing markers for hypoxia in their stroma, rather than in the epithelial compartment. Preserved BNIP3 function in CRC cells lowers apoptosis, and may thus be involved in alternative cell death pathways, such as autophagic cell death. However, BNIP3 silencing in tumour cells does not impact on hypoxia-driven poorer prognosis

Presence of HIF-1 and related genes in normal mucosa, adenomas and carcinomas of the colorectum

Expression of the transcription factor hypoxia-inducible factor 1 (HIF-1), which plays a key role in cellular adaptation to hypoxia, was investigated in normal colorectal mucosa (ten), adenomas (61), and carcinomas (23). Tissue samples were analyzed for HIF-1α, its upstream regulators, von Hippel–Lindau factor, AKT, and mammalian target of rapamycin (mTOR) and its downstream targets glucose transporter 1 (GLUT1), carbonic anhydrase IX, stromal-cell-derived factor 1 (SDF-1) by immunohistochemistry. In normal colorectal mucosa, HIF-1α was observed in almost all nuclei of surface epithelial cells, probably secondary to a gradient of oxygenation, as indicated by pimonidazole staining. The same staining pattern was present in 87% of adenomas. In carcinomas, HIF-1α was present predominantly around areas of necrosis (78%). Active AKT and mTOR, were present in all adenomas, carcinomas, and in normal colorectal mucosa. GLUT1 and SDF-1 were present in the normal surface epithelium of all adenoma cases, whereas in the carcinoma GLUT1 was located around necrotic regions and SDF-1 was present in all epithelial cells. In conclusion, HIF-1α appears to be physiologically expressed in the upper part of the colorectal mucosa. The present observations support that upregulation of HIF-1α and its downstream targets GLUT1 and SDF-1 in colorectal adenomas and carcinomas may be due to hypoxia, in close interaction with an active phosphatidylinositol 3-kinases–AKT–mTOR pathway

Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer

BACKGROUND: Delta-like ligand 4 (Dll4) is a Notch ligand that is upregulated by hypoxia and vascular endothelial growth factor-A (VEGF-A) and is reported to have a role in tumor angiogenesis. Evidence from xenograft studies suggests that inhibiting Dll4-Notch signalling may overcome resistance to anti-VEGF therapy. The aim of this study was to characterise the expression of Dll4 in colon cancer and to assess whether it is associated with markers of hypoxia and prognosis. METHOD: In all, 177 colon cancers were represented in tissue microarrays. Immunohistochemistry was performed using validated antibodies against Dll4, VEGF, hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, prolyl hydroxylase (PHD)1, PHD2, PHD3 and carbonic anhydrase 9 (CA9). RESULTS: The expression of Dll4 was observed preferentially in the endothelium of 71% (125 out of 175) of colon cancers, but not in the endothelium adjacent to normal mucosa (none out of 107, P<0.0001). The expression of VEGF was significantly associated with HIF-2alpha (P<0.0001) and Dll4 (P=0.010). Only HIF-2alpha had a significant multivariate prognostic effect (hazard ratio 1.61, 95% confidence interval 1.01-2.57). Delta-like ligand 4 was also expressed by neoplastic cells, particularly neoplastic goblet cells. CONCLUSION: Endothelial expression of Dll4 is not a prognostic factor, but is significantly associated with VEGF. Assessing endothelial Dll4 expression may be critical in predicting response to anti-VEGF therapies

Tumour–stroma interactions in colorectal cancer: converging on β-catenin activation and cancer stemness

Sporadic cases of colorectal cancer are primarily initiated by gene mutations in members of the canonical Wnt pathway, ultimately resulting in β-catenin stabilisation. Nevertheless, cells displaying nuclear β-catenin accumulation are nonrandomly distributed throughout the tumour mass and preferentially localise along the invasive front where parenchymal cells are in direct contact with the stromal microenvironment. Here, we discuss the putative role played by stromal cell types in regulating β-catenin intracellular accumulation in a paracrine fashion. As such, the tumour microenvironment is likely to maintain the cancer stem cell phenotype in a subset of cells, thus mediating invasion and metastasis

Prognostic DNA methylation markers for sporadic colorectal cancer: a systematic review

Background Biomarkers that can predict the prognosis of colorectal cancer (CRC) patients and that can stratify high-risk early stage patients from low-risk early stage patients are urgently needed for better management of CRC. During the last decades, a large variety of prognostic DNA methylation markers has been published in the literature. However, to date, none of these markers are used in clinical practice. Methods To obtain an overview of the number of published prognostic methylation markers for CRC, the number of markers that was validated independently, and the current level of evidence (LoE), we conducted a systematic review of PubMed, EMBASE, and MEDLINE. In addition, we scored studies based on the REMARK guidelines that were established in order to attain more transparency and complete reporting of prognostic biomarker studies. Eighty-three studies reporting on 123 methylation markers fulfilled the study entry criteria and were scored according to REMARK. Results Sixty-three studies investigated single methylation markers, whereas 20 studies reported combinations of methylation markers. We observed substantial variation regarding the reporting of sample sizes and patient characteristics, statistical analyses, and methodology. The median (range) REMARK score for the studies was 10.7 points (4.5 to 17.5) out of a maximum of 20 possible points. The median REMARK score was lower in studies, which reported a p value below 0.05 versus those, which did not (p = 0.005). A borderline statistically significant association was observed between the reported p value of the survival analysis and the size of the study population (p = 0.051). Only 23 out of 123 markers (17%) were investigated in two or more study series. For 12 markers, and two multimarker panels, consistent results were reported in two or more study series. For four markers, the current LoE is level II, for all other markers, the LoE is lower. Conclusion This systematic review reflects that adequate reporting according to REMARK and validation of prognostic methylation markers is absent in the majority of CRC methylation marker studies. However, this systematic review provides a comprehensive overview of published prognostic methylation markers for CRC and highlights the most promising markers that have been published in the last two decades