Prognostic value of apoptosis in rectal cancer patients of the dutch total mesorectal excision trial: radiotherapy is redundant in intrinsically high-apoptotic tumors.

PURPOSE: The combination of radiotherapy and good quality surgery reduces local recurrence rate for rectal cancer patients. This study assesses the prognostic value of both intrinsic and radiotherapy-induced apoptosis and evaluates the relevance of radiotherapy for outcome of rectal cancer patients. EXPERIMENTAL DESIGN: Tumor samples (1,198) were available from the Dutch Total Mesorectal Excision trial, in which rectal cancer patients were treated with standardized surgery and randomized for preoperative short-term radiotherapy or not. Tumor samples were obtained at time of surgery. Tissue microarrays were constructed and stained with the active caspase-specific M30 antibody to determine the amount of apoptotic epithelial tumor cells. RESULTS: Nonirradiated patients with a negative circumferential margin displaying lower than median levels of apoptosis developed more local recurrences (10.5% versus 6.1%; P=0.06) and more rapidly after surgery than patients with high intrinsic apoptosis in their tumors (median time to recurrence, 13.0 versus 21.3 months; P=0.04). In multivariate analysis, intrinsic apoptosis was an independent predictor for the development of local recurrences (hazard ratio, 2.0; P=0.05). Radiotherapy increased apoptosis level (11 versus 23 apoptotic cells/mm2 tumor epithelium; P<0.001), but this apoptosis did not influence patients' prognosis. CONCLUSIONS: Rectal cancer patients with low intrinsic apoptosis will benefit from radiotherapy with respect to the development of local recurrences. Because apoptosis is an inherent characteristic of tumors, patients who do not need radiotherapy may be selected based on the apoptotic index of the primary tumor.

Macrodissection versus microdissection of rectal carcinoma: minor influence of stroma cells to tumor cell gene expression profiles.

BACKGROUND: The molecular determinants of carcinogenesis, tumor progression and patient prognosis can be deduced from simultaneous comparison of thousands of genes by microarray analysis. However, the presence of stroma cells in surgically excised carcinoma tissues might obscure the tumor cell-specific gene expression profiles of these samples. To circumvent this complication, laser microdissection can be performed to separate tumor epithelium from the surrounding stroma and healthy tissue. In this report, we compared RNAs isolated from macrodissected, of which only surrounding healthy tissue had been removed, and microdissected rectal carcinoma samples by microarray analysis in order to determine the most reliable approach to detect the expression of tumor cell-derived genes by microarray analysis. RESULTS: As microdissection yielded low tissue and RNA quantities, extra rounds of mRNA amplification were necessary to obtain sufficient RNA for microarray experiments. These second rounds of amplification influenced the gene expression profiles. Moreover, the presence of stroma cells in macrodissected samples had a minor contribution to the tumor cell gene expression profiles, which can be explained by the observation that more RNA is extracted from tumor epithelial cells than from stroma. CONCLUSION: These data demonstrate that the more convenient procedure of macrodissection can be adequately used and yields reliable data regarding the identification of tumor cell-specific gene expression profiles.

High intrinsic apoptosis, but not radiation-induced apoptosis, predicts better survival in rectal carcinoma patients.

PURPOSE: An important feature of malignant tumors is the disturbance in the balance between proliferation and cell death. We evaluated the relevance of intrinsic and radiation-induced apoptosis and proliferation for prognosis in rectal cancer patients. METHODS AND MATERIALS: Patients were selected from a study that randomized for preoperative radiotherapy (RT). Apoptosis and proliferation were scored using specific antibodies in immunohistochemistry. The number of positive cells per square millimeter of carcinoma cells was determined in 98 randomly selected tumors, of which 45 had been irradiated. For the survival analyses, a cohort of 104 patients without positive circumferential resection margins was selected. RESULTS: In nonirradiated patients, high levels of intrinsic apoptosis correlated with better local control (p = 0.04) and better cancer-specific survival (p = 0.02). RT increased the median amount of apoptosis from 10.8 to 21.5 cells/mm(2) (p = 0.004), but this was not predictive for survival. The amount of proliferative cells was not altered after RT and had no influence on prognosis. CONCLUSIONS: Intrinsic apoptosis correlated with both local control and cancer-specific survival, but proliferation was not predictive for prognosis. However, although RT increased apoptosis, its prognostic value was lost after RT. This is possibly because in rectal cancer, the proliferative status of tumors is always high and the aggressiveness of the tumor is determined by the number of "spontaneous" apoptotic tumor cells.

p53 expression in human rectal tissue after radiotherapy: upregulation in normal mucosa versus functional loss in rectal carcinomas.

PURPOSE: In vitro, ionizing radiation of epithelial cells leads to upregulation of wild-type p53 and subsequent induction of p21(waf1). The effect of radiotherapy (RT) on the expression of these proteins in patients is unknown. We assessed the influence of RT on the expression of p53 and p21(waf1) in normal mucosa and rectal carcinomas in vivo. METHODS: Tumor and normal tissue samples were derived from rectal cancer patients randomized in a clinical trial in which the value of preoperative RT was evaluated. p53 and p21(waf1) expression was determined in 51 irradiated and 52 nonirradiated patients using immunohistochemistry. RESULTS: In normal mucosa, both p53 and p21(waf1) were strongly upregulated after RT compared with the expression in unirradiated normal tissue (p <0.001). In tumor cells, no significant difference in the expression of p53 or p21(waf1) was found in the irradiated vs. nonirradiated group. In the few rectal tumors with wild-type p53, induction of p53 after RT did not necessarily lead to upregulation of p21(waf1). CONCLUSION: These findings demonstrate that in normal mucosa, a functional p53-p21(waf1) pathway is present, whereas in tumor cells it is defective in almost all cases because of either p53 mutation or down- or upstream disruption in tumors with wild-type p53. Therefore, we believe that the role of p53 expression as a single prognostic marker in rectal cancer needs reconsideration.

c-Myc is able to sensitize human melanoma cells to diverse apoptotic triggers.

Compared with other types of tumours, malignant melanomas are highly refractory to radio- or chemotherapy. To support the search for possible sensitizers, we explored the effects of the cellular oncoproteins c-Myc and N-Ras, which can decrease the clonogenic potential of irradiated p53-negative IGR39D melanoma cells. Using stable transfectants of this cell line, we showed that mutant N-Ras decreased the proliferation rate by inducing a prolonged cell cycle arrest. In contrast, c-Myc made these melanoma cells more prone to radiation-induced cell death. Membrane blebbing, the formation of apoptotic bodies and caspase activation, as measured by cleavage of Asp-Glu-Val-Asp (DEVD) substrate and poly(ADP-ribose) polymerase (PARP), indicate that these cells die by an apoptotic process. c-Myc also sensitized these p53-deficient melanoma cells to treatment with various cytotoxic drugs and heat shock. Similar results were obtained in inducible c-Myc models of IGR39D and in another melanoma cell line, 9007, which expresses functional p53. Together, these findings indicate that c-Myc is capable of sensitizing typically resistant tumour cells and that this occurs irrespective of the functional status of the p53 protein. Our results should facilitate the identification of factors that can be exploited for the treatment of aggressive cancers.

Gene expression profiling reveals two separate mechanisms regulating apoptosis in rectal carcinomas in vivo.

The level of apoptosis in rectal carcinomas of patients treated by surgery only predicts local failure; patients with intrinsically high-apoptotic tumors develop less local recurrences than patients with low levels of apoptosis. To identify genes involved in this intrinsic apoptotic process in vivo, 47 rectal tumors with known apoptotic phenotype (24 low- and 23 high-apoptotic) were analyzed by oligonucleotide microarray technology. We identified several genes differentially expressed between low- and high-apoptotic tumors. Unsupervised clustering of the tumors based on expression levels of these genes separated the low-apoptotic from the high-apoptotic tumors, indicating a gene expression-dependent regulation. In addition, this clustering revealed two subgroups of high-apoptotic tumors. One high-apoptotic subgroup showed subtle differences in mRNA and protein expression of the known apoptotic regulators BAX, cIAP2 and ARC compared to the low-apoptotic tumors. The other subgroup of high-apoptotic tumors showed high expression of immune-related genes; predominantly HLA class II and chemokines, but also HLA class I and interferon-inducible genes were highly expressed. Immunohistochemistry revealed HLA-DR expression in epithelial tumor cells in 70% of these high-apoptotic tumors. The expression data suggest that high levels of apoptosis in rectal carcinoma patients can be the result of either slightly altered expression of known pro- and anti-apoptotic genes or high expression of immune-related genes.