Inflammatory infiltration, fibrosis, necrosis and mucinous content in relation to clinicopathological and molecular factors in rectal cancers with or without preoperative radiotherapy

The association between inflammatory infiltration, fibrosis, necrosis and mucinous content in rectal cancers, and their relationship to preoperative radiotherapy (RT) clinicopathological and biological factors (p53, apoptosis and Cox-2) is not fully characterised. We analysed these histopathological parameters and their relationships in rectal cancer patients who participated in a clinical trial of preoperative RT. One hundred and forty-eight preoperative biopsies and 153 surgically resected tumours were examined. Of the surgical specimens, 81 had surgery alone and 72 received RT before surgery. A higher grade of inflammatory infiltration was related to favourable survival in the whole group of patients (p=0.004, for multivariate analysis p=0.01) as well as in the subgroups of patients with (p=0.04) or without RT (p=0.01). After RT, tumours showed a decreased infiltration (p=0.0003) and increased necrosis (p=0.006), strong necrosis was related to favourable survival (p=0.046). Necrosis (p=0.054) and fibrosis (p=0.06) tended to be increased in p53-negative tumours after RT. Inflammatory infiltration was a strong prognostic factor in rectal cancer patients, regardless of RT. RT tended to induce necrosis and fibrosis in p53-negative tumours.     

Divergence between the high rate of p53 mutations in skin carcinomas and the low prevalence of anti-p53 antibodies.

Circulating anti-p53 antibodies have been described and used as tumoural markers in patients with various cancers and strongly correlate with the p53 mutated status of the tumours. No study has yet looked at the prevalence of such antibodies in skin carcinoma patients although these tumours have been shown to be frequently p53 mutated. Most skin carcinoma can be diagnosed by examination or biopsy, but aggressive, recurrent and/or non-surgical cases' follow up would be helped by a biological marker of residual disease. We performed a prospective study looking at the prevalence of anti-p53 antibodies using an ELISA technique in a series of 105 skin carcinoma patients in comparison with a sex- and age-matched control skin carcinoma-free group (n = 130). Additionally, p53 accumulation was studied by immunohistochemistry to confirm p53 protein altered expression in a sample of tumours. Anti-p53 antibodies were detected in 2.9% of the cases, with a higher prevalence in patients suffering from the more aggressive squamous cell type (SCC) of skin carcinoma (8%) than for the more common and slowly growing basal cell carcinoma type or BCC (1.5%). p53 protein stabilization could be confirmed in 80% of tumours studied by IHC. This low level of anti-p53 antibody detection contrasts with the high rate of p53 mutations reported in these tumours. This observation shows that the anti-p53 humoral response is a complex and tissue-specific mechanism.     

Determination of microsatellite instability, p53 and K-RAS mutations in hepatic metastases from patients with colorectal cancer: relationship with response to 5-fluorouracil and survival

In vitro and clinical studies have suggested that high-frequency microsatellite instability (MSI-H) phenotype, p53 and K-ras mutations might influence the response to chemotherapy in a variety of tumors, including primary colorectal cancers (CRC). Unresectable hepatic metastases from CRC are commonly treated with 5-fluorouracil (5FU) and folinic acid. Since several new active drugs are now used for treating CRC, molecular determinants predictive to response to 5FU would thus be crucial for optimizing indications of chemotherapy to those patients. MSI-H phenotype, p53 and K-ras status were characterized in a prospective study of 56 patients with CRC metastatic to the liver and treated with 5FU-based chemotherapy. The objective response rate after a 3-month treatment was 32.1%. The prevalence of p53 mutations, K-ras mutations and MSI-H phenotype was 62.5%, 30.3% and 1.8%, respectively. No significant association was found between response to chemotherapy and p53 mutations (78% mutated tumors in responders vs. 55% in nonresponders; p = 0.10) and K-ras mutations (39% mutated tumors in responders vs. 26% in nonresponders; p = 0.34). Survival was longer for patients with p53-mutated metastases than for patients with unresected wild-type p53 metastases (median survival 15 months vs. 17 months; p = 0.06). The determination of the MSI-H phenotype, p53 and K-ras status in hepatic metastases from CRC does not discriminate a group of patients that should preferentially benefit from 5FU-based chemotherapy. The prognosis of patients with treated liver metastases is better when p53 is mutated.     

Apoptosis and its correlation with proliferative activity in rectal cancer

BACKGROUND AND OBJECTIVES: Alterations in the normal control of apoptosis and cell proliferation are important factors in multistep colorectal carcinogenesis. The aim of this study was to determine the frequency of apoptosis and cell proliferation in rectal cancers and to examine their relationship to clinicopathological variables and expression of bcl-2 and p53. METHODS: Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining and immunohistochemical staining for Ki-67, bcl-2, and p53 were performed on paraffin-embedded tissue samples of 57 rectal cancers. RESULTS: There was a positive linear correlation between apoptotic index (AI) and proliferative index (PI) (gamma = 0.276, P = 0.038). Both apoptosis and cell proliferation were more frequently found in rectal cancers with lymph node metastasis (P = 0.045 and 0.010, respectively). However, the ratio of AI and PI was not different by nodal status. There was no association between Dukes stage and AI or PI. The frequency of apoptosis was inversely related to the expression of bcl-2, but was not related to the p53 status of rectal cancer. There were no association between cell proliferation and the expression of bcl-2 or p53. CONCLUSIONS: Our results suggest that the susceptibility to apoptosis in rectal cancer is clearly related to the proliferative activity and high turnover rate of tumor cells may contribute to lymph node metastasis.     

Suppression of tumorigenic and metastatic potentials of human melanoma cell lines by mutated (143 Val-Ala) p53.

Metastatic melanoma, compared with other cancers, appears to be unusual because of its low frequency of p53 mutations and prevalence of wild-type p53 protein in advanced malignancy. Here, we examined the effects of wild-type and mutated p53 (143 Val-Ala) on tumorigenic and metastatic potential of two human melanoma cell lines. The cell line UISO-MEL-4 contains wild-type p53 and is tumorigenic, whereas UISO-MEL-6 lacks p53 and produces lung and liver metastasis upon s.c. injection into athymic mice. Our study showed that UISO-MEL-4 stably transfected with wild-type p53 cDNA driven by cytomegalovirus promoter-enhancer sequences expressed high levels of p53 and p21 and formed s.c. tumours in vivo. Mutated p53 (143 Val-Ala) expression, on the other hand, inhibited tumour growth in 50% of cases and produced significantly slower growing non-metastatic tumours. Reduced tumour growth involved necrotic as well as apoptotic cell death. Inhibition of tumour growth was abrogated by the addition of Matrigel (15 mg ml(-1)). With UISO-MEL-6 cells, stably transfected with mutant p53, tumour growth was delayed and metastasis was inhibited. In soft agar colony formation assay, both wild-type and mutant p53 transfectants reduced anchorage-independent colony formation in vitro. These data suggest that mutated (143 Val-Ala) p53, which retains DNA binding and some of the transactivation functions of the wild-type p53 protein, suppresses tumorigenic and metastatic potentials of human melanoma cell lines in vivo.     

Targeting the MDM2-p53 interaction for cancer therapy

p53 is a powerful tumor suppressor and is an attractive cancer therapeutic target because it can be functionally activated to eradicate tumors. The gene encoding p53 protein is mutated or deleted in half of human cancers, which inactivates its tumor suppressor activity. In the remaining cancers with wild-type p53 status, its function is effectively inhibited through direct interaction with the human murine double minute 2 (MDM2) oncoprotein. Blocking the MDM2-p53 interaction to reactivate the p53 function is a promising cancer therapeutic strategy. This review will highlight the advances in the design and development of small-molecule inhibitors of the MDM2-p53 interaction as a cancer therapeutic approach.     

Nutlin-3, an Antagonist of MDM2, Enhances the Radiosensitivity of Esophageal Squamous Cancer with Wild-Type p53

Murine double minute 2 (MDM2) negatively regulates the activity of the p53 protein and plays a vital role in cell cycle arrest, apoptosis, and senescence mediated by p53. Nutlin-3, an antagonist of MDM2, is frequently used in anti-cancer studies. In many human tumors, nutlin-3 stabilizes p53 status and enhances p53 expression in cells with wild-type p53. However, the effect of nutlin-3 combined with radiotherapy on esophageal squamous cancer (ESCC) has not been reported. In this study, we examined whether nutlin-3 increases the radiosensitivity of ESCC in vitro and in vivo.We chose two cell lines, ECA-109 (wild-type p53) and TE-13 (p53 mutated), for the following experiments. Cell proliferation and clonogenic survival experiments showed that nutlin-3 inhibits the cell growth and colony formation of ECA-109 cells in a dose-dependent manner. Flow cytometry analysis showed that the apoptosis rate of ECA-109 cells co-treated with nutlin-3 and irradiation(IR) was significantly increased compared with cells treated with irradiation or nutlin-3 alone. Western blotting detected the expression of apoptosis-associated proteins in ECA-109 cells in response to nutlin-3 and irradiation. These effects were not evident in TE-13 cells. Xenograft mouse models indicated that nutlin-3 suppresses tumor growth and promotes radiosensitivity in the ESCC cell line ECA-109 in vivo. We have demonstrated that co-treatment of nutlin-3 with irradiation can significantly inhibit the growth and improve the radiosensitivity of ESCC cells with wild-type p53. The study suggests that nutlin-3 may be a potent therapeutic agent in conjunction with radiotherapy in ESCC.     

Meat consumption and K-ras mutations in sporadic colon and rectal cancer in The Netherlands Cohort Study

Case-cohort analyses were performed on meat and fish consumption in relation to K-ras mutations in 448 colon and 160 rectal cancers that occurred during 7.3 years of follow-up, excluding the first 2.3 years, and 2948 subcohort members of The Netherlands Cohort Study on diet and cancer. Adjusted incidence rate ratios and 95% confidence intervals were computed for colon and rectal cancer and for K-ras mutation status subgroups. Total fresh meat, most types of fresh meat and fish were not associated with colon or rectal cancer, neither overall nor with K-ras mutation status. However, several weak associations were observed for tumours with a wild-type K-ras, including beef and colon tumours, and an inverse association for pork with colon and rectal tumours; for meat products, an increased association was observed with wild-type K-ras tumours in the colon and possibly with G>A transitions in rectal tumours.     

Therapy effect of either paclitaxel or cyclophosphamide combination treatment in patients with epithelial ovarian cancer and relation to TP53 gene status.

Cell death after treatment with chemotherapy is exerted by activation of apoptosis, and the p53 protein has been shown to actively participate in this process. This recent focus on TP53 status as a possible determinant of cancer therapy response has raised the question of whether or not mutations in the TP53 gene have an influence on paclitaxel therapy. The TP53 status has been analysed at the DNA level in tumours from 45 ovarian cancer patients randomized to treatment with paclitaxel and cisplatin or cyclophosphamide and cisplatin. Therapy response was obtained for 38 patients with clinically evaluable disease after initial surgery. The positive response rate to the paclitaxel/cisplatin therapy was 85% vs 61% for the patients who received the cyclophosphamide/cisplatin regimen. A significant difference in relapse-free survival in favour of paclitaxel/cisplatin chemotherapy was found (P = 0.001). A total of 33 tumour samples (73%) had detectable sequence alterations in the TP53 gene. When relapse-free survival was estimated for all patients with TP53 alterations in their tumours, a significant better outcome for the paclitaxel/cisplatin group was found compared with the patient group receiving cyclophosphamide and cisplatin therapy (P = 0.002). We did not observe an association between TP53 tumour status and prognosis for patients who received paclitaxel/cisplatin combination treatment, indicating that the effect of this therapy is not influenced by this parameter.     

大肠癌培养细胞p53存在状态与化疗敏感性

目的　探讨人大肠癌培养细胞p5 3存在状态与化疗敏感性的相关性。方法　应用p5 3机能诊断法 ,确认 6株人大肠癌培养细胞p5 3的存在状态 ,以MTT方法比较了p5 3野生型和突变型两组肠癌细胞对化疗敏感性的差异 ,用流式细胞仪做细胞周期的进一步分析。结果　p5 3野生型肠癌细胞较p5 3突变型细胞的化疗敏感性高 5～ 10倍 ,并且在化疗药阿霉素诱导下可选择地发生细胞凋亡和G1阻滞。结论　人体大肠癌培养细胞p5 3存在状态直接与其化疗敏感性有关。     

Generation of a selectively cytotoxic fusion protein against p53 mutated cancers

ABSTRACT: BACKGROUND: A significant number of cancers are caused by defects in p21 causing functional defects in p21 or p53 tumour-suppressor proteins. This has led to many therapeutic approaches including restoration by gene therapy with wild-type p53 or p21 using viral or liposomal vectors, which have toxicity or side-effect limitations. We set out to develop a safer, novel fusion protein which has the ability to reconstitute cancer cell lines with active p21 by protein transduction. METHODS: The fusion protein was produced from the cell-translocating peptide Antennapedia (Antp) and wild-type, full-length p21 (Antp-p21). This was expressed and refolded from E. coli and tested on a variety of cell lines and tumours with differing p21 or p53 status. RESULTS: Antp-p21 penetrated and killed cancer cells that do not express wild type p53 or p21. This included cells that were matched to cogenic parental cell lines. Antp-p21 killed cancer cells selectively that were malignant as a result of mutations or nuclear exclusion of the p53 and p21 genes and over-expression of MDM2. Non-specific toxicity was excluded by showing that Antp-p21 penetrated but did not kill p53- or p21- wild-type cells. Antp-p21 was not immunogenic in rabbits. Recombinant Antp peptide alone was not cytotoxic, showing that killing was due to the transduction of the p21 component of Antp-p21. Antp-p21 was shown to penetrate cancer cells engrafted in vivo and resulted in tumour eradication when administered with conventionally-used chemotherapeutic agents, which alone were unable to produce such an effect. CONCLUSIONS: Antp-p21 may represent a new and promising targeted therapy for patients with p53-associated cancers supporting the concept that rational design of therapies directed against specific cancer mutations will play a part in the future of medical oncology.     

Resveratrol induces colon tumor cell apoptosis independently of p53 and precede by epithelial differentiation, mitochondrial proliferation and membrane potential collapse.

Resveratrol, a polyphenol present in wine and grapes, can inhibit tumor cell growth in vitro and tumorigenesis in vivo. Some of its effects have been linked to activation of the p53 tumor suppressor; however, p53 is frequently mutated in tumors, particularly in the common and often therapy-resistant colon cancers. Using the human wild-type p53-expressing HCT116 colon carcinoma cell line and HCT116 cells with both p53 alleles inactivated by homologous recombination, we show in the current study that resveratrol at concentrations comparable to those found in some foods can induce apoptosis independently of p53. The cell death is primarily mitochondria-mediated and not receptor-mediated. No cells survived in cultures continuously exposed to 100 microM resveratrol for 120 hr. When compared with 5-FU, resveratrol stimulated p53 accumulation and activity only weakly and with delayed kinetics and neither the increased levels nor the activity affected apoptosis detectably. The apoptosis agonist Bax was overproduced in response to resveratrol regardless of p53 status, yet the kinetics of Bax expression were influenced by p53. Remarkably, apoptosis was preceded by mitochondrial proliferation and signs of epithelial differentiation. Thus, resveratrol triggers a p53-independent apoptotic pathway in HCT116 cells that may be linked to differentiation.     

New p53-based anti-cancer therapeutic strategies

Thep53 gene is frequently mutated in human tumours and therefore an important target for therapeutic intervention. Several p53-based strategies for treatment of cancer are currently under development.p53 gene therapy has resulted in tumour regression in patients with lung cancer. A mutant adenovirus can obliterate tumour cells carrying mutant p53 or lacking p53, but is unable to replicate in normal cells. Furthermore, current studies suggest that reactivation of mutant p53 proteins in tumours using small p53-activating molecules may initiate p53-dependent apoptosis and thus eliminate the tumour.     

p53 gene status and response to platinum/paclitaxel-based chemotherapy in advanced ovarian carcinoma.

PURPOSE: The p53 gene plays a critical role in cellular response to DNA damage and has been implicated in the response to platinum compounds in ovarian carcinoma patients. Because taxanes could induce p53-independent apoptosis, we assessed the relevance of p53 gene status to response in ovarian carcinoma patients receiving paclitaxel and platinum-containing chemotherapy. PATIENTS AND METHODS: Forty-eight previously untreated patients with advanced disease received standard paclitaxel/platinum-based chemotherapy. In tumor specimens collected at the time of initial surgery, before therapy, p53 gene status and expression were examined by single-strand conformation polymorphism, sequence analysis, and immunohistochemical analysis. Microsatellite instability analysis was performed on available samples from 30 patients. RESULTS: Thirty-four (71%) of the 48 patients had a clinical response. Pathologic complete remission was documented in 13 (27%) of 48 patients. p53 mutations were detected in 29 (60%) of 48 tumors. Among the patients with mutant p53 tumors, 25 patients (86%) responded to chemotherapy. Only nine (47%) of 19 patients with wild-type p53 tumors responded to the same treatment. The overall response rate and the complete remission rate were significantly higher among patients with mutant p53 tumors than among patients with wild-type p53 tumors (P: =.008). Most of the tested tumors not associated with complete remission (10 of 12 tumors) were also characterized by microsatellite instability. The complete remission rate was higher among patients with tumors without microsatellite instability (five of seven patients). CONCLUSION: In contrast to the limited efficacy of treatment with paclitaxel in combination with standard platinum doses against wild-type p53 ovarian tumors, patients with mutant p53 ovarian tumors were more responsive to paclitaxel-based chemotherapy. The pattern of response to chemotherapy containing paclitaxel is different from that associated with high-dose cisplatin therapy. Determining p53 mutational status can be useful in predicting therapeutic response to drugs effective in ovarian carcinoma.     

COP1, the negative regulator of p53, is overexpressed in breast and ovarian adenocarcinomas

The tumor suppressor protein p53 plays a central role in protecting normal cells from undergoing transformation. Thus, it is fitting that cancer cells selectively dampen the p53 response to gain a selective growth advantage. In fact, the p53 gene is the most commonly mutated tumor suppressor gene in human cancers, and if the gene is not mutated, then other components of the p53 pathways are skewed to dampen the p53 response to stress. We recently identified COP1 as a novel and critical negative regulator of p53. COP1 is a RING finger-containing protein that targets p53 for degradation to the proteasome and is necessary for p53 turnover in normal and cancer cells. However, the association between COP1 and cancer remains to be determined. We performed expression analysis of COP1 in ovarian and breast cancer tissue microarrays. COP1 is significantly overexpressed in 81% (25 of 32) of breast and 44% (76 of 171) of ovarian adenocarcinoma as assessed by in situ hybridization and immunohistochemistry. Overexpression of COP1 correlated with a striking decrease in steady state p53 protein levels and attenuation of the downstream target gene, p21, in cancers that retain a wild-type p53 gene status. Overall, these results suggest that overexpression of COP1 contributes to the accelerated degradation of p53 protein in cancers and attenuates the tumor suppressor function of p53.     

Wild-type p53 gene transfection in human cultured sarcomas: effect of CDDP

We examined the susceptibility of five human bone and soft tissue sarcoma cell lines to transfection with recombinant p53 adenovirus vector (AxCA-p53). Transfection efficiency was more than 90% at 72 h with AxCA-lacZ at a multiplicity of infection (MOI) of 50 in all the cell lines, except for MG-63 (p53 gene mutated) cells. Western blot analysis showed overexpression of both P21/Waf1 and Bax protein in all the cell lines, implying sufficient and successful p53 gene transfection. AxCA-p53 transfection at MOI of 50 resulted in a significant decline of viable cells at 72 h, due to apoptosis, in NY (mutated) and Saos-2 (deletion), but not in the other three lines. The two apoptosis-induced cell lines showed a gradual increase in Bax expression up to 72 h and non-detectable expression of Bcl-XL from 48 h, suggesting the involvement of an apoptosis-inducing mechanism. Pre-treatment with cis-diamminedichloroplatinum (II) (CDDP) at 0.1 microg/ml significantly suppressed tumor cell viability in NY and HuO-3N1 (mutated), but not in the other three lines including HT-1080 carrying the wild-type p53 gene, implying the existence of different mechanisms for the tumor suppressive effect of p53 gene transfection and CDDP. These results indicate that wild-type p53 gene transfection with CDDP is a promising therapy for some, but not all, non-resectable bone-and soft tissue sarcomas, regardless of intrinsic p53 gene status.     

Prediction of TP53 status for primary cisplatin, fluorouracil, and leucovorin chemotherapy in ethmoid sinus intestinal-type adenocarcinoma.

PURPOSE: To assess the role of TP53 status in predicting pathologic complete remission after primary chemotherapy in patients with ethmoidal intestinal-type adenocarcinoma (ITAC). PATIENTS AND METHODS: Thirty patients with ethmoidal ITAC enrolled onto a phase II study received chemotherapy with cisplatin, fluorouracil, and leucovorin (PFL) followed by surgery and radiation. On surgical specimens, absence of viable tumor cells was defined as pathologic complete remission (pCR). TP53 status/p53 function, analyzed on pretreatment biopsies, were retrospectively correlated with pathologic results and patient outcome. RESULTS: Twelve patients achieved a pCR; 18 patients did not (overall response rate, 40%). In patients with wild-type (wt) TP53 or functional p53 protein, the pCRs were 83% and 80%, respectively; in patients with mutated TP53 or impaired p53 protein, pCRs were 11% and 0%, respectively (P < or = .0001). At a median 55-month follow-up, all pCR patients were disease-free; 44% of nonresponding patients experienced relapse (P = .0061). CONCLUSION: The results indicate the existence of two genetic ITAC subgroups, defined by differences in TP53 mutational status or protein functionality, that strongly influence pathologic response to primary chemotherapy and, ultimately, prognosis. PFL seems to be highly effective in terms of pCR and disease-free survival in the presence of a wt or a still-efficient p53 protein, even when encoded by a mutated TP53 gene (eg, early-stop codon mutation), but ineffective in ITACs carrying a disabled p53 protein. Whether this model is extensible to other head and neck cancers needs appropriate investigation.     

p53 mutations, protein expression and cell proliferation in squamous cell carcinomas of the head and neck.

Thirty-three patients with squamous cell carcinoma of the head and neck region were studied concerning p53 protein expression and mutations in exons 4-9 of the p53 gene using immunohistochemistry, polymerase chain reaction (PCR)-single strand conformation polymorphism analysis and DNA sequencing. Immunoreactivity was found in 64% and p53 gene mutations in 39% of the tumours. Thirty-three per cent of the immunopositive and 50% of the immunonegative tumours were mutated within exons 5-8. In one immunopositive tumour three variants of deletions were observed. Sequencing of the p53 mutated, immunonegative tumours revealed four cases with deletions, one case with a transversion resulting in a stop codon and one case with a splice site mutation which could result in omission of the following exon at splicing. All mutations in the immunonegative tumours resulted in a truncated p53 protein. No association between p53 gene status and expression of proliferating cell nuclear antigen (PCNA) or cell proliferation as judged by in vivo incorporation of the thymidine analogue iododeoxyuridine (IdUrd) was found.