Expression Of P53 Tumor Suppressor Gene Research Articles

Abstract 4131: Combination therapeutic strategy to fight against triple-negative breast cancer

Abstract Triple-negative breast cancers (TNBCs) lack estrogen-and progesteron receptors and human epidermal growth factor receptor-2 (HER2) which limits the therapeutic approaches. The high metastasizing capability of TNBCs is in correlation with the poor oxygenation of the cancerous breast tissue. This leads to the activation of the hypoxia-inducible-factor 1 (HIF-1), which induces multiple target genes that are involved in tumor progression and metastasis formation. This makes HIF-1 an attractive drug target, however, HIF-1 inhibitors failed if applied in monotherapy. Therefore, our aim is to investigate the combined therapeutic effect of HIF-1 inhibitor acriflavine and paclitaxel, a commonly used taxane in TNBC treatment. The synergy assessment of the two drugs was carried out by utilizing Sulforhodamine B assay on TNBC cell lines. The in vivo toxicity and efficacy of the drug combination was tested on NOD SCID mice with orthotopically inoculated MDA-MB-231 cell line. Quantitative and qualitative analysis of multiple genes involved in tumor progression was performed by mRNA and protein expression level assessment with qPCR, western blot and immunohistochemistry methods. HIF-1 stimulation was achieved by either incubating the cells at 1% oxygen level or through CoCl2 treatment. The in vitro mRNA expression studies demonstrated that acriflavin possesses a hypoxic-effect-mitigating property on the expression of metastasis-involved- and HIF-1 target genes. Acriflavine alleviated the epithelial-mesenchymal transition of TNBC cells by increasing the expression of the epithelial marker, E-cadherin. At protein level, we detected decrease in HIF-1alpha and Phospho-Protein kinase B (Akt) and increase in p53 tumor suppressor gene expression in response to acriflavine treatment. We further established MDA-MB-231 xenograft model in mice for investigating in vivo toxicity and efficacy. The drug combination efficiently reduced the tumor size and the macrometastasis formation compared to the control groups. The protein analysis of frozen tumor tissue samples indicated that the combination therapy decreased the HIF-1alpha expression and the amount of Phospho-Akt and Phospho-Mitogen-activated protein kinases (pMAPK/pERK) in CoCl2treated mice. Additionally, expression of epithelial markers increased and that of mesenchymal markers decreased. The combination therapy led to a decrease in the platelet-derived growth factor receptor beta (PDGFRB) as well. To conclude, we identified a novel drug combination which can efficiently combat TNBC by inhibiting HIF-1 signaling, mitigating cell migration and metastasis formation and suppressing the activation of the Phosphoinositide 3-kinase (PI3K) and MAPK pathways. This work was supported by the Hungarian Thematic Excellence Program (TKP2021-EGA-44) and the National Laboratories Program - National Laboratory for Tumor Biology (NLP-17). Citation Format: Laura Svajda, Ivan Ranđelović, Sára Eszter Surguta, Marcell Baranyi, István Kenessey, Mihály Cserepes, József Tóvári. Combination therapeutic strategy to fight against triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4131.

Anticancer Potential of Hesperidin against HEp-2 Laryngeal Carcinoma Cell Line in Comparison to Doxorubicin

BACKGROUND: Doxorubicin (DOX) is a drug that is frequently used to treat a variety of cancers. Unfortunately, in many situations, it is ineffective, and raising the dosage is restricted due to systemic toxicity. An important strategy to minimize the toxic effects of the above cited drug is to use co-adjuvant. A citrus flavonoid hesperidin (Hesp) has emerged as promising anticancer natural product and proved to be potent antioxidant agent. It suppresses cancer cell replicating by triggering apoptosis and cell cycle arrest. AIM: The study’s goal was to investigate anticarcinogenic effects of Hesp in comparison with DOX against HEp-2 laryngeal carcinoma cell line. MATERIALS AND METHODS: Five groups of HEp-2 cell line were included, two groups were subjected to Hesp and the other two groups were subjected to DOX, which was used as a reference drug, in addition to a control untreated group. Expression of Bcl-2 and p53 genes was evaluated. Furthermore, the cell cycle arrest and apoptotic induction were assessed. RESULTS: Hesp exerted anti-proliferative effects against HEp-2 cells which increase in time dependent manner. Gene profile analysis revealed highly statistically significant decrease of anti-apoptotic Bcl-2 expression and highly statistically significant increase of tumor suppressor gene p53 expression (p ˂ 0.01 and p ˂ 0.0001, respectively) for both tested drugs. CONCLUSIONS: Hesp proved potential anticancer effects with reducing cancer cell viability in HEp-2 cell line through cell cycle arrest and apoptotic mechanism. It could be used as a prodrug or coadjuvant in treatment of oral cancer.

The role of estradiol metabolism in urogenital schistosomiasis-induced bladder cancer.

Urogenital schistosomiasis is a neglected tropical disease that can lead to bladder cancer. How urogenital schistosomiasis induces carcinogenesis remains unclear, although there is evidence that the human blood fluke Schistosoma haematobium, the infectious agent of urogenital schistosomiasis, releases estradiol-like metabolites. These kind of compounds have been implicated in other cancers. Aiming for enhanced understanding of the pathogenesis of the urogenital schistosomiasis-induced bladder cancer, here we review, interpret, and discuss findings of estradiol-like metabolites detected in both the parasite and in the human urine during urogenital schistosomiasis. Moreover, we predict pathways and enzymes that are involved in the production of these metabolites emphasizing their potential effects on the dysregulation of the tumor suppressor gene p53 expression during urogenital schistosomiasis. Enhanced understanding of these potential carcinogens may not only shed light on urogenital schistosomiasis-induced neoplasia of the bladder, but would also facilitate development of interventions and biomarkers for this and other infection-associated cancers at large.

Targeted p53 activation by saRNA suppresses human bladder cancer cells growth and metastasis.

BackgroundPrevious study showed that dsP53-285 has the capacity to induce tumor suppressor gene p53 expression by targeting promoter in non-human primates’ cells. And it is well known that TP53 gene is frequently mutant or inactivated in human bladder cancer. Hereby, whether this small RNA can activate the expression of wild-type p53 and inhibit human bladder cancer cells remains to be elucidated.MethodsOligonucleotide and lentivirus were used to overexpress dsP53-285 and dsControl. Real-time PCR and western blot were used to detect genes’ mRNA and protein expression, respectively. Cell proliferation assay, colony formation, flow cytometry, transwell assay and wound healing assay were performed to determine the effects on bladder cancer cells proliferation and migration/invasion in vitro. Animal models were carried out to analyze the effects on cells growth and metastasis in vivo.ResultsTransfection of dsP53-285 into human bladder cancer cell lines T24 and EJ readily activate wild-type p53 expression by targeting promoter. Moreover, dsP53-285 exhibited robust capacity to inhibit cells proliferation and colony formation, induce cells G0/G1 arrest, suppress migration and invasion. Besides, the Cyclin-CDK genes (Cyclin D1 and CDK4/6) were down-regulated and the EMT-associated genes (E-cadherin, β-catenin, ZEB1 and Vimentin) were also expressed inversely after dsP53-285 treatment. In addition, dsP53-285 could also significantly suppress the growth of bladder cancer xenografts and metastasis in nude mice. Most importantly, the anti-tumor effects mediated by dsP53-285 were mainly achieved by manipulating wild-type p53 expression.ConclusionOur findings indicate that the dsP53-285 can upregulate wild-type p53 expression in human bladder cancer cells through RNA activation, and suppresses cells proliferation and metastasis in vitro and in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0329-8) contains supplementary material, which is available to authorized users.

Abstract 3492: Metformin inhibits salivary gland tumor growth through cell cycle arrest and apoptosis

Abstract Human salivary gland cancer (SGC) is one of the common malignancies found in the head and neck area. The limitation of the treatment and poor prognosis are calling for more investigations into novel intervention to improve the treatment modality in SGCs. Metformin as a widely used anti-type-2 diabetes medicine had demonstrated its anti-tumor potential in many different types of cancers. Our previous work demonstrated that metformin effectively targeted c-Myc oncogene to prevent the initiation and progression of prostate cancer. Interestingly, the overexpression of c-Myc has also been shown in some SGCs, such as salivary gland adenoid cystic carcinoma. Therefore, it is intriguing to explore metformin's anti-neoplastic efficacy of metformin in SGC. In this study, we used a human salivary adenocarcinoma cell line, HSY cell line as a model for salivary gland tumor. Our in vitro results show metformin prominently suppresses the growth of HSY cell through stimulation of cell cycle arrest and apoptosis. Meanwhile, metformin significantly down-regulates cMyc expression and rescues p53 tumor suppressor gene expression. Considering that mTOR inhibitors resistance could occur when tumors over-express cMYC. PP242, a novel and potent mTOR inhibitor was used in combination with metformin to inhibit HSY cell growth. As expected, metformin enhanced the inhibitory effects of PP242 associated with reduced c-MYC levels in vitro. Further in HSY xenograft tumors, massive necrotic areas were observed inside the tumors from metformin treated mice while only limited necrotic area in the core of tumors from PBS treated mice. Staining of proliferation marker Ki-67 confirmed lower proliferation rate in tumors from metformin treated group compare to that from the PBS group. In conclusion, our novel finding of metformin efficacy in SGC not only proves metformin plays a pivotal role in suppressing SGCs through cell cycle arrest and apoptosis, but also suggest that a combination treatment using metformin and mTOR inhibitors together may yield better inhibitory efficacy. Citation Format: Yuqi Guo. Metformin inhibits salivary gland tumor growth through cell cycle arrest and apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3492. doi:10.1158/1538-7445.AM2015-3492

P53 mediated apoptosis by reduction sensitive shielding ternary complexes based on disulfide linked PEI ternary complexes

Reduction-sensitive hyaluronic acid derivatives (HA–SS–COOH) were shielded on the DNA/polyethylenimine (PEI) to construct ternary complexes (DNA/PEI/HA–SS–COOH, DPS ternary complexes) with efficient gene transfection. Details studied were conducted to investigation of factors influencing transfection efficiency, including the gene compression by fluorescence resonance energy transfer (FRET) spectrum and the intracellular fate of fluorescent labeled complexes by the confocal laser scanning microscope (CLSM). In the FRET study, DPS complexes were found to enhance condensation of DNA in preparation, while timely loosen gene under exposure to reductive reagent. Similar cellular uptake levels were observed for the designed reduction sensitive complexes and the stable one (DNA/PEI/HA, DPH ternary complexes), but the intracellular process was strikingly different for the two types of complexes. Only DPS showed obvious desired intracellular deshielding and endosomal escape, which contributed to highly efficient gene delivery. After loading with p53 plasmid, DPS complexes achieved significantly up-regulated p53 tumor suppressor gene expression at both mRNA and protein levels, as revealed by quantitative polymerase chain reaction (qPCR) and western blot investigations. Transgene induced apoptosis was evaluated by propidium iodide staining and flow cytometry analysis of cell cycle. Tumor cells transfected by DPS complexes containing p53 gene displayed almost 50% higher suppression in proliferation compared to those untreated cells, accompanied with a 46% elevation in the number of cells at sub-G1 phase and remarkable p53 dependent cell cycle perturbations prior to apoptosis. These results demonstrated that targeted delivery of p53 gene via reduction-sensitive DPS ternary complexes enabled up-regulated cellular p53 mRNA level through the exogenous p53 gene, inducing a significant p53-dependent anti-proliferative effect on tumor cells, which could be effective means of cancer treatment.

A cytohistological study of p53 overexpression in ovarian neoplasms

Background:The present study was undertaken to evaluate the diagnostic accuracy of imprint cytology in ovarian neoplasms, investigate the biological significance of p53 expression in malignant ovarian tumors and correlate it with histological type, grade and stage of tumor.Material and Methods:A total of 50 cases including 25 prospective and 25 retrospective cases were studied. Imprint cytology was performed on 25 ovarian tumors and compared with histopathological diagnosis. p53 immunohistochemistry was performed on all 50 cases.Results:On immunohistochemistry, all the benign tumors were negative for p53 while 42% of primary ovarian malignant tumors were positive. p53 expression was found to have a diagnostic value in differentiating benign from malignant tumors. p53 overexpression did not show any significant correlation with prognostic factors as stage of disease, grade of differentiation and type of tumor.Conclusion:The present study confirms the importance of p53 tumor suppressor gene expression as documented by immunohistochemistry in the differentiation of malignant and benign ovarian tumors.

MR Traceable Delivery of p53 Tumor Suppressor Gene by PEI-Functionalized Superparamagnetic Iron Oxide Nanoparticles

Cancer gene therapy involves the replacement of missing or altered genes with healthy ones. In this paper, we have proposed tumor suppressor gene-carrying superparamagnetic iron oxide nanoparticles (SPIONs) for anti-cancer gene therapy. Thermally crosslinked SPIONs (TCL-SPIONs) were conjugated with branched polyethylenimine (PEI 1800 Da) by EDC-NHS chemistry for p53 plasmid DNA delivery. The morphology of the bPEI conjugated TCL-SPIONs (bPEI-TCL-SPION) and pDNA-loaded bPEI-TCL-SPION nanoparticles was measured using transmission electron microscopy (TEM). The particle sizes of the pDNA-loaded bPEI-TCL-SPION nanoparticles were also confirmed by dynamic light scattering, and ranged from 100 to 130 nm, depending on the molar charge ratio. The fluorescently labeled pDNA was complexed with bPEI-TCL-SPION and its intracellular internalization was investigated using confocal microscopy. The p53 plasmid-loaded bPEI-TCL-SPION nanoparticles achieved significantly higher p53 tumor suppressor gene expression and cellular viability compared to positive controls. The expressed wild-type p53 protein suppressed tumor cell proliferation as compared to the mutant control. When transgene expression of the p53 tumor suppressor gene was evaluated at the mRNA level and quantified using real-time PCR, the results were highly dependent on the molar charge ratio (N/P) as well as the cancer cell type. SPIONs internalized within cancer cells were tracked by magnetic resonance (MR) imaging. It was concluded that bPEI-TCL-SPION could be used as efficient gene delivery carriers that can be tracked by MR imaging.

Clinicopathological Significance of Macrophage Migration Inhibitory Factor and its Relation with P53 in Gastric Cancer

Based on previous investigations, the progress of gastritis may lead to gastric carcinomas. In some epithelial tumors, macrophage migration inhibitory factor (MIF), which is an inflammatory cytokine may inactivate p53 and play a role in tumorigenesis process. We decided to evaluate clinicopathological significance of MIF expression and the relation between p53 and MIF expressions in gastric adenocarcinomas. Seventy-three consecutive cases of gastric adenocarcinomas, the tissue samples of which were available, were included in this study. Tissue sections were stained for MIF and p53 expression by immunohistochemistry and the expression was defined as positive (for more than 10%) and negative (for less than 10%) groups. Location of the tumor, histological subtypes, and grade of the tumor were determined by using routine H&E staining. Distant metastasis, lymph node involvement, and consequently the stage of tumor were specified. The patients' age and gender were obtained from their medical records. The relationship between expression of MIF and these variables was determined. Overexpression of MIF was observed in the cytoplasm of cancer cells in 46.6% (34/73) of cases and nuclear immunostaining of p53 was observed in 37% (27/73) of cases. Expression of MIF was significantly correlated with the location of tumor, but this expression has no statistically significant correlation with variables including: age, gender histological subtypes, distant metastasis, and lymph node involvement, stage and grade of the tumor, and p53 tumor suppressor gene expression. Our study suggests that MIF in gastric adenocarcinomas versus many other epithelial tumors cannot have a prominent role in tumor progress and inactivation of p53 tumor suppressor gene.

ANTICANCER ACTIVITY OF CALANONE ON HeLa CELL LINE

Calanone (coumarin derivate compound), isolated from Calophyllum sp. had been shown to have cytotoxic activity on leukemia L1210 cell line with IC50 = 59.40 mg/mL. Calanone presumed have anticancer activity on HeLa cervical carcinoma cell. This study was conducted to investigate the cytotoxic and apoptotic activity of calanone and its effect to p53 and p21 expression on HeLa cervical carcinoma cell. Cytotoxic assay of calanone was performed on HeLa cell line, using MTT assay. Apoptotic assay was performed on HeLa cell line incubated with calanone for 24 h, by immunofluororescence method, using fluorochromes ethidium bromide and acridine orange. Expression of p53 was examined on HeLa cell line, by PCR with p53 wild-type primer. Expression of p21 was examined on HeLa cell line, by immunohistochemistry method. 5-fluorourasil was used as positive control in cytotoxic, apoptotic assay, and p53 expression. The result showed that calanone has cytotoxic activity on HeLa cell line, with IC50 = 22.887 mg/mL, caused cytotoxicity through apoptotic mechanism, increase p53 tumor suppressor gene expression, while the p21 expression test showed a negative result. Keywords: Calanone, cytotoxic, HeLa cell line

A crystalline compound (BM-ANF1) from the Indian toad ( Bufo melanostictus, Schneider) skin extract, induced antiproliferation and apoptosis in leukemic and hepatoma cell line involving cell cycle proteins

In our earlier communication, it was reported that Indian toad ( Bufo melanostictus) skin extract (TSE) possesses antiproliferative and apoptogenic activity in U937 and K562 cells [Giri et al., 2006. Antiproliferative, cytotoxic and apoptogenic activity of Indian toad ( Bufo melanostictus, Schneider) skin extract in U937 and K562 cells. Toxicon 48 (4), 388–400]. In the present study, a compound (BM-ANF1) has been isolated from the TSE by alumina gel column chromatography, crystallized and evaluated for its antiproliferative and apoptogenic activity in U937, K562 and HepG2 cells. BM-ANF1 produced dose-dependent inhibition of U937, K562 and HepG2 cell growth. The antiproliferative activity was reflected by the MTT assay and demonstrated by the reduced expression of proliferative cell nuclear antigen (PCNA). Flow-cytometric analysis showed that BM-ANF1 arrested the cell cycle at G1 phase and enhanced annexin-V binding in U937 and K562 cells. Scanning electron microscopic and fluorescent microscopic analysis of U937 and K562 cells revealed the apoptogenic nature of the compound. Alkaline comet assay showed that BM-ANF1 produced DNA fragmentation. The dose-dependent expression of caspase 3 indicated that the apoptogenic properties of BM-ANF1 were mediated through the activation of downstream effector nucleases in the cancer cells. The increased expression of p53 and moderate expression of p21 Cip1/p27 Kip1 due to BM-ANF1 treatment in HepG2 cells supported that the apoptogenic activity of BM-ANF1 was mediated through p53 tumor-suppressor gene expression followed by the expression of p21 Cip1 and p27 Kip1 and it was likely to be linked with cell cycle arrest at G1 phase in cancer cells. From the present study, it may be suggested that the crystalline compound, BM-ANF1, was antiproliferative and apoptogenic in human leukemic and hepatoma cells.

Gallbladder carcinoma: the role of p53 protein overexpression and Ki‐67 antigen expression as prognostic markers

The overexpression of p53 protein and the expression of Ki-67 antigen may affect the survival of patients with gallbladder carcinoma. This association has been tested in a series of 41 patients with relatively early carcinoma of the gallbladder. Forty-one surgical specimens from patients with a postoperative histological diagnosis of gallbladder carcinoma were studied. All patients were operated by simple cholecystectomy only because the tumours were not advanced and/or their general condition was poor. Patients submitted to radical operations were excluded. p53 expression was calculated from nuclear staining according to the intensity and extent of positive cells, as graded on a scale from 1 to 3; a combined score of >3 was considered as overexpression. Ki-67 expression was calculated by the MIB-I index: the percentage of positively stained tumour cell nuclei out of the total tumour cells counted (n = 1000); >20% of stained cells was considered positive. Twenty-nine gallbladder carcinomas (71%) overexpressed p53 protein in the cell nuclei. No significant differences were found in relation to cell differentiation on the level of tumour infiltration through the gallbladder wall. Five-year survival of patients with gallbladder carcinoma overexpressing p53 was 17.2%, while survival of patients without p53 overexpression was 30% (not significant). Twenty-four cases (58.5%) were considered positive for the MIB-I index. There were no differences between the grade of cell differentiation and wall infiltration. Five-year survival of the patients with a MIB-I positive index was 9.2% as opposed to 27.7% for those with a negative index (not significant). p53 protein nuclear overexpression and Ki-67 protein expression in gallbladder carcinoma were not related to histological differentiation, level of gallbladder wall invasion or patient survival.

Polyozellus multiplex, a Korean wild mushroom, as a potent chemopreventive agent against stomach cancer

Polyozellus multiplex, a Korean wild mushroom, was extracted using methanol, and the extract was further fractionated with water and ethylacetate. Assay of each fraction with MTT revealed significant tumoristatic effects of the water fraction of Polyozellus multiplex against human gastric and other cancer cells but not normal human lymphocytes. Modifying effects of the water fraction on glandular stomach mucosa were investigated in male Wistar rats treated with N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). The dietary 0.5% or 1% water fraction of Polyozellus multiplex significantly increased glutathione S-transferase (GST) and superoxide dismutase (SOD) activities, and showed a tendency for increase in glutathione (GSH) levels, compared to the MNNG alone group. It also caused a significant reduction in proliferating cell nuclear antigen (PCNA)-labeling index of the glandular stomach epithelium, along with increase in p53 tumor suppressor gene expression. These results suggest that Polyozellus multiplex is a candidate for chemoprevention against gastric cancer.

Validation of p53 immunohistochemistry as a prognostic factor in breast cancer in clinical practice.

Abstract Context.—Abnormal p53 tumor suppressor gene expression as detected by immunohistochemistry is a possible prognostic factor in breast cancer. The difference in techniques used to evaluate the expression of mutated p53 protein is under intense scrutiny, as well as its uses either independently or in conjunction with other prognostic factors in breast cancer. Objective.—To determine whether p53 immunohistochemistry can be used as a reliable indicator of the presence of mutated nuclear p53 protein, and whether this method can be performed reliably in a community hospital's clinical practice. Design.—One hundred twenty-two cases of breast carcinoma were stained and analyzed for the presence of p53 protein using DO-7 (Dako Corporation, Carpinteria, Calif) p53 antibody. Results.—Of the 122 cases of invasive carcinoma studied, 23 (18.7%) were positive for p53, and 16 (16.3%) of 98 cases with coexisting ductal carcinoma in situ were positive for p53. This finding is in agreement with comparable published ...

Validation of p53 Immunohistochemistry as a Prognostic Factor in Breast Cancer in Clinical Practice

Abnormal p53 tumor suppressor gene expression as detected by immunohistochemistry is a possible prognostic factor in breast cancer. The difference in techniques used to evaluate the expression of mutated p53 protein is under intense scrutiny, as well as its uses either independently or in conjunction with other prognostic factors in breast cancer. To determine whether p53 immunohistochemistry can be used as a reliable indicator of the presence of mutated nuclear p53 protein, and whether this method can be performed reliably in a community hospital's clinical practice. ne hundred twenty-two cases of breast carcinoma were stained and analyzed for the presence of p53 protein using DO-7 (Dako Corporation, Carpinteria, Calif) p53 antibody. Of the 122 cases of invasive carcinoma studied, 23 (18.7%) were positive for p53, and 16 (16.3%) of 98 cases with coexisting ductal carcinoma in situ were positive for p53. This finding is in agreement with comparable published studies. Based on the results of this study, we conclude that p53 immunohistochemistry qualifies as a diagnostic technique suitable for clinical practice in a community hospital. Its detection may be particularly promising in clinical trials of new molecular therapies directed at the p53 tumor suppressor gene.

Serum p53 antibodies in patients with oral lesions: Correlation with p53/HSP70 complexes

The functional significance of alterations in expression of tumour-suppressor gene p53 and 70-kDa heat-shock protein (HSP70) in eliciting p53-specific humoral response in oral-cancer patients is not yet known. In this study, p53 auto-antibodies were analyzed in sera from oral-cancer patients by immunoblotting and results were correlated with clinicopathological features of the patients as well as with the levels of HSP70, p53 and p53-HSP70 complexes in matched patients' tumour tissue, for determining their diagnostic/prognostic significance and relationship to survival. Circulating anti-p53 antibodies were observed in 7 of 30 cancer patients and 3 of 25 patients with pre-malignant lesions. Over-expression of p53 protein in matched oral lesions was observed in 22 of these 30 cancer patients and 14 of 25 patients with dysplastic lesions. No detectable levels of p53 protein or anti-p53 antibodies were observed in normal subjects (15 cases). Elevated levels of HSP70 were observed in 23 of these 30 oral tumours and 17 of 25 dysplastic lesions. All the anti-p53-antibody-seropositive cases showed elevated levels of p53 and HSP70 proteins, as well as formation of p53-HSP70 complexes, in matched dysplastic or malignant lesions, suggesting that these molecular alterations may be early events in oral tumorigenesis and are implicated in eliciting p53-specific humoral immune response in these patients. Anti-p53-antibody-seropositive cases showed poor prognosis and significantly decreased overall disease-free survival in comparison with the seronegative cases. Detection of circulating anti-p53 antibodies may serve as a useful non-invasive marker for identifying oral tumours having poor prognosis.

The prognostic value of p53 nuclear overexpression and MIB-1 as a proliferative marker in transitional cell carcinoma of the bladder

There is controversy regarding the value of biologic markers as prognostic indicators independent of clinicopathologic parameters in transitional cell carcinoma (TCC) of the bladder. The authors examined the prognostic value of p53 tumor suppressor gene expression and the proliferative marker MIB-1 in TCC of the bladder. Fresh frozen samples from 114 TCCs of the bladder and 13 normal bladders were studied by immunohistochemistry, using monoclonal antibodies MIB-1 for proliferation and PAb 1801 for p53 nuclear overexpression. Scores were determined in each case by counting at least 500 nuclei per slide in 3-5 selected regions. Patients were stratified for both markers into two groups for time-event analysis, according to the median number of nuclei stained. Patients with nuclear staining below the median value of the score were considered negative in the statistical analysis. Quantitative immunostaining was analyzed in relation to the time to recurrence, progression, and cancer death, and compared with clinical and pathologic parameters for prognostic significance in univariate and multivariate analysis (stepwise logistic regression). Median nuclear overexpression of p53 was 22% and that of MIB-1 28%. There was a strong association between proliferation and p53 nuclear overexpression (P < 0.0001). Progression free and disease specific survival rate estimates (log rank test) were significantly lower in patients with p53 or MIB-1 scores above 22% and 28%, respectively. Multivariate analysis indicated that stage as well as p53 and MIB-1 immunostaining provided independent prognostic information. Quantitative immunohistochemical evaluation of nuclear p53 and MIB-1 immunostaining inexpensively provides prognostic indicators in cases of TCC of the bladder.

Biostatistical modeling using traditional variables and genetic biomarkers for predicting the risk of prostate carcinoma recurrence after radical prostatectomy

Approximately 50-60% of patients treated with radical prostatectomy for clinically localized prostate carcinoma are found to have microscopic disease that is not organ-confined, and a significant portion of these patients will relapse. Multiple studies have attempted to identify these high risk patients by evaluating many potential prognostic variables. These studies, however, have not included the more recent molecular biomarkers found to be independent predictors of disease recurrence. One hundred thirty-two patients who underwent radical prostatectomy at one center between 1986 and 1993 were subjected to a multivariable Cox regression analysis to determine the preoperative and postoperative variables that remain significant predictors for the likelihood of serologic recurrence. The preoperative variables included in the model were age, race, and prostate specific antigen(PSA); the postoperative variables were Gleason sum, nuclear grade, pathologic stage (capsular status), p53 tumor suppressor gene expression, bcl-2 protooncogene expression, and proliferative biomarker Ki-67 expression. Biomarkers were also evaluated separately. A model was developed using only variables that remained significant predictors for the likelihood of recurrence. The following equation calculated the relative risk of recurrence: Rw = exp [(0.70 x Race) + (0.79 x PSA[4.1-10]) + (1.34 x PSA[>10]) + ( 0.91 x Organ confinement) + (0.65 x p53[1,2+]) + (1.45 x p53[3,4+]) + (0.70 x bcl-2)]. This equation categorized men into 3 distinct risk groups (low: Rr < 5.0; intermediate: Rr = 5.0-15.0; high risk: Rr > 15.0). This equation allows patients at high risk for PSA recurrence to be identified shortly after radical surgery. These patients at high risk for serologic recurrence and eventual progression may be considered for currently accepted adjuvant therapy or enrollment in clinical trials for the newer investigational therapies for locally advanced prostate carcinoma.

Pituitary carcinoma: a clinicopathologic study of 15 cases.

Pituitary carcinomas are rare adenohypophysial neoplasms, the definition, diagnosis, therapy, and prognosis of which are controversial. Pituitary carcinomas were defined as primary adenohypophysial neoplasms with documented craniospinal and/or systemic metastases. The authors report a clinicopathologic study of 15 examples examined by light microscopy, immunohistochemistry, and image analysis. Both proliferative activity and p53 tumor suppressor gene expression were studied. The study group consisted of 15 patients, including 8 males and 7 females ranging in age from 34-71 years (mean, 56 years). Of these patients, seven had adrenocorticotropic hormone (ACTH)-producing tumors (four in the context of Nelson's syndrome), seven had prolactin-producing tumors, and one had a nonfunctioning tumor. No evidence of diabetes insipidus was seen in any case. Fourteen tumors were initially considered macroadenomas. Of the ten cases for whom tumor extent was known, all had invasive tumors. The interval from the initial diagnosis of adenoma to that of carcinoma ranged from 0.3 to 18.0 years (mean, 6.6 years; median, 5.0 years); the longest mean interval (15.3 years) occurred for patients with Nelson's syndrome. The latency was twice as long for ACTH-producing tumors as for prolactin (PRL) cell tumors (9.5 vs. 4.7 years). All carcinomas showed a greater tendency toward systemic metastasis than craniospinal metastasis; the rate of systemic metastasis was 71% for PRL cell tumors and 57% for ACTH-producing tumors. Thirteen percent of tumors showed both patterns of metastasis. Fully 50% of primary tumors and the majority of metastases showed nuclear pleomorphism and/or hyperchromasia. The mean mitotic, MIB-1, and proliferating cell nuclear antigen indices for primary tumors and metastases were as follows: 2/10 high-power field (hpf), 2.6% and 11%, respectively; 6/10 hpf, 7.8% and 16%, respectively. Staining for p53 protein was noted in 57% of primary tumors and 88% of metastatic tumors; a relative increase in p53 expression in metastases was noted in 83%. All but one of the primary and metastatic tumors were aneuploid. The most common treatments were radiation therapy and, for PRL cell carcinomas, dopamine agonist administration. Both treatments provided only palliation. Eighty percent of the patients died of metastatic disease 7 days to 8 years after the diagnosis of carcinoma; of these, 66% died within 1 year. At last follow-up, 20% of patients were alive with metastases 9-18 months after diagnosis. Nearly all pituitary carcinomas present as functioning, microscopically atypical or mitotically active, invasive macroadenomas. By definition, after an interval related to their immunotype, all metastasize. The tumors show a greater tendency toward systemic metastasis than craniospinal metastasis and are associated with poor prognosis. Radiation and dopamine agonist therapy generally provide only palliation. Proliferation indices and p53 expression tend to be higher in metastases than in primary tumors. The current definition of pituitary carcinoma requires the demonstration of metastasis; however, high mitotic and MIB-1 labeling indices as well as p53 immunoreactivity suggest the diagnosis and appear to be of prognostic significance. A redefinition of aggressive pituitary tumors is proposed--one that facilitates the recognition of tumors prone to metastasis.

Overexpression of p53 protein and histologic grades of dysplasia in colorectal adenomas.

To clarify the relation between tumor-suppressor gene p53 expression and histologic grades of dysplasia in colorectal adenomas, we performed immunohistochemical analysis in a series of 59 colorectal polyps and 40 advanced carcinomas. Adenomatous polyps were stained by hematoxylin and eosin and classified into mild, moderate, and severe dysplasia (intramucosal carcinoma), according to the World Health Organization's classification. p53 was positive in 7.1 percent (2/28) of mild, 29.4 percent (5/17) of moderate, and 62.5 percent (5/8) of severe dysplasia. In submucosal and advanced carcinomas, positivity rates were 75 percent (3/4) and 47.5 percent (19/40), respectively. Different staining patterns were found, according to grades of dysplasia. In the adenomas with mild or moderate dysplasia, a few focal crypts showed localized p53-positive staining. Adenomas with severe dysplasia had two different staining types. One was a focal staining type as shown in mild or moderate dysplasia; the other was a diffuse staining type, in which glands with mild or moderate dysplasia, surrounding severe dysplasia area, were also stained. Submucosal and advanced carcinomas showed a strong positive staining in cancer cells only. Overexpression of p53 protein in adenomas with mild or moderate dysplasia and existence of two types of expression in adenomas with severe dysplasia were observed. These facts suggested the possible existence of different pathways in the adenoma to carcinoma progression.