Expression of p53 gene in B-CLL peripheral lymphocytes.

The p53 tumor suppressor gene expression has been studied in 23 B-CLL cases at different clinical stages. The analysis failed to show a direct correlation with each stage, but the significantly lower frequency of a BglII RFLP in the pathologic population suggests a role of this gene in B-CLL. Northern Blot analysis showed the expression of p53 mRNA in all the B-CLL cases. A protocol for the RT-PCR methods was set up to study a very small amount of materials which should be better used for sequence analysis.     

p53 codon 72 polymorphism, DNA damage and repair, and risk of non-melanoma skin cancer

A very common polymorphism of p53, that of codon 72, codes either for a proline (P72) or an arginine (R72). The two alleles differ in their biological properties: P72 is a stronger inducer of p21, while R72 induces 5-10 times more apoptosis. It is not known, however, whether this polymorphism influences genome stability. The influence of p53 codon 72 polymorphism on cancer risk has been studied for different types of cancer with mixed and inconsistent results. With respect to sporadic non-melanoma skin cancer (NMSC), there are few studies, with small sample sizes, and none in a Latinoamerican population. These studies have found no association between p53 genotype at codon 72 and NMSC. We analyzed whether p53 codon 72 genotype influences genomic stability and the sensitivity of cells to UVB. We also carried out a case-control study of NMSC in a Mexican population which included 204 BCC cases, 42 SCC cases, and 238 controls. There was no association between p53 genotype and basal levels of DNA damage, oxidative DNA damage sensitivity, or DNA repair capacity. R72 dominantly increased the in vitro sensitivity of cells to UVB-induced apoptosis. There was no significant association either between p53 genotype and basal cell carcinoma (BCC), squamous cell carcinoma (SCC) or both combined.     

Simplified approaches for the development of an ELISA to detect circulating autoantibodies to p53 in cancer patients

Background  

The recognition that human tumors stimulate the production of autoantibodies has initiated the use of this immune response as serological markers for the early diagnosis and management of cancer. The enzyme-linked immunosorbent assay (ELISA) is the most common method used in detecting autoantibodies, which involves coating the microtiter plate with the tumor associated antigen (TAA) of interest and allowing serum antibodies to bind. The patient's sample is directly in contact with the coating antigen so the protein used for coating must be pure to avoid non-specific binding. In this study, a simplified method to selectively and specifically immobilize TAAs onto microtiter plates in order to detect circulating autoantibodies in cancer patients without prior purification process was described. Wild type full-length p53 protein was produced in fusion with biotin carboxyl carrier peptide (BCCP) or hexahistidine [(His)6] using pAK400 and pET15b(+) vectors, respectively. The recombinant p53 fusion protein produced was then subjected to react with either a commercial p53 monoclonal antibody (mAb) or sera from lung cancer patients and healthy volunteers in an enzyme-linked immunosorbent assay (ELISA) format.     

The p53 tumor suppressor gene. A preliminary clinical study in breast cancer patients.

p53 was originally considered to be a nuclear oncogene, but several convergent lines of research have indicated that the wild-type gene functions as a tumor suppressor gene negatively regulating the cell cycle. Mutations in the p53 gene have been detected in many tumor types and seem to be the most common genetic alterations in human cancer. In this preliminary study, sera of 92 patients (pts) with breast disease were analyzed for the presence of the mutant p53 protein (mp53) with a selective immunoenzyme assay employing a monoclonal antibody (PAb 240) specific for the majority of mammalian m p53 but not for the wild-type protein. Of the 10 patients with benign breast disease, only two (20%) showed detectable m p53 levels in the serum. In the breast cancer group, sera from 7 of the 30 pts (23%) without lymph node involvement were positive for m p53, as were 7 out of the 45 pts (15%) with metastatic lymph nodes and 1 out of the 7 pts (14%) with disseminated disease. The specificity of m p53 assay evaluated in 20 healthy controls was 100%. These preliminary results showed that serum positivity for m p53 is not related to breast disease extension. Further studies to assess the utility of m p53 as a possible prognosis factor in breast cancer are currently in progress.     

Increased damage of exon 5 of p53 gene in workers from an arsenic plant

Mutagenesis is a multistage process. Substitution mutations can be induced by base modified through alteration of pairing property. Mutations of exon 5 and 8 of p53 gene have been found in most arsenicosis patients with precarcinomas and carcinomas, but never in arsenicosis individuals without precarcinomas and carcinomas. This study investigates whether base modification exists in exon 5 and 8 of p53 gene, and explores the dose-effect relationship between damage of exon 5 of p53 gene and urinary arsenic. Concentrations of urinary 8-hydroxydeoxyguanine (8-OHdG) are analyzed to identify the occurrence of DNA damage. The real-time PCR developed by Sikorsky et al. is applied to detect base modification in exon 5 and 8 of p53 gene for apparently healthy participants. Our results show that the mean total arsenic concentrations of two exposed groups from an arsenic plant are significantly elevated compared with the control group, and the damage level of exon 5 of the high-exposed group is significantly higher than that of the control group, but which does not happen in exon 8. The closely correlation between the damage index of exon 5 and urinary organic arsenic concentration are found. Concentration of 8-OHdG of the high-exposed group is significantly higher than that of the control group. These results imply that base modification in exon 5 of p53 gene can be induced by arsenic. In addition, our study suggests that the damage level of exon 5 is a useful biomarker to assess adverse health effect levels caused by chronic exposure to arsenic.     

Socioeconomic status and non-melanoma skin cancer: A nationwide cohort study of incidence and survival in Denmark

Background: The two main types of non-melanoma skin cancer differ with the pattern of exposure to ultraviolet radiation (UVR): basal cell carcinoma (BCC) appears to be more closely related to intermittent solar exposure and sunburn, while the risk for squamous cell carcinoma (SCC) is a result of lifetime cumulated exposure to UVR. As these exposures may differ by social position, we investigated its role in the risk for and survival after BCC and SCC diagnosed in Denmark in 1994–2006 with follow-up through 2006. Methods: The analyses were based on 52,166 cases of BCC and 5033 cases of SCC in a cohort of 3.7 million people born between 1925 and 1976 and residing in Denmark in 1992–2006. Information on cancer cases and socioeconomic indicators were obtained from population-based registries. We used log-linear Poisson regression models to estimate incidence rate ratios and cumulative relative survival to estimate survival up to 10 years after the first incident cases of BCC and SCC. Results: High socioeconomic status, measured by both education and disposable income, was strongly associated with a higher risk for BCC, whereas there was no association between SCC and educational level and only a weak association with income. In general, relative survival after BCC was better than after SCC; the pattern of survival was not affected by socioeconomic indicators. Conclusions: The observed pattern of social status and risk for non-melanoma skin cancer differed substantially for the two cancer types, supporting the hypothesis that they may have different aetiologies.     

Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation.

Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation. The tumor suppressor gene, p53, is mutated in approximately 50% of human sporadic cancers and in an inherited cancer predisposition (Li-Fraumeni syndrome). We have analyzed the status of the wild-type p53 allele in tumors taken from p53-deficient heterozygous (p53+/-) mice. These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild-type p53. We present evidence that a high proportion of the tumors from the p53+/- mice retain an intact, functional, wild-type p53 allele. Unlike p53+/- tumors which lose their wild-type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following gamma-irradiation, activates p21(WAF1/CIP1) and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild-type p53), shows high levels of binding to oligonucleotides containing a wild-type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization. These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis.     

Expression of p53, p63 and p73 in the orofacial region of human embryos

We studied p53, p63, p73 protein expression in the orofacial region of five human embryos aged 7-18 weeks of intrauterine development using a three-step immunohistochemical method. Expression of proteins in various locations was evaluated semiquantitatively. A decrease in p53, p63 and p73 proteins occurred in the 13-week-old material with the exception of the tooth germ where a drop in p73 appeared in the ninth week.     

Trends in incidence and survival analysis in non-melanoma skin cancer from 1994 to 2012 in Girona,Spain: A population-based study

IntroductionWe present an epidemiological study focused on Non-melanoma skin cancers (NMSC), including squamous cell carcinoma (SCC), basal cell carcinoma (BCC), Merkel cell carcinoma (MCC), dermatofibrosarcoma protuberans (DFS) and adnexal and skin appendages neoplasm (ASAN), a neoplasm understudied in cancer registries.Material and methodsWe analyze trends of incidence and survival of NMSC registered with the Cancer Registry of Girona, Spain.ResultsWe found 14389 cases of NMSC, accounting 3,474 SCC, 10729 BCC, 33 MCC, 61 DFSP and 71 ASAN. Incidence increased significantly in SCC and BCC with annual percentage of change of 1.6 and 1.5, respectively, but not in MCC, DFS or ASAN. Five-year relative survival for both sexes was 90.1% in SCC, 99.8% in BCC, 44.2% in MCC, 93.7% in DFS and 84% in ASAN.ConclusionsOur study confirms the increasing incidence and good survival of SCC and BCC and enhances knowledge on the epidemiology of the less incidental NMSC.     

Distinct innate immune gene expression profiles in non-melanoma skin cancer of immunocompetent and immunosuppressed patients

Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the most frequent skin cancers in humans. An intact immune system is critical for protection against SCC since organ transplant recipients (OTR) have a 60- to 100-fold higher risk for developing these tumors. The role of the innate immune system in tumor immunosurveillance is unclear. Our aim was to determine the expression of selected innate immune genes in BCC and SCC arising in immunocompetent and OTR patients. Lesional and peri-lesional skin from 28 SCC and 19 BCC were evaluated for mRNA expression of toll-like receptors (TLR) 1-9, downstream TLR signaling molecules, and antimicrobial peptides. 11 SCC occurring in OTR patients were included in the analysis. We found that SCC but not BCC showed significantly elevated expression of TLRs 1-3, 5-8, TRIF and TRAF1. TNF was increased in SCC compared to normal skin. BCC showed increased IFNγ. hBD1, hBD2 and psoriasin mRNA and protein expression were significantly higher in SCC than in normal skin and higher than in BCC. SCC from OTR showed only an increase in hBD2 but no increase in hBD1 or psoriasin. We conclude that innate immune gene expression in SCC is distinct from normal skin and BCC. BCC shows lesser induction of innate immune genes. SCC from OTR patients have depressed expression of hBD1 and psoriasin compared to SCC from immunocompetent patients.     

A pilot study of recurrence of human glial tumours in light of p53 heterozygosity status

Genetic alterations in the p53 tumour suppressor gene of human chromosome 17 have been frequently found to be associated with various tumours. Glial tumours arise from the supporting cells of the brain. They have a poor outcome and often recur. The present study was undertaken to compare the loss of heterozygosity (LOH) of p53 gene in pairs of primary and recurrent gliomas and to try and correlate it to the degree of malignancy and recurrence interval. LOH of p53 was taken as an indicator of the inactivation of p53 due to genetic changes. Ten patients with recurrent gliomas were studied. Three patients had LOH at primary surgery and two of these had LOH at recurrent surgery. One patient had LOH of p53 only at recurrent surgery but not at primary surgery. No indicative correlation was found between p53 heterozygosity status on one hand and grade of malignancy (primary and recurrent) and recurrence interval on the other.     

A pilot study of circulating miRNAs as potential biomarkers of early stage breast cancer

Background

To date, there are no highly sensitive and specific minimally invasive biomarkers for detection of breast cancer at an early stage. The occurrence of circulating microRNAs (miRNAs) in blood components (including serum and plasma) has been repeatedly observed in cancer patients as well as healthy controls. Because of the significance of miRNA in carcinogenesis, circulating miRNAs in blood may be unique biomarkers for early and minimally invasive diagnosis of human cancers. The objective of this pilot study was to discover a panel of circulating miRNAs as potential novel breast cancer biomarkers.

Methodology/Principal Findings

Using microarray-based expression profiling followed by Real-Time quantitative Polymerase Cycle Reaction (RT-qPCR) validation, we compared the levels of circulating miRNAs in plasma samples from 20 women with early stage breast cancer (10 Caucasian American (CA) and 10 African American (AA)) and 20 matched healthy controls (10 CAs and 10 AAs). Using the significance level of p<0.05 constrained by at least two-fold expression change as selection criteria, we found that 31 miRNAs were differentially expressed in CA study subjects (17 up and 14 down) and 18 miRNAs were differentially expressed in AA study subjects (9 up and 9 down). Interestingly, only 2 differentially expressed miRNAs overlapped between CA and AA study subjects. Using receiver operational curve (ROC) analysis, we show that not only up-regulated but also down-regulated miRNAs can discriminate patients with breast cancer from healthy controls with reasonable sensitivity and specificity. To further explore the potential roles of these circulating miRNAs in breast carcinogenesis, we applied pathway-based bioinformatics exploratory analysis and predicted a number of significantly enriched pathways which are predicted to be regulated by these circulating miRNAs, most of which are involved in critical cell functions, cancer development and progression.

Conclusions

Our observations from this pilot study suggest that the altered levels of circulating miRNAs might have great potential to serve as novel, noninvasive biomarkers for early detection of breast cancer.     

Reversible anergy in circulating lymphocytes of cancer patients during interleukin-2 therapy

Interleukin-2 plays a crucial role in enhancing the antitumor immune response. Clinical trials, mainly in renal cell carcinoma and melanoma patients, have been carried out with encouraging results. Recent reports demonstrated that interleukin-2 therapy may depress the immune response either in vitro or in vivo. We decided to monitor, in nine renal cancer patients, the proliferative responses and the parallel variations in Ca²⁺ homeostasis of peripheral blood lymphocytes collected before, during and after the first cycle of a 3-day interleukin-2 systemic administration. The proliferative response to phytohemagglutinin or concanavalin A significantly dropped early during interleukin-2 infusion. Consistently, an impairment in mobilizing Ca²⁺, either from internal stores or via influx from outside, was observed. Results obtained with a mAb-CD3 molecular complex strongly suggested that the TCR/CD3 signal transduction pathway was defective. In contrast, no major variations were observed in the general machinery controlling Ca²⁺ homeostasis nor in the total Ca²⁺-releasable pool. Patients' lymphocytes, cultured in vitro for 3 days in medium alone, showed an almost complete recovery in their ability to respond to mitogens. In conclusion, we show that interleukin-2 administration in cancer patients induces a reversible state of anergy in circulating lymphocytes, assessed both by the reduction in the proliferative response and the block of the mitogen-activated intracellular Ca²⁺ signalling.This work was supported by grant CNR-ACRO 9202374     

Expression of Ki-67, p53 and p63 proteins in keratocyst odontogenic tumours: an immunohistochemical study

Aim To investigate the immunohistochemical expression of Ki-67, p53 and p63 in Keratocyst Odontogenic Tumours (KOTs) in order to contribute to the biological profile of this tumor. Methods Immunohistochemical technique was performed using the EnVision™ System in 37 cases of KOTs. Results Ki-67- and p53-immunostained cells were mainly located in the suprabasal layers. p63-positive cells were found throughout the lining cystic epithelium. No difference in the immunostaining for these proteins was observed between primary and recurrent KOTs (Ki-67: P = 0.5591; p53: P = 0.9847; p63: P = 0.9127), or between KOTs associated with Nevoid Basal Cell Carcinoma Syndrome (NBCCS) and sporadic KOTs (Ki-67: P = 0.7013; p53: P = 0.3197; p63: P = 0.2427). Conclusions It is possible that biological behavior of KOTs may be related to suprabasal proliferative compartment in the cystic epithelium as observed by high levels of Ki-67, p53 and p63. In addition, p63 immunostaining may represent immaturity of keratinocytes in KOTs, and suggests that this protein may participate in the regulation of epithelial cell differentiation. Taken together, these data may favor tumorigenesis on KOTs.     

p53 codon 72 polymorphism in cervical cancer patients and healthy women from Poland

A polymorphism at codon 72 of gene p53 results in the presence of either arginine or proline at this position. We investigated the distribution of p53 codon 72 polymorphism in cervical cancer patients and a control group of healthy women from Poland. Our results do not confirm the hypothesis that the p53 codon polymorphism could play a role as a factor for squamous carcinoma of the cervix.     

Absence of p53 germ-line mutations in bilateral breast cancer patients

Summary The cause of Li-Fraumeni syndrome, a rare group syndrome of familial cancers, has recently been identified. Patients with this inherited condition are highly susceptible to specific neoplasms, including early-onset breast cancers. The available evidence links Li-Fraumeni syndrome to inherited mutations of the tumor suppressor gene p53. Moreover, somatically acquired p53 mutations and gene deletions are common feature in breast cancer of sporadic origin. These findings suggest that germline p53 mutations are important in familial and, possibly sporadic, breast tumors. We have therefore screened lymphocyte DNA from 19 unrelated bilateral cancer patients for germline p53 mutations in exons 5, 6, 7 and 8. We have however detected no germline mutations by means of the single-strand confirmation polymorphism technique in any of the lymphocyte DNAs examined and conclude that p53 mutations are not generally involved in bilateral breast cancer.     

Spontaneous chromosomal anomalies in lymphocytes from xeroderma pigmentosum. A study of ten patients

A cytogenetic study of the lymphocytes from 6 classic and 4 variant forms of xeroderma pigmentosum is reported. This study performed on 978 R-banded metaphases shows that there is no specific chromosomal rearrangement in this disorder. In UDS-deficient forms, the rates of deletions, chromatid gaps and chromosome gaps are significantly increased. The preferential involvement of G-bands is discussed.     

Prognostic value of p53 overexpression in head and neck carcinomas: a pilot study

OBJECTIVE: To correlate p53 overexpression in squamous cell carcinoma of the head and neck region with the outcome of treatment. STUDY DESIGN: Twenty-five biopsy-proven squamous cell carcinomas of the head and neck region, locally advanced and untreated, were studied. Before treatment, all patients underwent fine needle aspiration from primary and/or metastatic lesions. Smears were prepared from the aspirate for immunostaining, and p53 overexpression was measured semiquantitatively. All patients received a radical dosage of radiation equivalent to 60 Gy for 6 weeks in 30 fractions from a 6-MV linear accelerator. Local-regional disease control was studied, and the mean follow-up duration was one year. The pretreatment values of p53 overexpression were correlated with the outcome of treatment. RESULTS: Overexpression of p53 was found in 36% patients. At the end of 1 year, 6/9 patients showing overexpression were disease free as compared to 5/16 patients without overexpression. The difference was not significant (chi2 test, P>.05). CONCLUSION: Response to radiation therapy is not dependent on p53 overexpression in squamous cell carcinoma of the head and neck region. However, this was only a pilot study, and a large number of cases are needed to establish the prognostic value of p53 overexpression in locally advanced head and neck squamous cell carcinoma.     

Immunohistochemical detection of p53 protein in basal cell skin cancer after microwave-assisted antigen retrieval

p53 is the most frequently altered tumor-suppressor gene in skin cancer. In normal tissues the p53 protein (wild type) has a very short half-life and it is not detectable immunohistochemically. In contrast, the mutant p53 protein has an extended half-life in tumor cells and can be detected by immunohistochemical methods. p53 is widely used as an indicator of tumor aggression and progression. Fixation methods especially formaldehyde based fixation may mask the immunohistochemical detection of p53 protein but antigen retrieval methods enhance the inmmunohistochemical detection of p53 protein by remodification of protein structure. This study was designed to evaluate the efficacy of different fixatives, of microwaving and microwave pretreatment method to retrieve p53 immunoreactivity in paraffin-embedded non-lesioned (adjacent normal tissue) human skin samples or pathological human skin samples diagnosed as basal cell carcinoma. The samples were fixed at RT and/or in microwave oven either in neutral buffered formalin or alcohol for different time periods. For antigen retrieval, the sections were irradiated in a microwave oven for 5 cycles in 10 mM citrate buffer (pH 6.00). In this study the effects of six different fixation methods on the immunohistochemical staining have been investigated in basal cell tumor specimens. The application of antigen retrieval method was also examined and compared. Optimal results were obtained using samples fixed in alcohol either at room temperature (24 h) or in microwave oven.