MicroRNAs (miRNAs) as the regulatory short noncoding RNAs are involved in a wide array of cellular and molecular processes. They negatively regulate gene expression and their dysfunction is correlated with cancer development through modulation of multiple signaling pathways. Therefore, these molecules could be considered as novel biomarkers and therapeutic targets for more effective management of human cancers. Recent studies have demonstrated that the miR-181 family is dysregulated in various tumor tissues and plays a pivotal role in carcinogenesis. They have been shown to act as oncomirs or tumor suppressors considering their mRNA targets and to be involved in cell proliferation, apoptosis, autophagy, angiogenesis and drug resistance. Additionally, these miRNAs have been demonstrated to exert their regulatory effects through modulating multiple signaling pathways including PI3K/AKT, MAPK, TGF-b, Wnt, NF-κB, Notch pathways. Given that, in this review, we briefly summarise the recent studies that have focused on the roles of miRNA-181 family as the multifunctional miRNAs in tumorigenesis and cancer development. These miRNAs may serve as diagnostic and prognostic biomarkers or therapeutic targets in human cancer gene therapy.

Mutations in the KRAS and TP53 genes have been found frequently in lung tumors and specimens from individuals at high risk for lung cancer and have been suggested as predictive markers for lung cancer. In order to assess the prognostic value of these two genes' mutations in lung cancer recurrence, we analyzed mutations in codon 12 of the KRAS gene and in hotspot codons of the TP53 gene in 176 bronchial biopsies obtained from 77 former lung cancer patients. Forty-seven patients (61.0%) showed mutations, including 35/77 (45.5%) in the KRAS gene and 25/77 (32.5%) in the TP53 gene, among them 13/77 (16.9%) had mutations in both genes. When grouped according to past or current smoking status, a higher proportion of current smokers showed mutations, in particular those in the TP53 gene (P = 0.07), compared with ex-smokers. These mutations were found in both abnormal lesions (8/20 or 40%) and histologically normal tissues (70/156 or 44.9%) (P = 0.812). They consisted primarily of G to A transition and G to T transversion in both the KRAS (41/56 or 73.2%) and TP53 (24/34 or 70.6%) genes, consistent with mutations found in lung tumors of smoking lung cancer patients. Overall, recurrence-free survival (RFS) among all subjects could be explained by age at diagnosis, tumor stage, tumor subtype, and smoking (P < 0.05, Cox proportional hazard). Therefore, KRAS and TP53 mutations were frequently detected in bronchial tissues of former lung cancer patients. However, the presence of mutation of bronchial biopsies was not significantly associated with a shorter RFS time. © 2016 Wiley Periodicals, Inc.

MicroRNAs (miRNAs) are short noncoding RNAs that act as post-transcriptional regulators. The low complementarity required between the sequences of a miRNA and its target mRNA enables a single miRNA to act on a large range of targets. Thus miRNAs have an intersecting complex effect that spans a multiplicity of pathways and processes. In this review, the different roles of a vital miRNA, miR-181a, in physiological and pathological developments are collated in an attempt to highlight the intersections of such processes and to show how the deregulation of miR-181a could in one context drive malignancy, whereas in another it can lead to autoimmunity. Such deregulation could be related to the faulty levels of one of its own targets, p53, which was recently reported to control an array of miRNAs, one of which is miR-181a. This sheds light on a hidden loop of chaos behind chronic diseases such as autoimmunity and cancer.

The molecular basis of lung carcinogenesis must be understood more fully and exploited to enhance survival rates of patients suffering from lung cancer. In this review we will discuss the major molecular alterations that occur in lung cancer. Emphasis is placed on alterations that occur early during carcinogenesis since they might be relevant for future screening programs. Finally we will shortly review new approaches that are used to study the molecular pathology of lung cancer and how they can be applied in a clinical setting.

A/J mice with genetic alterations in K-ras, p53, or Ink4a/Arf were employed to investigate whether mice carrying these germline mutations would be susceptible to tobacco smoke-induced lung tumorigenesis. Transgenic mice of both genders and their wild-type littermates were exposed to environmental cigarette smoke for 6 months, followed by recovery in air for 5 months. A significant increase of lung tumor multiplicity was observed in K-ras, p53, or Ink4a/Arf mutant mice when compared with wild-type mice. Furthermore, an additive effect was observed between the mice with a mutant p53 transgene and an Ink4A/Arf deletion during tobacco smoke-induced lung tumorigenesis. Sequence analysis of the K-ras gene indicated that the mutations had occurred at either codon 12/13 or 61 in both spontaneously occurring (air control) and tobacco smoke-induced lung tumors. K-ras mutations were found in 62% of the tumors from air-control animals and 83% in those exposed to tobacco smoke. The mutation spectrum found in tumors from mice exposed to tobacco smoke is somewhat similar to that in tumors from air-control mice. In addition, we identified three novel mutations at codon 12: GGT (Gly) --> TTT (Phe), ATT (Ile), and CTT (Leu). These findings provide evidence that K-ras, p53, and Ink4a/Arf mutations play a role in tobacco smoke-related lung carcinogenesis. The similarity of the mutation spectra in the K-ras oncogene observed in tobacco smoke-induced tumors, as compared to spontaneous tumors, suggests that tobacco smoke enhances lung tumorigenesis primarily through promoting spontaneously occurring K-ras mutations.

In this study, we used p53 transgenic mice to investigate whether mice carrying this germline mutation would be susceptible to tobacco smoke-induced lung tumorigenesis. We subjected male transgenic mice and their wild-type littermates to whole-body exposure to environmental cigarette smoke (ECS) for up to 9.5 months. K-ras gene expression was significantly increased, 28 days after ECS exposure, in the apparently healthy lung of p53 mutant mice. An increase of lung tumor incidence and multiplicity was observed in p53 transgenic mice after exposure to ECS for either 5 months, followed by recovery in air for 4.5 months, or 9.5 continuative months of exposure. Conversely, no tumorigenic effect was observed in their wild-type littermates. Sequence analysis of the K-ras gene indicated that mutations had occurred at codon 12, 13 or codon 61 in tumors both from the air control group and tobacco smoke treatment groups. K-ras mutations were found in 100 %, 100 % and 77 % of tumors from animals exposed to air, ECS for 5 months, followed by recovery in air for 4.5 months, and ECS for 9.5 continuative months, respectively. The K-ras mutations were seemingly not related to the p53 genotype of the animals or to ECS exposure. The mutation spectrum was similar in tumors from the different groups. An apparently higher incidence of K-ras codon 12 mutations in the 9.5 months ECS group was not statistically significant. These findings provide evidence that mice carrying a mutant p53 transgene appear to be more sensitive to ECS-induced lung tumors than the corresponding wild-type littermates. K-ras mutations seem to be independent of the p53 status but the early overexpression of this oncogene is related to the p53 status in ECS-exposed mice. These results suggest that tobacco smoke enhances lung tumorigenesis primarily through promoting spontaneously occurring K-ras mutations.

To evaluate p53 protein overexpression and to measure serum CA19.9 concentrations in cytological diagnosis of patients with suspected pancreatic cancer.

24 patients with suspected pancreatic cancer due to chronic pancreatitis, had a pancreatic mass determined by imaging methods. The serum CA19.9 concentration was measured by solid phase radioimmunoassay. On laparotomy, puncture biopsy was performed, and specimens were divided into two parts for cytological diagnosis and detection of p53 protein.

Cytology offered a sensitivity of 0.63, a specificity of 1.00, and an accuracy of 0.63. p53 protein analysis offered a sensitivity of 0.44, a specificity of 1.00, and an accuracy of 0.73. CA19.9 offered a sensitivity of 0.44, a specificity of 0.80, and an accuracy of 0.67. The combined cytology and p53 protein analysis showed a sensitivity of 0.78, a specificity of 1.00, and an accuracy of 0.92. Cytology and CA19.9 showed a sensitivity of 0.67, a specificity of 0.80, an accuracy of 0.67. combined cytology and p53 protein analysis and CA19.9 showed a sensitivity of 0.78, a specificity of 0.80,and an accuracy of 0.79.

Superior to any single test, the combined approach is helpful for the differential diagnosis of pancreatic cancer complicated with chronic pancreatitis. The combined cytology and p53 protein analysis offers the best diagnostic efficacy.

Abnormalities of fragile histidine triad (FHIT) and TP53 have been found frequently in nonsmall cell lung cancers. In the current study, 263 primary nonsmall cell lung cancers were investigated for the expressions of Fhit and p53 by immunohistochemistry. Marked reduction of Fhit immunoreactivity (<10% positivity) in 156 (59%) tumours and overexpression of p53 protein (>10% positivity) in 89 (34%) tumours were found. Reduced Fhit expression was also noted in most squamous cell carcinomas (80 out of 99, 81%), and in a smaller fraction of adenocarcinomas (76 out of 164, 46%; P<0.001). p53 nuclear staining was demonstrated in 54 out of 99 (55%) squamous cell carcinomas and in 35 out of 164 (21%) adenocarcinomas (P<0.001). The loss of Fhit expression and p53 overexpression was significantly more common in tumours occurring in smokers (93 out of 113, 82% and 56 out of 113, 50%) than in those of nonsmokers (63 out of 150, 42%; P<0.001 and 33 out of 150, 22%; P<0.001). Notably, p53 overexpression was associated with distant metastasis of patients in the whole series (P=0.027) and in adenocarcinoma (P=0.001). It was also associated with a poorer survival of patients with adenocarcinoma (P=0.032).

Endobronchial carcinoma develops through a continuum of morphologically recognizable pre-neoplastic changes. At present, no marker has been identified that can reliably predict the biological behavior of these lesions. Endobronchial lesions (n=39) sampled from patients (n=20) without clinically overt lung cancer, were analyzed by immunohistochemistry (IHC) for abnormal expression regarding the p53 protein, i.e. suprabasal p53 expression. Clear suprabasal p53 immunostaining was found in two (12%) of the hyperplastic or squamous metaplastic lesions, in one (10%) of the mildly or moderately dysplastic lesions and in nine (75%) of the severely dysplastic or carcinoma in situ (CIS) lesions. Suprabasal p53 immunostaining was found significantly more frequent in severe dysplasia or CIS (P<0.01). Of 17 patients follow-up data were available. After a median follow up of 7 months (range 2-37 months), six patients presented with bronchial carcinoma within the same lobe or bronchial spur where biopsies had been taken. Four of these patients revealed suprabasal p53 immunostaining in the biopsies obtained from the sites of future cancer. In three patients biopsies were obtained from future cancer sites as well as from distant sites in the ipsilateral lung. Suprabasal p53 immunostaining was found exclusively at future cancer sites of these patients (P=0.02). Suprabasal p53 immunostaining in addition to histology improved the specificity and the positive predictive value for bronchial carcinoma development in the same lobe or bronchial spur, compared with histology alone. These results suggest that suprabasal p53 immunostaining is associated with bronchial cancer and might have additive value to predict the biological behavior of pre-neoplastic endobronchial lesions in the population at risk of bronchial cancer.

To determine whether the nuclear accumulation of p53 in patients with early bronchial neoplasia represents an altered susceptibility for the development of lung cancer.

We evaluated the percentage of cells accumulating nuclear p53 immunohistochemically in squamous cell carcinoma (SCC) of the lung, the associated uninvolved bronchial mucosa, and epithelial hyperplasia in 60 archival lung specimens of smokers and in the normal bronchial epithelium and hyperplastic lesions of 60 smokers who had not developed lung cancer.

The percentage of cells accumulating p53 was significantly higher in SCC-associated uninvolved bronchial epithelia of (mean [+/- SD], 4 +/- 0.9%) and in specimens from patients with epithelial hyperplasia (mean, 9 +/- 2%) compared to the percentage of cells from the bronchial epithelia of (mean, 0.5 +/- 0.2%) and in specimens from patients with epithelial hyperplasia (mean, 1.5 +/- 0.5%) who were smokers who had not developed lung cancer (p = 0.0002 and p = 0.0004, respectively). We also observed a statistically significant stepwise increase in the percentage of cells accumulating p53 from SCC-associated uninvolved bronchial epithelium to those from a patient with epithelial hyperplasia to those from a patient with SCC (mean, 35 +/- 4%), suggesting the involvement of p53 accumulation in the development of SCC (p <or= 0.05 for all comparisons). The accumulation of p53 in SCC cells was not significantly associated with the size of the tumor, nodal involvement, the stage of the disease, the presence or absence of metastasis, the grade of differentiation, or survival of the disease, indicating its lack of association with the clinical progression of the disease.

These results suggested that p53 accumulation is an early event in lung carcinogenesis and potentially could be useful in the identification of smokers who are at risk of developing SCC, but not in the estimation of survival of the disease.

The objective of this study was to evaluate the possible prognostic significance of p53 protein overexpression and tumor angiogenesis (TA) in nasopharyngeal carcinoma (NPC) patients, together with other clinicopathological variables. Forty-two NPC patients were evaluated in relation to survival. Nuclear p53 overexpression in neoplastic and endothelial cells was detected by immunohistochemistry (IHC) with the monoclonal antibody DO-7 and the polyclonal antibody against factor VIII-related antigen, respectively. Thereafter, we evaluated p53 cases in order to determine their nuclear immunoreactivity from negative (-) to positive (+, ++, +++). In addition, microvessels were counted in the most active areas of tumor neovascularization or hotspots using an image computer analyzer (MicroImage). A Cox multiple regression survival analysis was used to determine the best prognostic indicators in NPC patients. As a result, tumor microvessel count, considered as a continuous variable, was the most important independent prognostic indicator in relation to survival (p = 0.0273), with a relative risk of death of 2,4399 [95% confidence interval = 1.1051 ; 5.3871] associated with the highest microvessel counts. Moreover, the only clinicopathological variable that demonstrated prognostic value in a Cox multiple regression survival analysis was histological type (p = 0.05). In addition, we did not observe any statistical association between intratumoral microvessel density (IMD), clinicopathological variables and p53 protein expression.

Forty-four specimens of non-malignant and malignant human lung tissue, taken from patients with non-small cell lung cancer (NSCLC), were examined for the expression of wild-type p53, mutant p53, and bcl-2 and the occurrence of programmed cell death (apoptosis). Wild-type p53 expression peaked in peritumoral and metaplastic samples, whereas mutant p53, bcl-2 and apoptosis were first detected in metaplasia and increased with progression to carcinoma. Bcl-2 positive samples had lower levels of apoptosis than bcl-2 negative samples and was independent of wild-type or mutant p53 expression. These results suggest that the over-expression of wild-type p53 may be an early cellular response to an alteration in normal cellular homeostasis. The ensuing increase in apoptosis appears to be relatively independent of mutant or wild-type p53 expression, but does not occur in cells expressing bcl-2.

Prognostic value of p53 and PCNA expression in non-small cell lung cancer (NSCLC) remains controversial. In this study we determined the relevance of these abnormalities in terms of overall survival and disease-free survival in 95 NSCLC patients who underwent curative pulmonary resection. Expression of p53 was found in 44 samples (45%), expression of PCNA-in 79 samples (83%), and expression of both markers-in 35 samples (36%). There was no relationship between expression of either protein and major clinicopathological characteristics. Median survival for patients with and without p53 expression was 36 and 33 months, respectively and 5-year survival probability-29 and 37%, respectively (P=0.73). Median survival for patients with and without PCNA expression was 36 and 27 months, respectively and 5-year survival probability-35 and 25%, respectively (P=0.60). There was no significant difference in overall survival between particular groups of patients with tumors carrying four possible p53/PCNA phenotypes. In multivariate analysis including patient age, sex, tumor stage, tumor type and differentiation, p53 and PCNA expression, the only variable important for survival was stage of disease. These results suggest the lack of prognostic relevance of p53 and PCNA expression in surgically treated NSCLC patients.

Southwest Metropolitan Mexico City (SWMMC) atmosphere is a complex mixture of air pollutants, including ozone, particulate matter, and aldehydes. Children in SWMMC are exposed chronically and sequentially to numerous toxicants, and they exhibit significant nasal damage. The objective of this study was to assess p53 accumulation by immunohistochemistry in nasal biopsies of SWMMC children. We evaluated 111 biopsies from 107 children (83 exposed SWMMC children and 24 control children residents in a pollutant-compliant Caribbean island). Complete clinical histories and physical examinations, including an ear-nose-throat (ENT) exam were done. There was a significant statistical difference in the upper and lower respiratory symptomatology and ENT findings between control and exposed children (p < 0.001). Control children gave no respiratory symptomatology in the 3 months prior to the study; their biopsies exhibited normal ciliated respiratory epithelium and were p53-negative. SWMMC children complained of epistaxis, nasal obstruction. and crusting. Irregular areas of whitish-gray recessed mucosa over the inferior and middle turbinates were seen in 25% of SWMMC children, and their nasal biopsies displayed basal cell hyperplasia, decreased numbers of ciliated and goblet cells, neutrophilic epithelial infiltrates, squamous metaplasia. and mild dysplasia. Four of 21 SWMMC children with grossly abnormal mucosal changes exhibited strong transmural nuclear p53 staining in their nasal biopsies (p 0.005, odds ratio 26). In the context of lifetime exposures to toxic and potentially carcinogenic air pollutants, p53 nasal induction in children could potentially represent. a) a checkpoint response to toxic exposures, setting up a selective condition for p53 mutation, or b) a p53 mutation has already occurred as a result of such selection. Because the biological significance of p53 nuclear accumulation in the nasal biopsies of these children is not clear at this point, we strongly suggest that children with macroscopic nasal mucosal abnormalities should be closely monitored by the ENT physician. Parents should be advised to decrease the children's number of outdoor exposure hours and encourage a balanced diet with an important component of fresh fruits and vegetables.

It has been reported that cases of ovarian endometriosis those with epithelial cytological atypia have potential for malignant transformation. This study was planned to determine the incidence of atypical endometriosis and its cytological criteria, to evaluate the malignant potential of atypical endometriosis via immunohistochemical methods (p53). In this study we evaluated 140 samples obtained from 120 cases of ovarian endometriosis and 10 ovarian endometrioid carcinomas that have been previously diagnosed histopathologically. We re-evaluated endometriosis cases with respect to their epithelial and stromal features, existence of acute or chronic inflammatory cells in endometriotic epithelium or stroma and other accompanying histological findings. We observed atypia in 7 (5.8%) cases; reactive atypia in 37 (30.8%) cases, no atypia in 76 (63.4%) cases. We evaluated immunohistochemical p53 expression in 7 atypical cases, 37 reactive atypical cases, and in 10 of those without atypia and in 10 endometrioid carcinoma cases. We noted no staining in cases with atypia, reactive atypia and without atypia while 3 cases of endometrioid carcinoma had positive staining for p53. We concluded that prominent nucleolus and angulation of nuclear contour could be added to criteria of atypia that were mentioned before in the literature. In our study, even though p53 expression could not be shown with immunohistochemical methods in atypical endometriotic cases; it can not be determined that atypical endometriosis lesions are not premalignant. Still, endometriosis cases should be evaluated carefully by the pathologist for foci of cytological atypia and it should be kept in mind that malignant transformation might occur in these atypical endometriosis cases.

The aim of this study was to investigate the frequency of three (epi)genetic alterations (p53 and K-ras mutations and p16(INK4a) promoter hypermethylation) in symptomatic chronic smokers compared with patients with lung cancer and to evaluate the use of exfoliative material for such analyses.

Fifty-one patients with histologically confirmed lung cancer and 25 chronic smokers (> 20 pack-years) were investigated for mutations in the K-ras (codon 12) and p53 (codons 248, 249, and 273) genes and for allelic hypermethylation of the p16(INK4a) gene. DNA was isolated from sputum and bilateral bronchial lavage, and brushings were taken at bronchoscopy.

Forty-one genetic lesions were detected within exfoliative material from the group of 51 patients with lung cancer and 10 lesions in the chronic smoker group. K-ras mutations occurred exclusively in the lung cancer group, whereas p53 mutations and p16(INK4a) promoter hypermethylation were also found in chronic smokers. Three of eight chronic smokers who harbored an (epi)genetic alteration were subsequently diagnosed with lung cancer. Analysis of sputum yielded information equivalent to that of samples obtained during bronchoscopy.

p16(INK4a) promoter hypermethylation and p53 mutations can occur in chronic smokers before any clinical evidence of neoplasia and may be indicative of an increased risk of developing lung cancer or of early disease. K-ras mutations occur exclusively in the presence of clinically detectable neoplastic transformation. Molecular analysis of sputum for such markers may provide an effective means of screening chronic smokers to enable earlier detection and therapeutic intervention of lung cancer.

CD40 is a cell surface receptor which, when ligated, modulates apoptosis in some cell types, perhaps via activation of the expression of members of the Bcl2 gene family. This study sought to determine whether expression of CD40 in a series of 134 squamous cell carcinomas (SCCs) of the lung was related to apoptosis or clinical parameters, either alone or in connection with Bcl2 expression. Sections of SCCs were stained for CD40 and Bcl2 by immunohistochemical techniques. An index was determined for these two markers by counting stained and unstained malignant cells under high power. Sections were also stained by haematoxylin and eosin for determination of the apoptotic index by counting apoptotic bodies. About 40 per cent of SCCs expressed CD40 in at least some of the malignant cells, with about 10 per cent essentially uniformly stained. Similar proportions expressed Bcl2, with a tendency for expression to be mutually excluisve. Apoptosis did not correlate with CD40 expression, nor was there evidence of a co-operative or an antagonistic effect with Bcl2. Bcl2 expression, on the other hand, correlated significantly with the apoptosis score. CD40 expression showed no significant relationship with survival or any other clinicopathological parameter except ploidy. Bcl2 expression, however, correlated with longer survival. It is concluded that CD40 expression in SCC of the lung does not by itself relate to apoptosis, nor is it a useful indicator of prognosis.

Alterations of the P53 or Rb gene are among the most frequently observed genetic changes in primary lung cancer. Nevertheless, there has been no final conclusion on the relationship between P53 or Rb protein expression and clinico-pathological parameters in primary lung cancer. A large-scale study was performed to examine the clinicopathological and prognostic significance of P53 and Rb expressions in 207 surgically resected non-small cell lung cancer (NSCLC) patients.

Tumor specimens were obtained from 207 primary NSCLC surgically resected from January 1990 through December 1994. The avidin-biotin-peroxidase method was used to determine the expression of P53 or Rb of tumor cells using anti-P53 or anti-Rb monoclonal antibodies. The relationships between P53 or Rb protein expression and the clinicopathological parameters were analyzed.

Expression of P53 or Rb protein was detected in 115 (55.6%) and 136 (65.7%) of the 207 lung tumors, respectively. P53 had significantly higher positive results in patients with regional lymph node metastasis and advanced tumor stage. Rb expression was significantly lower in lung cancers with a macro- or microscopic picture of tumor necrosis. Additionally, an inverse correlation between the expression of Rb and P53 was found. By multiple variate analysis, P53 expression and pathological stage were independent, significant prognostic factors. Further analysis demonstrated P53 expression was an independent prognostic factor in stage 1, but not in other stages.

P53 expression is especially useful as a prognostic factor in stage 1 lung cancer.

DNA aneuploidy, p53 overexpression, and high cell proliferation frequently occur in gastric cancer. However, little is known about the time of their appearance throughout cancer progression. Therefore, the objective of the present study was to determine when such abnormalities occur during gastric cancer progression. We classified the gastric cancers examined into intestinal (n = 65) and diffuse (n = 34) types. DNA ploidy was examined using flow cytometry and expression of MIB-1 and p53 immunoreactivity were studied using the avidin-biotin complex method in three stages of gastric cancer (mucosal, submucosal, deeply invasive cancer, i.e., advanced cancer). The incidence of DNA aneuploidy in intestinal-type mucosal cancers (15/27, 55.6%) was lower than that of submucosal invasive cancers (14/16, 87.5%) or advanced cancers (19/22, 86.4%), while a low incidence of DNA aneuploidy was observed in each diffuse-type cancer group (mucosal, 1/12, 8.3%; submucosal invasive, 3/9, 33.3%; advanced, 8/14, 57.1%). Although overexpression of the p53 gene in intestinal-type cancer was found in early stage, that in diffuse-type cancer was observed in advanced stage. Among the intestinal-type mucosal cancers, the MIB-1 percent positive was higher in aneuploid tumors than diploid ones. DNA aneuploidy and overexpression of the p53 gene may play an important role in the early tumorigenesis of intestinal-type gastric cancer and in the late event of tumorigenesis of diffuse-type gastric cancer.

Genomic instability is a driving force for tumorigenesis. p53 and telomerase play central roles in maintaining genomic integrity. The purpose of this study was to assess the associations among p53 protein overexpression, telomerase activity and genetic instability in lung cancer. We found that telomerase activity was detectable in 80% of 100 lung tumours, but only 7.7% of 91 paired adjacent normal tissues. p53 protein was overexpressed in 63% of the tumours but only 2% of the normal tissues. p53 was overexpressed in 56 of the 80 (70%) tumour tissues with telomerase activity but only seven of the 20 (35%) without telomerase activity. p53 protein overexpression carried a 6.7-fold (95% confidence interval, 1.7-27.7) increased risk for positive telomerase activity after adjustment by age, sex, ethnicity, smoking status and family history of lung cancer. The mean in vitro bleomycin-induced breaks per cell (a marker of cancer susceptibility) was significantly higher (0.92) for patients who overexpressed p53 in lung tumour tissue than that for patients with no detectable p53 expression in lung tumour tissue (0.65). Our data suggest that p53 protein overexpression may be common in individuals genetically susceptible to carcinogen exposure. p53 status may be related to telomerase expression.

The present study is aimed to evaluate the genetic evidence for multicentricity of synchronous and metachronous multiple lung carcinomas. Nineteen cases of synchronous multiple lung carcinomas and 11 cases of metachronous multiple lung carcinomas were analyzed for p53 protein overexpression by immunohistochemistry (DO-7) and for genetic abnormality of the p53 gene by loss of heterozygosity (LOH) at chromosome 17p and by polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis. They were also analyzed for K-ras mutation. DNA from three patients was also sequenced by the dideoxy sequencing method to confirm the presence of mutations and determine the base substitutions. Different spectrums of genetic changes, which were evaluated by a combination of p53 mutation, LOH at chromosome 17p and p53 overexpression, were observed in 11 of 19 cases of synchronous multiple lung carcinomas (57.9%) in the present study. Similarly, five of 11 cases of metachronous multiple lung carcinomas (45.4%) showed a different pattern of genetic changes. The present data suggest that some of the multiple carcinomas have different clonal origins, although their histological types are identical, and support the use of genetic markers in the differential diagnosis between metastasis and second primary carcinoma of the lung.

Accumulation of mutant p53 protein occurs frequently in human malignancies, including 40-60% of non-small cell lung carcinomas. The implications of such p53 over-expression, usually assessed by immunohistochemical techniques, for the prognosis of lung cancer patients remain undetermined. In this study, we used a time-resolved immunofluorometric assay to measure p53 protein concentrations in extracts prepared from 86 primary non-small cell lung tumours and examined the associations between p53 protein levels (corrected for total protein) and other clinico-pathologic variables, including post-surgical disease-free and overall survival. Contingency tables analysed by chi2 tests revealed no significant relationships between p53 status, defined by a median cut-off point, and patient gender, age, disease stage, histologic grade and type, lymph node extension, smoking history and administration of adjuvant chemotherapy or radiation. However, multivariate Cox proportional hazard regression analysis demonstrated a dose-response relationship between p53 concentration, expressed as a 4-level, quartile-divided variable, and increased risk of relapse (p = 0.010) and death (p = 0.016). Patients whose tumours contained p53 concentrations exceeding the median value had over 3-fold higher risk of relapse (p = 0.002) and death (p = 0.007) than those whose tumours had lower p53 concentrations. We also provide evidence suggesting that the impact of p53 on survival is greater in patients with squamous cell carcinoma than in those with adenocarcinoma. Although the latter finding needs confirmation, our results suggest that application of an immunoassay of p53 protein on non-small cell lung tumour extracts may identify patients at increased risk of unfavourable outcome.

The prognostic value of the mutation of the p53 tumor suppressor gene in non-small cell lung carcinomas (NSCLC) is controversial and a polymorphism of the p53 gene at codon 72 consisting of two alleles, arginine (Arg) and proline (Pro), has been reported to be associated with the incidence of smoking-related NSCLC. We have studied 67 cases of NSCLC in relation to the mutation of the p53 gene by polymerase chain reaction (PCR)-single-strand conformation polymorphism and 178 cases in relation to the polymorphism of the gene by PCR using allele-specific primers. The genetic alterations of the p53 gene (from exons 5 to 9) were found in 34% of the patients. Frequent mutations were observed among younger patients (less than 65 years old), however, there is no significant correlation of the mutation with smoking history, histopathological types, clinical stages or prognosis. We observed that Arg/Arg homozygotes were frequently found in non-smoking patients with NSCLC but Arg/Pro heterozygotes were infrequent in the group. There was no significant association of the polymorphic alleles with histopathological types, clinical stages or prognosis. Thus, the polymorphism of the p53 gene affects the predisposition of non-smokers to NSCLC, but the alteration of the p53 gene is independent of tumor progression and histopathology.

To investigate whether p53 and proliferating cell nuclear antigen (PCNA) are maintained at relapse of non-small cell lung cancer (NSCLC), we examined tumor materials from nine patients with NSCLC who had undergone resection for primary cancer and also a second resection for its relapse to the lung. In each case, histological types of primary and relapsing tumor were identical (eight adenocarcinomas and one squamous cell carcinoma). Immunohistochemical staining analysis for p53 oncoprotein expression revealed that seven of the nine cases had identical p53 expression in primary and relapsing tumor (p53 positive in three cases and negative in four) and that in the remaining two cases, p53 positive conversion during relapse was found in one case and negative conversion in one. Immunostaining for PCNA expression revealed that PCNA expression was observed in five primary tumors, and at relapse these cases were also PCNA positive. Three of the remaining four cases showed PCNA positive conversion during relapse. This study of a small number of patients indicates that results of p53 and PCNA immunostaining of resected materials of NSCLC seem to be of little significance for predicting future relapse.

Individuals with one aerodigestive tract malignancy have a high incidence of second primary aerodigestive tumors. The mechanism for this field effect has not been determined. We studied an individual with widespread dysplastic changes in the respiratory epithelium but no overt carcinoma. The entire tracheobronchial tree obtained at autopsy was embedded in paraffin, and bronchial epithelial cells were isolated by microdissection. DNA extracted from the microdissected cells was analyzed for point mutations in the p53 tumor suppressor gene. A single, identical point mutation consisting of a G:C to T:A transversion in codon 245 was identified in bronchial epithelium from 7 of 10 sites in both lungs. Epithelium at sites containing the p53 mutation was morphologically abnormal, exhibiting squamous metaplasia and mild to moderate atypia. No invasive tumor was found in the tracheobronchial tree or any other location. Cells from peripheral blood, kidney, liver, and lymph node exhibited no abnormality in the p53 gene. The widespread presence of a single somatic p53 point mutation in the bronchi of a smoker suggests that a single progenitor bronchial epithelial clone may expand to populate broad areas of the bronchial mucosa-a novel mechanism for field carcinogenesis in the respiratory epithelium that may be of importance in assessing individuals for risk of a second primary tumor as well as in devising effective strategies for chemoprevention of lung cancer.

One in ten tobacco smokers develops bronchogenic carcinoma over a lifetime. The study of susceptibility of an individual and a population to lung cancer traditionally has been limited to the study of tobacco smoke dose and family history of cancer. New insights into lung carcinogenesis have made the study of molecular markers of risk possible in human populations in the emerging field of molecular epidemiology. This review summarizes data addressing the relationships of human lung cancer to polymorphisms of phase I procarcinogen-activating and phase II-deactivating enzymes and intermediate biomarkers of DNA mutation, such as DNA adducts, oncogene and tumor suppressor gene mutation, and polymorphisms. These parameters are reviewed as they relate to tobacco smoke exposure, procarcinogen metabolizing polymorphisms, and the presence of lung cancer. Problem areas in biomarker validation, such as cross-sectional data interpretation; tissue source, race, statistical power, and ethical implications are addressed.

To document the frequency and extent of p53 gene product expression in paraffin sections of resected non-small cell carcinoma of the lung and in cytological preparations of the same tumours; to determine the effect of microwave antigen retrieval on antigen detection.

Representative paraffin sections of 50 non-small cell carcinomas were stained with an antibody to p53 gene product (DO-7) both with and without prior microwave antigen retrieval. Cytoblocks and cell smears obtained from 19 cases were similarly stained.

Using a histochemical scoring system (0-300) which takes into account staining intensity and extent, 78% (n = 39) of microwave pretreated paraffin sections and 52% (n = 26) of non-pretreated sections scored between 5 and 300; p = 0.001; 56% (n = 28) of microwave pretreated sections and only 2% (n = 1) of non-pretreated sections scored between 100 and 300 (p = 0.0001); 75% of direct smears of tumours and 80% of cytoblocks stained similarly to the paraffin sections of the resected specimens. No smears or cytoblocks stained positively when the sections of the resected specimen were negative.

As up to 78% of non-small cell lung carcinomas overexpress p53 gene product, this may prove to be a valuable diagnostic method in biopsy or cytological material when the morphological diagnosis is uncertain. Microwave antigen retrieval is effective on formalin fixed tissue.

Following up-regulation of an angiogenesis inhibitor by the wild-type p53 protein proven recently, we have analysed on the one hand the prognostic impact of microvessel count (MC) and p53 protein overexpression in non-small-cell lung carcinoma (NSCLC) progression and, on the other hand, the inter-relation between the microvascular pattern and the p53 protein expression. Moreover, we assessed the expression of vascular endothelial growth factor (VEGF), one of the pivotal mediators of tumour angiogenesis, in order to investigate its relation to p53 protein expression and MC. Tumours from 73 patients resected for NSCLC between March 1991 and April 1992 (median follow-up 47 months, range 32-51 months) were analysed using an immunohistochemical method. In univariate analysis, MC and p53 accumulation were shown to affect metastatic nodal involvement, recurrence and death significantly. Multiple logistic regression analysis showed an important prognostic influence of MC and nodal status on overall (P = 0.0009; P = 0.01) and disease-free survival (P = 0.0001; P = 0.03). Interestingly, a strong statistical association was observed between p53 nuclear accumulation and MC (P = 0.0003). The same inter-relationship was found in non-squamous histotype (P = 0.002). When we analysed the concomitant influence of MC and p53 expression on overall survival, we were able to confirm a real predominant role of MC in comparison with p53. With regard to VEGF expression, p53-negative and lowly vascularized tumours showed a mean VEGF expression significantly lower than p53-positive and highly vascularized cancers (P = 0.02). These results underline the prognostic impact of MC and p53 protein accumulation in NSCLC and their reciprocal inter-relationship, supporting the hypothesis of a wild-type p53 regulation on the angiogenetic process through a VEGF up-regulation.

Proliferating cell nuclear antigen (PCNA) is a 36 kD nuclear protein involved in DNA replication that is believed to provide an indication of proliferation in some neoplasms. This study analyzes PCNA expression in 24 cases of primary non-small cell lung cancer using monoclonal PC-10 antibodies in paraffin embedded material. We found significant inter- and intra-tumoral variations in PCNA expression, and no statistically significant relation between the amount of PCNA expression and the size and location of tumors, index of mitosis, histological tumor type or patient age. We found a statistically significant relation (r = 0.47; p < 0.05) between survival and amount of PCNA expression in a sample of 19 cases, but no statistically significant differences in survival related to whether PCNA expression was slight (0-25), moderate (25%-50%) or high (> 50%), and no prognostic value for degree of PCNA expression.

Alterations in the p53 tumor suppressor gene have been implicated in the genesis and/or progression of the majority of human cancers, including osteosarcoma. Stabilization of the protein by mutation or interaction with other proteins prolongs its half-life, rendering it detectable by immunohistochemistry. Osteosarcoma is the most common primary canine bone tumor and is characterized by frequent early metastases. Multilobular tumors of bone involve primarily flat bones of the head and are low-grade malignancies with lower metastatic potential. The objectives of this study were to determine the prevalence of p53 protein overexpression in 106 osteogenic tumors of dogs using an indirect immunohistochemical method and to compare p53 overexpression between tumors with different clinical behavior. A polyclonal p53 antibody (CM-1) served as the primary antibody. Tumors were scored based upon an estimate of the percentage of tumor cells stained. Significant differences in the prevalence of overexpression were observed between osteosarcomas (72%) and multilobular tumors of bone (20%, P = 0.0020). Osteosarcomas of the appendicular skeleton had a significantly higher prevalence of p53 overexpression (84%) than did osteosarcomas of the axial skeleton (56%, P = 0.0060). Our results show that p53 tumor suppressor protein is overexpressed in the majority of canine osteosarcomas. The higher prevalence of overexpression in osteosarcomas versus multilobular tumors of bone and in osteosarcomas of the appendicular skeleton versus those of the axial skeleton suggests that alterations in p53 expression correlate with highly aggressive tumor behavior.

Abnormalities of the p53 oncogene in lung cancer have recently been reported to be more frequent in small cell lung cancer (SCLC) than in non-small cell lung cancer (non-SCLC), but their status in combined type SCLC is as yet unknown. In this study, immunohistochemical analysis using a polyclonal antibody against p53 protein was performed in 12 surgically resected specimens of combined type SCLC. Immunoreactivity of the p53 protein was found in 5 (42%) of the 12 cases, and the immunostaining pattern of the p53 protein in areas of the non-small cell carcinoma type was the same as in those of the small cell carcinoma type. Thus, it seems that the incidence of p53 abnormalities in combined type SCLC is slightly lower than in ordinary type SCLC. It is also suggested that abnormalities of the p53 oncogene in this histological type may not be a specific event related to the morphological difference between small cell carcinoma and non-small cell carcinoma.

Non-small-cell lung cancer (NSCLC) prognosis is strictly related to well-established clinicopathological parameters which have unfortunately become insufficient in the prognostic evaluation of this type of cancer. As p53 and bcl-2 gene deregulations are frequently involved in several types of epithelial malignancies, we investigated the Bcl-2 and p53 protein expression in 91 and 101 cases of NSCLC respectively. The expression was then compared with established indicators of prognosis and biological behaviour of the tumours. No relationship was observed between Bcl-2 and either clinicopathological or biological parameters such as histology, grading, tumour status, nodal metastasis and proliferative activity evaluated by scoring proliferating cell nuclear antigen expression and Ki-67 immunoreactivity. However, the mean Bcl-2 expression was significantly lower in patients who developed metastasis during follow-up or died of metastatic disease (P = 0.006 and P = 0.01 respectively). Moreover, survival probability was higher in patients who expressed the Bcl-2 protein (P = 0.0002). In contrast with this, p53 protein accumulation was observed in tumours with metastatic nodal involvement (P = 0.02) or in patients who developed metastasis during follow-up (P = 0.01), although no correlation was found between p53 expression and overall survival. An inverse relationship was also found between Bcl-2 and the anti-oncogene protein product p53 (P = 0.01). Thus, a high proportion of NSCLCs express p53 and Bcl-2 proteins and their expression may have prognostic importance.