Genetic alterations of lung adenocarcinoma in relation to smoking and ethnicity

Adenocarcinoma of the lung is now the most common histologic subtype of non-small-cell lung cancer (NSCLC) worldwide. In Chinese populations, the incidence of lung adenocarcinoma is amongst the highest worldwide and its development in non-smoking females is particularly striking. Information on the associated underlying genetic changes has been, however, minimal to date. The present study represents the first systematic analysis on the overall genetic changes in lung adenocarcinoma of Chinese female non-smokers. We undertook a genome-wide investigation into the abnormalities in lung adenocarcinomas of 18 life-long non-smoking Chinese females using the technique of comparative genomic hybridization (CGH). With a view to isolating the relative roles of gender, ethnicity and tobacco consumption, we recruited control groups of 10 Caucasian female non-smokers and 22 male Chinese smokers. Although gains on 1q, 5p, 7p and 8q, and regional losses on 8p, 17p, 13q and 18q were commonly seen, there were no significant differences between the Caucasian and Chinese non-smoking women. The observation suggests that lung adenocarcinomas, regardless of ethnic origin, adopt similar pathologic pathways during the accumulation of genetic events. Besides, genomic imbalances, particularly gains per tumor, were significantly more common among the tobacco-related tumors (P=0.006). In particular, regional over-representations of 13q21-q34 (P=0.044), 17q25 (P=0.015), 19q13.1 (P=0.044) and 22q (P=0.044) may have implications for the pathologic pathways associated with the tobacco-related lung adenocarcinoma.     

Evaluation of prognostic-significance of p53 gene alterations in patients with surgically resected lung-cancer

Clinical significance of p53 gene alterations, as a prognostic factor, was assessed in 69 patients with surgically resected lung cancer. The p53 gene alterations (exon 5-9) were examined by the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method of genomic DNA. The p53 gene alterations were detected in all histological types of lung cancer, with a positive rate of 45% (31/69). In the alteration-positive group, patients in the advanced stages of III and IV were seen more frequently than in the negative group (58% vs. 21%, p<0.05). Such a difference was not observed in other parameters such as age, gender, histological type and smoking habit. The prognosis was, on a whole, poorer in the alteration-positive group than for the -negative one (5-year survival rate: 19.3% vs. 40.6%, MST: 17 months vs. 36 months), but the difference did not reach statistical significance. However, in the case of females (p<0.05), adenocarcinoma (p<0.01), early stages of I and II (p<0.05) and non-smokers (p<0.005), a significantly poorer prognosis was observed in the gene alteration-positive group than for the -negative one. These results suggest that the p53 gene alteration may be a useful prognostic factor in certain subgroups with lung resected for cancer.     

Levels of p53 antigen in the serum of nonsmall cell lung-cancer patients correlate with positive p53 immunohistochemistry on tumor sections, tumor necrosis and nodal involvement

We examined 50 non-small cell lung cancers and the matching serum from the same patients for the levels of p53 antigen. Histological sections of the tumor specimens were stained with anti-p53 specific antibodies, and the sera were tested by ELISA. We observed a correlation between the expression of the p53 protein in the tumors and the presence of circulating p53 antigen, using either a wild type-(P=0.024) or a mutant specific (P=0.007) ELISA test. The p53 serum levels were also significantly associated (P=0.019) with the extent of tumor necrosis, suggesting that circulating p53 may be derived by shedding from dead tumor cells. The levels of mutant p53 detected in the serum were significantly higher in patients with lymph node involvement (P=0.012) and with late-staged disease. (P=0.05). No association was found with tumor size and extent. Testing for serum levels of mutant p53 could provide additional prognostic information and could be used as as novel molecular diagnostic tool in the management of non-small cell lung cancer.     

Microsatellite Instability and k-ras, p53 Mutations in Thyroid Lymphoma

Patho-epidemiological studies showed that thyroid lymphoma (TL) arises in inflammatory lesions of chronic lymphocytic thyroiditis (CLTH). Replication error (RER) is found in inflammatory lesions and associated cancer, suggesting that chronic inflammation could be a risk factor for neoplastic development through causing RER. To clarify whether RER is involved in the pathogenesis of TL, we examined the microsatellite instability (MSI) in 9 cases with CLTH and 19 with TL, including 10 diffuse large B-cell lymphoma (DLBL), 4 follicle center cell lymphoma, 3 marginal zone B-cell lymphoma of extranodal (MALT) type, and 2 lymphoplasmacytic type. Sixteen distinct microsatellite repeats were analyzed. Mutations of p53 and k- ras genes were also examined. When alterations at 2 or more microsatellite loci were judged as positive, only 5 DLBL cases exhibited MSI. The frequency of MSI in DLBL was significantly higher than that in other types of TL and CLTH ( P < 0.05). Four of 19 cases (21.1%) showed point mutation of the k- ras gene. The k- ras mutations occurred in the cases with DLBL with RER, and four of five cases with RER had a k- ras mutation, indicating a close association between RER and k- ras mutation. p53 mutations were not found in the CLTH. Two of 19 TL cases showed mutations of p53 gene. There was no significant association between RER and p53 mutation. These findings indicate that genomic instability contributes to the progression of TL from low grade to high grade, but not to the development of low grade lymphoma in CLTH lesions.     

Alterations of p53 and k-ras genes in human colorectal-cancer with ulcerative-colitis

Using polymerase chain reaction and single strand conformation polymorphism assay, we examined genetic alterations of p53 and K-ras genes in multiple lesions of colorectal cancers that developed in three patients with ulcerative colitis. p53 gene mutations were found in two lesions and a K-ms gene mutation at codon 12 was found only in one. Neither was found in non-cancerous lesions. The results indicate that mutation of both genes may not be so frequent among Japanese patients. They were found only in very few cancerous lesions of each patient. Therefore, it is possible that the oncogenic process in each lesion is a consequence of rather independent events. Our preliminary results suggest the potential usefulness of CD44 variant forms as markers for dysplastic and cancerous tissues in ulcerative colitis.     

Microsatellite instability and k-ras, p53 mutations in thyroid lymphoma.

Patho-epidemiological studies showed that thyroid lymphoma (TL) arises in inflammatory lesions of chronic lymphocytic thyroiditis (CLTH). Replication error (RER) is found in inflammatory lesions and associated cancer, suggesting that chronic inflammation could be a risk factor for neoplastic development through causing RER. To clarify whether RER is involved in the pathogenesis of TL, we examined the microsatellite instability (MSI) in 9 cases with CLTH and 19 with TL, including 10 diffuse large B-cell lymphoma (DLBL), 4 follicle center cell lymphoma, 3 marginal zone B-cell lymphoma of extranodal (MALT) type, and 2 lymphoplasmacytic type. Sixteen distinct microsatellite repeats were analyzed. Mutations of p53 and k-ras genes were also examined. When alterations at 2 or more microsatellite loci were judged as positive, only 5 DLBL cases exhibited MSI. The frequency of MSI in DLBL was significantly higher than that in other types of TL and CLTH (P < 0.05). Four of 19 cases (21.1%) showed point mutation of the k-ras gene. The k-ras mutations occurred in the cases with DLBL with RER, and four of five cases with RER had a k-ras mutation, indicating a close association between RER and k-ras mutation. p53 mutations were not found in the CLTH. Two of 19 TL cases showed mutations of p53 gene. There was no significant association between RER and p53 mutation. These findings indicate that genomic instability contributes to the progression of TL from low grade to high grade, but not to the development of low grade lymphoma in CLTH lesions.     

Ras and p53 expression in non-small-cell lung-cancer patients - p53 over-expression correlates with a poor prognosis

Expression of the tumor suppressor gene p53 and the ras oncogene were examined in 46 tumor and nodal specimens of non-small cell lung cancer (NSCLC) using the antibodies p53 pAb 240 and ras Y13-259 respectively. p53 expression was elevated in 46% and ras p21 was over-expressed in 85% of the tumor specimens analyzed. Fifteen cases of benign lessions were also assessed for both ras p21 and p53 expression; all were found to have negative staining. p53 over-expression was found to correlate with a poor prognosis in both the tumor specimens (p<0.05) and in the nodal tissues (p<0.005). Ras p21 over-expression was found to be associated with survival (p<0.1) in both the tumor and the nodal specimens. Stage of the disease correlated with survival; similarly both p53 and ras p21 over-expression correlated with stage. No correlations were found with the pathological grade of the tumors nor with a history of smoking or duration of smoking. No K-ras mutations at codon 12 were observed in a further 15 NSCLC specimens analyzed. These results indicate that the p53 gene in particular plays a role in the stages of NSCLC.     

Genetic alterations of the p53 gene are a feature of malignant mesotheliomas

A putative tumor suppressor gene, p53, has been shown to be altered in a variety of human tumor types. The primary mechanism of p53 inactivation is believed to be mutation of one allele followed by loss of the second allele. Malignant mesothelioma is a tumor that has been highly associated with exposure to asbestos fibers, which are known to cause chromosomal abnormalities in mesothelial cells. We have examined four mesothelioma cell lines for genetic abnormalities in p53. Cytogenetic analysis revealed that two of the four tumors had abnormalities (numerical and/or structural) of chromosome 17 (the locus of the p53 gene). Restriction fragment length polymorphism analysis using a chromosome 17p-specific probe (pYNZ22) revealed that two tumors had loss of heterozygosity in the region of 17p13. The relative level of p53 mRNA expression was examined by Northern analysis, with one tumor showing negligible expression of p53 mRNA. The complementary DNA of p53 was generated from the three tumors showing detectable mRNA expression, and the region between codons 70 and 319 was amplified by the polymerase chain reaction and sequenced. DNA single-base substitutions were detected in two of the tumor cell lines, each resulting in amino acid substitutions. One tumor had an arginine to histidine substitution at position 175, and one tumor had a glycine to aspartic acid substitution at position 245. The observed mutations took place in regions of high cross-species sequence homology, indicating that these regions may be functionally important. The correlation of chromosomal loss in 17p on the cytogenetic and molecular level along with p53 mRNA expression and DNA sequence data indicate that genetic alterations in p53 could be a feature of malignant mesotheliomas and may reveal an important role of asbestos fibers in tumor suppressor gene inactivation.     

ING1 and p53 tumor suppressor gene alterations in adenocarcinomas of the esophagogastric junction

The aim of this study was to characterize molecular alterations of the recently reported candidate tumor suppressor gene, ING1, and to explore the relationship between ING1 and p53 in a well-defined series of adenocarcinomas of the esophagogastric junction (AdEGJ). Polymerase chain reaction (PCR)-based assays were used to characterize ING1 and p53 alterations, relative to histologically normal esophageal mucosa. Two tumors were found to have ING1 mutations: one novel missense mutation (AGC(Ser)-->ATC(Ile)) at codon 147, and one silent mutation (TCG(Ser)-->TCA(Ser)) at codon 173. Reduced expression of the two major alternatively spliced ING1 messenger RNA variants, p47(ING1a) and p33(ING1b) was variable, but was reduced (1.2-10-fold) in 12 of 19 AdEGJs compared to normal esophageal epithelium. No association between p53 and ING1 alterations was apparent. We conclude that reduced ING1 expression is frequently associated with AdEGJ tumorigenesis, further supporting its role as a tumor suppressor gene, and that ING1 expression is independent of p53 status.     

The prognostic significance of p53 tumor suppressor gene alterations in ovarian carcinoma

BACKGROUND: The prognostic significance and nature of p53 dysfunction in ovarian carcinoma is unclear. The relation between p53 overexpression, p53 mutations, and their effects on overall survival in primary ovarian carcinoma is explored. METHODS: Tumor specimens from 171 consecutive epithelial ovarian carcinomas were examined for overexpression of p53 protein with DO7 antibody. P53 mutations were determined by direct sequencing. The influences of conventional histopathologic prognostic factors and various p53 molecular alterations on overall survival were assessed. RESULTS: Overall, 48.5% and 57.3% of the samples showed p53 overexpression and p53 mutation, respectively. Although neither p53 overexpression nor the mere presence of a p53 mutation impacted overall survival, the combination did prognosticate survival both in univariate and multivariate models. The authors' results suggest 4 mechanisms that may affect p53 dysfunction in nearly 100% of advanced stage ovarian carcinomas. These include null mutation, nonresponsive p53 (wild-type [wt] p53 sequence, DO7 negative), sequestration (wt p53 sequence, DO7 positive), and missense mutation. Median survival for these groups that constitute sequentially 21.3%, 20.5%, 12.3%, and 45.9% of the 122 Stage III or IV (International Federation of Gynecology and Obstetrics) cancers was 1.49, 1.31, 3.09, and 3.6 years, respectively. The nonresponsive p53 and null sequence tumors grouped together as functionally null convey the worst prognosis relative to missense mutations in a univariate model (P = 0.006). Functionally null p53 (P = 0.002), stage (P = 0.008), and optimal cytoreduction (P = 0.008) were independent prognostic factors by multivariate analysis. CONCLUSIONS: Sequestration of wt p53 is unique to advanced stage ovarian carcinoma. Functionally null p53 represents an independent molecular predictor of compromised survival.     

Genetic interactions between the Wilms' tumor 1 gene and the p53 gene

In recent years, a number of proteins have been identified that can modify the activities of the Wilms' Tumor 1 (WT1) proteins. One of these modifiers is the p53 protein. To investigate a genetic interaction between the p53 gene and the wt1 gene, we have crossed their respective knockout mice. The absence of p53 appears to have no gross effect on the phenotype of wt1-null mice. Both wt1-null and double-null embryos develop pericardial bleeding and die in utero. In adult p53-null mice, wt1-heterozygosity (wt1het) predisposes to an earlier onset of lymphomagenesis and the development of kidney abnormalities resembling oncocytoma in humans. wt1-heterozygosity alone predisposes to the development of glomerular sclerosis.     

Genetic background determines the response to adenovirus-mediated wild-type p53 expression in pancreatic tumor cells.

The development of new therapies is particularly urgent with regard to pancreatic tumors. Gene therapy approaches involving p53 replacement are promising due to the central role of p53 in the cellular response to DNA damage and the high incidence of p53 mutations in pancreatic tumors. Adenoviruses containing wild-type (wt) p53 cDNA (Ad5CMV-p53) were introduced into four human pancreatic cell lines to examine the impact caused by exogenous wt p53 on these cells. Introduction of wt p53 in mutant p53 cells (NP-9, NP-18, and NP-31) caused marked falls in cell proliferation and rises in the level of apoptosis. In contrast, overexpression of p53 did not induce apoptosis in NP-29 (wt p53). The presence of p16 contributes to the induction of apoptosis, as demonstrated by introduction of the wt p16 gene (Ad5RSV-p16). Analysis of cell cycle and apoptosis in etoposide-treated cells corroborated the inability of NP-29 to die by apoptosis, suggesting that this wt p53 cell line lacks p53 downstream functions in the apoptosis pathway. Taken together, our results indicate that the effects elicited by exogenous p53 protein depend upon the molecular alterations related to p53 actions on cell cycle and apoptosis. Therefore, knowledge of the genetic background of tumor cells is crucial to the development of efficient therapies based on the introduction of tumor suppressor genes.     

Cigarette smoking and other risk factors in relation to p53 expression in breast cancer among young women.

p53 mutations may be a fingerprint for cigarette smoking and other environmental carcinogens, including breast carcinogens. This study was undertaken to explore whether p53 mutations are associated with environmental or other suspected or established risk factors for breast cancer. p53 protein detection by immunohistochemistry (which is more easily quantified in large epidemiological studies than are mutations, and are highly correlated with them) was determined for 378 patients from a case-control study of breast cancer. In this population-based sample of women under the age of 45 years, 44.4% (168/378) of the cases had p53 protein detected by immunohistochemistry (p53+). Polytomous logistic regression was used to calculate the odds ratios (ORs) for p53+ and p53- breast cancer, as compared with the controls, in relation to cigarette smoking and other factors. The ratio of the ORs was used as an indicator of heterogeneity in risk for p53+ versus p53- cancer. The ratio of the ORs in a multivariate model was substantially elevated among women with a greater than high school education [2.39; 95% confidence interval (CI), 1.43-4.00], current cigarette smokers (1.96; 95% CI, 1.10-3.52), and users of electric blankets, water beds, or mattresses (1.78; 95% CI, 1.11-2.86). Nonsignificant heterogeneity was noted for family history of breast cancer and ethnicity but not for other known or suspected risk factors. Coupled with the strong biological plausibility of the association, our data support the hypothesis that in breast cancer, as with other tumors, p53 protein immunohistochemical detection may be associated with exposure to environmental carcinogens such as cigarette smoking.     

Detection of hepatitis-B virus-DNA and mutations in k-ras and p53 genes in human hepatocellular carcinomas

Hepatitis B virus (HBV) infection is considered as one of the major risk factors in the development of human hepatocellular carcinoma (HCC). Recent studies have also suggested the implication of oncogene and onco-suppressor genes in liver carcinogenesis. We studied 41 cases of HCC for the presence of HBV DNA and point mutations in codon 12 of K-ras and codon 249 of p53. We used 'nested' PCR for the amplification of HBV because of the expected low incidence of the virus DNA in the samples. PCR was also used for the amplification of K-ras and p53 regions that contain the codons of interest, followed by RFLP analysis for the detection of point mutations. HBV DNA was amplified in 22 cases (53.7%), while 5 cases (12.2%) appeared to carry mutations in codon 12 of K-ras and 7 cases (17.1%) had mutations in codon 249 of the p53 gene. These results further support the correlation between HBV infection and HCC and also indicate an implication of K-ras and p53 genes in hepatocarcinogenesis.     

Patterns of EGFR, HER2, TP53, and KRAS mutations of p14arf expression in non-small cell lung cancers in relation to smoking history

Mutations in the tyrosine kinase domain of the epidermal growth factor receptor EGFR are common in non-small cell lung cancer (NSCLC) of never smokers, whereas HER2 mutations are rare. We have analyzed EGFR and HER2 mutations and the expression of the two products of the CDKN2A gene (p14(arf) and p16(INK4a)) in 116 NSCLC that have been previously analyzed for TP53 and KRAS mutations in relation to smoking history of patients. EGFR mutations were detected in 20 of 116 (17%) tumors, whereas five (4.3%) tumors contained HER2 mutations. No tumor contained both mutations. Of tumors with EGFR or HER2 mutation, 72% were adenocarcinomas, 68% were from never smokers, and 32% were from former smokers. EGFR but not HER2 mutations were mutually exclusive with KRAS mutation. Among never smokers, 11 of 16 tumors with EGFR mutation also had TP53 mutation, in contrast with two of 17 tumors without EGFR mutation (P = 0.0008). Expression of p14(arf), but not p16(ink4a), was more frequently down-regulated in never smokers (62.5%) than ever smokers (35%; P = 0.008). All tumors with EGFR or HER2 mutations and wild-type TP53 showed down-regulation of p14(arf) expression. These observations suggest that functional inactivation of the p14(arf)/p53 connection is required in tumors with EGFR or HER2 mutations, consistent with the notion that these proteins are part of a fail-safe mechanism protecting cells against untimely or excessive mitotic signals.     

Interleukin-8 in non-small cell lung carcinoma: relation with angiogenic pattern and p53 alterations

Progression of solid tumors, including NSCLC, is associated with increase in MVC (microvessel count), as a measure of tumor angiogenesis resulting from an imbalance between angiogenic factors and inhibitors. However, since tumor angiogenesis is a multi-step process under the control of various molecules, the mechanism of angiogenesis has not been fully clarified. Interleukin (IL)-8 has been shown to have a potential angiogenic effect in vitro and in vivo, and is overexpressed in several human solid cancers. Among the various angiogenic factors, vascular endothelial growth factor (VEGF) has been shown to correlate with a high MVC and with adverse prognosis in several human cancers, including NSCLC. Alterations of p53 suppressor gene are the most common genetic changes found in malignant tumors; several studies examined the link between aberrant p53 and angiogenesis in lung cancer, but only a few studies report data regarding a relation between p53 mutations and IL-8 expression. In this study we observed a correlation between IL-8 mRNA expression, intratumoral MVC and VEGF mRNA expression levels; furthermore, an aberrant p53 status was related to IL-8 expression. However, in our samples IL-8 levels did not significantly affect prognosis of NSCLC; more studies are required to elucidate the precise role of IL-8 in a large series of patients with non-small cell lung carcinoma.     

Meta-analyses of p53 tumor suppressor gene alterations and clinicopathological features in resected lung cancers.

p53 alterations are the most common genetic lesions observed in lung cancers. Because of the limited size of individual studies, the distributions of p53 alterations by clinicopathological features have not been well characterized. Here, we present meta-analyses describing the occurrence of p53 alterations by patient/tumor characteristics in resected lung cancer. The association between p53 alterations (gene and/or protein) and a variety of variables were evaluated by calculating pooled odds ratios (ORs) and confidence intervals (CIs). p53 alterations were detected in 46.8% of 4684 non-small cell lung cancers. p53 alterations occurred more frequently in the more strongly smoking-associated histotypes: squamous cell (51.2%) and large cell (53.7%) carcinomas versus adenocarcinomas [38.8%; OR (squamous versus adenocarcinoma) = 1.81, 95% CI = 1.55-2.11]. p53 alterations were found to be associated with T1-4, N0-3, stage I-III, differentiation, and sex: OR (T3 versus T1) = 1.62 (95% CI = 0.99-2.65), OR (N1-3 versus N0) = 1.65 (95% CI = 1.27-2.15), OR (stage III versus stage I) = 1.98 (95% CI = 1.35-2.89), OR (poorly and moderately versus well-differentiated) = 3.04 (95% CI = 1.56-5.94), and OR (male versus female) = 1.39 (95% CI = 1.10-1.75). No strong associations between p53 and ras or aneuploidy were observed. Lung cancer studies of p53 and smoking need to consider the effect of histotype, and prognostic studies of p53 should adjust for the effects of T and N or stage and histotype. The apparent association between p53 and sex may be confounded by histotype and must be evaluated by multivariate studies.     

p53 over-expression and p53 mutations in colon carcinomas: relation to dietary risk factors.

Epidemiological studies have suggested that dietary factors may differently affect p53-dependent and p53-independent pathways to colon cancer. Results of such studies may depend on the method used to assess p53 status. This case-control study of 185 colon-cancer cases and 259 controls examines this relation, using both immunohistochemistry and SSCP(exons 5-8)/sequencing to detect p53 abnormalities. Of 185 carcinomas analyzed using immunohistochemistry, 81 (44%) were categorized as p53 over-expression. p53 mutations were detected in 59 tumors (32%). A slight increase in risk observed for intake of saturated fat was largely due to an increased risk in cases without p53 over-expression (OR per 16.1 g/day, 1.46; 95% CI, 1.08-1.97), and no association in cases with p53 over-expression (OR, 1.07, 95% CI, 0.78-1.47). However, findings were less pronounced when cases were classified by mutation analysis (wild-type OR, 1.33; 95% CI, 1.01-1.75; mutated OR, 1.16; 95% CI, 0.81-1.65). Similar results were observed for total fat intake. For other nutrients and for vegetable and meat food groups no differences in risk for either p53 pathway were observed, independent of the laboratory technique used. Interestingly, in cases with transversion mutations in the p53 gene, an increased risk was observed for saturated fat (OR, 2.00; 95% CI, 0.97-4.14), in contrast to those with mutations at CpG sites (OR, 0.93; 95% CI, 0.55-1.57). An increase in colon-cancer risk for the p53-independent pathway due to fat intake, is more pronounced when using immunohistochemistry. However, mutation analysis is needed to study the possible association with a small group of tumors with transversion mutations.