The expression of three genes in primary non-small cell lung cancer is associated with metastatic spread to the brain.

PURPOSE: Brain metastases affect 25% of patients with non-small cell lung cancer (NSCLC). We hypothesized that the expression of genes in primary NSCLC tumors could predict brain metastasis and be used for identification of high-risk patients, who may benefit from prophylactic therapy. EXPERIMENTAL DESIGN: The expression of 12 genes was measured by real-time quantitative reverse transcriptase PCR in 142 frozen NSCLC tissue samples. Univariate and multivariate Cox regression analysis was used to analyze the correlation between gene expression and the occurrence of brain metastasis. Immunohistochemistry on independent samples was used to verify the findings. RESULTS: A score based on the expression levels of three genes, CDH2 (N-cadherin), KIFC1, and FALZ, was highly predictive of brain metastasis in early and advanced lung cancer. The probability of remaining brain metastasis-free at 2 years after diagnosis was 90.0+/-9.5% for patients with stage I/stage II tumors and low score compared with 62.7+/-12% for patients with high score (P<0.01). In patients with more advanced lung cancer, the brain metastasis-free survival at 24 months was 89% for patients with low score compared with only 37% in patients with high score (P<0.02). These results were confirmed by immunohistochemical detection of N-cadherin in independent cohort of primary NSCLC. CONCLUSIONS: The expression levels of three genes in primary NSCLC tumors may be used to identify patients at high risk for brain metastasis who may benefit from prophylactic therapy to the central nervous system.

Development of an enzymatic DNA repair assay for molecular epidemiology studies: distribution of OGG activity in healthy individuals.

While the role of reduced DNA repair in susceptibility to hereditary cancers is well established, its role in sporadic cancer is less understood. One of the reasons is the lack of specific DNA repair assays that are suitable for epidemiology studies. Here we describe the development of the OGG test, an epidemiology-grade enzymatic assay for the activity of the base excision repair enzyme 8-oxoguanine DNA glycosylase, in protein extracts prepared from human blood cells. The assay is robust and reproducible, with a coefficient of variation of 10%. Using the OGG test we determined OGG activity in 120 healthy individuals. Our results show an inter-individual variation of 2.8-fold in OGG activity, from 3.6 up to 10.1units/microg protein, with a mean value of 7.2units/microg protein. There was no significant difference in OGG activity between males and females, or between smokers and non-smokers. Interestingly, there was a gender-specific effect of age: OGG activity was slightly but significantly lower in males older than the age of 55 years compared to younger males, but not in females at the same age groups. Analysis of OGG1 mRNA by quantitative real-time RT-PCR showed a group trend of an increase in OGG enzymatic activity with increasing mRNA expression, but the correlation between activity and mRNA in individuals was poor, indicating the importance of factors other than mRNA expression. The OGG test described is expected to be useful in studying the role of 8-oxoguanine repair in cancer, as recently demonstrated for non-small cell lung cancer [T. Paz-Elizur, M. Krupsky, S. Blumenstein, D. Elinger, E. Schechtman, Z. Livneh, J. Natl. Cancer Inst. 95 (2003) 1312-1319]. In addition, it may serve as a paradigm for the development of additional functional DNA repair tests, which are needed in order to gain further insight into the role of DNA repair in cancer risk and pathology.

Reduced repair of the oxidative 8-oxoguanine DNA damage and risk of head and neck cancer.

An increasing number of studies indicate that reduced DNA-repair capacity is associated with increased cancer risk. Using a functional assay for the removal of the oxidative DNA lesion 8-oxoguanine by the DNA-repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), we have previously shown that reduced OGG activity is a risk factor in lung cancer. Here, we report that OGG activity in peripheral blood mononuclear cells from 37 cases with squamous cell carcinoma of the head and neck (SCCHN) was significantly lower than in 93 control subjects, frequency matched for age and gender. Retesting of OGG activity 3 to 4 years after diagnosis and successful treatment of 18 individuals who recovered from the disease showed that OGG activity values were similar to those determined at diagnosis, suggesting that reduced OGG activity in case patients was not caused by the disease. Logistic regression analysis indicated that the adjusted odds ratio (OR) associated with a unit decrease in OGG activity was statistically significantly increased [OR, 2.3; 95% confidence interval (95% CI), 1.5-3.4]. Individuals in the lowest tertile of OGG activity exhibited an increased risk of SCCHN with an OR of 7.0 (95% CI, 2.0-24.5). The combination of smoking and low OGG was associated with a highly increased estimated relative risk for SCCHN. These results suggest that low OGG is associated with the risk of SCCHN, and if confirmed by additional epidemiologic studies, screening of smokers for low OGG activity might be used as a strategy for the prevention of lung cancer and SCCHN.

Lipoid pneumonia: a preventable complication.

BACKGROUND: Lipoid pneumonia is a pneumonitis resulting from the aspiration of lipids, and is commonly associated with the use of mineral oil as a laxative. LP is relatively unfamiliar to clinicians and is probably underdiagnosed. OBJECTIVES: To increase physicians' awareness of LP, its diagnosis and prevention. METHODS: We present two illustrative cases of LP and review the literature. RESULTS: Two cases of LP were diagnosed within half a year in an internal medicine ward. Both cases were elderly patients, and LP was associated with the use of mineral oil as a laxative agent. Computerized tomography revealed bilateral low attenuation infiltrates, associated with a "crazy paving" pattern in one case. Sudan Black staining was diagnostic in both cases--in one on a transbronchial biopsy specimen, and in the other on sputum cytologic examination. Both patients suffered from neurologic diseases and were at risk of aspiration. In both cases clinical symptoms and signs continued for several months prior to diagnosis but resolved after the mineral oil was discontinued. CONCLUSIONS: LP often occurs in elderly patients who are at risk of aspiration. The condition may be underdiagnosed. Since in most cases mineral oil cathartics are the causative agent, an effort at primary prevention is indicated. It is suggested that the licensing of mineral oil for internal use be changed.

Genetic analyses of non-small cell lung cancer in Jewish Israeli patients.

BACKGROUND: The contribution of the abnormal DNA mismatch repair system to non-small cell lung cancer tumorigenesis is controversial and has not been reported in Jewish Israeli patients. Similarly, the involvement of 3p deletions in NSCLC in the same population has not been assessed. OBJECTIVES: To assess the contribution of the DNA-MMR system to NSCLC pathogenesis by analyzing microsatellite instability, and evaluate loss of heterozygosity at 3p rates in Israeli NSCLC patients. METHODS: Paired DNA from tumorous and non-tumorous tissue was extracted, and genotyping for MSI determination was carded out using the five Bethesda markers and for determining LOH two 3p markers were used. Genotyping was performed using polymerase chain reaction amplification and size separation on an ABI semiautomatic DNA sequencer, and the allelic patterns of tumorous and non-tumorous tissue were compared. RESULTS: Forty-four NSCLCs from 35 smokers and 9 non-smokers were analyzed, with 26 of the 44 (59%) at stage I disease. Using five microsatellite markers (D17S250, D5S346, D2S123, BAT-25, BAT-26) (known as Bethesda markers) for MSI determination, 6 of the 44 tumors (13.6%) exhibited MSI in at least one marker. Similarly, genotyping for LOH at chromosome 3p was performed using two markers (D3S4103, D3S1234) located at 3p14.2 I. With D3S4103, 33 of the 44 patients successfully analyzed were homozygous and therefore non-informative with respect to LOH. Using D3S1234, 33 of 36 patients (91.7%) were heterozygous, and 23 of these individuals' tumors (69.7%) displayed LOH. Unexpectedly, 4 of 33 tumors (12.1%) genotyped by D3S4103, and 16 of 36 tumors (44.5%) genotyped by D3S1234 showed a pattern of MSI, even though only one of these tumors showed a similar pattern when genotyped with the five consensus markers. Overall, 23 of 44 tumors (52.3%) demonstrated MSI on at least one marker, and 5 of these 23 tumors (21.7%) had MSI on two or more markers. CONCLUSIONS: MSI using 3p markers and not the Bethesda markers occurs at a high rate and in early stages in Jewish NSCLC patients.

Sil overexpression in lung cancer characterizes tumors with increased mitotic activity.

Sil (SCL interrupting locus) was cloned from the most common chromosomal rearrangement in T-cell acute lymphoblastic leukemia. It is an immediate early gene whose expression is associated with cell proliferation. Sil protein levels are tightly regulated during the cell cycle, reaching peak levels in mitosis and disappearing on transition to G1. A recent study found Sil to be one of 17 genes whose overexpression in primary adenocarcinomas predicts metastatic spread. We hypothesized that Sil might have a role in carcinogenesis. To address this question, we utilized several approaches. Using a multitumor tissue array, we found that Sil protein expression was increased mostly in lung cancer, but also at lower levels, in a subset of other tumors. Microarray gene expression analysis and immunohistochemistry of lung cancer samples verified these observations. Sil gene expression in lung cancer correlated with the expression of several kinetochore check-point genes and with the histopathologic mitotic index. These observations suggest that overexpression of the Sil gene characterizes tumors with increased mitotic activity.

Craving to smoke in orthodox Jewish smokers who abstain on the Sabbath: a comparison to a baseline and a forced abstinence workday.

RATIONALE: Previous studies suggest that craving for cigarettes is substantially influenced by non-nicotine mechanisms such as habits, cues, and expectations. As orthodox Jews must refrain from smoking during the Sabbath, examining their craving levels during this habitual abstinence may be informative in separating smoking deprivation from other determinants of craving and withdrawal. OBJECTIVE: To examine the extent to which the habitual abstinence of Orthodox Jews during the Sabbath is associated with craving to smoke and with other reactions to smoking abstinence. METHODS: Twenty orthodox Jewish heavy smokers were assessed three times: on a workday when smoking as usual, on a Sabbath when they never smoke, and on a forced abstinence workday. Craving, irritability, and other commonly reported smoking withdrawal symptoms were assessed retrospectively at several time points during the preceding 24 h. RESULTS: Craving to smoke, and to a lesser extent, irritability, was lower during the Sabbath than during the two other test days. Self-reported difficulty in abstaining was also lower on the Sabbath than on the workday. Craving in the evening preceding the test day was always significantly higher than in the next morning, despite the overnight abstinence before the morning assessment. CONCLUSIONS: These results support previous findings in showing that craving to smoke is determined to a large extent by smoking-related habits, cues, and expectations.

Altered adenosine-to-inosine RNA editing in human cancer.

Adenosine-to-inosine (A-to-I) RNA editing was recently shown to be abundant in the human transcriptome, affecting thousands of genes. Employing a bioinformatic approach, we identified significant global hypoediting of Alu repetitive elements in brain, prostate, lung, kidney, and testis tumors. Experimental validation confirmed this finding, showing significantly reduced editing in Alu sequences within MED13 transcripts in brain tissues. Looking at editing of specific recoding and noncoding sites, including in cancer-related genes, a more complex picture emerged, with a gene-specific editing pattern in tumors vs. normal tissues. Additionally, we found reduced RNA levels of all three editing mediating enzymes, ADAR, ADARB1, and ADARB2, in brain tumors. The reduction of ADARB2 correlated with the grade of malignancy of glioblastoma multiforme, the most aggressive of brain tumors, displaying a 99% decrease in ADARB2 RNA levels. Consistently, overexpression of ADAR and ADARB1 in the U87 glioblastoma multiforme cell line resulted in decreased proliferation rate, suggesting that reduced A-to-I editing in brain tumors is involved in the pathogenesis of cancer. Altered epigenetic control was recently shown to play a central role in oncogenesis. We suggest that A-to-I RNA editing may serve as an additional epigenetic mechanism relevant to cancer development and progression.

Repair of the oxidative DNA damage 8-oxoguanine as a biomarker for lung cancer risk.

DNA repair has a major role in suppressing the rate of accumulation of mutations. Therefore, variations in DNA repair are likely to play an important role in determining cancer risk. While there is compelling evidence that defects in DNA repair cause high predisposition to several hereditary cancers, there is a paucity of data on the role of DNA repair in sporadic cancers. We present our approach of using functional DNA repair tests, rather than gene polymorphism, to study the potential of DNA repair enzymes to serve as biomarkers for lung cancer risk. We have previously developed a functional DNA repair blood test for the enzymatic repair of the oxidative DNA lesion 8-oxoguanine, and found that reduced OGG activity is a risk factor in non-small cell lung cancer. Moreover the combination of smoking and low OGG activity was associated with a greatly increased lung cancer risk (Paz-Elizur et al, JNCI 95 (2003) 1312-1319). The use of OGG activity as a potential biomarker for lung cancer risk is validated in collaboration with the M. D. Anderson Cancer Center, under the sponsorship of the Associate Members Program of the Early Detection Research Network (EDRN, NCI, NIH).

Comparative analysis of algorithms for signal quantitation from oligonucleotide microarrays.

MOTIVATION: Recent years' exponential increase in DNA microarrays experiments has motivated the development of many signal quantitation (SQ) algorithms. These algorithms perform various transformations on the actual measurements aimed to enable researchers to compare readings of different genes quantitatively within one experiment and across separate experiments. However, it is relatively unclear whether there is a 'best' algorithm to quantitate microarray data. The ability to compare and assess such algorithms is crucial for any downstream analysis. In this work, we suggest a methodology for comparing different signal quantitation algorithms for gene expression data. Our aim is to enable researchers to compare the effect of different SQ algorithms on the specific dataset they are dealing with. We combine two kinds of tests to assess the effect of an SQ algorithm in terms of signal to noise ratio. To assess noise, we exploit redundancy within the experimental dataset to test the variability of a given SQ algorithm output. For the effect of the SQ on the signal we evaluate the overabundance of differentially expressed genes using various statistical significance tests. RESULTS: We demonstrate our analysis approach with three SQ algorithms for oligonucleotide microarrays. We compare the results of using the dChip software and the RMAExpress software to the ones obtained by using the standard Affymetrix MAS5 on a dataset containing pairs of repeated hybridizations. Our analysis suggests that dChip is more robust and stable than the MAS5 tools for about 60% of the genes while RMAExpress is able to achieve an even greater improvement in terms of signal to noise, for more than 95% of the genes.

DNA repair activity for oxidative damage and risk of lung cancer.

BACKGROUND: Although smoking is a major cause of lung cancer, only a proportion of smokers develop lung cancer, suggesting a genetic predisposition in some individuals. Because tobacco smoking is associated with the increased formation of DNA lesions, including those induced from oxidative damage, we investigated whether the activity of the DNA repair enzyme 8-oxoguanine DNA N-glycosylase (OGG), which repairs the oxidative DNA lesion 8-oxoguanine, is associated with lung cancer. METHODS: We conducted a molecular epidemiologic case-control study that included 68 case patients with non-small-cell lung cancer and 68 healthy control subjects, frequency matched for age and sex. Enzymatic OGG activity was determined in protein extracts prepared from peripheral blood mononuclear cells or lung tissue by assaying the cleavage product of a radiolabeled synthetic DNA oligonucleotide containing an 8-oxoguanine residue. Odds ratios (ORs) and 95% confidence intervals (CIs) were determined by conditional logistic regression. All statistical tests were two-sided. RESULTS: OGG activity was lower in peripheral blood mononuclear cells from case patients than in those from control subjects. After adjustment for age and smoking status, individuals in the lowest tertile of OGG activity had an increased risk of non-small-cell lung cancer compared with individuals in the highest tertile (OR = 4.8, 95% CI = 1.5 to 15.9). The adjusted OR associated with a unit decrease in OGG activity was statistically significantly increased (OR = 1.9, 95% CI = 1.3 to 2.8). There was no interaction between OGG activity and smoking status. The estimated relative risk of lung cancer for smokers with low OGG activity was 34- or 124-fold higher for smokers with a low OGG activity of 6.0 or 4.0 U/ micro g protein, respectively, than for nonsmokers with a normal OGG activity of 7.0 U/ micro g protein, illustrating the cumulative effect of low OGG activity and smoking. CONCLUSIONS: Low OGG activity is associated with an increased risk of lung cancer. Although prospective studies are needed to validate the results, they suggest that smoking cessation in individuals with reduced OGG activity might be an effective strategy in lung cancer prevention.