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.

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.

Alterations in genomic content and changes in gene expression levels are central characteristics of tumors and pivotal to the tumorigenic process. We analyzed 23 non-small cell lung cancer (NSCLC) tumors by array comparative genomic hybridization (array CGH). Aberrant regions identified included well-characterized chromosomal aberrations such as amplifications of 3q and 8q and deletions of 3p21.31. Less frequently identified aberrations such as amplifications of 7q22.3-31.31 and 12p11.23-13.2, and previously unidentified aberrations such as deletion of 11q12.3-13.3 were also detected. To enhance our ability to identify key acting genes residing in these regions, we combined array CGH results with gene expression profiling performed on the same tumor samples. We identified a set of genes with concordant changes in DNA copy number and expression levels, i.e. overexpressed genes located in amplified regions and underexpressed genes located in deleted regions. This set included members of the Wnt/beta-catenin pathway, genes involved in DNA replication, and matrix metalloproteases (MMPs). Functional enrichment analysis of the genes both overexpressed and amplified revealed a significant enrichment for DNA replication and repair, and extracellular matrix component gene ontology annotations. We verified the changes in expressions of MCM2, MCM6, RUVBL1, MMP1, MMP12 by real-time quantitative PCR. Our results provide a high resolution map of copy number changes in non-small cell lung cancer. The joint analysis of array CGH and gene expression analysis highlights genes with concordant changes in expression and copy number that may be critical to lung cancer development and progression.

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).

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.(Cancer Res 2006; 66(24): 11683-9).

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/mug protein, with a mean value of 7.2units/mug 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.

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.

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.

We report a case where recurrent "pneumonia" was eventually diagnosed as lipoid pneumonia in an elderly patient with cerebrovascular disease. The discontinuation of paraffin oil laxative led to clinical improvement. Lipoid pneumonia, a foreign body-type reaction to the presence of lipid within lung parenchyma, is probably underdiagnosed and underreported, and paraffin oil laxative is the main causative agent. Paraffin oil is marketed as a food additive, and no information about its hazards is provided to clinicians or patients. We suggest that a change in paraffin oil licensing may decrease the incidence of lipoid pneumonia.