Enrichment of a neutrophil-like monocyte transcriptional state in glioblastoma myeloid suppressor cells.

Glioblastomas (GBM) are lethal central nervous system cancers associated with tumor and systemic immunosuppression. Heterogeneous monocyte myeloid-derived suppressor cells (M-MDSC) are implicated in the altered immune response in GBM, but M-MDSC ontogeny and definitive phenotypic markers are unknown. Using single-cell transcriptomics, we revealed heterogeneity in blood M-MDSC from GBM subjects and an enrichment in a transcriptional state reminiscent of neutrophil-like monocytes (NeuMo), a newly described pathway of monopoiesis in mice. Human NeuMo gene expression and Neu-like deconvolution fraction algorithms were created to quantitate the enrichment of this transcriptional state in GBM subjects. NeuMo populations were also observed in M-MDSCs from lung and head and neck cancer subjects. Dexamethasone (DEX) and prednisone exposures increased the usage of Neu-like states, which were inversely associated with tumor purity and survival in isocitrate dehydrogenase wildtype (IDH WT) gliomas. Anti-inflammatory ZC3HA12/Regnase-1 transcripts were highly correlated with NeuMo expression in tumors and in blood M-MDSC from GBM, lung, and head and neck cancer subjects. Additional novel transcripts of immune-modulating proteins were identified. Collectively, these findings provide a framework for understanding the heterogeneity of M-MDSCs in GBM as cells with different clonal histories and may reshape approaches to study and therapeutically target these cells.

DNA methylation as a pharmacodynamic marker of glucocorticoid response and glioma survival.

Assessing individual responses to glucocorticoid drug therapies that compromise immune status and affect survival outcomes in neuro-oncology is a great challenge. Here we introduce a blood-based neutrophil dexamethasone methylation index (NDMI) that provides a measure of the epigenetic response of subjects to dexamethasone. This marker outperforms conventional approaches based on leukocyte composition as a marker of glucocorticoid response. The NDMI is associated with low CD4 T cells and the accumulation of monocytic myeloid-derived suppressor cells and also serves as prognostic factor in glioma survival. In a non-glioma population, the NDMI increases with a history of prednisone use. Therefore, it may also be informative in other conditions where glucocorticoids are employed. We conclude that DNA methylation remodeling within the peripheral immune compartment is a rich source of clinically relevant markers of glucocorticoid response.

KRAS mutation, KRAS-LCS6 polymorphism, and non-small cell lung cancer.

The let-7 family of microRNAs are important regulatory molecules in lung cancer. One downstream target of let-7 is the RAS gene family, including KRAS, an important oncogene in the etiology and clinical outcome of lung adenocarcinoma. Recently, a SNP in the let-7 binding region of the KRAS 3' UTR was identified (termed LCS6). This functional polymorphism alters let-7 binding, resulting in both increased KRAS expression and decreased let-7 exposure. Further, this SNP has been reported as a risk trait for lung cancer among low-moderate smokers. Given the functionality of LCS6, we tested the hypothesis that this SNP is associated with the occurrence of KRAS mutation as well as patient survival. Here, we report there is no association between the LCS6 KRAS polymorphism and KRAS mutation. Further, we find no association between the LCS6 polymorphism and lung cancer survival. These unexpected findings imply that this newly reported KRAS-LCS6 polymorphism will have limited clinical utility for NSCLC.

Differentially expressed genes are marked by histone 3 lysine 9 trimethylation in human cancer cells.

Histone H3 lysine 9 trimethylation (H3K9Me3) has been associated with transcriptional repression, but recent findings implicate this chromatin modification in transcriptional activation and mRNA elongation by RNA polymerase II. Here, we applied immunoprecipitation (IP) with a custom DNA tiling microarray containing many transcription factors important in development and cancer (for example homeotic genes; N=683 total genes) to explore the relationship between H3K9Me3 and other histone modifications with the differential expression of genes. Cancer cell lines derived from different tissues (2 leukemia, 2 medulloblastoma) were characterized with IP antibodies to H3K9Me3, H3K4 dimethylation (H3K4Me2) and H3K9 acetylation (H3K9Ac). MV4-11 is known to overexpress the HOXA9 and MEIS1 genes, whereas D283 overexpresses the OTX2 homeobox gene. Gene expression was assessed by Affymetrix U133 array. Mapping the number and size of histone markings demonstrated significant colocalization of H3K9Ac and H3K4Me2 with H3K9Me3, indicating a pattern of putative 'activating' and 'repressive' markings. The median site size was 600-821 bp and 72-95% or 53-80% of chromatin signal sites were located within 1 kb or 500 bp of transcription start sites (TSS), respectively. A relatively small number of genes displayed additional H3K9Me3 sites in the 5'-region distant from the TSS. Comparing genes with modification sites to those without sites in their promoters confirmed the positive associations of H3K9Ac and H3K4Me2 with gene expression and revealed that H3K9Me3 is associated with active genes rather than being a repressive marking as previously thought. The positive regulatory effect of all three types of modifications were quantitatively correlated with site size, and applied to absolute gene expression within a single cell line as well as relative expression among pairs of cell lines. Extended patterns of H3K9Me3 upstream of some genes (for example HOXA9 and OTX2) may result from the action of multiple promoter elements. We found an inverse relationship between promoter DNA hypermethylation and H3K9Me3 in three studied genes (HOXA9, TMS1, RASSF1A). The localization of H3K9Me3 downstream of the TSSs of expressed genes and not within promoter regions of hypermethylated and silenced genes is consistent with the proposed coupling of H3K9Me3 with RNA polymerase II. Our results indicate a need for revising aspects of the histone code involving H3 lysine methylation. Awareness of H3K9Me3 as a mark of gene activity, not repression, is especially important for the classification of human cancer using chromatin and histone profiles.

DNA adducts among asphalt paving workers.

OBJECTIVE: Asphalt is used extensively in the highway construction industry and contains a complex mixture of polycyclic aromatic hydrocarbons, some of which are known or suspected to be human carcinogens. Though numerous epidemiologic studies have described an excess cancer risk among asphalt workers, a causal relationship has not been established. Accordingly, the primary objective of this study was to use DNA adducts as a biomarker of biologically effective dose and determine whether DNA damage resulted from occupational exposure to asphalt among paving workers. METHODS: Over a 12 month period, four peripheral blood samples (spring, summer, fall and winter) were obtained from 49 asphalt paving workers (169 samples) and 36 non-paving construction workers (103 samples). The spring, summer and fall samples were collected during the work-season, whereas the winter samples were collected during the off-season (due to the seasonality of paving work). Mononuclear white blood cells were isolated and analyzed for DNA adducts via the (32)P-postlabeling assay and generalized linear models were used to evaluate the DNA adduct data. RESULTS: Among paving workers during the work-season, DNA adducts increased during each day of the workweek such that mean adduct levels were lowest on Mondays (3 adducts per 10(10) nucleotides) and highest on Fridays (46 adducts per 10(10) nucleotides). Additionally, a 3-fold difference in adduct burden was observed by paving task such that mean adduct levels were lowest among roller operators (7 adducts per 10(10) nucleotides) and highest among screedmen (23 adducts per 10(10) nucleotides). Using adducts as a measure of biologically effective dose, these findings (weekday trend and task-based differences) were consistent with a previous evaluation of absorbed dose in the same population. Adduct levels were not, however, higher among paving workers than among non-pavers. Adducts were also highest during the winter months, suggestive of a seasonal effect that has been observed in previous studies. CONCLUSION: These findings indicate that adduct burden increased throughout the workweek among paving workers, suggesting that DNA damage may be associated with occupational exposure to hot-mix asphalt. However, the lack of contrast with non-paving workers, as well as the seasonal variation warrants additional investigation.

Interaction between the bone morphogenetic proteins and Ras/MAP-kinase signalling pathways in lung cancer.

Bone morphogenetic proteins (BMPs) are an integral component of the TGFbeta superfamily, responsible for regulation of cell proliferation, differentiation, migration and programmed cell death in a variety of cell types. The BMPs transduce their signals directly through the SMAD family of proteins but they also have been reported to interact with the MAPK and Erk pathways. Inactivation of the BMP pathway genes has been implicated as important in several cancers. Recent work has shown that BMP3b is epigenetically inactivated in cancer and suggests that BMP6 can be epigenetically inactivated. We investigated whether BMP6 is epigenetically inactivated in cell lines and whether BMP3b and BMP6 are epigenetically inactivated in non-small-cell lung cancer (NSCLC). We also studied the relationship between BMP methylation and k-ras mutation. Here, we demonstrate that the BMP3b and BMP6 genes are common targets of epigenetic inactivation in NSCLC, and that they are significantly more likely to be concurrently inactivated (P=0.009). Furthermore, this coinactivation of BMP3b and BMP6 is significantly associated with mutation of k-ras codon 12 in lung cancer (P=0.003); those with a k-ras mutation were six times more likely to have concurrent methylation of these BMP loci. Hence, these data suggest that concurrent inactivation of the BMP and activation of the Ras signalling pathways are important in lung carcinogenesis.

RAS mutation is associated with hyperdiploidy and parental characteristics in pediatric acute lymphoblastic leukemia.

We explored the relationship of RAS gene mutations with epidemiologic and cytogenetic factors in a case series of children with leukemia. Diagnostic bone marrow samples from 191 incident leukemia cases from the Northern California Childhood Leukemia Study were typed for NRAS and KRAS codon 12 and 13 mutations. A total of 38 cases (20%) harbored RAS mutations. Among the 142 B-cell acute lymphoblastic leukemia (ALL) cases, RAS mutations were more common among Hispanic children (P=0.11) or children born to mothers <30 years (P=0.007). Those with hyperdiploidy at diagnosis (>50 chromosomes) had the highest rates of RAS mutation (P=0.02). A multivariable model confirmed the significant associations between RAS mutation and both maternal age and hyperdiploidy. Interestingly, smoking of the father in the 3 months prior to pregnancy was reported less frequently among hyperdiploid leukemia patients than among those without hyperdiploidy (P=0.02). The data suggest that RAS and high hyperdiploidy may be cooperative genetic events to produce the leukemia subtype; and furthermore, that maternal age and paternal preconception smoking or other factors associated with these parameters are critical in the etiology of subtypes of childhood leukemia.

Urinary 1-hydroxypyrene and polycyclic aromatic hydrocarbon exposure among asphalt paving workers.

OBJECTIVES: Using urinary 1-hydroxypyrene (1-OHP) as a measure of total absorbed dose, the primary objective of this study was to evaluate the total effect of inhalation and dermal PAH exposures while considering other factors such as age, body mass index and smoking that may also have a significant effect on urinary 1-OHP. METHODS: The study population included two groups of highway construction workers: 20 paving workers and 6 milling workers. During multiple consecutive workshifts, personal air and dermal samples were collected from each worker and analyzed for pyrene. During the same work week, urine samples were collected pre-shift, post-shift and at bedtime each day and analyzed for 1-OHP. Distributed lag models were used to evaluate the independent effect of inhalation and dermal exposures that occurred at each of several preceding exposure periods and were used to identify the relevant period of influence for each pathway. RESULTS: The paving workers had inhalation (mean 0.3 micro g/m(3)) and dermal (5.7 ng/cm(2)) exposures to pyrene that were significantly higher than the milling workers. At pre-shift on Monday morning, following a weekend away from work, the pavers and millers had the same mean baseline urinary 1-OHP level of 0.4 micro g/g creatinine. The mean urinary 1-OHP levels among pavers increased significantly from pre-shift to post-shift during each work day, while the mean urinary 1-OHP levels among millers varied little and remained near the baseline level throughout the study period. Among pavers there was a clear increase in the pre-shift data during the work week, such that the average pre-shift level on day 4 (1.4 micro g/g creatinine) was 3.5 times higher than the average pre-shift results on day 1 (0.4 micro g/g creatinine). The results of the distributed lag model indicated that the impact of dermal exposure was approximately eight times the impact of inhalation exposure. Furthermore, dermal exposure that occurred during the preceding 32 h had a statistically significant effect on urinary 1-OHP, while the effect of inhalation exposure was not significant. CONCLUSIONS: We found that distributed lag models are a valuable tool for analyzing longitudinal biomarker data and our results indicate that dermal contact is the primary route of exposure to PAHs among asphalt paving workers. An exposure assessment of PAHs that does not consider dermal exposure may considerably underestimate cumulative exposure and control strategies aimed at reducing occupational exposure to asphalt-related PAHs should include an effort to reduce dermal exposure.

Daycare attendance and risk of childhood acute lymphoblastic leukaemia.

The relationship between daycare/preschool ("daycare") attendance and the risk of acute lymphoblastic leukaemia was evaluated in the Northern California Childhood Leukaemia Study. Incident cases (age 1-14 years) were rapidly ascertained during 1995-1999. Population-based controls were randomly selected from the California birth registry, individually matched on date of birth, gender, race, Hispanicity, and residence, resulting in a total of 140 case-controls pairs. Fewer cases (n=92, 66%) attended daycare than controls (n=103, 74%). Children who had more total child-hours had a significantly reduced risk of ALL. The odds ratio associated with each thousand child-hours was 0.991 (95% confidence interval (CI): 0.984-0.999), which means that a child with 50 thousand child-hours (who may have, for example, attended a daycare with 15 other children, 25 h per week, for a total duration of 30.65 months) would have an odds ratio of (0.991)(50)=0.64 (95% CI: 0.45, 0.95), compared to children who never attended daycare. Besides, controls started daycare at a younger age, attended daycare for longer duration, remained in daycare for more hours, and were exposed to more children at each daycare. These findings support the hypothesis that delayed exposure to common infections plays an important role in the aetiology of childhood acute lymphoblastic leukaemia, and suggest that extensive contact with other children in a daycare setting is associated with a reduced risk of acute lymphoblastic leukaemia.

Molecular characterization of genomic AML1-ETO fusions in childhood leukemia.

T(8;21) AML1(CBFA2)-ETO(MTG8) is the most common chromosomal translocation in acute myeloid leukemia (AML) in both children and adults. We sought to understand the structure and gain insight into the fusion process between AML1 and ETO by sequencing genomic fusions in 17 primary childhood AMLs and two cell lines with t(8;21). Reciprocal translocations were sequenced for seven of the 19 samples. We assumed a null hypothesis that the translocation breakpoints would be evenly distributed along the intronic breakpoint cluster regions. Testing for multimodality via smoothed bootstrap statistical methods suggested, however, the presence of two separate cluster regions within both the AML1 and ETO breakpoint cluster regions. ETObreakpoints were predominantly located in intron 1B in a defined cluster 5' of exon 1A (scan statistic P value = 0.00001). All patients with available RNA expressed an AML1-ETO mRNA fusion between exon 5 of AML1 and exon 2 of ETO. Since the structural restraints for the fusion protein of AML1-ETO exclude exon 1A, we reason that ETO intron 1B harbors a structural feature with propensity for breakage and/or recombination. Chromosomal breakpoints displayed evidence of fusion by a non-homologous end joining process, with microhomologies and nontemplate nucleotides at some fusion junctions. Breakpoints in general displayed similar complexity of duplications, deletions, and insertions to other common pediatric leukemia translocations (TEL-AML1, MLL-AF4, PML-RARA, CBFB-MYH11) that we and others have analyzed.

Whole genome amplification increases the efficiency and validity of buccal cell genotyping in pediatric populations.

The collection of buccal cells provides a noninvasive method for obtaining DNA for genetic studies. Here we report the results on buccal cell genotyping from our ongoing study of childhood leukemia in Northern California. We have collected buccal samples from children ranging in age from 4 months to 15 years using an interviewer- or nurse-administered protocol using a cytology brush. Initial results of the genotyping, including the glutathione S-transferase mu, glutathione S-transferase theta, NAD(P)H:quinone oxidoreductase, and methylenetetrahydrofolate reductase polymorphisms, were disappointing because many specimens contained little DNA, failed repeated attempts at PCR amplification, and produced unreliable results. Here we evaluate a solution to the problem that involves whole genome amplification using the improved primer extension preamplification methodology. Sixty cases of pediatric acute leukemia were studied; five PCR-based genotypes were attempted using buccal cell DNA and whole genome amplified (WGA) buccal DNA. Results were compared with genotyping results using DNA isolated from peripheral whole blood or bone marrow for each child. The standard buccal protocol failed to yield successful PCR reactions in 30-57% of specimens, whereas WGA-buccal was markedly more efficient (2-5% failed PCR). A success rate of 100% was achieved with one repeat test of the failed WGA-PCR reactions. Misclassification of genotype was common for the glutathione S-transferase theta marker using the standard buccal procedure. The WGA-buccal protocol, however, produced genotyping results fully concordant with the referent blood or bone marrow DNA results for all five loci. DNA yields were increased by WGA to allow for approximately 900 PCR reactions/brush. WGA is very useful for improving the efficiency and validity of PCR-based genotyping in pediatric populations.

Hemoglobin adducts and sister chromatid exchanges in hospital workers exposed to ethylene oxide: effects of glutathione S-transferase T1 and M1 genotypes.

Ethylene oxide (EtO) is a genotoxic carcinogen with widespread uses as an industrial chemical intermediate and sterilant. We examined the effects of glutathione S-transferase T1 (GSTT1) and M1 (GSTM1) genotypes on the levels of N-(2-hydroxyethyl)valine (HEV) adducts in the erythrocytes and sister chromatid exchange (SCE) in lymphocytes from a group of 58 operators of sterilizers that used EtO and nonexposed workers from nine hospitals in the United States and one hospital in Mexico City. Cumulative exposure to EtO was estimated during the 4-month period before the collection of blood samples. Results showed that EtO exposure was significantly associated with the levels of HEV adducts and SCE after adjusting for cigarette smoking and other potential confounders. A significantly higher HEV adduct level (0.17 +/- 0.03 versus 0.08 +/- 0.01, mean +/- SE; P = 0.02) but lower SCE frequency (5.31 +/- 0.39 versus 6.21 +/- 0.17; P = 0.04) was observed in subjects with homozygous deletion of the GSTT1 gene (null genotype) as compared with those with at least one copy of the gene (positive genotype). In multiple regression analysis, the GSTT1-null genotype was associated with an increase in HEV adduct level (beta = 1.62; P = 0.02) and a decrease in SCE frequency (beta = -1.25; P = 0.003) after adjusting for age, gender, race, education, cigarette smoking, and EtO exposure status. The inverse SCE-GSTT1 relationship remained unchanged when SCE was further examined in relation to HEV adducts as an indicator of the internal EtO dose. The GSTM1 genotype was not associated with the level of either HEV adduct or SCE. These data indicate that the GSTT1-null genotype is associated with increased formation of EtO-hemoglobin adducts in relation to occupational EtO exposure, suggesting that individuals with homozygous deletion of the GSTT1 gene may be more susceptible to the genotoxic effects of ETO: The unexpected finding of decreased SCEs, which is less clear, may be attributed to the nonchemical specificity of this end point and the lack of expression of the GSTT1 enzyme in lymphocytes.

Ethnicity delineates different genetic pathways in malignant glioma.

In the United States and the San Francisco Bay Area, whites are nearly twice as likely as non-whites to develop brain cancer. To test whether prevalence and types of alterations in the p53 pathway in brain tumor development may explain some of this difference in risk, we have analyzed the p53 status of astrocytic gliomas from a population-based sample of cases within our San Francisco Bay Area Adult Glioma Study. We identified mutations in exons 5-8 of p53 using DNA extracted from formalin-fixed paraffin-embedded tissue blocks from 146 whites and 26 non-whites with astrocytic glioma by PCR-single-strand conformation polymorphism and direct sequencing. Tumor P53 protein (TP53) immunohistochemistry (IHC) available for 164 of these cases showed that tumors from 50% (13 of 26) of non-whites and 32% (44 of 138) of whites contained intense IHC staining for TP53, indicating persistence of TP53 protein. Irrespective of IHC status, tumors from 42% (11 of 26) of non-whites versus 13% (19 of 146) of whites contained p53 mutations (age/gender-adjusted odds ratio, 5.7; 95% confidence interval, 2.2-15.1; P = 0.0004). Patients with p53 mutation-positive tumors were also significantly younger than patients with mutation-negative tumors and somewhat more likely to be female. A higher proportion of tumors from non-whites than from whites had transition mutations, but there were similar proportions of transversion mutations in tumors from whites and non-whites. Whites and non-whites also had similar proportions of tumors with p53 mutations that stained intensely for TP53 (78 and 82%, respectively). Because whites have higher risk for glioma than non-whites in this population, that the gliomas from whites were less likely than those from non-whites to have p53 mutation suggests that whites may be more likely than non-whites to be at risk for the more common type of astrocytic gliomas, which do not contain p53 mutations.

Promoter methylation of DAP-kinase: association with advanced stage in non-small cell lung cancer.

Death associated protein (DAP)-kinase is a 16 kDa calmodulin-dependent serine/threonine kinase that carries a death domain at its C-terminus. DAP-kinase functions as a positive mediator of apoptosis that is induced by interferon-gamma. Recent studies suggest that DAP-kinase is involved in tumor metastasis and that it can be inactivated by methylation of CpG islands in the promoter region of the gene in some human tumors. However, little is known about the factors that are associated with the occurrence of DAP-kinase promoter methylation. We investigated both the possible associations of tobacco carcinogen and asbestos exposure with DAP-kinase promoter methylation, and the demographic and clinical factors associated with DAP-kinase promoter methylation in non-small cell lung cancer (NSCLC). One hundred and eighty-five patients diagnosed with NSCLC undergoing surgical resection from June, 1992 through December, 1996 at Massachusetts General Hospital participated in this study. Methylation-Specific PCR (MSP), performed using fresh-frozen tissue, was used to determine the methylation status of the promoter region of the DAP-kinase gene. Forty-seven (25%) of 185 tumors showed DAP-kinase promoter methylation. There was a significant association between methylation and an advanced pathologic stage (P=0.003, Fisher's exact test). Methylation of the DAP-kinase promoter was also associated with an increase in tumor size (P=0.009, Fisher's exact test) and lymph node involvement (P=0.04). No association was found between promoter methylation of DAP-kinase and k-ras or p53 mutation. In addition there was no association with a history of exposure to tobacco or asbestos. Controlling for age, sex, and histology, the odds ratios describing the association of DAP-kinase hypermethylation with stage were 2.70 (1.13--6.45), 3.11 (1.37--7.08) and 7.77 (1.21--50.03) in stages II, III and IV, respectively. Stage I cases with DAP-kinase promoter methylation had worse overall survival, but with the small sample size and limited follow-up this did not reach statistical significance. Our findings suggest that methylation of the promoter region of the DAP-kinase gene is not associated with exposure to tobacco or asbestos. However, they strongly suggest that DAP-kinase may be important in the progression of non-small cell lung cancer from early to late stage disease.

p16(INK4a) and histology-specific methylation of CpG islands by exposure to tobacco smoke in non-small cell lung cancer.

The p16(INK4a) protein inhibits cyclin-dependent kinase 4, a key regulator of progression through the G(1) phase of the cell cycle. Methylation of CpG islands in the promoter region is an important avenue for inactivation of p16. The mechanism of methylation of the p16 promoter region, however, has not been elucidated. Recent reports investigating p16 methylation in non-small cell lung cancer (NSCLC) suggest that carcinogens in tobacco smoke induce the DNA methylation process. We investigated the association between methylation of the p16 promoter region and exposure to tobacco smoke in 185 primary NSCLCS: We also studied the relationship of p16 methylation with mutation of the K-ras and p53 genes, as well as with methylation at the DAP-kinase and p14(ARF) loci. Finally, we evaluated the prognostic significance of p16 methylation in NSCLC. The prevalence of p16 methylation was greater in squamous cell carcinoma (41%) compared with adenocarcinoma (22%; P = 0.03; Fisher's exact test). Methylation of p16 was significantly associated with pack-years smoked (P = 0.007; Wilcoxon rank sum test), duration of smoking (P = 0.0009; Wilcoxon rank sum test), and negatively with the time since quitting smoking (P = 0.03; Wilcoxon rank sum test). No methylation of the nearby p14(ARF) locus was detected, and methylation of the DAP-kinase locus was not associated with either p16 methylation or with exposure to tobacco smoke. In patients with stage 1 adenocarcinoma, p16 methylation was an independent risk factor predicting significantly shorter postsurgery survival (P = 0.03), controlling for the significant effects of other factors, including K-ras mutation. These findings suggest that methylation of CpG islands in tobacco-associated cancers occurs in a gene- and tissue-specific manner and is induced directly or indirectly by exposure to tobacco smoke in NSCLC.

Associations between ERCC2 polymorphisms and gliomas.

Xeroderma pigmentosum complementation group D/excision repair cross-complementing in rodents 2 (ERCC2) encodes a protein that is part of the nucleotide excision repair pathway and the transcription factor IIH transcription complex. Mutations in this gene have been shown to cause three distinct clinical diseases including xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. Several ERCC2 polymorphisms, the effects of which on gene function are not known, have been described. To investigate whether constitutive sequence variations might be associated with adult onset gliomas, blood specimens from a case-control study (187 cases and 169 controls) were genotyped for seven previously described polymorphisms (R156R, I199M, H201Y, D312N, A575A, D711D, and K751Q). A novel R616C polymorphism was also identified. Cases were significantly more likely than controls to be homozygous for the silent AA variant at codon 156 (odds ratio, 2.3; 95% confidence interval, 1.3-4.2). Although this was observed for patients in each of three histological subgroups of cases, (glioblastoma multiforme, astrocytoma, and oligoastrocytoma) compared with controls, the association was strongest for patients with oligoastrocytoma (odds ratio, 3.2; 95% confidence interval, 1.1-9.5). In contrast, cases were somewhat less likely than controls to carry variants at D312N, D711D, and K751Q, but not significantly so overall or for any subgroup after adjustment for age and gender. Individuals with variant nucleotides at D312N, D711D, and K751Q were significantly more likely to carry a variant at another of those three codons and less likely to carry a variant nucleotide at R156R, regardless of case or control status. Although the pattern of association observed here is consistent with a role of ERCC2 variants in the prevention or causation of glioma, these results are also consistent with the possibility that another gene linked to ERCC2 may be involved. This seems especially so because the strongest association was observed with a silent nucleotide variation.

Tobacco smoke-induced DNA damage and an early age of smoking initiation induce chromosome loss at 3p21 in lung cancer.

The short arm of chromosome 3 is thought to harbor a novel oncogenic locus that is important in the genesis of lung cancer. The region at 3p21 is believed to contain a distinct locus that is sensitive to loss from the action of tobacco smoke carcinogens and has been reported to be specifically targeted for deletion in lung cancer. To investigate whether 3p21 alteration in lung cancer is associated with carcinogen exposure, PCR-based analysis was performed to detect loss of heterozygosity (LOH) on chromosome 3 at 3p21 in non-small cell lung carcinoma (NSCLC). We also measured instability at the BAT-26 locus, because the mismatch DNA repair gene, hMLH1, is found at 3p21. LOH at 3p21 was analyzed for association with the clinical features of NSCLC, p53 mutation status, polynuclear aromatic hydrocarbon-DNA adduct levels (measured using 32P-postlabeling) and carcinogen exposure information including cigarette smoking and asbestos exposure. Of 219 lung cancers, 150 cases (68.5%) were informative at the D3S1478 locus, and 44.2% of squamous cell carcinoma cases and 30.2% of adenocarcinoma cases showed 3p21 LOH. None of the cancers showed BAT-26 instability. The prevalence of 3p21 LOH was higher in both current and former smokers compared with never smokers and was higher in p53 mutated cases. Among squamous cell carcinoma cases, there was a strong association of increased 3p21 LOH with increasing polynuclear aromatic hydrocarbon-DNA adducts levels (P = 0.03), as well as an increased prevalence LOH with earlier age of smoking initiation (P = 0.02). Our results confirm that 3p21 LOH is strongly associated with measures of biologically effective dose of exposure to tobacco carcinogens. Our results also suggest that alterations of hMLH1 are not related to any of the reported associations, because there was no evidence of microsatellite instability. Finally, LOH in 3p21 may be an early molecular event in NSCLC, because it is significantly associated with a tendency to start smoking at a young age.

Genetic resistance factor for HIV-1 and immune response to varicella zoster virus.

A 32 bp deletion in the chemokine receptor CCR5 gene modulates HIV-1 infection. However, whether this CCR5 gene variation modifies immunity to common herpesvirus infections is unknown. We investigated whole blood IgG concentrations of 157 normal adult blood donors. Also we assessed whether the 32 bp deletion of CCR5 (delta32CCR5) was associated with circulating IgG to four herpesviruses: varicella zoster virus, Epstein-Barr virus, cytomegalovirus, and herpes simplex virus type 1 and type 2. Individuals who carried delta32CCR5 were 9.2 times more likely to be seronegative for varicella zoster virus than non-carriers (95% CI 2.9-29.1), but no differences were seen for the other herpesviruses studied. Variation in CCR5 may modulate humoral immunity to varicella zoster virus.