James E. Browning

Cancer susceptibility variants and the risk of adult glioma in a US case–control study

Malignant gliomas are the most common and deadly brain tumors. Although their etiology remains elusive, recent studies have narrowed the search for genetic loci that influence risk. We examined variants implicated in recent cancer genome-wide association studies (GWAS) for associations with glioma risk in a US case–control study. Cases were identified from neurosurgical and neuro-oncology clinics at major academic centers in the Southeastern US. Controls were identified from the community or were friends or other associates of cases. We examined a total of 191 susceptibility variants in genes identified in published cancer GWAS including glioma. A total of 639 glioma cases and 649 controls, all Caucasian, were included in analysis. Cases were enrolled a median of 1 month following diagnosis. Among glioma GWAS-identified variants, we detected associations in CDKN2B, RTEL1, TERT and PHLDB1, whereas we did not find overall associations for CCDC26. Results showed clear heterogeneity according to histologic subtypes of glioma, with TERT and RTEL variants a feature of astrocytic tumors and glioblastoma (GBM), CCDC26 and PHLDB1 variants a feature of astrocytic and oligodendroglial tumors, and CDKN2B variants most prominent in GBM. No examined variant in other cancer GWAS was found to be related to risk after adjustment for multiple comparisons. These results suggest that GWAS-identified SNPs in glioma mark different molecular etiologies in glioma. Stratification by broad histological subgroups may shed light on molecular mechanisms and assist in the discovery of novel loci in future studies of genetic susceptibility variants in glioma.

Rare TP53 genetic variant associated with glioma risk and outcome

Validation of a recent finding linking a rare variant in TP53 to the risk of glioma, the most common primary brain tumour, is reported here. This study genotyped the single nucleotide polymorphism (SNP) rs78378222 in 566 glioma cases and 603 controls. The variant ‘C’ allele (with an allelic frequency of 1.1% in controls) was associated with a 3.5-fold excess in glioma risk (odds ratio 3.54; p=0.0001). Variant carriers had significantly improved survival (hazard ratio 0.52; p=0.009) when compared to non-carriers. The rs78378222 SNP is the first confirmed rare susceptibility variant in glioma. Results may shed light on the aetiology and progression of these tumours.

SSBP2 variants are associated with survival in glioblastoma patients.

Purpose: Glioblastoma is a devastating, incurable disease with few known prognostic factors. Here, we present the first genome-wide survival and validation study for glioblastoma. Experimental Design: Cox regressions for survival with 314,635 inherited autosomal single-nucleotide polymorphisms (SNP) among 315 San Francisco Adult Glioma Study patients for discovery and three independent validation data sets [87 Mayo Clinic, 232 glioma patients recruited from several medical centers in Southeastern United States (GliomaSE), and 115 The Cancer Genome Atlas patients] were used to identify SNPs associated with overall survival for Caucasian glioblastoma patients treated with the current standard of care, resection, radiation, and temozolomide (total n = 749). Tumor expression of the gene that contained the identified prognostic SNP was examined in three separate data sets (total n = 619). Genotype imputation was used to estimate hazard ratios (HR) for SNPs that had not been directly genotyped. Results: From the discovery and validation analyses, we identified a variant in single-stranded DNA-binding protein 2 (SSBP2) on 5q14.1 associated with overall survival in combined analyses (HR, 1.64; P = 1.3 × 10−6). Expression of SSBP2 in tumors from three independent data sets also was significantly related to patient survival (P = 5.3 × 10−4). Using genotype imputation, the SSBP2 SNP rs17296479 had the strongest statistically significant genome-wide association with poorer overall patient survival (HR, 1.79; 95% CI, 1.45-2.22; P = 1.0 × 10−7). Conclusion: The minor allele of SSBP2 SNP rs17296479 and the increased tumor expression of SSBP2 were statistically significantly associated with poorer overall survival among glioblastoma patients. With further confirmation, previously unrecognized inherited variations influencing survival may warrant inclusion in clinical trials to improve randomization. Unaccounted for genetic influence on survival could produce unwanted bias in such studies. Clin Cancer Res; 18(11); 3154–62. ©2012 AACR.

SWI/SNF gene variants and glioma risk and outcome.

BACKGROUND The human SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex plays essential roles in a variety of cellular processes and has been implicated in human cancer. However, the role of germline genetic variants in this complex in relation to cancer risk is not well studied. METHODS We assessed the association of 16 variants in the catalytic subunits (SMARCA2 and SMARCA4) of the SWI/SNF complex with the risk of glioma subtypes (lower grade astrocytoma, oligodendroglioma and glioblastoma [GBM]) and with mortality from high-grade tumors (GBM) in a multicenter US case-control study that included 561 cases and 574 controls. Associations were estimated with odds ratios (OR, for risk) or hazards ratios (HR, for mortality) with 95% confidence intervals (CI). False discovery rate (FDR-q) was used to control for multiple testing in risk associations. RESULTS None of the investigated SNPs was associated with overall glioma risk. However, analyses according to histological subtypes revealed a statistically significant increased risk of oligodendroglioma in association with SMARCA2 rs2296212 (OR = 4.05, 95% CI = 1.11-14.80, P = 0.030, q = 0.08) and rs4741651 (OR = 4.68, 95% CI = 1.43-15.30, P = 0.011, q = 0.08) and SMARCA4 rs11672232 (OR = 1.90, 95% CI = 1.01-3.58, P = 0.048, q = 0.08) and rs12232780 (OR = 2.14, 95% CI = 1.06-4.33, P = 0.035, q = 0.08). No significant risk associations were observed for GBM or lower grade astrocytoma. Suggestive associations with GBM mortality were not validated in the Cancer Genome Atlas. CONCLUSION Our findings suggest that genetic variants in SMARCA2 and SMARCA4 influence the risk of oligodendroglioma. Further research is warranted on the SWI/SNF complex genes and epigenetic mechanisms more generally in the development of glioma in adults.

Brain tumor risk according to germ-line variation in the MLLT10 locus

Genome-wide association studies have recently identified a cancer susceptibility locus at 10p12 mapping to MLLT10 associated with the onset of diverse tumors. We genotyped two tightly linked single-nucleotide polymorphisms (SNPs) at MLLT10 associated with meningioma (rs12770228) or ovarian cancer (rs1243180), and tested for associations among 295 meningioma cases, 606 glioma cases and 646 noncancer controls, all of European descent. The variant ‘A’ allele in MLLT10 rs12770228 was associated with an increased risk of meningioma (per allele odds ratio: 1.25; 95% confidence interval: 1.02, 1.53; P=0.031). Similar associations were observed for rs1243180. MLLT10 variants were unrelated to glioma. Functional investigation identified 22 candidate functional SNPs mapping to this region. The present study further validates 10p12 as a meningioma risk locus.

Abstract 104: Toenail iron, genetic variation in iron status, and the risk and outcome of glioma .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Elevated iron stores can trigger overproduction of reactive oxygen species and induce oxidative DNA damage. To our knowledge, no studies have investigated the association of body iron stores with the risk of glioma. In this investigation, we examined single nucleotide polymorphisms (SNPs) identified as markers of iron status in genome-wide association studies, and also measured iron stores in toenail samples in a clinic-based case-control study conducted at medical centers in the southeastern US. Genotyping was performed in 622 newly diagnosed, nonrecurrent glioma cases (including 341 WHO grade IV glioblastomas (GBM); 146 WHO grade II or III astrocytomas, 94 mixed oligoastrocytomas (MOAs) and oligodendrogliomas, and 41 gliomas with unspecified histology) and 628 healthy controls with no history of brain tumors. Illumina GoldenGate and Taqman OpenArray assays were used to genotype oral DNA samples. A total of 24 SNPs associated with markers of iron status were genotyped. Iron levels in toenail samples were measured in 200 glioma cases and 200 controls using neutron-activation analysis. Logistic regression was used to estimate age and gender-adjusted odds ratios (OR) and 95% confidence intervals (CI) for glioma risk according to examined genotypes and toenail iron levels. Proportional hazards regression was used to estimate age and gender-adjusted hazard ratios (HR) for glioma-related death among 320 patients with GBM or high grade astrocytomas all treated with the current standard of care for high grade glioma (eg. surgery, radiation and temozolomide) (248 deaths; median Kaplan-Meier survival: 15.0 months). We observed no overall association with glioma risk or patient outcome for SNPs in ARSB, BTN1A1, C7ORF10, [FLJ43390][1], GHR, GTSCR1, HFE, HIST1H2BJ, KRT18P33, LRRC16, SCGN, SLC17A1, TOPBP1, and WTAP. Among non-GBM astrocytomas, borderline risk associations were observed for [rs236918][2] in PCSK7 (G>C; minor allele frequency (MAF) = 0.11) (per variant allele OR = 0.50; 95% CI: 0.29 to 0.88; p for trend = 0.01) and with rs1049296 in TF (C>T; MAF = 0.17) (recessive model OR = 3.03; 95% CI: 1.11 to 8.27; p = 0.03). Among oligodendrogliomas/MOAs, risk associations were observed for rs4820268 in TMPRSS6 (A>G; MAF = 0.43) (recessive model OR = 1.91; 95% CI: 1.14 to 3.22; p = 0.01) and rs12216125 in TRIM38 (C>T; MAF = 0.35) (dominant model OR = 1.67; 95% CI: 1.03 to 2.72; p = 0.04). No SNPs were associated with the risk of GBM. One SNP, [rs972275][3] in RSPO3 (G>C; MAF = 0.38), was associated with shorter patient survival (dominant model HR = 1.40; 95% CI: 1.06, 1.87; p=0.02). Increasing levels of toenail iron was associated with a non-significant decrease in glioma risk (OR = 0.88; 95% CI: 0.77 to 1.02; p = 0.08). Iron levels were not associated with survival. To our knowledge this is the first report suggesting that genetically determined variation in iron status may affect glioma risk and patient outcome. Further studies are needed to confirm these results. Citation Format: Gabriella M. Anic, Reid C. Thompson, L. Burton Nabors, Jeffrey J. Olson, Melissa H. Madden, James E. Browning, John D. Brockman, Peter A. Forsyth, Kathleen M. Egan. Toenail iron, genetic variation in iron status, and the risk and outcome of glioma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 104. doi:10.1158/1538-7445.AM2013-104 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=FLJ43390&atom=%2Fcanres%2F73%2F8_Supplement%2F104.atom [2]: /lookup/external-ref?link_type=GEN&access_num=rs236918&atom=%2Fcanres%2F73%2F8_Supplement%2F104.atom [3]: /lookup/external-ref?link_type=GEN&access_num=rs972275&atom=%2Fcanres%2F73%2F8_Supplement%2F104.atom

Abstract 714: Single cell phenotypic heterogeneity as a prognostic factor in glioblastoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background – Glioblastoma Multiforme (GBM) is a highly aggressive and heterogeneous disease in which survival of patients is measured only in months. Pathologic features associated with patient outcome are still incompletely understood. Until recently, phenotypic features of single cells have not been investigated due to technological limitations. However, with the advent of high-content slide scanning coupled with cognitive and scriptable algorithms, researchers are now positioned to identify and quantify single cell features that may provide unique insight on tumor behavior. In this project we sought to identify variations in cellular phenotypes which correlate with time of post diagnostic survival in patients with GBM. Methods – A total of 157 GBM patients were selected for study on the basis of short (SS) (median K-M survival = 6 months; N=81) or long (LS) (median K-M survival = 33 months, N=76) survival times. Median age was 56 years and all patients underwent current standard of care therapy for GBM (surgery, radiation and temozolomide). Among the 157 cases, 11 were excluded due to poor sample quality (final n=146). For each case, the diagnostic H&E slide was digitally scanned using the ScanScope XT (Aperio, Vista, CA, USA) with a 200x/0.8NA objective lens. Definiens TissueStudio v3.0 (Munich, Germany) was used to identify viable tumor regions. Individual cells were segmented in areas of viable tumor and twenty separate features extracted from each tumor cell (subcellular compartmentalization, nucleus to cytoplasm area ratio, nuclear size and shape, etc). In total, the 20 features were extracted in thousands of single cells for each GBM case. Output was evaluated by Matlab (The MathWorks, Inc,, Natick, MA) using a heatmap approach by first normalizing the scales of each feature to a range of 0-1, and assigning a color from green (0) to red (1) (5 classes) for each cell or compartment (x) and each feature (y). Results – This study was completed in a series of three stages including training followed by two replication sets. In the training set (N=50), four of the evaluated features associated with the size and shape of the cancer cell nuclei (i.e. width [μm], circularity, ellipticity and hematoxylin intensity), were found to distinguish the SS group (15/25) from the LS group (6/25) based on supervised classification. A similar pattern was observed in replication set 1 (15/24 and 8/28, respectively) and replication set 2 (15/23 and 8/21, respectively). Overall, 66% of cases were correctly classified with respect to survival time on the basis of these cellular features (p=0.0001). Conclusions – Quantitative image analysis may be useful in the identification of novel prognostic features in GBM with potential for gaining new biological insights on the behavior of these tumors. Citation Format: Mark C. Lloyd, Melissa H. Madden, L. Burton Nabors, Reid C. Thompson, Jeffrey J. Olson, Steven L. Carroll, James Browning, Tamir Epstein, Robert A. Gatenby, Kathleen M. Egan. Single cell phenotypic heterogeneity as a prognostic factor in glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 714. doi:10.1158/1538-7445.AM2013-714

Abstract 644: Premorbid body weight and survival in high grade glioma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Greater adiposity has been linked to an increased risk and/or poorer survival in a variety of cancers. However, the relationship of body fat mass to glioma outcome is not well studied. In the present analysis, we examined whether premorbid body weight determined 1 to 5 years prior to diagnosis is associated with glioma patient survival. Methods: The analysis was based on 619 patients with high grade glioma (84% glioblastoma multiforme (GBM) and16% anaplastic astrocytoma (AA)) enrolled in a US-based multicenter case-control study between December 2004 and March 2011. In a structured interview, subjects provided information on height at age 21, weight at age 21 and weight 5 years prior to the study interview (1 year during the pilot study phase; N=95). Subjects were interviewed a median of 1.2 months following glioma diagnosis (interquartile range: 3 weeks to 2.5 months). A total of 408 deaths occurred, all glioma-related, 2 months to 4.8 years following diagnosis (median: 9.4 months). Proportional hazards regression was used to estimate Hazard Ratios (HR) and 95% Confidence Intervals (CIs) for glioma-related death according to categories of body mass index (BMI) (kg/m2) defining obesity (BMI >=30; N=178), overweight (BMI 25-29.9; N=254), normal weight (BMI 18.5-24.9; N=179) and underweight (BMI <18.5; N=8). All results were adjusted for age, gender and glioma subtype (GBM versus AA). Results: Patients with a premorbid BMI of 30 or greater had an elevated rate of glioma-related death when compared to patients with a normal BMI (HR=1.39; 95% CI: 1.07-1.82) whereas no significant excess mortality was observed in patients defined as overweight (HR=1.15; 95% CI: 0.89-1.49). Patients underweight 1-5 years prior to diagnosis had significantly poorer outcomes when compared to patients of normal weight (HR=2.43; 95% CI: 1.06-5.57), and weight loss of 10 pounds or greater between age 21 and 1-5 years prior to diagnosis (N=27) was significantly associated with a worse outcome (HR=1.92; 95% CI: 1.18-3.13) when compared to stable weight (within 10 pounds). All results were consistent in men and women, and were unchanged in analysis restricted to patients treated with the current standard of care including surgery, radiation and temozolomide (not shown). Conclusions: In this large well-characterized patient series, premorbid obesity was significantly associated with poorer survival following glioma diagnosis. Low body weight was also associated with a poorer outcome possibly reflecting effects of preclinical disease-associated changes on body weight. Findings for obesity support the hypothesis that elevated pre-diagnostic body weight is detrimental to glioma survival and suggest a role for insulin resistance, aberrant IGF signaling, and/or low-level chronic inflammation in the progression of high grade tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 644. doi:1538-7445.AM2012-644

Abstract 2629: Common genetic variants in the vitamin D pathway including genome-wide associated variants in relation to glioma risk and outcome

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Experimental and clinical evidence suggest that vitamin D protects against several types of cancer by promoting cell differentiation and apoptosis and by inhibiting cell proliferation and angiogenesis. In vitro evidence supports a similar protective function in glioma; however, no study has examined whether common genetic variants in the vitamin D pathway are related to glioma risk or patient outcome. We evaluated these potential associations in a clinic-based case-control study conducted at medical centers in the southeastern US. Genotyping was performed in 623 newly diagnosed (eg. nonrecurrent) glioma cases (including 343 WHO grade IV glioblastomas (GBM); 148 WHO grade II or III astrocytomas, 95 oligoastrocytomas and oligodendrogliomas, and 37 gliomas with unspecified histology) and 631 healthy controls with no history of brain tumors. A total of 7 candidate tagging single nucleotide polymorphisms (SNPs) were genotyped in the vitamin D receptor (VDR at 12q13) including rs2107301, rs2238135, rs4516035, rs731236 (Taq1), rs1544410 (Bsm1), rs11568820 (Cdx2), and rs2228570 (Fok1). SNPs associated with serum concentrations of 25-hydroxy vitamin D in genome-wide association studies (GWAS) were also evaluated including rs1155563, rs12512631, rs2282679, and rs7041 in GC (4q12-q13), rs10741657 in CYP2R1 (11p15), rs6013897 in CYP24A1 (20q13), rs3829251 in NADSYN1 (11q13), and rs6599638 at C10orf88 (10q26). Genotyping was performed in oral DNA samples using Illumina GoldenGate and Taqman OpenArray assays. Logistic regression was used to estimate age and gender-adjusted odds ratios (OR) and 95% confidence intervals (CI) for glioma risk according to vitamin D genotypes. Proportional hazards regression was used to estimate age and gender-adjusted hazard ratios (HR) for glioma-related death among 439 patients with high grade tumors including GBM and high grade astrocytomas (331 deaths; median Kaplan-Meier survival: 15.0 months). GWAS SNPs in NADSYN1, GC, and C10ORF88 were not associated with glioma risk or patient survival. Risk associations limited to GBM were observed for rs2238135 in the VDR (G>C; minor allele frequency (MAF) = 0.20) (per variant allele OR = 1.31; 95% CI: 1.01 to 1.71; p for trend = 0.04) and for GWAS SNP rs10741657 located near CYP2R1 (G>A; MAF = 0.40) (per variant allele OR = 0.79; 95% CI: 0.63 to 0.98; p for trend = 0.03). The variant allele in CYP24A1 rs6013897 (T>A; MAF = 0.20) was associated with prolonged survival among patients with high grade tumors (per variant allele HR = 0.79; 95% CI: 0.59 to 0.97; p for trend = 0.03) in patients uniformly treated with the current standard of care (surgery, radiation and temozolomide). To our knowledge this is the first report suggesting that genetic variation in vitamin D related genes may be a determinant of glioma risk and outcome. Further studies are needed to confirm these results and identify the putative causal variant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2629. doi:1538-7445.AM2012-2629

Erratum to: An exploratory analysis of common genetic variants in the vitamin D pathway including genome-wide associated variants in relation to glioma risk and outcome

Unfortunately, given name and family name of two authors (Nabors LB and F. Reed Murtagh) have been wrongly identified in the original article. The correct representation has been provided in this erratum.