JoEllen Weaver

Common variation in KITLG and at 5q31.3 predisposes to testicular germ cell cancer

Testicular germ cell tumors (TGCT) have been expected to have a strong underlying genetic component. We conducted a genome-wide scan among 277 TGCT cases and 919 controls and found that seven markers at 12p22 within KITLG (c-KIT ligand) reached genome-wide significance (P < 5.0 × 10−8 in discovery). In independent replication, TGCT risk was increased threefold per copy of the major allele at rs3782179 and rs4474514 (OR = 3.08, 95% CI = 2.29–4.13; OR = 3.07, 95% CI = 2.29–4.13, respectively). We found associations with rs4324715 and rs6897876 at 5q31.3 near SPRY4 (sprouty 4; P < 5.0 × 10−6 in discovery). In independent replication, risk of TGCT was increased nearly 40% per copy of the major allele (OR = 1.37, 95% CI = 1.14–1.64; OR = 1.39, 95% CI = 1.16–1.66, respectively). All of the genotypes were associated with both seminoma and nonseminoma TGCT subtypes. These results demonstrate that common genetic variants affect TGCT risk and implicate KITLG and SPRY4 as genes involved in TGCT susceptibility.

A second independent locus within DMRT1 is associated with testicular germ cell tumor susceptibility

Susceptibility to testicular germ cell tumors (TGCT) has a significant heritable component, and genome-wide association studies (GWASs) have identified association with variants in several genes, including KITLG, SPRY4, BAK1, TERT, DMRT1 and ATF7IP. In our GWAS, we genotyped 349 TGCT cases and 919 controls and replicated top hits in an independent set of 439 cases and 960 controls in an attempt to find novel TGCT susceptibility loci. We identified a second marker (rs7040024) in the doublesex and mab-3-related transcription factor 1 (DMRT1) gene that is independent of the previously described risk allele (rs755383) at this locus. In combined analysis that mutually conditions on both DMRT1 single nucleotide polymorphism markers, TGCT cases had elevated odds of carriage of the rs7040024 major A allele [per-allele odds ratio (OR) = 1.48, 95% confidence interval (CI) 1.23, 1.78; P = 2.52 × 10(-5)] compared with controls, while the association with rs755383 persisted (per allele OR = 1.26, 95% CI 1.08, 1.47, P = 0.0036). In similar analyses, the association of rs7040024 among men with seminomatous tumors did not differ from that among men with non-seminomatous tumors. In combination with KITLG, the strongest TGCT susceptibility locus found to date, men with TGCT had greatly elevated odds (OR = 14.1, 95% CI 5.12, 38.6; P = 2.98 × 10(-7)) of being double homozygotes for the risk (major) alleles at DMRT (rs7040024) and KITLG (rs4474514) when compared with men without TGCT. Our findings continue to corroborate that genes influencing male germ cell development and differentiation have emerged as the major players in inherited TGCT susceptibility.

Allelic Imbalance in BRCA1 and BRCA2 Gene Expression Is Associated with an Increased Breast Cancer Risk

The contribution of BRCA1 and BRCA2 to familial and non-familial forms of breast cancer has been difficult to accurately estimate because of the myriad of potential genetic and epigenetic mechanisms that can ultimately influence their expression and involvement in cellular activities. As one of these potential mechanisms, we investigated whether allelic imbalance (AI) of BRCA1 or BRCA2 expression was associated with an increased risk of developing breast cancer. By developing a quantitative approach utilizing allele-specific real-time PCR, we first evaluated AI caused by nonsense-mediated mRNA decay in patients with frameshift mutations in BRCA1 and BRCA2. We next measured AI for BRCA1 and BRCA2 in lymphocytes from three groups: familial breast cancer patients, non-familial breast cancer patients and age-matched cancer-free females. The AI ratios of BRCA1, but not BRCA2, in the lymphocytes from familial breast cancer patients were found to be significantly increased as compared to cancer-free women (BRCA1: 0.424 versus 0.211, P = 0.00001; BRCA2: 0.206 versus 0.172, P = 0.38). Similarly, the AI ratios were greater for BRCA1 and BRCA2 in the lymphocytes of non-familial breast cancer cases versus controls (BRCA1: 0.353, P = 0.002; BRCA2: 0.267, P = 0.03). Furthermore, the distribution of under-expressed alleles between cancer-free controls and familial cases was significantly different for both BRCA1 and BRCA2 gene expression (P < 0.02 and P < 0.02, respectively). In conclusion, we have found that AI affecting BRCA1 and to a lesser extent BRCA2 may contribute to both familial and non-familial forms of breast cancer.

Quantification of excision repair cross-complementing group 1 and survival in p16-negative squamous cell head and neck cancers.

Purpose: Multimodality treatment of squamous cell carcinoma of the head and neck (SCCHN) often involves radiotherapy and cisplatin-based therapy. Elevated activity of DNA repair mechanisms, such as the nucleotide excision repair (NER) pathway, of which ERCC1 is a rate-limiting element, are associated with cisplatin and possibly RT resistance. We have determined excision repair cross-complementing group 1 (ERCC1) expression in human papillomavirus (HPV)-negative SCCHN treated with surgery [±adjuvant radiotherapy/chemoradiation (CRT)]. Experimental Design: We assessed ERCC1 protein expression in archival tumors using immunofluorescence staining and automatic quantitative analysis (AQUA) with three antibodies to ERCC1 (8F1, FL297, and HPA029773). Analysis with Classification and Regression Tree (CART) methods ascertained the cutoff points between high/low ERCC1 expression. Multivariable analysis adjusted for age, T, and N stage. Kaplan–Meier curves determined median survival. ERCC1 expression at initial tumor presentation and in recurrent disease were compared. Performance characteristics of antibodies were assessed. Results: ERCC1 low/high groups were defined on the basis of AQUA analysis with 8F1/2009, FL297, and HPA029773. Among patients treated with surgery plus adjuvant radiotherapy/CRT, longer median survival was observed in ERCC1-low versus ERCC1-high tumors (64 vs. 29 months; P = 0.02; HPA029773). Data obtained with HPA029773 indicated no survival difference among patients treated only with surgery. Recurrent cancers had lower ERCC1 AQUA scores than tumors from initial presentation. Extensive characterization indicated optimal specificity and performance by the HPA029773 antibody. Conclusions: Using AQUA, with the specific ERCC1 antibody HPA029773, we found a statistical difference in survival among high/low-ERCC1 tumors from patients treated with surgery and adjuvant radiotherapy. Clin Cancer Res; 19(23); 6633–43. ©2013 AACR.

Abstract 2804: ERCC1 (excision repair cross complementing group 1) is a predictive biomarker of survival in squamous cell carcinoma of the head and neck (SCCHN)

Introduction: We analyzed SCCHN archival tissue in order to determine if baseline ERCC1 levels as measured by AQUA is associated with prognosis, benefit from adjuvant treatment or T stage in patients with resectable SCCHN. Experimental Procedures: Tissue microarrays containing head and neck squamous cell carcinoma samples obtained from patients treated at Fox Chase Cancer Center from 1990-2002 were constructed. Slides were stained by a modified indirect immunofluorescence method. Sections were incubated with ERCC1 antibody (8F1, Lab Vision) and wide-spectrum screening rabbit cytokeratin antibody (Dako Z0622). Prolong Gold mounting medium (P36931; Molecular Probes) containing 4,6-Diamidino-2-phenylindole (DAPI) was used to define tissue nuclei. A binary image (tumor mask) was created from the cytokeratin image of each histospot. ERCC1 levels were measured using fluorescent immunohistochemistry on the HistoRx PM-2000 image analysis platform and the data analyzed using AQUA algorithms. The in situ biomarker profiling system generated quantitative measurements of patient protein levels based on AQUA scores derived from fluorescent output of labeled markers. Tumor mask, nuclear, and cytoplasmic ERCC1 expression were measured. Baseline demographic and prognostic information was compared using Student9s t-test for continuous variable or Fisher9s exact tests for categorical variable. Analysis for overall survival was performed using Kaplan-Meier method, with comparisons using the log-rank test. Cox proportional hazard models were also used to examine the effect of ERCC1 after adjusted for the pathological T stage. Results: Tissue was analyzed from 109 patients (70 male, 39 females). Primary sites included tongue (34), glottic (15), retromolar trigone (10), tonsil (5), pyriform sinus (3), oral cavity (36), other (6). 33 were treated with surgery. 76 received adjuvant radiation or platinum based chemoradiation. The ERCC1 expression by AQUA score was defined as high versus low at the 30 th percentile (262.37 in nuclear compartment, 161.31 in cytoplasmic compartment, 361.09 in tumor mask). In the 33 patients treated with surgery alone, there was no association with ERCC1 status and survival. In this group, low ERCC1 levels in all 3 compartments was associated with a higher T stage and tumor size (p=0.05 in nuclear, p=0.003 in tumor mask, p=0.05 in cytoplasm). Among the 76 patients who received adjuvant radiation/chemoradiation, low ERCC1 measured in the nuclear compartment was associated an increased survival (p=0.02). Conclusions: Low nuclear ERCC1 expression as defined by AQUA score in SCCHN is associated with greater T stage and benefit to adjuvant therapy with radiation/chemoradiation. We are developing a study to guide treatment selection for recurrent/metastatic disease based on ERCC1 status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2804.