Genone-wide Discovery of Molecular Alterations in Head and Neck Cancer

描述（由申请人提供）：对于该ARRA RFA-OD-09-004 GO申请，我们建议使用编码基因的高通量序列分析结合拷贝数、基因表达和甲基化分析，对口腔癌进行整合的全基因组突变分析。遗传改变代表了人类癌症的根本原因，包括口腔肿瘤。在美国，口腔癌占46，000例头颈癌病例的约50%。口腔癌影响身体外观和重要功能，包括味觉，吞咽和言语/发声。除了显著的发病率外，头颈癌导致约12，000人死亡，其中口腔癌将导致约5，400人死亡。鉴于这些肿瘤的显著发病率和死亡率，对口腔癌的新临床方法存在深刻的需求。该项目将确定口腔癌治疗、诊断和预后干预的潜在新途径，并将作为其他头颈癌基因组分析的模型。对于突变筛查，我们将采用两阶段，高通量DNA测序方法，我们最近已经完善和用于识别乳腺癌，结肠癌，胰腺癌和胶质母细胞瘤肿瘤中的新的肿瘤相关突变。该策略显著提高了功率并降低了大规模肿瘤测序的成本，提供了对来自超过20，000个编码基因的约200，000个外显子的>95%的碱基的高灵敏度突变分析。在该方法的第一阶段，将对一组24个临床注释的口腔癌样本进行外显子测序分析。将在来自同一患者的正常DNA样本中检查所有突变，以鉴定和确认肿瘤特异性突变。在第二阶段，将通过对至少48个肿瘤的更大集合进行测序来分析突变基因。先前开发的生物统计学标准将用于区分肿瘤相关的驱动突变和不相关的乘客突变。在检测突变的同一组口腔癌样本中，我们还将使用高密度SNP微阵列进行拷贝数分析，通过下一代测序和基因表达系列分析（SAGE）组合进行基因表达分析，并通过Infinium甲基化微阵列进行基因甲基化分析。生物信息学分析将这些发现与突变数据相结合，遵循在我们最近的研究中已被证明是强大的信号通路的观点。基于其他肿瘤类型的经验，我们的假设是，启动子甲基化引起的拷贝数变化和表达缺失，与突变协同作用，将在对口腔癌发展重要的途径中富集。与其他癌症类型一样，识别这些改变的信号通路可能为口腔癌提供潜在的治疗策略。预计这笔赠款将产生重大的经济影响，导致从事口腔癌基因组研究的个人就业，并在随后的一般头颈癌研究中就业。 公共卫生相关性：我们建议使用高通量编码基因序列分析结合拷贝数、基因表达和甲基化分析来进行口腔癌的全基因组综合突变分析。

DESCRIPTION (provided by applicant): For this ARRA RFA-OD-09-004 GO application, we propose to perform an integrated genome-wide mutational analysis of oral cancers using high throughput sequence analysis of coding genes combined with analyses of copy number, gene expression, and methylation. Genetic alterations represent the underlying cause of human cancer, including tumors of the oral cavity. In the United States, oral cancers represent ~50% of the 46,000 cases of head and neck cancers. Oral cancer affects physical appearance and vital functions, including taste, swallowing, and speech/phonation. In addition to significant morbidity, head and neck cancer result in ~12,000 deaths, of which oral cancer will be responsible for ~5,400 deaths. Given the significant morbidity and mortality of these tumors, there is a profound need for novel clinical approaches for oral cancer. This project will identify potential new avenues for therapeutic, diagnostic and prognostic intervention in oral cancer, and will serve as a model for genomic analyses of other head and neck cancers. For mutational screening, we will employ the two stage, high throughput DNA sequencing approach that we have recently refined and used to identify novel tumor-relevant mutations in breast, colon, pancreatic, and glioblastoma tumors. This strategy significantly increases the power and reduces the cost of large scale tumor sequencing, providing highly sensitive mutational analysis of >95% of bases of ~200,000 exons from over 20,000 coding genes. In the first stage of this approach, exon sequencing analysis will be performed on a set of 24 clinically annotated oral cancer samples. All mutations will be examined in a normal DNA sample from the same patient to identify and confirm tumor-specific mutations. In the second stage, the mutated genes will be analyzed by sequencing a larger set of at least 48 tumors. Previously developed biostatistical criteria will be applied to discriminate between tumor-relevant driver and irrelevant passenger mutations. In the same set of oral cancer samples examined for mutations, we will also perform copy number analysis using high density SNP microarrays, gene expression analysis by combination of next generation sequencing and serial analysis of gene expression (SAGE), and gene methylation analysis by Infinium methylation microarrays. Bioinformatics analyses will integrate these findings with mutational data, following a signaling pathways perspective that has proved to be powerful in our recent studies. Our hypothesis, based on experience in other tumor types, is that copy number changes and expression loss by promoter methylation, acting in concert with mutations, will be enriched in pathways that are important for development of oral cancer. As has been the case in other cancer types, identification of these altered signaling pathways is likely to provide potential therapeutic strategies for oral cancer. This grant is expected to have a significant economic effect, resulting in the employment of individuals performing genomic research on oral cancer and in subsequent studies of head and neck cancers in general. PUBLIC HEALTH RELEVANCE: We propose to perform an integrated genome-wide mutational analysis of oral cancers using high throughput sequence analysis of coding genes combined with analyses of copy number, gene expression, and methylation.