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

描述(申请人提供)：对于这个ARRA RFA-OD-09-004 GO应用，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来对口腔癌进行整合的全基因组突变分析。基因改变是人类癌症的根本原因，包括口腔肿瘤。在美国，口腔癌约占46,000例头颈癌病例的50%。口腔癌会影响身体外观和重要功能，包括味觉、吞咽和言语/发音。除了严重的发病率外，头颈部癌症还导致约12,000人死亡，其中口腔癌将导致约5,400人死亡。鉴于这些肿瘤的发病率和死亡率很高，迫切需要新的临床方法来治疗口腔癌。该项目将确定口腔癌治疗、诊断和预后干预的潜在新途径，并将作为其他头颈部癌症基因组分析的模型。对于突变筛查，我们将使用我们最近改进的两阶段高通量DNA测序方法，并用于在乳腺、结肠、胰腺和胶质母细胞瘤肿瘤中识别与肿瘤相关的新突变。这一策略显著提高了大规模肿瘤测序的能力并降低了成本，提供了对来自20,000多个编码基因的200,000个外显子中95%的碱基进行高度敏感的突变分析。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。所有突变都将在同一患者的正常DNA样本中进行检测，以识别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的基因。以前开发的生物统计学标准将被应用于区分与肿瘤相关的司机和无关的乘客突变。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，使用下一代测序和基因表达序列分析(SAGE)相结合的方法进行基因表达分析，以及使用Infinium甲基化微阵列进行基因甲基化分析。生物信息学分析将把这些发现与突变数据结合起来，遵循信号通路的观点，这在我们最近的研究中被证明是强大的。我们的假设是，基于对其他肿瘤类型的经验，由启动子甲基化引起的拷贝数变化和表达丢失，与突变一起作用，将在对口腔癌发展至关重要的途径中得到丰富。正如在其他癌症类型中的情况一样，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。这笔赠款预计将产生重大的经济影响，导致雇用个人进行口腔癌症的基因组研究以及随后对头颈癌的一般研究。 公共卫生相关性：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，对口腔癌进行完整的全基因组突变分析。

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.