CANCER AND INFLAMMATION GENETICS

少数民族妇女乳腺癌的分子分析乳腺癌仍然是美国女性最常见的恶性肿瘤，是一个主要的公共卫生问题。尽管在预防、早期发现和治疗方面取得了进展，但每年仍有37，500名妇女死于这种恶性肿瘤。虽然少数民族妇女的乳腺癌发病率较低，但在死亡率、诊断延误和三阴性疾病方面存在着显著的健康差距。对乳腺癌家族的分析导致了两个主要位点的鉴定，BRCA 1和BRCA 2。这些基因和几个次要的高外显基因座（PTEN、CHEK 2、TP 53）一起占多重家族中疾病的45-60%。因此，仍然存在额外的基因赋予乳腺癌的风险。通过建立少数民族乳腺癌妇女队列，我们希望解决其中一些问题。我们已经开始收集来自霍华德大学的非裔美国人样本和来自波多黎各的西班牙裔样本。此外，我们启动了一个全国性的西班牙裔乳腺癌队列，并已开始招募。还与危地马拉城癌症研究所建立了合作关系，并提交了一份收集样本和数据的议定书。2.前列腺癌：外显子组测序和应用的验证我们应用Roche/454外显子组测序技术对单个前列腺癌患者的正常DNA和来自五个不同转移病灶的DNA进行测序。在每个正常和转移性肿瘤上获得超过300万个读数，导致超过25倍的覆盖率。在每个样品中进行变体的预测，并且鉴定了存在于三个或更多个转移性病变中的超过500个变体。我们确定了TET 2基因的突变，TET 2基因是一种以前只在造血系统癌症中突变的基因。启动子区的分析已经确定了基因的起始位点和主要的启动子元件。此外，我们还进行了结合伴侣分析，并确定了新的蛋白质相互作用。3.肾脏肿瘤外显子组和转录组测序为了将高通量测序技术应用于肾癌，我们使用Illumina Solexa仪器进行转录组和外显子组测序。Illumina外显子组方法使用Agilent溶液捕获技术。对来自两个散发性透明细胞肿瘤的DNA进行外显子组捕获并测序至平均深度75倍。从序列分析中，我们确定了101个基因的列表，其中一个新描述的变体影响编码区，在消除某些大的，可变的基因家族，如嗅觉受体。我们通过分析以下特征进一步完善了这一列表：使用Polyphen，SIFT和其他程序预测突变的严重程度;在分析的五个基因组中，有五分之三或更多的基因发生突变;在COSMIC癌症相关突变数据库中该基因存在突变;该基因与癌症相关蛋白相互作用的参与;以及在测序的肿瘤中基因区域中存在杂合性缺失。从这种组合分析中，我们选择了16个基因作为后续分析的最高优先级。我们跟踪了PBRM 1基因的突变，并显示该基因在大约40%的肾脏肿瘤中发生突变。我们对这些相同的肿瘤进行了表达分析，以了解PBRM 1和VHL突变在肾癌表达中的作用。 4.视网膜母细胞瘤是墨西哥和中美洲最常见的儿科实体瘤之一，占所有诊断病例的10%，而在美国和欧洲占所有病例的23%。对墨西哥视网膜母细胞瘤发病率的计算表明，该国各地区的发病率各不相同，与危地马拉接壤的恰帕斯州发病率最高。为了进一步了解患病率较高的相关因素，我们对危地马拉唯一的儿科癌症医院Unidad Nacional de Oncologia Pediatrica（UNOP）治疗超过8年的246例连续病例进行了分析。收集了诊断时的年龄、出生地区、偏侧性、种族和父亲职业的数据，并将该队列与所有急性淋巴细胞白血病病例的队列进行比较，并将儿童检查并发现无癌症作为对照。根据这些数据，我们计算出危地马拉城地区14岁以下儿童视网膜母细胞瘤的发病率为8.1例/百万。这一发病率比美国和欧洲的发病率高两倍，在首都地区的土著和混合人群中相似。发病率的升高不是由于家族性病例的增加，而是环境因素造成的。对土著和混合人口视网膜母细胞瘤发病率的分析表明，远离首都的农村地区土著儿童发病率较低。这种差异在急性淋巴细胞白血病中更为明显。单侧视网膜母细胞瘤占72%。诊断的平均年龄和诊断阶段提前，导致生存率降低。 为了了解危地马拉视网膜母细胞瘤病例的突变谱，我们对14例视网膜母细胞瘤中RB 1基因的所有27个外显子进行了突变扫描和测序。在患者中检测到五种不同的生殖系致癌突变。此外，我们开发了一种灵敏的RB 1启动子甲基化检测方法，并在组织样本上验证了该方法。

Molecular Analysis of Breast Cancer in Minority Women Breast cancer remains the most common malignancy in females in the United States and is a major public health problem. Although progress has been made in prevention, early detection, and therapy, 37,500 women die of this malignancy annually. Although incidence of breast cancer is lower in minority women, significant health disparities exist for mortality, late diagnosis and triple negative disease. Analysis of breast cancer families resulted in the identification of two major loci, BRCA1 and BRCA2. These genes and several minor high-penetrant loci (PTEN, CHEK2, TP53) together account for 45-60% of disease in multiplex families. Therefore, there remain additional genes conferring risk for breast cancer. By establishing cohorts of minority women with breast cancer we hope to address some of these issues. e have begun collecting African American samples from Howard University and Hispanic samples from Puerto Rico. In addition we initiated a nationwide cohort of Hispanic breast cancer and have initiated recruitment. A collaboration with the Instituto Cancerologia in Guatemala City has also been established and a protocol submitted to collect samples and data. 2. Prostate Cancer: Validation of Exome Sequencing and Application We applied Roche/454 exome sequencing technology to the sequencing of normal DNA and DNA from five different metastatic lesions in a single prostate cancer patient. More than 3 million reads were obtained on each of the normal and metastatic tumors resulting in greater than 25-fold coverage. Prediction of variants was carried out in each sample, and more than 500 variants that were present in three or more of the metastatic lesions were identified. We identified mutations in the TET2 gene, a gene previously know to be mutated solely in hematopoetic cancers. Analysis of the promoter region has identified the start site of the gene and major promoter elements. In addition we have performed binding partner analysis and identified new protein interactors. 3. Kidney Tumor Exome and Transcriptome Sequencing To apply the technologies of high-throughput sequencing to renal cancer, we have used Illumina Solexa instrument for both transcriptome and exome sequencing. The Illumina exome approach uses an Agilent solution capture technology. DNA from two sporadic clear cell tumors were exome captured and sequenced to an average depth of 75-fold. From the sequence analysis, we identified a list of 101 genes with a newly described variant affecting the coding region, after eliminating genes from certain large, variable gene families, such as olfactory receptors. We further refined this list by analyzing the following characteristics: predicted severity of the mutation using Polyphen, SIFT, and other programs; presence of the same gene mutated in three out of five or more of the five genomes analyzed; presence of a mutation in that gene in the COSMIC database of cancer-related mutations; involvement of the gene in interactions with cancer-related proteins; and presence of loss of heterozygosity in the gene region in the tumors sequenced. From this combined analysis, we selected 16 genes as top priority for follow-up analysis. We have followed up on mutations of the PBRM1 gene and shown the gene to be mutated in approximately 40% of kidney tumors. We have undertaken expression analysis of these same tumors to understand the role of mutations in PBRM1 and VHL in expression in renal cancer. 4. Retinoblastoma in Latin America-Epidemiology and Genetic Analysis Retinoblastoma is one of the most common pediatric solid tumors in Mexico and Central America, accounting for up to 10% of all diagnosed cases, as opposed to 23% of all cases in the United States and Europe. Incidence calculations of retinoblastoma in Mexico have shown that the incidence varies within regions of the country and is highest in the Chiapas region bordering Guatemala. To further understand the factors involved in the higher prevalence, we performed an analysis of 246 consecutive cases treated over 8 years at the Unidad Nacional de Oncologia Pediatrica (UNOP), the sole pediatric cancer hospital in Guatemala. Data on age at diagnosis, birth region, laterality, ethnicity, and fathers occupation were captured, and this cohort was compared to a cohort of all cases with acute lymphocytic leukemia and as controls, children examined and found to be cancer-free. From this data we calculated the incidence of retinoblastoma to be 8.1 cases/million children under the age of 14 in the Guatemala City region. This incidence is elevated twofold over the incidence in the United States and Europe, and is similar in indigenous and admixed populations in the capital region. The elevated incidence is not due to an increase in familial cases, suggesting an environmental contributor. Analysis of retinoblastoma incidence in indigenous and admixed populations demonstrates a lower incidence in indigenous children in rural areas farther from the capitol. This disparity is even more pronounced in acute lymphocytic leukemia. Unilateral retinoblastoma accounted for 72% of cases. The average age of diagnosis and stage at diagnosis are advanced, resulting in reduced survival. To understand the spectrum of mutations in Guatemalan retinoblastoma cases, we mutation scanned and sequenced all 27 exons of the RB1 gene in 14 retinoblastoma tumors. Five different germline oncogenic mutations were detected in patients. In addition we developed a sensitive assay for methylation of the RB1 promoter and validated this assay on tissue samples.