CANCER AND INFLAMMATION GENETICS

乳腺癌仍然是美国女性中最常见的恶性肿瘤，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、晚期诊断和三阴性疾病方面存在巨大的健康差距。对乳腺癌家族的分析发现了两个主要的基因座，BRCA1和BRCA2。这些基因和几个次要的高渗透位点（PTEN, CHEK2, TP53）共同占多家族疾病的45-60%。因此，还有其他基因会增加患乳腺癌的风险。通过建立少数民族乳腺癌妇女队列，我们希望解决其中的一些问题。我们已经开始从霍华德大学收集非裔美国人样本，从波多黎各收集西班牙裔样本。此外，我们启动了一个全国性的西班牙裔乳腺癌队列，并开始招募。还与危地马拉城的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. 我们将Roche/454外显子组测序技术应用于单个前列腺癌患者的正常DNA和来自五个不同转移灶的DNA测序。在每个正常和转移性肿瘤上获得了超过300万次读取，导致超过25倍的覆盖率。在每个样本中进行变异预测，并确定了存在于三个或更多转移性病变中的500多种变异。我们发现了TET2基因的突变，这个基因以前只在造血癌症中发生突变。通过对启动子区域的分析，确定了该基因的起始位点和主要启动子元件。此外，我们还进行了结合伙伴分析，并确定了新的蛋白质相互作用物。3. 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外显子组方法使用安捷伦溶液捕获技术。从两个散发性透明细胞肿瘤中捕获DNA外显子组并测序至平均深度75倍。从序列分析中，我们确定了101个具有新描述的影响编码区变异的基因，在排除某些大型可变基因家族（如嗅觉受体）的基因后。通过分析以下特征，我们进一步完善了这个列表：使用Polyphen、SIFT和其他程序预测突变的严重程度；在被分析的5个基因组中，5个或更多的基因组中有3个存在相同的基因突变；在COSMIC癌症相关突变数据库中存在该基因突变；参与基因与癌症相关蛋白的相互作用；在肿瘤序列中存在基因区域的杂合性缺失。从这个综合分析中，我们选择了16个基因作为后续分析的重中之重。我们对PBRM1基因的突变进行了跟踪研究，发现该基因在大约40%的肾肿瘤中发生突变。我们对这些肿瘤进行了表达分析，以了解PBRM1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童实体肿瘤之一，占所有诊断病例的10%，而在美国和欧洲，这一比例为23%。墨西哥视网膜母细胞瘤的发病率计算表明，该国不同地区的发病率各不相同，在与危地马拉接壤的恰帕斯地区发病率最高。为了进一步了解高患病率的相关因素，我们对危地马拉唯一的儿科癌症医院——国家儿科肿瘤医院（UNOP） 8年来连续治疗的246例病例进行了分析。收集了诊断年龄、出生地区、侧卧、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，并将检查后发现无癌症的儿童作为对照。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口中也类似。发病率的升高不是由于家族病例的增加，而是环境因素。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的发病率较低。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%。平均诊断年龄和诊断阶段较早，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例视网膜母细胞瘤肿瘤的RB1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种系致癌突变。此外，我们开发了一种敏感的RB1启动子甲基化检测方法，并在组织样本上验证了该检测方法。

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.