CANCER AND INFLAMMATION GENETICS

1. 乳腺癌和前列腺癌BRCA1和BRCA2基因分析。乳腺癌仍然是美国女性中最常见的恶性肿瘤，也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。对乳腺癌家族的分析发现了两个主要的基因座，BRCA1和BRCA2。我们设计了一项临床研究来解决其中的一些问题，通过社区组织、有大量西班牙裔人口的诊所、公共活动和互联网招募患有乳腺癌的西班牙裔妇女。(http://clinicaltrials.gov/show/NCT01251900)。我们从10个不同的州共招募了200名西班牙裔乳腺癌受试者，完成同意书、问卷、唾液样本和病理报告的收集成功率为88%。使用一种可以通过普通邮件发送的唾液收集设备，可以将收集、运输和DNA提取的材料成本降至25美元以下。总共鉴定出7个明显有害的等位基因，包括一个新描述的等位基因，BRCA1的单核苷酸缺失（6005delT）。其他6个等位基因是独特的，除了BRCA2中的E1308X外，所有等位基因都代表了这些基因中不常见的突变。七个突变中有六个是移码或终止密码子。一个错义变体是一个复合等位基因C1787S和G1788D。这些等位基因在乳腺信息核心（BIC） （http://research.nhgri.nih.gov/projects/bic）中被报道了5次，并被认为是顺式的，在同一个等位基因上；下一代测序证实了这一点。同样的方法被用于对来自波多黎各两个中心的300名未选择的乳腺癌患者进行测序。表2显示了识别的变体。有趣的是，波多黎各乳腺癌主要有BRCA2突变和高频率的E1308X突变。BRCA1和BRCA2突变携带者患前列腺癌以及复发性和致命性疾病的风险更高。因此，我们对沃尔特里德医院的800名前列腺癌患者进行了测序。与brca12相比，这两个基因的变异被鉴定为BRCA2等位基因的比例略高。膀胱癌是美国最常见的肿瘤之一，也是最昂贵的治疗方法。为了了解这种恶性肿瘤的分子基础，我们对99例中国和14例美国原发性膀胱尿路上皮肿瘤进行了外显子组测序。在美国肿瘤中观察到67个基因的体细胞变异，包括10个缺失基因，22个无义基因，78个错义基因和2个剪接突变。在1例肿瘤中有20个基因发生突变，包括已知在膀胱癌中发生突变的基因，如FGFR3、TP53和TSC1。8个基因在3个或更多肿瘤中发生改变，被称为频繁突变基因（FMG），包括先前鉴定的基因KDM6A和ARID1A，分别在4个和3个肿瘤中发生改变。与其他研究相比，四个改变的基因是新的膀胱癌fmg: BRCA1相关蛋白-1 (BAP1)，染色质结构域解旋酶DNA结合蛋白1 (CHD1)，染色质结构域解旋酶DNA结合蛋白1样（CHD1L）和GCN1总控制氨基酸合成1样1 （GCN1L1）。由于这些基因编码的蛋白质在染色质重塑（CRM）中具有不同的作用，因此对这些基因特别感兴趣。拉丁美洲儿童癌症流行病学和遗传分析我们现在有1250多例患者样本，代表了危地马拉国家儿科肿瘤协会（UNOP）现有患者和幸存者的80%。2014年，尼加拉瓜的“La Mascota”医院也开展了类似的工作，该医院正在招募癌症和儿科遗传病患者。这些联合研究是拉丁美洲儿童肿瘤学领域最广泛的研究之一。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%。为了进一步了解导致发病率较高的因素，我们对危地马拉唯一的儿科癌症医院项目厅10年来连续治疗的327例病例进行了分析。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，土著儿童和远离首都的农村地区的发病率较低。这种差异在急性淋巴细胞白血病中更为明显。为了支持我们的假设，即农村土著视网膜母细胞瘤发病率较低是由于诊断晚或未诊断，我们比较了土著和非土著混合病例的死亡率和诊断阶段。我们发现本地病例的死亡率明显更高（65比40%，P= 0.00011）。从诊断开始的生存分析显示本地病例的差异非常显著[危险比2.66 (1.86-3.82)P = 2.45 × 10-8]。4. 子宫颈癌基因组学。宫颈癌是生活在贫困中的妇女中最常见的致命恶性肿瘤，85%以上的死亡发生在世界上最贫穷的妇女中。美洲有3万多名妇女死于宫颈癌。为了确定拉丁美洲宫颈肿瘤妇女的生活方式因素、HPV毒株和分子特征，我们前瞻性地招募了受试者，并在危地马拉的癌症研究所和委内瑞拉的中央大学医院采集了肿瘤组织和血液。293例危地马拉宫颈癌患者平均年龄50岁，33例委内瑞拉宫颈癌患者平均年龄47岁。危地马拉患者的怀孕率明显更高（6.1 vs 4.4），使用口服避孕药或烟草制品的可能性更低，但在家用木头做饭的可能性是前者的10倍。为了鉴定潜在的癌症基因，对23个危地马拉CC和相应的正常dna进行外显子组捕获和测序。在两种或两种以上的肿瘤中预测会发生突变的基因变异，在已知的体细胞癌基因中，或在宫颈癌中先前显示会发生突变的基因中。在已知的癌症基因中发现了预测的体细胞突变，包括PIK3CA、RB1、TP53、MAPK1、HRAS、KRAS、TSC1、BRCA1、BRCA2、BAP1和ATM。使用AmpliSeq Exome协议，对所有外显子扩增相同的肿瘤和正常dna，并进行拷贝数测定。正如之前在宫颈癌中看到的，几乎所有危地马拉肿瘤都有3-5个3q染色体拷贝，14/23在含有TP53位点的17p染色体缺失。共有9/23的宫颈肿瘤表现为广泛的染色体重排或染色体断裂。

1. Analysis of the BRCA1 and BRCA2 genes in Breast and Prostate Cancer. 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. Analysis of breast cancer families resulted in the identification of two major loci, BRCA1 and BRCA2. We designed a clinical study to address some of these issues, with recruitment of Hispanic women with breast cancer through community organizations, clinics with large Hispanic populations, public events, and the internet. (http://clinicaltrials.gov/show/NCT01251900). We recruited a total of 200 Hispanic breast cancer subjects from 10 different states, and had an 88% success rate in completion of collection of consent, questionnaire, saliva sample, and pathology report. The use of a saliva collection device that can be sent by regular mail allowed the materials cost of collection, shipping, and DNA extraction to be under $25. A total of seven clearly deleterious alleles were identified, including a newly described allele, a single nucleotide deletion in BRCA1 (6005delT). The other six alleles were unique and, except for E1308X in BRCA2, all represent mutations uncommonly seen in these genes. Six of the seven mutations are frameshift or termination codons. The one missense variant is a compound allele C1787S and G1788D. These alleles have been reported 5 times in the Breast Information Core (BIC) (http://research.nhgri.nih.gov/projects/bic) and have been proposed to be in cis, on the same allele; next-generation sequencing confirmed this. The same approach was used to sequence 300 unselected breast cancer patients from two centers in Puerto Rico. Table 2 displays the variants identified. Interestingly, Puerto Rican breast cancer has predominately BRCA2 mutations and a high frequency of the E1308X mutation. BRCA1 and BRCA2 mutation carriers have a higher risk of prostate cancer as well as recurrent and lethal disease. Therefore we sequenced 800 prostate cancer patients from Walter Reed Hospital. Variants in both genes were identified with a somewhat higher rate of BRCA2 alleles compared to BRCA1 2. Bladder Cancer: Whole Exome Sequencing and Application Bladder cancer is one of the most common tumors in the US and is the most expensive to treat. To understand the molecular basis of this malignancy we subjected 99 Chinese and 14 US primary urothelial bladder tumors to exome sequencing. Validated somatic variants in US tumors were observed in 67 genes including 10 indels, 22 nonsense, 78 missense, and 2 variants in splice junctions. Twenty genes were mutated in 1 tumor, including genes known to be mutated in bladder cancer such as FGFR3, TP53, and TSC1. Eight genes were altered in 3 or more tumors and are referred to as frequently mutated genes (FMG), including previously identified genes, KDM6A and ARID1A, altered in four and three tumors, respectively. Four altered genes are novel bladder cancer FMGs when compared to other studies: BRCA1 associated protein-1 (BAP1), chromodomain helicase DNA binding protein 1 (CHD1), chromodomain helicase DNA binding protein 1-like (CHD1L), and GCN1 general control of amino-acid synthesis 1-like 1 (GCN1L1). These genes are of particular interest as they encode proteins that have distinct roles in chromatin remodeling (CRM) 3. Pediatric cancer in Latin America-Epidemiology and Genetic Analysis We now have over 1250 patient samples representing 80% of the current patients and survivors from the Unidad Nacional de Oncologia Pediatrica (UNOP), in Guatemala. A similar effort was started in 2014 at the Hospital "La Mascota" in Nicaragua where both cancer and cases of pediatric genetic disease are being recruited. These combined studies represent one of the most extensive for pediatric oncology in Latin America. Retinoblastoma is one of the most common pediatric solid tumors in Mexicoand Central America, accounting for up to 10% of all diagnosed cases. To further understand the factors involved in the higher prevalence, we performed an analysis of 327 consecutive cases treated over 10 years at UNOP the sole pediatric cancer hospital in Guatemala. 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 two-fold 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 ascertainment in indigenous children and in rural areas farther from the capitol. This disparity is even more pronounced in acute lymphocytic leukemia. To support our hypothesis that the lower observed incidence of rural indigenous retinoblastoma cases is due to late or absent diagnosis, we compared the mortality and stage of diagnosis of indigenous and non-indigenous admixed cases. We found a significantly higher mortality of indigenous cases (65 vs. 40%, P= 0.00011). Survival analysis from diagnosis demonstrated a highly significant difference [Hazard Ratio 2.66 (1.86-3.82) P = 2.45x10-8] for indigenous cases. 4. Cervical cancer genomics. Cervical cancer is the most frequently fatal malignancy in women living in poverty with over 85% of deaths occurring in the poorest women of the world. Over 30,000 women die of cervical cancer in the Americas. To determine the lifestyle factors, HPV strains and molecular characteristics of Latin American women with cervical tumors, subjects were prospectively enrolled and tumor tissue and blood collected at the Instituto de Cancerologia (Guatemala) and the Hospital Central Universitario (Venezuela). The 293 patients with cervical cancer (CC) from Guatemala have an average age of 50 years and 33 subjects from Venezuela with CC an average age of 47. The Guatemalan patients have significantly more pregnancies (6.1 vs 4.4) and are less likely to use oral contraceptives or tobacco products, but are 10 times more likely to cook in the home with wood. To identify potential cancer genes, 23 Guatemalan CC and corresponding normal DNAs were subjected to exome capture and sequencing. Variants in genes predicted to be mutated in two or more tumors, in known somatic cancer genes or in genes previously shown to be mutated in cervical cancer. Predicted somatic mutations were found in known cancer genes including PIK3CA, RB1, TP53, MAPK1, HRAS, KRAS, TSC1, BRCA1, BRCA2, BAP1, and ATM. The same tumor and normal DNAs were also amplified for all exons, using the AmpliSeq Exome protocol, and copy number determination performed. As seen previously in cervical cancer, nearly all Guatemalan tumors have 3-5 copies of chromosome 3q, and 14/23 have chromosome loss at 17p containing the TP53 locus. In total 9/23 cervical tumors show evidence of extensive chromosomal rearrangement or chromothripsis.