Transcriptomic and Proteomic Approaches to Discovering Viral Causes for Human Hem

描述（由申请人提供）：全球五分之一的癌症病例是由感染引起的（国际癌症研究机构，2002年），但只有七种病毒被确定会导致人类癌症。我们的实验室发现并鉴定了其中两种病原体，KS疱疹病毒（KSHV/HHV8）和默克尔细胞多瘤病毒（MCV），这两种病毒都是通过高度定向的基因组搜索发现的。这些发现开辟了癌症生物学的新领域，并为改进由这些病毒引起的癌症的诊断、治疗和预防提供了新的基础。癌症遗传学的传统方法几乎完全集中在体细胞突变和大规模测序研究上，可能会错过可能导致某些人类肿瘤的病毒的发现。在过去的四年里，发现了一组七种新的人类多瘤病毒（包括MCV，已知引起默克尔细胞癌（MCC）），它们编码T抗原癌蛋白，是可靠的候选肿瘤病毒。虽然大多数成年人慢性感染这些病毒，但通过癌症基因组解剖计划测序研究没有发现。NCI主任的挑衅性问题倡议#12源于我们最近与MCV相关的MCC研究，表明MCV是一种常见的共生皮肤感染，在病毒基因组发生特定突变后引发肿瘤。这是一种新的致癌机制，除非以有序的方式对非人类病毒序列进行高度定向的搜索，否则不一定能通过癌细胞基因组测序项目发现。为了鉴定人类癌症病毒，我们开发了数字转录组减法（DTS），这是一种深度测序方法，依赖于高保真序列数据库的生成，并提供肿瘤细胞转录的定量数据。虽然标准的深度测序已经被其他人用来搜索病毒，但成功的分析高度依赖于特定的技术技能和假设。2008年，我们使用DTS在MCC中发现了MCV mRNA序列，从而允许随后的全病毒基因组测序和验证性研究。在本应用中，我们通过定向和无偏见的方法，最大限度地提高了在血液恶性肿瘤中识别新型癌症病毒的可能性。我们的定向方法将调查血液学恶性肿瘤的一个大面板的存在，新的人类多瘤病毒。利用从MCV生物学中获得的信息，我们将确定肿瘤特异性病毒突变模式是否存在，以区分因果病毒感染和偶然病毒感染。我们的无偏倚方法将使用DTS检查高度选择的ebv阴性移植后淋巴细胞增生性疾病（EN-PTLD）的金标准病例，以检测新病毒转录物的存在。将对EN-PTLD肿瘤进行高phred等效测序，达到<1转录本/百万（TPM）水平，以鉴定新的病毒转录本。这些数据将与EBV阳性PTLD和CD19阳性外周B细胞的DTS进行比较，以1)确认病毒转录物检测，2)确定EBV阴性和EBV阳性疾病之间细胞基因表达模式的差异。我们还将启动与太平洋西北国家实验室的探索性合作，利用整个EN-PTLD组织样本的无监督LC- MS/MS蛋白质组学开发下一代肿瘤病毒发现技术。从相同组织样本中获得的DTS（转录）和LC-MS/MS（肽）数据的比较，将允许更精确地减去人类序列数据，以识别肿瘤中存在的新型病毒。随着这些目标的完成，我们期望能够回答六种新的人类多瘤病毒中的一种是否有助于淋巴细胞恶性肿瘤，我们将确定EN- PTLD是否含有一种新的病毒。这种系统的方法为发现新的人类癌症病毒提供了最高的可能性，我们将开发新的技术方法，可广泛用于未来寻找人类癌症感染。

DESCRIPTION (provided by applicant): One in 5 cancer cases worldwide is caused by infection (International Agency for Research on Cancer, 2002) and yet only seven viruses have been established to cause human cancers. Our laboratory discovered and characterized two of these agents, KS herpesvirus (KSHV/HHV8) and Merkel cell polyomavirus (MCV), both of which were discovered through highly-directed genomic searches. These findings initiated new fields in cancer biology and provided new bases for improved diagnosis, treatment and prevention of cancers caused by these viruses. Traditional approaches to cancer genetics have focused almost exclusively on somatic cell mutations and large scale sequencing studies are likely to miss discovery of viruses that might be causing some human tumors. Over the past four years, a group of seven new human polyomaviruses (including MCV, known to cause Merkel cell carcinoma (MCC)) were discovered that encode T antigen oncoproteins and are credible candidate tumor viruses. Although most adults are chronically infected with these viruses, none were found through cancer genome anatomy project sequencing studies. The NCI Director's Provocative Question Initiative #12 arises from our recent MCV-related MCC studies showing that MCV, a common commensal skin infection, initiates tumors after specific mutations to the viral genome. This is a new mechanism for carcinogenesis that will not necessarily be found through cancer cell genome sequencing projects unless a highly directed search for nonhuman viral sequences is performed in a orderly fashion. To identify human cancer viruses, we developed digital transcriptome subtraction (DTS), a deep sequencing approach that depends on generation of high-fidelity sequence databases and provides quantitative data on tumor cell transcription. Although standard deep sequencing has been used by others to search for viruses, successful analysis is highly-dependent on specific technical skills and assumptions. In 2008, we used DTS to discover MCV mRNA sequences in MCC, allowing subsequent full viral genome sequencing and confirmatory studies. In this application, we maximize the likelihood for identifying a novel cancer virus (es) in hematologic malignancies through both directed and unbiased approaches. Our directed approach will survey a large panel of hematologic malignancies for presence of the new human polyomaviruses. Using information gained from the biology of MCV, we will determine whether tumor-specific viral mutation patterns are present that differentiate causal from incidental viral infections. Our unbiased approach will use DTS to examine highly-selected gold-standard cases of EBV-negative post-transplant lymphoproliferative disorder (EN-PTLD) for presence of novel viral transcripts. High PHRED-equivalent sequencing of EN-PTLD tumors to <1 transcript per million (TPM) level will be performed to identify novel viral transcripts. These data will be compared to DTS on EBV-positive PTLD and CD19+ peripheral B cells to 1) confirm virus transcript detection and 2) determine differential cellular gene expression patterns between EBV-negative and EBV- positive disease. We will also initiate an exploratory collaboration with the Pacific Northwest National Laboratory to develop the next generation of tumor virus discovery technology using unsupervised LC- MS/MS proteomics of whole EN-PTLD tissue samples. Comparisons of DTS (transcript) and LC-MS/MS (peptide) data, obtained from the same tissue samples, will allow a more precise subtraction of human sequence data to identify novel viruses present in tumors. With the completion of these aims, we anticipate being able to answer whether one of the six new human polyomaviruses contributes to hematolymphoid malignancies and we will determine whether or not EN- PTLD harbors a novel virus. This systematic approach provides the highest probability to find a new human cancer virus and we will develop new technologic approaches that can be widely used in future searches for infections in human cancer. PUBLIC HEALTH RELEVANCE: Cancer viruses cause a large but underappreciated fraction of human cancer cases. New viruses causing cancer can be discovered with new sequencing technologies but are usually overlooked unless the samples are analyzed in a highly-specialized manner. Once a new tumor virus is found, a major obstacle to overcome is determining whether the virus is the cause for the cancer or is a simply a coincidental infection that plays no role in cancer formation. We plan to approach these problems in two ways. We will first perform a broad search for members of a group of newly-discovered viruses, called polyomaviruses, in hematologic cancers. These viruses all encode oncoproteins ("T antigens") that target cellular pathways controlling cell proliferation. For these viruses to cause cancer, they must undergo specific mutations within the cancer cell. These mutations prevent the polyomaviruses from replicating and so finding these mutations distinguishes tumor-causing from coincidental polyomavirus infections in samples that are positive for one or more of the new polyomaviruses. We will next examine in detail one type of hematologic cancer highly likely to be caused by viral infection. Most cases of this malignancy, called post-transplant lymphoproliferative disorder, are caused by a known tumor virus (Epstein-Barr virus, EBV). We will examine cases of post-transplant lymphoproliferative disorder that are negative for EBV and compare them to EBV-positive samples to search for gene products from a new virus. The ability to find EBV fragments in known EBV-positive tumors will serve as a quality- control reference for our studies. If a new virus fragment is found, the whole virus can be rapidly acquired and then tested in a variety of tumors to determine if it is also present in other, more common cancers. We will first use a genomic methodology developed in our laboratory and successfully used to discover one new human cancer virus called Merkel cell polyomavirus (MCV). This method detects messenger RNA from viruses in tumors. We will next couple this technology with a method to detect peptides made in tumor cells through collaboration with scientists at the DOE Pacific Northwest National Laboratory. We anticipate that this combined approach will detect viruses in tumors if they are present and rule out certain classes of viral cause when no virus is found. The data generated by this project can be repurposed to also measure cellular gene expression from tumors so that even if no new viruses are discovered, the critical nonviral genetic changes that contribute to a cancer can be studied.