Fujiko Duke

Sequence analysis of mutations and translocations across breast cancer subtypes

Breast carcinoma is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis and responses to available therapy. Recurrent somatic alterations in breast cancer have been described, including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration. Previous DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA, TP53, AKT1, GATA3 and MAP3K1, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3–AKT3 fusion enriched in triple-negative breast cancer lacking oestrogen and progesterone receptors and ERBB2 expression. The MAGI3–AKT3 fusion leads to constitutive activation of AKT kinase, which is abolished by treatment with an ATP-competitive AKT small-molecule inhibitor.

Genomic analysis identifies association of Fusobacterium with colorectal carcinoma

The tumor microenvironment of colorectal carcinoma is a complex community of genomically altered cancer cells, nonneoplastic cells, and a diverse collection of microorganisms. Each of these components may contribute to carcinogenesis; however, the role of the microbiota is the least well understood. We have characterized the composition of the microbiota in colorectal carcinoma using whole genome sequences from nine tumor/normal pairs. Fusobacterium sequences were enriched in carcinomas, confirmed by quantitative PCR and 16S rDNA sequence analysis of 95 carcinoma/normal DNA pairs, while the Bacteroidetes and Firmicutes phyla were depleted in tumors. Fusobacteria were also visualized within colorectal tumors using FISH. These findings reveal alterations in the colorectal cancer microbiota; however, the precise role of Fusobacteria in colorectal carcinoma pathogenesis requires further investigation.

Landscape of genomic alterations in cervical carcinomas

Cervical cancer is responsible for 10–15% of cancer-related deaths in women worldwide. The aetiological role of infection with high-risk human papilloma viruses (HPVs) in cervical carcinomas is well established. Previous studies have also implicated somatic mutations in PIK3CA, PTEN, TP53, STK11 and KRAS as well as several copy-number alterations in the pathogenesis of cervical carcinomas. Here we report whole-exome sequencing analysis of 115 cervical carcinoma–normal paired samples, transcriptome sequencing of 79 cases and whole-genome sequencing of 14 tumour–normal pairs. Previously unknown somatic mutations in 79 primary squamous cell carcinomas include recurrent E322K substitutions in the MAPK1 gene (8%), inactivating mutations in the HLA-B gene (9%), and mutations in EP300 (16%), FBXW7 (15%), NFE2L2 (4%), TP53 (5%) and ERBB2 (6%). We also observe somatic ELF3 (13%) and CBFB (8%) mutations in 24 adenocarcinomas. Squamous cell carcinomas have higher frequencies of somatic nucleotide substitutions occurring at cytosines preceded by thymines (Tp*C sites) than adenocarcinomas. Gene expression levels at HPV integration sites were statistically significantly higher in tumours with HPV integration compared with expression of the same genes in tumours without viral integration at the same site. These data demonstrate several recurrent genomic alterations in cervical carcinomas that suggest new strategies to combat this disease.

Sequence-Based Discovery of Bradyrhizobium enterica in Cord Colitis Syndrome

BACKGROUND Immunosuppression is associated with a variety of idiopathic clinical syndromes that may have infectious causes. It has been hypothesized that the cord colitis syndrome, a complication of umbilical-cord hematopoietic stem-cell transplantation, is infectious in origin. METHODS We performed shotgun DNA sequencing on four archived, paraffin-embedded endoscopic colon-biopsy specimens obtained from two patients with cord colitis. Computational subtraction of human and known microbial sequences and assembly of residual sequences into a bacterial draft genome were performed. We used polymerase-chain-reaction (PCR) assays and fluorescence in situ hybridization to determine whether the corresponding bacterium was present in additional patients and controls. RESULTS DNA sequencing of the biopsy specimens revealed more than 2.5 million sequencing reads that did not match known organisms. These sequences were computationally assembled into a 7.65-Mb draft genome showing a high degree of homology with genomes of bacteria in the bradyrhizobium genus. The corresponding newly discovered bacterium was provisionally named Bradyrhizobium enterica. PCR identified B. enterica nucleotide sequences in biopsy specimens from all three additional patients with cord colitis whose samples were tested, whereas B. enterica sequences were absent in samples obtained from healthy controls and patients with colon cancer or graft-versus-host disease. CONCLUSIONS We assembled a novel bacterial draft genome from the direct sequencing of tissue specimens from patients with cord colitis. Association of these sequences with cord colitis suggests that B. enterica may be an opportunistic human pathogen. (Funded by the National Cancer Institute and others.)

Oncogenic RIT1 mutations in lung adenocarcinoma.

Lung adenocarcinoma is comprised of distinct mutational subtypes characterized by mutually exclusive oncogenic mutations in RTK/RAS pathway members KRAS, EGFR, BRAF and ERBB2, and translocations involving ALK, RET and ROS1. Identification of these oncogenic events has transformed the treatment of lung adenocarcinoma via application of therapies targeted toward specific genetic lesions in stratified patient populations. However, such mutations have been reported in only ∼55% of lung adenocarcinoma cases in the United States, suggesting other mechanisms of malignancy are involved in the remaining cases. Here we report somatic mutations in the small GTPase gene RIT1 in ∼2% of lung adenocarcinoma cases that cluster in a hotspot near the switch II domain of the protein. RIT1 switch II domain mutations are mutually exclusive with all other known lung adenocarcinoma driver mutations. Ectopic expression of mutated RIT1 induces cellular transformation in vitro and in vivo, which can be reversed by combined PI3K and MEK inhibition. These data identify RIT1 as a driver oncogene in a specific subset of lung adenocarcinomas and suggest PI3K and MEK inhibition as a potential therapeutic strategy in RIT1-mutated tumors.

The tumor virus landscape of AIDS-related lymphomas

Immunodeficiency dramatically increases susceptibility to cancer as a result of reduced immune surveillance and enhanced opportunities for virus-mediated oncogenesis. Although AIDS-related lymphomas (ARLs) are frequently associated with known oncogenic viruses, many cases contain no known transforming virus. To discover novel transforming viruses, we profiled a set of ARL samples using whole transcriptome sequencing. We determined that Epstein-Barr virus (EBV) was the only virus detected in the tumor samples of this cohort, suggesting that if unidentified pathogens exist in this disease, they are present in <10% of cases or undetectable by our methods. To evaluate the role of EBV in ARL pathogenesis, we analyzed viral gene expression and found highly heterogeneous patterns of viral transcription across samples. We also found significant heterogeneity of viral antigen expression across a large cohort, with many patient samples presenting with restricted type I viral latency, indicating that EBV latency proteins are under increased immunosurveillance in the post-combined antiretroviral therapies era. Furthermore, EBV infection of lymphoma cells in HIV-positive individuals was associated with a distinct host gene expression program. These findings provide insight into the joint host-virus regulatory network of primary ARL tumor samples and expand our understanding of virus-associated oncogenesis. Our findings may also have therapeutic implications, as treatment may be personalized to target specific viral and virus-associated host processes that are only present in a subset of patients.

In search of a candidate pathogen for giant cell arteritis: sequencing-based characterization of the giant cell arteritis microbiome.

To characterize the microbiome of the temporal artery in patients with giant cell arteritis (GCA), and to apply an unbiased and comprehensive shotgun sequencing‐based approach to determine whether there is an enrichment of candidate pathogens in the affected tissue.

Malawi Polyomavirus Is a Prevalent Human Virus That Interacts with Known Tumor Suppressors

ABSTRACT Malawi polyomavirus (MWPyV) is a recently identified human polyomavirus. Serology for MWPyV VP1 indicates that infection frequently occurs in childhood and reaches a prevalence of 75% in adults. The MWPyV small T antigen (ST) binds protein phosphatase 2A (PP2A), and the large T antigen (LT) binds pRb, p107, p130, and p53. However, the MWPyV LT was less stable than the simian virus 40 (SV40) LT and was unable to promote the growth of normal cells. This report confirms that MWPyV is a widespread human virus expressing T antigens with low transforming potential.

DNA copy number analysis of metastatic urothelial carcinoma with comparison to primary tumors

BackgroundTo date, there have been no reports characterizing the genome-wide somatic DNA chromosomal copy-number alteration landscape in metastatic urothelial carcinoma. We sought to characterize the DNA copy-number profile in a cohort of metastatic samples and compare them to a cohort of primary urothelial carcinoma samples in order to identify changes that are associated with progression from primary to metastatic disease.MethodsUsing molecular inversion probe array analysis we compared genome-wide chromosomal copy-number alterations between 30 metastatic and 29 primary UC samples. Whole transcriptome RNA-Seq analysis was also performed in primary and matched metastatic samples which was available for 9 patients.ResultsBased on a focused analysis of 32 genes in which alterations may be clinically actionable, there were significantly more amplifications/deletions in metastases (8.6% vs 4.5%, p < 0.001). In particular, there was a higher frequency of E2F3 amplification in metastases (30% vs 7%, p = 0.046). Paired primary and metastatic tissue was available for 11 patients and 3 of these had amplifications of potential clinical relevance in metastases that were not in the primary tumor including ERBB2, CDK4, CCND1, E2F3, and AKT1. The transcriptional activity of these amplifications was supported by RNA expression data.ConclusionsThe discordance in alterations between primary and metastatic tissue may be of clinical relevance in the era of genomically directed precision cancer medicine.

A121: In Search of Infectious Triggers of Periodic Fever, Aphthous Stomatitis, Pharyngitis and Adenitis Syndrome

Periodic fever, aphthous stomatitis, pharyngitis and adenitis (PFAPA) syndrome is the most prevalent pediatric autoinflammatory fever disorder. As there is no known genetic cause or confirmatory test, diagnosis of PFAPA is challenging. Clockwork interval between episodes is a characteristic feature, which aids in diagnosis. The true prevalence and the etiology of PFAPA are not known, but genetic factors leading to immune dysregulation as well as infectious agents have been suggested as causative factors. Oral corticosteroids abort the episodes in majority of patients, and in recent years, tonsillectomy has been shown to be effective in inducing remission. Our center has a significant success rate with tonsillectomy in PFAPA patients. Using unbiased next‐generation sequencing, we investigated the presence of potential infectious agents and gene expression signatures in tonsils from patients with PFAPA, chronic tonsillitis and obstructive sleep apnea (OSA).