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Dysfunctional immune activation accumulates during chronic viral infection and contributes to disease pathogenesis. In HIV-1, immune activation is exacerbated by concurrent infection with hepatitis C virus (HCV), accelerating depletion of CD4+ T cells. HIV-1 suppression with antiretroviral therapy (ART) generally reconstitutes CD4+ T cell counts, while also reducing the proportion that is activated. Whether this immune reconstitution also reduces the complexity of the CD4+ T cell population is unknown. We sought to characterize the relationship between activated CD4+ T cell repertoire diversity and immune reconstitution following ART in HIV-1/HCV coinfection. We extracted T cell receptor (TCR) sequences from RNA sequencing data obtained from activated CD4+ T cells of HIV-1/HCV coinfected individuals before and after treatment with ART (clinical trial NCT01285050). There was notable heterogeneity in both the extent of CD4+ T cell reconstitution and in the change in activated CD4+ TCR repertoire diversity following ART. Decreases in activated CD4+ TCR repertoire diversity following ART were predictive of the degree of CD4+ T cell reconstitution. The association of decreased activated CD4+ TCR repertoire diversity and improved CD4+ T cell reconstitution may represent loss of nonspecifically activated TCR clonotypes, and possibly selective expansion of specifically activated CD4+ clones. These results provide insight into the dynamic relationship between activated CD4+ TCR diversity and CD4+ T cell recovery of HIV-1/HCV coinfected individuals after suppression of HIV-1 viremia.

Despite advancements in targeting the immune checkpoints program cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T lymphocyte–associated protein 4 (CTLA-4) for cancer immunotherapy, a large number of patients and cancer types remain unresponsive. Current immunotherapies focus on modulating an antitumor immune response by directly or indirectly expanding antitumor CD8 T cells. A complementary strategy might involve inhibition of Tregs that otherwise suppress antitumor immune responses. Here, we sought to identify functional immune molecules preferentially expressed on tumor-infiltrating Tregs. Using genome-wide RNA-Seq analysis of purified Tregs sorted from multiple human cancer types, we identified a conserved Treg immune checkpoint signature. Using immunocompetent murine tumor models, we found that antibody-mediated depletion of 4-1BB–expressing cells (4-1BB is also known as TNFRSF9 or CD137) decreased tumor growth without negatively affecting CD8 T cell function. Furthermore, we found that the immune checkpoint 4-1BB had a high selectivity for human tumor Tregs and was associated with worse survival outcomes in patients with multiple tumor types. Thus, antibody-mediated depletion of 4-1BB–expressing Tregs represents a strategy with potential activity across cancer types.

Mutations in mitochondrial DNA (mtDNA) have been linked to risk, progression, and treatment response of head and neck squamous cell carcinoma (HNSCC). Due to their clonal nature and high copy number, mitochondrial mutations could serve as powerful molecular markers for detection of cancer cells in bodily fluids, surgical margins, biopsies and lymph node (LN) metastasis, especially at sites where tumor involvement is not histologically apparent. Despite a pressing need for high-throughput, cost-effective mtDNA mutation profiling system, current methods for library preparation are still imperfect for detection of low prevalence heteroplasmic mutations. To this end, we have designed an ultra-deep amplicon-based sequencing library preparation approach that covers the entire mitochondrial genome. We sequenced mtDNA in 28 HNSCCs, matched LNs, surgical margins and bodily fluids, and applied multiregional sequencing approach on 14 primary tumors. Our results demonstrate that this quick, sensitive and cost-efficient method allows obtaining a snapshot on the mitochondrial heterogeneity, and can be used for detection of low frequency tumor-associated mtDNA mutations in LNs, sputum and serum specimens. These findings provide the foundation for using mitochondrial sequencing for risk assessment, early detection, and tumor surveillance.

We have recently performed the characterization of alternative splicing events (ASEs) in head and neck squamous cell carcinoma, which allows dysregulation of protein expression common for cancer cells. Such analysis demonstrated a high ASE prevalence among tumor samples, including tumor-specific alternative splicing in the GSN gene.In vitro studies confirmed that overall expression of either ASE-GSN or wild-type GSN (WT-GSN) isoform inversely correlated with cell proliferation, whereas the high ratio of ASE-GSN to WT-GSN correlated with increased cellular invasion. Additionally, a change in expression of either isoform caused compensatory changes in expression of the other isoform. Our results suggest that the overall expression and the balance between GSN isoforms are mediating factors in proliferation, while increased overall expression of ASE-GSN is specific to cancer tissues. As a result, we propose ASE-GSN can serve not only as a biomarker of disease and disease progression, but also as a neoantigen for head and neck squamous cell carcinoma treatment, for which only a limited number of disease-specific targeted therapies currently exist.

Accurately measuring mitochondrial DNA copy number (mtDNAcn) is important for improving our understanding of cancer biology. Prior studies have revealed differences in mtDNAcn between matched bulk tumor-normal pairs from a range of different organs. However, there is little consistency across tumor types; compared to their normal tissue counterparts, some cancers appear to have increased mtDNAcn, others appear to have decreased mtDNAcn, and in others, such as prostate cancer, studies have shown contradictory results. The lack of consistency of mtDNAcn changes in tumor-normal pairs may reflect a failure to address the marked heterogeneity in mtDNAcn across different cell types within normal tissues. In addition, tumor cell heterogeneity in mtDNAcn has also not been taken into account in most prior studies, which can further confound tumor-normal comparisons. Finally, little is known about mtDNAcn alterations in cancer precursor lesions. To address these technical limitations, we combined a recently introduced quantitative in situ hybridization method with immunohistochemistry in a multiplex assay. This enabled us to measure mtDNAcn using digital image analysis in specific cell populations of interest on formalin fixed paraffin embedded (FFPE) and frozen tissue samples. We determined that normal prostate basal cells have approximately 3-fold higher mtDNA levels than normal prostate luminal cells. Compared to the adjacent normal prostate glands, we found markedly increased levels of mtDNA in high-grade prostatic intraepithelial neoplasia (HGPIN), the presumptive precursor lesions to most prostate cancers. We also found increased levels as well as heterogeneity of mtDNAcn in invasive prostate cancer lesions, and more consistently elevated levels in castration-resistant metastatic prostate cancer (CRPC). Increased mtDNA copy number was also identified by whole genome sequencing and quantitative PCR from laser capture micro-dissected human prostate tumor-normal pairs. We also observed higher mtDNAcn in two other precancer lesion types, pancreatic intraepithelial neoplasia and colorectal adenomas. Our approach represents a technological advance as it facilitates mtDNAcn measurements in a manner that preserves morphology, allowing for the evaluation of specific cell populations of interest. These findings raise the hypothesis that increased mitochondrial mass and function may drive the development of precancerous lesions in the prostate, pancreas and colon, as well as invasive prostate cancer development and progression. Citation Format: Jiayu Chen, Qizhi Zheng, Jessica L. Hicks, Levent Trabzonlu, Emmanuel S. Antonarakis, Mark C. Markowski, Samuel R. Denmeade, Busra Ozbek, Anuj Gupta, Tatianna C. Larman, Ralph H. Hruban, Sarah Wheelan, Srinivasan Yegnasubramanian, Angelo M. De Marzo. Increased mitochondrial DNA copy number occurs during prostate cancer progression and in cancer precursor lesions across multiple organs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2404.

Chromatin alterations mediate mutations and gene expression changes in cancer. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) has been utilized to study genome-wide chromatin structure in human cancer cell lines, yet numerous technical challenges limit comparable analyses in primary tumors. Here we have developed a new whole-genome analytic pipeline to optimize ChIP-Seq protocols on patient-derived xenografts from human papillomavirus–related (HPV+) head and neck squamous cell carcinoma (HNSCC) samples. We further associated chromatin aberrations with gene expression changes from a larger cohort of the tumor and normal samples with RNA-Seq data. We detect differential histone enrichment associated with tumor-specific gene expression variation, sites of HPV integration in the human genome, and HPV-associated histone enrichment sites upstream of cancer driver genes, which play central roles in cancer-associated pathways. These comprehensive analyses enable unprecedented characterization of the complex network of molecular changes resulting from chromatin alterations that drive HPV-related tumorigenesis. Cancer Res; 77(23); 6538–50. ©2017 AACR.

Purpose: Although most human cancers display a single histology, there are unusual cases where two or more distinct tissue types present within a primary tumor. One such example is metaplastic breast carcinoma, a rare but aggressive cancer with a heterogeneous histology, including squamous, chondroid, and spindle cells. Metaplastic carcinomas often contain an admixed conventional ductal invasive or in situ mammary carcinoma component, and are typically triple-negative for estrogen receptor, progesterone receptor, and HER-2 amplification/overexpression. An unanswered question is the origin of metaplastic breast cancers. While they may arise independently from their ductal components, their close juxtaposition favors a model that postulates a shared origin, either as two derivatives from the same primary cancer or one histology as an outgrowth of the other. Understanding the mechanism of development of these tumors may inform clinical decisions. Experimental Design: We performed exome sequencing for paired metaplastic and adjacent conventional invasive ductal carcinomas in 8 patients and created a pipeline to identify somatic variants and predict their functional impact, without having normal tissue. We then determined the genetic relationships between the histologically distinct compartments. Results: In each case, the tumor components have nearly identical landscapes of somatic mutation, implying that the differing histologies do not derive from genetic clonal divergence. Conclusions: A shared origin for tumors with differing histologies suggests that epigenetic or noncoding changes may mediate the metaplastic phenotype and that alternative therapeutic approaches, including epigenetic therapies, may be required for metaplastic breast cancers. Clin Cancer Res; 23(16); 4875–84. ©2017 AACR.

The Cancer Genome Atlas (TCGA) sequencing analysis of head and neck squamous cell carcinoma (HNSCC) recently reported on gene fusions, however, few human papillomavirus (HPV) positive samples were included, and the functional relevance of identified fusions was not explored. We therefore performed an independent analysis of gene fusions in HPV-positive oropharyngeal SCC (OPSCC). RNA sequencing was performed on 47 HPV-positive OPSCC primary tumors and 25 normal mucosal samples from cancer unaffected controls on an Illumina TruSeq platform. MapSplice2 was used for alignment and identification of fusion candidates. Putative fusions with less than five spanning reads, detected in normal tissues, or that mapped to the same gene were filtered out. Selected fusions were validated by RT-PCR and Sanger sequencing. Within 47 HPV-positive OPSCC tumors, 282 gene fusions were identified. Most fusions (85.1%) occurred in a single tumor, and the remaining fusions recurred in 2-16 tumors. Gene fusions were associated with significant up regulation of 16 genes (including EGFR and ERBB4) and down regulation of four genes (PTPRT, ZNF750, DLG2, SLCO5A1). Expression of these genes followed similar patterns of up regulation and down regulation in tumors without these fusions compared to normal tissue. Five of six gene fusions selected for validation were confirmed through RT-PCR and sequencing. This integrative analysis provides a method of prioritizing functionally relevant gene fusions that may be expanded to other tumor types. These results demonstrate that gene fusions may be one mechanism by which functionally relevant genes are altered in HPV-positive OPSCC.

BACKGROUND/PURPOSE: Tumor heterogeneity is a now well-recognized phenomenon that can affect the classification, prognosis and treatment of human cancers. Heterogeneity is often described in primary breast cancers based upon histologic subtypes, hormone- and HER2-receptor status, and immunolabeling for various markers, which can be seen within a single tumor as mixed cellular populations, or as separate discrete foci. EXPERIMENTAL DESIGN/METHODS: Here we present a case report of a patient’s primary breast cancer that had two separate but adjacent histologic components, one that was estrogen receptor (ER) positive, and the other ER negative. Each component was subjected to whole exome sequencing and compared for gene identity to determine clonal origin. RESULTS: Using prior bioinformatic tools, we demonstrated that both the ER positive and negative components shared many variants, including passenger and driver alterations. Copy number variations also supported the two components were derived from a single common clone. CONCLUSIONS: These analyses strongly suggest that the two ER components of this patient’s breast cancer were derived from the same clonal origin. Our results have implications for the evolution of breast cancers with mixed histologies, and how they might be best managed for optimal therapy.

Heterogeneity within and among tumors in a metastatic cancer patient is a well-established phenomenon that may confound treatment and accurate prognosis. Here, we used whole-exome sequencing to survey metastatic breast cancer tumors from 5 patients in a rapid autopsy program to construct the origin and genetic development of metastases. Metastases were obtained from 5 breast cancer patients using a rapid autopsy protocol and subjected to whole-exome sequencing. Metastases were evaluated for sharing of somatic mutations, correlation of copy number variation and loss of heterozygosity, and genetic similarity scores. Pathological features of the patients' disease were assessed by immunohistochemical analyses. Our data support a monoclonal origin of metastasis in 3 cases, but in 2 cases, metastases arose from at least 2 distinct subclones in the primary tumor. In the latter 2 cases, the primary tumor presented with mixed histologic and pathologic features, suggesting early divergent evolution within the primary tumor with maintenance of metastatic capability in multiple lineages. We used genetic and histopathological evidence to demonstrate that metastases can be derived from a single or multiple independent clones within a primary tumor. This underscores the complexity of breast cancer clonal evolution and has implications for how best to determine and implement therapies for early- and late-stage disease.

Telomerase consists of at least two essential elements, an RNA component hTR or TERC that contains the template for telomere DNA addition and a catalytic reverse transcriptase (TERT). While expression of TERT has been considered the key rate-limiting component for telomerase activity, increasing evidence suggests an important role for the regulation of TERC in telomere maintenance and perhaps other functions in human cancer. By using three orthogonal methods including RNAseq, RT-qPCR, and an analytically validated chromogenic RNA in situ hybridization assay, we report consistent overexpression of TERC in prostate cancer. This overexpression occurs at the precursor stage (e.g. high-grade prostatic intraepithelial neoplasia or PIN) and persists throughout all stages of disease progression. Levels of TERC correlate with levels of MYC (a known driver of prostate cancer) in clinical samples and we also show the following: forced reductions of MYC result in decreased TERC levels in eight cancer cell lines (prostate, lung, breast, and colorectal); forced overexpression of MYC in PCa cell lines, and in the mouse prostate, results in increased TERC levels; human TERC promoter activity is decreased after MYC silencing; and MYC occupies the TERC locus as assessed by chromatin immunoprecipitation (ChIP). Finally, we show that knockdown of TERC by siRNA results in reduced proliferation of prostate cancer cell lines. These studies indicate that TERC is consistently overexpressed in all stages of prostatic adenocarcinoma and that its expression is regulated by MYC. These findings nominate TERC as a novel prostate cancer biomarker and therapeutic target. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

OBJECTIVE To assess if the reduction in HIV-1 RNA in CD4 T cells is correlated with the persistence of immune activation following early antiretroviral therapy (ART). DESIGN Clinical trial (NCT01285050). METHODS Next-generation sequencing was used to study total RNA from activated CD4 T cells (CD38 and human leukocyte antigen - antigen D related (HLA-DR) expressing) collected from 19 treatment-naive HIV-1/hepatitis C virus-infected patients before and early after ART initiation (≥12 weeks after plasma HIV-1 RNA

Pancreatic ductal adenocarcinoma (PDAC) is nearly uniformly lethal, with a median overall survival in 2014 of only 6 months. The genetic progression of sporadic PDAC (SPC) is well established, with common somatic alterations in KRAS, p16/CDKN2A, TP53, and SMAD4/DPC4. Up to 10 % of all PDAC cases occur in families with two or more affected first-degree relatives (familial pancreatic cancer, FPC), but these cases do not appear to present at an obviously earlier age of onset. This is unusual because most familial cancer syndrome patients present at a substantially younger age than that of corresponding sporadic cases. Here we collated the reported age of onset for FPC and SPC from the literature. We then used an integrated approach including whole exomic sequencing, whole genome sequencing, RNA sequencing, and high density SNP microarrays to study a cohort of FPC cell lines and corresponding germline samples. We show that the four major SPC driver genes are also consistently altered in FPC and that each of the four detection strategies was able to detect the mutations in these genes, with one exception. We conclude that FPC undergoes a similar somatic molecular pathogenesis as SPC, and that the same gene targets can be used for early detection and minimal residual disease testing in FPC patients.

Dyskeratosis congenita is a telomere-mediated syndrome defined by mucocutaneous features. The X-linked mode of inheritance accounts for half the cases, and is thought to predominantly manifest in childhood as bone marrow failure. We identified two male probands who presented in the fifth decade with idiopathic pulmonary fibrosis and cancer. Their pedigrees displayed consecutively affected generations. Five of six females (83%) manifested mucocutaneous features of dyskeratosis congenita, and two had wound-healing complications. No mutations in autosomal dominant telomere genes were present, but exome sequencing revealed novel variants in the X-chromosome DKC1 gene that predicted missense mutations in conserved residues, p.Thr49Ser and p.Pro409Arg. Variants segregated with the telomere phenotype, and affected females were heterozygotes showing skewed X-inactivation. Telomerase RNA levels were compromised in cells from DKC1 mutation carriers, consistent with their pathogenic role. These findings indicate that females with heterozygous DKC1 mutations may be at increased risk for developing telomere phenotypes that, at times, may be associated with clinical morbidity.

The human microbiome may play a role in prostate health and disease as both direct and indirect interactions. Direct interactions between microbiota and prostate cancer include prostate infections, inflammation, prostatitis, and potentially interactions with the urinary microbiome. Indirectly, the gastrointestinal (GI) microbiota may influence prostate cancer via xenobiotic metabolism, augmentation of treatment response, and contributions to systemic inflammation and cytokines. In the present study, we are seeking to understand how different prostate cancer treatments may be affected by the microbial composition of the GI tract. We are currently developing a biorepository of fecal specimens from prostate cancer patients in different clinical states of disease. A key question during the planning phase of this biorepository was if samples can be collected and banked in the form of a rectal swab, instead of the more traditional method of a stool sample. Rectal swabs provide an advantage over collection of stool specimens in that they are easily collected (the patient can self-collect) in a standardized, “on demand” fashion during routine patient visits. Therefore, a pilot study was undertaken to compare the microbial profile of samples collected via both methods from the same individual. We concomitantly collected both stool samples and rectal swabs from 6 patients undergoing active surveillance for prostate cancer at the Johns Hopkins Hospital. All samples were stored at -80°C until we were ready to isolate bacterial DNA. DNA was extracted with a phenol:chloroform based protocol that we have optimized for microbiome studies that includes multiple enzyme digest and bead beating. We conducted Illumina amplicon sequencing of PCR products amplified with universal primers designed against the V6 hypervariable region of the bacterial 16S rRNA gene. We tested our sequencing strategy against Microbiome Reference Standards obtained from American Type Culture Collection (ATCC). The results of our analysis from prostate cancer patients indicated high similarity of bacterial profiles obtained for matched stool and swabs in 4 of the 6 patients. In both of the cases that were dissimilar, there was a greater representation of Enterobacteriaceae in the stool sample versus the swab. We conclude that collection of rectal swabs is a more feasible and convenient means of sampling the GI microbiota in prostate cancer patients, especially when aiming to conduct longitudinal studies with multiple sample collections to correlate microbiota profiles to treatments and/or treatment response. Due to the differences that were identified, we recommend deciding on the sample collection method at the onset of biobanking, and keeping the sampling method consistent throughout. Citation Format: Sarah E. Ernst, Mark C. Markowski, Anuj Gupta, Sarah J. Wheelan, H. Ballentine Carter, Alan W. Partin, Cynthia L. Sears, Karen S. Sfanos. Biobanking and feasibility considerations for prostate cancer gastrointestinal microbiome studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 129.

Although most human cancers display a single histology, there are unusual cases where two or more distinct tissue types present within a primary tumor. One such example is metaplastic breast carcinoma. Metaplastic breast carcinoma is a rare but aggressive cancer with a heterogenous histology, including squamous, chondroid, and spindle cells. Metaplastic carcinomas often contain an admixed conventional ductal invasive or in situ mammary carcinoma component, and are typically triple-negative for estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER-2) amplification/overexpression. An unanswered question is the origin of metaplastic breast cancers. While they may arise independently from their ductal components, their close juxtaposition favors a model that postulates a shared origin, either as two derivatives from the same primary cancer, or one histology as a clonal outgrowth of the other. Understanding the mechanism of development of these tumors may inform clinical decisions. We performed exome sequencing for paired metaplastic and adjacent conventional invasive ductal carcinomas in eight patients and created a pipeline to identify somatic variants and predict their functional impact in these samples, without having matched normal tissue. We then determined the genetic relationships between the histologically distinct compartments. In each case, the tumor components have nearly identical landscapes of somatic variation, implying that the differing histologies do not derive from genetic clonal divergence, suggesting that epigenetic or noncoding changes mediate the metaplastic phenotype. This indicates that alternative therapeutic approaches, including epigenetic therapies, may render metaplastic breast cancers susceptible to current and future therapies. Citation Format: Bracha E. Avigdor, Katie Beierl, Christopher D. Gocke, Daniel J. Zabransky, Karen Cravero, Kelly Kyker-Snowmana, Berry Button, David Chu, Sarah Croessmann, Rory L. Cochran, Roisin M. Connolly, Ben H. Park, Ashley Cimino-Mathews, Sarah J. Wheelan. Whole exome sequencing of metaplastic breast carcinoma indicates monoclonality with associated ductal carcinoma component [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-278. doi:10.1158/1538-7445.AM2017-LB-278

PURPOSE: Analyze the functional role of alternative splicing in HPV-related (HPV+) Head and Neck Squamous Cell Carcinoma (HNSCC) biology and oncogenesis. HPV+ HNSCC incidence is rapidly increasing, while its oncogenic mechanisms, demographic, and clinico-pathological characteristics are largely uncharacterized and discrete from those of HPV- HNSCC. Investigation into the molecular biology of HPV pathogenesis in HNSCC may help with creation of more specific clinical tools for diagnosis and treatment, which are limited and lack accuracy. Alternative Splicing Events (ASEs), post-transcriptional modifications resulting in variant transcripts leading to alternative protein isoforms, have been under-investigated as putative causes of gene expression dysregulation in HPV+ HNSCC. Using RNA-Seq data, a genome-wide ASE discovery analysis was performed on a JHU HPV+ HNSCC cohort, which has expanded HPV+ HNSCC transcriptional characterization by identifying novel ASE biomarkers implicated in carcinogenic progression. Outlier Gene Set Analysis (OGSA) was used to identify genes with high rates of ASE and results were validated in TCGA samples from seven different cancer types. To verify the functional implications of differential isoform expression, vectors for knock-in and knock-down of WT-GSN and ASE-GSN isoforms were developed to evaluate changes in cell proliferation and invasion. ASE-GSN/WT-GSN ratio outliers were identified by OGSA in tumor samples from discovery JHU HPV+ HNSCC cohort, as well as in TCGA public domain data from HNSCC, Lung Squamous Cell Carcinoma, Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma, Bladder Urothelial Carcinoma, Breast Invasive Carcinoma, and Colon Adenocarcinoma. The increased ASE-GSN/WT-GSN ratio was also observed in a cohort of HPV+ Head and Neck Cancer Cell lines. SCC61 and UM-SCC-22B HNSCC cell lines were used to evaluate the function GSN ASEs in vitro. Overall expression levels of both GSN isoforms inversely correlated with cell proliferation whereas the high ratio of ASE-GSN to WT-GSN correlated with increased cellular invasion of stroma in vitro. Additionally, a decrease or increase in expression of one isoform is seen to cause a compensatory increase or decrease in the other isoform respectively. ASE-GSN outliers correlated with HPV+ cancers indicate that expression ratio between GSN isoforms is related to tumor behavior, which is reinforced in vitro where WT-GSN is inversely correlated and ASE-GSN is directly correlated to with cell invasion. These relationships suggest that both overall expression and balance between the two GSN isoforms are mediating factors in cell death or proliferation, while shorter ASE-GSN is more likely to indicate progression to cancer. Finally, we propose a mechanism for the role of GSN isoforms in apoptosis and metastasis, in which trends in expression between WT- and ASE-GSN isoforms can alternatively affect rates of apoptosis or metastatic ability. Citation Format: Dylan Z. Kelley, Emily L. Flam, Theresa Guo, Craig Bohrson, Michael Considine, Ludmila V. Danilova, Justin A. Bishop, Chi Zhang, Wayne M. Koch, David Sidransky, William Westra, Sarah Wheelan, Liliana Florea, Elana J. Fertig, Joseph A. Califano, Daria A. Gaykalova. Functional characterization of alternatively spliced GSN in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4474. doi:10.1158/1538-7445.AM2017-4474

Loss-of-heterozygosity (LOH) analysis of archival tumor tissue can aid in determining the clinical significance of BRCA variants. Here we describe an approach for assessing LOH in formalin-fixed, paraffin-embedded (FFPE) tissues using variant-specific probes and droplet digital polymerase chain reaction (ddPCR). We evaluated LOH in 2 related breast cancer patients harboring a rare missense BRCA2 variant of unknown clinical significance (c.6966G>T; M2322I). Conventional PCR followed by Sanger sequencing suggested a change in allelic abundance in the FFPE specimens. However, we found no evidence of LOH as determined by the allelic ratio (wild type-variant) for BRCA2 in both patients' archival tumor specimens and adjacent normal control tissues using ddPCR. In summary, these experiments demonstrate the utility of ddPCR to quickly and accurately assess LOH in archival FFPE tumor tissue.

Alternative splice events (ASES) are significant components of potential oncogenic pathways alterations and play a critical role in malignant cell transformation in a variety of solid and liquid tumors, including head and neck squamous cell carcinoma (HNSCC). However, high throughput analyses performed to date have not considered ASEs. Therefore, they have detected a limited number of genetic alterations for HNSCC, which incompletely describe the HNSCC specific pathway alterations. The heterogeneous nature of these alterations has made the discovery of reliable HNSCC biomarkers and therapeutic targets for this disease challenging. We performed alternative splice events (ASEs) analysis to enhance our understanding of HNSCC biology. To define ASEs specific to HNSCC we designed a novel bioinformatics pipeline from RNA-sequencing data of HNSCC tumors and independent normal samples. Evaluating the top scoring candidates, we have found several highly promising ASE candidates, including GSN, Gelsolin, an actin-binding protein, a key regulator of actin filament assembly and disassembly. Previously published literature proposes that GSN demonstrates tumor-suppressor properties by reducing cell proliferation in vivo and in vitro via suppression of protein kinase C (PKC, part of the PI3K pathway which is altered in HNSCC). The alternative splicing event involves an insertion of 110 bp from the 14th intron. This insertion contains a stop codon in frame, and the splice variant gives rise to a truncated (562 amino acids(aa)) protein with only 4 Gelsolin domains (instead of the full-length 731 aa protein with 6 Gelsolin domains). Using RNA-Seq data we demonstrated that 40% of tumor samples harbor the GSN-ASE. QRT-PCR confirmed that while total expression of GSN is decreased in HNSCC samples, GSN is expressed in the alternative truncated form only in HNSCC tumors and not normal tissues. Accordingly, total GSN expression is also seen to be downregulated in breast, lung and colon cancers. Evaluation of TCGA data confirmed the pre-dominant expression of the truncated GSN isoform over the wt GSN in HNSCC, bladder urothelial carcinoma, colon adenocarcinoma, lung SCC, breast invasive carcinoma, cervical SCC and endocervical adenocarcinoma. Moreover, we confirmed that that the expression of the truncated GSN is important for the migration and invasion of the cancer cells in vitro. These data suggest that alternative splicing plays an important role in the GSN gene for multiple tumor types. Citation Format: Daria A. Gaykalova, Dylan Kelley, Theresa Guo, Craig Bohrson, Ilse Tiscareno, Veronika Zizkova, Michael Considine, Ludmila Danilova, Emily Flam, Justin Bishop, Julie Ahn, Samantha Merritt, Marla Goldsmith, Chi Zhang, Wayne Koch, William Westra, Zubair Khan, Michael Ochs, Sarah Wheelan, Elana Fertig, Joseph Califano. The discovery of novel GSN alternative splicing in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2880.

L1 retrotransposons comprise 17% of the human genome and are its only autonomous mobile elements. Although L1-induced insertional mutagenesis causes Mendelian disease, their mutagenic load in cancer has been elusive. Using L1-targeted resequencing of 16 colorectal tumor and matched normal DNAs, we found that certain cancers were excessively mutagenized by human-specific L1s, while no verifiable insertions were present in normal tissues. We confirmed de novo L1 insertions in malignancy by both validating and sequencing 69/107 tumor-specific insertions and retrieving both 5′ and 3′ junctions for 35. In contrast to germline polymorphic L1s, all insertions were severely 5′ truncated. Validated insertion numbers varied from up to 17 in some tumors to none in three others, and correlated with the age of the patients. Numerous genes with a role in tumorigenesis were targeted, including ODZ3, ROBO2, PTPRM, PCM1, and CDH11. Thus, somatic retrotransposition may play an etiologic role in colorectal cancer.

The Sleeping Beauty ( SB ) transposon mutagenesis system is a powerful tool that facilitates the discovery of mutations that accelerate tumorigenesis. In this study, we sought to identify mutations that cooperate with MYC , one of the most commonly dysregulated genes in human malignancy. We performed a forward genetic screen with a mouse model of MYC-induced liver cancer using SB-mediated mutagenesis. We sequenced insertions in 63 liver tumor nodules and identified at least 16 genes/loci that contribute to accelerated tumor development. RNAi-mediated knockdown in a liver progenitor cell line further validate three of these genes, Ncoa2/Src-2, Zfx, and Dtnb , as tumor suppressors in liver cancer. Moreover, deletion of Ncoa2/Src-2 in mice predisposes to diethylnitrosamine-induced liver tumorigenesis. These findings reveal genes and pathways that functionally restrain MYC-mediated liver tumorigenesis and therefore may provide targets for cancer therapy.

Human cancer cell lines and xenografts are valuable samples for whole-genome sequencing of human cancer. Tumors can be maintained by serial xenografting in athymic (nude) or severe combined immunodeficient (SCID) mice. In the current study, we developed a molecular assay to quantify the relative contributions of human and mouse in mixed DNA samples. The assay was designed based on deletion/insertion variation between human and mouse genomes. The percentage of mouse DNA was calculated according to the relative peak heights of PCR products analyzed by capillary electrophoresis. Three markers from chromosomes 9 and 10 accurately predicted the mouse genome ratio and were combined into a multiplex PCR reaction. We used the assay to quantify the relative DNA amounts of 93 mouse xenografts used for a recently reported integrated genomic analysis of human pancreatic cancer. Of the 93 xenografts, the mean percentage of contaminating mouse DNA was 47%, ranging from 17% to 73%, with 43% of samples having >50% mouse DNA. We then comprehensively compared the human and mouse genomes to identify 370 additional candidate gene loci demonstrating human-mouse length variation. With increasing whole-genome sequencing of human cancers, this assay should be useful to monitor strategies to enrich human cancer cells from mixed human-mouse cell xenografts. Finally, we discuss how contaminating mouse DNA affects next-generation DNA sequencing.

Background Infection with multiple CMV strains is common in immunocompromised hosts, but its occurrence in normal hosts has not been well-studied. Methods We analyzed CMV strains longitudinally in women who acquired CMV while enrolled in a CMV glycoprotein B (gB) vaccine trial. Sequencing of four variable genes was performed in samples collected from seroconversion and up to 34 months thereafter. Results 199 cultured isolates from 53 women and 65 original fluids from a subset of 19 women were sequenced. 51 women were infected with one strain each without evidence for genetic drift; only two women shed multiple strains. Genetic variability among strains increased with the number of sequenced genetic loci. Nevertheless, 13 of 53 women proved to be infected with an identical CMV strain based on sequencing at all four variable genes. CMV vaccine did not alter the degree of genetic diversity amongst strains. Conclusions Primary CMV infection in healthy women nearly always involves shedding of one strain that remains stable over time. Immunization with CMVgB-1 vaccine strain is not selective against specific strains. Although 75% of women harbored their unique strain, or a strain shared with only one other woman, 25% shared a single common strain, suggesting that this predominant strain with a particular combination of genetic loci is advantageous in this large urban area.

Human cancer cell lines and xenografts are valuable samples for whole-genome sequencing of human cancer. Tumors can be maintained by serial xenografting in athymic (nude) or severe combined immunodeficient (SCID) mice. Critical to next generation based sequencing is the relative proportions of human and mouse DNA. In the current study, we developed a molecular assay to quantify the relative contributions of human and mouse in mixed DNA samples. The assay was designed based on deletion/insertion variation between human and mouse genomes. The percentage of mouse DNA was calculated according to the relative peak heights of PCR products analyzed by capillary electrophoresis. Three markers from chromosomes 9 and 10 accurately predicted the mouse genome ratio and were combined into a multiplex PCR reaction. We used the assay to quantify the relative DNA amounts of 93 mouse xenografts used for a recently reported integrated genomic analysis of human pancreatic cancer. Of the 93 xenografts, the mean % of contaminating mouse DNA was 47%, ranging from 17% to 73%, with 43% of samples having greater than 50% mouse DNA. We then comprehensively compared the human and mouse genomes to identify 370 additional candidate gene loci demonstrating human-mouse length variation. With increasing use of whole genome sequencing of human cancers, this assay should be useful to monitor strategies to enrich human cancer cells from mixed human-mouse cell xenografts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2210.

Summary Female eutherian mammals use X chromosome inactivation (XCI) to epigenetically regulate gene expression from ∼4% of the genome. To quantitatively map the topography of XCI for defined cell types at single cell resolution, we have generated female mice that carry X-linked, Cre-activated, and nuclear-localized fluorescent reporters—GFP on one X chromosome and tdTomato on the other. Using these reporters in combination with different Cre drivers, we have defined the topographies of XCI mosaicism for multiple CNS cell types and of retinal vascular dysfunction in a model of Norrie disease. Depending on cell type, fluctuations in the XCI mosaic are observed over a wide range of spatial scales, from neighboring cells to left versus right sides of the body. These data imply a major role for XCI in generating female-specific, genetically directed, stochastic diversity in eutherian mammals on spatial scales that would be predicted to affect CNS function within and between individuals.