Faculty Bibliography

Background/UNASSIGNED:Transposable elements make up a significant portion of the human genome. Accurately locating these mobile DNAs is vital to understand their role as a source of structural variation and somatic mutation. To this end, laboratories have developed strategies to selectively amplify or otherwise enrich transposable element insertion sites in genomic DNA. Results/UNASSIGNED:Here we describe a technique, Transposon Insertion Profiling by sequencing (TIPseq), to map Long INterspersed Element 1 (LINE-1, L1) retrotransposon insertions in the human genome. This method uses vectorette PCR to amplify species-specific L1 (L1PA1) insertion sites followed by paired-end Illumina sequencing. In addition to providing a step-by-step molecular biology protocol, we offer users a guide to our pipeline for data analysis, TIPseqHunter. Our recent studies in pancreatic and ovarian cancer demonstrate the ability of TIPseq to identify invariant (fixed), polymorphic (inherited variants), as well as somatically-acquired L1 insertions that distinguish cancer genomes from a patient's constitutional make-up. Conclusions/UNASSIGNED:TIPseq provides an approach for amplifying evolutionarily young, active transposable element insertion sites from genomic DNA. Our rationale and variations on this protocol may be useful to those mapping L1 and other mobile elements in complex genomes.

BACKGROUND:Chagas disease is still prevalent in rural areas of South America. In endemic areas of Bolivia, school children are screened for the program of Chagas disease eradication of the Ministry of Health, and positive children are treated. Here, we compared the fecal, oral and skin microbiomes of children with or without Chagas disease, and before and after benznidazol treatment of infected children. METHODS:A total of 543 Bolivian children (5-14 years old) were tested for Chagas disease, and 20 positive children were treated with Benznidazole. Fecal samples and oral and skin swabs were obtained before and after treatment, together with samples from a group of 35 uninfected controls. The 16S rRNA genes were sequenced and analyzed using QIIME to determine Alpha diversity differences and community distances, and linear discriminant analyses to determine marker taxa by infection status or treatment. RESULTS:Twenty out of 543 children screened were seropositive for Chagas disease (3.7%) and were included in the study, together with 35 control children that were seronegative for the disease. Fecal samples, oral and skin swabs were taken at the beginning of the study and after the anti-protozoa therapy with Benznidazole to the chagasic children. Infected children had higher fecal Firmicutes (Streptococcus, Roseburia, Butyrivibrio, and Blautia), and lower Bacteroides and also showed some skin -but not oral- microbiota differences. Treatment eliminated the fecal microbiota differences from control children, increasing Dialister (class Clostridia) and members of the Enterobacteriaceae, and decreasing Prevotella and Coprococcus, with minor effects on the oral and skin bacterial diversity. CONCLUSIONS:The results of this study show differences in the fecal microbiota associated with Chagas disease in children, and also evidence that treatment normalizes fecal microbiota (makes it more similar to that in controls), but is associated with oral and skin microbiota differences from control children. Since microbiota impacts in children, it is important to determine the effect of drugs on the children microbiota, since dysbiosis could lead to physiological effects which might be avoidable with microbiota restoration interventions.

BACKGROUND:In Latin America (LA), there is a high incidence rate of breast cancer (BC) in premenopausal women, and the genomic features of these BC remain unknown. Here, we aim to characterize the molecular features of BC in young LA women within the framework of the PRECAMA study, a multicenter population-based case-control study of BC in premenopausal women. METHODS:Pathological tumor tissues were collected from incident cases from four LA countries. Immunohistochemistry (IHC) was performed centrally for ER, PR, HER2, Ki67, EGFR, CK5/6, and p53 protein markers. Targeted deep sequencing was done on genomic DNA extracted from formalin-fixed, paraffin-embedded tumor tissues and their paired blood samples to screen for somatic mutations in eight genes frequently mutated in BC. A subset of samples was analyzed by exome sequencing to identify somatic mutational signatures. RESULTS:The majority of cases were positive for ER or PR (168/233; 72%), and 21% were triple-negative (TN), mainly of basal type. Most tumors were positive for Ki67 (189/233; 81%). In 126 sequenced cases, TP53 and PIK3CA were the most frequently mutated genes (32.5% and 21.4%, respectively), followed by AKT1 (9.5%). TP53 mutations were more frequent in HER2-enriched and TN IHC subtypes, whereas PIK3CA/AKT1 mutations were more frequent in ER-positive tumors, as expected. Interestingly, a higher proportion of G:C>T:A mutations was observed in TP53 in PRECAMA cases compared with TCGA and METABRIC BC series (27% vs 14%). Exome-wide mutational patterns in 10 TN cases revealed alterations in signal transduction pathways and major contributions of mutational signatures caused by altered DNA repair pathways. CONCLUSIONS:These pilot results on PRECAMA tumors give a preview of the molecular features of premenopausal BC in LA. Although the overall mutation burden was as expected from data in other populations, mutational patterns observed in TP53 and exome-wide suggested possible differences in mutagenic processes giving rise to these tumors compared with other populations. Further -omics analyses of a larger number of cases in the near future will enable the investigation of relationships between these molecular features and risk factors.

Focal radiation therapy enhances systemic responses to anti-CTLA-4 antibodies in preclinical studies and in some patients with melanoma^1-3, but its efficacy in inducing systemic responses (abscopal responses) against tumors unresponsive to CTLA-4 blockade remained uncertain. Radiation therapy promotes the activation of anti-tumor T cells, an effect dependent on type I interferon induction in the irradiated tumor^4-6. The latter is essential for achieving abscopal responses in murine cancers⁶. The mechanisms underlying abscopal responses in patients treated with radiation therapy and CTLA-4 blockade remain unclear. Here we report that radiation therapy and CTLA-4 blockade induced systemic anti-tumor T cells in chemo-refractory metastatic non-small-cell lung cancer (NSCLC), where anti-CTLA-4 antibodies had failed to demonstrate significant efficacy alone or in combination with chemotherapy^7,8. Objective responses were observed in 18% of enrolled patients, and 31% had disease control. Increased serum interferon-β after radiation and early dynamic changes of blood T cell clones were the strongest response predictors, confirming preclinical mechanistic data. Functional analysis in one responding patient showed the rapid in vivo expansion of CD8 T cells recognizing a neoantigen encoded in a gene upregulated by radiation, supporting the hypothesis that one explanation for the abscopal response is radiation-induced exposure of immunogenic mutations to the immune system.

BACKGROUND:In lung cancer, upregulation of the PI3K pathway is an early event that contributes to cell proliferation, survival, and tissue invasion. Upregulation of this pathway was recently described as associated with enrichment of the lower airways with bacteria identified as oral commensals. We hypothesize that host-microbe interactions in the lower airways of subjects with lung cancer affect known cancer pathways. METHODS:Airway brushes were collected prospectively from subjects with lung nodules at time of diagnostic bronchoscopy, including 39 subjects with final lung cancer diagnoses and 36 subjects with non-cancer diagnosis. Additionally, samples from 10 healthy control subjects were included. 16S rRNA gene amplicon sequencing and paired transcriptome sequencing (RNAseq) were performed on all airway samples. In addition, an in vitro model with airway epithelial cells exposed to bacteria/bacterial products was performed. RESULTS:The composition of the lower airway transcriptome in the cancer patients was significantly different from the controls, which included upregulation of ERK and PI3K signaling pathways. The lower airways of lung cancer patients were enriched for oral taxa (Streptococcus and Veillonella), which was associated with upregulation of the ERK and PI3K signaling pathways. In vitro exposure of airway epithelial cells to Veillonella, Prevotella, and Streptococcus led to upregulation of these same signaling pathways. CONCLUSIONS:The data presented here shows that several transcriptomic signatures previously identified as relevant to lung cancer pathogenesis are associated with enrichment of the lower airway microbiota with oral commensals.

Venous thromboembolism (VTE), comprised of deep vein thrombosis and pulmonary embolism, is a common health problem both in the United States and worldwide, with significant associated morbidity and mortality. Despite multiple known genetic and situational risk factors, an estimated 30% of all events remain classified as idiopathic, demonstrating a significant knowledge gap in the pathophysiology VTE. While platelets are well established as an essential contributor to thrombus formation and there has been recent interest in the role of neutrophil extracellular traps, specific cell types and pathways involved in the pathogenesis of VTE remain uncertain. In this study, our primary aims were to define a unique transcriptional signature for VTE and to identify the types of cells and specific pathways involved in development of VTE. Whole blood was collected in PAX gene tubes and RNA sequencing for coding mRNA was performed in an unbiased manner in 201 patients with prevalent VTE as well as 43 healthy controls. We used a bioinformatics approach to develop a unique signature for VTE by identifying differentially expressed genes, developing cell-type modules, and ascertaining pathways driving differentially expressed transcripts. We performed additional analyses on subgroups of patients with idiopathic VTE, patients with incident VTE, and VTE patients matched to healthy controls by age and sex. We went on to use machine learning methods to learn models that best differentiate VTE patients from healthy controls and validated it on a left out test set within our VTE population. Genes specific to neutrophils, erythrocytes, and platelets, in that order, were most significantly upregulated in patients with VTE compared to healthy controls. Genes related to T-cells were downregulated. Pathway analysis revealed upregulated neutrophil activation and degranulation, erythrocyte differentiation and homeostasis, and platelet degranulation. A gene signature of 217 transcripts was outstanding at differentiating patients with VTE versus healthy controls (AUC 0.94). Following adjustment for age, sex, and race/ethnicity our genetic signature remained significantly robust at differentiating patients with VTE versus controls (AUC 0.83). Our expression signature remained stable across patients with idiopathic VTE (AUC 0.93), and in patients who went on to develop future VTE events (AUC 0.95). In summary, we have demonstrated a whole blood transcriptional signature for prevalent and incident VTE. Genes related to neutrophils, erythrocytes, and platelets are upregulated in patients with VTE and genes related to T-cells were downregulated. These findings suggest an active role of cell types once thought to be passively entrapped within thrombus and provide new areas of study to establish the pathophysiology of VTE

Unveiling the transcriptome of human blastocysts can provide a wealth of important information regarding early embryonic ontology. Comparing the mRNA production of embryos with normal and abnormal karyotypes allows for a deeper understanding of the protein pathways leading to viability and aberrant fetal development. In addition, identifying transcripts specific for normal or abnormal chromosome copy number could aid in the search for secreted substances that could be used to non-invasively identify embryos best suited for IVF embryo transfer. Using RNA-seq, we characterized the transcriptome of 71 normally developing human blastocysts that were karyotypically normal vs. trisomic or monosomic. Every monosomy and trisomy of the autosomal and sex chromosomes were evaluated, mostly in duplicate. We first mapped the transcriptome of three normal embryos and found that a common core of more than 3,000 genes is expressed in all embryos. These genes represent pathways related to actively dividing cells, such as ribosome biogenesis and function, spliceosome, oxidative phosphorylation, cell cycle and metabolic pathways. We then compared transcriptome profiles of aneuploid embryos to those of normal embryos. We observed that non-viable embryos had a large number of dysregulated genes, some showing a hundred-fold difference in expression. On the contrary, sex chromosome abnormalities, XO and XXX displayed transcriptomes more closely mimicking those embryos with 23 normal chromosome pairs. Intriguingly, we identified a set of commonly deregulated genes in the majority of both trisomies and monosomies. This is the first paper demonstrating a comprehensive transcriptome delineation of karyotypic abnormalities found in the human pre-implantation embryo. We believe that this information will contribute to the development of new pre-implantation genetic screening methods as well as a better understanding of the underlying developmental abnormalities of abnormal embryos, fetuses and children.

Purpose: Acrylamide, a probable human carcinogen (Group 2A), is ubiquitously present in the human environment. The compound is found in heated starchy foods, coffee, as well as cigarette smoke. Humans are also exposed to acrylamide occupationally. The carcinogenicity of acrylamide has been attributed to the effects of glycidamide, its reactive and mutagenic epoxide metabolite shown to be an inducer of rodent tumors at various anatomical sites. Covalent adducts with haemoglobin were reported in humans exposed to acrylamide, and DNA adducts in experimental systems. Methods: In order to characterize the pre-mutagenic DNA lesions and global mutation spectra induced by acrylamide and glycidamide, we combined DNA-adduct and wholeexome sequencing analyses in an established exposureclonal immortalization system based on mouse embryonic fibroblasts. Results: Sequencing and computational analysis of cell clones immortalized after exposure revealed a unique mutational signature of glycidamide, characterized by predominant T:A>A:T transversions, T:A>C:G transitions and C:G>A:T transversions in specific trinucleotide contexts, and with significant transcription strand bias. Computational interrogation of the human pan-cancer genome sequencing data (PCAWG data set) using experimentally derived glycidamide signature revealed the presence of the glycidamide signature in lung adenocarcinomas, lung squamous cell carcinomas and liver cancer, manifesting as a sub-component of the COSMIC tobacco smoking-related signature 4, as well as possibly associated with dietary intake of acrylamide. Conclusions: Our study demonstrates how the controlled experimental characterization of specific genetic damage associated with glycidamide exposure facilitates identifying corresponding patterns in cancer genome data, thereby underscoring how mutational signature laboratory experimentation contributes to the elucidation of cancer causation

Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. Here, we analyzed pediatric and adult pineoblastoma samples (n = 23) using a combination of genome-wide DNA methylation profiling and whole-exome sequencing or whole-genome sequencing. Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower-grade pineal tumors and normal pineal gland. Recurrent variants were found in genes involved in PKA- and NF-κB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expresion of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain.