Faculty Bibliography

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/PURPOSE/OBJECTIVE:To search for a transmissible agent involved in lupus pathogenesis, we investigated the faecal microbiota of patients with systemic lupus erythematosus (SLE) for candidate pathobiont(s) and evaluated them for special relationships with host immunity. METHODS:In a cross-sectional discovery cohort, matched blood and faecal samples from 61 female patients with SLE were obtained. Faecal 16 S rRNA analyses were performed, and sera profiled for antibacterial and autoantibody responses, with findings validated in two independent lupus cohorts. RESULTS:strain-restricted antibodies were detected in those with active nephritis (including Class III and IV) in the discovery cohort, with findings validated in two independent cohorts. CONCLUSION/CONCLUSIONS:These findings suggest a novel paradigm in which specific strains of a gut commensal may contribute to the immune pathogenesis of lupus nephritis.

Here, we report the draft genome of Streptococcus halitosis sp. nov. strain VT-4, a novel bacterium isolated from the dorsal part of the tongue of a patient with halitosis. The genome comprised 1,880,608 bp with a GC content of 41.0%. There were 1,782 predicted protein-coding genes, including those associated with virulence and antibiotic resistance.

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.

Microglandular adenosis (MGA) is a rare breast lesion reported to be associated with invasive carcinoma in up to 20-30% of cases, and has been proposed as a non-obligate precursor to basal-like breast cancers. We identified a case of matrix-producing metaplastic carcinoma with morphologic and immunohistochemical evidence of progression from MGA to atypical MGA (AMGA), carcinoma in situ (CIS) and invasive carcinoma. We performed whole exome sequencing of each component (MGA, AMGA, CIS and cancer) to characterize the mutational landscape of these foci. There was significant copy number overlap between all foci, including a segmental amplification of the CCND1 locus (partial chromosome 11 trisomy) and MYC (8q24.12-13). Using a bioinformatics approach, we were able to identify three putative mutational clusters and recurrent, stop-gain non-synonymous mutations in both ZNF862 and TP53 that were shared across all foci. Finally, we identified a novel deleterious splice-acceptor site mutation of chr5:5186164G>T (chromosome 5p15) encoding the gene, ADAMTS16, in the invasive component.

Introduction The analysis of somatic mutational signatures in a single tumour can reveal clues on the natural history of this tumour, including past exposures to carcinogens. Characterising the mutational signatures of potential carcinogens used in industry may thus help identify cancers that are due to occupational exposure. Here, we used experimental models to define the mutational signatures of dichloromethane (DCM) and 1,2- dichloropropane (DCP), two solvents suspected to cause cholangiocarcinomas in occupational settings. Material and methods Experimentally induced tumours and matching normal tissues from mice exposed to DCP (by gavage), DCM (by gavage or inhalation) or DCP +DCM (by gavage), and spontaneous tumours from vehicle/sham-exposed mice were analysed by whole-exome sequencing. Somatic mutation calling was performed to define exome-wide mutation patterns induced by DCP and DCM in these assays. Mutation data obtained in biliary tract cancers of workers in the printing industry who have been exposed to DCP and DCM as single agents or as mixtures, as well as with public somatic mutation data from biliary tract cancers were mined for the presence of the experimentally defined DCP and DCM mutational signatures. Results and discussions Liver tumours from DCP and DCM exposed mice had distinct somatic mutations patterns compared to spontaneous liver tumours from vehicle/sham-exposed mice. While mutations in DCP-exposed mice were dominated by C:G>T:A transitions, the most frequent types of mutations in DCM-exposed mice were T:A>A:T and T:A>C:G substitutions. An average of 10.3 somatic mutations was observed per Mb in tumours from DCM-exposed mice, approximately 3- fold higher than in DCP-exposed or sham-treated mice. The mutation patterns found in DCP-exposed mice, but not DCMexposed mice, presented some similarities with the mutational signature observed in cholangiocarcinomas of Japanese patients with occupational DCP/DCM exposure history. Conclusion These results show that the analysis of tumour genomes from mouse carcinogenicity assays can support the characterisation of mutational signatures of carcinogenic compounds relevant to human exposures

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

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.