Genomics reveals historic and contemporary transmission dynamics of a bacterial disease among wildlife and livestock

Whole-genome sequencing has provided fundamental insights into infectious disease epidemiology, but has rarely been used for examining transmission dynamics of a bacterial pathogen in wildlife. In the Greater Yellowstone Ecosystem (GYE), outbreaks of brucellosis have increased in cattle along with rising seroprevalence in elk. Here we use a genomic approach to examine Brucella abortus evolution, cross-species transmission and spatial spread in the GYE. We find that brucellosis was introduced into wildlife in this region at least five times. The diffusion rate varies among Brucella lineages (∼3 to 8 km per year) and over time. We also estimate 12 host transitions from bison to elk, and 5 from elk to bison. Our results support the notion that free-ranging elk are currently a self-sustaining brucellosis reservoir and the source of livestock infections, and that control measures in bison are unlikely to affect the dynamics of unrelated strains circulating in nearby elk populations

Effects of Inactivated \u3ci\u3eMycobacterium bovis\u3c/i\u3e Vaccination on Molokai-Origin Wild Pigs Experimentally Infected with Virulent \u3ci\u3eM. bovis\u3c/i\u3e

The wild pig population on Molokai, Hawaii, USA is a possible reservoir for bovine tuberculosis, caused by Mycobacterium bovis, and has been implicated in decades past as the source of disease for the island’s domestic cattle. Heat-inactivated vaccines have been effective for reducing disease prevalence in wild boar in Spain and could prove useful for managing M. bovis in Molokai wild pigs. We designed an experiment to test this vaccine in wild pigs of Molokai genetics. Fifteen 3–4-month-old pigs were orally administered 106–107 colony forming units (cfu) of heat-inactivated M. bovis (Vaccinates; n = 8; 0.2 mL) or phosphate buffered saline (Controls; n = 7; 0.2 mL). Each dose was administered in a 0.5 mL tube embedded in a fruit candy/cracked corn mix. Boosters were given seven weeks post-prime in the same manner and dose. Nineteen weeks post-prime, pigs were orally challenged with 1 × 106 cfu of virulent M. bovis. Twelve weeks post-challenge, pigs were euthanized and necropsied, at which time 23 different tissues from the head, thorax, and abdomen were collected and examined. Each tissue was assigned a lesion score. Ordinal lesion score data were analyzed using non-zarametric Wilcoxon Signed Rank test. Effect size was calculated using Cohen’s d. Four of eight Vaccinates and four of seven Controls had gross and microscopic lesions, as well as culture-positive tissues. Vaccinates had statistically lower lesion scores than Controls in the following areas: gross thoracic lesion scores (p = 0.013 Cohen’s d = 0.33) and microscopic thoracic lesion scores (p = 0.002, Cohen’s d = 0.39). There were no differences in head lesion scores alone, both gross and microscopic, nor were there differences when comparing combined gross and microscopic head and thoracic lesion scores. These results are indicative that this vaccination protocol affords a modest degree of infection containment with this vaccine in Molokai wild pigs

Genomics of Brucellosis in Wildlife and Livestock of the Greater Yellowstone Ecosystem

Brucellosis, a disease caused by the bacterium Brucella abortus, has recently been expanding its distribution in the Greater Yellowstone Ecosystem (GYE), with increased outbreaks in cattle and rising seroprevalence in elk (Cervus elaphus) over the past decade. Genetic studies suggest elk are a primary source of recent transmission to cattle. However, these studies are based on Variable Number Tandem Repeat (VNTR) data, which are limited in assessing and quantifying transmission among species. The goal of this study was to (i) investigate the introduction history of B. abortus in the GYE, (ii) identify B. abortus lineages associated with host species and/or geographic localities, and (iii) quantify transmission across wildlife and livestock host species and populations. We sequenced B. abortus whole genomes (n= 207) derived from isolates collected from three host species (bison, elk, cattle) over the past 30 years, throughout the GYE. We identified genetic variation among isolates, and applied a spatial diffusion phylogeographic modeling approach that incorporated temporal information from sampling. Based on these data, our results suggest four divergent Brucella lineages, with a time to most recent common ancestor of ~130 years ago, possibly representing a minimum of four brucellosis introductions into the GYE. Two Brucella lineages were generally clustered by geography. Evidence for cross-species transmission was detected among all species, though most events occur within species and herds. Understanding transmission dynamics is imperative for implementing effective control measures and may assist in identifying source populations responsible for past and future brucellosis infections in wildlife and outbreaks in livestock

Whole-genome sequencing of chronic lymphocytic leukemia identifies subgroups with distinct biological and clinical features

The value of genome-wide over targeted driver analyses for predicting clinical outcomes of cancer patients is debated. Here, we report the whole-genome sequencing of 485 chronic lymphocytic leukemia patients enrolled in clinical trials as part of the United Kingdom's 100,000 Genomes Project. We identify an extended catalog of recurrent coding and noncoding genetic mutations that represents a source for future studies and provide the most complete high-resolution map of structural variants, copy number changes and global genome features including telomere length, mutational signatures and genomic complexity. We demonstrate the relationship of these features with clinical outcome and show that integration of 186 distinct recurrent genomic alterations defines five genomic subgroups that associate with response to therapy, refining conventional outcome prediction. While requiring independent validation, our findings highlight the potential of whole-genome sequencing to inform future risk stratification in chronic lymphocytic leukemia

Addressing challenges of molecular precision diagnostics for cancer patients in the genomics era

Genomic technologies have proven successful in research-led studies and their potential for characterising prognostic and predictive biomarkers in cancer patients has been recognised. However, such techniques and findings are yet to be applied in routine diagnostic environments. Indeed, many challenges remain before being able to establish a comprehensive genomics classification for all cancer patients, not least pre-analytical difficulties and variants interpretation. One of the most important pre-analytical challenges is the use of formalin-fixed paraffin-embedded (FFPE) tissues which are far more widely available in diagnostics compared with alternative fresh-frozen (FF) tissue samples. The key issue is that FFPE treatment of the tissue greatly impacts on the DNA quality and downstream analysis. This is important because genomic studies have discovered a significant number of mutations in multiple cancer driver genes. The presence of these mutations informs on patients' prognoses or responses to treatment and guides clinical management. With an increasing number of targeted therapies available it is critical to stratify patients using genomics technologies. However, current clinically available tests fail to identify known driver mutations in the majority of patients. The overall objective of the work presented in this thesis was to address current challenges and limitations of FFPE testing by improving knowledge in genomic technology and by providing a means to offer genomic-guided precision medicine to a greater number of cancer patients. The specific aims were (i) to explore the feasibility of using FFPE tissue from patients with solid tumour for clinical grade whole genome sequencing (WGS) and (ii) to use WGS and targeted sequencing data to search for novel genomic biomarkers of aggressive disease in CLL patients. In Chapter 3, a prospective WGS study of cancer patients using 52 matching FF and FFPE samples was performed. This highlighted 71&percnt; agreement in somatic single nucleotide variants and revealed sub-optimal copy number alteration (CNA) detection from FFPE samples (44&percnt; median correlation with FF) due to non-uniform coverage. In Chapter 4, after pre-analytical investigation and experimental optimisation, CNA detection was improved significantly using lower reverse crosslinking temperature in FFPE DNA extraction (80&deg;C or 65&deg;C depending the methods) or by controlling formalin fixation. The results showed that WGS data derived from FFPE tissue derived DNA is capable of detecting 96&percnt; of clinically actionable variants (including 97&percnt; of CNAs) and therefore the approach is considered suitable for implementation in the routine clinical diagnostic environment. Furthermore, the study of several clinical trial CLL cohorts presented in this thesis highlighted multiple novel genomic biomarkers of aggressive disease in CLL patients. In Chapter 5 relapse/refractory patients (R/R) were clustered according to the driver mutations identified using targeted sequencing; a multiple-hit profile defined as recurrent combinations of &ge; 2 mutations of TP53, SF3B1 and ATM was identified. This multiple-hit profile, found in 19&percnt; of patients was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (p=0.003). Other potential biomarkers were identified by exploring the genome of 320 CLL patients, such as mutational signatures. Collectively, the work performed and the data generated provide new insights to overcome current challenges linked to the establishment of genomics technologies in the clinic in order to offer precision medicine to cancer patients.</p

Addressing challenges of molecular precision diagnostics for cancer patients in the genomics era

Genomic technologies have proven successful in research-led studies and their potential for characterising prognostic and predictive biomarkers in cancer patients has been recognised. However, such techniques and findings are yet to be applied in routine diagnostic environments. Indeed, many challenges remain before being able to establish a comprehensive genomics classification for all cancer patients, not least pre-analytical difficulties and variants interpretation. One of the most important pre-analytical challenges is the use of formalin-fixed paraffin-embedded (FFPE) tissues which are far more widely available in diagnostics compared with alternative fresh-frozen (FF) tissue samples. The key issue is that FFPE treatment of the tissue greatly impacts on the DNA quality and downstream analysis. This is important because genomic studies have discovered a significant number of mutations in multiple cancer driver genes. The presence of these mutations informs on patients' prognoses or responses to treatment and guides clinical management. With an increasing number of targeted therapies available it is critical to stratify patients using genomics technologies. However, current clinically available tests fail to identify known driver mutations in the majority of patients. The overall objective of the work presented in this thesis was to address current challenges and limitations of FFPE testing by improving knowledge in genomic technology and by providing a means to offer genomic-guided precision medicine to a greater number of cancer patients. The specific aims were (i) to explore the feasibility of using FFPE tissue from patients with solid tumour for clinical grade whole genome sequencing (WGS) and (ii) to use WGS and targeted sequencing data to search for novel genomic biomarkers of aggressive disease in CLL patients. In Chapter 3, a prospective WGS study of cancer patients using 52 matching FF and FFPE samples was performed. This highlighted 71% agreement in somatic single nucleotide variants and revealed sub-optimal copy number alteration (CNA) detection from FFPE samples (44% median correlation with FF) due to non-uniform coverage. In Chapter 4, after pre-analytical investigation and experimental optimisation, CNA detection was improved significantly using lower reverse crosslinking temperature in FFPE DNA extraction (80°C or 65°C depending the methods) or by controlling formalin fixation. The results showed that WGS data derived from FFPE tissue derived DNA is capable of detecting 96% of clinically actionable variants (including 97% of CNAs) and therefore the approach is considered suitable for implementation in the routine clinical diagnostic environment. Furthermore, the study of several clinical trial CLL cohorts presented in this thesis highlighted multiple novel genomic biomarkers of aggressive disease in CLL patients. In Chapter 5 relapse/refractory patients (R/R) were clustered according to the driver mutations identified using targeted sequencing; a multiple-hit profile defined as recurrent combinations of ≥ 2 mutations of TP53, SF3B1 and ATM was identified. This multiple-hit profile, found in 19% of patients was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (p=0.003). Other potential biomarkers were identified by exploring the genome of 320 CLL patients, such as mutational signatures. Collectively, the work performed and the data generated provide new insights to overcome current challenges linked to the establishment of genomics technologies in the clinic in order to offer precision medicine to cancer patients.</p

Effects of Inactivated \u3ci\u3eMycobacterium bovis\u3c/i\u3e Vaccination on Molokai-Origin Wild Pigs Experimentally Infected with Virulent \u3ci\u3eM. bovis\u3c/i\u3e

The wild pig population on Molokai, Hawaii, USA is a possible reservoir for bovine tuberculosis, caused by Mycobacterium bovis, and has been implicated in decades past as the source of disease for the island’s domestic cattle. Heat-inactivated vaccines have been effective for reducing disease prevalence in wild boar in Spain and could prove useful for managing M. bovis in Molokai wild pigs. We designed an experiment to test this vaccine in wild pigs of Molokai genetics. Fifteen 3–4-month-old pigs were orally administered 106–107 colony forming units (cfu) of heat-inactivated M. bovis (Vaccinates; n = 8; 0.2 mL) or phosphate buffered saline (Controls; n = 7; 0.2 mL). Each dose was administered in a 0.5 mL tube embedded in a fruit candy/cracked corn mix. Boosters were given seven weeks post-prime in the same manner and dose. Nineteen weeks post-prime, pigs were orally challenged with 1 × 106 cfu of virulent M. bovis. Twelve weeks post-challenge, pigs were euthanized and necropsied, at which time 23 different tissues from the head, thorax, and abdomen were collected and examined. Each tissue was assigned a lesion score. Ordinal lesion score data were analyzed using non-zarametric Wilcoxon Signed Rank test. Effect size was calculated using Cohen’s d. Four of eight Vaccinates and four of seven Controls had gross and microscopic lesions, as well as culture-positive tissues. Vaccinates had statistically lower lesion scores than Controls in the following areas: gross thoracic lesion scores (p = 0.013 Cohen’s d = 0.33) and microscopic thoracic lesion scores (p = 0.002, Cohen’s d = 0.39). There were no differences in head lesion scores alone, both gross and microscopic, nor were there differences when comparing combined gross and microscopic head and thoracic lesion scores. These results are indicative that this vaccination protocol affords a modest degree of infection containment with this vaccine in Molokai wild pigs

The Road to Dissemination: The Concept of Oligometastases and the Barriers for Widespread Disease.

Over the last years, the oligometastatic disease state has gained more and more interest, and randomized trials are now suggesting an added value of stereotactic radiotherapy on all macroscopic disease in oligometastatic patients; but what barriers could impede widespread disease in some patients? In this review, we first discuss the concept of oligometastatic disease and some examples of clinical evidence. We then explore the route to dissemination: the hurdles a tumoral clone has to overtake before it can produce efficient and widespread dissemination. The spectrum theory argues that the range of metastatic patterns encountered in the clinic is the consequence of gradually obtained metastatic abilities of the tumor cells. Tumor clones can obtain these capabilities by Darwinian evolution, hence early in their genetic progression tumors might produce only a limited number of metastases. We illustrate selective dissemination by discussing organ tropism, the preference of different cancer (sub)types to metastasize to certain organs. Finally we discuss biomarkers that may help to distinguish the oligometastatic state.info:eu-repo/semantics/publishe

Transmission of Brucellosis from Elk to Cattle and Bison, Greater Yellowstone Area, USA, 2002–2012

Bovine brucellosis has been nearly eliminated from livestock in the United States. Bison and elk in the Greater Yellowstone Area remain reservoirs for the disease. During 1990–2002, no known cases occurred in Greater Yellowstone Area livestock. Since then, 17 transmission events from wildlife to livestock have been investigated