BACKGROUND:While liquid biopsy has potential to transform cancer diagnostics through minimally-invasive detection and monitoring of tumors, the impact of preanalytical factors such as the timing and anatomical location of blood draw is not well understood. METHODS:To address this gap, we leveraged pet dogs with spontaneous cancer as a model system for liquid biopsy of plasma cell-free DNA (cfDNA), as their compressed disease timeline facilitates rapid diagnostic benchmarking. We examined key cfDNA metrics, including DNA concentration in plasma, as well as tumor fraction and fragment size ratio derived from ultra low pass whole genome sequencing. RESULTS:We show that liquid biopsy metrics in dogs are consistent with reported human metrics. The tumor content of samples is slightly higher when blood is obtained from a central vein closer to the tumor. Metrics also differ between lymphoma and non-hematopoietic cancers, supporting cancer-type-specific interpretation. Disease status tracks with liquid biopsy findings over the course of treatment over both short (hours to days) and long (weeks to months) time frames, and trends of increased tumor fraction and other metrics are observed prior to clinical relapse in dogs with lymphoma and osteosarcoma. CONCLUSIONS:Together, these data support the utility of pet dogs with cancer as a relevant system for advancing liquid biopsy platforms.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive hematopoietic neoplasm that affects humans as well as dogs. While previous studies on canine DLBCL (cDLBCL) have significantly advanced our understanding of the disease, the majority of this research has relied on whole-exome sequencing, which is limited in its ability to detect copy number aberrations and other genomic changes beyond coding regions. Furthermore, many of these studies lack sufficient clinical follow-up data, making it difficult to draw meaningful associations between genetic variants and patient outcomes. Our study aimed to characterize the mutational landscape of cDLBCL using whole-genome sequencing of matched tumor-normal samples obtained from a cohort of 43 dogs previously enrolled in a clinical trial for which longitudinal follow-up was available. We focused on identifying genes that were significantly or recurrently mutated with coding point mutations, copy number aberrations, and their associations with patient outcomes. We identified 26 recurrently mutated genes, 18 copy number gains, and 8 copy number losses. Consistent with prior studies, the most commonly mutated genes included TRAF3, FBXW7, POT1, TP53, SETD2, DDX3X and TBL1XR1. The most prominent copy number gain occurred on chromosome 13, overlapping key oncogenes such as MYC and KIT, while the most frequent deletion was a focal loss on chromosome 26, encompassing IGL, PRAME, GNAZ, RAB36, RSPH14, and ZNF280B. Notably, our set of recurrently mutated genes was significantly enriched with genes involved in epigenetic regulation. In particular, we identified hotspot mutations in two histone genes, H3C8, and LOC119877878, resulting in H3K27M alterations predicted to dysregulate gene expression. Finally, a survival analysis revealed that H3K27M mutations in H3C8 were associated with increased hazard ratios for progression-free survival. No copy number aberrations were associated with survival. These findings underscore the critical role of epigenetic dysregulation in cDLBCL and affirm the dog as a relevant large animal model for interrogating the biological activity of novel histone-modifying treatment strategies.

Background: To explore the safety and utility of combining low dose single-agent doxorubicin with a canine specific anti-CD20 monoclonal antibody (1E4-cIgGB) in client owned dogs with untreated B-cell lymphoma. Animals: Forty-two client-owned dogs with untreated B-cell lymphoma. Methods: A prospective, single arm, open label clinical trial of dogs with B-cell lymphoma were enrolled to receive 1E4-cIgGB and doxorubicin in addition to 1 of 3 immunomodulatory regimens. B-cell depletion was monitored by flow cytometry performed on peripheral blood samples at each visit. Results: Dogs demonstrated a statistically significant depletion in CD21+ B-cells 7 days following the first antibody infusion (median fraction of baseline at 7 days = 0.04, P < .01) that persisted throughout treatment (median fraction of baseline at 21 days = 0.01, P < .01) whereas CD5+ T-cells remained unchanged (median fraction of baseline at 7 days = 1.05, P = .88; median fraction of baselie at 7 days = 0.79, P = .42; Figure 1; Supplemental Table 3). Recovery of B-cells was delayed, with at Day 196, only 6/17 dogs (35%) remaining on the study had CD21+ counts >0.5 of baseline, indicating sustained B cell depletion at 4+ months after the final treatment. 1E4-cIgGB was well tolerated with only 1 dog exhibiting a hypersensitivity event within minutes of the last antibody infusion. Conclusions: The canine 1E4-cIgGB anti-CD20 monoclonal antibody is apparently safe when administered with doxorubicin and effectively depletes B-cells in dogs with DLBCL.

Pet dogs develop spontaneous diffuse large B cell lymphoma (DLBCL), and veterinary clinical trials have been employed to treat canine DLBCL and to inform clinical trials for their human companions. A challenge that remains is selection of treatment to improve outcomes. The dogs in this study were part of a larger clinical trial evaluating the use of combinations of doxorubicin chemotherapy, anti-CD20 monoclonal antibody, and one of three small molecule inhibitors: KPT-9274, TAK-981, or RV1001. We hypothesized that significant differential expression of genes (DEGs) in the tumors at baseline could help predict which dogs would respond better to each treatment based on the molecular pathways targeted by each drug. To this end, we evaluated gene expression in lymph node aspirates from 18 trial dogs using the NanoString nCounter Canine Immuno-oncology (IO) Panel. We defined good responders as those who relapsed after 90 days, and poor responders as those who relapsed prior to 90 days. We analyzed all dogs at baseline and compared poor responders to good responders, and found increased CCND3 correlated with poor prognosis and increased CD36 correlated with good prognosis, as is observed in humans. There was minimal DEG overlap between treatment arms, prompting separate analyses for each treatment cohort. Increased CREBBP and CDKN1A for KPT-9274, increased TLR3 for TAK-981, and increased PI3Kδ , AKT3 , and PTEN , and decreased NRAS for RV1001 were associated with better prognoses. Trends for selected candidate biomarker genes were confirmed via qPCR. Our findings emphasize the heterogeneity in DLBCL, similarities and differences between canine and human DLBCL, and ultimately identify biomarkers that may help guide the choice of chemoimmunotherapy treatment in dogs.

Table S1: KTN0158 Dosing Cohorts Table S2: Summary of Anti-KTN0158 Antibody Titers in Dogs with Mast Cell Tumors Following KTN0158 Treatment

Cutaneous T-cell lymphoma (CTCL) is an uncommon type of lymphoma involving malignant skin-resident or skin-homing T cells. Canine epitheliotropic lymphoma (EL) is the most common form of CTCL in dogs, and it also spontaneously arises from T lymphocytes in the mucosa and skin. Clinically, it can be difficult to distinguish early-stage CTCLs apart from other forms of benign interface dermatitis (ID) in both dogs and people. Our objective was to identify novel biomarkers that can distinguish EL from other forms of ID, and perform comparative transcriptomics of human CTCL and canine EL. Here, we present a retrospective gene expression study that employed archival tissue from biorepositories. We analyzed a discovery cohort of 6 canines and a validation cohort of 8 canines with EL which occurred spontaneously in client-owned companion dogs. We performed comparative targeted transcriptomics studies using NanoString to assess 160 genes from lesional skin biopsies from the discovery cohort and 800 genes from the validation cohort to identify any significant differences that may reflect oncogenesis and immunopathogenesis. We further sought to determine if gene expression in EL and CTCL are conserved across humans and canines by comparing our data to previously published human datasets. Similar chemokine profiles were observed in dog EL and human CTCL, and analyses were performed to validate potential biomarkers and drivers of disease. In dogs, we found enrichment of T cell gene signatures, with upregulation of IFNG, TNF, PRF1, IL15, CD244, CXCL10, and CCL5 in EL in dogs compared to healthy controls. Importantly, CTSW, TRAT1 and KLRK1 distinguished EL from all other forms of interface dermatitis we studied, providing much-needed biomarkers for the veterinary field. XCL1/XCL2 were also highly specific of EL in our validation cohort. Future studies exploring the oncogenesis of spontaneous lymphomas in companion animals will expand our understanding of these disorders. Biomarkers may be useful for predicting disease prognosis and treatment responses. We plan to use our data to inform future development of targeted therapies, as well as for repurposing drugs for both veterinary and human medicine.

Serum KTN0158 concentration over time

Supplementary Table 1: Adverse Events

Osteosarcoma constitutes a rare childhood cancer that is associated with dismal outcomes. As such, there is a need to develop non-invasive immunotherapeutic approaches to treat this aggressive malignancy. To address this need, we described the cellular heterogeneity in spontaneously occurring canine osteosarcoma tumors then evaluated how treatment with adjuvant immunotherapy altered the tumor microenvironment. We investigated the effects of a triple drug immunotherapeutic (orally administered losartan, ladarixin, and toceranib) that was designed to deplete immune suppressive macrophages and promote antitumor immunity. The treated cohort of dogs were given the adjuvant immunotherapy for 14 days prior to amputation, while naïve samples were collected from unrelated dogs undergoing amputation to treat a primary osteosarcoma tumor. In total, we completed single cell RNA sequencing on 6 naïve dogs and 6 treated dogs to profile just under 100,000 cells. Unsupervised clustering revealed 30 unique cell populations with T cells, macrophages, and neutrophils making up the majority of immune infiltrates. Evaluation of changes to the cellular proportions following treatment revealed a marked reduction in macrophages, which suggests the combination immunotherapy was able to alter the tumor microenvironment. Overall, this study provides unique insights into the heterogeneity within canine osteosarcoma tumors and highlights the impacts of adjuvant immunotherapy on tumor infiltrating immune cell transcriptional programs. T32 OD012201 U01 CA5341458 Shipley family foundation

Pet dogs develop spontaneous cancers at a rate estimated to be five times higher than that of humans, providing a unique opportunity to study disease biology and evaluate novel therapeutic strategies in a model system that possesses an intact immune system and mirrors key aspects of human cancer biology. Despite decades of interest, effective utilization of pet dog cancers has been hindered by a limited repertoire of necessary cellular and molecular reagents for both in vitro and in vivo studies, as well as a dearth of information regarding the genomic landscape of these cancers. Recently, many of these critical gaps have been addressed through the generation of a highly annotated canine reference genome, the creation of several tools necessary for multi-omic analysis of canine tumours, and the development of a centralized repository for key genomic and associated clinical information from canine cancer patients, the Integrated Canine Data Commons. Together, these advances have catalysed multidisciplinary efforts designed to integrate the study of pet dog cancers more effectively into the translational continuum, with the ultimate goal of improving human outcomes. The current review summarizes this recent progress and provides a guide to resources and tools available for comparative study of pet dog cancers.

Signal transducer and activator of transcription 3 (STAT3) dysregulation has been characterized in canine OS, with previous data suggesting that constitutive STAT3 activation contributes to survival and proliferation in OS cell lines in vitro. Recently, the contribution of STAT3 to tumour metabolism has been described across several tumour histologies, and understanding the metabolic implications of STAT3 loss may elucidate novel therapeutic approaches with synergistic activity. The objective of this work was to characterize metabolic benchmarks associated with STAT3 loss in canine OS. STAT3 expression and activation was evaluated using western blotting in canine OS cell lines OSCA8 and Abrams. STAT3 was deleted from these OS cell lines using CRISPR-Cas9, and the effects on proliferation, invasion and metabolism (respirometry, intracellular lactate) were determined. Loss of STAT3 was associated with decreased basal and compensatory glycolysis in canine OS cell lines, without modulation of cellular proliferation. Loss of STAT3 also resulted in diminished invasive capacity in vitro. Interestingly, the absence of STAT3 did not impact sensitivity to doxorubicin in vitro. Our data demonstrate that loss of STAT3 modulates features of aerobic glycolysis in canine OS impacting capacities for cellular invasions, suggesting a role for this transcription factor in metastasis.

The characterization of immortalized canine osteosarcoma (OS) cell lines used for research has historically been based on phenotypic features such as cellular morphology and expression of bone specific markers. With the increasing use of these cell lines to investigate novel therapeutic approaches prior to in vivo translation, a much more detailed understanding regarding the genomic landscape of these lines is required to ensure accurate interpretation of findings. Here we report the first whole genome characterization of eight canine OS cell lines, including single nucleotide variants, copy number variants and other structural variants. Many alterations previously characterized in primary canine OS tissue were observed in these cell lines, including TP53 mutations, MYC copy number gains, loss of CDKN2A, PTEN, DLG2, MAGI2, and RB1 and structural variants involving SETD2, DLG2 and DMD. These data provide a new framework for understanding how best to incorporate in vitro findings generated using these cell lines into the design of future clinical studies involving dogs with spontaneous OS.

Background We reported recently in a spontaneous canine model of metastatic osteosarcoma (OS) that oral treatment with two repurposed agents targeting monocyte migration (losartan) and MDSC and Tregs (toceranib) induced partial responses in 4/16 dogs (25%) with another 4 dogs experiencing durable stable disease (SD), for a clinical benefit rate of 50%. In the current study, we evaluated the utility of incorporating ladarixin (an orally bioavailable allosteric CXCR1/2 antagonist) into the losartan/toceranib regimen in 15 dogs with OS metastatic to the lungs. Methods The effects of treatment on lung metastases, circulating cytokine concentrations, and gene expression profiles in PBMC were assessed in this new study. Studies are also ongoing to determine whether this same approach has activity in the adjuvant setting in dogs with appendicular OS treated prior to and continuously following amputation, in lieu of standard cytotoxic adjuvant chemotherapy. To date, 18 animals have been enrolled in the adjuvant trial, and analysis of the impact on MFI and OST are ongoing as data matures. Results Of enrolled dogs with greater than 60 days of follow up, 1 dog underwent a complete response (400+ days) and another 4 dogs experienced PR, for an objective response rate of 36%; another 5 dogs exhibited durable SD, for an overall clinical benefit rate of 71%. The 3-drug protocol was generally well-tolerated, with no adverse events other than those associated with toceranib alone (gastrointestinal signs). Transcriptomic analysis of PBMCs from 8 dogs on days 0 and 14 using Nanostring revealed significant downregulation of 30 immune genes, including IRF2, IRF8, CTLA4, NFKB, and CCR2, and upregulation of 176 genes, including IL21, CCL19, B7-H3, IL17A/B. Pathway analysis demonstrated significant downregulation of IFN-γ and IFN-α response pathways, and upregulation of β-catenin signaling pathways. Conclusions In summary, these findings in a canine spontaneous osteosarcoma model indicate that the combined losartan/ladarixin/toceranib immunotherapy protocol is biologically active and can effectively modify the immune suppressive tumor microenvironment to generate spontaneous antitumor activity in dogs with advanced OS lung metastases.

BACKGROUND:Ethiopia's high neonatal mortality rate led to the government's 2013 introduction of Community-Based Newborn Care (CBNC) to bring critical prevention and treatment interventions closer to communities in need. However, complex behaviors that are deeply embedded in social and cultural norms continue to prevent women and newborns from getting the care they need. A demand creation strategy was designed to create an enabling environment to support appropriate maternal, newborn, and child health (MNCH) behaviors and CBNC. We explored the extent to which attitudes and behaviors during the prenatal and perinatal periods varied by the implementation strength of the Demand Creation Strategy for MNCH-CBNC.METHODS:Using an embedded, multiple case study design, we purposively selected four kebeles (villages) from two districts with different levels of implementation strength of demand creation activities. We collected information from a total of 150 key stakeholders across kebeles using multiple qualitative methods including in-depth interviews, focus group discussions, and illness narratives; sessions were transcribed into English and coded using NVivo 10.0. We developed case reports for each kebele and a final cross-case report to compare results from high and low implementation strength kebeles.RESULTS:We found that five MNCH attitudes and behaviors varied by implementation strength. In high implementation strength kebeles women felt more comfortable disclosing their pregnancy early, women sought antenatal care (ANC) in the first trimester, families did not have fatalistic ideas about newborn survival, mothers sought care for sick newborns in a timely manner, and newborns received care at the health facility in less than an hour. We also found changes across all kebeles that did not vary by implementation strength, including male engagement during pregnancy and a preference for giving birth at a health facility.CONCLUSIONS:Findings suggest that a demand creation approach-combining participatory approaches with community empowering strategies-can promote shifts in behaviors and attitudes to support the health of mothers and newborns, including use of MNCH services. Future studies need to consider the most efficient level of intervention intensity to make the greatest impact on MNCH attitudes and behaviors.

Abstract Pet dogs are a powerful spontaneous model for human cancers. Dogs develop the same cancers that humans do, with striking similarities at the clinical, histopathologic, and genomic levels. These cancers have an accelerated clinical course compared with their human counterparts, and canine patients undergo many of the same interventions and treatments as human patients. The dog model thus offers the opportunity to implement rapid clinical trials informative for human medicine. We are developing liquid biopsy in dogs to further refine its clinical use by addressing questions that are difficult to assess in human patients. We have four ongoing pilot studies examining several different aspects of blood biopsy in canine patients. (1) We characterized the yield and tumor fraction of cell-free DNA (cfDNA) in nine canine cancer histologies using prospectively collected and banked plasma samples. (2) We tested the correlation between large-scale somatic copy number aberrations (SCNAs) in 24 osteosarcoma tumor samples and matched plasma samples and are now preparing to perform whole-exome sequencing (WES) of these samples to assess the concordance of SNVs and INDELs. (3) We investigated the use of cfDNA as a biomarker for response to therapy in four dogs with multicentric lymphoma treated with L-asparaginase via longitudinal blood biopsy. (4) We are optimizing phlebotomy techniques and characterizing short-term cfDNA dynamics by testing the effect of blood draw site and time of day on yield and tumor fraction. Samples from three sites (jugular, saphenous, and cephalic veins) were collected in a cohort of ten patients, and collection of samples at three time points throughout the day in a separate cohort is ongoing. (1) The majority (93%) of plasma samples yielded or were projected to yield sufficient DNA (>2ng) for ultra-low-pass whole-genome sequencing, and 37% had a tumor fraction ≥10%, allowing for WES given sufficient input DNA. (2) We found a strong correlation (Spearman coefficient of 0.74) between SCNAs in 13 tumor/plasma pairs with tumor fraction ≥10%. We are moving forward with WES of these cfDNA samples to examine simple somatic mutations. (3) Longitudinal cfDNA yield and tumor fraction correlated with response to therapy in three of four lymphoma patients. (4) Sample collection and sequencing are under way for the studies on the effect of blood draw site and time of day. Our studies demonstrated the feasibility of blood biopsy in dogs, the high concordance between cfDNA and tumor samples, and suggested that blood biopsy is a promising method for monitoring response to therapy. Emerging results will help to optimize phlebotomy protocols and characterize cfDNA dynamics over short time scales, as well as assessing in more detail the concordance between cfDNA and matched tumor samples. Further development of blood biopsy in dogs has the potential to improve and accelerate clinical application of this technology in human medicine and to facilitate further high-impact comparative translational studies in the dog model. Citation Format: Kate Megquier, Kan Xiong, Heather L. Gardner, Justin Rhoades, Viktor Adalsteinsson, Cheryl A. London, Elinor K. Karlsson. Advancing blood biopsy through the canine comparative model [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A58.

Cancer progression is an evolutionary process. During this process, evolving cancer cell populations encounter restrictive ecological niches within the body, such as the primary tumor, circulatory system, and diverse metastatic sites. Efforts to prevent or delay cancer evolution-and progression-require a deep understanding of the underlying molecular evolutionary processes. Herein we discuss a suite of concepts and tools from evolutionary and ecological theory that can inform cancer biology in new and meaningful ways. We also highlight current challenges to applying these concepts, and propose ways in which incorporating these concepts could identify new therapeutic modes and vulnerabilities in cancer.

Introduction: A well-functioning, responsive referral system relies on patient compliance in order to address newborn health problems that cannot be managed by lower level health facilities. Given that early identification and management of young infants with possible serious bacterial infection is fundamental to community-based newborn care. Objective:Thispaper presents the findings of an assessment ofadherence to referral. Methods:Usinga cross-sectional design with mixed qualitative and quantitative methods in five zones in two regions this study looked at sick young infants (0-2 months) whose caretakers sought care for them from a health post and who were classified with possible serious bacterial infectionin the 12 months prior to the study. We reviewed clinical records of possible serious bacterial infectioncases at health posts and health centers and conducted interviews with caretakers of referred possible serious bacterial infection cases and health workers.

Osteosarcoma (OS) is a rare, metastatic, human adolescent cancer that also occurs in pet dogs. To define the genomic underpinnings of canine OS, we performed multi-platform analysis of OS tumors from 59 dogs, including whole genome sequencing ( n = 24) and whole exome sequencing (WES; n = 13) of primary tumors and matched normal tissue, WES ( n = 10) of matched primary/metastatic/normal samples and RNA sequencing ( n = 54) of primary tumors. We found that canine OS recapitulates features of human OS including low point mutation burden (median 1.98 per Mb) with a trend towards higher burden in metastases, high structural complexity, frequent TP53 (71%), PI3K pathway (37%), and MAPK pathway mutations (17%), and low expression of immune-associated genes. We also identified novel features of canine OS including putatively inactivating somatic SETD2 (42%) and DMD (50%) aberrations. These findings set the stage for understanding OS development in dogs and humans, and establish genomic contexts for future comparative analyses.

The formation of the CTSI One Health Alliance (COHA) network has generated the infrastructure necessary to support "Big Data" collaborative comparative and translational research in veterinary medicine. We describe the first step in the design, implementation, and dissemination of a collaborative information technology infrastructure that will serve the public and clinicians (COHA public/member based web site at https://ctsaonehealthalliance.org/) and its research focused COHA Research Workbench application. The core research infrastructure, TRANSLATOR (TRanslational ANimal Shared ColLAboraTive Observational Research), represents the foundation of a federated research-capable network to enable pooling large datasets from both electronic health records and publications. The public facing COHA website is a mechanism for both the dissemination of knowledge to the public and to foster collaborations amongst veterinary clinician scientists and the greater medical research community.